BILAYER BIONIC DRUG-LOADED HYDROGEL, AND PREPARATION AND APPLICATION THEREOF

Abstract

Disclosed is a bilayer bionic drug-loaded hydrogel, and preparation and application thereof. The hydrogel includes: an outer-layer hydrogel and an inner-layer hydrogel. The outer-layer hydrogel is prepared by: forming a polyvinyl alcohol hydrogel by directionally freezing a polyvinyl alcohol aqueous solution, soaking the polyvinyl alcohol aqueous solution in a sodium sulfate solution, and removing salt ions after soaking; and the inner-layer hydrogel is prepared by components of: a loaded drug, polyvinyl alcohol, chitosan, genipin, water, and a pH adjuster. The hydrogel of the present disclosure can be applied to deeply infected areas of wounds in open war wounds, and has protective, anti-inflammatory, haemostatic, reparative, anti-drug resistant bacterial effects, and other properties.

Claims

1. A bilayer bionic drug-loaded hydrogel, comprising: an outer-hydrogel layer and an inner-layer hydrogel; wherein the outer-hydrogel layer is prepared by: forming a polyvinyl alcohol hydrogel by directionally freezing a polyvinyl alcohol aqueous solution, soaking the polyvinyl alcohol hydrogel in a 0.5-1.5 mol/L sodium sulfate solution, and removing salt ions after soaking, wherein a mass fraction of the polyvinyl alcohol aqueous solution in the outer-layer hydrogel is in the range of 5% to 10%; the inner-layer hydrogel is prepared from components of: a loaded drug, polyvinyl alcohol, chitosan, genipin, water, and a pH adjuster, wherein mass fractions of the polyvinyl alcohol, the chitosan, and the genipin are in the range of 5% to 10%, in the range of 2% to 4%, and in the range of 0.01% to respectively, and the loaded drug is an antibacterial drug; the outer-layer hydrogel and the inner-layer hydrogel are physically cross-linked to form intermolecular hydrogen bonds and microcrystals, and thus seamlessly bonded; the inner-layer hydrogel is in a double-network structure, the double-network structure comprising a first network and a second network, wherein 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, and the second network is formed by chemically cross-linking molecules of the genipin and the chitosan; and the bilayer bionic drug-loaded hydrogel is obtained by placing the outer-layer hydrogel in a mould, adding an inner-layer solution into the mould, and freezing and thawing several times under aseptic and light-proof conditions.

2. The bilayer bionic drug-loaded hydrogel according to claim 1, wherein the mass fraction of the polyvinyl alcohol aqueous solution in the outer-layer hydrogel is 5%.

3. The bilayer bionic drug-loaded hydrogel according to claim 1, wherein the pH adjuster is a weak acid.

4. The bilayer bionic drug-loaded hydrogel according to claim 1, wherein the antibacterial drug is vancomycin.

5. A method for preparing a bilayer bionic drug-loaded hydrogel as defined in claim 1, the method comprising: preparation of an outer-layer hydrogel: forming a polyvinyl alcohol hydrogel by directionally freezing a polyvinyl alcohol aqueous solution, soaking the polyvinyl alcohol aqueous solution in a sodium sulfate solution, and removing salt ions after soaking to obtain an outer-layer polyvinyl alcohol hydrogel; preparation of an inner-layer hydrogel precursor solution: taking chitosan, adding water and stirring uniformly, adding a glacial acetic acid and stirring until the chitosan is dissolved, adding polyvinyl alcohol, heating and stirring to dissolve to obtain a polyvinyl alcohol/chitosan mixed solution; and adding a vancomycin aqueous solution into the prepared polyvinyl alcohol/chitosan mixed solution, followed by stirring, and adding a genipin aqueous solution, followed by stirring in a dark environment to obtain an inner-layer solution.

6. The method according to claim 5, wherein the chitosan and the polyvinyl alcohol are sterilized by ultraviolet irradiation, and the water is sterilized by autoclaving.

7. The method according to claim 5, wherein in the preparation of the inner-layer hydrogel precursor solution, adding the polyvinyl alcohol, heating to 90 C., and stirring to dissolve.

8. An application of a bilayer bionic drug-loaded hydrogel as defined in claim 1 in medical materials.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1a is a structural diagram of a bilayer drug-loaded hydrogel according to the present disclosure;

[0035] FIG. 1B is an optical photograph of a bilayer drug-loaded hydrogel according to the present disclosure;

[0036] FIG. 2a is a cross-sectional view of an outer-layer hydrogel observed by a scanning electron microscope according to the present disclosure;

[0037] FIG. 2b is a longitudinal cross-sectional view of an outer-layer hydrogel observed by a scanning electron microscope according to the present disclosure;

[0038] FIG. 3 is an image of an inner-layer hydrogel observed by a scanning electron microscope according to the present disclosure;

[0039] FIG. 4 is an image of a bilayer hydrogel observed by a scanning electron microscope according to the present disclosure;

[0040] FIG. 5a is a stress-strain curve of an outer-layer hydrogel in tension according to the present disclosure;

[0041] FIG. 5b is a relation diagram between Young's modulus of hydrogel and Young's modulus of skin (the largest circle area represents a Young's modulus range of the hydrogel) according to the present disclosure;

[0042] FIG. 6a shows mechanical properties of an outer hydrogel according to the present disclosure;

[0043] FIG. 6b shows mechanical properties of an inner hydrogel according to the present disclosure;

[0044] FIG. 7 shows a Fourier transform infrared spectroscopy (FTIR) of an inner-layer hydrogel according to the present disclosure;

[0045] FIG. 8 shows cytotoxicity evaluation of a bilayer hydrogel according to the present disclosure;

[0046] FIG. 9 shows an antioxygenic property (DPPH) of an inner-layer hydrogel according to the present disclosure;

[0047] FIG. 10a shows an antibacterial effect of a bilayer hydrogel on Escherichia coil according to the present disclosure;

[0048] FIG. 10b shows an antibacterial effect of a bilayer hydrogel on Staphylococcus epidermidis according to the present disclosure;

[0049] FIG. 10c shows an antibacterial effect of a bilayer hydrogel on Staphylococcus aureus according to the present disclosure;

[0050] FIG. 11a shows an adhesive property of a bilayer hydrogel to a hand according to the present disclosure;

[0051] FIG. 11b shows an adhesive property of a bilayer hydrogel to an elbow joint according to the present disclosure;

[0052] FIG. 11c shows an adhesive property of a bilayer hydrogel to a glass according to the present disclosure; and

[0053] FIG. 11d shows an adhesive property of a bilayer hydrogel to a plastic according to the present disclosure.

DETAILED DESCRIPTION

[0054] 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.

Example 1

[0055] Preparation of an Outer Polyvinyl Alcohol (PVA) Hydrogel [0056] (1) 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. [0057] (2) 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 in step (1) was placed on the directional freezing device; after the PVA solution was fully frozen, the mould was 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 an 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.

[0058] Preparation of an Inner-Layer Hydrogel Precursor Solution [0059] (1) 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. [0060] (2) 5 g of PVA was added into the chitosan solution prepared in step (1), 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. [0061] (3) 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.

[0062] 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.

[0063] Preparation of a bilayer bionic drug-loaded hydrogel [0064] (1) The PVA/chitosan solution (a mass ratio of PVA to chitosan is 5:2) prepared in step (2) of the inner-layer hydrogel precursor solution was added into a sterilized beaker, the vancomycin solution (each milliliter of hydrogel contains 8 mg of vancomycin) prepared in step (3) of the inner-layer hydrogel precursor solution was added and stirred uniformly, followed by adding the genipin solution (each milliliter of hydrogel contains 0.1 mg of genipin) prepared in step (3) of the inner-layer hydrogel precursor solution and stirred uniformly under dark condition, and an inner-layer solution was obtained. [0065] (2) 4 mL solution prepared in step (2) of the outer-layer polyvinyl alcohol (PVA) hydrogel was placed in a mould after removing excessive UP water, 4 mL solution prepared in step (1) of inner-layer hydrogel precursor solution was added into the mould, placed in a sterile and light-proof container, frozen and thawed three times, followed by standing for 3 days at room temperature, and the bilayer bionic drug-loaded hydrogel was obtained.

[0066] 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 an inner layer. Meanwhile, the genipin made the chitosan form a second network by chemical cross-linking. 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.

[0067] A structural diagram of a bilayer drug-loaded hydrogel of the present disclosure was shown in FIG. 1a and FIG. 1B. As shown in FIG. 1a, a structural diagram of the bilayer hydrogel indicated that the inner and outer layers had different microporous structures; as shown in FIG. 1B, an optical photograph of the bilayer hydrogel indicated that the inner and outer-layer hydrogels were tightly bonded together.

Experimental Example 1 Microscopic Morphology

[0068] The microscopic morphology of cross section a and longitudinal section b of the outer-layer hydrogel of the present disclosure, as shown in FIG. 2a and FIG. 2b respectively, demonstrated that the prepared outer-layer hydrogel had a directional microporous structure.

[0069] An image of a cross section of the inner-layer hydrogel under a scanning electron microscope, as shown in FIG. 3, indicated that an irregular porous structure of the inner-layer hydrogel provided a stronger water absorption property.

[0070] Images of a cross section and a longitudinal section of the bilayer hydrogel under a scanning electron microscope, as shown in FIG. 4, proved that the inner and outer layers of the bilayer hydrogel were tightly bonded together, and also proved significant structural differences between the outer and inner layers of the bilayer hydrogel as the outer layer had a directional microporous structure while the inner layer had an irregular porous structure.

Experimental Example 2: Mechanical Properties

[0071] Tensile properties of materials were measured with a universal mechanical testing machine.

[0072] Mechanical properties of a hydrogel of the present disclosure were shown in FIG. 5a and FIG. 5b. FIG. 5a showed a stress-strain curve of an outer-layer hydrogel in tensile after removing salt ions by soaking in different concentrations of sodium sulfate solution for 4 days; and FIG. 5b showed a relationship between Young's modulus of hydrogel and Young's modulus of skin (the largest circle area represented a Young's modulus range of hydrogels). In FIG. 5a, an ultimate tensile strength of the outer-layer PVA hydrogel was 1.5440.273 Mpa, a maximum elongation was 906.344153.9486%, and hydrogels with different mechanical properties could be prepared by adjusting concentrations of salt ions when performing salting-out. Therefore, the directional freezing and salting-out employed in the present disclosure had a synergistic effect. In FIG. 5b, the maximum Young's modulus of hydrogel was 170.3547 Kpa, and the adjustable range covered the Young's modulus of skin. The water content was about 75%, which was equivalent to the water content (71.77%) of skin. Therefore, the outer-layer hydrogel of the bilayer bionic drug-loaded hydrogel of the present disclosure had mechanical properties similar to those of skin and could withstand high intensity squeezing, pulling and rubbing, thereby protecting the wound of skin from negative external stimulation while keeping the wound surface dry and clean.

[0073] Mechanical properties of inner and outer hydrogels of the present disclosure were shown in FIG. 6a and FIG. 6b. As seen from FIG. 6a and FIG. 6b, the mechanical properties between the outer hydrogel and the inner hydrogel were different. As a structure determines properties, and the properties reflect the structure, the significant difference in structures between the outer and inner layer was verified again, which was consistent with the results of FIG. 4. Therefore, it was concluded from FIG. 6a and FIG. 6b that the directional microporous structure of the outer layer significantly improved the mechanical properties of hydrogel.

Experimental Example 3: Infrared Spectroscopy

[0074] Materials are performed total reflection scanning using an infrared spectrometer. Infrared spectroscopy of an inner-layer hydrogel of the present disclosure is shown in FIG. 7. The infrared spectroscopy of the pure PVA hydrogel showed absorption peaks respectively caused by extensional vibration of oxhydryl (OH) at 3200-3600 cm.sup.1, asymmetric and symmetrical extensional vibrations of alkyl (CH) at 2937 and 2917 cm.sup.1, in-plane bending vibration of alkyl (CH) at 1425 cm.sup.1, in-plane bending vibration of oxhydryl (OH) at 1329 cm.sup.1, extensional vibration of COC at 1092 cm.sup.1, and extensional vibration of CC at 848 cm.sup.1. The infrared spectroscopy of the chitosan showed absorption peaks respectively caused by stretching of CO of peptide bond at 1634 cm.sup.1, bending of NH of the peptide bond at 1554 cm.sup.1, and stretching of CN of the peptide bond at 1408 cm.sup.1. A carbohydrate structure of the chitosan is at 1073 cm.sup.1, and pyranose ring of the chitosan is at 1073 cm.sup.1. The infrared spectroscopy of CS/PVA composite hydrogel showed that characteristic peaks of the chitosan and the polyvinyl alcohol appear in the chitosan/PVA composite hydrogel. The infrared spectroscopy of the chitosan/PVA composite hydrogel after cross-linked by genipin was relatively stronger at 1634 cm1 compared to the infrared spectroscopy of the chitosan/PVA composite hydrogel, it was due to the formation of a large amount of amides after cross-linking between the chitosan and the genipin, which indicated that the chitosan and the genipin had a cross-linking reaction. In addition, the blue color of the composite hydrogel after the addition of genipin also proved that the genipin reacts with the chitosan.

Experimental Example 4: Cytotoxicity

[0075] 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%, 109.31% 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.

[0076] The cytocompatibility evaluation of the bilayer bionic drug-loaded hydrogel of the present disclosure was shown in FIG. 8. Combining results of FIG. 8 with the evaluation of the degree of cytotoxicity of samples according to rating criteria listed in Table 1, it was concluded that bilayer bionic drug-loaded hydrogel specimens of the present disclosure were rated as grade 1 and non-cytotoxic, and could be used as medical materials.

TABLE-US-00001 TABLE 1 Evaluation of RGR according to IS010993-5:1999 and GB/T 16886. 5/12-2003 Rating RGR Explanation Grade 0 100 Non-cytotoxic Grade 1 75-99 Non-cytotoxic Grade 2 50-74 May be cytotoxic Grade 3 25-49 Cytotoxic Grade 4 1-25 Cytotoxic Grade 5 0 Cytotoxic [00001]$\mathrm{RGR}\left(\%\right)=\frac{\mathrm{average}\mathrm{absorbance}\mathrm{values}\mathrm{of}\mathrm{experimental}\mathrm{groups}}{\mathrm{average}\mathrm{absorbance}\mathrm{values}\mathrm{of}\mathrm{negative}\mathrm{control}\mathrm{groups}}100.$

Experimental Example 5: Antioxidant Property

[0077] The antioxidant property of the hydrogel was evaluated using a DPPH radical scavenging method, the result was shown in FIG. 9. As shown in FIG. 9, with the increasing of hydrogel contents, an absorption peak at 517 nm of the hydrogel treatment group diminished, indicating that the hydrogel had a strong scavenging effect on DPPH radicals.

Experimental Example 6: Antibacterial Property

[0078] The antibacterial property of 5 mg/ml of hydrogel loading with vancomycin was evaluated using an inhibition zone method, as shown in FIG. 10a to FIG. 10c. FIG. showed an antibacterial effect of the hydrogel on Escherichia coli; FIG. 10b showed an antibacterial effect of the hydrogel on Staphylococcus epidermidis; and FIG. 10c showed an antibacterial effect of the bilayer hydrogel on Staphylococcus aureus. According to results shown in FIG. 10a to FIG. 10c, a loaded-drug hydrogel group had a strong inhibitory effect on the Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus, while an unloaded-drug hydrogel had no obvious inhibitory effect. The weak antibacterial effect of the unloaded-drug hydrogel only relied on the component of the chitosan, and therefore inhibition effects on bacteria with higher concentrations were not obvious.

Experimental Example 7: Adhesion Property

[0079] Adhesion effects of the hydrogel of the present disclosure on different materials surface were shown in FIG. 11a to FIG. 11d. FIG. 11a showed an adhesion of the hydrogel on hands; FIG. 11b showed an adhesion of the hydrogel on elbow joints; FIG. 11c showed an adhesion of a bilayer hydrogel on glass; and FIG. 11d showed an adhesion of a bilayer hydrogel on plastic. As seen from FIG. 11a to FIG. 11d, the hydrogel had a good adhesion property, showing vertical adhesion to the back of hands, elbow joints, plastics and glass without falling off.

[0080] 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.