Design flexibility influencing the in vitro behavior of cationic SLN as a nonviral gene vector

Several advanced in vitro and in vivo studies have proved the broad potential of cationic solid lipid nanoparticles (SLN) as nonviral vectors. However, a few data are available about the correlation between lipid component of the SLN structure and in vitro performance in terms of cell tolerance and transfection efficiency on different cell lines. In this paper SLN were prepared using stearic acid as main lipid component, stearylamine as cationic agent and protamine as transfection promoter and adding phosphatidylcholine (PC), cholesterol (Chol) or both to obtain three different multicomponent SLN (SLN–PC, SLN–Chol and SLN–PC–Chol, respectively). Cytotoxicity and transfection efficiency of the obtained SLN:pDNA complexes were evaluated on three different immortalized cell lines: COS-I (African green monkey kidney cell line), HepG2 (human hepatocellular liver carcinoma cell line) and Na1300 (murine neuroblastoma cell line).     

Development of nanosomes using high‐pressure homogenization for gene therapy

Objectives The aim of this project was to develop a novel lipid‐based formulation suitable for gene therapy. Methods Novel nanosize liposome (nanosome) formulations containing pDNA (plasmid DNA) were developed using high‐pressure homogenization (HPH). The effect of lipid concentration was studied at two levels: 3 mm and 20 mm . The preformed nanosomes were incubated for 18–20 h with pDNA or pDNA/protamine sulfate (PS) complex. The physical properties of the pDNA nanosomes were compared by particle size distribution and zeta‐potential measurements. Their biological properties were also compared by pDNA efficiency of encapsulation/complexation, integrity, nuclease digestion, transfection efficiency and cell cytotoxicity. Key findings pDNA nanosomes prepared with 20 mm lipid (nanosomes : pDNA : PS at a ratio of 8.6 : 1 : 2) had particle sizes of 170–422 nm (90% confidence). The zeta‐potential of the formulation was 49.2 ± 1.5 mV, and the pDNA encapsulation/complexation efficiency was ～98%. pDNA nanosomes prepared with 3 mm lipid (nanosomes : pDNA : PS at a ratio of 2.09 : 1 : 2) had particle sizes of 140–263 nm (90% confidence). The zeta‐potential of this formulation was 36.4 ± 1.2 mV, and the pDNA encapsulation/complexation efficiency was ～100%. However, a comparison of the efficiency of transfection indicated that pDNA nanosomes prepared with low‐concentration lipids (3 mm ) showed significantly higher transfection efficiency compared with the pDNA nanosomes prepared with high‐concentration lipids (20 mm ), as well as those prepared with Fugene‐6 (a commercially available transfection reagent). This particular formulation (pDNA nanosomes, 3 mm lipids) also showed significantly less cytotoxicity compared with the other pDNA nanosome formulations. Conclusions To conclude, these results indicate that condensing pDNA with PS followed by subsequent complexation with low‐concentration nanosomes generated from HPH can produce a pDNA nanosome formulation that will boost transfection efficiency, while minimizing cytotoxicity. This new technology appears to be an efficient tool for future commercial or large‐scale manufacture of DNA delivery systems for gene therapy.     

Gelucire-stabilized nanoparticles as a potential DNA delivery system

Clinical viability of gene delivery systems has been greatly impacted by potential toxicity of the delivery systems. Recently, we reported the nanoparticle (NP) preparation process that employs biocompatible materials such as Gelucire® 44/14 and cetyl alcohol as matrix materials. In the current study, the NP preparation was modified for pDNA loading through: (i) inclusion of cationic lipids (DOTAP or DDAB) with NP matrix materials; or (ii) application of cationic surfactants (CTAB) to generate NPs with desired surface charges for pDNA complexation. Colloidal stability and efficiency of loading pGL3-DR4X2-luciferase plasmid DNA in NPs were verified by gel permeation chromatography. Compared to pDNA alone, all the NPs were effective in preserving pDNA from digestion by DNase. While pDNA loading using CTAB-NPs involved fewer steps compared to DOTAP-NPs and DDAB-NPs, CTAB-NPs were greatly impacted by elevated cytotoxicity level which could be ascribed to the concentrations of CTAB in NP formulations. In vitro transfection studies (in HepG2 cells) based on luciferase expression showed the ranking of cell transfection efficiency as DOTAP-NPs?>?DDAB-NPs?>?CTAB-NPs. The overall work provided an initial assessment of gelucire-stabilized NPs as a potential platform for gene delivery.     

“Intelligent” nanoassembly for gene delivery: In vitro transfection and the possible mechanism

A new “intelligent” nanoassembly (INA), consisting of a condensed core of pDNA with protamine sulfate (PS) and a dioleoylphosphatidyl ethanolamine (DOPE)-based lipid envelope containing poly(ethylene glycol)-disulfide-DOPE (PSD), was designed and investigated. The in vitro release experiment was carried out in solution containing 10 mM of Glutathione, which reflected the redox potential of the intracellular environment. The experimental result indicated that PSD possessed a good ability of self-dePEGylation and could result in efficient release of content in the reductive environment. INAs showed higher transfection efficiency and much lower cytotoxicity compared with Lipofectamine? 2000 on HEK 293 cells. Cellular uptake and subcellular localization, as well as the quantitation of nuclear transfer demonstrated that the superior transfection efficiency of INAs could result from both enhanced cellular uptake mediated by DOPE and efficient nuclear delivery mediated by PS. The biodistribution of INAs in nude mice bearing tumor implied that this PSD-based nanoassembly loading PS/DNA could be a promising gene delivery system for tumor therapy.     

VEGF receptor binding peptide-linked amphiphilic peptide with arginines and valines for endothelial cell-specific gene delivery

An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch (R3V6) has been previously reported to deliver plasmid DNA (pDNA) into cells with no toxicity. Here, the vascular endothelial growth factor receptor binding peptide (VRBP) was linked to R3V6 to promote endothelial-specific gene delivery. The pDNA/VRBP-linked R3V6 (VRBP-R3V6) complex was physically characterized via various methods. In a gel retardation assay, pDNA was completely retarded by VRBP-R3V6 at a weight ratio of 1:2 (pDNA:peptide). VRBP-R3V6 also protected pDNA from DNase I for longer than 60 min. Heparin competition assay showed that the pDNA/VRBP-R3V6 complex did not release pDNA when heparin was introduced at a two-fold weight excess of pDNA. In vitro transfection showed that VRBP-R3V6 had transfection efficiency into endothelial cells approximately 200 times greater than that of R3V6. In addition, the transfection efficiency was further enhanced into hypoxic endothelial cells. However, in human embryonic kidney 293 and neuroblastoma N2A cells, VRBP-R3V6 only achieved a transfection rate 10 times higher than R3V6, indicating that VRBP-R3V6 has high specificity for endothelial cells. VRBP-R3V6 was also shown to be nontoxic in a cytotoxicity assay. The data presented here suggest that VRBP-R3V6 may prove useful for specific gene delivery to endothelial cells.     

VEGF receptor binding peptide-linked amphiphilic peptide with arginines and valines for endothelial cell-specific gene delivery

An amphiphilic peptide with a 3-arginine stretch and a 6-valine stretch (R3V6) has been previously reported to deliver plasmid DNA (pDNA) into cells with no toxicity. Here, the vascular endothelial growth factor receptor binding peptide (VRBP) was linked to R3V6 to promote endothelial-specific gene delivery. The pDNA/VRBP-linked R3V6 (VRBP-R3V6) complex was physically characterized via various methods. In a gel retardation assay, pDNA was completely retarded by VRBP-R3V6 at a weight ratio of 1:2 (pDNA:peptide). VRBP-R3V6 also protected pDNA from DNase I for longer than 60 min. Heparin competition assay showed that the pDNA/VRBP-R3V6 complex did not release pDNA when heparin was introduced at a two-fold weight excess of pDNA. In vitro transfection showed that VRBP-R3V6 had transfection efficiency into endothelial cells approximately 200 times greater than that of R3V6. In addition, the transfection efficiency was further enhanced into hypoxic endothelial cells. However, in human embryonic kidney 293 and neuroblastoma N2A cells, VRBP-R3V6 only achieved a transfection rate 10 times higher than R3V6, indicating that VRBP-R3V6 has high specificity for endothelial cells. VRBP-R3V6 was also shown to be nontoxic in a cytotoxicity assay. The data presented here suggest that VRBP-R3V6 may prove useful for specific gene delivery to endothelial cells.     

鱼精蛋白／DNA复合物的构建与生物性能评价

目的研究非病毒基因载体鱼精蛋白／DNA复合物的制备方法及对其细胞毒性、不同量鱼精蛋白对pDNA的结合能力、体外细胞转染率。方法不同量鱼精蛋白与DNA在室温孵育后,得到鱼精蛋白／DNA复合物;用MTT法检测鱼精蛋白对HeLa子宫颈癌细胞的毒性作用,同时与PEI进行比较;利用琼脂糖电泳实验测定不同N／P比形成复合物时对DNA的阻滞情况;用结合沉淀试验比较不同量鱼精蛋白对包裹DNA的能力的影响;在荧光显微镜下观察比较它们对BEL-7402肝癌细胞转染率的大小。结果当鱼精蛋白／DNA复合物浓度升高时,对He—La子宫颈癌细胞的毒性均为0级,而聚乙烯亚胺（PEI）浓度升高时,对细胞的毒性明显增加;鱼精蛋白与质粒DNA形成复合物时所需的N／P比为1．5：1,较PEI／DNA复合物形成时所需的N／P比2：1要小;鱼精蛋白包裹DNA的能力随N／P比增大而增强,并且包裹DNA的能力要比PEI／DNA复合物转变得快;鱼精蛋白／DNA复合物对BEL-7402肝癌细胞的转染率较PEI／DNA复合物低,但毒性较低。结论鱼精蛋白／DNA复合物是一种制备工艺简单、细胞毒性小、对pDNA包裹能力高、转染率相对较高,具有一定应用潜力的非病毒基因载体。     

Establishment of evaluation method for siRNA delivery using stable cell line carrying the luciferase reporter gene

We determined the influence of siRNA (short interfering RNA) for expression of plasmid DNA (pDNA), when mismatched siRNA and pDNA encoding beta-galactosidase (beta-gal) were transfected into HeLa cells by the cotransfection method in which they were simultaneously added to the cells. Cationic liposomes (Lipofectamine2000) were used as a gene transfection reagent. The knockdown effect on beta-gal was observed even when mismatched siRNA was used, and the effect depended on the amount of added mismatched siRNA. But, there was not a distinct difference of introduction of pDNA into cells between using mismatched siRNA and without using it. We considered that the cotransfection method should be avoided when we confirm RNAi efficiency. The reliable evaluation method for siRNA delivery in vitro was thus established by using NFAT reporter HeLa stable cell line or CHO (pMAM-luc) cell line that had DNA encoding luciferase. The following experimental conditions for each cell line were optimized: cell numbers seeded, total incubation times, concentrations of added inducers, and incubation times after addition of inducers. Transfection performance was compared for six commercially available reagents by this method. No commercially available transfection reagent, however, could reduce luciferase activity by less than one tenth without causing cellular cytotoxicity. Development of novel reagents providing higher transfection effects without cytotoxicity is needed.     

Surfactant protein A (SP-A)-tacrolimus complexes have a greater anti-inflammatory effect than either SP-A or tacrolimus alone on human macrophage-like U937 cells

Protamine has attracted much attention as DNA condenser and nuclear transfer enhancer although the excess of hydrophilicity and the strong DNA pack restrain its potentialities. In order to overcome this limitation, we added Protamine in the composition of solid lipid nanoparticles (SLN-Protamine) and we compared this carrier with the same kind of SLN containing Esterquat 1 instead of Protamine (SLN-EQ1). Carriers cytotoxicity was assessed on COS-I cells evaluating the cell cycle by propidium iodide test, while the transfection efficiency was studied using pEGFP as plasmid model. The cell penetrating activity of Protamine inside the lipid vectors was evaluated studying cell internalization by confocal microscopy using Red Nile-labeled carriers. SLN-Protamine:pDNA showed a mean diameter five-times smaller than the size of SLN-EQ1:pDNA and a remarkably lesser cytotoxicity. Transfection by SLN-Protamine:pDNA was seven-times more effective compared with the Protamine:pDNA polyplexes while no transfection capacity was observed for SLN-EQ1:pDNA complexes due to their inability to be internalized owing to their larger dimension. Red Nile-SLN-Protamine were localized in endocytic-like vesicles into the nuclear membrane suggesting the inclusion of Protamine in nano-lipophilic systems may enhance the reduction in the complex dimensions, the nuclear pDNA translocation and the pDNA release in the cells.     

Dextran-protamine-solid lipid nanoparticles as a non-viral vector for gene therapy: in vitro characterization and in vivo transfection after intravenous administration to mice

The aim of present work is to evaluate the transfection capacity of a new multicomponent system based on dextran (Dex), protamine (Prot), and solid lipid nanoparticles (SLN) after intravenous administration to mice. The vectors containing the pCMS-EGFP plasmid were characterized in terms of particle size and surface charge. In vitro transfection capacity and cell viability were studied in four cell lines, and compared with the transfection capacity of SLN without dextran and protamine. Transfection capacity was related to the endocytosis mechanism: caveolae or clathrin. The Dex-Prot-DNA-SLN vector showed a higher transfection capacity in those cells with a high ratio of activity of clathrin/caveolae-mediated endocytosis. However, the complex prepared without dextran and protamine (DNA-SLN) was more effective in those cells with a high ratio of activity of caveolae/clathrin-mediated endocytosis. The interaction with erythrocytes and the potential hemolytic effect were also checked. The Dex-Prot-DNA-SLN vector showed no agglutination of erythrocytes, probably due to the presence of dextran. After intravenous administration to BALB/c mice, the vector was able to induce the expression of the green fluorescent protein in liver, spleen and lungs, and the protein expression was maintained for at least 7 days. Although additional studies are necessary, this work reveals the promising potential of this new gene delivery system for the treatment of genetic and non-genetic diseases through gene therapy.     

Lipoplexes versus nanoparticles: pDNA/siRNA delivery

Abstract

Small interfering RNA (siRNA) has been widely used as potential therapeutic for treatment of various genetic disorders. However, rapid degradation, poor cellular uptake and limited stability in blood limit the effectiveness of the systemic delivery of siRNA. Therefore, an efficient delivery system is required to enhance its transfection and duration of therapeutics. In the present study, plasmid DNA (pEGFPN3) expressing green fluorescent protein (GFP) was used as a reporter gene. Chitosan nanoparticles/polyplexes and cationic liposomes/lipoplexes were developed and compared for their transfectivity and therapeutic activity in mammalian cell line (HEK 293). The nanoparticulates were first characterized by assessing the surface charge (zeta potential), size (dynamic light scattering) and morphology (transmission electron microscope) followed by evaluation for their DNA retardation ability, transfection efficiency and cytotoxicity on HEK 293 cell line. The chitosan nanoparticles/plasmid DNA (pDNA) complex and liposomes/pDNA complex were co-transfected with GFP-specific siRNA into HEK 293 cells and it was found that both are efficient delivery vehicles for siRNA transfection, resulting in ～57% and ～70% suppression of the targeted gene (GFP), respectively, as compared with the mock control (cells transfected with nanocarrier/pDNA complexes alone). This strong inhibition of GFP expression indicated that cationic liposomes are better than chitosan nanoparticles and can be used as an effective carrier of siRNA in mammalian cells.     

Polybutylcyanoacrylate nanoparticles as novel vectors in cancer gene therapy

To make progress toward an efficient gene vector for cancer gene therapy, a novel nonviral vector of polybutylcyanoacrylate nanoparticles (PBCA NPs) was developed. Cetyltrimethyl ammonium bromide (CTAB) was used to modify the surface of PBCA NPs, and then the plasmid DNA (pDNA) of pAFP-TK was wrapped into PBCA-CTAB NPs. Atomic force microscopy and zeta potential demonstrated that PBCA-CTAB NPs were 80-200 nm in diameter and had +15.6 mV positive surface charges. Assay using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide showed that PBCA-CTAB NPs had less cytotoxicity to 3T3 cells than HepG2 cells. The analysis of PBCA-CTAB-DNA complexes could not only protect DNA from degradation by DNase I, it could also transfer pDNA into targeted cells with high transfection efficiency. Furthermore, when PBCA-CTAB NPs combined with suicide gene pAFP-TK, alpha-fetoprotein-positive cells transfected by it were highly sensitive to ganciclovir treatment, and cell survival declined precipitously. Therefore, this target strategy using a pAFP-TK/GCV suicide gene therapy system in which PBCA-CTAB NPs serve as gene delivery vectors explores a promising area for alpha-fetoprotein-positive hepatocellular carcinoma and associated carcinoma therapy.     

Effect of Solid Lipid Nanoparticles Formulation Compositions on Their Size,Zeta Potential and Potential for <Emphasis Type="BoldItalic">In Vitro</Emphasis> pHIS-HIV-Hugag Transfection

Purpose This work was conducted to determine model equations describing the effect of solid lipid nanoparticles (SLN) formulation compositions on their size and zeta potential using the face-centered central composite design and to determine the effect of SLN formulation compositions on the potential for in vitro pHIS-HIV-hugag transfection. Materials and Methods SLN were prepared by the hot high pressure homogenization technique using cetylpalmitate as lipid matrix at varying concentrations of Tween 80 and Span 85 mixture, dimethyldioctadecyl ammonium bromide (DDAB) and cholesterol. Size and zeta potential used as responses of the design were measured at pH 7.0. The model equations were accepted as statistical significance at p value of less than 0.05. Ability of SLN to form complex with pHIS-HIV-hugag was evaluated by electrophoretic mobility shift assay. In vitro cytotoxicity of SLN was studied in HeLa cells using alamar blue bioassay. The potential of SLN for in vitro pHIS-HIV-hugag transfection was also determined in HeLa cells by western blot technique. Results SLN possessed diameter in a range of 136–191 nm and zeta potential 11–61 mV depending on the concentrations of surfactant mixture, DDAB and cholesterol. The regression analysis showed that the model equations of responses fitted well with quadratic equations. The ability of SLN to form complex with pHIS-HIV-hugag was also affected by formulation compositions. In vitro cytotoxicity results demonstrated that HeLa cells were not well tolerant of high concentrations of SLN but still survived in a range of 100–200 μg/ml of SLN in culture medium. The results of transfection study showed ability of SLN to use as a vector for in vitro pHIS-HIV-hugag transfection. However, their potential for in vitro transfection was lower than the established transfection reagent. Conclusions Size and zeta potential of SLN could be predicted from their quadratic model equations achieved by combination of three variables surfactant, DDAB and cholesterol concentrations. In addition, these variables also affected the potential of SLN as a vector for in vitro pHIS-HIV-hugag transfection. The results here provide the framework for further study involving the SLN formulation design for DNA delivery.     

壳聚糖纳米粒作为基因载体的研究:粒径对转染效率的影响

研究粒径对壳聚糖(chitosan，CS)纳米粒介导的转染效率的影响。通过调整CS溶液加入质粒基因(plasmid DNA，pDNA)溶液的速度和涡旋时间制备250，580和1 300 nm粒径pDNA/CS纳米粒，研究粒径对CS介导的细胞转染效率的影响。为深入探讨粒径对转染效率的影响，考察了3种粒径pDNA/CS纳米粒的药剂学性质，对抗核酸酶作用和细胞对纳米粒的吸附和摄取行为。结果表明：本文制备的3种粒径纳米粒的药剂学性质和凝聚pDNA的能力等特性基本无差别，均能有效保护pDNA免受核酸酶降解；在HEK293细胞中的转染效率无显著差异；与细胞共孵育4 h，流式细胞仪测定的三者细胞摄取率与摄取量相似；荧光显微图像显示3种粒径纳米粒均以小聚集体形式吸附于细胞表面，激光扫描共聚焦显微图像显示直径约为2 μm小聚集体较易被细胞内吞入胞。因此粒径在250～1 300 nm中对壳聚糖纳米粒介导的细胞转染率基本无影响。     

Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose

The successful control of intracellular trafficking (i.e., endosomal escape and nuclear delivery) is prerequisite for the development of a gene delivery system. In the present study, we developed an in vivo hepatic gene delivery system using a plasmid DNA (pDNA)-encapsulating lipid envelope-type nanoparticle, to which we refer as a multifunctional envelope-type nanodevice (MEND). The critical structural elements of the MEND are a DNA/protamine condensed core coated with lipid bilayers including serum-resistant cationic lipids. Intravenous administration of bare MEND represents minimal transfection activity. For the surface modification of functional devices, hydrophobic moieties were chemically attached, which are shed in the spontaneous orientation outward from the MEND surface by anchoring to the lipid bilayers. Modification of the pH-dependent fusogenic peptide GALA as an endosome escape induced transfection activity by 1 and 2 orders of magnitude. In an attempt to induce the nuclear delivery of pDNA, maltotriose, a recently characterized nuclear localization signal, was additionally modified. As a result, transfection activity further enhanced by 1 order of magnitude, and it reached to the higher level obtained for a conventional lipoplex and an in vivo jetPEI-Gal, with less hepatic toxicity. The data show that the combination of GALA and maltotriose results in a highly potent functional device that shows an enhanced endosomal escape and nuclear delivery in vivo.     

Physical stability and in-vitro gene expression efficiency of nebulised lipid-peptide-DNA complexes

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid-DNA complexes. Complexes prepared from the cationic lipid 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145+/-3 nm pre-nebulisation vs. 142+/-2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102+/-1 nm pre-nebulisation vs. 113+/-2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.     

Examination of the biophysical interaction between plasmid DNA and the polycations, polylysine and polyornithine, as a basis for their differential gene transfection in-vitro

The impetus to develop non-viral gene delivery vectors has led to examination of synthetic polycationic polymers as plasmid DNA (pDNA) condensing agents. Previous reports have highlighted superiority (up to x 10-fold) in the in-vitro transfection of pDNA complexes formed by poly-(L)-ornithine (PLO) compared to those formed with poly-(L)-lysine (PLL). The apparent basis for this consistent superiority of PLO complexes remains to be established. This comparative study investigates whether physico chemical differences in the supramolecular properties of polycation:pDNA complexes provide a basis for their observed differential gene transfection. Specifically, particle size distribution and zeta potential of the above complexes formulated over a wide range of polycation:pDNA ratios were found to be consistent with a condensed (150-200 nm) cationic ( + 30-40 mV) system but not influenced by the type of cationic polymer used. A spectrofluorimetric EtBr exclusion assay showed that polycation:pDNA complexes display different pDNA condensation behaviour, with PLO able to condense pDNA at a lower polycation mass compared to both polylysine isomers, and form complexes that were more resistant to disruption following challenge with anionic counter species, i.e. poly-(L)-aspartic acid and the glycosaminoglycan molecule. heparin. We conclude that particle size and surface potential as gross supramolecular properties of these complexes do not represent, at least in a non-biological system, the basis for the differential transfection behaviour observed between these condensing polymers. However, differences in the ability of the polylysine and polyornithine polymers to interact with pDNA and to stabilise the polymer-pDNA assembly could have profound effects upon the cellular and sub-cellular biological processing of pDNA molecules and contribute to the disparity in cell transfection efficiency observed between these complexes.     

Fabrication,characterization and in vitro evaluation of poly(D,L-lactide-co-glycolide) microparticles loaded with polyamidoamine–plasmid DNA dendriplexes for applications in nonviral gene delivery

We report, for the first time, on the preparation, characterization and in vitro testing of poly(D,L-lactide-co-glycolide) (PLGA) microparticles loaded with polyamidoamine (PAMAM)–plasmid DNA (pDNA) dendriplexes. Loading of pDNA into the PLGA microparticles increased by 150% when pDNA was first complexed with PAMAM dendrimers relative to loading of pDNA alone. Scanning electron microscopy (SEM) showed that the presence of PAMAM dendrimers in the PLGA microparticles created porous features and indentations on the surface of the microparticles. Loading PLGA microparticles with PAMAM–pDNA dendriplexes lowered the average PLGA microparticle size and changed the surface charge of the microparticles from negative to positive when compared to PLGA microparticles loaded with pDNA alone. The zetapotential and buffering capacity of the microparticles increased as the generation of the PAMAM dendrimer loaded in the PLGA microparticles increased. Gel electrophoresis assays showed that all the PLGA microparticle formulations were able to entrap the pDNA within the PLGA matrix. There was no significant difference in the cytotoxicity of PLGA microparticles loaded with PAMAM–pDNA dendriplexes when compared to PLGA microparticles loaded with pDNA alone. Furthermore, and in contrast to PAMAM dendrimers alone, the generation of the PAMAM dendrimer loaded in the PLGA microparticles had no significant impact on cytotoxicity or transfection efficiencies in human embryonic kidney (HEK293) or Monkey African green kidney fibroblast-like (COS7) cells. The transfection efficiency of PLGA microparticles loaded with generation 3 (G3) PAMAM–pDNA dendriplexes was significantly higher than PLGA microparticles loaded with pDNA alone in HEK293 and COS7 cells. PLGA microparticles loaded with G3 PAMAM–pDNA dendriplexes generated equivalent transfection efficiencies as (G3 to G6) PAMAM–pDNA dendriplexes alone in COS7 cells when the transfection was carried out in serum containing media. The delivery system developed in this report has low toxicity, high pDNA loading efficiencies and high transfection efficiencies that are not reduced in the presence of serum. A delivery system with these characteristics is expected to have significant potential for translational applications. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:368–384, 2010     

Emerging significance of plasmid DNA nuclear import in gene therapy

The signal-mediated import of plasmid DNA (pDNA) into nondividing mammalian cell nuclei is one of the key biological obstacles to nonviral therapeutic pDNA delivery. Overcoming this barrier to pDNA transfer is thus an important fundamental objective in gene therapy. Here, we outline the rationale behind current and future strategies for signal-mediated pDNA nuclear import. Results obtained from studies of the nuclear delivery of pDNA coupled to experimentally defined nuclear localisation signal (NLS) peptides, in conjunction with detergent-permeabilised reconstitution cell assays, direct intracellular microinjection, cell-based transfection, and a limited number of in vivo experiments are discussed.