本研究闡述如何使用硫酸軟骨素 (Chondroitin sulfate)及幾丁聚醣 (Chitosan)作為基材，透過靜電噴霧技術 (Electrospray technique)包覆化療用藥-阿黴素 (Doxorubicin)，以及超順磁氧化鐵 (Superparamagnetic iron oxide)，製備形成新型次微米等級之藥物載體粒子 (Drug delivery systems)之實驗方法。研究探討材料特性、體外釋放試驗、體外抗腫瘤試驗，以及活體動物模式之成效。結果顯示，此DOX-SPIO-CS/CHI 次微米粒子之平均粒徑為110.7 nm (SD = 30.23)，針對Doxorubicin之包覆率約為31% (SD = 8.07)，並具備緩慢釋放之特性及人類癌細胞株之體外試驗阻殺效果。在實際投藥劑量為0.06, 0.12, 0.25 nM之條件下，DOX-SPIO-CS/CHI MPs懸浮藥劑針對Hep G2之肝癌細胞存活率試驗結果分別為0.06 nM: 20.5%, 0.12 nM: 4.6%, 0.25 nM: 6.3%，DOX溶液則為0.06 nM: 70.7%, 0.12 nM: 49.9%, 0.25 nM: 21.3%；針對Huh-6之肝癌細胞存活率試驗結果分別為0.06 nM: 52.2%, 0.12 nM: 12.8%, 0.25 nM: 23.4%，DOX溶液則為0.06 nM: 71.9%, 0.12 nM: 62.5%, 0.25 nM: 43.9%。動物實驗結果顯示，針對Hep G2試驗別治療後，Exp.試驗組以68.6%之腫瘤抑制效果優於P.C.試驗組之50.8%；針對Huh-6試驗別治療後，Exp.試驗組以40%之腫瘤抑制效果優於P.C.試驗組之36.5%。此外，在兩個動物試驗別，結果皆顯示使用DOX-SPIO-CS/CHI MPs懸浮藥劑進行治療而造成較少之體重流失 (Hep G2: Exp.= -0.59%, P.C.= -5.88%; Huh-6: Exp.= +10.93%, P.C.= -0.78%)。

In this study, we describes we synthesized a novel polyelectrolyte micorparticle, DOX-SPIO-CS/CHI MPs, as drug delivery systems (DDS) for hepatic cancer treatment. Also, we investigated the properties of these micropar-ticles, from material characterizations, formulation tests, in vitro study to in vivo study. The results show that our DOX-SPIO-CS/CHI MPs displays an average diameter of 110.7 nm (SD = 30.23), and reveals a spherical shape. The encapsu-lation efficiency of doxorubicin is about 31% (SD = 8.07) based on our spec-trometer measurement. In the results of release profile test, we found a sus-tained-release behavior of DOX-SPIO-CS/CHI MPs, which released 51.5% of DOX within 48 h of testing time. Based on the results of cell viability assay and animal study, the DOX-SPIO-CS/CHI MPs was found to show stronger ability than that of free DOX, when given to Hep G2 and Huh-6 human liver cancer cell line for treatment and nude mice of Hep G2/Huh-6-induced tumor model.

1. Jemal, A., et al., Global cancer statistics. CA Cancer J Clin, 2011. 61(2): p. 69-90.
2. Raza, A. and G.K. Sood, Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World J Gastroenterol, 2014. 20(15): p. 4115-27.
3. Klein, J. and L.A. Dawson, Hepatocellular carcinoma radiation therapy: review of evidence and future opportunities. Int J Radiat Oncol Biol Phys, 2013. 87(1): p. 22-32.
4. H.T.et al., Liver Cancer and its Prevention. Asian Pacific J Cancer Prev, 2005. 6: p. 244-250.
5. Minotti, G., et al., Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol Rev, 2004. 56(2): p. 185-229.
6. Cutts, S.M., et al., The power and potential of doxorubicin-DNA adducts. IUBMB Life, 2005. 57(2): p. 73-81.
7. Chlebowski, R.T., et al., Influence of nandrolone decanoate on weight loss in advanced non-small cell lung cancer. Cancer, 1986. 58(1): p. 183-6.
8. Tacar, O., P. Sriamornsak, and C.R. Dass, Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol, 2013. 65(2): p. 157-70.
9. Strother, R. and D. Matei, Pegylated liposomal doxorubicin in ovarian cancer. Ther Clin Risk Manag, 2009. 5(3): p. 639-50.
10. Kiviharju, K., M. Leisola, and T. Eerikainen, Optimization of Streptomyces peucetius var. caesius N47 cultivation and epsilon-rhodomycinone production using experimental designs and response surface methods. J Ind Microbiol Biotechnol, 2004. 31(10): p. 475-81.
11. Tsoneva, Y., et al., Molecular structure and pronounced conformational flexibility of doxorubicin in free and conjugated state within a drug-peptide compound. J Phys Chem B, 2015. 119(7): p. 3001-13.
12. Bagheri, S., S.M. Hassani, and G. Naderi, Theoretical study on physicochemical and geometrical properties of Doxorubicin and Daunorubicin conjugated to PEO-b-PCL nanoparticles. Euro. J. Exp. Bio, 2012. 2(3): p. 641-645.
13. Alakhova, D.Y., et al., Effect of doxorubicin/pluronic SP1049C on tumorigenicity, aggressiveness, DNA methylation and stem cell markers in murine leukemia. PLoS One, 2013. 8(8): p. e72238.
14. Yokochi, T. and K.D. Robertson, Doxorubicin inhibits DNMT1, resulting in conditional apoptosis. Mol Pharmacol, 2004. 66(6): p. 1415-20.
15. Dejeux, E., et al., RDesNearAch methylation profiling in doxorubicin treated primary locally advanced breast tumours identifies novel genes associated with survival and treatment response. Dejeux et al. Molecular Cancer 2010, 9:68, 2010. 9: p. 68-80.
16. Kumar, S., et al., Doxorubicin-Induced Cardiomyopathy 17 Years after Chemotherapy. Texas Heart Institute Journal, 2012. 39(3): p. 424-427.
17. Octavia, Y., et al., Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol, 2012. 52(6): p. 1213-25.
18. Chatterjee, K., et al., Doxorubicin cardiomyopathy. Cardiology, 2010. 115(2): p. 155-62.
19. Branco, A.F., et al., Differentiation-dependent doxorubicin toxicity on H9c2 cardiomyoblasts. Cardiovasc Toxicol, 2012. 12(4): p. 326-40.
20. Smola, M., T. Vandamme, and A. Sokolowski, Nanocarriers as pulmonary drug delivery systems to treat and to diagnose respiratory and non respiratory diseases. International Journal of Nanomedicine, 2008. 3(1): p. 1-19.
21. Naidu, M.U., et al., Chemotherapy-induced and/or radiation therapy-induced oral mucositis--complicating the treatment of cancer. Neoplasia, 2004. 6(5): p. 423-31.
22. Sol Silverman, J., Diagnosis and Management of Oral Mucositis. J Support Oncol, 2007. 5: p. 013-021.
23. Ross, P.J., et al., Do patients with weight loss have a worse outcome when undergoing chemotherapy for lung cancers? Br J Cancer, 2004. 90(10): p. 1905-11.
24. Petruson, K.M., E.M. Silander, and E.B. Hammerlid, Quality of life as predictor of weight loss in patients with head and neck cancer. Head Neck, 2005. 27(4): p. 302-10.
25. Maeng, J.H., et al., Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer. Biomaterials, 2010. 31(18): p. 4995-5006.
26. Yeh, M.K., et al., Novel protein-loaded chondroitin sulfate-chitosan nanoparticles: preparation and characterization. Acta Biomater, 2011. 7(10): p. 3804-12.
27. Hu, C.S., et al., Influence of charge on FITC-BSA-loaded chondroitin sulfate-chitosan nanoparticles upon cell uptake in human Caco-2 cell monolayers. Int J Nanomedicine, 2012. 7: p. 4861-72.
28. Xiao, K., et al., The effect of surface charge on in vivo biodistribution of PEG-oligocholic acid based micellar nanoparticles. Biomaterials, 2011. 32(13): p. 3435-46.
29. He, C., et al., Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials, 2010. 31(13): p. 3657-66.
30. Yamamoto, Y., et al., Long-circulating poly(ethylene glycol)–poly(d,l-lactide) block copolymer micelles with modulated surface charge. Journal of Controlled Release, 2001. 77: p. 27-38.
31. Tsai, H.Y., et al., Antitumor efficacy of doxorubicin released from crosslinked nanoparticulate chondroitin sulfate/chitosan polyelectrolyte complexes. Macromol Biosci, 2011. 11(5): p. 680-8.
32. S, K.V., B.K.D. , and R.S. S, Natural Polymers – A Comprehensive Review. J Pharm Biomed Sci., 2012. 3(4): p. 1597-1613.
33. A. P. Anwunobi, M.O.E., Recent Applications of Natural Polymers in Nanodrug Delivery. Journal of Nanomedicine & Nanotechnology, 2011. s4(01).
34. Golla, K., et al., Biocompatibility, absorption and safety of protein nanoparticle-based delivery of doxorubicin through oral administration in rats. Drug Deliv, 2013. 20(3-4): p. 156-67.
35. Henrotin, Y., et al., Chondroitin sulfate in the treatment of osteoarthritis: from in vitro studies to clinical recommendations. Ther Adv Musculoskelet Dis, 2010. 2(6): p. 335-48.
36. Giri, T.K., et al., Modified chitosan hydrogels as drug delivery and tissue engineering systems: present status and applications. Acta Pharmaceutica Sinica B, 2012. 2(5): p. 439-449.
37. Bhattarai, N., J. Gunn, and M. Zhang, Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliv Rev, 2010. 62(1): p. 83-99.
38. Marei, M.K., et al., Principles, Applications, and Technology of Craniofacial Bone Engineering, in Integrated Biomaterials in Tissue Engineering. 2012, John Wiley & Sons, Inc. p. 183-234.
39. Zhao, L., et al., Fatigue and human umbilical cord stem cell seeding characteristics of calcium phosphate-chitosan-biodegradable fiber scaffolds. Biomaterials, 2010. 31(5): p. 840-7.
40. VandeVord, P.J., et al., Evaluation of the biocompatibility of a chitosan scaffold in mice. J Biomed Mater Res, 2002. 59(3): p. 585-90.
41. Elena Udrea, L., et al., Preparation and characterization of polyvinyl alcohol—chitosan biocompatible magnetic microparticles. Journal of Magnetism and Magnetic Materials, 2011. 323(1): p. 7-13.
42. Rinaudo, M., Chitin and chitosan: Properties and applications. Progress in Polymer Science, 2006. 31(7): p. 603-632.
43. Vivek, R., et al., Oxaliplatin-chitosan nanoparticles induced intrinsic apoptotic signaling pathway: a "smart" drug delivery system to breast cancer cell therapy. Int J Biol Macromol, 2014. 65: p. 289-97.
44. Jain, N., et al., Lactosaminated-N-succinyl chitosan nanoparticles for hepatocyte-targeted delivery of acyclovir. Journal of Nanoparticle Research, 2013. 16(1).
45. Bei, Y.Y., et al., Novel self-assembled micelles based on palmitoyl-trimethyl-chitosan for efficient delivery of harmine to liver cancer. Expert Opin Drug Deliv, 2014. 11(6): p. 843-54.
46. Clegg, D.O., et al., Glucosamine, chondroitin sulfate, and the two in combination for painful knee osteoarthritis. N Engl J Med, 2006. 354(8): p. 795-808.
47. Zhao, Q., et al., Magnetic nanoparticle-based hyperthermia for head & neck cancer in mouse models. Theranostics, 2012. 2: p. 113-21.
48. Kim, M.H., et al., Magnetic nanoparticle targeted hyperthermia of cutaneous Staphylococcus aureus infection. Ann Biomed Eng, 2013. 41(3): p. 598-609.
49. Wang, Y.X., S.M. Hussain, and G.P. Krestin, Superparamagnetic iron oxide contrast agents: physicochemical characteristics and applications in MR imaging. Eur Radiol, 2001. 11(11): p. 2319-31.
50. Wang, Y.X., Superparamagnetic iron oxide based MRI contrast agents: Current status of clinical application. Quant Imaging Med Surg, 2011. 1(1): p. 35-40.
51. Yu, M.K., et al., Drug-loaded superparamagnetic iron oxide nanoparticles for combined cancer imaging and therapy in vivo. Angew Chem Int Ed Engl, 2008. 47(29): p. 5362-5.
52. Thorek, D.L., et al., Superparamagnetic iron oxide nanoparticle probes for molecular imaging. Ann Biomed Eng, 2006. 34(1): p. 23-38.
53. Cloupeau, M. and B. Prunetfoch, Electrohydrodynamic Spraying Functioning Modes - a Critical-Review. Journal of Aerosol Science, 1994. 25(6): p. 1021-1036.
54. Xu, Y. and M.A. Hanna, Electrospray encapsulation of water-soluble protein with polylactide. Effects of formulations on morphology, encapsulation efficiency and release profile of particles. Int J Pharm, 2006. 320(1-2): p. 30-6.
55. Valo, H., et al., Electrospray encapsulation of hydrophilic and hydrophobic drugs in poly(L-lactic acid) nanoparticles. Small, 2009. 5(15): p. 1791-8.
56. Hong, Y.L., et al., Electrohydrodynamic atomization of quasi-monodisperse drug-loaded spherical/wrinkled microparticles. Journal of Aerosol Science, 2008. 39(6): p. 525-536.
57. Gaskell, S.J., Electrospray- Principles and Practice. J. Mass Spectrom, 1997. 32: p. 677-688.
58. Seo, H., et al., Preparation of polysaccharide nanofiber fabrics by electrospray deposition: Additive effects of poly(ethylene oxide). Polymer Journal, 2005. 37(6): p. 391-398.
59. Lee, Y.H., et al., Release profile characteristics of biodegradable-polymer-coated drug particles fabricated by dual-capillary electrospray. J Control Release, 2010. 145(1): p. 58-65.
60. Lee, Y.H., M.Y. Bai, and D.R. Chen, Multidrug encapsulation by coaxial tri-capillary electrospray. Colloids Surf B Biointerfaces, 2011. 82(1): p. 104-10.
61. Jayasinghe, S.N. and A. Townsend-Nicholson, Stable electric-field driven cone-jetting of concentrated biosuspensions. Lab Chip, 2006. 6(8): p. 1086-90.
62. Kien Nguyen, T., et al., Control and improvement of jet stability by monitoring liquid meniscus in electrospray and electrohydrodynamic jet. Journal of Aerosol Science, 2014. 71: p. 29-39.
63. Yin, H., L. Liao, and J. Fang, Enhanced Permeability and Retention (EPR) Effect Based Tumor Targeting: The Concept, Application and Prospect. JSM Clin Oncol Res, 2014. 2(1): p. 1010-1014.
64. Ahmed, F., et al., Biodegradable polymersomes loaded with both paclitaxel and doxorubicin permeate and shrink tumors, inducing apoptosis in proportion to accumulated drug. J Control Release, 2006. 116(2): p. 150-8.
65. Fouad, A.A., et al., Cardioprotective effect of cannabidiol in rats exposed to doxorubicin toxicity. Environ Toxicol Pharmacol, 2013. 36(2): p. 347-57.
66. Ravasco, P., I. Monteiro Grillo, and M. Camilo, Cancer wasting and quality of life react to early individualized nutritional counselling! Clin Nutr, 2007. 26(1): p. 7-15.
67. Van Cutsem, E. and J. Arends, The causes and consequences of cancer-associated malnutrition. Eur J Oncol Nurs, 2005. 9 Suppl 2: p. S51-63.
68. Introduction to Energy Dispersive X-ray Spectrometry (EDS).
69. Ai, W., et al., A novel graphene-polysulfide anode material for high-performance lithium-ion batteries. Sci Rep, 2013. 3: p. 2341.
70. Louis, K.S. and A.C. Siegel, Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol, 2011. 740: p. 7-12.
71. Swinehart, D.F., The Beer-Lambert Law. J. Chem. Educ., 1962. 39 (7): p. 333-335.
72. Terry L Riss, P., et al., Assay Guidance Manual: Cell Viability Assays. Assay Guidance Manual, 2013: p. 1-23.
73. Morton, C.L. and P.J. Houghton, Establishment of human tumor xenografts in immunodeficient mice. Nat Protoc, 2007. 2(2): p. 247-50.
74. Jensen, M.M., et al., Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper. BMC Med Imaging, 2008. 8: p. 16.
75. Dey, S., S. Pramanik, and A. Malgope, Formulation and optimization of sustained release Stavudine microspheres using response surface methodology. ISRN Pharm, 2011. 2011: p. 627623.
76. Ramasamy, T., et al., Formulation and evaluation of chondroitin sulphate tablets of aceclofenac for colon targeted drug delivery. Iran J Pharm Res, 2012. 11(2): p. 465-79.
77. Kumirska, J., et al., Application of spectroscopic methods for structural analysis of chitin and chitosan. Mar Drugs, 2010. 8(5): p. 1567-636.
78. Zangi, R. and B.J. Berne, Aggregation and dispersion of small hydrophobic particles in aqueous electrolyte solutions. J Phys Chem B, 2006. 110(45): p. 22736-41.
79. Desset, S., O. Spalla, and B. Cabane, Redispersion of alumina particles in water. Langmuir, 2000. 16(26): p. 10495-10508.
80. Nguyen, T.H., et al., Nanostructured liquid crystalline particles provide long duration sustained-release effect for a poorly water soluble drug after oral administration. J Control Release, 2011. 153(2): p. 180-6.
81. Benachour, N., et al., Time- and dose-dependent effects of roundup on human embryonic and placental cells. Arch Environ Contam Toxicol, 2007. 53(1): p. 126-33.
82. von Meyenfeldt, M., Cancer-associated malnutrition: an introduction. Eur J Oncol Nurs, 2005. 9 Suppl 2: p. S35-8.
83. Donohoe, C.L., A.M. Ryan, and J.V. Reynolds, Cancer cachexia: mechanisms and clinical implications. Gastroenterol Res Pract, 2011. 2011: p. 601434.
84. Chen, Z.C., L.J. Chen, and J.T. Cheng, Doxorubicin-Induced Cardiac Toxicity Is Mediated by Lowering of Peroxisome Proliferator-Activated Receptor delta Expression in Rats. PPAR Res, 2013. 2013: p. 456042.
85. MJ., T., Mechanisms of Cancer Cachexia. Physiol Rev, 2009. 89: p. 381-410.
86. Bosaeus, I., et al., Dietary intake and resting energy expenditure in relation to weight loss in unselected cancer patients. Int J Cancer, 2001. 93(3): p. 380-3.