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Technological challenges in the preclinical development of an HIV nanovaccine candidate


Despite a very active research in the field of nanomedicine, only a few nano-based drug delivery systems have reached the market. The “death valley” between research and commercialization has been partially attributed to the limited characterization and reproducibility of the nanoformulations. Our group has previously reported the potential of a peptide-based nanovaccine candidate for the prevention of SIV infection in macaques. This vaccine candidate is composed of chitosan/dextran sulfate nanoparticles containing twelve SIV peptide antigens. The aim of this work was to rigorously characterize one of these nanoformulations containing a specific peptide, following a quality-by-design approach. The evaluation of the different quality attributes was performed by several complementary techniques, such as dynamic light scattering, nanoparticle tracking analysis, and electron microscopy for particle size characterization. The inter-batch reproducibility was validated by three independent laboratories. Finally, the long-term stability and scalability of the manufacturing technique were assessed. Overall, these data, together with the in vivo efficacy results obtained in macaques, underline the promise this new vaccine holds with regard to its translation to clinical trials.

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  1. 1.

    Niu Z, Conejos-Sánchez I, Griffin BT, O’Driscoll CM, Alonso MJ. Lipid-based nanocarriers for oral peptide delivery. Adv Drug Deliv Rev. 2016;106:337–54.

  2. 2.

    Yu M, Wu J, Shi J, Farokhzad OC. Nanotechnology for protein delivery: overview and perspectives. J Control Release. 2016;240:24–37.

  3. 3.

    Santalices I, Gonella A, Torres D, Alonso MJ. Advances on the formulation of proteins using nanotechnologies. J Drug Deliv Sci Technol. 2017;42:155–80.

  4. 4.

    Samaridou E, Alonso MJ. Nose-to-brain peptide delivery – the potential of nanotechnology. Bioorg Med Chem. 2018;26:2888–905.

  5. 5.

    Li Z, Rana TM. Therapeutic targeting of microRNAs: current status and future challenges. Nat Rev Drug Discov. 2014;13:622–38.

  6. 6.

    Liu Y, Xu C-F, Iqbal S, Yang X-Z, Wang J. Responsive nanocarriers as an emerging platform for cascaded delivery of nucleic acids to cancer. Adv Drug Deliv Rev. 2017;115:98–114.

  7. 7.

    Saraiva SM, Castro-López V, Pañeda C, Alonso MJ. Synthetic nanocarriers for the delivery of polynucleotides to the eye. Eur J Pharm Sci. 2017;103:5–18.

  8. 8.

    Kaczmarek JC, Kowalski PS, Anderson DG. Advances in the delivery of RNA therapeutics: from concept to clinical reality. Genome Med. 2017;9:60.

  9. 9.

    Irvine DJ, Hanson MC, Rakhra K, Tokatlian T. Synthetic nanoparticles for vaccines and immunotherapy. Chem Rev. 2015;115:11109–46.

  10. 10.

    Cordeiro AS, Alonso MJ. Recent advances in vaccine delivery. Pharm Pat Anal. 2015;5:49–73.

  11. 11.

    Dacoba TG, Olivera A, Torres D, Crecente-Campo J, Alonso MJ. Modulating the immune system through nanotechnology. Semin Immunol. 2017;34:78–102.

  12. 12.

    Gause KT, Wheatley AK, Cui J, Yan Y, Kent SJ, Caruso F. Immunological principles guiding the rational design of particles for vaccine delivery. ACS Nano. 2017;11:54–68.

  13. 13.

    Bobo D, Robinson KJ, Islam J, Thurecht KJ, Corrie SR. Nanoparticle-based medicines: a review of FDA-approved materials and clinical trials to date. Pharm Res. 2016;33:2373–87.

  14. 14.

    Anselmo AC, Mitragotri S. Nanoparticles in the clinic. Bioeng Transl Med. 2016;1:10–29.

  15. 15.

    Ventola CL. Progress in nanomedicine: approved and investigational nanodrugs. P T. 2017;42:742–55.

  16. 16.

    Desai N. Challenges in development of nanoparticle-based therapeutics. AAPS J. 2012;14:282–95.

  17. 17.

    Ragelle H, Danhier F, Préat V, Langer R, Anderson DG. Nanoparticle-based drug delivery systems: a commercial and regulatory outlook as the field matures. Expert Opin Drug Deliv. 2017;14:851–64.

  18. 18.

    Hua S, de Matos MBC, Metselaar JM, Storm G. Current trends and challenges in the clinical translation of nanoparticulate nanomedicines: pathways for translational development and commercialization. Front Pharmacol. 2018;9:1–14.

  19. 19.

    Dormont F, Rouquette M, Mahatsekake C, Gobeaux F, Peramo A, Brusini R, et al. Translation of nanomedicines from lab to industrial scale synthesis: the case of squalene-adenosine nanoparticles. J Control Release. 2019;307:302–14.

  20. 20.

    Gabizon A, Bradbury M, Prabhakar U, Zamboni W, Libutti S, Grodzinski P. Cancer nanomedicines: closing the translational gap. Lancet. 2014;384:2175–6.

  21. 21.

    Yu LX, Amidon G, Khan MA, Hoag SW, Polli J, Raju GK, et al. Understanding pharmaceutical quality by design. AAPS J. 2014;16:771–83.

  22. 22.

    Wicki A, Witzigmann D, Balasubramanian V, Huwyler J. Nanomedicine in cancer therapy: challenges, opportunities, and clinical applications. J Control Release. 2015;200:138–57.

  23. 23.

    Agrahari V, Agrahari V. Facilitating the translation of nanomedicines to a clinical product: challenges and opportunities. Drug Discov Today. 2018;23:974–91.

  24. 24.

    Zamboni WC, Torchilin V, Patri AK, Hrkach J, Stern S, Lee R, et al. Best practices in cancer nanotechnology: perspective from NCI nanotechnology alliance. Clin Cancer Res. 2012;18:3229–41.

  25. 25.

    Pallagi E, Ambrus R, Szabó-Révész P, Csóka I. Adaptation of the quality by design concept in early pharmaceutical development of an intranasal nanosized formulation. Int J Pharm. 2015;491:384–92.

  26. 26.

    Rose F, Wern JE, Ingvarsson PT, van de Weert M, Andersen P, Follmann F, et al. Engineering of a novel adjuvant based on lipid-polymer hybrid nanoparticles: a quality-by-design approach. J Control Release. 2015;210:48–57.

  27. 27.

    Shah B, Khunt D, Bhatt H, Misra M, Padh H. Intranasal delivery of venlafaxine loaded nanostructured lipid carrier: risk assessment and QbD based optimization. J Drug Deliv Sci Technol. 2016;33:37–50.

  28. 28.

    Raina H, Kaur S, Jindal AB. Development of efavirenz loaded solid lipid nanoparticles: risk assessment, quality-by-design (QbD) based optimisation and physicochemical characterisation. J Drug Deliv Sci Technol. 2017;39:180–91.

  29. 29.

    Marto J, Ruivo E, Lucas SD, Gonçalves LM, Simões S, Gouveia LF, et al. Starch nanocapsules containing a novel neutrophil elastase inhibitor with improved pharmaceutical performance. Eur J Pharm Biopharm. 2018;127:1–11.

  30. 30.

    Simões A, Veiga F, Figueiras A, Vitorino C. A practical framework for implementing quality by design to the development of topical drug products: nanosystem-based dosage forms. Int J Pharm. 2018;548:385–99.

  31. 31.

    Faria M, Björnmalm M, Thurecht KJ, Kent SJ, Parton RG, Kavallaris M, et al. Minimum information reporting in bio–nano experimental literature. Nat Nanotechnol. 2018;13:777–85.

  32. 32.

    Vicente S, Peleteiro M, Díaz-Freitas B, Sanchez A, González-Fernández Á, Alonso MJ. Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: a needle-free vaccination strategy. J Control Release. 2013;172:773–81.

  33. 33.

    Correia-Pinto JF, Csaba N, Schiller J, Alonso MJ. Chitosan-poly (I:C)-PADRE based nanoparticles as delivery vehicles for synthetic peptide vaccines. Vaccines. 2015;3:730–50.

  34. 34.

    González-Aramundiz JV, Presas E, Dalmau-Mena I, Martínez-Pulgarín S, Alonso C, Escribano JM, et al. Rational design of protamine nanocapsules as antigen delivery carriers. J Control Release. 2017;245:62–9.

  35. 35.

    Crecente-Campo J, Lorenzo-Abalde S, Mora A, Marzoa J, Csaba N, Blanco J, et al. Bilayer polymeric nanocapsules: a formulation approach for a thermostable and adjuvanted E. coli antigen vaccine. J Control Release. 2018;286:20–32.

  36. 36.

    Li H, Nykoluk M, Li L, Liu LR, Omange RW, Soule G, et al. Natural and cross-inducible anti-SIV antibodies in Mauritian cynomolgus macaques. PLoS One. 2017;12:e0186079.

  37. 37.

    Dacoba TG, Omange RW, Li H, Crecente-Campo J, Luo M, Alonso MJ. Polysaccharide nanoparticles can efficiently modulate the immune response against an HIV peptide antigen. ACS Nano. 2019;13:4947–59.

  38. 38.

    Li H, Omange RW, Liang B, Toledo N, Hai Y, Liu LR, et al. bioRxiv. 2019.

  39. 39.

    Rathore AS, Winkle H. Quality by design for biopharmaceuticals. Nat Biotechnol. 2009;27:26–34.

  40. 40.

    European Medicines Agency. ICH guideline Q8 (R2) on pharmaceutical development [Internet]. 2017 [cited 2019 Dec 5]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/09/WC500002872.pdf.

  41. 41.

    Li H, Omange RW, Plummer FA, Luo M. A novel HIV vaccine targeting the protease cleavage sites. AIDS Res Ther. 2017;14:51.

  42. 42.

    England RJA, Homer JJ, Knight LC, Ell SR. Nasal pH measurement: a reliable and repeatable parameter. Clin Otolaryngol. 1999;24:67–8.

  43. 43.

    U.S. Food and Drug Administration. Guidance for industry: nasal spray and inhalation solution, suspension, and spray drug products — chemistry, manufacturing, and controls documentation [Internet]. 2002 [cited 2019 Dec 1]. Available from: https://www.fda.gov/media/70857/download.

  44. 44.

    May JC, Wheeler RM, Etz N, Del Grosso A. Measurement of final container residual moisture in freeze-dried biological products. Dev Biol Stand. 1992;74:153–64.

  45. 45.

    International Organization for Standardization. Particle size analysis — Dynamic light scattering (DLS) (ISO/DIS Standard No. 22412). 2017.

  46. 46.

    Hartig SM. Basic image analysis and manipulation in ImageJ. Curr Protoc Mol Biol. 2013;102:1–12.

  47. 47.

    Klein M, Menta M, Dacoba TG, Crecente-Campo J, Alonso MJ, Dupin D, et al. Advanced nanomedicine characterization by DLS and AF4-UV-MALS: application to a HIV nanovaccine. J Pharm Biomed Anal. 2020;179:113017.

  48. 48.

    Rapalli VK, Khosa A, Singhvi G, Girdhar V, Jain R, Dubey SK. Application of QbD principles in nanocarrier-based drug delivery systems. In: Beg S, Hasnain MS, editors. Pharm Qual by Des. Amsterdam: Elsevier; 2019. p. 255–96.

  49. 49.

    Cordeiro AS, Alonso MJ, de la Fuente M. Nanoengineering of vaccines using natural polysaccharides. Biotechnol Adv. 2015;33:1279–93.

  50. 50.

    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;28:2607–14.

  51. 51.

    Rose F, Wern JE, Gavins F, Andersen P, Follmann F, Foged C. A strong adjuvant based on glycol-chitosan-coated lipid-polymer hybrid nanoparticles potentiates mucosal immune responses against the recombinant Chlamydia trachomatis fusion antigen CTH522. J Control Release. 2018;271:88–97.

  52. 52.

    Sharma S, Mukkur TK, Benson HA, Chen Y. Enhanced immune response against pertussis toxoid by IgA-loaded chitosan–dextran sulfate nanoparticles. J Pharm Sci. 2012;101:233–44.

  53. 53.

    Correia-Pinto JF, Csaba N, Alonso MJ. Vaccine delivery carriers: insights and future perspectives. Int J Pharm. 2013;440:27–38.

  54. 54.

    Stano A, Nembrini C, Swartz MA, Hubbell JA, Simeoni E. Nanoparticle size influences the magnitude and quality of immune response after intranasal immunization. Vaccine. 2012;30:7541–6.

  55. 55.

    Kaur IP, Kakkar V, Deol PK, Yadav M, Singh M, Sharma I. Issues and concerns in nanotech product development and its commercialization. J Control Release. 2014;193:51–62.

  56. 56.

    Maguire CM, Rösslein M, Wick P, Prina-Mello A. Characterisation of particles in solution – a perspective on light scattering and comparative technologies. Sci Technol Adv Mater. 2018;19:732–45.

  57. 57.

    Caputo F, Clogston J, Calzolai L, Rösslein M, Prina-Mello A. Measuring particle size distribution of nanoparticle enabled medicinal products, the joint view of EUNCL and NCI-NCL. A step by step approach combining orthogonal measurements with increasing complexity. J Control Release. 2019;299:31–43.

  58. 58.

    European Nanomedicine Characterisation Laboratory. Measuring batch mode DLS [Internet]. 2016 [cited 2019 Dec 5]. Available from: http://www.euncl.eu/about-us/assay-cascade/PDFs/Prescreening/EUNCL-PCC-001.pdf?m=1468937875&.

  59. 59.

    Shang J, Gao X. Nanoparticle counting: towards accurate determination of the molar concentration. Chem Soc Rev. 2014;43:7267–78.

  60. 60.

    Lakkireddy HR, Bazile D. Building the design, translation and development principles of polymeric nanomedicines using the case of clinically advanced poly(lactide(glycolide))–poly(ethylene glycol) nanotechnology as a model: an industrial viewpoint. Adv Drug Deliv Rev. 2016;107:289–332.

  61. 61.

    European Medicines Agency. Questions and answers on the supply situation of Caelyx [Internet]. 2013 [cited 2019 Dec 5]. Available from: http://www.ema.europa.eu/docs/en_GB/document_library/Medicine_QA/2013/04/WC500142510.pdf.

  62. 62.

    Vetten MA, Yah CS, Singh T, Gulumian M. Challenges facing sterilization and depyrogenation of nanoparticles: effects on structural stability and biomedical applications. Nanomedicine. 2014;10:1391–9.

  63. 63.

    Tsukada Y, Hara K, Bando Y, Huang CC, Kousaka Y, Kawashima Y, et al. Particle size control of poly(DL-lactide-co-glycolide) nanospheres for sterile applications. Int J Pharm. 2009;370:196–201.

  64. 64.

    Masson V, Maurin F, Fessi H, Devissaguet JP. Influence of sterilization processes on poly(ε-caprolactone) nanospheres. Biomaterials. 1997;18:327–35.

  65. 65.

    U.S. Food and Drug Administration. Guidance for industry Q1A(R2) stability testing of new drug substances and products [Internet]. ICH Guidel. 2003 [cited 2019 Dec 5]. Available from: https://www.fda.gov/media/71707/download.

  66. 66.

    Szymańska E, Winnicka K. Stability of chitosan – a challenge for pharmaceutical and biomedical applications. Mar Drugs. 2015;13:1819–46.

  67. 67.

    Valencia PM, Farokhzad OC, Karnik R, Langer R. Microfluidic technologies for accelerating the clinical translation of nanoparticles. Nat Nanotechnol. 2012;7:623.

  68. 68.

    Stroock AD. Chaotic mixer for microchannels. Science. 2002;295:647–51.

  69. 69.

    Samaridou E, Walgrave H, Salta E, Álvarez DM, Castro-López V, Loza M, et al. Nose-to-brain delivery of enveloped RNA - cell permeating peptide nanocomplexes for the treatment of neurodegenerative diseases. Biomaterials. 2020;230:119657.

  70. 70.

    Roces CB, Christensen D, Perrie Y. Translating the fabrication of protein-loaded poly(lactic-co-glycolic acid) nanoparticles from bench to scale-independent production using microfluidics. Drug Deliv Transl Res. 2020.

  71. 71.

    Paliwal R, Babu RJ, Palakurthi S. Nanomedicine scale-up technologies: feasibilities and challenges. AAPS PharmSciTech. 2014;15:1527–34.

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Authors would like to thank the RIAIDT-USC analytical facilities, for the microscopy imaging. All the icons used in the graphical abstract were designed by Freepick at www.flaticon.com.


This work was supported by the European Union’s Horizon 2020 research program (NanoPilot project – grant agreement number 646142) and by Xunta de Galicia’s Grupos de referencia competitiva (grant number ED431C 2017/09). T.G. Dacoba acknowledges a predoctoral FPU grant from the Spanish Ministry of Education, Culture and Sports (grant number FPU14/05866).

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Correspondence to María J. Alonso or José Crecente-Campo.

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Dacoba, T.G., Ruiz-Gatón, L., Benito, A. et al. Technological challenges in the preclinical development of an HIV nanovaccine candidate. Drug Deliv. and Transl. Res. (2020). https://doi.org/10.1007/s13346-020-00721-8

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  • HIV
  • Quality-by-design
  • Scale-up
  • Microfluidics
  • Industrial translation
  • Nanoparticles