近年來，愈來愈多的研究表明薄荷醇具有明顯的抗癌效果，可抑制多種惡性腫瘤的生長。已知薄荷醇具有多種藥理活性，如抗炎鎮痛、清涼止癢、抗真菌等，目前在臨床上被廣泛應用。研究發現薄荷醇其受體TRPM8在腫瘤細胞中高度表達，薄荷醇能激活腫瘤細胞上的TRPM8通道，允許鈣離子進入腫瘤細胞，增加細胞內的鈣濃度，降低細胞活力從而誘導腫瘤細胞死亡。然而，薄荷醇具有高揮發性、難溶於水等的局限性。本研究使用人類血清白蛋白作為藥物載體，將薄荷醇與人類血清白蛋白結合製作成包覆薄荷醇之白蛋白微氣泡對比劑 (Menthol-MBs)，以提升薄荷醇的穩定度。結合施打超音波能量可誘發包覆薄荷醇之白蛋白微氣泡破裂釋放藥物並產生穴蝕效應以增強藥物輸送效率，促進藥物導致的腫瘤細胞死亡。
本研究將薄荷醇與人類血清白蛋白結合，製作出包覆薄荷醇之白蛋白微氣泡 (Menthol-MBs)，經測量分析顯示平均粒徑為3.45 ± 0.25 µm，藥物包覆率為26.93 ± 0.39%。為探討包覆薄荷醇之白蛋白微氣泡結合超音波能量對腫瘤細胞生存率的影響，本研究採用實驗組別 (1) 控制組 (Control)；(2) 微氣泡對比劑 (MBs)；(3) 薄荷醇 (Menthol)；(4) 薄荷醇混合微氣泡 (Menthol+MBs)；(5) 薄荷醇包覆於微氣泡 (Menthol-MBs) 分別進行有無施打超音波能量之細胞實驗。實驗結果證實了薄荷醇與人類血清白蛋白結合能夠穩定薄荷醇，增強毒殺效果。加上超音波能量施打造成微氣泡破裂導致穴蝕效應使腫瘤細胞通透度增加，有助包覆於微氣泡球殼上的薄荷醇釋放進入細胞，誘發細胞死亡。因此包覆薄荷醇之白蛋白微氣泡結合超音波能量施打可達到治療效果最佳化 (p<0.001)。本研究將首創探討白蛋白微氣泡包覆薄荷醇結合超音波能量誘發穴蝕效應以提升薄荷醇於頭頸癌治療效果的可行性。

Recently, many evidences show the obvious anticancer effect of menthol which can inhibit the growth of malignant tumors. It has been found that menthol has many pharmacological properties such as anti-inflammatory, analgesic, cooling and relieving itching etc. Menthol is currently widely used in clinical applications. TRPM8, the receptor of menthol, is a nonselective cation channel which is highly expressed in tumor cells. It can be activated by menthol to allow calcium ions entering tumor cells. Increased calcium ion concentration in tumor cells result in reducing tumor cell viability, and inducing tumor cell death. However, limitations of menthol are high volatility and low solubility in water. In this thesis, human serum albumin (HSA) is used as a drug carrier to encapsulate menthol and produce menthol-loaded microbubbles (Menthol-MBs). Combined with ultrasound energy application, the menthol-loaded microbubbles could expanse and then collapse, resulting a cavitation effect to enhance the tumor cells death.
The average particle diameter of the menthol-loaded microbubbles is 3.45 ± 0.25 µm, and the encapsulation efficiency is 26.93 ± 0.39%. In order to investigate the feasibility of ultrasound mediated menthol-loaded microbubbles in cancer treatment, our thesis designed the in vitro cell experiments in five groups: (1) no treatment (Control), (2) microbubbles contrast agents (MBs), (3) Menthol, (4) Menthol mixed with microbubbles (Menthol+MBs), (5) Menthol-loaded microbubbles (Menthol-MBs) with or without ultrasound energy. The experimental results proved that the combination of menthol and HSA can stabilize menthol and enhance its pharmacological activity. In addition, ultrasound energy induces the collapsion of the microbubbles, causing the cavitation effect. It increases the permeability of the cancer cells, which enhances drug delivery into the tumor cells, hence induces cell death significantly. Therefore, the combination of menthol-loaded microbubbles and ultrasound energy can achieve the best therapeutic effect (p < 0.001). The feasibility of menthol-loaded microbubbles combined with ultrasound energy inducing cavitation to enhance the therapeutic effect on hypopharyngeal cancer was firstly investigated.

[1] World Health Organization. (2018). Cancer. Retrieved from https://www.who.int/en/news-room/fact-sheets/detail/cancer. Retrieved 21 Feb, 2021.

[2] Cancer Council NSW. (n.d.). What is cancer? Retrieved from
https://www.cancercouncil.com.au/cancer-information/understanding-cancer/what-is-cancer/. Retrieved 21 Feb, 2021.

[3] National Cancer Institute. (2017). Head and Neck Cancers. Retrieved from
https://www.cancer.gov/types/head-and-neck/head-neck-fact-sheet. Retrieved 21 Feb, 2021.

[4] Cancer Council NSW. (2019). Understanding Head and Neck Cancers. ISBN 978 1 925651 66 9.

[5] G. Zachary White. (2020). 10 Symptoms of Head and Neck Cancer. Retrieved from
https://www.bronsonhealth.com/news/10-symptoms-of-head-and-neck-cancer/
. Retrieved 21 Feb, 2021.

[6] Sabatini, M.E., Chiocca, S. (2020). Human papillomavirus as a driver of head and neck cancers. Br J Cancer 122, 306–314.

[7] Höckel, M. (2012). Cancer permeates locally within ontogenetic compartments: clinical evidence and implications for cancer surgery. Future Oncology, 8(1), 29–36. doi:10.2217/fon.11.128

[8] Sudhakar, A. (2009). History of cancer, ancient and modern treatment methods. Journal of cancer science & therapy, 1(2), 1.

[9] Malhotra, V., & Perry, M. C. (2003). Classical chemotherapy: mechanisms, toxicities and the therapeutc window. Cancer biology & therapy, 2(sup1), 1-3.

[10] National Cancer Institute. (2021). Targeted Cancer Therapies Fact Sheet.Retrieved from https://www.cancer.gov/about-cancer/treatment/types/targeted-therapies
/targeted-therapies-fact-sheet. Retrieved 28 Feb, 2021.
 
[11] Chen, C., Liu, Z.-G., Wang, S.-L., Sheng, X.-B., Wer, Z.-H., Yang, Z.-C., Chen, W.-X,, Wang, A.-Y., Zheng, S.-Z., Jiang, G.-R., Lu, Y. (2015). Research progress in the role of menthol and its receptor TRPM8 in tumor. Chinese Pharmacological Bulletin, 31(3): 312-314.

[12] Nathan, M., & Scholten, R. (1999). The Complete German Commission E Monographs: Therapeutic Guide to Herbal Medicines.

[13] A. Farco, J., & Grundmann, O. (2013). Menthol - Pharmacology of an Important Naturally Medicinal “Cool.” Mini Reviews in Medicinal Chemistry, 13(1), 124–131.

[14] Werley, M. S., Coggins, C. R., & Lee, P. N. (2007). Possible effects on smokers of cigarette mentholation: a review of the evidence relating to key research questions. Regulatory Toxicology and Pharmacology, 47(2), 189-203.

[15] Kim, S. H., Nam, J. H., Park, E. J., Kim, B. J., Kim, S. J., So, I., & Jeon, J. H. (2009). Menthol regulates TRPM8-independent processes in PC-3 prostate cancer cells. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1792(1), 33-38.

[16] Li, Q., Wang, X., Yang, Z., Wang, B., & Li, S. (2009). Menthol induces cell death via the TRPM8 channel in the human bladder cancer cell line T24. Oncology, 77(6), 335-341.

[17] Faridi, U., Sisodia, B. S., Shukla, A. K., Shukla, R. K., Darokar, M. P., Dwivedi, U. N., & Shasany, A. K. (2011). Proteomics indicates modulation of tubulin polymerization by L‐menthol inhibiting human epithelial colorectal adenocarcinoma cell proliferation. Proteomics, 11(10), 2115-2119.

[18] Yee, N. S. (2016). TRPM8 ion channels as potential cancer biomarker and target in pancreatic cancer. Advances in protein chemistry and structural biology, 104, 127-155.

[19] Liu, Z., Wu, H., Wei, Z., Wang, X., Shen, P., Wang, S. & Lu, Y. (2016). TRPM8: a potential target for cancer treatment. Journal of cancer research and clinical oncology, 142(9), 1871-1881.

[20] Balakrishnan, A. (2015). Therapeutic uses of peppermint-a review. Journal of pharmaceutical sciences and research, 7(7), 474.

[21] 台灣兒科醫學會. (2015) 薄荷醇與類似物質的兒童使用建議. 檢自https://www.pediatr.org.tw/people/edu_info.asp?id=20. Retrieved 19 March, 2021. 

[22] Fanali, G., Di Masi, A., Trezza, V., Marino, M., Fasano, M., & Ascenzi, P. (2012). Human serum albumin: from bench to bedside. Molecular Aspects of Medicine, 33(3), 209-290.

[23] Smith, G. S., G. L. Walter, and R. M. Walker. (2013). Chapter 18-Clinical Pathology in Non-Clinical Toxicology Testing. Haschek and Rousseaux's Handbook of Toxicologic Pathology (Third Edition), Boston: Academic Press, 565–594.

[24] Yang, F., Zhang, Y., & Liang, H. (2014). Interactive Association of Drugs Binding to Human Serum Albumin. International Journal of Molecular Sciences, 15(3), 3580-3595.

[25] Kratz, F., Müller-Driver, R., Hofmann, I., Drevs, J., & Unger, C. (2000). A Novel Macromolecular Prodrug Concept Exploiting Endogenous Serum Albumin as a Drug Carrier for Cancer Chemotherapy. Journal of Medicinal Chemistry, 43(7), 1253-1256.

[26] Yeggoni, D. P., Rachamallu, A., Dubey, S., Mitra, A., & Subramanyam, R. (2018). Probing the interaction mechanism of menthol with blood plasma proteins and its cytotoxicity activities. Journal of Biomolecular Structure and Dynamics, 36(2), 465-474.

[27] Morel, D. R., Schwieger, I., Hohn, L., Terrettaz, J., LLULL, J. B., CORNIOLEY, Y. A., & Schneider, M. (2000). Human pharmacokinetics and safety evaluation of SonoVue™, a new contrast agent for ultrasound imaging. Investigative Radiology, 35(1), 80.

[28] Goldberg, B. B., Liu, J. B., & Forsberg, F. (1994). Ultrasound contrast agents: a review. Ultrasound in Medicine & Biology, 20(4), 319-333.

[29] Quaia, E. (2007). Microbubble ultrasound contrast agents: an update. European Radiology, 17(8), 1995-2008.

[30] Zhao, Y. Z., Du, L. N., Lu, C. T., Jin, Y. G., & Ge, S. P. (2013). Potential and problems in ultrasound-responsive drug delivery systems. International Journal of Nanomedicine, 8, 1621.

[31] Tinkov, S., Bekeredjian, R., Winter, G., & Coester, C. (2009). Microbubbles as ultrasound triggered drug carriers. Journal of Pharmaceutical Sciences, 98(6), 1935–1961.
 
[32] New World Encyclopedia. Ultrasound, (n.d.), Retrieved from http://www.newworldencyclopedia.org/entry/Ultrasound. Retrieved 5 April, 2021

[33] Chan, V., & Perlas, A. (2011). Basics of ultrasound imaging. In Atlas of ultrasound-guided procedures in interventional pain management. Springer, New York, NY, 13-19.

[34] Dubinsky, T. J., Cuevas, C., Dighe, M. K., Kolokythas, O., & Hwang, J. H. (2008). High-intensity focused ultrasound: current potential and oncologic applications. American journal of roentgenology, 190(1), 191-199.

[35] Wang, T. Y., E Wilson, K., Machtaler, S., & K Willmann, J. (2013). Ultrasound and microbubble guided drug delivery: mechanistic understanding and clinical implications. Current pharmaceutical biotechnology, 14(8), 743-752.

[36] Kong, G., & Dewhirst, M. W. (1999). Review hyperthermia and liposomes. International Journal of Hyperthermia, 15(5), 345-370.

[37] Lefor, A. T., Makohon, S., & Ackerman, N. B. (1985). The effects of hyperthermia on vascular permeability in experimental liver metastasis. Journal of surgical oncology, 28(4), 297-300.

[38] Izadifar, Z., Babyn, P., & Chapman, D. (2019). Ultrasound cavitation/microbubble detection and medical applications. Journal of Medical and Biological Engineering, 39(3), 259-276.

[39] Leong, T., Ashokkumar, M., & Kentish, S. (2011). The fundamentals of power ultrasound-a review.

[40] Church, C. C., & Carstensen, E. L. (2001). “Stable” inertial cavitation. Ultrasound in medicine & biology, 27(10), 1435-1437.

[41] Misono, T. (2019). Dynamic Light Scattering (DLS). Measurement Techniques and Practices of Colloid and Interface Phenomena, 65-69.

[42] Xu, R. (2008). Progress in nanoparticles characterization: Sizing and zeta potential measurement. Particuology, 6(2), 112-115.
 
[43] Liao, Q. C. (2000). Principle and prospect to improve the resolution of field emission scanning electron microscope. JOURNAL-CHINESE ELECTRON MICROSCOPY SOCIETY, 19(5), 709-716.

[44] Kushwah, H., Hans, T., Chauhan, M., Mittal, G., & Sandal, N. (2020). Development and Validation of the Spectrophotometric Method for the Determination of Menthol. Journal of Applied Spectroscopy, 87(3), 563-567.

[45] The Privalsky Lab, U. Cell Counting with a Hemocytometer. n.d.; Retrieved from http://microbiology.ucdavis.edu/privalsky/hemocytometer. Retrieved 21 May, 2021.

[46] Byth, H. A., Mchunu, B. I., Dubery, I. A., & Bornman, L. (2001). Assessment of a simple, non‐toxic alamar blue cell survival assay to monitor tomato cell viability. Phytochemical Analysis: An International Journal of Plant Chemical and Biochemical Techniques, 12(5), 340-346.

[47] Rampersad, S. N. (2012). Multiple applications of Alamar Blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors, 12(9), 12347-12360.