A reverse transfection technology to genetically engineer adult stem cells

A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.     

Delivery of plasmid IGF-1 to chondrocytes via cationized gelatin nanoparticles

The objective of the present study was to investigate the use of gelatin and cationized-gelatin nanoparticles for the nonviral delivery of the plasmid DNA encoding for insulin-like growth factor (IGF)-1 to adult canine articular chondrocytes in vitro; plasmid for enhanced green fluorescence protein (EGFP) was used as a marker gene. The spherical cationized gelatin nanoparticles were on average 172 nm in diameter, compared with the often ellipsoid-shaped unmodified (noncationized) gelatin particles that generally appeared to be 10 mum to greater than 20 mum in length. The zeta potential of the positively charged cationized gelatin nanoparticles containing the plasmid was around 20 mV compared with about 2 mV for the unmodified gelatin particles. There was no noticeable fluorescence from the cells treated with the nanoparticles prepared with the original (noncationized) gelatin particles containing the pEGFP. In contrast, numerous cells in the group transfected with the cationized gelatin-pEGFP nanoparticles were found to fluoresce demonstrating the transfection of the cells. There was five-fold elevation in the amount of IGF-1 produced by the cells treated with the cationized gelatin nanoparticles containing the IGF-1 plasmid compared with the unmodified (noncationized) gelatin particles. There was a clear effect of varying the weight ratio of plasmid IGF-1 in the cationized gelatin nanoparticles on the IGF-1 in the medium of cells exposed to the nanoparticles for 5 h. A peak in the amount of released IGF-1 was detected at a gelatin:IGF-1 weight ratio of 250:1.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin-plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin-plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.     

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin–plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin–plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.     

Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds

Angiogenesis is critical in the early stage of reparative processes and tissue regeneration, but the persistence of a vascular network may interfere with later transformation/maturation in naturally avascular tissues such as articular cartilage. Our supposition is that the timed delivery of an anti-angiogenic factor in cartilage tissue engineering may facilitate the formation of hyaline cartilage by inducing the regression of vascularization. To this end our overall goal is to prepare an off-the-shelf scaffold containing the gene for a potent anti-angiogenic factor. The objective of this study was to investigate the use of a type I/III collagen scaffold for the non-viral transfection of marrow stromal cells (MSCs, also referred to as mesenchymal stem cells) with the plasmid encoding endostatin. Caprine MSCs were transfected by the naked plasmid alone and plasmid incorporated into a cationic lipid complex in three experiments: 1) cells were transfected in monolayer; 2) monolayer-transfected cells were grown in a collagen sponge-like scaffold; and 3) non-transfected cells were grown in a collagen scaffold containing the naked plasmid and endostatin lipoplex. Independent variables were the passage number of the cells and the plasmid loading. The amount of endostatin released by the cells into the medium was measured using an ELISA. The results demonstrated the overexpression of endostatin by MSCs growing in the endostatin lipoplex-supplemented collagen scaffolds. Endostatin released by the cell-seeded scaffolds reached a peak of 13 ng/ml for scaffolds incorporating as little as 20 μg of plasmid, at the 3-day collection period ending 5 days post-seeding. The accumulated endostatin synthesis over a 2-week period began to achieve what may be a therapeutic level. MSCs transfected with the endostatin gene in monolayer continued to express the gene when grown in the collagen scaffolds. The results demonstrate the promise of the non-viral delivery of the gene for this potent anti-angiogenic protein to MSCs via a collagen scaffold.     

Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers

Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.     

In vitro transfection of plasmid DNA by cationized gelatin prepared from different amine compounds

The objective of this paper is to compare the in vitro transfection efficiency of a luciferase plasmid DNA using cationized gelatin prepared from different amine compounds. The compounds used here were ethylenediamine, putrescine, spermidine and spermine, chemically introduced to the carboxyl group of gelatin for the cationization. Complexation of the cationized gelatin with the plasmid DNA was performed by simply mixing the two materials at various N+/P- mixing ratios (the molar number ratio of amino groups of gelatin to the phosphate groups of DNA) in aqueous solution. Gel retardation studies revealed that the formation of cationized-gelatin-plasmid DNA complexes depended on the N+/P- mixing ratio. The stronger interaction of plasmid DNA with the cationized gelatin of spermine compared to the other cationized gelatins was observed by an ethidium bromide intercalation assay and Scatchard binding analysis. When the transfection efficiency of plasmid DNA complexed with the various cationized gelatins at different N+/P- mixing ratios was evaluated for mouse L929 fibroblasts, the highest transfection efficiency was observed for the complex prepared from the cationized gelatin of spermine at a N+/P- mixing ratio of 2. The present study indicates that there is an optimal N+/P- mixing ratio and a type of amine compound or cationization extent of cationized gelatin to enhance the transfection efficiency of plasmid DNA.     

交联明胶材料制备及细胞毒性的实验研究

目的:制备交联明胶,并检测其微球物理性质和细胞毒性。方法:采用双相乳化冷凝聚合法制备交联明胶,采用光镜和扫描电镜观察微球外形和分散度,紫外分光光度法测绘交联明胶体外戊二醛释放曲线,MTT实验检测其细胞毒性。结果:所制备的交联明胶微球表面光滑、直径均匀,干粉微球直径为21.13±1.42μm,溶胀后为26.72±1.74μm,溶胀率为127%。戊二醛释放实验和MTT实验证明其无细胞毒性。结论:交联明胶微球能在体外缓释超过30天,能有效吸附溶液、细胞毒性低,是下一步以交联明胶为载体进行基因转染实验的基础。     

Hydrophilized 3D porous scaffold for effective plasmid DNA delivery

In this study, hydrophilic PLGA/Pluronic F127 scaffolds loaded with a pDNA/PEI-PEG complex were prepared to estimate their potential use as a polymeric matrix for pDNA delivery. The scaffold was fabricated by a novel precipitation/particulate leaching method. The prepared pDNA/PEI-PEG complex-loaded PLGA/Pluronic F127 scaffold exhibited a highly porous (porosity, 93-95%) and open pore structure, as well as hydrophilicity, which can provide the good environment for cell adhesion and growth. The pDNA/PEI-PEG complexes were efficiently loaded into the PLGA/Pluronic F127 scaffold and continuously released from the scaffolds up to ~90% of the initial loading amount over a period of 8 wk, which may lead to continuous gene transfection into human bone marrow mesenchymal stem cells (hBMMSCs). From the in vitro cell culture in the scaffolds for transfection, it was observed that the pDNA/PEI-PEG complex-loaded hydrophilic PLGA/Pluronic F127 scaffold has a higher transfection efficiency of the pDNA/PEI-PEG complexes into hBMMSCs than the hydrophobic PLGA ones. The cell viability associated with the pDNA/PEI-PEG complexes released from the PLGA/Pluronic F127 scaffold was not significantly different from that of the PLGA/Pluronic F127 scaffold without pDNA, indicating its low cytotoxicity, probably due to the sustained release of the pDNA/PEI-PEG complex from the scaffolds. From these results, we could suggest that the pDNA/PEI-PEG complex-loaded hydrophilic PLGA/Pluronic F127 scaffold can be an effective gene delivery system for 3D tissue formation.     

Impregnation of plasmid DNA into three-dimensional scaffolds and medium perfusion enhance in vitro DNA expression of mesenchymal stem cells

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.     

Gene transfection of human mesenchymal stem cells with a nano‐hydroxyapatite–collagen scaffold containing DNA‐functionalized calcium phosphate nanoparticles

This study aimed to fabricate a growth factor‐releasing biodegradable scaffold for tissue regeneration. We prepared multishell calcium phosphate (CaP) nanoparticles functionalized with DNA, polyethyleneimine (PEI), protamine and octa‐arginine (R8) and compared their respective transfection activity and cell viability measures using human mesenchymal stem cells. DNA–protamine complexes improved the transfection efficiency of CaP nanoparticles with the exception of those functionalized with R8. These complexes also greatly reduced the cytotoxicity of PEI. In addition, we also fabricated DNA–protamine‐functionalized CaP nanoparticle‐loaded nano‐hydroxyapatite–collagen scaffolds and investigated their gene transfection efficiencies. These experiments showed that the scaffolds were associated with moderate hMSC cell viability and were capable of releasing the BMP‐2 protein into hMSCs following gene transfection. In particular, the scaffold loaded with protamine‐containing CaP nanoparticles showed the highest cell viability and transfection efficiency in hMSCs; thus, it might be suitable to serve as an efficient growth factor‐releasing scaffold.     

Controlled release of complexed DNA from polycaprolactone film: comparison of lipoplex and polyplex release

This report investigates the comparative in vitro controlled release and transfection efficiencies of pDNA-lipofectamine complex (lipoplex) and pDNA-poly(ethylene imine) complex (polyplex), from a biodegradable polycaprolactone (PCL) film. The effect of molecular weight of gelatin used as a porogen on in vitro release and transfection efficiency was also studied. A sustained release profile was obtained for naked pDNA and lipoplex from polymeric films for a month, while the release of polyplexes (PEI/DNA) is simply a burst at day 5, with little or no release thereafter. The release of polyplexes from PCL films is retarded due to interaction between the polyplexes and the polymer. A high burst release was seen for naked pDNA which was suppressed in the presence of gelatin. The extent of suppression of the burst effect by gelatin increased with its molecular weight. For complexed pDNA (lipoplex), the release was slow, but could be accelerated using gelatin; again the acceleration in release is dependant on the molecular weight of the gelatin used. The addition of gelatin as a porogen has no effect on the release of polyplexes from PCL films. The bioactivity of released plasmid DNA and complexes was studied by in vitro transfection using COS-7 cells. Transfection was observed from released lipoplexes samples till day 9 from PCL film with lower MW gelatin and till day 18 in the case of PCL films with higher MW gelatin. The results also showed that the bioactivity of released lipoplexes was superior to that of the naked pDNA.     

Gelatin coating to stabilize the transfection ability of nucleic acid polyplexes

Amphiphilic polymers are effective in complexing and delivering therapeutic nucleic acids, such as plasmid DNA (pDNA) and short interfering RNA (siRNA). However, long-term stability of the complexes is not desirable, as it may have an impact on the transfection efficiency in vivo. To develop a method to preserve complex stability we first showed that pDNA complexes formed with the amphiphilic polymer linoleic acid-substituted polyethylenimine (PEI–LA) and incubated at 37 °C lost ～90% of their transfection efficiency after only 24 h of complex formation. Polyethyleneglycol modification of complexes to control the increase in complex size and incubation in scaffolds used for implantation did not preserve the transfection ability of the complexes. Among a variety of approaches explored, gelatin coating of complexes was found to be the best at maintaining the original transfection efficiency. Mechanistic studies suggested that improved complex uptake, not size stability, was responsible for retention of the transfection efficiency. Similarly to the results with pDNA, gelatin coating also prevented the decreases in uptake and silencing efficiency of siRNA complexes observed following incubation at 37 °C. Gelatin-stabilized complexes were, furthermore, effective in vivo and led to subcutaneous transgene expression with a low pDNA dose that was otherwise ineffective. We conclude that a simple gelatin coating approach offers an efficient means to preserve the transfection efficiency of polyplexes.     

N-亚甲基磷酸化壳聚糖基因纳米粒子的体外细胞毒性及基因转染实验研究

目的 考察N-亚甲基磷酸化壳聚糖( NMPCS)基因纳米粒子的体外细胞毒性及基因转染效率.方法 采用均相反应法制备了NMPCS,用复凝聚法制备了NMPCS/DNA纳米粒子;通过MTT实验考察了NMPCS及其与DNA复合物对HeLa细胞的细胞毒性,以荧光索酶质粒为报告基因考察了NMPCS及NMPCS-CaZ+载体介导的体外基因转染效率.结果 NMPCS及其与DNA的复合物在体外表现出很小的细胞毒性,远远低于同等浓度时聚乙烯亚胺(PEI)的毒性.通过对壳聚糖进行亚甲基磷酸化修饰后,可大幅提高载体的基因转染效率.结论 N-亚甲基磷酸化壳聚糖有望成为一种新型、安全、高效的非病毒基因载体.     

Expression profile of plasmid DNA obtained using spermine derivatives of pullulan with different molecular weights

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 x 10(3). Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.     

Expression profile of plasmid DNA obtained using spermine derivatives of pullulan with different molecular weights

The objective of this study was to prepare a novel gene carrier from pullulan, a polysaccharide with an inherent affinity for the liver, and evaluate the feasibility in gene transfection. Pullulan with different molecular weights was cationized by chemical introduction of spermine. The cationized pullulan derivative was complexed with a plasmid DNA and applied to HepG2 cells for in vitro gene transfection. The level of gene expression depended on the molecular weight of cationized pullulan derivatives and the highest level was observed for the cationized pullulan derivative with a molecular weight of 47.3 × 10³. Pre-treatment of cells with asialofetuin decreased the level of gene expression by the complexes. These findings indicate that the cationized pullulan derivative is a promising non-viral carrier of plasmid DNA which is internalized in a receptor-mediated fashion.     

Effect of lipopolysaccharide addition on the gene transfection of spermine-introduced pullulan-plasmid DNA complexes for human mesenchymal stem cells

Abstract

The objective of this study is to investigate the effect of lipopolysaccharide (LPS) addition on the gene transfection of human mesenchymal stem cells (hMSC). hMSC were treated with the LPS at different concentrations and the complex of spermine-introduced pullulan and luciferase plasmid DNA for 3?h. The maximum level of gene expression was observed for hMSC treated with a certain concentration range of LPS. In addition, the cytotoxicity, cellular internalization of complexes, and cell cycle after LPS treatment were investigated. The cytotoxicity increased with an increase in the LPS concentration treated. On the other hand, the cellular internalization of complexes increased with the increased LPS concentration, although the internalization was sharply reduced at the high concentration. The LPS treatment increased the actin polymerization of cells to allow to spread more. The enhanced cells spreading would enhance the cellular internalization of complexes. In addition, the LPS treatment increased the rate of cell cycle. It is possible that the balance of cytotoxicity, cellular internalization, and cell cycle caused by the LPS addition results in the enhanced gene transfection at a certain LPS concentration. It is concluded that LPS treatment positively modified the cellular internalization and the cell cycle, resulting in the enhanced gene transfection.     

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.     

Efficacy and cytotoxicity of cationic-agent-mediated nonviral gene transfer into osteoblasts

Ex vivo gene transfer into osteoblastic cells is an advantageous strategy for bone tissue engineering. This study investigated the efficacy and cytotoxicity of in vitro cationic-agent-mediated nonviral gene transfer into osteoblasts. Various cationic agents, lipid, gelatin, and polyethylenimine (PEI) were tested. Each was formulated in various concentrations to form a complex with plasmid DNA encoding red fluorescent protein. The cationic agent/DNA complexes were transfected into human fetal osteoblastic cell line and rat bone-marrow-derived primary osteoblasts, as well as NIH 3T3 fibroblast controls. Rat primary osteoblasts were transfected more with cationic lipid and PEI agents than with gelatin carrier, yielding transfection efficacy up to 18.1% and 12.7 %, respectively. In contrast, human fetal osteoblastic cell line was transfected more with cationic lipid and gelatin than with PEI. There was a positive correlation between the lipid and PEI doses and cytotoxicity. When the lipid and PEI were used to transfect the rat primary osteoblasts in a dose that yielded the highest transfection efficacy, cell survival rates decreased as low as 40%. When their transfection efficacies into primary osteoblasts were compromised at two thirds of the highest value, that is, 12.6% and 8.3% for the lipid and PEI, respectively, the cell survival rate was nearly 80%. Cationic gelatin was associated with cell survival rates over 60 % in any cell type, regardless of the doses tested. These results suggest that different types of osteoblastic cells may possess different ability to the uptake and expression of cationic-agent-bound DNA. There seemed to be agent-specific threshold doses that dropped the cell survival rate. Cationic-agent-mediated nonviral gene transfer into osteoblastic cells may be successful when the agent- and dose-dependent transfection efficacy and cytotoxicity are optimized.     

Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.