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The messenger RNA (mRNA)-based therapy, especially mRNA vaccines, has shown its superiorities in versatile design, rapid development and scale production, since the outbreak of coronavirus disease 2019 (COVID-19). Although the Pfizer-BioNTech and Moderna COVID-19 mRNA vaccines had been approved for application, unexpected adverse events were reported to be most likely associated with the mRNA delivery systems. Thus, the development of mRNA delivery system with good efficacy and safety remains a challenge. Here, for the first time, we report that the neutral cytidinyl lipid, 2-(4-amino-2-oxopyrimidin-1-yl)-N-(2,3-dioleoyl-oxypropyl) acetamide (DNCA), and the cationic lipid, dioleoyl-3,3′-disulfanediylbis-[2-(2,6-diaminohexanamido)] propanoate (CLD), could encapsulate and deliver the COVID-19 mRNA-1096 into the cytoplasm to induce robust adaptive immune response. In the formulation, the molar ratio of DNCA/CLD to a single nucleotide of COVID-19 mRNA-1096 was about 0.9: 0.5: 1 (the N/P ratio was about 7: 1). The DNCA/CLD-mRNA-1096 lipoplexes were rationally prepared by the combination of the lipids DNCA/CLD with the aqueous mRNA solution under mild sonication to stimulate multiple interactions, including H-bonding, π-stacking and electrostatic force between the lipids and the mRNA. After intramuscular applications of the DNCA/CLD-mRNA-1096 lipoplexes, robust neutralizing antibodies and long-lived Th1-biased SARS-CoV-2-specific cell immunity were detected in the immunized mice, thus suggesting the DNCA/CLD a promising mRNA delivery system. Moreover, our study might also inspire better ideas for developing mRNA delivery systems., Graphical abstract Unlabelled Image

Lipids spontaneously aggregate in an aqueous environment forming supramolecular structures of various architectures known as liquid crystalline mesophases. Their thermodynamic properties determined by dual polar/apolar nature coupled with the possibility to modulate the structural parameters, phase geometry and stability are challenging for applications in drug delivery systems. We review a few examples of functionality of lipid bilayers.

Cationic liposomes (CLs) have been regarded as the most promising gene delivery vectors for decades with the advantages of excellent biodegradability, biocompatibility, and high nucleic acid encapsulation efficiency. However, the clinical use of CLs in cancer gene therapy is limited because of many uncertain factors in vivo. Extracellular barriers such as opsonization, rapid clearance by the reticuloendothelial system and poor tumor penetration, and intracellular barriers, including endosomal/lysosomal entrapped network and restricted diffusion to the nucleus, make CLs not the ideal vector for transferring extrinsic genes in the body. However, the obstacles in achieving productive therapeutic effects of nucleic acids can be addressed by tailoring the properties of CLs, which are influenced by lipid compositions and surface modification. This review focuses on the physiological barriers of CLs against cancer gene therapy and the effects of lipid compositions on governing transfection efficiency, and it briefly discusses the impacts of particle size, membrane charge density, and surface modification on the fate of CLs in vivo, which may provide guidance for their preclinical studies., Graphical Abstract, The concept of lipoplexes applied to cancer gene therapy has been popular for decades; however, no such medicine has been marketed. Deng and colleagues highlight the physiological barriers against the clinical success of the cationic liposomal gene delivery system in cancer therapy. They suggest that the rational design of clinically applicable lipoplexes requires designers to pay close attention to the barriers and the impact of physicochemical properties of cationic liposomes on the therapeutic outcome.

In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal.

The design of nanovectors able to overcome biological barriers is one of the main challenges in biomedicine. Gemini cationic lipids are considered potential candidates for gene therapy due to their high biocompatibility and capacity to condense nucleic acids safely in the form of lipoplexes. However, this approach presents difficulties regarding genetic unpacking and, therefore, control over this process becomes crucial to ensure successful transfection. In this work, gemini cationic lipoplexes were prepared in the presence of plasmonic gold nanostars (AuNSs) to afford a nanovector that efficiently releases plasmid DNA (pDNA) upon irradiation with near-infrared femtosecond laser pulses. A critical AuNSs concentration of 50 pM and optimized laser power density of 400 mW led to successful pDNA release, whose efficiency could be further improved by increasing the irradiation time. Agarose gel electrophoresis was used to confirm pDNA release. UV-Vis-NIR spectroscopy and transmission electron microscopy studies were performed to monitor changes in the morphology of the AuNSs and lipoplexes after irradiation. From a physicochemical point of view, this study demonstrates that the use of AuNSs combined with gemini cationic lipoplexes allows control over pDNA release under ultrafast laser irradiation.

The interaction between calf-thymus DNA, ctDNA, and various single-chained surfac-tants with different functional groups at the end of hydrophobic tail was studied with the goal of investigating the influence of the functional group nature on surfactant DNA compacting effi-ciency. The surfactants investigated were dodecyltriethylammonium bromide (DTEABr), triethyl(1-phenoxydodecyl)ammonium bromide (12PhBr), triethyl(2-naphthoxydodecyl)ammonium bromide (12NBr) and 11-(isonicotinoyloxy)-N,N,N-triethyl-1-undecanaminium bromide (11PyBr). Results made evident that the surfactants’ tendencies to self-aggregation is the key factor determining their efficiency to compact the nucleic acid. Subsequently, DOPE/12NBr/pEGFP-C1 lipoplexes, with different cationic surfactant molar fractions (α) and mass ratios (L/D), were prepared and charac-terized. DOPE is a zwitterionic phospholipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the plasmid pEGFP-C1 carries a GFP coding sequence with the necessary regulatory elements for constitutive expression of the gene in human cells. 12NBr was chosen because it was the most efficient DNA compacting agent among the surfactants investigated. Finally, the cytotoxicity and transfection efficiency (TE) of DOPE/12NBr/pDNA lipoplexes, with different compositions, were investigated., This work was financed by the Consejería de Educación y Ciencia de la Junta de Andalucía (FQM-206 and FQM-274, and PI-0005-2018), the VI Plan Propio Universidad de Sevilla (PP2019/00000748), and the European Union (Feder Funds).

Cancer is an extremely complex disease, typically caused by mutations in cancer-critical genes. By delivering therapeutic nucleic acids (NAs) to patients, gene therapy offers the possibility to supplement, repair or silence such faulty genes or to stimulate their immune system to fight the disease. While the challenges of gene therapy for cancer are significant, the latter approach (a type of immunotherapy) starts showing promising results in early-stage clinical trials. One important advantage of NA-based cancer therapies over synthetic drugs and protein treatments is the prospect of a more universal approach to designing therapies. Designing NAs with different sequences, for different targets, can be achieved by using the same technologies. This versatility and scalability of NA drug design and production on demand open the way for more efficient, affordable and personalized cancer treatments in the future. However, the delivery of exogenous therapeutic NAs into the patients’ targeted cells is also challenging. Membrane-type lipids exhibiting permanent or transient cationic character have been shown to associate with NAs (anionic), forming nanosized lipid-NA complexes. These complexes form a wide variety of nanostructures, depending on the global formulation composition and properties of the lipids and NAs. Importantly, these different lipid-NA nanostructures interact with cells via different mechanisms and their therapeutic potential can be optimized to promising levels in vitro. The complexes are also highly customizable in terms of surface charge and functionalization to allow a wide range of targeting and smart-release properties. Most importantly, these synthetic particles offer possibilities for scaling-up and affordability for the population at large. Hence, the versatility and scalability of these particles seem ideal to accommodate the versatility that NA therapies offer. While in vivo efficiency of lipid-NA complexes is still poor in most cases, the advances achieved in the last three decades are significant and very recently a lipid-based gene therapy medicine was approved for the first time (for treatment of hereditary transthyretin amyloidosis). Although the path to achieve efficient NA-delivery in cancer therapy is still long and tenuous, these advances set a new hope for more treatments in the future. In this review, we attempt to cover the most important biophysical and physicochemical aspects of non-viral lipid-based gene therapy formulations, with a perspective on future cancer treatments in mind.

Lipid-based carriers represent the most widely used alternative to viral vectors for gene expression and gene silencing purposes. This class of non-viral vectors is particularly attractive for their ease of synthesis and chemical modifications to endow them with desirable properties. Despite combinatorial approaches have led to the generation of a large number of cationic lipids displaying different supramolecular structures and improved behavior, additional effort is needed towards the development of more and more effective cationic lipids for transfection purposes. With this review, we seek to highlight the great progress made in the design of each and every constituent domain of cationic lipids, that is, the chemical structure of the headgroup, linker and hydrophobic moieties, and on the specific effect on the assembly with nucleic acids. Since the complexity of such systems is known to affect their performances, the role of formulation, stability and phase behavior on the transfection efficiency of such assemblies will be thoroughly discussed. Our objective is to provide a conceptual framework for the development of ever more performing lipid gene delivery vectors.

International journal of molecular sciences 20(22), 5546 - (2019). doi:10.3390/ijms20225546, Dimeric cationic surfactants (gemini-type) are a group of amphiphilic compounds with potential use in gene therapy as effective carriers for nucleic acid transfection (i.e., siRNA, DNA, and plasmid DNA). Our studies have shown the formation of lipoplexes composed of alkanediyl-α,ω-bis[(oxymethyl)dimethyldodecylammonium] chlorides and selected 21-base-pair nucleic acid (dsDNA and siRNA) oligomers. To examine the structure and physicochemical properties of these systems, optical microscopy, circular dichroism spectroscopy (CD), small-angle X-ray scattering of synchrotron radiation (SR-SAXS), and agarose gel electrophoresis (AGE) were used. The lengths of spacer groups of the studied surfactants had a significant influence on the surfactants’ complexing properties. The lowest charge ratio (p/n) at which stable lipoplexes were observed was 1.5 and the most frequently occurring microstructure of these lipoplexes were cubic and micellar phases for dsDNA and siRNA, respectively. The cytotoxicity tests on HeLa cells indicated the non-toxic concentration of surfactants to be at approximately 10 µM. The dicationic gemini surfactants studied form complexes with siRNA and dsDNA oligomers; however, the complexation process is more effective towards siRNA. Therefore these systems could be applied as transfection systems for therapeutic nucleic acids, Published by Molecular Diversity Preservation International, Basel

Purpose: Cationic polymers and cationic liposomes have shown to be effective non-viral gene delivery vectors. In this study, we tried to improve the transfection efficiency by employing the advantages of both. Methods: For this purpose, modified polyallylamines (PAAs) were synthesized. These modifications were done through the reaction of PAA (15 KDa) with acrylate and 6-bromoalkanoic acid derivatives. Liposomes comprising of these cationic polymers and cationic lipid were prepared and extruded through polycarbonate filters to obtain desired size. Liposome-DNA nanocomplexes were prepared in three carrier to plasmid (C/P) ratios. Size, zeta potential and DNA condensation ability of each complex were characterized separately and finally transfection efficiency and cytotoxicity of prepared vectors were evaluated in Neuro2A cell line. Results: The results showed that mean particle size of all these nanocomplexes was lower than 266 nm with surface charge of 22.0 to 33.9 mV. Almost the same condensation pattern was observed in all vectors and complete condensation was occurred at C/P ratio of 1.5. The lipoplexes containing modified PAA 15 kDa with 10% hexyl acrylate showed the highest transfection efficacy and lowest cytotoxicity in C/P ratio of 0.5. Conclusion: In some cases nanocomplexes consisting of cationic liposome and modified PAA showed better transfection activity and lower cytotoxicity compared to PAA.

In autoimmune diseases as Type 1 diabetes, the actual treatment that provides the missing hormones is not able, however, to interrupt the underlining immunological mechanism. Importantly, novel immunotherapies are exploited to protect and rescue the remaining hormone producing cells. Among probable targets of immunotherapy, the C1858T mutation in the PTPN22 gene, which encodes for the lymphoid tyrosine phosphatase (Lyp) variant R620W, reveals an autoimmunity related pathophysiological role. Our scope was to establish new C1858T PTPN22 siRNA duplexes delivered by liposomal carriers (lipoplexes) to patients' PBMC. Following lipoplexes treatment, CD3+ and CD3- immunotypes were efficiently transfected; cell integrity and viability were preserved. Specific target mRNA down-modulation was observed. After T cell receptor stimulation, in lipoplexes-treated PBMC Lyp function was restored by increased release of IL-2 in cultures. Results set-up the stage for ultimate trials in the treatment of autoimmunity based on the specific inhibitory targeting of C1858T PTPN22 by lipoplexes.

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1β secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants., SCOPUS: ar.j, info:eu-repo/semantics/published

Delivery of genes, including plasmid DNAs, short interfering RNAs (siRNAs), and messenger RNAs (mRNAs), using artificial non-viral nanotherapeutics is a promising approach in cancer gene therapy. However, multiple physiological barriers upon systemic administration remain a key challenge in clinical translation of anti-cancer gene therapeutics. Besides extracellular barriers including sequestration of gene delivery nanoparticles from the bloodstream by resident organ-specific macrophages, and their poor extravasation and tissue penetration in tumors, overcoming intracellular barriers is also necessary for successful delivery of nucleic acids. Whereas for RNA delivery the endosomal barrier holds a key importance, transfer of DNA cargo additionally requires translocation into the nucleus. Better understanding of crossing membrane barriers by nucleic acid nanoformulations is essential to the improvement of current non-viral carriers. This review aims to summarize relevant literature on intracellular trafficking of non-viral nanoparticles and determine key factors toward surmounting intracellular barriers. Moreover, recent data allowed us to propose new interpretations of current hypotheses of endosomal escape mechanisms of nucleic acid nanoformulations.

New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25&ndash, 80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.

Therapeutics based on small interfering RNA (siRNA) have promising potential as antiviral and anti-inflammatory agents. To deliver siRNA across cell membranes to reach the RNAi pathway in the cytosol of target cells, non-viral nanoparticulate delivery approaches are explored. Recently, we showed that encapsulation of siRNA in lipid-polymer hybrid nanoparticles (LPNs), based on poly(DL-lactic-co-glycolic acid) (PLGA) and cationic lipid-like materials (lipidoids), remarkably enhances intracellular delivery of siRNA as compared to siRNA delivery with LPNs modified with dioleoyltrimethylammoniumpropane (DOTAP) as the lipid component. However, the potential immune modulation by these cationic lipids remains unexplored. By testing lipidoids and DOTAP for innate immune-receptor-activating properties in vitro, we found that neither lipidoids nor DOTAP activate human Toll-like receptor (TLR) 2, 3, 7, and 9. However, in contrast to DOTAP, lipidoids are strong agonists for TLR4 and activate murine antigen-presenting cells in vitro. This agonistic effect was further confirmed in silico using a prediction model based on crystal structures. Also, lipidoids formulated as lipoplexes or as stable nucleic acid lipid particles, which was the reference formulation for siRNA delivery, proved to activate TLR4. However, by combining lipidoids with PLGA into LPNs, TLR4 activation was abrogated. Thus, lipidoid-mediated TLR4 activation during siRNA delivery may be modulated via optimization of the formulation design., Graphical Abstract

The need for synthetic gene therapy or gene silencing vehicles that can insert therapeutic nucleic acids (DNA or siRNA) into cells (so-called transfection) has focused interest on lipid-nucleic acid assemblies (lipoplexes). This paper reviews the kinetics pathways leading to lipoplex formation and structure. The process is qualitatively comparable to those of cluster nucleation and growth and to the adsorption of polyelectrolytes on colloidal particles: Initially is a rapid stage where the nucleic acid binds onto the surface of the cationic lipid aggregate (adsorption, or nucleation). This is followed by an intermediate step where the lipid/nucleic acid complexes flocculate to form larger structures (growth). The last and final step involves internal rearrangement, where the overall global structure remains constant while local adjustment of the nucleic acid/lipid organization takes place until the equilibrium lipoplex characteristics are obtained. This step can require unusually long time scales of order hours or longer. Understanding the kinetics of lipoplex formation is not only of fundamental interest as a multi-component, multi-length scale and multi-time scale process, but also has significant implications for the utilization of lipoplexes as carriers for gene delivery and gene silencing agents.

A promising strategy to design safer and more effective cationic lipids for gene delivery with inherent antibacterial properties is to covalently tether a lipophilic moiety with oligomeric aminoglycosides (AGs), a large family of Gram-negative-active antibiotics. Herein, we reported the development of a new class of multicationic-head AG-based amphiphiles built on the tetramino-tetrahexyloxycalix[4]arene (4A4Hex-calix-calix[4]) scaffold. Three different conjugates, namely 4A4Hex-calix-calix[4]-neomycin, -neamine, and -paromomycin, were synthesized and characterized. Due to the inherent multivalency of AGs and the amphiphilic behaviour, every 4A4Hex-calix-calix[4]-AG exhibited greater DNA binding ability than the gold standard transfectant 25 kDa bPEI and striking DNA packing ability. DNA/4A4Hex-calix-calix[4]-AG complexes at charge ratios (CRs, +/−) used for transfections displayed good colloidal stability, with a hydrodynamic diameters of ≈150 nm and an overall surface charges of ≈+30 mV. DNA/4A4Hex-calix[4]-AGs nanoassemblies, everyone tested at the optimal CR, invariably showed good transfection efficiency in two cell lines, along with low-to-negligible cytotoxicity. Besides, DNA/4A4Hex-calix-calix[4]-AG complexes exhibited appreciable antimicrobial activity against Gram-negative bacteria, even greater than uncomplexed 4A4Hex-calix-calix[4]-AGs. Altogether, these results disclose 4A4Hex-calix[4]-AGs as promising gene delivery tools with unique antibacterial properties.

Recent advances in biochemical and biophysical research have been achieved through the employment of microfluidic devices. Microfluidic mixing of therapeutic agents with biomaterials yields systems with finely tuned physical-chemical properties for applications in drug and gene delivery. Here, we investigate the role of preparation technology (microfluidic mixing vs. bulk self-assembly) on the transfection efficiency (TE) and cytotoxicity of multicomponent cationic liposome/DNA complexes (lipoplexes) in live Chinese hamster ovarian (CHO) cells. Decoupling TE and cytotoxicity allowed us to combine them in a unique coherent vision. While bulk self-assembly produces highly efficient and highly toxic MC lipoplexes, microfluidics manufacture leads to less efficient, but less cytotoxic complexes. This discrepancy is ascribed to two main factors controlling lipid-mediated cell transfection, i.e. the lipoplex concentration at the cell surface and the lipoplex arrangement at the nanoscale. Further research is required to optimize microfluidic manufacturing of lipoplexes to obtain highly efficient and not cytotoxic gene delivery systems.

Peritoneal carcinomatosis is a severe form of cancer in the abdomen, currently treated with cytoreductive surgery and intravenous chemotherapy. Recently, nebulization has been proposed as a less invasive strategy for the local delivery of chemotherapeutic drugs. Also, RNA interference has been considered as a potential therapeutic approach for treatment of cancer. In this study, Lipofectamine RNAiMAX/siRNA complexes and cyclodextrin/siRNA complexes are evaluated before and after nebulization. Nebulization of the siRNA complexes does not significantly lower transfection efficiency when compared to non-nebulized complexes. After incubation in ascites fluid, however, the cyclodextrin/siRNA complexes show a drastic decrease in transfection efficiency. For the Lipofectamine RNAiMAX/siRNA complexes, this decrease is less pronounced. It is concluded that nebulization is an interesting technique to distribute siRNA complexes into the peritoneal cavity, providing the complexes are stable in ascites fluid which might be present in the peritoneal cavity.

Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the uncoated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration.

Topical vaginal sustained delivery of siRNA presents a significant challenge due to the short residence time of formulations. Therefore, a drug delivery system capable to adhere to the vaginal mucosa is desirable, as it could allow a prolonged delivery and increase the effectiveness of the therapy. The aim of this project is to develop a polymeric solid mucoadhesive system, loaded with lipoplexes, able to be progressively rehydrated by the vaginal fluids to form a hydrogel and to deliver siRNA to vaginal tissues. To minimize adhesive interactions with vaginal mucus components, lipoplexes were coated with different derivatives of polyethylene glycol: DPSE-PEG2000, DPSE-PEG750 and ceramide-PEG2000. Based on stability and diffusion properties in simulated vaginal fluids, lipoplexes containing DSPE-PEG2000 were selected and incorporated in hydroxyethyl cellulose (HEC) hydrogels. Solid systems, called sponges, were then obtained by freeze-drying. Sponges meet acceptable mechanical characteristics and their hardness, deformability and mucoadhesive properties are not influenced by the presence of lipoplexes. Finally, mobility and stability of lipoplexes inside sponges rehydrated with vaginal mucus, mimicking in situ conditions, were evaluated by advanced fluorescence microscopy. The release rate was found to be influenced by the HEC concentration and consequently by the viscosity after rehydration. This study demonstrates the feasibility of entrapping pegylated lipoplexes into a solid matrix system for a prolonged delivery of siRNA into the vagina.

International audience; Several siRNA (small interfering RNA) therapeutics are undergoing clinical trials for cancer, respiratory diseases or macular degeneration, but most are administrated locally. In order to overcome the different barriers to attain an efficient siRNA action after systemic administration, nanocarriers able to carry and protect siRNA are awaited. With this aim, we developed a new platform of siRNA lipid nanocapsules (LNCs) using different cationic lipids, combining the properties of LNCs (siRNA protection and targeting) and lipoplexes (efficient siRNA delivery into the cell). The formulation was revealed to contain different compartments. A siRNA quantification method based on UV spectroscopy was developed to locate and quantify siRNA in each compartment. All in all, these novel siRNA LNCs presented sizes of about 55nm with a neutral surface charge and siRNA encapsulation efficiencies up to 65% representing appropriate characteristics for systemic administration.

The concept that the effective unit of interest in the cell-nanomaterial interaction is the particle and its corona of associated proteins is emerging. Here we investigate the compositional evolution of the protein corona of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) cationic liposomes (CLs) and DOTAP/DNA lipoplexes over a wide range of plasma concentrations (2.5-80%). The composition of the hard corona of lipoplexes is quite stable, but that of CLs does evolve considerably. We show that the protein corona of CLs is made of both low-affinity and competitive-binding proteins whose relative abundance changes with the plasma concentration. This result may have deep biological implications for the application of lipid-based gene vectors both in vitro and in vivo.

Gene silencing by siRNA (synthetic dsRNA of 21-25 nucleotides) is a well established biological tool in gene expression studies and has a promising therapeutic potential for difficult-to-treat diseases. Five fatty acids of various chain length and oxidation state (C12:0, C18:0, C18:1, C18:2, C22:1) were conjugated to the naturally occurring polyamine, spermine, and evaluated for siRNA delivery and gene knock-down. siRNA delivery could not be related directly to gene silencing efficiency as N4,N9-dierucoyl spermine resulted in higher siRNA delivery compared to N4,N9-dioleoyl spermine. GFP silencing in HeLa cells showed that the unsaturated fatty acid amides are more efficient than saturated fatty acid amides, with N4,N9-dioleoyl spermine resulting in the most efficient gene silencing in the presence of serum. The alamarBlue cell viability assay showed that fatty acid amides of spermine have good viability (75%–85% compared to control) except N4,N9-dilauroyl spermine which resulted in low cell viability. These results prove that unsaturated fatty acid amides of spermine are efficient, non-toxic, non-viral vectors for siRNA mediated gene silencing.

The interactions of nucleic acids with lipid membranes are of great importance for biological mechanisms as well as for biotechnological applications in gene delivery and drug carriers. The optimization of liposomal vectors for clinical use is absolutely dependent upon the formation mechanisms, the morphology, and the molecular organization of the lipoplexes, that is, the complexes of lipid membranes with DNA. Differential scanning calorimetry (DSC) has emerged as an efficient and relatively easy-to-operate experimental technique that can straightforwardly provide data related to the thermodynamics and the kinetics of the DNA—lipid complexation and especially to the lipid organization and phase transitions within the membrane. In this review, we summarize DSC studies considering nucleic acid—membrane systems, accentuating DSC capabilities, and data analysis. Published work involving cationic, anionic, and zwitterionic lipids as well as lipid mixtures interacting with RNA and DNA of different sizes and conformations are included. It is shown that despite limitations, issues such as DNA- or RNA-induced phase separation and microdomain lipid segregation, liposomal aggregation and fusion, alterations of the lipid long-range molecular order, as well as membrane-induced structural changes of the nucleic acids can be efficiently treated by systematic high-sensitivity DSC studies.

Purpose Tumor gene expression after the intravenous (i.v.) administration of current polymer-based gene delivery systems is generally low and short-lived. Immune stimulatory CpG dinucleotides, present within the plasmid DNA of the polyplexes are likely to contribute to this. The effect of CpG replacement on the levels of transgene expression was studied, after the i.v. administration of polyethylenimine (PEI) polyplexes. Methods Tumor transfection and immune stimulation of PEI polyplexes containing plasmid DNA encoding for luciferase and rich in CpG motifs was monitored and compared to polyplexes containing the same gene but devoid of CpG motifs. Lipoplexes based on 1,2-dioleyl-3-trimethylammonium-propane/dioleoylphosphatidylethanolamine liposomes were included as a control. Results The replacement of CpGrich DNA by CpGfree DNA did neither affect the physical properties of the DNA complexes nor did it affect their in vitro transfection activity or cytotoxicity. The immune stimulation (interleukin-12) after i.v. administration of the PEI DNA complexes was low and unaffected by the presence of CpG motifs. The absence of CpG motifs within the different DNA complexes improved the degree and the duration of organ and tumor gene expression. Conclusion The depletion of CpG dinucleotides within the plasmid DNA of polyplexes enhances the degree and duration of in vivo transgene expression.

International audience; Even though RNA interference is a highly specific technique to selectively silence the expression of any gene, the delivery of RNAi molecules remains a challenge. Recently, we developed a clinically acceptable efficient formulation of siRNA. This delivery system consisted of the cationic lipid 2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methyl-acetamide, or DMAPAP, (ii) the neutral lipid 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine or DOPE and (iii) an anionic polymer that enhances lipoplexes efficiency. We show here that this enhancement of efficiency is due to higher stability of polymer containing-siRNA lipoplexes, leading to longer retention of siRNA integrity. We assayed siRNA integrity upon incubation of siRNA lipoplexes in various biological media using electrophoresis and Fluorescence Correlation Spectroscopy. We show that the addition of anionic polymer provided enhanced stability of incorporated siRNA mainly in saline buffer at room temperature and in human serum at 37 degrees C and did not interfere with the release of siRNA from lipoplexes in modeled cellular media. (C) 2015 Elsevier B.V. All rights reserved.

Two double-tailed pyridinium cationic amphiphiles, differing only in the degree of unsaturation of the alkyl chains, have been selected for a detailed study of their aggregation behavior, under conditions employed for transfection experiments. The transfection efficiencies of the two molecules are remarkably different, especially when combined with 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine ( DOPE) as helper lipid. The phase behavior of the cationic amphiphile/ DOPE mixtures have been studied using P-31- and H-2-NMR ( on deuterated cationic amphiphiles) as main techniques, to monitor independently the behavior of the two components. In water, the lamellar organization is dominant for both the surfactants in their mixtures with the helper lipid. In HEPES saline buffer (HBS), the mixtures of the unsaturated surfactant form inverted phases and, in particular, stable H-II phases for DOPE contents greater than or equal to 30 mol %. By contrast, the saturated surfactant does not form homogeneously mixed inverted phases in mixtures with DOPE at room temperature. However, mixed inverted phases are observed for this system at higher temperatures and, after mixing has been achieved by heating, the metastable mixed phases remain present for several hours at 5 degreesC. At 35 degreesC the dominant phase is the cubic phase. The lipoplex composed of equimolar mixtures of the unsaturated surfactant with DOPE and plasmid DNA was found to be organized in highly curved bilayers.

Dioctadecyldimethylammoniumbromide (DODAB):Monoolein (MO) lipoplexes have mainly been studied within the range of high molar ratios of DODAB, with noticeable transfection efficiencies in the Human Embryonic Kidney (HEK, a.k.a. 293T) cell line. In thiswork,we intend to study the effect of highMOcontent on the structure and physicochemical properties of pDNA/DODAB:MOlipoplexes to achieve some correlationwith their transfection efficiency. Static/Dynamic Light Scattering and Cryo-TEM imaging were used to characterize the size/ morphology of DNA/DODAB:MO lipoplexes at different DODAB:MO contents (2:1, 1:1, 1:2) and charge ratios (CRs) (+/−). Nile Red fluorescence emission was performed to detect changes in microviscosity, hydration and polarity of DNA/DODAB:MO systems. Lipoplexes stability at physiological pH values and in the presence of anionic lipids was evaluated by Förster Resonance Energy Transfer (FRET). Physicochemical/structural data were complemented with transfection studies in HEK cells using the β-galactosidase reporter gene activity assay. This work reports the coexistence of multilamellar and non-lamellar inverted phases in MO-richer lipoplexes (DODAB:MO 1:2 and 1:4), leading to transfection efficiencies comparable to those of multilamellar (DODAB-richer) lipoplexes, but at higher charge ratios [CR (+/−) = 6.0] and without dose-effect response. These results may be related to the structural changes of lipoplexes promoted by high MO content., FEDER (037291) through POFC — COMPETE, Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/46968/2009 (PhD grant), PEst-C/BIA/UI4050/2011(CBMA), PEst-C/FIS/UI0607/2011 (CFUM).

Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of helper lipids on the transfection efficiency with cationic liposomes, the effect of the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of similar to 150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000 (R) (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1-2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective 'packaging' of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

Bleomycin is a chemotherapeutic agent barely diffusible through the plasmatic membrane. We evaluated DNA/cationic lipids complexes (lipoplexes) as mediators of its uptake in four spontaneous canine melanoma derived cell lines (Ak, Bk, Br and Rkb). Cell survival after lipofection plus or minus bleomycin was determined by the acid phosphatase method and the cellular uptake of lipoplexes, carrying the E. coli β-galactosidase gene, was evidenced by SYBR Green I staining. The four cell lines resulted sensitive to the bleomycin/lipoplexes system in both spatial configurations. Survival rates values were lower than 20% in monolayers of the four tested lines and lower than 30% in three lines (Ak, Bk and Rkb) when grown as spheroids. The sensitization to bleomycin depended on lipoplexes in Ak and Rkb while Bk (in both spatial configurations) and Br (as monolayers) were sensitive to bleomycin alone. Although some degree of sensitivity to bleomycin was induced by cationic lipids alone in Ak and Rkb monolayers, the maximal bleomycin effects appeared in the presence of lipoplexes. The sensitization was independent of transcriptional activity. The co-administration of lipoplexes diminished bleomycin IC50: 10-fold in Ak and Rkb monolayers; and sensitized the Ak and Rkb resistant spheroids. The bleomycin cytotoxic effects depended on lipoplexes concentration and diminished when cells were incubated at 8 °C. Our results suggest that lipoplexes sensitize cells to bleomycin, increasing its uptake by an active transport mechanism, such as endocytosis. The bleomycin/lipoplexes system appears as a promising combination of chemotherapy and non-viral cancer gene therapy. Fil: Gil Cardeza, Maria Lourdes. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rossi, Ursula Amaranta. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Villaverde, Marcela Solange. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Glikin, Gerardo Claudio. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Finocchiaro, Liliana Maria Elena. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Dr. Ángel Roffo". Unidad de Transferencia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

© 2013 IOP Publishing Ltd. Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol-DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm2s-1). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity v ≈ 0.03 μm2s-1and random motion. The method permitted us to generate an intracellular transport map showing several regions of concerted motion of lipoplexes.

International audience; Amphiphilic block copolymers are molecules composed of hydrophilic and hydrophobic segments having the capacity to spontaneously self-assemble into a variety of supramolecular structures like micelles and vesicles. Here, we propose an original way to self-assemble amphiphilic block copolymers into a supported bilayer membrane for defined coating of nanoparticles. The heart of the method rests on a change of the amphiphilicity of the copolymer that can be turned off and on by varying the polarity of the solvent. In this condition, the assembly process can take advantage of specific molecular interactions in both organic solvent and water. While the concept potentially could be applied to any type of charged substrates, we focus our interest on the design of a new type of polymer assembly mimicking the virus morphology. A capsid-like shell of glycoprotein-mimic amphiphilic block copolymer was self-assembled around a positively charged complex of siRNA and polyethyleneimine. The process requires two steps. Block copolymers first interact with the complexes dispersed in DMSO through electrostatic interactions. Next, the increase of the water content in the medium triggers the hydrophobic effect and the concomitant self-assembly of free block copolymer molecules into a bilayer membrane at the complex surface. The higher gene silencing activity of the copolymer-modified complexes over the complexes alone shows the potential of this new type of nanoconstructs for biological applications, especially for the delivery of therapeutic biomolecules.

The association behavior of different nucleic acids with cationic liposomes has been monitored, in order to find out how the polymer length, the type of base and the charge density affect the lipoplex formation. In particular the associative features displayed by the homopolymer 20-mer of adenine, Oligo (dA), of timine, Oligo (dT), and of guanine, Oligo (dG), were compared to understand the role of the base. The effects of the nucleic acid length and of the charge density were evaluated taking account of the association of the polyadenylic acid and of the DNA onto the liposomes. The results show that the homopolymer Oligo (dG) is able to interact with the cationic liposomes to the same extent as DNA, in spite of the fact that Oligo (dG) is a short polymer made of 20 residues and DNA is a longer and dual strand polymer having a higher charge density.

International audience; The mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand.

Most lipid formulations require cholesterol for successful transfection, but the precise reason remains to be more clearly understood. Here, we have studied the effect of cholesterol on the transfection efficiency (TE) of lipoplexes in vitro. Addition of cholesterol to highly effective DC-Chol-DOPE/DNA lipoplexes increases TE, with 40 mol% cholesterol yielding about 10-fold improvement. The transfection mechanisms of cholesterol-containing lipoplexes have been investigated by combining dynamic light scattering, synchrotron small angle X-ray scattering, laser scanning confocal microscopy and transfection efficiency measurements. Our results revealed that cholesterol-containing lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for efficient endosomal escape. We also found evidence that formulations with high cholesterol content are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency. © 2012 Elsevier B.V. All rights reserved.

Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments. © 2011 American Chemical Society.

Gene-based therapeutic approaches are based upon the concept that, if a disease is caused by a mutation in a gene, then adding back the wild-type gene should restore regular function and attenuate the disease phenotype. To deliver the gene of interest, both viral and nonviral vectors are used. Viruses are efficient, but their application is impeded by detrimental side-effects. Among nonviral vectors, cationic liposomes are the most promising candidates for gene delivery. They form stable complexes with polyanionic DNA (lipoplexes). Despite several advantages over viral vectors, the transfection efficiency (TE) of lipoplexes is too low compared with those of engineered viral vectors. This is due to lack of knowledge about the interactions between complexes and cellular components. Rational design of efficient lipoplexes therefore requires deeper comprehension of the interactions between the vector and the DNA as well as the cellular pathways and mechanisms involved. The importance of the lipoplex structure in biological function is revealed in the application of synchrotron small-angle X-ray scattering in combination with functional TE measurements. According to current understanding, the structure of lipoplexes can change upon interaction with cellular membranes and such changes affect the delivery efficiency. Recently, a correlation between the mechanism of gene release from complexes, the structure, and the physical and chemical parameters of the complexes has been established. Studies aimed at correlating structure and activity of lipoplexes are reviewed herein. This is a fundamental step towards rational design of highly efficient lipid gene vectors.

Three-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome-DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A 3, which was close to unity, denoting the preponderant occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (latrunculin B and nocodazole, respectively). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin microfilaments. Relevance of the results for enhanced gene delivery is discussed. © The Royal Society of Chemistry 2012.

The interactions between cationic liposomes doped with the anionic nucleolipid 1,2-dipalmitoyl-sn-glycero-3-cytidine diphosphate (DP-Cyt) and deoxyribonucleic acid (DNA) were investigated. Toward this goal, new liposomal and lipoplex formulations characterized by the presence of the anionic amphiphile DP-Cyt were proposed. The effects of incorporation of the cytosine functionalized lipid DP-Cyt into the cationic bilayers were analyzed by means of electrophoretic mobility, dynamic light scattering (DLS) and fluorescence spectroscopy techniques. These approaches allowed us to follow the DNA condensation process and to identify specific electrokinetic characteristics of liposome and DNA-liposome complexes formation. Specifically, DP-Cyt liposomes and DNA were shown to form electrically stable or unstable complexes depending on the charge ratio between the phosphate group of DNA and the cationic lipid. Remarkably, a prominent role for DP-Cyt in enhancing the DNA binding capacity on liposomes was demonstrated. Zeta potential experiments performed on systems with different liposomes/DNA ratio showed that the value of the charge neutralization point is a function of the content of the incorporated DP-Cyt. As a whole, our data demonstrate that the association of cationic DP-Cyt doped liposomes with DNA is driven by both electrostatic interaction and additional specific interactions at the polar head level based on the cytidine nucleobase.

Complexes formed by DNA and lipid mixtures have great interest for the assessment of self-assembling mechanisms in open biological systems. X-ray diffraction has revealed that minor alterations of the cationic/neutral lipid composition produced major alterations to the liquid crystalline structure and the hydration of the complexes. We have extended to these systems by an approach based on the identification of a fundamental repeating unit that grows according to the general principles of supramolecular polymerisation and liquid crystallinity. Structural reorganisations that optimise the electrostatic and hydrophobic compensation are enhanced by competition between the rigidity of the polyelectrolyte and the cohesion of the lipid assembly. The non-hydrated hexagonal structure revealed by X-ray examination is represented by a dendritic-type supramolecular polymerisation of DNA units decorated by the aliphatic tails of dissociated liposomes. An increase in the cationic/neutral lipid component ratio enhances the stability of planar bilayers, favouring the formation of the partly hydrated lamellar structure revealed by X-ray diffraction

A shotgun proteomics approach was used to compare human plasma protein binding capability with cationic liposomes, DNA–cationic lipid complexes (lipoplexes), and lipid–polycation–DNA (LPD) complexes. Nano-high-performance liquid chromatography coupled with a high-resolution LTQ Orbitrap XL mass spectrometer was used to characterize and compare their protein corona. Spectral counting and area under curve methods were used to perform label-free quantification. Substantial qualitative and quantitative differences were found among proteins bound to the three different systems investigated. Protein variety found on lipoplexes and LPD complexes was richer than that found on cationic liposomes. There were also significant differences between the amounts of protein. Such results could help in the design of gene-delivery systems, because some proteins could be more selectively bound rather than others, and their bio-distribution could be driven in vivo for more efficient and effective gene therapy.

The 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. Fil: Muñoz Úbeda, Mónica. Universidad Complutense de Madrid. Facultad de Ciencias Químicas; España Fil: Misra, Santosh K.. Indian Institute of Science; India Fil: Barrán Berdón, Ana L.. Universidad Complutense de Madrid. Facultad de Ciencias Químicas; España Fil: Aicart Ramos, Clara. Universidad Complutense de Madrid. Facultad de Ciencias Químicas; España Fil: Sierra, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentina Fil: Biswas, Joydeep. Indian Institute of Science; India Fil: Kondaiah, Paturu. Indian Institute of Science; India Fil: Junquera, Elena. Universidad Complutense de Madrid. Facultad de Ciencias Químicas; España Fil: Bhattacharya, Santanu. Indian Institute of Science; India Fil: Aicart, Emilio. Universidad Complutense de Madrid. Facultad de Ciencias Químicas; España

Four guanidine derivatives of N4, N9-diacylated spermine have been designed, synthesized, and characterized. These guanidine-containing cationic lipids bound siRNA and formed nanoparticles. Two cationic lipids with C18 unsaturated chains, N1,N12-diamidino-N4, N9-dioleoylspermine and N1,N12-diamidino-N4-linoleoyl-N9-oleoylspermine, were more efficient in terms of GFP expression reduction compared to the other cationic lipids with shorter C12 (12:0) and very long C22 (22:1) chains. N1,N12-Diamidino-N4-linoleoyl-N9-oleoylspermine siRNA lipoplexes resulted in GFP reduction (26%) in the presence of serum, and cell viability (64%). These data are comparable to those obtained with TransIT TKO. Thus, cationic lipid guanidines based on N4, N9-diacylated spermines are good candidates for non-viral delivery of siRNA to HeLa cells using self-assembled lipoplexes.

International audience; Non-viral gene therapy requires innovative strategies to achieve higher transfection efficacy. A few years ago, our group proposed bioinspired lipids whose interaction with DNA was not based on ionic interactions, but on hydrogen bonds. We thus developed lipids bearing a thiourea head which allowed an interaction with DNA phosphates through hydrogen bonds. After a proof of concept with a lipid bearing three thiourea functions, a molecular and cellular screening was performed by varying all parts of the lipids: the hydrophobic anchor, the spacer, the linker, and the thiourea head. Two lipothiourea-based structures were identified as highly efficient in vitro transfecting agents. The lipothioureas were shown to reduce non specific interactions with cell membranes and deliver their DNA content intracellularly more efficiently, as compared to cationic lipoplexes. These lipids could deliver siRNA efficiently and allowed specific cell targeting in vitro. In vivo, thiourea lipoplexes presented a longer retention time in the blood and less accumulation in the lungs after an intravenous injection in mice. They also induced luciferase gene expression in muscle and tumor after local administration in mice. Therefore, these novel lipoplexes represent an excellent alternative to cationic lipoplexes as transfecting agents. In this review we will focus on the structure activity studies that permitted the identification of the two most efficient thiourea lipids.

In this work unilamellar liposomes doped with a novel non-ionic 5'-Uridine-head nucleolipid, Lauroyl Uridine (LU), were prepared and characterized for their ability to interact with the polynucleotide polyadenylic acid (poly-A). Vesicles, were made up of the cationic lipid DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), the zwitterionic lipid DOPE (1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine), and the novel amphiphile Lauroyl Uridine. The influence of the non-ionic nucleolipid on essential liposomes properties, such as the structure and net charge was first investigated by a comparative analysis performed on the different lipoplex preparations by means of ζ-potential and size measurements. Both structure and net charge of liposomes were shown to be not modified by the presence of the non-ionic nucleolipid. The role of the synthetic lipid inserted as anchor in the liposome bilayer in the condensation process between vesicles and the polynucleotide poly-A was then analyzed by UV-vis, Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The data presented comparative UV-vis analyses that evidenced the occurrence of staking interactions in the poly-A only in LU containing lipoplexes. CD and NMR studies indicated the presence of H-bonding interaction between Lauroyl Uridine containing vesicles and the polynucleotide poly-A. The results presented in this work support a role for Lauroyl Uridine in A-U molecular recognition, thus, suggesting that cationic liposomes doped with the non-ionic nucleolipid Lauroyl Uridine could represent a model system to study molecular interactions among single stranded polynucleotides and lipid anchor bearing the complementary bases.

The first interaction between lipoplexes and cells is charge-mediated and not specific. Endocytosis is considered to be the main pathway for lipoplex entry. Upon interaction between lipoplexes and the plasma membrane, intermixing between lipoplex and membrane lipids is necessary for efficient endocytosis. Here we study the mechanism of the different endocytic pathways in lipid-mediated gene delivery. We show that DC-Chol-DOPE/DNA lipoplexes preferentially use a raft-mediated endocytosis, while DOTAP-DOPC/DNA systems are mainly internalized by not specific fluid phase macropinocitosys. On the other hand, most efficient multicomponent lipoplexes, incorporating different lipid species in their lipid bilayer, can use multiple endocytic pathways to enter cells. Our data demonstrate that efficiency of endocytosis is regulated by shape coupling between lipoplex and membrane lipids. We suggest that such a shape-dependent coupling regulates efficient formation of endocytic vesicles thus determining the success of internalization. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.