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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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.
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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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
- phase separation