在近十年中，治療性蛋白質藥物及其相關產品快速增長。蛋白質藥物具有特異性強、耐受性高、藥效高、毒性低等優勢等，被公認為是治療相關疾病的良好候選藥物。然而，基於蛋白質的藥物也受到一些缺點的限制，例如通過粘膜的通透性差、在胃腸道中快速降解、血漿半衰期短等。因此如何提高蛋白質藥物的效率、可持續性和緩釋性是此類藥物發展的關鍵。為了解決這些問題，確保藥物穩定一致和延長釋放及吸收的時間，可使用溶解性的微針或用聚合物與微乳液封裝的蛋白質藥物。然而，這些方法都有其缺點，例如每次治療時加載藥物量的限制、微針製造過程中的汙染或微脂粒的低免疫相容性。相比之下，水凝膠與低免疫原性受體結合方面具特異性、高度生物相容性和可生物降解的等優勢。考量前述等缺點，我們選擇透明質酸 (HA)用於本研究，透明質酸為一種糖胺聚醣，廣泛用於長放緩釋型藥物。在本論文中，為了模擬 HA應用於化妝品化合物緩釋藥物的能力，我們選擇一種存在於淚液中，可檢測糖尿病視網膜病變的生物標誌物Lipocalin-1(LCN-1)與HA作混和。混合後LCN-1會從中釋放，和捕獲LCN-1抗體接合的聚苯乙烯 (PS) 微珠形成目標抗原捕獲的三明治結構。此相互作用將導致微珠的有效粒徑改變，根據斯托克斯-愛因斯坦方程式(Stoke-Einstein equation)，擴散率與微珠直徑成反比，粒徑改變會導致其擴散率發生變化，藉此擴散率的變化我們可以瞭解LCN-1於HA中釋放的情形。本研究追蹤三天LCN-1與HA混和後釋放的過程，為了防止 HA 在 LCN-1 和 PS 相互作用中的干擾，提出了帶有過濾膜的PMMA 試片作為支架和滲透屏障，允許 LCN-1 通過，同時減少HA的干擾。校準曲線將由在使用 HA 背景下對 LCN-1 進行定性和定量的評估。這項研究開發了一種快速靈敏的體外模擬藥物釋放裝置，不僅可以研究基於HA的 LCN-1 釋放效率，未來還可以評估其他基於水凝膠蛋白質藥物釋放的替代方案。

Therapeutic protein drugs and their related products have displayed rapid growth over the past decades. Protein drugs are well known as good candidates for treating various diseases owing to their advantages over the current products, such as high specificity, high tolerance, high potency, and low toxicity. Nevertheless, protein-based drugs are also limited by some disadvantages, such as poor permeability through mucosal membranes, fast degradability in the GI tract, short plasma half-life, and so on. Therefore, increasing the efficiency, sustainability, and sustained release of protein drugs is the pivotal point. In order to tackle these problems, multiple attempts have been conducted in both external and internal aspects of protein drugs to ensure their consistent and long-term release, such as the use of dissolvable microneedle or encapsulate therapeutic protein drugs with polymer and microemulsion. However, these methods have their disadvantages, such as the limitation in loaded active compounds per treatment, the tidiness process in the manufacture of the microneedle, or low immuno-compatible of micro bubble capsulation. By contrast, hydrogels show advantages in performing a specific receptor binding with low immunogenicity, highly biocompatibility, and biodegradable. All of those disadvantages lead to our selection of Hyaluronic Acid (HA), a glycosaminoglycan for this study. HA is widely used for the sustained release of drug compounds and can be applied in various fields. In this thesis, to mimic the ability of HA in its ability of sustained release drug compound for cosmetic application, a combination of HA with LCN-1, a biomarker presents in tear fluid to detect diabetic retinopathy as a target protein for this proof-of-concept real-time protein tracking method. In order to monitor the process throughout 3 days, a bead-based sandwich immunoassay has been selected. This bead-based includes conjugated antibody in polystyrene (PS) particles as the method for detection and for quantitatively evaluating the release of LCN-1 in HA based on the Brownian motion phenomenon. The interaction between the released LCN-1 from HA and PS will result in the change in particle diameter and hence, its diffusivity. To prevent the interference of the HA in the LCN-1 and PS interaction, this study proposed an approach in which a PMMA based chip with a filter can act as a holder and semi-permeable barrier that allows LCN-1 to pass through while minimizing the pass-through of HA. The calibration curves will be prepared for qualitative and quantitative assessment of LCN-1 under the use of HA. Overall, this research successfully developed a rapid and sensitive detection in vitro device not only to investigate the efficiency of Hyaluronic Acid-based LCN-1 release but also to provide an alternative to evaluating other hydrogel-based protein drugs.

[1] R. J. Keizer, A. D. Huitema, J. H. Schellens, and J. H. Beijnen, "Clinical pharmacokinetics of therapeutic monoclonal antibodies,", Clinical pharmacokinetics, vol. 49, no. 8, pp. 493-507, 2010.

[2] B. Albarran, A. S. Hoffman, and P. S. Stayton, "Efficient intracellular delivery of a pro-apoptotic peptide with a pH-responsive carrier," Reactive & Functional Polymers, vol. 71, no. 3, pp. 261-265, 2011.

[3] J. Li and D. J. Mooney, "Designing hydrogels for controlled drug delivery," Nature Reviews Materials, vol. 1, no. 12, p. 16071, 2016.

[4] R. Aliperta, P. B. Welzel, R. Bergmann, U. Freudenberg, N. Berndt, A. Feldmann, C. Arndt, S. Koristka, M. Stanzione, M. Cartellieri, A. Ehninger, G. Ehninger, C. Werner, J. Pietzsch, J. Steinbach, M. Bornhäuser, and M. P. Bachmann,"Cryogel-supported stem cell factory for customized sustained release of bispecific antibodies for cancer immunotherapy," Scientific reports, vol. 7, pp. 42855-42855, 2017.

[5] D. Wang, H. Dong, M. Li, X. Meng, Y. Cao, K. Zhang, W. Dai, C. Wang, and X. Zhang,"Hyaluronic acid encapsulated CuS gel-mediated near-infrared laser-induced controllable transdermal drug delivery for sustained therapy," ACS Sustainable Chemistry & Engineering, vol. 5, no. 8, pp. 6786-6794, 2017.

[6] R. Parhi, "Cross-linked hydrogel for pharmaceutical applications: a review," Advanced Pharmaceutical Bulletin, vol. 7, no. 4, pp. 515-530, 2017.

[7] M. B. Brown and S. A. Jones, "Hyaluronic acid: a unique topical vehicle for the localized delivery of drugs to the skin," Journal of the European Academy of Dermatology and Venereology, vol. 19, no. 3, pp. 308-18, 2005.

[8] E. A. Balazs and J. L. Denlinger, "Viscosupplementation - a new concept in the treatment of osteoarthritiS," The Journal of Rheumatology, Article vol. 20, pp. 3-9, 1993.

[9] E. Maheu, X. Ayral, and M. Dougados, "A hyaluronan preparation (500-730 kDa) in the treatment of osteoarthritis: a review of clinical trials with Hyalgan," International Journal of Clinical Practice, vol. 56, no. 10, pp. 804-13, 2002.

[10] P. Ghosh and D. Guidolin, "Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent?" Seminars in Arthritis and Rheumatism, vol. 32, no. 1, pp. 10-37, 2002.

[11] P. Baranczewski, A. Stańczak, K. Sundberg, R. Svensson, A. Wallin, J. Jansson, P. Garberg, and H. Postlind, "Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development," Pharmacological Reports, vol. 58, no. 4, pp. 453-72, 2006.

[12] M. L. Frisk, W. H. Tepp, E. A. Johnson, and D. J. Beebe, "Self-assembled peptide monolayers as a toxin sensing mechanism within arrayed microchannels," Analytical Chemistry, vol. 81, no. 7, pp. 2760-2767, 2009.

[13] R. Victor, F. Gamez, W. Keener, J. White, and M. Poli, "Rapid detection of Clostridium botulinum toxins A, B, E, and F in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection," Analytical Biochemistry, vol. 353, pp. 248-56, 2006.

[14] I. Cavero, J.-M. Guillon, and H. H. Holzgrefe, "Human organotypic bioconstructs from organ-on-chip devices for human-predictive biological insights on drug candidates," Expert Opinion on Drug Safety, vol. 18, no. 8, pp. 651-677, 2019.

[15] Y.-T. Yang, J.-C. Wang, and H.-S. Chuang, "Developing rapid antimicrobial susceptibility testing for motile/non-Motile bacteria treated with antibiotics covering five bactericidal mechanisms on the basis of bead-based optical diffusometry," Biosensors, vol. 10, no. 11, p. 181, 2020.

[16] W.-L. Chen and H.-S. Chuang, "Trace biomolecule detection with functionalized Janus Particles by rotational diffusion," Analytical Chemistry, vol. 92, no. 19, pp. 12996-13003, 2020.

[17] H.-P. Cheng and H.-S. Chuang, "Rapid and sensitive nano-immunosensors for Botulinum," ACS Sensors, vol. 4, no. 7, pp. 1754-1760, 2019.
[18] A. M. Ahmed, "History of diabetes mellitus," Saudi Medical Journal, vol. 23, no. 4, pp. 373-8, 2002.

[19] J. A. C. Sterne, S. Murthy, J. V. Diaz, A. S. Slutsky, J. Villar, D. C. Angus, D. Annane, L. C. P. Azevedo, O. Berwanger, A. B. Cavalcanti, P. F. Dequin, B. Du, J. Emberson, D. Fisher, B. Giraudeau, A. C. Gordon, A. Granholm, C. Green, R. Haynes, N. Heming, J. P. T. Higgins, P. Horby, P. Jüni, M. J. Landray, A. Le Gouge, M. Leclerc, W. S. Lim, F. R. Machado, C. McArthur, F. Meziani, M. H. Møller, A. Perner, M. W. Petersen, J. Savovic, B. Tomazini, V. C. Veiga, S. Webb, and J. C. Marshall, "Association between administration of systemic corticosteroids and mortality among critically ill patients with COVID-19: a meta-analysis," (in eng), Journal of the American Medical Association, vol. 324, no. 13, pp. 1330-1341, 2020.

[20] G. Kokic, H. S. Hillen, D. Tegunov, C. Dienemann, F. Seitz, J. Schmitzova, L. Farnung, A. Siewert, C. Höbartner, and P. Cramer,"Mechanism of SARS-CoV-2 polymerase stalling by remdesivir," Nature Communications, vol. 12, no. 1, p. 279, 2021.

[21] E. P. Herrero, M. J. Alonso, and N. Csaba, "Polymer-based oral peptide nanomedicines," Therapeutic delivery, vol. 3, no. 5, pp. 657-68, 2012.

[22] M. K. Tiwari, R. Singh, R. K. Singh, I.-W. Kim, and J.-K. Lee, "Computational approaches for rational design of proteins with novel functionalities," Computational and Structural Biotechnology Journal, vol. 2, pp. e201209002-e201209002, 2012.

[23] D. S.S., " Global markets for bioengineered protein drugs. 2017," Business Communications Company Research, 2018.

[24] T. Bratt, "Lipocalins and cancer," Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, vol. 1482, no. 1, pp. 318-326, 2000.

[25] L. Lögdberg and L. Wester, "Immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses," Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology, vol. 1482, no. 1, pp. 284-297, 2000.

[26] É. Csősz, P. Boross, A. Csutak, A. Berta, F. Tóth, S. Póliska, Z. Török, and J. Tőzsér, "Quantitative analysis of proteins in the tear fluid of patients with diabetic retinopathy, " (in eng), Journal of proteomics, vol. 75, no. 7, pp. 2196-204, 2012.

[27] B. Munos, "Lessons from 60 years of pharmaceutical innovation," Nature Reviews Drug Discovery, vol. 8, no. 12, pp. 959-68, 2009.

[28] K. Smietana, M. Siatkowski, and M. Moller, "Trends in clinical success rates," Nature Reviews Drug Discovery, vol. 15, no. 6, pp. 379-80, 2016.

[29] Y. C. Kim, J. H. Park, and M. R. Prausnitz, "Microneedles for drug and vaccine delivery," Advanced Drug Delivery Reviews, vol. 64, no. 14, pp. 1547-68, 2012.

[30] H. L. Quinn, M. C. Kearney, A. J. Courtenay, M. T. McCrudden, and R. F. Donnelly, "The role of microneedles for drug and vaccine delivery," Expert opinion on drug delivery, vol. 11, no. 11, pp. 1769-80, 2014.

[31] H. S. Gill and M. R. Prausnitz, "Coated microneedles for transdermal delivery," (in eng), Journal of Controlled Release, vol. 117, no. 2, pp. 227-37, 2007.

[32] M. Dangol, S. Kim, C. G. Li, S. Fakhraei Lahiji, M. Jang, Y. Ma, I. Huh, and H. Jung, "Anti-obesity effect of a novel caffeine-loaded dissolving microneedle patch in high-fat diet-induced obese C57BL/6J mice," Journal of Controlled Release, vol. 265, pp. 41-47, 2017.

[33] W. Martanto, J. S. Moore, O. Kashlan, R. Kamath, P. M. Wang, J. M. O'Neal, and M. R. Prausnitz, "Micro infusion using hollow microneedles," Pharmaceutical Research, vol. 23, no. 1, pp. 104-13, 2006.

[34] W. Martanto, J. S. Moore, O. Kashlan, R. Kamath, P. M. Wang, J. M. O'Neal, and M. R. Prausnitz, "Microinfusion using hollow microneedles," Pharmaceutical research, vol. 23, no. 1, pp. 104-13, Jan 2006.
[34] R. F. Donnelly, K. Mooney, M. T. McCrudden, E. M. Vicente-Pérez, L. Belaid, P. González-Vázquez, J. C. McElnay, and A. D. Woolfson, "Hydrogel-forming microneedles increase in volume during swelling in skin, but skin barrier function recovery is unaffected," Journal of Pharmaceutical Sciences, vol. 103, no. 5, pp. 1478-86, 2014.

[35] S. Mitragotri, "Devices for overcoming biological barriers: the use of physical forces to disrupt the barriers," Advanced Drug Delivery Reviews, vol. 65, no. 1, pp. 100-3, 2013.

[36] C. Nastiti, T. Ponto, E. Abd, J. E. Grice, H. A. E. Benson, and M. S. Roberts, "Topical nano and microemulsions for skin delivery," Pharmaceutics, vol. 9, no. 4, 2017.

[37] U. A. Shinde, S. H. Modani, and K. H. Singh, "Design and development of repaglinide microemulsion gel for transdermal delivery," AAPS PharmSciTech, vol. 19, no. 1, pp. 315-325, 2018.

[38] S. Banerji, A. J. Wright, M. Noble, D. J. Mahoney, I. D. Campbell, A. J. Day, and D. G. Jackson, "Structures of the Cd44–hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction," Nature Structural & Molecular Biology, vol. 14, no. 3, pp. 234-239, 2007.

[39] J. R. E. Fraser, T. C. Laurent, and U. B. G. Laurent, "Hyaluronan: its nature, distribution, functions and turnover," Journal of Internal Medicine, vol. 242, no. 1, pp. 27-33, 1997.

[40] T. C. Laurent and J. R. E. Fraser, "Hyaluronan1," The Federation of American Societies for Experimental Biology Journal, vol. 6, no. 7, pp. 2397-2404, 1992.

[41] T. C. Laurent, U. B. Laurent, and J. R. Fraser, "Functions of hyaluronan," Annals of the Rheumatic Diseases, vol. 54, no. 5, p. 429, 1995.

[42] R. Stern, A. A. Asari, and K. N. Sugahara, "Hyaluronan fragments: An information-rich system," European Journal of Cell Biology, vol. 85, no. 8, pp. 699-715, 2006.

[43] R. Stern, "Hyaluronidases in cancer biology," Seminars in Cancer Biology, vol. 18, no. 4, pp. 275-280, 2008.

[44] A. J. Day and J. K. Sheehan, "Hyaluronan: polysaccharide chaos to protein organisation," Current Opinion in Structural Biology, vol. 11, no. 5, pp. 617-622, 2001.

[45] M. A. Selyanin, P. Y. Boykov, V. N. Khabarov, and F. Polyak, "Hyaluronic Acid: Preparation, Properties, Application in Biology and Medicine," John Wiley & Sons, Ltd., Book p. 215, 2015.

[46] G. Ma, Y. Liu, D. Fang, J. Chen, C. Peng, X. Fei, and J. Nie, "Hyaluronic acid/chitosan polyelectrolyte complexes nanofibers prepared by electrospinning," Materials Letters, vol. 74, pp. 78-80, 2012.

[47] R. C. Polexe and T. Delair, "Elaboration of stable and antibody functionalized positively charged colloids by polyelectrolyte complexation between chitosan and hyaluronic acid," Molecules, vol. 18, no. 7, pp. 8563-8578, 2013.

[48] T. Pouyani, G. S. Harbison, and G. D. Prestwich, "Novel hydrogels of Hyaluronic acid: synthesis, surface morphology, and solid-state NMR," Journal of the American Chemical Society, vol. 116, no. 17, pp. 7515-7522, 1994.

[49] J. E. Scott and F. Heatley, "Hyaluronan forms specific stable tertiary structures in aqueous solution: A 13C NMR study," Proceedings of the National Academy of Sciences, vol. 96, no. 9, p. 4850, 1999.

[50] J. E. Scott, C. Cummings, A. Brass, and Y. Chen, "Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient network-forming polymer," Biochemical Journal, vol. 274, no. 3, pp. 699-705, 1991.

[51] N. Pannacciulli, D. S. Le, A. D. Salbe, K. Chen, E. M. Reiman, P. A. Tataranni, and J. Krakoff, "Postprandial glucagon-like peptide-1 (GLP-1) response is positively associated with changes in neuronal activity of brain areas implicated in satiety and food intake regulation in humans," NeuroImage, vol. 35, no. 2, pp. 511-7, 2007.

[52] T. C. LAURENT, U. B. LAURENT, and J. R. E. FRASER, "The structure and function of hyaluronan: An overview," Immunology & Cell Biology, vol. 74, no. 2, pp. a1-a7, 1996.

[53] I. Danishefsky and E. Siskovic, "Conversion of carboxyl groups of mucopolysaccharides into amides of amino acid esters," Carbohydrate Research, vol. 16, no. 1, pp. 199-205, 1971.

[54] P. Bulpitt and D. Aeschlimann, "New strategy for chemical modification of hyaluronic acid: Preparation of functionalized derivatives and their use in the formation of novel biocompatible hydrogels," Journal of Biomedical Materials Research, vol. 47, no. 2, pp. 152-169, 1999.

[55] J. C. Antunes, J. M. Oliveira, R. L. Reis, J. M. Soria, J. L. Gómez-Ribelles, and J. F. Mano, "Novel poly(L-lactic acid)/hyaluronic acid macroporous hybrid scaffolds: Characterization and assessment of cytotoxicity," Journal of Biomedical Materials Research Part A, vol. 94A, no. 3, pp. 856-869, 2010.

[56] J. Liang, L. Cheng, J. J. Struckhoff, and N. Ravi, "Investigating triazine-based modification of hyaluronan using statistical designs," Carbohydrate Polymers, vol. 132, pp. 472-480, 2015.

[57] J. J. Young, K. M. Cheng, T. L. Tsou, H. W. Liu, and H. J. Wang, "Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide,", Journal of Biomaterials Science. Polymer edition, vol. 15, no. 6, pp. 767-80, 2004.

[58] C. E. Schanté, G. Zuber, C. Herlin, and T. F. Vandamme, "Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications," Carbohydrate Polymers, vol. 85, no. 3, pp. 469-489, 2011.

[59] I. Pasquali-Ronchetti, D. Quaglino, G. Mori, B. Bacchelli, and P. Ghosh, "Hyaluronan-phospholipid interactions," Journal of structural biology, vol. 120, no. 1, pp. 1-10, 1997.

[60] M. M. Rieger, Surfactants in cosmetics. New York: Dekker, 1985.

[61] E. Kroner, R. Maboudian, and E. Arzt, "Adhesion characteristics of PDMS surfaces during repeated pull-off force measurements," Advanced Engineering Materials, vol. 12, no. 5, pp. 398-404, 2010.

[62] R. Crowder, "8 - Stepper motors," Electric Drives and Electromechanical Systems (Second Edition), R. Crowder, Ed.: Butterworth-Heinemann, pp. 209-226, 2020.

[63] C. Y. Chung, J. C. Wang, and H. S. Chuang, "Simultaneous and quantitative monitoring of co-cultured Pseudomonas aeruginosa and Staphylococcus aureus with antibiotics on a diffusometric platform," Scientific Reports, vol. 7, p. 46336, 2017.

[64] J. C. Wang, Y. C. Tung, K. Ichiki, H. Sakamoto, T. H. Yang, S. Suye, and H. S. Chuang,"Culture-free detection of methicillin-resistant Staphylococcus aureus by using self-driving diffusometric DNA nanosensors," Biosensors and Bioelectronics, vol. 148, 2020.

[65] G. E. Uhlenbeck and L. S. Ornstein, "On the theory of the Brownian motion," Physical Review, vol. 36, no. 5, pp. 0823-0841, 1930.

[66] Y. J. Fan, H. J. Sheen, C. J. Hsu, C. P. Liu, S. Lin, and K. C. Wu, "A quantitative immunosensing technique based on the measurement of nanobeads' Brownian motion", Biosensors and Bioelectronics, vol. 25, no. 4, pp. 688-94, 2009.

[67] Y.-S. Sie and H.-S. Chuang, "A micro-volume viscosity measurement technique based on μPIV diffusometry," Microfluidics and Nanofluidics, vol. 16, no. 1, pp. 65-72, 2014.

[68] J.-C. Wang, S.-W. Chi, D.-B. Shieh, and H.-S. Chuang, "Development of a self-driving bioassay based on diffusion for simple detection of microorganisms," Sensors and Actuators B: Chemical, vol. 278, pp. 140-146, 2019.

[69] F. Capone, E. Guerriero, A. Sorice, G. Colonna, G. Ciliberto, and S. Costantini, "Serum Cytokinome profile evaluation: a tool to define new diagnostic and prognostic markers of cancer using multiplexed bead-based immunoassays," Mediators of Inflammation, vol. 2016, p. 3064643, 2016.

[70] S. Sable and A. Pandit, "Mapping the cavitation intensity in an ultrasound bath using acoustic emission," AIChE Journal, vol. 46, pp. 684-694, 2000.

[71] C. Berteau, O. Filipe-Santos, T. Wang, H. E. Rojas, C. Granger, and F. Schwarzenbach, "Evaluation of the impact of viscosity, injection volume, and injection flow rate on subcutaneous injection tolerance," Medical Devices, vol. 8, pp. 473-484, 2015.

[72] R. W. Johnstone, S. M. Andrew, M. P. Hogarth, G. A. Pietersz, and I. F. C. McKenzie, "The effect of temperature on the binding kinetics and equilibrium constants of monoclonal antibodies to cell surface antigens," Molecular Immunology, vol. 27, no. 4, pp. 327-333, 1990.

[73] A. S. Hoffman, "Hydrogels for biomedical applications," Advanced Drug Delivery Reviews, vol. 54, no. 1, pp. 3-12, 2002.