CN114306281B

CN114306281B - Visual heterozygous cell membrane nano-delivery system and preparation method and application thereof

Info

Publication number: CN114306281B
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: 2023-04-07
Anticipated expiration: 2042-01-25
Also published as: CN114306281A

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 grafted 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 molecular 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 an M2 phenotype to an 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 the guidance of fluorescence images to remodel the tumor microenvironment and induce anti-tumor immunity, and can be matched with near-infrared laser to carry out photothermal treatment to generate a tumor in-situ vaccine to prevent the recurrence and metastasis of tumors.

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 medicine, a hybrid cell membrane adsorbed on the nanoparticle medicine 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) Subjecting the platelets and the neutral particles to hypotonic lysis, repeated freezing and thawing and gradient centrifugation 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 to 3, the time of each ultrasonic crushing is 4 to 6min, the ultrasonic power is 750W, and the frequency is 20KHz.

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 to 18mg/mL.

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

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 to the reaction solution and stirring for a period of time specifically comprises:

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 hybrid cell membrane fragments by using the positively charged R848 nanoparticles, wherein the hybrid cell membrane and the NHS modified near-infrared fluorescent reagent can enable the NHS modified near-infrared fluorescent reagent with the active group to be combined with amino on the cell membrane under the incubation condition, and finally a visualized hybrid cell membrane nano delivery system is formed. 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 drugs and improve the drug administration safety.

The preparation method is simple to realize, 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 particle size distribution 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 part 1 of the experimental example;

FIG. 3 is a confocal imaging diagram of RNP @ PNM in part 2 of the experimental example;

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

FIG. 5 is the targeted fluorescence imaging of RNP and RNP @ PNM in section 4 of the experimental example in different organs and tumors in mice after surgery;

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

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

FIG. 8 shows the relative fluorescence intensities of RNP and RNP @ PNM under photothermal stimulation in the different organs and tumors of mice in section 5 of the experimental example;

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 the part 6 of the experimental example;

FIG. 11 is a photograph of the ex vivo tumors of the 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 is a graph showing 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 for 40-120 min at room temperature, adding a polyethyleneimine 600 solution and 2,2'- [ propane-2, 2-diylbis- (thio) ] diacetic acid into the reaction solution after the reaction is finished, 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-diylbis- (thio) ] diacetic acid is 5: 1;

(b) Preparation of platelet cell membranes (denoted PM) and neutrophilic granulosa cell membranes (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,

preparing PM: adding hypotonic solution (pure water solution containing 10ug/mL protease inhibitor) into platelet, cracking at 4 deg.C for 24h, repeatedly freezing and thawing the cell suspension with liquid nitrogen and 37 deg.C water bath twice (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 1640 culture medium containing 100ng/mL LPS for 3 hours for activation, centrifuging to remove the culture medium, adding hypotonic solution (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 water bath at 37 ℃, centrifuging at 4 ℃ for 10 minutes at 800Xg, 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 a platelet cell membrane and a neutral particle cell membrane, carrying out ultrasonic treatment for 5min at 4 ℃ by adopting an ultrasonic crusher uninterruptedly at 750W and 20KHz, and carrying out ultrasonic crushing twice to obtain a nanoscale hybrid cell membrane (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 Xg 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. RNP surface charge is positive, PNM surface charge is negative, 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 granulosa membranes were labeled with DIO and DID, respectively, and R848 was replaced with nile red. After the heterozygosis cell membrane nanoparticles are prepared, fixing the heterozygosis cell membrane nanoparticles in glycerol, and observing 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

Cytotoxicity of RNP and RNP @ PNM in example 1 was examined as follows:

RAW264.7 cells were plated at 1X 10 per well ⁴ Cell density in 96-well plates, after 12 hours of culture, the concentration of RNP or RNP @ PNM was varied (1, 2.5, 5, 10. Mu.g/mL) at 37 ℃ and 5% CO ₂ Incubating for 24h under the condition, 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 of RNP and RNP @ PNM to postoperative tumors, BALB/c mice were inoculated subcutaneously on the right side 1X 10 ⁶ 4T1 cells. When the tumor volume increases to about 200mm ³ Meanwhile, a part of the tumor is removed by surgery to establish a postoperative tumor model. RNP or RNP @ PNM, R848 by tail vein injection was replaced with Nile Red (15 ug). 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. 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 effect on postoperative tumor targeting, 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 10 ⁶ 4T1 cells. When the tumor volume increases to about 200mm ³ In 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 (15 ug) 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 tumor targeting effect of RNP @ PNM is better 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 of PBS, IR820-NHS (noted IR), RNP-IR and RNP @ PNM-IR (IR 820-NHS content 15. Mu.g) were each piped in EP. 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 rise to and be maintained at around 45 ℃ within 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 back with 1X 10 ⁶ 4T1 cells, tumor volume of about 150mm in 4T1 tumor-bearing mice ³ 90% 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 injection and irradiation treatmentsRepeating the steps once at intervals of one week; 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 combination of rnp @ pnm-IR formulation 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

After 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 back ⁵ 4T1 cells, and establishing a remote tumor model. The grouping comprises PBS, RNP @ PNM-IR, PNM-IR + Laser, RNP @ PNM-IR + Laser + aCD47; 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 again after irradiation; these injections and irradiation treatments were repeated once, one week apart; 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 the 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 is inoculated subcutaneously by luciferase-labeled 4T1 (4T 1-Luc), after two rounds of drug injection and laser treatment, all tumors are removed by operation, and a tumor recurrence and metastasis model is established. The packet includes PBS, RNP @ PNM-IR + Laser + aCD47. 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 of mice, with the number n =8 for each group, and survival curves were 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 fig. 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 mouse 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;

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

the nanoparticle medicament is an R848 nanoparticle formed by taking bovine serum albumin as a carrier.

2. The method for preparing the visual hybrid cell membrane nano delivery system according to claim 1, characterized by comprising the following steps:

(a) Carrying out hypotonic lysis, repeated freezing and thawing and gradient centrifugation on the platelet and the neutrophil granulocytes respectively to obtain a platelet cell membrane and a neutrophil granulocytic membrane;

(b) Mixing platelet cell membranes and neutral granulocyte cell membranes and carrying out ultrasonic crushing to obtain nanoscale heterozygous cell membranes;

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

3. The method according to claim 2, wherein the step (c) further comprises:

4. The method for preparing the nanoparticle medicament of claim 2, wherein the nanoparticle medicament is an R848 nanoparticle formed by taking bovine serum albumin as a carrier, and the R848 nanoparticle is prepared by the following method:

5. The method according to claim 2, wherein in the step (b), the number of ultrasonication is 1 to 3, each ultrasonication time is 4 to 6min, the ultrasonic power is 750W, and the frequency is 20KHz.

6. The method according to claim 3, wherein the incubation temperature is 35 to 39 ℃ and the incubation time is 1 to 3 hours.

7. The use of the visualized hybrid cell membrane nano-delivery system of claim 1 in the preparation of a drug for treating tumor by immune combination with photothermal therapy.

8. An immuno-combination photothermal tumor therapy drug comprising the visualized hybrid cell membrane nano delivery system of claim 1.