Catalytic modulation of the tumor microenvironment using cascade enzymes for synergistic boron neutron capture therapy in the treatment of gliomas

doi:10.21203/rs.3.rs-4939120/v1

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Catalytic modulation of the tumor microenvironment using cascade enzymes for synergistic boron neutron capture therapy in the treatment of gliomas

https://doi.org/10.21203/rs.3.rs-4939120/v1

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Glioma is the most common primary malignant tumor of the central nervous system. Despite traditional treatments such as surgical resection, combined postoperative chemotherapy, and radiotherapy, it remains challenging to significantly improve the long-term survival rate of patients. This is primarily due to the incomplete surgical removal of the tumor and its resistance to radiotherapy. Boron neutron capture therapy (BNCT) is a radiotherapy that can selectively kill tumor cells. Current research has demonstrated that BNCT offers effective local disease control for gliomas and head and neck tumors. However, the existing boron-containing drugs are still unsatisfactory due to their low boron content and poor targeting ability. The synergistic treatment has provided new ideas for the development of BNCT, and the emergence of nanosystems offers the possibility of prolonged retention and pinpoint delivery of boron drugs in the tumor cites. The unique tumor microenvironment(TME) of gliomas, characterized by the blood brain barrier (BBB), oxidative stress, hypoxia and angiogenesis, renders conventional treatments ineffective and poor prognosis. Therefore, to combine the TME regulation and BNCT therapy, we prepared a stable nanosystem in this study. It is a borane-contained cationic liposome modified with cRGD peptide which enhances the tumor-targeting ability. And the enzymes Lactate oxidase(LOX) and Catalase(CAT) are absorbed on the surface of the nanosystem which reduce the concentration of lactic acid through a cascade reaction to generate O₂ and decrease the protein expression level of HIF-1α. This nanosystem exhibited a more potent anti-tumor effect both in vitro and in vivo. Also it reduced tumor stemness in vivo, which improves the prognosis. Therefore, the novel nanosystem combined microenvironment regulation therapy and BNCT shows the great potential application in anti-tumor treatment.

boron neutron capture therapy

glioma

nano-delivery system

tumor microenvironment

Glioma is a type of central nervous system tumors originating from neuroglial cells which can extensively infiltrate the brain parenchyma (1)and accounts for approximately 80–85% of malignant brain tumors in adults(2). Postoperative radiotherapy with 60 Gy ray can significantly improve survival rates of gliomas, but also increases the toxicity in patients(3). Besides, due to the transcriptional plasticity and heterogeneity of gliomas, radiation resistance inevitably occurs, thus limiting the effectiveness of radiation therapy in treating gliomas.(4) Therefore, there is an urgent need to explore new treatment methods for gliomas.

Boron Neutron Capture Therapy (BNCT) is a type of radiation therapy that can selectively kill tumor cells (5). ¹⁰B is a natural, non-radioactive element that undergoes a fission reaction when irradiated with low-energy (0.025 eV) thermal neutrons or epithermal neutrons (10,000 eV), producing high linear energy transfer (LET) alpha particles (⁴He) and recoil lithium-7 (⁷Li) nuclei (6, 7). These particles are characterized by high energy and short paths. The LET of these particles is higer than 100 keV/µm, which means that just a few alpha particles can have a lethal effect on tumor cells. Since their range of destruction is 5–9 µm, approximately equals to the diameter of a tumor cell, they cause minimal damage to surrounding normal cells, making BNCT an ideal method of radiation therapy (8).

The success of BNCT hinges on the selective accumulation of boron agents in tumor cells. Currently, two boron-containing drugs are frequently used in clinical studies: boronophenylalanine (BPA) and sodium borocaptate (BSH) (9, 10). Although BPA(11) has good tumor targeting properties, its low boron content and poor solubility hinder its further use in clinical and research applications. Despite containing twelve ¹⁰B atoms, BSH enters tumor cells through passive diffusion across the plasma membrane and cannot cross the BBB (12, 13), leading to unsatisfactory treatment outcomes, especially for malignant tumors such as gliomas. Therefore, to enhance the effectiveness of BNCT, it is crucial to develop more efficient and selective boron delivery agents.

Boron-containing nanoparticles greatly improve boron transport efficiency and demonstrate substantial tumor inhibition in animal models. Over the past decades, boron-loaded nanocarriers have been extensively studied.(14–17) Boron nitride nanoparticles (18, 19), boric acid-enriched nanoparticles (20), boron-containing carbon dots (21), and boron-carrying liposomes (22) have been utilized to accomplish boron drug enrichment and delivery, and are regarded as promising nanoparticles for future research. BNCT has also now been used in combination with a number of other therapies.(23–26) Our group has conducted numerous studies on the development of novel boron carriers and the synergistic treatment of BNCT, primarily focusing on brain targeting and combination with immunotherapy,(27) utilizing carboranes, which are widely used in various fields such as nanomaterials and medical pharmacology (28).

Tumor cells must produce sufficient ATP and biosynthetic precursors to sustain the demands of cell proliferation. Otto Warburg first studied the reasons for increased metabolic rates in several tumors in 1926 (29), demonstrating that tumor cells absorb large amounts of glucose as their primary energy source and produce excess lactate even in the presence of oxygen, a phenomenon known as the Warburg effect (30). Lactate is primarily produced in the TME, and recent studies have shown that lactate is not only a secondary byproduct of tumor cell metabolism but also a key molecule involved in tumor development and immune evasion (31). The accumulation of lactate creates an acidic environment in tumors, with a pH of 6.0–6.5, which is closely associated with tumor metastasis, angiogenesis and treatment resistance, and also enhances the invasiveness of the tumor (32, 33).

In the TME, the accumulation of lactate complements the formation of hypoxia. The hypoxic microenvironment of tumors can increase the genetic instability of tumor cells by inducing mutations and inhibiting DNA repair, playing a significant role in enhancing resistance to radiation therapy (34). Particularly in malignant gliomas, hypoxia can inhibit the transition of tumor cells from proliferative arrest to cellular senescence, thus diminishing the efficacy of DNA-damaging agents (35). The impact of hypoxia in TME is mediated by the activity of several transcription factors, among which Hypoxia-inducible factor 1 (HIF-1) plays the most crucial role (36). A significant portion of lactate accumulation within tumors is the result of metabolic reprogramming mediated by HIF-1 (37): hypoxia promotes anaerobic glycolysis, increasing the production and release of lactate and the accompanying acidification of the extracellular environment. In turn, lactate can indirectly stabilize one of the subunits of HIF-1, HIF-1α, thus enabling HIF-1 activation to be unaffected by the presence of hypoxia (38). Hypoxia is also inextricably linked to tumor stemness(39). It has been shown that there is a positive correlation between HIF-1α and stemness in a variety of cells(40–43), including gliomas(44–46).

LOX, by consuming lactate and O₂, produces pyruvate and H₂O₂, showing therapeutic potential in reducing tumor lactate and inducing TME modulation (47–49). However, the hypoxic conditions in the TME only provide a limited oxygen supply, resulting in reduced LOX activity and lactate consumption in tumor tissues (50). Furthermore, the production of H₂O₂ in normal tissues can cause severe side effects to damaged tissues and organs (51). CAT catalyzes the intermediate product H₂O₂ into H₂O and O₂ through a peroxidation reaction. Therefore, LOX can be utilized to catalyze the production of H₂O₂ from lactate in the TME, and further combined with CAT to cascade decompose H₂O₂ into O₂. The generated O₂ can then enhance the catalytic efficiency of LOX, thereby achieving efficient consumption of lactate in the TME and improving the hypoxic conditions of the tumor.

Herein, this study utilized thin-film hydration technique to prepare cRGD peptide-modified cationic liposomes containing carborane, which adsorb LOX and CAT enzymes via electrostatic attraction of positive and negative charges. The cRGD peptide is widely used due to its ability to bind to αvβ3 integrin. Integrin αvβ3 plays a crucial role in angiogenesis and is overexpressed in the endothelial cells of tumors (52), hence the modification with cRGD helps in the precise delivery of the nanocarriers to the tumor site. As shown in Fig. 1, the nanocarriers initially target the tumor through the action of the tumor-targeting peptide cRGD. Upon arrival, the LOX adhered to the liposomes consumes lactate in the microenvironment, thus enhancing tumor starvation therapy and increasing the tumor's uptake of carborane (CB). Simultaneously, the released CAT catalyzes the decomposition of H₂O₂ in the microenvironment, leading to the substantial release of O₂. This not only alleviates the hypoxic condition of gliomas but also provides sufficient energy for the continued consumption of lactate, thereby exacerbating the energy shortage in tumor cells. Finally, combined with the neutron capture reaction of BNCT, this nanosystem shows powerful anti-tumor effect.

Materials and Reagents

1-Bromomethyl-o-carborane (CB) was purchased from YL Biochem Co., Ltd. (Zhengzhou, China). c(RGDfK) cyclo (Arg-Gly-Asp-D-Phe-Lys) was customized by Shanghai Apeptide Co., Ltd. (Shanghai, China). 1,2-Dioleoyl-3-trimethylammonium-propane chloride (DOTAP), 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), N-(carbonyl-methoxypolyethyleneglycol 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG5000) was purchased from CordenPharma Co., Ltd. (Switzerland). Lactate oxidase (LOX) and Catalase (CAT) was purchased from Yuanye Biochemical Co., Ltd. (China). DiR perchlorate was provided from Meilun Biochemical Co., Ltd. (China). [Ru(dpp)₃]Cl₂ was purchased from Aladdin Biochemical Co., Ltd. (China).

Synthesis of CLC@lipo-cRGD

Weigh out various phospholipid materials and cholesterol according to the following molar ratios: DOTAP:DOPE:CHOL:DSPE-PEG5000 = 0.8:1:0.5:0.023. Weigh a total mass of 30 mg, consisting of approximately 13.31 mg DOTAP, 10.00 mg DOPE, 3.45 mg CHOL, and 2.35 mg DSPE-PEG5000. Additionally, weigh 5 mg of CB and dissolve all substances in chloroform, assisted by ultrasonic bath sonication. Under reduced pressure at 40°C, rotate the solution in a rotary evaporator for 1 hour until the organic solvent is completely removed, forming a uniform thin film at the bottom of the round-bottom flask. Concurrently, prepare a 5% sucrose solution by dissolving 0.5 g of sucrose in 10 mL of DEPC water, preheating it to 50°C in a water bath. Add 10 mL of this solution to the previously formed thin film and sonicate in a water bath until a uniform emulsion is formed. Then, process the emulsion in an ultrasonic disruptor (250 W) for 5 minutes until a clear, transparent liquid is formed. Finally, filter the solution twice through a 0.22 µm MCE water membrane to obtain the drug-loaded cationic liposome solution (CB@lipo).

Accurately weigh cRGD and prepare it into an aqueous solution, then mix it thoroughly with the previously prepared drug-loaded cationic liposome CB@lipo using a pipette tip. After allowing the mixture to stand at room temperature for 30 minutes, the resulting drug-loaded cationic liposome and peptide complex, CB@lipo-cRGD, is obtained, where the molar ratio of cRGD peptide to the nanoliposome is 5%. Add 40 U of LOX to the prepared nanocarrier solution CB@lipo-cRGD and stir at 4°C for 2 hours. Then, centrifuge at 15,000 rpm for 15 minutes at 4°C to wash off the unbound LOX, resulting in CB/LOX@lipo-cRGD. Next, add 40 U of CAT to the obtained CB/LOX@lipo-cRGD and stir at 4°C for another 2 hours. Centrifuge again at 15,000 rpm for 15 minutes at 4°C to wash off the unbound CAT, obtaining the final product CLC@lipo-cRGD.

Characterization of multifunctional nanoliposomes

Apply a suitable amount of CB@lipo and CLC@lipo-cRGD solutions onto a copper grid, allow to adsorb for 1 minute, then use filter paper to remove excess liquid and thoroughly dry the samples. Observe their external morphology using Transmission Electron Microscopy (TEM). Measure the average hydrodynamic diameter (size), Zeta potential, and polydispersion index (PDI) of each sample using a Dynamic Light Scattering (DLS) instrument. Use the BCA and SDS-PAGE to determine the loading efficiency of LOX and CAT.

Cell Lines

The cancer cell lines GL261 was purchased from Beina Chuanglian Biotechnology Institute (Beijing, China) and the luciferase transfected GL261 (Luc-GL261) were cultured in complete growth media (DMEM medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin) at 37°C in a humid atmosphere maintained of 5% CO₂.

Cell Viability Assay

Inoculate GL261 cells in the logarithmic growth phase at a density of 3000 cells per well onto a 96-well cell culture plate, and incubate overnight at 37°C in a cell culture incubator with 21% O₂ and 5% CO₂. Remove the old culture medium, and add 100 µL of blank medium or medium containing the drugs CB, CB@lipo-cRGD, and CLC@lipo-cRGD to each well (CB is dissolved in DMSO, while the other nanocarriers are dissolved in 5% sucrose water).After drug administration, continue incubation overnight in a cell culture incubator with 21% O₂ and 5% CO₂. Remove the medium and wash the wells with PBS. Add culture medium containing 10% CCK-8 to each well and incubate for 1–2 hours. Finally, measure the optical density (OD) using an ELISA reader at a wavelength of 450 nm.

Inoculate GL261 cells in the logarithmic growth phase at a density of 3000 cells per well onto a 96-well cell culture plate. Place the plate in a cell culture incubator at 37°C with 21% O₂ and 5% CO₂ and incubate overnight. Remove the old culture medium and add 100 µL of the drug-containing medium CLC@lipo-cRGD to each well.Set up normoxic and hypoxic groups: for the normoxic group, continue incubation under 21% O₂ conditions; for the hypoxic group, transfer the cells to a hypoxia chamber at 1% O₂ for 12 hours prior to drug administration to simulate a hypoxic environment. After adding the drug, incubate the cell culture plates overnight in incubators set at 21% O₂ and 1% O₂, respectively.After incubation, aspirate the culture medium and wash the wells with PBS. Add culture medium containing 10% CCK-8 to each well and incubate for 1–2 hours. Finally, measure the optical density (OD) using an ELISA reader at a wavelength of 450 nm.

Cellular Uptake of Nanoparticles in Cell

Harvest GL261 cells in the logarithmic growth phase and switch to serum-free medium 12 hours before drug administration. Aspirate the old culture medium and wash the cells three times with PBS. Set up normoxic (21% Oc conditions) and hypoxic groups (1% O₂ conditions). For the hypoxic group, transfer the cells to a hypoxia chamber 12 hours before drug administration to simulate a hypoxic environment. To each culture flask, add drugs with equal boron concentrations of CB, CB@lipo, CB@lipo-cRGD, and CLC@lipo-cRGD. CB is dissolved in DMSO and added to the medium, maintaining a final DMSO concentration of 1% and a boron concentration of 50 µg/mL. Incubate the flasks in a cell culture incubator at 21% O₂ and 5% CO₂ for 4 hours. Aspirate the culture medium and wash the cells three times with PBS to remove residual drug-containing medium. Collect the cells for cell counting. Add a volume ratio of 3:1 of concentrated nitric acid and hydrogen peroxide, along with 5 µL of phosphoric acid to stabilize the boron elements in the solution. Mix well and digest in a 80°C water bath using the wet heating method for 2 hours. Afterward, perform ultrasonic water bath treatment for 1 hour, and dilute to volume with ultrapure water. Filter the solution through a 0.22 µm MCE water membrane before sending it for boron content determination by ICP-MS.

Uptake by cancer cells observed by Confocal Microscopy

Harvest GL261 cells in the logarithmic growth phase and digest them with trypsin. Seed the cells at a density of 10,000 cells per dish in special glass-bottomed dishes suitable for confocal microscopy. Incubate the dishes overnight at 37°C in a 5% CO₂ environment. Set up normoxic (21% O₂ conditions) and hypoxic groups (1% O₂ conditions). For the hypoxic group, transfer the cells to a hypoxia chamber 12 hours before drug administration to simulate a hypoxic environment. After incubation, remove the old culture medium and add drug-containing media with CB-DiR@lipo, CLC-DiR@lipo, CB-DiR@lipo-cRGD, or CLC-DiR@lipo-cRGD, keeping the DiR concentration consistent at 5 µmol/L in each group. Continue to incubate the cells in a 21% O₂, 5% CO₂ cell culture incubator for 4 hours. Remove the cells from the incubator, aspirate the drug-containing medium, and wash three times with PBS pre-cooled to 4°C. Fix the cells with 4% paraformaldehyde for 30 minutes, followed by three washes with pre-cooled PBS. Incubate the cells with ready-to-use DAPI for 10 minutes, then wash three more times with PBS. Observe the cellular fluorescence images using a laser confocal microscope. The DiR fluorescence excitation channel should be set at 640 nm and the DAPI fluorescence excitation channel at 405 nm. Use the Image J software for semi-quantitative analysis of cellular uptake across the different groups.

Live and dead cell staining

Set up groups without drug irradiation (Control + Neutron) and with drug irradiation (CB + Neutron, CB@lipo + Neutron, CLC@lipo-cRGD + Neutron) (n = 3). Add 5 mL of blank medium, CB, CB@lipo, or CLC@lipo-cRGD containing drug medium (boron concentration of 50 µg/mL) to each bottle. The CLC@lipo-cRGD + Neutron group is divided into normoxic and hypoxic groups, and placed in a cell culture incubator at 21% O₂ and 5% CO₂ for 4 hours before drug withdrawal. After washing three times with PBS, each group is irradiated with a neutron dose, with a total thermal neutron flux for the experiment of 1.1376×10¹² n/cm². After irradiation, the cells are seeded at a density of 10,000 cells per confocal dish. Add 1 mL of prepared Calcein AM/PI staining solution to resuspend the cells, and incubate in the dark for 15 minutes. After washing with PBS and resuspending in PBS, transfer 20 µL of the cell suspension to a 96-well plate. Use a confocal microscope to capture images of live and dead cells stained with Calcein AM/PI.

Detection of apoptosis levels by flow cytometry

Resuspend 1×10⁵ cells from the aforementioned groups in 100 µL of 1× Binding Buffer in a 5 mL flow cytometry tube. Add 5 µL of Annexin V-FITC solution and 10 µL of PI solution, and gently mix. Incubate the mixture in the dark at room temperature for 15 minutes. After incubation, add 200 µL of 1× Binding Buffer to each tube. Finally, perform fluorescence detection using a flow cytometer.

Lactic acid catalytic capacity assay and H₂O₂ generation capacity assay

Standard Curves Establishment

Resuspend 100,000 cells from the aforementioned groups in 100 µL of 1× Binding Buffer in a 5 mL flow cytometry tube. Add 5 µL of Annexin V-FITC solution and 10 µL of PI solution, and gently mix. Incubate the mixture in the dark at room temperature for 15 minutes. After incubation, add 200 µL of 1× Binding Buffer to each tube. Finally, perform fluorescence detection using a flow cytometer.

Prepare lactic acid standard solutions at concentrations of 0.125, 0.25, 0.5, 1, 2, and 3 mM. Transfer these solutions into a 96-well plate. After using a hydrogen peroxide (H₂O₂) test kit, measure the absorbance values of these lactic acid standard solutions using an M5 full-spectrum multifunctional microplate reader at a wavelength of 406 nm. Use these absorbance values to plot the standard curve for hydrogen peroxide.

Solution level

Place 1 mL of PBS, Free LOX, CB@lipo-cRGD, and CLC@lipo-cRGD solutions (each with a LOX concentration of 1.21 µg/mL) into dialysis bags with a molecular weight cutoff of 2000 Da. Transfer these into centrifuge tubes containing 5 mL of lactic acid solution at a concentration of 0.15 mg/mL. Incubate in a constant temperature shaker set at 37°C and 60 rpm. At 0, 15, 30, 45, and 60 minutes, remove 100 µL of the external solution from each dialysis bag and transfer to a 96-well plate. Measure the absorbance of the lactic acid standard solution using a lactic acid test kit in an M5 spectrophotometer (wavelength of 530 nm). Use the resulting values to calculate the concentration of lactic acid based on the standard curve.

Transfer 0.5 mL of lactic acid solutions at different concentrations (0, 5, 10, 20 mg/mL) into 2 mL centrifuge tubes. Add 0.5 mL of PBS, Free LOX, CB@lipo-cRGD, and CLC@lipo-cRGD solutions to each tube (each with a LOX concentration of 1.21 µg/mL) (n = 3). Place the tubes in a constant temperature shaker incubator set at 37°C and shake at 60 rpm for 20 minutes. Centrifuge at 15,000 rpm and 4°C for 15 minutes. Withdraw 100 µL of the supernatant from each tube and transfer it to a 96-well plate. Add the reagents from the H₂O₂ test kit. Measure the absorbance of the solutions at a wavelength of 406 nm using an M5 full-spectrum multifunctional microplate reader. Calculate the concentration of hydrogen peroxide (H₂O₂) using the standard curve derived from the absorbance values.

Cancer cell level

Add 0.5 mL of PBS, Free LOX, Free CAT, CB@lipo-cRGD, CB/LOX@lipo-cRGD, CB/CAT@lipo-cRGD, and CLC@lipo-cRGD solutions to each well, each containing 20,000 cells. The LOX concentration in each solution is 1.21 µg/mL, and the CAT concentration is 1.64 µg/mL. After incubating for 6 hours, withdraw 100 µL of cell culture medium from each well and transfer it to a 96-well plate. Using a lactic acid test kit, measure the absorbance of the lactic acid standard solution at a wavelength of 530 nm with an M5 full-spectrum multifunctional microplate reader. Use the resulting values to calculate the lactic acid concentration based on the standard curve.

The operation was the same as above, the culture solution of cells of each well (100 µL/well) was dropped into 96-well plate, and the absorbance value of lactic acid standard solution (wavelength 406 nm) was measured in M5 full-band multifunctional enzyme labeling instrument after adding H₂O₂ detection kit, and then substituted into the standard curve to calculate the H₂O₂ concentration.

Tumor sphere tissue level

Prepare 2% agarose gel and sterilize it at high temperature. Use a Pasteur pipette to add an appropriate amount of agarose gel into a U-shaped 96-well plate, and allow it to cool. When the tumor cell density reaches 80–90%, digest with trypsin and resuspend in DMEM high glucose medium containing 10% serum, then seed onto the agarose gel. Centrifuge at 1500 rpm for 15 minutes, then place on a shaker at 120 rpm for 1 hour. Change half of the medium the next day. When the tumor spheroids form spherical structures with a diameter of about 400 µm under the microscope, set up normoxic (21% O₂ incubation conditions) and hypoxic groups (1% O₂ incubation conditions) (n = 3). For the hypoxic group, cells are transferred to the hypoxia chamber 12 hours before drug treatment to simulate an anoxic environment. Add different drug-containing culture media to each well (with LOX concentration at 1.21 µg/mL and CAT concentration at 1.64 µg/mL), and incubate for 6 hours. Then, withdraw 100 µL of cell culture medium from each well and transfer it to a 96-well plate. Measure the absorbance of the lactic acid standard solution using a lactic acid test kit in an M5 full-spectrum multifunctional plate reader at a wavelength of 530 nm. Calculate the lactic acid concentration using the values obtained from the standard curve.

The culture solution (100 µL/well) of tumor spheres in each well was dropped into 96-well plate, and the absorbance value of lactic acid standard solution (wavelength 406 nm) was measured in M5 full-band multifunctional enzyme marker after adding H₂O₂ detection kit, and then substituted into the standard curve to calculate the concentration of H₂O₂.

CLC@lipo-cRGD Enhances the Suppression of HIF-1α Expression

Add 0.5 mL of blank culture medium, CB@lipo-cRGD, and CLC@lipo-cRGD drug-containing culture medium to each well, respectively, with LOX concentration at 2.42 µg/mL and CAT concentration at 3.28 µg/mL. Transfer the hypoxic group cells to a hypoxia chamber to simulate the tumor hypoxic microenvironment and promote the accumulation of lactic acid in tumor cells. After incubating for 4 hours, remove the drugs, wash with PBS, and then add buffer lysis solution to each well to fully lyse the cells. Finally, use Western Blot technology to quantitatively evaluate the expression levels of HIF-1α in the cells and perform semi-quantitative analysis using ImageJ software.

Inoculate GL261 cells at 80% confluency subcutaneously into the right flank of male C57BL/6 mice to establish an in vivo model of subcutaneous glioma-bearing animals. Select mice with appropriately sized and consistent tumors for further experiments. On days 1, 3, and 5, inject 400 µL different preparations into the subcutaneous tumor tissues. On day 7, harvest the tumor tissues from each group, wash off the surface impurities with PBS, and then dry the tissues. Fix the tumor tissues from each group in 4% paraformaldehyde, embed them in paraffin, prepare tumor sections, and perform immunofluorescence staining for HIF-1α to evaluate the efficacy of CLC@lipo-cRGD in suppressing HIF-1α expression in the tumors. Perform semi-quantitative analysis using ImageJ software.

Oxygen production efficiency of CLC@lipo-cRGD

To evaluate the oxygen production efficiency of CLC@lipo-cRGD, 0.5 mL of a 10 mM H₂O₂ solution and 10 µL of [Ru(dpp)₃]Cl₂ solution (10 mg/L) were added to each well of a 24-well plate. After 12 hours, 0.5 mL of PBS, Free CAT, CB/LOX@lipo-cRGD, CB/CAT@lipo-cRGD, and CLC@lipo-cRGD solutions were added to the wells, with LOX concentration at 1.21 µg/mL and CAT concentration at 1.64 µg/mL. The plate was then incubated at 37°C for 15 minutes. Fluorescence was recorded using a small animal fluorescence imaging system (IVIS, Ex/Em = 488/620 nm), and semi-quantitative analysis was performed.

In a 24-well plate, add 0.5 mL of a 10 mM H₂O₂ solution to each well, followed by 0.5 mL of PBS, CB@lipo-cRGD, CB/LOX@lipo-cRGD, CB/CAT@lipo-cRGD, and CLC@lipo-cRGD solutions (with LOX concentration at 1.21 µg/mL and CAT concentration at 1.64 µg/mL). Use a dissolved oxygen meter to quantitatively measure the oxygen concentration produced at specific time points. Add 0.5 mL of either blank culture medium, CB@lipo-cRGD, or CLC@lipo-cRGD containing medium (with LOX concentration at 2.42 µg/mL and CAT concentration at 3.28 µg/mL) to each well containing 5 × 10⁴ cells. Transfer some of the cells to a hypoxia chamber to simulate the tumor hypoxic environment and promote lactate accumulation in the tumor cells. Use the dissolved oxygen meter (Model JPB-607A) to quantitatively measure the oxygen concentration produced every 2 hours.

Mice and animal models

C57BL/6 mice (male, 5-week-old) purchased from Slaccas (Shanghai, China) were adaptive fed for more than one week for subsequent experiments. The animals were maintained under standard housing conditions and all related experiments were conducted followed by the guidelines which has been evaluated and approved by the Ethics Committee of Zhejiang University.

Orthotopic Glioma Mice Model: To establish the glioma model, 5 week-old male C57BL/6 mice (about 20 g) were anesthetized by intraperitoneal injection of 0.08 mL/10 g 1% pentobarbital solution. After the mice were fixed and the surgical area was exposed, 3×10⁵ cells/5 µL Luc-GL261 cells were trypsinized and resuspended in sterile PBS were slowly injected into the left corpus striatum (2 mm lateral, 1 mm posterior to the bregma, and 3.0 mm in depth) by a stereotactic fixation device using a mouse adaptor. Six days later, the orthotopic glioma mice model were imaged and evaluated by IVIS spectrum (PerkinElmer, USA) after 5 min postintraperitoneal injection of d-luciferin (150 mg∙kg^− 1, Gold Biotechnology, USA). The MRI (4.7 T/30 cm Bruker Biospec scanner, Germany) was observed too. The T/N ratios and BNCT experiments were executed in the orthopotic glioma mice model. The T/N ratios were the ratios of the boron concentration in the tumor to that in the surrounding normal tissue measured by ICP-MS.

Evaluation of in vivo targeting distribution

To investigate the enhancement of tumor targeting capability of nanocarriers by cRGD targeting peptides, fluorescently similar tumor-bearing mice were selected and randomly divided into two groups, each containing six mice. Each group was intravenously injected with 200 µL of either CLC-DiR@lipo or CLC-DiR@lipo-cRGD solution. The bioluminescence of Luc-GL261 and the distribution of DiR fluorescence in the brain were observed at 0, 4, 6, 8, 12, and 24 hours post-injection using a small animal in vivo imaging system (DiR has a near-infrared fluorescence, Ex/Em = 740/780 nm). At 12 hours post-injection, three mice from each group were euthanized by decapitation, and the brain, heart, liver, spleen, lung, and kidney tissues were harvested for further fluorescence imaging. Semi-quantitative analysis of the fluorescence intensity at the tumor site was performed using ImageJ software.

In Vivo Anti Tumor Effect of BNCT

Fluorescently similar tumor-bearing mice were selected and randomly divided into ten groups (n = 8): non-irradiation groups included Control (PBS group), CB group, CB@lipo group, CB@lipo-CRGD group, and CLC@lipo-CRGD group; BNCT irradiation groups included Control + Neutron group, CB + Neutron group, CB@lipo + Neutron group, CB@lipo-CRGD + Neutron group, and CLC@lipo-CRGD + Neutron group. On day 11 post-modeling, the aforementioned formulations were administered intravenously. For the BNCT groups, neutron irradiation was applied to the tumor site on day 12 (with only the head exposed to shield the rest of the body from radiation). The cumulative thermal neutron flux for the experiment was 5.15 × 10⁸ n/cm²/s, with an irradiation time of 20 minutes per group(53, 54). Post-irradiation, the mice were continued to be housed, and their body weight was measured every other day to plot curves of body weight and survival rate changes. Tumor size was monitored periodically using small animal in vivo imaging. Mice experiencing a weight loss greater than 20% of their total body weight were removed from the experimental groups and euthanized. On day 21 of treatment, whole blood was collected from the orbital vein to assess routine blood indices and biochemical markers of liver and kidney function. After euthanasia by decapitation, brain, heart, lung, liver, spleen, and kidney tissues were harvested from each group, washed with PBS, fixed in 4% paraformaldehyde, and processed for histological examination. Tissue sections were stained with H&E and Ki67 immunohistochemistry for the evaluation of organ toxicity and tumor tissue analysis.

Histopathological Evaluation

For histological analysis, the brain, heart, liver, spleen, lung, and kidney samples of the mice model in the different groups were fixed in 4% paraformaldehyde for 24 h and washed twice with PBS. Paraffin-embedded tissue sections were stained with H&E and observed on a microscope. The brain tumors were also detected by immunohistochemistry.

Statistics

Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, CA, USA). Data were presented as means ± S.D. Statistical evaluation of differences between experimental groups was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. Statistical significance was considered at least at p < 0.05.

Preparation and characterization of CLC@lipo-cRGD

We first characterized the prepared liposomes using Dynamic Light Scattering (DLS) to analyze the particle size and zeta potential. The results shown in Fig. 1A,B indicate that CLC@lipo-cRGD had an average particle size of 134.39 nm, with a polydispersity index (PDI) of 0.16, displaying a monodisperse distribution. The average surface zeta potential was − 6.57 mV. It had been reported that nanoparticles with a size less than 200 nm can more effectively accumulate in tumors through the Enhanced Permeability and Retention (EPR) effect. Additionally, nanoparticles with a negatively charged surface can avoid binding with negatively charged substances in the bloodstream, thereby extending their circulation time and favorably increasing drug accumulation in the target area(55). We next evaluated the stability of the CLC@lipo-cRGD nanoparticle carriers in aqueous solutions and serum. As shown in the Fig. 1C,D, the stability results indicated that the CLC@lipo-cRGD nanoparticles can maintain stability for up to 6 days in aqueous solutions, with only minor changes in particle size and dispersity. In simulated physiological conditions, the nanoparticle size and dispersity of CLC@lipo-cRGD remain stable for up to 2 days.

Further examination of CLC@lipo-cRGD was conducted using Transmission Electron Microscopy (TEM). As shown in Fig. 1E,F, CB@lipo displayed a regular lipid bilayer morphology with uniformly dispersed particle sizes, consistent with the results from the DLS particle size and zeta potential analyzer. Compared to CB@lipo, the size of CLC@lipo-cRGD after enzyme adsorption and peptide binding was not significantly different, although the morphology was less regular than that of CB@lipo, probably because of the irregular adsorption of enzymes. Nevertheless, it exhibited uniform dispersity, aligning with the aforementioned results. BCA and SDS-PAGE experiments were conducted to verify the successful binding of LOX, CAT, and cRGD to CLC@lipo-cRGD. As shown in Fig. S1 and Fig. 1G, with the addition of enzymes and the peptide cRGD, there was a gradual increase in the protein content of the nanoparticle carriers, indicating successful binding of the enzymes and cRGD peptide to the lipid carriers.

The enzyme activity assay of CLC@lipo-cRGD In Vitro

Due to the high demand for nutrients during the active proliferation of tumor cells, they rely heavily on non-oxidative phosphorylation and glycolysis, leading to the production of a large amount of lactate, which characterizes the TME with high levels of lactic acid. As shown in Fig. 2A, the LOX catalysis of lactic acid generates an unstable EFMNH₂-pyruvate complex, where the pyruvate readily dissociates, resulting in the decomposition of the reduced intermediate EFMNH₂ into H₂O₂. Therefore, we measured the standard curves for lactic acid and H₂O₂ (Fig. S2), then evaluated the catalysis of lactic acid and the generation of H₂O₂ by LOX-loaded nanoparticles from multiple perspectives, including in lactic acid solutions, tumor cells, and tumor spheroids.

As shown in Fig. 2B,D, CLC@lipo-cRGD demonstrated a lactic acid reduction effect consistent with that of Free LOX after 30 minutes. Additionally, due to the presence of CAT, which can break down the metabolic intermediate product H₂O₂ into H₂O and O₂, CLC@lipo-cRGD further reduces lactic acid concentration compared to Free LOX. The concentration of H₂O₂ in the CLC@lipo-cRGD group was significantly higher than that in the Control group and the formulation group without LOX. However, due to the catalytic action of CAT on H₂O₂ in CLC@lipo-cRGD, the concentration of H₂O₂ in its solution was lower than in the Free LOX group. At the tumor cell level, as Fig. 2C,E-I indicate, compared to the PBS group, the lactate concentration in the CLC@lipo-cRGD group significantly decreased (***p < 0.001), consistent with the effect of Free LOX. Consistent with the solution level results, when CAT and LOX are present together, the lactic acid concentration is further reduced compared to the Free LOX group, indicating that CAT's breakdown of intermediate products can also promote the decomposition of lactic acid, and this effect is more pronounced under hypoxic conditions, possibly due to hypoxic cells producing more lactic acid. Compared to the PBS control group, the concentration of H₂O₂ in the tumor cell culture medium of the CLC@lipo-cRGD group was significantly increased (***p < 0.001), and the concentration of H₂O₂ was higher in hypoxic cells than in normoxic cells. This was consistent with the lactic acid production results, as tumor cells under hypoxic conditions accumulated more lactic acid. In summary, the nanoparticle carrier loaded with LOX and CAT can efficiently catalyze lactic acid and generate H₂O₂, providing raw materials for the subsequent oxygen-producing process of CLC@lipo-cRGD, which is beneficial for improving the hypoxic state of tumors, promoting tumor cell uptake of nanoparticles, and alleviating the harm caused by the high lactic acid state in the microenvironment. Additionally, the production of H₂O₂ in normal tissues can cause serious side effects to damaged tissues and organs, thus reducing the accumulation of H₂O₂ through CAT can reduce the toxic effects on tissues surrounding the tumor in the body.

Oxygen generation by CLC@lipo-cRGD

As mentioned earlier, hypoxia is a typical characteristic of the TME, especially in gliomas, and is associated with cell invasion, drug resistance, and angiogenesis. Hypoxic cells are less sensitive to irradiation and more likely to develop radiation resistance, affecting the efficacy of radiotherapy. Therefore, improving the hypoxic TME can enhance the sensitivity of gliomas to radiotherapy, thereby enhancing the effectiveness of BNCT. CAT has excellent catalytic activity for converting H₂O₂ produced by LOX metabolism of lactic acid into oxygen through peroxidation reactions, further oxidizing various toxic substrates while catalyzing the accumulation of excess H₂O₂ into H₂O and O₂. Using [Ru(dpp)₃]Cl₂ as a dissolved oxygen probe to detect oxygen generation, Fig. 3E,F show that the fluorescence intensity of [Ru(dpp)₃]Cl₂ is reduced in the CAT group and the CAT-loaded nanoparticle carriers compared to the enzyme-free or LOX-only loaded nanoparticle carriers, indicating that the production of dissolved oxygen in the solution leads to the oxidative degradation of the molecular structure of [Ru(dpp)₃]Cl₂, confirming that CAT catalysis of H₂O₂ is a key component in oxygen production. Using a portable dissolved oxygen meter, the concentration of dissolved oxygen in the solution and in tumor cells under normoxic and hypoxic conditions was measured in real-time, as shown in Fig. 3G and Fig. S3. After adding CLC@lipo-cRGD to the H₂O₂ solution, the concentration of dissolved oxygen continuously increased, and the concentration of dissolved oxygen produced by hypoxic cells was higher, consistent with the previous results of lactic acid consumption and H₂O₂ production experiments. This indicates that CLC@lipo-cRGD has good catalytic oxygen-producing performance, capable of resolving the hypoxic conditions in the tumor area, and further enhancing the effectiveness of BNCT.

Reduction of HIF-1a expression by CLC@lipo-cRGD

Given the exceptional ability of CLC@lipo-cRGD to catalyze lactic acid, generate H₂O₂, and produce oxygen, aims to verify the effectiveness of the CLC@lipo-cRGD nanoparticle carrier in mitigating tumor hypoxia. This verification is achieved by measuring the expression levels of HIF-1α in tumor cells and subcutaneous tumor tissues. Initially, the expression levels of HIF-1α in GL261 cells, under both normoxic and hypoxic conditions, were examined after treatment with different formulations using Western Blot techniques. As shown in Fig. 3A,B, compared to the Control group and the CB@lipo-cRGD group, CLC@lipo-cRGD loaded with LOX and CAT demonstrated a more potent effect in suppressing the expression of HIF-1α in tumor cells, especially under hypoxic conditions, as indicated by narrower protein bands and lower semi-quantitative data. Subsequently, different formulations were injected intratumorally in a mouse subcutaneous glioma model, and the expression levels of HIF-1α in tumor tissues were examined through HIF-1α immunofluorescence staining. The results, as shown in Fig. 3C,D, indicate that CLC@lipo-cRGD, demonstrate that CLC@lipo-cRGD effectively utilizes the high lactic acid and low oxygen levels in the TME through the cascade catalytic effects of LOX and CAT. This process significantly reduces the expression of HIF-1α in tumor tissues (***p < 0.001).

Cellular Uptake and Distribution of Nanoparticles.

The safety results of CLC@lipo-cRGD, as determined by the CCK-8 assay (Fig. S4), indicate that CLC@lipo-cRGD functions as a low-toxicity drug carrier. After incubating it with GL261 cells for 48 h, the cell viability at various concentrations was close to 100%, indicating that it did not exert toxic effects on the cells. The binding of the cRGD peptide with the two enzymes still maintained the low toxicity of the drug carrier to cells. Therefore, the CLC@lipo-cRGD does not cause cytotoxic effects on the GL261 cells at boron concentrations below 100 µg/mL and can be further used for subsequent in vitro and in vivo experiments.

The effective uptake of drug carriers by tumors is a crucial prerequisite for the carriers to function. We first evaluated the uptake ability using a Confocal Laser Scanning Microscope. As shown in Fig. 4B,C, after 4 h’s incubation, CLC-DiR@lipo-cRGD was effectively taken up by GL261 cells. The success of BNCT relies on the specific accumulation of boron capture agents in tumor cells, and the ideal therapeutic effect requires that the boron content per gram of tumor tissue reaches 20 µg, equivalent to a concentration of 20 ng of boron element per 10⁶ cells. Therefore, using ICP-MS, we quantitatively analyzed the boron content in GL261 cells treated with CB, CB@lipo, CB@lipo-cRGD, and CLC@lipo-cRGD to verify if the drug carriers meet the BNCT requirements. Results shown in Fig. 4A indicate that under normoxic conditions, the boron uptake by GL261 cells treated with CLC@lipo-cRGD reached 1160.11 ng per 10⁶ cells, and under hypoxic conditions, it reached 953.10 ng per 10⁶ cells. Liposome encapsulation of CB increased cellular uptake compared to free CB. The ICP-MS results are consistent with the confocal images and semi-quantitative data, showing that modification with cRGD significantly increased the cellular uptake of liposomes, demonstrating the potent targeting ability of the cRGD peptide. For hypoxic cells, the addition of LOX and CAT enzymes improved the hypoxic condition of the tumor, thereby increasing cellular uptake. Hence, we found that modifying cRGD enhanced the cellular uptake of nanoparticles and achieved tumor targeting. And the modification with the two enzymes increased the uptake of nanoparticles by hypoxic cells, further enhancing the uptake of nanoparticles by tumor cells.

The antitumor effect of CLC@lipo-cRGD In Vitro

To investigate the cytotoxic effect of CLC@lipo-cRGD on GL261 cells in vitro after BNCT treatment, we utilized flow cytometry to assess the level of apoptosis in GL261 cells and employed Calcein AM/PI staining to evaluate the ratio of live and dead cells post-treatment, further verifying that CLC@lipo-cRGD can effectively kill tumor cells in vitro. Fig. S5 shows that compared to the only neutron irradiation group, the CLC@lipo-cRGD group displayed a significant increase in the number of dead cells marked by PI and a significant decrease in the number of live cells marked by Calcein AM. The number of live cells marked by Calcein AM also decreased in the hypoxic group compared to the normoxic group, which may be due to the poor growth conditions of GL261 cells under hypoxia. Figure 5D shows that after treatment with CLC@lipo-cRGD combined with BNCT, the level of apoptosis in tumor cells increased. The apoptosis rate of CB was 35.7%, while after modification with enzymes and cRGD, the apoptosis rate increased to 70.6% in the normoxic group and to 83.0% in the hypoxic group.

Biodistribution of CLC@lipo-cRGD In Vivo

The surface modification of tumor-targeting peptide cRGD has been one of the important strategies to enhance the tumor-targeting capability of nanoparticle formulations in recent years. In this study, on top of the passive targeting via the EPR effect, the active targeting function of cRGD is combined to achieve higher formulation delivery efficiency, aiming to realize enhanced in vivo anti-tumor effects. Therefore, we established an in situ brain glioma-bearing mouse model and used small animal in vivo fluorescence imaging technology to investigate the in vivo targeting distribution of CLC-DiR@lipo-cRGD, exploring the effects of cRGD peptide modification on enhancing tumor targeting capabilities. As shown in Fig. S6, compared to the CLC-DiR@lipo group, the intravenous injection of cRGD-modified CLC-DiR@lipo-cRGD began showing brain fluorescence signals at 6 hours, observed significant DiR fluorescence signals at the brain tumor site at 8 hours, and maintained high fluorescence signals at 12 h. Semi-quantitative fluorescence analysis using ImageJ showed significant differences at 12 h (***p < 0.001), demonstrating the powerful tumor-targeting ability of cRGD. Additionally, as shown in Fig. S7A,B, at 12 h, brain tissues (including tumors) and major organs were extracted for bioluminescence and fluorescence imaging. The results also verified that cRGD modification could promote drug carrier targeting to tumor, and the dual targeting function of the EPR effect and cRGD peptides helps to enhance the tumor delivery efficiency of the formulation, creating favorable conditions for improving in vivo anti-tumor effects subsequently.

The antitumor effect of CLC@lipo-cRGD In Vivo

This study establishes an in situ brain glioma-bearing mouse model to comprehensively evaluate the therapeutic effects of CLC@lipo-cRGD in inhibiting tumor growth in vivo. As shown in Fig. 5A, on the 9th day after model establishment, all mice were imaged for tumor fluorescence using small animal in vivo imaging. Mice with approximately the same amount of fluorescence were randomly divided into 10 groups (n = 8): the non-irradiation groups consisted of ① Control group (PBS group), ② CB group, ③ CB@lipo group, ④ CB@lipo-CRGD group, ⑤ CLC@lipo-cRGD group; the BNCT irradiation groups consisted of ① Control + Neutron group, ② CB + Neutron group, ③ CB@lipo + Neutron group, ④ CB@lipo-CRGD + Neutron group, ⑤ CLC@lipo-cRGD + Neutron group. On the 11th day post-modeling, different formulations were injected intravenously, and the BNCT groups received neutron radiation at the tumor site on the 12th day. After irradiation, mice were continuously bred, and their body weight was measured every other day, with curves plotted for changes in mouse body weight and survival rate. Tumor volume changes were observed and photographed using small animal in vivo imaging at regular intervals. Results of body weight and survival curves are shown in Fig. 5B,C. It is evident that the survival rate in the irradiation groups was significantly higher than that in the non-irradiation groups, with a noticeable slowdown in tumor suppression speed, and all mice in the non-irradiation groups died within 30 days, demonstrating that this in situ brain glioma model effectively simulates the rapid progression characteristic of clinical brain gliomas. Treatment with boron-containing drugs combined with BNCT could inhibit tumor progression and enhance mouse survival rates, The survival rate in the experimental group was 70% and the median survival rate in the experimental group compared to the control group was 100%.

As seen in Fig. S5D, compared to the irradiation groups ①, ②, and ③ without cRGD modification, the survival rate significantly increased in the irradiation groups ④ and ⑤ with cRGD modification, and tumor size was notably reduced. This is due to the dual targeting action of EPR and cRGD peptides on the drug carriers, effectively increasing the concentration of drug carriers at the tumor site, thereby enhancing the therapeutic effect of BNCT irradiation. The irradiation group ⑤, loaded with LOX and CAT, showed improved mouse survival rate and tumor volume suppression compared to group ④, proving that by utilizing the high lactic acid levels characteristic of the TME, CLC@lipo-cRGD can reduce the concentration of lactic acid in tumor tissues and increase oxygen production through a cascade reaction. This improves the hypoxic state of the tumor, thereby increasing tumor cell uptake, reducing tumor tissue resistance to radiation, eliminating the adverse regulatory effects of lactic acid and H₂O₂ in the TME, promoting anti-tumor action in vivo, and further inhibiting tumor progression.

Subsequently, we further evaluated the changes in tumor growth through the results of Ki67 immunohistochemistry in tumor tissues. As shown in Fig. S8, on top of the CB@lipo-cRGD + Neutron group, the CLC@lipo-cRGD + Neutron group exhibited a stronger effect in inhibiting cell proliferation and promoting cell apoptosis, as indicated by a further reduction in Ki67 expression levels. Overall, the combined treatment of CLC@lipo-cRGD with BNCT achieved more effective anti-tumor effects in vivo.

Both SOX2 and CD133 are important indicators of tumor stemness (56–58), and it has been shown in many studies that the stemness of gliomas is inextricably linked to the upstream and downstream regulatory roles of SOX2 and CD133(59–61). Therefore, we evaluated the changes in tumor stemness after BNCT treatment using SOX2 and CD133 immunofluorescence staining. The results are shown in Fig. S9, tumors in the CB@lipo-cRGD + Neutron group and the CLC@lipo-cRGD + Neutron group showed reduced CD133 expression after treatment, which could indicate that multifunctional liposomes may be able to reduce tumor cell stemness in certain pathways, thus helping to improve prognosis.

To investigate the in vivo safety of the formulations, on day 32 or when the mice met the criteria for euthanasia, the brain, heart, liver, spleen, lungs, and kidneys of the mice from each group were extracted and weighed. The results, as shown in Fig. S10, indicate that there were no significant changes in the mass of any organs. Additionally, an H&E staining evaluation of the organs was conducted, and Fig. S11 shows that no significant organ damage was observed. Furthermore, this chapter also includes a complete blood count and biochemical analysis of the mice after treatment. Fig. S12A shows that all blood count indicators were normal, and Fig. S12B indicates that liver function markers (AST, ALT, ALP) and kidney function markers (BUN, CREA) showed no significant abnormalities after treatment, demonstrating good in vivo safety.

To address the high lactate and hypoxia characteristics of the TME and increase the uptake of boron-containing drugs by tumor cells, we designed and constructed a multifunctional nanoliposome drug delivery system, CLC@lipo-cRGD, co-loaded with LOX and CAT enzymes and modified with cRGD peptide. We explored the effects of utilizing the TME combined with BNCT treatment in an in situ glioma mouse model. With the passive targeting capability of the EPR effect and the active targeting of the cRGD peptide, CLC@lipo-cRGD achieves high efficiency in tumor delivery. Leveraging the high lactate levels in the TME, LOX catalyzes the conversion of lactate to increase H₂O₂ concentration. Subsequently, CAT consumes the generated H₂O₂ to produce O₂, enhancing the catalytic efficiency of LOX and improving the hypoxic conditions. Through this cascade reaction, further uptake of the nanoparticles by tumor cells is facilitated. By suppressing the expression of HIF-1α protein in tumors, combined with the reduction of lactate and hypoxia characteristics, radiation resistance due to hypoxia is lowered, resulting in safer and more effective anti-tumor effects both in vitro and in vivo. The results showed that we successfully prepared multifunctional boron-containing liposomes, and the modification of cRGD peptide enhanced the uptake of drug carriers by tumor cells to 1160.11 ng/10⁶ cells, which far exceeded the standard of BNCT. The loading of LOX and CAT enabled the nanoparticles to be well taken up by tumor cells even in low oxygen conditions. The cascade reaction reduced the lactate concentration and generated oxygen at three different levels: solution, tumor cells, and tissues, which further reduced the protein expression level of HIF-1α. BNCT co-treatment enhanced the apoptosis of GL261 cells, which showed stronger anti-tumor effects. The in vivo results demonstrated that CLC@lipo-cRGD combined with BNCT treatment significantly increased the survival rate of hormonal mice, with a median survival rate of 100% in the experimental group, and reduced tumor stemness. Thus, this multifunctional nanoliposome would be a promising and safe nanodrug delivery system in BNCT.

Ethics approval and consent to participate

The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Ethics Committee of Zhejiang University. Written informed consent was obtained from individual or guardian participants.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 82373206), National Key Research and Development Program of China (No. 2022YFE0107800, No. 2023YFA1008600) and Zhejiang Provincial Natural Science Foundation of China under Grant No. LHDMZ24H300002. We thank Haixi Wang, Lu Zhang and Xingcai Guan at School of Nuclear Science and Technology, Lanzhou University for providing assistance for BNCT irradiation of animals. We thank Mengjie Yu at Bio-Ultrastructure Analysis Lab. of Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University for her technical assistance on transmission electron microscopy; Jianyang Pan (Research and Service Center,College of Pharmaceutical Sciences, Zhejiang University) for performing NMR spectrometry for structure elucidation; Yingying Huang at Zhejiang University School of Medicine for her technical assistance on flow cytometry; Qin Han, Chenyu Yang and Dandan Song at the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University for their technical assistance on confocal laser scanning microscopy and transmission electron microscopy; Junli Xuan and Yanwei Li at the Core Facilities of Zhejiang University School of Medicine for technical assistance of confocal laser scanning microscopy and flow cytometry analysis.

Author Contributions

Min Han and Qichun Wei conceived the project and designed the experiments. Min Han and Qichun Wei supervised the project, discussed and commented on the manuscript. Qi Dai and Qiyao Yang performed the majority of the experiments and data analysis. Zhicheng Zhang, Qin Yaxin and Xiaoyan Sunassisted with cell culture of GL261 and the establishment of animal models. Xiaoyan Bao, Linjie Wu, Ruolin Jiang, Zhiqing Ben, Xufang Ying and Xin Tan helped with the animal experiments. Benhua Xu, Hefa Huang, Rui Quan and Ruirong helped provide BNCT equipment and assisted in conducting animal irradiation experiments. Zhuang Qiyao Yang, Xiaoyan Bao, Zhicheng Zhang, Qin Yaxin and Xiaoyan Sun proofread the manuscript. Qi Dai wrote the manuscript.

Competing Interests statement

All the authors declare no conflicting interests.

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No competing interests reported.

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Graphical Abstract Schematic diagram of the construction of CLC@lipo-cRGD nanocarriers and its synergistic anti-tumor mechanism in combination with BNCT and TME therapy.
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Catalytic modulation of the tumor microenvironment using cascade enzymes for synergistic boron neutron capture therapy in the treatment of gliomas

Status:

Version 1

Abstract

Figures

Introduction

Methods and Materials

Materials and Reagents

Synthesis of CLC@lipo-cRGD

Characterization of multifunctional nanoliposomes

Cell Lines

Cell Viability Assay

Cellular Uptake of Nanoparticles in Cell

Uptake by cancer cells observed by Confocal Microscopy

Live and dead cell staining

Detection of apoptosis levels by flow cytometry

Lactic acid catalytic capacity assay and H₂O₂ generation capacity assay

Standard Curves Establishment

Solution level

Cancer cell level

Tumor sphere tissue level

CLC@lipo-cRGD Enhances the Suppression of HIF-1α Expression

Oxygen production efficiency of CLC@lipo-cRGD

Mice and animal models

Histopathological Evaluation

Statistics

Result and Discussion

Preparation and characterization of CLC@lipo-cRGD

Oxygen generation by CLC@lipo-cRGD

Reduction of HIF-1a expression by CLC@lipo-cRGD

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Catalytic modulation of the tumor microenvironment using cascade enzymes for synergistic boron neutron capture therapy in the treatment of gliomas

Status:

Version 1

Abstract

Figures

Introduction

Methods and Materials

Materials and Reagents

Synthesis of CLC@lipo-cRGD

Characterization of multifunctional nanoliposomes

Cell Lines

Cell Viability Assay

Cellular Uptake of Nanoparticles in Cell

Uptake by cancer cells observed by Confocal Microscopy

Live and dead cell staining

Detection of apoptosis levels by flow cytometry

Lactic acid catalytic capacity assay and H2O2 generation capacity assay

Standard Curves Establishment

Solution level

Cancer cell level

Tumor sphere tissue level

CLC@lipo-cRGD Enhances the Suppression of HIF-1α Expression

Oxygen production efficiency of CLC@lipo-cRGD

Mice and animal models

Histopathological Evaluation

Statistics

Result and Discussion

Preparation and characterization of CLC@lipo-cRGD

Oxygen generation by CLC@lipo-cRGD

Reduction of HIF-1a expression by CLC@lipo-cRGD

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Lactic acid catalytic capacity assay and H₂O₂ generation capacity assay