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Particle Size Influences the Immune Response Produced by Hepatitis B Vaccine Formulated in Inhalable Particles

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ABSTRACT

Purpose

To test the hypothesis that particle size influences the magnitude of immune response produced by hepatitis B surface antigen (HBsAg) encapsulated in poly (lactic-co-glycolic acid) (PLGA) microspheres.

Methods

Microspheres were prepared by a double-emulsion-solvent-evaporation method, and the particles were characterized for size, morphology, porosity and antigen content. Immunogenicity of encapsulated antigen and safety were studied in rats. Uptake of fluorescent-labeled particles by rat alveolar macrophages was studied by confocal microscopy.

Results

With increasing internal aqueous phase (IAP) volume of the microsphere, an increase in particle size and a decrease in particle density were observed. Particles with varying geometric diameters showed aerodynamic diameters between 1 and 6 μ. Addition of poly vinyl alcohol to the IAP resulted in particles with a porous surface. The integrity of HBsAg was maintained upon encapsulation in microspheres. Continuous release of the antigen was observed for formulations incubated in phosphate-buffered saline for 28 days. Immunogenicity increased as a function of particle size upon pulmonary administration. HBsAg encapsulated in ∼5 μm particles elicited a significantly higher immune response compared to that encapsulated in ∼12 μm particles. Similar to in vivo immune response data, fluorescent-labeled microspheres of smaller size were taken up more efficiently by rat alveolar macrophages compared to those of larger size. No significant increase in either tumor necrosis factor alpha level in bronchoalveolar lavage fluid or wet lung weight, indicators of inflammation, was observed in rats that received optimized formulations via the pulmonary route.

Conclusions

HBsAg delivered in PLGA microspheres elicited an increase in immunogenicity, and the magnitude of immune response was more pronounced with smaller particles. Inhalable particles of HBsAg could be a viable approach to needle-free vaccination.

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REFERENCES

  1. Borges O, Cordeiro-da-Silva A, Romeijn SG, Amidi M, de Sousa A, Borchard G, et al. Uptake studies in rat Peyer’s patches, cytotoxicity and release studies of alginate coated chitosan nanoparticles for mucosal vaccination. J Control Release. 2006;114:348–58.

    Article  PubMed  CAS  Google Scholar 

  2. Zhou X, Liu B, Yu X, Zha X, Zhang X, Chen Y, et al. Controlled release of PEI/DNA complexes from mannose-bearing chitosan microspheres as a potent delivery system to enhance immune response to HBV DNA vaccine. J Control Release. 2007;121:200–7.

    Article  PubMed  CAS  Google Scholar 

  3. O’Hagan DT, Rahman D, McGee JP, Jeffery H, Davies MC, Williams P, et al. Biodegradable microparticles as controlled release antigen delivery systems. Immunology. 1991;73:239–42.

    PubMed  Google Scholar 

  4. Singh M, Kazzaz J, Ugozzoli M, Chesko J, O’Hagan DT. Charged polylactide co-glycolide microparticles as antigen delivery systems. Expert Opin Biol Ther. 2004;4:483–91.

    Article  PubMed  CAS  Google Scholar 

  5. Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev. 2005;57:391–410.

    Article  PubMed  CAS  Google Scholar 

  6. Singh M, Li XM, McGee JP, Zamb T, Koff W, Wang CY, et al. Controlled release microparticles as a single dose hepatitis B vaccine: evaluation of immunogenicity in mice. Vaccine. 1997;15:475–81.

    Article  PubMed  CAS  Google Scholar 

  7. O’Hagan DT, McGee JP, Holmgren J, Mowat AM, Donachie AM, Mills KH, et al. Biodegradable microparticles for oral immunization. Vaccine. 1993;11:149–54.

    Article  PubMed  Google Scholar 

  8. Jaganathan KS, Vyas SP. Strong systemic and mucosal immune responses to surface-modified PLGA microspheres containing recombinant hepatitis B antigen administered intranasally. Vaccine. 2006;24:4201–11.

    Article  PubMed  CAS  Google Scholar 

  9. Bivas-Benita M, Ottenhoff TH, Junginger HE, Borchard G. Pulmonary DNA vaccination: concepts, possibilities and perspectives. J Control Release. 2005;107:1–29.

    Article  PubMed  CAS  Google Scholar 

  10. Lu D, Hickey AJ. Pulmonary vaccine delivery. Expert Rev Vaccines. 2007;6:213–26.

    Article  PubMed  CAS  Google Scholar 

  11. Steinman RM. Some interfaces of dendritic cell biology. APMIS. 2003;111:675–97.

    Article  PubMed  CAS  Google Scholar 

  12. Zuckerman JN, Zuckerman AJ. Current topics in hepatitis B. J Infect. 2000;41:130–6.

    Article  PubMed  CAS  Google Scholar 

  13. Thomas C, Gupta V, Ahsan F. Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses. Int J Pharm. 2009;379:41–50.

    Article  PubMed  CAS  Google Scholar 

  14. Thomas C, Rawat A, Bai S, Ahsan F. Feasibility study of inhaled hepatitis B vaccine formulated with tetradecylmaltoside. J Pharm Sci. 2008;97:1213–23.

    Article  PubMed  CAS  Google Scholar 

  15. Rawat A, Majumder QH, Ahsan F. Inhalable large porous microspheres of low molecular weight heparin: in vitro and in vivo evaluation. J Control Release. 2008;128:224–32.

    Article  PubMed  CAS  Google Scholar 

  16. Ungaro F, De Rosa G, Miro A, Quaglia F, La Rotonda MI. Cyclodextrins in the production of large porous particles: development of dry powders for the sustained release of insulin to the lungs. Eur J Pharm Sci. 2006;28:423–32.

    Article  PubMed  CAS  Google Scholar 

  17. Pandit S, Cevher E, Zariwala MG, Somavarapu S, Alpar HO. Enhancement of immune response of HBsAg loaded poly (L-lactic acid) microspheres against hepatitis B through incorporation of alum and chitosan. J Microencapsul. 2007;24:539–52.

    Article  PubMed  CAS  Google Scholar 

  18. Evora C, Soriano I, Rogers RA, Shakesheff KN, Hanes J, Langer R. Relating the phagocytosis of microparticles by alveolar macrophages to surface chemistry: the effect of 1, 2-dipalmitoylphosphatidylcholine. J Control Release. 1998;51:143–52.

    Article  PubMed  CAS  Google Scholar 

  19. Li M, Rouaud O, Poncelet D. Microencapsulation by solvent evaporation: state of the art for process engineering approaches. Int J Pharm. 2008;363:26–39.

    Article  PubMed  CAS  Google Scholar 

  20. Reich G. Ultrasound-induced degradation of PLA and PLGA during microsphere processing: influence of formulation variables. Eur J Pharm Biopharm. 1998;45:165–71.

    Article  PubMed  CAS  Google Scholar 

  21. Sansdrap P, Moes AJ. Influence of manufacturing parameters on the size characteristcis and the release profiles of nifedipine from poly(DL-lactide-co-glycolide) microspheres. Int J Pharm. 1993;98:157–64.

    Article  CAS  Google Scholar 

  22. Scholes PD, Coombes AGA, Illum L, Davis SS, Vert M, Davies MC. The preparation of sub-200 nm poly (lactide-co-glycolide) microspheres for site-specific drug delivery. J Control Release. 1993;25:145–53.

    Article  CAS  Google Scholar 

  23. Yang YY, Chung TS, Ng NP. Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials. 2001;22:231–41.

    Article  PubMed  CAS  Google Scholar 

  24. Crotts G, Park TG. Preparation of porous and nonporous biodegradable polymeric hollow microspheres. J Control Release. 1995;35:91–105.

    Article  CAS  Google Scholar 

  25. Igartua M, Hernandez RM, Esquisabel A, Gascon AR, Calvo MB, Pedraz JL. Stability of BSA encapsulated into PLGA microspheres using PAGE and capillary electrophoresis. Int J Pharm. 1998;169:45–54.

    Article  CAS  Google Scholar 

  26. Mao S, Xu J, Cai C, Germershaus O, Schaper A, Kissel T. Effect of WOW process parameters on morphology and burst release of FITC-dextran loaded PLGA microspheres. Int J Pharm. 2007;334:137–48.

    Article  PubMed  CAS  Google Scholar 

  27. Hussain A, Majumder QH, Ahsan F. Inhaled insulin is better absorbed when administered as a dry powder compared to solution in the presence or absence of alkylglycosides. Pharm Res. 2006;23:138–47.

    Article  PubMed  CAS  Google Scholar 

  28. Xu H, Verbeken E, Vanhooren HM, Nemery B, Hoet PH. Pulmonary toxicity of polyvinyl chloride particles after a single intratracheal instillation in rats. Time course and comparison with silica. Toxicol Appl Pharmacol. 2004;194:111–21.

    Article  PubMed  CAS  Google Scholar 

  29. Toews GB. Cytokines and the lung. Eur Respir J Suppl. 2001;34:3s–17.

    Article  PubMed  CAS  Google Scholar 

  30. Edwards DA, Hanes J, Caponetti G, Hrkach J, Ben-Jebria A, Eskew ML, et al. Large porous particles for pulmonary drug delivery. Science. 1997;276:1868–71.

    Article  PubMed  CAS  Google Scholar 

  31. Amidi M, Pellikaan HC, Hirschberg H, de Boer AH, Crommelin DJ, Hennink WE, et al. Diphtheria toxoid-containing microparticulate powder formulations for pulmonary vaccination: preparation, characterization and evaluation in guinea pigs. Vaccine. 2007;25:6818–29.

    Article  PubMed  CAS  Google Scholar 

  32. Amorij JP, Saluja V, Petersen AH, Hinrichs WL, Huckriede A, Frijlink HW. Pulmonary delivery of an inulin-stabilized influenza subunit vaccine prepared by spray-freeze drying induces systemic, mucosal humoral as well as cell-mediated immune responses in BALB/c mice. Vaccine. 2007;25:8707–17.

    Article  PubMed  CAS  Google Scholar 

  33. O’Hagan DT, Singh M, Ulmer JB. Microparticle-based technologies for vaccines. Methods (San Diego, Calif). 2006;40:10–9.

    Google Scholar 

  34. Vyas SP, Singh RP, Jain S, Mishra V, Mahor S, Singh P, et al. Non-ionic surfactant based vesicles (niosomes) for non-invasive topical genetic immunization against hepatitis B. Int J Pharm. 2005;296:80–6.

    Article  PubMed  CAS  Google Scholar 

  35. Eldridge JH, Staas JK, Meulbroek JA, Tice TR, Gilley RM. Biodegradable and biocompatible poly(DL-lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhances the level of toxin-neutralizing antibodies. Infect Immun. 1991;59:2978–86.

    PubMed  CAS  Google Scholar 

  36. Jaganathan KS, Singh P, Prabakaran D, Mishra V, Vyas SP. Development of a single-dose stabilized poly(D, L-lactic-co-glycolic acid) microspheres-based vaccine against hepatitis B. J Pharm Pharmacol. 2004;56:1243–50.

    Article  PubMed  CAS  Google Scholar 

  37. Saraf S, Mishra D, Asthana A, Jain R, Singh S, Jain NK. Lipid microparticles for mucosal immunization against hepatitis B. Vaccine. 2006;24:45–56.

    Article  PubMed  CAS  Google Scholar 

  38. O’Hagan DT, Jeffery H, Davis SS. Long-term antibody responses in mice following subcutaneous immunization with ovalbumin entrapped in biodegradable microparticles. Vaccine. 1993;11:965–9.

    Article  PubMed  Google Scholar 

  39. Feng L, Qi XR, Zhou XJ, Maitani Y, Wang SC, Jiang Y, et al. Pharmaceutical and immunological evaluation of a single-dose hepatitis B vaccine using PLGA microspheres. J Control Release. 2006;112:35–42.

    Article  PubMed  CAS  Google Scholar 

  40. Katare YK, Muthukumaran T, Panda AK. Influence of particle size, antigen load, dose and additional adjuvant on the immune response from antigen loaded PLA microparticles. Int J Pharm. 2005;301:149–60.

    Article  PubMed  CAS  Google Scholar 

  41. Fifis T, Gamvrellis A, Crimeen-Irwin B, Pietersz GA, Li J, Mottram PL, et al. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol. 2004;173:3148–54.

    PubMed  CAS  Google Scholar 

  42. Brewer JM, Pollock KG, Tetley L, Russell DG. Vesicle size influences the trafficking, processing, and presentation of antigens in lipid vesicles. J Immunol. 2004;173:6143–50.

    PubMed  CAS  Google Scholar 

  43. Kanke M, Sniecinski I, DeLuca PP. Interaction of microspheres with blood constituents: I. Uptake of polystyrene spheres by monocytes and granulocytes and effect on immune responsiveness of lymphocytes. J Parenter Sci Technol. 1983;37:210–7.

    PubMed  CAS  Google Scholar 

  44. Jain AK, Goyal AK, Gupta PN, Khatri K, Mishra N, Mehta A, et al. Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B. J Control Release. 2009;136:161–9.

    Article  PubMed  CAS  Google Scholar 

  45. Lombry C, Marteleur A, Arras M, Lison D, Louahed J, Renauld JC, et al. Local and systemic immune responses to intratracheal instillation of antigen and DNA vaccines in mice. Pharm Res. 2004;21:127–35.

    Article  PubMed  CAS  Google Scholar 

  46. Mishra D, Mishra PK, Dubey V, Nahar M, Dabadghao S, Jain NK. Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes. Eur J Pharm Sci. 2008;33:424–33.

    Article  PubMed  CAS  Google Scholar 

  47. Oh YK, Park JS, Yoon H, Kim CK. Enhanced mucosal and systemic immune responses to a vaginal vaccine coadministered with RANTES-expressing plasmid DNA using in situ-gelling mucoadhesive delivery system. Vaccine. 2003;21:1980–8.

    Article  PubMed  CAS  Google Scholar 

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ACKNOWLEDGMENTS

The authors are grateful to Shantha Biotechnics (Hyderabad, India) for providing HBsAg. Special thanks to Mr. Charles Linch of the Medical Photography and Electron Microscopy Department in Lubbock for his help in scanning electron microscopy studies. We also gratefully acknowledge the assistance of Drs. Bickel, Arumugam and Rawat of Texas Tech University Health Sciences Center in Amarillo for their inputs in conducting the particle uptake and HBsAg integrity studies and formulation development.

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  1. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 S. Coulter St., Amarillo, Texas, 79106, USA

    Chandan Thomas, Vivek Gupta & Fakhrul Ahsan

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  1. Chandan Thomas
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Correspondence to Fakhrul Ahsan.

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Thomas, C., Gupta, V. & Ahsan, F. Particle Size Influences the Immune Response Produced by Hepatitis B Vaccine Formulated in Inhalable Particles. Pharm Res 27, 905–919 (2010). https://doi.org/10.1007/s11095-010-0094-x

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