PENETRATION OF HYDROPHILIC SULFORHODAMINE B ACROSS THE PORCINE CORNEA EX-VIVO

  • Wanachat Chaiyasan Department of Optometry, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand,
  • Sangly P. Srinivas School of Optometry, Indiana University, Bloomington, IN, United States,
  • Pattravee Niamprem Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand,
  • Waree Tiyaboonchai The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on High-Education, Ministry of Education, Thailand, The Center of Excellence in Medical Biotechnology, Naresuan University, Phitsanulok, Thailand

Abstract

Objective: Sulforhodamine B (SRB) is a hydrophilic tracer whose fluorescence is unaffected by pH unlike that of carboxyfluorescein. Therefore, SRB may serve as a better tracer when there are significant changes in pH. Thus, in this study, the suitability of SRB to assess the barrier properties of the cellular layers of the cornea was examined using a custom-built confocal scanning micro-fluorometer (CSMF).

Methods: The dye solution (0.1% SRB) was prepared in PBS-Ca2+and three experiments were performed ex vivo using freshly isolated porcine eyes. First, we investigated the penetration of SRB across the endothelium by injection of the dye into the anterior chamber. Next, we measured the penetration of SRB across the epithelium after exposing the ocular surface to the dye. Finally, we examined the penetration after exposure to the dye with detergent (Tween 20) and exposure to the dye concomitant with microneedle injuries. The dye concentration profiles across the cornea were measured using CSMF.

Results: SRB penetrated the corneal endothelium readily into the stroma following injection into the anterior chamber in a time-dependent manner. Despite accumulation in the stroma, SRB did not partition into the epithelium. In agreement with these findings, the dye did not cross the epithelium after topical administration. Co-administration with Tween 20 and injury to the epithelium with microneedles, however, led to penetration of the dye into the stroma.

Conclusions: SRB is a hydrophilic dye that can be used as an alternative fluorescent tracer to assess the barrier function of the cellular layers of the cornea.

Keywords: Sulforhodamine B, Cornea, Endothelium, Epithelium, Confocal scanning microfluorometry

Author Biographies

Wanachat Chaiyasan, Department of Optometry, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand,
Department of Optometry
Faculty of Allied Health Sciences
Naresuan University
Sangly P. Srinivas, School of Optometry, Indiana University, Bloomington, IN, United States,
School of Optometry, Indiana University
Pattravee Niamprem, Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand,
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University
Waree Tiyaboonchai, The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on High-Education, Ministry of Education, Thailand, The Center of Excellence in Medical Biotechnology, Naresuan University, Phitsanulok, Thailand
Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University

References

1. Srinivas SP. Dynamic regulation of barrier integrity of the corneal endothelium. Optom Vis Sci 2010;87:239-54.
2. Srinivas SP. Cell signaling in regulation of the barrier integrity of the corneal endothelium. Exp Eye Res 2012;95:8-15.
3. Srinivas SP, Goyal A, Talele DP, Mahadik S, Sudhir RR, Murthy PP, et al. Corneal epithelial permeability to fluorescein in humans by a multi-drop method. PLoS One 2018;13:e0198831.
4. Nagataki S, Brubaker RF, Grotte DA. The diffusion of fluorescein in the stroma of rabbit cornea. Exp Eye Res 1983;36:765-71.
5. Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. Teer measurement techniques for in vitro barrier model systems. J Lab Autom 2015;20:107-26.
6. Araie M, Maurice D. The rate of diffusion of fluorophores through the corneal epithelium and stroma. Exp Eye Res 1987;44:73-87.
7. Gupta C, Chauhan A, Srinivas SP. Penetration of fluorescein across the rabbit cornea from the endothelial surface. Pharm Res 2012;29:3325-34.
8. Robertson TA, Bunel F, Roberts MS. Fluorescein derivatives in intravital fluorescence imaging. Cells 2013;2:591-606.
9. Araie M. Carboxyfluorescein. A dye for evaluating the corneal endothelial barrier function in vivo. Exp Eye Res 1986;42:141-50.
10. Mordon S, Devoisselle JM, Maunoury V. In vivo ph measurement and imaging of tumor tissue using a ph-sensitive fluorescent probe (5,6-carboxyfluorescein): Instrumental and experimental studies. Photochem Photobiol 1994;60:274-9.
11. Amato DV, Lee H, Werner JG, Weitz DA, Patton DL. Functional microcapsules via thiol-ene photopolymerization in droplet-based microfluidics. ACS Appl Mater Interfaces 2017;9:3288-93.
12. Behrendorff N, Shukla A, Schwiening C, Thorn P. Local dynamic changes in confined extracellular environments within organs. Clin Exp Pharmacol Physiol 2009;36:1010-5.
13. Schulz A, Hornig S, Liebert T, Birckner E, Heinze T, Mohr GJ. Evaluation of fluorescent polysaccharide nanoparticles for ph-sensing. Org Biomol Chem 2009;7:1884-9.
14. Chodosh J, Dix RD, Howell RC, Stroop WG, Tseng SC. Staining characteristics and antiviral activity of sulforhodamine b and lissamine green b. Invest Ophth Vis Sci 1994;35:1046-58.
15. Maurice D, Singh T. A permeability test for acute corneal toxicity. Toxicol Lett 1986;31:125-30.
16. Hara H, Cooper DKC. Xenotransplantation–the future of corneal transplantation? Cornea 2011;30:371-8.
17. Elsheikh A, Alhasso D, Rama P. Biomechanical properties of human and porcine corneas. Exp Eye Res 2008;86:783-90.
18. Wang Y, Gabe K, Kompella UB, Barbieri B, Srinivas SP. Confocal scanning microfluorometer for transcorneal fluorescence lifetime measurements. Invest Ophth Vis Sci 2012;53:3093.
19. Srinivas SP, Chaiyasan W, Niamprem P, Keefer K, Tiyaboonchai W, Kompella UB. Penetration of polar sulforhodamine b into the cornea. Invest Ophth Vis Sci 2015;56:4138.
20. Maurice DM, Srinivas SP. Fluorometric measurement of light absorption by the rabbit cornea. Exp Eye Res 1994;58:409-13.
21. Srinivas SP, Maurice DM. A microfluorometer for measuring diffusion of fluorophores across the cornea. IEEE Trans Biomed Eng 1992;39:1283-91.
22. Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. AAPS J 2010;12:348-60.
23. Niamprem P, Srinivas SP, Tiyaboonchai W. Development and characterization of indomethacin-loaded mucoadhesive nanostructured lipid carriers for topical ocular delivery. Int J Appl Pharm 2018;10:91-6.
24. Chaiyasan W, Srinivas SP, Tiyaboonchai W. Development and characterization of topical ophthalmic formulations containing lutein-loaded mucoadhesive nanoparticles. Int J Pharm Pharm Sci 2016;8:261-6.
25. Morsi N, Ghorab D, Refai H, Teba H. Preparation and evaluation of alginate/chitosan nanodispersions for ocular delivery. Int J Pharm Pharm Sci 2015;7:234-40.
26. Chaiyasan W, Praputbut S, Kompella UB, Srinivas SP, Tiyaboonchai W. Penetration of mucoadhesive chitosan-dextran sulfate nanoparticles into the porcine cornea. Colloids Surf B 2017;149:288-96.
27. Marquez-Curtis LA, McGann LE, Elliott JAW. Expansion and cryopreservation of porcine and human corneal endothelial cells. Cryobiology 2017;77:1-13.
28. Sanchez I, Martin R, Ussa F, Fernandez Bueno I. The parameters of the porcine eyeball. Graefes Arch Clin Exp Ophthalmol 2011;249:475-82.
29. Jay L, Brocas A, Singh K, Kieffer JC, Brunette I, Ozaki T. Determination of porcine corneal layers with a high spatial resolution by simultaneous second and third harmonic generation microscopy. Opt Express 2008;16:16284-93.
30. Stiemke MM, McCartney MD, Cantu-Crouch D, Edelhauser HF. Maturation of the corneal endothelial tight junction. Invest Ophth Vis Sci 1991;32:2757-65.
31. Gupta C, Chauhan A, Mutharasan R, Srinivas SP. Measurement and modeling of diffusion kinetics of a lipophilic molecule across rabbit cornea. Pharm Res 2010;27:699-711.
32. Annear MJ, Petersen-Jones SM. Surgery of the ocular surface. In: Auer JA, Stick JA. editors. Chapter 57. Equine surgery (fourth edition). Saint Louis: W. B. Saunders; 2012. p. 770-92.
33. Marsh RJ, Maurice DM. The influence of non-ionic detergents and other surfactants on human corneal permeability. Exp Eye Res 1971;11:43-8.
34. Ujhelyi Z, Fenyvesi F, Varadi J, Feher P, Kiss T, Veszelka S, et al. Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug delivery systems and their effects on paracellular transport in caco-2 cell monolayer. Eur J Pharm Sci 2012;47:564-73.
Statistics
138 Views | 110 Downloads
How to Cite
Chaiyasan, W., Srinivas, S. P., Niamprem, P., & Tiyaboonchai, W. (2018). PENETRATION OF HYDROPHILIC SULFORHODAMINE B ACROSS THE PORCINE CORNEA EX-VIVO. International Journal of Applied Pharmaceutics, 10(6), 94-102. https://doi.org/10.22159/ijap.2018v10i6.28505
Section
Original Article(s)