Lipid nanoparticle drug delivery system and application thereof in improving tumor vascular normalization

Abstract

The application discloses a kind of lipid nano-drug delivery system and its preparation method in the application of improving tumor vascular normalization, the lipid nano-drug delivery system includes drug, CaO2 Nanoparticle and anionic liposome, the drug and CaO2 Nanoparticle are co-loaded in the anionic liposome;The surface of the CaO2 Nanoparticle is with positive charge, and the preparation raw material of the anionic liposome contains 1,2-dioleoyl-sn-glycerol-3-phosphoric acid sodium salt. 2+ The lipid nano-drug delivery system can enhance endothelial cell-derived NO production by mediating eNOS activation of Met and other drugs and Ca

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of a lipid nanoparticle drug delivery system and its preparation method in improving tumor angiogenesis normalization. Background Technology

[0002] The growth and expansion of all solid tumors are highly dependent on their vascular networks, which are responsible for delivering oxygen, essential nutrients, and growth factors to the tumor tissue. Abnormal tumor angiogenesis, stemming from a disruption of the delicate balance between pro-angiogenic and anti-angiogenic signals, is a core mechanism driving rapid tumor proliferation and malignant transformation. These abnormal new blood vessels are characterized by disordered structure, dysfunctional processes, easy leakage, and frequent compression by surrounding tissues. They not only provide convenient channels for tumor cell migration and spread but also collectively shape a unique tumor microenvironment (TME). This environment, characterized by hypoxia, acidity, and high interstitial pressure, significantly inhibits the infiltration and function of immune cells, thereby weakening the body's anti-tumor immune response.

[0003] Given the central role of abnormal angiogenesis in tumor progression, anti-angiogenic therapy has become an important treatment strategy. This therapy mainly targets key molecules such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR), using antibodies or small molecule antagonists to block pro-angiogenic signals. Initially, it can significantly reduce tumor blood vessel density, but high-dose treatment is often accompanied by side effects, such as promoting the activation of alternative angiogenesis pathways and exacerbating tumor hypoxia, forming a vicious cycle of treatment resistance.

[0004] To overcome this challenge, researchers have proposed a vascular normalization strategy, which involves using low-dose anti-angiogenic drugs to induce a more regular and mature transformation of tumor vascular structures. This process not only helps alleviate tumor hypoxia and reduce interstitial pressure, but also improves the immunosuppressive properties of the tumor microenvironment (TME), creating conditions for effective infiltration and function of immune cells. However, achieving and maintaining the window of vascular normalization requires precise control of drug dosage and timing to avoid over-pruning of the vascular network, which places higher demands on the design of clinical treatment protocols.

[0005] Building upon this foundation, nitric oxide (NO), as a key vascular regulatory molecule, is increasingly recognized for its role in tumor angiogenesis and maturation. Restoring or enhancing the perivascular NO concentration gradient can promote vascular homeostasis and normalize tumor angiogenesis, thereby optimizing the tumor microenvironment (TME), improving drug delivery efficiency, and activating anti-tumor immune responses. However, current methods relying on exogenous NO donors are limited by the unsustainability of their concentration gradient within the TME. Therefore, developing novel therapeutic strategies is of great significance. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a lipid nanoparticle drug delivery system that exhibits high stability and can promote tumor angiogenesis normalization to improve tumor vascularization.

[0007] The present invention also proposes a method for preparing the above-mentioned lipid nanocarrier system.

[0008] The present invention also proposes an application of the above-mentioned lipid nanocarrier system in improving tumor angiogenesis normalization.

[0009] According to a first aspect of the present invention, a lipid nanoparticle drug delivery system includes a drug, CaO2 nanoparticles and anionic liposomes, wherein the drug and CaO2 nanoparticles are co-loaded in the anionic liposomes;

[0010] The drug includes metformin (Met), the CaO2 nanoparticles have a positively charged surface, and the raw materials for preparing the anionic liposomes contain sodium 1,2-dioleoyl-sn-glycerol-3-phosphate (DOPA).

[0011] The lipid nanoparticle drug delivery system according to embodiments of the present invention has at least the following beneficial effects: The present invention cleverly integrates DOPA-containing anionic liposomes with positively charged CaO2 nanoparticles, significantly improving the overall stability of the drug delivery system through their electrostatic interaction. This design ensures the integrity of the drug during delivery, laying the foundation for efficient targeted delivery. The system can precisely reach the tumor vascular region via the circulatory system, and then, utilizing the unique membrane fusion mechanism of liposomes, first release drugs such as Met and CaO2 nanoparticles into the tumor vascular endothelial cells. This process not only achieves initial drug localization but also promotes transcytosis of these active ingredients by endothelial cells, thereby effectively delivering the drug and nanoparticles to the tumor core region. Within the tumor vascular endothelial cells, drugs such as Met can activate the AMPK-eNOS signaling pathway, and some CaO2 degradation produces Ca... 2+ By activating the activity of calmodulin (CaM) and using Ca 2+ The / CaM complex binds to the binding site on the eNOS subunit, thereby activating eNOS. The key role of these two pathways is to upregulate the activity of endothelial nitric oxide synthase (eNOS), thus significantly enhancing NO (nitric oxide) production. The local concentration gradient of NO around blood vessels not only helps normalize the tumor vascular system and improve the hypoxic state of the tumor microenvironment (TME), but also promotes the infiltration and function of immune cells, creating favorable conditions for subsequent immunotherapy.

[0012] This invention utilizes the transcytosis of nanomedicines in tumor endothelial cells. Specifically, aside from a portion of the lipid nanoparticles taken up by vascular endothelial cells and subsequently degraded, the majority of the lipid nanoparticles enter the tumor region via transcytosis and are taken up by tumor cells. Within the tumor cells, the lipid nanoparticles release drugs such as Met and CaO2 nanoparticles. The CaO2 nanoparticles degrade in the acidic tumor cell microenvironment, producing a large amount of Ca2+. 2+ and H2O2. These products not only induce Ca 2 Overload and oxidative stress damage directly kill tumor cells and can also trigger immunogenic cell death (ICD), activating the body's anti-tumor immune response. Furthermore, drugs such as Met also reduce the production of the immunosuppressive metabolite kynurenine by inhibiting the uptake of tryptophan by tumor cells, thereby alleviating the immunosuppressive state of the tumor mechanoimmune (TME) and further enhancing the anti-tumor immune response.

[0013] In summary, the lipid nanoparticle drug delivery system of the present invention, through precise delivery, local activation and synergistic effects of multiple mechanisms, not only directly kills tumor cells, but also improves the tumor microenvironment and enhances the immune response, providing a novel strategy and approach for tumor treatment.

[0014] According to some embodiments of the present invention, the raw materials for preparing the anionic liposomes further include 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), cholesterol (Cholesterol), and 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000)(DSPE-PEG2000).

[0015] According to some embodiments of the present invention, the mass ratio of 1,2-dioleoyl-sn-glycerol-3-phosphate choline, sodium 1,2-dioleoyl-sn-glycerol-3-phosphate, cholesterol, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-(methoxy(polyethylene glycol)-2000) in the raw materials for preparing the anionic liposomes is 1:0.8 to 1:0.1 to 0.4:0.2 to 0.5. For example, DOPC:DOPA:cholesterol:DSPE-PEG2000 = 3.93:3.62:0.97:1.41.

[0016] According to some embodiments of the present invention, the mass ratio of 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), the drug, and CaO2 nanoparticles is 1:1.2-1.4:1.3-1.5, such as 3.93:5.2:5.6.

[0017] According to some embodiments of the present invention, the average particle size of the CaO2 nanoparticles is 80–90 nm. For example, a particle size of 83.08 ± 0.73 nm controls the particle size of the calcium superoxide nanoparticles within a smaller range, further improving the stability of the liposomes.

[0018] According to some embodiments of the present invention, the average particle size of the lipid nanoparticle drug delivery system is 100–120 nm, such as 112.37 ± 0.71 nm.

[0019] According to some embodiments of the present invention, the method for preparing the CaO2 nanoparticles includes the following steps:

[0020] A soluble calcium salt, a dispersant, and an alcohol solvent are mixed to obtain a precursor solution; H2O2 solution is added dropwise to the precursor solution, followed by the addition of a precipitant, and the reaction proceeds to obtain the final product.

[0021] According to some embodiments of the present invention, the alcohol solvent includes at least one of methanol and ethanol.

[0022] According to some embodiments of the present invention, the dispersant includes at least one selected from sodium citrate, cetyltrimethylammonium bromide (CTAB), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) 200, and PEG 400.

[0023] According to some embodiments of the present invention, the soluble calcium salt and the dispersant are dissolved in an alcohol solvent at a mass ratio of 1:2 to 4. For example, a ratio of 1:3.

[0024] According to some embodiments of the present invention, the precipitant includes at least one of NH3·H2O, NaOH and KOH.

[0025] According to some embodiments of the present invention, the soluble calcium salt includes at least one of CaCl2, Ca(NO3)2 and their hydrates.

[0026] According to some embodiments of the present invention, in the precursor solution, Ca 2+ The concentration is 0.01–0.1 mol / L.

[0027] According to some embodiments of the present invention, the mass ratio of the soluble calcium salt to the dispersant is 1:2 to 5.

[0028] According to some embodiments of the present invention, the precursor solution contains Ca 2+ The molar ratio with H2O2 is 1:0.2-3.

[0029] According to some embodiments of the present invention, the mass concentration of the H2O2 solution is 5% to 30%.

[0030] According to some embodiments of the present invention, the dropping rate of the H2O2 solution is 0.05 to 5 mL / min.

[0031] According to some embodiments of the present invention, the method for preparing the CaO2 nanoparticles further includes a step of stirring before adding the precipitant.

[0032] According to some embodiments of the present invention, the stirring time is 20 min to 60 min, preferably 30 min.

[0033] According to some embodiments of the present invention, the method for preparing the CaO2 nanoparticles further includes a step of stirring after adding a precipitant.

[0034] According to some embodiments of the present invention, the stirring time is 10 min to 30 min, preferably 20 min.

[0035] According to some embodiments of the present invention, the method for preparing the CaO2 nanoparticles further includes a post-processing step, specifically including a step of solid-liquid separation of the product after the reaction is completed, collection of the solid phase, and washing.

[0036] According to some embodiments of the present invention, the solid-liquid separation is specifically a centrifugal process, wherein the centrifugation speed is 14,000 rpm to 16,000 rpm and the time is 10 to 20 minutes. For example, the speed is 15,000 rpm and the time is 15 minutes.

[0037] The preparation method according to a second aspect embodiment of the present invention includes the following steps:

[0038] A lipid nanoparticle drug delivery system was prepared by using a thin-film dispersion method to combine raw materials for anionic liposome preparation with drugs and CaO2 nanoparticles.

[0039] The preparation method according to the embodiments of the present invention has at least the following beneficial effects: the preparation method of the present invention is simple to operate and has good prospects for industrial application.

[0040] According to some embodiments of the present invention, the thin film dispersion method specifically includes the following operations: dissolving anionic liposome raw materials (including DOPC, DOPA, cholesterol and DSPE-PEG2000) and Met in chloroform, mixing with an ethanol solution of CaO2 nanoparticles, and ultrasonically dispersing; and preparing the lipid nanoparticle drug delivery system by rotary evaporation of the uniformly dispersed anionic liposome raw materials, Met and CaO2 nanoparticles.

[0041] According to some embodiments of the present invention, the temperature during the ultrasonic dispersion process is room temperature.

[0042] According to some embodiments of the present invention, the room temperature is 25±5℃, such as 25℃.

[0043] According to some embodiments of the present invention, the water bath temperature during the rotary evaporation process is 42-45°C.

[0044] According to some embodiments of the present invention, the rotary evaporation time is greater than 35 min.

[0045] According to some embodiments of the present invention, the preparation method of the lipid nanoparticle drug delivery system further includes the following step: removing unloaded CaO2 nanoparticles and unloaded drugs.

[0046] According to some embodiments of the present invention, the unencapsulated CaO2 nanoparticles are removed by low-speed centrifugation.

[0047] According to some embodiments of the present invention, the low-speed centrifugation method uses a rotation speed of 800–1200 rpm and a centrifugation time of 4–6 min. For example, the rotation speed is 1000 rpm and the time is 5 min.

[0048] According to some embodiments of the present invention, the unencapsulated drug is removed by ultrafiltration.

[0049] According to some embodiments of the present invention, the ultrafiltration method specifically includes the following operations: transferring the remaining mixture after the removal of uncoated CaO2 nanoparticles to an ultrafiltration tube, and centrifuging at a speed of 4000-5000 rpm for 20-50 min. For example, the speed is 4500 rpm and the time is 30 min.

[0050] According to the third aspect of the present invention, the application of the above-described lipid nanocarrier system in the preparation of antitumor drugs is specifically described.

[0051] According to the embodiments of the present invention, the application has at least the following beneficial effects: Antitumor drugs containing the lipid nanoparticle drug delivery system of the present invention can cleverly utilize the membrane fusion effect of liposomes to first partially release the active drug components (including Met, etc.) and CaO2 into vascular endothelial cells. Subsequently, the endothelial cells transcapitate the remaining majority of the lipid nanoparticle drug delivery system into the tumor region. The drug released in the vascular endothelial cells and the partially generated CaO2... 2+ It can mediate eNOS activation, thereby enhancing endothelial cell-derived NO production, forming a NO concentration gradient around tumor blood vessels to normalize tumor blood vessels, reversing the tumor immunosuppressive microenvironment, and improving the therapeutic effect of tumor calcium overload.

[0052] According to some embodiments of the present invention, the antitumor drug utilizes the transcytosis of tumor vascular endothelial cells, via Met and Ca... 2+Activating vascular endothelial cell eNOS improves the therapeutic effect of tumor angiogenesis normalization. Improving tumor angiogenesis further enhances calcium overload tumor therapy. The antitumor strategy of this antitumor drug is to promote tumor angiogenesis normalization and synergistically enhance oxidative stress damage in tumor cells with calcium overload therapy, thereby weakening their immune evasion ability.

[0053] The present invention also provides an antitumor drug comprising the lipid nanocarrier system described above.

[0054] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0055] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0056] Figure 1 Transmission electron microscopy (TEM) images (B) of CaO2 nanoparticles A and lipid nano-drug delivery system prepared in the embodiments of the present invention;

[0057] Figure 2 This is a particle size distribution diagram of the CaO2 nanoparticles and lipid nano-drug delivery system prepared in the embodiments of the present invention;

[0058] Figure 3 The image shows the Zeta potential diagram of the CaO2 nanoparticles and lipid nanocarrier system prepared in the embodiments of the present invention.

[0059] Figure 4 The lipid nanoparticle drug delivery system prepared in the embodiments of the present invention under different pH conditions Ca 2+ Release result;

[0060] Figure 5 This is the result of changes in the phosphorylated protein expression level of eNOS in HUVEC cells after different drug treatments in the test examples of this invention;

[0061] Figure 6 The results show the Cy5 content in fresh serum-free culture medium at different time points after HUVEC cells were co-incubated with Cy5-C@L (liposomes encapsulating Cy5 and CaO2) for 6 hours in the test examples of this invention.

[0062] Figure 7 The test examples of this invention show scratch marks (A) on HUVEC cells after treatment with different drugs and related quantitative results (B).

[0063] Figure 8 This is a laser confocal microscope image showing the calcium content of 4T1 cells after different drug treatments in the test examples of this invention.

[0064] Figure 9 This is the result of the change in tryptophan levels in the culture medium of 4T1 cells after different drug treatments in the test examples of this invention. Detailed Implementation

[0065] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available. Unless otherwise specified, the same parameter value is the same in all embodiments. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0066] In the description of this invention, references to terms such as "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0067] In the description of this invention, the use of terms such as "first," "second," etc., is for the purpose of distinguishing technical features only and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0068] Example

[0069] This example provides a lipid nanoparticle drug delivery system, which is prepared from a drug, CaO2 nanoparticles and anionic liposomes. The drug is Met, and Met and CaO2 nanoparticles are co-loaded on the anionic liposomes. The CaO2 nanoparticles have a positive charge on their surface, and the raw materials for preparing the anionic liposomes are composed of DOPA, DOPC, cholesterol and DSPE-PEG2000.

[0070] The specific steps for preparing CaO2 nanoparticles are as follows:

[0071] (1) Dissolve 200 mg CaCl2·2H2O and 600 mg PVP in 60 mL methanol under ultrasonic conditions in a water bath, then add 0.4 mL H2O2 dropwise and stir magnetically for 30 minutes.

[0072] (2) Add 1 mL of ammonia water and continue stirring for 2 minutes;

[0073] (3) The product was transferred to a 50 mL centrifuge tube and centrifuged at 15000 rpm for 15 minutes. The supernatant was discarded, and the precipitate was washed three times with 20 mL of methanol to obtain CaO2 nanoparticles. The nanoparticles were then dispersed in anhydrous ethanol for storage.

[0074] The specific steps for preparing the lipid nanoparticle drug delivery system are as follows:

[0075] (1) Dissolve the anionic liposomes in 7 mL of chloroform with raw materials (3.93 mg DOPC, 3.62 mg DOPA, 0.965 mg cholesterol and 1.405 mg DSPE-PEG2000) and 5.2 mg Met, and add them together with 1 mL of CaO2 nanoparticle (5.6 mg) ethanol solution into a round-bottom flask. Disperse the liposomes in a water bath for 2 minutes by sonication.

[0076] (2) Using the thin film dispersion method, the solution that is uniformly dispersed in the eggplant-shaped flask is rotary evaporated for 35 minutes to prepare anionic liposomes;

[0077] (3) Remove the eggplant-shaped bottle from which the film has been successfully formed, add 2 mL of ultrapure water to it, sonicate it in a water bath for 5 minutes to completely hydrate it, and then transfer the product to a 2 mL EP tube.

[0078] (4) Centrifuge at 1000 rpm for 5 minutes, collect the supernatant and transfer it to an ultrafiltration tube, centrifuge at 4500 rpm for 30 minutes, and collect the concentrated product to obtain the lipid nanoparticle drug delivery system (denoted as MC@L).

[0079] The morphology of the CaO2 nanoparticles and lipid nanocarrier systems prepared by the above operation was observed using transmission electron microscopy (TEM). The results are as follows: Figure 1 As shown, the CaO2 nanoparticles and lipid nanoparticle drug delivery system exhibit a relatively obvious spherical structure and uniform particle size. The hydrodynamic particle size of the CaO2 nanoparticles and lipid nanoparticle drug delivery system was measured using a Malvern particle size analyzer, and the results are as follows. Figure 2 As shown, the average particle sizes of CaO2 nanoparticles and lipid nanoparticle drug delivery systems are approximately 83.08 ± 0.73 nm and 112.37 ± 0.71 nm, respectively. Figure 2 The potential measurement results are as follows: Figure 3 As shown, from Figure 3 As can be seen, CaO2 nanoparticles are positively charged, and when they are co-loaded with Met onto liposomes, the potential of the resulting lipid nanoparticle drug delivery system changes from positive to negative, proving the successful encapsulation by liposomes.

[0080] Compare with Example 1

[0081] This example provides a lipid nanoparticle drug delivery system prepared from the drug Met and anionic liposomes (denoted as M@L). The preparation process is consistent with the previous examples, except that CaO2 nanoparticles are not encapsulated.

[0082] Compare with Example 2

[0083] This example provides a lipid nanoparticle drug delivery system prepared from CaO2 nanoparticles and anionic liposomes (denoted as C@L). The preparation process is consistent with the previous examples, except that the drug is not encapsulated.

[0084] Test case

[0085] This example tests the performance of the drug delivery systems prepared in the above embodiments and control examples.

[0086] (1) In vitro Ca2+ of lipid nanocarrier systems 2+ Release condition test

[0087] MC@L (a lipid nanoparticle drug delivery system simultaneously encapsulating Met and CaO2) solution was loaded into dialysis bags and immersed in PBS solutions of different pH values ​​(7.4, 6.5, or 5.5). The bags were incubated at 37°C with shaking. 0.5 mL of the buffer solution outside the dialysis bag was collected at different time points to detect CaO2. 2+ The concentration (determined using a calcium colorimetric assay kit) was then adjusted, and 0.5 mL of fresh PBS buffer at a different pH was added.

[0088] like Figure 4 As shown, the Ca of MC@L decreases with decreasing pH. 2+ The release rate was significantly accelerated, and Ca was released at pH 5.5. 2 + The highest total release was observed at pH 6.5, followed by pH 7.4. This is because acidic conditions favor the degradation of CaO2 nanoparticles into Ca. 2+ And H2O2.

[0089] (2) Test of phosphorylated protein expression level of eNOS in HUVEC cells

[0090] HUVEC cells were seeded into six-well plates and cultured for 24 h. The culture medium was then replaced with fresh complete medium (DMEM). M@L (liposomes encapsulating Met only), C@L (liposomes encapsulating CaO2 only), and MC@L were added for drug treatment. A control group without drug treatment was set up. Cells were then transferred to a CO2 incubator and cultured for another 48 h. After removing the old medium, the cells were washed once with PBS, and proteins were extracted. Western blotting was used to detect the phosphorylated protein expression levels of eNOS in each group. The test results are as follows: Figure 5 As shown.

[0091] Depend on Figure 5 It was found that both the Met-containing group (M@L) and the CaO2 nanoparticle-containing group (C@L) could increase the phosphorylated protein expression level of eNOS in HUVEC cells (eNOS-P). Ser1177 These are respectively composed of Met and Ca. 2+ This is caused by Met increasing the phosphorylation and activity of eNOS by activating protein kinase (AMPK), further promoting eNOS-mediated NO production. However, since eNOS itself is affected by Ca... 2+ The regulation thus reduces the amount of Ca produced by the degradation of CaO2 nanoparticles. 2+ It can also regulate eNOS and enhance NO production.

[0092] (3) Transcytosis assay of HUVEC cells

[0093] HUVEC cells were seeded in 6-well plates and cultured for 24 hours. The culture medium was then replaced with fresh complete DMEM, and liposomes (Cy5-C@L) encapsulating Cy5 (Cyanine 5) and CaO2 nanoparticles were added. The cells were then transferred to a CO2 incubator and cultured for another 6 hours. The old medium was removed and replaced with fresh serum-free medium. The supernatant was collected at different time points to determine the Cy5 content. The experimental results are as follows: Figure 6 As shown.

[0094] Depend on Figure 6 It can be seen that when the drug-containing culture medium is discarded and replaced with fresh serum-free culture medium, the Cy5 content in the culture medium is extremely low at 0h. However, as the incubation time increases, the Cy5 content in the culture medium gradually increases, indicating that HUVEC cells exocytose the drug they have taken up into the culture medium, which proves the transcytosis of the lipid nanoparticle drug delivery system in vascular endothelial cells.

[0095] (4) Scratch assay of HUVEC cells

[0096] HUVEC cells were seeded into six-well plates and cultured for 24 hours. A sterile yellow pipette tip was used to evenly and straightly scratch the monolayer of cells. The cells were washed once with PBS to remove any floating cell debris. Drug-containing culture medium was then added, and microscopic images of the scratches were taken at different time points. The experimental results are as follows: Figure 7 As shown.

[0097] Depend on Figure 7 The results of the cell scratch assay in A showed that the group containing Met (M@L) and the group containing CaO2 nanoparticles (C@L) could inhibit the migration of HUVEC cells to varying degrees, proving that their angiogenesis ability was inhibited.

[0098] (5) Experiment on changes in calcium content in 4T1 cells

[0099] 4T1 cells were seeded into laser confocal microscopy dishes and cultured for 24 h. The culture medium was then replaced with fresh complete DMEM, and M@L, C@L, and MC@L were added for drug treatment. A control group without drug treatment was set up. The cells were then transferred to a CO2 incubator and cultured for another 24 h. The old medium was removed, and the cells were washed once with PBS. Then, 5 μM PBS solution containing Fluo-4AM was added, and the cells were incubated at 37°C for 30 min. After washing once with PBS, the cells were incubated at 37°C for another 20 min. Finally, the cells were observed using a laser confocal microscope. The results are as follows: Figure 8 As shown.

[0100] Depend on Figure 8 As shown, the Met-containing group (M@L) and the CaO2 nanoparticle-containing group (C@L) can inhibit HUVEC cell migration to different degrees. Among them, the scratch healing of the MC@L group is the least, which proves that its angiogenesis ability is inhibited to the greatest extent.

[0101] (6) Detection of tryptophan uptake in 4T1 cells

[0102] 4T1 cells were seeded into six-well plates and cultured for 24 hours. The culture medium was then replaced with fresh complete DMEM, and M@L, C@L, and MC@L were added for drug treatment. A control group without drug treatment was set up. The cells were then transferred to a CO2 incubator and cultured for another 24 hours. The old culture medium was collected into EP tubes, centrifuged at 1000 rpm for 5 minutes, and the supernatant was collected. The tryptophan content was detected by ELISA. The experimental results are as follows: Figure 9 As shown.

[0103] Depend on Figure 9It was found that the residual extracellular tryptophan in the Met-containing group (M@L) was significantly higher than that in the control group, proving that Met treatment can effectively inhibit the uptake of tryptophan by tumor cells. This is because Met inhibits the uptake of tryptophan by 4T1 tumor cells by downregulating MYC, leading to a reduction in the tryptophan transporter SLC7A5. Furthermore, since CaO2 nanoparticles cause calcium overload in tumor cells, promoting oxidative stress damage or cell death, the tryptophan uptake in the C@L group also decreased to some extent.

[0104] In summary, the lipid nanoparticle drug delivery system of this invention is prepared by co-loading Met and CaO2 nanoparticles onto anionic liposomes. The raw materials used in the anionic liposomes include DOPC, DOPA, cholesterol, and DSPE-PEG2000. By introducing the anionic lipid DOPA, utilizing its electrostatic interaction with the positively charged CaO2 nanoparticles, and controlling the particle size of the CaO2 nanoparticles within a small range, the stability of the liposomes can be enhanced, which is beneficial for drug storage and in vivo circulation. This lipid nanoparticle drug delivery system cleverly utilizes the membrane fusion effect of liposomes to first release Met and CaO2 nanoparticles into vascular endothelial cells, which then transcapitulate the Met and CaO2 nanoparticles to the tumor region. Within tumor vascular endothelial cells, Met-mediated eNOS activation enhances endothelial cell-derived NO production, thereby creating a NO concentration gradient around tumor vessels to normalize tumor vascularity and alleviate the hypoxic microenvironment. Through transcytosis of vascular endothelial cells and extravasation mediated by the endothelial space, Met, CaO2 nanoparticles, and other lipid nanoparticle drug delivery systems can accumulate within tumor cells and achieve calcium overload, inducing oxidative stress damage and immunogenic cell death in tumor cells. Furthermore, Met can inhibit tumor cell uptake of tryptophan, thereby reducing the production of immunosuppressive kynurenine, reversing the immunosuppressive microenvironment, and improving the therapeutic effect of tumor treatment, showing promising application prospects in the field of tumor therapy.

[0105] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A lipid nanoparticle drug delivery system, characterized in that: The invention comprises a drug, CaO2 nanoparticles, and anionic liposomes, wherein the drug and CaO2 nanoparticles are co-loaded in the anionic liposomes; the drug comprises metformin; the CaO2 nanoparticles have a positively charged surface; the raw materials for preparing the anionic liposomes contain sodium 1,2-dioleoyl-sn-glycerol-3-phosphate; the raw materials for preparing the anionic liposomes also contain 1,2-dioleoyl-sn-glycerol-3-phosphate choline, cholesterol, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-(methoxy(polyethylene glycol)-2000); The mass ratio of 1,2-dioleoyl-sn-glycerol-3-phosphate choline, sodium 1,2-dioleoyl-sn-glycerol-3-phosphate, cholesterol, and 1,2-distearate-sn-glycerol-3-phosphate ethanolamine-N-(methoxy(polyethylene glycol)-2000) in the raw materials for preparing the anionic liposomes is 1:0.8~1:0.1~0.4:0.2~0.

5. The method for preparing the CaO2 nanoparticles includes the following steps: A soluble calcium salt, a dispersant, and an alcohol solvent are mixed to obtain a precursor solution. H₂O₂ solution is first added dropwise to the precursor solution, followed by the addition of a precipitant, and the reaction proceeds to obtain the final product. The dispersant includes at least one of polyvinylpyrrolidone, sodium citrate, hexadecyltrimethylammonium bromide, PEG 200, and PEG 400. The mass ratio of 1,2-dioleoyl-sn-glycerol-3-phosphocholine, the drug, and CaO₂ nanoparticles is 1:1.2~1.4:1.3~1.

5.

2. The lipid nanoparticle drug delivery system according to claim 1, characterized in that: The average particle size of the CaO2 nanoparticles is 80-90 nm; and / or, the average particle size of the lipid nanoparticle drug delivery system is 100-120 nm.

3. The method for preparing the lipid nanoparticle drug delivery system according to any one of claims 1 to 2, characterized in that: Includes the following steps: A lipid nanoparticle drug delivery system was prepared by using a thin-film dispersion method to combine raw materials for anionic liposome preparation with drugs and CaO2 nanoparticles.

4. The application of the lipid nanoparticle drug delivery system according to any one of claims 1 to 2 in the preparation of antitumor drugs, characterized in that: The anti-tumor drug is an anti-breast cancer drug.

5. The application according to claim 4, characterized in that: The antitumor drug utilizes endocytosis of endothelial cells, via metformin and Ca2+. 2+ Drugs that activate nitric oxide synthase in vascular endothelial cells and improve tumor vascular abnormalities.

6. An antitumor drug, characterized in that: The antitumor drug includes the lipid nanocarrier system as described in any one of claims 1 to 2, wherein the antitumor drug is an anti-breast cancer drug.

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