Modified deacetylated chitosan composition as well as preparation method and application thereof

Abstract

The invention discloses a modified deacetylated chitosan composition as well as a preparation method and application thereof. The modified deacetylated chitosan composition comprises a modified deacetylated chitosan carrier and an active matter, the modified deacetylated chitosan carrier comprises deacetylated chitosan of which the surface is modified with amino acid and / or hydroxyethyl; the active matter carries negative charge groups, and the negative charge groups comprise at least one of carboxyl, sulfo, phosphate and sulfydryl; the mass ratio of the modified deacetylated chitosan carrier to the active matter is 1: (0.1-100). The modified deacetylated chitosan composition provided by the invention can enter the interior of the skin through pores of the skin by utilizing the specific physicochemical properties of the nanoparticles, achieves a good transdermal effect under the condition of not damaging the skin structure, and is natural, safe and high in transdermal efficiency.

Description

Technical Field

[0001] The invention belongs to the technical field of nanomaterials, and in particular relates to a modified deacetylated chitosan composition and a preparation method and application thereof. Background Art

[0002] Nanoparticles of polymer matrix can be used as carriers for active substances. The size can range from micrometers to nanometers. They can be applied to biomedicine, daily chemicals, food and other technical fields. Deacetylated chitosan is obtained by deacetylation of natural chitin. It has good biocompatibility and has been widely used in the pharmaceutical and medical fields. Many hydrophilic molecules, such as polyethylene glycol, trimethylammonium, phosphoryl, sulfonyl, etc., have been used to develop amphiphilic chitosan derivatives, and further form self-assembled nano-chitosan.

[0003] In recent years, proteins and peptides have been widely used in cosmetics. Some proteins have good moisturizing properties, can absorb and lock water, form a moisturizing film, and improve the moistness and softness of the formula; in addition, many proteins have antioxidant properties, can neutralize free radicals, reduce oxidative damage, and delay skin aging, and are widely used in anti-aging cosmetics. In addition, proteins can promote the repair and regeneration of skin cells, accelerate wound healing, improve skin texture, reduce wrinkles and fine lines, and make the skin firmer and younger.

[0004] Polypeptides, also known as peptides, are a class of compounds with a molecular structure between amino acids and proteins. They are composed of one or more amino acids bonded by peptide bonds in a certain order. Usually, peptides composed of 2 to 10 amino acids are called oligopeptides (small molecule peptides); peptides composed of 11 to 50 amino acids are called polypeptides; and peptides composed of more than 50 amino acids are called proteins. Polypeptides have a high homology with body substances, so they are more compatible with the skin. For example, acetyl hexapeptide-8 has good antioxidant properties, can scavenge free radicals, promote collagen nuclear magnetic resonance, and improve skin elasticity and gloss. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a modified deacetylated chitosan composition, which can utilize the unique physical and chemical properties of nanoparticles to enter the skin through the pores of the skin, achieve a good transdermal effect without destroying the skin structure, and is natural, safe, and has high transdermal efficiency.

[0006] The invention also provides a method for preparing the modified deacetylated chitosan composition.

[0007] The invention also provides the application of the preparation method.

[0008] The invention also provides a skin care product.

[0009] According to a first aspect of the present invention, a modified deacetylated chitosan composition is provided, wherein the modified deacetylated chitosan composition comprises a modified deacetylated chitosan carrier and an active substance;

[0010] The modified deacetylated chitosan carrier comprises deacetylated chitosan with positively charged groups modified on the surface;

[0011] The active substance carries a negatively charged group, and the negatively charged group includes at least one of a carboxyl group, a sulfonyl group, a phosphate group, and a thiol group;

[0012] The mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(0.1-100).

[0013] In some embodiments of the present invention, the deacetylated chitosan with positively charged groups on the surface includes deacetylated chitosan with amino acids and / or hydroxyethyl groups on the surface.

[0014] In some preferred embodiments of the present invention, the molar ratio of the amino acid to deacetylated chitosan is 1:(1-10).

[0015] In some preferred embodiments of the present invention, the molar ratio of the hydroxyethyl group to the deacetylated chitosan is 3:(1-30).

[0016] In some embodiments of the present invention, the mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(0.3-20).

[0017] In some preferred embodiments of the present invention, the mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(3-15).

[0018] In some embodiments of the present invention, the active substance includes at least one of collagen, fibronectin, elastin, acetyl hexapeptide-8, snake venom peptide, acetyl tetrapeptide-9 and palmitoyl tripeptide-5.

[0019] In some preferred embodiments of the present invention, the active ingredients include collagen, acetyl hexapeptide-8 and snake venom peptide.

[0020] In some embodiments of the invention, the amino acids include at least one of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, histidine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and arginine.

[0021] In some preferred embodiments of the present invention,

[0022] In some embodiments of the present invention, the modified deacetylated chitosan carrier is amphiphilic and positively charged.

[0023] In some embodiments of the present invention, the modified deacetylated chitosan carrier is prepared by a preparation method comprising the following steps:

[0024] A1: dissolving chitosan in acetic acid solution, adding an oxidant under ultrasound assistance to carry out oxidation reaction, adjusting the pH of the reaction system to 10-12 after the reaction, collecting the precipitate, and obtaining deacetylated chitosan;

[0025] A2: adding ethylene oxide to the deacetylated chitosan obtained in step A1 to react and obtain hydroxyethyl-modified deacetylated chitosan substance A;

[0026] A3: Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to the amino acid solution to react and obtain a transition state ester;

[0027] A4: Add the substance A obtained in step A2 to the transition state ester obtained in step A3, and react to obtain a modified deacetylated chitosan carrier.

[0028] In some preferred embodiments of the present invention, the final concentration of chitosan in the acetic acid solution in step A1 is 5-15 g / L.

[0029] In some more preferred embodiments of the present invention, the final concentration of chitosan in the acetic acid solution in step A1 is 10 g / L.

[0030] In some preferred embodiments of the present invention, the concentration of the acetic acid solution in step A1 is 8 g acetic acid per 100 mL.

[0031] In some preferred embodiments of the present invention, the oxidant in step A1 comprises hydrogen peroxide.

[0032] In some preferred embodiments of the present invention, the final concentration of the oxidant in the reaction system in step A1 is 10 to 20 mmol / L.

[0033] In some preferred embodiments of the present invention, the reaction temperature of the oxidation reaction in step A1 is 50-70°C.

[0034] In some more preferred embodiments of the present invention, the reaction temperature of the oxidation reaction in step A1 is 60°C.

[0035] In some preferred embodiments of the present invention, the oxidation reaction time in step A1 is 3 to 7 hours.

[0036] In some more preferred embodiments of the present invention, the oxidation reaction time in step A1 is 5 hours.

[0037] In some preferred embodiments of the present invention, the reaction temperature in step A2 is 50-70°C.

[0038] In some more preferred embodiments of the present invention, the reaction temperature in step A2 is 60°C.

[0039] In some preferred embodiments of the present invention, the volume mass ratio of the ethylene oxide in step A2 to the chitosan in step A1 is 10:(1-2) mL / g.

[0040] In some preferred embodiments of the present invention, the reaction time in step A2 is 5 to 7 hours.

[0041] In some more preferred embodiments of the present invention, the reaction time in step A2 is 6 hours.

[0042] In some preferred embodiments of the present invention, the reaction in step A2 is carried out under stirring conditions at a stirring speed of 1200 to 1500 rpm.

[0043] In some preferred embodiments of the present invention, the concentration of the amino acid solution in step A3 is 0.5-2 mol / L.

[0044] In some more preferred embodiments of the present invention, the concentration of the amino acid solution in step A3 is 1 mol / L.

[0045] In some preferred embodiments of the present invention, the solvent of the amino acid solution in step A3 is phosphate buffered saline (PBS).

[0046] In some preferred embodiments of the present invention, the final concentration of EDC in the reaction system in step A3 is 0.5-2 mol / L.

[0047] In some more preferred embodiments of the present invention, the final concentration of EDC in the reaction system in step A3 is 1 mol / L.

[0048] In some preferred embodiments of the present invention, the final concentration of NHS in the reaction system in step A3 is 0.5-2 mol / L.

[0049] In some more preferred embodiments of the present invention, the final concentration of NHS in the reaction system in step A3 is 1 mol / L.

[0050] In some preferred embodiments of the present invention, the reaction temperature in step A3 is 2-6°C.

[0051] In some more preferred embodiments of the present invention, the reaction temperature in step A3 is 4°C.

[0052] In some preferred embodiments of the present invention, the reaction time in step A3 is 2 to 6 hours.

[0053] In some more preferred embodiments of the present invention, the reaction time in step A3 is 4 hours.

[0054] In some preferred embodiments of the present invention, the reaction temperature in step A4 is 18-28°C.

[0055] In some preferred embodiments of the present invention, the reaction time in step A4 is 0.5 to 2 hours.

[0056] In some more preferred embodiments of the present invention, the reaction time in step A4 is 1 h.

[0057] In some preferred embodiments of the present invention, the step A4 further comprises dialysis separation and freeze drying after the reaction is completed.

[0058] In some embodiments of the present invention, the average particle size of the modified deacetylated chitosan composition is 100-300 nm.

[0059] According to a second aspect of the present invention, a method for preparing a modified deacetylated chitosan composition is provided, the preparation method comprising the following steps:

[0060] S1: Under a protective gas environment, the modified deacetylated chitosan carrier and the active substance are mixed in a solvent to obtain a mixed solution;

[0061] S2: Stir the mixed solution obtained in step S1 at a speed of 500 to 2500 rpm for 1 to 6 hours to obtain a modified deacetylated chitosan composition.

[0062] In some embodiments of the present invention, the protective gas in step S1 includes nitrogen or argon.

[0063] In some embodiments of the present invention, the mass ratio of the modified deacetylated chitosan carrier to the active substance in step S1 is 1:(0.1-100).

[0064] In some preferred embodiments of the present invention, the mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(0.3-20).

[0065] In some more preferred embodiments of the present invention, the mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(3-15).

[0066] In some embodiments of the present invention, the solvent in step S1 includes any one of phosphate buffer, citric acid / sodium citrate buffer, sodium hydrogen phosphate / disodium hydrogen phosphate buffer and acetic acid / sodium acetate buffer.

[0067] In some embodiments of the present invention, the final concentration of the modified deacetylated chitosan carrier in the solvent in step S1 is 3-5 g / L.

[0068] In some preferred embodiments of the present invention, the final concentration of the modified deacetylated chitosan carrier in the solvent in step S1 is 4 g / L.

[0069] In some embodiments of the present invention, the stirring speed in step S2 is 1000-2000 rpm.

[0070] In some embodiments of the present invention, the stirring time in step S2 is 2 to 4 hours.

[0071] In some embodiments of the present invention, the step S2 further comprises performing ultracentrifugation and collecting the precipitate after the stirring is completed.

[0072] In some preferred embodiments of the present invention, the speed of the ultracentrifugation is 55000-60000 rpm.

[0073] In some more preferred embodiments of the present invention, the speed of the ultracentrifugation is 58000 rpm.

[0074] In some preferred embodiments of the present invention, the ultracentrifugation time is 20 to 40 minutes.

[0075] In some more preferred embodiments of the present invention, the ultracentrifugation time is 30 min.

[0076] According to the third aspect of the present invention, the use of the preparation method according to the second aspect of the present invention in the preparation of a transdermal composition is proposed.

[0077] In some embodiments of the present invention, the active ingredient in the transdermal composition comprises an antioxidant.

[0078] In some preferred embodiments of the present invention, the antioxidant comprises a polypeptide.

[0079] In some embodiments of the present invention, the transdermal composition can be used to prepare skin care products and medicines.

[0080] According to a fourth aspect of the present invention, a product is provided, wherein the product comprises the modified deacetylated chitosan composition according to the first aspect of the present invention.

[0081] In some embodiments of the present invention, the product comprises a skin care product.

[0082] In some preferred embodiments of the present invention, the skin care product includes at least one of a facial cleanser, a toner, a skin care lotion, a facial mask, a facial cream, a foundation, a primer or a sunscreen.

[0083] The present invention has at least the following beneficial effects:

[0084] 1) The modified chitosan carrier used in the modified chitosan composition provided by the present invention is modified with hydroxyethyl and amino acid, has amphiphilicity and positive charge, has high compatibility with organisms, is easily biodegradable, and is environmentally friendly;

[0085] 2) In the modified deacetylated chitosan composition provided by the present invention, the modified deacetylated chitosan carrier can form a self-assembled nanostructure with an active substance (polypeptide) with a negatively charged group through the action of positive and negative charges. The average particle size after assembly is about 100 to 300 nm, and large molecules and poorly permeable protein / polypeptide active substances can be delivered to the subcutaneous tissue to exert their effects;

[0086] 3) The modified deacetylated chitosan composition provided by the present invention utilizes the unique physical and chemical properties of nanoparticles to enter the skin through the pores of the skin, achieving an excellent transdermal effect without destroying the skin structure. It is natural, safe, and has a high transdermal efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0088] Figure 1 This is the infrared spectrum result diagram of the modified deacetylated chitosan carrier prepared in Example 1 of the present invention;

[0089] Figure 2 This is the result of H NMR spectrum of the modified deacetylated chitosan carrier prepared in Example 1 of the present invention;

[0090] Figure 3 This is a graph showing the Zeta potential results of the modified deacetylated chitosan carrier prepared in Example 1 of the present invention;

[0091] Figure 4 This is a graph showing the Zeta potential results of the active ingredient mixture (10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide) used in Example 1 of the present invention;

[0092] Figure 5 This is a graph showing the Zeta potential result of the modified deacetylated chitosan composition prepared in Example 1 of the present invention;

[0093] Figure 6 This is a test result diagram of the skin permeability of the modified deacetylated chitosan composition provided in Example 1 in the test example of the present invention; wherein FITC is a fluorescent dye for nano inclusions, DAPI is a cell nucleus stain, Merge is a fusion of the two, and the scale bar is 1 μm. DETAILED DESCRIPTION

[0094] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0095] Example 1

[0096] This embodiment provides a modified deacetylated chitosan composition, and the preparation method of the modified deacetylated chitosan composition specifically comprises the following steps:

[0097] 1) Preparation of modified deacetylated chitosan carrier:

[0098] ① Dissolve 1.5 g of chitosan in 150 mL of acetic acid solution (8 g of acetic acid per 100 mL), and carry out mixed oxidation reaction with 2 mL of oxidant hydrogen peroxide aqueous solution (10-20 mM) at 60 ° C for 5 h; after the reaction is completed, adjust the pH of the reaction system to 11 (10-12 is acceptable), and collect the precipitate, i.e., deacetylated chitosan;

[0099] ② Add the deacetylated chitosan obtained in step ① into 10 mL of ethylene oxide, stir at 60° C. for 6 h at a stirring speed of 1300 rpm (1200 to 1500 rpm), to obtain a hydroxyethyl-modified deacetylated chitosan derivative A;

[0100] ③ Dissolve arginine in PBS solution in advance to obtain a 1 mol / L arginine solution; add 1 mol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 1 mol / L N-hydroxysuccinimide (NHS), the mixed volume ratio of arginine solution, NHS solution and EDC solution is 1:1:2, react at 4°C for 4 hours to obtain a transition state ester;

[0101] ④ Add the derivative A obtained in step ② to the transition state ester obtained in step ③ at a final concentration of 1 mol / L and react at room temperature for 1 hour. The modified deacetylated chitosan carrier is obtained by dialysis separation and freeze drying. The dialysate used in the dialysis is PBS buffer, and the molecular weight cutoff of the semipermeable membrane used in the dialysis is about 1000Da. The infrared spectrum test results of the modified deacetylated chitosan carrier are as follows: Figure 1 The results of the H NMR spectrum of the modified deacetylated chitosan composition are shown in Figure 2 As shown; the above freeze drying is performed at -80°C for 7h (6-8h is acceptable);

[0102] NMR results: δ8.19(s,1H),6.14-6.19(m,1H),5.09(s,1H),4.81(s,3H),4.58(s,5H),4.34(s,3H),3.61-3.77(m,7H),3.54–3.51(m,4H),3.48(s,7H),3.42(d,J=5.8Hz,7H),3.39(s,15H),3.13-3.22(m,2H),2.81(s,5H),2.39–2.35(m,2H),2.24-2.30(t,1H).

[0103] 2) Weigh 10 mg of the modified chitosan carrier obtained in step 1) and dissolve it in 2.5 mL PBS (pH = 7.4). Under nitrogen conditions, add 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide to the above modified chitosan carrier solution, and stir at a stirring speed of 1500 rpm for 2 hours; then collect the precipitate by ultracentrifugation (58000 rpm ultracentrifugation for 30 minutes, control the temperature at 4°C), wash the precipitate with PBS for 3 times, and freeze-dry the precipitate to obtain a modified chitosan composition. The above polypeptide is encapsulated in the modified chitosan carrier through the interaction between positive and negative charges.

[0104] Example 2

[0105] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide are replaced with 1 mg of type III human collagen, 1 mg of acetyl hexapeptide-8 and 1 mg of snake venom peptide, and the remaining steps are the same.

[0106] Example 3

[0107] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide are replaced with 50 mg of type III human collagen, 50 mg of acetyl hexapeptide-8 and 50 mg of snake venom peptide, and the remaining steps are the same.

[0108] Example 4

[0109] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide are replaced with 30 mg of type III human collagen, 30 mg of acetyl hexapeptide-8 and 40 mg of snake venom peptide, and the remaining steps are the same.

[0110] Example 5

[0111] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide are replaced with 6 mg of type III human collagen, 6 mg of acetyl hexapeptide-8 and 8 mg of snake venom peptide, and the remaining steps are the same.

[0112] Example 6

[0113] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that 10 mg of type III human collagen, 10 mg of acetyl hexapeptide-8 and 10 mg of snake venom peptide are replaced with 20 mg of type III human collagen, 20 mg of acetyl hexapeptide-8 and 20 mg of snake venom peptide, and the remaining steps are the same.

[0114] Example 7

[0115] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of embodiment 1 only in that the stirring speed in step 2) is adjusted to 500 rpm and the stirring time is adjusted to 1 h, and the other steps are the same.

[0116] Example 8

[0117] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of embodiment 1 only in that the stirring speed in step 2) is adjusted to 2500 rpm and the stirring time is adjusted to 6 h, and the other steps are the same.

[0118] Example 9

[0119] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of embodiment 1 only in that the stirring speed in step 2) is adjusted to 2500 rpm, and the remaining steps are the same.

[0120] Example 10

[0121] This embodiment provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of embodiment 1 only in that the stirring time in step 2) is adjusted to 6 hours, and the other steps are the same.

[0122] Comparative Example 1

[0123] This comparative example provides a modified deacetylated chitosan composition. The preparation method of the modified deacetylated chitosan composition is different from that of Example 1 only in that the stirring operation in step 2) is adjusted to stirring at a speed of 1500 rpm until dissolved and then ultracentrifugation is immediately performed to collect the precipitate. The stirring time is less than 1 hour, and the remaining steps are the same.

[0124] Comparative Example 2

[0125] This comparative example provides a modified chitosan composition. The amounts of the modified chitosan carrier and the active substance in the modified chitosan composition are the same as those in Example 1. However, the preparation method is the same as that described in Example 3 of Chinese patent document CN115778844A.

[0126] Test example

[0127] This test example tests the performance of the modified deacetylated chitosan composition and liposome encapsulation provided by Examples 1 to 10 and Comparative Examples 1 to 2, including the average particle size, skin permeability, in vitro antioxidant properties and effects on collagen-related genes of various samples; the specific test methods and test results are as follows:

[0128] 1. Zeta potential detection:

[0129] In this test example, the Zeta potential of the modified deacetylated chitosan carrier prepared in Example 1, the active substance mixture before being encapsulated in the carrier, and the modified deacetylated chitosan composition obtained after encapsulation were measured. The results are as follows: Figure 3 to Figure 5 shown.

[0130] Depend on Figure 3 to Figure 5It can be seen that the Zeta potential of the modified deacetylated chitosan carrier prepared in Example 1 is 33.2 mV, the Zeta potential of the active substance mixture before being encapsulated into the carrier is -30.1 mV, and the Zeta potential of the modified deacetylated chitosan composition obtained after encapsulation is 27.5 mV; after encapsulation, the Zeta potential of the composition migrates, indicating that the modified deacetylated chitosan composition prepared by the present invention successfully encapsulates the active substance.

[0131] 2. Average particle size test:

[0132] This test example adopts wet dispersion technology, mechanically stirs at a speed of 1500-2000rpm to evenly disperse the sample, and ultrasonic high-frequency oscillation at 750kW power, 80%-100% amplitude and 50kHz-1MHz frequency is used to fully disperse the agglomerated particles. The electromagnetic circulation pump makes the large and small particles evenly distributed in the entire circulation system. The computer program is operated according to the Malvern particle size analyzer instruction manual to test the particle size curve distribution of each group of package samples and obtain the average particle size. The results are shown in Table 1. Among them, the average particle size of the modified deacetylated chitosan carrier itself is about 218.10nm.

[0133] Table 1 Average particle size of each group of inclusion samples

[0134]

[0135] As can be seen from Table 1, the average particle sizes of Examples 2, 7 and 8 are smaller than those of Examples 1 and 3, indicating that the encapsulation amounts of Examples 2, 7 and 8 are not large; the average particle sizes of Examples 1 and 3 are close, indicating that increasing the amount of active substance in Example 3 cannot enhance the encapsulation amount; Comparative Example 1 is a simple physical mixing, and the active substance is basically not encapsulated into the carrier, so there is almost no difference in particle size before and after encapsulation.

[0136] 3. Skin permeability test:

[0137] It is common to use animal skin as test material in transdermal absorption studies. In this experiment, Kunming mouse skin was selected as the transdermal absorption research model.

[0138] The skin sample was taken from the back of the mouse; approximately 1 cm 2 The test area was evenly smeared with 0.5 mL of the sample with FITC fluorescent agent, and the stratum corneum cells were embedded, sliced, and stained with DAPI at 2h and 8h after smearing. The morphology of the slices was observed under a confocal microscope, and the penetration rate was calculated (penetration rate = subcutaneous fluorescence penetration / 0.5 mL total sample volume * 100%). The results are shown in Table 2;

[0139] The preparation method of the above sample with FITC fluorescent agent (Marsshall method) is as follows:

[0140] (1) Materials: modified deacetylated chitosan compositions obtained in Examples 1 to 10 and Comparative Examples 1 to 2, 0.5 mol / L (pH 9.0) carbonate buffer, sterile physiological saline, fluorescein isothiocyanate, 1% thimerosal aqueous solution, 50 mL beaker, 4° C. refrigerator, electromagnetic stirrer, dialysis bag, glass rod, 0.01 mol / L PBS buffer at pH 7.2 or 3.0.

[0141] (2) Methods and steps:

[0142] ① Preparation of sample solution: Take the modified deacetylated chitosan composition provided in each embodiment and comparative example in a beaker, add physiological saline and carbonate buffer to make the final concentration of each group of samples 20 mg / mL, the carbonate buffer volume is 1 / 10 of the total amount, mix well, place the flask on an electromagnetic stirrer (at a speed suitable for no foaming) for 5 to 10 minutes to obtain a sample solution.

[0143] ② Preparation of fluorescein: According to the total amount of sample to be labeled, add 0.01 mg of fluorescent pigment per mg of sample and use an analytical balance to accurately weigh the required fluorescein isothiocyanate powder.

[0144] ③ Binding (or labeling): Gradually add the weighed fluorescent dye to the sample solution while stirring to avoid the fluorescent dye sticking to the wall of the flask (add it within about 5 to 10 minutes). After adding, continue stirring in the dark for about 12 hours. During the binding period, the mixed system should be kept at about 4°C, so the beaker and stirrer need to be moved to a 4°C refrigerator.

[0145] ④ Dialysis: After binding, centrifuge the labeled sample solution (2500r / min) for 20min to remove a small amount of precipitate, put it into a dialysis bag, and then place it in a beaker and dialyze it with pH 8.0 buffered saline (0-4°C) overnight.

[0146] ⑤ Column: Take the marker that has been dialyzed overnight and pass it through a Sephadex G-25 or G-50 column to separate the free fluorescein and collect the fluorescently labeled samples for testing; the eluent used is 0.01 mol / L phosphate buffer (pH 7.2).

[0147] The penetration effect of the modified deacetylated chitosan composition sample provided in Example 1 after 2h and 8h of penetration is as follows: Figure 6 shown.

[0148] Table 2 Permeability of each group of wrapping samples

[0149]

[0150] According to Table 2, at 2h and 8h of penetration, the percutaneous permeability of Example 1 is the highest among all groups, which are 10.23% and 38.41%, respectively, followed by Example 3 and Example 8; the lower concentration of active substance in Example 2, the lower stirring speed and reaction time in Example 7 may be the main factors affecting the encapsulation and permeability; Comparative Example 1 is a simple physical mixing, and the active substance is almost not encapsulated, so it cannot penetrate the skin; it can be seen that the permeability of the sample in Example 1 in mouse skin is the best. The modified deacetylated chitosan composition provided by the present invention has good skin permeability and can be used to deliver a variety of active substances that are difficult to penetrate the skin.

[0151] 4. In vitro antioxidant capacity test:

[0152] This test example uses the DPPH method to determine the antioxidant activity of the sample. DPPH, also known as 1,1-diphenyl-2-trinitrophenylhydrazine, is a very stable nitrogen-centered free radical. Its stability mainly comes from the steric hindrance of the three benzene rings of resonance stabilization, which prevents the unpaired electrons on the nitrogen atom in the middle from playing their due electron pair role. The anhydrous ethanol solution of DPPH is purple, with maximum absorption at a wavelength of 517nm, and a linear relationship between absorbance and concentration; when a free radical scavenger is added to the DPPH solution, it can combine with or replace the DPPH free radical, so that the number of free radicals is reduced, the absorbance is reduced, and the color of the solution becomes lighter, thereby evaluating the ability to scavenge free radicals, that is, the effect of the sample in scavenging DPPH free radicals is obtained by the absorbance at a wavelength of 517nm, which is further used to evaluate the antioxidant capacity.

[0153] 1) Test equipment and materials:

[0154] The required materials are DPPH, deionized water, and anhydrous ethanol; the required instrument is a spectrophotometer.

[0155] 2) Test method:

[0156] ① Preparation of DPPH stock solution:

[0157] Accurately weigh 3.5 mg of DPPH, dissolve it with anhydrous ethanol, transfer it to a 10 mL volumetric flask, make up to 10 mL with anhydrous ethanol, then take 2 mL of the solution from the 10 mL volumetric flask and transfer it to a 100 mL volumetric flask, make up to 100 mL with anhydrous ethanol, shake well to obtain a DPPH stock solution with a final concentration of 0.0178 mmol / L, and refrigerate it for later use.

[0158] ② Preparation of test solution:

[0159] The inclusions prepared in Examples 1 to 8 and Comparative Examples 1 to 2 were respectively dissolved in water to a final concentration of 5 wt % to obtain solutions to be tested.

[0160] ③Determination of DPPH free radical scavenging rate:

[0161] Add 4.0 mL of the DPPH stock solution obtained in step ① and 1 mL of the solution to be tested into a 10 mL colorimetric tube, mix well, and measure the absorbance (A) at a wavelength of 517 nm using a colorimetric dish. The absorbance is recorded as A. i Then, store it at room temperature and avoid light for 30 minutes, then measure its absorbance, which is recorded as A j The control group solution is a single DPPH ethanol solution, and its absorbance is A c The formula for calculating the free radical scavenging rate K is: K (%) = [1-(A i -A j ) / A c ]*100%; the experiment was repeated three times and the average value of K was taken. The results are shown in Table 3.

[0162] Table 3 DPPH free radical scavenging rate of each group of package samples

[0163]

[0164]

[0165] Since the coated active substance has certain antioxidant and free radical scavenging functions, the DPPH free radical scavenging rate test can also indirectly reflect the coating condition of the inclusion body. As shown in Table 3, under the condition of 5wt% inclusion, the free radical scavenging rate of Example 1 is the highest at 68.5%, followed by Example 3 and Example 8, which are 59.21% and 55.40% respectively, and Example 2 and Example 4 are slightly lower; in the comparative example, the scavenging rate of Comparative Example 1 is very low, only 5.29%, indicating that the content of active substances in the encapsulated carrier is very small and almost negligible.

[0166] 5. Collagen-related gene mRNA expression test:

[0167] The modified deacetylated chitosan composition provided by the present invention is mainly applied to daily anti-aging skin care products, and the focus of efficacy research is mainly whether the package can affect collagen-related genes. COL1A1 is the type I α chain of collagen, which is the most abundant collagen in the human body and widely exists in human skin; MMP-1 (matrix metalloproteinase-1) is the most important enzyme for degrading type I and type III collagen. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, destroys the normal structure of collagen fibers and elastic fibers, and causes wrinkles and other aging manifestations on the skin; overexpression of MMP-9 (matrix metalloproteinase-9) will cause degradation of type IV collagen and destroy the basement membrane structure of the skin.

[0168] Therefore, this test example evaluates whether the modified deacetylated chitosan composition provided by the present invention can exert an anti-aging effect on the skin by detecting the mRNA expression of genes related to collagen synthesis and degradation.

[0169] 1) Primers used:

[0170] The COL1A1 upstream primer sequence was: 5′-GCTCCTCTTAGGGGCCACT-3′ (SEQ ID NO: 1), and the COL1A1 downstream primer sequence was 5′-ATTGGGGACCCTTAGGCCAT-3′ (SEQ ID NO: 2);

[0171] The sequence of the upstream primer of MMP-1 is 5′-GATTCGGGGAGAAGTGATGTTC-3′ (SEQ ID NO: 3), and the sequence of the downstream primer of MMP-1 is 5′-CTCCTTTGGCTTCCCTAGAACT-3′ (SEQ ID NO: 4);

[0172] The sequence of the upstream primer of MMP-9 is 5'-GCACCACCACAACATCACCTAT-3' (SEQ ID NO: 5), and the sequence of the downstream primer of MMP-9 is 5'-GATGACGAGTTGTGGTCCCTG-3' (SEQ ID NO: 6).

[0173] 2) Test method:

[0174] Human fibroblasts (NHDF) were cultured in vitro. The blank group was added with PBS buffer solution, and the experimental group was irradiated under UV conditions for 3h (300mJ / cm 2 , 2 to 4 hours), the experimental group was added with the inclusion solution of Examples 1 to 8 and Comparative Examples 1 to 2 to a final concentration of 250 μg / mL, incubated for 16 hours (12 to 24 hours), the cell pellets were collected and 1 mL of 10% (v / v) DMSO (the solvent is 1 mol / L NaOH solution) was added respectively, lysed at 80°C for 1 hour, the lysed liquid was collected, transferred to a 96-well plate, and the OD value at 470 nm was measured with a microplate reader.

[0175] In the collagen-related gene content determination experiment, after the experimental treatment was completed, 1 mL of Trizol was added to each group of cell samples, and the cells were lysed by vigorous shaking. The total RNA was extracted by phenol-chloroform extraction. The ReverTra AceqPCR RT Master Mix with gDNA Remover kit was used to reverse transcribe and synthesize cDNA. GreenRealtime PCR Master Mix kit was used to detect gene expression levels, and 2 -△△CT The relative expression of collagen-related genes was analyzed by the method (the gene expression of the blank non-radiation group was set to 1.0), and the results are shown in Table 4.

[0176] Table 4 Relative mRNA expression of fibroblasts after treatment with each group of wraps

[0177]

[0178] As shown in Table 4, after UV radiation, the relative expression of COL1A1 gene in the model group decreased to 0.5 (the blank non-radiation group was 1.0), and MMP-1 and MMP-2 increased to 1.5 and 3.5, respectively, and the UV radiation was effective; after adding 250 μg / mL concentration of each sample, Example 1 enhanced the mRNA expression of COL1A1 collagen gene to 1.5, and the expression of MMP-1 and MMP-9 genes decreased to 0.8 and 1.5, respectively; followed by Example 3 and Example 8, and the improvement rates of Examples 2 and 7 were relatively low; in the comparative examples, the difference between Comparative Example 1 and the model group UV radiation was small.

[0179] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A modified deacetylated chitosan composition, characterized in that: The modified deacetylated chitosan composition comprises a modified deacetylated chitosan carrier and an active substance; The modified deacetylated chitosan carrier comprises deacetylated chitosan with positively charged groups modified on the surface; The active substance carries a negatively charged group, and the negatively charged group includes at least one of a carboxyl group, a sulfonic acid group, a phosphoric acid group, and a thiol group; The mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(0.1-100).

2. The modified deacetylated chitosan composition according to claim 1, characterized in that The active substance includes at least one of collagen, fibronectin, elastin, acetyl hexapeptide-8, snake venom peptide, acetyl tetrapeptide-9 and palmitoyl tripeptide-5.

3. The modified deacetylated chitosan composition according to claim 1, characterized in that The deacetylated chitosan with positively charged groups on the surface includes deacetylated chitosan with amino acids and / or hydroxyethyl groups on the surface; Preferably, the amino acids include at least one of isoleucine, leucine, lysine, methionine, phenylalanine, threonine, valine, histidine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine and arginine.

4. A method for preparing a modified deacetylated chitosan composition, characterized in that: The preparation method comprises the following steps: S1: Under a protective gas environment, the modified deacetylated chitosan carrier and the active substance are mixed in a solvent to obtain a mixed solution; S2: Stir the mixed solution obtained in step S1 at a speed of 500 to 2500 rpm for 1 to 6 hours to obtain a modified deacetylated chitosan composition.

5. The preparation method according to claim 4, characterized in that: The protective gas in step S1 includes nitrogen or argon.

6. The preparation method according to claim 4, characterized in that: In step S1, the mass ratio of the modified deacetylated chitosan carrier to the active substance is 1:(0.1-100).

7. The preparation method according to claim 4, characterized in that: The solvent in step S1 includes any one of phosphate buffer, citric acid / sodium citrate buffer, sodium hydrogen phosphate / disodium hydrogen phosphate buffer and acetic acid / sodium acetate buffer.

8. The preparation method according to claim 4, characterized in that: The step S2 further comprises performing ultracentrifugation after the stirring is completed and collecting the precipitate.

9. Use of the preparation method according to any one of claims 4 to 8 in the preparation of a transdermal composition.

10. A product, characterized in that The product comprises the modified deacetylated chitosan composition as claimed in any one of claims 1 to 3.

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