Biomimetic Small-Molecule Self-Assembly of PI3K inhibitor integrated with immunomodulator to amplify anticancer efficacy

https://doi.org/10.1016/j.cej.2021.133747Get rights and content

Highlights

  • Combine kinase-inhibiting therapeutics with immunotherapy to amplify anticancer efficacy.

  • Carrier-free nanoparticles based on PI3K inhibitor ZSTK were prepared.

  • Hybridized biomimetic nanoparticles displayed specific targeting ability and significant anticancer efficacy.

  • Nanosystem is designed to target macrophage in the microenvironment.

Abstract

Overactivation of PI3K signaling has been identified in prostate cancer. ZSTK is an effective pan-PI3K inhibitor, but it was withdrawn from early-phase clinical trials because of its poor solubility, on-target/off-tumor toxicity, and recruitment and polarization of tumor-associated macrophages (TAMs). Indomethacin (IND) reduces the polarization of TAMs, increases the proinflammatory macrophage (M1 phenotype) ratio in the tumor microenvironment, and enhances the immune response in tumor cells. Herein, we developed ZSTK carrier-free nanoparticle (ZNP)-loaded biomimetic hybrid indomethacin liposomes (I@Lip) with PC3 cell membranes (termed ZNPs/I@CML). ZNPs/I@CML displayed amplified synergistic therapeutic effects on zebrafish and nude mouse PC3 xenograft models. Their homologous targeting ability can deliver ZSTK to tumor tissue and elicit effective antiproliferative effects on PC3 cancer cells. Meanwhile, the released IND improved the immunosuppressive microenvironment, inducing immunological activation against cancer cells. Additionally, ZNPs/I@CML did not cause liver toxicity compared with other treatment groups, as shown by histological analysis, suggesting their improved safety. Our data demonstrate that biomimetic hybrid liposome-coated carrier-free nanoparticles are a promising nanoplatform for amplifying the antitumor efficacy of kinase-inhibiting therapeutics with immunotherapy, but more preclinical and clinical evidence is required.

Introduction

Prostate cancer is the most common cancer diagnosed in men in 2021[1]. Overactivation of phosphatidylinositol 3-kinase (PI3K) signaling has been identified in multiple malignancies, including prostate cancer. The PI3K pathway is altered in approximately 50% of metastatic castration-resistant prostate cancers (mCRPCs) and 20–40% of primary prostate cancers [2], [3]. Multiple PI3K inhibitors have been investigated to assess their therapeutic potential in various malignancies, including prostate cancer [4].

ZSTK474 (ZSTK), a PI3K inhibitor that we identified using the JFCR39 (Japanese Foundation for Cancer Research 39) drug discovery system, is under clinical evaluation in patients with advanced solid malignancies [5], [6], [7]. However, it was withdrawn from early-phase clinical trials because of its poor therapeutic response and resistance as well as on-target/off-tumor side effects, similar to various PI3K inhibitors [8], [9], [10]. These effects may result from PI3K inhibition increased macrophage infiltration and induced the expression of macrophage-associated cytokines and chemokines in the tumor environment as well as their toxicity in normal tissue [11].

Macrophages and other myeloid subsets constitute up to 70% of tumor immune subsets in prostate cancer [12]. Macrophages have a leading position in the tumor microenvironment of prostate cancer. They can polarize into classically activated macrophages (M1) and alternatively activated macrophages (M2). M1 macrophages exhibit antitumor activity by activating the adaptive immune response, while M2 macrophages exert protumor activity by suppressing immune function in the tumor microenvironment [13], [14]. Macrophages can be regulated by prostaglandin E2 (PGE2) to shift toward the M2 phenotype in the tumor [15], [44]. Indomethacin (IND), a nonsteroidal anti-inflammatory drug that inhibits the synthesis of PGE2, has the ability to reduce the PGE2-mediated M2 polarization of macrophages, increase the proinflammatory macrophage (M1 phenotype) ratio in the tumor microenvironment, and enhance the immune response in tumor cells [17], [18].

Above all, IND has great potential to be employed as an immunotherapy agent to enhance the anticancer effect of ZSTK on prostate cancer. However, the poor selectivity and off-target toxicity in normal tissues, such as gastrointestinal toxicities of ZSTK and IND, compromise their therapeutic efficacy [8], [19]. To overcome these drawbacks, we tried to incorporate ZSTK and IND into nanomaterials.

Cell membrane-based biomimetic nanoparticles (CMBNs) can specifically bind to the source cancer cells. Their homotypic binding properties achieve clearance escape, enabling longer blood circulation and effective tumor targeting [20], [21]. However, the limited source of cells restricts the development of CMBNs [22], [23]. Fortunately, liposomes, as a successful commercial preparation, can compensate for CMBNs since their components are similar to the lipid bilayer of cells [24], [25] Our previous work constructed cancer cell membrane-based biomimetic nanoparticles with carrier-free nanoparticles as the core. The results demonstrated that this system may become an innovative strategy for cancer chemotherapy [26].

The combination of kinase-inhibiting therapy and immunotherapy is a promising approach in the treatment of cancer. Current existing nanosystems of kinase-inhibitors are first prepared using PEG-PLGA or liposomes, and then administered in combination with PD-1 antibody which targets T cells [27], [28]. In our work, biomimetic nanoparticles are constructed to deliver both kinase-inhibitor and immunomodulator in one nanosystem, which may be more convenient for application. In addition, our nanosystem is designed to target macrophage in the microenvironment, not T cells. We first synthesized ZSTK (a PI3K inhibitor) into nanoparticles (termed ZNPs) as a core by self-assembly. Then, we successfully encapsulated IND in the lipid bilayer of liposomes and hybridized it with the PC3 cell membrane to generate I@CML. Finally, ZNPs were modified with I@CML to build cancer-derived membrane/liposome-coated nanoparticles (termed ZNPs/I@CML). The preparation and application of ZNPs/I@CML are shown in Fig. 1. The physicochemical properties, biological characteristics, cellular uptake efficacy, in vivo therapeutic efficacy, and biosafety of ZNPs/I@CML were evaluated in our study. These results demonstrated that the hybridized biomimetic nanoparticles displayed specific targeting ability and significant antitumor efficacy, indicating that the combination of kinase-inhibiting therapeutics and immunotherapy could emerge as an alternative approach in the treatment of cancer.

Section snippets

Reagents.

ZSTK474 was purchased from Selleck (London, Canada). Indomethacin was purchased from Yunpeng Pharmaceutical Group (Shanxi, China). Hydrogenated Soybean Phosphotidylcholine (HSPCs), cholesterol, and DSPE-mPEG2000 were purchased from AVT (Shanghai) Pharmaceutical Tech Co., Ltd. Anti-sodium potassium ATPase, anti-pancadherin, anti-histone H3, and anti-GAPDH antibodies were supplied by Abcam (Cambridge, USA). Anti-CD24 antibody was supplied by ZEN Bio (Chengdu, China). Anti-CD68, anti-CD86, and

Preparation and characterization of ZNPs/I@CML.

The ZNPs were prepared using the nanoprecipitation method. It is a universal, flexible, and green method for preparing carrier-free nanodrugs, which provide high drug loadings, increase the half-life of anticancer drugs, and minimize the use of carriers and excipients to overcome the toxicity induced by the carrier. Although carrier-free nanodrugs have been widely used in the combination of cytotoxic anticancer agents, photosensitizer/photothermal agents, and immunotherapeutic agents, only a

Conclusions

We successfully constructed ZSTK carrier-free nanoparticle (ZNP)-loaded biomimetic hybrid indomethacin liposomes (I@Lip) with PC3 cell membranes, termed ZNPs/I@CML, to improve the treatment of prostate cancer. We demonstrated that ZNPs/I@CML could not only increase active targeting efficacy but also regulate the polarization of TAMs to improve the immune response on tumor cells, resulting in enhanced therapeutic outcomes in vivo. Importantly, ZNPs/I@CML exhibited minimal systemic side effects

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This study was supported by grants from National Natural Science Foundation of China (82061148017, 82073890, and 81501578), and the Natural Science Foundation for Young Scientists of Tianjin (18JCQNJC83500).

Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

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