Liposome wrapping L-arginine / Prussian blue nanoparticles and preparation method and application thereof

Abstract

The invention provides lipidosome wrapping L-arginine / Prussian blue nano particles and a preparation method and application thereof, and belongs to the technical field of biological agents. L-arginine is deposited on hPB through iron chelation, then the lipidosome is coated to avoid leakage of water-soluble L-arginine in circulation, and thus the L-arginine / Prussian blue nano particles are obtained. The liposome coated with the L-arginine / Prussian blue nano particles is obtained. Due to the EPR effect, the hPLL-coated Lipo can be efficiently transferred and distributed into tumor tissues, heat is generated under the near-infrared light irradiation condition to ablate a large number of tumor cells, the utilization degree of the tumor cells to L-arginine is greatly limited, meanwhile, immunogenic death is induced, DAMPS is released in situ, infiltration and activation of anti-tumor T cells are further promoted, and the anti-tumor effect is achieved. The tumor immunotherapy is enhanced. And under illumination, the liposome disintegrates and releases the L-arginine, so that nutritional support is provided for immunological competence cells in competitive disadvantage, and activation of an immune microenvironment is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of biological preparations, and in particular to a liposome encapsulating L-arginine / Prussian blue nanoparticles, a preparation method and application thereof. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors in clinical practice. Even though surgical intervention and chemotherapy are relatively mature, its prognosis is still not ideal, and other methods need to be found to improve the efficiency of colorectal cancer treatment. L-arginine plays a key role in metabolism and immune response, and plays a bridging role in the synthesis of various metabolites necessary for cell survival and proliferation, including nitric oxide, polyamines, amino acids, and pyrimidines. However, due to abnormal blood perfusion and material transport disorders caused by vascular malformations in tumor tissues, L-arginine supply is insufficient; rapidly proliferating CT26 colon cancer cells lowly express ASS1 (an enzyme for self-synthesis of L-arginine), and M2 macrophages highly express arginase 1 (Arginase 1, ARG1, which metabolizes L-arginine into urea and L-ornithine). Both of them take up a large amount of L-arginine from TME, resulting in L-arginine depletion and promoting tumor growth and metastasis. Under the condition of L-arginine deficiency, T cells at a competitive disadvantage show reduced expression of CD3ζ chain, reduced cytokine production, and cell cycle arrest; M1 macrophages lack L-arginine as a substrate for synthesizing NO, and their tumor killing function is impaired. Therefore, L-arginine supplementation therapy helps activate immune cells in the tumor microenvironment, restores T cell function, promotes the production of central memory cells, improves the tumor killing function of M1 macrophages, and prolongs the survival time of tumor-bearing mice.

[0003] There are literatures that supplement L-arginine by oral administration, intraperitoneal injection, intratumoral injection, etc. to treat colorectal cancer. However, oral supplementation of L-arginine may lead to strong gastrointestinal reactions; intraperitoneal injection of L-arginine solution may have a low efficiency in delivering L-arginine to the tumor site due to poor selectivity; intratumoral injection of L-arginine solution is easier to diffuse outside the tumor tissue and has a shorter retention time in the tumor. Therefore, nanosystems can be used to solve the problems of poor targeting and low bioavailability of L-arginine. Although supplementing L-arginine can activate the immune microenvironment and promote the function of immune cells, it will also provide nutritional support to tumor cells with competitive advantages, which has potential treatment risks. Therefore, it is particularly important to limit the utilization of L-arginine by tumor cells.

[0004] In addition, platinum drugs are the first-line chemotherapy drugs for the clinical treatment of various tumors, which lack tumor targeting. During chemotherapy, patients usually have to endure severe adverse reactions, including drug-induced nephrotoxicity, ototoxicity and neurotoxicity. And the commonly used nanoparticles in tumor photothermal therapy include gold nanorods, graphene oxide, etc. As nanocarriers, gold nanorods can convert light energy into heat energy under the irradiation of near-infrared light to kill cancer cells. However, gold nanorods also have certain limitations in their applications. For example, the biocompatibility and toxicity of gold nanorods are issues that need attention. Gold nanorods with a size of less than 5nm can be quickly excreted through the urinary system, while larger gold nanorods may accumulate in the body, causing potential toxicity. In addition, the production cost of gold nanorods is relatively high, which limits their promotion and application in the market, and the photothermal cycle stability of gold nanorods is poor. Photothermal therapy based on graphene oxide also has certain limitations. For example, under low-power NIR irradiation, the photothermal conversion efficiency needs to be further improved; although graphene oxide can be used as a drug carrier, in practical applications, how to improve drug delivery efficiency and accelerate drug release in tumor cells is still a challenge. Summary of the invention

[0005] The purpose of the present invention is to provide a liposome encapsulating L-arginine / Prussian blue nanoparticles and its preparation method and application. The liposome encapsulating L-arginine / Prussian blue nanoparticles prepared by the method provided by the present invention (abbreviated as hPFL@Lipo) can be efficiently delivered and distributed to tumor tissues, and a large number of tumor cells are ablated by heat under near-infrared light irradiation conditions, which greatly limits the utilization of L-arginine by tumor cells, and at the same time induces immunogenic death, releases DAMPS in situ, further promotes anti-tumor T cell infiltration and activation, and enhances tumor immunotherapy. And under light irradiation, the liposome disintegrates and releases L-arginine, providing nutritional support for immune-active cells at a competitive disadvantage, and promoting the activation of the immune microenvironment.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing liposomes encapsulating L-arginine / Prussian blue nanoparticles, comprising the following steps:

[0008] (1) mixing Prussian blue nanocubes, PVP and hydrochloric acid, and etching them in a high pressure reactor to obtain hollow Prussian blue nanoparticles;

[0009] (2) after mixing the hollow Prussian blue nanoparticles obtained in step (1) and an aqueous solution of L-arginine, under heating and stirring conditions, adding dropwise an ethanol solution of FeCl3·6H2O, thereby depositing L-arginine on the Prussian blue nanoparticles through iron chelation, to obtain Prussian blue nanoparticles depositing L-arginine through iron chelation, referred to as hPFLNPs;

[0010] (3) mixing the hPFLNPs obtained in step (2) with ethanol, adding a solution containing DOPA, and sequentially performing ultrasound, a first centrifugation, and redissolution to obtain a pretreated hPFLNPs solution;

[0011] (4) The pretreated hPFLNPs solution obtained in step (3) is mixed with a chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and pegylated-distearoylphosphatidylethanolamine, followed by first stirring and distillation, and then ultrapure water is added and shaken for hydration to obtain liposomes encapsulating L-arginine / Prussian blue nanoparticles.

[0012] Preferably, the method for preparing Prussian blue nanocubes in step (1) comprises the following steps:

[0013] (S1) mixing K3[Fe(CN)6], PVP, hydrochloric acid and ultrapure water to obtain a mixed solution; sequentially performing a second stirring, heating and a second centrifugation on the mixed solution to obtain nano seeds;

[0014] (S2) Using the nano seeds obtained in step (S1) as raw materials, repeating the operation in step (S1) to obtain Prussian blue nanocubes.

[0015] Preferably, in the step (S1), the mass ratio of K3[Fe(CN)6] to PVP is 1.1:(22-28).

[0016] Preferably, the heating temperature in the step (S1) is 70 to 90° C., and the heating time is 18 to 24 hours.

[0017] Preferably, in step (1), the mass ratio of Prussian blue nanocubes to PVP is 1:(3-8).

[0018] Preferably, the temperature of the etching treatment in step (1) is 120-160° C., and the time of the etching treatment is 2-6 hours.

[0019] Preferably, in step (2), the ratio of the mass of hollow Prussian blue nanoparticles in the solution containing hollow Prussian blue nanoparticles, the mass of L-arginine in the aqueous solution of L-arginine, and the mass of FeCl3 in the ethanol solution of FeCl3 is (1-2):(8-25):(8-25).

[0020] Preferably, in the chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine in step (4), the molar ratio of dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine is (2-8):(2-8):(0.5-4).

[0021] The present invention also provides liposomes encapsulating L-arginine / Prussian blue nanoparticles prepared by the preparation method described in the above technical solution.

[0022] The present invention also provides the use of the liposome encapsulating the L-arginine / Prussian blue nanoparticles described in the above technical solution in anti-tumor drugs.

[0023] The present invention provides a method for preparing liposomes encapsulating L-arginine / Prussian blue nanoparticles, constructing an integrated nanosystem to destroy the metabolic competitive advantage between tumor cells and immune cells, greatly limit the uptake of L-arginine by tumor cells, and improve the activity of anti-tumor immune response. Specifically, the present invention deposits L-arginine on hPB through iron chelation to obtain hPFLNPs, and then encapsulates liposomes to avoid leakage of water-soluble L-arginine in the circulation, and finally obtains liposomes encapsulating L-arginine / Prussian blue nanoparticles, namely hPFL@Lipo. Due to the EPR effect, hPFL@Lipo in the present invention can be efficiently delivered and distributed to tumor tissues, generate heat and ablate a large number of tumor cells under near-infrared light irradiation conditions, greatly limit the utilization of L-arginine by tumor cells, and at the same time induce immunogenic death, release DAMPS in situ, further promote anti-tumor T cell infiltration and activation, and enhance tumor immunotherapy. Furthermore, under light, the liposomes disintegrate to release L-arginine, providing nutritional support for immune active cells that are at a competitive disadvantage, and promoting the activation of the immune microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the process for preparing hPFL@Lipo dispersion according to the present invention;

[0025] Figure 2 This is a high-resolution X-ray photoelectron spectroscopy (XPS) diagram of hPB and hPFL prepared in Example 1 of the present invention, wherein: Figure 2 The upper figure in a is the N1s XPS spectrum, and the upper figure in a is hPFL, and the lower figure in a is hPB. Figure 2 b is the Fe 2p XPS spectrum, and the upper picture in b is hPFL, and the lower picture in b is hPB, Figure 2 c is the C1s XPS spectrum, and the upper figure in c is hPFL, and the lower figure in c is hPB;

[0026] Figure 3 Fourier transform infrared spectra of hPB and hPFL prepared in Example 1 of the present invention;

[0027] Figure 4 This is a thermogravimetric analysis (TGA) curve of hPB and hPFL prepared in Example 1 of the present invention at a heating rate of 10°C / min, wherein: Figure 4 a is the curve of mass changing with temperature, Figure 4 b is the UV-visible absorption spectrum, and the inset in b is the actual picture of hPB and hPFL, with hPB on the left and hPFL on the right;

[0028] Figure 5 The scanning electron microscope (SEM) images of PB, hPB and hPFL prepared in Example 1 of the present invention are shown in FIG. Figure 5 a, b, and c are PB, hPB, and hPFL, respectively;

[0029] Figure 6 This is a transmission electron microscope (TEM) image of the hPFL and hPFL@Lipo dispersions prepared in Example 1 of the present invention, wherein: Figure 6 a and b are hPFL and hPFL@Lipo dispersions, respectively;

[0030] Figure 7 The zeta potential (n=3) diagram of hPB, hPF, hPFL and hPFL@Lipo prepared in Example 1 of the present invention, the hydrodynamic size distribution diagram of hPB, hPFL and hPFL@Lipo in Example 1, and the size change diagram of hPFL@Lipo prepared in Example 1 after being immersed in RPMI1640 medium for 48 hours, wherein, Figure 7 a in the figure is the Zeta potential (n=3) diagram, Figure 7 b is the hydrodynamic size distribution diagram, Figure 7 In the figure c is the size change diagram;

[0031] Figure 8 The hPFL@Lipo prepared in Example 1 of the present invention releases Fe without near-infrared light irradiation and with near-infrared light irradiation (808 nm laser irradiation). 3+ The release curves of (m) and L-Arg (n), wherein Figure 8 a in the formula is Fe 3+ (m), Figure 8 Wherein b is L-Arg(n);

[0032] Fig. 9The figure is a flowchart of anti-tumor treatment of the PBS group, hPFL@Lipo group and hPFL@Lipo+L group in the tumor-bearing mouse model, a tumor volume growth curve in the tumor-bearing mouse model and a statistical graph of mouse survival rate in the tumor-bearing mouse model, wherein: Fig. 9 Figure a is a flowchart of anti-tumor treatment. Fig. 9 b is the tumor volume growth curve. Fig. 9 Middle c is a statistical chart of mouse survival rate;

[0033] Fig.10 H&E staining images of the heart, liver, spleen, lung and kidney cells of tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group of the present invention after 7 days of treatment;

[0034] Fig.11 The flow cytometry results of CD8+T cells infiltrating immune cells in tumor tissues of tumor-bearing mice in the PBS group, hPFL@Lipo group, and hPFL@Lipo+L group of the present invention are shown, wherein: Fig.11 Figure a is a flow cytometry graph of tumor-infiltrating CD8+T cells. Fig.11 Middle b is a statistical chart of CD8+T cells;

[0035] Fig.12 The flow cytometry results of IFN-γ+T cells infiltrating immune cells in tumor tissues of tumor-bearing mice in the PBS group, hPFL@Lipo group, and hPFL@Lipo+L group of the present invention are shown, wherein: Fig.12 Middle a is the flow cytometry graph of tumor-infiltrating IFN-γ+T cells. Fig.12 Middle b is the statistical graph of IFN-γ+T cells;

[0036] Fig.13 The flow cytometry results of the M1 macrophages infiltrating immune cells in tumor tissues of tumor-bearing mice in the PBS group, hPFL@Lipo group, and hPFL@Lipo+L group of the present invention are shown, wherein: Fig.13 Middle a is the flow cytometry graph of tumor-infiltrating M1 macrophages. Fig.13 Middle b is the cell statistics chart of M1 macrophages. DETAILED DESCRIPTION

[0037] The present invention provides a method for preparing liposomes encapsulating L-arginine / Prussian blue nanoparticles, comprising the following steps:

[0038] (1) Prussian blue nanocubes, PVP and hydrochloric acid are mixed and then etched in a high pressure reactor to obtain hollow Prussian blue nanoparticles, referred to as hPB;

[0039] (2) after mixing the hollow Prussian blue nanoparticles obtained in step (1) and an aqueous solution of L-arginine, adding dropwise an ethanol solution of FeCl3·6H2O under heating and stirring conditions, thereby depositing L-arginine on the Prussian blue nanoparticles through iron chelation to obtain hPFLNPs;

[0040] (3) mixing the hPFLNPs obtained in step (2) with ethanol, adding a solution containing DOPA, and sequentially performing ultrasound, a first centrifugation, and redissolution to obtain a pretreated hPFLNPs solution;

[0041] (4) The pretreated hPFLNPs solution obtained in step (3) is mixed with a chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and pegylated-distearoylphosphatidylethanolamine, followed by first stirring and distillation, and then ultrapure water is added and shaken for hydration to obtain liposomes encapsulating L-arginine / Prussian blue nanoparticles.

[0042] In the present invention, unless otherwise specified, the raw materials used are conventional commercial products in the art.

[0043] The present invention mixes Prussian blue nanocubes, PVP and hydrochloric acid, and then performs etching treatment in a high-pressure reactor to obtain hollow Prussian blue nanoparticles;

[0044] In the present invention, the method for preparing the Prussian blue nanocubes preferably comprises the following steps:

[0045] (S1) mixing K3[Fe(CN)6], PVP, hydrochloric acid and ultrapure water to obtain a mixed solution; sequentially performing a second stirring, heating and a second centrifugation on the mixed solution to obtain nano seeds;

[0046] (S2) Using the nano seeds obtained in step (S1) as raw materials, repeating the operation in step (S1) to obtain Prussian blue nanocubes, also known as PB NPs.

[0047] In the present invention, the mass ratio of K3[Fe(CN)6] to PVP is preferably 1.1:(22-28), more preferably 1.1:(23-26), and further preferably 1.1:24. The present invention utilizes PVP to be adsorbed on the surface of the prepared nanoseeds to form protection and prevent the nanoseeds from flocculating; by regulating the amount of PVP, the morphology and size of the nanoseeds can be controlled. Within a certain concentration range, the greater the amount of PVP, the smaller the size of the prepared nanoseeds.

[0048] In the present invention, the concentration of the hydrochloric acid is preferably 12M; the ratio of the volume of the hydrochloric acid to the mass of K3[Fe(CN)6] is preferably (1-4) mL:1.1 g, and more preferably (1.5-3.5) mL:1.1 g. The present invention uses hydrochloric acid to adjust the pH value of the reaction system, inhibit the hydrolysis of iron ions in K3[Fe(CN)6] and FeCl3, promote the reaction, avoid too high or too low pH resulting in too low reaction yield, or fail to form nano seeds, and obtain target nano seeds with high yield.

[0049] In the present invention, the second stirring time is preferably 20 to 40 minutes. The present invention promotes uniform mixing of the components through the second stirring.

[0050] In the present invention, the heating temperature is preferably 70 to 90° C., more preferably 75 to 85° C.; the heating time is preferably 18 to 24 hours, more preferably 19 to 22 hours. In the present invention, the heating is preferably carried out under stirring. The present invention controls the heating temperature and time within the above range to ensure that the reaction is fully carried out, thereby improving the crystallinity and yield of the prepared Prussian blue nanoseeds.

[0051] In the present invention, the speed of the second centrifugation is preferably 10000-14000 rpm; the time of the second centrifugation is preferably 3-8 min. In the present invention, after the second centrifugation is completed, it is preferred that the product of the second centrifugation is washed with ultrapure water for 2-5 times to obtain nano seeds.

[0052] In the present invention, the average particle size of the nano seeds is preferably 30 to 90 nm, more preferably 50 to 70 nm. The synthesis of the nano seeds in the present invention provides a basis for the subsequent growth of Prussian blue. These nano seeds will serve as cores to promote the growth of Prussian blue crystals, and can further regulate the size and shape of the final Prussian blue crystals. The nucleation process can be controlled to avoid the formation of small particles caused by excessive nucleation, which is crucial for the subsequent growth of Prussian blue particles of uniform size and morphology.

[0053] In the present invention, the average particle size of the Prussian blue nanocubes is preferably 90 to 150 nm, more preferably 110 to 130 nm. The Prussian blue nanocubes in the present invention have good biosafety, a large specific surface area and a high photothermal conversion efficiency. The present invention controls the average particle size of the Prussian blue nanocubes within the above range so that they have a size suitable for systemic circulation, and can be passively targeted to the tumor site through the high permeability and retention effect (EPR) in solid tumors, which is conducive to the precise treatment of tumors; its porous and easy surface functionalization properties give it the function of efficiently binding or loading drug molecules, which helps to overcome the weaknesses of conventional drugs such as poor solubility, insufficient specific targeting ability and large toxic side effects.

[0054] In the present invention, the mass ratio of the Prussian blue nanocube to PVP is preferably 1: (3-8). The present invention controls the mass ratio of the Prussian blue nanocube to PVP within the above range to give full play to the role of PVP as a stabilizer, prevent the Prussian blue nanocube from agglomerating during the etching process, ensure uniform etching of the Prussian blue nanocube, and finally obtain hollow Prussian blue nanoparticles with good dispersibility.

[0055] In the present invention, the concentration of the hydrochloric acid is preferably 1M; the ratio of the volume of the hydrochloric acid to the mass of the Prussian blue nanocube is preferably (15-30) mL:20 mg. The present invention uses hydrochloric acid as an etchant to promote the dissolution of the inside of the Prussian blue nanocube, avoiding excessive use of hydrochloric acid, which will lead to its final complete dissolution, and avoiding too low a use of hydrochloric acid, which will lead to the inability to effectively etch the Prussian blue nanocube.

[0056] In the present invention, the temperature of the etching treatment is preferably 120 to 160° C., more preferably 130 to 150° C., and the time of the etching treatment is preferably 2 to 6 hours, more preferably 3 to 5 hours. The present invention controls the temperature and time of the etching treatment within the above range to promote the acid etching process, avoids the poor etching effect caused by too low temperature or too short time, and avoids the complete dissolution of the Prussian blue nanocube caused by too high temperature or too long time, and obtains hollow Prussian blue nanoparticles with the best shell thickness, which is conducive to the subsequent loading of L-arginine.

[0057] After the etching treatment is completed, the present invention sequentially centrifuges and washes the product of the etching treatment with ultrapure water to obtain hollow Prussian blue nanoparticles.

[0058] In the present invention, the centrifugal speed is preferably 10000-14000 rpm; the centrifugal time is preferably 3-8 min.

[0059] After obtaining the hollow Prussian blue nanoparticles, the present invention mixes the hollow Prussian blue nanoparticles with an aqueous solution of L-arginine, and then drops an ethanol solution of FeCl3·6H2O under heating and stirring conditions, so that L-arginine is deposited on the Prussian blue nanoparticles through iron chelation to obtain hPFL NPs.

[0060] In the present invention, the mass ratio of the hollow Prussian blue nanoparticles, the mass ratio of L-arginine in the aqueous solution of L-arginine, and the mass ratio of FeCl3 in the ethanol solution of FeCl3 is preferably (1-2):(8-25):(8-25), and more preferably 2:25:25. The present invention controls the usage ratio of the three within the above range to ensure that a sufficient amount of L-arginine is deposited on the PB through iron chelation, and to ensure that the loading amount of L-arginine is sufficiently high.

[0061] In the present invention, the temperature of the heating and stirring is preferably 40 to 60° C., more preferably 50° C. The present invention controls the temperature of the heating and stirring within the above range to promote the reaction and ensure the yield, avoid the reaction being slow due to too low a temperature, and avoid the rapid generation of iron oxides due to too high a temperature, resulting in a reduction in the L-arginine loading.

[0062] In the present invention, after the completion of the dropwise addition of the FeCl3 ethanol solution, the process preferably includes: centrifuging and washing the system solution after the dropwise addition of the FeCl3 ethanol solution in sequence to obtain hPFLNPs.

[0063] After obtaining hPFL NPs, the present invention mixes the hPFLNPs with ethanol, then adds a solution containing DOPA, and sequentially performs ultrasound, first centrifugation and redissolution to obtain a pretreated hPFLNPs solution.

[0064] In the present invention, the mass ratio of hPFLNPs to DOPA in the DOPA-containing solution is preferably (10-20):(1-3). The present invention controls the mass ratio of hPFLNPs to DOPA in the DOPA-containing solution within the above range to ensure that sufficient hPFLNPs are combined with DOPA, on the one hand to avoid wasting DOPA, and on the other hand to avoid DOPA forming liposomes by itself and affecting its efficiency of encapsulating hPFL NPs, so as to avoid the formed nanoparticles having many impurities.

[0065] In the present invention, the ultrasonic time is preferably 15 to 30 minutes. In the present invention, the re-dissolution is preferably to dissolve the product of the first centrifugation in chloroform to obtain a pre-treated hPFLNPs solution.

[0066] After obtaining the pretreated hPFLNPs solution, the present invention mixes the pretreated hPFLNPs solution with a chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and pegylated-distearoylphosphatidylethanolamine, and then sequentially performs first stirring and distillation, and then adds ultrapure water, and hydrates by shaking to obtain liposomes encapsulating L-arginine / Prussian blue nanoparticles, also known as hPFL@Lipo dispersion.

[0067] In the present invention, the molar ratio of the mass of the pretreated hPFLNPs in the pretreated hPFLNPs solution to the mass of dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine in the chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine is 40 mg:(2-8) mM:(2-8) mM:(0.5-4) mM. In the present invention, dipalmitoylphosphatidylcholine (DPPC) is a synthetic phospholipid. Compared with natural phospholipids, synthetic phospholipids are not easily oxidized, have a clear phase transition temperature, and are stable in nature and have strong antioxidant properties. The cholesterol is an amphiphilic substance, which can adjust the fluidity of the bilayer, reduce the permeability of the liposome membrane, reduce drug leakage, and at the same time, enable the lipid membrane to maintain a certain flexibility, and enhance the ability of the liposome vesicle to resist changes in external conditions. Therefore, the present invention can prepare stable liposomes by mixing cholesterol with phospholipids. DSPE-PEG (polyethylene glycol distearoylphosphatidylethanolamine) is a PEG-derivatized phospholipid, which forms a hydration film on the surface of the liposome, reduces the combination of the liposome with proteins, enzymes and other components in the plasma, and enhances the stability of the liposome in the blood. At the same time, the PEG chains are staggered on the surface of the liposome to form a soft brush-like conformation, which hinders the adsorption and adhesion of proteins, antibodies, cells and the like, effectively avoids the recognition and phagocytosis of the reticuloendothelial system (RES), thereby prolonging the circulation time of the liposome in the body, and at the same time has good biocompatibility and biodegradability. The present invention improves the stability, biocompatibility, targeting and drug release efficiency of liposomes by adding a certain amount of DPPC, cholesterol and DSPE-PEG at the same time, thereby playing a better effect in the drug delivery system, and the synergistic effect of these raw material components makes the prepared liposomes become an effective drug carrier, which can protect the drug, improve the stability and bioavailability of the drug, and realize the directional release of the drug. And the present invention controls the quality of pretreated hPFLNPs, and the molar ratio of dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine within the above range, which can optimize the drug encapsulation rate and drug loading of the liposomes, thereby affecting the particle size, potential and polydispersity index (PDI), etc. These physicochemical properties are crucial for the in vivo distribution and release behavior of the liposomes, thereby improving the efficiency and effect of drug delivery.

[0068] In the present invention, the distillation method is preferably vacuum rotary evaporation. In the present invention, chloroform is evaporated by vacuum rotary evaporation to form a uniform lipid film.

[0069] In the present invention, the oscillation hydration time is preferably 0.5 to 3 hours, more preferably 1 hour. The present invention uses oscillation hydration to hydrate and detach the lipid film, thereby obtaining a hPFL@Lipo dispersion with good dispersibility and uniform particle size.

[0070] The present invention also provides liposomes encapsulating L-arginine / Prussian blue nanoparticles prepared by the preparation method described in the above technical solution.

[0071] The present invention also provides the use of the liposome encapsulating the L-arginine / Prussian blue nanoparticles described in the above technical solution in anti-tumor drugs.

[0072] In the present invention, the anti-tumor drug is preferably an anti-colorectal cancer drug.

[0073] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0074] Example 1

[0075] A method for preparing liposomes encapsulating L-arginine / Prussian blue nanoparticles, comprising the following steps:

[0076] (1) mixing hydrochloric acid (20 mL, 1 M), PB NPs (20 mg) and PVP (100 mg) and sealing them in a Teflon-lined reactor, performing an etching treatment at 140° C. for 4 h, collecting the precipitate by centrifugation (12,000 rpm, 5 min) and washing it twice with ddH2O to obtain hollow Prussian blue nanoparticles, also known as hollow PB NPs (abbreviated as hPB), dissolving the hollow Prussian blue nanoparticles in ddH2O to obtain a solution containing hollow Prussian blue nanoparticles for later use;

[0077] The mass ratio of the Prussian blue nanocubes PB NPs to PVP is 1:4; the volume ratio of the hydrochloric acid to the mass ratio of the Prussian blue nanocubes is 20 mL:20 mg;

[0078] The preparation method of the Prussian blue nanocube PB NPs comprises the following steps:

[0079] (S1) K3[Fe(CN)6] (1.1 g), PVP (24 g), hydrochloric acid solution (12 M, 2.67 mL) and 320 mL ddH2O were mixed in a flask and magnetically stirred for 30 min until the mixture was uniformly mixed to obtain a mixed solution; the mixed solution was placed in an oil bath at 80°C and heated for 20 h, and the mixture was centrifuged for the second time (12,000 rpm, 5 min), and the residue was washed three times with ddH2O to obtain nanoseeds with an average particle size of 60 nm;

[0080] In the step (S1), the mass ratio of K3[Fe(CN)6] to PVP is 1.1:24; the volume ratio of hydrochloric acid to the mass ratio of K3[Fe(CN)6] is 2.67 mL:1.1 g;

[0081] (S2) K3[Fe(CN)6] (1.1 g), PVP (24 g), hydrochloric acid solution (12 M, 0.267 mL), the nanoseeds with an average particle size of 110 nm (80 mg) and 320 mL ddH2O were mixed in a flask, and the operation in step (S1) was repeated to obtain PB NPs with an average particle size of 120 nm;

[0082] (2) In 5 mL of L-Arg solution (100 mg mL -1 ) was dispersed with 40 mg of the solution containing hollow Prussian blue nanoparticles prepared in step (1), stirred at 50°C, and 20 mL of ethanol solution of FeCl3 (25 mg mL -1 ), the precipitate was collected by centrifugation (12,000 rpm, 5 min), and washed twice with ddH2O to obtain Prussian blue nanoparticles depositing L-arginine by iron chelation, referred to as hPFLNPs;

[0083] The mass ratio of the hollow Prussian blue nanoparticles in the solution containing the hollow Prussian blue nanoparticles, the mass ratio of the L-arginine in the aqueous solution of L-arginine, and the mass ratio of the FeCl3 in the ethanol solution of FeCl3·6H2O is 2:25:25;

[0084] (3) 40 mg of the hPFLNPs obtained in step (2) was mixed with 40 mL of ethanol (the concentration of hPFLNPs in the obtained hPFLNPs ethanol solution was 1 mg mL -1 ), then add 2 ml of DOPA chloroform solution (the concentration of DOPA is 2 mg mL -1 ) mixed under ultrasound for 20 min, centrifuged to remove free DOPA, and then the precipitate was redispersed in chloroform to obtain a pretreated hPFLNPs solution;

[0085] (4) mixing the pretreated hPFLNPs solution obtained in step (3) with a chloroform solution containing dipalmitoylphosphatidylcholine (DPPC), cholesterol and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG) in a round-bottom flask, and vigorously stirring overnight, evaporating the chloroform by vacuum rotary evaporation to form a uniform lipid film, and finally adding ddH2O to the lipid film, and hydrating and shedding the lipid film by shaking for 1 hour to obtain liposomes encapsulating L-arginine / Prussian blue nanoparticles with an average particle size of 145 nm, also known as hPFL@Lipo dispersion;

[0086] The molar ratio of dipalmitoylphosphatidylcholine, cholesterol and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG) in the chloroform solution containing dipalmitoylphosphatidylcholine (DPPC), cholesterol and PEGylated distearoylphosphatidylethanolamine (DSPE-PEG) is 4:4:2;

[0087] The molar ratio of the mass of the pretreated hPFLNPs in the pretreated hPFLNPs solution to the dipalmitoylphosphatidylcholine (DPPC), cholesterol and PEGylated-distearoylphosphatidylethanolamine (DSPE-PEG) in the chloroform solution containing dipalmitoylphosphatidylcholine (DPPC), cholesterol and PEGylated-distearoylphosphatidylethanolamine (DSPE-PEG) is 40 mg: 8 mM: 4 mM: 4 mM.

[0088] Figure 1 Schematic diagram of the process for preparing hPFL@Lipo dispersion according to the present invention, as shown in Figure 1 As shown, the present invention prepares hPFL by loading L-Arg on the surface of hollow Prussian blue nanoparticles. In order to prevent the leakage of L-Arg, hPFL is encapsulated in liposomes to obtain hPFL@Lipo.

[0089] Figure 2 This is a high-resolution X-ray photoelectron spectroscopy (XPS) diagram of hPB and hPFL prepared in Example 1 of the present invention, wherein: Figure 2 The upper figure in a is the N1s XPS spectrum, and the upper figure in a is hPFL, and the lower figure in a is hPB. Figure 2 b is the Fe 2p XPS spectrum, and the upper picture in b is hPFL, and the lower picture in b is hPB, Figure 2 Figure c is the C1s XPS spectrum, and the upper figure in c is hPFL, and the lower figure in c is hPB. Figure 2 It can be seen that the N 1s XPS spectrum shows that the Fe-N signal in hPFL is enhanced ( Figure 2 (a) Figure 2 The Fe 2p XPS spectrum in (b) shows two binding energies at 709 and 722 eV, corresponding to the Fe 2p 3 / 2 and Fe 2p 1 / 2 states, respectively, representing Fe in the +3 oxidation state. Figure 2 Panel c shows the carbonyl carbon signals of hPFL compared with hPB.

[0090] Figure 3 The Fourier transform infrared spectra of hPB and hPFL prepared in Example 1 of the present invention are shown in FIG. Figure 3 It can be seen that the hPFL prepared in Example 1 has a wavelength of 1700-1750 cm -1 The nearby infrared absorption band can be assigned to C=O stretching vibration, indicating the successful loading of L-Arg.

[0091] In order to further verify the drug loading capacity of hPFL, the present invention performed thermogravimetric analysis on the hPB and hPFL prepared in Example 1, and obtained the thermogravimetric analysis (TGA) curves of the hPB and hPFL prepared in Example 1 at a heating rate of 10°C / min as shown in the following figure: Figure 4 As shown, Figure 4 a is the curve of mass changing with temperature, Figure 4 b is the UV-visible absorption spectrum, and the inset in b is the actual picture of hPB and hPFL. The left side of the inset is hPB, and the right side of the inset is hPFL. According to the thermogravimetric analysis (TGA) curves of hPB and hPFL, the mass percentage of Fe in hPFL is 17.32% ( Figure 4 In addition, the loading capacity of the formed hPFL was evaluated as 177 μg L-Arg per mg hPB according to the naphthol-diethylhydrazine method. The UV-visible spectrum of hPFL showed a smaller red shift compared to hPB ( Figure 4 b), The inset in b shows the color difference between hPB and hPFL.

[0092] The scanning electron microscope (SEM) images of PB, hPB and hPFL prepared in Example 1 of the present invention are as follows: Figure 5 As shown, Figure 5 a, b and c are PB, hPB and hPFL respectively. The transmission electron microscopy (TEM) images of hPFL and hPFL@Lipo dispersions prepared in Example 1 are shown in FIG. Figure 6 As shown, Figure 6 a and b are hPFL and hPFL@Lipo dispersions, respectively. Figure 5 and Figure 6 It can be seen that the PB, hPB and hPFL prepared in Example 1 all showed a cubic structure with a uniform size distribution of 110, 120, 120 nm ( Figure 5 Medium ac, Figure 6 (a) Figure 6 TEM images of hPFL@Lipo show nanoscale spheres of approximately 145 nm ( Figure 6 (b)

[0093] Figure 7 The Zeta potential (n=3) of hPB, hPF, hPFL and hPFL@Lipo prepared in Example 1 is shown in FIG. Figure 7 The hydrodynamic size distribution of hPB, hPFL and hPFL@Lipo in Example 1 was obtained by dynamic light scattering (DLS). Figure 7b; the size change of hPFL@Lipo prepared in Example 1 after being immersed in RPMI 1640 medium for 48 hours is shown in FIG. Figure 7 As shown in Figure c. The liposomes modified on the hPFL surface resulted in an increase in hydrodynamic size and a change in surface Zeta potential from positive to negative ( Figure 7 Middle a, Figure 7 In addition, hPFL@Lipo dispersions can maintain stability and dispersibility in biological culture medium for 48 hours ( Figure 7 (c).

[0094] The present invention further studies the Fe 3+ hPFL@Lipo prepared in Example 1 releases Fe without near-infrared light irradiation and with near-infrared light irradiation (808 nm laser irradiation) 3+ The release curves of (m) and L-Arg (n) are shown in Figure 8 As shown, Figure 8 a in the formula is Fe 3+ (m), Figure 8 Where b is L-Arg(n), Figure 8 It can be seen that after infrared light irradiation, the Fe 3+ The release of α-HPO increased from 1.89 mg to 14.55 mg, and the release of L-Arg increased from 0.07 mg to 1.28 mg. The release after infrared laser treatment was much higher than that without infrared laser treatment. The above results prove the successful synthesis of hPFL@Lipo in Example 1.

[0095] In order to study the anti-tumor effect of the hPFL@Lipo dispersion prepared by the present invention in a mouse tumor-bearing model, an anti-tumor treatment experiment was performed on the mouse tumor-bearing model according to the following method:

[0096] First, a mouse tumor model was established: 6-week-old female BALB / c mice were anesthetized and inoculated with CT26 tumor cells (100 μL, 10 7 cells / mL) to construct a tumor-bearing mouse model;

[0097] Then when the tumor volume is about 100mm 3 The mice were randomly divided into three groups (n=4): PBS, hPFL@Lipo, and hPFL@Lipo+L. Then, each group of mice was injected with 100 μL of PBS, hPFL@Lipo (1 mg mL -1 ). 24 h later, the mice in the illumination group were anesthetized, and the tumor tissues were exposed to 1.5 W / cm 2The day of nanoparticle injection was recorded as D1, and the longest diameter (L) and shortest diameter (W) of each mouse tumor were measured every 2 days thereafter, and the tumor volume V was calculated according to the following formula 肿瘤 =1 / 2×W 2 × L, using GraphPadPrism software to draw the tumor growth curve. The tumor volume of mice exceeded 2000mm 3 The end point event (death) was recorded, and the survival curve was drawn using GraphPad Prism software. Fig. 9 .

[0098] The flow chart of anti-tumor treatment of PBS prepared in Example 1, hPFL@Lipo prepared in Example 1, and hPFL@Lipo+L prepared in Example 1 in a tumor-bearing mouse model is shown in FIG. Fig. 9 As shown in a, the tumor volume growth curve in the tumor-bearing mouse model is as follows Fig. 9 As shown in b, the statistical graph of mouse survival rate in the tumor-bearing mouse model is as follows Fig. 9 As shown in c. Fig. 9 It can be seen that hPFL@Lipo plus light (i.e., hPFL@Lipo+L group) can inhibit the growth of mouse tumors and prolong the survival of tumor-bearing mice.

[0099] In order to study the biocompatibility of PBS and hPFL@Lipo dispersions prepared in Example 1 of the present invention with tumor-bearing mice, in the above anti-tumor treatment experiment, the heart, liver, spleen, lung and kidney cells of the important organs of the tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group after 7 days of treatment were respectively taken for H&E staining, and the H&E staining images of the heart, liver, spleen, lung and kidney cells of the tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group after 7 days of treatment in the present invention were obtained as shown in the figure. Fig.10 As shown by Fig.10 It can be seen that PBS and hPFL@Lipo in the present invention have no significant damage to important organs and no obvious toxic side effects, which proves that both have good biocompatibility.

[0100] In order to study the effect of hPFL@Lipo prepared in Example 1 of the present invention on the tumor immune microenvironment of mice, in the above anti-tumor treatment experiment, the tumor tissues of tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group were respectively taken, digested into single cell suspensions with digestive enzymes, and red blood cells were removed by red blood cell lysis solution. The surface and intracellular antibody staining of tumor-infiltrating macrophages and T cells was performed with flow cytometry antibodies, and finally flow cytometry detection was performed. The flow cytometry results of tumor tissue infiltrating immune cells CD8+T cells, IFN-γ+T cells and M1 macrophages of tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group of the present invention were obtained, respectively. Figures 11 to 13 As shown, Fig.11 Figure a is a flow cytometry graph of tumor-infiltrating CD8+T cells. Fig.11 Middle b is a statistical chart of CD8+T cells. Fig.12 Middle a is the flow cytometry graph of tumor-infiltrating IFN-γ+T cells. Fig.12 Middle b is the IFN-γ+T cell statistics chart, Fig.13 Middle a is the flow cytometry graph of tumor-infiltrating M1 macrophages. Fig.13 Middle b is the M1 macrophage cell statistics chart, Figures 11 to 13 It can be seen that the hPFL@Lipo combined with near-infrared light in the present invention can increase the infiltration of CD8 + T cells, IFN-γ + T cells, promoting polarization of macrophages toward the M1 phenotype.

[0101] In summary, the hPFL@Lipo dispersion prepared by the method provided by the present invention promotes CD8 + T cells and IFN-γ + The infiltration of T cells in tumors increases the proportion of M1 macrophages, inhibits the growth of tumor cells, and prolongs the survival of tumor-bearing mice. The scheme provided by the present invention provides a paradigm for regulating adverse metabolic competition to promote immunotherapy. The hPFL@Lipo dispersion in the present invention has been shown to have good biocompatibility, the hollow Prussian blue nanoparticles have strong drug loading capacity, high photothermal stability, high photothermal conversion efficiency, low production cost, easy mass production, and simple synthesis process; in animal treatment, after intravenous administration, it can be located to the tumor tissue through the high permeability and retention effect (EPR) effect, and a large number of tumor cells are ablated by heat under near-infrared light irradiation conditions, which greatly limits the utilization of L-arginine by tumor cells, and induces immunogenic death, releases DAMPS in situ, further promotes anti-tumor T cell infiltration and activation, and enhances tumor immunotherapy. And under light, the liposome disintegrates and releases L-arginine, providing nutritional support for immune-active cells at a competitive disadvantage, and promoting the activation of the immune microenvironment.

[0102] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing liposomes encapsulating L-arginine / Prussian blue nanoparticles, characterized in that: The following steps are involved: (1) mixing Prussian blue nanocubes, PVP and hydrochloric acid, and etching them in a high pressure reactor to obtain hollow Prussian blue nanoparticles; (2) after mixing the hollow Prussian blue nanoparticles obtained in step (1) and an aqueous solution of L-arginine, adding dropwise an ethanol solution of FeCl3·6H2O under heating and stirring conditions, thereby depositing L-arginine on the Prussian blue nanoparticles through iron chelation to obtain hPFLNPs; (3) mixing the hPFLNPs obtained in step (2) with ethanol, adding a solution containing DOPA, and sequentially performing ultrasound, a first centrifugation, and redissolution to obtain a pretreated hPFLNPs solution; (4) The pretreated hPFLNPs solution obtained in step (3) is mixed with a chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and pegylated-distearoylphosphatidylethanolamine, followed by first stirring and distillation, and then ultrapure water is added and shaken for hydration to obtain liposomes encapsulating L-arginine / Prussian blue nanoparticles.

2. The preparation method according to claim 1, characterized in that: The method for preparing the Prussian blue nanocubes in step (1) comprises the following steps: (S1) mixing K3[Fe(CN)6], PVP, hydrochloric acid and ultrapure water to obtain a mixed solution; sequentially performing a second stirring, heating and a second centrifugation on the mixed solution to obtain nano seeds; (S2) Using the nano seeds obtained in step (S1) as raw materials, repeating the operation in step (S1) to obtain Prussian blue nanocubes.

3. The preparation method according to claim 2, characterized in that: In the step (S1), the mass ratio of K3[Fe(CN)6] to PVP is 1.1:(22-28).

4. The preparation method according to claim 2, characterized in that: The heating temperature in step (S1) is 70-90°C and the heating time is 18-24h 。 5. The preparation method according to claim 1, characterized in that: In the step (1), the mass ratio of Prussian blue nanocubes to PVP is 1:(3-8).

6. The preparation method according to claim 1, characterized in that: The temperature of the etching treatment in the step (1) is 120 to 160° C., and the time of the etching treatment is 2 to 6 hours.

7. The preparation method according to claim 1, characterized in that: The mass ratio of the hollow Prussian blue nanoparticles in step (2), the mass ratio of L-arginine in the aqueous solution of L-arginine, and the mass ratio of FeCl3 in the ethanol solution of FeCl3 is (1-2): (8-25): (8-25) 。 8. The preparation method according to claim 1, characterized in that: In the chloroform solution containing dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine in step (4), the molar ratio of dipalmitoylphosphatidylcholine, cholesterol and PEGylated-distearoylphosphatidylethanolamine is (2-8):(2-8):(0.5-4).

9. Liposomes encapsulating L-arginine / Prussian blue nanoparticles prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the liposome encapsulating L-arginine / Prussian blue nanoparticles according to claim 9 in anti-tumor drugs.

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