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

Abstract

The present invention provides a liposome encapsulating L-arginine / Prussian blue nanoparticles and its preparation method and application, which belongs to the field of biological preparation technology. The present invention deposits L-arginine on hPB through iron chelation, and then coats liposomes to avoid leakage of water-soluble L-arginine in the circulation, thereby obtaining liposomes encapsulating L-arginine / Prussian blue nanoparticles. Due to the EPR effect, hPFL@Lipo in the present invention can be efficiently delivered and distributed to tumor tissue, 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, while inducing immunogenic death, releasing DAMPS in situ, further promoting anti-tumor T cell infiltration and activation, and enhancing 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.

Description

Technical Field

[0001] The present 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 an application thereof. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors in clinical practice. Even though surgical 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 acts as a bridge in the synthesis of various metabolites necessary for cell survival and proliferation, including nitric oxide, polyamines, amino acids, and pyrimidines. However, due to vascular malformations in tumor tissues leading to abnormal blood perfusion and material transport disorders, the supply of L-arginine is insufficient; rapidly proliferating CT26 colon cancer cells lowly express ASS1 (an enzyme used to self-synthesize L-arginine), while 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 the TME, leading to L-arginine depletion and promoting tumor growth and metastasis. Under conditions of L-arginine deficiency, T cells are at a competitive disadvantage, exhibiting reduced CD3ζ chain expression, decreased cytokine production, and cell cycle arrest. M1 macrophages, lacking L-arginine as a substrate for NO synthesis, are impaired in their tumor-killing function. Therefore, L-arginine supplementation therapy can help activate immune cells in the tumor microenvironment, restore T cell function, promote the generation of central memory cells, enhance the tumor-killing function of M1 macrophages, and prolong the survival of tumor-bearing mice.

[0003] Literature has documented the use of L-arginine supplementation for the treatment of colorectal cancer through oral, intraperitoneal, and intratumoral administration. However, oral L-arginine supplementation may result in significant gastrointestinal side effects; intraperitoneal injection of L-arginine solutions may result in inefficient delivery of L-arginine to the tumor site due to poor selectivity; and intratumoral injection of L-arginine solutions tends to diffuse beyond the tumor tissue and have a shorter retention time within the tumor. Therefore, nanosystems could be used to address the poor targeting and bioavailability of L-arginine. Although L-arginine supplementation can activate the immune microenvironment and promote immune cell function, it can also provide nutritional support to tumor cells that have a competitive advantage, posing potential therapeutic risks. Therefore, limiting L-arginine utilization by tumor cells is particularly important.

[0004] Furthermore, platinum-based drugs are clinically used as first-line chemotherapy drugs for various tumors, but they lack tumor targeting. During chemotherapy, patients often experience severe adverse reactions, including drug-induced nephrotoxicity, ototoxicity, and neurotoxicity. Commonly used nanoparticles in tumor photothermal therapy include gold nanorods and graphene oxide. Gold nanorods, as nanocarriers, can convert light energy into heat energy under near-infrared light irradiation, thereby killing cancer cells. However, gold nanorods also have limitations in their application. For example, the biocompatibility and toxicity of gold nanorods are issues that require attention. Gold nanorods smaller than 5 nm are rapidly excreted through the urinary system, while larger ones may accumulate in the body, causing potential toxicity. Furthermore, the relatively high production cost of gold nanorods has limited their market adoption and application, and they also exhibit poor photothermal cycling stability. Graphene oxide-based photothermal therapy also has limitations. For example, the photothermal conversion efficiency under low-power NIR irradiation needs to be further improved. While graphene oxide can be used as a drug carrier, improving drug delivery efficiency and accelerating drug release within tumor cells remain challenges in practical applications. Summary of the Invention

[0005] The purpose of the present invention is to provide a liposome encapsulating L-arginine / Prussian blue nanoparticles, its preparation method, and application. The liposome encapsulating L-arginine / Prussian blue nanoparticles (abbreviated as hPFL@Lipo) prepared by the method provided by the present invention can be efficiently delivered and distributed to tumor tissues. Under near-infrared light irradiation conditions, it produces heat to ablate a large number of tumor cells, significantly limiting the tumor cells' utilization of L-arginine, while inducing their immunogenic death, releasing DAMPs in situ, further promoting anti-tumor T cell infiltration and activation, and enhancing tumor immunotherapy. Moreover, under light irradiation, the liposomes disintegrate and release L-arginine, providing nutritional support for immune-active cells that are 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) Prussian blue nanocubes, PVP, and hydrochloric acid are mixed and then etched 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, an ethanol solution of FeCl3·6H2O is added dropwise under heating and stirring conditions, thereby depositing L-arginine on the Prussian blue nanoparticles through iron chelation, thereby obtaining Prussian blue nanoparticles deposited with 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 sonication, 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 hydrated by shaking 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; and sequentially performing a second stirring, heating, and a second centrifugation on the mixed solution to obtain nanoseeds;

[0014] (S2) Using the nanoseeds 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 the 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 liposomes 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 disrupt the metabolic competitive advantage between tumor cells and immune cells, significantly limiting the uptake of L-arginine by tumor cells, and improving the activity of anti-tumor immune responses. Specifically, the present invention deposits L-arginine on hPB through iron chelation to obtain hPFLNPs, which are then coated with liposomes to prevent leakage of water-soluble L-arginine in the circulation, ultimately obtaining liposomes encapsulating L-arginine / Prussian blue nanoparticles, namely hPFL@Lipo. Due to the EPR effect, the hPFL@Lipo in the present invention can be efficiently delivered and distributed to tumor tissues, generating heat under near-infrared light irradiation to ablate a large number of tumor cells, significantly limiting the utilization of L-arginine by tumor cells, and at the same time inducing their immunogenic death, releasing DAMPS in situ, further promoting the infiltration and activation of anti-tumor T cells, and enhancing tumor immunotherapy. Furthermore, under light irradiation, the liposomes disintegrate and 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 Schematic diagram of the process for preparing hPFL@Lipo dispersion of 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 image in a is the N1s XPS spectrum, and the lower image in a is the hPFL, and the lower image in a is the hPB. Figure 2 b is the Fe 2p XPS spectrum, and the upper image in b is hPFL, and the lower image in b is hPB, Figure 2 c is the C1s XPS spectrum, and the upper image in c is hPFL, and the lower image 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 This is a scanning electron microscope (SEM) image of PB, hPB, and hPFL prepared in Example 1 of the present invention, wherein Figure 5 a, b, and c are PB, hPB, and hPFL, respectively;

[0029] Figure 6 This is a transmission electron microscopy (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) of hPB, hPF, hPFL, and hPFL@Lipo prepared in Example 1 of the present invention, the hydrodynamic size distribution of hPB, hPFL, and hPFL@Lipo in Example 1, and the size change of hPFL@Lipo prepared in Example 1 after being immersed in RPMI1640 medium for 48 hours are shown. Figure 7 Figure a is the Zeta potential (n=3) diagram, Figure 7 b in the middle 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+ (m) and L-Arg (n) release curves, where Figure 8 a in the equation is Fe 3+ (m), Figure 8 Where b is L-Arg(n);

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

[0033] Figure 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 after 7 days of treatment in the present invention;

[0034] Figure 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: Figure 11 Middle a is the flow cytometry graph of tumor-infiltrating CD8+T cells. Figure 11 Middle b is a statistical graph of CD8+ T cells;

[0035] Figure 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 in the present invention are shown, wherein: Figure 12 Middle a is the flow cytometry graph of tumor-infiltrating IFN-γ+T cells. Figure 12 Middle b is the statistical graph of IFN-γ+T cells;

[0036] Figure 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: Figure 13 Middle a is the flow cytometry graph of tumor-infiltrating M1 macrophages. Figure 13 Middle b is the M1 macrophage cell statistics chart. 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 were 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, an ethanol solution of FeCl3·6H2O is added dropwise under heating and stirring, 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 sonication, 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 hydrated by shaking 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 field.

[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; and sequentially performing a second stirring, heating, and a second centrifugation on the mixed solution to obtain nanoseeds;

[0046] (S2) Using the nanoseeds 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 even more preferably 1.1:24. The present invention utilizes PVP adsorbed on the surface of the prepared nanoseeds to provide protection and prevent nanoseed aggregation. By regulating the amount of PVP, the morphology and size of the nanoseeds can be controlled. Within a certain concentration range, a greater amount of PVP results in smaller nanoseeds.

[0048] In the present invention, the concentration of the hydrochloric acid is preferably 12M; the volume ratio of the hydrochloric acid to the mass of K3[Fe(CN)6] is preferably (1-4) mL:1.1 g, more preferably (1.5-3.5) mL:1.1 g. The present invention utilizes hydrochloric acid to adjust the pH of the reaction system, inhibiting the hydrolysis of iron ions in K3[Fe(CN)6] and FeCl3, promoting the reaction, and avoiding excessively high or low pH values ​​that result in low reaction yields or the inability to form nanoseeds, thereby obtaining a high yield of target nanoseeds.

[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 70-90°C, more preferably 75-85°C; the heating time is preferably 18-24 hours, more preferably 19-22 hours. In the present invention, the heating is preferably performed under stirring. The present invention controls the heating temperature and time within the above ranges to ensure sufficient reaction progress and improve the crystallinity and yield of the prepared Prussian blue nanoseeds.

[0051] In the present invention, the second centrifugation speed is preferably 10000-14000 rpm; the second centrifugation time is preferably 3-8 min. In the present invention, after the second centrifugation is completed, the process preferably further comprises: washing the product of the second centrifugation with ultrapure water 2-5 times to obtain nanoseeds.

[0052] In the present invention, the average particle size of the nanoseeds is preferably 30 to 90 nm, more preferably 50 to 70 nm. The synthesis of the nanoseeds in the present invention provides a foundation for the subsequent growth of Prussian blue. These nanoseeds will serve as cores to promote the growth of Prussian blue crystals, further regulating the size and shape of the final Prussian blue crystals. By controlling the nucleation process, the formation of small particles caused by excessive nucleation can be avoided, 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. They can be passively targeted to the tumor site through the high permeability and retention effect (EPR) in solid tumors, which is beneficial for 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 nanocubes to PVP is preferably 1:(3-8). The present invention controls the mass ratio of the Prussian blue nanocubes to PVP within the above range to fully utilize the function of PVP as a stabilizer, prevent the Prussian blue nanocubes from agglomerating during the etching process, ensure uniform etching of the Prussian blue nanocubes, and ultimately obtain hollow Prussian blue nanoparticles with good dispersion.

[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 nanocubes is preferably (15-30) mL:20 mg. The present invention utilizes hydrochloric acid as an etchant to promote the dissolution of the interior of the Prussian blue nanocubes, avoiding the use of excessive amounts of hydrochloric acid, which would lead to their complete dissolution, and the use of insufficient amounts of hydrochloric acid, which would result in ineffective etching of the Prussian blue nanocubes.

[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 ranges to promote the acid etching process, avoids poor etching effects caused by too low a temperature or too short a time, and avoids complete dissolution of the Prussian blue nanocubes caused by too high a temperature or too long a time, thereby obtaining hollow Prussian blue nanoparticles with an optimal shell thickness, which are conducive to subsequent loading of L-arginine.

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

[0058] In the present invention, the centrifugal speed is preferably 10,000 to 14,000 rpm; and the centrifugal time is preferably 3 to 8 minutes.

[0059] After obtaining hollow Prussian blue nanoparticles, the present invention mixes the hollow Prussian blue nanoparticles with an aqueous solution of L-arginine, and then dropwise adds 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 hPFL NPs.

[0060] In the present invention, the mass ratio of the hollow Prussian blue nanoparticles, the mass of L-arginine in the L-arginine aqueous solution, and the mass ratio of FeCl3 in the FeCl3 ethanol solution 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, ensuring a sufficiently high L-arginine loading.

[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 too low a temperature which will slow the reaction, and avoid too high a temperature which will promote the rapid formation of iron oxides, resulting in a reduced 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 the system solution after the dropwise addition of the FeCl3 ethanol solution and washing with ultrapure water 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 ultrasonication, a 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 sufficient hPFLNPs bind to DOPA, thereby avoiding DOPA waste and preventing DOPA from forming liposomes and affecting its efficiency in encapsulating hPFLNPs. This prevents the formed nanoparticles from being contaminated with impurities.

[0065] In the present invention, the ultrasonication 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 pretreated 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, followed by first stirring and distillation, and then adding ultrapure water and hydrating 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 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 less susceptible to oxidation, have a clear phase transition temperature, and are stable and have strong antioxidant properties. Cholesterol is an amphiphilic substance that can regulate bilayer fluidity, reduce liposome membrane permeability, and reduce drug leakage. It can also maintain a certain degree of flexibility in the lipid membrane and enhance the ability of liposome vesicles to withstand changes in external conditions. Therefore, the present invention can prepare stable liposomes by mixing cholesterol with phospholipids. DSPE-PEG (PEGylated distearoylphosphatidylethanolamine) is a PEG-derivatized phospholipid that forms a hydration film on the liposome surface, reducing the binding of liposomes to proteins, enzymes, and other components in plasma, thereby enhancing the stability of liposomes in blood. At the same time, the PEG chains interlaced on the liposome surface form a soft brush-like conformation, which hinders the adsorption and adhesion of proteins, antibodies, cells, etc., effectively avoiding recognition and phagocytosis by the reticuloendothelial system (RES), thereby extending the circulation time of the liposomes in the body, while also having good biocompatibility and biodegradability. The present invention improves the stability, biocompatibility, targeting and drug release efficiency of liposomes by simultaneously adding a certain amount of DPPC, cholesterol and DSPE-PEG, thereby playing a better effect in the drug delivery system. The synergistic effect of these raw materials makes the prepared liposomes an effective drug carrier, which can protect the drug, improve the stability and bioavailability of the drug, and achieve targeted release of the drug. In addition, 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 efficiency 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 liposomes 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 following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some 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 making creative efforts 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) Hydrochloric acid (20 mL, 1 M), PB NPs (20 mg), and PVP (100 mg) were mixed and sealed in a Teflon-lined reactor, and etched at 140°C for 4 h. The precipitate was collected by centrifugation (12,000 rpm, 5 min) and washed twice with ddH2O to obtain hollow Prussian blue nanoparticles, also known as hollow PB NPs (abbreviated as hPB). The hollow Prussian blue nanoparticles were dissolved in ddH2O to obtain a solution containing hollow Prussian blue nanoparticles, which was set aside;

[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 of ddH2O were mixed in a flask and magnetically stirred for 30 min until uniformly mixed to obtain a mixture. The mixture was heated in an 80°C oil bath for 20 h, centrifuged a second time (12,000 rpm, 5 min), and the residue was rinsed 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 of hydrochloric acid and the mass ratio of K3[Fe(CN)6] are 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 nanoparticles with an average particle size of 110 nm (80 mg) and 320 mL of 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 in 40 mg of the solution containing hollow Prussian blue nanoparticles prepared in step (1), stirred at 50 ° C, and 20 mL of FeCl3 ethanol solution (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 of L-arginine in the aqueous solution of L-arginine, and the mass ratio of FeCl3 in the ethanol solution of FeCl3·6H2O is 2:25:25;

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

[0085] (4) The pretreated hPFLNPs solution obtained in step (3) was mixed with a chloroform solution containing dipalmitoylphosphatidylcholine (DPPC), cholesterol, and polyethylene glycol distearoylphosphatidylethanolamine (DSPE-PEG) and placed in a round-bottom flask. After vigorous stirring overnight, the chloroform was evaporated by vacuum rotary evaporation to form a uniform lipid film. Finally, ddH2O was added to the lipid film, and the lipid film was hydrated and detached 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 of the present invention, as shown in FIG. Figure 1 As shown, the present invention prepared hPFL by loading L-Arg on the surface of hollow Prussian blue nanoparticles. To prevent the leakage of L-Arg, hPFL was 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 image in a is the N1s XPS spectrum, and the lower image in a is the hPFL, and the lower image in a is the hPB. Figure 2 b is the Fe 2p XPS spectrum, and the upper image in b is hPFL, and the lower image 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 -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 conducted thermogravimetric analysis on hPB and hPFL prepared in Example 1, and obtained thermogravimetric analysis (TGA) curves of 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 image 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. Compared with hPB, the UV-visible spectrum of hPFL showed a smaller red shift ( 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 Where 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 and 120 nm ( Figure 5 Middle AC, Figure 6 (a) Figure 6 TEM images of hPFL@Lipo show nanoscale spheres around 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 7As shown in b; the size change of hPFL@Lipo prepared in Example 1 after being immersed in RPMI 1640 culture 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+ The 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 2. Figure 8 As shown, Figure 8 a in the equation 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 amounts after infrared laser treatment were much higher than those 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 in 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-bearing 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 100 mm 3 The mice were randomly divided into 3 groups (n=4): PBS, hPFL@Lipo, and hPFL@Lipo+L. Then, 100 μL of PBS, hPFL@Lipo (1 mg mL -1 After 24 h, 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. 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, tumor growth curve was drawn using GraphPadPrism software. The tumor volume of mice exceeded 2000 mm 3 The endpoint event (death) was recorded, and the survival curve was drawn using GraphPad Prism software. Figure 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 tumor-bearing mouse models is shown in FIG. Figure 9 As shown in a, the tumor volume growth curve in the tumor-bearing mouse model is as follows Figure 9 As shown in b, the statistical graph of mouse survival rate in the tumor-bearing mouse model is as follows Figure 9 As shown in c. Figure 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-mentioned 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. 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 are shown in the figure below. Figure 10 As shown by Figure 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-mentioned anti-tumor treatment experiment, tumor tissues of tumor-bearing mice in the PBS group, hPFL@Lipo group and hPFL@Lipo+L group were respectively obtained, digested into single-cell suspensions with digestive enzymes, and red blood cells were removed by red blood cell lysis solution. Surface and intracellular antibody staining of tumor-infiltrating macrophages and T cells was performed using 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 11-13 As shown, Figure 11 Middle a is the flow cytometry graph of tumor-infiltrating CD8+T cells. Figure 11 Middle b is the CD8+T cell statistics chart, Figure 12 Middle a is the flow cytometry graph of tumor-infiltrating IFN-γ+T cells. Figure 12 Middle b is the IFN-γ+T cell statistics chart, Figure 13 Middle a is the flow cytometry graph of tumor-infiltrating M1 macrophages. Figure 13 Middle b is the M1 macrophage cell statistics chart, Figures 11-13 It can be seen that hPFL@Lipo combined with near-infrared light in the present invention can increase the infiltration of CD8 + T cells, IFN-γ + T cells, promoting macrophage polarization 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 the tumor 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 in 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, while inducing their immunogenic death, releasing DAMPS in situ, further promoting anti-tumor T cell infiltration and activation, and enhancing tumor immunotherapy. And under light irradiation, the liposomes disintegrate and release 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 principles of the present invention. These improvements and modifications should also be regarded as within 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) Prussian blue nanocubes, PVP, and hydrochloric acid were mixed and then etched 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, an ethanol solution of FeCl3·6H2O is added dropwise under heating and stirring conditions, thereby depositing L-arginine on the Prussian blue nanoparticles through iron chelation to obtain hPFL NPs; (3) mixing the hPFL NPs obtained in step (2) with ethanol, adding a solution containing DOPA, and sequentially performing ultrasonic treatment, a first centrifugation, and redissolution to obtain a pretreated hPFL NPs solution; (4) The pretreated hPFL NPs 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 hydrated by shaking 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 nanoseeds; (S2) Using the nanoseeds 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 the step (S1) is 70-90° C., and the heating time is 18-24 hours.

5. The preparation method according to claim 1, characterized in that The mass ratio of Prussian blue nanocubes to PVP in step (1) is 1:(3-8).

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

7. The preparation method according to claim 1, characterized in that In the step (2), the mass ratio of the hollow Prussian blue nanoparticles, the mass of L-arginine in the L-arginine aqueous solution, and the mass ratio of FeCl3 in the FeCl3 ethanol solution 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 the preparation of anti-tumor drugs.

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