Skip to main content

Advertisement

Log in

Whole-Eye Perfusion Model for Screening of the Ocular Formulations via Confocal Laser Scanning Microscopy

  • Rapid Communication
  • Published:
AAPS PharmSciTech Aims and scope Submit manuscript

Abstract

Various physiological, anatomical barriers make ocular drug delivery very challenging. Hence, better in vitro screening models are needed for rapid screening of the formulations. In this study, a simple whole-eye perfusion model was designed and its application was explored for screening targeted formulation across the full-thickness cornea using confocal laser scanning microscopy. PEG-cholecalciferol-based integrin targeted coumarin-6 micelles (TC6M) and non-targeted coumarin-6 micelles (NTC6M) were developed by solvent diffusion evaporation technique. The formulations NTC6M and TC6M had particles size 23.5 ± 5 nm and 28.5 ± 6 nm respectively and osmolality of 294–300 mOsml/Kg. The whole-eye perfusion model was developed using porcine eye. TC6M and NTC6M were instilled on the excised porcine eyes as well as in the eyes of NZW rabbits. Corneas were excised from the experimental eyes; coumarin-6 penetration across the corneas was analyzed using confocal microscope. Coumarin-6-loaded micelles had particle size below 50 nm. NTC6M formulations showed penetration to the deeper layers up to 500 μm porcine eyes and up to 50 μm in rabbit corneas. However, TC6M formulations exhibited superior retention, as higher fluorescent intensities were observed in upper layers up to 50 μm depth in the porcine eye and 20 μm depth in rabbit eye. Hence, applicability of whole-eye perfusion model in preliminary screening of the formulations was successfully demonstrated. Whole-eye perfusion model when combined with confocal microscopy has potential to be used as an efficient tool for rapid screening and optimization of various ophthalmic formulations.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  1. Agrahari V, Mandal A, Agrahari V, Trinh HM, Joseph M, Ray A, et al. A comprehensive insight on ocular pharmacokinetics. Drug Deliv Transl Res. 2016;6(6):735–54.

    Article  CAS  Google Scholar 

  2. Patel A, Cholkar K, Agrahari V, Mitra AK. Ocular drug delivery systems: an overview. World J Pharmacol. 2013;2(2):47–64.

    Article  CAS  Google Scholar 

  3. Iaccheri B, Torroni G, Cagini C, Fiore T, Cerquaglia A, Lupidi M, et al. Corneal confocal scanning laser microscopy in patients with dry eye disease treated with topical cyclosporine. Eye. 2017;31(5):788–94.

    Article  CAS  Google Scholar 

  4. Zhang M, Chen J, Luo L, Xiao Q, Sun M, Liu Z. Altered corneal nerves in aqueous tear deficiency viewed by in vivo confocal microscopy. Cornea. 2005;24(7):818–24.

    Article  Google Scholar 

  5. Villani E, Galimberti D, Viola F, Mapelli C, Mojana F, Pirondini C, et al. The cornea in Sjögren’s syndrome: an in vivo confocal study. Invest Ophthalmol Vis Sci. 2006;47(13):1365.

    Google Scholar 

  6. Malhotra M, Majumdar DK. Effect of preservative, antioxidant and viscolizing agents on in vitro transcorneal permeation of ketorolac tromethamine. Indian J Exp Biol. 2002;40(5):555–9.

    CAS  PubMed  Google Scholar 

  7. Ahuja M, Sharma SK, Majumdar DK. In vitro corneal permeation of diclofenac from oil drops. Yakugaku Zasshi. 2007;127(10):1739–45.

    Article  CAS  Google Scholar 

  8. Hämäläinen KM, Kananen K, Auriola S, Kontturi K, Urtti A. Characterization of paracellular and aqueous penetration routes in cornea, conjunctiva, and sclera. Invest Ophthalmol Vis Sci. 1997;38(3):627–34.

    PubMed  Google Scholar 

  9. Olsen TW, Edelhauser HF, Lim JI, Geroski DH. Human scleral permeability. Effects of age, cryotherapy, transscleral diode laser, and surgical thinning. Invest Ophthalmol Vis Sci. 1995;36(9):1893–903.

    CAS  PubMed  Google Scholar 

  10. Liu Q, Wang Y. Development of an ex vivo method for evaluation of precorneal residence of topical ophthalmic formulations. AAPS PharmSciTech. 2009;10(3):796–805.

    Article  CAS  Google Scholar 

  11. Dutescu RM, Panfil C, Merkel OM, Schrage N. Semifluorinated alkanes as a liquid drug carrier system for topical ocular drug delivery. Eur J Pharm Biopharm. 2014;88(1):123–8.

    Article  CAS  Google Scholar 

  12. Thiel MA, Morlet N, Schulz D, Edelhauser H, Dart J, Coster DJ, et al. A simple corneal perfusion chamber for drug penetration and toxicity studies. Br J Ophthalmol. 2001;85(4):450–3.

    Article  CAS  Google Scholar 

  13. Pescina S, Govoni P, Potenza A, Padula C, Santi P, Nicoli S. Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: histological and permeability evaluation. J Pharm Sci. 2015;104(1):63–71.

    Article  CAS  Google Scholar 

  14. Morrison PW, Connon CJ, Khutoryanskiy VV. Cyclodextrin-mediated enhancement of riboflavin solubility and corneal permeability. Mol Pharm. 2013;10(2):756–62.

    Article  CAS  Google Scholar 

  15. Morrison PW, Khutoryanskiy VV. Enhancement in corneal permeability of riboflavin using calcium sequestering compounds. Int J Pharm. 2014;472(1–2):56–64.

    Article  CAS  Google Scholar 

  16. Kompella UB, Sundaram S, Raghava S, Escobar ER. Luteinizing hormone-releasing hormone agonist and transferrin functionalizations enhance nanoparticle delivery in a novel bovine ex vivo eye model. Mol Vis. 2006;12(134–135):1185–98.

    CAS  PubMed  Google Scholar 

  17. Kutlehria S, Behl G, Patel K, Doddapaneni R, Vhora I, Chowdhury N, et al. Cholecalciferol-PEG conjugate based nanomicelles of doxorubicin for treatment of triple-negative breast cancer. AAPS PharmSciTech. 2017.

  18. Kutlehria S, Vhora I, Bagde A, Chowdhury N, Behl G, Patel K, et al. Tacrolimus loaded PEG-cholecalciferol based micelles for treatment of ocular inflammation. Pharm Res. 2018;35(6):117.

    Article  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. Acad J First Med Coll PLA. 2004;24(8):892–6 903.

    CAS  Google Scholar 

  20. Vukovic L, Khatib FA, Drake SP, Madriaga A, Brandenburg KS, Král P, et al. Structure and dynamics of highly PEG-ylated sterically stabilized micelles in aqueous media. J Am Chem Soc. 2011;133(34):13481–8.

    Article  CAS  Google Scholar 

  21. Dave V, Paliwal S. A novel approach to formulation factor of aceclofenac eye drops efficiency evaluation based on physicochemical characteristics of in vitro and in vivo permeation. Saudi Pharm J. 2014;22(3):240–5.

    Article  Google Scholar 

  22. Bonferoni M, Rossi S, Ferrari F, Caramella C. A modified Franz diffusion cell for simultaneous assessment of drug release and washability of mucoadhesive gels. Pharm Dev Technol. 1999;4(1):45–53.

    Article  CAS  Google Scholar 

  23. Richman JB, Tang-Liu DD. A corneal perfusion device for estimating ocular bioavailability in vitro. J Pharm Sci. 1990;79(2):153–7.

    Article  CAS  Google Scholar 

  24. Agarwal P, Rupenthal ID. In vitro and ex vivo corneal penetration and absorption models. Drug Deliv Transl Res. 2016;6(6):634–47.

    Article  CAS  Google Scholar 

  25. Luschmann C, Tessmar J, Schoeberl S, Strau O, Luschmann K, Goepferich A. Self-assembling colloidal system for the ocular administration of cyclosporine a. Cornea. 2014;33(1):77–81.

    Article  Google Scholar 

  26. Wang W, Qian X, Song H, Zhang M, Liu Z. Fluid and structure coupling analysis of the interaction between aqueous humor and iris. Biomed Eng Online. 2016;15(Suppl 2):133.

    Article  Google Scholar 

  27. Van den Berghe C, Guillet M, Compan D. Performance of porcine corneal opacity and permeability assay to predict eye irritation for water-soluble cosmetic ingredients. Toxicol in Vitro. 2005;19(6):823–30.

    Article  Google Scholar 

  28. Farris RL, Stuchell RN, Mandel ID. Basal and reflex human tear analysis: I. Physical measurements: osmolarity, basal volumes, and reflex flow rate. Ophthalmology. 1981;88(8):852–7.

    Article  CAS  Google Scholar 

  29. Camber O, Rehbinder C, Nikkila T, Edman P. Morphology of the pig cornea in normal conditions and after incubation in a perfusion apparatus. Acta Vet Scand. 1987;28(2):127–34.

    CAS  PubMed  Google Scholar 

  30. K-i E, Nakamura T, Kawasaki S, Kinoshita S. Porcine corneal epithelial cells consist of high-and low-integrin β1–expressing populations. Invest Ophthalmol Vis Sci. 2004;45(11):3951–4.

    Article  Google Scholar 

  31. Weng Y-H, Ma X-W, Che J, Li C, Liu J, Chen S-Z, et al. Nanomicelle-assisted targeted ocular delivery with enhanced antiinflammatory efficacy in vivo. Adv Sci. 2017;5(1):1700455.

    Article  Google Scholar 

  32. Carter RT. The role of integrins in corneal wound healing. Vet Ophthalmol. 2009;12:2–9.

    Article  CAS  Google Scholar 

  33. Stepp MA. Corneal integrins and their functions. Exp Eye Res. 2006;83(1):3–15.

    Article  CAS  Google Scholar 

  34. Elner SG, Elner VM. The integrin superfamily and the eye. Invest Ophthalmol Vis Sci. 1996;37(5):696–701.

    CAS  PubMed  Google Scholar 

  35. Sanchez I, Martin R, Ussa F, Fernandez-Bueno I. The parameters of the porcine eyeball. Graefes Arch Clin Exp Ophthalmol. 2011;249(4):475–82.

    Article  Google Scholar 

  36. Latvala T, Päällysaho T, Tervo K, Tervo T. Distribution of α6 and β4 integrins following epithelial abrasion in the rabbit cornea. Acta Ophthalmol Scand. 1996;74(1):21–5.

    Article  CAS  Google Scholar 

  37. Schulz D, Iliev ME, Frueh BE, Goldblum D. In vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low coherence reflectometer. Vis Res. 2003;43(6):723–8.

    Article  Google Scholar 

  38. Whittaker AL, Williams DL. Evaluation of lacrimation characteristics in clinically normal New Zealand white rabbits by using the Schirmer tear test I. J Am Assoc Lab Anim Sci. 2015;54(6):783–7.

    PubMed  PubMed Central  Google Scholar 

  39. Varga M. Textbook of rabbit medicine. Elsevier Health Sciences; 2013.

Download references

Acknowledgments

We thank Dr. Imran Vohra for his technical guidance. We are also thankful to Dr. Dmitry for providing access to the Confocal Laser Scanning Microscope in Maglab (FSU) and Mr. Howell (owner of Jhonston’s meat market Monticello, Fl) for providing porcine eyes.

Funding

This project is supported by NSF-CREST Center for Complex Materials Design for Multidimensional Additive Processing (CoManD) award # 1735968 and Research Center in Minority Institute (RCMI) U54, 2454MD007582-34A1 grant

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Mandip Singh.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kutlehria, S., Bagde, A., Patel, N. et al. Whole-Eye Perfusion Model for Screening of the Ocular Formulations via Confocal Laser Scanning Microscopy. AAPS PharmSciTech 20, 307 (2019). https://doi.org/10.1208/s12249-019-1493-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1208/s12249-019-1493-x

KEY WORDS

Navigation