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

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

Chitosan lactate as a nonviral gene delivery vector in COS-1 cells

The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate, [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (2∶1, 4∶1, 8∶1, 12∶1, 24∶1) formed complexes with pSV β-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 12∶1, but less efficient than PEI (P<.05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was ≈50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity. Published: August 4, 2006     

Synthesis and characterization of a head-tail type polycation block copolymer as a nonviral gene vector

A head-tail type polycation block copolymer, which is composed of the polyamidoamine (PAMAM) dendron and poly(L-lysine) (PLL) blocks, was newly designed as a nonviral gene vector in this study. This block copolymer (PAMAM dendron-PLL) was successfully synthesized in two steps: the synthesis of the PAMAM dendron block and the polymerization of the PLL block from the PAMAM dendron block. PAMAM dendron and PLL blocks in block copolymer showed independent deprotonation behavior, and their pK(a) were determined to be 6.8 and 9.0, respectively. The complexation with pDNA was evaluated by gel retardation assay and dye exclusion assay, and both assays indicated that pDNA was selectively complexed with PLL block of block copolymer. Also, the PAMAM dendron-PLL poplyplexes showed 10(2) fold higher transfection efficiency to HeLa cells as that for PLL polyplexes. This might be due to the buffering effect of the PAMAM dendron block. This block copolymer could produce a function share in each block, i.e., tail block complexed with pDNA and head block showed a buffering effect. This molecular design of the head-tail type block copolymer might provide a new approach for realizing in vivo gene therapy.     

Nucleofection as an efficient nonviral transfection method for human monocytic cells

Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available. A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results. As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity. Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2–6 h. The technique is suitable not only for monocytes but also for other hard-to-transfect cells.     

Acid‐activated nonviral peptide vector for gene delivery

Nonviral vector–based gene therapy is a promising strategy for treating a myriad of diseases. Cell‐penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl‐TH by attaching a stearyl moiety to the N‐terminus of the acid‐activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl‐TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl‐TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl‐TH and the complexes formed by stearyl‐TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl‐TH a potential nucleic acid delivery vector for gene therapy.     

Alkyl chain moieties of polyamidoamine dendron-bearing lipids influence their function as a nonviral gene vector

We recently developed a novel family of cationic lipids consisting of a polyamidoamine (PAMAM) dendron and two dodecyl chains. Their transfection activity increases with increasing generation of the dendron moiety [Takahashi et al. (2003) Bioconjugate Chem. 14, 764-773]. In the present study, to elucidate the effect of hydrophobic tail moieties of the dendron-bearing lipids, two kinds of PAMAM G3 dendron-bearing lipids were synthesized with different alkyl lengths, DL-G3-2C18 and DL-G3-2C12. Their functions as gene vectors were compared. Irrespective of their different alkyl chain lengths, these dendron-bearing lipids formed complexes with plasmid DNA with similar efficiency. However, their complex sizes differed markedly: DL-G3-2C18 lipoplexes exhibited much smaller diameters than DL-G3-2C12 lipoplexes. Interaction of the lipoplexes with heparin revealed that the DL-G3-2C18 lipoplexes required more heparin than DL-G3-2C12 lipoplexes to cause dissociation of plasmid DNA from the lipoplexes. Although the DL-G3-2C12 lipoplexes and DL-G3-2C18 lipoplexes transfected CV1 cells with similar efficiency in the absence of serum, only the latter retained high transfection activity in the presence of serum. These results indicate that hydrophobic interaction of alkyl chain moieties plays an important role in the increment of stability and the serum-resistant transfection activity for dendron-bearing lipid lipoplexes.     

Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy

This paper investigates a series of dendrons based on the Newkome dendritic scaffold that displays a naturally occurring polyamine (spermine) on their surface. These dendrons have previously been shown to interact with DNA in a generation dependent manner with the more highly branched dendrons exhibiting a strong multivalency effect for the spermine surface groups. In this paper, we investigate the ability of these dendrons to transfect DNA into cells (human breast carcinoma cells, MDA-MB-231, and murine myoblast cells, C2C12) as determined by the luciferase assay. Although the dendrons are unable to transfect DNA in their own right, they are capable of delivering DNA in vitro when administered with chloroquine, which assists with escape from endocytic vesicles. The cytotoxicity of the dendrons was determined using the XTT assay, and it was shown that the dendrons were nontoxic either alone or in the presence of DNA. However, when administered with DNA and chloroquine, the most highly branched dendron did exhibit some cytotoxicity. This paper elucidates the relationship between in vitro transfection efficiency and toxicity. While transfection efficiencies are modest, the low toxicity of the dendrons, both in their own right, and in the presence of DNA, provides encouragement that this type of building block, which has a relatively high affinity for DNA, will provide a useful starting point for the further synthetic development of more effective gene transfection agents.     

PAMAM conjugated chitosan through naphthalimide moiety for enhanced gene transfection efficiency

Development of efficient and safe gene carrier is the main hurdle for successful gene therapy till date. Poor water solubility and low transfection efficiency of chitosan are the main drawbacks to be efficient gene carrier for successful gene therapy. In this work, PAMAM conjugated chitosan was prepared through naphthalimide moiety by simple substitution reaction. The synthesis of the chitosan conjugates was confirmed by FTIR, ¹H NMR and XRD analyses. The conjugates showed enhanced DNA binding capability compared to that of unmodified chitosan. Moreover, the conjugates showed minimal cytotoxicity compared to that of polyethyleneimine (PEI, 25 kDa) and also showed good blood compatibility with negligible haemolysis. The transfection efficiency of the conjugate was significantly increased compared to that of unmodified chitosan and it also surpassed the transfection efficiency by PEI. Therefore, PAMAM conjugated chitosan can be used safely as alternate efficient gene delivery vector in gene therapy.     

Polyamidoamine dendron-bearing lipids as a nonviral vector: influence of dendron generation

Recently, we demonstrated that octadecyl chains are important as alkyl chain moieties of polyamidoamine (PAMAM) dendron-bearing lipids for their serum-resistant transfection activity [Bioconjugate Chem.2007, 18, 1349-1354]. Toward production of highly potent vectors, we examined the influence of the generation of dendron moiety on transfection activity of PAMAM dendron-bearing lipids having two octadecyl chains. We synthesized dendron-bearing lipids with PAMAM G1, G2, and G3 dendrons, designated respectively as DL-G1-2C(18), DL-G2-2C(18), and DL-G3-2C(18). The DL-G2-2C(18) and DL-G3-2C(18) interacted with plasmid DNA effectively and formed stable lipoplexes with small sizes and spherical shape. However, DL-G1-2C(18) interacted with plasmid DNA less effectively and formed tubular-shaped lipoplexes with lower stability and larger size. Cells took up DL-G2-2C(18) and DL-G3-2C(18) lipoplexes efficiently, but cellular uptake of the DL-G1-2C(18) lipoplexes was less efficient. Nevertheless, DL-G1-2C(18) lipoplexes achieved 100-10?000 times higher levels of transgene expression, which was evaluated using luciferase gene as a reporter gene. Confocal scanning laser microscopic analysis of intracellular behaviors of the lipoplexes revealed that DL-G1-2C(18) lipoplexes generated free plasmid DNA molecules in the cytosol more effectively than other lipoplexes did. Moderate binding ability of DL-G1-2C(18) might be responsible for generation of lipoplexes which deliver plasmid DNA into cells, liberate it in the cytoplasm, and induce efficient transgene expression.     

Polycation nanostructured lipid carrier, a novel nonviral vector constructed with triolein for efficient gene delivery

A novel nonviral gene transfer vector was developed by modifying nanostructured lipid carrier (NLC) with cetylated polyethylenimine (PEI). Polycation nanostructured lipid carrier (PNLC) was prepared using the emulsion-solvent evaporation method. Its in vitro gene transfer properties were evaluated in the human lung adenocarcinoma cell line SPC-A1 and Chinese Hamster Ovary (CHO) cells. Enhanced transfection efficiency of PNLC was observed after the addition of triolein to the PNLC formulation and the transfection efficiency of the optimized PNLC was comparable to that of Lipofectamine™2000. In the presence of 10% serum the transfection efficiency of the optimal PNLC was not significantly changed in either cell line, whereas that of Lipofectamine™2000 was greatly decreased in both. Thus, PNLC is an effective nonviral gene transfer vector and the gene delivery activity of PNLC was enhanced after triolein was included into the PNLC formulation.     

人转铁蛋白偶联交联聚乙烯亚胺体外靶向转染肿瘤细胞

为了提高聚乙烯亚胺(Polythylenimine,PEI)类载体对肿瘤细胞的靶向性同时降低其细胞毒性,先用1800DaPEI制备了交联低分子量PEI,然后将人转铁蛋白与之偶联,得到了新型肿瘤靶向性人转铁蛋白偶联交联聚乙烯亚胺基因载体(TCP)。对所得的TCP的理化特性经行了表征,并检测了其细胞毒性。采用TCP介导pGL-3和pEGFP分别对293T、HepG2和Hela细胞系进行体外转染实验。结果表明:TCP是一种低毒高效的基因载体,在肿瘤细胞中的转染效率显著增强,因为其二硫键可在细胞内还原降解,而且通过偶联的转铁蛋白配体与肿瘤细胞表达的转铁蛋白受体间的相互作用,可增强该载体对肿瘤细胞的转染效率和靶向性。     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

A thermoresponsive chitosan-NIPAAm/vinyl laurate copolymer vector for gene transfection

A carboxyl-terminated N-isopropylacrylamide/vinyl laurate (VL) copolymer was prepared and coupled with chitosan (molecular weight = 2000) to produce a chitosan-NIPAAm/VL copolymer (PNVLCS) vector. The aqueous solution of PNVLCS displayed an obvious thermoresponsive behavior with a lower critical solution temperature (LCST) about 26 degrees C. The transmission electron microscopy (TEM) showed that the size of PNVLCS/DNA complexes varied with charge ratios (+/-), and the smaller nanoparticles were formed at higher charge ratios. DLS revealed that the size of complex particles was dependent on temperature. The results of temperature-variable circular dichroism (CD), UV, and electrophoresis retardation indicated that at lower charge ratios, DNA in the complexes assume a B conformation, whereas increasing charge ratios caused B --> C type conformation transformation; the dissociation-formation of PNVLCS/DNA complexes could be tuned by varying temperature: at 37 degrees C, the collapse of PNIPAAm in PNVLCS was favorable for the formation of compact complexes, shielding more DNA from exposure; at 20 degrees C, the hydrated and extended PNIPAAm chains facilitated the unpacking of DNA from PNVLCS, increasing the exposure of DNA. PNVLCS was used to transfer plasmid-encoding beta-galactosidase into C2C12 cells. The level of gene expression could be controlled by varying incubation temperature. The transfection efficiency of PNVLCS was well improved by temporarily reducing culture temperature to 20 degrees C, whereas naked DNA and Lipofectamine 2000 did not demonstrate the characteristics of thermoresponsive gene transfection.     

Synthesis of N-alkylated noeurostegines and evaluation of their potential as treatment for Gaucher's disease

The potent and selective inhibitor of β-glucosidases, noeurostegine, was evaluated as an inhibitor of glucocerebrosidase (GCase) to give an IC₅₀ value of 0.4 μM, being 250- and 150-fold better than N-butyl and N-nonyl noeurostegine, respectively. The parent noeurostegine and its N-butyl and N-nonyl alkylated congeners were also tested as pharmacological chaperones against a N370S GCase mutant. Of these, only noeurostegine, was found to increase enzyme activity, which in potency was comparable to that previously reported for isofagomine.     

阳离子聚合物在非病毒基因转染中的研究进展

基因治疗为治疗先天性遗传疾病和严重后天获得性疾病提供了一条新途径.目前,基因载体分为两类:病毒载体和非病毒载体.病毒载体转染效率高,但由于某些病毒载体存在免疫原性、致癌性、宿主DNA插入整合等缺点,从而限制了它们的应用.非病毒载体具有价格低、制备简单、安全有效、无免疫原性等优点,成为基因载体研究的热点.阳离子多聚物是非病毒载体的典型代表.文中综述近年来阳离子多聚物作为基因载体的研究现状和进展,重点介绍了阳离子多聚物基因载体的分类和与DNA的相互作用和传递机制.     

AAV as a viral vector for human gene therapy

Investigation of the adeno-associated virus (AAV) life cycle has enabled the establishment of methodology and identification of critical cis-acting sequences required for recombinant AAV production. Vectors derived from the defective human parvovirus (AAV) have been used for successful gene transfer and expression in many diverse mammalian cell types, such as erythroid, airway epithelium, and neuronal cells. One of the crucial steps in the continued case of AAV as a vector is the development of packaging systems that will allow efficient encapsidation of foreign genes into AAV virions. For this reason, the focus of this article will be generation of recombinant AAV vectors.     

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.