Open Access.© 2017 D. Pedraza et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 License Biomed. Glasses 2017; 3:111–122 Research Article Daniel Pedraza, Jaime Díez, Isabel-Izquierdo-Barba, Montserrat Colilla, and María Vallet-Regí* Amine-Functionalized Mesoporous Silica Nanoparticles: A New Nanoantibiotic for Bone Infection Treatment https://doi.org/10.1515/bglass-2017-0011 Received Oct 27, 2017; revised Dec 04, 2017; accepted Dec 16, 2017 Abstract: This manuscript reports an effective new al- ternative for the management of bone infection by the development of an antibiotic nanocarrier able to pene-5 trate bacterial biofilm, thus enhancing antimicrobial effec- tiveness. This nanosystem, also denoted as “nanoantibi- otic”, consists in mesoporous silica nanoparticles (MSNs) loaded with an antimicrobial agent (levofloxacin, LEVO) and externally functionalized with N-(2-aminoethyl)-3-10 aminopropyltrimethoxysilane (DAMO) as targeting agent. This amine functionalization provides MSNs of positive charges, which improves the affinity towards the nega- tively charged bacteria wall and biofilm. Physical and chemical properties of the nanoantibiotic were studied15 using different characterization techniques, including X- ray diffraction (XRD), transmission electron microscopy (TEM), N2 adsorption porosimetry, elemental chemical analysis, dynamic light scattering (DLS), zeta (ζ )-potential and solid-state nuclear magnetic resonance (NMR). “In20 vial” LEVO release profiles and the in vitro antimicrobial effectiveness of the different released doses were investi- gated. The efficacyof thenanoantibiotic against a S. aureus biofilmwas also determined, showing the practically total destruction of the biofilm due to the high penetration abil-25 ity of the developed nanosystem. These findings open up promising expectations in the field of bone infection treat- ment. Keywords: Mesoporous Silica Nanoparticles; Amine- functionalization; Bacteria and Biofilm targeting; Antimi-30 crobial release; Bone infection treatment *Corresponding Author: María Vallet-Regí: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Uni- versidad Complutense de Madrid, Instituto de Investigación Sani- taria Hospital 12 de Octubre i+12, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedic- ina, CIBER-BBN, Madrid, Spain; Email: Email: vallet@ucm.es; Tel.: +34 91 394 18 61; Fax: +34 91 394 17 86 1 Introduction Bone infection is a potentially dangerous affection that keeps defying the scientific community due to its status as a clinical pathology with important involvements in the 35 socioeconomic field [1–3]. The main trigger agent of this issue is the Staphylococcus aureus (S. aureus) pathogen [4, 5], and according to databases of PubMed and Scopus, the annual incidence of invasive infections of S. aureus varies from 1.6 to 29.7 cases of each 100,000, depending on the 40 studied location. Actually, they come to be the 2.8 from 43% of total infection in bone and joint [6]. Bone infection remains a substantial challenge be- cause of the shortcomings found in current therapies, which relies on the systemic administration of antibiotics 45 and surgery. They are commonly associated to high rate of side effects in the patients, long hospital stays and high morbidity [7]. The development of new strategies that permit to opti- mize the antimicrobials concentration in the infection site 50 without increasing toxicity would represent a significant advance in the management us such infections. In this context, the entering of nanomedicine into this scenario has revolutionized the scientific researchand it is expected that in the forthcoming decades transforms the pharma- 55 ceutical and biotechnological industries. The reason un- derlying this revolution relies on the possibility of circum- venting the main limitations of conventional medicine, such as the lack of specificity, narrow therapeutic window, Daniel Pedraza, Jaime Díez: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Universidad Complutense de Madrid, Instituto de Investigación Sanitaria Hospital 12 de Oc- tubre i+12, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain Isabel-Izquierdo-Barba, Montserrat Colilla: Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Univer- sidad Complutense de Madrid, Instituto de Investigación Sanitaria Hospital 12 de Octubre i+12, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain; CIBER de Bioingeniería, Biomateriales y Nanomedicina, CIBER-BBN, Madrid, Spain https://doi.org/10.1515/bglass-2017-0011 112 | D. Pedraza et al. Figure 1: Representative depiction of the mechanism of action of the nanoantibiotic against bacterial biofilm. low solubility and stability, unappropriated pharmacoki- netics and diverse side effects of drugs [8]. Albeit during the last 40 years promising results have been achieved, the clinical application of nanotechnology, in its broad- est sense, remains an enormous defy. This fact can be ex-5 plained because nanomedicine requires complex and spe- cific technological solutions that make difficult its phar- maceutical development [9]. The application of nanoma- terials in medicine has provided more than 250 products already approved or in different phases of clinical trials.10 Nowadays, one of the most important challenges in bone infection therapy is the design of nanocarriers able to protect, transport and release antimicrobial agents in a controlled way once they reached the target (bacteria and biofilm) [8, 10]. Among nanocarriers, mesoporous silica15 nanoparticles (MSNs) are excellent candidates to develop targeted stimuli-responsive drug delivery systems [9, 11– 19]. They exhibit unique properties such as high specific surface area, large pore volume, tunable pore structures andeasilymodifiable surfacedue to thepresence of silanol20 groups (Si-OH) [10, 20].Moreover,MSNshavebeendemon- strated to be biocompatible both in vitro and in vivo [21–23] and exhibit adequate stability in different biological me- dia [24, 25]. Herein, we designed a nanosystem named “nanoan- 25 tibiotic” as a new therapeutic alternative to fight against bone infection. This nanoantibiotic, which is based on MSNs, combines an antimicrobial agent with an amine functionalization as targeting agent to recognize the bac- teria. Levofloxacin (LEVO) antibiotic, a wide range fluo- 30 roquinolone usually employed in osteomyelitis and im- plant associated bone infections due to its strong cortical bone affinity [26] was used. The used targeting agent, N-(2- aminoethyl)-3-aminopropyltrimethoxysilane (DAMO), de- ploys positively charged amine groups on the MSN sur- 35 face that enables attractive electrostatic interactions with the bacteria wall and/or biofilm, exhibiting negative den- sity charge. The hypothesis of the current research work is schematically depicted in Figure 1. Amine-Functionalized Mesoporous Silica Nanoparticles | 113 Figure 2: Scheme of the DAMO grafting to the Si-OH present on the MSN external surface. 2 Materials and Methods 2.1 Synthesis and functionalization of MSNs Pristine MSNs, denoted as MSN, were synthesized via the modified Stöber method [27]. Briefly, 1 g of cetyl trimethylammonium bromide (CTAB, Sigma-Aldrich) was5 dissolved in 480 mL Milli-Q water (resistivity 18.2 MΩ cm) containing 3.5 mL of a 2M solution of NaOH (99%, Sigma- Aldrich). The mixture was kept under magnetic stirring (400-600 rpm) at 80∘C for 45 min. Afterwards, 5 mL of tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich) were10 addedwith a pump injector NE-300 (Just Infusion) at a rate of 0.33 mL/min, maintaining the same conditions of tem- perature and stirring for 2 h. Later, the flask was cooled down rapidly and its content was centrifuged in a cen- trifuge Heraeus Multifugue X3 (Thermo Fisher Scientific)15 at 10,000 rpm and 10∘C for 10 min in 3 cycles. Between each cycle, the content was resuspended with 80 mL of absolute ethanol (99.5%, PanReac), for its washing. Fi- nally, the obtained product was dried in a vacuum stove (Vaciotemp-T JP Selecta, ICT) at 30∘C. To provide theMSNs20 fluorescent properties for detecting them using confocal microscopy, this synthesis route was modified by adding fluorescein. For this purpose, 1 mg of fluorescein isoth- iocyanate (FITC, Sigma-Aldrich) was added to 2.2 µL of (3-aminopropyl)triethoxysilane (APTES, 99%, ABCR) and25 100 µL of absolute ethanol under constant stirring at 200 rpm for 2h at room temperature (RT) and protected from light. After this time, 5 mL of TEOS were added until com- plete homogenization was obtained. This solution was in- jected in the synthesis flask containing the aqueous solu-30 tion of the structure-directing agent in basic medium, as indicated for the preparation of the MSN. To provide MSN of targeting properties towards bacteria, their surface was functionalized with N-(2- aminoethyl)-3-aminopropyltriethoxysilane (DAMO, 95%,35 ABCR) by means of a post-synthesis grafting under an- hydrous conditions and inert atmosphere [28]. The reac- tion was produced through the condensation between the DAMO molecules and the silanol groups on the MSN sur- face, as indicated in Figure 2. The functionalization was 40 carried out before the surfactant removal from the meso- porous cavities, in such a way that the material is only functionalized in the exterior, leaving the pores free after the extraction process [29]. The amount of DAMO used for the functionalization process was calculated taking into 45 account the average superficial density of silanol groups in theMSNs (4.9 SiOH/nm2) [30] and the fact that eachDAMO molecule mainly condenses with three Si-OH groups (in good agreement with the results derived from 29Si NMR, vide infra). Briefly, 1 g ofMSNwas dried in a three-mouthed 50 flask with round bottom at 80∘C and vacuum conditions for 24 h. Then, three cycles alternating vacuum and N2 were performed. 200 mL of anhydrous toluene (99.8%, Sigma-Aldrich) were added and the flask was immersed in a ultrasound bath for 1 h. Afterwards, 0.8 mL of DAMO 55 were added and the flask was left at 110∘C with reflux for 24 h. Finally, the product was centrifuged by successive washing cycles with isopropanol, ethanol and methanol, respectively. The product was left to dry for 24 h under vac- uum at 30∘C. The surfactant extraction was performed via 60 cationic exchange. For this purpose, an extracting solution containing 100mL of deionized water, 1.9 L of ethanol and 20 g of NH4NO3 (99.5%, Sigma-Aldrich) was made. Then, 1 g of MSNs was added to 600 mL of extracting solution. The suspension was kept during 24 h with a reflux (80∘C) 65 and magnetic stirring. Finally, the product was washed 3 times with ethanol and was left to dry for 24 h at 30∘C in a vacuum oven. 2.2 Materials Characterization The structural characterizationwas performed using X-ray 70 diffraction (XRD) in a diffractometer Philips X’pert Plus working with Kα radiation of Cu (λ = 1.54 Å) at 40 kV and 20 mA, in the range 0.6-7∘ with a contact time of 5 s and transmission electronic microscopy (TEM) in a JEOL JEM 1400 microscope with a voltage accelerator of 120 kV 75 with a CCD of 2048 x 2048 pixels. The textural proper- ties were determined using porosimetry of N2 adsorption at −196∘C using a Micromeritics ASAP 2020 analyzer (Mi- 114 | D. Pedraza et al. Figure 3: Sequential process of functionalization, extraction and LEVO loading of MSNs affording MSN-DAMO@LEVO. cromeritics Co.). The surface areawas calculated using the Brunauer-Emmet-Tellet (BET) method [31]. The total pore volume (VT) was estimated from the N2 adsorbed at a rel- ative pressure of 0.97. The pore diameter (Dp) was deter- mined using the Barret-Joyner-Halenda (BJH)method. The5 hydrodynamic sizewas determined via dynamic light scat- tering (DLS) in a Zetasizer Nano ZS (Malvern Instruments Ltd.) equipped with a 633 nm laser. The same equipment was used tomeasure the ζ -potential. The chemical compo- sition was determined via elemental chemical analysis in10 a Perkin-Elmer 2400 CHNS thermoanalyzer. The changes in the chemical environments of the Si and C atoms of the different samples were studied through 29Si (single pulse, SP, and cross-polarization, CP) and 1H→ 13C solid- state nuclear magnetic resonance (NMR) in a Bruker AV-15 400-WB spectrometer, using spinning speeds of 10 and 12 kHz and observation frequencies of 79.49 and 100.62 MHz, respectively. The 29Si spectra were obtained using a sin- gle pulse sequence (29Si SP magic angle spinning, MAS, NMR), whilst the 13C ones were obtained using a 1H→13C20 CP MAS NMR sequence. The time periods between succes- sive collections were 5 and 3 ms for 29Si and 13C, respec- tively, and the total number of scans was approximately 1,000 for both cases. 2.3 Levofloxacin loading and drug release25 The loading of LEVO (≥ 98.0%, Sigma-Aldrich) inside the matrix of MSN and MSN-DAMO was done via the impreg- nation method [32]. 125 mg of MSN and MSN-DAMO was suspended in 25 mL of a drug solution (26.1 mg/mL) in the absence of light under magnetic stirring during 24 h at RT,30 and the resulting samples were denoted as MSN@LEVO and MSN-DAMO@LEVO, respectively. Figure 3 schemat- ically shows the full process followed to obtain MSN- DAMO@LEVO. The release of LEVO from each of the matrices was 35 done under in vitro conditions at 37∘C and physiological pH (pH 7.4). A solution of 4mg of the loadedmaterials was prepared in 500 µL of phosphate buffered saline (PBS 1x, Sigma Aldrich). 170 µL of this suspension was introduced in a release cap and placed in a bucket containing 3.6 mL 40 of PBS separated by a dialysis membrane that only allows for the diffusion of LEVO molecules. The system was kept at 37∘C under magnetic stirring and the concentration of released drug was monitored via fluorescence in a Biotek- Powerwave XSwith the programme Gen5 (v.1.00.14), using 45 a λ(exc)=292 nm y λ(em)=494 nm. To determine the LEVO concentration a calibration curve in the 12 to 0.02 µg/mL range was performed. The “in vial” release study was car- ried out by renewing the media at given times. With the aim of determining the effectiveness of the different re- 50 leased LEVOdoses against bacteria growth, 100 µL of each dose was inoculated in 900 µL of suspension contain- ing 108 bacteria/mL in PBS, which was later incubated overnight at 37∘C and under orbital stirring at 200 rpm. This study was performed with Gram-positive (S. aureus) 55 and Gram-negative (E. coli) bacteria. The presence (or ab- sence) of bacteria, along with its quantification was done by means of counting the colony forming units (CFUs) on tryptic soy agar (TSA, Sigma-Aldrich) plates. For this pur- pose 10 µL of this solution was seeded in TSA plates, be- 60 ing incubated at 37∘C overnight, with a later counting of the CFUs. All the assays were done in triplicate with their respective control samples. 2.4 In vitro antimicrobial tests The nanoantibiotic effectiveness was determined using 65 three different approaches: targeting to the bacteria wall, targeting to bacterial biofilm, and antimicrobial activity against preformed S. aureus biofilm. Amine-Functionalized Mesoporous Silica Nanoparticles | 115 Targeting to bacteria: In this study Gram-negative bac- teria E. coli ATCC25922 collection strain was used. Before the assays, disk-shaped cover glasses (9 mm diameter) were sterilized by UV-light and then incubated with poly- D-lysine (0.3 mL per well from a 0.1 mg/mL stock solution5 in Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich) for 90 min at 37 ∘C. Then, the excess poly-D-lysine was re- moved by washing with sterilized Milli-Q water and the cover glasses were left to dry overnight in a sterile envi- ronment. Poly-D-Lysine treated cover glasses were placed10 in 24 well culture plates (CULTEK). Then, 500 µL of the 108 bacteria/mL solutionwas added onto each cover glass and subsequently 500 µL of FMSNs and FMSNs-DAMO suspen- sions in PBS at different concentrations (5 and 10 µg/mL) were also inoculated and incubated at 37∘C and 200 rpm15 under orbital stirring during 90min. Then, each glass-disk was washed twice with sterile Hank’s balanced salt solu- tion (HBSS, Sigma-Aldrich) and 1mL of HBSS was added. FM 4-64FX dye (0.2 mL from a 5 µg/mL stock solution in HBSS, Invitrogen) was added to stain the bacteria wall in20 red and incubated on ice for 10 min under orbital stirring. Later on, the cover glasses were washed with HBSS and fixed in 2% wt/vol paraformaldehyde in PBS for 10 min at RT. The fixative was removed with HBSS twice and 0.5 mL HBSS was added to each well before imaging. The sam-25 ples were examined in an Olympus FV1200 confocal mi- croscope. Targeting to bacterial biofilm: S. aureus ATCC28213 strain was used for these studies. After treating with poly- D-lysine, as abovedescribed, the cover glasseswereplaced30 in a 24 well culture plate and 500 µL of a solution of bac- teria with 108 bacteria per mL were added to each plate during 48 h. Then, 500 µL of fluorescent MSN and MSN- DAMO suspensions at different concentrations (10 and 20 µg/mL) in PBS was added and incubated at 37∘C under35 orbital stirring at 200 rpm. Themediumusedwas 66%TSB + 0.2% glucose (wt/vol) to promote robust biofilm forma- tion. After 90 min, the treated cover glasses were washed three times with sterile PBS, stained with 5 µL/mL cal- cofluor (calcofluor white, Sigma-Aldrich) and incubated40 during 15 min. The calcofluor stained the mucopolysac- charides of the biofilm (extracellular matrix in blue). The samples were examined in an Olympus FV1200 confocal microscope. Control biofilms without nanoparticles was also observed.45 Antimicrobial effects against Gram positive S. aureus biofilm: Effectiveness of the complete nanosystems con- taining both targeting and antimicrobial LEVO agents against bacterial biofilm was determined. For these pur- pose, S. aureus biofilms were developed, as above de- 50 scribed. Then, 0.5 mL of the suspension of nanoparti- cles in PBS at a concentration 10 µg/mL was added. Af- ter 90 min of incubation, the cover glasses were washed three times with sterile PBS, stained with a 3 µL/mL of Live/Deadr Bacterial Viability Kit (BacklightTM, Invitro- 55 gen) and 5 µL/mL of calcofluor solution was added. Both reactants were incubated during 15 min at RT. Biofilm formation was examined in an Olympus FV1200 confo- cal microscope. Bacterial biofilm control was also stud- ied. Eight photographs (60x magnification) of each sam- 60 ple were taken. The surface area covered by adhered bac- teria was calculated using ImageJ software (National Insti- tute of Health, Bethesda, MD). The experiments were per- formed in triplicate. 3 Results and discussion 65 3.1 Sample Characterization The structural and textural characterization of MSNs be- fore and after DAMO functionalization was performed us- ing TEM, XRD and N2 adsorption porosimetry (Figure 4). TEM images showa sphericalmorphology of nanopar- 70 ticles sized ca. 120 nm, showing a honeycomb disposition of the inner channels that follow a hexagonal symmetry. XRD diagrams confirm that structure, showing diffraction maxima at 2.3∘, 3.9∘ and 4.5∘ which can be indexed as the 10, 11 and 20 reflections, respectively, of a 2D hexag- 75 onal structure p6mm plain group. In terms of textural properties, the adsorption-desorption N2 isotherms pro- files are associated to mesoporous MCM-41 type materials with cylindrical parallel pores. Table 1 contains structural parameters (surface area(SBET), pore volume (VP), pore di- 80 ameter (DP)) of samples before andafter functionalization, showingadecrease of themafterDAMOgrafting.Addition- ally, using the lattice parameter (a0) obtained by XRD and Dp, we can obtainwall thickness,which increases after the functionalization process. We can observe a reduction on 85 hydrodynamic size (DH) of MSN-DAMO against MSN due to DAMO’s hydrophilic behavior, which provides a higher colloidal stability. The ζ -potential changemeans a change on the sign of the surface charge density, which confers MSN-DAMO of potential bacterial targeting capability. 90 Quantitative determination of functional groups, per- formed by elemental chemical analysis, indicated that the amount of DAMO incorporated into MSN-DAMO was 286 mg/g. With the aim of evaluating the modification in the chemical environments of MSNs we carried out solid state 95 116 | D. Pedraza et al. Figure 4: (A) TEM images, (B) XRD patterns and (C) N2 adsorption isotherms of MSN and MSN-DAMO. Table 1: Lattice parameter (a0), hydrodynamic size (DH ), textural properties, ζ -potential and weight (%) of DAMO and LEVO for the different samples. Sample a0 (nm) DH(nm) SBET (m2/g) VP (cm3/g) DP (nm) twall (nm) ζ -pot. (mV) % DAMO % LEVO MSN 4.55 190 937 0.85 2.4 2.1 −36.4 0 0 MSN-DAMO 4.68 141 670 0.40 2.1 2.5 37.4 28.6 0 MSN@LEVO 4.50 183 798 0.73 2.6 1.9 −37.0 0 3.18 MSN-DAMO@LEVO 4.51 139 294 0.25 1.9 2.6 +35.1 28.6 5.03 NMR studies. Figure 5 shows CP-MAS and SP-MAS of 29Si spectra of samples and Table 2 displays the relative abun- dance of Si species in the different environments derived from the deconvolution and subsequent integration of the resulting peaks. Both MSNs spectra show resonances at5 −94, −102 y −111 ppm, which are assigned to silicon atoms in Q2, Q3 and Q4 environments. Q2 and Q3 structural units are ascribed to the Si-OH groups present on the MSN sur- face. The relative abundances of Si atoms in Q2 and Q3 environments decrease in the case of functionalized sam- 10 ple, due to the condensation reaction of DAMO moieties with the Si-OH groups of MSN surface. Furthermore, MSN- DAMO spectrum shows two additional peaks associated to organosiloxane groups T2 and T3 at ca. −59 and −68 ppm, respectively. These data confirm the presence of covalent 15 unions between the MSN surface and DAMOmolecules. Amine-Functionalized Mesoporous Silica Nanoparticles | 117 Figure 5: Left) 1H→29Si-CP-MAS NMR results of MSN and MSN-DAMO that indicate the maxima position of Qn y Tm. Right) 1H→29Si-SP-MAS NMR results of MSN and MSN-DAMO. Table 2: Relative abundance of species of Si on Qn and Tm environ- ments obtained from the deconvolution of the 29Si SP-MAS NMR signals. Sample % T2 % T3 %Q2 %Q3 %Q4 MSN 0 0 13 31 56 MSN-DAMO 5 12 6 19 58 In MSN spectrum maxima corresponding to remain- ing CTAB are observed, while in MSN-DAMO signals asso- ciated to various C atoms of DAMO appear, whose assigna- tion is also included in Figure 6. 3.2 Levofloxacin loading and release assays5 The amount of LEVO incorporated to samples was de- termined by elemental chemical analysis, resulting in 31.8 mg/g and 50.3 mg/g for MSN@LEVO and MSN- DAMO@LEVO, respectively. Figure 7 represents the release curves of LEVO from the different matrices. The results show that LEVO release from MSN is slower than from MSN-DAMO, which also produces a partial retention of the drug in the former. On the contrary, drug release profile from MSN-DAMO follows a typical diffusion model, where almost the total loaded LEVO is released after 72 h of as- say. Both release curveswere analyzed and comparedwith Chapman equation (Eq. 1) [28, 33]: w(t) w0 = A (︁ 1 − e−kt )︁δ (1) where w(t) is the amount of LEVO released at t time, w0 is the initial amount of loaded drug,A is themaximumLEVO release, k is the release kinetic constant and δ is a dimen- sionless parameter of non-ideality that characterizes the release. 10 118 | D. Pedraza et al. Figure 6: 1H→13C CP-MAS NMR spectra of MSN and MSN-DAMO. On the top, the assignation to different carbon atoms of DAMO is shown. In both cases, δ is minor than 1, showing that the be- havior is different from a first order kinetic. The k values varies from 0.021 to 0.51 h−1 for MSN@LEVO and MSN- DAMO@LEVO, respectively,which couldpoint to strong at- tractive interactions of the LEVOmolecules with the Si-OH5 groups present in thematrix of pristineMSN, as it has been demonstrated elsewhere [34]. The effectiveness of the different released LEVO doses at several times from both MSNs against E. coli and S. aureus bacterial growth was performed. The counting of10 CFUs after 1, 3 and 24 h showed the efficacy from both ma- trices against the two tested strains (data not shown). Figure 7: Cumulative LEVO release profiles from MSN and MSN- DAMO samples and comparison with Chapman equation. 3.3 Targeting to bacteria wall and biofilm Regarding targeting to bacteria wall (Figure 8), confocal images reveal that fluorescentMSN-DAMOare locatednear 15 the E. coli bacteria wall resulting in aggregates of nanopar- ticles (green dots) on the bacteria’s surface for both tested concentration. Note than in the case of MSN such green dots are not observed, probably due to the lack of inter- action with the bacteria wall and that would be easily re- 20 moved during the washing process. Concerning targeting to bacterial biofilm, Figure 9 rep- resents the effect of DAMO functionalization (MSN-DAMO, green), to E. coli biofilm (blue) investigated by using con- focal microscopy at different depths. It can be appreci- 25 ated noticeable differences betweenMSN andMSN-DAMO. Thus, in the case of MSN, the nanoparticles are localized in the vicinity of the biofilm. However, for MSN-DAMO it can be observed that the nanoparticles are internalized within biofilm, demonstrating the capability of this amine- 30 functionalized nanosystem to penetrate the mucopolysac- charide matrix. 3.4 Nanoantibiotic eflcacy Once demonstrated the ability of MSN-DAMO nanopar- ticles to target the bacteria wall and internalize into 35 the biofilm, we measured the effectiveness of the com- plete nanosystem (MSN-DAMO@LEVO) against a previ- ously formed S. aureus biofilm. We chose such bacterial strain since it is the most common responsible of bone in- fection processes [4]. Figure 10 represents the confocal mi- 40 croscopy study corresponding to the preformed S. aureus biofilm before and after being treated with MSN@LEVO Amine-Functionalized Mesoporous Silica Nanoparticles | 119 Figure 8: Targeting to E. coli bacteria wall studies by confocal microscopy. Figure 9: Targeting to E. coli biofilm studies by using confocal microscopy. 120 | D. Pedraza et al. Figure 10: Left: Control biofilm. Right: Biofilm treated with MSN and MSN-DAMO nanosystems. and MSN-DAMO@LEVO. The results show that the un- treated biofilm is composed of live bacteria multilayer (green) with a small amount of dead bacteria (red) coated by a polysaccharide matrix (blue) with an average thick- ness of 35 µm. After treatment with MSN@LEVO a remain-5 ing bacteria layer mainly formed by live cells is observed. Nonetheless, in the case of the full MSN-DAMO@LEVO nanosystem the complete destruction of biofilm is appreci- ated, appearing only isolated bacteria, almost negligible. These results evidence that the incorporation of a target-10 ing agent onto the MSNs platform triggers the complete biofilm destruction in combination with antibiotics. 4 Conclusions We have developed a new nanoantibiotic consisting on mesoporous silica nanoparticles decorated with amine 15 groups on their surface and loaded with levofloxacin into their mesopores. Amine-functionalization provides this nanocarrier of a positive charge density that improves the targeting capability of the nanosystem to bacteria wall and bacterial biofilm, meanwhile improving the antibi- 20 otic release by achieving faster release kinetics. Microbi- ological studies against S. aureus biofilms show the com- plete destruction of biofilm when both elements (antibi- otic and targeting agents) are synergistically combined in auniquenanoplatform.Weenvision this nanoantibiotic as 25 a promising alternative to current therapies for the treat- ment of bone infection. Amine-Functionalized Mesoporous Silica Nanoparticles | 121 Acknowledgement: MVR acknowledges funding from the European Research Council (Advanced Grant VERDI; ERC- 2015- AdG Proposal No.694160). The authors also thanks to SpanishMINECO (CSO2010-11384- E,MAT2013-43299- R, MAT2015-64831- R, MAT2016-75611- R AEI/FEDER, E). The5 authors wish to thank the ICTS Centro Nacional de Mi- croscopia Electrónica (Spain), CAI X-ray Diffraction, CAI NMR, CAI Flow Cytometry and Fluorescence Microscopy of the Universidad Complutense de Madrid (UCM, Spain) for the assistance.10 References [1] Campoccia D., Montanaro L., Arciola CR., The significance of in- fection related to orthopedic devices and issues of antibiotic re- sistance, Biomaterials, 2006, 27, 2331-2339. [2] Lew D.P., Waldvogel F.A., Osteomyelitis, Lancet, 364 (2004)15 369-379. [3] Vila J., Soriano A., Mensa J., Molecular basis of microbial ad- herence to prosthetic materials. Role of biofilms in prosthesis- associated infection, Enferm. 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[34] García-Álvarez R., Izquierdo-Barba I., Vallet-Regí M., 3D scaf- foldwith effectivemultidrug sequential release against bacteria biofilm, Acta Biomaterialia 49, 2016, 113-126. 5 1 Introduction 2 Materials and Methods 2.1 Synthesis and functionalization of MSNs 2.2 Materials Characterization 2.3 Levofloxacin loading and drug release 2.4 In vitro antimicrobial tests 3 Results and discussion 3.1 Sample Characterization 3.2 Levofloxacin loading and release assays 3.3 Targeting to bacteria wall and biofilm 3.4 Nanoantibiotic efficacy 4 Conclusions