Herre Talsma

Effect of Size and Serum Proteins on Transfection Efficiency of Poly ((2-dimethylamino)ethyl Methacrylate)-Plasmid Nanoparticles

AbstractPurpose. The aim of this study was to gain insight into the relation between the physical characteristics of particles formed by a plasmid and a synthetic cationic polymer (poly(2-dimethylamino)ethyl methacrylate, PDMAEMA) and their transfection efficiency. Methods. The PDMAEMA-plasmid particles were characterized by dynamic light scattering (size) and electrophoretic mobility measurements (charge). The transfection efficiency was evaluated in cell culture (COS-7 cells) using a pCMV-lacZ plasmid coding for β-galactosidase as a reporter gene. Results. It was shown that the optimal transfection efficiency was found at a PDMAEMA-plasmid ratio of 3 (w/w), yielding stable and rather homogeneous particles (diameter 0.15 µm) with a narrow size distribution and a slightly positive charge. Particles prepared at lower weight ratios, showed a reduced transfection efficiency and were unstable in time as demonstrated by DLS measurements. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. Interestingly, the transfection efficiency of the particles was not affected by the presence of serum proteins. Conclusions. PDMAEMA is an interesting vector for the design of in vivo and ex vivo gene transfection systems.

2-(dimethylamino)ethyl methacrylate based (co)polymers as gene transfer agents

Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is able to bind to DNA by electrostatic interactions. At a polymer/plasmid ratio above 2 (w/w) positively charged complexes were formed with a size around 0.2 microm. The transfection efficiency of polymer/plasmid complexes was evaluated in cell culture (COS-7 and OVCAR-3 cells) using a pCMV-lacZ plasmid, encoding for beta-galactosidase, as a reporter gene. The optimal transfection efficiency was found at a PDMAEMA/plasmid ratio of 3-5 (w/w). Under these conditions 3-6% of the cells were actually transfected. Like other cationic polymers, PDMAEMA is slightly cytotoxic. This activity was partially masked by complexing the polymer with DNA. A pronounced effect of the molecular weight of the polymer on the transfection efficiency was observed. An increasing molecular weight resulted in an increasing number of transfected cells. Dynamic light scattering experiments showed that high molecular weight polymers (Mw>300 kDa) were able to condense DNA effectively (particle size 0.15-0.20 microm). In contrast, when plasmid was incubated with low molecular weight PDMAEMA, large complexes were formed (size 0.5-1.0 microm). Copolymers of DMAEMA with methyl methacrylate (MMA), ethoxytriethylene glycol methacrylate (triEGMA) or N-vinyl-pyrrolidone (NVP) also acted as transfection agents. A copolymer with 20 mol % of MMA showed a reduced transfection efficiency and a substantial increased cytotoxicity compared with a homopolymer of the same molecular weight. A copolymer with triEGMA (48 mol %) showed both a reduced transfection efficiency and a reduced cytotoxicity, whereas a copolymer with NVP (54 mol %) showed an increased transfection efficiency and a decreased cytotoxicity as compared to a DMAEMA homopolymer.

Relation between transfection efficiency and cytotoxicity of poly(2-(dimethylamino)ethyl methacrylate)/plasmid complexes

Abstract Poly(2-(dimethylamino)ethyl methacrylate), p(DMAEMA), with varying molecular weight was synthesized, complexed with pCMV-LacZ plasmid and evaluated as transfection agent in COS-7 and OVCAR-3 cells. Both the transfection efficiency and cell viability were monitored after two days culturing. The transfection efficiency of p(DMAEMA)/plasmid complexes was studied as a function of polymer/plasmid (w/w) ratio and compared with two known carriers: poly( l -lysine) and DEAE dextran. For all polymeric carriers a maximum in transfection efficiency was found when cell viability was 40–70%. This maximum was found at polymer/plasmid ratio 5/1, 6/1–13/1 and 50/1–200/1 (w/w) for poly( l -lysine), p(DMAEMA) and DEAE dextran respectively. The transfection efficiency of p(DMAEMA)/plasmid complexes was two-fold higher than DEAE dextran/plasmid complexes and eight times higher than poly( l -lysine)/plasmid complexes, although the carrier/plasmid complexes had about the same physicochemical characteristics. This indicates that differences in the transfection efficiency can be ascribed to differences in structural properties of the transfectant. At a sub-optimum p(DMAEMA)/plasmid ratio (5/1, w/w) a tenfold increase in number of transfected cells (after 48 h culturing) was found with increasing incubation time of the cells with plasmid (from 5 to 240 min) and was associated with a high number of viable cells (75–100%). On the other hand, at high polymer/plasmid ratio (20/1, w/w) the transfection efficiency passed through a maximum after 30 min incubation. Longer incubation times resulted in a substantial reduction of living cells. The same trends were observed in the presence of chloroquine and serum. However, the viability of the cells was substantially reduced, probably due to the toxicity of chloroquine. Transfection efficiency was also studied as a function of p(DMAEMA) molecular weight. Polymers with molecular weight >300 kDa were better transfection agents in both COS-7 and OVCAR-3 cells than low molecular weight polymers. Dynamic light scattering measurements showed that high molecular weight polymers were able to condense DNA effectively resulting in particles of 0.17–0.21 μm. In contrast, when plasmid was incubated with low molecular weight p(DMAEMA), large complexes were formed (size of approximately 1.0 μm). Obviously, smaller complexes have an advantage over larger polymer/plasmid complexes in cell entry. Taken together the results of transfection efficiency and cytotoxicity, we hypothesize that complexes enter cells by membrane destabilization, either at the cell surface or within vesicles.

Controlled release of proteins from dextran hydrogels

Dextran hydrogels were investigated as matrices for the controlled release of proteins. The hydrogels were prepared by a free radical polymerization of aqueous solutions of glycidyl methacrylate derivatized dextran (dex-GMA). The release of three model proteins (lysozyme, BSA and IgG) from hydrogels varying in water content and degree of GMA-substitution was studied. The release rate was dependent on the size of the proteins and the equilibrium water content of the gels. It was shown that the release of the proteins was independent of the degree of GMA substitution of gels at high equilibrium water contents. On the other hand, the release was strongly affected by the degree of GMA substitution of the gels at low water contents. Some of these gels did not show any significant protein release, which suggests that the hydrogel mesh size was smaller than the protein diameter. In hydrogels where no screening occurred, the diffusion of the proteins could be effectively described by the free volume theory. Hydrogel mesh sizes were estimated from swelling data using the Flory-Rehner theory. This approach, however, resulted in an underestimation of the actual hydrogel mesh as derived from release experiments. Possible explanations for this discrepancy are discussed.

Dextran hydrogels for the controlled release of proteins

Abstract The release of proteins from non-degrading and enzymatically degrading dextran hydrogels was investigated. These dextran hydrogels were obtained by radical polymerization of aqueous solutions of glycidyl methacrylate derivatized dextran (dex-GMA). It was shown by FTIR analysis that by a proper selection of the polymerization conditions, hydrogels could be obtained in which more than 95% of the methacrylate groups had reacted within 20 min. The release of three model proteins (lysozyme, albumin, IgG) from non-degrading dextran gels with a varying initial water content and crosslink density was studied. For gels with a high equilibrium water content, the amount of protein released was proportional to the square root of time. The diffusion of proteins in these highly hydrated gels could be effectively described by the free volume theory. On the other hand, the release of the proteins from hydrogels with a low hydration level was marginal and did not follow the free volume theory, indicating that in these gels screening occurred. Dextran hydrogels can be made degradable by coencapsulation of dextranase. The degradation rate could be established by determination of reducing oligosaccharides and was dependent on the amount of dextranase in the gel. The release of a model protein (IgG) from degrading dextran hydrogels was investigated as a function of the amount of dextranase present in the gel and the gel characteristics. In the absence of dextranase no significant release of IgG from the hydrogels occurred, indicating that the protein diameter is larger than the hydrogel mesh size. However, in the presence of dextranase the release of IgG from the gels abruptly increased after a certain time when the enzyme had degraded the hydrogel to a certain extent.

Polyurethane-based drug delivery systems

Polyurethanes (PUs) are formed by a reaction between isocyanates and diols to yield polymers with urethane bonds (-NH-COO-) in their main chain. A great variety of building blocks is commercially available that allows the chemical and physical properties of PUs to be tailored to their target applications, particularly for the biomedical and pharmaceutical fields. This article reviews the synthesis and characterization of PUs and PU-copolymers, as well as their in vitro and in vivo biodegradability and biocompatibility. Particular emphasis is placed on the use of PUs for the controlled release of drugs and for the (targeted) delivery of biotherapeutics.

Effect of DNA topology on the transfection efficiency of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes.

In this paper the effect of the topology of plasmid DNA (supercoiled, open-circular and linear) on its binding characteristics with the polymeric transfectant poly((2-dimethylamino)ethyl methacrylate) was studied. The formed polyplexes were also evaluated for their transfection properties in vitro in two different cell lines. Anion-exchange chromatography was used for the separation of supercoiled and open-circular plasmid from a plasmid stock solution. Linear plasmids were prepared by endonucleases that cleaved the plasmid either in the promoter region or in a region not specific for expression (ampicillin resistance region). Plasmid DNA was also heat-denatured for 6 h at 70 degrees C, resulting in DNA mainly in the open-circular and oligomeric forms. The transfection of two different cell lines was dependent on the topology of the DNA in the order supercoiled>open-circular approximately heat-denatured>linear DNA prepared by cleaving in the nonspecific region>linear DNA prepared by cleaving in the promoter region. No differences in the size of the complexes or in the quenching of the DNA-intercalating fluorophore acridine orange were found as function of the topology. However, circular dichroism spectroscopy revealed differences between the topological plasmid species, both in the free form and in the presence of excess of cationic polymer.

Characterization of liposomes. The influence of extrusion of multilamellar vesicles through polycarbonate membranes on particle size, particle size distribution and number of bilayers

Abstract The influence of extrusion on number of bilayers, particle size (ps) and particle size distribution (psd) of multilamellar vesicles (composition on a molar basis: phosphatidylcholine/phosphatidylserine/cholesterol, 10:1:4) is investigated. Ps and psd are investigated with freeze-fracture electron microscopy, average number of bilayers 〈 N 〉, with small angle X-ray scattering and 31 P-NMR. The encapsulated volume ( E ) is determined using carboxyfluorescein as marker. From E and the volume/surface ratio of the liposomes, determined from the psd, 〈 N 〉 was also calculated. Determined and calculated number of bilayers are in reasonable agreement. Extrusion reduces 〈 N 〉.

Stabilization of gene delivery systems by freeze-drying

Freeze-drying of three different forms of gene delivery systems was performed using a controlled two-step drying process and 10% sucrose as lyoprotectant. Complexes of pCMVL plasmid with transferrin-conjugated polyethylenimine, adenovirus-enhanced transferrinfection consisting of pCMVL/transferrin-polylysine complexes linked to inactivated adenovirus particles, and a recombinant, E1-defective adenovirus expressing a luciferase reporter gene were tested. Three weeks after freeze-drying the reagents were rehydrated with water and tested for transfection activity. Luciferase gene expression levels were retained at high levels in all three systems, in contrast to reagents stored in solution. The use of the lyoprotectant was essential. In the absence of sucrose the transfection activities dropped by a factor of 100-1000. The data suggest freeze-drying as a useful method for stabilization and storage of standardized batches of transfection agents.

Formation of dextran hydrogels by crystallization

In this paper, a novel method is presented for the preparation of dextran hydrogels and microspheres, based on crystallization. Although dextrans are known to be well soluble in water, precipitation was observed in concentrated aqueous solutions of low molecular weight dextran (dextran 6000), whereas for solutions of dextran with higher molecular weights (dextran 40,000 and 220,000) no precipitation was observed in the time-frame studied. The kinetics of the precipitation process were studied and showed that precipitation was faster when more concentrated dextran solutions were used. Furthermore, the precipitation process was accelerated by stirring and by the presence of salts. Depending on the precipitation time, microspheres or gels were obtained. The precipitates were insoluble in water at room temperature, but readily dissolved in boiling water or DMSO. IR spectroscopy and (modulated) differential scanning calorimetry ((M)DSC) demonstrated that the precipitates were crystalline. We hypothesize that crystallization is due to association of the chains through hydrogen bonding, induced by the large polymer/water ratio in concentrated dextran 6000 solutions.