Bioengineered hybrid dual-targeting nanoparticles reprogram the tumour microenvironment for deep glioblastoma photodynamic therapy

Abstract

Glioblastoma (GBM) poses significant therapeutic challenges due to its hypoxic and immunosuppressive tumour microenvironment (TME), low immunogenicity and physical barriers. While combining photodynamic therapy (PDT) with immunotherapy holds promise, its efficacy is hampered by insufficient immune activation. In this study, we develop a multifunctional photodynamic-enhanced biomimetic intelligent nanoplatform (FBFO@HM@aOPN) responsive to the TME. The nanoplatform consists of a dual-enzyme nanozyme encapsulated in a prokaryotic-eukaryotic hybrid membrane, further modified with a pH-sensitive tumor-targeting antibody. After systemic administration, FBFO@HM@aOPN selectively accumulates in the GBM through vascular regulation and extracellular matrix (ECM) remodelling while generating oxygen to alleviate hypoxia. Crucially, the platform concurrently induces immunogenic death in tumour cells and reprograms protumoral macrophages to antitumor phenotypes. This dual action robustly activates both innate and adaptive immunity, significantly inhibiting GBM growth. Furthermore, when combined with anti-PD1 immunotherapy, the nanoplatform dramatically boosts the treatment effect and effectively prevents postsurgical tumour recurrence. Therefore, our work offers a multimodal platform for stimulating anti-tumour immunity, with potential applicability for GBM patients.

Fig. 1. Schematic illustration of the synthesis process of FBFO@HM@aOPN NPs and the treatment mechanism for immune-remodelled photodynamic therapy against GBM in vivo.

a Schematic diagram of the synthetic route of FBFO NPs. b Preparation of a prokaryotic-eukaryotic hybrid vesicle membrane (HM) formed by the fusion of bacterial outer membrane vesicles (OMVs) and M1 macrophage-derived exosomes (M1EVs). c Preparation of anti-OPN (aOPN) attached to a pH-sensitive benzoic acid‒imide bond. d Schematic illustration of FBFO@HM@aOPN NPs for immunity-remodelled photodynamic therapy against GBM in vivo.

Fig. 2. Preparation and characterization of FBFO@HM@aOPN NPs.

a Transmission electron microscopy (TEM) images of FBFO NPs of uniform size and morphology. Scale bar, 100 nm. b Element mapping of the FBFO NPs. Scale bar, 100 nm. c X-ray diffraction (XRD) patterns of the Fe₃O₄ and FBFO NPs. d UV–vis diffuse reflectance spectra of the Fe₃O₄ and FBFO NPs. e Schematic illustration of the working mechanisms of FBFO. f Degradation of methylene blue (MB) by the Fe₃O₄ and FBFO NPs. g Oxygen (O₂) generation curves of Fe₃O₄ and FBFO NPs. h Electron spin resonance (ESR) spectra of different groups. i Representative flow cytometry and quantification analysis of azide (N3)-modified M1EVs after incubation with DBCO-Cy5.5 (n = 3 independent experiments). The data are presented as the means ± SDs. p values were calculated via Student’s t test for two-group comparisons; ****p < 0.0001. j Confocal laser scanning microscopy (CLSM) analysis of azide (N3)-modified M1EVs after incubation with DBCO-Cy5.5. Scale bar, 50 nm. k CLSM analysis showing the colocalization of M1EVs and OMVs. Scale bar, 50 nm. l Western blotting revealed that the HMs inherited components from M1EVs (TSG101, CD63, CD81 and integrins α4β1, as well as the chemokine receptor CXCR4) and OMVs (FtsZ). m CLSM image analysis showing the colocalization of HM and aOPNs. Scale bar, 100 nm. n TEM images of FBFO@HM@aOPN NPs of uniform size and morphology. Scale bar, 100 nm. o ELISA quantification of anti-OPN release at different pH values (n = 3 independent experiments). The data are presented as the means ± SDs. p values were calculated via Student’s t test for two-group comparisons; ****p < 0.0001. p Degradation of MB and q O₂ generation curves of FBFO@HM@aOPN under the indicated conditions. Source data are provided as a Source Data file.

Fig. 3. Catalytic activity and therapeutic effect of FBFO@HM@aOPN in GBM cells in vitro.

a Flow cytometry analysis showing the internalisation of Cy5.5-labelled FBFO by CT2A GBM cells. The right image shows the quantification of Cy5.5-positive cells (n = 3 independent experiments). b CLSM image of CT2A GBM cells stained with DCFH-DA to indicate NP-induced ROS generation. Scale bar, 200 μm. Cell viability of CT2A GBM cells treated with different formulations (c) with/without 660 nm irradiation. (d) Under hypoxia/normoxia conditions (n = 3 independent experiments). e Annexin V-FITC/PI apoptosis analysis of CT2A GBM cells treated with different formulations under 660 nm irradiation by flow cytometry (n = 3 independent experiments). f Lipid peroxidation level of CT2A GBM cells treated with different formulations under 660 nm irradiation, as determined with a malondialdehyde (MDA) assay kit (n = 3 independent experiments). g CLSM image of CALR and Hoechst costained CT2A cells treated with different formulations under 660 nm irradiation. Scale bar, 50 μm. Released h HMGB1 and i ATP detected in the cell culture supernatant of CT2A GBM cells treated with different formulations under 660 nm irradiation (n = 3 independent experiments). j Schematic illustration of ICD induced by FBFO@HM@aOPN and the potential mechanism by which it enhances immunotherapy. All the data are presented as the means ± SDs. The p values were determined via two-tailed one-way ANOVA with a Tukey post hoc test (a, c–f, h, i), p > 0.05, no significance (ns), *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 4. FBFO@HM@aOPN NPs significantly polarized M0- and M2-like macrophages towards the M1-like phenotype in vitro.

a Flow cytometry analysis showing the internalisation of Cy5.5-labelled FBFO by Raw264.7 cells (n = 3 independent experiments). b CLSM image of Raw264.7 cells stained with DCFH-DA to indicate NP-induced ROS generation. Scale bar, 200 μm. c Volcano plot showing the expression profile related to DEGs related to macrophage polarisation in Raw264.7 cells treated with PBS or FBFO@HM@aOPN. d GO BP and (e) GSEA enrichment analyses of the DEGs between the PBS- and FBFO@HM@aOPN-treated Raw264.7 cells. Flow cytometry analysis of the proportions of f CD80⁺CD86⁺ (n = 3 independent experiments), g MHC I⁺ (n = 4 independent experiments), h H-2Kb/SIINFEKL⁺ (n = 4 independent experiments), and i CD206⁺ (n = 3 independent experiments) among Raw264.7 cells treated with different formulations under 660 nm irradiation. j Establishment of BMDMs. Flow cytometry analysis of the proportions of k CD80⁺CD86⁺, l MHC I⁺, m H-2Kb/SIINFEKL⁺, and n CD206⁺ cells in BMDMs treated with different formulations under 660 nm irradiation (n = 3 independent experiments). o GSEA enrichment analyses of the DEGs between the PBS- and FBFO@HM@aOPN-treated Raw264.7 cells. p Western blot analysis of STING-IRF3 and NF-κB p65 phosphorylation in Raw264.7 cells and BMDMs treated with different formulations under 660 nm irradiation. q Schematic diagram of the mechanism by which FBFO@HM@aOPN reprograms macrophages to the M1-like phenotype. All the data are presented as the means ± SDs. The p values were determined via two-tailed one-way ANOVA with a Tukey post hoc test (f–i, k–n), p > 0.05, no significance (ns), *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 5. Tumour suppression in a 3D tumour spheroid model.

a CLSM image of the killing of CT2A GBM cells (red) by repolarized M1 macrophages (green). Scale bar, 100 μm. b Flow cytometry analysis of the killing of CT2A GBM cells by repolarized M1 BMDMs (n = 3 independent experiments). Schematic illustration of in vitro three-dimensional (3D) tumour model (c) preparation and (d) potential for assessing the penetration effect. e Z-stack CLSM images of the penetration of Cy5.5-labelled FBFO NPs in CT2A-BMDM multicellular spheroids after 6 h of incubation. Scale bar, 250 μm. f Z-stack CLSM images of CT2A-BMDM multicellular spheroids (MCTSs) dyed with hypoxia green reagent to indicate the degree of hypoxia. Scale bar, 250 μm. g Representative photographs of MCTSs at a certain time (left) and the volume histogram of MCTSs treated with different formulations on day 7 (right, n = 3 independent experiments). Scale bar, 200 μm. h Flow cytometry analysis of the proportions of CD206⁺ BMDMs in MCTSs treated with different formulations under 660 nm irradiation (n = 3 independent experiments). i Secreted IFN-β, IL-6, TNF-α, CCL5 and CXCL10 concentrations in the cell culture supernatants of MCTSs subjected to various treatments, as measured by ELISA (n = 3 independent experiments). All the data are presented as the means ± SDs. The p values were determined via two-tailed one-way ANOVA with a Tukey post hoc test (b, g–i), p > 0.05, no significance (ns), *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 6. Biodistribution, biocompatibility, and antitumour effects in vivo.

a Schematic diagram of the in vitro BBB model. b The transendothelial electrical resistance (TEER, Ω·cm²) values in the in vitro BBB model at different time points after incubation with formulations (n = 3 independent experiments). c Transport ratios of different formulations determined by detecting Cy5.5 fluorescence intensity in the apical chamber and basolateral chamber after 4 h of incubation in the in vitro BBB model (n = 3 independent experiments). d In vivo Cy5.5 fluorescence images of CT2A-bearing mice at different time points after intravenous injection of different formulations. Ex vivo (e) Cy5.5 fluorescence images and (f) quantitative histogram of the fluorescence intensity of both the CT2A-bearing brain and major organs (including the heart, liver, spleen, lung, and kidney; n ≥ 3 mice for each group). g Experimental design of therapeutic FBFO@HM@aSPP intervention in a CT2A tumour model (n = 5 mice per group). h Representative bioluminescence images (left) and fluorescence intensity quantification (right), as well as (i) Kaplan–Meier survival curves of luciferase-expressing CT2A-bearing mice subjected to different treatments (n = 5 mice per group). j H&E staining of brain tumour tissues from CT2A tumour-bearing mice subjected to various treatments. Scale bar, 2 mm. CLSM images of (k) Ki67 (scale bar, 50 μm), (l) CD44 (scale bar, 20 μm) and (m) CALR (scale bar, 20 μm) staining of brain tumour tissues from CT2A tumour-bearing mice subjected to various treatments. All the data are presented as the means ± SDs. The p values were determined via two-tailed one-way ANOVA with a Tukey post hoc test (c, h). For i, p values are calculated via the log-rank (Mantel‒Cox) test. p > 0.05, not significant (ns), *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data are provided as a Source Data file.

Fig. 7. ScRNA-seq analysis revealed increased immune remodelling.

a Uniform manifold approximation and projection (UMAP) plot showing the 13 major cell types. The dots represent individual cells, and the colours represent different cell populations. The samples were separated according to their origin (PBS- and FBFO@HM@aSPP-treated GBM). b Cell density distribution across two tissue groups. A high relative cell density indicates bright magma. c Bar graphs displaying the relative cellular fractions of the major cell types across the two tissue groups. d Differential gene expression analysis showing up- and downregulated genes across malignant and malignant IFN subpopulations in the treated group compared with the PBS group. e Bar plot showing the results of the KEGG enrichment analyses of the DEGs between FBFO@HM@aSPP-treated and PBS-treated malignant cells. f UMAP plot showing the 3 BMDM subpopulations. The dots represent individual cells, and the colours represent different cell populations. g Cell density distribution across two tissue groups. A high relative cell density indicates bright magma. h Dot plot showing the gene expression of M1-like and M2-like markers. i Bar plot showing HALLMARK enrichment analyses of the DEGs between FBFO@HM@aSPP-treated and PBS-BMDM-treated cells. j GSEA showing that the upregulated genes were significantly enriched in antigen processing and presentation and positive regulation of phagocytosis in the treated group. k UMAP plot showing the 6 lymphocyte subpopulations. The dots represent individual cells, and the colours represent different cell populations. l Cell density distribution across two tissue groups. A high relative cell density indicates bright magma. m Cell density showing the expression of effector-related genes. A high relative cell density indicates bright magma. n Enriched pathways of DEGs across different lymphocyte subpopulations. Colours from blue to red indicate the absolute value of the DE score from low to high. o Dot plot showing the gene expression of immune checkpoints and effectors. p Visualisation of the main senders and receivers of cell clusters in the 2D space of the (CD80/CD86) and (MHC-I and MHC-II) pathways.

Fig. 8. FBFO@HM@aOPN enhanced antitumour immunity in vivo.

a Flow cytometry analysis of the proportions of (left) CD86⁺ (middle) MHCI⁺ and (right) CD206⁺ among f4/80⁺ CD45⁺ cells from CT2A-bearing brain tissues treated with different formulations under 660-nm irradiation (n = 5 mice for each group). Flow cytometry analysis of the proportions of (b) CD3⁺ T cells, (c) NK1.1⁺ CD3- NK cells, (d) CD3⁺ CD8⁺ TILs, and (e) GZMB⁺ TILs among the CD45⁺ cells from CT2A-bearing brain tissues treated with different formulations under 660 nm irradiation (n = 5 mice for each group). Flow cytometry analysis of the proportions of (f) central memory T cells (TCM, CD3⁺ CD44⁺ CD62L⁺) and (g) effector memory T cells (TEM, CD3⁺ CD44⁺ CD62L^-) in CT2A-bearing spleens treated with different formulations under 660-nm irradiation (n = 5 mice for each group). h Bubble chart showing the interactions between PD-1/PD-L1 activity (released by TAMs and the parasite) and PD-L1 receptor expression (expressed by lymphocyte cell subtypes) in treated and PBS-treated samples. i Schematic illustration of the experimental design for postsurgical recurrence challenge in CT2A GBM tumours. (j) Representative bioluminescence image and (k) fluorescence intensity quantification (right), as well as (l) Kaplan–Meier survival curve of luciferase-expressing CT2A-bearing mice subjected to different treatments under 660-nm irradiation (n = 5 mice in each group). m Flow cytometry analysis of the proportions of (left) CD3⁺ T cells, (middle) IFN-γ + T cells, and (right) GZMB⁺ TILs among CD45⁺ cells from CT2A-bearing brain tissues treated with different formulations under 660-nm irradiation (n = 5 mice for each group). All the data are presented as the means ± SDs. The p values were determined via two-tailed one-way ANOVA with a Tukey post hoc test (a–g, k, m). For l, p values are calculated via the log-rank (Mantel‒Cox) test. p > 0.05, not significant (ns), *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Source data are provided as a Source Data file.