Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Physicochemical and biological characterization of polyethylenimine-graft-poly(ethylene glycol) block copolymers as a delivery system for oligonucleotides and ribozymes

Two different series of polyethylenimine (PEI) block copolymers grafted with linear poly(ethylene glycol) (PEG) were investigated as delivery systems for oligodeoxynucleotides (ODN) and ribozymes. The resulting interpolyelectrolyte complexes were characterized with respect to their physicochemical properties, protection efficiency against enzymatic degradation, complement activation, and biological activity under in vitro conditions. The effect of PEG molecular weight and the graft density of PEG blocks on complex characteristics was studied with two different series of block copolymers. The resulting ODN complexes were characterized by photon correlation spectroscopy (PCS) and laser Doppler anemometry (LDA) to determine complex size and zeta potential. Electrophoresis was performed to study the protective effects of the different block copolymers against enzymatic degradation of ODN. Intact ODN was quantified via densitometric analysis. Ribozymes, a particularly unstable type of oligonucleotides, were used to examine the influence of block copolymer structure on biological activity. The stabilization of ribozymes was also characterized in a cell culture model. Within the first series of block copolymers, the grafted PEG chains (5 kDa) had marginal influence on the complex size. Two grafted PEG chains were sufficient to achieve a neutral zeta potential. Within the second series, size and zeta potential increased with an increasing number of PEG chains. A high number of short PEG chains resulted in a decrease in complex size to values comparable to that of the homopolymer PEI 25 kDa and a neutral zeta potential, indicating a complete shielding of the charges. Complement activation decreased with an increasing number of short PEG 550 Da chains. Ribozyme complexes with PEG-PEI block copolymers achieved a 50% down-regulation of the target mRNA. This effect demonstrated an efficient stabilization and biological activity of the ribozyme, which was comparable to that of PEI 25 kDa. PEGylated PEI block copolymers represent a promising new class of drug delivery systems for ODN and ribozymes with increased biocompatibility and physical stability.     

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.     

Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility

Soluble supramolecular inclusion complexes were formed by threading alpha-cyclodextrin (alpha-CD) molecules over poly(ethylene glycol) (PEG) and poly(epsilon-caprolactone) (PCL) chains of ternary block copolymers of PEG, PCL and polyethylenimine (PEI). Characteristic shifts of PCL absorptions in FTIR, (1)H NMR and UV spectra strongly suggest that alpha-CD is threaded over PEG and PCL blocks. Due to the reduced hydrophobic interaction between PCL blocks, the resulting supramolecular complexes displayed a dramatically increased solubility, in comparison with the ternary block copolymers. Their ability to complex DNA was almost as efficient as that of branched PEI 25 kDa, as shown in the ethidium bromide fluorescence quenching experiments. Resulting DNA polyplexes displayed a size of around 200 nm and a neutral surface charge. Microscopy studies in 3T3 fibroblasts revealed an efficient cellular uptake. Transfection efficiencies of inclusion complexes were in the same order of magnitude as PEI. In contrast to PEI a 100x lower toxicity was observed by MTT-assay, allowing the administration of nitrogen-to-phosphate ratios of up to 20. These new gene delivery systems merit further characterization under in vivo conditions.     

Efficient suppression of secretory clusterin levels by polymer-siRNA nanocomplexes enhances ionizing radiation lethality in human MCF-7 breast cancer cells in vitro

Small interfering RNA molecules (siRNA) hold great promise to specifically target cytoprotective factors to enhance cancer therapy. Like antisense RNA strategies, however, the use of siRNA is limited because of in vivo instability. As a first step to overcome delivery issues, a series of graft copolymers of polyethylene glycol and polyethylenimine (PEI-g-PEG) were synthesized and investigated as nontoxic carriers for delivery of siRNA targeting the signaling peptide of secretory clusterin (sCLU), a prosurvival factor that protects cells from ionizing radiation (IR) injury, as well as chemotherapeutic agents. Three copolymers with different PEG grafting densities were tested for their abilities to bind and form nanocomplexes with siRNA. A copolymer composed of 10 PEG grafts (2 kDa each) per PEI polymer (2k10 copolymer) gave the highest binding affinity to siRNA by ethidium bromide exclusion assays, and had the smallest nanocomplex size (115 +/- 13 nm diameter). In human breast cancer MCF-7 cells, 2k10-siRNA-sCLU nanocomplexes suppressed both basal as well as IR-induced sCLU protein expression, which led to an over 3-fold increase in IR-induced lethality over 2k10-siRNA scrambled controls. In summary, this study demonstrates the proof-of-principle in using nanoparticle-mediated delivery of specific siRNAs to enhance the lethality of IR exposure in vitro, opening the door for siRNA-mediated knockdown of specific cytoprotective factors, such as DNA repair, anti-apoptotic, free radical scavenging, and many other proteins.     

Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation

Low molecular weight N-maleated chitosan-graft-PAMAM (polyamidoamine) copolymer was prepared through N-maleated chitosan (NMC) by Michael type addition reaction to enhance its solubility in water as well as its cationic character for enhancement of DNA complexation. FTIR, (1)H NMR, XRD and GPC were used to characterize the graft copolymers. The copolymer showed better DNA complexation ability at low N/P ratio than that of chitosan due to increased surface charge density by the incorporation of PAMAM molecule on to chitosan backbone. The copolymer can effectively protect the DNA toward anionic surfactant. In vitro release study showed efficient DNA release occurred at physiological pH (pH 7.4). In vitro cell cytotoxicity test indicated toward less cytotoxicity of NMC-graft-PAMAM copolymers compared to that of 25kDa PEI. Thus, the synthesized NMC-graft-PAMAM copolymers have great potential of finding application in drug and gene delivery.     

Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.     

Polycationic block copolymers of poly(ethylene oxide) and poly(propylene oxide) for cell transfection

A facile, one-step synthesis of cationic block copolymers of poly(2-N-(dimethylaminoethyl) methacrylate) (pDMAEMA) and copolymers of poly(propylene oxide) (PPO) and poly(ethylene oxide) (PEO) has been developed. The PEO-PPO-PEO-pDMAEMA (L92-pDMAEMA) and PEO-pDMAEMA copolymers were obtained via free radical polymerization of DMAEMA initiated by polyether radicals generated by cerium(IV). Over 95% of the copolymer fraction was of molecular mass ranging from 6.9 to 7.1 kDa in size, indicating the prevalence of the polyether-monoradical initiation mechanism. The L92-pDMAEMA copolymers possess parent surfactant-like surface activity. In contrast, the PEO-pDMAEMA copolymers lack significant surface activity. Both copolymers can complex with DNA. Hydrodynamic radii of the complexes of the L92-pDMAEMA and PEO-pDMAEMA with plasmid DNA ranged in size from 60 to 400 nm, depending on the copolymer/DNA ratio. Addition of Pluronic P123 to the L92-pDMAEMA complexes with DNA masked charges and decreased the tendency of the complex to aggregate, even at stoichiometric polycation/DNA ratios. The transfection efficiency of the L92-pDMAEMA copolymer was by far greater than that of the PEO-pDMAEMA copolymer. An extra added Pluronic P123 further increased the transfecton efficacy of L92-pDMAEMA, but did not affect that of PEO-pDMAEMA.     

Ternary Interpolyelectrolyte Complexes DNA–Polycation–Polyanion: A New Approach to Optimization of Mixtures for Transfection of 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 M30–40 kDa as polycation and polyacrylic acid (PA) with M20 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 -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.     

Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

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.     

Galactosyl conjugated N-succinyl-chitosan-graft-polyethylenimine for targeting gene transfer

Through incorporating lactobionic acid (LA) bearing a galactose group to N-succinyl-chitosan-graft-polyethylenimine (NSC-g-PEI), NSC-g-PEI-LA copolymers were synthesized as gene vectors with hepatocyte targeting properties. The molecular weight and composition of NSC-g-PEI-LA copolymers were characterized using gel permeation chromatography (GPC) and (1)H nuclear magnetic resonance spectroscopy ((1)H NMR) respectively. Agarose gel electrophoresis assays showed good DNA binding ability of NSC-g-PEI-LA, and the particle size of the NSC-g-PEI-LA/DNA complexes were between 150 and 400 nm as determined by a Zeta sizer. The NSC-g-PEI-LA/DNA complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The zeta potentials of these complexes were increased with the weight ratio of NSC-g-PEI-LA/DNA. NSC-g-PEI-LA has a lower cytotoxicity than PEI (25 kDa) and the toxicity decreased with increasing substitution of LA. The transfection efficiency of different complexes was evaluated by luciferase assay. Compared with PEI (25 kDa) and NSC-g-PEI/DNA, NSC-g-PEI-LA showed good transfection activity and cell specificity to HepG2 cells. The results suggested that NSC-g-PEI-LA has the potential to be used as a safe and effective targeting gene vector.     

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.     

Novel thermoresponsive nonviral gene vector: P(NIPAAm-co-NDAPM)-b-PEI with adjustable gene transfection efficiency

A thermoresponsive cationic copolymer, poly( N-isopropylacrylamide- co- N-(3-(dimethylamino)propyl)methacrylamide)- b-polyethyleneimine (P(NIPAAm- co-NDAPM)- b-PEI), was designed and synthesized as a potential nonviral gene vector. The lower critical solution temperature (LCST) of P(NIPAAm- co-NDAPM)- b-PEI in water measured by UV-vis spectroscopy was 38 degrees C. P(NIPAAm- co-NDAPM)- b-PEI as the gene vector was evaluated in terms of cytotoxicity, buffer capability determined by acid-base titration, DNA binding capability characterized by agarose gel electrophoresis and particle size analysis, and in vitro gene transfection. P(NIPAAm- co-NDAPM)- b-PEI copolymer exhibited lower cytotoxicity in comparison with 25 kDa PEI. Gel retardation assay study indicated that the copolymer was able to bind DNA completely at N/P ratios higher than 30. At 27 degrees C, the mean particle sizes of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes decreased from 1200 to 570 nm corresponding to the increase in N/P ratios from 10 to 60. When the temperature changed to 37 degrees C, the mean particle sizes of complexes decreased from 850 to 450 nm correspondingly within the same N/P ratio range due to the collapse of thermoresponsive PNIPAAm segments. It was found that the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes was higher than or comparable to that of 25 kDa PEI/DNA complexes at their optimal N/P ratios. Importantly, the transfection efficiency of P(NIPAAm- co-NDAPM)- b-PEI/DNA complexes could be adjusted by altering the transfection and cell culture temperature.     

Monomolecular assembly of siRNA and poly(ethylene glycol)-peptide copolymers

In this work, we design and investigate the complex formation of highly uniform monomolecular siRNA complexes utilizing block copolymers consisting of a cationic peptide moiety covalently bound to a poly(ethylene glycol) (PEG) moiety. The aim of the study was to design a shielded siRNA construct containing a single siRNA molecule to achieve a sterically stabilized complex with enhanced diffusive properties in macromolecular networks. Using a 14 lysine-PEG (K14-PEG) linear diblock copolymer, formation of monomolecular siRNA complexes with a stoichiometric 1:3 grafting density of siRNA to PEG is realized. Alternatively, similar PEGylated monomolecular siRNA particles are achieved through complexation with a graft copolymer consisting of six cationic peptide side chains bound to a PEG backbone. The hydrodynamic radii of the resulting complexes as measured by fluorescence correlation spectroscopy (FCS) were found to be in good agreement with theoretical predictions using polymer brush scaling theory of a PEG decorated rodlike molecule. It is furthermore demonstrated that the PEG coating of the siRNA-PEG complexes can be rendered biodegradable through the use of a pH-sensitive hydrazone or a reducible disulfide bond linker between the K14 and the PEG blocks. To model transport under in vivo conditions, diffusion of these PEGylated siRNA complexes is studied in various charged and uncharged matrix materials. In PEG solutions, the diffusion coefficient of the siRNA complex is observed to decrease with increasing polymer concentration, in agreement with theory of probe diffusion in semidilute solutions. In charged networks, the behavior is considerably more complex. FCS measurements in fibrin gels indicate complete dissociation of the diblock copolymer from the complex, while transport in collagen solutions results in particle aggregation.     

Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system

BACKGROUND: Polyethylenimines (PEIs) with high molecular weights are effective nonviral gene delivery vectors. However, the in vivo use of these PEIs can be hampered by their cellular toxicity. In the present study we developed and tested a new PEI polymer synthesized by linking less toxic, low molecular weight (MW) PEIs with a commonly used, biocompatible drug carrier, beta-cyclodextrin (CyD). METHODS AND RESULTS: The terminal CyD hydroxyl groups were activated by 1,1'-carbonyldiimidazole. Each activated CyD then linked two branched PEI molecules with MW of 600 Da to form a CyD-containing polymer with MW of 61 kDa, in which CyD served as a part of the backbone. The PEI-CyD polymer developed was soluble in water and biodegradable. In cell viability assays with sensitive neurons, the polymer performed similarly to low-MW PEIs and displayed much lower cellular cytotoxicity compared to PEI 25 kDa. The gene delivery efficiency of the polymer was comparable to, and at higher polymer/DNA ratios even higher than, that offered by PEI 25 kDa in neural cells. Attractively, intrathecal injection of plasmid DNA complexed by the polymer into the rat spinal cord provided levels of gene expression close to that offered by PEI 25 kDa. CONCLUSIONS: The polymer reported in the current study displayed improved biocompatibility over non-degradable PEI 25 kDa and mediated gene transfection in cultured neurons and in the central nervous system effectively. The new polymer would be worth exploring further as an in vivo delivery system of therapeutic genetic materials for gene therapy of neurological disorders.     

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.     

Novel shielded transferrin-polyethylene glycol-polyethylenimine/DNA complexes for systemic tumor-targeted gene transfer

Tumor-targeting DNA complexes which can readily be generated by the mixing of stable components and freeze-thawed would be very advantageous for their subsequent application as medical products. Complexes were generated by the mixing of plasmid DNA, linear polyethylenimine (PEI22, 22 kDa) as the main DNA condensing agent, PEG-PEI (poly(ethylene glycol)-conjugated PEI) for surface shielding, and Tf-PEG-PEI (transferrin-PEG-PEI) to provide a ligand for receptor-mediated cell uptake. Within the shielding conjugates, PEG chains of varying size (5, 20, or 40 kDa) were conjugated with either linear PEI22 (22 kDa) or branched PEI25 (25 kDa). The three polymer components were mixed together at various ratios with DNA; particle size, surface charge, in vitro transfection activity, and systemic gene delivery to tumors was investigated. In general, increasing the proportion of shielding conjugate in the complex reduced surface charge, particle size, and in vitro transfection efficiency in transferrin receptor-rich K562 cells. The particle size or surface charge of the complexes containing the PEG-PEI conjugate did not significantly change after freeze-thawing, while complexes without the shielding conjugate aggregated. Complexes containing PEG-PEI conjugate efficiently transfected K562 cells after freeze-thawing. Furthermore the systemic application of freeze-thawed complexes exhibited in vivo tumor targeted expression. For complexes containing the luciferase reporter gene the highest expression was found in tumor tissue of mice. An optimum formulation for in vivo application, PEI22/Tf-PEG-PEI/PEI22-PEG5, containing plasmid DNA encoding for the tumor necrosis factor (TNF-alpha), inhibited tumor growth in three different murine tumor models. These new DNA complexes offer simplicity and convenience, with tumor targeting activity in vivo after freeze-thawing.     

Novel branched poly(ethylenimine)-cholesterol water-soluble lipopolymers for gene delivery

A novel water-soluble lipopolymer was synthesized by linking cholesteryl chloroformate to the secondary amino groups of branched poly(ethylenimine) (PEI) of 1,800 and 10,000 Da. Conjugation through PEI secondary amines gives this newly synthesized lipopolymer (abbreviated as PEI-Chol) special advantage over our previously synthesized lipopolymers, which utilized the primary amino groups for conjugation, as the primary amino groups have a significant role in DNA condensation. Also, significantly, only one cholesterol molecule was grafted onto each PEI molecule (confirmed by (1)H NMR and MALDI-TOF mass spectrometry), leaving enough space for the steric interactions of the PEI's primary amines with the DNA. The PEI-Chol lipopolymer was characterized for the critical micellar concentration (cmc), buffer capacity, DNA condensation (by band retardation and circular dichroism), in vitro transfection efficiency, and cell viability. The cmcs of PEI-Chol 1,800 and PEI-Chol 10,000 were 496.6 and 1,330.5 microg/mL, respectively. The acid-base titration indicated high buffering capacity of the polymers around the pH range of 5-7, which indicated their potential for buffering in the acidic pH environment of the endosomes. The band retardation studies indicated that efficient condensation of the plasmid DNA could be achieved using these lipopolymers. The circular dichroism spectra indicated a change in DNA conformation and adoption of lower energy state upon condensation with these lipopolymers when an N/P ratio of 2.5/1 or above was formulated. The mean particle size of these complexes was in the range 110-205 nm, except for the complexes prepared using PEI of 1,800 Da, which had a mean particle size of 384 +/- 300 nm. The zeta potential of DNA complexes prepared using PEI-Chol 1,800, PEI-Chol 10,000 and PEI of 1,800, 10,000, and 25,000 Da at an N/P ratio of 15/1 was in the range 23-30 mV and was dependent on the N/P ratios. The in vitro transfection of PEI-Chol/pCMS-EGFP complexes in Jurkat cells showed high levels of expressed Green Fluorescent Protein (GFP) with little toxicity as determined by flow cytometry. These novel water-soluble lipopolymers provided good transfection efficiency with other desirable characteristics such as water solubility, free primary amino groups for efficient DNA condensation and high buffer capacity that indicated the possibility of efficient endosomal release.     

Synthesis and characterization of polyrotaxanes consisting of cationic alpha-cyclodextrins threaded on poly[(ethylene oxide)-ran-(propylene oxide)] as gene carriers

Cationic polymers have been receiving growing attention as gene delivery carriers. Herein, a series of novel cationic supramolecular polyrotaxanes with multiple cationic alpha-cyclodextrin (alpha-CD) rings threaded and blocked on a poly[(ethylene oxide)-ran-(propylene oxide)] (P(EO-r-PO)) random copolymer chain were synthesized and investigated for gene delivery. In the cationic polyrotaxanes, approximately 12 cationic alpha-CD rings were threaded on the P(EO-r-PO) copolymer with a molecular weight of 2370 Da and an EO/PO molar ratio of 4:1, while the cationic alpha-CD rings were grafted with linear or branched oligoethylenimine (OEI) of various chain lengths and molecular weights up to 600 Da. The OEI-grafted alpha-CD rings were only located selectively on EO segments of the P(EO-r-PO) chain, while PO segments were free of complexation. This increased the mobility of the cationic alpha-CD rings and the flexibility of the polyrotaxanes, which enhanced the interaction of the cationic alpha-CD rings with DNA and/or the cellular membrane. All cationic polyrotaxanes synthesized in this work could efficiently condense plasmid DNA to form nanoparticles that were suitable for delivery of the gene. Cytotoxicity studies showed that the cationic polyrotaxanes with all linear OEI chains of molecular weights up to 423 Da exhibited much less cytotoxicity than high-molecular-weight branched polyethylenimine (PEI) (25 kDa) in both HEK293 and COS7 cell lines. The cationic polyrotaxanes displayed high gene transfection efficiencies in a variety of cell lines including HEK293, COS7, BHK-21, SKOV-3, and MES-SA. Particularly, the gene delivery capability of the cationic polyrotaxanes in HEK293 cells was much higher than that of high-molecular-weight branched PEI (25 k).