Ultrasonic controlled-release hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ultrasonic controlled-release hydrogel as well as a preparation method and application thereof, and relates to the technical field of medical instruments. The hydrogel provided by the invention can realize ultrasonic controlled release, can realize effective control of drug release, can adjust the dosage according to clinical requirements, and improves the clinical transformation potential of a drug controlled release system. The composition is especially suitable for comprehensive immunoregulation of various pathogenic factors of chronic wounds. The preparation method provided by the invention is simple and easy to implement, low in cost and good in biological safety.

Description

Technical Field

[0001] The present invention relates to the technical field of medical devices, and in particular to an ultrasound-controlled release hydrogel and a preparation method and application thereof. Background Art

[0002] Currently, existing hydrogel dressings all passively release drugs, primarily through passive diffusion and natural degradation, with no way to control the release rate. Existing nanoparticles suffer from low drug loading, the need for repeated dosing, and sudden drug release, hindering their rapid translation into clinical practice. There is an urgent need to develop a novel controlled-release drug system that effectively controls drug release and enhances its potential for clinical translation.

[0003] In view of this, the present invention is proposed. Summary of the Invention

[0004] The purpose of the present invention is to provide an ultrasound-controlled release hydrogel and its preparation method and application to solve the above technical problems.

[0005] The present invention is achieved in that:

[0006] In a first aspect, the present invention provides a method for preparing an aqueous dispersion, comprising the following steps:

[0007] (1) preparing nanoparticles: covalently linking a calcitonin gene-related peptide and a material having an active oxygen scavenging ability through a chemical reaction; the material having an active oxygen scavenging ability is selected from at least one of metalloporphyrins, natural enzymes, vitamins, polyphenols, coenzyme Q10, and melatonin;

[0008] (2) The organic solution containing the nanoparticles is then mixed with the DSPE-PEG-modified targeting agent to prepare an organic mixed solution, wherein the final concentrations of the nanoparticles and the DSPE-PEG-modified targeting agent in the organic mixed solution are 10-20 mg / mL and 0.5-4 mg / mL, respectively; under ultrasonic water bath conditions, the mixed solution is mixed with water at a volume ratio of 1:7-10, ultrasonically treated, and then the ultrasonicated liquid is dialyzed to obtain an aqueous dispersion; the targeting agent can target cell surface receptors.

[0009] In a second aspect, the present invention further provides a method for preparing an ultrasound-controlled release hydrogel, comprising the following steps:

[0010] The aqueous dispersion prepared by the above aqueous dispersion preparation method is mixed with natural or synthetic polymer materials and subjected to cross-linking reaction to obtain the aqueous dispersion.

[0011] In a third aspect, the present invention further provides an aqueous dispersion prepared by the above-mentioned method for preparing the aqueous dispersion.

[0012] In a fourth aspect, the present invention further provides an ultrasound-controlled release hydrogel, which is prepared by the above-mentioned method for preparing the ultrasound-controlled release hydrogel.

[0013] In a fifth aspect, the present invention further provides use of the aqueous dispersion or the above-mentioned ultrasound-controlled release hydrogel in wound repair products.

[0014] The present invention has the following beneficial effects:

[0015] The aqueous dispersion or ultrasonically controlled release hydrogel provided by the present invention comprises nanoparticles, which carry redox active materials and have unique catalase-like and superoxide dismutase-like activities, and can remove hydrogen peroxide and superoxide free radicals (·O2 - ), protecting cells from oxidative stress damage, thus exhibiting excellent antioxidant capacity. The aqueous dispersion or hydrogel also includes a targeting agent that can target cell surface receptors, thereby modulating the cell phenotype. For example, when the targeting agent is folic acid (FA), the high affinity of its FA moiety for the cell surface receptor folate receptor (FR) enables the labeled nanoparticles to target FR-expressing cells.

[0016] The aqueous dispersion or hydrogel provided by the present invention can be ultrasonically controlled for release, effectively controlling drug release and adjusting dosage according to clinical needs, thereby enhancing the clinical translational potential of the controlled-release drug system. It is particularly suitable for comprehensive immune regulation of various pathogenic factors in chronic wounds.

[0017] The aqueous dispersion or ultrasonically controlled-release hydrogel provided by the present invention is easy to prepare, low in cost, and has good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 The graph shows the nanoparticle release rate results under different ultrasonic powers;

[0020] Figure 2 The scanning electron microscope images of the hydrogel surface morphology after ultrasound at different powers;

[0021] Figure 3 The experimental results of hydrogel promoting wound healing in diabetic mice (left is the appearance of the wound, right is the cross-section of the wound);

[0022] Figure 4The experimental results show that the hydrogel regulates the transformation of mouse macrophages into an anti-inflammatory phenotype (A animal experiment, B cell experiment) and eliminates the oxidative stress state in macrophages (C);

[0023] Figure 5 The figure shows the results of detecting the number of FITC-positive macrophages in each treatment group by flow cytometry. DETAILED DESCRIPTION

[0024] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0025] In a first aspect, the present invention provides a method for preparing an aqueous dispersion, comprising the following steps:

[0026] (1) preparing nanoparticles: covalently linking a calcitonin gene-related peptide and a material having an active oxygen scavenging ability through a chemical reaction; the material having an active oxygen scavenging ability is selected from at least one of metalloporphyrins, natural enzymes, vitamins, polyphenols, coenzyme Q10, and melatonin;

[0027] (2) then mixing the organic solution containing the nanoparticles with the DSPE-PEG-modified targeting agent to prepare an organic mixed solution, wherein the final concentrations of the nanoparticles and the DSPE-PEG-modified targeting agent in the organic mixed solution are 10-20 mg / mL and 0.5-4 mg / mL, respectively; under ultrasonic water bath conditions, the mixed solution is mixed with water at a volume ratio of 1:7-10, ultrasonically treated, and then the ultrasonicated liquid is dialyzed to obtain an aqueous dispersion; the targeting agent can target cell surface receptors.

[0028] The aqueous dispersion provided by the present invention includes nanoparticles, and the nanoparticles are provided with a material having active oxygen scavenging ability. The material has unique active oxygen scavenging properties and biomimetic catalytic ability, and can scavenge hydrogen peroxide and superoxide free radicals (·O2 - ), protecting cells from oxidative damage, thus exhibiting excellent resistance to oxidative stress. The aqueous dispersion also includes a targeting agent that can target cell surface receptors, thereby modulating the cell phenotype. For example, when the targeting agent is folic acid (FA), the high affinity of its FA moiety for the cell surface receptor folate receptor (FR) enables the labeled nanoparticles to target macrophages.

[0029] The aqueous dispersion provided by the present invention can be ultrasonically controlled for release, effectively controlling drug release and adjusting dosage according to clinical needs, thereby enhancing the clinical translational potential of the controlled-release drug system. It is particularly suitable for comprehensive immune regulation of various pathogenic factors of chronic wounds.

[0030] The aqueous dispersion provided by the present invention is easy to prepare, low in cost and has good biosafety.

[0031] Calcitonin gene-related peptide (CGRP) is a neuropeptide composed of 37 amino acids, with a disulfide-bonded ring at its N-terminus. There are two main isoforms: α-CGRP (primarily found in the central and peripheral nervous systems) and β-CGRP (primarily found in the enteric nervous system and pituitary gland). These two isoforms have similar structures and biological functions. The CGRP in the present invention can be either α-CGRP or β-CGRP.

[0032] In a preferred embodiment of the present invention, the chemical reaction for covalently linking the calcitonin gene-related peptide and the material having active oxygen scavenging ability is selected from at least one of the following: an amide reaction, a Schiff base reaction, a click chemistry reaction, and a thiol-disulfide exchange reaction;

[0033] In a preferred embodiment of the present invention, the click chemistry reaction is a thiol-ene / alkyne click reaction or a copper-catalyzed azide-alkyne cycloaddition;

[0034] In a preferred embodiment of the present invention, the chemical reaction is an amide reaction, using an EDC / NHS combination system, DCC, DIC, or CMC, to link the CGRP and the redox-active material into an amphiphilic molecule.

[0035] In a preferred embodiment of the present invention, an EDC / NHS combined system is used for amide reaction, and the mixing molar ratio of the material having active oxygen scavenging ability, calcitonin gene-related peptide, EDC, and NHS is: 1:1.2-2:1.2-2:1.5-2.

[0036] In a preferred embodiment of the present invention, the mixing molar ratio of the redox-active material, calcitonin gene-related peptide, EDC, and NHS is: 1:1.2:1.2:1.5, 1:1.5:1.5:1.8, or 1:1.8:1.8:2.

[0037] In a preferred embodiment of the present invention, the DSPE-PEG modified target is selected from at least one of the following DSPE-PEG modified targets: folic acid, mannose, hyaluronic acid and phosphatidylserine;

[0038] Preferably, the DSPE-PEG modified target is DSPE-PEG modified folic acid;

[0039] Preferably, the molecular weight of PEG in the DSPE-PEG modified target is selected from 2000-6000 KD.

[0040] DSPE-PEG-FA is composed of DSPE (distearylphosphatidylethanolamine), PEG (polyethylene glycol), and FA (folic acid). DSPE provides good lipophilicity, PEG increases water solubility and improves biocompatibility, and the FA portion has a high affinity for the cell surface receptor folate receptor (FR).

[0041] In a preferred embodiment of the present invention, the metalloporphyrin is manganese porphyrin, iron porphyrin, cobalt porphyrin, zinc porphyrin, copper porphyrin, nickel porphyrin or rare earth metal porphyrin; the natural enzyme is selected from superoxide dismutase or catalase; the vitamin is selected from vitamin A, vitamin D, vitamin K, vitamin B1, vitamin B9, vitamin B12, vitamin C or vitamin E;

[0042] In a preferred embodiment of the present invention, the vitamins are selected from vitamin C or vitamin E.

[0043] In a preferred embodiment of the present invention, the ultrasonic treatment time is 2-5 min, for example, the ultrasonic treatment time is 2, 3, 4 or 5 min.

[0044] In a second aspect, the present invention further provides a method for preparing an ultrasound-controlled release hydrogel, comprising the following steps:

[0045] The aqueous dispersion prepared by the above aqueous dispersion preparation method is mixed with natural or synthetic polymer materials and subjected to cross-linking reaction to obtain the aqueous dispersion.

[0046] In a preferred embodiment of the present invention, the natural or synthetic polymer material is sodium alginate.

[0047] In a preferred embodiment of the present invention, during the cross-linking reaction, the aqueous dispersion is diluted to 0.1-5 μg / mL, the natural or synthetic polymer material is dissolved in the diluted aqueous dispersion at a mass fraction of 2-5%, and then mixed with a 1-3% by mass calcium chloride solution or calcium sulfate suspension;

[0048] In a preferred embodiment of the present invention, the mixing ratio of the diluted aqueous dispersion to the calcium chloride solution or calcium sulfate suspension is 3-5:1, and the cross-linking reaction time is 5 minutes.

[0049] In a third aspect, the present invention further provides an aqueous dispersion prepared by the above-mentioned method for preparing the aqueous dispersion.

[0050] In a fourth aspect, the present invention further provides an ultrasound-controlled release hydrogel, which is prepared by the above-mentioned method for preparing the ultrasound-controlled release hydrogel.

[0051] In a fifth aspect, the present invention further provides the use of the aqueous dispersion or the above-mentioned ultrasound-controlled release hydrogel in wound repair products;

[0052] In a preferred embodiment of the present invention, the wound repair product is selected from chronic wound repair products. Wound repair materials include but are not limited to artificial skin materials, wound dressings, and the like.

[0053] In one embodiment of the present invention, when the hydrogel is used, the hydrogel is applied to the wound surface or injected into the wound. 2 Ultrasound irradiation at a power, 10% to 50% duty cycle, and 1-3 MHz frequency was performed for 3 minutes to half an hour to achieve different drug release rates.

[0054] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0055] Example 1

[0056] This embodiment provides a method for preparing an aqueous dispersion and a hydrogel.

[0057] 1. The preparation method of the aqueous dispersion is as follows:

[0058] (1) Preparation of nanoparticles with macrophage phenotype regulation and reactive oxygen species scavenging capabilities. The specific preparation method is as follows:

[0059] Calcitonin gene-related peptide (Guangzhou Carbon Water Technology Co., Ltd.), N-hydroxysuccinimide (NHS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), and 5-(4-carboxyphenyl)-10,15,2-triphenylmanganese porphyrin were dissolved in N-dimethylformamide (DMF) solution at a molar ratio of 1.2:2:1.2:1, mixed sequentially, and stirred overnight. The reaction product was evaporated to dryness using a rotary evaporator to produce nanoparticles.

[0060] (2) The nanoparticles were dissolved in dimethyl sulfoxide (DMSO) at a theoretical concentration of 10 mg / mL, and 1 mg / mL of DSPE-PEG2K-FA (Hangzhou Chuangshi Biotechnology Co., Ltd., AT11226) was added to prepare a mixed solution. The mixed solution was then mixed with ultrapure water at a volume ratio of 1:7 under ultrasonication (the mixed solution was injected into the ultrapure water), and ultrasonication was continued for 3 minutes. The resulting solution was dialyzed for 4 hours using a dialysis bag with a cutoff value greater than 5 kD to obtain an aqueous dispersion of nanoparticles with macrophage phenotype modulation and reactive oxygen species scavenging capabilities.

[0061] 2. Preparation of Hydrogel

[0062] The aqueous dispersion prepared above is dissolved in a sodium alginate solution, and a certain amount of calcium chloride solution or calcium sulfate suspension is added to form a calcium-crosslinked sodium alginate hydrogel.

[0063] The aqueous dispersion was diluted to 1 μg / mL, and the sodium alginate solution was dissolved in the diluted aqueous dispersion at a mass fraction of 4%, and then mixed with a 1% by mass calcium sulfate suspension; the mixed volume ratio of the diluted aqueous dispersion and the calcium chloride solution suspension was 4:1, and the cross-linking reaction time was 5 minutes.

[0064] Comparative Example 1

[0065] Compared with Example 1, the only difference is that when preparing the aqueous dispersion, the DSPE-PEG2K-FA in Example 1 is replaced by DSPE-PEG2K (i.e., MC group), that is, the aqueous dispersion does not contain folic acid, and the other steps are the same.

[0066] Each treatment group was labeled with FITC: blank control (no aqueous dispersion added, Blank group), MC group aqueous dispersion, aqueous dispersion with folic acid prepared in Example 1 (MCF group), and pre-blocking macrophage surface receptors with free folic acid before adding MCF (i.e., MCF+FA group); each treatment group was mixed with macrophages.

[0067] The number of FITC-labeled positive macrophages was detected by flow cytometry. Figure 5 As shown, compared with the MC group or the MCF+FA group in which free FA was used to block macrophage surface receptors in advance, the macrophages in the MCF group had the highest uptake of nanoparticles, demonstrating the targeting effect of FA on macrophages.

[0068] Experimental Example 1

[0069] The release rate of nanoparticles from the hydrogel prepared in Example 1 was experimentally measured under different ultrasonic powers.

[0070] The hydrogel prepared in Example 1 was placed in 3 volumes of normal saline and the water was heated at 0, 0.1, 0.3, 0.5 and 1 W / cm 2 The nanoparticle release rate was tested by ultrasound at 1 MHz frequency, 20% power and 20% duty cycle for 3 minutes.

[0071] Test results reference Figure 1 As shown, the results show that compared with 0-0.5W / cm 2 Power, 1W / cm 2 The release rate of nanoparticles was the highest after ultrasonic treatment with high power.

[0072] The surface morphology of the hydrogels after ultrasonic treatment at different powers was characterized. The scanning electron microscopy results of the surface morphology of the hydrogels after ultrasonic treatment at different powers were referred to Figure 2 The results showed that the nanoparticles had uniform morphology and an average particle size of 23.50nm±4.92nm.

[0073] Experimental Example 2

[0074] The hydrogel prepared in Example 1 was used to treat chronic wounds.

[0075] The specific experimental method is as follows: a 6mm diameter punch was used to punch holes in the back of diabetic mice (8-week-old male mice), and a 10mm inner diameter silicone ring was fixed around the wound using animal tissue glue to prevent wound closure caused by surrounding skin contraction. The hydrogel was injected into the wound surface at a rate of 0.5mL / wound and a 0.3W / cm 2 Ultrasound was applied at 1 MHz frequency for 3 minutes with a power of 20% duty cycle. This was repeated every 3 days. Mice were sacrificed on the 10th day, and full-thickness skin samples were taken from the wounds for HE and MASSON staining.

[0076] Figure 3 The results showed that the hydrogel can promote wound healing in diabetic mice (left is the appearance of the wound, right is the cross-section of the wound).

[0077] Experimental Example 3

[0078] Test of the immunomodulatory ability of hydrogel dressing. Mouse bone marrow-derived macrophages (BMDM) were extracted from 6-8 week old female C57BL / 6N mice and induced to differentiate for 5 days with 20 ng / mL macrophage colony-stimulating factor (M-CSF). They were then treated with 100 ng / mL lipopolysaccharide (LPS) for 24 hours to induce polarization to M1 type. The M2 type control group was treated with 50 ng / mL IL-4 for 48 hours. Subsequently, the cells were incubated with hydrogel-treated culture medium for 48 hours. Macrophages were stained with fluorescently labeled antibodies, and the expression levels of cell surface macrophage phenotype markers CD86 (pro-inflammatory phenotype) and CD206 (anti-inflammatory phenotype) were detected by flow cytometry. For the characterization of reactive oxygen species levels, the hydrogel-treated cells were incubated with the reactive oxygen species fluorescent probe DCFH-DA (1 μM), and the intracellular reactive oxygen species levels were observed using a fluorescence microscope.

[0079] Figure 4The results showed that the hydrogel dressing provided by the present invention has a high immunomodulatory capacity. The hydrogel regulates the transformation of mouse macrophages into an anti-inflammatory phenotype (A animal experiment, B cell experiment) and eliminates oxidative stress in macrophages (C), exerting an immunomodulatory function.

[0080] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing an aqueous dispersion, characterized in that: It includes the following steps: (1) preparing nanoparticles: covalently linking a calcitonin gene-related peptide and a material having an active oxygen scavenging ability through a chemical reaction; the material having an active oxygen scavenging ability is selected from at least one of metalloporphyrins, natural enzymes, vitamins, polyphenols, coenzyme Q10, and melatonin; (2) then mixing the organic solution containing the nanoparticles with the DSPE-PEG-modified targeting agent to prepare an organic mixed solution, wherein the final concentrations of the nanoparticles and the DSPE-PEG-modified targeting agent in the organic mixed solution are 10-20 mg / mL and 0.5-4 mg / mL, respectively; under ultrasonic water bath conditions, the mixed solution is mixed with water at a volume ratio of 1:7-10, ultrasonically treated, and then the ultrasonicated liquid is dialyzed to obtain an aqueous dispersion; the targeting agent can target cell surface receptors.

2. The method for preparing an aqueous dispersion according to claim 1, wherein The chemical reaction for covalently linking the calcitonin gene-related peptide and the material having active oxygen scavenging ability is selected from at least one of the following: an amide reaction, a Schiff base reaction, a click chemistry reaction, and a thiol-disulfide exchange reaction; Preferably, the click chemistry reaction is a thiol-ene / alkyne click reaction or a copper-catalyzed azide-alkyne cycloaddition; Preferably, the chemical reaction is an amide reaction, using an EDC / NHS combined system, DCC, DIC or CMC; Preferably, an EDC / NHS combined system is used for the amide reaction, and the molar ratio of the material having active oxygen scavenging ability, calcitonin gene-related peptide, EDC, and NHS is: 1:1.2-2:1.2-2:1.5-2.

3. The method for preparing an aqueous dispersion according to claim 1, wherein The DSPE-PEG modified target is selected from at least one of the following DSPE-PEG modified targets: folic acid, mannose, hyaluronic acid and phosphatidylserine; Preferably, the DSPE-PEG-modified target is DSPE-PEG-modified folic acid; Preferably, the molecular weight of PEG in the DSPE-PEG modified target is selected from 2000-6000 KD.

4. The method for preparing an aqueous dispersion according to claim 1, wherein The metalloporphyrin is manganese porphyrin, iron porphyrin, cobalt porphyrin, zinc porphyrin, copper porphyrin, nickel porphyrin or rare earth metal porphyrin; the natural enzyme is superoxide dismutase or catalase; the vitamin is vitamin A, vitamin D, vitamin K, vitamin B1, vitamin B9, vitamin B12, vitamin C or vitamin E; The vitamins are selected from vitamin C or vitamin E; Preferably, the ultrasonic treatment time is 2-5 min.

5. A method for preparing an ultrasound-controlled release hydrogel, characterized in that: It includes the following steps: The aqueous dispersion prepared by the method for preparing the aqueous dispersion according to any one of claims 1 to 4 is mixed with a natural or synthetic polymer material and subjected to a cross-linking reaction to obtain the aqueous dispersion.

6. The method for preparing the ultrasound-controlled release hydrogel according to claim 5, characterized in that: The natural or synthetic polymer material is selected from sodium alginate.

7. The method for preparing the ultrasound-controlled release hydrogel according to claim 6, characterized in that: During the cross-linking reaction, the aqueous dispersion is diluted to 0.1-5 μg / mL, the natural or synthetic polymer material is dissolved in the diluted aqueous dispersion at a mass fraction of 2-5%, and then mixed with a calcium chloride solution or calcium sulfate suspension at a mass percentage of 1-3%; Preferably, the mixing ratio of the diluted aqueous dispersion to the calcium chloride solution or calcium sulfate suspension is 3-5:1, and the cross-linking reaction time is 5-10 minutes.

8. An aqueous dispersion, characterized in that The aqueous dispersion is prepared by the preparation method of any one of claims 1 to 4.

9. An ultrasound-controlled release hydrogel, characterized in that: The hydrogel is prepared by the method for preparing the ultrasound-controlled release hydrogel according to any one of claims 5 to 7.

10. Use of the aqueous dispersion according to claim 8 or the ultrasound-controlled release hydrogel according to claim 9 in wound repair products; Preferably, the wound repair product is selected from chronic wound repair products.

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