Synthesis of Calcium Phosphates in the Presence of Ionic Surfactants, Phospholipids and Mesoporous Silica Nanoparticles to Control the Bioceramic Mesostructure 50 nm Universidad Complutense de Madrid Facultad de Farmacia Departamento de Química Inorgánica y Bioinorgánica Síntesis de Fosfatos de Calcio en Presencia de Surfactantes Iónicos, Fosfolípidos y Nanopartículas de Sílice Mesoporosa para Controlar la Mesoestructura de la Biocerámica TESIS DOCTORAL Madrid, Septiembre 2015 Okan Mersinlioğlu Universidad Complutense de Madrid Facultad de Farmacia Departamento de Química Inorgánica y Bioinorgánica TESIS DOCTORAL Síntesis de Fosfatos de Calcio en Presencia de Surfactantes Iónicos, Fosfolípidos y Nanopartículas de Sílice Mesoporosa para Controlar la Mesoestructura de la Biocerámica Memoria presentada por Okan Mersinlioğlu para optar al grado de Doctor por la Universidad Complutense de Madrid Directores Dr. Antonio J. Salinas Sánchez y Dra. Blanca González Ortiz Madrid, Septiembre 2015 Universidad Complutense de Madrid Facultad de Farmacia Departamento de Química Inorgánica y Bioinorgánica DOCTORAL THESIS Synthesis of Calcium Phosphates in the Presence of Ionic Surfactants, Phospholipids and Mesoporous Silica Nanoparticles to Control the Bioceramic Mesostructure A dissertation presented by Okan Mersinlioğlu to apply for the degree of Doctor by Universidad Complutense de Madrid Supervisors Dr. Antonio J. Salinas Sánchez and Dra. Blanca González Ortiz Madrid, September 2015 i Universidad Complutense de Madrid Dr. Antonio J. Salinas Sánchez y Dra. Blanca González Ortiz, Profesores Titulares del Departamento de Química Inorgánica y Bioinorgánica de la Facultad de Farmacia de la Universidad Complutense de Madrid, en calidad de directores de esta Tesis Doctoral, CERTIFICAN: Que la presente memoria titulada “Synthesis of Calcium Phosphates in the Presence of Ionic Surfactants, Phospholipids and Mesoporous Silica Nanoparticles to Control the Bioceramics Mesostructure” ha sido realizado por Okan Mersinlioğlu bajo nuestra dirección, en el Departamento de Química Inorgánica y Bioinorgánica de la Facultad de Farmacia de la Universidad Complutense de Madrid, y que reúne las condiciones necesarias para ser presentada como requisito para optar al grado de Doctor por la Universidad Complutense de Madrid. Madrid, 22 de Septiembre de 2015 Dr. Antonio J. Salinas Sánchez Dra. Blanca González Ortiz iii Universidad Complutense de Madrid Dr. Antonio J. Salinas Sánchez and Dr. Blanca González Ortiz, Associate Professors of the Department of Inorganic and Bioinorganic Chemistry of Faculty of Pharmacy at Universidad Complutense de Madrid, Spain, as supervisors of this Doctoral Thesis, CERTIFICATE: That the present PhD dissertation entitled “Synthesis of Calcium Phosphates in the Presence of Ionic Surfactants, Phospholipids and Mesoporous Silica Nanoparticles to Control the Bioceramics Mesostructure” has been developed by Okan Mersinlioğlu under our supervision, at the Department of Inorganic and Bioinorganic Chemistry of Faculty of Pharmacy at Universidad Complutense de Madrid, and that it fulfils all the requirements to apply for the degree of Doctor by Universidad Complutense de Madrid. Madrid, September 22nd 2015 Dr. Antonio J. Salinas Sánchez Dr. Blanca González Ortiz v To Hanife, the most beautiful woman in my life&heart. Hayatımda ki & Kalbimde ki en güzel kadına, Hanifeme. To my family, Aileme, vii Acknowledgements This thesis would not have been possible without the support and advice of many of my colleagues and friends, and I want to thank them. A scientific work is never the result of individual efforts but always the result of teamwork. I thank my supervisors Dr. Antonio J. Salinas Sánchez and Dr. Blanca González Ortiz for all their support and patience during all of my study years. Thank you for believing in me. I thank Prof. Dr. María Vallet-Regí, head of the Research Group in Intelligent Biomaterials (GIBI), for the opportunity of doing this project and for all her support and ideas. Her work has been an inspiration for me. Also, I thank Dr. Juan Carlos Doadrio, head of the Department, for let me conclude this thesis in the department. I thank Prof. Dr. Matthias Epple for accepting me in his group in my three months stay at Institute of Inorganic Chemistry of the University of Duisburg−Essen, Germany. I also thank all my new friends at Institute of Inorganic Chemistry. I also thank all my friends and colleagues at Department of Inorganic and Bioinorganic Chemistry of the Universidad Complutense de Madrid. You are great and make my life richer and funnier than ever. Montse, your help is also unforgettable! Thanks for all those funny conversations. You are always a hard worker. Dani, María Teresa, África, Antonio Luis and Isabel, thank you for your time and help, and for sharing your energy. Ha acido un placer conoceros y gracias por ser tan amables. Jesus, Juan Peña and Vicky thank you for your time and for making me laugh every time. Alejandro Baeza, call me back if you ever go to Turkey again, if I’m not available, my expensive guide book will help you in Istanbul better than me. It’s unique and you know it. Miguel eres muy español tío, you are awesome and a very friendly person. And your son, Rodriguito, es más guapo que tú. Dear Merche, may be you don’t remember it but your help was unforgettable when we met at the commercial centre of Mar Negro region, Thank you! Ana Fontecha, I’ve never known anyone as optimistic and positive as you! Your help is unforgettable. Thank you very much! Sandra, enhorabuena por tu boda, wishing you the best in your new life. Edu Ruiz, thank you for your time and sharing your ideas! Thank you Jose, Pilar and Ana Martín for your support and help all time. Marina, there are no words to explain what a person you are. Thank you for being in my life and my life wouldn’t be the same without you. You are lucky because you have a great family in Murcia! You and your family are unforgettable! Mónica, Rafael, Nati, Paris, Rocío, Sandra Moltalvo and Gonzalo I couldn’t possibly have funnier friends than all of you. I was very happy with you guys, thank you all. Edu G and Ángel, thank you for hosting me in your house and making a ninja of me. It was very funny to be a part of your family, Thank you! Chico, you know that nunca me caes bien tío. Pero tuve muchos momentos increíbles contigo. Eres grande, muy grande colega, un abrazote muy fuerte. Dr. Fernando Conde, evet evet, eres muy grande, educado, abierto y muy internacional. I had a great time with you here, and in Turkey when we drank “Çay” and “Ayran”. ix Teşekkürler Acknowledgements in Turkish for my family and Turkish friends: Sekiz yıl önce İspanya’ya ilk adımlarımı attığımda, yeni doğmuş bebek gibiydim herşeyden habersiz, dilsiz, tecrübesiz, ailesiz ve eğitimsiz. Uçağa binerken nasılsa geri dönerim düşüncesiyle dokuz yıl geçirdiğim İspanya’da, hayatımın ikinci dönemini adlandırdığım bu bambaşka bölümde, bu emekleyen çocuğu büyüttüm ve eğittim… Zorlukları öğrendi, zoru başarmayı, imkansızı basitleştirdi, çok çalışmanın zeki olmaktan daha akıllıca bir hamle olduğunu keşfetti, her insanın fikrini dinlemeyi öğrendi, zoluklara göğüs görmeyi, sorumluluklarını…Geliştikçe yeni yerler keşfetti, keşfettikçe yeni insanlar tanıdı, tanıdıkça yeni hayatlar öğrendi, öğrendikçe insanlara güvenmeyide. Nasıl yaşarsa öyle insanlarla karşılasacağını öğrendi… Hayatta herseyin zor olduğunu ama pes etmemek kaydıyla her şeyın imkansız olmadığını, Ve herkesin değil, kendi yaptıklarının daha doğru olduğunu keşfetti… Hiçbir başarı tesadüf değildir ve her başarı mutlaka görünmeyen bir ekibin eseridir… Yürüdüğüm bu yolda bana destek olan, güvenen ve asla unutamayacağım insanları saymaya kalksam sayfalar yetmez… Saygı değer Hocam Yrd.Doç.Dr. M. Galip İçduygu, laboratuvarda bi şeker karıştırmanın kimyasal bir deney olduğunu bilmeden çıktığım bu yolda, destek olduğunuz, ve hayatımın en değerli anlarında İspanya macerasında bana cesaret verdiğiniz, önderlik ettiğiniz ve en önemlisi güvendiğiniz için sonsuz teşekkür ederim… En başta Aileme teşekkürler, Annem babama, Onur abim, Emine Yengeme ve şirin kızları Şevval- Ecrine, İsa kardeşim ve Ailesine, bana her daim destek oldunuz, Ailemden uzaklaştıkça geri dönülmez bir yola çıktığımı anladım… Kardeşim Abdullah, abisinin en değerlisi, abisini tanıyamasada onu hiç yalnız bırakmadı... Manevi ailem; Ahmet Yılmaz abim, Hatun yenge ve kızları… Mersinlioğlu ailesi; çınar ağacımız Hayati, Baştacımız Yadigar, abimiz Hakan, mühendisimiz Gökhan, kardeşlerim Yahya, Meryem, Esra, kalecimiz Osman ve aileleri... Bana kendi ailem gibi destek çıktığınız, inandığınız ve güvendiğiniz için teşekkürler… Siz olmasaydınız bu öyküm yarıda kalırdı, eminim… Sak Ailesi; Havva yengem, Mehmet abim, Kick bokscumuz Rıdvan ve Eşi Hanife, Furkan ve Sena, kardeşim Engin ve ailesine, Hollanda‘dan Hasan abim ve ailesine, Ayhan amcama ve beni hayata hazırlayarak hep güvenen Miyase yengeme, Mevlüt ve Zehra‘ya ; Ne kadar teşekkür etsem azdır. Beni sürekli kardeşiniz olarak gördüğünüz ve sürekli güleryüzlü davrandığınız için çok teşekkür ederim… Ömrümde tanıdığım tek ve en değerli Halama (unutulmaz böreklerine), Eniştem Ferhat abime (ve 20 lirasına), pırlanta gibi çocukları Fatih’e, Miyase ve şeyma’ya… Her daim güler yüzünüzü eksik etmediniz… Yılmaz Ailesine; Bayram dayım, Nurgül yengem, Bünyamin-Ensar ve Fezyullah kardeşlerim… Diğer büyük ailem… Her zaman destekçim oldunuz, kapınızı açık tuttunuz, ekmeğinizi paylaştınız… Ne kadar teşekkür etsem azdır. Bu zorlu yolda hep yanımda oldunuz ve güvendiniz… Teşekkür demek az kalır ama manevi anlamı büyüktür anlayana… Sebahittin hocam, Genç Nuran yengemiz ve değerli kızları… Gülüşünüze neşenize benide ortak ettiniz, teşekkürler… DJ Gökhan Tüysüz, gülüşünü esirgemedin, kardeşten öteydin, Nutellalı ekmeğini bölüştün ve en önemlisi İspanya’da evime misafirim oldun. Her zaman kardeşten öte sıcak samimi içten doğallığın ve desteklerin için teşekkür ederim.. Ailem ne kadarda büyükmüş… Bir çınar gibi sağlam, güçlü ve büyük…! Tübitak’ın saygı değer Yüksek mühendis ve Doktor araştırmacıları Yasemin, Ayşegül, Meral ve Ayşen… Her daim misafirliğinizden çok mutlu oldum, sizi tanımak çok güzeldi. Yasemin, çekinerek kapınızdan girdiğim gün ailenin inanılmaz saygın misafirperverliği ve bana içten samimi davranışları beni mahçup etti, asla unutmıycam! Mühendis Hanım Eda Elmas arkadaşım… Elmas gibi bir kalbe sahip dost ve arkadaşım. İçten samimi davranışların ve ögrencilik sıralarından beri devam eden dostluğumuz hiç solmasın... Senin gibi daha nice iyi dostlarıma, Betül Güneş Mercan, Sinem Mercan Kalaycı, dostum Ahmet Gökhan Taşpınar, taze anne Gündem, Ünlü fizikçim Adem Öztürk, Milli sporcumuz Aynur Samat, Murat bostan ve dahası… Daha isimlerini sayamayacağım ve anlıkta olsa hayatıma giren herkes; Malaga’dan Meltem, İstanbul’dan Kemal Dalyan, Madrit’ten Ankara’ya uzanan Ramazan çınar, Zaragoza’dan Ankara’ya uzanan kısa hikâyede Burçin ve Gökhan Güzel ailesi, Portekiz’deki esrarengiz rehberime, İtalya Venedik’te ki beni misafir eden arkadaşlara ve saçları limonlu İtalyan otobüs şoförüne, Slovenya’da ki esrarengiz tren istasyonuna, eski erasmus arkadaşlarıma ve Slovenya’da kaybettiğim fotoğraf makineme, Fransa’da ki Eyfel kulesine, Estonya’da ki arkadaşıma, Brüksel’de ki dostlara, Hollanda’nın esrar-engiz kokusuna, Almanya’daki arkadaşlara, Essen’de ki soğuk alman polislerine ve laboratuvardaki çalışma arkadaşlarıma, İsveç Stockholm ve İsviçre Zürih‘de ki tanımadığım içten sıcak insanlara ve unutulmaz doğasına, küçücük şirin ülke olan Andorra Prensliği’nin dağlarına, Bilbao’daki gezgin dostum Deniz Derin ve annesine, Huelva’da hiç tanımadığım ve beni evinde misafir eden dostlarıma, Barcelona-San sebastian-Sevilya ve Salamanca sokaklarına, Mayorca adasındaki yoldaşım dostum Sinan‘a, Avusturya Viyana’nın türklerine, uykusuz gecelerime, herkese selam olsun… Hayatıma bir anlıkta olsa neşe kattınız… Ve İspanya… Endülis Huelva’da sıradan bir erasmusla başlayıp, Madrid Başkentte çınar gibi büyüyen bir profille, Araştırmacı-mühendis-doktor ve tercümanlık iş tercübelerini elde ettiğim dokuz yıl boyunca her yerini gezdiğim, tabiri caizse, ikinci evim, vatanım yurdum dediğim ve acısıyla tatlısıyla gençliğimin en güzel ve unutulmaz yıllarını yaşadığım bu sıcacık ülkem… Bana kattıkların, verdiğin hayat deneyimi, iş tecrübesi ve eşsiz eğitim için ne kadar teşekkür etsem azdır… Ve…hayatımın geriye kalanını emanet ettiğim en değerlime, Eşime, ve bana güvenip destek olan Atmaca ailesinin reisi Günaydın babam, patronu Emine annem ve pırlanta gibi kardeşlerim Ümit- Derya-Uğur-Yağmur‘a sevgilerimle… Hayatımda bana engel olanlara, kötü davrananlara veya beni görmezden gelenlere… Sizede teşekkürler, bilmeden beni doğru yola yönlendirdiğiniz için… Hayatımda, bir dönemi kapatıp, yeni döneme Doktor olarak girdiğim bugünüme sayenizde ulaşabildim… Hiçbir başarı tesadüf değildir, aksine bedeli ödenmiştir… Sevgiler Okan xi Acknowledgements Okan Mersinlioğlu is grateful to the Ministry of Science and Innovation of Spain (MICINN) for the financial support through a predoctoral fellowship, FPI BES-2009-027503. I thank the following for funding this work: the Ministry of Science and Innovation of Spain (MICINN) through project MAT2008−00736, the Ministry of Economy and Competitiveness of Spain (MINECO) through project MAT2012−35556 and Community of Madrid (CAM) through Consortium BITI, ref S2009/MAT−1472. This work was also supported by the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Spain. I also thank Dr. Mª Luisa Ruiz González (Department of Inorganic Chemistry, UCM) for the acquisition of TEM images of samples prepared by precipitation of CaP during the synthesis of mesoporous silica nanospheres (MSNSs), Dr. Fernando Conde (X−ray Diffraction CAI, UCM), Isabel Salido Herranz (Library of Faculty of Pharmacy, UCM), Dr. Francisco Javier García García (National Center of Electron Microscopy, UCM) and Dr. Adrián Gómez Herrero (National Center of Electron Microscopy, UCM) for their kind technical support. xiii Communications derived from this Thesis • POSTER – Precipitation of Calcium phosphates in the presence of Ionic Surfactants Blanca González, Antonio J. Salinas, Okan Mersinlioğlu, María Vallet-Regí 23rd European Conference on Biomaterials, 11–15 September, 2010, Tampere, Finland.  RAPID-FIRE PRESENTATION AND POSTER – Nanostructured Calcium Phosphates from Phospholipids Templates Okan Mersinlioğlu, Antonio J. Salinas, Blanca González, María Vallet-Regí 24th European Conference on Biomaterials, 4–8 September, 2011, Dublin, Ireland.  ORAL PRESENTATION – Apatite coatings on MCM–41 nanospheres Okan Mersinlioğlu, Blanca González, Antonio J. Salinas, María Vallet-Regí EUROMAT2013– European Congress and Exhibit on Advanced Materials and Processes, 8−13 September 2013, Sevilla, Spain. Manuscripts in preparation: - Lamellar discontinuous mesostructured hydroxyapatite in the presence of sodium dodecyl benzene sulfonate (Chapter II) - Mesostructured calcium phosphates in the presence of phospholipids (Chapter III) - Mesoporous silica nanospheres coated by apatite nanoparticles (Chapter IV) xv List of abbreviations Abbreviation Full Name ° / °C Degree, Degree celsius (centigrade) ζ Zeta Potential U Electrophoretic mobility Η Viscosity of water e Dielectric constant of water λ Wavelength θ Theta [ ] Concentration α−TCP α−tricalciumphosphate β−TCP β−tricalciumphosphate β−CPP β−Calcium pyrophosphate ao Unit cell parameter Å Angstrom ACP Amorphous calcium phosphate ATR Attenuated total reflectance BET Brunauer−Emmett−Teller BJH Barrett−Joyner−Halenda Co Spontaneous curvature CaP Calcium phosphate CDHA Calcium−deficient hydroxyapatite CMC Critical micelle concentration CTAB Cetyltrimethylammonium bromide d Days dhkl Interplanar distance DCPA Dicalcium phosphate anhydrous, mineral monetite DCPD Dicalcium phosphate dihydrate, DCPD, mineral brushite DEPETES Diethylphosphatethyltriethoxy silane DLS Dynamic light scattering DNA Deoxyribonucleic acid Dp Pore diameter ED Electron diffraction EDS Energy dispersive X−ray spectroscopy EPG / EPG®BB Empigen®BB detergent EtOH Ethanol FA Fluorapatite FHA Fluorescent hydroxyapatite FTIR Fourier transform infrared spectroscopy H Hour HA Hydroxyapatite HLB Hydrophilic lipophilic balance HMM Hybrid mesoporous material HRTEM High resolution transmission electron microscopy ICDD International centre for diffraction data IUPAC International union of pure and applied chemistry LDHASi Lanthanide-doped HA/silica composite MAP Monoalkyl phosphate List of abbreviations MBGs Mesoporous bioactive glasses MCM Mobil composition of matter: class of mesoporous silica materials MCM−41 Mobil composition of matter No. 41 MCM−48 Mobil composition of matter No. 48 MCM−50 Mobil composition of matter No. 50 MCPA Monocalcium phosphate anhydrous MCPM Monocalcium phosphate monohydrate MSNSs Mesoporous silica nanospheres NPs Nanoparticles OCP Octacalcium phosphate P/Po Relative pressure PC Phosphatidylcholine PC33 Asolectin from soybean, about 33 % of PC PC60 L-α-Lecithin from egg yolk, about 60 % of PC PC94 Lipoid-S100 from soybean, about 94 % of PC PEI Poly(ethyleneimine) PLs Phospholipids SBET BET surface area SBA−15 Santa Barbara amorphous type material−15 SBF Simulated body fluid SDS Sodium dodecyl sulfate SDBS Sodium dodecyl benzene sulfonate SEM Scanning electron microscopy SA−XRD Small angle X−ray diffraction t Time T Temperature TEM Transmission electron microscopy TEOS Tetraethyl orthosilicate TEP Triethyl phosphate TG/DTA Thermogravimetric / differential thermal analysis TIP Triethyl phosphite THSMP Trihydroxysilylpropyl methylphosphonate TTCP Tetracalcium phosphate UV Ultra violet WA−XRD Wide angle X−ray diffraction xvii List of samples synthesized in thesis Sample Code Full name NHCaP Nanostructured hybrid calcium phosphates NHHA Nanostructured hybrid hydroxyapatite MHCaP Multistructured hybrid calcium phosphate HMM Hybrid mesostructured material LCaP Lamellar structured calcium phosphate CaPX / PCaX Nano−structured calcium phosphate PC33 Hybrid material synthesized in the presence of Asolectin from soybean PC60 Hybrid material synthesized in the presence of L-α-Lecithin from egg yolk PC94−1 & PC94−2 Hybrid materials synthesized in the presence of lipoid-S100 from soybean MSNSs Mesoporous silica nanospheres MSNS-HA-X Mesoporous silica nanospheres coated with HA NSFD MSNSs functionalized with DEPETES NSFT MSNSs functionalized with THSMP NSFD-HA MSNSs functionalized with DEPETES and then coated with HA NSFT-HA MSNSs functionalized with THSMP and then coated with HA LDHASix Lanthanide-doped HA/silica composite INDEX xix SUMMARY 1 RESUMEN 9 I. INTRODUCTION 17 I.1 Biomaterials for hard tissues regeneration 19 I.2 Calcium phosphate bioceramics 21 I.2.a Synthesis of calcium phosphates by precipitation 21 I.2.b Hydroxyapatite 22 I.2.c Lanthanide-doped hydroxyapatite 23 I.3 Mesoporous Silica Nanoparticles 24 I.3.a Functionalization of mesoporous silica phases 25 I.4 Surfactants for mesophases formation 27 I.4.a Hydrophilic-lipophilic balance 29 I.4.b Critical micelle concentration 29 I.4.c Nanostructure formation 30 I.5 Synthesis of mesostructured hybrid materials 31 I.6 Sol−gel processing of bioceramics 31 I.7 Nanoparticles 32 I.8 Objectives and distribution of the Memory 33 I.9 References 35 II. PRECIPITATION OF MESOSTRUCTURED CALCIUM PHOSPHATES IN THE PRESENCE OF IONIC SURFACTANTS 43 II.1 Introduction 45 II.2 Calcium phosphate in the presence of ionic surfactants 47 II.2.a Calcium phosphate in the presence of sodium dodecyl sulphate 47 II.2.b Calcium phosphate in the presence of sodium dodecyl benzene sulphonate 57 II.3 Calcium phosphate in the presence of surfactant mixture: anionic and cationic 61 II.3.a Calcium phosphate in mixed surfactants of sodium dodecyl sulphate and cetyltrimethyl ammonium bromide 62 II.3.b Calcium phosphate in mixed surfactants of sodium dodecyl benzene sulphonate and cetyltrimethyl ammonium bromide 65 II.3.c Calcium phosphate in mixed surfactants of mono-n-dodecyl phosphate and cetyltrimethyl ammonium bromide 68 II.4 Calcium phosphate in the presence of a zwitterionic surfactant 70 II.5 Conclusions of Chapter II 74 II.6 References 76 INDEX xx III. PRECIPITATION OF MESOSTRUCTURED CALCIUM PHOSPHATES IN THE PRESENCE OF PHOSPHOLIPIDS 79 III.1 Introduction 81 III.2 Precipitation of calcium phosphates in the presence of phospholipids 83 III.3 Conclusions of Chapter III 89 III.4 References 90 IV. HYDROXYAPATITE COATINGS ON MESOPOROUS SILICA NANOSPHERES 93 IV.1 Introduction 95 IV.2 Synthesis of mesoporous silica nanospheres, MSNSs sample 96 IV.3 Precipitation of calcium phosphate simultaneous to the formation of mesoporous silica nanospheres, MSNS−HA−1 sample 99 IV.4 Soaking mesoporous silica nanospheres into a sol precursor of calcium phosphate, MSNS−HA−2 sample 103 IV.5 Wetting mesoporous silica nanospheres into a sol precursor of calcium phosphate, MSNS−HA−3 sample 109 IV.6 Functionalization of nanospheres with phosphate–like groups and subsequent wetting into a sol precursor of CaP 112 IV.6.1 Functionalization of nanospheres with diethylphosphatoethyltriethoxysilane: by post-synthesis for NSFD sample, and by co-condensation for NSFD1 and NSFD2 samples 113 IV.6.2 Wetting of DEPETES functionalized MSNSs via post-synthesis into a sol precursor of CaP, NSFD−HA sample 116 IV.6.3 Functionalization of nanospheres with 3−trihydroxysilylpropyl methylphosphonate by co-condensation, NSFT sample 118 IV.6.4 Wetting of THSMP functionalized MSNSs with a sol precursor of CaP, NSFT−HA sample 121 IV.7 Summary and conclusions of Chapter IV 123 IV.8 References 125 V. COATING OF MESOPOROUS SILICA NANOSPHERES WITH FLUORESCENT HYDROXYAPATITE NANOPARTICLES 131 V.1 Introduction 133 V.2 Materials synthesis 133 V.3 Conclusions of Chapter V 139 V.4 References 140 INDEX xxi VI. CONCLUSIONS 143 VII. APPENDIX: EXPERIMENTAL PART 149 VII.1 Tables for the interpretation of FTIR spectra of synthetized materials 151 VII.2 Characterization Techniques 153 VII.3 Commercial Products 155 VII.4 Preparation of Simulated Body Fluid 156 VII.5 Description of Synthesis 157 VII.5.a Synthesis of Calcium Phosphate in the Presence of Surfactants (Chapter II) 157 VII.5.b Synthesis of Calcium Phosphate in the Presence of Phospholipids (Chapter III) 159 VII.5.c Synthesis of Hydroxyaptite coatings on Mesoporous Silica Nanospheres (Chapter IV) 160 VII.5.d Synthesis of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles (Chapter V) 163 VII.6 References 164   Summary / Resumen 1 SUMMARY   Summary / Resumen 3 Synthesis of Calcium Phosphates in the Presence of Ionic Surfactants, Phospholipids and Mesoporous Silica Nanoparticles to Control the Bioceramic Mesostructure Introduction Currently, there is a great interest in the development of new biomaterials because of their foreseeable enormous contribution for the demands of an aging society that needs alternatives in the treatment of hard tissues diseases or cancer. First biomaterials developed were as bioinert as possible. Then, in the years 70´s and 80´s of the past Century, bioactive and biodegradable biomaterials, including calcium phosphates and bioactive glasses, started to be investigated. At present, the interest is focused towards the synthesis of biomaterials with controlled mesostructure and functionalized surface to improve and tailor at demand their behaviour into the body. Moreover, for bone tissue engineering applications, biomaterials are processed as 3D scaffolds able to be decorated with biochemical signals and cells. Nevertheless, for cancer therapy some recent strategies are based on mesoporous nanoparticles able to transport drugs or genes when injected in the blood stream, without being recognised and withdrawn by macrophages. Calcium phosphates (CaPs) exhibit chemical and structural similarities to the mineral component of hard tissues (biological apatite). Thus, they are biocompatible and bioactive and were widely investigated as bioceramics for over 40 years, mainly for applications in Orthopedics or Dentistry. In that time the research effort was focused in aspects such as the phase obtained, the microstructure, the particle size or the calcium deficiency. Very recently, it was proposed that a control of the mesostructure during the synthesis of CaPs would improve its current performance as well as allow adjusting their properties for the requirements of new clinical applications. On the other hand, in 2001 ordered mesoporous silicas were proposed as matrices in drug delivery systems. More recently, these mesoporous systems synthesized as nanoparticles have gained much attention for applications in nanomedicine, such as cell targeted drug/gene delivery nanosystems or nanosystems incorporating moieties for diagnosis. Mesoporous silica nanospheres (MSNSs), with a diameter ranging 50-300 nm and mesopores of approximately 2-3 nm, use to be ordered in the 2D hexagonal arrangement of MCM-41 material. Moreover, they possess high pore volume and surface area rich in silanol groups (Si–OH) that can be functionalized with a great variety of organic moieties. The high number of mesoporous silica families makes one wonders if it would be possible to synthesise an analogous family of mesoporous materials based in CaPs. Hereafter, the first approaches can be to use analogous strategies to those used in the syntheses of mesoporous silicas. Thus is, to employ organic amphiphilic molecules which could act as a template of the CaPs mesostructure via supramolecular assemblies. Up to now, the efforts made in this line had little success because the chemistry of essentially ionic compounds, such as CaPs, is far from the chemistry of essentially network covalent compounds, such as silica. Consequently, the interactions between the surfactants and calcium and phosphate ions in solution are quite different than with silicate species. However, the synthesis of new CaP-based mesostructured materials could allow new clinical applications or improve current performance. Moreover, the new knowledge acquired in this effort could also bring new bioceramics and organic-inorganic hybrids based in CaP. Summary / Resumen 4 Objectives The first main objective of this thesis has been to investigate the synthesis of CaPs in the presence of different types of ionic surfactants, pure and mixtures, and in the presence of different concentrations of phosphatidylcholine, trying to obtain mesostructured CaPs (Chapters II and II). The second essential objective has been to investigate the synthesis of CaPs in the presence of mesoporous silica nanospheres to obtain core@shell systems of the type mesoporous silica@hydroxyapatite (Chapters IV and V). This multiple approach was attempted to show if it is possible to obtain CaPs with controlled mesostructure. Chapter I contains the Introduction of this thesis. Therefore, it describes the background and current status of the subject matters. Chapter II addresses the synthesis and characterization of CaPs in the presence of various ionic surfactants, pure and mixture of them, aimed to behave as structure directing agents. The investigated surfactants were: sodium dodecyl sulphate (SDS), sodium dodecyl benzene sulphonate (SDBS), cetyltrimethyl ammonium bromide (CTAB) and mono-n-dodecyl phosphate (MAP). Moreover, EMPIGEN®BB detergent (EPG), a zwitterionic surfactant, was tested as mesostructure template for the first time as an alternative approach. Surfactants in water can be organized in different mesostructures such as micellar, lamellar, hexagonal or cubic. Synthesis conditions and surfactants have been optimized looking for interactions with the precipitated CaP, i.e., to obtain materials with mesostructural characteristics different to materials prepared in the absence of surfactant. In Chapter III we aimed at synthesizing HA based hybrids with a sponge-like mesostructure via precipitation of CaP in the presence of three phospholipid (PL) sources with substantially different contents of phosphatidylcholine (PC): asolectin 33%, lecithin 60% and lipoid 94% of PC. The different structures of the nanohybrids as a function of the synthesis conditions, pH of solution (basic or neutral) and Ca/P molar ratio (1.0 or 1.67) and concentration of PC, were investigated. In this case, given the biocompatibility of PLs, it arises the synthesis of CaP-PLs hybrids, i.e., PLs were not removed from the final material. The new CaP-PLs hybrid materials were characterized and the mechanisms of formation for the nanostructured materials were proposed. Given the difficulty found in the previous chapters to obtain CaP systems as ordered mesoporous materials, in Chapter IV various strategies were investigated to obtain CaP coatings, namely HA, on mesoporous silica nanospheres (MSNSs) with MCM-41 structure. In this case our approach implies the use of a well-defined mesoporous material as the basis of the final nanosystem. The purpose of this study was to design multifunctional core@shell nanoparticles that can be applied in bone regeneration, as well as specific intracellular carriers of drugs, proteins or agents for image, which could be used for applications such as the treatment of cancer. The nanospheres coated by CaP would exhibit well interconnected pore structures and excellent in vitro bioactivity that made them useful for several biomedical applications. For instance, an HA coating could act as a nanogate allowing the controlled release of the biomolecules enclosed in the silica mesopores under specific conditions, for instance a pH decrease. The attempted approaches to obtain MSNSs coated by CaPs were: (i) co-synthesis of CaP and MSNSs, (ii) soaking and (iii) wetting MSNSs in a sol precursor of CaP, Summary / Resumen 5 and (iv) functionalization of MSNSs with organic moieties containing phosphate-like groups to facilitate the CaP coating after wetting them into a sol precursor of CaP. Except in the first approach, in which both processes are simultaneous, the synthesis of materials is performed in two steps: (1) the synthesis of MSNSs ranging 150-250 nm of diameter and around 2 nm of pore diameter and (2) the step to coat MSNSs with CaP. An improvement of these systems is dealt with in Chapter V, where the subject investigated is the coating of MSNSs with HA doped with europium as fluorescent element via the precipitation of fluorescent CaP on previously synthetized MSNSs, which would give greater visibility and traceability of the nanosystem during the biological application, such as cell trafficking or tumour localization. Chapter VI summarizes the principal conclusions derived from this thesis. Finally, the general conditions and detailed experimental work, as well as structural characterization and analysis techniques employed, are included in Chapter VII of this report. Results and Conclusions Chapter II. Precipitation of Mesostructured Calcium Phosphates in the Presence of Ionic Surfactants - Ionic surfactants, namely anionic, containing S or P atoms in their polar head, such as SDS, SDBS and MAP, can be used to obtain mesostructured organic-inorganic hybrids based in CaPs. - The interaction of amphiphilic molecules of surfactant with calcium and phosphate ions, during the CaP precipitation from aqueous solutions, yield hybrid materials exhibiting lamellar or discontinuous lamellar mesostructures, but no mesoporous order. - In the synthesis conditions, i.e., Ca/P molar ratio 1 and 1.67, surfactant concentration from 15 to 90 mM and 240 mM and pH not buffered, the CaP most common phases present in the lamellar hybrids were brushite, hydroxyapatite or monetite. - SDS is a suitable surfactant to obtain highly mesostructured lamellar CaP hybrids. - Relatively high SDBS concentrations and Ca/P molar ratio of 1 yield discontinuous lamellar or curved lamellar mesostructures, forming meshes, of poorly crystallized HA. The formation of HA at this low Ca/P molar ratio is attributed to the pH increase (7 to 9) of medium due to the protonation of the sulphate polar heads of SDBS. - The mixture of an anionic surfactant, such as SDS, SDBS or MAP, with the cationic cetyltrimethyl ammonium bromide (CTAB) produces hybrids with mesostructure analogous to that obtained with the pure anionic surfactants, suggesting a non-effective interaction of CTAB with calcium and phosphate ions. - TEM analysis of materials obtained using a surfactant mixture shows two types of particles: (i) rod-like particles exhibiting lamellar mesostructure and (ii) sheet-like particles unstable under the electron beam. The bubbles formed when these sheet-like particles were investigated, attributed to the organic matter combustion and to the removal of crystallization water Summary / Resumen 6 molecules in brushite, avoid the identification of the possible mesostructure in the sheet-like particles. - The use of energetic synthesis procedures, such as ageing in an autoclave or refluxing after the CaP precipitation, leads to monetite, a CaP phase derived from brushite after remove the two water molecules of crystallization. - The use of EPG, a zwitterionic surfactant containing one carboxybetaine group, as mesostructure template for CaP fails and no interactions with calcium and phosphate ions are detected. For the future, the investigation of another zwitterionic surfactant containing a sulfobetaine groups is suggested. Chapter III. Precipitation of Mesostructured Calcium Phosphates in the Presence of Phospholipids - The precipitation of CaPs in phospholipids (PLs) suspensions containing different concentrations of phosphatidylcholine (PC) yields to the synthesis of CaP-PLs hybrid materials. - The inorganic components of the hybrids are poorly crystallized HA, when pH is basic and Ca/P molar ratio 1.67, and brushite at pH neutral and Ca/P = 1. - The organic matter content is similar in both hybrid types, around 20% in brushite-containing hybrids and around 17% in HA-containing ones. CaP-PLs hybrid materials subjected to a thermal process to remove the organic matter evolve to non-porous HA. Since no mesoporous materials were obtained after calcination, this Chapter has been focused in the characterization of the CaP-PLs hybrid materials. - TEM analysis of hybrids revealed four types of nanostructures: (i) lamellar bilayer with 4.2 nm in thickness of layer (apatite, asolectin), (ii) bilayer vesicles around 35 nm in diameter of vesicles and 4.7 nm in thickness of bilayer (brushite, lecithin), (iii) micelles of about 4.3 nm in diameter (brushite, lipoid) and (iv) bilayer sponge−like or worm−like tubular mesostructures with 4.3 nm in thickness of disordered bilayer sponge−like structure (apatite, lipoid). - Since both components of CaP-PLs hybrid materials are biocompatible they can be investigated as drug delivery platforms via the encapsulation of a drug within the phospholipid structure of the hybrid. Chapter IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres In this chapter, MCM−41 mesoporous silica nanospheres (MSNSs) were tried to be coated with HA nanoparticles or with a HA layer, to expand the clinical applications of both materials. According to this goal, the conclusions derived from each synthetic route attempted are: • Co-synthesis of CaP and MSNSs. The CaP precipitation simultaneous to the sol-gel synthesis of MSNSs yield a mixture of independent particles of poorly crystallized HA rods and polydisperse MSNSs (SBET 594 m2/g). Summary / Resumen 7 • Soaking of MSNSs into a sol precursor of CaP. First, pure SiO2 MSNSs (SBET = 1230 m2/g) were synthesized. After soaking them in the sol precursor of CaP for 3 hours, the nanospheres (SBET = 250 m2/g) convert in ellipsoids with the elements Si, Ca, P and O in their composition. The ellipsoids exhibit a radial mesopore channels arrangement close to the particles surface. Moreover, the obtained material exhibits a moderate in vitro bioactive response in simulated body fluid. • Wetting of MSNSs by a sol precursor of CaP. In this approach, pure SiO2 MSNSs (SBET = 1230 m2/g) were synthesized first, then soaked for 30 minutes in the sol precursor of CaP and subsequently subjected to a washing process mimicking a dip-coating procedure with the sol. TEM images after wetting confirm the successful formation of HA NPs covering the MSNSs surface. In this case, the SBET of the coated material suffer a drastic decrease of ≈90 % compared to the initial MSNSs. Unlike the previous method, in this approach MSNSs are not altered in shape neither in composition. • Functionalization of MSNSs with phosphate-like groups and subsequent wetting with a sol precursor of CaP. MSNSs were functionalized with diethylphosphatoethyl triethoxysilane (DEPETES) or 3-trihydroxysilylpropylmethylphosphonate (THSMP) to facilitate the subsequent interaction of their phosphate-like groups with calcium ions and then phosphate ions in the sol. o DEPETES-functionalized MSNSs exhibiting MCM-41 hexagonal arrangement and high SBET (878 to 1190 m2/g) were synthesized by co-condensation or post-synthesis. After functionalization, P-containing MSNSs were wetted with a sol precursor of CaP. A homogeneous distribution of a HA layer around the MSNSs and some HA NPs are observed by TEM. SBET of 335 m2/g indicates a partial coating of the MSNSs surface. o MSNSs were functionalized with THSMP by a co-condensation method. The obtained material exhibits a high SBET of 1316 m2/g. THSMP-functionalized MSNSs were wetted with a sol precursor of CaP. TEM analysis showed that a core@shell structure MSNS@HA was obtained. Coated MSNSs are non-porous showing a decrease in SBET of around 97% regarding the initial MSNSs. Chapter V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles - The precipitation of Eu3+ doped HA (fluorescent HA, FHA) in the presence of MCM-41 type MSNSs yields to the synthesis of FHA/MSNSs composites. - Some FHA/MSNSs composites exhibit the red luminescence of lanthanides under UV light. The luminous intensity is dependent on the amount of Eu3+ ion in the composite. - A core@shell structure (MSNS@HA) is observed by TEM for one of the samples investigated. However, this sample does not show a fluorescent emission due to its low Eu3+ content.   Summary / Resumen 9 RESUMEN   Summary / Resumen 11 Síntesis de Fosfatos de Calcio en Presencia de Surfactantes Iónicos, Fosfolípidos y Nanopartículas de Sílice Mesoporosa para Controlar la Mesoestructura de la Biocerámica Introducción Actualmente, hay un gran interés en el desarrollo de nuevos biomateriales debido a su enorme contribución a las demandas de una sociedad cada vez más envejecida que necesita alternativas para el tratamiento del cáncer o las enfermedades de los tejidos duros. Los primeros biomateriales se desarrollaron para que fuesen lo más bioinertes posible. A partir de los años 70´s y 80´s del siglo XX, comenzaron a investigarse biomateriales bioactivos o biodegradables, incluyendo algunos fosfatos de calcio y los vidrios bioactivos. En la actualidad, el interés se enfoca hacia la síntesis de biomateriales con mesoestructura controlada y funcionalizados para mejorar y adaptar su comportamiento en el cuerpo. Por otra parte, para aplicaciones de ingeniería de tejido óseo, los biomateriales se procesan como andamios 3D capaces de ser funcionalizados con señales bioquímicas y células. Asimismo, algunas estrategias recientes para la terapia del cáncer se basan en nanopartículas mesoporosas que son capaces de transportar drogas o material genético cuando se inyectan en el torrente sanguíneo, sin ser retiradas de la circulación por los macrófagos. Los fosfatos de calcio (CaPs) exhiben muchas semejanzas químicas y estructurales con el componente mineral de los tejidos duros (la apatita biológica). Por ello, son biocompatibles y bioactivos y han sido ampliamente investigados como biocerámicas durante más de 40 años, principalmente para aplicaciones en Ortopedia u Odontología. A lo largo de ese tiempo el esfuerzo investigador se ha centrado en aspectos como la fase de fosfato de calcio, su microestructura, el tamaño de partícula o la deficiencia en calcio. Más recientemente, se ha propuesto que un control de la mesoestructura durante la síntesis de los CaPs permitiría mejorar sus prestaciones actuales así como ajustar sus propiedades a las necesidades de nuevas aplicaciones clínicas. Por otra parte, en 2001 se propuso la utilización los materiales de sílice mesoporosa ordenada como matrices en sistemas de liberación controlada de fármacos. Estos sistemas mesoporosos sintetizados en forma de nanopartículas están recibiendo recientemente mucha atención para aplicaciones en nanomedicina, tales como nanosistemas dirigidos a células para el suministro de fármacos o material genético y también en nanosistemas que incorporen componentes de diagnóstico. Las nanoesferas de sílice mesoporosa (MSNSs), con un diámetro entre 50-300 nm y mesoporos de unos 2-3 nm, ordenados en una simetría hexagonal 2D típica del material MCM-41. Además, poseen elevado volumen de poros y una alta superficie específica que es rica en grupos silanol (Si-OH) que pueden ser funcionalizados con una gran variedad de moléculas orgánicas. El elevado número de familias de sílice mesoporosa existentes lleva a preguntarse si sería posible sintetizar una familia análoga de materiales mesoporosos basados en CaPs. En este sentido, los primeros intentos podrían basarse en estrategias análogas a las utilizadas en la síntesis de sílices mesoporosas, es decir, emplear moléculas orgánicas anfifílicas para que actúen como plantilla de la mesoestructura de los CaPs mediante ensamblaje supramolecular. Hasta ahora, los esfuerzos realizados en esta línea han tenido poco éxito debido fundamentalmente a que la química de compuestos esencialmente iónicos, tales como los CaPs, difiere mucho de la química de compuestos esencialmente covalentes en red, como la sílice. En consecuencia, las interacciones entre los surfactantes y los iones calcio y fosfato en disolución son bastante diferentes que con las especies silicato. Sin embargo, la síntesis de nuevos materiales mesoestructurados basados en CaPs podría Summary / Resumen 12 permitir nuevas aplicaciones clínicas o mejorar las actuales. Por otra parte, los nuevos conocimientos adquiridos en este esfuerzo también podrían llevar a la obtención de nuevas biocerámicas e híbridos orgánico-inorgánicos basados en CaPs. Objetivos El primer objetivo de esta tesis ha sido investigar la síntesis de CaPs en presencia de diferentes tipos de surfactantes iónicos, puros y mezclas, así como de diferentes concentraciones de fosfatidilcolina, intentando obtener CaPs mesoestructurados (Capítulos II y II). El segundo objetivo fundamental ha sido investigar la síntesis de CaPs en presencia de nanoesferas de sílice mesoporosa para obtener sistemas núcleo@corteza del tipo MSNSs@HA (Capítulos IV y V). Con este enfoque múltiple se ha intentado demostrar si es posible conseguir CaPs con mesoestructura controlada. El Capítulo I contiene la introducción de esta tesis. Por lo tanto, describe los antecedentes y el estado actual de las materias de estudio. El Capítulo II aborda la síntesis y caracterización de los CaPs en presencia de varios surfactantes iónicos, puros y una mezcla de ellos, destinados a comportarse como agentes directores de estructura. Los surfactantes investigados han sido: dodecil sulfato de sodio (SDS), dodecil benceno sulfonato de sodio (SDBS), bromuro de cetiltrimetil amonio (CTAB) y mono-n-dodecil fosfato (MAP). Por otra parte, un surfactante zwitteriónico, EMPIGEN ® BB (EPG), ha sido probado por primera vez, en un enfoque alternativo, como plantilla de la mesoestructura. Los surfactantes en agua se organizan en diferentes mesostructuras formando micelas, lamelas, o fases de simetría hexagonal o cúbica. Se han optimizado el tipo de surfactante y las condiciones de síntesis buscando interacciones con el CaP que se obtiene por precipitación, es decir, para obtener materiales con características mesostructurales diferentes a los sintetizados en ausencia de surfactante. En el Capítulo III se han intentado sintetizar híbridos basados en HA con una mesoestructura tipo esponja mediante la precipitación de CaPs en presencia de tres fuentes de fosfolípidos (PL) con distinto contenido en fosfatidilcolina (PC): Asolectin 33%, Lecitina 60% y Lipoid 94%. Se han investigado las diferentes estructuras obtenidas para los nanohíbridos en función de: las condiciones de síntesis, el pH de la disolución (básico o neutro), la relación molar Ca/P (1.0 o 1.67) y la concentración de PC. En este caso, dada la biocompatibilidad de los PLs, se presenta la síntesis de híbridos CaP-PLs, es decir, sin eliminar los fosfolípidos del material. Se han caracterizado los nuevos híbridos CaP-PLs y se han propuesto los mecanismos de formación de las nanoestructuras formadas. Dada la dificultad encontrada en los capítulos anteriores para obtener materiales de CaPs como sistemas mesoporosos ordenados, en el Capítulo IV se investigan diversas estrategias para obtener recubrimientos de CaP, principalmente HA, en nanoesferas de sílice mesoporosa (MSNSs) con estructura tipo MCM-41. En este caso nuestro enfoque implica el uso de un material mesoporoso bien definido que actúe como base de los nanosistemas finales. El propósito de este estudio ha sido diseñar nanopartículas núcleo@corteza multifuncionales que puedan ser aplicadas en la regeneración ósea, así como nanosistemas portadores de drogas, proteínas o agentes de imagen a nivel intracelular que podrían ser utilizados para aplicaciones como el tratamiento del cáncer. Las nanoesferas recubiertas por CaPs exhibirían estructuras de poros bien interconectados y excelente bioactividad in vitro que les haría útiles para diversas aplicaciones biomédicas. Por ejemplo, una capa de HA podría actuar como una nanocompuerta que permitiese la liberación controlada de Summary / Resumen 13 biomoléculas alojadas los mesoporos de la sílice bajo condiciones específicas, por ejemplo la disminución del pH. Los procedimientos que se han intentado para obtener MSNSs recubiertas por CaP han sido: (i) co-síntesis de CaP y MSNSs, inmersión (ii) e (iii) impregnación de las MSNSs en un sol precursor de CaP y (iv) funcionalización de MSNSs con moléculas orgánicas que contienen grupos fosfato para facilitar el recubrimiento durante la impregnación con un sol precursor de CaP. Excepto en la primera ruta, en la que ambos procesos son simultáneos, la síntesis de los materiales se realiza en dos pasos: (1) síntesis de las MSNSs de 150-250 nm de diámetro y unos 2 nm de diámetro de poro y (2) recubrimiento de las MSNSs con una fase de CaP. En el Capítulo V se aborda una mejora de estos sistemas, donde el tema investigado es el recubrimiento de las MSNSs con HA dopada con un elemento fluorescente como el europio, mediante la precipitación de CaP fluorescente sobre MSNSs sintetizadas previamente. Se busca dar una mayor visibilidad y trazabilidad de los nanosistemas durante la aplicación de biológica, permitiendo la localización del recorrido de las nanopartículas por el interior de la célula o la acumulación de las mismas en el tumor. El Capítulo VI resume las principales conclusiones derivadas de esta tesis. Por último, las condiciones generales y el trabajo experimental detallado, así como las técnicas de caracterización y análisis estructurales empleados, se incluyen en el Capítulo VII de esta memoria. Resultados y Conclusiones Capítulo II. Precipitación de fosfatos de calcio mesoestructurados en presencia de surfactantes iónicos - Los surfactantes iónicos, principalmente aniónicos, que contienen átomos de S o P en su cabeza polar, como el SDS, SDBS y MAP, pueden utilizarse para obtener híbridos orgánico- inorgánicos mesoestructurados basados en CaPs. - La interacción de las moléculas anfifílicas de surfactante con los iones calcio y fosfato, durante la precipitación de CaPs de soluciones acuosas, produce materiales híbridos que exhiben mesostructura lamelar o lamelar discontinua, pero sin orden mesoporoso. - En las condiciones de síntesis, es decir, relación molar Ca/P 1 o 1,67, concentración de surfactante de 15 a 90 mM o 240 mM y sin tamponar el pH, las fases de CaP más comunes que presentan los híbridos lamelares formados son brushita, hidroxiapatita o monetita. - El SDS es un surfactante adecuado para obtener CaPs híbridos con mesoestructura lamelar. - Concentraciones relativamente altas de SDBS y una relación molar Ca/P de 1 dan lugar a mesostructuras discontinua lamelar o curvada lamelar, formando redes de HA poco cristalina. La formación de HA a pesar de la baja relación molar Ca/P se atribuye al aumento de pH (de 7 a 9) del medio debido a la protonación del grupo sulfato de la cabeza del SDBS en la síntesis. - La mezcla de un surfactante aniónico, como SDS, SDBS o MAP, con el catiónico cetiltrimetil bromuro de amonio (CTAB) produce híbridos con mesoestructura análoga a la obtenida con los surfactantes aniónicos puros, lo que sugiere una interacción no efectiva del CTAB con los iones calcio y fosfato. Summary / Resumen 14 - El análisis mediante TEM de los materiales obtenidos con mezcla de surfactantes muestra dos tipos de partículas: (i) tipo-barra con mesoestructura lamelar y (ii) tipo-hoja que son inestables bajo el haz de electrones. Las burbujas que se forman cuando se enfocan estas partículas, atribuidas a la combustión de la materia orgánica y la eliminación del agua de cristalización de la brushita, evitan la identificación de su posible mesoestructura. - La utilización de procedimientos de síntesis más enérgicos, como el envejecimiento en autoclave o el reflujo después de la precipitación del CaP, lleva a la formación de monetita, una fase de CaP derivada de brushita tras eliminar las dos moléculas de agua de cristalización. - La utilización de EPG, surfactante zwitteriónico que contiene un grupo de carboxibetaína, como plantilla de la mesoestructura del CaP no tiene éxito ya que no se detecta su interacción con los iones calcio y fosfato. Para investigaciones futuras, se sugiere la utilización de otro surfactante zwitteriónico que contenga grupos sulfobetaína. Capítulo III. Precipitación de fosfatos de calcio mesoestructurados en presencia de fosfolípidos - La precipitación de CaPs en suspensiones de fosfolípidos (PLs) con diferentes concentraciones de fosfatidilcolina (PC) conduce a la síntesis de materiales híbridos CaP-PLs. - Los componentes inorgánicos de los híbridos son HA poco cristalina, cuando el pH es básico y la relación molar Ca/P = 1.67 y brushita a pH neutro y Ca/P = 1. - El contenido de materia orgánica en ambos tipos de híbridos es semejante: alrededor del 20% en los que contienen brushita y alrededor del 17% en los que contienen HA. Los híbridos CaP-PLs sometidos a un proceso térmico para eliminar la materia orgánica evolucionan a HA no porosa. Puesto que ninguno de los materiales obtenidos después de la calcinación presenta mesoporosidad, este capítulo se ha centrado en la caracterización de los híbridos CaP-PLs. - El análisis mediante TEM de los híbridos reveló cuatro tipos de nanoestructuras: (i) bicapa lamelar de 4.2 nm de espesor de capa (apatita, Asolectin), (ii) vesículas bicapa alrededor de 35 nm de diámetro y 4.7 nm de espesor de pared bicapa (brushita, Lecitina), (iii) micelas de unos 4.3 nm de diámetro (brushita, Lipoid) y (iv) mesostructuras tipo esponja o gusano con bicapas tubulares de 4.3 nm de grosor de bicapa de la estructura desordenada (apatita, Lipoid). - Puesto que ambos componentes de los híbridos CaP-PLs son biocompatibles, estos materiales son susceptibles de utilizar como plataformas de liberación de fármacos mediante la encapsulación del fármaco en la estructura de los fosfolípidos que forman el híbrido. Capítulo IV. Recubrimientos de hidroxiapatita en nanoesferas de sílice mesoporosa En este capítulo, se intentó recubrir nanoesferas de sílice mesoporosa tipo MCM-41 (MSNSs) con NPs de HA o con una capa de HA, para ampliar las aplicaciones clínicas de ambos materiales. Según este objetivo, las conclusiones derivadas de cada ruta sintética empleada son: • Co-síntesis de CaP y MSNSs. La precipitación de CaP simultánea a la síntesis sol-gel de MSNSs produce una mezcla de partículas independientes de HA poco cristalina y en forma de barras y MSNSs con una amplia dispersión de tamaños y una SBET de 594 m2/g. Summary / Resumen 15 • Immersion de MSNSs en un sol precursor de CaP. En primer lugar se sintetizaron MSNSs de SiO2 pura (SBET = 1230 m2/g). Después de sumergirlas en el sol precursor de CaP durante 3 horas, las nanoesferas se transforman en elipsoides con Si, Ca, P y O en su composición y una SBET de 250 m2/g. Las partículas elipsoidales exhiben una disposición radial de canales mesoporosos cerca de su superficie. Además, el material obtenido exhibe una moderada respuesta bioactiva in vitro en fluido corporal simulado (SBF). • Impregnación de MSNSs por un sol precursor de CaP. En esta aproximación, las MSNSs de SiO2 pura (SBET = 1230 m2/g), se sumergieron 30 minutos en el sol precursor de CaP y posteriormente se sometieron a un proceso de lavado repetidas veces con el sol, imitando un procedimiento de recubrimiento por inmersión. Las imágenes de TEM tras la impregnación confirman la formación de NPs de HA que recubren la superficie de las MSNSs. En este caso, la SBET del material recubierto sufre una drástica disminución del ≈90% comparado con las MSNSs iniciales. A diferencia del método anterior, con este método de síntesis las MSNSs no se alteran, ni en su forma ni en su composición. • Funcionalización de MSNSs con grupos fosfato y posterior impregnación con un sol precursor de CaP. Las MSNSs se funcionalizaron con dietilfosfatoetil trietoxisilano (DEPETES) o con 3- trihidroxisililpropilmetilfosfonato (THSMP) para facilitar la posterior interacción de sus grupos tipo fosfato con los iones calcio y fosfato del sol. o Las MSNSs funcionalizadas con DEPETES mediante co-condensación o post-síntesis mostraron una mesoporosidad ordenada tipo MCM-41 y altas SBET (878 y 1190 m2/g, respectivamente). Tras la impregnación con un sol precursor de CaP, el análisis por TEM muestra una distribución homogénea de una capa HA alrededor de las MSNSs junto con NPs de HA. La elevada SBET de 335 m2/g indica que la superficie de las MSNSs sólo se ha recubierto parcialmente. o Se funcionalizaron MSNSs con THSMP por co-condensación. El material obtenido que presenta una alta SBET de 1316 m2/g fue impregnado con un sol precursor de CaP. El análisis TEM muestra una estructura MSNS@HA tipo núcleo@corteza. Las MSNSs recubiertas son no porosas mostrando una disminución en SBET de alrededor del 97% con respecto a las MSNSs iniciales. Capítulo V. Recubrimiento de Nanoesferas de sílice mesoporosa con nanopartículas de hidroxiapatita fluorescente - La precipitación de HA dopada con Eu3+ (HA fluorescente, FHA) en presencia de MSNSs tipo MCM-41 conduce a la síntesis de materiales compuestos FHA/MSNSs. - Algunos materiales compuestos FHA/MSNSs exhiben luminiscencia roja propia de los elementos de lantánidos irradiados con luz UV. La intensidad luminosa depende de la cantidad de iones Eu3+ en el material. - En una de las muestras investigadas se observa por TEM una estructura núcleo@corteza (MSNS@HA). Sin embargo, esta muestra no presenta emisión fluorescente debido a su bajo contenido en Eu3+.   I. Introduction 17 I. INTRODUCTION   I. Introduction 19 I.1 Biomaterials for hard tissues regeneration Biomaterials are materials that play their role in contact with biological systems. Therefore, they must be biocompatible because their implantation creates interfaces between the biological and the physical worlds. That way, they must interact with biological systems and should be biologically active within the human body. Figure I.1 displays some examples of biomaterials improving the quality of life of people. The current ageing of the population due to the increase of the life span and the subsequent increase in degenerative problems, arthritis, osteoporosis and others, as well as the incidence of road traffic accidents, justify the large demand of biomaterials to sustain and improve the quality of life of thousands of people. For this reason they have experienced a great development in the last decades due to the multidisciplinary effort of investigators from different areas, including: Science and Engineering of Materials, Chemistry, Physics, Medicine, Biology or Pharmacy [1-3]. Figure I.1 Biomaterials in the human body (Source: www.bilgiustam.com/biyomalzeme-vucutta- kullanilan-yapay-malzemeler-2/). Different definitions of “biomaterial” can be found in the literature. Several ones are included below. In 1982 Hench and Erthridge stated in their book [4]: A biomaterial is used to make the devices to replace a part or a function of the body in a safe, reliable, economic, and physiologically acceptable manner. Perhaps the most popular definition of biomaterials is the one endorsed in 1987 by a consensus of experts [5]. A biomaterial is a nonviable material used in a medical device, intended to interact with biological systems. In 2004 Ratner et al in the second edition of their noteworthy book “Biomaterials Science” removed from the previous definition the words “medical” and “nonviable”, to address the new tissue engineering and hybrid artificial organ applications, where living cells are used [6]. Therefore the actual simpler definition of a biomaterial is: Material used in a device, intended to interact with biological systems. The science of biomaterials has shown continuous and rapid growth in the last 50 years, and biomaterials are now in direct interaction with the fields of medicine, chemistry and materials I. Introduction 20 science [7]. In medical applications, biomaterials are rarely used as they are, but integrated into medical devices or implants. Compulsory characteristics of biomaterials are to be non−toxic, non−carcinogenic, non−allergenic, and non−inflammatory. In addition they must be biocompatible, applicable and biofunctional throughout their lifetime [3, 7]. The new generation of biomaterials can be produced from metallic, polymeric, ceramic, or composite components. In the last 20 years, humans have realized that ceramics and their composites can be used to replace various parts of the body, particularly for bone regeneration applications [8]. In this sense, it is very common to classify the bioceramics in terms of their reactivity within the human body as bioinert, usually considered as first generation bioceramics, or as biorebsorbable and bioactive, i.e. able to bond directly with the living tissues, as second generation bioceramics. However in the last years a different type of biomaterials is being studied. These biomaterials include a second generation bioceramic that behaves just as a scaffold and exhibits a hierarchical porosity able to host cells and/or osteoinductor agents. Such materials are considered nowadays as third generation biomaterials [9]. Thus, the ceramics that are used in the body are classified as bioceramics and they are used as biomaterials in the medical science. Furthermore, they are considered as inorganic materials in chemistry science. Their relative inertness to the body fluids, high compressive strength, and esthetically pleasant appearance led to the use of bioceramics in dentistry as dental filling and also in medicine. These materials can be used in granulated form, with predefined shapes or in porous form, for coating and artificial bone filling applications. Porous calcium phosphate (CaP) bioceramics have more potential applications in bone tissue engineering because of its large surface area and pore volume [10, 11]. Bioceramics are biocompatible with living cells and with the body, and they are suitable for bone regeneration and tissue engineering. Besides, porous bioceramics have received much attention because their porosity allows vascularization and cell growth in body by chemical self−interaction. For this reason calcium phosphate bioceramics have been under constant study in the last forty years as potential candidates for hard tissues replacement. Nowadays, the advent of Nanotechnology to the Biomaterials world has evidenced the importance of designing the new implants not only at the macrometric scale but also at the mesoscopic scale and even at the molecular level. In fact, the control of the biomaterials mesostructure is revealing as a requisite for an effective control of their in vivo behavior, including their bone regeneration capability [12, 13]. Depending on this, it is possible to synthesize porous materials in varying pore sizes: microporous, containing pores with diameters less than 2 nm; mesoporous, containing pores between 2 and 50 nm, and macroporous containing pores larger than 50 nm. This memory is devoted towards the design and characterization of mesostructured calcium phosphates to be used for the design of implants aimed at repairing hard tissues, and their employ as biomaterials. For this purpose, in this thesis calcium phosphate and silica based bioceramics were developed as third generation bioceramics by novel methods. In the future, these novel methods and synthesized hybrid materials may play an important role in the development of new bioceramics based on calcium phosphate and mesoporous silica materials [14]. I. Introduction 21 I.2 Calcium phosphate bioceramics In the last 50 years, there is increased interest in the use of calcium phosphates as bioceramic material. CaP bioceramics are widely being investigated and applied in diverse areas of bone regeneration and hard tissue engineering. Their chemical and structural similarities to biological apatite make them perfect candidates in bone filling applications [14, 15]. Bone structure is naturally formed by 60 − 75 % of inorganic components (mainly calcium phosphates), 25 % of organic components (principally collagen) and 5 − 8 % water [16, 17]. Hence, calcium phosphates are often used for bone substitution because: (i) their chemical similarity to the mineral component of hard tissues (bone and teeth) [18, 19]; (ii) they are bioactive, biocompatible and biologically form a strong bond with the surrounding bone tissue [20, 21]; and (iii) they integrate into living tissue and induce the remodeling of natural bone [22-25]. The most habitual CaPs used as biomaterials are listed in Table I.1 [26]. From this list, HA is selected to be used for application as bioceramics in the field of bone and hard tissue regeneration, because its similarity with the main crystalline component of the mineral phase of bone, and also because it can be adequately synthesized at the laboratory by chemical methods [27-30]. CaPs can be obtained in different crystalline forms by changing experimental synthesis conditions (such as temperature, pressure and pH) and the Ca/P molar ratio. Thus, by changing synthesis conditions, other crystalline calcium phosphates besides HA can be synthesized such as tetracalcium phosphate (TTCP), brushite (or dicalcium phosphate dehydrate, DCPD), monetite (dicalcium phosphate anhydrous, DCPA), α−TCP or β−TCP [31-33]. Table I.1 Calcium phosphates used as biomaterials [26]. Ca/P molar ratio Compound Acronym Formula 0.5 Monocalcium phosphate monohydrate MCPM Ca(H2PO4)2⋅H2O 0.5 Monocalcium phosphate anhydrous MCPA Ca(H2PO4)2 1.0 Dicalcium phosphate dihydrate, brushite DCPD CaHPO4⋅2H2O 1.0 Dicalcium phosphate anhydrous, monetite DCPA CaHPO4 1.33 Octacalcium phosphate OCP Ca8(HPO4)2(PO4)4⋅5H2O 1.5 α−Tricalcium phosphate α−TCP α−Ca3(PO4)2 1.5 β−Tricalcium phosphate β−TCP β− Ca3(PO4)2 1.2-2.2 Amorphous calcium phosphate ACP CaxHy(PO4)z⋅nH2O 1.5-1.67 Calcium−deficient hydroxyapatite CDHA Ca10−x(HPO4)x(PO4)6−x(OH)2−x(0 60mM, the third peak of the lamellar structure (300) was observed at 2θ = 6.9° by WA−XRD (see Figure II.19). II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 64 The 100 reflexion of the lamellar mesostructure was observed as a low intensity peak for samples MHCaP-1, MHCaP-2 and MHCaP-3 prepared with [SDS+CTAB] = 5, 15 and 30 mM, respectively. A more intense peak was observed in MHCaP-4, MHCaP-5, MHCaP-6 and MHCaP-7 samples prepared from [SDS+CTAB] = 45, 60, 75 and 90 mM, respectively. Therefore, an increase in intensity of lamellar peak was observed as surfactant concentration increased during synthesis of materials. Regarding the CaP phase obtained in the hybrid materials, the WA-XRD patterns show in all cases the characteristic diffraction peaks of brushite [23] (Figure II.19). Transmission Electron Microscopy − Electron Diffraction TEM micrographs show two different morphological features in MHCaP-7 sample because rods and sheets or patches of around 300 nm in size are identified (see Figure II.20.a). Inset in Figure II.20.a shows the corresponding ED pattern exhibiting rings that can be assigned to a rather poorly crystallized brushite-like phase. The rods possess a lamellar mesostructure with 1.7 nm layer spacing and its formation may be explained due to the presence of SDS surfactant (see Figure II.20.c). Unfortunately, a deep analysis of the sheets was not possible with the transmission electron microscope since these particles rapidly decompose under the electron beam. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 65 In Figure II.20.b it can be observed a bubbles formation which was appreciable in a very short time, probably due to the combustion and evaporation of organic matter form the hybrid sample, as well as evaporation of crystalline water from the brushite phase. Conclusion of Section II.3.a An equimolar mixture of SDS and CTAB in increasing concentrations (from 5 to 90 mM) has been used as structure directing agent to obtain a lamellar mesostructured brushite calcium phosphate. Lamellar mesostructured brushite rods were identified by TEM-ED, although the presence of sheets with no clear mesostructure was also observed. II.3.b Calcium phosphate in mixed surfactants of sodium dodecyl benzene sulphonate and cetyltrimethyl ammonium bromide This section describes the synthesis of mesostructured hybrid calcium phosphates by precipitation in the presence of the mixture of SDBS and CTAB. Two samples were prepared with Ca/P molar ratio 1 and surfactant mixture concentration of 30 mM with 3:1 and 1:3 SDBS/CATB molar ratios, respectively. According to Section II.2.b, we expect to obtain HA in samples from highly concentrated SDBS surfactant in aqueous media. In highly concentrated CTAB surfactant solution, with the Ca/P molar ratio 1, we expect to obtain brushite. Synthesized organic-inorganic hybrid mesostructured materials were shortly named as HMM31 for SDBS+CTAB (3:1) and HMM13 for SDBS+CTAB (1:3). Thermogravimetric and Differential Thermal Analysis TG and DTA curves for both hybrid materials are consistent with the presence of brushite because the sharp weight loss around 200 °C is associated with the endothermic process for crystalline water loosing, as previously commented. Surfactant combustion is confirmed by DTA exothermic processes in the 300-600 °C temperature range. The exothermic peak around 500 °C is usually present in CTAB containing samples, therefore it is intense in the hybrid material prepared with the higher ratio in CTAB, HMM13 sample. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 66 Fourier Transform Infrared Spectroscopy FTIR spectra of HMM13 and HMM31 samples prepared with SDBS+CTAB in molar ratios 1:3 and 3:1 respectively (Figure II.22) are compatible with brushite and apatite−like profiles, respectively. However, pure HA cannot be assigned for HMM31 sample only with FTIR data since the presence of brushite is pointed out from the TG/DTA data. Small and Wide angle X−ray Diffraction The X−ray diffraction patterns of the hybrid samples are shown in Figure II.23. By SA−XRD no clear mesostructure is detected in any of the two samples, although two broad reflections centred at 2.9° and 4.5° seem to emerge in the SA diffractogram for the sample with higher SDBS content. Figure II.23 Small angle (left) and wide angle (right) XRD patterns of hybrid materials HMM13 and HMM31, where * and ◊ indicate HA and brushite, respectively. By WA−XRD, a brushite phase was clearly confirmed for HMM13 (1:3 mol ratio of SDBS+CTAB). On the other hand, a mixture of low crystalline HA and low crystalline brushite is identified for sample HMM31 (3:1 mol ratio of SDBS+CTAB). The formation of HA in this sample prepared with a Ca/P II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 67 molar ratio 1 can be explained due to a higher amount of SDBS surfactant than CTAB surfactant. The presence of higher amount of SDBS allows the pH of the medium increase during the synthesis due to the protonation of sulphonate polar head in phosphate aqueous solution (see section II.2.b). Therefore, two different CaPs have been obtained in the presence of SDBS and CTAB mixture by only changing the surfactant proportions. Transmission Electron Microscopy TEM images of HMM31 sample (Figure II.24) reflect the mixture of apatite and brushite phases found in the WA-XRD analysis. Two different particle morphologies are observed in the sample, nanorods identified as a low crystalline apatite in their ED pattern rings (Figures II.24 a and b, respectively) and sheets with a prismatic shape typical for brushite (Figure II.24.e). TEM images at higher magnification of the nanorods reveal a hybrid material with lamellar mesostructure having a 1.6 nm of layer spacing. The difference with the hybrid material obtained in the presence of pure SDBD (sample NHA1-3, layer spacing 2.5 nm, Figure II.14) can be attributed to the simultaneous presence of CTAB in this sample. Unfortunately, the analysis at higher magnification of the prismatic particles results in their quick decomposition, probably due to the combustion and evaporation of the organic matter and evaporation of the crystalline water of brushite under the electron beam. Figure II.24 TEM images of different regions in hybrid material HMM31 (SDBS+CTAB = 3:1, mol/mol). (a) Region of nanorods identified as HA by ED, (b) and (c) HRTEM images of the nanorods, (d) ED pattern of the nanorods region, (e) region of sheets with a prismatic shape typical for brushite and (f) HRTEM images of the sheets where bubbles due to decomposition are observed. Conclusion of Section II.3.b Two phases of calcium phosphate were obtained from Ca/P molar ratio 1 and [SDBS+CTAB] = 30 mM (1:3 and 3:1, mol:mol) by precipitation in aqueous media. The different CaP phases were obtained by II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 68 changing the molar ratio of surfactants. According to WA-XRD, crystalline brushite was obtained when SDBS+CTAB (1:3) and a mixture of low crystalline hydroxyapatite and brushite when SDBS+CTAB (3:1). Hence, surfactants ratio plays an important role on the obtained CaP phase. A higher presence of SDBS in the surfactant mixture favours the obtaining of HA, since protonation features of SDBS in aqueous media is responsible of a pH increase and HA growth, as it was justified in the case of CaP precipitation in the presence of SDBS alone. Also a lamellar mesostructure is favoured in the presence of a higher ratio of SDBS. II.3.c Calcium phosphate in mixed surfactants of mono-n-dodecyl phosphate and cetyltrimethyl ammonium bromide The synthesis of calcium phosphates by precipitation in the presence of the surfactants mixture MAP and CTAB is here presented. The synthesized hybrid material was prepared with [MAP+CTAB] = 240 mM, surfactant mixture in 1:1 molar ratio and Ca/P molar ratio 1. After the precipitation of the CaP the material was subjected to an autoclave process. The sample is termed LCaP. Thermogravimetric and Differential Thermal Analysis A total weight loss of 27% took place between RT and 500 °C. This weight loss is attributed mainly to organic matter due to the temperature range in which is produced (200- 500 °C), as well as the absence in this case of a clear endothermic peak around 200 °C. This fact would exclude the presence of brushite in this sample. Fourier Transform Infrared Spectroscopy The FTIR spectrum of this material is shown in Figure II.26 and is similar to that of monetite together with an appreciable amount of organic matter. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 69 Small and wide angle X−ray Diffraction SA−XRD and WA−XRD patterns of this material are shown in Figure II.27. SA−XRD pattern showed the first and second maxima of lamellar structure (L−marked in the figure) observed at 2θ = 2.4 and 4.9°. The third maximum of the lamellar structure was observed at 2θ = 7.3° in the WA diffractogram. The calculated distances are 3.60, 1.82 and 1.21 nm for these three reflexions, which are very similar to the reported ones for lamellar HA prepared in the presence of pure MAP [5]. Intense diffraction maxima, attributable to monetite, are observed in the WA−XRD pattern at 2θ = 13.1°, 20.6°, 26.5°, 28.5°, 30.1°, 32.6°, 35.9°, 38.8°, 40.1°, 41°, 44.5°, 45.7°, 47.5°, 49.2°, 50.6° and 53.0° that can be assigned to (010), (120), (220), (1 11), (112), (102), (022), (120), (030), (003), (132), (340), (322), (322), (122) and (312) reflections, respectively. Figure II.27 Lamellar mesostructure (left) and crystalline monetite (right) as detected by SA−XRD and WA−XRD measurements respectively. • indicate monetite diffraction maxima. Scanning Electron Microscopy The surface morphology of the hybrid material was examined by SEM, see Figure II.28. Two different morphologies, needle and cloud−like, were observed in Figure II.28.a. Moreover a cloud-like structure formed by shell layers can be seen in Figure II.28.b. Figure II.28 SEM images of hybrid calcium phosphate obtained with MAP+CTAB (1:1) and Ca/P = 1. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 70 Transmission Electron Microscopy The observation of this sample by TEM was not fully useful since the sample mostly decomposes under the electron beam and a rapid bubble formation is produced (Figure II.29, right). Nonetheless, a lamellar structure with 3.5 nm of layer distance was observed in some stable prismatic rod particles. This mesostructure is probably due to the presence of MAP because of its lamellar formation features in calcium phosphates already reported [5]. Figure II.29 TEM images at different magnifications of hybrid LCaP sample. Conclusion of Section II.3.c On one hand, as already found in section II.2.a, the use after the CaP precipitation of an energetic process, such as synthesis in an autoclave, leads to the achieving of monetite, a calcium phosphate phase derived from brushite but without the two water molecules. On the other hand, the hybrid sample is not stable under the TEM examination. Only particles with a clear lamellar mesostructure seem to be stable, which could point to the accomplishing of stable phases only in the case of mesostructure formation. Lamellar mesostructure would be due to the presence of MAP. The presence of CTAB does not modified the spacing distances with respect to samples prepared in the presence of pure MAP. II.4 Calcium phosphate in the presence of a zwitterionic surfactant A zwitterion (formerly called a dipolar ion) is a neutral molecule with a simultaneous positive and a negative electrical charge at different locations within the molecule. A zwitterionic surfactant such as EPG®BB exhibits neutral charge arising from the presence of a quaternary ammonium group and a deprotonated carboxylic acid group in its “polar” head. The aim in this section was to synthesize CaP http://en.wikipedia.org/wiki/Molecule http://en.wikipedia.org/wiki/Electrical_charge http://en.wikipedia.org/wiki/Zwitterion II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 71 materials in the presence of zwitterionic surfactant by precipitation to check whether mesostructural order could be obtained. Four materials were synthesized in 30 and 45 mM concentrations of EPG®BB surfactant by adding the solutions containing Ca2+ and HPO4 2− ions in different order. Therefore, two synthesis methods have been assayed as follows: in the first one, the calcium salt was dissolved in EPG®BB solution and then hydrogen phosphate salt was added to it and, in the second case, hydrogen phosphate salt was dissolved in EPG®BB solution and then the calcium salt was added to the solution. Two materials synthesized with [EPG] = 30 and 45 mM with Ca2+ in the solution were denominated CaP1 and CaP2, respectively. The other two materials synthesized with [EPG] = 30 and 45 mM with hydrogen phosphate ions in the solution were called PCa1 and PCa2, respectively. In all cases the Ca/P molar ratio was 1. Thermogravimetric and Differential Thermal Analysis PCa1 and PCa2 samples showed similar results to CaP1 and CaP2 samples. Thus, TGA curves for just two samples, PCa1 and PCa2, are shown in Figure II.30. DTA curves for all the samples were also very similar and only that of PCa1 is represented in the figure. TGA curves of samples PCa1 and PCa2 show a sharp weight losses of 23 and 33% around 200 °C respectively, which occurs simultaneously with an endothermic peak in DTA. This process is attributed to the removal of crystalline water from brushite. Phase change from brushite to monetite occurred at about 200 °C when removing crystallization water molecules. Remarkably there is not apparent loss of organic matter in these samples. Fourier Transform Infrared Spectroscopy FTIR analysis of the four samples shows brushite characteristic spectrum for each of them (see Figure II.31). As already suspected from the TG/DTA data, no bands due to ν(CH) modes from EPG®BB in the samples were observed around 2900 cm–1, therefore, the absence of organic matter points to a lack of interaction between CaP and EPG®BB surfactant. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 72 Small and wide angle X−ray Diffraction Small and wide angle X−ray diffraction patterns of these materials are shown in Figure II.32. By WA- XRD analysis crystalline brushite was observed in all samples. Brushite peaks were detected at 2θ = 11.6°, 20.9°, 23.3°, 29.2°, 30.4°, 34.0°, 41.5°, 42.0°, 45.2°, 48.4 and 50.1° for (020), (021), (040), (041), (221), (220), (151), (242), (171), (260) and(241), respectively. By SA-XRD one broad peak is observed at 2θ = 4.6° in all samples, which could be attributable to the first diffraction maxima of the octacalcium phosphate phase (OCP) [30]. However, the width of the peak is not characteristic for a crystalline OCP phase. Furthermore, no peaks attributable to OCP were found in the WA-XRD analysis. In fact, in the diffractograms of CaP1 and CaP2 samples there appear two broad maxima, at 2θ = 1° and 4.6°, that are not clearly indexable to a mesostructure, taking into account as well that there is not a surfactant template present in these samples, in agreement with TG/DTA and FTIR analysis. Figure II.32 Small angle (left) and wide angle (right) X−ray diffraction patterns of materials prepared in the presence of EPGBB surfactant, where ◊ indicates brushite. In order to better clarify the possible assignment of these two maxima an additional sample was prepared. The synthesis of the calcium phosphate phase was performed in the same experimental conditions that for samples CaP1 and CaP2, but in the absence of surfactant. The results analyzed by XRD (Figure II.33) show the same diffraction pattern in both angle regions, i.e., two broad maxima at 2θ = 1° and 4.7°, and the calcium phosphate phase of brushite. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 73 Figure II.33. Small angle (left) and wide angle (right) X−ray diffraction patterns of calcium phosphate prepared in the absence of surfactant, where ◊ indicates brushite. In fact, these two broad maxima were already observed for samples prepared in very low surfactant concentration, such as [SDS] = 15 and 30 mM (Figure II.6, II.7 and II.8) and [SDBS] = 15 mM (Figure II.12). By now we do not have a satisfactory explanation for these two broad maxima at such a low diffraction angle, since a deeper analysis of these samples by TEM was not possible, as explained below. Transmission Electron Microscopy In an attempt to clarify structural aspects the CaP2 sample was subjected to TEM observation (Figure II.34). Unfortunately, this sample was not stable under the electron beam. A very quick bubble formation was observed and taking into account that this sample does not contain organic matter this fact is probably caused from the evaporation of water from the crystalline brushite. Figure II.34 TEM images of the CaP2 sample. A, B and C are different magnifications of the same particle showing rapid evolution with time. D is an image of another particle in the sample. Conclusion of Section II.4 EPG®BB surfactant was assayed as a structure template to synthesize mesostructured calcium phosphate for the first time. However, FTIR results did not show any stretching bands from CH2 groups of EPG®BB and no weight loss due to organic matter was detected by TGA. Therefore, we can conclude that there is no interaction between CaP and this zwitterionic surfactant. As a consequence, no defined mesostructure was observed by small angle X−ray diffraction. Crystalline brushite was identified by wide angle X−ray diffraction and this phase was not stable under the TEM analysis conditions. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 74 II.5 Conclusions of Chapter II The interaction of several amphiphilic molecules with silica in aqueous media has led, through the confluence between the sol-gel and supramolecular chemistries, to a variety of mesoporous silica materials which are thoroughly investigated for many industrial applications and biomedicine. In this field, the synthesis of mesostructured calcium phosphates could bring new applications due to its likely bioactivity and biocompatibility. For this reason, several authors have treated to obtain them by using similar strategies of synthesis to those used for mesostructured silica. However, the chemical behaviour in aqueous solution of both families of compounds is completely different. Therefore, at the start of this thesis, the reported data on mesostructured calcium phosphates were limited to the obtaining of apatites with lamellar structure through its precipitation in the presence of two anionic surfactants: SDS and MAP. In this chapter precipitation of calcium phosphates was investigated in the presence of: 1) Anionic surfactants: SDS (deepening on already published) and SDBS. 2) Mixture of an anionic surfactant: SDS, SDBS, or MAP, and one cationic: CTAB. 3) Zwiterionic surfactant: EPG Table II.3 summarizes the families of compounds here investigated, including the results previously reported, the most significant results of this investigation and some suggestions for the future work. Table II.3. Main results derived from the precipitation of calcium phosphates in the presence of ionic surfactants studied in Chapter II. Surfactant Previous work of others Main results of this thesis Suggested work for the future SDS Lamelar apatite [reference 6] • Lamelar CaPs mixture • Lamelar brushite, rods SDBS ---- • Discontinuous lamelar apatite (or curved lamellar), meshes Studies of bioactivity and biocompatibility of this new phase MAP Lamelar apatite [refs. 5,21,22] ---- ---- SDS + CTAB ---- • Lamelar brushite, rods + non- mesostructured sheets (or plates) CTAB does not improve the structural characteristics of the hybrids obtained with pure SDS, SDBS or MAP. CTAB does not seem to interact during the formation of the CaP lamellar phase. SDBS + CTAB ---- • Low [SDBS]: non lamelar brushite • High [SDBS]: Lamelar HA + brushite (rods – prismatic lamelas) MAP + CTAB ---- • Lamelar monetite prismatic rods EPG (carboxybetaine) ---- • Brushite (no organic matter) The use other zwitterionic surfactant containing sulfobetaines could be a promising approach II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 75 In our study, as well as the type of surfactant(s), it was investigated the effect of: the surfactant concentration, the pH of precipitation, the Ca/P molar ratio (set in most cases to 1.0 or 1.67), and other parameters of synthesis. The main conclusions derived from the present study are summarized as follows:  When high concentration of SDBS was used, a poorly crystallized HA was obtained even when the Ca/P molar ratio used during synthesis was 1. This result was attributed to the increase of the pH during the synthesis from 7 up to 9, due to the protonation of sulphate polar heads of SDBS in phosphate aqueous solution.  SDBS plays a double role, it acts as mesostructure template as well as a base.  The use of energetic processes, such as ageing in autoclave or refluxing the material after the CaP precipitation, leads to monetite, anhydrous dicalcium phosphate.  The hybrid materials obtained by precipitation in the presence of surfactant mixtures showed two types of particles: (i) rod particles with lamellar mesostructure and (ii) sheet-like particles with no clear mesostructure. These sheet-like particles were not stable under the TEM analysis conditions. A rapid bubble formation process is explained by the combustion of the organic matter and the evaporation of the crystallization water from brushite. However, this fact points to a stabilization of the CaP phases in the case of clear mesostructure formation, because only the lamellar rods are stable under the TEM examination.  The lamellar mesostructure formation mechanism is showed in Figure II.35. Firstly, surfactants were added into the hydrogen phosphate solution to obtain a structure template before adding Ca2+ solution. By this method, brushite and poorly crystallized HA with lamellar structure were obtained with SDS, SDBS and with the mixtures of SDS+CTAB, SDBS+CTAB and MAP+CTAB. However, in the case of mixtures of surfactants, the lamellar phase seems to be due just to SDS, SDBS or MAP, and CTAB does not seem to interact during the formation of the CaP lamellar phase. Figure II.35 Proposed mechanism for the synthesis of lamellar structured CaP in presence of SDS, SDBS or MAP surfactants. In summary, ionic surfactants, especially anionic containing S or P in their polar heads, are useful to obtain mesostructured CaPs in lamellar mesostructure. In the future, the development of the synthesis techniques and methods for such a particular structures may be useful in the biomedical field for bone regeneration applications. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 76 II.6 References: 1. J. B. Thompson, H. J. Kindt, B. Drake, H. G. Hansma, D.E. Morse, P. K. Hansma. Nature 2001, 414, 773-776. Bone indentation recovery time correlates with bond reforming time. 2. C. T. Kresge, M. E. Leonowicz, W. J. Roth, J. C. Vartuli, J. S. Beck. Nature 1992, 359, 710-712. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. 3. N. Kawa, Y. Oumi, T. Kimura, T. Ikeda, T. Sano. J. Mater. Sci. 2008, 43, 4198-4207. 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Surfactant based assembly of mesoporous patterned calcium phosphate micron-sized rods. 9. H. Wang, L. Zhai, Y. Li, T. Shi. Mater. Res. Bull. 2008, 43, 1607-1614. Preparation of irregular mesoporous hydroxyapatite. 10. Y-H. Yang, C-H. Liu, Y-H. Liang, F-H: Lin, K. C. W. Wu. J. Mater. Chem. B. 2013, 1, 2447-2450. Hollow mesoporous hydroxyapatite nanoparticles (hmHANPs) with enhanced drug loading and pH-responsive release properties for intracellular drug delivery. 11. M. Vallet-Regí, F. Balas, D. Arcos. Angew. Chem. Int. Ed. 2007, 46, 7548-7558. Mesoporous materials for drug delivery. 12. F. Shao, L. Liu, K. Fan, Y. Cai, J. Yao. J. Mater. Sci. 2012, 47, 1054-1058. Ibuprofen loaded porous calcium phosphate nanospheres for skeletal drug delivery system. 13. C-. X. Zhao, L. Yu, A. P. J. Middelberg. J. Mater. Chem. B. 2013, 1, 4828-4833. Magnetic mesoporous silica nanoparticles end-capped with hydroxyapatite for pH-responsive drug release. 14. Y. Hong, H. Fan, B. Li, B. Guo, M. Liu, X. Zhang. Mater. Sci. Eng. R. 2010, R70, 225-242. Fabrication, biological effects and medical applications of calcium phosphate nanoceramics. 15. S. Recillas, V. Rodriguez-Lugo, M.L. Montero, S. Viquez-Cano, L. Hernandez, V. M. Castano. J. Ceram. Process Res. 2012, 13, 5-10. Studies on the precipitation behaviour of calcium phosphate solutions. 16. Sergey V. Dorozhkin. Front. Nanobiomed. Res. 2014, 2, 219-341. Nanodimensional and nanocrystalline calcium orthophosphates. II. Precipitation of mesostructured calcium phosphates in the presence of ionic surfactants 77 17. N. Kawa, H. Hori, K. Tatsuo, Y. Oumi, T. Sano. Micropor. Mesopor. Mat. 2011, 141, 56-60. Unique surface property of surfactant-assisted mesoporous calcium phosphate. 18. J . Jinhua, F. Yong, Z. Lirong, Y. Hong, C. Yanli, Z. Dazhou, Z. Ping. J. Mater. Sci. 2011, 46, 3828- 3834. Synthesis and characterization of multi-lamellar mesostructured hydroxyapatites using a series of fatty acids. 19. Q. Shen, H. Wei, Y. Zhao, D.-J. 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Synthesis of rod- like mesoporous silica using mixed surfactants of cetyltrimethylammonium bromide and cetyltrimethylammonium chloride as templates. 25. R. Z. LeGeros. KARGER 1991, Vol.15. Calcium phosphates in oral biology and medicine. 26. G. Cheng, C. Liu. Mater. Chem. Phys. 2003, 77, 359-364. Preparation of lamellar mesoporous silica microspheres via SDS templates. 27. J. C. Elliott. Elsevier Science B. V. 1994, 387. Structure and chemistry of the apatites and other calcium orthophosphates. 28. H. Zhao, W. He, Y. Wang, Y. Yue, X. Gao, Z. Li, S. Yan, W. Zhou, X. Zhang. Mater. Chem. Phys. 2008, 111, 265-270. Biomineralizing synthesis of mesoporous hydroxyapatite-calcium pyrophosphate polycrystal using ovalbumin as biosurfactant. 29. M. P. Ferraz, F. J. Monteiro, C. M. Manuel. J. Appl. Biomater. Biomech. 2004, 2, 74-80. Hydroxyapatite nanoparticles: a review of preparation methodologies. 30. Y. Honda, T. Anada, S. Morimoto, Y. Shiwaku, O. Suzuki. Cryst. Growth Des. 2011, 11, 1462- 1468. Effect of Zn2+ on the physicochemical characteristics of octacalcium phosphate and Its hydrolysis into apatitic phases.   III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 79 III. PRECIPITATION OF MESOSTRUCTURED CALCIUM PHOSPHATES IN THE PRESENCE OF PHOSPHOLIPIDS   III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 81 III.1 Introduction As it was previously indicated, in recent years particular attention has been paid to the preparation of mesostructured CaPs by using a templating route [1-9]. In this chapter, phospholipids (PLs) are used as structure directing agents, specifically phosphatidylcholine (PC) because is a biocompatible substance that exhibits self-assembly characteristics in water [5, 10-12]. PLs are widely distributed throughout the human body such as structural components in the cell and nucleus membranes. Due to their high biocompatibility they are receiving increasing attention in the synthesis of new biomimetic biomaterials [13-15]. PC plays an important role in many biological processes [15-18]. However until now, few studies were published about the synthesis of CaPs in the presence of PLs or PC [7, 19-22]. PC can be obtained from different sources such as egg yolk or soy bean [10]. Figure III.1 shows the schematic depiction of a PC molecule showing its main structural features: hydrophobic tails, glycerol and polar head groups. PC is an amphiphilic molecule that exhibits a tendency to self-assemble in water solutions. Polar head groups are forming outside and hydrophilic tails collected inside to form sandwich sheets called bilayers. These bilayers can form micelles and hollow spheres denoted vesicles. Critical micelle concentration and the molecular polarity and size of PC molecule determine the mesophase formed. Figure III.1 Double tail, hydrophobic tails, glycerol and polar head group of phosphatidylcholine. The idea for the experimental conditions and precipitation of CaPs in the presence of PLs was inspired by those used to obtain sponge mesoporous silica materials through a self-assembly process of PLs reported by A. Galarneau et al [23, 24]. In this study, phosphatidylcholine (PC) was used as template of mesoporous silica materials. Lecithin (containing 60% of PC) leaded to lamellar materials evoking multilamellar systems and vesicles formation. Then, these authors used dodecylamine as co- surfactant since the alkyl-amine was anticipated to play a role in the formation of the mesostructure given by the PLs. It promoted the decrease of the spontaneous curvature of the phospholipid bilayers. The phase transition from multilamellar mesostructures to sponge−like mesostructure was driven by the presence of dodecylamine which packs the head groups of the lecithin. Figure III.2 shows this phase transition and the sponge-like structure of the silica material obtained. In this chapter, we aimed at synthesizing HA based hybrids with a sponge-like mesostructure, looking for a stable mesostructure that could lead to a mesoporous CaP material after removing the organic part. The precipitation of CaPs in aqueous suspensions of PLs was used to obtain new mesostructured hybrid materials CaP-PLs. The influence of several parameters of synthesis: (i) pH of III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 82 solutions (basic or neutral), (ii) Ca/P molar ratio (1.0 or 1.67) and (iii) the PLs source (asolectin, lecithin or lipoid) was investigated. The mesostructure of the new hybrids is characterized and mechanisms of formation of the nanostructured materials are proposed [25]. Figure III.2 Left: Vesicle to sponge-like phase transition by addition of dodecylamines/alcohol and silica source to lecithin vesicles. Co denotes the spontaneous curvature of each phase. Right: TEM image of sponge-like silica materials showing the sponge structure. Inset: Schematic representation of the pore structure of sponge-like silica materials. Figure taken from ref 23: Micropor. Mesopor. Mater. 2007, 104, 103-114. Three PLs sources with increasing contents of PC were used: Asolectin from soybean, which is a mixture of mainly three PLs containing about 33 % of PC (PC33); L-α-Lecithin from egg yolk, which contains about 60 % of PC (PC60) and Lipoid-S100 from soybean, containing 94 % of PC (PC94). Table III.1 includes the experimental synthesis conditions, reactant and solvent amounts, Ca/P molar ratio and pH of the four samples discussed in this section. Hybrid materials were named as PC33, PC60, PC94−1 and PC94−2 with the numbers indicating the percentage of PC in the commercial source, respectively. Actually, many samples using different experimental conditions were synthesized but only the four most significant ones are presented in this chapter. Table III.1 Amount of phospholipids (PLs), moles of reactants, solvents and NaOH used in the synthesis of hybrids CaP−PLs. To analyze the textural properties of the inorganic materials and to check whether this method could lead to mesoporous apatite-like CaPs, hybrids were calcined at high temperature to remove the organic PLs. As a representative example of calcined sample, PC33 calcined material is presented in Samples (PLs source, % of PC) HPO4 2– (mol) Ca2+ (mol) PLs (g) H2O (mol) EtOH (mol) NaOH (mol) PC33 (Asolectin, 33) 1 1.67 0.5 126 38 2 PC60 (Lecithin, 60) 1 1 0.5 69 38 – PC94–1 (Lipoid, 94) 1 1 0.5 194 – – PC94–2 (Lipoid, 94) 1 1.67 0.5 223 – 1 III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 83 this chapter together with the other four hybrid samples. However, N2 adsorption-desorption isotherms of hybrid samples, as well as calcined samples, indicated that no significant surface area and porosity was obtained in any case (results not shown). III.2 Precipitation of calcium phosphates in the presence of phospholipids The detailed experimental procedure of synthesis of the hybrid materials is given in section VII.4.b. The general synthetic procedure was the following: CaPs were precipitated from calcium and phosphate ions in the presence of PLs, using a modification of a method described by Galarneau et al for the synthesis of sponge mesoporous silica [23, 26, 27]. Next, the most relevant results obtained for physical-chemical characterization of samples are presented. Thermogravimetric and Differential Thermal Analysis TG and DTA results for all the hybrid materials, are shown in Figure III.3. TGA curves of samples showed continuous weight losses mostly in the interval from 225 to 500 °C. In addition, TGA curves exhibited a slight weight loss between room temperature and 125 °C that was attributed to the loss of physisorbed water. TGA of PC94−2 and PC33 samples have dissimilar curves that PC60 and PC94−1 samples. PC33 and PC94−2, samples obtained in basic medium, present a continuous weight loss in all the interval with weigh losses of 18 % (PC33) and 14 % (PC94−2) in the 170 to 420 °C interval. These weight losses were observed together with exothermic peaks at 320 °C in the DTA curves that were assigned to the PLs combustion. For PC60 and PC94−1 samples, obtained at neutral pH, TGA curves exhibited sharp weight losses of 7 and 14 % (PC60 and P94−1, respectively) in the 170 to 225 °C range, associated with an endothermic peak in the DTA curve. This behaviour is characteristic for crystalline water loss in CaP materials consisting in brushite phase. In the 225 to 470 °C interval weight losses of 21 and 16 % (PC60 and P94−1, respectively) were observed together with exothermic peaks at 380 °C in the DTA curves, which were assigned to the PLs combustion. Therefore, the observed differences may be attributed to different CaP phases in the hybrid materials. III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 84 Fourier Transform Infrared Spectroscopy The FTIR spectra of the four samples studied are shown in Figure III.4, where in addition FTIR spectra of the calcined PC33 sample and for the pure PC are also included. Concordant with the information obtained by TG/DTA, two types of FTIR spectra were obtained. Thus, for PC33 and PC94−2 the spectra were analogous to those of apatite−like phases. However, the PC60 and PC94−1 FTIR spectra were similar to that of brushite [28]. In addition, FTIR spectroscopy is sensitive to the presence of specific functional groups. First, the presence of organic matter was confirmed in the four samples of hybrid materials by the bands at 2920 cm–1 and 2851 cm–1 assigned to asymmetric and symmetric stretching modes mainly from −CH2− groups. The bands of carbonyl group (C=O) present in the PLs structure appears at 1728-35 cm–1 in samples PC60 and PC94−1. However, the carbonyl bands are not visible in PC33 and PC94−2 spectra, i. e., in the apatite containing hybrids obtained in basic media. This fact could be related to an hydrolisis of the ester group, which is catalyzed at basic pH. Moreover, the FTIR spectra allow visualizing the relative amount of water in the hybrids. Again substantial differences were observed as a function of whether the hybrids might contain either apatite or brushite. PC60 and PC94−1 spectra exhibited the well-known bands at 3531 cm−1 and 3470 cm−1 of ν(OH) from HPO4 2−, at 3260 cm–1 and 3154 cm–1 of ν(OH) and at 1647 cm–1 of δ(HOH) from the H2O crystallization molecules in the brushite structure. However, in the apatite containing hybrids, PC33 and PC94−2, just a minimum amount of water, as humidity, detected by the presence of a very broad and low intensity band at about 3300 cm–1 was observed. FTIR spectrum of the calcined PC33 sample exhibits the bands corresponding to vibration modes for the following structural units: crystalline PO4 3– group observed at 1015, 600 and 550 cm–1; and CO3 2– groups observed at 1423 and 860 cm–1. The FTIR spectra of the samples also contain the bands corresponding to the vibration modes of –OH, PO4 3–, CO3 2– and HPO4 2– groups. In general terms the following facts can be indicated: - CO3 2– bands are only present at 1470 cm–1 in the apatite−containing hybrids spectra, i.e., the calcined PC33, PC33 and PC94−2, with more intensity in the PC33 sample. - –OH bands from hydroxyl group of HA appear at wave numbers 3642 cm–1 in PC33 and 3567 cm–1 in PC 94−2. Figure III.4 FTIR analysis of hybrid materials synthesized in the presence of PLs. III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 85 - P–OH, P–O and O–P–OH bands are quite similar in the spectra of the brushite-containing hybrids, i.e., PC60 and PC94−1. FTIR spectra of both hybrids are practically identical. - However, PC33 and PC94−2 spectra present some differences: the mentioned variations in the OH bands position and two bands at 531 cm–1 and 1560 cm–1 only visible in the calcined PC33 and PC33 spectrum. Besides, the PO4 3– band at 1020 cm–1 is broader for the calcined PC33 and PC33. These findings could be attributed to a lower homogeneity of PC33 obtained with a PLs mixture being the PC content of 33 % whereas PC94−2 was synthesised with PC of 94 % of purity. Small and wide angle X−ray Diffraction Figure III.5 shows the small angle (0.6 ° to 5 ° in 2θ) and wide angle (5 ° to 50 ° in 2θ) X−ray diffractograms of the four hybrid samples investigated and the calcined PC33. The small angle region was studied to detect the possible mesostructure of the hybrid materials. This fact would be confirmed by the presence of diffraction maxima in this region indicative of repetitive distances of a few nanometres in the sample structure. The wide angle XRD region was investigated to identify the CaP crystalline phases formed. The WA-XRD patterns of samples can be explained as follows: on the one hand the two hybrids synthesised at basic pH, i.e., PC33 and PC94−2 correspond to poorly crystallized apatite-like phases. No extra diffraction maxima of additional phases were obtained. The calcined PC33 sample exhibits a diffractogram concordant with an apatite-like phase with narrow maxima typical for samples subjected to a thermal treatment. The presence of a small amount of CaO was also detected in this sample. On the other hand, those diffractograms of hybrids synthesised at neutral pH were assigned to a brushite phase, although in both cases the most intense XRD reflection of monetite (002) was observed, indicative of the presence of a small amount of this phase in the hybrid materials. In PC33 and PC60 samples less information was found in the low angle region. However, a poorly defined shoulder centred at 1.4°, equivalent to a distance of around 6 nm, was observed for PC33 pattern and a broad diffraction at 2° for PC60 that corresponds to around 4.7 nm. This weak reflections are typical of nano-ordered mesostructures with lamellar bilayer and vesicles formation [5, 29], and this will be confirmed by TEM. As observed in Figure.III5, clear diffraction maxima are present in the small angle region of PC94−1 and PC94−2 patterns at 2.0° and 1.0°, respectively that correspond to distances of 4.3 nm and 8.6 nm respectively. Observed results can be summarized as follows: • At neutral pH: d ≤ 4.7 nm, d value is smaller and the observed CaP phase is brushite, • At basic pH: d ≥ 6 nm, d value is bigger and the observed CaP phase is HA. III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 86 Figure III.5 Small and wide angle X−ray diffraction patterns of four hybrid materials and the calcined PC33, where * and ◊ indicate hydroxyapatite and brushite, respectively. High Resolution Transmission Electron Microscopy High Resolution Transmission Electron Microscopy (HRTEM) images showed that PLs and CaP are arranged with different mesostructure in four samples depending on pH and type of solutions, as well as on Ca/P molar ratio and PLs source (see Figure III.6). The calcined PC33 sample, identified by WA-XRD mainly as a HA phase, was observed according to TEM as non porous particles with an eroded surface morphology. Obtained images of four samples PC33, PC60, PC94−1 and PC94−2 by HRTEM are shown in Figure III.7 together with a proposed scheme to explain how PLs form a mesostructure, and what kind of mesostructures were obtained. HRTEM images are interpreted as follows: (i) discontinuous lamellar bilayer structured hydroxyapatite with 4.2 nm size for sample PC33 [30]; (ii) bilayer vesicles of brushite with ~35 nm of diameter (media average) and 4.7 nm of bilayer thickness for sample PC60; III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 87 (iii) micelles structured brushite with 4.3 nm for sample PC94−1, where said micelles structure is confirmed according to Nabakumar P. et al.[5]; (iv) bilayer sponge−like [30] or worm−like structured hydroxyapatite observed with 4.3 nm for sample PC94−2. Figure III.6 HRTEM images of all materials. The CaP phases previously identified by XRD are described in the TEM images as non-porous HA (calcined PC33), HA with lamellar structures (PC33), brushite with vesicle structure (PC60), brushite with micelle structure (PC94−1) and HA phase with sponge−like structure (PC94−2). In case of PC33, several bilayers can pile up with a spacing nano-layer of water solution separating one another; such a structure is referred to as a discontinued lamellar phase [29, 30]. For sample PC60 prepared in water/ethanol solution, the polar characteristic of water provokes the formation of bilayer vesicles of PLs with around 35 nm in diameter. The thickness of the bilayer is 4.7 nm and inside the bilayer vesicles CaP with brushite structure is present. A mechanism for the mesostructure formation in sample PC94−1 is based on micelles behavior in water solution. In a first step, PC molecules form a micelle layer in the solution. After the addition of an aqueous PO4 3– solution to the aqueous PC solution, the PC−PO4 3– complex, formed by many PO4 3– species interacting with the surface of the micelle, is obtained. After an aqueous Ca2+ solution is added, a PC−calcium phosphate phase is formed because of the conformational compatibility between the micelle and the calcium phosphate. The micelle acts as a nucleating point for the growth of brushite crystals at an adequate aging temperature and time. III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 88 Figure III.7 HRTEM micrographs of calcium phosphate hybrids in the presence of PLs (left). Schematic representation of the PL and CaP arrangements (right). III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 89 Figure III.8 Temporal evolution of PC94-2 sample analyzed by TEM operating at 200 kV. (g) Schematic representation of the CaP-PLs arrangement as sponge-like hybrid material, PC94-2. III.3 Conclusions of Chapter III Organic−inorganic hybrid materials were synthesised by precipitation of calcium phosphates in phospholipids (PLs) suspensions. As PLs source, different commercial preparations were used, all of them containing phosphatidylcholine (PC) as main PL in proportions of 33% (asolectin), 60% (lecithin) and 94% (lipoid). Ca2+ and HPO4 2− ions were managed by different purity of PLs in ethanol and water dispersions: the PLs mixtures (i.e. asolectin and lecithin) were prepared in a mixture of water and ethanol. Lipoid was suspended in pure distilled water. The inorganic components of the hybrids were To observe the mesostructure in all hybrid samples we used HR-TEM operating at 200 kV. PC94-2 sample was analyzed in detail. During the analysis by TEM, samples are subjected to a high energy electron beam able to decompose organic matter. Figure III.8 shows the time evolution of PC94-2 sample during the analysis. As a difference with samples analyzed in Chapter II, in this case the time required to appreciate changes in the sample was somewhat longer. While in the first image (Figure III.8 (a)) different areas are not appreciated, along the time under the electron beam (ca. 2 minutes from a to e images) combustion of the organic matter occurs and it is removed from the sample. However, bubble formation was not observed here. This fact allows the visualization of the sponge-like type mesostructure formation that was initially present in the hybrid material. III. Precipitation of mesostructured calcium phosphates in the presence of phospholipids 90 poorly crystallized HA, when pH of medium was basic and the Ca/P molar ratio was 1.67 for samples PC33 and PC94-2. The observed CaP phase was brushite (CaHPO4·2H2O) for neutral pH and Ca/P = 1.0. No mesoporous materials were obtained by removing the organic matter via calcination. Instead, dense HA phase was obtained. FTIR analysis reveals a possible hydrolysis of the ester group of PC in samples prepared under basic pH. However, all samples present in FTIR the bands corresponding to CH2 groups, and moreover, thermogravimetric analysis data confirmed the presence of organic matter in the materials showing weight losses in the range of 220 °C and 470 °C around 20% in brushite containing hybrids, and around 17% in apatite ones. Furthermore, TEM analysis reveals that four different nanostructures were obtained: (i) lamellar bilayer with a spacing of 4.2 nm (apatite, asolectin), (ii) bilayer vesicles around of 35 nm in diameter (brushite, lecithin) and 4.7 nm in thickness of vesicles, (iii) micelles with sizes of approximately 4.3 nm (brushite, lipoid), and (iv) bilayer sponge−like or worm−like tubular mesostructures with 4.3 nm thickness of disordered bilayer sponge−like structure (apatite, lipoid). 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The multiple faces of self-assembled lipidic systems. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 93 IV. HYDROXYAPATITE COATINGS ON MESOPOROUS SILICA NANOSPHERES   IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 95 IV.1 Introduction Mesoporous silica nanospheres (MSNSs) are widely investigated in recent years for biomedical applications including the loading of anticancer drugs or the incorporation of magnetic thermoseeds to be used in hyperthermia treatments [1]. Nowadays, the drug delivery from MSNSs injected in the bloodstream is considered as an important approach for targeted tumor therapy [2-6]. The most relevant information on MSNSs has been presented in Chapter I. On the other hand, SiO2–CaO–P2O5 mesoporous bioactive glasses (MBGs) have been proposed for bone regeneration applications. These glasses exhibit the excellent textural properties (high surface area and pore volume) and mesopore order similar to pure silica mesoporous materials, together with a remarkable quick of the in vitro bioactive response [7]. MBGs have been also investigated for other clinical applications including gene transfection [8], encapsulation of proteins [9], tumor treatment [10], scaffold processing [11, 12] or drug delivery [13]. In some of these applications their use as nanoparticles (NPs) could bring considerable advantages. For instance, their use as drug nanocarriers for intracellular delivery due to cellular uptake of NPs, and the possibility of systemic administration through the blood stream. However, to our best knowledge no ternary SiO2–CaO– P2O5 MBG−NPs have been synthetized. In recent years, several close approaches in which we found inspiration were reported, including: (i) The synthesis of bioactive binary SiO2–CaO template glasses by M. Vallet-Regí [14], (ii) The synthesis of binary mesoporous SiO2–CaO NPs, by Wu et al [15], (ii) The coating of hydroxyapatite (HA) NPs by mesoporous silica, by Anderson et al [16], (iii) The functionalization of MSNSs with phosphonic acid, by Jin et al [17], (iv) The synthesis of ternary SiO2–CaO–P2O5 MBGs adding biologically relevant elements, by Shruti et al [18]. The present study tries to coat MCM−41 type MSNSs with HA NPs which would greatly improve their bioactivity and biocompatibility when used for bone therapy [19-22]. In addition, the HA coating could act as a nanogate allowing the controlled release under specific conditions – a pH decrease, for instance – of the biomolecules contained in the mesopores [23]. However, the synthesis of bioactive silica-based materials with such multifunctional properties still remains a significant challenge. The nanospheres coated by CaP NPs would exhibit well interconnected pore structures and excellent in vitro bioactivity useful for biomedical applications [24-28]. As it was indicated, the main objective of this memory was the synthesis of mesostructured calcium phosphates. The synthesis and characterization of CaPs obtained in the presence of ionic surfactants and phospholipids were described in Chapters II and III. In this Chapter, we changed the strategy by using MSNSs with hexagonal MCM–41 structure as nano-ordered structure core to lead Ca2+ and HPO4 2− ions to obtain HA NPs coatings as the nanospheres shell. The procedure attempted to obtain MSNSs coated by HA NPs involved two steps: (1) the synthesis of MSNSs ranging 150 – 250 nm of diameter and around 2 nm of pore diameter; (2) the coating of MSNSs by HA by using several approaches including: (i) the simultaneous precipitation of CaP on IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 96 MSNSs surface during the MSNSs sol-gel synthesis [29], (ii) to mantain the MSNSs soaked in a sol precursor of CaP and (iii) to wet the MSNSs several times with a sol precursor of CaP [30], and (iv) to functionalize the MSNSs with organic chains containing phosphate-like groups and then to wet them with a sol precursor of CaP [31]. For only one case (section IV.3), the co-synthesis of CaP and MSNSs, the strategy used does not implie the use of presynthesized MSNSs. Table IV.1 shows the approaches of synthesis and the sample codes. Table IV.1 Coating strategies, sample code and section of this Chapter where they will be described. Section Strategy of Synthesis Sample Code IV.2 Synthesis of mesoporous silica nanospheres MSNSs IV.3 Co-synthesis of CaP and MSNSs MSNS−HA−1 IV.4 Soaking MSNSs into a sol precursor of CaP MSNS−HA−2 IV.5 Wetting several times the MSNSs by a sol precursor of CaP MSNS−HA−3 IV.6 Functionalization of MSNSs with phosphate-like groups and subsequent wetting with a sol precursor of CaP IV.6.1 Co-condensation and post-synthesis with diethylphosphatoethyl-triethoxysilane, DEPETES NSFD1, NSFD2, NSFD IV.6.2 Wetting of DEPETES functionalized MSNSs NSFD−HA IV.6.3 Co-condensation with 3-trihydroxysilylpropyl methylphosphonate, THSMP NSFT IV.6.4 Wetting of THSMP functionalized MSNSs NSFT−HA IV.2 Synthesis of mesoporous silica nanospheres, MSNSs sample Regarding all the experimental strategies investigated in this Chapter to obtain core@shell MSNS@CaP composites, Sections IV.4, IV.5, IV.6.1 and IV.6.2 use MSNSs as starting material to be coated with CaP by different methods: soaking, wetting and functionalization and wetting, respectively. On the other hand, in Section IV.3, the sol-gel synthesis of MSNSs was performed simultaneously to the precipitation of the CaP. Therefore, we will start describing the synthesis and characterization of MSNSs that were used as reactants in Sections IV.4, IV.5 and IV.6. There are many methods to synthesize the MSNSs [2, 17]. In the present study our method was based on hydrolysis and condensation of silicon alkoxydes in the presence of structure directing agents and following a modified Stöber method. The synthesis procedure is detailed in Chapter VII.4.c. Briefly, CTAB was mixed with basic aqueous solution at 80 °C and stirred until a homogeneous solution was obtained. After that, TEOS as silica source was slowly added with stirring for two hours. Finally, the mixture was filtered, washed thoroughly with water/ethanol (1:1) and dried. The surfactant was removed by calcination. Next step was the physical and chemical characterization of the obtained MSNSs material. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 97 Fourier Transform Infrared Spectroscopy FTIR spectrum of MSNSs is shown in Figure IV.1. A typical spectrum of silica was observed exhibiting the following bands: at 1044 cm–1 a strong band of the asymmetric stretching mode νs(SiO) of SiO2; a shoulder at around 970 cm–1 attributed to the stretching mode of the Si-O bonds of the silanol groups of the silica surface; a medium band at 800 cm–1 for the symmetric stretching mode νs(SiO) of SiO2; and a strong band at 432 cm–1 for the bending mode δ(SiOSi) of SiO2. Small and wide angle X−ray Diffraction Figure IV.2 shows the SA-XRD and WA-XRD patterns of MSNSs. SA-XRD pattern shows three diffraction maxima at 2.6°, 4.4° and 5.1° in 2θ, which can be respectively indexed to (10), (11) and (20) reflections of a 2D hexagonal arrangement, being the unit cell parameter ao= 39.7 Å (ao= 2d100/√3, for d100 = 34.3 Å). These positions are easily indexable, q1, q2 and q3 are positions of the peaks 1, 2 and 3. We observed following relations q2=q1x√3, q3=q1x√4... For WA-XRD the MSNSs diffractogram shows only a diffuse maximum centred at 23o characteristic of amorphous silica. Figure IV.2 SA−XRD (left) and WA−XRD (right) patterns of MSNS. The diffraction patterns were assigned to a 2D-hexagonally arranged mesoporous lattice and to amorphous silica, respectively. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 98 N2 Adsorption−Desorption Isotherms Figure IV.3 shows the N2 adsorption- desorption isotherm, and as an inset, the pore diameter distribution of MSNSs [5]. Through these measurements it was obtained a maximum in the pore size distribution at 2.27 nm and a BET surface area of 1230 m2/g. MSNSs sample exhibits type IV BET isotherms with no observed hysteresis loop, confirming the presence of a cylindrical, one-dimensional channel-like mesoporous structure in the nanospheres. The N2 isotherms present a sharp inflection at a relative pressure of 0.2−0.3, which corresponds to the capillary condensation and evaporation within channel-type uniform mesopores. In addition, a secondary step at a P/Po above 0.95 is observed, attributed to condensation in textural porosity, i.e., in the macropores formed between the nanoparticles after the drying process. Scanning Electron Microscopy The spherical morphology of particles in MSNS sample is revealed by the SEM micrograph in Figure IV.4. SEM image shows homogenously dispersed nanospheres with sizes ranging between 150 and 320 nm in diameter. The mean value of 100 nanospheres measured is 228 ± 30 nm. Transmission Electron Microscopy Figure IV.5 shows two TEM micrographs of MSNSs. As shown in the left side micrograph, the silica nanoparticles exhibit spherical shape, while the micrograph in the right side details their ordered mesopores hexagonal arrangement. Spacing between mesoporous silica channels was measured at 2.5 nm. 320 nm 150 nm 1 μm Figure IV.4 Surface morphology of MSNSs observed by SEM. MSNS IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 99 Figure IV.5 TEM micrographs of MSNS showing the nanospheres (left) and a higher magnification image to observe better their pore arrangement. Conclusion of Section IV.2: Synthesis of mesoporous silica nanospheres, MSNSs sample MSNSs were obtained as monodispersed particles with sizes between 150 and 320 nm of diameter and a high surface area of 1230 m2/g. These MSNSs were used as reference to compare with modified MSNSs investigated in the next sections. IV.3 Precipitation of calcium phosphate simultaneous to the formation of mesoporous silica nanospheres, MSNS−HA−1 sample In this approach MSNSs were treated to coat by HA−NPs in one step involving two simultaneous processes as follows: (i) precipitation of HA and (ii) synthesis of MSNSs. Accordingly, Ca2+ and phosphate ions were added into the solution containing TEOS at the onset of silica condensation (for a more detailed description of the synthesis method, see Chapter VII). Hence, the aim was to provide interactions between silica, Ca2+ and phosphate ions during the silica condensation to synthesize homogeneous silica@CaP composites with the silica MSNSs coated by nanocrystalline HA. Calcium and phosphate precursor solutions were added in a Ca/P molar ratio of 1.67. Furthermore, the Stöber method uses sodium hydroxide to catalyze the silicate precursor hydrolysis and this basic medium favours the HA formation. On the other hand, if Ca2+ and phosphate ions penetrated into the silica mesopores they could increase their interaction with silica matrix. This would increase the homogeneity of silica-HA composites. The main goal was to achieve ionic interactions between silicate, Ca2+ and phosphate ions. The following text describes the characterization of the sample obtained, MSNS-HA-1. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 100 Fourier Transform Infrared Spectroscopy FTIR spectrum of MSNS−HA−1 is shown in Figure IV.6. The spectrum shows overlapping bands in the 1100-1000 cm–1 range corresponding to the stretching mode of ν(SiO) and ν(PO) for SiO2 and PO4 3– groups, respectively. For silica also appear the bands for the stretching mode ν(SiO) at 806 cm–1 and at 950 cm–1 for the silanol surface groups, as well as the δ(SiOSi) bending mode at 443 cm–1. Finally, bands at 601 and 563 cm–1 that were assigned to δ(OPO) bending modes of PO4 3– in crystalline environment were visible. These two peaks of phosphate are characteristic of crystalline phosphate [32]. Hence, the FTIR spectrum of MSNS−HA−1 contains both bands of silica and bands of phosphate in a crystalline environment which hints at the simultaneous formation of SiO2 and HA. Small and wide angle X−ray Diffraction Figure IV.7 shows the SA−XRD and WA−XRD patterns of MSNS−HA−1. SA−XRD pattern shows four diffraction maxima at 2θ = 2.2 °, 3.9 °, 4.5 ° and 6.0 ° , which can be respectively indexed to (100), (110), (200) and (210) reflections of a 2D hexagonal arrangement in the MSNSs [33], being the unit cell parameter ao= 45.8 Å (ao= 2d100/√3, for d100 = 39.7 Å). The first four peaks of MSNS−HA−1 sample by SA−XRD confirm that the 2D mesoporous hexagonal arrangement is present, but do not confirm if some interaction was established between SiO2, Ca2+ and phosphate ions. Figure IV.7 SA−XRD (left) and WA−XRD (right) patterns of MSNS−HA−1. The diffraction maxima in the SA-XRD pattern were indexed to a 2D hexagonally arranged mesoporous material. The poorly defined diffraction maxima in the WA-XRD pattern were assigned to a very poorly crystallized HA phase. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 101 WA−XRD pattern can be indexed as a poorly crystalline HA. As it can be observed in Figure IV.8 (right) the diffuse diffraction maxima in the pattern were assigned to the (002), (211), (112), (300), (202), (310) and (222) reflections of HA (International Centre for Diffraction Data (ICDD) Powder Diffraction File No. 9-432). The calcination temperature selected for the synthesis or this material was 550 °C, enough to remove the surfactant whereas the hexagonal order in the MSNSs is maintained, avoiding at the same time further modifications in the HA phase formed if higher temperatures were used. Differently from WA-XRD data of MSNSs where a diffuse maximum centred at 23° characteristic of amorphous silica is registered, here we observed poorly crystalline HA in MSNS-HA-1 sample. N2 Adsorption−Desorption Isotherms Figure IV.8 shows nitrogen adsorption and desorption isotherm of MSNS−HA−1. The Figure also includes the SBET surface area and the pore size diameter distribution. This isotherm is analogous to previously reported for MCM−41 materials [34]. The SBET was 594 m2/g and the pore size distribution exhibited two maxima at 0.6 nm and 2.6 nm, respectively. In general, isotherm displays a decrease of N2 adsorption capacity in the presence of the HA phase compared with pure silica MSNSs with surface area 1235 m2/g (see section IV.2). However, the surface area of the silica nanospheres is not totally coated because the composite material still shows mesoporous features with a remarkable surface area of 594 m2/g. This result indicated that HA is not completely absorbed within the accessible mesopores of material. Hence, after simultaneous synthesis of MSNSs and HA, the SBET of the sample decreased around 52 %. Transmission Electron Microscopy In Figure IV.9 TEM micrographs of MSNS−HA−1 are shown. Particles with three different features can be distinguished as follows: (i) uncoated MSNSs, (ii) nanorods and (iii) nanoparticles which are identified as HA. The silica particles exhibited spherical shape with ordered mesopore channels arrangement. However, great differences in particle size were found, MSNSs around 60 nm and around 320 nm of diameter. Spacing between mesoporous silica channels were 2.5 nm as can be observed in Figure IV.9.d. Moreover, Figures IV.9.b and IV.9.d shows some HA nanoparticles and HA nanorods. These needle- like HA nanorods formed are similar to those reported by Zhao, Grigoriadou and Verma [35-37]. To sum up, TEM results show that the precipitation method of CaP simultaneously with the synthesis of IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 102 ordered mesoporous silica nanospheres yield MSNSs with high dispersity in particle diameters and a CaP phase with a fairly heterogeneous distribution of the HA phase. Figure IV.9 TEM micrographs of MSNS−HA−1 showing MSNSs and HA nanorods. Conclusion of Section IV.3: Precipitation of calcium phosphate simultaneous to the formation of mesoporous silica nanospheres, MSNS−HA−1 sample Poorly crystallized HA nanorods and mesoporous silica spheres with a high polydispersity in particle diameter were separately observed by this method. The objective of using the hexagonal structure of MSNSs as core was not reached because the interactions between CaP (HA) and silicate ions were poor. However, the presence of HA could confer bioactive features to the material obtained with this approach. According to the analyses, the presence of HA NPs in the sample decreased the textural properties of MSNSs (surface area and porosity). Figure IV.10, schematically depicts the synthesis of MSNS−HA−1 sample, showing how HA nanorods and MSNSs were separately obtained. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 103 Figure IV.10 Schematic depiction of MSNS−HA−1 synthesis by simultaneous addition of Ca2+ and phosphate ions together with the silica precursor TEOS. IV.4 Soaking mesoporous silica nanospheres into a sol precursor of calcium phosphate, MSNS−HA−2 sample In this approach the synthesis of silica@CaP nanocomposites was attempted by soaking pure silica MSNSs in a sol precursor of CaP. After that, the material was filtered and characterized to check if silica@CaP composites were formed. This strategy of synthesis is here described for the first time. In detail, the synthesis procedure can be divided in the next steps, as it is schematically explained in Figure IV.11: Figure IV.11 Schematic representation of method used showing how the CaP sol precursor is prepared and then used to soak the MSNSs. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 104 (1) Synthesis of the silica nanospheres MSNSs to be used as core, (2) Soaking of MSNSs for 3 h with stirring in the sol precursor of CaP followed by a filtration. We had speculated that this process could be enough to obtain an appropriate interaction between Ca2+ and HPO4 2− ions and the silica matrix. (3) The MSNSs soaked in the sol precursor of CaP were heated in air at 550 °C trying to obtain HA. The sample obtained after this process was termed as MSNS−HA−2. Fourier Transform Infrared Spectroscopy FTIR spectrum of MSNS−HA−2 in the range between 2000 and 400 cm–1 is shown in Figure IV.12. The assignation of the bands present in the spectrum is as follows: • SiO2: at 1050 cm–1 for νas(SiO) and at 800 cm–1 for νs(SiO) stretching modes; at 435 cm–1 for δ(SiOSi) bending mode. • PO4 3−: at 548 cm–1 for δ(OPO) of PO4 3− in an amorphous environment. • H2O: at 1630 cm–1 for δ(HOH) of physisorbed water. Therefore, the presence of CaP in the sample can be deduced by the band at 548 cm–1, which was not visible in the MSNSs spectrum (Figure IV.1). Small and wide angle X−ray Diffraction Figure IV.13 shows the SA−XRD and WA−XRD patterns of MSNS−HA−2 sample. SA−XRD pattern shows two diffuse diffraction maxima at 2θ = 2.44 ° and 4.2 °, which can be respectively indexed to (100) and (110) reflections of a 2D hexagonal arrangement in the MSNSs, being the unit cell parameter ao= 42.8 Å (ao= 2d100/√3, for d100 = 37 Å). Figure IV.13 SA−XRD and WA−XRD patterns of MSNS−HA−2 sample. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 105 Thus, after soaking the silica nanospheres in sol precursor of CaP, only two XRD maxima with very low intensity were visible in the XRD pattern. This fact reveals a decrease in the hexagonal order of MSNS−HA−2 compared with the initial MSNSs, see Figure IV.2. Regarding the WA−XRD pattern in Figure IV.13 (right), no clear diffraction maxima were observed, indicating the absence of crystalline phases in MSNS−HA−2. Although some maxima for poorly crystallized HA seem to emerge from the background of the wide diffuse band between 20 and 37 o in 2θ, the WA−XRD diagram is not conclusive at all in this regard. N2 Adsorption−Desorption Isotherms Figure IV.14 shows nitrogen adsorption and desorption isotherm of MSNSs and MSNS−HA−2. The Figure also includes the SBET surface area value and the pore size diameter distribution (inset). The analysed sample is compared with the initial untreated MSNSs used as reference. For MSNS−HA−2 SBET was 250 m2/g and the pore size distribution exhibited two maxima for Dp of 0.6 nm and 2.5 nm, respectively. As observed in the Figure, MSNS−HA−2 sample displays a decrease of N2 adsorption capacity after the soaking and filtration treatment compared with pure silica MSNSs, which exhibit a SBET of 1230 m2/g and a maximum in the pore size distribution at Dp of 2.27 nm. However, the surface area of the silica nanospheres of MSNS−HA−2 sample should not be totally coated by HA, because it still shows mesoporous features and a quite remarkable SBET of 250 m2/g. This result could indicate that CaP particles are not completely located within the accessible mesopores of material. Hence, after the treatment with the sol precursor for calcium phosphate, the SBET decreased around 80 % with respect to pure MSNSs. Transmission Electron Microscopy − Energy Dispersive X−ray Spectroscopy Figure IV.15 shows several TEM micrographs of the MSNS−HA−2 sample. TEM images show that the initial nanospheres have changed their morphology, from spherical NPs to ellipsoid shaped NPs. Also, the 2D hexagonal array of mesopores has disappeared, although a radial porosity is observed. These ellipsoid shaped NPs seem to be formed by a unique phase with mean elemental composition in Si:P:Ca of 50:25:25, analyzed by EDS in several NPs. These results indicate that the composition of the initial SiO2 nanospheres has also changed. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 106 A few particles with different morphology were identified by TEM in some regions of the sample and their analysis by EDS gave values mainly for Ca and P, being Si in a minimum proportion. Therefore, some particles of a CaP phase were also formed. Figure IV.15 TEM micrographs of MSNS−HA−2 showing ellipse−like MSNSs and few particles identified as a CaP phase (up, right). The values of Si, Ca and P elemental composition (atomic %) analyzed by EDS are indicated as insets. In vitro bioactivity test Simulated body fluid (SBF) solution, proposed by Kokubo et al [38, 39], is used to perform in vitro simulations of in vivo conditions. Several revisions of the SBF solution have been proposed, including one in 2003 which took into account the fact that a large proportion of calcium and magnesium species present in serum is bound to proteins and hence unavailable for apatite precipitation [40]. According to Kokubo and Takadama’s paper [38], a bioactive material is a material on which bone−like hydroxyapatite will form selectively after it is immersed in a serum−like 100 nm 100 nm 100 nm100 nm Ca:74 P:22 Si:4 P:25 Si:61 Ca:14 Si:54 P:20 Ca:26 MSNS−HA−2 MSNS−HA−2 IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 107 solution. Since 1990, the use of SBF for in vitro bioactivity testing has been reported [41]. In the present study, the bioactivity tests in SBF were performed to confirm the apatite formation ability on the MSNS−HA−2 surface. The in vitro bioactivity of MSNS−HA−2, i.e., its ability to be coated by an apatite−like layer after soaking in a simulated body fluid (SBF), was tested as follows: 42 mg of MSNS−HA−2 compacted into a pellet were soaked in 8.4 mL of SBF for three different time intervals up to 15 days. After the soaking period, the pellet was gently washed with water and ethanol and dried at 80 °C for 24 h. Figure IV.16 shows the variation of Ca2+ concentration and pH in the SBF with the soaking time of MSNS−HA−2 measured in an ILyte Electrolyte Analyzer. As observed in the Figure, calcium levels increased quickly during the first 4 days, after that it increased slowly until 8 days. Thereafter, the calcium concentration remained stable until the end of the assay. The pH evolution follows a profile different to that of [Ca2+] showing in the first 6 days a slight decrease from the initial 7.40 to 7.35 and it is unchanged in a value of 7.37 until the end of the assay. These results could indicate that a Ca2+−H+ exchange occurs between the sample and the SBF solution, mainly in the first 6 days of assay. FTIR spectroscopy was carried out to evaluate the changes in the sample surface as a function of soaking time. Figure IV.17 collects the FTIR spectra of MSNS−HA−2 before and after different times in SBF. Before soaking (t=0) the spectrum shows characteristic absorption bands of amorphous phosphate groups at 600 cm−1 with low intensity, and silica bands at 1040, 800 and 470 cm−1 already observed in Fig IV.12. An increase in the intensity of the amorphous phosphate absorption band is observed after 4 days in SBF combined with a decrease in the definition of the silicate bands. Both effects are more visible when the soaking time increased to 8 and 15 days. Figure IV.18 shows the SEM micrographs of the sample before and after soaking in SBF for 8 days. MSNSs are clearly seen as spheres before soaking in SBF (Figure IV.18, left), t = 0. SEM micrograph after 8 days in SBF reveals that the sample surface undergoes some changes because new particles are formed (Figure IV.18, right). IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 108 Figure IV.18 SEM micrographs of MSNS−HA−2 surface before and after 8d in SBF. Conclusion of Section IV.4: Soaking mesoporous silica nanospheres into a sol precursor of calcium phosphate The MSNSs were soaked for 3 h in a sol precursor of CaP and then filtered. 2D hexagonal ordered mesoporous silica NPs were intended to be used as structure core to be covered by HA. The synthesis procedure was schematically explained in Figure IV.11. By this procedure the sample obtained exhibited the following features: ellipse-like NPs and an elemental composition different from the initial SiO2, containing Si, Ca and P. Moreover, a significant decrease in porosity and decrease in surface area of ca. 80% was found by N2 adsorption, although the surface area is 250 m2/g, which is still valid for bioapplications. Therefore, this procedure affects mesoporosity, composition and shape of mesoporous silica nanospheres. Also, this material shows a moderate in vitro bioactivity in SBF. Figure IV.19 Schematic representation of method used showing the wetting process of the MSNSs into the precursor sol of CaP. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 109 IV.5 Wetting mesoporous silica nanospheres into a sol precursor of calcium phosphate, MSNS−HA−3 sample In this case, the core@shell material (MSNSs@CaP) was treated to be obtained by soaking the MSNSs in the sol precursor of CaP for a short period of time and subsequently performing a filtration and repeated washings with the sol for 20 times. That is, the filtered solution was poured again over the filter containing the nanospheres, repeating the process for 20 times. With this procedure, schematically depicted in Figure IV.19 (previous pag), we were trying to mimic a dip-coating method. The obtained sample was called MSNS−HA−3. The main difference with the previous study was the soaking time in sol precursor of CaP (30 min), avoiding a long soaking time which seems to affect in a high extend the MSNSs, as described in the previous section. Fourier Transform Infrared Spectroscopy The FTIR spectrum of MSNS−HA−3 is shown in Figure IV.20 and compared with FTIR spectrum of uncoated MSNS. The bands were assigned as follows: • SiO2: at ca. 1050 and at 802 cm–1 the bands for stretching modes ν(SiO). At 432 cm–1 bending mode δ(SiOSi). • PO4 3–: at ca. 1050 cm–1 the band for stretching mode ν(PO) and at 600 and 565 cm–1 for δ(OPO). The HA presence in the sample can be confirmed by phosphate bands observed at 600 and 565 cm–1 revealing the presence of PO4 3− in a crystalline environment [42]. Small and wide angle X−ray Diffraction Figure IV.21 (left) shows the SA−XRD pattern of MSNS-HA-3 to analyze the possible hexagonal order in the nanospheres, compared with MSNSs. Figure IV.21 The possible nanostructural order shown by SA−XRD (left) and diffuse maxima indexed for poorly crystallized HA shown by WA−XRD (right) for MSNS-HA-3. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 110 SA−XRD pattern of sample shows three diffraction maxima with low intensity at 2θ = 2.6°, 4.4° and 5.1°, which can be respectively indexed to (100), (110) and (200) reflections of a 2D hexagonal arrangement. For WA−XRD, the sample shows the poorly defined diffraction maxima assigned to a very low crystalline HA phase. As it can be observed in the Figure, the diffuse diffraction maxima in the diffractogram were assigned to the (002), (211), (112), (300), (202), (301), (310) and (222) reflections of a HA phase (ICDD Powder Diffraction File No. 9-432). Thus, while the WA−XRD pattern for MSNSs shows only a diffuse maximum centred at 25 o characteristic of amorphous silica, WA−XRD pattern of sample MSNS−HA−3 can be assigned to a poorly crystallized HA. N2 Adsorption−Desorption Isotherms Figure IV.22 shows N2 adsorption and desorption isotherms of MSNS−HA−3. The Figure also includes the SBET surface area and the pore size diameter distribution. As observed in the Figure, the SBET was 136 m2/g and the pore size distribution exhibited maxima at 2.21 nm. In general, isotherms display a decrease of N2 adsorption capacity after coating compared with pure silica MSNSs with a SBET of 1235 m2/g. However, the pore diameter is maintained, from 2.27 nm to 2.21 nm, indicating that the accessible pores preserve the initial diameter. This fact would be indicative of a partial coverage of the silica nanospheres, leaving pores without an entrance blockage. This fact also discard occupation of the cylindrical channels or internal surface by the HA. The composite material still shows mesoporous features with quite enough surface area. Hence, after the treatment with the sol, the SBET decreased around 90 %. Transmission Electron Microscopy Figure IV.23 shows TEM images of MSNSs and MSNS−HA−3 samples. For MSNSs, SiO2 nanospheres in the range 150-250 nm of diameter, exhibiting highly ordered 2D hexagonal mesoporous structure are observed. After sol filtration repeated times and thermal treatment (MSNS−HA−3 sample), the nanospheres appeared fully coated by HA nanoparticles as identified by the XRD analysis. Nanospheres of silica coated by hydroxyapatite phase are clearly observed in MSNS−HA−3 sample with homogeneous size of around 250 nm of diameter. The sol filtration method allows a fairly homogeneous distribution of HA nanoparticles layer throughout the mesoporous silica nanospheres surface. Elemental composition analyzed by EDS gives values for Si:P:Ca of 59:17:24 (atomic %) when registered in the centre of the covered nanospheres. However, for the particles near the borders of the nanospheres values for Si:P:Ca of 2:38:60 (atomic %) were obtained. The silicon content IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 111 decreased as expected for a calcium phosphate composition, which in addition possesses a Ca/P molar ratio of 1.58, very close to the corresponding ratio in HA, 1.67. Figure IV.23 TEM images of MSNSs (before wetting by sol precursor of CaP) and MSNS−HA−3 (after wetting by sol precursor of CaP). Conclusion of Section IV.5: Wetting mesoporous silica nanospheres into a sol precursor of calcium phosphate MSNS coated with HA were synthesized by wetting MSNSs in a sol precursor of CaP for a short time and subsequently filtering and washing the nanospheres with the sol for 20 times, resembling a dip−coating method. The differences with our previous study in section IV.4 were (i) the soaking time of the MSNSs in sol precursor of CaP that was now only 30 min instead of 3 h and (ii) the wetting procedure with the sol up to 20 times. Final sample was calcined in air at 550 °C to obtain HA phase. Our goal here was to check if the soaking time plays an important role on the shape, composition and mesoporosity of MSNSs. For MSNS−HA−3, the presence of HA and its interaction with the silica mesoporous structure were confirmed by TEM, FTIR and WA−XRD. By SA−XRD, the very low intense diffraction maxima indicated the presence of a 2D hexagonal mesoporous order still present after HA coating. The SBET surface area was 136 m2/g, indicating a decrease of N2 adsorption capacity of around 90% with respect to uncoated MSNS with a SBET surface area of 1235 m2/g. The pore diameter is maintained in the sample treated with the sol, therefore, the formed HA is not inside the pores but capping the entrance in 200 nm 100 nm 100 nm 100 nm ̴ 250 nm 225 nm MSNS−HA−3 MSNS IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 112 most of them. TEM images confirm the formation of HA NPs covering the surface of MSNSs. Moreover, homogenously formed nanospheres were observed by TEM. As opposed to MSNS−HA−2, in this case no ellipse−like nanospheres were observed. These results confirm the effect of soaking time over nanospheres composition, shape and mesoporosity. This new route could be used to improve the bioactivity and biocompatibility of nanospheres designed for clinical applications in bone regeneration. Specific applications will require wetting levels that allow us to reach different textural properties. The composite is thought to be suitable as bone filler material where a biodegradable and osteoconductive implant with a localized release of biologically active substances is required. IV.6 Functionalization of nanospheres with phosphate–like groups and subsequent wetting into a sol precursor of CaP The application of nanotechnology in the field of drug delivery has brought many innovations on the surface functionalization of inorganic nanomaterial-based delivery vehicles, such as MSNSs. The ability to functionalize the surface of silica groups of mesoporous nanospheres with organic entities make them suitable as a new platform for various biotechnological and biomedical applications [13, 43-57]. Here, the abundant silanol groups (Si−OH) on the silica surface of MSNSs were functionalized with molecules containing phosphate-like groups such as diethylphosphatoethyltriethoxysilane (DEPETES) [17, 58-60] and 3-trihydroxysilylpropyl methylphosphonate (THSMP), see Figure IV.24. Figure IV.24 Chemical structure of DEPETES and THSMP. The following two subsequent steps were used to achieve the main goal of this study: (i) functionalization of MSNSs with phosphate−like groups and (ii) using these groups as binding moieties between silica surface and Ca2+ and HPO4 2− ions to coat the MSNSs with HA NPs. This novel approach gives us an opportunity to synthesize the nanostructured HA onto the surface of prefunctionalized silica. Co-condensation and post-synthesis methods were used to obtain the MSNSs functionalized with phosphate−like groups. Co-condensation method (direct synthesis) allows synthesizing the silica phases functionalized with phosphate−like groups in the presence of surfactant. By this method, it is possible to prepare mesostructured silica phases with DEPETES and THSMP in the presence of surfactants leading to materials with organic residues covalently anchored to the pore walls. Post−synthesis method allows preparing the pre-synthesized MSNS with phosphate-like groups. This method carries out in two steps: (i) first MSNSs are synthesized by addition of the silica source (TEOS) into a surfactant Si P O O O O O O EtO DEPETES EtO EtO HO HO Si HO O P O O― Na+ CH3 THSMP IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 113 template in water solution and, once the surfactant is removed, (ii) MSNSs are then functionalized with the alkoxysilanes. Regarding the silanes used for phosphate-like functionalization, DEPETES can be used in co- condensation as well as in post-synthesis methods. However, THSMP is only appropriate for co- condensation because it already possesses the silanol groups or hydrolized alkoxysilane moiety. Therefore, the following samples were prepared: Two samples with DEPETES obtained by co-condensation containing 10 and 20% mol of DEPETES with respect to TEOS, NSFD1 and NSFD2 samples, and one sample with DEPETES obtained by post- synthesis, NSFD sample. The amount of DEPETES used for each sample is explained in section VII.5.c. The obtained samples are named NSFD1, NSFD2 and NSFD (Nanospheres prefunctionalized by DEPETES). The sample functionalized with THSMP was prepared by co-condensation and it is named NSFT (Nanospheres prefunctionalized by THSMP). The functionalized samples were extracted with acidified ethanol solution to remove surfactant as described in section VII.5.c. Then, all functionalized samples were subjected to a wetting process in a sol precursor of CaP and they are named NSFD−HA and NSFT−HA for DEPETES and THSMP, respectively. IV.6.1 Functionalization of nanospheres with diethylphosphatoethyltriethoxysilane: by post-synthesis for NSFD sample, and by co-condensation for NSFD1 and NSFD2 samples Thermogravimetric and Differential Thermal Analysis The TG/DTA curves, as well as weight loses, for the samples obtained with DEPETES are shown in Figure IV.25. TGA curves show continuous weight loses from 180 to ~450 °C, due to organic matter combustion. Moreover, TGA curves exhibit a small weight loss between room temperature and 150 °C that was attributed to the physisorbed water loss. Samples functionalized by co-condensation are subjected to a solvent extraction to remove the surfactant. With this method it is not usual the complete removal of the surfactant, being common a remained amount of around 5% wt. Samples NSFD1 and NSFD2 present different TGA curves, as well as a bigger amount of organic matter, that the sample prepared by post-synthesis NSFD, in which the surfactant is completely removed by calcination before organic functionalization. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 114 Fourier Transform Infrared Spectroscopy The FTIR spectra for all samples functionalized with DEPETES are shown in Figure IV.26. Typical spectrum of silica was obtained. The presence of a small amount of DEPETES in all samples was confirmed by the low intensity bands of ν(CH) from CH2 groups and δ(OPO) from phosphonate groups. Small angle X−ray Diffraction Figure IV.27 shows the SA−XRD patterns of these three samples. In total four XRD maxima were observed that can be indexed on a hexagonal lattice typical of MSNSs [33]. An interplanar spacing was around d100 ≈ 40 for (dhkl = 100). As shown in the figure, other three peaks were observed around d(Å) = 22 and 19 and 15 nm for 2θ = 2.2°, 3.8°, 4.4 and 5.8°, respectively. N2 Adsorption−Desorption Isotherms Pore surface area and pore diameter data of the samples were obtained by nitrogen adsorption and desorption isotherms (Figure IV.28). All samples are based on a model mesoporous adsorbent exhibiting a pore structure in the form of hexagonal arrays of uniform tubular channels of controlled width [61]. The well-defined reversible type IV isotherms obtained in all samples correspond to MCM−41 type. This is a type IV BET isotherm of mesoporous materials with an adsorption curve step between relative pressures P/Po of 0.2 and 0.4 [62]. The following values of BJH pore diameter (Dp) and BET surface area (SBET) were obtained: • NSFD Dp = 0.8 and 1.9 nm ; SBET = 878 m2/g • NSFD1, 10 % Dp = 2.5 nm ; SBET = 1190 m2/g • NSFD2, 20 % Dp = 0.6 and 2.3 nm ; SBET = 920 m2/g IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 115 Figure IV.28 N2 adsorption-desorption isotherms (left) and pore diameter data (right) of the samples obtained by nitrogen adsorption and desorption isotherms. Scanning Electron Microscopy and Energy Dispersive X−ray Spectroscopy The morphology of the samples is observed by SEM images in Figure IV.29. These micrographs show that all samples consist on monodisperse spherical nanoparticles. The particle size seems bigger in the case of co-condensation using 20% of DEPETES, being between 170 and 250 nm for all the samples. DEPETES presence was confirmed by phosphorous detection in the EDS analysis, being the P content very similar for the three samples although slightly bigger for the sample obtained by post- synthesis. Figure IV.29 SEM micrographs of the DEPETES functionalized MSNSs. Conclusion of Section IV.6.1: Functionalization of mesoporous silica nanospheres with diethylphosphatoethyltriethoxysilane MSNSs functionalized with phosphate groups of DEPETES were prepared via two functionalization methods, co-condensation and post-synthesis. The presence of DEPETES was confirmed by the content in organic matter, EDS and FTIR analysis, observing phosphonate groups and hydrocarbon chains. All samples show a high specific surface area according to BET model. These results confirm that the DEPETES functionalization has not decreased the surface area to a high extent. In addition, the hexagonal arrangement was observed by WA−XRD. Therefore, the typical mesoporous silica nanospheres were no structurally affected. The sample selected for the next step is NSFD, the obtained via post-synthesis, due to its slightly higher content in phosphorous. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 116 IV.6.2 Wetting of DEPETES functionalized MSNSs via post-synthesis into a sol precursor for CaP, NSFD−HA sample Fourier Transform Infrared Spectroscopy The FTIR spectra of the NSFD−HA sample is shown in Figure IV.30 together with NSFD sample, before sol treatment. The band attributed to the bending mode δ(OPO) from phosphate groups is more intense after sol treatment. C−H stretching bands in NSFD-HA sample could not be observed due to calcinations in the HA preparation process. Small and Wide X−ray Diffraction SA−XRD and WA−XRD analysis were used to determine the mesostructure and crystalline phase in NSFD-HA sample, see Figure IV.31. Due to the wetting of the MSNSs functionalized with DEPETES with the sol precursor of CaP, the three peaks of hexagonal structure were observed with very low intensity by SA−XRD (Figure IV.30, left). These three peaks can be indexed on the hexagonal arrangement of MCM−41 material, corresponding to a interplanar spacing d100 ≈ 37.5 Å (dhkl = 100). Besides the broad maximum of amorphous SiO2, a potential nanocrystallized hydroxyapatite phase was detected in the WA-XRD pattern after wetting in the sol precursor of CaP. Poorly defined diffraction maxima can be assigned to the (002), (112) and (222) reflections of an HA−like phase. Figure IV.31 SA−XRD (left) and WA−XRD (right) of NSFD−HA sample, where * indicates HA. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 117 N2 Adsorption−Desorption Isotherms Nitrogen adsorption−desorption isotherms of NSFD after being soaked in the CaP precursor sol are shown in Figure IV.32. The sample exhibit similar type IV isotherm that before being soaked, demonstrating the features of typical mesoporous materials. The surface area was SBET was 335 m2/g, which is lower than the typical for MSNSs materials. It has decreased around 62% compared with NSFD. Pore size distribution exhibited a maximum at 1.8 nm. Transmission Electron Microscopy TEM micrographs of different regions of the NSFD−HA sample presented in Figure IV.33 show nanospheres possessing mesopore ordered structure. The shape of nanospheres is not modified after wetting them with the sol precursor of CaP. The nanospheres seem to be covered by a phase rich in Ca and P, as the EDS analysis in several nanospheres indicates. There is also presence of other nanoparticles in which the 0.7 nm spacing could be attributed to the interplanar distance (001) of hydroxyapatite. Therefore, the sol coating method allows a fairly homogeneous distribution of an HA layer throughout the ordered MSNSs. Conclusion of Section IV.6.2: HA coating of mesoporous silica nanospheres functionalized with diethylphosphatoethyltriethoxysilane by post−synthesis In this study, an easy synthesis route for making multifunctional silica@HA composite material is presented. Our goal was to coat MSNSs with HA and make it a bioactive material for biomedical applications. The MSNSs with HA particles were prepared by wetting MSNSs functionalized with DEPETES into a precursor sol for CaP. Then, they were subjected to a thermal treatment at high temperature to obtain the HA phase of interest for its bioactive properties. The obtained product could be useful in biomedical applications. This system exhibits a potential application in the fields of drug delivery based on its possible bioactive and mesoporous properties. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 118 Figure IV.33 TEM images of NSFD−HA sample. IV.6.3 Functionalization of nanospheres with 3−trihydroxysilylpropyl methylphosphonate by co-condensation, NSFT sample During synthesis the molar ratios of used reactants were as follows: 1 TEOS / 0.3 THSMP. Thermogravimetric and Differential Thermal Analysis TG/DTA were used to detect the presence of surfactant and THSMP and the results are shown in Figure IV.34. TG curve exhibited a weight loss between room temperature and ~100 °C that was IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 119 attributed to physisorbed water. The organic matter loss of THSMP and residual surfactant from the extraction method was estimated around 13 %. Fourier Transform Infrared Spectroscopy The FTIR spectrum is shown in Figure IV.35. A typical spectrum of silica was obtained for this sample. In addition, the following bands confirming the presence of THSMP are observed: • Phosphonate bending region: at 577 cm−1 for δ(OPO). • CH stretching region for THSMP: at 2981 and 2926 cm–1 for νas(CH) and νs(CH), respectively. Small angle X−ray Diffraction SA−XRD analysis was used to determine the mesostructure of the nanospheres functionalized with THSMP (Figure IV.36). An interplanar spacing d100 ≈ 39.8 Å was observed (dhkl = 100). Four peaks observed in SA−XRD 2θ region can be indexed on a hexagonal lattice typical of MCM−41 type, confirming that the 2D hexagonal order is achieved during the co-condensation with the used TEOS/THSMP proportion. N2 Adsorption−Desorption Isotherms BET surface area and pore diameter data of the sample were calculated from the nitrogen adsorption and desorption isotherms in Figure IV.37. The sample exhibits a hexagonal array of uniform tubular mesoporous channels that is confirmed the Type IV isotherms with no observed hysteresis loop. It shows a well−defined step in the adsorption curve between partial pressures P/Po of 0.2 and 0.4. Pore diameter distribution calculated by BJH showed two maxima at 0.8 and 2.7 nm. A BET surface area of 1316 m2/g was calculated. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 120 Scanning Electron Microscopy and Energy Dispersive X−ray Spectroscopy Morphology of particles in sample is revealed by SEM micrographs in Figure IV.38. SEM image indicates that NSFT sample is formed by monodisperse spherical nanoparticles with 235 nm in average diameter. THSMP presence was confirmed by EDS analysis where a peak for phosphorous is observed. Transmission Electron Microscopy Figure IV.39 shows the TEM micrographs of NSFT. The nanospheres, homogeneous in size, with an average diameter of 200 nm, exhibit a highly ordered hexagonal mesoporous structure. Therefore, functionalization with THSMP by co-condensation in the TEOS/THSMP molar ratio used of 1:0.3 did not affect the mesoporous structure of MSNSs. The presence of THSMP phosphate−like groups was again confirmed by EDS with phosphorous and silicon values of 2 and 97 atomic %, respectively, dismissing the content in carbon and oxygen (see inset, Figure IV.39). Figure IV.39 TEM images of MSNSs functionalized with THSMP via co-condensation method. Conclusion of Section IV.6.3: Functionalization of mesoporous silica nanospheres with 3−trihydroxysilylpropyl methylphosphonate by co−condensation Hexagonally ordered MSNSs were functionalized using phosphate-like groups of THSMP by co- condensation (molar ratios of reactants: TEOS/THSMP = 1/0.3). According to N2 adsorption analysis, the synthesized sample shows a typical mesoporous isotherm with 1316 m2/g of BET surface area and pore diameter of 2.7 nm. The diameters of nanospheres are quite homogeneous around 200 nm as observed by TEM. TEM micrographs also show the 2D hexagonal order. The presence of THSMP in the nanospheres was confirmed by EDS and FTIR studies. The MSNSs have been functionalized with phosphate−like groups to facilitate the interaction with Ca2+ ions to obtain HA NPs on the MSNSs external surface, which was attempted in the next section. IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 121 IV.6.4 Wetting of THSMP functionalized mesoporous silica nanospheres with a sol precursor of CaP, NSFT−HA sample Fourier Transform Infrared Spectroscopy The FTIR spectrum of sample NSFT−HA is shown in Figure IV.40. In this case the FTIR spectrum is compatible with silica as well as a calcium phosphate phase possessing PO4 3− in crystalline environment. Presence of carbonate groups is also identified in the spectrum. X−ray Diffraction SA−XRD and WA−XRD patterns of NSFT−HA sample are shown in Figure IV.41. Mesostructure was not observed in SA−XRD analysis. Figure IV.41 SA/WA−XRD analysis of HA coating of MSNSs functionalized with THSMP, where * indicates hydroxyapatite. According to WA−XRD, a nanocrystallized HA phase was observed in NSFT−HA sample. The diffraction maxima can be assigned to the (100), (101), (002), (102), (210), (211), (112), (300), (202), (310), (322), (312) and (213) reflections of an HA−like phase. N2 Adsorption−Desorption Isotherms Figure IV.42 shows N2 adsorption-desorption isotherms of NSFT−HA. NSFT−HA sample exhibits a non-porous behaviour, with a surface area of 30 m2/g, as a great decrease of N2 adsorption capacity. The IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 122 decrease in surface area compared to the uncoated sample was around 97 %. This fact could be indicating a pore blocking due to the HA coating of the MSNSs in the external surface. Transmission Electron Microscopy − Energy Dispersive X−ray Spectroscopy TEM micrographs of the functionalized MSNSs (NSFT) and the functionalized MSNS subjected to soaking and wetting treatment by the precursor sol of CaP (NSFT-HA) are shown in Figure IV.43. Figure IV.43 TEM−EDS of the functionalized NSFT sample (a, b) and the NSFT−HA sample (c, d, e, f). TEM images of NSFT, just described in the previous section, show nanospheres monodisperse in size were the 2D hexagonal arrangement of mesopores is clearly observed (Figures IV.43.a and b). Nanoparticles more irregular in size were observed for NSFT−HA sample, which could be explained considering that the soaking process may deform the nanospheres, as it was discussed for sample MSNS-HA-2 (Figure IV.43.c and d). The nanoparticles seem to be covered by a new layer, although the presence of the channels is still perceptible underneath this layer (Figure IV.43.d. and f). IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 123 The presence of a CaP phase in NSFT−HA sample was confirmed by EDS analysis. The measured contents (atomic %) of Ca, P and Si were 14, 29 and 57 for Figure IV.43.c, and 35, 15 and 50 for Figure IV.43.d, respectively. Conclusion of Section IV.6.4: Hydroxyapatite coating of mesoporous silica nanospheres functionalized with 3−trihydroxysilylpropyl methylphosphonate by co−condensation MSNSs have been functionalized with phosphate−like groups of THSMP by a co-condensation method, and then coated with an HA layer by soaking and wetting them in a precursor sol for CaP, as explained in the scheme of Figure IV.44. As observed by TEM images with this approach core@shell structure of MSNS@HA has been successfully obtained. The nanoparticles were observed with a non- porous surface area, as confirmed by N2 adsorption measurements. The presence of HA would improve the bioactivity and biocompatibility properties of the mesoporous silica nanospheres. Figure IV.44 Schematic depiction of the overall process and detailed steps of wetting of the MSNS in the sol to obtain NSFT−HA. IV.7 Summary and conclusions of Chapter IV The present study tried to coat MCM−41 type MSNS with HA NPs or HA layer which would greatly improve their bioactivity and biocompatibility. Mesoporous silica nanospheres with 2D hexagonal MCM−41 structure of were attempted to be used as nano-ordered structure core to obtain HA coatings as shell onto the external surface of the MSNSs. According to this goal, the conclusions derived from each synthetic route are as follows: • MSNSs were synthesized as silica nanospheres exhibiting 2D hexagonal MCM-41 type mesoporous order and high surface area (SBET = 1230 m2/g). IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres 124 • Co-synthesis of CaP and MSNSs, MSNS−HA−1 sample. The precipitation of CaP method was used simultaneously to the synthesis of MSNSs by sol-gel chemistry. Poorly crystallized HA rods and silica nanospheres polydisperse in size were separately observed as a mixture of independent particles (SBET 594 m2/g). Thus, with this approach our objective was not reached because no interactions between CaP and silicate ions took place. • Soaking of MSNSs into a sol precursor of CaP, MSNS−HA−2 sample. In this case the soaking time into the sol affected the shape, composition and mesoporosity of the original MSNSs. Pure SiO2 mesoporous nanospheres become ellipsoids with a Si, Ca and P composition and a radial mesoporosity around 80% lower. The obtained material revealed a slow bioactive response in SBF. • Wetting of MSNSs by a sol precursor of CaP, MSNS−HA−3 sample. MSNS coated with HA NPs were successfully obtained when the soaking time into the sol is minimized to 30 min and subsequently the nanoparticles are subjected to a wetting process trying to mimic a dip- coating process. With this wetting process the MSNSs are not altered in shape neither in composition. TEM images confirm the formation of HA NPs covering the surface of MSNSs. The surface area reduction of sample is around 90 % (SBET 136 m2/g). • Functionalization of MSNSs with phosphate-like groups and subsequent wetting into a sol precursor of CaP. Last section was designed to ascertain the functionalization of MSNS with phosphate-like groups such as DEPETES or THSMP. The functionalized MSNSs exhibited 2D hexagonal arrangement and highly porous materials were obtained with specific surface areas between 878 and 1316 m2/g. The goal was to facilitate the interaction of phosphate- like groups of MSNSs with Ca2+ ions to obtain HA NPs. This would give a possible interaction between silica, Ca2+ and phosphate ions in order to obtain HA NPs or a HA layer coating the surface of the MSNSs. The following results were obtained: o MSNSs were successfully functionalized with DEPETES by co-condensation for NSFD1 and NSFD2 and by post-synthesis for NSFD samples. o The functionalized NSFD exhibiting a slightly higher P content was wetted by sol filtration. This method allows a fairly homogeneous distribution of an HA layer throughout the MSNSs, as well as some HA nanoparticles. Specific surface area is still 335 m2/g, indicating that the surface is not totally covered. o MSNSs were functionalized with THSMP by co-condensation with a BET surface area of 1316 m2/g, NSFT sample. o NSFT sample was wetted by sol filtration. The nanospheres were coated with a HA layer. As observed by TEM with this approach core@shell structure of MSNS@HA has been successfully obtained. 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Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 131 V. COATING OF MESOPOROUS SILICA NANOSPHERES WITH FLUORESCENT HYDROXYAPATITE NANOPARTICLES   V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 133 V.1 Introduction Materials based on hydroxyapatite (HA) exhibit good biocompatibility and osteoconductivity [1, 2]. Thus, they are widely used in Orthopaedics and Dentistry [3]. HA structure can accept numerous ionic substitutions including lanthanide ions. Therefore, in recent years several strategies have been developed to produce luminescent HA nanoparticles (NPs) doped with Eu3+, Eu2+, Ce3+, Eu3+/Y3+ or Pb2+ ions [4-7], that could be used as luminescent labels or luminescent drug carriers [8]. Moreover, small amounts of a doping ion (0.5–2 mol %) induce luminescence in HA−based materials [4]. The emitted wavelength (colour) can be modified by selecting a specific dopant or a combination of dopants. In general, the luminescence intensity of a material is dependent on the concentration of the doping ions, the crystal structure and the degree of crystallinity of the host material. In this study nanocrystalline HA is the host material and Eu3+ and Y3+ the doping ions. On the other hand, as it was explained in Chapters I.3 and IV, silica mesoporous matrices are potential drug carriers because of their textural features including: (i) ordered pore network, homogeneous in size (pore diameter, Dp ≈ 2-10 nm); (ii) high pore volume; (iii) high surface area; and (iv) silanol (Si–OH) rich surface. The functionalization of Si–OH groups allows tailoring at demand the drug loading and release. These unique features make mesoporous materials excellent candidates for controlled drug-delivery systems (DDSs), and intensive research was carried out in this topic in the last decade [9-14]. As a continuation of the research developed in Chapter IV, the aim in this chapter is the coating of MCM−41 mesoporous silica nanospheres (MSNSs) by europium−doped HA NPs, to develop new biocompatible luminescent HA/MSNSs based materials. If successful results are achieved, MSNSs coated by europium-doped HA NPs would be promising biomaterials with fluorescent properties that after optimization could be highly appropriate for biomedical applications [7, 15, 16]. Such applications include: luminescent labels, luminescent drug carriers, biological probes, biological labelling and red fluorescent probes that follow an excitation in the visible domain [17-19]. V.2 Materials synthesis Experimental conditions to obtain the MSNSs coated with europium−doped HA NPs were inspired in those used to obtain lanthanide−doped HA and silica materials reported by S. Dembski [7, 20]. First, HA was precipitated in the presence of Eu3+ ions in a suspension of previously prepared MSNSs. The final step was completed by addition of Poly(ethyleneimine), PEI, (Mw=750.000 g/mol), that is used to stabilize the particles [7]. Citric acid was used in the preparation of one of the samples as chelating ligand of metal ions [20]. This chapter was developed during a short research stay and therefore, the syntheses were carried out at the Institute of Inorganic Chemistry of the University of Duisburg Essen. Synthesized lanthanide (Eu+3)−doped HA/MSNSs composites were shortly named as LDHASix where x indicates the number of synthesized sample. Four samples were synthesised by using the synthesis conditions detailed in Table VII.3 of the Chapter VII and briefly described in Table V.1. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 134 Table V.1 Calcium, monohydrogen phosphate and europium ion concentrations used to coat 0.25 g of MSNPs. As far as possible, all synthesized samples of FHA (Fluorescent HA)/MSNSs composites were characterized during the period of stay at the mentioned German institution. Therefore, the most representative samples were analysed with the results presented below. These analysed FHA/MSNSs samples are compared with MSNSs used as reference. Fourier Transform Infrared Spectroscopy The FTIR spectra of the four samples investigated and that of reference MSNSs are shown in Figure V.1. A typical spectrum of mesoporous silica was obtained for MSNSs. For FHA/MSNSs samples, the spectra were analogous to those of apatite–like phases. Thus, the FTIR spectra of FHA/MSNSs samples exhibit the following bands of the characteristic functional groups: • SiO2: At 1054 cm–1 for νas(SiO) symmetric stretching mode, at 968 cm–1 for υ(SiO) stretching mode of Si-OH groups, at 805 cm–1 for νs(SiO) and at 440 cm–1 for δ(SiOSi) bending mode. • PO4 3−: At 1024 for νas(PO) stretching mode; 605 and 553 cm–1 for δ(OPO) bending mode. • CO3 2−: In the 1450-1400 cm–1 interval for ν(CO). The HA presence in synthesized samples of FHA/MSNSs can be confirmed by the observed FTIR bands of phosphate in LDHASi2 and LDHASi4. In addition, these samples present in their FTIR spectra normal vibration bands of carbonate groups, suggesting that the apatite phase is hydroxycarbonate apatite (HCA). Samples MSNPs (g) Ca2+ (mM) HPO4 2– (mM) Eu3+ (mM) LDHASi1 0.25 19.7 11.94 0.2 LDHASi2 0.25 2462.5 1492.5 25 LDHASi3 0.25 0.1905 0.116 0.00075 LDHASi4 0.25 2437.5 1458.8 50 V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 135 The coexistence of the adsorption bands coming from phosphate and silica can be observed in the spectra of LDHASi1 and LDHASi3. That way, LDHASi1 spectrum is very similar to that of the reference, although bands that can be assigned to PO4 3− bending modes are also present. In LDHASi3 the situation is analogous but with an increase in the intensity of the phosphate bands. Finally, in the FTIR spectrum of LDHASi4 certain anomalies in the shape of the bands suggest that another calcium phosphate phase such as brushite could co−precipitate with the HA phase that, in any case, would be the majority calcium phosphate phase. Small and wide angle X − ray Diffraction Figure V.2 shows the small and wide angle XRD patterns of pure MCM−41 silica nanospheres, used as reference, and also the patterns of the FHA/MSNSs composites synthesised in this chapter. The MSNSs XRD pattern, registered in the small angle region, shows three diffraction maxima at 2θ = 2.3 °, 4.07 ° and 4.7 °, which can be respectively indexed to the (10), (11) and (20) reflections of a 2D hexagonal arrangement with MCM−41 structure [21]. However, the diffractogram of LDHASi2 shows only a low intensity maximum at 2θ = 2.3 ° that can be assigned to the (10) reflection of the hexagonal phase. This could indicate that after the process to coat the silica nanospheres with Eu3+−doped HA NPs, only the highest intensity maximum remained visible. For LDHASi3 and LDHASi4, no diffraction maxima were observed, indicative of the absence of hexagonal order. The MSNSs XRD pattern, registered in the wide angle region, shows only a diffuse maximum centred at 25 o characteristic of amorphous silica. A low crystalline HA phase was observed in the diffractogram of LDHASi1. The diffraction maxima can be assigned to the (100), (101), (111), (002), (210), (211), (202), (310), (222) and (213) reflections of HA (ICDD Powder Diffraction File No. 9-432). However in LDHASi2, two weak reflections were observed at 2θ = 26.0 o and 31.7 o which could be indexed to the (002) and (211) reflections of an apatite−like phase [22]. For LDHASi3 and LDHASi4, very low crystalline HA was observed because the diffraction maxima of HA are wider and a lower number of reflections, assigned to (002), (211), (310) and (222), were now observed. Therefore, in terms of increasing HA crystallinity, the four samples would be classified as follows: LDHASi2, LDHASi3, LDHASi4, and LDHASi1. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 136 Figure V.2 Small and wide angle XRD patterns of reference (MSNSs with MCM-41 structure) and the synthesized materials, where * indicates HA. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 137 Scanning Electron Microscopy, Energy Dispersive X−ray Spectroscopy and Elemental Analysis The surface morphology of MSNSs before and after coating with europium−doped HA was studied by SEM (Figure V.3). Micrograph of the uncoated material shows the typical pseudo−spherical shape of mesoporous MCM−41 NPs obtained via sol-gel in the presence of structure directing agents and using the Stöber modified method. However, the morphology of samples containing FHA NPs is completely different. For LDHASi1, two morphologies of HA NPs and nanospherical MSNSs with average diameter of 170 nm were observed, and the presence of calcium, phosphorous and silicon in LDHASi1 (left) was confirmed by EDS with 46.3, 37.2 and 16.5 %, respectively. According to EDS analysis, we found major proportion of silicon in LDHASi1. The Ca/P ratio was calculated as 1.24 which could corresponds to a calcium-deficient HA (CDHA) phase together with amorphous CaP. As a difference from the other samples, the surface morphology of LDHASi2 shows homogeneous rod−like HA nanoparticles with around 60 nm in length and 15 nm in width. No visible nanospheres of silica in LDHASi2 were observed by SEM. Moreover the presence of calcium, phosphorous and silicon was confirmed by EDS analysis with 55.9, 40.7 and 3.5 %, respectively. Accordingly to this, LDHASi2 includes a low content of silicon. The Ca/P ratio was 1.36 which corresponds to a CDHA phase. The content in PEI, (H(NHCH2CH2)nNH2), in the samples was measured from the nitrogen present in its amino groups, by using CHN elemental analysis as shown Table V.2. This Table also contains the calcium, phosphorous and silicon percentages in the samples measured by EDS analysis in the SEM microscope. Table V.2 Ca, P and Si contents obtained by SEM−EDS (at-%) and N content (wt-%) by CHN analysis. Element LDHASi1 LDHASi2 Method Ca 46.3 55.9 SEM-EDS (At-%) P 37.2 40.7 Si 16.5 3.5 N 1.1 2.4 CHN Elemental Analysis (wt %) P Ca Si 170 nm 1 μm LDHASi1 500 nm 150 nm LDHASi1 LDHASi2MSNSs 1 μm 1 μm 180 nm CaP Si Figure V.3 Surface morphology of samples MSNSs, LDHASi1 and LDHASi2 observed by SEM. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 138 In the synthesis of LDHASi1 sample the relative proportion of silica NPs respect to Ca2+ and HPO4 2− ions was very high, and consequently the silicon content is high. And, as it was expected, LDHASi2 contains higher amounts of Ca and P because in this case the proportion of these elements was very high compared with the amount of silica NPs added during the synthesis. Transmission Electron Microscopy In Figure V.4 TEM micrographs of MSNSs, LDHASi1 and LDHASi2 are shown. Hexagonally meso−structured MSNSs were shown as reference for comparison. In the micrograph of sample LDHASi1 it can be visualized that MSNSs were completely coated by fluorescent HA. Therefore, a stronger interaction between europium- doped HA NPs and MCM-41 nanospheres can be inferred. TEM image of LDHASi2 sample shows that the nanospheres have not been coated by nanocrystalline HA, in other words, ellipse-shaped MSNSs and europium−doped HA phase are not interacting with each other. In any case HA nanocrystals are homogeneous in size and forming groups into similar shapes. In this Chapter and Chapter IV.4, similar materials were obtained by different synthetic methods, and similar results were observed. However, in this study, the difference is the synthesis of CaP in the presence of europium ions. In both chapters, MSNSs coated HA NPs were observed, and the morphology of the obtained HA phase was compared with that reported by Linden et al [23]. Separated nanospheres and HA phase were observed in some of the samples according to TEM images. In any case (before or after the coating process), some nanospheres were ellipsoid shaped due to the synthesis conditions. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 139 Ultraviolet Spectroscopy The luminescent properties of samples were analysed by UV–light absorption at λ = 254 nm [20]. According to the low Eu3+ contents showed in Table V.1, LDHASi1 and LDHASi3 did not show fluorescent emission when exposed to the ultraviolet light source, and this fact could be explained in one case due to the low concentration of europium ions when a solution of europium−doped CaP (≤ 0.2 mM) was prepared. In LDHASi2 and LDHASi4, the luminescent characteristic (red−light emission) was observed because the concentration of prepared solution of europium−doped CaP was 25 and 50 mM, respectively. V.3 Conclusions of Chapter V A new method for coating silica materials with europium-doped HA was proposed. The luminescent FHA/MSNSs composites were synthesized including different amounts of Eu3+ ions via a modified precipitation method for HA. Some FHA/MSNSs composites exhibited red luminescence of lanthanides under UV light; luminous intensity was dependent on the amount of Europium ion. Furthermore, biocompatible and bioactive properties should be expected for these nanocomposites due to their HA coating. The fluorescent characteristics and XRD results of the synthesized samples are summarized in Table V.3. Table V.3 Fluorescent characteristics and XRD results of samples. The synthesis procedure of MSNSs coated with fluorescent doped HA is schematically depicted in Figure V.5. Four samples of MSNSs and europium-doped HA composites were synthesised by modified precipitation, and the observed results were compared with pure MSNSs (MCM–41 type) used as reference. According to Figure V.5, the MSNSs were coated by precipitation with fluorescent HA (red−light emission). Then the MSNSs were stabilized with PEI (shown by drawing). LDHASi2 and LDHASi3 were synthesized as FHA/MSNSs composites. Moreover, the presence of moderately crystalline HA was confirmed by WA-XRD. In summary, MSNSs of MCM-41 type has been used as intended core during the precipitation of Europium−doped HA as shell component. HA crystallinity in the samples increased in the following sequence: LDHASi2, LDHASi3, LDHASi4 and LDHASi1. FHA/MSNSs (red-light emission) composites have been confirmed by UV light in LDHASi2 and LDHASi4. According to these results, Europium−doped HA/MSNSs composites were successfully synthesized reaching the main objective of this Chapter. The structure as core@shell (MSNS@HA) was observed in sample LDHASi1 by TEM. However, it did not show fluorescent emission due to lower proportion of Eu3+ in its synthesis. Samples Fluorescence Crystalline phase (XRD) LDHASi1 Colourless Crystalline HA LDHASi2 Red-light Apatite−like phase LDHASi3 Colourless Low crystalline HA LDHASi4 Red-light Low crystalline HA V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 140 Figure V.5 Proposed mechanisms for the coating of MSNSs with FHA and subsequent stabilization with a polycationic polymer such as PEI. V.4 References: 1. H. A. Lowenstam, S. Weiner. Oxford University Press: New York 1989. On Biomineralization. 2. S. Mann. Chem. Unserer Zeit 1986, 20, 69-76. Biomineralization: A new branch of bioinorganic chemistry. 3. S. V. Dorozhkin, M. Epple. Angew. Chem. Int. Edit. 2002, 41, 3130-3146. Biological and medical significance of calcium phosphates. 4. A. Doat, F. Pelle, A. Lebugle. J. Solid State Chem. 2005, 178, 2354-2362. Europium-doped calcium pyrophosphates: Allotropic forms and photoluminescent properties. 5. R. Ternane, M. T. Cohen-Adad, G. Panczer, C. Goutaudier, C. Dujardin, G. Boulon, N. Kbir- Ariguib, M. Trabelsi-Ayedi. Solid State Sci. 2002, 4, 53-59. Structural and luminescent properties of new Ce3+ doped calcium borophosphate with apatite structure. 6. M. Mehnaoui, R. Ternane, G. Panczer, M. Trabelsi-Ayadi, G. Boulon. J. Phys. Condens. Matter. 2008, 20, 275227/1-275227/6. Structural and luminescent properties of new Pb2+-doped calcium chlorapatites Ca(10-x)Pbx(PO4)6.Cl2 (0≤x≤10). 7. M. Neumeier, A. L. Hails, A. S. Davis, S. Mann, M. Epple. J. Mater. Chem. 2011, 21, 1250-1254. Synthesis of fluorescent core-shell hydroxyapatite nanoparticles. 8. S. P. Mondejar, A. Kovtun, M. Epple. J. Mater. Chem. 2007, 17, 4153-4159. Lanthanide-doped calcium phosphate nanoparticles with high internal crystallinity and with a shell of DNA as fluorescent probes in cell experiments. 9. S. Wang. Micropor. Mesopor. Mater. 2008, 117, 1-9. Ordered mesoporous materials for drug delivery. V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles 141 10. M. Vallet-Regí, F. Balas, D. Arcos. Angew. Chem. Int. Ed. 2007, 46, 7548-7558. Mesoporous materials for drug delivery. 11. M. Vallet-Regí, A. Ramila, R. P. del Real, J. Perez-Pariente. Chem. Mater. 2001, 13, 308-311. A new property of MCM-41: drug delivery system. 12. F. Tang, L. Li, D. Chen. Advanced Materials 2012, 24, 1504-1534. Mesoporous Silica Nanoparticles: Synthesis, Biocompatibility and Drug Delivery. 13. D. Zhao, Y. Wan, W. Zhou. Wiley-Vch. 2013, 517. Ordered mesoporous materials. 14. C. Argyo, V. Weiss, C. Bräuchle, T. Bein. Chem. Mater. 2013, 26, 435-451. Multifunctional Mesoporous Silica Nanoparticles as a Universal Platform for Drug Delivery. 15. W. Wang, D. Shi, J. Lian, Y. Guo, G. Liu, L. Wang, R. C. Ewing. Appl. Phys. Lett. 2006, 89, 183106/1-183106/3. Luminescent hydroxylapatite nanoparticles by surface functionalization. 16. A. Doat, M. Fanjul, F. Pelle, E. Hollande, A. Lebugle. Biomaterials 2003, 24, 3365-3371. Europium-doped bioapatite: a new photostable biological probe, internalizable by human cells. 17. F. Chen, Y.- J. Zhu, K.- H. Zhang, J. Wu, K.-W. Wang, Q.-L. Tang, X.- M. Mo. Nanoscale Res. Lett. 2011, 6, 67-69. Europium-doped amorphous calcium phosphate porous nanospheres: preparation and application as luminescent drug carriers. 18. A. Lebugle, F. Pelle, C. Charvillat, I. Rousselot, J. Y. Chane-Ching. Chem. Commun. 2006, 606- 608. Colloidal and monocrystalline Ln3+ doped apatite calcium phosphate as biocompatible fluorescent probes. 19. A. Doat, F. Pelle, N. Gardant, A. Lebugle. J. Solid State Chem. 2004, 177, 1179-1187. Synthesis of luminescent bioapatite nanoparticles for utilization as a biological probe. 20. S. Dembski, M. Milde, M. Dyrba, S. Schweizer, C. Gellermann, T. Klockenbring. Langmuir 2011, 27, 14025-14032. Effect of pH on the synthesis and properties of luminescent SiO2/calcium phosphate: Eu3+ core-shell nanoparticles. 21. J. S. Beck, J. C. Vartuli, W. J. Roth, M. E. Leonowicz, C. T. Kresge, K. D. Schmitt, C. T. W. Chu, D. H. Olson, E. W. Sheppard. J. Am. Chem. Soc. 1992, 114, 10834-10843. A new family of mesoporous molecular sieves prepared with liquid crystal templates. 22. S. V. Dorozhkin. J. Mater. Sci. 2007, 42, 1061-1095. Calcium orthophosphates. 23. J. Andersson, S. Areva, B. Spliethoff, M. Linden. Biomaterials 2005, 26, 6827-6835. Sol-gel synthesis of a multifunctional, hierarchically porous silica/apatite composite.   VI. Conclusions 143 VI. CONCLUSIONS   VI. Conclusions 145 VI. Conclusions Chapter II. Precipitation of Mesostructured Calcium Phosphates in the Presence of Ionic Surfactants - Ionic surfactants, namely anionic, containing S or P atoms in their polar head, such as SDS, SDBS and MAP, can be used to obtain mesostructured organic-inorganic hybrids based in CaPs. - The interaction of amphiphilic molecules of surfactant with calcium and phosphate ions, during the CaP precipitation from aqueous solutions, yield hybrid materials exhibiting lamellar or discontinuous lamellar mesostructures, but no mesoporous order. - In the synthesis conditions, i.e., Ca/P molar ratio 1 and 1.67, surfactant concentration from 15 to 90 mM and 240 mM and pH not buffered, the CaP most common phases present in the lamellar hybrids were brushite, hydroxyapatite or monetite. - SDS is a suitable surfactant to obtain highly mesostructured lamellar CaP hybrids. - Relatively high SDBS concentrations and Ca/P molar ratio of 1 yield discontinuous lamellar or curved lamellar mesostructures, forming meshes, of poorly crystallized HA. The formation of HA at this low Ca/P molar ratio is attributed to the pH increase (7 to 9) of medium due to the protonation of the sulphate polar heads of SDBS. - The mixture of an anionic surfactant, such as SDS, SDBS or MAP, with the cationic cetyltrimethyl ammonium bromide (CTAB) produces hybrids with mesostructure analogous to that obtained with the pure anionic surfactants, suggesting a non-effective interaction of CTAB with calcium and phosphate ions. - TEM analysis of materials obtained using a surfactant mixture shows two types of particles: (i) rod-like particles exhibiting lamellar mesostructure and (ii) sheet-like particles unstable under the electron beam. The bubbles formed when these sheet-like particles were investigated, attributed to the organic matter combustion and to the removal of crystallization water molecules in brushite, avoid the identification of the possible mesostructure in the sheet-like particles. - The use of energetic synthesis procedures, such as ageing in an autoclave or refluxing after the CaP precipitation, leads to monetite, a CaP phase derived from brushite after remove the two water molecules of crystallization. - The use of EPG, a zwitterionic surfactant containing one carboxybetaine group, as mesostructure template for CaP fails and no interactions with calcium and phosphate ions are detected. For the future, the investigation of another zwitterionic surfactant containing a sulfobetaine groups is suggested. VI. Conclusions 146 Chapter III. Precipitation of Mesostructured Calcium Phosphates in the Presence of Phospholipids - The precipitation of CaPs in phospholipids (PLs) suspensions containing different concentrations of phosphatidylcholine (PC) yields to the synthesis of CaP-PLs hybrid materials. - The inorganic components of the hybrids are poorly crystallized HA, when pH is basic and Ca/P molar ratio 1.67, and brushite at pH neutral and Ca/P = 1. - The organic matter content is similar in both hybrid types, around 20% in brushite-containing hybrids and around 17% in HA-containing ones. CaP-PLs hybrid materials subjected to a thermal process to remove the organic matter evolve to non-porous HA. Since no mesoporous materials were obtained after calcination, this Chapter has been focused in the characterization of the CaP-PLs hybrid materials. - TEM analysis of hybrids revealed four types of nanostructures: (i) lamellar bilayer with 4.2 nm in thickness of layer (apatite, asolectin), (ii) bilayer vesicles around 35 nm in diameter of vesicles and 4.7 nm in thickness of bilayer (brushite, lecithin), (iii) micelles of about 4.3 nm in diameter (brushite, lipoid) and (iv) bilayer sponge−like or worm−like tubular mesostructures with 4.3 nm in thickness of disordered bilayer sponge−like structure (apatite, lipoid). - Since both components of CaP-PLs hybrid materials are biocompatible they can be investigated as drug delivery platforms via the encapsulation of a drug within the phospholipid structure of the hybrid. Chapter IV. Hydroxyapatite Coatings on Mesoporous Silica Nanospheres In this chapter, MCM−41 mesoporous silica nanospheres (MSNSs) were tried to be coated with HA nanoparticles or with a HA layer, to expand the clinical applications of both materials. According to this goal, the conclusions derived from each synthetic route attempted are: • Co-synthesis of CaP and MSNSs. The CaP precipitation simultaneous to the sol-gel synthesis of MSNSs yield a mixture of independent particles of poorly crystallized HA rods and polydisperse MSNSs (SBET 594 m2/g). • Soaking of MSNSs into a sol precursor of CaP. First, pure SiO2 MSNSs (SBET = 1230 m2/g) were synthesized. After soaking them in the sol precursor of CaP for 3 hours, the nanospheres (SBET = 250 m2/g) convert in ellipsoids with the elements Si, Ca, P and O in their composition. The ellipsoids exhibit a radial mesopore channels arrangement close to the particles surface. Moreover, the obtained material exhibits a moderate in vitro bioactive response in simulated body fluid. • Wetting of MSNSs by a sol precursor of CaP. In this approach, pure SiO2 MSNSs (SBET = 1230 m2/g) were synthesized first, then soaked for 30 minutes in the sol precursor of CaP and subsequently subjected to a washing process mimicking a dip-coating procedure with the sol. TEM images after wetting confirm the successful formation of HA NPs covering the MSNSs VI. Conclusions 147 surface. In this case, the SBET of the coated material suffer a drastic decrease of ≈90 % compared to the initial MSNSs. Unlike the previous method, in this approach MSNSs are not altered in shape neither in composition. • Functionalization of MSNSs with phosphate-like groups and subsequent wetting into a sol precursor of CaP. MSNSs were functionalized with diethylphosphatoethyl triethoxysilane (DEPETES) or 3-trihydroxysilylpropylmethylphosphonate (THSMP) to facilitate the subsequent interaction of their phosphate-like groups with calcium ions and then phosphate ions in the sol. o DEPETES-functionalized MSNSs exhibiting MCM-41 hexagonal arrangement and high SBET (878 to 1190 m2/g) were synthesized by co-condensation or post-synthesis. After functionalization, P-containing MSNSs were wetted with a sol precursor of CaP. A homogeneous distribution of a HA layer around the MSNSs and some HA NPs are observed by TEM. SBET of 335 m2/g indicates a partial coating of the MSNSs surface. o MSNSs were functionalized with THSMP by a co-condensation method. The obtained material exhibits a high SBET of 1316 m2/g. THSMP-functionalized MSNSs were wetted with a sol precursor of CaP. TEM analysis showed that a core@shell structure MSNS@HA was obtained. Coated MSNSs are non-porous showing a decrease in SBET of around 97% regarding the initial MSNSs. Chapter V. Coating of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles - The precipitation of Eu3+ doped HA (fluorescent HA, FHA) in the presence of MCM-41 type MSNSs yields to the synthesis of FHA/MSNSs composites. - Some FHA/MSNSs composites exhibit the red luminescence of lanthanides under UV light. The luminous intensity is dependent on the amount of Eu3+ ion in the composite. - A core@shell structure (MSNS@HA) is observed by TEM for one of the samples investigated. However, this sample does not show a fluorescent emission due to its low Eu3+ content.   VII. Appendix: Experimental Part 149 VII. APPENDIX: EXPERIMENTAL PART   VII. Appendix: Experimental Part 151 VII.1 Tables for the interpretation of FTIR spectra of synthetized materials The next tables resume the assignments used to analyze the FTIR results of the materials prepared in this thesis. The following abbreviations are used: w: weak, m: medium; s: strong; vs: very strong; sh: shoulder; shp: sharp; br: broad. Organic matter from surfactants Wavenumber (cm−1) Vibration mode Functional group Structural unit Assignment 2950 (w) νas (CH) CH3 group Asymmetric stretching mode of the C-H bonds of the methyl group 2916 (s) νas (CH) CH2 group Asymmetric stretching mode of the C-H bonds of the methylene group 2850 (m) νs (CH) CH2 group Symmetric stretching mode of the C-H bonds of the methylene group Silica Wavenumber (cm−1) Vibration mode Functional group Structural unit Assignment 3500-3300 (br) ν (OH) H2O Stretching mode of the O-H bonds of the physisorbed water 1630 δ (HOH) H2O Bending mode of the H-O-H bonds of the physisorbed water 1050 (vs) νas (SiO) SiO2 Asymmetric stretching mode of the Si-O bonds of the silica network 950-910 (sh) ν (SiO) Si-OH Stretching mode of the Si-O bonds of the silanol groups of the silica surface 800 (m) νs (SiO) SiO2 Symmetric stretching mode of the Si-O bonds of the silica network 440 (s) δ (SiOSi) SiO2 Bending mode of the Si-O-Si bonds of the silica network (siloxane bonds) Calcium phosphates: HAp Ca10(PO4)6(OH)2 Wavenumber (cm−1) Vibration mode Functional group Structural unit Assignment 3800-3000 (br) 3400 (br) ν (OH) H2O Stretching mode of the O-H bonds of the adsorbed water 3500 (w) or 3572 (w) ν (OH) OH Stretching mode of the O-H bond of the hydroxyl group 1630 δ (HOH) H2O Bending mode of the H-O-H bonds of the adsorbed water 1087 (s) 1046 (s) 1032 (s) νas (PO) PO4 3− Asymmetric stretching mode of the P-O bonds of the phosphate groups 960 (w) νs (PO) PO4 3− Symmetric stretching mode of the P-O bonds of the phosphate groups 600 (s) 574 (m) 560 (m) Two or three bands δ (OPO) PO4 3− in crystalline environment Bending mode of the O-P-O bonds of the phosphate groups in crystalline environment 470 (w) or 460 δ (OPO) PO4 3− Bending mode of the O-P-O bonds of the phosphate groups VII. Appendix: Experimental Part 152 Calcium phosphates: Carbonated HAp Ca10(PO4,CO3)6(OH)2 Wavenumber (cm−1) Vibration mode Functional group Structural unit Assignment 1550 1500 1465 1430 1410 ν (CO) CO3 2− Stretching mode of the C-O bonds of the carbonate group 1087 (s) 1046 (s) 1032 (s) νas (PO) PO4 3− Asymmetric stretching mode of the P-O bonds of the phosphate groups 970 (w) νs (PO) PO4 3− Symmetric stretching mode of the P-O bonds of the phosphate groups 883 875 870 (m) δ (OCO) CO3 2− Bending mode of the C-O bonds of the carbonate group 600 (s) 574 (m) 560 (m) Two or three bands δ (OPO) PO4 3− in crystalline environment Bending mode of the O-P-O bonds of the phosphate groups in crystalline environment 470 (w) or 460 δ (OPO) PO4 3− Bending mode of the O-P-O bonds of the phosphate groups Calcium phosphates: Brushite CaHPO4⋅2H2O Wavenumber (cm−1) Vibration mode Functional group Structural unit Assignment 3540 (s) νas (OH) OH Asymmetric stretching mode of the O-H bonds of the monohydrogenphosphate group 3460 (s) νs (OH) OH Symmetric stretching mode of the O-H bonds of the monohydrogenphosphate group 3260 (m) νas (OH) H2O Asymmetric stretching mode of the H-O-H bonds of the crystalline water 3150 (m) νs (OH) H2O Symmetric stretching mode of the H-O-H bonds of the crystalline water 1650-1640 (shp, s) δ (HOH) H2O Bending mode of the H-O-H bonds of the crystalline water 1092 (s) 1060 (sh) 1020 (s) νas (PO) HPO4 2− Asymmetric stretching mode of the P-O bonds of the phosphate groups 970 (m) 870 (m) νs (PO) HPO4 2− Symmetric stretching mode of the P-O bonds of the phosphate groups 790 (m) 660 (m) δ (OPO) HPO4 2− Bending mode of the O-P-O bonds of the phosphate groups 580 (w) 520 (w) δ (OPO) HPO4 2− Bending mode of the O-P-O bonds of the phosphate groups VII. Appendix: Experimental Part 153 VII.2 Characterization Techniques Thermogravimetric and Differential Thermal Analysis (TG/DTA) Thermogravimetric analyses were used to investigate the weight losses of synthesized materials during the temperature treatment. Calcium phosphate phase changes as well as exothermic/endothermic processes of synthesized materials were observed by DTA. In the cases in which TGA curve exhibited a small weight loss between room temperature and 125 °C this fact was attributed to the loss of water physisorbed in samples. TG/DTA was performed in air at a flow rate of 100 ml min–1 and a heating rate of 10 °C min–1 between 30 and 1000 °C on a Perkin–Elmer Pyris Diamond thermobalance. Platinum crucible was used as sample holder and alumina as reference. Elemental Analysis of Carbon, Hydrogen and Nitrogen Elemental analysis was performed on a Perkin–Elmer 2400 CHN thermo analyzer at the Elemental Microanalysis CAI of Universidad Complutense de Madrid. During the short research stay at the Inorganic Chemistry Center of the University of Duisburg−Essen, the contents of carbon, hydrogen and nitrogen were determined by standard combustion analysis with EA 1110 instrument (CE Instruments). Fourier Transform Infrared Spectroscopy (FTIR) FTIR was used to identify the functional groups of synthesized materials. FTIR spectra were collected by using a Nicolet iS10 spectrometer equipped with a Goldengate® attenuated total reflectance (ATR) device. FTIR is typically operated in the range between 400 and 4000 cm–1. OMNIC spectroscopy software package from Nicolet was used for the treatment of the FTIR spectra. During the short research stay, FTIR spectra were collected by using Bruker−Vertex 70 FTIR spectrometer in the range between 400 and 4000 cm–1. In this case, OPUS spectroscopy software package for FTIR was used. Powder X−ray Diffraction (XRD) The XRD analyses were carried out using a Philips X´Pert MPD diffractometer (Philips Electronics NV, Eindhoven, Netherlands) with Cu Kα radiation (λ=1.5418 Å) at the X−ray diffraction CAI of Universidad Complutense de Madrid operated by Dr. Fernando Conde. Small−angle XRD was used to examine the mesostructures in synthesized materials and wide−angle XRD was used to examine the crystalline phases of CaP in synthesized samples. Wide−angle X−ray was collected over the range 2θ = 5–50°, with a step size of 2θ = 0.04° and a counting time per step of 1 s. Small−angle X−ray diffractions were collected over the range 2θ = 0.6–6.5°, with a step size of 2θ = 0.02° and a counting time per step of 5 s. VII. Appendix: Experimental Part 154 During the short research stay, synthesized materials were characterised by using X−ray diffraction (Bruker D8 Advance: Cu Kα radiation, λ=1.5418 Å). The diffractograms were collected over the range 2θ = 10–50°, with a step size of 2θ = 0.01 ° and a counting time of 102 s per step for wide−angle XRD. The range of small−angle XRD was between 0.95 and 6 °. Nitrogen Adsorption Isotherms The surface area and pore size measurements of hybrid materials were performed by nitrogen adsorption/desorption analysis at –196 °C on ASAP 2010 and 2020 porosimeter (Micromeritics Co, Norcross GA, USA). Prior to the analysis, powder samples were degassed at 125 °C for 24 h under a vacuum lower than 5 µmHg. The surface area was determined by Brunauer–Emmett–Teller (BET) method [1,2]. The pore diameter distribution was obtained by the Barrett–Joyner–Halenda (BJH) method [1,2]. Transmission Electron Microscopy − Electron Diffraction (TEM−ED) TEM was performed to analyze the mesostructures and crystalline phases in synthesized materials on a JEOL JEM−2100 microscope operating at 200 kV in the National Centre of Electron Microscopy at Universidad Complutense de Madrid. I want to thank Dr. Mª Luisa Ruiz González for measuring samples prepared by precipitation of CaP during the synthesis of mesoporous silica nanospheres (MSNSs). All synthesized samples were prepared by dispersing a small amount of powder materials in butanol or water with an ultrasonicator bath. After sonication, suspensions were deposited by 2−3 drops of suspension on a perforated carbon−coated copper grid. During my short research stay in Germany, synthesized materials were examined by JEOL 1200 microscope operating at 120 kV. Scanning Electron Microscopy and Energy Dispersive X−ray Spectroscopy (SEM–EDS) The surface morphology of synthesized materials was studied by Scanning Electron Microscopy coupled with Energy Dispersive X−ray Spectroscopy. SEM–EDS analysis was carried out using a JEOL JSM 6335F Microscope at the National Center of Electron Microscopy of Universidad Complutense de Madrid. For this purpose, synthesized samples were mounted onto a copper stud, dried at 70 °C for 24 h under vacuum, and coated with a carbon film. During my short research stay in Germany, the surface morphology of mesoporous silica coated with fluorescent HA was studied by SEM−EDS. This analysis was carried out using a FEI ESEM Quanta 400 FEG coupled with EDX detector S−UTW−Si(Li). For this purpose, the surface morphology of mesoporous silica coated with fluorescent HA samples were mounted onto a copper stud and coated with a film of gold–palladium (80:20). VII. Appendix: Experimental Part 155 Determination of Ca2+ concentration and pH value after SBF test Variation in the Ca2+ concentration and pH value in solution were determined on an ILyte Na+, K+, Ca2+ pH analyzer. Ultraviolet spectroscopy During the short research stay, the intense luminescence of europium−doped hybrid materials was observed by using a UV lamp (λ = 254 nm) [3]. VII.3 Commercial Products For the synthesis of the materials here described, the following products have been commercially acquired from different companies. Sigma−Aldrich: Calcium chloride dihydrate, CaCI2·2H2O; calcium nitrate tetrahydrate, Ca(NO3)2·4H2O; sodium phosphate dibasic dihydrate, Na2HPO4·2H2O; sodium dodecyl benzene sulphonate, C18H29O3SNa; sodium dodecyl sulphate, C12H25NaO4S; cetyltrimethyl ammonium bromide, C19H42NB; Empigen®BB surfactant 35%, CH3(CH2)8−14CH2N+(CH3)2CH2COO–; asolectin from soybean (mixture of phospholipids); L-α-Lecithin from egg yolk; tetraethyl orthosilicate, Si(OC2H5)4; triethylphosphite, (C2H5O)3P; 3-trihydroxysilylpropyl methylphosphonate 42%, CH3P(O)(ONa)O(CH2)3Si(OH)3; sodium hydroxide, NaOH; Toluene; sodium chloride, NaCl; sodium hydrogen carbonate, NaHCO3; potassium chloride, KCl; di-potassium hydrogen phosphate trihydrate, K2HPO4·3H2O; magnesium chloride hexahydrate, MgCl2·6H2O; calcium chloride, CaCl2; sodium sulfate, Na2SO4; tris-hydroxymethyl aminomethane, (HOCH2)3CNH2 (Tris); poly(ethyleneimine), H(NHCH2CH2)nNH2; yttrium chloride hexahydrate 99.9%, YCl3·6H2O. Merck: Diammonium hydrogenphosphate, (NH4)2HPO4. Riedel–de Haën: Calcium nitrate tetrahydrate, Ca(NO3)2·4H2O. ABCR GmbH: Diethylphosphatoethyltriethoxysilane 92% (DEPETES), C12H29O6PSi. Lipoid GmbH: Lipoid-S100 from soybean. Alfa–Aesar: Mono-alkyl phosphate (MAP), C12H27O4P; Europium (III) nitrate hexahydrate 99.9%, Eu(NO3)3·6H2O. Panreac: Ethanol absolute 99.5 %; ammonia 25 %; hydrochloric acid 37 %. Deionized water was further purified by passage through a Milli-Q Advantage A-10 Purification System (Millipore Corporation) to a final resistivity of 18 MΩ cm or above. http://en.wikipedia.org/wiki/Sodium VII. Appendix: Experimental Part 156 VII.4 Preparation of Simulated Body Fluid Simulated Body Fluid (SBF) was prepared by Kokubo method . The following powder reagent grade chemicals have to be stocked in a desiccator. Milli-Q water is used for the preparation of SBF [4]. (1) sodium chloride, (2) sodium hydrogen carbonate, (3) potassium chloride, (4) di-potassium hydrogen phosphate trihydrate, (5) magnesium chloride hexahydrate, (6) calcium chloride, (7) sodium sulfate, (8) tris-hydroxymethyl aminomethane, (9) 1M (mol/1) hydrochloric acid, (10) pH standard solutions, (pH 4, 7 and 9) Protocol for preparing SBF: 1- All bottles and wares were washed with 1N−HCl solution, neutral detergent, and ion−exchanged and distilled water, and then dried. 2- 500 ml of ion−exchanged and distilled water was put into one liter polyethylene bottle, and the bottle was then covered with a watch glass. 3- The water in the bottle was stirred with a magnetic stirrer, dissolving the reagents one by one in the order as given in Table VII.1 (each one once the previous reagent was completely dissolved). 4- The temperature of the solution in the bottle was adjusted at 36.5 °C with a water bath, and solution pH was adjusted at pH 7.40 by stirring the solution and titrating 1N−HCl solution (when the pH electrode is removed from the solution, the water used for washing the electrode was added to the solution). 5- The solution was transferred from the polyethylene bottle to a volumetric glass flask. 6- Total volume of solution was then adjusted to one liter by adding ion−exchanged and distilled water and shaking the flask at 20 °C. 7- The solution was transferred from the flask to a polyethylene or polystylene bottle, and the bottle was stored in a refrigerator at 5–10 °C (if any substance precipitated during storage, the SBF solution was rendered invalid and not used). Table VII.1 Reagents for preparing SBF (1 liter) Order Reagent Amount 1 NaCl 7.996 g 2 NaHCO3 0.350 g 3 KCl 0.224 g 4 K2HPO4·3H2O 0.228 g 5 MgCl2·6H2O 0.305 g 6 1N-HCl (about 90% of total amount of HCl to be added) 40 mL 7 CaCl2 0.278 g 8 Na2SO4 0.071 g 9 (HOCH2)3CNH2 6.057 g VII. Appendix: Experimental Part 157 VII.5 Description of Synthesis VII.5.a Synthesis of Calcium Phosphate in the Presence of Surfactants (Chapter II) First, 0.4175 M or 0.25 M CaCl2·2H2O and 0.25 M Na2HPO4·2H2O solutions were prepared in volumetric flask as stock solutions. 25 ml of each solution were taken from stocks when necessary. Calcium Phosphate in the Presence of Sodium Dodecyl Sulphate Nanostructured CaPs were synthesized by precipitation as previously reported [5]. Fast crystallization and precipitation of CaP is achieved by this method due to the rapid interaction between Ca2+ and HPO4 2− ions in water solution. SDS was dissolved in the Na2HPO4⋅2H2O solution and surfactant amounts used are as follows: 0.2163 g, 0.4326 g, 0.6489 g, 0.8651 g, 1.0814 g and 1.2977 g for 15 mM, 30 mM, 45 mM, 60 mM, 75 mM and 90 mM of SDS concentration in the final reaction volume, respectively. Samples were classified in two series as a function of the Ca/P molar ratio relation of 1 and 1.67. All samples were synthesized without buffering the pH. The molar ratios of materials are respectively: 1 Na2HPO4·2H2O / 1 and 1.67 CaCl2·2H2O / 0.12 – 0.24 – 0.36 – 0.48 – 0.6 and 0.72 of SDS / 445 H2O. The general procedure is described as follows: over 25 ml of Na2HPO4·2H2O solution (0.25 M) heated at 40 °C, the corresponding amount of SDS was slowly added under stirring. The resulting solution was stirred at 40 °C during 20 min, and then 25 ml of CaCl2·2H2O solution (0.25 or 0.4175 M) was added drop by drop. The mixture was stirred at 40 °C for 1 h and then was left to stay at 37 °C for 24 h. The processes of reflux and autoclave were performed respectively at 80 and 125 °C for 24 h for 30 mM concentration of SDS surfactant. Finally, the mixture was filtered, washed with Milli-Q water, and dried in the oven. Calcium Phosphate in the Presence of Sodium Dodecyl Benzene Sulphonate The starting materials are calcium chloride dehydrate, sodium phosphate dibasic dihydrate and sodium dodecyl benzene sulphonate as surfactant. SDBS was dissolved in the Na2HPO4⋅2H2O solution and surfactant amounts used are as follows: 0.2613 g, 0.5227 g, 1.0454 g and 1.5681 g for 15 mM, 30 mM, 60 mM and 90 mM of SDBS concentration in the final reaction volume, respectively. All samples were prepared with molar ratio relation of Ca/P = 1 and synthesized without buffering the pH. The molar ratios of materials are: 1 Na2HPO4·2H2O / 1 CaCI2·2H2O / 0.12 − 0.24 − 0.48 − 0.72 of SDBS / 445 H2O, respectively. The general procedure is described as follows: over 25 ml of Na2HPO4·2H2O solution (0.25 M) heated at 40 °C, the corresponding amount of SDBS was slowly added under stirring. The resulting solution was stirred at 40 °C during 20 min, and then 25 ml of CaCI2·2H2O (0.25 M) were added drop by drop. The mixture was stirred at 40 °C for 1 h and then was left to stay at 37 °C for 24 h. Finally, the mixture was filtered, washed with Milli-Q water, and dried in the oven. VII. Appendix: Experimental Part 158 Calcium Phosphate in the Presence of the Surfactants Mixture: Sodium Dodecyl Sulphate/CetylTrimethyl Ammonium Bromide SDS and CTAB surfactants were separately dissolved in half the volume of Na2HPO4·2H2O solution (12.5 mL, 0.25 M) and surfactant amounts used are as follows: 0.007 g, 0.216, 0.432, 0.65 g, 0.865, 1.08 g and 1.3 g of SDS and 0.1 g, 0.27 g, 0.546, 0.82 g, 1.1 g, 1.37 g and 1.64 g of CTAB surfactant for 0.5 mM, 0.15 mM, 30 mM, 45 mM, 60 mM, 75 mM and 90 mM of [SDS + CTAB, 1:1] in the final reaction volume, respectively. All samples were prepared with molar ratio relation of Ca/P = 1 and synthesized without buffering the pH. The molar ratios of materials are: 1 Na2HPO4·2H2O / 1 CaCI2·2H2O / 0.004 – 0.012 – 0.024 – 0.036 – 0.048 – 0.06 and 0.072 of SDS + CTAB (1:1) / 445 H2O, respectively. The general procedure is described as follows: over 12.5 ml of Na2HPO4·2H2O solution (0.25 M) heated at 40 °C, the corresponding amount of SDS or CTAB was slowly added under stirring. These solutions were mixed and the resulting solution was stirred at 40 °C during 20 min, and then 25 ml of CaCI2·2H2O (0.25 M) was added drop by drop. The mixture was stirred at 40 °C for 1 h and then was left to stay at 37 °C for 24 h. Finally the mixture was filtered, washed with Milli-Q water, and dried in the oven. Calcium Phosphate in the Presence of the Surfactants Mixture: Sodium Dodecyl Benzene Sulphonate/CetylTrimethyl Ammonium Bromide SDBS and CTAB surfactants were separately dissolved in half the volume of Na2HPO4·2H2O solution (12.5 mL, 0.25 M) and the amounts of surfactants used for [SDBS+CTAB] 30 mM are as follows: 0.39204 g of SDBS and 0.1366 g of CTAB for 3:1 molar ratios; 0.13068 g of SDBS and 0.41001 g of CTAB for 1:3 molar ratios. All samples were prepared with molar ratio relation of Ca/P = 1 and synthesized without buffering the pH. The molar ratios of materials are: 1 Na2HPO4·2H2O / 1 CaCI2·2H2O / 0.18 and 0.06 of SDBS / 0.06 and 0.18 of CTAB / 445 H2O, respectively. The general procedure is described as follows: over 12.5 ml of Na2HPO4.2H2O solution (0.25 M) heated at 40 °C, the corresponding amount of SDBS or CTAB was slowly under stirring. These solutions were mixed and the resulting solution was stirred at 40 °C during 20 min, and then 25 ml of CaCI2·2H2O (0.25 M) was added drop by drop. The mixture was stirred at 40 °C for 1 h and then was left to stay at 37 °C for 24 h. Finally, the mixture was filtered, washed with Milli-Q water and dried in the oven. VII. Appendix: Experimental Part 159 Calcium Phosphate in the Presence of the Surfactants Mixture: Monoalkyl Phosphate/CetylTrimethyl Ammonium Bromide Several samples with different CTAB/MAP molar ratio and increasing surfactants mixture concentration were prepared. The sample described in the chapter was prepared with the following molar ratios of reactants: 1 Na2HPO4·2H2O / 1 CaCI2·2H2O / 0.12 MAP / 0.12 CTAB / 52 H2O and synthetized without buffering the pH. Final surfactant mixture concentration in the reaction volume is 240 mM. An autoclave process was performed after the synthesis by precipitation method. The procedure is described as follows: 0.3654 g of MAP and 0.5 g of CTAB were dissolved in Na2HPO4·2H2O solution (2.0349 g in 7.5 mL of H2O). The resulting solution was stirred at 40 °C during 30 min, and then CaCl2·2H2O (1.6809 g in 2.5 mL of H2O) were added drop by drop. The mixture was slowly stirred at 40 °C for 1 h and then stirred at R.T. for 16 hours (overnight). Finally, a process of autoclave was performed at 100 °C for 24 h. The mixture was filtered, washed with Milli-Q water, and dried in the oven. Calcium Phosphate in the Presence of the Zwitterionic Surfactant EMPIGEN BB 35% surfactant (EPG) was dissolved in water to a concentration of 60 and 90 mM (30 and 45 mM of surfactant in the final reaction volume, respectively) and surfactant amounts used were 1.17 and 1.75 ml of EPG 30% to prepare 25 mL of solution. Sample preparation solutions had the Ca/P molar ratio 1 and pH value was not buffered. The molar ratios of material: 1 Na2HPO4·2H2O / 1 CaCI2·2H2O / 0.68 and 1.02 of EPG / 445 H2O, respectively. Four samples were synthesized by adding calcium and phosphate sources as follows: (i) For samples CaP1 and CaP2: over 25 mL of the EPG solution heated a 40 °C, 0.92 g of CaCI2·2H2O were slowly added under stirring. The resulting solution was stirred at 40°C during 20 min, and then 25 ml of Na2HPO4·2H2O (0.25 M) were added drop by drop. (ii) For samples PCa1 and PCa2: over 25 mL of the EPG solution heated at 40 °C, 1.11 g of Na2HPO4·2H2O were slowly added under stirring. The resulting solution was stirred at 40⋅°C during 20 min, and then 25 ml of CaCI2·2H2O (0.25 M) were added drop by drop. The mixture was stirred at 40°C for 1h and then was left to stay at 37 °C for 24 h. Finally, the mixture was filtered, washed with Milli-Q water, and dried in the oven. VII.5.b Synthesis of Calcium Phosphate in the Presence of Phospholipids (Chapter III) The reactants used in the syntheses were: calcium chloride dihydrate, sodium phosphate dibasic dihydrate and sodium hydroxide. Three Phospholipids (PLs) sources with increasing contents of Phosphatidylcholine (PC) were used: Asolectin from soybean, containing about 33% of PC (PC33), L−α−Lecithin from egg yolk, about 60% of PC (PC60) and Lipoid−S100 from soybean, containing 94% of PC (PC94). In some cases ethanol absolute 99.5% was used as co-solvent. http://pub.panreac.com/ds/161086IN.HTM VII. Appendix: Experimental Part 160 Table VII.2 shows amounts of solvents, reactants and Ca/P molar ratio. Synthesized materials were named as PC33, PC60, PC94−1 and PC94−2 with the numbers indicating the percentage of PC in the commercial reagent, respectively. Table VII.2 Amount of phospholipids (PLs), moles of reactants, solvents and NaOH used in the synthesis of hybrids CaP−PLs. Ca/P molar ratios are also included. The general synthetic procedure was the following: CaPs were precipitated from calcium and phosphate ions in the presence of PLs, using a modification of a method described by Galarneau et al for the synthesis of sponge mesoporous silica [6]. The experimental procedure is described as follows: Firstly, 0.5 g of PC33 or PC60 was stirred in a solvent mixture of ethanol and water at room temperature and stirring at 1200 rpm overnight, until a homogeneous suspension was formed. Similarly, 0.5 g of PC94−1 or PC94−2 was stirred in water. Amounts of solvent mixture of ethanol−water and water were given in Table VII.2. Secondly, Na2HPO4·2H2O was dissolved in deionised water by stirring at R.T. CaCl2·2H2O was dissolved in EtOH or water by stirring until obtaining a homogeneous solution. Phosphate, calcium, and PLs solutions were separately prepared from each other. Finally, calcium and phosphate solutions were added into the PLs solution drop by drop under the stirring of 500 rpm at R.T. for 15 min, and the mixture solution was left under stirring 15 min to obtain a homogenous solution. The mixture was left 24 h at room temperature for aging and then it was filtered and washed five times with 50 ml of H2O/EtOH (1/1;v/v) and dried at 50 °C for 24 h. PC33 sample was calcinated at 700 °C for 8h and subsequently designated calcinated PC33. VII.5.c Synthesis of Hydroxyaptite coatings on Mesoporous Silica Nanospheres (Chapter IV) - Synthesis of mesoporous silica nanospheres, MSNSs sample Synthesis of the nanospherical particles of mesoporous silica MCM-41 type with hexagonal pore arrangement, was performed by sol-gel chemistry in the presence of structure directing agents and following a modified Stöber method [7], as follows: 1g of CTAB was dissolved in 480 ml of water containing 3.5 ml of NaOH [2M] under stirring. The solution was heated at 80 °C and then 5 ml of TEOS as SiO2 source was slowly added into mixture at 0.25 ml. min–1 using a syringe pump. The mixture was stirred at 700 rpm at 80 °C for 2 h. The material was filtered, washed thoroughly with water/ethanol (v/v : 50 ml / 50 ml) and dried in the vacuum oven at 80°C overnight. The surfactant Samples (PLs source, % of PC) HPO4 2– (mol) Ca2+ (mol) PLs (g) H2O (mol) EtOH (mol) NaOH (mol) PC33 (Asolectin, 33) 1 1.67 0.5 126 38 2 PC60 (Lecithin, 60) 1 1 0.5 69 38 – PC94–1 (Lipoid, 94) 1 1 0.5 194 – – PC94–2 (Lipoid, 94) 1 1.67 0.5 223 – 1 VII. Appendix: Experimental Part 161 template was removed by heating at 550 °C for 6 h using a heating ramp of 2.5 °C/min. The obtained hexagonal mesoporous nanospherical silica particles were named as MSNSs sample. - Co−precipitation of calcium phosphate on mesoporous silica nanospheres, MSNS−HA−1 sample In the synthesis, the following molar ratios of reactants were used: 1 TEOS / 0.1223 CTAB / 0.3125 NaOH / 1190 H2O / 0.4175 Ca / 0.25 TIP, respectively. 1 g of CTAB was mixed with 480 ml of water and added 3.5 ml of NaOH (2M) under stirring. The mixture was heated at 80 °C and stirred until colourless homogeneous mixture for 45 min. 5 ml of TEOS was slowly added into mixture at 0.25 ml min–1 by a syringe pump. 5 min later after adding TEOS, 20 ml of CaCl2·2H2O 0.4175 M and 20 ml of Na2HPO4·2H2O 0.25 M were simultaneously added drop by drop into the solution. Then the mixture was mixed at 700 rpm at 80 °C for 2 hours. The sample was obtained after filtering, washing thoroughly with water−ethanol (v/v: 50/50) and drying in the vacuum oven at 80 °C overnight. To remove surfactant and keep the hexagonal form, a calcination process was performed at 550 °C for 6 h and heating rate of 2.5 °C min–1. - Soaking and Wetting of silica nanospheres into a sol precursor of calcium phosphate, MSNS−HA−2 and MSNS−HA−3 samples Molar ratios of reactants were used such as: 1 TIP / 1.67 CaCl2·2H2O / 4 TEOS / 0.5 CTAB / 1.25 NaOH / 4762 H2O, respectively. Preparation of hexagonal mesoporous silica nanospheres (MSNSs): 1 g of CTAB was mixed with 480 ml of water and added 3.5 ml of NaOH (2M) under stirring. The mixing was heated at 80 °C and stirred until colourless homogeneous mixture for 45 min. After that, 5 ml of TEOS was slowly added into mixture at 0.25 ml min–1 by a syringe pump. Then the mixture was mixed at 700 rpm at 80 °C for 2 hours. The ordered nanospherical silicate was obtained after filtering, washing thoroughly with water/ethanol (v/v: 50/50) and drying in the vacuum oven at 80 °C overnight. To remove surfactant, a calcination process was performed at 550 °C for 6 h and heating rate of 2.5 °C min–1. The obtained hexagonal mesoporous nanospherical silica particles were named as MSNSs. The CaP sol preparation for coating MSNSs: The sol solution was prepared following a reported method [8]. Firstly, TIP was hydrolysed in water (in a 1:4 molar ratio) for 24 hours. Secondly, 4 M calcium nitrate aqueous solution was added to hydrolyzed phosphite solution, in a Ca/P ratio equal to 1.67, which corresponds to HA phase. The mixture was stirred for 15 min and subsequently was aged at 60 °C for 5 h and 30 min. The CaP sol solution is prepared for coating the MSNSs. Coating MSNSs with nano−crystalline Apatite by CaP Sol Filtration: For soaking the nanospheres into a CaP sol, 0.3 g of pre−synthesized MSNSs (as white powder) was stirred for 3 h and filtered by sol precursor, and for wetting the nanospheres with a CaP sol, same 0.3 g of pre−synthesized MSNSs was stirred in sol for 30 min and filtered by the same sol solution for 20 times using 0.45 μm Tecnokroma® VII. Appendix: Experimental Part 162 cellulose membrane filter. Then, MSNSs coated with HA was dried at 60 °C overnight. To obtain hydroxyapatite, final sample was calcinated in air at 550 °C for 6 h with 2.5 °C min–1. - Prefunctionalization of nanospheres with phosphate–like groups and subsequent wetting into a sol precursor of CaP The synthesis was prepared by Y. G. Jin method [9] and the following molar ratios of reactants were used: 1 TEOS / 0.1225 CTAB/ 0.3125 NaOH / 1205 H2O. By co−condensation method: The corresponding amount of THSMP or DEPETES were used: 1TEOS / 0.3 THSMP / 0.1107 DEPETES 10% / 0.25 DEPETES 20 %. 1 g of CTAB was mixed with 480 ml of water and added 3.5 ml of NaOH (2M) under stirring. The mixing was heated at 80 °C and stirred until colourless homogeneous mixture for 45 min. After that, 5 ml of TEOS was slowly added into mixture at 0.25 ml min–1 by a syringe pump and stirred for 15 min. Then, 0.8 and 1.78 ml of DEPETES 10 % and 20 % or 0.5 ml of THSMP was added and the mixture was stirred at 700 rpm at 80 °C for 2 h. The sample of MSNSs functionalized with DEPETES or THSMP was obtained after filtering, washing thoroughly with water/ethanol (v/v: 250/250) and drying overnight in the vacuum oven at 80 °C. To remove the CTAB surfactant by J. I. Zink method [10], as−synthesized particles were suspended in 60 ml of methanol and 2.3 ml of 12 M HCl. The solution was refluxed for 10 h, and the synthesized samples were filtered and washed with 100 ml of methanol: water (v:v). By post−synthesis method: The corresponding amount of DEPETES was used: 1 TEOS / 0.07 for DEPETES. 0.5 g of pre−synthesized MSNSs as powder was mixed in 30 ml of toluene under nitrogen gas and stirred 2 hours. Then, 0.5 ml of DEPETES was added under nitrogen gas, and the mixture was refluxed at 110 °C overnight. The sample was filtered and washed by toluene: ethanol (50:50 / v:v), and dried overnight at 50 °C. Soaking and wetting of mesoporous silica nanospheres into a sol precursor of CaP, NSFD−HA and NSFT−HA samples The sol precursor for coating the nanospheres functionalized with DEPETES and THSMP has been prepared as previously explained in the section “The CaP sol preparation for coating MSNSs”. The coating of prefunctionalized nanospheres can be explained as follows: 0.35 g of MSNSs functionalized with DEPETES (as white powder) was stirred in sol for 15 min and filtered by the same sol precursor for 5 times; similarly, 0.40 g of MSNSs functionalized with THSMP VII. Appendix: Experimental Part 163 was stirred in sol for 30 min and filtered by the same sol solution for 20 times using 0.45 μm Tecnokroma® cellulose membrane filter. Then, the final samples were dried at 40 °C overnight. To obtain hydroxyapatite, final samples were calcinated in air at 550 °C for 8 h with 2.5 °C min–1. VII.5.d Synthesis of Mesoporous Silica Nanospheres with Fluorescent Hydroxyapatite Nanoparticles (Chapter V) Europium−doped hybrid materials were synthesized by S. Dembski and M. Epple methods [3, 11]. The starting materials used in this work included Ca(NO3)2·4H2O and (NH4)2HPO4 as calcium and phosphate source, respectively. PEI, Poly(ethyleneimine) (Mw 750.000 g mol–1, 2 g L–1), is used to stabilize the particles [11]. Europium source is Eu(NO3)3·6H2O. Milli-Q water and ethanol absolute 99.5%, were used for prepare the solution in this study. As silica source, MSNSs (type MCM−41) as structure template were synthesized previously following a modified Stöber method described in Section VII.3.d, preparation of hexagonal mesoporous silica nanospheres (MSNSs), and used during the synthesis [7]. On the other hand, hexagonally structured MSNS coated by fluorescent HA was synthesized by precipitation of CaP in the presence of Eu3+ ions in a suspension of MSNSs [11-13]. Table VII.3 Amounts of MSNSs, solvents and reactants used for the synthesis of MSNSs coated by fluorescent HA. MCM–41 mesoporous silica material as white powder was mixed in EtOH solution and sonicated for 15 min. Secondly, aqueous solutions of Ca2+:Eu3+ and HPO4 2− were added, separately. The mixture was stirred for 1 h at R.T. Then, to remove supernatants, the mixture was centrifuged at 3500 rpm for 15 min and re−dispersed in enough amount of water. PEI was added into mixture and sonicated for 15 min, and then the mixture was centrifuged again and dried overnight at 60 °C. In all synthesis, Ca/P molar ratio was kept as 1.67 and pH value is 10−12 by addition of 2 M of NaOH. The amounts and concentration of reactants used are presented in Table VII.3. MSNPs (g) Ca2+ (mM) HPO4 2– (mM) Eu3+ (mM) PEI (2mg mL–) Citric acid anhydrous H2O (mL) EtOH (mL) LDHASi1 0.25 19.7 11.94 0.2 10 mL 75 5 LDHASi2 0.25 2462.5 1492.5 25 10 mL 40 10 LDHASi3 0.25 0.1905 0.116 0.00075 375 mg in15 mL 11.6 mmol 40 25 LDHASi4 0.25 2437.5 1458.8 50 10 mL 40 5 VII. Appendix: Experimental Part 164 VII.6 References: 1. S. J. Gregg, K. S. W. Sing. Adsorption Surface Area and Porosity, 2nd ed. Academic Press, New York, 1982. 2. S. Lowell, J. E. Shields, M. A. Thomas, M. Thommes. Characterization of Porous Solids and Powders: Surface Area, Pore Size and Density. Particle Technology Series, Vol. 16, 2004, Springer Science+Business Media B.V. 3. S. Dembski, M. Milde, M. Dyrba, S. Schweizer, C. Gellermann, T. Klockenbring. Langmuir 2011, 27, 14025-14032. Effect of pH on the synthesis and properties of luminescent SiO2/calcium phosphate: Eu3+ core-shell nanoparticles. 4. L. B. A. Macon, T. B. Kim, E. M. Valliant, K. Goetschius, R. K. Brow, D. E. Day, A. Hoppe, A. R. Boccaccini, I. Y. Kim, C. Ohtsuki, T. Kokubo, A. Osaka, M. Vallet-Regí, D. Arcos, L. Fraile, A. J. Salinas, A. V. Teixeira, Y. Vueva, R. M. Almeida, M. Miola, C. Vitale-Brovarone, E. Verné, W. Höland, R. J. Jones. J. Mater. Sci.: Mater. Med. 2015, 26, 1-10. A unified in vitro evaluation for apatite-forming ability of bioactive glasses and their variants. 5. C. Liu, X. Ji, G. Cheng. Appl. Surf. Sci. 2007, 253, 6840-6843. Template synthesis and characterization of highly ordered lamellar hydroxyapatite. 6. A. Galarneau, G. Renard, M. Mureseanu, A. Tourrette, C. Biolley, M. Choi, R. Ryoo, F. R. Di, F. Fajula. Micropor. Mesopor. Mater. 2007, 104, 103-114. Synthesis of sponge mesoporous silicas from lecithin/dodecylamine mixed-micelles in ethanol/water media: A route towards efficient biocatalysts. 7. W. Stöber, A. Fink, E. Bohn. J. Colloid Interf. Sci. 1968, 26, 62. Controlled growth of monodisperse silica spheres in the micron size range. 8. S. Sánchez-Salcedo, M. Vila, I. Izquierdo-Barba, M. Cicuendez, M. Vallet-Regí. J. Mater. Chem. 2010, 20, 6956-6961. Biopolymer-coated hydroxyapatite foams: a new antidote for heavy metal intoxication. 9. Y. G. Jin, S. Z. Qiao, Z. P. Xu, C. J. C. Diniz, G. Q. Lu. J. Phys. Chem. C. 2009, 113, 3157-3163. Porous silica nanospheres functionalized with phosphonic acid as Intermediate-temperature proton conductors. 10. H. Meng, M. Xue, T. Xia, Z. Ji, D. Y. Tarn, J. I. Zink, A. E. Nel. ACS Nano 2011, 5, 4131-4144. Use of size and a copolymer design feature to Improve the biodistribution and the enhanced permeability and retention effect of doxorubicin-loaded mesoporous silica nanoparticles in a murine xenograft tumor model. 11. M. Neumeier, A. L. Hails, A. S. Davis, S. Mann, M. Epple. J. Mater. Chem. 2011, 21, 1250-1254. Synthesis of fluorescent core-shell hydroxyapatite nanoparticles. 12. V. Sokolova, M. Epple. Nanoscale 2011, 3, 1957-1962. Synthetic pathways to make nanoparticles fluorescent. 13. L. Armelao, S. Quici, F. Barigelletti, G. Accorsi, G. Bottaro, M. Cavazzini, E. Tondello. Coord. Chem. Rev. 2010, 254, 487-505. Design of luminescent lanthanide complexes: From molecules to highly efficient photoemitting materials. 200 nm 50 nm 0 2 Portada Interna Departamento de Química Inorgánica y Bioinorgánica Departamento de Química Inorgánica y Bioinorgánica 0 3 Varios, portada etc 0 4 Índice tesis 0 5 Summary-Resumen 1 Chapter I Introduction 2 Chapter II Surfactants 3 Chapter III Phospholipids 4 Chapter IV HA Coatings on MSNS 5 Chapter V FHA Coating on MSNS 6 Chapter VI Conclusions 7 Chapter VII Experimental Part Página en blanco