Biodegradable cyclen-based linear and cross-linked polymers as non-viral gene vectors

Several 1,4,7,10-tetraazacyclododecane (cyclen)-based linear (3a-c) and cross-linked (8a-d) polymers containing biodegradable ester or disulfide bonds were described. These polymeric compounds were prepared by ring-opening polymerization from various diol glycidyl ethers. The molecular weights of the title polymers were measured by GPC. Agarose gel retardation assays showed that these compounds have good DNA-binding ability and can completely retard plasmid DNA (pDNA) at weight ratio of 20 for linear polymers and 1.2 for cross-linked polymers. The degradation of these polymers was confirmed by GPC. The formed polyplexes have appropriate sizes around 400 nm and zeta-potential values about 15-40 mV. The cytotoxicities of 8 assayed by MTT are much lower than that of 25 KDa PEI. In vitro transfection toward A549 and 293 cells showed that the transfection efficiency (TE) of 8c-DNA polyplex is close to that of 25 kDa PEI at 8c/DNA weight ratio of 4. Structure-activity relationships (SAR) of these linear and cross-linked polymers were discussed in their DNA-binding, cytotoxicity, and transfection studies. In addition, in the presence of serum, the TE of 8/DNA polyplexes could be improved by introducing chloroquine or Ca(2+) to pretreated cells.     

Controlled delivery of plasmid DNA and siRNA to intracellular targets using ketalized polyethylenimine

A new polyethylenimine (PEI)-derived biodegradable polymer was synthesized as a nonviral gene carrier. Branches of PEI were ketalized, and capabilities of nucleic acid condensation and delivery efficiency of the modified polymers were compared with ones of unketalized PEI. Ketalized PEI was able to efficiently compact both plasmid DNA and siRNA into nucleic acids/ketalized PEI polyplexes with a range of 80-200 nm in diameter. Nucleic acids were efficiently dissociated from the polyplexes made of ketalized PEI upon hydrolysis. In vitro study also demonstrated that ketalization enhanced transfection efficiency of the polyplexes while reducing cytotoxicity, even at high N/ P ratios. Interestingly, transfection efficiency was found to be inversely proportional to molecular weights of ketalized PEI, while RNA interference was observed in the opposite way. This study implies that selective delivery of plasmid DNA and siRNA to the nucleus and the cytoplasm can be achieved by tailoring the structures of polymeric gene carriers.     

Effective delivery of siRNA to transgenic rice cells for enhanced transfection using PEI-based polyplexes

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.     

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.     

Oligoethylenimines grafted to PEGylated poly(β-amino ester)s for gene delivery

Novel polymers composed of net-like PEGylated poly(β-amino ester) (N-P-1, M(w) = 6900 or N-P-2, M(w) = 21,400) and oligoethylenimine (OEI) (OEI423 or OEI600) were synthesized and evaluated as gene carriers. The molecular weights of these polymers were well-controlled by the concentration of the cross-linking reaction. The synthesized polymers showed high biodegradability, less cytotoxicity, and efficient DNA retard ability. The N-P-1-OEI600/DNA complex showed much slower aggregation in the presence of 10 and 20% serum solutions. In vitro transfection assays, N-P-2-OEI423, N-P-1-OEI600, and N-P-2-OEI600 showed enhanced transfection efficiency compared with the PEI25K control in the presence or in the absence of serum in different cell lines. In particular, in Cos-7 cells, the transfection efficiency of N-P-1-OEI600 was 20.9 times higher than that of PEI25K in the presence of serum. The polymer/DNA complex stability, lower cytotoxicity, and higher transfection efficiency in the presence of serum revealed that N-P-1-OEI600 could be a potential nonviral gene carrier for In Vivo application.     

Cross-linked polyethylenimine as potential DNA vector for gene delivery with high efficiency and low cytotoxicity

Polyethylenimine (PEI) has been known as an efficient gene carrier with the highest cationiccharge potential.High transfection efficiency of PEI,along with its cytotoxicity,strongly depends on itsmolecular weight.To enhance its gene delivery efficiency and minimize cytotoxicity,we have synthesizedsmall cross-linked PEI with biodegradable linkages and evaluated their transfection efficiencies in vitro.Inthis study,branched PEI with a molecular weight of 800 Da was cross-linked by small diacrylate[1,4-butanediol diacrylate or ethyleneglycol dimethacrylate (EGDMA)] for 2-6 h.The efficiencies of thecross-linked PEI in in vitro transfection of plasmid DNA containing enhanced green fluorescent protein(EGFP) reporter gene were assessed in melanoma B 16F10 cell line and other cell lines.Flow cytometrywas used to quantify the cellular entry efficiency of plasmid and the transgene expression level.Thecytotoxicities of the cross-linked PEI in these cells were evaluated by MTT assay.EGDMA-PEI 800-4h,atypical cross-linked PEI reported here,mediated a more efficient expression of reporter gene than thecommercially available 25-kDa branched PEI control,and resulted in a 9-fold increase in gene deliveryin B16F10 cells and a 16-fold increase in 293T cells,while no cytotoxicity was found at the optimizedcondition for gene delivery.Furthermore,the transfection activity of polyplexes was preserved in thepresence of serum proteins.     

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

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

Poly(ethylene oxide) grafted with short polyethylenimine gives DNA polyplexes with superior colloidal stability, low cytotoxicity, and potent in vitro gene transfection under serum conditions

Poly(ethylene oxide) grafted with 1.8 kDa branched polyethylenimine (PEO-g-PEI) copolymers with varying compositions, that is, PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI, were prepared and investigated for in vitro nonviral gene transfer. Gel electrophoresis assays showed that PEO(13k)-g-10PEI, PEO(24k)-g-10PEI, and PEO(13k)-g-22PEI could completely inhibit DNA migration at an N/P ratio of 4/1, 4/1, and 3/1, respectively. Dynamic light scattering (DLS) and zeta potential measurements revealed that all three graft copolymers were able to effectively condense DNA into small-sized (80-245 nm) particles with moderate positive surface charges (+7.2 ～ +24.1 mV) at N/P ratios ranging from 5/1 to 40/1. The polyplex sizes and zeta-potentials intimately depended on PEO molecular weights and PEI graft densities. Notably, unlike 25 kDa PEI control, PEO-g-PEI polyplexes were stable against aggregation under physiological salt as well as 20% serum conditions due to the shielding effect of PEO. MTT assays in 293T cells demonstrated that PEO-g-PEI polyplexes had decreased cytotoxicity with increasing PEO molecular weights and decreasing PEI graft densities, wherein low cytotoxicities (cell viability >80%) were observed for polyplexes of PEO(13k)-g-22PEI, PEO(13k)-g-10PEI, and PEO(24k)-g-10PEI up to an N/P ratio of 20/1, 30/1, and 40/1, respectively. Interestingly, in vitro transfection results showed that PEO(13k)-g-10PEI polyplexes have the best transfection activity. For example, PEO(13k)-g-10PEI polyplexes formed at an N/P ratio of 20/1, which were essentially nontoxic (100% cell viability), displayed over 3- and 4-fold higher transfection efficiencies in 293T cells than 25 kDa PEI standard under serum-free and 10% serum conditions, respectively. Confocal laser scanning microscopy (CLSM) studies using Cy5-labeled DNA confirmed that these PEO-g-PEI copolymers could efficiently deliver DNA into the perinuclei region as well as into nuclei of 293T cells at an N/P ratio of 20/1 following 4 h transfection under 10% serum conditions. PEO-g-PEI polyplexes with superior colloidal stability, low cytotoxicity, and efficient transfection under serum conditions are highly promising for safe and efficient in vitro as well as in vivo gene transfection applications.     

Purification of polyethylenimine polyplexes highlights the role of free polycations in gene transfer

BACKGROUND: Nonviral vectors based on polyethylenimine (PEI) usually contain an excess of PEI that is not complexed to DNA. Since unbound PEI contributes to cellular and systemic toxicity, purification of polyplexes from unbound PEI is desirable. METHODS: Size exclusion chromatography (SEC) was used to purify PEI polyplexes of free PEI. Transfection properties of purified polyplexes and the effect of free PEI on gene delivery were studied in vitro and in vivo after systemic application into mice. RESULTS: SEC did not change the size and zeta-potential of polyplexes. Independent of the amount of PEI used for complex formation, purified PEI polyplexes had the same final PEI nitrogen/DNA phosphate ratio of 2.5. Notably, purified PEI polyplexes demonstrated low cellular and systemic toxicity. High transfection efficiency was achieved with purified polyplexes at high DNA concentrations (8-15 microg/ml). At low DNA concentrations (2-4 microg/ml) gene transfer with purified particles was less efficient than with polyplexes containing free PEI both in vitro and in vivo. Mechanistic studies showed that free PEI partly blocked cellular association of DNA complexes but was essential for the following intracellular gene delivery. Adding free PEI to cells treated with purified particles with a delay of up to 4 h resulted in significantly enhanced transfection efficiency compared with non-purified particles or purified particles without free PEI. CONCLUSIONS: This study presents an efficient method to remove free PEI from PEI polyplexes by SEC. Our results from transfection experiments demonstrate that free PEI substantially contributes to efficient gene expression but also mediates toxic effects in a dose-dependent manner. Purified polyplexes without free PEI have to be applied at increased concentrations to achieve high transfection levels, but exhibit a greatly improved toxicity profile.     

Polymer-DNA hybrid nanoparticles based on folate-polyethylenimine-block-poly(L-lactide)

The ability of amphiphilic block copolymers that consist of polyethylenimine (PEI) and poly(L-lactide) (PLLA) to modulate the delivery of plasmid DNA was evaluated. Folate-polyethylenimine-block-poly(l-lactide) (folate-PEI-PLLA) was synthesized by linking folic acid and PLLA to PEI diamine. Water-soluble polycation PEI provides gene-loading capability. Additionally, PEI is considered to exhibit high transfection efficiency and endosomal disrupting capacity. Hydrophobic PLLA that is incorporated into the gene delivery vector is believed to enhance the cell interactions and tissue permeability of the delivery system. Polymeric carrier containing folic acid is expected to be able to identify tumor surface receptors and transfect cells by receptor-mediated endocytosis. The results of agarose retardation assay indicated that the folate-PEI-PLLA began to form polyplexes at a polymer/DNA weight ratio (P/D) of over 10, whereas branched polyethylenimine (B-PEI) formed polyplexes with DNA at a ratio of above 1. The spherical particle morphology was supplemented with a particle size of approximately 100 nm at 10 P/D ratio. The results indicated that folate-PEI-PLLA with proper PEI/PLLA ratio effectively reduced cytotoxicity and maintained acceptable transfection efficiency. Low cytotoxicity of the folate-PEI-PLLA gives an advantage to high-dose administration.     

Branched polyethylenimine derivatives with reductively cleavable periphery for safe and efficient in vitro gene transfer

Twenty-five kDa polyethylenimine (PEI) is one of the most efficient nonviral gene transfer agents currently applied as a golden standard for in vitro transfection. In this study, novel 25 kDa PEI derivatives with reductively cleavable cystamine periphery (PEI-Cys) were designed to reduce carrier-associated cytotoxicity and to enhance further the transfection activity. The Michael-type conjugate addition of 25 kDa PEI with N-tert-butoxycarbonyl-N'-acryloyl-cystamine (Ac-Cys-(t)Boc) and N-tert-butoxycarbonyl-N'-methacryloyl-cystamine (MAc-Cys-(t)Boc) followed by deprotection readily afforded PEI-Cys derivatives, denoted as PEI-(Cys)x(Ac) and PEI-(Cys)x(MAc), with degree of substitution (DS) ranging from 14 to 34 and 13 to 38, respectively. All PEI-Cys derivatives had higher buffer capacity than the parent 25 kDa PEI (21.2 to 23.1% versus 15.1%). Gel retardation and ethidium bromide exclusion assays showed that cystamine modification resulted in largely enhanced interactions with DNA. PEI-(Cys)x(Ac) could condense DNA into small-sized particles of 80-90 nm at and above an N/P ratio of 5/1, which were smaller than polyplexes of 25 kDa PEI (100-130 nm). In comparison, PEI-(Cys)x(MAc) condensed DNA into somewhat larger particles (100-180 nm at N/P ratios from 30/1 to 5/1). Gel retardation and dynamic light scattering (DLS) measurements showed that PEI-Cys polyplexes were quickly unpacked to release DNA in response to 10 mM dithiothreitol (DTT). These PEI-Cys derivatives revealed markedly decreased cytotoxicity as compared with 25 kDa PEI with IC(50) values of >100 mg/L and 50-75 mg/L for HeLa and 293T cells, respectively (corresponding IC(50) data of 25 kDa PEI are ca. 11 and 3 mg/L). The in vitro transfection experiments in HeLa and 293T cells using pGL3 as a reporter gene showed that gene transfection activity of PEI-Cys derivatives decreased with increasing DS and PEI-(Cys)x(MAc) exhibited higher transfection activity than PEI-(Cys)x(Ac) at similar DS. Notably, polyplexes of PEI-(Cys)14(Ac) and PEI-(Cys)13(MAc) showed significantly enhanced gene transfection efficiency (up to 4.1-fold) as compared with 25 kDa PEI formulation at an N/P ratio of 10/1 in both serum-free and 10% serum-containing conditions. The modification of PEI with reductively cleavable periphery appears to be a potential approach to develop safer and more efficient nonviral gene vectors.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

Disulfide cross-linked polyethylenimines (PEI) prepared via thiolation of low molecular weight PEI as highly efficient gene vectors

Ring-opening reaction of low molecular weight polyethylenimine with an Mw of 800 Da (800 Da PEI) with methylthiirane produced thiolated polyethylenimine (PEI-SHX ). The thiolation degree X, which is the average number of thiol groups on a PEI molecule, was readily adjusted by the methylthiirane/PEI ratio. Oxidation of the thiolated PEIs with DMSO afforded disulfide cross-linked PEIs (PEI-SSX ). The molecular weights of PEI-SS X were estimated by viscosity measurement to be 7100, 8000, and 8400 for X=2.6, 6.5, and 9.4, respectively. The PEI-SSX series can bind and condense plasmid DNAs effectively forming nanosized polyplexes. The size of dry polyplexes is less than 100 nm on the TEM pictures. In solution, the size of the polyplexes was measured by DLS to be about 400 nm. In vitro experiments showed that the PEI-SS X series have a lower cytotoxicity and higher gene transfection efficiency compared with the high molecular weight PEI with Mw of 25 KDa. The presence of fetal bovine serum did not decrease the transfection efficiency. The results proved the hypothesis that reductively degradable disulfide-containing PEIs could possesses simultaneously higher gene transfection efficiency and lower cytotoxicity than the nondegradable ones.     

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.     

Extracellular barriers to in Vivo PEI and PEGylated PEI polyplex-mediated gene delivery to the liver

Polyplex-mediated gene therapy is a promising alternative to viral gene therapy. One challenge to these synthetic carriers is reduced transfection efficiencies in vivo compared to those achieved in vitro. Many of the intracellular barriers to gene delivery have been elucidated, but similar quantification of extracellular barriers to gene delivery remains a need. In this study, the unpackaging of polyplexes by serum proteins, soluble glycosaminoglycans, and an extracellular matrix extract was demonstrated by a YOYO-1 fluorescence quenching assay. Additionally, exposing polyplexes to serum or proteoglycans before in vitro transfection caused decreased cellular uptake of DNA. Lastly, PEI polyplexes and PEGylated PEI polyplexes were injected into the portal vein of mice, and the intrahepatic distributions of labeled DNA and polymer were assessed by confocal microscopy. PEI polyplexes delivered DNA to the liver, but extensive vector unpackaging was observed, with PEI primarily colocalized with the extracellular matrix. PEGylated polyplexes mediated less DNA delivery to the liver, possibly due to premature vector unpackaging in the blood. Through this work, both the blood and the extracellular matrix have been determined to be significant extracellular barriers to polyplex-mediated in vivo gene delivery to the liver.     

Reducible poly(amido ethylenimine)s designed for triggered intracellular gene delivery

Poly(amido ethylenimine) polymers, a new type of peptidomimetic polymer, containing multiple disulfide bonds (SS-PAEIs) designed to degrade after delivery of plasmid DNA (pDNA) into the cell were synthesized and investigated as new carriers for triggered intracellular gene delivery. More specifically, three SS-PAEIs were synthesized from Michael addition reactions between cystamine bisacrylamide (CBA) and three different ethylene amine monomers, i.e., ethylenediamine (EDA), diethylenetriamine (DETA), or triethylenetetramine (TETA). Complete addition reactions were confirmed by (1)H NMR. The molecular weight, buffer capacity, and relative degree of branching for each SS-PAEI was determined by gel permeation chromatography (GPC), acid-base titration, and liquid chromatography-mass spectroscopy (LC-MS), respectively. Physicochemical characteristics of polymer/pDNA complexes (polyplexes) were analyzed by gel electrophoresis, particle size, and zeta-potential measurements. All three SS-PAEIs effectively complex pDNA to form nanoparticles with diameters less than 200 nm and positive surface charges of approximately 32 mV. The in vitro gene transfer properties of SS-PAEIs were evaluated using mouse embryonic fibroblast cell (NIH3T3), primary bovine aortic endothelial cell (BAEC), and rat aortic smooth muscle cell (A7R5) lines. Interestingly, polyplexes based on all three SS-PAEIs exhibited remarkably high levels of reporter gene expression with nearly 20x higher transfection efficiency than polyethylenimine 25k. The high transfection efficiency was maintained in the presence of 10% serum in the transfection medium. Furthermore, confocal microscopy experiments using labeled pDNA indicated that polyplexes of SS-PAEI displayed greater intracellular distribution of pDNA as compared to PEI, most likely due to environmentally triggered release. Therefore, SS-PAEIs are a new class of transfection agents that facilitate high gene expression while maintaining a low level of toxicity.     

Synthesis of biodegradable cross-linked poly(beta-amino ester) for gene delivery and its modification, inducing enhanced transfection efficiency and stepwise degradation

Biodegradable cross-linked poly(beta-amino ester) (CLPAE) was synthesized by Michael addition of pentaerythritol triacrylate and N,N-dimethylethylenediamine and modified with aminohexanoic acid and lysine to CLPAE-Ahx and CLPAE-Lys, respectively, for a gene delivery system. They could self-assemble with plasmid DNA, forming nanosized polyplexes, and CLPAE-Ahx polyplex released plasmid DNA slowly during a week through stepwise degradation. The polymers showed minimal cytotoxicity on 293 cells due to their biodegradability and biocompatibility. Transfection efficiencies of CLPAE-Ahx and CLPAE-Lys were comparable to that of PEI in 293 cells and C2C12 cells. Additionally, high transfection of CLPAE-Ahx on primary rat aorta vascular smooth muscle cells (SMC) and primary mouse embryonic fibroblast cells (MEF) shows a potential for a gene delivery system on primary cells, restenosis treatment of human SMC, and MEF cell function research. In conclusion, CLPAE-Ahx could be used as a nontoxic and highly efficient gene delivery system.     

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

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

Low molecular weight polyethylenimine grafted N-maleated chitosan for gene delivery: properties and in vitro transfection studies

To develop chitosan-based efficient gene vectors, chitosans with different molecular weights were chemically modified with low molecular weight polyethylenimine. The molecular weight and composition of polyethylenimine grafted N-maleated chitosan (NMC-g-PEI) copolymers were characterized using gel permeation chromatography (GPC) and (1)H NMR, respectively. Agarose gel electrophoresis assay showed that NMC-g-PEI had good binding ability with DNA, and the particle size of the NMC-g-PEI/DNA complexes was 200-400 nm, as determined by a Zeta sizer. The nanosized complexes observed by scanning electron microscopy (SEM) exhibited a compact and spherical morphology. The NMC-g-PEI copolymers showed low cytotoxicity and good transfection activity, comparable to PEI (25 KDa) in both 293T and HeLa cell lines, except for NMC 50K-g-PEI. The results indicated that the molecular weight of NMC-g-PEI has an important effect on cytotoxicity and transfection activity, and low molecular weight NMC-g-PEI has a good potential as efficient nonviral gene vectors.