Targeting peptide modified nano-micelle co-loaded with near-infrared fluorescent dye and immunologic adjuvant as well as preparation method and application of targeting peptide modified nano-micelle

Abstract

The invention relates to the technical field of biology, in particular to a targeting peptide modified nano-micelle co-loaded with near-infrared fluorescent dye and an immunologic adjuvant, a preparation method and an application of the targeting peptide modified nano-micelle co-loaded with the near-infrared fluorescent dye and the immunologic adjuvant. A nano-carrier of the nano-micelle is a PLGA-PEG nano-carrier, a targeting peptide is a TMTP1 targeting peptide, a near-infrared fluorescent dye is indole green, and an immunologic adjuvant is MPLA. The nano-micelle can target ovarian cancer primary lesion and metastatic lesion, increase aggregation of micelles in tumor tissues, generate PTT effect through laser irradiation under the guidance of near-infrared fluorescence, cause immune death of tumor cells, release tumor-associated antigens, and promote DC cell recognition of presentation antigens and DC cell activation under the assistance of an immunologic adjuvant, so that the tumor-associated antigens can be degraded, and the tumor-associated antigens can be degraded. In addition, macrophages and NK cells can be collected, T cell immune response is induced, secretion of immunomodulatory factors is promoted, other tumor cells not irradiated by laser are recognized and killed, immune memory is generated, and tumor relapse is inhibited.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a targeting peptide-modified nano micelle co-loaded with a near-infrared fluorescent dye and an immune adjuvant, a preparation method and an application thereof. Background Art

[0002] Ovarian cancer is one of the leading causes of death from gynecological tumors, and its incidence has been on the rise in recent years. Global cancer statistics show that there are approximately 2,387,700 new cases of ovarian cancer and 151,900 deaths each year. Due to the lack of early and effective screening methods and obvious clinical manifestations, most patients are diagnosed in the advanced stage. Despite active first-line treatment with surgery and adjuvant chemotherapy, the 5-year survival rate is still as low as 45%. Although more than 80% of patients respond positively in the early stages of treatment, most patients will eventually relapse and develop chemotherapy resistance. How to improve the current status of ovarian cancer treatment has been a difficult problem that has puzzled the gynecological oncology community for many years. New treatment strategies for ovarian cancer patients are urgently needed.

[0003] Immunotherapy is the newest treatment method after the three major conventional tumor treatments, and it has developed rapidly in the past decade. Ovarian cancer can be recognized by the host immune system. Tumor-reactive T cells and antibodies produced by spontaneous anti-tumor immune responses can be detected in the peripheral blood, tumor tissues and ascites of ovarian cancer patients, and the infiltration of cytotoxic T cells in ovarian cancer is positively correlated with the improvement of overall survival. Tumor-mediated recruitment of suppressive leukocytes and iatrogenic effects such as lymphocytotoxic drugs are the main factors leading to immune escape of ovarian cancer. In order to stimulate the body's immune response, immune checkpoint inhibitors, adoptive T cell therapy, especially the newly emerging chimeric antigen receptor T (CAR-T) and cancer vaccines have been gradually applied.

[0004] In recent years, with the rapid development of nanotechnology and biomaterials, drug delivery by nanocarriers has attracted much attention in the treatment of cancer and other diseases. Nanocarriers can significantly change drug pharmacokinetics, target tumor tissues, and release drugs in a controlled manner to enhance treatment outcomes and reduce toxic side effects on normal tissues. Cancer vaccines or immune checkpoint inhibitors that rely on nanocarriers have become a hot topic in cancer immunotherapy research in the past decade. Tumor vaccine delivery by nanocarriers can increase the uptake of DC cells and enhance antigen-specific immune responses.

[0005] Recent studies have shown that hyperthermic intraperitoneal chemotherapy (HIPEC) can improve the prognosis of patients with advanced ovarian cancer, but it still has limitations. Because HIPEC acts on all organs in the abdominal and pelvic cavities without tumor targeting, it inevitably damages normal organs and causes complications such as intestinal adhesions and obstruction. In addition, the treatment and perfusion cycle is long, and some patients cannot tolerate it and have to terminate the treatment. If targeted hyperthermia can be used, the survival rate of patients can be greatly improved.

[0006] Photothermal therapy (PTT) has attracted widespread attention worldwide as a new treatment method that can achieve precise individualized intervention in treatment. PTT uses the photothermal effect of photothermal converters to convert light energy into heat energy, increase the temperature of the surrounding environment, and cause tumor cell apoptosis and necrosis. Compared with traditional treatment methods, PTT has obvious advantages. The use of adjustable doses of external laser irradiation can accurately locate tumors and minimize damage to surrounding healthy tissues.

[0007] Nanocarriers have also been widely used to deliver photothermal converters to improve the efficiency of photothermal conversion and the targeting of photothermal therapy, as well as in combined therapy. PTT can enhance other therapeutic effects by directly killing cancer cells or by improving drug release efficiency, stimulating drug release, regulating the tumor immune microenvironment, and triggering the release of tumor-specific antigens.

[0008] TMTP1 can target the primary lesions and micrometastases of highly metastatic tumors. SPECT imaging agents labeled with the radioactive element technetium can specifically image highly metastatic tumors of ovarian cancer and can be used in the diagnosis and treatment of ovarian cancer. Indocyanine green (ICG) is the only near-infrared fluorescent dye approved by the FDA. In addition to being a good near-infrared fluorescent imaging agent, the most noteworthy property of ICG is the PTT effect. MPLA vaccines have been approved for clinical use or are undergoing phase III clinical trials, such as (hepatitis), (HPV types 16 and 18), Quattro (pollen allergy), etc.

[0009] However, free ICG is unstable in aqueous solution, easily cleared, and lacks targeting. Unencapsulated MPLA is quickly degraded and cleared in vivo. These shortcomings seriously affect the clinical application of photothermal therapy.

[0010] Currently, there is no carrier that can transport ICG and MPLA into the body and enable them to exist stably in the body. Summary of the invention

[0011] The technical problem to be solved by the present invention is to provide a targeting peptide-modified nano micelle co-loaded with a near-infrared fluorescent dye and an immune adjuvant, a preparation method and an application thereof.

[0012] The technical solution of the present invention to solve the above technical problems is as follows:

[0013] The invention provides a targeting peptide-modified nano micelle co-loaded with a near-infrared fluorescent dye and an immune adjuvant, wherein the nanocarrier of the nano micelle is a PLGA-PEG nanocarrier, the targeting peptide is a TMTP1 targeting peptide, the near-infrared fluorescent dye is indocyanine green, and the immune adjuvant is MPLA.

[0014] Based on the above technical solution, the present invention can also be improved as follows.

[0015] Furthermore, the TMTP1 targeting peptide is a cyclic TMTP1 polypeptide, and the amino acid sequence of the cyclic TMTP1 polypeptide is NVVRQC.

[0016] The present invention provides a method for preparing the nano micelle as described above, firstly modifying the carrier precursor of the nano carrier with a targeting peptide, then loading the near-infrared fluorescent dye and the immune adjuvant onto the modified nano carrier to obtain the nano micelle.

[0017] Further, the following steps are included:

[0018] S1, mixing and dissolving the TMTP1 targeting peptide and the carrier precursor material, and after reaction, obtaining the modified nanocarrier;

[0019] S2, mixing ICG, MPLA and cationic materials with the modified nanocarrier, and slowly dripping into the continuously stirred water, centrifuging after continued stirring, and collecting the precipitate;

[0020] S3, dissolving the precipitate collected in step S2 in water, and centrifuging at least once more. After the centrifugation is completed, the precipitate finally collected is the nano micelle.

[0021] Furthermore, the cationic material is poly(β-amino ester).

[0022] Furthermore, in step S2, the mass ratio of ICG, MPLA and poly(β-amino ester) is 1:0.75:2.

[0023] Furthermore, the preparation method of the carrier precursor material is as follows: maleimidobutyric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide are dissolved in dichloromethane and then stirred for activation; polyethylene glycol diamine and triethylamine are added for further activation to obtain a MA-modified PEG solution; PLGA50 / 50-COOH, EDC and NHS are dissolved in DCM and then stirred for activation to obtain a PLGA activated product; the MA-modified PEG solution and the PLGA activated product are mixed, stirred and reacted, and then added dropwise to a mixed solution of ether and methanol to obtain a precipitate, and the precipitate is dried to obtain the carrier precursor material.

[0024] Furthermore, the mass ratio of the TMTP1 targeting peptide to the carrier precursor substance is 1:20.

[0025] The present invention also provides the application of nano micelles, wherein the nano micelles can be used to prepare preparations for treating endogenous tumors, wherein the preparations are drugs, pharmaceutical raw materials, vaccines or targeted inhibitors.

[0026] The present invention also provides a preparation for treating ovarian cancer, comprising the nano micelles as described above.

[0027] The beneficial effects of the present invention are:

[0028] The targeting peptide-modified nanomicelles co-loaded with near-infrared fluorescent dye and immune adjuvant of the present invention can target primary foci and metastatic foci of ovarian cancer, increase the aggregation of micelles in tumor tissues, produce PTT effect by laser irradiation under the guidance of near-infrared fluorescence, induce immune death of tumor cells, release tumor-associated antigens and, with the assistance of immune adjuvants, promote DC cells to recognize and present antigens and activation of DC cells, exert the function of vaccines, recruit macrophages and NK cells, induce T cell immune response, promote the secretion of immune regulatory factors, recognize and kill other tumor cells that have not been irradiated by laser, and produce immune memory to inhibit tumor recurrence.

[0029] The targeting peptide-modified nanomicelles co-loaded with near-infrared fluorescent dye and immune adjuvant of the present invention solve the problems in the prior art that free ICG is unstable in aqueous solution and unencapsulated MPLA is quickly degraded and cleared in vivo, providing a new option for the diagnosis and treatment of ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The preparation flow chart of the targeting peptide-modified nanomicelles co-loaded with near-infrared fluorescent dye and immune adjuvant of the present invention, Example 1;

[0031] Figure 2The nanomicelles modified with the targeting peptide of the present invention and co-loaded with the near-infrared fluorescent dye and the immune adjuvant are the appearance diagrams of the prepared nanomicelles in Example 2;

[0032] Figure 3 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with the near-infrared fluorescent dye and the immune adjuvant, in Example 3, the size distribution diagram of four nanomicelles was measured by dynamic light scattering, Figure 3 A in the middle is Con-IM, Figure 3 B in the middle is TP1-ICG, Figure 3 C in the middle is TP1-MP, Figure 3 D in the middle is TP1-IM;

[0033] Figure 4 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with near-infrared fluorescent dye and immune adjuvant, TEM morphology of four nanomicelles in Example 3, the scale bar is 200 nm; Figure 4 A in the middle is Con-IM, Figure 3 B in the middle is TP1-ICG, Figure 3 C in the middle is TP1-MP, Figure 3 D in the middle is TP1-IM;

[0034] Figure 5 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with near-infrared fluorescent dye and immune adjuvant, in Example 4, are the in vitro photothermal performance test diagrams of the TP1-IM nanomicelles; Figure 5 A in the middle is a representative image of infrared thermal imaging; Figure 5 B is the temperature change diagram within 5 minutes under NIR irradiation;

[0035] Figure 6 The targeting peptide-modified nanomicelles of the present invention co-loaded with near-infrared fluorescent dye and immune adjuvant, in Example 5, the in vitro cellular uptake results of each nanomicelle; Figure 6 A in the middle is the flow cytometry detection result of each nanomicelle at different time. Figure 6 B is the comparison of the average fluorescence intensity of each nanomicelle at different times. Figure 6 C is the electrophoresis diagram of the protein expression level of XPNPEPE in 3T3 cells and ID8 cells detected by western blot experiment;

[0036] Figure 7 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with near-infrared fluorescent dye and immune adjuvant, in Example 5, the combined fluorescence microscopy images of Con-CM and TP1-CM in vitro and 3T3 and ID8 cells, Figure 7 A in the middle is 3T3 cells, Figure 7 Middle B is ID8 cell;

[0037] Figure 8 The targeting peptide-modified nanomicelles of the present invention co-loaded with near-infrared fluorescent dye and immune adjuvant, in Example 6, the results of cell expression of HSP70 and CRT protein in each nanomicelle after NIR laser irradiation, Figure 8 A in the middle is the immunofluorescence staining image of HSP70 protein of ID8 cells after incubation of ID8 cells with each nanomicelle and NIR laser irradiation. Figure 8 B is the average fluorescence intensity of HSP70 protein on the surface of ID8 cells in each nanomicelle. Figure 8 Middle C is the immunofluorescence image of CRT protein of ID8 cells in each nanomicelle. Figure 8 Middle B is the average fluorescence intensity of CRT protein on the surface of ID8 cells in each nanomicelle;

[0038] Fig. 9 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with the near-infrared fluorescent dye and the immune adjuvant, in Example 7, the immune activation of DC in vivo by each nanomicelle under NIR laser irradiation, Fig. 9 A is the flow cytometry detection of mature DCs (CD11c + CD80 + CD86 + ), Fig. 9 B is the quantitative result of the mature proportion of DC cells in TDLNs of tumor-bearing mice after different treatments. Fig. 9 C in the middle is the content of cytokine IL-12p70 in the serum of tumor-bearing mice, Fig. 9 D in the figure is the content of cytokine TNF-α in the serum of tumor-bearing mice. Fig. 9 E in the middle is the content of cytokine IFN-γ in the serum of tumor-bearing mice;

[0039] Fig.10 The nanomicelles modified with the targeting peptide of the present invention and co-loaded with the near-infrared fluorescent dye and the immune adjuvant, in Example 8, each nanomicelle has an inhibitory effect on subcutaneous tumors of ovarian cancer under NIR laser irradiation, Fig.10 A in the middle is the imaging picture of subcutaneous tumor in mice at different time points. Fig.10 Middle B is the tumor volume growth curve of ID8 subcutaneous tumor model mice after different treatments. Fig.10 Survival curve of C mice;

[0040] Fig.11 The targeting peptide-modified nanomicelles of the present invention co-loaded with near-infrared fluorescent dye and immune adjuvant, HE staining image of mouse tumor tissue corresponding to each nanomicelle in Example 8;

[0041] Fig.12The targeting peptide-modified nanomicelles of the present invention co-loaded with near-infrared fluorescent dye and immune adjuvant, in Example 8, are TUNEL staining images of mouse tumor tissues corresponding to each nanomicelle. DETAILED DESCRIPTION

[0042] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0043] The targeting peptide-modified nano micelles co-loaded with near-infrared fluorescent dye and immune adjuvant of the present invention comprises a PLGA-PEG nano carrier, a TMTP1 targeting peptide, an indocyanine green (ICG) near-infrared fluorescent dye and an immune adjuvant.

[0044] The targeting peptide-modified nanomicelles (TP1-IM micelles) co-loaded with near-infrared fluorescent dye and immune adjuvant of the present invention can target primary foci and metastatic foci of ovarian cancer, increase the aggregation of micelles in tumor tissues, produce PTT effect by laser irradiation under the guidance of near-infrared fluorescence, induce immune death of tumor cells, release tumor-associated antigens and, with the assistance of immune adjuvants, promote DC cells to recognize and present antigens and activation of DC cells, exert the function of vaccines, recruit macrophages and NK cells, induce T cell immune response, promote the secretion of immune regulatory factors, recognize and kill other tumor cells that have not been irradiated by laser, and produce immune memory to inhibit tumor recurrence.

[0045] Specifically, the nano-micelles can effectively kill ovarian cancer cells in vitro, induce immunogenic death of ID8 cells, and stimulate BMDCs maturation under NIR laser irradiation. In vivo, they can actively target ID8 subcutaneous tumors, and TP1-IM micelle-mediated photothermal therapy and immunotherapy under NIR laser irradiation can significantly promote the maturation of DCs in tumor-draining lymph nodes, effectively inhibit the growth of ovarian cancer, and prolong the survival of tumor-bearing mice.

[0046] Preferably, the TMTP1 targeting peptide is a cyclic TMTP1 polypeptide, the amino acid sequence of the cyclic TMTP1 polypeptide is NVVRQC, and the cyclic structure thereof is a connection of the head and tail amino acids.

[0047] The method for preparing the nano micelle of the present invention comprises the following steps: firstly modifying the carrier precursor of the nano carrier with a targeting peptide, and then loading a near-infrared fluorescent dye and an immune adjuvant onto the modified nano carrier to obtain the nano micelle.

[0048] The following steps are involved:

[0049] First, a carrier precursor material is prepared. The preparation method is as follows: maleimidobutyric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide are dissolved in dichloromethane and then stirred for activation; polyethylene glycol diamine and triethylamine (NH2-PEG-NH2) are added for further activation to obtain a MA-modified PEG solution; PLGA50 / 50-COOH, EDC and NHS are dissolved in DCM and then stirred for activation to obtain a PLGA activated product; the MA-modified PEG solution and the PLGA activated product are mixed, stirred and reacted, and then added dropwise to a mixed solution of ether and methanol to obtain a precipitate, and the precipitate is dried to obtain the carrier precursor material.

[0050] S1. Mixing and dissolving the TMTP1 targeting peptide and the carrier precursor material, and after reaction, obtaining a modified nanocarrier.

[0051] Preferably, the cyclic TMTP1 polypeptide and MAL-PEG-PLGA are ultrasonically dissolved in N,N-dimethylformamide (DMF) and then mixed, and placed on a shaker at room temperature for 8 hours under light-proof conditions to obtain a cTMTP1 polypeptide-modified cTMTP1-PEG-PLGA reactant solution.

[0052] Further preferably, the mass ratio of cyclic TMTP1 to MAL-PEG-PLGA is 1:20.

[0053] S2. Mix ICG, MPLA and cationic materials with the modified nanocarriers and slowly drip them dropwise into the continuously stirred water. Continue stirring and centrifuge to collect the precipitate.

[0054] Preferably, the cationic material is poly(β-amino ester) (PBAE).

[0055] Preferably, the mass ratio of ICG, MPLA and poly(β-amino ester) is 1:0.75:2.

[0056] Preferably, ICG, MPLA and cationic material PBAE are added to the above-synthesized cTMTP1-PEG-PLGA solution and ultrasonically mixed. The mixture is slowly dripped into the continuously stirred deionized water, and after stirring for 30 minutes, the mixture is poured into a centrifuge tube, and the mixture is centrifuged at 12000 rpm for 30 minutes at 4°C.

[0057] S3, dissolving the precipitate collected in step S2 in water, and centrifuging at least once more. After the centrifugation is completed, the precipitate finally collected is nano micelles.

[0058] Preferably, the centrifugation condition in this step is centrifugation at 12000 rpm for 30 minutes at 4°C.

[0059] The nano micelle of the present invention can be used to prepare a preparation for treating endogenous tumors, and the preparation is a drug, a pharmaceutical raw material, a vaccine or a targeted inhibitor; and provides a new option for the diagnosis and treatment of ovarian cancer.

[0060] The preparation for treating ovarian cancer of the present invention comprises the nano micelle as claimed in claim 1 or 2.

[0061] The present invention is described below by means of specific examples.

[0062] Example 1 Effect of different PBAE and ICG dosages on ICG encapsulation efficiency

[0063] This example specifically investigates the effect of the PBAE / ICG mass ratio on the ICG encapsulation efficiency to determine the optimal PBAE and ICG dosage. Specifically, in this example, MAL-PEG-PLGA is first synthesized, and then the TP1-IM nano-micelles of the present invention are prepared. The preparation process is as follows: Figure 1 As shown, the specific steps are as follows:

[0064] (1) Synthesis of MAL-PEG-PLGA

[0065] 28mg maleimidobutyric acid (MA), 21mg 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) (EDC) and 15.3mg N-hydroxysuccinimide (NHS) were dissolved in 3ml dichloromethane (DCM) and stirred on a magnetic stirrer for activation for 5 hours; 400mg polyethylene glycol diamine (NH2-PEG-NH2) and 16.5μl triethylamine were weighed and added to the above reaction bottle and continued to activate for 5 hours to obtain MA-modified PEG solution. 1.8mg PLGA50 / 50-COOH, 16mg EDC and 12mg NHS were dissolved in 3ml DCM and stirred on a magnetic stirrer for activation for 5 hours to obtain PLGA activated product. The MA-modified PEG solution was mixed with the PLGA activated product and continued to react on a magnetic stirrer for 48 hours. The reactant was dropped into a 5:1 mixture of ether and methanol to precipitate the product MAL-PEG-PLGA, and then the obtained product was dried in a vacuum drying oven for 12 hours.

[0066] (2) Preparation and optimization of TP1-IM nanomicelles

[0067] Weigh 1 mg of cyclic TMTP1 (cyclic-TMTP1, cTMTP1, NVVRQC, the first amino acids are connected to form a cyclic polypeptide) polypeptide and 20 mg of MAL-PEG-PLGA, dissolve them by ultrasonication in 1 ml of N,N-dimethylformamide (DMF) and mix them evenly, place them on a shaker at room temperature for 8 hours under light-proof conditions to obtain a cTMTP1 polypeptide-modified cTMTP1-PEG-PLGA reaction product solution.

[0068] This embodiment specifically adopts the univariate method to determine the optimal experimental conditions for improving the ICG encapsulation efficiency. The dosages of ICG and MPLA are fixed at 200 μg and 150 μg, and the dosages of the cationic material PBAE are set to 0 μg, 200 μg, and 400 μg, respectively (i.e., the PBAE / ICG mass ratios are 0, 1:1, and 2:1).

[0069] 200 μg ICG, 150 μg MPLA and different amounts of cationic material PBAE were added to the above-synthesized cTMTP1-PEG-PLGA solution and ultrasonically mixed. The mixture was slowly dripped into 15 ml of deionized water with continuous stirring, and after stirring for 30 minutes, the mixture was poured into a 50 ml centrifuge tube, and then the mixture was centrifuged at 12000 rpm for 30 minutes at 4 ° C. After centrifugation, the supernatant was retained for the detection of ICG encapsulation efficiency, and the green precipitate at the bottom of the centrifuge tube was ultrasonically dissolved with 15 ml of deionized water, and then centrifuged again at 12000 rpm for 30 minutes at 4 ° C. After centrifugation, the green precipitate at the bottom of the centrifuge tube is the TP1-IM nanomedicine. The precipitate is ultrasonically dissolved with 1 ml of deionized water and stored in a 4°C refrigerator away from light. Some samples are taken to detect the hydrated particle size of the nanomicelles. The optimal experimental conditions for synthesizing TP1-IM nanomicelles are comprehensively determined by detecting the encapsulation rate and hydrated particle size of ICG in the TP1-IM micelles. The test results are shown in Table 1.

[0070] Table 1 Optimization of preparation conditions for TP1-IM nanoparticles

[0071]

[0072] It can be seen from Table 1 that when the mass ratio of PBAE / ICG is 2:1, the ICG coverage rate can reach more than 78%, the hydrated particle size can reach more than 160 nm, and the dispersibility can reach more than 0.1, indicating that this dosage has the best ICG coverage rate.

[0073] According to the above experimental results, when preparing TP1-IM nanomicelles, the mass ratio of PBAE / ICG was specifically selected to be 2:1.

[0074] Example 2 Preparation of comparative nano-micelle materials

[0075] In order to conduct a detailed comparative verification of the specific effects of the TP1-IM nanomicelles of the present invention, this embodiment prepared nanomicelles TMTP1@PEG-PLGA-ICG (TP1-ICG for short) that only encapsulates the photothermal material ICG, nanomicelles TMTP1@PEG-PLGA-MPLA (TP1-MP for short) that only encapsulates the immune adjuvant MPLA, and co-loaded nano-nanomicelles PEG-PLGA-ICG / MPLA (Con-IM for short) whose surface is not modified with the targeting peptide cTMTP1.

[0076] The steps for preparing TP1-ICG and TP1-MP nanomicelles are the same as those for preparing TP1-IM nanomicelles in Example 1. In the process of drug administration, TP1-ICG nanomicelles are administered with 200 μg of ICG and a corresponding mass of cationic material PBAE, while TP1-MP nanomicelles are administered with 150 μg of MPLA.

[0077] The specific preparation process of Con-IM is as follows: first, weigh 20 mg mPEG5000-PLGA50 / 50, dissolve it with 1 ml DMF by ultrasound and mix it. Then add 200 μg ICG, the corresponding mass of cationic material PBAE and 150 μg MPLA to the above solution and mix it by ultrasound. The mixture is slowly dripped into 15 ml of deionized water that is continuously stirred, and stirring is continued for 30 minutes. The remaining steps are the same as the preparation of TP1-IM nano micelles in Example 1.

[0078] The appearance of TP1-IM prepared in Example 1, TP1-ICG, TP1-MP and Con-IM prepared in this example are as follows: Figure 2 shown.

[0079] Example 3 Physicochemical Characterization of Each Nanomicelle

[0080] In this example, the peaks of four nanomicelles, Con-IM, TP1-ICG, TP1-MP and TP1-IM, were measured by dynamic light scattering (DLS). Figure 3 As shown, the morphology of the four nanomicelles was also observed by transmission electron microscope (TEM). Figure 4 The average hydrated particle size, dispersibility, Zeta potential and ICG encapsulation efficiency of each nanomicelle are shown in Table 2.

[0081] according to Figure 3As can be seen from Table 2, the four nanomicelles are all single distribution peaks, and their corresponding average hydrated particle sizes are 157.30±4.80nm, 167.20±8.20nm, 167.50±8.70nm, and 165.50±8.20nm, respectively. The four nanomicelles have good dispersibility, and their corresponding PDIs are 0.06±0.05, 0.12±0.05, 0.06±0.02, and 0.07±0.05, respectively. The zeta potentials of the four nanomicelles Con-IM, TP1-ICG, TP1-MP, and TP1-IM are -18.99±2.47mV, -13.25±1.34mV, -23.97±2.41mV, and -9.93±1.52mV, respectively, and all carry negative charges. In addition, the encapsulation efficiency of ICG in the three nanomicelles, Con-IM, TP1-ICG and TP1-IM, was maintained at about 80%, which was high enough to meet the needs of subsequent tumor imaging and treatment experiments.

[0082] according to Figure 4 It can be seen that the four micelles of Con-IM, TP1-ICG, TP1-MP and TP1-IM all appear as round structures with uniform size and regular morphology under the microscope. The size of the four nanomicelles under transmission electron microscopy is about 100nm, slightly smaller than the hydrated particle size.

[0083] Table 2 Hydration particle size, Zeta potential and ICG encapsulation efficiency of nanomicelles in each group

[0084]

[0085] Example 4 In vitro photothermal performance of TP1-IM nanomicelles

[0086] This example aims to evaluate whether the heat generation performance of ICG is affected after it is encapsulated by PLGA-PEG carrier. Specifically, this example uses an infrared thermal imaging camera to record the heat generation performance of free ICG and three nanomicelles of Con-IM, TP1-ICG, and TP1-IM at the same ICG concentration (10 μg / ml) under NIR irradiation (808 nm, 2 W / cm 2 ) The temperature changes within 5 minutes, the results are as follows Figure 5 shown.

[0087] according to Figure 5 Thermal imaging of A and Figure 5From the temperature change curve in Figure B, it can be seen that the temperature of free ICG and Con-IM, TP1-ICG, and TP1-IM nanomicelles increased rapidly 1 minute after NIR irradiation, and reached 46.6±0.3℃, 49.6±1.5℃, 49.6±0.3℃, and 49.8±1.1℃, respectively, 5 minutes after irradiation, indicating that the heat generation capacity of the nanomicelles encapsulating ICG is slightly better than that of free ICG.

[0088] In contrast, the temperature of PBS, which served as a blank control, increased from 28.5±0.4℃ to 36.5±1.6℃ after 5 minutes of NIR irradiation, an increase of only about 8℃.

[0089] The above results indicate that the ICG-loaded nanomicelles have good photothermal properties and can be used in subsequent photothermal therapy experiments.

[0090] Example 5 In vitro cellular uptake of TP1-IM nanomicelles

[0091] This example specifically tests the in vitro targeting of cells by TP1-IM nanomicelles obtained after linking TMTP1 cyclic polypeptide to PLGA-PEG nanocarriers.

[0092] In order to more conveniently observe the uptake of nanoparticles by cells in vitro, the fluorescent probe coumarin 6 (Cou6) was used instead of the dye ICG to synthesize the control group nanoparticles (Con-CM) encapsulating Cou6 and MPLA and the experimental group nanoparticles (TP1-CM) encapsulating Cou6 and MPLA.

[0093] The cells used in this example are normal mouse embryonic fibroblasts (3T3 cells) and mouse ovarian cancer cells (ID8 cells). The specific steps of the experiment are: 3T3 cells and ID8 cells are incubated with the same concentration (2.5ng / ml) of Con-CM and TP1-CM nanomedicines for 0.5 hours, 1 hour, 2 hours, and 4 hours, and flow cytometry is used to detect the uptake of the two nanomedicines in 3T3 and ID8 cells and the mean fluorescence intensity (MFI). Protein immunoblotting experiments are used to detect the expression level of TMTP1 receptor protein XPNPEPE in 3T3 cells and ID8 cells.

[0094] This example also uses a fluorescence microscope to further visually observe the uptake of nanomedicine in cells and its sub-localization in cells. The specific steps are as follows: 3T3 cells and ID8 cells were incubated with the same concentration (5 ng / ml) of Con-CM and TP1-CM nanomedicines for 2 hours, and then the uptake of nanomedicine by cells was observed under a fluorescence microscope.

[0095] Test results such as Figure 6 and Figure 7 shown. Figure 6 In the table, ns means no statistical difference, *** means P < 0.001, and **** means P < 0.0001. Figure 7 In the blocking treatment group, cTMTP1 cyclic peptide was added for 30 minutes before TP1-CM nanoparticles were added. DAPI was used to locate the cell nucleus, and the scale bar is 20 μm.

[0096] according to Figure 6 As can be seen from A and B, as the incubation time of nanoparticles with cells increases, their uptake by cells is gradually increasing. Under the same incubation time conditions, there is no significant difference in the uptake of Con-CM and TP1-CM nanomedicines in 3T3 cells. However, compared with Con-CM, TP1-CM can be taken up more by ovarian cancer cell ID8, and the uptake of TP1-CM nanomicelles in ID8 cells is greater than that in 3T3 cells. After TP1-CM nanomicelles were incubated with ID8 cells for 2 hours, their uptake in cells was higher, and there was still a significant difference in the uptake of Con-CM nanomedicine in cells compared with the control group.

[0097] according to Figure 6 As can be seen in Figure C, compared with 3T3 cells, the XPNPEP2 protein level was significantly upregulated in ID8 cells, which also indicates that TP1-CM can achieve specific targeting of ID8 cells through ligand-receptor-mediated active binding.

[0098] according to Figure 7 Middle A and Figure 7 As can be seen in Figure B, TP1-CM micelles were taken up more in ID8 cells compared with Con-CM. However, only a small amount of both nanodrugs were taken up in 3T3 cells, and there was no significant difference in the uptake of the two nanodrugs in 3T3 cells. In order to further evaluate whether the uptake of TP1-IM micelles in ID8 cells was mediated by TMTP1 targeting peptide, this study treated the cells with cTMTP1 for 30 minutes before incubating them with TP1-IM nanodrugs. The uptake of TP1-IM nanodrugs in ID8 cells was partially blocked by cTMTP1 peptide. In addition, after merging the cell nucleus image with the green fluorescence images of Con-CM and TP1-CM nanodrugs, it was found that the two nanodrugs were mainly located in the cytoplasm after being taken up by cells.

[0099] The above experimental results of this example show that the TMTP1 targeting peptide-modified PLGA-PEG nanocarrier constructed by the present invention can specifically recognize ID8 cells, and this targeting effect is mediated by the XPEPEP2 receptor, and the nanomedicine is mainly located in the cytoplasm after being taken up by the cells.

[0100] Example 6 TP1-IM nanomicelles induce immunogenic cell death in ID8 cells under NIR laser irradiation

[0101] PTT can induce anti-tumor immune response by inducing immunogenic cell death (ICD). After PTT treatment, necrotic or apoptotic cells will release tumor-associated antigens (TAAs) and express damage-associated molecular patterns (DAMPs). In this process, heat shock protein 70 (HSP70) is translocated from the nucleus to the cell surface, and calreticulin (CRT) is translocated from the endoplasmic reticulum to the cell surface and then binds to DC to exert its immune activation effect.

[0102] Therefore, this example further explored whether TP1-IM nanomicelles can induce ID8 cells to express HSP70 and CRT proteins under NIR laser irradiation.

[0103] The specific experimental process of this embodiment is:

[0104] ID8 cells were incubated with PBS and Con-IM, TP1-ICG, and TP1-IM nanomedicines containing the same ICG concentration (25 μg / ml) for 2 hours and then illuminated under 808 nm laser (2 W / cm 2 ) for 5 minutes. After the cells were cultured for 12 hours, they were stained with HSP70 antibody and CRT antibody, respectively. The expression of HSP70 protein and CRT protein on the cell surface was detected by laser confocal microscopy. The results are shown in Figure 7 shown.

[0105] Figure 8 Middle A and Figure 8 C in the middle shows the immunofluorescence staining of ID8 cells expressing HSP70 and CRT, +L indicates that the group was treated with NIR laser irradiation, and DAPI was used to locate the cell nucleus. The scale bar is 15μm. Figure 8 Medium B and Figure 8 D in the figure are the quantitative analysis results of the mean fluorescence intensity of HSP70 protein and CRT protein on the cell surface, ns means no significant statistical difference, * means P < 0.05, **** means P < 0.0001

[0106] according to Figure 8 Middle A and Figure 8As can be seen in B, after NIR irradiation, there was no obvious HSP70 protein expression on the surface of ID8 cells in the negative control group PBS+L. Compared with the PBS+L control group, the expression of HSP70 protein in the TP1-IM treatment group without NIR laser irradiation did not change significantly. In contrast, the protein expression of HSP70 on the cell surface in the TP1-ICG+L and TP1-IM+L treatment groups after NIR laser irradiation increased significantly. However, the expression of HSP70 in the Con-IM+L group after NIR laser irradiation only increased slightly, which was attributed to the low uptake of Con-IM+L nanoparticles in cells, and also indirectly verified the targeting of TMTP1 targeting peptide-modified nanomedicines to tumor cells.

[0107] according to Figure 8 Middle C and Figure 8 As can be seen in D, consistent with the results of the changes in HSP70 protein on the cell surface of the above-mentioned treatment groups, after NIR irradiation, there was no obvious CRT protein expression on the surface of ID8 cells in the negative control group PBS+L group. Compared with the PBS+L control group, the expression of CRT protein in the TP1-IM treatment group without NIR laser irradiation did not change significantly. In contrast, the protein expression of CRT on the cell surface of the TP1-ICG+L and TP1-IM+L treatment groups after NIR laser irradiation increased significantly. However, the expression of HSP70 in the Con-IM+L group after NIR laser irradiation only increased slightly.

[0108] The above experimental results of this example show that TP1-IM nanomicelles can effectively induce ICD in ID8 cells under NIR laser irradiation, laying a foundation for promoting DC maturation.

[0109] Example 7 TP1-IM nanomicelles promote DC immune activation in vivo under NIR laser irradiation

[0110] Based on Example 6, this example further evaluated whether TP1-IM nanomicelles could promote immune activation of DCs in tumor draining lymph nodes (TDLNs) and secretion of cytokines in mouse serum under NIR laser irradiation.

[0111] In this example, tumor-bearing mice were used for the experiment. The specific experimental process was as follows: when the tumor volume of ID8 subcutaneous tumor model mice grew to 100 mm 3 At about 14 h, PBS, Con-IM, TP1-ICG and TP1-IM nanomedicine aqueous solutions were injected into the tail vein, respectively. Twelve hours later, the tumor site was illuminated by 808 nm laser (2 W / cm 2) for 5 minutes. The mice were divided into the following five groups according to the drugs given and whether they received laser irradiation: PBS+L, Con-IM+L, TP1-ICG+L, TP1-IM, TP1-IM+L, +L means receiving NIR laser irradiation. TDLNs were isolated 3 days after NIR laser irradiation, and mature DCs (CD11c + CD80 + CD86 + ) ratio, and also determined the DC cell maturation ratio in TDLNs of tumor-bearing mice after different treatments, and used CBA method to detect the content of cytokines IL-12p70, TNF-α and IFN-γ in the serum of tumor-bearing mice after different treatments. Fig. 9 As shown, Fig. 9 In the table, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

[0112] according to Fig. 9 Middle A and Fig. 9 As can be seen in B, compared with the PBS+L group, the Con-IM+L group only slightly increased the proportion of DC maturation, while the TP1-ICG+L and TP1-IM treatment groups could significantly promote the maturation of DC in TDLNs, indicating that effective photothermal therapy and immune adjuvants can induce anti-tumor immune responses in vivo. It is worth noting that the proportion of DC maturation promoted by TP1-IM+L treatment was significantly better than that of the photothermal therapy group TP1-ICG+L and the immune adjuvant group TP1-IM.

[0113] according to Fig. 9 As can be seen from C to E, compared with the PBS+L group, the Con-IM+L group only slightly increased the cytokines IL-12p70, TNF-α and IFN-γ, while the TP1-ICG+L and TP1-IM treatment groups were able to significantly promote the increase of these three cytokines in mouse serum. It is worth noting that the proportion of cytokine increase in the TP1-IM+L treatment group was significantly better than that in the TP1-ICG+L and TP1-IM groups.

[0114] The above experimental results of this example show that in tumor-bearing mice, TP1-IM nano-micelles can cooperate with photothermal therapy and immune adjuvant-mediated immune activation under NIR laser irradiation, and effectively stimulate the body to produce anti-tumor immune response.

[0115] Example 8 Inhibitory effect of TP1-IM nanomicelles on subcutaneous tumors of ovarian cancer under NIR laser irradiation

[0116] Considering that TP1-IM nano-micelles can cooperate with photothermal therapy and immunotherapy to induce a strong immune response under NIR laser irradiation, this example further investigated whether TP1-IM nano-micelles can inhibit the growth of subcutaneous tumors of ovarian cancer under NIR laser irradiation.

[0117] The specific experimental process of this embodiment is as follows: when the tumor volume of ID8 subcutaneous tumor model mice grows to 50-100 mm 3 Around 24 hours after administration, PBS, Con-IM, TP1-ICG and TP1-IM nanowater solution were injected into the tail vein. Twelve hours after administration, the tumor site of the mice was illuminated by 808 nm laser (2 W / cm 2 ) for 5 minutes. The experiment was divided into the following five treatment groups: PBS+L, Con-IM+L, TP1-ICG+L, TP1-IM, TP1-IM+L, +L means that the tumor site of the mouse was treated with NIR laser irradiation. The day of NIR laser irradiation was recorded as day 0, and a small animal imaging device was used to take pictures to detect the killing of tumor cells by PTT in vivo. Thereafter, pictures were taken every 14 days to detect the tumor growth of each group, and the survival status of the mice was recorded. The experimental results are shown in the figure. Fig.10 shown. Fig.10 A in the middle is the mouse tumor imaging at different time points. Fig.10 Middle B is the tumor volume growth curve of ID8 subcutaneous tumor model mice after different treatments.

[0118] In addition, since effective tumor elimination is an important prerequisite for inhibiting tumor progression, this example also observed the survival of the mice in the treatment group for 90 days. Fig.10 As shown in C.

[0119] according to Fig.10 As can be seen in A, 2 days after NIR irradiation, the fluorescence signal of the tumor site in the TP1-ICG+L and TP1-IM+L treatment groups was significantly reduced, indicating that PTT mediated by TP1-ICG and TP1-IM can quickly and effectively kill ID8 subcutaneous tumors. From the bioluminescence imaging results at different time points, it can be observed that compared with the PBS+L control group, the other treatment groups can all show a certain inhibitory effect on tumor growth. However, the inhibitory effect of the TP1-IM+L treatment group on tumor growth was significantly better than that of the Con-IM+L group, the single photothermal therapy group TP1-ICG+L, and the immune adjuvant group TP1-IM.

[0120] according to Fig.10As can be seen in B, compared with the PBS+L control group, the Con-IM+L treatment group, the single photothermal treatment group TP1-ICG+L and the immune adjuvant group TP1-IM can all show a certain inhibitory effect on tumor growth, but none of them can completely inhibit tumor growth. Compared with these treatment groups, the TP1-IM+L treatment group can almost completely eliminate the tumor 6 days after NIR laser irradiation, and this tumor inhibition effect lasts until 27 days after NIR irradiation treatment. Unfortunately, starting from 27 days after NIR irradiation treatment, the growth of the tumor site of the mice in the TP1-IM+L treatment group began to rebound.

[0121] according to Fig.10 As shown in C, compared with the control group PBS+L, the photothermal therapy combined with immunotherapy mediated by the TP1-IM+L treatment group can significantly prolong the survival of ID8 subcutaneous tumor model mice, while the Con-IM+L treatment group, the single photothermal therapy group TP1-ICG+L and the immune adjuvant group TP1-IM can not significantly prolong the survival of tumor-bearing mice.

[0122] In order to further visually observe the tumor killing effect induced by TP1-IM+L treatment in vivo, this example randomly selected three tumor-bearing mice from each group on the 6th day after NIR laser irradiation treatment, and the mice were killed and their tumor tissues were separated for hematoxylin-eosin (HE) staining and TUNEL staining analysis. The results are shown in Figure 2. Fig.11 and Fig.12 As shown, compared with the other treatment groups, HE staining of the TP1-ICG+L and TP1-IM+L treatment groups showed a large range of tumor cell nuclear condensation and fragmentation in the tumor tissues, while TUNEL staining showed a significant increase in apoptosis and cells in the tumor tissues.

[0123] The above experimental results of this example show that the photothermal therapy mediated by TP1-IM nanoparticles has a rapid and obvious killing effect on tumor cells, and it can cooperate with the immune effect induced by the immune adjuvant MPLA encapsulated in the carrier to effectively inhibit the growth of ovarian cancer.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A targeting peptide-modified nanomicelle co-loaded with a near-infrared fluorescent dye and an immune adjuvant, characterized in that: The nanocarrier of the nanomicelle is a PLGA-PEG nanocarrier, the targeting peptide is a TMTP1 targeting peptide, the near-infrared fluorescent dye is indocyanine green, and the immune adjuvant is MPLA.

2. The targeting peptide-modified nanomicelles co-loaded with near-infrared fluorescent dye and immune adjuvant according to claim 1, characterized in that: The TMTP1 targeting peptide is a cyclic TMTP1 polypeptide, and the amino acid sequence of the cyclic TMTP1 polypeptide is NVVRQC.

3. A method for preparing nano micelles as claimed in claim 1 or 2, characterized in that: The carrier precursor material of the nanocarrier is firstly modified with a targeting peptide, and then the near-infrared fluorescent dye and the immune adjuvant are loaded onto the modified nanocarrier to obtain the nanomicelle.

4. The method for preparing nano micelles according to claim 3, characterized in that: The following steps are involved: S1, mixing and dissolving the TMTP1 targeting peptide and the carrier precursor material, and after reaction, obtaining the modified nanocarrier; S2, mixing ICG, MPLA and cationic materials with the modified nanocarrier, and slowly dripping into the continuously stirred water, centrifuging after continued stirring, and collecting the precipitate; S3, dissolving the precipitate collected in step S2 in water, and centrifuging at least once more. After the centrifugation is completed, the precipitate finally collected is the nano micelle.

5. The method for preparing nano micelles according to claim 4, characterized in that: The cationic material is a poly(β-amino ester).

6. The method for preparing nano micelles according to claim 5, characterized in that: In step S2, the mass ratio of ICG, MPLA and poly(β-amino ester) is 1:0.75:

2.

7. The method for preparing nano micelles according to claim 5, characterized in that: The preparation method of the carrier precursor material is as follows: dissolving maleimidobutyric acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide in dichloromethane and stirring to activate; adding polyethylene glycol diamine and triethylamine to continue activation to obtain a MA-modified PEG solution; PLGA50 / 50-COOH, EDC and NHS are dissolved in DCM and then stirred for activation to obtain a PLGA activated product; the MA-modified PEG solution and the PLGA activated product are mixed, stirred and reacted, and then added dropwise to a diethyl ether and methanol mixed solution to obtain a precipitate, and the precipitate is dried to obtain the carrier precursor material.

8. The method for preparing nano micelles according to claim 7, characterized in that: The mass ratio of the TMTP1 targeting peptide to the carrier precursor material is 1:

20.

9. The use of the nano micelles according to claim 1 or 2, characterized in that: The nano micelles can be used to prepare preparations for treating endogenous tumors, and the preparations are drugs, pharmaceutical raw materials, vaccines or targeted inhibitors.

10. A preparation for treating ovarian cancer, characterized in that: Comprising the nano micelles as described in claim 1 or 2.

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