CN113563642A

CN113563642A - A kind of composite microneedle for biological analysis and sampling and preparation method thereof

Info

Publication number: CN113563642A
Application number: CN202110719334.4A
Authority: CN
Inventors: 崔淑芬; 杨文龙; 陈永丽; 张宇豪; 刘崇杰; 冯景隆; 张珝
Original assignee: Shenzhen Polytechnic
Current assignee: Shenzhen Vocational And Technical University
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2021-10-29
Anticipated expiration: 2041-06-28
Also published as: CN113563642B

Abstract

The invention discloses a preparation method of a composite microneedle for biological analysis and sampling, comprising: providing a chitosan solution and a trehalose solution, mixing the chitosan solution and the trehalose solution to prepare a mixed solution; and providing a polyvinyl alcohol solution; Mix the mixed solution and the polyvinyl alcohol solution evenly, remove the foam, and obtain a composite solution; fill the composite solution into the mold, dry it once, then fill the mold with the composite solution, and carry out two steps. The composite microneedle is obtained by drying for three times, and then adding the base material, drying for three times, and demoulding. In the invention, polyvinyl alcohol and chitosan are compounded to prepare microneedles, and the antibacterial properties, mechanical properties, penetration properties and absorption properties of microneedles are effectively improved by optimizing and improving the preparation method and its parameter conditions. The needle tip is tall and straight, the needle body is complete and smooth, no hollow, air bubbles, and warpage occur, and it has a good subcutaneous sampling effect, and has a good application prospect in biological analysis and sampling.

Description

Composite microneedle for biological analysis sampling and preparation method thereof

Technical Field

The invention belongs to the technical field of biological diagnosis and instrument and meter analysis, and particularly relates to a composite microneedle for biological analysis and sampling and a preparation method thereof.

Background

The micro-needle is a needle-shaped micron-scale fine array structure, and the length of the micro-needle is generally 25-1000 mu m. Microneedles have rapidly developed over the past few decades and have been developed vigorously in the fields of transdermal drug delivery and the like. In recent years, the micro-needle is taken as a detection tool, blood and tissue fluid are extracted by the micro-needle for detection and analysis, and the micro-needle is taken as an electrode to directly detect blood sugar, melanoma, pH value and the like in body fluid in real time, and the micro-needle shows good real-time detection application prospect in these aspects. The micro-needle sampling is combined with some real-time analysis methods, blood, tissue fluid and the like can be obtained in a painless and minimally invasive mode, and further real-time detection can be realized. However, microneedles are still in the beginning stage as sampling tools, almost all microneedle detections adopt hollow microneedles at present, and no microneedles for sampling by utilizing the adsorption property of high-molecular polymers are available.

Artificially synthesized polymers such as polylactic acid, polyglycolide, natural polymer Chitosan (Chitosan, CS) and the like are common microneedle materials, and can be used as drug-loaded sustained-release microneedles with a single component. In addition, CS is a high molecular polymer which is widely available, natural and nontoxic, and has excellent bioactivity and biodegradability. Chen and the like prepare a CS microneedle coated with bovine serum albumin by a pouring method, and the CS microneedle can release drugs slowly and effectively within 8 days. It can be seen that CS is not easily dissolved in interstitial fluid, has a characteristic of being easily swollen by absorbing water, and is a condition for a detection-type microneedle.

However, CS itself has disadvantages of brittleness and poor mechanical properties, thereby limiting its application in the field of microneedles.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a composite microneedle for biological analysis and sampling and a preparation method thereof. The composite microneedle has good mechanical property and absorption property, and has good absorption and collection effect on skin tissue fluid.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

a method for preparing a composite microneedle for bioanalysis sampling, comprising:

providing a chitosan solution and a trehalose solution, and mixing the chitosan solution and the trehalose solution to prepare a mixed solution;

providing a polyvinyl alcohol solution;

uniformly mixing the mixed solution with the polyvinyl alcohol solution, and removing bubbles to obtain a composite solution;

filling the composite solution into a mold, carrying out primary drying, then filling the mold with the composite solution, carrying out secondary drying, then filling a substrate material, carrying out tertiary drying, and demolding to obtain the composite microneedle.

The addition of trehalose increases the fluidity of the chitosan solution, thereby accelerating the concentration of the solution and helping to obtain a solution of the desired concentration. Polyvinyl alcohol (PVA) is a water-soluble polymer prepared by hydrolyzing polyvinyl acetate, and has the advantages of no toxicity, stable chemical properties and the like. PVA has high mechanical strength and toughness and good biocompatibility, and can become an ideal biological material. The inventor finds that a large number of hydroxyl groups are arranged on the molecular chain of PVA and can be combined with CS to form hydrogen bonds, so that the swelling performance of CS can be further improved after the hydroxyl groups and the CS are mixed, and the composite microneedle with excellent absorption performance is obtained. Meanwhile, due to the good toughness of PVA, the defect of brittleness of CS can be effectively improved by blending the PVA and the PVA, and the composite microneedle with excellent mechanical property is obtained.

After primary drying, the composite solution is filled into the mold, secondary drying is carried out, and then the substrate material is filled to prepare the microneedle substrate. Therefore, the micro-needle can be prevented from being hollow, so that the micro-needle has higher mechanical hardness, and the integrity of the needle body is kept. Preferably, the base material is polyvinylpyrrolidone.

Preferably, the chitosan solution is made of insoluble chitosan. It will be understood by those skilled in the art that insoluble chitosan refers to chitosan that is insoluble or nearly insoluble in water. The inventor finds that compared with water-soluble chitosan, the insoluble chitosan is adopted to prepare the microneedle, so that the microneedle has more excellent antibacterial performance, can better prevent the microneedle from introducing pollutants such as bacteria and the like into a sampling object during transdermal sampling, is not easy to shrink in the drying process, is more beneficial to keeping the integrity of the microneedle body, has better pullout property, is not easy to warp and has a smoother needle surface.

Preferably, the preparation method of the chitosan solution comprises the following steps:

dissolving insoluble chitosan in a glacial acetic acid solution to obtain transparent and clear viscous liquid;

and (II) dialyzing the viscous liquid to remove acid, so that glacial acetic acid is completely permeated, and the chitosan solution is obtained.

Preferably, the glacial acetic acid solution is a mixed solution of ultrapure water and glacial acetic acid, and the addition amount of the glacial acetic acid is 5-15 mu L/mL in terms of the volume of the ultrapure water. More preferably, the amount of glacial acetic acid added to the glacial acetic acid solution is 10 μ L/mL based on the volume of ultrapure water; glacial acetic acid is added in the amount to fully dissolve the insoluble chitosan.

The addition of glacial acetic acid changes the properties of the insoluble chitosan, making it soluble in water and finally displacing the glacial acetic acid by dialysis.

Preferably, the concentration of the insoluble chitosan in the mixed solution is 8 wt%.

Preferably, the addition amount of the trehalose is 10% of the mass of the insoluble chitosan.

Preferably, the concentration of the polyvinyl alcohol solution is 20 wt%.

Preferably, in the polymer composite solution, the volume ratio of the mixed solution to the polyvinyl alcohol solution is 4: 1.

The absorption performance can influence the subcutaneous sampling effect of the microneedle, and the better the absorption performance is, the better the absorption effect of the microneedle on subcutaneous tissue fluid is, so that the sampling analysis is facilitated. The concentrations of the insoluble chitosan and the polyvinyl alcohol can influence the mechanical property and the absorption property of the composite microneedle, and the composite microneedle with better mechanical property and absorption property can be obtained by adopting the preparation under the conditions.

The composite microneedle with the best mechanical property and absorption property can be obtained by adopting the preparation conditions.

Preferably, the polyvinylpyrrolidone is PVPK 60.

Preferably, the temperature of the primary drying, the secondary drying and the tertiary drying is 40 ℃, and the total drying time is 9 h; in some embodiments, the time for the first drying is 1h, the time for the second drying is 2h, and the time for the third drying is 6 h. The inventor finds that the temperature and time for drying and curing are one of the key factors for determining the success of microneedle preparation. If the drying time is insufficient or the drying temperature is too low, the microneedle material is incompletely polymerized, so that the microneedle is softened and has insufficient hardness, and the use requirement cannot be met. If the drying temperature is too high, the microneedle body can be caused to generate bubbles, and the microneedle substrate can be warped, so that the shape of the microneedle body cannot meet the use requirement. The conditions provided by the invention are adopted for drying, the conditions are mild, the properties of the microneedle material cannot be damaged, the curing effect is good, the microneedle has high hardness, no bubbles are generated, the microneedle cannot be warped, and the using requirements can be better met.

Preferably, the preparation method of the mold comprises the following steps: and (3) preparing a Polydimethylsiloxane (PDMS) female die by taking the metal microneedle body as a male die.

Preferably, the preparation of the PDMS negative mold comprises: (1) mixing the non-crosslinked and cured PDMS prepolymer with a curing agent according to the mass ratio of 10:1 to obtain a PDMS mixed solution; (2) placing the PDMS mixed solution in a vacuum drier to remove air bubbles; (3) and (3) casting the male mold by using the PDMS mixed solution with bubbles removed, and drying and curing at 90 ℃ for 1h to obtain a PDMS female mold.

The invention also provides a compound microneedle for biological analysis sampling, which is prepared by the preparation method of the compound microneedle for biological analysis sampling.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, the polyvinyl alcohol and the insoluble chitosan are compounded to prepare the microneedle, and the preparation method and the parameter conditions thereof are optimized and improved, so that the antibacterial property, the mechanical property, the penetrating property and the absorption property of the microneedle are effectively improved, the needle point of the microneedle is straightened, the needle body is complete and smooth, the situations of hollowness, bubbles, warping and the like do not occur, the subcutaneous sampling effect is better, and the application prospect in biological analysis and sampling is good.

Drawings

Fig. 1 is a microscopic image of microneedles prepared in example 1 and comparative example 1;

FIG. 2 is a microscopic image of the microneedles prepared in example 1 and comparative examples 2 to 3;

FIG. 3 is a microscopic image of the microneedles prepared in comparative examples 4-5;

FIG. 4 is a graph showing the swelling curves of microneedles prepared in examples 1 to 3 and comparative example 6;

FIG. 5 is a graph showing the absorption curve of FITC by microneedles prepared in examples 1 to 3 and comparative example 6;

FIG. 6 is a microscopic image of the microneedles prepared in examples 1-3 and comparative example 6 after a stress test;

FIG. 7 is a graph showing the penetration effect of the micro-needle prepared in examples 1 to 3 and comparative example 6 on pigskin;

FIG. 8 is a graph showing the antibacterial effect of the microorganisms prepared in comparative examples 6 and 7 against Escherichia coli;

FIG. 9 is a graph showing the antibacterial effects of the microorganisms prepared in comparative examples 6 and 7 against Staphylococcus aureus.

Detailed Description

The invention provides a preparation method of a composite microneedle for biological analysis sampling, which comprises the following steps:

(1) preparation of the Mixed solution

Dissolving insoluble chitosan powder in glacial acetic acid and ultrapure water to obtain transparent and clear viscous liquid; wherein the addition amount of the glacial acetic acid is 5-15 mu L/mL (more preferably 10 mu L/mL) by volume of the ultrapure water;

putting the viscous liquid into a dialysis bag, and then putting the viscous liquid into ultrapure water for dialysis so that glacial acetic acid can be completely permeated out to obtain a chitosan solution;

dissolving trehalose powder in water to obtain a trehalose solution;

uniformly mixing the chitosan solution and the trehalose solution according to the mass ratio of trehalose to insoluble chitosan of 1:10 to obtain a clear colorless solution, and concentrating to obtain a mixed solution with the insoluble chitosan concentration of 8 wt%.

(2) Preparation mould

Taking a commercial metal microneedle body as a male die;

mixing the non-crosslinked and cured PDMS prepolymer with a curing agent according to the mass ratio of 10:1 to obtain a PDMS mixed solution;

placing the PDMS mixed solution in a vacuum drier to remove air bubbles;

and (3) casting the male mold by using the bubble-removed PDMS mixed solution, and drying and curing for 1h at 90 ℃ to obtain a PDMS female mold, wherein the PDMS female mold is a mold for preparing the microneedles.

(3) Preparation of PVA solution

PVA is added into ultrapure water, and the mixture is heated in a water bath until the PVA is completely dissolved, so that a PVA solution with the concentration of 20 wt% is obtained.

(4) Preparation of composite solution

And (3) uniformly mixing the mixed solution obtained in the step (1) and the PVA solution obtained in the step (3) according to the volume ratio of 4:1, and removing bubbles to obtain a composite solution.

(5) Preparation of composite microneedles

And (3) filling the composite solution obtained in the step (4) into the mold obtained in the step (2), putting the mold into a drying oven for primary drying at 40 ℃ for 1h, then adding the composite solution into the mold to fill the mold groove, performing secondary drying, drying at 40 ℃ for 2h, then adding 50% of PVPK60 in mass fraction into the mold to fill the mold groove, using the mold groove as a substrate of the microneedle, performing tertiary drying, drying at 40 ℃ for 6h, and demolding to obtain the composite microneedle.

The present invention will be described in detail with reference to specific embodiments in order to make the above objects, features and advantages more comprehensible. It is apparent that the following examples are only a part of the embodiments of the present invention, and not all of them. It should be understood that the embodiments of the present invention are only for illustrating the technical effects of the present invention, and are not intended to limit the scope of the present invention. The methods employed in the examples of the present invention are all conventional in the art unless otherwise specified, and the starting materials, equipment and equipment used are commercially available. Wherein the curing agent is Dow Corning 184 glue (SYLGARD 184 PDMS).

An experimental instrument:

rotary evaporator, zhengzhou great wall industrial and trade company; centrifuge 5810R Centrifuge, Eppendorf, germany; nikon ECLIPSE NI scientific grade upright fluorescence microscope, Nikon corporation, japan; dialysis bag, Chunpo office stationery special camp; DHG-9245A electric heating constant temperature blast drier, Shanghai-constant technology Instrument Co., Ltd; the Ono Honour constant temperature oscillator, Tianjin Ono instruments, Inc.; 96 well plates, corning; research plus pipette, Eppendorf, Germany; sartorius BSA224S analytical balance, Sartorius scientific instruments (beijing) ltd; YH-C electronic balance, raynangyingji; EPED-E2-20TS laboratory-grade ultrapure water instrument, Nanjing Yipu Dada science and technology development Co., Ltd; VGT-1990QTD ultrasonic cleaner, Suzhou Jiangdong precision instruments ltd; a standard hand-held magnifier, an eye optical shop;

experimental reagent: the water-soluble chitosan is added into the mixture,chengdu Akda chemical reagent, Inc.; insoluble chitosan, Shanghai Michelin Biochemical technology, Inc.; glacial acetic acid, Shanghai Michelin Biotech, Inc.; d-trehalose, anhydrous, Shanghai Michelin Biochemical technology Ltd; polyvinylpyrrolidone (PVPK60), yokodada chemical agents ltd; staphylococcus aureus (1)

6538D-5), the american ATCC biosignal resource center; escherichia coli (

8739D-5), the american ATCC biosignal resource center; trypticase Soy peptone liquid Medium, Kyoto Tokay Microbiol technologies, Inc.; LB BROTH (LB BROTH), guangdong cika microbiology ltd; LB AGAR (LB AGAR), Kyork, Guangdong, Microscience and technology Co., Ltd; trypticase soy peptone agar medium, Kyodoku Kogyo Microscience Co., Ltd; technical agar POWDER (ACAR POWDER), guangdong cyclokay microbiological science co; polydimethylsiloxane (PDMS), Dow Corning, usa; ultrapure water (conductivity 18.2 M.OMEGA.. cm)^-1) The water purifier is self-made in a laboratory.

Example 1

A preparation method of a composite microneedle comprises the following steps:

1. preparation of the Mixed solution

Weighing 1.0g of insoluble chitosan powder, adding 50mL of ultrapure water and 0.5mL of glacial acetic acid in sequence, fully stirring until the solution is transparent and clear and sticky, pouring the solution into a dialysis bag, clamping the front end and the rear end of the dialysis bag by using clamps, soaking the dialysis bag in the ultrapure water for 6 hours to ensure that the glacial acetic acid is completely penetrated out, measuring the pH value of the solution, and pouring the solution into a round-bottom flask. Trehalose powder 0.1g was dissolved in 1.5mL of water to prepare a trehalose solution. Uniformly mixing a trehalose solution and a chitosan solution subjected to deacidification according to the mass ratio of trehalose to insoluble chitosan of 1:10 to improve the fluidity of the chitosan solution so as to generate a uniform and clear colorless solution, concentrating the solution by using a rotary evaporator to obtain a mixed solution with the concentration of insoluble chitosan of 8 wt%, and sealing and storing at 4 ℃ for later use.

2. Preparation of microneedle molds

A PDMS female mold is prepared by taking a commercial metal microneedle body as a male mold, and the preparation process comprises the following steps: mixing the non-crosslinked and cured PDMS prepolymer with a curing agent according to a mass ratio of 10: 1; removing air bubbles in the PDMS mixed solution in a vacuum dryer; casting the PDMS mixed solution with bubbles removed into a male mold; and (3) placing the mixture in an oven at 90 ℃ for 1h for curing, and obtaining a PDMS female die after curing.

3. Preparation of composite microneedles

200mg of PVA was weighed into 3 10mL centrifuge tubes, 800. mu.L of ultrapure water was added, and the mixture was dissolved completely at 90 ℃ in a water bath to prepare a 20 wt% PVA solution. Mixing the prepared mixed solution with a PVA solution according to the volume ratio of 4:1, stirring for 1h in a water bath kettle at 60 ℃ to fully mix and dissolve the solution, and then centrifuging for 10min by using a centrifuge 6000r/min to remove bubbles until the solution is clear and free of bubbles, thus obtaining the composite solution. Filling the composite solution into a mold (namely a female mold), drying for 1h at 40 ℃ in an oven, then adding the composite solution into the mold to fill the mold groove, drying for 2h at 40 ℃, then adding 50% of PVPK60 in mass fraction into the mold to fill the mold groove, using the mold groove as a substrate of the microneedle, drying for 6h at 40 ℃ to completely form the microneedle, and demolding to obtain the composite microneedle.

Example 2

A method of manufacturing a composite microneedle, which is different from example 1 only in the concentration of the PVA solution, and is otherwise the same as example 1. The concentration of the PVA solution of example 2 was 10% by weight.

Example 3

A method of manufacturing a composite microneedle, which is different from example 1 only in the concentration of the PVA solution, and is otherwise the same as example 1. The PVA solution of example 3 had a concentration of 30% by weight.

Comparative example 1

Comparative example 1 differs from example 1 only in that: comparative example 1 the composite microneedle was prepared without adding the composite solution, but with PVPK60 having a mass fraction of 50%, and the same as in example 1.

The specific operation of comparative example 1 is as follows:

preparing a composite solution and a PDMS female die according to the method of the embodiment 1, filling the composite solution into a die, drying the die in an oven at 40 ℃ for 1h, then adding 50% of PVPK60 in mass fraction into the die to be used as a substrate of the microneedle, filling the groove of the die, drying the die at 40 ℃ for 8h to completely form the microneedle, and demolding to obtain the composite microneedle.

Comparative example 2

The comparative example 2 is different from the example 1 only in the drying time, and the total drying time of the comparative example 2 is 5h (primary drying 1h, secondary drying 2h, tertiary drying 2 h).

Comparative example 3

Comparative example 3 is different from example 1 only in the drying temperature, and comparative example 3 has a drying temperature of 60 ℃.

Comparative example 4

Comparative example 4 differs from example 1 only in the concentration of insoluble chitosan. In comparative example 4, the concentration of insoluble chitosan in the mixed solution used to prepare microneedles was 6 wt%.

Comparative example 5

Comparative example 5 differs from example 1 only in the drying time, and the total drying time of comparative example 5 was 10 hours (primary drying 1 hour, secondary drying 2 hours, tertiary drying 7 hours).

Comparative example 6

A preparation method of chitosan micro-needle comprises the following steps:

1. preparation of the Mixed solution

2. Preparation of microneedle molds

3. Preparation of microneedles

Filling the mixed solution into a mold (namely a female mold), drying for 1min at 40 ℃ in an oven, then adding the mixed solution into the mold to fill the mold groove, drying for 1h at 40 ℃, then adding 50% of PVPK60 in mass fraction into the mold to fill the mold groove, using the mold groove as a substrate of the microneedle, drying for 4h at 60 ℃ to completely form the microneedle, and demolding to obtain the microneedle. Since the microneedle is prepared by using only the chitosan solution in the comparative example, and the chitosan is easy to concentrate and thin after being dried, the drying time is shortened. And because no polymerization reaction exists, even if the drying temperature is 60 ℃, the performance of the chitosan microneedle is not adversely affected.

Comparative example 7

A preparation method of chitosan micro-needle comprises the following steps:

1. preparation of the Mixed solution

0.6g of water-soluble chitosan powder is weighed into a 5mL centrifuge tube, 3mL of ultrapure water is added, and the mixture is stirred to be completely dissolved to prepare the chitosan hydrogel with the concentration of 20 wt%.

2. Preparation of microneedle molds

3. Preparation of microneedles

Filling chitosan hydrogel into a mold (namely a female mold), drying in an oven at 40 ℃ for 1min, then adding the chitosan hydrogel into the mold to fill the mold groove, drying at 40 ℃ for 1h, adding 50% of PVPK60 in mass fraction into the mold to fill the mold groove, using the mold groove as a substrate of the microneedle, drying at 60 ℃ for 4h to completely form the microneedle, and demolding to obtain the microneedle.

Experimental testing

First, observation by microscope

1. The composite microneedles prepared in example 1 and comparative example 1 were observed under a microscope, and the results are shown in fig. 1. In fig. 1, a drawing a shows a composite microneedle prepared in comparative example 1, and a drawing B shows a composite microneedle prepared in example 1. As can be seen from fig. 1, the composite microneedle of example 1 has a complete shape, a smooth needle body, a straight needle tip, and no hollow core, and meets the requirements. In the comparative example 1, the prepared microneedle has a complete shape but a hollow interior of the microneedle body because the PVPK60 is directly added and supplemented instead of the composite solution as the substrate, so that the microneedle has poor mechanical property, insufficient penetrating power and undesirable use effect.

2. The composite microneedles prepared in example 1 and comparative examples 2 to 5 were observed under a microscope, and the results are shown in fig. 2 to 3. In fig. 2, a drawing a is a composite microneedle prepared in comparative example 2, a drawing B is a composite microneedle prepared in comparative example 3, and a drawing C is a composite microneedle prepared in example 1. As can be seen from fig. 2, the composite microneedle of example 1 has a complete shape, a smooth needle body, a straight needle tip, no hollow core, no air bubbles, and no substrate warpage, and meets the requirements. In contrast, in comparative example 2, the microneedle was not polymerized completely due to insufficient drying time, and was softened, and the hardness was not satisfactory. In comparative example 3, due to the excessively high drying temperature, bubbles appear inside the microneedle body, the substrate warps, and the shape of the microneedle body does not meet the requirement. In fig. 3, the left image is the composite microneedle prepared in comparative example 4, and the right image is the composite microneedle prepared in comparative example 5. As can be seen from fig. 3, in comparative example 4, the needle body of the microneedle was bent and not stiff, particularly, the needle tip was not satisfactory due to the excessively low concentration (6 wt%) of the insoluble chitosan. In comparative example 5, the needle body of the microneedle was not smooth and uneven due to the excessively long drying and curing time (10 hours). Therefore, the needle body can obtain better curing effect by adopting the drying and curing condition (drying at 40 ℃ for 9h) of the invention, the condition is better for preparing the microneedle, and the condition is mild, and even if the microneedle is loaded with the heat-instable medicine and the like, the microneedle can not be damaged.

Second, testing absorption Properties

Microneedles prepared in examples 1-3 and comparative example 6 were used to test swelling performance by the following method:

firstly, 1.5g of agarose is weighed in a conical flask, then 100mL of ultrapure water is added, the agarose is fully dissolved by microwave heating, the mixture is kept warm in a water bath kettle at 60 ℃ to prevent solidification, 40 microliter of FITC mother liquor is added while the mixture is hot, and the mixture is fully stirred to be uniformly distributed. 5mL of agarose solution was added to each of the four dishes and allowed to cool at room temperature to coagulate into a solid. Five microneedles each prepared in examples 1 to 3 and comparative example 6 were measured for their initial weight. The microneedle was vertically inserted into the agarose solid with tweezers, and the microneedle was removed at 1, 2, 3, 4, and 5min for timing, and the mass of the microneedle after imbibition and swelling was immediately measured, and the microneedle swelling degree was calculated using the following formula.

In the formula, W₁Is the initial weight of the microneedle, W₂The weight of the microneedle after imbibition and swelling.

The test results are shown in FIG. 4, where pure CS represents the microneedle prepared in comparative example 6, 10% PVA-CS represents the microneedle prepared in example 2, 20% PVA-CS represents the microneedle prepared in example 1, and 30% PVA-CS represents the microneedle prepared in example 3 (the same applies to the other figures). As can be seen from fig. 4, the swelling degree of the CS/PVA composite microneedles fabricated showed an increasing tendency as the PVA concentration increased, but the swelling degree of example 3 having the PVA concentration of 30 wt% was rather decreased compared to that of example 1 having the PVA concentration of 20 wt%. The reason is that: on one hand, PVA is a water-soluble high molecular compound, the molecular side chain of the PVA has a large number of hydroxyl groups, and the hydrophilicity is excellent, so the swelling degree of the CS/PVA composite micro-needle is correspondingly improved along with the increase of the concentration of the PVA; on the other hand, since hydroxyl groups in PVA can form hydrogen bonds with CS, the increase in PVA content greatly increases the number of aggregation entanglement points of the hydrogen bonds, which is disadvantageous to swelling. Therefore, the final swelling degree of the CS/PVA composite microneedle is determined by both of the above-described factors. Therefore, the concentration of PVA is preferably 20% by weight.

Third, testing the adsorption capacity of water-soluble drugs

In order to explore the adsorption capacity of the microneedles prepared in examples 1-3 and comparative example 6 on water-soluble drugs, the fluorescent dye FITC is uniformly distributed in the agarose solution to simulate the microneedle to absorb the water-soluble drugs in subcutaneous tissues. Firstly, 5mL of ultrapure water is added into each small hole of two prepared twelve-hole plates, the weighed microneedle is entirely soaked in water for 12h, and all obtained samples are detected to be the FITC content through an enzyme-labeling instrument under the excitation wavelength of 490nm and the emission wavelength of 520 nm.

The good absorption capacity of the micro-needle for the water-soluble drugs can enable the micro-needle to be used for detecting the drug concentration of subcutaneous tissue fluid, PVA-CS micro-needles with different concentrations are prepared, 1.5% agarose solution is used for simulating the subcutaneous tissue fluid, a certain amount of FITC is used for simulating the water-soluble drugs, and the absorption amount of the drugs is calculated by measuring the fluorescence value of the drugs. The FITC Linear equation is: y is 3.3287x +103.15, and FITC has a good linear relationship in the concentration range of 3.8ng/mL to 760 ng/mL.

The results of the absorption amount of the FITC by the microneedles with different PVA concentrations are shown in FIG. 5, and from the general trend, the absorption amount of the FITC by the microneedles gradually increases along with the increase of the PVA concentration in the microneedles, which shows that the absorption amount of the water-soluble drugs by the microneedles can be increased after the chitosan solution is added into the PVA. From the results, the 20% PVA-CS composite microneedles (i.e., the microneedles of example 1) had the best absorption effect for FITC, and the pure chitosan microneedles (i.e., the microneedles of comparative example 6) had the worst absorption effect. Compared with the pure chitosan micro-needle, the composite micro-needle has obviously improved imbibing effect and can be used for detecting the concentration of the soluble drug in subcutaneous tissue fluid.

Fourthly, testing mechanical properties

Sufficient mechanical strength is important for microneedles to penetrate the skin to create channels for drug transport. Firstly, placing the prepared microneedle with the needle point facing upwards on a plane, clamping a weight by using forceps, vertically and lightly placing the weight on the microneedle for 5min, and observing the shape of the microneedle under a microscope. The microneedles prepared in examples 1 to 3 and comparative example 6 were subjected to a pressure test for 5min using a 500g weight, and then the shapes of the microneedles after the test were observed under a microscope. As a result, as shown in fig. 6, a is a form of the microneedle of comparative example 6 after the stress test, B is a form of the microneedle of example 2 after the stress test, C is a form of the microneedle of example 1 after the stress test, and D is a form of the microneedle of example 3 after the stress test. As can be seen from fig. 6, the microneedles of comparative example 6 were deformed to the greatest extent, and the deformation of the microneedles was gradually reduced as the concentration of PVA increased, indicating that the mechanical properties of the microneedles were improved by the combination of CS and PVA.

Fifth, evaluation of in vitro skin penetration

Firstly, taking the abdominal skin of an in-vitro domestic pig, wiping the skin surface with 75% alcohol, then absorbing redundant moisture on the skin surface with filter paper, pressing the micro needle with a thumb with proper force after the micro needle is slightly dried to puncture the skin of the in-vitro domestic pig with smooth and undamaged surface, staying for 5min, taking out the micro needle, and immediately observing whether the punctured skin surface has a pinhole matrix. The results are shown in fig. 7, in which a is a graph showing the penetrating effect of the microneedle of comparative example 6 on the pigskin, B is a graph showing the penetrating effect of the microneedle of example 2 on the pigskin, C is a graph showing the penetrating effect of the microneedle of example 1 on the pigskin, and D is a graph showing the penetrating effect of the microneedle of example 3 on the pigskin. As can be seen from FIG. 7, all four microneedles can successfully puncture the skin of the in vitro domestic pig and have a micro channel.

Sixth, antibacterial experiment

Selecting two bacterial strains, i.e. Staphylococcus aureus and Escherichia coli, wherein Staphylococcus aureus is a gram-positive bacteria representative, Escherichia coli is a gram-negative bacteria representative, and the strains are respectively used for Escherichia coli and Staphylococcus aureusThe bacteria are subjected to in vitro bacteriostatic activity research. The antibacterial performance of insoluble chitosan microneedle (comparative example 6) and water-soluble chitosan microneedle (comparative example 7) were studied separately, and two bacterial strains were recovered from a freezer at-80 deg.C, inoculated into a liquid medium with an inoculating loop, and isolated colonies of Staphylococcus aureus were inoculated into a fresh trypticase soy peptone liquid medium, and Escherichia coli was inoculated into a fresh LB broth medium, which was placed in a constant temperature shaker (37 deg.C, 180r/min) and cultured overnight. Diluting the bacterial liquid with culture solution according to gradient, adding water-soluble chitosan microneedle and insoluble chitosan microneedle when the growth reaches logarithmic prophase (OD 600 nm-0.1-0.2) by using enzyme-labeling instrument, adding a blank group without microneedle for culture comparison, culturing in a constant temperature oscillator, and measuring OD by using enzyme-labeling instrument at 0h, 1h, 3h, 5h, 7h, 9h, 12h, 24h and 36h₆₀₀And (4) performing three groups of values in parallel, making a line graph, and performing comparative analysis.

The test results are shown in fig. 8 to 9, where fig. 8 shows the antibacterial activity against escherichia coli, and fig. 9 shows the antibacterial activity against staphylococcus aureus. As can be seen from the figure, the water-soluble chitosan and the insoluble chitosan both have the bacteriostatic action on escherichia coli and staphylococcus aureus, and the inhibitory effect of the insoluble chitosan microneedle on the two bacteria is particularly obvious.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. a preparation method of composite microneedle for biological analysis sampling, is characterized in that, comprises:

Provide chitosan solution and trehalose solution, mix chitosan solution and trehalose solution to make mixed solution;

Provide polyvinyl alcohol solution;

Mixing the mixed solution and the polyvinyl alcohol solution uniformly, defoaming, to obtain a composite solution;

Filling the composite solution into the mold, drying for one time, then filling the mold with the composite solution, drying for the second time, then adding the base material, drying for three times, and demoulding, the composite solution is obtained. Microneedles.

2 . The method for preparing composite microneedles for biological analysis and sampling according to claim 1 , wherein the chitosan solution is made of insoluble chitosan. 3 .

3. The method for preparing composite microneedles for biological analysis and sampling as claimed in claim 1, wherein the method for preparing the chitosan solution comprises:

(1) dissolving insoluble chitosan in glacial acetic acid solution to obtain a transparent and clear viscous liquid;

(2) The viscous liquid is dialyzed to remove acid, so that the glacial acetic acid is completely penetrated to obtain the chitosan solution.

4. The preparation method of the composite microneedle for biological analysis and sampling as claimed in claim 3, wherein the glacial acetic acid solution is a mixed solution of ultrapure water and glacial acetic acid, and by the volume of ultrapure water, ice The amount of acetic acid added was 5 to 15 μL/mL.

5 . The method for preparing composite microneedles for biological analysis and sampling according to claim 2 , wherein the concentration of the insoluble chitosan in the mixed solution is 8 wt %, and the added amount of the trehalose is 5 . 10% of the mass of the insoluble chitosan.

6 . The method for preparing composite microneedles for biological analysis and sampling according to claim 1 , wherein the concentration of the polyvinyl alcohol solution is 20 wt %. 7 .

7 . The method for preparing composite microneedles for biological analysis and sampling according to claim 1 , wherein, in the polymer composite solution, the volume ratio of the mixed solution to the polyvinyl alcohol solution is 4:1. 8 .

8. The method for preparing composite microneedles for biological analysis and sampling according to claim 1, wherein the temperature of the primary drying, secondary drying and tertiary drying is 40°C, and the total drying time is 9h .

9 . The method for preparing composite microneedles for biological analysis and sampling according to claim 1 , wherein the method for preparing the mold comprises: using the metal microneedle needle body as a male mold to prepare polydimethylsiloxane. 10 . A negative mold; the preparation steps of the PDMS negative mold include: (1) mixing the uncrosslinked and cured PDMS prepolymer and the curing agent at a mass ratio of 10:1 to obtain a PDMS mixed solution; (2) mixing the The PDMS mixed solution was placed in a vacuum desiccator to remove air bubbles; (3) the positive mold was cast with the air bubble-removed PDMS mixed solution, and dried at 90°C for 1 h to obtain a PDMS negative mold.

10 . A composite microneedle for biological analysis and sampling, characterized in that it is prepared by the method for preparing a composite microneedle for biological analysis and sampling according to any one of claims 1 to 9 .