Copolymers of 2-(dimethylamino)ethyl methacrylate with ethoxytriethylene glycol methacrylate or N-vinyl-pyrrolidone as gene transfer agents.

Random copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate (triEGMA) or N-vinylpyrrolidone (NVP) of different molecular weights and compositions were synthesized, characterized and evaluated as polymeric transfectants in vitro. All synthesized copolymers (comonomer fraction up to 66 mol%) were able to bind to DNA, yielding polymer-plasmid complexes (polyplexes). However, the polymer-plasmid ratio at which small complexes (size 0.2-0.3 microm) were formed, increased with increasing mole fraction of the comonomer. zeta-Potential measurements revealed that the polymer-plasmid ratio where charge neutralization of DNA occurred, increased with increasing mole fraction of triEGMA. The cytotoxicity of the copolymers, either complexed with DNA or in the free form, decreased with increasing mole fraction of both comonomers (triEGMA and NVP). This reduction was even more than what could be expected based on the DMAEMA mole fraction in the copolymer. The copolymers with a molecular weight up to 170¿ omitted¿000 had the same transfection capability as a homopolymer of comparable molecular weight. However, higher molecular weight copolymers showed a reduced transfection capability compared to the homopolymer, which was ascribed to the reduced capability to condense the size of plasmid. Transfection efficiency of polyplexes composed of copolymers with a low triEGMA content increased with increasing molecular weight. Although the copolymers with 50 mol% triEGMA were also better transfectants than the homopolymer, the transfection efficiency did not increase further with increasing molecular weight. Interestingly, NVP-DMAEMA copolymers synthesized by polymerization to high conversion showed both excellent DNA binding and condensing characteristics (polyplex size <0.3 microm) and transfection capabilities. This is ascribed to a synergistic effect of DMAEMA-rich copolymers and NVP-rich copolymers present in this system on the complex formation with plasmid DNA.

Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery.

A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.3 micron and a slightly positive zeta-potential, which can be taken up by cells, e.g., via endocytosis. However, the transfection efficiency and the cytotoxicity of the polymers differed widely: the highest transfection efficiency and cytotoxicity were observed for poly[2-(dimethylamino)ethyl methacrylate], p(DMAEMA). Assuming that polyplexes enter cells via endocytosis, p(DMAEMA) apparently has advantageous properties to escape the endosome. A possible explanation is that, due to its average pK(a) value of 7.5, p(DMAEMA) is partially protonated at physiological pH and might behave as a proton sponge. This might cause a disruption of the endosome, which results in the release of both the polyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol. On the other hand, the analogues of p(DMAEMA) studied here have a higher average pKa value and have, consequently, a higher degree of protonation and a lower buffering capacity. This might be associated with a lower tendency to destabilize the endosome, resulting in both a lower transfection efficiency and a lower cytotoxicity. Furthermore, molecular modeling showed that, of all studied polymers, p(DMAEMA) has the lowest number of interactions with DNA. We therefore hypothesized that the superior transfection efficiency of p(DMAEMA) containing polyplexes can be ascribed to an intrinsic property of p(DMAEMA) to destabilize endosomes combined with an easy dissociation of the polyplex once present in the cytosol and/or the nucleus.

A versatile method for the conjugation of proteins and peptides to poly[2-(dimethylamino)ethyl methacrylate].

Random copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) with aminoethyl methacrylate (AEMA) were synthesized by radical polymerization. The amount of incorporated primary amino groups could be controlled by the feed ratio of AEMA to DMAEMA, and was varied from 2 to 6 mol %. Subsequently, protected thiol groups were introduced in a derivatization step with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and subsequent treatment with dithiothreitol (DTT). The obtained thiolated p(DMAEMA-co-AEMA) was conjugated to transferrin (Tf) or the F(ab') fragment of mAb 323/A3 via a disulfide linkage. Moreover, the maleimide derivative of the nuclear localization signal (NLS) decapeptide Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Asp-NH(2) was coupled to the thiolated polymer via a thioether linkage. The coupling efficiency, as determined by GPC (Tf), SDS-PAGE [F(ab')], or (1)H NMR (NLS peptide) was 90-95% for the Tf conjugate, and more than 95% for the F(ab') conjugate and the NLS conjugate. The synthetic strategy described in this paper is a universal method for the preparation of conjugates of proteins and peptides with pDMAEMA in particular. This method can possibly be used to synthesize protein-polymethacrylate conjugates in general.

Thermosensitive polymers as carriers for DNA delivery.

Copolymers of 2-(dimethylamino) ethyl methacrylate (DMAEMA) and N-isopropylacryl amide (NIPAAm) of various monomer ratios and molecular weights were evaluated as carrier systems for DNA delivery. All copolymers, even with a low DMAEMA content of 15 mol%, were able to bind to DNA at 25 degrees C. Light-scattering measurements indicate that complexation is accompanied by precipitation of the (co)polymer in the complex caused by a drop of the lower critical solution temperature of the (co)polymer. The (co)polymer/plasmid ratio at which complexes with a size of around 200 nm were formed increased with increasing NIPAAm content of the copolymer and was independent of molecular weight of the (co)polymer. However, complexes containing (co)polymers of low molecular weight or high NIPAAm content prepared at 25 degrees C aggregated rapidly when the temperature was raised to 37 degrees C, whereas complexes containing (co)polymers of high molecular weight or lower NIPAAm content were relatively stable at 37 degrees C. The zeta potential of the complexes was also independent of molecular weight of the (co)polymer and increased with increasing (co)polymer/plasmid ratio until a plateau value was reached. The (co)polymer/plasmid ratio at which this plateau was reached increased with increasing NIPAAm content. The plateau values decreased from around 26 mV to around 13 mV when the NIPAAm content of the copolymer was increased from 0 to 85 mol%. The cytotoxicity of the complexes strongly decreased with increasing NIPAAm content and was independent of molecular weight of the (co)polymer. The transfection efficiency of complexes with poor stability was in general much lower than that of complexes with good stability. The transfection efficiency as a function of the (co)polymer/plasmid ratio showed a bell-shaped curve. The (co)polymer/plasmid ratio at which the transfection efficiency was maximal increased with increasing NIPAAm content, while the maximum transfection efficiency strongly decreased with increasing NIPAAm content of the copolymer. The results of this study show that the formation of stable (co)polymer/plasmid complexes with a size of around 200 nm is a prerequisite for efficient transfection. Furthermore, the transfection efficiency and cytotoxicity strongly decreased with decreasing zeta potential. Therefore, besides the size, the zeta potential can also be used as a characteristic to predict the behavior of this type of (co)polymer/plasmid complexes in transfection. Copolymers of DMAEMA and NIPAAm provided with a homing device may be interesting carrier systems for gene targeting because these copolymers can condense DNA to small particles, and the resulting complexes show a low cytotoxicity and aspecific transfection.

Comparative transfection studies of human ovarian carcinoma cells in vitro, ex vivo and in vivo with poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes.

BACKGROUND: Poly(2-(dimethylamino)ethyl methacrylate) (p(DMAEMA)) can be used successfully for in vitro transfection of different cell lines, including the OVCAR-3 human ovarian carcinoma cell line. The aim of this study was to investigate whether it is possible to transfect OVCAR-3 cells in vivo with polyplexes containing p(DMAEMA). METHODS: In order to understand the generally observed gap between in vitro and in vivo transfection, we gradually went from in vitro to in vivo transfection of OVCAR-3 cells, while keeping the exposure conditions the same, as far as possible. To find the reason for the negligible degree of in vivo transfection, in vitro cultured OVCAR-3 cells were transfected in the presence of peritoneal ascites fluid. Next, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. RESULTS: P(DMAEMA)-containing polyplexes can transfect OVCAR-3 cells in vitro with an overall transfection efficiency of 10%. Cells grown in vivo can be transfected ex vivo with p(DMAEMA)/plasmid complexes with an overall transfection efficiency of approximately 1-2%. When transfection complexes are injected i.p. into nude mice bearing OVCAR-3 cells in the peritoneal cavity, the degree of in vivo transfection efficiency achieved is negligible. In vitro cultured OVCAR-3 cells were also transfected with polyplexes in the presence of peritoneal ascites fluid. The results indicate that one or more components of ascites had a negative effect on the transfection efficiency of p(DMAEMA)-containing polyplexes. To elucidate which component(s) of ascites may have interfered, the influence of hyaluronic acid, one of the ascites components, on the transfection efficiency was studied. The outcome suggests that hyaluronic acid may have induced a negative effect on the transfection capability of p(DMAEMA)-containing polyplexes. CONCLUSION: P(DMAEMA) is an efficient transfectant in vitro and ex vivo. However, transfected cells were not detected in vivo which may be caused by a negative influence of components of the ascites fluid.

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.

Effect of size and serum proteins on transfection efficiency of poly ((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles.

PURPOSE: 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 beta-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 micron) 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.