Tacrolimus Loaded PEG-Cholecalciferol Based Micelles for Treatment of Ocular Inflammation
- 12 Downloads
Abstract
Purpose
Poor corneal permeability, nasolacrimal drainage and requirement of chronic administration are major drawbacks of existing therapies for ocular inflammation. Hence, we designed topical micelles of PEG2000 conjugated with cholecalciferol (PEGCCF).
Methods
Integrin targeted tacrolimus loaded PEGCCF micelles (TTM) were prepared by solvent diffusion evaporation method and characterized for particle size, osmolality, encapsulation efficiency and drug loading. Therapeutic potential of TTM was evaluated in benzalkonium chloride induced ocular inflammation model in BALB/c mice. Corneal flourescein staining and histopathological analysis of corneal sections was performed.
Results
TTM had a particle size of 45.3 ± 5.3 nm, encapsulation efficiency (88.7 ± 0.9%w/w) and osmolality of 292–296 mOsmol/Kg. TTM significantly reduced the corneal fluorescence as compared to tacrolimus suspension (TACS). H&E staining showed that TTM could restore corneal epithelial thickness, reduce stromal edema (p < 0.05) and decrease number of inflammatory cells (p < 0.01) compared with TACS. Immunohistochemistry analysis demonstrated lower expression of Ki67 + ve cells (p < 0.05) and IL-6 throughout the cornea against TACS (p < 0.01) and the control (p < 0.001).
Conclusions
TTM is an innovative delivery system for improving ocular inflammation due to a) integrin targeting b) PEGCCF in the form of carrier and c) anti-inflammatory and synergistic effect (due to Pgp inhibition) with TAC.
KEY WORDS
micelles ocular inflammation tacrolimus targetedABBREVIATIONS
- APC
Antigen presenting cells
- BKC
Benzalkonium chloride
- CRP
C-reactive protein
- DCC
Dicyclohexylcarbodiimide
- DMAP
4-dimethylaminopyridine
- DSPE-PEG2000
1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]
- HCEC
Human corneal epithelial cells
- IFN- γ
Interferon-γ
- IHC
Immunohistochemistry
- iOP
Intraocular pressure
- LC
Langerhans cells
- mPEG2000
Polyethylene glycol methyl ether (average Mn~2000)
- NFAT
Nuclear factor of activated T cells
- PDI
Poly-dispersity index
- PEG
Polyethylene glycol
- PEGCCF
PEG2000 conjugated with cholecalciferol
- Pgp
P-glycoprotein
- RGD
Arg-Gly-Asp
- SF
Sodium fluorescein
- TAC
Tacrolimus
- TACS
Tacrolimus suspension
- TEA
Triethylamine
- TNF-α
Tumor necrosis factor
- TTM
Targeted tacrolimus loaded micelles
- VD3
Cholecalciferol
References
- 1.Kymionis GD, Bouzoukis DI, Diakonis VF, Siganos C. Treatment of chronic dry eye: focus on cyclosporine. Clin Ophthalmol (Auckland, NZ). 2008;2(4):829.CrossRefGoogle Scholar
- 2.Thakur A, Xue M, Stapleton F, Lloyd A, Wakefield D, Willcox M. Balance of pro-and anti-inflammatory cytokines correlates with outcome of acute experimental Pseudomonas aeruginosa keratitis. Infect Immun. 2002;70(4):2187–97.CrossRefPubMedPubMedCentralGoogle Scholar
- 3.Wakefield D, Lloyd A. The role of cytokines in the pathogenesis of inflammatory eye disease. Cytokine. 1992;4(1):1–5.CrossRefPubMedGoogle Scholar
- 4.De Paiva CS, Corrales RM, Villarreal AL, Farley WJ, Li D-Q, Stern ME, et al. Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye. Exp Eye Res. 2006;83(3):526–35.CrossRefPubMedGoogle Scholar
- 5.Isawi H, Dhaliwal DK. Corneal melting and perforation in Stevens Johnson syndrome following topical bromfenac use. J Cataract Refract Surg. 2007;33(9):1644–1646.Google Scholar
- 6.Dua HS, Attre R. Treatment of post-operative inflammation following cataract surgery: a review. Eur Ophthal Rev. 2012;6(2):98–103.CrossRefGoogle Scholar
- 7.Dinning W. Steroids and the eye--indications and complications. Postgrad Med J. 1976;52(612):634–8.CrossRefPubMedPubMedCentralGoogle Scholar
- 8.Waris A, Nagpal G, Nagpal G, Akhtar N. Tacrolimus in ophthalmology. Indian J Clin Exp Ophthalmol. 2015;1(1):3–6.Google Scholar
- 9.Kheirkhah A, Zavareh M, Farzbod F, Mahbod M, Behrouz M. Topical 0.005% tacrolimus eye drop for refractory vernal keratoconjunctivitis. Eye. 2011;25(7):872.CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Vichyanond P, Kosrirukvongs P. Use of cyclosporine A and tacrolimus in treatment of vernal keratoconjunctivitis. Curr Allergy Asthma Rep. 2013;13(3):308–14.CrossRefPubMedGoogle Scholar
- 11.Park J-H, Joo C-K, Chung SK. Comparative study of tacrolimus and bevacizumab on corneal neovascularization in rabbits. Cornea. 2015;34(4):449–55.CrossRefPubMedGoogle Scholar
- 12.Fukushima A, Ohashi Y, Ebihara N, Uchio E, Okamoto S, Kumagai N, et al. Therapeutic effects of 0.1% tacrolimus eye drops for refractory allergic ocular diseases with proliferative lesion or corneal involvement. Br J Ophthalmol. 2014; https://doi.org/10.1136/bjophthalmol-2013-304453.
- 13.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.CrossRefPubMedPubMedCentralGoogle Scholar
- 14.Dey S, Patel J, Anand BS, Jain-Vakkalagadda B, Kaliki P, Pal D, et al. Molecular evidence and functional expression of P-glycoprotein (MDR1) in human and rabbit cornea and corneal epithelial cell lines. Invest Ophthalmol Vis Sci. 2003;44(7):2909–18.CrossRefPubMedGoogle Scholar
- 15.Chang C, Bahadduri PM, Polli JE, Swaan PW, Ekins S. Rapid identification of P-glycoprotein substrates and inhibitors. Drug Metab Dispos. 2006;34(12):1976–84.CrossRefPubMedGoogle Scholar
- 16.Johnson JA, Grande JP, Roche PC, Campbell RJ, Kumar R. Immuno-localization of the calcitriol receptor, calbinclin-d28k and the plasma membrane calcium pump in the human eye. Curr Eye Res. 1995;14(2):101–8.CrossRefPubMedGoogle Scholar
- 17.Yin Z, Pintea V, Lin Y, Hammock BD, Watsky MA. Vitamin D enhances corneal epithelial barrier function. Invest Ophthalmol Vis Sci. 2011;52(10):7359–7364.Google Scholar
- 18.Suzuki T, Sano Y, Kinoshita S. Effects of 1α, 25-dihydroxyvitamin D3 on Langerhans cell migration and corneal neovascularization in mice. Invest Ophthalmo Vis Sci. 2000;41(1):154–158.Google Scholar
- 19.Dang S, Lu X, Zhou J, Bai L. Effects of 1alpha, 25-dihydroxyvitamin D3 on the acute immune rejection and corneal neovascularization in high-risk penetrating keratoplasty in rats. Di 1 jun yi da xue xue bao= Academic journal of the first medical college of PLA. 2004;24(8):892–6. 903PubMedGoogle Scholar
- 20.Kawabata Y, Wada K, Nakatani M, Yamada S, Onoue S. Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: basic approaches and practical applications. Int J Pharm. 2011;420(1):1–10.CrossRefPubMedGoogle Scholar
- 21.Kasimova AO, Pavan GM, Danani A, Mondon K, Cristiani A, Scapozza L, et al. Validation of a novel molecular dynamics simulation approach for lipophilic drug incorporation into polymer micelles. J Phys Chem B. 2012;116(14):4338–45.CrossRefPubMedGoogle Scholar
- 22.Tomoda K, Terashima H, Suzuki K, Inagi T, Terada H, Makino K. Enhanced transdermal delivery of indomethacin using combination of PLGA nanoparticles and iontophoresis in vivo. Colloids Surf B: Biointerfaces. 2012;92:50–4.CrossRefPubMedGoogle Scholar
- 23.Bucolo C, Drago F, Salomone S. Ocular drug delivery: a clue from nanotechnology. Front Pharmacol. 2012;3Google Scholar
- 24.Weng Y, Liu J, Jin S, Guo W, Liang X, Hu Z. Nanotechnology-based strategies for treatment of ocular disease. Acta Pharm Sin B. 2017;7(3):281–91.CrossRefPubMedGoogle Scholar
- 25.Mun EA, Morrison PWJ, Williams AC, Khutoryanskiy VV. On the Barrier Properties of the Cornea: A Microscopy Study of the Penetration of Fluorescently Labeled Nanoparticles, Polymers, and Sodium Fluorescein. Mol Pharm. 2014;11(10):3556–64.CrossRefPubMedGoogle Scholar
- 26.Lauweryns B, van den Oord JJ, Volpes R, Foets B, Missotten L. Distribution of very late activation integrins in the human cornea. An immunohistochemical study using monoclonal antibodies. Invest Ophthalmol Vis Sci. 1991;32(7):2079–2085.Google Scholar
- 27.Stepp MA. Corneal integrins and their functions. Exp Eye Res. 2006;83(1):3–15.CrossRefPubMedGoogle Scholar
- 28.Chu Y, Chen N, Yu H, Mu H, He B, Hua H, et al. Topical ocular delivery to laser-induced choroidal neovascularization by dual internalizing RGD and TAT peptide-modified nanoparticles. Int J Nanomedicine. 2017;12:1353–68.CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Singh SR, Grossniklaus HE, Kang SJ, Edelhauser HF, Ambati BK, Kompella UB. Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV. Gene Ther. 2009;16(5):645–59.CrossRefPubMedPubMedCentralGoogle Scholar
- 30.Kutlehria S, Behl G, Patel K, Doddapaneni R, Vhora I, Chowdhury N, et al. Cholecalciferol-PEG conjugate based micelles of doxorubicin for treatment of triple-negative breast cancer. AAPS Pharm Sci Tech. 2017;Google Scholar
- 31.Lin Z, Liu X, Zhou T, Wang Y, Bai L, He H, et al. A mouse dry eye model induced by topical administration of benzalkonium chloride; 2011. 257–264 p.Google Scholar
- 32.Taravati P, Lam DL, Leveque T, Van Gelder RN. Postcataract surgical inflammation. Curr Opin Ophthalmol 2012;23(1):12–18.Google Scholar
- 33.Kymionis GD, Bouzoukis DI, Diakonis VF, Siganos C. Treatment of chronic dry eye: focus on cyclosporine. Clin Ophthalmol. 2008;2(4):829–36.CrossRefPubMedPubMedCentralGoogle Scholar
- 34.Dey S, Gunda S, Mitra AK. Pharmacokinetics of Erythromycin in Rabbit Corneas after Single-Dose Infusion: Role of P-Glycoprotein as a Barrier to in Vivo Ocular Drug Absorption. J Pharmacol Exp Ther. 2004;311(1):246–55.CrossRefPubMedGoogle Scholar
- 35.Kawazu K, Yamada K, Nakamura M, Ota A. Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-Glycoprotein Transport Activity and Binding to Cyclophilin. Invest Ophthalmol Vis Sci. 1999;40(8):1738–1744.Google Scholar
- 36.Saha P, Yang JJ, Lee VH. Existence of a p-glycoprotein drug efflux pump in cultured rabbit conjunctival epithelial cells. Invest Ophthalmol Vis Sci. 1998;39(7):1221–1226.Google Scholar
- 37.Xue M-L, Zhu H, Thakur A, Willcox M. 1α, 25-Dihydroxyvitamin D3 inhibits pro-inflammatory cytokine and chemokine expression in human corneal epithelial cells colonized with Pseudomonas aeruginosa. Immunol Cell Biol. 2002;80(4):340–5.CrossRefPubMedGoogle Scholar
- 38.Ley K, Rivera-Nieves J, Sandborn WJ, Shattil S. Integrin-based Therapeutics: Biological Basis, Clinical Use and New Drugs. Nat Rev Drug Discov. 2016;15(3):173–83.CrossRefPubMedPubMedCentralGoogle Scholar
- 39.Kunath K, Merdan T, Hegener O, Häberlein H, Kissel T. Integrin targeting using RGD-PEI conjugates for in vitro gene transfer. J Gene Med. 2003;5(7):588–99.CrossRefPubMedGoogle Scholar
- 40.Barot M, Bagui M, Gokulgandhi MR, Mitra AK. Prodrug Strategies in Ocular Drug Delivery. Medicinal chemistry (Shariqah (United Arab Emirates)). 2012;8(4):753–68.CrossRefGoogle Scholar
- 41.Duvvuri S, Majumdar S, Mitra AK. Role of metabolism in ocular drug delivery. Curr Drug Metab. 2004;5(6):507–15.CrossRefPubMedGoogle Scholar
- 42.Foroutan SM, Watson DG. Synthesis and characterisation of polyethylene glycol conjugates of hydrocortisone as potential prodrugs for ocular steroid delivery. Int J Pharm. 1997;157(1):103–11.CrossRefGoogle Scholar
- 43.Foroutan SM, Watson DG. The in vitro evaluation of polyethylene glycol esters of hydrocortisone 21-succinate as ocular prodrugs. Int J Pharm. 1999;182(1):79–92.CrossRefPubMedGoogle Scholar
- 44.Hao T, Chen D, Liu K, Qi Y, Tian Y, Sun P, et al. Micelles of d-α-Tocopheryl Polyethylene Glycol 2000 Succinate (TPGS 2K) for Doxorubicin Delivery with Reversal of Multidrug Resistance. ACS Appl Mater Interfaces. 2015;7(32):18064–75.CrossRefPubMedGoogle Scholar
- 45.Dintaman JM, Silverman JA. Inhibition of P-glycoprotein by D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS). Pharm Res. 1999;16(10):1550–6.CrossRefPubMedGoogle Scholar
- 46.Epstein SP, Chen D, Asbell PA. Evaluation of biomarkers of inflammation in response to benzalkonium chloride on corneal and conjunctival epithelial cells. J Ocul Pharmacol Ther: Offic J Assoc Ocul Pharmacol Ther. 2009;25(5):415–24.CrossRefGoogle Scholar
- 47.Warcoin E, Clouzeau C, Roubeix C, Raveu AL, Godefroy D, Riancho L, et al. Hyperosmolarity and Benzalkonium Chloride Differently Stimulate Inflammatory Markers in Conjunctiva-Derived Epithelial Cells in vitro. Ophthalmic Res. 2017;58(1):40–8.CrossRefPubMedGoogle Scholar
- 48.Thomson AW, Bonham CA, Zeevi A. Mode of action of tacrolimus (FK506): molecular and cellular mechanisms. Ther Drug Monit. 1995;17(6):584–91.CrossRefPubMedGoogle Scholar
- 49.Lin Z, Liu X, Zhou T, Wang Y, Bai L, He H, et al. A mouse dry eye model induced by topical administration of benzalkonium chloride. Mol Vis. 2011;17:257–64.PubMedPubMedCentralGoogle Scholar
- 50.De Saint Jean M, Brignole F, Bringuier AF, Bauchet A, Feldmann G, Baudouin C. Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells. Invest Ophthalmol Vis Sci. 1999;40(3):619–630.Google Scholar
- 51.Li C, Song Y, Luan S, Wan P, Li N, Tang J, et al. Research on the Stability of a Rabbit Dry Eye Model Induced by Topical Application of the Preservative Benzalkonium Chloride. PLoS One. 2012;7(3):e33688.CrossRefPubMedPubMedCentralGoogle Scholar
- 52.Ebihara N, Ohtomo K, Tokura T, Ushio H, Murakami A. Effect of tacrolimus on chemokine production by corneal myofibroblasts via toll-like receptors, compared with cyclosporine and dexamethasone. Cornea. 2011;30(6):702–8.CrossRefPubMedGoogle Scholar
- 53.Dickie LJ, Church LD, Coulthard LR, Mathews RJ, Emery P, McDermott MF. Vitamin D3 down-regulates intracellular Toll-like receptor 9 expression and Toll-like receptor 9-induced IL-6 production in human monocytes. Rheumatology (Oxford, England). 2010;49(8):1466–71.CrossRefGoogle Scholar
- 54.Carvalho JTG, Schneider M, Cuppari L, Grabulosa CC, Aoike TD, QBM R, et al. Cholecalciferol decreases inflammation and improves vitamin D regulatory enzymes in lymphocytes in the uremic environment: A randomized controlled pilot trial. PLos One. 2017;12(6):e0179540.CrossRefPubMedPubMedCentralGoogle Scholar
- 55.Brumback RA, Leech RW. Neuropathology and basic neuroscience: Springer Science & Business Media; 2012.Google Scholar