Hydrogel microneedle of bionic siderophore as well as preparation method and application of hydrogel microneedle

Abstract

The invention relates to the technical field of biological materials, and provides a bionic siderophore hydrogel microneedle and a preparation method and application thereof.The preparation method comprises the steps that carboxymethyl chitosan is dissolved in water, a carboxyl activating reagent is added and stirred, siderophore or bionic siderophore components are added, stirring continues, and a first mixed solution is formed; dialyzing the first mixed solution to remove the carboxyl activation reagent, and then drying to obtain a dried product; dissolving the dried substance in a glacial acetic acid solution, and stirring to obtain a second mixed solution; mixing and stirring the second mixed solution and a polyvinyl alcohol solution to form a third mixed solution; and injecting the third mixed solution into a polydimethylsiloxane mold, vacuumizing, drying, and demolding to obtain the hydrogel microneedle. A siderophore or a bionic siderophore is combined with carboxymethyl chitosan and attached to the inner wall of the microneedle, bacteria are captured and adsorbed in a targeted mode through the siderophore, painless and noninvasive extraction is achieved with the help of the microneedle technology, and therefore precise detection of subcutaneous bacteria or fungi is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a bionic iron carrier hydrogel microneedle and a preparation method and application thereof. Background Art

[0002] Subcutaneous bacterial detection is of great significance for early detection of bacterial infection, judgment of infection severity, and assessment of infection status. However, current detection technologies mainly rely on acupuncture and biopsy, which are highly invasive and may not only lead to infection risks, but also cause significant pain and discomfort to patients. At the same time, they have high requirements for the operating environment and personnel, increasing the cost and complexity of detection. In addition, the sensitivity and specificity of these methods are also limited, especially during the sample collection process, which is prone to contamination, resulting in inaccurate test results.

[0003] Existing epidermal detection technologies have a weak ability to reflect deep infections and are difficult to meet clinical needs. At the same time, the dependence of bacterial growth on iron provides a potential target for detection, but current detection tools have not effectively utilized the bacterial iron carrier mechanism to improve targeting and detection efficiency. Summary of the invention

[0004] The purpose of the present invention is to solve at least one technical problem in the background technology and provide a bionic iron carrier hydrogel microneedle and a preparation method and application thereof.

[0005] To achieve the above object, the present invention provides a method for preparing a biomimetic siderophore hydrogel microneedle, comprising:

[0006] Dissolving carboxymethyl chitosan in water, adding a carboxyl activation reagent and stirring, then adding an iron carrier or a biomimetic iron carrier component and continuing to stir to form a first mixed solution;

[0007] The first mixed solution is dialyzed to remove the carboxyl activation reagent, and then dried to obtain a dried product;

[0008] The dried product is dissolved in glacial acetic acid solution and stirred to obtain a second mixed solution;

[0009] The second mixed solution is mixed with the polyvinyl alcohol solution and stirred to form a third mixed solution;

[0010] The third mixed solution is injected into a polydimethylsiloxane mold, vacuumed and dried, and demolded to obtain a hydrogel microneedle.

[0011] According to one aspect of the present invention, the concentration of the aqueous solution of carboxymethyl chitosan is 12.00-20.00 mg / mL.

[0012] According to one aspect of the present invention, the siderophore is enterobactin, salmonellin, ferrochrome, pseudomonadin or circin;

[0013] The bionic iron carrier is dopamine hydrochloride or deferoxamine mesylate.

[0014] According to one aspect of the present invention, the carboxyl activation reagent is N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole or phenylphosphonodioxide benzyl imide.

[0015] According to one aspect of the present invention, the concentration of the glacial acetic acid solution is 0.08-0.16 mol / L, and the concentration of the polyvinyl alcohol solution is 150-250 mg / mL.

[0016] According to one aspect of the present invention, the volume ratio of the glacial acetic acid solution to the polyvinyl alcohol solution in the third mixed solution is 1:4-1:5.

[0017] According to one aspect of the present invention, the polydimethylsiloxane mold is a mold made of polydimethylsiloxane, and the parameters of the mold are: aperture area of ​​2cm×2cm, 1.5cm×1.5cm or 1cm×1cm, thickness of 5mm, 3mm or 2mm, microneedle array parameters: needle length of 800μm, 600μm or 300μm, needle bottom diameter of 500μm, 300μm or 100μm, surface roughness Ra≤0.5μm, Shore A hardness of 50-60.

[0018] According to one aspect of the present invention, the vacuum degree of the evacuation and drying is -0.09 to -0.1 MPa.

[0019] To achieve the above object, the present invention also provides a biomimetic siderophore hydrogel microneedle prepared according to the above method for preparing a biomimetic siderophore hydrogel microneedle.

[0020] To achieve the above object, the present invention also provides an application of the hydrogel microneedle of the above bionic iron carrier in bacteria and fungi detection.

[0021] According to the scheme of the present invention, the hydrogel microneedle of the present invention is made of carboxymethyl chitosan and polyvinyl alcohol. Among them, polyvinyl alcohol is an inert polymer material that is hydrophilic, biocompatible, non-toxic, non-carcinogenic, and non-immunogenic. It has high compatibility with human tissues and will not cause serious immune reactions or toxicity problems when used; and it has unique phase change characteristics, that is, it hardens when dry, which enhances the mechanical strength, and softens after absorbing water. Carboxymethyl chitosan also has good biocompatibility and antibacterial properties. The large number of hydroxyl groups on the polyvinyl alcohol molecular chain can form hydrogen bonds with the amino and hydroxyl groups in the carboxymethyl chitosan molecules. Polyvinyl alcohol can also destroy the regularity of the chitosan molecular chain and reduce its crystallinity. The two work together to achieve a close combination of the two, and also help to improve the performance of the material.

[0022] Iron is an essential nutrient for the growth and reproduction of pathogens. To meet the acquisition of iron, bacteria compete with host cells for iron resources by secreting iron carriers, and this mechanism provides a potential target for anti-infection. Iron carriers can be natural iron carriers and synthetic iron carriers, and iron carriers are mainly divided into catecholamines, hydroxamic acids, phenolic acid esters, and carboxylic acids. Among them, dopamine hydrochloride and deferoxamine structures are biomimetic of catecholamines and hydroxamic acids and iron action sites, which can form complexes with iron ions, hinder bacteria from obtaining iron, and thus can capture bacteria. Therefore, the present invention combines iron carriers or biomimetic iron carriers with carboxymethyl chitosan, attaches to the inner wall of microneedles, uses iron carriers to target and capture and adsorb bacteria, and then uses microneedle technology to achieve painless, non-invasive and efficient extraction, so as to achieve the purpose of accurate detection of subcutaneous bacteria or fungi.

[0023] The present invention achieves non-invasive and efficient subcutaneous bacterial detection by integrating biomimetic iron carriers into hydrogel microneedles. The microneedles can painlessly penetrate the skin, target and capture bacteria, significantly improve detection sensitivity and specificity, and use composite materials such as carboxymethyl chitosan and polyvinyl alcohol to provide excellent biocompatibility and mechanical properties. This invention not only reduces the discomfort of patients, but also reduces the risk of infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematically showing the SEM image of the hydrogel microneedle of the biomimetic siderophore obtained in Example 1;

[0025] Figure 2 The infrared spectrum of the chitosan with dopamine hydrochloride attached in Test Example 1 is schematically shown;

[0026] Figure 3 A graph schematically showing the mechanical properties of the microneedle of Test Example 2;

[0027] Figure 4 , Figure 5 and Figure 6The coated plate diagrams schematically represent the targeted bacterial capture capabilities of the experimental group, control group and blank group of Test Example 3 respectively;

[0028] Figure 7 and Figure 8 The coated plate images schematically show the targeted capture capability of bacteria on the skin of animals in the experimental group and the control group of Test Example 4. DETAILED DESCRIPTION

[0029] The content of the present invention will now be discussed with reference to exemplary embodiments. It should be understood that the embodiments discussed are only to enable those skilled in the art to better understand and thus implement the content of the present invention, rather than implying any limitation on the scope of the present invention.

[0030] As used herein, the term “including” and variations thereof are to be interpreted as open-ended terms meaning “including, but not limited to.” The term “based on” is to be interpreted as “based, at least in part, on.” The terms “one embodiment” and “an embodiment” are to be interpreted as “at least one embodiment.”

[0031] According to one embodiment of the present invention, a method for preparing a biomimetic siderophore hydrogel microneedle comprises:

[0032] Dissolving carboxymethyl chitosan (CS) in water, adding a carboxyl activation reagent and stirring, then adding an iron carrier or a biomimetic iron carrier component, and continuing to stir to graft the iron carrier or the biomimetic iron carrier onto the carboxymethyl chitosan to form a first mixed solution;

[0033] The first mixed solution is dialyzed to remove the carboxyl activation reagent, and then dried (lyophilized or oven-dried, preferably lyophilized) to obtain a dried product;

[0034] The dried product is dissolved in glacial acetic acid solution and stirred to obtain a second mixed solution;

[0035] The second mixed solution is mixed with a polyvinyl alcohol (PVA) solution and stirred to form a third mixed solution;

[0036] The third mixed solution is injected into a polydimethylsiloxane mold, vacuumed and dried, and demolded to obtain a hydrogel microneedle.

[0037] Further, according to one embodiment of the present invention, the concentration of the aqueous solution of carboxymethyl chitosan is 12.00-20.00 mg / mL, preferably 14.00 mg / mL.

[0038] Further, according to one embodiment of the present invention, the iron carrier is mainly divided into catecholamines (such as enterobactin, salmonellin), hydroxamic acids (such as ferrochrome), phenolic acid esters (such as pseudomonadin), carboxylic acids (such as cyclobactin), preferably catecholamines;

[0039] The biomimetic iron carrier is dopamine hydrochloride (DA) or deferoxamine mesylate (DFO), preferably DA.

[0040] Further, according to one embodiment of the present invention, the carboxyl activation reagent is N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) or N-hydroxysuccinimide (NHS) or N,N'-dicyclohexylcarbodiimide (DCC) or 1-hydroxybenzotriazole (HOBt) or phenylphosphonyl dioxybenzyl imide (BOP), preferably EDC and NHS.

[0041] Further, according to one embodiment of the present invention, the time of adding the carboxyl activator and stirring is 18-36 hours, preferably 24 hours;

[0042] After adding the siderophore or biomimetic siderophore component, stirring for 18-36 hours, preferably 24 hours;

[0043] The dialysis time is 18-48 hours, preferably 48 hours.

[0044] Further, according to one embodiment of the present invention, the concentration of the glacial acetic acid solution is 0.08-0.16 mol / L, preferably 0.1 mol / L, and the dried product is dissolved in the glacial acetic acid and stirred for 18-36 hours, preferably 24 hours;

[0045] The concentration of the polyvinyl alcohol (PVA) solution is 150-250 mg / mL, preferably 200 mg / mL. The volume ratio of the glacial acetic acid solution to the polyvinyl alcohol (PVA) solution after mixing and stirring is 1:4-1:5, preferably 1:4. The polyvinyl alcohol (PVA) solution is added and mixed and stirred for 18-36 hours, preferably 24 hours.

[0046] Further, according to one embodiment of the present invention, the polydimethylsiloxane (PDMS) mold is a polydimethylsiloxane mold prepared by a template casting method, and the parameters of the mold are: aperture area of ​​2cm×2cm, 1.5cm×1.5cm or 1cm×1cm, thickness of 5mm, 3mm or 2mm, microneedle array parameters: needle length of 800μm, 600μm or 300μm, needle bottom diameter of 500μm, 300μm or 100μm, surface roughness Ra≤0.5μm, Shore A hardness of 50-60. Preferred mold parameters are: aperture area of ​​1.5cm×1.5cm, thickness of 3mm, microneedle array parameters: needle length of 600μm, needle bottom diameter of 300μm, surface roughness Ra=0.5μm, Shore A hardness of 50.

[0047] Further, according to one embodiment of the present invention, the vacuum degree of evacuation and drying is -0.09 to -0.1 MPa, preferably -0.1 MPa; the vacuum drying time is 18-36 hours, preferably 24 hours.

[0048] Furthermore, to achieve the above object, the present invention also provides a biomimetic siderophore hydrogel microneedle prepared according to the above method for preparing a biomimetic siderophore hydrogel microneedle. The specific preparation method is as described above and will not be repeated here.

[0049] Furthermore, to achieve the above-mentioned purpose, the present invention also provides an application of a biomimetic iron carrier hydrogel microneedle in the detection of bacteria and fungi. The application method is, for example:

[0050] The hydrogel microneedle patch of the bionic iron carrier is applied to the skin surface. After pressing for a period of time, the microneedle patch is removed and placed in a solution. The bacterial content is detected using the spread plate counting method.

[0051] The size of the microneedle patch is 1 cm×1 cm, 1.5 cm×1.5 cm, or 2 cm×2 cm, preferably 1 cm×1 cm.

[0052] The pressing time is 10-60 seconds, preferably 20-30 seconds.

[0053] According to the above scheme of the present invention, the hydrogel microneedle of the present invention is made of carboxymethyl chitosan and polyvinyl alcohol. Among them, polyvinyl alcohol is an inert polymer material that is hydrophilic, biocompatible, non-toxic, non-carcinogenic, and non-immunogenic. It has high compatibility with human tissues and will not cause serious immune reactions or toxicity problems when used; and it has unique phase change characteristics, that is, it hardens when dry, which enhances the mechanical strength, and softens after absorbing water. Carboxymethyl chitosan also has good biocompatibility and antibacterial properties. The large number of hydroxyl groups on the polyvinyl alcohol molecular chain can form hydrogen bonds with the amino and hydroxyl groups in the carboxymethyl chitosan molecules. Polyvinyl alcohol can also destroy the regularity of the chitosan molecular chain and reduce its crystallinity. The two work together to achieve a close combination of the two, and also help to improve the performance of the material.

[0054] Iron is an essential nutrient for the growth and reproduction of pathogens. To meet the acquisition of iron, bacteria compete with host cells for iron resources by secreting iron carriers, and this mechanism provides a potential target for anti-infection. Iron carriers can be natural iron carriers and synthetic iron carriers, and iron carriers are mainly divided into catecholamines, hydroxamic acids, phenolic acid esters, and carboxylic acids. Among them, dopamine hydrochloride and deferoxamine structures are biomimetic of catecholamines and hydroxamic acids and iron action sites, which can form complexes with iron ions, hinder bacteria from obtaining iron, and thus can capture bacteria. Therefore, the present invention combines iron carriers or biomimetic iron carriers with carboxymethyl chitosan, attaches to the inner wall of microneedles, uses iron carriers to target and capture and adsorb bacteria, and then uses microneedle technology to achieve painless, non-invasive and efficient extraction, so as to achieve the purpose of accurate detection of subcutaneous bacteria or fungi.

[0055] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail in the form of specific embodiments in conjunction with the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0056] Example 1

[0057] A method for preparing a hydrogel microneedle of a biomimetic iron carrier comprises:

[0058] Preparation of chitosan solution: 700 mg of carboxymethyl chitosan (CS) was dissolved in 50 mL of deionized water to prepare a solution with a concentration of 14.00 mg / mL; 0.07 g of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 0.07 g of N-hydroxysuccinimide (NHS), as well as 5 mL of 2% MES solution were added to the solution and stirred at room temperature (25±2°C) for 24 hours; then 5 mg of dopamine hydrochloride (DA) was added and stirring was continued for 24 hours;

[0059] Freeze drying and dialysis: The above solution was put into a 3500 kDa dialysis bag, placed in a 1000 mL beaker, and dialyzed for 48 hours with 800 mL of ultrapure water to remove the activation reagent. The water was replaced every 3 hours during the dialysis. After the dialysis was completed, the obtained solution was freeze dried for 24 hours to obtain a chitosan solid with a biomimetic iron carrier.

[0060] Preparation of microneedle preparation solution: Weigh 0.10 g of freeze-dried chitosan solid, dissolve in 5 mL of 0.1 mol / L glacial acetic acid solution, stir for 24 hours to obtain a uniform solution; mix this solution with 20 mL of 200 mg / mL polyvinyl alcohol (PVA) solution at a volume ratio of 1:4, continue stirring for 24 hours to obtain a microneedle preparation solution;

[0061] Microneedle molding: The prepared solution was injected into a PDMS (polydimethylsiloxane) mold, placed under vacuum conditions (-0.1 MPa) to extract bubbles and dried for 24 hours; after demolding, hydrogel microneedles were obtained, such as Figure 1 shown.

[0062] Test Example 1

[0063] Infrared test:

[0064] The freeze-dried biomimetic iron carrier loaded chitosan solid was ground and tested by infrared spectroscopy. The results showed that the carboxymethyl chitosan molecules were successfully grafted with the biomimetic iron carrier (dopamine hydrochloride). The specific spectral results are as follows Figure 2 shown.

[0065] Test Example 2

[0066] Microneedle mechanical properties test:

[0067] The mechanical properties of the hydrogel microneedles obtained in Example 1 were measured using a compression tester. The experimental results show that under a force of 0.2 N, the microneedles deformed but did not break, and could be stably inserted into the simulated skin surface. The mechanical property curve is shown in FIG. Figure 3 shown.

[0068] Test Example 3

[0069] Targeted capture of bacteria capability verification:

[0070] (1) Experimental design: 5 μL of Staphylococcus aureus bacterial solution with a concentration of OD600 = 1 was added to a 96-well plate. The experiment was divided into a blank group (no microneedles), a control group (microneedles without biomimetic iron carriers) and an experimental group (hydrogel microneedles of Example 1) were placed; after culturing at 37°C for 24 hours, the bacterial solution was taken out and spread on the plate to observe the number of colonies.

[0071] (2) Experimental results: The number of colonies in the experimental group was significantly less than that in the control group and the blank group, which verified the ability of the hydrogel microneedles obtained in Example 1 to target and capture bacteria. Figure 4-Figure 6 shown.

[0072] Test Example 4

[0073] Targeted capture of bacteria on animal skin:

[0074] (1) Experimental design: After disinfecting a 2 cm × 2 cm pig skin, 10 μL of Staphylococcus aureus liquid with an OD600 concentration of 1 was injected. The experimental group was inserted with the microneedles containing the biomimetic siderophore obtained in Example 1, and the control group was inserted with blank microneedles without biomimetic siderophore; after culturing at 37°C for 24 hours, the microneedles were transferred to NB liquid culture medium and cultured for another 24 hours, and then the plate was coated to observe the number of colonies.

[0075] (2) Experimental results: The results showed that the number of colonies in the experimental group was significantly higher than that in the control group, indicating that the microneedles containing biomimetic iron carriers have a high efficiency in capturing bacteria in the skin. Figure 7 and Figure 8 shown.

[0076] Finally, it should be noted that the above embodiments are only preferred embodiments, which are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing a biomimetic siderophore hydrogel microneedle, characterized in that: include: Dissolving carboxymethyl chitosan in water, adding a carboxyl activation reagent and stirring, then adding an iron carrier or a biomimetic iron carrier component and continuing to stir to form a first mixed solution; The first mixed solution is dialyzed to remove the carboxyl activation reagent, and then dried to obtain a dried product; The dried product is dissolved in glacial acetic acid solution and stirred to obtain a second mixed solution; The second mixed solution is mixed with the polyvinyl alcohol solution and stirred to form a third mixed solution; The third mixed solution is injected into a polydimethylsiloxane mold, vacuumed and dried, and demolded to obtain a hydrogel microneedle.

2. The method for preparing the biomimetic siderophore hydrogel microneedle according to claim 1, characterized in that: The concentration of the aqueous solution of carboxymethyl chitosan is 12.00-20.00 mg / mL.

3. The method for preparing the biomimetic siderophore hydrogel microneedle according to claim 1, characterized in that: The iron carrier is enterobactin, salmonellin, ferrochrome, pseudomonadin or circin; The bionic iron carrier is dopamine hydrochloride or deferoxamine mesylate.

4. The method for preparing the biomimetic siderophore hydrogel microneedle according to claim 1, characterized in that: The carboxyl activation reagent is N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole or phenylphosphonyldioxide benzyl imide.

5. The method for preparing the hydrogel microneedle of the biomimetic siderophore according to claim 1, characterized in that: The concentration of the glacial acetic acid solution is 0.08-0.16 mol / L, and the concentration of the polyvinyl alcohol solution is 150-250 mg / mL.

6. The method for preparing the biomimetic siderophore hydrogel microneedle according to claim 1, characterized in that: The volume ratio of the glacial acetic acid solution to the polyvinyl alcohol solution in the third mixed solution is 1:4-1:

5.

7. The method for preparing the biomimetic siderophore hydrogel microneedle according to claim 1, characterized in that: The polydimethylsiloxane mold is a mold made of polydimethylsiloxane, and the parameters of the mold are: aperture area of ​​2cm×2cm, 1.5cm×1.5cm or 1cm×1cm, thickness of 5mm, 3mm or 2mm, microneedle array parameters: needle length of 800μm, 600μm or 300μm, needle bottom diameter of 500μm, 300μm or 100μm, surface roughness Ra≤0.5μm, and Shore A hardness of 50-60.

8. The method for preparing the hydrogel microneedle of the biomimetic siderophore according to any one of claims 1 to 7, characterized in that: The vacuum degree of the evacuation and drying is -0.09 to -0.1Mpa. 9 . The biomimetic siderophore hydrogel microneedle prepared by the method for preparing the biomimetic siderophore hydrogel microneedle according to any one of claims 1 to 8 .

10. Use of the biomimetic iron carrier hydrogel microneedle according to claim 9 in bacteria and fungi detection.

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