Skip to main content
SpringerLink
Log in

Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration

  • Original Article
  • Published:
Drug Delivery and Translational Research Aims and scope Submit manuscript

Abstract

The present study reports the tetanus toxoid (TT)-loaded layer-by-layer nanoassemblies (layersomes) with enhanced protection, permeation, and presentation for comprehensive oral immunization. The stable and lyophilized TT-loaded layersomes were prepared by a thin-film hydration method followed by alternate layer-by-layer coating of an electrolyte. The developed system was assessed for in vitro stability of antigen and formulation, cellular uptake, ex vivo intestinal uptake, and immunostimulatory response using a suitable experimental protocol. Layersomes improved the stability in simulated biological media as well as protected the integrity/conformation and native 3D structure of TT as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorescence spectroscopy, respectively. The cell culture studies demonstrated a 3.8-fold higher permeation of layersomes in Caco-2 cells and an 8.5-fold higher uptake by antigen-presenting cells (RAW 264.7). The TT-loaded layersomes elicited a complete immunostimulatory profile consisting of higher systemic (serum IgG titer), mucosal (sIgA titer), and cellular (interleukin-2 (IL-2) and interferon-γ (IFN-γ) levels) immune response after peroral administration in mice. The modified TT inhibition assay further confirmed the elicitation of complete protective levels of anti-TT antibody (>0.1 IU/mL) by layersomes. In conclusion, the proposed strategy is expected to contribute significantly in the field of stable liposome technology for mass immunization through the oral route.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Rieux AD, Fievez V, Garinot M, Schneider YJ, Préat V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release. 2006;116:1–27.

    Article  PubMed  Google Scholar 

  2. Jain S, Harde H, Indulkar A, Agrawal AK. Improved stability and immunological potential of tetanus toxoid containing surface engineered bilosomes following oral administration. Nanomedicine. 2014;10:431–40.

    Article  CAS  PubMed  Google Scholar 

  3. Jain S, Khomane K, Jain AK, Dani P. Nanocarriers for transmucosal vaccine delivery. Curr Nanosci. 2011;7:160–77.

    Article  CAS  Google Scholar 

  4. Isaka M, Yasuda Y, Kozuka S, Miura Y, Taniguchi T, Matano K, et al. Systemic and mucosal immune responses of mice to aluminium-adsorbed or aluminium-non-adsorbed tetanus toxoid administered intranasally with recombinant cholera toxin B subunit. Vaccine. 1998;16:1620–6.

    Article  CAS  PubMed  Google Scholar 

  5. Westerink J, Louise Smithson S, Srivastava N, Blonder J, Coeshott C, Rosenthal GJ. ProJuvant™ (Pluronic F127®/chitosan) enhances the immune response to intranasally administered tetanus toxoid. Vaccine. 2001;20:711–23.

    Article  CAS  PubMed  Google Scholar 

  6. Tafaghodi M, Sajadi Tabassi 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.

    Article  CAS  PubMed  Google Scholar 

  7. Jung T, Kamm W, Breitenbach A, Hungerer K-D, Hundt E, Kissel T. Tetanus toxoid loaded nanoparticles from sulfobutylated poly(vinyl alcohol)-graft-poly(lactide-co-glycolide): evaluation of antibody response after oral and nasal application in mice. Pharm Res. 2001;18:352–60.

    Article  CAS  PubMed  Google Scholar 

  8. Mann JF, Scales HE, Shakir E, Alexander J, Carter KC, Mullen AB, et al. Oral delivery of tetanus toxoid using vesicles containing bile salts (bilosomes) induces significant systemic and mucosal immunity. Methods. 2006;38:90–5.

    Article  CAS  PubMed  Google Scholar 

  9. Benediktsdottir BE, Baldursson O, Masson M. Challenges in evaluation of chitosan and trimethylated chitosan (TMC) as mucosal permeation enhancers: from synthesis to in vitro application. J Control Release. 2014;173:18–31.

    Article  CAS  PubMed  Google Scholar 

  10. Agrawal AK, Harde HP, Thanki K, Jain S. Improved stability and antidiabetic potential of insulin containing folic acid functionalized polymer stabilized multilayered liposomes following oral administration. Biomacromolecules. 2014;15:350–60.

    Article  CAS  PubMed  Google Scholar 

  11. Jain S, Kumar D, Swarnakar NK, Thanki K. Polyelectrolyte stabilized multilayered liposomes for oral delivery of paclitaxel. Biomaterials. 2012;33:6758–68.

    Article  CAS  PubMed  Google Scholar 

  12. Harde H, Agrawal AK, Jain S. Trilateral ‘3P’ mechanics of stabilized layersomes technology for efficient oral immunization. J Biomed Nanotechnol. 2015;11:363–81.

    Article  CAS  PubMed  Google Scholar 

  13. Harde H, Siddhapura K, Agrawal AK, Jain S. Divalent toxoids loaded stable chitosan–glucomannan nanoassemblies for efficient systemic, mucosal and cellular immunostimulatory response following oral administration. Int J Pharm. 2015;487:292–304.

    Article  CAS  PubMed  Google Scholar 

  14. Jain S, Rathi VV, Jain AK, Das M, Godugu C. Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin. Nanomedicine. 2012;7:1311–37.

    Article  CAS  PubMed  Google Scholar 

  15. Xing DK-L, Crane DT, BolgianoT B, Corbel MJ, Jones C, Sesardic D. Physicochemical and immunological studies on the stability of free and microsphere-encapsulated tetanus toxoid in vitro. Vaccine. 1996;14:1205–13.

    Article  CAS  PubMed  Google Scholar 

  16. Jain S, Indulkar A, Harde H, Agrawal AK. Oral mucosal immunization using glucomannosylated bilosomes. J Biomed Nanotechnol. 2014;10:932–47.

    Article  CAS  PubMed  Google Scholar 

  17. Kapoor R, Harde H, Jain S, Panda AK, Panda BP. Downstream processing, formulation development and antithrombotic evaluation of microbial nattokinase. J Biomed Nanotechnol. 2015;11:1213–24.

    Article  CAS  PubMed  Google Scholar 

  18. Harde H, Singh RP, Agrawal AK, Jain S. Disease-based selection of nanocarriers in drug delivery based on hydrophobicity and surface charge. J Nanogen Nanomed. 2013;1:1–10.

    Google Scholar 

  19. Harde H, Agrawal AK, Jain S. Development of stabilized glucomannosylated chitosan nanoparticles using tandem crosslinking method for oral vaccine delivery. Nanomedicine. 2014;9:2511–29.

    Article  CAS  PubMed  Google Scholar 

  20. Jain AK, Thanki K, Jain S. Co-encapsulation of tamoxifen and quercetin in polymeric nanoparticles: implications on oral bioavailability, antitumor efficacy, and drug-induced toxicity. Mol Pharm. 2013;10:3459–74.

    Article  CAS  PubMed  Google Scholar 

  21. Sarti F, Perera G, Hintzen F, Kotti K, Karageorgiou V, Kammona O, et al. In vivo evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A. Biomaterials. 2011;32:4052–7.

    Article  CAS  PubMed  Google Scholar 

  22. Singh M, Li X-M, Wang H, McGee J, Zamb T, Koff W, et al. Immunogenicity and protection in small-animal models with controlled-release tetanus toxoid microparticles as a single-dose vaccine. Infect Immun. 1997;65:1716–21.

    PubMed Central  CAS  PubMed  Google Scholar 

  23. Men Y, Thomasin C, Merkle HP, Gander B, Corradin G. A single administration of tetanus toxoid in biodegradable microspheres elicits T cell and antibody responses similar or superior to those obtained with aluminum hydroxide. Vaccine. 1995;13:683–9.

    Article  CAS  PubMed  Google Scholar 

  24. Harde H, Agrawal AK, Jain S. Tetanus toxoids loaded glucomannosylated chitosan based nanohoming vaccine adjuvant with improved oral stability and immunostimulatory response. Pharm Res. 2015;32:122–34.

    Article  CAS  PubMed  Google Scholar 

  25. Jain AK, Thanki K, Jain S. Solidified self-nanoemulsifying formulation for oral delivery of combinatorial therapeutic regimen: part I. Formulation development, statistical optimization, and in vitro characterization. Pharm Res. 2014;31:923–45.

    Article  CAS  PubMed  Google Scholar 

  26. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58:1688–713.

    Article  CAS  PubMed  Google Scholar 

  27. Manning MC, Patel K, Borchardt RT. Stability of protein pharmaceuticals. Pharm Res. 1989;6:903–18.

    Article  CAS  PubMed  Google Scholar 

  28. Thomasin C, Corradin G, Men Y, Merkle HP, Gander B. Tetanus toxoid and synthetic malaria antigen containing poly(lactide)/poly(lactide-co-glycolide) microspheres: importance of polymer degradation and antigen release for immune response. J Control Release. 1996;41:131–45.

    Article  CAS  Google Scholar 

  29. Li X, Aldayel AM, Cui Z. Aluminum hydroxide nanoparticles show a stronger vaccine adjuvant activity than traditional aluminum hydroxide microparticles. J Control Release. 2014;173:148–57.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Harde H, Das M, Jain S. Solid lipid nanoparticles: an oral bioavailability enhancer vehicle. Expert Opin Drug Deliv. 2011;8:1407–24.

    Article  CAS  PubMed  Google Scholar 

  31. Mathiowitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, et al. Biologically erodable microspheres as potential oral drug delivery systems. Nature. 1997;386:410–4.

    Article  CAS  PubMed  Google Scholar 

  32. Ogra PL, Faden H, Welliver RC. Vaccination strategies for mucosal immune responses. Clin Microbiol Rev. 2001;14:430–45.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Mackay IR, Rosen FS, Ada G. Vaccines and vaccination. N Engl J Med. 2001;345:1042–53.

    Article  Google Scholar 

  34. O’Hagan DT, Rappuoli R. Novel approaches to vaccine delivery. Pharm Res. 2004;21:1519–30.

    Article  PubMed  Google Scholar 

  35. Borrow R, Balmer P, Roper M. The immunological basis for immunization series. Module 3: tetanus. Department of Immunization, Vaccines and Biologicals. World Health Organization, Geneva, Switzerland. . 2006. 8–10.

  36. Plotkin SA. Correlates of protection induced by vaccination. Clin Vaccine Immunol. 2010;17:1055–65.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors are grateful to the Department of Biotechnology (DBT), Government of India, New Delhi, India, for providing financial support and to the Director, NIPER, for providing the necessary infrastructure facilities. The authors are also grateful to Dr. M. L Mago and Lavit Jambu for gift samples of tetanus toxoid and training facility provided at Panacea Biotech Ltd., India. Technical assistance provided by Mr. Rahul Mahajan in SEM analysis and Mr. Vinod Kumar in TEM analysis is also duly acknowledged.

Conflict of interest

The authors declare that they have no competing interests.

Compliance with Ethical Standards

All animal protocols were duly approved by the Institutional Animal Ethics Committee and Institutional Biosafety Committee of NIPER, SAS Nagar, India, and completed under the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Author information

Authors and Affiliations

  1. Centre for Pharmaceutical Nanotechnology, Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab, 160 062, India

    Harshad Harde, Ashish Kumar Agrawal & Sanyog Jain

Authors
  1. Harshad Harde
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashish Kumar Agrawal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sanyog Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sanyog Jain.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(DOC 148 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Harde, H., Agrawal, A.K. & Jain, S. Tetanus toxoid-loaded layer-by-layer nanoassemblies for efficient systemic, mucosal, and cellular immunostimulatory response following oral administration. Drug Deliv. and Transl. Res. 5, 498–510 (2015). https://doi.org/10.1007/s13346-015-0247-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13346-015-0247-x

Keywords

Search

Navigation