Deferoxamine mesylate response type nickel-iron layered double hydroxide and application thereof

Abstract

The invention provides deferoxamine mesylate response type nickel-iron layered double hydroxide and application thereof, and belongs to the technical field of biological medicine. The deferoxamine mesylate response type ferro-nickel layered double hydroxide provided by the invention comprises DFOM and NiFe-LDH (layered double hydroxide). By utilizing the characteristic that DFOM is chelated with iron ions, the iron ions in NiFe-LDH are chelated, and nickel ion release is promoted to realize effective killing of malignant tumors, so that the tumor inhibition capability of deferoxamine mesylate response type ferro-nickel layered double hydroxide is improved. Experimental results show that the tumor inhibition rate of the deferoxamine mesylate responsive type ferro-nickel layered double hydroxide provided by the invention reaches 76.2%.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a deferoxamine mesylate-responsive nickel-iron layered double hydroxide and an application thereof. Background Art

[0002] Malignant tumors are among the most threatening diseases and pose a persistent and severe challenge to public health. Therefore, finding effective cancer treatment strategies remains of vital importance.

[0003] In cancer treatment, nanomaterials are used as drug carriers, with advantages such as high drug loading capacity, precise tumor targeting, and flexible surface modification to achieve responsive drug release. However, drug-loaded nanocarriers also have many limitations such as acquired drug resistance caused by the drug itself, uneven tissue distribution, unexpected leakage, and inability to achieve efficient responsive release, which can easily weaken the therapeutic effect. Therefore, how to improve the tumor inhibition effect of materials has become a technical problem that needs to be solved urgently in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a deferoxamine mesylate-responsive nickel-iron layered double hydroxide and its application. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide provided by the present invention has an excellent tumor inhibition effect.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a deferoxamine mesylate-responsive nickel-iron layered double hydroxide, comprising deferoxamine mesylate (DFOM) and nickel-iron layered double hydroxide (NiFe-LDH).

[0007] The present invention also provides a deferoxamine mesylate-responsive nickel-iron layered double hydroxide, comprising DFOM and a targeting peptide-modified NiFe-LDH (denoted as pNiFe-LDH).

[0008] Preferably, the targeting peptide is a DSPE-PEG-modified PDGFR-β binding cyclic peptide.

[0009] Preferably, the preparation method of the DSPE-PEG modified PDGFR-β binding cyclic peptide comprises:

[0010] The PDGFR-β binding cyclic peptide (amino acid sequence: C*SRNLIDC*, denoted as PDGFB cyclic peptide), coupling reagent, organic solvent and DSPE-PEG-NH2 are mixed to carry out the first coupling reaction to obtain DSPE-PEG modified PDGFB cyclic peptide.

[0011] Preferably, the coupling reagents are EDC and NHS.

[0012] Preferably, the mass ratio of the PDGFB cyclic peptide to DSPE-PEG-NH2 is 1:(2-3).

[0013] Preferably, the temperature of the first coupling reaction is room temperature, and the time of the first coupling reaction is 1-3 h.

[0014] Preferably, the preparation method of the pNiFe-LDH includes:

[0015] Mix NiFe-LDH, an organic solvent and a targeting peptide, and carry out a second coupling reaction to obtain pNiFe-LDH.

[0016] Preferably, the temperature of the second coupling reaction is room temperature, and the time of the second coupling reaction is 10-14 h.

[0017] The present invention also provides the application of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide described in the above technical solution or the deferoxamine mesylate-responsive nickel-iron layered double hydroxide described in the above technical solution in the preparation of anti-tumor drugs.

[0018] The present invention provides a deferoxamine mesylate-responsive nickel-iron layered double hydroxide, which includes DFOM and NiFe-LDH. The present invention utilizes the property of DFOM to chelate iron ions, chelates the iron ions in NiFe-LDH and promotes the release of nickel ions to achieve effective killing of malignant tumors, thereby improving the ability of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide to inhibit tumors. Experimental results show that the tumor inhibition rate of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide provided by the present invention reaches 76.2%. Description of the Drawings

[0019] Figure 1 Cell viability diagrams after treating PC3 cells with Comparative Examples 1-4 for 24 h;

[0020] Figure 2 Cell viability diagrams after treating PC3 cells with the NiFe-LDH group of Comparative Example 4 for 0 h, 24 h, 36 h and 48 h respectively;

[0021] Figure 3 Cell viability diagrams after treating PC3 cells with Comparative Examples 1 and 5-8 for 24 h;

[0022] Figure 4 Cell viability diagrams after treating PC3 cells with Comparative Example 1 and the NiFe-LDH+DFOM groups of Application Examples 1-6 for 24 h;

[0023] Figure 5 Cell viability and death level diagrams evaluated by the Hoechst33342 / propidium iodide (HO / PI) double staining assay for Comparative Example 1, 4, 8 and Application Example 6;

[0024] Figure 6 PC3 cells were treated with the control group, NiFe-LDH + DFOM group, NiFe-LDH + DFOM + Z-VAD-FMK (40 μM) group, and NiFe-LDH + DFOM + Z-VAD-FMK (80 μM) group respectively, and then the apoptosis ratio of the cells was detected by flow cytometry;

[0025] Figure 7 It is the volcano plot of the distribution of up-regulated and down-regulated proteins in the NiFe-LDH + DFOM group compared with the control group for proteomic sequencing;

[0026] Figure 8 It is the difference in IGFBP3 expression between adjacent normal tissues and cancer tissues;

[0027] Figure 9 It is the representative immunoblotting images of the expression of key proteins in the PI3K / AKT / mTOR pathway after treating prostate cancer PC3 cells with the control group, DFOM group, NiFe-LDH group, and NiFe-LDH + DFOM group for 24 h;

[0028] Figure 10 It is the expression levels of key proteins in the PI3K / AKT / mTOR pathway (n = 3) after treating prostate cancer PC3 cells with the control group, DFOM group, NiFe-LDH group, and NiFe-LDH + DFOM group for 24 h;

[0029] Figure 11 It is the representative immunoblotting images of the expression of apoptosis-related proteins after treating prostate cancer PC3 cells with the control group, DFOM group, NiFe-LDH group, and NiFe-LDH + DFOM group for 24 h;

[0030] Figure 12 It is the expression levels of apoptosis-related proteins (n = 3) after treating prostate cancer PC3 cells with the control group, DFOM group, NiFe-LDH group, and NiFe-LDH + DFOM group for 24 h;

[0031] Figure 13 It is the representative immunoblotting images of the expression of IGFBP3, unphosphorylated and phosphorylated PI3K proteins after treating PC3 cells transfected with si-NC or si-IGFBP3 with the NiFe-LDH + DFOM group;

[0032] Figure 14 It is the expression levels of IGFBP3, unphosphorylated and phosphorylated PI3K proteins (n = 3) after treating PC3 cells transfected with si-NC or si-IGFBP3 with the NiFe-LDH + DFOM group;

[0033] Figure 15 Cell viability levels after treatment of the control group and the NiFe-LDH + DFOM group in prostate cancer PC3 cells transfected with si-NC or si-IGFBP3

[0034] Figure 16 Flow chart of animal experiments

[0035] Figure 17 Statistical chart of the average body weight of mice in each group after injection with nine groups: normal saline (NS), DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.)

[0036] Figure 18 Statistical chart of the average tumor volume of mice in each group after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.)

[0037] Figure 19 Statistical chart of the tumor volume of each of the 5 mice after injection with NS

[0038] Figure 20 Statistical chart of the tumor volume of each of the 5 mice after injection with DFOM (i.v.)

[0039] Figure 21 Statistical chart of the tumor volume of each of the 5 mice after injection with DFOM (i.t.)

[0040] Figure 22 Statistical chart of the tumor volume of each of the 5 mice after injection with NiFe-LDH

[0041] Figure 23 Statistical chart of the tumor volume of each of the 5 mice after injection with pNiFe-LDH

[0042] Figure 24 Statistical chart of the tumor volume of each of the 5 mice after injection with NiFe-LDH + DFOM (i.v.)

[0043] Figure 25Statistical graphs of tumors of 5 mice each after injection with NiFe-LDH+DFOM(i.t.);

[0044] Figure 26 Statistical graphs of tumors of 5 mice each after injection with pNiFe-LDH+DFOM(i.v.);

[0045] Figure 27 Statistical graphs of tumors of 5 mice each after injection with pNiFe-LDH+DFOM(i.t.);

[0046] Figure 28 Digital pictures of tumor masses dissected from euthanized mice in each group after injection with nine groups: NS, DFOM(i.v.), DFOM(i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH+DFOM(i.v.), NiFe-LDH+DFOM(i.t.), pNiFe-LDH+DFOM(i.v.), pNiFe-LDH+DFOM(i.t.);

[0047] Figure 29 Data of serum alanine aminotransferase (ALT) of euthanized mice in each group after injection with nine groups: NS, DFOM(i.v.), DFOM(i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH+DFOM(i.v.), NiFe-LDH+DFOM(i.t.), pNiFe-LDH+DFOM(i.v.), pNiFe-LDH+DFOM(i.t.);

[0048] Figure 30 Data of serum aspartate aminotransferase (AST) of euthanized mice in each group after injection with nine groups: NS, DFOM(i.v.), DFOM(i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH+DFOM(i.v.), NiFe-LDH+DFOM(i.t.), pNiFe-LDH+DFOM(i.v.), pNiFe-LDH+DFOM(i.t.);

[0049] Figure 31 Data of serum blood urea nitrogen (BUN) of euthanized mice in each group after injection with nine groups: NS, DFOM(i.v.), DFOM(i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH+DFOM(i.v.), NiFe-LDH+DFOM(i.t.), pNiFe-LDH+DFOM(i.v.), pNiFe-LDH+DFOM(i.t.);

[0050] Figure 32 Data of serum creatinine (CREA) of mice euthanized after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t).

[0051] Figure 33 Hematoxylin-eosin (H&E) staining images of major organs of mice euthanized after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t). Detailed implementation mode

[0052] The present invention provides a deferoxamine mesylate-responsive nickel-iron layered double hydroxide, comprising DFOM and NiFe-LDH.

[0053] The present invention has no special limitation on the sources of the raw materials, and commercially available products well-known to those skilled in the art can be used.

[0054] The present invention has no special limitation on the size of the NiFe-LDH, and NiFe-LDH well-known to those skilled in the art can be used.

[0055] As an implementation mode, the particle size of the NiFe-LDH can be 30 nm.

[0056] The present invention has no special limitation on the ratio of the DFOM and NiFe-LDH, and it can be adjusted according to needs. In the present invention, the DFOM is a chelating agent, which can destroy the stable structure of NiFe-LDH, thereby converting the "biologically inert" NiFe-LDH into a "biologically active" nano-therapeutic agent, and then exhibiting the expected tumor killing effect.

[0057] As an implementation mode, the molar ratio of the DFOM to the mass of the NiFe-LDH can be (1-2) μmol:(1-4) mg, or can also be (1-2) μmol:(1-2) mg.

[0058] The present invention has no special limitation on the preparation method of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide, as long as it contains DFOM and NiFe-LDH.

[0059] The present invention utilizes the property of DFOM to chelate iron ions, chelates the iron ions in NiFe-LDH and promotes the release of nickel ions to achieve effective killing of malignant tumors, thereby improving the ability of deferoxamine mesylate-responsive nickel-iron layered double hydroxide to inhibit tumors.

[0060] The present invention also provides a deferoxamine mesylate-responsive nickel-iron layered double hydroxide, which comprises DFOM and pNiFe-LDH. In the present invention, by using pNiFe-LDH, the deferoxamine mesylate-responsive nickel-iron layered double hydroxide can actively target and aggregate at the cancer cell site in response to DFOM, reduce the toxic and side effects on non-target organs, and achieve precise and effective treatment of cancer cells.

[0061] The present invention has no special limitation on the sources of the various raw materials, and commercially available products well-known to those skilled in the art can be used.

[0062] In the present invention, the targeting peptide is preferably a DSPE-PEG-modified PDGFB cyclic peptide; the preparation method of the DSPE-PEG-modified PDGFB cyclic peptide preferably comprises:

[0063] Mixing PDGFB cyclic peptide, a coupling reagent, an organic solvent and DSPE-PEG-NH2, and carrying out a first coupling reaction to obtain the DSPE-PEG-modified PDGFB cyclic peptide.

[0064] In the present invention, by connecting the PDGFB cyclic peptide, an important regulator of prostate cancer proliferation and metastasis, to the surface of NiFe-LDH, pNiFe-LDH is prepared, realizing specific targeting of prostate cancer.

[0065] In the present invention, the coupling reagent is preferably EDC and NHS; the mass ratio of EDC to NHS is preferably 1:1. In the present invention, the coupling reagent can activate carboxyl groups and promote the first coupling reaction.

[0066] In the present invention, the mass ratio of the PDGFB cyclic peptide to DSPE-PEG-NH2 is preferably 1:(2-3), more preferably 1:2.

[0067] In the present invention, the mass ratio of the coupling reagent to the PDGFB cyclic peptide is preferably 1:(18-22), more preferably 1:20.

[0068] In the present invention, the organic solvent is preferably DMSO. The present invention has no special limitation on the dosage of the organic solvent, as long as the raw materials can be completely dissolved.

[0069] In the present invention, the mixing of the PDGFB cyclic peptide, the coupling reagent, the organic solvent and DSPE-PEG-NH2 preferably involves mixing the PDGFB cyclic peptide, the coupling reagent and the organic solvent, and then adding DSPE-PEG-NH2. By adopting the above mixing method, the degree of mixing of the raw materials can be improved in the present invention.

[0070] The present invention has no special limitation on the operation of mixing the PDGFB cyclic peptide, the coupling reagent and the organic solvent and then adding DSPE-PEG-NH2, and the operations well-known to those skilled in the art can be adopted.

[0071] In the present invention, the temperature of the first coupling reaction is preferably room temperature; the time of the first coupling reaction is preferably 1 to 3 h, more preferably 2 h. Limiting the temperature and time of the first coupling reaction within the above ranges can further improve the degree of the first coupling reaction in the present invention.

[0072] In the present invention, the first coupling reaction is preferably carried out under stirring conditions. The present invention has no special limitation on the stirring operation, and the stirring operations well-known to those skilled in the art can be adopted.

[0073] In the present invention, the preparation method of the pNiFe-LDH preferably includes:

[0074] Mixing NiFe-LDH, an organic solvent and a targeting peptide, and carrying out a second coupling reaction to obtain pNiFe-LDH.

[0075] The present invention has no special limitation on the size of the NiFe-LDH, and the NiFe-LDH well-known to those skilled in the art can be adopted.

[0076] As an embodiment, the particle size of the NiFe-LDH can be 30 nm.

[0077] In the present invention, the organic solvent is preferably DMSO. The present invention has no special limitation on the dosage of the organic solvent, as long as the raw materials can be completely dissolved.

[0078] In the present invention, the mass ratio of NiFe-LDH to the PDGFB cyclic peptide is preferably (18 - 22):1, more preferably 20:1.

[0079] The present invention has no special limitation on the operation of mixing NiFe-LDH, the organic solvent and the targeting peptide, and the technical solutions for preparing the mixed material well-known to those skilled in the art can be adopted.

[0080] In the present invention, the temperature of the second coupling reaction is preferably room temperature; the time of the second coupling reaction is preferably 10 to 14 h, more preferably 12 h. Limiting the temperature and time of the second coupling reaction within the above ranges in the present invention can further improve the degree of the second coupling reaction.

[0081] In the present invention, the second coupling reaction is preferably carried out under stirring conditions. There are no special limitations on the stirring operation in the present invention, and the stirring operations well-known to those skilled in the art can be adopted.

[0082] After the second coupling reaction is completed, in the present invention, the product obtained from the second coupling reaction is preferably centrifuged, filtered, washed with water, and dispersed in water in sequence to obtain pNiFe-LDH.

[0083] There are no special limitations on the centrifugation and filtration operations in the present invention, and the operations well-known to those skilled in the art can be adopted.

[0084] In the present invention, the water used for the water washing is preferably ultrapure water. There are no special limitations on the number of times of the water washing in the present invention, and it can be judged according to common sense.

[0085] There are no special limitations on the operation of the water dispersion in the present invention, and the operations well-known to those skilled in the art can be adopted.

[0086] There are no special limitations on the ratio of the DFOM to pNiFe-LDH in the present invention, and it can be adjusted according to needs. In the present invention, the DFOM is a chelating agent, which can destroy the stable structure of pNiFe-LDH, thereby converting the "biologically inert" pNiFe-LDH into a "biologically active" nano-therapeutic agent, and then showing the expected tumor killing effect.

[0087] As an embodiment, the mass ratio of the DFOM to pNiFe-LDH can be (1 to 2):(1 to 2), and can also be 1:1.

[0088] There are no special limitations on the preparation method of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide in the present invention, and the preparation methods well-known to those skilled in the art can be adopted for preparation.

[0089] The deferoxamine mesylate-responsive nickel-iron layered double hydroxide provided by the present invention has the following four characteristics:

[0090] 1. Strong targeting: The surface of this material is modified with a PDGFB cyclic peptide, which can specifically recognize and efficiently bind to PDGFR-β on the surface of prostate cancer cells, and precisely target prostate cancer cells and tissues;

[0091] 2. Strong nickel ion release ability in DFOM response: pNiFe-LDH is stable under physiological conditions and can efficiently release nickel ions in a responsive manner in the DFOM environment;

[0092] 3. Significantly enhance the killing effect on prostate cancer cells in both in vivo and in vitro in response to DFOM: The material has strong targeting ability, high nickel ion release efficiency after responding to DFOM stimulation, and can significantly enhance the killing ability on prostate cancer cells both in vivo and in vitro;

[0093] 4. High biosafety: Neither pNiFe-LDH nor DFOM alone will have a significant impact on cell viability. Only when pNiFe-LDH and DFOM are used in combination will they significantly induce apoptosis of tumor cells.

[0094] In the present invention, pNiFe-LDH exhibits good stability and biocompatibility when used in combination with DFOM, and does not cause obvious damage to non-target organs; different from its high stability in the unstimulated state, whether by intravenous injection or intratumoral injection, DFOM can chelate trivalent iron (Fe 3+ ) to make pNiFe-LDH release Ni 2+ Thereby upregulating insulin-like growth factor binding protein 3 (IGFBP3) and inhibiting its downstream PI3K / AKT / mTOR signaling pathway, thus inducing apoptosis of prostate cancer cells both in vivo and in vitro; The present invention provides a clinically applicable drug-responsive LDH (pNiFe-LDH) that can actively target tumors and can switch from a "bio-inert" form to a "bio-active" form, providing a feasible and promising precision cancer treatment strategy.

[0095] The present invention also provides the application of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide described in the above technical solution or the deferoxamine mesylate-responsive nickel-iron layered double hydroxide described in the above technical solution in the preparation of anti-tumor drugs.

[0096] The present invention has no special limitation on the type of the tumor. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide provided by the present invention is applicable to any solid tumor.

[0097] As an implementation manner, the tumor can be prostate cancer cells.

[0098] In the present invention, the deferoxamine mesylate-responsive nickel-iron layered double hydroxide is preferably formulated into a solution for use when applied; the solvent used for formulating the solution is preferably deionized water. The present invention has no special limitation on the concentration of the solution, and it can be adjusted according to actual needs.

[0099] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0100] The main experimental reagents used in the examples and comparative examples are shown in Table 1.

[0101] Table 1 Main experimental reagents used in the examples and comparative examples

[0102]

[0103] The main experimental instruments used in the examples and comparative examples are shown in Table 2.

[0104] Table 2 Main experimental instruments

[0105] Instrument Model Manufacturer Electronic Analytical Balance OHAUS-CP114 Shanghai Ohaus Instrument Co., Ltd. High-Speed Centrifuge Avanti J26XP Beckman Coulter Mechanical Stirrer JJ-1 Guohua Electric Appliance Co., Ltd. Ultra-Pure Water Instrument Milli-Q Gradient Merck Millipore High-Speed Refrigerated Centrifuge 5430R Eppendorf Analytical Flow Cytometer DxFLEX Beckman Coulter Confocal Laser Scanning Microscope LSM800 Carl Zeiss

[0106] Example 1

[0107] The deferoxamine mesylate-responsive nickel-iron layered double hydroxide is composed of DFOM and NiFe-LDH;

[0108] The molar ratio of the amount of substance of DFOM to the mass of NiFe-LDH is 1 μmol: 2 mg;

[0109] The preparation method of the NiFe-LDH is as follows:

[0110] The metal salt solutions of Ni(NO3)2·6H2O and Fe(NO3)3·9H2O dissolved in 20.0 mL of ultrapure water were added to a flask. The concentration of Ni in the salt solution 2+ was 0.8 M, and the concentration of Fe 3+ was 0.2 M. Then the flask was placed on a stirrer and stirred evenly. After that, 100.0 mL of ammonia water with a concentration of 0.5 M was added dropwise to the continuously stirred mixed solution. At this time, the solution became turbid. Then it was placed in a water bath at 65 °C and continuously stirred for 18 h. The precipitate obtained by centrifuging the final product at 6000 rpm was washed 3 times with absolute ethanol and 5 times with ultrapure water to obtain NiFe-LDH with a particle size of about 30 nm.

[0111] Application Example 1

[0112] The DFOM and NiFe-LDH in Example 1 were mixed with deionized water to prepare a solution. The concentration of DFOM in the solution was 50 μM, and the concentration of NiFe-LDH in the solution was 100 μg / mL NiFe-LDH.

[0113] Example 2

[0114] On the basis of Example 1, the ratio of the amount of substance of DFOM to the mass of NiFe-LDH was replaced with 1 μmol: 4 mg, and the others were the same as in Example 1.

[0115] Application Example 2

[0116] Mix DFOM, NiFe-LDH in Example 2 with deionized water to prepare a solution. The concentration of DFOM in the solution is 50 μM, and the concentration of NiFe-LDH in the solution is 200 μg / mL NiFe-LDH.

[0117] Example 3

[0118] On the basis of Example 1, the ratio of the amount of substance of DFOM to the mass of NiFe-LDH was replaced with 1 μmol: 1 mg, and the others were the same as in Example 1.

[0119] Application Example 3

[0120] Mix DFOM, NiFe-LDH in Example 3 with deionized water to prepare a solution. The concentration of deferoxamine mesylate in the solution is 100 μM, and the concentration of NiFe-LDH in the solution is 100 μg / mL NiFe-LDH.

[0121] Application Example 4

[0122] Mix DFOM, NiFe-LDH in Example 1 with deionized water to prepare a solution. The concentration of DFOM in the solution is 100 μM, and the concentration of NiFe-LDH is 200 μg / mL.

[0123] Example 4

[0124] On the basis of Example 1, the ratio of the amount of substance of DFOM to the mass of NiFe-LDH was replaced with 2 μmol: 1 mg, and the others were the same as in Example 1.

[0125] Application Example 5

[0126] Mix DFOM, NiFe-LDH in Example 4 with deionized water to prepare a solution. The concentration of DFOM in the solution is 200 μM, and the concentration of NiFe-LDH is 100 μg / mL.

[0127] Application Example 6

[0128] Mix the DFOM, NiFe-LDH in Example 3 with deionized water to prepare a solution. The concentration of DFOM in the solution is 200 μM, and the concentration of NiFe-LDH in the solution is 200 μg / mL NiFe-LDH.

[0129] Comparative Example 1

[0130] Deionized water

[0131] Comparative Example 2

[0132] Mix NiFe-LDH with deionized water to prepare a 50 μg / mL NiFe-LDH solution.

[0133] Comparative Example 3

[0134] Mix NiFe-LDH with deionized water to prepare a 100 μg / mL NiFe-LDH solution.

[0135] Comparative Example 4

[0136] Mix NiFe-LDH with deionized water to prepare a 200 μg / mL NiFe-LDH solution.

[0137] Comparative Example 5

[0138] Mix DFOM with deionized water to prepare a 25 μM DFOM solution.

[0139] Comparative Example 6

[0140] Mix DFOM with deionized water to prepare a 50 μM DFOM solution.

[0141] Comparative Example 7

[0142] Mix DFOM with deionized water to prepare a 100 μM DFOM solution.

[0143] Comparative Example 8

[0144] Mix DFOM with deionized water to prepare a 200 μM DFOM solution.

[0145] Cytotoxicity experiment:

[0146] Cell viability detection experiments were conducted on the control group (Comparative Example 1), DFOM group (Comparative Examples 5-8), NiFe-LDH group (Comparative Examples 2-4), and NiFe-LDH + DFOM group (Application Examples 1-6): An appropriate amount of PC3 cells were inoculated into a 96-well plate. After the cells adhered and were cultured overnight, the materials of the above treatment groups were added respectively. After culturing for 24 h, MTT working solution was added. After incubating at 37 °C for 4 h, the supernatant in the plate was discarded. 150 μL of DMSO was added to each well to dissolve formazan, and the light absorption value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay detector to determine the effect of each treatment group on cell viability;

[0147] For the cell viability and death detection experiment: An appropriate amount of PC3 cells were inoculated into a 12-well plate. After the cells adhered and were cultured overnight, the materials of the above treatment groups were added respectively. After treatment for 24 h, the distribution of live cells and dead cells was determined by Hoechst33342 / propidium iodide (HO / PI) double staining assay.

[0148] Figure 1 Figure showing the cell viability after treating PC3 cells with Comparative Examples 1-4 for 24 h; Figure 2 Figure showing the cell viability after treating PC3 cells with the NiFe-LDH group of Comparative Example 4 for 0 h, 24 h, 36 h, and 48 h respectively; Figure 3 Figure showing the cell viability after treating PC3 cells with Comparative Examples 1 and 5-8 for 24 h; Figure 4 Figure showing the cell viability after treating PC3 cells with Comparative Example 1 and the NiFe-LDH + DFOM groups of Application Examples 1-6 for 24 h; Figure 5 Figure showing the evaluation of cell viability and death levels by using Hoechst33342 / propidium iodide (HO / PI) double staining assay for Comparative Example 1, 4, 8, and Application Example 6. The scale bar in the figure is 50 μm; ns in the figure indicates no significant difference; **p < 0.01; ***p < 0.001; ****p < 0.0001 (the same below).

[0149] From Figures 1-4 It can be seen that there was no obvious cytotoxicity after treating prostate cancer PC3 cells with NiFe-LDH or DFOM alone, and the anti-cancer effect was significant after co-treatment with DFOM and NiFe-LDH; From Figure 5 It can be seen that DFOM can significantly enhance the tumor-killing effect of NiFe-LDH on PC3 cells after response.

[0150] Application Example 7

[0151] Mix DFOM, NiFe-LDH, and Z-VAD-FMK in Example 3 with deionized water to prepare a solution. In the solution, the concentration of DFOM is 200 μM, the concentration of NiFe-LDH is 200 μg / mL, and the concentration of Z-VAD-FMK is 40 μM, denoted as the NiFe-LDH + DFOM + Z-VAD-FMK (40 μM) group.

[0152] Application Example 8

[0153] Mix DFOM, NiFe-LDH, and Z-VAD-FMK in Example 3 with deionized water to prepare a solution. In the solution, the concentration of DFOM is 200 μM, the concentration of NiFe-LDH is 200 μg / mL, and the concentration of Z-VAD-FMK is 80 μM, denoted as the NiFe-LDH + DFOM + Z-VAD-FMK (80 μM) group.

[0154] Detection of apoptosis level:

[0155] The groups are the control group (Comparative Example 1), the NiFe-LDH + DFOM group (Application Example 6), the NiFe-LDH + DFOM + Z-VAD-FMK (40 μM) group (Application Example 7), and the NiFe-LDH + DFOM + Z-VAD-FMK (80 μM) group (Application Example 8); an appropriate amount of PC3 cells are inoculated into a 24-well plate. After the cells are cultured overnight until they adhere to the wall, the above treatment group materials are added respectively and processed for 24 h. Then, the apoptosis level is determined by the AnnexinⅤ-APC / 7-AAD double staining assay.

[0156] Figure 6 For the control group (Comparative Example 1), the NiFe-LDH + DFOM group (Application Example 6), the NiFe-LDH + DFOM + Z-VAD-FMK (40 μM) group (Application Example 7), and the NiFe-LDH + DFOM + Z-VAD-FMK (80 μM) group (Application Example 8), after treating PC3 cells respectively, flow cytometry is used to detect the apoptosis ratio.

[0157] From Figure 6 It can be seen that NiFe-LDH responsive to DFOM exhibits a significant effect of inducing apoptosis in prostate cancer PC3 cells, and the apoptosis effect is significantly inhibited after combining with the apoptosis inhibitor Z-VAD-FMK.

[0158] Western blot:

[0159] Divided into 4 groups: control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), NiFe-LDH + DFOM group (Application Example 6); Take an appropriate amount of PC3 cells and inoculate them in a 12-well plate. After the cells are adherent and cultured overnight, add the control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), and NiFe-LDH + DFOM group (Application Example 6) to the treatment group cells respectively. After treating the cells for 24 h, use high-efficiency RIPA tissue / cell lysate to extract the total protein of each group of cells, and measure the protein concentration by the BCA method. Electrophorese the protein samples using 12% (the ratio of the total mass of acrylamide to the total volume of the polyacrylamide gel) polyacrylamide gel, and then transfer them to a nitrocellulose (NC) membrane. Block the membrane with 5 wt% bovine serum albumin, and incubate overnight at 4 °C with primary antibodies such as PARP, Pro Caspase-3, Cleaved PARP, and Cleaved Caspase-3. Then incubate the membrane with the corresponding secondary antibody at room temperature for 1 h, and use an enhanced chemiluminescence detection reagent to detect the protein signal to determine the protein expression level.

[0160] Figure 7 It is a volcano plot of the distribution of up-regulated and down-regulated proteins in the NiFe-LDH + DFOM group (Application Example 6) compared with the control group (Comparative Example 1) for proteomic sequencing; Figure 8 It is the expression difference of IGFBP3 between adjacent normal tissues and cancer tissues; Figure 9 It is a representative immunoblot image of the expression of key proteins in the PI3K / AKT / mTOR pathway after treating prostate cancer PC3 cells with the control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), and NiFe-LDH + DFOM group (Application Example 6) for 24 h respectively; Figure 10 It is the expression level of key proteins in the PI3K / AKT / mTOR pathway (n = 3) after treating prostate cancer PC3 cells with the control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), and NiFe-LDH + DFOM group (Application Example 6) for 24 h respectively; Figure 11 It is a representative immunoblot image of the expression of apoptosis-related proteins after treating prostate cancer PC3 cells with the control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), and NiFe-LDH + DFOM group (Application Example 6) for 24 h respectively; Figure 12 It is the expression level of apoptosis-related proteins (n = 3) after treating prostate cancer PC3 cells with the control group (Comparative Example 1), DFOM group (Comparative Example 8), NiFe-LDH group (Comparative Example 4), and NiFe-LDH + DFOM group (Application Example 6) for 24 h respectively; Figure 13Representative immunoblot images of IGFBP3, unphosphorylated and phosphorylated PI3K protein expression after the NiFe-LDH+DFOM group (Application Example 6) treated PC3 cells transfected with si-NC or si-IGFBP3, respectively; Figure 14 Levels of IGFBP3, unphosphorylated and phosphorylated PI3K protein expression (n = 3) after the NiFe-LDH+DFOM group (Application Example 6) treated PC3 cells transfected with si-NC or si-IGFBP3, respectively; Figure 15 Cell viability levels after the control group (Comparative Example 1) and the NiFe-LDH+DFOM group (Application Example 6) treated PC3 cells transfected with si-NC or si-IGFBP3, respectively;

[0161] From Figures 7-15 It can be seen that Ni 2 + released by DFOM-responsive NiFe-LDH can up-regulate IGFBP3, inhibit the activation of PI3K, thereby inhibiting the activity of downstream AKT kinase and a series of phosphorylation cascades; the inhibition of the PI3K / AKT / mTOR signaling pathway leads to the up-regulation of the expression of caspase-3 and PARP in the cleaved form, ultimately triggering apoptosis.

[0162] Example 5 pNiFe-LDH+DFOM group

[0163] The deferoxamine mesylate-responsive nickel-iron layered double hydroxide is composed of DFOM and pNiFe-LDH; the mass ratio of DFOM to pNiFe-LDH is 1:1;

[0164] The preparation method of the pNiFe-LDH is as follows:

[0165] (1) Dissolve 50 mg of PDGFB cyclic peptide, 500 mg of EDC, and 500 mg of NHS in 30 mL of DMSO, stir for 2 h, then add 100 mg of DSPE-PEG-NH2, and continue to stir for 2 h to obtain PDGFB-PEG-DSPE; wherein, the molecular weight of PEG is 2000 Da;

[0166] (2) Disperse 1 g of NiFe-LDH in 100 mL of DMSO, add the PDGFB-PEG-DSPE obtained in the step (1), stir for 12 h, then centrifuge at 25000 rpm and filter, collect the precipitate to obtain pNiFe-LDH;

[0167] The preparation method of the NiFe-LDH is as follows:

[0168] The metal salt solutions of Ni(NO3)2·6H2O and Fe(NO3)3·9H2O dissolved in 20 mL of ultrapure water were added to a flask. The concentration of Ni 2+ in the salt solution was 0.8 M, and the concentration of Fe 3+ was 0.2 M. Then, the flask was placed on a stirrer and stirred evenly. After that, 100 mL of 0.5 M ammonia water was added dropwise to the continuously stirred mixed solution. At this time, the solution became turbid. Then, it was placed in a water bath at 65 °C and continuously stirred for 18 h. The precipitate obtained by centrifuging the final product at 6000 rpm was washed 3 times with absolute ethanol and 5 times with ultrapure water to obtain NiFe-LDH with a particle size of about 30 nm.

[0169] Animal experiment: Mouse prostate cancer cells PC3 (5×10 6 cells) were inoculated into the right back of 6-week-old BALB / c nude mice. When the tumor volume reached about 200 mm 3 , the mice were randomly divided into nine groups: NS (normal saline), DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.) (5 mice in each group). The injection doses were DFOM: 30 mg / kg (prepared with normal saline), NiFe-LDH: 30 mg / kg (prepared with normal saline), pNiFe-LDH: 30 mg / kg (prepared with normal saline). The injection process was divided into two steps: First, NiFe-LDH or pNiFe-LDH was injected through the tail vein, and 3 h later, DFOM was injected through the tail vein (i.v.) or into the tumor (i.t.). The injection was performed once every three days for a total of four injections. The tumor volume and body weight were measured every three days. The treatment was terminated on the 12th day, and the mice were euthanized. Then, serum was collected for liver and kidney function analysis, and the main organs were collected and stained with hematoxylin-eosin (H&E). In addition, the tumor masses dissected from the euthanized mice in each group were photographed and weighed.

[0170] Figure 16 is the flow chart of the animal experiment; Figure 17 is the statistical chart of the average body weight of each group of mice after injection with nine groups: normal saline (NS), DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.); Figure 18Statistical graph of the average tumor volume of each group of mice after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.); Figure 19 Statistical graph of the tumor volume of each of the 5 mice after injection with NS; Figure 20 Statistical graph of the tumor volume of each of the 5 mice after injection with DFOM (i.v.); Figure 21 Statistical graph of the tumor volume of each of the 5 mice after injection with DFOM (i.t.); Figure 22 Statistical graph of the tumor volume of each of the 5 mice after injection with NiFe-LDH; Figure 23 Statistical graph of the tumor volume of each of the 5 mice after injection with pNiFe-LDH; Figure 24 Statistical graph of the tumor volume of each of the 5 mice after injection with NiFe-LDH + DFOM (i.v.); Figure 25 Statistical graph of the tumor volume of each of the 5 mice after injection with NiFe-LDH + DFOM (i.t.); Figure 26 Statistical graph of the tumor volume of each of the 5 mice after injection with pNiFe-LDH + DFOM (i.v.); Figure 27 Statistical graph of the tumor volume of each of the 5 mice after injection with pNiFe-LDH + DFOM (i.t.); Figures 19-27 The abscissa in is time (days), and the ordinate is tumor volume (mm 3 ); Figure 28 Digital pictures of the tumor masses dissected from each group of mice after euthanasia after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.); Figure 29 Data of serum alanine aminotransferase (ALT) of each group of mice after euthanasia after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.); Figure 30Data of serum aspartate aminotransferase (AST) of each group of euthanized mice after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.). Figure 31 Data of blood urea nitrogen (BUN) of each group of euthanized mice after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.). Figure 32 Data of serum creatinine (CREA) of each group of euthanized mice after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.). Figures 28-32 In it, I is NS, II is DFOM (i.v.), III is DFOM (i.t.), IV is NiFe-LDH, V is pNiFe-LDH, VI is NiFe-LDH + DFOM (i.v.), VII is NiFe-LDH + DFOM (i.t.), VIII is pNiFe-LDH + DFOM (i.v.), IX is pNiFe-LDH + DFOM (i.t.). Figure 33 Hematoxylin-eosin (H&E) staining images of major organs of each group of euthanized mice after injection with nine groups: NS, DFOM (i.v.), DFOM (i.t.), NiFe-LDH, pNiFe-LDH, NiFe-LDH + DFOM (i.v.), NiFe-LDH + DFOM (i.t.), pNiFe-LDH + DFOM (i.v.), pNiFe-LDH + DFOM (i.t.). The scale bar in the figure is 100 μm.

[0171] From Figures 16-28It can be seen that in the PC3 mouse prostate cancer tumor-bearing model, both the pNiFe-LDH + DFOM (i.v.) treatment group and the pNiFe-LDH + DFOM (i.t.) treatment group can effectively inhibit tumor growth. Among them, the pNiFe-LDH + DFOM (i.t.) treatment group has the most significant synergistic effect, and the tumor inhibition rate reaches 76.2%.

[0172] From Figures 29-33 It can be seen that in the PC3 mouse prostate cancer tumor-bearing model, the pNiFe-LDH + DFOM (i.t.) treatment group with the most significant synergistic effect did not cause obvious damage to non-target organs, and this combination administration method has good safety.

[0173] It can be seen from the above examples and comparative examples that the deferoxamine mesylate-responsive nickel-iron layered double hydroxide provided by the present invention has excellent tumor inhibitory effects.

[0174] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A deferoxamine mesylate-responsive nickel-iron layered double hydroxide, comprising DFOM and NiFe-LDH.

2. A deferoxamine mesylate-responsive nickel-iron layered double hydroxide, comprising DFOM and target peptide-modified NiFe-LDH.

3. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 2, wherein The target peptide is a DSPE-PEG-modified PDGFR-β binding cyclic peptide.

4. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 3, wherein, The preparation method of the DSPE-PEG-modified PDGFR-β binding cyclic peptide comprises: Mixing the PDGFR-β binding cyclic peptide, a coupling reagent, an organic solvent, and DSPE-PEG-NH2, and performing a first coupling reaction to obtain the DSPE-PEG-modified PDGFR-β binding cyclic peptide.

5. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 4, wherein The coupling reagent is EDC and NHS.

6. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 4, wherein The mass ratio of the PDGFR-β binding cyclic peptide to DSPE-PEG-NH2 is 1:(2-3).

7. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 4, wherein The temperature of the first coupling reaction is room temperature, and the time of the first coupling reaction is 1-3 h.

8. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 2, wherein, The preparation method of the target peptide-modified NiFe-LDH comprises: Mixing NiFe-LDH, an organic solvent, and the target peptide, and performing a second coupling reaction to obtain the target peptide-modified NiFe-LDH.

9. The deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 8, wherein The temperature of the second coupling reaction is room temperature, and the time of the second coupling reaction is 10-14 h.

10. Use of the deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to claim 1 or the deferoxamine mesylate-responsive nickel-iron layered double hydroxide according to any one of claims 2-9 in the preparation of an anti-tumor drug.

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