Nano-drug for regulating and controlling tumor immune microenvironment, preparation method, composition and application of nano-drug

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a nano-drug for regulating and controlling a tumor immune microenvironment, a preparation method, a composition and application of the nano-drug. The preparation method comprises the following steps: firstly, preparing mesoporous polydopamine nano-particles, modifying folic acid-polyethylene glycol on the surfaces of the mesoporous polydopamine nano-particles, and loading sorafenib and LY3200882 to obtain a nano-drug with an immunoregulation effect, and the nano-drug is used for regulating a tumor immunosuppressive microenvironment. Wherein the folic acid-polyethylene glycol modified nanoparticles can better target tumor tissues, then the photothermal performance of the polydopamine nanoparticles can enable tumor cells to generate immunogenic death and enhance immune response, and temperature rise caused by an acidic microenvironment and a photothermal effect is beneficial to release of sorafenib and LY3200882, so that the tumor cells can be better targeted to the tumor tissues. The sorafenib has the functions of inhibiting tumor cell proliferation, inhibiting tumor angiogenesis and promoting tumor blood vessel normalization, and the LY3200882 exerts the immunoregulation function by inhibiting a TGF-beta1 receptor, so that the tumor immunosuppression microenvironment is reversed together, and the tumor treatment effect is enhanced.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a nano-drug for regulating tumor immune microenvironment, a preparation method, a composition and an application thereof. Background Art

[0002] In recent years, immunotherapy has made remarkable progress in the field of tumor treatment. This therapy utilizes the patient's own immune system to more effectively kill tumor cells by activating immune cells, thereby improving the treatment effect. The tumor microenvironment, as the local ecosystem where tumor cells survive, encompasses various complex components such as tumor cells, neighboring cells, extracellular matrix, cytokines, chemokines, and metabolites. Its high heterogeneity has become a key challenge in immunotherapy. In particular, the inhibitory tumor microenvironment will significantly weaken the clinical benefits of immunotherapy.

[0003] To address this challenge, researchers have explored various strategies to improve the tumor-suppressive microenvironment in immunotherapy. For example, by regulating immune cells in the microenvironment, targeting the VEGF / VEGFR signaling pathway to inhibit tumor angiogenesis, promoting tumor vessel normalization, and enhancing the infiltration of immune cells. However, due to the complexity and diversity of the tumor microenvironment, single-pathway intervention often fails to comprehensively reverse its immunosuppressive properties. Therefore, adopting a multi-pathway combination strategy to improve the tumor immunosuppressive microenvironment is of great clinical significance for enhancing the effect of immunotherapy.

[0004] In this context, photothermal therapy has shown unique advantages. This therapy utilizes the photothermal conversion properties of specific materials to heat tumor cells and cause their death. This process can also trigger immunogenic cell death, prompting tumor cells to release damage-associated molecular patterns, thereby further activating the body's immune response. Combining vascular regulation means with immunomodulatory drugs, photothermal therapy can more effectively improve the tumor immunosuppressive microenvironment, and thus enhance the overall efficacy of immunotherapy. Therefore, combining photothermal therapy with immunomodulatory drugs to jointly regulate the tumor immune microenvironment has become a highly potential strategy aimed at enhancing the effect of immunotherapy and effectively inhibiting tumor growth. Summary of the Invention

[0005] To overcome the deficiencies of the prior art, the present invention innovatively proposes a method for preparing a nano-drug for regulating the tumor immune microenvironment, a preparation method, a composition and its application. The prepared drug-loaded photothermal nanoparticles SL@mPPF have excellent photothermal conversion performance, can effectively increase the temperature at the tumor site, thereby killing tumor cells and inducing their immunogenic death. In addition, the nanoparticles can also promote the precise release of the drugs sorafenib and LY3200882, and the synergistic effect of the two greatly promotes the infiltration of immune cells and enhances the body's immune response. These series of effects act on the tumor immunosuppressive microenvironment together, effectively improving its state, so as to achieve the purpose of inhibiting tumor growth.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a preparation method of a nano-drug for regulating the tumor immune microenvironment, comprising the following steps:

[0008] S1. Prepare mesoporous polydopamine nanoparticles (mPDA) by the self-assembly method of block copolymer-polydopamine composite micelles:

[0009] Dissolve dopamine hydrochloride and polyether F-127 in a mixed solvent, add trimethylbenzene to emulsify it; then add ammonia water,

[0010] Stir and react under heating conditions, and obtain mPDA after centrifugation and washing.

[0011] S2. Modify folic acid-polyethylene glycol (NH 2 -PEG 2000 -FA): Mix mPDA and NH 2 -PEG 2000 -FA in Tris-HCl solution, stir at room temperature for 12 h, and obtain mPDA-PEG 2000 -FA (mPPF) after centrifugation and washing.

[0012] S3. Load sorafenib: Dissolve sorafenib in an organic solvent, add mPPF and disperse it evenly, stir at room temperature for 24 h to load sorafenib into the mesopores of the nanoparticles, and obtain S@mPPF after centrifugation and washing.

[0013] S4. Load LY3200882: Dissolve LY3200882 in an organic solvent, then disperse the S@mPPF obtained in S3 in water, add the LY3200882 solution under ultrasound, then stir for 0.5 h, and obtain SL@mPPF after centrifugation and washing.

[0014] Preferably, the mixed solvent in step S1 is water and ethanol, and the volume ratio of water, ethanol, trimethylbenzene and ammonia water is 950 - 1050:950 - 1050:32:73 - 77.

[0015] Preferably, the mass ratio of dopamine hydrochloride to polyether F - 127 in step S1 is 1.4 - 1.6:1, and the concentration of dopamine hydrochloride in the mixed solvent composed of ethanol and water is 140 - 160 mg / mL.

[0016] Preferably, the heating temperature in step S1 is 50°C - 55°C, and the heating time is 2 h - 2.5 h.

[0017] Preferably, the concentration of Tris - HCl in step S2 is 9 - 11 mM, the pH is 8.4 - 8.6, and the mass ratio of mPDA to

[0018] NH 2 -PEG 2000 -FA is 40:1.8 - 2.2, and the concentration of mPDA is 38 - 42 mg / mL.

[0019] Preferably, the organic solvent in step S3 is methanol, the concentration of sorafenib is 4.8 - 5.2 mg / mL, and the concentration of mPPF is 38 - 42 mg / mL.

[0020] Preferably, the organic solvent in step S4 is dimethyl sulfoxide, the concentration of LY3200882 is 9 - 11 mg / mL, the concentration of S@mPPF solution is 9 - 11 mg / mL, and the volume ratio of LY3200882 to S@mPPF solution is 1:19 - 21.

[0021] The second aspect of the present invention provides a nano - drug for regulating the tumor immune microenvironment prepared by the preparation method described in the first aspect.

[0022] The nano - drug SL@mPPF for regulating the tumor immune microenvironment prepared by the method of the present invention contains mesoporous polydopamine nanoparticles (polydopamine has the ability of photothermal conversion, the nanoparticle surface has a mesoporous structure, and there are a large number of polyphenol groups, which can provide sites for drug loading and surface modification), NH 2 -PEG 2000 -FA (modified on the surface of mPDA, PEG 2000Modification can prolong the in - vivo circulation time. The folic acid group can bind to the folate receptor highly expressed on the surface of tumor cell membranes, enhancing the targeting ability to tumor cells), sorafenib (which can directly inhibit the proliferation of tumor cells and also inhibit tumor angiogenesis by inhibiting receptors such as VEGFR, promoting the normalization of tumor blood vessels to enhance immune cell infiltration), LY3200882 (a TGF - β1 receptor inhibitor that plays an immunomodulatory function).

[0023] In the third aspect of the present invention, there is provided the use of the nano - drug for regulating the tumor immune microenvironment described in the second aspect in the preparation of an anti - tumor drug. In - vivo and in - vitro results show that SL@mPPF has the ability of photothermal conversion, can target tumor tissues, heat up the tumor site and release drugs under near - infrared laser irradiation, kill tumor cells, trigger immunogenic cell death, increase immune cell infiltration in tumor tissues, improve the immunosuppressive microenvironment of tumors, and inhibit tumor growth.

[0024] In the fourth aspect of the present invention, there is provided an anti - tumor drug, which uses the nano - drug for regulating the tumor immune microenvironment described in the second aspect as the main active ingredient.

[0025] Preferably, the anti - tumor drug further comprises pharmaceutically acceptable excipients. The carrier is a functional pharmaceutical excipient obtainable in the pharmaceutical field, including surfactants, suspending agents, emulsifiers, and some novel pharmaceutical macromolecular materials, such as cyclodextrin, chitosan, polylactic acid, poly (lactic - glycolic acid) copolymer, poly (ethylene oxide - propylene oxide) block copolymer, polyether, polyethylene glycol, etc.

[0026] Preferably, the dosage form of the anti - tumor drug is any dosage form acceptable in the pharmaceutical field, including but not limited to powder, granule, injection, capsule, tablet, oral liquid.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] In the present invention, mesoporous polydopamine nanoparticles are first prepared, modified with folic acid - polyethylene glycol on the surface, and loaded with sorafenib and LY3200882 to obtain a nano - drug with immune regulatory effects for the regulation of the immunosuppressive microenvironment of tumors. Among them, the folic acid - polyethylene glycol - modified nanoparticles can better target tumor tissues. Subsequently, the photothermal properties of the polydopamine nanoparticles can cause immunogenic death of tumor cells, enhance the immune response, and the acidic microenvironment and the temperature increase caused by the photothermal effect are conducive to the release of sorafenib and LY3200882, so that sorafenib can play the functions of inhibiting tumor cell proliferation and tumor angiogenesis and promoting the normalization of tumor blood vessels, and LY3200882 can play an immunomodulatory function by inhibiting the TGF - β1 receptor, jointly reversing the immunosuppressive microenvironment of tumors and enhancing the tumor treatment effect. Description of the Drawings

[0029] Figure 1 It is the TEM image of mPDA;

[0030] Figure 2 It is the TEM image of SL@mPPF;

[0031] Figure 3 It is the Mapping image of SL@mPPF;

[0032] Figure 4 It is the UV-Vis absorption spectra of mPDA, mPPF, S@mPPF, SL@mPPF, LY (LY3200882), NH 2 -FA-PEG 2000 ;

[0033] Figure 5 It is the particle size distribution diagram of mPDA, mPPF, S@mPPF, SL@mPPF;

[0034] Figure 6 It is the hydrated particle size diagram of mPDA, mPPF, S@mPPF, SL@mPPF;

[0035] Figure 7 It is the zeta potential diagram of mPDA, mPPF, S@mPPF, SL@mPPF;

[0036] Figure 8 It is the curve diagram of the photothermal temperature change of SL@mPPF with different concentrations;

[0037] Figure 9 It is the photothermal temperature change diagram of SL@mPPF;

[0038] Figure 10 It is the cumulative release diagram of sorafenib in SL@mPPF under different in vitro conditions;

[0039] Figure 11 It is the cumulative release diagram of LY3200882 in SL@mPPF under different in vitro conditions;

[0040] Figure 12 It is the in vitro cytotoxicity diagram of 4T1 cells caused by different treatments;

[0041] Figure 13 It is the apoptosis and necrosis diagram of 4T1 cells caused by different treatments;

[0042] Figure 14 It is the average fluorescence intensity diagram of 4T1 cells after nanoparticle uptake under different conditions;

[0043] Figure 15Immunofluorescence staining images of HMGB1 in 4T1 cells caused by different treatments;

[0044] Figure 16 Immunofluorescence staining images of CRT in 4T1 cells caused by different treatments;

[0045] Figure 17 Fluorescence integral images of tumor tissues after tail vein injection of SL@mPDA-Cy5.5 and SL@mPPF-Cy5.5;

[0046] Figure 18 Temperature change graphs of tumor regions after laser irradiation in tumor-bearing mice after tail vein injection of different doses of SL@mPPF;

[0047] Figure 19 Images of mouse tumor sizes after treatment under different conditions;

[0048] Figure 20 After treatment under different conditions, CD8 + T cell ratio images in mouse tumor tissues;

[0049] Figure 21 Images of the proportion of Treg cells in mouse tumor tissues after treatment under different conditions. Detailed implementation manners

[0050] The following further describes the detailed implementation manners of the present invention. It should be noted here that the description of these implementation manners is used to help understand the present invention, but does not constitute a limitation to the present invention. In addition, the technical features involved in the various implementation manners of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0051] The experimental methods in the following examples are all conventional methods unless otherwise specified, and the test materials used in the following examples are all commercially available through conventional channels unless otherwise specified.

[0052] Example 1 Preparation method and characterization of drug-loaded photothermal nanoparticles SL@mPPF

[0053] 1. Experimental steps

[0054] (1) Prepare mesoporous polydopamine nanoparticles (mPDA) by the self-assembly method of block copolymer-polydopamine composite micelles:

[0055] Add 5 ml of ethanol and 5 ml of ultrapure water to a round-bottom flask, mix well, then add dopamine hydrochloride (150 mg) and polyether F-127 (100 mg) to the flask. Dissolve them by ultrasonic bath, and under stirring conditions, add 160 μL of trimethylbenzene dropwise, and continue stirring for 5 - 10 min to emulsify. Then, perform ultrasonic bath for 5 - 10 min for further emulsification. Add 375 μL of ammonia water dropwise under stirring, and stir and react at 50 °C in a water bath for 2 h. After the reaction, collect the obtained nanoparticle precipitate by centrifugation (12000 rpm, 10 min), and wash it alternately with ethanol and water three times to obtain mPDA nanoparticles, and disperse them in water by ultrasonic wave for storage.

[0056] (2) Modify folic acid-polyethylene glycol (NH 2 -PEG 2000 -FA): Disperse mesoporous polydopamine nanoparticles mPDA (40 mg) and NH 2 -PEG 2000 -FA (2 mg) in 1 mL of Tris-HCl (10 mM) buffer solution with pH = 8.5, stir in the dark at room temperature for 12 h, and centrifuge (12000 rpm, 10 min) to obtain the nanoparticle precipitate, and wash it three times with ultrapure water to obtain mPPF.

[0057] (3) Load sorafenib: Dissolve 5 mg of sorafenib by ultrasonic wave in 1 mL of methanol, then add mPPF

[0058] (40 mg) to it, disperse it evenly by ultrasonic wave, and stir in the dark at room temperature for 24 h to load sorafenib into the surface mesopores. Obtain the nanoparticle precipitate by centrifugation (12000 rpm, 10 min), and wash it three times with ultrapure water to obtain S@mPPF.

[0059] (4) Load LY3200882: Dissolve LY3200882 (0.5 mg) by ultrasonic wave in 50 μL of dimethyl sulfoxide to obtain LY3200882 solution (10 mg / mL); Disperse S@mPPF (10 mg) evenly in 1 mL of ultrapure water by ultrasonic wave. Under the condition of probe ultrasonic wave (150 W, 20 kHz), add 50 μL of LY3200882 solution (10 mg / mL, dimethyl sulfoxide) dropwise, perform ultrasonic wave for 5 min, then stir in the dark at room temperature for 30 min. Collect the nanoparticle precipitate by centrifugation (12000 rpm, 10 min), and wash it three times with ultrapure water to obtain SL@mPPF.

[0060] LY3200882 can bind to polydopamine through π-π stacking and other interactions to achieve the successful loading of LY3200882 on the surface of the nanoparticles.

[0061] 2. Experimental results:

[0062] The morphology of mPDA was observed by transmission electron microscopy (TEM). As Figure 1 shown, the scale bar is 100 nm. mPDA presents uniform spherical shapes with a size of approximately 150 nm, and has a mesoporous structure on the surface, showing the potential as a drug carrier. After surface modification and drug loading, SL@mPPF nanoparticles were obtained. As observed by TEM, as Figure 2 shown, the scale bar is 100 nm. Compared with mPDA, for SL@mPPF, the mesoporous surface of the nanoparticles is attached with substances, making the surface smoother. The central region has a darker color, while the attached layer on the surface has a lighter color, and the particle size increases to approximately 200 nm, which can prove the success of surface modification and drug loading. Elemental analysis of the SL@mPPF nanoparticles was further carried out by Mapping. As Figure 3 shown, the scale bar is 250 nm. Elements C, N, O, F, and Cl exist in the SL@mPPF nanoparticles, and sorafenib contains F and Cl elements, indicating the successful loading of sorafenib. The UV-Vis spectra of the nanoparticles are as Figure 4 shown. Compared with mPDA, for mPPF, smaller characteristic absorption peaks similar to those of NH 2 -PEG 2000 -FA appear at 200 nm - 230 nm and 270 nm - 300 nm, indicating the successful modification of NH 2 -PEG 2000 -FA; compared with S@mPPF, for SL@mPPF, characteristic absorption peaks similar to those of LY3200882 appear at 200 nm - 220 nm, indicating the successful loading of LY3200882.

[0063] The hydrodynamic diameters of mPDA, mPPF, S@mPPF, and SL@mPPF were measured by dynamic light scattering (DLS). As Figure 5 shown, the hydrodynamic diameter of mPDA is about 280 nm. After FA-PEG 2000 modification, the hydrodynamic diameter of mPPF increases to about 320 nm. After further loading of sorafenib, the hydrodynamic diameter of the obtained S@mPPF increases to about 390 nm. Finally, after further loading of LY3200882, the hydrodynamic diameter of the obtained SL@mPPF increases to about 450 nm. The increase in particle size after modification and drug loading also proves the success of modification and loading. By measuring the zeta potential of the nanoparticles, as Figure 6 shown, the zeta potentials of mPDA, mPPF, S@mPPF, and SL@mPPF are approximately -40 mV, -37 mV, -32 mV, and -24 mV respectively. The change in their potentials also indicates the success of modification and drug loading.

[0064] Example 2 Photothermal Performance and In Vitro Responsive Drug Release of SL@mPPF

[0065] 1. Experimental Procedures

[0066] (1) Photothermal Conversion Performance of SL@mPPF: Place SL@mPPF solutions with different concentrations in 1.5 mL centrifuge tubes and irradiate them with an 808 nm laser (1.5 W / cm 2 ), and record the change in temperature over time.

[0067] (2) Drug Release of SL@mPPF under Different Conditions: Before release, the light irradiation group is first treated by laser irradiation

[0068] (10 min, 1.5 W / cm 2 ). Then, place the differently treated SL@mPPF (0.34 mg) in a dialysis bag (Mw: 8 Kda - 14 Kda). Immerse the dialysis bag in 10 mL of buffer containing 0.5% Tween - 80 (pH = 5 and 7.4 respectively), then place it in a shaker at 37 °C. Take out a small amount of solution at different times and supplement an equal volume of the corresponding buffer. After concentrating and drying the sample solutions taken at different time points, add methanol and dissolve them by ultrasonic treatment. Determine the content by high - performance liquid chromatography and calculate the cumulative drug release amount.

[0069] 2. Experimental Results

[0070] (1) Photothermal Conversion Performance of SL@mPPF: As Figure 8 shown, compared with pure water, the temperature of SL@mPPF solutions with different concentrations increases significantly under 808 nm laser irradiation (1.5 W / cm 2 ), and the increase in temperature shows a concentration - dependence. As Figure 9 shown, the thermal imaging diagram of the temperature increase of 100 ug / mL SL@mPPF within 10 min under 808 nm laser (1.5 W / cm 2 )

[0071] irradiation.

[0072] (2) Drug Release of SL@mPPF under Different Conditions: As Figure 10 shown, under light irradiation and pH 5 conditions, sorafenib is released the fastest, and the cumulative release amount at 48 h is about 30%. As Figure 11 shown, both laser irradiation treatment and acidic pH conditions can promote the release of LY3200882, and the cumulative release amount of LY3200882 at 48 h is about 70%. It can be speculated that the acidic tumor microenvironment and local light - induced temperature increase are beneficial to the release of drugs in tumor tissues.

[0073] Example 3 In Vitro Cytotoxicity and Apoptosis

[0074] 1. Experimental procedures

[0075] In vitro cytotoxicity: 4T1 cells were seeded in 96-well plates (about 10,000 cells / well) and cultured overnight with high-glucose DMEM medium (10% FBS + 1% PS). After that, drugs at different concentrations (0, 6.25, 12.5, 25, 50, 100, 200 μg / mL) were added for treatment (calculated based on the concentration of SL@mPPF). After 4 h, the light irradiation group was irradiated with a laser (1.5 W / cm 2 , 10 min), and then incubated for another 24 h. After 24 h, the medium was aspirated, and the cells were washed once with PBS. Then, the medium containing CCK8 was added, and the absorbance at 450 nm was measured after incubation at 37 °C for an appropriate time to detect cell viability.

[0076] Cell apoptosis or necrosis: 4T1 cells were seeded in 12-well plates (about 100,000 cells / well) and cultured overnight with high-glucose DMEM medium (10% FBS + 1% PS). After that, different drugs were added for treatment (calculated based on the equivalent concentration of 100 μg / mL SL@mPPF). After incubation for 12 h, the light irradiation group was irradiated with a laser (1.5 W / cm 2 , 10 min), and then incubated for another 24 h. The medium was gently aspirated, and the cells were collected by trypsin digestion. The cells were stained with an (Annexin V-PI) cell apoptosis detection kit, and the apoptosis and necrosis of the cells were detected and analyzed by flow cytometry.

[0077] 2. Experimental results

[0078] In vitro cytotoxicity: As Figure 12 shown, unloaded mPPF had low cytotoxicity, and SL@mPPF after drug loading had certain cytotoxicity to cell proliferation, and the cytotoxicity increased with the increase of concentration. After laser irradiation, the cytotoxicity of the SL@mPPF and mPPF groups increased significantly, indicating that the increase in temperature after laser irradiation could directly inhibit the proliferation activity of 4T1 cells. The data were expressed as mean ± standard deviation.

[0079] Cell apoptosis or necrosis: As Figure 13 shown, SL@mPPF could induce apoptosis and necrosis of 4T1 cells, and after laser irradiation, the proportion of apoptosis and necrosis caused was higher. The data were expressed as mean ± standard deviation, and one-way ANOVA and Tukey's post hoc test were used to calculate the significance between groups. ****P < 0.0001.

[0080] Example 4 In vitro cell uptake experiment

[0081] 1. Experimental procedures

[0082] 4T1 cells were seeded in 12-well plates (about 100,000 cells / well) and cultured overnight in high-glucose DMEM medium (10% FBS + 1% PS). The folate-blocked group was pre-incubated with folic acid (1 mM) for 2 h, and then the cells were treated with rhodamine B-labeled SL@mPPF and SL@mPDA (100 μg / ml), respectively. At 6 h and 12 h, the medium was removed, the cells were washed and collected, and the fluorescence intensity of the cells was detected by flow cytometry.

[0083] 2. Experimental results

[0084] As Figure 14 shown, the average fluorescence intensity of folate-modified nanoparticles SL@mPPF taken up within the same time was higher than that of unmodified nanoparticles SL@mPDA; and folate pre-blocking treatment could reduce the uptake of SL@mPPF, indicating that folate modification could promote the uptake of nanoparticles by 4T1 cells. The data were expressed as mean ± standard deviation, and one-way ANOVA and Tukey's post hoc test were used to calculate the significance between groups. ****P < 0.0001.

[0085] Example 5 In vitro experiment on immunogenic cell death

[0086] 1. Experimental procedures

[0087] (1) Detection of HMGB1 (high mobility group box protein 1): 4T1 cells were seeded in confocal dishes (about 200,000 cells / dish) and cultured overnight in high-glucose DMEM medium (10% FBS + 1% PS). Different drugs were added for treatment (100 μg / ml, calculated based on the equivalent concentration of SL@mPPF). After 4 h, the light-irradiation group was irradiated with laser (1.5 W / cm 2 , 10 min) and then incubated for another 24 h. Then, HMGB1 immunofluorescence staining was performed and observed under a confocal microscope.

[0088] (2) Detection of CRT (calreticulin): 4T1 cells were seeded in confocal dishes (about 200,000 cells / dish) and cultured overnight in high-glucose DMEM medium (10% FBS + 1% PS). Different drugs were added for treatment (100 μg / ml, calculated based on the equivalent concentration of SL@mPPF). After 4 h, the light-irradiation group was irradiated with laser

[0089] (1.5 W / cm 2 , 10 min) and then incubated for another 6 h. Then, CRT immunofluorescence staining was performed and observed under a confocal microscope.

[0090] 2. Experimental results

[0091] (1) Detection of HMGB1 (high mobility group box protein 1): As Figure 15As shown, after SL@mPPF treatment, the HMGB1 protein in the cell nucleus decreased, and after laser irradiation treatment, the content of HMGB1 in the cell nucleus was even less, indicating that SL@mPPF after laser irradiation treatment can induce immunogenic cell death and promote the translocation of HMGB1 out of the cell nucleus.

[0092] (2) Detection of CRT (calreticulin): As Figure 16 shown, after SL@mPPF treatment, the CRT

[0093] protein on the cell surface increased, and after laser irradiation treatment, CRT increased significantly, indicating that SL@mPPF after laser irradiation treatment can induce immunogenic cell death and cause CRT to flip outwards.

[0094] Example 6 In vivo targeted tumor experiment of SL@mPPF

[0095] 1. Experimental procedure: SL@mPPF-Cy5.5 and SL@mPDA-Cy5.5 (50 mg / kg) modified with Cy5.5

[0096] were injected into 4T1 subcutaneous tumor-bearing mice via the tail vein (Modeling: Subcutaneously inject about 3 million 4T1 cells

[0097] / mouse; tumor volume is about 100 mm 3 ), and the mice were sacrificed 24 h later. The tumor tissues were taken out and the fluorescence content of the tumor tissues was measured with a small animal in vivo imager to determine the relative content of nanoparticles in the tumor tissues.

[0098] 2. Experimental results: As Figure 17 shown, SL@mPPF modified with NH 2 -PEG 2000 -FA can target tumor tissues more than SL@mPDA and increase the accumulation of nanoparticles in tumor tissues. The data are expressed as mean ± standard deviation, and one-way ANOVA and Tukey's post hoc test were used to calculate the significance between groups. ***P < 0.001.

[0099] Example 7 Photothermal conversion effect of SL@mPPF in in vivo tumor tissues

[0100] 1. Experimental procedure: Different doses of SL@mPPF were injected into 4T1 subcutaneous tumor-bearing mice via the tail vein (tumor volume is about 100 mm 3 ), and 4 h later, the tumor site was irradiated with 808 nm laser (1.5 W / cm 2 , 10 min), and a thermal imager was used to observe and record the temperature change of the tumor tissue.

[0101] 2. Experimental results: As Figure 18As shown, compared with the control group, the temperature at the tumor site in the SL@mPPF group increased significantly under laser irradiation, and the higher the drug dosage, the higher the temperature rise. This indicates that SL@mPPF can reach the tumor tissue and convert light energy into heat energy under near-infrared laser irradiation to achieve temperature rise at the tumor tissue site.

[0102] Example 8 Antitumor effect of SL@mPPF in vivo

[0103] 1. Experimental procedure: Establish a 4T1 subcutaneous tumor model in female BALB / c mice (subcutaneously inject about 3 million 4T1 cells per mouse). When the tumor volume grows to about 100 mm 3 ³, inject the drug via the tail vein (50 mg / kg, calculated based on the equivalent content of SL@mPPF). 4 hours later, irradiate the tumor tissue of the light irradiation group with 808 nm laser (1.5 W / cm 2 ², for 10 minutes). Administer the drug once every 3 days. The light irradiation group is treated with light irradiation. After administering the drug 3 times, continuously observe the tumor growth status. Sacrifice the mice on the 22nd day, take out the tumor tissue and photograph it to record the size.

[0104] 2. Experimental results: As Figure 19 shown, the excised tumor tissue taken out after treatment was significantly smaller than that of the control group, and the tumor in the SL@mPPF plus light irradiation group was the smallest, indicating that the treatment with SL@mPP plus light irradiation can play a good antitumor effect in vivo.

[0105] Example 9 Immune activation effect of SL@mPPF in vivo

[0106] 1. Experimental procedure: Establish a 4T1 subcutaneous tumor model in female BALB / c mice (subcutaneously inject about 3 million 4T1 cells per mouse). When the tumor volume grows to about 150 mm 3 ³, inject the drug via the tail vein (50 mg / kg, calculated based on the equivalent content of SL@mPPF). 4 hours later, irradiate the tumor tissue of the light irradiation group with 808 nm laser (1.5 W / cm 2 ², for 10 minutes). Administer the drug once every 3 days. The light irradiation group is treated with light irradiation. A total of 2 doses are administered. Sacrifice the mice 24 hours later, take out the tumor tissue, prepare it into a single-cell suspension, and perform antibody staining with CD3, CD4, CD8, CD25, and Foxp3. Detect the proportion of immune cells by flow cytometry.

[0107] 2. Experimental results: As Figure 20 shown, the proportion of CD8 + ⁺ T cells in the SL@mPPF group was significantly higher than that in the control group; as Figure 21As shown, the proportion of immunosuppressive Treg cells in the SL@mPPF group decreased significantly compared with the control group. This indicates that SL@mPPF can increase the proportion of immune killer cells in the tumor group, reduce the proportion of immunosuppressive cells, activate the immune response, and improve the immunosuppressive tumor microenvironment.

[0108] In summary, the nano-drug prepared by the present invention for regulating the tumor immune microenvironment has a uniform nano-size, has the ability of photothermal conversion, can successfully load drugs and achieve the responsive release of drugs, and can kill tumor cells; in vivo, it can target tumor tissues, raise the temperature of the tumor site through the photothermal conversion ability, kill and inhibit tumor growth, and release sorafenib and LY3200882 to achieve tumor vascular regulation, trigger immunogenic cell death, activate the immune response, increase the proportion of cytotoxic immune cells in the tumor tissue, reduce the proportion of immunosuppressive cells, jointly improve the tumor immunosuppressive microenvironment, and inhibit tumor growth.

[0109] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions, and variations of these embodiments still fall within the protection scope of the present invention.

Claims

1. A method for preparing a nano drug for regulating tumor immune microenvironment, characterized in that: The following steps are involved: S1. Preparation of mesoporous polydopamine nanoparticles (mPDA): dopamine hydrochloride and polyether F-127 are dissolved in a mixed solvent, trimethylbenzene is added for emulsification, ammonia water is added for heating and stirring reaction, and mPDA is obtained by centrifugation and washing; S2. Surface modification: mPDA obtained in step S1 was mixed with NH2-PEG 2000 -FA was mixed and stirred in Tris-HCl solution and centrifuged to obtain mPDA-PEG 2000 -FA(mPPF); S3, drug loading: dissolving sorafenib in an organic solvent and mixing with mPPF, and centrifuging and washing to obtain sorafenib-loaded S@mPPF; S4. Secondary drug loading: LY3200882 was dissolved in an organic solvent and then mixed with the S@mPPF aqueous solution, and then centrifuged and washed to obtain the dual-drug-loaded nanodrug SL@mPPF.

2. The method according to claim 1, characterized in that In step S1: The mixed solvent is water and ethanol, with a volume ratio of 950-1050:950-1050; The volume ratio of trimethylbenzene to ammonia water is 32:73-77; The mass ratio of dopamine hydrochloride to polyether F-127 is 1.4-1.6:1; The reaction conditions are stirring at 50°C-55°C for 2h-2.5h.

3. The method according to claim 1, characterized in that In step S2: The Tris-HCl buffer has a concentration of 9-11 mM and a pH of 8.4-8.6; The mPDA and NH2-PEG 2000 -FA mass ratio is 40:1.8-2.2; The mPDA concentration during the mixing was 38-42 mg / mL.

4. The method according to claim 1, characterized in that In step S3: The organic solvent is methanol; The concentration of the sorafenib solution is 4.8-5.2 mg / mL; The mPPF concentration is 38-42 mg / mL.

5. The method according to claim 1, characterized in that In step S4: The organic solvent is dimethyl sulfoxide; The concentration of the LY3200882 solution is 9-11 mg / mL; The concentration of the S@mPPF aqueous solution is 9-11 mg / mL; The volume ratio of the LY3200882 solution to the S@mPPF solution is 1:19-21.

6. A nanomedicine prepared according to the method according to any one of claims 1 to 5, characterized in that: Include: Mesoporous polydopamine nanoparticle carrier; NH2-PEG modified on the carrier surface 2000 -FA; Sorafenib loaded in mesopores; LY3200882 loaded on the surface of nanoparticles.

7. The nano drug according to claim 6, characterized in that: The mesoporous polydopamine nanoparticles contain phenolic hydroxyl groups on their surfaces and have an average particle size of 80-150 nm; The NH2-PEG 2000 -The modification amount of FA is 2.5%-4.0% of the carrier mass; The drug loading of sorafenib is 2.3%-2.7%, and the encapsulation efficiency is 18.4%-21.6%; The drug loading of LY3200882 is 4.2%-4.8%, and the encapsulation efficiency is 84%-96%.

8. The use of the nano drug according to claim 6 in the preparation of anti-tumor drugs, characterized in that It works through the following mechanisms: (a) Enrichment in tumor tissues through folate receptor-mediated targeting; (b) Producing photothermal effect under near-infrared light excitation and triggering drug release; (c) inhibiting tumor angiogenesis and promoting immune cell infiltration through sorafenib; (d) The photothermal effect-induced tumor immunogenic cell death and LY3200882 blocking the TGF-β1 signaling pathway together enhanced the infiltration of immune cells in tumor tissue and improved the immunosuppressive microenvironment.

9. An anti-tumor drug composition, characterized in that The invention comprises the nano drug according to any one of claims 6 to 7 as an active ingredient, and pharmaceutically acceptable excipients.

10. The pharmaceutical composition according to claim 9, characterized in that: The auxiliary material is selected from at least one of cyclodextrin, polyether F-127, and polyethylene glycol; The dosage form is selected from a nanosuspension or an intravenous injection; The mass proportion of the nano drug in the composition is 60%-90%.

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