Supramolecular Polymer-Nanomedicine Hydrogel Loaded with Tumor Associated Macrophage-Reprogramming polyTLR7/8a Nanoregulator for Enhanced Anti-Angiogenesis Therapy of Orthotopic Hepatocellular Carcinoma

Abstract

Anti-angiogenic therapies targeting inhibition of vascular endothelial growth factor (VEGF) pathway show clinical benefit in hypervascular hepatocellular carcinoma (HCC) tumors. However, HCC expresses massive pro-angiogenic factors in the tumor microenvironment (TME) in response to anti-angiogenic therapy, recruiting tumor-associated macrophages (TAMs), leading to revascularization and tumor progression. To regulate cell types in TME and promote the therapeutic efficiency of anti-angiogenic therapy, a supramolecular hydrogel drug delivery system (PLDX-PMI) co-assembled by anti-angiogenic nanomedicines (PCN-Len nanoparticles (NPs)) and oxidized dextran (DX), and loaded with TAMs-reprogramming polyTLR7/8a nanoregulators (p(Man-IMDQ) NRs) is developed for orthotopic liver cancer therapy. PCN-Len NPs target tyrosine kinases of vascular endothelial cells and blocked VEGFR signaling pathway. p(Man-IMDQ) NRs repolarize pro-angiogenic M2-type TAMs into anti-angiogenic M1-type TAMs via mannose-binding receptors, reducing the secretion of VEGF, which further compromised the migration and proliferation of vascular endothelial cells. On highly malignant orthotopic liver cancer Hepa1-6 model, it is found that a single administration of the hydrogel formulation significantly decreases tumor microvessel density, promotes tumor vascular network maturation, and reduces M2-subtype TAMs, thereby effectively inhibiting tumor progression. Collectively, findings in this work highlight the great significance of TAMs reprogramming in enhancing anti-angiogenesis treatment for orthotopic HCC, and provides an advanced hydrogel delivery system-based synergistic approach for tumor therapy.

Keywords: anti-angiogenic therapy; nanoregulator; orthotopic hepatocellular carcinoma; supramolecular hydrogel; tumor microenvironment.

Scheme 1

The design of TAMs‐targeted, acid‐sensitive supramolecular hydrogel for co‐delivery of TKI and TLR7/8 agonist (TLR7/8a) and its function in inducing a collaborative antitumor angiogenesis for HCC therapy. a) The structure of supramolecular hydrogels PLDX‐PMI consisting of drug‐loaded PCN‐Len NPs, hydrogel backbone chain DX, and TAMs‐targeted p(Man‐IMDQ) NRs. b) Schematic illustration of antitumor therapy toward tumor vascular network elicited by PLDX‐PMI‐mediated release of multikinase inhibitors targeting VEGFR and M2‐type TAMs repolarization. PCN‐Len NPs downregulates the expression of VEGF, inhibits tumor angiogenesis, and reduces the density of microvessels. Nanoregulators with macrophage‐targeting ligands repolarize pro‐angiogenic M2‐type TAMs to anti‐angiogenic M1‐types. This synergistically combined approach significantly enhanced the anti‐tumor angiogenesis effect of TKIs‐mediated nanomedicine in a highly malignant orthotopic HCC Hepa1‐6 model.

Figure 1

Characterization of PCN‐Len NPs, and tube formation and migration of HUVECs. a) Dynamic light scattering (DLS) profile of PCN‐Len NPs. b,c) Cytotoxicity of PCN‐Len NPs against Hepa1‐6 (b) and HUVECs (c) cells cultured with varied concentrations of lenvatinib. d) Cytotoxic effects of PCN NPs on 3T3 cells as a function of nanoparticle concentrations. e,f) Representative images and quantification of the inhibition of tubule formation of HUVECs treated with PCN‐Len NPs, lenvatinib and controls. g,h) Representative images and quantification of inhibition of HUVECs migration by transwell assay. Data represent mean ± SDs (n = 3). Statistical significance was analyzed using two‐tailed Student's t‐test between two groups. ***p < 0.001, between indicated groups.

Figure 2

Preparation and characterization of p(Man‐IMDQ) NRs, and macrophage repolarization to M1 phenotype. a) The chemical structure of p(Man‐IMDQ). b) The average particle size distribution of p(Man‐IMDQ) NRs determined by DLS at different pH values. c) Confocal images of p(Man‐IMDQ) NRs targeting macrophages. Macrophage nucleus was stained with DAPI. d) Flow cytometry analysis of MHCII and CD206 in BMDMs with different treatments (gated on F4/80⁺ cells). e) The percentage of F4/80⁺MHCII⁺ macrophages. f) Flow cytometry analysis of MHCII and CD206 expression in BMDMs that were pre‐polarized into M2 phenotype and then stimulated with different treatments (gated on F4/80⁺ cells). g) The percentage of repolarized F4/80⁺/CD206⁺ MHCII⁺ macrophages. h) ELISA analysis of proinflammatory (TNF‐α) and anti‐inflammatory cytokines (VEGF) expressed by M2 phenotype macrophages. Data represent mean ± SD (n = 3). Statistical significance was analyzed using two‐tailed Student's t‐test between two groups, ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 3

Characterization of supramolecular PLDX‐PMI hydrogel, and HUVECs migration and proliferation. a) The frequency sweep of PLDX‐PMI hydrogel detected in the angular frequency range of 1 to 100 rad s⁻¹. b) The strain sweep of PLDX‐PMI hydrogel at a fixed angular frequency (1 rad s⁻¹). c) The self‐healing analysis of PLDX‐PMI hydrogels. d) Representative live/dead staining and e) 3D construction of CLSM images of Hepa1‐6 cells treated by PDX, PCN‐Len NPs, and PLDX‐PMI hydrogel. f) Representative live/dead staining images of HUVECs treated by PDX, PCN‐Len NPs, and PLDX‐PMI. g) Representative images of migrated HUVECs by transwell assay. h) Protein expression level of p‐MEK1/2 and p‐Erk1/2 in HUVECs determined by western blotting. i) Schematic mechanism of PCN‐Len NPs and p(Man‐IMDQ) NRs in inhibiting endothelial cell proliferation.

Figure 4

Analysis of the gene expression profile dataset related to hepatocellular carcinoma (GSE146049). a) Volcano map. The red points represent upregulated genes selected based on the corrected p‐value < 0.05 and logFC > 1. The blue points represent downregulated genes selected based on the corrected p‐value < 0.05 and logFC ←1. The black points represent genes with no significant difference. b) Enrichment GO cluster analysis of DEGs. c) Functional enrichment analysis of DEGs. d) Hub genes calculated from PPI network of DEGs by MCC algorithm. e) KEGG enrichment analysis of hub genes. FC, fold change; GO, Gene Ontology; DEGs, differentially expressed genes; KEGG, Kyoto encyclopedia of genes and genomes; PPI, protein–protein interaction.

Figure 5

In vivo inhibition of orthotopic HCC progression by enhanced anti‐angiogenic therapy via co‐delivery of PCN‐Len NPs and p(Man‐IMDQ) NRs in supramolecular hydrogel. a) Schedule for orthotopic Hepa1‐6 HCC treatment. b) Curves of mouse body weight. Data represent mean ± SD (n = 5). c) The weight of tumors at the 7th and 14th day after treatment. Data represent mean ± SD (n = 3). d) Photographs of resected tumor‐bearing livers after treatment for 7 and 14 days. e) Representative images for H&E staining of tumor tissue sections. Statistical significance was analyzed using two‐tailed Student's t‐test between two groups, **p <0.01 and *p <0.05.

Figure 6

PCN‐Len NPs and p(Man‐IMDQ) NRs effectively decreased blood vessel density in HCC tumor tissues. Representative images of orthotopic Hepa1‐6 liver cancer tissue vasculature stained with CD31 (red) and α‐SMA (green), while nuclei were stained with DAPI.

Figure 7

PCN‐Len NPs and p(Man‐IMDQ) NRs induced M2‐type TAM reprogramming and promoted anti‐angiogenesis. a) Representative images of CD206 (red)/F4/80 (green) immunofluorescence staining of tumor tissues. b) Representative IHC images of VEGF and TNF‐α expressions in tumor sections from PBS, PDX, PDX‐PMI, PLDX, and PLDX‐PMI‐treated mice.