Keratin-tea polyphenol nanoparticles as well as preparation method and application thereof

Abstract

The invention provides keratin-tea polyphenol nanoparticles as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The keratin-tea polyphenol nanoparticles provided by the invention are characterized by comprising a keratin carrier and tea polyphenol which is embedded inside the keratin carrier and chemically bonded and self-assembled on the surface layer and inside the keratin carrier. According to the keratin-tea polyphenol nano-particles, formaldehyde serves as an initiator, keratin and tea polyphenol are subjected to nucleophilic substitution reaction, aldol condensation reaction and addition reaction are conducted between the keratin and between the tea polyphenol respectively, in the reaction process, polymerized keratin and tea polyphenol molecules further have a self-assembly effect, and the keratin-tea polyphenol nano-particles are obtained. Keratin-tea polyphenol nanoparticles are finally obtained, so that the stability of tea polyphenol is improved; and the keratin-tea polyphenol nanoparticles provided by the invention have nano sizes, so that the keratin-tea polyphenol nanoparticles can enter cancer cells more easily through cell permeation and endocytosis, thereby better inhibiting the growth of the cancer cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to keratin-tea polyphenol nanoparticles and a preparation method and application thereof. Background Art

[0002] Tea polyphenols is a general term for a class of polyhydroxy compounds in tea extracts. The main component of tea polyphenols is catechins, which contain 60-80%. Catechins can be further divided into four types: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). Their structures (see formula I) all contain a three-membered flavan ring structure containing rings A, B, and C. Due to the activity of the adjacent dihydroxy or trihydroxy groups of this type of polyhydroxy compound, especially the activity of EGCG, it is known to have preventive and therapeutic effects on cancer and many other diseases. Epidemiological studies have shown that drinking tea (≥4 cups) every day can reduce the risk of cancer in humans. Clinical studies have shown that regular tea drinking can inhibit malignant tumors in the stomach, revealing the auxiliary therapeutic effect of tea polyphenols in malignant tumors. A large number of animal model experiments have also confirmed that tea and tea polyphenols have preventive and therapeutic effects on cancer.

[0003]

[0004] However, under normal physiological pH conditions, EGCG has low stability and is easily degraded before being absorbed and utilized. Studies have shown that after oral intake of EGCG, 0.012% and 0.32% of oral EGCG were detected in the plasma of rats and humans, respectively. This has the defect of low bioavailability and is difficult to exert physiologically active functions. Summary of the invention

[0005] The purpose of the present invention is to provide a keratin-tea polyphenol nanoparticle and a preparation method and application thereof. The keratin-tea polyphenol nanoparticle provided by the present invention has excellent stability and has a high bioavailability when being absorbed and utilized.

[0006] In order to achieve the purpose of the present invention, the present invention provides the following technical solutions:

[0007] A keratin-tea polyphenol nanoparticle comprises a keratin carrier and tea polyphenol embedded in the keratin carrier and chemically bonded to the surface and inside of the keratin carrier.

[0008] Preferably, the mass ratio of the keratin carrier to tea polyphenols is 0.1-1:0.1-100.

[0009] Preferably, the particle size of the keratin-tea polyphenol nanoparticles is 80 to 130 nm.

[0010] Preferably, the keratin carrier comprises one or more of feather keratin, human hair keratin and wool keratin; the number average molecular weight of the keratin carrier is 20,000 to 45,000.

[0011] The present invention also provides a method for preparing the keratin-tea polyphenol nanoparticles described in the above technical solution, comprising the following steps:

[0012] The keratin carrier, tea polyphenols and formaldehyde are mixed, and chemical reaction and molecular self-assembly are carried out to obtain the keratin-tea polyphenols nanoparticles; the chemical reaction includes aldol condensation reaction, addition reaction and nucleophilic substitution reaction.

[0013] Preferably, the mass ratio of the keratin carrier to formaldehyde is 1:0.004-0.04.

[0014] Preferably, the keratin carrier is obtained by an extraction method comprising the following steps:

[0015] The keratin raw material is sequentially treated with an organic solvent and an acid to obtain a pretreated keratin raw material; the keratin raw material comprises one of feathers, wool and human hair;

[0016] The pretreated keratin raw material, an alkali agent, a surfactant, a denaturant, a reducing agent and water are mixed and subjected to reduction treatment to obtain the keratin carrier.

[0017] Preferably, the reduction treatment is carried out at a temperature of 67 to 73° C. for 8 to 12 hours; and the reduction treatment is carried out in a nitrogen or argon atmosphere.

[0018] Preferably, the temperature of the chemical reaction and molecular self-assembly is room temperature, and the time is 22 to 26 hours.

[0019] The present invention also provides the use of the keratin-tea polyphenol nanoparticles described in the above technical solution or the keratin-tea polyphenol nanoparticles prepared by the preparation method described in the above technical solution in preparing drugs for inhibiting the growth of cancer cells.

[0020] The present invention provides a keratin-tea polyphenol nanoparticle, comprising a keratin carrier and tea polyphenol embedded in the keratin carrier and chemically bonded to the surface and inside of the keratin carrier. The keratin-tea polyphenol nanoparticle of the present invention uses formaldehyde as an initiator, and the formaldehyde can react with the amino group on the side chain of keratin to produce hydroxymethyl derivatives, which can be dehydrated to form imines, i.e., Schiff bases; the imines are easy to react with the nucleophilic amino acid residues on the keratin peptide chain, resulting in the formation of methylene bridges in the keratin; the formaldehyde can also react with the catechins in the tea polyphenols to react with the aldol condensation type, including methylation, condensation and polymerization, to produce oligomeric catechins with methylene bridges. Furthermore, the thiol groups on the keratin of the present invention react with polyphenol quinone (polyphenol quinone is a highly active intermediate of the auto-oxidation of catechol-type or pyrogallol-type catechins) to form covalent thiol flavonoid adducts. The occurrence of the above reaction and the molecular assembly effect between keratin and oligomeric catechins jointly provide keratin-tea polyphenol nanoparticles with excellent stability, thereby improving the stability of tea polyphenols and thus improving the bioavailability of tea polyphenols.

[0021] Furthermore, the particle size of the keratin-tea polyphenol nanoparticles provided by the present invention is 80 to 130 nm, making it easier for the keratin-tea polyphenol nanoparticles to enter cancer cells through cell penetration and endocytosis, thereby better inhibiting the growth of cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 Schematic diagram of the preparation of keratin-tea polyphenol nanoparticles of the present invention;

[0024] Figure 2 This is the particle size distribution diagram of feather keratin-tea polyphenols nanoparticles obtained in Example 3;

[0025] Figure 3 This is a TEM image of feather keratin-tea polyphenols nanoparticles obtained in Example 3;

[0026] Figure 4 This is a graph showing the inhibition rate of feather keratin-tea polyphenol nanoparticles obtained in Example 3 on HepG2 cells;

[0027] Figure 5 This is a graph showing the inhibitory effect of tea polyphenol oligomers obtained in Comparative Example 1 on HepG2 cells;

[0028] Figure 6 This is a diagram showing the inhibitory effect of tea polyphenols raw materials on HepG2 cells. DETAILED DESCRIPTION

[0029] The invention provides a keratin-tea polyphenol nanoparticle, comprising a keratin carrier and tea polyphenol embedded in the keratin carrier and chemically bonded to the surface layer and the interior of the keratin carrier.

[0030] In the present invention, the mass ratio of the keratin carrier to tea polyphenols is 0.1-1:0.1-100.

[0031] In the present invention, the particle size of the keratin-tea polyphenol nanoparticles is 80-130 nm, and in a specific embodiment, it can be 100 nm, 110 nm or 90 nm. The particle size of the keratin-tea polyphenol nanoparticles provided by the present invention is controlled within the above range, so that it can enter cancer cells through cell penetration and endocytosis, thereby better inhibiting the growth of cancer cells.

[0032] In the present invention, the keratin carrier includes one or more of feather keratin, human hair keratin and wool keratin. In a specific embodiment, it can be feather keratin, wool keratin or human hair keratin. The keratin carrier is obtained by an extraction method comprising the following steps:

[0033] treating a keratin raw material with an organic solvent and an acid in sequence to obtain a pretreated keratin raw material; the keratin raw material comprises one or more of feathers, wool and human hair;

[0034] The pretreated keratin raw material, an alkali agent, a surfactant, a denaturant, a reducing agent and water are mixed and subjected to reduction treatment to obtain the keratin carrier.

[0035] The present invention sequentially treats the keratin raw material with an organic solvent and an acid to obtain a pretreated keratin raw material. In the present invention, the organic solvent includes one of acetone, petroleum ether and an alcohol reagent; the alcohol reagent can be ethanol, and in a specific embodiment, can be ethanol; the solid-liquid ratio of the keratin raw material to the organic solvent is 1.0g:200mL.

[0036] In the present invention, the organic solvent treatment includes: mixing the keratin raw material and the organic solvent, and performing a heating treatment to obtain the organic solvent pre-treated keratin raw material; the heating is water bath heating; the water bath heating temperature is 40°C, and the time is 2 hours; the water bath heating treatment is carried out under stirring conditions, and the present invention has no special restrictions on the stirring rate, which is based on the appearance of a vortex in the middle of the mixed solution during the stirring process. In the present invention, the heating treatment also includes solid-liquid separation; the solid-liquid separation method is not particularly limited, and in a specific embodiment, it can be suction filtration.

[0037] In the present invention, the reagent used for the acid treatment is hydrochloric acid; the concentration of the hydrochloric acid is 12M; the solvent used in the acid treatment process is a mixed solvent of water and an organic reagent, and the volume ratio of water to the organic reagent is 5:3; the organic reagent includes one of acetone, petroleum ether and an alcohol reagent; the alcohol reagent can be ethanol, and in a specific embodiment, it can be ethanol; the solid-liquid ratio of the keratin raw material treated with the organic solvent and the hydrochloric acid is 1.0g:1.0mL.

[0038] In the present invention, the acid treatment includes: placing the organic solvent pretreated keratin raw material into a mixed solvent, then adding hydrochloric acid to mix, and performing heating treatment to obtain a pretreated keratin raw material. In the present invention, the solid-liquid ratio of the organic solvent pretreated keratin raw material and the mixed solvent is 1.0g:80mL; the heating is water bath heating; the water bath heating temperature is 40°C and the time is 2h; the water bath heating treatment is carried out under stirring conditions, and the present invention has no special restrictions on the stirring rate, which is based on the appearance of a vortex in the middle of the mixed solution during the stirring process. In the present invention, the water bath heating treatment also includes washing and drying; the washing is performed until the filtrate after washing and filtration of the obtained product is neutral; the present invention has no special restrictions on the drying.

[0039] After obtaining the pretreated keratin raw material, the present invention mixes the pretreated keratin raw material, an alkali agent, a surfactant, a denaturant, a reducing agent and water, and performs a reduction treatment to obtain the keratin.

[0040] In the present invention, the alkaline reagent is tris(hydroxymethyl)aminomethane (Tris); the surfactant is sodium dodecyl sulfate (SDS); the denaturant is urea (U); the reducing agent is mercaptoethanol (ME); and the water is deionized water.

[0041] In the present invention, the alkaline reagent provides an alkaline environment for reduction extraction; the surfactant can interact with the extracted keratin to stably exist in the aqueous solution; the denaturant can accelerate the dissolution of keratin; and the reducing agent is used to break the disulfide bonds in the keratin raw material during the extraction process.

[0042] In the present invention, the mass ratio of the pretreated keratin raw material, Tris and SDS is 0.1-10:0.1:0.8, and in a specific embodiment, it can be 1:0.1:0.8, 3:0.1:0.8 or 5:0.1:0.8; the mass ratio of the pretreated keratin raw material, U, ME and water is 0.1-10:1:0.025-0.35:50; in a specific embodiment, it can be 1:1:0.35:50, 3:1:0.35:50 or 5:1:0.35:50.

[0043] In the present invention, the temperature of the reduction treatment is 67-73°C, and in a specific embodiment, it can be 70°C, 68°C or 72°C, and the time is 8-12h, and in a specific embodiment, it can be 10h, 9h or 11h; the reduction treatment is carried out in a nitrogen or argon atmosphere.

[0044] The invention adopts a reduction method to extract keratin from a keratin raw material. During the extraction process, a reducing agent reacts with a disulfide bond in the keratin raw material to form two thiols, thereby breaking the disulfide bond, destroying the dense cross-linked structure of the keratin raw material, and releasing the keratin.

[0045] In the present invention, the reduction treatment further includes filtering the obtained product, and dialyzing and drying the filtrate; the reagent used for the dialysis is deionized water, and the molecular weight cutoff of the dialysis bag is 8000-14000kDa; the time is 70 to 75h; the drying is freeze-drying, and the temperature is -40°C to -80°C, and in a specific embodiment, it can be -50°C or -60°C. The present invention has no special limitation on the drying time.

[0046] In the present invention, the number average molecular weight of the keratin carrier is 20,000 to 45,000, and in a specific embodiment, it can be 20,000, 30,000 or 45,000.

[0047] The present invention also provides a method for preparing the keratin-tea polyphenol nanoparticles described in the above technical solution, comprising the following steps:

[0048] The keratin carrier, tea polyphenols and formaldehyde are mixed, and chemical reaction and molecular self-assembly are carried out to obtain the keratin-tea polyphenols nanoparticles; the chemical reaction includes aldol condensation reaction, addition reaction and nucleophilic substitution reaction.

[0049] In the present invention, unless otherwise specified, all preparation raw materials are preferably commercially available products well known to those skilled in the art.

[0050] In the present invention, the content of tea polyphenols in the tea polyphenols is ≥90wt%, in a specific embodiment, the content of total catechins in the tea polyphenols is ≥80wt%; the content of epigallocatechin gallate (EGCG) in the catechins is ≥40wt%. In the present invention, the tea polyphenols are solid powders, which are prepared into tea polyphenols solution when used; the concentration of the tea polyphenols solution is 0.1-50mg / mL, and in a specific embodiment, it can be 5.0mg / mL, 10mg / mL or 20mg / mL.

[0051] In the present invention, the keratin carrier is used in the form of a keratin aqueous solution; the concentration of the keratin aqueous solution is 0.1 to 50 mg / mL, and in a specific embodiment, it can be 1.0 mg / mL, 10 mg / mL or 30 mg / mL.

[0052] In the present invention, the formaldehyde is used in the form of a formaldehyde solution; the concentration of the formaldehyde solution is 37%.

[0053] In the present invention, the mass ratio of the keratin carrier, tea polyphenols and formaldehyde is 1:0.1-100:0.004-0.04. In a specific embodiment, it can be 1:0.1:0.004, 1:10:0.04 or 1:20:0.04.

[0054] In the present invention, the temperature of the chemical reaction and the molecular self-assembly is room temperature, and the time is 22 to 26 hours. In a specific embodiment, it can be 24 hours or 25 hours.

[0055] In the present invention, after the chemical reaction and molecular self-assembly, the obtained product is dialyzed and dried; the reagent used for dialysis is deionized water, and the molecular weight cutoff of the dialysis bag is 8000-14000kDa; the time is 5 to 7 days; during the dialysis process of the present invention, the deionized water used for dialysis is replaced every 10 to 12 hours.

[0056] In the present invention, the drying is freeze drying, and the temperature is -40°C to -80°C. In a specific embodiment, it can be -50°C or -60°C. The present invention has no special limitation on the drying time.

[0057] The present invention also provides the use of the keratin-tea polyphenol nanoparticles described in the above technical solution or the keratin-tea polyphenol nanoparticles prepared by the preparation method described in the above technical solution in preparing drugs for inhibiting the growth of cancer cells.

[0058] In the present invention, the cancer cells include one or more of human liver cancer cells HepG2, human breast cancer cells MCF-7 and colon cancer cells HCT116.

[0059] In order to further illustrate the present invention, the keratin-tea polyphenol nanoparticles provided by the present invention and the preparation method and application thereof are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0060] Example 1

[0061] 1 g of wool was soaked in 200 mL of ethanol, heated to 40°C in a water bath, and mechanically stirred for 2 h, then filtered and dried to obtain ethanol-pretreated wool;

[0062] 1 g of the ethanol pretreated wool was soaked in 80 mL of a mixed solution of ethanol and water (the volume ratio of ethanol to water was 3:5), 1 mL of 12 M hydrochloric acid was added, and then heated in a water bath to 40°C and mechanically stirred for 2 hours, and then filtered, and the filtered wool was washed with distilled water until the filtrate was neutral, and then dried to obtain the hydrochloric acid pretreated wool;

[0063] 1 g of wool pretreated with hydrochloric acid was placed in a reaction container, and 50 mL of deionized water, 0.1 g of Tris, 0.8 g of SDS, 1 g of U, and 330 μL of ME were added. The reaction was stirred for 10 h at 70 ° C in a N2 atmosphere. After the reaction was completed, the filtrate was collected by suction filtration. The obtained filtrate was dialyzed with deionized water for 3 days (dialysis bag molecular weight cutoff MW: 3.5 kDa) to obtain wool keratin;

[0064] according to Figure 1 Schematic diagram of the preparation of wool keratin-tea polyphenol nanoparticles. The specific steps are as follows:

[0065] 45 mL of a 4 mg / mL wool keratin aqueous solution and 113 mL of a 20 mg / mL tea polyphenol aqueous solution were mixed at room temperature, and then 7.0 mL of a 37 wt% formaldehyde solution was added, and the mixture was mechanically stirred for reaction for 24 h at room temperature. After the reaction, the mixed solution was transferred into a dialysis bag with a molecular weight cutoff of 8000-14000 kDa, and then the dialysis bag was placed in a 2 L beaker filled with deionized water, and dialyzed for purification for 5 days. The deionized water in the beaker was changed every 12 h during the dialysis process. After the dialysis, the product was freeze-dried to obtain wool keratin-tea polyphenol nanoparticles (WK-TPNPs).

[0066] Example 2

[0067] 1 g of human hair was soaked in 200 mL of ethanol, heated to 40°C in a water bath, and then mechanically stirred for 2 h, and then filtered and dried to obtain ethanol-pretreated human hair;

[0068] 1 g of the ethanol pretreated human hair was soaked in 80 mL of a mixed solution of ethanol and water (the volume ratio of ethanol to water was 3:5), 1 mL of 12 M hydrochloric acid was added, and then heated in a water bath to 40° C. and mechanically stirred for 2 h, and then filtered, and the filtered human hair was washed with distilled water until the filtrate was neutral, and dried to obtain the hydrochloric acid pretreated human hair;

[0069] 1 g of human hair pretreated with hydrochloric acid was placed in a reaction container, and 50 mL of deionized water, 0.1 g of Tris, 0.8 g of SDS, 1 g of U, and 330 μL of ME were added. The reaction was stirred for 10 h at 70° C. under N2 atmosphere. After the reaction was completed, the filtrate was collected by suction filtration. The obtained filtrate was dialyzed with deionized water for 3 days (dialysis bag molecular weight cutoff MW: 3.5 kDa) to obtain human hair keratin;

[0070] according to Figure 1 Schematic diagram of the preparation of human hair keratin-tea polyphenol nanoparticles. The specific steps are as follows:

[0071] 45 mL of a 4 mg / mL human keratin aqueous solution and 113 mL of a 20 mg / mL tea polyphenol aqueous solution were mixed at room temperature, and then 3.5 mL of a 37 wt% formaldehyde solution was added, and the mixture was mechanically stirred for reaction for 24 h at room temperature. After the reaction, the mixed solution was transferred into a dialysis bag with a molecular weight cutoff of 8000-14000 Da, and then the dialysis bag was placed in a 2 L beaker filled with deionized water, and dialyzed for purification for 5 days. During the dialysis process, the deionized water in the beaker was changed every 12 h. After the dialysis, the product was freeze-dried to obtain human keratin-tea polyphenol nanoparticles (HK-TPNPs).

[0072] Example 3

[0073] 1 g of feathers were soaked in 200 mL of ethanol, heated to 40°C in a water bath, and mechanically stirred for 2 h, then filtered and dried to obtain ethanol-pretreated feathers;

[0074] 1 g of the ethanol pretreated feathers was immersed in 80 mL of a mixed solution of ethanol and water (the volume ratio of ethanol to water was 3:5), 1 mL of 12 M hydrochloric acid was added, and then heated to 40°C in a water bath and mechanically stirred for 2 h, and then filtered, and the filtered wool was washed with distilled water until the filtrate was neutral, and then dried to obtain the hydrochloric acid pretreated feathers;

[0075] 1 g of feathers pretreated with hydrochloric acid was placed in a reaction container, and 50 mL of deionized water, 0.1 g of Tris, 0.8 g of SDS, 1 g of U, and 330 μL of ME were added. The reaction was stirred for 10 h at 70 ° C in a N2 atmosphere. After the reaction was completed, the filtrate was collected by suction filtration. The obtained filtrate was dialyzed with deionized water for 3 days (dialysis bag molecular weight cutoff MW: 3.5 kDa) to obtain feather keratin;

[0076] according to Figure 1 Schematic diagram of the preparation of feather keratin-tea polyphenol nanoparticles. The specific steps are as follows:

[0077] 45 mL of a 2 mg / mL feather keratin aqueous solution and 113 mL of a 20 mg / mL tea polyphenol aqueous solution were mixed at room temperature, and then 3.5 mL of a 37 wt% formaldehyde solution was added, and the mixture was mechanically stirred for reaction for 24 h at room temperature. After the reaction, the mixed solution was transferred into a dialysis bag with a molecular weight cutoff of 8000-14000 kDa, and then the dialysis bag was placed in a 2 L beaker filled with deionized water, and dialyzed for purification for 5 days. During the dialysis process, the deionized water in the beaker was changed every 12 h. After the dialysis, the obtained product was freeze-dried to obtain feather keratin-tea polyphenol nanoparticles (FK-TPNPs).

[0078] The particle size of the FK-TP NPs obtained in Example 3 was measured by dynamic light scattering (DLS). Figure 2 As shown;

[0079] Figure 2 is the particle size distribution diagram of FK-TPNPs obtained in Example 3, Figure 2 The results showed that the particle size of FK-TP NPs was 100 nm.

[0080] The morphology of the FK-TP NPs obtained in Example 3 was observed using a JEM-1200EX / S transmission electron microscope (TEM). Figure 3 shown.

[0081] Figure 3 TEM image of FK-TP NPs obtained in Example 3. Figure 3 The results show that FK-TP NPs are regular spherical.

[0082] Comparative Example 1

[0083] A blank sample of tea polyphenol oligomers was prepared according to the preparation method described in Example 3, except that no feather keratin was added to obtain tea polyphenol oligomers OTP.

[0084] Test Case

[0085] The cytotoxicity of the feather keratin-tea polyphenol nanoparticles obtained in Example 3 and the tea polyphenol oligomers obtained in Comparative Example 1 was tested using HepG2 cells.

[0086] MTT experiment: HepG2 cells in the logarithmic growth phase were digested with 0.25% trypsin, counted on a hemocytometer, added to a 6-well plate, and 5 × 10 6cells / well; after the cells are completely attached to the wall, proceed according to the previously set concentration gradient. Collect the culture medium and cells in a 15mL centrifuge tube, centrifuge at 1500rpm / min for 5min, wash the cells once with PBS, centrifuge at 1500rpm for 5min to collect, centrifuge the cell suspension (1500rpm, 5min), remove the supernatant, then add 450μL PBS to the cell pellet to resuspend the cells, then add 50μL 0.4% trypan blue staining solution, incubate at room temperature for 5min, take a small amount of stained cells, count with a hemocytometer, dead cells are stained blue, swollen and dull, compared with dead cells, live cells will not be stained and maintain their normal shape and luster. Cell viability (%) = total number of live cells (TNLC) / (TNLC+total number of dead cells)×100%, the results are shown below.

[0087] Figure 4 This is the effect diagram of the inhibition rate of feather keratin-tea polyphenols nanoparticles on HepG2 cells obtained in Example 3. Figure 4 As shown in Table 1, when the concentration of FK-TP NPs was 20 μg / mL and FK-TP NPs were co-cultured with HepG2 cells for 24 h, the survival rate of HepG2 cells was 55.33%, that is, the inhibition rate of FK-TP NPs on HepG2 cells reached 44.67%.

[0088] Table 1 Inhibitory effect of feather keratin-tea polyphenol nanoparticles obtained in Example 3 on HepG2 cells

[0089] Concentration (μg / mL) HepG2 survival rate (%) 0 100 2.5 87.54941 5 80.23722 10 71.14625 20 55.33597

[0090] Figure 5 The inhibition rate of tea polyphenol oligomers on HepG2 cells obtained in Comparative Example 1 is shown in FIG. Figure 5 As shown in Table 2, when the concentration of OTP was 20 μg / mL and OTP was co-cultured with HepG2 cells for 24 h, the cell survival rate of HepG2 was about 64.28%, that is, the inhibition rate of OTP on HepG2 cells was 35.72%, indicating that the inhibitory effect of OTP on HepG2 cells was not as strong as that of FK-TPNP.

[0091] Table 2 Inhibitory effect of tea polyphenol oligomers obtained in comparative example 1 on HepG2 cells

[0092] Concentration (μg / mL) HepG2 survival rate (%) 0 100 2.5 90.21164 5 87.30159 10 73.01587 20 64.28571

[0093] Figure 6 This is the effect diagram of the inhibition rate of tea polyphenols on HepG2 cells. Figure 6As shown in the results of Table 3, when the concentration of tea polyphenols raw material (TP) reached 20 μg / mL, TP and HepG2 cells were co-cultured for 24 hours, the survival rate of HepG2 cells reached 94.7%, indicating that the inhibitory effect of pure TP raw material on HepG2 cells was relatively weak under the same experimental conditions.

[0094] Table 3 Effect diagram of the inhibition rate of tea polyphenols raw materials on HepG2 cells

[0095] Concentration (μg / mL) HepG2 survival rate (%) 0 100 2.5 100 5 98.73188 10 93.47826 20 94.74638

[0096] In summary, under the induction of formaldehyde, the present invention can effectively improve the stability of TP and prolong its circulation time in the body by the interaction between TP and feather keratin. It can passively target tumor tissues through the "enhanced permeability and retention effect" (EPR effect) and inhibit HepG2 cells, thereby improving the bioavailability of TP.

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A keratin-tea polyphenol nanoparticle, characterized in that: The invention comprises a keratin carrier and tea polyphenols embedded in the keratin carrier and chemically bonded and self-assembled in the interior and surface of the keratin carrier.

2. The keratin-tea polyphenol nanoparticles according to claim 1, characterized in that: The mass ratio of the keratin carrier to tea polyphenols is 0.1-1:0.1-100.

3. The keratin-tea polyphenol nanoparticles according to claim 1, characterized in that: The particle size of the keratin-tea polyphenol nanoparticles is 80-130 nm.

4. The keratin-tea polyphenol nanoparticles according to claim 1, characterized in that: The keratin carrier comprises one or more of feather keratin, human hair keratin and wool keratin; the number average molecular weight of the keratin carrier is 20,000-45,000.

5. The method for preparing keratin-tea polyphenol nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: The keratin carrier, tea polyphenols and formaldehyde are mixed, and chemical reaction and molecular self-assembly are carried out to obtain the keratin-tea polyphenols nanoparticles; the chemical reaction includes aldol condensation reaction, addition reaction and nucleophilic substitution reaction.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the keratin carrier to formaldehyde is 1:0.004-0.

04.

7. The preparation method according to claim 5, characterized in that: The keratin carrier is obtained by an extraction method comprising the following steps: treating a keratin raw material with an organic solvent and an acid in sequence to obtain a pretreated keratin raw material; the keratin raw material comprises one or more of feathers, wool and human hair; The pretreated keratin raw material, an alkali agent, a surfactant, a denaturant, a reducing agent and water are mixed and subjected to reduction treatment to obtain the keratin carrier.

8. The preparation method according to claim 7, characterized in that: The reduction treatment is carried out at a temperature of 67 to 73° C. for 8 to 12 hours; the reduction treatment is carried out in a nitrogen or argon atmosphere.

9. The preparation method according to claim 5, characterized in that: The temperature of the chemical reaction and the molecular self-assembly is room temperature, and the time is 22 to 26 hours.

10. Use of the keratin-tea polyphenol nanoparticles according to any one of claims 1 to 4 or the keratin-tea polyphenol nanoparticles prepared by the preparation method according to any one of claims 5 to 9 in preparing drugs for inhibiting the growth of cancer cells.

Images