CN114306281A

CN114306281A - Visual heterozygous cell membrane nano-delivery system and preparation method and application thereof

Info

Publication number: CN114306281A
Application number: CN202210086451.6A
Authority: CN
Inventors: 董霞; 吕丰; 盛书培; 张琳华; 马桂蕾; 朱敦皖; 张燕; 梅林�
Original assignee: Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2022-01-25
Filing date: 2022-01-25
Publication date: 2022-04-12
Anticipated expiration: 2042-01-25
Also published as: CN114306281B

Abstract

The invention discloses a visual heterozygous cell membrane nano-delivery system and a preparation method and application thereof, wherein the visual heterozygous cell membrane nano-delivery system comprises a nanoparticle medicament, a heterozygous cell membrane adsorbed on the nanoparticle medicament and an N-hydroxysuccinimide modified near-infrared fluorescent reagent; the hybrid cell membrane is formed by fusing a platelet cell membrane and a neutrophil cell membrane; the N-hydroxysuccinimide modified near-infrared fluorescent reagent is connected with amino on the surface of the hybrid cell membrane; the visual heterozygous cell membrane nano-delivery system realizes the double targeting effect on the tumor by utilizing the characteristics of two cell membranes of platelets and neutral particles, can accurately target the residual tumor part after operation, and effectively delivers the nano-particle medicine; in addition, the visual heterozygous cell membrane nano-delivery system can reduce the toxic and side effects of the drug and improve the drug administration safety.

Description

Visual heterozygous cell membrane nano-delivery system and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a visualized heterozygous cell membrane nano-delivery system and a preparation method and application thereof.

Background

Tumor immunotherapy is an innovative approach to cancer therapy that aims to activate or enhance the human immune system to recognize and kill cancer cells. Despite the continual improvement of surgical methods and other treatment modalities, residual microscopic tumors and Circulating Tumor Cells (CTCs) remain the major stumbling stones for cancer treatment. Therefore, there is a great need for a therapeutic strategy that enhances the ability to target residual tumors and CTCs after surgery, remodels the post-operative tumor microenvironment, and prevents tumor recurrence and metastasis.

R848 is a small molecule compound based on imidazoquinoline, can be recognized by T cell-like receptor (TLR)7/8, and can induce antitumor response. It is reported that recognition of R848 in the endosome results in activation and maturation of Antigen Presenting Cells (APC) and induces secretion of proinflammatory cytokines, type I Interferons (IFN) and chemokines. In addition to having an immunostimulatory function, R848 may also modulate immunosuppressive cells such as Myeloid Derived Suppressor Cells (MDSCs) and M2 macrophages. R848 may convert MDSCs into APCs, such as Dendritic Cells (DCs) and macrophages, and polarize Tumor Associated Macrophages (TAMs) from the M2 phenotype to the M1 phenotype. R848 modulates the immunosuppressive tumor microenvironment into an immunogenic microenvironment by polarizing the immunosuppressive TAMs and MDSCs into tumoricidal APCs rather than depleting them. Since TLR7/8 is present in the intracellular compartment, entry of R848 into the cell is essential for activation of immune cells. However, R848 has poor water solubility and low bioavailability when administered systemically. At the same time, it may cause side effects such as rapid leukopenia and short-term local immunodeficiency. Researchers typically formulate R848 as a nanoparticle to enhance their uptake by cells. However, R848 is a dose-dependent drug, but the passive targeting effect of nanoparticles is slightly insufficient when administered intravenously. Therefore, how to focus immunomodulators on the lesion through a precise delivery system, overcome local immune tolerance, and achieve effective treatment is an important issue.

Disclosure of Invention

The invention aims to provide a visual heterozygous cell membrane nano-delivery system and a preparation method and application thereof, wherein the visual heterozygous cell membrane nano-delivery system realizes double targeting effects on tumors by utilizing the characteristics of two cell membranes, and can accurately target postoperative residual tumor parts; in addition, the visual heterozygous cell membrane nano-delivery system can reduce the toxic and side effects of the drug, effectively deliver the immune drug R848 through fluorescence image guidance to remodel a tumor microenvironment, induce anti-tumor immunity, and perform photothermal treatment by matching with near-infrared laser to generate a tumor in-situ vaccine to prevent the recurrence and metastasis of the tumor.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

the invention provides a visual hybrid cell membrane nano delivery system, which comprises a nanoparticle drug, a hybrid cell membrane adsorbed on the nanoparticle drug and an N-hydroxysuccinimide modified near-infrared fluorescent reagent;

the hybrid cell membrane is formed by fusing a platelet cell membrane and a neutrophil cell membrane;

and the N-hydroxysuccinimide modified near-infrared fluorescent reagent is grafted with amino on the surface of the hybrid cell membrane.

The platelets and neutral particles have the characteristic of migrating to wounds and inflammatory parts, and actively target postoperative residual tumors; the cell membrane contains lipids and proteins of the source cell and inherits various unique capabilities from the source cell. The visual heterozygous cell membrane nano-delivery system realizes the double targeting effect on the tumor by utilizing the characteristics of two cell membranes of platelets and neutral particles, can accurately target residual tumor parts after operation, effectively delivers an immune drug R848 through the guidance of fluorescence images to remold a tumor microenvironment and induce anti-tumor immunity, and can be matched with near-infrared laser to carry out photo-thermal treatment to generate a tumor in-situ vaccine to prevent the recurrence and the metastasis of the tumor; in addition, the visual heterozygous cell membrane nano-delivery system can reduce the toxic and side effects of the drug and improve the drug administration safety.

Preferably, the nanoparticle drug is an R848 nanoparticle formed by taking bovine serum albumin as a carrier.

The second aspect of the present invention provides a preparation method of the above visualized hybrid cell membrane nano delivery system, wherein the preparation method comprises the following steps:

(a) carrying out hypotonic lysis, repeated freezing and thawing and gradient centrifugation on the platelets and the neutral particles respectively to obtain platelet cell membranes and neutral particle cell membranes;

(b) mixing platelet cell membranes and neutral particle cell membranes and carrying out ultrasonic crushing to obtain nano-scale hybrid cell membranes;

(c) and adsorbing the nanoscale heterozygous cell membrane onto the nanoparticle medicine in an electrostatic adsorption mode to obtain the visualized heterozygous cell membrane nano delivery system.

Preferably, the step (c) further comprises:

and carrying out mixed incubation and dialysis on the nanoparticle drug adsorbing the hybrid cell membrane and the N-hydroxysuccinimide modified near-infrared fluorescent reagent.

Preferably, the near-infrared fluorescent agent is indocyanine green or neoindocyanine green.

Preferably, in the step (a), the number of times of ultrasonic crushing is 1-3, the time of each ultrasonic crushing is 4-6 min, the ultrasonic power is 750W, and the frequency is 20 KHz.

Preferably, the incubation temperature is 35-39 ℃ and the incubation time is 1-3 h.

Preferably, the nanoparticle drug is an R848 nanoparticle formed by taking bovine serum albumin as a carrier, and the R848 nanoparticle is prepared by the following method:

(1) adding dithiothreitol into a bovine serum albumin solution, and stirring to obtain a mixed solution;

(2) and adding R848 into the mixed solution for reaction, adding polyethyleneimine and 2,2' - [ propane-2, 2-diyl bis- (thio) ] diacetic acid into the reaction solution after the reaction is finished, stirring for a period of time, and dialyzing to obtain R848 nanoparticles.

Preferably, in the step (1), the molar ratio of dithiothreitol to bovine serum albumin is (15-19): 1;

preferably, in the step (1), the concentration of the bovine serum albumin solution is 12-18 mg/mL.

Preferably, in the step (1), the stirring treatment time is 20-40 min.

Preferably, in the step (2), the polyethyleneimine is polyethyleneimine 600;

preferably, in the step (2), the reaction temperature is room temperature, and the reaction time is 40-120 min;

preferably, in the step (2), the adding R848 to the mixed solution for reaction specifically includes:

adding an ethanol solution containing R848 into the mixed solution for reaction, wherein the addition volume of the ethanol solution containing R848 is 1-1.2 times of the volume of the mixed solution, and the concentration of the ethanol solution containing R848 is 1.2-1.8 mg/mL;

adding polyethyleneimine and 2,2' - [ propane-2, 2-diylbis- (thio) ] diacetic acid into a reaction solution, and stirring for a period of time specifically comprises the following steps:

adding a polyethyleneimine solution and 2,2'- [ propane-2, 2-diyl bis- (thio) ] diacetic acid into a reaction solution, and stirring for 1.5-3 h, wherein the mass volume concentration of the polyethyleneimine solution is 1.2-1.8%, and the volume ratio of the reaction solution, the polyethyleneimine solution and the 2,2' - [ propane-2, 2-diyl bis- (thio) ] diacetic acid is 5: 0.6-1.2.

In the preparation process of the R848 nanoparticles, disulfide bonds of Bovine Serum Albumin (BSA) are reduced by Dithiothreitol (DTT), the conformation of the BSA is changed, the R848 nanoparticles enter cavities of the BSA through hydrophilic-hydrophobic interaction to form the R848 nanoparticles, and the R848 nanoparticles are positively charged through the crosslinking action of polyethyleneimine and 2-2' - [ propane-2-2-diyl-bis (thio) ] diacetic acid; and adsorbing the heterozygous cell membrane fragments by the positively charged R848 nanoparticles, and enabling the near-infrared fluorescent reagent with active groups after NHS modification to be jointed with amino on the cell membrane under the incubation condition of the heterozygous cell membrane and the NHS modified near-infrared fluorescent reagent to finally form a visual heterozygous cell membrane nano delivery system. The preparation method disclosed by the invention is simple to implement, high in efficiency and good in effect, and meets the application requirements.

The third aspect of the invention provides an application of the visualized hybrid cell membrane nano-delivery system in preparation of a medicine for treating tumors by immune combination and photothermal therapy.

In a fourth aspect, the invention provides an immune and photothermal combined tumor treatment drug, which comprises the visual hybrid cell membrane nano-delivery system.

Compared with the prior art, the invention has the beneficial effects that at least:

the visual heterozygous cell membrane nano-delivery system realizes the double targeting effect on the tumor by utilizing the characteristics of two cell membranes of platelets and neutral particles, can accurately target residual tumor parts after operation, effectively delivers an immune drug R848 to remold a tumor microenvironment, induces anti-tumor immunity, can be matched with near infrared laser to carry out photothermal treatment, generates a tumor in-situ vaccine, and prevents the recurrence and the metastasis of the tumor; in addition, the visual heterozygous cell membrane nano-delivery system can reduce the toxic and side effects of the drug and improve the drug administration safety.

The preparation method disclosed by the invention is simple to implement, high in efficiency and good in effect, and meets the application requirements.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.

FIG. 1 is a graph showing the particle size distributions of RNP, PNM and RNP @ PNM in part 1 of the experimental example;

FIG. 2 is the surface zeta potentials of RNP, PNM and RNP @ PNM in Experimental example section 1;

FIG. 3 is a confocal imaging of RNP @ PNM in Experimental section 2;

FIG. 4 shows the in vitro biocompatibility of RNP and RNP @ PNM in Experimental example part 3;

FIG. 5 is a fluorescent image of RNP and RNP @ PNM in section 4 of the experimental example, in different organs and tumors of mice after surgery;

FIG. 6 is a graph showing the relative fluorescence intensity of RNP and RNP @ PNM in the section 4 of the experimental example at different organs and tumors in mice after surgery;

FIG. 7 is a fluorescent image of RNP and RNP @ PNM under photothermal stimulation in different organs and tumors of mice in section 5 of the experimental example; (ii) a

FIG. 8 is a graph showing the relative fluorescence intensities of RNP and RNP @ PNM in part 5 of the experimental example in different organs and tumors of mice under photothermal stimulation;

FIG. 9 is a thermal imaging diagram in section 6 of the experimental example;

FIG. 10 is a graph showing a photothermal temperature increase curve in section 6 of the experimental example;

FIG. 11 is a photograph of isolated tumors from different treatment groups in section 7 of the experimental example; FIG. 12 is a graph of tumor growth for the different treatment groups of section 7 of the experimental example;

FIG. 13 is a photograph of the distal ex vivo tumors of different treatment groups in section 8 of the experimental example;

FIG. 14 is a graph of distal tumor growth for different treatment groups in section 8 of the experimental example;

FIG. 15 is a bioluminescence map of tumor recurrence and metastasis from the different treatment groups of section 9 of the experimental example;

FIG. 16 is a graph of lung staining for different treatment groups in section 9 of the experimental example;

FIG. 17 shows survival rates at different times for mice of different treatment groups in section 9 of the experimental example.

Detailed Description

The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.

It is to be noted that, unless otherwise specified, technical or scientific terms used herein shall have the ordinary meaning as understood by those skilled in the art to which the invention pertains.

Example 1

The embodiment is a preparation method of a visual hybrid cell membrane nano delivery system, which comprises the following steps:

(a) preparation of R848 nanoparticles (denoted RNP):

adding dithiothreitol into 15mg/mL bovine serum albumin solution, and stirring for 30min to obtain a mixed solution, wherein the molar ratio of the dithiothreitol to the bovine serum albumin is 17: 1;

adding an ethanol solution containing R848 into the mixed solution, reacting at room temperature for 40-120 min, after the reaction is finished, adding a polyethyleneimine 600 solution and 2,2'- [ propane-2, 2-diyl bis- (thio) ] diacetic acid into the reaction solution, stirring for 2h, and dialyzing to obtain RNP, wherein the addition volume of the ethanol solution containing R848 is 1.1 times of the volume of the mixed solution, the concentration of the ethanol solution containing R848 is 1.5mg/mL, the mass volume concentration of the polyethyleneimine 600 solution is 1.5%, and the volume ratio of the reaction solution, the polyethyleneimine solution and the 2,2' - [ propane-2, 2-diyl bis- (thio) ] diacetic acid is 5: 1;

(b) preparation of platelet cell membranes (denoted PM) and neutrophil membrane (denoted NM):

subjecting the platelet and neutral particles to hypotonic lysis, repeated freezing and thawing, and gradient centrifugation to obtain platelet cell membrane and neutral particle cell membrane, specifically,

preparation of PM: adding hypotonic solution (pure water solution containing 10ug/mL protease inhibitor) into platelet, cracking at 4 deg.C for 24 hr, repeatedly freezing and thawing the cell suspension twice with liquid nitrogen and 37 deg.C water bath (quick freezing for 5min, shaking for rapid dissolution), centrifuging at 4 deg.C for 15min at 800Xg, collecting supernatant, centrifuging at 4 deg.C for 30min at 10000Xg, and collecting precipitate to obtain PM;

preparing NM: culturing neutral particles in a 1640 culture medium containing 100ng/mL LPS for 3 hours for activation, centrifuging to remove the culture medium, adding a hypotonic solution (a pure water solution containing 10ug/mL protease inhibitor), cracking at 4 ℃ for 24 hours, repeatedly freezing and thawing the cell suspension twice (quick freezing for 5 minutes and quick dissolving by shaking) by using liquid nitrogen and a water bath at 37 ℃, centrifuging at 4 ℃ and 800Xg for 10 minutes, collecting supernatant, centrifuging at 4 ℃ and 7000Xg for 30 minutes, collecting supernatant, centrifuging at 4 ℃ and 20000Xg for 1 hour, and collecting precipitate to obtain NM;

(c) mixing platelet cell membranes and neutral particle cell membranes, carrying out ultrasonic treatment for 5min at 4 ℃ by adopting an ultrasonic disruptor uninterruptedly with 750W and 20KHz, and carrying out ultrasonic disruption twice to obtain nanoscale hybrid cell membranes (marked as PNM), wherein the mass ratio of PM to NM is 1: 3;

(d) adsorbing the nanoscale hybrid cell membrane onto the nanoparticle medicine in an electrostatic adsorption mode (the obtained material is recorded as RNP @ PNM), wherein the mass ratio of RNP to PNM is 5: and 2, mixing the obtained product with N-hydroxysuccinimide modified novel indocyanine green (recorded as IR-820-NHS) and incubating the obtained product at 37 ℃ for 2 hours, and dialyzing the obtained product to obtain the visual hybrid cell membrane nano delivery system (recorded as RNP @ PNM-IR).

Comparative example 1

The comparative example is a preparation method of R848 nano-particles (marked as RNP-IR) loaded with N-hydroxysuccinimide modified novel indocyanine green, and the preparation method comprises the following steps:

mixing RNP and IR-820-NHS at room temperature under stirring for 6 hr, and dialyzing to obtain RNP-IR.

Comparative example 2

The comparative example is a preparation method of a hybrid cell membrane (marked as PNM-IR) loaded with N-hydroxysuccinimide modified novel indocyanine green, and the preparation method comprises the following steps:

mixing PNM and IR-820-NHS, incubating at 37 deg.C for 2h, centrifuging at 20000Xg for 1h, and collecting precipitate to obtain PNM-IR.

Examples of the experiments

1. Determination of particle size and zeta Point the particle size and zeta potential of RNP, PNM, RNP @ PNM in example 1 were determined by atomic force microscopy and dynamic light scattering particle size analyzer, the particle size distribution of which is shown in FIG. 1; the results of the zeta potential analysis are shown in FIG. 2;

as can be seen from fig. 1 and 2: the average particle size of the R848 Nanoparticles (RNP) is about 150nm, the average particle size of the hybrid cell membrane (PNM) is about 200nm, and the average particle size of the R848 nanoparticles (RNP @ PNM) adsorbing the hybrid cell membrane is about 180 nm. The RNP surface charge is positive, the PNM surface charge is negative, and the RNP @ PNM surface charge is positive.

2. Confocal imaging of RNP @ PNM in example 1

Confocal fluorescence imaging was used to determine whether two cell membranes and the R848 nanoparticle fused. Before membrane fusion, platelet and neutrophilic membranes were labeled with DIO and DID, respectively, and R848 was replaced with nile red. After the heterozygosis cell membrane nanoparticles are prepared, the heterozygosis cell membrane nanoparticles are fixed in glycerol and observed under a laser confocal scanning microscope; the observation results are shown in FIG. 3, where red represents platelet cell membrane (PM), green represents R848 Nanoparticles (RNP), and blue represents neutrophil cell membrane (NM).

As can be seen from fig. 3: the co-localization of the three fluorescent signals determines the fusion of platelet membrane, neutrophil membrane and R848 nanoparticle.

3. Cytotoxicity assays

The cytotoxicity of RNP and RNP @ PNM in example 1 was determined as follows:

RAW264.7 cells were plated at 1X 10 per well⁴The cells were plated at a density in 96-well plates and cultured for 12 hours, after which time varying concentrations (1, 2.5, 5, 10. mu.g/mL) of RNP or RNP @ PNM were added at 37 ℃ with 5% CO₂Incubating for 24h under the condition, and detecting the cell viability by using MTS, wherein the detection result is shown in figure 4;

as can be seen from fig. 4: the RNP @ PNM is almost nontoxic to cells and has good in-vitro biocompatibility.

4. Study of postoperative tumor-targeted fluorescence imaging

To determine the targeting effect of RNP and RNP @ PNM on postoperative tumors, BALB/c mice were inoculated 1X 10 subcutaneously on the right side⁶4T1 cells. When the tumor volume increases to about 200mm³Meanwhile, a part of the tumor is removed by surgery to establish a postoperative tumor model. RNP or RNP @ PNM, R848 was replaced by nile red (15ug) by tail vein injection. Mice were subjected to in vivo fluorescence imaging at the indicated times using a small animal imaging system. Mice were then dissected and fluorescence profiles of major organs and tumors were obtained. The results of fluorescence imaging of different organs are shown in FIG. 5, and the relative fluorescence intensities of different organs are shown in FIG. 6;

as can be seen from fig. 5 and 6: compared with RNP, RNP @ PNM has better postoperative tumor targeting effect, and the drug has more accumulation at the tumor part and reduced distribution at the liver part.

5. Tumor targeted fluorescence imaging research under photothermal stimulation

To determine the targeting effect of RNP and RNP @ PNM on tumors following photothermal stimulation, BALB/c mice were inoculated subcutaneously on the right side with 1X 10⁶4T1 cells. When the tumor volume increases to about 200mm³In the treatment, PNM-IR was injected into the tail vein, and after 24 hours, laser irradiation was performed to expose the tumor site to 808nm laser (1W/cm)²) For 5 minutes. RNP or RNP @ PNM, R848 was then replaced by nile red (15ug) by tail vein injection. Mice were subjected to in vivo fluorescence imaging at the indicated times using a small animal imaging system. Mice were then dissected and fluorescence profiles of major organs and tumors were obtained. The fluorescence imaging results of different organs are shown in fig. 7, and the relative fluorescence intensities of different organs are shown in fig. 8;

as can be seen from fig. 7 and 8: compared with RNP, the RNP @ PNM has better tumor targeting effect after photo-thermal stimulation, and the medicine has more accumulation at the tumor part.

6. In vitro photothermal efficiency study

To determine the photothermal effect intensity of IR820-NHS in vitro, 100. mu.L PBS, IR820-NHS (noted IR), RNP-I were each placed in EP tubesR and RNP @ PNM-IR (IR820-NHS content 15. mu.g). Using a 808 laser (1W/cm)²) Irradiating and covering an EP tube, observing the temperature rise condition of the tumor within 5 minutes by using a thermal imager and drawing a curve, wherein the thermal imaging is shown in figure 9, and the temperature change curve is shown in figure 10;

as can be seen from fig. 9 and 10: both RNP-IR and RNP @ PNM-IR retain the photothermal efficiency of IR820-NHS and are able to be raised to and maintained at around 45 ℃ in 5 minutes in vitro.

7. Study of in vivo tumor suppression Effect

For treatment of primary tumors, BALB/c mice were inoculated subcutaneously on the right side of the back with 1X 10⁶4T1 cells, a tumor volume of about 150mm in 4T1 tumor-bearing mice³90% of tumors are removed by operation, and a postoperative tumor model is established. Treatment with different treatment modalities including PBS, RNP-IR, RNP @ PNM-IR, PNM-IR + Laser, RNP @ PNM-IR + Laser + aCD47 (R848: 40. mu.g per mouse); laser irradiation was carried out 48 hours after the injection, and the tumor site was exposed to 808nm laser light (1W/cm)²)5 minutes, and injecting the medicine once again after irradiation; these injections and irradiation treatments were repeated once, one week apart; measuring the tumor volume by a digital caliper, and calculating to be L multiplied by W/2(L, the longest dimension; W, the shortest dimension); monitoring tumor growth and plotting a curve; different groups of tumors were measured with digital calipers. Ex vivo tumors are shown in figure 11; the tumor growth curve is shown in fig. 12;

as can be seen from fig. 11 and 12: the RNP @ PNM-IR formulation in combination with photothermal was effective in inhibiting tumor growth and revealed potential for co-therapy with anti-CD 47 antibodies.

8. Remote tumor inhibition effect study

Following the first treatment of the primary tumor (i.e., on the day of 90% surgical resection of the primary tumor), mice were inoculated subcutaneously 5X 10 on the left side of the back⁵4T1 cells, and establishing a remote tumor model. The packet comprises PBS, RNP @ PNM-IR, PNM-IR + Laser, RNP @ PNM-IR + Laser + aCD 47; the primary tumor was injected for 48 hours and then irradiated with laser light to expose the tumor site to 808nm laser light (1W/cm)²)5 minutes, and injecting the medicine once again after irradiation; these injections and irradiation treatments were repeated once and once a week apart(ii) a Distal tumors were not treated therapeutically. Measuring the tumor volume by a digital caliper, and calculating to be L multiplied by W/2(L, the longest dimension; W, the shortest dimension); monitoring tumor growth and plotting a curve; different groups of tumors were measured with digital calipers, and the distal tumors ex vivo were as shown in fig. 13; the tumor growth curve is shown in fig. 14;

as can be seen from fig. 13 and 14: the combination of the RNP @ PNM-IR preparation and photo-thermal can effectively stimulate the systemic immune effect and inhibit the growth of remote tumors.

9. Study on tumor recurrence and metastasis inhibition effect

The primary tumor was inoculated subcutaneously with luciferase-labeled 4T1(4T1-Luc), and after two rounds of drug injection and laser treatment, all tumors were surgically removed to establish a tumor recurrence and metastasis model. The packet includes PBS, RNP @ PNM-IR + Laser + aCD 47. Tumor recurrence and metastatic burden were monitored by bioluminescence of tumor cells, and in vivo bioluminescence imaging was performed at the indicated time points using the IVIS system. The lung tissue was fixed and stained with a buff's solution and the lung metastasis was further observed. Survival was recorded daily for each group, and the number n of mice in each group was 8, and a survival rate curve was plotted. Tumor recurrence and metastasis at

day

33 and 42 are shown in FIG. 15; lung metastasis staining is shown in figure 16; the survival rate curve is shown in fig. 17;

as can be seen from FIGS. 15 to 17: after the combination treatment of the RNP @ PNM-IR preparation and the photothermal treatment, the tumor recurrence and metastasis can be effectively controlled, and the survival time of the mice is remarkably prolonged.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims

1. A visual hybrid cell membrane nano delivery system is characterized by comprising a nanoparticle drug, a hybrid cell membrane adsorbed on the nanoparticle drug and an N-hydroxysuccinimide modified near-infrared fluorescent reagent;

2. The visual hybrid cell membrane nano-delivery system according to claim 1, wherein the nanoparticle drug is an R848 nanoparticle formed by using bovine serum albumin as a carrier.

3. The preparation method of the visual hybrid cell membrane nano-delivery system according to any one of claims 1 to 2, characterized by comprising the following steps:

4. The method according to claim 3, wherein the step (c) further comprises:

5. The method for preparing the nanoparticle medicament of claim 3, wherein the nanoparticle medicament is R848 nanoparticles formed by taking bovine serum albumin as a carrier, and the R848 nanoparticles are prepared by the following method:

6. The preparation method according to claim 3, wherein in the step (a), the number of times of ultrasonication is 1 to 3, the time of each ultrasonication is 4 to 6min, the ultrasonic power is 750W, and the frequency is 20 KHz.

7. The preparation method according to claim 4, wherein the incubation temperature is 35-39 ℃ and the incubation time is 1-3 h.

8. The use of the visualized hybrid cell membrane nano-delivery system of any one of claims 1-2 in the preparation of a drug for treating tumors by immune combination with photothermal therapy.

9. An immune and photothermal combined tumor treatment drug, which comprises the visualized hybrid cell membrane nano delivery system according to any one of claims 1-2.