A nanoparticle PEG-CPP30 / QR@FT and its preparation method and application

Abstract

The present invention provides a nanoparticle PEG-CPP30 / QR@FT and a preparation method and application thereof, belonging to the technical field of nanoparticle preparation, and comprising the following steps: S1, preparing a metal organic framework material FT; S2, preparing a drug-loaded particle QR@FT; S3, preparing a nanoparticle PEG-CPP30 / QR@FT. The present invention adopts the above-mentioned nanoparticle PEG-CPP30 / QR@FT and a preparation method and application thereof, and the prepared nanoparticle PEG-CPP30 / QR@FT is a heat shock protein inhibitor with a definite effect.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticle preparation, and in particular to a nanoparticle PEG-CPP30 / QR@FT and a preparation method and application thereof. Background Art

[0002] Breast cancer refers to the phenomenon of uncontrolled proliferation of breast epithelial cells under the influence of multiple carcinogenic factors. Among female patients, breast cancer has surpassed lung cancer to become the most common diagnosed cancer, seriously threatening women's life and health. At present, the commonly used means of treating breast cancer are surgery, chemotherapy, radiotherapy, etc., but single treatment methods have great side effects and limited effects, so combined treatment has gradually become a new strategy for treating breast cancer.

[0003] Phototherapy is a very promising tumor treatment method with advantages such as strong specificity and few side effects, including photodynamic therapy (PDT) and photothermal therapy (PTT). PDT mainly kills tumor cells by producing ROS; PTT uses local high temperature to exert anti-tumor effects. Among them, mild-temperature photothermal therapy (mPTT) can achieve anti-tumor purposes under relatively low temperature conditions, with the advantages of high safety and few side effects on normal tissues. However, during phototherapy, the upregulation of heat shock protein expression seriously hinders the therapeutic efficiency of phototherapy. In PDT, the generation of ROS will aggravate the hypoxic environment in tumor cells and cause oxidative damage to macromolecular proteins in cells, such as cleavage, cross-linking, unfolding and aggregation. In this case, cells will deal with these injuries by upregulating heat shock proteins. In mPTT, cells will quickly respond to heat resistance, and cell damage can be easily repaired by heat shock proteins, thereby reducing the anti-tumor effect. Therefore, how to inhibit the heat shock protein response has become an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present invention is to provide a nanoparticle PEG-CPP30 / QR@FT and a preparation method and application thereof. The prepared nanoparticle PEG-CPP30 / QR@FT is an effective heat shock protein inhibitor.

[0005] To achieve the above object, the present invention provides a method for preparing nanoparticles PEG-CPP30 / QR@FT, comprising the following steps:

[0006] S1. Preparation of metal organic framework material FT;

[0007] S2. Preparation of drug-loaded particles QR@FT;

[0008] S3. Preparation of nanoparticles PEG-CPP30 / QR@FT.

[0009] Preferably, in step S1, the metal organic framework material FT is prepared by the following specific operations:

[0010] Prepare FeCl with DMF 3 6H 2 O, TCPP, benzoic acid solution, add the prepared solution into a penicillin bottle, add a polytetrafluoroethylene magnetic stirrer, stir, centrifuge, remove the supernatant, wash the obtained product with DMF, and dry it in a vacuum drying oven to obtain.

[0011] Preferably, in step S2, the drug loading process of the drug-loaded particles QR@FT is: drug loading time is 6 to 144 h; quercetin: FT is 0.5:1 to 5:1; drug concentration is 1 to 20 mg / mL.

[0012] Preferably, the drug loading is 54.90±1.39%.

[0013] Preferably, in step S3, the nanoparticles PEG-CPP30 / QR@FT are prepared by the following specific operations:

[0014] PEG-CPP30 was evenly dissolved in pure water, and QR@FT:PEG-CPP30 was added to QR@FT drug-loaded particles at a mass ratio of 20:1 to 0.5:1. After being evenly suspended, the mixture was placed on a magnetic stirrer and stirred at a speed of 100 to 1200 rpm for 0.25 to 5 h. After centrifugal freeze drying, nanoparticles PEG-CPP30 / QR@FT were obtained.

[0015] The present invention also provides a nanoparticle PEG-CPP30 / QR@FT prepared by the above-mentioned method for preparing the nanoparticle PEG-CPP30 / QR@FT.

[0016] The present invention also provides a use of the nanoparticle PEG-CPP30 / QR@FT as described above in the preparation of a drug for treating breast cancer.

[0017] The present invention also provides a use of the nanoparticle PEG-CPP30 / QR@FT as described above in inhibiting heat shock proteins.

[0018] Therefore, the present invention adopts the above-mentioned nanoparticle PEG-CPP30 / QR@FT and its preparation method and application, and the prepared nanoparticle PEG-CPP30 / QR@FT is an effective heat shock protein inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1is the scanning electron microscopy image of PEG-CPP30 / QR@FT;

[0020] Figure 2 is the change of particle size distribution of PEG-CPP30 / QR@FT;

[0021] Figure 3 This is the Zeta potential measurement diagram of PEG-CPP30 / QR@FT;

[0022] Figure 4 is the Fourier infrared spectrum of PEG-CPP30 / QR@FT;

[0023] Figure 5 is the DPBF remaining after laser irradiation of different preparations;

[0024] Figure 6 The in vitro temperature rise curves of the three preparations are shown;

[0025] Figure 7 is the temperature rise curve of PEG-CPP30 / QR@FT nanoparticles with different concentrations;

[0026] Figure 8 The temperature rise curves of PEG-CPP30 / QR@FT nanoparticles under different irradiation powers;

[0027] Fig. 9 The photothermal cycle diagram of PEG-CPP30 / QR@FT;

[0028] Fig.10 is the cell survival rate of MCF-7 cells after FT acted on them for different time periods; Fig.10 (A) is 24h; Fig.10 (B) is 48h;

[0029] Fig.11 The cell inhibition rate of different treatment groups on MCF-7 cells; *P<0.05, **P<0.01, ***P<0.001, comparison among groups;

[0030] Fig.12 The results of apoptosis detection of MCF-7 cells treated with different groups;

[0031] Fig.13 The percentage of normal cells, apoptotic cells and necrotic cells in different treatment groups is statistically shown; *P<0.05, **P<0.01, ***P<0.001, comparison between groups;

[0032] Fig.14 The photothermal imaging of the tumor site when each nanoparticle acts on mice;

[0033] Fig.15 The temperature rise curve of the tumor site when each nanoparticle acts on mice (n=5);

[0034] Fig.16 The changes in body weight over time after the nanoparticles in each group acted on tumor-bearing nude mice (n=5);

[0035] Fig.17 The changes of tumor volume over time after the nanoparticles in each group acted on tumor-bearing nude mice (n=5); among them, *P<0.05, **P<0.01, ***P<0.001, comparison between groups; #P<0.05, ##P<0.01, ###P<0.001, comparison with the control group;

[0036] Fig.18 The tumor images of each group of nanoparticles after 12 days of exposure to tumor in nude mice (n=5); Fig.18 (A) in the middle is the control group; Fig.18 (B) in the figure is the QR group; Fig.18 (C) in the figure is the QR@FT group; Fig.18 (D) in the middle is the PEG-CPP30 / QR@FT group; Fig.18 (E) in the figure is the FT+L group; Fig.18 (F) in the figure is the FT+QR+L group; Fig.18 (G) in the figure is the QR@FT+L group; Fig.18 (H) in the middle is the PEG-CPP30 / QR@FT+L group;

[0037] Fig.19 The tumor weight of tumor-bearing nude mice treated with nanoparticles in each group for 12 days (n=5); *P<0.05, **P<0.01, ***P<0.001, comparison between groups; #P<0.05, ##P<0.01, ###P<0.001, comparison with the control group;

[0038] Fig. 20 The expression of HSP70, HSP90 and HSP27 after different groups of nanoparticles acted on MCF-7 cells; *P<0.05, **P<0.01, ***P<0.001, comparison among groups; Fig. 20 (a) is the protein band diagram after different groups of nanoparticles acted on MCF-7; Fig. 20 (b) shows the expression of HSP70 after different groups of nanoparticles acted on MCF-7; Fig. 20 (c) shows the expression of HSP90 after different groups of nanoparticles acted on MCF-7; Fig. 20 (d) shows the expression of HSP27 after different groups of nanoparticles acted on MCF-7;

[0039] Fig.21 It is a fluorescence image of the expression of heat shock proteins after each nanoparticle acts on tumor tissue (×200, i.e., the magnification of the fluorescence microscope is 200 times);

[0040] Fig. 22 Statistics of heat shock protein fluorescence values ​​after each nanoparticle acted on tumor tissue; *P<0.05, **P<0.01, ***P<0.001, comparison among groups. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0042] Embodiment 1

[0043] The present invention provides a method for preparing nanoparticles PEG-CPP30 / QR@FT, and the specific steps are as follows:

[0044] S1. Preparation of metal organic framework material FT.

[0045] The synthesis was carried out by heating magnetic stirring method: FeCl 3 6H 2 O ((10-50 mg, 1-6 mL, preferably 30 mg, 2 mL), TCPP (tetrakis (4-carboxyphenyl) porphine) (5-25 mg, 1-6 mL, preferably 10 mg, 4 mL), benzoic acid solution (100-500 mg, 1-6 mL, preferably 280 mg, 4 mL), add the prepared solution to a 25 mL cillin bottle, add a 1.5 cm polytetrafluoroethylene magnetic stirrer, stir continuously at 100-1200 rpm (preferably 800 rpm) at 30-120° C. (preferably 90° C.) for 0.5-10 (preferably 5 h), centrifuge, remove the supernatant, wash the product with DMF 1-5 times (preferably 3 times), and dry it in a vacuum drying oven at 30-100° C. (preferably 60° C.) for 6-72 h (preferably 24 h). The synthesized sample is a dry purple-red powdery solid, odorless, and is stored in a desiccator at room temperature and away from light.

[0046] S2. Preparation of drug-loaded particles QR@FT.

[0047] The drug loading process was determined by orthogonal experiment, and the drug loading process was as follows: drug loading time was 6 to 144 h; quercetin: FT was 0.5:1 to 5:1; drug concentration was 1 to 20 mg / mL.

[0048] The process of quercetin loading on FT was stable and feasible, with a drug loading of 54.90±1.39%, and drug-loaded particles QR@FT were successfully synthesized.

[0049] S3. Preparation of nanoparticles PEG-CPP30 / QR@FT.

[0050] The surface of drug-loaded particles QR@FT was modified by reaction coating method. Firstly, QR@FT drug-loaded particles were prepared according to the optimal drug loading process selected above.

[0051] PEG-CPP30 is evenly dissolved in pure water, and QR@FT:PEG-CPP30 is added to QR@FT drug-loaded particles at a mass ratio of 20:1-0.5:1 (preferably 5:1). After being evenly suspended, the mixture is placed on a magnetic stirrer and stirred at a speed of 100-1200 rpm (preferably 600 rpm) for 0.25-5 h (preferably 1 h). After centrifugal freeze drying, nanoparticles PEG-CPP30 / QR@FT are obtained.

[0052] Characterization of nanoparticles PEG-CPP30 / QR@FT.

[0053] Scanning electron microscopy analysis of PEG-CPP30 / QR@FT.

[0054] Take an appropriate amount of PEG-CPP30 / QR@FT, add methanol to disperse, drop the sample on the surface of the silicon wafer, and evaporate it at room temperature. Before testing, fix the silicon wafer on the sample stage, vacuum and spray gold, take out the sample and place it in the sample exchange room, adjust the focus and observe and take pictures. The results are shown in Figure 1 .

[0055] Scanning electron microscopy results showed that the particle size of the modified drug-loaded particles was about 330nm and their morphology was olive-shaped.

[0056] Particle size determination of PEG-CPP30 / QR@FT.

[0057] Weigh an appropriate amount of PEG-CPP30 / QR@FT nanoparticles, add 3 mL of anhydrous ethanol and sonicate for 1 min to disperse them evenly, take 1 mL of the dispersion and dilute it with 9 mL of anhydrous ethanol to obtain a sample solution. Slowly inject the sample into the Malvern cuvette, select the dispersion medium, set the measurement temperature to 25 °C, and measure the same sample three times to obtain the particle size distribution of the sample. The results are shown in Figure 2 .

[0058] The particle size results showed that the particle size of nanoparticle PEG-CPP30 / QR@FT was 336.57±4.92nm, which was consistent with the scanning electron microscopy results.

[0059] Zeta potential determination of PEG-CPP30 / QR@FT.

[0060] Weigh a certain amount of PEG-CPP30 / QR@FT powder, add 4 mL of anhydrous ethanol and ultrasonicate for 1 min to disperse it evenly, take 1 mL of the dispersion and dilute it with 5 mL of pure water to obtain the sample solution. Slowly inject the sample solution into the Zeta potential measurement dish and put it into the instrument measurement tank. Select the dispersion medium, set the measurement temperature to 25°C, measure the same sample three times, and obtain the Zeta potential of the sample. The results are shown in Figure 3 .

[0061] The experimental results show that the Zeta potential of the nanoparticle PEG-CPP30 / QR@FT is -12.40±0.17mV.

[0062] Fourier transform infrared spectroscopy analysis of PEG-CPP30 / QR@FT.

[0063] Weigh appropriate amounts of PEG-CPP30, QR@FT, and PEG-CPP30 / QR@FT sample powders, disperse them with potassium bromide in a dry environment, grind them thoroughly, press them into thin sheets, and precipitate them at 4000-400 cm -1 The infrared spectrum is obtained by scanning within the range of Figure 4 .

[0064] Depend on Figure 4 It can be seen that the characteristic peaks of PEG-CPP30 / QR@FT are weaker and compared with the spectrum of QR@FT, the peaks in the range of 900-500 cm -1 The characteristic peak of the benzene ring vibration at 1630 cm-1 disappeared. The peak at this location is similar to that of PEG-CPP30. This may be due to the fact that the membrane-penetrating peptide covers up the vibration peak of the benzene ring. -1 The characteristic peak of PEG-CPP30 appeared at , which shows that PEG-CPP30 was successfully coated on the surface of QR@FT.

[0065] Evaluation of the optical properties of PEG-CPP30 / QR@FT.

[0066] Investigation of the photodynamic properties of nanoparticles in vitro.

[0067] DPBF was used as a singlet oxygen scavenger. DPBF has high specificity for singlet oxygen, can form endoperoxides and decompose into 1,2-dibenzoylbenzene. Once combined with singlet oxygen, DPBF is irreversibly oxidized, and the absorption intensity at UV-visible light (415nm) decreases rapidly. Therefore, the ability of nanoparticles to produce singlet oxygen can be examined by measuring the remaining amount of DPBF. In the experiment, 3 ml of DPBF solution was placed in a quartz dish, and pure DPBF was used as the control group. The experimental groups were added with 20 μL of QR@FT, PEG-CPP30 / QR@FT solution and 50 μg / mL FT solution containing QR at a concentration of 50 μg / mL, respectively, and the FT solution was heated at 0.40 W / cm 2 The solution was irradiated with a 660nm laser and the absorbance was measured every 15s. Figure 5 .

[0068] Investigation of the photothermal properties of nanoparticles in vitro.

[0069] In vitro temperature increase experiment investigation of nanoparticles.

[0070] PBS was used to prepare QR@FT, PEG-CPP30 / QR@FT solutions with a QR concentration of 50 μg / mL, and FT solutions with a concentration of 50 μg / mL. PBS solution was used as a control. 1 mL of each solution was placed in a 24-well plate and the power was 0.40 W / cm 2 The solution in the well was irradiated with a 660nm laser, and an infrared thermal imaging camera was used to record the temperature changes of the solution after laser irradiation for different times (0s, 30s, 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min), and a temperature rise curve was drawn. Figure 6 .

[0071] The experimental results show that the temperature rise performance of the nanoparticle PEG-CPP30 / QR@FT is relatively mild, reaching 44.7°C after 10 minutes of irradiation, and has mild photothermal performance.

[0072] Investigation of concentration dependence and power dependence of the photothermal performance of PEG-CPP30 / QR@FT.

[0073] In order to investigate the photothermal performance of PEG-CPP30 / QR@FT nanoparticles with different concentrations and the heating effect of PEG-CPP30 / QR@FT under different irradiation powers, PEG-CPP30 / QR@FT solutions with QR concentrations of 5, 15, 30, and 50 μg / mL were prepared respectively. 2 The power of irradiation was used to investigate the concentration dependence of the photothermal performance of the nanoparticles. At the same time, 0.16 W / cm 2 , 0.24W / cm2 , 0.32W / cm 2 , 0.40W / cm 2 Four powers were used to irradiate the PEG-CPP30 / QR@FT solution containing 50 μg / mL QR to investigate the power dependence of the photothermal properties of the nanoparticles. Figure 7 , Figure 8 .

[0074] The photothermal performance of PEG-CPP30 / QR@FT is concentration-dependent, time-dependent, and power-dependent. Within a certain range, the temperature of the nanoparticle solution gradually increases with increasing temperature, power, and time.

[0075] Investigation of photothermal stability of PEG-CPP30 / QR@FT.

[0076] In order to investigate the photothermal stability of PEG-CPP30 / QR@FT, the nanoparticles were subjected to 4 cycles of irradiation on and off. 1 mL of the solution was placed in a 24-well plate and the irradiation power was 0.40 W / cm 2 The solution in the well was irradiated with a 660nm laser for 240s, the power was turned off, and the irradiation was stopped for 330s. The cycle was repeated 4 times. During the irradiation process, the solution temperature was recorded every 30s with a thermal imaging camera and a cycle curve was drawn. Fig. 9 .

[0077] Depend on Fig. 9 It can be seen that with the increase in the number of illumination cycles, the maximum temperature of PEG-CPP30 / QR@FT nanoparticles can always be maintained at around 44°C, proving that PEG-CPP30 / QR@FT nanoparticles have good photothermal stability.

[0078] In vivo and in vitro cell and animal pharmacodynamic evaluation of nanoparticle PEG-CPP30 / QR@FT.

[0079] Human breast cancer MCF-7 cells were selected as the cell model for subsequent related experiments.

[0080] In vitro cellular safety evaluation of material FT.

[0081] Take the vigorously growing MCF-7 cells and inoculate them on a 96-well plate at 100 μL / well. After the cells adhere to the wall, remove the culture medium and add FT culture medium containing different concentrations (5, 10, 25, 50, 75, 100 μg / mL). Set up a control group and set up 6 replicate wells for each concentration. After culturing for 24 and 48 hours, respectively, accurately add 5 mg / mL MTT solution (20 μL / well) and continue incubating in the incubator for 4 hours. Remove the supernatant and add 100-150 μL of DMSO to each well. Shake in the dark for 10 minutes. Use an enzyme reader to set the excitation wavelength to 490 nm, measure the absorbance of each well, and calculate the cell survival rate. The experimental results are shown in Fig.10 .

[0082] Depend on Fig.10 It can be seen that, in the absence of light, when the action time is within 48 hours, the cell survival rate of MCF-7 cells treated with various doses of FT (5, 10, 25, 50, 75, 100 μg / mL) is above 90%, and there is no significant difference in the cell survival rate of the experimental group compared with the control group. This fully demonstrates that FT is highly safe in the concentration range of 0 to 100 μg / mL and has good biocompatibility.

[0083] MTT cell pharmacodynamic evaluation of nanoparticles PEG-CPP30 / QR@FT.

[0084] MCF-7 cells in the logarithmic growth phase were taken and inoculated on 96-well plates at 100 μL / well. The cells were incubated in an incubator overnight to allow the cells to adhere to the wall and grow. The experiment was divided into 8 groups, namely the control group, Laser group, QR group, QR@FT group, PEG-CPP30 / QR@FT group, FT+L group, QR@FT+L group and PEG-CPP30 / QR@FT+L group. The old culture medium was replaced with fresh cell culture medium containing preparations (QR, QR@FT, PEG-CPP30 / QR@FT, FT) at different concentrations (10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL). After incubation for 32 hours in the non-laser group, the culture medium was discarded and fresh culture medium without the preparation was added. In the laser group, after replacing the fresh culture medium, 0.4 W / cm 2 The cells were irradiated with 660nm laser for 3min. Each group was then incubated in the incubator for 16h. After the incubation, 20μL of 5mg / mL MTT solution was added to each well and incubated for 4h. Then 100-150μL of DMSO was added and the cells were shaken in the dark for 10min. The excitation wavelength was set to 490nm using a microplate reader, and the absorbance of each well was measured to calculate the cell survival rate. The results are shown in the table. Fig.11 .

[0085] The experimental results show that the cytotoxicity of each dosing group to MCF-7 cells is dose-dependent. Regardless of the presence or absence of light, the nanoparticle PEG-CPP30 / QR@FT showed the strongest pharmacodynamic effect. After laser irradiation, the efficacy of each group of preparations was significantly improved. The cell inhibition rate of the PEG-CPP30 / QR@FT+L group (containing QR concentration of 70μg / mL) reached 91.11%, which was much higher than the 44.71% of the QR group and 59.65% of the FT+L group at the same concentration. This shows that chemotherapy-phototherapy combined treatment is superior to chemotherapy or phototherapy alone, and achieves the purpose of combined treatment.

[0086] Evaluation of the apoptosis-promoting effect of nanoparticles PEG-CPP30 / QR@FT.

[0087] The grouping was consistent with the MTT experiment. The MCF-7 cells with vigorous growth were inoculated in a 24-well plate. After culturing for 12 hours, the culture medium was discarded and 1 mL of fresh cell culture medium containing 50 μg / mL quercetin preparations (QR, QR@FT, PEG-CPP30 / QR@FT, FT) was added. After culturing for 32 hours in the non-laser group, the fresh culture medium was replaced and cultured for another 16 hours. After the fresh culture medium was replaced in the laser group, 0.4 W / cm 2 Irradiate with 660nm laser for 3min, and continue to culture for 16h. After the culture is completed, the culture medium of each well is aspirated into a centrifuge tube for use, and then 0.3mL of 0.25% trypsin is added to each well, and the cells are placed in a 37°C incubator for digestion for 1-2min, and then observed under an inverted microscope. The digestion is terminated when the cell morphology becomes round and the gap becomes larger; the cell suspension is blown evenly with a pipette, and then transferred to a centrifuge tube, centrifuged at 1600rpm for 5min, and the supernatant is gently poured out. The cells are washed once with 1mL PBS buffer; 295μL Annexin V-FITC binding solution and 5μL Annexin V-FITC dye solution are added, and after incubation for 15min, 10μL of PI dye solution is added, mixed evenly, incubated for 5min, and then stored on ice, filtered through a nylon mesh (300 mesh), and cell apoptosis detection is completed within 1h. Flow cytometry results are shown in Figure 12-13 .

[0088] The apoptosis results were consistent with the MTT results. Regardless of whether it was illuminated or not, the PEG-CPP30 / QR@FT+L group showed the best pro-apoptotic effect. After illumination, the pro-apoptotic effect of each group was more obvious. Fig.13The results showed that the percentage of apoptotic cells in the PEG-CPP30 / QR@FT+L group (QR concentration was 50 μg / mL) reached 85.23%, which was much higher than that in other groups, proving once again that chemotherapy-phototherapy combination therapy is superior to chemotherapy or phototherapy alone.

[0089] In vivo animal pharmacodynamic evaluation of nanoparticles PEG-CPP30 / QR@FT.

[0090] Establishment of tumor-bearing nude mouse model.

[0091] Balb / C nude mice (4-5 weeks, 20 g) were used to construct a breast cancer animal model. The specific operation was as follows: the purchased nude mice were first adaptively raised in the animal room for 3 days, then the cultured MCF-7 cells were resuspended in PBS solution, and then 100 μL of MCF-7 cell suspension was slowly injected subcutaneously into the right upper limb of each nude mouse. The mice continued to be raised under standard conditions, and the tumor size was monitored at any time with a vernier caliper. Once the mice had tumors, they could be used for experiments.

[0092] Investigation of the in vivo photothermal properties of nanoparticles.

[0093] Tumor-bearing nude mice were randomly divided into 4 groups, namely control group, FT group, QR@FT group, and PEG-CPP30 / QR@FT group (all calculated as QR: 8 mg / kg). After a single administration, 0.4 W / cm 2 The 660nm laser was irradiated at 0min, 1min, 2min, 3min, 5min, and 7min, and the temperature of the tumor site was recorded with a thermal imaging camera, and a temperature rise curve was drawn to examine the photothermal conversion effect of each nanoparticle in the mouse body. The experimental results are shown in Figure 14-15 .

[0094] Effects of nanoparticles on body weight and tumor growth in tumor-bearing nude mice.

[0095] Tumor-bearing nude mice were randomly divided into 8 groups, namely control group, QR group, QR@FT group, PEG-CPP30 / QR@FT group, FT+L group, FT+QR+L group, QR@FT+L group, and PEG-CPP30 / QR@FT+L group. The control group was injected with 100 μL of normal saline via the tail vein, and the other groups were injected with 100 μL of the corresponding drugs (all calculated as QR: 8 mg / kg) via the tail vein, and the drugs were given every other day. The laser group was given 0.4 W / cm2 at the tumor site 9 hours after the drug was given on the same day. 2 The mice were irradiated with 660nm laser for 3min. The drugs were administered for 12 consecutive days. The mice were weighed and the tumor volume was measured once a day. After the last administration, the mice were killed by dislocation of the neck, and the tumors were dissected, weighed and photographed. The results are shown in Figure 16-19.

[0096] In the in vitro cell experiment, the prepared nanoparticles showed good anti-tumor effect. In order to further investigate its efficacy, a tumor-bearing nude mouse model was constructed and in vivo efficacy experiments were conducted. During the treatment, the weight changes of mice in each group were not much different, and there was no obvious downward trend. It can be shown that the nanoparticles have good in vivo safety and did not cause obvious harm to mice as a whole. The results of the tumor inhibition experiment showed that compared with the control group, the growth of tumors in mice in each administration group was inhibited to varying degrees. The modified preparation group was better than the unmodified preparation group, and the light group was better than the non-light group; the PEG-CPP30 / QR@FT+L group showed the strongest tumor inhibition effect. After the mice were killed, the tumors obtained by dissection were weighed. The tumor weight of the control group was 1.09±0.07g, while the tumor weight of the PEG-CPP30 / QR@FT+L group was only 0.20±0.02g, which was much less than the control group. This fully shows that the nanoparticles have a good anti-tumor effect, and after combining with phototherapy, the anti-tumor effect of the nanoparticles has been further improved, achieving the purpose of combined chemotherapy and phototherapy.

[0097] Evaluation of the anti-three heat shock proteins effects of nanoparticles PEG-CPP30 / QR@FT in vivo and in vitro.

[0098] Heat shock proteins HSP27, HSP70 and HSP90 were detected by Western blot.

[0099] 1.1 Experimental Grouping

[0100] The experiment was divided into 5 groups, namely control group, FT+L group, FT+QR+L group, QR@FT+L group, and PEG-CPP30QR@FT+L group. The expression levels of heat shock proteins HSP70, HSP90, and HSP27 in cells of each group were measured.

[0101] 1.2 Cell lysis

[0102] The original culture medium of each group of cells was discarded, and the cells were washed once with 1×PBS. Then, 300 μL / well of RIPA was added and shaken at 4°C for 30 min. Then, the cells in each well were repeatedly blown with a pipette, and the cell lysate was transferred to a 1.5 mL centrifuge tube to obtain a cell protein sample.

[0103] 1.3 Western blot detection of protein expression levels in cells

[0104] 1) Protein quantification: Take out the frozen supernatant for protein quantification.

[0105] a. Preparation of BSA standard

[0106] The preparation of BSA standard system is shown in Table 4.

[0107] Table 4 BSA dilution method

[0108] Vial Dilution volume (μL) 5mg / mL BSA volume (μL) BSA final concentration (μg / mL) A 0 100 5000 B 70 30 1500 C 30 Take 60 from tube B 1000 D 20 Take 60 from tube C 750 E 30 Take 60 from tube D 500 F 60 Take 60 from tube E 250 G 60 Take 60 from tube F 125 H 60 0 0 = Blank (blank hole)

[0109] bDetection method

[0110] ① Take 20 μL of different concentrations of standard and sample and add them into the reaction tube.

[0111] ②Add 1 mL of Bradford staining solution, mix well, and incubate at room temperature for 10 minutes.

[0112] ③Measure the absorbance at 595nm with a spectrophotometer, and calibrate the instrument to zero using a cuvette filled with water. Then measure the absorbance of the sample.

[0113] ④ Draw a standard curve based on the absorbance of the BSA standard. Calculate the protein concentration in the sample based on the standard curve and the dilution multiple of the sample.

[0114] 2) Glue making

[0115] a. Prepare SDS denatured 10% polyacrylamide gel (lower separation gel, single-sided). The preparation method is shown in Table 5.

[0116] Table 5 10% polyacrylamide gel formula

[0117] Element volume DDW 4.825mL 40% Acrylamide 2.475mL 1.5MTris-Cl (pH8.8) 2.5mL 10% SDS 100μL 10% ammonium persulfate 100μL TEMED 4.0μL

[0118] After mixing, quickly pour the gel to about 2 / 3 of the total height of the glass plate, then add 1 mL of water-saturated n-butanol on top of the gel to ensure the smoothness of the upper layer of the gel, and let it stand until the gel solidifies.

[0119] b. Prepare SDS denatured 5% polyacrylamide gel (upper concentrated gel, single-sided). The preparation method is shown in Table 6.

[0120] Table 65% polyacrylamide gel formula

[0121]

[0122]

[0123] After mixing, quickly pour the gel until the glass plate is filled, insert the comb, and let it stand until the gel solidifies. Remove the comb before electrophoresis, place the gel in 1× Tris-glycine electrophoresis buffer, and blow clean the sample well with a syringe needle.

[0124] 3) Electrophoresis

[0125] After mixing the protein sample with 5× loading buffer (containing β-mercaptoethanol), boil and denature for 5 minutes, and place on ice for 5 minutes. Take an appropriate amount of protein sample and load it on SDS denaturing 10% polyacrylamide gel electrophoresis (SDS-PAGE) until the target protein is effectively separated and then stop the electrophoresis.

[0126] 4) Transfer

[0127] After electrophoresis, the gel was taken out and placed in a special sandwich clip for membrane transfer. The gel was placed on the negative electrode and the PVDF membrane was placed on the positive electrode. The membrane was transferred in a transfer buffer at 4°C and 350mA constant current for 2 hours to transfer the proteins in the gel to the PVDF membrane to form a blot.

[0128] 5) Blocking and washing

[0129] Place the membrane in 5% skim milk powder and block it at room temperature for 2 hours. Then cut the membrane according to the protein blotting position and place it in an incubation box containing (1:1000) primary antibody and shake it at 4°C overnight. Place the membrane in 1×TBST solution and wash it for 5 minutes with shaking for a total of 4 times. Incubate the secondary antibody at room temperature for 1.5 hours, and continue to use 1×TBST solution, shake and rinse for 5 minutes, for a total of 4 times.

[0130] 6) Development and protein quantity statistics

[0131] Place the membrane in Western LightningTM chemiluminescent reagent for 30 seconds, immediately place the membrane in an exposure box, and expose the photosensitive film in a dark room for 1 minute, then develop and fix it. The film was scanned with an Epson PerfectionV39 scanner to analyze the brightness value of each group of protein bands. The method is to calculate the ratio of the brightness value of the protein band of each sample to the brightness value of the corresponding internal reference band, obtain the corrected protein band brightness value, and draw a bar graph. The results are shown in Fig. 20 .

[0132] The results of Western blot experiments show that simple phototherapy (FT+L) significantly upregulates the expression of three heat shock proteins in cells, seriously affecting the efficiency of phototherapy. When quercetin is present in the drug delivery system, the expression of heat shock proteins in cells is inhibited to varying degrees, and compared with free quercetin (FT+QR), the drug-loaded particles (QR@FT, PEG-CPP30 / QR@FT) prepared by the present invention show a more excellent heat shock protein inhibitory effect. The modified drug-loaded particles PEG-CPP30 / QR@FT have the strongest anti-heat shock protein effect, which may be because after modification, MCF-7 enhances the uptake of nanoparticles, thereby promoting their function. The experimental results show that nanoparticles can effectively solve the problem of heat shock protein response during phototherapy and improve the efficiency of phototherapy. At the same time, this also shows that nanoparticles can enhance the anti-tumor effect by downregulating the heat shock protein mechanism.

[0133] Immunofluorescence was used to detect HSP27, HSP70 and HSP90 in tumor tissues.

[0134] With the help of immunofluorescence technology, the heat shock proteins HSP27, HSP70, and HSP90 in the tumor tissues of mice in the Control group, FT+L group, FT+QR+L group, QR@FT+L group, and PEG-CPP30 / QR@FT+L group were selected for determination. The tumor tissues were prepared into dry sections, and then the sections were dewaxed and hydrated, immersed in citric acid antigen retrieval solution, and heated in a pressure cooker for 15 minutes. Place in Triton X-100 (mass fraction 0.5%) for 10 minutes, block with bovine serum albumin, and stand at room temperature for 1 hour. Incubate with primary antibody (the dilution ratio of HSP27, HSP70, and HSP90 is 1:500), incubate overnight at 4°C, and rinse with PBS three times. Incubate with secondary antibody (1:200), incubate at room temperature in the dark for 2 hours, and rinse with PBS. After drying, observe and photograph under a fluorescence microscope, and use ImageJ software to semi-quantitatively analyze the images and calculate the average fluorescence intensity. The experimental results are as follows Figure 21-22 .

[0135] The experimental results showed that phototherapy alone (FT+L group) significantly upregulated the expression of three heat shock proteins, HSP27, HSP70, and HSP90, in mouse tumor tissues, seriously hindering the effect of phototherapy. When quercetin was present in the drug delivery system, the expression of heat shock proteins in mouse tumor tissues was inhibited to varying degrees. The prepared drug-loaded particles (QR@FT, PEG-CPP30 / QR@FT) were more effective than quercetin monomers (FT+QR), and the modified nanoparticles PEG-CPP30 / QR@FT had the strongest anti-heat shock protein effect. This further proves that the prepared nanoparticles effectively solve the problem of heat shock protein response in phototherapy and weaken the self-protection mechanism of tumors.

[0136] Therefore, the present invention adopts the above-mentioned nanoparticle PEG-CPP30 / QR@FT and its preparation method and application, and the prepared nanoparticle PEG-CPP30 / QR@FT is an effective heat shock protein inhibitor.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing nanoparticles PEG-CPP30 / QR@FT, characterized in that: The following steps are involved: S1. Preparation of metal organic framework material FT; S2. Preparation of drug-loaded particles QR@FT; S3, preparation of nanoparticles PEG-CPP30 / QR@FT; In step S1, a metal organic framework material FT is prepared, and the specific operation is as follows: Prepare FeCl3·6H2O, TCPP and benzoic acid solutions respectively with DMF, add the prepared solutions into a vial, add a polytetrafluoroethylene magnetic stirrer, stir, centrifuge, remove the supernatant, wash the obtained product with DMF, and dry it in a vacuum drying oven to obtain; In step S2, the drug loading process of the drug-loaded particles QR@FT is as follows: drug loading time is 6 to 144 h; quercetin: FT is 0.5:1 to 5:1; drug concentration is 1 to 20 mg / mL; The drug loading was 54.90 ± 1.39%; In step S3, nanoparticles PEG-CPP30 / QR@FT are prepared, and the specific operation is as follows: PEG-CPP30 was evenly dissolved in pure water, and QR@FT:PEG-CPP30 was added to QR@FT drug-loaded particles at a mass ratio of 20:1 to 0.5:

1. After being evenly suspended, the mixture was placed on a magnetic stirrer and stirred at a speed of 100 to 1200 rpm for 0.25 to 5 h. After centrifugal freeze drying, nanoparticles PEG-CPP30 / QR@FT were obtained.

2. Nanoparticles PEG-CPP30 / QR@FT prepared by the method for preparing nanoparticles PEG-CPP30 / QR@FT as claimed in claim 1.

3. Use of the nanoparticle PEG-CPP30 / QR@FT as claimed in claim 2 in the preparation of a drug for treating breast cancer.

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