Magnesium-based nano particle as well as preparation method and application thereof

Abstract

The invention discloses magnesium-based nanoparticles as well as a preparation method and application thereof, and relates to the technical field of tumor drugs. The preparation method comprises the following steps: mixing and heating magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene to form a mixed solution; in an inert gas atmosphere, heating to 260-280 DEG C, keeping for 20-40 minutes, introducing carbon dioxide, keeping for 3-5 hours, adjusting the pH value to 7.5-8.5, and stirring for 1-3 hours to obtain a reaction solution; cooling to room temperature, adding an excessive mixed solution of ethanol and glycerol, standing, and centrifuging to obtain MgCO3 nanoparticles; the MgCO3 nanoparticles, bovine serum albumin and water are mixed, stirred and centrifuged, and the magnesium-based nanoparticles are obtained. The problem of precipitation of MgCO3 in a physiological environment is solved, the polymer can accurately target a tumor site due to the excellent acid responsiveness of the polymer, and the polymer is good in biocompatibility and stability.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor drugs, and in particular to magnesium-based nanoparticles and a preparation method and application thereof. Background Art

[0002] The human immune process is inseparable from immune cells. There are cell surface receptors on the surface of T cells. The receptors mediate the immune process by binding to ligands. There is an integrin on the surface of T cells: LFA-1, also known as lymphocyte function-associated antigen-1. As a signaling molecule, it can bind to the intercellular adhesion molecule ICAM-1 on the surface of target cells. The interaction between LFA-1 and ICAM-1 can regulate the migration, survival and proliferation of T cells, and also participate in the initiation of the formation of immune synapses, mediating lymphocyte homing and other physiological processes.

[0003] LFA-1 is a heterodimer composed of two subunits, α and β, which are non-covalently bonded. The α subunit is composed of approximately 1,000 amino acids and is divided into an extracellular region, a transmembrane region, and an intracellular region. The β subunit includes an N-terminal PSI-domain, a conserved region, and a C-terminal cysteine-rich region. There are several special domains at the N-terminus of the α subunit, including an I-domain domain, which has a cation binding site on the top. The bound cation plays a role in connecting the I-domain to the ligand. Cations stabilize the conformation of integrins and enhance the binding of integrin domain ligands by binding to acidic amino acids in the ligand. The cations bound by the I-domain of LFA-1 are mainly Mg. 2+ , Mn 2+ ,Mg 2+ With Mn 2+ It can change the conformation of integrin, activate integrin and stimulate the binding of LFA-1 and ICAM-1.

[0004] LFA-1 has three conformations: bent-closed, incompletely extended, and fully extended. The bent-closed conformation is a low-affinity conformation, and LFA-1 has a very weak binding ability to ligands such as ICAM-1. The incompletely extended conformation is an intermediate-affinity conformation, and the fully extended conformation has a strong binding ability to ligands and is a high-affinity conformation. 2+ The binding of divalent metal cations to the I-domain of LFA-1 will cause LFA-1 to switch to a high-affinity conformation, thereby promoting the binding of LFA-1 to ICAM-1. The binding of the two directly participates in the formation of immune synapses and promotes the proliferation of T cells.

[0005] Most of the MgCO3 reported in the literature is in the state of precipitation in water, so Mg 2+ How to stably deliver it to the body is an issue that needs to be addressed urgently.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a magnesium-based nanoparticle and a preparation method and application thereof. The present invention solves the precipitation problem of MgCO3 in a physiological environment by preparing MgCO3 nanoparticles. At the same time, the MgCO3 nanoparticles can accurately target tumor sites due to their excellent acid responsiveness. The surface of the MgCO3 nanoparticles is modified with bovine serum albumin (BSA), which can increase its biocompatibility, improve the stability of the nanoparticles, and prevent the nanoparticles from agglomerating and precipitating in a solution.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a method for preparing magnesium-based nanoparticles, comprising:

[0010] Mix magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene uniformly, then heat to 130-150° C., and stir at a stirring speed of 1800-2400 rpm to form a mixed solution; heat to 260-280° C. under an inert gas atmosphere, maintain for 20-40 min, then introduce excess carbon dioxide, maintain for 3-5 h, adjust the pH value to 7.5-8.5, and stir at a stirring speed of 1800-2200 rpm for 1-3 h to obtain a reaction solution;

[0011] The reaction solution was cooled to room temperature, and a mixture of excess ethanol and glycerol was added, and the mixture was allowed to stand for 8-20 minutes, and the precipitate was collected by centrifugation to obtain MgCO3 nanoparticles;

[0012] The MgCO3 nanoparticles, bovine serum albumin and water are mixed, stirred at a stirring speed of 1800-2200 rpm for 20-30 hours, and the precipitate is collected by centrifugation to obtain magnesium-based nanoparticles MgCO3@BSA.

[0013] In an optional embodiment, the mass ratio of the magnesium acetylacetonate dihydrate, the oleylamine, the oleic acid and the 1-octadecene is 1:9-10:2-3:11-12.

[0014] In an optional embodiment, the mixing volume ratio of the ethanol and the glycerol is 3.5-4.5:1.

[0015] In an optional embodiment, the mass volume ratio of the MgCO3 nanoparticles, the bovine serum albumin and the water is 1 mg: 1.01-1.2 mg: 1-3 mL.

[0016] In an optional embodiment, the centrifugal stirring speed is 14000-16000 rpm, and the centrifugation time is 10-20 min.

[0017] In a second aspect, the present invention provides a magnesium-based nanoparticle, which is prepared by the method for preparing the magnesium-based nanoparticle as described in any one of the aforementioned embodiments.

[0018] In an optional embodiment, the particle size of the magnesium-based nanoparticles is 90-100 nm.

[0019] In a third aspect, the present invention provides the use of the magnesium-based nanoparticles as described in the aforementioned embodiment in the preparation of a drug that can controllably release magnesium ions in the acidic environment of a tumor.

[0020] In a fourth aspect, the present invention provides the use of the magnesium-based nanoparticles as described in the above embodiments in the preparation of a drug for improving or treating tumors;

[0021] Preferably, the tumor is melanoma;

[0022] Preferably, the melanoma comprises melanoma B16F10 cells;

[0023] Preferably, the magnesium-based nanoparticles inhibit cancer cell proliferation by upregulating the expression of p-PKM2 and downregulating the expression of p-STAT3 in B16F10 cells.

[0024] In a fifth aspect, the present invention provides the use of the magnesium-based nanoparticles as described in the above embodiments in the preparation of a preparation for promoting a cellular immune response;

[0025] and / or, use of magnesium-based nanoparticles as described in the aforementioned embodiments in the preparation of a preparation for improving the affinity conformation of integrin LFA-1 protein on the surface of T cells;

[0026] And / or, use of magnesium-based nanoparticles as described in the aforementioned embodiments in the preparation of a preparation for promoting T cell proliferation.

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

[0028] The magnesium-based nanoparticles provided by the present invention are prepared by first preparing nano-sized MgO with magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene, then introducing carbon dioxide to prepare MgCO3 nanoparticles, and using a mixed solution of ethanol and glycerol to fully precipitate the MgCO3 nanoparticles, and then combining the MgCO3 nanoparticles with bovine serum albumin to form magnesium-based nanoparticles MgCO3@BSA. In the present invention, the precipitation problem of MgCO3 in a physiological environment is improved by preparing MgCO3 nanoparticles, and MgCO3 nanoparticles are realized. 2+ Stable delivery to the body, at the same time, CO3 2- The hydrolysis properties of MgCO3 nanoparticles can make them have excellent acid responsiveness, which can accurately target tumor sites and release Mg 2+ . Released Mg 2+ On the one hand, it can enter the cell through the TRPM7 channel on the tumor cell membrane. The increase of intracellular magnesium ions promotes the increase of downstream eEF2k, phosphorylation of downstream PKM2 protein, reduction of PKM2 dimer, weakening the protein kinase activity of PKM2 dimer, leading to a decrease in phosphorylation of downstream transcription activator STAT3, thereby inhibiting cancer cell proliferation. 2+ As a divalent metal ion, it binds to the metal ion binding site of LFA-1 on the surface of T cells, promoting the conversion of LFA-1 from a low-affinity conformation to a high-affinity conformation, promoting T cell proliferation and activation, and enhancing the body's immunity. Modifying the surface of MgCO3 nanoparticles with bovine serum albumin (BSA) can increase their biocompatibility, improve the stability of nanoparticles, prevent nanoparticles from agglomerating and precipitating in the solution, and help enhance the anti-cancer effect and cellular immune response of MgCO3 nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 Transmission electron micrographs of MgCO3 nanoparticles (a) and MgCO3@BSA nanoparticles (b) provided in Example 1 of the present invention;

[0031] Figure 2 The particle size distribution diagram of MgCO3 nanoparticles (a) and MgCO3@BSA nanoparticles (b) provided in Example 1 of the present invention;

[0032] Figure 3Potential diagram of BSA, MgCO3 nanoparticles and MgCO3@BSA nanoparticles provided in Example 1 of the present invention;

[0033] Figure 4 This is an elemental analysis diagram of MgCO3@BSA nanoparticles provided in Example 1 of the present invention;

[0034] Figure 5 Transmission electron micrographs of MgCO3 nanoparticles (a) and MgCO3@BSA nanoparticles (b) provided in Comparative Example 1 of the present invention;

[0035] Figure 6 This is a diagram showing the effect of ethanol and glycerol at different volume ratios on the precipitation of MgCO3@BSA nanoparticles in Experimental Example 1 of the present invention;

[0036] Figure 7 ion release curve of MgCO3@BSA nanoparticles in solvents at pH=5.5 and pH=7.4 in Experimental Example 2 of the present invention;

[0037] Figure 8 This is a graph showing the morphological changes of MgCO3@BSA nanoparticles in the solvents of pH=5.5 and pH=7.4 in Experimental Example 2 of the present invention;

[0038] Fig. 9 The figure (a) shows the survival rate of melanoma B16F10 cells under different concentrations of MgCO3@BSA nanoparticles and the figure (b) shows the survival rate of melanoma B16F10 cells under different treatment groups in Experimental Example 3 of the present invention;

[0039] Fig.10 Schematic diagram of the expression of eEF2k, PKM2, p-PKM2, STAT3 and p-STAT3 in melanoma B16F10 cells in Experimental Example 4 of the present invention;

[0040] Fig.11 This is a schematic diagram of the expression of high-affinity conformation LFA-1 on the surface of T cells in different treatment groups in Experimental Example 5 of the present invention;

[0041] Fig.12 Schematic diagram of the changes in tumor appearance (a), volume (b) and weight (c) of B16F10 model mice in different treatment groups in Experimental Example 6 of the present invention;

[0042] Fig.13 Schematic diagram of the changes in immune cells of B16F10 model mice in different treatment groups in Experimental Example 6 of the present invention. DETAILED DESCRIPTION

[0043] 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.

[0044] The present invention provides a method for preparing magnesium-based nanoparticles, which comprises:

[0045] S1. Preparation of MgCO3 nanoparticles.

[0046] Magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene are mixed uniformly under ultrasonic conditions, then heated to 130-150°C, and stirred at a stirring speed of 1800-2400rpm to form a mixed solution; the mass ratio of magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene is 1:9-10:2-3:11-12. In an inert gas atmosphere, heat to 260-280°C and maintain for 20-40 minutes.

[0047] In the present invention, high-speed stirring can make the mixture of magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene heated more evenly, and react to generate MgO nanoparticles in a nitrogen atmosphere. If the stirring speed is too low, uneven heating will result, and the size uniformity of the prepared magnesium oxide will be poor.

[0048] Then, an excess of carbon dioxide is introduced and maintained for 3-5 hours, the pH value is adjusted to 7.5-8.5, and the mixture is stirred at a stirring speed of 1800-2200 rpm for 1-3 hours to obtain a reaction solution.

[0049] In the present invention, carbon dioxide is continuously introduced during the entire reaction process, and the amount of carbon dioxide introduced is excessive. At this time, the nanoparticles of MgO react with the introduced carbon dioxide to generate MgCO3 nanoparticles. It should be understood that when carbon dioxide is introduced, carbon dioxide can be introduced independently, or a mixed gas of carbon dioxide and nitrogen can be introduced. The introduction ratio of carbon dioxide and nitrogen can be, for example, 90-100:0-10, and preferably the introduction ratio of carbon dioxide and nitrogen is 95:5. The reaction solution is cooled to room temperature, a mixed solution of excess ethanol and glycerol is added, and the mixture is allowed to stand for 8-20 minutes, centrifuged at 14000-16000rpm for 10-20 minutes, and the precipitate is collected to obtain MgCO3 nanoparticles. The mixed volume ratio of ethanol and glycerol is 3.5-4.5:1.

[0050] In the present invention, a mixture of ethanol and glycerol is used as a precipitation solution to precipitate MgCO3 nanoparticles in the reaction solution. The present invention has found that when the proportion of ethanol is too large or too small, the precipitation effect of MgCO3 nanoparticles will be affected, or effective precipitation will not be possible.

[0051] S2. Preparation of magnesium-based nanoparticles MgCO3@BSA.

[0052] MgCO3 nanoparticles, bovine serum albumin and water are mixed, stirred at a stirring speed of 1800-2200 rpm for 20-30 hours, centrifuged at 14000-16000 rpm for 10-20 minutes, and the precipitate is collected to obtain magnesium-based nanoparticles MgCO3@BSA.

[0053] The mass volume ratio of MgCO3 nanoparticles, bovine serum albumin and water is 1 mg: 1.01-1.2 mg: 1-3 mL.

[0054] BSA has abundant carboxyl, amino and thiol groups, and has a strong affinity for divalent metal cations in the body. MgCO3 nanoparticles bind to BSA through electrostatic or coordination interactions. Magnesium-based nanoparticles take advantage of the good biocompatibility and stability of BSA, enhancing the anti-cancer effect of nanoparticles.

[0055] The particle size of the magnesium-based nanoparticles prepared by the above-mentioned method for preparing magnesium-based nanoparticles is 90-100 nm.

[0056] The mechanism of the magnesium-based nanoparticles is as follows:

[0057] After local injection of magnesium-based nanoparticles, CO3 2- The hydrolysis characteristics of MgCO3@BSA nanoparticles can promote the release of MgCO3@BSA nanoparticles in the slightly acidic environment of tumors. 2+ On the one hand, it can enter the cell through the TRPM7 channel on the tumor cell membrane. The increase of intracellular magnesium ions promotes the increase of downstream eEF2k, phosphorylation of downstream PKM2 protein, reduction of PKM2 dimer, weakening the protein kinase activity of PKM2 dimer, leading to a decrease in phosphorylation of downstream transcription activator STAT3, thereby inhibiting cancer cell proliferation. 2+ As a divalent metal ion, it binds to the metal ion binding site of LFA-1 on the surface of T cells, promoting the conversion of LFA-1 from a low-affinity conformation to a high-affinity conformation, promoting T cell proliferation and activation, and enhancing the body's immunity. Magnesium-based nanoparticles utilize the good biocompatibility and stability of BSA, enhance the anti-cancer effect of nanoparticles, and can also promote cellular immune responses.

[0058] In this regard, the present invention correspondingly provides the use of the above-mentioned magnesium-based nanoparticles in the preparation of drugs that can controllably release magnesium ions in the acidic environment of tumors.

[0059] The present invention also provides the use of the above-mentioned magnesium-based nanoparticles in the preparation of drugs for improving or treating tumors;

[0060] Preferably, the tumor is melanoma;

[0061] Preferably, the melanoma comprises melanoma B16F10 cells;

[0062] Preferably, the magnesium-based nanoparticles inhibit cancer cell proliferation by upregulating the expression of p-PKM2 and downregulating the expression of p-STAT3 in B16F10 cells.

[0063] The present invention also provides the use of the above-mentioned magnesium-based nanoparticles in the preparation of a preparation for promoting cellular immune response;

[0064] and / or, use of the above-mentioned magnesium-based nanoparticles in the preparation of a preparation for improving the affinity conformation of the integrin LFA-1 protein on the surface of T cells;

[0065] And / or, use of the above-mentioned magnesium-based nanoparticles in the preparation of a preparation for promoting T cell proliferation.

[0066] The experimental reagents used in the present invention were all purchased from commercial sources, among which magnesium acetylacetonate dihydrate (Mg(acac)2·2H2O), oleic acid (OA), oleylamine (OM), and 1-octadecene (ODE) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Bovine serum albumin (BSA) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China). All chemicals were used as received without further treatment.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1

[0069] This embodiment provides a magnesium-based nanoparticle, and the preparation method thereof includes:

[0070] S1. Add 1.335g of magnesium acetylacetonate dihydrate, 12.864g of oleylamine (OM), 3.384g of oleic acid (OA), and 15.15g of 1-octadecene (ODE) to a three-necked flask and mix them evenly under ultrasonic conditions to form a milky white liquid. Use a magnetic stirring electric heating mantle to heat the mixture to 140°C, and form a uniform light yellow solution at a stirring rate of 2000rpm. Then, nitrogen was introduced into the three-necked flask, and the mixture was heated to 280°C and maintained at this temperature for 30 minutes. The solution temperature was maintained at 280°C, nitrogen was stopped, and carbon dioxide was continuously introduced for 4 hours. The pH value of the reaction sample was adjusted to 8.0 with NaOH (5.0M, 1μL), and a magnetic stirrer was used to stir at 2000rpm for 2 hours to obtain a reaction solution. The reaction solution was cooled to room temperature, and excess ethanol and glycerol (volume ratio of 4:1) were added and allowed to stand for 10 minutes. The mixture was centrifuged at 15000 rpm for 15 minutes, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and ultrapure water, respectively, to prepare MgCO3 nanoparticles (denoted as MgCO3NPs).

[0071] S2. Add 10 mg of MgCO3 nanoparticles and 10 mg of bovine serum albumin (BSA) into 20 mL of ultrapure water, stir at 2000 rpm using a magnetic stirrer for 24 hours, centrifuge at 15000 rpm for 15 minutes, collect the precipitate, and wash it three times with ultrapure water to prepare MgCO3@BSA nanoparticles as magnesium-based nanoparticles (denoted as MCB NPs).

[0072] The MgCO3 nanoparticles (denoted as MgCO3 NPs) and MgCO3@BSA nanoparticles (denoted as MCB NPs) prepared in Example 1 were characterized. Figure 1 , Figure 2 , Figure 3 and Figure 4 .

[0073] Figure 1 Figure a is a transmission electron microscope image of MgCO3 nanoparticles. Figure 1 Figure b is a transmission electron micrograph of MgCO3@BSA nanoparticles. By comparison, we can see that Figure 1 The surface of the MgCO3@BSA nanoparticles in (b) has a coating film, which proves that BSA is successfully loaded on the surface of the MgCO3 nanoparticles.

[0074] Figure 2 a in the figure is the particle size distribution of MgCO3 nanoparticles. Figure 2Figure b shows the particle size distribution of MgCO3@BSA nanoparticles. By comparison, it can be seen that the particle size of MgCO3 nanoparticles is 91.5nm, and after loading with BSA, the particle size of MgCO3@BSA nanoparticles increases to 96.0nm.

[0075] from Figure 3 It can be seen from the potential diagram that the potential of MgCO3@BSA nanoparticles is significantly different from that of BSA and MgCO3 NPs alone, proving that MgCO3@BSA nanoparticles were effectively synthesized in this example.

[0076] from Figure 4 It can be seen that the three elements Mg, C, and O in the synthesized MgCO3@BSA nanoparticles are evenly distributed in the nanoparticles.

[0077] Example 2

[0078] This embodiment provides a magnesium-based nanoparticle, and the preparation method thereof includes:

[0079] S1. Add 1.335g of magnesium acetylacetonate dihydrate, 12.015g of oleylamine (OM), 2.67g of oleic acid (OA), and 14.685g of 1-octadecene (ODE) to a three-necked flask and mix them evenly under ultrasonic conditions to form a milky white liquid. Use a magnetic stirring electric heating jacket to heat the mixture to 130°C, and form a uniform light yellow solution at a stirring rate of 2000rpm. Then, nitrogen was introduced into the three-necked flask, and the mixture was heated to 260°C and maintained at this temperature for 40 minutes. The solution temperature was maintained at 260°C, nitrogen was stopped, and carbon dioxide was continuously introduced for 3 hours. The pH value of the reaction sample was adjusted to 7.5 with NaOH (5.0M, 1μL), and a magnetic stirrer was used to stir at 1800rpm for 3 hours to obtain a reaction solution. The reaction solution was cooled to room temperature, and excess ethanol and glycerol (volume ratio of 3.5:1) were added and allowed to stand for 10 minutes. The mixture was centrifuged at 14000 rpm for 20 minutes, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and ultrapure water, respectively, to prepare MgCO3 nanoparticles (denoted as MgCO3 NPs).

[0080] S2. Add 10 mg of MgCO3 nanoparticles and 10.1 mg of bovine serum albumin (BSA) to 10 mL of ultrapure water, stir at 1800 rpm using a magnetic stirrer for 30 hours, centrifuge at 14000 rpm for 20 minutes, collect the precipitate, and wash it three times with ultrapure water to prepare MgCO3@BSA nanoparticles as magnesium-based nanoparticles (denoted as MCB NPs).

[0081] Example 3

[0082] This embodiment provides a magnesium-based nanoparticle, and the preparation method thereof includes:

[0083] S1. Add 1.335g of magnesium acetylacetonate dihydrate, 13.35g of oleylamine (OM), 4.005g of oleic acid (OA), and 16.02g of 1-octadecene (ODE) to a three-necked flask and mix them evenly under ultrasonic conditions to form a milky white liquid. Use a magnetic stirring electric heating jacket to heat the mixture to 150°C, and form a uniform light yellow solution at a stirring rate of 2400rpm. Then, nitrogen was introduced into the three-necked flask, and the mixture was heated to 280°C and maintained at this temperature for 20 minutes. The solution temperature was maintained at 300°C, nitrogen was stopped, and carbon dioxide was continuously introduced for 5 hours. The pH value of the reaction sample was adjusted to 8.5 with NaOH (5.0M, 1μL), and a magnetic stirrer was used to stir at 2200rpm for 1 hour to obtain a reaction solution. The reaction solution was cooled to room temperature, and excess ethanol and glycerol (volume ratio of 4.5:1) were added and allowed to stand for 10 minutes. The mixture was centrifuged at 16000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed three times with anhydrous ethanol and ultrapure water, respectively, to prepare MgCO3 nanoparticles (denoted as MgCO3NPs).

[0084] S2. Add 10 mg of MgCO3 nanoparticles and 12 mg of bovine serum albumin (BSA) to 30 mL of ultrapure water, stir at 2200 rpm using a magnetic stirrer for 20 hours, centrifuge at 16000 rpm for 10 minutes, collect the precipitate, and wash it three times with ultrapure water to prepare MgCO3@BSA nanoparticles as magnesium-based nanoparticles (denoted as MCB NPs).

[0085] Comparative Example 1

[0086] This comparative example provides a magnesium-based nanoparticle, and its preparation method is different from that of Example 1, and the specific operation is as follows:

[0087] Weigh 1.5g MgCl2·6H2O and 0.15g polyvinylpyrrolidone (PVP), measure 100mL anhydrous ethanol, place them in a clean 100mL beaker, stir at 1000rpm on a stirring table for 10 minutes to obtain a uniformly mixed solution, cover the beaker mouth with tin foil, and evenly punch holes in the tin foil. Weigh 15g dry NH4HCO3 and put it in another clean beaker. Place the two beakers in a vacuum dryer and react in a vacuum environment at 50℃ for 24 hours. After the reaction is completed, the resulting suspension is centrifuged at 12000rpm for 10 minutes, the precipitate is collected, and washed three times with ultrapure water to prepare MgCO3 nanoparticles (referred to as MgCO3 NPs).

[0088] 10 mg of MgCO3 nanoparticles and 10 mg of bovine serum albumin (BSA) were added to 20 mL of ultrapure water, stirred at 1800 rpm using a magnetic stirrer for 24 hours, centrifuged at 10000 rpm using a centrifuge for 10 minutes, the precipitate was collected, and washed three times with ultrapure water to prepare MgCO3@BSA nanoparticles as magnesium-based nanoparticles (denoted as MCB NPs).

[0089] The MgCO3 nanoparticles and MgCO3@BSA nanoparticles prepared in Comparative Example 1 were characterized. Figure 5 .

[0090] from Figure 5 As can be seen in Figure a, MgCO3 nanoparticles tend to aggregate and stack, with poor dispersion. Figure 5 As can be seen in b, the dispersibility of MgCO3@BSA nanoparticles is improved compared with that of MgCO3 nanoparticles, but it is still difficult to form nanoparticles with uniform particle size and stable morphology, which is not conducive to the biological application of nanoparticles.

[0091] Comparative Example 2

[0092] The present invention provides MgCl2.

[0093] Comparative Example 3

[0094] The present invention provides Na2CO3.

[0095] Experimental Example 1: Investigation of the Volume Ratio of Ethanol and Glycerol

[0096] After the reaction solution prepared in Example 1 was cooled to room temperature, it was allowed to stand for 10 min at a volume ratio of ethanol to glycerol of 1:1, 2:1, 3:1, 4:1, 5:1, and 10:1, respectively, and then the precipitation of MgCO3 nanoparticles was observed.

[0097] See also Figure 6 It can be seen that the nanoparticle precipitation rate is the highest when the ratio of ethanol to glycerol is 4:1. When the proportion of ethanol is too small, the amount of MgCO3 nanoparticles precipitated is small, and when the proportion of ethanol is too large, the MgCO3 nanoparticles cannot be effectively precipitated and the reaction solution cannot be effectively stratified.

[0098] Experimental Example 2: Ion release effect of MgCO3@BSA nanoparticles at different pH over time

[0099] The test method includes: placing the MgCO3@BSA nanoparticles prepared in Example 1 in PBS solutions of pH = 5.5 and pH = 7.4, respectively, detecting the ion release amount thereof at 0, 1, 2, 4, 8, 12, and 24 hours, respectively, and drawing the release curve thereof changing with time.

[0100] Test results can be found in Figure 7 .

[0101] from Figure 7 It can be seen that when pH = 7.4, MgCO3@BSA nanoparticles 2+ The release rate of Mg was low, with a 24-hour release rate of about 14.87%. Under acidic conditions of pH = 5.5, Mg 2+ The release rate is high, and the 24-hour release rate is about 85.8%. The above results show that MgCO3@BSA nanoparticles can be better released in the acidic environment of tumors.

[0102] Furthermore, this experimental example also tested the morphological changes of MgCO3@BSA nanoparticles under pH=7.4 and pH=5.5. Figure 8 It can be seen that under acidic conditions of pH = 5.5, MgCO3@BSA nanoparticles decompose rapidly, the particle size decreases and the morphology changes significantly, proving that its Mg 2+ The release rate of MgCO3@BSA nanoparticles was high. The morphology of MgCO3@BSA nanoparticles did not change significantly at pH = 7.4, which proved that its Mg 2+ The release rate is low.

[0103] Experimental Example 3 - Experiment on the killing effect of nanoparticles on melanoma B16F10 cells

[0104] The experimental method includes: adding different concentrations of MgCO3@BSA nanoparticles (MCB) provided in Example 1 to melanoma B16F10 cells, and detecting the effect of MgCO3@BSA nanoparticles on the survival rate of B16F10 cells as the concentration changes.

[0105] from Fig. 9 As can be seen in figure a, as the concentration of MgCO3@BSA increases, its ability to kill melanoma gradually increases. When the concentration of MgCO3@BSA reaches 100 μg / mL, its ability to kill melanoma reaches 79%.

[0106] In this experimental example, a killing experiment of different treatment groups on melanoma B16F10 cells was conducted in melanoma B16F10 cells.

[0107] This experimental example also sets up different treatment groups, which are respectively a blank control group (control), a negative control group (NaCl), a comparative example 2 group (MgCl2), a comparative example 3 group (Na2CO3) and an example 1 group (MCB) using a DMEM high-glucose medium containing 10% fetal bovine serum. The experimental method includes: using a DMEM high-glucose medium containing 10% fetal bovine serum to prepare the components of the above different treatment groups into 100 μg / mL reagents, adding 100 μg / mL to melanoma B16F10 cells to obtain different treatment groups, and detecting the effects of different treatment groups on the survival rate of B16F10 cells.

[0108] from Fig. 9 As can be seen in Figure b, the killing ability of MgCO3@BSA (100 μg / mL) nanoparticles against melanoma reached 79%, which is 4 times the killing ability of other treatment groups.

[0109] Experimental Example 4 - Western Blot Detection

[0110] This experimental example detects the expression of eEF2k, PKM2, p-PKM2, STAT3 and p-STAT3 in melanoma B16F10 cells. The different treatment groups are DMEM high glucose medium containing 10% fetal bovine serum as a blank control group (control), comparative example 2 group (MgCl2), comparative example 3 group (Na2CO3) and Example 1 group (MCB).

[0111] The experimental method included: nanoparticles in different treatment groups were co-incubated with melanoma B16F10 cells for 10 h, samples were collected, and the expression of eEF2k, PKM2, p-PKM2, STAT3 and p-STAT3 was measured by immunoblotting.

[0112] from Fig.10 It can be seen that the nanoparticles (MCB) provided in Example 1 can upregulate the expression of p-PKM2 and downregulate the expression of p-STAT3 in B16F10 cells. However, there is no significant change in the expression of p-PKM2 and p-STAT3 in Comparative Example 2 (MgCl2) and Comparative Example 3 (Na2CO3) compared with the blank control group (control).

[0113] Experimental Example 5

[0114] The experimental method includes: incubating B16F10 cells and mouse spleen T cells in a 1:1 ratio, adding a blank control group (control DMEM high-glucose medium containing 10% fetal bovine serum) and Example 1 group (MCB, MCB dissolved in DMEM high-glucose medium containing 10% fetal bovine serum) respectively, incubating in a cell culture incubator at 37°C for 30 minutes. In the last 10 minutes of incubation, adding LFA-1 protein m24 type monoclonal antibody, after the incubation, detecting the expression of LFA-1 protein with high affinity conformation on the surface of T cells.

[0115] Fig.11 The green fluorescence in the middle is B16F10 cells, the blue fluorescence is the T cell nucleus, and the red fluorescence is the high-affinity conformation of LFA-1 protein. After being treated with the Example 1 group (MCB), the expression of high-affinity conformation LFA-1 on the surface of T cells increased, proving that the MgCO3@BSA nanoparticles provided in Example 1 can increase the affinity conformation of the integrin LFA-1 protein on the surface of T cells, promote T cell proliferation and activation, and enhance the body's immunity.

[0116] Experimental Example 6 - Mouse Model Experiment

[0117] The mouse melanoma orthotopic model was established according to the conventional method: 100 μL of 5×10 5 When the tumor volume grew to about 100 mm3, the mice were randomly divided into 4 groups.

[0118] In this experimental example, the anti-cancer effect and cellular immune effect of melanoma B16F10 model mice under different treatment groups were tested. The different treatment groups were PBS as a blank control group (control), comparative example group 2 (MgCl2), comparative example group 3 (Na2CO3) and example group 1 (MCB).

[0119] (1) Anti-cancer experiments

[0120] The experimental method includes: intratumoral injection of nanoparticles in melanoma B16F10 model mice, respectively, in which the control group is 1×PBS; the drugs in the other treatment groups are dissolved in sterilized 1×PBS; the drug is administered once every two days, for a total of 7 treatments, the mice are weighed once every two days, the length and width of the mouse tumor are measured once, and the tumor volume is calculated using the formula: (length × width 2 )÷2. After treatment, the tumor tissues of mice were taken, photographed and weighed.

[0121] from Fig.12As can be seen from a, although the appearance of the tumor in the comparative example 2 group (MgCl2) and the comparative example 3 group (Na2CO3) has been reduced to a certain extent, it is not much different from the blank control group (control), while the appearance of the tumor in the example 1 group (MCB) has been significantly reduced. Fig.12 As can be seen from b and c, the effect of reducing the tumor volume and weight in Example 1 group (MCB) is also significantly better than that in the blank control group (control), comparative example 2 group (MgCl2), and comparative example 3 group (Na2CO3).

[0122] (2) Cellular immune effect

[0123] The experimental method includes: intratumoral injection of nanoparticles in melanoma B16F10 model mice, respectively, and the control group is 1×PBS; the drugs in the other treatment groups are dissolved in sterilized 1×PBS. After treatment of different treatment groups, mouse tumor tissues were taken for flow cytometry. The mouse tumor tissue was peeled off and 3mm 3 The tissue was cut into small cubes with sterile surgical scissors, homogenized in 1× PBS, and filtered through a 70μm cell filter to obtain a tumor single cell suspension. The cells were labeled with antibodies PE-anti-CD3, FITC-anti-CD4, APC-anti-CD8, and flow cytometry was used to evaluate the CD3 expression in tumor tissues. + 、CD4 + and CD8 + of T cells.

[0124] from Fig.13 It can be seen that the immune cells of the group treated with Example 1 (MCB) were significantly activated, and the number of activated immune cells in the MgCO3@BSA nanoparticle group was the largest. The measurement results showed that CD3 + 、CD4 + Compared with the blank control group, the number of T cells increased by about 36.7%, and CD3 + 、CD8 + T cells increased by about 8%.

[0125] In summary, the MgCO3@BSA nanoparticles provided by the present invention first prepare nano-sized MgO by using magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene, then introduce carbon dioxide to prepare MgCO3 nanoparticles, and fully precipitate the MgCO3 nanoparticles using a mixed solution of ethanol and glycerol, and then combine the MgCO3 nanoparticles with bovine serum albumin to form MgCO3@BSA nanoparticles. In the present invention, the precipitation problem of MgCO3 in a physiological environment is improved by preparing MgCO3 nanoparticles, and MgCO3 nanoparticles are realized. 2+ Stable delivery to the body, at the same time, CO3 2-The hydrolysis properties of MgCO3 nanoparticles can make them have excellent acid responsiveness, which can accurately target tumor sites and release Mg 2+ . Released Mg 2+ On the one hand, it can enter the cell through the TRPM7 channel on the tumor cell membrane. The increase of intracellular magnesium ions promotes the increase of downstream eEF2k, phosphorylation of downstream PKM2 protein, reduction of PKM2 dimer, weakening the protein kinase activity of PKM2 dimer, leading to a decrease in phosphorylation of downstream transcription activator STAT3, thereby inhibiting cancer cell proliferation. 2+ As a divalent metal ion, it binds to the metal ion binding site of LFA-1 on the surface of T cells, promoting the conversion of LFA-1 from a low-affinity conformation to a high-affinity conformation, promoting T cell proliferation and activation, and enhancing the body's immunity. Modifying the surface of MgCO3 nanoparticles with bovine serum albumin (BSA) can increase their biocompatibility, improve the stability of nanoparticles, prevent nanoparticles from agglomerating and precipitating in the solution, and help enhance the anti-cancer effect and cellular immune response of MgCO3 nanoparticles.

[0126] The above are only preferred embodiments of the present invention and are 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 method for preparing magnesium-based nanoparticles, characterized in that: It includes: Magnesium acetylacetonate dihydrate, oleylamine, oleic acid and 1-octadecene are uniformly mixed, and then heated to 130-150° C., and stirred at a stirring speed of 1800-2400 rpm to form a mixed solution; heated to 260-280° C. under an inert gas atmosphere, maintained for 20-40 min, and then introduced with excess carbon dioxide, maintained for 3-5 h, adjusted the pH value to 7.5-8.5, and stirred at a stirring speed of 1800-2200 rpm for 1-3 h to obtain a reaction solution; the reaction solution is cooled to room temperature, and a mixed solution of excess ethanol and glycerol is added, and the mixture is allowed to stand for 8-20 min, and the precipitate is collected by centrifugation to obtain MgCO3 nanoparticles; The MgCO3 nanoparticles, bovine serum albumin and water are mixed, stirred at a stirring speed of 1800-2200 rpm for 20-30 hours, and the precipitate is collected by centrifugation to obtain magnesium-based nanoparticles MgCO3@BSA.

2. The method for preparing magnesium-based nanoparticles according to claim 1, characterized in that: The mass ratio of the magnesium acetylacetonate dihydrate, the oleylamine, the oleic acid and the 1-octadecene is 1:9-10:2-3:11-12.

3. The method for preparing magnesium-based nanoparticles according to claim 1, characterized in that: The mixing volume ratio of the ethanol and the glycerol is 3.5-4.5:

1.

4. The method for preparing magnesium-based nanoparticles according to claim 1, characterized in that: The mass volume ratio of the MgCO3 nanoparticles, the bovine serum albumin and the water is 1 mg: 1.01-1.2mg:1-3mL.

5. The method for preparing magnesium-based nanoparticles according to claim 1, characterized in that: The stirring speed of the centrifugation is 14000-16000 rpm, and the centrifugation time is 10-20 min.

6. A magnesium-based nanoparticle, characterized in that: The magnesium-based nanoparticles are prepared by the method for preparing the magnesium-based nanoparticles according to any one of claims 1 to 5.

7. The magnesium-based nanoparticles according to claim 6, characterized in that The particle size of the magnesium-based nanoparticles is 90-100 nm.

8. Use of the magnesium-based nanoparticles according to claim 6 in preparing a drug capable of controllably releasing magnesium ions in an acidic environment of a tumor.

9. Use of the magnesium-based nanoparticles according to claim 6 in preparing drugs for improving or treating tumors; Preferably, the tumor is melanoma; Preferably, the melanoma comprises melanoma B16F10 cells; Preferably, the magnesium-based nanoparticles inhibit cancer cell proliferation by upregulating the expression of p-PKM2 and downregulating the expression of p-STAT3 in B16F10 cells.

10. Use of the magnesium-based nanoparticles according to claim 6 in preparing a preparation for promoting cellular immune response; And / or, use of the magnesium-based nanoparticles as claimed in claim 6 in the preparation of a preparation for improving the affinity conformation of the integrin LFA-1 protein on the surface of T cells; And / or, use of the magnesium-based nanoparticles as claimed in claim 6 in the preparation of a preparation for promoting T cell proliferation.

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