Microencapsulation of nanoparticulate complexes of DNA with cationic lipids and polymers in pectin beads for targeted gene delivery

Nanoparticulate complexes of plasmid DNA (pDNA) with cationic liposomes/polymer, of approx 200 nm diameter, were encapsulated with a high degree of efficiency within calcium pectinate gel beads. Electron microscopy showed the DNA nanocomplexes to be evenly distributed throughout the gel matrix. Controlled release of pDNA-lipid nanocomplexes was achieved by the action of pectinase enzymes, whereas release of naked and polymer-complexed DNA was found to be more greatly influenced by the swelling behavior of the polysaccharide matrices in buffer alone. Physical degradation of pDNA within pectin beads was found to be accelerated during bead drying, most probably as a result of shear forces generated within the gel matrices by the evaporation of water. Plasmid complexation with cationic liposomes provided a greater degree of protection for the DNA during bead drying than complexation with cationic polymer, and was shown to successfully transfect cultured cells after release from the beads, via the action of pectinase. Observations concerning the physical stability of nanocomplexed pDNA, and its encapsulation within and release from pectin gel beads, are discussed with reference to the electrostatic interactions existing between the various components.     

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.     

Physicochemical and transfection properties of cationic Hydroxyethylcellulose/DNA nanoparticles

In this study the physicochemical and transfection properties of cationic hydroxyethylcellulose/plasmid DNA (pDNA) nanoparticles were investigated and compared with the properties of DNA nanoparticles based on polyethylene imine (PEI), which is widely investigated as a gene carrier. The two types of cationic hydroxyethylcelluloses studied, polyquaternium-4 (PQ-4) and polyquaternium-10 (PQ-10), are already commonly used in cosmetic and topical drug delivery devices. Both PQ-4 and PQ-10 spontaneously interact with pDNA with the formation of nanoparticles approximately 200 nm in size. Gel electrophoresis and fluorescence dequenching experiments indicated that the interactions between pDNA and the cationic celluloses were stronger than those between pDNA and PEI. The cationic cellulose/pDNA nanoparticles transfected cells to a much lesser extent than the PEI-based pDNA nanoparticles. The low transfection property of the PQ-4/pDNA nanoparticles was attributed to their neutrally charged surface, which does not allow an optimal binding of PQ-4/pDNA nanoparticles to cellular membranes. Although the PQ-10/pDNA nanoparticles were positively charged and thus expected to be taken up by cells, they were also much less efficient in transfecting cells than were PEI/pDNA nanoparticles. Agents known to enhance the endosomal escape were not able to improve the transfection properties of PQ-10/pDNA nanoparticles, indicating that a poor endosomal escape is, most likely, not the major reason for the low transfection activity of PQ-10/pDNA nanoparticles. We hypothesized that the strong binding of pDNA to PQ-10 prohibits the release of pDNA from PQ-10 once the PQ-10/pDNA nanoparticles arrive in the cytosol of the cells. Tailoring the nature and extent of the cationic side chains on this type of cationic hydroxyethylcellulose may be promising to further enhance their DNA delivery properties.     

Biophysical characterization of the DNA binding and condensing properties of adenoviral core peptide mu.

Cationic peptides containing Lys and Arg residues interact with DNA via charge-charge interactions and are known to play an important role in DNA charge neutralization and condensation processes. In this paper, we describe investigations of the interaction of the cationic adenovirus core complex peptide mu with a dodecameric ODN (12 bp) and pDNA (7528 bp) using a combination of fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and photon correlation spectroscopy. Comparisons are made with protamine, a cationic peptide well-known for DNA charge neutralization and condensation. Equilibrium dissociation constants are derived independently by both CD and ITC methods for the interaction between protamine or mu with pDNA (K(d) = 0.6-1 microM). Thermodynamic data are also obtained by ITC, indicating strong charge-charge interactions. The interaction of protamine with pDNA takes place with decreasing entropy (-28.7 cal mol(-1) K(-1)); unusually, the interaction of mu with pDNA takes place with increasing entropy (Delta S degrees (bind) = 11.3 cal mol(-1) K(-1)). Although protamine and mu appear to destabilize pDNA double helix character to similar extents, according to CD thermal titration analyses, PCS studies show that interactions between mu and pDNA result in the formation of significantly more size-stable condensed particles than protamine. The enhanced flexibility and size stability of mu-DNA (MD) particles (80-110 nm) compared to protamine counterparts suggest that MD particles are ideal for use as a part of new nonviral gene delivery vectors.     

Poly(hydroxyethyl methacrylate) based affinity cryogel for plasmid DNA purification

The aim of this study is to prepare supermacroporous pseudospecific cryogel which can be used for the purification of plasmid DNA (pDNA) from bacterial lysate. N-methacryloyl-(l)-histidine methyl ester (MAH) was chosen as the pseudospecific ligand and/or comonomer. Poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-histidine methyl ester) [PHEMAH] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. Compared with the PHEMA cryogel (50 μg/g polymer), the pDNA adsorption capacity of the PHEMAH cryogel (13,350 μg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The amount of pDNA bound onto the PHEMAH cryogel disks first increased and then reached a saturation value (i.e., 13,350μg/g) at around 300 μg/ml pDNA concentration. pDNA adsorption amount decreased from 1137 μg/g to 160 μg/g with the increasing NaCl concentration. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 90%. The PHEMAH cryogel could be used 3 times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH cryogel disks promise high selectivity for pDNA.     

Plasmid DNA purification

The demand for efficient production methods of plasmid DNA (pDNA) has increased vastly in response to rapid advances in the use of pDNA in gene therapy and in vaccines since the advantageous safety concerns associated with non-viral over viral vectors.A prerequisite for the success of plasmid-based therapies is the development of cost-effective and generic production processes of pDNA. However, to satisfy strict regulatory guidelines, the material must be available as highly purified, homogeneous preparations of supercoiled circular covalently closed (ccc) pDNA. Large-scale production of pDNA for therapeutic use is a relatively new field in bioprocessing. The shift from small-scale plasmid production for cell transfection to large-scale production sets new constraints on the bacterial fermentation, processing of bacterial lysate and final purification and formulation of the plasmid DNA. The choice of bacterial strain used for plasmid cultivation affects the plasmid yield, the proportion of different isoforms and the amount of endotoxins in the starting material. The choice of bacterial strain will be greatly influenced by the production and purification procedures of pDNA. Master and working cell banks need to be characterised and established. Alkaline lysis of the bacteria damages the pDNA, resulting in a reduced recovery of ccc pDNA and an increase in partially denaturated ccc pDNA and open circular (oc) forms. Shear stress in these processes needs to be tightly controlled, and buffer composition and pH need to be optimised. To obtain a homogeneous plasmid DNA preparation, different pDNA purification strategies aim at capturing ccc pDNA and eliminating the oc isoform. A highly purified final product corresponding to the stringent recommendations set forth by health and regulatory authorities can be achieved by (i). different chromatography techniques integrated with ultra/diafiltration to achieve optimal purification results; (ii). the formulation of the final pDNA product, that requires a detailed study of the plasmid structure; and (iii). the development of sensitive analytical methods to detect different impurities (proteins, RNA, chromosomal DNA, and endotoxins). We present here a revue of the whole process to obtain such a plasmid DNA, and report an example of RNAse-free purification of ccc pDNA that could be used for gene therapy.     

High efficient encapsulation of plasmid DNA in PLGA microparticles by organic phase self-emulsification

To overcome the drawbacks of encapsulating plasmid DNA (pDNA) in poly (D,L-lactic-co-glycolic acid) (PLGA) by water-in-oil-in-water double-emulsion solvent-evaporation method, we have developed a novel procedure for encapsulating pDNA in PLGA microparticles called DNA organic phase self-emulsification (DOPSM). This method was based on both the extraction plasmid DNA from aqueous phase into organic phase and the spontaneous emulsification DNA in organic phase by solvent diffusion method. The efficiency of extraction plasmid DNA into organic phase is 99% and the concentration of pDNA in organic phase is up to 2.4 mg/ml. The efficiency of microencapsulation of plasmid DNA in PLGA is up to 76% and can be enhanced by lowering the pH of aqueous solution of emulsion. The microparticles size of PLGA of pDNA is in a narrow range of 1-2 microm. This procedure does not involve the high mechanical energy to emulsify which may damage the integrity of pDNA. This method can be applied to encapsulate the pDNA into microparticles of other biocompatible polymers with high efficiency.     

Simian virus 40 DNA replication: characterization of gaps in the termination region.

A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.     

Gene delivery using biodegradable polyelectrolyte microcapsules prepared through the layer-by-layer technique

Biodegradable and non‐biodegradable microcapsules were prepared via the layer‐by‐layer (LbL) technique consisting of the polyelectrolyte pairs of dextran sulphate/poly‐L ‐arginine and poly(styrene sulfonate)/poly(allylamine hydrochloride), respectively, in an attempt to encapsulate plasmid DNA (pDNA) for efficient transfection into NIH 3T3 cells. Results indicated the retention of bioactivity in the encased pDNA, as well as a correlation between the level of in vitro gene expression and biodegradability properties of polyelectrolyte. Furthermore, the incorporation of iron oxide nanoparticles within the polyelectrolyte layers significantly improved the in vitro transfection efficiency of the microcapsules. As a novel pDNA delivery system, the reported biodegradable microcapsules provide useful insight into plasmid‐based vaccination and where there is a prerequisite to deliver genes into cells capable of phagocytosis. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1088–1094, 2012     

Circular dichroism investigation of the effect of plasmid DNA structure on retention in histidine chromatography

The effect of temperature-induced changes in secondary and tertiary structures of plasmid DNA (pDNA) and on the retention behaviour of open circular (oc) and supercoiled (sc) isoforms in histidine-agarose chromatography was investigated by Circular dichroism (CD) spectroscopy. Chromatographic experiments performed with three plasmids (2.7, 6.1 and 7.4 kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0 M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 °C. Such behaviour was attributed to the temperature-induced removal of negative superhelical turns in sc pDNA which is accompanied by a decrease in the number of dissociated base pairs responsible for interaction with the histidine ligands. CD spectroscopy showed that temperature has an important effect on plasmid secondary structure if adenine-rich inserts are present in the plasmid structure. Chromatographic experiments also suggested that base composition could also be responsible for the induction of specific interactions with histidine ligands.     

阴离子交换晶胶层析分离质粒DNA

质粒DNA(pDNA)作为重要的基因治疗药物载体,其广泛应用受纯度和产量的限制。为了获得高纯度的pDNA,首先制备超大孔连续床晶胶基质,接枝二乙氨基乙基葡聚糖得到阴离子交换型晶胶介质;然后以pUC19质粒为例,将目标质粒转化至大肠杆菌,培养收集,碱液裂解和离心;最后用阴离子交换型晶胶介质从离心上清液中一步法层析分离pDNA。通过优化层析过程的pH值和洗脱条件,最终在pH值为6.6时,用0.5 mol/L的NaCl溶液洗脱,得到较高纯度的pDNA。整个分离过程中不使用动物源性酶,也不需常规分离中的高毒试剂,使获得pDNA的过程和产物更加安全。     

Evaluation of the high-pressure extrusion technique as a method for sizing plasmid DNA-containing cationic liposomes

A promising strategy to improve the immunogenic potential of DNA vaccines is the formulation of plasmid DNA (pDNA) with cationic liposomes. In this respect, particle size may be of crucial importance. This study aimed at the evaluation of high-pressure extrusion as a method for sizing cationic liposomes after entrapment of pDNA. This is a well-known sizing method for liposomes, but so far, it has not been applied for liposomes that are already loaded with pDNA. Liposomes composed of egg PC, DOTAP, and DOPE with entrapped pDNA were prepared by the dehydration-rehydration method and subjected to various extrusion cycles, comparing different membrane pore sizes and extrusion frequencies. At optimized extrusion conditions, liposome diameter (Zave) and polydispersity index (PDI) were reduced from 560 nm and 0.56-150 nm and 0.14 respectively, and 35% of the pDNA was retained. Importantly, gel electrophoresis and transfection experiments with pDNA extracted from these extruded liposomes demonstrated the preservation of the structural and functional integrity of the pDNA. The reduction in size resulted in enhanced transfection of HeLa cells, as detected by functional expression of the fluorescent protein, eGFP. In addition, these liposomes were able to stimulate Toll-like receptor 9, indicating efficient endosomal uptake and release of the included pDNA. In conclusion, high-pressure extrusion is a suitable technique to size cationic liposomes with entrapped pDNA and allows preparation of well-defined nanosized pDNA-liposomes, with preserved pDNA integrity. Their improved transfection efficiency and ability to activate an important pattern-recognition receptor are favorable properties for DNA vaccine delivery vehicles.     

Temperature-dependent selective purification of plasmid DNA using magnetic nanoparticles in an RNase-free process

Carboxyl group-functionalized magnetic nanoparticles were used to develop an RNase-free method for plasmid DNA (pDNA) purification directly from RNA-containing crude Escherichia coli lysates. This method takes advantage of differing adsorption behaviors of pDNA and RNA onto magnetic nanoparticle surfaces at different temperatures. Pure pDNA can be isolated between 70 and 80 °C without sacrificing DNA quality and quantity, as evidenced by comparison with that obtained using organic solvents or commercial kits. This RNase-free method is rapid, simple, cost-effective, and environmentally friendly, and it can be easily scaled up for the production of pharmacological-grade pDNA.     

Formulation and in vitro transfection efficiency of poly (D, L-lactideco-glycolide) microspheres containing plasmid DNA for gene delivery

The stability, in vitro release, and in vitro cell transfection efficiency of plasmid DNA (pDNA) poly (D,L.-lactide-co-glycolide) (PLGA) microsphere formulations were investigated. PLGA microspheres containing free and polylysine (PLL)-complexed pDNA were prepared by a water-oil-water solvent extraction/evaporation technique. Encapsulation enhanced the retention of the supereoiled structure of pDNA as determined by gel electrophoresis. PLL complexation of pDNA prior to encapsulation increased both the stability of the supercoiled form and the encapsulation efficiency. Free pDNA was completely degraded after exposure to DNase while encapsulation protected the pDNA from enzymatic degradation. Rapid initial in vitro release of pDNA was obtained from microspheres containing free pDNA. while the release from microspheres containing PLL-complexed pDNA was sustained for more than 42 days. Bioactivity of encapsulated pDNA determined by in vitro cell transfection using Chinese hamster ovary cells (CHO) showed that the bioactivity of encapsulated pDNA was retained in both formulations but to a greater extent with PLL-complexed pDNA microspheres. These results demonstrated that PLGA microspheres could be used to formulate a controlledrelease delivery system for pDNA that can protect the pDNA from DNase degradation without loss of functional activity.     

Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.     

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels.     

Creating a unique environment for selecting reactive enzymes with DNA: 'Sticky' binding of oligocation-grafted polymers to DNA

To provide colloidally stable polyplexes formed between pDNA and cationic polymers, cationic polymers have been modified with hydrophilic polymers to form a hydrophilic shell. Block copolymers of cationic and hydrophilic polymers and cationic polymers grafted with hydrophilic polymers are representative designs of such polymers. Here, we report a new design of cationic polymers and oligocationic peptide-grafted polymers. We synthesized 15 kinds of graft copolymers by varying the number of cationic charges of the peptides and their grafting density. We found that graft copolymers with less cationic peptides and less grafting density formed colloidally stable polyplexes. Interestingly, the less cationic graft copolymers bind to excess amounts of pDNA. We also found that the graft copolymers showed selectivity toward reactive enzymes affording the reaction of pDNA with nucleases, while suppressing both the replication of DNA by DNA polymerase and gene expression. The suppression of the replication and expression is considered to result from the high capacity of the graft copolymers for binding with pDNA. The polynucleotides produced by DNA polymerase or RNA polymerase would be captured by the graft copolymers to impede these enzymatic reactions.     

Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA

Reconstituted influenza virosomes (virus membrane envelopes) have been used previously to deliver pDNA (plasmid DNA) bound to their external surface to a variety of target cells. Although high transfection efficiencies can be obtained with these complexes in vitro, the virosome-associated DNA is readily accessible to nucleases and could therefore be prone to rapid degradation under in vivo conditions. In the present study, we show a new method for the production of DNA-virosomes resulting in complete protection of the DNA from nucleases. This method relies on the use of the short-chain phospholipid DCPC (dicaproylphosphatidylcholine) for solubilization of the viral membrane. The solubilized viral membrane components are mixed with pDNA and cationic lipid. Reconstitution of the viral envelopes and simultaneous encapsulation of pDNA is achieved by removal of the DCPC from the mixture through dialysis. Analysis by linear sucrose density-gradient centrifugation revealed that protein, phospholipid and pDNA physically associated to particles, which appeared as vesicles with spike proteins inserted in their membranes when analysed by electron microscopy. The DNA-virosomes retained the membrane fusion properties of the native influenza virus. The virosome-associated pDNA was completely protected from degradation by nucleases, providing evidence for the DNA being highly condensed and encapsulated in the lumen of the virosomes. DNA-virosomes, containing reporter gene constructs, transfected a variety of cell lines, with efficiencies approaching 90%. Transfection was completely dependent on the fusogenic properties of the viral spike protein haemagglutinin. Thus, DNA-virosomes prepared by the new procedure are highly efficient vehicles for DNA delivery, offering the advantage of complete DNA protection, which is especially important for future in vivo applications.     

Proliferating cell nuclear antigen loaded onto double-stranded DNA: dynamics, minor groove interactions and functional implications

Proliferating cell nuclear antigen (PCNA) acts as a biologically essential processivity factor that encircles DNA and provides binding sites for polymerase, flap endonuclease-1 (FEN-1) and ligase during DNA replication and repair. We have computationally characterized the interactions of human and Archaeoglobus fulgidus PCNA trimer with double-stranded DNA (ds DNA) using multi-nanosecond classical molecular dynamics simulations. The results reveal the interactions of DNA passing through the PCNA trimeric ring including the contacts formed, overall orientation and motion with respect to the sliding clamp. Notably, we observe pronounced tilting of the axis of dsDNA with respect to the PCNA ring plane reflecting interactions between the DNA phosphodiester backbone and positively charged arginine and lysine residues lining the PCNA inner surface. Covariance matrix analysis revealed a pattern of correlated motions within and between the three equivalent subunits involving the PCNA C-terminal region and linker strand associated with partner protein binding sites. Additionally, principal component analysis identified low frequency global PCNA subunit motions suitable for translocation along duplex DNA. The PCNA motions and interactions with the DNA minor groove, identified here computationally, provide an unexpected basis for PCNA to act in the coordinated handoff of intermediates from polymerase to FEN-1 to ligase during DNA replication and repair.