pDNA Complexes Research Articles

Role of scavenger receptors in peptide-based delivery of plasmid DNA across a blood–brain barrier model

Receptor-mediated transcytosis remains a major route for drug delivery across the blood–brain barrier (BBB). PepFect 32 (PF32), a peptide-based vector modified with targeting ligand (Angiopep-2) binding to low-density lipoprotein receptor-related protein-1 (LRP-1), was previously found to be a promising vector for plasmid delivery across an in vitro model of the BBB. Cellular uptake of PF32/plasmid DNA (pDNA) complexes was speculated the internalization via LRP-1 receptor. In this study, we prove that PF32/pDNA nanocomplexes are not only transported into brain endothelial cells via LRP-1 receptor-mediated endocytosis, but also via scavenger receptor class A and B (SCARA3, SCARA5, and SR-BI)-mediated endocytosis. SCARA3, SCARA5, and SR-BI are found to be expressed in the brain endothelial cells. Inhibition of these receptors leads to a reduction of the transfection. In conclusion, this study shows that scavenger receptors also play an essential role in the cellular uptake of the PF32/pDNA nanocomplexes.

Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy

Event Abstract Back to Event Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy Kishor Sarkar1, Harini V. Krishnan1, G Vinoth Kumar2, K Suresh Babu2, Vinayak Sant1 and Shilpa Sant1, 3, 4 1 University of Pittsburgh, Department of Pharmaceutical Sciences, United States 2 Pondicherry University, Centre for Nano Science and Technology, India 3 University of Pittsburgh, Department of Bioengineering, United States 4 University of Pittsburgh, McGowan Institute for Regenerative Medicine, United States Introduction: Gene therapy has become alternative therapeutic approach to treat genetic diseases such as cancer[1]. Nonviral vectors have gained tremendous attraction over the viral vector due to its low toxicity, non-immunogenicity and low production cost[2]-[4]. Recently, rare earth metal oxide especially cerium oxide nanoparticle (CNP) has widely been used in biomedical fields such as drug delivery, bio-imaging, nanomedicine and tissue engineering application because of its inherent antioxidant activity, free radical scavenging activity and radioprotection to normal cell[5],[6]. It is also reported that CNP increased the ROS (reactive oxygen species) in cancer cell[7]. Therefore, we hypothesized that therapeutic gene (like siRNA, p53 etc.) delivery by means of CNP may give the dual therapeutic effect in conjugation with increased ROS level in cancer cell for the treatment of cancer. Here we report the functionalization of CNP by low molecular weight polyethyleneimine (LPEI, 2 kDa) followed by conjugation of folic acid (FA) for targeted gene delivery. Methods: LPEI was conjugated with CNP (LPEI-conj-CNP) through carboxylation followed by amidation reaction. Then, FA was reacted with LPEI-conj-CNP by carbodiimide to get FA-LPEI-conj- CNP. CNP/pDNA (enhanced green fluorescent protein, EGFP encoding pDNA) complexes were prepared at various weight ratios and characterized by agarose gel electrophoresis assay and dynamic light scattering (DLS). In vitro transfection efficiency, toxicity, ROS and underlying mechanistic pathway were also evaluated in HeLa cell. Results: Poor water dispersibility of CNP at physiological pH is a major drawback for biological field especially gene/ drug delivery application. CNP forms stable dispersion in acidic pH only. Despite the positive zeta potential of CNP, it did not bind any DNA at any weight ratio due to poor dispersibility. But, the DNA complexation capability of CNP significantly increased after conjugation of LPEI and LPEI-conj-CNP complexed all pDNA at a very low weight ratio of 0.5:1. Due to presence of lots of primary amine groups in LPEI, the water dispersibility of CNP increased drastically and resulted better DNA complexation through the cationic primary amine groups. The transfection efficiency of CNP increased after conjugation of LPEI but the efficiency increased significantly after FA conjugation with LPEI-conj-CNP. FA-LPEI-conj-CNP/pDNA complex at weight ratio of 15:1 showed highest transfection efficiency which also surpassed the efficiency offered by PEI (25 kDa) (Figure 1). It was also observed that FA-LPEI-conj-CNP internalized into cell via folate receptor as well as caveolae mediated pathway and resulted enhanced transfection efficiency. While LPEI-conj-CNP followed clathrin mediated pathway followed by lysosomal compartment entrapment and as a result showed low transfection efficiency. It was found that green fluorescence (fluorescent dichlorofluorescein, DCF obtained through oxidation of nonfluorescent dichlorofluorescein diacetate, DCFA by ROS) increased drastically by FA-LPEI-conj-CNP compared to all CNPs and H2O2 treated cell indicating the increase in ROS (Figure 2). Conclusion: The transfection efficiency as well as ROS of CNP increased after functionalization of CNP especially after FA conjugation. Therefore, FA-LPEI-conj-CNP may be useful candidate in nonviral gene therapy for cancer treatment by using its dual property including gene delivery and ROS enhancement. This work was financially supported by Start-up funds from University of Pittsburgh School of Pharmacy

Development of a Multi-functional Nano-device for Safe and Effective Gene Delivery to Target Organs.

Nucleic acids are expected as novel effective medicines, although they require a drug delivery system (DDS). Complexes of nucleic acids with cationic liposomes and cationic polymers have been mainly used as DDS for clinical use. However, most cationic complexes have disadvantages such as strong cytotoxicity and low biocompatibility. We previously found that a plasmid DNA (pDNA) complex coated with biodegradable γ-polyglutamic acid (γ-PGA) provided adequate gene expression without cytotoxicity. Based on these results, we developed a new DDS (multi-functional Nano-device) of pDNA using biodegradable polyamino acids. A typical cationic polyamino acid, poly-L-lysine (PLL), was complexed with pDNA. The binary complexes, however, showed low gene expression and high cytotoxicity. Gene expression was enhanced by addition of poly-L-histidine (PLH) to the binary complexes. PLH can increase endosome escape of the complexes by inducing pH-buffering effects. The quaternary complexes (pDNA-PLL-PLH-γ-PGA complexes) exhibited high gene expression and low cytotoxicity. Furthermore, we used dendrigraft poly-L-lysine (DGL) instead of PLL and PLH to enhance gene expression. DGL had sterically congested cations and was biodegradable. The ternary complexes (pDNA-DGL-γ-PGA complexes) exhibited markedly high gene expression and low cytotoxicity. The pDNA-DGL-γ-PGA complexes also had high gene expression in the marginal zone (rich dendritic cells) of the spleen after intravenous injection into mice. These results indicate that pDNA-DGL-γ-PGA complexes may be useful as vaccine vectors. Therefore we prepared a novel malaria DNA vaccine using Plasmodium yoelii GPI8p-transamidase-related protein pDNA (PyTAM). The PyTAM-DGL-γ-PGA complexes markedly improved survival time of model mice infected with malaria.

Delivery of Plasmid DNA into Tumors by Intravenous Injection of PEGylated Cationic Lipoplexes into Tumor-Bearing Mice

For systemic injection of cationic liposome/plasmid DNA (pDNA) complexes (cationic lipoplexes), polyethylene glycol (PEG)-modification (PEGylation) of lipoplexes can enhance their systemic stability. In this study, we examined whether intravenous injection of PEGylated cationic lipoplexes into tumor-bearing mice could deliver pDNA into tumor tissues and induce transgene expression. PEGylation of cationic liposomes could prevent their agglutination with erythrocytes. However, when PEGylated cationic lipoplexes were injected intravenously into tumor-bearing mice, they accumulated in tumor vascular vessels and did not exhibit transgene expression in tumors with both poor and well-developed vascularization. Furthermore, PEGylated cationic lipoplexes of CpG- free pDNA could not increase transgene expression in tumors after intravenous injection. These results suggested that PEGylation could not extravasate cationic lipoplexes from vascular vessels in tumors and abolished transgene expression although it enhanced the systemic stability of cationic lipoplexes by avoiding interactions with blood components such as erythrocytes. Successful delivery of pDNA to tumors by PEGylated cationic liposomes will require a rational strategy and the design of liposomal delivery systems to overcome the issue associated with the use of PEG.

Effect of pDNA Complex Particle Size on Gene Expression: Difference between In Vitro and In Vivo Experiments

Effect of pDNA Complex Particle Size on Gene Expression: Difference between In Vitro and In Vivo Experiments We have developed a novel gene-transfection system comprising very small (70-150 nm) plasmid/polyethyleneimine/hyaluronan (HA) ternary complexes having negative surface charge, and reduced nonspecific interactions with bio-components. They showed high in vivo gene expression efficiency. However, in in vitro transfection experiments, such small ternary complexes often demonstrated rather much lower gene expression than the conventional DNA/ polycation complex. In this study, we investigated the influence of the complex particle size on gene expression efficiency, focusing on the difference between in vitro and in vivo transfection. Size of the DNA complex particles strongly depended on the concentration of the mixing solutions, and the higher concentration resulted in the larger particles. As for the in vitro transfection, DNA/ polyethyleneimine binary complexes could readily be bound to the cells through the electrostatic attractions, regardless of their size. Smaller binary complexes particles showed higher gene expression than the larger ones, likely due to their high internalization efficiency. On the other hand, small DNA/polyethyleneimine/HA ternary complexes having negative surface charge kept dispersing in the medium away from the cells, and demonstrated lower in vitro gene transfection efficiency than larger ones. In regards to in vivo transfection after intravenous injection, only small ternary complexes showed high gene expression, because reduced nonspecific interactions and small size of the complex particles are required to be distributed in the body, and accumulated in tumor. The superiority and inferiority of the vectors for in vivo transfection cannot be always correctly predicted by the results of in vitro transfection.

Polyethylenimine-polyacrylic acid nanocomposites: Type of bonding does influence the gene transfer efficacy and cytotoxicity

The main aim of the current study is to compare the physicochemical properties, cytotoxicity and gene-transfer ability of electrostatically and covalently linked nanocomposites of polyethylenimine (PEI) and polyacrylic acid (PAA) on mammalian cells. Two series of nanocomposites, ionic PEI-PAA (iPP) and covalent PEI-PAA (cPP), were synthesized by varying the amounts of polyacrylic acid (PAA). Physicochemical characterization revealed that iPP nanopcomposites were of bigger sized than cPP nanocomposites with zeta potential almost comparable. Nucleic acid binding assay displayed that iPP and cPP nanocomposites, having sufficient cationic charge, efficiently interacted with plasmid DNA and completely retarded its electrophoretic mobility on agarose gel. In vitro MTT assay showed slightly higher cell viability of cPP/pDNA complexes over their ionic counterparts. Both the series of nanocomposite/pDNA complexes exhibited considerably higher transfection efficacy compared to pDNA complexes of native bPEI and the standard transfection reagent, Lipofectamine, with cPP/pDNA complexes performed much better than iPP/pDNA complexes. Flow cytometry further confirmed these findings where cPP-4/pDNA complex showed transfection in ∼85% HEK293 cells, while iPP-2/pDNA complex transfected ∼67% HEK293 cells. Lipofectamine/pDNA and bPEI/pDNA complexes could transfect just ∼35% and ∼26% HEK293 cells. All these results demonstrate the superiority of covalently linked nanocomposites (cPP) which could be used as efficient carriers for nucleic acids in future gene delivery applications.

Tris(2-aminoethyl)amine-based α-branched fatty acid amides – Synthesis of lipids and comparative study of transfection efficiency of their lipid formulations

The synthesis of a new class of cationic lipids, tris(2-aminoethyl)amine-based α-branched fatty acid amides, is described resulting in a series of lipids with specific variations in the lipophilic as well as the hydrophilic part of the lipids. In-vitro structure/transfection relationships were established by application of complexes of these lipids with plasmid DNA (pDNA) to different cell lines. The α-branched fatty acid amide bearing two tetradecyl chains and two lysine molecules (T14diLys) in mixture with the co-lipid 1,2-di-[(9Z)-octadec-9-enoyl]-sn-glycero-3-phosphoethanolamine (DOPE) (1/2, n/n) exhibits effective pDNA transfer in three different cell lines, namely Hep-G2, A549, and COS-7. The presence of 10% serum during lipoplex incubation of the cells did not affect the transfection efficiency. Based on that, detailed investigations of the complexation of pDNA with the lipid formulation T14diLys/DOPE 1/2 (n/n) were carried out with respect to particle size and charge using dynamic light scattering (DLS), ζ-potential measurements, and transmission electron microscopy (TEM). Additionally, the lipoplex uptake was investigated by confocal laser scanning microscopy (CLSM). Overall, lipoplexes prepared from T14diLys/DOPE 1/2 (n/n) offer large potential as lipid-based polynucleotide carriers and further justify advanced examinations.

Efficient pDNA Delivery Using Cationic 2-Hydroxypropyl-β-Cyclodextrin Pluronic-Based Polyrotaxanes.

A family of cationic Pluronic-based polyrotaxanes (PR(+)), threaded with 2-hydroxypropyl-β-cyclodextrin (HPCD), was synthesized for pDNA delivery into multiple cell lines. All PR(+) formed highly stable, positively charged pDNA complexes that were < 250 nm in diameter. The cellular uptake and pDNA transfection efficiencies of the PR(+):pDNA complexes was enhanced relative to the commercial transfection standards L2K and bPEI, while displaying similar or lower toxicity profiles. Charge density and threading efficiency of the PR(+) agent significantly influenced the colloidal stability and physical properties of the complexes, which impacted their intracellular transfection efficiencies. Taken together, our results suggest that HPCD: Pluronic PR(+) can be used as potent vectors for pDNA-based therapeutics.

PAMAM Dendrimer/pDNA Functionalized-Magnetic Iron Oxide Nanoparticles for Gene Delivery.

Herein, we report an easy and ingenious method to functionalize magnetic iron oxide nanoparticles (MNPs) with plasmid DNA (pDNA) to obtain nanohybrid systems suitable for nucleic acid therapy. The nanohybrids were prepared by combining complexes of dendrimers and pDNA (dendriplexes) and poly(styrene) sulfonate-coated MNPs through electrostatic interactions. The effects of the dendrimer generation (generations 2, 4 and 6) and the amine to phosphate group (N/P) ratio on the hydrodynamic diameter, zeta potential, cell viability, cellular internalization and transfection efficiency of the nanohybrids were systematically investigated at different transfection conditions (including incubation time, pDNA concentration, presence or absence of an external magnetic field, and presence or absence of fetal bovine serum). The results confirmed that the nanohybrids were able to transfect NIH 3T3 cells, and that the level of gene expression (the luciferase protein reporter gene was used) was strongly dependent on the dendrimer generation, the N/P ratio, and the pDNA concentration. The best system was based on dendriplex-coated MNPs formed by generation 6 dendrimers at an N/P ratio of 10 that, at optimized conditions, led to a gene expression level which was not significantly different from that obtained only using dendriplexes. In summary, a coherent set of results was reached indicating the potential of the developed nanohybrids as effective gene delivery nanomaterials.

Highly Effective Non-Viral Antitumor Gene Therapy System Comprised of Biocompatible Small Plasmid Complex Particles Consisting of pDNA, Anionic Polysaccharide, and Fully Deprotected Linear Polyethylenimine.

We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.

Effects of dendrimer/cyclodextrin conjugates as gene transfer carriers on nitric oxide production from macrophages

The development of safe gene transfer carriers with high transfection efficiency, which does not affect the cell function, is a challenging issue. In this study, we examined the effects of α-cyclodextrin (α-CyD)/dendrimer conjugate (α-CDE (G3)) on nitric oxide (NO) production in murine macrophages J774.1 cells stimulated with toll-like receptors (TLR) ligands. NO production from macrophages stimulated with TLR ligands was determined by the Griess method. Transfection efficiency of α-CDE (G3)/plasmid DNA (pDNA) complex was quantified by a luminometer. α-CDE (G3) significantly inhibited NO production from J774.1 cells stimulated with TLR ligands. α-CyD molecules in α-CDE (G3) had no effect on NO production. The inhibitory effect of α-CDE (G3) on NO production might be attributed to the dendrimer (G3). Increasing the degree of substitution (DS) of α-CyD in the α-CDE (G3) molecule was accompanied by a significant decrease in the inhibition of NO production. Furthermore, higher gene transfection efficiency of α-CDE (G3)/pDNA complex was observed upon increasing the DS of α-CyD. α-CDE (G3) with high DS value of α-CyD may be considered as a safe gene transfer carrier that does not adversely affect NO production from macrophages stimulated with TLR ligands.

169. Polymer Functionalized Gold Nanoparticles in Gene Delivery In Vitro

Non-viral gene delivery approaches have been extensively studied as a basic tool for intracellular gene transfer and gene therapy especially for genetic aberrations including cancer. Gold nanoparticles have attracted strong biomedical interest for drug/gene delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. In the present study the synthesis of photoluminescent nanoparticles consisting of a gold core coated with polyethyleneimine, poly-L-lysine, cysteine and chitosan is reported. These functionalized gold nanoparticles (FAuNPs) were investigated at different pH values and ionic strength to identify the optimum conditions to produce stable monodisperse nanoparticles. FAuNPs showed good stability at low ionic strength which is important for the flexibility of the polymer chain. All nanoparticle/polymer formulations showed spherical particles in the size range 11.9-195 nm with narrow particle distributions and low PDI (<1.2). Nanoparticle and pDNA complexation was efficiently demonstrated in the band shift and ethidium bromide intercalation assays respectively, with serum nuclease digestion revealing partial protection of theResults: Our novel PEGylated PLGA nanoparticles (without siRNA) showed higher accumulation and extended residence time in the inflamed foot of lipopolysaccharide (LPS)-induced mouse model of chronic inflammation. TNF-α-siRNA-NPs were 176.6±15.8 nm in diameter, with a zeta potential of -25.2±2.0 mV and an entrapment efficiency of 31±4%. The nanoparticles were stable in simulated biological medium. In vitro, less than 40% of the siRNA was released with 72 h at 37oC. Finally, TNF-α-siRNA-NPs significantly decreased TNF-α release from J774A.1 cells.complexed plasmid DNA (pCMV-luc). MTT cytotoxicity experiments indicated that the FAuNPs elicited a dose dependent cytotoxic effect in four mammalian cell lines (HepG2, HEK293, HeLa and Caco2). Au-PEI/pDNA maintained over 80% cell viability across all cell lines, while the Au-cys/pDNA exhibited a significant 91.8% (p<0.001) in Caco2 cells, Au-Chit/pDNA 126% (p<0.01) in HepG2 cells and Au-PLL/pDNA 104% in Hela cells. Transfection studies were accomplished using the luciferase reporter gene assay. Results showed that the FAuNPs produced greater transgene activity than the cationic polymer/DNA complexes on their own. This was evident for the Au-PEI/pDNA complex which produced a 12 fold increase in the HEK293 cells and a 9 fold increase in the HepG2 cells, compared to the PEI/pDNA complexes. The results of this study suggest that FAuNP's low cytotoxicity coupled with the ability to parametrically control particle size and surface properties, make these nanoparticles suitable non-viral gene delivery vectors. However further engineering and modifications of the FAuNPs may be required to enable in vivo gene delivery.

Construction of Novel Thermosensitive Magnetic Cationic Liposomes as a Drug and Gene Co-Delivery System.

This study aimed to develop a novel system for co-delivery of both drugs and genes to the same cells. We described the construction of thermosensitive magnetic cationic liposomes (TSMCLs). Liposomes were prepared by thin film hydration method, then calcein release assay and DSC were used to determine the thermosensitivity of liposomes, and gel retardation experiment was performed to monitor the formation of Lipoplex (Liposomes and pDNA complex), finally the in vitro transfection experiments were performed to evaluate the transfection efficiency of the liposomes. The results showed that TSMCLs combined the features of physical targeting under magnetic guidance and hyperthermia triggered drug release upon the application of alternating magnetic field. Thus TSMCLs could be used for the co-delivery of drugs and genes and have potential application in combined chemotherapy and gene therapy for cancer.

Gelucire-stabilized nanoparticles as a potential DNA delivery system

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs > DDAB-NPs > CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.

Quaternary Complexes Modified from pDNA and Poly-l-Lysine Complexes to Enhance pH-Buffering Effect and Suppress Cytotoxicity

We developed a modified complex of pDNA and poly-l-lysine (PLL) by the addition of poly-l-histidine (PLH) and γ-polyglutamic acid (γ-PGA) to enhance its pH-buffering effect and suppress cytotoxicity. The binary and ternary complexes of pDNA with PLL or/and PLH showed particle sizes of approximately 52-76 nm with cationic surface charge. The ternary complexes showed much higher gene expression than the binary complexes with PLL. The mixed solution of PLL and PLH showed higher buffering capacity than PLL solution. The high gene expression of ternary complexes was reduced by bafilomycin A1 . These results indicated the addition of PLH to PLL complexes promoted endosomal escape by enhancing the pH-buffering effect. The binary and ternary complexes showed cytotoxicity and blood agglutination because of their cationic surface charge. We therefore developed quaternary complexes by the addition of anionic γ-PGA, which was reported to decrease the toxicity of cationic complexes. In fact, quaternary complexes showed no cytotoxicity and blood agglutination. Also, quaternary complexes showed higher gene expression than ternary complexes regardless of their anionic surface charge. Quaternary complexes showed selectively high gene expression in the spleen after their intravenous administration. Thus, we successfully developed the quaternary complexes with high gene expression and no toxicity.

Combining polyethylenimine and Fe(III) for mediating pDNA transfection

BackgroundThe potential use of Fe(III) ions in biomedical applications may predict the interest of its combination with pDNA–PEI polyplexes. The present work aims at assessing the impact of this metal on pDNA complex properties. MethodsVariations in the formation of complexes were imposed by using two types of biological buffers at different salt conditions. The incorporation of pDNA in complexes was characterised by gel electrophoresis and dynamic light scattering. Transfection efficiency and cytotoxicity were evaluated in HeLa and HUH-7 cell lines, supported by flow cytometry assays. ResultsFe(III) enhances pDNA incorporation in the complex, irrespective of the buffer used. Transfection studies reveal that the addition of Fe(III) to complexes at low ionic strength reduces gene transfection, while those prepared under high salt content do not affect or, in a specific case, increase gene transfection up to 5 times. This increase may be a consequence of a favoured interaction of polyplexes with cell membrane and uptake. At low salt conditions, results attained with chloroquine indicate that the metal may inhibit polyplex endosomal escape. A reduction on the amount of PEI (N/P 5) formed at intermediary ionic strength, complemented by Fe(III), reduces the size of complexes while maintaining a transfection efficiency similar to that obtained to N/P 6. ConclusionsFe(III) emerges as a good supporting condensing agent to modulate pDNA–PEI properties, including condensation, size and cytotoxicity, without a large penalty on gene transfection. General significanceThis study highlights important aspects that govern pDNA transfection and elucidates the benefits of incorporating the versatile Fe(III) in a gene delivery system.

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina.

Enhancement of chitosan-mediated gene delivery through combination with phiC31 integrase

Gene transfer efficiency and expression stability are key factors to a successful gene therapy approach. In the present work we have developed a combined system for gene transfer that integrates well established non-viral polymeric vectors based on chitosan particles with the properties of phiC31-integrase that promotes a relatively non-immunogenic, site-specific integration, with sustained gene expression. Simultaneously, to overcome one of the major limitations in adeno-associated virus mediated gene transfer – the delivery of large genes – we have tested the capacity of our non-viral vectors to incorporate a large (8Kb) transgene. Polyplexes were extensively characterized for their size, surface charge, morphology, pDNA complexation, transfection efficiency and transgene expression in vitro using HEK293 cells. Co-transfection with integrase was done by complexation in a single polyplex preparation or the use of two separate polyplex preparations. Transgene expression, GFP and CEP290 (1Kb and 8Kb, respectively), was evaluated by fluorescence microscopy, flow cytometry and Western blot analysis. DNA complexation efficiency, particle size and morphology were consistent with gene delivery for all formulations. In contrast, transfection efficiency and transgene expression varied with polymer and polyplex size. Following delivery by chitosan polyplexes, high levels of GFP expression were still visible 16weeks post-transfection and over-expression of the large transgene was detected at least 6weeks post-transfection. Polyplexes incorporating phiC1 integrase demonstrate prolonged gene expression of both small (GFP, 1Kb) and large genes (CEP290, 8Kb). This approach, using a combined strategy of polymers and integrase may overcome the size limitation found in commonly used adeno-associated virus mediated gene transfer techniques, while maintaining a high safety profile and prolonged, sustained gene expression, thus constituting an alternative for gene delivery.

原子转移自由基聚合(ATRP)阳离子化菊粉作为非病毒基因载体的研究

利用原子转移自由基聚合（ATRP）将聚甲基丙烯酸 N ， N -二甲氨基乙酯[P（DMAEMA）] 偶联到菊粉多糖（Inulin）上，合成了新型基因载体PDIN. 利用核磁共振仪、 动态光散射分析仪、 透射电子显微镜和凝胶电泳对PDIN及其与质粒pDNA的复合物进行了表征. 凝胶阻滞实验结果表明，PDIN可以通过静电相互作用稳定结合pDNA. 噻唑蓝（MTT）细胞毒性测试、 溶血实验、 绿色荧光蛋白表达质粒（pGFP）及 β 半乳糖苷酶表达质粒（p β gal）转染实验结果表明，PDIN对MCF-7，Hela，COS7和HepG2细胞的毒性较小；其溶血率低，具有良好的血液相容性；载体PDIN能有效将pGFP和p β gal带入COS7细胞并表达，在 N / P 为1时转染效率最高，其 β 半乳糖苷酶的酶活为（3.36±0.74） U/mg蛋白，比Lipo2000转染效率[（4.33±0.77） U/mg蛋白] 略低. 因此，所合成的载体PDIN是一种有潜在应用价值的非病毒基因载体.

Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

Non-viral gene delivery vectors are emerging as a safer alternative to viral vectors. Among natural polymers, chitosan (Ch) is the most studied one, and low molecular weight Ch, specifically, presents a wide range of advantages for non-viral pDNA delivery. It is crucial to determine the best process for the formation of Low Molecular Weight Chitosan (LMWC)-pDNA complexes and to characterize their physicochemical properties to better understand their behavior once the polyplexes are administered. The transfection efficiency of Ch based polyplexes is relatively low. Therefore, it is essential to understand all the transfection process, including the cellular uptake, endosomal escape and nuclear import, together with the parameters involved in the process to improve the design and development of the non-viral vectors. The aim of this review is to describe the formation and characterization of LMWC based polyplexes, the in vitro transfection process and finally, the in vivo applications of LMWC based polyplexes for gene therapy purposes.