Inhibition of growth of hepatocellular carcinoma by co-delivery of anti-PD-1 antibody and sorafenib using biomimetic nano-platelets

Abstract

Background: Traditional nanodrug delivery systems have some limitations, such as eliciting immune responses and inaccuracy in targeting tumor microenvironments.

Materials and methods: Targeted drugs (Sorafenib, Sora) nanometers (hollow mesoporous silicon, HMSN) were designed, and then coated with platelet membranes to form aPD-1-PLTM-HMSNs@Sora to enhance the precision of drug delivery systems to the tumor microenvironment, so that more effective immunotherapy was achieved.

Results: These biomimetic nanoparticles were validated to have the same abilities as platelet membranes (PLTM), including evading the immune system. The successful coating of HMSNs@Sora with PLTM was corroborated by transmission electron microscopy (TEM), western blot and confocal laser microscopy. The affinity of aPD-1-PLTM-HMSNs@Sora to tumor cells was stronger than that of HMSNs@Sora. After drug-loaded particles were intravenously injected into hepatocellular carcinoma model mice, they were demonstrated to not only directly activate toxic T cells, but also increase the triggering release of Sora. The combination of targeted therapy and immunotherapy was found to be of gratifying antineoplastic function on inhibiting primary tumor growth.

Conclusions: The aPD-1-PLTM-HMSNs@Sora nanocarriers that co-delivery of aPD-1 and Sorafenib integrates unique biomimetic properties and excellent targeting performance, and provides a neoteric idea for drug delivery of personalized therapy for primary hepatocellular carcinoma (HCC).

Keywords: Immunotherapy; Nanoparticles; Platelet membrane; Sorafenib; Tumor microenvironment.

Fig. 1

Characterization of nanoparticles. (A) SEM images: HMSNs. Scale bar, 300 nm. (B) TEM images: HMSNs particles. Scale bar, 200 nm. (C) The surface element (C, O, Si) mapping of HMSNs. (D) HMSNs particles size distribution by intensity. (E) Zeta potential of HMSNs (-NH₂), PLTM and PLTM-HMSNs (mean ± SD; n = 3). (F, G) TEM images: PLTM, Scale bar, 200 nm; PLTM-HMSNs, Scale bar, 100 nm. (H) Membrane proteins detected by western blot. (H, I) CD41 and P-selectin during the construction of PLTM-HMSNs, PLTM as a control (mean ± SD). (J) Confocal laser photo of aPD-1-PLTM-HMSNs@Sora (CY5.5 (red) stained aPD-1, FITC (green) labeled HMSNs)

Fig. 2

(A, B) CLSM images of HMSNs@Sora and aPD-1-PLTM-HMSNs@Sora uptaken by MHCC97H cells incubated with FITC-labelled HMSNs for different times. (C, D) CLSM images of HMSNs@Sora and aPD-1-PLTM-HMSNs@Sora uptaken by Hep-G2 cells incubated with FITC-labelled HMSNs for different times. (E, F) Corresponding quantified fluorescence intensity for MGCC97H and HepG2 cells. NPs, cell membrane and nucleus were stained with FITC (green), Dil (red) and Hoechst (blue), respectively. Scale bar in the last image can be applied to the others. Scale bar, 25 μm

Fig. 3

Cell viability of MHCC97H (A) and HepG2 (B) after incubation with different concentrations of HMSNs and PLTM-HMSNs for 24 h. (C) The liver and kidney function indexes of BALB/c mice. All results are presented as the mean ± SD. (D) HE-stained organs from tumor-free BALB/c mice after injected with nanoparticles. Scale bar, 20 μm

Fig. 4

Anti-tumor efficacy assays in vivo of tumor-bearing mice. (A) In Vivo fluorescence imaging of Cy5.5 labeled aPD-1-PLTM-HMSNs@Sora in H22 tumor-bearing mice. (B) Tumor macroscopic images (1: PBS; 2: Sora; 3: aPD-1; 4: aPD-1 + Sora; 5: aPD-1-PLTM-HMSNs@Sora). (C) Tumor weight. (D) Growth curves of tumors in each group. Error bars represent the SD (n = 5). (E) HE stained tumor slices in each treatment group. (F) Expression of VEGFR2 in tumor tissue. (G) Quantitative analysis of the inhibition by Sora, aPD-1, aPD-1 + Sora, aPD-1-PLTM-HMSNs@Sora on the VEGF-A of tumor (H-score means Histochemistry score). Scale bar, 20 μm. Error bars represent the SD (n = 3)

Fig. 5

aPD-1-PLTM-HMSNs@Sora triggered a robust, T-cell-mediated anti-tumor immune response. (A) Immunofluorescence of residual tumors showed CD4⁺ T cells and CD8⁺ T cells infiltration. Scale bar, 20 μm. (B, C) Quantitative analysis of the number of CD8⁺ T cells and CD4⁺ effector T cells of tumor after treatment. (D) Immunofluorescence of residual tumors showed Foxp3⁺ T cells infiltration. Scale bar, 20 μm (AOD means average optical density). (E) Quantitative analysis of the number of Foxp3⁺ T cells of tumor after treatment. Error bars represent the SD (n = 3). Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001