Structural effects of carbohydrate-containing polycations on gene delivery. 3. Cyclodextrin type and functionalization

Linear cationic beta-cyclodextrin (beta-CD)-based polymers can form polyplexes with plasmid DNA and transfect cultured cells. The effectiveness of the gene delivery and the cellular toxicity has been related to structural features in these polycations. Previous beta-CD polycations were prepared from the cocondensation of 6(A),6(D)-dideoxy-6(A),6(D)-diamino-beta-CD monomers with other difunctionalized monomers such as dimethyl suberimidate (DMS). Here, the type of CD and its functionalization are varied by synthesizing numerous 3(A),3(B)-dideoxy-3(A),3(B)-diamino-beta- and gamma-CD monomers. Both alkyl- and alkoxydiamines are prepared in order to vary the nature of the spacing between the CD and the primary amines in the monomers. These diamino-CD-monomers are polymerized with DMS to yield amidine-based polycations. The nature of the spacer between the CD-ring and the primary amines of each monomer is found to influence both molecular weight and polydispersity of the polycations. When these polycations are used to form polyplexes with plasmid DNA, longer alkyl regions between the CD and the charge centers in the polycation backbone increase transfection efficiency and toxicity in BHK-21 cells, while increasing hydrophilicity of the spacer (alkoxy versus alkyl) provides for lower toxicity. Further, gamma-CD-based polycations are shown to be less toxic than otherwise identical beta-CD-based polycations.     

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.     

新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Structure-activity examination of poly(glycoamidoguanidine)s: glycopolycations containing guanidine units for nucleic acid delivery

In this study we synthesized a new series of polymers known as poly(glycoamidoguanidine)s (PGAGs). These new polymer structures were synthesized by copolymerizing a carbohydrate monomer (diester; galatarate or tartarate) with a diamine incorporating guanidine or methylguanidine as a charge center to create a polyamide backbone. These materials were strategically designed and compared to our previously studied DNA delivery vehicles, poly(glycoamidoamine)s (PGAAs), which contain secondary amines as the charge groups along the polymer backbone to examine the effect of charge center type on the cellular delivery efficiency of plasmid DNA (pDNA). The guanidine moieties within the PGAGs facilitate electrostatic binding with the negatively charged phosphate backbone of plasmid DNA (pDNA). Stable polymer-pDNA complexes (polyplexes) with sizes in the range of 60-200 nm are formed at polymer/pDNA charge ratios (N/P) of 5 and above. When the PGAGs are complexed with Cy5-labeled pDNA (Cy5-pDNA) at N/P ratios of 10 and 25, between 80 and 95% of HeLa cells were positive for Cy5 fluorescence, indicating effective cellular internalization of the polyplexes. The toxicity of both PGAA and PGAG polyplexes was studied via MTT assays, and over 95% cell survival was observed at N/P ratios of 5, 10, 15, 20, 25, and 30 in HeLa cells. Transgene expression was examined via luciferase assays at various N/P ratios in the absence and presence of serum. In the absence of serum, the PGAG polyplexes revealed similar transgene expression when compared to polyplexes formed with their analogous PGAA structures. In the presence of serum, one analog (Gg) consisting of galactarate copolymerized with the guanidine monomer yielded gene expression similar to the positive control, Glycofect Transfection Reagent. This new series of guanidine-containing oligomers are promising as a new design strategy to incorporate an alternative charge center type within the backbone of glycopolymer-based nucleic acid delivery vehicles.     

Peptide-Based Multicomponent Oligonucleotide Delivery Systems: Optimisation of Poly-<Emphasis Type="SmallCaps">l</Emphasis>-lysine Dendrons for Plasmid DNA Delivery

Gene therapy is a promising means to treat or prevent diseases either through gene silencing or expression. Some of the most effective delivery agents are polycationic dendrimers, which are highly branched constructs incorporating many positively charged groups. Two of the most effective dendrimers are polyethyleneimine (PEI) and poly(amidoamine) (PAMAM), which show high proficiency at overcoming barriers to oligonucleotide delivery. However, because of their abundance of cationic charge, they are associated with severe toxicity. We have therefore aimed to develop a low toxicity oligonucleotide delivery system, incorporating multiple components that have been selected and optimised to overcome the barriers to efficient oligonucleotide delivery. In this work we have focused on improving the toxicity, cellular uptake, and condensation of plasmid DNA (pDNA) through the fusion of synthetic poly-l-lysine (PLL) dendrons with the cell penetrating peptide TAT(48-60). A library of dendron structures, from 4⁺ to 16⁺ charge, and constructs containing six histidine residues, were synthesised. The effects of each modification on pDNA binding and condensation; cellular uptake and toxicity; and the size and zeta-potential of the complexes were assessed to identify the optimum dendron for incorporation into our systems. This work concluded that increasing the dendron charge from 4⁺ to 16⁺ significantly improved cellular uptake and pDNA condensation, with no effect on toxicity, while PLL dendrons with greater than 16⁺ charge could not be efficiently produced. In comparison, the incorporation of six histidines into these constructs had a variable effect on cellular uptake, and generated larger sized complexes, but did not affect toxicity.     

PEGylation of poly(ethylene imine) affects stability of complexes with plasmid DNA under in vivo conditions in a dose-dependent manner after intravenous injection into mice

The influence of PEGylation on polyplex stability from poly(ethylene imine), PEI, and plasmid DNA was investigated both in vitro and after intravenous administration in mice. Polyplexes were characterized with respect to particle size (dynamic light scattering), zeta-potential (laser Doppler anemometry), and morphology (atomic force microscopy). Pharmacokinetics and organ accumulation of both polymers and pDNA were investigated using 125I and 32P radioactive labels, respectively. Furthermore gene expression patterns after 48 h were measured in mice. To elucidate the effect of different doses, all experiments were performed using ca. 1.5 microg and 25 microg of pDNA per mouse. Our studies demonstrated that both PEI and PEG-PEI form stable polyplexes with DNA with similar sizes of 100-130 nm. The zeta potential of PEI/pDNA polyplexes was highly positive, whereas PEG-PEI/pDNA showed a neutral surface charge as expected. The pharmacokinetic and organ distribution profiles after 2 h show similarities for both PEI and pDNA blood-level time curves from polyplexes at both doses indicative for significant stability in the bloodstream. A very rapid clearance from the bloodstream was observed and as major organs of accumulation liver and spleen were identified. PEG-PEI/pDNA complexes at a dose of approximately 25 microg exhibit similar profiles except a significantly lower deposition in the lung. At the lower dose of approximately 1.5 microg pDNA, however, for polyplexes from PEG-PEI, significant differences in blood level curves and organ accumulation of polymer and pDNA were found. In this case PEG-PEI shows a greatly enhanced circulation time in the bloodstream. By contrast, pDNA was rapidly cleared from circulation and significant amounts of radioactivity were found in the urine, suggesting a rapid degradation possibly by serum nucleases after complex separation. Regarding in vivo gene expression, no luciferase expression could be detected at approximately 1.5 microg dose in any organ using both types of complexes. At 25 microg only in the case of PEI/pDNA complexes were significant levels of the reporter gene detected in lung, liver, and spleen. This coincided with high initial accumulation of pDNA complexed with PEI and a high acute in vivo toxicity. For PEG-PEI, initial accumulation was much lower and no gene expression as well as a low acute toxicity was found. In summary, our data demonstrate that PEG-PEI used in this study is not suitable for low dose gene delivery. At a higher dose of approximately 25 microg, however, polyplex stability is similar to PEI/pDNA combined with a more favorable organ deposition and significantly lower acute in vivo toxicity. These findings have consequences for the design of PEG-PEI-based gene delivery systems for in vivo application.     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

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

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

Biotin-triggered release of poly(ethylene glycol)-avidin from biotinylated polyethylenimine enhances in vitro gene expression

Covalently poly(ethylene glycol) (PEG)-ylated polyethylenimine (PEI)/pDNA complexes display prolonged blood circulation profiles compared with PEI/pDNA complexes, but such PEGylated particles may not be suitable for tumor targeting due to low interaction with cell membranes, low internalization, and low gene expression. Noncovalent PEGylation of cationic particles via PEG-avidin/biotin-PEI is an attempt to bridge the gap between the positive attributes of PEG (prolonged particle circulation) and the positive attributes of nontoxic cationic polymers (enhanced cell interactions) for greater gene expression. Our polymer, 2PEG-avidin/biotin-PEI8, forms salt-stable particles ( approximately 100 nm) under physiologic conditions with a minimum of two 2PEG-avidin molecules bound per polymer chain (biotin-PEI8, 8 biotins/PEI). Following 10 days of incubation with 3000-fold excess biotin, 2PEG-avidin completely dissociated from biotin-PEI8, and gene expression was increased 2.1-32-fold in various cell lines when the desirable transfection feature of the cationic polymer was retained. This new PEGylation approach has implications for generally improving the clinical aspect of gene delivery via a two-step therapeutic strategy: (1) intravenous injection of noncovalent PEG-avidin/biotin-polycation nanoparticles for prolonged circulation, followed by (2) temporal release of PEG-avidin from biotin-polycation through either endogenous biotin or intravenous injection of biotin.     

Galactose-poly(ethylene glycol)-polyethylenimine for improved lung gene transfer

Polyethylenimine (PEI) is a potential gene transfer agent, but is limited by its poor transfection efficiency in vivo due to poor solubility and stability, pronounced toxicity and non-specific interaction with target cells. To improve its pulmonary gene transfection property, galactose (whose binding lectins are abundantly expressed in the lung) was selected as a ligand to improve the binding and uptake of the modified PEI/pDNA (plasmid DNA) polyplexes into lung cells. A novel protocol was developed to synthesize galactose-polyethylenglycol (PEG)-PEI copolymers. The resulting galactose-PEG-PEI/pDNA polyplexes showed improved solubility, stability, and reduced toxicity. Compared with that obtained by PEI/pDNA at a N/P ratio of 6, the transfection efficiency of 1% galactose-PEG-PEI/pDNA polyplexes at the N/P ratio of 36 was 4.5- and 11.6-fold in the A549 cell line and in mice lung, respectively. These data taken suggest that galactose-PEG-PEI may be a promising pulmonary gene delivery system.     

Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody

To achieve efficient systemic gene delivery to the lung with minimal toxicity, a vector was developed by chemically conjugating a cationic polymer, polyethylenimine (PEI), with anti-platelet endothelial cell adhesion molecule (PECAM) antibody (Ab). Transfection of mouse lung endothelial cells with a plasmid expression vector with cDNA to luciferase (pCMVL) complexed with anti-PECAM Ab-PEI conjugate was more efficient than that with PEI-pCMVL complexes. Furthermore, the anti-PECAM Ab-PEI conjugate mediated efficient transfection at lower charge plus-to-minus ratios. Conjugation of PEI with a control IgG (hamster IgG) did not enhance transfection of mouse lung endothelial cells, suggesting that the cellular uptake of anti-PECAM Ab-PEI-DNA complexes and subsequent gene expression were governed by a receptor-mediated process rather than by a nonspecific charge interaction. Conjugation of PEI with anti-PECAM Ab also led to significant improvement in lung gene transfer to intact mice after intravenous administration. The increase in lung transfection was associated with a decrease compared with PEI-pCMVL with respect to circulating proinflammatory cytokine (tumor necrosis factor-alpha) levels. These results indicate that targeted gene delivery to the lung endothelium is an effective strategy to enhance gene delivery to the pulmonary circulation while simultaneously reducing toxicity.     

Protein-polymer nanoparticles for nonviral gene delivery

Protein-polymer conjugates were investigated as nonviral gene delivery vectors. BSA-poly(dimethylamino) ethyl methacrylate (PDMA) nanoparticles (nBSA) were synthesized using in situ atom transfer radical polymerization (in situ ATRP) and BSA as a macroinitiator. The diameter and charge of nBSA was a function of the ATRP reaction time and ranged from 5 to 15 nm and +8.9 to +22.5, respectively. nBSA were able to condense plasmid DNA (pDNA) and form polyplexes with an average diameter of 50 nm. nBSA/pDNA polyplexes transfected cells with similar efficiencies or better as compared to linear and branched PEI. Interestingly, the nBSA particle diameter and charge did not affect pDNA complexation and transgene expression, indicating that the same gene delivery efficiency can be achieved with lower charge ratios. We believe that with the use of protein-polymer conjugates additional functionality could be introduced to polyplexes by using different protein cores and, thus, they pose an interesting alternative to the design of nonviral gene delivery vectors.     

Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer

We have developed a novel vector constructed with pDNA, polyethylenimine (PEI), and mucin 1 (MUC1) aptamer for tumor-targeted gene delivery. The MUC1 aptamer and non-specific aptamer were employed to coat the pDNA/PEI complexes electrostatically and stable nanoparticles were formed. The addition of a non-specific aptamer to the pDNA/PEI complex decreased gene expression in the human lung cancer cell line, A549 cells expressing MUC1 regularly. At the same time, the pDNA/PEI/MUC1 aptamer complex showed higher gene expression than pDNA/PEI/non-specific aptamer complex. Furthermore, the pDNA/PEI/MUC1 aptamer complex showed markedly high gene expression in tumor-bearing mice; thus, pDNA/PEI/MUC1 aptamer complexes are useful as a tumor-targeted gene delivery system with high transfection efficiency.     

Nuclear delivery of plasmid DNA determines the efficiency of gene expression

As a cationic non‐viral gene delivery vector, poly(agmatine/ N, N′‐cystamine‐bis‐acrylamide) (AGM‐CBA) showed significantly higher plasmid DNA (pDNA) transfection ability than polyethylenimine (PEI) in NIH/3T3 cells. The transfection expression of AGM‐CBA/pDNA polyplexes was found to have a non‐linear relationship with AGM‐CBA/pDNA weight ratios. To further investigate the mechanism involved in the transfection process of poly(AGM‐CBA), we used pGL3‐control luciferase reporter gene (pLUC) as a reporter pDNA in this study. The distribution of pLUC in NIH/3T3 cells and nuclei after AGM‐CBA/pLUC and PEI/pLUC transfection were determined by quantitative polymerase chain reaction (qPCR) analysis. The intracellular trafficking of the polyplexes was evaluated by cellular uptake and nuclei delivery of pLUC, and the intracellular availability was evaluated by the ratio of transfection expression to the numbers of pLUC delivered in nuclei. It was found that pLUC intracellular trafficking did not have any correlation with the transfection expression, while an excellent correlation was found between the nuclei pLUC availability and transfection expression. These results suggested that the intracellular availability of pLUC in nuclei was the rate‐limiting step for pLUC transfection expression. Further optimization of the non‐viral gene delivery system can be focused on the improvement of gene intracellular availability.     

Tris[2-(acryloyloxy)ethyl]isocyanurate cross-linked low-molecular-weight polyethylenimine as gene delivery carriers in cell culture and dystrophic mdx mice

Hyperbranched poly(ester amine)s (PEAs) were successfully synthesized by Michael addition reaction between tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) and low-molecular-weight polyethylenimine (LPEI, M(w) 0.8k, 1.2k, and 2.0k) and evaluated in vitro and in vivo as gene carriers. PEAs effectively condensed plasmid DNA with particle sizes below 200 nm and surface charges between 11.5 and 33.5 mV under tested doses [at the ratios 2-10:1 of polymer/pDNA(w/w)]. The PEAs showed significantly lower cytotoxicities when compared with PEI 25k in two different cell lines. The PEAs (C series) composed of PEI 2k showed higher transgene expression compared to PEAs of PEI 0.8k (A series) or 1.2k (B series). Highest gene transfection efficiency in CHO, C2C12 myoblast, and human skeletal muscle (HSK) cell lines was obtained with TAEI/PEI-2K (C12) at a ratio of 1:2. Both C12, C14(TAEI/PEI-2K at a ratio of 1:4) demonstrated 5-8-fold higher gene expression as compared with PEI 25k in mdx mice in vivo through intramuscular administration. No obvious muscle damage was observed with these new polymers. Higher transfection efficiency and lower toxicity indicate the potential of the biodegradable PEAs as safe and efficient transgene delivery vectors.     

Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans

BACKGROUND: Here we report on studies that probe whether the intracellular kinetics of plasmid DNA (pDNA) and cell surface glycosaminoglycans (GAGs) are modified during the cell cycle in a way that can be correlated with changes in gene transfer efficiency with poly(ethyleneimine) (PEI) and poly-L-lysine (PLL) polyplexes. METHODS: Synchronized D407 retinal cells were transfected with PEI and PLL polyplexes using a luciferase reporter. The free and/or loosely complexed nuclear pDNA was determined by real-time PCR, and compared with transgene expression, the rate of pinocytosis by FITC-dextran uptake and the content of cell surface GAGs. RESULTS: The amount of free and/or loosely complexed nuclear pDNA between cell cycle phases varied approximately 4-20 times (G1 < S < G2/M). Both carriers delivered pDNA in a similar way into the nucleus (PLL vs. PEI < or = 3.5-fold), but PEI was approximately 10-100 times more efficient in gene expression than PLL (G1 < G2/M < S). The rate of pinocytosis increased up to 70-fold from G1 to middle S phase. Cell surface heparan and chondroitin sulfate increased 50-80%, and hyaluronan decreased 50% when the cells went from G1 through S to G2/M. CONCLUSIONS: The data obtained indicates that no single parameter (pinocytosis, cell surface GAGs, nuclear uptake) solely accounts for the differential pDNA uptake or expression during cell cycle, and that the main difference in PLL- and PEI-mediated transfections seems to be at the nuclear level.     

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.     

Biophysical characterization of the DNA binding and condensing properties of adenoviral core peptide mu.

Cationic peptides containing Lys and Arg residues interact with DNA via charge-charge interactions and are known to play an important role in DNA charge neutralization and condensation processes. In this paper, we describe investigations of the interaction of the cationic adenovirus core complex peptide mu with a dodecameric ODN (12 bp) and pDNA (7528 bp) using a combination of fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and photon correlation spectroscopy. Comparisons are made with protamine, a cationic peptide well-known for DNA charge neutralization and condensation. Equilibrium dissociation constants are derived independently by both CD and ITC methods for the interaction between protamine or mu with pDNA (K(d) = 0.6-1 microM). Thermodynamic data are also obtained by ITC, indicating strong charge-charge interactions. The interaction of protamine with pDNA takes place with decreasing entropy (-28.7 cal mol(-1) K(-1)); unusually, the interaction of mu with pDNA takes place with increasing entropy (Delta S degrees (bind) = 11.3 cal mol(-1) K(-1)). Although protamine and mu appear to destabilize pDNA double helix character to similar extents, according to CD thermal titration analyses, PCS studies show that interactions between mu and pDNA result in the formation of significantly more size-stable condensed particles than protamine. The enhanced flexibility and size stability of mu-DNA (MD) particles (80-110 nm) compared to protamine counterparts suggest that MD particles are ideal for use as a part of new nonviral gene delivery vectors.     

Structural effects of carbohydrate-containing polycations on gene delivery. 1. Carbohydrate size and its distance from charge centers

Cationic polymers have the ability to bind plasmid DNA (pDNA) through electrostatic interactions and condense it into particles that can be readily endocytosed by cultured cells. The effects that polycation structure has on toxicity and gene delivery efficiency are investigated here by synthesizing a series of amidine-based polycations that contain the carbohydrates d-trehalose and beta-cyclodextrin (CD) within the polycation backbone. The carbohydrate size (trehalose vs CD) and its distance from the charge centers affect the gene delivery behavior in BHK-21 cells. It is found that as the charge center is further removed from the carbohydrate unit, the toxicity is increased. Also, as the size of the carbohydrate moiety is enlarged from trehalose to beta-cyclodextrin, the toxicity is reduced. The absence of a carbohydrate in the polycation produces high toxicity. All carbohydrate polycations transfect BHK-21 cells to approximately the same level of gene expression.