Nanopharmaceutical capable of slowly releasing NO and loading anticancer drugs, method thereof, and application thereof

Abstract

The present application discloses a nanopharmaceutical that can slowly release NO and load anticancer drugs, as well as its preparation method and application. The nanopharmaceutical of the present application slowly releases nitric oxide and loads anticancer drugs; the nanopharmaceutical is a nanoparticle formed by self-assembled polylactic-co-glycolic acid block copolymers embedding doxorubicin, nitrosoacetylpenicillamine and cationic liposomes. The nanopharmaceutical that slowly releases nitric oxide and loads anticancer drugs in the present application has the co-delivery of nitric oxide and doxorubicin, which not only realizes the combined treatment of nitric oxide therapy and chemotherapy, but also breaks the limitations of single treatment; moreover, the immunomodulatory effect of doxorubicin is increased by nitric oxide co-transmission, which can better regulate the tumor immunosuppressive microenvironment and improve the anti-tumor immune response. The nanopharmaceutical of the present application can reduce M2 macrophages, reverse the immunosuppressive tumor microenvironment, and thus achieve synergistic anti-tumor therapy.

Description

Technical Field

[0001] The present application relates to a sustained-release agent, in particular to a nanoparticle that releases NO and is loaded with anticancer drugs, and a preparation method and application thereof. Background Art

[0002] Cancer has become a serious health hazard to humanity today. Although chemotherapy is currently the most commonly used approach in clinical practice, it still faces many challenges. For example, different cancer cell types have varying sensitivities to chemotherapy drugs. Long-term use of chemotherapy drugs can damage the patient's immune system or induce drug resistance, and they can have severe toxic side effects, all of which can lead to chemotherapy failure.

[0003] In recent years, immunotherapy has made significant progress in the treatment of malignant tumors. Among these advances, the combination of immunotherapy and chemotherapy (hereafter referred to as "chemoimmunotherapy") represents an emerging approach. Chemoimmunotherapy has transformed the paradigm of traditional chemotherapy, eliciting anti-tumor immunity and improving cancer treatment efficacy while mitigating the high toxicity and side effects of chemotherapy. Nitric oxide (NO) is a versatile signaling molecule that has been shown to offer significant advantages in cancer therapy, serving as an immune adjuvant. NO can influence tumor proliferation in various ways, including directly killing tumor cells, enhancing cancer cell sensitivity to chemotherapeutic agents, and inhibiting tumor cell growth and migration. Importantly, NO production can modulate the immunosuppressive tumor microenvironment by promoting dendritic cell maturation and reducing the expression of M2 macrophages. Studies have shown that in situ NO release at the tumor site can ameliorate the immune deficiencies of the tumor microenvironment; however, how to effectively deliver NO to the tumor site and maintain a long-term, slow release remains a key challenge. Cancer therapy requires precise control of the rate and timing of NO release. A release rate that is too fast may lead to severe cytotoxicity, while a release time that is too long may reduce the therapeutic effect and increase side effects. Furthermore, nitric oxide has a short half-life in the body and is easily metabolized and cleared. Therefore, to improve the therapeutic effect, it is necessary to develop a stable nitric oxide delivery system that ensures sufficient bioavailability and prolongs the existence of nitric oxide.

[0004] Using nanoparticles to load chemotherapy drugs for efficient tumor treatment is a common method for reducing drug toxicity and improving drug targeting. Polymers have excellent biocompatibility and are therefore widely used for the delivery of anti-tumor drugs. Polyethylene glycol-polylactic acid-polyglycolic acid block copolymers (PEG-b-PLGA) are commonly used in the prior art to form nanomicelles that can encapsulate a variety of drug molecules. They also exhibit a certain degree of biodegradability, allowing for sustained release of drugs during degradation. Therefore, they are often used to load anti-tumor drugs.

[0005] Doxorubicin (DOX) is an antibiotic with a broad anti-tumor spectrum. When encapsulated in PEG-b-PLGA nanomicelles, its drug loading is very low. Chemical means are often needed to chemically connect DOX and PEG-b-PLGA molecular chains to increase the drug loading. However, these chemical means may affect drug activity and even cause toxic side effects.

[0006] S-Nitroso-N-acetyl-D-penicillamine (SNAP) is a common nitric oxide donor that spontaneously releases NO under physiological conditions and has low toxicity. However, like doxorubicin, its drug loading is also low when encapsulated in PEG-b-PLGA nanomicelles.

[0007] Currently, relatively little research has been conducted on combining NO therapy with other treatment modalities (such as chemotherapy, radiotherapy, or immunotherapy). Furthermore, the drug loading of either doxorubicin or NO alone is generally low, resulting in poor therapeutic efficacy. Therefore, effectively combining NO with other anticancer drugs remains a key and challenging area of research in the field of anticancer drugs. Summary of the Invention

[0008] The purpose of this application is to provide a new nanoparticle that slowly releases NO and loads anticancer drugs, as well as its preparation method and application.

[0009] This application adopts the following technical solutions:

[0010] The first aspect of the present application discloses a nanoparticle that slowly releases nitric oxide and carries anticancer drugs. The nanoparticle of the present application is a nanoparticle formed by self-assembled polylactic acid-glycolic acid block copolymer (abbreviated as PLGA) encapsulating doxorubicin, nitrosoacetylpenicillamine and cationic liposomes.

[0011] It should be noted that the present application creatively uses self-assembled polylactic acid-glycolic acid block copolymers to embed doxorubicin and nitrosoacetylpenicillamine, and uses cationic liposomes as drug delivery carriers. The nanoparticles formed can achieve the co-delivery of nitric oxide and doxorubicin. The dual-responsive NO and DOX co-delivery nanosystem achieves simultaneous chemotherapy and ICD-activated immunotherapy, breaking the limitations of single treatment. The immunomodulatory effect of DOX is increased by NO co-transmission, which can better regulate the tumor immunosuppressive microenvironment and enhance the anti-tumor immune response in the body. The nanoparticles of the present application can reduce M2 macrophages, reverse the immunosuppressive tumor microenvironment, and thus achieve synergistic anti-tumor therapy.

[0012] Preferably, the surface of the nanoparticles is modified with hyaluronic acid.

[0013] It should be noted that the present application loads hyaluronic acid HA on the surface of the nanoparticles. Since HA can bind to the CD44 receptor highly expressed by various tumor cells, the nanoparticles of the present application can penetrate into the cells and achieve high cellular uptake.

[0014] Preferably, the number average molecular weight of the polylactic acid-glycolic acid block copolymer is 5 to 30 kD.

[0015] Preferably, the number average molecular weight of the polylactic acid-glycolic acid block copolymer is 5 to 15 kD.

[0016] Preferably, the particle size of the nanoparticles is 50 to 200 nm.

[0017] Preferably, the cationic liposome is trimethyl-2,3-dioleoyloxypropylammonium bromide.

[0018] Preferably, the molar ratio of the polylactic acid-glycolic acid block copolymer, doxorubicin, nitrosoacetylpenicillamine and cationic liposome is 0.1-1:2-2.5:2-2.3:1-1.5.

[0019] The second aspect of the present application discloses the use of the nanopharmaceutical of the present application in the preparation of anti-tumor drugs.

[0020] The third aspect of the present application discloses a method for preparing the nanopharmaceutical of the present application, comprising the following steps:

[0021] (1) dissolving doxorubicin, nitrosoacetylpenicillamine, and cationic liposomes in a first organic solvent to obtain a mixture solution;

[0022] (2) adding polylactic acid-glycolic acid block copolymer in a light-proof environment, and evacuating the mixture solution until a colloidal mixture is formed;

[0023] (3) adding a first organic solvent dropwise to the colloidal mixture to dissolve it, thereby obtaining a new mixture solution;

[0024] (4) adding the new mixture solution of step (3) dropwise into ultrapure water to obtain an emulsion;

[0025] (5) ultrasonically treating the emulsion to volatilize the first organic solvent; alternatively, after volatilizing the first organic solvent, adding a hyaluronic acid solution and ultrasonically treating again to volatilize the solvent; it is understood that if hyaluronic acid modification is required, after ultrasonically treating the emulsion to volatilize the first organic solvent, adding a solution containing hyaluronic acid and ultrasonically treating again can be sufficient; if hyaluronic acid modification is not required, ultrasonically treating the emulsion to volatilize the first organic solvent before proceeding to the next step;

[0026] (6) After the ultrasonic treatment, an ice bath is placed to obtain a nanosuspension, and the nanosuspension is ultrafiltered to obtain nanoparticles, i.e., the nanopharmaceutical of the present application.

[0027] Preferably, in the preparation method of the present application, the first organic solvent is tetrahydrofuran.

[0028] Preferably, in the preparation method of the present application, doxorubicin is pre-dissolved in a second organic solvent.

[0029] Preferably, in the preparation method of the present application, ultrapure water is preheated to 37° C. before the dropwise addition of the new mixture solution in step (3) is performed.

[0030] Preferably, in the preparation method of the present application, the solvent of the hyaluronic acid solution is the first organic solvent.

[0031] Preferably, in the preparation method of the present application, the second organic solvent for dissolving doxorubicin is chloroform.

[0032] Preferably, in the preparation method of the present application, in step (4), ultrasonic treatment is performed using an ultrasonic water bath while the new mixture solution is added dropwise.

[0033] Preferably, in the preparation method of the present application, the temperature of the ultrasonic water bath in step (4) is 37° C. It is understood that the temperature of the ultrasonic water bath is actually the preheating temperature of the ultrapure water, that is, the temperature is ensured to be stable at 37° C. throughout the entire dropwise addition process to ensure the formation of nanoparticles of the desired size.

[0034] Preferably, in the preparation method of the present application, the power of the ultrasonic water bath for ultrasonic treatment in step (4) is 200W.

[0035] Preferably, in the preparation method of the present application, the power of the ultrasonic treatment in step (5) is 100-200 W, and the ultrasonic time is at least 10 minutes.

[0036] Preferably, in the preparation method of the present application, in step (5), no ice bath protection is performed during ultrasonic treatment, and the solvent is volatilized by ultrasonic heating.

[0037] Preferably, in the preparation method of the present application, the amount of hyaluronic acid used in step (5) is 0.1 to 1 part by weight of hyaluronic acid per 10 parts by weight of the polylactic acid-glycolic acid block copolymer.

[0038] Preferably, in the preparation method of the present application, in step (6), before ultrafiltration, the mixture is placed at room temperature, away from light, with the lid opened, after an ice bath, to allow the residual solvent to evaporate, and then stored at a temperature of less than or equal to 4° C. away from light to obtain a nanosuspension.

[0039] Preferably, in the preparation method of the present application, after ultrafiltration of the nanosuspension in step (6), ultrasonic washing is further performed, followed by ultrafiltration, and the washing and ultrafiltration are repeated at least twice.

[0040] The beneficial effects of this application are:

[0041] The nanopharmaceuticals described in this application, which release nitric oxide slowly and carry anticancer drugs, can co-deliver nitric oxide and doxorubicin. This not only enables combined nitric oxide therapy and chemotherapy, breaking the limitations of single-agent therapy, but also enhances the immunomodulatory effects of doxorubicin through the co-delivery of nitric oxide, thereby better regulating the tumor's immunosuppressive microenvironment and enhancing its anti-tumor and immunostimulatory effects in vivo. The nanopharmaceuticals described in this application provide a new combination therapy nanoparticle for tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 shows the particle size distribution of PLGA-SNAP-DOX nanoparticles of the embodiment of the present application;

[0043] Figure 2 shows the particle size distribution of different nanoparticles of the embodiment of the present application;

[0044] Figure 3 shows the zeta potential diagram of different nanoparticles of the present application embodiment;

[0045] Figure 4 shows the ultraviolet-visible absorption spectrum of the nanoparticles of the embodiment of the present application;

[0046] Figure 5 shows the NO sustained release graph of the nanoparticles of the embodiment of the present application;

[0047] Figure 6 The fluorescence confocal image of the cell uptake of nanoparticles in the embodiment of the present application is shown;

[0048] Figure 7 A fluorescence confocal image showing the release of NO in cells by the nanoparticles of the embodiment of the present application is shown;

[0049] Figure 8 The toxicity test graph of the nanoparticles containing different concentrations of DOX and NO on cells in the embodiment of the present application is shown;

[0050] Figure 9 The fluorescence confocal image of the ICD effect induced by the nanoparticles in the embodiment of the present application is shown;

[0051] Figure 10 A graph showing changes in CRT expression levels in 4T1 cells after treatment with nanoparticles according to an embodiment of the present application is shown;

[0052] Figure 11A graph showing changes in HMGB1 expression in 4T1 cells after treatment with nanoparticles according to an embodiment of the present application is shown;

[0053] Figure 12 The graph shows the changes in the expression of CD206 in macrophages after treatment with the nanoparticles of the embodiment of the present application;

[0054] Figure 13 The graph shows the changes in the expression of CD80 and CD86 in DC after treatment with the nanoparticles of the embodiment of the present application;

[0055] Figure 14 The figures show the monitoring results of tumor size in different experimental groups in the examples of the present application;

[0056] Figure 15 The figures show the monitoring results of the weight of mice in different experimental groups in the examples of the present application;

[0057] Figure 16 The fluorescence detection diagram of apoptosis of tumor tissue cells in vivo in the examples of the present application is shown. DETAILED DESCRIPTION

[0058] In view of the fact that there are relatively few studies on the combined use of NO therapy with other treatment methods in the existing technology, this application has developed a new dual-responsive NO and DOX co-delivery nanosystem, which realizes simultaneous chemotherapy and ICD-initiated immunotherapy, breaking the limitations of single treatment. The immunomodulatory effect of DOX is increased by NO co-delivery, including in vitro and in vivo ICD enhancement and increased DC maturation. It can also regulate the tumor immunosuppressive microenvironment and enhance the anti-tumor and immunostimulatory effects in vivo.

[0059] The nanopharmaceutical of the present application is a dual-responsive nanosystem for the co-delivery of NO and DOX, which realizes the chemoimmunotherapy of NO in collaboration with doxorubicin, and is used for simultaneous chemotherapy and ICD-activated immunotherapy. The present application prepares drug-loaded nanoparticles by thin film hydration method / improved multiple emulsification method, that is, the carrier material and the drug are dissolved in an organic solvent, and then the organic solvent is completely removed by thorough mixing. After redissolving in a trace amount of organic solvent, it is added to pure water, and after ultrasonic emulsification, nanoscale microspheres are formed by high-intensity ultrasound. During the high-intensity ultrasound treatment, the organic solvent is volatilized by ultrasonic heating, and finally the surface is modified with HA by electrostatic adsorption to obtain the nanoparticles loaded with hydrophobic drugs of the present application.

[0060] The nanopharmaceuticals described herein are self-assembled PLGA nanoparticles that encapsulate DOX and SNAP materials, and the nanoparticle surfaces are modified with hyaluronic acid (HA). Hyaluronic acid (HA) can bind to the CD44 receptor, which is highly expressed on various tumor cells, allowing the nanopharmaceutical to penetrate the cells, thereby achieving high cellular uptake. The immunomodulatory effects of DOX are also enhanced by NO co-transmission, including increased ICD and DC maturation in vitro and in vivo. It can also modulate the tumor immunosuppressive microenvironment, enhancing anti-tumor and immunostimulatory effects in vivo.

[0061] In one implementation of the present application, the nanopharmaceutical achieved excellent therapeutic effects in 4T1 (mouse breast cancer cell) tumors. This dual-responsive NO and DOX co-delivery nanosystem provides a new strategy for cancer chemoimmunotherapy.

[0062] The nanoparticles (or nanomedicines, nanopharmaceuticals) of the present application include doxorubicin, SNAP, DOTAP, PLGA encapsulated around the doxorubicin, SNAP, and DOTAP, and HA loaded onto the outer surface of the PLGA. The nanoparticles of the present application have a particle size of 50 to 200 nm. The number average molecular weight of the PLGA is 5 to 30 kDa. Preferably, the number average molecular weight of the PLGA is 5 to 15 kDa.

[0063] Compared with existing technologies, the advantages of the nanoparticles of this application are: 1) The nanoparticles of this application have a high drug loading rate and an appropriate particle size. 2) The nanoparticles of this application can downregulate M2 tumor-associated macrophages, reversing the tumor immunosuppressive microenvironment and enhancing anti-tumor immune responses. 3) The hyaluronic acid (HA) loaded on the surface of the nanoparticles of this application can bind to the CD44 receptor, which is highly expressed on various tumor cells, enabling the nanoparticles to penetrate into cells and achieve high cellular uptake.

[0064] The present application is further described in detail below through specific examples. The following examples are only used to further illustrate the present application and should not be understood as limiting the present application.

[0065] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0066] Example 1

[0067] The nanoparticles in this example are composed of PLGA encapsulated with doxorubicin, SNAP, and DOTAP, and the surface of the nanoparticles is modified with hyaluronic acid. The specific preparation method includes:

[0068] (1) Accurately weigh 10 mg of PLGA, 1.33 mg of doxorubicin, 0.5 mg of SNAP, and 1 mg of DOTAP; then dissolve all of doxorubicin, SNAP, and DOTAP in 2 mL of tetrahydrofuran (THF) to obtain a mixture solution; wherein the number average molecular weight of PLGA is 5 kD; wherein doxorubicin is dissolved in chloroform in advance to obtain a solution with a doxorubicin concentration of 2 mg / mL, and the solution is added at an amount of 1.33 mg of doxorubicin.

[0069] (2) Then, 10 mg of PLGA was added, and the mixture solution was transferred to a centrifuge tube, which was sealed and vacuumed using a liquid nitrogen cold well in a light-proof environment to quickly evaporate the organic solvent. The process was stopped when the mixture solution in the centrifuge tube formed a colloidal mixture.

[0070] (3) 100 μL of THF was added dropwise to the colloidal mixture to redissolve it and obtain a new mixture solution.

[0071] (4) Preheat 2 mL of ultrapure water to 37° C. in advance, and add the new mixture solution obtained in step (3) dropwise into the ultrapure water in an ultrasonic water bath (temperature 37° C., ultrasonic power 200 W) to form a preliminary emulsion.

[0072] (5) The resulting emulsion was then sonicated using an ultrasonic cell disruptor at a power of 200 W without ice bath protection. The tetrahydrofuran was volatilized by ultrasonic heating for 10 minutes. Then, 200 μL of tetrahydrofuran containing 0.2 mg of HA was added and sonicated for another 10 minutes.

[0073] (6) After the ultrasonication, the suspension was quickly placed on ice for an ice bath. After about 30 minutes of ice bathing, the suspension was kept at room temperature away from light and opened for one day to release the residual THF. The suspension was then kept away from light in a refrigerator at 4°C to obtain a clear and transparent nanosuspension. The nanosuspension was ultrafiltered in a 30KD ultrafiltration tube. After the ultrafiltration was completed, 2 mL of ultrapure water was added for washing and ultrafiltration was performed again. The washing and ultrafiltration were repeated 3 times to remove free substances including free DOX and SNAP, thereby obtaining the nanopharmaceutical of this example, which was labeled as PLGA-SNAP-DOX.

[0074] In addition, in order to facilitate a series of subsequent characterization experiments, the following nanoparticles were also prepared in this example: PLGA nanoparticles, PLGA-SNAP nanoparticles, and PLGA-DOX nanoparticles

[0075] The specific preparation method is:

[0076] Preparation of PLGA nanoparticles: The preparation is basically the same as in Example 1, except that in step 1, 1 mg of DOTAP and 0.1 mg of DIR-BOA were dissolved in 2 mL of tetrahydrofuran. The rest of the preparation is the same as in Example 1.

[0077] Preparation of PLGA-SNAP nanoparticles: The preparation was substantially the same as in Example 1, except that in step 1, 0.5 mg of SNAP, 1 mg of DOTAP, and 0.1 mg of DIR-BOA were dissolved in 2 mL of tetrahydrofuran. The remaining steps were the same as in Example 1.

[0078] Preparation of PLGA-DOX nanoparticles: The preparation is substantially the same as in Example 1, except that in step 1, 1.33 mg of doxorubicin, 1 mg of DOTAP, and 0.1 mg of DIR-BOA were dissolved in 2 mL of tetrahydrofuran. The rest of the preparation is the same as in Example 1.

[0079] The nanoparticles prepared above were subjected to the following tests:

[0080] (1) Characterization of particle size, potential and ultraviolet absorption

[0081] The particle size and zeta potential of PLGA, PLGA-SNAP, PLGA-DOX, and PLGA-SNAP-DOX were measured using a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK). The UV-visible absorption spectra of PLGA, PLGA-SNAP, PLGA-DOX, and PLGA-SNAP-DOX were measured using a Shimadzu UV-1700 spectrometer (Germany).

[0082] The test results are as follows Figure 1 and Figure 2 As shown in the figure, the particle sizes of PLGA and PLGA-DOX nanoparticles measured by dynamic light scattering were 58.5 nm and 69.7 nm, respectively, showing uniform and normally distributed particle sizes. Compared to the 71.4 nm particle size of PLGA-SNAP, the hydrated particle size of PLGA-SNAP-DOX nanoparticles increased to 76.1 nm, showing uniform and normally distributed particle sizes.

[0083] The zeta potential of nanoparticles is Figure 3 As shown, the potential of each nanoparticle varies. The potential of unmodified PLGA is 27.9 mV, while the potential of the synthesized PLGA-SNAP-DOX after SNAP modification is 37.3 mV. Characterization of the particle size and potential of each nanoparticle demonstrates that the PLGA-SNAP-DOX has a relatively uniform particle size and good dispersion.

[0084] The UV absorption spectra of each nanoparticle are as follows Figure 4 As shown, since the absorption peak of DOX is at 420-580 nm, compared with PLGA and PLGA-SNAP NPs, PLGA-DOX and PLGA-SNAP-DOX have obvious broad absorption bands at 420-580 nm, which indicates that PLGA-SNAP-DOX has been successfully synthesized in this example.

[0085] (2) Nanoparticle NO sustained release test

[0086] The release of NO was detected using a nitric oxide detection kit (Shanghai Biyuntian Co., Ltd.), and the release of NO in PLGA-SNAP and PLGA-SNAP-DOX was quantitatively analyzed using the Griess assay. The nanoparticles (NO concentration: 150 μM) were stored at 37°C for different times (0, 2, 4, 8, 12, 24, 48, 72 h), and then centrifuged at 13,000 rpm for 3 min to remove PLGA-SNAP-DOX or PLGA-SNAP. 50 μL of room temperature Griess Reagent I was first added to 50 μL of sample, followed by 50 μL of room temperature Griess Reagent I. The absorbance at 540 nm was detected using an enzyme reader and the amount of NO released was calculated. The results are shown in Figure 2. Figure 5 shown.

[0087] Figure 5 The results showed that the concentration of NO released from PLGA-SNAP-DOX continued to increase over time within 24 hours and reached a plateau after 48 hours, with a NO release efficiency of approximately 98%, which is similar to the results of PLGA-SNAP. These results indicate that at 37°C, PLGA-SNAP-DOX has the ability to slowly and continuously release NO, ensuring deep penetration and release of NO in the body.

[0088] (III) In vitro study of PLGA-SNAP-DOX cell uptake

[0089] DiR-BOA dye was used to detect the cellular uptake of different nanoparticles. This application also prepared nanoparticles based on fluorescent dyes:

[0090] Preparation of HA-PLGA@DiR-BOA nanoparticles: The preparation was basically the same as in Example 1, except that in step 1, 1 mg of DOTAP and 0.1 mg of DIR-BOA were dissolved in 2 mL of tetrahydrofuran. The rest of the preparation was the same as in Example 1.

[0091] Preparation of PLGA@DiR-BOA (without HA) nanoparticles: The preparation was essentially the same as in Example 1, except that in step 1, 1 mg of DOTAP and 0.1 mg of DIR-BOA were dissolved in 2 mL of tetrahydrofuran. In step 5, HA was not added, and the remaining steps were the same as in Example 1.

[0092] DiRBOA was used to quantitatively detect the cellular uptake of different nanoparticles. Confocal laser scanning microscope (CLSM) was used to detect HA-PLGA@DiR-BOA and PLGA@DiR-BOA in 4T1 cells. 4T1 cells were seeded in confocal culture dishes at a cell count of 2.0×10 5 / well, and then cultured under standard culture conditions (37 ° C, 5% CO2) for 24 hours to ensure uniform attachment and growth of cells. Afterwards, the culture medium in the dish was aspirated and discarded, and then the drug culture medium containing HA-PLGA@DiR-BOA and PLGA@DiR-BOA was added and cultured for 4 hours. The culture medium containing the drug was then removed and washed multiple times with PBS solution to remove residual drugs. The cells were stained with the prepared Hoechst 33342 dye (500 μL / dish) for 5 minutes. After staining, they were washed twice with PBS buffer to remove excess dye. Next, 500 μL of 4% paraformaldehyde solution was added to fix the cells for 15 minutes, and then washed twice with PBS buffer to remove the 4% paraformaldehyde solution. Finally, 100 μL of PBS buffer was added to the culture dish, and the cells were imaged using CLSM to observe the uptake of the drug.

[0093] The results are as follows Figure 6 As shown in the figure, the cell nucleus stained with Hoechst 33342 emits blue fluorescence, and the cytoplasm emits red fluorescence after the near-infrared dye DiR-BOA is taken up by the cells. The Merge figure shows that after culturing 4T1 cells for 4 hours, HA-PLGA@DiR-BOA can be observed in the cytoplasm with obvious red fluorescence. 4T1 cells take up significantly more HA-PLGA@DiR-BOA than PLGA@DiR-BOA, indicating that the HA-modified nanoparticles HA-PLGA@DiR-BOA can target 4T1 cells.

[0094] (IV) NO release at the cellular level

[0095] 4T1 cells (2.5×10 4Cells were seeded in 24-well plates (100 cells / well) and cultured at 37°C and 5% CO₂ for 24 hours. A drug-containing medium containing PLGA-SNAP-DOX (NO: 0.4 μM, DOX: 2.0 μM) was then added for 6 hours. The medium was aspirated, and the cells were gently washed three times with PBS. Following washes, cells were stained with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA) for 30 minutes at 37°C. The cells were then washed twice with PBS to remove any residual stain. Next, cells were fixed with 500 μL of 4% paraformaldehyde solution for 15 minutes and then washed twice with PBS to remove the 4% paraformaldehyde. DAPI dye was then stained for 30 minutes, followed by washing with PBS. Finally, 100 μL of PBS buffer was added to the culture dish, and imaging was performed using a confocal laser scanning microscope (DAF-FM Ex: 480 nm; DAPI Ex: 405 nm).

[0096] The results are as follows Figure 7 As shown, the specific NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (3-Amino,4-aminomethyl-2',7'-difluorescein, DAF-FM) was used to further detect the ability of PLGA-SNAP-DOX to release NO intracellularly. Since the PLGA-DOX group did not contain SNAP (NO donor), it had almost no green fluorescence signal. However, compared with PLGA-DOX, PLGA-SNAP-DOX produced a stronger green fluorescence signal in 4T1 cells. The results showed that PLGA-SNAP-DOX loaded with NO donor was effectively internalized by 4T1 cells and successfully released NO intracellularly.

[0097] (V) In vitro cytotoxicity evaluation of nanoparticles

[0098] The cytotoxicity of PLGA-SNAP-DOX was determined by CCK-8 assay. 4Cells / well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. 4T1 cells were incubated with different concentrations of DOX (0.2, 2, 20, 40, 80 μM) or NO (0.04, 0.4, 3.6, 7.2, 14.4 μM) of nanoparticles for 24 hours. After incubation, the culture medium in the well plate was removed, washed twice with 200 μL PBS buffer, and the cells were incubated with complete culture medium containing 10% CCK-8 reagent at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader (MOLECULAR DEVICES, Spectra Max Id3) to determine the percentage of remaining cell viability.

[0099] Figure 8 The results showed that after incubation of 4T1 cells with different concentrations of DOX (0.2, 2, 20, 40, 80 μM) and NO (0.04, 0.4, 3.6, 7.2, 14.4 μM), PLGA-DOX and PLGA-SNAP-DOX induced enhanced antitumor activity, and cell viability decreased in a concentration-dependent manner, while PLGA-SNAP exhibited mild cytotoxicity to 4T1 cells. In addition, the cytotoxicity of PLGA-DOX was much weaker than that of PLGA-SNAP-DOX, with cell viability ranging from 97% to 34% for PLGA-DOX and 98% to 18% for PLGA-SNAP-DOX. These results indicate that PLGA-SNAP-DOX has strong cytotoxicity against 4T1 cells, which can be attributed to the synergistic antitumor effect of DOX and NO.

[0100] (VI) Evaluation of the immunogenic death effect of nanoparticles

[0101] To investigate the immunogenic cell death effects in vitro, immunofluorescence staining was performed after cells were treated with various methods. 4T1 cells (2×10 5Cells / well) were seeded in confocal culture dishes and cultured at 37°C and 5% CO2 for 24 hours. The cells were incubated with culture medium containing different drugs PLGA, HA-S@PLGA, HA-D@PLGA, and HA-DS@PLGA for 6 hours. They were then fixed with 4% paraformaldehyde for 15 minutes, followed by washing three times with PBS buffer for 5 minutes each. They were then treated with blocking solution and blocked for 1 hour. Antibody dilution (calreticulin CRT) was added to the culture dish, placed in a 4°C refrigerator, incubated for 12 hours, and washed three times with PBS buffer for 5 minutes. The secondary antibody Alexa Fluor 488 fluorescent probe was then incubated for 1 hour. Washed three times with PBS buffer for 5 minutes. DAPI dye was stained at room temperature for 30 minutes, washed twice with PBS to wash away the residual dye, and observed with a confocal laser scanning microscope.

[0102] For the detection of high mobility group protein B1 (HMGB 1), after the cells are fixed, use 0.3% (v / v) TritonX-100 / PBS (200 μL per well) to permeabilize the cells for 10 minutes, wash with PBS buffer for 5 minutes, and wash three times. Then treat with blocking solution and block for 1 hour. Add the antibody dilution (high mobility group protein B1) to the culture dish, place it in a 4°C refrigerator, incubate for 12 hours, wash with PBS buffer for 5 minutes, and wash three times. Then incubate with the secondary antibody Alexa Fluor 594 fluorescent probe for 1 hour. Wash with PBS buffer for 5 minutes, and wash three times. Stain with DAPI dye at room temperature for 30 minutes, wash twice with PBS, wash away the residual dye, and observe with a confocal laser scanning microscope.

[0103] During the entire experimental procedure, direct light should be avoided to reduce fluorescence attenuation. For flow cytometric analysis of calreticulin expression, treated cells were fixed in 4% paraformaldehyde solution, incubated with anti-calreticulin antibody (Abcam, USA) for 30 minutes, and then incubated with goat anti-rabbit IgG (secondary antibody) conjugated to Alexa Fluor 488 fluorescent probe for 30 minutes. The cells were pelleted by centrifugation (3500 rpm, 5 minutes), and the supernatant was carefully discarded. The cell pellet was then resuspended in PBS buffer and washed three times to remove residual culture medium and other impurities. After washing, the cells were finally resuspended in 1000 μL of PBS. The calreticulin protein expression level of the cells was then quantified using a CytoFLEX flow cytometer. HMGB1 was detected using an enzyme-linked immunosorbent assay (ELISA) kit.

[0104] Figure 9Results showed that the ICD-inducing ability of PLGA-SNAP, PLGA-DOX, and PLGA-SNAP-DOX in 4T1 cells was further investigated by measuring CRT expression and HMGB1 release. Confocal imaging was used to analyze the membrane exposure of CRT in 4T1 cells after treatment with PLGA-SNAP, PLGA-DOX, and PLGA-SNAP-DOX. PLGA-DOX and PLGA-SNAP-DOX treatment induced significant CRT exposure in 4T1 cells, while PBS, PLGA, and PLGA-SNAP treatment induced negligible CRT exposure. HMGB1 secretion was also examined by confocal fluorescence imaging. Strong HMGB1 red fluorescence signals were observed in the PLGA-DOX and PLGA-SNAP-DOX groups, while PBS, PLGA, and PLGA-SNAP treatments showed negligible HMGB1 release.

[0105] like Figure 10 As shown in Figure 2, flow cytometry was used to quantitatively investigate the CRT exposure of 4T1 cells after different treatments. Compared to the PBS control group, the proportion of PLGA-SNAP and PLGA-DOX cells increased by 1.5 and 2.0 times, respectively. It was also noted that the proportion of PLGA-SNAP-DOX cells increased by 2.4 times. In addition, Figure 11 As shown in Figure 2, this example also used ELISA to quantify the concentration of HMGB1 in the cell supernatant after different treatments. The amount of HMGB1 released from the PLGA-SNAP-DOX-treated group was 3.0 times higher than that from the PLGA-DOX-treated group. This indicates that PLGA-SNAP-DOX can induce the ICD effect to the greatest extent, which is consistent with the above-mentioned CLSM results.

[0106] (VII) Immunomodulation of tumor microenvironment by nanoparticles in vitro

[0107] 1. Culture of Macrophages (BMDM) and Dendritic Cells (DCs)

[0108] Cell acquisition and isolation: Bone marrow was harvested from the femur and tibia of mice to obtain bone marrow-derived macrophages (BMDM) and dendritic cells (DCs), and macrophages and DCs were isolated.

[0109] Culture of BMDM macrophages:

[0110] (1) The isolated bone marrow mononuclear cells (referring to macrophages BMDM) were cultured at 4×10 6Cells were seeded / well in a 6-well plate and cultured in RPMI1640 medium supplemented with 10% FBS, 1% PS, and 20 ng / mL macrophage colony-stimulating factor (M-CSF, PEPROTECH) in an incubator at 37°C and 5% CO2.

[0111] (2) On day 5, the culture medium was replaced with fresh culture medium containing M-CSF at a concentration of 20 ng / mL.

[0112] (3) On day 7, macrophages were washed and cultured in fresh medium. They were co-stimulated with IL-4 (10 ng / mL) and IL-13 (10 ng / mL) for 48 hours to obtain M2 macrophages for subsequent studies.

[0113] Dendritic cell (DCs) culture:

[0114] Dendritic cells (DCs) were cultured in an incubator at 37°C and 5% CO2 in a medium supplemented with granulocyte-macrophage colony-stimulating factor (GM CSF).

[0115] 2. Immunomodulatory Effects of Nanoparticles on Tumor Microenvironment in Vitro

[0116] (1) Macrophages and DCs were divided into 5×10 5 Cells / well were seeded in a 24-well plate, and 4T1 cells (5×10 5 ) were placed in the well plate and incubated for 24 hours (ie, the mixture of nanomedicine and 4T1 cells was inoculated into the well plate containing macrophages and the well plate containing DCs, respectively).

[0117] (2) Cells were collected and stained with anti-CD45-FITC, anti-CD11c-BV421, anti-CD80-PE, anti-CD86-APC, anti-F4 / 80-AF488, and anti-CD206-BV421. Data were collected on a flow cytometer (Beckman Coulter, USA) and analyzed using FlowJo software. Figure 12 and Figure 13 shown. Figure 12 This is a graph showing changes in the expression of CD206 in macrophages after nanoparticle treatment. Figure 13 This is a graph showing the changes in CD80 and CD86 expression in DCs after nanoparticle treatment.

[0118] The results of flow cytometry analysis showed that the PLGA-SNAP and PLGA-SNAP-DOX treatment groups significantly reduced the expression of CD206, indicating that the M2 macrophage immunosuppressive cells were downregulated and the immunosuppressive tumor microenvironment was improved. To confirm the maturation of DCs, the nanoparticles were incubated with 4T1 cells for 4 hours and then incubated with immature DCs (iDCs) for 24 hours. Afterwards, flow cytometry analysis was performed using co-stimulatory molecules CD80 and CD86 as maturation markers to quantify the level of DCs maturation. The results showed that compared with PLGA-SNAP and PLGA-DOX, PLGA-SNAP-DOX greatly promoted the maturation of CDs, and CD80 + CD86 + The expression of PLGA-SNAP-DOX in the ICD-induced cells was significantly higher (2.0-fold for CD80 and 3.5-fold for CD86), which was consistent with the pattern observed in the ICD-induced results. These results indicate that PLGA-SNAP-DOX can promote the increase of ICD and DC maturation in vitro, reduce M2 macrophages, reverse the tumor immunosuppressive microenvironment, and enhance the anti-tumor immune response.

[0119] (8) In vivo anti-tumor experiments

[0120] (1) Female BALB / c mice aged 6–8 weeks were purchased from Hunan SJA Experimental Animal Co., Ltd. (Changsha, Hunan, China).

[0121] (2) For the construction of mouse orthotopic tumor model, 1×10 7 4T1 cells were injected subcutaneously into the right hind thigh of each mouse at a volume of 100 μL. The tumor volume was calculated by length (mm) × width (mm) × width (mm) / 2. The tumor grew to 80-100 mm. 3 Get treatment.

[0122] (3) Mice were randomly divided into different groups: PBS, PLGA, HA-S@PLGA, HA-D@PLGA, and HA-DS@PLGA. Tumor-bearing mice were injected intratumorally with the nanoparticle solution under anesthesia, and tumor growth and mouse body weight were monitored for up to 15 days after implantation (n = 5 mice).

[0123] (4) Starting from day 4, the tumors in the PBS control group reached a measurable tumor size, so the tumor volume and mouse body weight were measured from this date. The mice were euthanized on day 15 of the drug intervention, and tumor tissues were collected for further analysis. Tumor growth and mouse body weight were monitored during the treatment period. Figure 14 and Figure 15 As shown. Among them, Figure 14 The monitoring result of tumor size is Figure 15 This is the monitoring result of mouse body weight.

[0124] Figure 14 and Figure 15 The results showed that mice treated with PLGA-DOX had a moderate inhibition of tumor growth, while the PLGA-SNAP-DOX-treated group showed a more exciting tumor suppression effect compared to the PBS-treated group. This can be attributed to the synergistic effect caused by DOX and NO. It should be noted that at the end of treatment (7 days after injection), the tumor volume of mice receiving PLGA-SNAP-DOX was reduced to 37.8mm 3 The PLGA-SNAP-DOX group had a significantly lower mean tumor size than the PLGA-DOX group (8.6-fold and 18.0-fold smaller than the PLGA-DOX and PLGA-SNAP groups, respectively), indicating that the PLGA-SNAP-DOX group plays an important role in the treatment of tumors by reversing the tumor immune microenvironment and enhancing the immune response. Furthermore, no weight loss was observed in any of the 4T1 tumor-bearing mice during the entire treatment process, demonstrating the safety of PLGA-SNAP-DOX therapy.

[0125] (IX) In vivo apoptosis test in tumor tissue

[0126] (1) The collected tumor tissues were fixed in 4% paraformaldehyde at 4°C for 12 hours and then dehydrated with 30% sucrose solution. The tissues were then frozen in OCT gel (Sakura, Torrance, CA, USA) and sliced using a freezing microtome (Leica, Germany). They were then stained with TUNEL and DAPI in sequence, and data were collected using a fluorescence microscope (Olympus FV 3000, Tokyo, Japan) and analyzed using ImageJ software. The results are shown in Figure 2. Figure 16 shown.

[0127] Figure 16 Results from the TUNEL staining assay revealed strong green fluorescence in the PLGA-SNAP-DOX and PLGA-DOX-treated groups. In contrast, only a few apoptotic cells were observed in the PBS, PLGA, and PLGA-SNAP-treated groups. Overall, these results suggest that NO released by PLGA-SNAP-DOX and DOX maximized their synergistic therapeutic effect in vivo, significantly inhibiting tumor growth.

[0128] Based on the above examples, the present application further optimizes the dosage of doxorubicin, SNAP, and DOTAP. The results show that every 10 mg of PLGA can embed 0.1 to 10 mg of doxorubicin, 0.1 to 5 mg of SNAP, and 0.1 to 5 mg of DOTAP, that is, every 10 parts by weight of PLGA can embed 0.1 to 10 parts by weight of doxorubicin, 0.1 to 5 parts by weight of SNAP, and 0.1 to 5 parts by weight of DOTAP. In addition, the present application further tested the number average molecular weight of PLGA, and the results showed that PLGA with a number average molecular weight of 5 to 30 KD is suitable for this application, among which PLGA with a number average molecular weight of 5 to 15 KD has the best effect. As for the particle size of the nanoparticles, the optimal range is 50 to 200 nm.

[0129] The PLGA-SNAP-DOX nanoparticles prepared under these conditions can be used for the simultaneous delivery of DOX and SNAP, while also achieving the slow release of NO. By releasing NO, they synergistically enhance the ICD effect of DOX on tumor cells, reducing M2 TAMs and reversing the immunosuppressive tumor microenvironment. Nanoparticles prepared under these conditions can successfully reshape the immunosuppressive microenvironment and inhibit tumor growth, thereby achieving synergistic anti-tumor therapy.

[0130] The above content is a further detailed description of the present application in conjunction with specific implementation methods, and the specific implementation of the present application cannot be considered to be limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, several simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A nanopharmaceutical, characterized in that: The nanomedicine slowly releases nitric oxide and is loaded with anticancer drugs. The nanomedicine is a nanoparticle formed by self-assembled polylactic acid-glycolic acid block copolymer encapsulating doxorubicin, nitrosoacetylpenicillamine, and cationic liposomes. The surface of the nanoparticle is modified with hyaluronic acid. The cationic liposome is trimethyl-2,3-dioleoyloxypropylammonium bromide. The preparation method of the nanomedicine comprises: Step 1, dissolving doxorubicin, nitrosoacetylpenicillamine and cationic liposomes in a first organic solvent to obtain a mixture solution; Step 2: adding polylactic acid-glycolic acid block copolymer in a light-proof environment, and vacuuming the mixture solution until a colloidal mixture is formed; Step 3, adding a first organic solvent dropwise to the colloidal mixture to dissolve it, thereby obtaining a new mixture solution; Step 4, adding the new mixture solution dropwise into ultrapure water to obtain an emulsion; Step 5, ultrasonically treating the emulsion to volatilize the first organic solvent; or, after volatilizing the first organic solvent, adding the hyaluronic acid solution and ultrasonically treating again to volatilize the solvent; Step 6: After the ultrasonic treatment is completed, the mixture is placed in an ice bath to obtain a nanosuspension, and the nanosuspension is ultrafiltered to obtain nanoparticles, i.e., the nanopharmaceutical.

2. The nanopharmaceutical according to claim 1, wherein: The number average molecular weight of the polylactic acid-glycolic acid block copolymer is 5-30KD.

3. The nanopharmaceutical according to claim 1, wherein: The particle size of the nanoparticles is 50-200 nm.

4. The nanopharmaceutical according to any one of claims 3, wherein: The molar ratio of the polylactic acid-glycolic acid block copolymer, adriamycin, nitrosoacetylpenicillamine and cationic liposome is 0.1-1:2-2.5:2-2.3:1-1.

5.

5. The nanopharmaceutical according to claim 4, characterized in that: The first organic solvent is tetrahydrofuran; and / or, the doxorubicin is pre-dissolved in a second organic solvent; And / or, the ultrapure water is preheated to 37° C. before being added dropwise; and / or, the solvent of the hyaluronic acid solution is the first organic solvent; The second organic solvent is chloroform.

6. The nanopharmaceutical according to claim 5, characterized in that: In step 4, ultrasonic treatment is performed using an ultrasonic water bath while the new mixture solution is added dropwise; The temperature of the ultrasonic water bath was 37°C; The power of ultrasonic water bath for ultrasonic treatment was 200W; In step 5, the power of the ultrasonic treatment is 100-200 W, and the ultrasonic treatment time is at least 10 minutes; In step 5, the solvent is volatilized by ultrasonic heating without ice bath protection during ultrasonic treatment; In step 5, the amount of hyaluronic acid is 0.1 to 1 parts by weight of hyaluronic acid per 10 parts by weight of the polylactic acid-glycolic acid block copolymer; In step 6, before ultrafiltration, after the ice bath, the mixture is first placed at room temperature, away from light, with the lid open, to allow the residual solvent to evaporate, and then stored at a temperature of less than or equal to 4° C. away from light to obtain a nanosuspension; Step 6, after the nanosuspension is ultrafiltered, further includes ultrasonic washing, and then ultrafiltration, and the washing and ultrafiltration are repeated at least twice.

7. Use of the nanopharmaceutical according to any one of claims 1 to 6 in the preparation of anti-tumor drugs.

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