Preparation method of bovine serum albumin-metal nano-composite

Abstract

The invention discloses a preparation method of a bovine serum albumin-metal nano-composite, relates to the technical field of nano-materials, and particularly relates to a method for preparing a nano-material through biomineralization after coordination of protein and metal ions. Bovine serum albumin (BSA) serves as a common multifunctional protein carrier, and the molecular structure of the bovine serum albumin (BSA) is rich in amino acid residues. Specific groups on the residues can be coordinated with metal ions. After metal ions are coordinated with bovine serum albumin, the metal ions can be further mineralized under specific conditions to form nanoparticles. The formed nano material has good biological safety, and serum protein can be conveniently and chemically modified based on the residues with reaction activity in serum protein molecules. According to the invention, a metal ion-protein coordination process is combined with a biomineralization reaction in simulated body fluid, and a mechanism research basis is provided for interaction of exogenous / endogenous metal ions and protein in vivo.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to a method for preparing nanomaterials through biomineralization after coordination of proteins and metal ions. Background Art

[0002] With the rapid development of materials science and nanotechnology, various nanocarriers such as liposomes, polymer nanoparticles, inorganic nanoparticles and proteins have received extensive attention in the field of precision cancer nanomedicine. Among them, serum proteins have shown good safety in biomedical applications due to their extremely low toxicity and immunogenicity. This property makes them widely used as carriers of small molecule therapeutic drugs and imaging agents, and they are also ideal candidate materials for vaccine excipients and other drugs.

[0003] Bovine serum albumin (BSA) is a common multifunctional protein carrier, and its molecular structure is rich in amino acid residues. Specific groups on these residues can coordinate with metal ions. When metal ions coordinate with bovine serum albumin, they can be further mineralized to form nanoparticles under specific conditions. The formed nanomaterial is based on these reactive residues in serum protein molecules, and serum protein can be easily chemically modified. The preparation method of the present invention combines the metal ion-protein coordination process with the biomineralization reaction in simulated body fluids, providing a basis for the mechanism study of the interaction between exogenous / endogenous metal ions and proteins in vivo. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing nanomaterials by mineralization reaction after coordination of protein and metal ions.

[0005] Furthermore, the method for preparing the bovine serum albumin-metal nanocomposite comprises the following steps:

[0006] The BSA protein and the metal salt were stirred and dissolved in deionized water and stirred at room temperature for 1 hour to promote the coordination between the metal ions and the BSA protein.

[0007] Furthermore, the above solution was dissolved in simulated body fluid (SBF), Tris HCl was added, and the mixture was stirred and mixed uniformly at room temperature.

[0008] Furthermore, CaCl 2 Ultrasonic dissolution in deionized water to make CaCl 2 The solution was added dropwise to the above mixed solution, stirred for reaction at room temperature for 1 h, the supernatant was discarded, the product was obtained after washing with deionized water 3 times and freeze-dried.

[0009] Furthermore, the metal salt used in the present invention is MnCl 2 ·4H2 O、ZnCl 2 MgSO 4 7H 2 O, FeCl 2 ·4H 2 O.

[0010] Compared with the prior art, the present invention provides a bovine serum albumin-metal nanocomposite and a preparation method thereof. The present invention only reacts the bovine serum albumin solution and the metal ion source solution, and forms nanoparticles by mineralization under the action of calcium ions. The whole process only requires room temperature stirring, and no complex equipment or high temperature and high pressure conditions are required. The generated Fe, Mn, and Mg-based complexes have excellent biocompatibility due to the natural carrier characteristics of BSA, and can be adapted to biological systems such as drug delivery; and the Zn-based complexes show a killing effect on 4T1 tumor cells at a lower concentration (300 μg / mL), providing a new strategy for the development of low-toxic and high-efficiency metal anticancer drugs. In addition, the technology provides a basis for the mechanism study of the interaction between exogenous / endogenous metal ions and proteins in vivo by simulating the coordination-mineralization process of metal ions and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a transmission electron microscope image of the bovine serum albumin-calcium manganese nanocomposite prepared by the present invention;

[0012] Figure 2 It is a transmission electron microscope image of the bovine serum albumin-calcium zinc nanocomposite prepared by the present invention;

[0013] Figure 3 It is a transmission electron microscope image of the bovine serum albumin-calcium-magnesium nanocomposite prepared by the present invention;

[0014] Figure 4 It is a transmission electron microscope image of the bovine serum albumin-calcium iron nanocomposite prepared by the present invention;

[0015] Figure 5 It is the bovine serum albumin-metal nanocomposite prepared by the present invention, wherein (a) 150 μg / mL (b) 150 μg / mL of 3T3 / L929 normal fibroblast activity graph;

[0016] Figure 6 This is a 4T1 tumor cell activity diagram of the bovine serum albumin-metal nanocomposite prepared by the present invention. DETAILED DESCRIPTION

[0017] Specific embodiments of the present invention are given below to further illustrate the composition of the present invention.

[0018] Example 1

[0019] (1) 15 mg BSA protein and 16 mg MnCl 2 ·4H 2 O was dissolved in 5 mL of deionized water and stirred at room temperature for 1 h to promote the 2+ Coordinate with BSA protein. Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of TrisHCl (pH 8.0), and stir to mix evenly at room temperature. 2 Ultrasonic dissolution in 5 mL of deionized water, 1.5 mL of CaCl 2 The solution was added dropwise to the above mixed solution, stirred for reaction at room temperature for 1 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, washed with deionized water 3 times and freeze-dried to obtain the product.

[0020] (2) 15 mg BSA protein and 12.5 mg ZnCl 2 Stir and dissolve in 5 mL of deionized water and stir at room temperature for 1 h to promote Zn 2+ Coordinate with BSA protein. Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of Tris HCl (pH 8.0), and stir to mix well at room temperature. 2 Ultrasonic dissolution in 5 mL of deionized water, 1.5 mL of CaCl 2 The solution was added dropwise to the above mixed solution, stirred for reaction at room temperature for 1 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, washed with deionized water 3 times and freeze-dried to obtain the product.

[0021] (3) 15 mg BSA protein and 20.5 mg MgSO 4 7H 2 O was dissolved in 5 mL of deionized water and stirred at room temperature for 1 h to promote Mg 2+ Coordinate with BSA protein. Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of TrisHCl (pH 8.0), and stir to mix evenly at room temperature. 2 Ultrasonic dissolution in 5 mL of deionized water, 1.5 mL of CaCl 2 The solution was added dropwise to the above mixed solution, stirred for reaction at room temperature for 1 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, washed with deionized water 3 times and freeze-dried to obtain the product.

[0022] (4) 15 mg BSA protein and 16 mg FeCl 2 ·4H 2O was dissolved in 5 mL of deionized water and stirred at room temperature for 1 h to promote the 2+ Coordinate with BSA protein. Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of TrisHCl (pH 8.0), and stir to mix evenly at room temperature. 2 Ultrasonic dissolution in 5 mL of deionized water, 1.5 mL of CaCl 2 The solution was added dropwise to the above mixed solution, stirred for reaction at room temperature for 1 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, washed with deionized water 3 times and freeze-dried to obtain the product.

[0023] Example 2

[0024] L929, 3T3, and 4T1 cells were inoculated at 8,000 cells / well into a 96-well plate containing 200 μL DMEM medium. Cultured at 37°C for 24 hours, the cells adhered to the bottom of each well of the 96-well plate. The bovine serum albumin-metal nanocomplex was evenly dispersed in the DMEM medium, and the concentrations were configured to be 150 μg / mL and 300 μg / mL, respectively. The culture medium in the 96-well plate was aspirated, and the pre-configured DMEM solution containing different concentrations of bovine serum albumin-metal nanocomplex was added to the corresponding wells. After incubation for 24 hours, 20 μL CCK8 reagent was added, and after incubation for 1 hour, the liquid was transferred to a new 96-well plate, and the new plate was placed in an ELISA instrument to record the absorbance at a wavelength of 450 nm.

Claims

1. A method for preparing a bovine serum albumin-calcium-manganese nanocomposite, characterized in that: The following steps are involved: Dissolve 15 mg of bovine serum albumin (BSA) and 16 mg of MnCl2·4H2O in 5 mL of deionized water and stir for 1 h at room temperature; Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of Tris HCl (pH 8.0), and stir to mix evenly at room temperature; add 1.5 mL of 60 μg / mL CaCl2 solution dropwise into the above mixed solution, stir to react at room temperature for 1 h, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash 3 times with deionized water, and then freeze-dry to obtain the product.

2. A method for preparing a bovine serum albumin-calcium zinc nanocomposite, characterized in that: The following steps are involved: 15 mg of BSA protein and 12.5 mg of ZnCl2 were dissolved in 5 mL of deionized water and stirred at room temperature for 1 h; Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of Tris HCl (pH 8.0), and stir to mix evenly at room temperature; add 1.5 mL of 60 μg / mL CaCl2 solution dropwise into the above mixed solution, stir to react at room temperature for 1 h, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash 3 times with deionized water, and then freeze-dry to obtain the product.

3. A method for preparing a bovine serum albumin-calcium-magnesium nanocomposite, characterized in that: The following steps are involved: 15 mg of BSA protein and 20.5 mg of MgSO4·7H2O were dissolved in 5 mL of deionized water and stirred at room temperature for 1 h; Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of Tris HCl (pH 8.0), and stir to mix evenly at room temperature; add 1.5 mL of 60 μg / mL CaCl2 solution dropwise into the above mixed solution, stir to react at room temperature for 1 h, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash 3 times with deionized water, and then freeze-dry to obtain the product.

4. A method for preparing a bovine serum albumin-calcium iron nanocomposite, characterized in that: The following steps are involved: 15 mg of BSA protein and 16 mg of FeCl2·4H2O were dissolved in 5 mL of deionized water and stirred at room temperature for 1 h; Dissolve 400 μL of the above solution in 5 mL of SBF, add 400 μL of Tris HCl (pH 8.0), and stir to mix evenly at room temperature; add 1.5 mL of 60 μg / mL CaCl2 solution dropwise into the above mixed solution, stir to react at room temperature for 1 h, centrifuge at 12000 rpm for 10 min, discard the supernatant, wash 3 times with deionized water, and then freeze-dry to obtain the product.

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