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Formulation and In Vitro Characterization of PLGA/PLGA-PEG Nanoparticles Loaded with Murine Granulocyte-Macrophage Colony-Stimulating Factor

AAPS PharmSciTech volume 22, Article number: 191 (2021) Cite this article

Abstract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated notable clinical activity in cancer immunotherapy, but it is limited by systemic toxicities, poor bioavailability, rapid clearance, and instability in vivo. Nanoparticles (NPs) may overcome these limitations and provide a mechanism for passive targeting of tumors. This study aimed to develop GM-CSF-loaded PLGA/PLGA-PEG NPs and evaluate them in vitro as a potential candidate for in vivo administration. NPs were created by a phase-separation technique that did not require toxic/protein-denaturing solvents or harsh agitation techniques and encapsulated GM-CSF in a more stable precipitated form. NP sizes were within 200 nm for enhanced permeability and retention (EPR) effect with negative zeta potentials, spherical morphology, and high entrapment efficiencies. The optimal formulation was identified by sustained release of approximately 70% of loaded GM-CSF over 24 h, alongside an average size of 143 ± 35 nm and entrapment efficiency of 84 ± 5%. These NPs were successfully freeze-dried in 5% (w/v) hydroxypropyl-β-cyclodextrin for long-term storage and further characterized. Bioactivity of released GM-CSF was determined by observing GM-CSF receptor activation on murine monocytes and remained fully intact. NPs were not cytotoxic to murine bone marrow-derived macrophages (BMDMs) at concentrations up to 1 mg/mL as determined by MTT and trypan blue exclusion assays. Lastly, NP components generated no significant transcription of inflammation-regulating genes from BMDMs compared to IFNγ+LPS “M1” controls. This report lays the preliminary groundwork to validate in vivo studies with GM-CSF-loaded PLGA/PEG-PLGA NPs for tumor immunomodulation. Overall, these data suggest that in vivo delivery will be well tolerated.

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References

  1. 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(6):1693–702. https://doi.org/10.1084/jem.176.6.1693.

    Article  CAS  PubMed  Google Scholar 

  2. Metcalf D. Cell-cell signalling in the regulation of blood cell formation and function. Immunol Cell Biol. 1998;76(5):441–7. https://doi.org/10.1046/j.1440-1711.1998.00761.x.

    Article  CAS  PubMed  Google Scholar 

  3. Arellano M, Lonial S. Clinical uses of GM-CSF, a critical appraisal and update. Biologics. 2008;2(1):13–27.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Sanofi Pharmaceuticals. Sargramostim: Package Insert. U.S. Food and Drug Administration. 2017. https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/103362s5237lbl.pdf. Accessed 20 Nov 2020.

  5. Burgess AW, Metcalf D. The nature and action of granulocyte-macrophage colony stimulating factors. Blood. 1980;56(6):947–58.

    Article  CAS  Google Scholar 

  6. Metcalf D. The granulocyte-macrophage colony-stimulating factors. Science. 1985;229(4708):16–22. https://doi.org/10.1126/science.2990035.

    Article  CAS  PubMed  Google Scholar 

  7. Zhan Y, Lew AM, Chopin M. The pleiotropic effects of the GM-CSF rheostat on myeloid cell differentiation and function: more than a numbers game. Front Immunol. 2019;10:2679. https://doi.org/10.3389/fimmu.2019.02679.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. R OD. An update on GM-CSF and its potential role in melanoma management. Melanoma Manag. 2020;7(3):MMT49. https://doi.org/10.2217/mmt-2020-0011.

    Article  Google Scholar 

  9. Kaufman HL, Ruby CE, Hughes T, Slingluff CL Jr. Current status of granulocyte-macrophage colony-stimulating factor in the immunotherapy of melanoma. J Immunother Cancer. 2014;2:11. https://doi.org/10.1186/2051-1426-2-11.

  10. Hoeller C, Michielin O, Ascierto PA, Szabo Z, Blank CU. Systematic review of the use of granulocyte-macrophage colony-stimulating factor in patients with advanced melanoma. Cancer Immunol Immunother. 2016;65(9):1015–34. https://doi.org/10.1007/s00262-016-1860-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Quezada SA, Peggs KS, Curran MA, Allison JP. CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells. J Clin Invest. 2006;116(7):1935–45. https://doi.org/10.1172/JCI27745.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Seidel JA, Otsuka A, Kabashima K. Anti-PD-1 and anti-CTLA-4 therapies in cancer: mechanisms of action, efficacy, and limitations. Front Oncol. 2018;8(86). https://doi.org/10.3389/fonc.2018.00086.

  13. Luke JJ, Donahue H, Nishino M, Giobbie-Hurder A, Davis M, Bailey N, et al. Single institution experience of ipilimumab 3 mg/kg with sargramostim (GM-CSF) in metastatic melanoma. Cancer Immunol Res. 2015;3(9):986–91. https://doi.org/10.1158/2326-6066.CIR-15-0066.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Li B, VanRoey M, Wang C, Chen T-hT, Korman A, Jooss K. Anti–programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor–secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors. Clin Cancer Res. 2009;15(5):1623. https://doi.org/10.1158/1078-0432.CCR-08-1825.

    Article  CAS  PubMed  Google Scholar 

  15. Pisal DS, Kosloski MP, Balu-Iyer SV. Delivery of therapeutic proteins. J Pharm Sci. 2010;99(6):2557–75. https://doi.org/10.1002/jps.22054.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Greish K. Enhanced permeability and retention (EPR) effect for anticancer nanomedicine drug targeting. Methods Mol Biol. 2010;624:25–37. https://doi.org/10.1007/978-1-60761-609-2_3.

    Article  CAS  PubMed  Google Scholar 

  17. Soundararajan A, Bao A, Phillips WT, Perez R 3rd, Goins BA. [(186)Re]Liposomal doxorubicin (Doxil): in vitro stability, pharmacokinetics, imaging and biodistribution in a head and neck squamous cell carcinoma xenograft model. Nucl Med Biol. 2009;36(5):515–24. https://doi.org/10.1016/j.nucmedbio.2009.02.004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gabizon A, Shmeeda H, Barenholz Y. Pharmacokinetics of pegylated liposomal doxorubicin: review of animal and human studies. Clin Pharmacokinet. 2003;42(5):419–36. https://doi.org/10.2165/00003088-200342050-00002.

    Article  CAS  PubMed  Google Scholar 

  19. Cohen-Sela E, Chorny M, Koroukhov N, Danenberg HD, Golomb G. A new double emulsion solvent diffusion technique for encapsulating hydrophilic molecules in PLGA nanoparticles. J Control Release. 2009;133(2):90–5. https://doi.org/10.1016/j.jconrel.2008.09.073.

    Article  CAS  PubMed  Google Scholar 

  20. Grabowski N, Hillaireau H, Vergnaud J, Tsapis N, Pallardy M, Kerdine-Romer S, et al. Surface coating mediates the toxicity of polymeric nanoparticles towards human-like macrophages. Int J Pharm. 2015;482(1-2):75–83. https://doi.org/10.1016/j.ijpharm.2014.11.042.

    Article  CAS  PubMed  Google Scholar 

  21. Dudeck O, Jordan O, Hoffmann KT, Okuducu AF, Tesmer K, Kreuzer-Nagy T, et al. Organic solvents as vehicles for precipitating liquid embolics: a comparative angiotoxicity study with superselective injections of swine rete mirabile. AJNR. 2006;27(9):1900–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Bilati U, Allémann E, Doelker E. Strategic approaches for overcoming peptide and protein instability within biodegradable nano- and microparticles. Eur J Pharm Biopharm. 2005;59(3):375–88. https://doi.org/10.1016/j.ejpb.2004.10.006.

    Article  CAS  PubMed  Google Scholar 

  23. Haji Mansor M, Najberg M, Contini A, Alvarez-Lorenzo C, Garcion E, Jerome C, et al. Development of a non-toxic and non-denaturing formulation process for encapsulation of SDF-1a into PLGA/PEG-PLGA nanoparticles to achieve sustained release. Eur J Pharm Biopharm. 2018;125:38–50.

    Article  CAS  Google Scholar 

  24. Giteau A, Venier-Julienne MC, Marchal S, Courthaudon JL, Sergent M, Montero-Menei C, et al. Reversible protein precipitation to ensure stability during encapsulation within PLGA microspheres. Eur J Pharm Biopharm. 2008;70(1):127–36. https://doi.org/10.1016/j.ejpb.2008.03.006.

    Article  CAS  PubMed  Google Scholar 

  25. Lehtonen A, Matikainen S, Miettinen M, Julkunen I. Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation. J Leukoc Biol. 2002;71(3):511–9.

    CAS  PubMed  Google Scholar 

  26. Eubank TD, Roberts RD, Khan M, Curry JM, Nuovo GJ, Kuppusamy P, et al. Granulocyte macrophage colony-stimulating factor inhibits breast cancer growth and metastasis by invoking an anti-angiogenic program in tumor-educated macrophages. Cancer Res. 2009;69(5):2133–40. https://doi.org/10.1158/0008-5472.Can-08-1405.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Sharma N, Madan P, Lin S. Effect of process and formulation variables on the preparation of parenteral paclitaxel-loaded biodegradable polymeric nanoparticles: a co-surfactant study. Asian J Pharm Sci. 2016;11(3):404–16. https://doi.org/10.1016/j.ajps.2015.09.004.

    Article  Google Scholar 

  28. Krishnamachari Y, Madan P, Lin S. Development of pH- and time-dependent oral microparticles to optimize budesonide delivery to ileum and colon. Int J Pharm. 2007;338(1):238–47. https://doi.org/10.1016/j.ijpharm.2007.02.015.

    Article  CAS  PubMed  Google Scholar 

  29. Huang W, Tsui CP, Tang CY, Gu L. Effects of compositional tailoring on drug delivery behaviours of silica xerogel/polymer core-shell composite nanoparticles. Sci Rep. 2018;8(1):13002. https://doi.org/10.1038/s41598-018-31070-9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Abdelwahed W, Degobert G, Fessi H. Investigation of nanocapsules stabilization by amorphous excipients during freeze-drying and storage. Eur J Pharm Biopharm. 2006;63(2):87–94. https://doi.org/10.1016/j.ejpb.2006.01.015.

    Article  CAS  PubMed  Google Scholar 

  31. Vega E, Egea MA, Calpena AC, Espina M, García ML. Role of hydroxypropyl-β-cyclodextrin on freeze-dried and gamma-irradiated PLGA and PLGA-PEG diblock copolymer nanospheres for ophthalmic flurbiprofen delivery. Int J Nanomedicine. 2012;7:1357–71. https://doi.org/10.2147/ijn.S28481.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev. 2006;58(15):1688–713. https://doi.org/10.1016/j.addr.2006.09.017.

    Article  CAS  PubMed  Google Scholar 

  33. Hamilton JA. GM-CSF-dependent inflammatory pathways. Front Immunol. 2019;10(2055). https://doi.org/10.3389/fimmu.2019.02055.

  34. Na YR, Gu GJ, Jung D, Kim YW, Na J, Woo JS, et al. GM-CSF induces inflammatory macrophages by regulating glycolysis and lipid metabolism. J Immunol. 2016;197(10):4101–9. https://doi.org/10.4049/jimmunol.1600745.

    Article  CAS  PubMed  Google Scholar 

  35. ISO 10993-5:2009 biological evaluation of medical devices. Part 5: tests for in vitro cytotoxicity. International Organization for Standardization; Geneva Shwio.

  36. Pettit DK LJ, Huang WJ, Pankey SC, Nightlinger NS, Lynch DH, Schuh JA, Morrissey PJ, Gombotz WR. Characterization of poly(glycolide-co-D,L-lactide/poly(D,L-glycolide) microspheres for controlled release of GM-CSF. Pharm Res 1997;14(10):1422–30

  37. Hill HC, Conway TF, Sabel MS, Jong YS, Mathiowitz E, Bankert RB, et al. Cancer immunotherapy with interleukin 12 and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres. Cancer Res. 2002;62(24):7254–63.

    CAS  PubMed  Google Scholar 

  38. Vanitha S, Goswami U, Chaubey N, Ghosh SS, Sanpui P. Functional characterization of recombinant human granulocyte colony stimulating factor (hGMCSF) immobilized onto silica nanoparticles. Biotechnol Lett. 2016;38(2):243–9. https://doi.org/10.1007/s10529-015-1984-0.

    Article  CAS  PubMed  Google Scholar 

  39. Anderson PM, Hanson DC, Hasz DE, Halet MR, Blazar BR, Ochoa AC. Cytokines in liposomes: preliminary studies with IL-1, IL-2, IL-6, GM-CSF and interferon-gamma. Cytokine. 1994;6(1):92–101.

    Article  CAS  Google Scholar 

  40. Kedar E, Palgi O, Golod G, Babai I, Barenholz Y. Delivery of cytokines by liposomes. III. Liposome-encapsulated GM-CSF and TNF-alpha show improved pharmacokinetics and biological activity and reduced toxicity in mice. J Immunother. 1997;20(3):180–93. https://doi.org/10.1097/00002371-199705000-00003.

    Article  CAS  PubMed  Google Scholar 

  41. Babai I, Barenholz Y, Zakay-Rones Z, Greenbaum E, Samira S, Hayon I, et al. A novel liposomal influenza vaccine (INFLUSOME-VAC) containing hemagglutinin-neuraminidase and IL-2 or GM-CSF induces protective anti-neuraminidase antibodies cross-reacting with a wide spectrum of influenza A viral strains. Vaccine. 2001;20(3-4):505–15. https://doi.org/10.1016/s0264-410x(01)00326-7.

    Article  CAS  PubMed  Google Scholar 

  42. Duong HTT, Thambi T, Yin Y, Kim SH, Nguyen TL, Phan VHG, et al. Degradation-regulated architecture of injectable smart hydrogels enhances humoral immune response and potentiates antitumor activity in human lung carcinoma. Biomaterials. 2020;230:119599. https://doi.org/10.1016/j.biomaterials.2019.119599.

    Article  CAS  PubMed  Google Scholar 

  43. Mukherjee BSK, Pattnaik G, Ghosh S. Preparation, characterization and in-vitro evaluation of sustained release protein-loaded nanoparticles based on biodegradable polymers. Int J Nanomedicine. 2008;3(4):487–96.

    Article  CAS  Google Scholar 

  44. Feczkó T, Tóth J, Dósa G, Gyenis J. Optimization of protein encapsulation in PLGA nanoparticles. Chem Eng Process. 2011;50(8):757–65. https://doi.org/10.1016/j.cep.2011.06.008.

    Article  CAS  Google Scholar 

  45. Jiang X, Lin H, Jiang D, Xu G, Fang X, He L, et al. Co-delivery of VEGF and bFGF via a PLGA nanoparticle-modified BAM for effective contracture inhibition of regenerated bladder tissue in rabbits. Sci Rep. 2016;6(1):20784. https://doi.org/10.1038/srep20784.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. European Medicines Agency. International Conference on Harmonization (ICH) guidelines Q3C (R6) on impurities: guidelines for residual solvents. 2019. https://www.ema.europa.eu/en/documents/scientific-guideline/international-conference-harmonisation-technical-requirements-registration-pharmaceuticals-human-use_en-33.pdf. Accessed 22 Nov 2020.

  47. Yang A, Yang L, Liu W, Li Z, Xu H, Yang X. Tumor necrosis factor alpha blocking peptide loaded PEG-PLGA nanoparticles: preparation and in vitro evaluation. Int J Pharm. 2007;331(1):123–32. https://doi.org/10.1016/j.ijpharm.2006.09.015.

    Article  CAS  PubMed  Google Scholar 

  48. Coleman J, Lowman A. Biodegradable nanoparticles for protein delivery: analysis of preparation conditions on particle morphology and protein loading, activity and sustained release properties. J Biomater Sci Polym Ed. 2012;23(9):1129–51. https://doi.org/10.1163/092050611x576648.

    Article  CAS  PubMed  Google Scholar 

  49. Bilati U, Allémann E, Doelker E. Development of a nanoprecipitation method intended for the entrapment of hydrophilic drugs into nanoparticles. Eur J Pharm Sci. 2005;24(1):67–75. https://doi.org/10.1016/j.ejps.2004.09.011.

    Article  CAS  PubMed  Google Scholar 

  50. Swed ACT, Fleury F, Boury F. Protein encapsulation into PLGA nanoparticles by a novel phase separation method using non-toxic solvents. J Nanomed Nanotechnol. 2014;5(6):241.

    Google Scholar 

  51. Boongird A, Nasongkla N, Hongeng S, Sukdawong N, Sa-Nguanruang W, Larbcharoensub N. Biocompatibility study of glycofurol in rat brains. Exp Biol Med. 2011;236(1):77–83. https://doi.org/10.1258/ebm.2010.010219.

    Article  CAS  Google Scholar 

  52. Morille M, Van-Thanh T, Garric X, Cayon J, Coudane J, Noël D, et al. New PLGA-P188-PLGA matrix enhances TGF-β3 release from pharmacologically active microcarriers and promotes chondrogenesis of mesenchymal stem cells. J Control Release. 2013;170(1):99–110. https://doi.org/10.1016/j.jconrel.2013.04.017.

    Article  CAS  PubMed  Google Scholar 

  53. Swed A, Cordonnier T, Dénarnaud A, Boyer C, Guicheux J, Weiss P, et al. Sustained release of TGF-β1 from biodegradable microparticles prepared by a new green process in CO2 medium. Int J Pharm. 2015;493(1-2):357–65. https://doi.org/10.1016/j.ijpharm.2015.07.043.

    Article  CAS  PubMed  Google Scholar 

  54. Kandalam S, Sindji L, Delcroix GJ, Violet F, Garric X, André EM, et al. Pharmacologically active microcarriers delivering BDNF within a hydrogel: novel strategy for human bone marrow-derived stem cells neural/neuronal differentiation guidance and therapeutic secretome enhancement. Acta Biomater. 2017;49:167–80. https://doi.org/10.1016/j.actbio.2016.11.030.

    Article  CAS  PubMed  Google Scholar 

  55. Filipe V, Hawe A, Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by nanosight for the measurement of nanoparticles and protein aggregates. Pharm Res. 2010;27(5):796–810. https://doi.org/10.1007/s11095-010-0073-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Gross J, Sayle S, Karow AR, Bakowsky U, Garidel P. Nanoparticle tracking analysis of particle size and concentration detection in suspensions of polymer and protein samples: Influence of experimental and data evaluation parameters. Eur J Pharm Biopharm. 2016;104:30–41. https://doi.org/10.1016/j.ejpb.2016.04.013.

    Article  CAS  PubMed  Google Scholar 

  57. Haggag Y, Abdel-Wahab Y, Ojo O, Osman M, El-Gizawy S, El-Tanani M, et al. Preparation and in vivo evaluation of insulin-loaded biodegradable nanoparticles prepared from diblock copolymers of PLGA and PEG. Int J Pharm. 2016;499(1-2):236–46. https://doi.org/10.1016/j.ijpharm.2015.12.063.

    Article  CAS  PubMed  Google Scholar 

  58. Beletsi A, Panagi Z, Avgoustakis K. Biodistribution properties of nanoparticles based on mixtures of PLGA with PLGA–PEG diblock copolymers. Int J Pharm. 2005;298(1):233–41. https://doi.org/10.1016/j.ijpharm.2005.03.024.

    Article  CAS  PubMed  Google Scholar 

  59. Wei Q, Wei W, Tian R, Wang L-y, Su Z-G, Ma G-H. Preparation of uniform-sized PELA microspheres with high encapsulation efficiency of antigen by premix membrane emulsification. J Colloid Interface Sci. 2008;323(2):267–73. https://doi.org/10.1016/j.jcis.2008.04.058.

    Article  CAS  PubMed  Google Scholar 

  60. Santander-Ortega MJ, Csaba N, González L, Bastos-González D, Ortega-Vinuesa JL, Alonso MJ. Protein-loaded PLGA–PEO blend nanoparticles: encapsulation, release and degradation characteristics. Colloid Polym Sci. 2010;288(2):141–50. https://doi.org/10.1007/s00396-009-2131-z.

    Article  CAS  Google Scholar 

  61. Govender T, Stolnik S, Garnett MC, Illum L, Davis SS. PLGA nanoparticles prepared by nanoprecipitation: drug loading and release studies of a water soluble drug. J Control Release. 1999;57(2):171–85. https://doi.org/10.1016/s0168-3659(98)00116-3.

    Article  CAS  PubMed  Google Scholar 

  62. Tran MK, Swed A, Boury F. Preparation of polymeric particles in CO(2) medium using non-toxic solvents: formulation and comparisons with a phase separation method. Eur J Pharm Biopharm. 2012;82(3):498–507. https://doi.org/10.1016/j.ejpb.2012.08.005.

    Article  CAS  PubMed  Google Scholar 

  63. Swed A, Cordonnier T, Denarnaud A, Boyer C, Guicheux J, Weiss P, et al. Sustained release of TGF-beta1 from biodegradable microparticles prepared by a new green process in CO2 medium. Int J Pharm. 2015;493(1-2):357–65. https://doi.org/10.1016/j.ijpharm.2015.07.043.

    Article  CAS  PubMed  Google Scholar 

  64. White LJ, Kirby GTS, Cox HC, Qodratnama R, Qutachi O, Rose FRAJ, et al. Accelerating protein release from microparticles for regenerative medicine applications. Mater Sci Eng. 2013;33(5):2578–83. https://doi.org/10.1016/j.msec.2013.02.020.

    Article  CAS  Google Scholar 

  65. Li Y, Pei Y, Zhang X, Gu Z, Zhou Z, Yuan W, et al. PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats. J Control Release. 2001;71(2):203–11. https://doi.org/10.1016/s0168-3659(01)00218-8.

    Article  CAS  PubMed  Google Scholar 

  66. Giteau A, Venier-Julienne MC, Aubert-Pouëssel A, Benoit JP. How to achieve sustained and complete protein release from PLGA-based microparticles? Int J Pharm. 2008;350(1-2):14–26. https://doi.org/10.1016/j.ijpharm.2007.11.012.

    Article  CAS  PubMed  Google Scholar 

  67. Pakulska MM, Elliott Donaghue I, Obermeyer JM, Tuladhar A, McLaughlin CK, Shendruk TN, et al. Encapsulation-free controlled release: electrostatic adsorption eliminates the need for protein encapsulation in PLGA nanoparticles. Sci Adv. 2016;2(5):e1600519. https://doi.org/10.1126/sciadv.1600519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Crotts G, Sah H, Park TG. Adsorption determines in-vitro protein release rate from biodegradable microspheres: quantitative analysis of surface area during degradation. J Control Release. 1997;47(1):101–11. https://doi.org/10.1016/S0168-3659(96)01624-0.

    Article  CAS  Google Scholar 

  69. Wei Y, Wang YX, Wang W, Ho SV, Qi F, Ma GH, et al. Microcosmic mechanisms for protein incomplete release and stability of various amphiphilic mPEG-PLA microspheres. Langmuir. 2012;28(39):13984–92. https://doi.org/10.1021/la3017112.

    Article  CAS  PubMed  Google Scholar 

  70. Buske J, König C, Bassarab S, Lamprecht A, Mühlau S, Wagner KG. Influence of PEG in PEG–PLGA microspheres on particle properties and protein release. Eur J Pharm Biopharm. 2012;81(1):57–63. https://doi.org/10.1016/j.ejpb.2012.01.009.

    Article  CAS  PubMed  Google Scholar 

  71. Ratanji KD, Derrick JP, Dearman RJ, Kimber I. Immunogenicity of therapeutic proteins: influence of aggregation. J Immunotoxicol. 2014;11(2):99–109. https://doi.org/10.3109/1547691x.2013.821564.

    Article  CAS  PubMed  Google Scholar 

  72. Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2016;99(Pt A):28–51. https://doi.org/10.1016/j.addr.2015.09.012.

    Article  CAS  PubMed  Google Scholar 

  73. Klibanov AL, Maruyama K, Torchilin VP, Huang L. Amphipathic polyethyleneglycols effectively prolong the circulation time of liposomes. FEBS Lett. 1990;268(1):235–7. https://doi.org/10.1016/0014-5793(90)81016-h.

    Article  CAS  PubMed  Google Scholar 

  74. Mori A, Klibanov AL, Torchilin VP, Huang L. Influence of the steric barrier activity of amphipathic poly(ethyleneglycol) and ganglioside GM1 on the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett. 1991;284(2):263–6. https://doi.org/10.1016/0014-5793(91)80699-4.

    Article  CAS  PubMed  Google Scholar 

  75. Gref R, Lück M, Quellec P, Marchand M, Dellacherie E, Harnisch S, et al. 'Stealth' corona-core nanoparticles surface modified by polyethylene glycol (PEG): influences of the corona (PEG chain length and surface density) and of the core composition on phagocytic uptake and plasma protein adsorption. Colloids Surf B: Biointerfaces. 2000;18(3-4):301–13. https://doi.org/10.1016/s0927-7765(99)00156-3.

    Article  CAS  PubMed  Google Scholar 

  76. Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol. 2015;33(9):941–51. https://doi.org/10.1038/nbt.3330.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Maupas C, Moulari B, Béduneau A, Lamprecht A, Pellequer Y. Surfactant dependent toxicity of lipid nanocapsules in HaCaT cells. Int J Pharm. 2011;411(1):136–41. https://doi.org/10.1016/j.ijpharm.2011.03.056.

    Article  CAS  PubMed  Google Scholar 

  78. Le Roux G, Moche H, Nieto A, Benoit J-P, Nesslany F, Lagarce F. Cytotoxicity and genotoxicity of lipid nanocapsules. Toxicol in Vitro. 2017;41:189–99. https://doi.org/10.1016/j.tiv.2017.03.007.

    Article  CAS  PubMed  Google Scholar 

  79. Pallardy MJ, Turbica I, Biola-Vidamment A. Why the immune system should be concerned by nanomaterials? Front Immunol. 2017;8(544). https://doi.org/10.3389/fimmu.2017.00544.

  80. Składanowski M, Golinska P, Rudnicka K, Dahm H, Rai M. Evaluation of cytotoxicity, immune compatibility and antibacterial activity of biogenic silver nanoparticles. Med Microbiol Immunol. 2016;205(6):603–13. https://doi.org/10.1007/s00430-016-0477-7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was also supported by WVU Flow Cytometry and Single Cell Core and the following grants: TME CoBRE GM121322, S10 equipment grant #OD016165, Stroke CoBRE GM109098, and WV-CTSI grant #GM103434. NM is supported by Cell & Molecular Biology and Biomedical Engineering (CBTP) National Institute of General Medical Sciences (NIGMS) T32 training grant (T32GM133369). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We acknowledge the use of the WVU Shared Research Facilities and thank Dr. Marcela Redigolo for her collaboration in obtaining SEM images. We also acknowledge Tasneem Arsiwala and Dr. Marieta Gencheva for insightful discussions, and Kelly Monaghan for assistance with the pSTAT5 staining protocol.

Funding

This work was supported by NIH Grants (USA): R01CA194013 and R01CA192064 (to TDE), R00EB023990 (to BD), R21EB02855301A1 (to BD), WVCTSI Grant U54GM104942 (West Virginia State Startup Funds to TDE), WVCTSI/WVCI Open Award (to TDE), and P20GM103434 (WV-INBRE).

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Authors and Affiliations

  1. Department of Microbiology, Immunology, and Cell Biology, West Virginia University, PO Box 9177, Morgantown, West Virginia, 26506-9177, USA

    Nicole E. Mihalik & Timothy D. Eubank

  2. West Virginia University Cancer Institute, PO Box 9530, Morgantown, West Virginia, 26506, USA

    Nicole E. Mihalik, Sijin Wen, Benoit Driesschaert & Timothy D. Eubank

  3. West Virginia Clinical and Translational Science Institute, Morgantown, West Virginia, 26506, USA

    Sijin Wen, Benoit Driesschaert & Timothy D. Eubank

  4. West Virginia University School of Public Health Department of Biostatistics, Morgantown, West Virginia, 26506, USA

    Sijin Wen

  5. Department of Pharmaceutical Sciences, West Virginia University, Morgantown, West Virginia, 26506, USA

    Benoit Driesschaert

  6. In Vivo Multifunctional Magnetic Resonance (IMMR) center, Morgantown, West Virginia, 26506, USA

    Benoit Driesschaert & Timothy D. Eubank

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  1. Nicole E. Mihalik
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  2. Sijin Wen
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Correspondence to Benoit Driesschaert or Timothy D. Eubank.

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Mihalik, N.E., Wen, S., Driesschaert, B. et al. Formulation and In Vitro Characterization of PLGA/PLGA-PEG Nanoparticles Loaded with Murine Granulocyte-Macrophage Colony-Stimulating Factor. AAPS PharmSciTech 22, 191 (2021). https://doi.org/10.1208/s12249-021-02049-z

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KEY WORDS

  • PLGA-PEG
  • GM-CSF
  • nanoparticles
  • phase separation
  • macrophages