Pharmaceutical Research

, Volume 29, Issue 9, pp 2565–2577 | Cite as

Optimization of Stability, Encapsulation, Release, and Cross-Priming of Tumor Antigen-Containing PLGA Nanoparticles

  • Shashi Prasad
  • Virginia Cody
  • Jennifer K. Saucier-Sawyer
  • Tarek R. Fadel
  • Richard L. Edelson
  • Martin A. Birchall
  • Douglas J. Hanlon
Research Paper



In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP.


Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate.


Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate.


Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.

Key Words

antigen delivery dendritic cell immunotherapy molecular weight nanoparticles 





antigen presenting cell


American Type Culture Collection


bicinchonic acid


bone marrow dendritic cells


bovine serum albumin


cytometric bead array


cluster of differentiation


cytotoxic T cells


dendritic cell

FT + S

freeze-thaw + sonication


granulocyte macrophage-colony stimulating factor


head and neck squamous cell carcinoma










molecular weight




phosphate buffered saline


poly (lactic-co-glycolic acid)


polyvinyl alcohol


room temperature


sodium dodecyl sulfate polyacrylamide gel electrophoresis


scanning electron microscopy


Statistical Package for the Social Sciences


tumor associated antigen




tumor infiltrating lymphocytes


tumor necrosis factor-α


Western blot


Acknowledgments and Disclosures

The authors thank Prof Mark Saltzman and Dr Camille Solbrig in Biomedical Engineering for assistance and supply of reagents associated with NP encapsulation, Dr Michael Girardi and Kacie Carlson in Dermatology for their help in animal handling, and members of the Girardi and Cresswell laboratory for helpful discussions. We also thank Prof Sasaki and Shelley Jolie in Otolaryngology Head and Neck Surgery, Dr Diane Kowalski and Lori Patruno in Surgical Pathology, and members of the team of anaesthetists involved in care of patients recruited to this study. This work was partially funded by intra mural grant available to the Department of Dermatology, Yale University School of Medicine and partially by a NCI/NTRAC grant managed by Cancer Research UK. There is no perceived, potential or real conflict of interest.


  1. 1.
    Cruz LJ, Tacken PJ, Fokkink R, Joosten B, Stuart MC, Albericio F, et al. Targeted PLGA nano-but not microparticles specifically deliver antigen to human dendritic cells via DC-SIGN in vitro. J Control Rel. 118–126 (2010).Google Scholar
  2. 2.
    Thomas C, Gupta V, Ahsan F. Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses. Int J Pharm. 2009;379:41–50.PubMedCrossRefGoogle Scholar
  3. 3.
    Tel J, Lambeck AJA, Cruz LJ, Tacken PJ, de Vries IJM, Figdor CG. Human Plasmacytoid Dendritic Cells Phagocytose, Process, and Present Exogenous Particulate Antigen. J Immunol. 2010;184:4276–83.PubMedCrossRefGoogle Scholar
  4. 4.
    Tuncay M, Calis S, Kas HS, Ercan MT, Peksoy I, Hincal AA. Diclofenac sodium incorporated PLGA (50:50) microspheres: formulation considerations and in vitro/in vivo evaluation. Int J Pharm. 2000;195:179–88.PubMedCrossRefGoogle Scholar
  5. 5.
    Paolicelli P, Prego C, Sanchez A, Alonso MJ. Surface-modified PLGA-based nanoparticles that can efficiently associate and deliver virus-like particles. Nanomed. 2010;5:843–53.CrossRefGoogle Scholar
  6. 6.
    Rajapaksa TE, Lo DD. Microencapsulation of vaccine antigens and adjuvants for mucosal targeting. Curr Immunol Rev. 2010;6:29–37.CrossRefGoogle Scholar
  7. 7.
    Duan Y, Sun X, Gong T, Wang Q, Zhang Z. Preparation of DHAQ-loaded mPEG-PLGA-mPEG nanoparticles and evaluation of drug release behaviors in vitro/in vivo. J Mat Sci Mat Med. 2006;17:509–16.CrossRefGoogle Scholar
  8. 8.
    Astaneh R, Erfan M, Moghimi H, Mobedi H. Changes in morphology of in situ forming PLGA implant prepared by different polymer molecular weight and its effect on release behavior. J Pharm Sci. 2009;98:135–45.PubMedCrossRefGoogle Scholar
  9. 9.
    Fu X, Ping Q, Gao Y. Effects of formulation factors on encapsulation efficiency and release behaviour in vitro of huperzine A-PLGA microspheres. J Microencapsul. 2005;22:57–66.PubMedCrossRefGoogle Scholar
  10. 10.
    Murillo M, Grillo MJ, Rene J, Marin CM, Barberan M, Blasco JM, et al. A Brucella ovis antigenic complex bearing (poly-e-caprolactone) microparticles confer protection against experimental brucellosis in mice. Vaccine. 2001;19:4099–106.PubMedCrossRefGoogle Scholar
  11. 11.
    Ren JM, Zou QM, Wang FK, He Q, Chen W, Zen WK. PELA microspheres loaded H Pylori lysates and their mucosal immune response. World J Gastroenterol. 2002;8:1098–102.PubMedGoogle Scholar
  12. 12.
    Solbrig CM, Saucier-Sawyer JK, Cody V, Saltzman WM, Hanlon DJ. Polymer nanoparticles for immunotherapy from encapsulated tumor associated antigens and whole tumor cells. Mol Pharm. 2007;4:47–57.PubMedCrossRefGoogle Scholar
  13. 13.
    Jiang W, Gupta RK, Deshpande MC, Schwendeman SP. Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens. Adv Drug Deliv Rev. 2005;57:391–410.PubMedCrossRefGoogle Scholar
  14. 14.
    Li XY, Kong XY, Shi S, Zheng XL, Guo G, Wei YQ, et al. Preparation of alginate coated chitosan microparticles for vaccine delivery. BMC Biotechnol. 2008;8:89.PubMedCrossRefGoogle Scholar
  15. 15.
    Marazuela EG, Prado N, Moro E, Fernandez-Garcia H, Villalba M, Rodriguez R. Intranasal vaccination with poly (lactide-co-glycolide) microparticles containing a peptide of T of Ole 1 prevents mice against sensitization. Clin Exp Allerg. 2008;38:520–8.CrossRefGoogle Scholar
  16. 16.
    Zhou S, Liao X, Liang Z, Li X, Deng X, Li H. Preparation and characterization of biodegradable microspheres containing Hepatitis B surface antigen. Macromol Biosci. 2004;4:47–52.PubMedCrossRefGoogle Scholar
  17. 17.
    Costantino HR, Langer R, Kilbanov KM. Solid-phase aggregation of proteins under pharmaceutically relevant conditions. J Pharm Sci. 1994;83:1662–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Fu K, Kilbanov KM, Langer R. Protein instability in controlled-release systems. Nat Biotechnol. 2000;18:24–5.PubMedCrossRefGoogle Scholar
  19. 19.
    Jaganathan KS, Singh P, Prabhakaran D, Mishra V, Vyas SP. Development of a single-dose stabilized poly (D, L lactic-co-glycolic acid) microsphere-based vaccine against Hepatitis B. J Pharm Pharmacol. 2004;56:1243–50.PubMedCrossRefGoogle Scholar
  20. 20.
    Ganguli MJK, Maiti S. Nanoparticles from cationic coploymers and DNA that are soluble and stable in common organic solvents. J Am Chem Soc. 2004;126:26–7.PubMedCrossRefGoogle Scholar
  21. 21.
    Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med. 1992;176:1693–702.PubMedCrossRefGoogle Scholar
  22. 22.
    Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA, Vyth-Dreese FA, et al. Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells. J Exp Med. 2003;198:569–80.PubMedCrossRefGoogle Scholar
  23. 23.
    Johansen P, Gomez JMM, Gander B. Development of synthetic biodegradable microparticulate vaccines: a roller coaster story. Exp rev vaccines. 2007;6:471–4.CrossRefGoogle Scholar
  24. 24.
    Nestle FO, Farkas A, Conrad C. Dendritic-cell-based therapeutic vaccination against cancer. Curr Opinion Immunol. 2005;17:163–9.CrossRefGoogle Scholar
  25. 25.
    Johansen P, Men Y, Merkle HP, Gander B. Revisiting PLA/PLGA microspheres: an analysis of their potential in parenteral vaccination. Eur J Pharm and Biopharm. 2000;50:129–46.CrossRefGoogle Scholar
  26. 26.
    Sesardic D, Dobbelaer R. European union regulatory developments for new vaccine adjuvants and delivery systems. Vaccine. 2004;22:2452–6.PubMedCrossRefGoogle Scholar
  27. 27.
    Mittal G, Sahana DK, Bhardwaj V, Ravi MNV. Estradiol loaded PLGA nanoparticles for oral administration: effect of polymer molecular weight and copolymer composition on release behavior in vitro and in vivo. J Control Release. 2007;119:77–85.PubMedCrossRefGoogle Scholar
  28. 28.
    Mundargi RC, Babu VR, Rangaswamy V, Patel P, Aminabhavi TM. Nano/micro technologies for delivering macromolecular therapeutics using poly (d, l-lactide-co-glycolide) and its derivatives. J Control Release. 2008;125:193–209.PubMedCrossRefGoogle Scholar
  29. 29.
    Houchin M, Topp EM. Chemical degradation of peptides and proteins in PLGA: a review of reactions and mechanisms. J Pharm Sci. 2007;97:2395–404.CrossRefGoogle Scholar
  30. 30.
    Schwendeman SP. Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems. Crit Rev Ther Drug Carr Syst. 2002;19:73–98.CrossRefGoogle Scholar
  31. 31.
    Schwendeman SP, Costantino HR, Gupta RK, Tobio M, Chang AC, Alonso MJ, et al. Strategies for stabilizing tetanus toxoid toward the development of a single-does tetanus vaccine. Dev Biol Stand. 1996;87:293–306.PubMedGoogle Scholar
  32. 32.
    Kersten G, Donders D, Akkermans A, Beuvery EC. Single shot with tetanus toxoid in biodegradable microspheres protects mice despite acid-induced denaturation of the antigen. Vaccine. 1996;14:1627–32.PubMedCrossRefGoogle Scholar
  33. 33.
    Tamber H, Johansen P, Merkle HP, Gander B. Formulation as pects of biodegradable polymeric microspheres for antigen delivery. Adv Drug Deliv Rev. 2005;57:357–76.PubMedCrossRefGoogle Scholar
  34. 34.
    Jelvehgari M, Valizadeh H, Rezapour M, Nokhodchi A. Control of encapsulation efficiency in polymeric microparticle system of tolmetin. Pharm Dev Technol. 2010;15:71–9.PubMedCrossRefGoogle Scholar
  35. 35.
    Wang D, Robinson D, Kwona GS, Samuel J. Encapsulation of plasmid DNA in biodegradable poly(lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery. J Control Release. 1999;57:9–18.PubMedCrossRefGoogle Scholar
  36. 36.
    Fogeda C, Brodina B, Frokjaera S, Sundblad A. Particle size and surface charge affect particle uptake by human dendritic cells in an in vitro model. Int J Pharm. 2005;298:315–22.CrossRefGoogle Scholar
  37. 37.
    Jaraswekin S, Prakongpan S, Bodmeier R. Effect of poly(lactide-co-glycolide) molecular weight on the release of dexamethasone sodium phosphate from microparticles. J Microencapsul. 2007;24:117–28.PubMedCrossRefGoogle Scholar
  38. 38.
    Ravivarapu HB, Burton K, De Luca PP. Polymer and microsphere blending to alter the release of a peptide from PLGA microspheres. Eur J Pharm Biopharm. 2000;50:263–70.PubMedCrossRefGoogle Scholar
  39. 39.
    Mocellin S, Mandruzzato S, Zanovello P, Bronte V. Cancer rejection by the immune system: forcing the check-points of tumor immune escape. Drug Discov Today: Dis Mech. 2005;2:191–7.CrossRefGoogle Scholar
  40. 40.
    Bharali DJ, Mousa SA, Thanavala Y. Micro- and nanoparticle-based vaccines for hepatitis B. Adv Exp Med Biol. 2007;601:415–21.PubMedCrossRefGoogle Scholar
  41. 41.
    Elamanchili P, Lutsiak CME, Hamdy S, Diwan M, Samuel J. Pathogen-mimicking nanoparticles for vaccine delivery to dendritic cells. J Immunother. 2007;30:378–95.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Shashi Prasad
    • 1
  • Virginia Cody
    • 1
  • Jennifer K. Saucier-Sawyer
    • 2
  • Tarek R. Fadel
    • 2
  • Richard L. Edelson
    • 1
  • Martin A. Birchall
    • 3
  • Douglas J. Hanlon
    • 1
    • 4
  1. 1.Department of DermatologyYale UniversityNew HavenUSA
  2. 2.Department of Biomedical EngineeringYale UniversityNew HavenUSA
  3. 3.UCL Center for Stem Cells & Regenerative Medicine & UCL Ear InstituteRoyal National Throat Nose & Ear HospitalLondonUK
  4. 4.Yale UniversityNew HavenUSA

Personalised recommendations