Modified chitosan, transdermal penetration enhancer as well as preparation method and application of modified chitosan and transdermal penetration enhancer

Abstract

The invention relates to modified chitosan, a transdermal penetration enhancer as well as a preparation method and application of the transdermal penetration enhancer. The modified chitosan comprises a deacetylated chitosan main chain, the main chain is grafted with a # imgabs0 # group through an ether bond, and p is an integer between 0 and 200. The modified chitosan is cross-linked with sodium hyaluronate and then cross-linked with sodium hyaluronate again, nano sodium hyaluronate with the viscosity remarkably reduced can be obtained, and the nano sodium hyaluronate serving as a transdermal penetration enhancer can remarkably improve the skin permeability of hydroxyl pinacolone retinoate.

Description

Technical Field

[0001] The present invention relates to a modified chitosan, a transdermal penetration enhancer, and a preparation method and application thereof. Background Art

[0002] As the largest organ of the human body, the skin plays a barrier and protective role on the one hand, and on the other hand, it can also be used as a drug delivery method to avoid the first-pass effect of the liver and gastrointestinal inactivation that may occur during oral administration, attracting wide attention. However, due to the poor permeability of the stratum corneum of the skin, the bioavailability of most drugs absorbed through the skin is low, making it difficult to meet clinical applications. Therefore, how to improve the transdermal absorption of drugs is the key to skin drug delivery.

[0003] Currently, the main methods to improve the transdermal absorption of drugs are as follows: 1. Destroy the lipid ordered structure of the stratum corneum and expand the permeability between stratum corneum cells, such as azone, etc.; 2. Act on the proteins in keratinocytes to improve the permeability of intracellular channels, such as dimethyl sulfoxide, etc.; 3. Promote the distribution of drugs into the stratum corneum, such as propylene glycol or ethanol, etc. However, the above methods are all prone to damage the integrity of the stratum corneum of the skin, resulting in skin sensitivity.

[0004] Although the prior art reports a transdermal delivery system based on hydroxyethyl deacetylated chitosan linked with sodium hyaluronate (CN116574281A), it only detects the transdermal absorption of recombinant collagen and does not detect the transdermal absorption efficiency of other active substances; in addition, the structure does not contain a polyethylene glycol structure, so its biocompatibility is poor, resulting in a low transdermal absorption efficiency; Document CN117982372A uses carboxymethyl chitosan as a crosslinking agent to prepare crosslinked sodium hyaluronate. Due to the poor water solubility of carboxymethyl chitosan, when it reacts with sodium hyaluronate, the resulting product has a low crosslinking degree, resulting in a large particle size and a low transdermal absorption efficiency; in addition, the literature states that the obtained crosslinked sodium hyaluronate can penetrate into the skin through the dermis to achieve the effects of moisturizing and hydrating, but there is no evidence that the crosslinked hyaluronic acid enters the skin. Summary of the Invention

[0005] In order to promote the transdermal absorption efficiency of active substances, the present invention provides a modified chitosan, a transdermal penetration enhancer, and a preparation method and application thereof.

[0006] The present invention obtains a modified chitosan by grafting epoxyethyl groups with or without polyethylene glycol or epoxypropyl groups with or without polyethylene glycol onto chitosan deacetate. After crosslinking the modified chitosan with sodium hyaluronate, it can be used as a viscosity regulator to significantly reduce the viscosity of sodium hyaluronate. That is, after remixing the crosslinked modified chitosan - sodium hyaluronate with sodium hyaluronate, a nano - sodium hyaluronate with significantly reduced viscosity is obtained, which can effectively increase the water content of the skin, thereby causing the stratum corneum of the skin to undergo hydration, further increasing the skin permeability, and ultimately achieving the effect of bringing the active ingredient into the skin, increasing the amount of drug entering the skin, and improving the bioavailability of the active ingredient.

[0007] In a first aspect, the present invention provides a modified chitosan, which comprises a chitosan deacetate backbone and the backbone is grafted through an ether bond groups, where p is an integer between 0 and 200, such as 0, 1, 2, 3, 4, 5, 8, 10, 20, 25, 30, 50, 80, 100, 120, 150, 180, 200 or any value between them.

[0008] According to some embodiments of the present invention, p is an integer between 1 and 200. According to some embodiments of the present invention, p is an integer between 1 and 100. According to some embodiments of the present invention, p is an integer between 1 and 20. According to some embodiments of the present invention, p is an integer between 3 and 20.

[0009] According to some embodiments of the present invention, the modified chitosan comprises one or more of the structures shown in Formula 1, 2, 3, 4, 5 or 6 below:

[0010]

[0011] wherein, n and x are the same or different and are each independently selected from integers between 0 and 200; m is a positive integer.

[0012] In some embodiments, n and x are the same or different and are each independently selected from integers between 1 and 200. In some embodiments, n and x are the same or different and are each independently selected from integers between 1 and 100. In some embodiments, n and x are the same or different and are each independently selected from integers between 1 and 20. In some embodiments, n and x are the same or different and are each independently selected from integers between 3 and 20. In some embodiments, m is a positive integer between 20 and 40, such as 20, 25, 30, 35, 40 or any value between them.

[0013] In a second aspect, the present invention provides a method for preparing a modified chitosan as described in the first aspect of the present invention, which comprises the following steps:

[0014] React chitosan with an epoxidizing reagent to obtain the modified chitosan; wherein the epoxidizing reagent includes epichlorohydrin and / or polyethylene glycol chloride grafted with ethylene oxide.

[0015] In some embodiments, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 1 to 200, such as 1, 2, 3, 4, 5, 8, 10, 20, 25, 30, 50, 80, 100, 120, 150, 180, 200 or any value therebetween. In some embodiments, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 1 to 100. In some embodiments, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 1 to 20. In some examples, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 3 to 20. In some examples, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 3. In some examples, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 20.

[0016] In some embodiments, the average molecular weight Mw of the chitosan is 4000 to 5000.

[0017] In some embodiments, the reaction is carried out in a solvent, and the solvent includes amide solvents, such as N,N-dimethylformamide.

[0018] In some embodiments, the reaction is carried out in the presence of a first catalyst, and the first catalyst includes but is not limited to: one or more of alkali metal hydroxides and alkali metal carbonates. In some embodiments, the first catalyst includes one or more of sodium carbonate, cesium carbonate, potassium carbonate, sodium hydroxide, and potassium hydroxide.

[0019] In some embodiments, the molar ratio of the first catalyst to the epoxidizing reagent is 1:(1 - 3), such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3 or any value therebetween.

[0020] In some embodiments, the molar ratio of the chitosan to the epoxidizing reagent is 1:(0.1 - 1.0), such as 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1 or any value therebetween.

[0021] In some embodiments, the temperature of the reaction is 0 - 95°C, preferably 15 - 40°C.

[0022] In some embodiments, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours.

[0023] Third aspect, the present invention provides a cross-linked sodium hyaluronate, which comprises a first sodium hyaluronate cross-linked by a modified chitosan; wherein the modified chitosan comprises the modified chitosan described in the first aspect of the present invention or the modified chitosan prepared by the preparation method described in the second aspect of the present invention.

[0024] In some embodiments, the molecular weight of the first sodium hyaluronate is 800 Da - 1500 kDa, such as 800 Da, 1 kDa, 10 kDa, 30 kDa, 50 kDa, 80 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 800 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa or any value therebetween. In some embodiments, the molecular weight of the first sodium hyaluronate is 100 kDa - 1000 kDa. In some embodiments, the molecular weight of the first sodium hyaluronate is 200 kDa - 400 kDa.

[0025] In the present invention, the epoxy ethyl groups grafted on the modified chitosan are cross-linked with the hydroxyl groups of the first sodium hyaluronate after ring-opening.

[0026] In some embodiments, the molar ratio of the first sodium hyaluronate to the modified chitosan is 1:(0.1 - 1), such as 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1 or any value therebetween.

[0027] Fourth aspect, the present invention provides a preparation method of the cross-linked sodium hyaluronate as described in the third aspect of the present invention, which comprises the following steps:

[0028] Mix an aqueous solution of the modified chitosan with an aqueous solution of the first sodium hyaluronate and carry out a reaction to obtain the cross-linked sodium hyaluronate.

[0029] In some embodiments, each 100 mL of the aqueous solution of the modified chitosan contains 0.5 - 2 g, preferably 0.8 - 1.2 g, of the modified chitosan.

[0030] In some embodiments, each 100 mL of the aqueous solution of the first sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g, of the first sodium hyaluronate.

[0031] In some embodiments, the volume ratio of the aqueous solution of the modified chitosan to the aqueous solution of the first sodium hyaluronate is 1:(8 - 12).

[0032] In some embodiments, the reaction is carried out under the condition of pH 8.0 - 9.0.

[0033] In some embodiments, the temperature of the reaction is 0 - 80 °C, preferably 25 - 40 °C.

[0034] In some embodiments, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours.

[0035] In some embodiments, after the reaction, the pH is adjusted to 6.5 - 7.0.

[0036] In a fifth aspect, the present invention provides a transdermal penetration enhancer, which comprises a second sodium hyaluronate crosslinked by crosslinked sodium hyaluronate, and the crosslinked sodium hyaluronate comprises the crosslinked sodium hyaluronate described in the third aspect of the present invention or the crosslinked sodium hyaluronate obtained by the preparation method described in the fourth aspect.

[0037] The present invention discovers that by using the above crosslinked sodium hyaluronate as a crosslinking agent and crosslinking it with the second sodium hyaluronate again, the viscosity and transdermal effect of the second sodium hyaluronate can be significantly improved.

[0038] In some embodiments, the molecular weight of the second sodium hyaluronate is 800 Da - 1500 KDa, such as 800 Da, 1 KDa, 10 KDa, 30 KDa, 50 KDa, 80 KDa, 100 KDa, 200 KDa, 300 KDa, 400 KDa, 500 KDa, 800 KDa, 1000 KDa, 1100 KDa, 1200 KDa, 1300 KDa, 1400 KDa, 1500 KDa or any value therebetween. In some embodiments, the molecular weight of the second sodium hyaluronate is 100 KDa - 1500 KDa. In some embodiments, the molecular weight of the second sodium hyaluronate is 300 KDa - 1300 KDa. In some embodiments, the molecular weight of the second sodium hyaluronate is 1000 KDa - 1300 KDa.

[0039] In some embodiments, the dosage of the crosslinked sodium hyaluronate is 5 - 15% of the mass of the second sodium hyaluronate, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% or any value therebetween. In some embodiments, the dosage of the crosslinked sodium hyaluronate is 8 - 12% of the mass of the second sodium hyaluronate.

[0040] In a sixth aspect, the present invention provides a preparation method of the transdermal penetration enhancer described in the fifth aspect, which comprises the following steps:

[0041] Mix the aqueous solution of the crosslinked sodium hyaluronate with the aqueous solution of the second sodium hyaluronate and carry out a reaction to obtain the transdermal penetration enhancer.

[0042] In some embodiments, each 100 mL of the aqueous solution of the crosslinked sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g, of the crosslinked sodium hyaluronate.

[0043] In some embodiments, each 100 mL of the aqueous solution of the second sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g, of the second sodium hyaluronate.

[0044] In some embodiments, the volume ratio of the aqueous solution of the crosslinked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:(5 - 15), for example 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15 or any value therebetween, preferably 1:(8 - 12). In some examples, the volume ratio of the aqueous solution of the crosslinked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:10.

[0045] In some embodiments, the mass ratio of the crosslinked sodium hyaluronate to the second sodium hyaluronate is 1:

[0046] (0.01 - 1000), for example 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1.0, 1:1.5, 1:2, 1:

[0047] 5, 1:10, 1:20, 1:50, 1:80, 1:100, 1:200, 1:500, 1:800, 1:1000 or any value therebetween. In some embodiments, the mass ratio of the crosslinked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1 - 100). In some embodiments, the mass ratio of the crosslinked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1 - 10). In some embodiments, the mass ratio of the crosslinked sodium hyaluronate to the second sodium hyaluronate is 1:(1 - 10). In some embodiments, the mass ratio of the crosslinked sodium hyaluronate to the second sodium hyaluronate is 1:(0.1 - 1).

[0048] In some embodiments, the temperature of the reaction is 0 - 80 °C, preferably 25 - 40 °C.

[0049] In some embodiments, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours.

[0050] By mixing the above-mentioned cross-linked sodium hyaluronate with sodium hyaluronate, a transdermal penetration enhancer (nano-cross-linked sodium hyaluronate) is prepared. By adjusting the molecular weight of sodium hyaluronate and the addition ratio of cross-linked sodium hyaluronate, the size of the transdermal penetration enhancer particles is further adjusted, thereby changing the water absorption amount on the skin surface, and further improving the efficiency of promoting the penetration of the active ingredient into the skin as a transdermal delivery system.

[0051] In a seventh aspect, the present invention provides the use of the above-mentioned transdermal penetration enhancer or the transdermal penetration enhancer obtained by the above-mentioned preparation method in the preparation of skin care products.

[0052] In the present invention, the skin care products include but are not limited to: skin care lotions, skin care milks, skin care creams, skin care serums, skin care masks, etc.

[0053] According to some embodiments of the present invention, the active ingredient of the skin care product includes hydroxypinacolone retinoate.

[0054] The transdermal penetration enhancer provided by the present invention can effectively promote the transdermal absorption of the active ingredient (such as hydroxypinacolone retinoate) in skin care products, and has broad application prospects in the field of skin care products.

[0055] In an eighth aspect, the present invention provides a transdermal penetration composition, which includes hydroxypinacolone retinoate and the transdermal penetration enhancer of the present invention or the transdermal penetration enhancer obtained by the preparation method of the present invention, wherein the active ingredient includes one or more of retinol and its derivatives.

[0056] In some embodiments, in the composition, the concentration of the transdermal penetration enhancer is 0.1-5 wt%, such as 0.1 wt%, 0.2 wt%, 0.5 wt%, 0.8 wt%, 1.0 wt%, 1.5 wt%, 2.0 wt%, 3.0 wt%, 4.0 wt%, 5.0 wt% or any value therebetween. In some embodiments, in the composition, the concentration of the transdermal penetration enhancer is 0.1-2 wt%. In some embodiments, in the composition, the concentration of the transdermal penetration enhancer is 0.5-2 wt%.

[0057] In some embodiments, in the composition, the concentration of hydroxypinacolone retinoate is 0.05-0.5 wt%, such as 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.25 wt%, 0.3 wt%, 0.35 wt%, 0.4 wt%, 0.45 wt% or any value therebetween. In some embodiments, in the composition, the concentration of hydroxypinacolone retinoate is 0.05-0.2 wt%. In some embodiments, in the composition, the concentration of hydroxypinacolone retinoate is 0.1-0.2 wt%.

[0058] In some embodiments, in the composition, the mass ratio of the transdermal penetration enhancer to hydroxypinacolone retinoate is 1:(0.01 - 1000), such as 1:0.01, 1:0.05, 1:0.1, 1:0.5, 1:1.0, 1:1.5, 1:2, 1:

[0059] 5, 1:10, 1:20, 1:50, 1:80, 1:100, 1:200, 1:500, 1:800, 1:1000 or any value therebetween. In some embodiments, in the composition, the mass ratio of the transdermal penetration enhancer to hydroxypinacolone retinoate is 1:(0.1 - 10). In some embodiments, in the composition, the mass ratio of the transdermal penetration enhancer to hydroxypinacolone retinoate is 1:(0.1 - 1).

[0060] In a ninth aspect, the present invention provides a skin care product, which comprises the above-mentioned transdermal penetration enhancing composition, or is prepared from raw materials comprising the above-mentioned transdermal penetration enhancing composition.

[0061] In the present invention, the skin care product includes but is not limited to: skin care lotion, skin care milk, skin care cream, skin care essence, skin care mask, etc.

[0062] Compared with the prior art, the present invention has the following beneficial effects:

[0063] 1. The present invention creatively discovers that chitosan deacetate crosslinked sodium hyaluronate modified with glycidyl group or ethylene oxide polyethylene glycol group can be used as a crosslinking agent to crosslink sodium hyaluronate again, significantly reducing the viscosity of sodium hyaluronate, improving the skin permeability, effectively achieving the effect of bringing the active ingredient into the skin, increasing the amount of the active ingredient entering the skin, and improving the bioavailability of the active ingredient.

[0064] 2. The particle size distribution range of the transdermal penetration enhancer of the present invention is more concentrated, and it can form a film on the skin surface and play a penetration enhancing effect by improving the structure of the stratum corneum. Description of the Drawings

[0065] Figure 1 is the infrared spectrum of the products obtained in Example 2 and Example 3B.

[0066] Figure 2 is the infrared spectrum of the product obtained in Example 4B and sodium hyaluronate (300,000 Da).

[0067] Figure 3 is the total reflection infrared spectrum after treating mouse epidermis with the products of Example 3A, Example 3B, Example 3C and the product obtained by crosslinking the product of Example 4B with sodium hyaluronate with a molecular weight of 1.3 million Da respectively.

[0068] Figure 4 The water content of the epidermis after treating the epidermis with the products of Example 3A, Example 3B, and Example 4A, and a sodium hyaluronate solution with a molecular weight of 1 million Da, respectively. Detailed implementation manners

[0069] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and do not constitute any limitation to the present invention.

[0070] The present invention provides a modified chitosan, which includes a chitosan deacetate backbone, and the backbone is grafted through an ether bond group, and p is an integer between 0 and 200.

[0071] In some implementation manners, the structure of the modified chitosan of the present invention is shown in the following formulas 1, 2, 3, 4, 5, or 6:

[0072]

[0073] In Formulas 1 to 6, n and x are the same or different and are each independently selected from integers between 0 and 200, preferably integers between 1 and 200; m is a positive integer, preferably a positive integer between 20 and 40.

[0074] The present invention also provides a crosslinked sodium hyaluronate, which includes a first sodium hyaluronate crosslinked by the above-mentioned modified chitosan; the molecular weight of the first sodium hyaluronate is 800 Da - 1500 KDa, preferably 100 KDa - 1000 KDa, and more preferably 200 KDa - 400 KDa.

[0075] The present invention also provides a preparation method of the above-mentioned crosslinked sodium hyaluronate, which includes mixing an aqueous solution of the above-mentioned modified chitosan with an aqueous solution of the first sodium hyaluronate and reacting to obtain the crosslinked sodium hyaluronate.

[0076] Specifically, the reaction of the first sodium hyaluronate and the modified chitosan (taking the structure shown in Formula 4 as an example) is shown in the following formula:

[0077]

[0078] The present invention also provides a transdermal penetration enhancer, which includes a second sodium hyaluronate crosslinked by the above-mentioned crosslinked sodium hyaluronate, and the molecular weight of the second sodium hyaluronate is 800 Da - 1500 KDa, preferably 100 KDa - 1500 KDa, and more preferably 1000 KDa - 1300 KDa.

[0079] The above-mentioned cross-linked sodium hyaluronate of the present invention can be used as a cross-linking agent to cross-link with sodium hyaluronate again to obtain nano-sodium hyaluronate, which has a significantly reduced viscosity and a significantly increased transdermal rate. In particular, it has an excellent permeation-promoting effect on hydroxypinacolone retinoate and is suitable for preparing skin care products containing hydroxypinacolone retinoate.

[0080] The reagents used in the following experiments of the present invention are all conventional commercially available products or reagents prepared by conventional methods unless otherwise specified. The methods used in the experiments are all conventional experimental methods unless otherwise specified. The instruments used in the experiments can be obtained through commercial channels unless otherwise specified.

[0081] Example 1 Synthesis of Propylene Oxide Grafted Chitosan

[0082] Take 5 g of chitosan (average molecular weight Mw~4400) and dissolve it in N,N-dimethylformamide solution. Add 0.5 g of sodium carbonate, stir and then add 1 g of epichlorohydrin. React at room temperature for 24 hours. Subsequently, add 50 mL of water to quench the reaction, and after dialysis using a 500 Da dialysis membrane, a propylene oxide grafted chitosan solution is obtained. After freeze-drying, the product propylene oxide grafted chitosan is obtained.

[0083] Example 2 Synthesis of Ethylene Oxide Polyethylene Glycol Grafted Chitosan

[0084] Take 5 g of chitosan (average molecular weight Mw~4400) and dissolve it in N,N-dimethylformamide solution. Add 0.5 g of cesium carbonate, stir and then add 1 g of ethylene oxide grafted polyethylene glycol chloride (degree of polymerization = 20, CAS: 155101-67-0). React at room temperature for 24 hours. Subsequently, add 50 mL of water to quench the reaction, and after dialysis using a 500 Da dialysis membrane, an ethylene oxide polyethylene glycol grafted chitosan solution is obtained. After freeze-drying, the product ethylene oxide polyethylene glycol grafted chitosan is obtained. Analyzed by a Thermo FlashSmart elemental analyzer, the mass percentage content of each element is: N: 4.65%, C: 43.73%, H: 7.39%, O: 44.23%. The infrared spectrum of the product is as Figure 1 shown.

[0085] Example 3A Synthesis of Propylene Oxide Grafted Chitosan Cross-linked Sodium Hyaluronate (1 million Da)

[0086] Step 1: Take 0.1 g of the propylene oxide grafted chitosan prepared in Example 1, add 10 mL of water, and stir to dissolve.

[0087] Step 2: Take 1 g of sodium hyaluronate (1 million Da) and dissolve it in 100 mL of water.

[0088] Step 3: Add the solution prepared in Step 1 to the solution prepared in Step 2, adjust the pH to 8 - 9 using 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH to 6.5 - 7 using 10% HCl solution.

[0089] Step 4: After dialysis of the liquid obtained in Step 3 through a dialysis membrane with a molecular weight cut-off of 10,000 Da, freeze-dry to obtain the finished product of propylene oxide-grafted deacetylated chitosan cross-linked sodium hyaluronate (1 million Da).

[0090] Synthesis of propylene oxide-grafted deacetylated chitosan cross-linked sodium hyaluronate (100,000 Da) in Example 3B

[0091] The difference from Example 3A is that "sodium hyaluronate (1 million Da)" in Step 2 is replaced with "sodium hyaluronate (100,000 Da)".

[0092] Finally, the product of propylene oxide-grafted deacetylated chitosan cross-linked sodium hyaluronate (100,000 Da) is obtained. Analyzed by a Thermo Flash Smart elemental analyzer, the mass percentage of each element is as follows: N: 4.79%, C: 46.05%, H: 7.51%, O: 41.65%. The infrared spectrum of the product is as Figure 1 shown.

[0093] Synthesis of propylene oxide-grafted deacetylated chitosan cross-linked sodium hyaluronate (300,000 Da) in Example 3C

[0094] The difference from Example 3A is that "sodium hyaluronate (1 million Da)" in Step 2 is replaced with "sodium hyaluronate (300,000 Da)".

[0095] Synthesis of ethylene oxide polyethylene glycol-grafted deacetylated chitosan cross-linked sodium hyaluronate (100,000 Da) in Example 4A

[0096] Step 1: Take 0.1 g of ethylene oxide polyethylene glycol-grafted deacetylated chitosan prepared in Example 2, add 10 mL of water, and stir to dissolve.

[0097] Step 2: Take 1 g of sodium hyaluronate (100,000 Da) and dissolve it in 100 mL of water.

[0098] Step 3: Add the solution prepared in Step 1 to the solution prepared in Step 2, adjust the pH to 8 - 9 using 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH to 6.5 - 7 using 10% HCl solution.

[0099] Step 4: The liquid obtained in Step 3 is dialyzed through a dialysis membrane with a molecular weight cut-off of 10,000 Da, and then lyophilized to obtain the finished product of ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (100,000 Da).

[0100] Synthesis of ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (300,000 Da) in Example 4B

[0101] Step 1: Take 0.1 g of ethylene oxide polyethylene glycol grafted chitosan deacetate prepared in Example 2, add 10 mL of water, and stir to dissolve it;

[0102] Step 2: Take 1 g of sodium hyaluronate (300,000 Da) and dissolve it in 100 mL of water.

[0103] Step 3: Add the solution prepared in Step 1 to the solution prepared in Step 2, adjust the pH to 8 - 9 using 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH value to 6.5 - 7 using 10% HCl solution.

[0104] Step 4: The liquid obtained in Step 3 is dialyzed through a dialysis membrane with a molecular weight cut-off of 10,000 Da, and then lyophilized to obtain the finished product of ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (300,000 Da). Analyzed by Thermo FlashSmart elemental analyzer, the mass percentage of each element in the finished product is N: 3.22%, C: 36.66%, H: 5.36%; The infrared spectra of sodium hyaluronate (300,000 Da) and the product ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (300,000 Da) are as Figure 2 shown.

[0105] Synthesis of ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (1,000,000 Da) in Example 4C

[0106] Step 1: Take 0.1 g of ethylene oxide polyethylene glycol grafted chitosan deacetate prepared in Example 2, add 10 mL of water, and stir to dissolve it;

[0107] Step 2: Take 1 g of sodium hyaluronate (1,000,000 Da) and dissolve it in 100 mL of water.

[0108] Step 3: Add the solution prepared in Step 1 to the solution prepared in Step 2, adjust the pH to 8 - 9 using 10% NaOH solution, then stir at room temperature for 24 hours, and then adjust the pH value to 6.5 - 7 using 10% HCl solution.

[0109] Step 4: The liquid obtained in Step 3 is dialyzed through a dialysis membrane with a molecular weight cut-off of 10,000 Da, and then lyophilized to obtain the finished product of ethylene oxide polyethylene glycol grafted chitosan deacetate crosslinked sodium hyaluronate (1,000,000 Da).

[0110] Example 5 Synthesis of Chitosan Deacetate Grafted with Ethylene Oxide Polyethylene Glycol

[0111] The difference from Example 2 is that "ethylene oxide-grafted chlorinated polyethylene glycol (degree of polymerization = 20, CAS: 155101-67-0)" is replaced with "ethylene oxide-grafted chlorinated polyethylene glycol (degree of polymerization = 3, CAS: 155101-68-1)".

[0112] Example 6 Synthesis of Crosslinked Sodium Hyaluronate with Chitosan Deacetate Grafted with Ethylene Oxide Polyethylene Glycol (300,000 Da)

[0113] The difference from Example 4B is only that in Step 1, "chitosan deacetate grafted with ethylene oxide polyethylene glycol prepared in Example 2" is replaced with "chitosan deacetate grafted with ethylene oxide polyethylene glycol prepared in Example 5" to obtain crosslinked sodium hyaluronate with chitosan deacetate grafted with ethylene oxide polyethylene glycol (300,000 Da).

[0114] Comparative Example 1 Synthesis of Crosslinked Sodium Hyaluronate with Hydroxyethyl Chitosan

[0115] Dissolve 1 g of sodium hyaluronate (200,000 Da - 400,000 Da) in 50 mL of water, then add 3 g of prepared hydroxyethyl chitosan (average molecular weight Mw ~ 8000). After dissolution, add 0.1 mL of glutaraldehyde and react at room temperature for 12 hours. The reactant is dialyzed and freeze-dried to prepare 1.5 g of the product crosslinked sodium hyaluronate with hydroxyethyl chitosan (200,000 Da - 400,000 Da).

[0116] Comparative Example 2 Preparation of Crosslinked Sodium Hyaluronate with Carboxymethyl Chitosan as the Crosslinking Agent

[0117] Add 5 g of carboxymethyl chitosan (average molecular weight Mw ~ 160,000) to 30 mL of N,N-dimethylformamide. After stirring evenly, add 0.1 mL of thionyl chloride and react at 90 °C for 8 hours. Then continue to add 50 mL of N,N-dimethylformamide containing 5 g of sodium hyaluronate (1,000,000 Da) and continue to react for 6 hours. Add 300 mL of acetone to precipitate. After filtering off the solvent, wash with 3 × 30 mL of acetone to obtain crosslinked sodium hyaluronate with carboxymethyl chitosan (1,000,000 Da).

[0118] Example 7 Synthesis of Nanocrosslinked Sodium Hyaluronate

[0119] Step 1: Take 0.1 g of crosslinked sodium hyaluronate with ethylene oxide grafted chitosan (1,000,000 Da) prepared in Example 3A as the crosslinking agent, add 10 mL of water and stir to dissolve;

[0120] Step 2: Take 1 g of sodium hyaluronate (1.3 million Da) and dissolve it in 100 mL of water to obtain a sodium hyaluronate solution;

[0121] Step 3: Add the solution prepared in Step 1 to the sodium hyaluronate solution prepared in Step 2, and stir at room temperature for 48 hours to obtain a nano-crosslinked sodium hyaluronate solution;

[0122] Step 4: After dialysis of the nano-crosslinked sodium hyaluronate solution obtained in Step 3 through a dialysis membrane with a molecular weight cut-off of 10,000 Da, it is freeze-dried to obtain the finished product - nano-crosslinked sodium hyaluronate.

[0123] Synthesis of nano-crosslinked sodium hyaluronate in Examples 8 - 16

[0124] Using the same method as in Example 7 above, adjust the crosslinking agent used in Step 1 and the molecular weight of sodium hyaluronate used in Step 2 according to the formula in Table 1 below to obtain different nano-crosslinked sodium hyaluronic acids.

[0125] Table 1

[0126]

[0127]

[0128] The average particle size of the nano-crosslinked sodium hyaluronate product in each example in Table 1 was measured using a laser particle size analyzer, which is the Zetasizer Nano ZS produced by Malvern Instruments Ltd, UK. The measurement angle θ is 173°; the temperature of the sample cell can be adjusted in the range of 2 - 90 °C; the laser wavelength used is 633 nm.

[0129] Experiment 1 Viscosity test

[0130] Using the chitosan-grafted sodium hyaluronate crosslinked with propylene oxide / ethylene oxide polyethylene glycol prepared in Examples 1, 2, 3A, 3B, 3C, 4A, 4B, 4C, 5, 6 and Comparative Examples 1 - 2 above as a crosslinking agent, mix it with sodium hyaluronate solutions of different molecular weights to obtain nano-crosslinked sodium hyaluronate solutions, and investigate the viscosity changes of the nano-crosslinked sodium hyaluronate solutions.

[0131] In the following experiments, the bulk viscosity of the sodium hyaluronate solution was measured by an NDJ-8S digital display viscometer (Shanghai Precision Scientific Instruments Co., Ltd.) in an environment with a constant temperature of 298 K. The test method is as follows:

[0132] (1) Preparation of sodium hyaluronate solutions with different molecular weights: Weigh 1 g of sodium hyaluronate with a molecular weight of 1.3 million Da, 1 g of sodium hyaluronate with a molecular weight of 1 million Da, and 1 g of sodium hyaluronate with a molecular weight of 200,000 - 400,000 Da respectively, and dissolve them in 100 mL of water to prepare 1% sodium hyaluronate (1.3 million Da) solution, 1% sodium hyaluronate (1 million Da) solution, and 1% sodium hyaluronate (200,000 - 400,000 Da) solution;

[0133] (2) Preparation of cross - linker solution: Weigh 1 g of cross - linker and dissolve it in 100 mL of water to prepare a 1% cross - linker solution;

[0134] (3) Weigh 10 mL of 1% cross - linker solution and add it to 90 mL of 1% sodium hyaluronate solution prepared in step (1). After standing for 0.5 - 48 h, a nano - crosslinked sodium hyaluronate solution is obtained.

[0135] After testing, the viscosity of 1% sodium hyaluronate (1.3 million Da) solution is 13674 mPa·S;

[0136] The viscosity of 1% sodium hyaluronate (1 million Da) solution is 12109 mPa·S;

[0137] The viscosity of 1% sodium hyaluronate (200,000 - 400,000 Da) solution is 6832 mPa·S;

[0138] When the cross - linked sodium hyaluronate (300,000 Da) grafted with ethylene oxide - polyethylene glycol prepared in Example 4B is configured into a 1% cross - linker solution, the viscosity of this cross - linker solution is 126 mPa·S.

[0139] Some experimental results are shown in Table 2.

[0140] Table 2

[0141]

[0142]

[0143]

[0144] It can be concluded from Table 2 above that the cross - linked sodium hyaluronate prepared in Examples 3A - 3C, 4A - 4C, and Example 6 of this application can significantly reduce the viscosity of sodium hyaluronate solution as a cross - linker; while the sodium hyaluronate cross - linked with hydroxyethyl chitosan prepared in Comparative Example 1 and the sodium hyaluronate cross - linked with carboxymethyl chitosan prepared in Comparative Example 2 cannot change the viscosity and state of sodium hyaluronate.

[0145] Experiment 2 Skin epidermis experiment

[0146] (1) Analysis and research of mouse epidermis (SC) samples by ATR-FTIR

[0147] Step 1: Preparation of mouse epidermis layer

[0148] After sacrificing Kunming mice and removing the hair with an electric clipper, carefully shave it with a razor and obtain the mouse skin. The epidermis is gently peeled off by trimming the subcutaneous fat and immersing the full-thickness skin sample in water. The stratum corneum sheets are obtained by floating the newly prepared epidermis in an aqueous solution containing 0.0001% trypsin and 0.5% sodium bicarbonate for 24 hours. Remove the digest under the stratum corneum with filter paper, rinse the separated sheets in acetone for 30 seconds to remove any sebum or subcutaneous fat contamination, then freeze-dry and store in silica gel under vacuum.

[0149] Step 2: Preparation of different sodium hyaluronate samples

[0150] Sodium hyaluronate sample 1: Dissolve 1 g of sodium hyaluronate with a molecular weight of 1.3 million Da in 100 mL of water to prepare a 1% sodium hyaluronate (1.3 million Da) solution;

[0151] Sodium hyaluronate sample 2: Dissolve 1 g of the product prepared in Example 4B in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (0.3 million Da) solution; Take 10 mL of the 1% cross-linked sodium hyaluronate (0.3 million Da) solution and add it to 90 mL of the 1% sodium hyaluronate (1.3 million Da) solution (prepared in the same way as sodium hyaluronate sample 1). After standing for 0.5 - 48 h, obtain a nano-cross-linked sodium hyaluronate solution, denoted as 1% sodium hyaluronate (1.3 million Da) + 1% cross-linked sodium hyaluronate (0.3 million Da).

[0152] Sodium hyaluronate sample 3: Dissolve 1 g of the product prepared in Example 3A in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (1 million Da) solution;

[0153] Sodium hyaluronate sample 4: Dissolve 1 g of the product prepared in Example 3B in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (0.1 million Da) solution;

[0154] Sodium hyaluronate sample 5: Dissolve 1 g of the product prepared in Example 3C in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (0.3 million Da) solution.

[0155] Step 3: Testing

[0156] A 6 mm × 6 mm dry SC sample was incubated in 3 mL of each of the sodium hyaluronate samples 1 - 5 prepared in Step 2 above and a blank solvent (water, as a control) at room temperature for 24 hours. Subsequently, the SC sheets were carefully washed with distilled water to remove all residual solvents on their surfaces. After freeze-drying, spectroscopic measurements were performed using a Nicolet FTIR 5700 Fourier transform infrared spectrometer (Thermo Nicolet Corporation, USA) equipped with an ATR accessory under the following conditions: a single-crystal diamond crystal mirror, a scanning temperature range of 18 - 20 °C, 64 scans, a resolution of 4 cm⁻¹, and a scanning range of 800 - 4000 cm⁻¹. The infrared spectra are as Figure 3 shown. Each set of curves was recorded and analyzed using second derivative, deconvolution, and curve fitting. The specific method is as follows: Fourier self-deconvolution and curve fitting were performed on Figure 4 using Peakfit v4.12 software under the following conditions: a tolerance percentage of 9%, a deconvolution width of 3.00, a filter of 55.0, and a peak type of spectroscopic and Gaussian area. The height, width, and position of all peaks were optimized in sequence. The relative areas of the peaks in the amide I region were calculated based on the finally fitted peak areas, and the content of the secondary structure of the stratum corneum was determined based on the peak areas. The results are shown in Table 3.

[0157] Table 3 Changes in the secondary structure of the stratum corneum of the skin in different treatment groups

[0158]

[0159] As can be seen from Table 3, after the cross-linked sodium hyaluronate prepared in Example 4B was cross-linked again with sodium hyaluronate (130W), the β-sheet of the stratum corneum could be improved more significantly, promoting the skin absorption of the active ingredient.

[0160] (II): Analysis and research on water in mouse epidermis (SC) by ATR-FTIR

[0161] Step 1: Preparation of mouse epidermal layer

[0162] After sacrificing Kunming mice and removing the hair with an electric clipper, it was carefully shaved with a razor, and the skin of the mice was obtained. The epidermis was gently peeled off by trimming the subcutaneous fat and immersing the full-thickness skin sample in water. Stratum corneum sheets were obtained by floating the newly prepared epidermis in an aqueous solution containing 0.0001% trypsin and 0.5% sodium bicarbonate for 24 hours. The digest under the stratum corneum was removed with filter paper, and the separated sheets were rinsed in acetone for 30 seconds to remove any sebum or subcutaneous fat contamination, then freeze-dried and stored in silica gel under vacuum.

[0163] Step 2: Preparation of different sodium hyaluronate samples

[0164] Sodium hyaluronate sample 1: Take 1 g of sodium hyaluronate with a molecular weight of 1 million Da and dissolve it in 100 mL of water to prepare a 1% sodium hyaluronate (1 million Da) solution;

[0165] Sodium hyaluronate sample 2: Take 1 g of the product prepared in Example 3A and dissolve it in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (1 million Da) solution;

[0166] Sodium hyaluronate sample 3: Take 1 g of the product prepared in Example 3B and dissolve it in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (0.1 million Da) solution;

[0167] Sodium hyaluronate sample 4: Take 1 g of the product prepared in Example 4A and dissolve it in 100 mL of water to prepare a 1% cross-linked sodium hyaluronate (0.1 million Da) solution.

[0168] Step 3: Testing

[0169] Place a 6 mm × 6 mm dry SC sample at room temperature in 3 mL of the sodium hyaluronate samples 1 - 4 prepared in the above Step 2 and a blank solvent (water, as a control) and incubate. The operating steps are the same as described above. At predetermined time intervals (0, 2, 4, 6, 8, 12 hours), take out the SC sample and carefully wash it with distilled water to remove the residual solvent on the surface. Subsequently, blot the residual moisture on the surface of the SC with filter paper and detect the moisture content in the SC by ATR-FTIR under the above conditions. Determine the moisture content in the SC by calculating the ratio of the amide I absorption peak to the amide II absorption peak. The results are as Figure 4 shown. Compared with other examples, the chitosan cross-linked sodium hyaluronate grafted with ethylene oxide polyethylene glycol prepared in Example 4A can more significantly improve the water content of the stratum corneum and promote the skin absorption of the active ingredient.

[0170] Experiment 3: Transdermal effect test

[0171] All solvents and reagents are of chromatographic grade. The standard products are purchased from Shanghai Yuanye Bio-Technology Co., Ltd. See Tables 4 and 5 for details.

[0172] Table 4 Experimental reagents

[0173]

[0174] Table 5 Experimental instruments

[0175] Instrument Model and Specification Manufacturer Vortex Oscillator M16710-33 Thermo Scientific Desktop High-Speed Refrigerated Centrifuge Microfuge22R Beckman Coulter Chromatography waters 2695 waters

[0176] 1) Sample treatment

[0177] Add the penetration enhancer and the active ingredient to ultrapure water according to the formula in Table 6 below, and centrifuge at 12,000 r / min for 10 min; take the supernatant and transfer it to a lined tube.

[0178] 2) In vitro release and transdermal test method

[0179] Use a Franz diffusion cell and the excised skin of a small Bama pig. The upper chamber is the supply chamber, and the lower chamber is the receiving chamber. The volume of the receiving chamber is 15 mL, the inner diameter of the supply chamber is 1.5 cm, and the effective diffusion area is 1.766 cm 2 , (37 °C) constant temperature water bath, and stir magnetically at a constant speed of 200 r / min. During the test, fill the receiving chamber with the receiving solution (30% ethanol - physiological sodium chloride solution). Fix the excised skin of the small Bama pig between the two chambers with the stratum corneum facing up, so that the dermis is in full contact with the receiving solution, remove air bubbles, and add 1 mL of the test sample to the supply chamber (see Table 6 for the mass concentrations of the penetration enhancer and the active ingredient in the test sample). Precisely withdraw 1 mL of the receiving solution sample from the sampling tube at 6 h. After the withdrawn sample is filtered through a 0.45 μm microporous membrane, it is directly subjected to HPLC analysis.

[0180] The instrument used for HPLC analysis is a Waters 2695 high performance liquid chromatograph equipped with a Waters 2424 evaporative light scattering detector. The chromatographic column is Diamonsil C18(2) (150*4.6 mm, 5 μm), the mobile phase is acetonitrile: 0.3% TFA water / 7:93, the drift tube temperature is 85 °C, the nebulizer temperature is 30 °C, the nitrogen flow rate is 25 psi, the gain is 100, the mobile phase flow rate is 1 mL / min, the column temperature is 35 °C, and the injection volume is 10 μL.

[0181] Using the cumulative permeation amount as the monitoring index, calculate the cumulative permeation amount per unit area and the transdermal absorption percentage according to the following formula.

[0182]

[0183] Where: Q n is the cumulative release amount at the nth sampling time point; W% is the cumulative release percentage (i.e., the transdermal rate); V0 is the total volume of the release medium; C n is the drug concentration in the nth sampling solution; C i is the drug concentration in the ith sampling solution; V i is the volume of the ith sampling solution; S is the total mass of the drug in the dialysis bag; A is the effective diffusion area of the Franz diffusion cell.

[0184] The results are shown in Table 6.

[0185] Table 6

[0186]

[0187]

[0188] The results show that by using the nano-crosslinked sodium hyaluronate of the present invention as a penetration enhancer and mixing it with the active ingredient hydroxypinacolone retinoate, the penetration rate of the active ingredient can be significantly increased by 20%; moreover, under the same conditions, compared with other reference substances (such as water-soluble azone, glycerol, poloxamer, liposomes, etc.), the nano-crosslinked sodium hyaluronate of the present invention has a more excellent penetration enhancing effect.

[0189] The technical solution of the present invention is not limited to the limitations of the above specific embodiments, and any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. A modified chitin, which comprises a chitosan backbone, and the backbone is grafted through an ether bond group, and p is an integer between 0 and 200.

2. The modified chitin according to claim 1, wherein, p is an integer between 1 and 200, preferably an integer between 1 and 100, more preferably an integer between 1 and 20; and / or The modified chitosan includes one or more of the structures shown in the following formulas 1, 2, 3, 4, 5 or 6: Wherein, n and x are the same or different and are each independently selected from integers between 0 and 200, preferably integers between 1 and 200, more preferably integers between 1 and 100, and further preferably integers between 1 and 20; m is a positive integer, preferably a positive integer between 20 and 40.

3. A method for preparing the modified chitosan as claimed in claim 1 or 2, which comprises the following steps: Reacting chitosan deacetylate with an epoxidizing reagent to obtain the modified chitosan; wherein the epoxidizing reagent includes epichlorohydrin and / or polyethylene glycol chloride grafted with ethylene oxide; Preferably, the degree of polymerization of the polyethylene glycol chloride grafted with ethylene oxide is 1 to 200, preferably 1 to 100, more preferably 1 to 20; Preferably, the reaction is carried out in a solvent, and the solvent includes amide solvents, preferably including N,N-dimethylformamide; Preferably, the reaction is carried out in the presence of a first catalyst, and the first catalyst includes one or more of alkali metal hydroxides and alkali metal carbonates, more preferably including one or more of sodium carbonate, cesium carbonate, potassium carbonate, sodium hydroxide, and potassium hydroxide; Preferably, the molar ratio of the first catalyst to the epoxidizing reagent is 1:(1 - 3), preferably 1:(1.5 - 2.5); Preferably, the molar ratio of the chitosan deacetylate to the epoxidizing reagent is 1:(0.1 - 1.0), preferably 1:(0.1 - 0.5); Preferably, the temperature of the reaction is 0 - 95°C, preferably 15 - 40°C; Preferably, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours.

4. A crosslinked sodium hyaluronate, which includes a first sodium hyaluronate crosslinked by a modified chitosan; wherein the modified chitosan includes the modified chitosan as claimed in claim 1 or 2 or the modified chitosan prepared by the preparation method as claimed in claim 3; Preferably, the molecular weight of the first sodium hyaluronate is 800 Da - 1500 KDa, preferably 100 KDa - 1000 KDa, more preferably 200 KDa - 400 KDa; Preferably, the molar ratio of the first sodium hyaluronate to the modified chitosan is 1:(0.1 - 1).

5. A method for preparing the crosslinked sodium hyaluronate as claimed in claim 4, which comprises the following steps: Mixing an aqueous solution of the modified chitosan with an aqueous solution of the first sodium hyaluronate and reacting to obtain the crosslinked sodium hyaluronate; Preferably, every 100 mL of the aqueous solution of the modified chitosan contains 0.5 - 2 g, preferably 0.8 - 1.2 g of the modified chitosan; Preferably, every 100 mL of the aqueous solution of the first sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g of the first sodium hyaluronate; Preferably, the volume ratio of the aqueous solution of the modified chitosan to the aqueous solution of the first sodium hyaluronate is 1:(8 - 12); Preferably, the reaction is carried out under the condition of pH 8.0 - 9.0; Preferably, the temperature of the reaction is 0 - 80°C, preferably 25 - 40°C; Preferably, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours; Preferably, after the reaction, the pH is adjusted to 6.5 - 7.

0.

6. A transdermal penetration enhancer, which comprises a second sodium hyaluronate crosslinked by crosslinked sodium hyaluronate, and the crosslinked sodium hyaluronate comprises the crosslinked sodium hyaluronate described in claim 4 or the crosslinked sodium hyaluronate obtained by the preparation method described in claim 5; Preferably, the molecular weight of the second sodium hyaluronate is 800 Da - 1500 KDa, preferably 100 KDa - 1500 KDa, more preferably 1000 KDa - 1300 KDa; Preferably, the dosage of the crosslinked sodium hyaluronate is 5 - 15% by mass of the second sodium hyaluronate, preferably 8 - 12%.

7. A preparation method of a transdermal penetration enhancer as described in claim 6, which comprises the following steps: Mix the aqueous solution of the crosslinked sodium hyaluronate with the aqueous solution of the second sodium hyaluronate and carry out a reaction to obtain the transdermal penetration enhancer; Preferably, every 100 mL of the aqueous solution of the crosslinked sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g of the crosslinked sodium hyaluronate; Preferably, every 100 mL of the aqueous solution of the second sodium hyaluronate contains 0.5 - 2 g, preferably 0.8 - 1.2 g of the second sodium hyaluronate; Preferably, the volume ratio of the aqueous solution of the crosslinked sodium hyaluronate to the aqueous solution of the second sodium hyaluronate is 1:(5 - 15), preferably 1:(8 - 12); Preferably, the temperature of the reaction is 0 - 80°C, preferably 25 - 40°C; Preferably, the reaction time is 0.5 - 30 hours, preferably 12 - 24 hours.

8. Use of the transdermal penetration enhancer as described in claim 6 or the transdermal penetration enhancer obtained by the preparation method described in claim 7 in the preparation of skin care products; Preferably, the skin care products include skin care lotion, skin care milk, skin care cream, skin care essence, skin care mask.

9. A transdermal penetration composition, which comprises hydroxypinacolone retinoate and a transdermal penetration enhancer, and the transdermal penetration enhancer comprises the transdermal penetration enhancer described in claim 6 or the transdermal penetration enhancer obtained by the preparation method described in claim 7; Preferably, in the composition, the concentration of the transdermal penetration enhancer is 0.1 - 5 wt%, preferably 0.5 - 2 wt%; Preferably, in the composition, the concentration of hydroxypinacolone retinoate is 0.05 - 0.5 wt%, preferably 0.05 - 0.2 wt%; Preferably, in the composition, the mass ratio of the transdermal penetration enhancer to hydroxypinacolone retinoate is 1:(0.01 - 1000), preferably 1:(0.1 - 10), more preferably 1:(0.1 - 1).

10. A skin care product, which comprises the transdermal penetration enhancing composition as claimed in claim 9, or which is prepared from raw materials comprising the transdermal penetration enhancing composition as claimed in claim 9; Preferably, the skin care product comprises skin lotion, skin milk, skin cream, skin essence, and skin mask.

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