Fatty acid-containing block polymer, fatty acid surface-modified nanomaterial, and preparation method therefor and use thereof

Abstract

The present application provides a fatty acid-containing block polymer, a fatty acid surface-modified nanomaterial, and a preparation method therefor and the use thereof. In the present application, the hydrophilic block of the fatty acid-containing block polymer is a block copolymer made of fatty acyloxy acrylate and a hydrophilic monomer, and then a hydrophobic fatty acid monomer is introduced into the block copolymer, so that the hydrophobic fatty acid is positioned adjacent to the hydrophilic molecule. As a result, during the self-assembly process, the fatty acid can be oriented to the surface of the nanomaterial along with the hydrophilic molecule, so as to obtain the fatty acid surface-modified nanomaterial. The fatty acid-containing block polymer of the present application can be used in a drug delivery material. When the fatty acid surface-modified nanomaterial interacts with tumor cells, it can not only be efficiently taken up by the tumor cells for intracellular distribution but can also actively track tumor-derived extracellular vesicles, thereby exhibiting the characteristics of dual spatial distribution in tumors. It can be used for loading pharmaceutically active ingredients to act on tumors, thereby achieving the simultaneous inhibition of tumor growth and metastasis.

Description

A fatty acid block polymer, a fatty acid surface-modified nanomaterial, its preparation method and application Technical Field

[0001] This application relates to the field of polymer technology, specifically to a fatty acid block polymer, a fatty acid surface-modified nanomaterial, its preparation method, and its application. Background Technology

[0002] Tumor metastasis is a fatal characteristic of malignant tumors, the leading cause of high mortality rates in cancer patients, and a major challenge in clinical cancer treatment, considered a frontier of cancer research. Statistics show that 90% of cancer patients die from tumor metastasis or recurrence. Due to the uncertainty and systemic nature of tumor metastasis, metastatic tumors are difficult to cure completely with surgical resection or local radiotherapy, unlike locally benign tumors. Furthermore, the immunosuppressive effects formed in the tumor microenvironment not only promote tumor metastasis but also cause low immune responses and drug resistance, thus significantly reducing the effectiveness of cancer immunotherapy. In fact, tumor metastasis and recurrence are major challenges in clinical cancer treatment. Therefore, effectively controlling tumor spread and reducing metastasis and recurrence to prolong the survival of cancer patients is a major challenge in clinical cancer treatment. Developing novel treatment strategies to inhibit tumor growth, metastasis, and recurrence has become an important direction for development in the field of cancer prevention and treatment.

[0003] Current cutting-edge research primarily focuses on inhibiting tumor metastasis and recurrence through combined immunotherapy and other methods. This combined treatment strategy can help patients enhance their own immune system, precisely targeting cancer cells to achieve the effects of treating tumors and inhibiting tumor recurrence (T. Celià-Terrassa, et al. Nat. Cancer 2022, 3, 355-370). However, this combined treatment strategy is complex, requiring a long treatment period, and cancer cells are prone to developing drug resistance, leading to immune escape (JA Bluestone, et al. Nat. Med. 2017, 23, 540-547). More importantly, while activating the systemic immune system to improve anti-tumor recurrence, combined treatment strategies may also trigger numerous autoimmune side effects, including inflammation of multiple organs such as immune colitis, myocarditis, and immune nervous system inflammation, and even fatal side effects, posing serious uncertainties and safety risks to clinical treatment (AKSSalama, et al. CA-Cancer J. Clin. 2020, 70, 86-104). Therefore, developing safer and more effective treatment strategies to combat tumor metastasis and recurrence is a pressing problem in this field and has significant clinical implications.

[0004] Studies have shown that tumor metastasis and recurrence are inseparable from the support of specific microenvironments in distant organs, i.e., the pre-metastatic niche. Currently, an increasing number of studies have demonstrated that extracellular vesicles released by tumors can promote tumor progression through multiple mechanisms, including inducing / promoting metastasis and tumor tolerance (PD Vermeer, at al. Nat. Commun. 2018, 9, 4284-4298). For example, the Ren Guangwen research group at the Jackson Laboratory in the United States discovered that lung mesenchymal cells are rich in lipids, which can be transferred to tumor cells and NK cells through the secretion pathway of exosome-like vesicles, reshaping the pre-tumor microenvironment and promoting lung metastasis of breast cancer (GWRen, et al. Cell Metab. 2022, 34, 1960-1976). A study by Michele De Palma's group at the Swiss Federal Institute of Technology in Lausanne (EPFL) has found that two classes of cytotoxic drugs, taxanes and anthracyclines, widely used in preoperative (neoadjuvant) breast cancer treatment, induce tumors to release extracellular vesicles with enhanced metastatic potential (MDPalma, et al. Nat. Cell. Biol. 2019, 21, 190-202). Current research indicates that cancer cells utilize various strategies, such as aberrant gene expression, post-translational modifications, and altered signaling pathways, to regulate the biogenesis, composition, and ultimate function of extracellular vesicles, and promote tumor growth and metastasis through extracellular vesicle-mediated intercellular / tissue communication (T. Ochiya, et al. J. Clin. Invest. 2016, 126, 1163-1172). Therefore, if it is possible to inhibit the growth of primary tumors while effectively suppressing the formation of tumor extracellular vesicles or disrupting their biological function, it will effectively inhibit tumor metastasis and recurrence. Unfortunately, no antitumor drugs with this unique function have yet been discovered or reported to achieve this novel antitumor strategy. Summary of the Invention

[0005] To overcome the problems existing in the prior art, one objective of this application is to provide a fatty acid-containing block polymer. A second objective is to provide a method for preparing the aforementioned fatty acid-containing block polymer. A third objective is to provide a fatty acid-surface-modified nanomaterial. A fourth objective is to provide applications of the aforementioned fatty acid-surface-modified nanomaterial.

[0006] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0007] The first aspect of this application provides a fatty acid block polymer, the fatty acid block polymer comprising covalently linked hydrophobic blocks and copolymers derived from fatty acyl esters and hydrophilic monomers; wherein the fatty acyl esters are derived from fatty acids and hydroxy acrylates.

[0008] Preferably, the hydrophobic block is selected from polystyrene, polybutadiene, polylactic acid, polycaprolactone, or polylactide-glycolic acid.

[0009] Preferably, the fatty acid monomer is selected from acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, isohexanoic acid, octanoic acid, isooctanoic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, arachidic acid, eicosapentaenoic acid, arganic acid, docosahexaenoic acid, tetracosanoic acid, hexacosanoic acid, octacosanoic acid, or triacontanic acid.

[0010] Preferably, the hydrophilic monomer is selected from at least one of oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, acrylamide, N-isopropylacrylamide, or N,N-dimethylaminoethyl methacrylate.

[0011] More preferably, the structural formula of the fatty acid block polymer is selected from one of Formula I, Formula II, Formula III, Formula IV or Formula V:

[0012] In Formula I, Formula II, Formula III, Formula IV or Formula V, R1 is independently a formic acid fatty acyl oxy ester group, R2 is independently a hydrophilic group, R3 and R4 are independently hydrogen or methyl; m is independently 2 to 400, n is independently 2 to 400, m and n are positive integers, 0 < x < 1; in Formula V, y is 2 to 400, and y is a positive integer.

[0013] More preferably, R1 is selected from the following structural formula:

[0014] Where z = 1 to 20, and z is a positive integer.

[0015] More preferably, R2 is selected from the following structural formula:

[0016] Where n = 1 to 20, and z is a positive integer.

[0017] More preferably, the structural formula of the fatty acid block polymer is selected from one of formula VI, formula VII, formula VIII, formula IX, or formula X:

[0018] R1 is the polymer unit of oligo(ethylene glycol) methyl ether methacrylate, R2 is the formic acid fatty acyl ester group, R3 is the hydrophilic unit, and R4, R5, and R6 are independently hydrogen or methyl; in formulas VI, VII, VIII, and IX, m is 2 to 400, n is 2 to 400, m and n are positive integers, 0 < x + y < 1, and x and y are positive integers; in formula X, m is 2 to 400, n is 2 to 400, z is 2 to 400, m, n, and z are positive integers, 0 < x + y < 1, and x and y are positive integers.

[0019] The second aspect of this application provides a method for preparing the fatty acid block copolymer described in the first aspect, comprising the following steps:

[0020] The block copolymer is prepared by polymerizing fatty acid monomers, hydrophilic monomers, hydrophobic polymers, bifunctional chain transfer agents, and catalysts in a solvent.

[0021] Preferably, the bifunctional chain transfer agent is 4-cyano-4-[[(propenyl)thiomethyl]thio]valerate.

[0022] Preferably, the polymerization reaction temperature is 50-120°C.

[0023] More preferably, the polymerization reaction temperature is 60-70°C.

[0024] Preferably, the polymerization reaction takes 6-28 hours.

[0025] More preferably, the polymerization reaction takes 10-20 hours.

[0026] Preferably, the molar ratio of the fatty acid monomer to the hydrophilic monomer is 1:(1-20). More preferably, the molar ratio is 1:(3-12).

[0027] Preferably, the catalyst is an azo catalyst.

[0028] Preferably, the molar ratio of the fatty acid monomer to the catalyst is 1:(0.01-0.1).

[0029] Preferably, the solvent is selected from dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, tetrahydrofuran, dioxane, acetone, acetonitrile, ethylene glycol dimethyl ether, toluene, or xylene.

[0030] A third aspect of this application provides a fatty acid-surface-modified nanomaterial, which is assembled by the self-assembly of the fatty acid-containing block polymer described in the first aspect.

[0031] Preferably, the type of nanomaterial is selected from nanoparticles, vesicles, nanorods, nanofibers, or composite micelles.

[0032] The fourth aspect of this application provides the use of the fatty acid surface-modified nanomaterials described in the third aspect in drug loading.

[0033] Preferably, the drug is selected from antitumor drugs, antiviral drugs, antibacterial drugs, photosensitizing drugs, sound-sensitizing drugs, or radiosensitizing drugs.

[0034] Preferably, the raw materials for preparing a drug-loaded nanomaterial include the fatty acid surface-modified nanomaterial and the drug as described in the third aspect.

[0035] More preferably, the method for preparing the drug-loaded nanomaterial includes the following steps: mixing the fatty acid block polymer described in the first aspect with the active pharmaceutical ingredient in a solvent, and the fatty acid block polymer undergoing self-assembly to obtain the fatty acid surface-modified nanomaterial.

[0036] Preferably, the solvent consists of an organic phase and an aqueous phase.

[0037] The beneficial effects of this application are:

[0038] 1. This application provides a fatty acid block polymer, wherein the hydrophilic block of the polymer is a copolymer block obtained by synthesizing fatty acyl ester of acrylic acid and hydrophilic monomer. Then, through molecular design, hydrophobic fatty acid monomers are introduced into the block copolymer, so that the hydrophobic fatty acid is adjacent to the hydrophilic molecule. Thus, during the self-assembly process, the fatty acid can be oriented to the surface of the nanomaterial along with the hydrophilic molecule, thereby obtaining a fatty acid surface modified nanomaterial.

[0039] 2. The fatty acid-containing block polymer of this application can be used as a drug-loaded material. When the fatty acid-modified nanomaterial interacts with tumor cells, it can not only be efficiently taken up by tumor cells and distributed intracellularly, but also actively track extracellular vesicles of tumor cells, exhibiting a dual tumor spatial distribution characteristic that traditional nanomaterials cannot achieve. This allows it to be used to load active drug ingredients onto tumors, achieving simultaneous inhibition of tumor growth and metastasis. Furthermore, when combined with immunotherapy, this fatty acid-modified nanomaterial can significantly enhance the effect of synergistic immunotherapy. Attached Figure Description

[0040] Figure 1 is the 1H NMR spectrum of the PLGA-CTA compound in Example 1;

[0041] Figure 2 is the 1H NMR spectrum of the PA-MA compound in Example 1;

[0042] Figure 3 is the 1H NMR spectrum of the fatty acid block polymer in Example 1;

[0043] Figure 4 is the 1H NMR spectrum of the DA-MA compound in Example 2;

[0044] Figure 5 is the 1H NMR spectrum of the fatty acid block polymer in Example 2;

[0045] Figures 6a and 6b are the particle size and potential diagrams of the fatty acid surface-modified nanomaterials in Example 3, respectively.

[0046] Figures 7a and 7b are cryo-transmission electron microscopy (CTEM) images and CTEM images of the fatty acid surface-modified nanomaterials in Example 3, respectively.

[0047] Figure 8 is the 1H NMR spectrum of the fatty acid surface-modified nanomaterial in Example 3;

[0048] Figures 9a-b are confocal images (scale bar 5 μm) of the tumor dual distribution of the fatty acid surface-modified nanomaterials in Example 3;

[0049] Figures 10a-f show the cellular uptake results of the fatty acid surface-modified nanomaterials in Example 3;

[0050] Figure 11 shows the cellular uptake assay results of the fatty acid surface-modified nanomaterials in Example 3;

[0051] Figures 12a-b are the particle size and potential diagrams of the fatty acid surface-modified nanomaterials in Example 4, respectively.

[0052] Figure 13 is the 1H NMR spectrum of the fatty acid block polymer in Example 5;

[0053] Figures 14a-b are the particle size and potential diagrams of the fatty acid surface-modified nanomaterials in Example 5, respectively.

[0054] Figures 15a-b are cryogenic transmission electron microscopy (CTEM) images and transmission electron microscopy (TEM) images of the fatty acid surface-modified nanomaterials in Example 5, respectively.

[0055] Figures 16a-d show the in vitro photodynamic effects of the fatty acid surface-modified nanomaterials in Example 5;

[0056] Figure 17 shows the photodynamic effect of the fatty acid surface-modified nanomaterials in Example 5.

[0057] Figure 18 shows the cytotoxicity of the fatty acid surface-modified nanomaterials in Example 5;

[0058] Figures 19a-f show the in vivo antitumor properties of the fatty acid surface-modified nanomaterials in Example 5;

[0059] Figure 20 shows the in vivo anti-transfer properties of the fatty acid-modified nanomaterials in Example 5;

[0060] Figures 21a-f show the in vivo antitumor properties of the fatty acid surface-modified nanomaterials in Example 5 against 4T1 tumor-bearing mice.

[0061] Figure 22 shows the in vivo anti-metastasis performance of the fatty acid-modified nanomaterials in Example 5 against 4T1 tumor-bearing mice.

[0062] Figures 23a-f show the in vivo antitumor properties of the fatty acid surface-modified nanomaterials in Example 5 against B16F10 tumor-bearing mice.

[0063] Figure 24 shows the in vivo anti-metastasis performance of the fatty acid-modified nanomaterials in B16F10 tumor-bearing mice in Example 5.

[0064] Figures 25a-h illustrate the enhanced immune synergistic therapeutic effect of the fatty acid surface-modified nanomaterials in Example 5;

[0065] Figures 26a-f show the immune cell evaluation after enhanced immune synergistic therapy using the fatty acid surface-modified nanomaterials from Example 5;

[0066] Figure 27 is a flowchart of the preparation method of fatty acid block copolymer in the embodiments of this application;

[0067] Figure 28 is a flowchart of the preparation method of fatty acid surface-modified nanomaterials in the embodiments of this application. Detailed Implementation

[0068] The following specific embodiments further illustrate the content of this application in detail. Unless otherwise specified, the raw materials used in the following embodiments can be obtained from conventional commercial sources or prepared and isolated through simple synthesis; unless otherwise specified, the processes employed are conventional processes in the art.

[0069] The raw materials used in the following examples and experimental analyses are as follows:

[0070] Acrylic acid, styrene, 2,2′ azobisisobutyronitrile (AIBN), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), and thiazolyl blue (MTT) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0071] Decanoic acid, palmitic acid, and stannous 2-ethylhexanoate were purchased from Ailan Chemical Technology Co., Ltd.; ethyl isocyanate methacrylate, hydroxyethyl methacrylate, dihydroporphyrin Ce6, and 9,10-anthracitediylbis(methylene)dimalonic acid (ABDA) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0072] Oligo(ethylene glycol) methyl ether methacrylate and dibutyltin dilaurate were purchased from Sigma-Aldrich; anti-mouse PD-L1 antibody was purchased from BioXcell.

[0073] Human breast cancer cells MDA-MB-231, human liver cancer cells HepG2, mouse breast cancer cells 4T1, mouse melanoma cells B16F10, mouse embryonic fibroblasts NIH-3T3, mouse macrophages RAW264.7, and mouse T cells CTLL-2 were purchased from the Shanghai ATCC cell bank.

[0074] The preparation method of the fatty acid block copolymer in this application embodiment is shown in Figure 27, wherein S100 represents the polymerization reaction of fatty acid monomers, hydrophilic monomers, hydrophobic polymers, bifunctional chain transfer agents, and catalysts in a solvent; S200 represents the obtaining of the block copolymer. The preparation method of the fatty acid surface-modified nanomaterial in this application embodiment is shown in Figure 28, wherein S110 represents the mixing of the fatty acid block polymer with the organic phase of the pharmaceutical active ingredient and the aqueous phase; S120 represents the self-assembly of the fatty acid block polymer; S130 represents the obtaining of the fatty acid surface-modified nanomaterial.

[0075] Example 1

[0076] Example 1 provides a fatty acid block copolymer, and the specific preparation method of the fatty acid block copolymer is as follows:

[0077] 1) The preparation of PLGA-CTA follows the synthetic route as follows:

[0078] S1. 29.7 mg of chain transfer agent CTA-OH (0.09 mmol), 866 mg of lactide (6.01 mmol), and 270 mg of glycolide (2.31 mmol) were added to the polymerization tube, followed by a catalytic amount of stannous 2-ethylhexanoate (10 μL) and a small amount of toluene. The mixture was first degassed by three freeze-thaw cycles, then the moisture was removed by azeotropic distillation with toluene under oil pump evacuation, and finally the polymerization tube was sealed under vacuum. After confirming the vacuum in the polymerization tube, it was placed in an oil bath at 135 °C and stirred for 1 hour. After the reaction was completed, the reaction was stopped with liquid nitrogen. After it returned to room temperature, the polymerization tube was opened and dissolved with 1 mL of dichloromethane, and then the mixture was precipitated in excess cooled methanol. The above dissolution-precipitation cycle was repeated three times. The final product was dried overnight in a vacuum oven at room temperature, yielding 527 mg of a yellow viscous solid (yield: 45%).

[0079] 2) The preparation of PA-MA follows the synthetic route:

[0080] S2. Dissolve 1.2 g palmitic acid (4.6 mmol) in 5 mL dichloromethane, and add 0.8 g hydroxyethyl methacrylate (6.1 mmol), 1.1 g dicyclohexylcarbodiimide (DCC) (5.3 mmol, 1.15 mmol), and 0.056 g N,N-dimethyl-4-aminopyridine (DMAP) (0.4 mmol). Stir the resulting mixture at room temperature for 4 hours, and then filter to remove undissolved byproducts. Concentrate the filtrate under vacuum, and purify by silica gel column chromatography using dichloromethane / n-hexane (1 / 4) as the eluent to give 1.5 g of a white oily product (yield 83%).

[0081] 3) PLGA-bP(AA) 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 The preparation and synthetic route are as follows:

[0082] S3. 110 mg PA-MA (0.283 mmol), 20.8 mg acrylic acid (0.283 mmol), 385 mg oligo(ethylene glycol) methyl ether methacrylate (0.848 mmol), 185 mg PLGA-CTA (0.028 mmol), 8.2 mg rhodamine monomer RhB-MA (0.014 mmol), and 1.2 mg AIBN (0.007 mmol) were dissolved in 500 μL of 1,4-dioxane, then transferred to a polymerization tube. The mixture was degassed by three freeze-thaw cycles and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 12 h, the polymerization tube was briefly placed in liquid nitrogen, and the cap was opened to terminate the polymerization. The reaction mixture was precipitated in excess diethyl ether, and the precipitate was dissolved in a small amount of acetone and precipitated again in diethyl ether. After vacuum drying overnight, 447 mg PLGA-bP(AA) was obtained. 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 (Yield: 63%)

[0083] PLGA-bP(AA) prepared in Example 1 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32PLGA-CTA and PA-MA were confirmed by 1H NMR spectroscopy (Figures 1-3). Figure 1 shows that all hydrogen atoms at each position in PLGA-CTA have been assigned and integrated, and no other impurity peaks are observed, indicating that the synthesized PLGA-CTA structure is correct. Figure 2 shows that all hydrogen atoms at each position in PA-MA have been assigned and integrated, and no other impurity peaks are observed, indicating that the synthesized PA-MA structure is correct. Figure 3 shows that the composition ratio of the three components was calculated based on the peak areas of the characteristic peaks of PLGA (around 1.6, 4.8, and 5.2 ppm), polyethylene glycol (around 3.6 ppm), and PA-MA (around 1.3 ppm). The proportion of acrylic acid was calculated based on the conversion rate, confirming that the copolymer segment is PLGA-bP(AA). 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 .

[0084] In addition, as a control, a block polymer without fatty acid motifs was synthesized, with the chemical formula PLGA-bP(AA) 0.29 -co-OEG 0.70 -co-RhB 0.01 ) 28 Its structural formula and synthetic route are as follows:

[0085] Example 2

[0086] This embodiment 2 provides a fatty acid block copolymer, and the specific preparation method of the fatty acid block copolymer is as follows:

[0087] 1) The preparation of DA-MA follows the synthetic route:

[0088] S1. Dissolve 0.8 g of decanoic acid (4.6 mmol) in 5 mL of dichloromethane, and add 0.8 g of hydroxyethyl methacrylate (6.1 mmol), 1.1 g of DCC (5.3 mmol, 1.15), and 0.056 g of DMAP (0.4 mmol). Stir the resulting mixture at room temperature for 4 hours, and then filter to remove undissolved byproducts. Concentrate the filtrate under vacuum, and purify by silica gel column chromatography using dichloromethane / n-hexane (1 / 4) as the eluent to give 1.1 g of a white oily product (79% yield).

[0089] 2) PLGA-bP(AA) 0.25 -co-DA 0.25 -co-OEG 0.49 -co-RhB 0.01 )40 The preparation and synthetic route are as follows:

[0090] S2. 80.5 mg DA-MA (0.283 mmol), 20.8 mg acrylic acid (0.283 mmol), 385 mg oligo(ethylene glycol) methyl ether methacrylate (0.848 mmol), 185 mg PLGA-CTA (0.028 mmol), 8.2 mg rhodamine monomer RhB-MA (0.014 mmol), and 1.2 mg AIBN (0.007 mmol) were dissolved in 500 μL of 1,4-dioxane, then transferred to a polymerization tube. The mixture was degassed by three freeze-thaw cycles and finally sealed under vacuum. The polymerization tube was then immersed in a preheated 65°C oil bath. After stirring for 12 h, the polymerization tube was briefly placed in liquid nitrogen, and the cap was opened to terminate the polymerization. The reaction mixture was precipitated in excess diethyl ether, and the precipitate was dissolved in a small amount of acetone and precipitated again in diethyl ether. After vacuum drying overnight, 541 mg PLGA-bP(AA) was obtained. 0.25 -co-DA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 40 (Yield: 79%)

[0091] The DA-MA and PLGA-bP(AA) prepared in Example 2 0.25 -co-DA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 40 The 1H NMR spectrum was confirmed (see Figures 4-5). Figure 4 shows that all hydrogen atoms at each position in DA-MA were assigned and integrated, and no other extraneous peaks were observed, indicating that the synthesized DA-MA structure is correct. Figure 5 shows that the composition ratio of the three components was calculated based on the peak areas of the characteristic peaks of PLGA (around 1.6, 4.8, and 5.2 ppm), polyethylene glycol (around 3.6 ppm), and DA-MA (around 1.3 ppm). The proportion of acrylic acid was calculated based on the conversion rate, confirming that the copolymer segment is PLGA-bP(AA). 0.25 -co-DA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 40 .

[0092] Example 3

[0093] Example 3 provides a fatty acid surface-modified nanomaterial (NP1), prepared as follows: 3 mg of the fatty acid block polymer prepared in Example 1 was dissolved in 1 mL of acetone, and then added to 9 mL of deionized water in one go under magnetic stirring. After stirring at room temperature for 5 min, the suspension was transferred to a dialysis bag and dialyzed with deionized water for 8 h, with fresh deionized water replaced every 2 h to remove acetone.

[0094] The obtained fatty acid surface-modified nanomaterials (NP1) were characterized for particle size, potential, and morphology. The results are shown in Figures 6a-b, 7a-b, and 8. Figures 6a-b show the particle size and potential of NP1, respectively. The particle size distribution of NP1 is 33-52 nm, with an average particle size of 41 nm. The polydispersity index (PDI) is low (below 0.2), indicating that the nanoparticles are relatively uniform in size. The zeta potential results show that NP1 is negatively charged. Figure 7a shows a cryo-transmission electron microscope (CTEM) image of NP1. It can be seen from the image that NP1 is spherical and the particle size is relatively uniform. Figure 7b shows a CTEM image of NP1. It can be seen from the image that the size of NP1 is relatively uniform.

[0095] The fatty acid surface modification of the nanomaterial was then verified. 5 mg of the fatty acid-containing block polymer prepared in Example 1 was dissolved in 80 μL of deuterated chloroform, and then added to 500 μL of deuterated water in one go under magnetic stirring. After stirring for 5 min at room temperature, the suspension was transferred to an NMR tube for 1H NMR spectroscopy. The 1H NMR spectrum of the nanomaterial in deuterated water was compared with the 1H NMR spectrum of the fatty acid-containing block polymer prepared in Example 1 in deuterated chloroform. The results are shown in Figure 8. As can be seen from the figure, a clear palmitic acid signal peak appeared in the 1H NMR spectrum of the fatty acid-modified nanomaterial, indicating the presence of palmitic acid on the surface of the nanomaterial. The PLGA signal peak was not observed in the 1H NMR spectrum of the nanomaterial, indicating that PLGA is located in the hydrophobic core of the nanomaterial. This result confirms that the fatty acid-modified nanomaterial prepared in Example 3 has surface-modified fatty acids.

[0096] In addition, the tumor dual spatial distribution of the fatty acid surface-modified nanomaterial (NP1) prepared in Example 3 was determined.

[0097] Using unmodified fatty acid nanomaterials (Ctrl-NP1) as a control, the spatial distribution of NP1 in triple-negative breast cancer MDA-MB-231 cells was determined by laser confocal microscopy. Ctrl-NP1 was prepared as follows: 3 mg of the fatty acid-free block polymer PLGA-bP(AA) prepared in Example 1 was added... 0.29 -co-OEG 0.70 -co-RhB 0.01 ) 28Dissolved in 1 mL of acetone, then added to 9 mL of deionized water in one go under magnetic stirring. After stirring at room temperature for 5 min, the suspension was transferred to a dialysis bag and dialyzed with deionized water for 8 h, with fresh deionized water replaced every 2 h to remove acetone, thus obtaining nanomaterials without fatty acid surface modification (Ctrl-NP1).

[0098] As shown in Figure 9a, NP1 can be largely taken up by MDA-MB-231 tumor cells and accumulates on extracellular vesicles. Only a small amount of Ctrl-NP1 is taken up by MDA-MB-231 tumor cells, demonstrating that NP1 has a more efficient tumor cell uptake effect compared to Ctrl-NP1. Furthermore, the distribution of NP1 on extracellular vesicles after 3D reconstruction was investigated using confocal Z-stack mode. As shown in Figure 9b, in confocal 3D tomography imaging, the accumulation of NP1 on extracellular vesicles varies with the layer, demonstrating that NP1 has the ability to track vesicles. Therefore, these results indicate that when NP1 interacts with tumor cells, it can not only be efficiently taken up by tumor cells and distributed intracellularly, but also actively track extracellular vesicles, exhibiting a dual tumor spatial distribution capability that traditional nanomaterials cannot achieve.

[0099] Furthermore, the dual spatial distribution of NP1 in tumors was determined in 4T1 breast cancer cells, B16F10 melanoma cells, and HepG2 hepatocellular carcinoma cells, with normal cells (NIH-3T3 fibroblasts) and immune cells (RAW264.7 macrophages and CTLL-2 T lymphocytes) as controls. As shown in Figures 10a-f, 4T1, B16F10, and HepG2 cells incubated with NP1 exhibited strong red fluorescence in the cytoplasm (Figures 10a-c), while NIH-3T3, RAW264.7, and CTLL-2 cells exhibited weak red fluorescence (Figures 10d-f), indicating that NP1 has the ability to target cancer cells. More importantly, after 1 hour of incubation with NP1, 4T1, B16F10, and HepG2 cells showed a large number of nano- and micron-sized strong red fluorescent vesicles in the extracellular space. These vesicles are extracellular tumor vesicles (TEVs) actively tracked by NP1, demonstrating that NP1 exhibits a dual spatial distribution ability in tumors across multiple tumor cell types. Figure 11 shows the flow cytometry results after different cells were incubated with NP1 or Ctrl-NP1, which again demonstrates that NP1 can be specifically and efficiently taken up by tumor cells compared to Ctrl-NP1.

[0100] Example 4

[0101] Example 4 provides a fatty acid surface-modified nanomaterial (NP2), comprising the fatty acid-containing block polymer prepared in Example 2. The preparation method is as follows: 3 mg of the fatty acid-containing block polymer prepared in Example 2 is dissolved in 1 mL of acetone, and then added to 9 mL of deionized water in one go under magnetic stirring. After stirring at room temperature for 5 min, the suspension is transferred to a dialysis bag and dialyzed with deionized water for 8 h, replacing the deionized water every 2 h to remove acetone.

[0102] The obtained fatty acid surface-modified nanomaterial (NP2) was characterized by particle size and potential using a laser particle size analyzer. The particle size results are shown in Figure 12a. The hydrated particle size of NP2 is about 41 nm, and the PDI is small (below 0.2), indicating that the nanoparticles are relatively uniform in size. The zeta potential results in Figure 12b show that NP2 is negatively charged.

[0103] Example 5

[0104] Example 5 provides a fatty acid surface-modified nanomaterial (NP3), which is composed of Ce6-PLGA-bP(AA) prepared in Example 1, consisting of a fatty acid block polymer and a photosensitizer Ce6. 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 In Example 1, the fatty acid block polymer and the photosensitizer Ce6 are covalently linked. The synthesis equation and preparation method are as follows:

[0105] Dihydroporphyrin E6 (Ce6, 30 mg, 0.05 mmol, 2.5 equivalents) was reacted with PLGA-bP (AA) in DMSO (10 mL). 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 The terminal hydroxyl groups of DMSO (350 mg, 0.02 mmol, 1 equivalent) were coupled together, and DMAP (24 mg, 0.002 mmol, 0.1 equivalent) and DCC (4.5 mg, 1 mmol, 1.1 equivalent) were added. The mixture was stirred at room temperature for 24 h, filtered, and transferred to a dialysis bag. Dialysis with deionized water for 8 h removed impurities such as DMSO. Finally, the solution was lyophilized to obtain a dark purple, slightly viscous solid. The synthesized polymer was used... 1 Characterized by H-NMR.

[0106] 3mg Ce6-PLGA-bP(AA 0.25 -co-PA 0.25 -co-OEG0.49 -co-RhB 0.01 ) 32 Dissolve in 1 mL of acetone, then add to 9 mL of deionized water in one go with magnetic stirring. After stirring at room temperature for 5 min, transfer the suspension to a dialysis bag and dialyze with deionized water for 8 h, changing the deionized water every 2 h to remove acetone.

[0107] Ce6-PLGA-bP(AA) prepared in Example 5 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 The results were confirmed by 1H NMR spectroscopy (see Figure 13). The composition ratios of the four copolymers were calculated based on the peak areas of the characteristic peaks of PLGA (around 1.6 ppm, 4.8 ppm, and 5.2 ppm), polyethylene glycol (around 3.6 ppm), PA-MA (around 1.3 ppm), and Ce6 (around 6.5 ppm). The proportion of acrylic acid was calculated based on the conversion rate. Therefore, the copolymer segment was determined to be Ce6-PLGA-bP(AA). 0.25 -co-PA 0.25 -co-OEG 0.49 -co-RhB 0.01 ) 32 .

[0108] The fatty acid surface-modified nanomaterials (NP3) obtained in Example 5 were characterized for particle size, potential, and morphology. As shown in Figures 14a-b, the particle size distribution of NP3 was 42-67 nm, with an average particle size of 48 nm (Figure 14a). The zeta potential results showed that NP3 was negatively charged (Figure 14b). Figures 15a-b are cryo-transmission electron microscopy (CTEM) images (Figure 15a) and CTEM images (Figure 15b), respectively. It can be seen from the figures that the prepared NP3 is spherical and the particle size is relatively uniform.

[0109] In addition, as a control, a fatty acid-free surface-modified nanomaterial (Ctrl-NP3) was synthesized, which was Ce6-PLGA-bP(AA) composed of a fatty acid-free block polymer prepared in Example 1 and Ce6. 0.29 -co-OEG 0.70 -co-RhB 0.01 ) 28 The synthesis equation and preparation method are as follows:

[0110] 3mg Ce6-PLGA-bP(AA 0.29 -co-OEG 0.70 -co-RhB0.01 ) 28 Dissolved in 1 mL of acetone, then added to 9 mL of deionized water in one go under magnetic stirring. After stirring at room temperature for 5 min, the suspension was transferred to a dialysis bag and dialyzed with deionized water for 8 h, with fresh deionized water replaced every 2 h to remove acetone, to obtain the fatty acid-free surface-modified nanomaterial Ctrl-NP3.

[0111] Experimental Analysis

[0112] 1. The in vitro photodynamic effect of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5 was determined.

[0113] The in vitro photodynamic effect of the fatty acid-modified nanomaterial (NP3) prepared in Example 5 was evaluated using the unmodified nanomaterial (Ctrl-NP3) as a control. NP3 and Ctrl-NP3 contain the photosensitizer Ce6, and the singlet oxygen generated by them under light irradiation can be detected by ultraviolet absorption. ABDA, as a singlet oxygen scavenger, can be oxidized by the generated singlet oxygen, reducing its own ultraviolet absorption intensity, thereby characterizing the singlet oxygen generation efficiency. Appropriate amounts of NP3 and Ctrl-NP3 were dispersed in water to achieve a final nanoparticle concentration of 0.075 g / L (containing 5 μM Ce6), and a high-concentration ABDA dimethyl sulfoxide stock solution was prepared. The calculated volume of the high-concentration ABDA stock solution was added to the diluted nanophotosensitive agent, and the mixture was irradiated under a 660 nm light source (50 mW / cm²). 2 The solution was continuously irradiated for 0 min, 1 min, 2 min, 3 min, 4 min, and 5 min, and the sample was tested with a UV spectrometer after each irradiation. The obtained UV spectra were marked with the UV absorption intensity at the 361 nm peak of each curve. The ability of NP2 and Ctrl-NP2 to produce singlet oxygen under 660 nm light conditions could be calculated.

[0114] As shown in Figure 16a, the UV absorption intensity of ABDA gradually decreased after 5 min of NP3 illumination. This is because ABDA was degraded by singlet oxygen generated by NP3. Meanwhile, as shown in Figure 16b, Ctrl-NP3 also exhibited similar in vitro photodynamic effects to NP3. To prevent ABDA from being degraded under illumination, the ABDA solution was directly irradiated. Figure 16c shows that ABDA has strong stability and will not degrade in the absence of singlet oxygen. Finally, through statistical analysis of the above figures, as shown in Figure 16d, we calculated that NP3 and Ctrl-NP3 can degrade 60% of ABDA after 5 min of illumination, demonstrating good in vitro photodynamic effects.

[0115] 2. The photodynamic effect of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5 on tumor cells was determined.

[0116] First, healthy triple-negative breast cancer MDA-MB-231 cells were seeded into confocal culture dishes and cultured for 16 hours. Then, NP3 and Ctrl-NP3 were added to the confocal culture dishes at a final concentration of 0.1 g / L for incubation in the dark. After incubating the cells with nanoparticles at 37°C for 1 hour, the incubator was discarded, and the confocal culture dishes were washed three times with PBS. Then, 1 mL of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) diluted with DMEM cell culture medium was added, and the dishes were incubated at 37°C for 8 minutes. After dye fixation, the cells were washed twice with PBS, and 1 mL of freshly prepared DMEM basal culture medium was added to the confocal culture dishes. Next, a 660 nm laser at 50 mW / cm² was used. 2 Two minutes after power irradiation, MDA-MB-231 tumor cells were fluorescently imaged using a confocal microscope.

[0117] As shown in Figure 17, compared with the control group Ctrl-NP3, the NP3 group exhibited stronger fluorescence in MDA-MB-231 cells, indicating that under the same illumination conditions, the NP3 group could generate more singlet oxygen inside the cells. Furthermore, it was found that NP3 possesses a dual tumor spatial distribution function, capable of simultaneously generating singlet oxygen in tumor cells and extracellular vesicles.

[0118] 3. Determine the killing ability of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5 against tumor cells.

[0119] The killing ability of NP3, Ctrl-NP3, and Ce6 against tumor cells under 660 nm light irradiation was determined by the MTT assay. First, healthy triple-negative breast cancer MDA-MB-231 cells were cultured in 96-well plates (8×6), with the cell count controlled at 2×10⁶ cells per well. 4 Once the cells reached the logarithmic growth phase, the complete medium was replaced with PBS in columns 1 and 5, with 3.5 μg / mL Ce6 in columns 2 and 6, with 0.1 g / L Ctrl-NP3 in columns 3 and 7, and with 0.1 g / L NP3 in columns 4 and 8. After incubation at 37°C for 1 hour, the cells were washed with 1×PBS, and then incubated for another hour with basal medium. Next, a 660 nm laser (50 mW / cm²) was used to... 2Cells in rows 5, 6, 7, and 8 were irradiated with laser for 2 min. After laser irradiation, the cells were incubated again at 37°C for 12 h. After incubation, the cells were washed with PBS. During washing, 0.5 g of thiazolyl blue (MTT) was weighed, dissolved in 100 mL of PBS, and filtered through a 0.22 μm filter to remove bacteria, yielding a 5 mg / mL MTT stock solution. After washing, 180 μL of PBS and 20 μL of MTT stock solution were added to each well. After incubation at 37°C for 4 h, the supernatant was gently discarded with a pipette. Next, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was gently shaken for 10 min to dissolve any crystals. Finally, the absorbance of each well was measured at 490 nm using a microplate reader, and the results were recorded.

[0120] As shown in Figure 18, Ce6, Ctrl-NP3, and NP3 all exhibited negligible toxicity in the absence of laser irradiation, indicating that their dark cell toxicity is negligible and their biocompatibility is good. The laser irradiation conditions used were (660 nm, 50 mW / cm²). 2 The cytotoxicity of Ce6(L+), Ctrl-NP3(L+), and NP3(L+) was negligible. After 2 minutes of 660nm laser irradiation, Ce6(L+), Ctrl-NP3(L+), and NP3(L+) all produced ROS in tumor cells, further killing them. The cell survival rate decreased to 62% when co-incubated with Ce6, to 30% when co-incubated with Ctrl-NP3, and significantly decreased to 16% when co-incubated with NP3, indicating that NP3 has a superior anti-tumor effect compared to Ctrl-NP3 and Ce6.

[0121] 4. Determine the in vivo tumor-killing ability of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5.

[0122] To investigate the inhibitory effect of NP3 PDT photodynamic killing on primary and metastatic tumors, an MDA-MB-231 metastatic tumor model was established by seeding MDA-MB-231 cells into the subcutaneous fat pads of BALB / c nude mice. Eight groups of nude mice (n=4 per group) were prepared: ①PBS(L-), ②Ce6(L-), ③Ctrl-NP3(L-), ④NP3(L-), ⑤PBS(L+), ⑥Ce6(L+), ⑦Ctrl-NP3(L+), and ⑧NP3(L+). When the tumor volume in the mice reached 100 mm², the tumor model was established. 3 Subsequently, tumor-bearing mice were intravenously injected with PBS, Ce6, Ctrl-NP3, or NP3 at a dose of 2.5 mg / kg Ce6. Six hours after injection, the mice were treated with a 660 nm laser (200 mW / cm²). 2The mice were irradiated for 5 minutes. The tumor treatment period in the mice was set at 21 days, and we treated the mice every three days.

[0123] As shown in Figure 19a, neither laser irradiation alone nor nanoparticle therapy without laser treatment could control tumor growth. Notably, after NP3-mediated PDT treatment, tumor growth was significantly inhibited within 21 days, with a tumor inhibition rate of 88%. Ce6 and Ctrl-NP3-mediated PDT showed moderate tumor growth inhibition of 36% and 46%, respectively (Figure 19c). To more intuitively observe the anti-tumor ability of different treatment groups, after tumor treatment, mice in each group were sacrificed and their tumor tissues were completely removed for photographing. As shown in Figure 19b, the NP3(L+) group had the smallest tumor volume, demonstrating that NP3(L+) had the best inhibitory effect on MDA-MB-231 tumors. The tumor tissues of each group were weighed after photographing (Figure 19d). Similar to the above conclusions, the tumor mass of the NP3(L+) group was only 0.6g, the smallest compared to the other groups. To determine whether NP3 and Ctrl-NP3 would have toxic side effects on the mice, the weight changes of the mice were measured every 3 days during the tumor treatment process. As shown in Figure 19e, the mice in each group gradually gained weight over the 21 days of treatment. The differences in weight among the groups were similar, indicating that the treatment had no side effects.

[0124] The inhibitory effect of NP3 phototherapy on metastasis was evaluated by studying metastatic nodules in lung tissue. As shown in Figures 19f and 20, no obvious lung metastatic nodules were observed in the lungs of mice in the NP3(L+) group, suggesting that NP3 PDT treatment can effectively inhibit tumor metastasis. However, obvious metastatic nodules were found in the lungs of mice in the other groups. This was further confirmed by quantitative analysis of the number of lung nodules in each group (Figure 19f). In addition, histological examination of the lung tissue of mice in each group was performed using hematoxylin and eosin (H&E) staining to further monitor lung metastasis (Figure 20). Metastatic lesions were clearly visible in the PBS, Ce6, Ctrl-NP3, NP3, PBS(L+), Ce6(L+), and Ctrl-NP3(L+) groups, while almost no metastatic lesions were visible in the NP3(L+) group. These results indicate that NP3 PDT therapy has a good anti-metastatic effect. It is noteworthy that while Ce6 and Ctrl-NP3 PDT therapy can partially inhibit the growth of primary tumors, they cannot inhibit tumor metastasis. Conversely, NP3's PDT therapy simultaneously leads to photodynamic inhibition of the primary tumor and inhibition of tumor metastasis, due to NP3's active tracking and disabling of TEV to block intercellular / tissue communication.

[0125] 5. Determine the inhibitory effect of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5 on primary and metastatic tumors in 4T1 tumor-bearing mice.

[0126] A 4T1 metastatic tumor model was established by seeding 4T1 cells into the subcutaneous fat pads of BALB mice. Eight groups of mice (n=4 per group) were prepared: ①PBS(L-), ②Ce6(L-), ③Ctrl-NP3(L-), ④NP3(L-), ⑤PBS(L+), ⑥Ce6(L+), ⑦Ctrl-NP3(L+), and ⑧NP3(L+). Tumors were classified into eight groups based on their tumor size (100 mm²). 3 Subsequently, tumor-bearing mice were intravenously injected with PBS, Ce6, Ctrl-NP3, or NP3 at a dose of 2.5 mg / kg Ce6. Six hours after injection, the mice were treated with a 660 nm laser (200 mW / cm²). 2 The mice were irradiated for 5 minutes. The tumor treatment period in the mice was set at 21 days, and we treated the mice every three days.

[0127] As shown in Figure 21a, neither laser irradiation alone nor nanoparticle therapy without laser treatment could control tumor growth. Notably, tumor growth was significantly inhibited within 21 days after NP3-mediated PDT treatment, with a tumor inhibition rate of 86% (Figure 21c). Ce6 and Ctrl-NP3-mediated PDT showed moderate tumor growth inhibition of 44% and 56%, respectively. Tumor weight and photographs further confirmed this (Figures 21b and 21d). No significant weight loss was observed in any treatment group (Figure 21e). Furthermore, optical images of lung tissue and quantitative statistics of lung nodules at the end of treatment, as well as H&E staining results of lung tissue, all indicated that NP3-mediated PDT treatment effectively inhibited tumor metastasis (Figures 21f and 22).

[0128] 6. Determine the inhibitory effect of the fatty acid surface-modified nanomaterial (NP3) prepared in Example 5 on primary and metastatic tumors in B16F10 tumor-bearing mice.

[0129] To establish a B16F10 tumor-bearing mouse model, B16F10 cells were injected subcutaneously into the tissue at -6 days and -2 days (before the experiment) (1×10⁻⁶ cells per cell). 6 (each mouse) and tail vein (5 × 10) 5(Number of mice per group). Subsequently, on days 0, 2, 4, 6, and 8, mice were intravenously injected with PBS, Ce6, Ctrl-NP3, or NP3 at a dose of 2.5 mg / kg Ce6 (n=4 per group). Six hours after each administration, mice were either exposed to light (660 nm, 200 mW / cm², 5 minutes) or not. During treatment, tumor volume and body weight were measured every two days. After 10 days of treatment, 8 groups of mice were sacrificed, and tumor tissue was weighed and photographed. Lung tissue was also photographed and analyzed using H&E staining.

[0130] As shown in Figure 23a, neither laser irradiation alone nor nanoparticle therapy without laser treatment could control tumor growth. Notably, tumor growth was significantly inhibited within 10 days after NP3-mediated PDT treatment, with a tumor inhibition rate of 81%. Ce6 and Ctrl-NP3-mediated PDT showed moderate tumor growth inhibition of 34% and 52%, respectively (Figure 23c). Tumor weight and photographs further confirm this (Figures 23b and 23d). No significant weight loss was observed in any treatment group (Figure 23e). Furthermore, optical images of lung tissue and quantitative statistics of lung nodule numbers in each group at the end of treatment, as well as H&E staining results of lung tissue, all indicate that NP3-mediated PDT treatment effectively inhibits tumor metastasis (Figures 23f and 24).

[0131] 7. Determine the immunomodulatory effect of PDT combined with anti-PD-L1 mediated immune checkpoint blockade (ICB) therapy using fatty acid surface-modified nanomaterials (NP3) prepared in Example 5.

[0132] To establish primary and distant tumor models, mice were injected with 4T1 cells twice. The specific procedure was as follows: 4T1 cells (5 × 10⁻⁶ cells) were injected into the left and right legs of BALB / c mice on days -7 and -4, respectively. 6 A dual-tumor model was established (n=4 mice per group). On days 0, 3, 6, and 9, mice were intravenously injected with PBS, Ce6, Ctrl-NP3, or NP3 (n=4 per group), at a dose of Ce6 2.5 mg / kg. Six hours later, the mice were treated with a laser (660 nm, 200 mW / cm²). 2The tumor in the left hind leg of mice was irradiated for 5 minutes, while the tumor in the right hind leg was left untreated. Mice were intravenously injected with αPD-L1 (programmed cell death-ligand 1) antibody (1 mg / kg) on ​​days 1, 4, 7, and 10. Tumor volume and body weight were measured every two days during treatment. Mice were sacrificed on day 11, and tumor tissue was collected, weighed, photographed, and analyzed by immunoflow cytometry. The primary and distant tumor tissues were ground, digested with collagenase IV, hyaluronidase, and DNase I, filtered through a 70 μm filter, stained with fluorescent antibodies, and analyzed by flow cytometry. Flow cytometry was used to detect the proportions of dendritic cells (DCs, CD11b+CD80+CD86+), cytotoxic T cells (CTLs, CD45+CD3+CD8+), and myeloid suppressor cells (MDSCs, CD11b+Gr-1+) in the primary and distant tumors.

[0133] Figures 25a-d show the experimental results for primary tumors. As shown in Figures 25a-d, PDT treatment with NP3 significantly inhibited the growth of the primary tumor, with a tumor inhibition rate of 82%. Alone αPD-L1 immunotherapy inhibited tumor growth by 48%. More importantly, the combined use of NP3 and αPD-L1 under light irradiation achieved the highest tumor inhibition effect, with a tumor inhibition rate of 92%, which is a result of the synergistic effect of ICB and PDT therapy. Furthermore, excitingly, NP3 also showed inhibitory effects on distant tumors under light irradiation (Figures 25e-h show the experimental results for distant tumors), with a tumor inhibition rate of 31%, while Ce6 and Ctrl-NP3 had no significant effect on distant tumors under light irradiation. More importantly, the combined use of αPD-L1 and NP3 under light irradiation was found to significantly inhibit the growth of distant tumors, with a tumor inhibition rate of 86%, further emphasizing the superiority of NP3 synergistic immunotherapy.

[0134] The immune status in vivo was assessed by analyzing immune cells in primary and distant tumor tissues after various treatments. As shown in Figures 26a–f, the proportions of mature dendritic cells (DCs) and cytotoxic T cells (CTLs) in primary and distant tumors of mice treated with NP3(L+), αPD-L1, and NP3(L+)+αPD-L1 were significantly increased, indicating immune activation at the tumor site (Figures 26a, 26b, 26d, and 26e). Simultaneously, the number of myeloid suppressor cells (MDSCs) infiltrating the tumor was significantly reduced, indicating that the immunosuppressive tumor microenvironment was reversed (Figures 26c and 26f). Combined treatment resulted in the highest infiltration of mature DCs and CTLs within the tumor, while MDSC infiltration was the lowest. These results indicate that NP3, upon photoexcitation, activates a strong immune response in mice, not only inducing an in situ immune response in the tumor but also improving immunosuppression at the tumor site, reversing the tumor immune microenvironment, and enhancing the efficacy of synergistic immunotherapy.

[0135] The above embodiments are preferred embodiments of this application, but the implementation of this application is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this application shall be considered equivalent substitutions and shall be included within the protection scope of this application.

Claims

1. A fatty acid block polymer, characterized in that, The fatty acid block polymer includes covalently linked hydrophobic blocks and copolymer blocks derived from fatty acyl esters and hydrophilic monomers; the fatty acyl esters are prepared from fatty acids and hydroxy acrylates.

2. The fatty acid block polymer according to claim 1, characterized in that, The hydrophobic block is selected from polystyrene, polybutadiene, polylactic acid, polycaprolactone, or polylactide-glycolic acid; And / or, the fatty acid monomer is selected from acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, isohexanoic acid, octanoic acid, isooctanoic acid, capric acid, undecanoic acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, arachidic acid, eicosapentaenoic acid, arganic acid, docosahexaenoic acid, tetracosanoic acid, hexacosanoic acid, octacosanoic acid, or triacontanic acid; And / or, the hydrophilic monomer is selected from at least one of oligo(ethylene glycol) methyl ether methacrylate, acrylic acid, methacrylic acid, acrylamide, N-isopropylacrylamide or N,N-dimethylaminoethyl methacrylate.

3. The fatty acid block polymer according to claim 2, characterized in that, The structural formula of the fatty acid block polymer is selected from one of Formula I, Formula II, Formula III, Formula IV or Formula V: In Formula I, Formula II, Formula III, Formula IV or Formula V, R1 is independently a formic acid fatty acyl oxy ester group, R2 is independently a hydrophilic group, R3 and R4 are independently hydrogen or methyl; m is independently 2 to 400, n is independently 2 to 400, m and n are positive integers, 0 < x < 1; in Formula V, y is 2 to 400, and y is a positive integer.

4. The fatty acid block polymer according to claim 3, characterized in that, R1 is selected from the following structural formula: Where z = 1 to 20, and z is a positive integer.

5. The fatty acid block polymer according to claim 3, characterized in that, R2 is selected from the following structural formula: Where n = 1 to 20, and z is a positive integer.

6. The fatty acid block polymer according to claim 3, characterized in that, The structural formula of the fatty acid block polymer is selected from one of formula VI, formula VII, formula VIII, formula IX, or formula X: R1 is the polymer unit of oligo(ethylene glycol) methyl ether methacrylate, R2 is the formic acid fatty acyl ester group, R3 is the hydrophilic unit, and R4, R5, and R6 are independently hydrogen or methyl; m is 2 to 400, n is 2 to 400, z is 2 to 400, m, n, and z are positive integers, 0 < x + y < 1, and x and y are positive integers.

7. The method for preparing the fatty acid block copolymer according to any one of claims 1 to 6, characterized in that, Includes the following steps: The block copolymer is prepared by polymerizing acrylate fatty acyl oxy ester, hydrophilic monomer, hydrophobic polymer, bifunctional chain transfer agent, and catalyst in a solvent.

8. A fatty acid-surface-modified nanomaterial, characterized in that, The nanomaterial is formed by the self-assembly of a fatty acid block polymer as described in any one of claims 1 to 6, and the type of the nanomaterial is selected from nanoparticles, vesicles, nanorods, nanofibers or composite micelles.

9. The application of the fatty acid surface-modified nanomaterials of claim 8 in drug loading.

10. The application according to claim 9, wherein the drug is selected from antitumor drugs, antiviral drugs, antibacterial drugs, photosensitizing drugs, sound-sensitizing drugs, or radiosensitizing drugs.

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