GSH-responsive and Mitochondria-targeting Multifunctional Nanoplatform for MR imaging-guided enhanced chemotherapy of Glioma by Reshaping the Tumor Immune Microenvironment

doi:10.21203/rs.3.rs-5276343/v1

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GSH-responsive and Mitochondria-targeting Multifunctional Nanoplatform for MR imaging-guided enhanced chemotherapy of Glioma by Reshaping the Tumor Immune Microenvironment

https://doi.org/10.21203/rs.3.rs-5276343/v1

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Despite considerable progress in glioma research, present therapeutic approaches continue to be insufficiently efficacious, predominantly owing to challenging hindrances in conveying chemotherapy drugs across the blood-brain barrier (BBB) and reshaping the immunosuppressive tumor microenvironment (TME). In this study, a multifunctional nanoplatform was developed comprising poly-lactide-co-glycolide (PLGA) encapsulated with MnO₂ nanoparticles, triphenylphosphonium (TPP) conjugated with doxorubicin (DOX), and Angiopep-2 (Ang) for the magnetic resonance imaging-guided enhanced chemotherapy of glioma. The role of Ang promotes BBB penetration and tumor cell targeting, while TPP allows for an increased concentration of the Ang-PMT NPs in the mitochondria. Upon exposure to high concentration of glutathione (GSH) within the TME, the Ang-PMT NPs disintegrate rapidly, resulting in the production of Mn²⁺ and the subsequent release of DOX. The released DOX directly eradicates tumor cells and catalyzes mitochondrial DNA release, leading to immunogenic cell death (ICD) and the activation of the cGAS-STING pathway. Furthermore, the produced Mn²⁺ also activates the cGAS-STING pathway, thereby reshaping the TME and enhancing chemotherapy for glioma. The multifunctional nanoplatform demonstrated a notable inhibition of tumor growth in comparison to the control groups. It is anticipated that this innovative approach may offer promising prospects for the management of malignant glioma in clinical management.

multifunctional nanoparticles

MR imaging

chemotherapy

glioma

tumor microenvironment

Glioma represents the most common primary malignant neoplasm of the central nervous system (CNS), exhibiting a high degree of lethality, disability, recurrence, and overall survival rates of less than 12–15 months [1, 2]. The current standard of treatment strategies in clinical practice includes surgical resection followed by fractionated radiotherapy with concomitant and adjuvant with temozolomide chemotherapy [3–5]. Despite significant efforts over the past decades, malignant glioma remains poor prognosis due to the surgical residue and severe side effects of chemotherapeutic drugs [6, 7]. Therefore, it is urgent to explore new strategies to improve the efficacy of chemotherapy in the treatment of glioma.

One of the major hurdles for glioma treatment is the inherent present of the blood brain barrier (BBB), which limits the few available chemotherapeutics infiltrate into the tumor region, leads to the ineffective tumor cell killing [8, 9]. Angiopep-2 (Ang), a family of Kunitz-domain-derived peptides, binds to the low-density lipoprotein receptor-related protein-1 (LRP1), shows great transcytosis ability and parenchyma accumulation [10–12]. Numerous studies have demonstrated that Ang, as a targeting ligand, can penetrate the BBB and specially target glioma, thus significantly increasing the accumulation of chemotherapy drugs in glioma [13]. Therefore, we hypothesize that the integration of Ang and chemotherapy drugs could dramatically increase the concentration of chemotherapy drugs at the glioma site. Furthermore, the efficacy of chemotherapy is limited by the immunosuppressive tumor microenvironment (TME), which is characterized by hypoxia, acidosis, and the overexpression of glutathione (GSH) and hydrogen peroxide (H₂O₂).

In recent years, various strategies have been explored to regulate the TME, such as the elimination of the hypoxic TME through the delivery or production of oxygen in the tumor region, and the depletion of GSH to upregulate the level of reactive oxygen species (ROS) to kill cancer cells [14]. Thus, the reshaping of the TME represents a promising strategy for enhancing the efficacy of chemotherapy in the treatment of glioma [15]. Manganese oxide (MnO₂) is highly sensitive to the TME due to its ability to consume GSH and decompose Mn⁴⁺ into Mn²⁺ through a Fenton-like catalytic reaction. This reaction involves the interaction of MnO₂ with hydrogen peroxide (H₂O₂) within the TME, resulting in the generation of ROS such as hydroxyl radical (·OH) and singlet oxygen (¹O₂) [16, 17]. Furthermore, the Mn²⁺ generated in a stimulus-responsive manner exhibits compelling T₁-magnetic resonance (MR) imaging performance, making it an effective contrast agent for tumor detection and imaging [18]. In addition, Mn²⁺ has been shown to enhance the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, thereby amplifying the STING signal and increasing type I interferon (IFN) production [19–21]. Moreover, the Mn element is a vital trace element for the human body, whose metabolism can be controlled effectively, thereby contributing to good biocompatibility and the low long-term toxicity of Mn²⁺[22]. Thus, MnO₂ is regarded as a safe, TME-responsive, biocompatible, and ideal contrast agent for adjuvant tumor diagnosis and treatment.

In addition, chemotherapeutic agents such as doxorubicin (DOX) can induce apoptosis in tumor cells through the damage of DNA and mitochondrial function, as well as elicit immunogenic cell death (ICD) in tumor cells [23]. The chemotherapeutics-induced ICD is accompanied by the release of damage-associated molecular patterns (DAMPs), including calreticulin (CRT), adenosine triphosphate (ATP), and high mobility group protein B1 (HMGB1). Moreover, evidence indicates that chemotherapeutics-induced mitochondrial DNA release can activate the cGAS-STING pathway and initiate the production of IFNs. However, the delivery efficiency of DOX is frequently constrained by the absence of tumor-specific targeting, poor water solubility, and rapid metabolism [24, 25]. Nevertheless, it is urgent to improve the delivery efficiency of chemotherapeutics in a safe and effective manner for enhanced cancer chemotherapy. Moreover, mitochondrial targeting represents a promising approach for addressing the current challenges in clinical application of chemotherapy. Triphenylphosphonium (TPP), a well-known mitochondrial targeting moiety, is wildly used to achieve passive targeting of the mitochondrial membrane.

Herein, in this study, we rationally designed and systematically synthesized a multifunctional nanoplatform enhances the chemotherapy and exhibits tumor-targeting, mitochondria-targeting, and GSH consumption capabilities under the guidance of MRI imaging (Scheme 1). This multifunctional nanoplatform is composed of poly-lactide-co-glycolide (PLGA) loaded with MnO₂ nanoparticles, a chemotherapeutic drug, doxorubicin (DOX), which has been conjugated with TPP and Ang (Ang-TPP-DOX-MnO₂-PLGA), referred to as Ang-PMT NPs, for highly effective enhancing glioma chemotherapy (Scheme 1a). Following intravenous administration, the Ang-PMT NPs rapidly penetrate the BBB and target the tumor region with the assistance of the Ang modification (Scheme 1b). Subsequently, the mitochondriotropic TPP, which is encapsulated in the nanoparticles, concentrates further at the mitochondria of tumor cells. Consequently, the Ang-PMT NPs disintegrate owing to the reaction between MnO₂ NPs and the TME (mainly H⁺, H₂O₂ and GSH), thereby facilitating the Mn²⁺ production for MR imaging and reshaping the TME. Meanwhile, the release of DOX results in the direct killing of tumor cells and the triggering of mitochondrial DNA release, which in turn induces ICD and activates the cGAS-STING pathway (Scheme 1c). This study demonstrates that the Ang-PMT NPs effectively enhance the efficacy of chemotherapy for glioma tumors in vivo by engaging the ICD and cGAS-STING pathway. This multifunctional nanoplatform may prove valuable in the management of intracranial tumors in general.

Materials and reagents

TPP-DOX was purchased from MACKLIN (China), MnO₂ nanoparticles (5 nm, 5 mg/ mL) were purchased from Xi^’an ruixi Biological Technology Co. Ltd (USA). PLGA-PEG-COOH (50: 50, MW: 15 000) was purchased from Daigang BIO Engineer Ltd, Co. (China). JC-1 Mitochondrial Membrane Potential Assay Kit were purchased from KeyGEN Bio TECH (China), cell counting kit (CCK-8) and calcein & propidium iodide (PI) apoptosis assay kit were purchased from Dojindo Laboratories (Japan). GSH assay kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd (China).

Synthesis of Ang-PMT NPs

Ang-PMT NPs were fabricated by a well-established double emulsion (water/oil/water: W/O/W) method. First, 100 mg PLGA-PEG-COOH was dissolved in 2 mL CHCl₃. Then, 0.04 mL solution of MnO₂ NPs and 0.4 mL solution of TPP-DOX (100 mg mL^− 1) were added sequentially and the mixture was sonicated with ultrasonic probe (USA) at a parameter of 105 W for 2 min (W/O); subsequently, 3 mL of PVA solutions (w/v = 4%) was added into the mixture to emulsify again at a sonication intensity of 105 W for 5 min (W/O/W). 8 mL of isopropyl alcohol solution (w/v = 2%) was then added into the emulsion and the mixture was stirred to volatilize CHCl₃. Finally, the prepared NPs were re-suspended in PBS after centrifugation (10 000 rpm, 10 min) and stored at 4°C for further use. Ang-PM NPs and Ang-PT NPs were prepared by a similar procedure without adding DOX-TPP or MnO₂ NPs and with equivalent deionized water replaced. Fluorescent NPs were also prepared in a similar manner, with DiI or DiR or DiD adding to the mixture first and sealed with aluminum foil to avoid fluorescence quenching.

Characterization

The structure of Ang-PMT NPs were acquired through transmission electron microscopy (TEM) (HT-7800, Hitachi, Japan). Dynamic light scattering (DLS) measurements were carried out on Malvern Zetasizer. The UV–vis spectrophotometer was recorded by SHIMADZU UV-3600Plus with sweeping at 1 nm s^− 1. CLSM images were captured using a Nikon optical microscope (Japan). The flow cytometry assay was used by Beckman Coulter (American). Inductively coupled plasma optical emission spectrometry (ICP) was used by Aglient 7850 (American).

Cell culture and CT2A tumor-bearing mouse model

The murine breast cancer CT2A cells and brain-derived Endothelial cells.3 (bEdn.3) cells were purchased from the Chinese Academy of Sciences Cell Bank (China). The cells were cultured at 37°C with 5% CO₂ in DMEM supplemented with 10% FBS and 1% streptomycin/penicillin. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Chongqing Medical University and approved by the Animal Ethics Committee of Chongqing Medical University (IACUC-CQMU-2024 -0603).

Female C57BL/6J mice aged 6–8 weeks and weighing 20–25 g was obtained from the Experimental Animal Center of Chongqing Medical University. The mice were fed a standard pellet diet and had unrestricted access to water. They were housed in individual cages under standard environmental conditions, with a 12 h light/dark cycle and a temperature of 25 ± 2°C. To establish the mouse subcutaneous xenograft model, 100 µL PBS solutions containing CT2A cells (1 × 10⁶) were subcutaneously injected into the left flank of female C57BL/6J mice. The volume of the tumor was calculated as [0.5 × length × (width)²]. Orthotopic glioma-bearing mouse models were established according to the previously described protocol. C57BL/6J mice were anesthetized and immobilized on a brain stereotactic fixation device. Totally, 4 µL of CT2A cells at a density of 3 × 10⁶ was implanted into the right striatum of C57BL/6J mice (2 mm lateral, 1 mm longitudinal, and 2.5 mm depth).

In vitro GSH-responsive cytotoxicity assay

To evaluate the GSH responsive behavior of Ang-PMT NPs, CT2A cells and bEnd.3 (1 × 10⁴) were seeded into 96-well plates and allowed to adhere overnight respectively. Then, the medium was replaced by serum-free DMEM containing Ang-PMT NPs, Ang-PT NPs and Ang-PM NPs with different concentrations (0, 25, 50, 75, 100, and 150 µg/mL) for 2 h. Then, the cell viability was determined by CCK8 assay. To evaluate the cytotoxicity and the growth inhibition of various NPs, Ang-PMT NPs, Ang-PT NPs and Ang-PM NPs at the concentration of 100 µg/mL were incubated with CT2A cells for 30 min, 60 min, 90 min, 120 min, and 150 min, respectively. The cell viability was determined by CCK8 assay. Five replicates were conducted for each group.

Mitochondrial targeting capacity of Ang-PMT NPs

To evaluate the mitochondrial targeting capacity of Ang-PMT NPs, CT2A cells (1 × 10⁵) were cultured in confocal cell-culture dishes overnight, the cells were cocultured with Ang-PMT NPs and PMT NPs (100 µg/mL) for 1 h. After three-time washing with PBS, the cells were incubated with Mito-Tracker Green (λ_excitation/λ_emission = 490/516 nm) and DAPI for 30 min, respectively. CLSM was used to confirm the mitochondrial targeting capacity of Ang-PMT NPs. Moreover, the corresponding colocalization analysis of Mito-Tracker and DOX was using the Image J software.

In vitro evaluation of mitochondrial apoptosis

To evaluate the mitochondria-mediated apoptosis induced by Ang-PMT NPs, CT2A cells (1 × 10⁵) were cultured in confocal cell-culture dishes overnight. Then, cells were cocultured with PBS, TPP-DOX, Ang-PM NPs, Ang-PT NPs, and Ang-PMT NPs for 1 h, respectively. The concentration of various NPs was 100 µg/mL, and the TPP-DOX concentrations was 65 µg/mL (consistent with drug-loaded groups). After three-time washing with PBS, the cells were labeled with JC-1 dye (J-aggregate, λ_excitation/λ_emission = 585/590 nm and J-monomer, λ_excitation/λ_emissio = 514/529 nm) for 20 min. The fluorescence images were captured using the CLSM. The fluorescence ratio of aggregate and monomer dyes was quantified via Image J software.

In vitro immunogenic cell death (ICD) evaluation

The expression of CRT and HMGB1 was investigated by immunofluorescence analysis. CT2A cells (1 × 10⁵) were cultured in confocal cell-culture dishes overnight. Afterward, the cells were cocultured with PBS, TPP-DOX, Ang-PM NPs, Ang-PT NPs, and Ang-PMT NPs for 1 h, respectively. After being washed thrice, the cells were subjected to fixation in formaldehyde for 15 min, treated with Trion X-100 for 20 min, and blocked for 120 min (surface exposure of CRT analysis without Trion X-100 treatment). The cells were then treated with Calreticulin antibody (1:150) or HMGB1 antibody (1:150) for the entire night, maintained at 4°C, and incubated with CoraLite® Plus 488 (1:400) at ambient temperature for 60 min. After a 10 min incubation period with DAPI, the fluorescence images were captured using the CLSM.

The targeting evaluation of Ang-PMT NPs

Orthotopic glioma-bearing C57BL/6J mice were randomly assigned into two groups (PMT NPs and Ang-PMT NPs) and intravenously injected with DiD (Ex/Em:644 nm/665 nm)-labeled NPs PBS solution (5 mg PLGA NPs/kg, 200 µL). Fluorescent images were acquired at pre-injection, 2 h, 6 h, 24 h, and 48 h post-injection by using an LB983 NC320 (Germany). At 48 h after administration, the tumors and major organs (heart, liver, spleen, lung and kidneys) were harvested for fluorescent imaging and the corresponding fluorescence intensity was measured. Also, the DiR (Ex/Em:748 nm/780 nm) -labeled PMT NPs and Ang-PMT NPs (5 mg PLGA NPs/kg, 200 µL) were administered intravenously to the xenograft glioma-bearing C57BL/6J mice to investigate the targeting ability of Ang-PMT NPs. As previously described, fluorescent images were acquired.

Magnetic resonance imaging of Ang-PMT NPs

For in vitro MRI, three different PBS solution were prepared firstly as follows: (1) pH 7.2; (2) pH 6.5; (3) pH 7.2 + 2 mM GSH. Then, Ang-PMT NPs were dispersed in the three PBS solution and diluted to the different concentrations ([Mn] = 0, 0.005, 0.01, 0.02, 0.04, and 0.06 mM). Next, the mixture PBS solution was added into 2 mL Eppendorf tubes for T₁-weighted and T₁-mapping MRI (Bruker, Germany). The T₁-weighted images and T₁ values were acquired from the 7T MRI scanner. The following parameters were used to obtain T₁ value: echo time (TE) = 6.0 ms; repetition time (TR) = 400.0 ms; thickness = 1.0 mm; field of view (FOV) = 40.0 × 40.0 mm²; slice = 5; image size = 256 × 256. The corresponding T₁ relaxation time within the region of interest (ROI) of each sample was measured, and the T₁ relaxation coefficient (r₁) was calculated.

For in vivo MRI, orthotopic and xenograft glioma-bearing C57BL/6J mice were injected intravenously with Ang-PMT NPs (5 mg PLGA NPs/kg, 200 µL) respectively, and T₁-weighted images were acquired at pre-injection, 2 h, 6 h, 24 h, and 48 h post-injection. Then, Image J software was used to perform pseudo-coloring on each image. The parameters were used as follows: TE = 6.2 ms; TR = 800.0 ms; thickness = 0.8 mm; FOV = 35.0 × 30.0 mm²; slice = 18, image size = 256 × 256. For comparing the T₁ imaging performance of the agents, we quantified and analyzed the imaging signal to obtain signal-to-noise ratio (SNR) with the following equation: ΔSNR=(SNRpost − SNRpre)/SNRpre×100% (SNRtumor = SItumor/SDnoise), where SI represents signal intensity in region of interest and SD represents standard deviation in MR images.

Evaluation of enhanced chemotherapy efficacy

For evaluating the in vivo enhanced chemotherapy efficacy of Ang-PMT NPs, twenty-five CT2A tumor-bearing C57BL/6J mice were randomly divided into five groups (five mice per group) when the tumor volume reached about 50–80 mm³. The first group of mice was intravenously injected with PBS, set as the control group; the second group was injected with TPP-DOX (1 mg/kg) solution, set as “TPP-DOX” group; the third group was injected with Ang-PM NPs (5 mg PLGA NPs/kg) solution, set as “Ang-PM” group; the fourth group was injected with Ang-PT NPs (5 mg PLGA NPs/kg), set as “Ang-PT” group; the fifth group was injected with Ang-PMT NPs (5 mg PLGA NPs/kg), set as “Ang-PMT” group. All the experimental reagents were dispersed in 200 µL PBS before intravenous administration. The tumor volumes and body weights were measured by an electronic caliper every day post-laser irradiation from day 0 to day 10 after the corresponding treatments. On the 10th day after treatment, mice were euthanized and the tumor tissues were harvested for weighing and staining with hematoxylin-eosin staining (H&E), proliferation marker (Ki67), terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL), and cytotoxic T lymphocytes marker (CD8) to observe necrosis, proliferation, apoptosis, and infiltration of lymphocytes.

Statistical analysis

All statistical analyses were performed using the Prism 10.0. Data reported herein are means ± standard deviation (SD). Differences between groups were compared by a one-way analysis or Student’s t-test of variance (ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Synthesis and Characterization of Ang-PMT NPs

Scheme 1a illustrates the preparation process of Ang-PMT NPs. We successfully encapsulate MnO₂ NPs and TPP-DOX into the PLGA NPs and link the Ang to synthesize the multifunctional nanoplatform (Ang-PMT NPs). Transmission electron microscopy (TEM) image shows a high contrast and uniform distribution, indicating that the MnO₂ NPs are efficiently loaded into the spherical shell (Fig. 1a). The average hydrodynamic diameter (D_h) of Ang-PMT NPs was 190.1 ± 2.2 nm, which is slightly larger than that of PMT NPs (175.0 ± 0.9 nm), PM NPs (172.2 ± 0.8 nm), and PT NPs (153.2 ± 0.1 nm) (Fig. 1b), demonstrating that the Ang was successfully conjugated and that MnO₂ NPs and TPP-DOX were incorporated into the NPs. Moreover, the zeta potential of Ang-PMT NPs, PMT NPs, PM NPs, and PT NPs was determined to be − 11.7 ± 0.7 mV, -14.2 ± 0.7 mV, -14.6 ± 0.1 mV, and − 18.9 ± 0.7 mV, respectively (Fig. 1c), further verifying the successful Ang conjugation and MnO₂ NPs and TPP-DOX incorporation from a different perspective. Moreover, nuclear magnetic resonance (NMR) spectroscopy results confirmed the successful Ang modification of PLGA NPs owing to the observation of characteristic peaks at 7.00 and 7.40 ppm present in the spectrum of Ang-PLGA NPs (Fig. S1), which confirmed that the Ang peptide binds covalently to PLGA through amide bonds.

To assess the in vitro biological stability, Ang-PMT NPs were dispersed in various solutions, including pure water (H₂O), phosphate buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco's modified Eagle medium (DMEM) (containing 10% FBS). Over a 24-hour period, no aggregation was observed, and the corresponding D_h of the dispersions was recorded as 106.4, 107.3, 103.4, and 102.4 nm, respectively (Fig. 1d). Then, the long-term colloidal stability of Ang-PMT NPs was also evaluated. The Ang-PMT NPs showed an excellent stability over a week in H₂O, as indicated by a relatively uniform D_h and zeta potentials (Fig. S2). A hemolysis test was conducted to evaluate the preliminary biocompatibility of Ang-PMT NPs. The hemolysis rate was found to be less than 5% at Ang-PMT NPs concentrations were up to 250 µg/mL (Fig. 1e), indicating that the multifunctional nanoplatform exhibits favorable biocompatibility for in vivo applications. Subsequently, the loading and encapsulation efficiencies of TPP-DOX and MnO₂ NPs were evaluated, respectively. The UV-vis-NIR spectra exhibited the absorption of TPP-DOX at 488 nm (Fig. S3). The drug loading efficiencies and encapsulation efficiencies of TPP-DOX were quantified and calculated according to the equation, resulting in a drug loading efficiency of 10.2 wt% and an encapsulation efficiency of 87.1%, respectively (Fig. 1f). The carrier rate of MnO₂ NPs in Ang-PMT NPs, PMT NPs, and PM NPs was approximately 75.70 ± 2.58% and 67.00 ± 3.08%, respectively (Fig. 1g).

MnO₂ is known to deplete GSH and reduce pH values to decompose Mn⁴⁺ into Mn²⁺ [26, 27]. Therefore, we detected the GSH depletion and pH value reduction characteristics of Ang-PMT NPs were investigated. As shown in Fig. 1h, Ang-PMT NPs exhibited the capacity to deplete 1.0 mM GSH following a 30-minute incubation period with a GSH solution, suggesting a great GSH consumption ability of Ang-PMT NPs. We further assessed the drug release behavior of TPP-DOX from Ang-PMT NPs in PBS at various pH values or GSH concentrations. As shown in Fig. 1i, Ang-PMT NPs exhibited a sustained release of encapsulated DOX within 20 minutes under 2 mM GSH, due to the rapid decomposition of MnO₂ NPs. The release rates of TPP-DOX were observed to be significantly accelerated in mild acidic conditions (pH 6.5 with 2 mM GSH). And, the cumulative amount of TPP-DOX released from the Ang-PMT NPs reached 83.94%.

Intracellular GSH-responsive cytotoxicity, Phagocytosis ability, and Mitochondrial targeting capacity of Ang-PMT NPs

High levels of GSH have been observed in the majority of tumor cells [28, 29]. We first tested the content of GSH in CT-2A cells and bEnd.3 cells. As shown in Fig. S4a, the GSH content in CT-2A cells is markedly higher (2.75 mM) than that in the normal bEnd.3 cells (0.31mM) (***p < 0.001). Then, the capacity of Ang-PMT NPs to deplete intracellular GSH was assessed using CT-2A cells. The intracellular GSH content was reduced by 1.04 mM following incubation with Ang-PMT NPs (100 µg/mL) (Fig. S4b), suggesting a notable GSH consumption ability. To further investigate the GSH responsive behavior of Ang-PMT NPs inside cells, the CT-2A cells and bEnd.3 cells were incubated with Ang-PMT NPs at various concentrations (0, 25, 50, 75, 100, and 150 µg/mL) for 60 minutes, respectively. As shown in Fig. 2a, the viability of CT-2A cells exhibited an apparent cytotoxicity, especially when the concentration up to 100 µg/mL, revealing the MnO₂ NPs can react with GSH in the tumor cells and release DOX to kill tumor cells. In contrast, the viability of bEnd.3 cells did not show a noticeable reduction even the concentration of Ang-PMT NPs up to 150 µg/mL owing to the low levels of GSH, demonstrating the good biocompatibility of our nanoplatform in normal cells. Then, the CT-2A cells and bEnd.3 cells were incubated with Ang-PMT NPs (100 µg/mL) at different time points (0, 30, 60, 90,120,150, and 180 min), respectively. The viability of CT-2A cells less than 50% after the incubation time over 90 min, while the viability of bEnd.3 cells showed a negligible reduction even the incubation time extended to 180 min (Fig. 2b).

The BBB represents a significant obstacle to the effective delivery of drugs to the CNS [30]. To assess the efficacy of Ang-PMT NPs in traversing the BBB and targeting the tumor cells, we established an in vitro BBB model using mouse cerebral microvascular endothelial bEnd.3 cells in the upper compartment and CT-2A cells in the lower compartment of a transwell chamber (Fig. 2c). The BBB crossing and tumor cell targeting capabilities of Ang-PMT NPs were visualized using confocal laser scanning microscopy (CLSM) and analyzed by flow cytometry (FCM). As shown in Fig. 2d, the red fluorescence intensity of Ang-PMT NPs in CT-2A cells was much stronger than that of PMT NPs, owing to the enhanced targetability of Ang. The FCM results quantified that the red fluorescence intensity of Ang-PMT NPs in CT-2A cells (66.4%) was significantly higher than that of PMT NPs (20.8%) (Fig. 2e), demonstrating the effective penetration of the BBB and targeting of glioma. Next, the intracellular uptake behavior of Ang-PMT NPs on CT-2A cells was examined. The CLSM results indicated that the red fluorescence intensity of Ang-PMT NPs in the cytoplasm remarkedly increased with the prolongation of incubation time (Fig. 2f). FCM results revealed that the intracellular uptake rate of Ang-PMT NPs reached 92.9% at 45 minutes post-incubation and approached 100% at 60 minutes post-incubation (Fig. 2g). These results demonstrate the effective phagocytosis of Ang-PMT NPs by CT-2A cells following a 60-minute incubation period.

It is well known that TPP exhibits a good capacity to target mitochondrial membranes [31, 32]. The mitochondrial targeting capability of Ang-PMT NPs was visualized by CLSM using MitoTracker. The CT-2A cells were incubated with Ang-PMT NPs and PMT NPs for 60 minutes, respectively. As shown in Fig. 2h and 2i, the red fluorescence intensity of Ang-PMT NPs was effectively internalized by tumor cells and retained in mitochondria with green fluorescence intensity after 60 minutes of incubation, but a relatively poor accumulation of PMT NPs was observed in the mitochondrial areas, affirming the preferential targeting of mitochondria by cationic TPP⁺ due to the high mitochondrial membrane potential, [33] which leads to effective accumulation in mitochondria.

In vitro Evaluation of Ang-PMT NPs Induced Apoptosis

Mitochondria play a pivotal role in apoptotic cell death [34]. The active mitochondrial targeting capacity of Ang-PMT NPs can enhance the accumulation of chemotherapeutic drugs around mitochondria, leading to enhanced mitochondrial damage and dysfunction. To evaluate the mitochondria-mediated apoptosis induced by Ang-PMT NPs, the membrane-permeant probe, JC-1 dye was used to monitor the mitochondrial membrane potential. As shown in Fig. 3a and Fig. S5, the CT-2A cells that were incubated with Ang-PMT NPs exhibited the most pronounced green fluorescence intensity compared to the other groups (*p < 0.05), representing mitochondrial depolarization and early apoptosis. This result demonstrates the capacity of Ang-PMT NPs to elicit a more robust and expeditious apoptotic response compared to the controls. To further assess the in vitro therapeutic effect of Ang-PMT NPs, calcein-AM and PI were used to identify live cells (green fluorescence intensity) and dead cells (red fluorescence intensity). As shown in Fig. 3b, CLSM images showed that the vast majority of cells exhibited red fluorescence intensity following incubation with Ang-PMT NPs for 60 minutes, indicating significant cell apoptosis/necrosis. In comparison, cells treated with Ang-PM or Ang-PT exhibited minimal green fluorescence intensity, suggesting no obvious cell death. Moreover, the apoptotic rate induced by Ang-PMT NPs was analyzed by FCM, which yielded results that were consistent with the CLSM observations (Fig. 3c).

The intracellular release of the chemotherapeutic drug DOX induces apoptosis and ICD in tumor cells. This consequently leads to a substantial release of DAMPs including CRT, HMGB1, and ATP [35]. This process fundamentally alters the TME. To reveal the nature of the ICD induced by DOX, the expression of CRT and HMGB1 on CT-2A cells was observed via CLSM. As anticipated, the CRT expression of CT-2A cells incubated with Ang-PMT NPs was markedly enhanced, while minimal changes were observed in the other groups (Fig. 3d). Similarly, Ang-PMT NPs were observed to cause the highest extracellular release of HMGB1 from the nucleus, with the associated green fluorescence intensity in CT-2A cells being the lowest (Fig. 3d). In addition, the level of extracellular ATP was significantly increased in the Ang-PMT NPs group (Fig. 3e).

As a topoisomerase inhibitor, DOX has been shown to impede DNA replication and elevate cytoplasmic dsDNA levels [36]. Furthermore, Mn²⁺ has been demonstrated to activate the cGAS-STING pathway and enhance IFN production [37]. Consequently, the expression of the associated proteins in the cGAS-STING pathway was detected in CT-2A cells by western blot analysis following different treatments. As shown in Fig. 3e and Fig. S6, the expression of phosphorylated TBK1 (p-TBK1), phosphorylated IRF3 (p-IRF3), and STING protein was significantly higher in the Ang-PMT NPs group compared to the control groups, confirming the successful activation of STING signaling and demonstrating that dsDNA induced by DOX and Mn²⁺ uptake by tumor cells synergistically activated the cGAS-STING pathway through Ang-PMT NPs treatment.

Biosafety of Ang-PMT NPs

The promising biocompatibility and biosafety of Ang-PMT NPs are of significant importance for further clinical translation. The biosafety of Ang-PMT NPs was evaluated in healthy female C57BL/6J mice through biochemical testing and hematological analysis (H&E) staining. As shown in Fig. 4a, all the evaluated blood biochemical indices exhibited no discernible alterations at any time point when compared to the control group following intravenous administration of Ang-PMT NPs. Furthermore, H&E staining of major organs (e.g., the heart, liver, spleen, lung, and kidney) revealed no obvious acute or chronic adverse effects (Fig. 4b). The results confirmed the favorable biocompatibility and safety profile of Ang-PMT NPs, paving the way for further in vivo investigations.

The targeting efficacy and MR imaging of Ang-PMT NPs

To evaluate the tumor targeting efficacy of Ang-PMT NPs, we evaluated the distribution of fluorescent dye-labeled Ang-PMT NPs in tumor-bearing mice by fluorescence imaging. As shown in Fig. 5a and Fig. S7a, the fluorescence signals were observed at the tumor site at 2 h post-injection, peaked at 24 h post-injection, and were continuously observed in the tumor region up to 48 h post-injection, indicating that the NPs could remain in the tumor site for a long time. Moreover, the fluorescence intensity in tumor-bearing mice treated with Ang-PMT NPs was stronger than that observed in mice treated with PMT NPs (Fig. 5b and Fig. S7b), confirming the ability of Ang to cross the BBB and target tumor sites. The tumor and major organs were excised at 48 h post-injection to confirm the accumulation of NPs in tumor tissues. As shown by ex vivo imaging and quantitative analysis (Fig. 5a, 5c, and Fig. S7c), both two groups showed abundant fluorescence intensity in the liver and spleen, which can be attributed to the phagocytosis of the reticuloendothelial system. Moreover, the signals in the Ang-PMT NPs group were significantly stronger than those in the PMT NPs group (***p < 0.001), suggesting that Ang facilitates BBB penetration to a greater extent than the control formulation.

MR imaging plays an important role in the clinical diagnosis of glioma due to its high spatial resolution and deep tissue penetration [38, 39]. It is well known that Mn²⁺ are ideal contrast agents for T₁-weighted MRI [40]. To investigate the GSH-activated MRI performance of Ang-PMT NPs, we compared the T₁ imaging and relaxivity under different TME conditions, in the presence and absence of GSH. As shown in Fig. 5d, the T₁-weighted MR signals exhibited a rapid increase with elevated Ang-PMT NPs concentrations, particularly in the presence of GSH. However, the T₁-weighted MR signals demonstrated minimal change in the physiological environment (pH 7.2), despite the high concentration of Ang-PMT NPs, confirming the ability of Ang-PMT NPs to function as a GSH responsive T₁-weighted contrast agent. Furthermore, a linear relationship was identified between the 1/T₁ values and the concentration of Mn. In comparison to the pH 7.2 group, the T₁ relaxation coefficient (r₁) was markedly elevated in the other two groups. The r₁ value in the pH 6.5 + 2 mM GSH group reached 5.801 (Fig. 5e), which is likely attributed to the degradation of Mn⁴⁺ into Mn²⁺ in the presence of GSH.

In light of the high r₁ value exhibited by Ang-PMT NPs in response to GSH in vitro, we further investigated the GSH-activated MRI capabilities of Ang-PMT NPs in vivo. As shown in Fig. 5f and 5g, following intravenous administration of Ang-PMT NPs, a notable increase in brightness was evident within the tumor region, accompanied by a gradual enhancement over time. The signal-to-noise ratio (SNR) was used to quantitatively analyze the MR signal intensity over time, with the maximum value observed at 24 h post-injection. These findings suggest that Ang-PMT NPs may serve as a promising candidate for GSH-responsive T₁ MRI contrast agents.

In Vivo Enhanced Chemotherapy Efficacy

Motivated by the encouraging in vitro anti-tumor outcomes, the in vivo enhanced chemotherapy efficacy of Ang-PMT NPs was further evaluated in five groups of CT2A tumor-bearing mice (n = 5 per group): 1) PBS as the control group, 2) free TPP-DTX (1 mg/kg), 3) Ang-PM NPs (5 mg PLGA NPs/kg, same dose for the subsequent groups), 4) Ang-PT NPs, and 5) Ang-PMT NPs. The treatment protocol was illustrated in Fig. 6a. All tumor-bearing mice were administered the treatment via intravenous injection on day 0. Tumor volumes and body weights were assessed on a daily basis following the administration of the treatment. As displayed in Fig. 6b and Fig. S8, the free TPP-DOX group exhibited minimal inhibitory effects on tumor growth due to its rapid clearance from the blood circulation. In contrast, the Ang-PMT NPs and Ang-PT NPs groups exhibited significant tumor growth inhibition compared to other groups (***p < 0.001), suggesting enhanced chemotherapy efficiency. Particularly, the Ang-PMT NPs group exhibited the most pronounced inhibition of tumor growth inhibition compared to other groups (***p < 0.001), which could be attributed to the efficacious combination of antitumor activity stemming from the chemotherapy and the catalytic function of MnO₂ NPs and GSH within the tumor TME. On the 10th day following treatment, all tumors were extracted, and the tumor in the Ang-PMT NPs group displayed the most obvious inhibition of tumor growth in comparison to other treatment groups (Fig. 6c). Nevertheless, a slight inhibition of tumor growth was observed in the free TPP-DOX group and the other groups. Notably, negligible weight loss was observed during the therapeutic period (Fig. 6d), suggesting that Ang-PMT NPs do not induce systemic toxicity.

To elucidate the comprehensive antitumor capability of Ang-PMT NPs, H&E, Ki67, TUNEL, and CD8 T cells staining assays were performed. As shown in Fig. 6e, the Ang-PMT NPs group displayed the most significant tumor cell damage, as evidenced by the presence of numerous deformed nuclei (wrinkles, a reduced number of nuclei, karyopyknosis, and karyolysis) and an increase lymphocyte infiltration in the H&E of tumor tissues. Consistently, immunofluorometric assay on Ki-67, TUNEL, and CD8 T cells revealed that the Ang-PMT NPs group exhibited a significantly lower number of Ki67-positive proliferative cells, a higher number of apoptosis-positive cells and a notable increase in CD8-positive T cells within the tumor tissues than the other groups, suggesting a significant inhibitory effect on tumor aggressiveness and a beneficial influence on TME alterations.

In conclusion, we have efficaciously devised and synthesized a multifunctional nanoplatform incorporating PLGA NPs, MnO₂ NPs, TPP-DOX, and the targeting ligand Ang, that may significantly enhance the efficiency of glioma chemotherapy. The multifunctional nanoplatform exhibited notable efficacy in tumor therapy through the following mechanisms: i) Successful crossing of the BBB and phagocytosis by tumor cells owing to dual targets (Ang and mitochondriotropic TPP); ii) Immediate release of DOX and Mn²⁺ production due to the efficient reaction between GSH and MnO₂ NPs; iii) Besides directly killing tumor cells, DOX and Mn²⁺ further stimulated the process of ICD and activated the cGAS-STING pathway, thereby enhancing the efficacy of chemotherapy; iv) Assisted by MR imaging, the efficacy of treatment is visible and trackable. This innovative strategy provides a new avenue for glioma treatment. Furthermore, the synthesis process of Ang-PMT NPs is straightforward with a high yield, making it a prospective contender for mass production, ensuring scalability and cost-effectiveness, thus promoting clinical translation. Therefore, the developed multifunctional nanoplatform devised holds potential for clinical applications in managing glioma.

Supporting Information

Supporting Information is available from the online version.

Author Contributions

GZ: Conceptualization, Investigation, Methodology, Validation, Writing original draft. TK: Conceptualization, Methodology. WZ: Conceptualization, Investigation. HS: Investigation, Analyze data. GL: Methodology, Validation. XY: Conceptualization, Investigation. ZO: Analyze data. HW: Investigation, Methodology, Funding acquisition. HY: Funding acquisition, Conceptualization, Supervision, Writing review & editing. CL: Conceptualization, Supervision, Funding acquisition. All authors read and approved the final manuscript.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (82160340), the Yunnan Talents Support Program (XDYC-MY-2022-0064), the Chongqing Medical Youth Top-Notch Talent (YXQN202446), Chongqing Postdoctoral Science Foundation (2023CQBSHTB1005), Natural Science Foundation of Chongqing (CSTB2024NSCQ-MSX0102), and Graduate Innovation Foundation of Kunming Medical University (Grant No. 2024B013 and 2024S122).

Data availability

No datasets were generated or analysed during the current study.

Competing interests

The authors declare no competing interests.

Author details

¹ Department of Radiology, Yan’an Hospital Affiliated to Kunming Medical University (Kunming Yan'an Hospital), Kunming, 650051, China

² Department of Radiology, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital，Peking University Cancer Hospital Yunnan Campus), Kunming, 650106, China

³Department of Medical Imaging, The First Affiliated Hospital of Kunming Medical University, Kunming, 650032, China

⁴Department of Head and Neck Surgery, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Peking University Cancer Hospital Yunnan Campus), Kunming, 650106, China

⁵Department of neurology, The Second Hospital of Dalian Medical University, Dalian, 116000, China

⁶ Department of Ultrasound, Chongqing General Hospital, Chongqing University, Chongqing, 401147, China

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Scheme 1 is available in the Supplementary Files section.

No competing interests reported.

floatimage1.png
Scheme 1. Schematic illustration of multifunctional nanoplatform consisting of poly-lactide-co-glycolide (PLGA) loaded with manganese dioxide (MnO₂) and doxorubicin (DOX), conjugated with TPP and Ang for MR imaging-guided enhanced chemotherapy of glioma and reshaping the tumor immune microenvironment (TME): the preparation of Ang-PMT NPs (a) and the penetration of Ang-PMT NPs through the BBB and targeting of the tumor region with the assistance of Ang modification (b) and the use of Ang-PMT NPs for MR imaging-guided enhanced chemotherapy of glioma and reshaping of the TME (c).
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GSH-responsive and Mitochondria-targeting Multifunctional Nanoplatform for MR imaging-guided enhanced chemotherapy of Glioma by Reshaping the Tumor Immune Microenvironment

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