Hyaluronic acid and chitosan-DNA complex multilayered thin film as surface-mediated nonviral gene delivery system

Sustained release of DNA from the surface of materials represents a promising approach to combine the gene therapy and implantable biomaterials. The nonviral chitosan-DNA complexes were incorporated into the multilayer via layer-by-layer deposition with hyaluronic acid (HA). The UV–vis spectroscopy and atomic force microscopy (AFM) results showed the successful construction of the nonviral complex contained multilayers. The complexes were releasable in physiological condition and a sustained release manner was gained when the multilayer was crosslinked. The cell viability test and the gene transfection assay showed that the natural polyelectrolyte-based nonviral complex incorporated multilayer not only had good cytocompatibility, but also possessed the in vitro gene transfection ability. This kind of surface-mediated nonviral complex incorporated multilayer may have great potential in the localized and controlled delivery of DNA in biomedical implants and tissue engineering application.     

Lanthanide-doped chitosan nanospheres as cell nuclei illuminator and fluorescent nonviral vector for plasmid DNA delivery

Lanthanide-doped chitosan nanospheres (LDCNs) and lanthanide-Fe(3)O(4)-doped chitosan nanospheres (Fe(3)O(4)-LDCNs) are fabricated and show fluorescence, MRI effectiveness and desirable biocompatibility. Superior to most nanoparticles that were found retained in cytoplasmic organelles rather than the nucleus, the prepared chitosan nanospheres preferentially enter and illuminate the cell nuclei. Complexation of plasmid DNA (pDNA) to the nanospheres was accomplished via electrostatic forces between positively charged chitosan and negatively charged pDNA. Satisfactory results of the complexation indicate that the prepared chitosan nanospheres can serve as a potential fluorescent nonviral vector for pDNA delivery that can fulfill gene delivery and transfer efficiency assessment simultaneously, without an additional step of tagging fluorophores to the vectors carried out in fabrications of currently available pDNA delivery vectors.     

Novel supramolecular gelation route to in situ entrapment and sustained delivery of plasmid DNA

In this work, cationic block copolymer (F-68-PLL) composed of Pluronic F-68 and poly(L-lysine) segments was first prepared for the binding with plasmid DNA due to the electrostatic interaction between poly(L-lysine) segments and plasmid DNA, and subsequently used to interact with α-cyclodextrin (α-CD) in aqueous system for the supramolecular gelation by the inclusion complexation between Pluronic F-68 segments and α-CD. It was found that such a fabrication process could lead to the in situ entrapment of plasmid DNA into the supramolecular hydrogel matrix under mild conditions. Depending on the amounts of F-68-PLL and α-CD, the resultant hybrid hydrogel was found to have adjustable gelation time and mechanical strength. For the plasmid DNA complexes released from the supramolecular hydrogel, controlled release and sustained gene transfection were confirmed.     

Novel biodegradable polymers as gene carriers

This study investigated two new biodegradable polymers as gene controlled-released coatings for gene transfer. Poly(ethylene glycol)-co-poly(D,L-lactic acid) (PELA) and poly(ethylene glycol)-co-poly(lactic acid)-co-poly(glycolic acid) random copolymer (PELGA) were synthesized and used as microspheres matrices with encapsulated plasmid pCH110. The plasmid loading efficiency, cytotoxicity, transfection efficiency and in vitro degradation and release profiles of microsphere complexes were evaluated in details. The biodegradable polymers showed high DNA loading efficiency and low cytotoxicity as gene controlled-released coatings, and the poly(ethylene glycol) (PEG) contents of polymer matrices influenced the diameter, loading efficiency and transfection efficiency of plasmid DNA within the microspheres. The average diameters of PELA and PELGA microspheres were between 0.5 and 1.5 microm, and the plasmid loading efficiency was 62 and 73% for PELA and PELGA microspheres with 10% PEG content, respectively. In vitro testing showed a gradual release profile of DNA from polymeric matrices. The polymers/DNA microspheres had high transfection efficiency and early gene expression and maintenance of gene expression level for up to 96 h, although transfection efficiency were slightly lower than that of liposome in the initial 24 h. The biodegradable polymeric materials possess potential superiority as gene carriers.     

Improvements in transfection efficiency with chitosan modified poly(DL-lactide-co-glycolide) nanospheres prepared by the emulsion solvent diffusion method, for gene delivery

This study sought to evaluate the in vitro transfection efficiency of plasmid DNA (pDNA)-loaded chitosan-modified poly(DL-lactide-co-glycolide) nanospheres (CS-PLGA NS) in a gene-delivery system. Using the emulsion solvent diffusion (ESD) method, pDNA-loaded PLGA NS was prepared and the surface of the PLGA NS was modified by binding to CS. Gene transfection ability of CS-PLGA NS was examined in A549 cells. The luciferase gene was used as a reporter gene. The pattern of luciferase activity by pDNA-loaded CS-PLGA NS was initially weak, but gradually grew stronger before decreasing activity. These phenomena should be in accordance with the sustained-release profile of pDNA from PLGA NS in the cytosol and the pDNA protection against DNase. Positively charged CS-PLGA NS was found, by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, not to exhibit cytotoxicity on A549 cells. These results suggest that CS-PLGA NS are potential contributors to efficient pDNA delivery due to their increased interactions with cells and lack of cytotoxic effects.     

Mechanisms involved in gene electrotransfer using high- and low-voltage pulses--an in vitro study

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase permeability of cell membrane and thus enables transfer of genes. Currently, majority of research is focused on improving in vivo transfection efficiency, while mechanisms involved in gene electrotransfer are not completely understood. In this paper we analyze the mechanisms of gene electrotransfer by using combinations of high-voltage (HV) and low-voltage pulses (LV) in vitro. We applied different combinations of HV and LV pulses to CHO cells and determined the transfection efficiency. We obtained that short HV pulses alone were sufficient to deliver DNA into cells for optimal plasmid concentrations and that LV pulse did not increase transfection efficiency, in contrast to reported studies in vivo. However, for sub-optimal plasmid concentrations combining HV and LV pulses increased transfection rate. Our results suggest that low-voltage pulses increase transfection in conditions where plasmid concentration is low, typically in vivo where mobility of DNA is limited by the extracellular matrix. LV pulses provide additional electrophoretic force which drags DNA toward the cell membrane and consequently increase transfection efficiency, while for sufficiently high concentrations of the plasmid (usually used in vitro) electrophoretic LV pulses do not have an important role.     

Self‐Assembled Fluorodendrimers Combine the Features of Lipid and Polymeric Vectors in Gene Delivery

An ideal vector in gene therapy should exhibit high serum stability, excellent biocompatibility, a desired transfection efficacy and permeability into targeted tissues. Here, we describe a class of low‐molecular‐weight fluorodendrimers for efficient gene delivery. These materials self‐assemble into uniform nanospheres and allow for efficient transfection at low charge ratios and very low DNA doses with minimal cytotoxicity. Our results demonstrate that these vectors combine the features of synthetic gene vectors such as liposomes and cationic polymers and present promising potential for clinical gene therapy.     

Lipoic Acid-Modified Oligoethyleneimine-Mediated miR-34a Delivery to Achieve the Anti-Tumor Efficacy

MiR-34a, an important tumor suppressor, has been demonstrated to possess great potential in tumor gene therapy. To achieve the upregulation of miR-34a expression level, an oligoethyleneimine (OEI) derivative was constructed and employed as the carrier through the modification with lipoic acid (LA), namely LA-OEI. In contrast to OEI, the derivative LA-OEI exhibited superior transfection efficiency measured by confocal laser scanning microscopy and flow cytometry, owing to rapid cargo release in the disulfide bond-based reduction sensitive pattern. The anti-proliferation and anti-migration effects were tested after the miR-34a transfection to evaluate the anti-tumor response, using human cervical carcinoma cell line HeLa as a model. The delivery of LA-OEI/miR-34a nanoparticles could achieve obvious anti-proliferative effect caused by the induction of cell apoptosis and cell cycle arrest at G1 phase. In addition, it could inhibit the migration of tumor cells via the downregulation of MMP-9 and Notch-1 level. Overall, the LA-OEI-mediated miR-34a delivery was potential to be used as an effective way in the tumor gene therapy.     

(Coixan polysaccharide)-graft-polyethylenimine folate for tumor-targeted gene delivery

CP-PEI-FA was prepared as an effective vector for in vitro and in vivo tumor-targeted gene delivery. The structures of the polymers were characterized, and their DNA condensation capability, particle sizes, zeta potentials, cytotoxicity and in vitro/in vivo transfection were examined. The cytotoxicity of CP-PEI-FA was significantly lower than that of PEI 25 kDa and close to that of PEI 1200. The in vitro transfection of CP-PEI-FA was tested in C6 and HeLa cells (FR-positive cells) and A549 cells (FR-negative cells). CP-PEI-FA showed a high targeting specificity and good gene transfection efficiency in FR-positive cells. These results indicate that CP-PEI-FA is a safe and effective polyplex-forming agent for both in vitro and in vivo transfection of plasmid DNA.     

壳聚糖修饰PLGA阳离子型纳米微球的制备与表征

采用单乳化-溶剂(O/W)挥发技术制备表面带正电荷的壳聚糖(CHS)修饰聚乙/丙交酯(PLGA)纳米微球(PLGA/CHS), 通过正交试验优化了纳米微球的制备条件. 结果表明, 微球粒径可控制在150～200 nm内, 在pH=4时, 纳米微球表面电位最高为55 mV. 影响微球粒径的主要因素是聚合物的浓度, CHS的分子量和浓度以及介质的pH值对微球表面电位也有明显影响. 制备粒径较小而表面电位较高的PLGA/CHS纳米微球条件为: ρ(CHS)=3 mg/mL, ρ(PLGA)=10 mg/mL, Vo/Va=1/4. SEM图像显示经CHS修饰的PLGA的纳米微球形状规整, 荧光显微观察和XPS分析结果证实CHS包覆于微球表面.     

Dimerizable cationic detergents with a low cmc condense plasmid DNA into nanometric particles and transfect cells in culture

The size of condensed DNA particles is a key determinant for in vivo diffusion and gene delivery to cells. Gene molecules can be individually compacted by cationic thiol detergents into nanometric particles that are stabilized by oxidative conversion of the detergent into a gemini lipid. To reach the other goal, gene delivery, a series of cationic thiol detergents with various chain lengths (C(12)-C(16)) and headgroups (ornithine or spermine) was prepared, using a versatile polymer-supported synthetic strategy. Critical micelle concentrations and thiol oxidation rates of the detergents were measured. The formation and stability of complexes formed with plasmid DNA, as well as the size, xi-potential, morphology, and transfection efficiency of the particles were investigated. Using the tetradecane/ornithine detergent, a solution of 5.5 Kpb plasmid DNA molecules was converted into a homogeneous population of 35 nm particles. The same detergent, once oxidized, exhibited a typical lipid phase internal structure and was capable of effective cell transfection. The particle size did not increase with time. Surprisingly, the gel electrophoretic mobility of the DNA complexes was found to be higher than that of plasmid DNA itself. Favorable in vivo diffusion and intracellular trafficking properties may thus be expected for these complexes.     

Electrospray doxorubicin-loaded polycaprolactone-polyethylene glycol nanospheres/poloxamer-chitosan thermogel: A promising candidate for clinical applications

In recent years, polymer nanospheres have been considered as one of the most common materials in the drug delivery domain. In this research, polycaprolactone-polyethylene glycol (PCL-PEG) blend nanospheres were produced using the electrospray method to load doxorubicin. Also, these nanospheres can be used for injection in the treatment site by poloxamer-chitosan thermogel. In this research, PCL and PEG were used as raw materials to produce nanospheres. Then, doxorubicin was used for loading in nanospheres. The electrospray method was chosen as the method of nanosphere production. In the next step, poloxamer-chitosan thermogel was used for injection at the treatment site. In this method, Fourier transform infrared spectroscopy, scanning electron microscopy, and rheometer techniques were used to identify the compounds and properties of the obtained specimens. Also, the MTT test was used to investigate toxicity. The results showed that PCL-PEG polymer nanospheres were produced by loading doxorubicin using the electrospraying method with a diameter of 185 ± 23 nm. Also, these nanospheres were used for injection in the treatment site using poloxamer-chitosan thermogel. The amount of drug release in the PLX-CS (DOX-PCL-PEG)NSs was 63% in 144 h at medium pH 5.5. In the drug release system, the in-vitro method was utilized to study the release of PLX-CS (DOX-PCL-PEG) NSs. PCL-PEG nanospheres combined in poloxamer-chitosan thermogel polymer showed the controlled release of doxorubicin, therefore, the evaluated drug release system is considered a valuable perspective as an efficient and safe route for drug delivery in the target tissue and treating various types of cancer. This research can be used as a new method in drug delivery systems.     

High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents

In order to enhance the gene delivery efficiency and decrease cytotoxicity of polyplexes, copolymers consisting of branched polyethyleneimine (PEI) 25 kDa grafted with Pluronic (F127, F68, P105) were successfully synthesized using a simple two-step procedure. The copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge and transfection efficiency. The complex sizes were below 300 nm, which implicated their potential for intracellular delivery. The Pluronic-g-PEI exhibited better condensation and complexation properties than PEI 25 kDa. The cytotoxicity of PEI was strongly reduced after copolymerization. The Pluronic-g-PEI showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in HeLa cells. Compared with unmodified PEI 25 kDa Pluronic-g-PEI showed much higher transfection efficiency. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and grafted with Pluronic seem to provide promising properties as stable, high transfection efficiency vectors.     

Folate-equipped pegylated archaeal lipid derivatives: synthesis and transfection properties

We have previously shown that synthetic archaeal lipid analogues are useful vectors for drug/gene delivery. We report herein the synthesis and gene transfer properties of a series of novel di- and tetraether-type archaeal derivatives with a poly(ethylene glycol) (PEG) chain and further equipped with a folic acid (FA) group. The synthetic strategy and the purification by dialysis ensured complete removal of free FA. The lipids were mixed with a conventional glycine betaine-based cationic lipid and the resulting formulations were tested in transfection assays after complexation with plasmid DNA. All four novel co-lipids afforded efficient in vitro gene transfection. Moreover, the FA-equipped derivatives permitted ligand/receptor-based targeted transfection; their activity was inhibited when free FA was added to the transfection medium. These novel archaeal derivatives equipped with FA-PEG moieties may thus be of great interest for targeted in vivo transfection.     

Peptide-Based Nanoparticles for αvβ3 Integrin-Targeted DNA Delivery to Cancer and Uterine Leiomyoma Cells

Uterine leiomyoma is the most common benign tumor of the reproductive system. Current therapeutic options do not simultaneously meet the requirements of long-term efficiency and fertility preservation. Suicide gene delivery can be proposed as a novel approach to uterine leiomyoma therapy. Non-viral vehicles are an attractive approach to DNA delivery for gene therapy of both malignant and benign tumors. Peptide-based vectors are among the most promising candidates for the development of artificial viruses, being able to efficiently cross barriers of DNA transport to cells. Here we described nanoparticles composed of cysteine-crosslinked polymer and histidine-arginine-rich peptide modified with iRGD moiety and characterized them as vehicles for plasmid DNA delivery to pancreatic cancer PANC-1 cells and the uterine leiomyoma cell model. Several variants of nanoparticles were formulated with different targeting ligand content. The physicochemical properties that were studied included DNA binding and protection, interaction with polyanions and reducing agents, size, structure and zeta-potential of the peptide-based nanoparticles. Cytotoxicity, cell uptake and gene transfection efficiency were assessed in PANC-1 cells with GFP and LacZ-encoding plasmids. The specificity of gene transfection via αvβ3 integrin binding was proved in competitive transfection. The therapeutic potential was evaluated in a uterine leiomyoma cell model using the suicide gene therapy approach. The optimal formulation was found to be at the polyplex with the highest iRGD moiety content being able to transfect cells more efficiently than control PEI. Suicide gene therapy using the best formulation resulted in a significant decrease of uterine leiomyoma cells after ganciclovir treatment. It can be concluded that the application of iRGD-modified peptide-based nanoparticles has a high potential for cellular delivery of DNA therapeutics in favor of uterine leiomyoma gene therapy.     

Self‐Assembled and Size‐Controllable Oligonucleotide Nanospheres for Effective Antisense Gene Delivery through an Endocytosis‐Independent Pathway

The development of efficient gene delivery vectors has faced two major challenges, namely endo‐ and lysosomal escape and intracellular release. To address these problems, we developed an oligonucleotide (ON)‐template‐assisted polymerization approach to create ON nanospheres as gene vectors. Guanidinium‐containing disulfide monomers were organized on the ON templates to increase their effective local concentrations. Consequently, ring‐opening disulfide‐exchange polymerization between monomers was accelerated, further facilitating the self‐assembly of ON nanospheres. The size of these nanospheres was controlled by varying the length of the ON templates. Importantly, the nanospheres can be directly delivered into the cytosol through an endocytosis‐independent pathway, which is followed by intracellular depolymerization in the reductive cytosolic environment to release the packaged ONs, resulting in efficient gene silencing. The ON nanospheres thus hold great promise as candidates for gene therapy.     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.     

Improved GFP gene transfection mediated by polyamidoamine dendrimer-functionalized multi-walled carbon nanotubes with high biocompatibility

The multi-walled carbon nanotubes (MWCNTs)-polyamidoamine (PAMAM) hybrid was prepared by covalent linkage approach, and characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and ultraviolet-visible spectrometry. The PAMAM dendrimers were present on the surface of MWCNTs in high density, and the MWCNT-PAMAM hybrid exhibited good dispersibility and stability in aqueous solution. The interaction between MWCNT-PAMAM with plasmid DNA of enhanced green fluorescence protein (pEGFP-N1), intracellular trafficking of the hybrid, transfection performance and cytotoxicity to HeLa cells were evaluated in detail. We found that the MWCNT-PAMAM hybrid possessed good pEGFP-N1 immobilization ability and could efficiently delivery GFP gene into cultured HeLa cells. The surface modification of MWCNTs with PAMAM improved the transfection efficiency 2.4 and 0.9 times, and simultaneously decreased cytotoxicity by about 38%, as compared with mixed acid-treated MWCNTs and pure PAMAM dendrimers. The MWCNT-PAMAM hybrid can be considered as a new carrier for the delivery of biomolecules into mammalian cells. Therefore, this novel system may have good potential applications in biology and therapy, including gene delivery systems.     

Cationic PLA nanoparticles for DNA delivery: comparison of three surface polycations for DNA binding, protection and transfection properties

Biodegradable cationic nanoparticles (cNP) made of poly(lactide) (PLA) have been shown to be promising carrier systems for in vivo DNA delivery and immunization. In previous work, we have described a versatile approach for the elaboration of cationic PLA cNP based on the use of pre-formed particles and subsequent adsorption of a model polycation, the poly(ethylenimine) (PEI). Here, we evaluated two more polycations, chitosan and poly(2-dimethyl-amino)ethyl methacrylate (pDMAEMA)) to determine the most suitable one for the development of PLA cNP as DNA carriers. Cationic PLA-PEI, PLA-chitosan and PLA-pDMAEMA nanoparticles were compared for interaction with plasmid DNA and, more importantly, with regards to the biological properties of bound DNA. pDMAEMA coating yielded the most positively charged nanoparticles with the highest DNA binding capacity (32 mg/g). Loaded with DNA, all three cNP were in the same size range ( approximately 500 nm) and had a negative zeta potential (-50 mV). PLA-chitosan was the only cNP that released DNA at pH 7; the two others required higher pH. Adsorption and release from cNP did not alter structural and functional integrity of plasmid DNA. Moreover, DNA coated onto cNP was partially protected from nuclease degradation, although this protection was less efficient for PLA-chitosan than others. The highest transfection efficiency in cell culture was obtained with PLA-pDMAEMA carriers. We have shown that at least three different cationic polymers (chitosan, PEI, pDMAEMA) can be used for the production of PLA-based particulate DNA carriers and most probably other cationic polymers can also be used in the same purpose. PLA-pDMAEMA cNP were the most promising system for DNA delivery in this in vitro study. Our future work will focus on the in vivo evaluation of these gene delivery systems.     

纳米阳离子多聚物在基因载体系统的应用

阳离子多聚物能与DNA通过静电吸附作用而自组装成纳米微粒,防止DNA被核酸酶降解.阳离子多聚物由于具备合成简便、储存稳定、基因荷载率高、靶向性强、免疫原性低等优点而被用作基因载体.阳离子多聚物按特性可分为两类：合成型和天然型.经典的人工合成型阳离子多聚物基因载体主要有：多聚乙烯亚胺、多聚左旋赖氨酸和树状大分子等;天然生物型阳离子多聚物基因载体主要有壳聚糖及其衍生物和明胶等.本文详细论述了各种阳离子聚合物用作基因载体的性能特点、自身缺陷、介导基因进入细胞的机理和靶向性策略,并对非病毒基因载体的发展作出展望.