CN114306622A

CN114306622A - Fibrin gel containing adriamycin-entrapped platelet exosome and PD-L1 monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN114306622A
Application number: CN202210011585.1A
Authority: CN
Inventors: 孙进; 赵健; 叶皓; 何仲贵
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2022-01-06
Filing date: 2022-01-06
Publication date: 2022-04-12
Anticipated expiration: 2042-01-06
Also published as: CN114306622B

Abstract

Compared with an adriamycin solution, the adriamycin-loaded platelet exosome can be better combined with tumor cells, induce immunogenic tumor cell death and promote anti-tumor immune response. Meanwhile, the platelet exosome loaded with adriamycin can enter blood circulation through damaged blood vessels, track tumor cells in the circulation and eliminate the tumor cells. aPD-L1 is released at the tumor part at the same time, so that the PD-1/PD-L1 pathway can be blocked, and the tumor killing effect of cytotoxic T cells can be recovered. The combination of the two strategies triggers a stronger T cell immune response, significantly improving the tumor immune microenvironment. The method provides a new strategy and more choices for the combination of chemotherapy and immunotherapy, and meets the urgent need of high-efficiency preparations in clinic.

Description

Fibrin gel containing adriamycin-entrapped platelet exosome and PD-L1 monoclonal antibody, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to fibrin gel containing a platelet exosome and a PD-L1 monoclonal antibody entrapped with adriamycin, a preparation method thereof and application thereof in preparing a medicine for treating tumor metastasis diseases.

Background

Surgery is an effective method of treating melanoma, but unfortunately, local residual tumor microaneurysms and systemic CTCs continue to cause tumor recurrence, leading to patient death. Immune checkpoint inhibitors, particularly the PD-L1 monoclonal antibody, enhance the efficacy of melanoma therapy and produce a durable clinical response in some patients. However, systemic administration of immune checkpoint inhibitors promotes a sustained clinical response in less than 20% of patients with immunogenic tumors. Due to the lack of immunogenic antigens and various immune resistance mechanisms, single-drug therapy with immune checkpoint inhibitors (e.g., aPD-L1 therapy) has limited clinical efficacy.

Chemotherapeutic drugs (such as doxorubicin DOX) can directly kill tumor cells, induce ICD to produce tumor antigens or danger signals; subsequently, an anti-tumor immune response can be induced by co-stimulation with tumor antigens and ICI. However, safe and effective targeted delivery of chemotherapeutic drugs remains challenging, in part due to poor bioavailability and non-specific targeting. Therefore, the ability to combine safe and effective delivery of chemotherapeutic drugs with immune checkpoint blockade is critical to prevent postoperative tumor recurrence and metastasis.

Platelet exosomes have a great deal of roles in tumor metastasis and development, and some studies report that platelet exosomes promote proliferation and metastasis by protecting tumor cells from host immune monitoring, and can be combined with circulating tumor cells to form relatively large emboli so as to protect the circulating tumor cells from immune system attack and in-vivo shear force. The molecular mechanism of adhesion is mainly that the protein P-selectin overexpressed on its membrane can bind to CD44 receptor which is up-regulated in tumor cells with high specificity. Biomimetic drug delivery systems offer a new opportunity to mimic biological particles in vivo.

At present, platelet exosomes mainly explore the behaviors of the platelet exosomes in blood, and relevant researches and reports for eliminating in-situ tumors, inhibiting remote tumors and capturing and clearing circulating tumor cells in a targeted manner by using the platelet exosomes to entrap adriamycin and combining PD-L1 monoclonal antibodies are absent temporarily.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fibrin gel containing a platelet exosome and a PD-L1 monoclonal antibody which entrap adriamycin, a preparation method thereof and application thereof in preparing a medicament for treating tumor metastasis diseases. The fibrin gel (also called an immunotherapy bio-gel) not only serves as a local drug delivery reservoir for efficient delivery of therapeutic agents, but also simultaneously considers the effectiveness of natural targeted delivery of doxorubicin to circulating tumor cells using platelet exosomes in the gel and the promotion of therapy in combination with chemotherapy and immunotherapy. Doxorubicin (PexD) was entrapped with platelet exosomes, PexD was added to the thrombin solution, PD-L1 monoclonal antibody (aPD-L1) was added to the fibrinogen solution, and the spray was applied using a dual-barrel nebulizer. After surgical resection of the tumor, the spray gel served as a drug reservoir to concentrate and gradually release PexD and aPD-L1. PexD induces the tumor to produce ICD effects providing antigens and promoting anti-tumor immune responses, while also activating capture and clearing circulating tumor cells of lymph and blood circulation by high affinity between P-selectin and the CD44 receptor through in situ damaged vascular access to blood circulation. aPD-L1 was also released to block the PD1/PD-L1 pathway. The combination of the two strategies eliminates residual tumor cells and circulating tumor cells in situ, inhibits the growth of distant tumors and thus prevents tumor recurrence. The gel has remarkable inhibitory effect on lung and liver metastasis of melanoma.

In particular, the amount of the solvent to be used,

the first purpose of the invention is to provide a fibrin gel containing a platelet exosome and a PD-L1 monoclonal antibody, wherein the platelet exosome and the PD-L1 monoclonal antibody are used for encapsulating doxorubicin, can target circulating tumor cells to inhibit tumor metastasis, and are combined with a PD-L1 monoclonal antibody immunotherapy biogel, wherein the exosome and the doxorubicin are as follows by mass percent: 10% -30% of adriamycin and 70% -90% of exosome, wherein the adriamycin is adriamycin or a derivative of the adriamycin; the exosome is a platelet exosome extracted from murine platelets. The exosome and the adriamycin are further as follows by mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome.

Also provides a dosage form of the fibrin Gel containing the platelet exosome carrying the adriamycin and the PD-L1 monoclonal antibody, wherein the dosage form is a preparation that the platelet exosome carries the adriamycin bionic nanoparticles and the fibrinogen/thrombin Gel is used for loading aPD-L1-PexD-Gel;

the fibrinogen/thrombin gel comprises fibrinogen and thrombin with the mass ratio of 100: 1;

the adriamycin bionic nano particles are nano carriers of platelet exosomes entrapping adriamycin; wherein the adriamycin and platelet exosome comprises the following components in percentage by mass: 10% -30% of adriamycin and 70% -90% of platelet exosome; the further platelet exosomes and adriamycin are as follows by mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome;

the adriamycin is adriamycin or a derivative of the adriamycin;

the particle size of the adriamycin bionic nano particle is 142-172 nm;

the platelet exosome is of murine origin and is extracted from murine platelets.

The therapeutic delivery vehicle is intended for administration to a subject to reduce recurrence and/or metastasis following surgical resection. Overcomes the defects of immune clearance and incapability of clearing circulating tumor cells and new metastases, and achieves better treatment effect.

The second purpose of the invention is to provide a preparation method of the immunotherapy biological gel.

The third purpose of the invention is to provide the application of the immunotherapy biological gel and the dosage form thereof in preparing antitumor drugs.

In order to achieve the first purpose, the invention adopts the technical scheme that: provides fibrin gel containing adriamycin bionic nanoparticles and PD-L1 monoclonal antibody which are carried by an exosome membrane. The fibrin gel is quickly formed by simultaneously spraying and mixing equal volumes of a solution containing PexD bionic nanoparticle thrombin and a fibrinogen solution of PD-L1 monoclonal antibody.

In order to realize the second purpose, the invention adopts the technical scheme that: simultaneously spraying and mixing thrombin containing the platelet exosome carrying the adriamycin and a fibrinogen solution containing the PD-L1 monoclonal antibody with equal volume to form the biological gel for the immunotherapy.

The method specifically comprises the following steps:

(1) preparing a platelet exosome;

(2) preparing a platelet exosome-entrapped adriamycin bionic nanoparticle;

(3) simultaneously spraying and mixing the thrombin containing the platelet exosome entrapping adriamycin and the fibrinogen solution containing the PD-L1 monoclonal antibody with equal volume to form the immunotherapy biogel.

The above preparation method, wherein:

the platelet exosome in the step 1 is prepared by the following steps:

fresh mouse blood was centrifuged, and the blood was suspended in the same volume of ACD solution (citric acid-glucose) and then centrifuged to obtain platelets. Dilution of platelets with Tyrode-HEPES buffer and binding of Ca²⁺Ionophore, incubation and then centrifugation. The collected supernatant was further ultracentrifuged, and extracellular vesicles were ultracentrifuged to concentrate the particles. After resuspension, the extracellular vesicles are passed through a microfiltration membrane to obtain exosomes.

The adriamycin bionic nanoparticles entrapped by the platelet exosomes in the step 2 are prepared by the following steps:

and (3) dissolving the exosome and adriamycin in PBS, incubating for 1 hour at 37 ℃, and then centrifuging the mixed solution to obtain the PexD bionic nanoparticles.

The step 3 immunotherapy biological gel is prepared by the following steps:

immunotherapeutic biogels were obtained by spraying equal volumes of PexD-containing thrombin and fibrinogen containing PD-L1 monoclonal antibody.

In the preparation method, the adriamycin and the platelet exosome are prepared from the following components in percentage by mass: 10% -30% of adriamycin and 70% -90% of platelet exosome; the further platelet exosomes and adriamycin are as follows by mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome.

The adriamycin is adriamycin or a derivative of the adriamycin.

The particle size of the prepared adriamycin bionic nano particles is 142-172 nm.

The platelet exosome is of murine origin and is extracted from murine platelets; the platelet exosomes entrap doxorubicin biomimetic nanoparticles.

In order to achieve the third object, the invention adopts the technical scheme that: a fibrin gel containing PexD and PD-L1 monoclonal antibody for targeting tumor cells to prevent tumor metastasis and recurrence and its dosage form application in preparing medicine for treating tumor metastasis are provided.

The tumor metastasis especially refers to lung or liver metastasis of breast melanoma.

The invention has the beneficial effects that: the research result of the invention shows that the immunotherapy biological gel can 'wake up' the innate immune system of a host, and the platelet exosome containing the adriamycin can capture blood ring tumor cells, and is combined with the PD-L1 monoclonal antibody to inhibit postoperative local tumor recurrence and metastasis potential, so that the immunotherapy biological gel can be used as a promising method for preventing tumor recurrence.

Drawings

FIG. 1 is a schematic diagram of the preparation of 1 aPD-L1-PexD-Gel. The schematic shows a bioreactive fibrin gel comprising PexD biomimetic nanoparticles and aPD-L1 sprayed in situ in the tumor bed after surgery. The combination of chemotherapy and immunotherapy eliminates residual in situ tumor cells and captures circulating tumor cells, preventing melanoma recurrence and metastasis.

FIG. 2 characterization of PexD and gel.

(A) Appearance photos of Pex and PexD.

(B) TEM images of Pex and PexD. Scale bar (left): 500 nm. Scale bar (right): 100 nm.

(C) Particle size and potential of Pex and PexD.

(D) SDS-PAGE protein analysis of platelets, Pex and PexD.

(E) Western blot analysis was performed on platelets of Pex and PexD, Pex, PexD-labeled CD41, P-selectin and CD61, CD9, CD63 and TSG101, and CD44 on B16-F10 cells.

(F) Photographs of the appearance of platelets, Pex and PexD.

(G) aPD-L1-PexD-Gel in PBS at pH 6.5. Data are presented as mean ± sem. (n-3).

(H) cryo-Scanning Electron Microscope (SEM) images of fibrin gels containing PexD nanoparticles and aPD-L1. Scale bar (left): 40 μm. Scale bar (right): 20 μm.

FIG. 3PexD assessed in vitro adhesion, cellular uptake, cytotoxicity and ICD.

(A) Confocal microscopy images of B16-F10 cells incubated with free DOX and PexD for 0.5 and 2 hours, respectively. Scale bar: 10 μm.

(B) B16-F10 cells were incubated with free DOX and PexD for 0.5 and 2 hours of flow cytometry measurement. Scale bar: 10 μm.

(C) Fluorescence intensity analyzed by flow cytometry. Data are presented as mean ± standard deviation. (n-3).

(D) Adhesion of DiR-labeled Pex to B16-F10 cells was observed by confocal laser scanning microscopy. Scale bar (upper): 10 μm. Scale bar (lower): 5 μm.

(E) Relative HMGB1 release from B16-F10 cells treated with PexD or DOX (3 μ g/mL) for 24 hours.

(F) Relative ATP release from B16-F10 cells treated with PexD or DOX (3. mu.g/mL) for 24 hours.

(G) Representative flow cytometric analysis of CD80+ CD86+ cells. (C) (E), (F) P < 0.01; p < 0.0001.

FIG. 4 the B16-F10 tumor immune response induced by aPD-L1-PexD-Gel was harvested from mice 6 days after treatment.

(A) Schematic diagram of tumor model treatment by remote operation. (B16-F10-WT: wild-type B16-F10 melanoma cells; B16-F10-luc: luciferase-labeled B16-F10 melanoma cells; IVIS: in vivo imaging System; FC: flow cytometry analysis; DC imaging: digital Camera imaging).

(B) Representative flow cytometric analysis images of M2 macrophages (CD206hi) and (C) M1 macrophages (CD80hi) on gated F4/80+ CD11b + CD45+ cells.

(D) Representative flow cytometry analysis of CD4+ Foxp3+ T cells on CD3+ cells.

(E) Representative flow cytometric analysis of CD8+ T cells on CD3+ cells.

(F) Relative quantization in graph (B). Data are presented as mean ± sd. (n-4).

(G) The relative quantification in graph (C) is expressed as mean ± sd. (n-4).

(H) The relative quantification in graph (D) is expressed as mean ± sd. (n-4).

(I) The relative quantification in graph (E) is expressed as mean ± sd. (n-4). (F) (G) (H) (I) P < 0.05; p < 0.01; p < 0.001; p < 0.0001.

The gel of FIG. 5 prevented postoperative recurrence of the B16-F10 tumor.

(A) In vivo bioluminescence imaging of B16-F10 tumors after primary tumor resection. Four representative mice are shown per treatment group. The image associated with day 10 was taken before surgery.

(B) (C) mean tumor growth kinetics of the different groups. Growth curves stopped when the first mouse in each group died. Data are expressed as mean ± s.e.m. (n-6). Statistical significance was obtained by multiple comparisons between the analysis of single variance and Tukey post hoc tests.

(D) After various treatments, the survival rate (n ═ 6) corresponded to the tumor size of the mice.

(E) Change in body weight of mice in different groups. Data are expressed as mean ± s.e.m. (n-4).

(F) (H) tumors and spleen after different treatments (n ═ 5). Scale bar: 1 cm.

(G) (I) quantification of tumor and spleen weight. (G) (I) × P < 0.05; p < 0.01; p < 0.0001.

FIG. 6aPD-L1-PexD-Gel local treatment of systemic anti-tumor immune response.

(A) Schematic diagram of tumor model treatment by remote operation. (B16-F10-WT: wild-type B16-F10 melanoma cells; B16-F10-luc: luciferase-labeled B16-F10 melanoma cells; DC imaging: digital camera imaging).

(B) B16-F10 in vivo tumor bioluminescence imaging in response to local aPD-L1-PexD-Gel treatment.

(C) Growth curves of left and right tumors in untreated and treated mice. Data are presented as mean ± SD (n ═ 5).

(D) Representative flow cytometric analysis images of F4/80+ CD11b + CD45+ cells M2 macrophages (CD206hi) and M1 macrophages (CD80hi) were gated.

(E) Representative flow cytometric analyses of CD3+ cells CD4+ Foxp3+ T and CD3+ cells CD4+ CD8+ T cells.

(F) (G) relative quantization in graph (D). Data are presented as mean ± s.d. (n-4).

(H) (I) relative quantification in Panel (E). Data are presented as mean ± sd. (n-4).

(J) After various treatments, photographs of whole lung indian ink staining and H & E staining of tumor, kidney, lung, spleen, liver and heart sections were collected. Yellow and red arrows represent liver and lung metastases, respectively. Scale bar: 1 mm. (F) (G) (H) (I) P < 0.0001.

Figure 7 HE staining after lung metastasis treatment.

FIG. 8 HE staining of various organs after in situ tumor treatment.

Figure 9 pictures and quantification of tumor and spleen after distal tumor treatment.

FIG. 10 cytotoxicity profiles.

Fig. 11 western blot of tumor HMGB 1.

Fig. 12 dendritic cell CD80+, CD86+ quantification.

Figure 13 gel rheology test.

Detailed Description

The technical solutions of the present invention are further disclosed below by way of examples, but the present invention is not limited to the scope of the examples.

The fibrin Gel containing the platelet exosome entrapping adriamycin and the PD-L1 monoclonal antibody is prepared in the embodiment of the invention, the properties of the fibrin Gel are examined, meanwhile, the fibrin Gel (Gel), the Gel containing the adriamycin (DOX-Gel), the Gel containing the PexD (PexD-Gel) and the Gel containing the PD-L1 monoclonal antibody (aPD-L1-Gel) are used as a control, and the detailed examination contents are as follows:

1) preparing exosome, encapsulating the exosome with adriamycin through incubation and centrifugation, and characterizing physicochemical properties of the exosome, such as particle size, potential, morphology, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Western Blot for characterizing protein condition, gel physicochemical properties, a drug release curve and the like.

2) Platelet exosomes were examined for cytotoxicity, cellular uptake, adhesion to B16-F10 cells, and adhesion to tumor cells encapsulating doxorubicin.

3) The behavior and anti-metastasis therapeutic effect of fibrin Gel (aPD-L1-PexD-Gel) containing the platelet exosome entrapped adriamycin and the PD-L1 monoclonal antibody in C57BL/6 mice at the resection position after the right abdomen is inoculated with tumor by spraying are examined.

4) And (3) simulating a far-end tumor by inoculating the tumor to the left abdomen on the seventh day after inoculating the tumor to the C57BL/6 right abdomen, and spraying fibrin Gel (aPD-L1-PexD-Gel) containing platelet exosome entrapping adriamycin and PD-L1 monoclonal antibody on the C57BL/6 mouse body after the excision part of the right abdomen, wherein the fibrin Gel has the behavior and the anti-metastasis treatment effect.

Example 1

Preparation and characterization of platelet-entrapped doxorubicin and bioreactive fibrin gel.

The synthetic scheme is shown in figure 1.

1) Preparing the platelet exosome.

After the platelet-rich plasma is anticoagulated by EDTA, 100g of the platelet-rich plasma is centrifuged for 20min to remove erythrocytes, the supernatant is added into the ACD solution to prevent the activation of platelets, and 800g of the platelet-rich plasma is centrifuged for 20min to prepare a platelet membrane. Using Tyrode-HEPES buffer (1mM MgCl) at 30 deg.C₂、2mM CaCl₂And 3mM KCl₂) Dilution of platelets to 250X 10⁶platelets/mL, and binding Ca²⁺Ionophore (10mM, Sigma-Aldrich), incubated for 30 min, then centrifuged at 800g for 10 min. The collected supernatant was further ultracentrifuged, and the extracellular vesicles were ultracentrifuged at 100000g for 90 minutes to concentrate the particles. After resuspension, the extracellular vesicles were passed through a 220nm microporous membrane to obtain platelet exosomes (Pex).

2) Preparing the platelet exosome entrapping adriamycin bionic nanoparticles.

PexD is prepared by mixing exosomes with DOX. DOX was diluted appropriately with sterile saline for injection. 500 μ LDOX (1mg mL)^-1) And 500. mu.L of exosome solution (2mg mL)^-1) Prepared and incubated at 37 ℃ for 1 hour, and then the mixed solution was added to the centrifuge tube at 4 ℃. PexD was obtained by centrifugation at 100000rpm for 90 minutes. The platelet exosome and the adriamycin are as follows by mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome.

Pex obtained after ultracentrifugation was a white precipitate, and PexD appeared red due to DOX loading (FIG. 2A). Morphological studies of exosomes before and after DOX loading were performed by Transmission Electron Microscopy (TEM) (fig. 2B). Both Pex and PexD showed a characteristic disc-like bilayer membrane structure, indicating that Pex remained intact after loading with DOX. The Malvern particle size analyzer (fig. 2C) showed that the particle size distribution of the exosomes was relatively narrow, with the mean diameter of the free exosomes being about 115 nm. After loading with DOX, the average particle size increased to 157 nm. The average zeta potential of PexD is higher than Pex due to positively charged DOX (fig. 2C). We used SDS-PAGE and western blot detection as shown in fig. 2D and 2E, which both show the same protein composition, indicating that both essential proteins of platelets are efficiently retained.

3) By spraying equal volumes of thrombin (200U/mL) containing PexD (40. mu.g doxorubicin) and fibrinogen (10mg mL) containing PD-L1 monoclonal antibody (40. mu.g PD-L1 monoclonal antibody)^-1) Obtaining the immunotherapy biological gel.

After spraying fibrin gel containing PexD and aPD-L1, a red hydrogel was formed and the cover layer exhibited the characteristic color of orange PexD and clear aPD-L1 (FIG. 2F). The morphology of the fibrin gel containing PexD nanoparticles and aPD-L1 was verified by rheological tests. In the dynamic time scan (fig. 13A), the value of the storage modulus (G') was consistently greater than the value of the loss modulus (G "), indicating that the hydrogel is a stable soft material. In the dynamic strain scan (fig. 13B), the value of G' dominates the value of G ", the critical strain value of the gel is 68.35%, indicating robust gel formation. In the dynamic frequency sweep (FIG. 13C), the gel behavior was independent of frequency in the range of 0.1-100 rads-1. The excellent mechanical properties ensure the stability of the in vivo gel. Gel morphology was characterized by scanning electron microscopy (fig. 2H). The gel exhibits a three-dimensional porous structure, which is a prerequisite for drug release. We further explored the DOX release profile of the gel. We added PexD to the gel, incubated the drug-loaded gel in the tumor microenvironment (PBS at pH 6.5), and then quantified the release of DOX in the gel at different time points. According to the release profile (fig. 2G), a cumulative release of 43.4% DOX in the gel was observed over 24 hours. The results indicate that release of DOX from the drug loaded gel system is a time dependent delivery process.

Example 2

Adhesion of platelet exosomes to tumor cells.

We explored adhesion between DiR-labeled Pex and B16-F10 cells by confocal laser scanning microscopy and observed co-localization of DiR-labeled Pex and CD44 in B16-F10 cells (fig. 3D).

Example 3

PexD uptake by B16-F10 cells and cytotoxicity.

We observed cellular internalization of PexD by B16-F10 through confocal (fig. 3A), with higher cellular uptake efficiency of PexD than free DOX at the same incubation time, probably due to binding of Pex to B16-F10 cells. We further used flow cytometry to quantify the cellular uptake of PexD and free DOX solutions by B16-F10 cells. As shown in fig. 3B and 3C, PexD showed enhanced uptake of DOX by B16-F10 cells compared to free DOX solution. These results are consistent with those observed with a fluorescence microscope.

PexD and free DOX solution pair B16-F10 fineThe cytotoxicity of cells in vitro was determined by using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay. PexD had significantly higher cytotoxicity compared to free DOX solution (FIG. 10). Calculated half maximal Inhibitory Concentration (IC) of PexD₅₀) Values as low as 0.115. mu.g mL^-1. Overall, based on the higher cellular uptake of PexD, the results indicated that PexD was highly cytotoxic to B16-F10 cells.

Example 4

PexD elicits B16-F10 producing the ICD effect.

We examined the ability of PexD to induce ICD in B16-F10 cancer cells in vitro by assessing cell surface expression of calreticulin and extracellular secretion of HMGB1 and ATP. Importantly, 3. mu.g mL was used 24 hours after incubation^-1Free DOX-treated B16-F10 cells induced lower calreticulin expression levels than PexD, indicating that PexD can induce stronger ICD in cancer cells (fig. 11). PexD induced higher calreticulin expression in B16-F10 cells compared to DOX solution. Furthermore, the amount of HMGB1 released in the cell culture medium of B16-F10 cells at 24 hours after culture was higher after treatment with PexD than after treatment with free DOX (fig. 3E). The amount of ATP released into the cell culture medium by B16-F10 cells treated with PexD for 24 hours was also higher than that released by B16-F10 cells treated with free DOX (FIG. 3F). These results indicate that PexD induces stronger ICD in B16-F10 cancer cells than free DOX, in good agreement with the above cellular uptake and cytotoxicity results.

We then examined ICD-induced Dendritic Cell (DC) maturation. To assess the maturation status of DCs, bone marrow-derived immature DCs isolated from C57BL/6 mice bearing B16-F10 tumors were co-cultured with B16-F10 cancer cells pretreated with PexD or DOX. Importantly, the frequency of mature DCs (CD11c +/CD80+/CD86+) was significantly increased compared to DCs co-cultured with untreated B16-F10 cancer cells, indicating that PexD effectively promotes DC maturation by ICD of cancer cells (FIG. 3G, FIG. 12). These findings indicate that PexD triggers ICD in cancer cells and effectively promotes DC maturation.

Example 5

Circulating Tumor Cells (CTCs) in the circulatory system are cleared in vivo.

CTCs in the blood are the major cause of tumor metastasis. To test the ability of PexD to capture CTCs, saline, DOX, platelet-DOX, and PexD (40 μ g DOX per mouse) were injected intravenously into individual mice, and B16-F10 cancer cells were then mimicked into CTCs through the tail vein. Mice were sacrificed after 12d and lungs were isolated. As shown in fig. 7, pulmonary micrometastases were most common in mice treated with physiological saline. Mice treated with PexD showed little metastatic pulmonary nodules compared to mice treated with platelet-DOX, indicating a higher capture efficiency of CTCs. This result is attributed to the small particle size of PexD, which can penetrate better into tumor tissue. In the circulation, the high affinity between P-selectin and CD44 helps PexD capture CTCs and eliminate these cells by subsequent release of DOX.

Example 6

The behavior of the bioreactive fibrin gel in right abdominal tumored C57BL/6 mice, the anti-metastatic therapeutic effect and the resulting immune effect were examined.

To verify the therapeutic efficacy of aPD-L1-PexD-Gel, we used an incomplete tumor resection model. Different types of fibrin gels, including Gel (Gel), DOX-Gel, aPD-L1-Gel, PexD-Gel, and aPD-L1-PexD-Gel (40 μ g DOX per mouse, 40 μ g aPD-L1 per mouse) were sprayed into the tumor resection in situ (FIG. 4A). We observed a significant decrease in the proportion of M2-like macrophages in the aPD-L1-PexD-Gel treated group (25.2%), a significant increase in the proportion of M1-like macrophages (44.5%) and a Gel control group (FIG. 4B, C, F, G) compared to other treatments. As shown in FIGS. 4D, E, H and I, we observed reduced levels of regulatory T cells (Treg cells: CD4+ Foxp3+ T cells, also known as suppressor T cells) and increased levels of tumor infiltrating cytotoxic T lymphocytes (CD8+ T cells) in the groups treated with aPD-L1-Gel or PexD-Gel. These findings indicate that both the PD-L1 blocking strategy using aPD-L1 therapy and the PexD strategy relying on tumor destruction can trigger T cell mediated immune responses.

Notably, the combination of these two strategies elicits a stronger T cell immune response. The best results, i.e.maximal rejuvenation of T-cells, were obtained when aPD-L1-PexD-Gel was used. Since the initial release of DOX in the gel induces tumor ICD, and then dying tumor cells behave like a "tumor vaccine", aPD-L1 can reactivate non-functional T cells by blocking the PD-1/PD-L1 signaling pathway.

Tumor growth was monitored by measuring bioluminescent signals from B16-F10-luc cancer cells (FIG. 5A). Three-sixteenths of the mice showed no detectable tumor after treatment with aPD-L1-PexD-Gel, which means relatively good control of tumor growth (FIG. 5B, C). The image and weight of the recurrent tumor (FIG. 5F, G) also showed that aPD-L1-PexD-Gel showed an advantage in preventing local tumor recurrence. 50% of mice treated with aPD-L1-PexD-Gel survived for at least 50 days (FIG. 5D), and the body weight of these mice was unaffected by the treatment (FIG. 5E). Furthermore, we found aPD-L1-PexD-Gel treated group to have minimal tumor, and tumor, spleen and lung collected on day 22 showed that tumor metastasis had abolished in this group (FIG. 8). These results are consistent with the in vivo bioluminescence imaging results shown in figure 5A. Therefore, aPD-L1-PexD-Gel is considered to be a very effective immune Gel drug, which can prevent tumor recurrence and metastasis. In addition, there was a significant difference in spleen weight in tumor-bearing mice compared to healthy mice, which was caused by an abnormal immune function. Tumor-bearing mice often exhibit compensatory splenomegaly. Therefore, we performed euthanasia on experimental mice and harvested spleens to compare the degree of splenomegaly among groups. aPD-L1-PexD-Gel has good therapeutic effect, and the spleen of the mouse treated by the method is close to that of a healthy mouse. The spleen size of the mice in other groups was significantly increased. Spleen weight was further quantified. The spleen weight of the mice treated with physiological saline was 1.7 times that of the mice treated with aPD-L1-PexD-Gel (FIG. 5H, I), further demonstrating that aPD-L1-PexD-Gel produced good anti-tumor immunity.

Example 7

The behavior, anti-metastatic therapeutic effect and the resulting immune effect of the bioreactive fibrin gel were examined in C57BL/6 mice with distal tumors.

We investigated whether local treatment with aPD-L1-PexD-Gel triggered a systemic immune response to suppress distant tumors. The B16-F10 cancer cells were seeded on the side opposite the primary tumor to mimic tumor metastasis. The primary tumor was partially excised and fibrin gel containing PexD nanoparticles and aPD-L1 (40. mu.g DOX, 40. mu.g aPD-L1 per mouse) was sprayed at the site of excision (FIG. 6A). We observed aPD-L1-PexD-Gel inhibited local tumor recurrence and tumor growth at distant sites (FIG. 6B). The tumor growth curve (fig. 6C) and the images and weights of the relapsed tumors (fig. 9A, C) indicate that aPD-L1-PexD-Gel can trigger a systemic immune response and produce significant tumor recurrence inhibition.

For flow cytometry analysis, distant tumors and blood were collected and pooled to form single cell suspensions for testing. Consistent with the above findings, the blood levels of CD8+ T cells were significantly elevated in aPD-L1-PexD-Gel sprayed mice, while the levels of Foxp3+ T cells were significantly reduced (FIG. 6D, F, G). The number of M1-like TAMs increased while the number of M2-like TAMs decreased in distant tumors (fig. 6E, H, I). Compared to the saline group, the aPD-L1-PexD-Gel group mice had normal spleens (FIG. 9B, D), hematoxylin and eosin (H & E) staining (FIG. 6J) showing essentially no tumor metastasis in the major organs after aPD-L1-PexD-Gel treatment. These findings are consistent with the results of the tumor resection model described above, indicating that aPD-L1-PexD-Gel can inhibit tumor recurrence at the primary site, and inhibit tumor at the distant site, thereby further confirming activation of the immune system.

The result shows that the fibrin gel containing the platelet exosome entrapped adriamycin and the PD-L1 monoclonal antibody has obvious effect on treating tumor recurrence and metastasis, particularly lung or liver metastasis of melanoma, and can be used for preparing antitumor drugs, particularly antitumor metastasis drugs.

Claims

1. The fibrin gel containing the platelet exosome and the PD-L1 monoclonal antibody, which are loaded with adriamycin, is a platelet exosome and immunotherapy biogel combined with PD-L1 monoclonal antibody, wherein the platelet exosome and the adriamycin can target circulating tumor cells to inhibit tumor metastasis, and the exosome and the adriamycin are as follows by mass percent: 10% -30% of adriamycin and 70% -90% of exosome, wherein the adriamycin is adriamycin or a derivative of the adriamycin; the exosome is a platelet exosome extracted from murine platelets.

2. The fibrin gel comprising platelet exosomes and PD-L1 monoclonal antibodies entrapped doxorubicin according to claim 1, wherein said exosomes and doxorubicin are in the following mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome.

3. Use of a fibrin gel according to claim 1 or 2, comprising a platelet exosome loaded with doxorubicin and a PD-L1 monoclonal antibody, for inhibiting and/or treating post-melanoma recurrence.

4. A method for preparing the fibrin gel comprising the doxorubicin-loaded platelet exosome and the PD-L1 monoclonal antibody according to claim 1 or 2, comprising the steps of:

(1) preparing a platelet exosome;

centrifuging fresh mouse blood, suspending the blood in the same volume of ACD solution, centrifuging to obtain platelets, diluting the platelets with Tyrode-HEPES buffer, and binding Ca²⁺The method comprises the following steps of (1) performing incubation and centrifugation on an ionophore, further ultracentrifuging collected supernate, ultracentrifuging extracellular vesicles to concentrate particles, and after heavy suspension, passing the extracellular vesicles through a microporous filter membrane to obtain exosomes;

(2) preparing a platelet exosome-entrapped adriamycin bionic nanoparticle;

dissolving exosome and adriamycin into PBS, incubating at 37 ℃, and centrifuging the mixed solution to obtain platelet exosome-loaded adriamycin bionic nanoparticles;

5. The method for preparing the fibrin gel containing the platelet exosome and the PD-L1 monoclonal antibody loaded with doxorubicin according to claim 4, wherein the platelet exosome and the doxorubicin are prepared by the following steps: 16.7 percent of adriamycin and 83.3 percent of exosome.

6. The preparation method of the fibrin gel containing the platelet exosome and the PD-L1 monoclonal antibody carrying doxorubicin according to claim 4, wherein the prepared platelet exosome carrying doxorubicin biomimetic nanoparticles are applied to the preparation of antitumor drugs or the preparation of antitumor transfer drugs.

7. A dosage form of the fibrin Gel comprising the platelet exosome loaded with doxorubicin and the PD-L1 monoclonal antibody of claim 1 or 2, wherein the dosage form is the fibrin Gel aPD-L1-PexD-Gel comprising the platelet exosome loaded with doxorubicin biomimetic nanoparticles and the PD-L1 monoclonal antibody;

the adriamycin bionic nano particles are nano carriers of platelet exosomes entrapping adriamycin; wherein the adriamycin and platelet exosome comprises the following components in percentage by mass: 10% -30% of adriamycin and 70% -90% of platelet exosome;

the adriamycin is adriamycin or a derivative of the adriamycin;

the particle size of the adriamycin bionic nano particle is 142-172 nm;

8. The dosage form of the fibrin gel containing the platelet exosome and the PD-L1 monoclonal antibody loaded with doxorubicin according to claim 7, wherein the platelet exosome and the doxorubicin are present in the following mass percent: 16.7 percent of adriamycin and 83.3 percent of exosome.

9. Use of a dosage form of a fibrin gel comprising doxorubicin-loaded platelet exosomes and PD-L1 monoclonal antibody according to claim 7 or 8 for inhibiting and/or treating post-melanoma recurrence.