Hyejung Mok

siRNA Conjugate Delivery Systems

Small interfering RNA (siRNA) has been chemically conjugated to a variety of bioactive molecules, lipids, polymers, peptides, and inorganic nanostructured materials to enhance their pharmacokinetic behavior, cellular uptake, target specificity, and safety. To efficiently deliver siRNAs to the target cells and tissues, many different siRNA bioconjugates were synthesized and characterized, and their gene silencing efficiencies were tested in vitro and in vivo. In this review, recent developments of siRNA bioconjugates are summarized.

Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing

Small interfering RNA (siRNA) with 19-21 base pairs has been recently recognized as a new therapeutic agent for effectively silencing a specific gene on a post-transcription level. For siRNA therapeutics, safe and efficient delivery issues are significant hurdles to clinical applications. Here we present a new class of biologically active siRNA structure based on chemically self-crosslinked and multimerized siRNA through cleavable disulphide linkages. The multimerized siRNA can produce more stable and compact polyelectrolyte complexes with less cytotoxic cationic carriers than naked siRNA because of substantially increased charge densities and the presence of flexible chemical linkers in the backbone. The cleavable and multimerized siRNA shows greatly enhanced gene-silencing efficiencies in vitro and in vivo through a target-messenger-RNA-specific RNA interference processing without significantly eliciting immune induction. This study demonstrates that the multimerized siRNA structure complexed with selected cationic condensing agents can serve as potential gene-silencing therapeutics for treating various diseases.

Synthesis, characterization, antitumor activity of pluronic mimicking copolymer micelles conjugated with doxorubicin via acid-cleavable linkage.

Pluronic mimicking poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer having multiple hydroxyl groups in the PPO middle segment (core-functionalized Pluronic: CF-PLU) was synthesized for conjugation of doxorubicin (DOX). DOX was conjugated on the multiple hydroxyl groups of CF-PLU via an acid-labile hydrazone linkage (CF-PLU-DOX). In aqueous solution, CF-PLU-DOX copolymers self-assembled to form a core/shell-type micelle structure consisting of a hydrophobic DOX-conjugated PPO core and a hydrophilic PEO shell layer. The conjugated DOX from CF-PLU-DOX micelles was released out more rapidly at pH 5 than pH 7.4, indicating that the hydrazone linkage was cleaved under acidic condition. CF-PLU-DOX micelles exhibited greatly enhanced cytotoxicity for MCF-7 human breast cancer cells compared to naked DOX, while CF-PLU copolymer itself showed extremely low cytotoxicity. Flow cytometry analysis revealed that the extent of cellular uptake for CF-PLU-DOX micelles was greater than free DOX. Confocal image analysis also showed that CF-PLU-DOX micelles had a quite different intracellular distribution profile from free DOX. CF-PLU-DOX micelles were mainly distributed in the cytoplasm, endosomal/lysosomal vesicles, and nucleus, while free DOX was localized mainly within the nucleus, suggesting that CF-PLU-DOX micellar formulation might be advantageously used for overcoming the multidrug resistance (MDR) effect, which gradually develops in many tumor cells during repeated drug administration.

Self‐crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery.

Enhanced Intracellular Delivery of Quantum Dot and Adenovirus Nanoparticles Triggered by Acidic pH via Surface Charge Reversal

Quantum dot (QD) and adenovirus (ADV) nanoparticles were surface-modified with graft copolymers that exhibited a charge reversal behavior under acidic condition. Poly(L-lysine) (PLL) was grafted with multiple biotin-PEG chains (biotin-PEG-PLL graft copolymer), and the remaining primary amine groups in the PLL backbone were postmodified using citraconic anhydride, a pH-sensitive primary amine blocker, to generate carboxylate groups. The surfaces of streptavidin-conjugated QDs were modified with citraconylated biotin-PEG-PLL copolymer, producing net negatively charged QD nanoparticles. Under acidic conditions, citraconylated amide linkages were cleaved, resulting in the recovery of positively charged amine groups with subsequent alteration of surface charge values. Intracellular delivery of QD nanoparticles was greatly enhanced in an acidic pH condition due to the surface charge reversal. The surface of avidin-conjugated adenovirus (ADV-Avi) encoding an exogenous green fluorescent protein (GFP) gene was also modified in the same fashion. The expression extent of GFP was significantly increased at more acidic pH than pH 7.4. This study demonstrates that various nanosized drug carriers, imaging agents, and viruses could be surface-engineered to enhance their cellular uptake specifically at a low pH microenvironment like solid tumor tissue.

PEGylated and MMP-2 Specifically DePEGylated Quantum Dots: Comparative Evaluation of Cellular Uptake

Polyethylene glycol (PEG)-immobilized quantum dot (QD) nanoparticles, which could be specifically dePEGylated in response to the presence of the matrix metalloprotease-2 (MMP-2) enzyme, were prepared. The degree of PEGylation (MW 3400) on the surface of 12 nm streptavidin-coated QDs was stoichiometrically controlled by varying the feed amount of a biotin-substrate-PEG conjugate, where the substrate contained an MMP-2 cleavable peptide sequence. A biotin-cell penetrating peptide (CPP) conjugate was also immobilized onto the surface of the PEGylated QD surface to enhance the cellular uptake after dePEGylation. It was found that more than nine PEG chains per single QD were required to effectively inhibit the cellular uptake of modified QD particles down to around 20%, as compared with that of QD without PEG chains. However, the treatment of MMP-2 enzyme in the medium resulted in a substantial enhancement in the extent of QD cellular uptake by dePEGylation with concomitant resurfacing of sterically hidden CPP moieties. This study analyzed the effects of surface PEGylation density and MMP-2 specific dePEGylation on the cellular uptake of CPP-QD nanoparticles in a quantitative manner.

Synthesis, characterization, and intracellular delivery of reducible heparin nanogels for apoptotic cell death

Reducible heparin nanogels cross-linked with disulfide linkages were developed for efficient cellular uptake of therapeutic heparin to induce apoptotic cell death. The heparin nanogels were synthesized by forming nanocomplexes between thiolated heparin and poly(ethylene glycol) in a selected organic solvent, and subsequently producing intermolecular disulfide bonds between thiolated heparin molecules by ultrasonication. The resultant heparin nanogels had a stable structure with an average diameter of 248.7+/-26.8nm in aqueous solution. However, they rapidly disintegrated and released free heparin molecules under reductive environments, such as intracellular cytosol, through the cleavage of disulfide cross-links within their network structure. Confocal laser scanning microscopy and flow cytometric analysis revealed that these heparin nanogels significantly inhibited proliferation of mouse melanoma cells by inducing caspase-mediated apoptotic cell death. The present study suggested that the reducible heparin nanogels exhibiting a remarkable apoptotic activity could be potentially applied for cancer cell targeted delivery when combined with various therapeutic and diagnostic agents.

Self-assembled siRNA-PLGA conjugate micelles for gene silencing

Biodegradable poly(D,L-lactic-co-glycolic acid) (PLGA) was conjugated to the 3 end of small interfering RNA (siRNA) via a disulfide bond to synthesize siRNA-PLGA hybrid conjugates. siRNA-PLGA conjugates were spontaneously self-assembled to form a spherical core/shell type micellar structure of ~20 nm in an aqueous environment, probably by hydrophobic interaction of PLGA blocks in the core surrounded by an siRNA shell layer. When linear polyethylenimine was added to the siRNA-PLGA micelles in aqueous solution, stable siRNA-PLGA/LPEI micelles with a size of ~30 nm were produced via ionic complexation between siRNA and LPEI in the outer shell. The cationic siRNA-PLGA/LPEI micelles showed superior intracellular uptake and enhanced gene silencing effect, compared to naked siRNA/LPEI complexes. The hybrid micelle structure based on siRNA and PLGA can be potentially used as an efficient siRNA delivery system for gene silencing.

Gene silencing efficiency of siRNA-PEG conjugates: Effect of PEGylation site and PEG molecular weight

Small interfering RNA (siRNA) was conjugated with poly(ethylene glycol) (PEG) at four different terminal ends (sense 3, sense 5, antisense 3, and antisense 5) via cleavable disulfide and noncleavable thioether linkages to evaluate their gene silencing efficiencies upon complexation with Lipofectamine2000. The PEGylation site at the four siRNA termini and PEG molecular weight were not critical factors to significantly affect gene silencing activities. Cleavable siRNA-PEG conjugates showed comparable gene silencing activities to naked siRNA, and exhibited sequence-specific degradation of a target mRNA. Interestingly, noncleavable siRNA-PEG conjugates were processed by Dicer, enabling to exert RNAi effect without showing a target sequence-specific manner. However, only cleavable siRNA-PEG conjugates significantly reduced the extent of INF-alpha release as compared to noncleavable siRNA-PEG conjugates, suggesting that they can be potentially used for therapeutic siRNA applications.

Multifunctional siRNA delivery system: Polyelectrolyte complex micelles of six‐arm PEG conjugate of siRNA and cell penetrating peptide with crosslinked fusogenic peptide

For therapeutic applications of small interfering RNA (siRNA), serum stability, enhanced cellular uptake, and facile endosome escape are key issues for designing carriers. In this study, green fluorescent protein (GFP) siRNA was conjugated to a six‐arm polyethylene glycol (PEG) derivative via a reducible disulfide linkage (6PEG‐siRNA). The 6PEG‐siRNA conjugate was also functionalized with a cell penetrating peptide, Hph1 to enhance its cellular uptake property (6PEG‐siRNA‐Hph1). The 6PEG‐siRNA‐Hph1 conjugate was electrostatically complexed with cationic self‐crosslinked fusogenic KALA peptide (cl‐KALA) to form multifunctional polyelectrolyte complex micelles for gene silencing. The resultant siRNA complex formulation with multiple PEG chains showed superior physical stability and resistance to enzymatic degradation. The 6PEG‐siRNA‐Hph1/cl‐KALA complexes exhibited enhanced GFP gene silencing efficiency for MDA‐MB‐435 cells in the serum containing condition. The current reducible and multifunctional polyelectrolyte complex micelles are expected to have high potential for efficient delivery of therapeutic siRNA.