Application of Curcumin for Targeting Tumor Receptor: Gene Delivery

다국어 초록 (Multilingual Abstract)

Despite advances in tumor diagnosis and chemotherapy development, cancer has still maintained a top cause of disease-related human death worldwide. Although many advanced tumor patients are necessary the debulking surgery, extensive chemotherapy is also recommended because micronodular and floating tumor colonies cannot be removed by surgery or radiation. Therefore, the exact delivery of anti-tumor drugs (chemical drugs, genetic materials, and proteins) to tumor tissues or cells is very important to reduce cytotoxicity and dosage. Currently, targeted drug delivery systems (DDS) using nanotechnologies have presented as a solution. The aim of this study is to investigate the potential of curcumin and its derivatives as a novel vi
cancer-targeting ligand and suitable structure among phenylalanine (Ph), cinnamic acid (Ci), coumaric acid (Co), ferulic acid (Fe), and curcumin (Cu) which are produced in biosynthesis pathway of curcumin. Dextran grafted with branched polyethylenimine (bPEI) (DP) and lysine dendrimer peptide conjugated without (dK) or with PEGylated targeting ligands (dKPh, dKCi, dKCo, dKFe and dKCu) were successfully synthesized and their mixtures formulated in 9.5:0.5, 9:1, and 8:2 w/w ratios were used as a targeted gene carrier (DPK, DPKPh, DPKCi, DPKCo, DPKFE, and DPKCu). All tested gene carriers showed excellent DNA binding capability, DNA protection ability from nuclease, and exhibited condensation ability to compact DNA into uniform nanoparticles. In vitro gene transfection efficiency showed that DPKCi, DPKCo and DPKCu polyplexes were higher than DPK without targeting ligands in MCF-7 and SKBr3 breast cancer cells, but not in other cancer cell lines containing SKOV-3, PC3 and DLD-1. Moreover, gene transfection of their polyplexes were significantly inhibited after pre-incubation of curcumin or 17α-Estradiol in dose-dependent manner. Uptake and endosomal escape of polyplexes was qualitatively studied using confocal laser scanning microscope (CLSM), resulted in their targeting specificity. In vitro excellent anti-tumor activity of them was due to induced apoptosis via specific delivery of psiRNA-hBCL2, which expresses anti-bcl-2 shRNA, and vii
proved by increased caspase-9 and caspase-3. Presented results highlighted that curcumin and coumaric acid can specifically target membrane estrogen receptor α and target receptor of cinnamic acid is a membrane estrogen receptor α and β. This study demonstrated that DPKCi, DPKCo, DPKCu gene delivery system would have great potential for targeted therapy to breast cancer cells, at least, MCF-7 and SKBr3.

목차 (Table of Contents)

• I. Introduction 1
• II. Materials and Methods 8
• II-1. Materials 8
• II-2. Cell culture 9
• II-3. Peptide synthesis 9
• II-4. Preparation of copolymers 9
• II-5. Proton-nuclear magnetic resonance (1H-NMR) spectra 10
• II-6. Dynamic light scattering (DLS) of polyplexes 11
• II-7. Measurements of DNA binding affinity 11
• II-8. Transfection efficiency of polyplexes in various cell lines 12
• II-9. In vitro cytotoxicity and antitumor activity 12
• II-10. Titration efficacy of copolymer/pDNA complexes 13
• II-11. Cellular uptake of nanoparticles 13
• II-12. Endosome escape of nanoparticles 14
• II-13. Statistical analysis 14
• III. Results and discussion 16
• III-1. Designation and characterization of synthesized copolymers 16
• III-2. Nanoparticle formation of pDNA/copolymers complexes 26
• III-3. Transfection efficiency of polyplexes with pDNA 31
• III-4. Hemolytic and cytotoxic effects of polyplexes with pDNA 37
• III-5. Dose-dependent inhibition of gene transfection after pre- incubation of curcumin of estradiol 39
• III-6. Cellular uptake and cellular distribution 45
• III-7. Anti-tumor effects of polyplexes in tumor cell lines 48
• III-8. Transfected psiRNA-hBCL2 induces apoptosis via over-expression of caspase 9 and 3 51
• IV. Conclusion 53
• V. Reference 54