A hyaluronic acid derivative or salt with dual responsiveness to nitric oxide and reactive oxygen species, and its preparation method and application

Abstract

The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a hyaluronic acid derivative or salt having dual responsiveness to nitric oxide and reactive oxygen species, a preparation method thereof, and an application thereof; the present invention prepares HA-o-phenylenediamine-maleic acid-Vc by chemically grafting hyaluronic acid; it has environmental responsiveness, good biocompatibility, excellent injection properties, and good biological environment responsiveness, can perform intelligent response and scavenging according to the concentrations of reactive oxygen species and nitric oxide in the environment, reduce the concentration level of excessive environmental adverse factors, and has excellent stability, and can be used in the fields of drug intelligent delivery and cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry, and particularly relates to a hyaluronic acid derivative or salt with dual response characteristics to nitric oxide and reactive oxygen species, a preparation method thereof, and applications thereof. Background Art

[0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide, also known as hyaluronic acid. It is an unbranched high-molecular glycosaminoglycan composed of disaccharide repeating units of N-acetylglucosamine and D-glucuronic acid. It exists in the intercellular matrix of animal tissues and the capsules of certain bacteria, and is an important component of the cell matrix and various tissues, having a variety of important physiological functions. The unique viscoelasticity, biocompatibility, and biodegradability of sodium hyaluronate enable it to have a wide range of applications in the biomedical field, including as an ophthalmic surgery aid, an anti-adhesion agent after surgery, a skin wound healing and regeneration aid, a drug carrier, a tissue engineering scaffold, etc. In addition, the hydroxyl, carboxyl, and other polar groups in the hyaluronate molecule can form hydrogen bonds with water molecules to bind a large amount of water, and the water retention effect is obvious. As a water-containing gel-like substance, it has excellent performance in maintaining the viscoelasticity of biological tissues (especially skin tissues) and moisturizing biological tissues, and is widely used in the cosmetics field for moisturizing, lubricating, antioxidant, anti-inflammatory, etc.

[0003] Hyaluronic acid has a unique molecular structure and physical and chemical properties, and plays a key role in joint lubrication. As an important component of synovial fluid, hyaluronic acid can promote the production of synovial fluid and act as a lubricant and shock absorber in the joint structure, which can reduce the friction between joints, enhance joint lubrication, thereby protecting joints and reducing wear during joint movement. With the increase of age, the content of hyaluronic acid in the human body will gradually decrease, which may lead to insufficient joint lubrication and increase the risk of joint wear. In order to maintain joint health, hyaluronic acid or its salts, such as sodium hyaluronate, are widely used in the treatment of joint-related diseases. On the one hand, it can effectively lubricate joints, relieve symptoms such as joint pain and limited mobility, and has a certain effect on the treatment of diseases such as osteoarthritis. In addition, hyaluronic acid or its salts also have a certain anti-infective effect, which helps to improve joint inflammation and other conditions.

[0004] However, sodium hyaluronate is easily degraded and absorbed in the body, and the retention time is relatively short (after injection into the skin or joint, its half-life does not exceed 24 h). Its anti-inflammatory effect is mainly achieved through its moisturizing ability and the promotion of cell metabolism, but it has no ability to clear inflammatory factors in the inflammatory environment and does not have the special function of clearing oxygen and nitric oxide (NO), which affects the curative effect and application. Summary of the Invention

[0005] The object of the present invention is to solve the deficiencies in the prior art and provide a hyaluronic acid derivative or salt with dual responsiveness to nitric oxide and reactive oxygen species, as well as its preparation method and application.

[0006] To achieve the above object, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides a hyaluronic acid derivative or salt with dual responsiveness to nitric oxide and reactive oxygen species, and the structure of the hyaluronic acid derivative is shown in Formula I:

[0008]

[0009] The salt is sodium salt, potassium salt, zinc salt or calcium salt.

[0010] In the second aspect, the present invention provides a preparation method of a hyaluronic acid derivative or salt with dual responsiveness to nitric oxide and reactive oxygen species, and the method includes the following steps:

[0011] Step S1: Dissolve hyaluronic acid in water, add methyl 3,4-diaminobenzoate and a condensing agent, and stir for 6 - 30 h at a temperature of 10 - 50 °C to carry out a condensation reaction. After the reaction is completed, dialyze and purify, and then freeze-dry to obtain the product HA-o-phenylenediamine; the condensing agent is 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1∶1 - 3∶1 - 4∶1 - 4; the reaction formula is:

[0012]

[0013] Step S2: Dissolve the product HA-o-phenylenediamine in a solvent, dropwise add a solution of maleic anhydride, and carry out an esterification reaction by heating at a temperature of 25 - 60 °C for 6 - 30 h. After the reaction is completed, dialyze and freeze-dry to obtain the product HA-o-phenylenediamine-maleic acid; the molar ratio of HA-o-phenylenediamine to maleic anhydride is 1∶1 - 5; the reaction formula is:

[0014]

[0015] Step S3: The product HA-o-phenylenediamine-maleic acid is dissolved in a solvent and water, a condensing agent and vitamin C are added, and condensation reaction occurs with stirring at a temperature of 10 - 50 °C for 6 - 30 h. After the reaction is completed, dialysis and freeze-drying are carried out to obtain the product HA-o-phenylenediamine-maleic acid-Vc; the condensing agent is 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the molar ratio of the product HA-o-phenylenediamine-maleic acid, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and vitamin C is 1∶1 - 5∶1 - 5∶1 - 5; the reaction formula is:

[0016]

[0017] Further, in step S2, the solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and N-methylpyrrolidone.

[0018] In a third aspect, the present invention provides a composition comprising the hyaluronic acid derivative or salt described above.

[0019] The composition exists in the form of a gel-like aqueous solution.

[0020] In a fourth aspect, the present invention provides an application of the hyaluronic acid derivative or salt described above in the preparation of cosmetics and drugs having lubricating, anti-inflammatory, antioxidant, or moisturizing effects.

[0021] 1. The method of the present invention endows hyaluronic acid and its salts with more functions, such as intelligently responding to high-concentration adverse factors in the microenvironment, scavenging high-concentration factors, and playing a synergistic therapeutic role.

[0022] 2. The present invention grafts and introduces branched chains to form a cross-linked structure, reduces the degradation rate of hyaluronic acid and its salts, and prolongs the residence time in vivo.

[0023] 3. The branched chains grafted and introduced by the present invention undergo a structural change through environmental response. At the same time, drug release is completed, realizing the speed regulation after the drug is delivered to the target site and playing a targeted release role.

[0024] 4. After graft modification, the existing natural properties such as biocompatibility, gel properties, and transdermal absorption properties are retained, and at the same time, the lubricating, anti-inflammatory, antioxidant, and controlled release properties are enhanced.

[0025] 5. After graft modification of hyaluronic acid, through the condensation of the hydroxyl group in Vc by a chemical bond, the characteristic that the hydroxyl group is easily oxidized due to being exposed is reduced, and the stability of the antioxidant performance and the slow release characteristic of reactive oxygen species scavenging are significantly improved.

[0026] The present invention has the following beneficial effects: (1) By structurally modifying hyaluronic acid molecules, the present invention can be used for the treatment of arthritis or skin tissues and for drug delivery. After being delivered to the target site, the hyaluronic acid derivative or its salt can achieve an intelligent response to the microenvironment, scavenge excessive inflammatory factors such as reactive oxygen species and nitric oxide in the environment. As the introduced functional groups are consumed, the delivery carrier gradually degrades and the drug is slowly released, and the free hyaluronic acid or its salt exerts effects such as lubrication, moisturization, anti-inflammation, and anti-aging, achieving multiple therapeutic effects.

[0027] (2) The present invention prepares hyaluronic acid and its salts with environmental response characteristics, which have good biocompatibility, excellent injection characteristics, and good biological environment responsiveness. They can achieve an intelligent response according to the concentrations of reactive oxygen species and nitric oxide in the environment and scavenge them, reducing the concentration level of excessive environmental adverse factors, and have excellent stability. They can be used for intelligent drug delivery and the cosmetics field. Description of the Drawings

[0028] Figure 1 : NMR characterization of hyaluronic acid grafted with vitamin C in the comparative example;

[0029] Figure 2 : IR spectrum characterization of hyaluronic acid grafted with vitamin C in the comparative example;

[0030] Figure 3 : NMR characterization of hyaluronic acid grafted with o-phenylenediamine and vitamin C in Example 1;

[0031] Figure 4 : NMR characterization of hyaluronic acid grafted with o-phenylenediamine and vitamin C in Example 2;

[0032] Figure 5 : NMR characterization of hyaluronic acid grafted with o-phenylenediamine in Example 3;

[0033] Figure 6 : NMR characterization of hyaluronic acid grafted with o-phenylenediamine and vitamin C in Example 3;

[0034] Figure 7 : IR spectrum characterization of hyaluronic acid grafted with o-phenylenediamine in Example 3;

[0035] Figure 8 : IR spectrum characterization of hyaluronic acid grafted with o-phenylenediamine and vitamin C in Example 3;

[0036] Figure 9 : IR spectrum characterization of hyaluronic acid;

[0037] Figure 10:Viscosity and injectability of hyaluronic acid solutions with different concentrations (a. 20 mg / ml HA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right: representation of injectability), b. 40 mg / ml HA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right: representation of injectability), c. 60 mg / ml HA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right: representation of injectability), d. 80 mg / ml HA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right: representation of injectability), e. 100 mg / ml HA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right: representation of injectability);

[0038] Figure 11 :Viscosity and injectability of hyaluronic acid (VHA) solutions of o-phenylenediamine and vitamin C after grafting (70 mg / ml VHA solution (left: representation of inverted EP tube, middle: representation of tilted vial, right:

[0039] representation of injectability));

[0040] Figure 12 :Response and scavenging ability of grafted and blank hyaluronic acid to reactive oxygen species;

[0041] Figure 13 :Response and scavenging ability of grafted and blank hyaluronic acid to NO;

[0042] Figure 14 :Stability of reactive oxygen species scavenging ability of grafted hyaluronic acid VHA and physical mixture of hyaluronic acid and Vc (n = 3);

[0043] Figure 15 :Effect of hyaluronic acid grafted with o-phenylenediamine and vitamin C on the viability of RAW264.7 cells (n = 3);

[0044] Figure 16 :Effect of hyaluronic acid grafted with o-phenylenediamine and vitamin C on the viability of MH7A cells (n = 3);

[0045] Figure 17 :Detection of NO concentration level in RAW 264.7 cells polarized by LPS by Griess method (n = 3);

[0046] Figure 18 :Observation of NO fluorescence intensity in RAW 264.7 cells polarized by LPS under inverted fluorescence microscope;

[0047] Figure 19 :Statistical analysis of fluorescence intensity for NO concentration level in RAW 264.7 cells polarized by LPS;

[0048] Figure 20 : Observe the fluorescence intensity of ROS in RAW 264.7 cells polarized by LPS under an inverted fluorescence microscope;

[0049] Figure 21 : Statistically analyze the fluorescence intensity to determine the ROS concentration level in RAW 264.7 cells polarized by LPS. Specific implementation mode

[0050] The present invention is further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art will fall within the scope of the claims of the present invention.

[0051] Grafting reaction design and preparation process:

[0052] (a) Completely dissolve hyaluronic acid (HA) in water, with the ratio of HA to water (m / V) being 1:(50 - 200). Add methyl 3,4-diaminobenzoate, and add two condensing agents 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI). The molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, HOBT, and EDCI is 1∶1 - 3∶1 - 4∶1 - 4. Stir and react at a temperature of 10 - 50 °C for 6 - 30 h, dialyze with a dialysis bag for 24 h - 48 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine (abbreviated as ODP-HA).

[0053] (b) Dissolve the obtained solid HA-o-phenylenediamine in DMSO (100V), dissolve maleic anhydride in 10V DMSO, and dropwise add the maleic anhydride solution to the system. The molar ratio of HA-o-phenylenediamine to maleic anhydride is 1:1 - 5. After dropping, raise the temperature to 20 - 60 °C and react for 6 - 30 h. Dialyze with a dialysis bag for 24 h - 48 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine-maleic acid.

[0054] (c) Dissolve the obtained solid HA-o-phenylenediamine-maleic acid in DMSO (200V) and water (100V), add HOBT (1 - 5eq), EDCI (1 - 5eq), vitamin C (Vc) (the feeding amount is 1 - 5eq), stir and react at 10 - 40 °C for 6 - 30 h, dialyze with a dialysis bag for 24 - 48 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine-maleic acid-Vc (abbreviated as VHA).

[0055] Comparative example:

[0056] Dissolve hyaluronic acid (HA) in DMSO (200V) and water (100V), add HOBT (1 - 5eq), EDCI (1 - 5eq), and vitamin C (Vc) (the feeding amount is 1 - 5eq), stir and react at room temperature (preferably 10 - 40 °C) for 6 - 30 h, dialyze with a dialysis bag for 24 - 48 h (change water 2 - 3 times), and freeze-dry to obtain a solid, that is, hyaluronic acid-Vc (abbreviated as HA-Vc) is prepared. The nuclear magnetic resonance spectrum shows that the chemical shift δ = 4.6 - 4.7, which is the chemical shift of the hydrogen (CH) on the Vc ring, indicating that VC and hyaluronic acid have successfully reacted. The nuclear magnetic data is as Figure 1 shown. For the infrared spectrum of hyaluronic acid grafted with vitamin C, it can be seen from the spectrum that 1650 cm -1 ~1750 cm -1 is the C=O stretching vibration and C=C alkene stretching vibration spectrum of the ester group, which matches the lactone and double bond structures in the Vc structure, as Figure 2 shown.

[0057] Example 1

[0058] (a) Completely dissolve hyaluronic acid (HA) in water, the ratio of HA to water (m / V) is 1:(50 - 200), add methyl 3,4-diaminobenzoate, and add two condensing agents 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), where the molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, HOBT, and EDCI is 1∶1∶1∶1; stir and react at 20 °C for 10 h, dialyze with a dialysis bag for 24 h (change water 2 - 3 times), and freeze-dry to obtain a solid, that is, HA-o-phenylenediamine (abbreviated as ODP-HA).

[0059] (b) Dissolve the obtained solid HA-o-phenylenediamine in DMSO (100V), dissolve maleic anhydride in 10V DMSO, and dropwise add the maleic anhydride solution to the system, where the molar ratio of HA-o-phenylenediamine to maleic anhydride is 1:1. After dropping, raise the temperature to 35 °C and react for 10 h, dialyze with a dialysis bag for 24 h (change water 2 - 3 times), and freeze-dry to obtain a solid, that is, HA-o-phenylenediamine-maleic acid.

[0060] (c) Dissolve the obtained solid HA-o-phenylenediamine-maleic acid in DMSO (200V) and water (100V), add HOBT, EDCI, and vitamin C (Vc), where the molar ratio of HA-o-phenylenediamine-maleic acid, HOBT, EDCI, and vitamin C is 1:1:1:1. Stir and react at 20 °C for 10 h, dialyze with a dialysis bag for 24 h (change water 2 - 3 times), and freeze-dry to obtain a solid, that is, HA-o-phenylenediamine-maleic acid-Vc (abbreviated as VHA).

[0061] Example 2

[0062] (a) Completely dissolve hyaluronic acid (HA) in water. The ratio of HA to water (m / V) is 1:(50 - 200). Add methyl 3,4-diaminobenzoate, and add two condensing agents 1-hydroxybenzotriazole (HOBT) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI). The molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, HOBT, and EDCI is 1∶2∶2∶2. Stir and react at 30 °C for 15 h, dialyze with a dialysis bag for 36 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine (abbreviated as ODP-HA).

[0063] (b) Dissolve the obtained solid HA-o-phenylenediamine in DMSO (100V), dissolve maleic anhydride in 10V DMSO, and dropwise add the maleic anhydride solution to the system. The molar ratio of HA-o-phenylenediamine to maleic anhydride is 1:4. After dropping, raise the temperature to 45 °C and react for 15 h. Dialyze with a dialysis bag for 36 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine-maleic acid.

[0064] (c) Dissolve the obtained solid HA-o-phenylenediamine-maleic acid in DMSO (200V) and water (100V), add HOBT, EDCI, and vitamin C (Vc). The molar ratio of HA-o-phenylenediamine-maleic acid, HOBT, EDCI, and vitamin C is: 1:4:4:4. Stir and react at 30 °C for 15 h, dialyze with a dialysis bag for 36 h (change water 2 - 3 times), and freeze-dry to obtain a solid, namely HA-o-phenylenediamine-maleic acid-Vc (abbreviated as VHA).

[0065] Example 3

[0066] Compared with Example 2,

[0067] In step (a), the molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, HOBT, and EDCI is 1∶3∶3∶3; stir and react at 40 °C for 25 h, and dialyze with a dialysis bag for 48 h (change water 2 - 3 times).

[0068] In step (b), the molar ratio of HA-o-phenylenediamine to maleic anhydride is 1:5. After dropping, raise the temperature to 55 °C and react for 25 h. Dialyze with a dialysis bag for 48 h (change water 2 - 3 times).

[0069] In step (c), the molar ratio of HA-o-phenylenediamine-maleic acid, HOBT, EDCI, and vitamin C is: 1:5:5:5. Stir and react at 40 °C for 25 h, and dialyze with a dialysis bag for 48 h (change water 2 - 3 times). Others are the same as in Example 2.

[0070] In Example 1, the NMR characterization of the product of reaction step c: As Figure 3As shown, the ratio of hyaluronic acid, o-phenylenediamine and Vc is close to 1:1:1.

[0071] In Example 2, the NMR characterization of the product of reaction step c: As Figure 4 shown, the ratio of hyaluronic acid, o-phenylenediamine and Vc is 1:1:4.

[0072] 1H-NMR (D2O): The chemical shift δ 1.90 (3H) is the hydrogen of the acetyl group of hyaluronic acid; δ 6.8 (3H) is the hydrogen on the benzene ring of o-phenylenediamine; the chemical shift δ = 5.8, 6.4 (2H) is the peak of the carbon-carbon double bond (CH=CH) of maleic acid; the chemical shift δ = 4.6 - 4.7 is the chemical shift of the hydrogen (CH) on the Vc ring.

[0073] In Example 3, the NMR characterization of the product of reaction step a: As Figure 5 shown, 1H-NMR (D2O): The chemical shifts δ 6.8 (1H), 7.7 (2H) are the hydrogens on the benzene ring of o-phenylenediamine. From the number of hydrogens, the molar ratio of hyaluronic acid to methyl 3,4-diaminobenzoate is 1:1.

[0074] The NMR characterization of the product of reaction step c: As Figure 6 shown, 1H-NMR (D2O): The chemical shift δ 1.96 (3H) is the hydrogen of the acetyl group of hyaluronic acid; δ 6.8 (3H) is the hydrogen on the benzene ring of o-phenylenediamine; the chemical shift δ = 5.8, 6.4 (2H) is the peak of the carbon-carbon double bond (CH=CH) of maleic acid; the chemical shift δ = 4.6 - 4.7 is the chemical shift of the hydrogen (CH) on the Vc ring.

[0075] Through the number of hydrogens in the characteristic NMR of hyaluronic acid, o-phenylenediamine, maleic acid and Vc, the NMR characterization of the final product in Example 3 shows that the ratio of hyaluronic acid, o-phenylenediamine and Vc is 1:1:4.

[0076] In Example 3, the infrared spectrum characterization of the product of reaction step a, as Figure 7 shown, compared with Figure 9 the infrared spectrum characterization of hyaluronic acid, it can be seen from the spectrum that 1550 cm -1 ~1600 cm -1 is about the N-H bending vibration spectrum of the formed amide bond; 700 cm -1 ~800 cm -1 is the out-of-plane bending vibration spectrum of the benzene ring C-H, and 1210 cm -1 ~1300 cm -1 is the C-O stretching vibration spectrum of the ester group. From the above data, it shows that a new amide bond is formed, and at the same time, the spectrum containing the benzene ring and the ester group is shown, which matches the structure of o-phenylenediamine benzoate.

[0077] The infrared spectrum of the product of reaction step c, asFigure 8 As shown, it can be seen from the spectrum that at 1650 cm -1 ~1700 cm -1 are the C=O stretching vibration and C=C alkene bond stretching vibration spectra of the ester group. At about 1550 cm -1 ~1600 cm -1 is the N-H bending vibration spectrum of the formed amide bond. At 1350 cm -1 -1450 cm -1 is the cis-butene C-H vibration. At 700 cm -1 ~800 cm -1 is the out-of-plane bending vibration spectrum of the benzene ring C-H, which is matched with the structures of o-phenylenediamine benzoate, maleic acid and Vc.

[0078] Example 4: Performance evaluation of the product prepared in Example 3

[0079] (1) Evaluation of injectability

[0080] Deionized water was used to dissolve hyaluronic acid (HA). When necessary, ultrasonic assistance was used for dissolution to prepare a series of blank HA solutions with different concentrations. The vial inversion test was used to observe their states. A 1 mL syringe (24G needle) was used to suck in the prepared HA solution and inject it into an empty 5 mL sample bottle. Photos were taken to observe its injectability. The best concentration of HA was screened based on injectability and viscosity. The results showed that when the concentration reached 20 mg / ml, it showed good injectability but slightly poor viscosity. When the concentration reached 100 mg / ml, it showed good viscous characteristics but slightly poor injectability. At a concentration of 40 - 80 mg / ml, the HA solution had both good viscous characteristics and injectability. As Figure 10 shown, the viscous and injectable characteristics of 20 - 100 mg / ml HA solution.

[0081] According to the molar ratio of hyaluronic acid in the HA solution as a reference, the concentrations of hyaluronic acid solutions modified with o-phenylenediamine (ODP-HA), vitamin C (Vc-HA), o-phenylenediamine and vitamin C (VHA) were converted. The grafted hyaluronic acid was prepared, and the vial inversion test was used to observe its viscous state, and a 1 mL syringe (24G needle) was used to observe its injectability. The best concentration was screened based on injectability and viscosity.

[0082] The research results show that after grafting o-phenylenediamine and / or Vc groups, the viscosity of hyaluronic acid is significantly improved. When converted based on the HA concentration of 40 - 80 mg / ml, for the hyaluronic acid modified with o-phenylenediamine (ODP-HA), vitamin C (Vc-HA), and o-phenylenediamine and vitamin C (VHA), when converted based on the HA concentration reaching 60 - 80 mg / ml, the prepared gel is highly viscous and difficult to inject. However, when converted based on the HA concentration of 40 mg / ml, at a VHA concentration of 70 mg / ml, it has both injectability and viscosity, as Figure 11 shown.

[0083] (2) Investigation on the response and scavenging ability of grafted hyaluronic acid to reactive oxygen species in the microenvironment

[0084] The response and scavenging ability of VHA to ROS was evaluated using the sulfopeptide colorimetric method. 24% Ti(SO 4 ) 2 and H 2 SO 4 were dissolved in distilled water to prepare the Ti(SO 4 ) 2 precursor solution. 100 μL of 1.0 mM H 2 O 2 was respectively mixed with 100 μL of the aqueous solutions of HA, ODP-HA, Vc-HA, VHA, Vc, the physical mixture of Vc and HA (Vc+HA), and the blank aqueous medium, incubated at 25 °C for 30 min, centrifuged at 12000 rpm for 2 min, 50 μL of the hydrogel supernatant was collected, 100 μL of the Ti(SO 4 ) 2 precursor solution was added, and the absorbance was measured at 405 nm in a 96-well plate to determine the H 2 O 2 concentration. The results show that HA, ODP-HA, and the blank solution did not show the response and scavenging ability to reactive oxygen species, while Vc, Vc-HA, Vc+HA, and VHA all showed good response and scavenging ability to reactive oxygen species, as Figure 12 shown.

[0085] The above results prove that the HA modified with Vc has obvious scavenging ability to reactive oxygen species. The physical mixture of HA and Vc does not affect the scavenging ability of Vc to reactive oxygen species. Only ODP grafted with o-phenylenediamine functional groups has no response and scavenging characteristics to reactive oxygen species. Compared with the Vc group and the Vc+HA physical mixture group, VHA after chemical grafting modification has the same response and scavenging ability to reactive oxygen species, indicating that chemical grafting can effectively graft Vc groups onto hyaluronic acid molecules, and the introduced o-phenylenediamine does not affect the response and scavenging ability of Vc to reactive oxygen species.

[0086] (3) Investigation on the response and scavenging ability of grafted hyaluronic acid to nitric oxide in the microenvironment

[0087] The Griess method was used to evaluate the response and scavenging ability of VHA to NO. Preparation of NO saturated solution: The NO stock solution was prepared by the Cu and dilute nitric acid method. The reaction mechanism is 3Cu + 8HNO 3 (dilute) → 3Cu(NO3)2 + 2NO↑ + 4H 2 O. The specific reaction operation is as follows: 200 ml of dilute nitric acid (volume ratio 1:4) was added to a glass flask containing 12.7 g of Cu chips and heated with an alcohol lamp. Since O 2 will rapidly oxidize NO to form NO 2 , all devices were degassed with nitrogen for 30 minutes to remove O 2 . The gas was passed through 30% NaOH solution twice to capture NO 2 produced by the reaction of NO with trace O 2 . To obtain a saturated NO solution (1.8 mM, 20 °C) as the stock solution, 10 ml of deoxygenated deionized water was bubbled with NO for 30 minutes and stored under NO gas. The stock solution for each experiment was freshly prepared and stored in a glass flask with a rubber septum.

[0088] Evaluation of the scavenging ability of different types of hyaluronic acid to NO by the Griess method: 20 μL of NO solution was added to 120 μL of HA, ODP-HA, Vc-HA, VHA aqueous solutions, and blank water medium for mixing. After incubation at 25 °C for 30 min, the remaining NO concentration was measured using the Griess assay according to the reagent supplier's protocol. The results showed that HA, Vc-HA, and the blank solution did not show NO response and scavenging ability, while ODP-HA and VHA both showed good NO response and scavenging ability, as Figure 13 shown.

[0089] The above results prove that unmodified HA, the blank medium, and Vc-HA grafted only with Vc functional groups have no response and scavenging characteristics to nitric oxide. There is no significant difference in the nitric oxide response and scavenging ability between ODP-HA grafted only with o-phenylenediamine and VHA grafted with Vc and o-phenylenediamine by chemical grafting, indicating that the introduction of Vc does not affect the response and scavenging ability of the o-phenylenediamine group to NO, further proving that chemical grafting can effectively graft groups onto hyaluronic acid molecules, and the introduced Vc does not affect the response and scavenging ability of o-phenylenediamine to NO.

[0090] (4) Evaluation of the stability of grafted hyaluronic acid materials

[0091] The stability of the carrier material was evaluated by the stability of the reactive oxygen species scavenging ability. First, an aqueous solution of VHA and an aqueous solution containing a physical mixture of Vc and HA were prepared, stored at 4 °C for 7, 4, 2, 1, 0 days, and the scavenging ability of the two groups of preparations against ROS was evaluated by the sulfopeptide colorimetric method, the same as before.

[0092] Mix 24% Ti(SO 4 ) 2 and H 2 SO 4 in distilled water to prepare a Ti(SO 4 ) 2 precursor solution. Mix 100 μL of 1.0 mM H 2 O 2 and 100 μL of the VHA, Vc and HA mixed solution respectively. After incubation at 25 °C for 30 min, centrifuge at 12,000 rpm for 2 min, collect 50 μL of the hydrogel supernatant, add 100 μL of the Ti(SO 4 ) 2 solution, and measure the absorbance at 405 nm in a 96-well plate to determine the H 2 O 2 concentration.

[0093] The results showed that the scavenging ability of the VHA group against reactive oxygen species did not change significantly with the storage time, while the scavenging ability of the physical mixture group of Vc and HA decreased significantly with the extension of the storage time, and there was basically no reactive oxygen species scavenging ability after storing for 1 day, which proved that after grafting the Vc group onto the HA monomer molecule, the oxidation rate of the Vc group could be significantly inhibited. The mechanism was mainly that there were multiple hydroxyl groups in the Vc molecule that were easily oxidized. Through chemical reactions, they were condensed with the hydroxyl groups to play a space-occupying role, reducing the oxidation probability, thereby improving the stability of the Vc group and the stability of the reactive oxygen species scavenging ability, as Figure 14 shown.

[0094] (5) Evaluation of the cytotoxicity of the grafted hyaluronic acid material

[0095] Culture of mouse mononuclear macrophages (RAW 264.7): Take out the cryopreserved cells and place them in a 37 °C constant temperature water bath for thawing. After disinfection with 75% ethanol, place them in the ultra-clean bench for standby. Take out a sterilized 15 mL centrifuge tube, add 5 mL of DMEM medium (containing 10% fetal bovine serum, 100 U / mL streptomycin and 100 U / mL penicillin), then add 1 mL of the thawed cell cryopreservation solution and mix well. After centrifugation at 1500 rpm for 5 min, discard the supernatant. Re-add 5 mL of DMEM medium to resuspend the cells and transfer them to a T25 culture flask. Add 5 mL of medium, and gently shake the culture flask in a cross shape to evenly distribute the cell suspension. Subsequently, place the culture flask in an incubator with 5% CO2 Cultured in an incubator, and the fresh DMEM medium was replaced regularly for culture, and reserved for use.

[0096] Culture of arthritis synovial fibroblasts (MH7A cells): Take out the cryopreserved cells and place them in a 37°C constant temperature water bath for thawing. After disinfecting with 75% ethanol, place them in the ultra-clean bench for standby. Take out a sterilized 15 mL centrifuge tube, add 5 mL of DMEM medium (containing 15% fetal bovine serum, 100 U / mL streptomycin and 100 U / mL penicillin), then add 1 mL of the thawed cell cryopreservation solution and mix evenly. After centrifuging at 1500 rpm for 5 min, discard the supernatant. Re-add 5 mL of DMEM medium to resuspend the cells and transfer them to a T25 culture flask. Add 5 mL of medium to supplement. Gently shake the culture flask in a cross shape to evenly distribute the cell suspension. Subsequently, place the culture flask in a 5% CO 2 Cultured in an incubator, and the fresh DMEM medium was replaced regularly for culture, and reserved for use.

[0097] Study on the viability of RAW 264.7 and MH7A cells: Inoculate the cultured RAW 264.7 and MH7A cells in sufficient numbers into 96-well plates, with 3 replicates in each group, and place them in a 37°C, 5% CO 2 Incubate in an incubator for 24 h. When the cell state is stable, discard the medium. Prepare HA solutions with concentrations of 250 μg / mL, Vc-HA solutions, ODP-HA solutions, VHA solutions, and physically mixed (Vc + ODP + HA) solutions using the corresponding medium. The concentrations of each group are calculated based on HA. Add them to the 96-well plates and co-incubate with the above cells. Use the same volume of DMEM as the blank control. After 24 h, measure the survival rate of each cell using the CCK-8 method.

[0098] The results showed that after the solutions in each group were co-incubated with MH7A cells and RAW 264.7 cells for 24 h respectively, there was no significant difference in the cell viability of each group of solutions compared with the control group, and the survival rate of each group of cells was greater than 95%, proving that the toxicity of each group of materials HA, Vc-HA, ODP-HA, and VHA to cells was negligible, as Figure 15 、 Figure 16 shown.

[0099] (6) Evaluation of the response and scavenging ability of grafted hyaluronic acid materials to nitric oxide in cells

[0100] Prepare an HA solution with a concentration of 250 μg / ml, and calculate Vc-HA, ODP-HA, VHA, and the physically mixed group (Vc + ODP + HA) based on the concentration of HA. Use the Griess method and inverted fluorescence microscopy to detect the concentration of NO in cells respectively.

[0101] Measurement of NO concentration by Griess method: RAW264.7 cells were seeded in 24-well plates at a density of 3.5×10 5 cells / well and incubated overnight in DMEM. The medium was replaced with fresh medium containing 1 μg / mL LPS. After 24 h of incubation, different formulations of DMEM medium (i.e., (1) Control+LPS (C), (2) HA, (3) ODP-HA, (4) Vc-HA, (5) VHA, (6) Vc+ODP+HA) were used to treat the cells for another 24 h. The culture medium was collected and centrifuged (3000 rpm, 10 minutes, 4 °C). The supernatant was taken and the content of NO was quantitatively detected using a Griess kit (Beyotime Biotechnology) according to the instructions.

[0102] Observation under an inverted fluorescence microscope: RAW 264.7 cells were seeded on coverslips in 6-well plates at a density of 1×10 5 cells per well and incubated at 37 °C under DMEM conditions overnight. Then the medium was replaced with fresh medium containing 1 μg / mL -1 LPS. Different groups of samples (1) Control (C), (2) C+LPS, (3) HA, (4) ODP-HA, (5) Vc-HA, (6) VHA, and (7) Vc+ODP+HA were used for another 24 h of incubation. After incubation, the sample medium was replaced with fresh medium containing 5×10 -6 M DAF-FM DA (NO-specific fluorescent dye). After another 20 min of incubation, the cells were washed three times with PBS, and observed under an inverted fluorescence microscope. Statistical analysis of the fluorescence intensity was performed.

[0103] Results of the investigation by Griess method showed that the scavenging ability of ODP-HA, VHA, and the physical mixture group (Vc+ODP+HA) for NO in the cell microenvironment reached about 50%, which was significantly different from the control group (**** in the figure represents P<0.0001), indicating good scavenging ability. There was no significant difference between VHA and the ODP-HA and physical mixture groups (Vc+ODP+HA). HA and Vc-HA had no NO scavenging ability, as Figure 17 shown.

[0104] The results of fluorescence intensity analysis of the inverted fluorescence microscope showed that there was no significant difference in the fluorescence intensity of ODP-HA, VHA, and the physical mixture group (Vc+ODP+HA) compared with the control group, and the fluorescence intensity was significantly lower compared with the LPS group, indicating that ODP-HA, VHA, and the physical mixture group (Vc+ODP+HA) all had good ability to scavenge NO in cells, while HA and Vc-HA had no NO scavenging ability. There was a significant difference in the NO scavenging ability between VHA and HA, Vc-HA, as shown in Figure 18 and Figure 19 shown (*** in the figure represents P<0.0001).

[0105] (7) Evaluation of the response and scavenging ability of grafted hyaluronic acid materials to reactive oxygen species in cells

[0106] The intracellular ROS scavenging ability of materials in each group was detected using the ROS probe DCFH-DA. RAW264.7 cells were seeded on coverslips in 24-well plates at a density of 1×10 5 cells per well, and DMEM medium was added. They were incubated at 37°C. After incubation overnight, the medium was replaced with fresh medium containing 1 μg / mL LPS. After incubation for 24 h, different formulations of DMEM medium (i.e., (1) Control, (C) (2) C+LPS, (3) HA, (4) ODP-HA, (5) VC-HA, (6) VHA, (7) VC+ODP+HA) were used. After culturing for 24 h, DCFH-DA (10 μM, DMEM without fetal bovine serum) was added to each experimental group and incubated for 20 min, and the fluorescence intensity was observed under an inverted microscope.

[0107] The results of fluorescence intensity analysis showed that there was no significant difference in the fluorescence intensity of VC-HA, VHA, and the physical mixture group (Vc+ODP+HA) compared with the control group, and the fluorescence intensity was significantly lower compared with the LPS group, indicating that VC-HA, VHA, and the physical mixture group (Vc+ODP+HA) could all scavenge ROS in cells well. However, the fluorescence intensity of ODP-HA and HA was significantly higher than that of the control group and there was no significant difference compared with the LPS group, indicating that the ODP-HA and HA groups had no ability to scavenge reactive oxygen species. There was a significant difference in the reactive oxygen species scavenging ability between VHA and ODP-HA, HA, as shown in Figure 20 and Figure 21 shown (*** in the figure represents P<0.0001).

[0108] VHA can scavenge both reactive oxygen species and nitric oxide and has stability, playing multiple roles compared to single vitamin C-modified hyaluronic acid and ODP-modified hyaluronic acid, and is more stable than the physical mixture group.

[0109] The basic principles, main features and advantages of the present invention have been shown and described above. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other embodiments obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A hyaluronic acid derivative or salt having dual response to nitric oxide and reactive oxygen species, characterized in that: The structure of the hyaluronic acid derivative is as shown in Formula I: The salt is a sodium salt, a potassium salt, a zinc salt or a calcium salt; The preparation method of the hyaluronic acid derivative or salt comprises the following steps: Step S1, dissolving hyaluronic acid in water, adding methyl 3,4-diaminobenzoate and a condensing agent, stirring for 6 to 30 hours at a temperature of 10 to 50° C. to cause a condensation reaction, and after the reaction is completed, dialysis purification and freeze-drying to obtain a product HA-o-phenylenediamine; the condensing agent is 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, wherein the molar ratio of hyaluronic acid, methyl 3,4-diaminobenzoate, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1 to 3:1 to 4:1 to 4; Step S2, dissolving the product HA-o-phenylenediamine with a solvent, adding a solution of maleic anhydride dropwise, heating at a temperature of 25-60° C. for esterification reaction for 6-30 hours, and after the reaction is completed, dialyzing and freeze-drying to obtain the product HA-o-phenylenediamine-maleic acid; wherein the molar ratio of HA-o-phenylenediamine to maleic anhydride is 1:1-5; Step S3, dissolving the product HA-o-phenylenediamine-maleic acid with a solvent and water, adding a condensing agent and vitamin C, stirring for 6 to 30 hours at a temperature of 10-50° C. to cause a condensation reaction, and after the reaction is completed, dialyzing and freeze-drying to obtain the product HA-o-phenylenediamine-maleic acid-Vc; the condensing agent is 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, wherein the molar ratio of the product HA-o-phenylenediamine-maleic acid, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and vitamin C is 1:1-5:1-5:1-5.

2. The hyaluronic acid derivative or salt according to claim 1, characterized in that In step S2, the solvent is selected from one or more of dimethyl sulfoxide, dimethylformamide, and N-methylpyrrolidone.

3. A composition, characterized in that The invention comprises the hyaluronic acid derivative or salt according to claim 1.

4. A composition according to claim 3, characterized in that The composition is in the form of a gel-like aqueous solution.

5. Use of the hyaluronic acid derivative or salt according to claim 1 in the preparation of cosmetics or medicines having lubricating, anti-inflammatory, antioxidant or moisturizing effects.

Images