新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Poly[Lys-(AEDTP)]: a cationic polymer that allows dissociation of pDNA/cationic polymer complexes in a reductive medium and enhances polyfection.

Polyplexes of high stability resulting from the condensation of a plasmid DNA by a cationic polymer are widely used to develop polymer-based gene delivery systems. However, the plasmid must be released from its vector once inside the cells for an efficient expression of the exogenous gene in the cell nucleus. We have designed a disulfide-containing cationic polymer termed poly[Lys-(AEDTP)] which allowed for the formation of polyplexes and the release of the plasmid in a reductive medium. The amino groups of polylysine were substituted with 3-(2-aminoethyldithio)propionyl residues in order to have each amino group of poly[Lys-(AEDTP)] interacting with a phosphate DNA linked to the polymer backbone via a disulfide bond. As evidenced by agarose gel electrophoresis and ethidium bromide/pDNA fluorescence restoration, poly[Lys-(AEDTP)] polyplexes were decondensed and the plasmid released upon treatment with either dithiothreitol, glutathione in the presence of glutathione reductase, or the thioredoxin reductase. Electron microscopy showed that polyplexes exhibiting spherical particles of a mean size at about 100 nm were decondensed in the presence of glutathione and exhibited filamentous aggregates. Finally, we found that the transfection of 293T7 and HepG2 cells was 10- and 50-fold more efficient with poly[Lys-(AEDTP)] polyplexes, respectively, than with poly[Lys] polyplexes. These results indicate that disulfide-containing cationic polymers must be borne in mind for developing polymer-base gene delivery systems.     

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).     

Effect of polymer ionization on the interaction with DNA in nonviral gene delivery systems

The optimization of DNA-cationic polymer complexation is crucial for nonviral gene delivery. Although physicochemical characterization of the interaction between DNA and cationic polymers has recently attracted more attention in the nonviral DNA delivery field, the literature on the effect of varying polycation charge density on DNA-cationic polymer complexation is still scarce. Thus, the aim of this study was to systematically assess the influence of the degree of ionization of a weak cationic polyelectrolyte (poly[2-(dimethylamino)ethyl methacrylate] or DMAEMA homopolymer) on its ability to form complexes with DNA. This was achieved by varying the solution pH from 4.0 to 8.0 and analyzing the resulting effects on the binding affinity, thermodynamic properties, complex size, and morphology. Lowering the solution pH led to higher degrees of ionization for the cationic polymer and hence greater binding affinities with DNA, as judged by the increased propensity of the former to displace ethidium bromide from DNA and also by relatively low monomer:nucleotide molar ratio (0.8:1) required to retard the migration of free DNA. Isothermal titration microcalorimetry studies further confirmed that a stronger interaction occurred at low pH than at high pH. By decreasing the pH from 8.0 to 6.6, K(obs) increased from 7.8 x 10(5) to 20.4 x 10(5) M(-1). More efficient condensation at low pH was demonstrated by the reduction of ethidium bromide fluorescence in the loading wells from gel electrophoresis, decreased complex sizes without agglomeration occurring at high polymer/DNA ratios, together with discrete and dense spherical complexes observed in TEM studies. This may be attributed to the presence of electrostatic stabilization from excess cationic polymer chains, which provide a repulsive shell around the polymer/DNA complex. The physicochemical data indicate that the increased degree of ionization for the DMAEMA homopolymer at lower pH results in higher binding affinity, smaller and more compact complexes, and more efficient condensation. These findings therefore highlight the importance of the degree of ionization on DNA complex formation for weak cationic polyelectrolytes.     

Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection

A series of amphiphilic star and linear block copolymers were synthesized using ATRP. The core consisted of either polystyrene (PS) or poly(n-butyl acrylate) (PBuA), having different glass-transition (T(g)) values. These polymers were used as macroinitiators in the polymerization of the cationic 2-(dimethylamino)ethyl methacrylate (DMAEMA). The polymers were used to study the effects of polymer architecture and flexibility on the self-assembling properties, DNA complexation, and transfection. All polymers formed core-shell micelles in aqueous solutions and condensed plasmid DNA. Linear PDMAEMA-PBuA-PDMAEMA has transfection efficiency comparable to PEI25K in ARPE19 cell line. Glassy state of the micellar core and star-shaped architecture decreased the DNA transfection compared with the rubbery and linear polymer structures. The polymers showed low cellular toxicity at low nitrogen/phosphate (n/p) ratios.     

Poly(DMAEMA-NVP)-b-PEG-galactose as gene delivery vector for hepatocytes

A block copolymer composed of cationic polymer and poly(ethylene glycol) (PEG) was used as a DNA carrier. Poly(2-(dimethylamino)ethyl methacrylate (DMAEMA)-co-N-vinyl-2-pyrrolidone (NVP)) having a terminal carboxylic group was synthesized by free radical polymerization using an initiator, 4,4'-azobis(4-cyanovaleric acid). The terminal carboxylic acid was activated by N-hydroxysuccinimide (NHS) with dicyclohexylcarbodiimide (DCC) and then conjugated with PEG-bis(amine). For specific gene targeting to asialoglycoprotein receptor of hepatocytes, a galactose moiety was incorporated into the PEG terminal end of poly(DMAEMA-NVP)-b-PEG by reductive coupling using lactose and sodium cyanoborohydride. RSV luciferase plasmid was used as a reporter gene, and in vitro gene transfection efficiency was measured in HepG2 human hepatocarcinoma cells. Poly(DMAEMA-NVP)-b-PEG-galactose/DNA complexes formed at 0.5-2 polymer/plasmid weight ratio had compacted structures around 200 nm particle size and exhibited slightly negative surface charge. These complexes were coated with a cationic, pH sensitive, endosomolytic peptide, KALA, to generate positively charged poly(DMAEMA-NVP)-b-PEG-galactose/DNA/KALA complex particles. In the presence of serum proteins, both the PEG block and the galactose moiety of poly(DMAEMA-NVP)-b-PEG-galactose greatly enhanced the gene transfection efficiency, which was very close to that of Lipofectamine plus. Irrespective of the presence of serum proteins, as the KALA/DNA weight ratio increased, the transfection efficiency of poly(DMAEMA-NVP)-b-PEG-galactose was enhanced due to the pH dependent endosomal disruptive property of KALA. This study demonstrates that sufficient transfection efficiency as high as that of commercial agent could be attained by judicious formulation of molecular engineered poly(DMAEMA-NVP)-b-PEG-galactose in combination with an endosomolytic peptide, KALA.     

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.     

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.     

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.     

D-甘露糖修饰聚合物胶束的制备及其在靶向药物输送中的应用

聚合物胶束作为药物载体具有良好的稳定性和生物相容性,提高疏水性药物溶解性等优势,是一类很有应用潜力的药物传输系统。本研究以合成的共价键连D-甘露糖的双亲性聚合物分子(PGMA-Mannose)为药物载体,包载抗癌药物阿霉素(DOX)制备具有甘露糖受体靶向性和pH敏感药物释放特性的新型载药聚合物胶束。利用激光共聚焦显微镜和MTT细胞毒性评价方法对载药胶束的细胞内吞摄取和毒性进行评价。实验结果表明,载药胶束能特异性识别人乳腺癌细胞MDA-MB-231表面过度表达的甘露糖受体,被癌细胞大量摄取并在细胞溶酶体酸性环境内释放药物,而载药胶束在表面甘露糖受体低表达的HEK293细胞中只有少量摄取。与原药DOX相比,该载药胶束对癌细胞的毒性显著提高,而对正常细胞的毒性较低。因此,该PGMA-Mannose聚合物胶束有望成为一种新型的靶向药物输送系统应用于癌症的治疗。     

Melittin enables efficient vesicular escape and enhanced nuclear access of nonviral gene delivery vectors

Entry of exogenously applied DNA into the cytoplasm and subsequent transport into the nucleus are major cellular barriers for nonviral gene delivery vectors. To overcome these barriers, we have covalently attached the cationic peptide melittin to poly(ethylenimine) (PEI). This conjugate condensed DNA into small, discrete particles (<100 nm in diameter), and the membrane lytic activity of melittin enabled efficient release of the DNA into the cytoplasm, as monitored by fluorescence microscopy and flow cytometry. Compared with PEI, the transfection activity was strongly increased within a broad range of cell lines and types tested, including different tumor cell lines but also primary hepatocytes and human umbilical vein endothelial cells. The early onset of gene expression (within 4 h, reaching maximal values after 12 h) and the high reporter gene expression achieved in slowly dividing or confluent cells suggested a further role of melittin after releasing the DNA into the cytoplasm. Intracytoplasmic microinjection of melittin-containing PEI.DNA complexes into fibroblasts produced 40% cellular frequency of reporter gene expression that was inhibitable by co-injection of wheat germ agglutinin, whereas simple PEI.DNA complexes showed only 10%. These data suggest that melittin enables release of nonviral gene transfer particles into the cytoplasm and also enhances their transport into the nucleus, possibly via the cationic cluster KRKR near the C terminus of the peptide.     

Spectroscopic and molecular docking investigation of polynucleotides and DNA binding affinity to Nile blue dye

We used UV-vis absorption spectroscopy, fluorescence spectrophotometry and molecular docking calculations to investigate intermolecular interaction between the cationic dye, Nile blue (NB), and synthetic polynucleotides, poly(A-T), poly(G-C) and calf thymus DNA (Ct-DNA) at physiological pH. Strong hypsochromic absorbance and fluorescence quenching were observed that showed strong binding of NB to these polynucleotides and DNA. The binding affinity values derived from maximum absorption of the spectra of NB bound to various polynucleotides and Ct-DNA concentrations suggests that NB exhibits greater binding affinity to poly(G-C) than to poly(A-T). The thermodynamic parameters suggested that hydrogen bonds and van der Waals forces might play a major role in the binding of NB to DNA. The molecular docking results suggested that NB was an intercalator of the stacked base pairs of Ct-DNA.     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Surface-tethered DNA complexes for enhanced gene delivery

Overcoming the barriers to efficient gene transfer is a fundamental goal of biotechnology. A versatile approach to enhance the delivery of nonviral DNA involves complexation with cationic polymers, which can be designed to overcome the barriers to effective gene transfer. More recently, DNA release from a polymer substrate or scaffold has been shown to enhance gene transfer, likely by increasing DNA concentrations in the cell microenvironment. We propose a novel approach that combines these two strategies in which cationic polymer/DNA complexes are tethered to a substrate that supports cell adhesion. The cationic polymers package the DNA for efficient internalization and the surface tethering functions to maintain elevated concentrations in the cell microenvironment for cells adhered to the substrate. The cationic polymer polylysine (degree of polymerization equal to 19 or 150) was modified with biotin groups, which was confirmed by mass spectrometry and biochemical analysis. Complex formation of DNA with biotinylated-polylysine, or mixtures of biotinylated and nonbiotinylated polylysines, was confirmed by gel electrophoresis. Plasmid DNA encoding for the reporter gene beta-galactosidase was complexed with different mixtures of biotinylated and nonbiotinylated polylysine and incubated on neutravidin (nonglycosylated avidin)-coated surfaces. DNA surface densities ranging from 0.1 to 4.3 microg/cm2 were observed and found to be a function of the number of biotin groups, the molecular weight of the polylysine, and the amount of DNA. HEK293T or NIH/3T3 cells were then seeded onto the DNA-modified surfaces, and transfection was quantified at 48 and 96 h. Transfection by the DNA surfaces was observed with both cell lines, and expression levels up to 100 fold greater than bulk delivery of the complexes was obtained. Transfection was found to be a function of the surface DNA quantities and the number of tethers on the complex. Transfected cells were observed only in the region in which DNA complexes were tethered, suggesting that the location of transfected cells can be specifically controlled. Surface tethering of DNA represents a promising approach to enhancing gene transfer and spatially controlling gene delivery, which may have applications to a multitude of fields ranging from tissue engineering to functional genomics.     

Endosomal escape of polymeric gene delivery complexes is not always enhanced by polymers buffering at low pH

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes ("polyplexes") from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH ("proton sponge"). We synthesized a new polymer with two tertiary amine groups in each monomeric unit [poly(2-methyl-acrylic acid 2-[(2-(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA]. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis.     

Gene delivery properties of end-modified poly(beta-amino ester)s

Here, we present the synthesis of a library of end-modified poly(beta-amino ester)s and assess their utility as gene delivery vehicles. Polymers were synthesized using a rapid, two-step approach that involves initial preparation of an acrylate-terminated polymer followed by a postpolymerization amine-capping step to generate end-functionalized polymers. Using a highly efficient poly(beta-amino ester), C32, we show that the terminal amine can greatly affect and improve polymer properties relevant to gene delivery. Specifically, the in vitro transfection levels can be increased by 30% and the optimal polymer:DNA ratio lowered 5-fold by conjugation of the appropriate end group. The most effective modifications were made by grafting primary diamine molecules to the chain termini. The added charge and hydrophobicity of some derivatives enhanced DNA binding and resulted in the formation of polymer-DNA complexes less than 100 nm in diameter. In addition, cellular uptake was improved 5-fold over unmodified C32. The end-modified poly(beta-amino ester)s presented here are some of the most effective gene-delivery polycations, superior to polyethylenimine and previously reported poly(beta-amino ester)s. These results show that the end-modification of poly(beta-amino ester)s is a general strategy to alter functionality and improve the delivery performance of these materials.     

Development and evaluation of a novel nano‐scale vector for siRNA

To synthesize a lipid‐cationic polymer (LCP) containing brassidic acid side chain and to investigate its transfection efficiency and characteristics as a siRNA gene vector. The LCP was chemically synthesized and its nucleic acid binding capacity was determined by gel electrophoresis. HeLa‐EGFP and TH1080‐EGFP cell lines were transfected with siRNA against enhanced green fluorescent protein (EGFP) gene using a LCP to investigate the transfection efficiency. An MTT assay was performed to evaluate the cellular toxicity of the LCP vector. Its degradability and stability under acidic conditions were also investigated. The LCP vector possessed high DNA binding capacity. More than 73% of the cellular fluorescence was inhibited by the LCP‐mediated transfection of siRNA against EGFP gene, indicating that vector had high transfection efficiency. Cellular viability was about 95% at the optimum transfection efficiency of LCP, suggesting that the cellular toxicity of LCP was very low. The LCP was also observed to be degradable; moreover, it could be easily stored at normal temperature. A gene vector used for the transfection of siRNA was successfully fabricated from synthesized LCP. Its numerous excellent properties entitle values for further scientific research. J. Cell. Biochem. 111: 881–888, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of the incorporation of a hydrophobic middle block into a PEG-polycation diblock copolymer on the physicochemical and cell interaction properties of the polymer-DNA complexes

One-component homopolymers of cationic monomers (polycations) and diblock copolymers comprising poly(ethylene glycol) (PEG) and a polycation block have been the most widely used types of polymers for the formulation of polymer-based gene delivery systems. In this study, we incorporate a hydrophobic middle block into the conventional PEG-polycation architecture and investigate the effects of this hydrophobic modification on the physicochemical and cell-level biological properties of the polymer-DNA complexes that are relevant to gene delivery applications. The ABC-type triblock copolymer used in this study consists of (A) PEG, (B) hydrophobic poly( n-butyl acrylate) (PnBA), and (C) cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) component polymers. The properties of the triblock copolymer/DNA complexes are compared with those of two other more conventional DNA carriers derived, respectively, using a PDMAEMA homopolymer and a PEG-PDMAEMA diblock copolymer that had comparable molecular weights for individual blocks. In aqueous solution, the PEG-PnBA-PDMAEMA polymer forms positively charged spherical micelles. The electrostatic complexation of these micelles with plasmid DNA molecules results in the formation of stable small-sized DNA particles that are coated with a micelle monolayer, as confirmed by agarose gel electrophoresis, dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). Proton nuclear magnetic resonance ( (1)H NMR) spectroscopy measurements indicate that the whole micelle-DNA assembly (named "micelleplex" for convenience) is shielded predominantly by the PEG chains. DLS and optical microscopy imaging measurements indicate that compared with PDMAEMA-DNA polyplexes, the micelleplexes have a significantly lower tendency to aggregate under physiological salt concentrations and show reduced interactions with negatively charged components in serum such as albumin and erythrocytes. While the micelleplexes are comparable to the PEG-PDMAEMA-based DNA polyplexes in terms of their stability against aggregation under high salt concentrations and in the presence of the albumin protein, they have a slightly higher tendency to interact with erythrocytes than the diblock copolymer polyplexes. Agarose gel electrophoresis measurements indicate that relative to the PEG-PDMAEMA polyplexes, the micelleplexes provide better protection of the encapsulated DNA from enzymatic degradation and also exhibit greater stability against disintegration induced by polyanionic additives; in these respects, the PDMAEMA homopolymer-based polyplexes show the best performance. In vitro studies in HeLa cells indicate that the PDMAEMA polyplexes show the highest gene transfection efficiency among the three different gene delivery systems. Between the micelleplexes and the PEG-PDMAEMA polyplexes, a higher gene transfection efficiency is observed with the latter system. All three formulations show comparable levels of cytotoxicity in HeLa cells.     

PGS支架的合成、特性及在生物医学中应用的研究进展

聚癸二酸甘油酯(PGS)是一种生物可降解的高分子聚合弹性体,因其良好的性能,在许多生物医学研究中应用广泛。PGS支架的机械性能与机体软组织相似,依从性好,降解时以表面侵蚀的方式降解,不伴有膨胀或变形,周围组织炎症反应、纤维变性轻,与多种细胞相容性好。基于PGS良好的性能,主要应用于软组织替代和软组织工程,比如心肌、血管、神经、软骨、视网膜、鼓膜,另外也有用于药物转运载体、组织粘附材料的研究。     

Synthesis and evaluation of vitamin D-based cationic lipids for gene delivery in vitro

A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.