Co-delivery of EGCG and melittin with self-assembled fluoro-nanoparticles for enhanced cancer therapy

Preparation and characterization of FEGCG@MPI NPs

FEGCG and MPI are bonded by hydrogen bond. As expected, the NPs could be self-assembled. FEGCG/MPI or EGCG/MPI, and their particle size was about 299, 499 nm, Zeta potential was 21, 38 mv (Figure 1A). Transmission electron microscope (TEM) results confirmed the successful formation of FEGCG/MPI (Figure 1B). These results demonstrated that FEGCG/MPI system has the potential to deliver biomolecules ranging from small molecule drugs to larger macromolecules.

Cytotoxicity, apoptosis and cellular uptake of FEGCG@MPI NPs

Haberman and Zhan qi obtain melittin from bee venom. Because of its strong toxicity (mainly hemolytic activity), its clinical application is limited. It is generally that the hemolytic toxins of melittin are mainly based on the transmission control mechanism. Melittin can directly form holes in the biofilm to cause hemolysis, and can also be degraded by phospholipase on the activated red cell membrane. We guess FEGCG@MPI NPs can reduce the hemolytic reaction of MPI, which may be due to the formation of nanoparticles that encapsulate phospholipids to avoid the attack of MPI perforation. Hemolytic activity assay indicated that (100μg/mLFEGCG+3μg/mL MPI) mixed administration group had less hemolytic reaction than free MPI (Supplementary Figure 1).

For the exploration of the activity of FEGCG, MPI and its synergy on the growth of Hepatoma cells, CCK-8 assays were used to detect the viability of Hepatoma cell (Hep 3B cell) after treatment with various concentrations of FEGCG, MPI and FEGCG@MPI NPs separately. As presented in (Figure 2A, 2C), FEGCG inhibited the growth of Hep3B cell in a dose-dependent manner. Up to 100μg/mL of FEGCG shows significant decrease in viability in Hep3B cell. In addition, melittin also shows a significant inhibitory effect on Hep3B cell after incubation for 24 h. As shown in (Figure 2B), cell activity was significantly reduced when it was treated with MPI. However, the concentration was 3μg/mL, cell activity no longer changes with concentration. Finally, to explore the effect on cell activity after preparing nanoparticles by mixing two single drugs, which is shown in (Figure 2C), a mixture of 100μg/mL FEGCG and 3μg/mL MPI was added to Hep3B cells, killing effect on tumor cells is the most obvious (Figure 2C) and apoptosis peaks were significantly increased (Figure 2D, 2E).

Confocal microscopy and flow cytometry experiments demonstrated that FEGCG/MPI achieved the highest cell uptake. It is further confirmed that FEGCG@MPI NPs has better efficiency in Hep3B cells (liver cancer) (Figure 2F). These results indicated that the FEGCG/MPI was a feasible and effective strategy to overcome the limitations in biomolecule delivery (Figure 2F, 2G).

Regulation of PD-L1 and apoptosis of FEGCG@MPI NPs in vitro

Bcl-2 family proteins are major regulators of apoptosis in health and disease. In the present study, we evaluated the expression of Bax and Bcl-2 genes in hepatocellular carcinoma to investigate the regulatory effect of FEGCG and MPI on the Bcl-2 family. Bcl-2 protein family is a special family. 25 homologous proteins of Bcl-2 family have been found. Bad and Bax promote apoptosis, while Bcl-2 and Bcl-2 inhibit apoptosis. It can be seen from (Figure 3A) that FEGCG can regulate the increase of Bax and the decrease of Bcl-2, and melittin can significantly reduce the protein content of Bcl-2. From the study, it can be concluded that mixed administration can significantly reduce the protein content of Bcl-2 and increase the protein content of Bad. Programmed death ligand 1 (PD-L1) is the ligand of programmed death 1 (PD-1) and an important immune checkpoint protein, which is responsible for negatively regulating the stability and integrity of T cell immune function. However, the response rate of patients to current anti PD-1 or anti PD-L1 treatment is still very low, and many initial responders eventually develop drug resistance to these treatments. FEGCG, the raw material of this study, can reduce the content of PD-L1 through transcriptional level, thereby effectively inhibiting the growth of liver cancer, thereby reducing the expression of PD-L1. At the same time, it can be seen from (Figure 3B) that mixed administration can enhance the effect of FEGCG in reducing PD-L1, and cooperate with MPI to play an anti-tumor role.

Migration and invasion abilities of FEGCG@MPI NPs in vitro

Cancer cell invasion refers to local invasion or distant metastasis of tumor cells. In 1889, Paget put forward the theory of “seed and soil”, which believed that only when appropriate tumor cells (seeds) had special affinity with the growth environment provided by specific tissues or organs (soil), they would transfer. In recent years, there has been a new understanding of this theory. In 1977, Fidler et al. first confirmed the heterogeneity of tumor cells, that is, tumor cells in primary tumor tissues have different biological characteristics, including some cells with metastatic potential. Under the selective pressure of the host, these cells have the ability to migrate and infiltrate, form tumor thrombi, survive in the circulation, stay in the distant capillary bed, infiltrate the organ parenchyma and proliferate, and finally form a monoclonal metastasis. The main cause of death in HCC patients is the invasion and migration of cancer cells. MPI can well inhibit the invasion of liver cancer cells. This article mainly explored whether FEGCG@MPI NPs can reduce more the invasion and migration of tumor cells. FEGCG@MPI NPs significantly inhibited cell migration (Figure 4A, 4B) and decreased wound healing (Figure 4C, 4D) compared with other control cells. These results indicated that compared with MPI and FEGCG, FEGCG@MPI NPs can significantly reduce cell invasion and migration.

Antitumor efficacy of FEGCG@MPI NPs in vivo

Next, the in vivo efficacy was examined, and the treatment regime was showed in (Figure 5A). When the tumor model was established, different drugs were injected into tumor on 1st, 2nd, 4th, 6th, 8th, 10th, 12th, and 15th days. On the 16th day, all mice were killed for tumor weight and tumor volume (Figure 5B). The growth of tumor volume and weight of FEGCG/MPI NPs in the treatment group was significantly slower than that of the other three groups (Figure 5C, 5D). The results of immunohistochemistry are consistent with the in vitro experiments. The content of PD-L1 in control group was relatively high, and FEGCG@MPI NPs was relatively low (Supplementary Figure 2). These results indicated the superior antitumor efficacy of FEGCG@MPI NPs. The flow chart of our article is summarized in Supplementary Figure 3.

Chemicals and reagents

(−)-epigallocatechin-3-O-gallate (EGCG) was purchased from Energy Chemical (Shanghai, China). 2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoro-1,8-octanediol (12F) were purchased from Macklin Biochemical Co. Ltd. (Shanghai, China). RIPA lysis buffers and Phenylmethanesulfonyl fluoride (PMSF) were obtained from Solarbio (China). The primary antibodies to Bax, Bcl-2, PD-L1, STAT-1, P-STAT-1, IRF and β-actin were purchased from Cell Signaling Technology (United States). The secondary antibodies to HRP AffinPure goat anti-rabbit IgG and HRP AffinPure goat anti-mouse IgG were purchased from Proteintech (United States). The Alexa Fluor®488 goat anti-mouse/rabbit IgG and Alexa Fluor®568 goat anti-mouse/rabbit IgG were purchased from Invitrogen (United States). PBS, DMEM, and EDTA were purchased from WISENT. Triton X-100 and 4, 6-dimethyl-2-phenylindole (DAPI) were purchased from Abcam (United Kingdom). Human hepatocellular carcinomas (Hep3B) were obtained from American type culture collection (ATCC). All other reagents were obtained from Beyotime Biotechnology Co., Ltd. (Shanghai, China) unless otherwise stated. Experimental data were obtained from Beiyu Biotechnology Co., Ltd. (Shanghai, China).

Synthesis, preparation, and characterization of FECGC and FEGCG@MPI NPs

The fluoropolymer of FEGCG was synthesized through the reaction of fluorinated compound and EGCG with the ROS-sensitive linker of oxalyl chloride. FEGCG and MPI are combined by a hydrogen bond. The complexes were prepared by complexing the carrier with FEGCG@MPI NPs at w/w = 1 for 30 mins (FEGCG@MPI NPs concentrations = 10 μg mL⁻¹). The particle size and zeta potential of FEGCG@MPI NPs complexes were determined by DLS. The loading efficiency of FEGCG@MPI NPs was determined using a FITC fluorescence quenching assay (excitation at 450 nm, emission at 480–600 nm).

Cell toxicity, apoptosis and cellular uptake

Fresh blood samples from mice were placed in EDTA-containing tube. The blood was centrifuged at 3000 rpm at 4° C for 10 minutes to collect red blood cells (RBC), and then divided into five groups, which were: (i) PBS, (ii) MPI, (iii) FEGCG, (iv) FEGCG@MPI NPs, (v) Triton. After incubating these samples at 37° C for 60 minutes, the blood samples were centrifuged at 3000 rpm for 5 minutes to get the supernatant and the concentration of hemoglobin was measured at 545 nm. Data (means ± SD) were from three independent experiments.

Human Hepatoma cells were cultured in DMEM culture medium at 37° C with 5% CO2 atmosphere.

For testing the proliferative activity of cells, 2x10³ Hep3B cells were seeded into 96-well plate per well in 200 μl DMEM medium. On the second day, the cells were treated with various concentrations (10, 20, 50, 100, and 200μg/ml) of FEGCG and MPI (4, 6, 8, 12, and 24μg/ml) for 24 hours. Subsequently, the previous medium was removed, and 100 μl DMEM medium containing 10 μl Cell Counting Kit (CCK)-8 solution (Vazyme) were added to each well. The plate was incubated for 1 h, and the absorbance values per well were recorded at 450 nm. The resulting data were analyzed from three independent experiments and then normalized to the absorbance of the wells containing culture medium alone.

Hep3B cells (5×10⁵ cells) were seeded in six-well plates and incubated with PBS, MPI, FEGCG, and FEGCG@MPI NPs separately for 24h. Then the cells were digested with trypsin without EDTA and washed with PBS twice, suspended in binding buffer and stained with Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme). The cells were cultured in darkness (37° C) for 20 minutes and apoptotic cells were observed using FACSC calibur flow cytometry (Sakuri, USA) within 1 h.

For cell delivery, Hep3B cells were cultured for confocal imaging or flow cytometry measurement. For EGCG delivery, cells were incubated with free MPI for 4 hours. For peptide delivery, cells were incubated with EGCG/MPI-FITC and FEGCG/MPI-FIT for 4 hours. After incubation, cells were washed with PBS, and DAPI was stained before confocal microscope, and cells were collected by flow cytometry.

Cell migration and invasion

Briefly, Hep3B cells were seeded into 6-well plates containing complete growth media with 10% FBS. When the 98%-100% of the tissue culture dished was occupied by cells, scratch with the sterile 10-200 μl pipette tips was done. The scratch-wound healing cum cell migration was monitored over time and images were captured under a light microscope with 10× objective lens at the 0, 12, and 24 h time-points after denudation, and then images were analyzed with the ImageJ software.

For an invasion assay, 2 × 10⁵ Hep3B cells were seeded into 100 μL serum-free DMEM medium in the upper surface and allowed to invade toward the underside of the membrane in 24-well tissue culture plates containing 600 μL complete growth media with 10% FBS for 24 h. Then PBS, FEGCG, MPI, and FEGCG@MPI NPs were added to lower chamber for 24h. Finally, the invaded cells on the underside of the membrane were fixed with 4% methanol, and then stained with crystal violet dye for 30 minutes. After that, the sample is observed under an optical microscope.

Proteins of PD-L1 and apoptosis pathway determination

Hep3B cells were lysed with Cell lysis buffer for Western and IP (Beyotime Biotechnology, Jiangsu, China) containing 1 % PMSF. The protein content of different fractions was detected via the bicinchoninic acid (BCA) method. The obtained protein samples were subjected to sodium dodecyl sulfonate polyacrylamide gel electrophoresis (SDS-PAGE) with 10% gel concentration, and transferred onto PVDF membranes with pre-cast gels using wet transfer with 250 mA for 1 h. Membranes were put in 5% skimmed milk and seal it for 2h, then the first antibody (anti-β-actin, 1:1000 PD-L1 (Abmart, Shanghai, China)), 1:1000 anti-STAT-1 (Abcam, MA, USA), anti-P-STAT1-1 (Abcam, USA), anti-IRF, Bcl-2 and Bad (Cell Signaling, USA) were incubated for 4° C overnight. The next day, membranes were probed with second antibody (HRP-anti-mouse/rabbit IgG Cat#7076, Cat#7074, antibody, CST, Beijing, China). After another three washes, signals were detected by LuminataTM Western HRP substrates (Millipore Corporation, Billerica, MA, USA) and Chemi-DocTM MP Imaging System (v5.2.1, Bio-Rad Laboratories, Inc, Hercules, CA, USA).

Animals and tumor models

Athymic BALB/c nude mice (male, 6-8weeks) were purchased from the Gempharmatech Co., Ltd. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School (NO. 2018-289-01). For establishing a Hep3B tumor bearing mouse model, 2x10⁶ cells/0.2 mL PBS was subcutaneously implanted into the left armpit of the mice. When the tumor volume grew to approximated 200 mm³, the mice were randomly divided into four groups (n = 5) : (i) PBS, (ii) MPI, (iii) FEGCG, (iv) FEGCG@MPI NPs. The treatment was performed on days 1, 2, 4, 6, 8, 10, 12, 15 by intratumor injection, and the tumor volume and body weight of the mice were recorded every two days (Supplementary Figure 2). Tumor volume (mm³) = length × width²/2. On day 15, all mice were sacrificed and the tumors were collected for weight recording. After tumor resection, the tumor was weighed and sent to sections for immunohistochemical staining according to a standard procedure.

Statistical analysis

Statistical analysis was performed by two-sided Student’s t-test for two groups and one-way ANOVA analysis of variance for multiple groups (when P < 0.05, the data were considered statistically significant).

Availability of data and materials

The data used in the study are not public data. The datasets used in the study are available from the corresponding authors on reasonable requests.