Nanoparticles embedded with peptidomidol and preparation method of nanoparticles

Abstract

The invention discloses a nano-particle embedded with peptomidol and a preparation method of the nano-particle. The preparation method comprises the following steps: adding cyclodextrin, chitosan, polyvinyl alcohol and peptomidol into a metal ion salt solution, and carrying out a cross-linking reaction to obtain the peptomidol-embedded nanoparticles. According to the invention, by constructing the cyclodextrin-PVA-chitosan coated peptomidol nano-particles and introducing PVA into a cyclodextrin-chitosan embedding system, the embedding efficiency and the drug loading capacity of peptomidol are remarkably improved, and meanwhile, the solubility and the dispersity are improved. In addition, the nanoparticles provided by the invention can regulate the release kinetics of the embedding material, realize the controlled release effect, improve the thermal stability and reduce the degradation of active ingredients.

Description

Technical Field

[0001] The invention relates to the technical field of polymer materials, and in particular to a nanoparticle encapsulating a peptide Amido and a preparation method thereof. Background Art

[0002] In recent years, with the increasing demand for skin whitening, various whitening ingredients have been developed and applied in the cosmetics industry. Among them, Thiamidol is considered to be a whitening ingredient with potential application value due to its excellent whitening effect. Although Thiamidol shows a significant whitening effect, its application in cosmetics is subject to certain restrictions, mainly due to its poor solubility. Although Thiamidol can be dissolved in alcohol solvents, it will precipitate quickly once mixed with sufficient water. This characteristic not only affects the uniform distribution of Thiamidol in cosmetics, but also has a negative impact on its absorption capacity in the skin. Therefore, seeking technical means to improve the solubility of Thiamidol and improve its absorption effect in the skin has become an important direction of current research.

[0003] With the continuous development of functional cosmetics and pharmaceutical preparations, how to effectively improve the bioavailability and stability of poorly water-soluble compounds has become one of the research hotspots. Cyclodextrin and chitosan are widely used in drug delivery and food industries, and their encapsulation technology is often used to improve the solubility, stability and bioavailability of active ingredients. Common encapsulation methods include physical mixing, solvent evaporation, spray drying and cross-linking. The physical mixing method is simple to operate, but may lead to uneven release of active ingredients; the solvent evaporation method can improve the encapsulation rate, but has strict requirements on the use of solvents; the spray drying method is suitable for large-scale production, but may cause degradation of heat-sensitive ingredients; the cross-linking method can enhance stability, but the selection of cross-linking agents is more difficult. In addition, the above processes still have some disadvantages and limitations, such as the solubility limitation of cyclodextrin, strict requirements on operating conditions, uneven encapsulation efficiency, biocompatibility issues and high production costs. Therefore, although the encapsulation system of cyclodextrin and chitosan has good application potential, it still needs to overcome its solubility limitation, improve encapsulation efficiency and improve environmental stability. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a nanoparticle encapsulating peptide Anmido and a preparation method thereof to improve the water solubility of peptide Anmido.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is:

[0006] In one aspect, the present invention provides a method for preparing nanoparticles encapsulating the peptide Anmido, comprising the following steps:

[0007] Cyclodextrin, chitosan, polyvinyl alcohol (PVA) and peptide anamido are subjected to cross-linking reaction in a solution to which metal ion salt is added to obtain the nanoparticles encapsulating the peptide anamido.

[0008] As a preferred embodiment, the metal ion salt is calcium chloride;

[0009] And / or, the cyclodextrin is β-cyclodextrin;

[0010] And / or, the chitosan is carboxymethyl chitosan;

[0011] Preferably, the mass ratio of cyclodextrin, chitosan, polyvinyl alcohol and peptide anmito is 4-8:3-6:1-10:1-4;

[0012] Preferably, the mass of the metal ion salt is 3% to 10% of the mass of chitosan.

[0013] As a preferred embodiment, the cross-linking reaction time is 2 to 12 hours, more preferably 8 hours;

[0014] Preferably, the temperature of the cross-linking reaction is 20 to 80°C, more preferably 65°C;

[0015] Preferably, the solution to which the metal ion salt is added is an acidic solution; in certain specific embodiments, the pH of the solution to which the metal ion salt is added is adjusted to be acidic by adding acetic acid.

[0016] In certain specific embodiments, the cross-linking reaction is carried out under stirring conditions; the cross-linking reaction also includes post-processing of centrifugation or filtration, and freeze-drying.

[0017] As a preferred embodiment, the preparation method specifically comprises the following steps:

[0018] S1: dissolving chitosan and polyvinyl alcohol (PVA) in an acetic acid aqueous solution and stirring evenly; adding metal ion salt;

[0019] Alternatively, dissolve chitosan in an acetic acid aqueous solution and stir evenly; add a metal ion salt;

[0020] S2: adding cyclodextrin, peptide anamido and polyvinyl alcohol to carry out cross-linking reaction;

[0021] Preferably, in step S1, the amount of chitosan and the acetic acid aqueous solution is in the following relationship: 3-6 g of chitosan is dissolved in 100 mL of acetic acid aqueous solution with a mass fraction of 1% to 3%.

[0022] In another aspect, the present invention provides nanoparticles encapsulating the peptide Anmido obtained by the above preparation method.

[0023] The main advantages of the present invention are:

[0024] (1) The present invention constructs cyclodextrin-PVA-chitosan coated peptide Anmido nanoparticles, introduces PVA into the cyclodextrin-chitosan embedding system, significantly improves the embedding efficiency and drug loading of peptide Anmido, improves the solubility and dispersibility of the active ingredient, and promotes the formation of a more uniform carrier structure, thereby improving the fixation efficiency of the active ingredient. In addition, the nanoparticles provided by the present invention can adjust the release kinetics of the embedding material, achieve a controlled release effect, and improve thermal stability and reduce the degradation of the active ingredient.

[0025] (2) The nanoparticles constructed by the present invention can achieve the slow release of peptide Anmido, which can not only provide immediate moisturizing and repairing effects in the short term, but also continuously release active ingredients to achieve long-term care effects. It is particularly suitable for skin care products that need to exert effects continuously (such as facial masks, moisturizing creams, etc.).

[0026] (3) The nanoparticle preparation method provided by the present invention has low cost, and the chemical reagents used are safe and have no toxic side effects, and are suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The peptide Amito standard curve measured in Example 7 of the present invention.

[0028] Figure 2 A graph showing the inhibition rate of tyrosinase by nanoparticles encapsulating the peptide Anmido measured in Example 9 of the present invention.

[0029] Figure 3 The release curve of the peptide Anmido measured in Example 10 of the present invention. DETAILED DESCRIPTION

[0030] The following embodiments are only some embodiments of the present invention, rather than all embodiments. Therefore, the detailed description in the embodiments of the present invention provided below is not intended to limit the scope of the present invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative work belong to the protection scope of the present invention.

[0031] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0032] In the following examples, β-cyclodextrin was purchased from Shanghai Aladdin (C104384); carboxymethyl chitosan was purchased from Shanghai Aladdin (C304738); and polyvinyl alcohol was purchased from Shanghai Aladdin (P105126).

[0033] Example 1

[0034] Weigh 3 g of carboxymethyl chitosan, dissolve it in 100 mL of 1% acetic acid solution, and stir it magnetically until it is completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir it thoroughly to dissolve it, and obtain a chitosan solution;

[0035] Slowly add 4 g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol (PVA) solution containing 1 g of peptide Amido (containing 1 g of polyvinyl alcohol); stir magnetically at 800 rpm at 65°C for 8 hours; remove unencapsulated peptide Amido by ultrafiltration, and lyophilize the clarified liquid to obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Amido.

[0036] Example 2

[0037] Weigh 3 g of carboxymethyl chitosan and 1 g of polyvinyl alcohol, dissolve them in 100 mL of 1% acetic acid solution, and stir them magnetically until they are completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir and dissolve them thoroughly to obtain a chitosan-PVA solution;

[0038] Slowly add 4g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol solution containing 1g of peptide Anmido (containing 1g polyvinyl alcohol); magnetically stir at 800rpm at 65°C for 8h; ultrafiltration removes unencapsulated peptide Anmido, and lyophilizes the clarified liquid to obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Anmido.

[0039] Example 3

[0040] Weigh 6 g of carboxymethyl chitosan and 3 g of polyvinyl alcohol, dissolve in 100 mL of 1% acetic acid solution, and stir magnetically until completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir thoroughly to dissolve, and obtain a chitosan-PVA solution;

[0041] Slowly add 8g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol solution containing 2g of peptide Anmido (containing 2g of polyvinyl alcohol); magnetically stir at 800rpm at 65°C for 8h; ultrafiltration removes unencapsulated peptide Anmido, and lyophilizes the clarified liquid to obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Anmido.

[0042] Example 4

[0043] Weigh 6 g of carboxymethyl chitosan and 3 g of polyvinyl alcohol, dissolve them in 100 mL of 2% acetic acid solution, and stir them magnetically until they are completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir and dissolve them thoroughly to obtain a chitosan-PVA solution;

[0044] Slowly add 8g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol solution containing 3g of peptide Anmido (containing 3g of polyvinyl alcohol); magnetically stir at 800rpm at 65°C for 8h; ultrafiltration removes unencapsulated peptide Anmido, and lyophilizes the clarified liquid to obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Anmido.

[0045] Example 5

[0046] Weigh 6 g of carboxymethyl chitosan and 3 g of polyvinyl alcohol, dissolve in 100 mL of 1% acetic acid solution, and stir magnetically until completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir thoroughly to dissolve, and obtain a chitosan-PVA solution;

[0047] Slowly add 8g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol solution containing 4g of peptide Anmido (containing 4g of polyvinyl alcohol); magnetically stir at 800rpm at 65°C for 8h; ultrafiltration removes unencapsulated peptide Anmido, and after freeze-drying the clarified liquid, obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Anmido.

[0048] Example 6

[0049] Weigh 4 g of carboxymethyl chitosan and 5 g of polyvinyl alcohol, dissolve them in 100 mL of 3% acetic acid solution, and stir them magnetically until they are completely dissolved to form a stable mixed solution; then slowly add 200 mg of calcium chloride, stir and dissolve them thoroughly to obtain a chitosan-PVA solution;

[0050] Slowly add 8g of cyclodextrin and stir thoroughly to dissolve; add polyvinyl alcohol solution (containing 5g of polyvinyl alcohol) containing 4g of peptide Anmido; magnetically stir at 800rpm at 65°C for 8h; ultrafiltration removes unencapsulated peptide Anmido, and lyophilizes the clarified liquid to obtain cyclodextrin-PVA-chitosan nanoparticles loaded with peptide Anmido.

[0051] Example 7

[0052] 1. Draw the standard curve of peptide Anmido:

[0053] Peptide Anmido solutions with concentrations of 2, 10, 20, 50, and 100 ppm were prepared with acetonitrile solvent; the solutions were filtered with a 0.22 μm membrane and then subjected to HPLC-DAD detection, and a standard curve was drawn.

[0054] Chromatographic conditions: Waters-DAD system was used, the chromatographic column was VenusilASB-C18 (250×4.6mm; id, 5μm), the injection volume was 10μL, and the measurement was at 215nm; the column temperature was 35℃; the mobile phase was acetonitrile; isocratic elution was adopted, and the total time was 30min.

[0055] Figure 1This is the standard curve of peptide Anmido obtained by high performance liquid chromatography in this example. When the concentration of peptide Anmido is in the range of 2 to 100 ppm, the working curve shows a good linear relationship. The equation of the standard curve is:

[0056] y=110.0530x-211.0504, R 2 =0.99922.

[0057] In the above formula, y is the corresponding peak area measured at the maximum absorption wavelength, and x is the concentration of peptide Anmido (ppm).

[0058] 2. Test the embedding efficiency of nanoparticles

[0059] 10 mg (m0) of the nanoparticles encapsulating the peptide Anmido prepared in the above example was dissolved in ethanol, and the peptide Anmido encapsulated in the nanoparticles was replaced and released by ethanol. Then, the weight (m1) of the peptide Anmido in the solution was determined by HPLC according to the standard curve, and the peptide Anmido encapsulation rate was calculated according to the following formula. The specific results are shown in Table 1.

[0060] Embedding rate (%) = (m1 / m0) * 100%

[0061] Table 1

[0062] <![CDATA[m1(mg)]]> Embedding rate Example 1 3.862 38.62％ Example 2 4.325 43.25％ Example 3 6.461 64.61％ Example 4 6.189 61.89％ Example 5 5.862 58.62％ Example 6 6.073 60.73％

[0063] Example 8 Solubility Test

[0064] 5 g of the nanoparticles encapsulating the peptide Anmido obtained in Examples 1 to 5 were dispersed in 100 mL of water at room temperature and stirred. The dissolution was observed after standing. The test results are shown in Table 2.

[0065] Table 2

[0066] sample Characteristics Solubility Example 1 White granules or powder Partially dissolved Example 2 White granules or powder Completely dissolved Example 3 White granules or powder Completely dissolved Example 4 White granules or powder Completely dissolved Example 5 White granules or powder Completely dissolved Example 6 White granules or powder Completely dissolved

[0067] Example 9 In vitro whitening efficacy test

[0068] Using PBS buffer solution as solvent, the substrate L-DOPA was prepared into a 1 mg / mL reaction solution (L-DOPA solution); tyrosinase was prepared into an enzyme solution with an activity of 100 U / mL for later use;

[0069] The experiment was divided into a reaction group, a reaction control group, a blank group and a blank control group:

[0070] (1) 25 μL of 1000 ppm aqueous dispersion of nanoparticles encapsulating peptide Anmido was added to the reaction group and the reaction control group respectively; an equal volume of PBS buffer solution was added to the blank group and the blank control group;

[0071] (2) Add 25 μL of the prepared tyrosinase solution to the reaction group and blank group respectively, and add an equal volume of PBS buffer solution to the reaction control group and blank control group, mix well, and incubate in a 37°C incubator for 5 min;

[0072] (3) All experimental groups were added with 100 μL of the prepared L-DOPA solution and incubated in a 37°C incubator for 5 min. The absorbance (A) value was measured at a wavelength of 475 nm. The above experiment was repeated 3 times, and the tyrosinase inhibition rate was calculated according to the following formula:

[0073] Inhibition rate = 1-(A 反应组 -A 反应对照组 ) / (A 空自组 -A 空自对照组 )

[0074] The tyrosinase inhibition rate measured in this example is as follows Figure 2 As shown in the figure, it can be seen that the inhibition rate of tyrosinase is positively correlated with the embedding effect of each embodiment. Among them, Example 4 has the highest inhibition rate of tyrosinase and the best whitening effect.

[0075] Example 10 Release Test

[0076] Prepare 100 mL of PBS buffer (pH 7.4) as a release medium; at 37° C., take 1 g of the nanoparticles encapsulating peptide Anmido prepared in Example 3 and disperse them in the release medium; set different time points of 1 h, 2 h, 4 h, 8 h, 24 h, and 48 h for sampling, and use high performance liquid chromatography to determine the concentration of peptide Anmido in the buffer at each time point. The measurement results are as follows: Figure 3 As shown in the figure, it can be seen that the concentration of peptide amine in the buffer solution increases with time, indicating that the nanoparticles embedded with peptide amine prepared by the present invention have a sustained release function at 37°C and pH 7.4, and can continuously release the active ingredient.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing nanoparticles encapsulating peptide Anmido, characterized in that: The steps include: Cyclodextrin, chitosan, polyvinyl alcohol and peptide anmido are subjected to cross-linking reaction in a solution added with metal ion salt to obtain the nanoparticles embedded with the peptide anmido.

2. The preparation method according to claim 1, characterized in that: The metal ion salt is calcium chloride; And / or, the cyclodextrin is β-cyclodextrin; And / or, the chitosan is carboxymethyl chitosan.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the cyclodextrin, chitosan, polyvinyl alcohol and peptide anmito is 4-8:3-6:1-10:1-4.

4. The preparation method according to claim 1, characterized in that: The mass of the metal ion salt is 3% to 10% of the mass of the chitosan.

5. The preparation method according to claim 1, characterized in that: The cross-linking reaction time is 2 to 12 hours, preferably 8 hours; Preferably, the temperature of the cross-linking reaction is 20-80°C, more preferably 65°C.

6. The preparation method according to claim 1, characterized in that: The solution to which the metal ion salt is added is an acidic solution; the pH of the solution to which the metal ion salt is added is adjusted to be acidic by adding acetic acid.

7. The preparation method according to claim 1, characterized in that: The cross-linking reaction is carried out under stirring conditions; the cross-linking reaction also includes post-processing of centrifugation or filtration and freeze-drying.

8. The preparation method according to claim 1, characterized in that: The preparation method specifically comprises the following steps: S1: dissolving chitosan and polyvinyl alcohol in an acetic acid aqueous solution and stirring evenly; adding metal ion salt; Alternatively, dissolve chitosan in an acetic acid aqueous solution and stir evenly; add a metal ion salt; S2: Add cyclodextrin, peptide anmido and polyvinyl alcohol; carry out cross-linking reaction.

9. The preparation method according to claim 8, characterized in that: In step S1, the amount of chitosan and acetic acid aqueous solution is as follows: 3-6 g of chitosan is dissolved in 100 mL of acetic acid aqueous solution with a mass fraction of 1% to 3%.

10. Nanoparticles encapsulating the peptide Anmido obtained by the preparation method according to any one of claims 1 to 9.

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