Hydrogel microneedle of biomimetic iron carrier and preparation method and application thereof

Abstract

The present application relates to the technical field of biological materials, and provides a hydrogel microneedle of a biomimetic iron carrier as well as a preparation method and application thereof, the preparation method comprising the following steps: 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; dialyzing the first mixed solution to remove the carboxyl activation reagent, and then drying to obtain a dry substance; stirring the dry substance in an acetic acid solution to obtain a second mixed solution; mixing and stirring the second mixed solution with a polyvinyl alcohol solution to form a third mixed solution; injecting the third mixed solution into a polydimethylsiloxane mold, vacuumizing and drying, and demolding to obtain the hydrogel microneedle. The present application combines the iron carrier or the biomimetic iron carrier with the carboxymethyl chitosan, and the iron carrier is attached to the inner wall of the microneedle, targets and adsorbs bacteria, and is extracted by the microneedle technology, so that the precise detection of subcutaneous bacteria or fungi is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomaterials technology, and in particular to a biomimetic iron carrier hydrogel microneedle, its preparation method, and its application. Background Technology

[0002] Subcutaneous bacterial testing is crucial for early detection of bacterial infections, assessment of infection severity, and evaluation of infection status. However, current testing techniques primarily rely on needle puncture and biopsy, which are highly invasive, potentially posing infection risks and causing significant pain and discomfort to patients. These methods also require specific operating environments and personnel, increasing testing costs and complexity. Furthermore, the sensitivity and specificity of these methods are limited, especially as they are susceptible to contamination during sample collection, leading to inaccurate results.

[0003] Existing epidermal detection technologies have limited ability to reflect deeper infections, making it difficult to meet clinical needs. Meanwhile, the iron-dependent nature of bacterial growth provides a potential target for detection, but current detection tools have not effectively utilized bacterial siderophore mechanisms to improve targeting and detection efficiency. Summary of the Invention

[0004] The purpose of this invention is to solve at least one technical problem in the background art and to provide a biomimetic iron carrier hydrogel microneedle, its preparation method and application.

[0005] To achieve the above objectives, the present invention provides a method for preparing biomimetic iron carrier hydrogel microneedles, comprising:

[0006] Dissolve carboxymethyl chitosan in water, add carboxyl activating reagent and stir, then add iron carrier or biomimetic iron carrier component and continue stirring to form the first mixed solution;

[0007] The first mixed solution was dialyzed to remove the carboxyl activating agent, and then dried to obtain the dried product;

[0008] The dried substance was 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 the third mixed solution;

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

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

[0012] According to one aspect of the invention, the siderophore is enterobacterin, salmonin, ferrochromium, pseudomonocytocin, or violetin;

[0013] The biomimetic siderophore is dopamine hydrochloride or deferroamine mesylate.

[0014] According to one aspect of the present invention, the carboxyl activating agent is N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, or phenylphosphonyl dioxide benzylimide.

[0015] According to one aspect of the 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 invention, the volume ratio of the glacial acetic acid solution to the polyvinyl alcohol solution in the third mixed solution is 1:4 to 1:5.

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

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

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

[0020] To achieve the above objectives, the present invention also provides an application of the above-mentioned biomimetic iron carrier hydrogel microneedles in the detection of bacteria and fungi.

[0021] According to the present invention, the hydrogel microneedles are made of carboxymethyl chitosan and polyvinyl alcohol. Polyvinyl alcohol is a hydrophilic, biocompatible, non-toxic, non-carcinogenic, and non-immunogenic inert polymer material with high compatibility with human tissues, and will not cause serious immune reactions or toxicity problems during use; it also has unique phase transition characteristics, hardening upon drying to increase mechanical strength, and softening upon absorbing water. Carboxymethyl chitosan also has good biocompatibility and antibacterial properties. The numerous 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 disrupt the regularity of the chitosan molecular chain, reducing its crystallinity. The combined effect of these two components achieves a tight bond between them and also helps improve the material's performance.

[0022] Iron is an essential nutrient for the growth and reproduction of pathogens. To meet their iron requirements, bacteria compete with host cells for iron resources by secreting siderophores, a mechanism that provides a potential target for anti-infection. Siderophores can be natural or synthetic, and are mainly classified into catecholamines, hydroxamic acids, phenolic esters, and carboxylic acids. Dopamine hydrochloride and deferoxamine, with their biomimetic structures mimicking the iron-binding sites of catecholamines and hydroxamic acids, can form complexes with iron ions, hindering bacterial iron acquisition and thus capturing bacteria. Therefore, this invention combines siderophores or biomimetic siderophores with carboxymethyl chitosan, attaching them to the inner wall of microneedles. The siderophores target and adsorb bacteria, and microneedle technology enables painless, non-invasive, and highly efficient extraction, achieving precise detection of subcutaneous bacteria or fungi.

[0023] This invention achieves non-invasive and highly efficient subcutaneous bacterial detection by integrating a biomimetic siderophore into a hydrogel microneedle. The microneedle can painlessly penetrate the skin, targeting and capturing bacteria, significantly improving detection sensitivity and specificity. Furthermore, it utilizes a composite material of carboxymethyl chitosan and polyvinyl alcohol to provide excellent biocompatibility and mechanical properties. This invention not only reduces patient discomfort but also lowers the risk of infection. Attached Figure Description

[0024] Figure 1 SEM image of the biomimetic iron carrier hydrogel microneedles obtained in Example 1 is shown schematically.

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

[0026] Figure 3 A graph illustrating the mechanical properties of the microneedles in Test Example 2;

[0027] Figure 4 , Figure 5 and Figure 6The plating plates schematically represent the targeted bacterial capture capabilities of the experimental group, control group, and blank group in Test Example 3, respectively.

[0028] Figure 7 and Figure 8 The images schematically represent the targeted bacterial capture capabilities of the skin of the animals in the experimental and control groups of Test Example 4. Detailed Implementation

[0029] The invention will now be discussed with reference to exemplary embodiments. It should be understood that the described embodiments are merely intended to enable those skilled in the art to better understand and thus implement the invention, and are not intended to imply any limitation on the scope of the invention.

[0030] As used herein, the term "comprising" and its variations are to be interpreted as open-ended terms meaning "including but not limited to". The term "based on" is to be interpreted as "at least partially based 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 biomimetic iron carrier hydrogel microneedles includes:

[0032] Dissolve carboxymethyl chitosan (CS) in water, add carboxyl activating reagent and stir, then add iron carrier or biomimetic iron carrier component and continue stirring to graft the iron carrier or biomimetic iron carrier onto carboxymethyl chitosan to form the first mixed solution.

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

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

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

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

[0037] Furthermore, 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] Furthermore, according to one embodiment of the present invention, the siderophores are mainly classified into catecholamines (such as enterobacterin and salmonin), hydroxamic acids (such as ferrochromium), phenolic esters (such as pseudomonocytin), and carboxylic acids (such as piracetam), with catecholamines being preferred;

[0039] The biomimetic siderophore is dopamine hydrochloride (DA) or deferromethylamine methanesulfonate (DFO), with DA being preferred.

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

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

[0042] After adding the iron carrier or biomimetic iron carrier components, stir for 18-36 hours, preferably 24 hours;

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

[0044] Furthermore, 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 material is dissolved in 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 mixed and stirred glacial acetic acid solution to the polyvinyl alcohol (PVA) solution is 1:4-1:5, preferably 1:4. After adding the polyvinyl alcohol (PVA) solution and mixing, stir for 18-36 hours, preferably 24 hours.

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

[0047] Furthermore, according to one embodiment of the present invention, the vacuum degree of vacuuming 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 objectives, the present invention also provides a biomimetic siderophore hydrogel microneedle prepared according to the above-described method for preparing biomimetic siderophore hydrogel microneedles. The specific preparation method is as described above and will not be repeated here.

[0049] Furthermore, to achieve the above objectives, the present invention also provides an application of biomimetic siderophore hydrogel microneedles in the detection of bacteria and fungi. Application methods include:

[0050] A biomimetic iron carrier hydrogel microneedle patch was applied to the skin surface and pressed for a period of time. The microneedle patch was then removed and placed in a solution. The bacterial content was detected using the plate count method.

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

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

[0053] According to the above-described scheme of the present invention, the hydrogel microneedles of the present invention are made of carboxymethyl chitosan and polyvinyl alcohol. Polyvinyl alcohol is a hydrophilic, biocompatible, non-toxic, non-carcinogenic, and non-immunogenic inert polymer material with high compatibility with human tissues, and will not cause serious immune reactions or toxicity problems during use; it also has unique phase transition characteristics, namely, it hardens when dry, increasing its mechanical strength, and softens after absorbing water. Carboxymethyl chitosan also has good biocompatibility and antibacterial properties. The numerous 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 disrupt the regularity of the chitosan molecular chain, reducing its crystallinity. The combined effect of these two factors achieves a tight bond between the two and also helps to improve the material's performance.

[0054] Iron is an essential nutrient for the growth and reproduction of pathogens. To meet their iron requirements, bacteria compete with host cells for iron resources by secreting siderophores, a mechanism that provides a potential target for anti-infection. Siderophores can be natural or synthetic, and are mainly classified into catecholamines, hydroxamic acids, phenolic esters, and carboxylic acids. Dopamine hydrochloride and deferoxamine, with their biomimetic structures mimicking the iron-binding sites of catecholamines and hydroxamic acids, can form complexes with iron ions, hindering bacterial iron acquisition and thus capturing bacteria. Therefore, this invention combines siderophores or biomimetic siderophores with carboxymethyl chitosan, attaching them to the inner wall of microneedles. The siderophores target and adsorb bacteria, and microneedle technology enables painless, non-invasive, and highly efficient extraction, achieving precise detection of subcutaneous bacteria or fungi.

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0056] Example 1

[0057] The preparation method of biomimetic iron-supported hydrogel microneedles includes:

[0058] Preparation of chitosan solution: 700 mg of carboxymethyl chitosan (CS) was dissolved in 50 mL of deionized water to obtain 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) and 5 mL of 2% MES solution were added to the solution, and the mixture was stirred at room temperature (25±2℃) for 24 hours; then 5 mg of dopamine hydrochloride (DA) was added, and the mixture was stirred for another 24 hours.

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

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

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

[0062] Test Example 1

[0063] Infrared test:

[0064] The freeze-dried biomimetic siderophore loaded with chitosan solid was ground and subjected to infrared spectroscopy. The results showed that carboxymethyl chitosan molecules were successfully grafted with the biomimetic siderophore (dopamine hydrochloride). Specific spectral results are as follows: Figure 2 As shown.

[0065] Test Example 2

[0066] Microneedle mechanical property testing:

[0067] The mechanical properties of the hydrogel microneedles obtained in Example 1 were determined using a compression tester. Experimental results showed 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 curves are shown below. Figure 3 As shown.

[0068] Test Example 3

[0069] Validation of targeted bacterial capture capability:

[0070] (1) Experimental design: 5 μL of Staphylococcus aureus bacterial suspension with a concentration of OD600 = 1 was added to each well of a 96-well plate. The experiment was divided into a blank group (without microneedles), a control group (with microneedles without biomimetic iron carriers) and an experimental group (with hydrogel microneedles from Example 1). After incubation at 37°C for 24 hours, the bacterial suspension was removed, spread on plates, and the colony count was observed.

[0071] (2) Experimental Results: The number of bacterial colonies in the experimental group was significantly lower than that in the control group and the blank group, verifying the ability of the hydrogel microneedles obtained in Example 1 to target and capture bacteria. The experimental results are as follows: Figures 4-6 As shown.

[0072] Test Example 4

[0073] Validation of targeted bacterial capture on animal skin:

[0074] (1) Experimental design: After disinfecting a 2cm×2cm piece of pigskin, 10μL of Staphylococcus aureus bacterial suspension with an OD600 of 1 was injected. The experimental group was inserted with microneedles containing biomimetic siderophores obtained in Example 1, while the control group was inserted with blank microneedles without biomimetic siderophores. After incubation at 37℃ for 24 hours, the microneedles were transferred to NB liquid medium and incubated for another 24 hours. Subsequently, the colony count was observed by plating.

[0075] (2) Experimental Results: The results showed that the number of bacterial colonies in the experimental group was significantly higher than that in the control group, indicating that the microbeads containing biomimetic siderophores have a highly efficient ability to capture bacteria in the skin. The experimental results are as follows: Figure 7 and Figure 8 As shown.

[0076] Finally, it should be noted that the above embodiments are merely preferred embodiments, and are only used to illustrate the technical solutions of the present invention and not to limit it. 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A method for preparing biomimetic iron-supported hydrogel microneedles, characterized in that, include: Dissolve carboxymethyl chitosan in water, add carboxyl activating reagent and stir, then add iron carrier or biomimetic iron carrier component and continue stirring to form the first mixed solution; The first mixed solution was dialyzed to remove the carboxyl activating agent, and then dried to obtain the dried product; The dried substance was 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 the third mixed solution; The third mixed solution was injected into a polydimethylsiloxane mold, vacuumed and dried, and then demolded to obtain hydrogel microneedles.

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

3. The method for preparing biomimetic iron carrier hydrogel microneedles according to claim 1, characterized in that, The siderophore is enterobacterin, salmonin, ferrochromium, pseudomonocytocin, or violetin. The biomimetic siderophore is dopamine hydrochloride or deferroamine mesylate.

4. The method for preparing the biomimetic iron carrier hydrogel microneedles according to claim 1, characterized in that, The carboxyl activating agent is N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, or phenylphosphonodioxide benzylimide.

5. The method for preparing biomimetic iron carrier hydrogel microneedles 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 biomimetic iron carrier hydrogel microneedles 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 to 1:

5.

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

8. The method for preparing hydrogel microneedles with biomimetic iron carriers according to any one of claims 1-7, characterized in that, The vacuum degree during the evacuation and drying process is -0.09 to -0.1 MPa.

9. The biomimetic siderophore hydrogel microneedles prepared by the method of any one of claims 1-8.

10. The application of the biomimetic siderophore hydrogel microneedles according to claim 9 in the detection of bacteria and fungi.

Images