Development of a Nonviral Gene Delivery Vehicle for Systemic Application

Abstract

Polycation vehicles used for in vitro gene delivery require alteration for successful application in vivo. Modification of polycations by direct grafting of additional components, e.g., poly(ethylene glycol) (PEG), either before or after DNA complexation, tend to interfere with polymer/DNA binding interactions; this is a particular problem for short polycations such as linear, beta-cyclodextrin-containing polycations (betaCDPs). Here, a new method of betaCDP polyplex (polycation/DNA composite structures) modification is presented that exploits the ability to form inclusion complexes between cyclodextrins and adamantane. Surface-PEGylated betaCDP polyplexes are formed by self-assembly of the polyplexes with adamantane-PEG conjugates. While unmodified polyplexes rapidly aggregate and precipitate in salt solutions, the PEGylated betaCDP polyplexes are stable at conditions of physiological salt concentration. Addition of targeting ligands to the adamantane-PEG conjugates allows for receptor-mediated delivery; galactosylated betaCDP-based particles reveal selective targeting to hepatocytes via the asialoglycoprotein receptor. Galactosylated particles transfect hepatoma cells with 10-fold higher efficiency than glucosylated particles (control), but show no preferential transfection in a cell line lacking the asialoglycoprotein receptor. Thus, surface modification of betaCDP-based polyplexes through the use of cyclodextrin/adamantane host/guest interactions endows the particles with properties appropriate for systemic application.

A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene

“…27 However, to date, most targeting sterically stabilized gene delivery vehicles have been studied in vitro. [3][4][5][6][7][8][9] As the ultimate goal of any potential gene therapy is delivery in vivo, a comparison study of TsPLP both in vitro and in vivo was undertaken to elucidate the influence of the PEGylation on gene delivery efficiency. PLP showed the lowest in vitro transfection activity (Figure 2), and this is likely due to the PEG molecule preventing association of the lipoplex with the target cell 2,12 and/or due to PEG inhibition of fusion between lipid and endosomal membrane, which is necessary for the intracellular release of exogenously delivered DNA.…”

“…2 The application of this modification (PEGylation) to nonviral gene delivery systems has drawn considerable interest. [3][4][5][6][7][8][9] Benefits of PEGylation have been described and include improvement of systemic circulation of lipoplexes or polyplexes; 1,10 improvement of the solubility of polyplexes thereby allowing the preparation of higher therapeutic concentrations without subsequent aggregation; 10,11 and reduction of in vivo toxicity of the polyplex. 10 However, disadvantages of PEGylation have also been observed.…”

Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles

“…We have previously shown that polyplexes can be stabilized against salt-induced aggregation by formulation with AD-PEG at 1:1 AD-PEG to cyclodextrin ratios (mole to mole). 16 In addition, polyplex surface charge can be tuned by the addition of an adamantane-PEG modifier containing anionic residues such as AD-gluglu-PEG-gal. For example, polyplexes formulated at a charge ratio of 3 and modified with 100% AD-PEG have positive zeta potential (~+15 mV) while modification with increasing ratios of AD-gluglu-PEG-gal to AD-PEG results in particles with negative zeta potentials (Fig.…”

“…[29][30][31] For example, DNAzymes targeting the translation initiation region of c-myc RNA have been reported to site-selectively cleave full length RNA and downregulate c-myc gene expression in smooth muscle cells. 30,31 The objective of this work is to assess the ability of the cyclodextrincontaining polycation delivery system 9,11,16 to provide for the in vivo delivery of anti-c-myc DNAzymes 30,31 to subcutaneous tumors in mice. The study focuses on the preparation of transferrin-modified polyplexes and their biodistribution in tumor-containing nude mice.…”

“…2B and C). 16 The adamantane (AD) forms an inclusion species with exposed cyclodextrins on the polyplexes by complexation, an enthalpically driven physical phenomenon based on van der Waal's interactions between the cyclodextrin cavity interior and the molecular surface of the adamantane derivatives. 16 Transferrin-modified, poly (ethylene glycol) (PEG)-stabilized polyplexes (Tf-PEG-polyplexes) are prepared by polyplex modification with transferrin-PEG-AD and PEG-AD conjugates (Fig.…”