褐藻醣膠是源自於褐藻中所萃取的硫酸化多醣，在研究中發現褐藻醣膠具有抗癌特性、抑制血管新生以及活化免疫系統等作用，目前研究發展以抗癌作用為主，對於其佐劑效果並沒有深入探討，因此本研究主要探討褐藻醣膠的免疫能力，並結合疫苗微針(microneedle, MN)作為主題，評估褐藻醣膠是否能成為輔助疫苗效果之佐劑。本研究使用善笙科技股份有限公司所提供之兩款褐藻醣膠，高分子褐藻醣膠(high molecular fucoidan, HFU)與全分子褐藻醣膠(whole-profile fucoidan, WFU)，將兩款褐藻醣膠與巨噬細胞Raw264.7共培養，檢測主要組織相容性複合體(major histocompatibility complex, MHC)是否有表達，發現WFU相較於HFU更能促進細胞表達MHC Ⅰ與MHC Ⅱ，使輔助T細胞被活化，顯示WFU能提升細胞介導免疫與體液免疫能力的潛力。其中將WFU混合卵白蛋白(ovalbumin, OVA)製成可溶式微針(dissolving MN)，OVA為模擬抗原，並使用polyvinylpyrrolidone/polyvinyl alcohol (PVP/PVA)幫助微針成型，以離心法製成OVA-loaded WFU MN。體外穿刺豬皮結果顯示穿刺深度約為367 μm，穿刺10分鐘後可傳輸27.5 ± 3.6 μg OVA(n=5)與141.1 ± 7.4 μg WFU(n=5)進入真皮層中，傳遞效率各為42.4% ± 5.6%(n=5)與37.9% ± 2.0%(n=5)。將OVA-loaded WFU MN施打於大鼠背部，並追蹤皮膚上的FITC-OVA螢光，微針造成之微傷口約4-5小時內可完全癒合，且FITC-OVA能滯留於皮膚中至少6天。大鼠免疫試驗中分析血清中OVA-specific total IgG濃度，發現於第二週時，OVA-loaded WFU MN組相較於皮下注射OVA組有更高的抗體表現，且於第六週強度達最高峰，證明本實驗室製作之含褐藻醣膠之疫苗微針確實能提升免疫反應之效果。

We used the fucoidan, whole-profile fucoidan (WFU) and high molecular fucoidan (HFU), as an adjuvant from SIMPSON BIOTECH CO., LTD and combined with ovalbumin (OVA) to examine the effect of immune response. We observe that the cytotoxicity was not shown on WFU and HFU, but the effect of immune response can be found on WFU. Therefore, we used WFU blending with OVA to fabricate microneedles (MNs). MNs were applied to porcine skin, and the penetration depth was about 367 μm. The delivered dose was about 27.5 ± 3.6 μg OVA (n=5) and 141.1 ± 7.4 μg WFU (n=5). After calculation, the delivery efficiency was 42.4% ± 5.6% and 37.9% ± 2.0% (n=5), respectively. In vivo study was assessed with Sprague Dawley (S.D.) rats. Upon MN treatment on S.D. rat, the skin can be recovered within 4-5 hours, and the FITC-OVA can’t be tracked for about 6 days, which means it could be retained by S.D. rats for about 6 days. Serum analysis showed that compared with subcutaneous OVA vaccination, OVA-specific total IgG test in the 2nd week could be obviously detected by using OVA-loaded WFU MN and the highest intensity could be examined in the 6th week. It proved that the first fucoidan-containing microneedles produced by our laboratory can indeed promote the effect of immune responses.

[1] J. Wang, L. Geng, Y. Yue, Q. Zhang, Use of fucoidan to treat renal diseases: A review of 15 years of clinic studies, Prog Mol Biol Transl Sci, 163 (2019) 95-111.
[2] B. Li, F. Lu, X. Wei, R. Zhao, Fucoidan: structure and bioactivity, Molecules, 13 (2008) 1671-1695.
[3] L. Wu, J. Sun, X. Su, Q. Yu, Q. Yu, P. Zhang, A review about the development of fucoidan in antitumor activity: Progress and challenges, Carbohydr Polym, 154 (2016) 96-111.
[4] G. van Weelden, M. Bobinski, K. Okla, W.J. van Weelden, A. Romano, J.M.A. Pijnenborg, Fucoidan Structure and Activity in Relation to Anti-Cancer Mechanisms, Mar Drugs, 17 (2019) 32.
[5] S.R. Khil’chenko, T.S. Zaporozhets, N.M. Shevchenko, T.N. Zvyagintseva, U. Vogel, P. Seeberger, B. Lepenies, Immunostimulatory Activity of Fucoidan from the Brown AlgaFucus evanescens: Role of Sulfates and Acetates, Journal of Carbohydrate Chemistry, 30 (2011) 291-305.
[6] S.Y. Wu, W.Y. Yang, C.C. Cheng, K.H. Lin, B.P. Sampurna, S.M. Chan, C.H. Yuh, Low molecular weight fucoidan inhibits hepatocarcinogenesis and nonalcoholic fatty liver disease in zebrafish via ASGR/STAT3/HNF4A signaling, Clin Transl Med, 10 (2020) e252.
[7] H. Maruyama, H. Tamauchi, M. Hashimoto, T. Nakano, Antitumor activity and immune response of Mekabu fucoidan extracted from Sporophyll of Undaria pinnatifida, In Vivo, 17 (2003) 245-249.
[8] N.E. Ustyuzhanina, M.I. Bilan, N.A. Ushakova, A.I. Usov, M.V. Kiselevskiy, N.E. Nifantiev, Fucoidans: pro- or antiangiogenic agents?, Glycobiology, 24 (2014) 1265-1274.
[9] J.Y. Kwak, Fucoidan as a marine anticancer agent in preclinical development, Mar Drugs, 12 (2014) 851-870.
[10] J.O. Jin, W. Zhang, J.Y. Du, K.W. Wong, T. Oda, Q. Yu, Fucoidan can function as an adjuvant in vivo to enhance dendritic cell maturation and function and promote antigen-specific T cell immune responses, PLoS One, 9 (2014) e99396.
[11] W. Zhang, T. Oda, Q. Yu, J.O. Jin, Fucoidan from Macrocystis pyrifera Has Powerful Immune-Modulatory Effects Compared to Three Other Fucoidans, Marine Drugs, 13 (2015) 1084-1104.
[12] K. Hayashi, T. Nakano, M. Hashimoto, K. Kanekiyo, T. Hayashi, Defensive effects of a fucoidan from brown alga Undaria pinnatifida against herpes simplex virus infection, Int Immunopharmacol, 8 (2008) 109-116.
[13] H. Maruyama, H. Tamauchi, F. Kawakami, K. Yoshinaga, T. Nakano, Suppressive Effect of Dietary Fucoidan on Proinflammatory Immune Response and MMP-1 Expression in UVB-Irradiated Mouse Skin, Planta Med, 81 (2015) 1370-1374.
[14] H. Maruyama, H. Tamauchi, M. Hashimoto, T. Nakano, Suppression of Th2 immune responses by mekabu fucoidan from Undaria pinnatifida sporophylls, International archives of allergy and immunology, 137 (2005) 289-294.
[15] E. Apostolova, P. Lukova, A. Baldzhieva, P. Katsarov, M. Nikolova, I. Iliev, L. Peychev, B. Trica, F. Oancea, C. Delattre, V. Kokova, Immunomodulatory and Anti-Inflammatory Effects of Fucoidan: A Review, Polymers (Basel), 12 (2020) 2338.
[16] T.A. Seimon, A. Obstfeld, K.J. Moore, D.T. Golenbock, I. Tabas, Combinatorial pattern recognition receptor signaling alters the balance of life and death in macrophages, Proceedings of the National Academy of Sciences, 103 (2006) 19794-19799.
[17] T. Areschoug, S. Gordon, Scavenger receptors: role in innate immunity and microbial pathogenesis, Cell Microbiol, 11 (2009) 1160-1169.
[18] H. Yu, T. Ha, L. Liu, X. Wang, M. Gao, J. Kelley, R. Kao, D. Williams, C. Li, Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages, Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1823 (2012) 1192-1198.
[19] D. Wang, B. Sun, M. Feng, H. Feng, W. Gong, Q. Liu, S. Ge, Role of scavenger receptors in dendritic cell function, Hum Immunol, 76 (2015) 442-446.
[20] T. Tian, H. Chang, K. He, Y. Ni, C. Li, M. Hou, L. Chen, Z. Xu, B. Chen, M. Ji, Fucoidan from seaweed Fucus vesiculosus inhibits 2,4-dinitrochlorobenzene-induced atopic dermatitis, Int Immunopharmacol, 75 (2019) 105823.
[21] Z. Lin, X. Tan, Y. Zhang, F. Li, P. Luo, H. Liu, Molecular Targets and Related Biologic Activities of Fucoidan: A Review, Mar Drugs, 18 (2020) 376.
[22] S. Jozefowski, R. Biedron, M. Srottek, M. Chadzinska, J. Marcinkiewicz, The class A scavenger receptor SR-A/CD204 and the class B scavenger receptor CD36 regulate immune functions of macrophages differently, Innate Immun, 20 (2014) 826-847.
[23] A. Sette, S. Crotty, Adaptive immunity to SARS-CoV-2 and COVID-19, Cell, 184 (2021) 861-880.
[24] M.F. Bachmann, G.T. Jennings, Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns, Nature Reviews Immunology, 10 (2010) 787-796.
[25] D. Li, D. Hu, H. Xu, H.K. Patra, X. Liu, Z. Zhou, J. Tang, N. Slater, Y. Shen, Progress and perspective of microneedle system for anti-cancer drug delivery, Biomaterials, 264 (2021) 120410.
[26] K.M. Kwon, S.M. Lim, S. Choi, D.H. Kim, H.E. Jin, G. Jee, K.J. Hong, J.Y. Kim, Microneedles: quick and easy delivery methods of vaccines, Clin Exp Vaccine Res, 6 (2017) 156-159.
[27] R.F. Donnelly, T.R.R. Singh, D.I.J. Morrow, M.A.D. Woolfson, Microneedle-mediated Transdermal and Intradermal Drug Delivery, Wiley-Blackwell, (2012) p.20-21.
[28] H.R. Jeong, J.Y. Bae, J.H. Park, S.K. Baek, G. Kim, M.S. Park, J.H. Park, Preclinical study of influenza bivalent vaccine delivered with a two compartmental microneedle array, Journal of Controlled Release, 324 (2020) 280-288.
[29] L. Niu, L.Y. Chu, S.A. Burton, K.J. Hansen, J. Panyam, Intradermal delivery of vaccine nanoparticles using hollow microneedle array generates enhanced and balanced immune response, Journal of Controlled Release, 294 (2019) 268-278.
[30] S.C. Balmert, C.D. Carey, G.D. Falo, S.K. Sethi, G. Erdos, E. Korkmaz, L.D. Falo, Jr., Dissolving undercut microneedle arrays for multicomponent cutaneous vaccination, Journal of Controlled Release, 317 (2020) 336-346.
[31] M. Leone, J. Monkare, J.A. Bouwstra, G. Kersten, Dissolving Microneedle Patches for Dermal Vaccination, Pharm Res, 34 (2017) 2223-2240.
[32] J.K. Kaushik, R. Bhat, Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose, J Biol Chem, 278 (2003) 26458-26465.
[33] P. Van Dijck, D. Colavizza, P. Smet, J.M. Thevelein, Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells, Applied and Environmental Microbiology, 61 (1995) 109.
[34] A. Hassanzadeh-Barforoushi, A.M.K. Law, A. Hejri, M. Asadnia, C.J. Ormandy, D. Gallego-Ortega, M. Ebrahimi Warkiani, Static droplet array for culturing single live adherent cells in an isolated chemical microenvironment, Lab on a Chip, 18 (2018) 2156-2166.
[35] R. González-Pinzón, R. Haggerty, D.D. Myrold, Measuring aerobic respiration in stream ecosystems using the resazurin-resorufin system, Journal of Geophysical Research: Biogeosciences, 117 (2012) G00N06.
[36] D.L. Jaye, R.A. Bray, H.M. Gebel, W.A. Harris, E.K. Waller, Translational applications of flow cytometry in clinical practice, J Immunol, 188 (2012) 4715-4719.
[37] E. Zhang, F. Chu, T. Zhao, Y. Chai, H. Liang, S. Song, A. Ji, Determination of fucoidan in rat plasma by HPLC and its application in pharmacokinetics, Pakistan journal of pharmaceutical sciences, 33 (2020).
[38] S. Jaworski, M. Wierzbicki, E. Sawosz, A. Jung, G. Gielerak, J. Biernat, H. Jaremek, W. Łojkowski, B. Woźniak, J. Wojnarowicz, L. Stobiński, A. Małolepszy, M. Mazurkiewicz-Pawlicka, M. Łojkowski, N. Kurantowicz, A. Chwalibog, Graphene Oxide-Based Nanocomposites Decorated with Silver Nanoparticles as an Antibacterial Agent, Nanoscale Research Letters, 13 (2018) 116.