Hwa Yeon Jeong

Leukemia-specific siRNA delivery by immunonanoplexes consisting of anti-JL1 minibody conjugated to oligo-9 Arg-peptides

Targeted mRNA degradation by short interfering RNAs (siRNAs) offers a great potential to treat cancers. siRNA therapeutics for leukemias are, however, hindered by poor intracellular uptake, limited blood stability and nonspecific delivery. To solve these problems, we developed an anti-JL1 immunonanoplex (antibody-coupled nanocomplex) for siRNA delivery using anti-JL1 minibody (leukemia cell-specific minibody) conjugated to oligo-9-Arg peptide (9R) for effective siRNA delivery to leukemic cells. The anti-JL1 immunonanoplexes were able to deliver siRNA specifically to leukemic cells (CEM and Jurkat), but not to control cancer cells (H9). According to FACS and confocal microscopic analysis, siRNAs delivered by immunonanoplex particles were rapidly taken up by the JL1-positive cancer cells in 2 h. Furthermore, we showed that the anti-JL1 immunonanoplexes were effectively targeted to JL1-positive cells (CEM) inoculated in the mouse bone marrow. These results suggest that the anti-JL1 immunonanoplex is a powerful siRNA delivery system for human leukemia therapies.

Cancer-targeted Nucleic Acid Delivery and Quantum Dot Imaging Using EGF Receptor Aptamer-conjugated Lipid Nanoparticles

Co-application of fluorescent quantum dot nanocrystals and therapeutics has recently become a promising theranostic methodology for cancer treatment. We developed a tumor-targeted lipid nanocarrier that demonstrates notable efficacy in gene delivery as well as tumor bio-imaging. Coupling of aptamer molecules against the EGF receptor (EGFR) to the distal termini of lipid nanoparticles provided the carrier with tumor-specific recognition capability. The cationic lipid component, referred to as O,O’-dimyristyl-N-lysyl glutamate (DMKE), was able to effectively complex with anionic small-interfering RNA (siRNA). The hydrophobic quantum dots (Q-dots) were effectively incorporated in hydrophobic lipid bilayers at an appropriate Q-dot to lipid ratio. In this study, we optimized the liposomal formula of aptamer-conjugated liposomes containing Q-dots and siRNA molecules (Apt-QLs). The anti-EGFR Apt-QLs exhibited remarkable EGFR-dependent siRNA delivery as well as fluorescence imaging, which were analyzed in cultured cancer cells and tumor xenografts in mice. These results imply that the formulation of Apt-QLs could be widely utilized as a carrier for tumor-directed gene delivery and bio-imaging.

Sendai viroplexes for epidermal growth factor receptor-directed delivery of interleukin-12 and salmosin genes to cancer cells.

The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor‐directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off‐target effects of gene transfection.

Sendai Viroplexes for EGF Receptor‐directed Delivery of IL12 and Salmosin Genes to Cancer Cells

The effective delivery of therapeutic genes to target cells has been a fundamental goal in cancer gene therapy because of its advantages with respect to both safety and transfection efficiency. In the present, study we describe a tumor‐directed gene delivery system that demonstrates remarkable efficacy in gene delivery and minimizes the off‐target effects of gene transfection.

Tumor-specific delivery of therapeutic siRNAs by anti-EGFR immunonanoparticles

Background Efficient target-specific siRNA delivery has always been a primary concern in the field of siRNA clinical application. Purpose In this study, four different types of anti-epidermal growth factor receptor (EGFR) antibody-conjugated immunonanoparticles were prepared and tested for cancer cell-targeted therapeutic siRNA delivery. Materials and methods The prepared nanoparticles encapsulating siRNAs were character-ized by gel retardation and particle analysis using a Zetasizer. In vitro transfection and reduction of target genes, vimentin and JAK3, were determined using quantitative reverse transcription polymerase chain reaction. In vivo tumor targeting and antitumoral efficacies of the nanoparticles were evaluated in mice carrying tumors. Results Among these immunonanoparticles, anti-EGFR immunolipoplexes and immunoviroplexes exhibited remarkable cell binding and siRNA delivery to EGFR-expressing tumor cells compared to immunoliposomes and immunovirosomes. Especially, the anti-EGFR immunoviroplexes exhibited the most efficient siRNA transfection to target tumor cells. Therefore, antitumoral vimentin and Janus kinase-3 siRNAs were loaded in the anti-EGFR immunolipoplexes and immunoviroplexes, which were tested in mice carrying SK-OV-3 tumor xenografts. In fact, the therapeutic siRNAs were efficiently delivered to the tumor tissues by both delivery vehicles, resulting in significant inhibition of tumor growth. Moreover, administration of doxorubicin in combination with anti-EGFR immunoviroplexes resulted in remarkable and synergistic tumor growth inhibition. Conclusion This study provides experimental proof that cancer cell-targeted immunoviroplexes are an efficient siRNA delivery system for cancer therapy. Moreover, this study also suggests that a combination of conventional chemotherapy and tumor-directed anticancer siRNA therapy would be a better modality for cancer treatment.

155. Cancer-Specific Theranosis Using Quantum Dots and siRNA Co-Loaded in Micellar Nanoparticles

Theranostics is a proposed process of diagnostic therapy for an individual patient. It has been considered a powerful tool of personalized medicine. In this study, we have prepared lipid micellar nanoparticles encapsulating quantum dots (QDs) as a diagnostic agent and incorporating cholesterol-siRNA (Cho-siRNA) as a therapeutic agent. Furthermore, for targeted delivery of QDs and Cho-siRNA, anti-EGFR antibodies or aptamers were conjugated to the surface of the nanoparticles. First of all, for stable nanostructure of lipid micelles, we determined an optimal molar ratio of lipids, QDs, and Cho-siRNA as 350:1:0.35. The size of QD micelles was approximately 50 nm that is suitable for efficient localization in tumors via enhanced permeability and retention (EPR) effect. Their serum stability and pH stability suggest that the QD micelle structure is stable enough to be applied under biological conditions. The RNase protection assay showed that Cho-siRNA molecules in the QD micelles were effectively protected by steric shielding of hydrophilic polyethylene glycol molecules. The analyses with a flow cytometry and a confocal microscope showed that the nanoparticles can effectively deliver Cho-siRNA to the target cells, followed by effective residing of siRNA molecules in the cytoplasm. A further systematic evaluation of the micellar nanoparticles established in this study would verify their value as a theranostic vehicle.

590. Anti-EGFR Aptamer-Conjugated Lipoplexses, a Novel Gene Delivery Strategy for Triple Negative Breast Cancer Therapy

Basal-like breast cancer, one of the subtypes of breast cancer, have been focused because of their pathologic features like absence of three common markers such as estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu). The basal subtype is also called triple negative breast cancer (TNBC) and has shown a higher rate of metastasis, poor prognosis, unresponsive to therapy. Especially, their lack of HER2 expression and aggressive progression make it difficult to utilize HER2-targeted therapeutics and diagnostics to the metastatic TNBC. In this study, theranostic cationic liposomes consisting of quantum dots (QDs) and O,O’-dimyristyl-N-lysyl glutamate (DMKE) were prepared. The quantum dot-incorporating cationic liposomes (QCLs) were then complexed with Bcl-2 and PKC- λ/ι small interfering RNA (siRNA) for silencing of genes inhibiting apoptosis, and promoting cell proliferation, and cell migrations. The cationic characteristics of the QCLs provided an effective siRNA transfection in vitro, but it had limitations in in vivo applications. To enhance cancer-directed delivery, epidermal growth factor receptor (EGFR) aptamer-polyethylene glycol conjugates were inserted into the prepared QCLs. The advantages of the two-step strategy of QCL preparation include effective QD formulation, in vivo long circulation, and TNBC specific gene silencing. This study suggests that EGFR aptamer-conjugated QCL can be utilized as a useful theranostic agent for TNBC subtype breast cancers.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

149. Theranostic Aptamer-Conjugated Liposomal Nanoparticle for Triple Negative Breast Cancer

Triple-negative breast cancers (TNBC) have been focused because of their pathologic features like absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/neu) expressions and a higher rate of metastasis. Especially, their lack of HER2 expression and aggressive progression make it difficult to utilize HER2-targeted therapeutics and diagnostics to the metastatic TNBC. In this study, first of all quantum dots were incorporated into cationic liposomes consisting of O,O’-dimyristyl-N-lysyl glutamate (DMKE). The quantum dot-incorporating cationic liposomes (QCLs) were then complexed with therapeutic small interfering RNA (siRNA) for sequence-specific RNA interference. The cationic characteristics of the QCLs provided an effective siRNA transfection in vitro, but it had limitations in in vivo applications. To enhance cancer-directed delivery, epidermal growth factor receptor (EGFR) aptamer-polyethylene glycol conjugates were inserted into the prepared QCLs. The advantage of the two-step strategy of QCL preparation includes enhanced diagnostic feature, in vivo long circulation, and TNBC specific gene silencing. This research suggests that aptamer-conjugated QCL can be utilized as a useful theranostic agent for TNBC subtype breast cancers.

151. EGFR-Directed Micellar Nanoparticles for Cancer Theranosis

In this study, we have prepared lipid micellar nanoparticles encapsulating quantum dots (QDs) as a diagnostic agent and incorporating cholesterol-siRNA (Cho-siRNA) as a therapeutic agent. Furthermore, for targeted co-delivery of QDs and Cho-siRNA, anti-EGFR antibodies or aptamers were conjugated to the surface of the nanoparticles. Firstly, to optimize the nanostructure of lipid micelles, we determined an optimal molar ratios of lipids, QDs, and Cho-siRNA as 350:1:0.35. The size of QD micelles was about 50 nm that is suitable for efficient localization in tumors via enhanced permeability and retention (EPR) effect. Their serum stability and pH stability suggest that the QD micelle structure is stable enough to be applied to biological conditions. The RNase protection assay showed that Cho-siRNA molecules in the QD micelles were effectively protected by sterically shielding of hydrophilic polyethylene glycol molecules. The analyses with a flow cytometry and a confocal microscope showed that the nanoparticles can effectively deliver Cho-siRNA to the target cells, followed by effective residing of siRNA molecules in the cytoplasm. These results suggest that the micellar nanoparticles established in this study would be widely utilized as a theranostic vehicle.