臺灣博碩士論文加值系統

藥物載體會根據不同應用使用不同製備方式而有不同的結構，如實心球體、中空球體、多孔球體、單孔球體、殼核球體等。其中最常見的製備方式是乳化法，它是利用乳化劑將高分子油相溶於水相中，乳化劑是作為穩定並讓兩相共存的界面活性劑。乳化劑的濃度會直接影響到兩相的穩定性以及微球的粒徑大小，而不同的高分子溶劑會直接影響到微球表面的粗糙度等。生物可降解的高分子，如PLA、PCL、PLGA等，由於其降解特性可穩定緩慢釋放藥物，長時間對癌細胞造成傷害，因此成為現今作為藥物載體材料的首要選擇。有文獻指出結晶高分子載體可以利用材料熱力學之特性使其自行將孔洞密合。本篇研究是將聚左旋乳酸以乳化法配合溶劑蒸發法一步製備出單孔空心微球載體，粒徑可小至6-9微米，並利用材料本身熱力學特性以簡單的方式包覆阿黴素作為藥物載體並對小鼠乳腺癌細胞JC (CRL-2116)進行細胞毒性測試，在24小時藥物釋放後對癌細胞達到約48%的殺滅效果。

According to applications, carriers is prepared by different methods to product many kinds of structures, such as solid, hollow, porous, single-hole, shell-core, etc. Emulsification is the most common method of product drug carrier, which uses an emulsifier to dissolve and stabilize the both of oil and water phase. The stability of emulsion and the particle sizes are directly affected by the concentration of the emulsifier, and the surface roughness of polymer microspheres are directly affected by the solvents for the polymer. Biodegradable polymers, such as PLA, PCL, PLGA, etc., are the best choice for medical materials currently, because of its degradation characteristics, the drug release will be continuously and steadily that lethal to cancer cells for a long time. The literatures point out that crystalline polymer can be use of their thermodynamics properties to make the pores closing by itself. In this study, we prepare of poly (L-lactic acid) hollow microspheres carrier by an emulsification and a solvent evaporation method, the particle size can be as small as 6-9 um. Using the traditional cancer treatment drug, doxorubicin was loaded in a simple way by the thermodynamic properties of PLLA, and was used for mouse breast cancer cells JC (CRL-2116) to test for cytotoxicity. It achieved approximately 48% killing effect on JC cells after 24 hours of drug release.

目錄

中文摘要I
ABSTRACTII
誌謝III
目錄IV
圖目錄VI
表目錄IX
縮寫表1
第一章、緒論2
1.1前言2
1.2研究動機與目的3
1.3實驗設計4
第二章、文獻回顧5
2.1藥物載體5
2.1.1微粒(Microparticles)6
2.1.1.1聚合物微粒8
2.1.2 脂質體（Liposomes）9
2.1.3乳液(Emulsion)12
2.2聚左旋乳酸(Poly lactic acid, PLLA)13
2.2.1聚乳酸降解原理與分析13
2.2.1.1水解13
2.2.1.2光降解14
2.2.1.3微生物降解14
2.2.1.4酶降解15
2.2.1.5調節改善降解16
2.3單孔中空載體製備原理方法17
2.3.1單孔中空微球製備17
2.3.1.1乳化作用17
2.3.1.2溶脹法(Swelling)21
2.3.2裝載藥物及閉孔形成藥物載體製備23
2.3.2.1熱退火(Annealing)23
2.3.2.2溶劑升溫法23
2.4乳腺癌24
2.4.1阿黴素(DOX)對乳腺癌的研究25
第三章、材料與方法27
3.1藥品及材料27
3.1.1細胞實驗相關27
3.1.2 藥物載體相關28
3.2儀器29
3.2.1 細胞實驗相關29
3.2.2 藥物載體相關29
3.3器材30
3.4電腦軟體30
3.5實驗方法31
3.5.1聚己內酯顆粒表面開孔方法31
3.5.2聚乳酸單孔空心微球製備31
3.5.3藥物載體製備32
3.5.4細胞培養基本操作32
3.5.4.1細胞活化32
3.5.4.2細胞繼代33
3.5.4.3細胞計數方法33
3.5.4.4小鼠乳腺癌細胞培養液配製34
3.5.5藥物釋放測試35
3.5.6細胞毒性測試35
3.5.7載體降解測試36
3.6 儀器方法37
3.6.1 SEM639037
3.6.2 OM(包含螢光影像)37
3.6.3 HPLC37
3.7 數據統計分析方法38
第四章、實驗結果與討論39
4.1載體表面型態鑑定39
4.1.1 PCL顆粒39
4.1.2 PLLA單孔空心微球41
4.2利用螢光顯微鏡觀察Rhodamine與DOX裝載情形44
4.2.1 Rhodamine44
4.2.2 Doxorubicin44
4.3 HMPs-DOX藥物釋放結果45
4.4 HMPs-DOX對JC細胞之毒性測試46
4.5 HMPs在體外降解之結果48
第五章、結論49
第六章、未來展望50
第七章、參考文獻51

參考文獻
1.Tyler, B., et al., Polylactic acid (PLA) controlled delivery carriers for biomedical applications. Advanced drug delivery reviews, 2016. 107: p. 163-175.
2.Nugroho, R.W.N., et al., Highlighting the importance of surface grafting in combination with a Layer-by-Layer approach for fabricating advanced 3D Poly (L-lactide) microsphere scaffolds. Chemistry of Materials, 2016. 28(10): p. 3298-3307.
3.Jeong, U., et al., Microscale fish bowls: a new class of latex particles with hollow interiors and engineered porous structures in their surfaces. Langmuir, 2007. 23(22): p. 10968-10975.
4.Kim, T.H. and K.C. Song, Preparation of Poly-L-Lactic Acid (PLLA) Microspheres by Solvent-Evaporation Method. Korean Chemical Engineering Research, 2018. 56(4): p. 461-468.
5.Kanamala, M., et al., Mechanisms and biomaterials in pH-responsive tumour targeted drug delivery: a review. Biomaterials, 2016. 85: p. 152-167.
6.Zhu, Y.J. and F. Chen, pH‐responsive drug‐delivery systems. Chemistry–An Asian Journal, 2015. 10(2): p. 284-305.
7.Hamedi, S. and M. Koosha, Designing a pH-responsive drug delivery system for the release of black-carrot anthocyanins loaded in halloysite nanotubes for cancer treatment. Applied Clay Science, 2020. 197: p. 105770.
8.Goodman, A.M., et al., Near-infrared remotely triggered drug-release strategies for cancer treatment. Proceedings of the National Academy of Sciences, 2017. 114(47): p. 12419-12424.
9.García-Hevia, L., et al., Magnetic lipid nanovehicles synergize the controlled thermal release of chemotherapeutics with magnetic ablation while enabling non-invasive monitoring by MRI for melanoma theranostics. Bioactive materials, 2022. 8: p. 153-164.
10.Yang, G., et al., Near-infrared-light responsive nanoscale drug delivery systems for cancer treatment. Coordination Chemistry Reviews, 2016. 320: p. 100-117.
11.Raza, A., et al., “Smart” materials-based near-infrared light-responsive drug delivery systems for cancer treatment: a review. Journal of Materials Research and Technology, 2019. 8(1): p. 1497-1509.
12.Song, W., et al., Efficient synthesis of folate-conjugated hollow polymeric capsules for accurate drug delivery to cancer cells. Biomacromolecules, 2020. 22(2): p. 732-742.
13.Chandra, S., et al., Dendritic magnetite nanocarriers for drug delivery applications. New Journal of Chemistry, 2010. 34(4): p. 648-655.
14.Jia, Q., et al., PEGMA-modified bimetallic NiCo Prussian blue analogue doped with Tb (III) ions: Efficiently pH-responsive and controlled release system for anticancer drug. Chemical Engineering Journal, 2020. 389: p. 124468.
15.Zhang, K., et al., PEG–PLGA copolymers: Their structure and structure-influenced drug delivery applications. Journal of Controlled release, 2014. 183: p. 77-86.
16.Ninan, N., et al., Natural polymer/inorganic material based hybrid scaffolds for skin wound healing. Polymer Reviews, 2015. 55(3): p. 453-490.
17.Chapman, D., Physico-Chemical Properties of Phospholipids and Lipid-Water Systems, in Liposome technology. 2019, CRC Press. p. 1-18.
18.Daraee, H., et al., Application of liposomes in medicine and drug delivery. Artificial cells, nanomedicine, and biotechnology, 2016. 44(1): p. 381-391.
19.Wang, A.Z., R. Langer, and O.C. Farokhzad, Nanoparticle delivery of cancer drugs. Annual review of medicine, 2012. 63: p. 185-198.
20.Szulc, J., et al., Liposomes--therapeutic progress and technological problems. Polski Merkuriusz Lekarski: Organ Polskiego Towarzystwa Lekarskiego, 2002. 12(68): p. 164-168.
21.Kalepu, S. and V. Nekkanti, Insoluble drug delivery strategies: review of recent advances and business prospects. Acta Pharmaceutica Sinica B, 2015. 5(5): p. 442-453.
22.Kang, D.I., et al., Liposome composition is important for retention of liposomal rhodamine in P-glycoprotein-overexpressing cancer cells. Drug delivery, 2009. 16(5): p. 261-267.
23.Lehtinen, J., et al., Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma. PloS one, 2012. 7(7): p. e41410.
24.Khalifa, A.M., et al., Current strategies for different paclitaxel-loaded Nano-delivery Systems towards therapeutic applications for ovarian carcinoma: A review article. Journal of Controlled Release, 2019. 311: p. 125-137.
25.Zia, A., et al., Advances and opportunities of oil-in-oil emulsions. ACS applied materials & interfaces, 2020. 12(35): p. 38845-38861.
26.Costa, C., et al., Emulsion formation and stabilization by biomolecules: The leading role of cellulose. Polymers, 2019. 11(10): p. 1570.
27.Nour, A.H., Emulsion types, stability mechanisms and rheology: A review. International Journal of Innovative Research and Scientific Studies (IJIRSS), 2018. 1(1).
28.Wang, B., H. Tian, and D. Xiang, Stabilizing the oil-in-water emulsions using the mixtures of Dendrobium officinale polysaccharides and gum arabic or propylene glycol alginate. Molecules, 2020. 25(3): p. 759.
29.Barkat, A.K., et al., Basics of pharmaceutical emulsions: A review. African Journal of Pharmacy and Pharmacology, 2011. 5(25): p. 2715-2725.
30.Fingas M, F.B., Water-in-oil emulsions: formation and prediction. Handbook of Oil Spill Science and Technology. 2015: p. 225-270.
31.Fingas, M. and B. Fieldhouse, Formation of water-in-oil emulsions and application to oil spill modelling. Journal of hazardous materials, 2004. 107(1-2): p. 37-50.
32.Chen, G. and D. Tao, An experimental study of stability of oil–water emulsion. Fuel processing technology, 2005. 86(5): p. 499-508.
33.Premlal Ranjith HM, W.U., Lipid emulsifiers and surfactants in dairy and bakery products. Modifying Lipids for Use in Food. . 2006: p. 393–428.
34.Mohamed, A.I., et al., Influence of surfactant structure on the stability of water-in-oil emulsions under high-temperature high-salinity conditions. Journal of Chemistry, 2017. 2017.
35.Bhatia, N., et al., A review on multiple emulsions. International Journal of Pharmaceutical Erudition, 2013. 3(2): p. 22-30.
36.Yaqoob Khan, A., et al., Multiple emulsions: an overview. Current drug delivery, 2006. 3(4): p. 429-443.
37.Feczkó, T., J. Tóth, and J. Gyenis, Comparison of the preparation of PLGA–BSA nano-and microparticles by PVA, poloxamer and PVP. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2008. 319(1-3): p. 188-195.
38.Kemala, T., E. Budianto, and B. Soegiyono, Preparation and characterization of microspheres based on blend of poly (lactic acid) and poly (ɛ-caprolactone) with poly (vinyl alcohol) as emulsifier. Arabian Journal of Chemistry, 2012. 5(1): p. 103-108.
39.Nampoothiri, K.M., N.R. Nair, and R.P. John, An overview of the recent developments in polylactide (PLA) research. Bioresource technology, 2010. 101(22): p. 8493-8501.
40.Castro-Aguirre, E., et al., Poly (lactic acid)—Mass production, processing, industrial applications, and end of life. Advanced drug delivery reviews, 2016. 107: p. 333-366.
41.Iñiguez-Franco, F., et al., Effect of nanoparticles on the hydrolytic degradation of PLA-nanocomposites by water-ethanol solutions. Polymer Degradation and Stability, 2017. 146: p. 287-297.
42.Tawakkal, I.S., et al., A review of poly (lactic acid)‐based materials for antimicrobial packaging. Journal of food science, 2014. 79(8): p. R1477-R1490.
43.Park, K. and M. Xanthos, A study on the degradation of polylactic acid in the presence of phosphonium ionic liquids. Polymer Degradation and Stability, 2009. 94(5): p. 834-844.
44.Karamanlioglu, M., R. Preziosi, and G.D. Robson, Abiotic and biotic environmental degradation of the bioplastic polymer poly (lactic acid): A review. Polymer Degradation and stability, 2017. 137: p. 122-130.
45.Hung, K.-C., Y.-L. Chen, and J.-H. Wu, Natural weathering properties of acetylated bamboo plastic composites. Polymer Degradation and Stability, 2012. 97(9): p. 1680-1685.
46.Auras, R.A., et al., Poly (lactic acid): synthesis, structures, properties, processing, and applications. 2011: John Wiley & Sons.
47.Elsawy, M.A., et al., Hydrolytic degradation of polylactic acid (PLA) and its composites. Renewable and Sustainable Energy Reviews, 2017. 79: p. 1346-1352.
48.Casalini, T., et al., A perspective on polylactic acid-based polymers use for nanoparticles synthesis and applications. Frontiers in bioengineering and biotechnology, 2019: p. 259.
49.Zaaba, N.F. and M. Jaafar, A review on degradation mechanisms of polylactic acid: Hydrolytic, photodegradative, microbial, and enzymatic degradation. Polymer Engineering & Science, 2020. 60(9): p. 2061-2075.
50.Saadi, Z., et al., Fungal degradation of poly (l-lactide) in soil and in compost. Journal of Polymers and the Environment, 2012. 20(2): p. 273-282.
51.Thanasak, L., et al., Co-production of poly (llactide)-degrading enzyme and raw starch-degrading enzyme by Laceyella sacchari LP175 using agricultural products as substrate, and their efficiency on biodegradation of poly (l-lactide)/thermoplastic starch blend film. Int. Biodeterior. Biodegrad, 2015. 104: p. 401-410.
52.Lipsa, R., et al., Biodegradation of poly (lactic acid) and some of its based systems with Trichoderma viride. International journal of biological macromolecules, 2016. 88: p. 515-526.
53.Pattanasuttichonlakul, W., N. Sombatsompop, and B. Prapagdee, Accelerating biodegradation of PLA using microbial consortium from dairy wastewater sludge combined with PLA-degrading bacterium. International Biodeterioration & Biodegradation, 2018. 132: p. 74-83.
54.Yagi, H., et al., Mesophilic anaerobic biodegradation test and analysis of eubacteria and archaea involved in anaerobic biodegradation of four specified biodegradable polyesters. Polymer degradation and stability, 2014. 110: p. 278-283.
55.Qi, X., Y. Ren, and X. Wang, New advances in the biodegradation of Poly (lactic) acid. International Biodeterioration & Biodegradation, 2017. 117: p. 215-223.
56.Lee, S.H., I.Y. Kim, and W.S. Song, Biodegradation of polylactic acid (PLA) fibers using different enzymes. Macromolecular Research, 2014. 22(6): p. 657-663.
57.Martin, R.T., L.P. Camargo, and S.A. Miller, Marine-degradable polylactic acid. Green Chemistry, 2014. 16(4): p. 1768-1773.
58.Kawai, F., Polylactic acid (PLA)-degrading microorganisms and PLA depolymerases, in Green Polymer Chemistry: Biocatalysis and Biomaterials. 2010, ACS Publications. p. 405-414.
59.Hanphakphoom, S., et al., Characterization of poly (L-lactide)-degrading enzyme produced by thermophilic filamentous bacteria Laceyella sacchari LP175. The Journal of General and Applied Microbiology, 2014. 60(1): p. 13-22.
60.Kawai, F., et al., Different enantioselectivity of two types of poly (lactic acid) depolymerases toward poly (L-lactic acid) and poly (D-lactic acid). Polymer degradation and stability, 2011. 96(7): p. 1342-1348.
61.Satti, S.M., et al., Isolation and characterization of bacteria capable of degrading poly (lactic acid) at ambient temperature. Polymer Degradation and Stability, 2017. 144: p. 392-400.
62.Husárová, L., et al., Identification of important abiotic and biotic factors in the biodegradation of poly (l-lactic acid). International journal of biological macromolecules, 2014. 71: p. 155-162.
63.Bubpachat, T., N. Sombatsompop, and B. Prapagdee, Isolation and role of polylactic acid-degrading bacteria on degrading enzymes productions and PLA biodegradability at mesophilic conditions. Polymer Degradation and Stability, 2018. 152: p. 75-85.
64.Von Recum, H.A., et al., Degradation of polydispersed poly (L-lactic acid) to modulate lactic acid release. Biomaterials, 1995. 16(6): p. 441-447.
65.Zan, J., et al., Dilemma and breakthrough of biodegradable poly-l-lactic acid in bone tissue repair. Journal of Materials Research and Technology, 2022.
66.Oyama, H.T., et al., Environmentally safe bioadditive allows degradation of refractory poly (lactic acid) in seawater: Effect of poly (aspartic acid-co-l-lactide) on the hydrolytic degradation of PLLA at different salinity and pH conditions. Polymer Degradation and Stability, 2020. 178: p. 109216.
67.Bentz, K.C. and D.A. Savin, Hollow polymer nanocapsules: synthesis, properties, and applications. Polymer Chemistry, 2018. 9(16): p. 2059-2081.
68.Bazylińska, U., et al., Polymeric nanocapsules and nanospheres for encapsulation and long sustained release of hydrophobic cyanine-type photosensitizer. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2014. 442: p. 42-49.
69.Kang, H.-J. and J.-H. Kim, Removal of residual chloroform from amorphous paclitaxel pretreated by alcohol. Korean Journal of Chemical Engineering, 2019. 36(12): p. 1965-1970.
70.Zhou, F.-L., et al., Hollow polycaprolactone microspheres with/without a single surface hole by co-electrospraying. Langmuir, 2017. 33(46): p. 13262-13271.
71.Spek, S., et al., Characterisation of PEGylated PLGA nanoparticles comparing the nanoparticle bulk to the particle surface using UV/vis spectroscopy, SEC, 1H NMR spectroscopy, and X-ray photoelectron spectroscopy. Applied Surface Science, 2015. 347: p. 378-385.
72.Sahoo, S.K., et al., Residual polyvinyl alcohol associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. Journal of controlled release, 2002. 82(1): p. 105-114.
73.Chaiyasat, P., et al., Influence of poly (L-lactic acid) molecular weight on the encapsulation efficiency of urea in microcapsule using a simple solvent evaporation technique. Polymer-plastics technology and engineering, 2016. 55(11): p. 1131-1136.
74.Hyuk Im, S., U. Jeong, and Y. Xia, Polymer hollow particles with controllable holes in their surfaces. Nature materials, 2005. 4(9): p. 671-675.
75.Bordes, C., et al., Determination of poly (ɛ-caprolactone) solubility parameters: Application to solvent substitution in a microencapsulation process. International journal of pharmaceutics, 2010. 383(1-2): p. 236-243.
76.Liu, Y., et al., Codelivery of chemotherapeutics via crosslinked multilamellar liposomal vesicles to overcome multidrug resistance in tumor. PLoS One, 2014. 9(10): p. e110611.
77.Beck, W.T., Role of Drug-Induced Fas Ligand Expression in Breast Tumor Progression. 2001, ILLINOIS UNIV AT CHICAGO.
78.Iqbal, N. and N. Iqbal, Human epidermal growth factor receptor 2 (HER2) in cancers: overexpression and therapeutic implications. Molecular biology international, 2014. 2014.
79.Khan, M.A., et al., PI3K/AKT/mTOR pathway inhibitors in triple-negative breast cancer: a review on drug discovery and future challenges. Drug Discovery Today, 2019. 24(11): p. 2181-2191.
80.Ruchi Sharma, V., et al., PI3K/Akt/mTOR intracellular pathway and breast cancer: factors, mechanism and regulation. Current pharmaceutical design, 2017. 23(11): p. 1633-1638.
81.Davies, E. and S. Hiscox, New therapeutic approaches in breast cancer. Maturitas, 2011. 68(2): p. 121-128.
82.Carvalho, C., et al., Doxorubicin: the good, the bad and the ugly effect. Current medicinal chemistry, 2009. 16(25): p. 3267-3285.
83.Nolan, M., et al., Chemotherapy‐related cardiomyopathy: a neglected aspect of cancer survivorship. Internal medicine journal, 2014. 44(10): p. 939-950.
84.Thorn, C.F., et al., Doxorubicin pathways: pharmacodynamics and adverse effects. Pharmacogenetics and genomics, 2011. 21(7): p. 440.
85.Rawat, P.S., et al., Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. Biomedicine & Pharmacotherapy, 2021. 139: p. 111708.
86.Alves, A.C., et al., Influence of doxorubicin on model cell membrane properties: insights from in vitro and in silico studies. Scientific reports, 2017. 7(1): p. 1-11.
87.林淑美, 黃柏叡, Resveratrol prevents doxorubicin-induced cardiomyopathy through mitochondrial modulation. 2019: p. 1-69.
88.Wigner, P., et al., Doxorubicin–transferrin conjugate alters mitochondrial homeostasis and energy metabolism in human breast cancer cells. Scientific Reports, 2021. 11(1): p. 1-14.
89.Pilco-Ferreto, N. and G.M. Calaf, Influence of doxorubicin on apoptosis and oxidative stress in breast cancer cell lines. International journal of oncology, 2016. 49(2): p. 753-762.
90.Tavares-Valente, D., et al., Disruption of pH dynamics suppresses proliferation and potentiates doxorubicin cytotoxicity in breast cancer cells. Pharmaceutics, 2021. 13(2): p. 242.
91.Lao, J., et al., Liposomal doxorubicin in the treatment of breast cancer patients: a review. Journal of drug delivery, 2013. 2013.
92.Sawasdee, N., et al., Doxorubicin sensitizes breast cancer cells to natural killer cells in connection with increased Fas receptors. International journal of molecular medicine, 2022. 49(3): p. 1-12.
93.Possinger, K., et al. Primary chemotherapy for locally advanced breast cancer (LABC) with gemcitabine (G) as prolonged infusion, liposomal doxorubicin (M) and Docetaxel (T): results of a phase I trial. in Proceedings of the American Society of Clinical Oncology. 2002.
94.Gogas, H., et al., Neoadjuvant chemotherapy with a combination of pegylated liposomal doxorubicin (Caelyx®) and paclitaxel in locally advanced breast cancer: a phase II study by the Hellenic Cooperative Oncology Group. Annals of Oncology, 2002. 13(11): p. 1737-1742.
95.Wen, S.-h., et al., Sulbactam-enhanced cytotoxicity of doxorubicin in breast cancer cells. Cancer cell international, 2018. 18(1): p. 1-18.
96.Lebourg, M., J.S. Antón, and J.G. Ribelles, Porous membranes of PLLA–PCL blend for tissue engineering applications. European polymer journal, 2008. 44(7): p. 2207-2218.
97.Todo, M., et al., Fracture micromechanisms of bioabsorbable PLLA/PCL polymer blends. Engineering fracture mechanics, 2007. 74(12): p. 1872-1883.
98.Bolourtchian, N., K. Karimi, and R. Aboofazeli, Preparation and characterization of ibuprofen microspheres. Journal of microencapsulation, 2005. 22(5): p. 529-538.
99.Lee, B.D., et al., Development of a syngeneic in vivo tumor model and its use in evaluating a novel P-glycoprotein modulator, PGP-4008. Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics, 2003. 14(1): p. 49-60.
100.Czauderna, M. and J. Kowalczyk, Lactic acid can be easily and precisely determined by reversed-phase high performance liquid chromatography with pre-column derivatization. Journal of Animal and Feed Sciences, 2008. 17(2): p. 268-279.