AAPS PharmSciTech

, Volume 19, Issue 8, pp 3763–3769 | Cite as

Dry Formulations Enhanced Mucoadhesive Properties and Reduced Cold Chain Handing of Influenza Vaccines

  • Nattika Saengkrit
  • Somsak Saesoo
  • Noppawan Woramongkolchai
  • Warayuth Sajomsang
  • Sarunya Phunpee
  • Tararaj Dharakul
  • Uracha Rungsardthong RuktanonchaiEmail author
Research Article


To alleviate concerns in health security, emergency flu vaccine stockpiles are required for ensuring rapid availability of vaccines when needed. Cold chain preservation, at high cost and risk, is necessary to maintain vaccine efficacy. This study aimed to develop a dry, easily storable formula for influenza vaccine preparation. The formulation with mucoadhesive properties is expected to facilitate rapid delivery via nasal administration. Chitosan, a cationic polymer, was used as cryo-protectant and to promote mucoadhesion. Optimal concentrations and molecular weights of chitosan polymers were screened, with short chain chitosan (10 kDa) being most suitable. H1N1 dry powder, in different formulations, was prepared via freeze-drying. A series of cryo-protectants, trehalose (T), chitosan (C), fetal bovine serum (FBS; F), or a combination of these (TCF), were screened for their effects on prolonging vaccine shelf life. Physicochemical monitoring (particle size and zeta potential) of powders complexed with mucin revealed that the order of cryo-protectant mixing during preparation was of critical importance. Results indicated that the TCF formula retains its activity up to 1 year as indicated by TCID50 analysis. This approach was also successful at prolonging the shelf life of H3N2 vaccine, and has the potential for large-scale implementation, especially in developed countries where long-term storage of vaccines is problematic.


dry formulation vaccine formulation influenza vaccine mucoadhesive nasal vaccine delivery 


Funding Information

This research was financially supported by National Nanotechnology Center (NANOTEC), Thailand.


  1. 1.
    Cox R, Brokstad K, Ogra P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol. 2004;59(1):1–15.CrossRefPubMedGoogle Scholar
  2. 2.
    Amorij J-P, Hinrichs WLJ, Frijlink HW, Wilschut JC, Huckriede A. Needle-free influenza vaccination. Lancet Infect Dis. 2010;10(10):699–711.CrossRefPubMedGoogle Scholar
  3. 3.
    Lu D, Hickey AJ. Pulmonary vaccine delivery. Expert Rev Vaccines. 2007;6(2):213–26.CrossRefPubMedGoogle Scholar
  4. 4.
    Sullivan VJ, Mikszta JA, Laurent P, Huang J, Ford B. Noninvasive delivery technologies: respiratory delivery of vaccines. Expert Opin Drug Deliv. 2006;3(1):87–95.CrossRefPubMedGoogle Scholar
  5. 5.
    Block SL, Yogev R, Hayden FG, Ambrose CS, Zeng W, Walker RE. Shedding and immunogenicity of live attenuated influenza vaccine virus in subjects 5–49 years of age. Vaccine. 2008;26(38):4940–6.CrossRefPubMedGoogle Scholar
  6. 6.
    Greenbaum E, Engelhard D, Levy R, Schlezinger M, Morag A, Zakay-Rones Z. Mucosal (SIgA) and serum (IgG) immunologic responses in young adults following intranasal administration of one or two doses of inactivated, trivalent anti-influenza vaccine. Vaccine. 2004;22(20):2566–77.CrossRefPubMedGoogle Scholar
  7. 7.
    Milstien JB et al. Temperature sensitivity of vaccines. 2006.Google Scholar
  8. 8.
    Yannarell DA, Goldberg KM, Hjorth RN. Stabilizing cold-adapted influenza virus vaccine under various storage conditions. J Virol Methods. 2002;102(1):15–25.CrossRefPubMedGoogle Scholar
  9. 9.
    Murugappan S, Patil HP, Kanojia G, ter Veer W, Meijerhof T, Frijlink HW, et al. Physical and immunogenic stability of spray freeze-dried influenza vaccine powder for pulmonary delivery: comparison of inulin, dextran, or a mixture of dextran and trehalose as protectants. Eur J Pharm Biopharm. 2013;85(3):716–25.CrossRefPubMedGoogle Scholar
  10. 10.
    Amorij J, et al. Development of stable influenza vaccine powder formulations: challenges and possibilities. Pharm Res. 2008;25(6):1256–73.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Block SL, Reisinger KS, Hultquist M, Walker RE, for the CAIV-T Study Group. Comparative immunogenicities of frozen and refrigerated formulations of live attenuated influenza vaccine in healthy subjects. Antimicrob Agents Chemother. 2007;51(11):4001–8.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Bhatnagar BS, Bogner RH, Pikal MJ. Protein stability during freezing: separation of stresses and mechanisms of protein stabilization. Pharm Dev Technol. 2007;12(5):505–23.CrossRefPubMedGoogle Scholar
  13. 13.
    Salo R, Cliver D. Effect of acid pH, salts, and temperature on the infectivity and physical integrity of enteroviruses. Arch Virol. 1976;52(4):269–82.CrossRefPubMedGoogle Scholar
  14. 14.
    Hansen L, et al. Freeze-drying of live virus vaccines: a review. Vaccine. 2015;33(42):5507–19.CrossRefPubMedGoogle Scholar
  15. 15.
    Tannock GA, Hierholzer JC, Bryce DA, Chee CF, Paul JA. Freeze-drying of respiratory syncytial viruses for transportation and storage. J Clin Microbiol. 1987;25(9):1769–71.PubMedPubMedCentralGoogle Scholar
  16. 16.
    Sarkar J, Sreenivasa BP, Singh RP, Dhar P, Bandyopadhyay SK. Comparative efficacy of various chemical stabilizers on the thermostability of a live-attenuated peste des petits ruminants (PPR) vaccine. Vaccine. 2003;21(32):4728–35.CrossRefPubMedGoogle Scholar
  17. 17.
    Zhai S, Hansen RK, Taylor R, Skepper JN, Sanches R, Slater NKH. Effect of freezing rates and excipients on the infectivity of a live viral vaccine during lyophilization. Biotechnol Prog. 2004;20(4):1113–20.CrossRefPubMedGoogle Scholar
  18. 18.
    Crowe LM, Crowe JH, Rudolph A, Womersley C, Appel L. Preservation of freeze-dried liposomes by trehalose. Arch Biochem Biophys. 1985;242(1):240–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Kumru OS, Joshi SB, Smith DE, Middaugh CR, Prusik T, Volkin DB. Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies. Biologicals. 2014;42(5):237–59.CrossRefPubMedGoogle Scholar
  20. 20.
    Prabhu M, Bhanuprakash V, Venkatesan G, Yogisharadhya R, Bora DP, Balamurugan V. Evaluation of stability of live attenuated camelpox vaccine stabilized with different stabilizers and reconstituted with various diluents. Biologicals. 2014;42(3):169–75.CrossRefPubMedGoogle Scholar
  21. 21.
    Nemoto T, Horiuchi M, Ishiguro N, Shinagawa M. Detection methods of possible prion contaminants in collagen and gelatin. Arch Virol. 1999;144(1):177–84.CrossRefPubMedGoogle Scholar
  22. 22.
    Chun B-H, Lee YK, Lee BC, Chung N. Development of a varicella virus vaccine stabilizer containing no animal-derived component. Biotechnol Lett. 2004;26(10):807–12.CrossRefPubMedGoogle Scholar
  23. 23.
    Prego C, Paolicelli P, Díaz B, Vicente S, Sánchez A, González-Fernández Á, et al. Chitosan-based nanoparticles for improving immunization against hepatitis B infection. Vaccine. 2010;28(14):2607–14.CrossRefPubMedGoogle Scholar
  24. 24.
    Sawaengsak C, Mori Y, Yamanishi K, Srimanote P, Chaicumpa W, Mitrevej A, et al. Intranasal chitosan-DNA vaccines that protect across influenza virus subtypes. Int J Pharm. 2014;473(1):113–25.CrossRefPubMedGoogle Scholar
  25. 25.
    Zhao K, Shi X, Zhao Y, Wei H, Sun Q, Huang T, et al. Preparation and immunological effectiveness of a swine influenza DNA vaccine encapsulated in chitosan nanoparticles. Vaccine. 2011;29(47):8549–56.CrossRefPubMedGoogle Scholar
  26. 26.
    Heffernan MJ, Zaharoff DA, Fallon JK, Schlom J, Greiner JW. In vivo efficacy of a chitosan/IL-12 adjuvant system for protein-based vaccines. Biomaterials. 2011;32(3):926–32.CrossRefPubMedGoogle Scholar
  27. 27.
    Lavertu M, Xia Z, Serreqi AN, Berrada M, Rodrigues A, Wang D, et al. A validated 1 H NMR method for the determination of the degree of deacetylation of chitosan. J Pharm Biomed Anal. 2003;32(6):1149–58.CrossRefPubMedGoogle Scholar
  28. 28.
    Takeuchi H, et al. Novel mucoadhesion tests for polymers and polymer-coated particles to design optimal mucoadhesive drug delivery systems. Adv Drug Deliv Rev. 2005;57(11):1583–94.CrossRefPubMedGoogle Scholar
  29. 29.
    Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol. 1938;27(3):493–7.CrossRefGoogle Scholar
  30. 30.
    Rakkhithawatthana V, Sanitrum P, Sajomsang W, Na Ubon P, Tencomnao T, Saengkrit N. Investigation of gene transferring efficacy through nano-polyplex consisting of methylated N-(4-pyridinylmethyl) chitosan chloride and poly (ethylenimine) in human cell lines. Carbohydr Polym. 2010;80(1):276–84.CrossRefGoogle Scholar

Copyright information

© American Association of Pharmaceutical Scientists 2018

Authors and Affiliations

  • Nattika Saengkrit
    • 1
  • Somsak Saesoo
    • 1
  • Noppawan Woramongkolchai
    • 1
  • Warayuth Sajomsang
    • 1
  • Sarunya Phunpee
    • 1
  • Tararaj Dharakul
    • 2
  • Uracha Rungsardthong Ruktanonchai
    • 1
    Email author
  1. 1.National Nanotechnology Center (NANOTEC)National Science and Technology Development Agency (NSTDA)PathumthaniThailand
  2. 2.Department of Immunology, Faculty of MedicineSiriraj Hospital, Mahidol UniversityBangkokThailand

Personalised recommendations