CN114191408A

CN114191408A - Preparation method and application of casein-curcumin drug-loaded nanoparticles

Info

Publication number: CN114191408A
Application number: CN202111460387.5A
Authority: CN
Inventors: 张沛; 孙莎莎; 韩琳
Original assignee: Luoyang Normal University
Current assignee: Luoyang Normal University
Priority date: 2021-12-02
Filing date: 2021-12-02
Publication date: 2022-03-18

Abstract

The invention relates to a preparation method of casein-curcumin drug-loaded nanoparticles, which comprises the following steps: s1, placing the casein powder in distilled water to be completely dissolved to obtain a casein sample solution; s2, adjusting the pH of the casein sample solution obtained in the S1 by using a NaOH solution; s3, adding curcumin into the solution obtained in the step S2, and stirring by using a magnetic stirrer to form a casein solution wrapping the curcumin for later use; and S4, carrying out ultrasonic disruption on the casein solution entrapped with curcumin obtained in S3 by using an ultrasonic cell disruption instrument, wherein cooling treatment is adopted in the ultrasonic disruption treatment process, and the casein-curcumin drug-loaded nanoparticles are obtained after the ultrasonic disruption. The casein drug-loaded nanoparticles prepared by the invention are easily captured by microvilli and adhered to a mucous membrane, can prolong the retention time of a carrier to promote absorption, and can realize curcumin delivery on the premise of ensuring good dispersion and good stability of curcumin, thereby improving the utilization rate of curcumin and reducing the loss of dosage.

Description

Preparation method and application of casein-curcumin drug-loaded nanoparticles

Technical Field

The invention relates to the technical field of drug-loaded nanoparticles, in particular to a preparation method and application of casein-curcumin drug-loaded nanoparticles.

Background

Casein is a special protein capable of self-assembling into nanoparticles at isoelectric points, has a special structure, and can be regarded as an amphiphilic block copolymer consisting of hydrophobic amino acid and hydrophilic amino acid. Based on the characteristics, the casein can be easily self-assembled in a solution to form micelles, and nanoparticles with excellent performance and uniform shape are formed. Meanwhile, casein is easily hydrolyzed by biological enzyme, participates in metabolism of organisms, and is favorable for releasing loaded drugs.

Curcumin (curculin) is a polyphenol compound extracted from the rhizome of turmeric, a plant of the family zingiberaceae, and is orange-yellow in color and crystalline in appearance. According to researches, curcumin has wide pharmacology, small toxicity, small adverse effect and obvious anticancer, anti-tumor, anti-inflammatory and antioxidant effects. However, curcumin also has certain limitations, such as low solubility in water, poor stability and poor oral availability, so that curcumin is greatly limited in the development of pharmaceutical research.

The drug-loaded nanoparticles are combined with drugs in a chemical bonding or physical adsorption mode, so that the drugs are dispersed in the interior or on the surface of the nanoparticles. The drug delivery system formed by edible protein as a drug carrier becomes a new idea and a new direction of drug development nowadays and is a powerful catalyst for promoting drug research. The drug-loaded carrier is formed in a self-assembly mode (namely, a group with high polymer activity is modified and then spontaneously forms nanoparticles through non-covalent bond interaction), the particle size range is 10-1000nm, and the nano carrier formed by edible protein always occupies an important position in the development of a pharmaceutical preparation by the identity of a pharmaceutical adjuvant. Currently, most of the edible proteins currently belong to the genus casein are used as nanocarriers. However, many raw material carriers are adopted for preparing the nanoparticles by adopting casein at present, so that the toxicity of the nanoparticles is easy to enhance, and the reaction conditions are high, so that the structural characteristics of the casein are not favorably exerted.

Disclosure of Invention

The invention aims to solve the technical problems and the defects and provides a preparation method and application of casein-curcumin drug-loaded nanoparticles.

In order to solve the technical problems, the invention adopts the technical scheme that: a preparation method of casein-curcumin drug-loaded nanoparticles comprises the following steps:

s1, placing the casein powder in distilled water to be completely dissolved to obtain a casein sample solution for later use;

s2, adjusting the pH of the casein sample solution obtained in the S1 with NaOH solution for later use;

s3, adding curcumin into the solution obtained in the step S2, and stirring by using a magnetic stirrer to form a casein solution wrapping the curcumin for later use;

and S4, carrying out ultrasonic disruption on the casein solution entrapped with curcumin obtained in S3 by using an ultrasonic cell disruption instrument, wherein cooling treatment is adopted in the ultrasonic disruption treatment process, and the casein-curcumin drug-loaded nanoparticles are obtained after the ultrasonic disruption.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticle, 20-40mg of casein powder is dissolved in 10ml of distilled water in S1.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticles, the concentration of the NaOH solution in S2 is 1M, and the pH value of a casein sample solution is adjusted to 5-5.5.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticles, the weight of curcumin added into S3 is 800 mug-1.2 mg.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticles, the magnetic stirring time in S3 is 24 h.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticles, the working power of the ultrasonic cell disruption instrument in S4 is 200-400W, the working time is 10-30min, and the working frequency is 2S for 4S stop at each work.

As a further optimization of the preparation method of the casein-curcumin drug-loaded nanoparticles, the cooling treatment in S4 is to cool by crushed ice.

Casein-curcumin drug-loaded nanoparticles are prepared by the method.

The entrapment rate of the casein-curcumin drug-loaded nanoparticles is 90-95%.

The casein-curcumin drug-loaded nanoparticle is applied to improving the tumor killing effect of curcumin.

The invention has the following beneficial effects:

firstly, the invention utilizes the characteristic that casein can be self-assembled at isoelectric points, adjusts PH to enable the casein to be self-assembled to form nanoparticles, and ensures the characteristic of the casein to the maximum extent; the invention only uses casein to prepare the nanoparticles, does not add other raw materials, has the advantages of safety, nutrition and low cost, and the phenomenon that the toxicity of the nanoparticles is enhanced due to side reaction generated by various raw materials is fully avoided by using a single raw material. The preparation method is simple, the technical process is reasonable, the operation is easy, no special requirement is required on equipment, the simple preparation process fully ensures the structural characteristics of the casein, and the casein can be more favorably exerted; the preparation condition is mild, and the good characteristics of the casein are ensured to the greatest extent.

The invention adopts ultrasonic treatment to increase the intermolecular surface activity, reduce the apparent viscosity, change the secondary structure of protein, increase the surface hydrophobicity of casein, reduce the structural extension and intermolecular interaction of the casein, and improve the solubility.

The casein drug-loaded nanoparticles prepared by the invention are easily captured by microvilli and adhered to a mucous membrane, the retention time of a carrier can be prolonged to promote absorption, and the delivery of curcumin can be realized on the premise of ensuring good dispersion and good stability of the curcumin, so that the utilization rate of the curcumin is improved, the loss of the dosage is reduced, and the bioavailability is 60-65%, thereby breaking through the limitation of the curcumin and providing a new idea and method for cancer treatment.

Drawings

FIG. 1 is a transmission electron micrograph of casein-curcumin drug-loaded nanoparticles (CC-NPs) of example 1 of the present invention;

fig. 2 is a particle size distribution diagram of the casein-curcumin nanoparticles prepared in example 1 of the present invention, with drug-loaded nanoparticles for 30 days at 4 ℃ storage;

FIG. 3 is a cell uptake experimental image of the casein-curcumin nanoparticles of the present invention;

FIG. 4 is a graph showing the inhibitory effect of curcumin on Hela cells at different concentrations and at different times;

FIG. 5 is a graph showing the inhibitory effect of CC-NPs on Hela cells at different concentrations and at different times;

FIG. 6 is a graph comparing bioavailability of free curcumin and casein-curcumin nanoparticles;

FIG. 7 is a preparation process of CS-NPs in a casein self-assembly nanoparticle effect verification experiment;

FIG. 8 is a transmission electron microscope photograph of CS-NPs;

FIG. 9 is a graph showing the comparison of the average particle size and the dispersion coefficient (PDI) of drug-loaded nanoparticles prepared from casein samples with different concentrations;

FIG. 10 is a graph showing the change of surface hydrophobicity indexes measured after different times of ultrasonic treatment of casein solutions with different concentrations;

FIG. 11 is a graph showing the results of toxicity tests of CS-NPs at different concentrations on Hela cells.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

The details of the drugs and reagents used in the examples are given in table 1 below:

the experimental equipment used in the examples is detailed in table 2 below:

example 1

A preparation method of casein-curcumin drug-loaded nanoparticles comprises the following steps:

s1, placing 20mg of casein powder in 10ml of distilled water for complete dissolution to obtain a casein sample solution for later use;

s2, adjusting the pH of the casein sample solution obtained in the S1 to 5-5.5 by using a NaOH solution with the concentration of 1M for later use;

s3, adding 800 mu g of curcumin into the solution obtained in the step S2, and stirring for 24 hours by adopting a magnetic stirrer to form a casein solution wrapping the curcumin for later use;

and S4, performing ultrasonic disruption on the casein solution entrapped with curcumin obtained in the step S3 by using an ultrasonic cell disruptor, wherein the working power of the ultrasonic cell disruptor is 200W, the working time is 10min, the working frequency is 2S for every 4S of work, crushed ice is adopted for cooling treatment in the ultrasonic disruption treatment process, and the casein-curcumin drug-loaded nanoparticles (CC-NPs) are obtained after the ultrasonic disruption is completed.

Taking a transmission electron micrograph of the casein-curcumin drug-loaded nanoparticles (CC-NPs) prepared in the embodiment, as shown in fig. 1, the casein-curcumin nanoparticles prepared in the embodiment have uniform size and spherical shape, and the diameter of the casein-curcumin nanoparticles is about 260 nm.

The casein-curcumin drug-loaded nanoparticles prepared by the preparation method have the entrapment rate of 95%.

Example 2

s1, placing 30mg of casein powder in 10ml of distilled water for complete dissolution to obtain a casein sample solution for later use;

s3, adding 1.0mg of curcumin into the solution obtained in the step S2, and stirring for 24 hours by using a magnetic stirrer to form a casein solution wrapping the curcumin for later use;

and S4, performing ultrasonic disruption on the casein solution entrapped with curcumin obtained in the step S3 by using an ultrasonic cell disruptor, wherein the working power of the ultrasonic cell disruptor is 300W, the working time is 20min, the working frequency is 2S for every 4S of work, crushed ice is adopted for cooling treatment in the ultrasonic disruption treatment process, and the casein-curcumin drug-loaded nanoparticles are obtained after the ultrasonic disruption.

The casein-curcumin drug-loaded nanoparticles prepared by the preparation method have the entrapment rate of 93%.

Example 3

s1, placing 40mg of casein powder in 10ml of distilled water for complete dissolution to obtain a casein sample solution for later use;

s3, adding 1.2mg of curcumin into the solution obtained in the step S2, and stirring for 24 hours by using a magnetic stirrer to form a casein solution wrapping the curcumin for later use;

and S4, performing ultrasonic disruption on the casein solution entrapped with curcumin obtained in the step S3 by using an ultrasonic cell disruptor, wherein the working power of the ultrasonic cell disruptor is 400W, the working time is 10min, the working frequency is 2S for every 4S of work, crushed ice is adopted for cooling treatment in the ultrasonic disruption treatment process, and the casein-curcumin drug-loaded nanoparticles are obtained after the ultrasonic disruption.

The casein-curcumin drug-loaded nanoparticles prepared by the preparation method have the entrapment rate of 90.8%.

Example Effect data

Determination of curcumin encapsulation rate and drug loading rate

The casein-curcumin nanoparticles prepared in example 1 were taken. Placing the freshly prepared casein-curcumin nanoparticles in a low-temperature refrigerator at-20 ℃ overnight for pre-freezing, and then quickly placing the casein-curcumin nanoparticles into a freeze dryer for freeze drying to obtain a freeze-dried powdery sample. Weighing a proper amount of freeze-dried curcumin nanoparticle powder, adding 2mL of absolute ethyl alcohol, performing vortex washing on free curcumin which is not wrapped in the nanoparticles, then centrifuging at 2000r for 10min, and taking out supernatant. And washing the centrifuged nanoparticle precipitate again by the same method, repeating the washing for three times, and combining the washed and centrifuged supernatants. Calculating the mass of the free curcumin, and calculating the encapsulation rate and the drug-loading rate of the nanoparticle system to the curcumin by the following formulas:

the results are as follows:

the encapsulation rate of the casein nanoparticles to curcumin can reach 90%, which shows that the high-efficiency encapsulation and loading of curcumin can be realized, and the water solubility of curcumin and the stability of curcumin are improved.

Two, 4 ℃ storage stability and gastrointestinal pH stability

Storing the casein-curcumin nano solution in a refrigerator at 4 ℃, observing whether a sample is precipitated at intervals, measuring the turbidity of the sample at the position of 450nm by using an ultraviolet-visible light spectrophotometer, and detecting the size and the distribution of the particle size by using a particle sizer. In addition, samples were taken at intervals to determine curcumin content.

Preparing simulated gastric fluid with pH value of 1.5 and simulated intestinal fluid with pH value of 7.4 without adding enzyme, mixing casein nanoparticles with the simulated intestinal fluid and the simulated gastric fluid in equal volume, and measuring the particle size and distribution of the sample by using a laser particle sizer.

The results are as follows:

the drug-loaded nano solution was left at 4 ℃ and the particle size was measured at intervals. As shown in fig. 2, no obvious precipitation occurs within 30 days of storage time, the particle size of the drug-loaded nanoparticles remains substantially stable, which indicates good storage stability of the drug-loaded nanoparticles, and shows a higher practical application value of the drug-loaded nanoparticles.

The particle size of the drug-loaded nanoparticles is changed in the gastrointestinal pH environment, after the drug-loaded nanoparticles enter the gastric pH environment, the particle size is increased from 260.2nm to 268.9nm, the particle size is basically kept stable, and the particle size distribution is shown to be narrow in the figure. And when the nanoparticles enter the environment of intestinal pH value, the measured average particle size of the nanoparticles is 354.5nm, and the comparison of the particle size distribution diagram in the environment of gastrointestinal pH value in the figure shows that the stability of the drug-loaded nanoparticles in the stomach is higher than that in the intestine.

Third, casein-curcumin nanoparticle solubility experiment

And (3) re-dissolving a certain amount of casein-curcumin nanoparticle freeze-dried powder in distilled water, and calculating the concentration of the re-dissolved nanoparticles to calculate the solubility of the casein-curcumin nanoparticles.

The results are as follows:

the solubility of the casein-curcumin nanoparticles can reach 0.2-0.5mg/ml, and the casein-curcumin nanoparticles have good solubility, and the bioavailability of curcumin can be improved due to the extremely high solubility.

Fourth, cell uptake assay

Labeled casein-curcumin nanoparticles labeled with Fluorescein were prepared according to example 1 by adding Fluorescein Isothiocyanate (FITC) to casein-curcumin.

Hela cells were cultured in cell plates (96-well plates) with 5% CO₂Culturing in an incubator at 37 ℃, removing the culture solution after 80 percent of cells adhere to the wall, adding FITC labeled casein-curcumin nanoparticles, and still culturing in the incubator. And then observing the ingestion condition of the casein-curcumin nanoparticles under a fluorescence microscope after the culture is finished for 4 h.

The results are as follows:

as can be seen from FIG. 3, there is a clear fluorescence, which proves that the nanoparticle is taken up by the cells and the uptake is good.

Fifthly, in-vitro inhibition effect of curcumin on tumor cells

Hela cells were cultured in cell plates (96-well plates) at 37 ℃ in 5% CO₂Culturing in incubator until 80% of cellsAfter the adherence, the culture solution is removed, curcumin samples with the concentration of 40 mug/mL, 80 mug/mL and 120 mug/mL are respectively added, 6 parallel holes are arranged at each concentration, and according to the experimental condition, the culture solution is added to the periphery of a 96-hole plate to serve as a control group and still placed in an incubator for culture (the culture condition is unchanged). Then observing by using an inverted microscope and taking a picture for subsequent comparison after the culture is finished for 24 h; then, the supernatant of each well was aspirated in a clean bench, 20. mu.L of MTT (5 mg/mL) was added thereto, the culture was continued for 4 hours under natural conditions, the stock solution was aspirated, and DMSO (dimethyl sulfoxide) was added to each well in a fixed amount of 150. mu.L, followed by shaking, and the cell viability was calculated with reference to the following equation.

Cell Viability(％)＝ODs/ODc×100％

The absorbance values at 490nm wavelength of the control group and the experimental group were expressed as ODc and ODs.

The results are as follows:

the experimental results show that the same trend is observed for the increase of the curcumin concentration and the increase of the number of dead cells, when the administration concentration is increased to 80 mug/mL, the survival rate of the tumor cells is sharply reduced within 24h, and when the survival rate is reduced to 120 mug/mL, the cells almost completely die, which indicates that the higher the curcumin concentration is, the higher the killing power on the tumor cells is (see figure 4).

Sixthly, the inhibition effect of CC-NPs on tumor cells, namely the application of the casein-curcumin drug-loaded nanoparticles in improving the tumor killing effect of curcumin.

And (3) inspecting the inhibition degree of the CC-NPs nanoparticles on the growth condition of in vitro tumor cells by using an MTT colorimetric method. Culturing Hela cells to adherent with 96-well plate, removing culture solution, adding CC-NPs solution, and placing in incubator (the conditions of incubator are 37 deg.C, 5% CO)₂) The culture time is 24h and 48 h. OD value at 490nm wavelength was measured by microplate reader, and relative survival rate of tumor cells was calculated.

The results are as follows:

at the same concentration, CC-NPs have stronger killing power on cells compared with curcumin, and the survival rate of the cells is extremely low after 48 hours of culture. The drug-loaded nanoparticles have a slow release effect, and the inhibition effect on cancer cells is gradually increased along with the increase of time (figure 5).

Experiment of transmembrane transport of cells

And (3) researching the transmembrane transport condition of the curcumin-casein nano dispersion liquid monolayer cells by adopting a Hela monolayer cell model.

To simulate the acidic environment of the small intestine, the upper compartment ph6.5 and the lower compartment ph7.4 were used. The upper and lower chambers were filled with 0.5mL of cell suspension and 1.5mL of complete medium, respectively. Hela cell plate with 5% CO at 37 deg.C₂Overnight. Replacing cell suspension without adherence, adding 1ml, 20mg/ml free curcumin solution and curcumin nano system into the upper chamber as supply pool, adding 1.5ml complete culture medium into the lower chamber as receiving pool, repeating the steps at 37 deg.C and 5% CO for 3 times₂Cultured in an incubator with 2 hours intervals between each transit. After the transport study, the upper chamber was washed twice with PBS, the fluorescence intensity was measured at a wavelength of excitation light of 425nm and emission light of 518nm, and the bioavailability of curcumin at the cellular level was calculated by the following formula.

The results are as follows:

as can be seen from fig. 6, the bioavailability of the nanoparticles prepared by loading curcumin on casein is higher than that of free curcumin, further proving that the casein-curcumin nanoparticles prepared by the invention adopt an ultrasonic technology to prepare a nano drug-loading system with higher bioavailability.

Casein self-assembly nanoparticle effect verification experiment

A preparation method of casein self-assembled nanoparticles comprises the following steps:

s1: 20mg, 30mg and 40mg of casein powder are respectively weighed and completely dissolved in 10mL of distilled water to prepare casein solutions of 2mg/mL, 3mg/mL and 4mg/mL respectively.

S2, adjusting the pH value of the casein solution to be about 5-5.5 by using 1M NaOH solution.

S3: and (3) carrying out ultrasonic disruption on the casein solution by using an ultrasonic cell disruption instrument, wherein the temperature is reduced by using crushed ice, the working power is adjusted to 200W, and the working time is 30min (working for 4s and stopping for 2s), so that the nano particles (CS-NPs) formed by self-assembly of casein are obtained.

The results are as follows:

as shown in fig. 7: preparation process of CS-NPs

It can be clearly observed that: the color of the casein solution after sonication and the solution before sonication showed a clear to milky color change.

As shown in fig. 8: transmission electron microscopy of CS-NPs: the casein solution has obvious change in state before and after ultrasonic treatment, and most casein is found to be aggregated into spherical particles, and the size and distribution of the casein particles are more uniform under the shearing and dispersing action of the ultrasonic wave.

Firstly, the average particle size and the dispersion coefficient of the nanoparticles prepared by the experiment are measured, and the results are shown in the following table 3:

as can be seen from the above table: the average particle diameters of the three nanoparticles are 251.8 +/-5.63 nm, 372.9 +/-4.15 nm and 468 +/-5.25 nm respectively, and the dispersion indexes are 0.221, 0.323 and 0.415 in sequence. The graph of the comparison results of the average particle size and the dispersion coefficient (PDI) of drug-loaded nanoparticles prepared from casein samples with different concentrations is shown in fig. 9. As can be seen from fig. 9 and table 3: as the concentration increases, the average particle size of the Cs-NPs increases, and the dispersion coefficient of the particle size also increases.

In conclusion, the three nanoparticles have a particle size of 240-470nm, are uniformly dispersed, and have good stability.

Second, contrast the formation of casein self-assembled nanoparticles before and after ultrasonic wave

And (3) investigating the influence of the ultrasonic power on the particle size of the formed nanoparticles, fixing the pH value to be 5.3, and the solution concentration to be 2mg/ml, and investigating the particle size of the nanoparticles obtained under the conditions of no ultrasonic operation and 200w of ultrasonic power. The changes in particle size and distribution coefficient of the casein solution before and after sonication are shown in table 4 below:

table 4: casein solution particle size and distribution coefficient change before and after ultrasonic treatment

As can be seen from Table 4:

when the pH value of the casein solution is close to the isoelectric point, some casein molecules begin to aggregate to form fine flocculent aggregates, so that the particle size is larger, and the aggregates are dispersed by ultrasound and participate in the assembly of the casein molecules in the solution. The decrease of the distribution coefficient of the casein nanoparticles after ultrasonic treatment shows that the particle size of the nanoparticles in the solution is more uniform after ultrasonic treatment. The particle size value after ultrasonic treatment is reduced compared with the particle size before ultrasonic treatment.

Thirdly, the measurement of the hydrophobicity of the nano surface of the casein

And (3) measuring the surface hydrophobicity of the casein nano micelle dispersion liquid by using a fluorescent probe. The casein nanomicelles dispersion was diluted with ultrapure water to five concentration gradients (0.0025% to 0.02%). Prepare 8mM ANS solution in 10mM PBS, pH7.2, pipette 0.04mL of the prepared ANS solution into 5mL of the diluted sample. The relative fluorescence intensity was measured by a fluorescence photometer. The excitation wavelength was 365nm and the emission wavelength was 484 nm. The surface hydrophobicity index is equal to the initial slope of the relative fluorescence intensity versus protein concentration.

The results are as follows:

FIG. 10 shows the change of surface hydrophobicity index measured after different times of ultrasonic treatment of casein solutions with different concentrations. The results show that low concentration protein solutions (20mg/ml) show a significant decrease in surface hydrophobicity after 5min of sonication, but the change in hydrophobicity levels off with further sonication. The particle size of the casein nano micelle is obviously reduced by the ultrasonic treatment, the specific surface area is increased, and the surface hydrophobicity is not increased or decreased. This phenomenon indicates that the sonication does not simply split the large casein micelles, but reassembles the casein nanoclusters to form new nanoclusters, and hydrophobic groups are accumulated in the newly formed nanoclusters, while the hydrophobic groups on the surface layer are greatly reduced. However, the protein concentration is increased, and the decrease of the surface hydrophobicity index of the nano-micelle after 5min ultrasonic treatment is more and more insignificant, probably because when the protein concentration is high, the nano-micelle formed by the nano-cluster spontaneously is the nano-micelle formed by hydrophobic aggregation. Because, at high concentrations, hydrophobic aggregation occurs more readily.

Cytotoxicity test of Casein nanoparticles

1. Culture of cells

Hela cells in DMEM high-sugar culture medium (containing 100U/mL penicillin and streptomycin) containing 10% calf serum at 37 deg.C and 5% CO₂And culturing in an incubator. Changing the liquid every other day, and digesting with pancreatin for passage every 3-4 days.

Toxicity test of Cs-NPs

First, the concentration of Hela cells cultured in the logarithmic growth phase was adjusted to 4000 cells/mL, 200. mu.L of the cells were inoculated into 96 wells, and the inoculated 96-well culture plate was cultured in an incubator (the conditions of the incubator were set at 37 ℃ C., 5% CO)₂) When 80% of the adherently grown cells were present in the well plate, the culture solution in the well plate was removed, followed by addition of culture solutions having CS-NPs concentrations of (sample 1) 100. mu.g/mL, (sample 2) 200. mu.g/mL, (sample 3) 400. mu.g/mL, and (sample 4) 600. mu.g/mL, respectively. 6 parallel wells were set for each concentration, and the periphery of the 96-well plate was supplemented with a simple culture solution as a control group according to the experimental conditions, and was still placed in the incubator (culture conditions were unchanged). Then observing by using an inverted microscope and taking a picture for subsequent comparison after the culture is finished for 24 h; then, the supernatant of each well was aspirated in a clean bench, 20. mu.L of MTT (5 mg/mL) was added thereto, the culture was continued for 4 hours under natural conditions, the stock solution was aspirated, and DMSO (dimethyl sulfoxide) was added to each well in a fixed amount of 150. mu.L, followed by shaking, and the cell viability was calculated with reference to the following equation.

Cell Viability(％)＝ODs/ODc×100％

From the experimental results, it can be seen that after 24 hours of culture, the survival rate of the cells under the Cs-NPs with different concentrations is higher, no obvious cell death occurs, and the observation experimental results preliminarily show that: casein has no obvious cytotoxicity, and the single casein raw material fundamentally ensures the safety and reliability of the nanoparticles. (see fig. 11).

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. A preparation method of casein-curcumin drug-loaded nanoparticles is characterized by comprising the following steps: the method comprises the following steps:

2. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1, wherein the preparation method comprises the following steps: in S1, 20-40mg of the casein powder was dissolved in 10ml of distilled water.

3. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1, wherein the preparation method comprises the following steps: the concentration of the NaOH solution in S2 is 1M, and the pH value of the casein sample solution is adjusted to 5-5.5.

4. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1, wherein the preparation method comprises the following steps: the weight of the curcumin added into the S3 is 800 mu g-1.2 mg.

5. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1 or 4, wherein the preparation method comprises the following steps: the magnetic stirring time in S3 was 24 h.

6. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1, wherein the preparation method comprises the following steps: the working power of the ultrasonic cell disruption instrument in the S4 is 200-400W, the working time is 10-30min, and the working frequency is 2S stop every 4S.

7. The preparation method of the casein-curcumin drug-loaded nanoparticle as claimed in claim 1 or 6, wherein the preparation method comprises the following steps: the temperature reduction process in S4 is to reduce the temperature by using crushed ice.

8. A casein-curcumin drug-loaded nanoparticle is characterized in that: prepared by the method of any one of claims 1 to 7.

9. The casein-curcumin drug-loaded nanoparticle of claim 8, wherein: the entrapment rate of the casein-curcumin drug-loaded nanoparticles is 90-95%.

10. The application of the casein-curcumin drug-loaded nanoparticle as claimed in claim 8 in improving the tumor killing effect of curcumin.