Documenta Ophthalmologica

, Volume 138, Issue 1, pp 3–19 | Cite as

Toxicity and in vivo release profile of sirolimus from implants into the vitreous of rabbits’ eyes

  • Mayara Rodrigues Brandão De Paiva
  • Nayara Almeida Lage
  • Maria Carolina Andrade Guerra
  • Marcos Paulo Gomes Mol
  • Marcela Coelho Silva Ribeiro
  • Gustavo De Oliveira Fulgêncio
  • Dawidson A. Gomes
  • Isabela Da Costa César
  • Sílvia Ligório FialhoEmail author
  • Armando Silva-Cunha
Original Research Article



To assess the in vivo release profile and the retinal toxicity of a poly (lactic-co-glycolic acid) (PLGA) sustained-release sirolimus (SRL) intravitreal implant in normal rabbit eyes.


PLGA intravitreal implants containing or not SRL were prepared, and the viability of ARPE-19 and hES-RPE human retinal cell lines was examined after 24 and 72 h of exposure to implants. New Zealand rabbits were randomly divided into two groups that received intravitreal implants containing or not SRL. At each time point (1–8 weeks), four animals from the SRL group were euthanized, the vitreous was collected, and drug concentration was calculated. Clinical evaluation of the eyes was performed weekly for 8 weeks after administration. Electroretinography (ERG) was recorded in other eight animals, four for each group, at baseline and at 24 h, 1, 4, 6, and 8 weeks after the injection. ERG was carried out using scotopic and photopic protocols. The safety of the implants was assessed using statistical analysis of the ERG parameters (a and b waves, a and b implicit time, B/A ratio, oscillatory potential, and Naka–Rushton analysis) comparing the functional integrity of the retina between the PLGA and SRL-PLGA groups. After the last electrophysiological assessment, the rabbits were euthanized and retinal histopathology was realized.


After 24 and 72 h of incubation with PLGA or SRL-PLGA implants, ARPE-19 and hES-RPE cells showed viability over 70%. The maximum concentration of SRL (199.8 ng/mL) released from the device occurred within 4 weeks. No toxic effects of the implants or increase in the intraocular pressure was observed through clinical evaluation of the eye. ERG responses showed no significant difference between the eyes that received PLGA or SRL-PLGA implants at baseline and throughout the 8 weeks of follow-up. No remarkable difference in retinal histopathology was detected in rabbit eyes treated with PLGA or SRL-PLGA implants.


Intravitreal PLGA or SRL-PLGA implants caused no significant reduction in cell viability and showed no evident toxic effect on the function or structure of the retina of the animals. SRL was released from PLGA implant after application in the vitreous of rabbits during 8 weeks.


Cell viability Electroretinography Intravitreal drug delivery Sirolimus Biodegradable implant Toxicity 



The authors thank CNPq/MCT (Brazil), Fapemig (Brazil), and CAPES/MEC (Brazil) for the financial support.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Informed consent

Informed consent was not applicable.

Statement of human rights

This article does not contain any studies with human participants performed by any of the authors.

Statement on the welfare of animals

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.


  1. 1.
    Nussenblatt RB (1990) The natural history of uveitis. Int Ophthalmol 14:303–308CrossRefGoogle Scholar
  2. 2.
    Suttorp-Schulten MS, Rothova A (1996) The possible impact of uveitis in blindness: a literature survey. Br J Ophthalmol 80:844–848. CrossRefGoogle Scholar
  3. 3.
    Gritz DC, Wong IG (2004) Incidence and prevalence of uveitis in Northern California: the Northern California epidemiology of uveitis study. Ophthalmology 111:491–500. CrossRefGoogle Scholar
  4. 4.
    London NJS, Rathinam SR, Cunningham ET (2010) The epidemiology of uveitis in developing countries. Int Ophthalmol Clin 50:1–17. CrossRefGoogle Scholar
  5. 5.
    Hwang DK, Chou YJ, Pu CY, Chou P (2012) Epidemiology of uveitis among the Chinese population in Taiwan: a population-based study. Ophthalmology 119:2371–2376. CrossRefGoogle Scholar
  6. 6.
    Tomkins-Netzer O, Talat L, Bar A et al (2014) Long-term clinical outcome and causes of vision loss in patients with uveitis. Ophthalmology 121:2387–2392. CrossRefGoogle Scholar
  7. 7.
    Chu DS, Johnson SJ, Mallya UG et al (2013) Healthcare costs and utilization for privately insured patients treated for non-infectious uveitis in the USA. J Ophthalmic Inflamm Infect 3:64. CrossRefGoogle Scholar
  8. 8.
    Castiblanco C, Foster CS (2014) Review of systemic immunosuppression for autoimmune uveitis. Ophthalmol Ther 3:17–36. CrossRefGoogle Scholar
  9. 9.
    Mérida S, Palacios E, Navea A, Bosch-Morell F (2015) New immunosuppressive therapies in uveitis treatment. Int J Mol Sci 16:18778–18795. CrossRefGoogle Scholar
  10. 10.
    Kanda T, Shibata M, Taguchi M et al (2014) Prevalence and aetiology of ocular hypertension in acute and chronic uveitis. Br J Ophthalmol 98:932–936. CrossRefGoogle Scholar
  11. 11.
    Wong GK, Griffith S, Kojima I, Demain AL (1998) Antifungal activities of rapamycin and its derivatives, prolylrapamycin, 32-desmethylrapamycin, and 32-desmethoxyrapamycin. J Antibiot 51(5):487–491. CrossRefGoogle Scholar
  12. 12.
    Guba M (2002) Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 8:128–135CrossRefGoogle Scholar
  13. 13.
    Dejneka NS, Kuroki AM, Fosnot J, Tang W, Tolentino MJ, Bennett J (2004) Systemic rapamycin inhibits retinal and choroidal neovascularization in mice. Mol Vis 10:964–972.
  14. 14.
    Stahl A, Paschek L, Martin G et al (2008) Rapamycin reduces VEGF expression in retinal pigment epithelium (RPE) and inhibits RPE-induced sprouting angiogenesis in vitro. FEBS Lett 582:3097–3102. CrossRefGoogle Scholar
  15. 15.
    Abdur Rouf M, Vural I, Bilensoy E et al (2011) Rapamycin-cyclodextrin complexation: improved solubility and dissolution rate. J Incl Phenom Macrocycl Chem 70:167–175. CrossRefGoogle Scholar
  16. 16.
    Terada N, Lucas JJ, Szepesi A et al (1993) Rapamycin blocks cell cycle progression of activated T cells prior to events characteristic of the middle to late G1 phase of the cycle. J Cell Physiol 154:7–15. CrossRefGoogle Scholar
  17. 17.
    Napoli KL, Taylor PJ (2001) From beach to bedside: history of the development of sirolimus. Ther Drug Monit 23:559–586. CrossRefGoogle Scholar
  18. 18.
    Cholkar K, Gunda S, Earla R et al (2014) Nanomicellar topical aqueous drop formulation of rapamycin for back-of-the-eye delivery. AAPS PharmSciTech 16:610–622. CrossRefGoogle Scholar
  19. 19.
    Simamora P, Alvarez JM, Yalkowsky SH (2001) Solubilization of rapamycin. Int J Pharm 213:25–29. CrossRefGoogle Scholar
  20. 20.
    MacDonald A, Scarola J, Burke JT, Zimmerman JJ (2000) Clinical pharmacokinetics and therapeutic drug monitoring of sirolimus. Clin Ther 22(Suppl B):B101–B121. CrossRefGoogle Scholar
  21. 21.
    Roberge FG, Xu D, Chan CC et al (1993) Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction. Curr Eye Res 12:197–203. CrossRefGoogle Scholar
  22. 22.
    Shanmuganathan VA (2005) The efficacy of sirolimus in the treatment of patients with refractory uveitis. Br J Ophthalmol 89:666–669. CrossRefGoogle Scholar
  23. 23.
    Ibrahim MA, Sepah YJ, Watters A et al (2015) One-year outcomes of the save study: sirolimus as a therapeutic approach for uveitis. Transl Vis Sci Technol 4:4. CrossRefGoogle Scholar
  24. 24.
    Perry I, Neuberger J (2005) Immunosuppression: towards a logical approach in liver transplantation. Clin Exp Immunol 139:2–10. CrossRefGoogle Scholar
  25. 25.
    Shi W, Gao H, Xie L, Wang S (2006) Sustained intraocular rapamycin delivery effectively prevents high-risk corneal allograft rejection and neovascularization in rabbits. Investig Ophthalmol Vis Sci 47:3339–3344. CrossRefGoogle Scholar
  26. 26.
    Douglas LC, Yi NY, Davis JL et al (2008) Ocular toxicity and distribution of subconjunctival and intravitreal rapamycin in horses. J Vet Pharmacol Ther 31:511–516. CrossRefGoogle Scholar
  27. 27.
    Hou H, Nieto A, Belghith A et al (2015) A sustained intravitreal drug delivery system with remote real time monitoring capability. Acta Biomater 24:309–321. CrossRefGoogle Scholar
  28. 28.
    Mudumba S, Bezwada P, Takanaga H et al (2012) Tolerability and pharmacokinetics of intravitreal sirolimus. J Ocul Pharmacol Ther 28:507–514. CrossRefGoogle Scholar
  29. 29.
    Nguyen QD, Ibrahim MA, Watters A et al (2013) Ocular tolerability and efficacy of intravitreal and subconjunctival injections of sirolimus in patients with non-infectious uveitis: primary 6-month results of the SAVE Study. J Ophthalmic Inflamm Infect 3:32. CrossRefGoogle Scholar
  30. 30.
    Nguyen QD, Merrill PT, Clark WL et al (2016) Intravitreal sirolimus for noninfectious uveitis: a phase III Sirolimus study Assessing double-masKed Uveitis tReAtment (SAKURA). Ophthalmology 123:2413–2423. CrossRefGoogle Scholar
  31. 31.
    Airody A, Heath G, Lightman S, Gale R (2016) Non-infectious uveitis: optimising the therapeutic response. Drugs 76:27–39. CrossRefGoogle Scholar
  32. 32.
    Kuno N, Fujii S (2011) Recent advances in ocular drug delivery systems. Polymers (Basel) 3:193–221. CrossRefGoogle Scholar
  33. 33.
    Kang-Mieler JJ, Osswald CR, Mieler WF (2014) Advances in ocular drug delivery: emphasis on the posterior segment. Expert Opin Drug Deliv 11:1647–1660. CrossRefGoogle Scholar
  34. 34.
    da Silva R, Fialho SL, Siqueira RC et al (2010) Implants as drug delivery devices for the treatment of eye diseases. Braz J Pharm Sci 46:585–595. CrossRefGoogle Scholar
  35. 35.
    Fialho SL, Rêgo MB, Siqueira RC et al (2006) Safety and pharmacokinetics of an intravitreal biodegradable implant of dexamethasone acetate in rabbit eyes. Curr Eye Res 31:525–534. CrossRefGoogle Scholar
  36. 36.
    Thomson JA, Itzkovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147. CrossRefGoogle Scholar
  37. 37.
    Klimanskaya I, Hipp J, Rezai KA et al (2004) Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells 6:217–245. CrossRefGoogle Scholar
  38. 38.
    Dunn KC, Aotaki-Keen AE, Putkey FR, Hjelmeland LM (1996) ARPE-19, a human retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62:155–169CrossRefGoogle Scholar
  39. 39.
    Fialho SL, Siqueira RC, Jorge R, Silva-Cunha A (2007) Biodegradable implants for ocular delivery of anti-inflammatory drug. J Drug Deliv Sci Technol 17:93–97. CrossRefGoogle Scholar
  40. 40.
    McCulloch DL, Marmor MF, Brigell MG et al (2015) ISCEV standard for full-field clinical electroretinography (2015 update). Doc Ophthalmol 130:1–12. CrossRefGoogle Scholar
  41. 41.
    Barar J, Asadi M, Mortazavi-Tabatabaei SA, Omidi Y (2009) Ocular drug delivery; impact of in vitro cell culture models. J Ophthalmic Vis Res 4:238–252Google Scholar
  42. 42.
    ISO 10993-5. (2009) Biological evaluation of medical devices—Part 5: tests for in vitro cytotoxicityGoogle Scholar
  43. 43.
    Forrest ML, Won CY, Malick AW, Kwon GS (2006) In vitro release of the mTOR inhibitor rapamycin from poly(ethylene glycol)-b-poly(ε-caprolactone) micelles. J Control Release 110:370–377. CrossRefGoogle Scholar
  44. 44.
    Onyesom I, Lamprou DA, Sygellou L et al (2013) Sirolimus encapsulated liposomes for cancer therapy: physicochemical and mechanical characterization of sirolimus distribution within liposome bilayers. Mol Pharm 10:4281–4293. CrossRefGoogle Scholar
  45. 45.
    Li G, Cao L, Zhou Z et al (2015) Rapamycin loaded magnetic Fe3O4/carboxymethylchitosan nanoparticles as tumor-targeted drug delivery system: synthesis and in vitro characterization. Colloids Surf B Biointerfaces 128:379–388. CrossRefGoogle Scholar
  46. 46.
    Wang J, Jiang A, Joshi M, Christoforidis J (2013) Drug delivery implants in the treatment of vitreous inflammation. Mediat Inflamm. Google Scholar
  47. 47.
    Souza MCM, Fialho SL, Souza PAF et al (2014) Tacrolimus-loaded PLGA implants: in vivo release and ocular toxicity. Curr Eye Res 39:99–102. CrossRefGoogle Scholar
  48. 48.
    Fernandes-Cunha GM, Gouvea DR, de Oliveira Fulgêncio G et al (2015) Development of a method to quantify clindamycin in vitreous humor of rabbits’ eyes by UPLC-MS/MS: application to a comparative pharmacokinetic study and in vivo ocular biocompatibility evaluation. J Pharm Biomed Anal 102:346–352. CrossRefGoogle Scholar
  49. 49.
    Fialho SL, Souza PAF, de Oliveira Fulgêncio G et al (2013) In vivo release and retinal safety of intravitreal implants of thalidomide in rabbit eyes and antiangiogenic effect on the chorioallantoic membrane. J Drug Target 21:837–845. CrossRefGoogle Scholar
  50. 50.
    Fialho SL, Rego MGB, Cardillo JA et al (2003) Implantes biodegradáveis destinados à administração intra-ocular. Arq Bras Oftalmol 66:891–896. CrossRefGoogle Scholar
  51. 51.
    Haller JA, Bandello F, Belfort R et al (2011) Dexamethasone intravitreal implant in patients with macular edema related to branch or central retinal vein occlusion: twelve-month study results. Ophthalmology 118:2453–2460. CrossRefGoogle Scholar
  52. 52.
    Klamann A, Böttcher K, Ackermann P et al (2017) Intravitreal dexamethasone implant for the treatment of postoperative macular edema. Ophthalmologica 236:181–185. CrossRefGoogle Scholar
  53. 53.
    Kivilcim M, Peyman GA, Kazi AA et al (2007) Intravitreal toxicity of high-dose etanercept. J Ocul Pharmacol Ther 23:57–62. CrossRefGoogle Scholar
  54. 54.
    Peters T, Kim S-W, Castro V et al (2017) Evaluation of polyesteramide (PEA) and polyester (PLGA) microspheres as intravitreal drug delivery systems in albino rats. Biomaterials. Google Scholar
  55. 55.
    Damico FM, Scolari MR, Ioshimoto GL et al (2012) Vitreous pharmacokinetics and electroretinographic findings after intravitreal injection of acyclovir in rabbits. Clinics (Sao Paulo) 67:931–937. CrossRefGoogle Scholar
  56. 56.
    Odom JV, Bach M, Brigell M et al (2016) ISCEV standard for clinical visual evoked potentials: (2016 update). Doc Ophthalmol. Google Scholar
  57. 57.
    Manzano RPDA, Peyman GA, Khan P et al (2009) Testing intravitreal toxicity of rapamycin in rabbit eyes. Arq Bras Oftalmol 72:18–22. CrossRefGoogle Scholar
  58. 58.
    Nieto A, Hou H, Moon SW et al (2015) Surface engineering of porous silicon microparticles for intravitreal sustained delivery of rapamycin. Investig Ophthalmol Vis Sci 56:1070–1080. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Mayara Rodrigues Brandão De Paiva
    • 1
  • Nayara Almeida Lage
    • 1
  • Maria Carolina Andrade Guerra
    • 2
  • Marcos Paulo Gomes Mol
    • 2
  • Marcela Coelho Silva Ribeiro
    • 1
  • Gustavo De Oliveira Fulgêncio
    • 1
  • Dawidson A. Gomes
    • 3
  • Isabela Da Costa César
    • 1
  • Sílvia Ligório Fialho
    • 2
    Email author
  • Armando Silva-Cunha
    • 1
  1. 1.Faculty of PharmacyFederal University of Minas GeraisBelo HorizonteBrazil
  2. 2.Pharmaceutical Research and DevelopmentEzequiel Dias FoundationBelo HorizonteBrazil
  3. 3.Department of Biochemistry and ImmunologyFederal University of Minas GeraisBelo HorizonteBrazil

Personalised recommendations