Physicochemical and biological evaluation of siRNA polyplexes based on PEGylated Poly(amido amine)s.

PURPOSE: Use of RNA interference as novel therapeutic strategy is hampered by inefficient delivery of its mediator, siRNA, to target cells. Cationic polymers have been thoroughly investigated for this purpose but often display unfavorable characteristics for systemic administration, such as interactions with serum and/or toxicity. METHODS: We report the synthesis of a new PEGylated polymer based on biodegradable poly(amido amine)s with disulfide linkages in the backbone. Various amounts of PEGylated polymers were mixed with their unPEGylated counterparts prior to polyplex formation to alter PEG content in the final complex. RESULTS: PEGylation effectively decreased polyplex surface charge, salt- or serum-induced aggregation and interaction with erythrocytes. Increasing amount of PEG in formulation also reduced its stability against heparin displacement, cellular uptake and subsequent silencing efficiency. Yet, for polyplexes with high PEG content, significant gene silencing efficacy was found, which was combined with almost no toxicity. CONCLUSIONS: PEGylated poly(amido amine)s are promising carriers for systemic siRNA delivery in vivo.

Improved synthesis strategy of poly(amidoamine)s for biomedical applications: catalysis by "green" biocompatible earth alkaline metal salts.

Poly(amidoamine)s (PAAs) have received significant attention due to their good biocompatibility and fast biodegradation profile which gives these polymers high potential in biomedical applications. Conventional synthesis of PAAs via aza-type Michael addition reaction of primary amines to bis-acrylamides often proceeds slowly and takes several days, which does not allow fast and extensive screening of PAA libraries for their bioactivity. Current investigation was dedicated to the development of catalytic synthesis procedures in order to decrease the polymerization times. The salts of several transition metals, as well as earth alkali metals were studied for their catalytic activity in the polymerization reaction. It was found that the salts of earth alkali metals showed the highest potential in the catalysis of polymerization, whereas the salts of transition metals showed either no effect or even resulted in slowing down the reaction. In particular, the addition of CaCl(2) to the reaction mixtures resulted in remarkable increase of the reaction rate as compared to the system without catalyst. PAAs synthesized by the conventional procedure and those obtained by using CaCl(2) as a catalyst showed no difference in physico-chemical properties as well as in biological activity. The novel synthetic method for PAAs, using catalysts based on earth alkali metals, represents an attractive alternative to currently applied methods. Characteristics of earth alkali metals such as low toxicity and good biocompatibility make them especially useful in the preparation of these polymers for biomedical applications.

Flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/DNA polyplexes.

Extensive research is currently performed on designing safe and efficient non-viral carriers for gene delivery. To increase their efficiency, it is essential to have a thorough understanding of the mechanisms involved in cellular attachment, internalization and intracellular processing in target cells. In this work, we studied in vitro the cellular dynamics of polyplexes, composed of a newly developed bioreducible poly(amido amine) carrier, formed by polyaddition of N,N-cystamine bisacrylamide and 1-amino-4-butanol (p(CBA-ABOL)) on retinal pigment epithelium (RPE) cells, which are attractive targets for ocular gene therapy. We show that these net cationic p(CBA-ABOL)/DNA polyplexes require a charge-mediated attachment to the sulfate groups of cell surface heparan sulfate proteoglycans in order to be efficiently internalized. Secondly, we assessed the involvement of defined endocytic pathways in the internalization of the polyplexes in ARPE-19 cells by using a combination of endocytic inhibitors, RNAi depletion of endocytic proteins and live cell fluorescence colocalization microscopy. We found that the p(CBA-ABOL) polyplexes enter RPE cells both via flotillin-dependent endocytosis and a PAK1 dependent phagocytosis-like mechanism. The capacity of polyplexes to transfect cells was, however, primarily dependent on a flotillin-1-dependent endocytosis pathway.

Disulfide-based poly(amido amine)s for siRNA delivery: effects of structure on siRNA complexation, cellular uptake, gene silencing and toxicity.

PURPOSE: RNA interference (RNAi) is a process by which small interfering RNAs (siRNA) induce sequence-specific gene silencing. Therefore, siRNA is an emerging promise as a novel therapeutic. In order to realize the high expectations for therapeutic applications, efficient delivery systems for siRNA are necessary. METHODS: In this study, a new series of biodegradable poly(amido amine)s with disulfide linkages in the backbone was synthesized out of N,N'-cystaminebisacrylamide (CBA), 4-amino-1-butanol (ABOL) and ethylene diamine (EDA). Effects of different percentages of butanolic side chains and protonatable fragments in the main chain on siRNA complexation, cellular uptake, gene silencing and toxicity were investigated. RESULTS: Incorporation of EDA in the polymer resulted in increased siRNA condensation. Efficient siRNA condensation was shown to be necessary for cellular uptake; however, excess of polymer decreased siRNA uptake for polymers with high amounts of EDA. Silencing efficiency did not correlate with uptake, since silencing increased with increasing w/w ratio for all polymers. More than 80% knockdown was found for polyplexes formed with polymers containing 25% or 50% EDA, which was combined with low cytotoxicity. CONCLUSIONS: Poly(amido amine)s with minor fractions of protonatable fragments in the main chain are promising carriers for delivery of siRNA.

A method for quantifying cellular uptake of fluorescently labeled siRNA.

Efficient intracellular delivery of siRNA is a significant hurdle to its therapeutic success. For biological studies on the efficiency of carrier-mediated uptake of siRNA, quantitative determination of the amount of internalized siRNA is required. In this study, when the apparent uptake of fluorescently labeled siRNA, formulated in different lipo- and polyplexes, was examined using different techniques, major differences were observed. Additional experiments showed that these differences could be explained by quenching phenomena that were dependent on interactions between siRNA and carrier and their intracellular environment. Differences in fluorescent signal of complexed siRNA due to quenching could be overcome by measuring the fluorescent signal after lysing the transfected cells in lysis buffer that contained 2% SDS to dissociate siRNA from the complexes. This method offers a simple approach for quantifying cellular uptake of siRNA, which might help in the development of more efficient delivery systems.

A newly developed chemically crosslinked dextran-poly(ethylene glycol) hydrogel for cartilage tissue engineering.

Cartilage tissue engineering, in which chondrogenic cells are combined with a scaffold, is a cell-based approach to regenerate damaged cartilage. Various scaffold materials have been investigated, among which are hydrogels. Previously, we have developed dextran-based hydrogels that form under physiological conditions via a Michael-type addition reaction. Hydrogels can be formed in situ by mixing a thiol-functionalized dextran with a tetra-acrylated star poly(ethylene glycol) solution. In this article we describe how the degradation time of dextran-poly(ethylene glycol) hydrogels can be varied from 3 to 7 weeks by changing the degree of substitution of thiol groups on dextran. The degradation times increased slightly after encapsulation of chondrocytes in the gels. The effect of the gelation reaction on cell viability and cartilage formation in the hydrogels was investigated. Chondrocytes or embryonic stem cells were mixed in the aqueous dextran solution, and we confirmed that the cells survived gelation. After a 3-week culturing period, chondrocytes and embryonic stem cell-derived embryoid bodies were still viable and both cell types produced cartilaginous tissue. Our data demonstrate the potential of dextran hydrogels for cartilage tissue engineering strategies.

Shielding the cationic charge of nanoparticle-formulated dermal DNA vaccines is essential for antigen expression and immunogenicity.

Nanoparticle-formulated DNA vaccines hold promise for the design of in vivo vaccination platforms that target defined cell types in human skin. A variety of DNA formulations, mainly based on cationic liposomes or polymers, has been investigated to improve transfection efficiency in in vitro assays. Here we demonstrate that formulation of DNA into both liposomal and polymeric cationic nanoparticles completely blocks vaccination-induced antigen expression in mice and ex vivo human skin. Furthermore, this detrimental effect of cationic nanoparticle formulation is associated with an essentially complete block in vaccine immunogenicity. The blocking of DNA vaccine activity may be explained by immobilization of the nanoparticles in the extracellular matrix, caused by electrostatic interactions of the cationic nanoparticles with negatively charged extracellular matrix components. Shielding the surface charge of the nanoparticles by PEGylation improves in vivo antigen expression more than 55 fold. Furthermore, this shielding of cationic surface charge results in antigen-specific T cell responses that are similar as those induced by naked DNA for the two lipo- and polyplex DNA carrier systems. These observations suggest that charge shielding forms a generally applicable strategy for the development of dermally applied vaccine formulations. Furthermore, the nanoparticle formulations developed here form an attractive platform for the design of targeted nanoparticle formulations that can be utilized for in vivo transfection of defined cell types.