CN113101053A - Wound dressing capable of releasing nitric oxide - Google Patents

Abstract

The present invention relates to a wound dressing capable of releasing nitric oxide, comprising a first dressing containing NO synthase and a second dressing containing a substrate that can interact with NO synthase; Isolation; when in use, the first dressing and the second dressing are stacked and covered on the wound surface, and the outflow of wound exudate makes the NO synthase on the first dressing mix with the substrate on the second dressing to generate NO and act on it. Wound repair. Compared with the prior art, the present invention generates NO by establishing the same physiological system as in the body, and has stronger adaptability to the physical, chemical and physiological conditions of the wound environment, and has the advantages of adjustable NO generation rate, convenient storage, etc. advantage.

Description

Wound dressing capable of releasing nitric oxide

Technical Field

The invention belongs to the technical field of medical materials, and relates to a wound dressing capable of releasing nitric oxide.

Background

Nitric Oxide (NO) is a very important biological messenger involved in the regulation of many important physiological activities in the body. It is a potent vasodilator, increases local blood flow, and also promotes angiogenesis in conjunction with other growth factors. It also has effects in killing pathogenic microorganisms and reducing inflammatory response. NO plays a very important role in wound repair, and is involved in regulating many steps of wound repair, such as improving collagen production, promoting blood vessel growth, regulating inflammatory response, promoting differentiation and proliferation of various skin cells, etc. Research also shows that the wound surface has low NO level to delay wound repair, the wound closing time of a mouse with NO synthase (NOS) removed is prolonged by 30%, and a certain amount of NO conveyed on the wound surface can achieve a good effect of promoting repair.

In the body, NO is produced by NO synthase through the process of oxidative conversion of arginine to citrulline. There are three kinds of NOS in the body, neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), and Inducible Nitric Oxide Synthase (iNOS) that is expressed inducibly after injury, each of which differs in its expression, activity and calcium dependence.

Currently there are NO wound repair products that locally generate or deliver NO. NO precursor compounds such as O-nitroso compounds, S-nitroso compounds, nonanoic acid compounds, inorganic nitroso compounds, sildenone imines and the like are used in the art to decompose under suitable chemical conditions to generate NO, but the chemical conditions of the wound site are not always suitable. Therefore, research and development of a system capable of stably generating and supplying NO for a long time under biological conditions has great therapeutic significance for wound repair.

Disclosure of Invention

The invention aims to provide a wound dressing capable of releasing nitric oxide, which is used for solving the problems that the existing NO-based wound repair has poor adaptability to physical, chemical and physiological conditions of wound environments and can not continuously and stably supply NO.

The purpose of the invention can be realized by the following technical scheme:

a wound dressing capable of releasing nitric oxide comprises

A first dressing comprising a first adjuvant carrier and NO synthase disposed on the first adjuvant carrier, and

a second dressing comprising a second dressing carrier and a substrate disposed on the second dressing carrier, the substrate being capable of interacting with NO synthase;

during storage, the NO synthase is isolated from the substrate; when the wound dressing is used, the first dressing and the second dressing are stacked and cover a wound surface, and the outflow of wound seepage liquid is respectively decomposed to release NO synthase on the first dressing and a substrate on the second dressing to the wound surface and mixed, so that NO is locally generated and acts on wound repair.

As a preferred technical scheme, the first dressing and the second dressing are stored in a dry environment.

Further, the NO synthase includes at least one of a neuronal type nitric oxide synthase, an endothelial type nitric oxide synthase or an inducible type nitric oxide synthase, and the loading amount of the NO synthase is 50-5000ng/g, preferably 100-2000ng/g, and more preferably 500-1000 ng/g.

Further, the first dressing and the second dressing further comprise an additive, and the additive specifically comprises at least one of a DNA/chitosan compound, a poly-L-lysine/polyethylene glycol block copolymer, sodium carboxymethylcellulose, microcrystalline cellulose or hypromellose.

Further, the substrate comprises arginine and Nicotinamide Adenine Dinucleotide Phosphate (NADPH), and the loading amounts of the substrate are 1.0-10.0mg/g (preferably 2.0-5.0mg/g) and 5.0-50.0mg/g (preferably 10.0-20.0mg/g), respectively.

Further, the first dressing carrier and the second dressing carrier both comprise a dry material or a wet material. If the first dressing carrier and the second dressing carrier are both made of dry materials, the dressing can be pre-compounded into a wound dressing for convenient storage and use; if at least one of the dressing carriers is in a wet form, it is desirable to use it in an overlapping manner in the actual application to avoid premature interaction of the NO-synthase with the substrate.

Furthermore, the dry materials comprise non-woven materials and woven materials such as foam materials or film materials; the wet material comprises at least one of hydrogel, hydrocolloid or hydrophilic high molecular polymer such as protein.

Further, the foam material comprises polyurethane foam material, the woven material comprises cotton yarn, and the protein comprises collagen.

Further, the wet material is preferably a porous wet material that facilitates the penetration of enzymes, substrates or gases into the wound bed.

Further, in use, the dressing contacting with the wound surface serves as a wound contact layer, the dressing stacked on the wound contact layer serves as a dressing upper layer, and the mass ratio of the wound contact layer to the dressing upper layer is (0.5-2): 1.

Preferably, the wound contact layer is a porous material, especially a porous material provided with wound exudate channels.

The invention provides a novel wound dressing capable of generating and releasing NO on the surface of a wound, and particularly relates to a novel wound dressing which is prepared by respectively loading NO synthase and a substrate in a double carrier form, and then combining the two carriers to form a composite dressing so as to establish an NO generation mechanism similar to a physiological system under the condition of wound exudate, thereby continuously generating and releasing NO gas on the surface of the wound and promoting wound repair.

Compared with the prior art, the invention has the following characteristics:

1) the invention generates NO by establishing a physiological system which is the same as an organism, and compared with a wound repair technology which adopts NO precursors or directly applies NO gas, the system has stronger adaptability to physical, chemical and physiological conditions of a wound environment;

2) because the NO synthase and the substrate are respectively loaded on two different carriers, the mutual isolation and storage are facilitated, and the problem that raw materials are easy to degrade easily in a wound repair technology adopting an NO precursor is solved by respectively storing the NO synthase and the substrate in a dry environment, so that the storage requirement is effectively reduced, and the storage life is prolonged;

3) the invention can also adjust the release time and rate of NO generation by respectively adjusting the combination mode of the enzyme and the carrier, the combination mode of the substrate and the carrier, or the physicochemical characteristics of the carrier so as to achieve the aim of continuously generating NO within a certain time, for example, nNOS and eNOS are calcium-dependent enzymes, and the NO generation can be adjusted by increasing or decreasing the calcium ion concentration.

Detailed Description

The present invention will be described in detail with reference to specific examples.

A nitric oxide releasable wound dressing includes a first dressing and a second dressing.

Wherein the first dressing comprises a first dressing carrier and NO synthase (preferably loading of 100-2000ng/g, more preferably 500-1000ng/g) loaded on the first dressing carrier at a loading of 50-5000ng/g, and the NO synthase comprises at least one of neuronal nitric oxide synthase, endothelial nitric oxide synthase or inducible nitric oxide synthase. In addition, the first dressing further comprises an additive, and the additive specifically comprises at least one of DNA/chitosan compound, poly-L-lysine/polyethylene glycol block copolymer, sodium carboxymethylcellulose, microcrystalline cellulose or hypromellose.

The second dressing comprises a second dressing carrier and a substrate loaded on the second dressing carrier, wherein the substrate can react with NO synthase and comprises arginine and Nicotinamide Adenine Dinucleotide Phosphate (NADPH), the loading amount of the arginine is 1.0-10.0mg/g (preferably 2.0-5.0mg/g), and the loading amount of the NADPH is 5.0-50.0mg/g (preferably 10.0-20.0 mg/g).

The first dressing carrier and the second dressing carrier each comprise a dry material or a wet material. Wherein the dry material comprises non-woven materials such as foam materials or film materials and woven materials such as cotton yarns; the wet material includes at least one of hydrogel, hydrocolloid, or hydrophilic high molecular polymer such as protein.

When the carrier is made of wet material, the structure of the wet material is preferably porous structure, so that the enzyme, substrate or gas can permeate to the wound surface, and the opening rate of the porous structure is preferably 20-40%.

During storage, the NO synthase is isolated from the substrate; when the wound dressing is used, the first dressing and the second dressing are stacked and cover a wound surface, NO synthase on the first dressing is mixed with a substrate on the second dressing through outflow of wound seepage, NO is generated, and the NO acts on wound repair.

And when in use, the dressing contacting with the wound surface of the wound is used as a wound contact layer, the dressing stacked on the wound contact layer is used as a dressing upper layer, and the mass ratio of the wound contact layer to the dressing upper layer is controlled to be (0.5-2): 1.

A method of making a first dressing having a dry material as a carrier, comprising: preparing a first mixed solution containing NO synthase and an additive, soaking the cleaned and dried dry material into the first mixed solution, or spraying the first mixed solution on the cleaned and dried dry material, and drying to obtain the first dressing.

Wherein, in the first mixed solution, when the additive is double-stranded DNA and chitosan, the preferable concentration of the DNA in the mixed solution is 0.01-0.05mg/mL, and the preferable concentration of the chitosan is 0.01-0.05 mg/mL; when the additive is poly-L-lysine/polyethylene glycol block copolymer, the preferable concentration is 1-3 mg/mL; when the additive is sodium carboxymethylcellulose, the preferred concentration is 0.2-0.8 wt%;

when eNOS or iNOS is used as the NO synthase, the concentration thereof is preferably 200-500 ng/mL.

Further, a preferable preparation method of the first mixed solution includes: and (3) dropwise adding the aqueous solution of the additive or the phosphate buffered physiological saline solution of the additive into the NO synthase solution, and uniformly mixing.

A method of making a first dressing in the form of a hydrogel comprising: mixing the first additive solution and the NO synthase solution, fully stirring, filtering to obtain a precipitate, washing, drying, adding a surfactant, and uniformly mixing to obtain an NO synthase mixture; then adding a second additive solution, mixing, and standing for 8-12h at 2-8 ℃ to obtain the first dressing in the form of hydrogel.

Wherein the first additive is preferably microcrystalline cellulose, the concentration of NO synthase after the first additive solution is mixed with the NO synthase solution is 0.5-5 mu g/mL, the concentration of the first additive is 5-15 wt%, the surfactant is preferably Tween-20, the addition amount is 0.5-2% relative to the mass of the precipitate, the second additive solution is preferably 1-5 wt% hydroxypropyl methylcellulose solution, and the concentration of the NO synthase in the obtained mixture is 500ng/mL after the second additive solution is added.

A method of making a second dressing having a dry material as a carrier, comprising: and preparing a second mixed solution containing a substrate and an additive, soaking the cleaned and dried dry material into the second mixed solution, or spraying the second mixed solution on the cleaned and dried dry material, and drying to obtain the second dressing.

Wherein, in the second mixed solution, the preferable concentration of arginine is 1-10mmol/L, and the preferable concentration of NADPH is 2-15 mmol/L; the additive is preferably sodium carboxymethylcellulose or sodium alginate, and the preferred concentration is 1-8 wt%.

A method of preparing a second dressing in the form of a hydrocolloid, comprising: and mixing the mixed sol containing the additive with a substrate solution, defoaming in vacuum, and placing on silicone oil paper for drying and forming to obtain the second dressing in the form of hydrocolloid. Wherein the additive comprises at least one of styrene-isoprene-styrene block copolymer (5-20 wt%), pectin (15-25 wt%), antioxidant 1010(1-5 wt%) or sodium carboxymethylcellulose (15-25 wt%).

The above-mentioned preparation steps involving NO synthase are preferably carried out at 2-8 ℃.

The following examples are given in detail to illustrate the embodiments and specific procedures of the present invention, but the scope of the present invention is not limited to the following examples.

Example 1:

an eNOS-bearing polyurethane foam dressing is prepared by the following method:

1) fully cleaning and drying the polyurethane foam;

2) mixing double-stranded DNA with a chitosan solution to obtain a DNA/chitosan solution, then adding an eNOS recombinant protein solution into the solution, and uniformly mixing to obtain a dip-coating solution containing 0.02mg/mL of DNA, 0.02mg/mL of chitosan and 200ng/mL of eNOS;

3) soaking the polyurethane foam in a dip-coating solution at 4 ℃ for 30min so as to fully dip-coat the DNA/chitosan containing eNOS on the polyurethane foam;

4) and taking the polyurethane foam out of the solution and air-drying to obtain the eNOS-bearing polyurethane foam dressing, wherein the eNOS load is 500ng/g, and storing the dressing in a dry environment at 4 ℃.

Similarly, this example also uses cotton yarn as the dressing matrix, and repeats the above preparation process to obtain the cotton yarn dressing bearing eNOS.

Wherein, the double-stranded DNA and the chitosan are purchased from Sigma-Aldrich (D8515, 448877), and the recombinant eNOS is purchased from Enzo Life Sciences (ALX-201-.

Example 2:

an iNOS-bearing polyurethane foam dressing is prepared by the following method:

1) fully cleaning and drying the polyurethane foam;

2) dissolving a poly-L-lysine/polyethylene glycol block copolymer in phosphate buffered saline (0.02mol/L, pH 7.4) at room temperature to obtain a mixed solution, dropwise adding the mixed solution into an iNOS recombinant protein solution, and uniformly mixing to obtain a dip-coating solution containing 2mg/mL of poly-L-lysine/polyethylene glycol block copolymer and 1000ng/mL of iNOS;

3) soaking the polyurethane foam in a dip-coating solution at 4 ℃ for 30min so as to fully dip-coat the iNOS-containing poly-L-lysine/polyethylene glycol block copolymer on the polyurethane foam;

4) and taking the polyurethane foam out of the solution and air-drying to obtain the iNOS-bearing polyurethane foam dressing, and storing the dressing in an environment at 4 ℃.

Similarly, in this example, cotton yarn is used as a dressing substrate, and the above preparation process is repeated to obtain a cotton yarn dressing carrying iNOS.

Wherein the Poly-L-lysine/polyethylene Glycol Block copolymers used were prepared as described by Vanderkerken (Vanderkerken S, et al. Synthesis and Evaluation of Poly (Ethylene Glycol) -Polymer Block copolymers as carriers for Gene delivery. journal of Bioactive and Compatible polymers.2000; 15(2): 115-.

Example 3:

an eNOS-bearing polyurethane foam dressing is prepared by the following method:

1) fully cleaning and drying the polyurethane foam;

2) dissolving sodium carboxymethylcellulose in phosphate buffered normal saline (0.02mol/L, pH 7.4) at room temperature to obtain a mixed solution, and then uniformly mixing the mixed solution with an eNOS recombinant protein solution to obtain a spraying solution containing 0.5 wt% of sodium carboxymethylcellulose and 200ng/mL eNOS;

3) spraying the polyurethane foam for 10min at room temperature by using the spraying liquid so as to fully coat the eNOS-containing sodium carboxymethyl cellulose on the polyurethane foam;

4) and (3) air-drying the polyurethane foam to obtain the eNOS-bearing polyurethane foam dressing, and storing the dressing in an environment at 4 ℃.

Similarly, in this example, cotton yarn is used as a dressing substrate, and the above preparation process is repeated to obtain a cotton yarn dressing carrying iNOS.

The recombinant eNOS and sodium carboxymethylcellulose were purchased from Sigma-Aldrich (D8515, C5013).

Example 4:

an iNOS-bearing hydrogel dressing is prepared by the following steps:

1) dissolving microcrystalline cellulose in phosphate buffer solution (0.05mol/L, pH 6.7) to obtain 10 wt% microcrystalline cellulose solution;

2) adding an iNOS recombinant protein solution into a microcrystalline cellulose solution according to the final concentration of 1 mu g/mL, fully stirring, filtering to obtain a precipitate, washing with deionized water, drying, adding Tween-20 accounting for 1% of the mass of the precipitate, and uniformly mixing to obtain an iNOS mixture;

3) heating purified water to 90 ℃, adding hydroxypropyl methylcellulose, stirring and dispersing uniformly to obtain a mixed solution containing 2 wt% of hydroxypropyl methylcellulose;

4) after the mixed solution is cooled to room temperature, adding an iNOS mixture to ensure that the final iNOS concentration is 200 ng/mL;

5) standing at 4 ℃ overnight to obtain the iNOS-bearing hydrogel dressing, and storing the dressing in an environment at 4 ℃.

Among them, recombinant iNOS was obtained from Enzo Life Sciences (ALX-201-852-0010) and microcrystalline cellulose (Avicel PC 105) was obtained from Earth Supplied Products.

Example 5:

an arginine and NADPH bearing hydrocolloid dressing is prepared by the following steps:

1) adding styrene-isoprene-styrene block copolymer, antioxidant 1010, pectin and sodium carboxymethylcellulose into water at 90 ℃ in sequence to obtain mixed sol containing 10 wt% of styrene-isoprene-styrene block copolymer, 2 wt% of antioxidant 1010, 20 wt% of pectin and 20 wt% of sodium carboxymethylcellulose;

2) adding arginine and NADPH to obtain a premixed sol containing 4mmol/L of arginine and 6mmol/L of NADPH;

3) and placing the premixed sol on silicone oil paper for drying and forming after vacuum defoaming to obtain the hydrocolloid dressing for bearing arginine and NADPH, wherein the load amounts of arginine and NADPH are 2.0mg/g and 13.0mg/g respectively.

Arginine and NADPH were purchased from Sigma-Aldrich (A5006, N5755).

Example 6:

a polyurethane foam dressing for bearing arginine and NADPH is prepared by the following steps:

1) adding arginine and NADPH into 5 wt% sodium carboxymethylcellulose solution to make the final concentrations of arginine and NADPH be 4mmol/L and 6mmol/L respectively, and mixing to obtain dip-coating solution;

2) soaking the polyurethane foam in a dip-coating solution at 4 ℃ for 30min so as to fully dip-coat arginine, NADPH and sodium carboxymethyl cellulose on the polyurethane foam;

3) and taking the polyurethane foam out of the solution and air-drying to obtain the iNOS-bearing polyurethane foam dressing, and storing the dressing in an environment at 4 ℃.

Similarly, in this embodiment, a 3 wt% sodium alginate solution is used to replace sodium carboxymethyl cellulose, and cotton yarn is used to replace polyurethane foam, and the above preparation process is repeated to obtain a cotton yarn dressing bearing arginine, NADPH, and sodium alginate.

Arginine and NADPH were purchased from Sigma-Aldrich (A5006, N5755).

Example 7:

a dry form of a composite NO-releasable wound dressing prepared by a process comprising:

the hydrocolloid dressing bearing arginine and NADPH prepared in example 5 was first perforated with a 2mm pore size and a 30% open porosity as a wound contact layer, and then the polyurethane foam dressing bearing eNOS prepared in example 1 was attached to the hydrocolloid dressing as an upper dressing layer, to obtain the above-described composite wound dressing capable of releasing NO.

Wherein the mass ratio of the dressing upper layer to the wound contact layer is 1: 1.5.

Example 8:

a dry form of a composite NO-releasable wound dressing prepared by a process comprising:

the composite wound dressing capable of releasing NO was obtained by first perforating the hydrocolloid dressing carrying arginine and NADPH prepared in example 5 (same as example 7) and using it as a wound contact layer, and then attaching the cotton yarn dressing carrying iNOS prepared in example 2 to the hydrocolloid dressing as a dressing upper layer.

Wherein the mass ratio of the dressing upper layer to the wound contact layer is 1: 2.

Example 9:

a wet-form composite wound dressing capable of releasing NO is prepared by the following steps:

firstly, the polyurethane foam dressing which is prepared in the embodiment 6 and is used for bearing arginine and NADPH is attached to a wound to be used as a wound contact layer, then the polyurethane foam dressing which is prepared in the embodiment 4 and is used for bearing iNOS is coated on the polyurethane foam dressing to be used as a dressing upper layer, and then the polyurethane foam dressing is covered with an adhesive transparent film dressing for wrapping, so that the composite wound dressing which can release NO is obtained.

Wherein the mass ratio of the dressing upper layer to the wound contact layer is 1:1.

Example 10:

a wet-form composite wound dressing capable of releasing NO is prepared by the following steps:

firstly, the cotton yarn dressing bearing arginine, NADPH and sodium alginate prepared in the embodiment 6 is pasted on a wound to be used as a wound contact layer, then the iNOS-bearing hydrogel dressing prepared in the embodiment 4 is coated on the cotton yarn dressing to be used as a dressing upper layer, and then the cotton yarn dressing is covered with an adhesive transparent film dressing for wrapping, so that the NO-releasing composite wound dressing is obtained.

Wherein the mass ratio of the dressing upper layer to the wound contact layer is 1: 1.8.

Example 11:

this example was used to test the NO release effect of the composite dressing of example 7 by the following method:

1) the composite wound dressing of example 7 was cut into a 5cm × 5cm size and placed in a petri dish;

2) injecting phosphate buffered normal saline containing 10% calf serum into the culture dish until the polyurethane foam layer is saturated by water, and then additionally injecting 10 mL;

3) the petri dish was covered and incubated at 37 ℃ for 4 h;

4) the culture broth in the dish was centrifuged for 10min and the supernatant was used to measure NO release of 346.7 μmol using Griess kit (R & D Systems, Inc.).

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims (10)

1. A nitric oxide releasable wound dressing comprising

A first dressing comprising a first dressing carrier and NO synthase disposed on the first dressing carrier, an

A second dressing comprising a second dressing carrier and a substrate disposed on the second dressing carrier, the substrate being capable of interacting with NO synthase;

during storage, the NO synthase is isolated from the substrate; when the wound dressing is used, the first dressing and the second dressing are stacked and cover a wound surface, NO synthase on the first dressing can be mixed with a substrate on the second dressing through outflow of wound seepage, NO is generated, and the NO acts on wound repair.

2. The nitric oxide-releasing wound dressing of claim 1, wherein said NO synthase comprises at least one of neuronal nitric oxide synthase, endothelial nitric oxide synthase or inducible nitric oxide synthase, and the loading of said NO synthase is 50-5000 ng/g.

3. The nitric oxide-releasing wound dressing of claim 1, further comprising an additive.

4. The nitric oxide-releasing wound dressing of claim 3, wherein said additive comprises at least one of DNA/chitosan complex, poly-L-lysine/polyethylene glycol block copolymer, sodium carboxymethylcellulose, microcrystalline cellulose or hypromellose.

5. The wound dressing of claim 1, wherein the substrate comprises arginine and nicotinamide adenine dinucleotide phosphate, and the loading amounts are 1.0-10.0mg/g and 5-50mg/g, respectively.

6. The nitric oxide-releasing wound dressing of claim 1, wherein the first dressing carrier and the second dressing carrier each comprise a dry material or a wet material.

7. The nitric oxide-releasing wound dressing of claim 6, wherein said dry material comprises a foam, a woven or a film material; the wet material comprises hydrophilic high molecular polymer.

8. The nitric oxide-releasing wound dressing of claim 7, wherein the foam material comprises polyurethane foam, the woven material comprises cotton yarn, and the hydrophilic polymer comprises at least one of hydrogel, hydrocolloid or protein.

9. The nitric oxide-releasing wound dressing of claim 6, wherein the moist material is a porous moist material.

10. The nitric oxide-releasing wound dressing of claim 1, wherein, in use, the dressing in contact with the wound bed serves as a wound contact layer, the dressing stacked on the wound contact layer serves as a dressing upper layer, and the mass ratio of the wound contact layer to the dressing upper layer is (0.5-2): 1.