A lymph node targeted tumor in situ vaccine and its preparation method and application

Abstract

The present invention discloses a lymph node targeted tumor in situ vaccine and a preparation method and application thereof, which belongs to the field of biomedicine technology. The immune adjuvant CpG is coated on mannose nanoparticles to obtain a CpG nanoadjuvant, and then an MMP responsive gel is used as a delivery system to deliver the CpG nanoadjuvant and trametinib together, thereby achieving sustained in situ antigen production, lymph node drainage and APCs presentation, and further activating anti-tumor immune response. The vaccine has a use in preparing vaccine drugs for treating tumors, and as an effective cancer vaccine for immunotherapy, achieves targeting to lymph nodes to induce anti-tumor immunity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a lymph node targeted tumor in situ vaccine and a preparation method and application thereof. Background Art

[0002] Ovarian cancer is the fifth leading cause of cancer-related death in women and the deadliest gynecological malignancy. Chemotherapy and surgery are the main clinical strategies for the treatment of ovarian cancer. However, these therapies remain ineffective for the treatment of ovarian cancer for several reasons: 1) Most patients are diagnosed at an advanced stage with extensive metastasis; 2) Adverse side effects hinder the use of many standard chemotherapy drugs; 3) Patients are increasingly multidrug-resistant (MDR) to chemotherapy. In addition, tumor cells dispersed during surgery and residual small tumor tissues can also promote the metastasis and recurrence of ovarian cancer. Therefore, there is an urgent need to develop new therapeutic strategies to inhibit the progression, recurrence, and metastasis of ovarian cancer.

[0003] Immunotherapy shows great potential in preventing tumor recurrence and metastasis by activating the immune system and enhancing anti-tumor immunity, thereby killing tumor cells. Among them, tumor vaccines that can produce specific anti-tumor immune responses are considered to be one of the most important immunotherapy strategies and have great therapeutic prospects. However, due to the inefficiency of its tumor vaccination cascade process (including antigen recognition and preparation, co-encapsulation of antigens and adjuvants, (lymph node) targeted delivery, DC internalization and maturation, antigen presentation and T cell activation), tumor vaccines have not achieved the expected clinical effects. In order to improve the efficacy of tumor vaccines, it is necessary to optimize the vaccine cascade process, especially antigen preparation, precise vaccine targeting and reshaping of the immunosuppressive microenvironment, thereby enhancing T cell activation.

[0004] Sufficient tumor-specific antigen load is a prerequisite for tumor vaccines to stimulate strong antigen-specific anti-tumor immune responses. Tumor antigens mainly include tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), and whole-cell tumor antigens. Since TAAs are co-expressed on the surface of normal cells and tumor cells, they are prone to central immune tolerance, which hinders the efficacy and clinical application of tumor vaccines based on TAAs. The production process of TSAs involves the collection, discovery, and generation of tumor samples, which is complex, costly, and time-consuming. Whole-cell tumor antigens, due to their broad spectrum of tumor antigens, can not only circumvent the shortcomings of the above two antigens, but also induce stronger anti-tumor immune responses, greatly reducing the chances of tumor escape and recurrence. However, traditional whole tumor antigens (such as whole cell lysates) have low immunogenicity and are rapidly degraded / cleared in the body. A more effective strategy recently developed is to induce whole-cell tumor antigens in situ, that is, to ablate tumors in vivo and release tumor antigens to continuously activate tumor-specific immune responses. This strategy usually induces immunogenic cell death through treatments such as radiotherapy, photothermal therapy, photodynamic therapy, and chemotherapy, prompting the dying cancer cells to release paracrine signals, which in turn transforms the tumor into an "in situ vaccine" and ultimately increases the type and number of tumor-infiltrating T cells. However, in most cases, free antigens are easily and rapidly cleared, and the APCs that process and present antigens are not fully activated, so the anti-tumor immune response generated is not sufficient. Therefore, it is very necessary to develop an effective platform that can continuously provide highly immunogenic in situ vaccines in combination with appropriate adjuvants.

[0005] In the prior art, the invention patent with publication number CN114096274A discloses an immunotherapy construct that can be administered simultaneously with anticancer therapy or used in combination with targeted therapeutic agents, specifically including delivery particles, at least one adjuvant, and one or more therapeutic agents / compounds that cause antigen release and / or regulate the immunosuppressive tumor microenvironment, wherein the delivery particles can be liposomes, liposome-based particles, polymer particles, inorganic particles or a mixture thereof; the adjuvant has immunostimulatory activity, and can be a CpG oligonucleotide, a DNA TLR agonist containing a CpG sequence, a non-CpG DNA TLR agonist, an RNA TLR agonist, an aluminum salt, an anti-CD40 antibody, a fusion protein, a cytokine, a small molecule TLR agonist, an oil- or surfactant-based adjuvant, a lipopolysaccharide, a plant extract or a derivative thereof; the therapeutic agent can be siRNA, miRNA, antisense oligonucleotide, mRNA, DNA, sgRNA (CRISPR-cas9 element), oligonucleotide, polynucleotide, peptide, protein, chemotherapeutic drug, toxin, antioxidant, small molecule inhibitor, antibody or radiotherapeutic agent. This patented method provides a non-responsive controlled release of adjuvant and antigen, with a limited amount of delivered particles reaching the tumor site.

[0006] The invention patent with publication number CN104284674A discloses a vaccine composition of an immunogenic PAA polypeptide or an isolated nucleic acid molecule encoding an immunogenic PAA polypeptide, which can be used to treat human prostate cancer. When administered, it can contain one or more immunosuppressive cell inhibitors and immune effector cell enhancers, wherein the immunosuppressive cell inhibitors can be selected from protein kinase inhibitors (such as imatinib, sorafenib, axitinib, etc.), CTLA-4 inhibitors (such as ipilimumab and tremelimumab), TLR agonists (such as CpG oligonucleotides), and the immune effector cell enhancer can be selected from CpG24555, CpG10103, CpG7909, CpG1018 or a TLR agonist of CpG1018. It can induce an immune response to a tumor-associated antigen associated with the neoplastic disease. The preparation steps of the patent method are complicated, the vaccine composition does not have lymph node targeting, its immunogenicity is limited, the tumor does not produce antigens in situ, and no in situ antigen reservoir is established. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a lymph node-targeted tumor in situ vaccine, wherein the method obtains a CpG nanoadjuvant by coating an immune adjuvant CpG on mannose nanoparticles, and then uses an MMP-responsive gel as a delivery system to co-deliver the CpG nanoadjuvant and trametinib, thereby achieving sustained in situ antigen production, lymph node drainage, and APCs presentation, thereby activating an anti-tumor immune response. To this end, the present invention also provides a lymph node-targeted tumor in situ vaccine prepared by the preparation method, and the use of the vaccine in the preparation of a vaccine drug for treating tumors.

[0008] The present invention is achieved through the following technical solution: A method for preparing a lymph node-targeted tumor in situ vaccine comprises the following steps:

[0009] S1. Preparation of mannose-modified CpG nanoadjuvant;

[0010] S2. reacting four-arm-maleimide-polyethylene glycol with a thiol-containing peptide to obtain a prepolymer solution;

[0011] S3. Add CpG nanoadjuvant and trametinib to the prepolymer solution to obtain an in situ tumor vaccine.

[0012] In the step S1, CpG is dissolved in PBS and then added to an organic solution containing mannose-modified polylactic acid. After dissolution, the solution is subjected to ultrasonic treatment and the organic solvent is removed to obtain a mannose-modified CpG nanoadjuvant.

[0013] The mass ratio of the CpG to mannose-modified polylactic acid is 1:1000.

[0014] During the ultrasonic treatment, an ultrasonic probe is used in an ice bath for 2 minutes with a power controlled at 10-20%, and then the mixed solution is added to the polyvinyl alcohol solution and ultrasonicated in an ice bath for 1-5 minutes with a power controlled at 10-40%. After the ultrasonic treatment is completed, an emulsion is formed.

[0015] In the step S2, the molar ratio of four-arm-maleimide-polyethylene glycol to the thiol-containing peptide is controlled to be 1:1.1.

[0016] In step S3, the mass ratio of the prepolymer solution, the CpG nanoadjuvant, and trametinib is controlled to be 135:4:1 or 135:1:1.

[0017] A lymph node-targeted tumor in situ vaccine is prepared by the above preparation method.

[0018] Application of a lymph node-targeted tumor in situ vaccine in the preparation of a vaccine drug for treating tumors.

[0019] The tumor includes ovarian cancer.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] (1) The tumor in situ vaccine provided by the present invention uses a specific gel delivery system as a carrier, that is, the MMP-responsive gel prepared by the addition reaction of Mal-arm-PEG and MMP-2 responsive peptide can be loaded with multiple active substances, such as antibodies, tumor antigens, cytokines and adjuvants, to jointly enhance the anti-tumor immune response; it can protect the active immunomodulator from being degraded in the body, thereby improving the therapeutic bioavailability; the release behavior of the active substance can respond to various environmental stimuli, such as pH, temperature, redox and enzymes, thereby achieving spatiotemporal specificity. At the same time, the tumor microenvironment can also specifically and highly express matrix metalloproteinases (MMPs). Therefore, the construction of MMP-responsive smart hydrogels can endow tumor vaccines with multiple functions, including ICD-induced in situ antigens, released NPs adsorbed antigens, targeted delivery of in situ vaccines, and sustained MMP response stimulation, thereby inducing effective anti-tumor immune responses.

[0022] (2) The present invention prepares mannose-modified polylactic acid (PLGA) nanoparticles and encapsulates the immune adjuvant CpG to activate TLR9. The mannose modification can enhance the recognition and absorption of nanoparticles by antigen presenting cells (APCs), which are then transported to the lymph nodes to enhance the antigen-specific immune response of the tumor in situ vaccine based on lymph node targeting.

[0023] (3) The present invention uses MMP-responsive gel to enable the co-delivery of mannose-modified CpG immune adjuvant and trametinib, achieving sustained in situ antigen production, lymph node drainage and APCs presentation, thereby activating anti-tumor immune response.

[0024] (4) The lymph node-targeted tumor in situ vaccine used in the present invention can greatly enhance the cascade process of tumor vaccines and synergize the inhibition of the PD-1 / PD-L1 axis, thereby inhibiting the recurrence and metastasis of ovarian cancer.

[0025] (5) The production process of the present invention is simple. Experiments have shown that the tumor in situ vaccine composed of MMP-responsive gel as a drug delivery system loaded with CpG nanoadjuvant and trametinib can simultaneously induce patient-specific immune response and counteract immune tolerance, and is expected to become an effective cancer vaccine for postoperative immunotherapy.

[0026] In summary, the present invention uses MMP-2 responsive gel to achieve co-delivery of chemotherapeutic drugs and lymph node targeted nanoadjuvants, induces continuous in situ production of tumor antigens through ICD, and adsorbs them to nanoadjuvants to form in situ vaccines, which can target lymph nodes to induce anti-tumor immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Particle size and morphology of CpG@Man-P (40nm, 100nm, 200nm).

[0028] Figure 2 Agarose gel electrophoresis diagram of CpG@Man-P.

[0029] Figure 3 The particle size change of CpG@Man-P after protein adsorption.

[0030] Figure 4 is the amount of adsorbed protein of CpG@Man-P.

[0031] Figure 5 This is the SDS-PAGE band diagram after CpG@Man-P adsorbed protein.

[0032] Figure 6 This is a scanning electron microscopy image of Tra / CpG@Man-P / Gel.

[0033] Figure 7 Swelling behavior of Tra / CpG@Man-P / Gel.

[0034] Figure 8 This is the rheological property curve of Tra / CpG@Man-P / Gel.

[0035] Fig. 9MMP-2 enzyme responsive degradation of Tra / CpG@Man-P / Gel.

[0036] Fig.10 This is the MMP-2 enzyme responsive release of Tra / CpG@Man-P / Gel.

[0037] Fig.11 The cytotoxicity of CpG@Man-P (40nm, 100nm, 200nm) (n≥3).

[0038] Fig.12 is the cytotoxicity of Tra (n≥3).

[0039] Fig.13 Tra induces immunogenic cell death (ICD) of ID8 cells.

[0040] Fig.14 In vitro uptake of CpG@Man-P (40nm, 100nm, 200nm) by BMDCs.

[0041] Fig.15 In vitro uptake of CpG@Man-P (40nm, 100nm, 200nm) by DC 2.4.

[0042] Fig.16 CpG@Man-P (40nm, 100nm, 200nm) induced BMDC maturation.

[0043] Fig.17 CpG@Man-P (40nm, 100nm, 200nm) induced DC2.4 cell maturation.

[0044] Fig.18 CpG@Man-P (40nm, 100nm, 200nm) induced T cell proliferation after adsorbing whole cell antigen.

[0045] Fig.19 The distribution of Dir@Man-P and Dir@P gels of different sizes in the tumor site and lymph nodes. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto.

[0047] Example 1: Synthesis of (40 nm) CpG@Man-P

[0048] 10 μg of CpG was dissolved in 100 μL of PBS, and the solution was added to 1 mL of acetone-dichloromethane (1:1, V / V) containing 10 mg / mL PLGA (L:A=50:50, Mw=10 kDa) as the organic phase. Ultrasonication was performed for 2 min using an ultrasonic probe in an ice bath at a power of 20%. The mixture was added to 5 mL of 5% PVA (203) solution and ultrasonicated for 5 min in an ice bath at a power of 40% to form an emulsion. The organic solvent was removed by reduced pressure evaporation at 37°C.

[0049] Example 2: Synthesis of (100 nm) CpG@Man-P

[0050] 10 μg of CpG was dissolved in 100 μL of PBS, and the solution was added to 1 mL of acetone-dichloromethane (1:1, V / V) containing 10 mg / mL PLGA (L:A=50:50, Mw=10 kDa) as the organic phase. Ultrasonication was performed for 2 min with an ultrasonic probe in an ice bath at a power of 15%. The mixture was added to 5 mL of 1% PVA (203) solution and ultrasonicated for 3 min in an ice bath at a power of 15% to form an emulsion. The organic solvent was removed by reduced pressure evaporation at 37°C.

[0051] Example 3: Synthesis of (200 nm) CpG@Man-P

[0052] 10 μg of CpG was dissolved in 100 μL of PBS, and the solution was added to 1 mL of acetone-dichloromethane (1:1, V / V) dissolved with 10 mg / mL PLGA (L:A=50:50, Mw=10 kDa) as the organic phase. Ultrasonication was performed for 2 min with an ultrasonic probe in an ice bath at a power of 10%. The mixture was added to 5 mL of 1% PVA (203) solution and ultrasonicated for 1 min in an ice bath at a power of 10% to form an emulsion. The organic solvent was removed by reduced pressure evaporation at 37°C.

[0053] The following analysis was performed on the CpG@Man-P of different sizes prepared in Examples 1 to 3:

[0054] 1. Particle size and morphology characterization

[0055] After CpG@Man-P (40nm, 100nm, 200nm) was diluted to appropriate multiples, its particle size and morphology were characterized. The particle size was measured by dynamic laser scattering particle size analyzer to obtain its particle size distribution diagram. The morphology diagram was obtained by scanning electron microscopy (×200nm). For details, see Figure 1 .

[0056] Depend on Figure 1It can be seen that the CpG@Man-P nanoparticles are spherical with regular morphology and are evenly dispersed. The particle sizes of the prepared nanoparticles are 36.4 ± 9.2 nm, 129.8 ± 6.6 nm, and 201.1 ± 9.5 nm, respectively.

[0057] (II) Investigation of CpG encapsulation efficiency and drug loading

[0058] Agarose gel electrophoresis was used to verify the loading of Man-P (40 nm, 100 nm, 200 nm) on the drug CPG, and the absorbance value was determined using an ultraviolet absorption spectrophotometer. The encapsulation efficiency and drug loading of the nanoparticles were calculated using the standard curve of CPG.

[0059] The drug loading ratio (DL) and encapsulation efficiency (ER) were calculated according to the following formula:

[0060] DL% = mass of encapsulated drug / (total mass of carrier and encapsulated drug) × 100%

[0061] ER% = encapsulated drug mass / total drug mass × 100%

[0062] The results of CpG drug loading and encapsulation efficiency are detailed in Figure 2 As shown in Table 1.

[0063] Depend on Figure 2 It can be seen that compared with the free CpG group, no obvious migration bands appeared in the CpG@Man-P (40nm, 100nm, 200nm) groups, indicating that CpG was successfully encapsulated by Man-P nanoparticles.

[0064] Table 1 CpG drug loading and encapsulation efficiency of CpG@Man-P

[0065]

[0066] As can be seen from Table 1, the CpG encapsulation efficiency of CpG@Man-P (40 nm, 100 nm, 200 nm) was measured to be approximately 78%, and the drug loading was approximately 0.08%, indicating that there was no significant difference in the CpG loading effect between nanoparticles of different particle sizes.

[0067] (III) Investigation of antigen protein adsorption capacity of nanoadjuvant CpG@Man-P

[0068] ID8 cell culture: ID8 cells were cultured in a constant temperature incubator containing 5% carbon dioxide and 37°C with DMEM complete medium (containing 10% high-quality fetal bovine serum and 1% double antibody), and the medium was changed every day. When the cells grew to about 80% in the culture dish, the subculture operation was performed. The operation method is as follows: discard the culture medium, add 5mL sterile PBS (pH 7.2), rinse 1-2 times and discard the rinse solution. Since ID8 cells are adherent cells, 2mL of trypsin solution containing EDTA needs to be added to digest in the incubator for 3min. After digestion, observe the cell digestion under a microscope. If most of the cells become round and fall off, add an equal volume of DMEM complete medium to terminate the digestion. Then collect the cells, centrifuge at 2000rpm for 5min, discard the supernatant, add 1-2mL of culture solution, blow evenly, and culture in separate dishes.

[0069] Extraction of total protein from ID8 cells: Pre-cool the centrifuge column and its receiving tube on ice. Wash the ID8 cells by adding pre-cooled PBS to the culture dish, shaking slightly, and discarding the supernatant. Then, add the corresponding volume of RIPA cell lysis buffer, repeatedly pipetting several times, transfer the lysed cells to the pre-cooled centrifuge column sleeve, centrifuge (14000-16000rpm, 30s), and immediately place the collection tube on ice and discard the centrifuge column.

[0070] Investigation of the adsorption capacity of CpG@Man-P on total protein of ID8 antigen: CpG@Man-P nanoparticles of different sizes (40nm, 100nm, 200nm) were incubated with total protein extract of ID8 cells for 1 h, and the CpG@Man-P nanoadjuvant adsorbed with antigen protein was separated by size exclusion chromatography, that is, agarose gel CL-4B was filled into a 1×15cm chromatographic column and eluted with PBS (pH 7.4).

[0071] After successfully separating nanoparticles of different sizes, the particle size changes of CpG@Man-P before and after protein adsorption were measured. Figure 3 .Depend on Figure 3 It can be seen that the particle size of the nanoparticles after adsorbing the antigen was determined by the DLS method, and the hydrated particle sizes of CpG@Man-P (40nm), CpG@Man-P (100nm), and CpG@Man-P (200nm) were approximately 63.18nm, 158.45nm, and 228.41nm, respectively. The size of the nanoparticles in each group increased, verifying the successful adsorption of the particles on the protein antigen.

[0072] The recovered CpG@Man-P nanoparticles were concentrated and the total protein content on the recovered nanoparticles was determined using a BCA protein quantification kit. Figure 4 .Depend on Figure 4It can be seen that through the BCA protein quantification method, the antigen adsorption amounts of CpG@Man-P (40nm), CpG@Man-P (100nm), and CpG@Man-P (200nm) were measured to be approximately 6.78μg / mg, 8.22μg / mg, and 12.68μg / mg, respectively, indicating that the increase in the particle size of CpG@Man-P may help to adsorb antigens.

[0073] Take 100 μL of CpG@Man-P nanoparticles of the same concentration and different particle sizes, add 25 μL of protein loading buffer, mix thoroughly, boil to completely denature the protein, and place in a -80℃ refrigerator for later use. Use commercially available 4% to 15% polyacrylamide gradient gel to separate the antigen protein adsorbed on the nanoparticles, and use Coomassie brilliant blue staining to observe the protein bands adsorbed on the CpG@Man-P nanoparticles. For details, see Figure 5 .Depend on Figure 5 It can be seen that compared with the TP group, CpG@Man-P (40 nm), CpG@Man-P (100 nm), and CpG@Man-P (200 nm) have similar characteristic bands, indicating that CpG@Man-P successfully captured the total protein of ID8 antigen.

[0074] Example 4: Synthesis of (40 nm) CpG@Man-P / Tra / Gel

[0075] Four-arm-maleimide-polyethylene glycol (4-arm-PEG 2000 -Mal, Mw = 20600.8) and a thiol-containing peptide (Ac-CRDGPQGIWGQDRC-NH2, Mw = 1631.75, Mal / SH molar ratio of 1:1.1) were dissolved in TEA buffer to obtain a prepolymer solution (4-arm-PEG 2000 -Mal concentration is 5%wt). The nanoparticles CpG@Man-P (40nm) prepared in Example 1 were washed and concentrated for later use. 0.4 mg CpG@Man-P nanoparticles (or 1.6 mg in other embodiments) and 0.4 mg trametinib (Tra) were added to 1 ml of the prepolymer solution in sequence, vortexed to mix thoroughly, and heated at 37°C for 30 min until the gel no longer flowed, to obtain the drug-loaded gel Tra / CpG@Man-P / Gel (40nm).

[0076] Example 5: Synthesis of (100 nm) CpG@Man-P / Tra / Gel

[0077] 4-arm-PEG 2000 -Mal (same as above) and thiol-containing peptide (same as above) were dissolved in TEA buffer to obtain a prepolymer solution (where 4-arm-PEG 2000-Mal concentration is 5%wt). The nanoparticles CpG@Man-P (100nm) prepared in Example 2 were washed and concentrated for use. 0.4 mg CpG@Man-P nanoparticles (or 1.6 mg in other embodiments) and 0.4 mg trametinib (Tra) were added to 1 ml of the prepolymer solution in sequence, vortexed to mix thoroughly, and heated at 37°C for 30 min until the gel no longer flowed to obtain the drug-loaded gel Tra / CpG@Man-P / Gel (100nm).

[0078] Example 6: Synthesis of (200 nm) CpG@Man-P / Tra / Gel

[0079] 4-arm-PEG 2000 -Mal (same as above) and thiol-containing peptide (same as above) were dissolved in TEA buffer to obtain a prepolymer solution (where 4-arm-PEG 2000 -Mal concentration is 5%wt). The nanoparticles CpG@Man-P (200nm) prepared in Example 3 were washed and concentrated for use. 0.4 mg CpG@Man-P nanoparticles (or 1.6 mg in other embodiments) and 0.4 mg trametinib (Tra) were added to 1 ml of the prepolymer solution in sequence, and heated at 37°C for 30 min until the gel no longer flowed, to obtain the drug-loaded gel Tra / CpG@Man-P / Gel (200nm).

[0080] Comparative Example 1: Synthesis of Tra / Gel

[0081] 4-arm-PEG 2000 -Mal (same as above) and thiol-containing peptide (same as above) were dissolved in TEA buffer to obtain a prepolymer solution (where 4-arm-PEG 2000 -Mal concentration is 5%wt). The prepolymer solution was vortexed and mixed, 0.4mg trametinib (Tra) was added to 1ml of the prepolymer solution, and heated at 37°C for 30min until the gel no longer flowed, thereby obtaining Tra / Gel gel.

[0082] Comparative Example 2: Synthesis of PEG gel

[0083] 4-arm-PEG 2000 -Mal (same as above) and thiol-containing peptide (same as above) were dissolved in TEA buffer to obtain a prepolymer solution (where 4-arm-PEG 2000 The prepolymer solution was vortexed and heated at 37°C for 30 min until the hydrogel no longer flowed, thereby obtaining a blank PEG gel.

[0084] The following performance tests were performed on the performance of Examples 4 to 6, and Comparative Examples 1 and 2:

[0085] 1. Morphological characterization

[0086] The drug-loaded gel Tra / CpG@Man-P / Gel (40 nm) prepared in Example 4 was freeze-dried and the volume change of the gel was observed. The freeze-dried gel was cut into pieces of about 3 mm 2 The pore structure inside the gel was observed by scanning electron microscope (SEM). Figure 6 (Scale bar is 50 μm).

[0087] Depend on Figure 6 It can be seen that the gel presents an interconnected porous multilayer network structure, and the pore size is distributed between 50-100 μm.

[0088] (II) Determination of equilibrium swelling degree

[0089] The equilibrium swelling ratio (SR) of the drug-loaded gels Tra / CpG@Man-P / Gel (40 nm, 100 nm, 200 nm) prepared in Examples 4 to 6 and the Tra / Gel prepared in Comparative Example 1 was measured by gravimetric method.

[0090] A certain mass (150 mg, m0) of gel was immersed in 10 mL of PBS at 37°C. After removing the free water on the surface of the gel with filter paper at a predetermined time, the mass of the gel (m0) was measured. t ), three parallel samples were performed for each experiment.

[0091] SR can be calculated using the following formula:

[0092] SR (%) = (m t -m0) / m0× 100%.

[0093] The results of the equilibrium swelling of different gels are shown in Figure 7 .Depend on Figure 7It can be seen that each group of gels showed rapid swelling before 15h. The gel containing CpG@Man-P (100nm) first reached equilibrium (or the highest) swelling degree at 15h, and the other three groups reached equilibrium at about 24h. The equilibrium swelling rates of the gels in the Tra / Gel, Tra / CpG@Man-P / Gel (40nm), Tra / CpG@Man-P / Gel (100nm), and Tra / CpG@Man-P / Gel (200nm) groups were about 228%, 304%, 246%, and 280%, respectively. The swelling rate of the gel group loaded with nanoparticles was generally higher than that of the Tra / Gel group, but the difference between the gel groups loaded with CpG@Man-P (40nm, 100nm, 200nm) of different sizes was not obvious. This proves that encapsulating nanoparticles may slightly increase the swelling rate of the gel, but the change in particle size does not significantly affect the swelling properties of the gel.

[0094] (III) Rheological properties test

[0095] The drug-loaded gels Tra / CpG@Man-P / Gel (40nm, 100nm, 200nm) prepared in Examples 4 to 6, the Tra / Gel gel prepared in Comparative Example 1, and the PEG prepared in Comparative Example 2 were tested by a rheological instrument. In order to study the viscoelastic properties of the gel, the storage modulus (G') and loss modulus (G") of the gel samples at different angular frequencies were measured, and the rheological performance curves of the gel samples were obtained. For details, see Figure 8 shown.

[0096] Depend on Figure 8 It can be seen that the G' of each group of gels is much larger than G", indicating that the hydrogel has strong elasticity, weak viscosity, solid-like characteristics, and is successfully prepared. Overall, with the increase of frequency, the G" of the sample increases slightly, while G' shows a trend of first slightly increasing and then decreasing, indicating that the drug-loaded gel has a weak response to stress changes. As can be seen from the figure, the loss modulus of each group did not show significant differences. For the storage modulus, the gel group loaded with CpG@Man-P (200nm) has the highest G' value, indicating that its intermolecular cross-linking density is more effective and has higher mechanical properties. In addition, the G' of each group is higher than that of the blank gel control group (PEG), which also reflects that the mechanical properties of the gel are enhanced after drug loading.

[0097] 4. Evaluation of MMP-2 enzyme responsiveness of PEG gel

[0098] MMP-2 activation: dilute rhMMP-2 to 100 μg / mL with Assay buffer and incubate at 37°C for 1 h.

[0099] Evaluation of MMP-2 responsiveness: rhMMP-2 was dissolved in PBS buffer to obtain buffers of different concentrations. PEG hydrogels with an accurate mass (m0) were placed in different buffer media (PBS, PBS + MMP-2 100ng / mL, PBS + MMP-2 200ng / mL, PBS + MMP-2 400ng / mL) and incubated at 37°C. After 5 days, the samples were removed from the buffer, rinsed with distilled water, and vacuum dried to obtain the gel mass after degradation (m t ).

[0100] The formula for calculating gel mass loss rate is:

[0101] Mass loss rate (%) = (m t - m0) / m0× 100%.

[0102] For details on the MMP-2 enzyme-responsive degradation effect on PEG gel, see Fig. 9 .Depend on Fig. 9 It can be seen that after the gel was treated with different concentrations of MMP-2 for 5 days, the gel mass of the 100 ng / mL, 200 ng / mL, and 400 ng / mL groups lost approximately 47%, 59%, and 93%, respectively, indicating that the drug-loaded gel can be degraded in a MMP-2-responsive manner.

[0103] 5. In vitro drug release experiment

[0104] Use Dil to fluorescently label CpG@Man-P (40nm, 100nm, 200nm): Dil solution was added to 10mg / mL PLGA (L∶A=50∶50, Mw=10kDa) dissolved in 1mL acetone-dichloromethane (1∶1, V / V) as the organic phase, and 100μL PBS was added to the organic phase. The rest of the process was the same as the synthesis method of CpG@Man-P. After the preparation, PVA and free Dil were removed using a 100kDa ultracentrifuge.

[0105] Prepare gels containing particles with different Dil fluorescence labels and Tra (particle concentration is 50 mg / Kg, trametinib (Tra) concentration is 1 Kg / mL). Incubate at MMP-2 (200 ng / mL), 37°C, pH 6.8. Take out 1 mL of liquid at 2h, 4h, 6h, 8h, 16h, 24h, 48h, 72h, 96h, and 120h, and measure the release of particles at 542nm excitation and 564nm emission. Measure the absorbance at 360nm to calculate the release of Tra.

[0106] The MMP-2 enzyme responsive release of CpG@Man-P (40nm, 100nm, 200nm) is shown in Fig.10 .

[0107] Depend on Fig.10 It can be seen that the release of nanoparticles (Dil@Man-P, 8A) and drugs (Tra, 8B) in the gel group containing MMP-2 enzyme was significantly higher than that in the corresponding control group, indicating that the prepared gel has the ability of MMP-2 responsive degradation and can responsively release the nanoadjuvant CpG@Man-P and ICD inducer Tra.

[0108] Furthermore, the CPG@Man-P / Tra / Gel of Examples 4 to 6 was evaluated at the cellular level.

[0109] 1. Cell safety evaluation

[0110] Cultivation of DC 2.4 cells: DC 2.4 cells were cultured in a 37°C constant temperature incubator containing 5% CO2 using RPMI-1640 complete medium, and the medium was changed every day. When the cells in the medium grew to 70% full, the subculture operation was performed. That is, the medium was discarded, 5 mL of sterile PBS (pH 7.2) was added for rinsing, and the rinsing solution was discarded. DC 2.4 cells are semi-adherent cells. 5 mL of RPMI-1640 complete medium was directly added and the cells were carefully blown to collect the cells. After centrifugation at 250g for 5 minutes, the supernatant was discarded, and RPMI-1640 complete medium was added to blow the cells evenly, and then cultured in separate dishes.

[0111] The MTT method was used to investigate the safety of CpG@Man-P with different particle sizes (40nm, 100nm, 200nm) on DC cells. The specific method is as follows:

[0112] DC 2.4 cells were cultured at 3 × 10 3 The cells were inoculated into a 96-well plate at a concentration of 100 / well. After 12 hours, 40nm, 100nm, and 200nm CpG@Man-P were added at a concentration of 20μg / mL, respectively, and incubated at 37°C for 24 hours. After that, the culture medium was replaced with 1640 medium containing 0.5mg / mL MTT. After incubation at 37°C for 4 hours, the culture medium was aspirated, and 100μl DMSO solution was added to each well. After constant temperature shaking at 37°C for 20 minutes, the ultraviolet absorption value at 570nm was detected using an enzyme marker. The survival rate was calculated according to the following formula:

[0113]

[0114] For test results, see Fig.11 .Depend on Fig.11It can be seen that after incubation with CpG@Man-P (40nm), CpG@Man-P (100 nm), and CpG@Man-P (200nm) for 24 hours, DC 2.4 cells still showed a high cell survival rate. Even when the concentration of CpG@Man-P was increased to 1 mg / mL, the cell survival rate was still as high as nearly 90%, indicating that the nanoparticles had no obvious killing or inhibitory effect on DC2.4 cells. In addition, the cell safety of CpG@Man-P of different particle sizes did not show significant differences. This shows that the size has no significant effect on the DC cytotoxicity of nanoparticles.

[0115] 2. Evaluation of cytotoxicity of the chemotherapy drug trametinib (Tra)

[0116] The MTT method was used to investigate the cytotoxicity of different concentrations of Tra to ID8 cells. The specific method is as follows:

[0117] ID8 cells were cultured at 3 × 10 3 The cells were inoculated into a 96-well plate at a concentration of XX / well. After 12 h, Tra at a concentration of XX was added and incubated at 37°C for 24 h. After that, the culture medium was replaced with 1640 medium containing 0.5 mg / mL MTT. After incubation at 37°C for 4 h, the culture medium was removed and 100 μl of DMSO solution was added to each well. After constant temperature shaking at 37°C for 20 min, the ultraviolet absorbance at 570 nm was detected using an ELISA reader and the survival rate was calculated.

[0118] For test results, see Fig.12 .Depend on Fig.12 It can be seen that the killing effect of Tra on ID8 cells is concentration-dependent, with an IC50 of 20.84 μg / mL.

[0119] 3. Immunogenic death of tumor cells

[0120] ID8 cells were seeded in 12-well plates at 6×104 and cultured at 5% CO2 and 37°C for 24 h. ID8 cells were incubated with 10 μg and 20 μg of Tra for 24 h. ID8 cells were incubated with 10 μg and 20 μg of Tra for 24 h. The expression of CRT was then observed by flow cytometry.

[0121] ID8 cells were seeded in a confocal dish at 6×104 and cultured at 5% CO2 and 37°C for 24 h. ID8 was incubated with 10 μg and 20 μg of Tra for 24 h. The cells were fixed with 4% paraformaldehyde and blocked. The cells were then incubated with rabbit anti-mouse CRT primary antibody overnight at 4°C. The primary antibody was discarded and washed with PBS. After adding the secondary antibody and incubating for 1 h in the dark, the secondary antibody was discarded and washed with PBS. Finally, the cells were stained with DAPI.

[0122] ID8 cells were seeded at 6×104 in a 12-well plate and cultured at 5% CO2 and 37°C for 24 h. ID8 cells were incubated with 10 μg and 20 μg of Tra for 24 h. ID8 cells were incubated with 10 μg and 20 μg of Tra for 24 h. The release amount in the upper culture medium was then measured using an ATP kit.

[0123] For detailed test results, see Fig.13 .Depend on Fig.13 It can be seen that treating ID8 with different doses of Tra can induce ID8 to release a large amount of ATP (as shown in Figure A); confocal (as shown in Figure B) and flow cytometry (as shown in Figure C and D) results show that ID8 will show a large amount of calreticulin (CRT) exposed on the cell membrane surface after being treated with Tra. These results indicate that Tra can induce immunogenic death of ID8 tumor cells and continuously produce whole tumor cell antigens in situ.

[0124] 4. Evaluation of BMDC cell uptake of nanoadjuvant CpG@Man-P

[0125] Extraction of BMDC cells: Take an adult C57 mouse, cut off the hind legs above the hip joint of the mouse, expose the femur and tibia, keep the knee joint and ankle joint immobile, remove muscles and tissues as much as possible, ensure the integrity of the bones during the process, and perform subsequent operations in a clean bench. Inject 75% ethanol into the culture dish, soak the clean bones in it for 3-5 minutes to disinfect and sterilize, and clean all bones. Use scissors to cut off both ends of the bone as close to the joint as possible. Inject PBS into the syringe, then insert the syringe needle into both ends of the bone, flush the bone marrow into the culture dish, and rinse 2-3 times until the bone turns completely white. Use a pipette to aspirate the bone marrow fluid several times to separate all clumps. Centrifuge the suspension at 1200rpm for 5min, discard the supernatant, add 2mL of red blood cell lysis buffer, resuspend the cells, incubate at room temperature for 5min, then add an equal volume of PBS, filter with a 70μm mesh, centrifuge and discard the supernatant. Wash 1-2 times with PBS, then resuspend the cells with RPMI-1640 complete medium, and adjust the cell concentration to 5×10 5 , add to the cell culture dish, place the dish in a 37°C incubator with 5% CO2, and culture it, which is recorded as day 0; on day 3, add 10 mL of culture medium and 20 ng / mL GM-CSF to the dish, and change the medium by half on days 6 and 8. On day 10, collect the cells, namely BMDC.

[0126] Incubation of BMDC cells: 8×10 BMDC cells were cultured per well. 5Cells were seeded in 12-well plates and incubated at 37°C and 5% CO2 for 24 hours. BMDCs were incubated with CpG@Man-P (40nm, 100nm, 200nm) of different particle sizes and CpG@P (40nm, 100nm, 200nm) without mannose modification at a concentration of 20μg / mL for 4 hours. The incubated cells were incubated with CD11c antibodies to distinguish BMDCs.

[0127] BMDC surface molecule staining for flow cytometry: resuspend BMDC with 600μl PBS, centrifuge (2000rpm, 5min), wash repeatedly several times, add appropriate amount of specific fluorescent antibody, incubate at 4℃ in the dark for 30min. After staining, centrifuge (2000rpm, 5min), and then resuspend the cells with 300μl flow staining buffer for flow cytometry detection.

[0128] For detailed test results, see Fig.14 .Depend on Fig.14 It can be seen that the cell uptake of the CpG@Man-P group was higher than that of the CpG@P group, indicating that the modification of mannose gave the nanoparticles the ability to target DC cells. In addition, the BMDC cell uptake of the CpG@Man-P (200nm) group was significantly higher than that of the other two groups, and the BMDC cell uptake decreased with the decrease in the particle size of CpG@Man-P, which may be due to the fact that DC cells have a higher uptake ability for larger-sized nanoparticles.

[0129] 5. Evaluation of the uptake capacity of DC 2.4 cells for nanoadjuvant CpG@Man-P

[0130] Cultivation of DC 2.4 cells: DC 2.4 cells were cultured in a 37°C constant temperature incubator containing 5% CO2 using RPMI-1640 complete medium, and the medium was changed every day. When the cells in the medium grew to 70% full, the subculture operation was performed. That is, the medium was discarded, 5 mL of sterile PBS (pH 7.2) was added for rinsing, and the rinsing solution was discarded. DC 2.4 cells are semi-adherent cells. 5 mL of RPMI-1640 complete medium was directly added and the cells were carefully blown to collect the cells. After centrifugation at 250g for 5 minutes, the supernatant was discarded, and RPMI-1640 complete medium was added to blow the cells evenly, and then cultured in separate dishes.

[0131] Incubation of DC 2.4 cells: 8 × 10 DC 2.4 cells were cultured per well. 5Cells were seeded in confocal dishes and incubated at 37°C, 5% CO2 for 24 h. DC 2.4 was incubated with fluorescently labeled CpG@Man-P (40 nm, 100 nm, 20 nm) of different sizes and CpG@P (40 nm, 100 nm, 200 nm) without mannose modification at a concentration of 20 μg / mL for 4 h.

[0132] Confocal microscopy observation of DC 2.4 cells uptake of CpG@Man-P: Fix with 4% paraformaldehyde for 10 min. Wash residual paraformaldehyde with appropriate amount of PBS. Stain with DAPI (1 μg / mL) for 10 min, wash excess dye with PBS. Then observe under confocal microscope.

[0133] For test results, see Fig.15 .Depend on Fig.15 It can be seen that the cell fluorescence intensity of the CpG@Man-P group is higher than that of the CpG@P group, indicating that the modification of mannose effectively enhances the DC uptake of CpG@Man-P, showing the active targeting ability of the particles. At the same time, the CpG@Man-P (200nm) treatment group showed the highest uptake compared with the CpG@Man-P (40 nm) group and the CpG@Man-P (100nm) group, confirming that DC cells have a higher uptake ability for larger-sized nanoparticles.

[0134] (VI) Examination of the ability to induce BMDC cell maturation

[0135] BMDC cells were plated at 8 × 10 per well. 5 Cells were seeded in 12-well plates and incubated at 37°C, 5% CO2 for 24 hours. BMDCs were co-incubated with CpG@Man-P (40nm, 100nm, 200nm), CpG, LPS, Man-P (40nm, 100nm, 20 nm) (CpG = 2ug / mL, LPS = 1ug / mL) for 48 hours. The incubated cells were incubated with CD11c, CD86, and CD80 antibodies for 30 minutes, and the mature BMDC cells were determined by flow cytometry after washing with PBS.

[0136] For test results, see Fig.16 .Depend on Fig.16 It can be seen that both free CpG and Man-P can induce BMDC maturation to a certain extent, showing CD86 + and CD80 + The cell ratio increased, and Man-P of different sizes loaded with CpG showed enhanced ability to promote DC maturation, indicating that CpG@Man-P nanoadjuvant integrated CpG adjuvant and Man-mediated TLR-4 pathway to induce BMDC maturation.

[0137] 7. Examination of the ability to induce DC cell maturation

[0138] DC2.4 cells were plated at 5×10 4 Cells were seeded in 12-well plates and incubated at 37°C, 5% CO2 for 24 hours. BMDCs were co-incubated with CpG@Man-P (40nm, 100nm, 200nm), CpG, LPS, Man-P (40nm, 100nm, 200nm) (CpG = 2ug / mL, LPS = 1ug / mL) for 48 hours. The incubated cells were incubated with CD11c, CD86, and CD80 antibodies for 30 minutes, washed with PBS, and mature DC2.4 cells were determined by flow cytometry.

[0139] For test results, see Fig.17 .Depend on Fig.17 It can be seen that free CpG induces DC2.4 cell maturation to a certain extent, showing CD86 + and CD80 + The cell ratio increased, and Man-P of different sizes loaded with CpG showed enhanced ability to promote DC maturation, indicating that the CpG@Man-P nanoadjuvant integrated the CpG adjuvant and the Man-mediated TLR-4 pathway, thereby inducing DC maturation.

[0140] Furthermore, the following experiments were conducted on the CPG@Man-P / Tra / Gel of Examples 4 to 6.

[0141] 1. T cell activation

[0142] The mice were killed, washed twice with PBS and then chopped. The cell mesh was then placed on a 6-well cell culture plate. The cell mesh was then ground with a 5 mL syringe plunger until no obvious solid tissue was present. PBS was added to wash the plunger and mesh. The cell suspension was collected and filtered twice with a mesh. The cell suspension was collected and centrifuged, the supernatant was discarded, 5 mL of red blood cell lysis buffer was added, the suspension was evenly blown and allowed to stand for 8 min, and the same volume of PBS was added to stop the lysis; the suspension was washed once. T cells were labeled with CFSE.

[0143] BMDCs were plated at 5 × 10 per well. 4 Cells were seeded in 12-well plates and incubated at 37°C, 5% CO2 for 24 hours. BMDCs were incubated with CpG@Man-P (40nm, 100nm, 200nm), CpG, (CpG=2ug / mL, LPS=1ug / mL) for 48 hours. Various reagents were added for two days of incubation, and CFBE-labeled spleen cells were added for another 3 days of incubation. The cells were incubated with CD8 antibodies for 30 minutes, and T cell proliferation was observed by flow cytometry after washing with PBS.

[0144] For test results, see Fig.18 .Depend on Fig.18 It can be seen that whole cell antigen (Protein) and free adjuvant CpG can induce T cell proliferation to a certain extent; CpG@Man-P can induce a large number of T cells to proliferate after adsorbing whole cell antigen, indicating that nanoadjuvants can adsorb antigens to form in situ vaccines and induce strong T cell proliferation and immune response.

[0145] 2. Lymph node targeting - in vivo imaging

[0146] The DiR solution was added to 10 mg / mL PLGA-Man or PLGA (L∶A=50∶50, Mw=10KDa) dissolved in 1 mL acetone-dichloromethane (1∶1, V / V) as the organic phase. 100 μL PBS was added to the organic phase and ultrasonicated for 2 min in an ice bath using an ultrasonic probe at a power of 10%. The mixture was added to 5 mL 1% PVA (203) solution and ultrasonicated for 1 min in an ice bath at a power of 10% to form an emulsion. The organic solvent was removed by reduced pressure evaporation at 37°C. PVA and free DiR were removed using a 100 KDa ultracentrifuge (DiR concentration 50 ug / m).

[0147] The mice were 8-10 weeks old and were shaved and 5 × 10 6 ID8 cells in a 50:50 mixture of PBS cell suspension and matrix gel were injected subcutaneously into the right flank. Once the tumor was approximately 200 mm 3 , mice were randomly divided into 6 treatment groups ( n = 6).

[0148] 100ul of CpG@Man-P and CpG@P PEG gels of different sizes were injected into the tumor site. The fluorescence changes of the tumor site of mice were observed using the IVIS system at the following time points, and 6 mice were randomly selected from each group. The tumor and lymph nodes were dissected out (to observe the degradation of the gel at the tumor site and the lymphatic reflux, respectively), placed in paraformaldehyde, and imaged under the IVIS system.

[0149] For test results, see Fig.19 .Depend on Fig.19 It can be seen that compared with Dir@P, the Man-modified nanoparticles Dir@Man-P accumulated more in the tumor (as shown in Figure A) and were more targeted to the lymph nodes (as shown in Figure B and C). At the same time, the 40nm Dir@Man-P showed the strongest tumor retention and lymph node targeted distribution, thanks to the faster release and better lymph node drainage of the small-particle nanoadjuvant.

[0150] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a lymph node-targeted tumor in situ vaccine, characterized in that: The following steps are involved: S1. Preparation of mannose-modified CpG nanoadjuvant; S2. reacting four-arm-maleimide-polyethylene glycol with a thiol-containing peptide to obtain a prepolymer solution; S3. Add CpG nanoadjuvant and trametinib to the prepolymer solution to obtain an in situ tumor vaccine. The thiol-containing peptide is Ac-CRDGPQGIWGQDRC-NH2; and the tumor is ovarian cancer.

2. The preparation method according to claim 1, characterized in that: In the step S1, CpG is dissolved in PBS and then added to an organic solution containing mannose-modified polylactic acid. After dissolution, the solution is subjected to ultrasonic treatment and the organic solvent is removed to obtain a mannose-modified CpG nanoadjuvant.

3. The preparation method according to claim 2, characterized in that: The mass ratio of the CpG to mannose-modified polylactic acid is 1:1000.

4. The preparation method according to claim 2, characterized in that: During the ultrasonic treatment, an ultrasonic probe is used in an ice bath for 2 minutes with a power controlled at 10-20%, and then the mixed solution is added to the polyvinyl alcohol solution and ultrasonicated in an ice bath for 1-5 minutes with a power controlled at 10-40%. After the ultrasonic treatment is completed, an emulsion is formed.

5. The preparation method according to claim 1, characterized in that: In the step S2, the molar ratio of four-arm-maleimide-polyethylene glycol to the thiol-containing peptide is controlled to be 1:1.

1.

6. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of the prepolymer solution, the CpG nanoadjuvant, and trametinib is controlled to be 135:4:1 or 135:1:

1.

7. A lymph node targeted tumor in situ vaccine, characterized in that: The method is prepared according to any one of claims 1 to 6.

8. Use of the tumor in situ vaccine obtained by the preparation method according to claim 1 in the preparation of a vaccine drug for treating ovarian cancer.

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