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Biomimetic GBM-targeted drug delivery system boosting ferroptosis for immunotherapy of orthotopic drug-resistant GBM

Abstract

Background

Clinical studies have shown that the efficacy of programmed cell death receptor-1/programmed cell death ligand-1 (PD-1/PD-L1) inhibitors on glioblastoma (GBM) is much lower than what is expected because of the low immunogenicity of GBM. Ferroptosis of cancer cells can induce the maturation of dendritic cells (DC cells) and increase the activity of T cell. The activated T cells release IFN-γ, which subsequently induces the ferroptosis of cancer cells. Thus, the aim of this paper is to set up a new GBM-targeted drug delivery system (Fe3O4-siPD-L1@M-BV2) to boost ferroptosis for immunotherapy of drug-resistant GBM.

Results

Fe3O4-siPD-L1@M-BV2 significantly increased the accumulation of siPD-L1 and Fe2+ in orthotopic drug-resistant GBM tissue in mice. Fe3O4-siPD-L1@M-BV2 markedly decreased the protein expression of PD-L1 and increased the ratio between effector T cells and regulatory T cells in orthotopic drug-resistant GBM tissue. Moreover, Fe3O4-siPD-L1@M-BV2 induced ferroptosis of GBM cells and maturation of DC cell, and it also increased the ratio between M1-type microglia and M2-type microglia in orthotopic drug-resistant GBM tissue. Finally, the growth of orthotopic drug-resistant GBM in mice was significantly inhibited by Fe3O4-siPD-L1@M-BV2.

Conclusion

The mutual cascade amplification effect between ferroptosis and immune reactivation induced by Fe3O4-siPD-L1@M-BV2 significantly inhibited the growth of orthotopic drug-resistant GBM and prolonged the survival time of orthotopic drug-resistant GBM mice.

Graphical Abstract

Background

Glioblastoma (GBM) is an aggressive intracranial malignant tumor with high mortality and morbidity, accounting for 80% of malignant tumors in central nervous system (CNS). The overall median survival for GBM patients is only about 15 months [1, 2]. At present, surgical resection followed by radiotherapy and temozolomide (TMZ) chemotherapy is considered to be the basic treatment for patients with newly diagnosed GBM [3,4,5]. However, it is frustrating that long-term use of TMZ in GBM patients inevitably leads to the overexpression of O6-methylguanine DNA methyltransferase (MGMT) in GBM cells, which results in the resistance of GBM cells to TMZ. Subsequently, the efficacy of TMZ is significantly reduced or even lost [6, 7]. Therefore, it is an urgent need to find new treatment methods for TMZ-resistant GBM.

Immunotherapy, a very promising cancer treatment method, inhibits tumor growth and metastasis by inducing systemic and sustained immune response [8]. However, the efficacy of immunotherapy on GBM is much lower than what is expected. This is resulted from the following reasons. Firstly, as compared with other cancer such as non-small cell lung cancer, GBM in most cases shows a lower tumor mutational burden [9], resulting in lower immunogenicity of GBM cells and less recruitment of effector T cells (Teff cell) in GBM tissue [10]. Secondly, GBM cells usually recruit regulatory T cell (Treg cell) into GBM tissue by secreting chemokines such as colony stimulating factor 1 (CSF1), C-X-C Motif Chemokine Ligand 12 (CXCL12), C-X-C Motif Chemokine Ligand 1 (CXCL1) and granulocyte–macrophage colony stimulating factor (GM-CSF) [11]. Treg cell inhibits the function of Teff cell, subsequently reducing the generation of interleukin-2 (IL-2) and interferon-γ (IFN-γ) [12, 13]. Finally, GBM cells are able to polarize anti-tumor M1 type microglia/macrophage into the immunosuppressive M2 type microglia/macrophage by secreting immunomodulatory cytokines [14, 15]. M2 type microglia/macrophage also inhibits the function of Teff cell and promotes the progression of GBM by secreting cytokines such as interleukin-6 (IL-6), interleukin-10 (IL-10) and C–C Motif Chemokine Ligand 2 (CCL2) [16].

Ferroptosis is a form of iron-dependent cell death. The essences of ferroptosis are the over-load of Fe2+, depletion of glutathione (GSH) and the decrease of glutathione peroxidase (GPX4) [17, 18]. Lipid oxides cannot be metabolized through GPX4. Subsequently, a large number of hydroxyl radicals are produced through Fenton reaction, leading to lipid peroxidation in cancer cells. This finally results in cancer cell death [19, 20]. Many studies have shown that ferroptosis also leads to the maturation of DC cells in cancer tissue in vivo, and the matured DC cells present antigen to T lymphocytes to activated Teff [21, 22]. Moreover, PD-1/PD-L1 inhibitor is able to activate Teff cell to secrete IFN-γ [23]. IFN-γ secreted by activated Teff cell inhibits the cysteine transporter (xCT) and subsequently prevents the cysteine from being taken up by cancer cells, resulting in the reduction of GSH synthesis in cancer cells. Then, ferroptosis is significantly enhanced in turn [24,25,26]. In theory, ferroptosis inducer and PD-1/PD-L1 inhibitor can mutually enhance each efficacy when they are simultaneously used to treat GBM.

siRNA shows high specificity and low toxicity in cancer treatment [27]. It has potential application value to interfere PD-L1 protein synthesis in drug-resistant GBM cells by using siPD-L1. However, lack of suitable siPD-L1 delivery vector and easy degradation of siPD-L1 in blood circulation are the main obstacles that limit the application of siPD-L1 in the treatment of GBM [28, 29]. As compared with other carriers, Fe3O4 nanoparticle is a promising siPD-L1 carrier [30]. Firstly, Fe3O4 nanoparticle displays good biocompatibility and biodegradability, and it is easily available. Fe3O4 nanoparticle has been approved for clinic use by the Food and Drug Administration (FDA) [31]. Secondly, Fe3O4 nanoparticle significantly increases the intracellular iron content especially Fe2+ [32, 33], which provides sufficient substrate for ferroptosis in drug-resistant GBM cells. Last, Fe3O4 nanoparticle shows super paramagnetism, which allows it to be directed delivery by an external magnetic field [34]. However, Fe3O4 nanoparticle is difficult to cross blood–brain barrier (BBB) [35, 36]. Recent studies have shown that GBM tissue can recruit microglia by secreting chemokines such as C-X3-C motif chemokine ligand 1 (CX3CL1) and CSF-1 [37,38,39]. In theory, microglia membrane coated Fe3O4 nanoparticle can be recruited to drug-resistant GBM.

In this study, disulfide bonds were used to connect thiolated siPD-L1 and thiolated Fe3O4 nanoparticles to increase the stability of siPD-L1 in blood circulation. Fe3O4 nanoparticles connected with siPD-L1 (Fe3O4-siPD-L1) are further coated with microglial membrane (M-BV2) to form a biomimetic brain-targeted nanoparticle Fe3O4-siPD-L1@M-BV2. After Fe3O4-siPD-L1@M-BV2 was taken up by orthotopic drug-resistant GBM cells, the disulfide bond between Fe3O4 nanoparticles and siPD-L1 was broken by intracellular GSH, releasing siPD-L1 and inhibiting the protein expression of PD-L1 in orthotopic drug-resistant GBM cells [40]. Subsequently, Teff cell was activated to enhance the killing effect on drug-resistant GBM cell [41]. At the same time, Fe3O4-siPD-L1@M-BV2 facilitated the ferroptosis of drug-resistant GBM cells, which further activated Teff cell by improving the maturation of DC cells. Moreover, activated Teff cell enhanced the ferroptosis of drug-resistant GBM cells in turn by secreting IFN-γ. Finally, there forms a cascade amplification effect between ferroptosis and immune activation in orthotopic drug-resistant GBM tissue (Scheme 1).

Scheme 1.
scheme 1

Reciprocal cascade amplification between ferroptosis and immunotherapy of Fe3O4-siPD-L1@M-BV2

Materials and methods

Materials

Thiolated Fe3O4 nanoparticle was bought from Ruixibio (Xi’an, China). Thiolated siPD-L1 (5’-GAAGGGAAAUGCUGCCCUUTT-3’, 5’-AAGGGCAUUUCCCUUCTT-3’) and cyanine5 labeled siPD-L1 were obtained from GenePharma (Shanghai, China). PD-L1 antibody, GPX4 antibody, xCT antibody, clusters of differentiation 31 (CD31) antibody, clusters of differentiation 44 (CD44) antibody, E-cadherin antibody, N-cadherin antibody, matrix metalloproteinases-9 (MMP-9) antibody, CX3CL1 antibody, colony-stimulating factor 1 receptor (CSF-1R) antibody, clusters of differentiation16/32 (CD16/32) antibody, clusters of differentiation 206 (CD206) antibody, ionized calcium binding adaptor molecule 1 (Iba-1) antibody were bought from Abcam (London, England). CSF-1 antibody and C-X3-C motif chemokine receptor 1 (CX3CR1) antibody were bought from Proteintech (Beijing, China). β-actin antibody were bought from Affinity (Colorado, USA). GM-CSF was bought from Peprotech (Suzhou, China). Anti-mouse clusters of differentiation 11 c-allophycocyanin (CD11c-APC), anti-mouse clusters of differentiation 80-fluorescein (CD80-FITC), anti-mouse clusters of differentiation86-phycoerythrin (CD86-PE), anti-mouse clusters of differentiation 3-allophycocyanin (CD3-APC), anti-mouse clusters of differentiation 8-fluorescein isothiocyanate (CD8-FITC), anti-mouse interferon-γ-phycoerythrin-sulfo-cyanine7 (IFN-γ-PE-Cy7), anti-mouse clusters of differentiation4-phycoerythrin-sulfo-cyanine7 (CD4-PE-Cy7), anti-mouse clusters of differentiation 25-fluorescein isothiocyanate (CD25-FITC), anti-mouse forkhead box p3-phycoerythrin (FoxP3-PE) were obtained from Biolegend (California, USA). Enzyme linked immunosorbent assay (ELISA) kits were obtained from Cloud-Clone corp (Wuhan, China). GSH detection kit and H2O2 detection kit were bought from Solarbio (Beijing, China). Reactive oxygen species (ROS) detection kit was bought from Bestbio (Shanghai, China). Boron difluoride pyrrole fluorescent dyes-C11 (BODIPY-C11) staining solution was bought from ThermoFisher (Massachusetts, USA).

GL261 cells, HT-22 cells, BV2 cells, RAW264.7 cells and bEnd3 cells were purchased from CytoBiotech (Xi’an, China). TMZ-resistant GL261 cells were induced in our lab. 6 weeks old C57 mice and 8 weeks old Sprague-Dawley (SD) male rats were provided by the Experimental Animal Center of Air Force Medical University (Xi’an, China).

Extraction of BV2 cell membrane

BV2 cells in logarithmic growth phase were collected and mixed with 3 mL low-osmotic lysate and 30 μL protease inhibitor. Then, BV2 cells suspension was immerged into liquid nitrogen. After BV2 cells suspension was frozen and thawed for 3 times, the cell lysate was centrifuged for 10 min at 4 ℃ (14,000 × g). The supernatant was discarded, and 3 mL sterilized deionized water was added into precipitate. The mixture was performed ultrasound for 2 min, and supernatant was collected by centrifuging mixture for 20 min at 4 ℃ (14,000 × g). Then BV2 cell membrane (M-BV2) was obtained by lyophilizing the supernatant.

Preparation of Fe3O4-siPD-L1@M-BV2

Thiolated siPD-L1 (150 μL, 0.264 mg/mL), H2O2 (30%, 55 μL) and thiolated Fe3O4 nanoparticles (120 μL, 5 mg/mL) were added into enzyme-free EP tube, and the mixture was stirred for 1 h at room temperature to connect siPD-L1 with Fe3O4 nanoparticles by disulfide bond. The reaction mixture was centrifuged for 10 min at 4 ℃ (9000 × g), and the supernatant was discarded. The precipitate was washed with DEPC water for 6 times to completely remove free siPD-L1. After that, precipitate was re-suspended into PBS buffer to get Fe3O4-siPD-L1 suspension. The mass ratio between thiolated Fe3O4 and thiolated siPD-L1 was optimized by agarose gel electrophoresis. Scramble siPD-L1 was used to prepare Fe3O4-siNC nanoparticle by using the same method in the preparation of Fe3O4-siPD-L1. Finally, 3 mL sterilized deionized water containing 10 mg M-BV2 was added into Fe3O4-siPD-L1 suspension. After being performed ultrasonic for 1 min, the mixture solution was incubated at 37 ℃ for 10 min. The mixture solution was extruded through 0.4 μm polycarbonate membrane for 21 times to obtain Fe3O4-siPD-L1@M-BV2. Fe3O4-FAM was prepared by reacting Fe3O4-siPD-L1 with maleimide modified carboxyfluorescein (FAM-MAL). To prepare Fe3O4-FAM@M-BV2, M-BV2 was coated on the surface of Fe3O4-FAM by using the same method in the preparation of Fe3O4-siPD-L1@M-BV2.

Characterization of Fe3O4-siPD-L1@M-BV2

Firstly, the morphology and element composition of Fe3O4-siPD-L1@M-BV2 were investigated by field emission transmission electron microscopy (TEM, FEI Talos F200X, Super-X, ThermoFisher, USA). Particle size, zeta potential and the stability of Fe3O4 nanoparticles and Fe3O4-siPD-L1@M-BV2 in deionized water, phosphate buffer solution (PBS) and fetal bovine serum (FBS, 10%) were determined by dynamic laser particle size analyzer (Delsa Nano C, Beckman, USA). Fe3O4-siPD-L1@M-BV2 dispersion was placed at 37 ℃ in a constant temperature and humidity incubator. After Fe3O4-siPD-L1 was labeled by FAM and M-BV2 was labeled by DiI, the membrane structure of Fe3O4-siPD-L1@M-BV2 was observed by laser scanning confocal microscope (LSCM, Nikon, Japan). The proteins maintained in Fe3O4-siPD-L1@M-BV2 were observed by SDS-PAGE [42]. Besides, proteins such as CX3CR1, CSF-1R, CX3CL1 and CSF-1 in Fe3O4-siPD-L1@M-BV2 were detected by western blot [37]. The hemolysis of Fe3O4-siPD-L1@M-BV2 was investigated by using rat erythrocytes [43]. The release of siPD-L1 from Fe3O4-siPD-L1@M-BV2 in GSH-containing solution was observed by gel retardation assay [44]. The stability of Fe3O4-siPD-L1@M-BV2 in RNase A-containing buffer was observed by gel retardation assay.

The selective uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell

GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells in logarithmic growth phase were separately inoculated into different 24-well plates containing cover glass at density of 2 × 105 cells/mL and incubated at 37 ℃ for 24 h. Four cover glass inoculated with different cells were transferred into one well of 6-well plate, and 2 mL of fresh serum-free dulbecco's modified eagle medium (DMEM) containing Fe3O4-FAM@M-BV2 (the equivalent Fe3O4 concentration was 200 μg/mL) was added into each well. Fe3O4-FAM was used as control. The cells were cultured for 1, 2 and 4 h, respectively. (1) The cells were collected and re-suspend in PBS. The uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell was detected by flow cytometer (Beckman, A00-1–1102, USA). (2) The cell culture medium was discarded, and the cells were fixed with 4% paraformaldehyde for 10 min. The cells were washed with PBS for 3 times. Then cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) solution (0.5 μg/mL) for 10 min. After the cells were washed with PBS for 3 times, the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell was observed by LSCM.

The uptake mechanism of Fe3O4-FAM@M-BV2 by GL261/TR cell

(1) Fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 (the equivalent Fe3O4 concentration was 200 μg/mL) was co-incubated with CX3CR1 antibody (1 μg/mL) for 2 h at 37 ℃, and then the mixture was added into a 24-well plate pre-inoculated with GL261/TR cells on a cover glass. The cell was culture at 37 ℃ for 4 h. (2) Fresh serum-free DMEM containing CX3CL1 antibody (1 μg/mL) was added into a 24-well plate pre-inoculated with GL261/TR cells on a cover glass. After co-incubation at 37 ℃ for 2 h, fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 (the equivalent Fe3O4 concentration was 200 μg/mL) was added into cell culture medium, and cell was cultured at 37 ℃ for 4 h. (3) Chlorpromazine solution (10 μg/mL), colchicine solution (800 μg/mL), methyl-β-cyclodextrin solution (5 μg/mL), and 2-deoxy-D-glucose (900 μg/mL) were added into 24-well plates pre-inoculated with GL261/TR cells on a cover glass. The cell was incubated at 37 ℃ for 2 h, and then fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 (equivalent Fe3O4 concentration was 200 μg/mL) was added into cell culture medium. The cell was cultured at 37 ℃ for 4 h. (4) The GL261/TR cell on a cover glass was incubated at 4 ℃ for 1 h, and then fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 (4 ℃, equivalent Fe3O4 concentration was 200 μg/mL) was added into cell culture medium. The cell was cultured at 4 ℃ for 4 h. After that, the cells were collected and re-suspend in PBS. The uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell, HT-22 cell, BV2 cell and RAW264.7 cell was detected by flow cytometer. Besides, the cells were fixed with 4% paraformaldehyde for 10 min. The cells were washed with PBS for 3 times. Then cells were stained with DAPI solution (0.5 μg/mL) for 10 min. After the cells were washed with PBS for 3 times, the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells was observed by LSCM.

The silencing effect of Fe3O4-siPD-L1@M-BV2 on PD-L1 protein in GL261 cells and GL261/TR cells

GL261 cells and GL261/TR cells in logarithmic growth phase were inoculated into 6-well plates at density of 5 × 105 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe3O4-siNC (150 nM scramble siPD-L1), siPD-L1@Lipo2000 (150 nM siPD-L1), Fe3O4-siPD-L1 (150 nM siPD-L1) and Fe3O4-siPD-L1@M-BV2 (150 nM siPD-L1) and cultured for 48 h. Then, cells were collected and proteins were extracted. Western blot was used to investigate the silencing effect of Fe3O4-siPD-L1@M-BV2 on PD-L1 protein in GL261 cells and GL261/TR cells.

Effects of Fe3O4-siPD-L1@M-BV2 on the viability of GL261/TR cells

GL261/TR cells in logarithmic growth phase were inoculated into 96-well plates at density of 3 × 104 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with serum-free DMEM medium containing Fe3O4, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 (equivalent Fe3O4 concentration was 10, 20, 50, 100, 200 μg/mL), and the cells were incubated at 37 ℃ for 48 h. 20 μL MTT solution (5 mg/mL) was added into each well and incubated at 37 ℃ for 4 h. The culture medium in well was discarded, and 150 μL DMSO was added into each well. The absorbance of each well was measured at 490 nm by enzyme linked immune-analyzer (Bio-Rad Laboratories, Inc. California, USA), and the cell survival rate was calculated.

MTT method was also used to illuminate whether Fe3O4-siPD-L1@M-BV2 could induce ferroptosis. Briefly, GL261/TR cells in logarithmic growth phase were inoculated into 96-well plates at density of 3 × 104 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe3O4-siPD-L1@M-BV2 (equivalent Fe3O4 concentration was 10, 20, 50, 100, 200 μg/mL) at the present of IFN- γ(10 ng/mL), ferrostatin1 (Fer-1, 10 μM) and deferoxamine (DFO, 100 μM). The cells were incubated at 37 ℃ for 48 h. 20 μL MTT solution (5 mg/mL) was added into each well, and cells were incubated at 37℃ for 4 h. The culture medium in well was discarded, and 150 μL DMSO was added into each well. The absorbance of each well was measured at 490 nm by enzyme linked immune-analyzer, and the cell survival rate was calculated.

Staining of living and dead cells

GL261/TR cells in logarithmic growth phase were inoculated into 6-well plates at density of 5 × 105 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe3O4-siNC, Fe3O4, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2 + Fer-1, Fe3O4-siPD-L1@M-BV2 + DFO and Fe3O4-siPD-L1@M-BV2 + IFN-γ. The equivalent Fe3O4 concentration was 200 μg/mL. The concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. Cells were incubated at 37 ℃ for 48 h. The cells were collected and washed twice with 1 × assay buffer. Then, 1 mL staining solution was used to re-suspended cells, and cell suspension was incubated at 37 ℃ for 20 min. After centrifugation (2000 × g, 5 min), the supernatant was discarded, and the cells were re-suspended in PBS. The living and dead cells was observed under a fluorescence microscope (Nikon, Japan).

Effects of Fe3O4-siPD-L1@M-BV2 on expression of GPX4 and xCT in GL261/TR cells

GL261/TR cells in logarithmic growth phase were inoculated into 6-well plates at density of 5 × 105 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing IFN-γ, Fe3O4, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2 + Fer-1, Fe3O4-siPD-L1@M-BV2 + DFO and Fe3O4-siPD-L1@M-BV2 + IFN-γ. The equivalent Fe3O4 concentration was 200 μg/mL. The concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. The cells were incubated at 37 ℃ for 48 h. Then, cells were collected and proteins were extracted. Western blot was used to detect protein expression of GPX4 and xCT in GL261/TR cells.

Effects of Fe3O4-siPD-L1@M-BV2 on GSH and H2O2 level in GL261/TR cells

GL261/TR cells in logarithmic growth phase were inoculated into cell culture bottle (25 cm2) at density of 5 × 105 cells/mL and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe3O4-siNC, Fe3O4, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2 + Fer-1, Fe3O4-siPD-L1@M-BV2 + DFO and Fe3O4-siPD-L1@M-BV2 + IFN-γ. The equivalent Fe3O4 concentration was 200 μg/mL. The concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. After the cells were incubated at 37 ℃ for 48 h, the cells were collected. The cells were frozen and thawed for 3 times. The cell lysate was centrifuged for 10 min (8000 × g, 15 min), and the supernatant was collected. The concentration of GSH and H2O2 in the supernatant was respectively detected by using GSH and H2O2 detection kit.

Effects of Fe3O4-siPD-L1@M-BV2 on ROS and lipid peroxidation level in GL261/TR cells

GL261/TR cells in logarithmic growth phase were inoculated into 6-well plates at density of 5 × 105 cells/mL per well and incubated at 37 ℃ for 24 h. The culture medium was replaced with fresh serum-free DMEM medium containing Fe3O4-siNC, Fe3O4, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2 + Fer-1, Fe3O4-siPD-L1@M-BV2 + DFO and Fe3O4-siPD-L1@M-BV2 + IFN-γ. The equivalent Fe3O4 concentration was 200 μg/mL. The concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively. The cells were incubated at 37 ℃ for 24 h. The cells were washed with serum-free DMEM medium for 3 times. (1) For detection ROS level, 1 mL of DHE dye solution (diluted 1000 times with serum-free DMEM medium) was added into each well. The cells were incubated at 37 ℃ for 1 h. Then cells were fixed with 4% paraformaldehyde and stained with DAPI solution (0.5 μg/mL). After the cells were washed with PBS for 3 times, the ROS in GL261/TR cells was observed under fluorescence microscope. (2) For detection lipid peroxidation level, 1 mL of C11 BODIPY dye solution (diluted 1000 times with serum-free DMEM medium) was added into each well. The cells were incubated at 37 ℃ for 0.5 h. Then cells were fixed with 4% paraformaldehyde and stained with DAPI solution (0.5 μg/mL). After the cells were washed with PBS for 3 times, the lipid peroxidation (LPO) in GL261/TR cells was observed under fluorescence microscope.

Effects of Fe3O4-siPD-L1@M-BV2 on DC cell maturation in vitro

Femur and tibia of C57 male mice were isolated and immersed in 75% ethanol for 5 min. Then, femur and tibia were immersed in serum-free roswell park memorial institute 1640 medium (RPMI1640). The ends of the femur and tibia were cut off with scissors, and the bone marrow cells were rinsed out from the femur and tibia by using serum-free RPMI1640 medium. The culture medium containing bone marrow cells was filtered (70 μm, BioFIL). The filtrate was centrifuged (1000×g, 3 min), and supernatant was discard. The cells were re-suspended with red blood cell lysate (R1010, Solarbio). After the cell suspension was placed at room temperature for 1.5 min, RPM1640 complete medium was added. The supernatant was discarded by centrifugation (1000×g, 3 min), and the cells were re-suspended by RPM1640 complete medium containing GM-CSF (20 ng/mL). The cells were inoculated into 6-well plates at a density of 3×106 cells/mL in each well (1 mL). 3 days later, 1 mL of RPMI1640 complete medium containing GM-CSF (20 ng/mL) was added into each well and cultured for another 2 days.

2 mL of cell culture medium containing Fe3O4, Fe3O4-siNC, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2+IFN-γ, Fe3O4-siPD-L1@M-BV2+DFO, and Fe3O4-siPD-L1@M-BV2+Fer-1 (the equivalent Fe3O4 concentration was 200 μg/mL, the concentration of IFN-γ, Fer-1 and DFO was 10 ng/mL, 10 μM and 100 μM, respectively) was added into transwell donor chamber planted GL261/TR cells, respectively. After incubation for 6 h, donor chamber was transferred to a 6-well plate inoculated with DC cells at the bottom and cultured for 24 h. DC cells were collected and re-suspended with PBS. APC-CD11c antibody (0.2 mg/mL), FITC-CD80 antibody (0.5 mg/mL) and PE-CD86 antibody (0.2 mg/mL) were added into cell culture medium, and cell was incubated at 4 ℃ for 30 min in dark room. The cells were collected and re-suspended with 200 μL PBS. The proportion of CD11c+, CD86+ and CD80+ DCs was detected by flow cytometer.

Efficiency of Fe3O4-siPD-L1@M-BV2 transport across the in vitro BBB

bEnd3 cells in the logarithmic growth phase were inoculated into the transwell donor chamber at a density of 5 × 105 cells/mL per well. Serum-free DMEM medium was added into the recipient chamber, and bEnd3 cells were cultured at 37 ℃. The complete medium was replaced every two days. The resistance between transwell donor chamber and recipient chamber was measured by using a resistance meter. When the resistance value exceeded 200 Ω/cm2, the in vitro BBB model was regarded to be successful established [45].

The transwell donor chamber was transferred into a 24-well plate inoculated with GL261/TR cells at the bottom. 400 μL of fresh serum-free DMEM medium containing Fe3O4-FAM, Fe3O4-FAM@M-BV2 were added into donor chamber (the equivalent Fe3O4 concentration was 200 μg/mL). 0.8 mL of DMEM medium was added into recipient chamber. At the same time, a group with a magnet outside of the 24-well plates (Fe3O4-FAM@M-BV2 + magnet) was designed. After cell was incubated for 1 h, 4 h, 8 h and 12 h, the resistance values between transwell donor chamber and recipient chamber was measured to evaluate the integrity of the in vitro BBB model. The fluorescence intensity of the medium in recipient chamber was determined by fluorescence spectrophotometer (HITACHI, F-2700, Japan), and the in vitro BBB transmission efficiency of Fe3O4-FAM@M-BV2 was calculated. Finally, GL261/TR cells at the bottom of recipient chamber were collected and re-suspend in PBS. The uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell was detected by flow cytometer. Besides, GL261/TR cells at the bottom of recipient chamber were fixed with 4% paraformaldehyde and then were stained with DAPI solution (0.5 μg/mL). After the cells were washed with PBS for 3 times, the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells was observed by LSCM after it penetrated in vitro BBB.

Effects of CX3CL1 and CSF-1 on the transport of Fe3O4-siPD-L1@M-BV2 across the in vitro BBB

(1) After the successful establishment of the in vitro BBB model, the transwell donor chamber was transferred into a 24-well plates inoculated with GL261/TR cells at the bottom, and 800 μL fresh serum-free DMEM was added into recipient chamber. (2) After the successful establishment of the in vitro BBB model, the transwell donor chamber was transferred into a 24-well plates without GL261/TR cells at the bottom, and 800 μL fresh serum-free DMEM containing CX3CL1 (200 ng/mL) or CSF-1 (100 ng/mL) was added into recipient chamber. After that, 400 μL of fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 was added into transwell donor chamber (the equivalent Fe3O4 concentration was 200 μg/mL). The 24-well plates without GL261/TR cell and chemokine was used as the control. After incubation for 4 h, the fluorescence intensity of the culture medium in recipient chamber was measured by fluorescence spectrophotometer.

After the successful establishment of the in vitro BBB model, transwell donor chamber was transferred into a 24-well plate inoculated with GL261/TR cells. (1) Fe3O4-FAM@M-BV2 (the equivalent Fe3O4 concentration was 200 μg/mL) was incubated with fresh serum-free DMEM containing CX3CR1 or CSF-1R antibody for 2 h at 37 ℃, and then they were added into transwell donor chamber. 800 μL of serum-free DMEM was added into recipient chamber. (2) Fresh serum-free DMEM containing CX3CL1 or CSF-1 antibody was added into recipient chamber and incubated with GL261/TR cells at 37 ℃ for 2 h. After that, 400 μL of fresh serum-free DMEM containing Fe3O4-FAM@M-BV2 was added into donor chamber (the equivalent Fe3O4 concentration was 200 μg/mL). The concentration of antibody was 1 μg/mL. Fe3O4-FAM@M-BV2 without incubation with antibody was used as control. After incubation for 4 h, the fluorescence intensity of the culture medium in recipient chamber was measured by fluorescence spectrophotometer.

Establishment of orthotopic drug-resistant GBM model in mice

Luciferase expressed GL261/TR cell (Luc-GL261/TR) in logarithmic growth phase were prepared as 2 × 107/mL cell suspension. C57 mice were anesthetized and fixed on the operation table, and a micro-injector was inserted into the skull at the right front 2 mm of the intersection of sagittal suture and coronal suture. 5 μL of cell suspension was slowly injected into the brain with the brain stereotactic locator to establish orthotopic drug-resistant GBM model in vivo.

Distribution and pharmacokinetics of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice

On the 10th day after plantation of Luc-GL261/TR cell, luciferase substrate was intraperitoneally injected (150 mg/kg). 5 min post injection, the mice were anesthetized with isoflurane. The GBM growth was observed by bioluminescence imaging (IVIS Lumina II, Caliper, USA), and the mice that the volume of orthotopic drug-resistant GBM did not meet the requirements were excluded.

The GBM-bearing mice were randomly divided into 4 groups. siPD-L1, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet (Additional file 1: Fig. S1) were injected into GBM-bearing mice via tail vein (siPD-L1 was labeled by Cy5, equivalent siPD-L1 dose was 0.3 mg/kg). 6 h and 12 h later, in vivo bioluminescence imaging was used to observe the fluorescence distribution in the whole body of orthotopic drug-resistant GBM mice. The integrated brain targeting efficiency was calculated with the following equation. The integrated brain targeting efficiency = (fluorescence intensity in brain organ/fluorescence intensity in whole body) × 100%. Blood, brain, heart, liver, spleen, lung and kidney tissues were collected at 0.08, 1, 3, 6, 12 and 24 h after drug administration. In vivo bioluminescence imaging was used to observe the distribution of Cy5 labeled Fe3O4-siPD-L1@M-BV2 in brain, heart, liver, spleen, lung and kidney. The brain tissue was immobilized in 4% paraformaldehyde for 24 h. After tissue was sectioned, DAPI was used to label the nuclei, CD31 antibody was used to label the tumor vessels, and the distribution of Fe3O4-siPD-L1@M-BV2 in drug-resistant glioma tissue was observed by LSCM. Fluorescence spectrophotometer was used to detect the Cy5 labeled Fe3O4-siPD-L1@M-BV2 concentration in plasma samples. The GBM tissue was isolation from brain tissue. The GBM tissue and normal brain tissue were respectively ground with 0.8 mL PBS buffer in an ice bath, and the tissue homogenate was centrifuged (9000 × g, 15 min, 4 ℃). The precipitation and supernatant were separated. (1) The concentration of Cy5 labeled Fe3O4-siPD-L1@M-BV2 in extracellular fluid of brain tissue was detected by fluorescence spectrophotometer. (2) The precipitation was ground with 0.6 mL RIPA cell lysate. The cell lysate was centrifuged (9000 × g, 20 min, 4 ℃). The intracellular Cy5 labeled Fe3O4-siPD-L1@M-BV2 concentration in GBM tissue was detected by fluorescence spectrophotometer. (3) The intracellular content of Fe2+ in GBM tissue was detected by using Perls stain kit. The precipitation was ground with 0.6 mL RIPA cell lysate. The cell lysate was centrifuged (9000 × g, 20 min, 4 ℃). 0.5 mL of supernatant was added into 0.5 mL NH4Fe(SO4)2 solution. The above mixture solution was shaken at room temperature for 30 min. Then, the absorbance value of mixture solution at 700 nm was detected by using UV spectrophotometer. After that, dilute nitric acid was added into mixture solution, and the absorbance value of mixture solution at 700 nm was detected again by using UV spectrophotometer. The content of Fe2+ can be calculated according to the change of absorbance value.

The therapeutic effect of Fe3O4-siPD-L1@M-BV2 on orthotopic drug-resistant GBM in mice

On the 10th day after plantation of Luc-GL261/TR cell, luciferase substrate was intraperitoneally injected (150 mg/kg). 15 min later, the mice were anesthetized with isoflurane. The GBM growth was observed by bioluminescence imaging, and the mice that the volume of orthotopic drug-resistant GBM did not meet the requirement was excluded.

The GBM-bearing mice were randomly divided into 10 groups: normal saline group, TMZ group (44 mg/kg), Fe3O4 group, Fe3O4-siNC group (equivalent siNC dose was 0.3 mg/kg), Fe3O4-siPD-L1 group (equivalent siPD-L1 dose was 0.3 mg/kg), Fe3O4-siPD-L1@M-BV2 group (equivalent siPD-L1 dose was 0.3 mg/kg), Fe3O4-siPD-L1@M-BV2 group (equivalent siPD-L1 dose was 1 mg/kg), Fe3O4-siNC@M-BV2 (equivalent siNC dose was 1 mg/kg), Fe3O4-siNC@M-BV2 + magnet (equivalent siNC dose was 1 mg/kg) and Fe3O4-siPD-L1@M-BV2 + magnet group (equivalent siPD-L1 dose was 1 mg/kg). Different formulations were injected into GBM-bearing mice through tail vein once every three days, for a total of 4 times. (1) The body weight and the death of GBM-bearing mice were recorded. The survival curve was drawn, and the median survival time was calculated. (2) On 10, 17, 20 and 23 day after plantation of Luc-GL261/TR cell, the mice were intraperitoneally injected with luciferase substrate (150 mg/kg), the orthotopic GBM growth was observed by in vivo bioluminescence imaging. On the 24th day after plantation of Luc-GL261/TR cell, the GBM-bearing mice were sacrificed. Blood of GBM-bearing mice was collected and serum was separated. The contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CREA) in serum were detected by automatic biochemical analyzer (Chemray 800, Shenzhen, China). The lymphocytes in orthotopic GBM tissue were isolated. The number of CD11c+CD86+CD80+ cells (mDCs), CD4+CD25+FoxP3+T cells (Treg cell) and CD3+CD8+IFN-γ+T cells (Teff cell) in orthotopic GBM tissue were detected by flow cytometer. The expressions of PD-L1, invasion-related proteins including E-cadherin, N-cadherin, CD44, matrix metalloproteinases-9 (MMP-9), tumor necrosis factor-α (TNF-α), ferroptosis-related proteins including GPX4 and xCT in orthotopic drug-resistant GBM tissue were detected by western blot. The TNF-α, IFN-γ, IL-6, IL-10 and IL-12 level in orthotopic drug-resistant GBM tissue was determined by ELISA kit. H&E staining, Ki67 staining and tdT-mediated dUTP nick-end labeling (TUNEL) staining was used to investigate the effects of Fe3O4-siPD-L1@M-BV2 on cell morphology, proliferation and apoptosis in orthotopic drug-resistant GBM tissue. The expression of CD16/32, CD206, Iba-1, GPX4 and PD-L1 in paraffin sections of orthotopic drug-resistant GBM tissue was observed by immunofluorescence staining method. Dihydroethidium (DHE) probe was used to stain ROS in frozen sections of drug-resistant GBM tissue. The content of GSH in orthotopic drug-resistant GBM tissue was determined by GSH detection kit. H&E staining was performed on heart, liver, spleen, lung and kidney tissues to investigate the in vivo toxicity of Fe3O4-siPD-L1@M-BV2 in GBM-bearing mice.

Statistical analysis

All data are expressed as mean ± standard deviation. The statistics analysis of each group was performed by using one-way ANOVA with SPSS 26.0 statistical software. p < 0.05 was considered statistically significant.

Results

Characterization of Fe3O4-siPD-L1@M-BV2

The schematic diagram of Fe3O4-siPD-L1@M-BV2 is showing in Fig. 1A. The agarose gel electrophoresis experiment showed that siPD-L1 and Fe3O4 were completely connected when mass ratio between siPD-L1 and Fe3O4 was 1:15 (Fig. 1B). The average particle size of Fe3O4 was 130 nm (Fig. 1C). Mapping results showed that Fe3O4 contained O, Fe and S elements. Beside O, Fe and S, Fe3O4-siPD-L1 contained N and P elements, indicating that siPD-L1 was successfully connected with Fe3O4 (Fig. 1D; Additional file 1: Fig. S2A). The average particle size and zeta potential of Fe3O4-siPD-L1@M-BV2 was 144 nm and − 27 mV (Fig. 1E; Additional file 1: Fig. S2B). The protein bands of Fe3O4-siPD-L1@M-BV2 were consistent with that of BV2 cell membrane (M-BV2) (Fig. 1F), indicating that the proteins on BV2 cells membrane were well retained in Fe3O4-siPD-L1@M-BV2. Western blot results indicated that microglial chemokines CX3CL1 and CSF-1 were highly expressed in GL261/TR cells, and their receptors CX3CR1 and CSF-1R were retained in M-BV2 and Fe3O4-siPD-L1@M-BV2 (Fig. 1G). Moreover, LSCM experiment showed that red color of M-BV2 was completely merged with green color of Fe3O4 (Fig. 1H). The above results demonstrated that M-BV2 was successfully coated on the surface of Fe3O4-siPD-L1. Fe3O4-siPD-L1@M-BV2 remained stable within 7 days in PBS and water, and it was stable within 3 days in 10%FBS (Additional file 1: Fig. S2C). TEM results showed that the appearance of Fe3O4 and Fe3O4-siPD-L1@M-BV2 was spherical (Additional file 1: Fig. S2D, E). GSH promoted the release of siPD-L1 from Fe3O4-siPD-L1@M-BV2 in concentration-dependent manner (Fig. 1I). The naked siPD-L1 was rapidly degraded in RNase A solution, while Fe3O4-siPD-L1@M

Fig. 1
figure 1

Characterization of Fe3O4-siPD-L1@M-BV2. A Diagram of the composition of Fe3O4-siPD-L1@M-BV2. B The optimized mass ratio between siPD-L1 and Fe3O4 screened by agarose gel electrophoresis. C Particle size distribution of Fe3O4. D Element mapping analysis diagram of Fe3O4-siPD-L1. E Particle size distribution of Fe3O4-siPD-L1@M-BV2. F SDS-PAGE protein analysis of Fe3O4-siPD-L1@M-BV2. G The protein expressions of CX3CL1, CSF-1, CX3CR1 and CSF-1R in GL261/TR cell, BV2 cell, GL261/TR cell membrane (M-GL261/TR), BV2 cell membrane (M-BV2) and Fe3O4-siPD-L1@M-BV2. H LSCM imaging of Fe3O4-siPD-L1@M-BV2. Green color (FAM) stands for Fe3O4-siPD-L1, and red color (DiI) stands for BV2 cell membrane. I GSH responsive of Fe3O4-siPD-L1@M-BV2 observed by agarose gel electrophoresis. J Stability of siPD-L1 in RNase A observed by agarose gel electrophoresis

-BV2 protected siPD-L1 from degradation by RNase A (Fig. 1J). Fe3O4-siPD-L1@M-BV2 did not cause hemolysis reaction (Additional file 1: Fig. S2F, G).

Cellular uptake and gene silence efficiency of Fe3O4-FAM@M-BV2

GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells at logarithmic growth stage were respectively planted into 24-well plates containing cover glass at the same density. Cells were incubated at 37 ℃ for 24 h, the densities of the four cells were basically the same (Additional file 1: Fig. S3). When GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells were co-cultured in a petri dish (Fig. 2A), they took up Fe3O4-FAM@M-BV2 in a time-dependent manner. BV2 cells and RAW264.7 cells took up more amount of Fe3O4-FAM than Fe3O4-FAM@M-BV2. As compared with Fe3O4-FAM, more amount of Fe3O4-FAM@M-BV2 was taken up by GL261/TR cells. Moreover, GL261/TR cells took up much more amount of Fe3O4-FAM@M-BV2 than HT-22 cells did (Fig. 2B–F; Additional file 1: Fig. S4). This indicated that M-BV2 coating significantly reduced the uptake of Fe3O4-FAM@M-BV2 by BV2 cells and RAW264.7 cells. Fe3O4-FAM@M-BV2 was specifically taken up by GL261/TR cells. Furthermore, low temperature and 2-deoxy-D-glucose pretreatment markedly reduced the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells, indicating that the endocytosis of Fe3O4-FAM@M-BV2 by GL261/TR cells required energy supply. Colchicine significantly inhibited the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells, suggesting that Fe3O4-FAM@M-BV2 was taken by GL261/TR cells mainly through macropinocytosis. CX3CR1 antibody and CX3CL1 antibody also significantly reduced the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells, indicating that CX3CR1 and CX3CL1 were involved in the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells (Fig. 2G, Additional file 1: Fig. S5). After nanoparticle was taken up by GL261/TR cells, the silence efficiency of siPD-L1@Lipo2000, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 on PD-L1 protein in GL261/TR cells were 69.18%, 58.74% and 74.81%, respectively (Fig. 2H). The similar results were observed in GL261 cells (Additional file 1: Fig. S6).

Fig. 2
figure 2

Uptake of Fe3O4-FAM@M-BV2 by GL261/TR cell. A Schematic diagram of co-culture of GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells in a petri dish. B The uptake of Fe3O4-FAM@M-BV2 by co-cultured GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells. CF The typical flow cytometer diagrams of Fe3O4-FAM@M-BV2 uptake by co-cultured GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells. G Uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells in the presence of different uptake inhibitors observed by LSCM. H The silence effect of Fe3O4-siNC, siPD-L1@Lipo2000, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 on PD-L1 protein in GL261/TR cells (n = 3, mean ± SD, *P < 0.05, **P < 0.01)

The ferroptosis induced by Fe3O4-siPD-L1@M-BV2

The essence of ferroptosis is Fenton reaction, which is triggered by Fe2+ and H2O2 (Fig. 3A). Firstly, MTT assay showed that Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 significantly reduced the activity of GL261/TR cells in a concentration-dependent manner. As compared with Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 displayed stronger inhibitory effect on the activity of GL261/TR cells (Fig. 3B). In the presence of IFN-γ, the cytotoxicity of Fe3O4-siPD-L1@M-BV2 on GL261/TR cell was significantly enhanced. However, in the existence of ferroptosis inhibitor such as Fer-1 and DFO, the cytotoxicity of Fe3O4-siPD-L1@M-BV2 on GL261/TR cell was markedly attenuated (Fig. 3C). The same results were observed by living and dead cells staining experiment (Additional file 1: Fig. S7). The above results confirmed that Fe3O4-siPD-L1@M-BV2 induced ferroptosis.

Fig. 3
figure 3

The ferroptosis induced by Fe3O4-siPD-L1@M-BV2 in GL261/TR cells. A Mechanism of ferroptosis. B Effect of Fe3O4-siPD-L1@M-BV2 on the viability of GL261/TR cells. C Effect of Fe3O4-siPD-L1@M-BV2 on the viability of GL261/TR cells in the presence of IFN-γ, Fer-1 and DFO. D Effect of Fe3O4-siPD-L1@M-BV2 on GSH level in GL261/TR cells. E Effect of Fe3O4-siPD-L1@M-BV2 on the protein expression of GPX4 in GL261/TR cells in the presence of Fer-1 and DFO. F Effect of Fe3O4-siPD-L1@M-BV2 on the protein expression of GPX4 in GL261/TR cells in the presence of IFN-γ. G Effect of Fe3O4-siPD-L1@M-BV2 on ROS (marked with DHE, red color) and LPO (marked with BODIPY-C11, green color) level in GL261/TR cells. H Statistic analysis of effect of Fe3O4-siPD-L1@M-BV2 on ROS level in GL261/TR cells. I Statistic analysis of effect of Fe3O4-siPD-L1@M-BV2 on LPO level in GL261/TR cells. J Effect of Fe3O4-siPD-L1@M-BV2 on H2O2 level in GL261/TR cells. (n = 3, mean ± SD, *P < 0.05, **P < 0.01; ns: no significant difference)

Secondly, Fe3O4-siPD-L1@M-BV2 significantly reduced GSH level in GL261/TR cells. In the existence of IFN-γ, the GSH level in GL261/TR cells was further decreased by Fe3O4-siPD-L1@M-BV2. However, in the presence of Fer-1 and DFO, the GSH depletion induced by Fe3O4-siPD-L1@M-BV2 was weakened in GL261/TR cells (Fig. 3D).

Thirdly, Fe3O4 had no significant effect on GPX4 protein expression in GL261/TR cells. Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 significantly reduced the protein expression of GPX4 in GL261/TR cells (Fig. 3E). In the existence of Fer-1 and DFO, the inhibitory effect of Fe3O4-siPD-L1@M-BV2 on GPX4 protein expression in GL261/TR cells was weakened. IFN-γ inhibited GPX4 and x-CT protein expression in GL261/TR cells, and the inhibitory effect of Fe3O4-siPD-L1@M-BV2 on GPX4 protein expression was enhanced in the presence of IFN-γ (Fig. 3F; Additional file 1: Fig. S8).

Finally, Fe3O4-siNC, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 significantly increased ROS, LPO and H2O2 level in GL261/TR cells. Moreover, in the presence of IFN-γ, ROS, LPO and H2O2 level in GL261/TR cells were further increased by Fe3O4-siPD-L1@M-BV2. However, in the presence of Fer-1 and DFO, the increase of ROS, LPO and H2O2 in GL261/TR cells induced by Fe3O4-siPD-L1@M-BV2 was attenuated (Fig. 3G–J).

The maturation of DC cell induced by Fe3O4-siPD-L1@M-BV2 in vitro

Flow cytometer experiment showed that the proportion of CD11c+CD80+CD86+ cells in Fe3O4, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 treatment groups was 9.67%, 45.26% and 68.2%, respectively, indicating that Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 significantly promoted the maturation of DC cells in vitro. As compared with Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 facilitated the maturation of DC cells more strongly. In addition, the proportion of CD11c+CD80+CD86+ cells in Fe3O4-siPD-L1@M-BV2 + IFN-γ groups was increased to 77.31%, suggesting the effect of Fe3O4-siPD-L1@M-BV2 on inducing maturation of DC cell was further enhanced in the presence of IFN-γ. However, the proportion of CD11c+CD80+CD86+ cells in Fe3O4-siPD-L1@M-BV2 + DFO and Fe3O4-siPD-L1@M-BV2 + Fer-1 groups was 54.4% and 50.21%, respectively. This indicated that ferroptosis inhibitors significantly reduced the maturation of DC cell induced by Fe3O4-siPD-L1@M-BV2 (Fig. 4).

Fig. 4
figure 4

Effect of Fe3O4-siPD-L1@M-BV2 on the maturation of DC cell in vitro. A Schematic diagram of co-culture of GL261/TR cells and iDC cells. B The maturation of DC cells in vitro analyzed by flow cytometer. C Statistic analysis of the matured DC cell (n = 3, mean ± SD, *P < 0.05, **P < 0.01, ns: no significant difference)

Transport of Fe3O4-FAM@M-BV2 across in vitro BBB

Within 12 h when drug is administered, there was no significant difference in resistance values between donor chamber and recipient chamber. It indicated that drug treatment did not damage the integrity of in vitro BBB model (Additional file 1: Fig. S9). As compared with Fe3O4-FAM, the transportation ratio of Fe3O4-FAM@M-BV2 across in vitro BBB was significantly higher, and it was further increased in the presence of external magnetic field (Fig. 5A, B). Compared with Fe3O4-FAM, the accumulation of Fe3O4-FAM@M-BV2 in GL261/TR cells was significantly increased after it penetrated in vitro BBB. In the presence of external magnetic field, the accumulation of Fe3O4-FAM@M-BV2 in GL261/TR cells was further increased after it penetrated in vitro BBB (Fig. 5C, D, Additional file 1: Fig. S10). Moreover, the transport ratio of Fe3O4-FAM@M-BV2 across in vitro BBB was significantly increased when GL261/TR cells were cultivated in recipient chamber in comparison with that without GL261/TR cells in recipient chamber. This suggested that GL261/TR cells facilitated the transport of Fe3O4-FAM@M-BV2 across in vitro BBB. When there were not GL261/TR cells in recipient chamber, the addition of CSF-1 and CX3CL1 in recipient chamber also significantly increased the transport ratio of Fe3O4-FAM@M-BV2 across in vitro BBB. After CSF-1 antibody and CX3CL1 antibody were added into recipient chamber, the transport ratio of Fe3O4-FAM@M-BV2 across in vitro BBB was significantly reduced. Moreover, when CSF-1R antibody and CX3CR1 antibody were added into donor chamber to block CSF-1R and CX3CR1 on the surface of Fe3O4-FAM@M-BV2, the transport ratio of Fe3O4-FAM@M-BV2 across in vitro BBB was also significantly decreased (Fig. 5E, F).

Fig. 5
figure 5

Transportation of Fe3O4-FAM@M-BV2 across in vitro BBB. A Schematic diagram of the in vitro BBB. B Transportation ratio of Fe3O4-FAM@M-BV2 across in vitro BBB. C The accumulation of Fe3O4-FAM@M-BV2 in GL261/TR cells after penetrating in vitro BBB detected by LCSM. D The typical flow cytometer diagrams of Fe3O4-FAM@M-BV2 accumulated in GL261/TR cells after Fe3O4-FAM@M-BV2 penetrated in vitro BBB. E Schematic diagram of mechanism study of Fe3O4-FAM@M-BV2 penetrating in vitro BBB. F The effect of CSF-1R, CX3CR1, CSF-1 and CX3CL1 on transportation ratio of Fe3O4-FAM@M-BV2 across the in vitro BBB. (n = 3, mean ± SD, *P < 0.05, **P < 0.01)

Distribution and pharmacokinetics of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice

After intravenous administration of Fe3O4-siPD-L1@M-BV2, the distribution of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice was observed by in vivo bioluminescence imaging. The results showed that as compared with Fe3O4-siPD-L1 group, fluorescence intensity in mice brain in Fe3O4-siPD-L1@M-BV2 group and Fe3O4-siPD-L1@M-BV2 + magnet group was significantly increased. Fe3O4-siPD-L1@M-BV2 + magnet group showed the strongest fluorescence intensity in mice brain (Fig. 6A; Additional file 1: Fig. S11). The integrated brain targeting efficiencies of Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet group at 12 h were 13.69%, 30.85% and 41.7%, respectively (Fig. 6B). Results of LSCM showed that a large amount of Fe3O4-siPD-L1@M-BV2 distributed in orthotopic drug-resistant GBM tissue, and little amount of Fe3O4-siPD-L1@M-BV2 distributed in the normal brain tissue. Moreover, Fe3O4-siPD-L1 mainly distributed in the blood vessels, while Fe3O4-siPD-L1@M-BV2 mainly distributed out of blood vessels in orthotopic drug-resistant GBM tissue (Fig. 6C), suggesting that Fe3O4-siPD-L1@M-BV2 penetrated deep region in GBM tissue.

Fig. 6
figure 6

Distribution and pharmacokinetics of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice. A The distribution of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice observed by in vivo bioluminescence imaging. B The integrated brain targeting efficiency of Fe3O4-siPD-L1@M-BV2. C Distribution of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM tissue observed by LSCM. CD31 staining: green color (stands for blood vessel). Cy5 staining: red color (stands for nanoparticle). T: tumor tissue; N: normal tissue. D Plasma siPD-L1 concentration–time curve in orthotopic drug-resistant GBM mice. E The content of siPD-L1 in orthotopic drug-resistant GBM tissue. F The content of siPD-L1 in normal brain tissue. G The siPD-L1 content ratio between in GBM tissue and in normal brain tissue. H The intracellular level of siPD-L1 in orthotopic drug-resistant GBM tissue. I The extracellular level of siPD-L1 in orthotopic drug-resistant GBM tissue. J The ratio of siPD-L1 content between in intracellular and in extracellular. K The intracellular Fe2+ level in orthotopic drug-resistant GBM tissue at 12 h after drug administration. (n = 3, mean ± SD, *P < 0.05, **P < 0.01, ns: no significant difference)

Pharmacokinetic experiment showed that the half-life of siPD-L1 in plasma in naked siPD-L1, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet groups was 1.52 h, 2.82 h, 8.88 h and 8.79 h, respectively (Fig. 6D). After administration of Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet, the content of siPD-L1 in orthotopic drug-resistant GBM tissue increased successively in comparison with naked siPD-L1 and Fe3O4-siPD-L1. Moreover, the content of siPD-L1 in orthotopic drug-resistant GBM tissue was significantly higher than that in normal brain tissue (Fig. 6E–G). As compared with the Fe3O4-siPD-L1 group, the intracellular level of siPD-L1 in drug-resistant GBM tissue was significantly increased in Fe3O4-siPD-L1@M-BV2 group and Fe3O4-siPD-L1@M-BV2 + magnet group (Fig. 6H–J). The intracellular level of Fe2+ in orthotopic drug-resistant GBM tissue was significantly increased in Fe3O4-siPD-L1@M-BV2 group and Fe3O4-siPD-L1@M-BV2 + magnet group as compared with that in Fe3O4-siPD-L1 group (Fig. 6K).

The inhibitory effect of Fe3O4-siPD-L1@M-BV2 on the growth of orthotopic drug-resistant GBM in mice

In vivo bioluminescence imaging was used to dynamic observe the growth of orthotopic drug-resistant GBM (Fig. 7A). As compared with normal saline, Fe3O4-siNC and Fe3O4-siNC@M-BV2 significantly inhibited the growth of orthotopic drug-resistant GBM. As compared with Fe3O4-siNC and Fe3O4-siNC@M-BV2, Fe3O4-siPD-L1 and Fe3O4-siPD-L1@M-BV2 significantly inhibited the growth of orthotopic drug-resistant GBM. Fe3O4-siPD-L1@M-BV2 displayed much stronger inhibitory effect on the growth of orthotopic drug-resistant GBM in comparison with Fe3O4-siPD-L1, suggesting that ferroptosis combined with immunotherapy further inhibited the growth of orthotopic drug-resistant glioma. In addition, in the presence of external magnetic field, the inhibitory effect of Fe3O4-siPD-L1@M-BV2 on orthotopic drug-resistant GBM was further enhanced (Fig. 7B, C).

Fig. 7
figure 7

The therapeutic effect of Fe3O4-siPD-L1@M-BV2 on orthotopic drug-resistant GBM in mice. A Schematic diagram of drug administration and observation of therapeutic effect. B The inhibitory effect of Fe3O4-siPD-L1@M-BV2 on the growth of orthotopic drug-resistant GBM observed by in vivo bioluminescence imaging. C Statistical analysis of orthotopic drug-resistant GBM growth. (n = 6, mean ± SD, *P < 0.05, **P < 0.01). D Effects of Fe3O4-siPD-L1@M-BV2 on the survival time of orthotopic drug-resistant GBM mice. (n = 10, mean ± SD, *P < 0.05, **P < 0.01). E H&E, Ki67 and TUNEL staining of orthotopic drug-resistant GBM tissue. F Semi-quantitative analysis of Ki67 expressed in orthotopic drug-resistant GBM tissue. (n = 3, mean ± SD, *P < 0.05, **P < 0.01). G Semi-quantitative analysis of TUNEL positive cells in orthotopic drug-resistant GBM tissue. (n = 3, mean ± SD, *P < 0.05, **P < 0.01)

On 26th, 28th, 27th, 29th, 32th and 41th day after the plantation of Luc-GL261/TR cell, all normal saline, TMZ, Fe3O4, Fe3O4-siNC, Fe3O4-siPD-L1 and low-dose Fe3O4-siPD-L1@M-BV2 treated orthotopic drug-resistant GBM mice died in succession. However, on 60 days after the plantation of Luc-GL261/TR cell, the survival rate was 10% and 30% in high-dose Fe3O4-siPD-L1@M-BV2 group and high-dose Fe3O4-siPD-L1@M-BV2 + magnet group, respectively (Fig. 7D). The median survival time of normal saline, TMZ, Fe3O4, Fe3O4-siNC, Fe3O4-siPD-L1, low-dose Fe3O4-siPD-L1@M-BV2, high-dose Fe3O4-siPD-L1@M-BV2 and high-dose Fe3O4-siPD-L1@M-BV2 + magnet treated GBM mice was 17.68, 21.39, 18.61, 22.28, 26.94, 31.56, 38.72 and 45.00 days, respectively.

H&E staining of orthotopic drug-resistant GBM tissue presented a long oval shape and a large number of pathological nuclear mitosis in normal saline, TMZ and Fe3O4 treated group. Fe3O4-siPD-L1@M-BV2 decreased the number of heteromorphic nuclear cells and nuclear division in orthotopic drug-resistant GBM tissue. The nucleus was regular spherical and the intercellular space became blurred or even disappeared in Fe3O4-siPD-L1@M-BV2 + magnet treated group (Fig. 7E). Moreover, Fe3O4-siNC@M-BV2, Fe3O4-siNC@M-BV2 + magnet, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet obviously inhibited Ki67 expression and increased the number of TUNEL positive cells in orthotopic drug-resistant GBM tissue as compared with normal saline, free TMZ and Fe3O4 (Fig. 7E–G). These results indicated that Fe3O4-siPD-L1@M-BV2 markedly inhibited proliferation of orthotopic drug-resistant GBM cells and promoted its apoptosis.

The body weight of orthotopic drug-resistant GBM mice in normal saline, TMZ, Fe3O4, Fe3O4-siNC, Fe3O4-siPD-L1 and low-dose Fe3O4-siPD-L1@M-BV2 group decreased gradually. The body weight of orthotopic drug-resistant GBM mice in high-dose Fe3O4-siPD-L1@M-BV2 group and Fe3O4-siPD-L1@M-BV2 + magnet group showed a trend of increasing (Additional file 1: Fig. S12), indicating the growth of orthotopic drug-resistant GBM was markedly blocked by Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet.

Mechanisms of Fe3O4-siPD-L1@M-BV2 on inhibition the growth of orthotopic drug-resistant GBM

Firstly, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet significantly increased the protein expression of E-cadherin and decreased the protein expression of TGF-β, MMP-9, CD44 and N-cadherin in orthotopic drug-resistant GBM tissue. (Additional file 1: Fig. S13), indicating Fe3O4-siPD-L1@M-BV2 inhibited the invasion and migration of orthotopic drug-resistant GBM cells through regulating invasion-related protein expression.

Secondly, Fe3O4-siPD-L1@M-BV2 decreased the protein expression of PD-L1, x-CT and GPX4 in orthotopic drug-resistant GBM tissue in dose-dependent manner. As compared with Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 reduced the protein expression of PD-L1, xCT and GPX4 more strongly. The protein expressions of PD-L1, xCT and GPX4 in orthotopic drug-resistant GBM tissue were less in Fe3O4-siPD-L1@M-BV2 + magnet group than that in Fe3O4-siPD-L1@M-BV2 group (Fig. 8A, B). At the same time, immunofluorescence staining was also used to investigate the effects of Fe3O4-siPD-L1@M-BV2 on the protein expression of GPX4 and PD-L1 in orthotopic drug-resistant GBM tissue, and the results were consistent with western blot experiment (Fig. 8C, Additional file 1: Fig. S14). In addition, Fe3O4-siPD-L1@M-BV2 dose-dependently reduced the content of GSH in orthotopic drug-resistant GBM tissue. The GSH content in orthotopic drug-resistant GBM tissue in Fe3O4-siPD-L1@M-BV2 + magnet group was lower in comparison with that in Fe3O4-siPD-L1@M-BV2 group (Fig. 8D). Meanwhile, Fe3O4-siPD-L1@M-BV2 dose-dependently increased the ROS level in orthotopic drug-resistant GBM tissue (Fig. 8C, E). Moreover, as compared with normal saline, Fe3O4-siNC and Fe3O4-siNC@M-BV2 also significantly inhibited the GPX4 expression, GSH and ROS level in orthotopic drug-resistant GBM tissue, indicating that Fe3O4-siNC and Fe3O4-siNC@M-BV2 induced ferroptosis and subsequently inhibited the growth of orthotopic drug-resistant glioma.

Fig. 8
figure 8

Effect of Fe3O4-siPD-L1@M-BV2 on immune microenvironment and ferroptosis in orthotopic drug-resistant GBM tissue. A The protein expression of PD-L1, xCT and GPX4 in orthotopic drug-resistant GBM tissue detected by western blot. B Statistic analysis of PD-L1, xCT and GPX4 protein expression. C The protein expression of PD-L1, GPX4 and ROS level in orthotopic drug-resistant GBM tissue observed by immunofluorescence staining. D The content of GSH in orthotopic drug-resistant GBM tissue. E Statistic analysis of ROS level in orthotopic drug-resistant GBM tissue. F The ratio of matured DC cell in orthotopic drug-resistant GBM tissue. G, H The proportion of Teff cell and Treg cell in orthotopic drug-resistant GBM tissue. IM The content of TNF-α, IFN-γ, IL-6, IL-10 and IL-12 in orthotopic drug-resistant GBM tissue. (n = 3, mean ± SD, *P < 0.05, **P < 0.01)

Thirdly, flow cytometer was used to investigate the effect of Fe3O4-siPD-L1@M-BV2 on the maturation of DC cell in orthotopic drug-resistant GBM tissue. The proportion of CD11c+CD80+CD86+ in normal saline, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 (0.3 mg/kg) and Fe3O4-siPD-L1@M-BV2 (1 mg/kg) treatment groups was 1.57%, 7.8%, 17.84%, 19.26%, respectively. As compared with Fe3O4-siPD-L1@M-BV2, the proportion of matured DC cells in Fe3O4-siPD-L1@M-BV2 + magnet group was significantly increased in orthotopic drug-resistant GBM tissue (Fig. 8F, Additional file 1: Fig. S15). Further study indicated that the proportion of CD3+CD8+IFN-γ+ T cells (Teff cell) in orthotopic drug-resistant GBM tissue in normal saline, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 (0.3 mg/kg) and Fe3O4-siPD-L1@M-BV2 (1 mg/kg) treatment group was 3.10%, 13.43%, 19.25% and 23.1%, respectively. Fe3O4-siPD-L1@M-BV2 + magnet obviously increased the number of Teff cell in orthotopic drug-resistant GBM tissue as compared with Fe3O4-siPD-L1@M-BV2 (Fig. 8G, Additional file 1: Fig. S16). The proportion of CD4+CD25+FoxP3+ T cells (Treg cell) in orthotopic drug-resistant GBM tissue in normal saline, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 (0.3 mg/kg) and Fe3O4-siPD-L1@M-BV2 (1 mg/kg) treatment group was 18.76%, 11.05%, 7.24% and 5.29%, respectively. As compared with Fe3O4-siPD-L1@M-BV2, Fe3O4-siPD-L1@M-BV2 + magnet markedly reduced the number of Treg cell in orthotopic drug-resistant GBM tissue (Fig. 8H, Additional file 1: Fig. S17).

Finally, Fe3O4-siPD-L1@M-BV2 dose-dependently increased the contents of TNF-α, IFN-γ, IL-6 and IL-12 but decreased the contents of IL-10 in orthotopic drug-resistant GBM cells. Fe3O4-siPD-L1@M-BV2 + magnet significantly increased the contents of TNF-α, IFN-γ, IL-6 and IL-12 in comparison with Fe3O4-siPD-L1@M-BV2 did (Fig. 8I–M). Moreover, immunofluorescence staining showed that a large amount of microglia was mainly distributed in orthotopic drug-resistant GBM tissue (Fig. 9A). Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet significantly increased the number of Iba-1+CD16/32+ cells (M1 type microglia) in drug-resistant GBM tissue in comparison with normal saline and Fe3O4-siNC. The number of Iba-1+CD16/32+ cells was the highest in Fe3O4-siPD-L1@M-BV2 + magnet treatment group (Fig. 9B, C). Furthermore, as compared with normal saline and Fe3O4-siNC, Fe3O4-siPD-L1, Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet significantly reduced the number of Iba-1+CD206+ cells (M2 type microglia) in drug-resistant GBM tissue. The number of Iba-1+CD206+ cells was the lowest in Fe3O4-siPD-L1@M-BV2 + magnet treatment group (Fig. 9B, D).

Fig. 9
figure 9

Effect of Fe3O4-siPD-L1@M-BV2 on polarization of microglia in orthotopic drug-resistant GBM tissue. A Distribution of microglia (marked with Iba-1, red color) in orthotopic drug-resistant GBM tissue. T: tumor tissue; N: normal tissue. B M1-type microglia (Iba-1 and CD16/32 co-positive cells) and M2-type microglia (Iba-1 and CD206 co-positive cells) in orthotopic drug-resistant GBM tissue. C, D Statistic analysis of M1 type and M2 type microglia in orthotopic drug-resistant GBM tissue. (n = 3, mean ± SD, *P < 0.05, **P < 0.01)

Preliminary safety evaluation of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice

On the 24th day after plantation of Luc-GL261/TR cell, H&E staining showed that no obvious abnormal morphological changes were observed in the heart, liver, spleen, lung and kidney in all treatment groups (Fig. 10A). Biochemical analysis further showed that ALT and AST activities, BUN and CREA contents in mice serum in each treatment group were all within the normal range (Fig. 10B–E).

Fig. 10
figure 10

Safety evaluation of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM mice. A H&E staining of heart, liver, spleen, lung and kidney tissue in orthotopic drug-resistant GBM mice. B, C Effects of Fe3O4-siPD-L1@M-BV2 on ALT and AST activity in serum of orthotopic drug-resistant GBM mice. D, E Effects of Fe3O4-siPD-L1@M-BV2 on content of BUN and CREA in serum of orthotopic drug-resistant GBM mice. The green area indicates the normal ranges. (n = 5, mean ± SD)

Discussion

The PD-1/PD-L1 signaling pathway plays an important role in the tumor immune microenvironment [46]. The activation of PD-1/PD-L1 signaling pathway induces the apoptosis of the Teff cell. Blocking the PD-1/PD-L1 signaling pathway activates Teff cell, thus inhibiting tumor growth [47]. However, clinical data show that only less than 10% of patients with GBM response to immunotherapy because of the low immunogenicity of GBM [10, 48]. It has been shown that ferroptosis of cancer cells release high-mobility group box 1 (HMGB1) in an autophagy dependent manner [49], which increases the immunogenicity of cancer cells and promotes the maturation of DC cells [50,51,52]. The matured DC cells presents antigen to T lymphocytes and activates innate and adaptive immunity.

The key features of ferroptosis are the overload of Fe2+, depletion of GSH, the decrease of GPX4 activity and the consequent lipid peroxidation of cell membrane [53]. The results of MTT assay showed that Fe3O4-siPD-L1@M-BV2 decreased GL261/TR cell activity in a concentration-dependent manner. However, the inhibitory effect of Fe3O4-siPD-L1@M-BV2 on GL261/TR cell activity was significantly reduced in the present of ferroptosis inhibitors. After GL261/TR cells were incubated with Fe3O4-siPD-L1@M-BV2, the content of GSH and the protein expression of GPX4 in GL261/TR cells significantly decreased, which attenuated the ability of GL261/TR cells to clear intracellular ROS. This led to the increase of ROS level and lipid peroxidation in GL261/TR cells. However, in the present of ferroptosis inhibitor, the lipid peroxidation induced by Fe3O4-siPD-L1@M-BV2 in GL261/TR cells was significantly attenuated. Moreover, in vivo experimental results indicated that as compared with Fe3O4, the GSH content and protein expression of GPX4 and xCT were markedly reduced, and the ROS level and Fe2+ content was significantly increased in orthotopic drug-resistant GBM tissue in Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + magnet treatment group. Those results demonstrated that ferroptosis was involved in the inhibitory effect of Fe3O4-siPD-L1@M-BV2 on GL261/TR cell activity. Fe3O4-siPD-L1@M-BV2 induced the ferroptosis of orthotopic drug-resistant GBM cells by increasing Fe2+ content and reducing the GPX4 protein expression.

When Fe3O4-siPD-L1@M-BV2 was co-cultured with GL261/TR cell, it significantly facilitated the maturation of DC cells. Ferroptosis inhibitors significantly inhibited the maturation of DC cells induced by Fe3O4-siPD-L1@M-BV2, suggesting that ferroptosis of GL261/TR cell induced by Fe3O4-siPD-L1@M-BV2 promoted the maturation of DC cells. Meanwhile, the in vitro experiment indicated that IFN-γ significantly enhanced ferroptosis of GL261/TR cells and the maturation of DC cells induced by Fe3O4-siPD-L1@M-BV2. Moreover, the in vivo experiment indicated that IFN-γ content and the number of matured DC cells in orthotopic drug-resistant GBM tissue significantly increased in Fe3O4-siPD-L1@M-BV2 group and Fe3O4-siPD-L1@M-BV2 + magnet group. Fe3O4-siPD-L1@M-BV2 reduced the protein expression of PD-L1 and increased the ratio between Teff cell and Treg cell in orthotopic drug-resistant GBM tissue. Those results suggested that Teff cell was activated by Fe3O4-siPD-L1@M-BV2 through blocking PD-1/PD-L1 signal pathway, resulting in the release of IFN-γ. IFN-γ released from activated Teff cell enhanced the ferroptosis of Fe3O4-siPD-L1@M-BV2, which promoted the maturation of DC cells in orthotopic drug-resistant GBM tissue. This activated the Teff cell in turn and subsequently inhibited the growth of orthotopic drug-resistant GBM.

Clinical studies have shown that there is a large number of microglia in GBM tissue [54, 55]. GBM tissue promotes the polarization of microglia toward anti-inflammatory M2 type [11]. Studies have also shown that PD-L1 antibody activate mice macrophages to secrete more TNF-α and IL-12, which induce polarization of macrophages toward a pro-inflammatory M1 type [56, 57]. The in vivo experimental results showed that Fe3O4-siPD-L1@M-BV2 significantly increased the content of IFN-γ, TNF-α and IL-12 in orthotopic drug-resistant GBM tissue. The ratio between M1 type microglia and M2 type microglia in orthotopic drug-resistant GBM tissue was significantly increased by Fe3O4-siPD-L1@M-BV2. The above results demonstrated that Fe3O4-siPD-L1@M-BV2 polarized M2 type microglia to M1 type microglia through increasing IFN-γ, TNF-α and IL-12 content in orthotopic drug-resistant GBM tissue.

Improving the stability in blood circulation and targeting to GBM is the key point for siPDL-L1 to play its role in vivo. Pharmacokinetic results showed that Fe3O4-siPD-L1@M-BV2 markedly increased the stability of siPD-L1 in blood. In vivo bioluminescence imaging results indicated that the coating of microglia membrane improved the brain targeting of Fe3O4 nanoparticles. LSCM observation indicated that Fe3O4-siPD-L1@M-BV2 selectively distributed in the orthotopic drug-resistant GBM tissue. In addition, as compared with Fe3O4-siPD-L1, the intracellular siPD-L1 in orthotopic drug-resistant GBM tissue was significantly increased after Fe3O4-siPD-L1@M-BV2 was administrated. Meanwhile, the content of Fe2+ in orthotopic drug-resistant GBM tissue was also significantly increased by Fe3O4-siPD-L1@M-BV2. Those results demonstrated that Fe3O4-siPD-L1@M-BV2 exhibited good targeting for orthotopic drug-resistant GBM tissue. siPD-L1 and Fe2+ were simultaneously delivered to orthotopic drug-resistant GBM by Fe3O4-siPD-L1@M-BV2. Due to the paramagnetism of Fe3O4 nanoparticle, the brain targeting rate and the accumulation of Fe3O4-siPD-L1@M-BV2 in orthotopic drug-resistant GBM tissue was further increased under external magnetic field.

The in vivo imaging showed that Fe3O4-siPD-L1@M-BV2 was mainly distributed in brain and liver, while less Fe3O4-siPD-L1@M-BV2 was distributed in kidney and other tissue after intravenous administration. The amount of Fe3O4-siPD-L1@M-BV2 in brain tissue reached its maximum value at 12 h after administration. The distribution of Fe3O4-siPD-L1@M-BV2 in liver and brain significantly decreased at 24 h after administration. The above results indicated that Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + megnet did not accumulate in the liver and kidney after a single tail vein injection. On the 24th day after plantation of Luc-GL261/TR cell, H&E staining and biochemical analysis indicated when Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + megnet were intravenously injected into orthotopic drug-resistant GBM mice for 4 times within 12 days, they did not cause significant damage to heart, liver, spleen, lung and kidney in orthotopic drug-resistant GBM mice. However, this just was a preliminary safety evaluation. It is not certain yet if hepatorenal toxicity can be caused by Fe3O4-siPD-L1@M-BV2 and Fe3O4-siPD-L1@M-BV2 + megnet when its dose is greater than 1 mg/kg or more than 4 times of administration. This needs further study.

Western blot results showed that CX3CL1 and CSF-1 were highly expressed in GL261/TR cells, while CX3CR1 and CSF-1R were highly expressed in the membrane of microglia and surface of Fe3O4-siPD-L1 nanoparticles. In vitro studies showed that in the existence of chemokine CSF-1 and CX3CL1, the efficiency of Fe3O4-siPD-L1@M-BV2 penetration in vitro BBB was significantly improved, and the accumulation of Fe3O4-siPD-L1@M-BV2 in GL261/TR cells was also markedly increased. In contrast, CSF-1 antibody, CX3CL1 antibody, CSF-1R antibody and CX3CR1 antibody significantly reduced the efficiency of Fe3O4-siPD-L1@M-BV2 penetration in vitro BBB and its accumulation in GL261/TR cells. The results demonstrated that the interaction between chemokines (CSF-1 and CX3CL1) secreted by GL261/TR cells and receptors (CSF-1R and CX3CR1) on the microglia membrane promoted Fe3O4-siPD-L1@M-BV2 to penetrate BBB and accumulate in GL261/TR cells.

Conclusion

Fe3O4-siPD-L1@M-BV2 was actively delivered to orthotopic drug-resistant GBM cell through the interaction between microglia membrane and GBM cell. Fe3O4-siPD-L1@M-BV2 activated Teff cell by blocking the PD-1/PD-L1 signaling pathway. The activated Teff cell released IFN-γ to promote ferroptosis of GBM cells and subsequently facilitated the maturation of DC cells in orthotopic drug-resistant GBM tissue. The matured DC cells presented GBM antigens to T lymphocytes to activate Teff cell in turn. Meanwhile, M2 type microglia was polarized to M1 type microglia by IFN-γ secreted via Teff cell in orthotopic drug-resistant GBM tissue. Finally, a virtuous cycle between ferroptosis and immunotherapy was formed and promoted each other by Fe3O4-siPD-L1@M-BV2, which exerted a synergistic effect on the treatment of orthotopic drug-resistant GBM.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information file.

Abbreviations

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

BBB:

Blood brain barrier

BODIPY-C11:

Boron difluoride pyrrole fluorescent dyes-C11

BUN:

Urea nitrogen

BSA:

Bovine albumin

CD11c-APC:

Clusters of differentiation 11 c-Allophycocyanin

CD25-FITC:

Clusters of differentiation 25-fluorescein isothiocyanate

CD3-APC:

Clusters of differentiation 3-Allophycocyanin

CD4-PE-Cy7:

Clusters of differentiation4-Phycoerythrin-Sulfo-Cyanine7

CD44:

Clusters of differentiation 44

CD8-FITC:

Clusters of differentiation 8-fluorescein isothiocyanate

CD80-FITC:

Clusters of differentiation 80-fluorescein

CD86-PE:

Clusters of differentiation86-Phycoerythrin

CREA:

Creatinine

CX3CL1:

C-X3-C motif chemokine ligand 1

CX3CR1:

C-X3-C motif chemokine receptor 1

DAPI:

4’,6-Diamidino-2-phenylindole dihydrochloride

DFO:

Deferoxamine

DHE:

Dihydroethidium

DMEM:

Dulbecco's modified eagle medium

DMSO:

Dimethyl sulfoxide

E-cadherin:

E-Ca2+dependent cell adhesion molecules

FoxP3-PE:

Forkhead box P3-Phycoerythrin

Fer-1:

Ferrostatin-1

GL261/TR:

TMZ-resistant glioma cells

GSH:

Glutathione

GM-CSF:

Granulocyte–macrophage colony stimulating factor

GPX4:

Glutathione Peroxidase 4

HMGB1:

Human high-mobility group box-1

Iba-1:

Ionized calcium binding adaptor molecule 1

HR:

Hemolysis rate

Iba-1:

Ionized calcium binding adaptor molecule 1

IL-6/10/12:

Interleukin-6/10/12

IFN-γ:

Interferon-γ

IFN-γ-PE-Cy7:

Interferon-γ-Phycoerythrin-Sulfo-Cyanine7

LPO:

Lipid peroxidation

LSCM:

Laser scanning confocal microscopy

M-BV2 :

Microglial cell membrane

MGMT:

O6-methylguanine-DNA-methyltransferase

MMP-9:

Matrix metalloproteinases-9

MTT:

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

N-cadherin:

N-Ca2+dependent cell adhesion molecules

PBS:

Phosphate buffer saline

PDI:

Polydispersity index

PD-L1:

Programmed cell death ligand-1

ROS:

Reactive oxygen species

RPMI1640:

Roswell Park Memorial Institute 1640

SDS-PAGE:

Sulfate–polyacrylamide gel electrophoresis

TEM:

Transmission electron microscope

TGF-β:

Transforming growth factor-β

TNF-α:

Tumor necrosis factor-α

Tris:

Trimethylol aminomethane

xCT:

Cystine/glutamic acid reverse transporter

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Funding

This work was financially supported by the National Natural Science Foundation of China (81872803, 82073775), Shaanxi Province Key Research and Development Projects of China (2021ZDLSF03-08) and Shaanxi Natural Science Foundation (2020JQ-458).

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BL, DL, and SZ conceived the manuscript. BL wrote the first draft. DL and SZ revised the first draft. BL and QJ performed the experiments. YC, ML, BZ, and QM provided grouping suggestions. All of the authors have read and approved the final manuscript.

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Correspondence to Daozhou Liu or Siyuan Zhou.

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All animal experiments were approved by the Air Force Medical University Institutional Animal Care and Utilization Committee (No: IACUC-20210403).

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Supplementary Information

Additional file 1: Figure S1.

Diagram of the applied magnetic field in orthotopic drug-resistant GBM mice brain after Fe3O4-siPD-L1@M-BV2 was injected into the tail vein. Figure S2. Characterization of Fe3O4-siPD-L1@M-BV2. (A) Element mapping analysis diagram of Fe3O4. (B) The zeta potential of Fe3O4, Fe3O4-siPD-L1, M-BV2 and Fe3O4-siPD-L1@M-BV2. (C) Stability of Fe3O4-siPD-L1@M-BV2 in water, PBS and 10% FBS solution. (D) TEM image of Fe3O4. (E) TEM image of Fe3O4-siPD-L1@M-BV2. (F) Hemolysis phenomenon of Fe3O4-siPD-L1@M-BV2. (G) Statistic analysis of hemolysis of Fe3O4-siPD-L1@M-BV2. (n = 3, mean ± SD). Figure S3. The density of GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells after the cells incubated at 37 ℃ for 24 h. (A) The density of GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells observed by optical microscope. (B) The density of GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells counted by using cell counters. (n = 3, mean ± SD, ns: no significant difference). Figure S4. Statistic analysis of Fe3O4-FAM@M-BV2 uptake by GL261/TR cells, HT-22 cells, BV2 cells and RAW264.7 cells detected by flow cytometer (n = 3, mean ± SD, *P < 0.05, **P < 0.01, ns: no significant difference). Figure S5. The uptake mechanism of Fe3O4-FAM@M-BV2 on GL261/TR cells detected by flow cytometer. (A) Effects of different inhibitors on the uptake of Fe3O4-FAM@M-BV2 by GL261/TR cells. (B) Statistic analysis of Fe3O4-FAM@M-BV2 uptake by GL261/TR cells (n = 3, mean ± SD, **P < 0.01). Figure S6. The effect of Fe3O4-siPD-L1@M-BV2 on protein expression of PD-L1 in GL261 cells. (n = 3, mean ± SD, *P < 0.05, **P < 0.01). Figure S7. The effect of Fe3O4-siPD-L1@M-BV2 on the viability of GL261/TR cells. (A) The effect of Fe3O4-siPD-L1@M-BV2 on the death and living GL261/TR cells. (B) Statistic analysis of death and living GL261/TR cells. (n = 3, mean ± SD, *P < 0.05, **P < 0.01, ns: no significant difference). Figure S8. The effect of IFN-γ on the xCT protein expression in GL261/TR cells. (n = 3, mean ± SD, **P < 0.01). Figure S9. The resistance values between transwell donor chamber and recipient chamber within 12 h after drug administration. (n = 3, mean ± SD, ns: no significant difference). Figure S10. Statistic analysis of Fe3O4-FAM@M-BV2 uptake by GL261/TR cells after Fe3O4-FAM@M-BV2 penetrated in vitro BBB detected by flow cytometer. (n = 3, mean ± SD, **P < 0.01). Figure S11. The distribution of Fe3O4-siPD-L1@M-BV2 in organs of orthotopic drug-resistant GBM mice observed by in vivo bioluminescence imaging. Figure S12. The effect of Fe3O4-siPD-L1@M-BV2 on body weight of orthotopic drug-resistant GBM mice. (n = 10, mean ± SD). Figure S13. The expression of invasion-related proteins in orthotopic drug-resistant GBM tissue after orthotopic drug-resistant GBM mice was treated with Fe3O4-siPD-L1@M-BV2. (A) The expression of invasion-related proteins in orthotopic drug-resistant GBM tissue detected by western blot. (B) Semi-quantitative analysis of invasion-related proteins. (n = 3, mean ± SD, *P < 0.05, **P < 0.01). Figure S14. Effect on the protein expression of GPX4 and PD-L1 in orthotopic drug-resistant GBM tissue after orthotopic drug-resistant GBM mice was treated with Fe3O4-siPD-L1@M-BV2. (A) Semi-quantitative analysis of GPX4 protein expression in orthotopic drug-resistant GBM tissue detected by immunofluorescence staining. (B) Semi-quantitative analysis of PD-L1 protein expression in orthotopic drug-resistant GBM tissue. (n = 3, mean ± SD, *P < 0.05, **P < 0.01, ns: no significant difference). Figure S15. Typical flow cytometric graph of the matured DC cells. Figure S16. Typical flow cytometric graph of CD3+CD8+IFN-γ+ T cells. Figure S17. Typical flow cytometric graph of CD4+CD25+FoxP3+ T cells.

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Liu, B., Ji, Q., Cheng, Y. et al. Biomimetic GBM-targeted drug delivery system boosting ferroptosis for immunotherapy of orthotopic drug-resistant GBM. J Nanobiotechnol 20, 161 (2022). https://doi.org/10.1186/s12951-022-01360-6

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