臺灣博碩士論文加值系統

眼部手術後或細菌感染時，都需要使用抗生素眼藥水來預防及治療。但眼藥水會受到病患依附性，藥物穿透力，以及藥物在局部組織留存的時間，影響到抗菌藥水的有效性。緩釋型藥物之議題日漸高張，而用於局部點藥有其好處，包括藥物留存時間長，藥物釋放時間較久，減少淚液過度攜帶藥物由鼻淚管流失等等。先前本團隊已經發表一篇以緩釋型溫敏感性幾丁聚醣水膠包覆Levofloxacin抗生素治療手術後眼內炎的體外研究，研究發現藥物經水膠包覆後有很好的抗菌以及生物利用性，減少點藥頻次以及提高病患依附性。本篇文章以同樣的緩釋型溫敏感性幾丁聚醣水膠包覆Levofloxacin，用活體外金黃色葡萄球菌性角膜炎模型，進行臨床前藥物有效性以及房水藥物濃度測試。實驗分成對照組(完全無感染無治療)，受感染組(接受金黃色葡萄球菌感染)，以及治療組(金黃色葡萄球菌感染24小時後接受水膠包覆藥物)。本篇結果顯示，感染組有明顯之角膜混濁以及間質霧化，但對照組以及治療組角膜並無間質霧化現象。我們接著將角膜組織進行細菌量化分析，治療組與受感染組比較下，治療組之菌量顯著無顯著上升且與對照組相當，代表治療組有顯著之抑菌效果。角膜組織分析HE染色中，受感染的角膜可見葛蘭氏染色呈現陽性，而對照組與治療組並無陽性反應。同時，在角膜組織發炎標記物(inflammatory marker)表現中，治療組在TNF-α, IL-1α, IL-6, and IL-8比起受感染組有顯著降低。在藥物濃度方面，水膠包覆Levofloxacin點在兔眼角膜24小時後抽取房水進行藥物濃度測試，有達到理想之藥物濃度。綜合結果顯示，溫敏感性幾丁聚醣水膠包覆Levofloxacin在理想藥物濃度之下，未來具有臨床眼部感染抑菌之應用性。

The application of topical ocular drugs for preventing post-operative ocular infections faces several challenges related to patient compliance, drug efficacy, and drug penetration. Developing a new way to deliver drugs to the eye is a fascinating yet difficult task. Existing eye medications have limitations like not staying in the eye for long, difficulty in passing through the outer layer of the eye, frequent blinking, quick tears production, and limited space in the eye for drugs to stay. These factors make it hard for drugs to reach the desired parts of the eye. To overcome these issues and ensure effective drug delivery, researchers are exploring new methods. Nanotechnology and controlled release devices have shown promise in improving drug delivery to the eye. These approaches use tiny carriers like nanoparticles and liposomes to improve drug absorption, release, and minimize side effects. These advancements offer hope for better treatment of eye diseases. In this study, we utilized a previously designed sustained-release system, namely the Levofloxacin-loaded chitosan/gelatin/β-glycerophosphate hydrogel, which exhibited excellent in vitro antibacterial efficacy and biocompatibility. To evaluate its preclinical efficacy and drug levels, we employed an ex-vivo corneal keratitis model infected with S. aureus. The results demonstrated that the ex-vivo corneal keratitis model with S. aureus infection exhibited mild opacity in the central cornea, but without significant stromal infiltration following treatment with the levofloxacin-loaded hydrogel. Quantitative analysis revealed a significant antibacterial activity, with no visible presence of S. aureus according to histological evidence. Additionally, the hydrogel treatment exhibited a notable anti-inflammatory effect. We further examined the drug concentration in the aqueous humor after instilling a single drop of the levofloxacin-loaded hydrogel, and it successfully achieved the desired therapeutic drug level even after 24 hours. These findings strongly suggest that the levofloxacin-loaded hydrogel, as validated by the ex-vivo corneal keratitis model, holds potential for the treatment of post-operative endophthalmitis or keratitis following ophthalmic surgery.

1.Introduction
1.1 Post-operative endophthalmitis………………………………………………… 2
1.2 Post-operative antibiotics ocular delivery …………………3
1.3 Fluoroquinolones………………………………………………………………4
1.4 Topical ocular drug delivery system……………………………6
1.5 Liposomes…………………………………………………………………………7
1.6 Nanoparticles………………………………………………………………………8
1.7 Thermosensitive gelling system…………………………………………8
1.8 Chitosan…………………………………10
1.9 Our previous work………………………………………………………………11
1.10 Aim of the current study………………………………………………14
2. Materials and Methods
2.1 Preparation of the thermosensitive levofloxacin-loaded hydrogel ………………17
2.2 Bacterial culture ……………………………………………………………18
2.3 Ex-vivo rabbit corneal culture……… ………………………………………18
2.4 Ex-vivo rabbit model of S. aureus keratitis ……………………19
2.5 S. aureus bacterial quantification in infected corneal tissues ……………………20
2.6 RNA extraction and quantification of inflammatory gene expression ……..……20
2.7 Histological analysis ………………………………………………………21
2.8 Aqueous humor drug concentrations ……………………………………………21
2.9 Statistical analysis ……………………………………………………..22
3. Results
3.1. External photography of dissected rabbit cornea with scleral rim …….24
3.2. Quantification of viable S. aureus bacteria from ex-vivo cornea model ….25
3.3. Anti-inflammatory effect of drug-loaded hydrogels ………26
3.4. Histological analysis ……………………………………………………………27
3.5. Levofloxacin drug delivery in aqueous humor …………………………29
4. Discussion …………………………………………….……………….…………31
5. Conclusion …………………………………………….………………………….37
6. Perspective…………………………………………….………………………….39
7. References…………………………………………….……………………….….41
Tables
Table 1 Gelation temperature, gelation time and osmolality of levofloxacin-containing hydrogel. 8

Figures
Figure 1 The Action of Levofloxacin 4
Figure 2 Chitosan-based thermo-reversible complex 8
Figure 3 In vitro release profile of levofloxacin from developed hydrogels 9
Figure 4 Images of antibacterial activities and inhibition zones of levofloxacin-containing hydrogel against (a) Staphylococcus aureus and (b) Staphylococcus epidermidis 9
Figure 5 In vitro biocompatibility of the levofloxacin-containing hydrogel. 10
Figure 6 Representative phase microscopic images show wound closure at 0, 4, 8, 24, 48 and 72 h.. 10
Figure 7 Flow chart of the current design 11
Figure 8 External photographic images of excised rabbit cornea. 17
Figure 9 The anti-bacterial activity of thermosensitive levofloxacin-loaded hydrogel. 18
Figure 10 Inflammatory marker expression was reduced with levofloxacin-loaded hydrogel treatment. 20
Figure 11 Histological analyses…………………………………………………21
Figure 12 Images of representative Gram stain sections of corneas in the infected group. 22
Figure 13 Representative chromatograms show baseline and 24-h levels of levofloxacin in aqueous humor.. 22

[1]. Behndig A, Montan P, Stenevi U, et al. One million cataract surgeries: Swedish National Cataract Register 1992e2009. J Cataract Refract Surg 2011;37:1539–45.
[2]. Gollogly HE, Hodge DO, St Sauver JL, Erie JC. Increasing incidence of cataract surgery: population-based study. J Cataract Refract Surg 2013;39:1383–9.
[3]. Keay L, Gower EW, Cassard SD, et al. Postcataract surgery endophthalmitis in the United States: analysis of the complete 2003 to 2004 Medicare database of cataract surgeries, Ophthalmology 2012; 119: 914-22.
[4]. Inoue T, Uno T, Usui N et al. Incidence of endophthalmitis and the perioperative practices of cataract surgery in Japan: Japanese Prospective Multicenter Study for Postoperative Endophthalmitis after Cataract Surgery, Japanese journal of ophthalmology 2018; 62: 24-30.
[5]. Daien V, Pape AL, Heve D, Carriere I, Villain M. Incidence and characteristics of cataract surgery in France from 2009 to 2012: a national population study. Ophthalmology 2015;122:1633-8.
[6]. Herrinton LJ, Shorstein NH, Paschal JF, et al. Comparative effectiveness of antibiotic prophylaxis in cataract surgery. Ophthalmology 2016;123:287-94.
[7]. Lalwani GA, Flynn HW, Scott IU, et al. Acute-onset endophthalmitis after clear corneal cataract surgery (1996–2005): clinical features, causative organisms, and visual acuity outcomes. Ophthalmology 2008;115:473-6.
[8]. Miller JJ, Scott IU, Flynn HW et al. Acute-onset endophthalmitis after cataract surgery (2000–2004): incidence, clinical settings, and visual acuity outcomes after treatment. American journal of ophthalmology 2005; 139: 983-7.
[9]. Rudnisky CJ, Wan D, Weis E. Antibiotic choice for the prophylaxis of post-cataract extraction endophthalmitis. Ophthalmology 2014; 121: 835-41.
[10]. Taban M, Behrens A, Newcomb RL et al. Acute endophthalmitis following cataract surgery: a systematic review of the literature. Archives of ophthalmology 2005; 123: 613-20.
[11]. Gower EW, Keay LJ, Stare DE, et al. Characteristics of endophthalmitis after cataract surgery in the United States Medicare population. 2015 Ophthalmology 122: 1625-32.
[12]. Friling E, Lundström M, Stenevi U, Montan P. Six-year incidence of endophthalmitis after cataract surgery: Swedish national study, Journal of Cataract & Refractive Surgery 2013; 39:15-21.
[13]. Shalaby A, Simplicio CSD, Lockwood A, Newsom R. Postoperative Endophthalmitis: Incidence, Causes and Comparison Between Medical and Surgical Treatment in a United Kingdom Region in the Last 10 Years. Acta Ophthalmologica 2015; 93:S255
[14]. Ng JQ, Morlet N, Pearman JW, et al. Management and outcomes of postoperative endophthalmitis since the endophthalmitis vitrectomy study: the Endophthalmitis Population Study of Western Australia (EPSWA)’s fifth report. Ophthalmology 2005; 112: 1199-206. e2.
[15]. Montan PG, Wejde G, Koranyi G, Rylander M. Prophylactic intracameral cefuroxime: efficacy in preventing endophthalmitis after cataract surgery, Journal of Cataract & Refractive Surgery 2002; 28: 977-81.
[16]. Ciulla TA, Starr MB, Masket S. Bacterial endophthalmitis prophylaxis for cataract surgery: an evidence-based update. Ophthalmology 2002;109:13-24.
[17]. Speaker MG, Menikoff JA. Prophylaxis of endophthalmitis with topical povidone-iodine. Ophthalmology 1991;98:1769-75.
[18]. Colleaux KM, Hamilton WK. Effect of prophylactic antibiotics and incision type on the incidence of endophthalmitis after cataract surgery. Can J Ophthalmol. 2000;35:373-8.
[19]. Smith A, Pennefather PM, Kaye SB, Hart CA. Fluoroquinolones: place in ocular therapy. Drugs 2001; 61:747–761.
[20]. Barry P. ESCRS Endophthalmitis Study Group. ESCRS study of prophylaxis of postoperative endophthalmitis after cataract surgery: Preliminary report of principal results from a European multicenter study, J Cataract Refract Surg 2006; 32:407-10.
[21]. Daien V, Papinaud L, Gillies MC, et al. Effectiveness and safety of an intracameral injection of cefuroxime for the prevention of endophthalmitis after cataract surgery with or without perioperative capsular rupture, JAMA ophthalmology 2016; 134: 810-6.
[22]. Gower, EW Lindsley K, Nanji AA, Leyngold I, McDonnell PJ. Perioperative antibiotics for prevention of acute endophthalmitis after cataract surgery, Cochrane Database Syst Rev 2013; 7.
[23]. Fintelmann RE, Naseri A. Prophylaxis of postoperative endophthalmitis following cataract surgery. Drugs 2010; 70;1395-409.
[24]. An JA, Kasner O, Samek, DA, Lévesque V. Evaluation of eyedrop administration by inexperienced patients after cataract surgery, Journal of Cataract & Refractive Surgery 2014; 40: 1857-61.
[25]. Hermann MM, Üstündag C, Diestelhorst M. Electronic compliance monitoring of topical treatment after ophthalmic surgery, International ophthalmology 2010; 30:385-90.
[26]. Blondeau JM. A review of clinical trials with fluoroquinolones with an emphasis on new agents. Expert Opin Investig Drugs 2000; 9: 383–413.
[27]. Blondeau JM. Fluoroquinolones: mechanism of action, classification, and development of resistance. Surv Ophthalmol 2004; 49(Suppl 2): S73–S78.
[28]. Bezwada P, Clark LA, Schneider S. Intrinsic cytotoxic effects of fluoroquinolones on human corneal keratocytes and endothelial cells. Curr Med Res Opin. 2008;24:419–24.
[29]. Watanabe R et al. Fluoroquinolone antibacterial eye drops: effects on normal human corneal epithelium, stroma, and endothelium. Clinical Ophthalmology 2010; 4: 1181–7.
[30]. Kupferman A, Leibowitz HM. Topically applied steroids in corneal disease. V. Dexamethasone alcohol. Arch Ophthalmol 1974;92:329–330
[31]. Leibowitz HM, Kupferman A. Kinetics of topically administered prednisolone acetate. Arch Ophthalmo 1976 ; 94: 1387–1389
[32]. Nair AB, Shah J, Al-Dhubiab BE, Jacob S, Patel SS, Venugopala KN, Morsy MA, Gupta S, Attimarad M, Sreeharsha N, Shinu P. Clarithromycin solid lipid nanoparticles for topical ocular therapy: optimization, evaluation and in vivo studies. Pharmaceutics 2021;13:523
[33]. Agrahari V, Mandal A, Agrahari V, Trinh HM, Joseph M, Ray A, Hadji H, Mitra R, Pal D, Mitra AK. Comprehensive insight on ocular pharmacokinetics. Drug Deliv Transl Res 2016;6:735–754
[34]. Patel A, Cholkar K, Agrahari V, et al. Ocular drug delivery systems: an overview. World J Pharmacol 2013; 2(2): 47–64.
[35]. Kaur IP, Garg A, Singla AK, et al. Vesicular systems in ocular drug delivery: an overview. Int J Pharm 2004; 269: 1–14.
[36]. Li N, Zhuang C, Wang M, et al. Liposome coated with low molecular weight chitosan and its potential use in ocular drug delivery. Int J Pharm 2009; 379: 131–138.
[37]. Qamar Z, Qizilbash FF, Iqubal MK, Ali A, Narang JK, Ali J, Baboota S. Nano- Based drug delivery system: recent strategies for the treatment of ocular disease and future perspective. Recent Pat Drug Deliv Formul 2019;13:246–254
[38]. Ako-Adounvo AM, Nagarwal RC, Oliveira L, Boddu SH, Wang XS, Dey S, Karla PK. Recent patents on ophthalmic nanoformulations and therapeutic implications. Recent Pat Drug Deliv Formul 2014;8:193–201
[39]. Gan L, Wang J, Jiang M, Bartlett H, Ouyang D, Eperjesi F, Liu J, Gan Y. Recent advances in topical ophthalmic drug delivery with lipid-based nano- carriers. Drug Discov Today 2013;18:290–297
[40]. Cheng YH, Yang SH, Su WY et al. Thermosensitive chitosan–gelatin–glycerol phosphate hydrogels as a cell carrier for nucleus pulposus regeneration: An in vitro study. Tissue Eng A 2009; 16:695–703.
[41]. Ganta S, Devalapally H, Shahiwala A, Amiji M. A review of stimuli-responsive nanocarriers for drug and gene delivery. J. Control. Release 2008; 12:187–204.
[42]. Hamidi M, Azadi A, Rafiei P. Hydrogel nanoparticles in drug delivery. Adv. Drug Deliv. Rev. 2008; 60: 1638–49.
[43]. Wichterle O, Lim D. Hydrophilic gels for biological use. 1960; Nature 185:117–8.
[44]. Qiu Y, Park K. Environment-sensitive hydrogels for drug delivery. Adv. Drug Deliv. Rev. 2001; 53: 321–39.
[45]. Cheng YH, Hung KH, Tsai TH et al. Sustained delivery of latanoprost by thermosensitive chitosan-gelatin-based hydrogel for controlling ocular hypertension.Acta Biometer. 2014; 10: 3460-6.
[46]. Cheng YH, Ko YC, Chang YF, Huang SH, and Liu CJ. Thermosensitive chitosan-gelatin-based hydrogel containing curcumin-loaded nanoparticles and latanoprost as a dual-drug delivery system for glaucoma treatment. Exp Eye Res. 2019; 179:179-87.
[47]. Muzzarelli RAA. Human enzymatic activities related to the therapeutic administration of chitin derivatives. Cell. Mol. Life Sci. 1997; 53: 131–40.
[48]. Salamat-MillerN, Chittchang M, Johnston TP. The use of mucoadhesive polymers in buccal drug delivery. Adv. Drug Deliv. Rev. 2005; 57 : 1666–91.
[49]. Khan F, Tare RS, Oreffo RO, Bradley M. Versatile biocompatible polymer hydrogels: scaffolds for cell growth. Angew. Chem. Int. Ed. Engl. 2009; 48:978–82.
[50]. Ahmadi R, Bruijn JD. Biocompatibility and gelation of chitosan-glycerol phosphate hydrogels. Journal of biomedical materials research. Part A 2008 86:824-32.
[51]. Tan JH, Ng E, Acharya UR. Evaluation of topographical variation in ocular surface temperature by functional infrared thermography. Infrared Phys Technol 2011;54:469–77.
[52]. McKenzie M, Betts D, Suh A, et al. Hydrogel-based drug delivery systems for poorly water-soluble drugs. Molecules 2015 ; 20: 20397–408.
[53]. Bhattarai N, Gunn J, Zhang M. Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliv Rev 2010;62: 83–99.
[54]. Cheng YH, Chang YF, Ko YC, Liu CJL. Sustained release of levofloxacin from thermosensitive chitosan-based hydrogel for the treatment of postoperative endophthalmitis, J. Biomed. Mater. Res. B Appl. Biomater. 2019; 108:1; 8–13.
[55]. Tomlinson A, Khanal S, Ramaesh K, Diaper C, McFadyen A. Tear film osmolarity: determination of a referent for dry eye diagnosis, Invest. Ophthalmol. Vis. Sci. 42006; 7:10: 4309–4315.
[56]. Pinnock A, Shivshetty N, Roy, S et al., Ex-vivo rabbit and human corneas as models for bacterial and fungal keratitis, Graefes Arch. Clin. Exp. Ophthalmol. 2017: 255; 2; 333–342.
[57]. Ameeduzzafar, S.S. Imam, S.N.A. Bukhari, J. Ahmad, A. Ali, Formulation and optimization of levofloxacin loaded chitosan nanoparticle for ocular delivery: in- vitro characterization, ocular tolerance and antibacterial activity, Int. Biol. Macromol. 2018; 108: 650–659.
[58]. Chandra J, Pearlman E, Ghannoum M. Animal models to investigate fungal biofilm formation, Methods Mol. Biol. 2014; 1147: 141–157.
[59]. Zaidi T, Yoong P, Pier GB. Staphylococcus aureus corneal infections: effect of the Panton-valentine leukocidin (PVL) and antibody to PVL on virulence and pathology, Invest. Ophthalmol. Vis. Sci. 2013; 54 (7): 4430–4438.
[60]. Alarcon I, Evans DJ, Fleiszig SM. The role of twitching motility in Pseudomonas aeruginosa exit from and translocation of corneal epithelial cells, Invest. Ophthalmol. Vis. Sci. 2009; 50 (5): 2237–2244.
[61]. Zhou Q, Chen H, Qu M, Wang Q, Yang L, Xie L. Development of a novel ex-vivo model of corneal fungal adherence, Graefes Arch. Clin. Exp. Ophthalmol. 2011; 249 (5): 693–700.
[62]. Hua X, Yuan X, Pietro AD, Wilhelmus KR. The molecular pathogenicity of Fusarium keratitis: a fungal transcriptional regulator promotes hyphal penetration of the cornea, Cornea 2010; 29 (12): 1440–1444.
[63]. Castro-Combs J, Noguera G, Cano M, et al. Corneal wound healing is modulated by topical application of amniotic fluid in an ex-vivo organ culture model, Exp. Eye Res. 2008; 87 (1): 56–63.
[64]. Yamada M, Mochizuki H, Kawai M, Mashima Y. Aqueous humor levels of topically applied levofloxacin in human eyes. Curr. Eye Res. 2002; 24 (5): 403–406.
[65]. Yamada M, Mochizuki H, Yamada K, Kawai M, Mashima Y. Aqueous humor levels of topically applied levofloxacin, norfloxacin, and lomefloxacin in the same human eyes, J. Cataract Refract. Surg. 2003; 29 (9): 1771–1775.
[66]. Bucci Jr. FA, Nguimfack IT, Fluet AT. Pharmacokinetics and aqueous humor penetration of levofloxacin 1.5% and moxifloxacin 0.5% in patients undergoing cataract surgery. Clin. Ophthalmol. 2016; 10: 783–789.