Intravenous fate of poly(2-(dimethylamino)ethyl methacrylate)-based polyplexes

The objective of this study was to assess the in vivo fate of poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA)-based polyplexes after intravenous administration into mice. Circulation kinetics and tissue distribution in terms of plasmid localization and transfection efficiency were assessed. To gain more insight into the observed biodistribution and gene expression profile, the interaction of pDMAEMA-based polyplexes with blood components (erythrocytes and albumin) was investigated in vitro. In the case of i.v. injection of positively charged polyplexes at a dose of 30 microg DNA most of the radioactivity was found in the lungs and the liver 60 min after injection. In the case of pDMAEMA/DNA polyplexes with a negative charge, uptake occurred mainly by the liver. Administration of positively charged complexes at a 30 microg DNA dose resulted in reporter gene expression primarily in the lungs. Injection of negatively charged complexes and naked plasmid did not result in luciferase expression in any of the organs examined. In vitro turbidity experiments showed the induction of a charge dependent aggregation process upon addition of albumin to the polyplexes pointing out to the involvement of aggregate formation in the dominant lung uptake of the positively charged polyplexes. Also, incubations of polyplexes after pre-incubation with a physiological concentration of albumin with washed erythrocytes confirmed that polyplexes induce the formation of extremely large structures. This paper underlines the need for the design of systems with reduced interaction with blood components to promote the delivery of DNA to target tissues outside the lungs.     

Synthesis and characterization of poly-L-lysine with controlled low molecular weight

N-Carboxy-(N^?-benzyloxycarbonyl)-L -lysine anhydride (Z-L -lysine NCA) was polymerized in dimethylformamide with triethylamine, diethylamine or hexylamine as initiator, at varying molar ratios of NCA to initiator (M/I ratio). After removal of the protecting Z-group the resulting poly-L -lysine was characterized with ¹H NMR and MALDI TOF MS. Both diethylamine- and hexylamine-initiated polymerization yielded poly-L -lysine in which the initiators were incorporated at the carboxylic end of the polymer. This indicates that the NCA polymerization occurred exclusively via nucleophilic attack of the initiator on the monomer. On the other, hand, when triethylamine was used as the initiator, poly-L -lysine was obtained in which no triethylamine-derived end group could be detected by MS. These polymer chains are most likely end-capped with an N-acyl-2,5-dioxopiperazine group at the carboxylic end of the polymer. Incorporation of diethylamine and hexylamine allowed determination of the degree of polymerization (DP) of the obtained polymers by ¹H NMR. The DP depended linearly on the M/I ratio, for both diethylamine and hexylamine, with higher DPs for the diethylamine-initiated poly-L -lysine at equal M/I ratio.     

Tissue reactions of in situ formed dextran hydrogels crosslinked by stereocomplex formation after subcutaneous implantation in rats

In this study, the in vivo biocompatibility of physically crosslinked dextran hydrogels was investigated. These hydrogels were obtained by mixing aqueous solutions of dextran grafted with L-lactic acid oligomers and dextran grafted with D-lactic acid oligomers. Gelation occurs due to stereocomplex formation of the lactic acid oligomers of opposite chirality. Since gelation takes some time, in situ gel formation is possible with this system. A number of sterilization methods was evaluated for their effect on the chemical and physical properties of the hydrogel. It was shown that of the investigated options (filtration, gamma irradiation, dry-heat and autoclaving) dry-heat sterilization was the preferred method to prepare sterile gels suitable for in vivo evaluations. Two types of stereocomplex gels were prepared and implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 30 days. A mild ongoing foreign body reaction was observed characterized by infiltration of macrophages. Giant cells were only scarcely formed and the low numbers of lymphocytes showed that priming of the immune system is hardly involved. Importantly, the gels fully degraded in vivo within 15 days, which is in good agreement with the in vitro degradation behaviour of these gels. In conclusion, stereocomplexed dextran-oligolactic gels showed good biocompatibility which makes them suitable candidates for the design of controlled release devices for pharmaceutically active proteins.     

Interaction of antithrombin III with preadsorbed albumin-heparin conjugates

The adsorption of antithrombin III (AT III) onto polystyrene surfaces preadsorbed with albumin or albuminheparin conjugates was studied using a two step enzyme immuno assay. When AT III-buffer solutions were used, the highest adsorption values were measured on high affinity albumin-heparin conjugate pretreated surfaces. Less AT III adsorption was found on nonfractionated albumin-heparin conjugate preadsorbed surfaces. AT III adsorption could also be detected on low affinity conjugate and albumin coated surfaces. When AT III was adsorbed from plasma or plasma dilutions with buffer, only AT III on surfaces preadsorbed with high affinity or nonfractionated albumin-heparin conjugate was found. These results demonstrate that the heparin moiety of the conjugate is directed to the solution phase whereas the albumin moiety contacts the polystyrene surfaca     

In vivo biocompatibility of dextran-based hydrogels

Dextran-based hydrogels were obtained by polymerization of aqueous solutions of methacrylated dextran (dex-MA) or lactate-hydroxyethyl methacrylate-derivatized dextran (dex-lactate-HEMA). Both nondegradable dex-MA and degradable dex-lactate-HEMA disk-shaped hydrogels, varying in initial water content and degree of substitution (DS, the number of methacrylate groups per 100 glucose units), were implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 6 weeks. The initial foreign-body reaction to the dex-MA hydrogels was characterized by infiltration of granulocytes and macrophages and the formation of fibrin, and exudate, as well as new blood vessels. This reaction depended on the initial water content as well as on the DS of the hydrogel and decreased within 10 days. The mildest tissue response was observed for the gel with the highest water content and intermediate DS. At day 21 all dex-MA hydrogels were surrounded by a fibrous capsule and no toxic effects on the surrounding tissue were found. No signs of degradation were observed. The initial foreign-body reaction to the degradable dex-lactate-HEMA hydrogels was less severe compared with the dex-MA gels. In general, the size of the dex-lactate-HEMA hydrogels increased progressively with time and finally the gels completely dissolved. Degradation of the dex-lactate-HEMA hydrogels was associated with infiltration of macrophages and the formation of giant cells, both of which phagocytosed pieces of the hydrogel. A good correlation between the in vitro and the in vivo degradation time was found. This suggests that extra-cellular degradation is not caused by enzymes but depends only on hydrolysis of the ester and/or carbonate bonds present in the crosslinks of the hydrogels. After 21 days, the degradable hydrogels, as such, could not be retrieved, but accumulation of macrophages and giant cells was observed, both of which contained particles of the gels intracellularly. As for the dex-MA hydrogels, no toxic effects on the surrounding tissue were found. The results presented in this study demonstrate that dextran-based hydrogels can be considered as biocompatible materials, making these hydrogels attractive systems for drug delivery purposes.     

Immunogenicity of meningococcal PorA formulations encapsulated in biodegradable microspheres.

The purpose of our study was to investigate the possibility to microencapsulate liposomes and meningococcal outer membrane vesicles (OMV), both containing neisserial pore protein A (PorA), in biodegradable dextran- and mannan-based microspheres and to study the immunogenicity of the encapsulated PorA formulations. PorA-liposomes and OMV were encapsulated in dextran- or mannan-based microspheres by using an aqueous two-phase system consisting of a polyethylene glycol solution and a methacrylated dextran or mannan solution. The formulations were characterized for size distribution, PorA structure and antigen recovery after release. Calcein-containing model liposomes were used to establish the encapsulation efficiency and release profiles from both types of microspheres. The immunogenicity of the PorA-containing formulations was determined in mice after subcutaneous immunization. Liposomes were encapsulated in dextran and mannan microspheres with a high efficiency (70-90%). Calcein liposomes, after a 5-day lag period, exhibited apparent zero-order release kinetics from both types of microspheres between Days 5 and 10 of incubation in vitro. The total release was 80 and 100% from mannan and dextran microspheres, respectively. The trimeric PorA conformation was preserved in the released liposomes and OMV and the antigen was partly recovered. The immunogenicity of PorA-liposomes and OMV encapsulated in dextran or mannan microspheres was preserved. In conclusion, PorA-liposomes and OMV could be encapsulated in dextran- and mannan-based microspheres with high efficiency. The immunogenicity of encapsulated antigen was preserved.