A targeted photosensitizer FA-BAT-NPs and its preparation method and application

Abstract

The present invention discloses a targeted photosensitizer, FA-BAT-NPs, and its preparation and application. First, the invention chemically links the photosensitizer 5-ALA with the mitochondrial targeting group TPP and modifies the Boc group to increase lipid solubility, thereby preparing a novel mitochondrial-targeted photosensitizer, BAT. The BAT is then prepared into BAT-NPs (nanoparticles) using an emulsification dispersion method. Finally, FA is modified onto the surface of the BAT-NPs using PEG as a connecting linker via an amide reaction, yielding the targeted photosensitizer, FA-BAT-NPs. The targeted photosensitizer, FA-BAT-NPs, possesses good water solubility and sustained release in vivo, exhibiting a strong inhibitory effect on breast cancer and producing virtually no toxic side effects.

Description

Technical Field

[0001] The present invention relates to the technical field of nano preparations, and in particular to a method for preparing a targeted photosensitizer FA-BAT-NPs. Background Art

[0002] After decades of research, clinicians have made great progress in the treatment of breast cancer (BC). Photodynamic therapy (PDT) is a treatment method that combines photosensitizers, lasers, and molecular oxygen to produce reactive oxygen species (ROS) to induce target cell death. Compared with traditional treatments such as surgery, chemotherapy, and radiation, PDT for breast cancer treatment has the advantages of safety, spatiotemporal selectivity, good efficacy, broad-spectrum response, and no scar formation. Although the therapeutic potential of PDT for chemotherapy-resistant tumors remains unclear, this treatment method has been considered by many to be a potential solution to the phenomenon of multidrug resistance in tumors.

[0003] Oxygen molecules are essential raw materials for the production of ROS by photosensitizers. Therefore, hypoxia in tumor tissues caused by uncontrolled cell proliferation, abnormal vascular perfusion, etc. will greatly reduce the number of ROS produced, thereby reducing the efficacy of PDT; at the same time, excessive production of ROS in normal tissues will lead to various toxic side effects. Therefore, it is necessary to design a new type of photosensitizer that has "zero release" in normal tissues to improve its biosafety. In addition, ROS can only act around the site of its generation (less than 20nm), which is much smaller than the size of our cells. In addition, its half-life is short (about 15s), so this is also a key limiting factor in improving the efficacy of PDT. In recent years, some teams have tried strategies such as self-sufficient oxygen molecules and photocatalytic decomposition of water to produce oxygen, but these methods all have certain limitations.

[0004] As our understanding of the tumor microenvironment gradually deepens, the hypoxic microenvironment of tumor tissue has been found to be associated with reduced drug efficacy, tumor recurrence and metastasis, and limited immune cell infiltration. This has often been overlooked by researchers in previous studies. Therefore, correcting the hypoxic microenvironment of tumor tissue may be the top priority for eradicating tumors. In recent years, some researchers have made some progress in this regard. Existing strategies to improve tumor microenvironment hypoxia mainly include: directly delivering oxygen to the hypoxic environment, but factors such as low oxygen loading, premature oxygen leakage, and high interstitial pressure severely limit the efficiency of oxygen delivery; or decomposing hydrogen peroxide (H2O2) in the tumor to generate oxygen, but due to the low content of available H2O2, the oxygen production is far from meeting the treatment requirements. Therefore, unilaterally promoting oxygen supply cannot effectively combat the hypoxic microenvironment of the tumor, and it is very necessary to seek a new solution.

[0005] As the "energy factory" of cells and the main regulator of cell apoptosis, mitochondria are the organelles that consume the most oxygen in cells. It has been reported that hypoxia in tumor tissues is mainly caused by excessive oxygen consumption due to the mitochondrial-related oxidative phosphorylation process. Therefore, preferentially destroying the mitochondria of tumor cells can inhibit their endogenous oxygen consumption. It is worth mentioning that because mitochondria are more sensitive to ROS, excessive exogenous ROS can cause changes in mitochondrial permeability, deformation, depolarization, and the release of cytochrome C, ultimately inducing cell apoptosis. Therefore, given the short effective duration and small effective range of ROS, the sensitivity of mitochondria to ROS and the ability of destroying mitochondria to improve hypoxia in the tumor microenvironment, it is necessary to develop a photosensitizer with mitochondrial targeting function. Summary of the Invention

[0006] The purpose of the present invention is to provide a targeted photosensitizer FA-BAT-NPs and a preparation method thereof. The targeted photosensitizer FA-BAT-NPs prepared using the preparation method of the present invention have good water solubility and sustained release ability in vivo, have a good inhibitory effect on breast cancer, and hardly produce any toxic side effects to the body.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In a first aspect of the present invention, a method for preparing a photosensitizer BAT is provided, comprising: using 5-ALA as a raw material to introduce a Boc group through an amide reaction to obtain Boc-ALA; and then coupling the Boc-ALA with TPP through an amide reaction to obtain Boc-ALA-TPP, referred to as BAT.

[0009] In a second aspect of the present invention, provided is the photosensitizer BAT prepared by the method.

[0010] In the third aspect of the present invention, a targeted photosensitizer FA-BAT-NPs is provided. The targeted photosensitizer FA-BAT-NPs has a core-shell structure, wherein the core-shell structure is composed of an outer shell wrapped around an inner core, the inner core is the photosensitizer BAT according to claim 4, and the outer shell is a folic acid-modified BSA carrier, wherein the folic acid and BSA in the folic acid-modified BSA carrier are bridged by PEG.

[0011] Furthermore, the targeted photosensitizer FA-BAT-NPs includes, by mass fraction, 1-3% photosensitizer BAT and 97-99% folic acid-modified BSA carrier; wherein the folic acid-modified BSA carrier includes 10-13% BSA carrier and 86-87% PEG-folic acid, so that the mass fraction of the folic acid-modified BSA carrier is 97-99%.

[0012] In a fourth aspect of the present invention, a method for preparing the targeted photosensitizer FA-BAT-NPs is provided, the method comprising:

[0013] The BAT is subjected to an emulsification dispersion method to obtain BAT-NPs nanoparticles;

[0014] Using PEG as a connecting chain, FA was modified on the surface of the BAT-NPs nanoparticles through an amide reaction to obtain the targeted photosensitizer FA-BAT-NPs.

[0015] In a fifth aspect of the present invention, provided is the use of the photosensitizer BAT or the targeted photosensitizer FA-BAT-NPs in the preparation of anti-breast cancer drugs.

[0016] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0017] 1. The photosensitizer BAT provided by the present invention chemically links the photosensitizer 5-ALA with the mitochondrial targeting group TPP and modifies the Boc group to increase lipid solubility, thereby preparing a novel mitochondrial-targeted photosensitizer BAT. Subsequent cell proliferation inhibition experiments and in vivo efficacy experiments demonstrated that the photosensitizer BAT is significantly more effective than 5-ALA. The anti-cancer effect of BAT is significantly stronger than that of 5-ALA, Diboc, and TPP alone. This is likely because the modification of 5-ALA with TPP and Boc groups gives it mitochondrial targeting function and lipid solubility that can penetrate biological membranes, significantly enhancing its efficacy compared to the use of 5-ALA alone in the treatment of breast cancer.

[0018] 2. The present invention prepared a novel drug delivery system, FA-BAT-NPs, and found that FA-BAT-NPs can significantly improve the therapeutic efficiency of breast cancer and have almost no toxicity to experimental mice. BAT-NPs were prepared by emulsification dispersion method, and then FA-BAT-NPs were prepared by chemical synthesis and proved to be able to significantly increase the apoptosis rate and cellular uptake of MCF-7 cells. The results of MTT experiment and cell apoptosis experiment showed that compared with BAT, FA-BAT-NPs enhanced the growth inhibition of MCF-7 cells and induced apoptosis of MCF-7 cells. The proliferation inhibition effects of BAT, BAT-NPs and FA-BAT-NPs on MCFv7 and MCF-10A breast cancer cells showed dose dependence. In in vivo pharmacodynamic experiments, the inhibitory effects of BAT, BAT-NPs and FA-BAT-NPs on breast cancer tumor growth showed time dependence. In addition, through routine blood tests and related organ tests, FA-BAT-NPs showed no toxicity to the test mice. The above experiments prove that it has great potential to be developed into an anti-cancer drug for clinical use.

[0019] 3. This study successfully synthesized a derivative of the photosensitizer 5-ALA, BAT, and developed a novel targeted photosensitizer, FA-BAT-NPs. Subsequent in vitro and in vivo experimental results demonstrated that: 1) FA was linked to the nanoparticle surface via a chemical reaction using functional PEG as a linker. In vitro experiments, including cell uptake and apoptosis experiments, and in vivo experiments, including animal efficacy experiments, demonstrated that FA significantly enhanced the targeting and inhibitory effect of the target drug delivery system on tumor tissues. Specifically, FA enhanced drug uptake by breast cancer cells; 2) Under the same conditions, BAT exhibited significantly stronger in vitro anti-tumor effects than 5-ALA. In addition, compared with BAT and BAT-NPs, FA-BAT-NPs have a significantly stronger in vitro anti-tumor effect; 3) Compared with BAT, BAT-NPs and FA-BAT-NPs significantly prolonged the half-life of the drug, indicating that FA-BAT-NPs have better in vivo sustained-release ability and improved the bioavailability of the drug; 4) FA-BAT-NPs have good tumor targeting and low toxicity to the body; 5) At the same drug concentration, BAT's inhibitory effect on breast cancer is significantly stronger than 5-ALA, and the inhibitory effect of FA-BAT-NPs is significantly better than that of BAT. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 Schematic diagram of the targeted drug delivery system FA-BAT-NPs. FA-BAT-NPs are created by loading nanoparticles with the photosensitizer BAT and modifying the nanoparticle surface with FA. FA is recognized by receptors on the surface of breast cancer cells, and BAT accumulates in mitochondria, generating ROS through related reactions.

[0022] Figure 2 Structural characterization of Boc-ALA-TPP. (A) H NMR spectrum and (B) C NMR spectrum of Boc-ALA-TPP.

[0023] Figure 3 Results of infrared spectroscopy analysis. (A) Comparison of BAT, NPs, BAT-NPs, and a physical mixture of BAT and NPs; (B) Comparison of FA, FA-BAT-NPs, and a physical mixture of BAT-NPs and FA.

[0024] Figure 4 In vitro characterization of FA-BAT-NPs. (A) Particle size distribution, (B) Transmission electron microscopy image, scale bar 100 nm, (C) In vitro release results. Cumulative release of BAT, BAT-NPs, and FA-BAT-NPs was measured in PBS at 37°C, pH 7.4. Results are expressed as mean ± SD (n = 3).

[0025] Figure 5 This is an in vitro cell proliferation inhibition experiment of FA-BAT-NPs. MCF-10A cells were co-incubated with various concentrations of FA-NPs, BAT, BAT-NPs, and FA-BAT-NPs solutions for (A) 24h, (C) 48h, and (E) 72h; MCF-7 cells were incubated under the same conditions for (B) 24h, (D) 48h, and (F) 72h; (G) MCF-10A and (H) MCF-7 cells were co-incubated with 150μg / mL of TPP, 5-ALA, Diboc, 5-FU, FA-NPs, BAT, BAT-NPs, and FA-BAT-NPs solutions for 48h (5-FU was the positive control group). After the two cell lines were incubated with each group of drugs for different time periods, the survival rate of cancer cells was detected (n=5). * P<0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 was used to compare statistical differences between the drug groups; # P<0.05, ## P < 0.01, ### P < 0.001, ####P<0.0001 was used for statistical comparison between each drug-treated group and the BAT group.

[0026] Figure 6 The results of FA-BAT-NPs uptake in cells. Uptake in MCF-10A cells: (A) confocal laser scanning detection, (C) semi-quantitative analysis. Uptake in MCF-7 cells: (B) confocal laser scanning detection, (D) semi-quantitative analysis. BAT-NPs and FA-BAT-NPs were fluorescently labeled with Coumarin 6 and treated with cells, and the green fluorescence of the cells was detected using a laser confocal microscope. Scale bar: 25 μm. The images were analyzed for mean fluorescence intensity using Image J software, and 2-way ANOVA ( ** P < 0.01, *** P < 0.001, **** The significance of the difference was evaluated by P < 0.0001. The data of each group are expressed as mean ± standard deviation (n = 3).

[0027] Figure 7 Results of mitochondrial membrane potential measurement in MCF-7 cells. (A) Confocal laser scanning and (B) semi-quantitative analysis of mitochondrial membrane potential measurement in MCF-7 cells. Scale bar: 10 μm. Images were analyzed for mean fluorescence intensity using Image J software, and two-way ANOVA ( *** P < 0.001, **** The significance of the difference was evaluated by P < 0.0001. The data of each group are expressed as mean ± standard deviation (n = 3).

[0028] Figure 8 Results of ROS content determination in MCF-7 cells. (A) Confocal laser scanning, (B) semi-quantitative analysis of ROS content in MCF-7 cells. Scale bar: 10 μm. Images were analyzed for mean fluorescence intensity using Image J software, and 2-way ANOVA ( **** The significance of the difference was evaluated by P < 0.0001. The data of each group are expressed as mean ± standard deviation (n = 3).

[0029] Figure 9 The results of cell apoptosis are shown in Figure 3. After MCF-7 cells were treated with each preparation, the percentages of early apoptosis, late apoptosis, and necrosis quadrants were detected by flow cytometry using the Annexin V / PI binding scatter plot.

[0030] Figure 10 Cell cycle results: MCF-7 cells were treated with each preparation for 48 h and then stained with propidium iodide. (A) Cell cycle analysis was performed by flow cytometry and (B) quantitative analysis was performed.** P < 0.01, *** P < 0.001, **** P<0.0001.

[0031] Figure 11 Figure 2 is the pharmacokinetic curve of BAT, BAT-NPs and FA-BAT-NPs in plasma. Figure 2 is the curve of the change of drug concentration in plasma of BALB / C nude mice over time after tail vein administration. The results are expressed as Mean ± SD (n = 3).

[0032] Figure 12 The tissue distribution results of each drug group at different time points. Tissue distribution results of BAT, BAT-NPs and FA-BAT-NPs in MCF-7 tumor-bearing nude mice at 1h, 3h, 8h and 12h. * P<0.05, ** P < 0.01, *** P < 0.001, **** The significance of the difference was evaluated at P<0.0001. The data of each group are expressed as mean ± SD (n=3).

[0033] Figure 13 Fluorescence imaging results of each drug group in nude mice. (A) Fluorescence distribution of each drug group at 3, 8, and 12 hours after tail vein administration; (B) Heart, liver, spleen, lung, kidney, and tumor tissues were dissected out of nude mice at 12 hours and photographed using an in vivo imaging system. Semi-quantitative fluorescence intensity analysis of (C) nude mice and (D) tissues was performed using Image software. Results are expressed as mean ± SD (n = 3). * P<0.1, ** P < 0.01, *** P < 0.001, **** P<0.0001 was used to evaluate statistical differences between groups.

[0034] Figure 14 The in vivo anti-tumor results of FA-BAT-NPs. MCF-7 tumor-bearing nude mice in each group began to receive treatment on the 10th day after inoculation. The specific method was to inject saline, FA-NPs, TPP, Diboc, 5-ALA, BAT, BAT-NPs, FA-BAT-NPs and 5-FU into the tail vein respectively. 4 hours after the administration, a 635nm (25mW / cm 2 ) laser irradiated the tumor site for 5 minutes. (A) Tumor volume was monitored every 2 days after the first treatment; (B) Tumor weight of nude mice in each group; (C) Representative tumor tissues of nude mice in each group. * P<0.05, ** P < 0.01,*** P < 0.001, **** P < 0.0001 was used to compare statistical differences between each drug administration; # P<0.05, ## P < 0.01, ### P < 0.001, #### P<0.0001 was used to compare the statistical differences between each drug group and the normal saline group.

[0035] Figure 15 Analysis of the anti-tumor mechanism of FA-BAT-NPs (×400). Tumor sections were fixed, deparaffinized, and assayed for Ki-67, a cell proliferation marker, and CD31 and VEGFR, endothelial cell markers. Necrosis was assessed by H&E staining, and apoptosis was detected by TUNEL. Scale bar: 40 μm.

[0036] Figure 16 Histopathological H&E results (×400). H&E staining of the heart, liver, lung, and kidney of mice treated with saline, FA-NPs, 5-ALA, TPP, Diboc, BAT, 5-FU, BAT-NPs, and FA-BAT-NPs for two weeks. Scale bar: 50 μm. DETAILED DESCRIPTION

[0037] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0038] Throughout this specification, unless otherwise specified, the terms used herein are to be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, this specification takes precedence. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the present invention can be purchased commercially or can be obtained by existing methods.

[0039] The present application will be described in detail below with reference to the examples, comparative examples and experimental data. FA-PEG-NH2 in the examples of the present invention is a commercially available product, R-1009-2K-3, manufactured by Xi'an Ruixi Biotechnology Co., Ltd.

[0040] Example 1. Preparation method of targeted photosensitizer FA-BAT-NPs

[0041] The present invention provides a method for preparing a targeted photosensitizer FA-BAT-NPs, comprising the following steps:

[0042] Step S1: Synthesis of photosensitizer BAT. The synthesis route is as follows:

[0043]

[0044] (1) Synthesis of Boc-ALA

[0045] 1.00 g (5.96 mmol) of 5-aminolevulinic acid hydrochloride, 1.30 g (5.96 mmol) of di-tert-butyl dicarbonate (Diboc, Di-tert-butyl dicarbonate) and 5.01 g (35.80 mmol) of sodium bicarbonate were placed in a 50 mL single-necked round-bottom flask. 20 mL of methanol was added and stirred at room temperature for 6 hours. The solid was removed by filtration, and the solvent was rotary evaporated to obtain an oily residue, which was dissolved by adding an appropriate amount of ultrapure water. The pH value of the solution was adjusted to 2 with 1 mol / L hydrochloric acid, and the product was immediately extracted with ethyl acetate. The organic layer was washed three times with an appropriate amount of saturated common salt water, dried over anhydrous magnesium sulfate, and finally the remaining organic liquid was removed by rotary evaporation. The product was allowed to crystallize to obtain a light yellow solid, Boc-ALA.

[0046] The NMR data of Boc-ALA compound are: 1 H NMR (400MHz, CDCl3) δ5.24 (s, 1H), 4.05 (s, 2H), 2.69 (s, 4H), 1.43 (s, 9H). 13 C NMR (101MHz, CDCl3) δ204.39(s), 177.13(s), 155.96(s), 80.27(s), 77.54(s), 7 7.22(s), 76.90(s), 50.45(s), 34.34(s), 28.50(s), 27.69(s).HRMS(ESI)Calcd for C 10 H 18 NO5, [M+H] + : m / z 232.3361, found: 232.1557. Productivity: 78%. The synthesis of the Boc-ALA sample was confirmed.

[0047] (2) Synthesis of TPP-NH2

[0048] 1.31 g (5.00 mmol) of triphenylphosphine and 1.10 g (5.00 mmol) of 3-bromopropylamine hydrobromide were placed in a 50 mL single-necked round-bottom flask, and 20 mL of anhydrous acetonitrile was added. The mixture was refluxed at 90°C for 12 h. After the reaction mixture cooled to room temperature, the acetonitrile was removed by rotary evaporation. The resulting concentrate was recrystallized from 50 mL of an organic mixture of acetonitrile: n-hexane: isopropanol: ether (v / v = 1:3:10:5) in a -20°C refrigerator overnight. Filter and vacuum dry for 12 h to obtain TPP-NH2 as a white powder.

[0049] The NMR data of compound TPP-NH2 are: 1 H NMR (400MHz, D2O) δ7.96-7.60 (m, 15H), 3.41 (t, J=15.0Hz, 2H), 3.14 (t, J=7.5Hz, 2H), 2.06 (d, J=7.3Hz, 2H). 13 C NMR (101MHz, DMSO) δ135.22 (d, J=2.8Hz), 133.73 (d, J=10.3Hz), 130.49 (d, J=12.5Hz), 118.57 (s), 117.71 (s), 40.23 (s), 40.02(s), 39.81(s), 39.60(s), 39.39(s), 39.18(s), 38.97(s), 20.18(s), 18.66(s), 18.13(s).HRMS(ESI)Calcd for C 21 H 24 NP + , [M-Br+H] 2+ : m / z 321.1629, found: 321.2254. Productivity: 92%. The synthesis of TPP-NH2 sample was confirmed.

[0050] (3) Synthesis of Boc-ALA-TPP

[0051] 0.48 mL (2.75 mmol) of N,N-diisopropylethylamine, 0.206 g (0.55 mmol) of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 0.127 g (0.55 mmol) of Boc-ALA were added to 12 mL of dichloromethane and stirred at room temperature for 15 minutes. 0.8 g (1.66 mmol) of TPP-NH2 and 2.7 mg (0.02 mmol) of 4-dimethylaminopyridine were then added and stirring continued for 5 hours. The reaction solution was filtered and washed with dichloromethane. The filtrate was rotary evaporated to dryness. The concentrate was dissolved in ethyl acetate and washed sequentially with saturated brine, 5% sodium bicarbonate solution, and 1 mol / L hydrochloric acid. Finally, the organic phase was rotary evaporated and recrystallized from n-hexane to obtain an oil. The oil was then vacuum dried for 12 hours to obtain Boc-ALA-TPP, a pale yellow powder, referred to as BAT.

[0052] Compound Boc-ALA-TPP 1 H NMR, 13 C NMR spectrum Figure 2 As shown, the synthesis of the Boc-ALA-TPP sample was confirmed. The characteristic peak of Boc-ALA-TPP appears in (j). Its NMR data is: 1 H NMR (400MHz, DMSO) δ7.92-7.75 (m, 15H), 7.01 (s, 1H), 6.00 (s, 1H), 3.52 (ddd, J=39.7, 23.3, 7.9Hz, 4H), 3.29-3.11(m, 2H), 3.02(s, 2H), 2.35-2.19(m, 2H), 1.80(dt, J=15.7, 7.8Hz, 2H), 1.34(s, 9H). 13 C NMR (101MHz, DMSO) δ156.07 (s), 135.08 (s), 133.53 (dd, J=32.2, 14.8Hz), 13 1.10-130.17(m), 129.36-128.78(m), 118.97(s), 118.11(s), 90.93(s), 78. 12(s), 45.41(s), 40.23(s), 40.02(s), 39.81(s), 39.60(s), 39.39(s), 39.1 8(s), 38.97(s), 30.97(s), 29.35(s), 28.26(s), 21.52(s).HRMS(ESI)Calcd for C 31 H 39 N2O4P + , [M+H] 2+: m / z 534.2637, found: 534.8617. Productivity: 58%. The synthesis of the Boc-ALA-TPP sample was confirmed.

[0053] Step S2: Preparation of drug-loaded BAT-NPs. In this experiment, BAT nanoparticles (BAT-loaded nanoparticles, abbreviated as BAT-NPs) were prepared using an emulsion dispersion method. 25.11 mg of lecithin, 25.11 mg of cholesterol, and 3.62 mg of BAT were weighed and dissolved in 2 mL of chloroform (an organic solvent) to form a drug-loaded oil phase. BSA was dissolved in ultrapure water to obtain an aqueous phase with a BSA concentration of 2%. The oil and aqueous phases were mixed and ultrasonicated for 12 minutes to obtain an emulsion. This emulsion was quickly transferred to a round-bottom flask, and the organic solvent was removed by rotary evaporation under reduced pressure to obtain BAT-NPs. For subsequent comparison, BAT was omitted and the remaining steps were identical to those described above to obtain the nanoparticles, abbreviated as NPs.

[0054] Step S3, preparation of FA-BAT-NPs; BAT-NPs (7.9 mg, 0.012 mmol) were weighed and dissolved in 3 mL of ultrapure water, NHS (1.4 mg, 0.0122 mmol) was added thereto, and stirred for 15 min. EDC (4.29 mg, 0.0224 mmol) was added and vigorously stirred for 3 min. 20 mg of FA-PEG-NH2 (0.01 mmol, Mr = 2,000) was weighed and dissolved in 1 mL of ultrapure water, and then added to the BAT-NPs reaction solution. The mixture was stirred in the dark and reacted at room temperature for 24 h. The mixture was separated by centrifugation at 3,000 × g for 12 min to obtain purified FA-BAT-NPs (folic acid-modified BAT nanoparticles).

[0055] Experimental Example 1: Characterization of FA-BAT-NPs and Performance Determination of Photosensitizer BAT

[0056] (1) Determination of particle size, PDI, and Zeta potential of targeted photosensitizer FA-BAT-NPs

[0057] An appropriate amount of FA-BAT-NPs solution was slowly injected into the sample cell to avoid bubbles and diluted with ultrapure water to an appropriate concentration. The sample was placed in the sample tank and the particle size, polydispersity index (PDI), and zeta potential were measured using a Malvern laser particle size analyzer until the particle size distribution stopped. The results are shown in Table 1.

[0058] Table 1. Characterization results of nanoparticles (n=3)

[0059]

[0060] As shown in Table 1, by comparing the particle sizes of NPs and BAT-NPs, it can be seen that the particle size of the unloaded nanoparticles is smaller than that of the drug-loaded nanoparticles. Based on the comparison of the particle sizes, it can be preliminarily proved that BAT was successfully encapsulated in the bovine serum albumin nanoparticles. This result is consistent with Figure 4 A. Consistent.

[0061] (2) Infrared spectroscopy analysis

[0062] Appropriate amounts of BAT, NPs, BAT-NPs, physical mixtures of BAT and NPs; FA, FA-BAT-NPs, and physical mixtures of FA and BAT-NPs were taken and scanned by infrared spectrometry to verify whether BAT was successfully encapsulated in the nanoparticles and whether the ligand FA was successfully connected. The results are shown in Figure 2. Figure 3 As shown in A, BAT has a characteristic peak of benzene ring skeleton structure (υ C＝C : 1601cm -1 ), a shoulder peak can be found in the results of physical mixing of BAT and NPs, while this characteristic peak is not seen in BAT-NPs. The results are not significantly different from those of NPs, which shows that BAT can be successfully encapsulated in bovine serum albumin nanoparticles through this experimental method. Figure 3 As shown in Figure B, the results of FA-BAT-NPs were compared with those of BAT-NPs and FA physical mixture. FA-BAT-NPs had obvious characteristic peaks of amide groups (υ C＝o : 1662cm -1 , δ N-H : 1638cm -1 ), proving that the ligand folic acid was successfully modified on the surface of drug-loaded nanoparticles.

[0063] (3) Determination of BAT nanoparticle encapsulation efficiency and drug loading

[0064] The prepared nanoparticle suspension was centrifuged at 3000×g for 10 min, and 0.5 mL of the supernatant was accurately measured and diluted to 5 mL. The suspension was then demulsified for 5 min and filtered through a 0.22 μm microporous filter membrane. The sample was then tested (chromatographic conditions: chromatographic column: Agilent ZORBAXSB-C18 column (4.6*250 mm, 5 μm); mobile phase: acetonitrile: 15 mmol / L KH2PO4 = 80:20; detection wavelength: 243 nm; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 20 μL). The chromatographic peak area of ​​BAT was recorded, and the BAT content in the nanoparticles (M) was calculated. loaded ).

[0065] Accurately pipette 0.5 mL of nanoparticle suspension into a 5 mL volumetric flask, dilute with methanol, and perform demulsification for 5 minutes. After filtering with a 0.22 μm microporous filter membrane, the sample is tested according to the above chromatographic conditions. According to the peak area of ​​BAT, the total drug dosage (M) injected into BAT can be calculated. total ).

[0066] The encapsulation efficiency (EE%) can be calculated based on the drug content in the BAT nanoparticles and the total amount of drug administered. Formula (1) is as follows:

[0067] EE(%)=M loaded / M total ×100%……(1)

[0068] The drug loading capacity (DL%) was determined as follows: the prepared nanoparticle suspension was freeze-dried to obtain nanoparticle powder. A certain amount of powder (this is the total mass of the nanoparticles, M) was taken. nanoparticles ), re-dissolved with PBS, centrifuged at 3000×g for 10 min, accurately aspirated 0.5 mL of the supernatant, fixed to volume with methanol in a 5 mL volumetric flask, and subjected to ultrasonic demulsification for 5 min. After filtering with a 0.22 μm microporous filter membrane, the sample was tested according to the above chromatographic conditions, and the chromatographic peak area of ​​BAT was recorded. The drug content in the nanoparticles (M loaded ). Formula (2) is as follows:

[0069] DL(%)=M loaded / M nanoparticles ×100%……(2)

[0070] The encapsulation efficiency of BAT-NPs was (94.82±0.80)%, and the drug loading was (7.85±0.65)%. The encapsulation efficiency of FA-BAT-NPs was (92.27±0.39)%, and the drug loading was (6.25±0.34)%. These results show that the encapsulation efficiency of BAT-NPs and FA-BAT-NPs is high. The results are shown in Table 1.

[0071] (4) Determination of BAT nanoparticle morphology

[0072] The surface morphology of NPs, BAT-NPs and FA-BAT-NPs was observed using a transmission electron microscope. The following method was used: After diluting an appropriate amount of the sample to be tested 100 times with distilled water, an appropriate amount of the diluent was added dropwise onto a dedicated copper grid. After leaving it for 10 minutes, the remaining diluent was removed. After staining for 3 minutes, the excess dye was removed with filter paper. After drying, the sample was observed using a transmission electron microscope (Jeol JEM-1400) and the morphological images of the nanoparticles were taken. Figure 4 As can be seen in Figure B, the nanoparticles are spherical. The particle sizes of BAT-NPs and FA-BAT-NPs are slightly larger than those of NPs. Therefore, based on the TEM results, it can be preliminarily determined that the drug BAT is successfully encapsulated in BSA nanoparticles.

[0073] (5) In vitro release of BAT nanoparticles

[0074] 4 mL each of BAT, BAT-NPs, and FA-BAT-NPs were placed in a sealed dialysis bag with a molecular weight cutoff of 3.0 kD. The bag was then placed in 50 mL of dissolution medium (PBS, pH = 7.4) containing 0.3% Tween-80 and stirred at a constant temperature (37°C). Three replicates were prepared for each formulation. Samples were taken at the following time points (0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h), with 1 mL of dissolution medium immediately added. The sample was diluted to 2 mL with methanol and filtered through a 0.22 μm microporous membrane. HPLC analysis was performed under the above-mentioned chromatographic conditions. The chromatographic peak area of ​​BAT was recorded, and the drug release was calculated using the standard curve.

[0075] Depend on Figure 4 C shows that the drug in the BAT group was basically completely released at 24 hours, and the cumulative release rate of the drug did not increase significantly after 24 hours; unlike the BAT group, the cumulative release rates of the BAT-NPs and FA-BAT-NPs groups increased slowly within 96 hours. The cumulative release rates of the two groups at 24 hours were approximately (50.73±1.70)% and (49.02±0.20)%, respectively, and the cumulative release rates at 96 hours were approximately (64.87±2.17)% and (63.92±2.11)%, respectively, indicating that BAT has the characteristics of slow release after being prepared into nanoparticles.

[0076] (6) Solubility determination of BAT nanoparticles

[0077] Excess BAT powder was dissolved in 5 mL of deionized water, anhydrous ethanol, and PBS buffer (pH 7.4). After sonication for 20 minutes, the mixture was centrifuged at 3,000 × g for 10 minutes and filtered through a 0.22 μm microporous filter. The filtrate was diluted with methanol and analyzed by HPLC under the above-mentioned chromatographic conditions. The chromatographic peak area of ​​BAT was recorded, and the equilibrium solubility was calculated using a standard curve. Lyophilized powders of BAT-NPs and FA-BAT-NPs were treated in the same manner, and the chromatographic peak area of ​​BAT was recorded and the equilibrium solubility was calculated using a standard curve.

[0078] Table 2. Solubility of samples in different solutions (n=5)

[0079]

[0080] As can be seen from Table 2, compared with BAT, the solubility of BAT-NPs (BAT-loaded nanoparticles) and FA-BAT-NPs (BAT-loaded nanoparticles modified with folic acid) in the three solutions was significantly improved, which shows that after BAT is encapsulated in nanoparticles, its solubility performance can be greatly improved, thereby overcoming the disadvantage of BAT's poor water solubility.

[0081] Experimental Example 2: In vitro study of FA-BAT-NPs nanoparticles

[0082] 1. Cell proliferation inhibition experiment

[0083] (1) In this study, MCF-10A and MCF-7 cells were used as model cells. The inhibitory effects of BAT, TPP, Diboc, 5-FU, BAT, FA-NPs, BAT-NPs, and FA-BAT-NPs on breast cancer cells were evaluated using the MTT assay. An appropriate amount of normally growing cells was prepared with DMEM complete medium to obtain a uniform cell suspension. The cell concentration was adjusted to 3.5×10 using an automatic cell counter. 4 cells / mL, 100 μL of the above cell suspension was inoculated into a 96-well plate, and an appropriate amount of PBS was added to the periphery of the 96-well plate to prevent excessive evaporation of the original liquid during culture and affect cell growth. Finally, the plate was placed in a 5% CO2 incubator for overnight culture until the cells adhered to the wall.

[0084] (2) The original culture medium was removed and different concentrations of BAT, FA-NPs, BAT-NPs, FA-BAT-NPs, as well as the raw materials 5-ALA, TPP, Diboc and the positive control 5-FU were added (five replicates per group), and the cells were cultured in a cell culture incubator. The cells were illuminated with 635 nm (25 mW / cm 2 ) laser irradiated the cells for 5 min.

[0085] (3) After 24 h, 48 h, and 72 h, 20 μL of 5 mg / mL MTT solution was added to each well of the 96-well plate. After incubation for 4 h, the liquid in the wells was slowly aspirated and 100 μL of DMSO was added to each well. The plates were shaken for 10 min to fully dissolve the crystals in the wells. The absorbance was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) and the measurement was repeated three times.

[0086] The survival rate of breast cancer cells in each group was calculated to evaluate its inhibitory effect on breast cancer cells.

[0087] Cell Viability (%) = (A samp1e -A blank ) / (A control-A blank )×100……(3)

[0088] The results are as follows Figure 5 As shown in the figure, this experiment used the MTT method to detect the inhibitory effect of each group of drugs on MCF-10A and MCF-7 cells after incubation for 24h, 48h, and 72h; drugs at concentrations of 150μg / mL and 200μg / mL showed better tumor cell inhibition. Figure 5 As shown in A, after incubation with 150 μg / mL and 200 μg / mL of each group of drugs for 24 hours, the survival rates of MCF-10A cells in the BAT-NPs group were (83.700±2.771)% and (70.566±4.260)% respectively; the cell survival rates in the FA-BAT-NPs group were (80.564±1.731)% and (71.962±3.368)% respectively; after incubation with MCF-7 cells for 24 hours, the survival rates of MCF-10A cells in the FA-BAT-NPs group were (83.700±2.771)% and (70.566±4.260)% respectively. Figure 5 B), the cell viability of the BAT-NPs group at drug concentrations of 150 μg / mL and 200 μg / mL was (80.408±1.611)% and (72.460±4.063)%; the cell viability of the FA-BAT-NPs group was (63.424±3.411)% and (47.504±2.795)%; for MCF-7 cells, the cell viability of the FA-BAT-NPs group was significantly reduced compared with that of the BAT group at the same concentration ( #### P < 0.0001), indicating that it has a stronger inhibitory effect on MCF-7 cells, while the inhibitory effect on human mammary epithelial cells MCF-10A is significantly smaller.

[0089] like Figure 5 As shown in C, after incubation of MCF-10A cells for 48 h, when the drug concentration was 150 μg / mL and 200 μg / mL, the cell viability of the BAT-NPs group was (71.074±4.027)% and (59.110±3.805)%, respectively, and the cell viability of the FA-BAT-NPs group was (68.532±2.520)% and (56.972±4.203)%, respectively; after incubation of MCF-7 cells with different preparations for 48 h ( Figure 5 D). When the drug concentration was 150 μg / mL and 200 μg / mL, the cell viability of the BAT-NPs group was (66.458±5.551)% and (62.190±4.655)%, respectively. The cell viability of the FA-BAT-NPs group was (36.970±4.438)% and (32.266±1.945)%, respectively. For MCF-7 cells, compared with the BAT group at the same concentration, FA-BAT-NPs at 150 μg / mL could more significantly reduce the cell viability (#### P<0.0001), which enhanced the inhibitory effect on cancer cells, and FA-BAT-NPs showed better in vitro anti-cancer activity.

[0090] like Figure 5 As shown in E, after incubation of MCF-10A cells for 72 hours, when the drug concentration was 150 μg / mL and 200 μg / mL, the cell viability of the BAT-NPs group was (67.690±2.344)% and (56.794±1.774)%, respectively. The cell viability of the FA-BAT-NPs group was (64.754±4.423)% and (55.236±4.723)%, respectively. There was no significant difference between the survival rate of MCF-10A cells treated with 150 μg / mL FA-BAT-NPs and the BAT group at the same concentration. After incubation of MCF-7 cells with different drugs for 72 hours ( Figure 5 F), when the drug concentration was 150μg / mL and 200μg / mL, the cell viability of BAT-NPs group was (66.438±2.589)% and (51.146±2.636)%, and the cell viability of FA-BAT-NPs group was (25.822±2.742)% and (24.210±5.024)%. Compared with the BAT group with the same concentration, FA-BAT-NPs at 200μg / mL significantly reduced the survival rate of MCF-7 cells ( #### P < 0.0001), which enhanced the inhibitory effect on cancer cells.

[0091] After each drug group acted on MCF-10A and MCF-7 cells for 24h, 48h and 72h respectively, the results showed that BAT, BAT-NPs and FA-BAT-NPs preparation groups had certain proliferation inhibitory effects on MCF-10A and MCF-7 cells. Compared with MCF-10A cells, the proliferation inhibition effect on MCF-7 cells was significantly stronger. This difference indicates that the target drug delivery system FA-BAT-NPs has a good inhibitory effect on breast cancer cells and is less toxic to normal breast cells. According to the experimental results at 48h, except for 5-FU (positive control group), the survival rate of cancer cells in the FA-BAT-NPs group (200μg / mL) was the lowest, and its inhibition rate reached 67.734%, indicating that FA-BAT-NPs had the most obvious inhibitory effect on the proliferation of cancer cells and showed good in vitro anti-cancer activity. At the same time, when the positive control group 5-FU acted on MCF-10A and MCF-7 cells ( Figure 5 H), significantly reduced the viability of cancer cells compared with the BAT group ( ####P < 0.0001), enhancing the inhibitory effect on tumor cells. After 48 hours of cell incubation, the cell viability of the 150 μg / mL BAT, 5-ALA, TPP, and Diboc groups was (62.654 ± 4.493)%, (81.302 ± 2.201)%, (91.964 ± 3.133)%, and (93.940 ± 4.438)%, respectively. This further demonstrates that the drug synthesized in this project, BAT, has a stronger inhibitory effect on tumor cell proliferation than 5-ALA, TPP, and Diboc alone.

[0092] 2. Cellular Uptake Experiment

[0093] The ability of FA-BAT-NPs to deliver drugs into target cells is an important indicator for evaluating their targeting properties. To investigate the cellular uptake of BAT-NPs and FA-BAT-NPs using MCF-7 and MCF-10A cells as models, green fluorescent BAT-NPs and FA-BAT-NPs were prepared using Coumarin 6.

[0094] Take an appropriate amount of normally growing MCF-7 and MCF-10A cells and prepare them with DMEM complete medium to obtain a concentration of 3×10 5 Cells / mL of cell suspension were added to each well of a 24-well culture plate, and 1 cell slide was placed in each well of the plate. 1 mL of the cell suspension was added, and 3 replicates were set up in each group. The cells were cultured in a cell incubator for 24 hours. After the cells adhered to the wall, the original culture medium was removed, and 1 mL of the above-mentioned BAT-NPs and FA-BAT-NPs dilutions were added. After culturing for 1 hour, 2 hours, and 4 hours, the original culture medium was discarded. The cells were washed 3 times with PBS and fixed for 30 minutes (4% paraformaldehyde), then washed twice with PBS, stained with DAPI staining solution for 5 minutes, and washed twice with PBS after the staining solution was removed. The cell slides in the plate were carefully removed, covered on a glass slide and sealed (50% glycerol), and stored at low temperature and in the dark. The distribution of green fluorescence in breast cancer cells was detected by laser confocal microscopy, and the cell pictures were semi-quantitatively analyzed using Image J software.

[0095] like Figure 6As shown, green fluorescence in the cells gradually increased over time, indicating that cellular internalization was dependent on incubation time. The fluorescence signal from MCF-7 cells was significantly stronger than that from MCF-10A cells, which may be due to the higher expression of folate receptors on the MCF-7 cell membrane surface. More interestingly, the intracellular green fluorescence from FA-BAT-NPs was much stronger than that from BAT-NPs, indicating that the targeted drug delivery system can promote the internalization of therapeutic drugs by MCF-7 cells. This significant difference may be related to the improved solubility and endocytosis of BAT-assembled nanomedicines, which also demonstrates the active targeting ability of FA-BAT-NPs.

[0096] 3. Mitochondrial membrane potential measurement experiment

[0097] BAT is a new photosensitizer with mitochondrial targeting. Its damage to tumor cell mitochondria is an important indicator for evaluating the mitochondrial targeting of FA-BAT-NPs. Based on the above experiments, this study selected MCF-7 cells as the experimental cell model and used the fluorescent agent Rhodamine 123 to detect the damage to tumor cell mitochondria, thereby reflecting the mitochondrial targeting of each group of drugs.

[0098] Take an appropriate amount of normally growing MCF-7 cells and prepare them with DMEM complete medium to obtain a concentration of 3×10 5 Cells / mL of cell suspension were placed in each well of a 24-well culture plate, and 1 mL of the above cell suspension was added. Three replicates were set up in each group and the cells were cultured in a cell culture incubator for 24 hours. After the cells adhered to the wall, the original culture medium was aspirated and 1 mL of blank solution, TPP, Diboc, 5-ALA, BAT, FA-NPs, BAT-NPs and FA-BAT-NPs dilution were added respectively. The cells were cultured in an incubator for 12 hours. The 635 nm (25 mW / cm 2 ) laser irradiation for 5 minutes. The culture medium was aspirated and the cells were thoroughly washed twice with PBS. The cells were then stained with 0.2 mL of Rhodamine 123 (2 μM) for 20 minutes and washed twice with serum-free medium. The cell slides were carefully removed from the plate, placed on a glass slide, and mounted with 50% glycerol. The slides were stored at low temperatures and protected from light. Fluorescence distribution within the breast cancer cells was examined using laser confocal microscopy, and semi-quantitative analysis of the cell images was performed using Image J software.

[0099] The results are as follows Figure 7As shown in the figure, this experiment used Rhodamine 123 to investigate the extent of mitochondrial damage in MCF-7 cells after 12 hours of incubation with different formulations. Bright green fluorescence was observed in cells treated with TPP, Diboc, FA-NPs, 5-ALA, or no treatment, indicating that mitochondrial function was intact. However, green fluorescence was significantly weaker in cells treated with BAT and BAT-NPs, indicating that BAT has mitochondrial targeting, allowing it to accumulate around mitochondria and produce damaging ROS, thereby reducing mitochondrial membrane potential. The FA-BAT-NPs group exhibited the weakest fluorescence intensity, as FA actively targets and is able to enter cancer cells.

[0100] IV. ROS content determination experiment

[0101] As a photosensitizer, the amount of ROS produced by FA-BAT-NPs is an important reference value for evaluating the therapeutic effect on tumor cells. Based on the above experiments, this study selected MCF-7 cells as the experimental cell model and used DCFH-DA as a fluorescent dye to detect ROS levels in tumor cells.

[0102] Take an appropriate amount of normally growing MCF-7 cells and prepare them with DMEM complete medium to obtain a concentration of 3×10 5 Cells / mL of cell suspension were prepared, and 1 cell slide was placed in each well of a 24-well culture plate. 1 mL of the above cell suspension was added, and 3 replicates were set up in each group. The cells were cultured in a cell culture incubator for 24 hours. After the cells adhered to the wall, the original culture medium was aspirated, and 1 mL of blank, TPP, Diboc, 5-ALA, BAT, FA-NPs, BAT-NPs and FA-BAT-NPs dilutions were added respectively and incubated for 12 hours. The original culture medium was removed, the cells were washed thoroughly with PBS, and the cells were incubated with DCFH-DA (1 mM) for another 2 hours. Finally, the cells were illuminated with 635 nm (25 mW / cm 2 ) laser irradiation for 5 minutes, then remove the culture medium and wash thoroughly twice with PBS. Carefully remove the cell slides from the plate, place them on glass slides, and seal the slides with 50% glycerol. Store at low temperature and in the dark. Fluorescence distribution within breast cancer cells was examined using laser confocal microscopy, and semi-quantitative analysis of cell images was performed using Image J software.

[0103] We used DCFH-DA as a fluorescent dye to detect the production of ROS in cells ( Figure 8). Like the blank group cells, the MCF-7 cells treated with TPP, Diboc, and FA-NPs showed almost no fluorescence. The related drug group using BAT as a photosensitizer showed obvious green fluorescence. When exposed to light, the cells reflected bright green fluorescence, indicating that a large amount of ROS were produced in the cells. It is worth noting that the fluorescence intensity of MCF-7 cells treated with 5-ALA was lower than that of the BAT group, indicating that its ability to produce ROS was weaker than that of BAT. This is most likely due to the mitochondrial targeting effect of BAT, which can alleviate the hypoxia of the tumor microenvironment by inhibiting the aerobic respiration of cancer cells, thereby generating more ROS, indicating that it has great potential for solving the problem of tumor hypoxia.

[0104] 5. Cell Apoptosis Experiment

[0105] Based on the above experiments, this study selected MCF-7 cells as the experimental cell model to investigate the cell apoptosis of MCF-7 cells after the action of 5-ALA, BAT, BAT-NPs and FA-BAT-NPs.

[0106] After 48 hours of exposure to drugs in each group, the cells were treated according to the instructions of the apoptosis kit. The original culture medium of the cells was carefully aspirated and temporarily stored in a centrifuge tube. The cells were digested with EDTA-free trypsin until the adherent cells could be gently blown off with a pipette tip, and then the previously collected cell culture medium was added, and the cells were gently blown evenly with a pipette tip. Centrifuge at 335×g for 5 minutes to precipitate the cells. Gently aspirate the supernatant and gently resuspend the cells with 1mL of pre-cooled PBS. The cells were centrifuged again and the supernatant was carefully removed. The cells were resuspended with Binding Buffer. Accurately aspirate 100μL of the above cell solution into a tube, add 5μL of Annexin V / FITC and mix well. Incubate in the dark for 5 minutes, then add PI solution (5μL), and finally add PBS (400μL). Be sure to store in an ice bath in the dark. Rapidly inject the sample using a flow cytometer, and calculate the cell apoptosis rate corresponding to each drug group according to formula (4). Repeat 3 times for each group and take the average value.

[30] .

[0107] Apoptosis Rate=Proportion of early apoptotic cells+Proportion of late apoptotic cells…(4)

[0108] like Figure 9As shown in the data, after different drugs acted on MCF-7 cells, compared with the Blank group, 5-ALA, BAT, BAT-NPs and FA-BAT-NPs significantly induced apoptosis of MCF-7 cells, among which the apoptosis rate induced by 5-ALA was (28.89±2.33)%, the apoptosis rate induced by BAT was (41.09±2.02)%, and the apoptosis rate induced by BAT-NPs was (32.58±3.43)%. Among them, FA-BAT-NPs induced the most obvious apoptosis, with a cell apoptosis rate of (48.08±3.00)%. This shows that after incubation, FA-BAT-NPs induced the most obvious apoptosis of MCF-7 cells.

[0109] VI. Cell Cycle Experiment

[0110] Based on the above experiments, this experiment selected MCF-7 cells as the experimental cell model to investigate the effects of 5-ALA, BAT, BAT-NPs and FA-BAT-NPs on the cell cycle. Take an appropriate amount of normally grown MCF-7 cells, prepare a cell suspension with DMEM complete medium, and place the suspension in a 6-well plate. Culture in an incubator. When enough cells adhere to the wall, the original culture medium is aspirated, and then 5-ALA, BAT, BAT-NPs and FA-BAT-NPs culture medium are added and cultured in an incubator for 48 hours. During this period, 635nm (25mW / cm 2 ) laser irradiation for 5 minutes. After completing the above steps, the cells were processed according to the operating procedures of the cell cycle kit: digestion, centrifugation, and cell collection were followed by washing with PBS once, centrifugation at 670 × g for 5 minutes to pellet the cells, and then the cell concentration was adjusted to 1 × 10 6 cells / mL. Take 1 mL of cell suspension, centrifuge and wash away the supernatant, add 500 mL of 70% pre-cooled ethanol and mix well, and fix in a 4°C refrigerator overnight. Aspirate the fixative and rinse once with PBS. Centrifuge and remove the supernatant, add 100 μL of RNase A solution to the cell pellet, resuspend the cells and place in a 37°C water bath for 30 minutes. Then add 400 mL of PI staining solution and mix well. Incubate at 4°C in the dark for 30 minutes. Store in a dark place and perform flow cytometry as soon as possible to measure the red fluorescence at an excitation wavelength of 488 nm.

[0111] like Figure 10 As shown in A&B, after DMSO (0.5%), 5-ALA, BAT, BAT-NPs and FA-BAT-NPs were co-incubated with MCF-7 cells for 48 h, compared with the Blank group, DMSO (0.5%) as a solvent had almost no effect on the MCF-7 cell cycle, while the percentage of cells in the G0 / G1 phase in the BAT, BAT-NPs and FA-BAT-NPs groups increased significantly (**** P<0.0001), and the number of cells in S phase decreased significantly, with significant difference ( *** P < 0.001), proving that the MCF-7 tumor cell cycle was arrested in the G0 / G1 phase; compared with the BAT and BAT-NPs groups, the FA-BAT-NPs group had a higher content of cells in the G0 / G1 phase and a lower content of cells in the S phase, proving that FA-BAT-NPs could arrest the cell cycle in the G0 / G1 phase to a greater extent than BAT and BAT-NPs, demonstrating that FA-BAT-NPs had a stronger effect on cell cycle arrest.

[0112] Experimental Example 3: Study on the in vivo distribution of FA-BAT-NPs nanoparticles

[0113] 1. Establishment and confirmation of breast cancer model

[0114] After approval from the Animal Ethics Committee, a nude mouse xenograft tumor model was established. Female BALB / C nude mice (3-4 weeks old) were housed in an SPF-grade indoor environment for acclimatization prior to the formal experiment. After 7 days of acclimatization, MCF-7 cells in the logarithmic growth phase and in good growth condition were obtained and prepared into a cell suspension in PBS for inoculation. The inoculation cell concentration was 9×10 7 cells / mL, with an inoculation volume of 0.15 mL per BALB / C nude mouse. Disinfect the nude mouse skin with iodine before inoculation. Draw out the cell suspension and inject it subcutaneously into the nude mouse. Observe the success of the desired nude mouse breast cancer model.

[0115] 2. Study on the in vivo distribution and targeting of FA-BAT-NPs

[0116] (1) Pharmacokinetic study of FA-BAT-NPs

[0117] Pharmacokinetic studies were conducted on established female BALB / C nude mice, with the drug administered via tail vein injection (25 mg / kg). The mice were randomly divided into three groups and fasted for 12 hours before the experiment.

[0118] After administration of the BAT group, BAT-NPs nanoparticle group, and FA-BAT-NPs nanoparticle group, 0.3 mL of blood was collected from the heart of the nude mice at 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h, respectively. The whole blood was quickly transferred and centrifuged for 5 min in a centrifuge (2012 × g). The upper plasma was obtained and processed according to the above-mentioned "biological sample processing method" and injected by HPLC. The drug concentration of PpIX in each group was obtained by peak area calculation. The graph was drawn using Graphpad 6.01 software. The area under the time-volume curve (AUC) of the animal plasma after administration of BAT, BAT-NPs, and FA-BAT-NPs was calculated using DAS ver. 2.1.1 software. 0-t (mg / L*h), peak drug concentration C max (mg / L), half-life T 1 / 2 Pharmacokinetic parameters such as z(h).

[0119] Figure 11 The pharmacokinetic curves shown in the Figure 10 demonstrate that in vivo drug effects in the BAT-NPs and FA-BAT-NPs groups were more sustained than in the BAT group in BALB / C nude mice. This result is consistent with the trends observed in the in vitro release experiments, further demonstrating the advantages of sustained-release and long-lasting effects of the nanoparticle-loaded drug delivery system, FA-BAT-NPs. Furthermore, the pharmacokinetic parameters shown in Table 10 demonstrate that the in vivo residence time and half-life of the FA-BAT-NPs group were significantly prolonged compared to the BAT-NPs group. The pharmacokinetic curves also demonstrate that the FA-BAT-NPs group significantly prolonged the drug's absorption process in vivo, demonstrating higher bioavailability.

[0120] (2) HPLC method to investigate the tissue distribution of nanoparticles

[0121] Thirty-six female BALB / C nude mice with established models were randomly divided into three groups of 12 mice each. Each nude mouse was injected with the drug via the tail vein at a dose of 25 mg / kg. The drug was administered according to the following regimen: the first group was the BAT group, consisting of four groups of three mice per group. The second group was the BAT-NPs group, consisting of four groups of three mice per group, and the third group was the FA-BAT-NPs group, consisting of four groups of three mice per group. The heart, liver, spleen, lung, kidney, and tumor tissues of the nude mice were dissected out from the three groups at 1 h, 3 h, 8 h, and 12 h after administration, and processed according to the above-mentioned "biological sample processing method". HPLC injection was performed, and the drug concentration of each sample was calculated by recording the peak area of ​​PpIX. Finally, the graph was drawn using the software Graphpad 6.01.

[0122] like Figure 12As shown in Figure 3, before 3 hours, the blood concentrations of BAT-NPs and FA-BAT-NPs groups were lower than those of the BAT group. Between 8 and 12 hours, the blood concentrations of BAT-NPs and FA-BAT-NPs groups were significantly higher than those of the BAT group ( **** P<0.0001), which shows that after BAT is encapsulated by NPs to make drug-loaded nanoparticles, the blood circulation time of the drug can be greatly prolonged. Figure 12 A) 3h( Figure 12 B) 8h( Figure 12 C) and 12h( Figure 12 At the D) time point, the drug in the BAT-NPs and FA-BAT-NPs groups was more distributed in the blood, liver, kidney and tumor tissues, and the drug content in the tumor tissue was higher than that in the BAT group ( **** P < 0.0001), among which the drug concentration of FA-BAT-NPs group was significantly higher than that of BAT-NPs group, which showed that FA-BAT-NPs had a certain targeting effect on tumor tissue.

[0123] (3) Fluorescence labeling method to investigate the in vivo distribution of FA-BAT-NPs drug delivery system

[0124] In vivo imaging was used to investigate the in vivo distribution and targeting of FA-BAT-NPs. DiR was first labeled into the nanoparticles and divided into control, saline, DiR, DiR-BAT-NPs, and FA-DiR-BAT-NPs groups. These nanoparticles were then injected into nude mice with breast cancer via the tail vein. Fluorescence was detected and captured using an IVIS imaging system. Fluorescence distribution was observed at 3, 8, and 12 hours.

[0125] After the experimental procedures were completed, the animals were immediately bled from the heart and sacrificed. Whole blood was centrifuged (1509 × g) for 5 minutes, and the upper plasma layer was collected and placed in an EP tube for later use. The hearts, livers, spleens, kidneys, lungs, and tumor tissues of the nude mice in each group were rapidly dissected. Fluorescence intensity of the organs and tumor tissues was analyzed using an IVIS imaging system 12 hours after the treatment, and photographs were recorded.

[0126] like Figure 13As shown in A, in nude mice, the fluorescence intensity of the DiR-BAT-NPs and FA-DiR-BAT-NPs groups loaded with the fluorescent probe DiR at 3h, 8h and 12h were stronger than those of the normal group and the saline group. At 3h, the fluorescence intensity around the tumor tissue was significantly enhanced. After 12h of filming, the heart, liver, spleen, lung, kidney and tumor tissues of the nude mice in the DiR, DiR-BAT-NPs and FA-DiR-BAT-NPs groups were dissected out and photographed, and the images were analyzed for average fluorescence intensity using Image J software. Figure 13 The results shown in B&D show that the average fluorescence intensity of the FA-DiR-BAT-NPs group in tumor tissue was significantly higher than that of the DiR and DiR-BAT-NPs groups ( **** P < 0.0001). Furthermore, fluorescence intensity was higher in the liver and kidneys in all groups, but lower in the kidneys in the FA-DiR-BAT-NPs group compared to the DiR-BAT-NPs group. Combining this experiment with the aforementioned pharmacokinetic results, it is clear that FA-modified nanoparticle carriers not only prolong the half-life of the drug in BAT and improve its in vivo processing, but also enhance tumor targeting capabilities of the targeted drug delivery system.

[0127] Experimental Example 4: In vivo pharmacodynamics of FA-BAT-NPs

[0128] 1. Experimental Grouping

[0129] Nude mouse breast cancer model was established according to the above-mentioned breast cancer model establishment and confirmation method. 45 female BALB / C nude mice with established models were randomly divided into 9 groups: saline group, blank nanoparticle group (FA-NPs), 5-aminolevulinic acid group (5-ALA), triphenylphosphine group (TPP), Diboc group, free drug group (BAT), positive control group (5-FU), drug-loaded nanoparticle group (BAT-NPs) and FA-modified drug-loaded nanoparticle group (FA-BAT-NPs). The drug was administered once a day through tail vein injection (25 mg / kg), and 4 hours after each administration, the drug was illuminated with 635 nm (25 mW / cm 2 ) laser irradiated the tumor site for 5 minutes and the drug was administered continuously for 14 days.

[0130] 2. Pharmacodynamic Evaluation

[0131] The disease model of this experiment was a breast cancer model with MCF-7 cells transplanted ectopically. The tumor volume of each group of nude mice was recorded 10 days after tumor inoculation. These nude mice were given the drug once a day and 4 hours after the drug administration, they were given a 635nm (25mW / cm 2) laser irradiated the tumor site for 5 minutes. Measurements were taken every 2 days after the start of treatment until the nude mice were sacrificed. Before administration, the body weight and tumor volume of the model mice were measured. The specific method for measuring the tumor volume was to use a vernier caliper to measure the longest axis (L) and the shortest axis (W) of each nude mouse tumor, and then calculate the results according to formula (6). The anti-tumor effect of each experimental group was expressed by tumor growth inhibition (TGI). TGI was the average tumor weight (MTW) ​​of the treatment group (TG) relative to the saline group (CG) on the 14th day. The average tumor weight (MTW) ​​can be calculated according to formula (5).

[0132] MTW(%)=(MTW TG -MTW CG ) / MTW CG ×100……(5)

[0133] V=L×W 2 ……(6)

[0134] MCF-7 tumor-bearing nude mice were killed on the 25th day after tumor inoculation. Figure 14 The tumor inhibition effect of each group of drugs on MCF-7 tumor-bearing nude mice is shown. Figure 14 A shows that compared with the saline group, the tumor volume of nude mice in the BAT and FA-BAT-NPs groups was significantly reduced ( **** P < 0.0001), indicating that it has a good tumor inhibitory effect. Among them, the tumor inhibitory effect of the FA-BAT-NPs group is significantly stronger than that of the BAT group. Figure 14 C shows that the tumor sizes of the other drug groups changed to varying degrees compared with the normal saline group. Figure 14 B shows the difference in the average tumor weight of nude mice in each group. The tumor inhibition rates of TPP, Diboc, 5-ALA, BAT, BAT-NPs, FA-BAT-NPs and 5-FU were (11.33±2.61)%, (27.11±4.65)%, (49.62±4.95)%, (70.08±3.35)%, (61.75±5.03)%, (82.81±2.30)%, and (84.69±4.05)%. Compared with the Saline group, the tumor weights of the 5-ALA group and the BAT group were significantly decreased, with significant differences ( #### P < 0.0001), and the inhibition rate of tumor was significantly improved. Among them, the tumor weight of the BAT group was smaller than that of the 5-ALA group, and the inhibition rate was lower, which proved that the BAT synthesized in this experiment improved the breast cancer inhibition effect of 5-ALA. Compared with the tumor weight of the BAT group and the BAT-NPs group, the FA-BAT-NPs group had a significant reduction ( ****P < 0.0001). Comparison of the tumor inhibition rates of the three groups also demonstrated that the FA-BAT-NPs group significantly enhanced its tumor inhibition. There was no significant difference between the targeted drug delivery system FA-BAT-NPs group and the positive control group 5-FU. Both groups demonstrated good in vivo tumor inhibition, which can serve as a basis for further research on the efficacy of FA-BAT-NPs against chemoresistant tumors. These results demonstrate that the targeted drug delivery system FA-BAT-NPs has a good inhibitory effect on MCF-7 xenograft tumors.

[0135] In addition, tumor tissues were histopathologically analyzed. Tumor tissues were harvested from all nude mice after sacrifice. Tumor tissues were subjected to TUNEL analysis and H&E staining. To investigate whether FA-BAT-NPs could affect tumor cell proliferation, we also examined the tissue expression of Ki-67, CD31, and VEGFR in tumors.

[0136] We performed pathological analysis of tumor tissues in nude mice using H&E staining to further confirm the antitumor activity of FA-BAT-NPs. Figure 15 The results showed that varying degrees of tumor cell necrosis and apoptosis were observed in nude mouse tumors treated with 5-ALA, TPP, Diboc, BAT, 5-FU, BAT-NPs, and FA-BAT-NPs. Compared with the saline control group, the average apoptosis and necrosis rates of the cell populations in the FA-BAT-NPs group and the 5-FU positive control group were the highest. The trends in the average apoptosis and necrosis rates of tumors in all experimental groups were generally consistent with the aforementioned in vivo tumor inhibition results, demonstrating that the FA-BAT-NPs targeted drug delivery system can inhibit tumor growth in nude mice and induce apoptosis and necrosis of tumor cells. TUNEL analysis revealed that compared with the saline control group, significant apoptosis was observed in tumor tissues of nude mice treated with 5-ALA, BAT, BAT-NPs, and FA-BAT-NPs. The highest average apoptosis rates were observed in tumors treated with the FA-BAT-NPs group and the 5-FU positive control group, at 44.88% and 44.67%, respectively.

[0137] We detected the tissue expression levels of Ki-67, CD31, and VEGFR in the tumors of nude mice in each group to investigate whether FA-BAT-NPs could inhibit the proliferation of tumor cells. Figure 15As shown in the figure, compared with the saline control group, the immunohistochemical analysis results of the nude mouse tumor tissues in the 5-ALA, TPP, Diboc, BAT, 5-FU, BAT-NPs, and FA-BAT-NPs groups showed that the proportion of Ki-67, CD31, and VEGFR positive cell populations decreased to varying degrees, among which the target drug delivery system FA-BAT-NPs and the 5-FU positive control group showed the most significant decrease. This indicates that the 5-FU and FA-BAT-NPs preparations have the strongest inhibitory effect on nude mouse tumors and the best therapeutic effect.

[0138] 3. In vivo safety studies

[0139] To determine the potential in vivo toxicity of FA-BAT-NPs and guide in vivo antitumor pharmacodynamic analysis, Kunming white mice were used as the experimental animal model. After 7 days of adaptive feeding, the mice were randomly divided into nine groups of five animals each: saline, FA-NPs, 5-ALA, TPP, Diboc, BAT, 5-FU, BAT-NPs, and FA-BAT-NPs. All animals were administered via tail vein injection (25 mg / kg) and subsequently monitored daily. Toxicity and mortality were monitored daily throughout the experiment, and routine changes, including activity, urine, and feces, were recorded. On the 15th day of treatment, all mice were sacrificed, and blood was collected from the heart. The blood was centrifuged at 1,000 × g for 10 minutes, and serum was collected. Serum levels of aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatinine (Cr), and alanine aminotransferase (ALT) were measured in each experimental group using an automated biochemical analyzer.

[0140] 1. Mouse body weight and organ index

[0141] We examined the in vivo toxicity of FA-BAT-NPs in Kunming mice. First, saline, FA-NPs, 5-ALA, TPP, Diboc, BAT, 5-FU, BAT-NPs, and FA-BAT-NPs were injected via the tail vein. The mice were then observed daily. Throughout the experiment, except for the 5-FU group, no significant weight loss, toxicity symptoms, abnormal behavior, or mortality were observed in any of the other groups. Diet, activity, and hair color remained normal in all mice.

[0142] Table 3. Survival rate and weight changes of mice

[0143]

[0144] Results were presented as mean±SD (n=3). *P<0.05, **** P<0.0001

[0145] As shown in Table 3, compared with the normal saline control group, the body weight of mice treated with 5-FU decreased by an average of 2.65%. **** P < 0.0001), the average body weight of the Diboc-treated group decreased slightly, and the body weights of the mice in the remaining 7 groups increased to varying degrees. The body weight increase on the 15th day was in the range of 0.4%-9%. There was no significant difference in the body weights of the mice in the remaining 8 groups ( * P>0.05). The above results showed that except for 5-FU, other drug groups had almost no toxicity to normal mice.

[0146] In addition, the heart, liver, lung, and kidney of the mice were immediately collected after being sacrificed, washed with saline, and then fixed with formalin (10%), weighed, and finally subjected to H&E pathological analysis. The visceral index was evaluated using formula (7):

[0147] Organ Index (%)=Organ Weight / Body Weight×100%......(7)

[0148] All experimental results were compared and analyzed using GraphPad Prism 8 software. Enumeration data are expressed as numbers or percentages, and measurement data are presented as means with standard deviations. Differences in mean values ​​between treatment groups were analyzed using one-way or two-way ANOVA for multiple comparisons. P < 0.05 was considered statistically significant.

[0149] The organ indices of mice in each group are shown in Table 4. It can be seen that the organ indices of the heart, liver, lung, and kidney after BAT and FA-BAT-NPs treatment did not change significantly compared with the saline group (P>0.05). However, the liver index of mice treated with 5-FU increased significantly (9.25±0.773)%, compared with the saline group (7.46±0.304)%, and the difference was statistically significant. Compared with 5-FU, FA-BAT-NPs treatment had no toxic effect on mice. These results indicate that BAT and FA-BAT-NPs have no toxic effect on mice, while the liver index of mice treated with 5-FU increased significantly compared with the saline group (P<0.05), indicating that 5-FU may be toxic to mice.

[0150] Table 4. Organ index of mice in each group

[0151]

[0152] Results were presented as mean±SD (n=3).**** P<0.0001

[0153] 2. Serum biochemical analysis

[0154] We also performed serum biochemical analysis on mice in each group, including AST, ALT, Cr, and BUN, to examine the potential toxic effects of BAT and FA-BAT-NPs on the liver and kidneys. The results are shown in Table 5. With the exception of the 5-FU group, all four serum biochemical parameters were within normal levels in the mice treated with the other groups, with no significant differences observed. AST and ALT in the 5-FU group were significantly higher than those in the saline group (P < 0.05), while Cr and BUN in the other groups were normal and showed no significant differences compared to the saline group (P > 0.05). These results indicate that all preparations had little to no toxicity to the mice's kidneys.

[0155] Table 5. Serum biochemical parameters of mice in each group

[0156]

[0157] Results were presented as mean±SD (n=3). **** P<0.0001

[0158] 3. H&E staining of organs

[0159] like Figure 16 As shown in the figure, the histological changes of the heart, liver, lung, and kidney collected 14 days after drug administration. Light microscopy showed that some liver tissues and hepatocytes of the experimental animals showed slight or mild vacuolar degeneration, which may be due to insufficient fasting of the animals during autopsy and excessive glycogen content in the liver. The lung tissues of the Saline, 5-FU, BAT-NPs, and FA-BAT-NPs-treated groups showed mild or moderate bleeding, which may be due to insufficient blood removal during autopsy and is considered to be unrelated to the drugs used in this experiment. In summary, there was basically no pathological damage related to the drug treatment in this experiment in the heart, liver, lung, and kidneys of all drug-treated groups, which further proves the low toxicity of FA-BAT-NPs.

[0160] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0161] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A method for preparing a photosensitizer BAT, characterized in that: The method comprises: using 5-ALA as a raw material to introduce a Boc group through an amide reaction to obtain Boc-ALA; then coupling the Boc-ALA with TPP through an amide reaction to obtain Boc-ALA-TPP, referred to as BAT; wherein the 5-ALA is 5-aminolevulinic acid and the TPP is triphenylphosphine. The method specifically comprises: 5-ALA and di-tert-butyl dicarbonate are subjected to amide reaction in methanol, and after the reaction is complete, Boc-ALA is obtained by post-treatment; TPP and 3-bromopropylamine hydrobromide are refluxed in anhydrous acetonitrile, and after the reaction is complete, TPP-NH2 is obtained by post-treatment; The Boc-ALA, TPP-NH2, N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 4-dimethylaminopyridine are subjected to a coupling reaction in dichloromethane to obtain BAT.

2. The method according to claim 1, characterized in that The molar ratio of the 5-ALA and di-tert-butyl dicarbonate is 1:1-6; the molar ratio of the TPP and 3-bromopropylamine hydrobromide is 1:1-3; the molar ratio of N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, Boc-ALA, TPP-NH2, and 4-dimethylaminopyridine is 5:1:1:3:0.

036.

3. A photosensitizer BAT, characterized in that The photosensitizer BAT is also known as Boc-ALA-TPP, and has the following structural formula:

4. A targeted photosensitizer FA-BAT-NPs, characterized in that: The targeted photosensitizer FA-BAT-NPs is a core-shell structure, wherein the core-shell structure is formed by an outer shell wrapped around an inner core, the inner core is the photosensitizer BAT according to claim 3, and the outer shell is a folic acid-modified BSA carrier, wherein folic acid and BSA in the folic acid-modified BSA carrier are bridged by PEG.

5. The targeted photosensitizer FA-BAT-NPs according to claim 4, characterized in that: Calculated by mass fraction, the targeted photosensitizer FA-BAT-NPs includes: 1-3% photosensitizer BAT, 97-99% folic acid-modified BSA carrier; wherein the folic acid-modified BSA carrier includes 10-13% BSA carrier and 86-87% PEG-folic acid.

6. A method for preparing the targeted photosensitizer FA-BAT-NPs according to claim 4 or 5, characterized in that: The method comprises: The BAT is subjected to an emulsification dispersion method to obtain BAT-NPs nanoparticles; Using PEG as a connecting chain, FA was modified on the surface of the BAT-NPs nanoparticles through an amide reaction to obtain the targeted photosensitizer FA-BAT-NPs.

7. The method according to claim 6, characterized in that The BAT is obtained by an emulsification dispersion method to obtain BAT-NPs nanoparticles, comprising: Phosphatidylcholine, cholesterol and BAT were dissolved in chloroform to form a drug-loaded oil phase; BSA was dissolved in ultrapure water to obtain an aqueous phase; mixing the oil phase and the water phase and crushing them by ultrasonication to obtain an emulsion; The organic solvent is removed from the emulsion by rotary evaporation under reduced pressure to obtain BAT-NPs nanoparticles.

8. The method according to claim 6, characterized in that The method uses PEG as a connecting chain to modify FA on the surface of the BAT-NPs nanoparticles through an amide reaction to obtain the targeted photosensitizer FA-BAT-NPs, specifically comprising: The BAT-NPs nanoparticles were dissolved in water, NHS and EDC were added and stirred evenly, and then FA-PEG-NH2 was added for reaction to obtain the targeted photosensitizer FA-BAT-NPs.

9. Use of the photosensitizer BAT according to claim 3 or the targeted photosensitizer FA-BAT-NPs according to claim 4 or 5 in the preparation of anti-breast cancer drugs.

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