Synthesis and gene transfection efficacies of PEI-cholesterol-based lipopolymers

Nine lipopolymers based on low molecular weight polyethyleneimines (PEI) and cholesterol via an ether linkage between the polymer amine and the cholesterol backbone have been synthesized. Different percentage of cholesterol moieties have been grafted on three types of PEI of molecular weights 800, 1200, and 2000. These lipopolymers were studied for gene transfection activities in HeLa cells. All the lipopolymers were first optimized for enhanced transfection efficacies as coliposomes with DOPE. All lipopolymers are better transfecting agents and highly serum compatible than commercially available PEI-25KDa. Transfection efficacies and serum compatibility of lipopolymers were found to be dependent upon the MW of PEI used for lipopolymer synthesis and percentage of cholesterol grafting on lipopolymers. Cell viability assay showed that PEI-25KDa is highly toxic as compared to all the lipopolymers. Lipopolyplexes were characterized by transmission electron microscopy, which showed the presence of spherical aggregates.     

Water-soluble lipopolymer for gene delivery

The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.     

Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy

Polyethylenimines (PEI) constitute efficient nonviral vectors for gene transfer. However, because free PEI shows some cytotoxicity and because intracellular dissociation of PEI/DNA complexes seems to be required for efficient transfection, it is important to monitor the concentrations of free and bound partners in the mixtures of DNA and PEI used for transfection. To reach this objective, we used fluorescence correlation spectroscopy with two-photon excitation to characterize the complexes formed with either rhodamine-labeled 25 kDa PEI or DNA plasmid molecules. At the molar ratios of PEI nitrogen atoms to DNA phosphate usually used for transfection, we found that approximately 86% of the PEI molecules were in a free form. The PEI/DNA complexes are composed on the average by 3.5 (+/-1) DNA plasmids and approximately 30 PEI molecules. From this composition and the pK(a) of PEI, it could be inferred that in contrast to DNA condensation by small multivalent cations, only a limited neutralization of the DNA phosphate groups is required for DNA condensation by PEI. Moreover, DNA appears only poorly compacted in the PEI/DNA complexes. As an application, fluorescence correlation spectroscopy was used to monitor the purification of PEI/DNA complexes by ultrafiltration as well as the heparin-induced dissociation of the complexes.     

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.     

Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.     

交联聚乙烯亚胺衍生物的构建及其转染COS-7 细胞的研究

目的:优化构建交联聚乙烯亚胺(Polyethylenemine,PEI)衍生物PEI-Bu,研究其对非洲绿猴肾成纤维细胞系(COS-7)的转染活性和细胞毒性。方法:以PEI 800Da为骨架,1,4-丁二醇二氯甲酸酯为连接剂制备聚合物PEI-Bu,琼脂糖凝胶电泳考察其复合质粒DNA的能力,MTT法检测PEI-Bu对COS-7的毒性,以荧光素酶质粒作为报告基因,测定PEI-Bu/DNA复合物在COS-7细胞的转染活性。结果:凝胶电泳表明PEI-Bu/DNA在质量比大于1时即具有复合DNA的能力,PEI-Bu的细胞毒性随浓度增大而增大,在同一浓度下PEI-Bu的细胞毒性小于PEI 25kDa,(P<0.05),PEI-Bu/DNA在质量比为5时达到最高转染活性,高于PEI 25kDa(P<0.01),并与Lipofectamine2000相当(P>0.05)。结论:PEI-Bu在COS-7细胞中是一种低细胞毒性、高转染活性的非病毒基因载体(与商业化的PEI 25kDa比较),其在基因治疗领域中具有潜在的应用前景。     

Hyperbranched poly(amino ester)s with different terminal amine groups for DNA delivery

Hyperbranched poly(amino ester)s containing tertiary amines in the core and primary, secondary, and tertiary amines in the periphery, respectively, were evaluated for DNA delivery in vitro. The same core structure facilitated the investigation on the effects of the terminal amine type on the properties of hyperbranched poly(amino ester)s for DNA delivery. The hydrolysis of the poly(amino ester)s was monitored using (1)H NMR. The results reflected that the terminal amine type had negligible effects on the hydrolysis rate but was much slower than that of linear poly(amino ester)s, probably due to the compact hyperbranched spatial structure preventing the accessibility of water. In comparison with PEI 25 K, the hyperbranched poly(amino ester)s showed much lower cytotoxicity in Cos7, HEK293, and HepG2 cells. Gel electrophoresis indicated that poly(amino ester)s could condense DNA efficiently, and the zeta potentials and sizes of the complexes formed with different weight ratios of hyperbranched poly(amino ester)s and DNA were measured. Remarkably, all the hyperbranched poly(amino ester)s showed DNA transfection efficiency comparable to PEI 25 K in Cos7, HEK293, and HepG2 cells regardless of the terminal amine type. Therefore, the terminal amine type had insignificant effects on the hydrolysis rate, cytotoxicity, DNA condensation capability, and in vitro DNA transfection efficiency of the hyperbranched poly(amino ester)s.     

Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine.

Polyethylenimine (PEI) is a polycation with potential application as a nonviral vector for gene delivery. Here we show that after conjugation with homobifunctional amine reactive reducible cross-linking reagents, low molecular weight polyethylenimine efficiently mediates in vitro gene delivery to Chinese hamster ovary (CHO) cells. Two cross-linking reagents, dithiobis(succinimidylpropionate) (DSP) and dimethyl.3,3'-dithiobispropionimidate*2HCl (DTBP), were utilized based on their reactivity and chemical properties. Both reagents react with primary amines to form reducible cross-links; however, unlike DSP, the DTBP cross-linker maintains net polymer charge through amidine bond formation. PEI with a reported weight-average molecular weight (M(w)) of 800 Da was reacted with either DSP or DTBP at PEI primary amine:cross-link reactive group ratios of 1:1 and 2:1. The transfection efficiencies of the resulting cross-linked products were evaluated in CHO cells using a luciferase reporter gene under a cytomegalovirus (CMV) promoter. Our results show that cross-linked polymers mediate variable levels of transfection depending on the cross-linking reagent, the extent of conjugation, and the N/P ratio. In general, we found conjugate size to be proportional to gene transfer efficiency. Using gel retardation analysis, we also evaluate the capacity of the cross-linked polymers to condense plasmid DNA before and after reduction with 45 mM dithiothreitol (DTT). DTT mediated reduction of intra-cross-link disulfide bonds and inhibited condensation of DNA by conjugates cross-linked with DSP at a ratio of 1:1, but had little effect on the remaining polymers. Analogous intracellular reduction of transfection complexes by reduced glutathione could facilitate uncoupling of PEI from DNA to enhance gene expression.     

PEI-g-chitosan, a novel gene delivery system with transfection efficiency comparable to polyethylenimine in vitro and after liver administration in vivo

Polyethylenimine-graft-chitosan (PEI-g-chitosan) was synthesized by performing cationic polymerization of aziridine in the presence of water-soluble oligo-chitosan (M(n) = 3400). The absolute molecular weight and chemistry of the PEI-g-chitosan obtained were characterized using GPC, 13C and 1H NMR, respectively. The results indicated that all the amines of chitosan were grafted with oligo-PEI, and the average length of the oligo-PEI side chains was determined by the feed molar ratio of aziridne/amine in chitosan. PEI-g-chitosan of M(n) = 7400 with a polydispersity index (PDI) of 1.50, and PEI side chains of M(n) = 206 was prepared for gene delivery. Gel electrophoresis showed that DNA migration was retarded completely at a N/P ratio of 2.5/1, indicating good DNA condensation capability of PEI-g-chitosan. The sizes and the zeta-potentials of the complexes of PEI-g-chitosan/DNA were characterized. The cytotoxicity of PEI-g-chiotsan was evaluated, and the results reflected that PEI-g-chitosan had a lower cytotoxicity than PEI (25 K). Gene transfection efficiency of PEI-g-chitosan in HepG2, HeLa, and primary hepatocytes cells and after administration in the common bile duct of rat liver was determined. Remarkably, PEI-g-chitosan showed a higher transfection efficiency than that of PEI (25 K) both in vitro and in vivo. The systematic distribution and the distribution in liver of the gene expression of the complexes of PEI-chitosan/DNA were determined as well.     

Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent

New lipopolymers were synthesized by conjugating cholic acid (ChA) to polyethylenimines (PEI; 2 and 25 kDa) and a polyallylamine (PAA; 15 kDa) via N‐acylation to develop effective gene delivery systems. The extent of ChA substitution linearly varied with the feed ratio during synthesis, indicating good control over grafting ratio. While ChA did not affect binding to plasmid DNA (pDNA) for higher molecular weight (MW) polymers, ChA substitution to 2 kDa PEI significantly affected the pDNA binding. Toxicity of the 2 kDa PEI was unaffected by ChA substitution, but it was improved for the higher MW polymers. Using immortal 293T cells and primary cord blood‐derived mesenchymal stem cells, low MW (2 kDa) PEI was shown to display much better transfection efficiency as a result of ChA substitution, unlike the higher MW polymers. We conclude that ChA could be a suitable substituent for non‐toxic (low MW) PEIs in order to improve their transfection efficiency. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1337–1341, 2013     

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) for gene delivery

Ruthenium(II) tris(bipyridine)-centered poly(ethylenimine) (Ru PEI) was synthesized via acid hydrolysis of Ru tris(bipyridine)-centered poly(2-ethyl-2-oxazoline) (Ru PEOX), and the luminescence, DNA entrapment, and transfection efficiencies were evaluated. Emission maxima for Ru PEI samples are red-shifted compared to Ru PEOX precursors, and the luminescence lifetimes are shorter in both methanol and aqueous solutions. Slower oxygen quenching of Ru PEOX and Ru PEI luminescence versus [Ru(bpy)3]Cl2 (bpy = bipyridine) is attributed to polymer shielding effects. Ru PEI luminescence is similar in the presence and absence of DNA. Ru PEI (7900 Da) and linear PEI (L-PEI; 22,000 Da) fully entrapped DNA (5.4 kb; pcDNA) at an N/P ratio of 2. LNCaP prostate cancer cells were transfected with a plasmid encoding for green fluorescent protein using Ru PEI and L-PEI vectors for comparison. For N/P = 48, the transfection efficiency for Ru PEI was approximately 50% relative to that of L-PEI.     

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.     

TACN-based cationic lipids with amino acid backbone and double tails: Materials for non-viral gene delivery

Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.     

Enhancing transfection efficiency using polyethylene glycol grafted polyethylenimine and fusogenic peptide

This study presents a new formulation method for improving DNA transfection efficiency using a fusogenic peptide and polyethylene glycol grafted polyethylenimine. Succinimidyl succinate polyethylene glycol (PEG-SSA) was conjugated with polyethylenimine (PEI). PEI is well known for a good endosomal escaping and DNA condensing agent. The positively charged synthetic fusogenic peptide, KALA, was coated on the negatively charged PEG-g-PEI/DNA and PEI/DNA complexes. The KALA/PEI/DNA complexes exhibited aggregation behavior at higher KALA coating amounts with an effective diameter of around 1,000 nm. However, the KALA/PEG-g-PEI/DNA complexes were 100–300 nm in size with a surface zeta-potential (ζ) value of about +20 mV. The conjugated PEG molecules suppressed any KALA-mediated inter-particle aggregation, and thereby improved the transfection efficiency. Consequently, the transfection efficiency of the KALA/PEG-g-PEI/DNA complexes was obtained by utilizing both the fusogenic activity of KALA and the steric repulsion effect of PEC.     

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions with l-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.     

Evaluation of hyperbranched poly(amino ester)s of amine constitutions similar to polyethylenimine for DNA delivery

New hyperbranched poly(amino ester)s were synthesized via A3 + 2BB'B' ' approach, represented by the Michael addition polymerization of trimethylol-propane triacrylate (TMPTA) (A3-type monomers) with a double molar 1-(2-aminoethyl)piperazine (AEPZ) (BB'B'-type monomer) performed in chloroform at ambient temperature. The results obtained by in situ monitoring the polymerization using NMR and MS indicated that hyperbranched poly(TMPTA1-AEPZ2) was formed via a A(B'B')2 intermediate, and the B' ' (the formed 2 degrees amine) was kept intact in the reaction. Therefore, poly(TMPTA1-AEPZ2) contained secondary and tertiary amines in the core and primary amines in the periphery similar to polyethylenimine (PEI). The chemistry of protonated poly(TMPTA1-AEPZ2) was further confirmed by 13C NMR, and the molecular weight, the radius of gyration (Rg), and the hydrodynamic radius (Rh) were determined using GPC, small-angle X-ray scattering (SAXS), and laser dynamic light scattering (LDLS), respectively. The ratio of Rg/Rh of ca. 1.1 verified the hyperbranched structure. Protonated hyperbranched poly(TMPTA1-AEPZ2) is degradable and less cytotoxic as compared with PEI (25 K). Gel electrophoresis reflected that stable complexes could be formed from protonated hyperbranched poly(TMPTA1-AEPZ2) and DNA, and the size and xi-potential of the complexes were characterized. Remarkably, protonated hyperbranched poly(TMPTA1-AEPZ2) showed transfection efficiency comparable to PEI (25 k) for in vitro DNA delivery.     

A method to increase the bioactivity of plasmid DNA by heat treatment

A method to increase the bioactivity of plasmid DNA by heat treatment has been developed. The structure of the heat treated plasmid DNA was investigated by electrophoresis assay and atomic force microscope (AFM) observation. Electrophoresis assay showed that the heat treated DNA consisted of three components: the supercoiled DNA (component I), the open circular DNA (component II) and the heat denatured DNA component. The bioactivity of the heat treated plasmid DNA was investigated by both DNA condensation experiments and gene transfection experiment with mammal cells. DNA condensation experiments showed that the heat denatured DNA component owned higher sensitivity to spermidine and polyethylenimine (PEI) than component I and component II DNA. Gene transfection experiment with PEI indicated that the heat treated DNA had higher gene transfection efficiency than untreated DNA. Our experiment not only shows an effective approach to increase the bioactivity of plasmid DNA but also leads a way to improve the bioactivity of DNA by physically modifying their structure.     

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.     

A rapid and inexpensive labeling method for microarray gene expression analysis

Background

Polyethyleneimine (PEI), a cationic polymer, is one of the successful and widely used vectors for non-viral gene transfection in vitro. However, its in vivo application was greatly limited due to its high cytotoxicity and short duration of gene expression. To improve its biocompatibility and transfection efficiency, PEI has been modified with PEG, folic acid, and chloroquine in order to improve biocompatibility and enhance targeting.

Results

Poly(ε-caprolactone)-Pluronic-Poly(ε-caprolactone) (PCFC) was synthesized by ring-opening polymerization, and PCFC-g-PEI was obtained by Michael addition reaction with GMA-PCFC-GMA and polyethyleneimine (PEI, 25 kD). The prepared PCFC-g-PEI was characterized by ¹H-NMR, SEC-MALLS. Meanwhile, DNA condensation, DNase I protection, the particle size and zeta potential of PCFC-g-PEI/DNA complexes were also determined. According to the results of flow cytometry and MTT assay, the synthesized PCFC-g-PEI, with considerable transfection efficiency, had obviously lower cytotoxicity against 293 T and A549 cell lines compared with that of PEI 25 kD.

Conclusion

The cytotoxicity and in vitro transfection study indicated that PCFC-g-PEI copolymer prepared in this paper was a novel gene delivery system with lower cytotoxicity and considerable transfection efficiency compared with commercial PEI (25 kD).     

Triple interpolyelectrolyte complexes DNA-polycation-polyanion: a new approach to optimization of mixtures for transfection in eukaryotic cells

A new approach to optimization of mixtures for the condensation and introduction of plasmid DNA into eukaryotic cells is proposed, which is based on the formation of ternary interpolyelectrolyte complexes (IPEC) DNA/polycation/polyanion. Polyethyleneimine (PEI) with M 30-40 kDa as polycation and polyacrylic acid (PA) with M 20 kDa or its grafted copolymer with polyethyleneglycol (PEG) as polyanion were used, and ternary complexes with various ratios of the components were prepared. The PA-PEG incorporation into a ternary complex (by itself or as a 1:1 mixture with PA) was shown to confer the solubility onto complexes in a wide range of DNA/PEI ratios. Incorporation of even minute amounts of PA-PEG (as a 1:9 mixture with PA), while not completely preventing the aggregation of ternary IPEC, drastically changed their sorption characteristics. Using a beta-galactosidase-encoding plasmid, efficiencies of transfection of the CHO-AA8 and 293 cells for different IPEC and DNA/lipofectin complex were compared. The maximum efficiency was exhibited by ternary complex DNA/PEI/polyanion where a 1:1 mixture of PA and PA-PEG was used as polyanion. Possible reasons for this effect and further ways of optimization of mixtures for expression of plasmid DNA in the context of the new approach are discussed.