Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations.

A robust strategy is reported to build perfectly monodisperse star polycations combining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) central core onto which oligoethyleneimine radial arms are installed. The architectural perfection of the compounds is demonstrated by a variety of physicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key to the strategy is the possibility of customizing the cavity size of the macrocyclic platform to enable/prevent the inclusion of adamantane motifs. These properties can be taken into advantage to implement sequential levels of stimuli responsiveness by combining computational design, precision chemistry and programmed host-guest interactions. Specifically, it is shown that supramolecular dimers implying a trimeric CT-tetraethyleneimine star polycation and purposely designed bis-adamantane guests are preorganized to efficiently complex plasmid DNA (pDNA) into transfection-competent nanocomplexes. The stability of the dimer species is responsive to the protonation state of the cationic clusters, resulting in dissociation at acidic pH. This process facilitates endosomal escape, but reassembling can take place in the cytosol then handicapping pDNA nuclear import. By equipping the ditopic guest with a redox-sensitive disulfide group, recapturing phenomena are prevented, resulting in drastically improved transfection efficiencies both in vivo and in vitro.

Trifaceted Mickey Mouse Amphiphiles for Programmable Self-Assembly, DNA Complexation and Organ-Selective Gene Delivery.

Instilling segregated cationic and lipophilic domains with an angular disposition in a trehalose-based trifaceted macrocyclic scaffold allows engineering patchy molecular nanoparticles leveraging directional interactions that emulate those controlling self-assembling processes in viral capsids. The resulting trilobular amphiphilic derivatives, featuring a Mickey Mouse architecture, can electrostatically interact with plasmid DNA (pDNA) and further engage in hydrophobic contacts to promote condensation into transfectious nanocomplexes. Notably, the topology and internal structure of the cyclooligosaccharide/pDNA co-assemblies can be molded by fine-tuning the valency and characteristics of the cationic and lipophilic patches, which strongly impacts the transfection efficacy in vitro and in vivo. Outstanding organ selectivities can then be programmed with no need of incorporating a biorecognizable motif in the formulation. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes by making cyclooligosaccharide patchiness the focus.

Tuning the Topological Landscape of DNA-Cyclodextrin Nanocomplexes by Molecular Design.

Original molecular vectors that ensure broad flexibility to tune the shape and surface properties of plasmid DNA (pDNA) condensates are reported herein. The prototypic design involves a cyclodextrin (CD) platform bearing a polycationic cluster at the primary face and a doubly linked aromatic module bridging two consecutive monosaccharide units at the secondary face that behaves as a topology-encoding element. Subtle differences at the molecular level then translate into disparate morphologies at the nanoscale, including rods, worms, toroids, globules, ellipsoids, and spheroids. In vitro evaluation of the transfection capabilities revealed marked selectivity differences as a function of nanocomplex morphology. Remarkably high transfection efficiencies were associated with ellipsoidal or spherical shapes with a lamellar internal arrangement of pDNA chains and CD bilayers. Computational studies support that the stability of such supramolecular edifices is directly related to the tendency of the molecular vector to form noncovalent dimers upon DNA templating. Because the stability of the dimers depends on the protonation state of the polycationic clusters, the coaggregates display pH responsiveness, which facilitates endosomal escape and timely DNA release, a key step in successful transfection. The results provide a versatile strategy for the construction of fully synthetic and perfectly monodisperse nonviral gene delivery systems uniquely suited for optimization schemes.

Click Synthesis of Size- and Shape-Tunable Star Polymers with Functional Macrocyclic Cores for Synergistic DNA Complexation and Delivery.

The architectural perfection and multivalency of dendrimers have made them useful for biodelivery via peripheral functionalization and the adjustment of dendrimer generations. Modulation of the core-forming and internal matrix-forming structures offers virtually unlimited opportunities for further optimization, but only in a few cases this has been made compatible with strict diastereomeric purity over molecularly diverse series, low toxicity, and limited synthetic effort. Fully regular star polymers built on biocompatible macrocyclic platforms, such as hyperbranched cyclodextrins, offer advantages in terms of facile synthesis and flexible compositions, but core elaboration in terms of shape and function becomes problematic. Here we report the synthesis and characterization of star polymers consisting of functional trehalose-based macrocyclic cores (cyclotrehalans, CTs) and aminothiourea dendron arms, which can be efficiently synthesized from sequential click reactions of orthogonal monomers, display no cytotoxicity, and efficiently complex and deliver plasmid DNA in vitro and in vivo. When compared with some commercial cationic dendrimers or polymers, the new CT-scaffolded star polymers show better transfection efficiencies in several cell lines and structure-dependent cell selectivity patterns. Notably, the CT core could be predefined to exert Zn(II) complexing or molecular inclusion capabilities, which has been exploited to synergistically boost cell transfection by orders of magnitude and modulate the organ tropism in vivo.

Plasmid-Templated Control of DNA-Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery.

Engineering self-assembled superstructures through complexation of plasmid DNA (pDNA) and single-isomer nanometric size macromolecules (molecular nanoparticles) is a promising strategy for gene delivery. Notably, the functionality and overall architecture of the vector can be precisely molded at the atomic level by chemical tailoring, thereby enabling unprecedented opportunities for structure/self-assembling/pDNA delivery relationship studies. Beyond this notion, by judiciously preorganizing the functional elements in cyclodextrin (CD)-based molecular nanoparticles through covalent dimerization, here we demonstrate that the morphology of the resulting nanocomplexes (CDplexes) can be tuned, from spherical to ellipsoidal, rod-type, or worm-like nanoparticles, which makes it possible to gain understanding of their shape-dependent transfection properties. The experimental findings are in agreement with a shift from chelate to cross-linking interactions on going from primary-face- to secondary-face-linked CD dimers, the pDNA partner acting as an active payload and as a template. Most interestingly, the transfection efficiency in different cells was shown to be differently impacted by modifications of the CDplex morphology, which has led to the identification of an optimal prototype for tissue-selective DNA delivery to the spleen in vivo.

Host-Guest-Mediated DNA Templation of Polycationic Supramolecules for Hierarchical Nanocondensation and the Delivery of Gene Material.

Only a few examples of monodisperse molecular entities that can compact exogenous nucleic acids into nanocomplexes, protect the cargo from the biological environment, facilitate cell internalization, and promote safe transfection have been reported up to date. Although these species open new venues for fundamental studies on the structural requirements that govern the intervening processes and their application in nonviral gene-vector design, the synthesis of these moieties generally requires a relatively sophisticated chemistry, which hampers further development in gene therapy. Herein, we report an original strategy for the reversible complexation and delivery of DNA based on the supramolecular preorganization of a ß-cyclodextrin-scaffolded polycationic cluster facilitated by bisadamantane guests. The resulting gemini-type, dual-cluster supramolecules can then undergo DNA-templated self-assembly at neutral pH value by bridging parallel DNA oligonucleotide fragments. This hierarchical DNA condensation mechanism affords transfectious nanoparticles with buffering capabilities, thus facilitating endosomal escape following cell internalization. Protonation also destabilizes the supramolecular dimers and consequently the whole supramolecular edifice, thus assisting DNA release. Our advanced hypotheses are supported by isothermal titration calorimetry, NMR and circular dichroism spectroscopic analysis, gel electrophoresis, dynamic light scattering, TEM, molecular mechanics, molecular dynamics, and transfection studies conducted in vitro and in vivo.

Trehalose-polyamine/DNA nanocomplexes: impact of vector architecture on cell and organ transfection selectivity.

A novel family of precision-engineered gene vectors with well-defined structures built on trehalose and trehalose-based macrocycles (cyclotrehalans) comprising linear or cyclic polyamine heads have been synthesized through procedures that exploit click chemistry reactions. The strategy was conceived to enable systematic structural variations and, at the same time, ensuring that enantiomerically pure vectors are obtained. Notably, changes in the molecular architecture translated into topological differences at the nanoscale upon co-assembly with plasmid DNA, especially regarding the presence of regions with short- or long-range internal order as observed by TEM. In vitro and in vivo experiments further evidenced a significant impact on cell and organ transfection selectivity. Altogether, the results highlight the potential of trehalose-polyamine/pDNA nanocomplex monoformulations to achieve targeting transfection without the need for any additional cell- or organ-sorting component.

Synthesis of cationic carbosilane dendrimers via click chemistry and their use as effective carriers for DNA transfection into cancerous cells.

New amine-terminated carbosilane dendrimers have been prepared by a Huisgen cycloaddition ("click chemistry" reaction) of azide-terminated carbosilane dendrimers with two different propargyl amines. The corresponding cationic derivatives with peripheral ammonium groups were obtained by subsequent addition of MeI. Quaternized dendrimers are soluble and stable in water or other protic solvents for long time periods, and have been studied as nonviral vectors for the transfection of DNA to cancer cells. In this study DNA-dendrimeric nanoparticles (dendriplexes) formulated with two different families of cationic carbosilane dendrimers (family 1 (G1, G2 and G3) and family 2 (G1, G2)) were characterized and evaluated for their ability to transfect cells in vitro and in vivo. Dendriplex derived from second generation dendrimer of family 1 (F1G2 5/1 (+/-)) increased the efficiency of plasmid-mediated gene transfer in HepG2 cells as compared to naked DNA and the commercial control dendrimer. Also, intravenously administered dendriplex F1G3 20/1 (+/-) is superior in terms of gene transfer efficiency in vivo.

Gemini Cationic Lipid-Type Nanovectors Suitable for the Transfection of Therapeutic Plasmid DNA Encoding for Pro-Inflammatory Cytokine Interleukin-12.

Ample evidence exists on the role of interleukin-12 (IL-12) in the response against many pathogens, as well as on its remarkable antitumor properties. However, the unexpected toxicity and disappointing results in some clinical trials are prompting the design of new strategies and/or vectors for IL-12 delivery. This study was conceived to further endorse the use of gemini cationic lipids (GCLs) in combination with zwitterionic helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine) as nanovectors for the insertion of plasmid DNA encoding for IL-12 (pCMV-IL12) into cells. Optimal GCL formulations previously reported by us were selected for IL-12-based biophysical experiments. In vitro studies demonstrated efficient pCMV-IL12 transfection by GCLs with comparable or superior cytokine levels than those obtained with commercial control Lipofectamine2000*. Furthermore, the nanovectors did not present significant toxicity, showing high cell viability values. The proteins adsorbed on the nanovector surface were found to be mostly lipoproteins and serum albumin, which are both beneficial to increase the blood circulation time. These outstanding results are accompanied by an initial physicochemical characterization to confirm DNA compaction and protection by the lipid mixture. Although further studies would be necessary, the present GCLs exhibit promising characteristics as candidates for pCMV-IL12 transfection in future in vivo applications.

Liposomes and lipopolymeric carriers for gene delivery.

In this work we have examined the ability of various lipopolyplexes to deliver genes into liver cancer cells. We evaluated different parameters such as the protocol of preparation, the lipid/DNA molar ratio, and the molecular weight and type of PEI, to optimize the formulation to achieve high transfection activity. Our hypothesis was that the association of PEI with cationic liposomes (lipopolyplexes) would increase luciferase expression compared to lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA) alone.

Targeted cationic poly(D,L-lactic-co-glycolic acid) nanoparticles for gene delivery to cultured cells.

We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.

Activated and non-activated PAMAM dendrimers for gene delivery in vitro and in vivo.

Nanotechnology, though not a new concept, has gained importance in medical breakthroughs. The preparation of nanosystems like polymeric nanoparticles can be used for drug and gene delivery. In this study dendrimeric nanoparticles prepared with generations 4 and 5 (G4, G5) polyamidoamine (PAMAM) dendrimers and plasmid DNA were characterized and their ability to transfect cells in vitro and in vivo evaluated. Additionally, the efficacy of these dendrimers on activation after heat treatment has been tested to attempt an enhancement in transfection activity over that of intact dendrimers. Measurements of the particle size and zeta potential as a function of the charge ratio and the generation of the polymer reveal that no significant differences were obtained in size by using G4 or G5 polymers in nonactivated dendriplexes prepared at different charge ratios. The zeta potentials of the dendriplexes are strongly positive and differ only slightly. Atomic force microscopy images of complexes showed that they are spherical, individualized, and homogeneously distributed. These vectors were also highly effective in protecting DNA from attack by DNase I and increased the efficiency of plasmid-mediated gene transfer in vitro to liver (HepG2) and colon (CT26) cancer cells as compared with naked DNA, even in the presence of 60% fetal bovine serum. Expression is enhanced at higher charge ratios with maximal values obtained at a charge ratio of 10:1 (+/-) and by increasing the dendrimer generation. Finally, the transfection activity of G4 and G5 dendriplexes was significantly enhanced in HepG2 and CT26 cells by activation of the dendrimers. In this respect we have optimized the time of activation to obtain the optimal levels of gene expression. Also, intravenously administered activated G4 and G5 dendrimer-DNA complexes are superior to the nonactivated ones in terms of gene transfer efficiency in vivo. In conclusion, our results showed that G4 and G5 PAMAM dendrimers are an effective nanosystem for gene delivery to colon and liver cancer cells in vitro, as well as for in vivo therapeutic applications. FROM THE CLINICAL EDITOR: This paper describes the synthesis and potential applications of mixed nanoparticles prepared with generations 4 and 5 (G4, G5) poly(amidoamine) (PAMAM) dendrimers and plasmid DNA. These mixed nanoparticles proved to be effective for gene delivery to colon and liver cancer cells in vitro, as well as in vivo.

A Non-Viral Plasmid DNA Delivery System Consisting on a Lysine-Derived Cationic Lipid Mixed with a Fusogenic Lipid.

The insertion of biocompatible amino acid moieties in non-viral gene nanocarriers is an attractive approach that has been recently gaining interest. In this work, a cationic lipid, consisting of a lysine-derived moiety linked to a C12 chain (LYCl) was combined with a common fusogenic helper lipid (DOPE) and evaluated as a potential vehicle to transfect two plasmid DNAs (encoding green fluorescent protein GFP and luciferase) into COS-7 cells. A multidisciplinary approach has been followed: (i) biophysical characterization based on zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and cryo-transmission electronic microscopy (cryo-TEM); (ii) biological studies by fluorescence assisted cell sorting (FACS), luminometry, and cytotoxicity experiments; and (iii) a computational study of the formation of lipid bilayers and their subsequent stabilization with DNA. The results indicate that LYCl/DOPE nanocarriers are capable of compacting the pDNAs and protecting them efficiently against DNase I degradation, by forming Lα lyotropic liquid crystal phases, with an average size of ~200 nm and low polydispersity that facilitate the cellular uptake process. The computational results confirmed that the LYCl/DOPE lipid bilayers are stable and also capable of stabilizing DNA fragments via lipoplex formation, with dimensions consistent with experimental values. The optimum formulations (found at 20% of LYCl content) were able to complete the transfection process efficiently and with high cell viabilities, even improving the outcomes of the positive control Lipo2000*.

Multidisciplinary Approach to the Transfection of Plasmid DNA by a Nonviral Nanocarrier Based on a Gemini-Bolaamphiphilic Hybrid Lipid.

A multidisciplinary strategy, including both biochemical and biophysical studies, was proposed here to evaluate the potential of lipid nanoaggregates consisting of a mixture of a gemini-bolaamphiphilic lipid (C6C22C6) and the well-known helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE) to transfect plasmid DNA into living cells in an efficient and safe way. For that purpose, several experimental techniques were employed, such as zeta potential (phase analysis light scattering methodology), agarose gel electrophoresis (pDNA compaction and pDNA protection assays), small-angle X-ray scattering, cryo-transmission electron microscopy, atomic force microscopy, fluorescence-assisted cell sorting, luminometry, and cytotoxicity assays. The results revealed that the cationic lipid and plasmid offer only 70 and 30% of their nominal positive () and negative charges (), respectively. Upon mixing with DOPE, they form lipoplexes that self-aggregate in typical multilamellar Lα lyotropic liquid-crystal nanostructures with sizes in the range of 100-200 nm and low polydispersities, very suitably fitted to remain in the bloodstream and cross the cell membrane. Interestingly, these nanoaggregates were able to compact, protect (from the degrading effect of DNase I), and transfect two DNA plasmids (pEGFP-C3, encoding the green fluorescent protein, and pCMV-Luc, encoding luciferase) into COS-7 cells, with an efficiency equal or even superior to that of the universal control Lipo2000*, as long as the effective +/- charge ratio was maintained higher than 1 but reasonably close to electroneutrality. Moreover, this transfection process was not cytotoxic because the viability of COS-7 cells remained at high levels, greater than 80%. All of these features make the C6C22C6/DOPE nanosystem an optimal nonviral gene nanocarrier in vitro and a potentially interesting candidate for future in vivo experiments.

Novel PAMAM-PEG-Peptide Conjugates for siRNA Delivery Targeted to the Transferrin and Epidermal Growth Factor Receptors.

The transferrin (TfR) and epidermal growth factor receptors (EGFR) are known to be overexpressed on the surface of a wide variety of tumor cells. Therefore, the peptides B6 (TfR specific) and GE11 (targeted to the EGFR) were linked to the PAMAM (polyamidoamine) structure via a polyethylenglycol (PEG) 2 kDa chain with the aim of improving the silencing capacity of the PAMAM-based dendriplexes. The complexes showed an excellent binding capacity to the siRNA with a maximal condensation at nitrogen/phosphate (N/P) 2. The nanoparticles formed exhibited hydrodynamic diameters below 200 nm. The zeta potential was always positive, despite the complexes containing the PEG chain in the structure showing a drop of the values due to the shielding effect. The gene silencing capacity was assayed in HeLa and LS174T cells stably transfected with the eGFPLuc cassette. The dendriplexes containing a specific anti luciferase siRNA, assayed at different N/P ratios, were able to mediate a mean decrease of the luciferase expression values of 14% for HeLa and 20% in LS174T cells, compared to an unspecific siRNA-control. (p < 0.05). In all the conditions assayed, dendriplexes resulted to be non-toxic and viability was always above 75%.

A Gemini Cationic Lipid with Histidine Residues as a Novel Lipid-Based Gene Nanocarrier: A Biophysical and Biochemical Study.

This work reports the synthesis of a novel gemini cationic lipid that incorporates two histidine-type head groups (C3(C16His)2). Mixed with a helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanol amine (DOPE), it was used to transfect three different types of plasmid DNA: one encoding the green fluorescence protein (pEGFP-C3), one encoding a luciferase (pCMV-Luc), and a therapeutic anti-tumoral agent encoding interleukin-12 (pCMV-IL12). Complementary biophysical experiments (zeta potential, gel electrophoresis, small-angle X-ray scattering (SAXS), and fluorescence anisotropy) and biological studies (FACS, luminometry, and cytotoxicity) of these C3(C16His)2/DOPE-pDNA lipoplexes provided vast insight into their outcomes as gene carriers. They were found to efficiently compact and protect pDNA against DNase I degradation by forming nanoaggregates of 120⁻290 nm in size, which were further characterized as very fluidic lamellar structures based in a sandwich-type phase, with alternating layers of mixed lipids and an aqueous monolayer where the pDNA and counterions are located. The optimum formulations of these nanoaggregates were able to transfect the pDNAs into COS-7 and HeLa cells with high cell viability, comparable or superior to that of the standard Lipo2000*. The vast amount of information collected from the in vitro studies points to this histidine-based lipid nanocarrier as a potentially interesting candidate for future in vivo studies investigating specific gene therapies.

Serum-resistant lipopolyplexes for gene delivery to liver tumour cells.

In this study, an efficient non-viral gene transfer system has been developed by employing polyethylenimine (PEI 800, 25 and 22kDa) and DOTAP and cholesterol (Chol) as lipids (lipopolyplex), at three different lipid/DNA molar ratios (2/1, 5/1 and 17/1) by using five different protocols of formulation. Condensation assays revealed that PEI of 800, 25 and 22kDa were very effective in condensing plasmid DNA, leading to a complete condensation at N/P ratios above 4. Addition of DOTAP/Chol liposomes did not further condense DNA. Increasing the molar ratio lipid/DNA in the complex resulted in higher positive values of the zeta-potential, while the particle size increased in some protocols, but not in others. High molecular weight PEI (800kDa) used in the formulation of lipopolyplexes lead to a bigger particle size, compared to that obtained with smaller PEI species, whether branched (25kDa) or linear (22kDa). These vectors were also highly effective in protecting DNA from attack by DNAse I. Transfection activity was maximal by using protocols 3 and 4 and a lipid/DNA molar ratio of 17/1. These complexes showed high efficiency in gene delivery of DNA to liver cancer cells, even in the presence of high concentration of serum (60% FBS). On the other hand, complexes formed with linear PEI (22kDa) were more effective than lipopolyplexes containing branched PEI (800 or 25kDa). The complexes resulted to be much more efficient than conventional lipoplexes (cationic lipid and DNA) and polyplexes (cationic polymer and DNA). The same behaviour was observed for complexes prepared in the presence of the therapeutic gene pCMVIL-12. Toxicity assays revealed a viability higher than 80% in all cases, independently of the protocol, molar ratio (lipid/DNA), molecular weight and type of PEI.

A biophysical study of gene nanocarriers formed by anionic/zwitterionic mixed lipids and pillar[5]arene polycationic macrocycles.

The use of divalent cations as mediators between anionic lipids (ALs) and nucleic acids has been explored for several years in gene therapy. However, a promising anionic lipid system which could surpass the outcomes of current cationic lipids (CLs) has not been found yet. One plausible reason for such poor efficiencies may be the impossibility of AL-DNA lipoplexes mediated by divalent cations to reach charge inversion, in contrast with the usual behavior of CL-DNA lipoplexes. In the present study, divalent bridge-cations have been replaced by a multivalent positively charged macrocycle in order to see whether charge reversal is reached and how this fact may improve transfection efficiency (TE). For that purpose, an extensive biophysical and biochemical study has been carried out on lipoplexes constituted by a mixture of: (i) an anionic lipid DOPG (sodium salt of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)); (ii) a zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine), which acts as a neutral helper lipid at physiological pH 7.4; (iii) a plasmid DNA (pDNA); and (iv) a polycationic macrocycle, pillar[5]arene (P10+), with the role of bridging the electrostatic interaction between the anionic mixed lipids and the pDNA, also negatively charged. The studies have been done at several DOPG molar compositions (α) and pillar[5]arene concentrations. Electrochemical experiments (zeta potential and gel electrophoresis) have revealed that, interestingly, DOPG/DOPE-P10+-pDNA lipoplexes show a charge inversion. Both studies have indicated that, at [P10+] ≥ 15 µM, pDNA is efficiently compacted by DOPG/DOPE mixed lipids, using P10+ as a bridge between the negative charge of the AL and anionic pDNA. SAXS diffractograms have shown the presence of two lyotropic liquid crystal phases: an inverted hexagonal one (H) found at low composition (α = 0.2), and a lamellar one (Lα) at medium composition (α = 0.5). Cryo-TEM and AFM experiments have confirmed these structures. Transfection and cell viability experiments using COS-7 cells in the presence of serum have reported moderate-to-high transfection levels and good cell viability results. The whole ensemble of the biophysical and biochemical results of the DOPG/DOPE-P10+-pDNA lipoplex indicates that this system may open up a novel and very promising route in the anionic non-viral gene vectors field.

Versatility of biodegradable poly(D,L-lactic-co-glycolic acid) microspheres for plasmid DNA delivery.

In this study, we have optimized different formulations of DNA encapsulated into PLGA microspheres by correlating the protocol of preparation and the molecular weight and composition of the polymer, with the main characteristics of these systems in order to design an efficient non-viral gene delivery vector. For that, we prepared poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles with an optimized water-oil-water double emulsion process, by using several types of polymers (RG502, RG503, RG504, RG502H and RG752), and characterized in terms of size, zeta potential, encapsulation efficiency (EE%), morphology, DNA conformation, release kinetics, plasmid integrity and erosion. The size of the particles ranged between 0.7 and 5.7 microm depending on the protocol of formulation and the molecular mass of the polymer used. The microspheres prepared by using in their formulation polymers of high molecular weight (RG503 and RG504) were bigger in size than in the case of using a lower molecular weight polymer (RG502). The EE (%) of plasmid DNA increased with increasing the molecular mass of the polymer and by using the most hydrophilic polymer RG502H, which contains terminal acidic groups in its structure. The plasmid could be encapsulated without compromising its structural and functional integrity. Also a protective effect of PLGA on endonuclease digestion is observed. Plasmid DNA release from microspheres composed of low molecular weight or hydrophilic polymers, like RG502H, was faster than from particles containing high molecular weight or hydrophobic polymers. These PLGA microspheres could be an alternative to the viral vectors used in gene therapy, given that may be used to deliver genes and other bioactive molecules, either very rapidly or in a controlled manner.

Antitumoral activity of transferrin-lipoplexes carrying the IL-12 gene in the treatment of colon cancer.

The present study aimed to establish an efficient targeted nonviral strategy for IL-12 gene transfer in colon carcinoma in vivo employing transferrin (Tf)-lipoplexes. Complexes for in vitro experiments were prepared at a 5/1(+/ - ) (lipid/DNA) charge ratio, with the ligand Tf (32 (microg/(microg DNA). Complexes for in vivo experiments contained 144 mM of total lipid (DOTAP/Chol), 60 (microg of pCMVLuc or pCMVIL-12 and 32 (microg of Tf-lipoplexes per microgram of plasmid. For intratumoral studies, CT26 (5 x 105 cells) in 50 microl of PBS were inoculated subcutaneously into the back of the mouse. Treatments began when tumor sizes reached 5-6 mm in diameter. Complexes were injected by a single intratumoral injection in a volume of 50 microl. Our in vitro results indicate that Tf-lipoplexes always mediate higher gene expression in colon (CT26) tumor cells, compared to plain-lipoplexes (without ligand) or naked plasmid. At the same time, CT26 tumor-bearing animals treated with Tf-lipoplexes containing the therapeutic gene IL-12, showed tumor growth inhibition, leading to a complete tumor regression in 75% of the treated mice (p < 0.001), without signs of recurrence. High levels of IL-12 and IFN-gamma were detected in the sera of treated mice. Mice survival also improved considerably by treatment with this system, with a survival rate of 88%, at 23 days post-administration. In summary, in this study we have developed an efficient, targeted cationic lipid-based system for the treatment of colon tumors. The vector has the advantages of ease of preparation and economy, in comparison with commercial transfection reagents, as well as, the possibility of a large scale production.