Polypeptide molecule for inducing spontaneous calcification of tumor cells and application thereof

Abstract

The invention provides a molecule for inducing spontaneous calcification of tumor cells and application thereof, the molecule for inducing spontaneous calcification of tumor cells at least contains two basic units, one basic unit is a targeting functional unit and is a targeting functional molecule fragment of tumor cells / tissues / microenvironment, and the other basic unit is a targeting functional molecule fragment of tumor cells / tissues / microenvironment. The other basic unit is a calcification induction functional unit; or the molecule for inducing spontaneous calcification of the tumor cells at least contains one basic unit, and the basic unit is a targeting functional unit and a calcification induction unit at the same time. After tumor cells are calcified, on one hand, calcium salt deposition on the surfaces of the cells can influence the physiological processes such as metabolism of the tumor cells, tumor cell apoptosis is further induced, and the effects of tumor inhibition and treatment are achieved; on the other hand, the contrast ratio of calcified tumor cells and tissues in clinical images is improved, and early diagnosis and accurate diagnosis of tumor focuses are facilitated.

Description

[0001] This invention patent application has the application number 202111570242.0, the filing date of December 21, 2021, and the invention title of "Molecules that Induce Spontaneous Calcification of Tumor Cells and Their Applications". The priority date is December 22, 2020, and the priority numbers are

[0002] the divisional applications of the invention patent applications with the priority numbers CN202011532338.3 and CN202011532304.4

[0003] Technical Field

[0004] The present invention relates to a class of molecules that selectively induce spontaneous calcification of tumor cells under physiological conditions, and their applications. Background Art

[0005] Chemotherapy is one of the most commonly used treatment methods for cancer patients. Even if surgery can completely remove all visible lesions, it is still necessary to use chemotherapeutic drugs to eliminate invisible cancer cells. Unfortunately, since most chemotherapeutic drugs act by affecting the cell growth and proliferation processes, they also cause damage to normal cells. The side effects of systemic chemotherapy for treating cancer are usually severe, and may lead to damage to the immune system, thereby causing neuropathy and neutropenia. Calcification is an important biological process in the human body. For example, the formation of bones and teeth is calcification, and it is also important in certain pathological diseases (such as atherosclerosis and kidney stones). Calcification has also been observed in cancer. Although its mechanism is unclear, researchers have reported that tumor calcification and regional lymph node calcification are favorable prognostic factors for colorectal cancer and lung cancer. In 2019, a research group at East China Normal University developed a method for inducing tumor calcification by systemic injection of calcium peroxide nanoparticles. The problem of liver accumulation of nanoparticles for drug delivery has always existed, so the clinical translation safety of this method deserves further exploration.

[0006] In 2016, the inventors reported a new strategy for treating tumors by forming cancer cell-targeted calcification through high-dose folic acid (FA) and Ca 2+ However, each FA molecule can only provide two carboxyl residues to bind Ca 2+ in biological fluids to promote the nucleation of calcium minerals. Therefore, this method relies on Ca 2+ levels higher than the physiological range, which may cause hypercalcemic crisis and limit the clinical application prospects. Moreover, systemic injection of folic acid has also been confirmed to promote tumor growth, significantly reduce the body weight of mice, and cause a sharp reduction in the survival time of mice. Summary of the Invention

[0007] ​To solve the problems existing in the prior art, the present invention provides a molecule that can selectively induce spontaneous calcification of tumor cells in the in vivo physiological environment. By inducing spontaneous calcification of tumors with this molecule, the growth of tumors can be inhibited, the therapeutic effects of radiotherapy, chemotherapy, and immunotherapy can be improved, and the contrast of imaging can be increased to achieve the goals of early diagnosis and precise diagnosis and treatment.

[0008] To achieve the object of the present invention, the present invention provides the following technical solutions:

[0009] The present invention discloses a molecule that induces spontaneous calcification of tumor cells. The molecule that induces spontaneous calcification of tumor cells contains at least two basic units, one of which is a targeting functional unit, which is a targeting functional molecular fragment of tumor cells / tissues / microenvironments, and the other basic unit is a calcification induction functional unit; or the molecule that induces spontaneous calcification of tumor cells contains at least one basic unit, and the basic unit is both a targeting functional unit and a calcification induction unit.

[0010] There is no particular limitation on the targeting functional unit in the present invention, as long as it can meet the requirement of being a targeting functional molecular fragment of tumor cells / tissues / microenvironments.

[0011] Further, the targeting functional unit is one or more of an antibody targeting a specific antigen on the surface of tumor cells, a ligand molecule targeting a highly expressed receptor on tumor cells, a polypeptide with specific tumor cell targeting or its cyclic peptide form, an aptamer with specific tumor cell targeting, a polysaccharide targeting tumor cells, and a molecule targeting a specific tumor microenvironment.

[0012] The polypeptide with specific tumor cell targeting in the present invention can be in a linear or cyclic form.

[0013] Further, the antibody targeting a specific antigen on the surface of tumor cells is an HER-2 antibody and / or an EGFR antibody.

[0014] Further, the ligand molecule targeting a highly expressed receptor on tumor cells is folic acid.

[0015] Further, the ligand molecule targeting a highly expressed receptor on tumor cells is urokinase-type plasminogen activator receptor (uPAR).

[0016] Further, the polypeptide with specific tumor cell targeting can be one or more of the SP94 polypeptide targeting liver cancer cells, the polypeptide TDSILRSYDWTY targeting lung cancer cells (such as shown in SEQ ID NO: 2), or the RGD peptide targeting tumor blood vessels, in a linear molecule or its cyclic peptide form.

[0017] Among them, the SP94 polypeptide sequence targeting liver cancer cells is SFSIIHTPILPL, as shown in SEQ ID NO:1.

[0018] If the number of free carboxyl groups in the targeting polypeptide molecule is greater than 5, it may simultaneously satisfy the two functions of targeting and calcification by itself and independently achieve the purpose of the present invention. However, the number of free carboxyl groups contained in the targeting polypeptide molecule is usually limited (n < 5), and it is generally difficult to have both the targeting and calcification functions simultaneously.

[0019] The targeting part in the polypeptide molecule is responsible for binding to the membrane proteins highly expressed on the surface of cancer cell membranes to prevent off-targeting. Therefore, due to the limited space of the present invention, not all of them are listed exhaustively. However, other targeting molecules not described in the present invention can also play the same role. The calcification functional end can be a repeating unit of polyglutamic acid containing free carboxyl groups (E n , n≥5), or a repeating sequence of casein phosphopeptide such as pS-pS-pS-pS. The calcification part is responsible for enriching calcium and phosphate ions in the microenvironment to generate calcification. Other molecules containing a large number of free carboxyl groups not detailed in the present invention can also play the same role.

[0020] Furthermore, the polysaccharide targeting tumor cells is hyaluronic acid targeting CD44 on the surface and / or fucoidan targeting P-selectin.

[0021] Furthermore, the molecule targeting the specific microenvironment of tumors is a polypeptide-hydrophobic hydrocarbon chain-hydrophilic chain cleaved by MMP response and / or a phosphate-hydrophobic hydrocarbon chain-hydrophilic chain cleaved by alkaline phosphatase response.

[0022] Furthermore, the calcification induction functional unit contains strongly negatively charged groups.

[0023] When the calcification induction functional unit satisfies the condition of retaining a large number of strongly negatively charged groups, other substitutions do not affect the calcification effect.

[0024] Furthermore, the strongly negatively charged group can be one or more of carboxyl group, sulfonic acid group, guanidine group, and phosphate group.

[0025] Furthermore, the calcification induction functional unit is a repeated arrangement of the same functional group containing strongly negatively charged genes or a combination of different functional groups containing strongly negatively charged genes.

[0026] The number of functional groups in the molecule can be changed as needed, that is, the number of monomers of the polymer macromolecule can be from 1 to infinity.

[0027] Furthermore, the calcification induction functional unit is polysialic acid and / or polyglutamic acid.

[0028] In the present invention, the molecule that induces spontaneous calcification of tumor cells can be any combination of the above-mentioned targeting functional unit and calcification induction functional unit.

[0029] Furthermore, the molecule is a folic acid-poly sialic acid cross-linked molecule. The targeting part can be folic acid, which is responsible for binding to the receptor protein highly expressed on the surface of cancer cell membranes to prevent off-target effects, or it can be other cancer targeting molecules. The calcification functional end is poly sialic acid, which contains a large number of free carboxyl groups and is responsible for enriching calcium and phosphate ions in the microenvironment to generate calcification.

[0030] In the present invention, the two functional regions (targeting + induction of calcification) of the molecule that induces spontaneous calcification of tumor cells can be achieved through two different molecular units (targeting functional domain + calcification induction functional domain), such as folic acid-poly sialic acid cross-linked molecule; or they can be combined into one, that is, one functional unit has both of these two functions (targeting + induction of calcification). For example, hyaluronic acid can both target the specific surface molecule CD44 of tumor stem cells and has the ability to induce calcification itself; fucoidan can both target the tumor-specific molecule P-selectin and has the ability to induce calcification itself.

[0031] The molecule that induces spontaneous calcification of tumor cells provided by the present invention is applicable to all types of cancers. The tumors can be selected from hematological system tumors such as leukemia, lymphoma, multiple myeloma; digestive system tumors such as esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct and gallbladder cancer; respiratory system tumors such as lung cancer, pleural tumor; nervous system tumors such as glioma, neuroblastoma, meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer; urinary system tumors such as kidney cancer, bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

[0032] The present invention also provides the use of the above-mentioned molecule that induces spontaneous calcification of tumor cells in the preparation of tumor drugs.

[0033] In addition, the present invention also provides a tumor drug, which contains the above-mentioned molecule that induces spontaneous calcification of tumor cells.

[0034] In addition to containing the molecule that induces spontaneous calcification of tumor cells of the present invention, the tumor drug also contains necessary excipients and can also contain other therapeutic drugs.

[0035] Furthermore, the tumor drug can administer the calcification induction molecule orally, intravenously, by intratumoral intervention, or by lymph node intervention.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention provides a molecule that targets and induces selective spontaneous calcification in tumor cells. This molecule generally consists of two functional regions. One part is the tumor-targeting functional region, and the other part is the calcification-inducing functional region. Its basic principle is that the targeting functional region of the molecule selects the cell type on which the molecule acts, and the calcification-inducing functional region exerts the induction of cell calcification in the physiological environment. After the tumor cells undergo calcification, on the one hand, the calcium salt deposition on the cell surface will affect physiological processes such as the metabolism of tumor cells, further inducing apoptosis of tumor cells, playing a role in tumor suppression and treatment; on the other hand, the contrast of calcified tumor cells and tissues in clinical imaging is increased, facilitating the early diagnosis and accurate diagnosis of tumor lesions.

[0038] The molecule provided by the present invention for inducing spontaneous calcification in tumor cells can achieve effects such as inhibiting tumor growth, reversing the drug resistance state of other tumor chemotherapy drugs, synergistically enhancing tumor radiotherapy, and synergistically enhancing tumor immunotherapy, and has the efficacy of a vaccine. Moreover, by administering the calcification-inducing molecule of the present invention through oral, intravenous, intratumoral intervention, lymph node intervention, etc., the clinical imaging, such as the imaging contrast of computed tomography (CT), ultrasound, positron emission tomography-computed tomography (PET-CT), and magnetic resonance imaging (MRI), can be changed (increased or decreased), enabling earlier detection of lesions or differentiation between benign and malignant tumor lesions.

[0039] Through animal experiments, it can be seen that this type of molecule of the present invention contains two functional regions. One is the targeting functional region of the receptor on the cancer cell membrane surface, which can selectively target the cancer cell membrane and has no targeting effect on normal cells. The other end is the functional region with the ability to induce calcification, which can selectively induce the deposition of calcium, phosphorus and other ions in the microenvironment on the cancer cell membrane, causing the tumor cells to undergo calcification. After calcification, the growth and metastasis of cancer cells are significantly inhibited, and the survival period of tumor-bearing mice is significantly extended. At the same time, the calcification of tumor cells and cancer tissues can improve the imaging contrast of clinical imaging, enabling earlier and more accurate detection and identification of small tumor lesions.

[0040] In summary, the molecule disclosed by the present invention selectively induces calcification in tumor cells, plays a role in tumor treatment, inhibits tumor growth, reverses tumor drug resistance, improves the effects of tumor radiotherapy and chemotherapy and immunotherapy, and prolongs the survival period of tumor patients; at the same time, the calcification of tumor cells and tissues can improve the imaging contrast of clinical imaging, enabling earlier and more accurate detection and identification of small tumor lesions. Brief Description of the Drawings

[0041] Figure 1 For detecting the situation of the targeting peptides bound to different cell surfaces by flow cytometry in Example 1, the right shift of the peak indicates that the targeting peptide molecule selectively binds to the surface of lung cancer cells and has no binding to normal epithelial cells.

[0042] Figure 2 Elemental spectra of scanning electron microscopy of lung cancer cells and normal lung epithelial cells after treatment with CiP peptide in Example 1. It can be seen that compared with normal lung epithelial cells, the calcium and phosphorus element peaks on the cell surface of lung cancer cells are significantly increased after treatment with CiP peptide, indicating that the targeting peptide molecule can selectively induce calcification of lung cancer cells and has no significant effect on normal epithelial cells.

[0043] Figure 3 Changes in the proliferation activities of different lung cancer cells and normal lung epithelial cells after treatment with targeting peptides at different concentrations in Example 1. It can be seen that the calcification induced by the targeting peptide can selectively inhibit the proliferation activities of various lung cancer cells and has no significant toxic and side effects on normal lung epithelial cells.

[0044] Figure 4 Imaging changes of lung nodules in mice before and after treatment with tail vein injection of targeting peptide in Example 1 for lung cancer and pulmonary nodules. It can be seen that the calcification of non-small cell lung cancer induced by the targeting peptide shows high-density calcification foci on ultrasound, while the boundaries of conventional lung cancer and pulmonary nodules are unclear, and the calcified lung cancer can be clearly distinguished from the surrounding tissues, indicating that lung cancer calcification can help early ultrasound imaging of lung cancer and differential diagnosis with pulmonary nodules.

[0045] Figure 5 Imaging changes of lung nodules in mice before and after treatment with tail vein injection of targeting peptide in Example 1 for lung cancer and pulmonary nodules. It can be seen that the calcification of non-small cell lung cancer induced by the targeting peptide has clear boundaries on CT and can be clearly distinguished from the surrounding tissues, indicating that calcification can help early imaging of lung cancer and differential diagnosis with pulmonary nodules.

[0046] Figure 6 Imaging changes of the tumor mass in the lungs of mice after treatment with tail vein injection of targeting peptide in Example 1 for lung cancer mice after calcification induction. It can be seen that the calcification of non-small cell lung cancer induced by the targeting peptide under physiological calcium and phosphorus conditions can effectively inhibit the growth and metastasis of lung cancer.

[0047] Figure 7 Scanning electron microscopy images of ovarian cancer cells HeLa and normal ovarian epithelial cells Etc / E6E7 after treatment with Folate-polySia composite molecules in Example 2. It can be seen that compared with normal ovarian epithelial cells, a calcified layer is significantly formed on the surface of HeLa cells after treatment of ovarian cancer cells HeLa with Folate-polySia molecules, while the surface of normal ovarian epithelial cells Etc / E6E7 is smooth. This indicates that the Folate-polySia molecule can selectively induce calcification of ovarian cancer cells HeLa and has no significant effect on normal epithelial cells.

[0048] Figure 8Changes in the proliferation activities of HeLa cells and normal ovarian epithelial cells after treatment with different concentrations of Folate-polySia molecules in Example 2. It can be seen that the calcification induced by Folate-polySia molecules can selectively inhibit the proliferation activity of HeLa cells, while having no significant toxic side effects on normal ovarian epithelial cells.

[0049] Figure 9 For the changes in the volume of the tumor in mice of the HeLa cell subcutaneous xenograft model after intraperitoneal injection of Folate-polySia molecules in Example 2. It can be seen that under physiological calcium and phosphorus conditions, the calcification of HeLa cells induced by Folate-polySia molecules can effectively inhibit the growth of ovarian cancer cells in vivo.

[0050] Figure 10 For the changes in the survival cycle of mice in the HeLa cell subcutaneous xenograft model after intraperitoneal injection of Folate-polySia molecules in Example 2. It can be seen that under physiological calcium and phosphorus conditions, the calcification of HeLa cells induced by Folate-polySia composite molecules can effectively extend the survival time of mice bearing ovarian cancer cells.

[0051] Figure 11 For the calcification scan of the tumor in mice of the HeLa cell subcutaneous xenograft model after intraperitoneal injection of Folate-polySia composite molecules in Example 2. It can be seen that under physiological calcium and phosphorus conditions, intraperitoneal injection of Folate-polySia composite molecules can induce significant white calcified masses in the tumor shown on micro-CT.

[0052] Figure 12 Changes in the proliferation activities of HeLa drug-resistant cells after treatment with different concentrations of Folate-polySia molecules for 48 h in Example 2. It can be seen that the calcification induced by Folate-polySia molecules can effectively inhibit the proliferation activity of HeLa drug-resistant cells, indicating that the calcification induced by Folate-polySia molecules can kill drug-resistant HeLa cells again.

[0053] Figure 13 Changes in the proliferation activities of HeLa drug-resistant cells after treatment with different concentrations of Folate-polySia molecules for 72 h in Example 2. It can be seen that the calcification induced by Folate-polySia molecules after 72 h of treatment can have a stronger killing effect on HeLa drug-resistant cells. Figure 14After treating HeLa cisplatin-resistant cells with different concentrations of Folate-polySia molecules and different concentrations of cisplatin for 48 hours in Example 2, the changes in the viability of the cells were observed. It can be seen that the treatment with Folate-polySia molecules can significantly enhance the killing effect of cisplatin on the originally drug-resistant HeLa cells.

[0054] Figure 15 For the change in the volume of the tumor in mice after intraperitoneal injection of Folate-polySia molecules into the subcutaneous xenograft tumor model mice of HeLa drug-resistant cells in Example 2. It can be seen that under physiological calcium and phosphorus conditions, the calcification of HeLa cells induced by Folate-polySia molecules can effectively inhibit the growth of ovarian cancer drug-resistant cells in vivo.

[0055] Figure 16 For the change in the survival period of mice after intraperitoneal injection of Folate-polySia composite molecules into the subcutaneous xenograft tumor model mice of HeLa drug-resistant cells in Example 2. It can be seen that under physiological calcium and phosphorus conditions, the calcification of HeLa cells induced by Folate-polySia molecules can effectively extend the survival time of tumor-bearing mice with ovarian cancer drug-resistant cells in vivo.

[0056] Figure 17 For the calcification scan of the tumor in mice after intraperitoneal injection of Folate-polySia molecules into the subcutaneous xenograft tumor model mice of HeLa drug-resistant cells in Example 2. It can be seen that under physiological calcium and phosphorus conditions, intraperitoneal injection of Folate-polySia molecules can induce significant white calcified masses in the tumor on micro-CT, indicating that Folate-polySia molecules can significantly induce calcification of ovarian cancer drug-resistant cells in vivo.

[0057] Figure 18 For the change in the proliferation activity of pancreatic cancer cells (KPC, Panc02), human hepatoma cells (Hep1-6), and normal pancreatic epithelial cells (HPDE) treated with different concentrations of fucoidan in Example 3. It can be seen that the calcification induced by fucoidan molecules can selectively inhibit the proliferation activity of cancer cells, while having no significant toxic side effects on normal epithelial cells. Detailed implementation methods

[0058] Example 1:

[0059] A polypeptide targeting non-small cell lung cancer (ExTDSILRSYDWTY (x is any integer)), through calcification, inhibits the proliferation and metastasis of lung cancer and helps achieve early diagnosis and differential diagnosis of lung cancer.

[0060] In the following experiments of this example, unless otherwise specified, the polypeptide molecular sequence used is E 24TDSILRSYDWTY. (1) The targeting peptide induces selective calcification of non-small cell lung cancer cells under physiological conditions

[0061] The free amino group in the lysine residue of the targeting peptide reacts with fluorescein isothiocyanate (FITC) in PBS solution for 12 h, dialyzes through a dialysis bag for 5 days, and after freeze-drying, the targeting peptide molecule modified by FITC is obtained. 100 μg / ml of the above-mentioned FITC-modified targeting peptide is used to culture human non-small cell lung cancer cells A549, H460, H1299, human lung epithelial cells Beas-2b, and human umbilical vein endothelial cells HUVECs in vitro when the Ca 2+ concentration reaches 2.75 mM and the phosphate concentration ranges from 1.61 mM for 30 min, washes 3 times with PBS, and detects the targeting peptide bound to the cell surface by flow cytometry. As Figure 1 shown: Under in vitro physiological conditions, the targeting peptide selectively binds to the surface of lung cancer cells and has a weak binding force to normal cells.

[0062] (2) The targeting peptide induces selective calcification of non-small cell lung cancer cells under physiological conditions

[0063] The targeting peptide at a concentration of 1 mg / ml is used to culture human non-small cell lung cancer cell A549 in vitro when the Ca 2+ concentration reaches 2.75 mM and the phosphate concentration ranges from 1.61 mM for 48 h, fixes with 2.5% glutaraldehyde for 48 h, dehydrates and dries, and then scans by electron microscopy to detect the calcification on the cell surface. EDX elemental scanning shows that there are crystalline calcified substances on the cell membrane surface of lung cancer cells after treatment with the targeting peptide, and the calcification peaks increase significantly, while the deposition of calcium and phosphorus elements on the surface of lung epithelial cells is less ( Figure 2 ). It shows that the targeting peptide can induce selective calcification of non-small cell lung cancer cells under physiological conditions.

[0064] (3) The calcification induced by the targeting peptide can selectively kill lung cancer cells and inhibit the proliferation of cancer cells.

[0065] The targeting peptide at appropriate concentration gradients (0, 0.05, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 4.0, 8.0, 10, 20 mg / kg) is used to culture human non-small cell lung cancer cells A549, H460, H1299, and human lung epithelial cells Beas-2b in vitro when the Ca 2+ concentration reaches 2.75 mM and the phosphate concentration ranges from 1.61 mM for 48 h, and the CCK8 cell proliferation experiment is used to detect the killing effect of the calcification of lung cancer cells induced by the targeting peptide on lung cancer cells. As Figure 3 shown: The calcification of lung cancer cells induced by the targeting peptide can selectively inhibit the proliferation activities of various lung cancer cells, and has no significant toxic side effects on normal lung epithelial cells.

[0066] (4) Calcification of non-small cell lung cancer induced by targeting peptides can assist in the early imaging of lung cancer and the differential diagnosis from pulmonary nodules.

[0067] 50 μg of Sod A peptide (AAAIAGAFGSFDKFR) was mixed with 0.25 ml of incomplete Freund's adjuvant and subcutaneously injected into the back of C57 mice. Two weeks later, 50 μg of Sod A peptide and 6,000 Sepharose 4B beads were dissolved in 0.2 ml of PBS, and after covalent coupling, they were injected into the tail vein of C57 mice to establish a pulmonary nodule model of C57 mice. 3×10 6 A549-Luci cells were injected into the tail vein to establish a lung metastasis tumor model of nude mice. Targeting peptides at a concentration of 200 mg / kg were injected into the tail vein to induce calcification in the lung cancer of some mice. At 4 weeks, ultrasound and CT scans were performed on normal lung cancer, calcified lung cancer, and pulmonary nodules simultaneously. The imaging results showed ( Figure 4 ): Lung cancer with calcification induced by targeting peptides could be clearly presented on ultrasound; Calcified lung cancer could be imaged on CT at an early stage, while conventional lung cancer could not ( Figure 5 ); Due to insignificant calcification, pulmonary nodules were not clearly imaged. This indicates that calcification of non-small cell lung cancer induced by targeting peptides can assist in the early imaging of lung cancer on CT and the differential diagnosis from pulmonary nodules, improving the sensitivity of CT diagnosis of lung cancer.

[0068] (5) Calcification of non-small cell lung cancer induced by targeting peptides under physiological calcium and phosphorus conditions can effectively inhibit the growth and metastasis of lung cancer.

[0069] 3×10 6 A549-Luci cells were injected into the tail vein to establish a lung metastasis tumor model of nude mice. Targeting peptide molecular drug (CiP) at a concentration of 200 mg / kg was injected into the tail vein. The control group was injected with PBS, 4 mg / kg of doxorubicin (Dox), and a control peptide (TP) with the same molar concentration as the CiP peptide to clarify whether calcification of human A549 cells induced by the CiP targeting peptide could inhibit the growth and metastasis of lung cancer. A small animal in vivo imager was used to track and monitor the growth of the tumor mass. As Figure 6 shown, compared with the control group, intravenous injection of the CiP targeting peptide could significantly inhibit the growth of lung metastasis tumors of A549 cells.

[0070] Repeat the above experimental method, and use E 6 TDSILRSYDWTY, E 10 TDSILRSYDWTY, E 16 TDSILRSYDWTY, E 30TDSILRSYDWTY, it can be found that the tail vein injection of CiP targeting peptide can significantly inhibit the growth of A549 cell lung metastatic tumors. Therefore, as long as ExTDSILRSYDWTY, where x > 5, can significantly inhibit the growth of A549 cell lung metastatic tumors.

[0071] Example 2:

[0072] Folic acid-poly sialic acid molecules that selectively induce spontaneous calcification of tumor cells under physiological conditions and their applications in cancer diagnosis and treatment.

[0073] (1) Structure of folate receptor-targeted poly sialic acid composite molecular drugs.

[0074] The preparation reaction of Folate-polySia composite molecular drugs is as follows: 2 mmol of folic acid (FA) is dissolved in 20 mL of dry DMSO, 1 mL of redistilled triethylamine is added to assist dissolution, and 4 mmol of N,N'-carbonyldiimidazole is added. Stir at room temperature for 1 h. Monitor the reaction of N,N'-carbonyldiimidazole with folic acid by thin-layer chromatography. Using dichloromethane:methanol = 3:1 as the developing agent, extract the reaction solution with ethyl acetate and develop color with an iodine cylinder, Rf CDI is 0.8. Dissolve 4 mmol of N-(2-aminoethyl)carbamic acid tert-butyl ester (EDA-Boc) in 1 mL of redistilled dichloromethane and drop it into the above reaction solution. Stir at room temperature and react overnight. Monitor the reaction of EDA-Boc by thin-layer chromatography, Rf Boc-NH2 is 0.5. Drop the reaction solution into ether for precipitation, wash it three times with ethyl acetate, and dry it with an oil pump. Crush the solid powder and dissolve it in 8.8 mL of dichloromethane, add 8.8 mL of trifluoroacetic acid, and bubbles are generated. After 2 h, detect the completion of the reaction of FA-(EDA-Boc)2 by thin-layer chromatography with methanol as the developing agent, and the Rf value is 5 / 6. Rotate and evaporate to remove dichloromethane and trifluoroacetic acid, drop the oily product into ether, crush it, wash it three times with ether, and dry it with an oil pump to obtain the product (N-(2-aminoethyl))2 folic acid (FA-(EDA) 2 ). Add 1.6 mmol of PSA-COOH to 10 mL of DMSO, add 1 mL of triethylamine to assist dissolution, add 2 mmol of N,N'-carbonyldiimidazole and react at room temperature for 24 h. Drop 0.5 mL of distilled water to quench the reaction, and add 1.6 mmol of the above-synthesized FA-(NH2) 2 , react at room temperature for 24 h, precipitate in ethanol, dialyze and freeze-dry to obtain the Folate-polySia composite molecule. The molecular formula of the composite molecule is shown in the figure: It contains a folate receptor targeting part and a calcification induction functional region, and the calcification functional region is a repeating sequence of multiple poly sialic acid monomers.

[0075]

[0076] (2) The polysialic acid composite molecule drug can selectively induce calcification in cervical cancer cell lines with high expression of folate receptor.

[0077] The Folate-polySia composite molecule at a concentration of 1 mg / ml was cultured with human cervical cancer cell line HeLa with high expression of folate receptor and normal cervical epithelium Ect1 / E6E7 cells for 48 hours in vitro under a Ca 2+ concentration of 2.75 mM and a phosphate concentration range of 1.61

[0078] mM. As Figure 7 shown: Under scanning electron microscopy, obvious crystalline calcifications were observed on the surface of the cervical cancer cell membrane, while no calcification occurred in normal cervical epithelial cells, indicating that the polysialic acid anti-tumor drug can selectively induce calcification in cervical cancer cell lines with high expression of folate receptor.

[0079] (3) Calcification induced by the polysialic acid composite molecule under in vitro physiological conditions can selectively kill cervical cancer cells.

[0080] The Folate-polySia composite molecule at concentration gradients (0, 0.001, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 μM) was cultured with human cervical cancer cell line HeLa with high expression of folate receptor and normal cervical epithelium Ect1 / E6E7 cells for 48 h in vitro under a Ca 2+ concentration of 2.75 mM and a phosphate concentration range of 1.61 mM. The CCK8 assay was used to detect the killing effect of the Folate-polySia composite molecule on cancer cells. As Figure 8 shown: Calcification induced by the polysialic acid composite molecule (Folate-polySia) can selectively kill cervical cancer cells and has no significant effect on normal cervical epithelium Ect1 / E6E7 cells.

[0081] (4) Under in vivo physiological conditions, the polysialic acid composite molecule drug can inhibit the proliferation of cervical cancer cells with high expression of folate receptor through calcification.

[0082] 5×10 6 HeLa cells were subcutaneously injected to establish a subcutaneous xenograft tumor model of the HeLa cell line with high expression of folate receptor in nude mice. Each mouse was intraperitoneally injected with 16.7 μmol / kg of the polysialic acid composite molecule drug, the control group was injected with PBS, equimolar concentration of Folate (folic acid) and 5 mg / kg of doxorubicin (Dox), to clarify whether the calcification induced by the polysialic acid composite molecule drug in cervical cancer can inhibit the growth of xenograft tumors in nude mice. As Figure 9 shown: The intraperitoneally injected polysialic acid composite molecule drug can significantly inhibit the growth of the xenograft tumor; significantly prolong the survival time of nude mice ( Figure 10) Micro-CT calcium scanning of the mouse tumor showed that significant calcification occurred in the tumors of the mice in the polysialic acid composite molecule drug injection group ( Figure 11 ).

[0083] (5) Under physiological conditions, the polysialic acid composite molecule drug can reverse the chemoresistance of cervical cancer cells with high folate receptor expression through calcification.

[0084] Folate-polySia composite molecule drugs with concentration gradients (0, 0.70, 1.05, 1.58, 2.37, 3.56, 5.33, 8.0, 12.0 mg / ml) were cultured in vitro in Ca 2+ When the concentration reached 2.75 mM and the phosphate concentration range was 1.61 mM, the human cervical cancer cisplatin-resistant strain (HeLa / DDP) with high folate receptor expression was cultured for 48 hours ( Figure 12 ) and 72 hours ( Figure 13 ), and the CCK8 experiment was used to detect the killing effect of Folate-polySia composite molecule drugs on cells; Folate-polySia composite molecule drugs with appropriate concentrations (0, 0.2, 0.5, 1.0 mg / ml) were cultured in vitro in Ca 2+ When the concentration reached 2.75 mM and the phosphate concentration range was 1.61 mM, after culturing the human cervical cancer cisplatin-resistant strain (HeLa / DDP) with high folate receptor expression for 48 hours, it was then treated with a certain concentration of cisplatin (10, 25, 50, 100 μg / ml). The Live / Dead staining experiment was used to observe the effect of cisplatin on the killing effect of calcified and non-calcified cells, and the Image J software was used to count the number of dead cells and calculate the killing effect ( Figure 14 ). As Figure 12 shown: Calcification induced by treatment with the polysialic acid composite molecule (Folate-polySia) for 48 hours can have a good killing effect on cisplatin-resistant cervical cancer cell lines; Calcification induced by treatment with the polysialic acid composite molecule (Folate-polySia) for 72 hours can have a stronger killing effect on cisplatin-resistant cervical cancer cell lines ( Figure 13 ); Low-concentration FA-PSA calcification induction treatment for 48 h can significantly increase the killing sensitivity of the chemotherapeutic drug cisplatin to cisplatin-resistant cell lines ( Figure 14 ). The polysialic acid composite molecule drug-induced calcification can significantly reverse the growth of subcutaneous transplanted tumors resistant to cisplatin in nude mice.

[0085] 5×10 6Subcutaneous injection of HeLa cells was performed to establish a subcutaneous xenograft model of HeLa cell line with high expression of folate receptor in nude mice. Each nude mouse was intraperitoneally injected with 16.7 μmol / kg of polysialic acid composite molecule drug (Folate-polySia) daily. The control group was injected with normal saline (Saline), 6.7 μmol / kg cisplatin once every five days, and 6.7 μmol / kg cisplatin plus 16.7 μmol / kg Folate-polySia daily by intraperitoneal injection, to clarify whether the calcification induced by Folate-polySia in human cervical cancer could increase the sensitivity of subcutaneous xenografts of cisplatin-resistant nude mice to the chemotherapeutic drug cisplatin. As Figure 15 shown, intraperitoneal injection of Folate-polySia at a certain concentration could significantly inhibit the growth of subcutaneous tumors of HeLa / DDP cells. The inhibitory effect of low-concentration Folate-polySia plus cisplatin on HeLa / DDP cells was more significant, and Folate-polySia increased the sensitivity to the chemotherapeutic drug cisplatin. In addition, the growth of subcutaneous tumors of HeLa / DDP cells inhibited by Folate-polySia could significantly prolong the survival time of nude mice( Figure 16 );

[0086] Micro-CT calcium scanning found that Folate-polySia could significantly induce calcification of drug-resistant HeLa / DDP cells( Figure 17 ).

[0087] Example 3:

[0088] Polysaccharide drug molecules that selectively induce tumor cell calcification under physiological conditions.

[0089] (1) Fucoidan structure targeting pancreatic cancer and liver cancer: Molecular formula (C 6 H 10 O 7 S) n , n depends on the molecular weight size,

[0090]

[0091] Fucoidan has a natural targeting property to P-selectin molecule, and P-selectin molecule is a tumor marker highly expressed in pancreatic cancer and liver cancer. This patent first reveals that by using the targeting property of fucoidan to P-selectin molecule highly expressed in pancreatic cancer and liver cancer, through the strongly negatively charged group (sulfonic acid group) in the fucoidan molecule, calcium and phosphate ions in the tumor microenvironment are adsorbed to achieve calcification encapsulation imaging and killing of cancer cells.

[0092] (2) Fucoidan can induce calcification of pancreatic cancer and liver cancer cells under in vitro physiological conditions

[0093] Fucoidan at a concentration of 2 mg / ml in vitro in Ca 2+ When cultured for 48 hours with a concentration of up to 2.75 mM and a phosphate concentration range of 1.61 mM, human pancreatic cancer cells Panc01 and normal pancreatic epithelial cells were fixed with 2.5% glutaraldehyde for 48 hours, dehydrated, dried, and then scanned by electron microscopy to detect cell surface calcification. EDX elemental scanning showed that after treatment with fucoidan, the calcification element peaks on the surface of the pancreatic cancer cell membrane of human pancreatic cancer cells Panc01 increased significantly, while the deposition of calcium and phosphorus elements on the surface of normal pancreatic epithelial cells HPDE was less (Table 1). This indicates that fucoidan can induce selective calcification of pancreatic cancer cells under physiological conditions.

[0094] Table 1 Changes in the content of main elements on the cell surface after treatment of cancer cells and normal epithelial cells with fucoidan

[0095] HPDE - fucoidantreatec

[0096]

[0097] Panc01 - fucoidantreated

[0098]

[0099] (3) Calcification induced by fucoidan molecules under in vitro physiological conditions can selectively kill pancreatic cancer and liver cancer cells.

[0100] Fucoidan molecules at appropriate concentration gradients (0, 0.5, 1.0, 2.0, 4.0 mg / ml) in vitro in Ca 2+ When cultured with a concentration of up to 2.75 mM and a phosphate concentration range of 1.61 mM, pancreatic cancer cells Panc02, liver cancer cells KPC and Hep1 - 6, and human normal pancreatic epithelial cells HPDE were cultured, and the CCK8 assay was used to detect the killing effect of fucoidan molecules on various cells. As Figure 18 shown:

[0101] Calcification induced by fucoidan molecules can significantly kill pancreatic cancer and liver cancer cells, and has no obvious toxic side effects on human normal pancreatic epithelial cells, indicating that calcification induced by fucoidan molecules has a good selective anti - tumor effect.

[0102] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present invention.

[0103]

[0104]

Claims

1. A molecule that selectively induces spontaneous calcification of tumor cells in the in vivo physiological environment, Characterized in that, The molecule that induces spontaneous calcification of tumor cells contains at least two basic units, one of which is a targeting functional unit, which is the targeting functional region of tumor cells, and the other basic unit is a calcification induction functional unit.

2. The molecule that induces spontaneous calcification of tumor cells according to claim 1, Characterized in that, The targeting functional unit is a polypeptide with specific tumor cell targeting or its cyclic peptide form.

3. The molecule that induces spontaneous calcification of tumor cells according to claim 1, Characterized in that, The polypeptide with specific tumor cell targeting is one or more of SP94 polypeptide targeting liver cancer cells, polypeptide TDSILRSYDWTY targeting lung cancer cells, or RGD peptide targeting tumor blood vessels.

4. The molecule that induces spontaneous calcification of tumor cells according to claim 1, Characterized in that, The calcification induction functional unit contains strongly negatively charged groups, and the strongly negatively charged groups are one or more of carboxyl group, sulfonic acid group, guanidine group, and phosphate group.

5. The molecule that induces spontaneous calcification of tumor cells according to claim 4, Characterized in that, The calcification induction functional unit is a repeated arrangement of the same functional groups containing strongly negatively charged groups or a combination of different functional groups containing strongly negatively charged groups.

6. The molecule that induces spontaneous calcification of tumor cells according to claim 5, Characterized in that, The calcification induction functional unit is a repeated unit of polyglutamic acid containing free carboxyl groups or a repeated sequence of casein phosphopeptides.

7. The molecule that induces spontaneous calcification of tumor cells according to claim 6, Characterized in that, The tumors include leukemia, lymphoma, multiple myeloma, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma and gallbladder cancer, lung cancer, pleural tumor; nervous system tumors such as glioma, neuroblastoma, meningioma, oral cancer, tongue cancer, laryngeal cancer, nasopharyngeal cancer, breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, penile cancer, kidney cancer, bladder cancer, skin cancer, melanoma, osteosarcoma, liposarcoma, thyroid cancer.

8. Use of the molecule that induces spontaneous calcification of tumor cells according to claims 1-7 in the preparation of tumor drugs.

9. A tumor drug, Characterized in that, The drug contains the molecule that induces spontaneous calcification of tumor cells according to claims 1-7.

10. A cancer vaccine, Characterized in that, The vaccine contains the molecule that induces spontaneous calcification of tumor cells according to claims 1-7.

Images