Skip to main content

Advertisement

SpringerLink
Log in

Nucleic Acid Aptamers as Stabilizers of Proteins: The Stability of Tetanus Toxoid

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (Mr ~150 kDa) with high affinity.

Methods

Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques.

Results

The total protein released was between 40–55% (24–45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid.

Conclusions

Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.

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

Similar content being viewed by others

References

  1. Van der Zeijst BA. Vaccines and global stability: achievements and challenges. Expert Rev Vaccine. 2008;7(10):1457–60.

    Article  Google Scholar 

  2. Kristensen D, Chen D. Stabilization of vaccines: lessons learned. Hum Vaccine. 2010;6(3):229–31.

    Article  Google Scholar 

  3. Brown DW, Burton A, Gacic-Dobo M, Karimov RI, Vandelaer J, Okwo-Bele JM. A mid-term assessment of progress towards the immunization coverage goal of the Global Immunization Vision and Strategy (GIVS). BMC Publ Health. 2011;11:806.

    Article  Google Scholar 

  4. O'Brien J. Advancing vaccinology in India. Expert Rev Vaccine. 2011;11(1):27–9.

    Article  Google Scholar 

  5. PATH. Immunization logistics and supply systems: From vision to action. Seattle: PATH; 2010.

  6. Breen ED, Curley JG, Overcashier DE, Hsu CC, Shire SJ. Effect of moisture on the stability of a lyophilized humanized monoclonal antibody formulation. Pharm Res. 2001;18(9):1345–53.

    Article  PubMed  CAS  Google Scholar 

  7. Flores-Fernández GM, Pagán M, Almenas M, Solá RJ, Griebenow K. Moisture-induced solid state instabilities in alpha-chymotrypsin and their reduction through chemical glycosylation. BMC Biotechnol. 2010;10:57.

    Article  PubMed  Google Scholar 

  8. Flores-Fernández GM, Solá RJ, Griebenow K. The relation between moisture-induced aggregation and structural changes in lyophilized insulin. J Pharm Pharmacol. 2009;61(11):1555–61.

    Article  PubMed  Google Scholar 

  9. Jiang W, Schwendeman SP. Formaldehyde-mediated aggregation of protein antigens: comparison of untreated and formalinized model antigens. Biotechnol Bioeng. 2000;70(5):507–17.

    Article  PubMed  CAS  Google Scholar 

  10. Jain NK, Roy I. Accelerated stability studies for moisture-induced aggregation of tetanus toxoid. Pharm Res. 2011;28(3):626–39.

    Article  PubMed  CAS  Google Scholar 

  11. Aggerbeck H, Heron I. Detoxification of diphtheria and tetanus toxin with formaldehyde. Detection of protein conjugates. Biologicals. 1992;20(2):109–15.

    Article  PubMed  CAS  Google Scholar 

  12. Schwendeman SP, Costantino HR, Gupta RK, Siber GR, Klibanov AM, Langer R. Stabilization of tetanus and diphtheria toxoids against moisture-induced aggregation. Proc Natl Acad Sci USA. 1995;92(24):11234–8.

    Article  PubMed  CAS  Google Scholar 

  13. Quintilio W, Takata CS, Sant'Anna OA, da Costa MH, Raw I. Evaluation of a diphtheria and tetanus PLGA microencapsulated vaccine formulation without stabilizers. Curr Drug Deliv. 2009;6(3):297–304.

    Article  PubMed  CAS  Google Scholar 

  14. Determan AS, Wilson JH, Kipper MJ, Wannemuehler MJ, Narasimhan B. Protein stability in the presence of polymer degradation products: consequences for controlled release formulations. Biomaterials. 2006;27(17):3312–20.

    Article  PubMed  CAS  Google Scholar 

  15. Jiang W, Schwendeman SP. Stabilization of tetanus toxoid encapsulated in PLGA microspheres. Mol Pharm. 2008;5(5):808–17.

    Article  PubMed  CAS  Google Scholar 

  16. Hermann T, Patel DJ. Adaptive recognition by nucleic acid aptamers. Science. 2000;287(5454):820–5.

    Article  PubMed  CAS  Google Scholar 

  17. Fickert H, Fransson IG, Hahn U. Aptamers to small molecules. In: Klussmann S, editor. The aptamer handbook. Weinheim: Wiley VCH; 2006. p. 95–115.

    Google Scholar 

  18. Cerchia L, de Franciscis V. Targeting cancer cells with nucleic acid aptamers. Trends Biotechnol. 2010;28(10):517–25.

    Article  PubMed  CAS  Google Scholar 

  19. Bunka DH, Platonova O, Stockley PG. Development of aptamer therapeutics. Curr Opin Pharmacol. 2010;10(5):557–62.

    Article  PubMed  CAS  Google Scholar 

  20. Keefe AD, Pai S, Ellington A. Aptamers as therapeutics. Nat Rev Drug Discov. 2010;9(7):537–50.

    Article  PubMed  CAS  Google Scholar 

  21. Schellekens H. Follow-on biologics: challenges of the “next generation”. Nephrol Dial Transplant. 2005;20(S4):iv31–6.

    Article  PubMed  Google Scholar 

  22. Kresse GB. Biosimilars - science, status, and strategic perspective. Eur J Pharm Biopharm. 2009;72(3):479–86.

    Article  PubMed  CAS  Google Scholar 

  23. Fox A. Biosimilar medicines–new challenges for a new class of medicine. J Biopharm Stat. 2010;20(1):3–9.

    Article  PubMed  Google Scholar 

  24. Hermanson GT, Mallia KA, Smith PK. Immobilized affinity ligand techniques. New York: Academic; 1992. p. 53–6.

    Google Scholar 

  25. Uversky VN, Li J, Fink AL. Evidence for a partially folded intermediate in alpha-synuclein fibril formation. J Biol Chem. 2001;276(14):10737–44.

    Article  PubMed  CAS  Google Scholar 

  26. Sambrook J, Russell D. Molecular cloning: A laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001. p. 1.31–8.

    Google Scholar 

  27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.

    Article  PubMed  CAS  Google Scholar 

  28. Jaganathan KS, Rao YU, Singh P, Prabakaran D, Gupta S, Jain A, et al. Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D, L-lactic-co-glycolic acid) versus chitosan microspheres. Int J Pharm. 2005;294(1–2):23–32.

    Article  PubMed  CAS  Google Scholar 

  29. 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(1):35–42.

    Article  PubMed  CAS  Google Scholar 

  30. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150(1):76–85.

    Article  PubMed  CAS  Google Scholar 

  31. Misra VK, Draper DE. The linkage between magnesium binding and RNA folding. J Mol Biol. 2002;317(4):507–21.

    Article  PubMed  CAS  Google Scholar 

  32. Nonaka Y, Sode K, Ikebukuro K. Screening and improvement of an anti-VEGF DNA aptamer. Molecules. 2010;15(1):215–20.

    Article  PubMed  CAS  Google Scholar 

  33. Lee J-H, Canny MD, De Erkenez A, Krilleke D, Ng Y-S, Shima DT, et al. A therapeutic aptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc Natl Acad Sci USA. 2005;102(52):18902–7.

    Article  PubMed  CAS  Google Scholar 

  34. Di Tommaso P, Moretti S, Xenarios I, Orobitg M, Montanyola A, Chang JM, et al. T-Coffee: a web server for the multiple sequence alignment of protein and RNA sequences using structural information and homology extension. Nucleic Acids Res. 2011;39(Web server issue):W13–7.

    Article  PubMed  Google Scholar 

  35. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673–80.

    Article  PubMed  CAS  Google Scholar 

  36. Edgar RC, Batzoglou S. Multiple sequence alignment. Curr Opin Struct Biol. 2006;16(3):368–73.

    Article  PubMed  CAS  Google Scholar 

  37. Chen CH, Chernis GA, Hoang VQ, Landgraf R. Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. Proc Natl Acad Sci USA. 2003;100(16):9226–31.

    Article  PubMed  CAS  Google Scholar 

  38. Soontornworajit B, Zhou J, Shaw MT, Fan TH, Wang Y. Hydrogel functionalization with DNA aptamers for sustained PDGF-BB release. Chem Commun (Camb). 2010;46(11):1857–9.

    Article  CAS  Google Scholar 

  39. Soontornworajit B, Zhou J, Snipes MP, Battig MR, Wang Y. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides. Biomaterials. 2011;32(28):6839–49.

    Article  PubMed  CAS  Google Scholar 

  40. Carrasquillo KG, Ricker JA, Rigas IK, Miller JW, Gragoudas ES, Adamis AP. Controlled delivery of the anti-VEGF aptamer EYE001 with poly(lactic-co-glycolic)acid microspheres. Invest Ophthalmol Vis Sci. 2003;44(1):290–9.

    Article  PubMed  Google Scholar 

  41. Tan W, Wang H, Chen Y, Zhang X, Zhu H, Yang C, et al. Molecular aptamers for drug delivery. Trends Biotechnol. 2011;29(12):634–40.

    Article  PubMed  CAS  Google Scholar 

  42. Choi SI, Han KS, Kim CW, Ryu KS, Kim BH, Kim KH, et al. Protein solubility and folding enhancement by interaction with RNA. PLoS One. 2008;3(7):e2677.

    Article  PubMed  Google Scholar 

  43. Choi SI, Ryu K, Seong BL. RNA-mediated chaperone type for de novo protein folding. RNA Biol. 2009;6(1):21–4.

    Article  PubMed  CAS  Google Scholar 

  44. Choi SI, Lim KH, Seong BL. Chaperoning roles of macromolecules interacting with proteins in vivo. Int J Mol Sci. 2011;12(3):1979–90.

    Article  PubMed  CAS  Google Scholar 

  45. Costantino HR, Schwendeman SP, Griebenow K, Klibanov AM, Langer R. The secondary structure and aggregation of lyophilized tetanus toxoid. J Pharm Sci. 1996;85(12):1290–3.

    Article  PubMed  CAS  Google Scholar 

  46. Abbas SA, Sharma VK, Patapoff TW, Kalonia DS. Opposite effects of polyols on antibody aggregation: thermal versus mechanical stresses. Pharm Res. 2012;29(3):683–94.

    Article  PubMed  CAS  Google Scholar 

  47. Solanki VA, Jain NK, Roy I. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against freeze-thawing. Int J Pharm. 2011;414(1–2):140–7.

    Article  PubMed  CAS  Google Scholar 

  48. Solanki VA, Jain NK, Roy I. Stabilization of tetanus toxoid formulation containing aluminium hydroxide adjuvant against agitation. Int J Pharm. 2012;423(2):297–302.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments AND DISCLOSURES

Financial support received from Indian Council of Medical Research (Govt. of India) is gratefully acknowledged. The authors are thankful to Mr. Mohinder Singh, Sophisticated Analytical Instrumentation Facility (SAIF), Panjab University, Chandigarh, India, for recording SEM images. NKJ acknowledges the award of senior research fellowship by Council for Scientific and Industrial Research (Govt. of India).

Author information

Authors and Affiliations

  1. Department of Biotechnology, National Institute of Pharmaceutical Education & Research (NIPER), Sector 67, S.A.S., Nagar, Punjab, 160 062, India

    Nishant Kumar Jain, Hardik C. Jetani & Ipsita Roy

Authors
  1. Nishant Kumar Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hardik C. Jetani
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ipsita Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ipsita Roy.

Electronic supplementary material

ESM 1

(DOCX 52 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jain, N.K., Jetani, H.C. & Roy, I. Nucleic Acid Aptamers as Stabilizers of Proteins: The Stability of Tetanus Toxoid. Pharm Res 30, 1871–1882 (2013). https://doi.org/10.1007/s11095-013-1030-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11095-013-1030-7

Key words

Search

Navigation