Drug bag for rapid mineralization of tumor cells, and preparation method and application thereof

Abstract

The invention relates to the technical field of chemical and biological materials and cell preparations, in particular to a medicine bag for rapid mineralization of tumor cells as well as a preparation method and application of the medicine bag. The drug for rapid mineralization of tumor cells comprises a first component and a second component, the first component comprises inorganic nanoparticles modified by a supramolecular host, and the second component comprises a supramolecular guest. The mineralizable inorganic ions in the medicine bag can be rapidly gathered on the surfaces of tumor cells through interaction of supramolecular main bodies, the tumor cells are induced to be rapidly mineralized, a mineralization blocking layer is formed, and medicine-free and low-side-effect treatment of tumors is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of chemistry, biological materials and cell preparations, and in particular to a drug package for rapid mineralization of tumor cells, a preparation method thereof and an application thereof. Background Art

[0002] Cancer seriously threatens human life and health, and achieving effective cancer treatment remains a major challenge facing the biomedical community. At present, the main clinical treatments for cancer are surgical resection, chemotherapy, and radiotherapy, but they all have certain limitations, such as surgical resection is traumatic and prone to tumor metastasis, chemotherapy has severe adverse reactions and is prone to drug resistance, and radiotherapy is prone to induce acute reactions and radiation damage.

[0003] Tumor cell mineralization can effectively block the exchange of nutrients and information between the tumor and the surrounding microenvironment, thereby inhibiting the proliferation, migration and invasion of tumor cells, and can achieve drug-free and low-side effect treatment of tumors. It is a new non-permeable tumor blocking treatment method with broad application prospects. The currently reported tumor cell mineralization methods mainly deliver mineralization carriers to the tumor site and induce the free calcium ions, iron ions and / or manganese ions in the blood to deposit and mineralize on the surface of tumor cells. However, due to the poor targeting of mineralization carriers, insufficient enrichment on the surface of tumor cells, and the low concentration of mineralizable ions such as calcium ions, iron ions and / or manganese ions in the blood, additional injection of high-concentration mineralizable ions as a mineralization source is required, resulting in a slow mineralization process, poor treatment effect, and the risk of inducing high blood mineralized ions, which limits its clinical application.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a drug package for rapid mineralization of tumor cells, a preparation method thereof and an application thereof. The mineralizable inorganic ions in the drug package provided in the embodiment of the present invention can rapidly aggregate on the surface of tumor cells through supramolecular host interactions, induce rapid mineralization of tumor cells and form a mineralization blocking layer, thereby achieving drug-free and low-side effect treatment of tumors.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a drug package for rapid mineralization of tumor cells, comprising a first component and a second component, wherein the first component comprises inorganic nanoparticles modified by a supramolecular host, and the second component comprises a supramolecular guest.

[0008] In an optional embodiment, the mass ratio of the first component to the second component is (10-100):1.

[0009] In an optional embodiment, the supramolecular guest is a polymer formed by guest molecules modifying a polymer matrix;

[0010] Preferably, the guest molecule is selected from any one of adamantane, ferrocene, phenylalanine and hexamethylenediamine;

[0011] Preferably, the polymer matrix is ​​selected from liposomes, preferably 1,2-distearoyl-sn-polyethylene glycol (DAPE-PEG) or cholesterol-polyethylene glycol (Chol-PEG);

[0012] Preferably, the supramolecular guest is selected from any one of adamantane-modified 1,2-distearoyl-sn-polyethylene glycol (DSPE-PEG-ADA) or ferrocene-modified 1,2-distearoyl-sn-polyethylene glycol (DSPE-PEG-Ferrocene).

[0013] In an optional embodiment, the inorganic ions in the supramolecular host-modified inorganic nanoparticles are derived from calcium, iron or manganese;

[0014] Preferably, the inorganic ions are derived from any one of calcium carbonate, calcium phosphate, calcium oxide, ferroferric oxide and manganese dioxide;

[0015] Preferably, the inorganic ions are derived from an inorganic ion source modified by a polymer containing hydroxyl and / or amino groups;

[0016] Preferably, the inorganic ions are derived from a calcium source modified by a polymer containing hydroxyl and / or amino groups;

[0017] Preferably, the inorganic ions are derived from any one of PDA-modified calcium carbonate, PLA-modified calcium carbonate, PEG-modified calcium carbonate, PDA-modified calcium phosphate, PLA-modified calcium phosphate, PEG-modified calcium phosphate, PDA-modified calcium oxide, PLA-modified calcium oxide, PEG-modified calcium oxide, PDA-modified ferroferric oxide, PLA-modified ferroferric oxide, PEG-modified ferroferric oxide, PDA-modified manganese dioxide, PLA-modified manganese dioxide and PEG-modified manganese dioxide.

[0018] In an optional embodiment, the host in the supramolecular host-modified inorganic nanoparticles is selected from any one of cyclodextrin, cucurbituril, calixarene, pillararene or crown ether.

[0019] In a second aspect, the present invention provides a use of the drug package for rapid mineralization of tumor cells described in the aforementioned embodiment in the preparation of a drug for treating tumors;

[0020] Preferably, the tumor includes any one of osteosarcoma cells, breast cancer cells, and melanoma cells.

[0021] In a third aspect, the present invention provides a method for preparing the drug package for rapid mineralization of tumor cells described in the aforementioned embodiment, comprising: forming the drug package using a first component and a second component.

[0022] In an optional embodiment, the preparation method of the first component comprises: mixing an inorganic ion source modified with a polymer containing hydroxyl groups and / or amino groups, a host and an activator to react;

[0023] Preferably, the inorganic ion source modified with the polymer containing hydroxyl and / or amino groups and the activator are mixed and stirred, and then added to the main body for reaction.

[0024] In an optional embodiment, the activator is selected from amine reagents, preferably at least one of EDC and DCC.

[0025] In an optional embodiment, the mass ratio of the inorganic ion source modified by the polymer containing hydroxyl groups and / or amino groups to the activator is 1:(2-5);

[0026] The mass ratio of the inorganic ion source modified by the polymer containing hydroxyl groups and / or amino groups to the main body is 1:(2-5).

[0027] The present invention has the following beneficial effects:

[0028] (1) The mineralizable inorganic ions in the drug package can rapidly aggregate on the surface of tumor cells through supramolecular interactions, inducing rapid mineralization of tumor cells and forming a mineralization blocking layer, thus achieving drug-free and low-side effect treatment of tumors.

[0029] (2) The embodiments of the present invention do not rely on mineralizable ions in the blood as a mineralization source, but directly aggregate the mineralizable ions on the surface of tumor cells, thereby avoiding a slow mineralization process and achieving rapid mineralization of tumor cells.

[0030] (3) The second component in the drug package provided in the embodiment of the present invention can be modified onto the surface of tumor cells through cell membrane intercalation, which is beneficial to the role of host and guest, and is beneficial to the enrichment of mineralizable ions, which is beneficial to improving the targeting of the drug package and reducing its therapeutic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0032] Figure 1It is the experimental result diagram of Experimental Example 1 of the present invention;

[0033] Figure 2 It is the experimental result diagram of Experimental Example 2 of the present invention;

[0034] Figure 3 It is the experimental result diagram of Experimental Example 3 of the present invention;

[0035] Figure 4 It is the experimental result diagram of Experimental Example 4 of the present invention;

[0036] Figure 5 This is a diagram of the experimental results of Experimental Example 5 of the present invention. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] The embodiment of the present invention provides a drug package for rapid mineralization of tumor cells, which includes a first component and a second component, wherein the first component includes inorganic nanoparticles modified by a supramolecular host, and the second component includes a supramolecular guest. Inorganic ions can rapidly aggregate on the surface of tumor cells through the strong interaction between the supramolecular host and the guest, induce rapid mineralization of tumor cells, and achieve tumor blocking treatment. The embodiment of the present invention improves the problem of poor tumor cell mineralization effect caused by low concentration of mineralizable ions in the blood through the above.

[0039] The above-mentioned inorganic ions refer to examples of mineralizable ions, including calcium ions, iron ions and manganese ions, etc., which then realize calcium mineralization, iron mineralization and manganese mineralization respectively.

[0040] When preparing the above-mentioned medicine package, the first component and the second component can be packaged separately, but form a medicine package. During use, the two components can be used separately or simultaneously, but the two components must be used together to achieve the efficacy of the calcium medicine package.

[0041] Among them, the inorganic ions in the inorganic nanoparticles modified by the supramolecular body are derived from calcium, iron or manganese; the inorganic ions are derived from any one of calcium carbonate, calcium phosphate, calcium oxide, ferroferric oxide and manganese dioxide; the inorganic ions are derived from an inorganic ion source modified by a polymer containing hydroxyl and / or amino groups; for example, a calcium source modified by a polymer containing hydroxyl and / or amino groups; further examples include but are not limited to any one of calcium carbonate modified by PDA, calcium carbonate modified by PLA, calcium carbonate modified by PEG, calcium phosphate modified by PDA, calcium phosphate modified by PLA, calcium phosphate modified by PEG, calcium phosphate modified by PDA, calcium oxide modified by PLA, calcium oxide modified by PEG, ferroferric oxide modified by PDA, ferroferric oxide modified by PLA, ferroferric oxide modified by PEG, manganese dioxide modified by PDA, manganese dioxide modified by PLA and manganese dioxide modified by PEG.

[0042] It should be noted that PEG, PDA, PLA, etc. may also be other polymers containing hydroxyl groups and / or amino groups.

[0043] Furthermore, the host in the inorganic nanoparticles modified with the supramolecular host is selected from any one of cyclodextrin, cucurbituril, calixarene, pillararene or crown ether.

[0044] That is to say, the inorganic nanoparticles modified by the supramolecular body are selected from any one of cyclodextrin, cucurbituril, calixarene, pillararene or crown ether and any one of calcium carbonate, calcium phosphate, calcium oxide, ferroferric oxide and manganese dioxide modified by any one of PEG, PDA and PLA.

[0045] Further, the supramolecular guest is a polymer formed by modifying a polymer matrix by a guest molecule; the guest molecule is selected from any one of adamantane, ferrocene, phenylalanine and hexamethylenediamine; the polymer matrix is ​​selected from liposomes, such as 1,2-distearoyl-sn-polyethylene glycol (DAPE-PEG) or cholesterol-polyethylene glycol (Chol-PEG). Specifically, the supramolecular guest is selected from any one of adamantane-modified 1,2-distearoyl-sn-polyethylene glycol (DSPE-PEG-ADA) or ferrocene-modified 1,2-distearoyl-sn-polyethylene glycol (DSPE-PEG-Ferrocene).

[0046] The mass ratio of the first component to the second component is (10-100): 1. For example, it is any value between (10-100): 1, such as 10: 1, 20: 1, 30: 1, 40: 1, 50: 1, 60: 1, 70: 1, 80: 1, 90: 1 and 100: 1.

[0047] Furthermore, an embodiment of the present invention provides an application of the above-mentioned drug package for rapid mineralization of tumor cells in the preparation of drugs for treating tumors; wherein the tumor cells are any one of osteosarcoma cells, breast cancer cells, and melanoma cells.

[0048] In a third aspect, an embodiment of the present invention provides a method for preparing the above-mentioned drug package for rapid mineralization of tumor cells, using a first component and a second component to form a drug package. Specifically, the preparation method of the first component includes: mixing and stirring an inorganic ion source modified with a polymer containing hydroxyl and / or amino groups and the activator, and then adding the main body to react.

[0049] Wherein, the activator is selected from amine reagents, for example, including but not limited to at least one of EDC and DCC. The mass ratio of the inorganic ion source modified by the polymer containing hydroxyl and / or amino groups to the activator is 1:(2-5); for example, any value between 1:2, 1:3, 1:4 and 1:5, etc. The mass ratio of the inorganic ion source modified by the polymer containing hydroxyl and / or amino groups to the main body is 1:(2-5); for example, any value between 1:2, 1:3, 1:4 and 1:5, etc.

[0050] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0051] Example 1

[0052] The present invention provides a method for preparing inorganic nanoparticles modified by a supramolecular host, namely, a method for preparing calcium carbonate nanoparticles (denoted as CaPC) modified by β-CD (that is, β-cyclodextrin), comprising:

[0053] 10 mg PDA-modified CaCO 3 (denoted as CaP) was dispersed in anhydrous dimethyl sulfoxide, 16.5 mg EDC and 12.4 mg DCC were added, and stirred at room temperature (RT) for 30 min. 25 mg β-CD-COOH was added to the above solution, and after reacting for 48 h, β-CD-modified CaP (denoted as CaPC) was obtained by centrifugation (5000 g).

[0054] The embodiment of the present invention provides a supramolecular guest, which is DSPE-PEG-ADA (purchased from Xi'an Ruixi Biotechnology Co., Ltd.).

[0055] Experimental Example 1

[0056] Verify whether supramolecular guests can act on tumor cells

[0057] Osteosarcoma cells (143B) were cultured in complete DMEM medium and then placed in a 37°C, 5% (V / V) CO2 The cells were cultured in a humidified incubator at 37°C with 5% CO. 2 The cells were cultured in an incubator for 1.5 h. Finally, the 143B cells were collected and washed by centrifugation with PBS to obtain ADA-modified 143B cells (denoted as 143B-ADA).

[0058] In this experiment, β-CD modified with green fluorescent dye FITC was used to label ADA modified on the cell membrane through the host-guest interaction between β-CD and ADA, and Hoechst was used to label the cell nucleus. CLSM fluorescence imaging was performed at different time points (0, 6, 12, and 24 h). The results are shown in Figure 1 ,according to Figure 1 It can be seen that DSPE-PEG-ADA has excellent insertion stability on the surface of 143B cells, indicating that supramolecular objects can be modified onto the surface of tumor cells through the cell insertion method.

[0059] Experimental Example 2

[0060] Verify whether the drug package can enrich calcium ions in tumor cells

[0061] The above 143B-ADA cells were cultured at a density of about 2.0×10 5 The cells were seeded in a 12-well plate at a density of 100 cells / mL, 1 mL of DMEM containing CaPC (10 mg / mL) of Example 1 was added, and incubated in a cell culture incubator at 25° C. for 24 h. The cells were then washed three times by centrifugation with PBS, and resuspended in 1.0 mL of PBS for later use.

[0062] The cells of the above-mentioned incubated materials and the unincubated cells (pure 143B cells) were detected by electron microscopy. The results are shown in Figure 2 ,in, Figure 2 A in the middle represents a schematic diagram of pure 143B cells. Figure 2 Figure B shows the cell image of 143B-ADA in Experimental Example 1. Figure 2 It can be seen that compared with the unmodified 143B cells with smooth surface, the modified 143B cells have a large amount of CaCO 3 The nanoparticles have a rough surface, indicating that the main effect of the supramolecular body can make mineralizable inorganic ions such as calcium ions directly accumulate on the surface of tumor cells, avoiding the slow mineralization process and achieving rapid mineralization of tumor cells.

[0063] Experimental Example 3

[0064] In vitro cytotoxicity assay

[0065] The cytotoxicity of 143B and 143B-ADA cells was analyzed by MTT method. First, 143B and 143B-ADA cells were cultured at 1.0×10 4 The cells were inoculated into 100 μL DMEM medium at a density of 10 cells / well. After culturing for 24 h, the cells were cultured for 24 h with 100 μL fresh DMEM medium containing CaP and CaPC (100 mg / mL). After discarding the medium, 100 μL of 10 μL MTT solution (5 mg mL -1 ) in fresh medium and cultured for 4 hours. After discarding the supernatant, 100 μL DMSO was added to dissolve the formazan. Finally, the cytotoxicity of the nanoparticles to 143B and 143B-ADA cells was evaluated using a microplate reader.

[0066] Results Figure 3 It can be seen that CaPC, which can induce cell mineralization, is more toxic to 143B-ADA cells than CaP, which cannot induce cell biomineralization.

[0067] Experimental Example 4

[0068] Targeted research

[0069] 143B cells (1×10 6 / 100μL PBS) was injected into the right leg of mice (6 weeks old, 17-21g) to obtain 143B tumor mice. When the tumor of the mouse grew to about 100mm 3 At the time of the experiment, they were randomly divided into four groups. The treatment of the control group mice consisted of peritumoral injection of 25.0 μL of saline, followed by another peritumoral injection of 25.0 μL of saline 15 minutes later. For the mice in the Ca group, the treatment consisted of peritumoral injection of 25.0 μL of saline, followed by a second peritumoral injection of 25.0 μL of saline containing CaP (50 mg / mL) 15 minutes later. For the mice in the ADA group, the treatment consisted of peritumoral injection of 25.0 μL of saline containing 0.1 mg / mL DSPE-PEG-ADA, followed by a second peritumoral injection of 25.0 μL of saline containing CaP (50 mg / mL) 15 minutes later. The mice were then anesthetized and captured using an in vivo imaging system (IVIS) at 0, 4, 8, 12, and 24 hours. All mice were sacrificed 24 hours later, and major organs and tumors were harvested for fluorescence imaging.

[0070] Results Figure 4 ,according to Figure 4 It can be seen that compared with CaP, CaPC can target tumors through the host-guest interaction between ADA and β-CD.

[0071] Experimental Example 5

[0072] Construction of mouse tumor model

[0073] Six-week-old BALB / c female nude mice (obtained from the Animal Center of the University of Macau, license number: UMARE-017-2024, housed under natural light cycle, fasted for 12 h before surgery, free access to water) were selected and 100.0 μL of 2.0×10 6 143B cells were injected into the subcutaneous part of the hind legs of mice. When the tumor volume reached about 80 mm 3 The osteosarcoma mouse model was constructed at 5 ℃ and 1 ℃. The mice were randomly divided into five groups (n=5). The drug dosage in each group was as follows:

[0074] Group 1 was first injected with 25.0 μL PBS intravenously, and then 25.0 μL PBS was injected intratumorally 30 minutes later.

[0075] Group 2 was first injected intravenously with 25.0 μL of CaP solution (50 mg / mL), and then injected intratumorally with 25.0 μL of PBS 30 minutes later.

[0076] Group 3 was first injected intravenously with 25.0 μL of CaP solution (50 mg / mL), and then injected intratumorally with 25.0 μL of DSPE-PEG-ADA (1 mg / mL) 30 minutes later.

[0077] Group 4 was first injected intravenously with 25.0 μL of CaPC solution (50 mg / mL), and then injected intratumorally with 25.0 μL of PBS 30 minutes later.

[0078] Group 5 was first injected intravenously with 25.0 μL of CaPC solution (50 mg / mL), and then injected intratumorally with 25.0 μL of DSPE-PEG-ADA (1 mg / mL) 30 minutes later.

[0079] After 3 treatments, mice were sacrificed. The vertical diameter of the tumor was measured using a caliper to monitor tumor growth. On day 14, mice were sacrificed, and tumors and major organs of all mice were harvested and photographed with a digital camera.

[0080] Results Figure 5 Compared with other groups, the tumor volume of mice in the 5th treatment group (5 mice in each group, parallel test) was the smallest, indicating that the drug package provided by the embodiment of the present invention can effectively reduce the volume of the tumor and can be used for the treatment of tumors.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug package for rapid mineralization of tumor cells, characterized in that: The invention comprises a first component and a second component, wherein the first component comprises inorganic nanoparticles modified by a supramolecular host, and the second component comprises a supramolecular guest.

2. The drug package for rapid mineralization of tumor cells according to claim 1, characterized in that: The mass ratio of the first component to the second component is (10-100):

1.

3. The drug package for rapid mineralization of tumor cells according to claim 1, characterized in that: The supramolecular guest is a polymer formed by guest molecules modifying a polymer matrix; Preferably, the guest molecule is selected from any one of adamantane, ferrocene, phenylalanine and hexamethylenediamine; Preferably, the polymer matrix is ​​selected from liposomes, preferably 1,2-distearoyl-sn-polyethylene glycol or cholesterol-polyethylene glycol; Preferably, the supramolecular guest is selected from any one of adamantane-modified 1,2-distearoyl-sn-polyethylene glycol and ferrocene-modified 1,2-distearoyl-sn-polyethylene glycol.

4. The drug package for rapid mineralization of tumor cells according to claim 1, characterized in that: The inorganic ions in the inorganic nanoparticles modified by the supramolecular body are derived from calcium, iron or manganese; Preferably, the inorganic ions are derived from any one of calcium carbonate, calcium phosphate, calcium oxide, ferroferric oxide and manganese dioxide; Preferably, the inorganic ions are derived from an inorganic ion source modified by a polymer containing hydroxyl and / or amino groups; Preferably, the inorganic ions are derived from a calcium source modified by a polymer containing hydroxyl and / or amino groups; Preferably, the inorganic ions are derived from any one of PDA-modified calcium carbonate, PLA-modified calcium carbonate, PEG-modified calcium carbonate, PDA-modified calcium phosphate, PLA-modified calcium phosphate, PEG-modified calcium phosphate, PDA-modified calcium oxide, PLA-modified calcium oxide, PEG-modified calcium oxide, PDA-modified ferrosoferric oxide, PLA-modified ferrosoferric oxide, PEG-modified ferrosoferric oxide, PDA-modified manganese dioxide, PLA-modified manganese dioxide and PEG-modified manganese dioxide.

5. The drug package for rapid mineralization of tumor cells according to claim 1, characterized in that: The host in the inorganic nanoparticles modified with supramolecular hosts is selected from any one of cyclodextrin, cucurbituril, calixarene, pillararene or crown ether.

6. Use of the drug package for rapid mineralization of tumor cells according to claim 1 in the preparation of drugs for treating tumors; Preferably, the tumor includes any one of osteosarcoma cells, breast cancer cells, and melanoma cells.

7. A method for preparing the drug package for rapid mineralization of tumor cells according to claim 1, characterized in that: include: A pharmaceutical package is formed using the first component and the second component.

8. The preparation method according to claim 7, characterized in that: The preparation method of the first component comprises: mixing an inorganic ion source modified by a polymer containing hydroxyl and / or amino groups, a main body and an activator to react; Preferably, the inorganic ion source modified with the polymer containing hydroxyl and / or amino groups and the activator are mixed and stirred, and then added to the main body for reaction.

9. The preparation method according to claim 8, characterized in that: The activator is selected from amine reagents, preferably at least one of EDC and DCC.

10. The preparation method according to claim 8, characterized in that: The mass ratio of the inorganic ion source modified by the polymer containing hydroxyl and / or amino groups to the activator is 1:(2-5); The mass ratio of the inorganic ion source modified by the polymer containing hydroxyl groups and / or amino groups to the main body is 1:(2-5).

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