CN114569578A

CN114569578A - Preparation and application of bionic nanoparticles with photo-chemotherapy function based on double-drug co-assembly

Info

Publication number: CN114569578A
Application number: CN202210289753.3A
Authority: CN
Inventors: 邵敬伟; 罗邦悦; 李林燕
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-12-31
Filing date: 2022-03-23
Publication date: 2022-06-03
Anticipated expiration: 2042-03-23
Also published as: CN114569578B

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to preparation and application of bionic nanoparticles with light-chemotherapy based on double-medicine co-assembly. The bionic nanoparticle consists of a shell and a nano inner core coated by the shell, wherein the shell is a HepG2 cell membrane, and the nano inner core is formed by co-assembling an anti-tumor hydrophobic drug sorafenib, an anti-cardiovascular disease hydrophobic drug propranolol and a photosensitizer indocyanine green. The bionic nanoparticles can be specifically accumulated at a liver cancer part through the homologous recognition effect of cell membranes, and accurately and efficiently release the medicine under the irradiation of 808 nm laser, so that the effect of combining multiple medicines and multiple therapies on resisting liver cancer is realized, the growth of liver cancer is remarkably inhibited, and the application prospect in preparing the anti-liver cancer medicine is wide.

Description

Preparation and application of bionic nanoparticles with light-chemotherapy function based on double-drug co-assembly

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to preparation and application of bionic nanoparticles with light-chemotherapy based on double-medicine co-assembly.

Background

Liver cancer is the most common primary liver cancer and can be caused by chronic hepatitis B virus, hepatitis C virus infection, diabetes and other diseases. Most patients have advanced stage in definite diagnosis due to their latent onset, difficult early diagnosis, high malignancy and other characteristics. In addition to the development of multidrug resistance, metastasis to adjacent or distant tissues and serious side effects during treatment, the mortality rate of liver cancer remains high. Currently, the main treatment means for liver cancer include surgical resection, liver transplantation, chemotherapy or radiotherapy, etc. Among them, surgical resection is the first choice for early liver cancer treatment, but only a few patients benefit from this because of the limited low early screening rate.

Sorafenib (SF) is a small-molecule targeted multi-kinase inhibitor, can block the Raf/MEK/ERK signal path from directly interfering the growth of liver cancer cells, and can inhibit the formation of liver cancer new vessels and indirectly inhibit the occurrence and development of tumors by inhibiting Vascular Endothelial Growth Factor Receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs). However, a great deal of clinical practice shows that even though SF greatly improves the disease progress of liver cancer patients, most patients are easy to have a drug resistance phenomenon after treatment, and therefore, the search for a proper drug sensitizer becomes a great challenge for treating liver cancer by SF. At present, researchers have explored various drug sensitizers to enhance the sensitivity of liver cancer cells to SF. Chinese patent 201010587253.5 discloses a composition containing epigallocatechin gallate (EGCG) and SF, wherein EGCG enhances the inhibitory effect of SF on liver cancer cells on one hand, and reduces the dosage of SF on the other hand, thereby reducing the use cost of the medicine. In addition, chinese patent 201910793921.0 discloses a combination drug for applying SF and sodium nitroprusside in liver cancer treatment, which also increases the inhibitory effect of SF on liver cancer cells, and reduces the dosage of SF, thereby reducing toxic and side effects. Therefore, the combination of the medicines for enhancing the sensitivity of the liver cancer cells to SF and reducing the toxic and side effects of the liver cancer cells is an effective liver cancer treatment strategy.

Propranolol (pplanol, PPL) is a beta adrenergic receptor blocker and is widely applied to the treatment of cardiovascular diseases. In recent years, studies have pointed out that β adrenoreceptors are expressed in many tumor cells and are closely involved in the proliferation, metastasis, immune escape and other behaviors of tumor cells. At present, researches prove that PPL not only can inhibit the proliferation of liver cancer cells by inhibiting a beta adrenergic receptor signal pathway, but also can enhance the sensitivity of the liver cancer cells to SF and has a positive effect on relieving the drug resistance of the cancer cells. Therefore, the combined use of SF and PPL is an effective strategy for inhibiting the occurrence and development of liver cancer.

With the rapid development of nano science and technology, the construction of a drug delivery system by using nano biomaterials brings new opportunities and challenges for tumor treatment. Currently, researchers have developed a variety of SF-based nano-drug delivery systems. Chinese patent 201810781142.4 discloses a multifunctional nano-drug based on hollow mesoporous silica co-carried plasmid, which co-carries SF and plasmid in hollow mesoporous silica, improves the bioavailability of SF, improves the transfection efficiency of plasmid, and improves the anti-tumor effect of SF. Chinese patent 202110518445.9 discloses a nanoparticle loaded with near-infrared emission fluorescent molecules/SF and a preparation method thereof, the nanoparticle co-loads a pyrrolopyrrole aza-fluoroboron compound and SF in a high molecular copolymer, increases the water solubility of SF, and provides a fluorescence detection means for in vivo metabolism research. However, the cooperative treatment of liver cancer by constructing a nano-drug delivery system based on the co-construction of SF and PPL has not been reported.

Based on the background, the invention intends to construct a dual-drug nano delivery system based on SF and PPL co-assembly, the system is assembled to form a nano inner core by virtue of intermolecular force between two drugs, a near infrared fluorescence probe indocyanine green (ICG) is introduced to enable the nano drug to have the phototherapy synergistic effect, and meanwhile, a bionic cell membrane from liver cancer cells is wrapped outside the nano inner core, so that the nano drug has the functions of tumor homologous targeting and immune escape. The bionic nanoparticle with light-chemotherapy functions based on double-drug co-assembly provided by the invention integrates a multi-drug-multi-therapy combination strategy into the same drug delivery platform, effectively improves the limitation of a single chemotherapy means on tumor inhibition, and has positive application potential in the field of liver cancer treatment.

Disclosure of Invention

The invention aims to provide preparation and application of bionic nanoparticles with photo-chemotherapy based on double-drug co-assembly. The invention constructs a nano inner core with light-chemotherapy synergistic action through pi-pi accumulation among hydrophobic drug molecules, electrostatic acting force and hydrophobic interaction for co-assembly, and coats a cell membrane derived from tumor cells on the outer layer of the nano inner core, so that the nano inner core has good tumor recognition capability and immune escape function. The bionic nanoparticle integrates a multi-drug-multi-therapy combined application strategy, and realizes a better tumor inhibition effect.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a bionic nanoparticle with photo-chemotherapy function based on double-drug co-assembly, which consists of a shell and a nano inner core coated by the shell, wherein the shell is a eukaryotic cell membrane, the nano inner core is formed by co-assembling an anti-tumor hydrophobic drug, an anti-cardiovascular disease hydrophobic drug and a photosensitizer, the particle size of the bionic nanoparticle is 100-400 nm, and the bionic nanoparticle has a photodynamic effect under the irradiation of near infrared light.

Furthermore, in the bionic nanoparticle based on dual-drug co-assembly and having light-chemotherapy, the eukaryotic cell membrane is a HepG2 cell membrane.

Furthermore, in the bionic nanoparticle with light-chemotherapy function based on double-drug co-assembly, the anti-tumor hydrophobic drug is sorafenib.

Furthermore, in the bionic nanoparticle with photo-chemotherapy based on dual-drug co-assembly, the cardiovascular disease resistant hydrophobic drug is propranolol.

Further, in the bionic nanoparticle with photo-chemotherapy based on dual-drug co-assembly, the photosensitizer is indocyanine green.

The invention also provides a preparation method of the bionic nanoparticle based on double-medicine co-assembly and having light-chemotherapy functions, and the method comprises the following steps:

(1) dissolving a certain amount of anti-tumor hydrophobic drug in ethanol to obtain a solution A, wherein the concentration of the anti-tumor hydrophobic drug is 1-1000000 mu M;

(2) dissolving a certain amount of cardiovascular disease resistant hydrophobic drugs in ethanol to obtain a solution B, wherein the concentration of the cardiovascular disease resistant hydrophobic drugs is 1-1000000 muM;

(3) dissolving a certain amount of photosensitizer in deionized water to obtain a solution C, wherein the concentration of the photosensitizer is 1-1000000 muM;

(4) uniformly mixing 1 volume part of the solution A and 1 volume part of the solution B to obtain a mixed solution AB; adding 1 volume part of the solution C into 17 volume parts of ultrapure water to obtain a diluted solution C; adding 2 parts by volume of the mixed solution AB into 18 parts by volume of the diluted solution C in a vortex state to obtain a solution D;

(5) mixing 1 part by volume of the eukaryotic cell membrane dispersion liquid with 10 parts by volume of the solution D in proportion, and carrying out ultrasonic treatment for 10 min at room temperature under the conditions of ultrasonic power of 250W and ultrasonic frequency of 40 KHz to obtain an aqueous solution of the bionic nanoparticles;

The preparation method of the eukaryotic cell membrane dispersion comprises the following steps: extracting eukaryotic cell membranes by adopting a differential centrifugation method, and dissolving the eukaryotic cell membranes in ultrapure water according to the proportion of 0.002 g/1 mL to obtain the eukaryotic cell membrane dispersion liquid.

The invention also provides application of the bionic nanoparticle with light-chemotherapy based on double-drug co-assembly in anti-liver cancer drugs.

The invention has the advantages that:

(1) the inner core of the bionic nanoparticle prepared by the invention is formed by co-assembling sorafenib, propranolol which are first-line clinical drugs and indocyanine green which is a near-infrared fluorescent probe approved by FDA and has good safety, does not contain an exogenous synthetic carrier, and has good biological safety.

(2) The cell membrane derived from liver cancer cells HepG2 is used as the bionic nano-drug prepared by the invention, and the HepG2 cell membrane endows the nano-drug with good tumor targeting property and enhances the specific recognition effect of the nano-drug on tumor cells.

(3) The bionic nanoparticles prepared by the invention have good stability under various physiological conditions.

(4) The bionic nanoparticles prepared by the invention can effectively generate active oxygen free radicals under the irradiation of 808 nm laser to generate photodynamic effect, thereby interfering the proliferation of tumor cells and inhibiting the growth of tumors.

(5) The preparation process of the bionic nanoparticles prepared by the invention is simple and efficient, a multi-drug-multi-therapy strategy is integrated into the same nano-drug delivery system, the limitation of a single chemotherapy means on the tumor inhibition effect is effectively improved, and the bionic nanoparticles show positive application potential in the field of liver cancer treatment.

Drawings

FIG. 1 is a structural formula diagram of sorafenib, propranolol and indocyanine green in the invention. І, sorafenib; І І, propranolol; І І І, indocyanine green.

FIG. 2 is a graph of particle size of PSINPs.

FIG. 3 is a graph of particle size for CM @ PSINPs.

FIG. 4 is a graph showing the particle size and potential of CM, PSINPs and CM @ PSINPs.

FIG. 5 shows the stability of aqueous solutions of CM @ PSINPs in storage at room temperature for 7 days.

FIG. 6 is a graph of the stability of CM @ PSINPs under various physiological conditions.

FIG. 7 is a graph comparing the degradation rates of ICG versus PSINPs, CM @ PSINPs, in free ICG solution.

Figure 8 is the tyndall effect of different drugs. I, ICG; II, SF; III, PPL; IV, PSINPs; v, CM @ PSINPs.

FIG. 9 is a graph comparing fluorescence emission intensity of ICG, PSINPs nm, CM @ PSINPs nm, and free ICG.

FIG. 10 is a comparison graph of singlet oxygen generation detection at PSINPs nm, CM @ PSINPs nm, and free ICG.

FIG. 11 is a SDS-PAGE protein characterization of HepG2 cell lysates, HepG2 cell membranes, PSINPs, and CM @ PSINPs. Lane I, HepG2 cell lysate; lane II, HepG2 cell membrane; lane III, PSINPs; lane IV, CM @ PSINPs.

FIG. 12 is a graph showing the cell proliferation inhibitory effect of each group of nano-drugs.

FIG. 13 is the uptake pattern of different nano-drugs by hepatoma cell HepG2 and normal hepatocyte L02.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1

The embodiment provides a preparation method of propranolol-sorafenib-indocyanine green co-assembly nanoparticles (PSINPs), which comprises the following steps:

0.00400 g of propranolol (PPL) is precisely weighed and dissolved in 1 mL of ethanol to obtain 4 mg/mL of solution A; 0.00400 g of Sorafenib (SF) is precisely weighed and dissolved in 1 mL of ethanol to obtain a solution B of 4 mg/mL; 0.00400 g of indocyanine green (ICG) is precisely weighed and dissolved in 1 mL of ultrapure water to obtain 4 mg/mL of solution C; mixing 50 mu L of the solution A with 50 mu L of the solution B to obtain a mixed solution AB; dispersing 50 mu L of the solution C in 850 mu L of ultrapure water to obtain a diluted solution C; and dropwise adding 100 mu L of the mixed solution AB into 900 mu L of the diluted solution C under a vortex state to obtain the aqueous solution of the nano-particles PSINPs.

As shown in FIG. 2, the PSINPs prepared in this example had an average particle size of about 150 nm.

Example 2

This example provides methods for preparing HepG2 cell lysates and HepG2 cell membrane pellets as follows:

preparation of HepG2 cell lysate: culturing HepG2 cells until the growth density is 80-90%, digesting the cells for 3-5 min by using 0.25 wt% of pancreatin containing EDTA, centrifuging the cells for 5 min under the condition of 1500 rpm to obtain cell precipitates, washing the precipitates for 3 times by using normal saline, dispersing the precipitates in 0.25 multiplied by normal saline containing 1 mM phenylmethylsulfonyl fluoride (PMSF), and placing the precipitates in a refrigerator at 4 ℃ for 1-2 hours to obtain HepG2 cell lysate.

Preparation of HepG2 cell membrane pellet: culturing HepG2 cells until the growth density is 80-90%, digesting the cells for 3-5 min by using 0.25 wt% pancreatin containing EDTA, centrifuging the cells for 5 min at 1500 rpm to obtain cell precipitates, washing the precipitates for 3 times by using normal saline, dispersing the precipitates in 0.25 multiplied normal saline containing 1 mM PMSF, and placing the precipitates in a refrigerator at 4 ℃ for 1-2 hours; and then transferring the cell suspension into a precooled cell homogenizer, slowly grinding for 30 times to fully crack the cell suspension, centrifuging at a low speed of 4 ℃ and 1500 rpm for 10 min to remove cell fragment impurities and larger organelles, taking the supernatant, centrifuging at a high speed of 4 ℃ and 15000 rpm for 30 min to obtain a precipitate, namely HepG2 cell membrane precipitate (CM), and dispersing 0.002 g of CM in 1 mL of ultrapure water to obtain HepG2 cell membrane dispersion liquid for later use.

Example 3

This example provides a method for preparing cell membrane-encapsulated PSINPs (CM @ PSINPs) from HepG2, comprising the steps of:

the HepG2 cell membrane dispersion collected in example 2 and the aqueous solution of PSINPs nanoparticles prepared in example 1 were mixed in the following ratio of 1: 10, and performing ultrasonic treatment (250W, 40 KHz) for 10 min at normal temperature to obtain the aqueous solution of the nanoparticles CM @ PSINPs.

As shown in FIG. 3, the average particle size of CM @ PSINPs prepared in this example was about 160 nm.

Example 4

Particle size and potential were measured using a Malvern particle sizer for PSINPs obtained in example 1, CM obtained in example 2, and CM @ PSINPs obtained in example 3.

As shown in fig. 4, after cell membranes are wrapped on the surfaces of the PSINPs nanoparticles, the diameters of the nanoparticles are increased by about 10 nm, and the potential of the nanoparticles is obviously reduced and is closer to the potential of the cell membranes, which indicates that the cell membranes are successfully wrapped on the surfaces of the nanoparticles.

Example 5

CM @ PSINPs obtained in example 3 were stored in an aqueous solution at room temperature, and the change in particle size was measured for 7 consecutive days to observe the stability.

The result is shown in fig. 5, the particle size of the bionic nanoparticles CM @ PSINPs is not obviously changed within 7 days, and the bionic nanoparticles CM @ PSINPs have good stability.

Example 6

CM @ PSINPs prepared in example 3 were stored under different physiological conditions (DMEM cell culture medium + volume fraction 10% fetal bovine serum, RPMI 1640 cell culture medium + volume fraction 10% fetal bovine serum, DMEM cell culture medium, RPMI 1640 cell culture medium), and the particle size change was measured at different time periods to observe the stability thereof.

As shown in figure 6, the particle size of the bionic nanoparticles CM @ PSINPs has good stability under various physiological conditions, and the particle size of the nanoparticles is not obviously changed within 48 h of storage.

Example 7

Respectively placing ICG aqueous solution and PSINPs aqueous solution with ICG equivalent concentration of 200 mug/mL at 25 ℃ in a dark place for storage, continuously measuring ICG degradation speed in different groups for 7 days, and inspecting the ICG stability in the medicine.

As shown in fig. 7, compared with the degradation rates of ICG in the free ICG solution and the PSINPs nanoparticles, the degradation rate of ICG in the cell membrane-encapsulated nano-groups (CM @ PSINPs) was significantly slowed, indicating that the encapsulation of the cell membrane has a certain protective effect on the degradation of ICG, and can effectively maintain the stability of the nano-drug.

Example 8

200 mug/mL ICG aqueous solution, 200 mug/mL SF aqueous solution, 200 mug/mL PPL aqueous solution, the PSINPs nano-particle aqueous solution prepared in the example 1 and the CM @ PSINPs nano-particle aqueous solution prepared in the example 3 were respectively placed in different penicillin bottles and irradiated by a laser pen.

As shown in fig. 8, the aqueous solution of the PSINPs nanoparticles prepared in example 1 and the aqueous solution of the CM @ PSINPs nanoparticles prepared in example 3 can observe a significant tyndall phenomenon under laser irradiation, i.e., a uniform light path, indicating that both of them have significant nano-morphology.

Example 9

The fluorescence emission intensity of ICG aqueous solution, PSINPs aqueous solution and CM @ PSINPs aqueous solution with 0.4 mu g/mL ICG equivalent concentration are taken and measured.

As shown in FIG. 9, the ICG fluorescence emission intensity of PSINPs was significantly decreased compared to that of free ICG, because ICG was encapsulated in the nano-core of PSINPs during the co-assembly of SF and PPL, resulting in a decrease in fluorescence emission; meanwhile, the fluorescence emission intensity of CM @ PSINPs is further reduced compared with that of PSINPs, and the fact that cell membranes are effectively wrapped on the surfaces of nano-core PSINPs is proved.

Example 10

Respectively taking 5.4 mu g/mL ICG aqueous solution, the PSINPs nano-particle aqueous solution prepared in the example 1 and the CM @ PSINPs nano-particle aqueous solution prepared in the example 3, respectively adding 1, 3-diphenyl isobenzofuran (1, 3-DPBF) with the final concentration of 30 mu g/mL, and after different illumination time, measuring the ultraviolet absorption at 415 nm to judge the generation capacity of singlet oxygen in different medicines.

The results are shown in FIG. 10, where both PSINPs and CM @ PSINP exhibited better singlet oxygen generating capacity than the free ICG solution. After 10 min of illumination, the absorption of PSINPs at 415 nm decreased by 88.4%, the absorption of CM @ PSINP at 415 nm decreased by 91.1%, and the absorption of ICG at 415 nm decreased by only 20.2%. The preparation method shows that after the nano particles are prepared, the medicine has better singlet oxygen generation capacity, the process is not influenced by the wrapping of cell membranes, more singlet oxygen is generated after illumination, and stronger photodynamic treatment potential is expressed.

Example 11

The retention of cell membrane proteins in the CM @ PSINPs nanoparticles prepared in example 3 was examined by SDS-PAGE protein gel electrophoresis experiments. The method comprises the following specific steps:

(1) preparing polyacrylamide gel: respectively preparing 12% of separation glue and 5% of concentrated glue, inserting a comb to prepare a sampling hole before the concentrated glue is solidified, and carefully pulling out the comb after the concentrated glue is completely solidified;

(2) sample preparation: 1 mL of the HepG2 cell lysate, the HepG2 cell membrane dispersion prepared in example 2, the aqueous solution of PSINPs prepared in example 1 and the CM @ PSINPs prepared in example 3 were centrifuged at 4 ℃ and 15000 rpm for 30 min to remove the supernatant, the precipitate was concentrated in 100. mu.L of ultrapure water, 8. mu.L of each was thoroughly mixed with 2. mu.L of SDS loading buffer (5X) (available from Cultitute Biotech, Inc., Beijing), and the protein was thoroughly denatured after being subjected to metal bath at 95 ℃ for 10 min and then added to different wells;

(3) Electrophoresis: setting the electrophoresis apparatus at 80V for 20 min, 100V for 1 h, stopping electrophoresis after the sample proteins are fully separated, and taking out the gel;

(4) dyeing: and (3) placing the gel in a Coomassie brilliant blue staining solution, keeping out of the sun for staining for 20 min, and after the sample is fully stained, fully washing the gel with a destaining solution until protein bands are clear and no excess staining solution remains.

(5) Pictures were taken using a chemiluminescence apparatus.

As shown in fig. 11, the results show that, with reference to single HepG2 cell lysate and HepG2 cell membrane, CM @ PSINPs prepared in example 3 fully retained most of proteins of HepG2 cells, and significantly similar protein bands can be observed in a gel electrophoresis image, which indicates that proteins on cell membranes are substantially completely retained on the biomimetic nanoparticles CM @ PSINPs, and provides reliable basis for the homologous targeting and immune escape functions of nanoparticles.

Example 12

Through MTT cytotoxicity experiments, the proliferation inhibition effect of different groups of nano-drugs on liver cancer cells HepG2 is examined. The method comprises the following specific steps:

(1) logarithmic growth HepG2 cells were harvested after 0.25% trypsin-EDTA (1 mM) digestion of the cell pellet and resuspended in fresh MEM medium at 1.0X 10 ⁴The density of each well was seeded in 96-well plates and placed at 37 ℃ in 5% CO₂And continuously culturing for 24 hours in the incubator, and performing subsequent treatment when the cells are completely attached to the wall.

(2) The PSINPs group, the PSINPs plus light group, the CM @ PSINPs group and the CM @ PSINPs plus light group were set, respectively, and each group of nano-drugs was diluted to a range of concentrations (1, 4, 8, 16, 20. mu.g/mL) in culture medium. Adding the drugs of different groups into a 96-well plate cultured with HepG2 cells for incubation, wherein the PSINs illumination-adding group and the CM @ PSINPs illumination-adding group are added with drugs for 4 h and then are treated with 808 nm laser (1W/CM)²) Incubation was continued after 5 min of irradiation.

(3) After 24 h of drug action, the old drug-containing medium was removed, 0.05 mg/mL MTT solution was added to each well at 100. mu.L/well and incubation continued for 4 h. Then, blue-violet formazan crystals were dissolved by adding 100. mu.L of DMSO to each well, and the absorbance at a wavelength of 570 nm was measured by a microplate reader, and the cell proliferation inhibition rate was calculated.

As shown in FIG. 12, the results were calculated to show the IC of PSINPs, CM @ PSINPs, PSINPs + illumination group and CM @ PSINPs + illumination group₅₀The values were 9.34. mu.g/mL, 16.71. mu.g/mL, 5.10. mu.g/mL, and 3.41. mu.g/mL, respectively. The result shows that the PSINPs have obvious cell killing capacity, and the cytotoxicity of the PSINPs is further enhanced after the illumination is introduced, which indicates that the nanoparticles have a certain light-chemotherapy synergistic effect; after the outer layer of the PISNPs nano-particles is wrapped by cell membranes, the cytotoxicity of the drug is reduced, presumably because the drug release is reduced after the nano-particles are wrapped by the cell membranes; however, upon introduction of light on this basis, cell proliferation inhibition by CM @ PSINPs The preparation method is remarkably enhanced, supposing that under the illumination condition, the photosensitizer ICG causes the temperature of the nanoparticles to rise to cause cell membrane shedding, and further trigger the effective release of the nanoparticles, and is remarkable in that the cytotoxicity of the CM @ PSINPs + illumination group is even remarkably stronger than that of the PSINPs + illumination group, and because the HepG2 bionic membrane is wrapped at the outer layer of the CM @ PSINPs, the homologous uptake of HepG2 cells is increased, the drugs can be efficiently released in the cells after illumination, and the cell killing capability is remarkably enhanced.

Example 13

And monitoring the uptake condition of different cells to different nano-drugs by a laser confocal microscope. The method comprises the following specific steps:

(1) respectively mixing liver cancer cell HepG2 and normal liver cell L02 at a ratio of 2.0 × 10⁵The density of each well was seeded in 12-well plates and 5% CO at 37 deg.C₂Incubate in incubator for 24 h. And after the cells are completely attached to the wall, carrying out subsequent treatment.

(2) Preparing ICG solution, preparing PSINPs and CM @ PSINPs nano-drugs, respectively diluting the nano-drugs until ICG equivalent concentration is 2 mug/mL, and adding the diluted nano-drugs into different cells for incubation for 4 h.

(3) Removing the old culture medium, washing the cells for 3 times by using normal saline, adding Hoechst 33342 staining solution, incubating for 10 min, washing the cells for two times by using the normal saline, sealing the cells by using an anti-fluorescence quencher, and shooting the uptake condition of the cells by using a laser confocal microscope.

The results are shown in fig. 13, where HepG2 cells showed the strongest fluorescence after treatment with CM @ PSINPs, indicating that HepG2 cells took up CM @ PSINPs significantly more than free ICG and PSINPs at nanometer, indicating that CM @ PSINPs had good homologous targeting ability after HepG2 cell membranes wrapped on the surface of the nano-drug PSINPs; meanwhile, normal hepatocytes L02 have no obvious uptake to PSINPs and CM @ PSINPs, which shows that the nanoparticles have good safety to normal hepatocytes and lay the experimental foundation for the in vivo biological application of the nanoparticles.

The above description is only a preferred embodiment of the present invention, and all the equivalent changes and modifications made according to the claims of the present invention should be covered by the present invention.

Claims

1. The bionic nanoparticle is characterized by consisting of a shell and a nano inner core coated by the shell, wherein the shell is a eukaryotic cell membrane, the nano inner core is formed by assembling an anti-tumor hydrophobic drug, an anti-cardiovascular disease hydrophobic drug and a photosensitizer together, the particle size of the bionic nanoparticle is 100-400 nm, and the bionic nanoparticle has a photodynamic effect under the irradiation of near infrared light.

2. The biomimetic nanoparticle according to claim 1, wherein the eukaryotic cell membrane is HepG2 cell membrane.

3. The biomimetic nanoparticle according to claim 1, wherein the anti-tumor hydrophobic drug is sorafenib.

4. The biomimetic nanoparticle according to claim 1, wherein the anti-cardiovascular disease hydrophobic drug is propranolol.

5. The biomimetic nanoparticle according to claim 1, wherein the photosensitizer is indocyanine green.

6. The preparation method of the bionic nanoparticle based on double-drug co-assembly and having the function of photo-chemotherapy according to claim 1, characterized in that the method comprises the following steps:

1) dissolving a certain amount of anti-tumor hydrophobic drug in ethanol to obtain a solution A, wherein the concentration of the anti-tumor hydrophobic drug is 1-1000000 muM;

2) dissolving a certain amount of cardiovascular disease resistant hydrophobic drugs in ethanol to obtain a solution B, wherein the concentration of the cardiovascular disease resistant hydrophobic drugs is 1-1000000 muM;

3) dissolving a certain amount of photosensitizer in water to obtain a solution C, wherein the concentration of the photosensitizer is 1-1000000 mu M;

4) uniformly mixing 1 volume part of the solution A and 1 volume part of the solution B to obtain a mixed solution AB; adding 1 volume part of the solution C into 17 volume parts of ultrapure water to obtain a diluted solution C; adding 2 parts by volume of the mixed solution AB into 18 parts by volume of the diluted solution C in a vortex state to obtain a solution D;

5) Mixing 1 part by volume of the eukaryotic cell membrane dispersion liquid with 10 parts by volume of the solution D, and carrying out ultrasonic treatment for 10 min at room temperature under the conditions of ultrasonic power of 250W and ultrasonic frequency of 40 KHz to obtain the aqueous solution of the bionic nanoparticles.

7. The preparation method of the bionic nanoparticle based on dual-drug co-assembly and having the function of photo-chemotherapy according to claim 6, wherein the preparation method of the eukaryotic cell membrane dispersion liquid comprises the following steps: extracting eukaryotic cell membranes by adopting a differential centrifugation method, and dissolving the eukaryotic cell membranes in ultrapure water according to the proportion of 0.002 g/1 mL to obtain the eukaryotic cell membrane dispersion liquid.

8. The use of the biomimetic nanoparticle of claim 1 in the preparation of an anti-liver cancer medicament.