Lactic acid metabolism regulation nano preparation, preparation method and application

Abstract

The invention relates to the field of biological medicine, in particular to a lactic acid metabolism regulation nano preparation, a preparation method and application thereof.The lactic acid metabolism regulation nano preparation contains an oxygen-enriched phase change agent, the oxygen-enriched phase change agent serves as a core, the outer side of the oxygen-enriched phase change agent is wrapped with lipidosome, the lipidosome is cRGD peptide modified lipidosome, the lipidosome is loaded with a GLUT-1 inhibitor, the GLUT-1 inhibitor is BAY-876, and the oxygen-enriched phase change agent serves as the core. The oxygen-enriched phase change agent is perfluorohexane. After the HIFU operation, the nano preparation can regulate lactic acid and reduce the generation of lactic acid, so that the problems that cells in a sublethal heat stress area undergo metabolic disorder and a large amount of lactic acid is generated after the HIFU operation are solved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to lactic acid metabolism regulating nano-formulations, their preparation methods, and their applications. Background Technology

[0002] Hepatocellular carcinoma (HCC) is one of the most common solid malignant tumors worldwide and the third leading cause of cancer-related deaths. Patients are often diagnosed at an advanced stage due to the lack of early specific symptoms, which severely limits the suitability and success rate of surgical resection. Therefore, non-surgical local treatment has become the main clinical option.

[0003] High-intensity focused ultrasound (HIFU) has been proven to safely and effectively ablate liver cancer locally due to its non-invasiveness, precise positioning, and real-time monitoring. However, with increasing tissue penetration depth and blood perfusion rate, ultrasound energy decays exponentially within the tissue, leading to insufficient energy deposition in the target area and inadequate thermal damage in some tissues, forming a sublethal heat stress zone. While cells in this zone do not undergo coagulative necrosis, they experience metabolic disturbances, primarily manifested as excessive lactate (LA) production. High concentrations of lactate accumulating in the tumor microenvironment (TME) can weaken effector T cell proliferation and cytotoxicity, and by promoting macrophage polarization towards the pro-tumor M2 phenotype, create an immunosuppressive microenvironment, thereby promoting malignant proliferation and increasing the risk of recurrence.

[0004] Existing technologies for intervening in tumor lactate metabolism have significant shortcomings: First, early studies mostly focused on "passively clearing existing lactate," but such strategies cannot block tumor cells from continuously synthesizing lactate through the Warburg effect, making it difficult to inhibit continuous tumor growth. Second, some studies have attempted to actively intervene in the source of lactate production, such as X. Meng et al. using hexokinase 2 (HK2) inhibitors to block the first step of glycolysis, but simply inhibiting HK2 leads to intracellular glucose accumulation, triggering other metabolic compensation pathways, and cannot achieve complete energy deprivation of tumor cells. Third, other studies have used glucose oxidase (Gox) to oxidize glucose to reduce intracellular glucose levels, but the activity of Gox is highly dependent on environmental factors such as pH and temperature, and the hypoxic microenvironment inside the tumor significantly weakens its efficacy, limiting clinical translation.

[0005] Therefore, how to regulate lactic acid and reduce its production after HIFU treatment remains an urgent problem to be solved. Summary of the Invention

[0006] The present invention aims to provide a lactate metabolism regulating nano-formulation, preparation method and application, so as to regulate lactate after HIFU, reduce lactate production, and solve the problem of metabolic disorder and excessive lactate production in sublethal heat stress area cells after HIFU.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: a lactic acid metabolism regulating nano-formulation containing an oxygen-enriched phase change agent.

[0008] The principle and effects of this solution are as follows: The nano-formulation of this invention contains an oxygen-enriched phase change agent. Under HIFU focusing, the oxygen-enriched phase change agent undergoes a phase transition to generate microbubbles, enhancing the local acoustic field. This reduces the required output power while maintaining the same ablation volume, significantly alleviating sublethal heat stress and inhibiting heat-induced lactate production. Simultaneously, the oxygen released during the phase transition of the oxygen-enriched phase change agent significantly improves tumor oxygenation levels, leading to downregulation of HIF-1α expression within the tumor and a decrease in the transcriptional level of glucose transporter 1 (GLUT1). This also helps reduce lactate production, achieving the goal of blocking lactate production at its source and preventing tumor hypoxia from causing an increase in HIF-1α and GLUT1 levels, leading to increased glucose uptake and amplified Warburg effect.

[0009] This approach is the first to combine the intervention target with the unique pathological state after HIFU: on the one hand, by triggering the phase transition of the oxygen-enriched phase change agent through ultrasound to reduce the output power required for HIFU, the formation of the sublethal heat stress region is directly reduced, blocking heat-induced lactate production from the upstream, which is a key link that has not been addressed in existing lactate metabolism intervention studies; on the other hand, by releasing oxygen through the oxygen-enriched phase change agent to improve tumor hypoxia, the upregulation of glucose transporter 1 (GLUT1) mediated by hypoxia-inducible factor-1α (HIF-1α) is inhibited, suppressing the Warburg effect from the "hypoxia-driven" level, thus achieving a source-based and comprehensive blockade of lactate production.

[0010] Preferably, as an improvement, the oxygen-enriched phase change agent is used as the core, and the oxygen-enriched phase change agent is wrapped with liposomes on the outside.

[0011] Preferably, as an improvement, the liposomes are cRGD peptide-modified liposomes.

[0012] Therefore, cRGD-mediated active targeting significantly increases the cumulative concentration of the agent at the tumor site, thereby improving the therapeutic effect.

[0013] Preferably, as an improvement, the liposomes are loaded with a GLUT-1 inhibitor.

[0014] Therefore, this approach not only improves tumor hypoxia by releasing oxygen through oxygen-enriched phase change agents and inhibits the upregulation of glucose transporter 1 (GLUT1) mediated by hypoxia-inducible factor-1α (HIF-1α), but also directly blocks glucose uptake by combining GLUT1-specific inhibitors. This dual inhibition of the Warburg effect from both the "hypoxia-driven" and "transport channel" levels avoids the metabolic compensation problem that is easily caused by single enzyme inhibitors, thus greatly achieving a source-based and comprehensive blockade of lactate production.

[0015] Preferably, as an improvement, the oxygen-enriched phase change agent is perfluorohexane.

[0016] Preferably, as an improvement, the GLUT-1 inhibitor is BAY-876.

[0017] To achieve the above objectives, the present invention adopts the following technical solution: the application of oxygen-enriched phase change agents in the preparation of drugs that reduce lactic acid production by alleviating sublethal heat stress under HIFU focusing.

[0018] Oxygen-enriched phase change agents generate microbubbles through phase transition under ultrasound triggering, enhancing the local acoustic field and reducing the required HIFU output power, thereby alleviating sublethal heat stress and inhibiting heat-induced lactate production. Simultaneously, the oxygen released by the oxygen-enriched phase change agent increases the tumor oxygen partial pressure, inhibits HIF-1α, and downregulates GLUT1, weakening tumor cell glucose uptake and thus reducing lactate production. Therefore, oxygen-enriched phase change agents have the effect of reducing lactate production under HIFU focusing. However, the use of oxygen-enriched phase change agents to reduce lactate production under HIFU focusing has not been reported in existing technologies. Therefore, this patented scheme applies the application of oxygen-enriched phase change agents to the preparation of drugs that reduce lactate production under HIFU focusing by alleviating sublethal heat stress.

[0019] Preferably, as an improvement, the oxygen-enriched phase change agent is perfluorohexane.

[0020] To achieve the above objectives, the present invention adopts the following technical solution: the application of lactate metabolism regulating nano-formulation drugs prepared under HIFU focusing to reduce lactate production by alleviating sublethal heat stress.

[0021] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing lactic acid metabolism regulating nano-formulations, comprising the following steps: S1, DPPC, cRGD-DSPE-PEG 2000 Cholesterol was dissolved in chloroform and added to BAY-876 and mixed well. S2. Remove organic solvents using a rotary evaporator, then add PBS for hydration and peel off the phospholipid membrane with the assistance of an ultrasonic cleaner; S3. Add perfluorohexane and use a sonic vibrator to obtain cRGD-Lip@PFH-BAY liposomes; S4. Oxygen-enriched cRGD-Lip@PFH-BAY can be obtained by oxygenating the obtained liposomes in an oxygen-enriched environment.

[0022] Preferably, as an improvement, DPPC, cRGD-DSPE-PEG 2000 It is soluble in chloroform at a mass ratio of 3:1:1 with cholesterol.

[0023] In summary, the lactate metabolism regulating nanoformulation cRGD-Lip@PFH-BAY in this application utilizes cRGD-mediated active targeting to increase the cumulative concentration of the formulation at the tumor site. PFH, under HIFU focusing, undergoes a phase transition to generate microbubbles, enhancing the local acoustic field and reducing the required output power while maintaining the same ablation volume, significantly alleviating sublethal heat stress and inhibiting heat-induced lactate production. The oxygen released during the phase transition significantly improves tumor oxygenation, leading to downregulation of HIF-1α expression within the tumor and a subsequent decrease in GLUT1 transcription levels. The release of BAY-876 further directly inhibits residual GLUT1. Through these triple mechanisms of action, intratumoral lactate levels are significantly reduced, directly promoting the maturation of dendritic cells (DCs) in lymph nodes, downregulating intratumoral immunosuppressive cell populations (including M2 tumor-associated macrophages and regulatory T cells), while simultaneously increasing M1 tumor-associated macrophages, NK cells, and CD8+. + The proportion of T cells effectively counteracts lactate-induced immunosuppression. Therefore, cRGD-Lip@PFH-BAY achieves lactate metabolism reprogramming through multiple mechanisms to reverse the immunosuppressive tumor microenvironment induced after HIFU, significantly improving the efficacy of liver cancer ablation and providing a feasible metabolic regulation strategy for the clinical translation of HIFU precision therapy. Attached Figure Description

[0024] Figure 1 Figures are attached to illustrate the preparation, characterization, and basic properties of cRGD-Lip@PFH-BAY. Among them, (A) schematic diagram of the synthesis of cRGD-Lip@PFH-BAY; (BC) transmission electron microscopy image of cRGD-Lip@PFH-BAY (scale bar = 1µm, 200nm); (DE) average particle size and zeta potential of Lip@PFH-BAY and cRGD-Lip@PFH-BAY; (F) UV-Vis absorption spectra of cRGD-Lip@PFH-BAY at different concentrations; (G) verification of successful loading of BAY-876 onto cRGD-Lip@PFH-BAY using UV-Vis absorption spectra of BAY-876, cRGD-Lip@PFH, and cRGD-Lip@PFH-BAY; (H) in vitro drug release curve of cRGD-Lip@PFH-BAY after HIFU irradiation; (I) cRGD-Lip@BAY and cRGD-Lip@PFH-BAY. (J) In vitro oxygen release curves of cRGD-Lip@PFH-BAY+HIFU; (K) Stability of Lip@PFH-BAY and cRGD-Lip@PFH-BAY; (J) Optical microscopic images of cRGD-Lip@PFH-BAY at different temperatures (56℃, 60℃, 70℃ and 80℃) (scale bar = 100µm).

[0025] Figure 2 The accompanying figures illustrate the study on the relief of sublethal heat stress from HIFU using perfluorohexane. (A) In vitro ultrasound images of PBS, cRGD-Lip@BAY, cRGD-Lip@PFH, and cRGD-Lip@PFH-BAY after HIFU irradiation; (B) Quantitative analysis results under contrast enhancement mode (n = 3); (C) In vivo ultrasound imaging of tumor-bearing mice after HIFU irradiation; (D) Corresponding quantitative analysis results under contrast enhancement mode (n = 3); (E) Visual images of the coagulative necrosis area in bovine liver after HIFU irradiation with different power intensities; (F) Comparison of ultrasound imaging and representative H&E staining images of Hepa1-6 tumors before and after HIFU ablation (scale bar = 100 μm); grayscale changes (G), volume quantification (H), and corresponding ultrasound energy efficiency factors (I) of ablated tumor tissues in each group (n = 3).

[0026] Figure 3 The accompanying figures illustrate the targeting and cytotoxicity studies of cRGD-Lip@PFH-BAY. Among them, (A) confocal laser scanning microscopy (CLSM) images showing the cellular uptake and tumor targeting characteristics of Lip@PFH-BAY and cRGD-Lip@PFH-BAY in Hepa1-6 cells (scale bar = 100 µm); (B) flow cytometry quantitative analysis of the targeting of Lip@PFH-BAY and cRGD-Lip@PFH-BAY, expressed as mean fluorescence intensity (n=3); (C) in vivo fluorescence imaging at different time points after intravenous injection of Lip@PFH-BAY or cRGD-Lip@PFH-BAY in Hepa1-6 tumor-bearing mice; (D) ex vivo fluorescence images of major organs (heart, liver, spleen, lung, and kidney) obtained from Hepa1-6 tumor-bearing mice 24 hours after intravenous injection of Lip@PFH-BAY or cRGD-Lip@PFH-BAY; (E) Hepa1-6 cells stained with calcein AM (green fluorescent labeling of live cells) and propidium iodide (red fluorescent labeling of dead cells) CLSM, showing cell states after different treatments (scale bar = 100 µm); (F) Flow cytometry analysis of apoptosis in Hepa1-6 cells in different experimental groups; (G) Flow cytometry quantification of apoptosis in Hepa1-6 cells under different treatment conditions (n=3).

[0027] Figure 4The accompanying figures illustrate the study on the reduction of lactate production by blocking GLUT1 in cRGD-Lip@PFH-BAY. (A) CLSM images of GLUT1 expression in Hepa1-6 cells after different treatments (scale bar = 60 µm); (B) Western blot analysis of GLUT1 expression on the cell surface of Hepa1-6 cells after different treatments; (CE) Quantitative analysis of intracellular lactate, extracellular glucose, and intracellular ATP in Hepa1-6 cells after 12 or 24 hours of different intervention methods (n=3).

[0028] Figure 5 The accompanying figures illustrate the study on how cRGD-Lip@PFH-BAY alleviates hypoxia and downregulates GLUT1. (A) Schematic diagram of the mechanism by which hypoxia is improved and lactate production is reduced; (B) CLSM images of Hepa1-6 cells after different treatments using the RDDP probe (scale bar = 100 µm); (C) Intracellular lactate levels in Hepa1-6 cells after different treatments (n=3); (D) Representative photoacoustic images of mouse tumor regions in oxyhemoglobin mode (750 nm / 850 nm) after different treatments; (E) Representative immunofluorescence and immunohistochemical images of HIF-1α, hypoxia probe, and GLUT1 expression in tumors of different treatment groups (scale bar = 100 µm); (F) Intracellular lactate levels in tumor tissues after different treatments (n=3).

[0029] Figure 6 The accompanying figures illustrate the research on cRGD-Lip@PFH-BAY-mediated lactate regulation and its effect on the immune microenvironment. (A) is a schematic diagram of the Transwell co-culture system; (BC) are representative flow cytometry images and CD80 levels of dendritic cell (DC) maturation induced after different treatments of Hepa1-6 cells. + CD86 +(n=3) Quantitative analysis of positive cells; (DE) Quantitative analysis of IL-6 and TNF-α levels in cell supernatant after treatment (n=3); (FI) Flow cytometry analysis showing the ratio of M2 (CD206) and M1 (CD86) tumor-associated macrophages in RAW264.7 cells induced by Hepa1-6 cells after different treatments (n=3); (J) Treatment regimens, timeline of treatment intervention and administration method analyzed by in vivo flow cytometry; (KN) Representative flow cytometry images and quantitative analysis of M2 tumor-associated macrophages (M2-TAMs), M1 tumor-associated macrophages (M1-TAMs), regulatory T cells (Tregs) and NK cells in tumor tissues of different treatment groups (n=3).

[0030] Figure 7 The accompanying figures illustrate the in vivo antitumor therapeutic effects of cRGD-Lip@PFH-BAY combined with HIFU. (A) Schematic diagram of the in vivo treatment regimen in Hepa1-6 tumor-bearing mice; (B) Tumor weight data of mice at the end of the treatment regimen (n=5); (C) Representative images of tumor volume changes in tumor-bearing mice throughout the treatment course; (D) Average tumor volume growth curves of mice in each treatment group (n=5); (E) Individual tumor volume change curves of mice in each treatment group (n=5); (F) Representative images of H&E, PCNA, and TUNEL staining of tumor tissue in each treatment group (scale bar = 100µm). Detailed Implementation

[0031] The following detailed description illustrates the specific implementation method: This embodiment discloses a lactate metabolism regulating nanoformulation containing an oxygen-enriched phase change agent, which is externally encapsulated in liposomes. Specifically, the liposomes are modified with cRGD peptides (cRGD peptides are short peptide molecules formed by cyclization of arginine (R), glycine (G), and aspartic acid (D) amino acids). In other embodiments, the cRGD peptide can be replaced with a membrane-penetrating peptide or other targeting peptide, such as tLyP-1. The liposomes are loaded with a GLUT-1 inhibitor, specifically BAY-876, but not limited to this; other commercially available drugs can also be used. In this embodiment, the oxygen-enriched phase change agent is further perfluorohexane (PFH), but other perfluorocarbons such as PFP can also be used.

[0032] This embodiment discloses a method for preparing a lactate metabolism regulating nanoformulation (cRGD-Lip@PFH-BAY) (refer to...). Figure 1 As shown in A), the following steps are included: S1, DPPC, cRGD-DSPE-PEG 2000Cholesterol was dissolved in 10 mL of chloroform at a mass ratio of 3:1:1 (specifically 6, 2, and 2 mg), and 1 mg of BAY-876 was added and mixed well. S2. The organic solvent was removed using a rotary evaporator (RE-52, Shanghai, 40℃, 75 rpm, 1h). Then, 4 mL of PBS was added for hydration and the phospholipid membrane was peeled off with the assistance of an ultrasonic cleaner (80W, 3 minutes). S3. Add 200µL of perfluorohexane and perform acoustic vibration using an acoustic vibrator (60W, 3min; ON 5s, OFF 5s; the entire acoustic vibration process must be conducted in an ice bath, and the vibrator probe must be below the liquid surface and not touch the tube wall) to obtain cRGD-Lip@PFH-BAY liposomes. S4. Oxygenate the obtained liposomes in an oxygen-enriched environment (take a 20mL syringe, fill it with oxygen, connect it to a two-way valve, and close the valve. Connect one end of the two-way valve to a container containing cRGD-Lip@PFH-BAY, open the valve, and let it stand at low temperature for 2-3 hours) to obtain oxygen-enriched cRGD-Lip@PFH-BAY.

[0033] In subsequent experiments, the only difference between the prepared cRGD-Lip@BAY (PFH-free) and cRGD-Lip@PFH (BAY-876-free) and the above-mentioned nano-formulations was the separate removal of PFH and BAY-876. The only difference between the prepared Lip@PFH-BAY and the above-mentioned nano-formulations was the cRGD-DSPE-PEG preparation method. 2000 Replace with DSPE-PEG 2000 .

[0034] In this embodiment, the lactate metabolism regulating nanoformation can be applied to the preparation of drugs that reduce lactate production by mitigating sublethal heat stress under HIFU focusing.

[0035] Oxygen-rich phase change agents, such as perfluorohexane (PFH), can be used to prepare drugs that reduce lactate production by mitigating sublethal heat stress under HIFU focusing.

[0036] The following series of experiments demonstrate the properties and effects of lactate metabolism-regulating nano-formulations.

[0037] I. Characterization Experiment Combination Figure 1 B- Figure 1 As shown in Figure C, optical microscopy and transmission electron microscopy revealed that the lactic acid metabolism regulating nanoparticles prepared in this embodiment exhibited good dispersibility and a uniform spherical morphology. Combined with... Figure 1 D- Figure 1As shown in Figure E, dynamic light scattering analysis revealed an average particle size of 264.88 ± 3.89 nm, a PDI of 0.16, and a surface potential of –39.47 ± 2.51 mV. During storage at 4 °C for 7 days, the binding... Figure 1 As shown in Figure J, continuous DLS monitoring revealed that the average particle size of cRGD-Lip@PFH-BAY remained essentially constant, with size fluctuations of less than 10% (approximately 264.88 ± 3.89 nm to 274.34 ± 5.43 nm), indicating good physical stability of its particle size during low-temperature storage. Figure 1 F- Figure 1 As shown in G, the UV-Vis spectrum of BAY-876 was subsequently measured using a UV-Vis spectrophotometer. The maximum absorption peak of BAY-876 appeared at approximately 280 nm, and the spectrum of cRGD-Lip@PFH-BAY also showed a distinct characteristic peak at this wavelength. Furthermore, the absorbance increased linearly with sample concentration, confirming the successful loading of BAY-876. Based on the standard curve of BAY-876, the encapsulation efficiency in cRGD-Lip@PFH-BAY, measured by UV spectrophotometry, was 75.38%, and the drug loading rate was 6.9%. Figure 1 As shown in Figure H, after HIFU irradiation (105 W, 100% duty cycle, 3 s), BAY-876 loaded on cRGD-Lip@PFH-BAY was released in large quantities, with an average cumulative release rate of 60.70 ± 2.50% in the first 4 hours. Conversely, the release rate of cRGD-Lip@PFH-BAY without HIFU treatment was slower. By 24 hours, the release rates of both groups were close to their respective peak values, but the cumulative release rate of the irradiated group was as high as 83.44 ± 2.88%, while that of the unirradiated group was only 20.04 ± 1.34%. Therefore, HIFU treatment is beneficial for the large-scale release of BAY-876.

[0038] II. Basic Attribute Research of cRGD-Lip@PFH-BAY Perfluorohexane (PFH) is a perfluorocarbon compound with a boiling point of approximately 56°C, which is chemically inert at room temperature, easy to store, and capable of dissolving large amounts of oxygen, making it an ideal carrier for alleviating tissue hypoxia. The inventors observed this under an optical microscope, combined with... Figure 1 As shown in K, cRGD-Lip@PFH-BAY remains liquid when heated to 55℃; a small number of microbubbles begin to appear when the temperature rises to 60℃, a large number of microbubbles are generated at 70℃, and microbubbles expand and rupture at 80℃. This series of temperature-dependent phase transition behaviors indirectly proves that PFH has been successfully encapsulated in lipid nanoparticles.

[0039] Based on the high oxygen solubility of PFH, we further used an oxygen electrode to measure the oxygen release under HIFU stimulation (105 W, 100% duty cycle, 3 s). The results showed that, combined with Figure 1 As shown in Figure I, cRGD-Lip@PFH-BAY significantly increased dissolved oxygen concentration during ultrasound irradiation, while cRGD-Lip@BAY lacking PFH released almost no oxygen, indicating that PFH acts as a highly efficient oxygen donor within the liposome. These properties enable this nanoformulation to replenish oxygen, thereby alleviating the side effects of tumor hypoxia.

[0040] III. Study on the reduction of lactic acid by perfluorohexane in alleviating sublethal heat stress of HIFU After HIFU stimulation (105 W, 100% duty cycle, 3 s), cRGD-Lip@PFH-BAY significantly enhanced the contrast-enhanced ultrasound imaging effect. Figure 2 A- Figure 2 As shown in Figure B, cRGD-Lip@PFH and cRGD-Lip@PFH-BAY showed the most significant enhancement, indicating that the microbubbles generated by the PFH phase transition can significantly improve the ultrasound imaging effect, and the addition of BAY-876 does not affect this effect. Furthermore, the phase transition explosion can generate high-intensity mechanical force, thereby synergistically enhancing the ablation efficacy of HIFU treatment. Figure 2 C- Figure 2 As shown in Figure D, the cRGD-Lip@PFH-BAY group showed the most significant increase in echo intensity after HIFU, indicating that PFH can effectively enhance the therapeutic effect of HIFU, leading to more thorough tumor necrosis. Furthermore, it reduces the power required for HIFU ablation, thereby reducing thermal stimulation and alleviating the Warburg effect.

[0041] To verify this synergistic mechanism, this study used fresh bovine liver as an ex vivo tissue model to systematically evaluate the enhancing effect of cRGD-Lip@PFH on HIFU ablation. Specifically, fresh bovine liver (150 mm × 150 mm × 100 mm) was degassed by a vacuum pump for 60 minutes and then placed in a HIFU system (JC-200, Chongqing HIFU Medical Technology Co., Ltd., China). 100 µL of PBS, cRGD-Lip@BAY, and cRGD-Lip@PFH (liposome concentration: 1 mg / mL) were injected into different bovine liver tissues, respectively. -1 Immediately after the procedure, HIFU irradiation was performed at the injection site, with parameters set to a duty cycle of 100%, and powers of 105W, 120W, and 135W, with a single irradiation time of 3 seconds. Afterward, the liver was removed, and sliced ​​layer by layer along the direction of the ultrasound beam until the area of ​​maximum damage was located and photographed. Experimental results showed that, combined with… Figure 2As shown in Figure E, with a similar ablation area of ​​bovine liver, the power required by the cRGD-Lip@PFH group with added PFH was significantly lower than that of the PBS group and the cRGD-Lip@BAY group loaded only with BAY-876. This indicates that the phase transition effect of PFH plays a crucial role in enhancing HIFU energy focusing, reducing operating power, and alleviating thermal stress. Therefore, adding PFH to nano-formulations can enhance HIFU energy focusing, reduce operating power, and alleviate thermal stress.

[0042] Subsequently, we further validated the HIFU ablation effect of cRGD-Lip@PFH in the Hepa1-6 mouse tumor model. Specifically, 200 µL of saline, cRGD-Lip@BAY, or cRGD-Lip@PFH (liposome concentration: 1 mg / mL) were injected into tumor-bearing mice via the tail vein. -1 24 hours later, HIFU treatment of the tumor was performed under ultrasound guidance (100% duty cycle, 105 W, 3 s). Combined with... Figure 2 F- Figure 2 As shown in Figure I, the ultrasound signal at the tumor site was significantly enhanced in the cRGD-Lip@PFH group after HIFU irradiation (105 W, 3 seconds), while no significant echogenicity changes were observed in the PBS group and the cRGD-Lip@BAY group at the same HIFU dose. Tumor specimens were then stained with 2,3,5-triphenyltetrazolium (TTC) to reveal necrosis and damage in the tumor tissue. Consistent with in vivo experimental results, the tumor necrosis volume was significantly increased in the cRGD-Lip@PFH group, and the target area ultrasound efficiency factor (EEF) was significantly reduced. H&E-stained sections showed obvious signs of nuclear fragmentation and dissolution in the cRGD-Lip@PFH group, with the largest affected area. This significantly increased tumor ablation volume indicates that PFH-containing cRGD-Lip@PFH can serve as a highly efficient HIFU ablation synergist, avoiding damage to surrounding tissues and lactic acid production caused by HIFU heating.

[0043] IV. Research on the targeting and cytotoxicity of cRGD-Lip@PFH-BAY To achieve efficient targeted inhibition of the GLUT1 pathway, the enrichment of BAY-876 in tumor cells is a crucial prerequisite. Studies have shown that solid tumors often overexpress integrins αvβ3 and αvβ5, and cRGD peptides can specifically bind to these receptors, thereby achieving precise localization of tumor blood vessels. Based on this, the inventors co-cultured DiI dye-labeled cRGD-Lip@PFH-BAY and Lip@PFH-BAY with Hepa1-6 cells (co-culture time: 0.5, 1, 2, 4 h; nanoparticle concentration: 200 μg / mL).-1 Cell uptake was observed under a laser confocal microscope after a corresponding time period. Results showed that... Figure 3 As shown in Figure A, the intracellular fluorescence aggregation level in the cRGD-Lip@PFH-BAY treatment group (G2) was significantly higher than that in the Lip@PFH-BAY group (G1), suggesting that cRGD modification significantly promoted the aggregation of nanocomplexes in Hepa1-6 cells. Figure 3 As shown in Figure B, further flow cytometry quantitative analysis confirmed that the targeting binding efficiency of cRGD-Lip@PFH-BAY to Hepa1-6 cells showed a time-dependent increase.

[0044] To further monitor the distribution of cRGD-Lip@PFH-BAY in mice (nanoformation injection dose: 200uL, 1mg / mL) -1 We used a live fluorescence imaging system to monitor its accumulation at the living tumor site in real time. The results showed that, combined with... Figure 3 As shown in Figure C, DiR-labeled cRGD-Lip@PFH-BAY showed a significant fluorescence signal at the tumor site 4 hours after injection, peaking at 24 hours and remaining at a high level for up to 48 hours. In contrast, the fluorescence signal of DiR-labeled Lip@PFH-BAY was weaker throughout the observation period, consistently lower than that of DiR-labeled cRGD-Lip@PFH-BAY. At 24 hours, major organs of the mouse were dissected for in vitro fluorescence imaging, combined with... Figure 3 As shown in Figure D, the results showed that both targeted and non-targeted nano-formulations accumulated in large quantities in the liver, but the fluorescence intensity in the tumor tissue of the targeted group was significantly higher than that of the non-targeted control group, verifying the aggregation and retention effect of the targeted nano-formulations at the tumor site, and demonstrating that it can effectively aggregate and retain tumors in vivo.

[0045] Based on the aforementioned targeting advantages, we further investigated the direct killing effect of cRGD-Lip@PFH-BAY on Hepa1-6 cells. We divided the cells into the following 5 groups: G1 control group, G2 Lip@PFH-BAY + HIFU group, G3 cRGD-Lip@PFH + HIFU group, G4 cRGD-Lip@BAY + HIFU group, and G5 cRGD-Lip@PFH-BAY + HIFU group. The dosage of the formulation in each group was 60 μg / mL. -11 mL. G2-G5 cells were pre-irradiated with HIFU (100% duty cycle, 105 W, 3 seconds) to release the drug. After the above treatment, dead and live cells were labeled with calcein-AM and propidium iodide (AM / PI), respectively, and cell viability was immediately observed under a laser confocal microscope. Figure 3 E- Figure 3 As shown in Figure G, the cRGD-Lip@PFH + HIFU group showed almost no cell damage, indicating that the lack of BAY-876 liposome loading does not cause cytotoxicity; BAY-876 can induce cell death to some extent. Notably, compared with the Lip@PFH-BAY + HIFU and cRGD-Lip@BAY + HIFU groups, the proportion of dead cells in the cRGD-Lip@PFH-BAY + HIFU group was significantly increased, indicating that cRGD peptide targeting combined with HIFU-released drugs has a better killing effect on tumor cells. In addition, flow cytometry showed anti-tumor results similar to the AM / PI double staining assay. These data indicate that cRGD-targeting peptide modification promotes more liposomes to enter the tumor cell interior, releasing BAY-876 and oxygen under the action of HIFU, targeting and blocking GLUT1, improving hypoxia, reducing glucose uptake, inhibiting tumor cell metabolism, and thus killing tumor cells.

[0046] V. Research on cRGD-Lip@PFH-BAY inhibiting the GLUT1 pathway and reducing lactate metabolism Based on the highly efficient tumor targeting achieved in vitro and in vivo by the aforementioned cRGD-modified multifunctional nanoparticles, we further investigated their role in inhibiting the GLUT1 pathway. Ultrasound-triggered phase transition rupture of cRGD-Lip@PFH-BAY releases BAY-876, which inhibits GLUT1, preventing glucose transport to tumor cells, suppressing glycolysis, and reducing ATP and lactate production. Building on this, we investigated the regulation of lactate metabolism by cRGD-Lip@PFH-BAY at the cellular level. First, we examined whether the nanoparticles could effectively inhibit GLUT1 expression on the cell surface, thereby reducing intracellular glucose uptake. Cells were divided into four groups: G1 control group, G2 cRGD-Lip@PFH + HIFU group (60 μg / mL). -1 G3 free BAY-876 group (4 μg mL) -1 ), G4 cRGD-Lip@PFH-BAY + HIFU group (60 μg mL -1For groups G2 and G4, different nano-formulations were first released using HIFU and then co-incubated with cells (cell culture incubator conditions: 5% CO2, 37℃). Twenty-four hours after treatment, the expression level of GLUT1 on the cell surface of different treatment groups was observed under a laser confocal microscope using immunofluorescence. Figure 4 As shown in Figure A, when cells were treated with free BAY-876, the surface fluorescence only showed a slight decrease, indicating that BAY-876 can effectively inhibit the expression of GLUT1 on the cell surface. However, after treatment with cRGD-Lip@PFH-BAY, the cRGD peptide led to a large accumulation of lipid complexes on the cell surface, resulting in more effective GLUT1 blockade, a significant decrease in fluorescence, and a weaker signal. Figure 4 As shown in Figure B, Western blot analysis further confirmed that the GLUT1 protein level in the cRGD-Lip@PFH-BAY treatment group was significantly lower than that in the other control groups. In summary, the cRGD-modified multifunctional lipid complex can more effectively inhibit GLUT1 expression and weaken the glycolysis pathway and glucose uptake by enhancing cellular uptake.

[0047] Next, we collected cells 12 and 24 hours after the different treatments described above and detected intracellular lactate levels in different groups of cells using a lactate assay kit. Figure 4 C- Figure 4 As shown in E, compared with the control group, at the same drug concentration (1 ng / mL) -1Both free BAY-876 and BAY-876-loaded nanoparticles reduced intracellular lactate levels. However, free BAY-876, lacking specific cell targeting, failed to effectively inhibit glucose uptake by tumor cells, resulting in a minimal inhibitory effect on lactate production. In contrast, targeted liposomes, through cRGD-mediated cell-specific binding, interacted more effectively with GLUT1, thereby more significantly reducing glucose uptake and lactate production. Lactate levels decreased by 39.35 ± 7.51% after 12 hours and by 63.90 ± 4.85% after 24 hours. Using the same method, we measured extracellular glucose-ATP levels. Compared to the control group, intracellular glucose levels remained essentially unchanged after cRGD-Lip@PFH + HIFU treatment; however, extracellular glucose levels slightly increased with BAY-876 alone, suggesting that glucose uptake was inhibited after GLUT1 inhibition. Treatment with cRGD-Lip@PFH-BAY + HIFU significantly reduced glucose uptake. After 12 hours, the glucose content in the supernatant increased by 20.58 ± 1.80%, and after 24 hours, it increased by 25.88 ± 3.09%. Consistent with the decreasing trend in lactate, the ATP value in the BAY-876 group decreased slightly, while the decrease was greatest in the cRGD-Lip@PFH-BAY + HIFU group, at 39.77 ± 2.53% after 12 hours and 59.13 ± 1.34% after 24 hours. These results demonstrate that cRGD-Lip@PFH-BAY can efficiently accumulate in Hepa1-6 tumor tissue, inhibit GLUT1, and reduce glucose uptake and lactate production.

[0048] VI. cRGD-Lip@PFH-BAY improves tumor hypoxia, downregulates GLUT1 expression, and reduces lactate accumulation (study). Besides directly blocking GLUT1's glucose uptake and reducing lactate production, enhancing the oxygenation level of tumor tissue can also promote the degradation of HIF-1α, thereby downregulating GLUT1 expression and ultimately reducing the accumulation of lactate within the tumor. We first used the hypoxia-indicating fluorescent probe Ru(dpp)3Cl2 to assess intracellular oxygen levels. This probe exhibits enhanced fluorescence under hypoxic conditions and is quenched in an oxygen-rich environment. Cells were divided into the following groups: G1 negative control group, G2 positive control group, G3 cRGD-Lip@PFH + HIFU group, G4 cRGD-Lip@BAY + HIFU group, and G5 cRGD-Lip@PFH-BAY + HIFU group. Specifically, Hepa1-6 cells were cultured at 5 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 12-well plates and incubated overnight. The negative control group was incubated for 24 hours under normal conditions in a cell incubator; the positive control and the nanoparticle-containing group were given PBS or nanoparticles (concentration: 30 μg / mL). -1 500 μL) and incubated in a hypoxic cell incubator for 24 hours. After 24 h, the hypoxia probe (Ru(dpp)3)Cl2 (10 mg / mL) was added. -1 Add the above groups and incubate for 4 h. Wash away excess (Ru(dpp)3)Cl2 under light-protected conditions, and observe the red fluorescence signal of each group under an inverted fluorescence microscope. From the fluorescence images ( Figure 5 B) As can be seen, red fluorescence was almost undetectable in tumor cells under normoxic culture conditions; however, in a hypoxic environment, intracellular red fluorescence was significantly enhanced, indicating that the oxygen indicator probe was fully activated. After adding PFH-loaded nanoparticles, the intensity of intracellular red fluorescence decreased significantly, indicating that the oxygen carried by PFH was rapidly released, significantly improving the hypoxic microenvironment. Subsequently, we used a lactate assay kit to measure the lactate concentration of Hepa1-6 cells under different treatment conditions to assess the effect of improved hypoxia on tumor lactate metabolism. The results showed ( Figure 5 C) Under hypoxic conditions, lactate levels in Hepa1-6 cells increased; after treatment with the GLUT1 inhibitor BAY-876, the lactate level in Hepa1-6 cells was effectively reduced. On this basis, the addition of PFH further reduced lactate levels, indicating that the synergistic effect of the two can more effectively inhibit the accumulation of glycolysis products.

[0049] In the Hepa1-6 xenograft model (logarithmically growing Hepa1-6 cells were collected and diluted with PBS to 1×10⁻⁶ cells), 6 A mouse tumor-bearing model was established by subcutaneously inoculating 100 μL of cell suspension into the right back of 5-week-old male C57BL mice at a density of 100 μL / 100 μL. We further investigated its ability to improve the hypoxic microenvironment of mouse tumors. Twenty-four hours after tail vein injection of 200 μL of different nano-formulations, the tumors were treated with HIFU to rupture liposomes, and then oxygen saturation (hemoglobin content) at the tumor site was analyzed using a photoacoustic imaging system. The results showed (…). Figure 5 D) After treatment with cRGD-Lip@BAY without PFH, sporadic blue hemoglobin signals were observed within the tumor, indicating that the tumor remained in a hypoxic state. However, after treatment with cRGD-Lip@PFH and cRGD-Lip@PFH-BAY, the tumor area showed a significant blue signal, indicating that the hypoxic state of the tumor had been significantly alleviated. Combined with... Figure 5As shown in Figure E, further validation of this result was achieved through HIF1-α immunofluorescence and GLUT1 immunohistochemical analysis of dissected tumor tissue. This indicates that cRGD-Lip@PFH-BAY+HIFU significantly improves tumor hypoxia, thanks to the cRGD-targeting peptide modification promoting the entry of more liposomes into the tumor, which, under the action of HIFU, releases the oxygen carried by PFH into the tumor. Figure 5 As shown in Figure F, the results of the lactate assay kit were consistent with the trends observed in in vitro experiments. Lactate levels in tumors dissected from mice in the cRGD-Lip@PFH-BAY + HIFU treatment group were significantly reduced. In conclusion, PFH not only effectively enhances HIFU through cavitation effects and reduces HIFU output power, but also carries oxygen, alleviating the hypoxic state of tumor cells and downregulating GLUT1 expression, thereby inhibiting lactate production.

[0050] VII. cRGD-Lip@PFH-BAY mediates lactate regulation to improve the immunosuppressive microenvironment Studies have shown that lactate induces an immunosuppressive phenotype in many immune cells, protecting tumors from immune attack and promoting tumor progression. For example, lactate can induce TAMs to polarize towards M2 and differentiate into Tregs, and inhibit DC maturation. This study first investigated the regulatory effects of lactate on the aforementioned immune cells at the cellular level, and the recovery of immune cell function after treatment with cRGD-Lip@PFH-BAY, including cell surface molecule expression and cytokine secretion.

[0051] Hepa1-6 cells were divided into five groups: G1 control group, G2 heat stress group, G3 cRGD-Lip@PFH group, G4 cRGD-Lip@BAY group, and G5 cRGD-Lip@PFH-BAY group. The specific procedure was as follows: First, Hepa1-6 cells treated with different methods (5 groups: control group, heat stress group, cRGD-Lip@PFH group, cRGD-Lip@BAY group, and cRGD-Lip@PFH-BAY group, with a nanoparticle concentration of 60 μg / mL) were... -1 Cells were placed in the upper chamber of a Transwell system, while DC 2.4 was seeded in the lower chamber. For the heat stress and liposome groups, cells were heated at 45°C for 15 minutes to simulate the thermal ablation effect of HIFU, and pre-treated with liposome HIFU (100% duty cycle, 105 W, 3 seconds) to achieve drug release. Figure 6 As shown in Figure A, we used a Transwell system to co-culture tumor cells and different nanoparticles in the upper chamber, and seeded dendritic cells (DCs) in the lower chamber. After 24 hours, we collected the DCs from the lower chamber and used flow cytometry to detect the cell surface co-stimulatory molecule CD80.+ CD86 + The results showed differences in expression. Figure 6 B- Figure 6 C), cRGD-Lip@PFH-BAY treatment of Hepa1-6 cells can promote DC maturation and CD80 + CD86 + The expression level was increased by 25.03 ± 2.6% compared to the heat stress group. Furthermore, elevated levels of TNF-α and IL-6 secreted by DC cells were detected in the supernatant. Figure 6 D- Figure 6 As shown in E. Simultaneously, using the same experimental method, Hepa1-6 cells treated with different methods were co-cultured with RAW264.7 cells (5% CO2, 37℃, 24h), and the polarization trend of M0 cells was analyzed using flow cytometry. The results showed ( Figure 6 F- Figure 6 I), cRGD-Lip@PFH-BAY treatment significantly reduced M2 type TAMs (CD206). + The polarization ratio increased the proportion of M1 type TAMs cells (CD86). + Laser confocal microscopy results showed the same experimental results, namely that cRGD-Lip@PFH-BAY can effectively promote the polarization of M0 cells to M1 cells and exert anti-tumor therapeutic effects.

[0052] Based on the above groupings, we verified the changes in intratumoral immune cells after regulation of lactate metabolism in mouse tumors. Figure 6 J illustrates the treatment regimen for improving the in vivo immune microenvironment using flow cytometry analysis, including the timeline of treatment intervention and administration method. Specifically, tumor-bearing Hepa1-6 mice were randomly divided into 5 groups: control group, HIFU group, cRGD-Lip@PFH + HIFU group, cRGD-Lip@BAY + HIFU group, and cRGD-Lip@PFH-BAY + HIFU group. The corresponding nanoparticles (concentration 1 mg / mL) were administered intravenously. -1 (200 μL). Twenty-four hours after injection, mice in the HIFU ablation group underwent HIFU irradiation (100% duty cycle, 105 W power, 3 seconds). Tumor tissue was harvested on days 4 and 7 post-treatment, prepared into single-cell suspensions, and analyzed after staining with different antibodies. Similar to in vitro results, combined with... Figure 6 K- Figure 6 As shown in Figure L, cRGD-Lip@PFH-BAY combined with HIFU treatment significantly increased the number of M1 phenotype TAMs (CD11b). + F4 / 80 +CD86 + The proportion of M2 phenotype TAM (sCD11b) was approximately 30.73 ± 0.80%, which was 18.20%, 12.20%, and 5.73% higher than that of the HIFU group (G2), cRGD-Lip@PFH + HIFU group (G3), and cRGD-Lip@BAY + HIFU group (G4), respectively; while the proportion of M2 phenotype TAM (sCD11b) was reduced. + F4 / 80 + CD206 + The proportion of macrophages (11.53 ± 1.07%) was reduced by 29.40%, 10.70%, and 5.20% compared to the HIFU group (G2), cRGD-Lip@PFH + HIFU group (G3), and cRGD-Lip@BAY + HIFU group (G4), respectively, confirming that cRGD-Lip@PFH-BAY-mediated lactate regulation can effectively induce macrophage polarization and improve the immunosuppressive state of the tumor microenvironment after HIFU. Furthermore, combined with... Figure 6 As shown in Figure M, we also found that the percentage of immunosuppressive T cells (Tregs) decreased after cRGD-Lip@PFH-BAY+HIFU treatment, dropping to 1.79%±0.15%, significantly lower than that in the HIFU group (G2, 8.99±0.60%), the cRGD-Lip@PFH+HIFU group (G3, 6.73±0.71%), and the cRGD-Lip@BAY+HIFU group (G4, 4.46±0.53%). This demonstrates that lactate-modulated therapy based on improved hypoxia and inhibited glucose transport has a regulatory effect on various immunosuppressive cells within the tumor. Simultaneously, combined with... Figure 6 As shown in N, this treatment also stimulates natural killer (NK) cells and CD8+ cells within the tumor. +The number of T cells increased, approximately 4.36 and 1.75 times higher than in the HIFU group, respectively. Furthermore, the proportion of mature dendritic cells (DCs) in lymph nodes also significantly increased. In addition, the serum levels of IL-10 and IFN-γ cytokines after different treatments were further assessed using an ELISA kit. Tumor cells can induce surrounding immune cells to secrete IL-10, forming an immunosuppressive microenvironment that helps tumor cells evade immune surveillance. The results showed that IL-10 levels in the combined treatment group were reduced by 56.3 ± 0.04% compared to the HIFU group, demonstrating its role in improving the immunosuppressive microenvironment. High expression of IFN-γ often indicates activation of the body's anti-tumor immune function; in this experiment, the IFN-γ concentration in the combined treatment group was 1.57 times higher than in the HIFU group, indicating that this treatment strategy can effectively induce a strong cellular immune response and enhance the killing effect on tumor cells. These results indicate that cRGD-Lip@PFH-BAY combined with HIFU treatment effectively reduces lactate production in the tumor microenvironment after HIFU, alleviates the immunosuppressive microenvironment, and induces anti-tumor immunity.

[0053] VIII. Research on cRGD-Lip @PFH-BAY combined with HIFU in vivo anti-tumor therapy Although we have demonstrated that lactate regulation strategies can effectively improve the tumor immune microenvironment, whether they can effectively inhibit tumor growth remains unknown. Therefore, we subsequently established the Hepa1-6 tumor-bearing mouse model as before ( Figure 7 A illustrates the in vivo treatment regimens in Hepa1-6 tumor-bearing mice to evaluate the anti-tumor therapeutic effect of cRGD-Lip@PFH-BAY + HIFU in vivo. Tumor-bearing mice were randomly divided into six groups for the experiment: G1 control group, G2 HIFU group, G3 Lip@PFH-BAY + HIFU group, G4 cRGD-Lip@PFH + HIFU group, G5 cRGD-Lip@BAY + HIFU group, and G6 cRGD-Lip@PFH-BAY + HIFU group. The treatment regimen involved a tail vein injection of 200 μL of the corresponding nano-formulation on day 0, followed by HIFU irradiation 24 hours later (100% duty cycle, 105 W power, 3 seconds). Body weight and tumor volume were monitored every two days during the experiment, and tumor images were taken every seven days. All mice were euthanized on day 16, and the tumors were subsequently dissected, photographed, and weighed. Figure 7 B- Figure 7 C). The mice's body weight remained stable throughout the treatment period, indicating that the dosage and treatment strategy used were safe and effective. This can be seen from the tumor growth curve (…). Figure 7 D- Figure 7In groups E), the tumors in groups G2-G3 grew rapidly, but the difference compared to the control group was not statistically significant, indicating that the effect of drug therapy alone is limited. Although the HIFU group showed some inhibitory effect on tumor growth, the tumors grew rapidly in the later stages of treatment. In contrast, the tumor growth rate of the cRGD-Lip@PFH+HIFU group and the cRGD-Lip@BAY+HIFU group was significantly inhibited, but their effect was not as good as that of the cRGD-Lip@PFH-BAY+HIFU group because the intratumoral lactate level was significantly inhibited after combined treatment. In addition, combined with Figure 7 As shown in Figure F, immunohistochemical examination of tumor tissue further validated the therapeutic efficacy of the cRGD-Lip@PFH-BAY+HIFU group. H&E staining sections showed significant signs of nuclear fragmentation and dissolution, with a marked increase in TUNEL immunofluorescence intensity, while the immunofluorescence intensity of proliferating cell nuclear antigen (PCNA) was significantly reduced, indicating that this treatment strategy can effectively induce cell damage and inhibit tumor cell proliferation. These results demonstrate that cRGD-Lip@PFH-BAY can effectively target tumor tissue, solving the problem of insufficient targeting in nanomedicine delivery. After HIFU irradiation, the liposome's liquid-gas phase transition enhances the targeted destruction of tumor cells by HIFU, reduces the required power, alleviates thermal stress, and simultaneously releases oxygen. BAY-876 regulates intratumoral lactate levels, relieving the inhibitory effect on the immune microenvironment and exerting a powerful anti-tumor therapeutic effect.

[0054] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A lactic acid metabolism regulating nano-formulation, characterized in that: It contains an oxygen-enriched phase change agent.

2. The lactic acid metabolism regulating nano-formulation according to claim 1, characterized in that: The oxygen-enriched phase change agent serves as the core, and liposomes are wrapped around the oxygen-enriched phase change agent.

3. The lactic acid metabolism regulating nano-formulation according to claim 2, characterized in that: The liposomes are modified with cRGD peptides or membrane-penetrating peptides.

4. The lactic acid metabolism regulating nano-formulation according to claim 2, characterized in that: The liposomes are loaded with GLUT-1 inhibitors.

5. The lactic acid metabolism regulating nano-formulation according to claim 1, characterized in that: The oxygen-enriched phase change agent is perfluorohexane or PFP.

6. The application of oxygen-enriched phase change agents in the preparation of drugs that reduce lactate production by alleviating sublethal heat stress under HIFU focusing.

7. The application according to claim 6, characterized in that: The oxygen-enriched phase change agent is perfluorohexane.

8. The application of the lactate metabolism regulating nano-formulation according to claims 1-5 in the preparation of drugs that reduce lactate production by alleviating sublethal heat stress under HIFU focusing.

9. A method for preparing a nano-formulation for regulating lactic acid metabolism, characterized in that: Includes the following steps: S1, DPPC, cRGD-DSPE-PEG 2000 Cholesterol was dissolved in chloroform and added to BAY-876 and mixed well. S2. Remove organic solvents using a rotary evaporator, then add PBS for hydration and peel off the phospholipid membrane with the assistance of an ultrasonic cleaner; S3. Add perfluorohexane and use a sonic vibrator to obtain cRGD-Lip@PFH-BAY liposomes; S4. Oxygen-enriched cRGD-Lip@PFH-BAY can be obtained by oxygenating the obtained liposomes in an oxygen-enriched environment.

10. The method for preparing the lactate metabolism regulating nano-formulation according to claim 9, characterized in that: DPPC, cRGD-DSPE-PEG 2000 It is soluble in chloroform at a mass ratio of 3:1:1 with cholesterol.

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