Method of preparing protein-coated zif structures loaded with chlorin e6 and manganese dioxide

Abstract

The application provides a preparation method of protein-coated ZIF structure loaded with chlorin e6 and manganese dioxide, which comprises the following steps: dissolving zinc nitrate hexahydrate and copper nitrate trihydrate in a methanol solution to obtain a mixed solution A; dissolving 2-methylimidazole in a methanol solution to obtain a solution B; stirring the solution B at room temperature, adding the mixed solution A into the solution B, continuing to react, adding chlorin e6, centrifuging, and forming a ZIF structure loaded with chlorin e6; dissolving a protein in water, adjusting pH to 7, adding potassium permanganate, uniformly stirring, and forming a protein-coated manganese dioxide compound aqueous solution; resuspending the ZIF structure loaded with chlorin e6, adding into the protein-coated manganese dioxide compound aqueous solution, uniformly stirring, centrifuging, resuspending, and forming protein-coated ZIF structure loaded with chlorin e6 and manganese dioxide nanoparticles.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, and specifically relates to a method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide. Background Technology

[0002] Zeolite-Imidazolate Framework (ZIF) is a subclass of Metal-Organic Framework (MOF) materials. It is formed through the self-assembly of transition metal cations (primarily zinc and cobalt ions) and organic ligands (such as imidazole or imidazole derivatives) as linkers. The imidazole anion mimics the bridging effect of oxygen in zeolite, forming at similar angles, but with a bond length greater than that of the zeolite structure. This results in ZIF structures with larger pore sizes, higher crystallinity, larger specific surface area, and tunable porosity, while also combining the thermal stability, chemical stability, and rich topological diversity of zeolite molecular sieves.

[0003] Currently, there are many methods for synthesizing ZIF particles, but the synthesis methods that can stably control the size of ZIF are relatively complex and unstable. It is also relatively difficult to modify the surface of ZIF structures, and most of them will self-aggregate in aqueous solutions.

[0004] Therefore, how to provide a method for preparing ZIF particles to stably control the size of the ZIF structure and enable the ZIF material to dissolve stably in aqueous solution is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] This invention provides a method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide, thereby solving at least one of the aforementioned technical problems.

[0006] This invention is achieved through the following technical solution:

[0007] This invention provides a method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide, comprising the following steps:

[0008] (1) Dissolve zinc nitrate hexahydrate and copper nitrate trihydrate in methanol to obtain mixed solution A; dissolve 2-methylimidazolium in methanol to obtain solution B;

[0009] (2) At room temperature, the solution B is stirred at a speed of not less than 700 rpm. At the stirring speed, the mixed solution A is added to the solution B and the reaction continues. Dihydroporphyrin e6 is added and centrifuged to form a ZIF structure loaded with dihydroporphyrin e6.

[0010] (3) Dissolve the protein in water, adjust the pH to 7, add potassium permanganate, and stir at a constant speed to form an aqueous solution of manganese dioxide complex encapsulated by the protein. The protein is natural bovine serum albumin.

[0011] (4) After resuspending the ZIF structure loaded with dihydroporphyrin e6, add the aqueous solution of the protein-encapsulated manganese dioxide complex to the ZIF structure loaded with dihydroporphyrin e6, stir at a constant speed, centrifuge, and resuspend to form a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles.

[0012] In the first aspect, the molar ratio of copper nitrate trihydrate to zinc nitrate hexahydrate in step (1) is 1:10 to 1:40, and the concentration of the methanol solution of copper nitrate trihydrate in the mixed solution A is 10 mM to 50 mM.

[0013] In the first aspect, the molar ratio of 2-methylimidazole to zinc nitrate hexahydrate in step (1) is 8:1.

[0014] In the first aspect, the concentration of solution B in step (1) is 240 mM to 360 mM.

[0015] In the first aspect, the reaction time described in step (2) is 15 min to 20 min.

[0016] In the first aspect, the molar ratio of 2-methylimidazole to the dihydroporphyrin E6 in step (2) is 4500:1.

[0017] In the first aspect, the concentration of the protein in step (3) is 20 mg / mL to 30 mg / mL.

[0018] In the first aspect, the concentration of potassium permanganate in step (3) is 20 mM.

[0019] In the first aspect, the uniform stirring speed in step (3) is 300 rpm and the uniform stirring time is 12 h.

[0020] In the first aspect, the uniform stirring speed in step (4) is 300 rpm and the uniform stirring time is 12 h.

[0021] Beneficial Effects: This invention proposes a method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide. First, zinc nitrate hexahydrate and copper nitrate trihydrate are dissolved in methanol to obtain a mixed solution A, and 2-methylimidazole is dissolved in methanol to obtain solution B. Mixed solutions A and B at fixed concentrations are prepared for later use. Second, at room temperature, mixed solution A is added to solution B under high-speed stirring to obtain the ZIF structure. High-speed stirring ensures that the ZIF particles are large enough to avoid significant loss during centrifugation due to small particle size. The size of the ZIF structure can also be controlled by varying the ratio of copper to zinc ions. Then, dihydroporphyrin e6 is added, and stirring continues to form a ZIF structure loaded with dihydroporphyrin e6. The ZIF structure utilizes the hydrophobicity of dihydroporphyrin E6 and the porosity of the ZIF structure to protect the dihydroporphyrin E6 within the pores of the ZIF structure. This allows the ZIF structure loaded with dihydroporphyrin E6 to not only reduce the toxicity of dihydroporphyrin E6 but also possess sound-sensitizing properties, making it suitable for medical applications. Finally, natural bovine serum albumin is used instead of chemical molecular stabilizers, reacting with potassium permanganate to form a protein-encapsulated manganese dioxide complex aqueous solution. After resuspending the dihydroporphyrin E6-loaded ZIF structure, the protein-encapsulated manganese dioxide complex aqueous solution is added to the ZIF structure. After uniform stirring, centrifugation, and resuspending, a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide nanoparticles is formed, resulting in even lower toxicity. In other words, the ZIF particle preparation method employed in this invention can achieve stable control of the ZIF structure size while ensuring stable dissolution of the ZIF material in aqueous solution. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this specification or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Transmission electron microscopy (TEM) image of a protein-encapsulated ZIF-8 structure loaded with dihydroporphyrin e6 (Ce6) and manganese dioxide;

[0024] Figure 2 UV-VIS image of manganese dioxide (MnO2) coated with natural bovine serum albumin (BSA);

[0025] Figure 3 UV-VIS absorption curve of protein-encapsulated ZIF-8 structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles;

[0026] Figure 4 Transmission electron microscopy (TEM) image of a metal-organic framework structure encapsulating an aqueous synthetic protein;

[0027] Figure 5 Transmission electron microscopy (TEM) image of a metal-organic framework structure encapsulated in polyvinylpyrrolidone. Detailed Implementation

[0028] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0029] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0031] Example 1:

[0032] Protein-encapsulated ZIF-8 structured nanoparticles loaded with dihydroporphyrin E6 and manganese dioxide were prepared according to the following method:

[0033] (1) The molar ratio of copper nitrate trihydrate to zinc nitrate hexahydrate is 1:20. Zinc nitrate hexahydrate and copper nitrate trihydrate are added to 30 mL of methanol solution. After dissolving and clarifying, mixed solution A is obtained. 2-methylimidazole is added to methanol solution. After dissolving and clarifying, methanol solution B of 2-methylimidazole with a concentration of 240 mM to 360 mM is obtained. The molar ratio of 2-methylimidazole to zinc nitrate is 8:1.

[0034] (2) At room temperature, add 120 mL of 360 mM 2-methylimidazolium methanol solution B to a 250 mL round bottom flask, set the stirring speed to not less than 700 rpm, and add 30 mL of mixed solution A under this high stirring speed. Continue stirring for 15 min to 20 min, then add 5 mg of dihydroporphyrin e6, centrifuge to form a ZIF-8 structure loaded with dihydroporphyrin e6.

[0035] (3) Add 20 mL of water and 500 mg of natural bovine serum albumin to a 50 mL round-bottom flask, stir, adjust the pH to 7, then add 63.2 mg of potassium permanganate, set the stirring speed to 300 rpm, and stir at a constant speed for 12 h to form an aqueous solution of protein-encapsulated manganese dioxide complex.

[0036] (4) After resuspending the ZIF-8 structure loaded with dihydroporphyrin e6, add the aqueous solution of the protein-encapsulated manganese dioxide complex to the ZIF structure loaded with dihydroporphyrin e6, set the stirring speed to 300 rpm, stir at a constant speed for 12 h, centrifuge, and resuspend to form a protein-encapsulated ZIF-8 structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles.

[0037] The resulting protein-encapsulated ZIF-8 structure was loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles and then characterized by sonication.

[0038] The transmission electron microscope (TEM) image of the protein-encapsulated ZIF-8 structure loaded with dihydroporphyrin E6 and manganese dioxide nanoparticles prepared in Example 1 is shown below. Figure 1 ,from Figure 1 It is known that the particle size of the nanoparticles is 62.5 nm; Figure 2 UV-VIS image of natural bovine serum albumin (BSA) coated with manganese dioxide (MnO2), from Figure 2 It can be seen that the characteristic absorption peak completely disappeared after potassium permanganate was reduced to MnO2 by BSA, indicating that the reaction was successful. Figure 3 The UV-VIS absorption spectrum of protein-encapsulated ZIF-8 structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles is shown below. Figure 3 It can be seen that dihydroporphyrin e6 was loaded into the ZIF-8 structure, and protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles was successfully prepared.

[0039] Example 2

[0040] ZIF-structured nanoparticles encapsulated in an aqueous synthetic protein were prepared using the following method:

[0041] (1) The molar ratio of copper nitrate trihydrate and zinc nitrate hexahydrate is 1:12. Add zinc nitrate hexahydrate and copper nitrate trihydrate to 15 mL of aqueous solution, dissolve and clarify to obtain mixed solution A.

[0042] (2) Add 15 mL of water, 1.08 g of dimethylimidazole, and 3.67 mg of cetyltrimethylammonium bromide to a 50 mL round-bottom flask. Set the stirring speed to no less than 700 rpm and add 15 mL of mixed solution A at this high stirring speed. Stir and react for 12 h, then centrifuge to form a ZIF structure.

[0043] (3) Add 20 mL of water and 500 mg of bovine serum albumin to a 50 mL round-bottom flask and stir at a constant speed to form an aqueous solution of bovine serum albumin.

[0044] (4) After resuspending the ZIF structure, add the aqueous solution of bovine serum albumin to the ZIF structure, set the stirring speed to 300 rpm, stir at a constant speed for 12 h, centrifuge, resuspend, and form protein-encapsulated ZIF structure nanoparticles.

[0045] The protein-encapsulated ZIF structure nanoparticles were ultrasonicated and then characterized. The transmission electron microscope (TEM) image of the aqueous-phase synthesized protein-encapsulated metal-organic framework structure prepared in this embodiment is shown below. Figure 4 ,from Figure 4 It can be seen that the nanoparticles are cubic structures with a particle size of no more than 90 nm.

[0046] Example 3

[0047] Polyvinylpyrrolidone-encapsulated gold ZIF-structured nanomaterials were prepared according to the following method:

[0048] (1) Add copper nitrate trihydrate to water, dissolve and clarify to obtain an aqueous solution of copper nitrate with a concentration of 24 mM;

[0049] (2) Add 15 mL of water, 1.08 g of dimethylimidazole, and 3.67 mg of hexadecyltrimethylammonium bromide to a 50 mL round-bottom flask. Set the stirring speed to no less than 700 rpm and add 15 mL of 24 mM copper nitrate aqueous solution at this high stirring speed. Stir the reaction for 12 h, centrifuge, and form a ZIF structure.

[0050] (3) Add 20 mL of water and 500 mg of polyvinylpyrrolidone to a 50 mL round-bottom flask and stir at a constant speed to form an aqueous solution of polyvinylpyrrolidone.

[0051] (4) After resuspending the ZIF structure, add the aqueous solution of polyvinylpyrrolidone to the ZIF structure, set the stirring speed to 300 rpm, stir at a constant speed for 12 h, centrifuge, resuspend, and form a ZIF structure nanomaterial encapsulated by polyvinylpyrrolidone.

[0052] The ZIF-structured nanomaterials encapsulated with polyvinylpyrrolidone were ultrasonicated and then characterized. The transmission electron microscope (TEM) spectra of the ZIF-structured nanomaterials encapsulated with polyvinylpyrrolidone prepared in this embodiment are shown below. Figure 5 ,from Figure 5 It can be seen that the nanomaterial is a two-dimensional nanosheet structure with a particle size of no more than 200 nm.

[0053] As can be seen from the TEM images of Examples 1-3, the different ratios of copper and zinc ions affect the size of the ZIF structure. In Example 3, which contains only copper ions, the particle size does not exceed 200 nm. In Examples 2 and 1, the particle size decreases as the proportion of zinc ions increases. In practice, ZIF structures tend to aggregate in aqueous solutions, therefore, stabilizers are needed to ensure that the ZIF structures can exist stably in aqueous solutions without aggregation. The bovine serum albumin used in Example 2 is less toxic and more biocompatible than the polyvinylpyrrolidone used in Example 3, and has good application prospects in drug loading.

[0054] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0055] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0056] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide, characterized in that, Includes the following steps: (1) Zinc nitrate hexahydrate and copper nitrate trihydrate are dissolved in methanol solution to obtain mixed solution A; 2-methylimidazolium is dissolved in methanol solution to obtain solution B; the molar ratio of copper nitrate trihydrate to zinc nitrate hexahydrate is 1:10 to 1:40, and the size of the ZIF structure is controlled by controlling the ratio of the two. (2) At room temperature, the solution B is stirred at a speed of not less than 700 rpm. At the stirring speed, the mixed solution A is added to the solution B and the reaction continues. Dihydroporphyrin e6 is added and centrifuged to form a ZIF structure loaded with dihydroporphyrin e6. (3) Dissolve the protein in water, adjust the pH to 7, add potassium permanganate, and stir at a constant speed to form an aqueous solution of manganese dioxide complex encapsulated by the protein. The protein is natural bovine serum albumin. (4) After resuspending the ZIF structure loaded with dihydroporphyrin e6, add the aqueous solution of the protein-encapsulated manganese dioxide complex to the ZIF structure loaded with dihydroporphyrin e6, stir at a constant speed, centrifuge, and resuspend to form a protein-encapsulated ZIF structure loaded with dihydroporphyrin e6 and manganese dioxide nanoparticles.

2. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 1, characterized in that, In step (1), the concentration of the methanol solution of copper nitrate trihydrate in the mixed solution A is 10 mM to 50 mM.

3. The method for preparing protein-encapsulated ZIF structures loaded with dihydroporphyrin E6 and manganese dioxide according to claim 2, characterized in that, The molar ratio of 2-methylimidazole to zinc nitrate hexahydrate in step (1) is 8:

1.

4. The method for preparing protein-encapsulated ZIF structures loaded with dihydroporphyrin E6 and manganese dioxide according to claim 3, characterized in that, The concentration of solution B in step (1) is 240 mM to 360 mM.

5. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 4, characterized in that, The reaction time described in step (2) is 15 min to 20 min.

6. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 5, characterized in that, In step (2), the molar ratio of 2-methylimidazole to the dihydroporphyrin E6 is 4500:

1.

7. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 6, characterized in that, The concentration of the protein mentioned in step (3) is 20 mg / mL to 30 mg / mL.

8. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 7, characterized in that, The concentration of potassium permanganate in step (3) is 20 mM.

9. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 8, characterized in that, The uniform stirring speed in step (3) is 300 rpm, and the uniform stirring time is 12 h.

10. The method for preparing a protein-encapsulated ZIF structure loaded with dihydroporphyrin E6 and manganese dioxide according to claim 9, characterized in that... The uniform stirring speed in step (4) is 300 rpm, and the uniform stirring time is 12 h.

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