Chitosan-graft-polyethylenimine as a gene carrier.

Chitosans have been proposed as biocompatible alternative cationic polymers that are suitable for non-viral delivery. However, the transfection efficiency of chitosan-DNA nanoparticles is still very low. To improve transfection efficiency, we prepared chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer by an imine reaction between periodate-oxidized chitosan and polyethylenimine (PEI). The molecular weight and composition of the CHI-g-PEI copolymer were characterized, using multi-angle laser scattering (GPC-MALS) and (1)H nuclear magnetic resonance ((1)H NMR), respectively. The copolymer was complexed with plasmid DNA (pDNA) in various copolymer/DNA (N/P) charge ratios, and the complex was characterized. CHI-g-PEI showed good DNA binding ability and high protection of DNA from nuclease attack. Also, with an increase in charge ratio, the sizes of the CHI-g-PEI/DNA complex showed a tendency to decrease, whereas the zeta potential of the complex showed an increase. The CHI-g-PEI copolymer had low cytotoxicity, compared to PEI 25K from cytotoxicity assays. At high N/P ratios, the CHI-g-PEI/DNA complex showed higher transfection efficiency than PEI 25K in HeLa, 293T and HepG2 cell lines. Our results indicate that the CHI-g-PEI copolymer has potential as a gene carrier in vitro.     

Peptide dendrimers as efficient and biocompatible gene delivery vectors: Synthesis and in vitro characterization

We report the synthesis and characterization of different generations of dendritic poly(l-lysine) vectors, and their use for in vitro gene transfection. Gel retardation assay revealed that the dendrimers could form complexes with plasmid DNAs (pDNAs), evident from the inhibition of the migration of pDNA at the N/P ratios of 0.5, 1 and 2 by G3, G4 and G5 dendritic generations, respectively. DNase I assay revealed the protection of pDNA acquired from the complexation with dendrimers from nuclease-catalyzed degradation, with the protection capacity of G5 being even stronger than poly(ethyleneimine) (PEI). Atomic force microscopy (AFM) revealed that all 4 generations of dendrimer/DNA complexes studied were of similar particle sizes within 100-200 nm. Zeta potential measurements showed that as the N/P ratio increased from 1 to 25, all dendrimer/pDNA complexes gradually changed from negative to positive charges. The higher generations tended to produce the greater positive potentials, indicating a stronger potency of the complexes to interact with negatively charged cell membranes. In vitro and in vivo cytotoxicity evaluations showed good biocompatibility of the dendrimers and their complexes over the different N/P ratios studied. In vitro gene transfection revealed higher efficiency of G5 than other dendrimers and insensitive variation to the presence of serum. Given its similar transfection efficiency to PEI but lower toxicity to cultured cells, dendrimer G5 could be a better candidate for gene delivery.     

Human serum albumin-polyethylenimine nanoparticles for gene delivery.

Nanoparticles consisting of DNA, human serum albumin (HSA) and polyethylenimine (PEI) were formed and tested for transfection efficiency in vitro with the aim of generating a nonviral gene delivery vehicle. HSA-PEI-DNA nanoparticles containing the pGL3 vector coding for luciferase as reporter gene were formed by charge neutralization. The particles were characterized by gel retardation assay, dynamic light scattering (size) and electrophoretic mobility measurements (charge). Stability was determined by spectrophotometric analysis and transfection efficiency was evaluated in cell culture using human embryonic epithelial kidney 293 cells. HSA-PEI-DNA nanoparticles were prepared by co-encapsulation of PEI as a lysosomotropic agent at varying nitrogen to phosphate (N/P) ratios. An optimum transfection efficiency was achieved when the particles were prepared at N/P ratios between 4.8 and 8.4. Furthermore, they displayed a low cytotoxicity when tested in cell culture. Our results show that HSA-PEI-DNA nanoparticles are a versatile carrier for DNA that may be suitable for i.v. administration.     

Polyethylenimine with acid-labile linkages as a biodegradable gene carrier.

Polyethylenimine (PEI) is a gene carrier with high transfection efficiency. However, PEI has high cytotoxicity, which depends on its molecular weight. To reduce the cytotoxicity, degradable PEIs with acid-labile imine linkers were synthesized with low molecular weight PEI1.8K (1.8 kDa) and glutadialdehyde. The molecular weights of the synthesized acid-labile PEIs were 23.7 and 13 kDa, respectively. The half-life of the acid-labile PEI was 1.1 h at pH 4.5 and 118 h at pH 7.4, suggesting that the acid-labile PEI may be rapidly degraded into nontoxic low molecular weight PEI in acidic endosome. In a gel retardation assay, plasmid DNA (pDNA) was completely retarded at a 3:1 N/P (nitrogen of polymer/phosphate of DNA) ratio. The zeta potential of the polyplexes was in the range of 46.1 to 50.9 mV and the particle size was in the range of 131.8 to 164.6 nm. In vitro transfection assay showed that the transfection efficiency of the acid-labile PEIs was comparable to that of PEI25K. In toxicity assay, the acid-labile PEI was much less toxic than PEI25K, due to the degradation of acid-labile linkage. Therefore, the acid-labile PEIs may be useful for the development of a nontoxic polymeric gene carrier.     

Enhancement Effect of Ultrasound-Induced Microbubble Cavitation on Branched Polyethylenimine-Mediated VEGF165 Transfection With Varied N/P Ratio

One isoform of the vascular endothelial growth factor, VEGF₁₆₅, has been reported to be a dominant mediator and regulator of angiogenic process, which plays an important role in treating cardiovascular diseases and chronically ischemic wounds. Branched polyethylenimine (bPEI) has been widely used as a non-viral delivery vector for gene therapy. Although bPEI-mediated DNA transfection efficiency can be raised by increasing the PEI nitrogen:DNA phosphate (N/P) ratio, cytotoxicity increases as well. In this study, the enhancement effect of microbubble inertial cavitation (IC) on bPEI-mediated VEGF₁₆₅ transfection was investigated, in an effort to optimize transfection efficiency using low N/P ratios. HEK 293T cells, mixed with bPEI:VEGF₁₆₅ complexes, were exposed to 1-MHz ultrasound pulses. The results show that: (1) IC activity induced by microbubble destruction can be quantified as an IC “dose” (ICD) and will increase with increasing acoustic driving pressure; (2) larger sonoporation pores can be generated by increasing ICD; (3) the transfection efficiency can be enhanced by increasing ICD until reaching a saturation level; and (4) microbubble IC activity has less cytotoxicity than bPEI, although a combinatorial effect of microbubble IC activity and bPEI could be observed on cell viability. The results suggest that, with appropriate ultrasound parameters, it is possible to optimize bPEI-mediated VEGF transfection efficiency using relatively low N/P ratios by employing ultrasound-induced microbubble inertial cavitation.     

New polyphosphoramidate with a spermidine side chain as a gene carrier.

A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly(1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD(50) of PPA-SP was 85 microg/ml in COS-7 cells, in contrast to 20 and 42 microg/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5 approximately 10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 microM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue.     

Galactosylated chitosan-graft-polyethylenimine as a gene carrier for hepatocyte targeting

Chitosans have been proposed as alternative, biocompatible cationic polymers for nonviral gene delivery. However, the low transfection efficiency and low specificity of chitosan need to be addressed before clinical application. We prepared galactosylated chitosan-graft-polyethylenimine (GC-g-PEI) copolymer by an imine reaction between periodate-oxidized GC and low-molecular-weight PEI. The molecular weight and composition were characterized using gel permeation chromatography column with multi-angle laser scattering and (1)H nuclear magnetic resonance, respectively. The copolymer was complexed with plasmid DNA in various copolymer/DNA (N/P) charge ratios, and the complexes were characterized. GC-g-PEI showed good DNA-binding ability and superior protection of DNA from nuclease attack and had low cytotoxicity compared to PEI 25K. GC-g-PEI/DNA complexes showed higher transfection efficiency than PEI 25K in both HepG2 and HeLa cell lines. Transfection efficiency into HepG2, which has asialoglycoprotein receptors, was higher than that into HeLa, which does not. GC-g-PEI/DNA complexes also transfected liver cells in vivo after intraperitoneal (i.p.) administration more effectively than PEI 25K. These results suggest that GC-g-PEI can be used in gene therapy to improve transfection efficiency and hepatocyte specificity in vitro and in vivo.     

Controlled release of cell-permeable gene complex from poly(L-lactide) scaffold for enhanced stem cell tissue engineering

The use of tissue engineering to deliver genes to stem cells has been impeded by low transfection efficiency of the inserted gene and poor retention at the target site. Herein, we describe the use of non-viral gene transfer by cell-permeable peptide (CPP) to increase the transfection efficiency. The combination of this technique with the use of a controlled release concept using a poly (l-lactide) scaffold allowed for prolonged uptake in stem cells. High transfection efficiency was obtained using a human-derived arginine-rich peptide denoted as Hph-1 (YARVRRRGPRR). The formation of complex between pDNA and Hph-1 was monitored using gel retardation tests to measure size and zeta potential. Complex formation was further assessed using a DNase I protection assay. A sustained gene delivery system was developed using a fibrous 3-D scaffold coated with pDNA/Hph-1 complexes. Transfection efficiency and the mean fluorescence intensity of human adipose-derived stem cells (hASCs) on the sustained delivery scaffold were compared to those of cells transfected via bolus delivery. Plasmid DNA completely bound Hph-1 at a negative-to-positive (N/P) charge ratio of 10. After complex formation, Hph-1 appeared to effectively protect pDNA against DNase I attack and exhibited cytotoxicity markedly lower than that of the pDNA/PEI complex. Plasmid DNA/Hph-1 complexes were released from the scaffolds over 14 days and were successfully transfected hASCs seeded on the scaffolds. Flow cytometry revealed that the transfection efficiency in hASCs treated with pDNA/Hph-1 complex was approximately 5-fold higher than that in cells transfected using Lipofectamine. The sustained delivery system showed a significantly higher transfection efficiency and remained able to transfect cells for a longer period of time than bolus delivery. These results suggest that cell-scaffold-based tissue regeneration can be further improved by transduction concept using CPP and controlled release using polymeric scaffold.     

Water-soluble lipopolymer as an efficient carrier for gene delivery to myocardium

Water-soluble lipopolymer (WSLP), which consisted of polyethylenimine (PEI, 1800 Da) and cholesterol, was characterized as a gene carrier to smooth muscle cells and myocardium. Acid-base titration showed that WSLP had a proton-buffering effect. The size of WSLP/plasmid DNA (pDNA) complex was around 70 nm. WSLP/pDNA complex was transfected to A7R5 cells, a smooth muscle cell line. WSLP showed the highest transfection at a 40/1 N/P ratio. WSLP has higher transfection efficiency than PEI (1800 and 25 000 Da), SuperFect, and lipofectamine. In addition, WSLP has less cytotoxicity than PEI (25 000 Da), SuperFect, and lipofectamine. Since WSLP has cholesterol moiety, it may utilize cellular cholesterol uptake pathway, in which low-density lipoprotein (LDL) is involved. An inhibition study with free cholesterol or low-density lipoprotein (LDL) showed that transfection was inhibited by cholesterol or LDL, suggesting that WSLP/pDNA complex is transfected to the cells through the cholesterol uptake pathway. To evaluate the transfection efficiency to myocardium, WSLP/pDNA complex was injected into the rabbit myocardium. WSLP showed higher transfection than PEI and naked pDNA. WSLP expressed the transgene for more than 2 weeks. In conclusion, WSLP is an efficient carrier for local gene transfection to myocardium, and useful in in vivo gene therapy.     

Evaluation of proinflammatory cytokine production induced by linear and branched polyethylenimine/plasmid DNA complexes in mice

The purpose of this study was to evaluate the cytokine response induced by linear and branched polyethylenimine (PEI)/plasmid DNA (pDNA) complex (polyplex) in relation to the ratio of PEI nitrogen and DNA phosphate (N/P ratio) of the polyplex, dose of pDNA, and structure and molecular weight of PEI, which are important for transfection efficacy of PEI polyplex. As a control, a N-[1-(2, 3-dioleyloxy) propyl]-n,n,n-trimethylammonium chloride/cholesterol liposome/pDNA complex (lipoplex) was selected for its high transfection efficacy in vivo. The concentration of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha were much lower after the administration of polyplex than lipoplex irrespective of the N/P ratio, dose of pDNA, or structure and molecular weight of PEI, although these factors affected the transfection efficacy in vivo. We demonstrated that the amount of activated nuclear factor-kappaB, which contributes substantially to the production of cytokines, was comparable with the control (no treatment) level, and significantly less than that obtained with lipoplex. Although the production of proinflammatory cytokines (TNF-alpha, interferon-gamma, and interleukin-12) was reduced on the administration of the linear PEI polyplex, serum alanine aminotransferase levels were significantly enhanced by pDNA in a dose-dependent manner, suggesting that such hepatic damage is not induced by proinflammatory cytokines.     

A novel environment-sensitive biodegradable polydisulfide with protonatable pendants for nucleic acid delivery

Clinical application of nucleic acid-based therapies is limited by the lack of safe and efficient delivery systems. The purpose of this study is to design and evaluate novel biodegradable polymeric carriers sensitive to environmental changes for efficient delivery of nucleic acids, including plasmid DNA and siRNA. A novel polydisulfide with protonatable pendants was synthesized by the oxidative polymerization of a dithiol monomer, which was readily prepared by solid phase chemistry. The polydisulfide exhibited good buffering capacity and low cytotoxicity. It formed stable complexes with both plasmid DNA and siRNA. The particle sizes of the complexes decreased with the increase of the N/P ratios in the range of 100 to 750 nm. The complexes were stable in the presence of salt and heparin under normal physiological conditions, but dissociated to release nucleic acids in a reductive environment similar to cytoplasm. The polydisulfide demonstrated N/P ratio dependent transfection efficiency for plasmid DNA and gene silencing efficiency for siRNA. The presence of an endosomal disrupting agent, chloroquine, did not affect the DNA transfection efficiency of the polydisulfide. The transfection or gene silencing efficiency of the polydisulfide/DNA or siRNA complexes was comparable to or slightly lower than that of corresponding PEI complexes. Moreover, the polydisulfide showed better serum-friendly feature than PEI when delivering either DNA or siRNA in the presence of 10% FBS. This novel polydisulfide is a promising lead for further design and development of safe and efficient delivery systems for nucleic acids.     

Polyethyleneimine-mediated gene delivery into rat pheochromocytoma PC-12 cells

In this study, we examined the use of polyethyleneimine (PEI) as a non-viral gene carrier and lipofectamine(trade mark) 2000 as control for rat pheochromocytoma PC-12 cells. The complex formation of PEI and DNA or lipofectamine and DNA was characterized by gel electrophoresis and measurement of particle size and surface charge. A gradual increase in surface charge (from 0.7 to 43 mV) and a gradual decrease in particle size (from 900 to 130 nm) was observed in the PEI-DNA complex with higher PEI concentrations. The cytotoxicity of PC-12 cells for lipofectamine-DNA complex was similar to PEI-DNA complex at N:P charge ratios of 4 and 8. Transfection efficiency was 14% for lipofectamine and 15% for PEI. At low N:P ratio, DNA condenses poorly, so the particle size tends to be large and polydispersed, resulting in poor transfection efficiency. Meanwhile, a high N:P ratio results in high transfection efficiency and cytotoxicity. Transfected PC-12 cells showed the generation of neurites from transfected PC-12 cells in the presence of NGF, indicating the differentiation of PC-12 cells. NGF-differentiated PC-12 cells were transfected by PEI-DNA complex of N:P charge ratio 8. From real-time imaging for transfection, the enhanced green fluorescent protein (EGFP) started to localize in the nuclei of PC-12 cells at 5 h and localized in the cytoplasm from 15 h. Our study demonstrates that PEI or lipofectamine may be applied as an effective gene carrier for PC-12 cells.     

Molecular weight-dependent gene transfection activity of unmodified and galactosylated polyethyleneimine on hepatoma cells and mouse liver

To optimize a receptor-mediated and cell-selective gene transfer with polyethyleneimine (PEI)-based vector, we synthesized three galactosylated PEIs (Gal-PEI) with different molecular weights (PEI₁₈₀₀, PEI_10,000, and PEI_70,000) and investigated their potential as a targetable vector to asialoglycoprotein receptor-positive cells. All PEI derivatives formed complexes with plasmid DNA (pDNA), whereas the particle size of the complex became smaller on increasing the molecular weight of PEI. Transfection efficiency in HepG2 cells with PEI was highest with PEI₁₈₀₀; efficiency was next highest with PEI_10,000, although the cellular association was similar. After galactosylation, Gal₁₉-PEI_10,000/pDNA and Gal₁₂₀-PEI_70,000/pDNA showed considerable agglutination with a galactose-recognizing lectin, but Gal₉-PEI₁₈₀₀ did not, suggesting that galactose units on the Gal₉-PEI₁₈₀₀-pDNA complex are not sufficiently available for recognition. Gal₁₉-PEI_10,000-pDNA and Gal₁₂₀-PEI_70,000-pDNA complexes showed galactose-inhibitable transgene expression in HepG2 cells. Transfection efficiency was greatest with Gal₁₉-PEI_10,000/pDNA, a result that highlights the importance of obtaining a balance between the cytotoxicity and the transfection activity, both of which are found to be a function of the molecular weight of PEI. After intraportal injection, however, Gal₁₅₃-PEI_70,000/pDNA having a low N/P ratio was most effective, suggesting that additional variables, such as the size of the complex, are important for in vivo gene transfer to hepatocytes.     

Combined delivery of dexamethasone and plasmid DNA in an animal model of LPS-induced acute lung injury

Dexamethasone was conjugated to low molecular weight polyethylenimine (2 kDa, PEI2k). Dexamethasone conjugated PEI2k (PEI2k-Dexa) was evaluated as a combined delivery carrier of dexamethasone and plasmid DNA (pDNA) in an animal model of lipopolysaccharide (LPS) induced acute lung injury (ALI). In vitro transfection of L2 lung epithelial cells, PEI2k-Dexa exhibited higher transfection efficiency than PEI2k or a simple mixture of PEI2k and dexamethasone. In addition, the PEI2k-Dexa/pβ-Luc complexes reduced the levels of pro-inflammatory cytokines in LPS activated Raw 264.7 macrophage cells. The anti-inflammatory effect of PEI2k-Dexa was higher than that of controls. The PEI2k-Dexa/pβ-Luc complexes were administered to mice via intratracheal injection. PEI2k-Dexa had higher pDNA delivery efficiency than PEI2k in the lung and decreased TNF-α and IL-6 in the lung homogenates and bronchoalveolar lavage (BAL) fluid compared with the controls. Furthermore, total protein and immunoglobulin M (IgM) concentrations in BAL fluid were reduced by the PEI2k-Dexa/pβ-Luc complexes. The intratracheal injection of the PEI2k-Dexa/pcDNA-EGFP complexes in the ALI model showed higher EGFP expression compared with PEI2k. Hematoxylin and eosin (H&E) staining showed that PEI2k-Dexa reduced inflammatory reaction in the lung. Therefore, PEI2k-Dexa may be useful for combination gene and drug therapy for ALI.     

Biodegradable poly(ethylenimine) for plasmid DNA delivery

Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential. High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight. Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery. Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI. Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs. Reaction conditions were carefully controlled to produce water soluble copolymers. Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-β-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8±0.9 to 151.8±3.4 nm. In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%.     

Revisit complexation between DNA and polyethylenimine — Effect of length of free polycationic chains on gene transfection

Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~ 3, irrespective of the PEI chain length and solvent. Each solution mixture with N:P > 3 contains two kinds of PEI chains: bound to DNA and free in the solution. It has been shown that it is those free PEI chains that play a vital role in promoting the gene transfection. The effects of the length of the bound and free chains on the gene transfection were respectively studied. Both short and long PEI chains are capable of condensing DNA completely at N:P ~ 3 but long ones are ~ 10²-fold more effective in the gene transfection, apparently due to their fast endocytosis and intracellular trafficking. The cellular uptake kinetics studied by flow cytometry reveals that long free chains increase the uptake rate constant of the DNA/PEI complexes. In the intracellular pathway, they are able to prevent the development of the later endolysosomes, and facilitate the subsequent release of the polyplexes from the endosomes. Our result shows that the “proton sponge” effect is not dominant because the shut-down of the proton pump only partially attenuates the transfection efficiency. A possible mechanism is speculated and presented.     

Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo

We have assessed whether magnetic forces (magnetofection) can enhance non-viral gene transfer to the airways. TransMAG(PEI), a superparamagnetic particle was coupled to Lipofectamine 2000 or cationic lipid 67 (GL67)/plasmid DNA (pDNA) liposome complexes. In vitro transfection with these formulations resulted in approximately 300- and 30-fold increase in reporter gene expression, respectively, after exposure to a magnetic field, but only at suboptimal pDNA concentrations. Because GL67 has been formulated for in vivo use, we next assessed TransMAG(PEI) in the murine nasal epithelium in vivo, and compared this to naked pDNA. At the concentrations required for in vivo experiments, precipitation of magnetic complexes was seen. After extensive optimization, addition of non-precipitated magnetic particles resulted in approximately seven- and 90-fold decrease in gene expression for naked pDNA and GL67/pDNA liposome complexes, respectively, compared to non-magnetic particles. Thus, whereas exposure to a magnetic field improved in vitro transfection efficiency, translation to the in vivo setting remains difficult.     

Degradable polyethylenimine-alt-poly(ethylene glycol) copolymers as novel gene carriers.

An ideal gene carrier requires both safety and transfection efficiency. Polyethylenimine (PEI) is a well-known cationic polymer, which has high transfection efficiency owing to its buffering capacity. But it has been reported that PEI is cytotoxic in many cell lines and non-degradable. In this study, we synthesized degradable PEI-alt-poly(ethylene glycol) (PEG) copolymers using Michael-type addition reactions as a new gene carrier and characterized them. These copolymers were complexed with plasmid DNA and the resulting complexes were characterized by dynamic light scattering, gel retardation and atomic force microscopy to determine particle sizes, complex formation and complex shape, respectively. Cytotoxicity and transfection efficiency of the copolymers were also checked in cultured HeLa human cervix epithelial carcinoma cells, HepG2 human hepatoblastoma cell line and MG63 human osteosarcoma cells. PEG to PEI ratio in the copolymers was near 1 and the molecular weight of the copolymer ranged from around 8000 to 12,900. These copolymers degraded rapidly at 37 degrees C in 0.1 M phosphate buffered saline (PBS, pH 7.4). The complete copolymer/DNA complex was formed at an N/P ratio of 12, producing a complex resistant to DNase I. Particle sizes decreased with increasing N/P ratio and PEG molecular weight, exhibiting a minimum value of 75 nm at an N/P ratio of 45 with PEI-alt-PEG (700). Cytotoxicity study showed that copolymers exhibited no cytotoxic effects on cells even at high copolymer concentration. Also, transfection efficiency was influenced by PEG molecular weight and, in case of PEI-alt-PEG (258), the transfection efficiency was higher than that for PEI 25 K in HepG2 and MG63, whereas it was lower than that for PEI 25K in HeLa cells.     

In vitro transfection of HeLa cells with temperature sensitive polycationic copolymers.

In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between -3.1 and +21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190-1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B. However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity.