Psoralen mannosylated chitosan polymer nanoparticles and preparation method and application thereof

Abstract

The present invention discloses a psoralen mannosylated chitosan polymer nanoparticle and its preparation method and application. The psoralen is composed of psoralen, poly(lactic-co-glycolic acid) copolymer, mannosylated chitosan and polyvinyl alcohol, wherein the mass ratio of psoralen, poly(lactic-co-glycolic acid) copolymer, mannosylated chitosan and polyvinyl alcohol is 2-10:10-30:16.5-38.5:1-9. The psoralen mannosylated chitosan polymer nanoparticle is composed of free psoralen drug and mannosylated chitosan polymer carrier. Compared with traditional psoralen, the psoralen has a more stable structure and better physicochemical properties, and has higher bioavailability and tumor targeting. It can be used as a regulator of tumor-associated macrophages and cytokines, and can also inhibit the growth of MDA-MB-231 breast cancer cells and can be used to prepare a drug for treating triple-negative breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to psoralen polymer nanoparticles and a preparation method and application thereof, and specifically relates to psoralen mannosylated chitosan polymer nanoparticles and a preparation method and application thereof. Background Art

[0002] Breast cancer is a common malignancy in women. Triple-negative breast cancer (TNBC), which is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounts for 10-15% of breast cancer patients. It is characterized by early onset, aggressiveness, metabolic remodeling, and poor prognosis. Currently, anti-cancer drug research primarily targets the tumor cells themselves. These include altering multidrug resistance genes, inhibiting the upregulation of efflux pump proteins to reduce drug efflux, inhibiting metabolic activity to enhance drug activity, and regulating apoptosis gene expression to induce tumor cell apoptosis. However, these approaches have been less than ideal. One reason for this is that tumor cell development stems not only from changes in the cells themselves but also from alterations in their microenvironment. With increasing understanding of TNBC tumor biology, therapeutic strategies targeting the tumor microenvironment (TME) are gaining increasing attention.

[0003] The tumor microenvironment (TME) refers to the local homeostatic environment composed of tumor cells, fibroblasts, mesenchymal cells, vascular endothelial cells, smooth muscle cells, inflammatory / immune cells, and the extracellular matrix. The TME differs significantly from the normal tissue microenvironment, characterized by hypoxia, weak acidity, a reducing environment, and low vascular density. The TME plays a crucial role in tumor development and progression, including proliferation, immune escape, and distant metastasis. The immune component of the TME is composed of tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), dendritic cells (DCs), and tumor-infiltrating lymphoid cells (TILs). The breast cancer immune microenvironment refers to the local internal environment formed by immune cells infiltrating breast tumors and the active mediators they secrete, along with breast cancer cells. This immunosuppressive microenvironment is primarily mediated by TAMs, TANs, MDSCs, and regulatory T cells (Tregs). The cytokines secreted by these cells, including vascular endothelial growth factor (VEGF), IL-1β, IL-6, IL-10, IL-17, TGF-β, tumor necrosis factor α (TNF-α), and matrix metalloproteinases (MMPs), are important components of the TME. They primarily monitor and defend tumor cells, directly or indirectly influencing tumor development and progression. Tumor-associated macrophages (TAMs) are among the most abundant immune cells infiltrating breast cancer. M2 macrophages exert immunosuppressive effects within the immune microenvironment, promoting breast cancer cell proliferation by activating immune checkpoints and secreting immunosuppressive factors.

[0004] TAMs originate from vascular mononuclear cells and account for 30% to 50% of the total TME cell population. Under the long-term influence of the TME, monocytes / macrophages undergo polarization. TAMs are primarily classified into two phenotypes. M1 TAMs secrete immunostimulatory factors such as IL-1β, TNF-α, and IFN-γ, and highly express tumor necrosis factor (TNF), iNOS, and MHC. They participate in polarized Th1 cell responses and activate cytotoxic T lymphocytes (CTLs) to exert anti-tumor immunity. M2 TAMs secrete immunosuppressive factors such as IL-6, IL-10, and TGF-β, and highly express Arg-1, IL-10, CD163, and CD206. They participate in tumor cell growth, metastasis, angiogenesis, immunosuppression, and tissue repair, mediating tumor immune escape and promoting tumor growth. Inhibiting the tumor-promoting phenotype of TAMs in the microenvironment, inducing the conversion of TAMs to a tumor-suppressing phenotype, or regulating the ratio and number of the two phenotypes of TAMs is expected to inhibit the growth of triple-negative breast cancer, but there is currently little research on TAMs regulators for triple-negative breast cancer.

[0005] Cytokines are important signaling molecules for intercellular transduction and play a crucial role in regulating the tumor microenvironment. Inflammatory cytokines are signaling molecules secreted by inflammatory cells. Studies have shown that regulating the expression of important immune / inflammatory cytokines in the tumor microenvironment is one strategy for treating breast cancer. However, current monoclonal antibody-based cytokine therapies are ineffective and often have side effects. Therefore, there is a need to find new therapeutic drugs that can simultaneously regulate multiple cytokines to significantly improve the tumor microenvironment.

[0006] Psoralen (PSO), a furanocoumarin compound, is one of the active ingredients in the traditional Chinese medicine (Bu Gu Zhi) and exhibits immunomodulatory, anti-inflammatory, antibacterial, antioxidant, anti-osteoporotic, and anti-tumor properties. PSO inhibits cell proliferation by inducing G0 / G1 arrest in tumor cells and regulating the Wnt / β-catenin pathway. It also suppresses tumor invasion and migration by inhibiting the NF-κB signaling pathway and epithelial-mesenchymal transition (EMT). It also reverses multidrug resistance by inhibiting Bc1-2 gene protein expression, ABCB1 promoter activity, and P-gp transporter efflux, making it a promising anti-tumor drug. However, PSO suffers from poor solubility, resulting in low bioavailability and limiting its anti-tumor application.

[0007] In view of this, it is necessary to provide a substance that can improve the poor solubility of PSO to enhance its bioavailability, tumor targeting and breast cancer treatment effect. Summary of the Invention

[0008] To this end, the technical problem to be solved by the present invention is that psoralen has poor solubility and biocompatibility and is difficult to be used in breast cancer treatment drugs, thereby proposing a psoralen mannosylated chitosan polymer nanoparticle with better solubility and biocompatibility, as well as a preparation method and application thereof.

[0009] In order to solve the above technical problems, the technical solution of the present invention is:

[0010] The first aspect of the present invention provides psoralen mannosylated chitosan polymer nanoparticles, which are composed of psoralen, poly(lactic acid-glycolic acid) copolymer, mannosylated chitosan and polyvinyl alcohol, wherein the mass ratio of psoralen, poly(lactic acid-glycolic acid) copolymer, mannosylated chitosan and polyvinyl alcohol is 2-10:10-30:16.5-38.5:1-9.

[0011] The second aspect of the present invention provides a method for preparing the psoralen mannosylated chitosan polymer nanoparticles, which comprises the following steps:

[0012] S1. Preparing mannosylated chitosan: weighing chitosan and mannose in a mass ratio of 1-5:5, dissolving chitosan in an acetic acid-sodium acetate buffer solution and stirring in the dark to obtain a chitosan solution; dissolving mannose in an acetic acid-sodium acetate buffer solution and stirring to ring-open the solution to obtain a mannose solution; adding the mannose solution and a catalyst to the chitosan solution, stirring uniformly, and dialyzing to obtain a mannosylated chitosan solution;

[0013] S2. Preparing psoralen polymer nanoparticles: dissolving poly(lactic acid-co-glycolic acid) copolymer in dichloromethane, adding a dehydrating agent and a modifier for reaction to obtain a pre-reaction product, adding psoralen to the pre-reaction product and mixing uniformly, adding the obtained mixed solution to a polyvinyl alcohol solution, performing low-temperature ultrasonication, stirring and mixing uniformly, performing an emulsification reaction, and centrifuging to obtain the supernatant to obtain a psoralen polymer nanoparticle solution;

[0014] S3. Preparation of psoralen mannosylated chitosan nanoparticles: adding the psoralen polymer nanoparticle solution to the mannosylated chitosan solution, stirring in the dark, and centrifuging. Filtering the supernatant after centrifugation to obtain psoralen mannosylated chitosan nanoparticles.

[0015] The third aspect of the present invention provides a use of the psoralen mannosylated chitosan nanoparticles in tumor-associated macrophages and cytokine regulators.

[0016] The fourth aspect of the present invention provides a use of the psoralen mannosylated chitosan nanoparticles in the preparation of a drug for treating triple-negative breast cancer.

[0017] The above technical solution of the present invention has the following advantages over the prior art:

[0018] The present invention provides psoralen mannosylated chitosan polymer nanoparticles, which are composed of psoralen, poly(lactic-co-glycolic acid) copolymer, mannosylated chitosan, and polyvinyl alcohol, wherein the mass ratio of psoralen, poly(lactic-co-glycolic acid) copolymer, mannosylated chitosan, and polyvinyl alcohol is 2-10:10-30:16.5-38.5:1-9. The psoralen mannosylated chitosan polymer nanoparticles, which are composed of free psoralen and a mannosylated chitosan polymer carrier, have a more stable structure and better physicochemical properties than traditional psoralens. They also have higher bioavailability and tumor targeting, can be used as regulators of tumor-associated macrophages and cytokines, can inhibit the growth of MDA-MB-231 breast cancer cells, and can therefore be used in the preparation of drugs for treating triple-negative breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0020] Figure 1 is a TEM image of psoralen mannosylated chitosan polymer nanoparticles provided in Example 1 of the present invention;

[0021] Figure 2 It is the Fourier infrared spectrum;

[0022] Figure 3 It is an X-ray diffraction test pattern;

[0023] Figure 4 is the differential scanning calorimetry spectrum;

[0024] Figure 5 is the thermogravimetric analysis spectrum;

[0025] Figure 6 This is the laser confocal test image of MCSPP NPs in MDA-MB-231 cells;

[0026] Figure 7 (a)-(b) are flow cytometric images of MCSPP NPs in MDA-MB-231 cells;

[0027] Figure 8 This is the laser confocal test image of MCSPP NPs in macrophages;

[0028] Figure 9 (a)-(b) are flow cytometric images of MCSPP NPs in macrophages;

[0029] Figure 10 This is a test graph of the effect of MCSPP NPs on cyclin expression detected by Western blot;

[0030] Figure 11 This is a test image of the effect of MCSPP NPs on MDA-MB-231 cell apoptosis detected by laser confocal microscopy;

[0031] Figure 12 This is a test graph of the effect of MCSPP NPs on MDA-MB-231 cell apoptosis detected by flow cytometry;

[0032] Figure 13 This is a test diagram of the effect of MCSPP NPs on the cell cycle of MDA-MB-231 cells and co-culture system;

[0033] Figure 14This is a test diagram of the effect of MCSPP NPs on the cell cycle of MDA-MB-231 cells and co-culture system;

[0034] Figure 15 This is a test graph of the effect of MCSPP NPs on apoptosis in the MDA-MB-231 cell co-culture system detected by flow cytometry;

[0035] Figure 16 is a graph of cell apoptosis detection after MCSPP NPs treated M2-CM co-cultured breast cancer cells;

[0036] Figure 17 (a)-(b) are test graphs showing the effects of MCSPP NPs on the secretion of IL-10, IL-12, TNF-α, and TGF-β in macrophages;

[0037] Figure 18 (a)-(b) are test diagrams of the regulatory effect of MCSPP NPs on TAMs phenotype. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise specified, the reagents, methods and equipment used in the examples of the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available.

[0040] Example 1

[0041] This embodiment provides psoralen mannosylated chitosan polymer nanoparticles, which are made of psoralen (PSO), poly(lactic-co-glycolic acid) (PLGA), mannosylated chitosan (MAN-CS), and polyvinyl alcohol. The carbon-nitrogen atomic ratio (C / N) of the mannosylated chitosan is 5.31 ± 0.03, the actual deacetylation degree of chitosan is 90.25%, and the amino substitution degree is 10.25%.

[0042] The psoralen mannosylated chitosan polymer nanoparticles (MCSPP NPs) provided in this example were prepared by the following method:

[0043] S1. Preparation of mannosylated chitosan (MAN-CS): Accurately weigh 19 mg of chitosan and dissolve it in 10 mL of acetic acid-sodium acetate (HAc-NaAc, pH 4.5) buffer solution. Stir in the dark at 25°C for 4 h to obtain a chitosan solution. Accurately weigh 15.2 mg of D-mannose and dissolve it in 1 mL of acetic acid-sodium acetate (HAc-NaAc, pH 4.5) buffer solution. Stir at 60°C for 2 h to ring-open mannose and obtain a mannose solution. Add the mannose solution and the catalyst sodium triacetoxyborohydride to the chitosan solution, stir at room temperature for 24 h, and dialyze the resulting product in a dialysis bag (Mw 12000-14000) for 48 h to obtain a mannosylated chitosan polymer solution.

[0044] S2. Preparation of psoralen polymer nanoparticles (PP NPs): Accurately weigh 15 mg of poly(lactic acid-co-glycolic acid) copolymer, add 2 mg of dicyclohexylcarbodiimide (DCC) and 2 mg of N-hydroxysuccinimide (NHS) into 5 mL of dichloromethane organic solvent, react at room temperature for 6 h, add 8 mg of psoralen (PSO), mix well and let stand for 10 min, slowly add the resulting mixed solution dropwise into 18 mL of 0.5% polyvinyl alcohol (PVA) solution; treat with ultrasound (power 257 W) for 8 min in an ice bath, then stir at 40°C and 480 rpm for 2 h for emulsification reaction, remove the organic solvent by rotary evaporation at 40°C, centrifuge at 3000 rpm for 10 min, and collect the supernatant to obtain psoralen polymer nanoparticle solution.

[0045] S3. Preparation of psoralen mannosylated chitosan nanoparticles (MCSPP NPs): The psoralen polymer nanoparticle solution obtained in step S2 was slowly added dropwise to the mannosylated chitosan solution obtained in step S1, stirred in the dark at room temperature for 2 h, centrifuged at 3000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain MCSPP NPs.

[0046] The psoralen mannosylated chitosan nanoparticles provided in this embodiment are prepared by loading free psoralen onto a mannosylated chitosan polymer carrier. Compared with traditional psoralen, the nanoparticles have a more stable structure, more diverse components, and better physical and chemical properties, and have higher bioavailability. At the same time, tests have shown that they also have high in vitro drug release, long release time, pH sensitivity, and sustained release properties. It can downregulate IL-10 and TGF-β secretion, upregulate IL-12 and TNF-α secretion, and downregulate the expression of CD206, a tumor-associated macrophage M2 marker, so that it can be applied to tumor-associated macrophages and cytokine regulators. Furthermore, it can also downregulate Cyclin D1, upregulate Cyclin A2 expression, induce MDA-MB-231 breast cancer cell cycle arrest and apoptosis, and thus can be used to prepare drugs for the treatment of triple-negative breast cancer.

[0047] Example 2

[0048] This embodiment provides psoralen mannosylated chitosan polymer nanoparticles, which are made of psoralen (PSO), poly(lactic-co-glycolic acid) (PLGA), mannosylated chitosan (MAN-CS) and polyvinyl alcohol. The carbon-nitrogen atomic ratio (C / N) of the mannosylated chitosan is 5.31 ± 0.03, the actual deacetylation degree of chitosan is 90.25%, and the amino substitution degree is 1.05%.

[0049] The psoralen mannosylated chitosan polymer nanoparticles (MCSPP NPs) provided in this example were prepared by the following method:

[0050] S1. Preparation of mannosylated chitosan (MAN-CS): Accurately weigh 25 mg of chitosan and dissolve it in 10 mL of acetic acid-sodium acetate (HAc-NaAc, pH 4) buffer solution. Stir in the dark at 20°C for 5 h to obtain a chitosan solution. Accurately weigh 10 mg of D-mannose and dissolve it in 1 mL of acetic acid-sodium acetate (HAc-NaAc, pH 4) buffer solution. Stir at 40°C for 3 h to ring-open mannose and obtain a mannose solution. Add the mannose solution and the catalyst sodium triacetoxyborohydride to the chitosan solution, stir at room temperature for 20 h, and dialyze the resulting product in a dialysis bag (Mw 12000-14000) for 40 h to obtain a mannosylated chitosan polymer solution.

[0051] S2. Preparation of psoralen polymer nanoparticles (PP NPs): Accurately weigh 10 mg of poly(lactic acid-co-glycolic acid) copolymer, dissolve it in 5 mL of dichloromethane organic solvent, add 1.33 mg of dicyclohexylcarbodiimide (DCC) and 1.33 mg of N-hydroxysuccinimide (NHS), react at room temperature for 3 h, add 2 mg of psoralen (PSO), mix well and let it stand for 5 min, slowly add the resulting mixed solution dropwise to 15 mL of 0.5% polyvinyl alcohol (PVA) solution, mix and emulsify; treat with ultrasound (power 150 W) in an ice bath for 10 min, then stir at 400 rpm at 20°C for 3 h, remove the organic solvent by rotary evaporation at 30°C, centrifuge at 2000 rpm for 15 min, and collect the supernatant to obtain the psoralen polymer nanoparticle solution.

[0052] S3. Preparation of psoralen mannosylated chitosan nanoparticles (MCSPP NPs): The psoralen polymer nanoparticle solution obtained in step S2 was slowly added dropwise to the mannosylated chitosan solution obtained in step S1. The mixture was stirred in the dark at room temperature for 1 h. The mixture was centrifuged at 2000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter to obtain MCSPP NPs.

[0053] Example 3

[0054] This embodiment provides psoralen mannosylated chitosan polymer nanoparticles, which are made of psoralen (PSO), poly(lactic-co-glycolic acid) (PLGA), mannosylated chitosan (MAN-CS), and polyvinyl alcohol. The carbon-nitrogen atomic ratio (C / N) of the mannosylated chitosan is 5.31 ± 0.03, the actual deacetylation degree of chitosan is 90.25%, and the amino substitution degree is 16.8%.

[0055] The psoralen mannosylated chitosan polymer nanoparticles (MCSPP NPs) provided in this example were prepared by the following method:

[0056] S1. Preparation of mannosylated chitosan (MAN-CS): Accurately weigh 20 mg of chitosan and dissolve it in 10 mL of acetic acid-sodium acetate (HAc-NaAc, pH 6) buffer solution. Stir in the dark at 30°C for 2 h to obtain a chitosan solution. Accurately weigh 16 mg of D-mannose and dissolve it in 1 mL of acetic acid-sodium acetate (HAc-NaAc, pH 6) buffer solution. Stir at 80°C for 1 h to ring-open the mannose and obtain a mannose solution. Add the mannose solution and the catalyst sodium triacetoxyborohydride to the chitosan solution and stir at room temperature for 30 h. Place the resulting product in a dialysis bag (Mw 12000-14000) and dialyze it for 55 h to obtain a mannosylated chitosan polymer solution.

[0057] S2. Preparation of psoralen polymer nanoparticles (PP NPs): Accurately weigh 30 mg of poly(lactic acid-co-glycolic acid) copolymer, add 4 mg of dicyclohexylcarbodiimide (DCC) and 4 mg of N-hydroxysuccinimide (NHS) into 5 mL of dichloromethane organic solvent, react at room temperature for 8 h, add 10 mg of psoralen (PSO), mix well and let stand for 15 min, slowly add the resulting mixed solution dropwise into 20 mL of 0.5% polyvinyl alcohol (PVA) solution; treat with ultrasound (power 300 W) for 2 min in an ice bath, then stir at 50°C and 500 rpm for 1 h for emulsification reaction, remove the organic solvent by rotary evaporation at 50°C, centrifuge at 4000 rpm for 5 min, and collect the supernatant to obtain psoralen polymer nanoparticle solution.

[0058] S3. Preparation of psoralen mannosylated chitosan nanoparticles (MCSPP NPs): The psoralen polymer nanoparticle solution obtained in step S2 was slowly added dropwise to the mannosylated chitosan solution obtained in step S1, stirred in the dark at room temperature for 3 h, centrifuged at 4000 rpm for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain MCSPP NPs.

[0059] Experimental example

[0060] The psoralen mannosylated chitosan nanoparticles (MCSPP NPs) used in the following experimental examples were all the psoralen mannosylated chitosan nanoparticles (MCSPP NPs) provided in Example 1.

[0061] 1. Morphology of psoralen mannosylated chitosan nanoparticles (MCSPP NPs)

[0062] The transmission electron microscopy (TEM) image of MCSPP NPs provided in Example 1 is as follows: Figure 1 As shown in the figure, at a magnification of 59000×, MCSPPNPs are spherical and have an obvious double-layer structure, indicating that the prepared nanoparticles have a "core-shell" structure, and mannosylated chitosan (MAN-CS) can be evenly adsorbed on the surface of psoralen polymer nanoparticles (PP NPs). There is no adhesion between the nanoparticles and the dispersion is good.

[0063] 2. Fourier transform infrared (FTIR) scanning test

[0064] Weigh appropriate amounts of PSO, CS, MAN, PLGA, PP NPs, MAN-CS, and MCSPP NPs, and mix them evenly with dried potassium bromide powder in a mortar at a weight ratio of 1:100. Fourier transform infrared spectrometer was used to analyze the morphology of the NPs at 4000-500 cm -1 Each sample was scanned.

[0065] The test results are as follows Figure 2 As shown, Figure 2 As can be seen from the middle curve (a), PSO is at 1716.89 cm -1 There is a strong absorption peak at 1633.37 cm, which is the characteristic peak of C=O. -1 The C=C stretching vibration absorption peak appears at 1576.30 cm -1 and 1448.60cm -1 The absorption peak at 1135.63 cm represents the characteristic peak of the benzene ring skeleton. -1 The strong absorption peak at 1021.63 cm indicates the presence of CO single bond stretching vibration of the ester group in PSO. -1 The absorption peak of α confirms the aromatic ether (=COC) structure in PSO, which is attributed to the symmetrical configuration of COC stretching vibration. Figure 2 The middle curve (b) is the infrared spectrum of PP NPs at 3405.61 cm -1 It can be seen that there are many hydroxyl groups in the nanoparticles, 2939.82 cm -1 This is a strong absorption peak caused by the deformation vibration of a large number of -CH3 in PLGA. Under the catalysis of DCC and NHS, the carboxyl group of PLGA reacts to form an ester group, 1737.38 cm -1 、1093.45 cm -1 The corresponding peaks are the stretching vibration absorption peaks of C=O and CO of PLGA. In addition, 1433.99 cm -1 The absorption peak of -CH3 in PLGA is 1376.52 cm -1 and 1260.17 cm -1 They are the OH in-plane bending vibration and CO stretching vibration of the secondary alcohol in PLGA. The characteristic peaks of olefin and benzene ring of PSO are at 1638.74 cm -1 and 1433.99 cm -1 A weak vibration peak can be seen at the bottom, indicating that PSO has not been chemically modified or degraded during the formation of nanoparticles. Figure 2 The middle curve (c) is the infrared absorption spectrum of CS, at 3423.97 cm -1 There are NH and OH stretching vibration absorption peaks at 1601.23 cm -1The in-plane bending vibration of NH is also the characteristic absorption peak of -NH2 primary amine group, 1382.26 cm -1 The stretching vibration peak of CN is at 2875.51cm, indicating that there are a large number of amino groups on CS. -1 The CH stretching vibration peak of methylene is at 1156.28 cm -1 The absorption peak at 1078.06 cm is the stretching vibration of CO in the primary alcohol on CS. The stretching vibration peak of CO in the six-membered monooxygen ring on CS is located at 1078.06 cm -1 There are many hydroxyl groups in mannose, such as Figure 2 As shown in the middle curve (d), at 3350.28 cm -1 and 1068.17 cm -1 Strong OH and CO stretching vibration absorption peaks appear at the , which is consistent with the chemical structure of mannose; it also shows that there are intermolecular hydrogen bonds in mannose, and -OH is associated in the form of multiple molecules. When mannose is coupled to CS, Figure 2 As shown in curve (e), at 3372.76 cm -1 The stretching vibration of OH at 1563.37 cm is significantly enhanced and the absorption peak becomes broad, indicating that after mannose is introduced into CS, the stretching vibration of OH in mannose and NH in CS overlap, and MAN-CS exists in the form of a polymer through hydrogen bonding. -1 、1408.98 cm -1 、1259.46 cm -1 The absorption peaks are NH in-plane bending vibration, C=N and CN stretching vibration, which confirm that after the ring-opening reaction of mannose, its aldehyde group reacts with the amino group of CS to form a Schiff base (RCH=NR`), and at the same time, it shows that there are still free primary amino groups on CS. -1 The vibration peak at is significantly enhanced, which is due to the overlap of CO stretching vibrations of COC of CS and CO stretching vibrations of hydroxyl groups on MAN. Figure 2 As shown in the middle curve (f), MCSPP NPs are at 3395.39 cm -1 and 2939.09 cm -1 Strong stretching vibration occurs at 1651.78 cm, which is consistent with the MAN-CS spectrum. However, OH, NH and CH shift to the long-wave direction, which may be due to the intermolecular hydrogen bond interaction between CS and PLGA. -1 、1569.95 cm -1 、1248.05 cm -1The stretching vibration of C=O (amide I band), the in-plane bending vibration of NH (amide II band), and the stretching vibration of CN (amide III band) appeared at 1736.77 cm, indicating that the free amino groups of CS and the ester groups of PLGA dehydrated and condensed to form amide bonds, and at the same time, electrostatic interactions were generated, and physical coupling occurred through the mutual attraction of anions and cations. -1 and 1090.14 cm -1 The strong absorption peaks at 1424.72 cm-1 are the characteristic peaks of C=O and COC in PLGA, which proves that PP NPs are encapsulated in MAN-CS without degradation. -1 and 1248.05 cm -1 The observed decrease in C=N stretching vibration and increase in CN stretching vibration at the 20 nm and 20 nm sites, respectively, indicate that the MCSPP NPs contain not only the Schiff base of MAN-CS but also amide bonds formed by dehydration condensation between PLGA and CS, and possibly residual N-acetyl groups from CS. In summary, infrared spectroscopy confirmed the interactions between MAN and CS, and between CS and PLGA.

[0066] 3. X-ray diffraction (XRD) analysis

[0067] A small amount of dry solid PSO, CS, MAN, PLGA, and MCSPP NPs were weighed and tested using an X-ray diffractometer. The X-ray diffraction patterns were drawn under the conditions of a tube voltage of 40 kV, Cu-Ka radiation, a scanning speed of 2° / min, and a scanning range of 3~40°.

[0068] The X-ray diffraction pattern test results are as follows Figure 3 As shown, Figure 3 The middle curve (a) shows that PSO has strong peaks at 2θ=9.28°, 16.14° and 27.82°. Figure 3 In the middle curve (e), broad diffraction peaks with low peak intensity were observed at 2θ=14.88° and 29.58°, indicating that PSO exists in the encapsulated nanoparticles in a crystalline structure. Figure 3 As shown in the middle curve (b), PLGA has a strong diffraction peak at 2θ=19.35°. Figure 3 As can be seen from curve (d), chitosan has two diffraction peaks at 2θ=13.06° and 19.72°, which are the characteristic peaks of its crystallization. PLGA and CS have peaks near 19°, while Figure 3As can be seen in curve (e), a broad diffraction peak appears at 2θ=19.09°, and the characteristic peak is significantly weakened, indicating that the introduction of PLGA on CS will affect its internal structure, which may destroy the hydrogen bonding between CS molecules and affect the formation of hydrogen bonds in the crystallization area, thereby destroying the CS crystal structure and reducing its crystallization performance, and some crystals become amorphous structures. Figure 3 As shown in the middle curve (c), MAN has a strong diffraction peak at 2θ=15.04°, which is similar to the diffraction peak of PSO, indicating that MAN may exist in the nanoparticles in an amorphous structure.

[0069] 4. Differential scanning calorimetry and thermogravimetric analysis

[0070] The thermal stability of the samples was analyzed by differential scanning calorimetry (DSC) and thermogravimetry (TG). A small amount of dry solid PSO, CS, MAN, PLGA, and MCSPP NPs was weighed and scanned in a thermal analyzer (TGA / DSC 3+) at a temperature of 37–400°C under nitrogen conditions at a heating rate of 10°C / min. The thermal analysis graphs were plotted. The test results are shown in Figure 2. Figure 4-5 shown.

[0071] Figure 4 The results of differential scanning calorimetry are shown in Figure 2. Figure 4 As can be seen from the curve (a), PSO has a strong peak at 167.2℃, corresponding to its melting point, and Figure 4 The MCSPP NPs represented by curve (e) have a weak peak at 168.8℃, indicating that the PSO peak intensity is reduced. The crystal form of PSO in the nanoparticles remains unchanged and exists in the polymer as a molecularly dispersed form. PLGA is an amorphous polymer, and its glass transition temperature is related to its molecular weight. The molecular weight of the PLGA used in the experiment is relatively small. Figure 4 There is a broad peak at 306.8°C in curve (b), and there is also a broad peak at 304.3°C in the nanoparticles, indicating that PLGA has high crystallinity and also maintains a crystalline form in the nanoparticles. Figure 4 In curve (c), MAN has a strong peak at 147.5℃, which corresponds to its melting point. No characteristic peak of MAN was observed in the nanoparticles, indicating that MAN is coupled to the CS surface in a non-crystalline form. Figure 4 In curve (d), CS has broad peaks at 77.3℃ and 270.8℃, respectively. Figure 4 There is a broad peak at 71.8 °C in curve (e), indicating that the peak of CS has shifted and exists in a crystalline form. However, the other peak of CS disappears in the nanoparticles, indicating that CS also exists in an amorphous form.

[0072] Figure 5It is the first-order differential curve of the mass percentage of the substance changing with temperature, that is, the detection result of the mass change rate changing with temperature. Figure 5 As can be seen from the curve (a), PSO has a strong peak at 276.3℃, indicating that its thermal weight loss rate changes significantly at this temperature, which is 48.1%. Figure 5 As can be seen from the curve (b), the thermal weight loss rate of PLGA at 304.8℃ is 49.5%. Figure 5 As shown in curve (c), MAN has two peaks at 205.7℃ and 303.3℃, ​​indicating that MAN has two weight loss stages, with thermal weight loss rates of 5.6% and 38.7% respectively. Figure 5 As can be seen from curve (d), the thermal weight loss rate of CS at 297.8℃ is only 28.8%, while Figure 5 As can be seen from curve (e), MCSPP NPs have a broad peak at 309.7 °C and a thermal weight loss rate of 35.4%. The results show that the temperature of the maximum degradation rate of the nanoparticles is higher than that of CS, PLGA, MAN and PSO, and the thermal stability is strong. The thermal weight loss rate of the nanoparticles is low. It may be that after CS modification, the intermolecular or intramolecular hydrogen bonds are enhanced, making the nanoparticles stable at high temperatures and not easily decomposed.

[0073] 5. Psoralen mannosylated chitosan polymer nanoparticles inhibit the growth of MDA-MB-231 breast cancer cells

[0074] 5.1 Sample preparation

[0075] (1) Preparation of culture medium (MDA-MB-231 cell culture medium): Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to DMEM culture medium, mix well, filter through a 0.22 μm sterile filter membrane, and store at 4°C until use.

[0076] (2) Preparation of cell freezing solution: Mix 1.1 mL of culture medium, 0.8 mL of fetal bovine serum, and 0.1 mL of cell-grade dimethyl sulfoxide thoroughly.

[0077] (3) Preparation of paclitaxel (PTX) solution: Take an appropriate amount of paclitaxel sample and use cell-grade dimethyl sulfoxide as solvent to prepare a 1 mg / mL paclitaxel solution. Dilute with fresh culture medium and filter through a 0.22 μm sterile filter membrane. Prepare and use immediately.

[0078] (4) Preparation of MTT solution: Weigh 250.0 mg of MTT powder, add 50 mL of PBS solution in a biosafety cabinet, dissolve under sonication in the dark, prepare a 5 mg / mL solution, filter through a 0.22 μm sterile filter membrane, and store at 4°C until use.

[0079] (5) Preparation of 1 μg / mL 4',6-diamidino-2-phenylindole (DAPI) staining solution: Pipette 10 μL of 5 mg / mL DAPI solution and dilute it with 4990 μL of PBS buffer to make a 10 μg / mL stock solution. Store at -20°C until use. When using, take 1 mL and dilute it with 9 mL of PBS buffer to obtain a 1 μg / mL DAPI solution.

[0080] (6) Preparation of 0.1% polyethylene glycol octylphenyl ether (Triton X-100) solution: Pipette 100 μL of 10% Triton X-100 solution and dilute it with 9.9 mL of PBS buffer to obtain 0.1% Triton X-100.

[0081] (7) Preparation of cell lysis buffer: RIPA lysis buffer, phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitors, and protease inhibitors were prepared in a ratio of 100:1:2:2.

[0082] (8) Preparation of electrophoresis buffer (10×): Accurately weigh 22.5 g of tris(hydroxymethyl)aminomethane (Tris-Base), 108 g of glycine, and 7.5 g of sodium dodecyl sulfate (SDS), and dissolve them in ultrapure water to 7.5 L, diluting 10 times when used.

[0083] (9) Preparation of transfer buffer (10×): Accurately weigh 15.1 g Tris-Base, 72 g glycine, and 1.85 g SDS sodium dodecyl sulfate, and dissolve and dilute to 500 mL with ultrapure water. When using, take 100 mL and add 200 mL methanol and 700 mL ultrapure water to make 1×.

[0084] (10) Preparation of phosphate buffered saline (PBST) membrane washing solution (10×): Accurately weigh 80 g NaCl, 2 g KCl, 36.3 g sodium dihydrogen phosphate dodecahydrate, 2.4 g potassium dihydrogen phosphate, and 5 mL Tween 20, dissolve in ultrapure water and dilute to 1 L. Dilute 10 times when using, and add 500 μL Tween 20.

[0085] 5.2 Cell culture

[0086] MDA-MB-231 cells and drug-treated cells were cultured in a 37°C incubator containing 5% CO2.

[0087] 5.3 Cellular uptake test

[0088] A coumarin-6 (C-6) concentration gradient (176, 352, 704, 1408, 2816 ng / mL) was prepared, and the absorbance was measured by fluorescence spectrometry in a microplate reader at an excitation wavelength of 466 nm and an emission wavelength of 504 nm, and a standard curve was drawn. CSPP NPs and MCSPP NPs were prepared using C-6 instead of psoralen, and the C-6 content in the nanoparticles was quantified using a microplate reader and the standard curve.

[0089] MDA-MB-231 cells and M2 macrophages were cultured in a laser confocal microscopy dish with culture medium containing 200 ng / mL C-6 CSPP NPs and MCSPP NPs, respectively. After incubation for 2 h, the original culture medium was discarded, the cells were washed with PBS, and fixed with 500 μL of 4% paraformaldehyde for 10 min. The cells were washed three times with PBS, and the cell nuclei were stained with 500 μL of 1 μg / mL DAPI staining solution for 10 min. The cells were washed three times with PBS, and 500 μL of PBS was added to the cells. The uptake of the cells by breast cancer cells and M2 macrophages was observed under a laser confocal microscope.

[0090] MDA-MB-231 cells and M2 macrophages were cultured in 6-well plates with culture medium containing 200 ng / mL C-6 CSPP NPs and MCSPP NPs, respectively. After incubation for 2 h, the cells were washed three times with PBS, digested with EDTA-free trypsin, washed with PBS, and finally 500 μL PBS was added to mix the cells. The cells were transferred to flow tubes and loaded into a flow cytometer to detect the mean fluorescence intensity.

[0091] To verify the role of mannose receptors in promoting nanoparticle uptake by macrophages under co-culture conditions, a competitive inhibition experiment with mannan was conducted. After 24 hours of culture, M2 macrophages were washed with PBS and an appropriate amount of mannan was added to a medium containing 2 mg / mL mannan. This medium was then incubated with the macrophages to allow for sufficient binding to cell surface mannose receptors. Two hours later, 1.5 mL of fresh medium containing 200 ng / mL C-6 was added to CSPP NPs and MCSPP NPs. Cellular uptake was monitored by laser confocal microscopy and flow cytometry. C-6 was loaded onto CSPP NPs and MCSPP NPs, and uptake of CSPP NPs and MCSPP NPs by MDA-MB-231 cells and M2 macrophages was observed by laser confocal microscopy to assess the cellular uptake of the nanoparticles. To ensure consistent concentration of C-6 loaded into the nanoparticles, the absorbance of C-6 was measured using the fluorescence channel of a microplate reader. Linear regression was performed with the absorbance OD value as the ordinate and the concentration of the C-6 solution as the abscissa. The standard curve fitting equation was y=372.33x+16533, with R2=0.9997. The results showed that C-6 had a good linear relationship in the concentration range of 176~2816 ng / mL.

[0092] Breast cancer cell uptake results Figure 6 As shown in the figure, the C-6 fluorescence intensity in the free C-6 group is weak, indicating that the nanoparticles can enter the cytoplasm and the content is small. Figure 7 As shown in (a)-(b), after incubation of cells with CSPP NPs and MCSPP NPs, there were significant differences compared with the control group (p<0.0001), while the cell uptake of CSPP NPs and MCSPPNPs was similar, with average fluorescence intensities of 1556 and 1597, respectively, indicating that the use of nanocarriers to load C-6 can significantly improve cellular uptake.

[0093] The mannose modified on MCSPP NPs can bind to the mannose receptor on the surface of M2 macrophages, which can promote the uptake of MCSPP NPs by M2 macrophages. The experimental results of laser confocal microscopy are shown in Figure 2. Figure 8 As shown in the figure, compared with the uptake of MDA-MB-231 cells, the C-6 fluorescence intensity of M2 macrophages in nanoparticle uptake was higher, and more nanoparticles were observed to enter the cytoplasm. Figure 9As shown in (a) and (b), M2 macrophages clearly exhibited the highest uptake of MCSPPNPs. After 2 hours of incubation with MCSPPNPs, the mean fluorescence intensity of M2 macrophages reached 4867, significantly different from that of the CSPPNPs group (p < 0.01). These results demonstrate that MCSPPNPs are extensively taken up by M2 macrophages, and that mannose modification of the nanoparticles significantly impacts their uptake.

[0094] To verify whether mannose receptors promote the uptake ability of M2 macrophages, a competitive inhibition experiment of mannan was performed. The results are shown in Figure 8-9 As shown in Figure 3, the C-6 fluorescence intensity of the MAN+CSPP NPs group and the MAN+MCSPP NPs group in M2 macrophages was weaker than that of the CSPP NPs group and the MCSPP NPs group. Figure 9 (b) shows that the average fluorescence intensities of MAN+CSPP NPs and MAN+MCSPP NPs are 2704 and 2804, respectively, with no significant difference between the two. The fluorescence intensity of the MAN+CSPP NPs group is similar to that of the CSPP NPs group, indicating that mannan can first interact with the mannose receptor, effectively reducing the active targeting of MCSPP NPs, thereby reducing the uptake of MCSPP NPs by M2 macrophages, showing the phenomenon of mannan competing with MCSPP NPs for binding to mannose receptors.

[0095] 5.4 Cell cycle detection

[0096] Western blot was used to examine the effect of MCSPP NPs on cyclin expression. MDA-MB-231 cells were seeded in 6-well plates and cultured overnight. The culture medium was removed and 2 mL of normal culture medium containing PBS, MCSPP NPs (10, 20, and 40 μg / mL, calculated as PSO), and M2-CM were added, respectively, and cultured for a further 24 hours. The cells were trypsinized, and the cell suspension was collected and centrifuged. The cells were washed twice with PBS and centrifuged. The cell pellet was collected and 100 μL of cell lysis buffer was added to each well. The cells were lysed on ice for 30 minutes, placed in a 1.5 mL EP tube, centrifuged, and the supernatant was collected and transferred to a fresh pre-chilled EP tube.

[0097] Protein quantification was performed using the BCA protein assay kit according to the instructions. The remaining sample was diluted with loading buffer, the protein was denatured in a metal bath, and stored at -20°C for later use. Gel was made and each sample and pre-stained marker were separated by electrophoresis. The process was stopped when the marker band separated to the bottom edge of the gel. Each sample and pre-stained marker band was transferred to a PVDF membrane by transfer. Blocked with 5% skim milk, washed with PBST, incubated with the primary antibody overnight, with β-actin as the control, washed with PBST, incubated with the secondary antibody for 2 h, washed with PBST, and ECL chemiluminescent solution was added. The images were imaged using a chemiluminescent gel imaging system. The results are shown in Figure 2. Figure 10 As shown in (a)-(e), compared with the control group, 40 µg / mL MCSPP NPs significantly increased Cyclin A2 protein expression in MDA-MB-231 cells (p<0.0001), with an average relative expression difference of 0.98 compared with the control group, indicating that high concentrations of MCSPP NPs can arrest cells in S phase. Treatment of MDA-MB-231 cells with 20 µg / mL MCSPP NPs minimized Cyclin D1 protein expression, with an average relative expression of 0.64. These results indicate that MCSPP NPs can alter the expression levels of Cyclin A2 and Cyclin D1, key proteins in the S phase, thereby blocking DNA replication and inhibiting breast cancer cell proliferation.

[0098] 5.5 Cell apoptosis test

[0099] The cell morphology after DAPI staining was observed by laser confocal microscopy: MDA-MB-231 cells were seeded in laser confocal microscopy dishes. After the cells attached to the wall, fresh culture medium was replaced and cultured. After overnight, the original culture medium was removed and 2 mL of fresh culture medium containing PBS, free PSO (20 μg / mL), free PTX (20 μg / mL), PP NPs (20 μg / mL), CSPP NPs (20 μg / mL), and MCSPP NPs (10, 20, and 40 μg / mL, calculated as PSO) were added, respectively, and incubated in an incubator for 24 h. After incubation, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, washed 3 times with PBS, and permeabilized with 0.1% Triton X-100 at room temperature for 10 min. The cells were washed 3 times with PBS, and 0.5 mL of DAPI staining solution with a final concentration of 1 μg / mL was added. The cells were stained in the dark for 15 min at room temperature, washed 3 times with PBS, and added with 50 1. Apply 1 μL of anti-fluorescence quenching mounting solution on the cells, cover with a coverslip, and observe the nuclear chromatin morphology under a laser confocal microscope.

[0100] Annexin-V / APC and PI double-stained flow cytometric analysis: MDA-MB-231 cells were seeded in 6-well plates. After overnight culture, the original culture medium was removed and 2 mL of fresh culture medium containing PBS, free PSO (20 μg / mL), free PTX (20 μg / mL), PP NPs (20 μg / mL), CSPP NPs (20 μg / mL), and MCSPP NPs (10, 20, and 40 μg / mL, calculated as PSO) were added, respectively, and culture was continued for 24 h. The culture plate was removed, the original culture medium was discarded, and the cells were washed three times with PBS. EDTA-free trypsin was added for digestion, and the cells were centrifuged. The cells were washed with PBS and centrifuged again. The cell suspension was collected and resuspended in 400 μL of cell staining buffer. The cells were transferred to a flow cytometer and 5 μL of Annexin-V / APC and PI staining solution were added. The cells were mixed and incubated at room temperature in the dark for 15 min. Normal cells (APC- / PI-), necrotic cells (APC- / PI+), early apoptotic cells (APC+ / PI-), and late apoptotic cells (APC+ / PI+) were detected by flow cytometry.

[0101] The effect of MCSPP NPs on MDA-MB-231 cell apoptosis was evaluated by changes in nuclear chromatin morphology. Breast cancer cells were fluorescently labeled with DAPI stain and the nuclear morphology was observed under a laser confocal microscope. Figure 11 As shown, in the control group, MDA-MB-231 cell nuclei were round, uniformly stained, and had clear edges, reflecting normal cell morphology. In the free PSO (20 µg / mL), PP NPs (20 µg / mL), and MCSPP NPs (10 µg / mL) treatment groups, MDA-MB-231 cell nuclei were condensed and marginalized, with chromatin accumulating in a crescent shape near the nuclear membrane. Blue fluorescence was evident, and vacuolated structures appeared in the cells, indicating that the breast cancer cells were in the early stage of apoptosis. However, in the free PTX (20 µg / mL), CSPP NPs (20 µg / mL), and MCSPP NPs (20 and 40 µg / mL) treatment groups, MDA-MB-231 cell nuclei underwent lysis, fragmenting into fragmented, round or oval bodies of varying sizes within the cell membrane, known as apoptotic bodies. These bodies exhibited strong blue fluorescence, indicating that the breast cancer cells were in the late stage of apoptosis.

[0102] Flow cytometry was used to detect apoptosis of MDA-MB-231 cells in drug-containing culture medium. The experimental results are as follows: Figure 12As shown in the results, compared with the control group, PSO induced apoptosis in 14.2% of cells. PP NPs and CSPP NPs increased the proportion of early apoptotic cells, with total apoptotic rates reaching 24.4% and 32.7%, respectively. MCSPP NPs at a concentration of 40 µg / mL increased the early apoptotic rate to 40.6%, demonstrating a significant apoptosis-inducing effect. At the same concentration of 20 µg / mL, PTX induced apoptosis significantly more than MCSPP NPs. These results suggest that MCSPP NPs primarily exert their effects by inducing early apoptosis.

[0103] 6. Testing of Psoralen Mannosylated Chitosan Polymer Nanoparticles as Tumor-Associated Macrophage and Cytokine Regulators for the Treatment of Triple-Negative Breast Cancer

[0104] 6.1 Sample preparation

[0105] (1) Preparation of culture medium (MDA-MB-231 cell culture medium): Add 10% fetal bovine serum and 1% penicillin-streptomycin solution to DMEM culture medium, mix well, filter through a 0.22 μm sterile filter, and store at 4°C until use. Culture medium for human monocytic leukemia (THP-1) cells: Add 10% fetal bovine serum, 1% penicillin-streptomycin solution and 0.05 mM β-mercaptoethanol solution to RPMI-1640 culture medium, mix well, filter through a 0.22 μm sterile filter, and store at 4°C until use.

[0106] (2) Preparation of cell freezing solution: Mix 1.1 mL of culture medium, 0.8 mL of fetal bovine serum, and 0.1 mL of cell-grade dimethyl sulfoxide thoroughly.

[0107] (3) Preparation of paclitaxel solution: Take an appropriate amount of paclitaxel sample and use cell-grade dimethyl sulfoxide as solvent to prepare 1 mg / mL paclitaxel solution. Dilute with fresh culture medium, filter through a 0.22 μm sterile filter membrane, and use immediately after preparation.

[0108] (4) Preparation of MTT solution: Weigh 250.0 mg of MTT powder, add 50 mL of PBS solution in a biosafety cabinet, dissolve under sonication in the dark, prepare a 5 mg / mL solution, filter through a 0.22 μm sterile filter membrane, and store at 4°C until use.

[0109] (5) Preparation of 1 μg / mL DAPI staining solution: Pipette 10 μL of 5 mg / mL DAPI solution and dilute it with 4990 μL of PBS buffer to make a 10 μg / mL stock solution. Store it at -20°C until use. When using, take 1 mL and dilute it with 9 mL of PBS buffer to obtain a 1 μg / mL DAPI solution.

[0110] (6) Preparation of 0.1% Triton X-100 solution: Pipette 100 μL of 10% Triton X-100 solution and dilute it with 9.9 mL of PBS buffer to obtain 0.1% Triton X-100.

[0111] (7) Preparation of (propylene glycol methyl ether acetate) PMA solution: Dissolve 1 mg of PMA in 1 mL of cell-grade dimethyl sulfoxide to prepare 1 mg / mL. Pipette 5 μL of the 1 mg / mL solution and add it to 50 mL of prepared THP-1 cell culture medium (100 ng / mL) to induce M0 macrophages.

[0112] (8) Preparation of lipopolysaccharide (LPS) solution: Dissolve 1 mg of LPS in 1 mL of cell-grade dimethyl sulfoxide to prepare 1 mg / mL. Pipette 5 μL of the 1 mg / mL solution and add it to 50 mL of prepared culture medium (100 ng / mL) to induce M1 macrophages.

[0113] (9) Preparation of interleukin-4 (IL-4) / interleukin-13 (IL-13) solution: Dissolve 50 μg of IL-4 / IL-13 in 5 mL of PBS buffer to prepare a 10 μg / mL stock solution, store at -20°C until use, and take 100 μL and add it to 50 mL of prepared culture medium (20 ng / mL) to induce M2 macrophages.

[0114] (10) Preparation of cell lysis buffer: RIPA lysis buffer, PMSF, phosphatase inhibitors, and protease inhibitors were prepared in a ratio of 100:1:2:2.

[0115] (11) Preparation of electrophoresis buffer (10×): Accurately weigh 22.5 g Tris-Base, 108 g glycine, and 7.5 g SDS, dissolve in ultrapure water and dilute to 7.5 L, diluting 10 times when used.

[0116] (12) Preparation of transfer buffer (10×): Accurately weigh 15.1 g Tris-Base, 72 g glycine, and 1.85 g SDS, dissolve and dilute to 500 mL with ultrapure water. Take 100 mL and add 200 mL methanol and 700 mL ultrapure water to make 1×.

[0117] (13) Preparation of PBST membrane washing solution (10×): Accurately weigh 80 g NaCl, 2 g KCl, 36.3 g sodium dihydrogen phosphate dodecahydrate, 2.4 g potassium dihydrogen phosphate, and 5 mL Tween 20, dissolve in ultrapure water and dilute to 1 L. Dilute 10 times when using, and add 500 μL Tween 20.

[0118] 6.2 Cell culture

[0119] MDA-MB-231 cells, THP-1 cells, and drug-treated or induced cells were cultured in a 37°C incubator containing 5% CO2.

[0120] 6.3 Cell cycle detection

[0121] MDA-MB-231 cells were seeded into 6-well plates and cultured overnight in M2-CM. The culture medium was then removed and 2 mL of fresh M2-CM containing PBS, free PSO (20 µg / mL), free PTX (20 µg / mL), PP NPs (20 µg / mL), CSPP NPs (20 µg / mL), and MCSPP NPs (10, 20, and 40 µg / mL, calculated as PSO) were added. The plates were then cultured for an additional 24 hours. The plates were removed, the original culture medium discarded, and the cells were washed three times with PBS. The cells were then digested with EDTA-free trypsin, centrifuged, washed with PBS, and centrifuged again. The cell suspension was collected and mixed with 0.5 mL of PBS. While vortexing, 1.5 mL of ice-cold ethanol was added to a final ethanol concentration of 75%, and the cells were fixed overnight at -20°C. The sample was removed, centrifuged, washed with pre-chilled PBS, centrifuged again, and resuspended in 0.5 mL PBS. The cells were transferred to a flow cytometer, and 300–500 μL of PI / RNAse staining solution was added. The cells were mixed and incubated at room temperature in the dark for 15 min. The cell cycle was detected by flow cytometry.

[0122] like Figure 13-14 As shown, when treated with drugs in M2-CM, the majority of breast cancer cells remained in the G0 / G1 phase. The average percentage of cells in the S phase remained between 15% and 20%, while that in the G0 / G1 phase remained between 64% and 76% across the different drug groups. However, in normal drug-treated culture medium, the S and G0 / G1 phases of breast cancer cells remained between 30% and 42%, respectively, and 44% and 63%, respectively. These results indicate that MCSPP NPs, when cultured in M2-CM, can induce cell cycle arrest in the S phase for a small fraction of MDA-MB-231 cells, while arresting the majority of cells in the G0 / G1 phase. The G0 / G1 phase was significantly different in the MCSPP NPs at a concentration of 40 μg / mL compared to the control group (p < 0.01). The presence of numerous tumor-promoting cytokines in M2-CM can accelerate the mitotic proliferation of breast cancer cells. MCSPP NPs primarily reduce cell proliferation by blocking the prophase of DNA synthesis in co-cultured breast cancer cells.

[0123] In addition, if Figure 10As shown, in M2-CM culture conditions, 20 and 40 µg / mL MCSPP NPs increased Cyclin A2 protein expression in MDA-MB-231 cells, with average relative expression levels of 3.18 and 3.78, respectively. In contrast, 40 µg / mL MCSPP NPs significantly decreased Cyclin D1 protein expression (p<0.01), with an average relative expression level of 0.83, a difference of 3.85 from the control group. These results indicate that in M2-CM co-cultures, high concentrations of MCSPP NPs significantly reduced Cyclin D1 protein expression, leading to cell cycle arrest at the G0 / G1 phase. In normal cultures, MCSPP NPs altered the expression of Cyclin A2, a key protein in the S phase, thereby blocking DNA replication and reducing breast cancer cell viability. These results are consistent with flow cytometry analysis.

[0124] 6.4 Apoptosis Detection

[0125] The cell morphology after DAPI staining was observed by laser confocal microscopy: MDA-MB-231 cells were seeded in laser confocal microscopy dishes, and after the cells attached to the wall, fresh M2-CM was replaced and cultured. After overnight, the original culture medium was removed, and 2 mL of M2-CM containing PBS, free PSO (20 μg / mL), free PTX (20 μg / mL), PP NPs (20 μg / mL), CSPP NPs (20 μg / mL), and MCSPP NPs (10, 20, and 40 μg / mL, calculated as PSO) were added, respectively, and incubated in an incubator for 24 h. After incubation, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 min, washed 3 times with PBS, and permeabilized with 0.1% Triton X-100 at room temperature for 10 min. The cells were washed 3 times with PBS, and 0.5 mL of DAPI staining solution with a final concentration of 1 μg / mL was added. The cells were stained in the dark at room temperature for 15 min. min, washed three times with PBS, and 50 μL of anti-fluorescence quenching mounting solution was added dropwise on the cells at room temperature in the dark. The cells were covered with a coverslip and the nuclear chromatin morphology was observed under a laser confocal microscope.

[0126] Annexin-V / APC and PI double-stained flow cytometric analysis: MDA-MB-231 cells were seeded in 6-well plates and cultured overnight in M2-CM. The original culture medium was removed and 2 mL of M2-CM containing PBS, free PSO (20 μg / mL), free PTX (20 μg / mL), PP NPs (20 μg / mL), CSPP NPs (20 μg / mL), and MCSPP NPs (10, 20, and 40 μg / mL, calculated as PSO) were added and cultured for another 24 h. The culture plate was removed, the original culture medium was discarded, and the cells were washed three times with PBS. EDTA-free trypsin was added for digestion, and the cells were centrifuged. The cells were washed with PBS and centrifuged again. The cell suspension was collected and resuspended in 400 μL of cell staining buffer. The cells were transferred to a flow cytometer and 5 μL of Annexin-V / APC and PI staining solution were added. The cells were mixed and incubated at room temperature in the dark for 15 min. Normal cells (APC- / PI-), necrotic cells (APC- / PI+), early apoptotic cells (APC+ / PI-), and late apoptotic cells (APC+ / PI+) were detected by flow cytometry.

[0127] like Figure 15 As shown in the figure, compared with normal culture medium, the apoptosis rate of MDA-MB-231 cells in each group decreased under the culture conditions of M2-CM, indicating that M2-CM can promote the proliferation activity of breast cancer. Under the conditions of M2-CM, the apoptosis effect of MCSPP NPs on breast cancer cells was reduced. MCSPP NPs had a smaller effect on promoting early apoptosis of breast cancer cells, but increased their late apoptosis rate. Figure 16As shown, compared with normal culture medium, treatment with MCSPP NPs at a concentration of 40 µg / mL resulted in a 32.3% apoptosis rate in breast cancer cells co-cultured with M2-CM, indicating that MCSPP NPs reduced the apoptosis-inducing effect of co-cultured cells and that M2-CM significantly inhibited apoptosis (p<0.01). At a concentration of 20 µg / mL, CSPP NPs and MCSPP NPs induced apoptosis in breast cancer cells cultured in M2-CM by 26.6% and 26.3%, respectively, indicating that mannose had no significant effect on breast cancer cells and that cellular uptake of CSPP NPs and MCSPP NPs was similar. MCSPP NPs at a concentration of 10 µg / mL showed no significant difference in apoptosis in breast cancer cells cultured in normal and M2-CM cultures. However, treatment with MCSPP NPs at a concentration of 20 µg / mL significantly increased apoptosis in cells cultured in normal culture (p<0.05). M2-CM contains a variety of cytokines secreted by M2 macrophages that stimulate the proliferation of TNBC, such as growth factor EGF, transforming growth factor TGF-β, arginase Arg-1, chemokine CCL-17 and inflammatory factor IL-6, which enable TNBC to continue to develop.

[0128] 6.5 Western blot analysis

[0129] MDA-MB-231 cells were seeded in a 6-well plate and cultured overnight in M2-CM. The culture medium was then removed and 2 mL of M2-CM containing PBS and MCSPP NPs (10, 20, and 40 µg / mL, calculated as PSO) was added. The cells were cultured for a further 24 hours. The cells were trypsinized, the cell suspension collected, and centrifuged. The cells were washed twice with PBS and centrifuged. The cell pellet was collected and 100 µL of cell lysis buffer was added to each well. The cells were lysed on ice for 30 minutes. The supernatant was placed in a 1.5 mL EP tube, centrifuged, and transferred to a fresh pre-chilled EP tube.

[0130] Protein quantification was performed using a BCA protein assay kit according to the manufacturer's instructions. The remaining sample was diluted with loading buffer, denatured in a metal bath, and stored at -20°C until needed. Gels were prepared and the samples and prestained markers were separated by electrophoresis, stopping when the marker bands reached the bottom edge of the gel. The samples and prestained marker bands were transferred to a PVDF membrane by transfer. The membranes were blocked with 5% skim milk, washed with PBST, and incubated with the primary antibody overnight. β-actin was used as a control. The membranes were washed with PBST and incubated with the secondary antibody for 2 h. After washing with PBST, ECL chemiluminescence solution was added and images were obtained using a chemiluminescence gel imaging system.

[0131] 6.6 Cytokine and Macrophage Phenotype Detection

[0132] The detection was performed using the method described in Experimental Example 6.3.

[0133] Different concentrations of MCSPP NPs acted on M2 macrophages, and the cytokine expression results were detected. Figure 17 As shown in the figure, compared with the control group, as the concentration of MCSPP NPs increased, the concentrations of various cytokines decreased or increased in turn. MCSPP NPs can inhibit the expression of IL-10 and TGF-β, and promote the expression of IL-12 and TNF-α. Figure 17 In (a), compared with the control group, 20 and 40 μg / mL MCSPP NPs had a significant inhibitory effect on IL-10 (p<0.001); 40 μg / mL MCSPP NPs could significantly reduce the concentration of TGF-β (p<0.001). Figure 17 In (b), 20 and 40 µg / mL MCSPP NPs significantly affected changes in IL-12 concentration (p<0.001), indicating that MCSPP NPs reshape the phenotype of macrophages or induce differentiation, inhibiting tumor-promoting M2 macrophages or re-regulating them to the M1 type to enhance the secretion and expression of immune stimulatory factors. The regulatory effect of MCSPP NPs on TNF-α increased with increasing MCSPP NP concentration. 10 µg / mL MCSPP NPs had no significant effect on M2 macrophages. The TNF-α concentration in the 40 µg / mL MCSPP NPs group was significantly different from that in the control group (p<0.0001), increasing by 395 pg / mL. The results showed that MCSPP NPs could regulate the secretion of cytokines by M2 macrophages, reduce the immunosuppressive factors IL-10 and TGF-β, and increase tumor necrosis factor and immunostimulatory factors TNF-α and IL-12, thereby changing the tumor-promoting phenotype of macrophages and inhibiting the growth of breast cancer cells.

[0134] like Figure 18 As shown, the average fluorescence intensities of 10, 20, and 40 μg / mL MCSPP NPs were 4198.3, 3540.3, and 2603, respectively, and the fluorescence intensity values ​​were negatively correlated with concentration. Compared with the control group, 20 μg / mL MCSPP NPs significantly reduced the fluorescence intensity of CD206 in TAMs (p < 0.01), while 40 μg / mL MCSPP NPs had an extremely significant regulatory effect on TAMs (p < 0.001), further demonstrating that MCSPP NPs can directly regulate the phenotype of TAMs, reduce the expression of CD206, a tumor-promoting phenotype marker of macrophages, inhibit M2 polarization, and thereby inhibit breast cancer cell growth.

[0135] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A psoralen mannosylated chitosan polymer nanoparticle, characterized in that: The psoralen mannosylated chitosan polymer nanoparticles are prepared from psoralen, poly(lactic acid-co-glycolic acid) copolymer, mannosylated chitosan, and polyvinyl alcohol, wherein the mass ratio of psoralen, poly(lactic acid-co-glycolic acid) copolymer, mannosylated chitosan, and polyvinyl alcohol is 2-10:10-30:16.5-38.5:1-9; the mannosylated chitosan has a carbon-nitrogen atomic ratio of 5.28-5.34, an actual deacetylation degree of chitosan of 90.25%, and an amino substitution degree of 1.04-16.81%. The psoralen mannosylated chitosan polymer nanoparticles are prepared by the following steps: S1. Preparing mannosylated chitosan: weighing mannose and chitosan in a mass ratio of 1-5:5, respectively, dissolving chitosan in an acetic acid-sodium acetate buffer solution and stirring in the dark to obtain a chitosan solution; dissolving mannose in an acetic acid-sodium acetate buffer solution and stirring to ring-open it to obtain a mannose solution; adding the mannose solution and a catalyst to the chitosan solution, stirring uniformly, and dialyzing to obtain a mannosylated chitosan solution, wherein the catalyst is sodium triacetoxyborohydride; S2. Preparing psoralen polymer nanoparticles: dissolving poly(lactic acid-co-glycolic acid) in dichloromethane, adding a dehydrating agent and a modifier to react together to obtain a pre-reaction product, wherein the dehydrating agent is dicyclohexylcarbodiimide and the modifier is N-hydroxysuccinimide; adding psoralen to the pre-reaction product and mixing uniformly; adding the obtained mixed solution to a polyvinyl alcohol solution, performing low-temperature ultrasonication, stirring and mixing uniformly, performing an emulsification reaction, and centrifuging to obtain the supernatant to obtain a psoralen polymer nanoparticle solution; S3. Preparation of psoralen mannosylated chitosan nanoparticles: adding the psoralen polymer nanoparticle solution to the mannosylated chitosan solution, stirring in the dark, and centrifuging. Filtering the supernatant after centrifugation to obtain psoralen mannosylated chitosan nanoparticles.

2. The psoralen mannosylated chitosan polymer nanoparticles according to claim 1, characterized in that: In step S1, the pH value of the acetic acid-sodium acetate buffer solution is 4-6.

3. The psoralen mannosylated chitosan polymer nanoparticles according to claim 2, characterized in that: In step S1, the chitosan is dissolved in an acetic acid-sodium acetate buffer solution and stirred in the dark at 20-30° C. for 2-5 hours; the mannose is dissolved in an acetic acid-sodium acetate buffer solution and stirred at 40-80° C. for 1-3 hours to ring-open the mannose; the mannose solution and the catalyst are added to the chitosan solution, stirred at room temperature for 20-30 hours, and dialyzed for 40-55 hours. During the dialysis treatment, a dialysis bag with a molecular weight cutoff of 12,000-14,000 is used.

4. The psoralen mannosylated chitosan polymer nanoparticles according to claim 3, characterized in that: In step S2, the mass ratio of the polylactic acid-glycolic acid copolymer to the dehydrating agent is 15:2, and the mass ratio of the polylactic acid-glycolic acid copolymer to the modifying agent is 15:2; the low-temperature ultrasonic process is ice bath ultrasonication, the ultrasonic power is 150-300W, and the ultrasonic time is 2-10min.

5. The psoralen mannosylated chitosan polymer nanoparticles according to claim 3, characterized in that: In step S2, the polylactic acid-glycolic acid copolymer reacts with the dehydrating agent and the modifier for 3-8 hours to obtain a pre-reaction product. After adding psoralen to the pre-reaction product, the mixture is mixed evenly and allowed to stand for 5-15 minutes. A polyvinyl alcohol solution with a volume concentration of 0.5% is added to the obtained mixed solution, and after mixing evenly, low-temperature ultrasonic treatment is performed. Then, the mixture is stirred at 20-50° C. and 400-500 rpm for 1-3 hours, and the organic solvent is removed by rotary evaporation at 30-50° C., and the mixture is centrifuged at 2000-4000 rpm for 5-15 minutes to obtain the supernatant.

6. The psoralen mannosylated chitosan polymer nanoparticles according to claim 3, characterized in that: In step S3, the psoralen polymer nanoparticle solution is added to the mannosylated chitosan solution, stirred in the dark at room temperature for 1-3 hours, and then centrifuged at 2000-4000 rpm for 5-15 minutes. The supernatant after centrifugation is filtered through a 0.22 μm filter membrane.

7. Use of the psoralen mannosylated chitosan nanoparticles according to claim 1 in the preparation of a drug for treating triple-negative breast cancer.

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