Composition for inhibiting tumors, nano-drug, preparation method and application

Abstract

The invention belongs to the technical field of nano medicine, and particularly relates to a composition for inhibiting tumors, a nano medicine, a preparation method and application, the composition for inhibiting the tumors comprises bufonid and a metal carbonyl compound, the molar ratio of the bufonid to the metal carbonyl compound is (0.2-0.5): (20-40), and the effect of inhibiting the tumors can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and specifically relates to a tumor-inhibiting composition, nanomedicine, preparation method and application. Background Art

[0002] Breast cancer is a common malignant tumor that threatens the health of women all over the world. In recent years, the incidence of breast cancer in my country has increased by 3% per year, and is showing a trend of younger age, which seriously threatens the physical and mental health of Chinese women. Chemotherapy, as one of the main means of clinical treatment of systemic breast cancer at this stage, can effectively alleviate the patient's condition and prolong the survival period. However, multidrug resistance (MDR) induced by long-term chemotherapy is one of the main reasons for the failure of breast cancer treatment at this stage. Therefore, actively exploring new treatment options to reverse / anti-MDR breast cancer has extremely important theoretical significance and wide application value for improving prognosis, reducing the recurrence and metastasis rate of patients, and improving survival rate.

[0003] CN115737594A discloses a bionic Prussian blue nanocomposite material loaded with cinobuccal venom, and a preparation method and application thereof. The bionic Prussian blue nanocomposite material comprises Prussian blue, cinobuccal venom and a bionic membrane. The cinobuccal venom is physically encapsulated in the Prussian blue, the bionic membrane is wrapped in the outer layer of the Prussian blue, and the particle size of the bionic Prussian blue nanocomposite material is 160nm to 180nm. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a tumor-inhibiting composition, a nanomedicine, a preparation method and an application to improve the tumor-inhibiting effect.

[0005] The embodiment of the present invention provides a tumor-inhibiting composition, comprising toad sterol and a metal carbonyl compound, wherein the molar ratio of toad sterol to the metal carbonyl compound is 0.2-0.5:20-40.

[0006] Preferably, the toad steroid is tofavin, bufotoxin, bufotoxin, bufotoxin, cinobufogenin and bufotoxin; the metal carbonyl compound is manganese carbonyl, vanadium carbonyl, chromium carbonyl, iron carbonyl, dicobalt carbonyl or nickel carbonyl.

[0007] More preferably, the bufasteroid is cinobufagin, and the metal carbonyl compound is carbonyl manganese.

[0008] An embodiment of the present invention provides a nanomedicine for inhibiting tumors, including a nanocarrier and a liposome coated on the nanocarrier, wherein the nanocarrier is loaded with toad sterol and a metal carbonyl compound; or, the liposome is loaded with toad sterol and a metal carbonyl compound; or, the nanocarrier is loaded with toad sterol and / or a metal carbonyl compound, and the liposome is loaded with a metal carbonyl compound and / or a toad sterol.

[0009] The toad steroid and the metal carbonyl compound of the present invention can be fully loaded in the nanocarrier, fully loaded on the liposome, or loaded on the nanocarrier and the liposome at the same time, or loaded on the nanocarrier and the liposome respectively. Preferably, the molar ratio of toad sterol to the metal carbonyl compound is 0.2-0.5:20-40, the nanocarrier is loaded with the metal carbonyl compound (the metal carbonyl compound is usually located inside the nanocarrier), the liposome is loaded with toad sterol (the toad sterol is doped between the phospholipid bilayers of the liposome); the surface of the liposome is connected with hyaluronic acid, the nanocarrier is a MOFs nanocarrier, more preferably Prussian blue nanoparticles, citric acid modified Prussian blue nanoparticles or hollow Prussian blue nanoparticles, more preferably Prussian blue nanoparticles, referred to as PB NPs, the liposome is selected from at least one of small unilamellar liposomes (0.02-0.08 μm), large unilamellar liposomes (0.1-1 μM), and multilamellar liposomes (1-5 μM), preferably large unilamellar liposomes, and the ratio is changed to make it flexible. The particle size is 70-170 nm, preferably 90-170 nm, and more preferably 80-120 nm.

[0010] The present invention provides a method for preparing the nano drug, comprising the following steps: The MOFs nanocarrier and the metal carbonyl compound are mixed and centrifuged to obtain gas-therapeutic MOFs nanoparticles; Mix toad sterol, lecithin, cholesterol, and DSPE-PEOz2K (DSPE-PEOz2K can give liposomes the ability to respond to decomposition and release under acidic pH), rotary evaporate, add gas to treat MOFs nanoparticle solution (solvent is water), ultrasonicate (30w ultrasonicate for 3 minutes), filter (0.22μm filter membrane), and obtain MOFs nanocomposite system; The aminated hyaluronic acid and the MOFs nanocomposite system are mixed to obtain a nano drug. The mass ratio of the aminated hyaluronic acid to the lecithin is 8-10:1-2.

[0011] Preferably, the solvent for the mixture of toad sterol, lecithin, cholesterol and DSPE-PEOz2K is chloroform; the mass ratio of cinobucoidin, lecithin, cholesterol and DSPE-PEOz2K is 0.2-1.5:6-8:1-2:0.5-1. The liposomes in this ratio are flexible and can be wrapped on the surface of MOFs nanoparticles.

[0012] The preparation method of amino-modified hyaluronic acid is to mix HA, EDC and NHS in PBS (buffer), add DSPE-PEG 2000 -NH 2, mixed evenly, dialyzed to obtain amino-modified hyaluronic acid. The mass ratio of HA, EDC, and NHS was 5:25:50, and the HA concentration was 5 mg / mL. DSPE-PEG 2000 -NH 2 The mass of HA is 5 times that of HA.

[0013] When the MOFs nanocarrier is Prussian blue nanoparticles, the preparation method is as follows: PVP solution with hydrochloric acid (pH 2.0-2.1) as the dissolving medium and K 3 [Fe(CN) 6 ] solution was mixed and stirred in a water bath. PVP and K 3 [Fe(CN) 6 ] is in a mass ratio of (7-10):(0.5-0.7), preferably 10:0.66, the water bath temperature is 50-80°C, preferably 70-80°C, more preferably 80°C, the stirring speed is 600-1000rpm, preferably 800rpm, the water bath time is 10-24h, preferably 16-24h, more preferably 20h.

[0014] When the metal carbonyl compound is carbonyl manganese, in the preparation method of the gas-treated MOFs nanoparticles, the mixed solvent is methanol, the mass ratio of the carbonyl manganese and the Prussian blue nanoparticles is (1-3):1, preferably 1:1, the concentration ratio of the Prussian blue nanoparticles and the carbonyl manganese in methanol is (0.5-1 mg / mL): (0.5-2 mg / ml), and the mixing is carried out by stirring for 6-12 hours.

[0015] The embodiment of the present invention provides a composition or a use of the nano drug, wherein the composition or the nano drug is used to prepare an anti-tumor drug. Preferably, the tumor is breast cancer.

[0016] The NHS is the abbreviation of N-hydroxysuccinimide, EDC is the abbreviation of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and HA is hyaluronic acid.

[0017] The beneficial effect of the present invention is that the present invention combines cinobufagin and carbonyl manganese, and experiments show that compared with single cinobufagin or single carbonyl manganese, it can significantly improve the effect of inhibiting tumors, especially breast cancer. Through the combination index, it is found that when the molar ratio is 0.2-0.5:20-40, the two have a synergistic effect, and the combination of the two can improve the effect of inhibiting tumors and reduce the dosage of a single substance.

[0018] The present invention effectively reduces the loss of nano-drugs during transportation in the body, significantly improves the utilization of drugs, and improves the technical problem of poor tumor treatment effects of existing chemotherapy drugs. Moreover, the "chemo-gas integrated" treatment has a significant synergistic effect compared with single treatment, and effectively inhibits the growth of multidrug-resistant breast cancer.

[0019] The hyaluronic acid molecules modified on the surface of the "chemo-gas integrated" dual therapeutic nanomedicine loaded with CS-1 in the present invention can actively target the tumor site, reduce the loss of the nanomedicine during transportation in the body, and significantly improve the utilization of the drug, thereby effectively inducing cell pyroptosis and achieving the purpose of "gas-chemotherapy" dual treatment of multidrug-resistant breast cancer. This liposome MOFs nanocomposite system that actively targets tumors can significantly inhibit the growth of multidrug-resistant breast cancer and reduce the toxic side effects of CS-1 and carbon monoxide themselves. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the preparation process of Example 1.

[0021] Figure 2 TEM images and particle size data of PBCO NPs provided in Example 1; (A) is the TEM image, and (B) is the particle size data.

[0022] Figure 3 TEM image and particle size data, CS-1 / MnCO encapsulation efficiency and acidic pH response CS-1 release curve of PBCO@Lip-CS-1[HA] NPs provided in Example 1; wherein (A) is a TEM image, (B) is particle size data, (C) is CS-1 / MnCO encapsulation efficiency, and (D) is an acidic pH response CS-1 release curve.

[0023] Figure 4 The CO release capacity of PBCO@Lip-CS-1[HA] NPs under the action of hydrogen peroxide and the CO release capacity in MCF-7 / ADR cells provided in Example 2; wherein (A) is the CO release capacity of PBCO@Lip-CS-1[HA] NPs under the action of hydrogen peroxide, and (B) is the CO release capacity of PBCO@Lip-CS-1[HA] NPs in MCF-7 / ADR cells.

[0024] Figure 5 The cell killing ability and pyroptosis inducing ability of PBCO@Lip-CS-1[HA] NPs provided in Example 2 in MCF-7 / ADR cells; wherein, (A) is the pyroptosis inducing ability, and (B) is the cell killing ability of PBCO@Lip-CS-1[HA] NPs in MCF-7 / ADR cells.

[0025] Figure 6The PBCO NPs and PBCO@Lip-CS-1[HA] NPs provided in Example 2 have synergistic ROS induction ability and synergistic cell death induction ability on MCF-7 / cell "gasification"; wherein (A) is the synergistic ROS induction ability of PBCO NPs and PBCO@Lip-CS-1[HA] NPs on MCF-7 / cell "gasification", and (B) is the synergistic cell death induction ability.

[0026] Figure 7 (A) Cell survival rate of breast cancer cells MCF-7 / ADR after combined treatment with CS-1 and MnCO. Figure 7 (B) Synergy index analysis of the combined use of CS-1 and MnCO.

[0027] Figure 8 The therapeutic effects of PBCO NPs and PBCO@Lip-CS-1[HA] NPs on MCF-7 / ADR solid tumors provided in Example 2. (A) is the tumor volume change curve of different experimental groups (control, CS-1, PBCO NP, PBCO@Lip-CS-1[HA]NP), (B) is the actual tumor image of different experimental groups, (C) is the tumor mass change of different experimental groups, (D) is the tumor inhibition rate of different experimental groups, and (E) is the weight change curve of mice in different experimental groups. DETAILED DESCRIPTION

[0028] The following will be combined Figure 1-8 The present invention is described in detail. The exemplary embodiments and descriptions of the present invention are used to explain the present invention but are not intended to limit the present invention.

[0029] Example 1 This embodiment provides a MOFs nanocomposite system capable of releasing carbon monoxide (hereinafter referred to as PBCO NPs), which is prepared according to the following steps: (1) Use hydrochloric acid at pH = 2.5 as the dissolving medium and add about 1 mg of K 3 [Fe(CN) 6 ] and 10 g PVP, stirred at room temperature for one hour, and heated in a water bath to 80 ° C; (2) Heat at 80°C for 24 h, transfer into a centrifuge tube, and centrifuge at 12,000 rpm at 25°C for 20 min. Discard the supernatant, add deionized water to reconstitute, centrifuge again, and repeat freeze-drying to obtain Prussian blue nanoparticles (PB NPs).

[0030] Test Example 1 1 mg of PB NPs and 1 mg of MnCO were taken and diluted to 2 mL with methanol solution, stirred at 800 rpm for 10 h at room temperature to synthesize PBCO NPs, and the concentration of MnCO in the supernatant after centrifugation was measured on a UV spectrophotometer. The MnCO encapsulation efficiency (EE) was calculated according to the following formula. Figure 2 The results showed that PB NPs could effectively load MnCO.

[0031]

[0032] Where MT is the total mass of added drug and MU is the mass of unencapsulated drug.

[0033] Example 2 This embodiment provides a dual-therapy nanomedicine (hereinafter referred to as PBCO@Lip-CS-1[HA]NPs), which is composed of PBCO NPs provided in Example 1, lecithin, cholesterol, DSPE-PEO Z 2K and cinobufolin are prepared, and the specific steps include: Phosphatidylcholine, cholesterol, DSPE-PEO Z 2K and cinobufagin were mixed in 5 mL chloroform at 8 mg: 1 mg: 0.5 mg: 0.5 mg, and the lipid film was obtained by rotary evaporation at 37 ° C. 0.5 mg of PBCO NPs was added and hydrated at 37 ° C for 1 h after water bath sonication. After water bath sonication again, the probe was used for 30w sonication, and 0.22 μM filter membrane was used for filtration and extrusion to obtain PBCO@Lip-CS-1 NPs. The mass ratio of PBCO NPs to lecithin was 1:16.

[0034] The compounds with a mass ratio of HA:EDC:NHS = 5:25:50 were mixed in PBS and stirred, and then DSPE-PEG with a mass 5 times that of HA was added. 2000 -NH 2 After stirring in a water bath for 24 hours, dialyze for 24 hours, and freeze-dry to obtain amino hyaluronic acid. The amino hyaluronic acid and lecithin were added to PBCO@Lip-CS-1 NPs at a mass ratio of 16:1, and the mixture was stirred in a water bath to obtain the hyaluronic acid-modified "chemical-gas integrated" dual-therapy nanomedicine, namely PBCO@Lip-CS-1[HA]NPs.

[0035] Example 3 This embodiment provides a PBCO@Lip-CS-1[HA]NPs, and its preparation method is different from that of Example 2 in that the mass ratio of PBCO NPs to lecithin is about 1:4.

[0036] Example 4 This embodiment provides a PBCO@Lip-CS-1[HA] NPs, and its preparation method is different from that of Example 2 in that the mass ratio of PBCO NPs to cinobufolin is about 1:2.

[0037] Example 5 This embodiment provides a PBCO@Lip-CS-1[HA] NPs, and its preparation method is different from that of Example 2 in that the mass ratio of PBCO NPs to cinobufagin is about 3:1.

[0038] Test Example 2 The PBCO@Lip-CS-1 NPs and PBCO@Lip-CS-1[HA] NPs provided in Example 2 were observed using a transmission electron microscope. Figure 3 Results A and B show that the particle size of PBCO@Lip-CS-1[HA]NPs is about 100nm, and it is square in shape like Prussian blue MOF material; and the surface of PBCO@Lip-CS-1[HA]NPs is covered with a thin film, indicating that the amino hyaluronic acid molecules are successfully loaded on the surface of PBCO@Lip-CS-1NPs. The CS-1 encapsulation efficiency was obtained by UV spectrophotometer. Figure 3 As shown in C, the material was placed in a dialysis bag in deionized water and dialyzed at 37°C with acidic and neutral pH deionized water as the external solution. Samples were taken at different time points to obtain the release curves under different pH conditions as shown in Figure 3 As shown in D.

[0039] Test Example 3 1 mL of PBCO@Lip-CS-1[HA] NPs PBS solution provided in Example 2 was placed in an EP tube. Different concentrations of H 2 O 2 and FL-CO-1 / PdCl 2 Materials (FL-CO-1 is an intracellular CO probe, PdCl 2 The probe catalyst) = 10 μM / 10 μM / 0.2 mg, and the fluorescence graph at 500-540 nm was measured using a fluorescence spectrophotometer. Figure 4 A The results show that PBCO@Lip-CS-1[HA] NPs have good CO release ability. PBS, CS-1, PBCO NPs and PBCO@Lip-CS-1[HA] NPs were applied to MCF-7 / ADR cells respectively. 2 Confocal imaging Figure 4 B, Results show CO release in MCF-7 / ADR cells.

[0040] Test Example 4 The killing of tumor cells by PBCO@Lip-CS-1[HA] NPs provided in Example 2, PBCO provided in Example 1, and cinobufagin were evaluated. Multidrug-resistant breast cancer cells MCF-7 / ADR were cultured, and PBS, CS-1, PBCO NPs, and PBCO@Lip-CS-1[HA] NPs were applied to MCF-7 / ADR cells, respectively. The survival rate of tumor cells was counted after 24 hours.

[0041] Figure 5 B The results show that the final material has a good cell killing effect. PBS, CS-1, PBCO NPs and PBCO@Lip-CS-1[HA]NPs were applied to MCF-7 / ADR cells respectively. PI was added 24 hours later, and confocal imaging was performed as shown in Figure 2. Figure 5 As shown in A, the results showed that MCF-7 / ADR cells exhibited pyroptotic cell morphology and cell membrane permeability was changed.

[0042] Test Example 5 The killing effects of PBCO@Lip-CS-1[HA] NPs (containing CS-1: 0.5-1 μM / PBCO: 10-20 μg / mL) provided in Example 2, PBCO (PB:MnCO=1:0.5-2) provided in Example 1, and cinobufagin on tumor cells were evaluated.

[0043] Multidrug-resistant breast cancer cells MCF-7 / ADR were cultured and PBS, CS-1, PBCONPs and PBCO@Lip-CS-1[HA]NPs were applied to MCF-7 / ADR cells. Flow cytometry was used to measure the ROS flow images after PI and calcein staining 12 hours later. Flow cytometry was used to measure the ROS flow images after DCFH-DA staining ( Figure 6 A) Green fluorescence was imaged using a confocal microscope.

[0044] The results showed that the combination of chemotherapy and gas in the nanomedicine for dual treatment of "chemical-gas integration" had a significant effect. Compared with the CS-1 chemotherapy group alone, the ROS increased by about 2 times and the number of dead cells increased by about 1.5 times ( Figure 6 B).

[0045] Test Example 6 The killing effect of PBCO and cinobufagin on tumor cells provided in Example 1 was evaluated ( Figure 7 A).

[0046] Multidrug-resistant breast cancer cells MCF-7 / ADR were cultured and the data were analyzed by Bliss synergy index using the Synergyfinder website ( Figure 7B), it can be seen that 0.2-0.5 μM CS-1 and 20-40 μM MnCO have a synergistic effect, achieving the purpose of enhancing the therapeutic effect through drug synergy.

[0047] Test Example 7 The animal model was established and analyzed according to the ethical requirements of animal experiments. The mice used to establish the model were 4-6 weeks old female BALB / c nude mice, and 5×10 7 MCF-7 / ADR cells were first grown in mice to form an average volume of 100 mm 3 The mice were randomly divided into 4 groups and treated by intravenous injection every 2 days (Ⅰ: Control group; Ⅱ: CS-1 group; Ⅲ: PBCO NPs group; Ⅳ: PBCO@Lip-CS-1[HA]NPs group). The tumor volume and body weight of the mice during the observation period were recorded, and the tumor tissues were subjected to H&E staining and Ki67 immunohistochemistry. Figure 8 As shown. After the injection of the nanomedicine of the present invention, obvious necrotic areas appeared in the tumor. The Ki67 immunohistochemistry results showed that the Ki67 level of the tumor was significantly reduced after the injection of the composite system of the present invention, and no abnormalities were found in other organs. It can be seen that the "chemical-gas integrated" dual-treatment nanomedicine of the present invention can effectively treat multidrug-resistant breast cancer.

[0048] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0049] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.

Claims

1. A tumor-inhibiting composition, characterized in that: The invention comprises toad sterol and a metal carbonyl compound, wherein the molar ratio of the toad sterol and the metal carbonyl compound is 0.2-0.5:20-40.

2. The composition according to claim 1, characterized in that The toad steroid is tofagin, bufalin, bufalin, bufalin, cinobufogenin and bufalin; the metal carbonyl compound is carbonyl manganese, carbonyl vanadium, carbonyl chromium, carbonyl iron, carbonyl dicobalt or carbonyl nickel.

3. The composition according to claim 2, characterized in that The toad steroid is cinobufagin, and the metal carbonyl compound is carbonyl manganese.

4. A nano drug for inhibiting tumors, characterized in that: It comprises a nanocarrier and a liposome coated on the nanocarrier, wherein the nanocarrier is loaded with toad sterol and a metal carbonyl compound; or the liposome is loaded with toad sterol and a metal carbonyl compound; or the nanocarrier is loaded with toad sterol and / or a metal carbonyl compound, and the liposome is loaded with a metal carbonyl compound and / or a toad sterol.

5. The nano drug according to claim 4, characterized in that: The molar ratio of the toad sterol to the metal carbonyl compound is 0.2-0.5:20-40, the nanocarrier is loaded with the metal carbonyl compound, and the liposome is loaded with toad sterol; the surface of the liposome is connected with hyaluronic acid, and the nanocarrier is a MOFs nanocarrier.

6. The nano drug according to claim 5, characterized in that: The MOFs nanocarrier is Prussian blue nanoparticles, citric acid modified Prussian blue nanoparticles or hollow Prussian blue nanoparticles.

7. A method for preparing the nano drug according to claim 5 or 6, characterized in that: The steps include: The MOFs nanocarrier and the metal carbonyl compound are mixed and centrifuged to obtain gas-therapeutic MOFs nanoparticles; Mix toad sterol, lecithin, cholesterol and DSPE-PEOz2K, perform rotary evaporation, add gas to treat MOFs nanoparticle solution, perform ultrasound, and filter to obtain a MOFs nanocomposite system; The amino-modified hyaluronic acid and the MOFs nanocomposite system are mixed to obtain a nanomedicine.

8. The preparation method according to claim 7, characterized in that: The solvent for the mixture of toad sterol, lecithin, cholesterol, and DSPE-PEOz2K is chloroform; The preparation method of amino hyaluronic acid is as follows: HA, EDC, and NHS are mixed in PBS, and DSPE-PEG is added. 2000 -NH2, mix well, dialyze to obtain amino hyaluronic acid.

9. A composition according to any one of claims 1 to 3, or a use of a nano drug according to any one of claims 4 to 6, characterized in that: The composition or nanomedicine is used for preparing anti-tumor drugs.

10. The use according to claim 9, characterized in that: The tumor is breast cancer.

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