Elsevier

Biomaterials

Volume 189, January 2019, Pages 48-59
Biomaterials

Gemcitabine nanoparticles promote antitumor immunity against melanoma

https://doi.org/10.1016/j.biomaterials.2018.10.022Get rights and content

Abstract

Myeloid-derived suppressor cells (MDSCs) promote tumor-mediated immunosuppression and cancer progression. Gemcitabine (Gem) is a MDSC-depleting chemotherapeutic agent; however, its clinical use is hampered by its drug resistance and inefficient in vivo delivery. Here we describe a strategy to formulate a Gem analogue gemcitabine monophosphate (GMP) into a lipid-coated calcium phosphate (LCP) nanoparticle, and investigate its antitumor immunity and therapeutic effects after systemic administrations. In the syngeneic mouse model of B16F10 melanoma, compared with free Gem, the LCP-formulated GMP (LCP-GMP) significantly induced apoptosis and reduced immunosuppression in the tumor microenvironment (TME). LCP-GMP effectively depleted MDSCs and regulatory T cells, and skewed macrophage polarization towards the antitumor M1 phenotype in the TME, leading to enhanced CD8+ T-cell immune response and profound tumor growth inhibition. Thus, engineering the in vivo delivery of MDSC-depleting agents using nanotechnology could substantially modulate immunosuppressive TME and boost T-cell immune response for enhanced antitumor efficacy.

Introduction

Among many pro-tumor mediators, the prominent presence of myeloid-derived suppressor cells (MDSCs), known as myeloid cells with potent suppressive activity, promotes tumor-mediated immunosuppression and correlates with reduced survival [1]. Two major subsets of MDSCs are monocytic MDSC (M-MDSC) and polymorphonuclear MDSC (PMN-MDSC). MDSCs in peripheral lymphoid organs are largely represented by PMN-MDSCs, which contribute to tumor-specific T-cell tolerance. In tumors, M-MDSCs are more prominent and have higher suppressive activity than PMN-MDSCs [2]. M-MDSCs differentiate to immune suppressive tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) [2,3], suggesting that targeting MDSCs may deliver superior antitumor therapeutic benefits. In addition, MDSCs promote the development and accumulation of immunosuppressive Foxp3+ regulatory T cells (Tregs) in tumors [3], which indirectly influences T-cell function and hampers antitumor immunity [[4], [5], [6]]. MDSCs regulate PD-L1 expression on tumor cells, decreasing antitumor immune responses mediated by T lymphocytes [7].

Chemotherapeutic agent gemcitabine (Gem), a cytidine nucleoside analogue, has been shown to inhibit MDSCs in tumor-bearing mice, leading to improved CD8+ T-cell antitumor activity accompanied by the inhibition of tumor growth [[8], [9], [10]]. However, repeated administrations of Gem cause drug resistance, presumably due to the dysfunction of nucleoside transporters required for the cellular uptake of Gem [11]. The Gem resistance may also result from the dysfunction of intracellular deoxycytidine kinase, which blocks the phosphorylation process of the prodrug Gem to its bioactive form gemcitabine triphosphate (GTP) [12]. In addition, due to rapid deamination, the elimination half-life of Gem is short, less than 30 min [13]. To address these clinically important issues associated with the drug resistance and short blood circulation time of Gem, we designed a lipid-coated calcium phosphate (LCP) nanoparticle platform to encapsulate Gem derivatives for improved pharmacokinetic profile and bioactivity. It is established that addition of the first phosphate group on Gem is the rate-limiting step to form GTP intracellularly [14]. We therefore encapsulated gemcitabine monophosphate (GMP) into LCP to promote the Gem-mediated MDSC modulation and antitumor activities. The LCP-formulated GMP (LCP-GMP) could bypass nucleoside transporters and enter into cells by endocytosis before efficient endosome release and sequential phosphorylation to its bioactive form GTP. This LCP nanocarrier could lead to prolonged blood circulation time of the encapsulated Gem derivatives compared with the unformulated drug [15].

In this study, we focused on an aggressive B16F10 melanoma model in which the suppressive leukocytes, MDSCs in particular, contribute to the creation of a highly immunosuppressive TME, leading to impaired antitumor immune responses and thereby enhancing tumor progression [16]. We evaluated the antitumor responses of LCP-GMP and free Gem on B16F10 tumor-bearing mice after repeated systemic administrations. We assessed the LCP-GMP-mediated modulations of immunosuppressive TME involving myeloid cells and tumor cells, and investigated the immunological effects of LCP-GMP against melanoma.

Section snippets

Materials

GMP disodium salt was synthesized by HDH Pharm Inc (Morrisville, NC). 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-distearoryl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol-2000) ammonium salt (DSPE-PEG2000) and 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (25-NBD cholesterol) were purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, cyclohexane and Igepal CO-520 were from

Preparation and characterization of nanoparticles

LCP was composed of a lipid-inorganic hybrid core-shell nanostructure. The phosphorylated drug GMP was precipitated in the calcium phosphate (Ca-P) nano-scaffold coated with a lipid bilayer, followed by grafting a high density of polyethylene glycol (PEG) chains. LCP-GMP was spherical in shape and monodispersed, with a particle size of ∼30 nm, as measured by TEM (Fig. 1A) and DLS (Fig. 1B). The zeta potential of LCP-GMP was around −20 mV, as measured by DLS. The encapsulation efficiency of GMP

Discussion

Gem is used to treat a number of types of cancer such as breast cancer, non-small cell lung cancer and pancreatic cancer. While it delivers reasonable efficacy, this chemotherapeutic agent suffers two major disposition barriers. In the plasma, Gem undergoes rapid deamination by cytidine deaminase and deamination represents inactivation. In addition, Gem is hydrophilic and its entrance into cells requires active transporters such as SLC28A1 and SLC29A1 [41]. After entering the cells, Gem

Conclusion

In the present study, we formulated chemotherapeutic and MDSC-depleting agent GMP into a lipid-coated calcium phosphate (LCP) nanoparticle. The Ca-P can act as a carrier scaffold for monophosphate metabolite of Gem through the formation of microprecipitates. This is of significance as many anti-cancer and anti-viral agents require intracellular phosphorylation for their therapeutic activity. We have shown that LCP-GMP can (1) trigger significant apoptosis in tumors; (2) largely deplete

Declaration of interest

No potential conflicts of interest were disclosed.

Author contributions

Y.Z. designed the experiments. Y.Z., X.B., J.A.C. performed the experiments. Y.Z., J.A.C., X.B., B.Y. analyzed the data. Y.Z. supervised the studies and wrote the manuscript.

Data availability

All relevant data are available from the authors on request and are included within the manuscript.

Acknowledgements

This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) Advance-CTR pilot grant (U54GM115677), Rhode Island Foundation – Medical Research Fund (#20164344) to Y.Z. as well as R01GM61988 and R01ES07965 to B.Y. We would like to thank URI Institute for Immunology and Informatics (iCubed) Cell Analysis and Sorting Core supported by COBRE grant NIH P20 GM104317, Lifespan Molecular Pathology Core, URI Genomics and Sequencing Center.

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