CN116570760B - Multifunctional slow-release dressing for promoting chronic wound healing and preparation method and application thereof - Google Patents
Multifunctional slow-release dressing for promoting chronic wound healing and preparation method and application thereof Download PDFInfo
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- CN116570760B CN116570760B CN202310840448.3A CN202310840448A CN116570760B CN 116570760 B CN116570760 B CN 116570760B CN 202310840448 A CN202310840448 A CN 202310840448A CN 116570760 B CN116570760 B CN 116570760B
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0004—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing inorganic materials
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
- A61L26/0014—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/0066—Medicaments; Biocides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
- A61L26/0061—Use of materials characterised by their function or physical properties
- A61L26/008—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
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- A—HUMAN NECESSITIES
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- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
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- A—HUMAN NECESSITIES
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- A61L2400/00—Materials characterised by their function or physical properties
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Abstract
The invention belongs to the field of medicines, relates to wound dressing, and in particular relates to a medicine for promoting chronic wound healingMultifunctional slow-release dressing, preparation method and application thereof, wherein the multifunctional slow-release dressing comprises hydrogel and active ingredients loaded in the hydrogel, and the hydrogel is prepared from polyvinyl alcohol, sodium alginate, carboxymethyl chitosan and CaO 2 PSC oxygen release hydrogel prepared from nano particles; the active ingredient is TAX@HKUST-1 nano particles, namely: copper metal organic framework material nano particles with dihydroquercetin doped in the inner cavity. The multifunctional slow-release dressing has outstanding oxygen release characteristics and anti-inflammatory and antibacterial effects, remarkably promotes wound healing, and has great potential in the treatment of chronic wounds such as diabetes.
Description
Technical Field
The invention belongs to the field of medicines, relates to wound dressing, and in particular relates to multifunctional slow-release dressing for promoting chronic wound healing, and a preparation method and application thereof.
Background
Chronic wounds such as diabetic ulcers, traumatic ulcers, pressure sores, etc., bacterial infection or inflammation is difficult to avoid during in vivo wound healing, and serious skin damage may even be life threatening. Especially for Diabetic Foot Ulcer (DFU), hyperglycemia can cause angiogenesis disorder, insufficient oxygen supply of wound surface, massive release of inflammatory factors, hypoxia of wound surface, bacterial growth, inflammation, septicemia and necrosis. At present, the treatment of diabetic foot ulcer patients mainly comprises debridement, autograft and the use of various dressings, but the treatment of traumatic hypoxia, bacterial infection and inflammation is relatively single, and the treatment effect is not ideal, so that the development of a wound dressing for releasing oxygen, resisting bacteria and inflammation and promoting wound healing is urgently needed.
Disclosure of Invention
In view of the above technical problems, the invention aims to provide a multifunctional slow-release dressing for promoting the healing of chronic wounds, which is PSC/TAX@HKUST-1 composite hydrogel, has outstanding oxygen release characteristics and anti-inflammatory and antibacterial effects, remarkably promotes the healing of wound surfaces, and has great potential in the treatment of chronic wounds such as diabetes.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a multifunctional slow-release dressing for promoting chronic wound healing comprises hydrogel and active ingredients loaded in the hydrogel; the improvement is that the hydrogel is prepared from polyvinyl alcohol, sodium alginate, carboxymethyl chitosan and CaO 2 PSC oxygen release hydrogel prepared from nano particles; the active ingredient is TAX@HKUST-1 nano particles, namely: copper metal organic framework material nano particles with dihydroquercetin doped in the inner cavity.
The invention also provides a preparation method of the multifunctional slow-release dressing for promoting chronic wound healing, which comprises the following steps:
step 1, doping dihydroquercetin into an inner cavity of a copper metal organic framework material to prepare TAX@HKUST-1 nano particles;
step 2, mixing polyvinyl alcohol, sodium alginate and carboxymethyl chitosan to prepare PVA/SA/CMCS mixed aqueous solution; subsequently, caO is added 2 Preparing PSC oxygen-releasing aqueous solution by the nano particles; and finally, adding the TAX@HKUST-1 nanoparticle aqueous solution into the PSC oxygen release aqueous solution, and preparing the multifunctional slow-release dressing by adopting a cyclic freeze thawing method, wherein the multifunctional slow-release dressing is PSC/TAX@HKUST-1 composite hydrogel.
As the preferable preparation method of the TAX@HKUST-1 nano particle in the step 1, the preparation method comprises the following steps: mixing copper-based metal organic framework material nano particles with a dihydroquercetin ethanol solution with the concentration of 100mg/mL, and stirring overnight in the dark; the mixture was then allowed to stand, the supernatant was discarded and dried under vacuum to give TAX@HKUST-1 nanoparticles.
As the preferable method of the invention, the preparation method of the PSC/TAX@HKUST-1 composite hydrogel in the step 2 comprises the following steps: polyvinyl alcohol, sodium alginate and carboxymethyl chitosan are respectively prepared into 10 percent, 2 percent and 4 percent aqueous solutions, and the aqueous solutions are mixed according to the volume ratio of 1.5:1:1 to prepare PVA/SA/CMCS mixed aqueous solutions; subsequently, caO is added 2 Nanoparticle to 1The concentration of mg/mL is evenly stirred at room temperature to prepare PSC oxygen-releasing aqueous solution; then, adding TAX@HKUST-1 aqueous solution with the concentration of 4mg/mL into the PSC oxygen-releasing aqueous solution in a volume ratio of 1:3, and stirring in the adding process; after homogenization, the resulting solution was poured into a mold, frozen at-20℃for 2 hours, thawed at room temperature for 1 hour, and repeated 2-3 cycles to obtain a PSC/TAX@HKUST-1 composite hydrogel.
As a further preferred aspect of the invention, the mass ratio of the copper-based metal organic framework material nanoparticles to the dihydroquercetin in the step 1 is 1:10.
As a further preferred aspect of the present invention, caO in step 2 2 The preparation method of the nanoparticle comprises the following steps: 53g of CaCl 2 ·2H 2 O was added to 200mL of distilled water, followed by 80mL of 1M sodium hydroxide solution, and stirred at room temperature; subsequently, 100mL of H was added 2 O 2 Dropwise adding into the mixture, stirring until light yellow precipitate is observed, filtering the solution, and washing the particles with sodium hydroxide and distilled water respectively; finally, the washed particles are dried at 80 ℃ and then homogenized into powder by using mortar to obtain CaO 2 And (3) nanoparticles.
In the test of the invention, it is unexpectedly found that after HKUST-1 is added into PSC oxygen-releasing hydrogel, copper ions and O 2 The synergistic effect can promote the angiogenesis of the wound surface, the number of blood vessels in the PSC/HKUST-1 group is obviously more than that of the PSC/TAX group, and the PSC/HKUST-1 hydrogel can promote the angiogenesis and accelerate the wound healing; therefore, the PSC/TAX@HKUST-1 composite hydrogel and the PSC/HKUST-1 hydrogel can be applied to the preparation of medicaments for promoting the generation of new blood vessels of wound surfaces; the preparation method of the PSC/HKUST-1 hydrogel comprises the following steps: mixing polyvinyl alcohol, sodium alginate and carboxymethyl chitosan to prepare PVA/SA/CMCS mixed aqueous solution; subsequently, caO is added 2 Preparing PSC oxygen-releasing aqueous solution by the nano particles; finally, the aqueous solution of HKUST-1 with the concentration of 2.4mg/mL is added into the aqueous solution of PSC oxygen release, and the PSC/HKUST-1 hydrogel is prepared by adopting a circulating freeze thawing method.
In the experiment of the invention, it is unexpectedly found that after HKUST-1 is added into PSC oxygen-releasing hydrogel, the release of copper ions promotes new collagen fibrilsHKUST-1, in synergy with TAX, promotes wound remodeling by enhancing collagen synthesis; therefore, the PSC/HKUST-1 hydrogel can be applied to the preparation of medicines for promoting the generation of new collagen fibrils of wound surfaces, and the preparation method of the PSC/HKUST-1 hydrogel comprises the following steps: mixing polyvinyl alcohol, sodium alginate and carboxymethyl chitosan to prepare PVA/SA/CMCS mixed aqueous solution; subsequently, caO is added 2 Preparing PSC oxygen-releasing aqueous solution by the nano particles; finally, the aqueous solution of HKUST-1 with the concentration of 2.4mg/mL is added into the aqueous solution of PSC oxygen release, and the PSC/HKUST-1 hydrogel is prepared by adopting a circulating freeze thawing method.
Advantages and beneficial effects of the invention
(1) After TAX and HKUST-1 are loaded into hydrogel, the sustained-release dressing provided by the invention releases Cu from HKUST-1 2+ The ions can be crosslinked with sodium alginate in the hydrogel, so that Cu is further delayed 2+ Thereby achieving the desired sustained release characteristics; in addition, due to the slow release of Cu by HKUST-1 2+ The release of copper ions is not only beneficial to CaO 2 Can also make the composite hydrogel have catalytic activity, and the activity can make the composite hydrogel decompose H 2 O 2 And generate O 2 The sustained release dressing can release oxygen more permanently and completely, optimize the anoxic environment of the wound and reduce H at the same time 2 O 2 Accumulation of toxicity, avoiding serious damage to cells or organs.
(2) The TAX in the slow-release dressing provided by the invention is degraded and released through the pores of HKUST-1 or HKUST-1, has a good slow-release effect, and obviously enhances the antioxidation capability of the hydrogel after the TAX is loaded into the hydrogel, thereby promoting the sustained release of dihydroquercetin (TAX) and accelerating the healing of diabetic wounds.
(3) The slow-release dressing provided by the invention has the advantages that the TAX and HKUST-1 cooperate to realize antibacterial effect, the antibacterial activity on escherichia coli is 90.93 +/-3.17%, the antibacterial activity on staphylococcus aureus is 88.88+/-2.35%, and the harm of bacterial invasion is effectively reduced.
(4) The slow-release dressing provided by the invention can inhibit the release of pro-inflammatory cytokines by macrophages, keratinocytes, endothelial cells and stromal cells, lighten inflammatory reaction, and has lasting anti-inflammatory, antibacterial and oxygen-generating effects; furthermore, the addition of HKUST-1 can promote the generation of blood vessels and collagen, and the release of TAX can also promote the formation of new collagen fibrils, and simultaneously promote the wound healing by inducing the proliferation of endothelial cells, and the synergistic effect of HKUST-1 and TAX can accelerate the chronic wound healing.
(5) The slow-release dressing provided by the invention is safe to contact with blood, does not have a hemolysis phenomenon, has no toxic or side effect, is beneficial to cell proliferation, can promote cell growth, and improves the overall activity, thereby accelerating wound healing.
(6) The slow-release dressing provided by the invention has outstanding oxygen release characteristics and anti-inflammatory and antibacterial effects, remarkably promotes wound healing, and has great potential in the treatment of chronic wounds such as diabetes.
(7) The slow-release dressing provided by the invention has excellent viscoelasticity and good swelling property, can be adhered to a wound, absorbs wound exudates, reduces bacterial infection and promotes wound healing.
(8) The preparation method provided by the invention can successfully load the TAX into the inner cavity of HKUST-1, and the loading quantity of the TAX in the TAX@HKUST-1 is up to 81.94 +/-2.60%.
(9) In the test of the invention, it is unexpectedly found that after HKUST-1 is added into the hydrogel, copper ions and O 2 The synergistic effect can promote the angiogenesis of the wound surface, and the PSC/TAX@HKUST-1 composite hydrogel and the PSC/HKUST-1 hydrogel can promote the angiogenesis of the wound surface and can be applied to the preparation of medicaments for promoting the angiogenesis of the wound surface.
(10) When the invention is tested, the unexpected discovery is that after HKUST-1 is added into the hydrogel, the release of copper ions promotes the formation of new collagen fibrils, and the PSC/HKUST-1 hydrogel can be applied to the preparation of medicines for promoting the generation of new collagen fibrils of wound surfaces.
Drawings
FIG. 1 is a representation of Nanoparticles (NPs) made in accordance with the present invention; wherein A) is the FT-IR spectrum of HKUST-1, TAX and TAX@HKUST-1; b) XRD spectra for HKUST-1, TAX and TAX@HKUST-1The method comprises the steps of carrying out a first treatment on the surface of the C) BET patterns for HKUST-1 and TAX@HKUST-1; D-F) HKUST-1, TAX@HKUST-1 and CaO, respectively 2 SEM images of (a).
FIG. 2 is a graph showing the morphology, structure and swelling property analysis of hydrogels prepared according to the present invention; wherein A-D) are SEM images of PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1, respectively; e) FT-IR spectra characterized for PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1; f) Swelling ratios for PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 hydrogel samples.
FIG. 3 is a test chart of hydrogels made in accordance with the present invention; wherein A) is the angular frequency dependent storage modulus (G ') and loss modulus (G') of the hydrogel sample; b) Cu in PBS solution for PSC/HKUST-1 2+ Releasing; c) Oxygen release in PBS for PSC/TAX@HKUST-1 and PSC; d) In vitro 48h H for PSC and PSC/TAX@HKUST-1 2 O 2 A release amount; e) A test chart for drug release in PBS for 24h from PSC/TAX@HKUST-1 and PSC/TAX; f) In vitro oxidation resistance comparison graphs of different hydrogels against ABTS.
FIG. 4 is a graph showing the analysis of the hydrogel produced by the present invention; wherein, A) is the antibacterial activity of PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 to staphylococcus aureus and escherichia coli; b) A quantitative analysis chart for inhibiting escherichia coli for the hydrogel sample; c) A quantitative analysis chart for inhibiting staphylococcus aureus for the hydrogel sample; d) A cell compatibility analysis chart of the hydrogel; e) Is a hydrogel blood compatibility analysis chart.
FIG. 5 is a test chart of the hydrogel prepared by the invention for repairing an in vivo wound surface; wherein A) is a representative image of the 16-day dermal wound healing process in PSC, PSC/TAX, PSC/HKUST-1 and PSC/TAX@HKUST-1; b) The wound closure rate is different in treatment.
FIG. 6 is a graph showing staining of skin tissue after treatment with the hydrogel prepared according to the present invention; wherein A) is an H & E stained image (scale: 100 μm) of each group of treated skin tissue; b) The nascent collagen fibers (scale: 100 μm) are displayed for trichromatic wound tissue images of masson.
FIG. 7 is a diagram of a day 16 skin tissue immunohistochemical analysis; wherein A) is an immunohistochemical image of skin tissue after each group of treatment (scale: 50 μm); b) Quantitative analysis of CD 31; c) Is a quantitative analysis chart of IL-6.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific examples, to which embodiments of the invention are not limited. For process parameters not specifically noted, reference may be made to conventional techniques. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The sources of the materials used in the embodiment of the invention are as follows:
copper acetate (Cu (CH) 3 COO) 2 Purity 99.0%); 1,3, 5-benzenetricarboxylic acid (H3 BTC, purity 98.0%); absolute ethyl alcohol (C) 2 H 5 OH, purity not less than 99 percent); SA (viscosity 200+ -20 mpa.s), purchased from Shanghai microphone Lin Shenghua Co., ltd. (Shanghai, china); polyvinyl alcohol (PVA, 1799) was purchased from shanghai alaa Ding Shenghua technologies limited (shanghai, china); chitosan (CS, deacetylation degree not less than 80.0%), molecular weight 5 ten thousand, purchased from Shandong Qingdao Bozhishui Co., ltd; acetic acid was purchased from vinca biotechnology limited (vinca china); dihydroquercetin (TAX) is purchased from China food and drug administration institute, with lot number 111816-201102 and purity 98.0%; hematoxylin-eosin (H)&E) And the masson staining kit was purchased from the institute of bioengineering (south Beijing, china); mouse platelet endothelial cell adhesion molecule precursor-1 (cd 31) and interleukin-6 (IL-6) were purchased from proteontech (chinese marchan); all chemical reagents were of analytical purity.
Example 1 of the present invention:
(1) Preparation of TAX@HKUST-1 nanoparticles
Copper metal organic framework (HKUST-1) nanoparticles were synthesized using reported methods. Briefly, copper acetate (0.3 g,1.5 mmol) dissolved in distilled water (10 mL) was slowly added dropwise to trimesic acid (H) 3 BTC) in ethanol (0.21 g,1 mmol) solution, stirred at room temperature for 30min to form a blue suspension; the product was centrifuged and then washed twice with ethanol/water (1:1 v/v) solution to eliminate any residual H 3 BTC and copper acetate;the obtained crystals were soaked in methanol for 72 hours, during which time the solvent was changed every 12 hours, and subjected to vacuum drying and thorough grinding to obtain HKUST-1 nanoparticles.
Subsequently, HKUST-1 nanoparticles (20 mg) were mixed with dihydroquercetin (TAX) ethanol solution (100 mg/mL,2 mL) and stirred overnight in the dark; then, the mixture was allowed to stand for 1 hour, and the supernatant was discarded to remove TAX attached to the surface of HKUST-1; finally, drying under vacuum condition to obtain a dry product of TAX@HKUST-1.
（2）CaO 2 Preparation of NPs
53g of CaCl 2 ·2H 2 O was added to 200mL of distilled water, followed by 80mL of 1M sodium hydroxide solution, and stirred at room temperature; subsequently, H is 2 O 2 (100 mL) was slowly added dropwise to the mixture and stirred for an additional 2 hours; gradually a pale yellow precipitate was observed to form, indicating that CaO was synthesized 2 A nanoparticle; the solution was filtered and the particles were then washed three times with sodium hydroxide and distilled water, respectively. Finally, the washed particles were dried at 80 ℃ for two hours and then homogenized into a powder using mortar.
(3) Preparation of PSC/TAX@HKUST-1 composite hydrogel
The cyclic freeze thawing method is adopted: polyvinyl alcohol (PVA), sodium Alginate (SA) and carboxymethyl chitosan (CMCS) are respectively prepared into 10 percent, 2 percent and 4 percent of water solution, and the water solution is mixed and prepared into PVA/SA/CMCS mixed water solution according to the volume ratio of 1.5:1:1; subsequently, caO is added 2 NPs to 1mg mL -1 Stirring uniformly at room temperature to obtain PSC oxygen-releasing aqueous solution; then, adding TAX@HKUST-1 aqueous solution (4 mg/mL) into the PSC oxygen-releasing aqueous solution in a volume ratio of 1:3, and stirring during the adding process; after homogenization, the resulting solution was poured into a mold, frozen at-20℃for 2 hours, thawed at room temperature for 1 hour, and repeated three cycles to obtain a PSC/TAX@HKUST-1 composite hydrogel.
PSC (PSC oxygen-releasing aqueous solution is circularly frozen and thawed), PSC/TAX (PSC oxygen-releasing aqueous solution is circularly frozen and thawed by adding 1.6mg/mL TAX aqueous solution into PSC oxygen-releasing aqueous solution in a volume ratio of 1:3), PSC/HKUST-1 (PSC oxygen-releasing aqueous solution is circularly frozen and thawed by adding 2.4mg/mL HKUST-1 aqueous solution into PSC oxygen-releasing aqueous solution in a volume ratio of 1:3) and PSC/TAX@HKUST-1 hydrogel are prepared by adopting the method, and freeze-drying is carried out.
Example 2 of the present invention:
the invention uses Fourier transform infrared spectrum (FT-IR) to characterize HKUST-1 and TAX@HKUST-1, and the scanning wave number is 400-4000cm -1 The method comprises the steps of carrying out a first treatment on the surface of the The crystal mode of the nano material HKUST-1, TAX@HKUST-1 is analyzed by X-ray diffraction (XRD), and the 2 theta angle is 5-45 ◦ ，CaO 2 An angle of 10 to 70 ◦ The method comprises the steps of carrying out a first treatment on the surface of the The specific surface areas of the samples HKUST-1 and TAX@HKUST-1 were determined by nitrogen adsorption/desorption at 77K using a specific surface area analyzer (BET); observing the morphology of the sample by using a Scanning Electron Microscope (SEM), and analyzing the granularity of the sample; detecting the loading amount of TAX in TAX@HKUST-1 by High Performance Liquid Chromatography (HPLC); the drug loading is calculated according to the following formula:
；
wherein W is 1 Is total drug in solution (TAX); w (W) 2 Is free drug (TAX) loaded; w (W) I Is the weight of TAX@HKUST-1.
The results of the FT-IR spectrum characterization of HKUST-1, TAX and TAX @ HKUST-1 of the present invention are shown in FIG. 1A, from which it can be seen that HKUST-1 has characteristic peaks at 1641, 1565cm-1, related to the stretching vibration of phenylcarboxyl group, 3400cm -1 The characteristic peak at this point is related to the stretching vibration of-OH, 490cm -1 As characteristic peaks of Cu-O, these are standard characteristic peaks of HKUST-1, which demonstrate the synthesis of HKUST-1. TAX has characteristic peaks at 1621cm-1, 1167cm-1 and 1073cm-1, and the prepared TAX@HKUST-1 is shown as the original peak type of HKUST-1, and the characteristic peak of TAX is added, so that the TAX is contained in the HKUST-1.
XRD characterization of HKUST-1, TAX and TAX@HKUST-1 as shown in FIG. 1B was performed, and it was found that the prepared samples of HKUST-1 and TAX@HKUST-1 were found to be 2 theta=6.9 ◦ 、9.5 ◦ 、11.6 ◦ 、13.4 ◦ 、17.5 ◦ 、19.0 ◦ The characteristic peaks at the positions are consistent with simulation and literature reports. Further demonstrating that the present invention has successfully synthesized HKUST-1, and TAXUniformly dispersed in the cavity of HKUST-1.
The BET of HKUST-1 and TAX@HKUST-1 were tested in the present invention as shown in FIG. 1C, and the results showed that the BET of HKUST-1 was 847.69m 2 g-1 and TAX@HKUST-1 having a BET of 118.28m 2 g-1, it can be seen that TAX has been successfully loaded into the lumen of HKUST-1.
After the TAX@HKUST-1 is successfully prepared, the invention synthesizes CaO 2 The characterization is carried out, and the result accords with the standard card and literature report, which shows that the invention successfully prepares CaO 2 。
The invention aims at verifying whether the nano-scale material is synthesized or not, and the nano-scale material is prepared from HKUST-1, TAX@HKUST-1 and CaO 2 The samples were examined by electron microscopy, as shown in FIGS. 1D-F, to obtain the microstructure of NPs. As a result, it was found that the three materials were uniform in structure and uniform in particle size distribution, the particle size and shape of HKUST-1 after drug delivery were not significantly changed, and CaO was used 2 The particle size of (2) is smaller.
In order to better obtain the particle size of NPs, the invention carries out the particle size distribution of HKUST-1 on the sample, the particle size distribution of the HKUST-1 is between 71.61+/-21.59 nm, the particle size of TAX@HKUST-1 is increased to 95.65+/-22.39 nm, caO 2 The above results demonstrate that the present invention successfully prepares the nanomaterial with a particle size of 51.60.+ -. 19.10 nm.
The invention uses high performance liquid chromatography to measure the drug-loading rate of TAX@HKUST-1, and the drug-loading percentage is 81.94 +/-2.60% by adopting the formula.
Example 3 of the present invention:
(1) Morphological analysis
PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 hydrogels were subjected to morphological analysis using Scanning Electron Microscopy (SEM), and the pore structure of the hydrogels was analyzed.
The internal structure of the cross section of the hydrogel is observed through a scanning electron microscope, and cross section scanning electron microscope diagrams of different PSC hydrogel wound dressings are shown in figures 2A-D. The results show that the 4 hydrogels all have porous structures, the PSCs are orderly crosslinked, and the pore channels are uniform. However, the addition of HKUST-1 and TAX disrupts the crosslinking of PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 hydrogels, resulting in their interaction with the hydrogels to create different pore sizes.
(2) Structural analysis
Characterization of the lyophilized hydrogels by Fourier transform Infrared Spectroscopy (FT-IR) and scanning wavenumbers of 400-4000cm -1 。
The IR spectra of the PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 hydrogel wound dressings of the present invention are shown in FIG. 2E. PSC hydrogel at 3000-3700cm -1 There is a broad absorption peak around, mainly the stretching vibration of hydrogen bond between molecular chains, and the stretching vibration of C-O at 1100cm < -1 >. Hydrogel hydrogen bond valleys of PSC/TAX blue shifted to 3570cm -1 This is related to the characteristic peak of the TAX itself. However, the hydrogen bond peaks and valleys of the loaded HKUST-1 and TAX@HKUST-1 were both located at 3440cm -1 This is due to the fact that the-OH peak of HKUST-1 is at 3440cm -1 Resulting in blue shift of hydrogen bond, whereas PSC at 1650 and 1460cm after addition of TAX or HKUST-1 -1 The c=c peak appears at this point, so it can be demonstrated that both TAX and HKUST-1 are loaded into the hydrogel.
(3) Swelling Property
In order to promote wound healing and avoid wound infection, the wound dressing has the characteristics of absorbing wound exudates and providing a moist environment, and the swelling performance is detected by the invention.
Weighing lyophilized hydrogel (W) d ) Placing in deionized water at 25deg.C, wiping off excessive water from the swollen hydrogel surface with filter paper at intervals, and weighing the hydrogel (W t ) The expansion ratio (SR) is calculated as follows:
；
wherein W is d And W is t The weight of the lyophilized hydrogel and the weight of the swollen hydrogel at different times, respectively.
Fig. 2F is a swelling curve for different PSC hydrogel wound dressings. From the swelling curve, the swelling rate of the wound dressing of different hydrogels is higher than 1000%, the wound dressing has good water absorption capacity, the swelling trend is basically consistent, and the swelling equilibrium is achieved within about 24 hours. Of these, HKUST-1 hydrogels had the highest swelling properties, because of the largest pore size, highest water absorption, and second PSC/TAX swelling ratios, PSC and TAX@HKUST-1 swelling properties were similar, consistent with their electron micrograph and water vapor transmission trend. The results prove that the composite hydrogel has good swelling performance, can absorb wound exudates, reduce bacterial infection and promote wound healing.
(4) Rheological properties
Hydrogels with good viscoelasticity are important in order to ensure good performance of the wound dressing. Thus, the present invention measures the rheological properties of PSC hydrogel wound dressings.
Rheological measurements of hydrogels were measured with a rotational rheometer and dynamic viscoelasticity measurements were performed at strain (0.01-100%) and frequency (1-100 HZ) to test the law of changes in hydrogels with strain and frequency to develop viscoelasticity.
According to the invention, through rheological property measurement, change curves of storage modulus and loss modulus under different stress conditions are obtained, as shown in fig. 3A, the storage modulus (G') represents energy stored by the material due to reversible elastic deformation, and the elastic property of the material is reflected; and loss modulus (G ") represents the amount of energy dissipated when the material is irreversibly deformed, reflecting the viscous nature of the material. The storage modulus and loss modulus curves of the PSC, PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 hydrogel wound dressings were consistent with the trend of change in strain, indicating that the hydrogels underwent both elastic deformation and viscous deformation throughout deformation. This suggests that hydrogels have excellent viscoelasticity and that the incorporation of NPs does not affect their mechanical properties.
(5) Copper ion release
To examine Cu 2+ The release content of HKUST-1 in the hydrogel was examined in vitro.
PSC/HKUST-1 samples were immersed in a 37℃PBS solution, shaken at 100rpm, and 2mL samples were taken at various time intervals for analysis, and an equal amount of fresh release medium was added. Copper content determination Using inductively coupled plasma Mass Spectrometry (ICP-MS), PSC/HKUST-1 was detected in PBS (pH7.4) Cu in 2+ Release content of (2).
The invention tests Cu of HKUST-1 in PSC at different time intervals 2+ The content is shown in FIG. 3B. HKUST-1 releases Cu cumulatively under PSB (pH 7.4) for 2d 2+ 31.29%,7d cumulative release of Cu 2+ 41.66% showing good retardation. This is probably due to the slow release effect of HKUST-1 itself; in addition, released Cu 2+ The ion and sodium alginate are crosslinked to further delay Cu 2+ Thereby achieving desired sustained release characteristics and possibly as H 2 O 2 The slow catalyst of (2) can prolong the oxygen precipitation time.
(6) Oxygen release amount
PSC, PSC/TAX@HKUST-1 hydrogel oxygen release was determined using a Lei Ci dissolved oxygen meter (JPSJ-606L). 1mL of hydrogel is prepared into a cylindrical sheet, the cylindrical sheet is placed into a glass bottle containing 10mLPBS solution, an oxygen measuring probe is inserted, the bottle mouth is sealed by a sealing film, the oxygen release condition of the hydrogel is measured, and PBS solution is used for zeroing.
Oxygen release analysis was performed on PSC and PSC/HKUST-1 hydrogels according to the present invention, and the long-term oxygen release profile of the dressing over 3 days is shown in FIG. 3C. By detection and analysis of oxygen release DO levels, it was found that the hydrogel could maintain a sustained oxygen release for 72 hours, the cumulative oxygen release of PSC group was 2.39.+ -. 0.09mg/L, the cumulative oxygen release of PSC/TAX@HKUST-1 group was 2.99.+ -. 0.09mg/L, and the HKUST-1-containing hydrogel had a higher and better oxygen release due to the slow release of Cu by HKUST-1 2+ The release of copper ions is beneficial to CaO 2 And can promote H 2 O 2 Is decomposed. Thus, the addition of HKUST-1 can promote more durable and complete oxygen release from PSC hydrogels.
(7) Hydrogen peroxide (H) 2 O 2 ) Quantification of
Although H 2 O 2 Can be decomposed into O 2 Promote wound healing but with high concentrations of H 2 O 2 (200 um) can cause serious damage to cells or organs.
To according to CaO 2 Concentration to measure H in PSC hydrogels 2 O 2 The content of the copper-based compound is measured by the method, 50uL of 0.01M phosphate buffer solution is added into a 96-well plate, and 50uLH is added 2 O 2 Standard solutions or hydrogel extracts were mixed into each well. Subsequently, 50ml of 0.01 mM CUSO was added 4 The solution and 0.01M fresh copper reagent solution were incubated for 30 minutes at room temperature. Placing 1mL of prepared oxygen-releasing hydrogel with different compositions into 15mL microtubes, adding 10mL of LPBS, incubating at 37 ℃, and taking the hydrogel extract at regular intervals to measure H 2 O 2 The content is as follows. The invention measures H 2 O 2 The absorbance of the standard solution (10-70 uM) and the hydrogel extract at 454nm is utilized to generate 10-70uM H by utilizing the absorbance difference between the sample and the blank solution 2 O 2 Calibration curve and calibrating H in hydrogel according to standard curve 2 O 2 Concentration.
The invention detects H in hydrogels at different times according to standard curves 2 O 2 The results are shown in FIG. 3D. At the initial time point (< 8H), H in the hydrogel 2 O 2 Rapidly released from the gel matrix, and reached a maximum at 8H, PSC set at 42.96+ -3.00 uM and PSC/HKUST-1 set at 22.33+ -1.74 uM due to swelling of the hydrogel and H 2 O 2 Is a cumulative sum of (a) and (b). The content then began to drop (> 8H) and reached a minimum at 48H with PSC set of 21.22.+ -. 3.27. Mu.M and PSC/HKUST-1 set of 8.88.+ -. 2.84. Mu.M, probably because the hydrogel was in near equilibrium at 8H, H 2 O 2 The large amount of released and decomposed, resulting in a great reduction of the subsequent release amount and an increase of the decomposed amount. PSC and PSC/HKUST-1H 2 O 2 The release process proves that H is added after HKUST-1 is added 2 O 2 The content is obviously reduced due to Cu released by HKUST-1 2+ Promote H 2 O 2 Decompose, thus indicating that HKUST-1 can be used as H 2 O 2 Is a catalyst for promoting the decomposition and reducing H 2 O 2 Accumulation of toxicity.
(8) In vitro drug release
In vitro release of TAX in PSC/TAX, PSC/TAX@HKUST-1 hydrogels was investigated using phosphate buffered saline (PBS, pH=7.4) as release medium. 1mL of the hydrogel (diameter 1.5cm, thickness 5 mm) was placed in a centrifuge tube containing 50mL of PBS solution, which was then immediately placed in a constant temperature shaker, the temperature of the instrument was set to 37℃and the shaking speed was 100 rpm. At intervals (0.5,1,2,4,8, 10, 12, 16 and 24 h) 1mL of release medium was withdrawn from the centrifuge tube and finally 1mL of fresh release medium was added immediately isothermally. TAX was detected by High Performance Liquid Chromatography (HPLC) (Waters 2695, USA) at 290nm by UV detection. Methanol (20%) and water (80%) were used as mobile phases, the flow rate was 1.0mL/min, and a COSMILC 18-PAQ column was used as stationary phase (4.6 mm. Times.250 mm,5 μm).
In order to examine the drug release effect, the invention compares the in vitro drug release conditions of PSC/TAX and PSC/TAX@HKUST-1. The in vitro drug release results of the hydrogels are shown in figure 3E. After 8 hours, PSC/TAX in vitro release reaches balance, and the cumulative release rate is 79.50 +/-2.75%; the in vitro release of PSC/TAX@HKUST-1 reaches equilibrium at 24 hours, and the cumulative release rate is 74.62 +/-2.49%. This is probably the reason that TAX in TAX@HKUST-1 can only be released through the pores of HKUST-1 or the degradation of HKUST-1 itself, and experimental results show that the addition of TAX in HKUST-1 has a good slow release effect.
Example 4 of the present invention:
(1) Antioxidant experiment
The invention adopts an ABTS free radical scavenging colorimetry to measure the in vitro antioxidant activity of the hydrogel. The working solution was prepared by mixing equal amounts of 7.4mM ABTS solution and 2.6mM potassium persulfate solution and reacting for 12 hours in the dark at room temperature, and after completion of the reaction, the solution was diluted with absolute ethanol until absorbance at 734nm was 0.70.+ -. 0.02, to give the final ABTS solution. 200uL of the hydrogel (diameter 1cm, thickness 2 mm) was placed in anhydrous methanol, and 800 uL of ABTS solution was added. The absorbance of the reaction solution was measured at an ultraviolet wavelength of 734nm after incubating the reaction mixture in the dark for 5 minutes. ABTS radical scavenging (ABTS scavenging activity) was calculated as follows:
；
wherein A is 0 The absorbance of the ABTS solution without the sample is a, and the absorbance of the ABTS solution with the sample is a.
The results of ABTS radical scavenging by different hydrogels are shown in fig. 3F. The clearance of PSC to ABTS free radical is 61.40+ -2.34%, the clearance of PSC/HKUST-1 to ABTS free radical is 48.29 + -1.46%, the clearance of PSC/TAX to ABTS free radical is 90.65+ -1.51%, and the clearance of PSC/TAX@HKUST-1 to ABTS free radical is 89.17+ -1.10%. The in vitro antioxidation results show that after TAX is added, the antioxidation capability of the hydrogel is obviously enhanced, and the TAX in TAX@HKUST-1 is not affected, so that the sustained release of dihydroquercetin is promoted, and the accelerated healing of diabetic wounds is promoted.
(2) Antibacterial property
The antibacterial property of the hydrogel is measured by adopting a plate colony counting method, and gram-positive staphylococcus aureus and gram-negative escherichia coli are selected for bacterial plants for experiments. Firstly, measuring OD values of two mother bacteria solutions at 600nm by an enzyme-labeled instrument, diluting the two mother bacteria solutions to 0.5 by using a sterile culture solution, and then diluting the bacterial mother solution to 1.0X10 by adopting a gradual dilution mode 5 CFU/mL. The sterilized hydrogel (diameter 1.5cm, thickness 5 mm) was placed in a 24-well plate, then 20. Mu.L of bacterial suspension was pipetted onto the sterile hydrogel, the blank without hydrogel was incubated in a thermostatic shaker at 37℃and a rotational speed of 200rpm for 2 hours. After the time was reached, the well plate was taken out, 1mL of the bacterial culture solution was added, and the bacterial culture solution was purged 3 times to homogenize the bacterial culture solution, and 20. Mu.L of the bacterial culture solution was dropped on an agar plate by a pipette to perform plating. The coated plates were labeled and the plates were incubated in an incubator at 37℃for 12 hours with inversion, and the bacterial colony count on the agar plates was counted to calculate the antibacterial ratio (Antibacterial rate). Each experiment was performed three times and the average value was calculated.
；
Where W is the bacterial count of the blank group and Q is the average bacterial count of the hydrogel group.
The inhibition results of the hydrogel on staphylococcus aureus and escherichia coli are shown in fig. 4A, and the experimental group has obvious inhibition effect on bacteria. Fig. 4B and 4C provide quantitative analysis of the performance of hydrogels against e.coli and s.aureus, respectively. For E.coli (FIG. 4B), the inhibitory activity of PSC hydrogels was 38.08.+ -. 2.66%, while the inhibitory activities of PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 were 74.28.+ -. 1.87%, 57.41.+ -. 3.56% and 90.93.+ -. 3.17%, respectively, indicating that the inhibitory effects on E.coli were significantly enhanced upon addition of HKUST-1 and TAX to the hydrogels. For staphylococcus aureus (FIG. 4C), the antibacterial activity of PSC hydrogel is 38.57+ -3.19%, PSC/TAX is 67.34 + -3.32%, PSC/HKUST-1 is 58.72 + -4.45%, PSC/TAX@HKUST-1 is 88.88+ -2.35%, and the above results show that the addition of HKUST-1 and TAX significantly enhances the antibacterial effect of hydrogel, wherein PSC/TAX@HKUST-1 has the best antibacterial effect; probably due to the synergistic antibacterial effect of TAX and HKUST-1, the harm of bacterial invasion is effectively reduced.
(3) Cytotoxicity of cells
In order to investigate the toxicity of hydrogels, the MTT method was used for evaluation. Cytotoxicity assays were performed on different hydrogels using HacaT cells. Cells were cultured in a 5% carbon dioxide incubator at 37 ℃. Firstly, sterilizing four hydrogels by ultraviolet irradiation, and then soaking the hydrogels in a cell culture medium for 24 hours to obtain a hydrogel leaching solution. Then 1X 10 4 Is seeded in 96-well plates and when the cell density is 80%, the medium is discarded and replaced with hydrogel extract. Culturing was continued for 24 hours. The MTT solution was then added to the 96-well plate and reacted for 4 hours. After 4h the MTT solution was removed and the reaction was quenched by addition of DMSO. Absorbance was measured at 490 nm. Cell viability (Cell viability) was calculated according to the following formula:
；
wherein A is s Is the absorbance of the sample, A b Is the absorbance of the blank group, A c Is the absorbance of the control group.
The effect of different hydrogels on HaCaT cell viability was evaluated using MTT method according to the present invention as shown in figure 4D. The results showed that the four hydrogels were fineCell viability was higher than 80% and no cytotoxicity was observed. Cell viability exceeded 100% for both the PSC/HKUST-1 group and the PSC/TAX@HKUST-1 group, indicating that HKUST-1 released Cu 2+ Is favorable for cell proliferation, promotes cell growth and improves overall activity.
(4) Rate of hemolysis
One successful wound dressing was not hemolytic (5% hemolysis), and the present invention evaluated the rate of hemolysis of various PSC hydrogels. Diluting rabbit blood stock to 4%, taking 400uL, adding into a centrifuge tube, adding 400uL of hydrogel leaching solution (A n ) Incubation in a 37℃constant temperature water bath for 3 hours, followed by centrifugation, and the supernatant was measured for absorbance (UV-1750, shimadzu Co., ltd., japan) with an ultraviolet-visible spectrophotometer at a wavelength of 545 nm. Positive control group (A) p ) Triton-X-100 was added, and the negative control group (A c ) Physiological saline was added and each sample was averaged 3 times in parallel to calculate the Hemolysis Rate (HR):
；
wherein A is n For the absorbance of each hydrogel sample, A c To add the absorbance of physiological saline, A p Absorbance for addition of Triton-X-100 solution.
As shown in FIG. 4E, the digital photograph of the hemolysis phenomenon of the hydrogel of the present invention shows that the hemolysis rate of the positive control group is 100.+ -. 1.20%, PSC is 0.99.+ -. 0.07%, PSC/HKUST-1 is 0.86.+ -. 0.09%, PSC/TAX is 1.68.+ -. 0.07%, PSC/TAX@HKUST-1 is 0.39.+ -. 0.05%. The hemolysis rate of different hydrogels is less than 5% (national standard), and the results show that the material is safe to contact with blood and has no hemolysis phenomenon.
Example 5 of the present invention:
(1) Establishment of diabetic mouse model and in-vivo wound repair
Mice were kept for one week (20 ℃,65% humidity, alternate light day and night) followed by five consecutive weeks of administration of a high-glucose high-fat diet to induce insulin resistance. No water withdrawal was performed for 12h after 3 days of fasting, and diabetes (T2D) was induced by intraperitoneal injection of streptozotocin citrate solution (0.1 m, ph=4.3) at doses of 80mg/kg, 70mg/kg, 60mg/kg in sequence. After one week of the third streptozotocin injection, mice with fasting glucose at 11.1mM or more and exhibiting typical polydipsia, polyphagia, polyuria and weight loss were screened as T2D models. Diabetic mice were randomly divided into control groups, PSC hydrogel experimental group, PSC/TAX hydrogel experimental group, PSC/HKUST-1 hydrogel experimental group and PSC/TAX@HKUST-1 hydrogel experimental group (n=10). After anesthetizing a mouse by intraperitoneal injection of pentobarbital sodium (0.1 mL/10 g), the back hair of the mouse was shaved off with a depilatory cream, and the skin was rubbed with 0.9% physiological saline to cause a circular wound of 1cm diameter on the back of the mouse. A control group was treated with 0.9% physiological saline, and the remaining four groups of skin wounds were given PSC hydrogel, PSC/TAX hydrogel, PSC/HKUST-1 hydrogel and PSC/TAX@HKUST-1 hydrogel, respectively. All hydrogels were sterilized under uv light for 1h before use. The wound sites were photographed on days 0, 4,8, 12, 16 and the wound diameters were measured. The following formula was used to quantify the percentage of wound closure (Wound healing rate):
；
wherein S is 0 Is the initial area of the wound and Sn is the wound area on day n.
To evaluate the effect of PSC/TAX@HKUST-1 administration on promoting wound healing of skin of diabetic mice, photographs of diabetic wounds were taken with a digital camera at predetermined time intervals, and wound healing rates were quantified with imageJ software. The results shown in fig. 5A demonstrate significant improvement in wound healing (p < 0.01) in diabetic mice over the 16-day treatment period for all four experimental groups compared to the control group. After 16d of treatment, the area of the wound surface of each treatment group is reduced, new epidermis appears, and the wound surface is reduced. The difference of wound healing rates between groups at the initial stage of wound healing (on the 4 th day) is small, and the difference between groups after the 4 th day is obvious. The PSC/HKUST-1, PSC/TAX and PSC/TAX@HKUST-1 groups had significant (p < 0.01) treatment compared to the control and PSC groups, PSC/TAX@HKUST-1 group had the best effect, with a wound closure area of 97.03.+ -. 1.25% (FIG. 5B). These wound healing rate dataSuggesting that the acceleration of wound healing may be due to dihydroquercetin and Cu 2+ They have long lasting anti-inflammatory, antibacterial and oxygen-generating effects, establish a friendly microenvironment, participate in normal blood glucose levels, overcome oxidative stress, promote the production of blood vessels and collagen, and promote the expression of growth factors.
(2) Histopathological staining
New blood vessels and new epidermis are important indicators of wound healing. The blood vessels provide nutrition and oxygen for tissue repair, promote migration of keratinocytes, and promote epithelialization. At the same time, the epidermis serves as a barrier to isolate the wound from external elements, while protecting the subcutaneous tissue and maintaining moisture levels. Inflammatory factors are the primary criteria for assessing tissue inflammation, and excessive inflammatory factors can hinder wound recovery. To assess the safety and efficacy of PSC/TAX@HKUST-1 in vivo, hematoxylin-eosin (H & E) staining was performed on wound skin histopathology samples.
Half of mice in each group on days 9 and 18 were euthanized, skin wound tissues of the mice were taken, fixed in 10% formalin solution, paraffin embedded, and cut into 5 μm thick sections. Sections were H & E and Masson stained and observed under an optical microscope (Bio-Rad, hercules, USA).
As shown in FIG. 6A (magnified 100 times), the model group had the highest inflammatory factor level, and the PSC/TAX@HKUST-1 group had the lowest inflammatory factor level, with a downward trend from left to right, indicating that TAX within TAX@HKUS-1 still retained the effective anti-inflammatory properties. The developing epidermis after PSC treatment shows that the wound healing effect of the experimental group is more obvious than that of the model group, the epidermis and the new blood vessels of PSC/HKUST-1 group show an increasing trend, the wound recovery after HKUST-1 is added is improved, and the formation of the new blood vessels is promoted. The number of vessels in the PSC/HKUST-1 group was greater than PSC/TAX due to the Cu released by HKUST-1 2+ While favoring angiogenesis, TAX promotes wound healing by further inducing endothelial cell proliferation. The results indicated that the PSC/TAX@HKUST-1 group performed best, probably because HKUST-1 and TAX acted together to promote wound healing.
Collagen is an important component in maintaining skin function and structure, and its production in the local wound layer is an important indicator of damaged skin tissue remodeling. Therefore, the invention carries out masson trichromatic staining on the damaged skin tissues of the diabetic mice, and detects the collagen deposition condition after treatment. The blue color observed in the mahalanobis stained image indicates newly synthesized collagen, and the dark color indicates higher levels of collagen. As shown in FIG. 6B (100-fold magnification), PSC/TAX@HKUST-1 group had the highest collagen content, while model group had the lowest collagen content. The collagen deposition rates were quantitatively determined, and as a result, the collagen deposition rates of the PSC/TAX@HKUST-1, PSC/TAX and PSC/HKUST-1 groups were 76.02.+ -. 2.33%, 63.67.+ -. 5.51% and 46.99.+ -. 2.43%, respectively, which were significantly higher than those of the model group (23.82.+ -. 3.63%) and PSC group (38.61.+ -. 3.83%). These findings indicate that release of copper ions and TAX promotes the formation of new collagen fibrils, thereby promoting wound remodeling by enhancing collagen synthesis.
(3) Immunohistochemical staining
To better assess the extent of wound healing in T2D mice, immunohistochemistry was used to measure the expression levels of platelet endothelial cell adhesion molecule-31 (CD 31), mouse interleukin (IL-6).
Angiogenesis is an important component of the skin wound healing process and is characterized by the initial burst of many immature blood vessels, eventually developing into a mature vascular network. CD31 is an important index for assessing angiogenesis and is a typical marker for endothelial cells. By assessing its expression, the formation of new tissue in the wound surface can be assessed. IL-6 plays a key role in wound inflammation, is rapidly released in the injury response, stimulates tissue macrophages, keratinocytes, endothelial cells and stromal cells to secrete pro-inflammatory cytokines, exacerbates wound inflammation and inhibits healing. Therefore, reducing the inflammatory response of diabetic wounds is critical to promoting wound healing. The results of immunohistochemical analysis are shown in FIG. 7. The present invention quantitatively analyzes the expression of CD31 and IL-6 proteins in skin tissue. Experimental results showed that the expression of CD31 was significantly increased in the wound surface of diabetic mice treated with PSC/HKUST-1 and PSC/TAX@HKUST-1, as compared to the control group (FIG. 7B). Suggesting HKUST-1 release of Cu 2+ Can promoteThe wound surface is angiogenic, and the wound surface is healed. The expression of IL-6 at the wound site was significantly reduced after PSC/TAX hydrogel treatment (fig. 7C), suggesting that TAX may inhibit release of pro-inflammatory cytokines by macrophages, keratinocytes, endothelial cells and stromal cells, thereby reducing the inflammatory response.
All statistical results are expressed in mean ± standard deviation, data analysis adopts single factor analysis of variance, tukey test is then performed, significance level is expressed in mean ± SD as compared to control group with p < 0.05 and p < 0.01.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements within the spirit and scope of the present invention.
Claims (7)
1. A multifunctional slow-release dressing for promoting chronic wound healing comprises hydrogel and active ingredients loaded in the hydrogel; the hydrogel is prepared from polyvinyl alcohol, sodium alginate, carboxymethyl chitosan and CaO 2 PSC oxygen release hydrogel prepared from nano particles; the active ingredient is TAX@HKUST-1 nano particles, namely: copper metal organic framework material nano particles with dihydroquercetin doped in the inner cavity.
2. A method of preparing a multifunctional slow-release dressing for promoting healing of chronic wounds according to claim 1, comprising the steps of:
step 1, doping dihydroquercetin into an inner cavity of a copper metal organic framework material to prepare TAX@HKUST-1 nano particles;
step 2, mixing polyvinyl alcohol, sodium alginate and carboxymethyl chitosan to prepare PVA/SA/CMCS mixed aqueous solution; subsequently, caO is added 2 Preparing PSC oxygen-releasing aqueous solution by the nano particles; and finally, adding the TAX@HKUST-1 nanoparticle aqueous solution into the PSC oxygen release aqueous solution, and preparing the multifunctional slow-release dressing by adopting a cyclic freeze thawing method, wherein the multifunctional slow-release dressing is PSC/TAX@HKUST-1 composite hydrogel.
3. The method for preparing the multifunctional slow-release dressing for promoting the healing of chronic wounds according to claim 2, wherein the method for preparing the TAX@HKUST-1 nano particles in the step 1 is as follows: mixing copper-based metal organic framework material nano particles with a dihydroquercetin ethanol solution with the concentration of 100mg/mL, and stirring overnight in the dark; the mixture was then allowed to stand, the supernatant was discarded and dried under vacuum to give TAX@HKUST-1 nanoparticles.
4. The method for preparing the multifunctional slow-release dressing for promoting the healing of chronic wounds according to claim 2, wherein the preparation method of the PSC/TAX@HKUST-1 composite hydrogel in the step 2 is as follows: polyvinyl alcohol, sodium alginate and carboxymethyl chitosan are respectively prepared into 10 percent, 2 percent and 4 percent aqueous solutions, and the aqueous solutions are mixed according to the volume ratio of 1.5:1:1 to prepare PVA/SA/CMCS mixed aqueous solutions; subsequently, caO is added 2 The nano particles reach the concentration of 1mg/mL, and are stirred uniformly at room temperature to prepare PSC oxygen-releasing aqueous solution; then, adding TAX@HKUST-1 aqueous solution with the concentration of 4mg/mL into the PSC oxygen-releasing aqueous solution in a volume ratio of 1:3, and stirring in the adding process; after homogenization, the resulting solution was poured into a mold, frozen at-20℃for 2 hours, thawed at room temperature for 1 hour, and repeated 2-3 cycles to obtain a PSC/TAX@HKUST-1 composite hydrogel.
5. The method for preparing a multifunctional slow-release dressing for promoting chronic wound healing according to claim 3, wherein the mass ratio of the copper-based metal organic framework material nanoparticles to the dihydroquercetin in the step 1 is 1:10.
6. The method for preparing a multifunctional slow-release dressing for promoting chronic wound healing according to claim 4, wherein CaO in step 2 2 The preparation method of the nanoparticle comprises the following steps: 53g of CaCl 2 ·2H 2 O was added to 200mL of distilled water, followed by 80mL of 1M sodium hydroxide solution, and stirred at room temperature; subsequently, 100mL of H was added 2 O 2 The mixture was added dropwise to the mixture,stirring until a pale yellow precipitate is observed, filtering the solution, and washing the granules with sodium hydroxide and distilled water respectively; finally, the washed particles are dried at 80 ℃ and then homogenized into powder by using mortar to obtain CaO 2 And (3) nanoparticles.
7. The use of a multifunctional sustained-release dressing for promoting chronic wound healing according to claim 1 in the preparation of a medicament for promoting wound neovascularization.
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