CN111821283A - A kind of cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and preparation method thereof - Google Patents

Abstract

The invention discloses a Prussian blue nanoparticle coated with zinc glutamate and loaded with triphenylphosphine-lonidamine by a cancer cell membrane and a preparation method thereof. A zinc glutamate layer, the surface of the outermost zinc glutamate layer has a load layer loaded with triphenylphosphine-lonidamine, and the load layer is coated with a tumor cell membrane layer. The invention has the ability to target tumor cells, has a long circulation time in the body, can accumulate in mitochondria and cause dysfunction, reduce the synthesis of ATP, down-regulate the synthesis of various heat shock proteins, cause cell apoptosis at the same time, and effectively enhance the low temperature light of tumors. Efficacy of heat therapy.

Description

A Cancer Cell Membrane Encapsulated Zinc Glutamate Loaded with Triphenylphosphine-Lonidamine Prussian blue nanoparticles and preparation method thereof

technical field

The invention belongs to the technical field of drug carriers, in particular to a zinc glutamate-coated Prussian blue nanoparticle coated with a cancer cell membrane and loaded with triphenylphosphine-lonidamine and a preparation method thereof.

Background technique

Cancer, a type of malignant tumor, is a non-communicable disease with very high morbidity and mortality worldwide. According to the International Institute for Cancer Research, there will be about 20 million new cancer cases worldwide in 2025. At present, traditional cancer treatment methods still occupy the main position, such as chemotherapy, radiotherapy and surgery, which have disadvantages such as high recurrence rate and strong side effects. As a new type of treatment, photothermal therapy is a treatment method that uses photothermal conversion agents to rapidly convert light energy into heat energy to kill tumor cells. Compared with traditional treatment methods, photothermal therapy has many advantages, but limited by the penetration depth of the laser, the deeper part of the tumor tissue is less heated, which is usually achieved by increasing the dosage of photothermal conversion agents and/or laser power. satisfactory treatment effect. However, due to non-selective thermal diffusion, high temperature may cause irreversible damage to normal tissues near the tumor, and may cause a series of side effects such as inflammation and tumor metastasis. Although low temperature does not cause normal tissue damage or other side effects near the tumor, its therapeutic effect is poor. Therefore, achieving low temperature photothermal therapy is an urgent problem to be solved in its clinical transformation.

Compared with normal cells, tumor cells overexpress heat shock proteins, which reduces their thermal sensitivity and maintains the activity of tumor cells at high temperature. Therefore, to enhance the heat sensitivity of tumor cells, it is an effective approach to combine heat shock protein inhibitors and photothermal conversion agents in the same nanosystem for photothermal therapy. Wiley Database (Advanced Materials, 2017, Volume 29, Page 1703588) reported that Liu Zhuang's group designed and constructed a one-dimensional nano-MOF material with indocyanine green as a ligand, loaded with gambogic acid to inhibit heat shock The effect of protein 90 achieved an excellent antitumor effect under low temperature (~43°C) photothermal therapy. Wiley database (advanced functional materials, 2016, volume 26, pages 3480-3489) reported that Zhou Jing's group used upconversion nanoparticles as carriers, siRNA blocked the synthesis of heat shock protein 70, and improved the effect of photothermal conversion preparations on tumors. the role of cells. Although these two solutions solve the problem of heat shock protein activity and expression, they can only target one heat shock protein, and there are many kinds of heat shock proteins and can play a role in photothermal therapy. Heat treatment effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects of the prior art, and to provide a cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticle loaded with triphenylphosphine-lonidamine.

Another object of the present invention is to provide a method for preparing the above-mentioned cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine.

Another object of the present invention is to provide the application of the above-mentioned cancer cell membrane-wrapped zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine.

The technical scheme of the present invention is as follows:

A cancer cell membrane-wrapped zinc glutamate-coated nanoparticle loaded with triphenylphosphine-lonidamine, comprising a Prussian blue nano-core, the surface of the Prussian blue nano-core is coated with at least one zinc glutamate layer, and is in the The surface of the outermost zinc glutamate layer has a load layer loaded with triphenylphosphine-lonidamine, and the load layer is coated with a tumor cell membrane layer.

In a preferred embodiment of the present invention, the tumor cell membrane layer is made of extracted HepG2 cell membrane.

In a preferred embodiment of the present invention, the triphenylphosphine-lonidamine is prepared by (2-bromomethyl)dimethylamine hydrobromide linking triphenylphosphine and lonidamine.

In a preferred embodiment of the present invention, the particle size is 100-200 nm.

Further preferably, the particle size of the Prussian blue nanocore is 70-90 nm.

As shown in Figure 1, the preparation method of the above-mentioned cancer cell membrane-wrapped zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine includes the following steps:

(1) Prussian blue nanoparticles are prepared by hydrothermal synthesis;

(2) Preparation of zinc glutamate-coated Prussian blue nanoparticles, including:

a. After mixing the above-mentioned Prussian blue nanoparticles with the polyvinylpyrrolidone aqueous solution, dropwise add zinc nitrate solution to react, and disperse after centrifugation and washing with water;

b, dropwise adding disodium glutamate solution to the material obtained in step a to react, and centrifugal washing to collect precipitation, repeating this for 1-3 times, and centrifugal collection to obtain zinc glutamate-coated Prussian blue nanoparticles;

(3) Preparation of zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine: The zinc glutamate-coated Prussian blue nanoparticles obtained in step (2) were dispersed in methanol, and then triphenyl glutamate was added. The methanol solution of triphenylphosphine-lonidamine was fully stirred, centrifuged and washed with water, and then the disodium glutamate solution was added for the reaction to obtain the triphenylphosphine-lonidamine-loaded zinc glutamate coated Prussian blue Nanoparticles;

(4) Preparation of cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles: the triphenylphosphine-lonidamine-loaded zinc glutamate obtained in step (3) was The encapsulated Prussian blue nanoparticles were dispersed in ultrapure water by ultrasonic, and the extracted tumor cell membrane was added dropwise during the ultrasonic process, collected by centrifugation after 2-5 minutes, and washed with water.

In a preferred embodiment of the present invention, the step (1) is: dissolving ferric chloride hexahydrate and citric acid monohydrate in ultrapure water to prepare a ferric chloride solution; then ferricyanide trihydrate Potassium and citric acid monohydrate are dissolved in ultrapure water to prepare potassium ferrocyanide solution; at 58-62 ℃, slowly add potassium ferrocyanide solution dropwise to the ferric chloride solution while stirring; Then continue stirring for 0.8-1.2 min at a constant temperature of 58-62 °C, then transfer to room temperature and stir for 4-6 min, then slowly pour 120 mL of acetone to induce crystallization, and sequentially collect by centrifugation, fully wash with water and vacuum freeze-drying to obtain the described Prussian blue nanoparticles.

In a preferred embodiment of the present invention, the frequency of the ultrasonic dispersion in the step (4) is 35-45 kHz.

Another technical scheme of the present invention is as follows:

The application of the above-mentioned cancer cell membrane-wrapped zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine in the preparation of low-temperature photothermal therapy drugs for tumors.

Another technical scheme of the present invention is as follows:

The application of the above-mentioned cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine in the preparation of a drug targeting mitochondria.

The beneficial effects of the present invention are:

1. The present invention has the ability to target tumor cells, has a long circulation time in the body, and can accumulate in mitochondria and cause dysfunction, reduce the synthesis of ATP, down-regulate the synthesis of various heat shock proteins, cause apoptosis at the same time, and effectively enhance the tumor. Efficacy of low temperature photothermal therapy.

2. The present invention can prolong the circulation time in the nanoparticle through the CD47 protein and phospholipid bilayer components on the cancer cell membrane, and the surface adhesion factor on the cancer cell membrane can actively target the same cancer cells.

3. The present invention can improve the problem of slow diffusion of lonidamine in the cell matrix, which leads to low efficacy. The high potential of mitochondria rapidly accumulates triphenylphosphine-lonidamine through electrostatic attraction, thereby increasing the efficacy.

4. The present invention has excellent photothermal performance, which can not only diagnose the tumor area in mice by photoacoustic imaging, but also judge the location of nanoparticles in tumor sites by photothermal imaging.

Description of drawings

Figure 1 is a schematic diagram of the preparation principle of the present invention.

Fig. 2 is the X-ray diffraction pattern of the Prussian blue nanoparticles and the zinc glutamate-coated Prussian blue nanoparticles in Example 2 of the present invention.

3 is a transmission electron microscope photograph of Prussian blue nanoparticles in Example 2 of the present invention.

FIG. 4 is a transmission electron microscope photograph of zinc glutamate-coated Prussian blue nanoparticles in Example 2 of the present invention.

FIG. 5 is a transmission electron microscope photograph of Prussian blue nanoparticles coated with lonidamine-loaded zinc glutamate by the cancer cell membrane in Example 4 of the present invention.

FIG. 6 is a transmission electron microscope photograph of the Prussian blue nanoparticles coated with zinc glutamate loaded with triphenylphosphine-lonidamine by the cancer cell membrane in Example 4 of the present invention.

Figure 7 shows the cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles in Example 5 at different pH (7.4 and 5.0) and different temperatures (42°C and 37°C). ) under the drug release profile.

Figure 8 shows the co-culture of HepG2 cells with lonidamine in Example 6 of the present invention for (a) 24h and (b) 48h, and co-culture with triphenylphosphine-lonidamine for (c) 24h and (d) Result graph of relative viability at 48h (first at 37°C for 10h, then at 42°C for 0, 0.5, 1, 2h, and finally at 37°C).

9 is a graph showing the experimental results of the uptake of zinc glutamate-coated Prussian blue nanoparticles and cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles by HepG2 cells in Example 7 of the present invention.

Figure 10 is a graph showing the results of cell viability of HepG2 cells under different conditions in Example 8 of the present invention (1.0W/cm2 power density 808nm laser irradiation for 10min, drug concentration is 20μg/mL)

Figure 11 shows (a) Western Blot and (b) relative expression levels of HSP90 and HSP70 expressed by HepG2 cells after different conditions in Example 9 of the present invention.

FIG. 12 is a photoacoustic imaging image of the tumor site of the tumor-bearing nude mouse in Example 10 of the present invention at 0, 4, 8, 12, and 24 h.

Figure 13 is a graph of (a) tumor volume change curve and (b) nude mouse body weight change graph every other day within 16 days of treatment in Example 11 of the present invention.

Figure 14 is a comparison chart of (a) the average weight of tumors and (b) a photo of tumor size after 16 days of treatment in Example 12 of the present invention (n=5, *p<0.05, **P<0.01, ** *P＜0.001)

Detailed ways

The technical solutions of the present invention will be further illustrated and described below through specific embodiments in conjunction with the accompanying drawings.

Example 1: Preparation of Prussian Blue Nanoparticles

Weigh 135mg of ferric chloride hexahydrate and 2625mg of citric acid monohydrate to dissolve in 500mL of ultrapure water to prepare a ferric chloride solution; then weigh 210mg of potassium ferrocyanide trihydrate and 2625mg of citric acid monohydrate to dissolve. Prepare a solution in 500 mL of ultrapure water. Measure 60 mL of ferric chloride solution in a beaker, and slowly add 60 mL of potassium ferrocyanide solution dropwise with stirring in a water bath at 60°C. After the dropwise addition, continue stirring for 1 min at a constant temperature of 60 °C, then transfer to room temperature and stir for 5 min, then slowly pour 120 mL of acetone to induce crystallization, and finally collect by centrifugation and wash with water for 3 times.

Example 2: Preparation of zinc glutamate-coated Prussian blue nanoparticles

3 mg of the Prussian blue nanoparticles in Example 1 were weighed and dispersed in ultrapure water, 10 mL of a 0.5 mg/mL polyvinylpyrrolidone solution was added, and the mixture was stirred for 30 min to fully mix. Then, 8 mL of zinc nitrate solution with a concentration of 5 mol/L was added dropwise and stirred for 30 min to assemble the first layer. After centrifugation, it was washed with water and dispersed again. 5 mL of 5 mol/L disodium glutamate solution was added dropwise, stirred for 30 min to assemble the second layer, and centrifuged and washed to collect the precipitate. A total of 4 layers were assembled in this way, collected by centrifugation, washed twice with water, and vacuum freeze-dried to obtain zinc glutamate-coated Prussian blue nanoparticles.

According to the X-ray diffraction patterns of the products of Example 1 and Example 2 (Fig. 2), all diffraction peak positions correspond to the diffraction surfaces of Prussian blue nanoparticles and zinc glutamate-coated Prussian blue nanoparticles, respectively, showing that zinc glutamate is coated with Prussian blue nanoparticles. The formation of Prussian blue nanoparticles; the transmission electron microscope photo of Prussian blue nanoparticles (Figure 3) shows that the Prussian blue nanoparticles have a uniform square structure; the transmission electron microscope photo of Prussian blue nanoparticles coated with zinc glutamate (Figure 4) can be It can be seen that it is a uniform core-shell structure, and the size distribution of the particles is statistically calculated by dynamic light scattering.

Example 3: Loading of Drugs

Weigh 5 mg of the zinc glutamate-coated Prussian blue nanoparticles obtained in Example 2 and disperse them in methanol, add a methanol solution of zinc nitrate to assemble the fifth layer, then add 2 mg of a methanol solution of lonidamine, stir for 2 h, centrifuge and remove Washed twice with water to obtain lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles.

38.1 mmol of triphenylphosphine and 2-aminobromoethane hydrobromide were respectively weighed into a 100 mL round-bottomed flask, and 50 mL of acetonitrile was added to the reaction at 82° C. under reflux for 24 h. After the reaction is completed, the precipitate is dried to obtain the product triphenylphosphine-aminoethane hydrobromide. Weigh 3 mmol of lonidamine, 3 mmol of 4-dimethylaminopyridine and 3 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride into a round-bottomed flask, add 10 mL of dimethyl sulfoxide was stirred at room temperature in the dark for 6 h. Then 3 mmol of triphenylphosphine-aminoethane hydrobromic acid was added, and the mixture was continuously stirred for 72 h in the dark at room temperature. After the reaction, extract with dichloromethane and ultrapure water (volume ratio of 5:1) to obtain triphenylphosphine-lonidamine.

5 mg of the zinc glutamate-coated Prussian blue nanoparticles obtained in Example 2 were weighed and dispersed in methanol, 2 mg of the methanol solution of the above triphenylphosphine-lonidamine was added, stirred for 2 h, centrifuged and washed twice with water. Then, 5 mL of 5 mol/L disodium glutamate solution was added to assemble the fifth layer to obtain the zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine.

Measure the absorbance of unloaded Lonidamine and Triphenylphosphine-Lonidamine in the supernatant, calculate the concentration according to the standard curve, and calculate the drug loading and encapsulation efficiency of Lonidamine to be 21.0± 2.2% and 51.8±3.5%, the drug loading and encapsulation efficiency of triphenylphosphine-lonidamine were 23.5±2.9% and 59.3±2.5%, respectively.

Example 4: Preparation of drug-loaded nanoparticles encapsulated by cancer cell membranes

Take 2 mg of lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles prepared in Example 3 and triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles in ultrapure water, and It was placed in a centrifuge tube, placed in an ultrasonic cleaner, and dispersed by ultrasonic at a frequency of 40 kHz. During the ultrasonic process, 50 μL of the extracted HepG2 cell membrane was added. After 3 minutes, centrifugation and washing twice with water were used to obtain cancer cell membranes wrapped with lonidamine-loaded glutamate. Zinc acid-coated Prussian blue nanoparticles and cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles.

According to the TEM photos (Fig. 5 and Fig. 6), the product prepared in this example has a cubic structure, and it can be seen that the outer surface is covered with a layer of cell membrane, with a thickness of about 10 nm; the particle size distribution is calculated by dynamic light scattering. It was found that the average particle diameter of the nanoparticles was 150 nm.

Example 5: Drug release under different conditions

2 mg of the triphenylphosphine-lonidamine-loaded zinc glutamate prepared in Example 3 was coated with Prussian blue nanoparticles and 2 mg of the cancer cell membrane prepared in Example 4 was coated and loaded with triphenylphosphine-lonidamine. The zinc glutamate-coated Prussian blue nanoparticles were put into a dialysis bag with a molecular weight cut-off of 3500 Da, clamped with a dialysis bag clip, and then loaded into a centrifuge tube, and 30 mL of pH 7.4 or 5.0 PBS was added. The shaker temperature was set at 43°C and 37°C, respectively, for drug release. At different time points 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48...h, 3 mL of drug-released PBS was taken to measure the absorbance and supplemented with 3 mL of new PBS, three times per group parallel. Calculate the corresponding concentration according to the standard curve, and calculate the cumulative release rate of the drug.

The drug release profile shown in Figure 7 shows that within 120 h, at pH 5.0 and 42 °C, the cancer cell membrane was coated with triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles p-triphenylphosphine - The cumulative release rate of lonidamine is about 50%, and its release rate is the fastest; the cumulative release rate is about 40% at pH 5.0 and 37°C; the cumulative release rate is about 35% at pH 7.4 and 42°C; The cumulative release rate was about 20% at pH 7.4 and 37 °C, and the release was the slowest, showing pH-responsive and temperature-responsive drug release.

Example 6: Antitumor effect of free drug

HepG2 cells were seeded into a 96-well plate at a cell density of 8×103 cells/well, 100 μL of DMEM complete medium was added to each well, and cultured at 37°C and 5% CO2 for 24h. Then, the complete DMEM medium containing lonidamine and triphenylphosphine-lonidamine was replaced, and the drug concentrations were 2, 5, 10, 20, and 50 μg/mL, respectively. The 96-well plate was first placed in a 37°C incubator for 10h, then transferred to a 42°C incubator, incubated at 42°C for 0.5h, 1h, and 2h, and finally transferred to a 37°C incubator. After the total culture time was 24h and 48h, the cell viability was determined by CCK-8 method.

In Figure 8, lonidamine, a drug with low antitumor efficacy, maintained a high cell viability after co-culture with HepG2 cells for 24h and 48h even when the concentration was as high as 50μg/mL; after 42 After incubation at ℃, the cell viability decreased by about 25%, indicating that the sensitivity of HepG2 cells to high temperature was significantly increased under the action of lonidamine, and it also confirmed that lonidamine has the effect of overcoming the heat resistance of tumor cells. . However, when the concentration of triphenylphosphine-lonidamine was 20 μg/mL, the inhibitory effect of lonidamine on HepG2 cells was higher than that of 50 μg/mL, indicating that triphenylphosphine-lonidamine has a higher inhibitory effect on HepG2 cells. The strong drug effect makes HepG2 cells more sensitive to high temperature; it shows that its anti-tumor effect is enhanced after connecting with the mitochondrial targeting group triphenylphosphine.

Example 7: Cellular Uptake

HepG2 cells were seeded on slides in a 24-well plate at a cell density of 1×105 cells/well. After 24 hours, the medium was replaced with a new medium containing 50 μg/mL of the zinc glutamate-coated Prussian blue nanoparticles of Example 2 and the zinc glutamate-coated Prussian blue nanoparticles of the product of Example 3. Continue to incubate for 2h and 4h in a 37°C incubator, then wash three times with PBS, and add 0.5mL of 4% paraformaldehyde to each well for 15min fixation. The nuclei were stained with EB dye in the dark for 5-10 minutes, and 10 μL of anti-fluorescence quencher was added dropwise to the slides.

In Figure 9, HepG2 cells were incubated with zinc glutamate-coated Prussian blue nanoparticles and cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles for 2 hours, respectively. After the tumor cells were stained with dye EB, they were observed under CLSM with a 405 nm laser. Prussian blue nanoparticles produced blue fluorescence under excitation. The blue fluorescence of the zinc glutamate-coated Prussian blue nanoparticle group was stronger, indicating that it entered HepG2 cells more, confirming that the Prussian blue nanoparticles encapsulated the HepG2 cell membrane and obtained the targeting ability.

Example 8: The effect of low temperature photothermal on tumor cells

HepG2 cells were seeded into a 96-well plate at a cell density of 8 × 103 cells/well, and cells containing triphenylphosphine-lonidamine and the product of Example 3 were added to encapsulate the cancer cell membrane loaded with triphenylphosphine-lonidamine. The culture medium of zinc glutamate-coated Prussian blue nanoparticles at concentrations of 10, 20, 50, 100, 150, and 200 μg/mL, respectively. After 10h, the cells were irradiated with 808nm laser at 1.0W/cm2 for 10min, and then incubated at 37℃. The total culture time was 24h, respectively, and were replaced with 100 μL of DMEM medium and 10 μL of CCK-8 reagent, and the blank control was CCK-8 reagent. Continue to culture in the incubator for 1-4h, detect by microplate reader at 450nm wavelength, and calculate the cell viability.

In Figure 10, under different treatment conditions, the equivalent concentration of triphenylphosphine-lonidamine remained unchanged at 20 μg/mL, and the effect on tumor cells was weak. Cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles under laser irradiation showed a survival rate of 39.7±6.1% for HepG2 cells, which was higher than that of triphenylphosphine-lonidamine and light. The effect of heat treatment alone was better on cytostatic. After laser irradiation of the cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles group, the temperature rise caused by photothermal action was limited, so a large number of cell death was not caused. However, in the group of Prussian blue nanoparticles coated with zinc glutamate loaded with triphenylphosphine-lonidamine on the cancer cell membrane, the laser irradiation temperature was the same, but a large number of cells died, indicating that the drug triphenylphosphine-lonidamine Damin makes HepG2 cells more sensitive to heat. These experimental results and the above experimental results fully demonstrate that the Prussian blue nanoparticles coated with zinc glutamate loaded with triphenylphosphine-lonidamine on the cancer cell membrane can enhance the effect of tumor photothermal therapy.

Example 9: ATP level detection

HepG2 cells were seeded in 6-well plates at 1×105 cells/well and cultured for 24h. The culture medium was removed by suction, and zinc glutamate-coated Prussian blue nanoparticles containing lonidamine, triphenylphosphine-lonidamine, the product of Example 4, cancer cell membrane-encapsulated and loaded lonidamine, and cancer cell membrane-encapsulated and loaded three were added. Phenylphosphine-Lonidamine zinc glutamate-coated medium for Prussian blue nanoparticles with a drug concentration of 20 μg/mL. After culturing for 12 h, 200 μL of lysate was added to each well, the cells were lysed by pipetting repeatedly, and then centrifuged at 12,000 g for 5 min at 4°C, and the supernatant was taken for determination. Add 100 μL of ATP detection working solution to the 96-well plate, then add 20 μL of sample, mix quickly, and use a chemiluminometer to measure the fluorescence signal.

In Figure 11, when the drug concentration was 20 μg/mL, the ATP level was higher in the lonidamine group and the cancer cell membrane-coated lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles group, while the triphenylphosphine- The ATP levels in the lonidamine group and the cancer cell membrane-wrapped triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles group were lower, indicating that after the targeting group triphenylphosphine was attached to lornidamine The potency of Damin is enhanced.

Example 10: Western Blot

HepG2 cells were seeded in 6-well plates. After the cells were cultured for 12 hours, they were added to each group of solutions for treatment. They were divided into the first group, the negative control group, without any treatment; the second group, incubated at 42°C for 30 minutes; The third group added zinc glutamate coated with cancer cell membrane to coat Prussian blue nanoparticles and then irradiated under 808 nm laser for 10 min; the fourth group added triphenylphosphine-lonidamine at a concentration of 20 μg/mL; The concentration of triphenylphosphine-lonidamine was 20 μg/mL. The culture was continued for 24 h, and then the old medium was carefully aspirated to avoid removal of poorly adherent cells, and the DMEM medium was removed by washing twice with PBS. Make up 1 mL of lysate to 10 μL of PMSF (100 mM) and shake well and place on ice. After adding lysate and fully lysing on ice, centrifuge at 4 °C for 5 min with a high-speed centrifuge. The supernatant is the whole protein of HepG2 cells extracted and stored in a -20 °C refrigerator. Western blot was used to detect heat shock protein 70 and Expression of heat shock protein 90.

In Figure 12, in the group of Prussian blue nanoparticles coated with zinc glutamate coated by laser irradiation at high temperature of 42 °C and the cancer cell membrane, with GADPH as the internal standard protein, the expressions of heat shock protein 70 and heat shock protein 90 increased by about 30%. %, indicating that heat shock proteins are synthesized in large quantities under the induction of heat stimulation to protect cells from damage. Under the action of triphenylphosphine-lonidamine, the content of ATP decreased, resulting in insufficient energy for the synthesis of heat shock protein in cells, and the content of heat shock protein 70 and heat shock protein 90 was greatly reduced, which was reduced to the negative control group. about 1/4 of . The results of Western Blot experiments corresponded to changes in mitochondrial membrane potential and ATP levels, which confirmed the mechanism of action of triphenylphosphine-lonidamine. By reducing ATP content, the synthesis of heat shock proteins was reduced, and the effect of photothermal therapy was improved. the goal of.

Example 11: In vivo photoacoustic imaging

When the tumor volume grew to about 200 mm3, the BALB/c nude mice inoculated with HepG2 cell tumors were injected with 200 μL of PBS, the product of Example 3, and the triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanometers into the tail vein respectively. The PBS suspension of Prussian blue nanoparticles and the PBS suspension of Prussian blue nanoparticles coated with zinc glutamate loaded with triphenylphosphine-lonidamine by the cancer cell membrane of the product of Example 4, the relative concentration of Prussian blue nanoparticles was 1 mg/mL, each Group of three. At different time points 0, 4, 8, 12, and 24 h, the photoacoustic signals at the tumor site were detected by a small animal photoacoustic imager to observe the enrichment in tumor tissue.

Figure 13 After tail vein injection of triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles and cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles Within 12 h, the photoacoustic signal in tumor tissue gradually increased with the blood circulation of nanoparticles in mice. By 12h, the area of photoacoustic signal was the largest and strongest, and after 24h, both the intensity and area of photoacoustic signal were weakened. In addition, the tumor tissue produces a strong photoacoustic signal, showing the shape of the tumor tissue, thus distinguishing it from the normal tissue, greatly increasing the signal-to-noise ratio of the photoacoustic signal, and providing a certain reference for tumor diagnosis. After encapsulating the cancer cell membrane, the photoacoustic signal in the tumor tissue is stronger and more distributed, which confirms the targeting ability of the cancer cell membrane.

Example 12: Animal-level verification of enhanced photothermal therapy effect

Thirty BALB/c nude mice inoculated with HepG2 cell tumor were randomly divided into 6 groups with 5 mice in each group. When the tumor volume grew to about 200mm3, 200μL of solution was injected into the tail vein after different treatment methods: the first group was injected with PBS in the tail vein; the second group was injected with PBS and then irradiated with 808 nm laser for 5 minutes; the third group was injected with cancer Cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles; the fourth group was injected with cancer cell membrane-coated zinc glutamate-coated Prussian blue nanoparticles through tail vein and then irradiated with 808 nm laser for 5 minutes; Prussian blue nanoparticles coated with zinc glutamate of phenylphosphine-lonidamine; the sixth group was injected into the tail vein of cancer cell membrane coated with zinc glutamate loaded with triphenylphosphine-lonidamine and then irradiated 5min of 808nm laser. It was treated again every other day, for a total of 2 times, and the body weight and tumor volume changes of the nude mice were recorded. After 16 days of treatment, the mice were dissected to obtain tumor tissue, weighed, and the tumor inhibition rate was calculated.

Figure 14 After 16d treatment, the tumor volume in the first group increased from about 200mm3 to 985.5±165.5mm3, and the tumor volume in the second group increased to 964.7±119.9mm3, indicating that the growth of tumor tissue under near-infrared laser irradiation was almost No effect. To observe the inhibitory effect of triphenylphosphine-lonidamine on tumor cells, the tumor volume of the cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles group was 874.4±189.9mm3 , the tumor volume was slightly reduced, indicating that triphenylphosphine-lonidamine has little effect on tumor inhibition when used alone. After the treatment, the mice were euthanized and the tumor tissue was dissected out, weighed and photographed. The average tumor weight in the PBS control group was 1.02 g, and the average tumor mass in the sixth group was 0.11 g, and the tumor inhibition rate was 89.2%. The average mass of the Prussian blue nanoparticles coated with triphenylphosphine-lonidamine by laser irradiation and cancer cell membrane alone was 0.58g and 0.89g. There was no significant change in the body weight of each group of mice before and after treatment, which also indicated that the nano-drug loading system had good biological safety. Judging from the combined treatment results of tumor-bearing mice, the cancer cell membrane-coated triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles had a good therapeutic effect on HepG2 tumor-bearing mice, indicating that its The field of biomedicine has certain application potential.

The above descriptions are only preferred embodiments of the present invention, so the scope of implementation of the present invention cannot be limited accordingly. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the description should still be covered by the present invention. In the range.

Claims (10)

1. A zinc glutamate wrapped Prussian blue nanoparticle loaded with triphenylphosphine-lonidamine and wrapped by a cancer cell membrane is characterized in that: the tumor cell membrane comprises a Prussian blue nano core, wherein the surface of the Prussian blue nano core is coated with at least one zinc glutamate layer, the surface of the outermost zinc glutamate layer is provided with a loading layer loaded with triphenylphosphine-lonidamine, and the loading layer is coated with a tumor cell membrane layer.

2. The method for coating zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine on cancer cell membranes according to claim 1, which is characterized in that: the tumor cell membrane layer is made of extracted HepG2 cell membrane.

3. The method for coating zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine on cancer cell membranes according to claim 1, which is characterized in that: the triphenylphosphine-lonidamine is prepared by connecting triphenylphosphine and lonidamine through (2-bromomethyl) dimethylamine hydrobromide.

4. The zinc glutamate-coated Prussian blue nanoparticle loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane according to any one of claims 1 to 3, wherein: the particle size is 100-200 nm.

5. The zinc glutamate-coated Prussian blue nanoparticle loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane according to claim 4, wherein: the particle size of the Prussian blue nano-core is 70-90 nm.

6. The preparation method of zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane according to any one of claims 1 to 5, which is characterized in that: the method comprises the following steps:

(1) preparing prussian blue nanoparticles by a hydrothermal synthesis method;

(2) the preparation method of the zinc glutamate coated Prussian blue nanoparticles comprises the following steps:

a. mixing the prussian blue nanoparticles with a polyvinylpyrrolidone aqueous solution, dropwise adding a zinc nitrate solution for reaction, centrifuging, washing with water, and dispersing;

b. dropwise adding a glutamic acid disodium solution into the material obtained in the step a for reaction, centrifugally washing, collecting precipitates, repeating the steps for 1-3 times, and centrifugally collecting to obtain zinc glutamate-coated prussian blue nanoparticles;

(3) preparing zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine: dispersing the zinc glutamate-coated Prussian blue nanoparticles obtained in the step (2) in methanol, adding a triphenylphosphine-lonidamine methanol solution, fully stirring, centrifuging, washing with water, and adding a disodium glutamate solution for reaction to obtain triphenylphosphine-lonidamine-loaded zinc glutamate-coated Prussian blue nanoparticles;

(4) preparing zinc glutamate wrapped Prussian blue nanoparticles with cancer cell membranes wrapped and loaded with triphenylphosphine-lonidamine: ultrasonically dispersing the zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine obtained in the step (3) into ultrapure water, dropwise adding the extracted tumor cell membranes in the ultrasonic process, centrifuging and collecting after 2-5min, and fully washing to obtain the zinc glutamate-coated Prussian blue nanoparticle.

7. The method of claim 6, wherein: the step (1) is as follows: dissolving ferric chloride hexahydrate and citric acid monohydrate in ultrapure water to prepare a ferric chloride solution; dissolving potassium ferrocyanide trihydrate and citric acid monohydrate in ultrapure water to prepare a potassium ferrocyanide solution; slowly dropwise adding a potassium ferrocyanide solution into the ferric chloride solution at 58-62 ℃ while stirring; and after the dripping is finished, continuously stirring for 0.8-1.2min at the constant temperature of 58-62 ℃, then transferring to room temperature, stirring for 4-6min, slowly pouring acetone for inducing crystallization, and sequentially carrying out centrifugal collection, full water washing and vacuum freeze drying to obtain the Prussian blue nanoparticles.

8. The method of claim 6, wherein: the frequency of the ultrasonic dispersion in the step (4) is 35-45 kHz.

9. The use of zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane as claimed in any one of claims 1 to 5 in the preparation of low-temperature tumor photothermal therapy medicine.

10. The use of zinc glutamate-coated Prussian blue nanoparticles loaded with triphenylphosphine-lonidamine and wrapped by cancer cell membrane as claimed in any one of claims 1-5 in the preparation of mitochondria-targeted drugs.

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