A lipid droplet nanocomplex for treating tumors and its preparation method and application

Abstract

The present invention discloses a lipid droplet nanocomposite for treating tumors, and a preparation method and application thereof. In the present invention, NRF‑2 protein inhibitor and ferritin targeting peptide are first loaded onto a two-dimensional nanosheet, and then the lipid droplets are wrapped on the surface of the two-dimensional nanosheet, and then the tumor targeting peptide is modified on the surface of the lipid droplets to obtain the nanocomposite for treating tumors. The nanocomposite can effectively inhibit tumor proliferation, metastasis and postoperative recurrence of tumors by cooperating with multiple therapies such as photodynamic therapy, ferroptosis and oxidative stress, and can effectively promote the death of tumor cells under hypoxic conditions, improve the effect of tumor treatment, and provide a new strategy for the treatment of cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-drugs, and particularly relates to a lipid droplet nano-complex for treating tumors, and a preparation method and application thereof. Background Art

[0002] The field of cancer treatment has been seeking more effective and safer methods. Although chemotherapy and radiotherapy are common treatments, they will inevitably cause damage to normal cells during treatment and may cause cancer cells to develop drug resistance, thus limiting the effectiveness of treatment. Therefore, it is particularly important to explore new treatment strategies.

[0003] Photodynamic therapy is a promising treatment modality that inhibits the growth of cancer cells by generating reactive oxygen species (ROS). However, the main challenge of this approach is that the generation of ROS requires sufficient oxygen, but the microenvironment of many tumors is hypoxic, which limits the generation of ROS and the efficacy of photodynamic therapy. In addition, when faced with oxidative stress, tumor cells will protect themselves by upregulating antioxidant proteins such as NRF-2, further reducing the efficacy of treatment.

[0004] Luteolin is a natural flavonoid compound that can exert anti-tumor effects by inhibiting NRF-2, but its targeting and bioavailability are low. Based on the high self-adaptability of tumors, tumor treatment requires a multimodal synergistic treatment strategy. This strategy not only inhibits the antioxidant stress response of tumor cells, but also exacerbates the oxidative stress of tumor cells by enhancing the ability to produce ROS.

[0005] Ferroptosis is also a treatment method that induces oxidative stress in tumors. By inducing the release of excess iron ions from the endogenous iron pool, it can not only induce ferroptosis, but also promote the Fenton reaction, providing the necessary oxygen for photodynamic therapy. In addition, lipid droplets exist in large quantities in adipose tissue, which can not only provide energy for the growth of tumor cells and have certain tumor targeting, but excessive lipid droplet uptake can also enhance tumor cell oxidative stress through lipid peroxidation, thereby inducing tumor cell death. The therapeutic effect can be significantly improved by combining treatment methods such as photodynamic therapy, ferroptosis, and lipid droplet targeted delivery. However, the successful implementation of this multimodal synergistic treatment strategy requires precise targeted drug delivery to ensure its safety and effectiveness.

[0006] Therefore, developing a drug that can effectively promote tumor cell death under hypoxic conditions and combining it with a multimodal synergistic treatment strategy to target tumor cells is a very promising research direction. This approach can not only overcome the limitation of oxygen supply, but also enhance the attack on tumor cells through multiple mechanisms, thus providing a new and more effective cancer treatment method. This method is expected to play an important role in future cancer treatment and bring new hope to patients. Summary of the invention

[0007] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a nanocomposite for treating tumors.

[0008] Another object of the present invention is to provide a nanocomposite prepared by the method for treating tumors.

[0009] Another object of the present invention is to provide application of the nanocomplex for treating tumors.

[0010] The purpose of the present invention is achieved through the following technical solutions:

[0011] A method for preparing a nanocomposite for treating tumors comprises the following steps: firstly loading an NRF-2 protein inhibitor and a ferritin targeting peptide onto a two-dimensional nanosheet, then wrapping lipid droplets on the surface of the two-dimensional nanosheet, and then modifying the tumor targeting peptide onto the surface of the lipid droplets to obtain the nanocomposite for treating tumors.

[0012] The NRF-2 inhibitor is preferably luteolin.

[0013] The ferritin targeting peptide is preferably HKNKGKKNGKHNGWK-PEG-NH2, wherein the molecular weight of PEG is preferably 1K.

[0014] The two-dimensional nanosheets are preferably black phosphorus nanosheets.

[0015] The tumor targeting peptide is preferably phospholipid polyethylene glycol hyaluronic acid (DSPE-PEG-HA), wherein the molecular weight of PEG is preferably 2K, and the molecular weight of HA is preferably 50K.

[0016] The coating of the two-dimensional nanomaterial surface by the lipid droplets can be prepared by any one of ultrasonic crushing, enzymatic hydrolysis, mechanical crushing and the like.

[0017] The method for preparing the nanocomposite for treating tumors specifically comprises the following steps:

[0018] (1) dispersing the two-dimensional nanosheets in water to obtain a two-dimensional nanosheet dispersion; dissolving the NRF-2 protein inhibitor in an organic solvent to obtain an NRF-2 protein inhibitor solution; then uniformly mixing the two-dimensional nanosheet dispersion and the NRF-2 protein inhibitor solution, stirring and reacting at room temperature in a dark environment, and after the reaction is completed, centrifuging, taking the precipitate and redispersing it in water to obtain a complex A;

[0019] (2) mixing the complex A obtained in step (1) with the ferritin targeting peptide solution, stirring and reacting at room temperature in a dark environment, and after the reaction is completed, centrifuging and taking the precipitate and redispersing it in water to obtain complex B;

[0020] (3) extracting lipid droplets from adipose tissue and dispersing them into a sodium chloride solution to obtain a lipid droplet mixture; then mixing the lipid droplet mixture with the complex B obtained in step (2) and subjecting the mixture to ultrasonic treatment at 0° C. in the dark, and after the ultrasonic treatment is completed, centrifuging the mixture and re-dispersing the precipitate into water to obtain a complex C;

[0021] (4) The complex C obtained in step (3) is mixed evenly with the tumor targeting peptide solution, and stirred to react at room temperature in the dark. After the reaction is completed, the mixture is centrifuged, and the precipitate is redispersed in water to obtain the complex D, i.e., the nanocomposite for treating tumors.

[0022] The two-dimensional nanosheets described in step (1) are preferably black phosphorus nanosheets (BP).

[0023] The water described in steps (1), (2), (3) and (4) is oxygen-free water, which can be obtained by deoxidizing ultrapure water.

[0024] The mass ratio of the two-dimensional nanosheets and the NRF-2 protein inhibitor described in step (1) is 0.2 to 1:1, preferably 0.2:1.

[0025] The concentration of the two-dimensional nanosheet dispersion in step (1) is 0.1-0.5 mg / mL, preferably 0.2 mg / mL.

[0026] The NRF-2 protein inhibitor described in step (1) is preferably luteolin.

[0027] The concentration of the NRF-2 protein inhibitor solution in step (1) is 0.1 to 2.5 mg / mL, preferably 1 mg / mL.

[0028] The volume ratio of the two-dimensional nanosheet dispersion and the NRF-2 protein inhibitor solution in step (1) is preferably 1:1.

[0029] The organic solvent described in step (1) is preferably ethanol.

[0030] The reaction time in step (1) is 12 to 24 hours, preferably 18 hours.

[0031] The centrifugation conditions described in steps (1), (2), (3) and (4) are: 4°C, (10,000-1,200)×g, and centrifugation for 15 minutes.

[0032] The ferritin targeting peptide described in step (2) is preferably: HKNKGKKNGKHNGWK-PEG-NH2, wherein the molecular weight of PEG is preferably 1K.

[0033] The mass ratio of the two-dimensional nanosheets in the complex A described in step (2) to the ferritin targeting peptide is 0.2 to 1:1, preferably 0.2:1.

[0034] The concentration of the two-dimensional nanosheets in the complex A in step (2) is 0.2-1 mg / mL, preferably 0.2 mg / mL.

[0035] The concentration of the ferritin targeting peptide solution in step (2) is 0.5-1 mg / mL, preferably 1 mg / mL.

[0036] The volume ratio of the complex A to the ferritin targeting peptide solution in step (2) is preferably 1:1.

[0037] The reaction time in steps (2) and (4) is 2 to 6 hours, preferably 4 hours.

[0038] The adipose tissue described in step (3) is preferably abdominal adipose tissue of mice.

[0039] The lipid droplets described in step (3) can be extracted by conventional methods in the art or by using a lipid droplet extraction kit; preferably, they are obtained by the following method: taking abdominal adipose tissue from mice, extracting lipid droplets using a lipid droplet extraction kit, and then dispersing the lipid droplets in a sodium chloride solution to obtain a lipid droplet mixture.

[0040] The concentration of the sodium chloride solution described in step (3) is 9 g / L.

[0041] The concentration of lipid droplets in the lipid droplet mixture described in step (3) is 3.5 million to 4 million / mL, preferably 3.6 million / mL.

[0042] The concentration of the two-dimensional nanosheets in the complex B in step (3) is 0.2-1 mg / mL, preferably 0.2 mg / mL.

[0043] The volume ratio of the lipid droplet mixture and complex B described in step (3) is preferably 1:1.

[0044] The conditions of the ultrasonic treatment in step (3) are: ultrasonic power 300W, ultrasonic frequency 4000Hz, ultrasonic treatment for 10 minutes, stop for 5 minutes, and cycle 5 times.

[0045] The tumor targeting peptide described in step (4) is preferably phospholipid polyethylene glycol hyaluronic acid (DSPE-PEG-HA), wherein the molecular weight of PEG is 2K and the molecular weight of HA is 50K.

[0046] The concentration of the tumor targeting peptide solution in step (4) is 0.5 mg / mL to 1 mg / mL, preferably 1 mg / mL.

[0047] The mass ratio of the two-dimensional nanosheets to the tumor targeting peptide in the complex C described in step (4) is 0.2 to 1:1, preferably 0.2:1.

[0048] The concentration of the two-dimensional nanosheets in the complex C in step (4) is 0.2-1 mg / mL, preferably 0.2 mg / mL.

[0049] The volume ratio of the complex C and the tumor targeting peptide solution described in step (4) is preferably 1:1.

[0050] A nanocomposite for treating tumors is prepared by any of the methods described above.

[0051] The nanocomposite for treating tumors is used in preparing drugs for preventing and / or treating tumors.

[0052] The nanocomplex can inhibit the growth and proliferation of tumor cells, increase the level of ROS in tumor cells, and promote the dissociation of ferritin in tumor tissue to release iron ions, thereby achieving the purpose of anti-tumor.

[0053] The nanocomplex for treating tumors is used in preparing drugs for preventing and / or treating tumor metastasis.

[0054] The nanocomplex for treating tumors is used in preparing drugs for preventing and / or treating postoperative recurrence of tumors.

[0055] The tumor is at least one of solid tumors (solid tumors); preferably breast cancer.

[0056] Compared with the prior art, the present invention has the following advantages and effects:

[0057] 1. The present invention provides a lipid droplet nanocomposite for treating tumors, which is composed of lipid droplets, black phosphorus nanosheets, luteolin, hyaluronic acid and ferritin targeting peptides, etc.; wherein the lipid droplets have tumor targeting and can also induce lipid peroxidation in tumor cells; black phosphorus nanosheets improve the water solubility of luteolin, and can also produce peroxides under 660nm laser irradiation, inducing oxidative stress in tumor cells; luteolin is an inhibitor of NRF-2 protein, which effectively inhibits the antioxidant response of tumor cells; hyaluronic acid can target tumor cells; ferritin targeting peptides target ferritin by making the nanocomposite target ferritin, and promote ferritin dissociation to release iron ions, thereby inducing endogenous ferroptosis. The application of the obtained nanocomposite in tumor treatment can significantly improve the therapeutic effect, especially in the case of hypoxia in the tumor microenvironment.

[0058] 2. The nanocomplex prepared by the present invention has multiple targeting properties, which effectively improves the therapeutic effect and accuracy of the nanocomplex. In the nanocomplex, hyaluronic acid plays an active tumor targeting effect; lipid droplets induce tumor uptake and increase the accumulation of nanocomplexes inside the tumor; after entering the cell, the ferritin targeting peptide promotes the enrichment of the nanocomplex near ferritin.

[0059] 3. The nanocomplex prepared by the present invention can inhibit tumor proliferation by coordinating multiple therapies such as photodynamic therapy, ferroptosis and oxidative stress, and can be used to treat solid tumors such as breast cancer. In the nanocomplex, black phosphorus is used as a photosensitizer to generate ROS by 660nm light irradiation, but the generation of ROS depends on sufficient oxygen supply; by targeting ferritin and inducing the Fenton reaction, not only additional iron ions are generated to promote ferroptosis of tumor cells, but also the free radicals generated by the Fenton reaction increase the level of cellular oxidative stress, and the oxygen generated also provides support for the photodynamic effect of black phosphorus; luteolin inhibits the expression of NRF-2 protein and reduces the antioxidant capacity of cancer cells; at the same time, excessive accumulation of lipid droplets in cells can induce lipid peroxidation, which not only aggravates the oxidative stress in cells, but also enhances the degree of ferroptosis of cells.

[0060] 4. The nanocomplex prepared by the present invention can inhibit tumor metastasis by cooperating with multiple therapies such as photodynamic therapy, ferroptosis and oxidative stress, and can be used to treat metastasis of solid tumors such as breast cancer.

[0061] 5. The nanocomplex prepared by the present invention cooperates with multiple therapies such as photodynamic therapy, ferroptosis and oxidative stress to inhibit postoperative tumor recurrence and can be used to treat postoperative recurrence of solid tumors such as breast cancer.

[0062] 6. The nanocomposite prepared by the present invention improves the problem that the hypoxic environment of the tumor limits the anti-cancer effect of photodynamic therapy. In the nanocomposite, by targeting ferritin and inducing the Fenton reaction, not only additional iron ions are generated to promote ferroptosis, but the oxygen generated by the Fenton reaction also provides support for the photodynamic effect of black phosphorus, solving the problem that photodynamic therapy is limited in hypoxic environments.

[0063] 7. The present invention introduces the targeted release of iron ions. By targeting ferritin and inducing the Fenton reaction, not only additional iron ions are produced to promote iron-dependent cell death (ferroptosis), but also the free radicals generated by the Fenton reaction increase the level of ROS, and the oxygen produced also provides support for the photodynamic effect of black phosphorus. In addition, in order to further aggravate the oxidative stress of cancer cells, the present invention uses luteolin to inhibit the expression of NRF-2 protein and reduce the antioxidant capacity of cancer cells; at the same time, excessive accumulation of lipid droplets in cells can induce lipid peroxidation, which not only aggravates the oxidative stress in cells, but also enhances the degree of ferroptosis of cells. The present invention adopts a multimodal synergistic treatment strategy to effectively promote the death of tumor cells under hypoxic conditions, providing a new strategy for cancer treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 It is the particle size distribution diagram of BP / LUT / HK / LD / HA of the present invention.

[0065] Figure 2 It is the Zeta potential diagram of BP / LUT / HK / LD / HA of the present invention.

[0066] Figure 3 It is a transmission electron microscope image of BP / LUT / HK / LD / HA of the present invention.

[0067] Figure 4 It is the element distribution diagram of BP / LUT / HK / LD / HA of the present invention.

[0068] Figure 5 This is a diagram showing the generation of ROS by BP / LUT / HK / LD / HA of the present invention after being irradiated with light in vitro.

[0069] Figure 6 This is a diagram showing the effect of BP / LUT / HK / LD / HA of the present invention on inhibiting tumor cell growth in vitro.

[0070] Figure 7 This is a diagram showing how BP / LUT / HK / LD / HA of the present invention promotes ROS levels in mouse tumor tissues.

[0071] Figure 8 This is a diagram showing how BP / LUT / HK / LD / HA of the present invention promotes the dissociation of ferritin in mouse tumor tissue to produce iron ions.

[0072] Fig. 9 This is a diagram showing the effect of BP / LUT / HK / LD / HA of the present invention on inhibiting tumor proliferation in mice.

[0073] Fig.10 This is a diagram showing the effect of BP / LUT / HK / LD / HA of the present invention in inhibiting lung metastasis in mice.

[0074] Fig.11 This is a diagram showing the effect of BP / LUT / HK / LD / HA of the present invention in inhibiting postoperative recurrence in tumor-bearing mice.

[0075] Fig.12 This is a graph showing the safety test results of BP / LUT / HK / LD / HA of the present invention on mice. DETAILED DESCRIPTION

[0076] The present invention will be described in further detail below in conjunction with the examples, but it should be understood that these examples are only for the purpose of describing in more detail and specifically, and should not be understood as being used to limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. The test methods for which specific experimental conditions are not specified in the following examples are usually carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0077] Example 1 Preparation of Nanocomposite

[0078] (1) Black phosphorus nanosheets (BP) (Xi'an Ruixi Biotechnology Co., Ltd., black phosphorus nanosheets BPNSs, R-BP-002) were dispersed in oxygen-free water at a black phosphorus concentration of 200 μg / mL. 1 mL of 200 μg / mL black phosphorus was mixed with 1 mL of 1 mg / mL luteolin (LUT) ethanol solution in a volume ratio of 1:1, and stirred at room temperature for 18 hours in the dark.

[0079] (2) After stirring, the mixture was centrifuged at 10,000 × g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was redispersed with 200 μL of oxygen-free water to obtain 200 μL of BP / LUT (BP concentration was 1 mg / mL).

[0080] (3) The above 200 μL BP / LUT was diluted with oxygen-free water to 1 mL BP / LUT (BP concentration was 200 μg / mL), and then mixed with 1 mg / mL ferritin targeting peptide (HKNKGKKNGKHNGWK-PEG-NH2; HK for short, where the molecular weight of PEG is 1K) (Xi'an Ruixi Biotechnology Co., Ltd.) oxygen-free aqueous solution at a volume ratio of 1:1, and stirred at room temperature for 4 hours in the dark.

[0081] (4) After stirring, the mixture was centrifuged at 10,000 × g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was redispersed with 200 μL of anaerobic water to obtain 200 μL of BP / LUT / HK (BP concentration was 1 mg / mL).

[0082] (5) Referring to the instructions of the lipid droplet extraction kit (Lipid Droplet Isolation Kit, ab242290), abdominal adipose tissue was obtained from female BALB / c mice (weight 18-22 g, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.), and the adipose tissue was homogenized, and then solution A and solution B in the kit were added successively, and lipid droplets were obtained after centrifugation. Subsequently, the lipid droplets were dispersed in 9 g / L sodium chloride solution and counted at 3.6 million / mL. BP / LUT / HK (BP concentration was 1 mg / mL) was diluted with oxygen-free water to 1 mL of BP / LUT / HK (BP concentration was 200 μg / mL); then, lipid droplets (LD) and BP / LUT / HK (BP concentration was 200 μg / mL) were mixed in a volume ratio of 1:1, and then ultrasonicated for 10 minutes at 0 degrees Celsius, stopped for 5 minutes, and cycled 5 times, wherein the ultrasonic conditions were 300W, 4000 Hz, 0 degrees Celsius, and protected from light.

[0083] (6) After the sonication, the mixture was centrifuged at 10,000 × g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was redispersed with 200 μL of oxygen-free water to obtain 200 μL of BP / LUT / HK / LD (BP concentration was 1 mg / mL).

[0084] (7) The above 200 μL BP / LUT / HK / LD was diluted with oxygen-free water to 1 mL BP / LUT / HK / LD (BP concentration was 200 μg / mL), and then mixed with 1 mg / mL tumor targeting peptide (phospholipid polyethylene glycol hyaluronic acid, DSPE-PEG-HA, molecular weight: PEG2K, HA50K) (Xi'an Ruixi Biotechnology Co., Ltd.) oxygen-free aqueous solution at a volume ratio of 1:1, and stirred at room temperature for 4 hours in the dark.

[0085] (8) After stirring, the mixture was centrifuged at 10,000 × g for 15 min at 4 °C, the supernatant was discarded, and the precipitate was redispersed with 200 μL of anaerobic water to obtain 200 μL of BP / LUT / HK / LD / HA (BP concentration was 1 mg / mL).

[0086] Example 2 Characterization and detection (particle size distribution diagram, Zeta potential diagram, transmission electron microscope diagram, element distribution diagram)

[0087] (1) The particle size and potential of BP / LUT and BP / LUT / HK / LD / HA prepared in step (2) and step (8) of Example 1 were measured respectively. At the same time, the BP in step (1) of Example 1 was dispersed in oxygen-free water as a control, with a concentration of 200 μg / mL. The instrument used was NanoBrook 90Plus PALS (Brookhaven Instruments, USA). The results are shown in Figure 1 and Figure 2 As shown: the average particle size of BP is 95.48nm, and the potential is -28.62mV; the particle size of BP / LUT in water is 132.03nm, and the potential is -3.79mV; the particle size of BP / LUT / HK / LD / HA in water is 349.57nm, and the potential is -6.37mV.

[0088] (2) The BP / LUT / HK / LD / HA prepared in step (8) of Example 1 was tested by transmission electron microscopy. The results are as follows: Figure 3 As shown: The transmission electron microscope image shows that the prepared nanoformulation is irregular in shape and has a size of about 300 nm.

[0089] (3) The element distribution test was performed on the BP / LUT / HK / LD / HA prepared in step (8) of Example 1. The results are as follows: Figure 4 As shown: The element distribution diagram results show that the main element of the prepared nanoformulation is phosphorus (P), and it also contains oxygen (O), carbon (C) and nitrogen (N).

[0090] Example 3 Cell culture

[0091] BALB / c mouse mammary tumor cells 4T1 (Jiangsu Keygen Biotechnology Co., Ltd.) were inoculated into DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 1% penicillin (100 U / mL) and streptomycin (100 μg / mL), and then placed in an incubator at 37 degrees Celsius, 5% CO2 and 95% relative humidity. Fresh medium was replaced every 24 hours, and cells were passaged when they grew to 70-80% under a microscope.

[0092] Example 4: Production of reactive oxygen species (ROS) by BP / LUT / HK / LD / HA after in vitro illumination

[0093] The BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 were respectively tested for the generation of ROS in 4T1 cells after illumination. The specific steps are as follows:

[0094] The 4T1 cells in Example 3 were cultured at a rate of 1×105 The cells were seeded in 6-well plates at the number of cells / well and placed in a 37-degree Celsius CO2 incubator overnight. When the cell growth density reached about 70%, BP, BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA (each experimental group was given an equal dose of black phosphorus with a final concentration of 0.1 mg / mL) were added to intervene in the cells, and an equal volume of PBS buffer was used as a control. Two hours after the drug intervention, laser irradiation with a wavelength of 660nm (light conditions: 50mW / cm 2 , continuous irradiation for 10 minutes). After the illumination ended, the cells were returned to the 37°C CO2 incubator for 8 hours. The reactive oxygen species ROS fluorescent probe DCFH-DA was diluted with serum-free culture medium at 1:1000 (v / v) to a final concentration of 10 μmol / L. The cell culture medium was removed and 1 mL of the diluted DCFH-DA was added. The cells were returned to the 37°C cell culture incubator and incubated for 20 minutes. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. Laser confocal microscopy was used to observe the effects of different drug groups on the level of ROS in 4T1 cells after intervention in 4T1 cells using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The experiment was repeated three times.

[0095] The results are as follows Figure 5 As shown: BP / LUT / HK / LD / HA after 660nm laser irradiation can significantly increase the ROS level in 4T1 cells.

[0096] Example 5 Effect of BP / LUT / HK / LD / HA on inhibiting tumor cell growth in vitro

[0097] The effects of BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 on the activity of 4T1 cells after illumination were detected respectively. The specific steps are as follows:

[0098] The 4T1 cells in Example 3 were cultured at 5×10 3 The cells were seeded in 96-well plates at the number of cells / well and placed in a 37-degree Celsius CO2 incubator overnight. When the cell growth density reached about 70%, BP, BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA were added to intervene in the cells (each experimental group was given an equal dose of black phosphorus with a final concentration of 0.1 mg / mL), and an equal volume of PBS buffer was used as a control. Two hours after the addition of drug intervention, laser irradiation with a wavelength of 660 nm was given (the illumination conditions were: 50 mW / cm 2, and continue irradiation for 10 minutes). After the illumination ends, return to the 37°C CO2 incubator and culture for 24 hours. Dilute CCK8 with serum-free culture medium at a volume ratio of 1:10. Remove the cell culture medium and add 100μL of diluted CCK8 to each empty space. Return to the 37°C cell culture incubator and incubate for 60 minutes. Then place the plate in an ELISA reader to detect the OD value of each well of the 96-well plate at a wavelength of 450nm. The experiment was repeated three times.

[0099] The results are as follows Figure 6 As shown: BP / LUT / HK / LD / HA after 660nm laser irradiation can significantly inhibit the activity of 4T1 cells.

[0100] Example 6 Animal breeding

[0101] Four-week-old female Balb / c mice weighing 18-22 g were used in the experiment and purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All experimental treatments of mice were reviewed and approved by the Experimental Animal Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine, and the animal research conducted was conducted in accordance with internationally recognized principles for the use and care of laboratory animals. All mice were fed in the SPF environment of the Experimental Animal Center of Guangdong Provincial Hospital of Traditional Chinese Medicine, maintained at an ambient temperature of 20-25°C and a relative humidity of 45-50%, and given sterilized food and water. After the experimental animals adapted to the breeding environment, 4T1 tumor masses were transplanted on the fourth pair of right breast pads.

[0102] When the tumor grows to 50 mm 3 The experimental animals were randomly divided into 13 groups, with 10 animals in each group. The administration methods of 8 groups were as follows: saline (injected into the tail vein), Erastin (30 mg / kg, intraperitoneal injection), black phosphorus without laser irradiation (BP-, BP = 5 mg / kg, injected into the tail vein), black phosphorus + laser irradiation (BP+, BP = 5 mg / kg, injected into the tail vein), BP / LUT + laser irradiation (BP / LUT+, BP = 5 mg / kg, injected into the tail vein), BP / LUT / HK + laser irradiation (BP / LUT / HK+, BP = 5 mg / kg, injected into the tail vein), BP / LUT / HK / LD + laser irradiation (BP / LUT / HK / LD+, BP = 5 mg / kg, injected into the tail vein) and BP / LUT / HK / LD / HA + laser irradiation (BP / LUT / HK / LD / HA+, BP = 5 mg / kg, injected into the tail vein), wherein the laser irradiation conditions were all laser irradiation with a wavelength of 660 nm (50 mW / cm 2, irradiation for 10 minutes, duty cycle 30%). The first administration was recorded as day 0. The observation of 10 experimental animals in each group ended 18 days after administration. They were killed according to animal ethics requirements, and the tumor tissues of the experimental animals were taken for subsequent research. The other 5 groups of experimental animals were administered as follows: saline (Saline, tail vein injection), Erastin (Erastin, 30 mg / kg, intraperitoneal injection) BP / LUT / HK / LD / HA+ laser irradiation (BP / LUT / HK / LD / HA+, BP=2.5 mg / kg, tail vein injection), BP / LUT / HK / LD / HA+ laser irradiation (BP / LUT / HK / LD / HA+, BP=5 mg / kg, tail vein injection), BP / LUT / HK / LD / HA+ laser irradiation (BP / LUT / HK / LD / HA+, BP=10 mg / kg, tail vein injection), wherein the laser irradiation conditions were laser irradiation with a wavelength of 660nm (50mW / cm 2 , irradiation for 10 minutes, duty cycle 30%). The first administration was recorded as day 0, and 10 experimental animals in each group underwent tumor resection surgery on the 21st day after administration, and the postoperative tumor recurrence was observed. The experimental animals were killed in accordance with animal ethics requirements on the 42nd day after administration.

[0103] Example 7: BP / LUT / HK / LD / HA promotes the level of reactive oxygen species (ROS) in mouse tumor tissue

[0104] The effects of BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 on the level of ROS in the tumor tissue of experimental animals after illumination were respectively detected. After 18 days of drug treatment in Example 6, the tumor tissue of the experimental animal was taken, and the tumor tissue was immediately placed in an OCT embedding agent and frozen into blocks by liquid nitrogen; the tumor tissue was cut into 4 μm thick slices and attached to a glass slide, and after returning to room temperature, the tumor tissue was circled with an immunohistochemical pen; DCFH-DA was diluted with PBS buffer at 1:1000 (v / v) to a final concentration of 10 micromoles / liter; the diluted DCFH-DA was dropped on the surface of the tumor tissue, and placed in a 37 degree Celsius constant temperature incubator for 30 minutes, and then the DCFH-DA was removed and washed 3 times with PBS buffer, each time for 5 minutes.

[0105] The results are as follows Figure 7 As shown: The ROS level in the tumor tissue of experimental animals irradiated with BP / LUT / HK / LD / HA and 660nm laser was significantly higher than that in the other experimental groups.

[0106] Example 8: BP / LUT / HK / LD / HA promotes the dissociation of ferritin in mouse tumor tissue to produce iron ions

[0107] The BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 were respectively tested for promoting the dissociation of ferritin in tumor tissues of experimental animals to produce iron ions after illumination. After 18 days of drug treatment in Example 6, tumor tissues of experimental animals were taken, and 100 mg of tumor tissues were uniformly weighed from each group to extract primary cells. Appropriate cells were centrifuged at room temperature for 5 minutes, the supernatant was discarded, and an appropriate volume of Calcein AM staining working solution was added to make the cell density about 1×10 6 / mL. Incubate the cells at 37 degrees Celsius for 30 minutes. After the incubation, centrifuge for 5 minutes, remove the supernatant, and slowly add PBS buffer containing 1% (v / v) serum (Fetal Bovine Serum, Gibco, US Origin) preheated at 37°C to resuspend the cells. Wash the cells 3 times with PBS buffer containing 1% (v / v) serum to fully remove the residual staining solution. Replace with fresh PBS buffer preheated at 37 degrees Celsius containing 1% (v / v) serum, and incubate at 37 degrees Celsius for another 30 minutes in the dark; centrifuge at room temperature for 5 minutes, and place in a fluorescent microplate reader for detection. The excitation light wavelength is 494nm, and the emission light wavelength is 514nm.

[0108] The results are as follows Figure 8 As shown: the degree of ferritin dissociation and iron ion release in the tumor tissue of experimental animals irradiated with BP / LUT / HK / LD / HA and 660nm laser was significantly higher than that in the other experimental groups.

[0109] Example 9 Effect of BP / LUT / HK / LD / HA in inhibiting tumor proliferation in mice

[0110] The effects of BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 on inhibiting tumor growth in experimental animals after irradiation with light were tested respectively. After 18 days of drug treatment, tumor tissues of experimental animals in different drug treatment groups were obtained in Example 6.

[0111] The results are as follows Fig. 9 As shown, BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA combined with 660nm laser irradiation have the effect of inhibiting tumor growth; among them, BP / LUT / HK / LD / HA combined with 660nm laser irradiation can more significantly inhibit tumor growth.

[0112] Example 10 Effect of BP / LUT / HK / LD / HA in inhibiting lung metastasis in mice

[0113] The effects of BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 on inhibiting lung metastasis of tumors in experimental animals after irradiation with light were tested respectively. After 18 days of drug treatment, lung tissues of experimental animals in different drug treatment groups were obtained in Example 6.

[0114] The results are as follows Fig.10 As shown, BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA combined with 660nm laser irradiation can reduce the number of lung metastases; among them, there were no lung metastases in the lung tissues of experimental animals in the BP / LUT / HK / LD and BP / LUT / HK / LD / HA combined with 660nm laser irradiation groups.

[0115] Example 11 Effect of BP / LUT / HK / LD / HA in inhibiting postoperative recurrence in tumor-bearing mice

[0116] The effect of BP / LUT / HK / LD / HA prepared in step (8) of Example 1 on inhibiting postoperative recurrence of tumors in experimental animals after illumination was detected. The concentration of BP / LUT / HK / LD / HA was adjusted with oxygen-free water. Taking the black phosphorus BP concentration as a representative, the BP concentration was adjusted to 0.5 mg / mL, 1 mg / mL and 2 mg / mL respectively. The dosage volume was calculated according to the weight of the mice. The dosage of black phosphorus in the low-dose group (low) was 2.5 mg / kg, the dosage of black phosphorus in the medium-dose group (medium) was 5 mg / kg, and the dosage of black phosphorus in the high-dose group (high) was 10 mg / kg. The specific dosage method is shown in Example 6. After the experimental animals received drug treatment for 21 days, their tumor tissues were removed and continued to be fed for 21 days. On the 42nd day, tumor tissues of experimental animals in different drug treatment groups were taken.

[0117] The results are as follows Fig.11 As shown, BP / LUT / HK / LD / HA combined with 660nm laser irradiation can significantly inhibit the probability of postoperative recurrence of breast cancer in experimental animals.

[0118] Example 12 In vivo safety of BP / LUT / HK / LD / HA

[0119] The safety of BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA prepared in steps (2), (4), (6) and (8) of Example 1 on major organs in experimental animals after exposure to light was tested. After 18 days of drug treatment, the liver, heart, spleen and kidney of the experimental animals in different drug treatment groups in Example 6 were taken for hematoxylin-eosin staining.

[0120] The results are as follows Fig.12 As shown, there were no obvious pathological changes in the main organs of the experimental mice after 660nm laser irradiation of BP, BP / LUT, BP / LUT / HK, BP / LUT / HK / LD and BP / LUT / HK / LD / HA combined.

[0121] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a nanocomposite for treating tumors, characterized in that: The method comprises the following steps: firstly loading the NRF-2 protein inhibitor and the ferritin targeting peptide onto the two-dimensional nanosheet, then wrapping the lipid droplets on the surface of the two-dimensional nanosheet, and then modifying the tumor targeting peptide onto the surface of the lipid droplets to obtain the nanocomplex for treating tumors; The NRF-2 protein inhibitor is luteolin; The ferritin targeting peptide is HKNKGKKNGKHNGWK-PEG-NH2, wherein the molecular weight of PEG is 1K; The two-dimensional nanosheet is a black phosphorus nanosheet; The tumor targeting peptide is phospholipid polyethylene glycol hyaluronic acid DSPE-PEG-HA, wherein the molecular weight of PEG is 2K and the molecular weight of HA is 50K.

2. The method for preparing a nanocomposite for treating tumors according to claim 1, characterized in that: The specific steps include: (1) dispersing the two-dimensional nanosheets in water to obtain a two-dimensional nanosheet dispersion; dissolving the NRF-2 protein inhibitor in an organic solvent to obtain an NRF-2 protein inhibitor solution; then mixing the two-dimensional nanosheet dispersion and the NRF-2 protein inhibitor solution evenly, stirring and reacting at room temperature in the dark, and after the reaction is completed, centrifuging, taking the precipitate and redispersing it in water to obtain a complex A; (2) mixing the complex A obtained in step (1) with the ferritin targeting peptide solution, stirring and reacting at room temperature in a dark environment, and after the reaction is completed, centrifuging and taking the precipitate and redispersing it in water to obtain complex B; (3) extracting lipid droplets from adipose tissue and dispersing them into a sodium chloride solution to obtain a lipid droplet mixture; then mixing the lipid droplet mixture with the complex B obtained in step (2) and subjecting the mixture to ultrasonic treatment at 0°C in the dark; after the ultrasonic treatment is completed, centrifuging the mixture and re-dispersing the precipitate into water to obtain a complex C; (4) The complex C obtained in step (3) is mixed evenly with the tumor targeting peptide solution, and the mixture is stirred to react at room temperature in the dark. After the reaction is completed, the mixture is centrifuged, and the precipitate is redispersed in water to obtain the complex D, i.e., the nanocomposite for treating tumors.

3. The method for preparing a nanocomposite for treating tumors according to claim 2, characterized in that: The mass ratio of the two-dimensional nanosheet and the NRF-2 protein inhibitor described in step (1) is 0.2 to 1:1; The concentration of the two-dimensional nanosheet dispersion in step (1) is 0.1-0.5 mg / mL; The concentration of the NRF-2 protein inhibitor solution in step (1) is 0.1 to 2.5 mg / mL; The mass ratio of the two-dimensional nanosheets to the ferritin targeting peptide in the complex A described in step (2) is 0.2 to 1:1; The concentration of the two-dimensional nanosheets in the complex A described in step (2) is 0.2-1 mg / mL; The concentration of the ferritin targeting peptide solution in step (2) is 0.5-1 mg / mL; The concentration of the sodium chloride solution in step (3) is 9 g / L; The concentration of lipid droplets in the lipid droplet mixture described in step (3) is 3.5 million / mL to 4 million / mL; The concentration of the two-dimensional nanosheets in the complex B in step (3) is 0.2 mg / mL to 1 mg / mL; The volume ratio of the lipid droplet mixture and complex B in step (3) is 1:1; The concentration of the tumor targeting peptide solution in step (4) is 0.5 mg / mL to 1 mg / mL; The mass ratio of the two-dimensional nanosheets to the tumor targeting peptide in the complex C described in step (4) is 0.2 to 1:1; The concentration of the two-dimensional nanosheets in the complex C described in step (4) is 0.2-1 mg / mL.

4. The method for preparing a nanocomposite for treating tumors according to claim 3, characterized in that: The mass ratio of the two-dimensional nanosheet and the NRF-2 protein inhibitor described in step (1) is 0.2:1; The mass ratio of the two-dimensional nanosheets to the ferritin targeting peptide in the complex A described in step (2) is 0.2:1; The concentration of lipid droplets in the lipid droplet mixture described in step (3) is 3.6 million / mL; The concentration of the two-dimensional nanosheets in the complex B described in step (3) is 0.2 mg / mL; The mass ratio of the two-dimensional nanosheets to the tumor targeting peptide in the complex C described in step (4) is 0.2:

1.

5. The method for preparing a nanocomposite for treating tumors according to claim 2, characterized in that: The organic solvent in step (1) is ethanol; The reaction time in step (1) is 12 to 24 hours; The water in steps (1), (2), (3) and (4) is oxygen-free water; The centrifugation conditions described in steps (1), (2), (3) and (4) are: 4°C, 10,000×g, for 15 minutes; The reaction time in steps (2) and (4) is 2 to 6 hours; The conditions of the ultrasonic treatment described in step (3) are: ultrasonic power 300 W, ultrasonic frequency 4000 Hz, ultrasonic treatment for 10 minutes, stop for 5 minutes, and cycle 5 times.

6. A nanocomposite for treating tumors, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the nanocomposite for treating tumors according to claim 6 in preparing a drug for treating tumors, characterized in that: The tumor is breast cancer.

8. Use of the nanocomposite for treating tumors according to claim 6 in the preparation of a drug for treating tumor metastasis or for treating postoperative tumor recurrence, characterized in that: The tumor is breast cancer.

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