Preparation method and application of albumin vitepofen nanoparticles

Abstract

The invention relates to the technical field of biological medicine, in particular to a preparation method and application of albumin vitepofen nanoparticles. The invention relates to an application of albumin vitepofen nanoparticles in tumor treatment. The preparation method of the albumin and vitepofen nanoparticles comprises the following steps: stirring an aqueous solution of an albumin polymer and vitepofen at a high speed to react, and purifying through a 100kDa ultrafiltration tube. According to the preparation method and application of the albumin and vitepofen nanoparticles, the prepared albumin and vitepofen nanoparticles can only obviously increase the amount of medicine penetrating through cells, and the photodynamic effect of vitepofen after the albumin and vitepofen nanoparticles enter the cells further has the anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a preparation method of albumin verteporfin nanoparticles and application thereof. Background Art

[0002] Verteporfin (VP), as a second-generation porphyrin photosensitizer, was initially used to treat ophthalmic diseases such as macular degeneration. However, in recent years, with the continuous deepening of its research, researchers have found that verteporfin has shown significant potential in the field of anti-tumor. As a photosensitizer, verteporfin can produce cytotoxicity under light conditions, which makes it a potential anti-tumor drug. Verteporfin can achieve anti-tumor effects by interfering with the metabolic process of tumor cells, inhibiting the growth and proliferation of tumor cells, and inducing their apoptosis. However, when verteporfin is used alone for anti-tumor treatment, the cell uptake is low and the cell penetration ability is limited, making it difficult to achieve the expected effect. Using albumin as a carrier to carry VP can significantly increase the amount of drug entering the cell and significantly improve its anti-tumor effect.

[0003] To this end, a preparation method of albumin verteporfin nanoparticles and application thereof are designed to provide another technical solution to the above technical problems. Summary of the invention

[0004] Based on this, it is necessary to provide a preparation method and application of albumin verteporfin nanoparticles to solve the technical problems raised in the above-mentioned background technology.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] Application of albumin verteporfin nanoparticles in the treatment of tumors.

[0007] The preparation method of albumin verteporfin nanoparticles comprises the following steps:

[0008] 1) Take an appropriate amount of albumin and dissolve it in pure water to prepare BSA solution;

[0009] 2) Take an appropriate amount of KOH solution and slowly add it dropwise to the BSA solution obtained in step 1);

[0010] 3) taking an appropriate amount of verteporfin and adding it dropwise to the solution obtained in step 2), and reacting the solution under light-proof and magnetic stirring conditions, and purifying and removing the unreacted raw materials to obtain albumin verteporfin nanoparticles.

[0011] As a preferred embodiment of the method for preparing the albumin verteporfin nanoparticles provided by the present invention, the concentration of the BSA solution is 25 mg / mL; the concentration of the KOH solution is 1 M; and the concentration of VP is 5 mM.

[0012] As a preferred embodiment of the method for preparing the albumin verteporfin nanoparticles provided by the present invention, the input amount of the BSA solution is 100 mg; the input amount of the KOH solution is 200 μL; and the input amount of the VP is 100 μL.

[0013] As a preferred embodiment of the method for preparing albumin verteporfin nanoparticles provided by the present invention, the reaction condition is 25° C. for 2 hours.

[0014] As a preferred embodiment of the method for preparing albumin verteporfin nanoparticles provided by the present invention, the magnetic stirring condition is stirring at 1300 rpm / min.

[0015] As a preferred embodiment of the method for preparing the albumin verteporfin nanoparticles provided by the present invention, the purification is to transfer the reaction solution into a 100kDa ultrafiltration tube, centrifuge at 4°C and 5000rpm / min for 10min to remove unreacted raw materials.

[0016] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0017] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0018] 1. The present invention provides a method for preparing albumin verteporfin nanoparticles and applications thereof. The albumin polymer aqueous solution is reacted with verteporfin by high-speed stirring and purified by a 100 kDa ultrafiltration tube. The prepared albumin verteporfin nanoparticles can not only significantly increase the amount of drug passing through cells, but also the photodynamic effect of verteporfin after entering the cells has an anti-tumor effect.

[0019] 2. The nanoparticles prepared by the present invention have high uniformity and high encapsulation rate; on the other hand, the loading of verteporfin and albumin makes the nanoparticles have excellent cell penetration ability, laying a foundation for photodynamic anti-tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 It is a schematic diagram of the synthesis process of albumin verteporfin of the present invention;

[0022] Figure 2 Schematic diagram of the morphology characterization of BSA@VP of the present invention;

[0023] Figure 3 Schematic diagram of particle size characterization of BSA@VP of the present invention;

[0024] Figure 4 Schematic diagram of the UV-visible absorption spectrum of BSA@VP of the present invention;

[0025] Figure 5 Schematic diagram of UV-visible absorption of VP of the present invention;

[0026] Figure 6 Schematic diagram of investigating the in vitro anti-tumor effect of BSA@VP of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0029] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0030] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0031] Embodiment 1

[0032] Reference Figure 1-Figure 4 , a preparation method of albumin verteporfin nanoparticles and application thereof, the steps are as follows:

[0033] A certain amount of albumin (BSA) was weighed and dissolved in pure water to prepare a BSA solution of a certain concentration; an appropriate amount of KOH solution was slowly added dropwise to the above solution; a certain amount of verteporfin (VP) was then added dropwise to the above system, and the reaction was carried out under light-proof and magnetic stirring conditions, and the albumin verteporfin nanoparticles were obtained after purification and removal of unreacted raw materials.

[0034] Preferably, the concentration of the BSA solution is 25 mg / mL; the concentration of the KOH solution is 1 M; and the concentration of the VP is 5 mM.

[0035] Preferably, the reaction conditions are 25° C. for 2 h.

[0036] Embodiment 2

[0037] The disclosure is made on the basis of the above-mentioned embodiment 1.

[0038] At room temperature, weigh 100 mg of BSA and add it to 4 mL of pure water, stir and dissolve into a uniform solution; slowly add 200 μL of KOH solution to the above system under high-speed stirring to adjust the pH of the system; then take 100 μL of VP and add it dropwise to the above system under light-proof conditions. React at room temperature with magnetic stirring at 1300 rpm / min. After reacting for 2 hours, centrifuge and purify using a 100 kDa ultrafiltration tube at 4°C, 5000 rpm / min, and 10 minutes to obtain BSA@VP nanoparticles. The synthesis process is as follows Figure 1 As shown; the transmission electron microscopy (SEM) test image of the obtained BSA@VP nanoparticles is shown Figure 2 As shown, Figure 2 a is a negatively stained transmission electron microscope of protein, scale bar: 200 nm, Figure 2 b is a negatively stained transmission electron microscope, scale bar: 50 nm, showing that the method can produce BSA@VP nanoparticles of uniform size; the hydrated particle size in aqueous solution is as follows Figure 3 As shown in Figure 2, the average particle size is 11.33 nm and the dispersion is good; its UV-visible absorption spectrum shows a VP characteristic absorption peak at around 680 nm, verifying the successful loading of VP (results are shown in Figure 2). Figure 4 shown).

[0039] Embodiment 3

[0040] refer to Figure 5 , the present invention investigated the encapsulation efficiency of BSA@VP.

[0041] The solution in the filtrate collection tube after centrifugation was taken to investigate the encapsulation efficiency. For the encapsulation efficiency of BSA@VP, VP standards of different concentrations (5, 10, 20, 40, 60, 80 μmol / L) were taken to establish a standard curve, and the regression equation y=0.0206x–0.0208 was obtained. The VP content in the filtrate collection tube was determined to be 0.90 μmol, and the encapsulation efficiency of BSA@VP was 81.82% (the results are shown in Figure 5 a UV-visible absorption spectra of VP at different concentrations, 5b standard curve of VP).

[0042] Embodiment 4

[0043] refer to Figure 6 The present invention investigated the in vitro photodynamic anti-tumor effect of BSA@VP.

[0044] Using breast cancer cell 4T1 as a model, the CCK-8 method was used to investigate the inhibitory effect of BSA@VP on cancer cells. The culture medium of 4T1 cells is a high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution (double antibody). The cells in good condition were digested with 0.25% trypsin, centrifuged, re-selected with fresh culture medium, and diluted to a cell suspension with a concentration of 1.5×105 / mL, and 100μL was inoculated in a 96-well plate. After 12 h of culture in an incubator at 37°C and 5% CO2, the cells adhered to the wall and the old culture medium was discarded. 100 μL of culture medium containing different concentrations of VP (2, 10, 20, 40 μmol / L) and BSA@VP culture medium containing different concentrations of VP (2, 10, 20, 40 μmol / L) were added. After 12 h of continuous culture, the NI R group was irradiated with a 660 nm laser at a power density of 31.6 mW / cm2 for 60 s. After 12 h of continuous culture, the cell survival rate of each group was tested by the CCK-8 method.

[0045] The results are as follows Figure 6 As shown. Figure 6 As shown in a, for 4T1 cells, the cell survival rate was almost unchanged when VP was used alone without illumination, but after illumination, the cell survival rate decreased due to the photodynamic effect of VP, which had a certain anti-tumor effect. Figure 6 b It can be seen that when VP is loaded with albumin, the survival rate of cancer cells is greatly reduced. This is because VP enters the cells more and more with albumin, exerting the maximum photodynamic effect, proving that the material has a strong ability to destroy and kill cancer cells.

[0046] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of albumin verteporfin nanoparticles in the treatment of tumors.

2. A method for preparing albumin verteporfin nanoparticles, characterized in that: Here are the steps: 1) Take an appropriate amount of albumin and dissolve it in pure water to prepare BSA solution; 2) Take an appropriate amount of KOH solution and slowly add it dropwise to the BSA solution obtained in step 1); 3) taking an appropriate amount of verteporfin and adding it dropwise to the solution obtained in step 2), and reacting the solution under light-proof and magnetic stirring conditions, and purifying and removing the unreacted raw materials to obtain albumin verteporfin nanoparticles.

3. The method for preparing albumin verteporfin nanoparticles according to claim 2, characterized in that: The concentration of the BSA solution is 25 mg / mL; the concentration of the KOH solution is 1 M; and the concentration of VP is 5 mM.

4. The method for preparing albumin verteporfin nanoparticles according to claim 2, characterized in that: The amount of BSA solution input was 100 mg; the amount of KOH solution input was 200 μL; and the amount of VP input was 100 μL.

5. The method for preparing albumin verteporfin nanoparticles according to claim 2, characterized in that: The reaction condition is 25° C. for 2 h.

6. The method for preparing albumin verteporfin nanoparticles according to claim 2, characterized in that: The magnetic stirring condition is stirring at 1300 rpm / min.

7. The method for preparing albumin verteporfin nanoparticles according to claim 2, characterized in that: The purification is to transfer the reaction solution into a 100 kDa ultrafiltration tube, and centrifuge at 4°C and 5000 rpm / min for 10 min to remove unreacted raw materials.

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