CN115501383A

CN115501383A - Preparation and application of near-infrared two-region response nano composite temperature-sensitive hydrogel

Info

Publication number: CN115501383A
Application number: CN202210528977.5A
Authority: CN
Inventors: 郑磊; 陈金香; 潘炜伦; 吴柏灯; 聂城涛; 李博; 刘春辰
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2022-05-16
Filing date: 2022-05-16
Publication date: 2022-12-23
Anticipated expiration: 2042-05-16
Also published as: CN115501383B

Abstract

The invention discloses preparation and application of a near-infrared two-region response nano composite temperature-sensitive hydrogel. The invention prepares imipenem @ liposome compound IL by filling antibiotic imipenem IMP into liposome vesicles, then modifies gold nanoshell G on an IL outer membrane, is connected with aptamer A of targeted gram-negative bacteria lipopolysaccharide to synthesize ILGA, and further coats the ILGA in temperature-sensitive hydrogel to obtain the ILGA @ gel composite temperature-sensitive hydrogel. The hydrogel has good fluidity at room temperature, and is suitable for wounds of various shapes; under the irradiation of laser with the wavelength of 1064nm in a near-infrared region II, the temperature can be rapidly increased to release imipenem, and efficient sterilization is realized; meanwhile, the hydrogel is converted from a liquid state to a solid state by temperature rise, and a gel film is formed on the surface of the wound in situ to protect the wound from secondary infection; the product of the invention also has good effects of stopping bleeding, reducing inflammation, moisturizing and accelerating wound repair, and is a gel dressing suitable for complex infection wound management.

Description

Preparation and application of near-infrared two-region response nano composite temperature-sensitive hydrogel

Technical Field

The invention relates to the technical field of medicines, in particular to preparation and application of a near-infrared two-region response nano composite temperature-sensitive hydrogel.

Background

Bacterial infections have become the most common problem impeding wound healing and skin formation. The conventional strategy for clinically infecting wounds is to use antibiotic therapy, but its anti-infective efficacy has been compromised due to antibiotic resistance (AMR). Antibiotics have been reported to be combined with photothermal therapy (PTT) to synergistically eradicate multidrug resistant (MDR) bacteria. Photothermal therapy (PTT) uses a photosensitizer to convert light energy into heat energy to remove pathogens, and has attracted much attention in the anti-infectious field because of its good therapeutic effect. However, conventional photothermal materials excited in the non-Near Infrared (NIR) region or the first near infrared (NIR-I) region have limited tissue penetration and low skin tolerance thresholds. Although PTT nanomaterials activated in the near infrared region two (NIR-II) can improve this situation, they often require cumbersome synthetic procedures. In addition, many of the reported photothermal platforms lack bacterial targeting, which reduces therapeutic efficacy and may cause undesirable damage to adjacent normal tissues. Therefore, there is an urgent need to develop a bacterial-targeted, NIR-II responsive nanococktail for PTT synergistic antibiotic therapy of wound infections.

Meanwhile, clinical studies indicate that wound infection is often accompanied by bleeding, long-term inflammation and bacterial secondary infection, causing unnecessary pain and additional medical burden to patients. Therefore, elimination of bacteria while providing an ideal wound healing environment is critical for wound healing. Hydrogels with tissue adhesive and moisturizing capabilities are widely considered to be one of the most attractive wound dressings. In particular, temperature-sensitive hydrogels are receiving much attention because they are transformed from a solution (sol) to a gel (gel) state once the temperature reaches their Lower Critical Solution Temperature (LCST). Due to this unique property, temperature sensitive hydrogels have great potential in wound-related applications (hemostasis, anti-infection, anti-inflammation, avoidance of re-infection, etc.). However, the preparation of such multifunctional temperature-sensitive hydrogels for wound repair by commonly used crosslinking methods is laborious and complicated. Therefore, the strategy of hybridizing the nano material with the temperature-sensitive hydrogel is a more efficient way for constructing the multifunctional wound dressing.

Disclosure of Invention

In view of the defects of the prior art, the invention aims to provide preparation and application of a near-infrared two-region response nano composite temperature-sensitive hydrogel, a novel all-in-one wound dressing is obtained by heterozygously constructing a nano material and the temperature-sensitive hydrogel in a more efficient mode, and the preparation method is used for solving the problems of labor waste, complexity and the like of the preparation method of the multifunctional temperature-sensitive hydrogel for wound repair in the prior art.

In order to achieve the above objects and other related objects, the present invention provides a method for preparing a near-infrared two-region-responsive nanocomposite temperature-sensitive hydrogel, comprising the steps of:

(1) Dispersing the liposome in imipenem, namely IMP aqueous solution, extruding the solution through a polycarbonate porous membrane filter by using an extruder, and removing free Imipenem (IMP) to obtain IMP @ liposome, namely IL;

(2) The method comprises the steps of constructing an IL outer membrane through in-situ reduction on the basis of a gold nanoshell to obtain IMP @ Lipsome @ gold nanoshell, namely ILG;

(3) Mixing and stirring the ILG solution and lipopolysaccharide Aptamer (marked as A) to react to synthesize IMP @ Lipsome @ gold nanoshell @ lipopolysaccharide Aptamer, namely ILGA;

(4) Dispersing lipopolysaccharide ILGA in a biocompatible temperature-sensitive hydrogel solution to synthesize IMP @ Lipsome @ gold nanoshell @ lipopolysaccharide Aptamer @ gel, namely ILGA @ gel, namely the near-infrared two-region-response nano composite temperature-sensitive hydrogel.

Further, in the step (1), the liposome is prepared by a membrane dispersion method.

Further, in the step (1), the film dispersion method includes the steps of: dissolving phospholipid and cholesterol in organic solvent, mixing, and removing organic solvent by rotary evaporation under reduced pressure to obtain lipid film.

Further, in the step (1), the phospholipid is selected from natural phospholipid and/or synthetic phospholipid, the natural phospholipid is selected from at least one of soybean phospholipid and lecithin, and the synthetic phospholipid is selected from at least one of 1,2-distearoyl-sn-glycerol-3-phosphocholine, namely DSPC, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ carboxyl (polyethylene glycol) ], namely DSPE-PEG-COOH (MW = 2000-3400 Da); preferably, the phospholipid is selected from synthetic phospholipids; more preferably, the synthetic phospholipid is selected from DSPC and DSPE-PEG-COOH.

Further, in the step (1), the mass ratio of the phospholipid to the cholesterol is (7-10): (1 to 3), preferably (7 to 9): 2.

further, in the step (1), the organic solvent is selected from any one of chloroform, diethyl ether and ethanol.

Further, in the step (1), the rotary evaporation temperature is 35-50 ℃, and the rotary evaporation speed is 50-80 rpm; preferably, the rotary evaporation temperature is 35-45 ℃, and the rotary evaporation speed is 60-70 rpm; more preferably, the rotary evaporation temperature is 40 ℃ and the rotary evaporation speed is 65rpm.

Further, in the step (1), dissolving phospholipid and cholesterol in an organic solvent, and uniformly mixing the phospholipid and the cholesterol through ultrasonic treatment; preferably, the sonication time is 2 to 5 minutes, more preferably 3 minutes.

Further, in the step (1), the concentration of imipenem, i.e., IMP aqueous solution is 0.5-2 mg/mL, preferably 1mg/mL.

Further, in the step (1), the mass ratio of the liposome to the IMP is 1: (1-2), preferably 1: (1.1-1.5), more preferably 1 (1.1-1.2).

Further, in the step (1), the polycarbonate porous membrane is a polycarbonate porous membrane of 100 to 300nm, preferably a polycarbonate porous membrane of 200 nm.

Further, in the step (1), free IMP is removed by ultrafiltration.

Further, in the step (2), the synthesis process of the ILG includes the following steps: adding the gold compound solution into the IL dispersion liquid, stirring uniformly, adding ascorbic acid, and stirring for reaction; after the reaction, the reaction mixture was centrifuged to wash the precipitate to obtain ILG.

Further, in the step (2), the gold compound is selected from HAuCl ₄ 。

Further, in the step (2), the dosage ratio of the gold ions to the IL is (1-2): 1 (c/m) (mol/mg), preferably 1.25.

Further, in the step (2), the concentration of IL in the IL dispersion is 0.5 to 2mg/mL, preferably 1mg/mL.

Further, in the step (2), the concentration of gold ions in the gold compound solution is 1 to 2mM, preferably 1.25mM.

Further, in the step (2), the gold compound solution is added to the IL dispersion, stirred at room temperature for 10 to 30 seconds, and then ascorbic acid is added.

Further, in the step (2), after the ascorbic acid is added, the reaction time is stirred for 2 to 5 minutes, preferably 3 minutes.

Further, in the step (2), the concentration of the ascorbic acid is 2 to 8mM, preferably 4 to 6mM, and more preferably 5mM.

Further, in the step (2), the centrifugation conditions are as follows: centrifuging at 4000-6000 g at 4 deg.c for 2-5 min; preferably at 4 ℃ and 5000g, and centrifuged for 3 minutes.

Further, in the step (3), the lipopolysaccharide aptamer is selected from a LA27 aptamer.

Further, in the step (3), the dosage ratio of the ILG to the lipopolysaccharide aptamer is (1-2): 1 (m/c) (mg/. Mu.mol), preferably 1:1 (m/c) (mg/. Mu.mol).

Further, in the step (3), the ILG solution is prepared with water at a concentration of 0.5 to 2mg/mL, preferably 1mg/mL.

Further, in the step (3), the concentration of the lipopolysaccharide aptamer is 0.5 to 2. Mu.M, preferably 1. Mu.M.

Further, in the step (3), the mixing and stirring reaction of the ILG solution and the lipopolysaccharide aptamer is performed under a dark condition, and the stirring reaction time is 6 to 12 hours, preferably 8 to 10 hours.

Further, in the step (3), a sodium chloride solution is added into the mixture obtained by reacting the ILG solution with the lipopolysaccharide aptamer, and the reaction is continued to be stirred, so that the modification amount of the lipopolysaccharide aptamer on the ILG surface is increased.

Further, in the step (3), the concentration of the sodium chloride solution is 0.5 to 2mM, preferably 1mM.

Further, in the step (3), the molar ratio of the sodium chloride to the lipopolysaccharide aptamer is (500-2000): 1, preferably 1000.

Further, in the step (3), the solution obtained by the reaction is centrifuged to collect the precipitate, and then centrifuged at a low temperature, and washed to obtain IMP @ Lipsome @ gold nanoshell @ aptamer, i.e. ILGA.

Further, in the step (3), the centrifugation conditions are as follows: 4000-6000 g, centrifuging for 2-5 minutes; preferably 5000g, and centrifuged for 3 minutes.

Further, in the step (3), the low-temperature centrifugation temperature is 4 ℃.

Further, in the step (4), the biocompatible temperature-sensitive hydrogel is selected from PLGA-PEG-PLGA.

Further, in the step (4), the dosage ratio of the ILGA to the hydrogel is 0.1: (15 to 20), preferably 0.1: 17.5.

Further, in the step (4), the concentration of the hydrogel solution is 15 to 20% (w/v), preferably 17.5% (w/v).

In a second aspect, the invention provides a near-infrared two-region response nano-composite temperature-sensitive hydrogel prepared by the method in the first aspect.

Further, the near-infrared two-region response nano-composite temperature-sensitive hydrogel can trigger ILGA to kill bacteria under the radiation of NIR-II light through the synergistic effect of PTT and IMP, reduce inflammation and accelerate wound repair.

In a third aspect, the invention provides the use of a near-infrared two-zone responsive nanocomposite, temperature-sensitive hydrogel according to the second aspect as and/or in the manufacture of a wound dressing.

In a fourth aspect, the invention provides a wound dressing comprising a near-infrared two-zone responsive nanocomposite, temperature-sensitive hydrogel according to the second aspect.

As mentioned above, the preparation and application of the near-infrared two-region response nano composite temperature-sensitive hydrogel of the invention have the following beneficial effects:

the invention designs an effective antibacterial system based on an Imipenem (IMP) -coated liposome and gold-shell nano hybrid and modified targeting lipopolysaccharide aptamer (shown as ILGA), and is used for NIR-II photoresponse cooperative PTT/antibiotic treatment. Wherein ILGA has excellent NIR-II (1064 nm) activated PTT properties, inducing its destruction to release the antibiotic IMP to synergistically eliminate bacteria; meanwhile, the biocompatible temperature-sensitive hydrogel is used as a matrix to load the ILGA so as to construct the integrated ILGA @ gel, the ILGA @ gel has good fluidity, and can perfectly match with the shape of a wound under the irradiation of NIR-II light to form an adhesive film layer, so that the bleeding is stopped firstly, the complicated infected wound symptoms are solved, and the wound healing is promoted.

The ILGA @ gel provided by the invention can trigger ILGA to kill bacteria under the radiation of NIR-II light through the synergistic effect of PTT and IMP, and then the ILGA induces macrophage polarization into M2 type macrophages to reduce inflammation, thereby accelerating wound repair. Meanwhile, the gel serves as a protective layer in the tissue generation process, can resist invasion of exogenous pathogenic bacteria, avoids reinfection and provides a moist microenvironment for wound healing.

The ILGA @ gel prepared by the invention has an outstanding effective antibacterial effect, and can also solve the accompanying complications of infected wounds.

Drawings

FIG. 1 shows a TEM image (scale bar 100 nm) of ILGA in example 1 of the present invention.

FIG. 2 shows thermal infrared images of LGA, nano-gold (Au), liposome and aqueous solution under laser irradiation in example 1 of the present invention.

FIG. 3 is a TEM image of ILGA after laser irradiation in example 1 of the present invention, in which red and blue arrows represent the fragmented gold nanoshells and destroyed liposomes on ILGA, respectively.

FIG. 4 is a sol-gel phase transition diagram showing ILGA @ gel and PLGA-PEG-PLGA hydrogels in example 1 of the present invention.

FIG. 5 shows a schematic diagram of 5mL of ILGA @ gel stored in a medical spray bottle for spraying in example 1 of the present invention.

FIG. 6 shows the antibacterial and anti-biofilm activities of ILGA @ gel in example 1 of the present invention (green for active bacteria and red for dead bacteria).

FIG. 7 shows representative pictures of incised skin wounds treated with ILGA @ gel on

days

0, 7, 11, and 15 in example 1 of the present invention.

FIG. 8 shows photographs of a mouse tail amputation model treated with gauze and ILGA @ gel + NIR in example 1 of the present invention, wherein: a is a photograph of a mouse tail amputation model treated with gauze and ilga @ gel + NIR, using mice without additional intervention as controls; b is a rabbit abdominal bleeding model before and after ILGA @ gel + NIR treatment; c is model of rabbit ear venous bleeding before and after ILGA @ gel + NIR treatment.

FIG. 9 is a graph showing the results of the in vivo antibacterial and wound healing characteristics evaluation experiments by ILGA @ gel in example 1 of the present invention, in which: a is the thermal infrared image of mice treated with ILGA @ gel + NIR, ILGA + NIR and NIR, respectively; b is the quantification of the temperature increase in three groups according to fig. 6.

FIG. 10 is a graph showing the changes in infected wounds after the ILGA @ gel group and the control group in example 1 of the present invention were treated, in which: a is a representative photograph of MDR-PA infected wounds with different formulation treatments, scale bar =1 cm; b is a quantification of wound contraction area for mice treated with PBS, ILGA + NIR or ILGA @ gel + NIR on

days

5, 7, 9 and 11.

Figure 11 shows H & E and Masson trichrome staining (scale bar =50 μm) of skin tissue obtained from mice with different dressings on day 5 and day 15, respectively, in example 1 of the invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The invention designs an effective antibacterial system based on an Imipenem (IMP) -coated liposome and a gold-shell nano hybrid and modified targeting lipopolysaccharide aptamer (shown as ILGA), and the effective antibacterial system is used for NIR-II (near infrared-infrared) photoresponse cooperative PTT/antibiotic therapy. The specific process is as follows:

first, IMP is loaded into liposome vesicles to obtain IL. Then, gold nanoshells (denoted as G) were formed on the outer membrane of IL, which was then modified with lipopolysaccharide aptamers (denoted as a) to synthesize ILGA. ILGA has excellent NIR-II (1064 nm) activating PTT properties, inducing its destruction to release the antibiotic IMP to synergistically eliminate bacteria. Further, the biocompatible temperature-sensitive hydrogel (PLGA-PEG-PLGA) is used as a matrix to load ILGA so as to construct integrated ILGA @ gel, so that the complicated infected wound symptoms are solved, and the wound healing is promoted. ILGA @ gel with good fluidity can perfectly match the shape of a wound under the irradiation of NIR-II light to form an adhesive film layer, and hemostasis is firstly realized. Meanwhile, NIR-II light triggers ILGA killing of bacteria through the synergistic effect of PTT and IMP, and then ILGA induces macrophage polarization into M2-type macrophages to reduce inflammation, thereby accelerating wound repair. The gel membrane acts as an active layer during tissue generation, avoiding reinfection, while providing a moist microenvironment for wound healing.

The integrated ILGA @ gel prepared by the invention has an outstanding effective antibacterial effect, and can also solve the accompanying complications of infected wounds. The present invention will be described in detail with reference to the following specific examples. It should also be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention, and that numerous insubstantial modifications and adaptations of the invention described above will occur to those skilled in the art. The specific process parameters and the like of the following examples are also merely one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.

Example 1

1. Composite material

1,2-distearoyl-sn-glycerol-3-phosphocholine, i.e. DSPC, 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ carboxyl (polyethylene glycol)]DSPE-PEG-COOH (MW =2000 Da), cholesterol and imipenem, IMP, were purchased from Allantin Biotechnology Ltd (Shanghai, china); tetrachloroauric acid trihydrate (HAuCl) ₄ ) Purchased from mclin biochemistry technology limited (shanghai, china); ascorbic acid and broth powders were purchased from solibao technologies ltd (beijing); lipopolysaccharide aptamer LA27 (SH-TTTTTTTTTTCCTTCTAACAGAATGTTGTTAGATAGC) was purchased from Biotechnology engineering, inc. (Shanghai, china); triblock temperature sensitive hydrogel PLGA-PEG-PLGA was purchased from Guangzhou carbohydrate science and technology, inc. (Guangzhou, china).

2. Synthesis of IL

IL was prepared by a thin film dispersion method, DSPC, cholesterol and DSPE-PEG-COOH were dissolved in chloroform (10 mL) at a molar ratio of 8. The mixture was then dried in a rotary evaporator under gradually reduced pressure and at 40 ℃ at a rotational speed of 65rpm, and the lipid film attached to the bottle was dispersed in an aqueous IMP solution (1 mg/mL). Next, the solution was extruded 20 times through a 200nm polycarbonate porous membrane filter using an extruder (Advanced Thermal Solutions), and free IMP was removed by ultrafiltration to obtain IL. The loading efficiency of IMP was calculated by (weight of IMP loaded)/(total weight of IL) x 100% and the absorption of IMP was measured by a microplate reader (Infinite M1000 Pro, tecan).

3. Synthesis of ILG

ILGs are constructed by in situ reduction of gold nanoshells on the IL outer membrane. Briefly, IL (1 mg/mL) was dispersed in water, followed by HAuCl ₄ (1.25 mM) solution was added rapidly to IL and stirred at room temperature for 15 seconds; ascorbic acid was then injected into the mixture and stirred for 3 minutes to give a final concentration of 5mM. The ILG was collected by centrifugation (5000 g,3 minutes) at 4 ℃ and washing three times with pure water.

4. Synthesis of ILGA

ILGA was synthesized by mixing an ILG solution (1 mg/mL, prepared with purified water) with LA27 aptamer (1. Mu.M, prepared with DEPC treated water) and stirring overnight in the dark. To increase the amount of modification of LA27 on the ILG surface, a sodium chloride solution (1 mM) was added to the mixture for 1 hour, and the resulting solution was centrifuged at 5000g for 3 minutes to collect a precipitate. The ILGA was collected by centrifugation (5000 g,3 minutes) at 4 ℃ and washed three times with pure water.

5. Synthesis of ILGA @ gel

ILGA was redispersed in 17.5% (w/v) PLGA-PEG-PLGA hydrogel solution to an ILGA concentration of 1mg/mL to obtain ILGA @ gel.

6. Experiment and experimental results and analysis

(1) The liposome is prepared by a film dispersion method, IMP is loaded to form IL, after gold shell and lipopolysaccharide aptamer LA27 are added, the ILGA is in a form that a liposome membrane is coated with a layer of irregular shell, and the shape is shot by a transmission electron microscope and is shown in figure 1.

(2) As shown in fig. 2, with Au solution (simply mix HAuCl) ₄ The temperature of the ILGA solution was significantly increased under 1064nm laser irradiation, indicating a good photothermal effect, compared to ascorbic acid), liposomes and water.

(3) As shown in fig. 3, with the benefit of photothermal response, ILGA breaks down and releases encapsulated IMP, with a significant change in the morphology of the ILGA after laser irradiation.

(4) In order to simultaneously address complications of wound infection (bleeding, long-term inflammation and reinfection), further research was conducted on ILGA. Specifically, a biodegradable and biocompatible temperature sensitive hydrogel PLGA-PEG-PLGA was selected as the matrix to load ILGA and form ILGA @ gel. As can be seen from the solution-gel (sol-gel) phase transition diagram in FIG. 4, the gel temperature of ILGA @ gel (17.5 wt%) was about 37 ℃.

The 5mL medical spray bottle was filled with ILGA @ gel, and the spraying process was as shown in FIG. 5, and the ILGA @ gel exhibited good fluidity at 37 ℃ or below.

(5) The antibacterial and anti-biofilm activity of the ILGA @ gel is verified by co-incubation of the bacterial liquid of the ILGA @ gel and the spectrum drug-resistant pseudomonas aeruginosa (MDR-PA) with the biofilm. As shown in FIG. 6 (green for active bacteria and red for dead bacteria), under NIR irradiation, ILGA @ gel was effective in killing MDR-PA and removing the biofilm formed.

(6) The wound closure performance of ilga @ gel was evaluated using a full-thickness skin incision model, skin incisions (2 cm, full thickness) were made in the right abdomen of the mice, and the mice were divided into two groups, one group was sprayed with ilga @ gel, and the other group was left untreated to serve as a control. As shown in FIG. 7, the wound width was significantly narrowed in the ILGA @ gel group as compared with the untreated group.

(7) Due to the gel properties of ilga @ gel, hemostatic performance can be assessed by mouse tail and rabbit bleeding models.

As shown in FIG. 8a, mice treated with ILGA @ gel stopped bleeding (less than 15 seconds) immediately after gelation occurred, and the amount of blood lost (22.6. + -. 7.7 mg) was significantly reduced in mice treated with ILGA @ gel as compared with gauze group (72.1. + -. 10.5 mg) and control group (130.2. + -. 16.6 mg).

A rabbit bleeding model was constructed by making a 20mm long and 5mm deep incision in the rabbit abdomen, as shown in FIG. 8b, and blood flow was immediately blocked after applying ILGA @ gel, and the wound was coagulated within a few minutes. As shown in FIG. 8c, ILGA @ gel stopped bleeding within 15 seconds even in areas with abundant blood flow, such as rabbit ear vein.

The above results indicate that ilga @ gel has excellent hemostatic effects in vivo.

(8) The in vivo antibacterial and wound healing properties of ILGA @ Gel were further evaluated by a full-thickness skin model of MDR-PA infection.

As shown in FIGS. 9a, b, the temperature of the ILGA @ gel group was increased from 32 ℃ to 51.3 ℃ within 100s, while the temperature of the ILGA @ gel group was increased from 31.3 ℃ to 48.6 ℃ after 3 minutes of NIR irradiation, and the ILGA @ GEL showed excellent photothermal effect characteristics.

As shown in fig. 10a, infected wounds after the ILGA @ gel group treatment had no residual bacterial biofilm from the first day, while the control group (ILGA) still had a significant biofilm, and fig. 10b shows the quantification of the area of wound healing.

Figure 11 shows H & E and Masson trichrome staining (scale bar =50 μm) of skin tissue obtained from mice with different dressings on day 5 and day 15, respectively. FIG. 11 shows that all three groups of skin tissues showed inflammatory responses at day 5, whereas the ILGA @ gel group showed less inflammatory infiltration; on day 15, epithelialization was complete, with no inflammation in the ilga @ gel group, while the control group had only skin tissue regenerating the basic structure of epithelium and dermis; furthermore, the regenerated skin tissue of the ILGA @ gel group showed higher M2-type macrophage expression compared to the control.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

SEQUENCE LISTING

<110> southern hospital of southern medical university

<120> preparation and application of near-infrared two-region response nano composite temperature-sensitive hydrogel

<130> PCQNF2111589-HZ

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 37

<212> DNA

<213> Artificial

<220>

<223> LA27

<400> 1

tttttttttt ccttctaaca gaatgttgtt agatagc 37

Claims

1. A preparation method of a near-infrared two-region response nano composite temperature-sensitive hydrogel is characterized by comprising the following steps:

(1) Dispersing the liposome in an aqueous solution of imipenem, extruding the solution through a polycarbonate porous membrane filter by using an extruder, and removing free imipenem to obtain IMP @ lipsome, namely IL;

(2) The method comprises the following steps of (1) constructing an IMP @ Lipsome @ gold nanoshell, namely ILG, on an IL outer membrane by in-situ reduction of a gold nanoshell;

(3) Mixing and stirring the ILG solution and lipopolysaccharide Aptamer to react to synthesize IMP @ Lipsome @ gold nanoshell @ lipopolysaccharide Aptamer, namely ILGA;

(4) And dispersing the ILGA in a temperature-sensitive hydrogel solution, namely Gel, and synthesizing the near-infrared two-region response nano composite temperature-sensitive hydrogel IMP @ Lipsoome @ gold nanoshell @ lipopolysaccharide Aptamer @ Gel, namely ILGA @ Gel.

2. The method of claim 1, wherein: in the step (1), the liposome is prepared by a film dispersion method.

3. The method of claim 2, wherein: in the step (1), the film dispersion method includes the steps of: dissolving phospholipid and cholesterol in organic solvent, mixing, and removing organic solvent by rotary evaporation under reduced pressure to obtain lipid film.

4. The production method according to claim 1, characterized in that: in the step (2), the synthesis process of the ILG comprises the following steps: adding the gold compound solution into the IL dispersion liquid, stirring uniformly, adding ascorbic acid, and stirring for reaction; after the reaction, the reaction mixture was centrifuged to wash the precipitate to obtain ILG.

5. The method of claim 4, wherein: in the step (2), the gold compound is selected from chloroauric acid, namely HAuCl ₄ ；

And/or in the step (2), adding the gold compound solution into the IL dispersion liquid, stirring at room temperature for 10-30 seconds, and then adding ascorbic acid; and/or sodium borohydride, wherein in the step (2), the centrifugation conditions are as follows: centrifuging at 4000-6000 g for 2-5 min at 4 deg.C.

6. The method of claim 1, wherein: in the step (3), the lipopolysaccharide aptamer is selected from a LA27 aptamer;

and/or in the step (3), the mixing and stirring reaction of the ILG solution and the lipopolysaccharide aptamer is carried out under the condition of keeping away from light, and the stirring reaction time is 6-12 hours;

and/or, in the step (3), adding a sodium chloride solution into the mixture obtained by the reaction of the ILG solution and the lipopolysaccharide aptamer, and continuing stirring for reaction.

7. The method of claim 1, wherein: the biocompatible temperature-sensitive hydrogel is selected from PLGA-PEG-PLGA.

8. The near-infrared two-region response nano-composite temperature-sensitive hydrogel prepared by the method according to any one of claims 1 to 7.

9. The use of the near-infrared two-region-responsive nanocomposite temperature-sensitive hydrogel according to claim 8 as and/or in the preparation of a wound dressing.

10. A wound dressing is characterized in that: a nanocomposite, temperature-sensitive hydrogel comprising the near-infrared two-zone response of claim 8.