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In Vitro Characterization of Chitosan-Based Particles as Carrier of Influenza Viral Antigens

Year 2018, Volume: 77 Issue: 1, 1 - 10, 22.06.2018

Abstract

DOI: 10.26650/EurJBiol.2018.0004


Objective: Chitosan is a natural polysaccharide derived from chitin by deacetylation. It is a non-toxic, biocompatible and biodegradable polymer, and has attracted considerable interest in a wide range of pharmaceutical applications including drug and vaccine delivery. The immune-stimulating activity of chitosan microparticles such as the activation of macrophages and polymorphonuclear leukocytes has been reported. In this work, we have investigated the releasing properties of influenza virus antigens from the chitosan microparticles and beads.

Materials and Methods: Chitosan microparticles and beads were prepared by coacervation and ionotropic gelation method, respectively. The microparticles and beads were loaded with the viral antigens by passive adsorption and/or entrapment into the microparticles. The titration of the viruses was defined by haemagglutination assay or by quantitation of viral proteins using the Bradford method.

Results: The results showed that the loading efficiency and the loading capacity of chitosan microparticles/beads with the viral antigens and the releasing profiles of the antigens from the particles changed depending on the type of chitosan, the pH of the loading buffer and the methods used to prepare the particles. The influenza viral antigens, passively adsorbed onto microparticles/beads, were released within 2 hours to 5 days. In contrast, the viral antigens entrapped into the chitosan microparticles were released more slowly and continued for up to 30 days.

Conclusion: It was concluded from the viral antigen releasing profiles of chitosan particles that the viral antigens entrapped into the microparticles are more suitable for in vivo applications as a potential mucosal vaccine.

References

  • 1. Tharanathan RN, Kittur FS. Chitin-the undisputed biomolecule of great potential. Crit Rev Food Sci Nutr 2003; 43(1): 61-87.
  • 2. Kurita K. Chitin and chitosan: functional biopolymers from marine crustaceans. Mar Biotechnol (NY) 2006; 8(3): 203-26.
  • 3. White SA, Farina PR, Fulton I. Production and isolation of chitosan from Mucor rouxii. Appl Environ Microbiol 1979; 38(2): 323-8.
  • 4. Elieh-Ali-Komi D, Hamblin MR. Chitin and chitosan: Production and application of versatile biomedical nanomaterials. Int J Adv Res (Indore) 2016; 4(3): 411-27.
  • 5. Synowiecki J, Al-Khateeb NA. Production, properties, and some new applications of chitin and its derivatives. Crit Rev Food Sci Nutr 2003; 43(2): 145-71.
  • 6. Felt O, Baeyens V, Buri P, Gurny R. Delivery of antibiotics to the eye using a positively charged polysaccharide as vehicle. AAPS PharmSci 2001; 3(4): E34.
  • 7. Turan K, Nagata K. Chitosan-DNA nanoparticles: the effect of cell type and hydrolysis of chitosan on in vitro DNA transfection. Pharm Dev Technol 2006; 11(4): 503-12.
  • 8. Dastan T, Turan K. In vitro characterization and delivery of chitosan-DNA microparticles into mammalian cells. J Pharm Pharm Sci 2004; 7(2): 205-14.
  • 9. Muanprasat C, Chatsudthipong V. Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacol Ther 2017; 170: 80-97.
  • 10. Borchard G. Chitosans for gene delivery. Adv Drug Deliv Rev 2001; 52(2):145-50.
  • 11. van der Lubben IM, Verhoef JC, Borchard G, Junginger HE. Chitosan and its derivatives in mucosal drug and vaccine delivery. Eur J Pharm Sci 2001; 14(3): 201-7.
  • 12. Muzzarelli RA. Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers. Mar Drugs 2010; 8(2): 292-312.
  • 13. Porporatto C, Bianco ID, Correa SG. Local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration. J Leukoc Biol 2005; 78(1): 62-9.
  • 14. Li D, Fu D, Kang H, Rong G, Jin Z, Wang X, et al. Advances and Potential Applications of Chitosan Nanoparticles as a Delivery Carrier for the Mucosal Immunity of Vaccine. Curr Drug Deliv 2017; 14(1): 27-35.
  • 15. Elderfield R, Barclay W. Influenza pandemics. Adv Exp Med Biol 2011; 719: 81-103.
  • 16. Kilbourne ED. Influenza pandemics of the 20th century. Emerg Infect Dis 2006; 12(1): 9-14.
  • 17. Cunha BA. Influenza: historical aspects of epidemics and pandemics. Infect Dis Clin North Am 2004; 18(1): 141-55.
  • 18. Robertson JS, Inglis SC. Prospects for controlling future pandemics of influenza. Virus Res 2011; 162(1-2): 39-46.
  • 19. Marcelin G, Sandbulte MR, Webby RJ. Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines. Rev Med Virol 2012; 22(4): 267-79.
  • 20. Sawaengsak C, Mori Y, Yamanishi K, Mitrevej A, Sinchaipanid N. Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine. AAPS Pharm Sci Tech 2014; 15(2): 317-25.
  • 21. Bitter C, Suter-Zimmermann K, Surber C. Nasal drug delivery in humans. Curr Probl Dermatol 2011; 40: 20-35.
  • 22. Shakya AK, Chowdhury MYE, Tao W, Gill HS. Mucosal vaccine delivery: Current state and a pediatric perspective. J Control Release 2016; 240: 394-413.
  • 23. Salatin S, Barar J, Barzegar-Jalali M, Adibkia K, Milani MA, Jelvehgari M. Hydrogel nanoparticles and nanocomposites for nasal drug/vaccine delivery. Arch Pharm Res. 2016; 39(9): 1181-92.
  • 24. Doavi T, Mousavi SL, Kamali M, Amani J, Fasihi Ramandi M. Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7. Iran Biomed J 2016; 20(2): 97-108.
  • 25. Rauw F, Gardin Y, Palya V, Anbari S, Gonze M, Lemaire S, et al. The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine. Vet Immunol Immunopathol 2010; 134(3-4): 249-58.
  • 26. Volkova MA, Irza AV, Chvala IA, Frolov SF, Drygin VV, Kapczynski DR. Adjuvant effects of chitosan and calcium phosphate particles in an inactivated Newcastle disease vaccine. Avian Dis 2014; 58(1): 46-52.
  • 27. Vasiliev YM. Chitosan-based vaccine adjuvants: incomplete characterization complicates preclinical and clinical evaluation. Expert Rev Vaccines 2015; 14: 37-53.
  • 28. Sonaje K, Chuang EY, Lin KJ, Yen TC, Su FY, Tseng MT, et al. Opening of epithelial tight junctions and enhancement of paracellular permeation by chitosan: microscopic, ultrastructural, and computed-tomographic observations. Mol Pharm 2012; 9(5): 1271-9.
  • 29. Sonaje K, Lin KJ, Tseng MT, Wey SP, Su FY, Chuang EY. Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins. Biomaterials 2011; 32(33): 8712-21.
  • 30. Barrett T, Inglis SC. Growth, purification and titration of influenza viruses. Ed. Mahy BWJ: Virology: A practical approach. Oxford, IRL Press, 1985; pp 119-50.
  • 31. Chrystie IL. Electron Mycroscopy. Eds. Mahy BWJ, Kangro HO. Virology methods manual. London: Academic Press, 1996; pp 91-107.
  • 32. Sanders B, Koldijk M, Schuitemaker H. Inactivated Viral Vaccines. Eds. In Nunnally BK, Turula VE, Sitrin RD. Vaccine Analysis: Strategies, Principles, and Control. Berlin Heidelberg: Springer-Verlag Berlin Heidelberg, 2015; pp 45-79.
  • 33. Shu XZ, Zhu KJ. Chitosan/gelatin microspheres prepared by modified emulsification and ionotropic gelation. J Microencapsul 2001; 18(2): 237-45.
  • 34. Lim ST, Martin GP, Berry DJ, Brown MB. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J Control Release 2000; 66(2-3): 281-92.
  • 35. Mi FL, Shyu SS, Chen CT, Schoung JY. Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release. Biomaterials 1999; 20(17): 1603-12.
  • 36. Global Health Estimates Technical Paper: Mathers C, Stevens G, Mahanani WR, et al.. WHO methods and data sources for country‐level causes of death 2000‐2015. 2017. WHO/HIS/IER/GHE/2016.3.
  • 37. Howard CR, Fletcher NF. Emerging virus diseases: can we ever expect the unexpected? Emerg Microbes Infect 2012; 1(12): e46.
  • 38. Kaur L, Sharma A, Yadav AK, Mishra N. Recent advances on biodegradable polymeric carrier-based mucosal immunization: an overview. Artif Cells Nanomed Biotechnol 2018; 46(3): 452-64.
  • 39. MuZikova G, Laga R. Macromolecular systems for vaccine delivery. Physiol Res 2016; 65:S203-S216.
  • 40. Mahapatro A, Singh DK. Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines. J Nanobiotechnology 2011; 9: 55.
  • 41. Arthanari S, Mani G, Peng MM, Jang HT. Chitosan-HPMC-blended microspheres as a vaccine carrier for the delivery of tetanus toxoid. Artif Cells Nanomed Biotechnol 2016; 44(2): 517-23.
  • 42. Ali A, Ahmed S. A review on chitosan and its nanocomposites in drug delivery. Int J Biol Macromol 2018; 109: 273-86.
  • 43. Mitra A, Dey B. Chitosan microspheres in novel drug delivery systems. Indian J Pharm Sci 2011; 73(4): 355-66.
  • 44. Kreuter J. Nanoparticles and microparticles for drug and vaccine delivery. J Anat 1996; 189 (Pt 3): 503-5.
  • 45. Vila A, Sanchez A, Evora C, Soriano I, McCallion O, Alonso MJ. PLA-PEG particles as nasal protein carriers: the influence of the particle size. Int J Pharm 2005; 292(1): 43-52.
  • 46. Oliveira CR, Rezende CM, Silva MR, Pêgo AP, Borges O, Goes AM. A new strategy based on SmRho protein loaded chitosan nanoparticles as a candidate oral vaccine against schistosomiasis. PLoS Negl Trop Dis 2012; 6(11): e1894.
  • 47. Gupta S, Kesarla R, Omri A. Formulation strategies to improve the bioavailability of poorly absorbed drugs with special emphasis on self-emulsifying systems. ISRN Pharm 2013; 2013: 848043.
  • 48. Ito Y, Kusawake T, Ishida M, Tawa R, Shibata N, Takada K. Oral solid gentamicin preparation using emulsifier and adsorbent. J Control Release 2005; 105(1-2): 23-31.
  • 49. Gupta KC, Ravi Kumar MN. Drug release behavior of beads and microgranules of chitosan. Biomaterials 2000; 21(11): 1115-9.
  • 50. Orienti I, Aiedeh K, Gianasi E, Bertasi V, Zecchi V. Indomethacin loaded chitosan microspheres. Correlation between the erosion process and release kinetics. J Microencapsul 1996; 13(4): 463-72.
  • 51. Kas HS. Chitosan: properties, preparations and application to microparticulate systems. J Microencapsul 1997; 14(6): 689-711.
  • 52. Demento SL, Cui W, Criscione JM, Stern E, Tulipan J, Kaech SM. Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype. Biomaterials 2012; 33(19): 495764.

In Vitro Characterization of Chitosan-Based Particles as Carrier of Influenza Viral Antigens

Year 2018, Volume: 77 Issue: 1, 1 - 10, 22.06.2018

Abstract

DOI:
10.26650/EurJBiol.2018.0004


Objective: Chitosan is a natural
polysaccharide derived from chitin by deacetylation. It is a non-toxic,
biocompatible and biodegradable polymer, and has attracted considerable
interest in a wide range of pharmaceutical applications including drug and
vaccine delivery. The immune-stimulating activity of chitosan microparticles
such as the activation of macrophages and polymorphonuclear leukocytes has been
reported. In this work, we have investigated the releasing properties of
influenza virus antigens from the chitosan microparticles and beads.



Materials and Methods: Chitosan
microparticles and beads were prepared by coacervation and ionotropic gelation
method, respectively. The microparticles and beads were loaded with the viral
antigens by passive adsorption and/or entrapment into the microparticles. The
titration of the viruses was defined by haemagglutination assay or by
quantitation of viral proteins using the Bradford method.



Results: The results showed that the
loading efficiency and the loading capacity of chitosan microparticles/beads
with the viral antigens and the releasing profiles of the antigens from the
particles changed depending on the type of chitosan, the pH of the loading
buffer and the methods used to prepare the particles. The influenza viral
antigens, passively adsorbed onto microparticles/beads, were released within 2
hours to 5 days. In contrast, the viral antigens entrapped into the chitosan
microparticles were released more slowly and continued for up to 30 days.



Conclusion: It was concluded from the viral
antigen releasing profiles of chitosan particles that the viral antigens
entrapped into the microparticles are more suitable for in vivo applications as
a potential mucosal vaccine.

References

  • 1. Tharanathan RN, Kittur FS. Chitin-the undisputed biomolecule of great potential. Crit Rev Food Sci Nutr 2003; 43(1): 61-87.
  • 2. Kurita K. Chitin and chitosan: functional biopolymers from marine crustaceans. Mar Biotechnol (NY) 2006; 8(3): 203-26.
  • 3. White SA, Farina PR, Fulton I. Production and isolation of chitosan from Mucor rouxii. Appl Environ Microbiol 1979; 38(2): 323-8.
  • 4. Elieh-Ali-Komi D, Hamblin MR. Chitin and chitosan: Production and application of versatile biomedical nanomaterials. Int J Adv Res (Indore) 2016; 4(3): 411-27.
  • 5. Synowiecki J, Al-Khateeb NA. Production, properties, and some new applications of chitin and its derivatives. Crit Rev Food Sci Nutr 2003; 43(2): 145-71.
  • 6. Felt O, Baeyens V, Buri P, Gurny R. Delivery of antibiotics to the eye using a positively charged polysaccharide as vehicle. AAPS PharmSci 2001; 3(4): E34.
  • 7. Turan K, Nagata K. Chitosan-DNA nanoparticles: the effect of cell type and hydrolysis of chitosan on in vitro DNA transfection. Pharm Dev Technol 2006; 11(4): 503-12.
  • 8. Dastan T, Turan K. In vitro characterization and delivery of chitosan-DNA microparticles into mammalian cells. J Pharm Pharm Sci 2004; 7(2): 205-14.
  • 9. Muanprasat C, Chatsudthipong V. Chitosan oligosaccharide: Biological activities and potential therapeutic applications. Pharmacol Ther 2017; 170: 80-97.
  • 10. Borchard G. Chitosans for gene delivery. Adv Drug Deliv Rev 2001; 52(2):145-50.
  • 11. van der Lubben IM, Verhoef JC, Borchard G, Junginger HE. Chitosan and its derivatives in mucosal drug and vaccine delivery. Eur J Pharm Sci 2001; 14(3): 201-7.
  • 12. Muzzarelli RA. Chitins and chitosans as immunoadjuvants and non-allergenic drug carriers. Mar Drugs 2010; 8(2): 292-312.
  • 13. Porporatto C, Bianco ID, Correa SG. Local and systemic activity of the polysaccharide chitosan at lymphoid tissues after oral administration. J Leukoc Biol 2005; 78(1): 62-9.
  • 14. Li D, Fu D, Kang H, Rong G, Jin Z, Wang X, et al. Advances and Potential Applications of Chitosan Nanoparticles as a Delivery Carrier for the Mucosal Immunity of Vaccine. Curr Drug Deliv 2017; 14(1): 27-35.
  • 15. Elderfield R, Barclay W. Influenza pandemics. Adv Exp Med Biol 2011; 719: 81-103.
  • 16. Kilbourne ED. Influenza pandemics of the 20th century. Emerg Infect Dis 2006; 12(1): 9-14.
  • 17. Cunha BA. Influenza: historical aspects of epidemics and pandemics. Infect Dis Clin North Am 2004; 18(1): 141-55.
  • 18. Robertson JS, Inglis SC. Prospects for controlling future pandemics of influenza. Virus Res 2011; 162(1-2): 39-46.
  • 19. Marcelin G, Sandbulte MR, Webby RJ. Contribution of antibody production against neuraminidase to the protection afforded by influenza vaccines. Rev Med Virol 2012; 22(4): 267-79.
  • 20. Sawaengsak C, Mori Y, Yamanishi K, Mitrevej A, Sinchaipanid N. Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine. AAPS Pharm Sci Tech 2014; 15(2): 317-25.
  • 21. Bitter C, Suter-Zimmermann K, Surber C. Nasal drug delivery in humans. Curr Probl Dermatol 2011; 40: 20-35.
  • 22. Shakya AK, Chowdhury MYE, Tao W, Gill HS. Mucosal vaccine delivery: Current state and a pediatric perspective. J Control Release 2016; 240: 394-413.
  • 23. Salatin S, Barar J, Barzegar-Jalali M, Adibkia K, Milani MA, Jelvehgari M. Hydrogel nanoparticles and nanocomposites for nasal drug/vaccine delivery. Arch Pharm Res. 2016; 39(9): 1181-92.
  • 24. Doavi T, Mousavi SL, Kamali M, Amani J, Fasihi Ramandi M. Chitosan-Based Intranasal Vaccine against Escherichia coli O157:H7. Iran Biomed J 2016; 20(2): 97-108.
  • 25. Rauw F, Gardin Y, Palya V, Anbari S, Gonze M, Lemaire S, et al. The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine. Vet Immunol Immunopathol 2010; 134(3-4): 249-58.
  • 26. Volkova MA, Irza AV, Chvala IA, Frolov SF, Drygin VV, Kapczynski DR. Adjuvant effects of chitosan and calcium phosphate particles in an inactivated Newcastle disease vaccine. Avian Dis 2014; 58(1): 46-52.
  • 27. Vasiliev YM. Chitosan-based vaccine adjuvants: incomplete characterization complicates preclinical and clinical evaluation. Expert Rev Vaccines 2015; 14: 37-53.
  • 28. Sonaje K, Chuang EY, Lin KJ, Yen TC, Su FY, Tseng MT, et al. Opening of epithelial tight junctions and enhancement of paracellular permeation by chitosan: microscopic, ultrastructural, and computed-tomographic observations. Mol Pharm 2012; 9(5): 1271-9.
  • 29. Sonaje K, Lin KJ, Tseng MT, Wey SP, Su FY, Chuang EY. Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins. Biomaterials 2011; 32(33): 8712-21.
  • 30. Barrett T, Inglis SC. Growth, purification and titration of influenza viruses. Ed. Mahy BWJ: Virology: A practical approach. Oxford, IRL Press, 1985; pp 119-50.
  • 31. Chrystie IL. Electron Mycroscopy. Eds. Mahy BWJ, Kangro HO. Virology methods manual. London: Academic Press, 1996; pp 91-107.
  • 32. Sanders B, Koldijk M, Schuitemaker H. Inactivated Viral Vaccines. Eds. In Nunnally BK, Turula VE, Sitrin RD. Vaccine Analysis: Strategies, Principles, and Control. Berlin Heidelberg: Springer-Verlag Berlin Heidelberg, 2015; pp 45-79.
  • 33. Shu XZ, Zhu KJ. Chitosan/gelatin microspheres prepared by modified emulsification and ionotropic gelation. J Microencapsul 2001; 18(2): 237-45.
  • 34. Lim ST, Martin GP, Berry DJ, Brown MB. Preparation and evaluation of the in vitro drug release properties and mucoadhesion of novel microspheres of hyaluronic acid and chitosan. J Control Release 2000; 66(2-3): 281-92.
  • 35. Mi FL, Shyu SS, Chen CT, Schoung JY. Porous chitosan microsphere for controlling the antigen release of Newcastle disease vaccine: preparation of antigen-adsorbed microsphere and in vitro release. Biomaterials 1999; 20(17): 1603-12.
  • 36. Global Health Estimates Technical Paper: Mathers C, Stevens G, Mahanani WR, et al.. WHO methods and data sources for country‐level causes of death 2000‐2015. 2017. WHO/HIS/IER/GHE/2016.3.
  • 37. Howard CR, Fletcher NF. Emerging virus diseases: can we ever expect the unexpected? Emerg Microbes Infect 2012; 1(12): e46.
  • 38. Kaur L, Sharma A, Yadav AK, Mishra N. Recent advances on biodegradable polymeric carrier-based mucosal immunization: an overview. Artif Cells Nanomed Biotechnol 2018; 46(3): 452-64.
  • 39. MuZikova G, Laga R. Macromolecular systems for vaccine delivery. Physiol Res 2016; 65:S203-S216.
  • 40. Mahapatro A, Singh DK. Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines. J Nanobiotechnology 2011; 9: 55.
  • 41. Arthanari S, Mani G, Peng MM, Jang HT. Chitosan-HPMC-blended microspheres as a vaccine carrier for the delivery of tetanus toxoid. Artif Cells Nanomed Biotechnol 2016; 44(2): 517-23.
  • 42. Ali A, Ahmed S. A review on chitosan and its nanocomposites in drug delivery. Int J Biol Macromol 2018; 109: 273-86.
  • 43. Mitra A, Dey B. Chitosan microspheres in novel drug delivery systems. Indian J Pharm Sci 2011; 73(4): 355-66.
  • 44. Kreuter J. Nanoparticles and microparticles for drug and vaccine delivery. J Anat 1996; 189 (Pt 3): 503-5.
  • 45. Vila A, Sanchez A, Evora C, Soriano I, McCallion O, Alonso MJ. PLA-PEG particles as nasal protein carriers: the influence of the particle size. Int J Pharm 2005; 292(1): 43-52.
  • 46. Oliveira CR, Rezende CM, Silva MR, Pêgo AP, Borges O, Goes AM. A new strategy based on SmRho protein loaded chitosan nanoparticles as a candidate oral vaccine against schistosomiasis. PLoS Negl Trop Dis 2012; 6(11): e1894.
  • 47. Gupta S, Kesarla R, Omri A. Formulation strategies to improve the bioavailability of poorly absorbed drugs with special emphasis on self-emulsifying systems. ISRN Pharm 2013; 2013: 848043.
  • 48. Ito Y, Kusawake T, Ishida M, Tawa R, Shibata N, Takada K. Oral solid gentamicin preparation using emulsifier and adsorbent. J Control Release 2005; 105(1-2): 23-31.
  • 49. Gupta KC, Ravi Kumar MN. Drug release behavior of beads and microgranules of chitosan. Biomaterials 2000; 21(11): 1115-9.
  • 50. Orienti I, Aiedeh K, Gianasi E, Bertasi V, Zecchi V. Indomethacin loaded chitosan microspheres. Correlation between the erosion process and release kinetics. J Microencapsul 1996; 13(4): 463-72.
  • 51. Kas HS. Chitosan: properties, preparations and application to microparticulate systems. J Microencapsul 1997; 14(6): 689-711.
  • 52. Demento SL, Cui W, Criscione JM, Stern E, Tulipan J, Kaech SM. Role of sustained antigen release from nanoparticle vaccines in shaping the T cell memory phenotype. Biomaterials 2012; 33(19): 495764.
There are 52 citations in total.

Details

Primary Language English
Journal Section Research Articles
Authors

Mehmet Karaaslan This is me

Kadir Turan

Publication Date June 22, 2018
Submission Date April 10, 2018
Published in Issue Year 2018 Volume: 77 Issue: 1

Cite

AMA Karaaslan M, Turan K. In Vitro Characterization of Chitosan-Based Particles as Carrier of Influenza Viral Antigens. Eur J Biol. June 2018;77(1):1-10.