CN117982667B

CN117982667B - Delivery system targeting TLR4-MD2/MyD88 signal pathway, preparation method and application thereof

Info

Publication number: CN117982667B
Application number: CN202410391679.5A
Authority: CN
Inventors: 朱熠; 张国楠; 黄建鸣
Original assignee: Sichuan Cancer Hospital
Current assignee: Sichuan Cancer Hospital
Priority date: 2024-04-02
Filing date: 2024-04-02
Publication date: 2024-06-21
Anticipated expiration: 2044-04-02
Also published as: CN117982667A

Abstract

The invention discloses a delivery system of a targeting TLR4-MD2/MyD88 signal path, a preparation method and application thereof, and belongs to the field of tumor targeting drugs; the exosome delivery carrier can encapsulate antidepressant drugs, is an exosome delivery system which has targeting, good stability and capability of improving tumor immunity microenvironment, encapsulates the antidepressant drugs into tumor cell exosomes, effectively internalizes the drug-carrying exosomes through a cell uptake endocytic mechanism, and increases the permeability and accumulation of the drug-carrying exosomes in tumors in a targeting way, thereby effectively improving the bioavailability and reducing toxic and side effects.

Description

Delivery system targeting TLR4-MD2/MyD88 signal pathway, preparation method and application thereof

Technical Field

The invention relates to the field of tumor targeting drugs, in particular to a delivery system for targeting a TLR4-MD2/MyD88 signal path, a preparation method and application thereof.

Background

Ovarian epithelial cancer, namely ovarian cancer (EOC), is one of the three common malignant tumors of the female reproductive system, and its recurrence rate and death rate are high in the top. Since the onset is hidden, early symptoms are not obvious, 70% of patients have reached a clinically advanced stage with a missed opportunity for radical treatment, and thus ovarian cancer is also known as "silent killer". There are currently two main approaches to the initial treatment of advanced ovarian cancer: (1) Adjuvant chemotherapy based on platinum/paclitaxel after initial tumor cell reduction surgery (PDS) +; (2) Platinum/paclitaxel drug based neoadjuvant chemotherapy (NACT) + intermittent tumor cell debulking (IDS) + platinum/paclitaxel based postoperative adjuvant chemotherapy. Clinical remission can be obtained in up to 80% of ovarian cancer patients after initial treatment, regardless of PDS or NACT +ids, but about 70% relapse within 3 years, even multiple times, with overall 5-year survival rates loitering around 30% without significant improvement. Mortality in ovarian cancer patients has not been substantially reduced to date. Thus, in an attempt to improve the outcome of current ovarian cancer patients, there is an urgent need for innovative, definitive and accessible therapeutic strategies, such as immune-directed combination therapies.

Ovarian cancer is a typical immunosuppressive tumor. In recent years, with the rise of immunotherapy, various immunotherapies have been involved in ovarian cancer, and the progress of "silent killers" has been strived for to change the clinical treatment pattern in which ovarian cancer has not benefited. Immune Checkpoint Inhibitor (ICI) -apoptosis receptor 1 (, PD-1 and its ligand (PD-L1)) inhibitors are one of the most successful immunotherapies, showing superior effects over conventional therapies in a variety of solid tumors, and the therapeutic efficacy is not satisfactory and the response rate is only 7-15% as compared to traditional chemotherapy of ovarian cancer, as seen from the clinical study results of single drug treatment of ovarian cancer with PD-1/PD-L1 inhibitors, ICI treatment has not shown advantages and no indication of related ovarian cancer immunotherapy has been approved yet.

As is known, the mechanism of tumor cell-induced immunosuppression under immune pressure is extremely complex. Ovarian cancer expresses its immunosuppressive phenotype and secretes various immunosuppressive cytokines during its development and progression through genetic mutation, clonal evolution and immune editing, such as: toll-like receptor 4 (TLR 4), myeloid differentiation factor 88 (MyD 88), indoleamine 2, 3-dioxygenase 1 (IDO 1), PD-L1/PD-1, interleukin (IL-) 6 and Aromatic Hydrocarbon Receptor (AHR) induce an upregulation of immune cells of the suppressive phenotype, thereby suppressing immune cell function and immune response. Paclitaxel is an activator of TLR4/MyD88/NF- κB signaling pathway, and researches report that the combination of TLR4/MyD88 signaling pathway blocker and IDO inhibitor can inhibit paclitaxel-induced mediated immunosuppression on one hand and increase the cytotoxic effect of paclitaxel on the other hand. This is a strategy for immunotherapy in combination with chemotherapy that is distinct from pure immunotherapy.

Amitriptyline, chemical name: n, N-dimethyl-3- [ 1o, 11-dihydro-5H-dibenzo [ a, d ] cycloheptatriene-5-subunit ] -1-propylamine is a tricyclic antidepressant and is not reported to be used for resisting tumors, especially ovarian cancer at present.

Disclosure of Invention

It is an object of the present invention to provide a delivery system targeting the TLR4-MD2/MyD88 signaling pathway to address the above-mentioned problems.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: a delivery system targeting the TLR4-MD2/MyD88 signaling pathway, the delivery system loading an exosome of tumor cells with an antidepressant, the tumor cells being ovarian cancer cells.

As a preferred technical solution, the antidepressant is amitriptyline.

As a preferable technical scheme, the encapsulation efficiency of the antidepressant is 36.6+/-6.7%, and the drug loading rate is 915.96 +/-278.96 pg/mug.

The second object of the present invention is to provide a method for preparing the delivery system, which adopts the technical scheme that the method comprises the following steps: exosomes in the mouse ID8 ovarian cancer cell supernatant were first extracted, and then the antidepressant was loaded into the extracted exosomes using electroporation.

As a preferable technical scheme, the extraction method of the exosomes is a precipitation method.

As a further preferable technical scheme, the extraction method of the exosome specifically comprises the following steps:

(1) Inoculating the mouse ID8 ovarian cancer cells into a cell culture dish, and when the cells are cultured in a complete culture medium containing exosomes until the cell density is 60-70%, removing the original culture medium and replacing the original culture medium with a new complete culture medium without exosomes;

(2) Continuously culturing the cells, collecting cell supernatant, and centrifuging for 10 min; sucking the supernatant;

(3) The cell supernatant is centrifuged again to ensure that the cells or cell debris are removed cleanly;

(4) Collecting supernatant, filtering with filter to remove vesicle and other impurities above 0.22 μm, and packaging in centrifuge tube;

(5) Taking out the centrifuge tube, adding the corresponding ExoQuick-TC reagent into the centrifuge tube, and then mixing;

(6) Placing the centrifuge tube in a refrigerator with the temperature of 4 ℃ above 12 h;

(7) Taking out the centrifuge tube containing ExoQuick-TC reagent from the refrigerator, and centrifuging;

(8) Taking out the centrifuge tube, and sucking the supernatant by a pipette for discarding;

(9) Centrifuging the centrifuge tube again;

(10) Taking out the centrifuge tube, sucking the residual supernatant by using a micropipette, discarding the residual supernatant, and obtaining a precipitate at the bottom of the tube, namely an exosome secreted by the cells;

(11) Adding sterile Phosphate Buffer (PBS) into each tube, re-suspending exosomes, mixing, packaging, and storing in a refrigerator at-80deg.C.

As a preferred technical solution, the specific method for loading the antidepressant into the extracted exosomes by using electroporation method is as follows:

(1) Dissolving an antidepressant in sterile Phosphate Buffer (PBS) to prepare an antidepressant solution;

(2) Taking exosomes and thawing;

(3) Adding the antidepressant solution prepared in the step (1) and the electroporation buffer solution into the exosome, and then uniformly mixing to obtain a mixed solution;

(4) Adding the uniformly mixed liquid obtained in the step (3) into an electric shock cup, and placing into an electric shock groove;

(5) Adjusting parameters of the electroporation apparatus, voltage: 600 V, pulse length: 90.μs, pulse number: 5 times, pulse interval: 1 s;

(6) Confirm the electric shock parameter, click the "confirm key" of the electroporation apparatus, finish electroporation medicament loading;

(7) Taking out the electric shock cup, sucking the mixed liquid in an aseptic EP tube by a liquid transfer device, adding a corresponding ExoQuick-TC reagent into the EP tube, and then mixing;

(8) Placing the EP pipe above a refrigerator 12 h at 4 ℃;

(9) And slowly taking the EP tube out of the refrigerator, centrifuging for a plurality of times, and taking supernatant, wherein the precipitate left at the bottom of the tube is the exosome loaded antidepressant product.

The invention further provides an application of the delivery system in preparing targeted drugs for treating ovarian cancer.

Through a great deal of research, the inventor of the application discovers that amitriptyline can target and block a TLR4-MD2/MyD8 signal path, reverse tumor immune escape and taxol-mediated survival response, and is used for immunotherapy of ovarian cancer.

Compared with the prior art, the invention has the advantages that: the exosome delivery carrier can encapsulate antidepressant drugs, is an exosome delivery system which has targeting, good stability and capability of improving tumor immunity microenvironment, encapsulates the antidepressant drugs into tumor cell exosomes, effectively internalizes the drug-carrying exosomes through a cell uptake endocytic mechanism, and increases the permeability and accumulation of the drug-carrying exosomes in tumors in a targeting way, thereby effectively improving the bioavailability and reducing toxic and side effects.

Drawings

FIG. 1 is an electron micrograph of amitriptyline @ exosomes of example 2 of the present invention;

FIG. 2 is a graph showing particle size and surface potential of amitriptyline @ exosomes of example 2 of the present invention;

FIG. 3 is a graph showing the results of cell viability assay according to example 5 of the present invention;

FIG. 4 is a graph showing the effect of ultrasound on amitriptyline @ exosome drug release in vitro according to example 7 of the present invention;

FIG. 5 is a graph of targeting results for example 8 of the present invention;

FIG. 6 is a graph of fluorescence signals from each group of in vivo fluorescence imaging of mice according to example 10 of the present invention;

FIG. 7 shows the change in tumor volume for each treatment group in example 11 of the present invention;

FIG. 8 is a graph showing the weight change of nude mice in each treatment group according to example 11 of the present invention;

FIG. 9 is a quantitative analysis of apoptosis index for each treatment group in example 11 of the present invention;

Fig. 10 is a graph of survival (n=6) of different treatment group EOC engrafted tumor mice in example 11 of the present invention.

Detailed Description

The invention will be further described with reference to the accompanying drawings.

Example 1:

A delivery system targeting TLR4-MD2/MyD88 signaling pathway, the method of making comprising the steps of:

(1) Extracting exosomes in the supernatant of ovarian cancer cells by a precipitation method;

1) Inoculating the mouse ID8 ovarian cancer cells into a cell culture dish according to the ratio of 1X 10 ⁶/mL, and when the cell density is 60-70% in a complete culture medium containing exosomes, removing the original culture medium and replacing the original culture medium with a new complete culture medium without exosomes;

2) Culturing the cells 48 h, collecting cell supernatant, centrifuging at 4deg.C for 10min, and collecting the supernatant; aspiration of supernatant, taking care to avoid aspiration of cells or cell debris;

3) The cell supernatant was centrifuged again at 3000 Xg, 4℃for 15 min to ensure that the cells or cell debris were removed;

4) Collecting supernatant, filtering with 0.22 μm pore size filter, removing vesicle and other impurities above 0.22 μm, and packaging in centrifuge tube;

5) Taking out the centrifuge tube, adding corresponding ExoQuick-TC reagent into the centrifuge tube according to the ratio of cell supernatant to ExoQuick-TC=5 to 1 (v/v), and then mixing by gently upside down;

6) Placing the centrifuge tube in a refrigerator with the temperature of 4 ℃ above 12 h;

7) Slowly remove the centrifuge tube containing ExoQuick-TC reagent from the refrigerator and centrifuge 1500 Xg for 30: 30 min;

8) Taking out the centrifuge tube, sucking the supernatant by using a pipettor, discarding, and cutting the precipitate attached to the bottom of the centrifuge tube;

9) The tube was centrifuged again at 1500 Xg for 5 min;

10 Taking out the centrifuge tube, sucking the residual supernatant by using a micropipette, discarding the residual supernatant, and obtaining a precipitate at the bottom of the tube, namely an exosome secreted by the cells;

11 100-150 mu L of sterile Phosphate Buffer (PBS) is added into each tube to resuspend the exosomes, and after uniform mixing, the exosomes are packaged by 1.5 mL sterile EP tubes and placed in a refrigerator at the temperature of minus 80 ℃ for standby.

(2) Loading exogenous amitriptyline into an exosome by adopting an electroporation method, and preparing the antidepressant-loaded exosome (amitriptyline@exosome);

1) 15mg amitriptyline is dissolved in 1 ml sterile Phosphate Buffer (PBS) to prepare amitriptyline solution (15 mg/ml);

2) The exosomes are taken out from a refrigerator at the temperature of-80 ℃ and thawed at the temperature of 4 ℃;

3) 100. Mu.L of amitriptyline solution and 100. Mu.L of electroporation buffer were added to 100. Mu.L of exosomes, and then mixed well (total volume 300. Mu.L);

4) Adding the above mixed solution into a 400 mu L electric shock cup, and placing into an electric shock groove;

5) Adjusting parameters of the electroporation apparatus, voltage: 600 V, pulse length: 90. μs, pulse number: 5 times, pulse interval: 1 s;

6) Confirm the electric shock parameter, click the "confirm key" of the electroporation apparatus, finish electroporation medicament loading;

7) Taking out the electric shock cup, sucking the mixed solution in a 1.5 mL sterile EP tube by a liquid transfer device, adding a corresponding ExoQuick-TC reagent into the EP tube according to the ratio of mixed solution: exoQuick-TC=5:1 (v/v), and then mixing the mixed solution by gently upside down;

8) Placing the EP pipe above a refrigerator 12h at 4 ℃;

9) The EP tube was slowly removed from the refrigerator and centrifuged at 1500 Xg for 30 min;

10 Taking out the EP tube, sucking the supernatant (comprising amitriptyline solution which is not loaded into the exosome and electroporation buffer solution) by a pipette, and collecting the supernatant in a new 1.5 mL sterile EP tube, wherein the precipitate adhered to the bottom of the centrifuge tube cannot be scattered;

11 Re-centrifuging the original EP tube at 1500 Xg for 5 min;

12 Sucking the residual supernatant in the original EP pipe by using a micropipette, collecting the supernatant in a new EP pipe with the supernatant in the step (10), and storing the EP pipe in a refrigerator at the temperature of-20 ℃ for later use; the precipitate left at the bottom of the tube is amitriptyline@exosome;

13 100-150 mu L of sterile Phosphate Buffer (PBS) is added into each tube of the original EP tube to resuspend amitriptyline@exosome, and the mixture is placed in a refrigerator at the temperature of minus 80 ℃ for standby.

It should be noted that the consumables and reagents used in the above method are commercially available, for example ExoQuick-TC reagent is available from SBI, USA, and electroporation buffer is available from BTX, USA.

Example 2:

And (3) physical characteristic detection: the size, morphology and distribution of the exosomes obtained in step (1) of example 1 were observed by transmission electron microscopy. Measuring the grain diameter and the potential of the exosome and amitriptyline@exosome obtained in the step (2) during preparation and after 90 days of preservation at-80 ℃ by a Malvern (Markov) laser grain size measuring instrument;

Detecting the mass of amitriptyline in the supernatant by using an HPLC (high Performance liquid chromatography), detecting the mass of amitriptyline@exosome by using a dioctyl phthalate (BCA) method, and calculating the encapsulation rate (%) of amitriptyline@exosome=detected amount/total administration amount multiplied by 100%; amitriptyline @ exosome drug loading (%) = detection amount/exosome total mass;

Results: the amitriptyline@exosome is observed to be in a concave disc vesicle shape under a transmission electron microscope, and the dispersibility is good as shown in figure 1; the average particle diameter of the Malvern (Markov) laser particle size measuring instrument is 171.1+/-17.8 nm, and the surface potential is-35.3+/-0.6 mV, as shown in figure 2; amitriptyline encapsulation efficiency is 36.6+/-6.7%, and drug loading rate is 915.96 +/-278.96 pg/. Mu.g.

Example 3:

Detecting the expression condition of exosome marker proteins TSG101 and CD63 in exosome and amitriptyline@exosome by a western immunoblotting method;

Results: by detection of western immunoblotting, it can be seen that exosomes and amitriptyline@exosomes were positive for exosome marker proteins TSG101 and CD63, and that the cell lysate calreticulin was negative, suggesting that both were consistent with exosome expression, and that there were no cellular components in them.

Example 4:

Laser confocal microscopy (CLSM) observation (red fluorescent mark is made on exosome film, and amitriptyline is made green fluorescent mark) and HPLC tandem mass spectrum to judge whether amitriptyline is successfully loaded into exosome;

Results: and (3) observing by a laser confocal microscope, wherein red fluorescence is visible in cells of all other groups except the PBS control group, and after co-localization, the red exosomes and the green cytoskeleton are overlapped and then displayed as orange, so that the exosomes and amitriptyline@exosomes can enter ovarian cancer cells in a targeted manner.

Example 5:

Cytotoxicity test (CCK 8 method and fluorescent staining observation)

Verifying whether amitriptyline@exosomes have cytotoxicity on mouse ovarian epithelial cancer ID8 cell lines and Human Umbilical Vein Endothelial Cells (HUVECs) by a knot CCK8 method and live/dead cell fluorescence (FDA/PI) staining;

Results: after 24 hours of action of exosomes or amitriptyline @ exosomes on ovarian cancer ID8 cell lines and HUVECs, the average survival rate of each group was greater than 95%, the differences were statistically significant (P > 0.05), indicating no significant cytotoxic effect of exosomes or amitriptyline @ exosomes (fig. 3).

Example 6:

Hemolysis experiment

Experimental grouping: ① Sterile PBS Buffer (PBS) control; ② An exosome group; ③ Amitriptyline @ exosome group;

Results: compared with a positive control (PBS group), the amitriptyline@exosome group has almost no erythrocyte destruction phenomenon within 3 hours, and the hemolysis rate is less than 5%; after shaking, the sinking red blood cells are uniformly dispersed, and the phenomenon of agglutination of the red blood cells does not occur, which indicates that exosomes or amitriptyline@exosomes have no obvious hemolysis effect;

Example 7:

researching the in-vitro drug release characteristics of amitriptyline@exosomes by using a dialysis method, and exploring the influence of ultrasound on the release of amitriptyline@exosomes;

The specific method comprises the following steps: selecting a low-frequency ultrasonic therapeutic instrument, and referring to irradiation parameters: the power is 1W/cm ², the frequency is 1 MHz, the irradiation time is 1 min, and the duty ratio is 10%. Each group was sampled at 0.5 mL, added with dithiothreitol (DL-Dithiothreitol, DTT) to prevent protein aggregation, and the samples were then placed in dialysis bags with a theoretical molecular weight cut-off of 50 kDa, and the dialysis bags were placed in petri dishes containing 30 mL sterile PBS buffer. Group ③ was subjected to ultrasonic irradiation. At various time points, 0.2 mL buffer was removed and the supernatant was checked for amitriptyline quality using HPLC. And calculating the accumulated release amount of amitriptyline in each group, and comparing the release conditions of the medicines in each group.

Experimental grouping: ① Amitriptyline group ② amitriptyline @ exosome group ③ amitriptyline @ exosome + ultrasound group.

Results: as shown in fig. 4, the cumulative drug release amount of each group gradually increases with the lapse of time. After 24 hours, the cumulative drug release (%) of amitriptyline group, amitriptyline @ exosome group and amitriptyline @ exosome+ultrasound group were 96.1±3.0, 79.1±2.5 and 82.1±2.5, respectively. Compared with amitriptyline@exosome group, the amitriptyline@exosome+ultrasonic group has higher drug release amount, which suggests that ultrasonic can promote the in vitro drug release of amitriptyline@exosome.

Example 8:

In vitro cell targeting ability assay

Laser confocal microscopy (CLSM) and Flow Cytometry (FCM) detection were used to verify the targeting of ultrasound-coordinated amitriptyline @ exosomes to ovarian cancer cells.

Experimental grouping: ① PBS control group; ② An exosome group; ③ Amitriptyline @ exosome group; ④ Amitriptyline @ exosome + ultrasound group;

The specific method comprises the following steps:

(1) Inoculating the OVCAR3 cells in a logarithmic growth phase pair into a 12-pore plate, and placing the cells in a 5% CO ₂ and 37 ℃ cell incubator for incubation;

(2) Preparing a storage solution with the concentration of 5mM DiI mu M and a DiI working solution with the concentration of 100 mu M, adding the DiI working solution into an exosome and an amitriptyline@exosome, and dyeing for 30min with the concentration of 10 mu M;

(3) Adding 30 mu L of sterile PBS buffer solution into ① th group of cells, adding 15 mu L of exosomes and 15 mu L of PBS buffer solution into ② th group of cells, adding 15 mu L of amitriptyline@exosomes and 15 mu L of PBS buffer solution into ③ th group of cells, adding 15 mu L of amitriptyline@exosomes and 15 mu L of PBS buffer solution into ④ th group of cells, and carrying out ultrasonic irradiation on the cells at the bottom of a culture dish, wherein the irradiation parameters are that the power is 1W/cm ², the frequency is 1 MHz, the irradiation time is 1 min and the duty ratio is 10%;

(4) The cells are put back into the incubator and are further incubated for 5 h;

(5) The medium was aspirated, washed 3 times with PBS, cells were fixed 10min at room temperature with 1mL of 4% paraformaldehyde solution added to each well, and washed 30 s with PBS;

(6) 200 mu L of 100 nM SF 488-labeled phalloidin (phalloidin) working solution (green fluorescence) is added, incubated at room temperature in a dark place for 30min, and cytoskeleton is dyed;

(7) Washing the well plate with PBS for 2 times, adding 1mL of 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI blue fluorescence) working solution into each well, incubating for 5 min at room temperature in a dark place, and staining cell nuclei;

(8) The well plate was washed 2 times with PBS. An appropriate amount of PBS solution was added to each well and observed under a laser confocal microscope.

(9) Sucking out the culture medium, washing the cells with PBS for 1-2 times, digesting the cells with pancreatin, centrifuging (800X rpm,4 min), and re-suspending 500 mu L of ice PBS solution in each tube into 1.5 mL EP tubes;

(10) FCM detection is performed.

The results are shown in FIG. 5: it can be seen that the red fluorescence of amitriptyline@exosome+ultrasound group is more than that of the exosome group and amitriptyline@exosome group, which suggests that ultrasound or ultrasound-combined microbubbles can improve the in vitro targeting of amitriptyline@exosome. The fluorescence carrying rates of the exosome group, the amitriptyline@exosome group and the amitriptyline@exosome+ultrasonic group are 96.8%, 97.2% and 99.5% respectively, except that the PBS control group has no obvious fluorescence in cells. And (3) analyzing the average fluorescence intensity, wherein the average fluorescence intensities of the PBS control group, the exosome group, the amitriptyline@exosome group and the amitriptyline@exosome+ultrasonic group are 738.6 +/-39.6, 22377.2 +/-1119.7, 22879.3 +/-1634.5 and 40727.8 +/-1425.9 respectively. It can be seen that the average fluorescence intensity of amitriptyline @ exosome + ultrasound is higher than that of the other groups; the difference in mean fluorescence intensity between the exosome and amitriptyline @ exosome groups was not statistically different (P > 0.05), but was higher than that of the PBS control group (P < 0.05). The results suggest that exosomes and amitriptyline@exosomes can enter ovarian cancer cells in a targeted manner, and that ultrasound can improve in vitro targeting of amitriptyline@exosomes.

Example 9:

a mouse ovarian epithelial cancer ID8 cell strain tumor-bearing mouse (C57 BL/6) model is established by adopting a method of subcutaneous injection at the back of buttocks.

The method comprises the following specific steps:

(1) Digesting ovarian cancer cells in logarithmic growth phase, centrifuging, counting, adjusting cell concentration (1×10 ⁶/ml), and mixing with matrigel at a certain proportion;

(2) Dehairing, alcohol disinfection, and subcutaneous inoculation of 100 μl of cell suspension on the back of the buttocks of the mice.

Example 10:

In vivo targeting concentration experiments

Experimental grouping: ① PBS control group; ② Amitriptyline group; ③ An exosome group; ④ Amitriptyline @ exosome group; ⑤ Amitriptyline @ exosome + ultrasound group. After the tumor-bearing mice of the ovarian epithelial cancer ID8 cell strain obtained in the example 9 are injected with the medicines through tail veins, ⑤ is subjected to tumor tissue ultrasonic irradiation, and 1 MHz ultrasonic irradiation frequency, 3W/cm ² output power, 20% duty ratio and 3 minutes duration are adopted as ultrasonic treatment conditions. Assessing in vivo targeting of ultrasound synergistic amitriptyline@exosomes by in vivo fluorescence imaging detection either after drug injection or immediately after ultrasound irradiation, 2h, 4h and 24 h;

Results: the in-vivo fluorescence imaging of the mice can be seen that the PBS control group and amitriptyline group tumor areas have no fluorescence display; the exosome group, amitriptyline@exosome group and amitriptyline@exosome+ultrasound group can be displayed by fluorescence in the tumor area at various time points (immediately after the completion of drug injection or after the completion of ultrasound irradiation, 2h, 4 h and 24 h), and the fluorescence intensity of the tumor area is higher than that in the viscera; the amitriptyline@exosome+ultrasound group is less distributed in the viscera than the exosome group and the amitriptyline@exosome group; along with the extension of time, amitriptyline, exosomes, amitriptyline@exosomes and amitriptyline@exosomes+ultrasound tumor regions are in a stronger continuous fluorescence signal within 24 hours, and the fluorescence intensity is gradually enhanced; in addition, the tumor zone fluorescence signal was higher in the amitriptyline @ exosome + ultrasound group than in the exosome group (fig. 6).

Example 11

Comparison of therapeutic Effect

Experimental grouping: control group: physiological saline; experimental group: ① Paclitaxel + PD-1 monoclonal antibody (IgG 4); ② Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline; ③ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosomes; ④ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound;

(1) Therapeutic dose: paclitaxel: 0.5 mg/ip only; amitriptyline and amitriptyline @ exosomes (100 μl/min): 50 μg/ip only; pamumab: 5 μg/ip. The total treatment was 3 times per week 1 time.

(2) Immediately after group ④ drug injections, the tumor area of the mice was irradiated with ultrasound. The irradiation parameters are as follows: the frequency is 1MHz, the output power is 3W/cm ², the duty ratio is 30%, and the ultrasonic irradiation is carried out for 3min;

(3) Immune evaluation index (mice were sacrificed 1 week after treatment): detecting the level expression change of MyD88, IDO1, AHR, PD-1 and PD-L1 genes by adopting the existing well-known qRT-PCR; immunohistochemistry is adopted to analyze the differential expression condition of MyD88, PD-L1, AHR and IDO1 in tumor tissues on the protein level; determination of Try metabolites in tumor bearing mice peripheral serum using HPLC tandem mass spectrometry included L-kynurenine, tryptophan expression and Kyn/Trp ratio:

Results: 1) Tumor tissues before and after treatment of EOC transplanted tumor mice are subjected to qRT-PCR detection on the level expression change of MyD88, IDO1, AHR, PD-1 and PD-L1 genes. Research results show that compared with a taxol+PD-1 monoclonal antibody (IgG 4) group, the taxol+PD-1 monoclonal antibody (IgG 4) +amitriptyline group, the taxol+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome group and the taxol+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome+therapeutic ultrasonic groups MyD88, PD-L1, AHR and IDO1 mRNA levels have a decreasing trend, and have statistical significance; wherein, the reduction of the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome group and the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome + therapeutic ultrasound group is most obvious. Thus, amitriptyline acts as a MyD88 inhibitor, and targets a key molecular target taking MyD88, IDO1 and AHR as a center in two related signal paths of MyD88/NF- κB-IL-6 and IL-6-IDO1-KYN/AHR-IL-6 signal loops, and blocking the paths possibly eliminates the immunosuppressive state of ovarian cancer.

2) To further evaluate the differential expression of MyD88, PD-L1, AHR and IDO1 at the protein level in tumor tissues of EOC-transplanted tumor mice treated with different groups, immunohistochemical analysis was performed on tumor tissues of EOC-transplanted tumor mice from different treatment groups. The research result shows that the immune and constitutive positive ratio of MyD88, IDO1 and AHR in the taxol+PD-1 monoclonal antibody (IgG 4) group is the highest; the positive ratios of paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline group, paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome group and paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound group MyD88, AHR and IDO1 immunohistochemical all showed a decreasing trend, with statistical significance (table 1). PD-L1 was expressed less in tumor tissue of EOC transplanted tumor mice with no significant statistical difference in expression between groups (P > 0.05).

Table 1 comparison of IHC positive proportion (%) of tumor tissue in differently treated group EOC-transplanted tumor mice

In Table 1, "blank" refers to paclitaxel+PD-1 monoclonal antibody (IgG 4) using normal saline ①; ② Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline; ③ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosomes; ④ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound.

3) The serum of EOC engrafted tumor mice from different treatment groups was used to detect L-kynurenine, tryptophan expression and Kyn/Trp ratio. The study results showed that the paclitaxel + PD-1 monoclonal antibody (IgG 4) group had a different increase in expression and Kyn/Trp ratio compared to the control group L-kynurenine, tryptophan, whereas the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline group, the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome group, and the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound group had a decreasing trend in expression and Kyn/Trp ratio, which was statistically significant (table 2).

TABLE 2 comparison of serum Kyn, trp (ng/ml) expression and Kyn/Trp ratios of mice with different treatment groups EOC transplants

In table 2: ① Paclitaxel + PD-1 monoclonal antibody (IgG 4); ② Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline; ③ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosomes; ④ Paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound.

(4) Treatment evaluation index: ① Recording the growth condition (tumor size) of the tumor, drawing a growth curve, and calculating the tumor inhibition rate; ② Comparing tumor cell proliferation (PCNA staining) and apoptosis (tunel staining) conditions for each treatment group;

results: 1) Tumor growth in each treatment group from day 0 (treatment start date) to day 15. During treatment, the control tumor volume increased rapidly to 1522±19.22 mm ³, whereas treatment with paclitaxel+pd-1 monoclonal antibody (IgG 4) resulted in a slight increase in tumor volume. The combination of amitriptyline with different dosage forms on the basis of taxol+PD-1 monoclonal antibody (IgG 4) inhibits the growth of tumors to different degrees. The treatment effect of the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline and the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome is similar, slightly better than that of the paclitaxel+PD-1 monoclonal antibody (IgG 4) alone, the tumor volume is slightly reduced, but the treatment effect is not obvious as compared with the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome+treatment ultrasonic group, the tumor volume of the group is reduced to 190+/-28.55 mm ³, and the tumor inhibition rate is 87.5+/-2.3% (shown in figure 7). The treatment group started on day 3 after the end of the treatment, the tumors resumed growth at a very slow rate. These results indicate that different dosage forms of amitriptyline can improve the sensitivity of chemo-immune treatment;

2) During the treatment period, the body weight of EOC transplanted tumor mice was measured every 3 days, and the average body weight of the PD-1 monoclonal antibody (IgG 4) group and the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline group mice was reduced by 2g and 2.8 g except for the control group, considering the cytotoxicity caused by the combination of the drugs. The average body weight of mice in the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome group and the paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + treatment ultrasound group was slightly increased, indicating that amitriptyline @ exosome is expected to reduce the potential toxicity of the drug (fig. 8);

3) On day 15 of treatment, one nude mouse per treatment group was dissected and normal nude mice without tumor inoculation were used as controls. The important organ tissues were extracted for H & E staining, and the results suggested that no significant toxicity changes were seen in the heart, liver, spleen, lung and kidneys in the control group and all treatment groups.

4) Tumor cell proliferation was observed in each treatment group by PCNA staining. As suggested by H & E staining, the combination of paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + treatment ultrasound showed significant tumor cell necrosis, manifested as cell nucleus shrinkage, nuclear fragmentation and nuclear lysis, so the PCNA positive cell number of the group was very small; and the control group does not have obvious necrosis, so that the number of PCNA positive cells is obviously increased. TUNEL staining results suggest that each treatment group was able to increase apoptosis compared to the control group, paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + treatment ultrasound group showed the strongest positive staining, the other groups showed weak or moderate positive staining, respectively. The quantitative analysis of apoptosis index also suggests a consistent conclusion that the apoptosis index of the control group, the paclitaxel+pd-1 monoclonal antibody (IgG 4) +amitriptyline group, the paclitaxel+pd-1 monoclonal antibody (IgG 4) +amitriptyline@exosome group, the paclitaxel+pd-1 monoclonal antibody (IgG 4) +amitriptyline@exosome+therapeutic ultrasound group are: 5.8 1.2%, 29.4.+ -. 4.3%, 44.9.+ -. 2.1%, 48.1.+ -. 3.0% and 65.9.+ -. 1.8%, whereas the apoptosis index of the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome+therapeutic ultrasound group is significantly higher than that of the other therapeutic group (P < 0.0001) (FIG. 9). These results again demonstrate that ultrasound in combination with amitriptyline @ exosomes can inhibit TLR4/MyD88 signaling pathway by competitive binding to MD2, thereby significantly enhancing the efficacy of ovarian cancer treatment-free.

(5) Tumor-bearing mice survival analysis

After the treatment is finished, 6 EOC transplanted tumor mice in each group are randomly selected to record the general state and observe the survival period;

Results: the median survival times of the control, paclitaxel + PD-1 monoclonal antibody (IgG 4) +amitriptyline @ exosome + therapeutic ultrasound groups were 38, 46, 49, 52 and 67 days, respectively. The survival time of mice in each treatment group of paclitaxel+PD-1 monoclonal antibody (IgG 4) combined with amitriptyline with different dosage forms is obviously longer than that of the mice in the paclitaxel+PD-1 monoclonal antibody (IgG 4) group (P < 0.001) (as shown in figure 10, the curves from left to right in figure 10 represent, in sequence, the blank control group, the paclitaxel+PD-1 monoclonal antibody group, the paclitaxel+PD-1 monoclonal antibody+amitriptyline group, the paclitaxel+PD-1 monoclonal antibody+amitriptyline@exosome+treatment ultrasonic group), and the paclitaxel+PD-1 monoclonal antibody (IgG 4) +amitriptyline@exosome+treatment ultrasonic group) has the longest survival time. These results show that paclitaxel + PD-1 monoclonal antibody (IgG 4) is applied in combination with amitriptyline of different dosage forms, blocking TLR4/MyD88 signaling pathway eliminates the immunosuppressive state of ovarian cancer, significantly improves the effect of EOC engrafting tumor mice treatment-free, and prolongs the survival time of mice.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. A delivery system targeting a TLR4-MD2/MyD88 signaling pathway, wherein the delivery system loads an antidepressant for tumor cell exosomes, the tumor cells being ovarian cancer cells; the antidepressant is amitriptyline.

2. The delivery system of claim 1, wherein the antidepressant has an encapsulation efficiency of 36.6±6.7% and a drug loading of 915.96 ± 278.96 pg/μg.

3. A method of preparing a delivery system according to claim 1 or 2, characterized by the steps of: exosomes in the mouse ID8 ovarian cancer cell supernatant were first extracted, and then the antidepressant was loaded into the extracted exosomes using electroporation.

4. The method according to claim 3, wherein the exosome is extracted by precipitation.

5. The method according to claim 4, wherein the method for extracting exosomes comprises the following steps:

(9) Centrifuging the centrifuge tube again;

6. The preparation method according to claim 4, wherein the specific method for loading the antidepressant into the extracted exosomes by electroporation is as follows:

(2) Taking exosomes and thawing;

(3) Adding the exosomes into the antidepressant solution prepared in the step (1) and the electroporation buffer solution, and then uniformly mixing to obtain a mixed solution;

(8) Placing the EP pipe above a refrigerator 12 h at 4 ℃;

7. Use of a delivery system according to claim 1 or 2 for the preparation of a targeted drug for the treatment of ovarian cancer, characterized in that the release of an antidepressant loaded in said delivery system is facilitated by means of ultrasound.