Nanoparticle loaded with photosensitizer ce6 and targeting mitochondria of breast cancer cells, and preparation method therefor and use thereof

Abstract

The present invention provides a nanoparticle loaded with a photosensitizer Ce6 and targeting mitochondria of breast cancer cells, and a preparation method therefor and a use thereof. The nanoparticle uses hollow mesoporous silica as a carrier; a photosensitizer Ce6 is loaded at the hollow position of the carrier; a red cell membrane is encapsulated outside the carrier; and one side of the carrier after being encapsulated by the red cell membrane is modified with a Pt nanocluster, and the other side of the carrier is modified with an LXL-1 aptamer and TPP. The present invention also provides a preparation method for the nanoparticle and a use of the nanoparticle in preparation of a drug for treating breast cancer.

Description

Nanoparticles loaded with photosensitizer Ce6 targeting breast cancer cell mitochondria, preparation method, and application thereof

[0001] This application claims priority to the Chinese patent application filed with the Patent Office of China on December 12, 2023, with application number 202311714847.1 and invention name “A nanoparticle loaded with photosensitizer Ce6 targeting breast cancer cell mitochondria and its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to nanoparticles loaded with photosensitizer Ce6 and targeting breast cancer cell mitochondria, as well as a preparation method and application thereof. Background Art

[0003] Breast cancer is a type of malignant tumor that develops in breast tissue and continues to grow indefinitely due to genetic or hormonal imbalances. It is the most common cancer in women and primarily includes ductal carcinoma and lobular carcinoma. Treatment for breast cancer primarily includes surgical resection, chemotherapy, radiotherapy, or a combination of these. However, traditional chemotherapy and radiotherapy lack tumor cell targeting, leading to serious side effects.

[0004] Aptamers are single-stranded oligonucleotides screened from synthetic single-stranded DNA / RNA libraries that can bind to target molecules with high affinity and specificity. Aptamers utilize intermolecular forces such as hydrogen bonds, van der Waals forces, and hydrophobic interactions to form unique three-dimensional structures, such as hairpins, pseudoknots, convex loops, and G-tetramers, enabling them to specifically recognize target substances and influence their biological activity. Aptamer probes developed in recent years, particularly cell-targeted aptamers, can specifically identify cancer cells in complex samples, including whole blood, offering new hope and solutions for early, specific, and rapid diagnosis and targeted treatment of tumors. Nanoparticle drug delivery systems can improve the delivery efficiency of anticancer drugs and reduce drug side effects. Their primary purpose is to prevent non-targeted tissues or areas from being exposed to therapeutic drugs, or to prevent damage to targeted tissues caused by high doses of specific drugs.

[0005] Chlorin e6 (Ce6) is a widely used photosensitizer with a molecular weight of 596.67KD and a molecular formula of C 34 H 36N4O6 is a member of the chlorophyll family. Ce6 is an amphiphilic (water-soluble and lipid-soluble) molecule with absorption peaks around 400nm and 660nm. Compared with some traditional photosensitizers such as photoporphyrin, the 660nm laser has stronger tissue penetration. On the other hand, Ce6 has a high singlet oxygen quantum production rate and has a relatively good application in photodynamic therapy. However, due to its amphiphilicity, it is difficult to encapsulate it using common nanoparticle preparation methods to make stable nanoparticles. For example, it is difficult to encapsulate Ce6 using common carrier albumin methods. Therefore, how to prepare a nanoparticle drug delivery system encapsulated with Ce6 and combine it with a targeted nucleic acid aptamer is one of the key research issues in this field.

[0006] Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide a nanoparticle loaded with photosensitizer Ce6 targeting breast cancer cell mitochondria, which can target the nanoparticles into the mitochondria of MDA-MB-231 breast cancer cells and produce 1 O2 damages mitochondria and releases DNA, causing cell death.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a nanoparticle loaded with photosensitizer Ce6 and targeting breast cancer cell mitochondria. The nanoparticle uses hollow mesoporous silica as a carrier, the photosensitizer Ce6 is loaded at the hollow position of the carrier, the red blood cell membrane is encapsulated on the outside of the carrier, one side of the carrier after encapsulating the red blood cell membrane is modified with Pt nanoclusters, and the other side of the carrier is modified with LXL-1 aptamer and TPP.

[0010] Preferably, the erythrocyte membrane is a mouse erythrocyte membrane.

[0011] Preferably, the nucleotide sequence of the LXL-1 aptamer is shown in SEQ ID NO: 1.

[0012] Preferably, the 5' end of the LXL-1 aptamer is modified with a carboxyl group.

[0013] The present invention also provides a method for preparing the above-mentioned nanoparticles, comprising the following steps:

[0014] (1) Hollow mesoporous silica and Ce6 in dimethyl sulfoxide solution were ultrasonically mixed and freeze-dried to obtain hollow mesoporous silica loaded with Ce6 nanoparticles HC;

[0015] (2) HC nanoparticles in PBS solution were mixed with red blood cell membrane powder in PBS solution, and squeezed through filter membrane to obtain red blood cell membrane-coated HC nanoparticles RHC;

[0016] (3) RHC nanoparticles were dispersed with PBS solution, centrifuged, and precipitated onto the poly-L-lysine surface, incubated at room temperature, washed with PBS solution, and the supernatant was removed; then, a positively charged Pt nanocluster solution was added, incubated at room temperature, washed with PBS solution, and the supernatant was removed to obtain asymmetrically modified nanoparticles Pt@RHC;

[0017] (4) Pt@RHC nanoparticles were dispersed in a PBS solution containing LXL-1, TPP-COOH, EDC, and NHS, stirred at room temperature, centrifuged, and the precipitate was washed with PBS solution and redispersed in PBS solution to obtain modified nanoparticles Pt@RHC-LXL-1 / TPP.

[0018] Preferably, in step (1), the preparation method of hollow mesoporous silica comprises the following steps: solid silica is added to a high-purity aqueous solution of CTAC and triethanolamine to obtain a mixed solution, TEOS solution is added to the mixed solution and stirred in a water bath to form silica nanoparticles coated on the silica surface, and then etching to obtain hollow mesoporous silica.

[0019] Preferably, the solid silica is prepared by mixing anhydrous ethanol, water and ammonia water, stirring at room temperature, and then adding TEOS solution.

[0020] Preferably, the volume ratio of the anhydrous ethanol, water, ammonia water and TEOS solution is 35-36:4-6:1-1.2:1, and the mass fraction of the TEOS solution is 98%.

[0021] Preferably, the stirring time at room temperature is 5 to 10 minutes, and the reaction time is 50 to 70 minutes.

[0022] Preferably, in every 30 mL of high-purity water, the mass ratio of CTAC, triethanolamine, and solid silica is 2 g:20 mg:100 mg; the volume ratio of the mixed solution to the TEOS solution is 30:0.15, and the mass fraction of the TEOS solution is 98%.

[0023] Preferably, the water bath stirring temperature is 75-85° C., and the stirring time is 50-70 min.

[0024] Preferably, the etching method comprises the following steps: cooling the nanoparticles in a water bath to 45-50° C., adding Na 2 CO 3 , and continuously stirring to obtain hollow mesoporous silica.

[0025] Preferably, the prepared hollow mesoporous silica is washed with a mixed cleaning solution, wherein the mixed cleaning solution is obtained by mixing anhydrous ethanol and concentrated hydrochloric acid in a volume ratio of 1:10.

[0026] Preferably, in step (1), the mass ratio of hollow mesoporous silica to Ce6 is 4-5:0.1-0.2, the ultrasonic mixing time is 8-12 min, and the ultrasonic frequency is 40 KHz.

[0027] Preferably, the nanoparticles HC are washed with ultrapure water.

[0028] Preferably, in step (2), the mass ratio of HC nanoparticles to red blood cell membrane powder is 1:1.

[0029] Preferably, the method for preparing the red blood cell membrane powder comprises the following steps: adding water to the red blood cell suspension, allowing the suspension to stand, allowing the red blood cells to absorb water and burst, centrifuging to remove hemoglobin, washing and dispersing the precipitate with PBS solution, and freeze-drying to obtain the red blood cell membrane powder.

[0030] Preferably, the centrifugation condition is 14000 rpm for 10 min.

[0031] Preferably, the filter membrane extrusion method in step (2) is: extrusion is performed using 450nm and 200nm filter membranes in sequence, with each extrusion being performed 4 to 5 times.

[0032] Preferably, in step (3), the centrifugation condition is 900r, 3min; and the incubation time is 50 to 70min.

[0033] Preferably, the preparation method of the Pt nanocluster solution comprises the following steps: adding an H2PtCl6 aqueous solution to ultrapure water, mixing the protamine aqueous solution under vigorous stirring at room temperature, adding NaBH4 dropwise after 30 minutes, continuously stirring the mixture at room temperature, and dialyzing for 24 hours to obtain a positively charged Pt nanocluster solution.

[0034] Preferably, the concentration of the H2PtCl6 aqueous solution is 16 mM, the concentration of the protamine aqueous solution is 5 mg / mL, and the concentration of NaBH4 is 500 mM; the volume ratio of the H2PtCl6 aqueous solution, ultrapure water, protamine aqueous solution, and NaBH4 is 0.8:40:0.5:0.4.

[0035] Preferably, in step (3), the centrifugation conditions are 900r, 3min; and the incubation time is 50 to 70min.

[0036] Preferably, in step (4), the concentration of TPP-COOH is 0.05-0.15 g / L, the concentration of EDC is 0.7-0.9 g / L, the concentration of NHS is 0.3-0.5 g / L, and the concentration of LXL-1 is 100 nM.

[0037] Preferably, in step (4), the reaction time is 12 to 18 minutes; the stirring time is 11 to 13 hours; and the centrifugation condition is 12000r for 15 minutes.

[0038] The present invention also provides the use of the nanoparticles or the preparation method of the nanoparticles in preparing drugs for treating breast cancer.

[0039] The present invention also provides the use of the nanoparticles or the preparation method of the nanoparticles in treating breast cancer, wherein the nanoparticles are used in combination with 808 nm laser.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The nanoparticles of the present invention can achieve the special targeting of MDA-MB-231 breast cancer cells by LXL-1 aptamers, allowing the nanoparticles to enter the cells smoothly. The Pt nanoclusters are used as the power device of the nanoparticles to overcome the Brownian force in the environment and catalyze H2O2 to produce oxygen in the tumor tissue environment, thereby alleviating the hypoxic environment of the tumor cells. The TPP is used to target mitochondria. Under the action of light conditions and intracellular mechanisms, the photosensitizer Ce6 loaded at the hollow position of the carrier is leaked into the mitochondria and cytoplasm, producing oxygen under light conditions. 1 O2 damages mitochondria and releases DNA into the cytoplasm, causing cell damage and ultimately cell death. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1: TEM image and DLS data of solid silicon dioxide (dSiO2), A is the TEM image, B is the DLS data;

[0043] Figure 2: TEM image and DLS data of hollow mesoporous silica (HMSN), A is TEM image, B is DLS data;

[0044] Figure 3: Nitrogen adsorption / desorption curves and pore size distribution of hollow mesoporous silica (HMSN). A is the nitrogen adsorption / desorption curve, and B is the pore size distribution.

[0045] Figure 4: TEM image and DLS data of positively charged Pt nanoclusters (PtNPs), A is TEM image, B is DLS data;

[0046] Figure 5: Zeta potential of HMSN, HC, red blood cell membrane (RBCM), PtNPs, RHC, Pt@RHC, LXL-1, and Pt@RHC-LXL-1 / TPP;

[0047] Figure 6: UV absorption peak and fluorescence spectrum intensity of nanoparticles. A is the UV absorption curve of HMSN, Ce6 and HC, and B is the fluorescence intensity of Ce6 and HC.

[0048] Figure 7: Pt@RHC-LXL-1 / TPP produced under light conditions 1 O2 capacity, A is the fluorescence curve over time after laser irradiation, B is the UV absorption curve over time after laser irradiation, C is the UV absorption curve after incubation with different H2O2 concentrations and laser irradiation for 10 min, D is the UV absorption curve after incubation with different concentrations of Pt@RHC-LXL-1 / TPP nanoparticles and laser irradiation for 10 min;

[0049] Figure 8: Effects of nanoparticles on the survival rate of MDA-MB-231 cells. A to E are the cell survival rates of MDA-MB-231 cells co-incubated with HMSN, Pt@RHC, Pt@RHC-LXL-1, Pt@RHC-LXL-1 / TPP, and Pt@RH-LXL-1 / TPP, respectively, after illumination. F is the cell survival rate of MDA-MB-231 cells co-incubated with Pt@RHC-LXL-1 / TPP without illumination.

[0050] Figure 9: Targeting results of Pt@RH-LXL-1 / TPP nanoparticles under fluorescence microscopy. A is a fluorescence image of MCF-7 breast cancer cells co-incubated with Pt@RH-LXL-1 / TPP nanoparticles for 1 h, 2 h, and 3 h, respectively; B is a fluorescence image of MDA-MB-231 breast cancer cells co-incubated with Pt@RH-LXL-1 / TPP nanoparticles for 1 h, 2 h, and 3 h, respectively. Scale bars are 50 μm.

[0051] Figure 10: After co-incubation of different nanoparticles with MDA-MB-231 breast cancer cells, the cells produced 1 For O2, the scale bar is 20 μm. DETAILED DESCRIPTION

[0052] The present invention provides a nanoparticle (Pt@RHC-LXL-1 / TPP) loaded with the photosensitizer Ce6, targeting the mitochondria of breast cancer cells. The nanoparticle uses a hollow mesoporous silica carrier, with the photosensitizer Ce6 loaded in the hollow portion of the carrier. The carrier encapsulates the red blood cell membrane. One side of the carrier after encapsulation is modified with Pt nanoclusters, and the other side is modified with LXL-1 aptamers and TPP. The breast cancer cells are preferably MDA-MB-231 breast cancer cells.

[0053] The present invention uses red blood cell membranes to encapsulate a vector loaded with the photosensitizer Ce6, which can increase the vector's survival time in the body and prevent it from being phagocytosed by immune cells after entering the body. The present invention uses Pt nanoclusters to asymmetrically modify nanoparticles, acting as their power source to overcome the Brownian forces in the environment. Simultaneously, the other side of the nanoparticle is modified with an LXL-1 aptamer specifically targeting MDA-MB-231 breast cancer cells and a TPP targeting mitochondria, ultimately forming the nanoparticle Pt@RHC-LXL-1 / TPP. This enhances the nanoparticle's targeting of MDA-MB-231 breast cancer cells and increases the probability of the nanoparticle entering MDA-MB-231 cells.

[0054] Since the concentration of H2O2 in tumor cells is higher than that in normal cells, the Pt nanoclusters in the present invention can catalyze H2O2 to produce oxygen in the tumor tissue environment, thereby alleviating the hypoxic environment of tumor cells. After entering the cells, TPP targets the mitochondria of cancer cells and, under the action of light conditions and intracellular mechanisms, leaks the photosensitizer Ce6 loaded in the hollow position of the carrier into the mitochondria and cytoplasm. 1 O2 damages mitochondria and releases DNA into the cytoplasm, causing cell damage and ultimately cell death.

[0055] The nucleotide sequence of the LXL-1 aptamer of the present invention is 5'-GAATTCAGTCGGACAGCGAAGTAGTTTTCCTTCTAACCTAAGAACCCGCGGCAGTTTAATGTAGATGGACGAATACGTCTCCC-3', as shown in SEQ ID NO: 1. The 5' end of the LXL-1 aptamer is modified with a carboxyl group (-COOH).

[0056] The present invention also provides a method for preparing the above-mentioned nanoparticles, comprising the following steps:

[0057] (1) Hollow mesoporous silica and Ce6 in dimethyl sulfoxide solution were ultrasonically mixed and freeze-dried to obtain hollow mesoporous silica loaded with Ce6 nanoparticles HC;

[0058] (2) PBS solution of HC nanoparticles is mixed with PBS solution of red blood cell membranes, and then squeezed through a filter membrane to obtain HC nanoparticles coated with red blood cell membranes (RHC);

[0059] (3) RHC nanoparticles were dispersed with PBS solution, centrifuged, and precipitated onto the poly-L-lysine surface, incubated at room temperature, washed with PBS solution, and the supernatant was removed; then, a positively charged Pt nanocluster solution was added, incubated at room temperature, washed with PBS solution, and the supernatant was removed to obtain asymmetrically modified nanoparticles Pt@RHC;

[0060] (4) Pt@RHC nanoparticles were dispersed in a PBS solution containing LXL-1, TPP-COOH, EDC, and NHS, stirred at room temperature, centrifuged, and the precipitate was washed with PBS solution and redispersed in PBS solution to obtain modified nanoparticles Pt@RHC-LXL-1 / TPP.

[0061] In step (1), the mass ratio of hollow mesoporous silica to Ce6 is 4-5:0.1-0.2, preferably 5:0.133; the ultrasonic mixing time is 8-12 minutes, preferably 9-11 minutes, and more preferably 10 minutes; and the ultrasonic frequency is preferably 40 kHz. Preferably, the freeze-drying step is followed by washing with ultrapure water to obtain high-purity nanoparticles of HC.

[0062] As an optional embodiment, the preparation method of hollow mesoporous silica used in the present invention is as follows: 1. Mix anhydrous ethanol, water and ammonia water, stir at room temperature, then add TEOS solution, and react the mixture at room temperature to obtain solid silica (dSiO2). 2. Dissolve CTAC and triethanolamine in high-purity water, stir at room temperature, add dSiO2 aqueous solution, stir at room temperature, then add TEOS, and stir the mixture in a water bath to form silica coated with silica surface (dSiO2@MSN). 3. Cool the dSiO2@MSN in a water bath, then add Na2CO3 and continue stirring to form hollow mesoporous silica (HMSN).

[0063] In step 1, the volume ratio of anhydrous ethanol, water, ammonia, and TEOS solution is preferably 35-36:4-6:1-1.2:1; the stirring time at room temperature is 5-10 minutes; the mass fraction of the TEOS solution is 98%; and the reaction time at room temperature is 50-70 minutes. In step 2, the mass ratio of CTAC, triethanolamine, and dSiO2 is 2g:20mg:100mg, the volume of TEOS is 0.15mL, the water bath temperature is 75-85°C, and the stirring time is 50-70 minutes. In step 3, the solution is cooled to 45-50°C, and the HMSN is washed multiple times with a mixed cleaning solution of anhydrous ethanol and concentrated hydrochloric acid (volume ratio of 1:10) to remove CTAC attached to the HMSN surface.

[0064] In step (2) above, the mass ratio of the HC nanoparticles to the red blood cell membrane powder is 1:1; preferably, the volume ratio of the PBS solution of the HC nanoparticles to the PBS solution of the red blood cell membranes is 1:1. As an optional embodiment, the red blood cell membrane powder is prepared by adding water to the red blood cell suspension and allowing it to stand, allowing the red blood cells to absorb water and rupture, centrifuging to remove hemoglobin, washing and dispersing the precipitate with a PBS solution, and freeze-drying to obtain the red blood cell membrane powder; the centrifugation conditions are preferably 14,000 rpm for 10 minutes.

[0065] The filter membrane extrusion method in the above step (2) is: extrusion is performed using 450nm and 200nm filter membranes in sequence, each extrusion is performed 4 to 5 times.

[0066] In the above step (3), the centrifugation condition is 900r for 3 minutes; the incubation time at room temperature is 50 to 70 minutes, preferably 60 minutes. The method for rotating the RHC nanoparticles onto the poly-L-lysine surface is as follows: a solution of poly-L-lysine (PLL) with a concentration of 0.01% is evenly applied to the bottom surface of a 12-well plate at 50 microliters per square centimeter, and after standing at room temperature for 5 minutes, the excess solution is aspirated to obtain a 12-well plate coated with PLL. The PBS suspension of RHC nanoparticles is added to the 12-well plate coated with PLL, and centrifuged at 900r for 3 minutes. Under the action of centrifugal force, the nanoparticles are embedded in the PLL on the surface of the 12-well plate. After removing the supernatant, a positively charged Pt nanocluster solution is added to the plate. The concentration of the positively charged Pt nanocluster solution is preferably 20 mg / mL. The mass ratio of nanoparticles to Pt nanoclusters in the final solution is 4 to 6:1, preferably 5:1.

[0067] As an optional embodiment, the positively charged Pt nanoclusters are prepared by adding an aqueous solution of H2PtCl6 to ultrapure water, then mixing the protamine aqueous solution under vigorous stirring at room temperature. After 30 minutes, NaBH4 is added dropwise over 30 seconds to reduce the H2PtCl6. Subsequently, the mixture is continuously stirred at room temperature and dialyzed for 24 hours, and the crude product is purified to obtain PtNPs. Preferably, the concentration of the aqueous solution of H2PtCl6 is 16mM, the concentration of the aqueous solution of protamine is 5mg / mL, and the concentration of NaBH4 is 500mM; the volume ratio of the aqueous solution of H2PtCl6, ultrapure water, aqueous solution of protamine, and NaBH4 is 0.8:40:0.5:0.4.

[0068] In step (4), the concentration of TPP-COOH is 0.05-0.15 g / L, preferably 0.1 g / L; the concentration of EDC is 0.7-0.9 g / L, preferably 0.8 g / L; the concentration of NHS is 0.3-0.5 g / L, preferably 0.4 g / L; and the concentration of LXL-1 is 100 nM. The mass volume ratio of the Pt@RHC nanoparticles to the PBS solution containing LXL-1, TPP-COOH, EDC, and NHS is 4-6 mg:1 mL, preferably 5 mg:1 mL.

[0069] In the above step (4), the reaction time is 12 to 18 minutes, preferably 15 minutes; the stirring time is 11 to 13 hours, preferably 12 hours; and the centrifugation condition is 12000r, 15 minutes.

[0070] The present invention also provides the use of the nanoparticles or the preparation method of the nanoparticles in preparing drugs for treating breast cancer.

[0071] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0072] In the specific examples of the present invention, 1-carboxypropyltriphenylphosphonium bromide (TPP), 1,3-diphenylisobenzofuran (DPBF), coumarin, chlorin e6 (Ce6), and dimethyl sulfoxide (DMSO) were purchased from Macklin. Dialysis tubing and poly-L-lysine (PLL) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. LXL-1 aptamer, protamine, cetyltrimethylammonium chloride (CTAC), sodium N-hydroxysulfosuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), triethanolamine (TEA), and MTT cell proliferation and cytotoxicity assay kits were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. Chloroplatinic acid (HPtCl), sodium carbonate, tetraethyl orthosilicate (TEOS), sodium borohydride (NaBH), anhydrous ethanol, and ammonia solution were purchased from Sinopharm. 2% normal mouse erythrocytes were purchased from Shanghai Yuchun Biotechnology Co., Ltd. DAPI, reactive oxygen species detection kit, and red blood cell membrane far-infrared fluorescent probe (DID) were purchased from Beyotime. Fetal bovine serum was purchased from Solebol.

[0073] In a specific embodiment of the present invention, MDA-MB-231 breast cancer cells were purchased from Wuhan Punosai Life Science Co., Ltd. and cultured in a constant temperature and humidity chamber at 37°C, with a humidity of 95% and a CO2 concentration of 5%. The culture medium used for the cells was DMEM containing 10% fetal bovine serum and 1% double antibiotic (penicillin-streptomycin).

[0074] In the following examples, unless otherwise specified, all methods are conventional.

[0075] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0076] Example 1

[0077] 1. Preparation of Pt@RHC-LXL-1 / TPP nanoparticles:

[0078] In the first step, 35.7 mL of anhydrous ethanol was mixed with 5 mL of water and 0.8 mL of ammonia and stirred at room temperature for 5 to 10 minutes. Then, 1 mL of a 98% TEOS solution was added and the mixture was allowed to react at room temperature for 1 hour to produce solid silicon dioxide (dSiO2).

[0079] In the second step, the dSiO2 nanoparticles were washed three times with water and then ethanol, and 200 mg of dSiO2 nanoparticles were suspended in 20 mL of water. CTAC (2 g) and triethanolamine (20 mg) were dissolved in 20 mL of high-purity water and stirred at room temperature for 1 hour. Then, 10 mL of the dSiO2 aqueous solution was added, stirred at room temperature for 1 hour, and then 0.15 mL of LTEOS was added. The mixture was stirred in an 80°C water bath for 1 hour to form silica-coated silica, i.e., dSiO2@MSN.

[0080] The third step was to etch the dSiO2@MSNs to form hollow mesoporous silica (HMSNs). The mixture obtained in the second step was cooled to 50°C in a water bath, and then 636 mg of Na2CO3 was added. The mixture was stirred for 30 minutes to form HMSNs. To remove the CTAC attached to the HMSN surface, the HMSNs were washed multiple times with a mixture of anhydrous ethanol and concentrated hydrochloric acid (1:10 by volume).

[0081] Step 4: Ce6 photosensitizer encapsulated in hollow mesoporous silica. 133 μL of 1.0 mg / mL Ce6 dissolved in dimethyl sulfoxide was added to 5 mg of hollow mesoporous silica. The mixture was ultrasonically mixed (frequency 40 kHz) for 10 minutes, freeze-dried, and washed with ultrapure water to obtain hollow mesoporous silica-encapsulated Ce6, i.e., HC.

[0082] Step 5: Prepare the erythrocyte membrane: Add 5 volumes of deionized water to a 2% mouse erythrocyte suspension, mix thoroughly, and incubate at 4°C for 1 hour to allow the erythrocytes to absorb water and rupture. Then, centrifuge at 14,000 rpm for 10 minutes to remove hemoglobin from the suspension. Discard the supernatant, and wash the precipitate three times with PBS. The resulting pale pink erythrocyte membrane precipitate is dispersed with PBS, stored at -20°C, and freeze-dried to obtain an erythrocyte membrane powder. Store at -20°C for subsequent experiments.

[0083] Step 6: Coating of HC with red blood cell membranes: 0.5 mL of HC in PBS (2 mg / mL) was mixed with 0.5 mL of red blood cell membrane in PBS (2 mg / mL), and the mixture was squeezed through 450 nm and 200 nm filters, respectively, with each squeeze repeated 5 times to obtain red blood cell membrane-coated HC, i.e., RHC.

[0084] Step 7: Preparation of positively charged Pt nanoclusters: An aqueous solution of HPtCl (800 μL, 16 mM) was added to 40 mL of ultrapure water, followed by a protamine aqueous solution (500 μL, 5 mg / mL) under vigorous stirring at room temperature. After 30 minutes, NaBH (400 μL, 500 mM) was added dropwise over 30 seconds to reduce the HPtCl. The mixture was then stirred at room temperature for 2 hours, and the crude product was finally purified by dialysis for 24 hours to yield PtNPs.

[0085] Step 8: Asymmetrically modify RHC with PtNPs: A 0.01% polylysine (PLL) solution was applied evenly to the bottom of a 12-well plate at 50 μL per square centimeter. After standing at room temperature for 5 minutes, the excess solution was aspirated to obtain a PLL-coated 12-well plate. A PBS suspension of RHC nanoparticles (2 mL, 20 mg / mL) was added to the PLL-coated 12-well plate and centrifuged at 900 r / min for 3 minutes. The nanoparticles were embedded in the PLL on the surface of the 12-well plate under the action of centrifugal force. The plate was incubated at room temperature for 1 hour, the supernatant was removed, and the plate was washed three times with PBS to remove unattached nanoparticles. After rinsing with PBS, the PBS solution was removed, and 400 μL of a 20 mg / mL Pt nanocluster solution was added to the wells and incubated for 1 hour. The plate was then washed with PBS to obtain asymmetrically modified nanoparticles, Pt@RHC.

[0086] In the ninth step, the nanoparticles were modified with the aptamer LXL-1 and TPP: 50 mg of Pt@RHC nanoparticles were dispersed in 10 mL of PBS solution containing LXL-1 (100 nM), TPP-COOH (0.1 g / L), EDC (0.8 g / L) and NHS (0.4 g / L). The mixture was stirred at room temperature for 12 h and centrifuged (12000 r, 15 min). The obtained nanoparticles were washed with PBS and redispersed in PBS to obtain Pt@RHC-LXL-1 / TPP.

[0087] 2. Preparation of HMSN nanoparticles:

[0088] The preparation method is the same as the first to third steps in the preparation of Pt@RHC-LXL-1 / TPP nanoparticles.

[0089] 3. Preparation of Pt@RHC nanoparticles:

[0090] The preparation method is the same as the first to eighth steps in the preparation of Pt@RHC-LXL-1 / TPP nanoparticles.

[0091] 4. Preparation of Pt@RHC-LXL-1 nanoparticles:

[0092] The preparation method from the first step to the eighth step is the same as the preparation of Pt@RHC-LXL-1 / TPP nanoparticles.

[0093] In the ninth step, the nanoparticles were modified with the aptamer LXL-1 and TPP: 50 mg of Pt@RHC nanoparticles were added to 10 mL of PBS solution containing LXL-1 (100 μL, 100 nM). The mixture was stirred at room temperature for 12 h and centrifuged (12000 r, 15 min). The obtained nanoparticles were washed with PBS and redispersed in PBS to obtain Pt@RHC-LXL-1.

[0094] 5. Preparation of Pt@RH-LXL-1 / TPP nanoparticles:

[0095] Compared with the preparation of Pt@RHC-LXL-1 / TPP nanoparticles, the fourth step (no coating of photosensitizer Ce6) was discarded and the subsequent HC was replaced by HMSN.

[0096] Example 2

[0097] The nanoparticles prepared in Example 1 were tested:

[0098] 1. Characterization of nanomaterials

[0099] Particle size and zeta potential were measured at 25°C using a laser particle size analyzer (Zetasizer Nano ZS90). A Nano-ZS90 zeta potential analyzer (Malvern Instrument Ltd., UK) was used to measure the particle size of the nanomaterials. Material morphology was characterized using a transmission electron microscope (JEM2100). UV-visible spectra were recorded on a Cary 60 UV-vis-NIR spectrophotometer.

[0100] Figure 1 shows the TEM image (A) and DLS data (B) of solid silica (dSiO2). In Figure 1, the TEM data and EDS data show that the synthesized silica has a well-defined spherical structure with a diameter of approximately 100 nm and good dispersion.

[0101] Figure 2 shows the TEM image (A) and DLS data (B) of hollow mesoporous silica (HMSN). In Figure 2, the TEM data and EDS data show that the hollow mesoporous silica has a well-defined spherical structure with a diameter of approximately 150 nm.

[0102] Figure 3 shows the nitrogen adsorption / desorption curve (A) and pore size distribution (B) of hollow mesoporous silica (HMSN). In Figure 3, the nitrogen adsorption / desorption data show that the pore size of hollow mesoporous silica is approximately 3 nm.

[0103] Figure 4 shows the TEM image (A) and DLS data (B) of positively charged Pt nanoclusters (PtNPs). The data in Figure 4 show that the particle size of the PtNPs is approximately 25 nm and they have good dispersion.

[0104] Figure 5 shows the zeta potentials of HMSN, HC, red blood cell membrane (RBCM), PtNPs, RHC, Pt@RHC, LXL-1, and Pt@RHC-LXL-1 / TPP. The zeta potential results in Figure 5 show that the zeta potential of hollow mesoporous silica in aqueous solution is negative, at -16.87. After loading it with Ce6, the material's potential shows no significant change, at -14.37. Because the erythrocyte membrane has a negative potential, its zeta potential is -16.67. Even after coating the Ce6-loaded nanoparticles with the erythrocyte membrane, the potential remains negative, at -20.23. The positively charged Pt nanoclusters have a potential of 24.1. After the RHC nanoparticles are modified with these positively charged Pt nanoclusters, the zeta value changes significantly, to -0.35, indicating successful Pt modification of the material. The potential of TPP is positive, the potential of LXL-1 aptamer is -0.519, and the potential value of Pt@RHC is 0.48 after asymmetric modification, indicating that the modification is successful.

[0105] The above results show that the present invention first synthesizes RHC on the basis of synthesizing HMSN nanoparticles, and obtains Pt@RHC-LXL-1 / TPP by surface modification, and successfully synthesizes Pt@RHC-LXL-1 / TPP nanoparticles.

[0106] 2. Fluorescence detection

[0107] Fluorescence measurement was performed on the Ce6 sample (HC) coated with hollow mesoporous silica obtained in the fourth step of step 1 of Example 1 to study the photodynamic properties of Ce6 nanoparticles:

[0108] Each sample was diluted with PBS buffer to a final concentration of 10 μg / mL for subsequent analysis. Fluorescence spectra were then recorded from 600 nm to 700 nm using an F4600 fluorescence spectrophotometer, using a xenon lamp as the excitation source and an excitation wavelength of 404 nm.

[0109] The ultraviolet absorption peaks of Ce6, hollow mesoporous silica, and hollow mesoporous silica loaded with Ce6(HC) were measured by ultraviolet spectrophotometer, and the fluorescence spectra of Ce6 and hollow mesoporous silica loaded with Ce6(HC) were tested by fluorescence spectrometer. The results are shown in Figure 6.

[0110] In Figure 6, A shows the UV absorption curves of hollow mesoporous silica, Ce6, and HC. The results show that the UV absorption peak of HC is significantly lower than that of Ce6. This is due to the quenching of Ce6's fluorescence signal after the hollow mesoporous silica is loaded with Ce6, indicating that the hollow mesoporous silica has been successfully loaded with Ce6. B shows the fluorescence intensity of Ce6 and HC. The results show that the fluorescence spectrum of HC is lower than that of Ce6, indicating that the fluorescence intensity decreases after the hollow mesoporous silica is loaded with Ce6, indicating that the loading is successful and still has light response capability.

[0111] 3. Detection of singlet oxygen in nanoparticles

[0112] The ·OH content was determined by the fluorescence intensity of 7-hydroxycoumarin, a coumarin product. Coumarin (4 mM), H₂O₂ (8 mM), and 100 μg / mL Pt@RHC-LXL-1 / TPP were thoroughly mixed and incubated for 1 hour. The mixture was then irradiated for 10 minutes. The fluorescence curve of the solution was measured every 2 minutes, observing changes in fluorescence intensity at 455 nm. The results are shown in Figure 7, Panel A.

[0113] Pt@RHC-LXL-1 / TPP (100 μg / mL), H2O2 (8 mM), and DPBF (20 μM) were incubated together at room temperature for 10 minutes, irradiated with 808 nm laser for different times (2 to 10 minutes), and the absorption curve was recorded. The results are shown in Figure 7 B.

[0114] Different concentrations of H2O2 (2, 4, 8, 10 mM) were added to 100 μg / mL Pt@RHC-LXL-1 / TPP, and after incubation with 20 μM DPBF, the mixture was irradiated with 808 nm laser for 10 min, and the changes in light absorption intensity at 410-420 nm were observed. The results are shown in Figure 7, Panel C.

[0115] In order to evaluate the different concentrations of Pt@RHC-LXL-1 / TPP nanoparticles, 1 To investigate the O2 level, H2O2 (8 mM) and 20 μM DPBF were added to different concentrations of Pt@RHC-LXL-1 / TPP nanoparticles (5, 10, 20, 50, 100 μg / mL). After irradiation with 808 nm laser for 10 min, the changes in the light absorption intensity at 410-420 nm were observed. The results are shown in Figure 7D.

[0116] In Figure 7, Figure A shows that there is no obvious change in fluorescence intensity at 455nm, from which it can be concluded that the solution does not produce ·OH. As shown in Figure B, as the irradiation time increases, the absorption peak at 410-420nm continues to decrease, indicating that under this condition, 1 Figure C shows that as the concentration of H2O2 increases, the light absorption intensity at 410-420nm decreases, that is, the characteristic absorption peak of DPBF gradually decreases, which indicates that DPBF is 1 O2 is oxidized. This means that the concentration of H2O2 is related to the 1 Figure D shows that as the concentration of Pt@RHC-LXL-1 / TPP increases, the UV absorption peak of DPBF decreases more, indicating that 1 O2 is affected by the concentration of Pt@RHC-LXL-1 / TPP.

[0117] 4. Determination of cytotoxicity of nanoparticles by MTT assay

[0118] MDA-MB-231 breast cancer cells were seeded in 96-well plates and cultured for 24 hours. Experimental groups were set up as follows: 0, 5, 10, 20, 50, 100 μg / mL HMSN; 0, 5, 10, 20, 50, 100 μg / mL Pt@RHC; 0, 5, 10, 20, 50, 100 μg / mL Pt@RHC-LXL-1; 0, 5, 10, 20, 50, 100 μg / mL Pt@RHC-LXL-1 / TPP; 0, 5, 10, 20, 50, 100 μg / mL Pt@HC-LXL-1 / TPP. The cells were incubated in a cell culture incubator for 12 hours, irradiated with 808 nm laser for 10 minutes, and then incubated for 12 hours. Another experimental group without light exposure was set up: 0, 5, 10, 20, 50, and 100 μg / mL Pt@RHC-LXL-1 / TPP were added, respectively, and incubated in a cell culture incubator for 12 h.

[0119] Carefully aspirate the supernatant from each well, then add 10 μL of MTT and 90 μL of fresh culture medium to each well and incubate at 37°C in the dark for 4 hours. After 4 hours, aspirate the supernatant, add 100 μL of DMSO, and shake on a shaker at low speed for 10 minutes. Detect the cells at 490 nm using a microplate reader. Calculate cell viability based on the measurement data. The results are shown in Figure 8.

[0120] In Figure 8, A is the cell viability of MDA-MB-231 cells co-incubated with HMSNs and irradiated with light. As the concentration of HMSNs increases, the cell viability is better, indicating that HMSNs have good biosafety.

[0121] In Figure 8, B is the cell survival rate of MDA-MB-231 cells co-incubated with Pt@RHC and irradiated with light. As the concentration of Pt@RHC increases, the cytotoxicity of Pt@RHC increases after irradiation with 808nm laser compared with HMSN, indicating that after 808nm laser irradiation, Ce6 as a photosensitizer produces a certain amount of 1 O2 damages the mitochondria of breast cancer cells and causes the death of breast cancer cells.

[0122] In Figure 8, C is the cell survival rate of MDA-MB-231 cells co-incubated with Pt@RHC-LXL-1 and irradiated with light. It can be seen that the cytotoxicity of Pt@RHC-LXL-1 is further increased compared with the same concentration of Pt@RHC. This is because LXL-1 has a targeting effect on MDA-MB-231. More Pt@RHC-LXL-1 enters the MDA-MB-231 breast cancer cells, and more Ce6 is produced under the condition of light. 1 O2 further increases the cytotoxicity.

[0123] In Figure 8, D is the cell survival rate of MDA-MB-231 cells co-incubated with Pt@RHC-LXL-1 / TPP and irradiated with light. It can be seen that the same concentration of Pt@RHC-LXL-1 / TPP nanoparticles and the same concentration of Pt@RHC-LXL-1 nanoparticles are more toxic to cells after irradiation with 808nm laser. This is because TPP has the ability to target mitochondria in MDA-MB-231 breast cancer cells. Under the condition of light, the Ce6 produced 1 Further increases in O2 cause greater damage to mitochondrial DNA and lead to more severe cell death.

[0124] In Figure 8, E is the cell survival rate of MDA-MB-231 cells co-incubated with Pt@RH-LXL-1 / TPP and irradiated with light. Under light conditions, the cytotoxicity of Pt@RHC-LXL-1 / TPP is lower than that of Pt@RH-LXL-1 / TPP at the same concentration. This is because Pt@RH-LXL-1 / TPP does not contain Ce6 and will not produce Ce6 even under light conditions. 1 O2 does not cause mitochondrial damage in MDA-MB-231 breast cancer cells, so it has less cytotoxicity to cells.

[0125] In Figure 8, F is the cell viability of MDA-MB-231 cells co-incubated with Pt@RHC-LXL-1 / TPP without illumination. Under the condition of no illumination, the cell viability of Pt@RHC-LXL-1 / TPP nanoparticles remained basically unchanged over time, indicating that Ce6 can only be produced under the condition of illumination. 1 O2 causes mitochondrial damage in cells and further leads to cell death.

[0126] 5. Fluorescence microscopy to detect the targeting of nanoparticles

[0127] To investigate the targeting activity of Pt@RH-LXL-1 / TPP nanoparticles on MDA-MB-231 breast cancer cells, MCF-7 breast cancer cells were used as a control experimental group. MDA-MB-231 and MCF-7 breast cancer cells were inoculated and cultured for 24 hours for targeting analysis.

[0128] 100 μg of DID was incubated with 20 mg of Pt@RH-LXL-1 / TPP nanoparticles overnight. DID produced a red fluorescent signal. MDA-MB-231 and MCF-7 breast cancer cells were then incubated with DID-labeled Pt@RHC-LXL-1 / TPP nanoparticles for 3 hours. Finally, the cells were stained with DAPI and the fluorescence intensity was observed under a fluorescence microscope. The results are shown in Figure 9.

[0129] Figure 9 (A) shows fluorescence images of MCF-7 breast cancer cells after incubation with Pt@RH-LXL-1 / TPP nanoparticles for 1, 2, and 3 hours, respectively. (B) shows fluorescence images of MDA-MB-231 breast cancer cells after incubation with Pt@RH-LXL-1 / TPP nanoparticles for 1, 2, and 3 hours, respectively. Comparing Figures A and B, we can see that over the same time period, the red fluorescence intensity of MDA-MB-231 breast cancer cells is significantly stronger than that of MCF-7 breast cancer cells. This experimental data indicates that significantly more Pt@RH-LXL-1 / TPP nanoparticles enter MDA-MB-231 breast cancer cells than MCF-7 cells. These results demonstrate that Pt@RH-LXL-1 / TPP nanoparticles are targeted to MDA-MB-231 breast cancer cells.

[0130] 6. Fluorescence microscopy to detect the production of nanoparticles in cells 1 O2

[0131] MDA-MB-231 breast cancer cells were inoculated and cultured for 24 hours for the detection and analysis of intracellular reactive oxygen species. PBS solution, 50 μg / mL Pt@RHC nanoparticles, 50 μg / mL Pt@RH-LXL-1 nanoparticles, and 50 μg / mL Pt@RH-LXL-1 / TPP nanoparticles were co-incubated with MDA-MB-231 breast cancer cells for 3 hours, irradiated with 808 nm laser for 10 minutes, and the production of intracellular reactive oxygen species was detected using a DCFH-DA kit. 1 In the case of O2, the stronger the green fluorescence intensity, the more 1 The more O2, the better the results are shown in Figure 10.

[0132] Figure 10 shows that after 808nm laser irradiation, the green fluorescence intensity of Pt@RHC is stronger than that of the PBS group, indicating that the nanoparticles enter the cells, Ce6 produces reactive oxygen species under the conditions of light, and DCFH-DA produces green fluorescence. Compared with Pt@RHC, the fluorescence intensity of Pt@RHC-LXL-1 is further enhanced, which shows that LXL-1 has a targeting effect on MDA-MB-231 breast cancer cells. More Pt@RHC-LXL-1 nanoparticles enter the MDA-MB-231 breast cancer cells and produce more after 808nm laser irradiation. 1 O2 also increased further. The fluorescence intensity of Pt@RHC-LXL-1 / TPP and Pt@RHC-LXL-1 groups was compared and found to be similar. This is because TPP 1 O2 has no effect. It is finally proved that after Pt@RHC-LXL-1 / TPP nanoparticles are co-incubated with cells, after 808nm laser irradiation, the cells will produce 1 O2.

[0133] In summary, the Pt@RH-LXL-1 / TPP nanoparticles prepared by the present invention can successfully enter the cells through the special targeting of LXL-1 aptamer to MDA-MB-231 breast cancer cells, and utilize TPP to target mitochondria, Ce6 leaks into mitochondria and cytoplasm, and produces 1 O2 damages mitochondria and releases DNA, leading to cell damage and ultimately cell death.

[0134] The research work of this invention is funded by the National Natural Science Foundation of China (Project No.: 22276102), and the project name is: Nanomotors based on targeted motion for antifungal analysis and organelle imaging research in the environment.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nanoparticle loaded with photosensitizer Ce6 targeting breast cancer cell mitochondria, characterized in that: The nanoparticles use hollow mesoporous silica as a carrier, the hollow position of the carrier is loaded with photosensitizer Ce6, the red blood cell membrane is encapsulated outside the carrier, one side of the carrier after encapsulating the red blood cell membrane is modified with Pt nanoclusters, and the other side of the carrier is modified with LXL-1 aptamer and TPP.

2. The nanoparticle according to claim 1, characterized in that The erythrocyte membrane is a mouse erythrocyte membrane.

3. The nanoparticle according to claim 1, characterized in that The nucleotide sequence of the LXL-1 aptamer is shown in SEQ ID NO:

1.

4. The nanoparticle according to claim 3, characterized in that The 5' end of the LXL-1 aptamer is modified with a carboxyl group.

5. The method for preparing nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) hollow mesoporous silica and Ce6 dimethyl sulfoxide solution are ultrasonically mixed and freeze-dried to obtain hollow mesoporous silica loaded with Ce6 nanoparticles HC; (2) PBS solution of HC nanoparticles is mixed with PBS solution of red blood cell membrane powder, and then filtered to obtain HC nanoparticles coated with red blood cell membranes. (3) RHC nanoparticles were dispersed with PBS solution, centrifuged, precipitated and rotated onto the poly-L-lysine surface, incubated at room temperature, washed with PBS solution and the supernatant was removed; then a positively charged Pt nanocluster solution was added, incubated at room temperature, washed with PBS solution and the supernatant was removed to obtain asymmetrically modified nanoparticles Pt@RHC; (4) Pt@RHC nanoparticles were dispersed in a PBS solution containing LXL-1, TPP-COOH, EDC and NHS for reaction, stirred at room temperature, centrifuged, the precipitate was washed with PBS solution, and redispersed in PBS solution to obtain modified nanoparticles Pt@RHC-LXL-1 / TPP.

6. The preparation method according to claim 5, characterized in that: In the step (1), the preparation method of hollow mesoporous silica comprises the following steps: solid silica is added to a high-purity aqueous solution of CTAC and triethanolamine to obtain a mixed solution, a TEOS solution is added to the mixed solution and stirred in a water bath to form silica nanoparticles coated on the surface of silica, and then the hollow mesoporous silica is obtained by etching.

7. The preparation method according to claim 6, characterized in that: The solid silicon dioxide is prepared by mixing anhydrous ethanol, water and ammonia water, stirring at room temperature, and then adding TEOS solution.

8. The preparation method according to claim 7, characterized in that: The volume ratio of the anhydrous ethanol, water, ammonia water and TEOS solution is 35-36:4-6:1-1.2:1, and the mass fraction of the TEOS solution is 98%.

9. The preparation method according to claim 7, characterized in that: The stirring time at room temperature is 5 to 10 minutes, and the reaction time is 50 to 70 minutes.

10. The preparation method according to claim 6, characterized in that: In every 30 mL of high-purity water, the mass ratio of CTAC, triethanolamine and solid silicon dioxide is 2 g:20 mg:100 mg; the volume ratio of the mixed solution to the TEOS solution is 30:0.15, and the mass fraction of the TEOS solution is 98%.

11. The preparation method according to claim 6, characterized in that: The water bath stirring temperature is 75-85° C. and the stirring time is 50-70 min.

12. The preparation method according to claim 6, characterized in that: The etching method comprises the following steps: cooling the nanoparticles in a water bath to 45-50° C., adding Na 2 CO 3 , and continuously stirring to obtain hollow mesoporous silica.

13. The preparation method according to claim 6, characterized in that: The prepared hollow mesoporous silica was washed with a mixed cleaning solution, wherein the mixed cleaning solution was obtained by mixing anhydrous ethanol and concentrated hydrochloric acid in a volume ratio of 1:

10.

14. The preparation method according to claim 5, characterized in that: In the step (1), the mass ratio of hollow mesoporous silica to Ce6 is 4-5:0.1-0.2, the ultrasonic mixing time is 8-12 minutes, and the ultrasonic frequency is 40 KHz.

15. The preparation method according to claim 5, characterized in that: The nanoparticles HC are washed with ultrapure water.

16. The preparation method according to claim 5, characterized in that: In the step (2), the mass ratio of HC nanoparticles to red blood cell membrane powder is 1:

1.

17. The preparation method according to claim 5, characterized in that: The preparation method of the red blood cell membrane powder comprises the following steps: adding water to the red blood cell suspension, standing, and after the red blood cells absorb water and burst, centrifuging to remove hemoglobin, washing and dispersing the precipitate with a PBS solution, and freeze-drying to obtain the red blood cell membrane powder.

18. The preparation method according to claim 17, characterized in that: The centrifugal condition is 14000 rpm, 10 min.

19. The preparation method according to claim 5, characterized in that: The filter membrane extrusion method in step (2) is: extrusion is performed using 450nm and 200nm filter membranes in sequence, each extrusion being performed 4 to 5 times.

20. The preparation method according to claim 5, characterized in that: In the step (3), the centrifugation condition is 900r, 3min; and the incubation time is 50-70min.

21. The preparation method according to claim 5, characterized in that: The preparation method of the Pt nanocluster solution comprises the following steps: adding H2PtCl6 aqueous solution to ultrapure water, mixing with protamine aqueous solution under strong stirring at room temperature, adding NaBH4 dropwise after 30 minutes, continuously stirring the mixture at room temperature, and dialyzing for 24 hours to obtain a positively charged Pt nanocluster solution.

22. The preparation method according to claim 21, characterized in that: The concentration of the H2PtCl6 aqueous solution is 16 mM, the concentration of the protamine aqueous solution is 5 mg / mL, and the concentration of NaBH4 is 500 mM; the volume ratio of the H2PtCl6 aqueous solution, ultrapure water, protamine aqueous solution, and NaBH4 is 0.8:40:0.5:0.

4.

23. The preparation method according to claim 5, characterized in that: In the step (4), the concentration of TPP-COOH is 0.05-0.15 g / L, the concentration of EDC is 0.7-0.9 g / L, the concentration of NHS is 0.3-0.5 g / L, and the concentration of LXL-1 is 100 nM.

24. The preparation method according to claim 5, characterized in that: In the step (4), the reaction time is 12 to 18 minutes; the stirring time is 11 to 13 hours; and the centrifugal condition is 12000r, 15 minutes.

25. Use of the nanoparticles according to any one of claims 1 to 4 or the method for preparing the nanoparticles according to any one of claims 5 to 24 in preparing drugs for treating breast cancer.

26. Use of the nanoparticles according to any one of claims 1 to 4 or the method for preparing the nanoparticles according to any one of claims 5 to 24 in treating breast cancer, characterized in that: The nanoparticles are used in combination with 808 nm laser.

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