CN113563642B - Composite microneedle for biological analysis sampling and preparation method thereof - Google Patents

Abstract

The invention discloses a preparation method of a composite microneedle for biological analysis sampling, which comprises the following steps: 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. According to the invention, the polyvinyl alcohol and the chitosan are compounded to prepare the microneedle, and the preparation method and the parameter conditions thereof are optimized and improved, so that the antibacterial performance, the mechanical performance, the penetrating performance and the absorption performance of the microneedle are effectively improved, the needle point of the microneedle is straight, the needle body is complete and smooth, no hollow, bubble or warping occurs, the hypodermic sampling effect is good, and the application prospect in bioanalysis sampling is good.

Description

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

technical field

The invention belongs to the technical field of biological diagnosis and instrumentation analysis, and in particular relates to a composite microneedle for biological analysis and sampling and a preparation method thereof.

Background technique

A microneedle is a needle-like micron-scale fine array structure with a general length between 25 and 1000 μm. Microneedling has developed rapidly in the past few decades, and has flourished in fields such as transdermal drug delivery. In recent years, microneedles have begun to be used as detection tools to extract blood and tissue fluid for detection and analysis, and microneedles are used as electrodes to directly detect blood sugar, melanoma, pH, etc. in body fluids in real time. Microneedles are used in these aspects. All of them show good prospects for real-time detection applications. Microneedle sampling combined with some real-time analysis methods can obtain blood, tissue fluid, etc. in a painless and minimally invasive way, and further realize real-time detection. However, as a sampling tool, microneedles are still in their infancy. At present, almost all microneedle detection uses hollow microneedles, and there is no microneedle that utilizes the adsorption properties of high molecular polymers for sampling.

Synthetic polymers such as polylactic acid, polyglycolide, and natural polymer chitosan (Chitosan, CS) are commonly used microneedle materials, which can be used as drug-loaded sustained-release microneedles with a single component. Moreover, CS is a wide-ranging, natural, non-toxic high molecular polymer with excellent biological activity and biodegradability. Chen et al. prepared a CS microneedle encapsulated with bovine serum albumin by the pouring method, which could release the drug slowly within 8 days. It can be seen that CS is not easy to dissolve in tissue fluid, and has the characteristics of water absorption and easy swelling, and has the conditions to be used as detection-type microneedles.

However, CS itself has defects such as brittleness and poor mechanical properties, which limit its application in the field of microneedles.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned shortcomings and deficiencies in the prior art, the purpose of the present invention is to provide a composite microneedle for biological analysis and sampling and a preparation method thereof. The composite microneedle of the invention has better mechanical properties and absorption properties, and has better absorption and collection effect on skin tissue fluid.

For realizing its purpose, the technical scheme that the present invention takes is as follows:

A preparation method of a composite microneedle for biological analysis and sampling, comprising:

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.

The addition of trehalose can increase the fluidity of the chitosan solution, thereby accelerating the concentration of the solution and helping to obtain the solution of the desired concentration. Polyvinyl alcohol (PVA) is a water-soluble polymer hydrolyzed from polyvinyl acetate, which has the advantages of non-toxicity and stable chemical properties. PVA itself has high mechanical strength and toughness, and also has good biocompatibility, making it an ideal biomaterial. The inventors found through research that there are a large number of hydroxyl groups on the molecular chain of PVA, which can combine with CS to form hydrogen bonds. Therefore, after blending the two, the swelling performance of CS can be further improved, thereby obtaining composite microneedles with excellent absorption performance. At the same time, due to the good toughness of PVA, the blending of the two can effectively improve the brittleness of CS, and obtain composite microneedles with excellent mechanical properties.

After the primary drying, the present invention fills the mold with the composite solution, performs secondary drying, and then supplements the base material to prepare the microneedle base. In this way, the hollow of the microneedle can be avoided, the microneedle has high mechanical hardness, and the integrity of the needle body can be maintained. Preferably, the base material is polyvinylpyrrolidone.

Preferably, the chitosan solution is made of insoluble chitosan. Those skilled in the art should understand that the insoluble chitosan refers to chitosan that is insoluble or almost insoluble in water. After research and development, the inventors found that, compared with water-soluble chitosan, using insoluble chitosan to prepare microneedles can make the microneedles have more excellent antibacterial properties, and can better prevent the microneedles from killing bacteria during transdermal sampling. and other pollutants are introduced into the sampling object, and it is not easy to shrink during the drying process, which is more conducive to maintaining the integrity of the microneedle body, the microneedle has better uprightness, less warping, and a smoother needle surface.

Preferably, the preparation method of 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 glacial acetic acid is completely penetrated to obtain the chitosan solution.

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

The addition of glacial acetic acid can change the properties of insoluble chitosan, make it soluble in water, and finally replace the glacial acetic acid by dialysis.

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

Preferably, the added 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 will affect the subcutaneous sampling effect of the microneedle. The better the absorption performance, the better the absorption effect of the subcutaneous tissue fluid of the microneedle, which is more conducive to sampling and analysis. The concentration of insoluble chitosan and polyvinyl alcohol will have an impact on the mechanical properties and absorption properties of the composite microneedles. The composite microneedles with better mechanical properties and absorption properties can be obtained by preparing under the above conditions.

Prepared under the above conditions, the composite microneedles with the best mechanical properties and absorption properties can be obtained.

Preferably, the polyvinylpyrrolidone is PVPK60.

Preferably, the temperature of the first drying, the second drying and the third drying is 40°C, and the total drying time is 9h; wherein, in some embodiments, the first drying time is 1h, and the second drying The time of drying is 2h, and the time of three dryings is 6h. The inventor found through research that the temperature and time of drying and curing are one of the key factors that determine whether the microneedle can be successfully prepared. If the drying time is insufficient or the drying temperature is too low, the polymerization of the microneedle material will be incomplete, the microneedle will be softened, and the hardness will be insufficient to meet the requirements for use. If the drying temperature is too high, bubbles will appear in the microneedle body, and the microneedle base will warp, resulting in the shape of the microneedle body failing to meet the requirements for use. The conditions provided by the present invention are used for drying, the conditions are relatively mild, the properties of the microneedle material are not damaged, and the curing effect is good, the microneedles have high hardness, no bubbles are generated, and no warping occurs. , can better meet the requirements of use.

Preferably, the method for preparing the mold comprises: using the metal microneedle body as a male mold to prepare a polydimethylsiloxane (PDMS) female mold.

Preferably, the preparation steps of the PDMS negative mold include: (1) mixing the uncrosslinked and cured PDMS prepolymer and the curing agent in 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.

The present invention also provides a composite microneedle for biological analysis and sampling, which is prepared by the above-mentioned preparation method of the composite microneedle for biological analysis and sampling.

Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention prepares microneedles by compounding polyvinyl alcohol and insoluble chitosan, and by optimizing and improving the preparation method and its parameter conditions, the antibacterial properties of the microneedles are effectively improved. performance, mechanical properties, penetration performance and absorption performance, and make the needle tip of the microneedle upright and straight, the needle body is complete and smooth, without hollowing, air bubbles, warping, etc., and has a good subcutaneous sampling effect. good application prospects.

Description of drawings

1 is a microscope image of the microneedles prepared in Example 1 and Comparative Example 1;

2 is a microscope image of the microneedles prepared in Example 1 and Comparative Examples 2-3;

Figure 3 is a microscope image of the microneedles prepared in Comparative Examples 4-5;

4 is a graph showing the swelling degree curves of the microneedles prepared in Examples 1 to 3 and Comparative Example 6;

Fig. 5 is the absorption curve diagram of FITC of microneedle prepared in Examples 1-3 and Comparative Example 6;

6 is a microscope image of the microneedles prepared in Examples 1 to 3 and Comparative Example 6 after being subjected to a pressure test;

7 is a diagram showing the penetration effect of micro-acupuncture on pigskin prepared by Examples 1 to 3 and Comparative Example 6;

Fig. 8 is the antibacterial effect diagram of micro-against Escherichia coli prepared by comparative example 6 and comparative example 7;

FIG. 9 is a graph showing the antibacterial effect of micro-against Staphylococcus aureus prepared in Comparative Example 6 and Comparative Example 7. FIG.

Detailed ways

The invention provides a preparation method of a composite microneedle for biological analysis and sampling. The steps are as follows:

(1) Preparation of mixed solution

Dissolve the insoluble chitosan powder in glacial acetic acid and ultrapure water to obtain a transparent and clear viscous liquid; wherein, based on the volume of ultrapure water, the addition amount of glacial acetic acid is 5-15 μL/mL (preferably 10 μL). /mL);

The viscous liquid is put into a dialysis bag, and then put into ultrapure water for dialysis, so that the glacial acetic acid is completely penetrated to obtain a chitosan solution;

Dissolving trehalose powder in water to obtain trehalose solution;

According to the mass ratio of trehalose:insoluble chitosan=1:10, the chitosan solution and the trehalose solution were mixed uniformly to obtain a clear colorless solution, which was concentrated to obtain a solution with an insoluble chitosan concentration of 8wt% mixture.

(2) Prepare the mold

The commercial metal microneedle body is used as the male mold;

Mix the uncrosslinked and cured PDMS prepolymer and the curing agent in a mass ratio of 10:1 to obtain a PDMS mixed solution;

The PDMS mixed solution is placed in a vacuum desiccator to remove air bubbles;

The male mold was cast with the PDMS mixed solution from which air bubbles were removed, and was dried and cured at 90° C. for 1 h to obtain the PDMS negative mold, which was the mold used for preparing the microneedles.

(3) Preparation of PVA solution

PVA was added to ultrapure water, heated in a water bath until completely dissolved, and a PVA solution with a concentration of 20 wt% was obtained.

(4) Preparation of composite solution

The mixed solution obtained in step (1) and the PVA solution obtained in step (3) are uniformly mixed in a volume ratio of 4:1 to remove air bubbles to obtain a composite solution.

(5) Preparation of composite microneedles

The composite solution obtained in step (4) was filled into the mold obtained in step (2), put into an oven for drying once, and dried at 40° C. for 1 h, and then the composite solution was added to the mold to make the composite solution. The grooves of the mold were filled up and dried for a second time at 40°C for 2 hours. Then, PVPK60 with a mass fraction of 50% was added to the mold to fill the mold grooves. As the base of the microneedles, three times of drying were performed. , dried at 40° C. for 6 hours, and demolded to obtain the composite microneedles.

In order to make the above objects, features and advantages of the present invention more obvious and easy to understand, the following specific embodiments are used for detailed description. Obviously, the following embodiments are only some, but not all, embodiments of the present invention. It should be understood that the embodiments of the present invention are only used to illustrate the technical effects of the present invention, but are not used to limit the protection scope of the present invention. Unless otherwise specified, the methods used in the examples of the present invention are all conventional methods in the art, and the used raw materials, instruments and equipment can be obtained through commercial channels. Among them, the curing agent is Dow Corning 184 glue (SYLGARD 184 PDMS).

旋转蒸发仪，郑州长城工贸有限公司；Centrifuge 5810R离心机，德国Eppendorf公司；Nikon ECLIPSE NI科研级正置荧光显微镜，日本Nikon公司；透析袋，春博办公文具专营店；DHG-9245A电热恒温鼓风干燥机，上海一恒科技仪器有限公司；欧诺Honour恒温振荡器，天津欧诺仪器股份有限公司；96孔板，康宁公司；Researchplus移液枪，德国Eppendorf公司；Sartorius BSA224S分析天平，赛多利斯科学仪器(北京)有限公司；YH-C电子天平，瑞安英衡有限公司；EPED-E2-20TS实验室级超纯水仪，南京易普达科技发展有限公司；VGT-1990QTD超声波清洗仪，苏州江东精密仪器有限公司；标准款手持式放大镜，一眸光学店；laboratory apparatus:

Rotary evaporator, Zhengzhou Great Wall Industry and Trade Co., Ltd.; Centrifuge 5810R, Eppendorf, Germany; Nikon ECLIPSE NI scientific-grade upright fluorescence microscope, Nikon, Japan; dialysis bag, Chunbo office stationery franchise store; DHG-9245A electric heating constant temperature drum Air dryer, Shanghai Yiheng Technology Instrument Co., Ltd.; Honor Honour constant temperature oscillator, Tianjin Honor Instrument Co., Ltd.; 96-well plate, Corning Corporation; Researchplus pipette, Eppendorf, Germany; Sartorius BSA224S analytical balance, Sartorius Sri Lanka Scientific Instruments (Beijing) Co., Ltd.; YH-C electronic balance, Ruian Yingheng Co., Ltd.; EPED-E2-20TS laboratory-grade ultrapure water meter, Nanjing Yipuda Technology Development Co., Ltd.; VGT-1990QTD Ultrasonic Cleaner, Suzhou Jiangdong Precision Instrument Co., Ltd.; standard hand-held magnifying glass, one eye optical shop;

6538D-5)，美国ATCC生物生物标准品资源中心；大肠杆菌(

8739D-5)，美国ATCC生物生物标准品资源中心；胰酪大豆胨液体培养基，广东环凯微生物科技有限公司；LB肉汤(LB BROTH)，广东环凯微生物科技有限公司；LB琼脂(LB AGAR)，广东环凯微生物科技有限公司；胰酪大豆胨琼脂培养基，广东环凯微生物科技有限公司；技术琼脂粉(ACAR POWDER)，广东环凯微生物科技有限公司；聚二甲基硅氧烷(Polydimethylsiloxane，PDMS)，美国Dow Corning公司；超纯水(导电率18.2MΩ·cm^-1)实验室纯水机自制。Experimental reagents: water-soluble chitosan, Chengdu Aikeda Chemical Reagent Co., Ltd.; insoluble chitosan, Shanghai Maclean Biochemical Technology Co., Ltd.; glacial acetic acid, Shanghai Maclean Biochemical Technology Co., Ltd.; D-trehalose, anhydrous, Shanghai McLean Biochemical Technology Co., Ltd.; Polyvinylpyrrolidone (PVPK60), Chengdu Aikeda Chemical Reagent Co., Ltd.; Staphylococcus aureus (

6538D-5), American ATCC Biological Standards Resource Center; Escherichia coli (

8739D-5), American ATCC Biological Standards Resource Center; Tryptone Soy Peptone Liquid Medium, Guangdong Huankai Microbial Technology Co., Ltd.; LB Broth (LB BROTH), Guangdong Huankai Microbial Technology Co., Ltd.; LB Agar (LB AGAR), Guangdong Huankai Microbial Technology Co., Ltd; (Polydimethylsiloxane, PDMS), Dow Corning Company, USA; ultrapure water (conductivity 18.2MΩ·cm ⁻¹ ) laboratory water purifier made by ourselves.

Example 1

A preparation method of composite microneedles, the steps are as follows:

1. Preparation of mixed solution

Weigh 1.0 g of insoluble chitosan powder, add 50 mL of ultrapure water and 0.5 mL of glacial acetic acid successively, stir well until the solution is transparent and clear and viscous, pour it into the dialysis bag, and then use a clip to place the front and rear of the dialysis bag. Clamp the ends, soak in ultrapure water for 6 hours, make the glacial acetic acid penetrate completely, measure its pH, and then pour it into a round-bottomed flask. Weigh 0.1 g of trehalose powder and dissolve it in 1.5 mL of water to prepare a trehalose solution. According to the mass ratio of trehalose:insoluble chitosan=1:10, the trehalose solution and the acid-removed chitosan solution are mixed evenly to improve the fluidity of the chitosan solution and make it a uniform and clear colorless solution , using a rotary evaporator to concentrate it to obtain a mixed solution with an insoluble chitosan concentration of 8 wt %, which is sealed and stored at 4° C. for future use.

2. Preparation of the microneedle mold

Using commercial metal microneedles as the male mold, the PDMS female mold was prepared. The preparation process is as follows: the uncrosslinked and cured PDMS prepolymer and the curing agent are mixed at a mass ratio of 10:1; removed in a vacuum desiccator Air bubbles in the PDMS mixed solution; cast the PDMS mixed solution with the bubbles removed into a positive mold; place it in an oven at 90°C for 1 h for curing, and obtain a PDMS negative mold after curing.

3. Preparation of composite microneedles

Weigh 200 mg of PVA into three 10 mL centrifuge tubes, add 800 μL of ultrapure water, and in a water bath at 90° C. to dissolve completely to prepare a 20 wt % PVA solution. Mix the above-prepared mixed solution and PVA solution at a volume ratio of 4:1, stir in a 60°C water bath for 1 hour to make the solution fully miscible, and then use a centrifuge at 6000 r/min for 10 minutes to remove air bubbles until the solution is clear and no more bubbles to obtain a composite solution. Fill the composite solution into the mold (ie, the female mold), put it in an oven to dry at 40°C for 1 hour, then add the composite solution to the mold to fill the mold groove, dry at 40°C for 2 hours, and then add the composite solution to the mold. PVPK60 with a mass fraction of 50% was added to fill the grooves of the mold to serve as the base of the microneedles, and dried at 40° C. for 6 hours to make the microneedles completely formed and demolded to obtain the composite microneedles.

Example 2

A preparation method of composite microneedles, which differs from Example 1 only in the concentration of the PVA solution, and the other is the same as Example 1. The concentration of the PVA solution of Example 2 was 10 wt%.

Example 3

A preparation method of composite microneedles, which differs from Example 1 only in the concentration of the PVA solution, and the other is the same as Example 1. The concentration of the PVA solution of Example 3 was 30 wt%.

Comparative Example 1

The only difference between Comparative Example 1 and Example 1 is that in Comparative Example 1, when preparing the composite microneedle, the composite solution was not supplemented, but PVPK60 with a mass fraction of 50% was directly supplemented, and the others were the same as Example 1.

The specific operation of Comparative Example 1 is as follows:

After the composite solution and PDMS negative mold were prepared according to the method of Example 1, the composite solution was filled into the mold, put into an oven for drying at 40°C for 1 h, and then added PVPK60 with a mass fraction of 50% to the mold as a microneedle The base of the mold is filled, the mold groove is filled, and dried at 40 °C for 8 hours to make the microneedle completely formed, and then demolded to obtain the composite microneedle.

Comparative Example 2

The difference between Comparative Example 2 and Example 1 is only in that the drying time is different, and the total drying time of Comparative Example 2 is 5h (one-time drying for 1 h, secondary drying for 2 h, and third drying for 2 h).

Comparative Example 3

The difference between Comparative Example 3 and Example 1 is only that the drying temperature is different, and the drying temperature of Comparative Example 3 is 60°C.

Comparative Example 4

The difference between Comparative Example 4 and Example 1 is only the concentration of insoluble chitosan. In Comparative Example 4, the concentration of insoluble chitosan in the mixed solution used for preparing the microneedles was 6 wt%.

Comparative Example 5

The difference between Comparative Example 5 and Example 1 is only that the drying time is different, and the total drying time of Comparative Example 5 is 10h (1h drying for one time, 2h for secondary drying, and 7h for third drying).

Comparative Example 6

A preparation method of chitosan microneedles, the steps are as follows:

1. Preparation of mixed solution

Weigh 1.0 g of insoluble chitosan powder, add 50 mL of ultrapure water and 0.5 mL of glacial acetic acid successively, stir well until the solution is transparent and clear and viscous, pour it into the dialysis bag, and then use a clip to place the front and rear of the dialysis bag. Clamp the ends, soak in ultrapure water for 6 hours, make the glacial acetic acid penetrate completely, measure its pH, and then pour it into a round-bottomed flask. Weigh 0.1 g of trehalose powder and dissolve it in 1.5 mL of water to prepare a trehalose solution. According to the mass ratio of trehalose:insoluble chitosan=1:10, the trehalose solution and the acid-removed chitosan solution are mixed evenly to improve the fluidity of the chitosan solution and make it a uniform and clear colorless solution , using a rotary evaporator to concentrate it to obtain a mixed solution with an insoluble chitosan concentration of 8 wt %, which is sealed and stored at 4° C. for future use.

2. Preparation of the microneedle mold

Using commercial metal microneedles as the male mold, the PDMS female mold was prepared. The preparation process is as follows: the uncrosslinked and cured PDMS prepolymer and the curing agent are mixed at a mass ratio of 10:1; removed in a vacuum desiccator Air bubbles in the PDMS mixed solution; cast the PDMS mixed solution with the bubbles removed into a positive mold; place it in an oven at 90°C for 1 h for curing, and obtain a PDMS negative mold after curing.

3. Preparation of Microneedles

Fill the mixed solution into the mold (ie the female mold), put it in an oven to dry at 40°C for 1 min, then add the mixed solution to the mold to fill the mold groove, dry at 40°C for 1 hour, and then add the mixed solution to the mold. PVPK60 with a mass fraction of 50% was added to fill the mold grooves and used as the base of the microneedles, and dried at 60° C. for 4 hours to make the microneedles completely formed and demolded to obtain the microneedles. Since this comparative example only uses chitosan solution to prepare microneedles, and chitosan is easily concentrated and thinned after drying, the drying time is shortened. And because there is no polymerization reaction, even if the drying temperature is 60°C, there is no adverse effect on the performance of the chitosan microneedles.

Comparative Example 7

A preparation method of chitosan microneedles, the steps are as follows:

1. Preparation of mixed solution

Weigh 0.6 g of water-soluble chitosan powder into a 5 mL centrifuge tube, add 3 mL of ultrapure water, stir to dissolve it completely, and prepare a chitosan hydrogel with a concentration of 20 wt %.

2. Preparation of the microneedle mold

Using commercial metal microneedles as the male mold, the PDMS female mold was prepared. The preparation process is as follows: the uncrosslinked and cured PDMS prepolymer and the curing agent are mixed at a mass ratio of 10:1; removed in a vacuum desiccator Air bubbles in the PDMS mixed solution; cast the PDMS mixed solution with the bubbles removed into a positive mold; place it in an oven at 90°C for 1 h for curing, and obtain a PDMS negative mold after curing.

3. Preparation of Microneedles

Fill the chitosan hydrogel into the mold (ie, the female mold), put it in an oven to dry at 40°C for 1 min, and then add the chitosan hydrogel to the mold to fill the mold groove, and bake at 40°C. After drying for 1 h, PVPK60 with a mass fraction of 50% was added to the mold to fill the mold grooves to serve as the base of the microneedles. Dry the microneedles at 60°C for 4 hours to make the microneedles completely formed and demolded to obtain the microneedles.

Experimental test

1. Microscope observation

1. The composite microneedles prepared in Example 1 and Comparative Example 1 were observed under a microscope, and the results are shown in Figure 1 . In Fig. 1, picture A is the composite microneedle prepared in Comparative Example 1, and picture B is the composite microneedle prepared in Example 1. It can be seen from Fig. 1 that the composite microneedle of Example 1 has a complete shape, a smooth needle body, a straight needle tip, and no hollow, which meets the requirements. In Comparative Example 1, because the composite solution was not added, but PVPK60 was directly added as the base, the fabricated microneedle had a complete shape, but the inside of the needle body was hollow. In this case, the microneedle had poor mechanical properties and penetrating power Insufficient, the use effect is not ideal.

2. The composite microneedles prepared in Example 1 and Comparative Examples 2-5 were observed under a microscope, and the results were shown in Figures 2-3. In FIG. 2 , picture A is the composite microneedle prepared in Comparative Example 2, picture B is the composite microneedle prepared in Comparative Example 3, and picture C is the composite microneedle prepared in Example 1. It can be seen from Figure 2 that the composite microneedle of Example 1 has a complete shape, a smooth needle body, a straight needle tip, no hollow, no air bubbles, and no warping of the base, which meets the requirements. In Comparative Example 2, due to insufficient drying time, the microneedle polymerization was incomplete, softening occurred, and the hardness did not meet the requirements. In Comparative Example 3, because the drying temperature was too high, bubbles appeared inside the microneedle body, and the base was warped, and the shape of the needle body failed to meet the requirements. In FIG. 3 , the left picture is the composite microneedle prepared in Comparative Example 4, and the right picture is the composite microneedle prepared in Comparative Example 5. It can be seen from Fig. 3 that the concentration of insoluble chitosan in Comparative Example 4 is too low (6 wt%), resulting in the needle body of the microneedle being bent and not upright, especially the needle tip, which does not meet the requirements. In Comparative Example 5, because the drying and curing time is too long (10h), the needle body of the microneedle is not smooth and uneven. Therefore, using the drying and curing conditions of the present invention (drying at 40° C. for 9 h) can obtain better curing effect of the needle body, which is the best condition for preparing microneedles, and the condition is mild, even if the microneedle is loaded with thermal instability Sex drugs, etc., will not be damaged.

2. Absorption performance test

The microneedles prepared in Examples 1 to 3 and Comparative Example 6 were used to test the swelling properties, and the method was as follows:

First, weigh 1.5g of agarose into a conical flask, then add 100mL of ultrapure water, heat it with microwave to fully dissolve it, keep it in a water bath at 60°C to prevent coagulation, add 40μL of FITC mother solution while it's still hot, stir well to distribute it evenly. 5 mL of agarose solution was added to each of the four petri dishes, and allowed to cool at room temperature to solidify. Five of each of the microneedles produced in Examples 1 to 3 and Comparative Example 6 were taken and their initial weights were measured. Use tweezers to pierce the microneedle vertically into the solid agarose, time it, take out the microneedle at 1, 2, 3, 4, and 5 minutes, and immediately measure the mass of the microneedle after absorption and swelling, and use the following formula to calculate the microneedle swelling degree.

In the formula, W ₁ is the initial weight of the microneedle, and W ₂ is the weight of the microneedle after absorption and swelling.

The test results are shown in Figure 4. Pure CS represents the microneedle made in Comparative Example 6, 10% PVA-CS represents the microneedle made in Example 2, and 20% PVA-CS represents the microneedle made in Example 1. 30% PVA-CS represents the microneedles produced in Example 3 (same for other figures). It can be seen from Figure 4 that with the increase of PVA concentration, the swelling degree of the prepared CS/PVA composite microneedles showed an upward trend, but Example 3 with PVA concentration of 30wt% was compared with the implementation of PVA concentration of 20wt% On the contrary, the swelling degree of Example 1 decreased. The reason is: on the one hand, PVA is a water-soluble polymer compound, its molecular side chain has a large number of hydroxyl groups, and its hydrophilicity is excellent. Therefore, as the concentration of PVA increases, the swelling degree of CS/PVA composite microneedles will also increase accordingly; On the other hand, since the hydroxyl groups in PVA can form hydrogen bonds with CS, the increase of PVA content greatly increases the number of cohesion and entanglement points of hydrogen bonds, which is not conducive to swelling. Therefore, the final swelling degree of CS/PVA composite microneedles is determined by the above two factors. Therefore, the concentration of PVA is preferably 20 wt%.

3. Water-soluble drug adsorption capacity test

In order to explore the adsorption capacity of the microneedles prepared in Examples 1 to 3 and Comparative Example 6 for water-soluble drugs, the fluorescent dye FITC was evenly distributed in the agarose solution to simulate the absorption of water-soluble drugs by the microneedles in the subcutaneous tissue. First, add 5 mL of ultrapure water to each well of the prepared two twelve-well plates, and soak the weighed microneedles in water for 12 hours. The FITC content was detected at an emission wavelength of 520 nm.

The good absorption capacity of microneedle for water-soluble drugs can make the microneedle used to detect the drug concentration of subcutaneous tissue fluid. By preparing different concentrations of PVA-CS microneedle, 1.5% agarose solution is used to simulate subcutaneous tissue fluid, and a certain amount of FITC is used to simulate water-soluble Sex drugs, and their absorption is calculated by measuring their fluorescence values. The linear equation of FITC is: y=3.3287x+103.15, and the linear relationship of FITC is good in the concentration range of 3.8ng/mL～760ng/mL.

The results of the absorption of FITC by the microneedles under different PVA concentrations are shown in Figure 5. From the overall trend, with the increase of the PVA concentration in the microneedles, the absorption of FITC by the microneedles also gradually increased, indicating that in the chitosan After adding PVA to the solution, the absorption of water-soluble drugs can be improved. From the results, the 20% PVA-CS composite microneedle (ie the microneedle of Example 1) has the best absorption effect on FITC, and the pure chitosan microneedle (ie the microneedle of Comparative Example 6) has the worst absorption effect . Compared with the pure chitosan microneedle, the liquid absorption effect of the composite microneedle is significantly improved, and it can be used to detect the soluble drug concentration in the subcutaneous tissue fluid.

4. Mechanical performance test

Sufficient mechanical strength is very important for microneedles to penetrate the skin to create channels for drug delivery. First, place the prepared microneedle tip on a flat surface, pick up the weight with tweezers and place it vertically on the microneedle for 5 minutes, and observe the microneedle shape under a microscope. The microneedles prepared in Examples 1 to 3 and Comparative Example 6 were subjected to a pressure test for 5 minutes using a weight of 500 g, and then the morphology of the microneedles after the test was observed under a microscope. The results are shown in Figure 6. In Figure 6, Figure A shows the shape of the microneedle of Comparative Example 6 after the pressure test, Figure B shows the shape of the microneedle of Example 2 after the pressure test, and Figure C shows the shape of the microneedle of Example 1. The shape of the microneedle after the pressure test, and Figure D shows the shape of the microneedle of Example 3 after the pressure test. It can be seen from Figure 6 that the microneedle of Comparative Example 6 has the largest deformation degree, and with the increase of PVA concentration, the deformation degree of the microneedle gradually decreases, indicating that the composite of CS and PVA can improve the mechanical properties of the microneedle.

5. Evaluation of skin penetration in vitro

First, take the abdominal skin of the isolated domestic pig, wipe the skin surface with 75% alcohol, and then use filter paper to absorb the excess water on the skin surface. After it is slightly dry, press the microneedle with the thumb with an appropriate amount of force to pierce the surface of the skin without damage. Body pig skin and stay for 5 minutes, and immediately after taking out the microneedle, observe whether there is a pinhole matrix on the surface of the pierced skin. The results are shown in Figure 7. In Figure 7, Figure A is the penetration effect of the micro-acupuncture on pigskin of Comparative Example 6, Figure B is the penetration effect of the micro-acupuncture on pigskin of Example 2, and Figure C is the micro-acupuncture of Example 1. Penetration effect of pigskin, Figure D shows the penetration effect of microacupuncture on pigskin of Example 3. It can be seen from Fig. 7 that the four kinds of microneedles can successfully pierce the isolated pig skin and have fine pores.

6. Antibacterial test

Two bacterial strains were selected, namely Staphylococcus aureus and Escherichia coli, Staphylococcus aureus was a representative of Gram-positive bacteria, Escherichia coli was a representative of Gram-negative bacteria, and the bacteria were inhibited in vitro against Escherichia coli and Staphylococcus aureus respectively. activity studies. The antibacterial properties of insoluble chitosan microneedles (i.e., comparative example 6) and water-soluble chitosan microneedles (i.e., comparative example 7) were investigated respectively. Ring inoculated into the liquid medium, the isolated colonies of Staphylococcus aureus were inoculated into fresh tryptic soy liquid medium, and Escherichia coli was inoculated into fresh LB broth medium, and put into a constant temperature shaker (37 ° C, 180 r/ min) incubate overnight. Then use the culture medium to dilute the bacterial liquid by gradient, and measure it with a microplate reader when it grows to the early logarithmic stage (OD 600nm ~ 0.1-0.2), add water-soluble chitosan microneedles and insoluble chitosan microneedles, and A blank group without microneedles was cultured for comparison, placed in a constant temperature shaker for culture, and at 0h, 1h, 3h, 5h, 7h, 9h, 12h, 24h, 36h, its OD ₆₀₀ value was measured with a microplate reader. Do three groups in parallel, and make a line chart for comparative analysis.

The test results are shown in Figures 8-9, Figure 8 shows the antibacterial properties against Escherichia coli, and Figure 9 shows the antibacterial properties against Staphylococcus aureus. It can be seen from the figure that both water-soluble chitosan and insoluble chitosan have bacteriostatic effect on Escherichia coli and Staphylococcus aureus, and the inhibitory effect of insoluble chitosan micro-against the two bacteria is particularly significant.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. A method for preparing a composite microneedle for biological analysis sampling is characterized by comprising the following steps:

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 chitosan solution is prepared from insoluble chitosan; the concentration of the insoluble chitosan in the mixed solution is 8 wt%, and the addition amount of the trehalose is 10% of the mass of the insoluble chitosan;

the temperature of the primary drying, the secondary drying and the third drying is 40 ℃, and the total drying time is 9 h.

2. A method of preparing a composite microneedle for bioanalytical sampling according to claim 1, wherein the method of preparing the chitosan solution comprises:

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.

3. The method of manufacturing a composite microneedle for bioanalysis sampling according to claim 2, wherein the glacial acetic acid solution is a mixed solution of ultrapure water and glacial acetic acid, and the amount of glacial acetic acid added is 5 to 15 μ L/mL in terms of the volume of ultrapure water.

4. The method of manufacturing a composite microneedle for bioanalytical sampling according to claim 1, wherein the concentration of the polyvinyl alcohol solution is 20 wt%.

5. A method of preparing a composite microneedle for bioanalytical sampling according to claim 1, wherein the volume ratio of the mixed solution to the polyvinyl alcohol solution in the polymer composite solution is 4: 1.

6. A method of fabricating a composite microneedle for bioanalytical sampling according to claim 1, wherein the method of fabricating the mold comprises: preparing a polydimethylsiloxane female die by taking a metal microneedle body as a male die; the preparation method of the PDMS female mold comprises the following steps: (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.

7. A composite microneedle for biological analysis sampling, characterized by being prepared by the method for preparing a composite microneedle for biological analysis sampling according to any one of claims 1 to 6.

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