Application of cell-penetrating peptide modified virus nanoparticles in preparation of products for cryoablation therapy

Abstract

The invention discloses an application of cell-penetrating peptide modified virus nanoparticles in preparation of a product for cryoablation treatment. The cell-penetrating peptide modified virus nanoparticles have autoimmunogenicity and ice crystal nucleation effect, have high biological safety, can be used for preparing cryoablation treatment products, especially in cryoablation cancer therapy, can improve the killing effect on tumors and improve the whole-body immune effect, and can be used for preparing cryoablation treatment products. The tumor recurrence and metastasis are effectively delayed.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterial applications, and more specifically, to the use of cell-penetrating peptide-modified viral nanoparticles in the preparation of products for cryoablation therapy, particularly in products for tumor cryoablation therapy. Background Art

[0002] With the rapid development of modern imaging technology, ablation has garnered widespread attention as a simple, minimally invasive treatment method. Ablation therapies can be categorized into radiofrequency ablation, microwave ablation, cryoablation, and laser ablation. Cryoablation was first used in the 1940s to treat breast and uterine cancer. Cryoablation involves percutaneously inserting a cryoprobe into the tumor tissue. The throttling and expansion of a cryogenic medium (such as liquid nitrogen or liquid argon) at the probe tip cools the tumor tissue to temperatures as low as -196°C. At these low temperatures, ice crystals form inside and outside tumor cells, penetrating cell and organelle membranes and causing irreversible mechanical damage. During the warming process after freezing, the recrystallization of ice crystals transforms small ice crystals into larger ones, similarly damaging cells. Furthermore, because tumor tissue growth requires a rich blood supply to provide nutrients and metabolic components, the hypothermia caused by freezing can constrict blood vessels, resulting in insufficient blood supply and causing tumor cell necrosis due to hypoxia. Compared to other ablation methods, cryoablation can achieve conformity to tumor tissue by adjusting the size and shape of the ice ball, reducing damage to normal tissue. Furthermore, cryoablation provides a low-temperature anesthetic effect on the patient, alleviating surgical pain. Furthermore, compared to the high-temperature destruction of tumor antigens by hyperthermia, the low-temperature conditions of cryoablation can minimize the denaturation of proteins, including tumor antigens, thereby preserving the immunogenicity of tumor tissue, thereby promoting immune responses to a certain extent and forming a systemic immune effect. These multiple advantages have led to the clinical use of cryoablation for the treatment of malignant tumors in various locations, including the liver, kidney, lung, and breast.

[0003] Although cryoablation is easy to use and highly efficient, it still faces problems such as insufficient freezing, minimal tumor cell necrosis around the ablation zone, and damage to healthy tissue. These issues can lead to poor tumor killing and recurrence of residual tumors after ablation, preventing cryoablation from achieving its intended effect. Furthermore, the immune effects of ablation are uncertain and may lead to immunosuppression due to the massive production of apoptotic cells, resulting in increased tumor metastasis and poor prognosis. Therefore, improving tumor killing efficiency, enhancing treatment efficacy, and activating the immune effects of cryoablation are currently key research issues in cryoablation therapy. Summary of the Invention

[0004] In response to the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a cell-penetrating peptide-modified viral nanoparticle, which can be applied to the field of cryoablation treatment, especially the field of tumor cryoablation treatment, to improve tumor killing efficiency, enhance treatment effect and activate the immune effect of cryoablation.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides the use of cell-penetrating peptide-modified viral nanoparticles in the preparation of products for cryoablation therapy.

[0007] Furthermore, the product is an ice crystal nucleating agent.

[0008] The inventors of the present invention have discovered that some viral nanoparticles act as ice crystal nucleators, such as tobacco mosaic virus, cowpea chlorotic mottle virus, cowpea mosaic virus, and papaya mosaic virus. By utilizing cell-penetrating peptides modified on the outer surface of the viruses, the viral nanoparticles can cross the membrane and enter the cytoplasm, promoting the formation of ice crystals both inside and outside the cells, thereby enhancing the killing effect of freezing on tumor cells. Furthermore, because the cell-penetrating peptide-modified viral nanoparticles can increase the ice crystal nucleation temperature, during the cryoablation process, some cells that die by apoptosis at sublethal temperatures can be converted to cells that die by necrosis due to ice crystal formation, promoting the release of damage-related pattern molecules and tumor-specific antigens, thereby stimulating the body's immune response. Furthermore, the cell-penetrating peptide-modified viral nanoparticles have their own immunogenicity. Therefore, combining the cell-penetrating peptide with the viral nanoparticles can simultaneously enhance the killing and immune effects of cryoablation, achieving the purpose of enhancing the therapeutic effect of cryoablation.

[0009] In the present invention, cell-penetrating peptides can be modified on the outer surface of the virus by methods known in the art to form cell-penetrating peptide-modified viral nanoparticles, including but not limited to a method for coupling cell-penetrating peptides to viral nanoparticles by a click chemistry reaction between an alkynyl group and an azide catalyzed by monovalent copper, a copper-free click chemistry reaction between dibenzocyclooctyne (DBCO) and an azide, an amide reaction between an NHS ester and an amino group, and a Michael addition reaction between a maleimide and a thiol group.

[0010] Furthermore, in the present invention, the virus is selected from one of tobacco mosaic virus, cowpea chlorotic mottle virus, cowpea mosaic virus and papaya mosaic virus.

[0011] The cell membrane-penetrating agent is selected from one of TAT, MPG, Stearyl-R8, EB1, PF6, VP2 and PEP-1.

[0012] According to a specific embodiment of the present invention, the cell-penetrating peptide-modified viral nanoparticles are cell-penetrating peptide-modified tobacco mosaic virus nanoparticles.

[0013] According to a specific embodiment of the present invention, the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles are TAT-modified tobacco mosaic virus nanoparticles.

[0014] Furthermore, the function of the product is to promote the nucleation, growth and recrystallization of ice crystals.

[0015] Furthermore, the function of the product is to enter the cytoplasm rather than the lysosome through transmembrane action and nucleate ice crystals in the cell.

[0016] Furthermore, the function of the product is to enhance the killing of tumor tissue by cryoablation through intratumoral injection.

[0017] Furthermore, the function of the product is to increase the secretion of heat shock protein-70 in necrotic cells by increasing the proportion of necrotic cells in tumor tissue under freezing conditions, thereby stimulating the maturation of dendritic cells in tumor-draining lymph nodes and enhancing the immune effect of cryoablation.

[0018] When used, cell-penetrating peptide-modified viral nanoparticles are injected into the tumor 12 hours before cryoablation. During surgery, a cryoablation probe is inserted into the tumor to cool the tumor tissue to -120°C, forming ice balls throughout the tumor tissue to achieve a therapeutic effect, and then passively thawed to room temperature.

[0019] The beneficial effects of the present invention are as follows:

[0020] The cell-penetrating peptide-modified viral nanoparticles provided by the present invention have both autoimmunogenicity and ice crystal nucleation effects, and have high biosafety. They can be used to prepare cryoablation treatment products, especially in cryoablation cancer therapy, to enhance the killing effect on tumors, improve the systemic immune effect, and effectively delay tumor recurrence and metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] Figure 1 Polyacrylamide gel electrophoresis image showing cell-penetrating peptide-modified tobacco mosaic virus nanoparticles;

[0023] Figure 2 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the ice crystal formation temperature in solution;

[0024] Figure 3Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the growth curve of ice crystals in solution;

[0025] Figure 4 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on ice crystal recrystallization in solution;

[0026] Figure 5 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the freezing temperature of 4T1 cells under direct freezing conditions;

[0027] Figure 6 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the freezing ratio of 4T1 cells at different temperatures under seed ice conditions;

[0028] Figure 7 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the proportion of necrotic cells in 4T1 cells after freezing at different temperatures;

[0029] Figure 8 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on cryoablation of mouse tumors;

[0030] Figure 9 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the growth of recurrent tumors in mice;

[0031] Figure 10 Shows the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on serum heat shock protein-70 concentration on day 2 after tumor ablation;

[0032] Figure 11 Shown is the effect of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles on the proportion of mature dendritic cells in lymph nodes draining recurrent tumors. DETAILED DESCRIPTION

[0033] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and drawings. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0034] Example 1

[0035] Alkyne groups were grafted onto the outer surface of TMV by electrophilic substitution reaction between tyrosine at position 139 on the outer surface of TMV and acetylene aniline diazonium salt. The specific method was as follows: 800 μL of p-toluenesulfonic acid aqueous solution (57 mg / mL), 150 μL of acetylene aniline acetonitrile solution (78 mg / mL) and 50 μL of nitrite aqueous solution (207 mg / mL) were mixed in the dark to obtain a presynthesized salt; 10 mg of TMV was dissolved in 0.1 M boric acid-borax buffer (pH 8.8) containing 0.1 M sodium chloride to a final concentration of 2 mg / mL of TMV, and 190 μL of the presynthesized salt was added thereto. The mixture was reacted in the dark at 4°C for 1 hour. After the reaction, the mixture was placed in a 1 million dialysis bag and dialyzed against 10 mM phosphate buffer to obtain the alkyne-modified TMV.

[0036] The cell-penetrating peptide modified with alkynyl and azide was coupled by a monovalent copper-catalyzed click chemistry reaction. The specific method was as follows: 50 μL of TAT-N3 (10 mg / mL) aqueous solution was added to 1 mL of alkynyl-modified TMV solution (1 mg / mL), and the mixture was mixed evenly at 4°C. 10 μL of sodium ascorbate aqueous solution (37 mg / mL), 10 μL of aminoguanidine hydrochloride aqueous solution (22 mg / mL) and 10 μL of copper sulfate aqueous solution (25 mg / mL) were added thereto in sequence, and the mixture was reacted in the dark for 30 minutes at 4°C. 10 μL of ETA-sodium hydroxide aqueous solution (0.5 M) was added to the reaction solution, and the reaction was terminated by stirring in the dark for 10 minutes. After the reaction, the solution was placed in a 1 million dialysis bag and dialyzed in 10 mM phosphate buffer to obtain cell-penetrating peptide-modified tobacco mosaic virus nanoparticles.

[0037] The successful synthesis of TMV-TAT was confirmed by the addition of a new band in the lane of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles in polyacrylamide gel electrophoresis (Appendix Figure 1 ).

[0038] Example 2

[0039] 20 μL of a 100 μg / mL PBS solution of the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was placed in an aluminum crucible, and the effect of the cell-penetrating peptide-modified tobacco mosaic virus solution on the ice crystal nucleation temperature was measured using a low-temperature differential scanning calorimeter. The scanning program was set to: cool the sample temperature from 20°C to -50°C at a cooling rate of 10°C / min, and the starting temperature of the exothermic peak during the cooling process was used as the ice crystal nucleation temperature of the solution. The results showed that the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles could advance the ice crystal formation temperature of the PBS solution (see Figure 2). Figure 2 ).

[0040] Example 3

[0041] 20 μL of 100 μg / mL PBS solution of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was placed in the center of a quartz crucible. To facilitate the observation of ice crystal growth, a cover glass was placed on top of the solution to form a solution film. The quartz crucible was placed in a low-temperature cold stage system and the solution was cooled to -20°C at a cooling rate of 20°C / min to freeze the solution. The ice crystals were focused using a biological microscope and heated to 0°C at a heating rate of 1°C / min. When only a single ice crystal of 10 μm in size remained in the field of view, the solution was cooled at a cooling rate of 1°C / min for 20 seconds. The size of the ice crystals at different times was measured and an ice crystal growth curve was drawn. The results showed that cell-penetrating peptide-modified tobacco mosaic virus nanoparticles could increase the ice crystal growth rate of the PBS solution (see Figure 2). Figure 3 ).

[0042] Example 4

[0043] The quartz crucible was cooled to -80°C, and 10 μL of a PBS solution of 100 μg / mL cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was dripped into the center of the quartz crucible from a height of 1.5 meters. The solution quickly formed an ice crystal film. The quartz crucible was placed in a low-temperature cold stage system and heated to -10°C at a heating rate of 5°C / min, and maintained at this temperature for annealing for 30 minutes. The sizes of the three largest ice crystals in the field of view were statistically analyzed as a characterization of the ice crystal recrystallization effect. The results showed that the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles can improve the ice crystal recrystallization effect of the PBS solution (see Appendix). Figure 4 ).

[0044] Example 5

[0045] A certain amount of the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was added to the mouse breast cancer cell line 4T1 to a final concentration of 10 μg / mL, and cultured for 12 hours under conventional cell culture conditions (37°C, 5% CO2, saturated humidity). After the culture, 20 μL of the cell suspension was dropped into the center of the quartz crucible, a cover glass was placed on top of the cell suspension, and the cell suspension was cooled using a low-temperature cooling stage system at a cooling rate of 10°C / min. The cell was observed using a biological microscope and the temperature corresponding to the blackening of the interior of the cell was used as the cell freezing temperature. The results showed that the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles that entered the cytoplasm could advance the temperature of ice crystal formation in the cell (see Appendix). Figure 5 ).

[0046] Example 6

[0047] A certain amount of the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was added to the mouse breast cancer cell line 4T1 to a final concentration of 80 μg / mL, and cultured for 12 hours under conventional cell culture conditions (37°C, 5% CO2, saturated humidity). After the culture was completed, 20 μL of the cell suspension was dropped into the center of the quartz crucible, a cover glass was placed on top of the cell suspension, and the cell suspension was cooled to -1°C at a cooling rate of 10°C / min using a low-temperature cooling stage system. The cell suspension was seeded with ice using a syringe needle pre-cooled with liquid nitrogen. After the cell suspension was frozen, it was cooled to -60°C at a cooling rate of 10°C / min, and a biological microscope was used to observe and count the number of frozen cells in the field of view at each temperature. The results showed that the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles that entered the cytoplasm could advance the formation temperature of ice crystals in the cell under the condition of the presence of extracellular ice (see Figure 2). Figure 6 ).

[0048] Example 7

[0049] A certain amount of the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1 was added to the mouse breast cancer cell line 4T1 to a final concentration of 20 μg / mL, and cultured for 12 hours under conventional cell culture conditions (37°C, 5% CO2, saturated humidity). The cell suspension was cooled to -15°C, 60°C, and -120°C for 2 minutes using a low-temperature cooling stage system. The frozen cells were fluorescently stained with Annexin V-FITC and PI, and the proportion of apoptotic and necrotic cells was detected by flow cytometry. The results showed that the cell-penetrating peptide-modified tobacco mosaic virus nanoparticles that entered the cytoplasm could increase the proportion of necrotic cells at different cooling rates (see Appendix). Figure 7 ).

[0050] Example 8

[0051] 4T1 cells in the logarithmic growth phase were collected and prepared with sterile saline to a concentration of 5x10 6 The single cell suspension of cells / mL was prepared, and 100 μL of cell suspension was subcutaneously injected into the right armpit of female Balb / c mice on day -10 to establish a mouse subcutaneous tumor model. The mice were randomly divided into groups on day -1, and the groups were: control group, cryoablation group only, cryoablation + tobacco mosaic virus group, cryoablation + cell-penetrating peptide-modified tobacco mosaic virus nanoparticle group. The cryoablation + cell-penetrating peptide-modified tobacco mosaic virus nanoparticle group was injected intratumorally with 50 μL of 100 μg / mL cell-penetrating peptide-modified tobacco mosaic virus nanoparticles prepared in Example 1. The mice were treated with cryoablation on day 0. Starting from day 0, the long and short diameters of the tumors were measured every other day using an electronic vernier caliper, and the tumor growth curve was recorded. The tumor volume was calculated using the formula:

[0052] V=0.5×l×d2

[0053] Where V is the tumor volume, l is the long diameter of the tumor, and d is the short diameter of the tumor.

[0054] The results showed that compared with the other groups, the pre-intratumoral injection of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles could effectively enhance the killing effect of cryoablation on tumors and delay the growth of primary tumors (see Appendix). Figure 8 ).

[0055] Example 9

[0056] 4T1 cells in the logarithmic growth phase were collected and prepared with sterile saline to a concentration of 5x10 6 The mice in Example 8 were subcutaneously injected with 100 μL of the cell suspension at the left armpit on the 10th day to establish a recurrent tumor model. The long and short diameters of the recurrent tumors were measured every other day using an electronic vernier caliper, and the growth curve of the recurrent tumors was recorded. The results showed that compared with the other groups, the pre-intratumoral injection of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles can effectively enhance the immune effect of cryoablation and delay the growth of recurrent tumors (see Appendix). Figure 9 ).

[0057] Example 10

[0058] On the third day, blood was collected from the mice in Example 8. After coagulation at room temperature for 1 hour, the blood was centrifuged at 3000 rpm for 10 minutes at 4°C to obtain serum. The serum was tested for heat shock protein-70 concentration using an enzyme-linked immunosorbent assay kit. The results showed that compared with the other groups, the pre-intratumor injection of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles can effectively increase the concentration of heat shock protein released after tumor tissue ablation (Appendix Figure 10 ).

[0059] Example 11

[0060] On the 22nd day, the mice in Example 8 were euthanized, and the draining lymph nodes on the side of the recurrent tumor in the left axilla were dissected and obtained. The single cell suspension of the lymph nodes was obtained by grinding, and the cells were labeled with fluorescently labeled CD11c, CD80, and CD86 antibodies. The proportion of CD86 and CD80 double-positive mature dendritic cells in the CD11c-positive cell population was detected by flow cytometry. The results showed that compared with the other groups, the early intratumoral injection of cell-penetrating peptide-modified tobacco mosaic virus nanoparticles can increase the proportion of mature dendritic cells in the draining lymph nodes of the recurrent tumor (see Appendix). Figure 11 ).

[0061] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Application of cell-penetrating peptide-modified viral nanoparticles in the preparation of products for cryoablation therapy.

2. The use according to claim 1, characterized in that The product is an ice crystal nucleating agent.

3. The use according to claim 1, characterized in that The virus is selected from one of tobacco mosaic virus, cowpea chlorotic mottle virus, cowpea mosaic virus and papaya mosaic virus.

4. The use according to claim 1, characterized in that The cell membrane-penetrating agent is selected from one of TAT, MPG, Stearyl-R8, EB1, PF6, VP2 and PEP-1.

5. The use according to claim 1, characterized in that The cell-penetrating peptide modified virus nanoparticles are cell-penetrating peptide modified tobacco mosaic virus nanoparticles.

6. The use according to claim 5, characterized in that The cell-penetrating peptide-modified tobacco mosaic virus nanoparticles are TAT-modified tobacco mosaic virus nanoparticles.

7. The use according to claim 1, characterized in that The function of the product is to promote the nucleation, growth and recrystallization of ice crystals.

8. The use according to claim 1, characterized in that The function of the product is to enter the cytoplasm rather than the lysosome through transmembrane action and nucleate ice crystals in the cell.

9. The use according to claim 1, characterized in that The function of the product is to enhance the killing of tumor tissue by cryoablation through intratumoral injection.

10. The use according to claim 1, characterized in that The function of the product is to increase the secretion of heat shock protein-70 in necrotic cells by increasing the proportion of necrotic cells in tumor tissue under freezing conditions, thereby stimulating the maturation of dendritic cells in tumor-draining lymph nodes and enhancing the immune effect of cryoablation.

Images