PLGA Nanospheres Loaded with Autoclaved Leishmania Major (ALM) and CpG-ODN: Preparation and in vitro Characterization

Document Type: Original Article


1 School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

3 Pharmacological Research Center of Medicinal Plants, Mashhad University of Medical Sciences, Mashhad, Iran


Several antigens, adjuvants and delivery systems have been evaluated for induction of protective immune responses against Leishmaniasis, but most of them have been inefficient. In this study, PLGA nanospheres as antigen delivery system CpG-ODN as an immunoadjuvant for increasing the immune responses against Autoclaved Leishmania major (ALM) were prepared and characterized.
Materials and Methods
PLGA nanospheres prepared by a double-emulsion (W/O/W) technique. The internal aqueous phase contained ALM and CpG-ODN, while the oily phase contained the solution of PLGA in dichloromethan and the external aqueous phase was PVA 7.5% (W/V) solution. Particulate characteristics were studied by scanning electron microscopy and particle size analysis. The encapsulation efficiency was determined by Lowry method for ALM and UV spectroscopy at 260 nm for CpG-ODN. The release profiles of antigen and CpG-ODN from nanospheres evaluated for one week.
Nanospheres were spherical in shape, having smooth surfaces. Mean diameters for blank and ALM + CpG-ODN loaded nanospheres recorded as 302±129 and 300±128 nm respectively. Also, the encapsulation efficiencies of ALM and CpG-ODN were 71.6±8.8 and 49.1±2.4%, respectively. Evaluation of the release profiles of ALM and CpG-ODN from nanospheres showed that 44.8±0.8% of ALM and 29.5±0.2% of CpG-ODN released from nanospheres in one week.
The prepared nanospheres with desirable size, encapsulation efficiency, and slow rate of release, had acceptable features for future in vivo studies.


1. Kedzierski L, Zhu Y, Handman E. Leishmania vaccines: progress and problems. Parasitol   2006; 133:S87-S112.

2. Follador I, Araujo C, Orge G, Cheng LH, de Carvalho LP, Bacellar O, et al. Immune responses to an inactive vaccine against American cutaneous leishmaniasis together with granulocyte-macrophage colony-stimulating factor. Vaccine 2002; 20:1365-1368.

3. Satti IN, Osman HY, Daifalla NS, Younis SA, Khalil EA, Zijlstra EE, et al. Immunogenicity and safety of autoclaved Leishmania major plus BCG vaccine in healthy Sudanese volunteers. Vaccine 2001; 19:2100-2106.

4. Flynn B, Wang V, Sacks DL, Seder RA, Verthelyi D. Prevention and treatment of cutaneous leishmaniasis in primates by using synthetic type D/A oligodeoxynucleotides expressing CpG motifs. Infect Immun 2005; 73:4948-4954.

5. Iborra S, Carrion J, Anderson C, Alonso C, Sacks D, Soto M. Vaccination with the Leishmania infantum acidic ribosomal P0 protein plus CpG oligodeoxynucleotides induces protection against cutaneous leishmaniasis in C57BL/6 mice but does not prevent progressive disease in BALB/c mice. Infect Immun 2005; 73: 5842-5852.

6. Wu W, Weigand L, Belkaid Y, Mendez S. Immunomodulatory effects associated with a live vaccine against Leishmania major containing CpG oligodeoxynucleotides. Eur J Immunol 2006; 36: 3238-3247.

7. Tewary P, Pandya J, Mehta J, Sukumaran B, Madhubala R. Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection. FEMS Immunol Med Microbiol 2004; 42: 241-248.

8. Coelho EA, Tavares CA, Lima Kde M, Silva CL, Rodrigues JM, Fernandes AP. Mycobacterium hsp65 DNA entrapped into TDM-loaded PLGA microspheres induces protection in mice against Leishmania (Leishmania) major infection. Parasitol Res 2006; 98:568-575.

9. ShimizuY,Takagi H,Nakayama T,Yamakami K,Tadakuma T, Yokoyama N, Kojima N. Intraperitoneal immunization with oligomannose-coated liposome-entrapped soluble leishmanial antigen induces antigen-specific T-helper type immune response in BALB/c mice through uptake by peritoneal macrophages. Parasite Immunol 2007; 29:229-239.

10. Bhowmick S, Ravindran R, Ali N. Leishmanial antigens in liposomes promote protective immunity and provide immunotherapy against visceral leishmaniasis via polarized Th1 response. Vaccine 2007; 25:6544-6556.

11. Badiee A, Jaafari MR, Khamesipour A. Leishmania major: immune response in BALB/c mice immunized with stress-inducible protein 1 encapsulated in liposomes. Exp Parasitol 2007; 115:127-134.

12. Jaafari MR, Ghafarian A, Farrokh-Gisour A, Samiei A, Kheiri MT, Mahboudi F, et al. Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice. Vaccine 2006; 24:5708-5717.

13. Diwan M, Tafaghodi M, Samuel J. Enhancement of immune responses by co-delivery of a CpG oligodeoxynucleotide and tetanus toxoid in biodegradable nanospheres. J Controlled Rel 2002; 85:247-262.

14. Johansen P, Men Y, Merkle HP, Gander B. Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination. Eur J Pharm Biopharm 2000; 50:129-146.

15. Newman KD, Elamanchili P, Kwon GS, Samuel J. Uptake of poly (D,L-lactic-co-glycolic acid) microspheres by antigen-presenting cells in vivo. J Biomed Mater Res 2002; 60:480-486.

16. Newman KD, Sosnowski DL, Kwon GS, Samuel J. Delivery of MUC1 mucin peptide by Poly (d,l-lactic-co-glycolic acid) microspheres induces type 1 T helper immune responses. J Pharm Sci 1998; 87:1421-1427.

17. Venkataprasad N, Coombes AGA, Singh M, Rohde M, Wilkinson K, Hudecz F, et al. Induction of cellular immunity to a mycobacterial antigen adsorbed on lamellar particles of lactide polymers. Vaccine 1999; 17:1814-1819.

18. McCluskie MJ, Davis HL. CpG DNA is a potent enhancer of systemic and mucosal immune responses against hepatitis B surface antigen with intranasal administration to mice. J Immunol 1998; 161:4463-4466.

19. Gupta RK, Siber GR. Adjuvants for human vaccines--current status, problems and future prospects. Vaccine 1995; 13:1263-1276.

20. Krieg AM. Mechanisms and applications of immune stimulatory CpG oligodeoxynucleotides. Biochim Biophys Act (BBA) - Gene Structure and Expression 1999; 1489:107-116.

21. Mendez S, Tabbara K, Belkaid Y, Bertholet S, Verthelyi D, Klinman D, et al. Coinjection with CpG-containing immunostimulatory oligodeoxynucleotides reduces the pathogenicity of a live vaccine against cutaneous Leishmaniasis but maintains its potency and durability. Infect Immun 2003; 71:5121-5129.

22. Rhee EG, Mendez S, Shah JA, Wu CY, Kirman JR, Turon TN, et al. Vaccination with heat-killed leishmania antigen or recombinant leishmanial protein and CpG oligodeoxynucleotides induces long-term memory CD4+ and CD8+ T cell responses and protection against Leishmania major infection. J Exp Med 2002; 195:1565-1573.

23. Verthelyi D, Kenney RT, Seder RA, Gam AA, Friedag B, Klinman DM. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J Immunol 2002; 168:1659-1663.

24. Diwan M, Elamanchili P, Cao M, Samuel J. Dose sparing of CpG oligodeoxynucleotide vaccine adjuvants by nanoparticle delivery. Curr Drug Deliv 2004; 1: 405-412.

25. Diwan M, Elamanchili P, Lane H, Gainer A, Samuel J. Biodegradable nanoparticle mediated antigen delivery to human cord blood derived dendritic cells for induction of primary T cell responses. J Drug Target 2003; 11: 495-507.

26. Tafaghodi M, Sajadi Tabassi SA, Jaafari M-R, Zakavi SR, Momen Nejad M. Evaluation of the clearance characteristics of various microspheres in the human nose by gamma-scintigraphy. Int J Pharm 2004; 280:125-135.

27. Diwan M, Park TG. Pegylation enhances protein stability during encapsulation in PLGA microspheres. J Controlled Rel 2001; 73:233-244.

28. Waterborg JH. Quantitation of proteins. In: Walker J (Ed.).The Protein Protocols Handbook. Newjersey: Humana Press; 2002.p. 3-36.

29. Barman SP, Lunsford L, Chambers P, Hedley ML. Two methods for quantifying DNA extracted from poly(lactide-co-glycolide) microspheres. J Controlled Rel 2000; 69:337-344.

30. Sinha VR, Trehan A. Biodegradable microspheres for protein delivery. J Controlled Rel 2003; 90:261-280.

31. Sanchez A, Tobio M, Gonzalez L, Fabra A, Alonso MJ. Biodegradable micro- and nanoparticles as long-term delivery vehicles for interferon-alpha. Eur J Pharm Sci 2003; 18:221-229.

32. Gutierro I, Hernandez RM, Igartua M, Gascon AR, Pedraz JL. Size dependent immune response after subcutaneous, oral and intranasal administration of BSA loaded nanospheres. Vaccine 2002; 21:67-77.

33. Jeffery H, Davis SS, O'Hagan DT. The preparation and characterization of poly (lactide-co-glycolide) microparticles. II. The entrapment of a model protein using a (water-in-oil)-in-water emulsion solvent evaporation technique. Pharm Res  1993; 10:362-368.

34.  Tafaghodi M, Jaafari MR, Sajadi Tabassi sA, Diwan M, Samuel J. Utilization of particulate and immuno-modulating adjuvants for enhancement of immune response against tumors antigenic markers. Iran J B Med Sci 2004; 6:14-23.

35.  Tafaghodi M, Sajadi Tabasi SA, Jaafari MR. Nasal immunization study by PLGA nanospheres encapsulated with tetanus toxoid and CpG-ODN. Iran J Pharm Res 2007; 6:151-158.

36.  Tafaghodi M, Sajadi Tabasi SA, Jaafari MR. Induction of systemic and mucosal immune responses by intranasal administration of alginate microspheres encapsulated with tetanus toxoid and CpG-ODN. Int J Pharm 2006; 319:37-43.

37.  Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol associated with poly (D, L-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J Controlled Rel 2002; 82:105-114.

38.  Zambaux MF, Bonneaux F, Gref R, Maincent P, Dellacherie E, Alonso MJ, Labrude P, Vigneron C. Influence of experimental parameters on the characteristics of poly(lactic acid) nanoparticles prepared by a double emulsion method. J Controlled Rel 1998; 50:31-40.

39. Jeffery H, Davis SS, O'Hagan DT. The preparation and characterisation of poly(lactide-co-glycolide) microparticles. I: Oil-in-water emulsion solvent evaporation. Int J Pharm 1991; 77:169-175.

40. Rebelatto MC, Guimond P, Bowersock TL, HogenEsch H. Induction of systemic and mucosal immune response in cattle by intranasal administration of pig serum albumin in alginate microparticles. Vet Immunol Immunopathol 2001; 83:93-105.

41  .Esparza I, Kissel T. Parameters affecting the immunogenicity of microencapsulated tetanus toxoid. Vaccine 1992; 10:714-720.

42.  Wang YM, Sato H, Horikoshi I. In vitro and in vivo evaluation of taxol release from poly (lactic-co-glycolic acid) microspheres containing isopropyl myristate and degradation of the microspheres. J  Controlled Rel 1997; 49:157-166.

43.  Freiberg S, Zhu XX. Polymer microspheres for controlled drug release. Int J Pharm 2004; 282: 1-18.

44.  Seo S-A, Khang G, Rhee JM, Kim J, Lee HB.  Study on in vitro release patterns of fentanyl-loaded PLGA microspheres. J Microencapsul 2003; 20:569-579.

45.  Ravivarapu  HB, Lee  H, DeLuca  PP. Enhancing initial release of peptide from poly(d,l-lactide-co-glycolide) (PLGA) microspheres by addition of a porosigen and increasing drug load. Pharm Dev Technol 2000; 5:287-296.

46.  Sandor  M, Enscore  D, Weston  P, Mathiowitz  E. Effect of protein molecular weight on release from micron-sized PLGA microspheres. J Controlled Rel 2001; 76:297-311.