A nanoparticle for dual-targeted treatment of breast cancer, its preparation method and application

Abstract

The present invention discloses a nanoparticle for dual-targeted treatment of breast cancer, and its preparation method and application. The nanoparticle for dual-targeted treatment of breast cancer uses polysaccharide-cholesterol hemisuccinate amphiphilic conjugate of Dendrobium officinale as a drug carrier to encapsulate anti-breast cancer chemotherapeutic drugs, and the surface of the drug carrier is modified with folic acid ligands. By using polysaccharide of Dendrobium officinale with immunomodulatory activity as a carrier and combining with folic acid ligands with active targeting function, the present invention realizes dual-targeting of breast cancer and exerts the effect of chemoimmunotherapy. After the prepared nanoparticles are delivered to solid tumors through passive and active targeting, the tumor cells are killed by the released chemotherapeutic drug (doxorubicin), and the immunogenic cell death (ICD) is enhanced by polysaccharide of Dendrobium officinale, promoting the proliferation of natural killer cells (NK cells), stimulating the maturation of dendritic cells, and coordinately regulating various immune cells, while preventing breast cancer lung metastasis.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more particularly, to a nanoparticle for dual-targeted treatment of breast cancer, and a preparation method and application thereof. Background Art

[0002] Breast cancer has become the most commonly diagnosed cancer among women globally and ranks fifth in cancer-related deaths. The high lethality rate of breast cancer is mainly attributed to tumor metastasis, with distant metastasis accounting for more than 90% of breast cancer-related deaths. Therefore, there is an urgent need to design novel therapeutic strategies to prevent the growth and metastasis of breast cancer. In current clinical practice, chemotherapeutic drugs, such as doxorubicin (Dox), represent a commonly used cancer treatment method. However, during the treatment with doxorubicin, significant cardiotoxicity will be exhibited. Fortunately, previous studies have shown that encapsulation using nanoparticles can significantly reduce doxorubicin-related toxicity. However, based solely on the EPR effect of nanoparticles (passive targeting strategy), the efficiency of nanoparticle enrichment at the breast cancer site is not high.

[0003] Nanoparticles can reach the tumor site through passive and active targeting processes. Passive tumor targeting utilizes the enhanced permeability and retention effect, enabling the nanoparticles to have an extended circulation time within the tumor. Active targeting usually modifies the nanoparticle carrier to bind with molecules or antibodies that have high selectivity and specificity for tumors. This active targeting method has great potential for directing drug nanocarriers to tumor tissues.

[0004] Zhang G et al. disclosed a folic acid-modified Bletilla striata polysaccharide-loaded doxorubicin-mediated pH-responsive micelle (Zhang G, Huang L, Wu J, et al. Doxorubicin-loaded folate-mediated pH-responsive micelle based on Bletilla striata polysaccharide: Release mechanism, cellular uptake mechanism, distribution, pharmacokinetics, and antitumor effects - ScienceDirect[J]. International Journal of Biological Macromolecules, 2020, 164: 566-577. DOI: 10.1016 / j.ijbiomac.2020.07.123.). Specifically, the hydrophobic group stearic acid (SA) and the targeting group folic acid (FA) were coupled to the hydroxyl group of Bletilla striata polysaccharide through an ester bond to prepare a folate receptor-targeted (FA-BSPs-SA) derivative based on stearic acid-modified Bletilla striata polysaccharide (BSPs-SA). A complex of doxorubicin hydrochloride and sodium cholate was prepared through electrostatic interaction and directly encapsulated into the FA-BSP-SA micelle. The FA-BSP-SA micelle improved the absolute bioavailability of Dox (p < 0.01) and prolonged the mean residence time. However, the biocompatibility of the stearic acid used was relatively low, which might trigger certain biological reactions, and its inhibitory rate against tumors needed to be improved and it did not have the function of preventing and treating breast cancer lung metastasis. Summary of the Invention

[0005] The object of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art and provide a nanoparticle for dual-targeted treatment of breast cancer.

[0006] The second object of the present invention is to provide a preparation method of the nanoparticle for dual-targeted treatment of breast cancer.

[0007] The third object of the present invention is to provide the application of the nanoparticle for dual-targeted treatment of breast cancer.

[0008] The above objects of the present invention are achieved by the following technical solutions:

[0009] A kind of nanoparticle for dual-targeted treatment of breast cancer uses polysaccharide-cholesterol hemisuccinate amphiphilic conjugate (CHS-DOP) of Dendrobium officinale as a drug carrier to encapsulate anti-breast cancer chemotherapeutic drugs, and the surface of the drug carrier is modified with folic acid (FA) ligand. It has passive and active targeting functions; the structural formula of the polysaccharide-cholesterol hemisuccinate amphiphilic conjugate of Dendrobium officinale is shown as follows:

[0010] n is 1-2.

[0011] Dendrobium officinale is a traditional Chinese medicine with the effects of nourishing yin and clearing heat, and promoting the production of body fluid to benefit the stomach. It has been used as a Chinese medicinal material and a food therapy product (such as in soups) for thousands of years. In recent years, Dendrobium officinale has been listed in the "medicinal and edible homology" catalog by the National Health Commission of China, indicating its safety and non-toxicity. Among the key components of Dendrobium officinale, polysaccharide of Dendrobium officinale plays an important role in disease treatment. It has been proven in the prior art that polysaccharide of Dendrobium officinale has anti-cancer activity. Due to the advantages of good biocompatibility, no side effects, and easy modification of polysaccharides, they are widely used in nanocarriers. Polysaccharide of Dendrobium officinale as a carrier has the advantages of natural pollution-free, good biocompatibility, can be decomposed through normal metabolic pathways, and has immunological activity. In addition, polysaccharide of Dendrobium officinale itself has anti-tumor effects and can also stimulate the maturation of DCs, thereby further enhancing the effect of chemotherapeutic drugs. Folic acid receptors (FRs) are a class of cell surface receptors that play a role in transporting folic acid into cells. It has been observed that FRs are overexpressed in various tumor cells, including ovarian cancer, triple-negative breast cancer, endometrial cancer, mesothelioma, colon cancer, and lung cancer, while they are rarely expressed in normal cells. By binding the nanocarrier with a ligand targeting FRs, specific cell targeting can be achieved. Previous studies have also shown that in tumor models expressing FRs, the accumulation of FA-targeted drug delivery carriers is enhanced. Therefore, by using FA ligand for surface modification and adopting an active targeting strategy, the delivery efficiency of nanomedicine to the target tumor tissue can be improved.

[0012] The present invention utilizes Dendrobium officinale polysaccharide as a drug delivery carrier to enhance the anti-tumor effect. The structure of Dendrobium officinale polysaccharide (DOP) is mainly composed of mannose and glucose. Utilizing the beneficial properties of Dendrobium officinale, it is used as a hydrophilic group, and cholesteryl hemisuccinate (CHS) is used as a hydrophobic group. The amphiphilic conjugate of Dendrobium officinale polysaccharide-cholesteryl hemisuccinate (CHS-DOP) formed can encapsulate hydrophobic chemotherapeutic drugs and has passive targeting function. Since cholesteryl hemisuccinate is a natural component in the human body, cholesteryl hemisuccinate has good biocompatibility in biological systems, reducing potential toxicity and side effects. Through folic acid ligand modification, it thus has active targeting function, thereby exerting dual targeting function. More nanoparticles are transported into breast cancer cells through FA, further greatly enhancing the effect of chemotherapy drug chemotherapy immunopotentiation. The nanoparticles for dual-targeted treatment of breast cancer have remarkable efficacy, and can not only inhibit the growth of breast tumors, but also inhibit the lung metastasis of breast tumors.

[0013] Further, the nanoparticles are uniform in size, with a particle size of 210-220 nm and a Zeta potential of -11 to -14 mV.

[0014] Further, the anti-breast cancer chemotherapeutic drug is doxorubicin (Dox).

[0015] Further, the Dendrobium officinale polysaccharide has a main chain of (1→4)-linked β-mannopyranose and β-D-glucopyranose, and a branched chain composed of terminal mannopyranose, is composed of mannose and glucose in a ratio of 4.47:1, and has an average molecular weight of 80.32 kDa; each seven monosaccharide units of the CHS-DOP are modified by one CHS unit. It can be prepared according to the prior art (Tao S, Song Y, Ding S, et al. Dendrobium officinale polysaccharide-based carrier to enhance photodynamic immunotherapy. [J]. Carbohydrate polymers, 2023, 317:, 121089. DOI: 10.1016 / j.carbpol.2023.121089.).

[0016] The present invention also provides a preparation method of the nanoparticles for dual-targeted treatment of breast cancer as described in any one of the above, comprising the following steps:

[0017] S1. Under the action of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), react polysaccharide from Dendrobium officinale with cholesteryl hemisuccinate to connect the hydroxyl group of polysaccharide from Dendrobium officinale with the carboxyl group of cholesteryl hemisuccinate, forming an amphiphilic conjugate of polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate (CHS-DOP conjugate);

[0018] S2. Connect the amphiphilic conjugate of polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate obtained in step S1 with folic acid in an organic solvent under the action of EDC·HCl and 4-dimethylaminopyridine (DMAP), and prepare a folic acid-conjugated polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate conjugate (FA-DOP-CHS) by dialysis and alcohol precipitation;

[0019] S3. Dissolve an anti-breast cancer chemotherapeutic drug and the folic acid-conjugated polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate conjugate described in step S2 in an organic solvent, react, dialyze, centrifuge, and filter to obtain nanoparticles encapsulating the anti-breast cancer chemotherapeutic drug.

[0020] Furthermore, the mass ratio of polysaccharide from Dendrobium officinale to cholesteryl hemisuccinate in step S1 is 1.6 - 1.7:1.

[0021] Furthermore, the mass ratio of the amphiphilic conjugate of polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate to folic acid in step S2 is 2.2 - 2.3:1.

[0022] Preferably, the organic solvents described in steps S2 and S3 are dimethyl sulfoxide (DMSO).

[0023] Furthermore, the weight ratio of the anti-breast cancer chemotherapeutic drug to the folic acid-conjugated polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate conjugate is 1:4 - 6.

[0024] Preferably, the weight ratio of the anti-breast cancer chemotherapeutic drug to the folic acid-conjugated polysaccharide from Dendrobium officinale-cholesteryl hemisuccinate conjugate is 1:5.

[0025] Furthermore, the dialysis in step S2 is carried out using a dialysis bag with a molecular weight cut-off value of 3500 Da.

[0026] Furthermore, the dialysis in step S3 is carried out using a dialysis bag with a molecular weight cut-off value of 5000 Da.

[0027] Furthermore, the filtration in step S3 is carried out using a 0.45 μm microporous membrane.

[0028] Preferably, the preparation method of the polysaccharide from Dendrobium officinale is to precipitate the Dendrobium officinale extract after hot water extraction with ethanol, then oxidatively degrade it with H2O2 and FeCl2·4H2O, and then obtain the polysaccharide from Dendrobium officinale (DOP) by freeze-drying. The purity of DOP is 84.82%. DOP is composed of mannose and glucose in a ratio of 4.47:1, and the average molecular weight is 80.32 kDa.

[0029] The present invention utilizes the advantageous properties of DOP, uses it as a hydrophilic group, and uses cholesterol hemisuccinate (CHS) as a hydrophobic group to form an amphiphilic conjugate called CHS-DOP. Subsequently, an FA-conjugated lipid, FA-DOP-CHS, was synthesized. A carrier containing Dox was prepared, and FA-DOP-CHS@Dox (abbreviated as "FA@Dox" nanoparticles (NPs)) was prepared. Subsequently, various characterizations were carried out, including drug loading capacity, critical micelle concentration (CMC), drug release, cell uptake efficiency, and cytotoxicity evaluation against FR-rich cancer cells (4T1 and CT26 cells) and FR-poor normal cells (L929 cells). Then, in vivo imaging experiments were carried out, and the anti-tumor and anti-metastatic effects of FA@Dox NPs in a 4T1 mouse model were evaluated to assess the therapeutic effect. In addition, biosafety evaluations were carried out, and immune responses in vivo and in vitro were analyzed. The results showed that the obtained FA@Dox nanoparticles were delivered to solid tumors through passive and active targeting, the tumor cells were killed by the released doxorubicin, and DOP enhanced immunogenic cell death (ICD), promoted the proliferation of natural killer cells (NK cells), promoted dendritic cell maturation, and coordinately regulated various immune cells, while preventing breast cancer lung metastasis. In summary, the present invention prepared a breast cancer targeting preparation with dual-targeted treatment of breast cancer to treat breast cancer occurrence and lung metastasis.

[0030] Therefore, the present invention also provides the use of the nanoparticles for dual-targeted treatment of breast cancer as described above in the preparation of a pharmaceutical preparation for treating breast cancer.

[0031] The present invention also provides the use of the nanoparticles for dual-targeted treatment of breast cancer as described above in the preparation of a pharmaceutical preparation for preventing and / or treating breast cancer lung metastasis.

[0032] The present invention also provides a pharmaceutical preparation for treating breast cancer, and the pharmaceutical preparation contains the nanoparticles for dual-targeted treatment of breast cancer as described above.

[0033] Furthermore, the drug further includes pharmaceutically acceptable excipients.

[0034] Compared with Zhang's research, the present invention has the following advantages: ① Since cholesteryl hemisuccinate is a natural component in the human body, cholesteryl hemisuccinate has good biocompatibility in biological systems, reducing potential toxicity and side effects. The main advantages of cholesteryl hemisuccinate relative to stearic acid are its good biocompatibility, high chemical activity, multifunctional structural rigidity, and regulated hydrophobicity. The nanoparticles prepared in the present invention have good biocompatibility, no toxic side effects, and good blood compatibility; ② The inhibition rate of the FA@Dox nanoparticles prepared in the present invention against tumors is 1.4 times that of free Dox against tumors, higher than the 1.38 times reported in Zhang's article; ③ The FA@Dox nanoparticles prepared in the present invention can prevent and treat breast cancer metastasis, and the inhibition rate against breast cancer lung metastasis reaches 54%, while the nanoparticles prepared in Zhang's article do not have such an effect. ④ As a product included in the "medicinal and edible homology" catalog, polysaccharides from Dendrobium officinale have anti-tumor and immune-enhancing effects. Moreover, the content of polysaccharides from Dendrobium officinale is rich. The 2020 edition of the Chinese Pharmacopoeia stipulates that the content of polysaccharides from Dendrobium officinale exceeds 25%, while the content requirement for Bletilla striata (also known as Baiji) in the Chinese Pharmacopoeia is that the content of 1,4-bis[4-(glucosyloxy)benzyl]-2-isobutylmalic acid ester (C 34 H 40 O 17 ) shall not be less than 1.5%, and it is not a polysaccharide. Moreover, literature reports that the highest polysaccharide content of Bletilla striata from Anlong is 23.20%; the lowest is that of Bletilla striata from Shibing, only 11.70% (Han Xue, Zeng Qinghong, Niu Minmin, etc., Determination of polysaccharide content in fresh Bletilla striata from different origins [J], 2020, 37(12): 34-35.). Compared with polysaccharides from Bletilla striata, polysaccharides from Dendrobium officinale have greater industrialization potential.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The nanoparticles for dual-targeted treatment of breast cancer in the present invention use polysaccharide-cholesterol hemisuccinate amphiphilic conjugate of Dendrobium officinale as a drug carrier to encapsulate anti-breast cancer chemotherapeutic drugs, and the surface of the drug carrier is modified with folic acid ligands. By using DOP with anti-tumor and immunomodulatory activities as a carrier and combining with folic acid ligands with active targeting functions, the present invention realizes dual targeting of breast cancer, exerts the effect of chemoimmunotherapy, and at the same time, the prepared nanoparticles have good biocompatibility, no toxic and side effects, and good blood compatibility. After the obtained FA-DOP-CHS@Dox ("FA@Dox") nanoparticles are delivered to solid tumors through passive and active targeting, the tumor cells are killed by the released doxorubicin, and the immunogenic cell death (ICD) is enhanced by DOP, promoting the proliferation of natural killer cells (NK cells), further stimulating the maturation of dendritic cells, and synergistically regulating various immune cells to exert anti-tumor effects, while preventing breast cancer lung metastasis. Compared with the use of chemotherapy alone, the nanoparticles of the present invention have the effect of reducing toxicity and increasing efficacy; compared with the combined treatment strategy of chemotherapy and immunotherapy, the nanoparticles of the present invention do not need to additionally add immune adjuvants or immune checkpoint inhibitors, which is simpler. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 are characterization results. Among them, (A) FT-IR spectra of FA, CHS-DOP, and FA-DOP-CHS; (B) 1 1H-NMR spectra of CHS, DOP, CHS-DOP, FA, and FA-DOP-CHS; (C) particle size distribution and morphology of FA@Dox NPs detected by DLS and TEM; (D) Zeta potential of CHS@Dox NPs and FA@Dox NPs; (E) release curves of Dox in CHS@Dox NPs and FA@Dox NPs at pH 5.5 and 7.4.

[0038] Figure 2 The critical micelle concentration (CMC) of CHS-DOP (A) and FA-DOP-CHS (B) nanoparticles measured by the DLS method is represented by the change curve of the particle counting rate with the concentration.

[0039] Figure 3 is the standard curve of doxorubicin (Dox) for calculating the loading amount and loading rate of Dox in CHS@Dox nanoparticles and FA@Dox nanoparticles.

[0040] Figure 4 The hydrated particle size distribution and morphology of CHS@Dox nanoparticles were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively.

[0041] Figure 5Particle size changes of CHS@Dox nanoparticles and FA@Dox nanoparticles within 5 days.

[0042] Figure 6 Polydispersity index (PDI) changes of CHS@Dox nanoparticles and FA@Dox nanoparticles within 5 days.

[0043] Figure 7 Confocal images of cellular uptake of Dox, CHS@Dox, and FA@Dox NPs by 4T1, CT26, and L929 cells. FA / FA@Dox represents FA@Dox NPs pretreated with free FA. Scale bar represents 20 μm.

[0044] Figure 8 Cell viability of free Dox, CHS@Dox nanoparticles, and FA@Dox nanoparticles after treating (A) 4T1 cells, (B) CT-26 cells, and (C) L929 cells for 48 hours, respectively.

[0045] Figure 9 Evaluation of ICD: (A) CRT exposure and (B) HMGB1 release of 4T1 cells or CT-26 cells treated differently under CLSM. Scale bar: 20 μm; (C) ATP secretion of 4T1 cells and CT-26 cells treated differently. * P < 0.05, ** P < 0.01, *** P < 0.001 represents significant differences between the treatment group and the control group; ## P < 0.01 represents significant differences between the FA@Dox group and the free Dox or CHS@Dox group.

[0046] Figure 10 Fluorescence imaging in 4T1 tumor-bearing mice: (A) In vivo fluorescence images at different time points after intravenous injection (the dashed yellow line indicates the tumor location); (B) Fluorescence imaging of in vivo organs and tumor tissues 24 hours after intravenous injection; (C) Quantitative analysis of the average fluorescence intensity of tumor tissues at different time points (n = 3); (D) Quantitative analysis of the average fluorescence intensity of different organ regions (n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001 represents significant differences between the treatment group and the free Dir group; # P < 0.05 represents significant differences between the FA@Dox group and the CHS@Dox group.

[0047] Figure 11Results of in vivo anti-tumor research. (A) Schematic diagram of breast cancer model construction; (B-C) Tumor volume growth curve and representative pictures; (D) Tumor weight; (H) Mouse body weight; ** P < 0.01, *** P < 0.001, comparison between the treatment group and the control group; # P < 0.05, ### P < 0.001, comparison between the FA@Dox group and the free Dox group and the CHS@Dox group.

[0048] Figure 12 Representative images of H&E staining, Ki67 and TUNEL immunofluorescence of 4T1 tumor sections of different administration groups.

[0049] Figure 13 Representative images of tumor metastasis in the lung tissue of mice with 4T1 lung metastasis model (black circles: tumor nodules).

[0050] Figure 14 Results detection of anti-metastasis effect. Among them, (A) Schematic diagram of the establishment of breast cancer lung metastasis model. (B) Representative photos showing the appearance of lung metastasis tumor nodules after fixation with Bouin's solution (top, front; bottom, back). (C) Analysis of the average number of lung tumor nodules in different treatment groups. (D) Analysis of lung weight after treatment. * P < 0.05, and ** P < 0.01. (E) Representative histological examination of the entire lung lobe of each group measured by H&E staining. Black arrows indicate visible lesions in the lung, scale bar = 1.0 mm. (F) Magnified view of the above picture. Scale bar = 100 μm.

[0051] Figure 15 Fluorescence imaging of calreticulin exposure (CRT) and high-mobility group box 1 (HMGB1) release in lung tissue and tumor tissue, scale bar is 20 μm.

[0052] Figure 16 Results of immune response analysis. Among them, flow cytometry analysis of activated DC (CD80 + CD86 + ) in tumor (A) and TDLNs (B); (C) Flow cytometry analysis of the infiltration of immune cells CD8 + T cells and CD4 + T cells in the tumor; (D) Flow cytometry analysis of Tregs cells (CD25 + Foxp3 + T cells) in the tumor; (E) Flow cytometry analysis of spleen NK cells (CD3 - CD49 + ) in the tumor; (F) Perforin (CD8+ Perforin + ) Flow cytometry analysis of; (G) Granzyme B in the supernatant was measured by ELISA. Compared with the control group, * P < 0.05, ** P < 0.01, *** P < 0.001.

[0053] Figure 17 For flow cytometry analysis of the proportion of mature dendritic cells (CD80 + CD86 + ) in 4T1 tumors of different treatment groups.

[0054] Figure 18 For flow cytometry analysis of the proportion of mature dendritic cells (CD80 + CD86 + ) in lymph nodes of different treatment groups.

[0055] Figure 19 For flow cytometry analysis of the proportion of CD8 + T cells and CD4 + T cells in 4T1 tumors of different treatment groups.

[0056] Figure 20 For immunofluorescence microscopy observation of (A) CD4 + , (B) CD8 + and (C) Foxp 3 + cells in tumor sections of 4T1 tumor-bearing mice.

[0057] Figure 21 For immunofluorescence microscopy observation of (A) CD4 + and (B) CD8 + cells in lung sections of 4T1 lung metastasis model mice.

[0058] Figure 22 For flow cytometry analysis of the proportion of regulatory T cells (CD25 + Foxp3 + ) in 4T1 tumors of different treatment groups.

[0059] Figure 23 For flow cytometry analysis of the proportion of natural killer cells (CD3 - CD49 + ) in the spleens of different treatment groups of mice.

[0060] Figure 24 For flow cytometry analysis of the proportion of perforin (CD8 + Perforin + )-positive cells in 4T1 tumors of different treatment groups.

[0061] Figure 25 Results of the blood compatibility evaluation of the nanoparticles. Among them, (A) hemolysis pictures and (B) hemolysis rates were obtained using CHS@Dox and FA@Dox with 2% Triton X-100 as the positive control and PBS buffer as the negative control (n = 3). NPs at different concentrations of 12.5 to 50 μg / mL were maintained for 2 hours at 37°C.

[0062] Figure 26 Representative images of H&E-stained sections of the liver, heart, spleen, lung, and kidney. (Black arrow: necrotic cardiomyocytes and broken myocardial fibers).

[0063] Figure 27 Images of magnified heart H&E-stained sections of the Control group and the FA@Dox group from left to right. (Black arrow: necrotic cardiomyocytes and broken myocardial fibers).

[0064] Figure 28 Blood biochemical analysis of mice treated with free Dox, CHS@Dox NPs, and FA@Dox NPs.

[0065] Figure 29 Schematic diagram of the enhanced chemoimmunotherapy of FA@DOP nanoparticles of the present invention and inhibition of breast cancer growth and lung metastasis. Detailed implementation manners

[0066] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0067] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0068] Mouse mammary tumor cells (4T1), mouse colon cancer cells (CT-26), and mouse fibroblasts (L929) were all obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM (Gibco, California, USA) containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2.

[0069] Example 1 Preparation and characterization of CHS@Dox and FA@Dox

[0070] 1. Method

[0071] (1) Preparation of amphoteric polysaccharide

[0072] According to the inventors' previous research (Tao S, Song Y, Ding S, et al. Dendrobium officinale polysaccharide-based carrier to enhance photodynamic immunotherapy. [J]. Carbohydrate polymers, 2023, 317:, 121089. DOI: 10.1016 / j.carbpol.2023.121089.), Dendrobium officinale polysaccharide (DOP) and amphiphilic CHS-DOP conjugate were prepared. Briefly, the Dendrobium officinale extract after hot water extraction was precipitated with ethanol and then oxidized and degraded with H2O2 and FeCl2·4H2O. Then, Dendrobium officinale polysaccharide (DOP) was obtained by freeze-drying. The purity of DOP was 84.82%. DOP has a backbone of (1→4)-linked β-mannopyranose and β-D-glucopyranose, as well as branches composed of terminal mannopyranose. DOP is composed of mannose and glucose in a ratio of 4.47:1 and has an average molecular weight of 80.32 kDa. DOP has high water solubility. To endow it with the ability of self-assembly in water, in the presence of EDC·HCl, a hydrophobic moiety CHS with good biocompatibility was introduced by connecting the carboxyl group of cholesterol hemisuccinate (CHS) to the hydroxyl group on DOP. The synthesis route of CHS-DOP conjugate is shown as follows:

[0073]

[0074] n is 1-2; specifically, 121.68 mg of cholesterol hemisuccinate (CHEMS, CHS), 57.5 mg of EDC·Hcl (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 36.65 mg of dimethylaminopyridine (DMAP) and 200 mg of DOP were added to 10 mL of dimethyl sulfoxide (DMSO), and the reaction product was kept at 38 °C for 48 hours. Precipitation was obtained after adding cold absolute ethanol. After filtration and washing with cold absolute ethanol, CHS-DOP conjugate was obtained.

[0075] (2) Synthesis of FA-conjugated CHS-DOP (FA-DOP-CHS)

[0076] CHS-DOP (100.2 mg), EDC·HCl (92.3 mg), DMAP (48.8 mg) and FA (44.1 mg) were added to 4 mL of DMSO, and then reacted continuously at 38 °C for 48 h. After the reaction, dialysis was carried out for 48 h using a dialysis bag (molecular weight cut-off value of 3500 Da) in deionized water. Subsequently, the solution was collected and precipitated with cold absolute ethanol. Then, the precipitate of the FA-DOP-CHS polymer was lyophilized and characterized by FT-IR spectroscopy using a NicoletTM iS50 Fourier transform infrared spectrometer (Thermo Fischer Scientific, Waltham, MA, USA) and 1H nuclear magnetic resonance spectroscopy using a 400 MHz nuclear magnetic resonance spectrometer (AVIII, Bruker, Germany). 1 1 1 H nuclear magnetic resonance

[0077] (3) Preparation of CHS@Dox and FA@Dox nanoparticles

[0078] Doxorubicin (Dox), CHS-DOP or FA-DOP-CHS was dissolved in DMSO, respectively. Then the doxorubicin solution was dropped into the CHS-DOP or FA-DOP-CHS solution, stirred at room temperature and maintained for 2 h (the weight ratio of doxorubicin / CHS-DOP or FA-DOP-CHS was 1:5). Then the solution was transferred to a dialysis bag (molecular weight cut-off value of 5000 Da) in deionized water for 24 h to remove the unloaded drug and DMSO. Finally, the obtained solution was centrifuged at 3000 rpm for 5 min and filtered through a 0.45 μm microporous membrane to obtain CHS-DOP@Dox (abbreviated as 'CHS@Dox') or FA-DOP-CHS@Dox (abbreviated as "FA@Dox") NPs.

[0079] (4) Critical micelle concentration (CMC)

[0080] The CMC of CHS-DOP and FA-DOP-CHS was determined by dynamic light scattering (DLS) method. Briefly, CHS-DOP or FA-DOP-CHS was serially diluted with water at concentrations of 0.001, 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 10 μg / mL. The change in particle count rate caused by micelle formation was measured by NanoBrook 90Plus Zeta (Brookhaven Instruments Corporation, New York, USA). The CMC value was determined as the concentration at the inflection point of the DLS data curve.

[0081] (5) Nanoparticle characterization

[0082] The particle size and polydispersity index (PDI) of CHS@Dox and FA@Dox were measured using a NanoBrook 90Plus Zeta. Subsequently, the Zeta potential was determined using a Nano-ZS90 ZetaSizer (Malvern Panalytical Ltd., UK). The morphologies of DOP@3BCP and PEG@3BCP were observed using a JEM-2100 transmission electron microscope (TEM) (JEOL, Tokyo, Japan). The Dox content was determined by fluorescence measurement (excitation wavelength 480 nm) in the dark and combined with a standard curve. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated therefrom.

[0083] (6) In vitro Dox release curve

[0084] The dialysis method was used to determine the Dox release curve of CHS@Dox or FA@Dox NPs. Briefly, CHS@Dox or FA@Dox (300 μg Dox content) was encapsulated in a dialysis bag with a molecular weight cut-off of 3500 Da and immersed in PBS solution (15 mL, pH = 5.5 or 7.4), and then placed in a shaker at 37 °C. Finally, 1 mL of the PBS solution was replaced with the same volume of fresh PBS at regular intervals to maintain the same volume. The Dox content in the sample was determined by fluorescence measurement.

[0085] 2. Results

[0086] (1) Characterization of DOP and CHS-DOP

[0087] The characterization of DOP and CHS-DOP has been reported in the inventors' previous study (Tao S, Song Y, Ding S, et al. Dendrobium officinale polysaccharide-based carrier to enhance photodynamic immunotherapy. [J]. Carbohydrate polymers, 2023, 317:, 121089. DOI: 10.1016 / j.carbpol.2023.121089.). Briefly, the purity of DOP was 84.82%. DOP was composed of mannose and glucose in a ratio of 4.47:1, with an average molecular weight of 80.32 kDa. The backbone of DOP was mainly [→4)-β-D-Manp-(1→4)-β-D-Glcp-(1→] n . As for CHS-DOP, every seven monosaccharide units were modified by one CHS unit.

[0088] (2) Characterization of CHS-DOP and FA-DOP-CHS

[0089] FT-IR and 1 H NMR experiments were carried out to verify whether FA molecules were successfully grafted onto CHS-DOP. The results are as Figure 1 shown in A. CHS-DOP and FA-DOP-CHS exhibited broad absorption peaks at 3400 cm -1 , 2900 cm -1 and 1020 cm -1 , which were attributed to O-H stretching vibration, C-H stretching vibration, and C-O bending vibration, respectively. All of these are considered typical features of polysaccharides. Compared with CHS-DOP, two new characteristic peaks were shown at 1606 cm -1 and 1485 cm -1 , which could be attributed to the stretching vibration of the benzene ring skeleton of FA. These results indicated that FA was successfully combined with CHS-DOP. In addition, in 1 the 1H NMR spectrum ( Figure 1 B), the characteristic peaks from δ 0.5 ppm to δ 2.5 ppm were the peaks of the methylene and methyl protons of CHS. The hydroxyl protons of DOP were located in the region of δ 4.3 - 5.0 ppm. The signals at δ 6.65 and δ 7.61 ppm (phenyl) were attributed to the aromatic protons of FA. The peak appearing at δ 8.67 ppm was attributed to the piperidine protons of FA. These characteristic peaks of CHS and FA were also observed in the FA-DOP-CHS polymer.

[0090] (3) Critical micelle concentration (CMC)

[0091] The results of the critical micelle concentration (CMC) are as Figure 2 shown. The CMC values of CHS-DOP and FA-DOP-CHS in the medium were 1.28 and 1.12 μg / mL, respectively. These results indicated that CHS-DOP and FA-DOP-CHS could self-aggregate into micelles.

[0092] (4) Characterization of CHS@Dox NPs and FA@Dox NPs

[0093] The standard curve of doxorubicin was plotted ( Figure 3), and the doxorubicin concentrations encapsulated in CHS-DOP and FA-DOP-CHS (CHS@Dox and FA@Dox NPs) were determined according to the standard curve. The drug loading contents in CHS@Dox and FA@Dox NPs were 10.53% and 10.22%, respectively. The drug loading efficiencies were 63.20% and 61.31%, respectively. Dynamic light scattering (DLS) showed that the average diameters of CHS@Dox and FA@Dox NPs were 237.31 ± 1.63 nm and 212.53 ± 2.49 nm ( Figure 1 C and Figure 4 ). The morphologies of CHS@Dox and FA@Dox NPs were observed by transmission electron microscopy, and it was confirmed that they were spherical and had a relatively uniform size distribution. Notably, the sizes of CHS@Dox or FA@Dox NPs observed by transmission electron microscopy were smaller than the hydrodynamic radii measured by dynamic light scattering. This may be because transmission electron microscopy measured the sizes of CHS@Dox or FA@Dox NPs in the dry state, while dynamic light scattering measured the sizes in the hydrated state. The ζ-potentials of CHS@Dox and FA@Dox NPs were -8.74 ± 0.87 mV and -12.8 ± 0.17 mV ( Figure 1 D). In addition, the particle sizes and PDI (polydispersity index) of CHS@Dox and FA@Dox NPs were monitored for 5 days and remained stable. ( Figure 5 and Figure 6 ).

[0094] As Figure 1 shown in E, the in vitro release curves of doxorubicin in the nanoparticles were tested at pH 5.5 and 7.4. After incubation at pH 7.4 for 48 hours, the cumulative release amounts of doxorubicin in CHS@Dox and FA@Dox NPs were 41% and 55%, respectively. Compared with the neutral environment (pH 7.4), the acidic environment (pH 5.5) accelerated the release of doxorubicin, showing cumulative releases of approximately 60% and 72% within 48 hours, respectively. This indicates that the encapsulated doxorubicin is more easily released in the acidic tumor microenvironment.

[0095] Example 2 Cell Uptake Study

[0096] To study the uptake of Dox, CHS@Dox or FA@Dox nanoparticles by 4T1 and CT-26 cells with high expression of folate receptor (FR) and L929 cells lacking FR, confocal laser scanning microscopy (CLSM, Nikon A1, Nikon Corporation, Tokyo, Japan) was used for observation. All cells were in confocal dishes (1×10 per dish 5cultured for 24 hours. Then, the cells were treated with free Dox, CHS@Dox or FA@Dox nanoparticles (5 μg / mL) for 8 hours, and then stained with DAPI for 10 min and observed under CLSM. To confirm the FR-mediated uptake effect of the nanoparticles, a group of cells was pretreated with free folic acid (FA, 20 μg / mL). To evaluate the FR-mediated uptake efficiency of FA@Dox nanoparticles, each group of cells had a set pretreated with free FA (20 μg / mL) and then treated with FA@Dox nanoparticles.

[0097] The results are as Figure 7 shown. CHS@Dox or FA@Dox nanoparticles (red) were localized in the perinuclear region of the cell lines, indicating that these nanoparticles were distributed in the perinuclear cytoplasm. The uptake efficiency of FA@Dox nanoparticles in folate receptor-expressing cell lines (4T1 and CT-26) was significantly higher than that of CHS@Dox nanoparticles. This was not observed in the L929 cell line lacking folate receptors. In addition, in the presence of excess free folic acid, the uptake of FA-targeted nanoparticles by 4T1 and CT-26 cell lines was significantly inhibited. This indicates that the uptake of targeted nanoparticles depends on folate receptor-mediated endocytosis. In addition, the role of folate receptors in the cell uptake mechanism was further confirmed in L929 cells, and the results showed that there was no significant difference in the uptake of L929 cells with or without folate pretreatment. These experimental results indicate that for tumor cells with high FR expression on the cell surface, FA-mediated FA@Dox nanoparticles have an active targeting effect.

[0098] Example 3 Cytotoxicity experiment

[0099] In this example, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was used for the cytotoxicity experiment. Briefly, 4T1 or CT-26 cells were cultured in a 96-well plate at a density of 1×10 4 cells / well for 24 hours. Then, the cells were treated with different concentrations (0, 0.5, 1, 2, 4, 8 μg / mL) of free doxorubicin, CHS@Dox NPs or FA@Dox NPs for another 48 hours. Subsequently, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 hours. After aspirating the medium, the precipitated formazan crystals were dissolved in 100 μL of DMSO. Finally, the absorbance at 490 nm was measured using a microplate reader (Multiskan GO microplate spectrophotometer, Thermo Scientific, USA).

[0100] The half-maximal inhibitory concentration (IC 50 ) of free Dox, CHS@Dox NPs, and FA@Dox NPs was calculated using GraphPad Prism software. Based on the IC 50 values of normal cells (L929) and tumor cells (4T1 or CT-26 cells), the safety index of different formulations was calculated according to the following equation: Safety index = IC 50 (normal cells) / IC 50 (tumor cells).

[0101] The viability of cancer cells (4T1 and CT-26 cells) and normal cells (L929 cells) after in vitro treatment was evaluated by MTT assay, with treatment groups including free Dox, CHS@Dox nanoparticles, and FA@Dox nanoparticles. As Figure 8 shown, with the increase in Dox concentration, the viability of 4T1, CT-26, and L929 cells treated with different administration groups decreased. CHS@Dox nanoparticles and FA@Dox nanoparticles showed concentration-dependent inhibitory effects in all three cell types, similar to free Dox. As shown in Table 1, the IC 50 of CHS@Dox nanoparticles and FA@Dox nanoparticles against cancer cells (4T1 and CT-26 cells) was slightly higher than that of free Dox. A possible explanation is that Dox is slowly released from the drug-loaded nanoparticles, resulting in a lower concentration than free Dox under the same conditions. However, for the treatment of normal cells (L929 cells), the IC 50 of CHS@Dox nanoparticles and FA@Dox nanoparticles was much higher than that of free Dox. Therefore, compared with free Dox, CHS@Dox nanoparticles and FA@Dox nanoparticles have a higher safety index.

[0102] Table 1 Half-maximal lethal dose (IC 50S ) and safety index (SI) of free Dox, CHS@Dox NPs, and FA@Dox NPs

[0103]

[0104] Example 4 Detection of Key Immunogenic Cell Death (ICD) Biomarkers

[0105] The evaluation of key ICD biomarkers was performed as described in our previous study. Briefly, anti-calreticulin (CRT) and high-mobility group box 1 (HMGB1) were evaluated by immunofluorescence and analyzed by confocal laser scanning microscopy. The extracellularly released ATP was determined using an ATP detection kit.

[0106] CRT exposure, HMGB1 release, and ATP secretion were detected to investigate whether CHS@Dox and FA@Dox nanoparticles could trigger ICD. The results are as Figure 9 shown in A. Less CRT exposure (green) was observed on the surface of 4T1 and CT-26 cells treated with free Dox and CHS@Dox nanoparticles. However, significant cell surface CRT exposure was observed in FA@Dox nanoparticles, which was attributed to the targeting effect of FA-mediated FA@Dox nanoparticles. Figure 9 B and Figure 9 C show that compared with the control group, the FA@Dox group significantly enhanced the release of HMGB1 and the secretion of ATP in 4T1 and CT-26 cells. At the same time, more HMGB1 release and ATP secretion were observed in the FA@Dox group compared with the free Dox and CHS@Dox groups. These results indicate that FA-targeted nanoparticles can enhance the release of damage-associated molecular patterns (DAMPs) during cancer cell treatment.

[0107] Example 5 In Vivo Antitumor and Antimetastatic Effects

[0108] 1. Methods

[0109] (1) In Vivo Biodistribution Evaluation

[0110] First, CHS-DOP nanoparticles loaded with the dye DiR (CHS@DiR) and FA-DOP-CHS nanoparticles loaded with DiR (FA@DiR) were prepared using the same method as the nanoparticle preparation process in the present invention. To study the in vivo behavior of CHS@Dox nanoparticles and FA@Dox nanoparticles, BALB / c mice inoculated with 4T1 breast tumors were divided into 4 groups, with 3 mice in each group. When the tumor volume reached approximately 200 mm 3 , free DiR, CHS@DiR nanoparticles, and FA@DiR nanoparticles (DiR dose of 5 mg / kg) were injected via the tail vein, and detection was performed using a fluorescence imaging system (Xenogen IVIS-Spectrum, USA) at 1 h, 3 h, 6 h, 12 h, and 24 h after injection. At the end of the experiment, the mice were sacrificed, and the main organs (heart, liver, spleen, lung, kidney) were collected and imaged in vitro using the same parameters.

[0111] (2) In Vivo Antitumor Effect

[0112] Female Balb / c mice (4 - 6 weeks old) were used to evaluate the in vivo antitumor and antimetastatic effects of CHS@Dox and FA@Dox nanoparticles. For the 4T1 tumor growth model, a total of 5×10 6 4T1 cells were subcutaneously injected into the right hind leg area of BALB / c mice. When the tumor volume reached 60 - 80 mm 3At that time, mice (n = 6) were intravenously injected with PBS, free Dox, CHS@Dox, and FA@Dox nanoparticles, respectively, at a dose of 5 mg / kg of Dox. Treatments were carried out on days 0, 3, and 6 of the present invention. The tumor size was measured every 2 days using an electronic caliper, and the tumor volume was calculated as volume = length × width 2 × 0.5. On the 14th day after treatment, the mice were sacrificed, and the major organs (heart, liver, spleen, lung, kidney) and tumors were removed for blood and histological examinations. At the same time, the tumors were also subjected to TUNEL immunofluorescence staining and Ki67 immunohistochemistry. Sections of H&E, TUNEL, and Ki67 were observed and imaged using an Olympus microscope (Olympus BX53, Japan). In addition, the collected blood samples were analyzed for blood biochemistry (including ALT, AST, BUN, UA, CK, and LDH) and blood routine (WBC, lymphocyte count, monocyte count, granulocyte count, RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, and MPV).

[0113] (3) Anti-metastasis effect in vivo

[0114] For the 4T1 lung metastasis model, 4T1 cells (2 × 10 6 cells) suspended in PBS were injected into BALB / c mice via the tail vein. Subsequently, the subsequent treatments were the same as the above procedures. Finally, the mice were sacrificed 14 days after treatment. The lungs of the mice were removed, the number of metastatic nodules was examined, and then the sections were stained with H&E.

[0115] (4) Immunoreaction analysis

[0116] To examine the treatment-induced CRT expression, HMGB1 release, and in vivo T lymphocyte infiltration, tumors with a size of approximately 80 mm 3Mice with 4T1 tumors were intravenously injected (i.v.) with PBS, Dox, CHS@Dox nanoparticles, or FA@Dox nanoparticles every 3 days at a dose of 5 mg / kg each. Three days after the last treatment, tumors, inguinal lymph nodes (LNs), and spleens were collected. Treatment-induced CRT expression and HMGB1 release at the tumor site were examined by immunofluorescence analysis. In addition, the excised tumors were cut into small pieces, and single-cell suspensions were prepared using a MACS Tumor Dissociation Kit (Miltenyi Biotec, Auburn, CA, 130-096-730) according to the protocol of the kit instructions. After staining with anti-mouse CD3-FITC, anti-mouse CD4-PerCP-Cy5.5, anti-mouse CD8-PE-Cy7, anti-Perforin or-PE, or anti-mouse CD4-PerCP-Cy5.5, anti-mouse CD25-BV421, and anti-mouse Foxp3-Alexa Fluor 647 antibodies, the cell suspensions were analyzed by flow cytometry. In addition, to examine DC maturation in vivo, the maturation of DC cells in lymph nodes and tumors was examined by flow cytometry after immunofluorescence staining using anti-mouse CD11c-PE, anti-mouse CD86-FITC, and anti-mouse CD80-APC antibodies. To analyze the secretion of cytokines induced after treatment, the concentrations of pro-inflammatory cytokines (including Granzyme B) in serum were analyzed using an ELISA kit according to the manufacturer's instructions.

[0117] 2. Results

[0118] (1) Anti-tumor and anti-metastatic effects in vivo

[0119] The in vivo distribution characteristics of drugs depend on the nanocarrier rather than the drug itself. Fluorescence results showed that free DiR was mainly distributed in the liver rather than in tumors. As expected, CHS@DiR nanoparticles and FA@DiR nanoparticles were able to circulate, distribute, and selectively accumulate in tumors. Figure 10 Figure A shows that the fluorescence intensities of CHS@DiR nanoparticles and FA@DiR nanoparticles remained in tumors within 24 hours. And the fluorescence accumulation of FA@DiR nanoparticles in tumors was faster and stronger than that of CHS@DiR nanoparticles, indicating that FA@DiR nanoparticles have an advantage in tumor targeting. This result confirmed the important role of FA active targeting.

[0120] Subsequently, in vitro imaging results showed consistent targeting effects. CHS@DiR nanoparticles and FA@DiR nanoparticles were generally transported to the liver and spleen and significantly stayed in tumors, while free DiR had no ability to accumulate in tumors ( Figure 10B). Compared with CHS@DiR nanoparticles, FA@DiR nanoparticles accumulated more in lung tissue rather than in liver tissue, which might be related to the first-pass effect of the liver and the active targeting of FA, suggesting that FA@Dox nanoparticles might inhibit the metastasis of breast tumors to the lungs. Quantitative fluorescence results showed that the fluorescence intensity of FA@DiR nanoparticles in tumors and lungs was significantly stronger, with a significant difference from the free DiR group (p<0.001), verifying the good tumor accumulation effect of FA@DiR nanoparticles ( Figure 10 C-D). Therefore, the designed drug nanoparticles modified with FA achieved significant targeting function in 4T1 tumor-bearing mice.

[0121] In this invention, in vivo anti-tumor studies were carried out on 4T1 tumor-bearing BALB / C mice, and the experimental methods were as Figure 11 shown in A. The in vivo anti-tumor effects were as Figure 11 shown in B-D. The results showed that compared with the model group, the tumor inhibition rate of free Dox was 58.71%, while the tumor inhibition rates of CHS@Dox and FA@Dox nanoparticles were 64.96% and 82.52% respectively. The tumor inhibition rates of CHS@Dox and FA@Dox nanoparticles were 1.10 and 1.40 times that of free Dox respectively. The results indicated that compared with free Dox and CHS@Dox nanoparticles, FA@Dox nanoparticles had a better effect in restricting tumor growth in tumor-bearing mice, which should be attributed to the active targeting effect of FA. The body weights increased steadily during the treatment of each group, indicating low systemic toxicity of the therapeutic drugs ( Figure 11 E). As Figure 12 shown, compared with other groups, the FA@Dox nanoparticle group caused the most tumor cell death in H&E staining. In addition, immunohistochemistry and immunofluorescence analysis showed that the Ki67 expression in the tumor tissue of the FA@Dox nanoparticle group was the lowest and the TUNEL signal was the strongest, both suggesting that FA@Dox nanoparticles could inhibit tumor growth to the greatest extent.

[0122] In the in vivo anti-tumor experiment, after the mice were sacrificed and lung tissues were collected, the metastasis of tumors to lung tissues was observed ( Figure 13 ). According to the biodistribution of FA@DiR NPs ( Figure 10 B), we speculated that FA@DOP NPs could inhibit lung metastasis. Therefore, we verified the anti-metastatic effect of FA@Dox NPs by constructing a lung metastasis model. The schematic diagram of establishing the breast cancer lung metastasis model was as Figure 14 shown in A, and the experimental results were as Figure 14 shown in B-D. The number of lung metastasis nodules in the FA@Dox group was significantly less than that in other treatment groups ( Figure 14B). The average number of metastatic nodules in each lung of the FA@Dox group (28 ± 7) was much less than that of the control group (62 ± 2), the free Dox group (59 ± 7), and the CHS@Dox group (50 ± 11). Figure 14 C). Meanwhile, the weight of the lungs in the FA@Dox group was significantly less than that of the other groups. Figure 14 D). In addition, the anti - tumor metastasis effect of FA@Dox was verified by the images of the collected lungs and their sections. H&E staining further showed a significant decrease in the number and size of visible metastatic nodules in the FA@Dox group. Figure 14 E - F). Overall, these results indicate that FA@Dox has significant efficacy, not only inhibiting breast tumor growth but also inhibiting lung metastasis of breast tumors.

[0123] (2) Immune response analysis

[0124] The exposure of calreticulin (CRT) and the release of high - mobility group box 1 (HMGB1), as key indicators of ICD, promoted the maturation of dendritic cells (DCs) and subsequently triggered T - lymphocyte - mediated anti - tumor immunity. In the present invention, we analyzed the exposure of CRT and the release of HMGB1 in tumor tissues. Figure 15 The results shown in indicated that the doxorubicin (Dox) group could enhance the exposure of CRT and the release of HMGB1 to a certain extent, indicating that Dox could promote the infiltration of immune cells into tumors by inducing ICD. Further analysis showed that compared with the control group and the Dox group, the FA@Dox and CHS@Dox groups showed significantly enhanced exposure of CRT and release of HMGB1, which should be attributed to the passive targeting effect of the nanoparticles and the DOP - induced anti - tumor immune effect. We also analyzed the exposure of CRT and the release of HMGB1 in lung tissues. The results were consistent with those observed in Figure 10 B. Subsequently, we performed immune cell analysis to confirm the initiation of adaptive immunity. We collected tumor tissues and tumor - draining lymph nodes (TDLNs) and performed flow cytometry analysis of the percentage of mature DCs (CD11c + CD80 + CD86 + ). As shown in Figure 16 A - B and Figure 17 - 18 , the proportion of mature DCs in tumor tissues (23.38% ± 4.09%) and TDLNs (17.70% ± 0.82%) in the FA@Dox - treated group was significantly higher than that of the control group and the Dox - treated group.

[0125] It has been reported that tumor cells undergoing ICD can trigger a systemic immune response. Tumor-associated antigens (TAAs) released by dying tumor cells and damage-associated molecular patterns (DAMPs) released by ICD further stimulate the maturation of DCs and enhance lymphocyte infiltration, ultimately activating the adaptive anti-tumor immune response. Subsequently, flow cytometry was used to analyze the percentage of cytotoxic T lymphocytes in tumor tissues. The control group and the free Dox treatment group did not express CD4 and CD8, showing a low immune response. However, the FA@Dox and CHS@Dox treatment groups showed higher levels of CD4 and CD8 expression, indicating effective activation of the anti-tumor immune response( Figure 16 C、 Figure 19 and Figure 20 A-B). The FA@Dox treatment group showed higher levels of CD4 and CD8 expression than the CHS@Dox treatment group, which was attributed to the tumor-targeting ability of FA. The same phenomenon was also observed in the lung tissues with a lung metastasis model( Figure 21 ), indicating that FA@DOP nanoparticles target lung tissues and induce the ICD effect.

[0126] It is well known that regulatory T cells (Tregs, labeled as Foxp3) play an important role in preventing lymphocyte infiltration and inhibiting T cell activity, and CD4 and CD25 play a key role in regulating the immune environment and activating T cells. Therefore, we further evaluated the proportion of Treg cells (CD25 + Foxp3) by flow cytometry analysis and detected the levels of CD4 and Treg in tumor sections with CD4 / Treg antibodies. As Figure 16 D and Figure 22 shown, compared with the control group and free Dox, the proportion of Treg cells (CD25 + Foxp3) was significantly reduced after treatment with FA@Dox and CHS@Dox. And the reduction level in the FA@Dox treatment group was better than that in the CHS@Dox group. Correspondingly, as Figure 20 shown in B-C, the CD4 / Treg ratio in the FA@Dox and CHS@Dox treatment groups showed a significant increase compared with the control group and the free Dox group. In addition, the increase level in the FA@Dox treatment group was better than that in the CHS@Dox group, further demonstrating the effective activation of the immune response.

[0127] NK cells can recognize tumor cells, are not restricted by tumor neoantigens and major histocompatibility complex molecules, and are a supplement to T cell-based anti-tumor therapy. The present invention evaluated the distribution of NK cells in the spleen and explored the NK cell proliferation ability of FA@Dox nanoparticles to explain the related immune response. As Figure 16 E and Figure 23As shown, compared with the control group and the free Dox group, the treatments with CHS@Dox and FA@Dox significantly promoted the content of NK cells in the spleen (p<0.01). This indicates that the nanoparticles can strongly stimulate and enhance the proliferation of NK cells for tumor immunotherapy.

[0128] In addition, as important cell markers for activating NK cells, perforin and granzyme B are important mediators of cell-mediated cytotoxic reactions. NK cells produce and secrete perforin and granzyme B to weaken and destroy tumor cells. Perforin is an important mediator of the effector function of granzyme B and helps granzyme B enter target cells. After mice were treated with different drug groups, perforin was detected by flow cytometry and the secretion of granzyme B was detected by ELISA. As Figure 16 F-G and Figure 24 shown, the results indicate that compared with other groups, the groups associated with CHS@Dox and FA@Dox nanoparticles showed a significant enhancement in perforin expression and granzyme B release (p<0.01). Overall, these results suggest that CHS@Dox and FA@Dox can regulate the proliferation of NK cells, thereby enhancing their killing effect on tumor cells.

[0129] Example 6 In vitro and in vivo safety evaluation

[0130] The blood compatibility of CHS@Dox and FA@Dox nanoparticles was evaluated by hemolysis tests at concentrations of 12.5 - 50 μg / mL. The results are as Figure 25 shown in A, and negligible hemolytic activity of CHS@Dox and FA@Dox nanoparticles was observed. At the same time, Figure 25 B shows a hemolysis rate below 5%, indicating good blood compatibility. In addition, histopathological analysis was performed on the normal tissues of mice. As is well known, cardiotoxicity is one of the most serious side effects caused by Dox. As Figure 26 and Figure 27 shown, a large number of necrotic cardiomyocytes and ruptured myocardial fibers were observed in the free Dox group (black solid arrows in the heart samples). In contrast, there was no obvious necrosis in the hearts of the CHS@Dox and FA@Dox nanoparticle groups. These results indicate that CHS@Dox and FA@Dox nanoparticles significantly reduce the cardiotoxicity of Dox. The significantly reduced toxicity of FA@Dox nanoparticles is likely attributed to the polysaccharide structure with high stability and ultra-sustained release. These properties of the drug carrier can reduce the toxicity to normal tissues and prevent premature release of the drug during circulation.

[0131] To further investigate the safety of CHS@Dox and FA@Dox nanoparticles, blood biochemistry and blood routine analyses were performed. The results are as Figure 28As shown, compared with the control group, the serum CK and LDH levels in the free Dox group were significantly increased, which was caused by the cardiotoxicity induced by Dox. However, no obvious changes were observed in the CHS@Dox and FA@Dox nanoparticle groups, indicating that the nanoparticles significantly alleviated the cardiotoxicity caused by Dox. The results of blood routine analysis showed that white blood cells (WBC), lymphocytes (Lymph), granulocytes (Gran), and platelets (PLT) were significantly decreased due to Dox. And these changes were significantly improved in the nanoparticle groups (Table 2). These results were consistent with the above histopathological analysis in the present invention. In summary, the FA@Dox nanoparticles are safe and reliable. Due to the reduced toxicity and enhanced anti-tumor and anti-metastasis effects, the FA@Dox nanoparticles have obvious advantages in cancer treatment. The schematic diagram of the FA@DOP nanoparticles of the present invention enhancing chemoimmunotherapy and inhibiting breast cancer growth and lung metastasis is as Figure 29 described.

[0132] Table 2 Blood routine analysis of mice after administration of free Dox, CHS@Dox nanoparticles, and FA@Dox nanoparticles

[0133]

[0134]

Claims

1. A nanoparticle for dual-targeted treatment of breast cancer, characterized in that: The anti-breast cancer chemotherapy drug is encapsulated by using a Dendrobium officinale polysaccharide-cholesterol hemisuccinate amphiphilic conjugate as a drug carrier, and the surface of the drug carrier is modified with a folic acid ligand; the structural formula of the Dendrobium officinale polysaccharide-cholesterol hemisuccinate amphiphilic conjugate is as follows: n is 1 to 2; the nanoparticles are uniform in size, with a particle diameter of 210 to 220 nm and a Zeta potential of -11 to -14 mV; and the anti-breast cancer chemotherapy drug is doxorubicin.

2. The nanoparticle according to claim 1, characterized in that: The Dendrobium officinale polysaccharide has a main chain of (1→4)-linked β-pyranose and β-D-pyranose glucose, a side chain consisting of terminal pyranose, and is composed of mannose and glucose in a ratio of 4.47:1, with an average molecular weight of 80.32 kDa.

3. The method for preparing nanoparticles for dual-targeted treatment of breast cancer according to any one of claims 1 to 2, characterized in that: The steps include: S1. reacting the Dendrobium officinale polysaccharide with cholesterol hemisuccinate under the action of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, so that the hydroxyl group of the Dendrobium officinale polysaccharide is connected to the carboxyl group of cholesterol hemisuccinate to form a Dendrobium officinale polysaccharide-cholesterol hemisuccinate amphiphilic conjugate; S2. The amphiphilic conjugate of Dendrobium officinale polysaccharide-cholesterol hemisuccinate prepared in step S1 is subjected to a connection reaction with folic acid in an organic solvent under the action of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine, and dialyzed and precipitated with alcohol to obtain a folic acid-conjugated Dendrobium officinale polysaccharide-cholesterol hemisuccinate conjugate; S3. dissolving the anti-breast cancer chemotherapy drug and the folic acid-conjugated Dendrobium officinale polysaccharide-cholesterol hemisuccinate conjugate described in step S2 in an organic solvent, reacting, dialyzing, centrifuging, and filtering to obtain nanoparticles encapsulating the anti-breast cancer chemotherapy drug.

4. The preparation method according to claim 3, characterized in that: The organic solvent in steps S2 and S3 is dimethyl sulfoxide.

5. The preparation method according to claim 3, characterized in that: The weight ratio of the anti-breast cancer chemotherapy drug to the folic acid-conjugated Dendrobium officinale polysaccharide-cholesterol hemisuccinate is 1:4-6.

6. Use of the dual-targeted nanoparticles for treating breast cancer according to any one of claims 1 to 2 in the preparation of a pharmaceutical preparation for treating breast cancer.

7. Use of the dual-targeted nanoparticles for treating breast cancer according to any one of claims 1 to 2 in the preparation of a drug for preventing and / or treating lung metastasis of breast cancer.

8. A pharmaceutical preparation for treating breast cancer, characterized in that: Nanoparticles for dual-targeted treatment of breast cancer comprising any one of claims 1 to 2.

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