CN117018282A

CN117018282A - light/temperature/pH multiple sensitivity nano composite hydrogel and preparation method thereof

Info

Publication number: CN117018282A
Application number: CN202310998444.8A
Authority: CN
Inventors: 车玉菊; 刘晓楠; 孟凡君; 马庆林; 潘世会
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-08-09
Filing date: 2023-08-09
Publication date: 2023-11-10

Abstract

The invention discloses a light/temperature/pH multiple sensitivity nano composite hydrogel and a preparation method thereof, wherein the tunica is separated from a sea squirt, and the sea squirt nanocellulose TCNCs is obtained through acidolysis; wrapping TCNCs with polydopamine to form PDA@TCNCs; quaternizing chitosan to form quaternized chitosan QCS, and then grafting alpha-cyclodextrin to form QCS-alpha-CD; connecting AZO phenyl groups to two ends of F127 to form F127AZO; mixing QCS-alpha-CD, F127AZO and PDA@TCNCs overnight to obtain the final product. The invention has the advantages of easy operation of reaction and mild condition, and the prepared nano composite hydrogel has high-efficiency light/temperature/pH multiplex sensitivity, injectability, high mechanical strength, self-repairing property and antibacterial property, and biocompatibility, and has wide application prospect in the aspects of drug slow-release systems, medical wound dressings and the like.

Description

light/temperature/pH multiple sensitivity nano composite hydrogel and preparation method thereof

Technical Field

The invention belongs to the technical field of composite hydrogels, relates to a photo/temperature/pH multiple-sensitivity high-strength self-healing injectable nanocomposite hydrogel and a preparation method thereof, and in particular relates to an injectable photo/temperature/pH multiple-sensitivity nanocomposite hydrogel with high mechanical strength, self-healing property and antibacterial property and a preparation method thereof.

Background

The skin is the largest organ of human body, and is used as a barrier to protect the human body from invasion of pathogens, prevent excessive evaporation of water, maintain the internal balance and ensure that the organ can work normally. However, the skin is vulnerable to accidental trauma. Once severely injured (e.g., full-thickness skin defects), the skin loses its ability to protect the body. Wound healing is a complex and continuous process, affected by factors, and requires a long time for recovery. Thus, the design and synthesis of wound dressings, and in particular multifunctional dressings, is critical to meeting various wound healing requirements.

Hydrogels are a three-dimensional polymer network formed by physical or chemical crosslinking, and are ideal choices for wound dressings due to their high moisture content, breathability, and excellent biocompatibility. The injectable hydrogel can adhere to the wound in situ, perfectly cover the irregular cut shape, and protect the skin defect from the harmful substances of the external environment (such as dust and microorganisms). However, developing next generation injectable hydrogels remains a challenge. In this regard, supramolecular hydrogels can provide unusable new material properties based on traditional principles, and can control the hydrogel structure reversibly and dynamically.

The intelligent response hydrogel refers to a type of hydrogel with gel properties changed when the external environment changes (such as temperature, electric field, pH value, light intensity and wavelength, solvent properties, ionic strength, pressure, etc.). Smart responsive hydrogels can generally undergo dynamic changes under specific stimuli, such as pH, temperature, light, and redox. Wherein, the temperature sensitive hydrogel can change the property (such as light transmittance, swelling degree and the like) along with the change of the external temperature, and the polymer chain of the hydrogel contains a moderately hydrophobic chain segment or both a hydrophilic chain segment and a hydrophobic chain segment. When the temperature changes, the interaction between the groups changes, the state of the molecular chains changes, and the interaction between the high molecular chains and hydrogen bonds between the high molecular chains and water molecules is influenced, so that the swelling degree or the light transmittance of the hydrogel is changed, namely the hydrogel responds to the temperature.

In addition, when the pH value of the outside changes, the hydrogel polymer chain with pH sensitivity contains an ionizable acidic or basic group (such as carboxyl, amino or sulfonic acid group and the like) to ionize, so that the hydrogen bond interaction, the ion concentration inside and outside the polymer chain and the interaction between the polymer and the solvent change, thereby changing the gel network structure. In general, the skin temperature at the injured site is high and weakly acidic. Therefore, the hydrogel dressing with pH and temperature response can realize slow release of the medicine, and is more beneficial to actual needs. In addition, for hydrogels containing photosensitive molecules, light can be used to control their molecular structure remotely with high efficiency. Therefore, the supermolecular hydrogel with light, temperature and pH value responsiveness can be used as a novel multifunctional wound dressing with great potential in the medical field.

Chitosan (CS) is a natural polysaccharide with a wide range of antibacterial properties and biodegradability. Due to the abundant amino groups, the modified and crosslinked amino groups are easy. By introducing quaternary ammonium salt groups into CS, the synthesized quaternary ammonium salt chitosan (QCS) has stronger water solubility and stronger antibacterial ability than chitosan, and shows good wound dressing potential. The hydrogel wound dressing with inherent antibacterial property has lasting antibacterial activity, and thus becomes a hot spot for antibacterial material research. However, conventional chitosan-based hydrogels exhibit weak mechanical properties and rapid degradation behavior, which greatly limit their applications.

The appearance of the nano composite hydrogel brings new thought and method for solving the problem of low strength of the hydrogel. For example, nanomaterials (such as silicon, hydroxyapatite, carbon nanotubes, graphene oxide, etc.) can be incorporated into the hydrogels to increase the mechanical strength of the hydrogels. However, their potential toxicity and unknown metabolic pathways limit the further use of these inorganic nanoparticles in injectable hydrogels. Some nano fillers of natural polymers not only can improve the mechanical strength of the hydrogel, but also can improve the biocompatibility of the hydrogel, and become a big hot spot for research in recent years. For example, by physical doping, sea squirt nanocellulose (TCNCs) is introduced. TCNCs are coating capsules obtained from sea animals, such as ascidians, which have better biocompatibility, higher specific surface area, higher tensile strength and higher young modulus than nanocellulose from other sources, and can be introduced into high polymer materials as reinforcing agents; meanwhile, only the viscera of the sea squirt is taken for use in the life production, so that the envelope is wasted greatly, and the extraction of TCNCs from the sea squirt envelope can greatly reduce environmental pollution and improve economic benefit. TCNCs is thus a novel nanomaterial with great potential for development.

In conclusion, the preparation of TCNCs-enhanced photo/temperature/pH multisensitive injectable nanocomposite hydrogels with self-healing and antibacterial properties has great practical significance.

Disclosure of Invention

Accordingly, the present invention is directed to providing a high-strength self-healing injectable nanocomposite hydrogel having multiple sensitivity to light/temperature/pH, in view of the problems existing in the prior art.

The first technical aim of the invention is to provide a light/temperature/pH multiple sensitivity nano composite hydrogel. In order to achieve the above object, the technical scheme of the present invention is as follows:

the light/temperature/pH multiple sensitivity nano composite hydrogel is prepared by mixing three components under physiological conditions; quaternizing chitosan to form Quaternized Chitosan (QCS), grafting alpha-cyclodextrin (alpha-CD) on the QCS to generate quaternized chitosan grafted cyclodextrin (QCS-alpha-CD); grafting AZO phenyl groups (AZO) at two ends of PF127 to form F127AZO; wrapping TCNCs with polydopamine to form PDA@TCNCs; mixing QCS-alpha-CD, F127AZO and PDA@TCNCs with a certain concentration overnight to obtain a nano composite hydrogel product, and freeze-drying to obtain a solid product.

Specifically, AZO groups on F127AZO enter a cyclodextrin cavity on QCS-alpha-CD, and interaction between a host and a guest occurs; oxidizing dopamine in polydopamine to form carbonyl in a quinone structure and amino in chitosan to form Schiff base; in addition, polydopamine coated sea squirt nanocellulose (PDA@TCNCs) is uniformly dispersed in the hydrogel network and forms physical crosslinking with a large number of hydrogen bonds generated between molecular chains, so that the product nano composite hydrogel disclosed by the invention is obtained;

wherein the quaternized chitosan grafted alpha-cyclodextrin (QCS-alpha-CD) molecular chain is

p, l, q are natural numbers of 1, 2,3, 4 and …;

the structural formula of F127AZO is as follows:

further, the structural formula of the photo/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel disclosed by the invention is shown in the attached figure 1 of the specification. And as shown in figure 1, the nano composite hydrogel contains F127 AZO/alpha-CD host guest interaction with light sensitivity, F127AZO with temperature sensitivity and Schiff base structure with pH sensitivity, and the composite hydrogel has multiple sensitivity to light, temperature and pH due to the structure of the composite hydrogel.

Still further, the nanocomposite hydrogel photosensitivity comes mainly from the photosensitive molecule Azobenzene (AZO), which interacts with α -cyclodextrin (α -CD) in aqueous solution as a host guest, which is photo-responsive. AZO has two geometric isomers: trans and cis, which undergo structural transformations upon irradiation with light of different wavelengths. When the ultraviolet light with the wavelength of 320-370nm is irradiated, the configuration of the AZO is converted from trans to cis, and the AZO is taken out of the alpha-CD cavity; and when irradiated by 400-450nm visible light, the light returns to the trans configuration and interacts with the alpha-CD host again. This reversible, light-sensitive, non-covalent attachment gives the hydrogel network reversible changes in network density and good self-healing capabilities.

The temperature sensitivity of the nano composite hydrogel mainly comes from F127AZO, and the nano composite hydrogel can be self-assembled into micelles in water and can be used as drug-loaded micelles to wrap small molecular drugs such as curcumin, and the solubility of the drugs is improved. PF127 is a typical amphiphilic triblock copolymer that self-assembles in water to form micelles. PF127 aggregates into micelles due to hydrophobic interactions between polyoxypropylene (PPO) blocks. As the temperature increases, the self-assembled PF127 micelles align more tightly, causing the hydrogel structure to change.

The pH sensitivity of the nano composite hydrogel is mainly derived from Schiff base formed by amino groups on chitosan and carbonyl groups on polydopamine surface quinone, and formed dynamic Schiff base bonds are decomposed under acidic conditions, so that the pH responsiveness of the hydrogel is endowed.

It should be noted that, the self-repairing property of the nanocomposite hydrogel is derived from the dynamic reversible schiff base bond and the host-guest complex, when the hydrogel structure is damaged, the schiff base bond and the host-guest complex are broken, and meanwhile, new schiff base bond and host-guest complex are continuously generated without any external stimulus and energy, so that the self-healing function of the hydrogel can be realized. The conventional injectable hydrogel is a hydrogel material which can be used for in-situ gelation and fixation at a target site after a precursor solution (an aqueous solution formed by components forming the hydrogel) is injected to the target site by a syringe, so that the hydrogel disclosed by the invention is a novel injectable hydrogel with self-healing property.

The high mechanical strength of the nano composite hydrogel is mainly derived from the physical enhancement effect of ascidian nanocellulose (TCNCs), and compared with nanocellulose from other sources, the ascidian nanocellulose (TCNCs) has the characteristics of high specific surface area, high tensile strength, high Young modulus, high hydrophilicity, high crystallinity and the like, and is a novel nanomaterial with great development potential. When the sea squirt nanocellulose (TCNCs) is applied to hydrogel, a three-dimensional space network structure can be formed with the hydrogel, so that the sea squirt nanocellulose has better mechanical strength, the defects of poor performance, high brittleness, easy deformation and the like of a single-component polymer material are overcome, and meanwhile, the sea squirt nanocellulose (TCNCs) has the advantages of natural raw materials, and waste can be biodegraded. In recent years, ascidian nanocellulose (TCNCs) has been used to reinforce high molecular polymers.

The second technical aim of the invention is to provide a preparation method of the light/temperature/pH multiple sensitivity nano composite hydrogel.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a preparation method of a light/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel, which specifically comprises the following steps:

(1) Preparation of ascidian nanocellulose (TCNCs): separating the tunica media from the sea squirt, hydrolyzing with sulfuric acid, standing for 12-24h, centrifuging, dialyzing with deionized water to neutrality to obtain uniform sea squirt nanocellulose suspension, and concentrating and lyophilizing the prepared sea squirt nanocellulose suspension by rotary evaporation method;

(2) Preparation of polydopamine-coated ascidian nanocellulose (pda@tcncs): diluting the TCNCs suspension prepared in the step (1) to a certain concentration and uniformly dispersing, and then adjusting the pH value to 8.5 by using a Tris buffer solution; adding dopamine hydrochloride, stirring at room temperature for 12-36h, and changing the color of the suspension from white to pink and then black; centrifuging the reaction product (with the rotating speed of 10000 r/min) and repeatedly washing with deionized water for several times to be neutral (pH=7.4), obtaining PDA@TCNCs suspension, and concentrating for later use;

(3) Preparation of Quaternized Chitosan (QCS): dispersing chitosan in acetic acid solution, adding a proper amount of 2, 3-epoxypropyl trimethyl ammonium chloride (GTMAC), continuously stirring at 50-65 ℃ for reaction for 12-36 hours, centrifuging the reaction solution after the reaction is finished, collecting supernatant, dialyzing the supernatant for 5-7 days, concentrating by a rotary evaporation method, and freeze-drying to obtain a white cotton-like product;

(4) Preparation of quaternized chitosan grafted α -cyclodextrin (QCS- α -CD): dissolving alpha-CD in NaOH solution, slowly adding a crosslinking agent Epichlorohydrin (ECH) into the solution, fully stirring for 4-6 hours at room temperature, uniformly dispersing the QCS prepared in the step (3) in water, adding the water into a reaction system, continuously reacting for 4-6 hours, dialyzing the product for 5-7 days after the reaction is finished, and concentrating and freeze-drying to obtain a white cotton-like product;

(5) Preparation of PF127 grafted azobenzene (F127 AZO): PF127 was dried in vacuo at 110℃for 4 hours before the reaction, and then PF127, 4- (phenylazo) benzoic acid, a catalyst and a dehydrating agent were dissolved in anhydrous methylene chloride, and the reaction was continued with stirring at room temperature for 24-72 hours. Filtering to remove byproducts after the reaction is finished, precipitating with diethyl ether and collecting the products;

(7) Preparation of hydrogels: PDA@TCNCs, QCS-alpha-CD and F127AZO prepared in (2), 4 and 5) are prepared into a water solution with a certain concentration, and the water solution is mixed under physiological conditions to obtain the hydrogel.

By adopting the technical scheme, the invention has the following beneficial effects:

the preparation method disclosed by the invention is easy to control production operation and temperature, mild in reaction condition, low in cost, easy to operate and suitable for popularization.

Preferably, in step (1), the TCNCs suspension has a mass fraction of 1.0wt%.

Preferably, in the step (2), the mass fraction of the PDA@TCNCs solution is 1.0-5.0 wt%.

Preferably, in step (5), the catalyst is 4-Dimethylaminopyridine (DMAP) and the dehydrating agent is Dicyclohexylcarbodiimide (DCC).

Preferably, in the step (6), the PDA@TCNCs account for 0.5-2.5% of the total mass of the hydrogel system; the mass fraction of the QCS-alpha-CD in the total mass of the hydrogel system is 4.0-6.0%; the F127AZO accounts for 10.0-26.0% of the total mass of the hydrogel system.

Compared with the prior art, the invention provides the photo/temperature/pH multi-sensitivity nano composite hydrogel and the preparation method thereof, which have the following excellent effects:

1. the TCNCs disclosed by the invention has wide sources, good biocompatibility and degradability, and is environment-friendly; meanwhile, TCNCs have higher length-diameter ratio and mechanical modulus, and are ideal nano reinforcing fillers.

2. The preparation method disclosed by the invention is easy to control production operation and temperature, mild in reaction condition, low in cost and easy to operate.

3. The nano composite hydrogel prepared by the invention has multiple sensitivity to light, temperature and pH due to the interaction of F127 AZO/alpha-CD host guest with sensitivity to light, F127AZO with sensitivity to temperature and Schiff base structure with pH sensitivity.

4. The nano composite hydrogel prepared by the method disclosed by the invention has good injectability, self-repairing property, antibacterial property and biocompatibility, is suitable for industrial production, and has wide application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic structural diagram of a nanocomposite hydrogel prepared according to the present invention.

FIG. 2 is an infrared (FT-IR) spectrum of a reactive material and hydrogel of the present invention; (a) is the infrared (FT-IR) spectrum of the hydrogel, (b) is the infrared (FT-IR) spectrum of F127AZO, (c) is the infrared (FT-IR) spectrum of QCS-alpha-CD, and (d) is the infrared (FT-IR) spectrum of QCS.

FIG. 3 is an X-ray diffraction (XRD) pattern of the reactive material and hydrogel of the present invention; (a) is the X-ray diffraction (XRD) pattern of QCS-alpha-CD, (b) is the X-ray diffraction (XRD) pattern of PDA@TCNCs, (c) is the X-ray diffraction (XRD) pattern of hydrogel,

FIG. 4 is a thermogravimetric analysis (TGA) profile of a nanocomposite hydrogel prepared according to the present invention.

Fig. 5 is a frequency sweep plot of the rheological properties of various compositions of hydrogels prepared in accordance with the present invention.

FIG. 6 is a temperature sweep pattern of the rheological properties of various compositions of hydrogels prepared in accordance with the present invention.

FIG. 7 is a surface Scanning Electron Microscope (SEM) map of the nanocomposite hydrogels prepared according to the present invention under different illumination conditions.

FIG. 8 is a graph of macroscopic self-healing properties of nanocomposite hydrogels of the present invention.

FIG. 9 is a graph of the rheological recovery properties of a nanocomposite hydrogel of the present invention when the strain value is converted from 10% to 60%.

FIG. 10 is a graph of macroscopic injectability properties of the nanocomposite hydrogels of the present invention.

FIG. 11 is a graph of the bacteriostatic properties of the nanocomposite hydrogels of the present invention.

FIG. 12 is a graph showing changes in drug release rates of the nanocomposite hydrogels of the present invention at pH 7.4 and 6.0, respectively, at 25 ℃.

FIG. 13 is a graph showing changes in drug release rates of the nanocomposite hydrogels of the present invention at pH 7.4 and 6.0, respectively, at 37 ℃.

FIG. 14 is cell viability data of the extract culture mouse mammary epithelial cell line (MCF-10A) of the nanocomposite hydrogels of the invention.

FIG. 15 is an in vivo evaluation of the nanocomposite hydrogels of the present invention to promote wound healing: (a) wound photographs on days 0, 7 and 13, (b) skin wound healing follow-up map, (c) wound shrinkage (n=6, < P < 0.05)

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses a preparation method of a light/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel, which is mild in reaction condition, easy to control and low in cost.

The present invention will be further specifically illustrated by the following examples, which are not to be construed as limiting the invention, but rather as falling within the scope of the present invention, for some non-essential modifications and adaptations of the invention that are apparent to those skilled in the art based on the foregoing disclosure.

The technical scheme of the invention will be further described below with reference to specific embodiments.

The reagents and starting materials used in the examples were as follows:

reagent: sea squirt, shandong province flourishing into a city and searching mountain group;f127, analytically pure, shanghai aladine Biochemical technologies Co., ltd; chitosan (number average molecular weight 50000), analytically pure, sienna huakang biotechnology limited; dopamine hydrochloride, analytically pure, shanghai Ala Biochemical technologies Co., ltd; 2, 3-epoxypropyl trimethyl ammonium chloride, analytically pure, shanghai Ala Biochemical technologies Co., ltd; 4- (phenylazo) benzoic acid, analytically pure, shanghai Ala Biochemical technologies Co., ltd; n, N' -Dicyclohexylcarbodiimide (DCC), analytically pure, shanghai Ala Latin Biochemical technologies Co., ltd; 4-Dimethylaminopyridine (DMAP), analytically pure, shanghai Ala Biochemical technologies Co., ltd; epichlorohydrin (ECH), analytically pure, shanghai aladine Biochemical technologies Co., ltd; alpha-cyclodextrin (alpha-CD), biochemical grade, anhui Hirsche CorpThe method comprises the steps of carrying out a first treatment on the surface of the Tris (hydroxymethyl) aminomethane (Tris) hydrochloride, analytically pure, shanghai Ala Biochemical technologies Co., ltd.

Medicine: curcumin (CUR) analytically pure, beijing carboline technologies limited.

Other raw materials: dichloromethane, analytically pure, tabacco to double chemical industry limited; concentrated sulfuric acid, analytically pure, tabacco to double chemical company, inc; n-hexane, analytically pure, tianjin, fuyu fine chemical Co., ltd; distilled water (H) ₂ O), laboratory homemade.

Example 1

The preparation method of the photo/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel specifically comprises the following steps:

step (1): separating the tunica element from the sea squirt, hydrolyzing with acid, standing for 12h, centrifuging, and dialyzing with deionized water to neutrality to obtain uniform sea squirt nanocellulose suspension.

Step (2): diluting the ecteinascidia nanocellulose suspension prepared in the step (1) to 0.5 weight percent, uniformly dispersing, adjusting the pH value to 8.5 by using a Tris buffer solution, adding dopamine hydrochloride equivalent to nanocellulose, stirring for 24 hours at room temperature, repeatedly centrifuging and washing, and concentrating to obtain the PDA@TCNCs suspension.

Step (3): 0.5g of chitosan was dispersed in 18.0mL of acetic acid solution (0.5%, v/v), followed by addition of 0.47g of 2, 3-epoxypropyltrimethylammonium chloride (GTMAC), and the reaction was continued with stirring at 55℃for 18 hours; after the reaction, the reaction solution was centrifuged and the supernatant was collected, and the supernatant was dialyzed for 5 to 7 days, and then concentrated by rotary evaporation and lyophilized to give a white cotton-like product.

Step (4): dissolving alpha-CD in NaOH solution, slowly adding cross-linking agent Epichlorohydrin (ECH) into the solution, and fully stirring at room temperature for 4.5 hours; after uniformly dispersing the QCS prepared in the step (3) in water, adding the QCS into a reaction system, and continuously reacting for 5 hours; after the reaction is completed, the product is dialyzed for 5-7 days, then concentrated and lyophilized to a white cotton-like product.

Step (5): PF127 was dried in vacuo at 110℃for 4 hours before the reaction, and then PF127 (4.0 g,0.317 mmol), 4- (phenylazo) benzoic acid (0.215 g,0.951 mmol), DMAP as a catalyst (0.058 g,0.475 mmol) and DCC as a dehydrating agent (0.196 g,0.951 mmol) were dissolved in 20mL of anhydrous methylene chloride and the reaction was continued with stirring at room temperature for 48 hours; after the reaction was completed, the by-product was removed by filtration, precipitated with diethyl ether and the product was collected.

Step (7): PDA@TCNCs, QCS-alpha-CD and F127AZO prepared in the steps (2), (4) and (5) are prepared into aqueous solutions with certain concentration according to the amounts accounting for 0.7%, 5% and 15% (wt/vol) of the hydrogel system respectively, and the aqueous solutions are mixed and kept stand overnight under physiological conditions to obtain the hydrogel.

Example 2

As described in example 1, except that: the amount of F127AZO is changed to 25%, other preparation process conditions and process parameters are unchanged, and the nano composite hydrogel with multiple sensitivity of light/temperature/pH is prepared.

Example 3

As described in example 1, except that: the amount of PDA@TCNCs is changed to 1.4%, other preparation process conditions and process parameters are unchanged, and the nano composite hydrogel with light/temperature/pH multiple sensitivity is prepared.

Example 4

As described in example 1, except that: the amount of PDA@TCNCs is changed to 2.1%, other preparation process conditions and process parameters are unchanged, and the nano composite hydrogel with light/temperature/pH multiple sensitivity is prepared.

Example 5

As described in example 1, except that: the amount of F127AZO is changed to 10 percent, other preparation process conditions and process parameters are unchanged, and the prepared photo/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel is prepared.

Example 6

As described in example 1, except that: the amount of the QCS-alpha-CD solution is changed to 8.0 percent, and other preparation process conditions and process parameters are unchanged, so that the nano composite hydrogel with multiple sensitivity of light/temperature/pH is prepared.

Example 7

As described in example 1, except that: the QCS-alpha-CD amount is changed to 12.0%, and other preparation process conditions and process parameters are unchanged, so that the nano composite hydrogel with multiple sensitivity of light/temperature/pH is prepared.

Example 8

Curcumin and F127AZO prepared in step (5) were dissolved in dichloromethane with slow stirring, after which the solution was concentrated using a rotary evaporator. Then, adding proper deionized water under continuous slow stirring to self-assemble F127AZO into micelle. Finally, obtaining yellow Cur-F127AZO powder by a freeze drying method.

Example 9

As described in example 1, except that F127AZO was replaced with Cur-F127AZO carrying curcumin prepared in example 8, the other process conditions and process parameters were unchanged, and a photo/temperature/pH multiple sensitivity high-strength self-healing injectable drug-carrying nanocomposite hydrogel was prepared.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

To further verify the excellent effects of the present invention, the inventors have also performed the following characterization; the nomenclature and corresponding amount of the added materials of each hydrogel sample are shown in Table 1:

TABLE 1 nomenclature of hydrogel samples prepared according to the invention and the corresponding amounts of raw materials added

Infrared spectroscopic analysis of the reaction mass and hydrogel:

the reaction mass and the hydrogel were subjected to infrared testing as shown in fig. 2. In the spectrum of QCS, the methyl band of GTMAC is 1478cm ^-1 There is a significant characteristic peak, indicating that GTMAC was successfully coupled to the amino group of chitosan; in the sugar ring of the alpha-CD group, 3373cm ^-1 The broad peak as the center isThe O-H telescopic vibration absorption peak indicates that the α -CD was successfully grafted to the backbone of the QCS; the stretching vibration of the-C=O bond of F127AZO is 1700cm ^-1 Obvious peak appears at 1600-1500cm ^-1 The signal at the position is a benzene skeleton vibration absorption peak, which indicates that azo groups are successfully introduced into the tail end of F127; hydrogel at 3700cm ^-1 Has a wide peak value of O-H stretching vibration of 1600cm ^-1 Is the characteristic absorption peak of Schiff base bond formed by the amino group of QCS-alpha-CD and the quinone group of PDA@TCNCs, indicating successful formation of hydrogel network.

X-ray diffraction analysis of the reaction mass:

XRD analysis was performed on the reaction mass and the hydrogel, as shown in FIG. 3. Characteristic peaks of QCS-alpha-CD and PDA@TCNCs appear, namely II type crystals of chitosan and I beta type crystals of TCNCs at about 20 degrees, which indicate that hydrogel is successfully formed.

(III) thermogravimetric analysis of hydrogels:

the hydrogels were subjected to thermogravimetric analysis as shown in figure 4. The thermal stability of the hydrogels was determined, with the exception of the initial weight loss peak of bound water at about 71.67 ℃, which was mainly followed by two weight loss peaks. The first peak weight loss occurs at 250-350 c and the maximum weight loss occurs at 314.67 c due to TCNCs and PDA chain breakage and QCS-a-CD decomposition. The second weight loss peak is between 350 and 440 ℃ and the maximum weight loss is 401.83 ℃, probably due to the disruption of the polymer backbone of F127AZO, CS and TCNCs, and the carbonization process of the organic molecules.

(IV) frequency sweep analysis of rheological properties of hydrogels of different compositions:

the hydrogels of different compositions were subjected to modulus scans over a range of frequencies as shown in fig. 5. The oscillation frequency sweep is performed in a frequency range of 0.1-10.0 Hz. The G' value is significantly greater than the G "value over the entire frequency range, which means that the supramolecular hydrogels have predominantly elastic properties. Furthermore, as pda@tcncs content and F127 hydrogel AZO content increased, G' of these hydrogels gradually increased from 902.38Pa to 8982.23Pa and from 902.38Pa to 1209.54Pa, respectively, due to increased intermolecular hydrogen bonds, schiff base bonds, and host/guest crosslinks.

(V) temperature scanning analysis of rheological properties of hydrogels of different compositions:

hydrogels of different compositions were subjected to modulus scans over a range of temperatures as shown in fig. 6. As the temperature increases, the G' value of all hydrogels increases sharply at the critical temperature, indicating an increase in hydrogel crosslink density. Furthermore, as pda@tcncs and F127AZO concentrations increased, the critical temperature decreased from 42.43 ℃ to 37.23 ℃, as the increase in hydrogel crosslink density increased with the addition of the crosslinker.

Morphology analysis of hydrogels:

based on the nanocomposite hydrogel prepared in example 1, the morphology of the nanocomposite hydrogel under different illumination conditions was analyzed, as shown in fig. 7. After 5min of irradiation with 365nm light, the three-dimensional network portion of the hydrogel was observed to have disappeared, indicating that a structural change was induced due to the disruption of host-guest interactions. Under ultraviolet light, azo changes into a trans-form structure, which separates from the inner cavity of the CD, thereby significantly affecting the structural integrity of the hydrogel. When the hydrogel was irradiated under 420nm light for 20min, a similar three-dimensional crosslinked structure was again observed, indicating that the lattice structure of the hydrogel was also restored due to the restoration of the azo group to the cis structure.

(seventh) self-healing Properties of hydrogels:

macroscopic analysis of the self-healing properties of hydrogels was performed as shown in figure 8. The hydrogel is cut into two semicircular shapes, then the sections are closely placed together, the sections are placed at room temperature for 2 hours, the hydrogel is self-healed to form an original disc shape, and the hydrogel can be clamped by forceps, so that the gel can bear the gravity of the hydrogel after healing.

(eight) rheology recovery test of hydrogels:

the self-healing behavior of the hydrogels was evaluated using a rheology recovery test, as shown in figure 9. The hydrogel is first subjected to 10% strain in the linear viscoelastic region and then 100% strain is applied to the linear viscoelastic region. When the strain was increased to 100%, the G 'of the hydrogel was decreased from 1907.20Pa to 241.41Pa, and the hydrogel became a sol state with G "G > G', indicating collapse of the hydrogel network and shear thinning characteristics of the hydrogel. When the strain was reduced to 10%, the G' quickly recovered to its near initial value, indicating that the hydrogel had good repair properties. The self-healing capacity of hydrogels is mainly derived from the physical cross-linking action of pda@tcncs, the dynamic covalent schiff base bond between the amine groups of QCS- α -CD and the quinone groups of pda@tcncs, and the host-guest interactions between QCS- α -CD and F127 AZO.

Injectability of hydrogels:

the injectability of the hydrogels was analyzed macroscopically as shown in FIG. 10. It can be seen that the hydrogel can be extruded through a 23 gauge needle without causing any blockage and can be injected into the desired shape, indicating that it has good injectability.

Antibacterial properties of hydrogels:

the antibacterial properties of the hydrogels were analyzed as shown in fig. 11. Coli (gram negative bacteria) and staphylococcus aureus (gram positive bacteria) were used to evaluate the surface antimicrobial activity of the hydrogels. The hydrogel is contacted with bacteria for 3 hours at 37 ℃, and has good killing rate to staphylococcus aureus and escherichia coli, which shows that the hydrogel has excellent inherent antibacterial property.

Drug release properties of hydrogels:

drug release studies aimed at discussing the smart responsive release behavior of curcumin-loaded hydrogels, as shown in fig. 12, 13. The effect of pH, light irradiation, temperature, etc. on drug release behavior was studied. Drug (curcumin) loaded hydrogels have faster release rates at pH 6.0 than at pH 7.4 due to the destruction of the schiff base in acidic media. In addition, the release of the drug is also affected by light. The medicine is irradiated by ultraviolet (365 nm,50 mWcm) ^-2 ) The release speed is faster and the release amount is more. This result shows that the curcumin-loaded hydrogel has light-responsive controlled release characteristics and is suitable for use in light-controllable delivery systems when rapid release is required.

As can be seen from fig. 13, the hydrogel exhibited similar pH and light responsive drug release behavior at 37 ℃ as 25 ℃. The drug release rate at day 1 was faster than at 25 ℃, probably due to the higher temperature promoting the diffusion of curcumin. The cumulative drug release on day 9 was lower than at 25 c, probably due to the tighter F127AZO micelle arrangement as the temperature was increased, resulting in a tighter hydrogel structure.

Cell compatibility of (twelve) hydrogels:

cell viability of the mouse mammary epithelial cell line (MCF-10A) was cultured using the hydrogel extract as shown in fig. 14. During cell culture, the cell proliferation of each hydrogel group of the hydrogel group has a remarkable increasing trend, and after 4 days of culture, the cell activity reaches more than 400%, so that the hydrogel has good biocompatibility.

Analysis of wound healing promoting Properties of (thirteen) nanocomposite hydrogels:

the wound healing capacity of the hydrogels was evaluated by constructing a rat full-thickness skin defect model. Fig. 15 shows wound healing of wounds on day 0, day 7 and day 13 control, blank hydrogel, hydrogel-loaded (L-), hydrogel-loaded (l+) (fig. 15 a) and follow-up records of the healing process (fig. 15 b). It can be seen that the hydrogel-loaded group (l+) treated wounds recovered faster than the other groups. The final wound shrinkage was 99.6% for the hydrogel-loaded (L+) group, while the wound shrinkage was approximately 85.2%, 86.8% and 98.3% for the control, blank and hydrogel-loaded (L-). This is probably due to the increased release of curcumin by uv radiation, which has a good anti-inflammatory effect, and the control of inflammation contributes to wound healing.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The light/temperature/pH multiple sensitivity high-strength self-healing injectable nano composite hydrogel is characterized in that the composite hydrogel is prepared by mixing PDA@TCNCs, QCS-alpha-CD and F127AZO under physiological conditions;

the QCS-alpha-CD is formed by grafting quaternized chitosan QCS with alpha-cyclodextrin, and the molecular chain of the QCS-alpha-CD is as follows:

wherein, p, l and q are all 1, 2,3 and 4 … natural numbers;

the F127AZO is formed by grafting AZO phenyl groups AZO at two ends of the F127, and the structural formula of the F127AZO is as follows:

2. a method for preparing the light/temperature/pH multisensitive high-strength self-healing injectable nanocomposite hydrogel according to claim 1, comprising the steps of:

(1) Preparation of ascidian nanocellulose TCNCs: separating tunicalin from sea squirt, acidolysis, standing, centrifuging, and dialyzing with deionized water to neutrality to obtain uniform sea squirt nanocellulose suspension; evaporating, concentrating and freeze-drying the sea squirt nanocellulose suspension to obtain the sea squirt nanocellulose TCNCs for later use;

(2) Preparing polydopamine coated sea squirt nanocellulose PDA@TCNCs: preparing the TCNCs prepared in the step (1) into suspension with a certain concentration, uniformly dispersing, and then adjusting the pH value to 8.5 by using a Tris buffer solution; adding dopamine hydrochloride, stirring at room temperature for 12-36h for reaction, centrifuging and washing the reaction product after the reaction is finished to obtain PDA@TCNCs suspension, and concentrating for later use;

(3) Preparation of quaternized chitosan QCS: dispersing chitosan in acetic acid solution, adding 2, 3-epoxypropyl trimethyl ammonium chloride GTMAC, continuously stirring at 50-65 ℃ for reaction for 12-36 hours, centrifuging the reaction solution after the reaction is finished, and collecting supernatant; dialyzing the supernatant for 5-7 days, evaporating, concentrating, and lyophilizing to obtain the final product;

(4) Preparing quaternized chitosan grafted alpha-cyclodextrin QCS-alpha-CD: dissolving alpha-CD in NaOH solution, adding epoxy chloropropane ECH serving as a crosslinking agent into the solution, stirring for 4-6 hours at room temperature, and then adding QCS prepared in the step (3) for continuous reaction for 4-6 hours; after the reaction is finished, the product is obtained through dialysis, concentration and freeze-drying;

(5) Preparing PF127 grafted azobenzene F127AZO: PF127, 4- (phenylazo) benzoic acid, a catalyst and a dehydrating agent are dissolved in anhydrous dichloromethane, the mixture is continuously stirred at room temperature for reaction for 24 to 72 hours, and after the reaction is finished, the mixture is filtered, and the product is precipitated and collected;

(6) Preparation of hydrogels: preparing PDA@TCNCs prepared in the step (2), QCS-alpha-CD prepared in the step (4) and F127AZO prepared in the step (5) into aqueous solution, mixing and standing overnight under physiological conditions, and obtaining the light/temperature/pH multiple sensitivity nano composite hydrogel.

3. The method for preparing the light/temperature/pH multisensitive high-strength self-healing injectable nanocomposite hydrogel according to claim 2, wherein the step (1) is specifically performed as follows:

separating the tunica element from the sea squirt, hydrolyzing with sulfuric acid, standing for 12-24h, centrifuging, dialyzing with deionized water to neutrality to obtain uniform sea squirt nanocellulose suspension, and lyophilizing the obtained sea squirt nanocellulose suspension with a lyophilizing machine to obtain dried and white flocculent sea squirt nanocellulose.

4. A method of preparing a photo/temperature/pH multisensitive high strength self-healing injectable nanocomposite hydrogel according to claim 2 or 3, wherein in step (1) the mass fraction of the ascidian nanocellulose suspension is 1.0wt%.

5. The method for preparing the light/temperature/pH multisensitive high-strength self-healing injectable nanocomposite hydrogel according to claim 2, wherein the step (2) is specifically performed as follows:

and (3) regulating the pH value of the sea squirt nanocellulose suspension to 8.5 by using a Tris buffer solution, then adding dopamine hydrochloride, stirring at room temperature for 12-36 hours for reaction, centrifuging a reaction product after the reaction is finished, washing with water at the rotating speed of 10000r/min to obtain a neutral PDA@TCNCs suspension, and concentrating for later use.

6. The method of preparing a photo/temperature/pH multisensitive high strength self-healing injectable nanocomposite hydrogel according to claim 2 or 5, wherein in step (2), the mass fraction of pda@tcncs suspension is 1.0% -5.0%.

7. The light/temperature/pH multisensitive high strength self-healing injectable nanocomposite hydrogel according to claim 2, wherein the QCS in step (3) is prepared by: dispersing chitosan CS in acetic acid solution, adding 2, 3-epoxypropyl trimethyl ammonium chloride GTMAC, continuously stirring at 50-65 ℃ for reaction for 12-36 hours, centrifuging the reaction solution after the reaction is finished, and collecting supernatant; dialyzing the supernatant for 5-7 days, evaporating, concentrating, and lyophilizing.

8. The method for preparing a photo/temperature/pH multisensitive high strength self-healing injectable nanocomposite hydrogel according to claim 2, wherein the QCS- α -CD in step (4) is prepared by: dissolving alpha-CD in NaOH solution, adding epoxy chloropropane ECH serving as a crosslinking agent into the solution, stirring for 4-6 hours at room temperature, and then adding the prepared QCS to continuously react for 4-6 hours; after the reaction is finished, the product is obtained through dialysis, concentration and freeze-drying.

9. The method for preparing a photo/temperature/pH multiple sensitive high strength self-healing injectable nanocomposite hydrogel according to claim 2, wherein in step (5), the catalyst is 4-dimethylaminopyridine DMAP and the dehydrating agent is dicyclohexylcarbodiimide DCC.

10. The method for preparing a photo/temperature/pH multiple sensitive nanocomposite hydrogel according to claim 2, wherein in step (6), pda@tcncs is 0.5 to 2.5% of the total mass of the hydrogel system, QCS- α -CD is 4.0 to 6.0% by weight of the total mass of the hydrogel system, and F127AZO is 10.0 to 26.0% of the total mass of the hydrogel system.