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.     

Enhancement of gene delivery using novel homodimeric tat peptide formed by disulfide bond

Cationic liposomes have been actively used as gene delivery vehicle because of their minimal toxicity, but their relatively low efficiency of gene delivery is the major disadvantage of these vectors. Recently, cysteine residue incorporation to HIV-1 Tat peptide increased liposomemediated transfection compared with unmodified Tat peptide. Therefore, we designed a novel modified Tat peptide having a homodimeric (Tat-CTHD, Tat-NTHD) and closed structure (cyclic Tat) simply by using the disulfide bond between cysteines to develop a more efficient and safe nonviral gene delivery system. The mixing of Tat-CTHD and Tat-NTHD with DNA before mixing with lipofectamine increased the transfection efficiency compared with unmodified Tat peptide and lipofectamine only in MCF-7 breast cancer cells and rat vascular smooth muscle cells. However, cyclic Tat did not show any improvement in the transfection efficiency. In the gel retardation assay, Tat-CTHD and Tat-NTHD showed more strong binding with DNA than unmodified Tat and cyclic Tat peptide. This enhancement was only shown when Tat-CTHD and Tat-NTHD were mixed with DNA before mixing with lipofectamine. The effects of Tat- CTHD and Tat-NTHD were also valid in the experiment using DOTAP and DMRIE instead of lipofectamine. We could not find any significant cytotoxicity in the working concentration and more usage of these peptides. In conclusion, we have designed a novel transfection-enhancing peptide by easy homodimerization of Tat peptide, and the simple mix of these novel peptides with DNA increased the gene transfer of cationic lipids more efficiently with no additional cytotoxicity.     

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.     

Recent patents in cationic lipid carriers for delivery of nucleic acids

Gene therapy is a medical technique intended for treatment of disorders caused by defective, missing, or overexpressing genes. Efficient delivery vectors are necessary in order to transport genetic material to the target cells. Such vectors include viral and non-viral carriers. Viral vectors transfect cells efficiently, however risks associated with their use have limited their clinical applications. Nonviral delivery systems are safer, easier to prepare, more versatile and cost effective. However, their transfection efficiency still falls behind that of the viral vectors. Considerable research into nonviral gene delivery has been conducted in the last two decades on synthetic soft materials such as cationic lipids, polymers, surfactants, and dendrimers as prospective nucleotide carriers for gene delivery. So far, cationic lipids are the most widely used constituents of nonviral gene carriers, with multiple strategies employed to improve their in vitro and in vivo transfection. Efforts in synthesizing new cationic lipids were not fully successful in closing the gap between the efficiency of the viral vectors and that of binary cationic lipid/DNA complexes. Current efforts for improving lipofection efficiency are focused on the development of multicomponent carriers including cationic lipids as key constituents. This review summarizes the recent patents on new cationic lipids as well as on multicomponent formulations enhancing their efficiency as nucleotide carriers.     

Facilitation of gene transfection with well-defined degradable comb-shaped poly(glycidyl methacrylate) derivative vectors

Recently, we reported that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) vectors (PGEAs) can produce good transfection efficiency, while exhibiting very low toxicity. Further improvement in degradability and transfection efficiency of the PGEA vectors will facilitate their application in gene therapy. Comb-shaped cationic copolymers have been of interest and importance as nonviral gene carriers. Herein, the degradable high-molecular-weight comb-shaped PGEA vectors (c-PGEAs) composed of the low-molecular-weight PGEA backbone and side chains were prepared by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions. The PGEA side chains were linked with the PGEA backbones via hydrolyzable ester bonds. Such comb-shaped c-PGEA vectors possessed the degradability, good pDNA condensation ability, low cytotoxicity, and good buffering capacity. More importantly, the comb-shaped c-PGEA vectors could enhance the gene expression levels. Moreover, the PGEA side chains of c-PGEA could also be copolymerized with some poly(poly(ethylene glycol)ethyl ether methacrylate) species to further improve the gene delivery system.     

New cationic lipids for gene transfer with high efficiency and low toxicity: T-shape cholesterol ester derivatives

New degradable cationic ester lipids with 'T-shape' configurations were synthesized and tested for gene delivery carrier. Their transfection efficiency and toxicity were compared with commercially available cationic lipids, DOTMA, DOSPA, and DC-Chol. They showed efficient transfection activity and almost no toxicity on mammalian cell lines. Their ester bond degradation was monitored by (1)H NMR.     

New unsymmetrical bolaamphiphiles: synthesis, assembly with DNA, and application for gene delivery

The success in gene therapy relies strongly on new efficient gene delivery vectors. Nonviral vectors based on lipids and polymers constitute an important alternative to the viral vectors. However, the key problem with these vectors is the poor structural control of their DNA complexes. In the present work, following new design we synthesized unsymmetrical bolaamphiphiles, molecules bearing neutral sugar (gluconic acid) and dicationic ornithine head groups connected by different long hydrophobic spacers. Within this design, a positively charged headgroup is expected to bind DNA, the hydrophobic spacer is to drive the formation of a monolayer membrane shell around DNA, while the neutral group is to be exposed outside of the complex. Our fluorescence and gel electrophoresis data showed that self-assembly of bolas and their interaction with DNA depend strongly on the bola structure. The size of bola/DNA complexes (bolaplexes) estimated from dynamic light scattering data was ～100 nm at low N/P (cationic nitrogen/DNA phosphate molar ratio), while at higher N/Ps it was significantly larger due to neutralization of their surface charge. Atomic force microscopy studies revealed nanostructural rod-shaped or spherical morphology of the bolaplexes. Transfection efficiency of the bolaplexes in vitro was significant when either DOPE or chloroquine were used as helping agents, suggesting that the key barrier for their internalization is the endosomal escape. Finally, all bolas showed low cytotoxicity (cell viability >80%). The present results show that bolas are prospective candidates for construction of nonviral gene delivery vectors. We believe that further optimization of polar head groups and a hydrophobic spacer in the bolas will lead to vectors with controlled small size and high transfection efficiency.     

Dendritic cationic lipids with highly charged headgroups for efficient gene delivery

Gene therapy is expected to lead to powerful new approaches for curing many diseases, a potential that is currently explored in worldwide clinical trials. Nonviral DNA delivery systems are desirable to overcome the inherent problems of viral vectors, but their current efficiency requires improvement and the understanding of their mechanism of action is incomplete. We have synthesized new multivalent cationic lipids with highly charged dendritic headgroups to probe the structure-transfection efficiency relationships of cationic liposome (CL)-DNA complexes, a prevalent nonviral vector. The lipid headgroups are constructed from ornithine cores and ornithine or carboxyspermine endgroups. The dendritic lipids were prepared on a gram scale, using a synthetic scheme that permits facile variation of the lipid building blocks headgroup, spacer, and hydrophobic moiety. They carry four to sixteen positive charges in their headgroups. Complexes of DNA with mixtures of the dendritic lipids and neutral 1,2-dioleoyl-sn-glycero phosphatidylcholine (DOPC) exhibit novel structures at high contents of the highly charged lipids, while the well-known lamellar phase is formed at high contents of DOPC. DNA complexes of the new dendritic lipids efficiently transfect mammalian cells in culture without cytotoxicity and, in contrast to lamellar complexes, maintain high transfection efficiency over a broad range of composition.     

A pH-sensitive polymer that enhances cationic lipid-mediated gene transfer.

The efficient release of nonviral gene carriers from endosomes is an important step for the successful delivery of DNA into the cell nucleus. A synthetic pH-sensitive anionic polymer, poly(propylacrylic acid) (PPAA), was designed to aid in endosomal escape of nonviral vectors and improve the transfection efficiencies with these vectors. Transfection of NIH3T3 fibroblasts with ternary physical mixtures of the cationic lipid DOTAP, pCMVbeta plasmid DNA, and PPAA showed marked enhancement of both gene expression levels and fraction of cells transfected compared to binary control mixtures of DOTAP and DNA. PPAA also significantly improved the serum-stability of DOTAP/DNA vectors. The DOTAP/DNA/PPAA vectors maintained high levels of transfection in media containing up to 50% serum. The striking enhancement of transfection efficiency with cationic lipid/DNA/PPAA mixtures, along with the enhanced serum-stability, suggests that PPAA may provide significant improvements for the in vivo intracellular delivery of drugs such as DNA, oligonucleotides, proteins, and peptides.     

Plasmid DNA-encapsulating liposomes: Effect of a spacer between the cationic head group and hydrophobic moieties of the lipids on gene expression efficiency

We have synthesized a series of cationic amino acid-based lipids having a spacer between the cationic head group and hydrophobic moieties and examined the influence of the spacer on a liposome gene delivery system. As a comparable spacer, a hydrophobic spacer with a hydrocarbon chain composed of 0, 3, 5, 7, or 11 carbons, and a hydrophilic spacer with an oxyethylene chain (10 carbon and 3 oxygen molecules) were investigated. Plasmid DNA (pDNA)-encapsulating liposomes were prepared by mixing an ethanol solution of the lipids with an aqueous solution of pDNA. The zeta potentials and cellular uptake efficiency of the cationic liposomes containing each synthetic lipid were almost equivalent. However, the cationic lipids with the hydrophobic spacer were subject to fuse with biomembrane-mimicking liposomes. 1,5-Dihexadecyl-N-lysyl-N-heptyl-l-glutamate, having a seven carbon atom spacer, exhibited the highest fusogenic potential among the synthetic lipids. Increased fusion potential correlated with enhanced gene expression efficiency. By contrast, an oxyethylene chain spacer showed low gene expression efficiency. We conclude that a hydrophobic spacer between the cationic head group and hydrophobic moieties is a key component for improving pDNA delivery.     

Long-term expression with a cationic polymer derived from a natural polysaccharide: schizophyllan

Among the various synthetic gene carriers based on biomaterials, cationic polymers with polysaccharide backbones have long been studied as nonviral vectors due to their low immunogenicity and high water solubility. Schizophyllan, a beta-(1,3)-glucan, is one of the various polysaccharides that are clinically administered. Furthermore, its safety in the human body has already been confirmed. Various functional groups can be selectively introduced into the side chain, not into the main chain of schizophyllan. Therefore, we have synthesized various oligoamine conjugates from schizophyllan. It was confirmed that their in vitro transfection efficiencies are superior to that of polyethylenimine by adjusting the molecular weight and the degree of amination of cationic schizophyllan. While it was possible to reduce cytotoxicity by adjusting the amount of DNA complex per cell, as seen with poly-L-lysine, polyethylenimine, and chitosan, PEGylation was the most effective means of reducing toxicity. Furthermore, using cationic schizophyllan carriers, it was also possible to express a reporter protein for a long period of time due to a long residence time of plasmid DNA in cells.     

Poly(D,L-lactide-co-glycolide acid) nanoparticles for DNA delivery: waiving preparation complexity and increasing efficiency

When designing a nonviral gene delivery system based on polymeric nanoparticles (NPs), it is important to keep in mind obstacles associated with future clinical applications. Simplifying the procedure of NPs production and taking toxicity into account are the most important issues that need to be addressed. Toxicity concerns in clinical trials may be raised when using additives such as cationic polymers/lipids, buffering reagents, and proteins. Therefore, the aim of this study was to simplify the formulation of poly (lactide-co-glycolide) acid NPs by shortening steps such as sonication time and by avoiding the use of additives while preserving its efficiency. NPs (300 nm) were formulated using a modified w/o/w technique with DNA entrapment efficiency of 80%. Once achieving such NPs, formulation parameters such as DNA loading, release kinetics, DNA integrity and bioactivity, uptake by cells, and toxicity were addressed. The NPs were readily taken by several cell lines and were localized mostly in their endo-lysosomal compartments. The NPs did not affect cells viability. Most importantly, transfection studies in COS-7 and Cf2th cells resulted with a 250-fold protein expression levels when compared with the control. These expression levels are higher than ones achieved with more complicated NPs systems, demonstrating the efficiency of our simplified NPs for gene delivery.     

Synthesis and characterization of cationic micelles self-assembled from a biodegradable copolymer for gene delivery

We have recently reported biodegradable cationic micelles self-assembled from an amphiphilic copolymer, poly{(N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl)methyl bis(ethylene)ammonium bromide]sebacate} (P(MDS-co-CES)), which were utilized to deliver a drug and nucleic acid simultaneously, and a synergistic effect was achieved. In this paper, synthesis and characterization of the polymer is presented in details, focusing on micelle formation and DNA binding under various conditions, cytotoxicity, in-vitro degradation, and gene transfection in various cell lines. The polymer was degradable and formed micelles at very low concentrations even in an environment with high salt concentration. These micelles fabricated at pH 4.6 had an average size of less than 82 nm and zeta potential of up to 84 +/- 5 mV, displaying strong DNA binding ability. They induced high gene expression efficiency in various cell lines, which was significantly greater than poly(ethylenimine) (PEI) especially in 4T1 mouse and MDA-MB-231 human breast cancer cell lines, but they were less cytotoxic. These cationic micelles may provide a promising nonviral vector for gene delivery.     

Characterization of PLL-g-PEG-DNA nanoparticles for the delivery of therapeutic DNA

Local and controlled DNA release is a critical issue in current gene therapy. As viral gene delivery systems are associated with severe security problems, nonviral gene delivery vehicles were developed. Here, DNA-nanoparticles using grafted copolymers of PLL and PEG to increase their biocompatibility and stealth properties were systematically studied. Ten different PLL-based polymers with no, low, and high PEG grafting and PEG molecular weights as well as different PLL backbone lengths were complexed with plasmids containing 3200 to 10,100 base pairs. Stable complexes were formed and selected for cytotoxicity and transfection efficiency. Predominantly, PLL-g-PEG-DNA nanoparticles grafted with 4 or 5% PEG moieties of 5 kDa transfected 40% COS-7 cells without reduction of cell viability when formed at N/P ratios between 0.1 and 12.5. The molecular weight of PLL did not significantly affect transfection efficiency or cytotoxicity indicating that a specific cationic charge-density-to-PEG-ratio is important for efficient transfection and low cytotoxicity. The PLL-g-PEG-DNA nanoparticles were spherical with a diameter of approximately 100 nm and did not aggregate over 2 weeks. Moreover, they protected included plasmid DNA against serum components and DNase I digestion. Therefore, such storage stable and versatile PLL-g-PEG-DNA nanoparticles might be useful to deliver differently sized therapeutic DNA for in vivo applications.     

非病毒型基因载体研究进展

非病毒载体以其安全性、低毒性、低免疫反应、靶向性及易于组装等优点被寄予厚望 ,对非病毒载体的研究人们投入了很大的精力 ,以期在基因治疗方面有所突破。综述了近年来非病毒载体的研究现状 ,分别总结阐述了阳离子脂质体载体、多聚阳离子载体、壳聚糖载体、树状高分子载体以及无机壳类SiO2 纳米粒子载体。提出非病毒载体今后的发展方向及发展的必要性。     

Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives

The continually increasing wealth of knowledge about the role of genes involved in acquired or hereditary diseases renders the delivery of regulatory genes or nucleic acids into affected cells a potentially promising strategy. Apart from viral vectors, non-viral gene delivery systems have recently received increasing interest, due to safety concerns associated with insertional mutagenesis of retro-viral vectors. Especially cationic polymers may be particularly attractive for the delivery of nucleic acids, since they allow a vast synthetic modification of their structure enabling the investigation of structure-function relationships. Successful clinical application of synthetic polycations for gene delivery will depend primarily on three factors, namely (1) an enhancement of the transfection efficiency, (2) a reduction in toxicity and (3) an ability of the vectors to overcome numerous biological barriers after systemic or local administration. Among the polycations presently used for gene delivery, poly(ethylene imine), PEI, takes a prominent position, due to its potential for endosomal escape. PEI as well as derivatives of PEI currently under investigation for DNA and RNA delivery will be discussed.This review focuses on structure-function relationships and the physicochemical aspects of polyplexes which influence basic characteristics, such as complex formation, stability or in vitro cytotoxicity, to provide a basis for their application under in vivo conditions. Rational design of optimized polycations is an objective for further research and may provide the basis for a successful cationic polymer-based gene delivery system in the future.     

CFTR transgene expression in primary DeltaF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids

Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population. The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet. In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents. Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects. Consequently, all of these drawbacks brought forth the development of nonviral transfection systems. Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome. Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo. In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a F508 homozygous patient. The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex. A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection.     

Poly(beta-amino ester) and cationic phospholipid-based lipopolyplexes for gene delivery and transfection in human aortic endothelial and smooth muscle cells

Safe and effective nonviral gene delivery and transfection in primary human vascular endothelial cells (EC) and smooth muscle cells (SMC) has tremendous potential for cardiovascular diseases such as in the treatment of coronary restenosis. Using a combination of a cationic biodegradable polymer, poly(beta-amino ester) (PBAE), and a cationic phospholipid, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), we have engineered a lipopolyplex nanovector system that can transfect EC and SMC cells with reasonably high efficiency. For instance, upon addition of 1.0 microg DNA complexed in lipopolyplexes the transfection efficiency in SMC was 20% and in EC was 33%. The results of this study shows that PBAE-DOTAP-plasmid DNA lipopolyplexes are a promising nonviral vector system for gene delivery and transfection in EC and SMC.     

Visualization of the degradation of a disulfide polymer, linear poly(ethylenimine sulfide), for gene delivery

Polyethylenimine (PEI) shows high transfection efficiency and cytoxicity due to its high amine density. The new disulfide cationic polymer, linear poly(ethylenimine sulfide) (l-PEIS), was synthesized for efficient and safe gene delivery. As the amine density of l-PEIS increased, the transfection efficiency also increased. l-PEIS-6 and l-PEIS-8 show transfection efficiencies that are similar to that of PEI. However, cytotoxicity of l-PEIS was not observed due to the biodegradable disulfide bond. The disulfide bonds are stable in the oxidative extracellular condition and can be degraded rapidly in the reductive intracellular condition. The degradation of l-PEIS in HeLa cells was visualized by fluorescence microscopy using the probe-probe dequenching effect of BODIPY-FL fluorescence dye. l-PEIS was degraded completely within 3 h.     

Pyridinium cationic lipids in gene delivery: an in vitro and in vivo comparison of transfection efficiency versus a tetraalkylammonium congener

Cationic lipids provide a promising alternative to the use of viruses for delivering genes therapeutically. Among the several classes of lipidic vectors, those bearing a heterocyclic cationic head have shown important advantages, such as low cytotoxicity and improved efficiency across different cell lines. We recently reported a simple and efficient strategy for obtaining pyridinium cationic lipids, starting from pyrylium salts and primary amines. The present study is aimed to compare the cellular toxicity and transfection efficiency generated by the pyridinium polar head versus the tetramethylammonium one on several tumor cell lines and also in experimental animals, delivered via intratumor injections. Thus, the lead compound 1-(2,3-dioleoyloxypropyl)-2,4,6-trimethylpyridinium lipid (2Oc), coformulated with different helper lipids in various molar ratios, was tested against its ammonium congener DOTAP-a standard transfection reagent. The results revealed that when formulated with cholesterol at 1:1 molar ratio, the pyridinium lipid 2Oc was able to transfect several cancer cell lines with similar or better efficiency than its tetraalkylammonium congener DOTAP, while producing lower cytotoxicity. The NCI-H23 lung cancer cell line was found to be the most susceptible to be transfected. Therefore, we designed an in vivo assay based on this type of carcinoma in nude mice, which were injected intratumoral with 2Oc- and DOTAP-based lipoplexes. The red fluorescent protein reporter revealed that the pyridinium cationic lipid was superior to its tetraalkylammonium congener, transfecting the tissue on a higher area and with higher efficiency. These encouraging findings, together with the simple and efficient synthetic strategy, lay the foundation for further development of pyridinium lipids for gene therapy with improved transfection efficiency in vivo and even further reduced cytotoxicity.