CN114306622B

CN114306622B - Fibrin gel containing platelet exosome containing doxorubicin and PD-L1 monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN114306622B
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: 2024-01-30
Anticipated expiration: 2042-01-06
Also published as: CN114306622A

Abstract

A fibrin gel containing platelet exosomes loaded with doxorubicin and PD-L1 monoclonal antibodies, a preparation method and application thereof belong to the technical field of medicines, and compared with doxorubicin solution, the platelet exosomes loaded with doxorubicin can be better combined with tumor cells, induce death of immunogenic tumor cells and promote anti-tumor immune response. Meanwhile, the platelet exosomes loaded with doxorubicin can enter the blood circulation through the damaged blood vessel, track the tumor cells in the circulation and clear the tumor cells. Simultaneously releasing aPD-L1 at the tumor site can block the PD-1/PD-L1 pathway and restore the tumor killing effect of cytotoxic T cells. The combination of the two strategies triggers a stronger T cell immune response, and the tumor immune microenvironment is remarkably improved. The method provides a new strategy and more choices for combining chemotherapy and immunotherapy, and meets the urgent need of high-efficiency preparations in clinic.

Description

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

Technical Field

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

Background

Surgery is an effective method of treating melanoma, but unfortunately, localized residual tumor micro-infiltration and systemic CTCs continue to lead to tumor recurrence leading to patient death. Immune checkpoint inhibitors, particularly PD-L1 monoclonal antibodies, increase the efficacy of melanoma treatment and produce a sustained clinical response in some patients. However, systemic administration of immune checkpoint inhibitors promotes a sustained clinical response in less than 20% of immunogenic tumor patients. The clinical efficacy of immune checkpoint inhibitor single drug therapies (such as the treatment of aPD-L1) is limited due to the lack of immunogenic antigens and various immune resistance mechanisms.

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 antigen and ICI. However, safe and effective targeted delivery of chemotherapeutic agents remains challenging, in part due to poor bioavailability and non-specific targeting. Thus, the ability to combine safe and effective delivery of chemotherapeutic agents with immune checkpoint blockade is critical for preventing postoperative tumor recurrence and metastasis.

Platelet exosomes play a number of roles in tumor metastasis and development, and some studies have reported using established animal models that platelet exosomes promote proliferation and metastasis by protecting tumor cells from host immune monitoring, and can bind to circulating tumor cells to form relatively large emboli, thereby protecting circulating tumor cells from immune system attacks and in vivo shear forces. The molecular mechanism of its adhesion is mainly that the protein P-selectin overexpressed on its membrane can bind highly specifically to the CD44 receptor up-regulated in tumor cells. The biomimetic drug delivery system provides a new opportunity to mimic biological particles in vivo.

At present, platelet exosomes are mainly explored in the behavior of the platelet exosomes in blood, and related researches and related reports of eliminating in-situ tumors, inhibiting remote tumors and capturing and eliminating circulating tumor cells in a targeted manner by using the platelet exosomes to entrap doxorubicin combined with PD-L1 monoclonal antibodies are not used temporarily.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention provides a fibrin gel containing a platelet exosome containing doxorubicin and a PD-L1 monoclonal antibody, a preparation method thereof and application thereof in preparing a medicament for treating tumor metastasis diseases. The fibrin gel (also called immunotherapeutic biogel) not only serves as a local drug delivery reservoir for efficient delivery of therapeutic agents, but also allows for the use of the natural targeted delivery of doxorubicin to circulating tumor cells by platelet exosomes in the gel and the efficacy of combined chemotherapy and immunotherapy-facilitated therapies. Doxorubicin (PexD) was entrapped in the platelet exosomes, pexD was added to the thrombin solution, PD-L1 monoclonal antibody (aPD-L1) was added to the fibrinogen solution, and spraying was performed using a twin-tube nebulizer. After surgical removal of the tumor, the spray gel served as a drug depot to concentrate and gradually release PexD and aPD-L1.PexD induces tumor production ICD effects to provide antigens and promote anti-tumor immune responses while also activating circulating tumor cells that capture and clear lymph and blood circulation through high affinity between P-selectin and CD44 receptors through in situ damaged blood vessels into the blood circulation. The aPD-L1 is also released to block the PD1/PD-L1 pathway. The combination of the two strategies eliminates in situ residual tumor cells and circulating tumor cells, inhibits the growth of distant tumors and thus prevents tumor recurrence. The gel has remarkable inhibiting effect on lung and liver metastasis of melanoma.

In particular, the method comprises the steps of,

the invention provides a fibrin gel containing platelet exosomes coated with doxorubicin and a PD-L1 monoclonal antibody, which is a platelet exosomes coated with doxorubicin capable of targeting circulating tumor cells to inhibit tumor metastasis and is combined with a PD-L1 monoclonal antibody immunotherapeutic biological gel, wherein the exosomes and the doxorubicin comprise the following components in percentage by mass: 10% -30% of doxorubicin and 70% -90% of exosomes, wherein the doxorubicin is doxorubicin or a derivative of doxorubicin; the exosomes are platelet exosomes extracted from mouse-derived platelets. The exosomes and doxorubicin are further prepared from the following components in percentage by mass: doxorubicin 16.7% and exosome 83.3%.

The preparation form of the fibrin Gel containing the platelet exosome and the PD-L1 monoclonal antibody of the entrapped doxorubicin is also provided, wherein the preparation form is that the platelet exosome is entrapped with the doxorubicin bionic nano-particles, and the fibrinogen/thrombin Gel is used for loading the aPD-L1-PexD-Gel;

the fibrinogen/thrombin gel contains fibrinogen and thrombin in a mass ratio of 100:1;

the doxorubicin bionic nano-particles are nano-carriers of platelet exosomes coated with doxorubicin; wherein, the mass percentages of the doxorubicin and the platelet exosomes are as follows: 10-30% of doxorubicin and 70-90% of platelet exosomes; the further platelet exosomes and doxorubicin are in mass percent: doxorubicin 16.7%, exosomes 83.3%;

the doxorubicin is doxorubicin or a derivative of doxorubicin;

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

the platelet exosomes are murine and are extracted from murine platelets.

The therapeutic agent 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 incapacity of clearing circulating tumor cells and new metastasis, and achieves better treatment effect.

A second object of the present invention is to provide a method for preparing said immunotherapeutic biogel.

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

In order to achieve the first object, the invention adopts the following technical scheme: provides a fibrin gel containing exosome membrane-entrapped doxorubicin bionic nanoparticle and PD-L1 monoclonal antibody. The fibrin gel is formed by simultaneously spraying and mixing equal volumes of the thrombin solution containing the PexD bionic nano-particles and the PD-L1 monoclonal antibody fibrinogen solution.

In order to achieve the second object, the invention adopts the following technical scheme: simultaneously spraying and mixing equal volumes of thrombin containing platelet exosomes containing doxorubicin and fibrinogen solution containing PD-L1 monoclonal antibody to form the immunotherapeutic biogel.

The method specifically comprises the following steps:

(1) Preparing platelet exosomes;

(2) Preparing a platelet exosome-entrapped doxorubicin bionic nanoparticle;

(3) Simultaneously spraying and mixing equal volumes of thrombin containing the platelet exosomes and coated with doxorubicin and fibrinogen solution containing PD-L1 monoclonal antibody to form the immunotherapeutic biogel.

The preparation method comprises the following steps:

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

fresh mouse blood was centrifuged, 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 to Ca ²⁺ Ionophore, incubation, and centrifugation. The collected supernatant was further ultracentrifuged, and extracellular vesicles were ultracentrifuged to concentrate particles. After resuspension, the extracellular vesicles pass through a microporous filter membrane to obtain exosomes.

The platelet exosome-entrapped doxorubicin bionic nanoparticle in the step 2 is prepared by the following steps:

the exosomes and the doxorubicin are dissolved in PBS, incubated for 1 hour at 37 ℃, and then the mixed solution is centrifuged to obtain the PexD bionic nanoparticle.

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

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

In the preparation method, the mass percentages of the doxorubicin and the platelet exosomes are as follows: 10-30% of doxorubicin and 70-90% of platelet exosomes; the further platelet exosomes and doxorubicin are in mass percent: doxorubicin 16.7% and exosome 83.3%.

The doxorubicin is doxorubicin or a derivative of doxorubicin.

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

The platelet exosome is murine and is extracted from mouse platelets; the platelet exosomes are coated with doxorubicin bionic nano-particles.

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

The tumor metastasis refers to lung or liver metastasis of milk melanoma in particular.

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 the host, and the internally contained platelet exosomes coated with doxorubicin can capture blood ring tumor cells, and can be combined with PD-L1 monoclonal antibody to inhibit postoperative local tumor recurrence and metastatic potential, so that the invention can be used as a promising method for preventing tumor recurrence.

Drawings

FIG. 1A schematic representation of the preparation of aPD-L1-PexD-Gel. The schematic shows a bioreactive fibrin gel comprising PexD biomimetic nanoparticles and aPD-L1, sprayed in situ in tumor beds 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. 2PexD and characterization of gel.

(A) Appearance photographs of Pex and PexD.

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

(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) Appearance photographs of platelets, pex and PexD.

(G) DOX curve of aPD-L1-PexD-Gel in PBS at pH 6.5. Data are expressed as mean ± sem. (n=3).

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

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

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

(C) Fluorescence intensity of flow cytometry analysis. Data are expressed as mean ± standard deviation. (n=3).

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

(E) Relative HMGB1 release from B16-F10 cells treated with PexD or DOX (3. Mu.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 cytometry analysis of CD80+CD86+ cells. (C), (E), (F) P <0.01; * P <0.0001.

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

(A) Schematic of tumor model treatment for distal surgery. (B16-F10-WT: wild-type B16-F10 melanoma cells; B16-F10-luc: luciferase-labelled B16-F10 melanoma cells; IVIS: in vivo imaging system; FC: flow cytometry analysis; DC imaging: digital camera imaging).

(B) Representative flow cytometry analysis images of M2 macrophages (CD 206 hi) and (C) M1 macrophages (CD 80 hi) on gated F4/80+cd11b+cd45+ cells.

(D) Representative flow cytometry analysis of cd4+foxp3+ T cells on cd3+ cells.

(E) Representative flow cytometry analysis of cd8+ T cells on cd3+ cells.

(F) Relative quantization in graph (B). Data are expressed 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 prevents recurrence of B16-F10 tumor after surgery.

(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 prior to surgery.

(B) (C) average tumor growth kinetics for the different groups. When the first mice in each group died, the growth curve stopped. Data are expressed as mean ± s.e.m. (n=6). Statistical significance was obtained by multiple comparisons between analysis of single variance and Tukey post-hoc test.

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

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

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

(G) (I) quantitative graphs of tumor and spleen weights. (G) (I) P <0.05; * P <0.01; * P <0.0001.

FIG. 6 topical treatment of a systemic anti-tumor immune response by aPD-L1-PexD-Gel.

(A) Schematic of tumor model treatment for distal surgery. (B16-F10-WT: wild-type B16-F10 melanoma cells; B16-F10-luc: luciferase-labelled B16-F10 melanoma cells; DC imaging: digital camera imaging).

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

(C) Growth curves of untreated and treated mice with left and right tumors. Data are expressed as mean ± SD (n=5).

(D) Representative flow cytometry analysis images of gated F4/80+cd11b+cd45+ cells M2 macrophages (CD 206 hi) and M1 macrophages (CD 80 hi).

(E) Representative flow cytometry analysis of cd3+ cell cd4+foxp3+ T and cd3+ cell cd4+cd8+ T cells.

(F) (G) relative quantization in Panel (D). Data are expressed as mean ± s.d. (n=4).

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

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

FIG. 7 HE staining following lung metastasis therapy.

FIG. 8 in situ tumor treatment followed by HE staining of individual organs.

Figure 9 pictures and quantification of tumors and spleens after distal tumor treatment.

FIG. 10 is a cytotoxicity curve.

Figure 11 western blot of tumor HMGB 1.

FIG. 12 CD80+, CD86+ quantification of dendritic cells.

Figure 13 gel rheology test.

Detailed Description

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

The fibrin Gel containing platelet exosomes coated with doxorubicin and PD-L1 monoclonal antibody is prepared and the properties thereof are examined, meanwhile, the fibrin Gel (Gel) is prepared, the doxorubicin-containing Gel (DOX-Gel), the PexD-containing Gel (PexD-Gel) and the PD-L1 monoclonal antibody-containing Gel (aPD-L1-Gel) are taken as a control, and the detailed examination contents are as follows:

1) Exosomes are prepared, and the exosomes are entrapped with doxorubicin through incubation and centrifugation, and physical and chemical properties of the exosomes are characterized, such as particle size, potential, morphology, protein characterization by SDS-PAGE and Western Blot, gel physical and chemical properties, drug release curves and the like.

2) The cytotoxicity of doxorubicin-entrapped platelet exosomes, cellular uptake, adhesion to B16-F10 cells, and adhesion to tumor cells were examined.

3) The behavior and anti-metastasis treatment effect of fibrin Gel (aPD-L1-PexD-Gel) containing platelet exosomes coated with doxorubicin and PD-L1 monoclonal antibody in C57BL/6 mice sprayed at the excision site after tumor grafting in the right abdomen were examined.

4) Distal tumors were simulated in left abdominal tumor at seventh day after C57BL/6 right abdominal tumor, and fibrin Gel (aPD-L1-PexD-Gel) containing platelet exosomes coated with doxorubicin and PD-L1 mab was sprayed on the behavior and anti-metastasis therapeutic effects in C57BL/6 mice after right abdominal resection.

Example 1

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

The synthetic schematic diagram is shown in figure 1.

1) Platelet exosomes are prepared.

Platelet rich plasma was anticoagulated with EDTA, centrifuged at 100g for 20min to remove red blood cells, and the supernatant was added to ACD solution to prevent platelet activation, and centrifuged at 800g for 20min to prepare platelet membrane. At 30℃with Tyrode-HEPES buffer (1 mM MgCl) ₂ 、2mM CaCl ₂ And 3mM KCl ₂ ) Diluting platelets to 250X 10 ⁶ platelets/mL, and bind Ca ²⁺ Ionophore (10 mM, sigma-Aldrich) was incubated for 30 min and then centrifuged at 800g for 10 min. The collected supernatant was further ultracentrifuged, and extracellular vesicles were ultracentrifuged at 100000g for 90 minutes to concentrate particles. After resuspension, the extracellular vesicles were passed through a 220nm microporous membrane to give platelet exosomes (Pex).

2) Preparing the platelet exosome-entrapped doxorubicin bionic nanoparticle.

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

The Pex obtained after ultra-high speed centrifugation was a white precipitate and the PexD was red by loading with DOX (fig. 2A). Morphological studies were performed on exosomes before and after DOX loading by Transmission Electron Microscopy (TEM) (fig. 2B). Both Pex and PexD showed a unique dished bilayer membrane structure, indicating that Pex remains 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 115nm. After DOX loading, the average particle size increased to 157nm. Due to the positively charged DOX, the average zeta potential of PexD is higher than that of Pex (FIG. 2C). We used SDS-PAGE and Western blot assays as shown in FIGS. 2D and 2E, which both showed identical protein fractions, indicating that the critical proteins of platelets were effectively retained.

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

After spraying the fibrin gel containing PexD and aPD-L1, a red hydrogel was formed and the cover layer exhibited the characteristic colors of orange PexD and transparent aPD-L1 (fig. 2F). The morphology of fibrin gel containing PexD nanoparticles and aPD-L1 was verified by rheological testing. In the dynamic time sweep (fig. 13A), the value of the storage modulus (G') was always greater than the value of the loss modulus (G "), indicating that the hydrogel was a stable soft material. In dynamic strain scans (fig. 13B), the value of G' dominates the value of G ", the critical strain value of the gel was 68.35%, indicating that gel formation is robust. In dynamic frequency sweep (FIG. 13C), the gel behavior is independent of frequency in the range of 0.1-100 rads-1. The excellent mechanical properties ensure the stability of the in vivo gel. The 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 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% of DOX in the gel was observed over 24 hours. The results indicate that the 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 the adhesion between DiR-labeled Pex and B16-F10 cells by confocal laser scanning microscopy, and observed co-localization of DiR-labeled Pex with CD44 in B16-F10 cells (fig. 3D).

Example 3

Uptake of PexD by B16-F10 cells and cytotoxicity.

We observed cellular internalization of PexD by B16-F10 by confocal (fig. 3A), with cellular uptake efficiency of PexD being higher than free DOX at the same incubation time, probably due to binding of Pex to B16-F10 cells. We further used flow cytometry to quantitatively determine 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 the free DOX solution. These results are consistent with those observed by fluorescence microscopy.

The in vitro cytotoxicity of PexD and free DOX solutions on B16-F10 cells was determined by using 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay. PexD has significantly higher cytotoxicity than the free DOX solution (FIG. 10). Calculated, half maximal Inhibitory Concentration (IC) of PexD ₅₀ ) Values as low as 0.115 μg mL ^-1 . Overall, the results indicate that PexD has high cytotoxicity to B16-F10 cells based on higher cellular uptake of PexD.

Example 4

PexD initiates the ICD effect situation with B16-F10.

We examined the in vitro induction of PexD by assessing cell surface expression of calreticulin and extracellular secretion of HMGB1 and ATPAbility of B16-F10 cancer cells ICD. Importantly, at 24 hours post incubation, 3. Mu.g mL was used ^-1 Free DOX-treated B16-F10 cells induced lower calreticulin expression levels than PexD, indicating that PexD can induce stronger ICDs in cancer cells (fig. 11). PexD induces 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 24 hours after incubation 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, well fitting the cellular uptake and cytotoxicity results described above.

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 pre-treated with PexD or DOX. Importantly, the frequency of mature DCs (CD11c+/CD80+/CD86+) was significantly increased compared to untreated B16-F10 cancer cell co-cultured DCs, indicating that PexD is effective in promoting DC maturation by ICD of cancer cells (FIG. 3G, FIG. 12). These findings indicate that PexD triggers ICD in cancer cells and is effective in promoting DC maturation.

Example 5

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

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

Example 6

The behavior of the bioresponsive fibrin gel in the body of C57BL/6 mice with tumors on the right abdomen was examined, the anti-metastatic therapeutic effect and the resulting immune effect.

To verify the therapeutic effect of aPD-L1-PexD-Gel, we used an incomplete tumor resection model. Different types of fibrin Gel, including Gel (Gel), DOX-Gel, aPD-L1-Gel, pexD-Gel and aPD-L1-PexD-Gel (40 μg DOX per mouse, 40 μg aPD-L1 per mouse) were injected into tumor resection cavity 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 the other treatments. As shown in fig. 4D, E, H and I, we observed a decrease in the level of regulatory T cells (Treg cells: cd4+foxp3+ T cells, also called suppressor T cells), an increase in the level of tumor-infiltrating cytotoxic T lymphocytes (cd8+ T cells), and levels in the group treated with either agd-L1-Gel or PexD-Gel. These findings indicate that both PD-L1 blocking strategies with agd-L1 treatment and PexD strategies that rely 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.the maximum T-cell viability, were obtained when aPD-L1-PexD-Gel was used. Since the initial DOX release in the gel induces tumor ICD, then dying tumor cells act like a "tumor vaccine", agd-L1 can reactivate nonfunctional T cells by blocking the PD-1/PD-L1 signaling pathway.

Tumor growth was monitored by measuring bioluminescence signals from B16-F10-luc cancer cells (fig. 5A). Three-sixths of mice showed no detectable tumor after treatment with aPD-L1-PexD-Gel, which means relatively good tumor growth control (FIG. 5B, C). Images and weights of recurrent tumors (fig. 5F, G) also indicate that aPD-L1-PexD-Gel shows advantages 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 not affected by treatment (FIG. 5E). Furthermore, we found that the tumors in the aPD-L1-PexD-Gel treated group were minimal, and that the tumors, spleen and lung collected on day 22 showed that tumor metastasis in this group had been eliminated (FIG. 8). These results are consistent with the in vivo bioluminescence imaging results shown in fig. 5A. Therefore, aPD-L1-PexD-Gel is considered to be a very effective immune Gel drug, which can prevent tumor recurrence and metastasis. Furthermore, there was a significant difference in spleen weight in tumor-bearing mice compared to healthy mice, which was a result of immune dysfunction. Tumor-bearing mice often exhibit compensatory splenomegaly. Thus, we performed euthanasia and splenomegaly on experimental mice to compare the extent of splenomegaly between groups. The aPD-L1-PexD-Gel has good treatment effect, and the spleen size of the treated mice is close to that of healthy mice. The spleen size was significantly increased in the other groups of mice. Spleen weight was further quantified. The spleen weight of normal saline-treated mice was 1.7 times that of the aPD-L1-PexD-Gel-treated mice (FIG. 5H, I), further demonstrating that aPD-L1-PexD-Gel produced good anti-tumor immunity.

Example 7

The behavior of the bioresponsive fibrin gel in vivo on C57BL/6 mice with distal tumors was examined, as well as the anti-metastatic therapeutic effect and the resulting immune effect.

We studied whether local treatment with aPD-L1-PexD-Gel triggered systemic immune responses 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 resected and a fibrin gel containing PexD nanoparticles and aPD-L1 (40 μg DOX,40 μg aPD-L1 per mouse) was sprayed at the resection site (fig. 6A). We observed that aPD-L1-PexD-Gel inhibited local tumor recurrence and tumor growth at distant sites (FIG. 6B). The tumor growth curve (FIG. 6C) as well as the image and weight of recurrent 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 a single cell suspension for testing. Consistent with the above findings, CD8+ T cell levels were significantly increased and Foxp3+ T cell levels were significantly decreased in blood of aPD-L1-PexD-Gel-sprayed mice (FIG. 6D, F, G). The number of M1-like TAMs increased in distant tumors, while the number of M2-like TAMs decreased (fig. 6E, H, I). In comparison to the saline group, spleens of mice in the aPD-L1-PexD-Gel group were normal (FIG. 9B, D), hematoxylin and eosin (H & E) staining (FIG. 6J) showed substantially no tumor metastasis in the major organs after the 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 tumors at distant sites, thereby further confirming activation of the immune system.

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

Claims

1. A method for preparing a fibrin gel comprising an doxorubicin-entrapped platelet exosome and a PD-L1 monoclonal antibody, comprising the steps of:

(1) Preparing platelet exosomes;

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 combining Ca ²⁺ The ionophore is incubated, and then centrifuged, the collected supernatant is further ultracentrifuged, extracellular vesicles are ultracentrifuged to concentrate particles, and after being resuspended, the extracellular vesicles pass through a microporous filter membrane to obtain exosomes;

(2) Preparing a platelet exosome-entrapped doxorubicin bionic nanoparticle;

the exosomes and the doxorubicin are dissolved in PBS, incubated at 37 ℃, and then the mixed solution is centrifuged to obtain the platelet exosomes coated doxorubicin bionic nanoparticle; the weight percentage is as follows: 10% -30% of doxorubicin and 70% -90% of exosomes;

2. The method for preparing the fibrin gel containing the platelet exosomes and the PD-L1 monoclonal antibodies, which comprises the doxorubicin according to claim 1, wherein the platelet exosomes and the doxorubicin are as follows by mass percent: doxorubicin 16.7% and platelet exosomes 83.3%.

3. The method for preparing the fibrin gel containing the platelet exosomes coated with the doxorubicin and the PD-L1 monoclonal antibody according to claim 1, wherein the prepared platelet exosomes coated with the doxorubicin bionic nano-particles are applied to preparing antitumor drugs or antitumor metastasis drugs.

4. A preparation form of a fibrin Gel containing platelet exosomes coated with doxorubicin and PD-L1 monoclonal antibodies prepared by the preparation method of claim 3 is characterized in that the preparation form is a fibrin Gel aPD-L1-PexD-Gel containing platelet exosomes coated with doxorubicin bionic nanoparticles and PD-L1 monoclonal antibodies;

the doxorubicin bionic nano-particles are nano-carriers of platelet exosomes coated with doxorubicin;

the particle size of the doxorubicin bionic nano particles is 142-172nm.

5. The dosage form of fibrin gel containing platelet exosomes and PD-L1 monoclonal antibodies, which comprises doxorubicin according to claim 4, wherein the platelet exosomes and doxorubicin are in mass percent: doxorubicin 16.7% and exosome 83.3%.