Simultaneous delivery of multiple gene-targeting siRNAs/anticancer drug for synergistically enhanced treatment of prostate cancer

다국어 초록 (Multilingual Abstract)

Simultaneous silencing of multiple apoptosis-related genes is an attractive approach to treat cancer. In this paper, it is presented for a multiple gene-targeting siRNA/drug delivery system for prostate cancer treatment with high efficiency. Bcl-2, survivin and androgen receptor genes involved in the cell apoptosis pathways were chosen as silencing targets with three different siRNAs. The colloidal nanocomplex delivery system (<10 nm in size) was formulated electrostatically between anionic siRNAs and a cationic drug (BZT), followed by encapsulation with Pluronic F-68 polymer. The formulated nanocomplex system exhibited sufficient stability against nuclease-induced degradation, leading to successful intracellular delivery for the desired therapeutic performance. Silencing of targeted genes and apoptosis induction were evaluated in vitro on human prostate LNCaP-LN3 cancer cells by using various biological analysis tools (e.g., real-time PCR, MTT cell viability test, and flow cytometry). It was demonstrated that when the total loaded siRNA amounts were kept the same in the nanocomplexes, the simultaneous silencing of triple genes with co-loaded siRNAs (i.e., Bcl-2, survivin, and AR-targeting siRNAs) induced apoptosis of cancer cells more efficiently than the silencing of each single gene alone, offering a novel way of improving the efficacy of gene therapeutics of prostate cancer.

목차 (Table of Contents)

• List of Figures iv
• Abstract vi
• 1. Introduction 1
• 1.1. Characteristics of prostate 1
• 1.1.1. General information of prostate cancer 1
• 1.1.2. Androgen and prostate cancer 2
• 1.2. Relation of apoptotic proteins with prostate cancer proliferation 4
• 1.2.1. anti-apoptotic protein, Bcl-2 4
• 1.2.2. Survivin 5
• 1.2.3. Androgen receptor (AR) 5
• 1.3. The overview of siRNA and siRNA delivery system 7
• 1.3.1. Central dogma and mechanisms of siRNA in cells 7
• 1.3.2. The challenges for siRNA delivery system 8
• 1.3.3. General methods for siRNA delivery 9
• 1.3.4. Previous study for siRNA/drug co-delivery system 10
• 1.4. Design for multiple gene-targeting siRNA/drug nanocomplex 11
• 2. Materials and Methods 14
• 2.1. Materials 14
• 2.2. Characterization and evaluation of SDNC 14
• 2.2.1. Preparation and characterization of SDNC 14
• 2.2.2. Gel retardation assay 15
• 2.3. In vitro studies 15
• 2.3.1. Cell culture 15
• 2.3.2. Cellular uptake 16
• 2.3.3. Assessment of gene silencing 16
• 2.3.4. Cytotoxicity and apoptosis assay 17
• 3. Results & Discussion 18
• 3.1. Preparation and characterization of SDNC 18
• 3.1.1. Preparation of SDNC 18
• 3.1.2. Customizing of proper ratio of siRNA and BZT in SDNC 18
• 3.1.3. Evaluation of SDNC consistency 21
• 3.1.4. Stability evaluation of SDNC 23
• 3.2. In vitro studies 25
• 3.2.1. Cellular uptake 25
• 3.2.2. Assessment of gene silencing 27
• 3.2.3. Cytotoxicity for treating SDNCs 29
• 3.2.4. Apoptosis assay for treating SDNCs 32
• 4. Conclusions 34
• 5. Oligonucleotide sequences 36
• 6. References 37
• 국문초록 43