High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents

In order to enhance the gene delivery efficiency and decrease cytotoxicity of polyplexes, copolymers consisting of branched polyethyleneimine (PEI) 25 kDa grafted with Pluronic (F127, F68, P105) were successfully synthesized using a simple two-step procedure. The copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge and transfection efficiency. The complex sizes were below 300 nm, which implicated their potential for intracellular delivery. The Pluronic-g-PEI exhibited better condensation and complexation properties than PEI 25 kDa. The cytotoxicity of PEI was strongly reduced after copolymerization. The Pluronic-g-PEI showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in HeLa cells. Compared with unmodified PEI 25 kDa Pluronic-g-PEI showed much higher transfection efficiency. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and grafted with Pluronic seem to provide promising properties as stable, high transfection efficiency vectors.     

京尼平交联低聚乙烯亚胺智能基因载体的制备与表征

使用京尼平与分子量为1800的超支化低聚乙烯亚胺在70％的乙醇溶液中反应,合成了具有荧光的交联型聚合物.利用核磁、凝胶渗透色谱、粒度仪、zeta电位仪和凝胶阻滞电泳对聚合物载体及其与DNA复合物颗粒进行了表征.研究表明,聚合物载体与DNA复合物颗粒粒径为120 ～150 nm,zeta电位为+20～25 mV,聚合物/...     

Synthesis of poly(propylene glycol)-block-polyethylenimine triblock copolymers for the delivery of nucleic acids

LPEIs, which are efficient DNA transfection agents, were found to be far less effective for the delivery of siRNAs. Here, two amphiphilic triblock copolymers LPEI(50) -b-PPG(36) -b-LPEI(50) (2) and LPEI(14) -b-PPG(68) -b-LPEI(14) (4) have been synthesized. The transfection assays showed that compound 2 was efficient for DNA transfection whilst it was almost inactive for siRNA delivery. In contrast, polymer 4 was inefficient for DNA transfection while it showed capabilities for siRNA delivery. Taken together, our results indicate that the properties required for DNA and siRNA delivery are different. Moreover, we show that introduction of a hydrophobic segment that allows self-assembly confers siRNA delivery capacities.     

(Coixan polysaccharide)-graft-polyethylenimine folate for tumor-targeted gene delivery

CP-PEI-FA was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and their DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro/in vivo transfection were examined. The cytotoxicity of CP-PEI-FA was significantly lower than that of PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (FR-positive cells) and A549 cells (FR-negative cells). CP-PEI-FA showed a high targeting specificity and good gene transfection efficiency in FR-positive cells. These results indicate that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.     

Chemo-physical and biological evaluation of poly(L-lysine)-grafted chitosan copolymers used for highly efficient gene delivery

For the success of non-viral gene delivery, it is of great importance to develop gene vectors with high efficiency but low toxicity. We demonstrate that PLL-grafted chitosan copolymers combine the advantages of PLL with its good pDNA-binding ability and of chitosan with its good biocompatibility. The chemo-physical properties of the prepared Chi-g-PLL copolymers are thoroughly characterized. The in vitro transfection study shows that the copolymers have a much higher gene transfer ability than the starting materials chitosan and PLL. A positive correlation between PLL chain lengths and transfection efficiency of the copolymers is found. Our results suggest that these novel Chi-g-PLL copolymers are good candidates for gene delivery in vivo.     

Correlation of the Physicochemical and Structural Properties of pDNA/Cationic Liposome Complexes with Their in Vitro Transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R(±)) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R(±) = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R(±) is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R(±) to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R(±) value. Interestingly, for R(±) = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R(±)) in the system. This information is used to explain the transfection differences observed at an R(±) = 9 as compared to R(±) = 3 and 6. Close to the isoneutrality region (R(±) = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.     

Plasmid DNA complexation with phosphorylcholine diblock copolymers and its effect on cell transfection

We examined a series of novel cationic MPC-based (2-methacryloyloxyethyl phosphorylcholine) copolymers as vectors for gene delivery, with emphasis on the assessment of the effects of the charge ratio (administered via pH variation) on the complex (polyplex) formation and the subsequent transfection efficiency. A combination of electrophoresis, dynamic light scattering, and small angle neutron scattering was used to characterize the structure and charge distribution of the polyplexes formed between the copolymer and the luciferase plasmid DNA. Polymers with larger hydrophobic side chains had lower p K a values and tended to aggregate more strongly. For a given copolymer, electrostatic interaction was the main driving force for the formation of the nanopolyplexes. When the cationic copolymers were in excess, the majority of the polyplexes formed was neutral, and only a small faction of them carried net positive charges. Polyplexes formed under excess copolymer protected the DNA from restriction enzyme digestion. As the copolymers were weak polyelectrolytes, the pH had a distinct effect on the structure and charge distribution of the polyplexes formed. Below the p K a, the copolymers were found to bind with the plasmid DNA in the form of unimers, while above the p K a, the copolymers self-aggregated and complexed with DNA in the form of micelles. It was subsequently found that unimer/DNA polyplexes were far more effective in the transfection of HEK293 cells than micellar DNA polyplexes. The results thus revealed that different hydrophobicities of the side chains in the copolymer series led to different nanostructuring and charge characteristics, which had a consequential effect on the transfection efficiency. This study provided useful insight into the molecular processes underlying polyplex formation and demonstrated a strong link between structural and physical properties of polyplexes and cell transfection efficiency.     

Cationic methacrylate copolymers containing primary and tertiary amino side groups: Controlled synthesis via RAFT polymerization,DNA condensation,and in vitro gene transfection

A versatile family of cationic methacrylate copolymers containing varying amounts of primary and tertiary amino side groups were synthesized and investigated for in vitro gene transfection. Two different types of methacrylate copolymers, poly(2‐(dimethylamino)ethyl methacrylate)/aminoethyl methacrylate [P(DMAEMA/AEMA)] and poly(2‐(dimethylamino)ethyl methacrylate)/aminohexyl methacrylate [P(DMAEMA/AHMA)], were obtained by reversible addition‐fragmentation chain transfer (RAFT) copolymerization of dimethylaminoethyl methacrylate (DMAEMA) with N‐(tert‐butoxycarbonyl)aminoethyl methacrylate (Boc‐AEMA) or N‐(tert‐butoxycarbonyl)aminohexyl methacrylate (Boc‐AHMA) followed by acid deprotection. Gel permeation chromatography (GPC) measurements revealed that Boc‐protected methacrylate copolymers had M_n in the range of 16.1–23.0 kDa and low polydispersities of 1.12–1.26. The copolymer compositions were well controlled by monomer feed ratios. Dynamic light scattering and agarose gel electrophoresis measurements demonstrated that these PDMAEMA copolymers had better DNA condensation than PDMAEMA homopolymer. The polyplexes of these copolymers revealed low cytotoxicity at an N/P ratio of 3/1. The in vitro transfection in COS‐7 cells in serum free medium demonstrated significantly enhanced (up to 24‐fold) transfection efficiencies of PDMAEMA copolymer polyplexes as compared with PDMAEMA control. In the presence of 10% serum, P(DMAEMA/AEMA) and P(DMAEMA/AHMA) displayed a high transfection activity comparable with or better than 25 kDa PEI. These results suggest that cationic methacrylate copolymers are highly promising for development of safe and efficient nonviral gene transfer agents. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 2869–2877, 2010     

Polycationic graft copolymers of poly(N-vinylpyrrolidone) as non-viral vectors for gene transfection

Novel graft copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with N-vinylpyrrolidone (NVP) were designed and synthesized by the free radical copolymerization of DMAEMA with precursor polymers of vinyl-functionalized poly(N-vinylpyrrolidone) (PVP). The ability of the PVP- grafted copolymers to bind and condense DNA was confirmed by ethidium bromide displacement assay, agarose gel electrophoresis and transmission electron microscopy. The presence of PVP in the copolymers had a favorable effect on the biophysical properties of polymer/DNA complexes. Colloidal stable complexes obtained from the copolymer systems, were shown to be separate, uniformly spherical nanoparticles by transmission electron microscopy. The approximate diameter of the complexes was 150–200 nm, as determined by dynamic light scattering studies. These results confirm an important role played by the PVP grafts in producing compact stable DNA complexes. The ζ-potential measurements revealed that the incorporation of the PVP grafts reduced the positive surface charge of polymer/DNA complexes. The cytotoxicity of the copolymers decreased with an increasing fraction of PVP. Furthermore, in vitro transfection experiments with these copolymers showed improved ability of transfection in cell culture, demonstrating an important role for PVP grafts in enhancement of the transfection efficiency.      

Thermoresponsive gene carriers based on polyethylenimine-graft-poly[oligo(ethylene glycol) methacrylate

A family of thermoresponsive cationic copolymers (TCPs) that contain branched PEI 25 K as the cationic segment and poly(MEO(2)MA-co-OEGMA(475)) as the thermosensitive block (TP) is prepared. The DNA binding capability, physicochemical properties, and biological performance of the TCPs are studied. All of these TCPs can condense DNA to form polyplexes with diameters of 150-300 nm and zeta potentials of 7-32 mV at N/P ratios between 12 and 36. The length of TP block is a key factor for shielding the positive surface charge of the polyplexes and protecting them against protein adsorption. TCPs with a higher TP content have a lower cytotoxicity while the best transfection performance is achieved by the TCPs with longest TP length, reaching a level of the intact PEI 25 K in the presence of serum.     

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.     

Amphiphilic Block Copolymer Micelles for Gene Delivery

Gene therapy is a promising method to treat acquired and inherited diseases by introducing exogenous genes into specific recipient cells. Polymeric micelles with different nanoscopic morphologies and properties hold great promise for gene delivery system. Conventional cationic polymers, poly(ethyleneimine)(PEI), poly(L-lysine)(PLL), poly(2-dimethyla-minoethyl methacrylate)(PDMAEMA) and novel cationic polymers poly(2-oxazoline)s(POxs), have been incorporated into block copolymers and decorated with targeting moieties to enhance transfection efficiency. In order to minimize cytotoxicity, nonionic block copolymer micelles are utilized to load gene through hydrophilic and hydrophobic interactions or covalent conjugations, recently. From our perspective, properties(shape, size, and mechanical stiffness, etc.) of block copolymer micelles may significantly affect cytotoxicity, transfection efficiency, circulation time, and load capacity of gene vectors in vivo and in vitro. This review briefly sums up recent efforts in cationic and nonionic amphiphilic polymeric micelles for gene delivery.     

Improvement of transfection efficiency by galactosylated N-3-guanidinopropyl methacrylamide-co-poly (ethylene glycol) methacrylate copolymers

GalactosylatedN-3-guanidinopropylmethacrylamide-co-poly (ethylene glycol) methacrylate copolymers (galactosylated GPMA-co-PEGMA, GGP) were developed in order to promote transfection efficiency in the presence of serum in this report. First of all, the galactosylated PEGMA-co-GPMA copolymers were prepared via aqueous reversible addition – fragmentation chain transfer polymerization (RAFT) of poly (ethylene glycol) methacrylate (PEGMA) with long circulating chain segment and N-3-aminopropyl methacrylamide (APMA) followed by galactosylation and guanidinylation. After that, GGP/plasmid DNA complexes were examined by a dynamic light scattering and gel electrophoresis. It is showed that GGP copolymers have effective condensing ability. The cytotoxicity of GGP was measured by MTT assay. It was found that all the GGP/plasmid DNA complexes had less cytotoxic effects on HepG2 cells than HeLa cells, and the galactose groups reduced the cytotoxicity of complexes with high charge ratios to HepG2 cells. Finally, the transfection efficiency of the galactosylated PEGMA-co-GPMA copolymers was investigated by luciferase expression assay. The results revealed that the copolymers with galactose groups more than 5.83% could induce the asialoglycoprotein (ASGP) receptor mediated transfection, which improved the transfection efficiency in target cells. The GPMA-co-PEGMA copolymers with 54.57% hydrophilic chain segment PEG should prevent the aggregation of protein on the GGP/pDNA complexes, and GGP with 7.94% galactose graft exhibited the highest transfection in the presence of serum.     

Impact of Cationic Charge Density and PEGylated Poly(Amino Acid) Tercopolymer Architecture on Their Use as Gene Delivery Vehicles. Part 2: DNA Protection,Stability, Cytotoxicity,and Transfection Efficiency

The impact of the molecular architecture on the transfection efficiency of PEGylated poly(amino acid) block copolymers was investigated for PEG‐b‐p(l ‐Lys)_x‐b‐p(l ‐Leu)_y, PEG‐b‐p(l ‐Leu)_x‐b‐p(l ‐Lys)_y, and PEG‐b‐p((l ‐Leu)_x‐co‐(l ‐Lys)_y). The block lengths of p(l ‐Lys) and p(l ‐Leu) were varied between 10, 20, and 40; and 10 and 20, respectively, to study the influence of the ionic/hydrophobic balance. The results show that ABC triblock copolymers form smaller and more stable polyplexes with plasmid DNA than AB diblock copolymers—as verified by long‐term aggregation and ethidium bromide exclusion studies—protect the DNA more effectively against nucleases, and provide better transfection efficiencies, as indicated by total protein as well as luciferase expression. More detailed studies revealed that triblock copolymers with p(l ‐Leu) forming the C‐block were most efficient in DNA complexation with a 2.3 times higher transfection rate. Furthermore, increasing the cationic character by increasing the p(l ‐Lys) chain length led to up to 25% higher transfection but at the same time induced some cytotoxicity. Diblock copolymers, where the amino acid–building blocks exist as a random copolymer, bind more loosely with DNA leading to less compact and less stable aggregates with lower transfection efficiencies.     

Polyplex Particles Based on Comb‐Like Polyethylenimine/Poly(2‐ethyl‐2‐oxazoline) Copolymers: Relating Biological Performance with Morphology and Structure

The present contribution is focused on feasibility of using comb‐like copolymers of polyethylenimine with poly(2‐ethyl‐2‐oxazoline) (LPEI‐comb‐PEtOx) with varying grafting densities and degrees of polymerization of PEI and PEtOx to deliver DNA molecules into cells. The copolymers form small and well‐defined particles at elevated temperatures, which are used as platforms for binding and condensing DNA. The electrostatic interactions between particles and DNA result in formation of sub‐100 nm polyplex particles of narrow size distribution and different morphology and structure. The investigated gene delivery systems exhibit transfection efficiency dependent on the copolymer chain topology, shape of the polyplex particles, and internalization pathway. Flow cytometry shows enhanced transfection efficiency of the polyplexes with elongated and ellipsoidal morphology. The preliminary biocompatibility study on a panel of human cell lines shows that pure copolymers and polyplexes thereof are practically devoid of cytotoxicity.     

Poly(2-oxazoline)-Based Polyplexes as a PEG-Free Plasmid DNA Delivery Platform

The present study expands the versatility of cationic poly(2-oxazoline) (POx) copolymers as a polyethylene glycol (PEG)-free platform for gene delivery to immune cells, such as monocytes and macrophages. Several block copolymers are developed by varying nonionic hydrophilic blocks (poly(2-methyl-2-oxazoline) (pMeOx) or poly(2-ethyl-2-oxazoline) (pEtOx), cationic blocks, and an optional hydrophobic block (poly(2-isopropyl-2-oxazoline) (iPrOx). The cationic blocks are produced by side chain modification of 2-methoxy-carboxyethyl-2-oxazoline (MestOx) block precursor with diethylenetriamine (DET) or tris(2-aminoethyl)amine (TREN). For the attachment of a targeting ligand, mannose, azide-alkyne cycloaddition click chemistry methods are employed. Of the two cationic side chains, polyplexes made with DET-containing copolymers transfect macrophages significantly better than those made with TREN-based copolymer. Likewise, nontargeted pEtOx-based diblock copolymer is more active in cell transfection than pMeOx-based copolymer. The triblock copolymer with hydrophobic block iPrOx performs poorly compared to the diblock copolymer which lacks this additional block. Surprisingly, attachment of a mannose ligand to either copolymer is inhibitory for transfection. Despite similarities in size and design, mannosylated polyplexes result in lower cell internalization compared to nonmannosylated polyplexes. Thus, PEG-free, nontargeted DET-, and pEtOx-based diblock copolymer outperforms other studied structures in the transfection of macrophages and displays transfection levels comparable to GeneJuice, a commercial nonlipid transfection reagent.     

Temperature‐Sensitive Amphiphilic Non‐Ionic Triblock Copolymers for Enhanced In Vivo Skeletal Muscle Transfection

It is reported that low concentration of amphiphilic triblock copolymers of pMeOx‐b‐pTHF‐b‐pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co‐injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.     

Decorated rods: a "bottom-up" self-assembly of monomolecular DNA complexes

Fluorescence correlation spectroscopy (FCS) and gel electrophoresis measurements are performed to investigate both the number and size of complexes of linear double-stranded DNA (dsDNA) fragments with 1:1 diblock copolymers consisting of a cationic moiety, branched polyethyleneimine (bPEI) of 2, 10, or 25 kDa, covalently bound to a neutral shielding moiety, poly(ethylene glycol) (PEG; 20 kDa). By systematically decreasing the bPEI length, the PEG grafting density along the DNA chain can be directly controlled. For 25 and 10 kDa bPEI-PEG copolymers, severe aggregation is observed despite the presence of the shielding PEG. Upon decreasing the bPEI length to 2 kDa, controlled self-assembly of monomolecular DNA nanoparticles is observed. The resulting complexes are in quantitative agreement with a theoretical model based on a single DNA encased in a dense PEG polymer brush layer. The resulting PEGylated complexes show high stability against both salt and protein and hence are of potential use for in vivo gene delivery studies.     

Poly(N‐methylvinylamine)‐Based Copolymers for Improved Gene Transfection

Poly(N‐methylvinylamines) with secondary amines can form complexes with plasmid DNA (pDNA) and provide transfection efficiency in HeLa cells in the same order as linear polyethyleneimine but with higher cell viability. Chemical modifications of poly(N‐methylvinylamine) backbones are performed to further improve transfection efficiency while maintaining low degree of cytotoxicity. In a first type of polymer, primary amino groups are incorporated via a copolymerization strategy. In a second one, primary amino and imidazole groups are incorporated also via a copolymerization strategy. In a third one, secondary amino groups are substituted with methylguanidine functions through a postpolymerization reaction. Thus, novel polymers of various molecular masses are synthesized, characterized, and their interaction with pDNA studied. Then, their transfection efficiency and cytotoxicity are tested in HeLa cells. Two polymethylvinylamine‐based copolymers, one containing 20% of imidazole moieties and another one composed of 12% of guanidinyl units allow remarkable transfection efficiency of HeLa, pulmonary (16HBE), skeletal muscle (C2C12), and dendritic (DC2.4) cells. Overall, this work thus identifies new promising DNA carriers and chemical modifications that improve the transfection efficiency while maintaining low degree of cytotoxicity.