化療長久以來被視為有效的癌症治療方式。然而，癌症產生的抗藥性大大阻礙了化療藥物的效果。 因此，本研究的目的是開發一個癌症治療策略可同時抑制抗藥性以及腫瘤生長，改善化療藥物的投遞效果，並達成更有效的癌症治療成效。 為了達成此目標，研究中設計出可注射式水膠系統來同時搭載化療藥物以及抑制抗藥性的基因片段，該水膠可直接將藥物傳遞到患部，不僅能提高藥物傳輸效率，也可避免在血液中產生投遞藥物時造成的潛在副作用。 在本研究中，水膠結構是由十字形聚乙二醇組成，並且藉由雙股的去氧核醣核酸自組裝性質交聯而成; 此外，水膠中也加入了層狀矽酸鹽來提升水膠的強度與成膠性質。 由於上述兩項交聯劑皆利用弱鍵結交聯水膠結構，因此本水膠具有可注射性質以及自我修復能力。這些交聯劑因為其特殊結構，亦可用來鑲嵌搭載常見的化療藥物阿黴素; 另ㄧ方面，抑制抗藥性的基因片段則可藉由與正電分子的靜電作用力包覆於水膠中傳遞。 在水膠中我們亦設計了一段酵素敏感性鍵結作用於控制水膠降解，進而造成藥物的釋放，此設計是針對腫瘤微環境大量釋放的酵素作為刺激藥物釋放的因子。 研究結果證明我們成功合成出了水膠結構，此水膠具有原位成膠性質，可注射性，以及隨著腫瘤微環境分泌酵素變化的可降解性。 另外，由於阿黴素在水膠中分別鑲嵌於兩種搭載因子上，兩者不同的釋放曲線能達成前後順序性的藥物釋放，以提升抗癌效果。 最重要的是，本研究中採取抑制抗藥性基因與化療藥物的共同搭配系統，確實能有效降低具抗藥性的癌症細胞存活率。 結合上述精心設計，我們開發出能同時搭載多種抗癌分子的水膠系統，達到控制包覆藥物以及抑制抗藥性基因片段的釋放順序，有效提升抗癌效果與降低腫瘤細胞的抗藥性。

Multidrug resistance (MDR) has been considered as a main impediment to cancer chemotherapy. In order to simultaneously overcome drug resistance and inhibit tumor growth, it is essential to develop a drug delivery system able to carry multiple therapeutic agents. Injectable hydrogel has been widely used in this aspect due to its in situ gelation property and the ability to encapsulate different bioactive genetic substances and drug molecules. In this study, tetra-armed polyethylene glycol (PEG) is crosslinked by complementary nucleic acid sequences to form the network structure of hydrogel with an enzyme-cleavable peptide motif in between. Additionally, laponite nanoclay is added to finely tune the gelation and mechanical properties of the hydrogel. Due to specific hydrogen bonds formation between nucleobase pairing, the hydrogel is injectable and self-healable. In this hydrogel, both nucleic acid sequence and laponite nanoclay can be tailor-designed as loading agents for anti-cancer drug. Moreover, MDR-targeted siRNAs are complexed with stearyl-octaarginine (STR-R8) in the hydrogel to further enhance therapeutic efficacy by overcoming MDR. The enzyme-cleavable peptide between tetra-armed PEG and DNA deliberately controls hydrogel degradation; thus, resulting in the release of doxorubicin (Dox) and siRNA in response to tumor microenvironment. The results show the successful configuration of hydrogel network structure with in situ gelation property, injectability, and degradable with the existence of tumor-associated enzyme, MMP-2. Dox is released sequentially from intercalated DNA and nanoclay lattice structure. The synergistic effect by cooperating MDR-targeted siRNAs and Dox is observed with the enhanced anti-cancer effect on drug resistant breast cancer cells. We suggest that with the tailor-designed hydrogel system, recurrent multidrug resistance in tumor cells can be significantly inhibited by the co-delivery of multiple therapeutic agents with spatial-temporal control release.

CHAPTER 1. INTRODUCTION 1
1.1 MULTIDRUG RESISTANCE (MDR) 1
1.1.1 Pump resistance 1
1.1.2 Non-pump resistance 2
1.1.3 Pgp transporters and Bcl-2 proteins 3
1.2 SIGNIFICANCE AND OBJECTIVE 4
1.3 COMBINATION THERAPY OVERCOMING DRUG RESISTANCE 5
1.3.1 RNA interference technology 5
1.3.2 Co-delivery of chemotherapeutic drugs together with MDR-targeted siRNA 8
1.4 INJECTABLE HYDROGELS IN DRUG DELIVERY 9
1.4.1 Supramolecular hydrogels 10
1.4.2 Self-assembled DNA crosslinked hydrogel 10
1.4.3 Poly(ethylene glycol) (PEG)/Laponite nanocomposite hydrogel 12
1.5 DRUG DELIVERY THROUGH SUPRAMOLECULAR INTERACTIONS 13
1.5.1 Double stranded DNA/ Dox intercalation 14
1.5.2 Laponite/ Dox intercalation 15
1.5.3 STR-R8/ siRNA complex 17
1.6 DISEASE TRIGGERED DELIVERY SYSTEM--- MMP-RESPONSIVE SMART DRUG DELIVERY SYSTEM 17
1.6.1 Matrix metalloproteinase (MMP) 18
1.6.2 MMP-responsive smart drug delivery systems 19
1.7 MOTIVATION AND DESIGN STRATEGY OF HYDROGEL 21
1.7.1 Motivation 21
1.7.2 Design rationale of the hydrogel 21
1.8 THE NICHE OF THIS STUDY 23
CHAPTER 2. MATERIALS AND METHODS 25
2.1 MATERIAL LIST 25
2.2 EXPERIMENTAL DESIGN 27
2.3 SYNTHESIS OF HYDROGEL NETWORK STRUCTURE 27
2.3.1 Modification of maleimide functional groups on tetra-armed PEG 27
2.3.2 Synthesis of tetra-armed PEG-peptide 27
2.3.3 Synthesis of tetra-armed PEG-peptide-DNA 28
2.4 CHARACTERIZATION OF CHEMICAL STRUCTURE 29
2.4.1 Proton nuclear magnetic resonance (1H NMR) 29
2.4.2 Fourier transform infrared spectroscopy (FTIR) 29
2.4.3 TNBS assay 30
2.5 FORMATION AND CHARACTERIZATION OF SUPRAMOLECULAR HYDROGEL 30
2.5.1 Hydrogel preparation 30
2.5.2 Temperature responsive property of DNA crosslinked hydrogel 30
2.5.3 Mechanical properties of hydrogel 30
2.5.4 Cleavage test of MMP-cleavable peptide by type Ⅳ collagenase 31
2.6 ANALYSES OF DOXORUBICIN LOADING CAPACITY AND RELEASE PROFILE 31
2.6.1 Intercalation of doxorubicin into double stranded DNA 31
2.6.2 Intercalation of doxorubicin within Laponite crystal stack 32
2.6.3 Release profile of doxorubicin from dsDNA-Dox conjugates 32
2.6.4 Release profile of doxorubicin from Laponite-Dox complexes 33
2.7 IN VITRO STUDY 33
2.7.1 Culture of breast cancer cells and breast epithelial cell 33
2.7.2 Cytotoxicity and anti-cancer effect 33
2.7.3 Statistical analysis 34
CHAPTER 3. RESULTS 35
3.1 CHARACTERIZATION OF HYDROGEL NETWORK STRUCTURE 35
3.1.1 Characterization of maleimide modified tetra-armed PEG 35
3.1.2 Characterization of tetra-armed PEG-peptide 37
3.1.3 Characterization of tetra-armed PEG-peptide-DNA 37
3.2 EVALUATION OF THE SUPRAMOLECULAR INJECTABLE HYDROGEL 39
3.2.1 Temperature responsiveness of DNA-crosslinked hydrogel 39
3.2.2 Mechanical properties of the supramolecular injectable hydrogel 39
3.2.3 Cleavage of MMP-cleavable peptide by type Ⅳ collagenase 41
3.3 ANALYSES OF DOXORUBICIN-LOADED AGENTS 42
3.3.1 Quantification of loaded doxorubicin 42
3.3.2 Release kinetics of doxorubicin from double stranded DNA and Laponite 43
3.4 IN VITRO STUDY 44
3.4.1 Therapeutic effect of doxorubicin and MDR-targeted siRNAs 44
CHAPTER 4. DISCUSSION 48
4.1 PHYSIOCHEMICAL PROPERTIES OF SUPRAMOLECULAR HYDROGEL 48
4.2 THERAPEUTIC EFFECT OF THE COMBINATION OF DOX AND MDR-TARGETED SIRNAS 50
CHAPTER 5. CONCLUSION 52
5.1 CONCLUSION 52
REFERENCE 54

1. Kartal-Yandim, M., A. Adan-Gokbulut, and Y.J.C.r.i.b. Baran, Molecular mechanisms of drug resistance and its reversal in cancer. Crit Rev Biotechnol, 2016. 36(4): p. 716-726.
2. Chen, A.M., et al., Co‐delivery of doxorubicin and Bcl‐2 siRNA by mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug‐resistant cancer cells. Small, 2009. 5(23): p. 2673-2677.
3. Housman, G., et al., Drug resistance in cancer: an overview. Cancers, 2014. 6(3): p. 1769-1792.
4. Kathawala, R.J., et al., The modulation of ABC transporter-mediated multidrug resistance in cancer: a review of the past decade. Drug Resist Updates, 2015. 18: p. 1-17.
5. Gottesman, M.M., T. Fojo, and S.E. Bates, Multidrug resistance in cancer: role of ATP–dependent transporters. Nat Rev Cancer, 2002. 2: p. 48-58.
6. Bar-Zeev, M., Y.D. Livney, and Y.G. Assaraf, Targeted nanomedicine for cancer therapeutics: Towards precision medicine overcoming drug resistance. Drug Resist Updates, 2017. 31: p. 15-30.
7. Waghray, D. and Q. Zhang, Inhibit or Evade Multidrug Resistance P-Glycoprotein in Cancer Treatment. J Med Chem, 2018. 61(12): p. 5108-5121.
8. Kang, M.H. and C.P. Reynolds, Bcl-2 inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res, 2009. 15(4): p. 1126-1132.
9. Delbridge, A.R. and A. Strasser, The BCL-2 protein family, BH3-mimetics and cancer therapy. Cell Death Differ, 2015. 22(7): p. 1071-1080.
10. Sun, Y., et al., Co-delivery of dual-drugs with nanoparticle to overcome multidrug resistance. Eur J Bio Med Res, 2016. 2(2): p. 12-18.
11. Resnier, P., et al., A review of the current status of siRNA nanomedicines in the treatment of cancer. Biomaterials, 2013. 34(27): p. 6429-6443.
12. Wang, J., et al., Delivery of siRNA therapeutics: barriers and carriers. AAPS J, 2010. 12(4): p. 492-503.
13. Xu, C.F. and J. Wang, Delivery systems for siRNA drug development in cancer therapy. Asian J Pharm Sci, 2015. 10(1): p. 1-12.
14. Kanasty, R., et al., Delivery materials for siRNA therapeutics. Nat Mater, 2013. 12(11): p. 967-977.
15. Taratula, O., et al., Nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and siRNA. J Control Release, 2013. 171(3): p. 349-357.
16. Mathew, A.P., et al., Injectable hydrogels for delivering biotherapeutic molecules. Int J Biol Macromol, 2018. 110: p. 17-29.
17. Voorhaar, L. and R. Hoogenboom, Supramolecular polymer networks: hydrogels and bulk materials. Chem Soc Rev, 2016. 45(14): p. 4013-4031.
18. Shao, Y., et al., Supramolecular Hydrogels Based on DNA Self-Assembly. Acc Chem Res, 2017. 50(4): p. 659-668.
19. Roh, Y.H., et al., Engineering DNA-based functional materials. Chem Soc Rev, 2011. 40(12): p. 5730-5744.
20. Xiong, X., et al., Responsive DNA‐based hydrogels and their applications. Macromol. Rapid Commun, 2013. 34(16): p. 1271-1283.
21. Li, F., et al., Polymeric DNA hydrogel: Design, synthesis and applications. Prog Polym Sci, 2019. 98: p. 101163.
22. Nagahara, S. and T. Matsuda, Hydrogel formation via hybridization of oligonucleotides derivatized in water-soluble vinyl polymers. Polymer Gels and Networks, 1996. 4(2): p. 111-127.
23. Li, C., et al., A writable polypeptide-DNA hydrogel with rationally designed multi-modification sites. Small, 2015. 11(9-10): p. 1138-1143.
24. Haraguchi, K., Nanocomposite hydrogels. Curr Opin Solid State Mater Sci, 2007. 11(3-4): p. 47-54.
25. Shikinaka, K., et al., Structural and mechanical properties of Laponite-PEG hybrid films. J Colloid Interface Sci, 2012. 369(1): p. 470-476.
26. Tomas, H., C.S. Alves, and J. Rodrigues, Laponite(R): A key nanoplatform for biomedical applications? Nanomedicine, 2018. 14(7): p. 2407-2420.
27. Chang, C.W., et al., PEG/clay nanocomposite hydrogel: a mechanically robust tissue engineering scaffold. Soft Matter, 2010. 6(20).
28. Perez-Arnaiz, C., et al., New insights into the mechanism of the DNA/doxorubicin interaction. J Phys Chem B, 2014. 118(5): p. 1288-1295.
29. Jawad, B., et al., Molecular mechanism and binding free energy of doxorubicin intercalation in DNA. Phys Chem Chem Phys, 2019. 21(7): p. 3877-3893.
30. Kim, K.R., et al., Drug delivery by a self-assembled DNA tetrahedron for overcoming drug resistance in breast cancer cells. Chem Commun, 2013. 49(20): p. 2010-2012.
31. Gonçalves, M., et al., pH-sensitive Laponite®/doxorubicin/alginate nanohybrids with improved anticancer efficacy. Acta Biomater, 2014. 10(1): p. 300-307.
32. Futaki, S., et al., Stearylated arginine-rich peptides: a new class of transfection systems. Bioconjugate Chem, 2001. 12(6): p. 1005-1011.
33. Khalil, I.A., et al., Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation. Gene Ther, 2004. 11(7): p. 636-44.
34. Taira, K., K. Kataoka, and T. Niidome, Non-viral gene therapy gene design and delivery, 2005. Springer: Tokyo.
35. Loffek, S., O. Schilling, and C.W. Franzke, Series "matrix metalloproteinases in lung health and disease": Biological role of matrix metalloproteinases: a critical balance. Eur Respir J, 2011. 38(1): p. 191-208.
36. Gialeli, C., A.D. Theocharis, and N.K. Karamanos, Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J, 2011. 278(1): p. 16-27.
37. Egeblad, M. and Z. Werb, New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer, 2002. 2(3): p. 161-174.
38. Yao, Q., et al., MMP-Responsive 'Smart' Drug Delivery and Tumor Targeting. Trends Pharmacol Sci, 2018. 39(8): p. 766-781.
39. Hatakeyama, H., et al., Systemic delivery of siRNA to tumors using a lipid nanoparticle containing a tumor-specific cleavable PEG-lipid. Biomaterials, 2011. 32(18): p. 4306-4316.
40. Li, W., et al., MMP-responsive in situ forming hydrogel loaded with doxorubicin-encapsulated biodegradable micelles for local chemotherapy of oral squamous cell carcinoma. RSC Advances, 2019. 9(54): p. 31264-31273.
41. Debnath, J., S.K. Muthuswamy, and J.S. Brugge, Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods, 2003. 30(3): p. 256-268.
42. Amer, M.H., et al., Translational considerations in injectable cell-based therapeutics for neurological applications: concepts, progress and challenges. NPJ Regen Med, 2017. 2(23): p. 1-13.
43. Bagalkot, V., et al., An aptamer-doxorubicin physical conjugate as a novel targeted drug-delivery platform. Angew Chem Int Ed Engl, 2006. 45(48): p. 8149-8152.
44. Xiao, S., et al., Fine tuning of the pH-sensitivity of laponite-doxorubicin nanohybrids by polyelectrolyte multilayer coating. Mater Sci Eng C, 2016. 60: p. 348-356.