Tumor-targeted anti-tumor nano-drug HPCR as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological pharmacy, and relates to an anti-tumor nano-drug, in particular to a tumor-targeted anti-tumor nano-drug HPCR as well as a preparation method and application thereof. An immune checkpoint inhibitor anti-PD-L1, a chemotactic factor CCL5 and rutin with an ACE2 activation effect are respectively grafted to HA through a chemical synthesis method to synthesize HA-anti-PD-L1, HA-CCL5 and HA-RT, then the HA-anti-PD-L1, HA-CCL5 and HA-RT are synthesized into the anti-tumor nano-drug HPCR through a film hydration method, and in-vivo experiments prove that the drug has an excellent anti-tumor effect; the medicine has good T cell activation effect, T cell recruitment effect, tumor matrix elimination, T cell infiltration increase and medicine permeation effects, and provides a new method and a new thought for tumor treatment.

Description

Technical Field

[0001] The invention belongs to the field of biopharmaceuticals and relates to the preparation of anti-tumor nanomedicines. Background Art

[0002] Cancer is a major global public health issue that poses a serious threat to human health. The incidence and mortality of malignant tumors are increasing worldwide. According to the latest data from the Global Cancer Report, the global cancer incidence rate was 247.5 / 100,000 in 2020, and the mortality rate was 127.8 / 100,000. The cancer incidence rate in China reached 293.91 / 100,000, and the mortality rate reached 174.55 / 100,000, which are significantly higher than the average global cancer incidence and mortality rates. Cancer has become an important public health issue affecting the health of Chinese residents.

[0003] At present, surgery, radiotherapy, chemotherapy and molecular targeted drugs are the main means of treating cancer. Surgery is effective in treating tumors and is also the preferred treatment for most early tumors. However, surgery is risky, especially for tumors in sensitive areas, which is dangerous and has a low success rate. Radiotherapy, as an important method for local treatment of tumors, directly kills tumor cells by inducing DNA damage in tumor cells, which can reduce the activity of cancer tissues and shrink local lesions. However, radiotherapy is a double-edged sword. While killing tumor cells, it also causes damage to normal tissues, which affects the patient's prognosis to a certain extent. Chemotherapy is a systemic treatment that can prolong the survival time of most patients, but conventional chemotherapy kills the body's normal proliferating and differentiating cells, inhibits the immune function of patients with malignant tumors, and affects the patient's long-term survival rate and resistance to other diseases. Molecular targeted drugs can selectively act on key molecules in the process of tumorigenesis, inhibit tumor growth, have the advantages of high efficiency and low toxicity, and are the mainstream of new tumor drug research and development in recent years. Among them, immune targeted therapy, including immune checkpoint molecule inhibitors, vaccines, oncolytic virus therapy, chimeric antigen receptor T cell therapy, etc., has developed rapidly. To date, immune checkpoint inhibitor (ICI) drugs, such as anti-cytotoxic T lymphocyte-associated protein 4 and anti-programmed cell death-protein 1 (PD-1), have achieved considerable success in many cancer immunotherapies, including melanoma, lung cancer, and other commonly diagnosed cancers. Therefore, ICI-based treatment strategies are supported in cancer research and treatment.

[0004] PD-1 and its ligand PD-L1 are the main immune checkpoint proteins. As an immune checkpoint inhibitor, the specific mechanism of PD-L1 antibody is to reduce the suppression of the immune system by blocking the binding of ligands and immune checkpoint molecules. However, more and more studies have shown that the positive response rate of patients treated with immune checkpoint targeted drugs is still low, which is an issue to be resolved. More and more evidence strongly suggests that the tumor immune microenvironment (TIME) plays a more important role in tumor immune surveillance and immunity than ICI. One of the main factors limiting the effect of ICI treatment is the lack of pre-existing T cell infiltration in the tumor microenvironment. Therefore, improving T cell activity and strengthening T cell infiltration can greatly enhance the effect of anti-PD-L1 treatment. The trafficking and localization of immune cells are regulated by chemokine-chemokine receptor interactions. Chemokines can attract innate and adaptive immune cells to tumor sites. CCL5 is an inflammatory chemokine that promotes immune cell chemotaxis by interacting with CCR1 and / or CCR5 on the surface of activated T cells. Its expression in the tumor microenvironment is important for T cell infiltration into tumors. Therefore, increasing the content of CCL5 in the tumor microenvironment can enhance the anti-tumor effect of anti-PD-L1.

[0005] However, stromal cells have been shown to suppress the immune system by inhibiting T cell trafficking and function through direct and indirect mechanisms. For example, tumor stroma, including cancer-associated fibroblasts (CAFs), can inhibit T cell activation and T cell infiltration. In addition, excessive collagen and fibronectin in the extracellular matrix (ECM) construct a natural protective physical barrier that hinders the effective delivery of drugs or T cells, leading to immunosuppression. In addition, hypoperfusion caused by mechanical forces within the tumor leads to vascular compression, resulting in a decrease in the number of immune cells infiltrating the tumor. Therefore, depletion of dense matrix to increase immune cell infiltration and drug penetration is of great significance for tumor suppression. The renin-angiotensin system (RAS) plays an important role in the regulation and production of extracellular matrix components. RASi (RAS inhibitors) treatment leads to a decrease in type I collagen (collagenI) and smooth muscle actin (α-smooth muscle actin, α-SMA). RASi has shown anti-tumor effects and has a profound impact on cancer treatment. Similar to the effects of RASi, in many cases, ACE2 (angiotensin-converting enzyme 2) / Ang-(1-7) (angiotensin-(1-7)) / MAS plays a role in counteracting or regulating the classical RAS axis. A study reported that Ang-(1-7) as an important downstream mediator of ACE2 exerts anti-tumor activity in preclinical models (Khanna P, SohHJ, Chen CH, et al. ACE2 abrogates tumor resistance to VEGFRinhibitorssuggesting angiotensin-(1-7) as a therapy for clear cell renal cell carcinoma. Sci Transl Med. 2021 Jan 20;13(577):eabc0170.). Studies have shown that in preclinical cancer models such as non-small cell lung cancer and clear cell renal cell carcinoma, increased ACE2 content can prevent tumor growth and epithelial-mesenchymal transition. Rutin (RT) is a natural flavonoid from plant sources with multiple pharmacological activities, including anti-inflammatory, antioxidant, antiviral, antitumor and immunomodulatory effects.Recent studies have shown that RT has a strong affinity for ACE2, and experiments have confirmed that RT can activate ACE2 / Ang1-7 to protect against cerebral ischemia (Zhao T, He F, Zhao K, et al. A Triple-Targeted Rutin-Based Self-Assembled Delivery Vector for Treating Ischemic Stroke by Vascular Normalization and Anti-Inflammation via ACE2 / Ang1-7 Signaling. ACS Cent Sci. 2023 Jun 5;9(6):1180-1199.). However, whether rutin can reduce the extracellular matrix to promote T cell infiltration and enhance the anti-PD-L1 anti-tumor effect remains to be verified.

[0006] As a natural mucopolysaccharide, hyaluronic acid (HA) has been widely used in medicine, cosmetics and food. Due to its excellent biodegradability, non-toxicity and non-immunogenicity, it has been widely studied in the delivery of anticancer drugs. Conjugating HA with other drugs can improve the solubility, stability and efficacy of drugs. Studies have found that the specific receptor CD44 of HA is highly expressed on the surface of various cancer cells and special pathological areas. Using HA as a carrier, more drugs can be delivered to cancer cells at the same drug concentration level, thereby producing a stronger cell killing effect. In summary, using HA to target the overexpression of CD44 receptors in tumor sites, achieving tumor enrichment of drugs, and acting on tumor cells through multiple mechanisms to improve the therapeutic effect is a current research hotspot, and also provides new methods and new ideas for tumor treatment. Summary of the invention

[0007] In order to solve the above problems, the present invention proposes a tumor-targeted anti-tumor nanomedicine HPCR and a preparation method and application thereof.

[0008] The technical solution of the present invention is achieved in this way: On the one hand, the present invention provides a method for preparing a tumor-targeted anti-tumor nanomedicine HPCR, the steps of which are as follows: (1) Add an activator to the HA aqueous solution for activation, then add an anti-PD-L1 solution, where anti-PD-L1 is an immune checkpoint inhibitor, and react to completion under argon protection. The reaction solution is dialyzed and filtered to obtain the product HA-anti-PD-L1; (2) Adding an activator to the HA aqueous solution for activation, and then adding CCL5 solution. CCL5 is an inflammatory chemokine. Increasing the content of CCL5 can improve the anti-tumor effect of anti-PD-L1. The reaction is completed under argon protection. The reaction solution is dialyzed and filtered to obtain the product HA-CCL5; (3) Add an activator to the HA aqueous solution for activation, and then add RT solution. RT has an ACE2 activation effect. The increase in ACE2 content can prevent tumor growth. The reaction is completed under argon protection. The reaction solution is dialyzed and filtered to obtain the product HA-RT; (4) Prepare HPCR by thin film hydration method: add the above-mentioned products HA-anti-PD-L1, product HA-CCL5 and product HA-RT into solvent and completely dissolve them, remove the organic solvent by rotary evaporation, hydrate, filter and ultrasonically extrude to obtain nano drug HPCR.

[0009] Preferably, the molar ratio of HA, activator, and anti-PD-L1 in the above step (1) is 1:1.2-1.5:1.3×10 -4 -1.5×10 -4 ; In step (2), the molar ratio of HA, activator, and CCL5 is 1:1.2-1.5:2.5×10 -5 -4.0×10 -5 ; In step (3), the molar ratio of HA, activator and RT is 1:1.2-1.5:3-5.

[0010] Preferably, in the above step (4), the molar ratio of HA-anti-PD-L1, HA-CCL5 and HA-TR is 50-60:80-90:1, and the solvent is dichloromethane, which is used to dissolve HA-anti-PD-L1, HA-CCL5 and HA-TR.

[0011] Preferably, the activator is EDC‧HCl, which is used to activate the carboxyl group of HA for 2-4 h.

[0012] Preferably, the dialysate for the dialysis is double distilled water, and the diameter of the microporous filter membrane for filtration is 0.45 μm.

[0013] In the second aspect, the anti-tumor nanomedicine HPCR is prepared by the above preparation method.

[0014] Thirdly, the application of the above-mentioned anti-tumor nanodrug HPCR in inhibiting the growth of H22 liver cancer transplanted tumors.

[0015] Fourthly, the above-mentioned anti-tumor nanomedicine HPCR is used in activating T cells, recruiting T cells and increasing T cell infiltration.

[0016] Fifthly, the above-mentioned anti-tumor nanomedicine HPCR is used to eliminate tumor cell extracellular matrix and increase drug penetration.

[0017] The sixth aspect is the application of the above-mentioned anti-tumor nanomedicine HPCR in anti-tumor.

[0018] Preferably, the concentration of the anti-tumor nano drug HPCR in the above application is 80-90 mg / kg.

[0019] The present invention has the following beneficial effects: 1. In the present application, anti-PD-L1, CCL5 and RT are grafted onto HA by a chemical synthesis method to synthesize HA-anti-PD-L1, HA-CCL5 and HA-RT, and then the anti-tumor nanodrug HPCR is prepared by a thin film hydration method. In vivo experiments have shown that the compound has a good tumor inhibitory effect; it has good functions of promoting T cell recruitment, eliminating tumor matrix, and reversing the tumor immunosuppressive microenvironment.

[0020] 2. This application established a BALB / c male mouse model of H22 liver cancer transplanted tumors, and combined the prepared anti-tumor nanodrug HPCR with anti-OX40 antibodies for treatment. It was confirmed that HPCR+anti-OX40 has excellent anti-tumor effects and significantly inhibits the growth of H22 liver cancer transplanted tumors in BALB / c male mice. In addition, HPCR has good tolerance and safety in mice.

[0021] 3. Flow cytometry, immunofluorescence and immunohistochemistry were used to detect the T cell content in tumor tissue. After HPCR+anti-OX40 treatment, CD8 + T cells were significantly upregulated compared with CD8 + The T cell count was 2.60 times higher, confirming that HPCR has T cell recruitment and activation effects, increasing T cell infiltration, achieving effective relief of immunosuppression and enhancing anti-tumor immune response.

[0022] 4. Immunohistochemistry was used to detect the expression of ACE2 in tumor tissues. It was observed that the expression of ACE2 protein and CCL5 protein in the HPCR group was significantly upregulated compared with the M group and HPC group. The levels of type I collagen and α-SMA protein in matrix-rich tumors and the level of tumor PD-L1 protein were significantly downregulated, confirming that HPCR regulates the extracellular matrix of tumor tissue by activating ACE2 and enhancing the drug penetration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 It is the particle size potential statistical diagram of HPCR; Figure a is the particle size statistical diagram of HPCR, and Figure b is the potential statistical diagram of HPCR.

[0025] Figure 2 TEM image of HPCR.

[0026] Figure 3 Figure 5 is a statistical graph of the stability of HPCR; Figure a is a statistical graph of the particle size stability of HPCR in PBS (4°C), Figure b is a statistical graph of the potential stability of HPCR in PBS (4°C), Figure c is a statistical graph of the particle size stability of HPCR in PBS containing 10% FBS (37°C), and Figure d is a statistical graph of the potential stability of HPCR in PBS containing 10% FBS (37°C).

[0027] Figure 4 This is the release curve of RT in HPCR.

[0028] Figure 5 Statistical graphs of tumor volume, tumor weight and mouse body weight in the H22 liver cancer transplant tumor model treated with HPCR+anti-OX40; Figure a is the statistical graph of the average tumor volume of each group, Figure b is the statistical graph of the tumor volume of each mouse in each group, Figure c is the statistical graph of the average tumor weight of each group, and Figure d is the statistical graph of the weight of mice in each group.

[0029] Figure 6 These are the detection results of ACE2 protein expression in tumor tissue and indicators of tumor extracellular matrix levels; Figure a is a representative image and positive statistical graph of ACE2 immunohistochemistry in tumor tissue, Figure b is a representative image and positive statistical graph of type I collagen immunohistochemistry in tumor tissue, and Figure c is a representative image and positive statistical graph of α-SMA immunofluorescence in tumor tissue.

[0030] Figure 7 These are the detection results of CCL5 and PD-L1 protein expression in tumor tissues; Figure a is a protein immunoblot image of CCL5 and PD-L1 in tumor tissues, and Figure b is a statistical graph of protein expression of CCL5 and PD-L1 in tumor tissues.

[0031] Figure 8These are the results of T cell content detection in tumor tissues; Figure a is a representative flow cytometry graph of the percentage of T cells in tumor tissues, Figure b is a statistical graph of the percentage of T cells in tumor tissues, Figure c is a representative image of CD8 immunofluorescence in tumor tissues, Figure d is a representative image of CD8 immunohistochemistry in tumor tissues, and Figure e is a positive statistical graph of CD8 immunohistochemistry in tumor tissues. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following experimental examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0034] The main experimental reagents, instruments, equipment, animals and consumables used in the present invention are introduced as follows: Hyaluronic acid was purchased from Shandong Freda Company and used without further purification. All other reagents in the synthesis process were purchased from Aladdin. H22 cell line was purchased from Shanghai Institute of Cell Biology, and BALB / c male mice were purchased from Henan Experimental Animal Center. All experimental protocols were reported to Henan University and approved by the Ethics Committee and Experimental Animal Welfare Committee of Henan University School of Medicine.

[0035] The anti-PD-L1 antibody and anti-OX40 agonistic antibody were purchased from Bio X Cell; Collagenase IV, hyaluronidase, deoxyribonuclease type I, and percoll cell separation medium were purchased from Coolaber; CD16 / 32 antibody, CD3 antibody, CD4 antibody, and CD8 antibody were purchased from Elabscience and Bioss; The embedding instruments were purchased from Shitai; The constant temperature box was purchased from Shanghai Jinghong; High-speed low-temperature desktop centrifuge was purchased from Thermo Fisher Scientific; The flow cytometer was purchased from Beckman; The paraffin rotary slicer was purchased from Leica Microsystems, Germany; The remaining reagents, drugs, etc. not specified are all analytical grade products commonly used in laboratories and will not be described in detail.

[0036] Example 1 A method for preparing an anti-tumor nano drug HPCR, the steps are as follows: (1) First, HA (37.9 mg, 0.1 mmol) with a molecular weight of 98 KDa, EDC·HCl (23.7 mg, 0.12 mmol), and NHS (14.2 mg, 0.12 mmol) were dissolved in 5 mL H 2 O and then stirred at 25 °C for 2 h to activate the carboxyl groups of HA. 2 A solution containing anti-PD-L1 (2 mg, 13.2 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the 1 mL TEA solution. The reaction was continued for 48 h in argon. Afterwards, the 1000 Da dialysis bag was used to dialyze the H 2 The desired HA-anti-PD-L1 white powder product was obtained by freeze drying.

[0037] (2) HA (37.9 mg, 0.1 mmol), EDC·HCl (23.7 mg, 0.12 mmol) and NHS (14.2 mg, 0.12 mmol) were dissolved in 5 mL H 2 O and then stirred at 25 °C for 2 h to activate the carboxyl groups of HA. 2 A solution containing CCL5 (10 μg, 2.5 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the HO solution, and then 1 mL of TEA was added dropwise. The reaction was continued for 48 h under argon. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze the H 2 The desired HA-CCL5 white powder product was obtained by freeze drying.

[0038] (3) HA (37.9 mg, 0.1 mmol), EDC·HCl (23.7 mg, 0.12 mmol) and NHS (14.2 mg, 0.12 mmol) were dissolved in 5 mL H 2 O, and then stirred at 25 °C for 2 h to activate the carboxyl groups of HA. Subsequently, RT (183.2 mg, 0.3 mmol) was dissolved in 0.5 mL DMF. It was precooled in an ice bath for 10 min. The RT solution was added dropwise to the HA solution and reacted for 12 h under argon protection. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze H 2O was dialyzed. The desired HA-RT bright yellow powder product was obtained by freeze drying. The loading efficiency of anti-PD-L1 detected by BCA protein assay was 60%, the loading efficiency of CCL5 detected by ELISA experimental method was 37%, and the grafting rate of RT was 34%.

[0039] (4) Prepare HPCR by thin film hydration method. Briefly, HA-anti-PD-L1, HA-CCL5 and HA-RT were placed in a round-bottom flask at a molar ratio of 50:81:1. An appropriate amount of dichloromethane was added and ultrasonic treatment was performed for 5 minutes until completely dissolved. The organic solvent was removed by rotary evaporation, and a certain amount of distilled water was added at a certain temperature for hydration. After hydration, the volume was measured after cooling to room temperature. The solution was passed through a 0.45 μm membrane filter and extruded after ultrasonic treatment for 5 minutes to obtain the target HPCR nanoparticles.

[0040] Evaluation of various characteristics of HPCR: The particle size distribution of nanoparticles was observed by dynamic light scattering (DLS). Figure 1 As shown in Figure a, the particle sizes of HPC and HPCR were approximately 226.9 ± 3.3 nm and 239.2 ± 3.3 nm, respectively. HPC and HPCR nanoparticles showed low polydispersity indices (PDI < 0.100) of 0.035 and 0.006, respectively. These results indicate that the particle distribution is relatively uniform, which meets the requirements for drug delivery. In addition, the particle size of the HPCR group increased slightly compared with the HPC group, which may be due to the successful loading of RT. Figure 1 As shown in b, the Zeta potential results show that both HPC and HPCR surfaces have negative charges, measured at approximately -7.56 ± 0.25 and -8.29 ± 0.41, respectively. These negative charges may originate from the carboxyl groups of HA, making both HPC and HPCR negatively charged. In vivo, since most proteins are negatively charged, nanoparticles with negative surface charges may reduce nonspecific protein adsorption when interacting with blood components. This property helps to enhance the stability of nanoparticles in the bloodstream and increase the effective drug concentration. In addition, we used transmission electron microscopy to further observe the morphology of the nanoparticles. As Figure 2 As shown, these nanoparticles showed a spherical structure, were non-adhesive, and displayed relatively uniform size. The storage stability of drugs and their stability in blood circulation are crucial for the widespread application of nano-drug delivery systems. In this study, we measured the size changes of HPC and HPCR nanoparticles in PBS (4°C) and PBS containing 10% FBS (37°C) to investigate their stability. Figure 3As shown in the figure, after 48 h of culture, the nanoparticles did not show significant size changes under physiological conditions at 4°C and 37°C. After incubation in 10% FBS-containing PBS for 14 days, the size and Zeta potential of the nanoparticles did not show significant changes, indicating that these nanoparticles have good storage stability and remain unchanged in the blood circulation, thereby improving the drug delivery efficiency.

[0041] In tumor treatment, the intelligent controlled release of nanomedicine can increase the effective content of drugs in cancer tissues and reduce the toxic side effects on normal tissues. Since the tumor microenvironment is weakly acidic, has an abnormal redox state, and contains a large amount of HAase, this experiment explored the multiple biological response release characteristics of HA-RT in buffers under different conditions.

[0042] First, the in vitro release of HA-RT was evaluated in buffers with different pH values ​​(7.4, 6.8, and 5.5). Figure 4 As shown in a, in a buffer solution with a pH of 7.4, the cumulative release of RT in HA-RT within 96 h was only 15.2%, indicating that HA-RT retained most of the loaded drugs in the neutral medium. However, as the pH value of the buffer decreased, the release rate of RT from the nanoparticles significantly accelerated. In particular, after 96 h of incubation, in a buffer solution with a pH of 5.5, the cumulative release of RT in HA-RT reached 32.4%, which was significantly higher than that in the physiological environment. These results indicate that HA-RT exhibits pH-responsive release characteristics. In addition, HA can be degraded by HAase, which is present in large quantities in the tumor microenvironment, so HA-functionalized nanoparticles can be partially destroyed due to HA degradation, allowing the loaded drugs to be released. As Figure 4 As shown in Figure b, the release of RT from HA-RT in pH 7.4 buffer with or without HAase (50 IU / mL) is shown. With the addition of HAase, the cumulative release of RT increased significantly. In particular, after incubation in pH 7.4 buffer containing HAase for 96 h, the cumulative release of RT from HA-RT increased from 15.2% to 40%. These results indicate that HA-RT exhibits HAase-responsive release characteristics, and the effect of HAase-responsive release characteristics on in vivo release is greater than that of pH-responsive release characteristics. In particular, after incubation in pH 5.5 buffer containing HAase for 96 h, the cumulative release of RT from HA-RT increased from 15.2% to 60.1%. The above results indicate that both acidic media and HAase can induce rapid release of RT from HA-RT within 96 h. It is demonstrated that multiple stimulations have a synergistic effect on the release of drugs from HA-RT, which is beneficial to reduce the nonspecific release of drugs, thereby allowing most drugs to be accurately released at the tumor site.

[0043] Example 2 A method for preparing an anti-tumor nano drug HPCR, the steps are as follows: (1) First, HA (37.9 mg, 0.1 mmol) with a molecular weight of 98 KDa, EDC·HCl (29.625 mg, 0.15 mmol), and NHS (17.75 mg, 0.15 mmol) were dissolved in 5 mL of H 2 O and then stirred at 25 °C for 4 h to activate the carboxyl groups of HA. 2 A solution containing anti-PD-L1 (2.27 mg, 15 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the 1 mL TEA solution. The reaction was continued for 48 h in argon. Afterwards, the 1000 Da dialysis bag was used to dialyze the H 2 The desired HA-anti-PD-L1 white powder product was obtained by freeze drying.

[0044] (2) HA (37.9 mg, 0.1 mmol), EDC·HCl (29.625 mg, 0.15 mmol) and NHS (17.75 mg, 0.15 mmol) were dissolved in 5 mL H 2 O and then stirred at 25 °C for 4 h to activate the carboxyl groups of HA. 2 A solution containing CCL5 (16 μg, 4 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the 2% HO solution, and then 1 mL of TEA was added dropwise. The reaction was continued for 48 h under argon. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze the H 2 The desired HA-CCL5 white powder product was obtained by freeze drying.

[0045] (3) HA (37.9 mg, 0.1 mmol), EDC·HCl (29.625 mg, 0.15 mmol) and NHS (17.75 mg, 0.15 mmol) were dissolved in 5 mL H 2 O, and then stirred at 25 °C for 4 h to activate the carboxyl groups of HA. Subsequently, RT (305.33 mg, 0.5 mmol) was dissolved in 0.5 mL DMF. It was precooled in an ice bath for 10 min. The RT solution was added dropwise to the HA solution and reacted for 12 h under argon protection. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze H 2O was dialyzed. The desired HA-RT bright yellow powder product was obtained by freeze drying. The loading efficiency of anti-PD-L1 detected by BCA protein assay was 65%, the loading efficiency of CCL5 detected by ELISA experimental method was 42%, and the grafting rate of RT was 38%.

[0046] (4) HPCR was prepared by thin film hydration method. Briefly, HA-anti-PD-L1, HA-CCL5 and HA-RT were placed in a round-bottom flask at a molar ratio of 55:85:1. An appropriate amount of dichloromethane was added and ultrasonic treatment was performed for 5 minutes until completely dissolved. The organic solvent was removed by rotary evaporation, and a certain amount of distilled water was added at a certain temperature for hydration. After hydration, the volume was measured after cooling to room temperature. The solution was passed through a 0.45 μm membrane filter and extruded after ultrasonic treatment for 5 minutes to obtain the target HPCR nanoparticles.

[0047] Example 3 A method for preparing an anti-tumor nano drug HPCR, the steps are as follows: (1) First, HA (37.9 mg, 0.1 mmol) with a molecular weight of 98 KDa, EDC·HCl (25.675 mg, 0.13 mmol), and NHS (15.38 mg, 0.13 mmol) were dissolved in 5 mL of H 2 O and then stirred at 25 °C for 3 h to activate the carboxyl groups of HA. 2 A solution containing anti-PD-L1 (2.12 mg, 14 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the 1 mL TEA solution. The reaction was continued for 48 h in argon. Afterwards, the 1000 Da dialysis bag was used to dialyze the H 2 The desired HA-anti-PD-L1 white powder product was obtained by freeze drying.

[0048] (2) HA (37.9 mg, 0.1 mmol), EDC·HCl (25.675 mg, 0.13 mmol) and NHS (15.38 mg, 0.13 mmol) were dissolved in 5 mL H 2 O and then stirred at 25 °C for 3 h to activate the carboxyl groups of HA. 2 A solution containing CCL5 (12 μg, 3 nmol) and DMAP (1.1 mg, 7.8 nmol) was added to the 1% HCl solution, and then 1 mL of TEA was added dropwise. The reaction was continued for 48 h under argon. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze the H 2The desired HA-CCL5 white powder product was obtained by freeze drying.

[0049] (3) HA (37.9 mg, 0.1 mmol), EDC·HCl (25.675 mg, 0.13 mmol) and NHS (15.38 mg, 0.13 mmol) were dissolved in 5 mL H 2 O, and then stirred at 25 °C for 3 h to activate the carboxyl group of HA. Subsequently, RT (21.984 mg, 0.4 mmol) was dissolved in 0.5 mL DMF. It was placed in an ice bath for precooling for 10 min. The RT solution was added dropwise to the HA solution and reacted for 12 h under argon protection. Afterwards, a dialysis bag with a molecular weight cutoff of 1000 Da was used to dialyze H 2 O was dialyzed. The desired HA-RT bright yellow powder product was obtained by freeze drying. The loading efficiency of anti-PD-L1 detected by BCA protein assay was 62%, the loading efficiency of CCL5 detected by ELISA experimental method was 40%, and the grafting rate of RT was 36%.

[0050] (4) Prepare HPCR by thin film hydration method. Briefly, HA-anti-PD-L1, HA-CCL5 and HA-RT were placed in a round-bottom flask at a molar ratio of 60:90:1. An appropriate amount of dichloromethane was added and ultrasonic treatment was performed for 5 minutes until completely dissolved. The organic solvent was removed by rotary evaporation, and a certain amount of distilled water was added at a certain temperature for hydration. After hydration, the volume was measured after cooling to room temperature. The solution was passed through a 0.45 μm membrane filter and extruded after ultrasonic treatment for 5 minutes to obtain the target HPCR nanoparticles.

[0051] Application Example 1 To test the inhibitory effect of HPCR nanomedicine on the growth of H22 liver cancer transplanted tumors in BALB / c male mice, the following experiments were conducted: First, culture H22 cells: put frozen mouse liver cancer H22 cells in a 37℃ water bath for 1 minute, add 3 mL of pre-cooled saline after thawing, centrifuge at 1500 r / min for 5 minutes, discard the supernatant, add 3 mL of pre-cooled saline, resuspend and centrifuge, repeat the above operation 3 times, and then calculate the cell viability by trypan blue staining to determine that the cell viability is greater than 95%. Count the cells using a cell counting plate and adjust the cell number to 2.5×10 7 / mL, resuspended in pre-cooled saline. Draw the cell suspension with a 1 mL sterile syringe, disinfect the injection site with 75% alcohol, and inject 0.4 mL of cell suspension into the peritoneum of a male mouse, and feed it normally for about 6-8 days. When the mouse's abdominal cavity is filled with ascites and the abdomen is swollen, disinfect and draw the ascites in the clean bench, filter it through a 200-mesh stainless steel cell sieve, wash it with pre-cooled saline, and centrifuge it twice. The cells collected from the ascites of one mouse are injected into 3 mice on average, and continue to feed. Repeat the above steps for cell passage.

[0052] Establishment of H22 tumor animal model: Under the aseptic conditions of the clean bench, the full mouse ascites was extracted, filtered through a 200-mesh cell sieve, resuspended in saline and centrifuged 3-5 times until the cell suspension was white and free of red blood cells. The cell viability was determined to be greater than 95% using the trypan blue staining method, and the cell count was performed using a cell counting plate, and the cell number was adjusted to 1.5×10 7 The cell suspension was extracted with a 1 mL sterile syringe, and the injection site was disinfected with 75% alcohol. Each male mouse was subcutaneously injected with 0.2 mL of cell suspension (3×10 6 After inoculation, the right armpit showed no obvious changes in the first three days after modeling. On the fourth day, a mass began to bulge in the right armpit, indicating that the modeling was successful. When the average tumor size was 80-100 mm 3 The experimental groups can be carried out.

[0053] The experimental grouping was randomized into model group (M); anti-OX40 group; HPC group; HPCR group; HPC+anti-OX40 group; HPCR+anti-OX40, n=5. The model group was fed normally after grouping. Anti-OX40 was injected intraperitoneally on days 1, 3, and 5 at a dose of 5 mg / kg. HPCR was injected into the tail vein on days 2, 4, and 6 at a dose of 83.38 mg / kg. During the experiment, the tumor length, width, and body weight of the mice were monitored every other day, and the tumor volume was calculated using the formula: V=L × W 2 ×1 / 2 (V: volume; L: length; W: width). Tumor tissues and other major organs (including brain, heart, liver, spleen, lung and kidney) were collected on day 8 after grouping and the final organ weights were determined.

[0054] The experimental results are as follows Figure 5 shown. Figure 5 a, b The results show that the tumor volume of mice in the HPCR+anti-OX40 group was significantly smaller than that in the M group, but the tumor volume of mice in the anti-OX40 group alone was not significantly different from that in the M group. Figure 5c The results showed that the tumor weight of the HPCR+anti-OX40 group measured at the end of treatment was only 18.7% of that of the M group, and a significant anti-tumor response was observed, while no significant anti-tumor response was observed in the anti-OX40 group. The results also demonstrated that the therapeutic effect of HPCR+anti-OX40 was significantly enhanced compared with the HPC group (69.3% of the M group) or the HPCR group (37.6% of the M group). Figure 5 As shown in d, HPCR was demonstrated to be well tolerated and safe in mice, as no weight loss was observed during treatment.

[0055] Application Example 2 In order to detect the role of HPCR in activating ACE2, thereby reducing the extracellular matrix of tumor cells and enhancing drug penetration, the expression of ACE2 and type I collagen was detected by immunohistochemistry, the expression of α-SMA was detected by immunofluorescence, and the expression of CCL5 and PD-L1 in tumor tissues was detected by protein immunoblotting. Immunohistochemistry is a reaction between antigen and antibody, followed by color development with DAB, and counterstaining of cell nuclei with hematoxylin. Microscopes can be observed to show that the cell nuclei are stained light blue, and positive cells are stained brown or dark brown. The cumulative optical density (IOD) of the positive expression area on the slice is counted, and the high or low IOD value can reflect the expression of the protein. Immunofluorescence technology is to first bind to the tissue antigen through the primary antibody, and then mark the fluorescent pigment that does not affect the activity of the antibody on the antibody. After binding to the corresponding primary antibody, it presents a specific fluorescence under a fluorescence microscope. The fluorescence intensity is then quantified to reflect the expression of the protein. Western immunoblotting: Tumor tissues were collected and minced, dissolved with RIPA buffer (Solarbio, Beijing, China), and PMSF and protease inhibitor cocktail (Solarbio, Beijing, China) were added to a final concentration of 1%. Protein concentration was measured using BCA reagent (Solarbio, Beijing, China). Equal amounts of protein were loaded for SDS-polyacrylamide gel electrophoresis. Then, the proteins were transferred to a PVDF membrane and then blocked with 5% skim milk for 2 h at room temperature. The membrane was incubated with the primary antibody at 4 °C overnight. The membrane was then incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG for 2 h at room temperature. Protein expression was visualized using enhanced chemiluminescence reagent (Abbkine, Wuhan, China).

[0056] The experimental grouping was randomized into model group (M); anti-OX40 group; HPC group; HPCR group; HPC+anti-OX40 group; HPCR+anti-OX40, n=5. The model group was fed normally after grouping. Anti-OX40 was injected intraperitoneally on days 1, 3, and 5 at a dose of 5 mg / kg. HPCR was injected into the tail vein on days 2, 4, and 6 at a dose of 83.38 mg / kg. During the experiment, the tumor length, width, and body weight of the mice were monitored every other day, and the tumor volume was calculated using the formula: V=L × W 2 ×1 / 2 (V: volume; L: length; W: width). Tumor tissues and other major organs (including brain, heart, liver, spleen, lung and kidney) were collected on day 8 after grouping and the final organ weights were determined.

[0057] The experimental results are as follows Figure 6 shown. Figure 6 aThe results showed that the expression of ACE2 protein in the HPCR group was significantly upregulated compared with that in the M group and the HPC group. Figure 6 Results b and c showed that the levels of type I collagen and α-SMA proteins were significantly downregulated in matrix-rich tumors in the HPCR group. The above results suggest that HPCR regulates the extracellular matrix of tumor tissue by activating ACE2.

[0058] Drug penetration Figure 7 As shown. The expression of CCL5 protein was significantly upregulated in the HPCR group compared with the M group and the HPC group. The tumor PD-L1 protein level was significantly downregulated in the HPCR group, which was due to the pre-binding of anti-PD-L1 with PD-L1 in the HPCR nanomedicine. The above results suggest that the penetration ability of HPCR drugs is enhanced.

[0059] Application Example 3 To detect HPCR-enhanced T cell infiltration in tumor tissues, flow cytometry was used to detect the content of T cells in tumor tissues, and immunohistochemistry and immunofluorescence were used to detect CD8 expression in tumor tissues: Preparation of single cell suspension of tumor tissue: The mice were killed by cervical dislocation. In a sterile environment, the tumor tissue was carefully peeled off and then cut into small pieces with scissors. Type IV collagenase (1 mg.mL) was added to the tumor tissue. -1 )、Type I deoxyribonuclease (0.2 mg.mL -1 ) and hyaluronidase (0.2 mg.mL -1) was placed in a 37°C incubator for 30 minutes. During the digestion process, the tissue suspension was mixed every 10 minutes. After digestion, the culture medium was added to terminate the digestion. The tissue suspension was filtered through a 200-mesh cell strainer. The filtered cell suspension was centrifuged at 1500 r / min for 5 minutes. The supernatant was discarded, and the cell pellet was added with red blood cell lysis solution and lysed on ice in the dark for 5 minutes, followed by low-speed centrifugation at 1500 r / min for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in PBS, centrifuged at low speed for 5 minutes, the supernatant was discarded, and the cell pellet was washed twice. Lymphocytes were screened out by percoll density gradient centrifugation. The cell suspension was washed twice with PBS.

[0060] The prepared tumor cell suspension was centrifuged and the supernatant was discarded, then resuspended in flow cytometry buffer, blocked with anti-CD16 / CD32 antibody on ice for 15 min, and then antibodies with different fluorescent dyes were added. The cells were incubated on ice in the dark for 30 min. After the incubation, the cells were washed twice with flow cytometry buffer, and finally the cells were resuspended in 0.5 mL flow cytometry staining buffer. The samples were detected using Beckman flow cytometer and analyzed by FlowJo.

[0061] Immunohistochemistry and immunofluorescence were used to detect CD8 expression in tumor tissues. Immunohistochemistry is an antigen-antibody reaction, followed by color development with DAB, and counterstaining of the cell nucleus with hematoxylin. Under a microscope, it can be observed that the cell nucleus is stained light blue, and the positive cells are stained brown or dark brown, which can reflect the expression of the protein. Immunofluorescence technology is to first bind to the tissue antigen through the primary antibody, and then label the fluorescent pigment that does not affect the activity of the antibody on the antibody. After binding to the corresponding primary antibody, it presents a specific fluorescence under a fluorescence microscope, which can reflect the level of expression of the protein.

[0062] The experimental grouping was randomized into model group (M); anti-OX40 group; HPC group; HPCR group; HPC+anti-OX40 group; HPCR+anti-OX40, n=5. The model group was fed normally after grouping. Anti-OX40 was injected intraperitoneally on days 1, 3, and 5 at a dose of 5 mg / kg. HPCR was injected into the tail vein on days 2, 4, and 6 at a dose of 83.38 mg / kg. During the experiment, the tumor length, width, and body weight of the mice were monitored every other day, and the tumor volume was calculated using the formula: V=L × W 2 ×1 / 2 (V: volume; L: length; W: width). Tumor tissues and other major organs (including brain, heart, liver, spleen, lung and kidney) were collected on day 8 after grouping and the final organ weights were determined.

[0063] Figure 8 The experimental results showed changes in T cell infiltration within the tumor. Figure 8 a, b Flow cytometry results showed that the percentage of T cells in the tumors of the HPCR+anti-OX40 group was significantly increased compared with that of the HPC+anti-OX40 group. Figure 8 Immunofluorescence sections of H22 tumors from untreated mice in c show CD8 + T cell infiltration was extremely low, but after HPCR+anti-OX40 treatment, CD8 + T cells were significantly upregulated compared with CD8 + T cells were 2.60 times higher. This was consistent with the immunofluorescence results. Figure 8 d, e Immunohistochemistry revealed that tumor-infiltrating CD8 + The above results indicate that HPCR+anti-OX40 treatment upregulated the number of T cells in tumors, especially CD8 + T cells, effectively relieving immunosuppression and enhancing anti-tumor immune response.

[0064] 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 method for preparing a tumor-targeted anti-tumor nanomedicine HPCR, characterized in that: Here are the steps: (1) Add an activator to the HA aqueous solution for activation, then add the anti-PD-L1 solution, and react to completion under argon protection. The reaction solution is dialyzed and filtered to obtain the product HA-anti-PD-L1; (2) Add an activator to the HA aqueous solution for activation, then add CCL5 solution, and react until complete under argon protection. The reaction solution is dialyzed and filtered to obtain the product HA-CCL5; (3) Add an activator to the HA aqueous solution for activation, then add the RT solution, and react until complete under the protection of argon. The reaction solution is dialyzed and filtered to obtain the product HA-RT; (4) The above-mentioned products HA-anti-PD-L1, HA-CCL5 and HA-RT are added to a solvent for dissolution, rotary evaporation, filtration and ultrasonic extrusion to obtain the nano drug HPCR.

2. The method for preparing the anti-tumor nano drug HPCR according to claim 1, characterized in that: The molar ratio of HA, activator, and anti-PD-L1 in step (1) is 1:1.2-1.5:1.32×10 -4 -1.5×10 -4 ; In step (2), the molar ratio of HA, activator, and CCL5 is 1:1.2-1.5:2.5×10 -5 -4.0×10 -5 ; In step (3), the molar ratio of HA, activator and RT is 1:1.2-1.5:3-5.

3. The method for preparing the anti-tumor nano drug HPCR according to claim 2, characterized in that: In the step (4), the molar ratio of HA-anti-PD-L1, HA-CCL5 and HA-TR is 50-60:80-90:1, and the solvent is dichloromethane.

4. The method for preparing the anti-tumor nano drug HPCR according to claim 3, characterized in that: The activating agent is EDC‧HCl, and the activation time is 2-4 h; the dialysate for dialysis is double distilled water, and the diameter of the microporous filter membrane for filtration is 0.45 μm.

5. The anti-tumor nano drug HPCR prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the anti-tumor nano drug HPCR according to claim 5 in inhibiting the growth of H22 liver cancer transplanted tumors.

7. Use of the anti-tumor nano drug HPCR according to claim 5 in activating T cells, recruiting T cells and increasing T cell infiltration.

8. Use of the anti-tumor nano drug HPCR according to claim 5 in eliminating tumor cell extracellular matrix and increasing drug penetration.

9. Use of the anti-tumor nano drug HPCR according to claim 5 in anti-tumor treatment.

10. The use according to any one of claims 6 to 9, characterized in that: The use concentration of the anti-tumor nano drug HPCR is 80-90 mg / kg.

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