CN115025037A - Separable microneedle loaded with active chlorella and preparation method thereof - Google Patents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/02—Algae
- A61K36/05—Chlorophycota or chlorophyta (green algae), e.g. Chlorella
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0023—Drug applicators using microneedles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0046—Solid microneedles
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0053—Methods for producing microneedles
Abstract
The invention relates to a separable microneedle loaded with active chlorella for treating diabetic wounds and a preparation method thereof, and specifically the separable microneedle is prepared by uniformly mixing GelMA hydrogel, a photoinitiator and chlorella solution, adding the mixture into a microneedle mould, defoaming for 5 min under negative pressure, repeating the process for 3 times, and curing the mixture for 15 s by using an ultraviolet lamp to prepare a microneedle tip; and adding a PVA solution until the micro-needle tip is covered, centrifuging to remove bubbles, and drying at 37 ℃ for 12 h to obtain the separable micro-needle loaded with the active chlorella. The invention prepares the separable microneedle loaded with the chlorella by loading the active chlorella into the microneedle tip taking hydrogel as the material and taking PVA as the substrate. Such separable microneedles are made into patches, and when applied to a skin wound, the PVA rapidly dissolves within a few minutes, leaving the hydrogel tip on the wound. Under the irradiation of the near-infrared LED, the chlorella can continuously release dissolved oxygen through photosynthesis, and the proliferation, migration and angiogenesis of cells are promoted, so that an anoxic microenvironment at a wound is relieved.
Description
Technical Field
The invention relates to a preparation method of a separable microneedle, in particular to a separable microneedle loaded with active chlorella for treating diabetic wounds and a preparation method thereof.
Background
There are hundreds of millions of people worldwide with diabetes, many of which are at risk for diabetic foot ulcers, and critically ill will be at risk for amputation. Not only is the incidence and mortality of chronic wounds of diabetes high, but the associated medical costs also place a heavy economic burden on the medical system. Studies have shown that oxygen is essential for normal cell growth and respiration, and that sufficient oxygen is a key factor in the treatment of diabetic wounds. Therefore, solving the problem of oxygen deficiency is a key problem to be solved urgently in the treatment of chronic wounds of diabetes. However, existing clinical treatments do not readily deliver or maintain sufficient oxygen to diabetic wounds. For example, hyperbaric oxygen inhalation does not prevent diabetic wound ischemia; local gaseous oxygen therapy is not effective because of the limited penetration of external gases into the tissue.
Although various oxygen-carrying materials developed at present have certain curative effects on promoting the healing of diabetic wounds, most oxygen-carrying systems have low oxygen permeability and are difficult to maintain for long-term oxygen supply, and the wound dressing only can contact the surface of the wounds and cannot penetrate into the wounds, so that the curative effect is poor.
Disclosure of Invention
The invention aims to provide a separable microneedle loaded with active chlorella for treating diabetic wounds and a preparation method thereof, and aims to solve the problem that most oxygen-carrying systems are poor in curative effect due to low oxygen permeability.
The invention is realized by the following steps: a preparation method of separable microneedle loaded with active chlorella comprises mixing GelMA hydrogel, photoinitiator and chlorella solution uniformly, adding into microneedle mould, defoaming under negative pressure for 5 min, repeating for 3 times, and curing with ultraviolet lamp for 15 s to obtain microneedle tip; and adding a PVA solution until the micro-needle tip is covered, centrifuging to remove bubbles, and drying at 37 ℃ for 12 h to obtain the separable micro-needle loaded with the active chlorella.
The preparation method of the separable microneedle loaded with the active chlorella specifically comprises the following steps:
a. culturing chlorella: placing the culture medium in a sterilized culture bottle, inoculating the chlorella strain in the sterilized culture bottle, sealing, and performing aseptic culture;
b. preparing a micro-needle tip: uniformly mixing GelMA hydrogel 10% (w/v) and photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite 0.5% (w/v) to obtain GelMA hydrogel solution, washing the chlorella solution cultured in the step a with PBS buffer solution, centrifuging to obtain chlorella precipitate, uniformly mixing the chlorella precipitate and the GelMA hydrogel solution according to the weight ratio of 1: 1, adding the mixture into a microneedle mould, performing negative pressure defoaming treatment on the mixed solution in the microneedle mould, irradiating with an ultraviolet lamp to cure the microneedle tip, and finishing illumination to obtain the microneedle tip;
c. and (3) preparing a finished product: and adding the PVA solution into a microneedle mould with a prepared microneedle point, covering the microneedle point with the solution, centrifuging to remove bubbles, and drying to obtain the separable microneedle loaded with the active chlorella.
Further, the strain used in step a is the strain numbered FACHB-482Chlorella vulgaris。
Further, the sterile culture mode in the step a is that the sterilized culture bottle is placed in a light incubator with the culture temperature of 25 ℃ for static culture for 5-10 days, and light irradiation for 12 hours and dark treatment for 12 hours are given every day.
Further, the negative pressure defoaming treatment mode in the step b is to perform negative pressure defoaming on the mixed solution for 5 min by using a vacuum dryer, and repeating the steps for 3 times.
Further, the drying treatment in step c is drying at 37 ℃ for 12 h.
The separable microneedle loaded with the active chlorella for treating the diabetic wounds is prepared by uniformly mixing GelMA hydrogel, a photoinitiator and a chlorella solution, adding the mixture into a microneedle mould, defoaming for 5 min under negative pressure, repeating the process for 3 times, and curing the mixture for 15 s by using an ultraviolet lamp to prepare a microneedle tip; and adding PVA solution to cover the micro-needle tip, centrifuging to remove bubbles, and drying at 37 ℃ for 12 h to prepare the micro-needle.
Chlorella (Cv) is a tiny green alga rich in proteins, lipids, polysaccharides, dietary fibers, vitamins and trace elements, has good biocompatibility, and more importantly, has more than ten times of photosynthesis capacity of other plants, can produce a large amount of dissolved oxygen, and is called as 'canned sun'. Therefore, the chlorella can be used as an oxygen supply factory to continuously provide dissolved oxygen and relieve the hypoxic microenvironment at the diabetic wound. The separable microneedle has certain air permeability and can enter internal tissues in a minimally invasive, painless and efficient transdermal drug delivery mode.
The invention prepares a separable microneedle (CvMN) loaded with chlorella by loading active chlorella into the tip of a microneedle which takes hydrogel as a material and taking PVA as a substrate. When such separable microneedles are made into a patch and applied to a skin wound, the PVA dissolves rapidly within a few minutes, leaving the hydrogel tip on the wound. Under the irradiation of the near-infrared LED, the chlorella can continuously release dissolved oxygen through photosynthesis, and the proliferation, migration and angiogenesis of cells are promoted, so that an anoxic microenvironment at a wound is relieved. Meanwhile, the chlorella is rich in trace elements Zn 2+ And Mg 2+ The inherent antioxidation can effectively remove free radicals and reduce the inflammatory reaction of the diabetic wound, thereby accelerating the healing of the diabetic wound. Experiments show that the chlorella loaded inside the separable microneedle can keep activity for at least 6 days, thereby ensuring the continuity of oxygen generation. In a word, the separable microneedle loaded with the active chlorella prepared by the invention has high biocompatibility, reduces inflammatory reaction, induces angiogenesis, re-epithelialization and collagen deposition, and can effectively accelerate the healing of diabetic wounds.
Drawings
FIG. 1 is a flow chart of the preparation of separable microneedles loaded with active Chlorella species.
Fig. 2 is a schematic structural view of a separable microneedle loaded with active chlorella.
Fig. 3 is an SEM image of isolated microneedles loaded with active chlorella.
FIG. 4 is a schematic diagram of the separation ability of separable microneedles carrying active Chlorella species, wherein FIG. 4a is a graph of the dissolution performance of a PVA matrix layer; fig. 4b is a cross-sectional fluorescence image of a detachable microneedle tip remaining inside an agarose hydrogel; fig. 4c is a planar fluorescence image of a detachable microneedle after application to an agarose hydrogel.
FIG. 5 is a schematic of the oxygen producing capacity of Chlorella and a separable microneedle loaded with active Chlorella; wherein FIG. 5a is a graph comparing the release of dissolved oxygen for different concentrations of Cv; FIG. 5b is a graph comparing the release of dissolved oxygen by CvMN under light or dark conditions; FIG. 5c is Cv (1X 10) with different concentrations of glucose added 9 CFU/mL) comparative graph of released dissolved oxygen; FIG. 5d shows Cv solution and CvMN (1X 10) 9 CFU/mL) comparative graph of the release of dissolved oxygen between the two with or without the addition of 500 μ M glucose; figure 5e is a graph comparing the oxygen release from different groups over the week.
FIG. 6 is a graph showing the antioxidant capacity of Chlorella vulgaris.
Fig. 7 is a picture of the healing of wounds in diabetic mice using isolated microneedles loaded with active chlorella.
Fig. 8 is a pathological analysis of wound tissue after treatment.
Detailed Description
As shown in FIG. 1, the preparation process of the separable microneedle (CvMN) loaded with active Chlorella of the present invention comprises the following steps:
(1) culturing chlorella:
chlorella strain (Latin seed name)Chlorella vulgarisAnd the number is: FACHB-482) was purchased from a freshwater algae seed bank of the chinese academy of sciences. The prepared BG-11 culture medium is placed in a 50 mL sterile culture bottle, then the strain is inoculated in the sterile culture bottle in a super clean bench, the bottle mouth is sealed, the inoculation date is marked, and sterile culture is carried out. Placing the sterilized culture bottle in an illumination incubator at 25 deg.C, statically culturing for 5-10 days, and applying illumination for 12 hr and dark treatment for 12 hr per day. The prepared chlorella solution is ready for use.
(2) Preparing a micro-needle tip:
and uniformly mixing 10 percent (w/v) of GelMA hydrogel with 0.5 percent (w/v) of photoinitiator phenyl-2, 4, 6-trimethylbenzoyl lithium phosphite to obtain GelMA hydrogel solution. Washing Chlorella solution with PBS buffer solution for 3 times, and centrifuging (4000 rpm, 5 min) to obtain Chlorella precipitate. Mixing the chlorella precipitate and the prepared GelMA hydrogel solution uniformly, adding into a microneedle mould, defoaming the mixed solution for 5 min under negative pressure by using a vacuum drier, repeating for 3 times, and irradiating for 15 s by using an ultraviolet lamp to cure the pinpoint of the microneedle. And obtaining the micro-needle tip after the illumination is finished.
(3) And (3) preparing a finished product:
adding 20% (w/v) PVA solution into the microneedle mould with the prepared microneedle point, completely covering the microneedle point, centrifuging to remove air bubbles, and drying at 37 ℃ for 12 h to obtain the final product of the separable microneedle loaded with the active chlorella.
The prepared cvMN loaded with active chlorella was photographed by an optical microscope to obtain a product structure shown in FIG. 2. As can be seen from the results, the prepared CvMN has a 15 × 15 microneedle array with the same length and alignment of the tips, pyramidal tips and a height of 600 μ M.
And spraying gold on the surface of the microneedle, and observing the structure of the microneedle of the product by using a cold field emission Scanning Electron Microscope (SEM) to obtain the SEM image shown in figure 3. As can be seen from the figure, the prepared separable microneedle carrying active chlorella is dark green.
Dissolution properties of the PVA base layer: the PVA base layer was immersed in a beaker containing PBS buffer, placed on a magnetic stirrer, stirred at 400 rpm at 37 ℃ and the change in weight of the PVA base layer was measured at various time points (0, 1, 2, 3, 4, 5 min). For each weighing, the original PBS buffer was removed using a pipette gun and an equal amount of fresh PBS buffer was added. The dissolution rate was finally calculated by the ratio of the change in weight of the PVA base layer to the initial weight. As can be seen from fig. 4a, the basal layer of CvMN was completely dissolved within 5 min in PBS buffer at 37 ℃, indicating that when CvMN was applied to the skin, the PVA basal layer was rapidly dissolved within a short time, thereby achieving separation of the microneedle tip from the basal layer, leaving the tip inside the skin.
Determination of the separating capacity of CvMN: firstly, 3% (w/v) agarose powder is dissolved in deionized water, heated for 30 s by a microwave oven until boiling, taken out and quickly poured into a 24-hole plate, and the agarose solution is cooled to form the hydrogel. The FITC-labeled microneedle was grasped with forceps, pressed against the surface of the agarose gel, and after 5 min, the PVA backing layer dissolved on the surface of the agarose gel was wiped off with absorbent paper. The shape of the tip inside the agarose gel was then observed using a fluorescence microscope and images were taken. The results of using agarose hydrogel to mimic the skin surface, and applying a microneedle with a tip labeled with FITC to the hydrogel surface, where the basal layer rapidly dissolved, leaving only the fluorescent tip remaining inside the hydrogel (as shown in fig. 4b, 4 c), indicate that CvMN has ideal detachability.
As shown in fig. 5, oxygen production capacity of CvMN was measured:
firstly, the oxygen production capacity of chlorella is measured: collecting Chlorella precipitate (10) with different concentrations 5 、10 6 、10 7 、10 8 And 10 9 CFU/mL), PBS washed three times and resuspended in PBS. NIR LED (light dose 50 mW/cm) at 635 nm with PBS as measuring medium 2 ) Oxygen content was measured using a dissolved oxygen meter, with triplicate determinations for each group.
Secondly, the oxygen production capacity of the CvMN is measured: by CvMN (1X 10) 9 CFU/mL), the activity of chlorella within CvMN was examined. Placing CvMN at (635 nm, 50 mW/cm) 2 ) Irradiating the glass substrate under an NIR LED for 30 min, processing the glass substrate in a dark place for 30 min, circulating for 6 periods in total, detecting the oxygen content by using an oxygen dissolving instrument, and parallelly measuring each group for three times. Next, it was examined whether the increase of Cv (1X 10) in glucose was possible 9 CFU/mL) and CvMN (1X 10) 9 CFU/mL), adding 125, 250 and 500. mu.M glucose, irradiating with NIR LED (635 nm, 50 mW/cm 2) for 30 min, detecting oxygen content with dissolved oxygen meter, and determining three in parallel for each groupNext, the process is carried out. Chlorella and CvMN were dispersed in BG11 (pH = 7.4) medium at 4 ℃ and oxygen production was monitored daily for one week.
As shown in FIG. 5a, under the irradiation of near infrared LED, chlorella can generate a large amount of oxygen through photosynthesis, and the oxygen yield is in positive correlation with the concentration of chlorella. As shown in FIG. 5b, the dissolved oxygen concentration of CvMN increased gradually from 0 mg/L to 6.8 mg/L over 30 min under sufficient light. Under dark conditions, the dissolved oxygen gradually drops from 6.8 mg/L to 0 mg/L within 30 min, which shows that the chlorella loaded on the tip of the separable micro-needle has complete activity and can perform photosynthesis and respiration.
Glucose (0, 125, 250, 500 μ M) was added to chlorella solution or to cvMN at different concentrations to simulate a high sugar environment, as shown in FIGS. 5c and 5d, and the increase in sugar content increased the oxygen production of cvMN and chlorella without any significant difference between the oxygen production of cvMN and chlorella at the same concentrations. As shown in figure 5e, under the condition of illumination, the oxygen production of CvMN and chlorella is kept relatively stable, the stability is good, and oxygen can be continuously and controllably produced to meet the requirement of treating diabetic wounds.
Determination of the ability of DPPH to scavenge free radicals: 150 uL of chlorella solution of various concentrations was added to 150 uL (0.1 mmol/L) of 1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH) in methanol. After vigorous shaking and incubation in the dark at room temperature for 30 min, the absorbance of the sample was measured at a wavelength of 517 nm using a microplate reader and all experiments were performed in parallel three times to obtain the free radical scavenging ability as shown in FIG. 6. The chlorella is rich in various antioxidant substances such as vitamins, gamma-linolenic acid, linoleic acid and the like, and as shown in fig. 6, the efficiency of scavenging free radicals is increased along with the increase of the concentration of the chlorella, which indicates that the chlorella can further improve the wound healing process by effectively scavenging free radicals at wound tissues.
DB/DB diabetic mice were randomly equally divided into 4 groups (Con, DM-Con, CvMN + Dark, CvMN + Light), 6 mice per group. The Con group was normal mice and did not receive any treatment. The CvMN + Light group was diabetic mice, treated with CvMN and given Light daily for 90 min. The CvMN + Dark group was also treated with CvMN, but no light treatment was given. The DM-Con group was diabetic mice and did not receive any treatment, resulting in the therapeutic effect shown in fig. 7. The recovery effect was best for cvmn (light) compared to the other groups, with the wound healing almost completely at day 12, approaching that of normal mice.
To assess epidermal regeneration and inflammation in the wound area, collected skin specimens were fixed in 10% formaldehyde solution, dehydrated, made into 5 μm thick paraffin sections, followed by HE, Masson staining, with the results shown in fig. 8: the progress of wound epithelialization, collagen deposition and granulation tissue formation was observed by H & E staining and Masson trichrome staining. The cvmn (light) group provides sufficient dissolved oxygen to the diabetic wound through photosynthesis, so the thickness values of epithelial gaps and granulation tissue are highest, the epithelial structure of the skin wound is more intact, and the collagen content is highest, while the DM-Con and cvmn (dark) groups have relatively low wound epidermal thickness and collagen deposition. Since collagen deposition plays an extremely important role in wound tissue remodeling, cvmn (light) has the best therapeutic effect in wound healing.
Claims (8)
1. A preparation method of a separable microneedle loaded with active chlorella is characterized in that GelMA hydrogel, a photoinitiator and chlorella solution are uniformly mixed, added into a microneedle mould, defoamed under negative pressure for 5 min, repeated for 3 times, and cured by an ultraviolet lamp for 15 s to prepare a microneedle tip; and adding a PVA solution until the micro-needle tip is covered, centrifuging to remove bubbles, and drying at 37 ℃ for 12 h to obtain the separable micro-needle loaded with the active chlorella.
2. A preparation method of a separable microneedle loaded with active chlorella is characterized by comprising the following steps:
a. culturing chlorella: placing the culture medium in a sterilized culture bottle, inoculating the chlorella strain in the sterilized culture bottle, sealing, and performing aseptic culture;
b. preparing a micro-needle tip: uniformly mixing 10% (w/v) of GelMA hydrogel with 0.5% (w/v) of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphite to obtain GelMA hydrogel solution, washing the chlorella solution cultured in the step a with PBS buffer solution, centrifuging to obtain chlorella precipitate, uniformly mixing the chlorella precipitate with the GelMA hydrogel solution according to the weight ratio of 1: 1, adding the mixture into a microneedle mould, performing negative pressure defoaming treatment on the mixed solution in the microneedle mould, irradiating with an ultraviolet lamp to cure the needlepoint, and finishing irradiation to obtain the needlepoint;
c. and (3) preparing a finished product: and adding a 20% (w/v) PVA solution into a microneedle mould with a micro-needle point, covering the micro-needle point with the solution, centrifuging to remove bubbles, and drying to obtain the separable microneedle loaded with the active chlorella.
3. The method for preparing an active chlorella-carrying separable microneedle according to claim 1, wherein the chlorella strain used in the step a is FACHB-482Chlorella vulgaris。
4. The method for preparing an isolated microneedle loaded with active chlorella according to claim 1, wherein the sterile culture in step a is performed by placing a sterilized flask in a light incubator at a culture temperature of 25 ℃ for static culture for 5 to 10 days, and applying light for 12 hours and dark for 12 hours per day.
5. The method for preparing separable microneedles supporting active chlorella according to claim 1, wherein the negative pressure defoaming treatment in step b is performed by performing negative pressure defoaming on the mixed solution using a vacuum drier for 5 min and repeating the steps for 3 times.
6. The method for preparing an isolated microneedle capable of supporting active chlorella according to claim 1, wherein the drying treatment in the step c is drying at 37 ℃ for 12 hours.
7. A separable microneedle carrying an active chlorella, characterized by being produced by the method for producing a separable microneedle carrying an active chlorella according to any one of claims 2 to 6.
8. A separable microneedle loaded with active chlorella is characterized in that GelMA hydrogel, a photoinitiator and chlorella solution are uniformly mixed, added into a microneedle mould, defoamed under negative pressure for 5 min, repeated for 3 times, and cured for 15 s by an ultraviolet lamp to prepare a microneedle tip; and adding PVA solution to cover the micro-needle tip, centrifuging to remove bubbles, and drying at 37 ℃ for 12 h to prepare the micro-needle.
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| XIAOXUAN ZHANG等: "Black Phosphorus-Loaded Separable Microneedles as Responsive Oxygen Delivery Carriers for Wound Healing", 《ACS NANO》, vol. 14, pages 5901, XP055864326, DOI: 10.1021/acsnano.0c01059 * |
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