Zeta potential of transfection complexes formed in serum-free medium can predict in vitro gene transfer efficiency of transfection reagent

We have tested the zeta potential (zeta, the surface charge density) of transfection complexes formed in serum-free medium as a rapid and reliable technique for screening transfection efficiency of a new reagent or formulation. The complexes of CAT plasmid DNA (1 microgram) and DC-chol/DOPE liposomes (3-20 nmol) were largely negatively charged (zeta=-15 to -21 mV), which became neutral or positive as 0.5 microgram or a higher amount of poly-L-lysine (PLL, MW 29300 or MW 204000) was added (-3.16+/-3.47 to +6.04+/-2.23 mV). However, the complexes of CAT plasmid DNA (1 microgram) and PLL MW 29300 (0.5 microgram or higher) were neutral or positively charged (-3.22+/-2.3 to +6.55+/-0.64 mV), which remained the same as 6.6 nmol of the liposomes was added. The complexes formed between two positively charged compounds, PLL MW 29300 (0.5 microgram) and the liposomes (3-20 nmol), were as closely positively charged as DNA/PLL or DNA/liposomes/PLL complexes (+3.31+/-0.41 to 7.16+/-1.0 mV). These results indicate that PLL determined the overall charge of the DNA/liposome/PLL ternary complexes. The complexes formed with histone (0.75 microgram or higher) were also positively charged, whose transfection activity was as high as PLL MW 29300. However, the complexes formed with protamine or PLL MW 2400 remained negatively charged. These observations are in good agreement with the transfection activity of the formulation containing each polycationic polymer. The presence of PLL MW 29300 did not change the hydrodynamic diameter of DNA/liposome/PLL complexes (d(H)=275-312 nm). The complexes made of different sizes of PLL (MW 2400 and 204000) also did not significantly change their size. This suggests that DNA condensation may not be critical. Therefore, zeta of the transfection complex can predict the transfection efficiency of a new formulation or reagent.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

Water-soluble lipopolymer for gene delivery

The use of biocompatible polymeric gene carriers may overcome the current problems associated with viral vectors in safety, immunogenicity, and mutagenesis. Nontoxic water-soluble lipopolymer (WSLP), poly(ethylenimine)-co-[N-(2-aminoethyl) ethyleneimin]-co-N-(N-cholesteryloxycarbonyl-(2-aminoethyl)ethylenimine) was synthesized using branched poly(ethylenimine) (PEI, mw 1800) and cholesteryl chloroformate. Following synthesis and purification, the structure and molecular weight of WSLP were confirmed by (1)H NMR and MADI-TOF mass spectrometry, respectively. The percentage of cholesterol conjugated to PEI was about 47%, and the average molecular weight of WSLP was approximately 2000 Da. WSLP/pDNA complexes were prepared at different N/P (nitrogen atoms of WSLP/phosphate of plasmid DNA) ratios and characterized in terms of particle size, zeta potential, osmolarity, surface morphology, and cytotoxicity. WSLP condensed plasmid DNA when N/P ratio reached 2.5/1 and no free DNA was detected at N/P ratio of 5/1 and above, as determined by agarose gel electrophoresis. The mean particle size was in the range of 25.9 to 148.5 nm and was dependent on N/P ratios. Atomic force microscopy (AFM) showed complete condensation of plasmid DNA with spherical particles of approximately 50 nm in diameter. WSLP/pDNA complexes or WSLP itself were nontoxic to CT-26 colon adenocarcinoma and 293 T human embryonic kidney transformed cells when formulated at the N/P ratio of 10/1 and below as determined by MTT assay. In contrast, PEI25000/pDNA complexes were toxic to these cells. Erythrocytes aggregated when incubated with PEI25000/pCMV-Luc complexes at high DNA concentrations, but there was little aggregation with WSLP/pCMV-Luc complexes. WSLP/pCMV-Luc complexes demonstrated higher transfection efficiency in both CT-26 and 293 T cells compared to PEI25000- or PEI1800-based formulations. WSLP/pCMV-Luc complexes are nontoxic and showed enhanced in vitro transfection. Thus, WSLP will be a suitable carrier for in vivo gene delivery.     

Preparation, characterization and transfection efficiency of cationic PEGylated PLA nanoparticles as gene delivery systems

The cationic polylactic acid (PLA) nanoparticle has emerged as a promising non-viral vector for gene delivery because of its biocompatibility and biodegradability. However, they are not capable of prolonging gene transfer and high transfection efficiency. In order to achieve prolonged delivery of cationic PLA/DNA complexes and higher transfection efficiency, in this study, we used copolymer methoxypolyethyleneglycol-PLA (MePEG-PLA), PLA and chitosan (CS) to prepare MePEG-PLA-CS NPs and PLA-CS NPs by a diafiltration method and prepared NPs/DNA complexes through the complex coacervation of nanoparticles with the pDNA. The object of our work is to evaluate the characterization and transfection efficiency of MePEG-PLA-CS versus PLA-CS NPs. The MePEG-PLA-CS NPs have a zeta potential of 15.7 mV at pH 7.4 and size under 100 nm, while the zeta potential of PLA-CS NPs was only 4.5 mV at pH 7.4. Electrophoretic analysis suggested that both MePEG-PLA-CS NPs and PLA-CS NPs with positive charges could protect the DNA from nuclease degradation and cell viability assay showed MePEG-PLA-CS NPs exhibit a low cytotoxicity to normal human liver cells. The potential of PLA-CS NPs and MePEG-PLA-CS NPs as a non-viral gene delivery vector to transfer exogenous gene in vitro and in vivo were examined. The pDNA being carried by MePEG-PLA-CS NPs, PLA-CS NPs and lipofectamine could enter and express in COS7 cells. However, the transfection efficiency of MePEG-PLA-CS/DNA complexes was better than PLA-CS/DNA and lipofectamine/DNA complexes by inversion fluorescence microscope and flow cytometry. It was distinctively to find that the transfection activity of PEGylation of complexes was improved. The nanoparticles were also tested for their ability to transport across the gastrointestinal mucosa in vivo in mice. In vivo experiments showed obviously that MePEG-PLA-CS/DNA complexes mediated higher gene expression in stomach and intestine of BALB/C mice compared to PLA-CS/DNA and lipofectamine/DNA complexes. These results suggested that MePEG-PLA-CS NPs have favorable properties for non-viral gene delivery.     

Preparation and characterization of chitosan and trimethyl-chitosanmodified poly-(ε-caprolactone) nanoparticles as DNA carriers

The purpose of this research was to prepare poly-(ε-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. Published: August 10, 2005     

Bacterial magnetic particles (BMPs)-PEI as a novel and efficient non-viral gene delivery system

BACKGROUND: Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS: We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS: BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS: We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.     

Delipidated serum abolishes the inhibitory effect of serum on in vitro liposome-mediated transfection

All the standard in vitro lipofection has been routinely performed in serum-free medium as the transfection activity of liposome/DNA complexes is sensitive to the presence of serum. In this study, we have demonstrated that lipid-rich serum lipoprotein included in the transfection medium strongly inhibited the transfection activity of DC-chol liposome/DNA complexes in five different cell types (CHO, 293, A2780CP, A431 and SKBR3). The levels of inhibition by serum lipoprotein were rather greater than those by serum and varied with cell types. However, this inhibition was completely abolished by delipidation of serum. Thus, delipidated serum can be included in the transfection medium. The complexes formed in the presence of serum (zeta=-18.2+/-1.07 mV), delipidated serum (zeta=-19.6+/-0.54 mV), IgG (zeta=-21.6+/-1.92 mV) or serum lipoprotein (zeta=-10.5+/-2.33 mV) were as much negatively charged as those in serum-free medium (zeta=-21.3+/-1.60 mV). The results suggest that the inhibition of liposome-mediated transfection by serum was not associated with charges of serum proteins but with lipids or lipid-associated proteins present in serum.     

Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA

We examined changes in zeta potential (the surface charge density, zeta) of the complexes of liposome (nmol)/DNA (microg) (L/D) formed in water at three different ratios (L/D=1, 10 and 20) by changing the ionic strength or pH to find an optimum formulation for in vivo gene delivery. At high DNA concentrations, zeta of the complexes formed in water at L/D=10 was significantly lowered by adding NaCl (zeta=+8.44+/-3.1 to -27.6+/-3.5 mV) or increasing pH from 5 (zeta=+15.3+/-1.0) to 9 (zeta=-22.5+/-2.5 mV). However, the positively charged complexes formed at L/D=20 (zeta=+6.2+/-3.5 mV) became negative as NaCl was added at alkaline pH as observed in medium (zeta=-19.7+/-9.9 mV). Thus, the complexes formed in water under the optimum condition were stable and largely negatively charged at L/D=1 (zeta=-58.1+/-3.9 mV), unstable and slightly positively charged at L/D=10 (zeta=+8.44+/-3.7 mV), and unstable and largely positively charged at L/D=20 (zeta=+24.3+/-3.6 mV). The negatively charged complexes efficiently delivered DNA into both solid and ascitic tumor cells. However, the positively charged complexes were very poor in delivering DNA into solid tumors, yet were efficient in delivering DNA into ascitic tumors grown in the peritoneum regardless of complex size. This slightly lower gene transfer efficiency of the negatively charged complexes can be as efficient as the positively charged ones when an injection is repeated (at least two injections), which is the most common case for therapy regimes. The results indicate that optimum in vivo lipofection may depend on the site of tumor growth.     

核酸载体lipopolyplex 的制备及细胞毒性研究

目的：为降低聚阳离子基因载体polyplex 的正电荷和毒性,在其表面构建中性磷脂膜制备lipopolyplex,并测定lipopolyplex 对小鼠结肠癌细胞CT26 和人乳腺癌细胞MCF-7 的细胞毒性。方法：采用PEI25KDa与DNA 复合制备polyplex,在polyplex 体系 中加入中性脂质体和SADGE 制备lipopolyplex。采用琼脂糖凝胶电泳考察lipopolyplex 对质粒DNA的包裹能力;采用激光粒度 仪和zeta 电位分析仪测定lipopolyplex 的粒径与zeta 电位;采用透射电镜观察lipopolyplex 的形态;采用CCK-8 试剂盒考察 lipopolyplex 对CT26和MCF-7 的细胞毒性。结果：琼脂糖凝胶电泳显示lipopolyplex 可以完全包裹质粒DNA;lipopolyplex 的粒 径在200 nm 左右,电位在-20 mV 左右;透射电镜下为较为规则的球状颗粒;lipopolyplex 在CT26 和MCF-7 细胞中的毒性明显 低于聚阳离子基因载体polyplex。结论：在polyplex 表面成功构建中性磷脂膜制备的lipopolyplex,可以完全的包裹DNA 并且细 胞毒性明显低于polyplex,在基因输送载体领域具有潜在应用价值。     

Development of novel poly(ethylene glycol)-based vehicles for gene delivery

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.     

Cholesterol-Peptide Hybrids to Form Liposome-Like Vesicles for Gene Delivery

In this paper, four amphiphilic cholesterol-peptide conjugates (Ch-R5H5, Ch-R3H3, Ch-R5 and Ch-R5) were designed and synthesized, and their properties in gene delivery were evaluated in vitro with an aim of developing more efficient gene delivery carriers. These amphiphilic cholesterol-peptide conjugates are composed of hydrophobic cholesterol and positively charged peptides. They were able to self-assemble into micelles at low concentrations and their critical micelle concentrations in phosphate buffered saline (pH 7.4) are ≤85 µg/mL. Amphiphilic cholesterol-peptide conjugates condensed DNA more efficiently than a hydrophilic cationic oligoarginine (R10) peptide with no hydrophobic segment. Their transfection efficiencies were at least two orders of magnitude greater than that of R10 peptide in HEK-293 cells. Moreover, the introduction of histidine residues in cholesterol-peptide conjugates led to higher gene expression efficiency compared with cholesterol-peptides without histidine (Ch-R5 and Ch-R3), and the luciferase expression level was comparable or even higher than that induced by PEI at its optimal N/P ratio. In particular, Ch-R5H5 condensed DNA into smaller nanoparticles than Ch-R3H3 at higher N/P ratios, and the minimum size of Ch-R5H5/DNA complexes was 180 nm with zeta potential of 23 mV, achieved at the N/P ratio of 30. This liposome-like vesicle may be a promising gene delivery carrier for intravenous therapy.     

Intracellular delivery of nanometric DNA particles via the folate receptor

The size of condensed DNA particles is a key determinant for both diffusion to target cells in vivo and intracellular trafficking. The smallest complexes are obtained when each DNA molecule collapses individually. This was achieved using a designed cationic thiol-detergent, tetradecyl-cysteinyl-ornithine (C(14)COrn). The resulting particles were subsequently stabilized by air-induced dimerization of the detergent into a disulfide lipid on the DNA template. Particles are anionic (zeta potential = -45 mV), and their size (30 nm) corresponds to the volume of a single plasmid DNA molecule. The electrophoretic mobility of the condensed DNA, though quasi-neutralized, was found higher than that of the extended DNA. Moreover, the dimerized (C(14)COrn)(2) lipid was found to be an efficient transfection reagent for various cell lines. In an attempt to achieve extended circulation times and to target tumors by systemic delivery, we have coated the particles with PEG-folate residues. Plasmid DNA was condensed into monomolecular particles as described above and coated by simple mixing with DPPE-PEG-folate. Physicochemical measurements showed particles coated with 2% of DPPE-PEG(3400)-folate remain monomolecular and are stable in the cell-culture medium. Caveolae-mediated cell entry was demonstrated by ligand-dependence, by competition with excess folic acid as well as by confocal microscopy.     

Biophysical properties and supramolecular structure of self-assembled liposome/ε-peptide/DNA nanoparticles: correlation with gene delivery

Using solid-phase synthesis, lysine can be oligomerized by a reaction of the peptide carboxylate with the ε-amino group to produce nontoxic, biodegradable cationic peptides, ε-oligo(L-lysines). Here α-substituted derivatives of such ε-oligo(L-lysines) containing arginine and histidine in the side chain were tested as vectors for in vitro gene delivery. Combination of ε-oligolysines with the cationic lipid DOTAP and plasmid DNA resulted in transfection efficiency exceeding that of DOTAP alone, without significant increase in cytotoxicity. Synchrotron small-angle X-ray scattering studies revealed self-assembly of the DOTAP, ε-oligolysines, and DNA to ordered lamellar complexes. High transfection efficiency of the nanoparticles correlates with increase in zeta potential above +20 mV and requires particle size to be below 500 nm. The synergistic effect of branched ε-oligolysines and DOTAP in gene delivery can be explained by the increase in surface charge and by the supramolecular structure of the DOTAP/ε-oligolysine/DNA nanoparticles.     

Enhancement of transfection efficiency by protamine in DDAB lipid vesicle-mediated gene transfer.

We have previously developed a simple gene transfection procedure mediated by cationic lipid vesicles for animal cells, in which a commercially available cationic surfactant, dimethyldioctadecyl ammonium bromide (DDAB), was used for making lipid vesicles. In the present study, we examined enhancement of transfection efficiency for this method by adding protamine to plasmid DNA solution before the formation of DNA/lipid vesicle complexes. Both free-base protamine and protamine sulfate provided enhanced transfection efficiency and expression level, but the optimal amount of the two protamines was different. The enhancement in transfection efficiency and expression level by protamines was observed in all the cell lines (COS-7, Hela, NIH3T3, MDCK, and BHK-21C13) and all the plasmids (pCMVbeta, pmiwZ, and pCH110) tested. The enhancement in both transfection efficiency and expression level was at most 20-fold compared with that using only DDAB lipid vesicles. Protamines seemed to protect DNA from degradation by DNase and promote DNA delivery into a nucleus.     

Synthesis of novel biodegradable cationic polymer: N,N-diethylethylenediamine polyurethane as a gene carrier

A new cationic polymer, N,N-diethylethylenediamine-polyurethane (DEDA-PU), bearing tertiary amines in the backbone and side chains, was synthesized and used as a nonviral vector for gene delivery. The DEDA-PU readily self-assembled with the plasmid DNA (pCMV-betagal) in water and buffer at physiological pH, as determined by agarose gel retardation, dynamic light scattering, zeta potential, atomic force microscopy (AFM), and restriction endonuclease protection assays. The results revealed that DEDA-PU was able to bind with plasmid DNA, yielding positively charged complexes with a size around 100 nm at a DEDA-PU/DNA ratio of 50/1 (w/w). The DEDA-PU/DNA complexes were able to transfect HEK 293 cells in vitro with an efficiency comparable to a well-known gene carrier [poly(2-dimethylaminoethyl methacrylate), PDMAEMA]. The cytotoxicity of DEDA-PU was substantially lower than PDMAEMA. The degradation studies indicated that DEDA-PU degrades hydrolytically in 20 mM HEPES buffer at pH 7.4 with a half-life of approximately 60 h. This study shows that DEDA-PU holds promise as biodegradable polycations for gene delivery and is interesting candidate for further study.     

Cationic liposome and plasmid DNA complexes formed in serum-free medium under optimum transfection condition are negatively charged

In medium where in vitro transfection is routinely performed, DC-chol liposomes alone were nearly neutral, whereas the DC-chol liposome/DNA complexes were largely negatively charged which changed only slightly at all [liposome]/[DNA] ratios (zeta=-27.1 to -21.8 mV). Three other commercial transfection reagents, Lipofectin(R), LipofectAMINE 2000, and SuperFect, were also largely negatively charged when complexed with DNA. The aggregation of liposomes in medium was prevented by the addition of DNA. Incubation of the complexes in medium did not change their size, charge or lipofection activity for 30 min. These results suggest that, in medium, the liposome/DNA complexes were formed at the time of mixing with negative charges.     

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.     

Cationic lipids with increased DNA binding affinity for nonviral gene transfer in dividing and nondividing cells

Effect of headgroup structure on catonic lipid-mediated transfection was investigated with either a (i) tertiary amine, (ii) quaternary amine with a hydroxyl, or (iii) quaternary amine with mesylate as headgroups. Liposomes were formulated using cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) as colipids, and transfection efficiencies were determined in rapidly dividing colon carcinoma (CT 26) and rat aortic smooth muscle (RASM) cells as well as in nondividing human pancreatic islets using luciferase and green fluorescent protein expression plasmids, pcDNA3-Luc and pCMS-EGFP, respectively. Liposome/pDNA complexes were evaluated for DNA conformational state by circular dichroism (CD), DNA condensation by electrophoretic mobility shift assay (EMSA), particle size and zeta potential by laser diffraction technique, and surface morphology by transmission electron microscopy (TEM). Encouraging transfection results were obtained with the mesylate headgroup based lipid in liposome formulations with DOPE as a colipid, which were higher than the commercially available Lipofectamine formulation. We hypothesize that the additional hydrogen bonding or covalent interactions of the headgroup with the plasmid DNA, leading to higher binding affinity of the cationic lipids to pDNA, results in higher transfection. This hypothesis is supported by TEM observations where elongated complexes were observed and more lipid was seen associated with the DNA.     

Layer-by-layer deposition of oppositely charged polyelectrolytes on the surface of condensed DNA particles.

DNA can be condensed with an excess of poly-cations in aqueous solutions forming stable particles of submicron size with positive surface charge. This charge surplus can be used to deposit alternating layers of polyanions and polycations on the surface surrounding the core of condensed DNA. Using poly-L-lysine (PLL) and succinylated PLL (SPLL) as polycation and polyanion, respectively, we demonstrated layer-by-layer architecture of the particles. Polyanions with a shorter carboxyl/backbone distance tend to disassemble binary DNA/PLL complexes by displacing DNA while polyanions with a longer carboxyl/backbone distance effectively formed a tertiary complex. The zeta potential of such complexes became negative, indicating effective surface recharging. The charge stoichiometry of the DNA/PLL/SPLL complex was found to be close to 1:1:1, resembling poly-electrolyte complexes layered on macrosurfaces. Recharged particles containing condensed plasmid DNA may find applications as non-viral gene delivery vectors.     

Use of the nuclease inhibitor aurintricarboxylic acid (ATA) for improved non-viral intratumoral in vivo gene transfer by jet-injection

BACKGROUND: Stability, integrity and retention of the DNA within the targeted tissue is decisive for efficient gene transfer using naked DNA. Pre-clinical and clinical studies require reproducible transfection rates by preventing rapid degradation of naked DNA in the transduced tissue. Tumor tissues contain nuclease activity, which can affect DNA stability if naked DNA is used. Therefore, inhibition of nuclease-mediated DNA degradation by the nuclease inhibitor aurintricarboxylic acid (ATA) might lead to improved gene transfer efficiency in tumor tissues. METHODS: For both, DNA-degradation analysis and in vivo gene transfer experiments, the beta-galactosidase (LacZ)-expressing pCMVbeta and the cytosine deaminase (CD)-expressing pCMV-CD plasmid were used. Influence of the nuclease inhibitor ATA was determined in tumors, in which naked pCMVbeta or pCMV-CD DNA and ATA was co-administered by jet-injection. The nuclease activity and inhibition by ATA was analyzed using the DNase Alert detection system. The influence of ATA on LacZ expression was determined by specific ELISA and its effect on the therapeutic efficacy of CD gene transfer on tumor growth was determined in vivo. RESULTS: The screening of different human mammary and colon carcinoma models revealed strong nuclease activity rapidly degrading naked plasmid DNA. Co-administration of ATA with pCMVbeta or pCMV-CD for in vivo jet-injection of tumors prevented DNA from nuclease degradation associated with either increased LacZ gene expression or improved reduction in tumor growth. CONCLUSIONS: Tumor-associated nuclease activity is a notable hurdle in gene transfer of naked DNA and therefore inhibition of nucleolytic degradation of plasmid DNA facilitates intratumoral gene expression.