Patent Application
20250059333
The present disclosure belongs to the field of medical materials, and in particular, relates to a bilayer bionic drug-loaded hydrogel, and preparation and application thereof.
Currently, wound dressings applied to wound care mainly include traditional wound dressings and modern wound dressings. The traditional wound dressings such as gauzes, bandages are generally used for dry and clean wounds. However, the traditional wound dressings fail to keep pathogenic bacteria out of wounds, leading to wound infections. Meanwhile, when the traditional wound dressings are used to care the wounds, the gauzes or bandages are probably adhered to the wounds, causing pain and secondary injuries to patients during dressing changing. Compared to the traditional wound dressings, the modern wound dressings can wet wound area, exchange air, absorb exudates, are not adhere to the wound surface, and promote autolytic debridement of the wound surface. As the modern dressings, hydrogel dressing has good biocompatibility, better water absorption, moisture retention, good air permeability and other advantages, and is thereby the hotspot in modern wound dressings field.
After years of studies and developments, various researchers have studied and developed different types of hydro gel wound dressings according to different wound types. These wound dressings are capable of wetting the wound surface, exchanging air, absorbing exudates, are not adhered to the wound surface, promoting autolytic debridement of the wound surface, and are antibacterial as well. However, those wound dressings are generally weak in tensile strength and toughness, and are not resistant to mechanical environment such as friction, stretching, and extrusion when applied to wound repair, and are prone to be damages, thus losing effects of protecting and repairing wound. As an important performance index for wound dressings, the antimicrobial effect is very important for the wound dressings. Currently, the antimicrobial effect of the hydrogel wound dressings is mainly achieved by the modifying material or loading antimicrobial drugs, while the antimicrobial property of the material itself is weak against strong infectious and drug-resistant pathogenic bacteria, thus resulting in undesirable anti-infection.
Polyvinyl alcohol (PVA) is a high-molecular polymer obtained by hydrolysis of vinyl acetate, and is widely used in the biomedical field because of its good water solubility, non-toxicity, non-irritation, and good biocompatibility, as well as the cross-linking ability by repeated freeze-thawing, radiation, and chemical reaction. Currently, PVA hydrogels with ultra-high and broadly modifiable mechanical strength can be prepared using the synergy of directional freezing and Hofmeister effect. However, such hydrogels have yet not been developed for skin wound dressings due to that the hydrogels prepared in this way have a dense and uniform structure, and mechanical properties would be severely impacted if other components are loaded, such as drugs or active factors. In addition, PVA does not have antibacterial and anti-inflammatory properties.
Chitosan is alkaline amino polysaccharide after deacetylation by chitin, and features sterilization, hemostasis, cell proliferation promotion, high biocompatibility and etc. Therefore, chitosan is widely used in the field of wound dressing. Genipin is a product of hydrolysis of Gardenia glycosides from Eucommia or Gardenia by β-glucosidase, and its molecules contain a large number of active groups, such as hydroxyl and carboxyl, which are extremely prone to react with compounds containing free amino groups (such as chitosan and gelatin). Therefore, genipin is often used as a cross-linking agent. As a natural cross-linking agent, toxicity of genipin is 10000 times lower than that of glutaraldehyde, a commonly used chemical cross-linking agent. Moreover, genipin also has anti-inflammatory and anti-oxidant effects. However, as a hydrogel, genipin has poor mechanical properties to resist the impact or friction from strong external forces. Therefore, when applied in dressing, genipin is prone to breakage and loss of function. In addition, the chitosan hydrogel is difficult to be firmly bonded to other hydrogels or materials upon gelling, showing poor adhesive property.
Various embodiments of the present disclosure are intended to provide a method of preparing a bilayer bionic drug-loaded hydrogel and application thereof, to solve the problem that existing wound dressings are generally weak in tensile strength and toughness and are not resistant to mechanical environment such as friction, stretching and extrusion when applied to wound repair. The hydrogel of the present disclosure can be applied to deeply infected areas in open war wounds, and has protective, anti-inflammatory, haemostatic, reparative, drug-resistant bacterial cleared effects and other properties.
The present disclosure provides a method for preparing a bilayer bionic drug-loaded hydrogel, comprising:
In an embodiment, in step (S1), a concentration of the sodium sulfate solution is 0.5-1.5 mol/L, and a weight percentage of the polyvinyl alcohol aqueous solution in the outer-layer hydrogel is 5-10%.
In an embodiment, the weight percentage of the polyvinyl alcohol aqueous solution in the outer-layer hydrogel is 5%.
In an embodiment, in step (S2), the pH adjuster is a weak acid.
In an embodiment, the pH adjuster is glacial acetic acid.
In an embodiment, in step (S2), a weight percentage of the polyvinyl alcohol in the first mixture solution is 5-10%, and a weight percentage of the chitosan in the first mixture solution is 2-4%.
In an embodiment, the chitosan and the polyvinyl alcohol are pre-sterilized by ultraviolet irradiation, and the water is pre-sterilized by autoclaving.
In an embodiment, in step (S2), the heating is performed at 85-95° C.
In an embodiment, in step (S3), the antibacterial drug is vancomycin, and a weight percentage of the genipin in the second mixture solution is 0.01-0.05%.
In an embodiment, in step (S4), the freezing and thawing is performed 1-5 times at −20-0° C., each lasting for 0.5-2 h.
In an embodiment, the inner-layer hydrogel has a double-network structure. The double-network structure includes a first network and a second network. The first network is a three-dimensional network structure formed by repeatedly freezing and thawing the polyvinyl alcohol, and the first network structure is a combination of hydrogen bonds between PVA molecular chains, microcrystals, and water in different bonding states at different scales. The second network is formed by chemically cross-linking molecules of the genipin and the chitosan.
In an embodiment, in step (S1), the freezing is performed at −80-0° C. for 0.5-4 h.
In summary, the method of preparing the bilayer bionic drug-loaded hydrogel of the present disclosure solves the problem that existing wound dressings are generally weak in tensile strength and toughness, and are not resistant to mechanical environment such as friction, stretching and extrusion when applied to wound repair, and has the following advantages:
Technical solutions of the present disclosure will be described clearly and completely below. Obviously, the examples described are only some, rather than all examples of the present disclosure. Based on the examples of the present disclosure, all other examples obtained by those ordinary skilled in the art without creative efforts should fall within the scope of protection of the present disclosure.
Provided herein was a method for preparing a bilayer bionic drug-loaded hydrogel, which included the following steps.
5 g of PVA was weighed and poured into a beaker, 95 mL of ultrapure (UP) water was added into the beaker, fully dissolved in a 90° C. water bath under heating and stirring, cooled and ultrasonically deformed, and a PVA solution with a mass fraction of 5% was obtained.
Liquid nitrogen was added into a directional freezing device, and when the temperature of the directional freezing device was constant, a mould containing the PVA solution prepared was placed on the directional freezing device for directional freezing at −60° C. for 1.5 h; after the PVA solution was fully frozen, the mould was removed from the directional freezing device and demoulding was carried out; the demoulded PVA hydrogel was soaked in a sodium sulfate solution for 72 h, followed by soaking in the UP water for 48 h, and the UP water was changed every 4 h to remove salt ions from the PVA hydrogel, and the outer-layer PVA hydrogel was obtained.
The mechanical properties of the outer-layer PVA hydrogel were adjusted by adjusting the concentration of PVA and the concentration of salting-out liquid.
2 g of chitosan was weighed and poured into a beaker, 100 mL of UP water was added into the beaker, and mixed uniformly by stirring; 1 mL of glacial an acetic acid was added into the beaker and stirred until the chitosan was fully dissolved, and a chitosan solution was prepared.
5 g of PVA was added into the chitosan solution, stirred in a 90° C. water bath under heating until the PVA was completely dissolved, cooled and ultrasonically deformed, and a PVA/chitosan mixed solution with a mass ratio of 5:2 was obtained.
A genipin solution with 1% of mass fraction was prepared by dissolving the genipin in the UP water; and a vancomycin solution with 8% of mass fraction was prepared by dissolving the vancomycin in the UP water.
The chitosan, PVA, beaker, and stirrer were sterilized by ultraviolet irradiation, and the UP water was sterilized by autoclaving. The concentration of the genipin exceeded the required concentration for cross-linking to enhance anti-inflammatory effect.
The PVA/chitosan solution (a mass ratio of PVA to chitosan is 5:2) was added into a sterilized beaker, the vancomycin solution (each milliliter of hydrogel contains 8 mg of vancomycin) was added and stirred uniformly, followed by addition of the genipin solution (each milliliter of hydrogel contains 0.1 mg of genipin) and uniform stirring under dark condition to obtain the inner-layer hydrogel precursor solution.
The outer-layer PVA hydrogel was placed in a mould after removing excessive UP water, and 4 mL of the inner-layer hydrogel precursor solution was added into the mould and placed in a sterile and light-proof container for freezing and thawing at −18° C. for 1 h for three times, followed by standing for 3 days at room temperature to form an inner-layer hydrogel on the outer-layer PVA hydrogel, such that the bilayer bionic drug-loaded hydrogel was obtained.
The PVA in the inner-layer precursor solution and the PVA in the outer-layer hydrogel formed molecular linked by repeated freezing and thawing, and formed a first network by cross-linking in the inner-layer hydrogel. Meanwhile, the genipin made the chitosan form a second network by chemical cross-linking under the repeated freezing and thawing in the inner-layer hydrogel. A soft, moist, adherent, anti-inflammatory, haemostatic and pro-repair inner-layer hydrogel similar to subcutaneous tissue was obtained by adjusting cross-1 inking parameters of the double network system. The loading of vancomycin greatly improved the ability of hydrogel to resist infection by Gram-positive resistant bacteria and was suitable for the care of severely infected wounds such as war wounds.
A structural diagram of a bilayer drug-loaded hydrogel of the present disclosure was shown in
Provided herein was a method for preparing a bilayer bionic drug-loaded hydrogel, which included the following steps.
5 g of PVA was weighed and poured into a beaker, 95 mL of ultrapure (UP) water was added into the beaker, fully dissolved in a 90° C. water bath under heating and stirring, cooled and ultrasonically deformed, and a PVA solution with a mass fraction of 5% was obtained.
Liquid nitrogen was added into a directional freezing device, and when the temperature of the directional freezing device was constant, a mould containing the PVA solution prepared was placed on the directional freezing device for directional freezing at −40° C. for 2 h; after the PVA solution was fully frozen, the mould was removed from the directional freezing device and demoulding was carried out; the demoulded PVA hydrogel was soaked in a sodium sulfate solution for 72 h, followed by soaking in the UP water for 48 h, and the UP water was changed every 4 h to remove salt ions from the PVA hydrogel, and the outer-layer PVA hydrogel was obtained.
The mechanical properties of the outer-layer PVA hydrogel were adjusted by adjusting the concentration of PVA and the concentration of salting-out liquid.
2 g of chitosan was weighed and poured into a beaker, 100 mL of UP water was added into the beaker, and mixed uniformly by stirring; 1 mL of glacial an acetic acid was added into the beaker and stirred until the chitosan was fully dissolved, and a chitosan solution was prepared.
5 g of PVA was added into the chitosan solution, stirred in a 95° C. water bath under heating until the PVA was completely dissolved, cooled and ultrasonically deformed, and a PVA/chitosan mixed solution with a mass ratio of 5:2 was obtained.
A genipin solution with 1% of mass fraction was prepared by dissolving the genipin in the UP water; and a vancomycin solution with 8% of mass fraction was prepared by dissolving the vancomycin in the UP water.
The chitosan, PVA, beaker, and stirrer were sterilized by ultraviolet irradiation, and the UP water was sterilized by autoclaving. The concentration of the genipin exceeded the required concentration for cross-linking to enhance anti-inflammatory effect.
The PVA/chitosan solution (a mass ratio of PVA to chitosan is 5:2) was added into a sterilized beaker, the vancomycin solution (each milliliter of hydrogel contains 8 mg of vancomycin) was added and stirred uniformly, followed by addition of the genipin solution (each milliliter of hydrogel contains 0.1 mg of genipin) and uniform stirring under dark condition to obtain the inner-layer hydrogel precursor solution.
The outer-layer PVA hydrogel was placed in a mould after removing excessive UP water, and 4 mL of the inner-layer hydrogel precursor solution was added into the mould and placed in a sterile and light-proof container for freezing and thawing at −10° C. for 1.2 h for three times, followed by standing for 3 days at room temperature to form an inner-layer hydrogel on the outer-layer PVA hydrogel, such that the bilayer bionic drug-loaded hydrogel was obtained.
The PVA in the inner-layer precursor solution and the PVA in the outer-layer hydrogel formed molecular links by repeated freezing and thawing. And formed a first network by cross-linking in the inner-layer hydrogel. Meanwhile, the genipin made the chitosan form a second network by chemical cross-linking under the repeated freezing and thawing in the inner-layer hydrogel. A soft, moist, adherent, anti-inflammatory, haemostatic and pro-repair inner-layer hydrogel similar to subcutaneous tissue was obtained by adjusting cross-linking parameters of the double network system. The loading of vancomycin greatly improved the ability of hydrogel to resist infection by Gram-positive resistant bacteria and was suitable for the care of severely infected wounds such as war wounds.
The preparation of the outer-layer PVA hydrogel was the same as that in Example 1.
An inner-layer hydrogel precursor solution was prepared according to the method provided in Example 1. Then the inner-layer hydrogel precursor solution was added into a mould and placed in a sterile and light-proof container for freezing and thawing at −18° C. for 1 h for three times, followed by standing to form the inner-layer hydrogel.
(3) The inner-layer hydrogel was putted on the outer-layer hydrogel, and added into the mould and placed in a sterile and light-proof container for freezing and thawing at −18° C. for 1 for three times, followed by standing for 3 days at room temperature. It was found that the inner-layer hydrogel was still separated from the outer-layer hydrogel, as shown in
The microscopic morphology of cross section a and longitudinal section b of the outer-layer hydrogel, as shown in
An image of a cross section of the inner-layer hydrogel under a scanning electron microscope, as shown in
Images of a cross section and a longitudinal section of the bilayer hydrogel under a scanning electron microscope, as shown in
Tensile properties of materials were measured with a universal mechanical testing machine.
Mechanical properties of a hydrogel of the present disclosure were shown in
Mechanical properties of inner and outer hydrogels of the present disclosure were shown in
Materials are performed total reflection scanning using an infrared spectrometer. Infrared spectroscopy of an inner-layer hydrogel of the present disclosure is shown in
According to ISO 10993-5:1999 and GB/T 16886.5-2003, the biocompatibility of hydrogels with different drug-loading amounts was evaluated, wherein vancomycin (VCM)=x mg/ml represented the number of milligrams of vancomycin per milliliter hydrogel. Day 1: cell relative growth rates (RGR) in groups of VCM=0 mg/mL, VCM=2 mg/mL, VCM=5 mg/mL and VCM=8 mg/mL are 90.69%, 108.89%, 109.311% and 107.94% respectively. Day 3: the RGRs in groups of VCM=0 mg/mL, VCM=2 mg/mL, VCM=5 mg/mL and VCM=8 mg/mL are 74.23%, 83.13%, 99.27% and 95.68% respectively.
The cytocompatibility evaluation of the bilayer bionic drug-loaded hydrogel of the present disclosure was shown in
The antioxidant property of the hydrogel was evaluated using a DPPH radical scavenging method, the result was shown in
The antibacterial property of 5 mg/ml of hydrogel loading with vancomycin was evaluated using an inhibition zone method, as shown in
Adhesion effects of the hydrogel of the present disclosure on different materials surface were shown in
Although contents of the present disclosure have been described in detail with reference to the above preferred examples, it should be appreciated that the above description should not be considered as a limitation to the present disclosure. Various modifications and alternatives to the present disclosure will be apparent to those skilled in the art upon reading the foregoing. Accordingly, the scope of protection of the present disclosure should be defined by the attached claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210809586.0 | Jul 2022 | CN | national |
This application is a continuation-in-part of U.S. patent application Ser. No. 18/476,562, filed on Sep. 28, 2023, now pending, which is a continuation of International Patent Application No. PCT/CN2023/102580 with a filing date of Jun. 27, 2023, designating the United States, and further claims the benefit of priority from Chinese Patent Application No. 202210809586.0, filed on Jul. 11, 2022. The content of the aforementioned application, including any intervening amendments thereto, is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2023/102580 | Jun 2023 | WO |
| Child | 18937934 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18476562 | Sep 2023 | US |
| Child | 18937934 | US |
