Soo Hyeon Lee

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.

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.

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.

Controlled synthesis of PEI-coated gold nanoparticles using reductive catechol chemistry for siRNA delivery

Development of nano-sized gene delivery vehicles for small interfering RNA (siRNA) delivery is of great importance for their clinical applications such as cancer therapy. Herein, we demonstrate the controlled synthesis of polyethyleneimine (PEI)-coated gold nanoparticles (AuNPs) using catechol-conjugated PEI (PEI-C) for siRNA delivery. Since the conjugated catechol groups are reductive and moderately hydrophobic, PEI-C formed spherical multi-cored micelles in aqueous solution and served as reductive templates for the growth and synthesis of spherical AuNPs with tunable sizes and surface charges. PEI-C was stably anchored on the surface of growing crystal gold seeds with crosslinking, resulting in robust cationic AuNPs. The fabricated PEI-coated AuNPs formed stable complexes with siRNA, and the complexes showed an excellent gene silencing effect in cancer cells. Size and surface charge values of the synthesized AuNPs had a great influence on intracellular uptake and unpacking of siRNA, and the resultant gene silencing efficiency. The PEI-coated AuNPs exhibited an extremely low cytotoxicity due to the reduced density of primary amine groups and the absence of uncomplexed PEI fraction in aqueous solution.

Is there a future for cell-penetrating peptides in oligonucleotide delivery?

Cell-penetrating peptides have been widely investigated as delivery vehicles for oligonucleotides (e.g., siRNA and antisense oligonucleotides). Different delivery strategies can be used, such as co-incubation, direct conjugation, non-covalent complex, and modification on the surface of liposome or polymer complexes. However, several challenges remain for their preclinical and clinical development. Endosomal escape, lack of cell/tissue specificity, and toxicity are major concerns in the design of cell-penetrating peptide-mediated delivery systems. In this commentary, we highlight recent reports of cell-penetrating peptide incorporation into oligonucleotide delivery systems and underline the remaining challenges, particularly for preclinical and clinical 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.

Optically traceable solid lipid nanoparticles loaded with siRNA and paclitaxel for synergistic chemotherapy with in situ imaging.

Here, we report quantum dot-incorporating solid lipid nanoparticles (SLNs) for anticancer theranostics with synergistic therapeutic effects of paclitaxel-siRNA combination. The natural components of a low-density lipoprotein (LDL) are reconstituted to produce LDL-mimetic SLNs having a stable core/shell nanostructure incorporating quantum dots and paclitaxel within the lipid shell while anionic siRNA molecules are electrostatically complexed with the outer surface of SLNs. The produced SLN/siRNA complexes efficiently deliver both of paclitaxel and Bcl-2 targeted siRNA into human lung carcinoma cells and exhibit synergistic anticancer activities by triggering caspase-mediated apoptosis as determined by median effect plot analysis. Moreover, the strong fluorescence from quantum dots within SLNs enables in situ visualization of intracellular translocation of SLNs into cancer cells. Our study suggests that LDL-mimetic SLNs can be utilized as a multifunctional and optically traceable nanocarrier for efficient anticancer theranostics.

Small-Interfering RNA (siRNA)-Based Functional Micro- and Nanostructures for Efficient and Selective Gene Silencing

Because of RNAs ability to encode structure and functional information, researchers have fabricated diverse geometric structures from this polymer at the micro- and nanoscale. With their tunable structures, rigidity, and biocompatibility, novel two-dimensional and three-dimensional RNA structures can serve as a fundamental platform for biomedical applications, including engineered tissues, biosensors, and drug delivery vehicles. The discovery of the potential of small-interfering RNA (siRNA) has underscored the applications of RNA-based micro- and nanostructures in medicine. Small-interfering RNA (siRNA), synthetic double-stranded RNA consisting of approximately 21 base pairs, suppresses problematic target genes in a sequence-specific manner via inherent RNA interference (RNAi) processing. As a result, siRNA offers a potential strategy for treatment of many human diseases. However, due to inefficient delivery to cells and off-target effects, the clinical application of therapeutic siRNA has been very challenging. To address these issues, researchers have studied a variety of nanocarrier systems for siRNA delivery. In this Account, we describe several strategies for efficient siRNA delivery and selective gene silencing. We took advantage of facile chemical conjugation and complementary hybridization to design novel siRNA-based micro- and nanostructures. Using chemical crosslinkers and hydrophobic/hydrophilic polymers at the end of siRNA, we produced various RNA-based structures, including siRNA block copolymers, micelles, linear siRNA homopolymers, and microhydrogels. Because of their increased charge density and flexibility compared with conventional siRNA, these micro- and nanostructures can form polyelectrolyte complexes with poorly charged and biocompatible cationic carriers that are both more condensed and more homogenous than the complexes formed in other carrier systems. In addition, the fabricated siRNA-based structures are linked by cleavable disulfide bonds for facile generation of original siRNA in the cytosol and for target-specific gene silencing. These newly developed siRNA-based structures greatly enhance intracellular uptake and gene silencing both in vitro and in vivo, making them promising biomaterials for siRNA therapeutics.

Enzyme-mediated cross-linking of Pluronic copolymer micelles for injectable and in situ forming hydrogels

A new class of injectable and erodible hydrogels exhibiting highly robust gel strength at body temperature was fabricated by enzyme-mediated cross-linking between Pluronic copolymer micelles. Tyramine-conjugated Pluronic F-127 tri-block copolymers at two terminal ends of polyethylene oxide (PEO) side chains were synthesized and utilized to form self-assembled micelles in aqueous solution. Tyrosinase was employed to convert tyramine-conjugated micelles to highly reactive catechol conjugated micelles that could further cross-link individual Pluronic copolymer micelles to form a highly stable gel structure. The enzyme cross-linked Pluronic hydrogels showed far lower critical gelation concentration, concomitantly showing enhanced gel strength compared to unmodified Pluronic copolymer hydrogels, suitable for sustained delivery of bioactive agents. Rheological studies demonstrated that the enzyme cross-linked hydrogels exhibited a fast and reversible sol-gel transition in response to temperature while maintaining sufficient mechanical strength at the gel state. In situ formed hydrogels were eroded gradually, releasing FITC-labeled dextran in an erosion-controlled manner. Moreover, they showed tissue-adhesive properties due to the presence of unreacted catechol groups in the gel structure. Enzyme cross-linked Pluronic hydrogels could be potentially used for delivery applications of drugs and cells.

Activatable Cell Penetrating Peptide–Peptide Nucleic Acid Conjugate via Reduction of Azobenzene PEG Chains

The use of stimuli-responsive bioactive molecules is an attractive strategy to circumvent selectivity issues in vivo. Here, we report an activatable cell penetrating peptide (CPP) strategy ultimately aimed at delivering nucleic acid drugs to the colon mucosa using bacterial azoreductase as the local reconversion trigger. Through screening of a panel of CPPs, we identified a sequence (M918) capable of carrying a nucleic acid analogue payload. A modified M918 peptide conjugated to a peptide nucleic acid (PNA) was shown to silence luciferase in colon adenocarcinoma cells (HT-29-luc). Reversible functionalization of the conjugates lysine residues via an azobenzene self-immolative linkage abolished transfection activity, and the free CPP-PNA was recovered after reduction of the azobenzene bond. This activatable CPP conjugate platform could find applications in the selective delivery of nucleic acid drugs to the colon mucosa, opening therapeutic avenues in colon diseases.