Chitosan hydrogel containing tacrolimus-loaded nanoliposome for ocular drug delivery: Physicochemical analysis and stability evaluation

Document Type : Research Paper

Authors

1 Department Of Chemical Engineering Shahreza Branch, Islamic Azad University, Shahreza, Iran

2 Department Of Chemical Engineering, Najafabad Branch, Islamic Azad University, Najafabad, Iran

10.22038/nmj.2024.80007.1977

Abstract

Objective(s): Recently, the use of tacrolimus in treating eye diseases has received much attention. Although this drug is powerful in treating eye diseases, however for various reasons, it lacks the necessary efficacy for multiple reasons. This research investigated the development of Tacrolimus encapsulated liposomes, optimization, loading effectiveness, increasing drug efficiency through absorption, controlled release, drug targeting, and reducing drug side effects such as nephropathy.
Materials and Methods: Two agents, liposome and chitosan, have been chosen to transport the drugs used in this study. Nanoliposomes were synthesized through the heating method and chitosan nanoparticles were by reversing the micelle method. A field emission scanning electron microscope(FESEM) was used to prepare images and a zeta sizer was used to measure the average size and distribution of particles. Drug release for 18 days was checked by in vitro and ex-vivo(Franz diffusion) tests. The MTT method was used to evaluate the cytotoxic effect of nanoparticles loaded with tacrolimus drug.
Results: The molar ratio of the drug to liposome and chitosan was chosen to be 0.002. A drug loading effectiveness of (88-95%) was obtained. Tacrolimus drug loading efficiency in liposomes (EPC100, EPC80, DPPC60, DPPC100) value (88.95-95-74%) was obtained for its entrapment in liposome core with passive loading strategy. The difference in drug release rate for EPC 80/chitosan liposome and EPC 100/chitosan was 83.6% and 93.1%, respectively, and for DPPC60/chitosan and DPPC100/chitosan liposomes, 72.8% and 78.8%, respectively.
Conclusion: With this study, it can be concluded that DPPC liposome was good for drug loading. The results of the test (FT-IR) showed that the loading of the drug was successful. The results of electron microscope tests in both samples (EPC, DPPC) indicated the synthesis of drug delivery systems with a spherical morphology with a diameter of less than 100 nanometers. The release results showed that the highest release rate was related to EPC liposomes. In the MTT test, it was observed that nanocarriers without tacrolimus drugs do not show any toxic effect on cells.

Keywords


1. Barbarino JM, Staatz CE, Venkataramanan R, Klein  TE, Altman   RB. PharmGKB summary. cyclosporine and tacrolimus pathways. Pharmacogenet Genomics. 2013;23(10):563-585.
2. Barbarino JM, Staatz CE, Venkataramanan R, Klein TE, Altman RB. PharmGKB summary: cyclosporine and tacrolimus pathways. Pharmacogenet Genomics. 2013;(10):563-585. 
3. Dheer D, Gupta PN, Shankar R. Tacrolimus: An updated review on delivering strategies for multifarious diseases. Eur J Pharm Sci.2018;114:217-227. 
4. Canadas O, Guerrero R, García-Cañero R, Orellana G, Menéndez M, Casals C. Characterization of liposomal tacrolimus in lung surfactant-like phospholipids and evaluation of its immunosuppressive activity. Biochemistry. 2004;(30):9926-9938.
5.van Kooij B, Rothova A, de Vries P. The pros and cons of intravitreal triamcinolone injections for uveitis and inflammatory cystoid macular edema. Ocul Immunol Inflamm. 2006;(2):73-85.
6.Haupert CL, Jaffe GJ. New and emerging treatments for patients with uveitis. International Ophthalmology Clinics. 2000;(2):205-220.
7.Zeng W, Li Q, Wan T, Liu C, Pan W, Wu Z, Zhang G, Pan J, Qin M, Lin Y, Wu C. Hyaluronic acid-coated niosomes facilitate tacrolimus ocular delivery: Mucoadhesion, precorneal retention, aqueous humor pharmacokinetics, and transcorneal permeability. Colloids Surf B Biointerfaces. 2016;(141):28-35. 
8.Bochot A, Fattal E, Boutet V, Deverre JR, Jeanny JC, Chacun H, Couvreur P. Intravitreal delivery of oligonucleotides by sterically stabilized liposomes. Invest Ophthalmol Vis Sci. 2002; (1):253-259. 
9. Dai Y, Zhou R, Liu L, Lu Y, Qi J, Wu W. Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation. Int J Nanomedicine. 2013;)14(:1921-1933.
10. Patel P, Patel H, Panchal S, Mehta T. Formulation strategies for drug delivery of tacrolimus: An overview. Int J Pharm Investig. 2012;(4):169.
11. Nekkanti V, Rueda J, Wang Z, Betageri GV. Design, characterization, and in vivo pharmacokinetics of tacrolimus proliposomes. AAPS PharmSciTech. 2016;)17(:1019-1029. 
12. Shen J, Sun M, Ping Q, Ying Z, Liu W. Incorporation of liquid lipid in lipid nanoparticles for ocular drug delivery enhancement. Nanotechnology. 2009;21(2):025101.
13. Gan L, Han S, Shen J, Zhu J, Zhu C, Zhang X, Gan Y. Self-assembled liquid crystalline nanoparticles as a novel ophthalmic delivery system for dexamethasone: improving preocular retention and ocular bioavailability. Int J Pharm. 2010;396(1-2):179-187. 
14. Hagigit T, Abdulrazik M, Valamanesh F, Behar-Cohen F, Benita S. Ocular antisense oligonucleotide delivery by cationic nanoemulsion for improved treatment of ocular neovascularization: an in-vivo study in rats and mice. J Control Release. 2012;160(2):225-231.
15. Li N, Zhuang C, Wang M, Sun X, Nie S, Pan W. Liposome coated with low molecular weight chitosan and its potential use in ocular drug delivery. Int J Pharm. 2009;379(1):131-138.
16. Hu K, Huang X, Gao Y, Huang X, Xiao H, McClements DJ. Core–shell biopolymer nanoparticle delivery systems: Synthesis and characterization of curcumin fortified zein–pectin nanoparticles. Food chemistry. 2015;182:275-281. 
17. Hironaka K, Inokuchi Y, Tozuka Y, Shimazawa M, Hara H, Takeuchi H. Design and evaluation of a liposomal delivery system targeting the posterior segment of the eye. J Control Release. 2009;136(3):247-253. 
18. Camelo S, Lajavardi L, Bochot A, Goldenberg B, Naud MC, Fattal E, et al. Ocular and systemic bio-distribution of rhodamine-conjugated liposomes loaded with VIP injected into the vitreous of Lewis rats. Mol Vis. 2007;13:2263. 
19. Lee MK. Liposomes for enhanced bioavailability of water-insoluble drugs: In vivo evidence and recent approaches. Pharmaceutics. 2020;12(3):264. 
20. Szebeni J, Wahl SM, Betageri GV, Wahl LM, Gartner S, Popovic M, et al. Inhibition of HIV-1 in monocyte/macrophage cultures by 2′, 3′-dideoxycytidine-5′-triphosphate, free and in liposomes. AIDS Res Hum Retroviruses. 1990;6(5):691-702. 
21. Patel P, Patel H, Panchal S, Mehta T. Formulation strategies for drug delivery of tacrolimus: an overview. Int J Pharm Investig. 2012;2(4):169. 
22. Bisht R, Mandal A, Jaiswal JK, Rupenthal ID. Nanocarrier mediated retinal drug delivery: overcoming ocular barriers to treat posterior eye diseases. WIREs Nanomedicine and Nanobiotechnology. 2018;10(2):1473. 
23. Reza Mozafari M, Johnson C, Hatziantoniou S, Demetzos C. Nanoliposomes and their applications in food nanotechnology. J Liposome Res. 2008;18(4):309-327. 
24. Mozafari MR. Bioactive entrapment and targeting using nanocarrier technologies: an introduction. InNanocarrier technologies: Frontiers of Nanotherapy 2006;1-16. 
25. Babu R, Chen L, Kanikkannan. Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement. Modification of the Stratum Corneum Springer. 2015;133-150.
26. Mozafari MR. Nanoliposomes: preparation and analysis. Liposomes: Methods and protocols Smart Pharm. Nanocarriers. 2010;(1):29-50. 
27. Mozafari MR, Reed CJ, Rostron C. 5-Fluorouracil encapsulated in colloidal lipid particles: entrapment, release and cytotoxicity evaluation in an airway cell line. Aerosol Med Pulm Drug Deliv. 2004;17(1):100-100.
28. Rasti B, Jinap S, Mozafari MR, Abd-Manap MY. Optimization on preparation condition of polyunsaturated fatty acids nanoliposome prepared by Mozafari method. J Liposome Res. 2014;24(2):99-105. 
29. Kaur IP, Smitha R. Penetration enhancers and ocular bioadhesives: two new avenues for ophthalmic drug delivery. Drug Dev Ind Pharm. 2002;28(4):353-369. 
30. Dong Y, Dong P, Huang D, Mei L, Xia Y, Wang Z, Pan X, Li G, Wu C. Fabrication and characterization of silk fibroin-coated liposomes for ocular drug delivery. Pharm Biopharm. 2015;91:82-90. 
31. de Campos AM, Diebold Y, Carvalho EL, Sánchez A, José Alonso M. Chitosan nanoparticles as new ocular drug delivery systems: in vitro stability, in vivo fate, and cellular toxicity. Pharm Res. 2004;21:803-810. 
32. Zhou W, Wang Y, Jian J, Song S. Self-aggregated nanoparticles based on amphiphilic poly (lactic acid)-grafted-chitosan copolymer for ocular delivery of amphotericin B. Int J Nanomedicine. 2013;3715-3728.
33. Wilson B, Samanta MK, Santhi K, Kumar KS, Ramasamy M, Suresh B. Chitosan nanoparticles as a new delivery system for the anti-Alzheimer drug tacrine. Nanomedicine. 2010;6(1):144-152. 
34. Vichare R, Garner I, Paulson RJ, Tzekov R, Sahiner N, Panguluri SK, et al. Biofabrication of chitosan-based nanomedicines and its potential use for translational ophthalmic applications. Appl Sci. 2020;10(12):4189. 
35. Yanat M, Schroën K. Preparation methods and applications of chitosan nanoparticles; with an outlook toward reinforcement of biodegradable packaging. React Funct Polym. 2021;161:104849. 
36. Ohya Y, Shiratani M, Kobayashi H, Ouchi T. Release behavior of 5-fluorouracil from chitosan-gel nanospheres immobilizing 5-fluorouracil coated with polysaccharides and their cell specific cytotoxicity. J. Macromol. Sci. chemist. 1994;31(5):629-642.
37. Jameela SR, Kumary TV, Lal AV, Jayakrishnan A. Progesterone-loaded chitosan microspheres: a long acting biodegradable controlled delivery system. J Control Release. 1998;52(1-2):17-24.
38. Banerjee T, Mitra S, Singh AK, Sharma RK, Maitra A. Preparation, characterization and biodistribution of ultrafine chitosan nanoparticles. Int J Pharm X. 2002;243(1-2):93-105. 
39. Kafshgari MH, Khorram M, Mansouri M, Samimi A, Osfouri S. Preparation of alginate and chitosan nanoparticles using a new reverse micellar system. Iran Polym J. 2012;)21(:99-107.
40. El-Shabouri MH. Positively charged nanoparticles for improving the oral bioavailability of cyclosporin-A. Int J Pharm X. 2002;249(1-2):101-108. 
41. Tokumitsu H, Ichikawa H, Fukumori Y. Chitosan-gadopentetic acid complex nanoparticles for gadolinium neutron-capture therapy of cancer: preparation by novel emulsion-droplet coalescence technique and characterization. Pharm Res. 1999;16:1830-1835.
42. Ichikawa H, Tokumitsu H, Miyamoto M, Fukumori Y. Nanoparticles for neutron capture therapy of cancer. Nanotechnologies for the Life Sciences: Online. 2007. 
43. Calvo P, Remunan-Lopez C, Vila-Jato JL, Alonso MJ. Novel hydrophilic chitosan-polyethylene oxide nanoparticles as protein carriers. J Appl Polym Sci. 1997;63(1):125-132. 
44. Fan W, Yan W, Xu Z, Ni H. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic gelation technique. Colloids Surf B Biointerfaces. 2012;)90(:21-27. 
45. De Campos AM, Sánchez A, Alonso MJ. Chitosan nanoparticles: a new vehicle for the improvement of the delivery of drugs to the ocular surface. Application to cyclosporin A. Int J Pharm X. 2001;224(1-2):159-168. 
46.Kalam MA. Development of chitosan nanoparticles coated with hyaluronic acid for topical ocular delivery of dexamethasone. Int J Biol Macromol. 2016;)89(:127-136.
47. Marshall, T. “Differences between in vitro, in vivo, and in silico studies 2019.
48. Olejnik A, Goscianska J, Nowak I. Active compounds release from semisolid dosage forms. RGUHS J Pharm Sci. 2012;101(11):4032-4045. 
49. Chang RK, Raw A, Lionberger R, Yu L. Generic development of topical dermatologic products: formulation development, process development, and testing of topical dermatologic products. AAPS J. 2013;)15(:41-52.
50. Salamanca CH, Barrera-Ocampo A, Lasso JC, Camacho N, Yarce CJ. Franz diffusion cell approach for pre-formulation characterisation of ketoprofen semi-solid dosage forms. NLM Pharm. 2018;10(3):148.
51. Pharmacopoeia US. US Pharmacopoeia and National Formulary [USP39–NF34], Volume 3, Rockville, Md: United States Pharmacopeial Convention.2016. 
52. Ponnammal P, Kanaujia P, Yani Y, Ng WK, Tan RB. Orally disintegrating tablets containing melt extruded amorphous solid dispersion of tacrolimus for dissolution enhancement. RSC Pharm. 2018;10(1):35. 
53. Obaidat RM, Tashtoush BM, Awad AA, Al Bustami RT. Using supercritical fluid technology (SFT) in preparation of tacrolimus solid dispersions. AAPS PharmSciTech. 2017;18:481-493.
54. Ferraboschi P, Colombo D, De Mieri M, Grisenti P. Evaluation, synthesis and characterization of tacrolimus impurities. J Antibiot (Tokyo). 2012;65(7):349-354. 
55. Pretsch E, Bühlmann P, Affolter C, Pretzsch E, Bühlmann P, Affolter C. Structure determination of organic compounds. Hematol Cell Ther. 2000.
56. Silverstein RM, Bassler GC. Spectrometric identification of organic compounds. J Chem Educ. 1962;39(11):546. 
57. Camargo GD, Ferreira L, Schebelski DJ, Lyra AM, Barboza FM, Carletto B, Koga AY, Semianko BC, Dias DT, Lipinski LC, Novatski A. Characterization and in vitro and in vivo evaluation of tacrolimus-loaded poly (ε-caprolactone) nanocapsules for the management of atopic dermatitis. RSC Pharm. 2021;13(12):2013.
58. Hewitt MG, Morrison PW, Boostrom HM, Morgan SR, Fallon M, Lewis PN, et al. In vitro topical delivery of chlorhexidine to the cornea: Enhancement using drug-loaded contact lenses and β-cyclodextrin complexation, and the importance of simulating tear irrigation. Mol Pharm. 2020;17(4):1428-1441.
59. Amardeep A, Agarwal S. Validated spectrophotometric method for determination of tacrolimus in marketed formulation. IOSR J Pharm. 2012;2:317-321.
60. Skwierczynski R, Curry P, Gray V, Krämer J, Stippler E, Suggett J, et al. Revision of the dissolution procedure: Development and validation< 1092. Dissolution Technologies. 2014; 21(1):6-8.
61. Birnbaum DT, Kosmala JD, Henthorn DB, Brannon-Peppas L. Controlled release of β-estradiol from PLAGA microparticles: The effect of organic phase solvent on encapsulation and release. J Control Release. 2000;65(3):375-387. 
62. Chattaraj SC, Kanfer I. Release of acyclovir from semi-solid dosage forms: A semi-automated procedure using a simple plexiglass flow-through cell. Int J Pharm X. 1995;125(2):215-222.
63. Saudagar RB, Deore PS. Formulation and characterization of fluorometholone nanosuspension for ophthalmic drug delivery by precipitation method. Sch Acad J Pharm. 2016;5(7):268-276.
64. Naik P, Shah SM, Heaney J, Hanson R, Nagarsenker MS. Influence of test parameters on release rate of hydrocortisone from cream: study using vertical diffusion cell. Dissolut Technol. 2016;23(3):14-20. 
65. Gupta PK, Pancholi SS, Das P. In vitro drug release testing method for nepafenac ophthalmic suspension. J Pharm Sci. 2024;113(4):1061-1067. 
66. Lamoureux F, Mestre E, Essig M, Sauvage FL, Marquet P, Gastinel LN. Quantitative proteomic analysis of cyclosporine-induced toxicity in a human kidney cell line and comparison with tacrolimus. J Proteomics. 2011;75(2):677-694.