Lipid nano drug delivery system and application of preparation method thereof in improvement of tumor vessel normalization

Abstract

The invention discloses a lipid nano drug delivery system and application of a preparation method of the lipid nano drug delivery system in improvement of tumor vessel normalization, the lipid nano drug delivery system comprises a drug, CaO2 nanoparticles and an anionic liposome, and the drug and the CaO2 nanoparticles are jointly loaded on the anionic liposome; the surfaces of the CaO2 nanoparticles are provided with positive charges, and the preparation raw materials of the anion liposome contain 1, 2-dioleoyl-sn-glycerol-3-sodium phosphate. The lipid nano drug delivery system can enhance the generation of endothelial cell-derived NO through Met and other drugs and Ca < 2 + > mediated eNOS activation, so that NO concentration gradient is formed around tumor blood vessels to realize tumor blood vessel normalization, the tumor immunosuppression microenvironment is reversed, and the tumor calcium overload treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of a lipid nano drug delivery system and a preparation method thereof in improving normalization of tumor blood vessels. Background Art

[0002] The growth and expansion of all solid tumors are highly dependent on their vascular network, which is responsible for delivering oxygen, essential nutrients, and growth factors to tumor tissues. Abnormal tumor angiogenesis stems from the disruption of the delicate balance between pro-angiogenic and anti-angiogenic signals, and is the core mechanism that drives rapid tumor proliferation and malignant transformation. These abnormal new blood vessels are characterized by disorganized structure, dysfunctional function, easy leakage, and frequent compression by surrounding tissues. They not only provide convenient channels for the migration and spread of tumor cells, but also jointly shape a unique tumor microenvironment (TME), which is marked by hypoxia, acidic environment, and high interstitial fluid pressure, significantly inhibiting 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 one of the important treatment strategies. This therapy mainly blocks pro-angiogenic signals by targeting key molecules such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR), using antibodies or small molecule antagonists, which can significantly reduce tumor vascular density in the early stages, but high-dose treatment is usually accompanied by side effects, such as promoting the activation of alternative angiogenic 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 is to promote the transformation of tumor vascular structure to a more regular and mature direction through the use of low-dose anti-angiogenic drugs. This process not only helps to alleviate the hypoxia state in the tumor and reduce interstitial fluid pressure, but also improves the immunosuppressive nature of the TME, creating conditions for the effective infiltration and function of immune cells. However, achieving and maintaining the window period for vascular normalization requires precise control of drug dosage and timing of administration to avoid excessive pruning of the vascular network, which places higher demands on the design of clinical treatment plans.

[0005] On this basis, nitric oxide (NO), as a key vascular regulatory molecule, has received increasing attention for its role in tumor angiogenesis and maturation. By restoring or enhancing the concentration gradient of NO around blood vessels, the maintenance of vascular homeostasis and the normalization of tumor blood vessels can be promoted, thereby optimizing the TME, improving drug delivery efficiency and activating anti-tumor immune responses. However, current methods that rely on exogenous NO donors are limited by the unsustainability of their concentration gradients in the TME. Therefore, it is of great significance to develop new treatment strategies. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a lipid nano drug delivery system, which has high stability and can promote tumor vascular normalization to improve tumor vascular normalization.

[0007] The present invention also provides a method for preparing the lipid nano drug delivery system.

[0008] The present invention also proposes an application of the lipid nano drug delivery system in improving the normalization of tumor blood vessels.

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

[0010] The drug comprises metformin (Met), the surface of the CaO2 nanoparticles carries positive charges, and the raw materials for preparing the anionic liposomes contain 1,2-dioleoyl-sn-glycerol-3-sodium phosphate (DOPA).

[0011] The lipid nano drug delivery system according to the embodiment of the present invention has at least the following beneficial effects: the scheme of the present invention cleverly combines anionic liposomes containing DOPA and positively charged CaO2 nanoparticles, and through the electrostatic interaction between them, significantly improves the overall stability of the drug delivery system. This design ensures the integrity of the drug during the delivery process and lays the foundation for efficient targeted delivery. The system can accurately reach the tumor vascular area through the blood circulation system, and then use the membrane fusion mechanism unique to liposomes to first release drugs such as Met and CaO2 nanoparticles into tumor vascular endothelial cells. This process not only achieves the initial positioning of the drug, but also promotes the transcytosis of these active ingredients by endothelial cells, thereby effectively delivering the drug and nanoparticles to the core area of ​​the tumor. In tumor vascular endothelial cells, drugs such as Met can activate the AMPK-eNOS signaling pathway, and the Ca generated by partial CaO2 degradation 2+ By activating calmodulin (CaM) activity and 2+ The two pathways bind to the binding sites on the eNOS subunit in the form of a / CaM complex, thereby activating eNOS. The key role of these two pathways is to upregulate the activity of endothelial nitric oxide synthase (eNOS), thereby significantly enhancing the production of NO (nitric oxide). 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] The present invention utilizes the transcytosis of nano drugs in tumor endothelial cells, that is, except for a part of the lipid nano drug delivery system taken up by vascular endothelial cells that will be degraded by endothelial cells, most of the lipid nano drug delivery system will enter the tumor area through the transcytosis of vascular endothelial cells and be taken up by tumor cells. In tumor cells, the lipid nano drug delivery system releases drugs such as Met and CaO2 nanoparticles, and the CaO2 nanoparticles are degraded in the acidic tumor cell microenvironment to produce a large amount of Ca 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) and activate the body's anti-tumor immune response. In addition, drugs such as Met also reduce the production of immunosuppressive metabolites kynurenine by inhibiting the uptake of tryptophan by tumor cells, thereby alleviating the immunosuppressive state of TME and further enhancing the anti-tumor immune response.

[0013] In summary, the lipid nano-drug delivery system of the present invention not only directly kills tumor cells through precise delivery, local activation and synergistic effects of multiple mechanisms, but also provides a new strategy and idea for tumor treatment by improving the tumor microenvironment and enhancing the immune response.

[0014] According to some embodiments of the present invention, the raw materials for preparing the anionic liposomes also include 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol (Cholesterol) and 1,2-distearoyl-sn-glycero-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-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-sodium phosphate, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) in the raw materials for preparing the anionic liposome is 1:0.8-1:0.1-0.4:0.2-0.5. 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-glycero-3-phosphocholine (DOPC), 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 to 90 nm, such as 83.08 ± 0.73 nm, and the particle size of the calcium superoxide nanoparticles is controlled 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 nano 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 CaO2 nanoparticles comprises the following steps:

[0020] A soluble calcium salt, a dispersant and an alcohol solvent are mixed to obtain a precursor solution; H2O2 solution is firstly added dropwise to the precursor solution, and then a precipitant is added to react to obtain the 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 invention, the dispersant includes at least one of 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 in a mass ratio of 1:2 to 4, such as 1:3.

[0024] According to some embodiments of the present invention, the precipitant comprises 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 hydrates thereof.

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

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

[0028] According to some embodiments of the present invention, the Ca in the precursor solution 2+ The molar ratio to 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 dripping 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 CaO2 nanoparticles further comprises a stirring step before adding a 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 CaO2 nanoparticles further comprises the step of stirring after adding the 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 CaO2 nanoparticles further includes a post-processing step, specifically including the steps of performing solid-liquid separation on the product, collecting the solid phase, and washing after the reaction is completed.

[0036] According to some embodiments of the present invention, the solid-liquid separation is specifically centrifugal treatment, and the rotation speed of the centrifugal treatment is 14000 rpm to 16000 rpm, and the time is 10 to 20 minutes, such as the rotation speed is 15000 rpm, and the time is 15 minutes.

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

[0038] The thin film dispersion method is used to prepare the raw materials of anionic liposomes, drugs and CaO2 nanoparticles into a lipid nano drug delivery system.

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

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

[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°C, such as 25°C.

[0043] According to some embodiments of the present invention, the water bath temperature in 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 method for preparing the lipid nano drug delivery system further comprises the following step: removing unencapsulated CaO2 nanoparticles and unencapsulated drugs.

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

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

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

[0049] According to some embodiments of the present invention, the ultrafiltration method specifically comprises the following operations: transferring the remaining mixture after removing the unencapsulated CaO2 nanoparticles to an ultrafiltration tube, and centrifuging at a speed of 4000 to 5000 rpm for 20 to 50 minutes, such as a speed of 4500 rpm for 30 minutes.

[0050] The application according to the third aspect of the present invention is specifically the application of the above-mentioned lipid nano drug delivery system in the preparation of anti-tumor drugs.

[0051] The application of the embodiment of the present invention has at least the following beneficial effects: the anti-tumor drug containing the lipid nano drug delivery system of the present invention can cleverly utilize the membrane fusion of liposomes to first partially release the active ingredients of the drug (including Met, etc.) and CaO2 to the vascular endothelial cells, and then the endothelial cells transcytose the remaining lipid nano drug delivery system to the tumor area. The drugs released in the vascular endothelial cells and the part of CaO2 produced 2+ It can mediate eNOS activation, thereby enhancing endothelial cell-derived NO production and forming a NO concentration gradient around tumor blood vessels to achieve tumor vascular normalization, reverse the tumor immunosuppressive microenvironment, and enhance the therapeutic effect of tumor calcium overload.

[0052] According to some embodiments of the present invention, the anti-tumor drug utilizes the transcytosis of tumor vascular endothelial cells through Met and Ca 2+Activate endothelial cell eNOS and improve the therapeutic effect of tumor vascular normalization. Improve tumor vascular normalization to further enhance calcium overload tumor therapy. The anti-tumor strategy of the anti-tumor drug is to promote tumor vascular normalization and cooperate with calcium overload therapy to enhance oxidative stress damage of tumor cells and weaken their ability to escape immunity.

[0053] The present invention also provides an anti-tumor drug, which comprises the above-mentioned lipid nano drug delivery system.

[0054] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[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 systems prepared in the embodiments of the present invention;

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

[0058] Figure 3 The Zeta potential diagram of the CaO2 nanoparticles and lipid nano-drug delivery system prepared in the embodiment of the present invention;

[0059] Figure 4 The Ca content of the lipid nano drug delivery system prepared in the embodiment of the present invention under different pH conditions 2+ Release results;

[0060] Figure 5 The results of the changes in the phosphorylated protein expression levels of eNOS in the HUVEC cells after being treated with different drugs in the test example of the present invention;

[0061] Figure 6 The results are the Cy5 content in fresh serum-free 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 example of the present invention;

[0062] Figure 7 The scratch marks A and the related quantitative results B of HUVEC cells treated with different drugs in the test example of the present invention;

[0063] Figure 8 This is a laser confocal microscope observation diagram of calcium content in 4T1 cells after being treated with different drugs in the test example of the present invention;

[0064] Fig. 9 The results are the change of tryptophan levels in the culture medium of 4T1 cells after being treated with different drugs in the test example of the present invention. DETAILED DESCRIPTION

[0065] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can be reagents and materials obtained from commercial channels. Unless otherwise specified, the same parameter in each embodiment has the same value. The embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limitations on the present invention.

[0066] In the description of the present invention, the description with reference to the term "some embodiments" or the like means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0067] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0068] Example

[0069] This example provides a lipid nano 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 in the anionic liposomes. The surface of the CaO2 nanoparticles has a positive charge, and the raw materials for preparing the anionic liposomes are composed of DOPA, DOPC, cholesterol and DSPE-PEG2000.

[0070] The preparation of CaO2 nanoparticles, the specific steps are:

[0071] (1) 200 mg of CaCl2·2H2O and 600 mg of PVP were dissolved in 60 mL of methanol under water bath sonication, followed by the addition of 0.4 mL of H2O2 dropwise and magnetic stirring for 30 min;

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

[0073] (3) The product was transferred to a 50 mL centrifuge tube and centrifuged at 15,000 rpm for 15 min. The supernatant was discarded and the precipitate was washed three times with 20 mL of methanol to obtain CaO2 nanoparticles, which were dispersed in anhydrous ethanol for storage.

[0074] The preparation of lipid nano drug delivery system, the specific steps are:

[0075] (1) Dissolve the raw materials of anionic liposomes (3.93 mg DOPC, 3.62 mg DOPA, 0.965 mg cholesterol and 1.405 mg DSPE-PEG2000) and 5.2 mg Met in 7 mL chloroform, add them into an eggplant-shaped bottle together with 1 mL of CaO2 nanoparticles (5.6 mg) ethanol solution, and disperse them by ultrasound in a water bath for 2 min;

[0076] (2) using a thin film dispersion method, the uniformly dispersed solution in the eggplant-shaped bottle was evaporated by a rotary evaporator for 35 min to prepare anionic liposomes;

[0077] (3) Take out the eggplant-shaped bottle with successful film formation, add 2 mL of ultrapure water into it, and thoroughly hydrate it by water bath sonication for 5 minutes. Then transfer the product to a 2 mL EP tube;

[0078] (4) Centrifuge at 1000 rpm for 5 minutes, collect the upper solution and transfer it to an ultrafiltration tube, centrifuge at 4500 rpm for 30 minutes, and collect the concentrated product, which is the obtained lipid nano-drug delivery system (denoted as MC@L).

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

[0080] Comparative Example 1

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

[0082] Comparative Example 2

[0083] This example provides a lipid nano drug delivery system, which is prepared from CaO2 nanoparticles and anionic liposomes (denoted as C@L). The preparation process is consistent with the example, 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 examples and control examples.

[0086] (1) In vitro Ca2+ expression of lipid nanoparticle drug delivery system 2+ Release condition test

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

[0088] like Figure 4 As shown in Figure 2, the Ca content of MC@L decreases with decreasing pH. 2+ The release rate was significantly accelerated, and the Ca release rate at pH 5.5 was significantly increased. 2 + The total release was the highest at pH 6.5 and pH 7.4, respectively, because the acidic conditions favored the degradation of CaO2 nanoparticles into Ca 2+ and H2O2.

[0089] (2) Phosphorylation protein expression level test of eNOS in HUVEC cells

[0090] HUVEC cells were inoculated into six-well plates, and fresh complete medium (DMEM medium) was replaced after 24 hours of culture. M@L (liposomes encapsulating only Met), C@L (liposomes encapsulating only CaO2) and MC@L were added to the plates for drug treatment, and a blank group without drug treatment was set as a blank control (Control). The cells were then transferred to a carbon dioxide incubator and cultured for 48 hours. After removing the old medium, the cells were washed once with PBS, and the protein was extracted and Western blotting was used to detect the phosphorylated protein expression level of eNOS in each group. The test results are shown in Figure 2. Figure 5 shown.

[0091] Depend on Figure 5 It can be seen that both the Met group (M@L) and the CaO2 nanoparticle group (C@L) can increase the phosphorylated protein expression level of eNOS in HUVEC cells (eNOS-P Ser1177 ), which are respectively composed of Met and Ca 2+ Met increases the phosphorylation and activity of eNOS by activating protein kinase (AMPK), further promoting eNOS-mediated NO production. 2+ Therefore, the small amount of Ca produced by the degradation of CaO2 nanoparticles 2+ It can also regulate eNOS and enhance NO production.

[0092] (3) HUVEC cell transcytosis experiment

[0093] HUVEC cells were seeded in 6-well plates, and after 24 hours of culture, fresh complete medium (DMEM medium) was replaced, and liposomes (Cy5-C@L) loaded with Cy5 (Cyanine 5) and CaO2 nanoparticles were added. The cells were then transferred to a carbon dioxide incubator and cultured for another 6 hours. The old medium was removed and replaced with fresh serum-free medium, and the supernatant of the medium was collected at different time points to detect the Cy5 content. The experimental results are shown in Figure 2. Figure 6 shown.

[0094] Depend on Figure 6 It can be seen that when the drug-containing culture medium was discarded and replaced with fresh serum-free culture medium, the Cy5 content in the culture medium was extremely low at 0h, and as the incubation time increased, the Cy5 content in the culture medium gradually increased, indicating that the HUVEC cells exocytized the taken-in drugs out of the culture medium, proving the transcytosis of the lipid nano-drug delivery system in vascular endothelial cells.

[0095] (4) HUVEC cell scratch assay

[0096] HUVEC cells were inoculated into six-well plates. After culturing for 24 hours, the monolayer of cells was scratched evenly and straightly with a sterile yellow pipette tip, washed once with PBS, and after removing the floating cell matter, the drug-containing culture medium was added. The scratch photos were taken with a microscope at different time points. Figure 7 shown.

[0097] Depend on Figure 7 The results of the cell scratch experiment in A show that the Met group (M@L) and the CaO2 nanoparticle group (C@L) can inhibit the migration of HUVEC cells to varying degrees, proving that their angiogenesis ability is inhibited.

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

[0099] 4T1 cells were inoculated into a laser confocal dish, and after culturing for 24 hours, fresh complete medium (DMEM medium) was replaced, and M@L, C@L and MC@L were added for drug treatment, and a blank group without drug treatment was set as a control. The cells were then transferred to a carbon dioxide incubator and continued to be cultured for 24 hours. The old medium was removed, and after washing once with PBS, a PBS solution containing Fluo-4AM (5μM) was added, incubated at 37°C for 30 minutes, washed once with PBS, and continued to be incubated at 37°C for 20 minutes. Finally, the cells were observed by laser confocal microscopy. The observation results are as follows Figure 8 shown.

[0100] Depend on Figure 8 As shown, the Met-containing group (M@L) and the CaO2 nanoparticle-containing group (C@L) could inhibit HUVEC cell migration to different degrees, among which the scratch healing degree of the MC@L group was the smallest, proving that its angiogenesis ability was inhibited to the greatest extent.

[0101] (6) 4T1 cell tryptophan uptake detection experiment

[0102] 4T1 cells were inoculated into six-well plates, and fresh complete medium (DMEM medium) was replaced after 24 hours of culture. M@L, C@L and MC@L were added for drug treatment, and a blank group without drug treatment was set as a control. The cells were then transferred to a carbon dioxide incubator and cultured for another 24 hours. The old medium was collected into an EP tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was taken and its tryptophan content was detected by ELISA. The experimental results are shown in Figure 2. Fig. 9 shown.

[0103] Depend on Fig. 9It can be seen that the residual extracellular tryptophan in the Met-containing group (M@L) is significantly higher than that in the control group, proving that Met treatment can effectively inhibit tumor cell uptake of tryptophan. This is because Met downregulates MYC, leading to a decrease in the tryptophan transporter SLC7A5, thereby inhibiting the uptake of tryptophan by 4T1 tumor cells. Since CaO2 nanoparticles can cause calcium overload in tumor cells, causing oxidative stress damage or death in tumor cells, the tryptophan uptake in the C@L group also decreased to a certain extent.

[0104] In summary, the lipid nano drug delivery system of the embodiment of the present invention is prepared by co-loading Met and CaO2 nanoparticles in anionic liposomes, wherein the raw materials for anionic liposomes include DOPC, DOPA, cholesterol and DSPE-PEG2000. By introducing the anionic lipid DOPA, utilizing the electrostatic interaction between it and the positively charged CaO2 nanoparticles, and controlling the particle size of the CaO2 nanoparticles within a smaller range, the stability of the liposomes can be enhanced, which is beneficial to drug storage and circulation in the body. The lipid nano drug delivery system cleverly uses the membrane fusion of liposomes to first release Met and CaO2 nanoparticles to vascular endothelial cells, and then the endothelial cells transcytose Met and CaO2 nanoparticles to the tumor area. In tumor vascular endothelial cells, Met-mediated eNOS activation can enhance endothelial cell-derived NO production, thereby forming a NO concentration gradient around tumor blood vessels to achieve tumor vascular normalization and alleviate the hypoxic microenvironment; with the help of endothelial cell transcytosis and endothelial gap-mediated extravasation, Met, CaO2 nanoparticles and other lipid nanodrug delivery systems can be enriched in tumor cells and achieve calcium overload, inducing tumor cell oxidative stress damage and immunogenic cell death. In addition, Met can also inhibit tumor cell uptake of tryptophan, thereby reducing the production of immunosuppressive kynurenine, reversing the immunosuppressive microenvironment, and improving the tumor treatment effect. It has good application prospects in the field of tumor treatment.

[0105] The embodiments of the present invention are described in detail above, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A lipid nano drug delivery system, characterized in that: The invention comprises a drug, CaO2 nanoparticles and anionic liposomes, wherein the drug and the CaO2 nanoparticles are co-loaded in the anionic liposomes; the drug comprises metformin, the surface of the CaO2 nanoparticles carries a positive charge, and the raw materials for preparing the anionic liposomes contain 1,2-dioleoyl-sn-glycerol-3-sodium phosphate.

2. The lipid nano drug delivery system according to claim 1, characterized in that: The raw materials for preparing the anionic liposome also include 1,2-dioleoyl-sn-glycero-3-phosphocholine, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000).

3. The lipid nano drug delivery system according to claim 2, characterized in that: The mass ratio of 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-sodium phosphate, cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(methoxy(polyethylene glycol)-2000) in the raw materials for preparing the anionic liposome is 1:0.8-1:0.1-0.4:0.2-0.

5.

4. The lipid nano drug delivery system according to claim 2, characterized in that: The mass ratio of the 1,2-dioleoyl-sn-glycero-3-phosphocholine, the drug, and the CaO2 nanoparticles is 1:1.2-1.4:1.3-1.

5.

5. The lipid nanoparticle drug delivery system according to any one of claims 1 to 4, characterized in that: The average particle size of the CaO2 nanoparticles is 80 to 90 nm; and / or the average particle size of the lipid nano drug delivery system is 100 to 120 nm.

6. The lipid nanoparticle drug delivery system according to any one of claims 1 to 4, characterized in that: The preparation method of the CaO2 nanoparticles comprises 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, and then a precipitant is added and reacted to obtain a product, wherein the dispersant includes at least one of polyvinyl pyrrolidone, sodium citrate, hexadecyltrimethylammonium bromide, PEG 200 and PEG 400.

7. The method for preparing the lipid nano drug delivery system according to any one of claims 1 to 6, characterized in that: The steps include: The thin film dispersion method is used to prepare the raw materials of anionic liposomes, drugs and CaO2 nanoparticles into a lipid nano drug delivery system.

8. Use of the lipid nano drug delivery system according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The anti-tumor drug utilizes endothelial cell transcytosis to induce the release of metformin and Ca 2+ Drugs that activate nitric oxide synthase in vascular endothelial cells and improve tumor vascular abnormalities.

10. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the lipid nano drug delivery system according to any one of claims 1 to 6.

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