Person: Aicart Sospedra, Emilio
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First Name
Emilio
Last Name
Aicart Sospedra
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Ciencias Químicas
Department
Area
Química Física
Identifiers
11 results
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Item How does the spacer length of cationic gemini lipids influence thelLipoplex formation with plasmid DNA? Physicochemical and biochemical characterizations and their relevance in gene therapy(Biomacromolecules, 2012) Muñoz Úbeda, Mónica; Misra, Santosh K.; Barran-Berdón, Ana L; Datta, Sougata; Aicart-Ramos, Clara; Castro-Hartmann, Pablo; Kondaiah, Paturu; Junquera González, María Elena; Bhattacharya, Santanu; Aicart Sospedra, EmilioLipoplexes formed by the pEGFP-C3 plasmid DNA (pDNA) and lipid mixtures containing cationic gemini surfactant of the 1,2-bis(hexadecyl dimethyl ammonium) alkanes family referred to as C16CnC16, where n = 2, 3, 5, or 12, and the zwitterionic helper lipid, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) have been studied from a wide variety of physical, chemical, and biological standpoints. The study has been carried out using several experimental methods, such as zeta potential, gel electrophoresis, small angle X-ray scattering (SAXS), cryo-TEM, gene transfection, cell viability/cytotoxicity, and confocal fluorescence microscopy. As reported recently in a communication (J. Am. Chem. Soc. 2011, 133, 18014), the detailed physicochemical and biological studies confirm that, in the presence of the studied series lipid mixtures, plasmid DNA is compacted with a large number of its associated Na+ counterions. This in turn yields a much lower effective negative charge, qpDNA − , a value that has been experimentally obtained for each mixed lipid mixture. Consequently, the cationic lipid (CL) complexes prepared with pDNA and CL/DOPE mixtures to be used in gene transfection require significantly less amount of CL than the one estimated assuming a value of qDNA − = −2. This drives to a considerably lower cytotoxicity of the gene vector. Depending on the CL molar composition, α, of the lipid mixture, and the effective charge ratio of the lipoplex, ρeff, the reported SAXS data indicate the presence of two or three structures in the same lipoplex, one in the DOPE-rich region, other in the CL-rich region, and another one present at any CL composition. Cryo-TEM and SAXS studies with C16CnC16/DOPE-pDNA lipoplexes indicate that pDNA is localized between the mixed lipidm bilayers of lamellar structures within a monolayer of ∼2 nm. This is consistent with a highly compacted supercoiled pDNA conformation compared with that of linear DNA. Transfection studies were carried out with HEK293T, HeLa, CHO, U343, and H460 cells. The α and ρeff values for each lipid mixture were optimized on HEK293T cells for transfection, and using these values, the remaining cells were also transfected in absence (-FBS-FBS) and presence (-FBS+FBS) of serum. The transfection efficiency was higher with the CLs of shorter gemini spacers (n = 2 or 3). Each formulation expressed GFP on pDNA transfection and confocal fluorescence microscopy corroborated the results. C16C2C16/DOPE mixtures were the most efficient toward transfection among all the lipid mixtures and, in presence of serum, even better than the Lipofectamine2000, a commercial transfecting agent. Each lipid combination was safe and did not show any significant levels of toxicity. Probably, the presence of two coexisting lamellar structures in lipoplexes synergizes the transfection efficiency of the lipid mixtures which are plentiful in the lipoplexes formed by CLs with short spacer (n = 2, 3) thanm, those with the long spacer (n = 5, 12).Item Effects of a delocalizable cation on the headgroup of gemini lipids on the lipoplex-type nanoaggregates directly formed from plasmid DNA(Biomacromolecules, 2013) Misra, Santosh ; Muñoz Úbeda, Mónica; Datta, Sougata; Barrán-Berdón, Ana L.; Aicart-Ramos, Clara; Castro-Hartmann, Pablo; Kondaiah, Paturu; Junquera González, María Elena; Battacharya, Santanu; Aicart Sospedra, EmilioLipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure−biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10−25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprinttype, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.Item A delocalizable cationic headgroup together with an oligo-oxyethylene spacer in gemini cationic lipids improves their biological activity as vectors of plasmid DNA(Journal of Materials Chemistry B, 2015) Kumar, Krishan; Barrán-Berdón, Ana ; Datta,Sougata; Muñoz Úbeda, Mónica; Aicart-Ramos, Clara; Kondaiah, Paturu; Junquera González, María Elena; Bhattacharya, Santanu; Aicart Sospedra, EmilioLipoplex nano-aggregates constituted of plasmid DNA (pDNA) pEGFP-C3 and mixed cationic liposomes, consisting of several percentages of a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl imidazolium) oxyethylene series, referred to as (C16Im)2(C2O)n, with oxyethylene spacers (n = 1, 2 or 3) between the imidazolium cationic groups and the DOPE zwitterionic helper lipid, have been characterized by various biophysical and biological approaches carried out at several GCL compositions (α), and either the mass or the effective charge ratio of the lipoplex. The electrochemical study by ζ-potential confirms that the three GCLs yield a 10% lower effective charge than the nominal one, while compacted pDNA yields only a 25% effective negative charge. The SAXS study reveals, irrespective of the spacer length (n) and effective charge ratio (ρeff), the presence of two lamellar structures, i.e., one (Lα,main) in the whole GCL composition and another (Lα,DOPE,rich) with higher periodicity values that coexists with the previous one at low GCL composition (α = 0.2). The cryo-TEM analysis shows two types of multilamellar structures consisting of cationic lipidic bilayers with pDNA sandwiched between them: a cluster-type (C-type) at low α = 0.2 and a fingerprint-type (FP-type) at α ≥ 0.5, both with similar interlamellar spacing (d) in agreement with the Lα,main structure determined by SAXS. Transfection efficacies (TEs) of each lipid mixture were determined in four different cell lines (HEK293T, HeLa, Caco-2 and A549) at several α and ρeff values in the absence and presence of serum (FBS). The optimized formulations (α = 0.2 and ρeff = 2.0) substantially transfect cells much better than a commercial transfection reagent, Lipofectamine 2000 and previously studied efficient lipoplexes containing other cationic head groups or spacers both in the absence and presence of serum. The activity of optimized formulations may be attributed to the combination of several factors, such as: (a) the fusogenic character of DOPE which results in higher fluidity of the lipoplexes at α = 0.2, (b) the coexistence of two lamellar structures at α = 0.2 that synergizes the TE of these lipid vectors, and mainly (c) the higher biocompatibility of the GCLs reported in this work due to the presence of two imidazolium cationic groups together with an oligo-oxyethylene spacer. The length of the spacer in the GCL seems to have less impact, although (C16Im)2(C2O)n/DOPE-pDNA lipoplexes with n = 1 and 3 show higher gene transfection than n = 2. All the optimum formulations reported herein are all highly efficient with negligible levels of toxicity, and thus, may be considered as very promising gene vectors for in vivo applications.Item Why is less cationic lipid required to prepare lipoplexes from plasmid DNA than linear DNA in gene therapy?(Journal of the American Chemical Society, 2011) Muñoz Úbeda, Mónica; Misra, Santosh ; Barrán-Berdón, Ana; Aicart-Ramos, Clara; Sierra, María ; Biswas, Joydeep; Kondaiah, Paturu; Junquera González, María Elena; Bhattacharya, Santanu; Aicart Sospedra, EmilioThe most important objective of the present study was to explain why cationic lipid (CL)-mediated delivery of plasmid DNA (pDNA) is better than that of linear DNA in gene therapy, a question that, until now, has remained unanswered. Herein for the first time we experimentally show that for different types of CLs, pDNA, in contrast to linear DNA, is compacted with a large amount of its counterions, yielding a lower effective negative charge. This feature has been confirmed through a number of physicochemical and biochemical investigations. This is significant for both in vitro and in vivo transfection studies. For an effective DNA transfection, the lower the amount of the CL, the lower is the cytotoxicity. The study also points out that it is absolutely necessary to consider both effective charge ratios between CL and pDNA and effective pDNA charges, which can be determined from physicochemical experiments.Item Magnetic silica nanoparticle cellular uptake and cytotoxicity regulated by electrostatic polyelectrolytes DNA loading at their surface(ACS Nano, 2011) Davila-Ibanez, Ana ; Salgueirino, Veronica; Martinez-Zorzano, Vicenta; Marino-Fernández, Rosalía; García-Lorenzo, Andrés; Maceira-Campos, Melodie; Muñoz Úbeda, Mónica; Junquera González, María Elena; Aicart Sospedra, Emilio; Rivas, José; Rodriguez-Berrocal, Javier; Legido, JoseMagnetic silica nanoparticles show great promise for drug delivery. The major advantages correspond to their magnetic nature and ease of biofunctionalization, which favors their ability to interact with cells and tissues. We have prepared magnetic silica nanoparticles with DNA fragments attached on their previously polyelectrolyte-primed surface. The remarkable feature of these materials is the compromise between the positive charges of the polyelectrolytes and the negative charges of the DNA. This dual-agent formulation dramatically changes the overall cytotoxicity and chemical degradation of the nanoparticles, revealing the key role that surface functionalization plays in regulating the mechanisms involved.Item Experimental and theoretical approach to the sodium decanoate-dodecanoate mixed surfactant system in aqueous solution(Langmuir, 2010) Rodríguez-Pulido, Alberto; Casado, Aitor; Muñoz Úbeda, Mónica; Junquera González, María Elena; Aicart Sospedra, EmilioThe mixed system consisting of two anionic surfactants of identical headgroups but with 10 and 12 carbon atoms on the hydrophobic tail, sodium decanoate (C10Na) and sodium dodecanoate (C12Na), has been studied in aqueous solution at 298.15 K by means of conductivity and fluorescence spectroscopy experiments and from a theoretical point of view. The monomeric and micellar phases of the mixed aggregates were analyzed through the experimental determination of the total critical micelle concentration, cmc*, the degree of ionization of the mixed micelle, β, and the total aggregation number, N*. Results indicate that, compared to the ideal behavior, the mixed system with two anionic surfactants differing only in two methylenes in the hydrophobic tail shows a negative deviation in the cmc* and a positive one in N*. Pure surfactants (C10Na and C12Na) form spherical micelles, but mixed micelles must aggregate with a rodlike shape to allow more surfactant molecules than expected. In addition, rodlike micelles result in more compacted aggregation (i.e., less area per polar head). From the experimental data in this work, several theoretical models for mixed surfactant systems have been checked: Rubingh’s model predicts lower deviations from ideality than Motomura’s model. The stability of the micelles has been analyzed by computing the standard Gibbs energy of micelle formation, ΔGmic,0, of pure and mixed micelles. Results of this work reinforce the feature that mixed systems formed by alkylsurfactants with the same polar head that differ in the hydrocarbon length, usually admitted as roughly ideal systems, may show nonideal behavior. This deviation, being mostly related to the difference in the chain length, Δnc, between surfactants can be analyzed only when very accurate experimental techniques as well as adequate theoretical models are used.Item Gene vectors based on DOEPC/DOPE mixed cationic liposomes: a physicochemical study(Soft Matter, 2011) Muñoz Úbeda, Mónica; Rodríguez-Pulido, Alberto; Nogales, Aurora; Llorca, Oscar; Quesada-Pérez, Manuel; Martín-Molina, Alberto ; Aicart Sospedra, Emilio; Junquera González, María ElenaA double approach, experimental and theoretical, has been followed to characterize from a physicochemical standpoint the compaction process of DNA by means of cationic colloidal aggregates. The colloidal vectors are cationic liposomes constituted by a mixture of a novel cationic lipid, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt) (DOEPC) and a zwitterionic lipid, the 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE). A wide variety of high precision experimental techniques have been used to carry out the analysis: electrophoretic mobility, small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM) and fluorescence spectroscopy (ethidium bromide intercalation assays). On the other hand, a theoretical model that considers the renormalization of charges of both the polyelectrolyte and the colloidal aggregates sheds light as well on the characteristics of the compaction process. This global information reveals that the compaction of DNA by the cationic liposomes is mostly driven by the strong electrostatic interaction among the positively charged surfaces of the colloidal aggregates and the negatively charged DNA, with a potent entropic component. DOEPC/DOPE liposomes are mostly spherical, with a mean diameter of around 100 nm and a bilayer thickness of 4.4 nm. From a morphological viewpoint, an appreciable amount of multilamellar structures has been found not only on the lipoplexes but also on the parent liposomes. The isoneutrality of the lipoplexes is found at liposome/DNA mass ratios that decrease with the molar fraction of cationic lipid in the mixed liposome (a). This liposome composition has a clear effect as well on the lipoplex structure, which goes from an inverted hexagonal phase (HII), usually related to improved cell transfection efficiency, at low cationic lipid molar fraction (a z 0.2), to a lamellar structure (La) when the cationic lipid content in the mixed liposomes increases (a $ 0.4), irrespective of the lipoplex charge ratio. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work, by using renormalized charges. The model allows us to confirm and predict the experimental isoneutrality conditions as well as to determine the maximum magnitude of this charge as a function of the composition of the resulting lipoplexes.Item Effect of lipid composition on the structure and theoretical phase diagrams of DC-Chol/DOPE-DNA lipoplexes(Biomacromolecules, 2010) Muñoz Úbeda, Mónica; Rodríguez-Pulido, Alberto; Nogales, Aurora; Martín-Molina, Alberto; Aicart Sospedra, Emilio; Junquera González, María ElenaLipoplexes constituted by calf-thymus DNA (CT-DNA) and mixed cationic liposomes consisting of varying proportions of the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Chol) and the zwitterionic lipid, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) have been analyzed by means of electrophoretic mobility, SAXS, and fluorescence anisotropy experiments, as well as by theoretically calculated phase diagrams. Both experimental and theoretical studies have been run at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction, α, and either the mass or charge ratios of the lipoplex, respectively. The experimental electrochemical results indicate that DC-Chol/DOPE liposomes, with a mean hydrodynamic diameter of around (120 ± 10) nm, compact and condense DNA fragments at their cationic surfaces by means of a strong entropically driven electrostatic interaction. Furthermore, the positive charges of cationic liposomes are compensated by the negative charges of DNA phosphate groups at the isoneutrality L/D ratio, (L/D)(ϕ), which decreases with the cationic lipid content of the mixed liposome, for a given DNA concentration. This inversion of sign process has been also studied by means of the phase diagrams calculated with the theoretical model, which confirms all the experimental results. SAXS diffractograms, run at several lipoplex compositions, reveal that, irrespectively of the lipoplex charge ratio, DC-Chol/DOPE-DNA lipoplexes show a lamellar structure, L(α), when the cationic lipid content on the mixed liposomes α ≥ 0.4, while for a lower content (α = 0.2) the lipoplexes show an inverted hexagonal structure, H(II), usually related with improved cell transfection efficiency. A similar conclusion is reached from fluorescence anisotropy results, which indicate that the fluidity on liposome and lipoplexes membrane, also related with better transfection results, increases as long as the cationic lipid content decreases.Item Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants(Journal of Nanobiotechnology, 2021) Muñoz Úbeda, Mónica; Semenzato, Martina; Franco-Romero, Anais; Junquera González, María Elena; Aicart Sospedra, Emilio; Scorrano, Luca; López Montero, IvánLipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments.Item Ribbon-type and cluster-type lipoplexes constituted by a chiral lysine based cationic gemini lipid and plasmid DNA(Soft Matter, 2012) Barrán Berdón, Ana ; Muñoz Úbeda, Mónica; Aicart Ramos, Clara; Pérez, Louedes; Infante, María Rosa; Castro Hartmann, Pablo; Martín Molina, Alberto; Aicart Sospedra, Emilio; Junquera González, María ElenaLipoplexes constituted by plasmid DNA pEGFP-C3 (pDNA) or linear double-stranded calf thymus DNA (ctDNA) and mixed cationic liposomes consisting of several percentages of the cationic lysine derived lipid C6(LL)2 and the zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) have been analyzed by both experimental and theoretical approaches. Experimental studies, consisting of electrophoretic mobility/zeta potential, small angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), negatively stained transmission electron microscopy (NS-TEM), and GelRed f1uorescence intercalation assays, have been carried out at several liposome and lipoplex compositions, defined in terms of cationic lipid molar fraction and either the mass or charge ratios of the lipoplex. The electrochemical study confirms that, in the presence of the mixed lipids and in contrast with what has usually been found for linear DNA, the plasmid DNA is compacted with a large number of its Na+ counterions, thus yielding a much lower effective negative charge (q pDNA) than that for ctDNA (q ctDNA), as reported recently by us (J. Am. Chem. Soc., 2011) for other lipoplexes. This finding is revealed as crucial for an optimum and efficient lipoplex preparation, since a lower effective negative charge implies a lower quantity of cationic lipid and, accordingly, a potential lower cytotoxicity. TEM experiments reveal a complex scenario of multilamellar nanostructures, from ribbon-type (typically present for chiral lipids) to cluster-type structures (usually found in cationic lipid/ DOPE systems), the composition of the mixed liposome playing an important role in the final morphology of the lipoplex. SAXS diffractograms confirm the existence of these two types of multilamellar structures through a deconvolution process of the first peak of diffractograms into two overlapping bands. On the other hand, a theoretical complexation model is employed to determine the net charge of the lipoplexes studied in this work. The model allows analysis and comparison of the electrochemical behaviour of lipoplexes containing linear DNA vs. those constituted by a supercoiled DNA, confirming the experimental findings