Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is... more 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.
Electron micrographs, dynamic light-scattering, and osmotic measurements have been performed on m... more Electron micrographs, dynamic light-scattering, and osmotic measurements have been performed on monolayered vesicles obtained from two bolaform amphiphiles of recent synthesis. Monolayered vesicles of bipolar lipids constitute an interesting model for primordial membranes.
The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated... more The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-ß-cyclodextrin (HPßCD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56°C). For
Poly(2-(dimethylamino)ethyl methacrylate), p(DMAEMA), with varying molecular weight was synthesiz... more 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
European Journal of Pharmaceutics and Biopharmaceutics, 1999
The aim of this study was to gain insight into the formulation parameters affecting the size of p... more The aim of this study was to gain insight into the formulation parameters affecting the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes (polyplexes). Experimental designs were applied to screen and optimize several variables, which may influence the complex size. In a screening design, it was demonstrated that at a fixed concentration of plasmid (40 micrograms/ml) after incubation with polymer, the size of the resulting polyplexes was highly dependent on the polymer/plasmid ratio as well as on the pH, viscosity (i.e. sucrose concentration) and ionic strength of the aqueous solution. However, the temperature, PEG 600 (up to 5% (v/v)) and Tween 80 (up to 0.2%) had a marginal effect on the size of the polyplexes. In an optimization design, the effect of the pH, polymer/plasmid ratio and Tween on the size of the polymer/plasmid complexes prepared at relatively high concentration of plasmid (50-200 micrograms/ml) was evaluated. Based on the results of the optimization design, a mathematical model was derived, which describes the relationship between the size of the polyplexes and the different formulation parameters. This model shows that even at high plasmid concentration (200 micrograms/ml), small sized polyplexes were formed at low pH and ionic strength, especially when the solution contains 20% (w/v) sucrose. This concentrated polyplex dispersion (polymer/plasmid ratio > 3/1 (w/w), 200 micrograms plasmid/ml) can be diluted down to 5 micrograms/ml plasmid without significant changes in particle size and transfection potential. At lower ratios, a growth in particle size was observed upon dilution of the complexes, which might also explain the low transfection efficiency of these polyplexes in vitro.
Thermoporometry (TPM) was applied to hydrotalcite precipitates prepared with carbonate, bicarboxy... more Thermoporometry (TPM) was applied to hydrotalcite precipitates prepared with carbonate, bicarboxylic acids and chloride. It was used to measure the formation of an ice body between the hydrotalcite particles. Before TPM could be applied, the dried hydrotalcite precipitate had to be soaked for two weeks in water. The mean value of a factor F measured by TPM, which described the shape of the ice body in hydrotalcite, was 1.7. This value was between those of a purely cylindrical (F = 2) and a purely spherical ice body (F = 1), indicating the formation of ice lenses. From the radius of the ice body, Rn, ice volume, Vn and shape factor F, the corresponding specific surface area of the hydrotalcite particles could be assessed. The TPM indicated that the distance between the separate hydrotalcite crystals in water, which is equal to 2(Rn+0.9) nm, was a function of the type of anion incorporated at the interlayer, such as chloride and bicarboxylic acid. The pore volume and surface area of t...
Cryopreservation and low temperature biology in blood transfusion, 1990
Liposomes can be looked upon as synthetic cells, as they are closed vesicles consisting of phosph... more Liposomes can be looked upon as synthetic cells, as they are closed vesicles consisting of phospholipid bilayers which surround an internal aqueous volume. Therefore, they might mimic the behaviour of peripheral blood cells during cryopreservation in several aspects. Freezing and freeze-drying have been proposed as stabilization techniques for the long-term storage of peripheral blood cells. However, attempts to store these cells in freeze dried form with complete retention of contents and activity have not been very successful until now. Major problems encountered were leakage of cell contents and alterations in cell size. A number of potential damaging effects during cooling of cell dispersions have been suggested, including crystallization of the cryoprotectant, crystallization of internal ice and osmotic forces originating from freeze concentration. The objective of this study was to investigate the behaviour of negatively charged liposomes in a dispersion containing cryoprotectant during a freezing/thawing cycle. Freezing and melting processes were monitored by differential scanning (DSC). The extent of marker (5 mM carboxyfluorescein) encapsulation was measured before and after the cycle.
Biomedical Polymers and Polymer Therapeutics, 2002
A biodegradable and biocompatible dextran hydrogel system has been developed of which the degrada... more A biodegradable and biocompatible dextran hydrogel system has been developed of which the degradation time can be tailored between 2 days and 3 months. This chapter shows that hydrogels based on crosslinked dextrans have unique properties as protein releasing matrices. Both the release pattern (first-order, zero-order or biphasic) as well as the duration of the release can be controlled by
Poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) is a water-soluble cationic polymer, which is... more 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.
Electron micrographs, dynamic light-scattering, and osmotic measurements have been performed on m... more Electron micrographs, dynamic light-scattering, and osmotic measurements have been performed on monolayered vesicles obtained from two bolaform amphiphiles of recent synthesis. Monolayered vesicles of bipolar lipids constitute an interesting model for primordial membranes.
The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated... more The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-ß-cyclodextrin (HPßCD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56°C). For
Poly(2-(dimethylamino)ethyl methacrylate), p(DMAEMA), with varying molecular weight was synthesiz... more 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
European Journal of Pharmaceutics and Biopharmaceutics, 1999
The aim of this study was to gain insight into the formulation parameters affecting the size of p... more The aim of this study was to gain insight into the formulation parameters affecting the size of poly((2-dimethylamino)ethyl methacrylate)-plasmid complexes (polyplexes). Experimental designs were applied to screen and optimize several variables, which may influence the complex size. In a screening design, it was demonstrated that at a fixed concentration of plasmid (40 micrograms/ml) after incubation with polymer, the size of the resulting polyplexes was highly dependent on the polymer/plasmid ratio as well as on the pH, viscosity (i.e. sucrose concentration) and ionic strength of the aqueous solution. However, the temperature, PEG 600 (up to 5% (v/v)) and Tween 80 (up to 0.2%) had a marginal effect on the size of the polyplexes. In an optimization design, the effect of the pH, polymer/plasmid ratio and Tween on the size of the polymer/plasmid complexes prepared at relatively high concentration of plasmid (50-200 micrograms/ml) was evaluated. Based on the results of the optimization design, a mathematical model was derived, which describes the relationship between the size of the polyplexes and the different formulation parameters. This model shows that even at high plasmid concentration (200 micrograms/ml), small sized polyplexes were formed at low pH and ionic strength, especially when the solution contains 20% (w/v) sucrose. This concentrated polyplex dispersion (polymer/plasmid ratio > 3/1 (w/w), 200 micrograms plasmid/ml) can be diluted down to 5 micrograms/ml plasmid without significant changes in particle size and transfection potential. At lower ratios, a growth in particle size was observed upon dilution of the complexes, which might also explain the low transfection efficiency of these polyplexes in vitro.
Thermoporometry (TPM) was applied to hydrotalcite precipitates prepared with carbonate, bicarboxy... more Thermoporometry (TPM) was applied to hydrotalcite precipitates prepared with carbonate, bicarboxylic acids and chloride. It was used to measure the formation of an ice body between the hydrotalcite particles. Before TPM could be applied, the dried hydrotalcite precipitate had to be soaked for two weeks in water. The mean value of a factor F measured by TPM, which described the shape of the ice body in hydrotalcite, was 1.7. This value was between those of a purely cylindrical (F = 2) and a purely spherical ice body (F = 1), indicating the formation of ice lenses. From the radius of the ice body, Rn, ice volume, Vn and shape factor F, the corresponding specific surface area of the hydrotalcite particles could be assessed. The TPM indicated that the distance between the separate hydrotalcite crystals in water, which is equal to 2(Rn+0.9) nm, was a function of the type of anion incorporated at the interlayer, such as chloride and bicarboxylic acid. The pore volume and surface area of t...
Cryopreservation and low temperature biology in blood transfusion, 1990
Liposomes can be looked upon as synthetic cells, as they are closed vesicles consisting of phosph... more Liposomes can be looked upon as synthetic cells, as they are closed vesicles consisting of phospholipid bilayers which surround an internal aqueous volume. Therefore, they might mimic the behaviour of peripheral blood cells during cryopreservation in several aspects. Freezing and freeze-drying have been proposed as stabilization techniques for the long-term storage of peripheral blood cells. However, attempts to store these cells in freeze dried form with complete retention of contents and activity have not been very successful until now. Major problems encountered were leakage of cell contents and alterations in cell size. A number of potential damaging effects during cooling of cell dispersions have been suggested, including crystallization of the cryoprotectant, crystallization of internal ice and osmotic forces originating from freeze concentration. The objective of this study was to investigate the behaviour of negatively charged liposomes in a dispersion containing cryoprotectant during a freezing/thawing cycle. Freezing and melting processes were monitored by differential scanning (DSC). The extent of marker (5 mM carboxyfluorescein) encapsulation was measured before and after the cycle.
Biomedical Polymers and Polymer Therapeutics, 2002
A biodegradable and biocompatible dextran hydrogel system has been developed of which the degrada... more A biodegradable and biocompatible dextran hydrogel system has been developed of which the degradation time can be tailored between 2 days and 3 months. This chapter shows that hydrogels based on crosslinked dextrans have unique properties as protein releasing matrices. Both the release pattern (first-order, zero-order or biphasic) as well as the duration of the release can be controlled by
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