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] The patent application for this invention is application number 202111570242.0, application date December 21, 2021, and the name of the invention is “Induced tumor

[0002] Molecules for spontaneous calcification of tumor cells and their applications", the priority date is December 22, 2020, and the priority number is

[0003] Divisional application of invention patent applications CN202011532338.3 and CN202011532304.4 Technical Field

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

[0005] Chemotherapy is one of the most commonly used treatments 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 work by affecting cell growth and proliferation processes, they can also cause damage to normal cells. The side effects of systemic chemotherapy used to treat cancer are often severe and may lead to damage to the immune system, 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 still unclear, researchers have reported that tumor calcification and regional lymph node calcification are benign prognostic factors for colorectal cancer and lung cancer. In 2019, a research group at East China Normal University developed a method to induce tumor calcification using systemic injection of calcium peroxide nanoparticles. The problem of liver accumulation of nanoparticles used for drug delivery has always existed, so the safety of clinical translation of this method deserves further exploration.

[0006] In 2016, the inventors reported a method of treating leukemia with high-dose folic acid (FA) and Ca 2+ This may form a new strategy for cancer cell targeting calcification to treat tumors. However, each FA molecule can only provide two carboxyl residues to bind Ca in biological fluids. 2+ , to promote the nucleation of calcium minerals. Therefore, this method relies on Ca above the physiological range 2+ Levels, which may cause hypercalcemia crisis, and the prospects for clinical application are limited. Moreover, systemic injection of folic acid has also been shown to promote tumor growth, significantly reduce the weight of mice, and cause a sharp decrease in the survival time of mice. Summary of the invention

[0007] In order 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 through this molecule, tumor growth can be inhibited, the therapeutic effects of chemoradiotherapy and immunotherapy can be improved, and the contrast of imaging can be improved to achieve the goals of early diagnosis and precise diagnosis and treatment.

[0008] To achieve the purpose 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 / microenvironment, and the other basic unit is a calcification inducing functional unit; or the molecule that induces spontaneous calcification of tumor cells contains at least one basic unit, which is both a targeting functional unit and a calcification inducing unit.

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

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

[0012] The polypeptide having specific tumor cell targeting property in the present invention may be in a linear or circular form.

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

[0014] Furthermore, the ligand molecule targeting the receptor highly expressed in tumor cells is folic acid.

[0015] Furthermore, the ligand molecule targeting the receptor highly expressed in tumor cells is urokinase type plasminogen activator receptor (uPAR).

[0016] Furthermore, 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 (as shown in SEQ ID NO: 2), or the RGD peptide targeting tumor blood vessels, in the form of linear molecules or cyclic peptides thereof.

[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 contained in the targeting polypeptide molecule is greater than 5, it is possible to simultaneously satisfy both the targeting and calcification functions 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 simultaneously possess both the targeting and calcification functions.

[0019] The targeting portion of the polypeptide molecule is responsible for binding to the membrane protein highly expressed on the surface of the cancer cell membrane to prevent off-target effects. Therefore, due to the limited space of the present invention, it is not possible to list all of them. However, other targeting molecules not described in the present invention may also play the same role. The calcification functional end may be a repeating unit of polyglutamic acid containing a free carboxyl group (E n , n≥5), or it can be 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 produce calcification. Other molecules containing a large number of free carboxyl groups that are not elaborated in detail in the present invention can also play the same role.

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

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

[0022] Furthermore, the calcification-inducing functional unit contains a strong negatively charged group.

[0023] The calcification inducing functional unit retains a large number of strongly negatively charged groups, and other substitutions do not affect the calcification effect.

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

[0025] Furthermore, the calcification inducing functional unit is a repeated arrangement of the same functional group containing a strong negative charge gene or a combination of different functional groups containing a strong negative charge gene.

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

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

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

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

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

[0031] The molecules inducing spontaneous calcification of tumor cells provided by the present invention are suitable for all types of cancers, and the tumors can be selected from blood system tumors such as leukemia, lymphoma, and 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 and pleural tumors; nervous system tumors such as glioma, neuroblastoma, and meningioma; head and neck tumors such as oral cancer, tongue cancer, laryngeal cancer, and nasopharyngeal cancer; gynecological and reproductive system tumors such as breast cancer, ovarian cancer, cervical cancer, vulvar cancer, testicular cancer, prostate cancer, and penis cancer; urinary system tumors such as kidney cancer and bladder cancer, skin and other systems such as skin cancer, melanoma, osteosarcoma, liposarcoma, and thyroid cancer.

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

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

[0034] The tumor drug contains not only the molecule that induces spontaneous calcification of tumor cells of the present invention, but also necessary auxiliary materials and may also contain other therapeutic drugs.

[0035] Furthermore, the tumor drug can administer the calcification-inducing molecule orally, intravenously, intratumorally, or intravenously.

[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 of tumor cells. The molecule is generally composed of two functional regions, one of which is a tumor targeting functional region and the other is a calcification inducing functional region. The 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 exercises an inducing effect on cell calcification in a physiological environment. After tumor cells are calcified, on the one hand, calcium salt deposition on the cell surface will affect physiological processes such as the metabolism of tumor cells, further induce tumor cell apoptosis, and play a role in tumor inhibition and treatment; on the other hand, the contrast of calcified tumor cells and tissues in clinical images is improved, which is convenient for early diagnosis and accurate diagnosis of tumor lesions.

[0038] The molecules that induce spontaneous calcification of tumor cells provided by the present invention can inhibit tumor growth, reverse the drug resistance of other tumor chemotherapy drugs, synergistically improve tumor radiotherapy, synergistically improve tumor immunotherapy, and have the efficacy of vaccines. In addition, by administering the calcification-inducing molecules of the present invention orally, intravenously, intratumorally, or by lymph node intervention, the image contrast of clinical imaging, such as computed tomography (CT), ultrasound, positron emission tomography-computed tomography (PET-CT), and magnetic resonance imaging (MRI), can be changed (increased or reduced), so as to detect lesions at an earlier stage or distinguish between benign and malignant tumor lesions.

[0039] The present invention can be seen through animal experiments that this type of molecule contains two functional areas, one is the targeting functional area of ​​the cancer cell membrane surface receptor, which can selectively target the cancer cell membrane without targeting normal cells. The other end is a functional area with the ability to induce calcification, which can selectively induce calcium and phosphorus ions in the microenvironment to deposit on the cancer cell membrane, causing tumor cells to calcify. 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 images, and detect and identify tumor micro-lesions earlier and more accurately.

[0040] In summary, the molecules disclosed in the present invention selectively induce calcification of tumor cells, play a role in tumor treatment, inhibit tumor growth, reverse tumor resistance, improve the effects of tumor radiotherapy, chemotherapy and immunotherapy, and prolong the survival of tumor patients; at the same time, the calcification of tumor cells and tissues can improve the imaging contrast of clinical images, and detect and identify tiny tumor lesions earlier and more accurately. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The flow cytometer was used to detect the targeting peptides bound to the surfaces of different cells in Example 1. The rightward shift of the peak value indicates that the targeting peptide molecules selectively bind to the surface of lung cancer cells, but not to normal epithelial cells.

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

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

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

[0045] Figure 5 The imaging changes of lung nodules in mice treated with lung cancer and lung nodules by tail vein injection of the targeting peptide in Example 1 on CT. 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 from lung nodules.

[0046] Figure 6 The imaging changes of lung tumors in mice treated with tail vein injection of the targeting peptide in Example 1 on the small animal in vivo imaging device after calcification induction. It can be seen that the calcification of non-small cell lung cancer induced by the targeting peptide under physiological concentrations of calcium and phosphorus can effectively inhibit the growth and metastasis of lung cancer.

[0047] Figure 7 The scanning electron microscope images of ovarian cancer cells HeLa and normal ovarian epithelial cells Etc / E6E7 after being treated with the Folate-polySia compound molecule in Example 2. It can be seen that compared with normal ovarian epithelial cells, a calcification layer is significantly formed on the surface of ovarian cancer cells HeLa after being treated with the Folate-polySia molecule, 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, but has no significant effect on normal epithelial cells.

[0048] Figure 8The figure shows the changes in the proliferation activity 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] Fig. 9 This is the volume change of the tumor in mice with HeLa cell subcutaneous transplanted tumor model injected intraperitoneally with Folate-polySia molecules in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, the calcification of HeLa cells induced by Folate-polySia molecules can effectively inhibit the growth of ovarian cancer cells in vivo.

[0050] Fig.10 This is the change in the survival period of mice with HeLa cell subcutaneous transplanted tumor model injected intraperitoneally with Folate-polySia molecules in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, the HeLa cell calcification induced by the Folate-polySia composite molecule can effectively prolong the survival time of ovarian cancer cell-bearing mice in vivo.

[0051] Fig.11 This is a scan of the calcification of the tumor in mice with Hela cell subcutaneous transplanted tumor model injected intraperitoneally with Folate-polySia composite molecules in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, intraperitoneal injection of Folate-polySia composite molecules can induce tumors to show significant white calcification blocks on micro-CT.

[0052] Fig.12 The figure shows the changes in the proliferation activity of HeLa resistant cells after 48 hours of treatment with different concentrations of Folate-polySia molecules in Example 2. It can be seen that the calcification induced by Folate-polySia molecules can effectively inhibit the proliferation activity of HeLa resistant cells, indicating that the calcification induced by Folate-polySia molecules can kill the resistant HeLa cells again.

[0053] Fig.13 The figure shows the changes in the proliferation activity of HeLa drug-resistant cells after 72 hours of treatment with different concentrations of Folate-polySia molecules in Example 2. It can be seen that the calcification induced by Folate-polySia molecule treatment for 72 hours can produce a stronger killing effect on HeLa drug-resistant cells. Fig.14The changes in cell death and viability after 48 hours of treatment of HeLa cisplatin-resistant cells with different concentrations of Folate-polySia molecules and different concentrations of cisplatin in Example 2. It can be seen that Folate-polySia molecule treatment can significantly promote cisplatin to produce a stronger killing effect on the originally resistant HeLa cells.

[0054] Fig.15 This is the volume change of the tumor in mice with subcutaneous transplanted tumor model of HeLa resistant cells injected intraperitoneally with Folate-polySia molecules in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, the calcification of HeLa cells induced by Folate-polySia molecules can effectively inhibit the growth of ovarian cancer resistant cells in vivo.

[0055] Fig.16 This is the change in the survival period of mice bearing subcutaneous transplanted tumors of HeLa resistant cells after intraperitoneal injection of the Folate-polySia composite molecule in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, the calcification of HeLa cells induced by the Folate-polySia molecule can effectively prolong the survival time of mice bearing ovarian cancer resistant cells in vivo.

[0056] Fig.17 This is the calcification scan of the mouse tumor after intraperitoneal injection of Folate-polySia molecules into the HeLa resistant cell subcutaneous transplanted tumor model mouse in Example 2. It can be seen that under physiological concentrations of calcium and phosphorus, intraperitoneal injection of Folate-polySia molecules can induce the tumor to show significant white calcification blocks on micro-CT, indicating that Folate-polySia molecules can significantly induce calcification of ovarian cancer resistant cells in vivo.

[0057] Fig.18 The changes in the proliferation activity of pancreatic cancer cells (KPC, Panc02), human liver cancer 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 DESCRIPTION

[0058] Embodiment 1:

[0059] The peptide targeting non-small cell lung cancer (ExTDSILRSYDWTY (x is an arbitrary integer) inhibits the proliferation and metastasis of lung cancer through calcification, and helps to achieve early diagnosis and differential diagnosis of lung cancer.

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

[0061] The free amino groups in the lysine residues of the targeting peptides were reacted with fluorescein isothiocyanate (FITC) in PBS solution for 12 h, dialyzed in a dialysis bag for 5 days, and freeze-dried to obtain the FITC-modified targeting peptide molecules. 100 μg / ml of the FITC-modified targeting peptides were detected in vitro. 2+ When the concentration reached 2.75mM and the phosphate concentration range was 1.61mM, human non-small cell lung cancer cells A549, H460, H1299, human lung epithelial cells Beas-2b and human umbilical vein endothelial cells HUVECs were cultured for 30min, washed with PBS three times, and the targeting peptides bound to the cell surface were detected by flow cytometry. Figure 1 As shown: Under in vitro physiological conditions, the targeting peptide selectively binds to the surface of lung cancer cells and has weak binding to normal cells.

[0062] (2) Targeted peptides induce selective calcification of non-small cell lung cancer cells under physiological conditions

[0063] Targeting peptide at a concentration of 1 mg / ml was activated in vitro by Ca 2+ The human non-small cell lung cancer A549 cells were cultured for 48 hours at a concentration of 2.75mM and a phosphate concentration range of 1.61mM, and then fixed with 2.5% glutaraldehyde for 48 hours. After dehydration and drying, the cells were scanned by electron microscopy to detect the calcification on the cell surface. EDX element scanning showed that after lung cancer cells were treated with targeted peptides, crystalline calcification was present on the cell membrane surface, and the calcification peak increased significantly, while calcium and phosphate deposition on the surface of lung epithelial cells was less ( Figure 2 ). This indicates that the targeting peptide can induce selective calcification of non-small cell lung cancer cells under physiological conditions.

[0064] (3) Calcification induced by targeted peptides can selectively kill lung cancer cells and inhibit the proliferation of cancer cells.

[0065] The targeting peptides with appropriate concentration gradient (0, 0.05, 0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 4.0, 8.0, 10, 20 mg / kg) were used for in vitro Ca 2+ Human non-small cell lung cancer cells A549, H460, H1299 and human lung epithelial cells Beas-2b were cultured for 48 hours at a concentration of 2.75 mM and a phosphate concentration range of 1.61 mM. CCK8 cell proliferation assay was used to detect the killing effect of lung cancer cell calcification induced by the targeting peptide on lung cancer cells. Figure 3 As shown: Targeted peptide-induced lung cancer cell calcification can selectively inhibit the proliferation activity of a variety of lung cancer cells without significant toxic side effects on normal lung epithelial cells.

[0066] (4) Targeted peptide-induced calcification of non-small cell lung cancer can help with early imaging of lung cancer and differential diagnosis from lung nodules.

[0067] 50 μg Sod A peptide (AAAIAGAFGSFDKFR) was mixed with 0.25 ml of incomplete Freund's adjuvant and injected subcutaneously into the back of C57 mice. Two weeks later, 50 μg Sod A peptide was dissolved in 0.2 ml PBS with 6000 agarose 4B beads and covalently coupled to the tail vein of C57 mice to construct a lung nodule model in C57 mice. 6 The lung metastasis model of nude mice was established by tail vein injection of A549-Luci cells. Tail vein injection of 200mg / kg of targeted peptide induced calcification of lung cancer in some mice. Ultrasound and CT scans were performed on normal lung cancer, calcified lung cancer and lung nodules at 4 weeks. The imaging results showed that ( Figure 4 ): Calcified lung cancer induced by targeted peptides can be clearly presented on ultrasound; calcified lung cancer can be imaged on CT in the early stage, while conventional lung cancer cannot ( Figure 5 ); The calcification of lung nodules is not obvious and the imaging is not clear. This shows that the calcification of non-small cell lung cancer induced by targeted peptides can help the early imaging of lung cancer on CT and the differential diagnosis of lung nodules, thereby improving the sensitivity of CT diagnosis of lung cancer.

[0068] (5) Targeted peptide-induced non-small cell lung cancer calcification under physiological concentrations of calcium and phosphate conditions can effectively inhibit the growth and metastasis of lung cancer.

[0069] 3×10 6 A lung metastasis model of nude mice was constructed by tail vein injection of A549-Luci cells, and a targeted peptide molecule 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 doxorubicin (Dox) and a control peptide (TP) at an equimolar concentration to the CiP peptide to determine whether the calcification of human A549 cells induced by the CiP targeted peptide can inhibit the growth and metastasis of lung cancer. The small animal in vivo imaging device was used to track and monitor the tumor production. Figure 6 As shown, tail vein injection of CiP targeting peptides could significantly inhibit the growth of A549 cell lung metastases compared with the control group.

[0070] Repeat the above experimental method, using E 6 TDSILRSYDWTY,E 10 TDSILRSYDWTY,E 16 TDSILRSYDWTY,E 30TDSILRSYDWTY, it can be found that the tail vein injection of the CiP targeting peptide can significantly inhibit the growth of A549 cell lung metastases. Therefore, as long as ExTDSILRSYDWTY is satisfied, where x > 5, it can play a significant role in inhibiting the growth of A549 cell lung metastases.

[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 the Folate-polySia composite molecular drug 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 the 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 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 will be 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. 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. The calcification functional region is a repetitive sequence of multiple poly sialic acid monomers.

[0075]

[0076] (2) Polysialic acid complex molecule drugs can selectively induce calcification in cervical cancer cell lines with high expression of folate receptors.

[0077] 1mg / ml Folate-polySia complex molecule in vitro Ca 2+ Concentrations up to 2.75mM, phosphate concentration range 1.61

[0078] mM, human cervical cancer cell line HeLa with high expression of folate receptor and normal cervical epithelial Ect1 / E6E7 cells were cultured for 48 h. Figure 7 As shown: Scanning electron microscopy revealed obvious crystalline calcifications on the membrane surface of cervical cancer cells, while normal cervical epithelial cells showed no calcification, indicating that polysialic acid anti-tumor drugs can selectively induce calcification in cervical cancer cell lines with high expression of folate receptors.

[0079] (3) Under physiological conditions in vitro, calcification induced by polysialic acid complex molecules can selectively kill cervical cancer cells.

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

[0081] (4) Under physiological conditions in vivo, polysialic acid complex molecular drugs can inhibit the proliferation of cervical cancer cells with high expression of folate receptors through calcification.

[0082] 5×10 6 The subcutaneous injection of HeLa cells was used to construct a subcutaneous transplant tumor model of nude mice with high expression of HeLa cell line of folate receptor. Each mouse was given 16.7μmol / kg of polysialic acid compound molecule drug intraperitoneally. The control group was injected with PBS, equimolar concentration of Folate (folic acid) and 5mg / kg of doxorubicin (Dox) to determine whether the cervical cancer calcification induced by polysialic acid compound molecule drug can inhibit the growth of transplant tumors in nude mice. Fig. 9 As shown: Intraperitoneal injection of polysialic acid complex molecule drugs can significantly inhibit the growth of transplanted tumors and significantly prolong the survival time of nude mice ( Fig.10); micro-CT calcium scans of mouse tumors revealed that significant calcification occurred in the tumors of mice injected with polysialic acid complex molecules ( Fig.11 ).

[0083] (5) Under physiological conditions, polysialic acid complex molecular drugs can reverse the chemotherapy resistance of cervical cancer cells with high expression of folate receptors through calcification.

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

[0085] 5×10 6HeLa cells were injected subcutaneously to construct a subcutaneous transplant tumor model of nude mice with high expression of HeLa cell line of folate receptor. Each nude mouse was given 16.7μmol / kg of polysialic acid complex molecule drug (Folate-polySia) intraperitoneally every day. The control group was injected with saline (Saline), 6.7μmol / kg cisplatin once every five days, and 6.7μmol / kg cisplatin plus 16.7μmol / kg Folate-polySia intraperitoneally every five days to determine whether Folate-polySia-induced human cervical cancer calcification can increase the sensitivity of cisplatin-resistant nude mouse subcutaneous transplant tumors to the chemotherapy drug cisplatin. Fig.15 As shown, intraperitoneal injection of a certain concentration of Folate-polySia can significantly inhibit the growth of HeLa / DDP cell subcutaneous tumors. The inhibitory effect of low concentration Folate-polySia plus cisplatin on HeLa / DDP cells is more significant. Folate-polySia increases the sensitivity to the chemotherapy drug cisplatin. In addition, the growth of HeLa / DDP cell subcutaneous tumors inhibited by Folate-polySia can significantly prolong the survival time of nude mice ( Fig.16 );

[0086] Micro-CT calcium scanning revealed that Folate-polySia can significantly induce calcification in drug-resistant HeLa / DDP cells ( Fig.17 ).

[0087] Embodiment 3:

[0088] Polysaccharide drug molecules that selectively induce calcification of tumor cells 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,

[0090]

[0091] Fucoidan has natural targeting to P-selectin molecules, which are tumor markers highly expressed in pancreatic cancer and liver cancer. This patent first reveals that fucoidan can be used to target P-selectin molecules highly expressed in pancreatic cancer and liver cancer, and that the strongly negatively charged groups (sulfonic acid groups) in fucoidan molecules can be used to adsorb calcium and phosphorus ions in the tumor microenvironment, thereby achieving calcification, encapsulation, imaging, and killing of cancer cells.

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

[0093] 2mg / ml concentration of fucoidan in vitro Ca 2+ Human pancreatic cancer cells Panc01 and normal pancreatic epithelial cells were cultured for 48 hours at a concentration of 2.75mM and a phosphate concentration range of 1.61mM, and fixed with 2.5% glutaraldehyde for 48 hours. After dehydration and drying, electron microscopy was used to detect cell surface calcification. EDX element scanning showed that the calcification element peak on the surface of the pancreatic cancer cell membrane of human pancreatic cancer cells Panc01 increased significantly after being treated with fucoidan, while calcium and phosphorus elements were less deposited on the surface of normal pancreatic epithelial cells HPDE (Table 1). This shows that fucoidan can induce selective calcification of pancreatic cancer cells under physiological conditions.

[0094] Table 1 Changes in the contents of major elements on the cell surface of cancer cells and normal epithelial cells after HPDE-fucoidan treatment

[0095]

[0096] Panc01-fucoidan treated

[0097]

[0098] (3) Calcification induced by fucoidan molecules under in vitro physiological conditions can selectively kill pancreatic cancer and liver cancer cells.

[0099] The appropriate concentration gradient (0, 0.5, 1.0, 2.0, 4.0 mg / ml) of fucoidan molecules in vitro Ca 2+ When the concentration reached 2.75mM and the phosphate concentration range was 1.61mM, pancreatic cancer cells Panc02, liver cancer cells KPC and Hep1-6, and human pancreatic normal epithelial cells HPDE were cultured, and CCK8 experiments were used to detect the killing effect of fucoidan molecules on various cells. Fig.18 As shown:

[0100] The calcification induced by fucoidan molecules can significantly kill pancreatic cancer and liver cancer cells, and has no obvious toxic side effects on normal human pancreatic epithelial cells, indicating that the calcification induced by fucoidan molecules has a good selective anti-tumor effect.

[0101] In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to the present invention.

[0102]

[0103]

Claims

1. Molecules that induce spontaneous calcification of tumor cells, It is 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 a targeting functional area of ​​tumor cells, and the other is a calcification inducing functional unit; the targeting functional unit is one or more of an antibody targeting specific antigens on the surface of tumor cells, a ligand molecule targeting a receptor highly expressed by tumor cells, a nucleic acid aptamer with specific tumor cell targeting, and a molecule targeting a specific tumor microenvironment.

2. The molecule that induces spontaneous calcification of tumor cells according to claim 1, It is characterized in that The antibody targeting specific antigens on the surface of tumor cells is HER-2 antibody and / or EGFR antibody.

3. The molecule that induces spontaneous calcification of tumor cells according to claim 1, It is characterized in that The ligand molecule targeting the receptor highly expressed in tumor cells is urokinase-type plasminogen activator receptor.

4. The molecule that induces spontaneous calcification of tumor cells according to claim 1, It is characterized in that The molecules targeting the specific microenvironment of tumors are polypeptide-hydrophobic hydrocarbon chain-hydrophilic chain cleaved in response to MMP and / or phosphate-hydrophobic hydrocarbon chain-hydrophilic chain cleaved in response to alkaline phosphatase.

5. The molecule that induces spontaneous calcification of tumor cells according to claim 1, It is characterized in that The tumors include leukemia, lymphoma, multiple myeloma, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct 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, and thyroid cancer.

6. Use of the molecules inducing spontaneous calcification of tumor cells as claimed in claims 1 to 5 in the preparation of tumor drugs. 7.Tumor drugs, It is characterized in that The drug comprises the molecule that induces spontaneous calcification of tumor cells as described in claims 1-5.

8. The tumor drug according to claim 7, It is characterized in that The calcification-inducing molecule is administered orally, intravenously, intratumorally, or intravenously.

9. Cancer vaccines, It is characterized in that The vaccine comprises the molecules that induce spontaneous calcification of tumor cells as described in claims 1-8.

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