Sun Hwa Kim

Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer

For efficient cancer therapy, small interfering RNA (siRNA) should be stably and efficiently delivered into the target tissue and readily taken up by cancer cells. To address these needs, a polyelectrolyte complex (PEC) micelle-based siRNA delivery system was developed for anti-angiogenic gene therapy. The interaction between poly(ethylene glycol) (PEG)-conjugated vascular endothelial growth factor siRNA (VEGF siRNA-PEG) and polyethylenimine (PEI) led to the spontaneous formation of nanoscale polyelectrolyte complex micelles (VEGF siRNA-PEG/PEI PEC micelles), having a characteristic siRNA/PEI PEC inner core with a surrounding PEG shell layer. Intravenous as well as intratumoral administration of the PEC micelles significantly inhibited VEGF expression at the tumor tissue and suppressed tumor growth in an animal tumor model without showing any detectable inflammatory responses in mice. Upon examination of the PEC micelle distribution and in vivo optical imaging following intravenously injection, enhanced accumulation of the PEC micelles was also observed in the tumor region. This study demonstrates the feasibility of using PEC micelles as a potential carrier for therapeutic siRNAs in local and systemic treatment of cancer.

LHRH receptor-mediated delivery of siRNA using polyelectrolyte complex micelles self-assembled from siRNA-PEG-LHRH conjugate and PEI.

Polyelectrolyte complex (PEC) micelles modified with cancer cell targeting moieties were prepared for intracellular delivery of vascular endothelial growth factor (VEGF) small interfering RNA (siRNA). A luteinizing hormone-releasing hormone (LHRH) peptide analogue was coupled as a cancer targeting ligand to the distal end of the poly(ethylene glycol) (PEG)-siRNA conjugate. The siRNA-PEG-LHRH conjugate self-assembled to form nanosized PEC micelles upon mixing with poly(ethylenimine) (PEI) via ionic interactions. The PEC micelles showed spherical morphology with a hydrodynamic diameter of ca. 150 nm. For LHRH receptor overexpressing ovarian cancer cells (A2780), the PEC micelles with LHRH exhibited enhanced cellular uptake compared to those without LHRH, resulting in increased VEGF gene silencing efficiency via receptor-mediated endocytosis. This study showed that PEC micelles decorated with specific cell-recognizable targeting ligands could be used for targeted delivery of siRNA.

VEGF siRNA delivery system using arginine-grafted bioreducible poly(disulfide amine).

Small interfering RNAs (siRNAs) are able to silence their target genes when they are successfully delivered intact into the cytoplasm. Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics. We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)), a bioreducible cationic polymer, as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R) polyplexes, the reductive environment of the cytoplasm cleaves the disulfide linkages in the polymeric backbone, resulting in decomplexing of the siRNA/poly(CBA-DAH-R) polyplexes and release of siRNA molecules throughout the cytoplasm. The siRNA/poly(CBA-DAH-R) polyplexes, which demonstrate increased membrane permeability with arginine modification, have a similar level of cellular uptake as siRNA/bPEI polyplexes. The VEGF siRNA/poly(CBA-DAH-R) polyplexes, however, inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines. The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs. These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy.

Cardiomyocyte-targeted siRNA delivery by prostaglandin E2-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis

A cardiomyocyte-targeted Fas siRNA delivery system was developed using prostaglandin E(2) (PGE(2))-modified siRNA polyplexes formed by a reducible poly(amido amine) to inhibit cardiomyocyte apoptosis. PGE(2), which was used as a specific ligand for cardiomyocyte targeting, was conjugated to the terminal-end of the sense siRNA (PGE(2)-siRNA). The reducible cationic copolymer, synthesized via Michael-type polyaddition of 1,6-diaminohexane and cystamine bis-acrylamide (poly(DAH/CBA)), tightly condensed the PGE(2)-siRNA conjugate to form nanosize polyplexes having a diameter of 100-150 nm. The PGE(2)-siRNA/poly(DAH/CBA) polyplexes decomplexed to release PGE(2)-siRNA in a cytosolic reducing environment due to the degradation of the reducible poly(DAH/CBA). The cellular uptake of the PGE(2)-siRNA/poly(DAH/CBA) polyplex was increased in rat cardiomyocytes (H9C2 cells) due to PGE(2) receptor-mediated endocytosis. When H9C2 cells were transfected with siRNA against Fas, a key regulator of ischemia-induced apoptosis, the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex delivery system led to a significant increase in Fas gene silencing, resulting in inhibition of cardiomyocyte apoptosis. The PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex did not induce interferon-alpha in peripheral blood mononuclear cells. These results suggest that the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex formulation may be clinically applicable as a cardiomyocyte-targeted Fas siRNA delivery system to inhibit apoptosis in cardiovascular disease.

Folate Receptor Targeted Delivery of Polyelectrolyte Complex Micelles Prepared from ODN‐PEG‐Folate Conjugate and Cationic Lipids

A polyelectrolyte complex micelle (PECM)‐based delivery system for targeting folate (FOL) receptor overexpressing tumor cells is demonstrated using poly(ethylene glycol) (PEG)‐conjugated oligonucleotide (ODN). The tumor targeting property was conferred to the PECM by tethering a folate moiety to the distal end of the PEG segment in an anti‐sense green fluorescent protein (GFP) ODN‐PEG conjugate. Nanoscale PECMs were spontaneously produced from ionic interactions between the ODN‐PEG‐FOL conjugate and a cationic lipid, lipofectamine (Lf). When treated with FOL receptor overexpressing cells (KB), the PCEMs caused a significant reduction in GFP expression in a dose‐dependent manner. This effect was not observed in FOL receptor deficient cells (A549). The enhanced transfection of ODN‐PEG‐FOL/Lf PECMs to KB cells was caused by FOL receptor mediated endocytosis. The efficiency of target‐specific gene suppression by ODN‐PEG‐FOL/Lf PECMs was maintained even in the presence of 10% fetal bovine serum in the transfection medium.

In vivo tumor targeting of ODN-PEG-folic acid/PEI polyelectrolyte complex micelles

A tumor-targeting antisense oligodeoxynucleotide (ODN) delivery system based on polyelectrolyte complex (PEC) micelles is demonstrated. ODN-PEG-folic acid (ODN-PEG-FA) was synthesized using a heterofunctional PEG linker. The PEC micelles for the targeted ODN delivery to tumor cells were produced by ionic interactions between the ODN-PEG-FA and polyethylenimine (PEI). The in vivo targeting properties of the PEC micelles were assessed using a mouse tumor model. The size of ODN-PEG-FA/PEI PEC micelles was 92.3 nm with a relatively narrow distribution. Cellular uptake of the ODN-PEG-FA/PEI PEC micelles by folic acid receptor over-expressing cells (KB) was greatly enhanced compared to that of ODN-PEG/PEI PEC micelles. When the ODN-PEG-FA/PEI PEC micelles were systemically administered to the mice bearing KB cell xenograft tumor, ODN was accumulated to the solid tumor in a target specific manner. This study suggests that the PEC micelles with a receptor-recognizable targeting ligand on the surface have potential for passive and active targeted delivery of ODN drugs to cancer cells.

Penetration and efficacy of VEGF siRNA using polyelectrolyte complex micelles in a human solid tumor model in-vitro

A polyelectrolyte complex(PEC) micelle-based siRNA delivery system has been developed for vascular endothelial growth factor (VEGF), and its antitumor efficacy has been demonstrated using in-vivo animal models. Penetration and distribution through the avascular regions of human solid tumors after extravasation are important issues for antitumor efficacy, especially for macromolecules such as VEGF siRNA PEC micelles. Using an in-vitro solid tumor model, multicellular layers(MCL) culture of human colorectal cancer cells, we evaluated the penetration kinetics and efficacy of VEGF siRNA PEC micelles(PEC-siRNA) in comparison to unmodified siRNA(N-siRNA). The PEC-siRNA showed full penetration (15-17 layers of cells) with a unique punctuated distribution pattern at 48 h following initial accumulation in the top layers and a significant suppression of mRNA and protein expression in a dose-dependent manner after 72 h exposure. Although the initial penetration of N-siRNA was faster than that of PEC-siRNA, N-siRNA showed complete loss of activity due to its instability within 24 h. Our data support the idea that PEC micelle formulation may provide stable penetration tool through the multilayers of cancer cells and ensure the gene silencing effect of VEGF. This study also demonstrated that MCL could serve as a useful in-vitro model to evaluate the dose- and time-dependent profiles of penetration and efficacy of macromolecular delivery systems in human solid tumor avascular regions.

Reducible poly(amido ethylenimine)-based gene delivery system for improved nucleus trafficking of plasmid DNA.

In a nonviral gene delivery system, localization of a plasmid DNA in the nucleus is a prerequisite for expression of a desired therapeutic protein encoded in the plasmid DNA. In this study, a reducible polymer-based gene delivery system for improved intracellular trafficking and nuclear translocation of plasmid DNA is introduced. The system is consisted of two components, a plasmid DNA having repeated binding sequence for a karyophilic protein, NFkappaB, and a reducible polymer. A reducible poly(amido ethylenimine), poly(TETA-CBA), was synthesized by a Michael-type addition polymerization between cystamine bisacrylamide and triethyl tetramine. The polymer forming tight complexes with plasmid DNA could be degraded in the reductive cytosol to release the plasmid DNA. The triggered release mechanism in the cytosol could facilitate the interaction between cytosolic NFkappaB and the plasmid DNA having repeated NFkappaB biding motif. Upon activation of NFkappaB by interleukin-1beta (IL-1beta), most of the plasmid distributed in the cytoplasm was localized within the nucleus, resulting in significantly higher gene transfection efficiency than controls with nondegradable PEI. The current study suggests an alternative way of improving transfection efficiency by taking advantage of endogenous transport machinery for intracellular trafficking and nuclear translocation of a plasmid DNA.

Reductive degradation behavior of bioreducible poly(disulfide amine) for enhancing SiRNA efficiency.

Bioreducible cationic polymer poly(CBA-DAH) containing repeated disulfide linkages on the polymer backbone was synthesized through Michael-type polyadditions of CBA to DAH monomers. Poly(CBA-DAH) could spontaneously form nanoscale polyelectrolyte complexes through electrostatic interactions with siRNA in an aqueous phase. These nanoparticles were rapidly degraded under the reductive cytoplasmic environment with subsequently releasing the siRNA cargo into the cytoplasm where RNAi takes place, as a result of the breakdown of disulfide bonds in the polymers. The reductive degradation behavior of the poly(CBA-DAH)/siRNA polyplexes is more likely to increase RNAi activity with enhancing the cytoplasmic localization of siRNA molecules. Poly(CBA-DAH) may have great potential as a gene carrier especially for therapeutic applications of siRNAs owing to the reductive degradation characteristics.

Prostate cancer cell–specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer–binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI–PEG–PCP). The PEI–PEG–PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI–PEG–PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI–PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI–PEG–PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI–PEG–PCP polyplexes were delivered into PC-3 cells in a PCP ligand–specific manner.