Introduction

Tumor growth needs massive ATP as energy supply. And the ability of tumor cells to absorb grapes is dozens of times higher than that of normal tissue cells, which called the "Warburg effect" [1,2,3]. Even under aerobic conditions, glucose produces lactic acid in tumor cells through glycolysis. It is a new treatment strategy for anti-tumor therapy by targeting tumor energy metabolism currently [4,5,6]. Among them, 3-bromopyruvate (3-BPA) and CPI-613 are widely studied as metabolic blockers [7,8,9,10]. 3-BPA has high affinity with GADPH and HK-II [11, 12]. It can completely interdict the activities of GADPH and HK-II under very low concentration [13, 14]. However, 3-BPA is instable in the liquid state and easy to decompose [15, 16]. CPI-613 is an inhibitor of mitochondrial tricarboxylic acid cycle metabolism in tumor cells [17, 18]. It suppresses the α-ketoglutarate dehydrogenase complex (KGDH) to restrain the glutamine replenishment pathway [19, 20]. Whereas CPI-613 is lipid-soluble which easily soluble in organic solvents, and difficult to dissolve in water [21, 22].

In recent years, with the rapid development of nanotechnology and wide application, nanomaterials have been widely used in biomedicine. Nanoparticles can protect drugs, reduce drug systemic toxicity, improve drug uptake, slow-release, and targeted drug delivery [23,24,25]. Hence, the above problem of drug solubility and stability in application could be well resolved by using nanomaterial.

Cancer immunotherapy is another new approach of anti-tumor therapy that apply the host immune system to fight against cancer [26]. Stimulator of interferon genes (STING), also known as transmembrane protein 173 (TMEM173) and MPYS/MITA/ERIS, which can induce the production of a variety of pro-inflammatory cytokines and chemokines, is shown great potential in enhancing anti-tumor immunity [27]. Many STING agonists have been developed or tested in preclinical and clinical trials for immunotherapy of diseases such as cancer and infectious diseases [28,29,30]. And yet, In terms of the current clinical attempts, it mainly focus on modified cyclic dinucleotide (CDN) to simulate the STING’s endogenous ligand (cGAMP) to treat solid tumors by intratumoral injection [29]. Using the symmetry of STING, GSK designed a connection strategy to cooperate with two symmetrically related amide benzimidazole (ABZI) based compounds to produce a high affinity ligand that interacts with STING in a cGAMP competitive manner. What’s more, ABZI was the first nonnucleotide intravenous STING agonist recently. ABZI not only overcomes the disadvantages of intratumoral injection but also has a strong anti-tumor effect [30].

In this study, 3-BPA and CPI-613 are applied conjunctively to block the energy metabolism of Hepatocellular carcinoma (HCC) via a nano-drug delivery system for the first time. Meanwhile, we combine BCP NPs with anti-tumor immunotherapy innovatively. Our study sweeps the obstacle of poor solubility and easy decomposition in metabolic drugs combination. Moreover, paves a novel way to effectively restrict the growth and metastasis of HCC. We also provide a reliable basis for future research on the combined administration of metabolic and immune therapy.

Results

BCP NPs synthesis and characterizations

The BCP NPs synthesis procedure and its anti-tumor mechanism is illustrated in Scheme 1. The PLGA NPs were prepared by an emulsion solvent evaporation approach. Meanwhile, CPI-613 and 3-BPA were regarded as oil phase and water phase respectively, loaded in the PLGA NPs. A dynamic light scattering (DLS) was applied to measure the size and zeta potential of BCP NPs (Additional file 1: Table S1). The particle size and potential of different molar ratio BCP NPs were not much different. High performance liquid chromatograph (HPLC) and pyruvate assay kit were used to determine the drug load and encapsulation rate of the different BCP NPs (Additional file 1: Table S2).

Scheme 1
scheme 1

Illustration of the the anti-tumor mechanism of BCP NPs

Full size image

As reported, the synergistic effect of two or more drugs largely depended on a series of factors, such as combination ratios, drug concentrations, and treatment time etc. [Mice samples

All mice were purchased from Wuhan Institute of Biological Products Co. Ltd and Bei**g Vital River Laboratory Animal Technology Co., Ltd. 6–8 weeks female C57BL/6 mice were used for experiments. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Medical Research Institute, Wuhan University.

Routine hematology study in vivo

To evaluate the biocompatibility and potential immunogenicity, Blood routine and liver and kidney function were analyzed. The whole blood samples were collected from BALB/c mice, detected by automatic blood cell analyzer (BC-2800vet, Mindray, China) and automatic biochemical analyser (Chemray 240, Shenzhen Redu Life Technology, China).

In vivo biocompatibility assay

The biocompatibility of BCP NPs was explored by hemolysis test. Briefly, to obtain the pure erythrocytes, the whole blood samples that obtained from healthy C57BL/6 mice were washed five times with PBS by centrifuged at 3000 rpm for 5 min. The red blood cells were re-suspended in PBS for later use. BCP NPs dispersed in PBS at various concentrations (0.5, 1, 2, 5, 10, 20, 50 and 100 µM), and incubated at 37 °C. 100% hemolysis was deemed as the erythrocytes mixed with DI water. The erythrocytes mixed with PBS was regarded as positive control. Finally, optical absorbance at 540 nm was recorded by a microplate reader.

Establishment of 4T1 tumor model

To establish the Hepa1–6 hepatocellular carcinoma tumor model, C57BL/6 mice were subcutaneously with Hepa1–6 cells resuspended in DMEM (4 × 106 cells per mouse) on the right abdomen.

In vivo fluorescence imaging

CY7 was used to track the nanoparticles in tumor-bearing mice. Briefly, tumor-bearing C57BL/6 mice were given the PLGA NPs which loaded Cy7 intravenously at a concentration of 5 mg/kg. Normal mice were used as negative control. The signal of Cy7 was measured at appropriate time points with the Caliper IVIS Lumina II (IVIS, Xenogen, Caliper Instruments).

Tumor inhibition in vivo

Tumor volume was measured in real time and calculated based on Eq. (1) [48] as follows:

$${\text{Tumor volume}} = \left( {\text{tumor length}} \right) \times \left( {\text{tumor width}} \right)^{{2}} \times 0.{5},$$
(1)

When the tumor size reached approximately 100 mm3. Mice were randomly divided into six groups, and were intravenously injected with 100 µL of (1) saline, (2) PLGA NPs, (3) free drugs (3-BPA + CPI-613), (4) BPA NPs, (5) CPI NPs, and (6) BCP NPs. The equivalent total drug dosages of 15 mg/kg were used for all the groups, respectively. An injection of medicine was given every 3 days. Simultaneously. The tumor volume was and body weight were recorded every other day. Finally, sacrificed the mice and excised the tumors and organs. The hematoxylin and eosin (H&E) staining and immunofluorescent staining were all according to the standard protocols.

The combination of NPs and STING agonist

When average tumor volume was 100 mm3, mice were randomly divided into four groups, 10 mice each group. Mice were intravenously injected with 100 µL of (1) saline, (2) BCP NPs, (3) ABZI (STING agonist), and (4) BCP NPs + ABZI. For all the groups, the total drug dosages were equivalent. The total drugs of BCP NPs were 15 mg/kg, and was given every 3 days. Meanwhile, the dose of ABZI was 1.5 mg/kg. The mice’s body weight and tumor volume were measured daily. The hematoxylin and eosin (H&E) staining and immunofluorescent staining were all according to the standard protocols [52].

To explore whether ABZI activated the immune system in mice, the changes of CD8 molecules on the surface of mouse T cells after administration was examined. Briefly, all spleen samples were processed to cell suspension, re-suspended in the flow buffer, and incubated with mouse Fc blocker. Finally, before the flow cytometry analysis, cells were stained with CD45-PE, CD3-V421, and CD8-APC.

Statistical analysis

All statistical analyses were completed by one-way analysis of variance (ANOVA). A P-value smaller than 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001) was regarded as statistically significant. All the data analyses were used Origin (OriginLab, MA, USA) and shown as the mean ± standard deviation (SD).