CN114224867A

CN114224867A - Statin drug loaded silk fibroin nanoparticles with anti-tumor effect and preparation and application thereof

Info

Publication number: CN114224867A
Application number: CN202111528361.XA
Authority: CN
Inventors: 沈琦; 马思语; 杨捷
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2021-12-14
Filing date: 2021-12-14
Publication date: 2022-03-25
Anticipated expiration: 2041-12-14
Also published as: CN114224867B

Abstract

The invention relates to a statin-loaded silk fibroin nanoparticle with an anti-tumor effect and preparation and application thereof, wherein a copper sulfate solution and a silk fibroin solution are mixed to prepare a copper-silk fibroin nanoparticle; mixing the copper-silk fibroin nanoparticles and the statin drug solution, centrifuging the reaction product, and separating to obtain a precipitate product; the mass ratio of the copper-silk fibroin nanoparticles to the medicine is 10-30: 1; the drug is rosuvastatin; the centrifugation temperature of the reaction product is 4-10 ℃; centrifuging the reaction product for 5-20 min; the centrifugal rotating speed of the reaction product is 10000-12000 rpm; and re-dispersing the precipitation product, performing centrifugal washing and separation for multiple times, and adding water for ultrasonic re-dispersion to obtain the drug-loaded silk fibroin nanoparticles. Compared with the prior art, the Cu-SF (RSV) NPs can promote effective iron death and effectively block the transfer of TNBC.

Description

Statin drug loaded silk fibroin nanoparticles with anti-tumor effect and preparation and application thereof

Technical Field

The invention belongs to the technical field of nano-drugs, and relates to a statin drug loaded protein nanoparticle with an anti-tumor effect and a preparation method thereof.

Background

Breast cancer is a common malignancy, with Triple Negative Breast Cancer (TNBC) accounting for approximately one-fourth of breast cancers. The most reported function principle of TNBC antitumor drug is inducing apoptosis for anticancer therapy. However, the efficacy of conventional chemotherapeutic drugs is limited due to the phenotypic diversity, chemotherapeutic resistance and early metastasis of aggressive TNBC. Iron death is a unique non-apoptotic type and several studies have demonstrated that iron death can inhibit breast cancer without the apoptotic pathway. In recent years, the CoQ/FSP1 axis was discovered, which acts in parallel with the classical GSH/GPX4 pathway to regulate the fatality of iron death. Iron death suppressing egg 1(FSP1) is an oxidoreductase whose major substrate, ubiquinone (CoQ), is produced through the mevalonate pathway, and FSP1 reduces CoQ to ubiquinol (CoQH) at the plasma membrane₂)。CoQH₂Can act as an antioxidant to prevent iron death from occurring.

Statins are drugs widely used clinically in dyslipidemia and cardiovascular diseases, and can interfere metabolism, block the rate-limiting enzyme hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase and reduce the level of mevalonate pathway CoQ. Atorvastatin is one of the statins, and is primarily used in the treatment of hypercholesterolemia and coronary heart disease, by a mechanism that lowers the levels of Low Density Lipoprotein (LDL) and triglycerides through competitive inhibition of HMG-CoA reductase. FSP1 expression and CoQH₂The levels were decreased together, resulting in a reversal of the effect of resistance to iron death. In addition to anti-iron death, the high malignancy and metastatic potential limit the efficacy of TNBC therapy. Over 90% of TNBC patients reported to die from tumor metastasis formed by cancer cells spreading from the primary tumor to distant organs. Statins inhibit tumor metastasis by down-regulating a matrix metalloproteinase 9(MMP9) that promotes metastasis. Thus, statins as FDA approved drugs may be a potential therapeutic approach for TNBC because of their ability to reduce FSP 1-mediated iron death resistance and MMP 9-mediated tumor metastasis.

Compared with lipophilic statins, hydrophilic statins (such as rosuvastatin drugs) have weaker membrane permeability and high in-vivo clearing speed, and influence the application of the drugs as antitumor drugs.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the statin-loaded silk fibroin nanoparticles which can synergistically promote the death of cancer cells and have an anti-tumor effect and the preparation method thereof.

The purpose of the invention can be realized by the following technical scheme: a statin-loaded silk fibroin nanoparticle with an anti-tumor effect comprises a copper-silk fibroin nanoparticle and a statin in a mass ratio of 5: 1-5.

Further, the statin drug is rosuvastatin.

The chemical structural formula of rosuvastatin is shown as follows:

furthermore, the silk fibroin nanoparticles loaded with statins have an average particle size of less than 200nm and uniform particle size.

The invention also provides a preparation method of the statin-loaded silk fibroin nanoparticles with the anti-tumor effect, which comprises the following steps:

s01, mixing the copper sulfate solution and the silk fibroin solution to prepare a copper-silk fibroin nanoparticle solution;

s02, slowly dropping the copper-silk fibroin nanoparticle solution into the statin solution, mixing and stirring at 10-30 ℃ for 10-30 minutes, performing ultrasonic treatment after vortex, further stirring in ventilation equipment for 2 hours, centrifuging the reaction product, and separating to obtain a precipitate;

s03, re-dispersing the precipitation product, centrifuging, washing and separating for multiple times, and then adding water for ultrasonic re-dispersing to obtain the drug-loaded silk fibroin nanoparticles.

Further, the copper sulfate solution in the step S01 is prepared by dissolving copper sulfate pentahydrate in ultrapure water, and standing the solution for 4-6 hours at 20-25 ℃ to obtain a copper sulfate solution with a mass concentration of 0.01-0.1 g/ml.

Further, the silk fibroin solution is prepared by dissolving silk fibroin in ultrapure water, and standing for 4-6 hours at 4-10 ℃ to obtain the silk fibroin solution with the mass concentration of 0.01-0.1 g/ml.

Further, the mass ratio of copper to silk fibroin in the copper-silk fibroin nanoparticles is 5: 1-5.

Further, the statin solution in step S02 is prepared by dissolving rosuvastatin in an organic solvent, and standing at 20-25 ℃ for 4-6 hours to obtain a rosuvastatin organic solution with a mass concentration of 0.1-1 mg/ml; the organic solvent is acetone.

Further, in the step S02 and the step S03, the centrifugal speed is 10000-13000 g, the centrifugal time is 15-30 minutes, and the ultrasonic processing power is 50-200W.

The invention also provides application of the statin-loaded silk fibroin nanoparticles with an anti-tumor effect, and the statin-loaded silk fibroin nanoparticles are used as anti-tumor drugs, in particular to drugs for TNBC treatment.

Silk Fibroin (SF), an FDA-approved polymer, has been widely used in clinical stents and drug carriers due to its good biocompatibility and biodegradability.

In the invention, the silk fibroin is used as a carrier to prepare the statin-loaded drug nano-particles for TNBC treatment. SF and Cu²⁺The coordination forms a complex, and then Cu-SF nanoparticles encapsulated with hydrophilic Rosuvastatin (RSV) are manufactured by self-assembly. High expression of FSP1 in TNBC induces resistance to iron death, limiting the anti-tumor effects of iron death, which is just overcome by metabolic intervention of RSV. RSV inhibits the mevalonate pathway and decreases CoQH₂To mitigate the FSP 1-mediated attenuation of iron death. Furthermore, Cu²⁺Triggering fenton-like reaction to generate Reactive Oxygen Species (ROS) and SF to consume Glutathione (GSH) to decrease glutathione peroxidase 4(GPX4) expression, and thus the effect of iron death is enhanced. In addition, RSV reduced MMP9 tableAchieve and block the transfer of TNBC. In this way, metabolic intervention nanoparticle Cu-sf (rsv) NPs can effectively eradicate TNBC tumors and inhibit tumor metastasis with minimal systemic toxicity. The invention uses silk fibroin as a carrier, and can effectively slow down the speed of removing statins in vivo. In addition, due to the fact that tumor tissues have high permeability and retention (EPR) effects, statins can achieve passive targeted drug delivery at tumor sites along with SF nano-carriers.

Compared with the prior art, the invention has the following advantages:

(1) the statin-loaded silk fibroin nanoparticles with the anti-tumor effect are developed, the in-vivo clearing speed of the statins is effectively slowed down, and the EPR effect can be utilized to realize the passive targeted drug delivery of tumor parts.

(2) The preparation process of the silk fibroin nanoparticles is improved, the safety of the preparation is improved, and the preparation process is simple and controllable. We predict that metabolically intervening nanoparticle Cu-SF (RSV) NPs may serve as a promising therapeutic platform for clinical TNBC therapy.

(3) Some TNBC cells are less sensitive to iron death due to iron death resistance mediated by ferrodeath inhibitory protein 1(FSP 1). Rosuvastatin (RSV) was encapsulated in Silk Fibroin (SF) nanoparticles (Cu-SF (RSV) NPs) to inhibit TNBC by overcoming FSP1 mediated iron death resistance. RSV alleviates CoQ/FSP1 axis-mediated iron death resistance by interfering with the mevalonate metabolic pathway. Furthermore, Cu²⁺Reactive Oxygen Species (ROS) can be generated by fenton-like reactions, SF can consume Glutathione (GSH) to reduce expression of GPX4, and can drive iron death into town. Thus, Cu-SF (RSV) NPs may promote potent iron death. In addition, Cu-SF (RSV) NPs effectively block the transfer of TNBC, and the metabolic intervention nanoparticles Cu-SF (RSV) NPs are expected to serve as a promising treatment platform for clinical TNBC treatment.

Drawings

Fig. 1 is a TEM morphological signature image of silk fibroin nanoparticles.

FIG. 2 shows the range of standard curve of copper ion concentration.

Fig. 3 is a graph comparing the glutathione consumption capacity of different concentrations of the fibroin nanoparticles.

Fig. 4 is a graph of ultraviolet absorption spectrum for verifying ROS generation effect by silk fibroin nanoparticles.

Fig. 5 is a graph comparing the effect of ROS generation by silk fibroin nanoparticles.

Fig. 6 is a graph of MMP9 western blot of in vitro experiments after stimulation with different formulation groups.

FIG. 7 shows the results of in vitro detection of CoH2 content.

Detailed Description

The present invention will be described in detail with reference to specific examples. The present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the scope of the present invention is not limited to the following embodiments.

In the following examples, the specific sources of the reagents are as follows: the material sources in the following examples are:

and the rest of the raw material reagents or treatment techniques which are not specifically described are conventional commercial raw materials or conventional treatment techniques in the field. The silk fibroin-copper nanoparticle synthetic route is prepared by referring to the following documents: gou S, Huang Y, Wan Y, et al, Multi-biochemical talk-based nanoparticles with on-demand photoplastic drug release capacity for CD44-targeted exhibition of scientific [ J ] Biomaterials,2019,212.

Example 1

Preparation of silk fibroin nanoparticles and blank nanoparticles of statins

Silk fibroin (0.1g) was weighed into a 15mL centrifuge tube, and 5mL ultrapure water was added. And (3) placing the centrifugal tube at 4-10 ℃ for 6 hours, and slightly shaking the centrifugal tube to dissolve the centrifugal tube. If flocculent material which is not dissolved is still observed in the solution after shaking for 10min, filtering by using a 0.45 mu m filter membrane, and collecting filtrate.

Copper sulfate pentahydrate (0.2g) was weighed into a 15mL centrifuge tube and dissolved in 5mL of ultrapure water. Placing the centrifugal tube in an ultrasonic instrument, performing 100% power ultrasonic for 5min to promote complete dissolution, and standing for 6h at 4-10 ℃.

2mL of the aqueous copper sulfate solution prepared above was placed in a 10mL beaker, and 2mL of the freshly prepared aqueous silk fibroin solution was slowly added dropwise to the aqueous copper sulfate solution using a pipette. Stirring at room temperature at 200rpm for 5min to obtain light blue silk fibroin-copper ion solution.

Dissolving rosuvastatin (1mg) in 2mL acetone, and standing at 4-10 ℃ for 6h to obtain a rosuvastatin acetone solution with mass concentration of 0.5 mg/mL.

Diluting the obtained silk fibroin-copper ion solution by 4 times with ultrapure water to obtain the silk fibroin-copper ion solution with the concentration of the silk fibroin of 2.5 mg/mL. And slowly dripping 2mL of the diluted silk fibroin-copper ion solution into a beaker containing 2mL of rosuvastatin acetone solution at the speed of 0.5mL/min, and stirring at 200rpm for 5min at room temperature. Cu-SF (RSV) NPs are prepared by utilizing the self-assembly property of silk fibroin under the action of organic reagent acetone. At the moment of dropwise adding acetone into the silk fibroin aqueous solution, the phenomenon of emulsification of the reaction system can be observed, and white liquid drops are generated. The emulsified solution was collected and vortexed on a vortexer for 30s, and then transferred to an ultrasonic cell disruptor, and sonicated on ice at 20% power for 2 min. After the sonication was completed, the solution was again placed in a beaker and stirred at 400rpm for 3 hours at room temperature, and the acetone present in the system was evaporated.

The solution from which acetone had been evaporated was centrifuged at 10000g for 18min and the supernatant was discarded. The precipitate was visible to the naked eye and washed three times with ultrapure water. The washing conditions were 13000rcf for 18min and the supernatant was discarded. The washed pellet was resuspended in 250. mu.L of ultrapure water. Again transferred to the ultrasonic cell disruptor and sonicated on ice at 20% power for 2 min. Finally obtaining the target Cu-SF (RSV) NPs.

II, Cu-SF (RSV) NPs particle size determination

The fluid dynamic diameter and polydispersity index (PDI) of nanoparticles were measured on nanoparticle solutions using a Dynamic Light Scattering (DLS) mode of a nanoparticle sizer (Nanobrookomni, Brookhaven). The samples were prepared by diluting 20-fold prepared Cu-SF (RSV) NPs into four-sided clear quartz cuvettes and prepared for testing. Each sample was tested for 20 cycles, three times. The nanoparticle is repeatedly prepared for three times, and the particle size detection is carried out to obtain the average value of the particle size of the nanoparticle.

TABLE 1SF-Cu²⁺Particle size table of NPs

Experimental groups	Particle size(nm)	PDI
			1	192.6±3.5	0.163±0.037
2	205.1±4.9	0.160±0.046
			3	201.4±2.5	0.224±0.047
Mean value of	199.7±7.1	0.182±0.042

The particle size of the nanoparticles is detected by a particle size analyzer, the average value is obtained by repeating the preparation for three times by using the same method, and the particle size of Cu-SF (RSV) NPs is 199.7 +/-7.1 nm and the polydispersity PDI is 0.182 +/-0.042, which shows that the particle size is nano-sized and the particle size distribution is relatively uniform. The experimental result proves that the prepared nanoparticles not only can lay a foundation for promoting the death process of iron, but also are expected to start a passive targeting mechanism, and are beneficial to targeting tumor parts to deliver drugs.

III, Cu-SF (RSV) NPs form detection under transmission electron microscope

The morphology and size of the nanoparticles were further characterized by transmission electron microscopy (TEM, LIBRA200CS, CarlZeiss). Firstly, a copper net (200 meshes) attached with a carbon film is placed on a glass slide (with a matte surface facing upwards), then 1-2 mu L of the prepared silk fibroin-copper nanoparticles are dripped on the matte surface of the copper net, and the silk fibroin-copper nanoparticles are volatilized and dried overnight in a fume hood to be sent to a sample for detection.

The detection result of the silk fibroin-copper nanoparticle transmission electron microscope is consistent with the detection result of a nanometer particle size analyzer, as shown in figure 1, the shape of Cu-SF (RSV) NPs is spherical, the average particle size is 54.2 +/-5.6 nm, and the particle size distribution is relatively uniform.

Fourthly, investigating the copper ion loading condition in Cu-SF (RSV) NPs nanoparticles

(1) Copper content determination method

0.2mL of 0.05mol/L copper ion solution is taken, 1mL of 2mol/L ammonia water is added and diluted to 5mL, and the maximum absorption wavelength is determined after ultraviolet full-wavelength scanning.

The maximum absorption wavelength of the copper ions was measured to be 608 nm. The prepared copper ion concentration standard curve is shown in the following table 2 and fig. 3, and the correlation coefficient r of the standard curve is 0.9999, which meets the relevant specification requirements.

TABLE 2 copper ion Standard Curve solution preparation Table

(2) Determination of copper content

4mL of Cu-SF (RSV) NPs were centrifuged at 1200rpm at 4 ℃ for 15 min. Taking 0.32mL of supernatant as a sample to be detected, and adding 0.08mL of 2mol/L ammonia water. Detection was carried out using a microplate reader at the wavelength of maximum absorption of copper ions determined in the above experiment

4mL of Cu-SF (RSV) NPs solution theoretically contained 160mg of copper sulfate pentahydrate, which was calculated to contain 40.64mg of copper ions based on its relative molecular mass. 0.32mL of sample to be detected is taken, and 0.08mL of 2mol/L ammonia water is added. The OD at 608nm, measured using a microplate reader at the maximum absorption wavelength of copper ions determined in the above experiment, was 0.895. Substituting into a standard curve equation, and performing subsequent calculation to obtain the free copper ion content of 21.06mg in the silk fibroin-copper nanoparticle solution. The mass of coordinated copper ions/total copper ions is 48.17%, which indicates that the silk fibroin has better copper ion entrapment capability.

Fifth, investigation of glutathione depletion Capacity of Cu-SF (RSV) NPs

The glutathione consumption capacity of the fibroin protein-copper nanoparticles is determined by adopting an o-phthalaldehyde fluorescence method.

The thiol group of cysteine and the amino group of glutamic acid in glutathione can be condensed with 2 aldehyde groups on o-phthalaldehyde to form a derivative product with a tricyclic conjugated structure capable of emitting strong fluorescence, and the derivative reaction rate is high, so that the fluorescence intensity emitted by the derivative product and the concentration of GSH form a good linear relation, and the content of GSH in a sample can be measured by adopting a calibration method.

The specific experimental steps are that the nanoparticles are dissolved into different concentrations by PBS (phosphate buffer solution) with the pH value of 8.0, the nanoparticles are incubated for 20min with 10mM GSH and 80 mu g of o-phthalaldehyde, fluorescence is measured by an enzyme-labeling instrument, the excitation wavelength is 340nm when the fluorescence is measured, and the emission wavelength is 430 nm.

As shown in figure 3 by comparing the glutathione consumption capacity of the fibroin nanoparticle with different concentrations, it can be seen that the glutathione consumption capacity of the preparation increases with the increase of the concentration, and is a positive feedback.

Sixthly, investigation on condition that Cu-SF (RSV) NPs nanoparticles generate hydroxyl radicals

A crystal violet colorimetric method is selected to investigate the condition that the nanoparticles generate hydroxyl radicals, and the crystal violet is a triphenylmethane basic dye which has strong absorption at the wavelength position of ultraviolet light. It can react with OH to form a colorless substance, so that the absorbance in the system is reduced. The silk fibroin-copper nanoparticles designed and prepared have the capability of increasing the OH level in the solution, so that the absorbance of the solution is reduced. According to the principle, a method for investigating the generation of hydroxyl radicals by the silk fibroin-copper nanoparticles can be established.

The specific experimental steps are divided into four groups of solution systems and numbered, and the solution systems are respectively control groups, namely only containing crystal violet groups and crystal violet + H₂O₂Group, crystal violet + NPs + H₂O₂And (4) grouping. The crystal violet concentration is 12 mu M, the hydrogen peroxide concentration is 100mM, and 1mL of silk fibroin nanoparticles (2.5mg/mL SF) are added into the nanoparticle group.

Crystal violet, which absorbs at the uv wavelength, can react with OH to form a colorless material, resulting in a decrease in the absorbance of the system. The ultraviolet wavelength absorption value in the crystal violet solution system is inversely proportional to the hydroxyl radical level, and the lower the ultraviolet wavelength absorption value is, the higher the hydroxyl radical level in the system is. As shown in fig. 4-5, the maximum absorption wavelength of crystal violet is 590 nm. The control group, i.e., one group, had the highest uv absorption wavelength. The peroxide bond in the hydrogen peroxide has instability, and the homolytic cleavage can be promoted by illumination and heating to form hydroxyl free radical. This can be shown in that the two groups added with hydrogen peroxide in the system generate certain hydroxyl free radicals, so that the ultraviolet absorbance of the hydroxyl free radicals is lower than that of the control group. Three groups of Cu-SF (RSV) NPs added into the system have similar ultraviolet absorption wavelengths, and the ultraviolet absorbances are lower than those of a control group, which means that the prepared Cu-SF (RSV) NPs nanoparticles can also generate certain hydroxyl radicals autonomously. In the experiment, the hydrogen peroxide level in the in vivo tumor environment is simulated by adding 100mM hydrogen peroxide, and the capability of the nanoparticles for generating hydroxyl radicals is investigated. The ultraviolet absorption wavelength of the fourth group of crystal violet, nano-particles and hydrogen peroxide is obviously lower than that of a control group, and is also lower than that of a group which is added with hydrogen peroxide or Cu-SF (RSV) NPs nano-particles independently, and the ultraviolet absorption of the fourth group shows that the crystal violet in a system is nearly exhausted. The phenomenon shows that copper ions in the nanoparticles play a Fenton-like catalytic role to catalyze hydrogen peroxide to be rapidly converted into hydroxyl radicals. The Cu-SF (RSV) NPs can be proved to have the capability of generating hydroxyl radicals by combining the former three groups of controls.

Seven, MMP9 Western blot analysis

4T1 cells were exposed to RSV, Cu-SF NPs and Cu-SF (RSV) NPs (RSV: 60. mu.g/mL, NPs: 1200. mu.g/mL) for 24 hours. Then, cells were harvested and lysed in ice cold NP40 buffer containing protease inhibitor (PMSF). All samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.2 μm nitrocellulose membranes. The membranes were blocked with 5% skim milk at 25 ℃ for 1 hour and then incubated with MMP9 antibody at 4 ℃ overnight. After that, the membrane was washed with TBST and incubated with secondary antibody for 1 hour at room temperature. Specific proteins are visualized by enhanced chemiluminescence. The MMP9 western blot is shown in FIG. 6, in which reference numerals 1-4 are respectively for the Control group (Control), free drug group (RSV), blank formulation group (Cu-SF NPs) and final formulation group (Cu-SF (RSV) NPs). As can be seen in fig. 6, MMP9 expression was lower in the RSV and Cu-sf (RSV) NPs groups than in the other groups, indicating that RSV can block tumor invasion and metastasis. Cu-sf (RSV) NPs showed a greater ability to reduce MMP9 expression than free RSV, due to the increased efficacy of free hydrophilic drugs by the nano-drug delivery system.

Eight, in vitro detection of CoQH2 level

Measurement of in vitro CoQH₂Levels to evaluate the ability of Cu-SF (RSV) NPs to modulate the CoQ/FSP1 axis. 4T1 cells were treated with RSV, Cu-SF NPs and Cu-SF (RSV) NPs (RSV: 60. mu.g/mL, NPs: 1200. mu.g/mL) for 24 hours. Cells were then harvested and processed to detect cellular CoQH2 levels according to the manufacturer's instructions for the reduced coenzyme Q10 (panthenol) detection kit.

As shown in FIG. 7, the results show that RSV can obviously inhibit mevalonate pathway and reduce CoQH₂May further influence the expression of FSP 1. FSP1 used CoQ as a substrate to generate CoQH2, CoQH2 as an antioxidant to prevent iron death. The reduction in CoQH and down-regulation of FSP1 indicate that Cu-sf (rsv) NPs can intervene in metabolism to alleviate FSP 1-mediated iron death resistance and to effectively activate iron death.

Example 2 screening of Experimental conditions

Have studiedThe self-assembly of silk fibroin into nanoparticles under the action of acetone is proved, but more problems occur in the experimental process of the preliminary scheme, and in order to ensure that the prepared nanoparticles have smaller particle size and better dispersity, factors which possibly influence each link of the experiment are investigated, and the optimal experimental scheme is screened by respectively repeating the experiment for multiple times as shown in tables 1 and 2. The influence of silk fibroin with different concentrations on the final particle size of the nanoparticles is researched under the condition of determining the volume of a reaction system, and the SF solution of 2.5mg/mL has the minimum particle size value under the condition. When the volume of the optimal reaction system is determined under the condition of determining the concentration of silk fibroin, the smaller reaction system is more favorable for the particle size of the nanoparticle finally obtained by the experiment, and the 2mL reaction system has the best performance. Screening comprehensive experimental conditions, selecting 2.5mg/mL SF-Cu²⁺1mL reacted optimally with 1mL rosuvastatin in acetone. And the project design and preparation scheme has repeatability.

TABLE 32 optimal SF concentration screening Table under mL reaction System

TABLE 42.5 optimal reaction System screening Table under SF condition

Tables 3 and 4 demonstrate that in SF-Cu²⁺Under the condition that the mass concentration is 1-5 mg/ml, the silk fibroin nanoparticles loaded with statins and having an anti-tumor effect can be synthesized.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. The statin-loaded silk fibroin nanoparticle with the anti-tumor effect is characterized by comprising a copper-silk fibroin nanoparticle and a statin in a mass ratio of 5: 1-5.

2. A statin-loaded silk fibroin nanoparticle having an anti-tumor effect according to claim 1, wherein the statin is rosuvastatin.

3. A statin-loaded silk fibroin nanoparticle having an anti-tumor effect according to claim 1, wherein the statin-loaded silk fibroin nanoparticle has an average particle size of less than 200nm and a uniform particle size.

4. A method for preparing statin-loaded silk fibroin nanoparticles having an anti-tumor effect according to any one of claims 1-3, comprising the steps of:

5. The method for preparing statin-loaded silk fibroin nanoparticles having an anti-tumor effect according to claim 4, wherein the copper sulfate solution in step S01 is prepared by dissolving copper sulfate pentahydrate in ultrapure water, and standing at 20-25 ℃ for 4-6 h to obtain a copper sulfate solution with a mass concentration of 0.01-0.1 g/ml.

6. The preparation method of statin-loaded silk fibroin nanoparticles with an anti-tumor effect according to claim 4, wherein the silk fibroin solution is prepared by dissolving silk fibroin in ultrapure water, and standing at 4-10 ℃ for 4-6 h to obtain the silk fibroin solution with a mass concentration of 0.01-0.1 g/ml.

7. The preparation method of statin-loaded silk fibroin nanoparticles with an anti-tumor effect according to claim 4, wherein the mass ratio of copper to silk fibroin in the copper-silk fibroin nanoparticles is 5: 1-5.

8. The preparation method of statin-loaded silk fibroin nanoparticles with an anti-tumor effect according to claim 4, wherein the statin solution in step S02 is prepared by dissolving rosuvastatin in an organic solvent, and standing at 20-25 ℃ for 4-6 h to obtain a rosuvastatin organic solution with a mass concentration of 0.1-1 mg/ml;

the organic solvent is acetone.

9. The preparation method of statin-loaded silk fibroin nanoparticles having an anti-tumor effect according to claim 4, wherein the centrifugation speed in steps S02 and S03 is 10000-13000 g, the centrifugation time is 15-30 minutes, and the sonication power is 50-200W.

10. Use of statin-loaded silk fibroin nanoparticles according to any of claims 1-3, wherein the statin-loaded silk fibroin nanoparticles are used as an anti-tumor drug.