CN114668778B - Microneedle for promoting wound healing - Google Patents
Microneedle for promoting wound healing Download PDFInfo
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Abstract
The invention discloses a composition for promoting wound healing, which is prepared from the following raw materials in parts by weight: tannic acid 0.01-0.1 weight portions and AgNO 3 0.001-0.002 parts, and bletilla striata polysaccharide 4-10 parts. The microneedle for promoting wound healing is prepared by specific components and dosage, has good biocompatibility, can promote cell proliferation, can promote expression of Epidermal Growth Factor (EGF) and vascular growth factor (VEGF), has good effect of accelerating wound healing, has remarkable effect, and has clinical popularization and application values.
Description
Technical Field
The invention particularly relates to a microneedle for promoting wound healing.
Background
Wound healing refers to a series of pathophysiological processes in which local tissues are repaired by regeneration, repair and reconstruction due to tissue loss caused by the action of wound factors. The physiological process is an inherent defensive adaptive response of the body to tissue cell damage caused by various deleterious factors. Any one step in the overall healing process is affected by a number of factors (intrinsic or extrinsic) such that the subsequent healing process is disturbed. Some factors are beneficial to wound healing such as proper local treatment means of the wound, growth factors and the like; however, some factors may prevent the wound from healing. These interference factors include: functional status of the blood circulation system, potential or onset diseases, medication conditions, etc.
In recent years, polymeric microneedle patches have attracted extensive research interest as a drug delivery system. It can be used for transdermal administration of a variety of substances, including small molecule drugs, proteins and cytokines. Typically, the therapeutic agent is encapsulated in polymeric microneedles, wherein the biocompatible polymer rapidly dissolves and releases the encapsulating agent in the deep dermis. The polymer microneedle patch can continuously release therapeutic drugs for a long time, overcomes the problem of local drug delivery, furthest reduces pain and tissue injury, and reduces the exposure of wounds to the external environment.
Li Chao and the like, the preparation of chitosan biological microneedles [ J ], the national academy of high molecular papers report the abstract set of papers-subject B biological macromolecules in 2015, discloses a chitosan microneedle which utilizes good biocompatibility of chitosan and can be used for repairing skin, but experimental researches show that the effect of promoting wound healing of the microneedle only containing chitosan active ingredients is not ideal.
Disclosure of Invention
In order to solve the problems, the invention provides a composition for promoting wound healing, which is prepared from the following raw materials in parts by weight:
tannic acid 0.01-0.1 weight portions and AgNO 3 0.001-0.002 parts, and bletilla striata polysaccharide 4-10 parts.
Further, the composite material is prepared from the following raw materials in parts by weight:
tannic acid 0.012 parts, agNO 3 0.0012 parts by weight of bletilla striata polysaccharide and 6 parts by weight of bletilla striata polysaccharide.
Further, it is prepared from tannic acid and AgNO 3 And bletilla polysaccharide as active components, and adding medicinal carrier to obtain topical dressing; the external dressing is emulsion, ointment, powder or micro needle, preferably micro needle.
Further, the drug carrier of the microneedle is prepared from the following raw materials: 2-10 parts of chitosan and 1-3 parts of acetic acid, preferably 4 parts of chitosan and 2 parts of acetic acid.
The invention also provides a method of the aforementioned composition, comprising the steps of:
1) Weighing the raw materials according to the proportionTaking acetic acid, tannic acid and AgNO as raw materials 3 Adding water into rhizoma bletilla polysaccharide to obtain solution; dissolving chitosan in acetic acid solution to obtain chitosan solution;
2) Pouring the chitosan solution obtained in the step 1) into a microneedle mould, centrifuging, adding NaOH solution for curing, and removing the NaOH solution to obtain a chitosan microneedle scaffold;
3) Taking the chitosan microneedle scaffold obtained in the step 2), centrifuging, and sequentially adding the tannic acid solution and AgNO obtained in the step 1) 3 And (3) drying the solution and the bletilla striata polysaccharide solution to obtain the product.
Further, in the step 1), the concentration of the acetic acid solution in the solution prepared by adding water is 2%, the concentration of the tannic acid solution is 200 mug/ml, and the concentration of the bletilla striata polysaccharide solution is 4%, w/v; the chitosan solution concentration was 4%, w/v.
Further, the concentration of the NaOH solution in the step 2) is 10%; the curing time is 20min; the NaOH solution is removed by flushing the solid surface with ultrapure water and then soaking the solid with ultrapure water for 6-12 h; the speed of the centrifugation is 4000 revolutions per minute and the time is 20 minutes.
Further, in the step 3), the tannic acid solution is divided into two parts and added into a microneedle holder for centrifugation for 20min; the AgNO 3 Dividing the solution into two parts, adding the two parts into a microneedle holder, and centrifuging for 20min; the bletilla polysaccharide solution is added into a microneedle scaffold in three equal parts for centrifugation for 20min, and each part of the bletilla polysaccharide solution is added at intervals of 30 min.
Still further, step 3) the speed of centrifugation is 4000 revolutions per minute.
The invention also provides application of the microneedle in preparation of a medicament for promoting wound healing.
The microneedle for promoting wound healing is prepared by specific components and dosage, has good biocompatibility, can promote cell proliferation, can promote expression of Epidermal Growth Factor (EGF) and vascular growth factor (VEGF), has good effect of accelerating wound healing, has remarkable effect, and has clinical popularization and application values.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 4% (w/v) concentration Chitosan optical microscope view
FIG. 2 view under a 10% (w/v) concentration chitosan optical microscope
FIG. 3 FTIR spectra of CS, TA, BSP, CT/Ag-MNs and CT/AgB-MNs
FIG. 4 is a SEM morphology of the prepared C-MNs, CT/Ag-MNs, CT/AgB-MNs
FIG. 5 energy dispersive X-ray spectrometer detection in situ synthesized nanosilver production pattern (a is side view of rear CT/AgB-MNs, b is top view of rear CT/AgB-MNs)
FIG. 6 CT/AgB-MNs EDS Spectrum
FIG. 7 effects of C-MNs, CT/Ag-MNs and CT/AgB-MNs on LPS-induced TNF- α and IL-10 production in RAW 264.7 cells
FIG. 8 shows a graph of intracellular ROS scavenging
Fig. 9 fluorescence microscope image
FIG. 10 antimicrobial Activity of microneedles (images of residual viable bacteria by plate count method: E.coli, b Staphylococcus aureus, c West Lin Jia oxygen Staphylococcus aureus; antibacterial ratio of microneedles: d E.coli, e Staphylococcus aureus, f West Lin Jia oxygen Staphylococcus aureus)
FIG. 11 MRSA forms an antimicrobial film after co-cultivation with C-MNs, CT/Ag-MNs (20).
FIG. 12 evaluation of anti-biofilm in microneedles (a: pictures of treatment with different microneedles not treated and used the third day of biofilm development; b: scanning electron microscope images of C-MNs, CT-MNs and CT/Ag-MNs for 1 day of MRSA treatment)
FIG. 13 evaluation of infected wounds by microneedle therapy (a: images of untreated wounds, C-MNs, CT/Ag-MNs, ag-D treatment wounds on days 0, 3, 7, 15; b: wound healing rates at different times)
FIG. 14 is a technical drawing for preparing microneedles
Detailed Description
EXAMPLE 1 preparation of the wound healing microneedle of the invention
The formula comprises the following components: 4g of chitosan, 2g of acetic acid, 0.012g of tannic acid and AgNO 3 0.0012g and bletilla polysaccharide 6g.
The preparation method comprises the following steps:
1) Weighing the raw materials according to the proportion;
2) Respectively taking acetic acid, tannic acid and AgNO 3 And bletilla polysaccharide, adding water to make into 2%, g/ml acetic acid solution, 200 μg/ml tannic acid solution, 20 μg/ml AgNO 3 Solution, 4% g/ml bletilla striata polysaccharide solution;
3) Dissolving chitosan in 2% acetic acid solution obtained in step 2) to obtain 4% chitosan solution (g/ml);
4) Pouring the chitosan solution obtained in the step 3) into a microneedle mould with the rotating speed of 4000 rpm, centrifuging for 20 minutes, adding 1ml of 10 percent g/ml, solidifying for 20 minutes by using NaOH solution, flushing the NaOH solution on the surface by using ultrapure water, soaking for 6-12 hours in the ultrapure water, and removing the NaOH solution in the microneedle to obtain the chitosan microneedle scaffold (C-MNs);
5) Taking 1/2 volume of the tannic acid solution obtained in the step 2), pouring the tannic acid solution into the C-MNs obtained in the step 4) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and then taking the rest tannic acid solution and adding the rest tannic acid solution into the C-MNs in the same way to obtain chitosan tannic acid microneedle (CT-MNs);
6) Taking 1/2 volume of AgNO obtained in step 2) 3 Pouring the solution into CT-MNs obtained in the step 5) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and taking out the rest AgNO 3 Adding the solution into CT-MNs by the same method to obtain in-situ synthesized nano-silver microneedles (CT/Ag-MNs);
7) And (3) taking the bletilla polysaccharide solution obtained in the step (2), dividing the bletilla polysaccharide solution into three equal parts, sequentially pouring the three equal parts into the bletilla polysaccharide solution obtained in the step (6) with the rotating speed of 4000 revolutions per minute, centrifuging the bletilla polysaccharide solution in CT/Ag-MNs for 20 minutes, adding one part of bletilla polysaccharide solution at intervals of 30 minutes, and drying at room temperature to obtain the in-situ synthesized nano-silver bletilla polysaccharide microneedles (CT/AgB-MNs).
EXAMPLE 2 preparation of the wound healing microneedle of the invention
The formula comprises the following components: 2g of chitosan, 1g of acetic acid, 0.01g of tannic acid and AgNO 3 0.001g, and bletilla polysaccharide 4g.
The preparation method comprises the following steps:
1) Weighing the raw materials according to the proportion;
2) Respectively taking acetic acid, tannic acid and AgNO 3 And bletilla polysaccharide, adding water to make into 2%, g/ml acetic acid solution, 200 μg/ml tannic acid solution, 20 μg/ml AgNO 3 Solution, 4% g/ml bletilla striata polysaccharide solution;
3) Dissolving chitosan in 2% acetic acid solution obtained in step 2) to obtain 4% chitosan solution (g/ml);
4) Pouring the chitosan solution obtained in the step 3) into a microneedle mould with the rotating speed of 4000 rpm, centrifuging for 20 minutes, adding 1ml of 10 percent g/ml, solidifying for 20 minutes by using NaOH solution, flushing the NaOH solution on the surface by using ultrapure water, soaking for 6-12 hours in the ultrapure water, and removing the NaOH solution in the microneedle to obtain the chitosan microneedle scaffold (C-MNs);
5) Taking 1/2 volume of the tannic acid solution obtained in the step 2), pouring the tannic acid solution into the C-MNs obtained in the step 4) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and then taking the rest tannic acid solution and adding the rest tannic acid solution into the C-MNs in the same way to obtain chitosan tannic acid microneedle (CT-MNs);
6) Taking 1/2 volume of AgNO obtained in step 2) 3 Pouring the solution into CT-MNs obtained in the step 5) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and taking out the rest AgNO 3 Adding the solution into CT-MNs by the same method to obtain in-situ synthesized nano-silver microneedles (CT/Ag-MNs);
7) And (3) taking the bletilla polysaccharide solution obtained in the step (2), dividing the bletilla polysaccharide solution into three equal parts, sequentially pouring the three equal parts into the bletilla polysaccharide solution obtained in the step (6) with the rotating speed of 4000 revolutions per minute, centrifuging the bletilla polysaccharide solution in CT/Ag-MNs for 20 minutes, adding one part of bletilla polysaccharide solution at intervals of 30 minutes, and drying at room temperature to obtain the in-situ synthesized nano-silver bletilla polysaccharide microneedles (CT/AgB-MNs).
EXAMPLE 3 preparation of the wound healing microneedle of the invention
The formula comprises the following components: 10g of chitosan, 3g of acetic acid, 0.1g of tannic acid and AgNO 3 0.01g, bletilla polysaccharide 10g.
The preparation method comprises the following steps:
1) Weighing the raw materials according to the proportion;
2) Respectively taking acetic acid, tannic acid and AgNO 3 And bletilla polysaccharide, adding water to make into 2%, g/ml acetic acid solution, 200 μg/ml tannic acid solution, 20 μg/ml AgNO 3 Solution, 4% g/ml bletilla striata polysaccharide solution;
3) Dissolving chitosan in 2% acetic acid solution obtained in step 2) to obtain 4% chitosan solution (g/ml);
4) Pouring the chitosan solution obtained in the step 3) into a microneedle mould with the rotating speed of 4000 rpm, centrifuging for 20 minutes, adding 1ml of 10 percent g/ml, solidifying for 20 minutes by using NaOH solution, flushing the NaOH solution on the surface by using ultrapure water, soaking for 6-12 hours in the ultrapure water, and removing the NaOH solution in the microneedle to obtain the chitosan microneedle scaffold (C-MNs);
5) Taking 1/2 volume of the tannic acid solution obtained in the step 2), pouring the tannic acid solution into the C-MNs obtained in the step 4) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and then taking the rest tannic acid solution and adding the rest tannic acid solution into the C-MNs in the same way to obtain chitosan tannic acid microneedle (CT-MNs);
6) Taking 1/2 volume of AgNO obtained in step 2) 3 Pouring the solution into CT-MNs obtained in the step 5) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, and taking out the rest AgNO 3 Adding the solution into CT-MNs by the same method to obtain in-situ synthesized nano-silver microneedles (CT/Ag-MNs);
7) Dividing the bletilla polysaccharide solution obtained in the step 2) into three equal parts, sequentially pouring the three equal parts into the CT/Ag-MNs obtained in the step 6) with the rotating speed of 4000 revolutions per minute, centrifuging for 20 minutes, adding one part of the bletilla polysaccharide solution each time for 30 minutes, and drying at room temperature to obtain the in-situ synthesized nano-silver-bletilla polysaccharide microneedles (CT/AgB-MNs).
The advantageous effects of the present invention are described below by way of test examples.
Experimental example 1 study of microneedle for promoting wound healing
1. Concentration screening of chitosan scaffolds
1) After centrifugation of 1% (w/v), 4% (w/v) and 10% (w/v) chitosan solutions for 20 minutes at 4000 rpm, the solutions were solidified with 10% NaOH solution, and the microneedle condition was observed by freeze drying first:
results: microneedles at a concentration of 1% (w/v) were not formed, failed to completely demold, 4% (w/v) and 10% (w/v) could be completely demolded, and the tips were complete.
2) 1ml of 4% (w/v) and 10% (w/v) chitosan solutions were used, respectively, into which tannic acid and AgNO were centrifuged 3 4% (w/v) bletilla polysaccharide solution, dried at room temperature and then demolded.
Results: the needle shape with the concentration of 4% (w/v) is complete, the needle tip of 10% (w/v) is shrunken, the analysis reasons are probably that the space of the chitosan internal bracket with the concentration of 10% (w/v) is too small, so that bletilla polysaccharide cannot be centrifuged to the bottom of the needle tip, and after drying, only the bracket at the needle tip is shrunken. (see FIGS. 1 and 2)
2. Fourier Transform Infrared (FTIR) experiments prove that tannic acid and bletilla polysaccharide are added into chitosan scaffold to generate hydrogen bonds
1) The experimental steps are as follows:
to characterize the chemical reaction that occurs between each component of the sample, FTIR was used to detect the sample. Weighing appropriate amount of refined BSP (bletilla polysaccharide) and potassium bromide, placing into agate mortar, grinding into fine powder particles, placing appropriate amount of powder into tablet press, pressing into tablet, placing into Fourier infrared spectrum with the Fourier infrared spectrum of 4000-500cm -1 In-range scanning, the obtained data is used for analyzing the structure of the BSP.
In a mortar, a proper amount of Chitosan (CS), tannic Acid (TA), in-situ reduced nano silver microneedles (CT/Ag-MNs, prepared according to example 1) and in-situ reduced nano silver white and polysaccharide microneedles (CT/AgB-MNs, prepared according to example 1) are respectively weighed, added with potassium bromide and ground into powder forcefully. The subsequent process is the same as the BSP infrared spectroscopy analysis described above. The microneedles prepared for analysis were added with each component to change the structure between the components.
2) Experimental results
An infrared spectrogram of CS, TA, BSP, CT/Ag-MNs and CT/AgB-MNs is shown in FIG. 3. At 3449cm with CS and TA -1 And 3369cm -1 Absorption of CT/Ag-MNs compared with the hydroxyl absorption peak of (C)The peak is 3451cm -1 Showing a shift to higher wavelengths. It was verified that CS and TA form hydrogen bonds in CT/Ag-MNs. BSP at 885cm -1 And 810cm -1 The peak at this point shows typical mannose absorption. With the addition of BSP, characteristic absorption peaks also appear in CT/AgB-MNs, proving the successful preparation of CT/AgB-MNs.
3. Scanning Electron Microscope (SEM)
1) Experimental procedure
SEM was used to observe the surface and internal morphology of the antimicrobial microneedles. Firstly, preheating an SEM for 30min, after an instrument is stabilized, placing a small amount of prepared micro-needles on a sample table adhered with conductive adhesive, placing the sample table in a small ion sputtering instrument for metal spraying treatment, taking out the micro-needles after metal spraying, placing the micro-needles in the SEM, vacuumizing the machine, setting electron microscope observation parameters (5.00 kv,9.2 mm) after a stand-by device is stabilized, and observing the surface and internal forms of the sample.
2) Experimental results
The morphology structure of the microneedles (C-MNs, CT/Ag-MNs, CT/AgB-MNs) is observed by a scanning electron microscope for characterization, as shown in the figure. As shown in fig. 4, the microneedle structures in all samples were uniform in size and exhibited a complete quadrangular pyramid shape. CS in C-MNs is porous after alkali curing treatment. After TA addition and in situ synthesis of Ag NPs, a porous structure was still observed, indicating that the addition of tannic acid and silver nitrate by centrifugation in C-MNs did not affect the morphology of CT/Ag-MNs. After BSP filling, no voids were observed in CT/AgB-MNs.
4. Energy dispersive X-ray spectrometer (EDS)
After SEM observation, the microneedles were scored using EDS. The distribution of Ag NPs in the microneedles was observed.
Experimental results: after EDS scanning is performed on CT/AgB-MNs, the distribution of the main components of Ag, C and O in the microneedle is shown in figure 5. FIG. 5 (a) is a side view of the rear CT/AgB-MNs and FIG. 5 (b) is a top view of the rear CT/AgB-MNs. The results show that Ag element is uniformly distributed in CT/AgB-MNs, and AgNO can be successfully processed by centrifugation 3 Centrifugally enter the needle tip of the micro needle and are uniformly distributed, thereby proving thatThe Ag NPs can be synthesized in situ through TA, so that the problem that the Ag NPs are easy to aggregate in preparation can be effectively solved. As shown by the EDS energy spectrum in FIG. 6, the Ag element peaks appear in the CT/AgB-MNs, indicating that Ag has been uniformly loaded to the CT/AgB-MNs.
5. In vitro anti-inflammatory experiments:
1) Test drug:
LPS: lipopolysaccharide
C-MNs: a chitosan microneedle scaffold prepared as in example 1.
CT/Ag-MNs: an in situ nano silver microneedle prepared as in example 1.
CT/AgB-MNs: in situ nano silver white and polysaccharide microneedle prepared according to example 1
2) Experimental procedure
Microneedle anti-inflammatory was assessed using real-time quantitative polymerase chain reaction (RT-PCR) detection of gene expression. After the RAW 264.7 cell fluid was diluted to a certain concentration with DMEM, the diluted cell fluid was divided into each well of a 6-well plate. Cells were grown with or without 1. Mu.g/mL LPS overnight with cell adhesion to the bottom of the wells for more than 12 hours with cell coverage up to 80%. After 24h co-cultivation, LPS-free medium was added and different groups of microneedles were placed in different wells, including control (no drug blank), C-MNs, CT/Ag-MNs and CT/AgB-MNs. After 24 hours of treatment, the microneedles were removed. After washing the cells 2 times with PBS at low temperature under each well, the cells were collected with a cell scraper and centrifuged at 5000rpm for 5 minutes. According to the experimental protocol, total RNA was extracted with Trizol reagent and the concentration was determined with a Nano Drop 2000c spectrophotometer. 1 μg of total RNA was reverse transcribed using the first strand cDNA synthesis kit according to the manufacturer's instructions. The iQ5 kit was then subjected to RT-PCR.
3) Experimental results
The corresponding inflammatory and anti-inflammatory factors were evaluated by RT-PCR as shown in FIG. 7. When the Raw 264.7 cells are stimulated by LPS+, the expression of TNF-alpha is up-regulated and the expression of IL-10 is down-regulated compared with the control group. TNF- α was significantly down-regulated after co-incubation with CT/AgB-MNs. IL-10 was up-regulated to near normal levels. Both TA and BSP have been reported to reduce pro-inflammatory factors, promote anti-inflammatory factor production, and the results are consistent with those reported. Thus, CT/AgB-MNs have good anti-inflammatory effects in vitro.
6. Antioxidation test: intracellular ROS scavenging assay
1) Test drug:
LPS: lipopolysaccharide
C-MNs: a chitosan microneedle scaffold prepared as in example 1.
CT/Ag-MNs: chitosan tannic acid microneedle prepared as in example 1.
CT/AgB-MNs: in situ nano silver white and polysaccharide microneedle prepared according to example 1
2) Experimental procedure
The L929 cells are first resuscitated for later use in experiments, the supernatant is discarded after centrifugation of the cells in the cryopreservation vessel, and a vessel of cells is typically divided into a culture dish to ensure the number of cells. After observing the cells for complete adherent growth, excess dead cells were washed off with PBS and then replaced with fresh medium until the cells were grown to a dish. After cell culture, L929 cells were added to 12-well plates and grouped: control group (blank without drug), LPS group, C-MNs group, CT/Ag-MNs group and CT/AgB-MNs group, LPS stimulation was added except for control group after 12h of cell wall-attached growth, and in brief, lipopolysaccharide LPS (1. Mu.g mL) was added in equal amount to each well -1 ) Culturing was continued for 24 hours. After 24h, different microneedles were added separately and incubated for a further 12h. After the co-incubation was completed, the microneedles were removed and the upper medium in the 12-well plate was carefully aspirated using a pipette and washed once with PBS, then each group of cells was collected, then PBS containing the dye DCFH-DA was added, incubation was continued at 37 ℃ for 30min, then excess dye was washed off using PBS, and then the fluorescence intensity of the cells was observed using a fluorescence microplate reader and an inverted fluorescence microscope, thereby evaluating ROS-scavenging ability of the microneedles.
3) Experimental results
To generate excessive ROS, L929 cells were stimulated with lps+. ROS levels were detected in vitro after incubation with C-MNs, CT/Ag-MNs and CT/AgB-MNs. As shown in FIG. 8, CT/AgB-MNs can significantly scavenge ROS. Both TA and BSP are reported to have radical trapping and antioxidant effects. The addition of TA and BSP to the microneedles can effectively scavenge free radicals and ROS. As shown in the fluorescence microscopy image of fig. 9, the ROS fluorescence observed by the CT/AgB-MNs group was lower than that of the control group, probably because the presence of BSP and TA could be mutually promoted, resulting in stronger ROS elimination. The results show that CT/AgB-MNs have great potential in solving the problem of excessive ROS on bacterial infection wounds.
7. Antibacterial agent
1) Test drug:
C-MNs: a chitosan microneedle scaffold prepared as in example 1.
CT-MNs: chitosan tannic acid microneedle prepared as in example 1.
CT/Ag-MNs (10): agNO removal for preparing in-situ synthesized nano silver microneedle 3 Except that the concentration of (C) was 10. Mu.g/ml, the procedure was as in example 1.
CT/Ag-MNs (15): agNO removal for preparing in-situ synthesized nano silver microneedle 3 Except that the concentration of (C) was 15. Mu.g/ml, the procedure was as in example 1.
CT/Ag-MNs (20): agNO removal for preparing in-situ synthesized nano silver microneedle 3 Except that the concentration of (C) was 20. Mu.g/ml, the procedure was as in example 1.
2) The anti-planktonic bacteria experimental steps:
antibacterial activity was evaluated on C-MNs, CT/Ag-MNs (10), CT/Ag-MNs (15) and CT/Ag-MNs (20) using Staphylococcus aureus, escherichia coli and methicillin-resistant Staphylococcus aureus. Bacteria were recovered, inoculated onto a solid medium and cultured in a incubator at 37℃for 18 hours, and then a colony was selected and added to 4ml of the liquid medium and incubated in a shaker at 37℃for 4 hours. Bacterial count assessment was performed using ultraviolet-visible spectrophotometry (600 nm). After the bacteria were diluted to 106CFU/mL with sterile liquid medium, 200. Mu.l of the samples added with the bacteria solution (three samples per group were sterilized by ultraviolet irradiation for 30 min) were incubated in a constant temperature incubator at 37℃for 4h, and then 800. Mu.l of sterile liquid medium was added for continued co-incubation overnight. Finally, the solution is used for detecting the antibacterial capability of the sample by a turbidimetry method and a plate counting method.
And (3) diluting a blank group bacterial liquid to 102CFU/mL by using a sterile liquid culture medium, diluting the group bacterial liquid of C-MNs, CT/Ag-MNs (10), CT/Ag-MNs (15) and CT/Ag-MNs (20) by using the same dilution multiple, inoculating 100 mu l of the culture medium to the surface of a solid culture medium by using a dilution coating flat plate method, placing the solid culture medium into a 37 ℃ incubator for culturing for 18 hours, photographing and recording the colony number on each culture medium.
Experimental results: e.coli and s.aureus are the most common bacteria to infect wounds, MRSA being the most common drug resistant bacteria. The three bacteria are subjected to antibacterial evaluation by using the micro-needle, so that a more powerful convincing force is provided for promoting the clinical use of the micro-needle auxiliary material. As shown in FIG. 10 (a, b, c), the CT/Ag-MNs group has better antibacterial activity on three main pathogenic bacteria. The antibacterial effect of the C-MNs and CT-MNs groups was not significant. Addition of AgNO to CT-MNs microneedles 3 After that, the antibacterial effect of the microneedles is further enhanced, and the colonies gradually decrease as the concentration of the synthesized nano silver increases. As with the trend of the antibacterial rate of the microneedles of FIG. 10 (d, e, f), when AgNO was added 3 At a concentration of 20. Mu.g/ml, the bacteriostasis rate of CT/Ag-MNs is all over 97%. The CT/Ag-MNs microneedle can effectively prevent bacterial infection, which is mainly attributed to the fact that CS and Ag NPs in the CT/Ag-MNs play a certain synergistic antibacterial role, and the antibacterial activity of the microneedle is improved.
3) The anti-methicillin-resistant staphylococcus aureus biomembrane experimental steps comprise:
after resuscitating MRSA bacteria, diluting and spreading the bacteria on a solid culture medium, culturing at a constant temperature of 37 ℃ for 18 hours, picking a colony into a sterilized TSB culture medium, oscillating for 4 hours at the temperature of 37 ℃ in a dual-function gas bath constant temperature oscillator, diluting the bacteria concentration to 108CFU/mL, diluting the sterilized liquid culture medium to 106CFU/mL, taking 200 mu l of bacteria liquid into a 24-well plate, adding a sterilized circular glass plate (with the diameter of 8 mm) to place the bottom of the 48-well plate, adding 800 mu l of sterilized TSB culture medium, culturing in a constant temperature oven at the temperature of 37 ℃ for 3 days, and replacing the culture medium once per day until bacterial biofilms are formed. After biofilm growth, TSB medium was removed. Directly adding 1ml of sterilized TSB culture medium to incubate without any treatment; other groups were fixed with the microneedle tips down, the biofilm was pierced, and 1ml of sterilized TSB medium was added for co-incubation for 6h. And then washing the biological film for three times by using sterilized normal saline, washing off excessive impurities, and dyeing the biological film according to the steps of the Live & read bacterial dyeing kit instruction, so that the confocal laser scanning microscope is convenient to observe the state of the biological film.
Experimental results: the anti-biofilm ability of various microneedles was evaluated in vitro. As shown in fig. 11, MRSA biofilm structure was smooth and intact, with most bacteria surviving. Once the microneedles are used, the biofilm becomes rough and porous and the number of bacterial deaths varies. After CT/Ag-MNs treatment, most of the bacteria in the biofilm were killed, indicating that it has potent anti-biofilm properties in vitro.
8. In vivo anti-biofilm assay
1) Test drug:
C-MNs: a chitosan microneedle scaffold prepared as in example 1.
CT-MNs: chitosan tannic acid microneedle prepared as in example 1.
CT/Ag-MNs: in situ synthesized nano silver microneedle prepared according to example 1
2) Experimental protocol
Animals were randomly divided into 4 groups of 3 animals, and after anesthesia, the SD rats were dehaired, and a full-thickness skin defect wound with a diameter of 8mm was constructed on the backs of the rats using a punch. Mu.l of MRSA (108 CFU/ml) bacteria solution was transferred to each wound with a pipette and attached with a transparent dressing to prevent external influences. All rats were kept in a single cage and after three days, biofilm construction was observed. After the rat dorsal full-thickness defect wound biofilm construction effort, 4 groups were randomized for in vivo anti-biofilm evaluation. The 4 groups are: blank (without any treatment), C-MNs, CT-MNs and CT/Ag-MNs. After 24h of treatment, the cells were sacrificed. The defective wound and its tissues beside were removed, and after curing with 2.5% glutaraldehyde solution for 24 hours, gradient dehydration was performed. After freeze drying, the skin surface biofilm was observed using SEM for evaluation of in vivo anti-biofilm properties.
3) Experimental results
The removal effect of the prepared microorganisms on the biofilm was examined in SD rats. As shown in the SEM images of fig. 12, the wound surface of the mice can form a thicker and dense biofilm 3 days after MRSA infection. After C-MNs and CT-MNs are treated for 1 day, the biological film can still be adhered to wound tissues, which shows that the effectiveness of the antibacterial biological film is low. And after CT/Ag-MNs treatment, the wound surface has almost no bacteria, which proves that the CT/Ag-MNs can effectively eliminate the antibacterial biological film.
9. Infected wound healing experiments
1) Test drug:
control: physiological saline
C-MNs: a chitosan microneedle scaffold prepared as in example 1.
CT/Ag-MNs: in situ synthesized nano silver microneedle prepared according to example 1
CT/AgB-MNs: in situ synthesized nano silver white and polysaccharide microneedle prepared according to example 1
Ag-D: commercial nanometer silver burn and scald dressing (specifically purchased from Anxin biotechnology Co., ltd.)
2) Experimental protocol
Wound repair assessment was performed immediately after the infected wound model was constructed. The experimental wound treatments were divided into five subgroups: namely a control group (1 ml of physiological saline is smeared), a C-MNs group, CT/Ag-MNs (per cm) 2 The usage amount of Ag is 2.08X10 -6 g) CT/AgB-MNs (per cm) 2 The usage amount of Ag is 2.08X10 -6 g) And Ag-D (per cm) 2 The usage amount of Ag is 5.00 multiplied by 10 -3 g) A group. Grouping local treatment, feeding all rats in a single cage until the test is finished, photographing the back wound surfaces of the rats on 0, 3, 7 and 15 days after injury, calculating the sizes of the wound surfaces through Image J Image analysis software, analyzing the wound surface healing condition, and calculating the wound healing rate as follows:
A 0 area of original wound surface (cm) 2 )，A n Area of wound surface (cm) on day n 2 )。
3) Experimental results
To verify the wound healing effect of the prepared microneedles in vivo, we established bacterial infection wounds on the back skin of rats. Meanwhile, the commercial product nano silver burn and scald dressing (Ag-D) is used as a control. Wound images were taken for each group after 0, 3, 7, 15 days of treatment, respectively. As shown in fig. 13 (a, b), all wound areas become smaller gradually with increasing days. In particular to CT/AgB-MNs treatment group, the wound surface is basically completely healed after 15 days. The commercialized product nano silver has lower wound healing rate, which is probably due to the fact that the self property of Ag-D cannot be degraded, the wound cannot be scabbed in time, and the wound healing rate is reduced.
In conclusion, the microneedle (shown in fig. 14) prepared by specific components and the dosage has good biocompatibility, can promote cell proliferation, can promote the expression of Epidermal Growth Factor (EGF) and vascular growth factor (VEGF), has good effect of accelerating wound healing, and has remarkable effect and clinical popularization and application values.
Claims (8)
1. A microneedle for promoting wound healing, which is characterized in that: the composite material is prepared from the following raw materials in parts by weight:
tannic acid 0.01-0.1 weight portions and AgNO 3 0.001-0.002 parts, bletilla polysaccharide 4-10 parts, chitosan 2-10 parts, acetic acid 1-3 parts.
2. The microneedle according to claim 1, characterized in that: the composite material is prepared from the following raw materials in parts by weight:
tannic acid 0.012 parts, agNO 3 0.0012 parts by weight, 6 parts by weight of bletilla striata polysaccharide, 4 parts by weight of chitosan and 2 parts by weight of acetic acid.
3. A method of making the microneedle of any one of claims 1 or 2, characterized by: it comprises the following steps:
1) Weighing raw materials according to a proportion, and respectively taking acetic acid, tannic acid and AgNO 3 Adding water into rhizoma bletilla polysaccharide to obtain solution; dissolving chitosan in acetic acid solution to obtain chitosan solution;
2) Pouring the chitosan solution obtained in the step 1) into a microneedle mould, centrifuging, adding NaOH solution for curing, and removing the NaOH solution to obtain a chitosan microneedle scaffold;
3) Taking outCentrifuging the chitosan microneedle scaffold obtained in the step 2), and sequentially adding the tannic acid solution and AgNO obtained in the step 1) 3 And (3) drying the solution and the bletilla striata polysaccharide solution to obtain the product.
4. A method according to claim 3, characterized in that: the concentration of acetic acid solution in the solution prepared by adding water in the step 1) is 2 percent, the concentration of tannic acid solution is 200 mug/ml, and the concentration of bletilla striata polysaccharide solution is 4 percent, w/v; the chitosan solution concentration was 4%, w/v.
5. A method according to claim 3, characterized in that: the concentration of the NaOH solution in the step 2) is 10%; the curing time is 20min; the NaOH solution is removed by flushing the solid surface with ultrapure water and then soaking the solid with ultrapure water for 6-12 h; the speed of the centrifugation is 4000 revolutions per minute and the time is 20 minutes.
6. A method according to claim 3, characterized in that: step 3), dividing the tannic acid solution into two parts, adding the two parts into a microneedle holder, and centrifuging for 20min; the AgNO 3 Dividing the solution into two parts, adding the two parts into a microneedle holder, and centrifuging for 20min; the bletilla polysaccharide solution is added into a microneedle scaffold in three equal parts for centrifugation for 20min, and each part of the bletilla polysaccharide solution is added at intervals of 30 min.
7. A method according to claim 3 or 6, characterized in that: step 3) the speed of centrifugation is 4000 rpm.
8. Use of the microneedle according to claim 1 or 2 for the preparation of a medicament for promoting wound healing.
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| WO1995020967A1 (en) * | 1994-02-04 | 1995-08-10 | Cell Therapeutics, Inc. | Composition for wound healing, neuron growth and vascularization |
| CN112375250A (en) * | 2020-10-19 | 2021-02-19 | 重庆大学 | Nano-silver modified chitosan-polyvinyl alcohol antibacterial composite sponge and preparation method thereof |
| CN114159551A (en) * | 2021-11-25 | 2022-03-11 | 西南交通大学 | Soluble double-layer drug-loaded microneedle patch for chronic wound repair and preparation method thereof |
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| WO1995020967A1 (en) * | 1994-02-04 | 1995-08-10 | Cell Therapeutics, Inc. | Composition for wound healing, neuron growth and vascularization |
| CN112375250A (en) * | 2020-10-19 | 2021-02-19 | 重庆大学 | Nano-silver modified chitosan-polyvinyl alcohol antibacterial composite sponge and preparation method thereof |
| CN114159551A (en) * | 2021-11-25 | 2022-03-11 | 西南交通大学 | Soluble double-layer drug-loaded microneedle patch for chronic wound repair and preparation method thereof |
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