CN114081958B

CN114081958B - Functionalized apoptosis small drug delivery system and preparation method and application thereof

Info

Publication number: CN114081958B
Application number: CN202111293292.9A
Authority: CN
Inventors: 董霞; 吕丰; 朱敦皖; 张琳华; 盛书培; 张燕; 马桂蕾; 梅林�; 李稳
Original assignee: Institute of Biomedical Engineering of CAMS and PUMC
Current assignee: Institute of Biomedical Engineering of CAMS and PUMC
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2023-04-25
Anticipated expiration: 2041-11-03
Also published as: CN114081958A

Abstract

The invention discloses a functionalized apoptosis small body drug delivery system, a preparation method and application thereof, wherein the functionalized apoptosis small body drug delivery system comprises nanoparticle drugs and apoptosis small bodies; the nanoparticle drug is encapsulated within the apoptotic bodies; the functionalized apoptosis small drug delivery system improves the uptake efficiency of cells on drugs and the targeting efficiency of drugs on tumors, and can generate photo-thermal effect to kill the tumors through laser irradiation; realizes the effect of jointly resisting tumors by combining immunization and photothermal therapy.

Description

Functionalized apoptosis small drug delivery system and preparation method and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a functionalized apoptosis small drug delivery system, a preparation method and application thereof.

Background

Malignant tumor is one of the primary diseases causing human death, and how to realize tumor targeting accurate treatment and prevent tumor recurrence and metastasis is a great challenge and a conquer of difficulties faced by tumor treatment. The development of tumor immunotherapy technology opens up a new way for tumor treatment. Tumor immunotherapy can stimulate the immune response of a host by restarting and maintaining the immune system's recognition of tumor cells, producing systemic anti-tumor immunity. However, the administration of the immune therapeutic system has serious off-target side effects, the overall response rate of patients is not high (about 5-30%), the curative effects of treating tumors and preventing tumor recurrence and metastasis are limited, and the immune escape and the formation of local tumor immunosuppressive networks are main barriers for the complete eradication of tumors. It is particularly important how to re-induce tumor-associated antigens sufficient to restart the body's immunity against the tumor immunosuppressive microenvironment. Systemic immunotoxicity may be induced by systemic administration of immunotherapeutic agents with certain off-target side effects, such as severe anemia caused by hematological toxicity of CD47 inhibitors, and drug release may be difficult to control or achieve desired efficacy. How to concentrate the immunostimulant on the focus by a precise and effective delivery system to overcome local immune tolerance and achieve efficient treatment is an important issue.

The problem with accurate and efficient delivery systems is targeted delivery and image guided delivery of drugs. With the development of medical imaging technology, image guidance provides a "navigation" system for a drug delivery system. Fluorescent imaging has the advantages of nonionic low-energy radiation, high sensitivity, continuous real-time monitoring, noninvasive or minimally invasive performance, relatively low equipment price and the like, and has rapid development in the application aspect of image-guided drug delivery. Near infrared fluorescent agents such as indocyanine green (ICG), new indocyanine green (IR-820), and the like are clinically used as fluorescent photothermal agents, exhibit strong fluorescence in the Near Infrared Region (NIR), and obtain good therapeutic effects in photothermal therapy. However, they suffer from the disadvantages of rapid clearance, insufficient in vivo photostability, and lack of in vivo targeting, limiting their combined use in tumor therapy.

Disclosure of Invention

The invention aims to provide a functional apoptosis small body drug delivery system, a preparation method and application thereof, wherein the drug delivery system prepared by the preparation method adopts the apoptosis small body as a carrier, can be specifically ingested by macrophages and actively targets tumors in a mode of taking the macrophages for convenience, effectively improves the targeting rate of the drug on the tumors, and kills the tumors by a near infrared fluorescent reagent.

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

the first aspect of the present invention provides a functionalized apoptotic body drug delivery system comprising a nanoparticle drug and an apoptotic body;

the nanoparticle drug is encapsulated within the apoptotic bodies.

Preferably, the functionalized apoptotic body drug delivery system further comprises an N-hydroxysuccinimide modified near infrared fluorescent reagent that anneals to amino groups on the apoptotic body surface.

The functionalized apoptotic body drug delivery system can be specifically ingested by macrophages, and compared with free drugs and nano preparations, the functionalized apoptotic body drug delivery system improves the ingestion efficiency of cells on the drugs; meanwhile, the tumor is actively targeted by taking the macrophage for the convenience of the vehicle, so that the targeting efficiency of the medicine to the tumor is improved, and the medicine is accumulated to the tumor more and infiltrates the deep part of the tumor; the near infrared fluorescent reagent connected with the apoptosis body is a photosensitizer, and can generate photo-thermal effect to kill tumor through laser irradiation; realizes the combined anti-tumor effect of the immune and photothermal therapy, and makes up the defect of the monotherapy in anti-tumor aspect.

Preferably, the apoptotic bodies are prepared from RAW267.4 cells.

Preferably, the nanoparticle drug is an R848 nanoparticle.

The second aspect of the present invention provides a method for preparing the functionalized apoptotic body drug delivery system, comprising the steps of:

and incubating the nanoparticle medicine and RAW264.7 cells together, washing and radiating by ultraviolet rays, then incubating again, and then centrifuging at a differential speed to obtain the functionalized apoptotic body medicine delivery system.

Preferably, the preparation method further comprises adding an N-hydroxysuccinimide modified near infrared fluorescent reagent before re-incubation.

Preferably, the N-hydroxysuccinimide modified near infrared fluorescent reagent is added at a final concentration of 12 to 18. Mu.g/mL.

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

Preferably, the ratio of nanoparticle drug to RAW264.7 cells is 2mg to (10) ⁶ ～10 ⁸ )cell。

Preferably, the co-incubation temperature is 35-39 ℃ and the time is 6-12 h;

preferably, the intensity of ultraviolet light in the ultraviolet irradiation is 0.08-0.12J/cm ² The irradiation time is 25-35 min;

preferably, the re-incubation temperature is 35-39 ℃ and the time is 1.5-3 h;

preferably, the differential centrifugation specifically includes:

centrifuging for 8-12 min at 250-350 g, collecting supernatant, and centrifuging for 15-25 min at 1800-2200 g.

Preferably, the nanoparticle drug is an R848 nanoparticle, the R848 nanoparticle being prepared by:

(a) Adding dithiothreitol into the bovine serum albumin solution and stirring to obtain a mixed solution;

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

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

preferably, the polyethyleneimine is polyethyleneimine 600;

preferably, the stirring treatment time is 20-40 min;

preferably, the concentration of the bovine serum albumin solution is 12-18 mg/mL.

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

preferably, 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;

preferably, adding polyethyleneimine and 2,2' - [ propane-2, 2-diylbis- (thio) ] diacetic acid to the reaction liquid and stirring for a period of time specifically comprises:

adding a polyethyleneimine solution and 2,2'- [ propane-2, 2-diylbis- (thio) ] diacetic acid into the 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 to the polyethyleneimine solution to the 2,2' - [ propane-2, 2-diylbis- (thio) ] diacetic acid is 5:0.6-1.2:0.6-1.2.

R848 is a small molecular compound based on imidazoquinoline, can be recognized by a T cell-like receptor (TLR) 7/8, and can induce an anti-tumor response; recognition of R848 in the endosome results in activation and maturation of Antigen Presenting Cells (APCs) and induction of secretion of pro-inflammatory cytokines, type I Interferons (IFNs), and chemokines. In addition to having immunostimulatory functions, R848 may also modulate immunosuppressive cells, such as Myeloid Derived Suppressor Cells (MDSCs) and M2 macrophages; r848 can convert MDSCs to 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 to the immunogenic microenvironment by polarizing the immunosuppressive TAMs and MDSCs as 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 is a hydrophobic drug that is more difficult to directly enter cells to function. R848 is generally prepared into nano particles to improve the uptake of cells, however, the passive targeting effect of the nano particles is slightly insufficient when the nano particles are administrated intravenously, and meanwhile, the anti-tumor effect exerted by a single immune adjuvant is very limited.

In the preparation process of the R848 nano-particles, dithiothreitol (DTT) is used for reducing disulfide bonds of Bovine Serum Albumin (BSA), the conformation of the BSA is changed, R848 enters a BSA cavity through hydrophilic-hydrophobic interaction to form the R848 nano-particles, and then the R848 nano-particles are provided with positive points through the crosslinking effect of polyethyleneimine and 2-2' -propane-2-2-diyl-bis (thio) ] diacetic acid; then the affinity of the positively charged R848 nano particles to cells enters the cells, and then the cells undergo ultraviolet irradiation to cause apoptosis to generate drug-loaded apoptosis bodies, and the apoptosis bodies and the NHS modified near infrared fluorescent reagent enable the near infrared fluorescent reagent with active groups to be combined with amino groups on the apoptosis bodies after the NHS modification under the incubation condition, so that a functional apoptosis body drug delivery system is finally formed. The preparation method provided 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 functionalized apoptotic body drug delivery system in preparing an immune combined photothermal tumor treatment drug.

In a fourth aspect, the invention provides an immune-coupled photothermal tumor treatment drug comprising the functionalized apoptotic body drug delivery system described above.

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

The preparation method provided 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 embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale.

FIG. 1 is a particle size distribution of R848NP, AB, and R848 NP/AB;

FIG. 2 shows the surface zeta potential of R848NP, AB, and R848 NP/AB;

FIG. 3 is a flow cytometry analysis of Annexin V-FITC stained AB (purple) and RAW264.7 cells (red) for apoptotic body yield;

FIG. 4 is a flow cytometry analysis of apoptotic body drug loading for Annexin V-FITC stained AB (purple) and R848NP/AB (green);

FIG. 5 is a confocal scanning electron microscope image of RAW264.7 cells ingesting different R848 preparations;

FIG. 6 is a flow cytometry analysis of RAW264.7 cells uptake of different R848 formulations;

FIG. 7 is a graph showing statistics of mean fluorescence intensities of RAW264.7 cells ingesting different R848 formulations;

FIG. 8 is a flow cytometry analysis of DC maturation promotion of different R848 formulations in Experimental example section 5;

FIG. 9 is a diagram showing the detection of the secretion of the pro-inflammatory cytokine TNF- α by ELISA in Experimental example part 5;

FIG. 10 is a diagram showing the detection of secretion of the pro-inflammatory cytokine IL-12 by ELISA in Experimental example part 5;

FIG. 11 is a diagram showing the detection of secretion of proinflammatory cytokine IL-6 by ELISA in Experimental example part 5;

FIG. 12 is a flow cytometry analysis of macrophage polarization induced by different R848 formulations in experimental example section 6;

FIG. 13 is a sample of the detection of secretion of the pro-inflammatory cytokine TNF- α by ELISA in section 6;

FIG. 14 is a sample of the detection of secretion of the pro-inflammatory cytokine IL-12 by ELISA in section 6;

FIG. 15 is a diagram showing the detection of secretion of proinflammatory cytokine IL-6 by ELISA in Experimental example part 6;

FIG. 16 is a fluorescence image of major organs and tumors of mice 24 hours after intravenous injection of nile red labeled R848NP or R848 NP/AB;

FIG. 17 is a graph showing statistics of relative fluorescence intensities of major organs and tumors of mice;

FIG. 18 is a confocal scanning electron microscope image of the combination of R848 and aCD47 that promotes BMDM (green) phagocytosis 4T1 (red);

FIG. 19 is a diagram showing the detection of secretion of the pro-inflammatory cytokine TNF- α by ELISA in Experimental example section 8;

FIG. 20 is a graph showing the detection of HMGB1 secretion by different treatment 4T 1;

FIG. 21 is a flow cytometry analysis of DC maturation of different treatments 4T 1;

FIG. 22 is a photograph of in vivo fluorescence of 4T1 tumor bearing mice injected intravenously with IR820 or AB-IR820 at various time points as observed by a small animal imaging system;

FIG. 23 is a graph showing the fluorescence intensity of a mouse tumor over time;

FIG. 24 is a near infrared thermogram of 4T1 tumor-bearing mice injected intravenously with PBS, IR820 or AB-IR820, after 24 hours with 808nm near infrared laser irradiation for various times;

FIG. 25 is a graph showing the temperature rise of a laser irradiated tumor site;

FIG. 26 is an in vivo and ex vivo image of tumors from different treatment groups of 4T1 tumor-bearing mice;

FIG. 27 is a graph showing tumor growth in different treatment groups of 4T1 tumor-bearing mice.

Detailed Description

Embodiments of the technical scheme of the present invention will be described in detail below with reference to the embodiments. The following examples are only for more clearly illustrating the technical aspects of the present invention, and thus are merely examples, and are not intended to limit the scope of the present invention.

It is noted that unless otherwise indicated, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

Example 1

The embodiment is a preparation method of a functionalized apoptotic body drug delivery system, comprising the following steps:

(a) Adding dithiothreitol into a 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;

(b) 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 R848 nano-particles (marked as R848 NP), wherein the adding 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 5:1:1;

(c) According to the ratio of R848 nano particles to cells being 2mg to 10 ⁷ cell, R848 nanoparticle incubated with RAW264.7 cells for 9h at 37deg.C, washed 2 times with DPBS and with 0.1J/cm ² After 30min of ultraviolet irradiation, the mixture was incubated at 37℃for 2h, and then, the mixture was centrifuged at 300g for 10min and the supernatant was collected, and the supernatant was centrifuged at 2000g for 20min to obtain a functionalized apoptotic body drug delivery system (designated as R848 NP/AB).

Example 2

(a) Adding dithiothreitol into a 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 solution is 17:1;

(c) The R848 nanoparticles and RAW264.7 cells were incubated at 37℃for 9h at a ratio of 2 mg:107 cells, washed 2 times with DPBS and 0.1J/cm ² After 30min of ultraviolet irradiation, adding N-hydroxysuccinimide modified neoindocyanine green to the solution after ultraviolet irradiation according to the addition final concentration of the N-hydroxysuccinimide modified neoindocyanine green to 15ug/mL, incubating for 2 hours at 37 ℃, centrifuging at 300g for 10min and collecting supernatant, and centrifuging at 2000g for 20min to obtain a functionalized apoptotic body drug delivery system (marked as R848NP/AB-IR 820).

Comparative example 1

The comparative example is a preparation method of apoptotic bodies, comprising the following steps:

RAW264.7 cells were incubated at 37℃for 9h, washed 2 times with DPBS and incubated with 0.1J/cm ² After 30min of UV irradiation, the mixture was incubated at 37℃for a further 2h, after which the mixture was centrifuged at 300g for 10min and the supernatant was collected, and the supernatant was centrifuged at 2000g for 20min to give apoptotic bodies (designated AB).

Comparative example 2

The comparative example is a preparation method of a novel N-hydroxysuccinimide modified indocyanine green modified apoptotic body, which comprises the following steps:

RAW264.7 cells were incubated at 37℃for 9h, washed 2 times with DPBS and incubated with 0.1J/cm ² After 30min of ultraviolet irradiation, according to the addition amount of N-hydroxysuccinimide modified new indocyanine green of …, adding N-hydroxysuccinimide modified new indocyanine green into the solution after ultraviolet irradiation, incubating for 2h at 37 ℃, then centrifuging at 300g for 10min, collecting supernatant, centrifuging at 2000g for 20min, and obtaining apoptotic bodies (marked as AB-IR).

Experimental example

1. Determination of particle size and zata point location the particle size and zata potential of R848NP, R848NP/AB in example 1, and AB in comparative example 1 were measured by an atomic force microscope and a dynamic light scattering particle size analyzer, and the particle size measurement results are shown in FIG. 1; the zata potential analysis result is shown in figure 2;

as can be seen from fig. 1 and 2: the average particle diameter of the R848 nano-particle is about 190nm, the average particle diameter of the apoptosis small body is about 1450nm, and the average particle diameter of the apoptosis small body carrying the R848 nano-particle is about 1000 nm. The surface charge of the R848 nanoparticle is positive, and the surface charge of the apoptotic bodies and the apoptotic bodies carrying the R848 nanoparticle is negative.

2. Detection of Apoptotic Body (AB) yield

AB with Annexin V-FITC staining, then using confocal microscopy for fluorescence imaging, and flow cytometry analysis, using FlowJo software to calculate AB yield; the results of flow cytometry analysis and calculation are shown in fig. 3:

as can be seen from fig. 3: apoptotic bodies account for 39.7% of the total production of particles by the cells and express apoptotic signals.

3. Apoptotic body drug-loading assay

Preparing R848NP/AB, wherein R848 is replaced by nile red, the apoptotic body is stained by Annexin V-FITC, and the positive signal of the sample is detected by a flow cytometer, and the detection result is shown in figure 4;

as can be seen from fig. 4: 94.6% of apoptotic bodies all had a positive signal for nile red.

4. In vitro cell uptake assay

To compare the uptake efficiency of macrophages on R848, R848NP/AB in vitro, RAW264.7 cells were grown in 6-well plates (1X 10) ⁶ Cells/wells); RAW264.7 cells were treated with free R848 (5 ug/ml), R848NP or R848NP/AB for 6 hours, PBS as control, R848 replaced with nile red; staining with DAPI, observing and photographing by a laser confocal microscope, and measuring and quantitatively detecting the fluorescence intensity of the cells by using a flow cytometer; the photographing result is shown in fig. 5, the fluorescence intensity is shown in fig. 6, and the histogram of the fluorescence intensity is shown in fig. 7;

as can be seen from fig. 5, 6 and 7: the R848NP/AB formulation significantly improved the uptake of hydrophobic drugs by macrophages compared to free drug and nano-formulations.

5. In vitro DC maturity studies

To determine the effect of R848, R848NP/AB on DC maturation, BMDCs (1X 10) obtained from BALB/c mice ⁶ Individual cells) were incubated with free R848 (5 ug/ml), R848NP or R848NP/AB for 24 hours, PBS as a control; after antibody staining, DC maturity was analyzed by flow cytometry; secreted cytokines (TNF- α, IL-12 and IL-6) were detected using ELISA; the results of flow cytometry on DC maturity analysis are shown in fig. 8; ELISA detection results of TNF-alpha are shown in FIG. 9, IL-12 in FIG. 10, and IL-6 in FIG. 11;

as can be seen from fig. 8 to 11: the R848NP/AB formulation stimulated DC maturation best in vitro and significantly increased the expression of the pro-inflammatory cytokines TNF- α, IL-12 and IL-6 compared to the free drug and the nano-formulation.

6. In vitro macrophage polarization

M2 macrophages are induced by treatment of bone marrow-derived macrophages (BMDM) with IL-4 (20 ng/mL); after successful induction of M2 macrophages, cells were treated with soluble R848 (5 ug/ml), R848NP/AB or control (PBS) for 24 hours; after antibody staining, the expression of macrophage surface markers was analyzed by flow cytometry, quantitatively differentiating into the proportion of M1 macrophages. And collecting the supernatant to measure the levels of TNF- α, IL-12 and IL-6 by ELISA, wherein the results of flow cytometry analysis are shown in FIG. 12, ELISA detection of TNF- α is shown in FIG. 13, ELISA detection of IL-12 is shown in FIG. 14, and ELISA detection of IL-6 is shown in FIG. 15;

as can be seen from fig. 12 to 15: the R848NP/AB formulation has the best effect of promoting M2-type macrophage polarization to the M1 phenotype in vitro and significantly increased expression of the pro-inflammatory cytokines TNF- α, IL-12 and IL-6 compared to the free drug and nanoformulation.

7. Targeted fluorescence imaging studies

To determine drug distribution and tumor targeting in vivo, BALB/c mice were inoculated subcutaneously 1X 10 on the right ⁶ 4T1 cells; when the tumor volume increases to about 200mm ³ In the case of R848NP or R848NP/AB by tail vein injection, R848 was replaced with nile red (15 ug); mice were subjected to in vivo fluorescence imaging at designated times using a small animal imaging system. The mice were then dissected to obtain fluorescence profiles of the major organs and tumors. The results of fluorescence imaging of different organs are shown in FIG. 16, and the contents of R848NP and R848NP/AB at different positions are shown in FIG. 17;

as can be seen from fig. 16 and 17: compared with the nano preparation, the R848NP/AB preparation has better targeting effect on tumors, more accumulation at tumor sites and reduced distribution at liver sites.

8. In vitro macrophage phagocytosis Activity study

BMDM isolated from BALB/c mice was stained with CFSE and 4T1 cells were stained with Mito-Tracker Red CMXRos. The cell treatment modes are divided into four groups, namely PBS, igG, R848 and R848+aCD47; two groups of BMDMs were treated with R848 12 hours in advance; 4T1 cells (1.5X10) ⁵ ) First blocked with IgG or aCD47 for 30 min; then will be compatible with BMDMs (1.5X10) ⁵ ) Co-culturing in serum-free medium, incubating at 37deg.C for 2 hr, and analyzing phagocytic behavior of macrophage on tumor cells by confocal microscope and flow cytometry; in addition, the level of TNF- α in the supernatant was measured by ELISA kit, and the results of flow cytometry analysis are shown in FIG. 18, and the results of detection of the level of TNF- α are shown in FIG. 19;

as can be seen from fig. 18 and 19: the R848NP/AB preparation can act synergistically with the anti-CD 47 antibody to activate phagocytosis of tumor cells by macrophages.

9. Study of photothermal induced immunogenic cell death

To detect the laser induced release level of high mobility group box B1 (HMGB-1) in vitro, 4T1 cells (2×10 ⁵ Individual cells/well) were inoculated in a 12-well plate for 24 hours, followed by addition of AB-IR (IR 820: 10. Mu.g/mL) for 6 hours; irradiating cells with laser light, controlling at 45 ℃ for 5 minutes, and taking non-irradiated cells as a control; detecting the concentration of HMGB1 in the supernatant by using a mouse HMGB1 ELISA kit; the HMGB1 detection result is shown in figure 20;

to measure the effect of tumor cell immunogenic death on DC maturation, BMDCs obtained from BALB/c mice (1X 10 ⁶ Individual cells) were incubated with PBS, 4T1, 4T1+AB-IR or 4T1+AB-IR after laser irradiation for 24 hours; the maturity of the DCs was then determined by antibody staining using flow cytometry; the results of confirming DC maturity by loss cytometry are shown in FIG. 21;

as can be seen from fig. 20 and 21: the photo-thermal effect of the connected IR820 on the apoptosis body under the irradiation of laser can effectively trigger the immunogenic cell death of tumor cells and stimulate DC maturation.

10. Evaluation of in vivo residence time and photothermal Effect

To determine the residence time of IR-820 in vivo and its intensity of photothermal effect, BALB/c mice were inoculated subcutaneously 1X 10 on the right side ⁶ 4T1 cells; when the tumor volume increases to about 200mm ³ Free IR-820 or AB-IR820 (10 ug/mouse quantified as IR 820) was injected by tail vein; in vivo fluorescence imaging of mice at designated times using a small animal imaging system; the mice were then dissected to obtain fluorescence profiles of the major organs and tumors. 24 hours after injection, a 808 laser (1.5W/cm ² ) The tumor part is covered by irradiation, and the temperature rise of the tumor is observed by a thermal imager and a curve is drawn. The fluorescence imaging results are shown in fig. 22; the change in fluorescence intensity for tumors at different times is shown in fig. 23; fluorescence thermal imaging of tumors 24 hours after injection is shown in fig. 24, and temperature change curves are shown in fig. 25;

as can be seen from fig. 22 to 25: free IR820 is mainly accumulated in the liver and rapidly cleared, while IR820 attached to apoptotic bodies is carried to the tumor site and stays for a long time, the accumulation amount is maximum at 24 hours, and the laser has good photo-thermal effect in combination with 808, and can be maintained at 45-46 ℃ within five minutes.

11. In vivo antitumor effect

For primary tumor treatment, BALB/c mice were inoculated subcutaneously 1X 10 on the right ⁶ 4T1 cells. The tumor volume of the 4T1 tumor-bearing BALB/c mice is about 100mm ³ Treatment with different treatment regimens including PBS, R848NP/AB-IR820, AB-IR820+ laser, R848NP/AB-IR820+ laser (R848: 30 μg per mouse); the first laser irradiation was performed 24 hours after injection, the second laser irradiation was performed 72 hours after injection, and the tumor site was exposed to 808nm laser (1.5W/cm ² ) 5 minutes; these injections and irradiation treatments were repeated once, at intervals of one week; tumor volume was measured with a digital caliper and calculated as LXW XW/2 (L, longest dimension; W, shortest dimension); monitoring tumor growth and drawing a curve; the different tumor volume maps are measured by a digital caliper and are shown in figure 26; the tumor growth curve is shown in fig. 27;

as can be seen from fig. 26 and 27: the R848NP/AB-IR820 formulation was effective in inhibiting tumor growth in combination with photothermal therapy and revealed the potential for co-therapy with anti-CD 47 antibodies.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.

Claims

1. A functionalized apoptotic body drug delivery system comprising a nanoparticle drug and an apoptotic body and an N-hydroxysuccinimide modified near infrared fluorescent reagent;

the nanoparticle drug is encapsulated within the apoptotic bodies;

the N-hydroxysuccinimide modified near infrared fluorescent reagent is spliced with amino groups on the surface of the apoptotic body;

the apoptosis body is prepared from RAW267.4 cells;

the nanoparticle drug is an R848 nanoparticle.

2. The method of preparing a functionalized apoptotic body drug delivery system according to claim 1, comprising the steps of:

3. The method of claim 2, wherein the nanoparticle drug is an R848 nanoparticle, and wherein the R848 nanoparticle is prepared by:

4. The method of claim 2, further comprising adding an N-hydroxysuccinimide modified near infrared fluorescent reagent prior to re-incubation.

5. The method according to claim 4, wherein the N-hydroxysuccinimide modified near infrared fluorescent reagent is added at a final concentration of 12 to 18. Mu.g/mL.

6. The method of claim 2, wherein the ratio of nanoparticle drug to RAW264.7 cells is 2 mg:10 (10 ⁶ ～10 ⁸ )cell。

7. The method according to claim 2, wherein the co-incubation temperature is 35-39 ℃ for a period of 6-12 hours.

8. The method according to claim 2, wherein the intensity of ultraviolet rays in the ultraviolet irradiation is 0.08 to 0.12J/cm ² The irradiation time is 25-35 min.

9. The preparation method according to claim 2, wherein the re-incubation temperature is 35-39 ℃ for 1.5-3 hours;

10. the method according to claim 2, wherein the differential centrifugation specifically comprises:

11. Use of the functionalized apoptotic body drug delivery system according to claim 1 in the preparation of a medicament for the immuno-combined photothermal treatment of a tumor.

12. An immuno-photothermal combination tumor treatment drug comprising the functionalized apoptotic body drug delivery system according to claim 1.