Mixtures of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)-g-poly(ethylene glycol) for improved gene delivery

Branched disulfide-containing poly(amido ethyleneimines) (SS-PAEIs) are biodegradable polymeric gene carrier analogues of the well-studied, nondegradable, and often toxic branched polyethylenimines (bPEIs), but with distinct advantages for cellular transgene delivery. Clinical success of polycationic gene carriers is hampered by obscure design and formulation requirements. This present work reports synthetic and formulation properties for a graft copolymer of poly(ethylene glycol) (PEG) and a branched SS-PAEI, poly(triethylentetramine/cystaminebisacrylamide) (p(TETA/CBA)). Several laboratories have previously demonstrated the advantages of PEG conjugation to gene carriers, but have also shown that PEG conjugation may perturb plasmid DNA (pDNA) condensation, thereby interfering with nanoparticle formation. With this foundation, our studies sought to mix various amounts of p(TETA/CBA) and p(TETA/CBA)-g-PEG2k to alter the relative amount of PEG in each formulation used for polyplex formation. The influence of different PEG/polycation amounts in the formulations on polymer/nucleic acid nanoparticle (polyplex) size, surface charge, morphology, serum stability and transgene delivery was studied. Polyplex formulations were prepared using p(TETA/CBA)-g-PEG2k, p(TETA/CBA), and mixtures of the two species at 10/90 and 50/50 volumetric mixture ratios (wt/wt %), respectively. As expected, increasing the amount of PEG in the formulation adversely affects polyplex formation. However, optimal polymer mixtures could be identified using this facile approach to further clarify design and formulation requirements necessary to understand and optimize carrier stability and biological activity. This work demonstrates the feasibility to easily overcome typical problems observed when polycations are modified and thus avoids the need to synthesize multiple copolymers to identify optimal gene carrier candidates. This approach may be applied to other polycation-PEG preparations to alter polyplex characteristics for optimal stability and biological activity.     

Physiochemical properties of low and high molecular weight poly(ethylene glycol)-grafted poly(ethylene imine) copolymers and their complexes with oligonucleotides

Inefficient delivery of antisense oligonucleotides (AOs) to target cell nuclei remains as the foremost limitation to their usefulness. Copolymers of cationic poly(ethylene imine) (PEI) and poly(ethylene glycol) (PEG) have been well-studied for delivery of plasmids. However, the properties of PEG-PEI-AO polyplexes have not been comprehensively investigated. Therefore, we synthesized a series of PEG-PEI copolymers and evaluated their physiochemical properties alone and when complexed with AO. The M(w) of PEG was found to be the main determinant of polyplex size, via its influence on particle aggregation. DLS measurements showed that when PEG5000 was grafted to PEI2K and PEI25K, polyplex diameters were extremely small (range 10-90 nm) with minimal aggregation. In contrast, when PEG550 was grafted to PEI2K and PEI25K, polyplexes appeared as much larger aggregates (approximately 250 nm). As expected, the surface charge (zeta potential) was higher for polyplexes containing PEI25K than those containing PEI2K, but decreased with increased levels of PEG grafting. Surprisingly, within the physiological range (pH 7.5-5), the buffering capacity of all copolymers was nearly equivalent to that of unsubstituted PEI2K or PEI25K, and was barely influenced by PEGylation. The stability of polyplexes was evaluated using a heparin polyanion competition assay. Unexpectedly, polyplexes containing PEI2K showed stability equal to or greater than that of PEI25K polyplexes. The level of PEG grafting also had a dramatic effect on polyplex stability. The relationships established between molecular formulations and polyplex size, aggregation, surface charge, and stability should provide a useful guide for future studies aimed at optimizing polymer-mediated AO delivery in cell and animal studies. A summary of the relationships between polyplex structures and recent studies of their transfection capacity is provided.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes

Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.     

Cyclic RGD peptide-conjugated polyplex micelles as a targetable gene delivery system directed to cells possessing alphavbeta3 and alphavbeta5 integrins

A cyclic RGD peptide-conjugated block copolymer, cyclo[RGDfK(CX-)]-poly(ethylene glycol)-polylysine (c(RGDfK)-PEG-PLys), was synthesized from acetal-PEG-PLys under mild acidic conditions and spontaneously associated with plasmid DNA (pDNA) to form a polyplex micelle in aqueous solution. The cyclic RGD peptide recognizes alphavbeta3 and alphavbeta5 integrin receptors, which play a pivotal role in angiogenesis, vascular intima thickening, and the proliferation of malignant tumors. The c(RGDfK)-PEG-PLys/pDNA polyplex micelle showed a remarkably increased transfection efficiency (TE) compared to the PEG-PLys/pDNA polyplex micelle for the cultured HeLa cells possessing alphavbeta3 and alphavbeta5 integrins. On the other hand, in the transfection against the 293T cells possessing no alphavbeta3 and a few alphavbeta5 integrins, the TE of the c(RGDfK)-PEG-PLys/pDNA micelle showed no increase compared to the TE of the PEG-PLys/pDNA micelle. Flow cytometric analysis revealed a higher uptake of the c(RGDfK)-PEG-PLys/pDNA micelle than the PEG-PLys/pDNA micelle against HeLa cells, consistent with the transfection results. Furthermore, a confocal laser scanning microscopic observation revealed that the pDNA in the c(RGDfK)-PEG-PLys micelle preferentially accumulated in the perinuclear region of the HeLa cells within 3 h of incubation. No such fast and directed accumulation of pDNA to the perinuclear region was observed for the micelles without c(RGDfK) ligands. These results indicate that the increase in the TE induced by the introduction of the c(RGDfK) peptide ligand was due to an increase in cellular uptake as well as facilitated intracellular trafficking of micelles toward the perinuclear region via alphavbeta3 and alphavbeta5 integrin receptor-mediated endocytosis, suggesting that the cyclic RGD peptide-conjugated polyplex micelle has promising feasibility as a site-specifically targetable gene delivery system.     

Synthesis of a biocleavable polyrotaxane-plasmid DNA (pDNA) polyplex and its use for the rapid nonviral delivery of pDNA to cell nuclei

This protocol provides a method for synthesizing a biocleavable polyrotaxane/plasmid DNA (pDNA) polyplex and for using it to deliver pDNA into cell nuclei. The biocleavable polyrotaxane is synthesized in four steps: (i) introduction of disulfide linkages at both terminals of PEG, (ii) preparation of an inclusion complex between disulfide-containing PEG and alpha-cyclodextrins (alpha-CDs), (iii) synthesis of polyrotaxane and (iv) modification of alpha-CDs in the polyrotaxane with dimethylethylenediamine. A polyplex of pDNA with the polyrotaxane is formed when the two compounds are dissolved together in a phosphate buffer. Subcellular localization of rhodamine-labeled pDNA in fluorescently labeled organelles is quantified by Z-series of confocal images captured by confocal laser scanning microscopy. Significant amounts of pDNA delivered to the nucleus can be expected as well as high transfection activity of the polyplex. This protocol can be completed in 23-32 d.     

Simple Branched Arginine-Based Structures can Enhance the Cellular Uptake of Peptide Cargos

In an attempt to design delivery vehicles to enable epitope-based vaccine uptake, we investigated the properties of a variety of synthetic, branched cationic structures. We found that branched compounds based on arginine or lysine were able to facilitate internalization of peptide cargo into cells to different degrees. Branched constructs containing only two arginine residues (R₂) were not only able to bind to cells more efficiently than constructs with two lysine residues (K₂) but were also internalized within vesicle like compartments in the cell. The extent of binding and uptake was enhanced when additional arginine residues were incorporated to form a tetra arginine construct (R₄). An investigation into the kinetics and dose dependence of cellular uptake of these arginine-based constructs demonstrated that binding and internalization is a rapid and efficient event. We also found uptake of the peptide epitope TYQRTRALV was enhanced when it was coupled to R₄. This approach may prove useful for introducing peptide epitopes into antigen presenting cells as self-adjuvanting structures and also for delivery of other peptides into different specialized cells.     

pH-sensitive cationic polymer gene delivery vehicle: N-Ac-poly(L-histidine)-graft-poly(L-lysine) comb shaped polymer

Advancing biotechnology spurs the development of new pharmaceutically engineered gene delivery vehicles. Poly(L-histidine) ?PLH? has been shown to induce membrane fusion at endosomal pH values, whereas PLL has a well documented efficacy in polyplex formation. Therefore, N-Ac-poly(L-histidine)-graft-poly(L-lysine) ?PLH-g-PLL? was synthesized by grafting poly(L-histidine) to poly(L-lysine) ?PLL?. PLH-g-PLL formed polyplex particles by electrostatic interactions with plasmid DNA ?pDNA?. The mean particle size of the polyplexes was in the range of 117 +/- 6 nm to 306 +/- 77 nm. PLH-g-PLL gene carrier demonstrated higher transfection efficacy in 293T cells than PLL at all equivalent weight ratios with pDNA. The inclusion of chloroquine as an endosomolytic agent enhanced transfection for both PLL and PLH-g-PLL gene carriers. PLH-g-PLL enhanced beta-galactosidase expression compared to PLL, but still increased in efficacy when chloroquine was included.     

Amphiphilic block copolymers enhance cellular uptake and nuclear entry of polyplex-delivered DNA

This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IkappaB and subsequent NFkappaB nuclear localization as well as upregulate numerous NFkappaB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes ("polyplexes") in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation, and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFkappaB binding sites, but have no effect on import of pDNA without these sites. Altogether, our studies suggest that Pluronics rapidly activate NFkappaB, which binds cytosolic pDNA that possesses promoters containing NFkappaB binding sites and consequently increase nuclear import of pDNA through NFkappaB nuclear translocation.     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Enhancement of star vector-based gene delivery to endothelial cells by addition of RGD-peptide

This study aimed to investigate the feasibility of using a cationic nonviral gene carrier in endothelial cells for enhancing gene expression by the addition of an integrin-binding RGD peptide. A 4-branched cationic polymer of poly( N,N-dimethylaminopropylacrylamide) (star vector), developed as a gene carrier, could complex with the luciferase-encoding plasmid DNA under a charge ratio of 5 (vector/pDNA) to form polymer/DNA complexes (polyplexes). The addition of the RGD-containing peptide (GRGDNP) to the polyplex solution led to a decrease in the zeta-potential from ca. +30 to +20 mV along with the reduction in the particle size from ca. 300 to 200 nm. Additionally, a marked inhibition of polyplex aggregation was observed, indicating the coating of the polyplex surface with RGD peptides. A transfection study on endothelial cells showed that the luciferase activity increased with the amount of RGD peptides added to the polyplexes and exhibited minimal cellular cytotoxicity. The transfection activity further increased when cyclic RGD peptides (RGDFV) were used; the activity with RGD peptide addition was approximately 8-fold compared to that without RGD peptide addition. Gene delivery to endothelial cells was significantly enhanced by only the addition of RGD peptides to star vector-based polyplexes.     

Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

Amino acid uptake and incorporation not cell-specific peptides and evidence for intracellular peptide pools in Aplysia neurons R3-R14

1. Relationships between intracellular amino acid concentrations and uptake rates and their utilization in synthesis of cell-specific peptides in neurons R3-R14 in the Aplysia parietovisceral ganglion are explored. 2. The uptake rates and intracellular concentrations of most amino acids are positively correlated and inversely related to their degree of incorporation into the peptides. 3. The bulk cellular pool of arginine is probably utilized in the synthesis of R3-R14 peptides, but much of the glycine taken up appears not to be readily available for protein synthesis. 4. There are rapidly and slowly turning over pools of the peptides, and portions of the peptides stay in the cell bodies for days.     

Complement activation and cell uptake responses toward polymer-functionalized protein nanocapsules

Self-assembling protein nanocapsules can be engineered for various bionanotechnology applications. Using the dodecahedral scaffold of the E2 subunit from pyruvate dehydrogenase, we introduced non-native surface cysteines for site-directed functionalization. The modified nanoparticle's structural, assembly, and thermostability properties were comparable to the wild-type scaffold (E2-WT), and after conjugation of poly(ethylene glycol) (PEG) to these cysteines, the nanoparticle remained intact and stable up to 79.7 ± 1.8 °C. PEGylation of particles reduced uptake by human monocyte-derived macrophages and MDA-MB-231 breast cancer cells, with decreased uptake as PEG chain length is increased. In vitro C4-depletion and C5a-production assays yielded 97.6 ± 10.8% serum C4 remaining and 40.1 ± 6.0 ng/mL C5a for E2-WT, demonstrating that complement activation is weak for non-PEGylated E2 nanoparticles. Conjugation of PEG to these particles moderately increased complement response to give 79.7 ± 6.0% C4 remaining and 87.6 ± 10.1 ng/mL C5a. Our results demonstrate that PEGylation of the E2 protein nanocapsules can modulate cellular uptake and induce low levels of complement activation, likely via the classical/lectin pathways.     

The role of assembly parameters on polyplex poly(beta-amino ester) nanoparticle transfections

Intracellular delivery of nucleic acids to mammalian cells using polyplex nanoparticles (NPs) remains a challenge both in vitro and in vivo, with transfections often suffering from variable efficacy. To improve reproducibility and efficacy of transfections in vitro using a next-generation polyplex transfection material poly(beta-amino ester)s (PBAEs), the influence of multiple variables in the preparation of these NPs on their transfection efficacy was explored. The results indicate that even though PBAE/pDNA polyplex NPs are formed by the self-assembly of polyelectrolytes, their transfection is not affected by the manner in which the components are mixed, facilitating self-assembly in a single step, but timing for self-assembly of 5–20 min is optimal. In addition, even though the biomaterials are biodegradable in water, their efficacy is not affected by up to eight freeze-thaw cycles of the polymer. It was found that there is a greater stability of nucleic acid-complexed polymer as a polyplex nanoparticle compared with free polymer. Finally, by exploring multiple buffer systems, it was identified that utilization of divalent cation magnesium or calcium acetate buffers at pH 5.0 is optimal for transfection using these polymeric materials, boosting transfection several folds compared with monovalent cations. Together, these results can improve the reproducibility and efficacy of PBAE and similar polyplex nanoparticle transfections and improve the robustness of using these biomaterials for bioengineering and biotechnology applications.     

Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing

Nonviral vectors, with their low immunogenicity and lack of pathogenicity, offer significant promise for siRNA therapy with fewer safety concerns. Nonviral vectors were also preferred in most transient siRNA delivery due to their ease of preparation. Previously, we incorporated tertiary amines and polyethylene glycol (PEG) into poly(ester urethane) to synthesize a soluble poly(amino ester glycol urethane), PaE(G)U, as a novel DNA transfection reagent for transgene delivery. The aim of this study was to develop PaE(G)U/siRNA polyplexes for gene silencing. We characterized the properties of PaE(G)U/siRNA polyplexes and compared them with those of PaE(G)U/DNA polyplexes. Using the Alexa Fluor 488-labeled, nonsilencing control siRNA as the reporter, we visualized cellular uptake of PaE(G)U/siRNA polyplexes and optimized the mass ratio of PaE(G)U/siRNA for delivery at 80/1. At this ratio, the average diameter of polyplexes was 540 nm, which was significantly larger than the average diameter of PaE(G)U/DNA polyplexes at 155 nm for efficient DNA delivery. Using the optimized PaE(G)U/siRNA polyplexes, transient silencing of constitutive luciferase expression (up to 92%) was achieved in our recombinant human HT-1080 fibroblast model via anti-luciferase siRNA delivery. In conclusion, PaE(G)U/siRNA polyplexes were developed and optimized for cellular uptake to allow efficient gene silencing. Engineering of soluble biodegradable polymers to incorporate amino, ester, PEG, and urethane units in the backbone constitutes a useful approach for the future design of siRNA carriers.     

新型PEI 衍生物在COS-7 细胞中的转染与毒性研究

目的:寻找一种新型的转染效率高,毒性低的非病毒基因载体.方法:通过化学方法合成Polyimine-MPEI,然后以不同质量比包裹绿色荧光蛋白质粒,检测在COS-7细胞中的转染效率和毒性.结果:在比例从5到100之间,转染效率均比较理想,能达到1.00E+07以上,Polyimine-MPEI的毒性也很小,细胞的生长率均在80％以上,明显高于PEI25KDa对照组.结论:Polyimine-MPEI是一个很有研究前景的聚合物载体,具有高转染效率低毒性的特点,可以通过延长反应时间,增加分子量,增大转染能力.     

Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.     

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: To enhance the efficiency of recombinant DNA utilization

In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60–70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG‐containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl‐PEI, was chemically synthesized. Using Design of Experiments–Response Surface Modeling to optimize the transfection process, the function of propyl‐PEI was compared to that of unmodified PEI in both parental CHO‐S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl‐PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1161–1170, 2014