Anti-Toxoplasma activity and impact evaluation of lyophilization, hot molding process, and gamma-irradiation techniques on CLH-PLGA intravitreal implants

  • Gabriella M. Fernandes-Cunha
  • Cíntia M. F. Rezende
  • Wagner N. Mussel
  • Gisele R. da Silva
  • Elionai C. de L. Gomes
  • Maria I. Yoshida
  • Sílvia L. Fialho
  • Alfredo M. Goes
  • Dawison A. Gomes
  • Ricardo W. de Almeida Vitor
  • Armando Silva-Cunha
Biomaterials Synthesis and Characterization Original Research
Part of the following topical collections:
  1. Biomaterials Synthesis and Characterization


Intraocular delivery systems have been developed to treat many eye diseases, especially those affecting the posterior segment of the eye. However, ocular toxoplasmosis, the leading cause of infectious posterior uveitis in the world, still lacks an effective treatment. Therefore, our group developed an intravitreal polymeric implant to release clindamycin, a potent anti-Toxoplasma antibiotic. In this work, we used different techniques such as differential scanning calorimetry, thermogravimetry, X-ray diffraction, scanning electron microscopy, and fourier-transform infrared spectroscopy to investigate drug/polymer properties while manufacturing the delivery system. We showed that the lyophilization, hot molding process, and sterilization by gamma irradiation did not change drug/polymer physical-chemistry properties. The drug was found to be homogeneously dispersed into the poly lactic-co-glycolic acid (PLGA) chains and the profile release was characterized by an initial burst followed by prolonged release. The drug profile release was not modified after gamma irradiation and non-covalent interaction was found between the drug and the PLGA. We also observed the preservation of the drug activity by showing the potent anti-Toxoplasma effect of the implant, after 24–72 h in contact with cells infected by the parasite, which highlights this system as an alternative to treat toxoplasmic retinochoroiditis.

Graphical Abstract


Differential Scanning Calorimetry Gamma Irradiation Clindamycin Intravitreal Injection Lyophilization Process 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors would like to acknowledge the financial support received from the following institutions: (Brazil), FAPEMIG (Minas Gerais—Brazil), Pró-reitoria de Pesquisa da Universidade Federal de Minas Gerais (Minas Gerais—Brazil), CAPES (Bolsistas da CAPES-Brasília/Brazil). Also to the Center of Microscopy at the Universidade Federal de Minas Gerais ( for providing the equipment and technical support for experiments involving electron microscopy, the Foundation Ezequiel Dias for providing the equipment for thermal analysis, and to Professor Dr. Ricardo Alves for providing the equipment for FTIR analysis. The authors would especially like to thank to Raquel Maria Souza, Breno Barbosa Moreira, Rosálida Estevam Nazar Lopes, and Guilherme Augusto de Souza Tiago for their technical assistance.


  1. 1.
    Perkins ES. Ocular toxoplasmosis. Br J Ophthalmol. 1973;57(1):1–17.CrossRefGoogle Scholar
  2. 2.
    Rothova A, Suttorp-VanSchulten MS, Frits TW, Kijlstra A. Causes and frequency of blindness in patients with intraocular inflammatory disease. Br J Ophthalmol. 1996;80:332–6.CrossRefGoogle Scholar
  3. 3.
    Gaudana R, Ananthula HK, Parenky A, Mitra AK. Ocular drug delivery. AAPS J. 2010;12(3):348–60.CrossRefGoogle Scholar
  4. 4.
    Patel A, Cholkar K, Agrahari V, Mitra AK. Ocular drug delivery systems: an overview. World J Pharmacol. 2013;2(2):47–64.CrossRefGoogle Scholar
  5. 5.
    Rothova A, Meenken C, Buitenhuis HJ, Brinkman CJ, Baarsma GS, Boen-Tan TN, de Jong PT, Klaassen-Broekema N, Schweitzer CM, Timmerman Z. Therapy for ocular toxoplasmosis. Am J Ophthalmol. 1993;115:517–23.CrossRefGoogle Scholar
  6. 6.
    Schoenwald RD. Ocular drug delivery: pharmacokinetic considerations. Clin Pharmacokinet. 1990;18:255–69.CrossRefGoogle Scholar
  7. 7.
    Martinez CE, Zhang D, Conway MD, Peyman GA. Successful management of ocular toxoplasmosis during pregnancy using combined intraocular clindamycin and dexamethasone with systemic sulfadiazine. Int Ophthalmol. 1998;22:85–8.CrossRefGoogle Scholar
  8. 8.
    Kishore K, Conway MD, Peyman GA. Intravitreal clindamycin and dexamethasone for toxoplasmic retinochoroiditis. Ophthalmic Surg Lasers. 2001;32:183–92.Google Scholar
  9. 9.
    Sobrin L, Kump LI, Fosters CS. Intravitreal clindamycin for toxoplasmosis retinochoroiditis. Retina. 2007;27:952–7.CrossRefGoogle Scholar
  10. 10.
    Soheilian M, Ramezani A, Azimzadeh A, Sadoughi MM, Dehghan MH, Shahghdami R, Yaseri M, Peyman GA. Randomized trial of intravitreal clindamycin and dexamethasone versus pyrimethamine, sulfadiazine, and prednisolone in treatment of ocular toxoplasmosis. Ophthalmology. 2011;118:134–41.CrossRefGoogle Scholar
  11. 11.
    Fernandes-Cunha GM, Saliba JB, Siqueira RC, Jorge R, Silva-Cunha A. Determination of triamcinolone acetonide in silicone oil and aqueous humor of vitrectomized rabbits’eyes: application for a pharmacokinetic study with intravitreal triamcinolone acetonide injections (Kenalog®40). J Pharm Biomed Anal. 2014;89:24–7.CrossRefGoogle Scholar
  12. 12.
    Sampat KM, Garg SJ. Complications of intravitreal injections. Curr Opin Ophthalmol. 2010;21(3):178–83.CrossRefGoogle Scholar
  13. 13.
    Bochot A, Fattal E. Liposomes for intravitreal drug delivery: a state of the art. J Control Release. 2012;161:628–34.CrossRefGoogle Scholar
  14. 14.
    Lee SS, Hughes P, Ross AD, Robinson MR. Biodegradable implants for sustained drug release in the eye. Pharm Res. 2010;27:2043–53.CrossRefGoogle Scholar
  15. 15.
    Siqueira RC, dos Santos WF, Scott IU, Messias A, Rosa MN, Fernandes-Cunha GM, Silva-Cunha A, Jorge R (2014) Neuroprotective effects of intravitreal triamcinolone acetonide and dexamethasone implant in rabbit retinas after pars plana vitrectomy and silicone oil injection. Retina 1–7.Google Scholar
  16. 16.
    Bernards DA, Bhisitkul RB, Wynn P, Steedman MR, Lee OT, Wong F, Thoongsuwan S, Desai TA. Ocular biocompatibility and structural integrity of micro and nanostructured poly(caprolactone) films. J Ocul Pharmacol Ther. 2013;29(2):249–57.CrossRefGoogle Scholar
  17. 17.
    Makadia HK, Siegel SJ. Poly Lactic-co-Glycolic Acid (PLGA) as biodegradable controlled drug delivery carrier. Polymers. 2011;3:1377–97.CrossRefGoogle Scholar
  18. 18.
    Zhou T, Lewis H, Foster RE, Schwendeman SP. Development of a multiple-drug delivery implant for intraocular management of proliferative vitreoretinopathy. J Control Release. 1998;55:281–95.CrossRefGoogle Scholar
  19. 19.
    Garcia A, Tully J, Maynor B, Yerxa B. Precisely engineered biodegradable intraocular implants for the sustained release of dexamethasone. Investig Ophthalmol Vis Sci. 2013;54:1079.Google Scholar
  20. 20.
    Chennamaneni SR, Mamalis C, Archer B, Oakey Z, Ambati BK. Development of a novel bioerodible dexamethasone implant for uveitis and postoperative cataract inflammation. J Control Release. 2013;167(1):53–9.CrossRefGoogle Scholar
  21. 21.
    Fernandes-Cunha GM, Gouvea DM, Fulgencio GO, Rezende CMF, Silva GR, Bretas JM, Fialho SL, Lopes NP, Silva-Cunha A. 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. 2015;102:346–52.CrossRefGoogle Scholar
  22. 22.
    Tamaddon L, Mostafavi A, Riazi-esfahani M, Karkhane R, Aghazadeh S, Rafiee-Tehrani M, Dorkoosh FA, Amoli FA. Development, characterizations and biocompatibility evaluations of intravitreal lipid implants. Jundishapur J Nat Pharm Prod. 2014;9(2):e16414.CrossRefGoogle Scholar
  23. 23.
    Tamaddon L, Mostafavi SA, Karkhane R, Riazi-Esfahani M, Dorkoosh FA, Rafiee-Tehrani M. Design and development of intraocular polymeric implant systems for long-term controlled-release of clindamycin phosphate for toxoplasmic retinochoroiditis. Adv Biomed Res. 2015;4(32):1–22.Google Scholar
  24. 24.
    Chemburkar SR, Bauer J, Deming K, Spiwek H, Patel K, Morris J, Henry R, Spanton S, Dziki W, Porter W, Quick J, Bauer P, Donaubauer J, Narayanan BA, Soldani M, Riley D, McFarland K. Dealing with the impact of ritonavir polymorphs on the late stages of bulk drug process development. Org Process Res Dev. 2000;4(5):413–7.CrossRefGoogle Scholar
  25. 25.
    Franks F. Frezee-drying: from empiricism to predictability. The significance of glass transitions. Dev Biol Stand. 1992;74:9–19.Google Scholar
  26. 26.
    Sintzel MB, Merkli A, Tabatabay C, Gurny R. Influence of irradiation sterilization on polymers used as drug carriers—a review. Drug Dev Ind Pharm. 1997;23(9):857–78.CrossRefGoogle Scholar
  27. 27.
    Bittner B, Mäder K, Kroll C, Borchert HH, Kissel T. Tetracycline-HCl-loaded poly(DL-lactide-co-glycolide) microspheres prepared by a spray drying technique: influence of γ-irradiation on radical formation and polymer degradation. J Control Release. 1999;59(1):23–32.CrossRefGoogle Scholar
  28. 28.
    Fialho SL, Silva-Cunha A. Manufacturing techniques of biodegradable implants intended to intraocular application. Drug Deliv. 2005;12:109–16.CrossRefGoogle Scholar
  29. 29.
    Chapiro A. Chemical modifications in irradiated polymers. Nucl Instrum Methods Phys Res B. 1988;32:111–4.CrossRefGoogle Scholar
  30. 30.
    Food and Drug Administration( 2000) FDA guidance for industry: analytical procedures and methods validation, U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Rockville.Google Scholar
  31. 31.
    Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.CrossRefGoogle Scholar
  32. 32.
    Tanaka T, Omata Y, Saito A, Shimazaki K, Yamauchi K, Takase M, Kawase K, Igarashi K, Suzuki N. Toxoplasma gondii: parasiticidal effects of bovine lactoferricin against parasites. Exp Parasitol. 1995;81:614–7.CrossRefGoogle Scholar
  33. 33.
    Bharate SS, Bharate SB, Bajaj AN. Interactions and incompatibilities of pharmaceutical excipients with active pharmaceutical ingredients: a comprehensive review. J Excip Food Chem. 2010;1(3):3–26.Google Scholar
  34. 34.
    Vukomanović M, Zavašnik-Bergant T, Bračko I, Skapin SD, Ignjatović N, Radmilović V, Uskoković D. Poly(D,L-lactide-co-glycolide)/hydroxyapatite core-shell nanospheres. Part 3: properties of hydroxyapatite nano-rods and investigation of a distribution of the drug within the composite. Colloids Surf B Biointerfaces. 2011;87(2):226–35.CrossRefGoogle Scholar
  35. 35.
    Wegiel LA, Mauer LJ, Edgar KJ, Taylor LS. Crystallization of amorphous solid dispersions of resveratrol during preparation and storage—impact of different polymers. J Pharm Sci. 2013;102(1):171–84.CrossRefGoogle Scholar
  36. 36.
    Bonadies I, Ambrogi V, Ascione L, Carfagna CA. A hyperbranched polyester as antinucleating agent for Artemisinin in electrospun nanofibers. Eur Polymer J. 2014;60:145–52.CrossRefGoogle Scholar
  37. 37.
    Blasi P, Schoubben A, Giovagnoli S, Perioli L, Ricci M, Rossi C. Ketoprofen Poly(lactide-co-glycolide) Physical Interaction. AAPS Pharm Sci Tech. 2007;8(2):E1–8.CrossRefGoogle Scholar
  38. 38.
    Patel P, Patel P. Formulation and evaluation of clindamycin HCL in situgel for vaginal application. Int J Pharm Investig. 2015;5(1):50–6.CrossRefGoogle Scholar
  39. 39.
    Costa HS, Mansur AAP, Pereira MM, Mansur HS. Engineered hybrid scaffolds of poly(vinyl alcohol)/bioactive glass for potential bone engineering applications: synthesis, characterization, cytocompatibility, and degradation. J Nanomater. 2012;. doi: 10.1155/2012/718470.Google Scholar
  40. 40.
    Jenquin MR, McGinity JW. Characterization of acrylic resin matrix films and mechanisms of drug-polymer interactions. Int J Pharm. 1994;101:23–34.CrossRefGoogle Scholar
  41. 41.
    Evaluation of Medicines for Human Use CHMP Assessment report Ozurdex International Nonproprietary Name: dexamethasone Procedure No: EMEA/H/C/001140.Google Scholar
  42. 42.
    Çaliş S, Bozdağ S, Kaş HS, Tuncay M, Hincal AA. Influence of irradiation sterilization on poly(lactide-co-glycolide) microspheres containing anti-inflammatory drugs. II Farmaco. 2002;57(1):55–62.CrossRefGoogle Scholar
  43. 43.
    Wijsman JA, Dekaban GA, Rieder MJ. Differential toxicity of reactive metabolites of clindamycin and sulfonamides in HIV-infected cells: influence of HIV infection on clindamycin toxicity in vitro. J Clin Pharmacol. 2005;45(3):346–51.CrossRefGoogle Scholar
  44. 44.
    Fichera ME, Bhopale MK, Roos DS. In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma Gondii. Antimicrob Agents Chemother. 1995;39(7):1530–7.CrossRefGoogle Scholar
  45. 45.
    Blais J, Tardif C, Chamberland S. Effect of clindamycin on intracellular replication, protein synthesis, and infectivity of Toxoplasma gondii. Antimicrob Agents Chemother. 1993;37(12):2571–7.CrossRefGoogle Scholar
  46. 46.
    Pfefferkorn ER, Nothnagel RF, Borotz SE. Parasiticidal effect of clindamycin on Toxoplasma gondii grow in culture cells and selection of a drug-resistant mutante. Antimicrob Agents Chemother. 1992;36(5):1091–6.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Gabriella M. Fernandes-Cunha
    • 1
  • Cíntia M. F. Rezende
    • 2
  • Wagner N. Mussel
    • 3
  • Gisele R. da Silva
    • 4
  • Elionai C. de L. Gomes
    • 5
  • Maria I. Yoshida
    • 5
  • Sílvia L. Fialho
    • 6
  • Alfredo M. Goes
    • 2
  • Dawison A. Gomes
    • 2
  • Ricardo W. de Almeida Vitor
    • 7
  • Armando Silva-Cunha
    • 1
  1. 1.Faculty of Pharmacy of the Federal University of Minas GeraisBelo HorizonteBrazil
  2. 2.Department of Biochemistry and Immunology DepartmentInstitute of Biological Science of the Federal University of Minas GeraisBelo HorizonteBrazil
  3. 3.Chemistry Department of the Federal University of Minas GeraisBelo HorizonteBrazil
  4. 4.School of PharmacyFederal University of Sao Joao del-ReiDivinópolisBazil
  5. 5.Laboratory of Thermal AnalysisChemistry Department of the Federal University of Minas GeraisBelo HorizonteBrazil
  6. 6.Ezequiel Dias FoundationBelo HorizonteBrazil
  7. 7.Department of ParasitologyInstitute of Biological Science of the Federal University of Minas GeraisBelo HorizonteBrazil

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