Nanometer needling instrument and preparation method and application thereof

Abstract

The invention discloses a nanometer needling instrument and a preparation method and application thereof. The nanoneedle comprises an acupuncture needle with the surface modified with host molecules, and the host molecules comprise annular molecules capable of forming an inclusion compound structure. According to the acupuncture needle, the surface of the acupuncture needle is ingeniously modified to form annular molecules (such as cyclodextrin) of an inclusion compound structure, a cavity can be formed, the acupuncture needle is endowed with the capacity of loading and releasing functional molecules, and the dual effects of acupuncture and medicine delivery are effectively achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of acupuncture needles, and relates to a nano needle tool and a preparation method and application thereof. Background Art

[0002] Acupuncture is the general term for acupuncture and moxibustion. Acupuncture refers to inserting needles (usually filiform needles) into the patient's body at a certain angle under the guidance of traditional Chinese medicine theory, using acupuncture techniques such as twisting and lifting to stimulate specific parts of the human body to achieve the purpose of treating diseases. Needles are made of metal, and stainless steel is the most commonly used needle material. Stainless steel filiform needles have high strength and toughness, straight and smooth needle bodies, heat resistance and rust resistance, and are not easily corroded by chemicals, so they are widely used in clinical practice. There are also filiform needles made of other metals, such as gold needles and silver needles. Although their electrical conductivity and heat transfer properties are significantly better than stainless steel filiform needles, their needle bodies are thicker, and their strength and toughness are not as good as stainless steel needles. In addition, they are expensive and are generally rarely used in clinical practice. Acupoint injection is a method of injecting Chinese and Western medicines into relevant acupoints to treat diseases.

[0003] The improvement of needles is one of the hot topics in the field of acupuncture, including the development of needles that can combine acupuncture and acupoint injection. For example, CN111719113A discloses a surface modification method for acupuncture needles, including contacting the acupuncture needles with a nitrogen-containing compound in the presence of a supercritical fluid, so that the nitrogen element in the nitrogen-containing compound adheres to the surface of the acupuncture needles, so as to solve the problem that the needles made by the existing surface processing methods of acupuncture needles have a weak sense of Qi in patients; Feng Lin et al. used nanotechnology to combine baicalin into hydrogel, and then loaded the hydrogel onto special acupuncture needles, and achieved drug release by directly inserting the needles into the knee joint to reach the subchondral bone. However, it only uses the needle as a carrier to load and release drugs, and does not achieve the synergy of acupuncture and drug delivery (Feng Lin, et.al. Advanced Science, 2022, DOI: 10.1002 / advs.202200079); CN214859218U, CN210583110U and CN208598803U disclose sleeve acupuncture needle technology, which loads the medicinal liquid in the sleeve outside the needle, or at the end of the needle or inside the hollow needle. During the acupuncture process, the medicinal liquid flows along the needle into the acupuncture site, but the medicinal liquid usually remains on the skin surface and is difficult to enter the deep tissue.

[0004] In summary, how to develop effective needles that can perform acupuncture and drug delivery simultaneously is one of the problems that urgently need to be solved in the field of acupuncture treatment. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a nano needle and a preparation method and application thereof. The present invention designs a new acupuncture needle to achieve the dual effects of acupuncture and drug delivery.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a nanoneedle, comprising an acupuncture needle having a surface modified with a main molecule, wherein the main molecule comprises a cyclic molecule capable of forming an inclusion compound structure.

[0008] In the present invention, a cyclic molecule (such as cyclodextrin, etc.) capable of forming an inclusion complex structure is cleverly designed to modify the surface of acupuncture needles to form a cavity, thereby giving the acupuncture needles the ability to load and release functional molecules, effectively achieving the dual effects of acupuncture and drug delivery.

[0009] It can be understood that cyclic molecules and their derivatives that can form inclusion complexes are suitable for the present invention.

[0010] Preferably, the cyclic molecule capable of forming an inclusion complex structure includes any one of cyclodextrin, cyclodextrin derivatives, cucurbituril or cucurbituril derivatives, or a combination of at least two thereof.

[0011] Preferably, the cyclodextrin includes any one of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin, or a combination of at least two of them.

[0012] Preferably, the cyclodextrin derivative includes any one of SBE-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, β-cyclodextrin hydrate, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, acetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, triacetyl-γ-cyclodextrin, succinyl-α-cyclodextrin-β-cyclodextrin, carboxymethyl-β-cyclodextrin, heptyl (2,3,6-tri-O-methyl)-β-cyclodextrin or 6-monodeoxy-6-monoamino-β-cyclodextrin hydrochloride, or a combination of at least two thereof.

[0013] Preferably, the cucurbituril derivatives include any one of sugammadex sodium, hydrated cucurbituridine, etc., or a combination of at least two thereof.

[0014] It can be understood that acupuncture needles made of different materials (such as iron, gold, silver or alloys, etc.) in the art are all suitable for the present invention.

[0015] Preferably, the host molecule is connected to the surface of the acupuncture needle via a chemical linker or a chemical bond.

[0016] Preferably, the chemical linker comprises any one of phenol, phenolic derivatives or adhesive peptides, or a combination of at least two of them.

[0017] Preferably, the phenolic derivative includes any one of methyl salicylate, ethyl salicylate, methyl anthranilate or methyl cinnamate, or a combination of at least two thereof.

[0018] Preferably, the adhesion peptide includes Trp-Lys-Tyr-Met-Val-d-Met (WKYMVm) and the like.

[0019] In a second aspect, the present invention provides a method for preparing the nanoneedle according to the first aspect, the preparation method comprising:

[0020] The host molecule is connected to the surface of an acupuncture needle.

[0021] Preferably, the connection method comprises an electrochemical method or a spray method.

[0022] Preferably, the preparation method further comprises the step of modifying the main molecule with a chemical linker.

[0023] Preferably, the method for modifying the chemical linker specifically comprises:

[0024] The solution of the main molecule is mixed with the chemical linker and reacted to obtain the main molecule modified with the chemical linker.

[0025] In the present invention, different chemical synthesis methods can be used to complete the transesterification process of the main molecule and the linker, including fine-tuning of the reaction temperature (such as changing the reaction temperature to obtain a higher yield), adjustment of the reaction solvent (such as replacing it with another organic solvent), use of different purification methods, use of catalysts, etc.

[0026] Preferably, the method for preparing the nanoneedle comprises the following steps:

[0027] (1) mixing a solution of a host molecule with a chemical linker and reacting the mixture to obtain a host molecule modified with a chemical linker;

[0028] (2) Using a solution of a main molecule modified with a chemical linker as an electrolyte solution and an acupuncture needle as a working electrode, an electrochemical process is performed using an electrochemical workstation to obtain the nanoneedle.

[0029] Preferably, Ag / AgCl electrode (or other reference electrode such as saturated calomel electrode, etc.) is used as reference electrode, and Pt wire electrode (or other counter electrode such as graphite electrode, etc.) is used as counter electrode (cathode). Electrochemical process is carried out using electrochemical workstation (such as Chenhua electrochemical workstation, GAMRY electrochemical workstation, etc.). Scanning range is set to 0.6-1.0mV (range can be expanded and changed, different scanning ranges should be selected according to the reference electrode, such as Ag / AgCl electrode, 0.868mV should be included in the range), scanning rate can be set to 1mV / s-50mV / s, and the rate exceeding 50mV / s will affect the uniformity of electroplating process, and the number of cycles is at least 1 time, for example, the rate is set to 5mV / s, and circulated 10 times.

[0030] In a third aspect, the present invention provides a nanoneedle delivery system, comprising the nanoneedle described in the first aspect and a functional molecule, wherein the functional molecule is loaded on a main molecule of the nanoneedle.

[0031] Preferably, the functional molecules include any one or a combination of at least two of drugs (such as small molecule drugs, nucleic acid drugs), fluorescent probe molecules or small molecule proteins or drug combinations.

[0032] In a fourth aspect, the present invention provides a method for preparing the nanoneedle delivery system according to the third aspect, the method comprising:

[0033] The nanoneedle described in the first aspect is contacted with a solution of functional molecules.

[0034] In the present invention, the application scenarios of the nanoneedle include but are not limited to acupuncture injection. Small molecules that can be delivered by nanoneedles include: fluorescent probe molecules, FDA-approved small molecule drugs, small molecule drug combinations, nucleic acid molecules, single or multiple active products of traditional Chinese medicine, traditional Chinese medicine extracts, and other active substances that meet the loading conditions in the preclinical and clinical trial process.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) Advantages and uniqueness of the types, concentrations and release processes of loaded molecules. In the process of clinical drug administration (such as acupuncture injection), the preparation of the drug solution requires that the active drug molecules have good water solubility and stability, and the types of drugs that can be selected are limited. The covalently linked cyclic molecules (such as cyclodextrin, etc.) on the surface of the nanoneedle of the present invention can load loaded molecules with various solubilities (such as water-soluble, alcohol-soluble, organic reagent-soluble, etc.) and different dissolution states (such as suspensions, solutions, etc.). For this reason, similar needle modification techniques can be used to introduce specific cyclic molecules to meet the chemical structure characteristics of the drug to be loaded. In addition, the nanoneedle combines acupuncture and drug delivery processes into one, and has the advantages of a wide range of applicable drug molecules and their combinations, and controllable drug concentration in a confined space. In addition, the nanoneedle provides sustained release performance to a certain extent, avoiding the occurrence of acute toxicity, and the safety of drug administration can be fully guaranteed. The corresponding acupuncture injection has problems such as difficulty in unifying dosage and concentration;

[0037] (2) Advantages and uniqueness in clinical operation. Acupoint injection lacks corresponding medical devices and needs to be developed and researched. There is no standard for the selection of needles for acupoint injection. The present invention combines acupuncture technology and drug delivery for the first time, achieving the dual functions of acupuncture treatment and drug delivery in a controllable manner within the same treatment point and time period;

[0038] (3) Advantages and uniqueness in safety. During the clinical operation of acupuncture injection, incorrect acupuncture injection location may cause damage to surrounding tissues, and mistakes are prone to occur in the preparation, suction and injection of injection drugs. The nanoneedle device of the present invention can simultaneously administer drugs during acupuncture, avoiding mistakes in the preparation of drug solutions, avoiding repeated damage to acupuncture points and surrounding tissues, and ensuring operational safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the design of the nano needle of the present invention;

[0040] Figure 2 This is a schematic diagram of the chemical modification principle of the main molecule in Example 1;

[0041] Figure 3 Schematic diagram of the electrochemical process in Example 1;

[0042] Figure 4 This is a schematic diagram of the structure of the nanoneedle prepared in Example 2;

[0043] Figure 5 This is a schematic diagram of the structure of the nanoneedle prepared in Example 3;

[0044] Figure 6The figure is the result of in vitro verification of the loading of fluorescent molecules on nanoneedles in Example 4; taking fluorescent dyes as an example, the nanoneedle platform technology is used for loading small molecules; the fluorescent dyes from left to right are: Hoechst 33324, Fluorescein Isothiocyanate (FITC), DiOC18 (3) (Dio), DilC18 (3) (Dil) and DiIC18 (5) (DiD);

[0045] Figure 7 This is a graph showing the release of fluorescent molecules in mice after acupuncture injection using nano needles loaded with fluorescent molecules FITC in Example 4;

[0046] Figure 8 This is a step-by-step diagram of FITC fluorescence in tissue sections in Example 4. DETAILED DESCRIPTION

[0047] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0048] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0049] A key design feature of the nanoneedle of the present invention is the use of an inclusion complex structure for drug encapsulation, which requires chemical modification of the host molecule and subsequent electrochemical attachment of the host molecule to the needle surface ( Figure 1 ). The hydrophobic cavity of the main molecule, such as cyclodextrin, provides the possibility of loading drug molecules. As shown in Table 1, various cyclodextrin molecules with different internal spaces and sugar ring sizes are suitable for this design, allowing the loading of various effective drug molecules with different physicochemical properties. In order to prove the principle, the pharmaceutical excipient β-cyclodextrin (β-CD) was used as the main molecule in the specific embodiment of the present invention for research and demonstration. The cyclodextrin modified with methyl salicylate was connected by electrochemical means. The chemical modification principle diagram of the main molecule is shown in FIG. Figure 2 As shown, and used for subsequent loading of single or multiple drug molecules.

[0050] Table 1

[0051] Sugar ring Inner diameter(nm) <![CDATA[Binding stability constant K 1:1 > Sugar ring molecular weight The molecular weight range of the drug that can be loaded α-CD 0.50 130±8 972.84 200-550 β-CD 0.62 490±8 1134.98 200-700 γ-CD 0.80 350±9 1297.12 unknown HPβCD 0.62 1010 1400 300-700 SBEβCD 0.62 4560 2163 350-600

[0052] Example 1

[0053] This example prepares a nanoneedle.

[0054] (1) Chemical modification of the main molecule

[0055] Dissolve 11.3 g of β-cyclodextrin (CAS#7585-39-9, McLean) in a proper amount of a mixed solution of ethanol and water: 33.3 mL of ethanol and 66.7 mL of distilled water, and stir magnetically at 60°C for 1 hour to fully dissolve the main molecule in the solvent. Add 6 g of methyl salicylate to the fully dissolved solution containing the main molecule, and continue to stir the mixture to allow the ester exchange reaction to proceed. Stir at 40°C for 4 hours to reach a chemical equilibrium state, forming a main molecule-phenol derivative precipitate (β-cyclodextrin-methyl salicylate). The reaction mixture is allowed to stand at 4°C overnight to allow the precipitate to settle out.

[0056] (2) Purification of the main molecule-phenol derivative

[0057] The main molecule-phenol derivative (β-cyclodextrin-methyl salicylate) precipitate was collected by suction filtration and washed with anhydrous ethanol. Finally, the collected sediment was dried (dried in an oven at 50° C. for 4 hours).

[0058] (3) Electrochemical deposition of the main molecule-phenol derivative on the acupuncture needle

[0059] The main molecule obtained by chemical modification, phenol derivative (300 mg of β-cyclodextrin-methyl salicylate), was dissolved in an appropriate amount of 0.01% NaOH solution to form an electrolyte solution;

[0060] Acupuncture needles (Hua Tuo brand acupuncture needles, 13 mm long and 0.25 mm in diameter) were used as working electrodes (anodes), Ag / AgCl electrodes were used as reference electrodes, and Pt wire electrodes were used as counter electrodes (cathodes). Electrochemical processes were performed using an electrochemical workstation (Chenhua electrochemical workstation, GAMRY electrochemical workstation) (schematic diagram as shown in Figure 3 The scanning range was set to 0.6-1.0 mV, the rate was set to 5 mV / s, and the cycle was repeated 10 times.

[0061] Example 2

[0062] This example prepares a nanoneedle.

[0063] (1) Design of drug-device combination using three peptides, as shown in the schematic diagram Figure 4As shown, one end of one of the peptides is used to connect to the surface of the acupuncture needle (such as: dermaseptin S1 (ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ)), another peptide is used to connect our drug molecules (such as: Thanatinpeptide (GSKKPVPIIYCNRRTGKCQRM)), and another peptide is used to connect the above two peptides (such as WKYMVm peptide). These combinations utilize the adhesion of polypeptides, which is a physical adsorption with good adsorption strength.

[0064] Example 3

[0065] This example prepares a nanoneedle.

[0066] Because the molecular composition and spatial structure of cucurbituril are similar to those of β-cyclodextrin, cucurbituril can be used to replace β-cyclodextrin to prepare nanoneedles. The result is shown in the schematic diagram. Figure 5 shown.

[0067] Example 4

[0068] This embodiment performs needle loading and drug release.

[0069] (1) In vitro nanoneedles loaded with fluorescent molecules

[0070] In order to facilitate the evaluation and characterization of the drug loading and release performance of the nanoneedle of the present application, fluorescent molecules and their combinations that meet the loading requirements are used for the test. The nanoneedle prepared in Example 1 was immersed in 200 μL of anhydrous ethanol solution, which contained the following fluorescent molecules (Hoechst 33342, FITC, DiO, Dil, DiD) at a concentration of 1 mg / mL. The loading process was carried out in a 1.5 mL Eppendorf tube and placed at 25°C in the dark for 24 hours. After that, the needle was dried at 25°C in the dark. The needle loaded with different fluorescent dyes was observed by single-photon confocal microscopy (Zeiss 710, Germany). The results are as follows: Figure 6 As shown, this demonstrates the needle's loading capacity for fluorescent small molecules.

[0071] (2) In vitro nanoneedles release fluorescent molecules

[0072] In order to obtain visual evidence of the release of fluorescent molecules, the nanoneedles loaded with fluorescent molecules were immersed in 200 μL of ethanol and photographed under ultraviolet light (365 nm). Since most dyes are colored, bright field photos can also be taken under natural light conditions.

[0073] In order to carry out quantitative analysis, the FITC-loaded nanoneedles were immersed in 200 μL, 1× PBS solution at 25°C for a period of time and taken out from the solution after the following time points (30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes to 30 minutes), and the fluorescence intensity of FITC in the solution was measured by a microplate reader (excitation wavelength: 495 nm, emission wavelength: 525 nm).

[0074] (3) The ability of in vitro nanoneedles to simultaneously load and release multiple molecules

[0075] For co-loading of two fluorescent molecules, the needle was immersed in 200 μL of dye ethanol solution containing 1 mg / mL of FITC and DiD, and the rest of the study observation method was similar to the above method.

[0076] (4) Mouse model to study the release of fluorescent molecules by nanoneedles in vivo

[0077] Eight-week-old female C57BL / 6 mice were used to observe the release of FTIC from nanoneedles in vivo. The mice were treated with electroacupuncture (EA) using nanoneedles loaded with FITC according to the acupuncture model of traditional Chinese medicine. The acupoints were the right "Zusanli" (ST-36) and the right "Yanglingquan" (GB-34). As a control, mice were injected with 20 μg FITC (20 μL 1×PBS) by intramuscular injection. The images of the release of fluorescent molecules in vivo were obtained by IVIS imaging system (IVIS Spectrum, PerkinElmer). The images were observed at 0, 3, 5, 15, and 30 minutes, respectively. The results are shown in Figure 2. Figure 7 As shown in the figure, FITC dye is gradually released over time and reaches a peak about 15 minutes after acupuncture. Compared with intramuscular injection, nanoneedle administration has better in vivo delivery and enrichment effects.

[0078] In order to observe the distribution of FITC fluorescence molecules in deep tissues, mice were killed after IVIS imaging, then embedded in OCT, and sliced ​​into 20 μm thick sections using a frozen tissue slicer. The distribution of FITC fluorescence in tissue sections was observed using an inverted fluorescence microscope (DM IL LED, Leica). Figure 8 As shown in the figure, after acupuncture, FITC dye gradually penetrated into the muscle tissue over time. After 15 minutes of acupuncture, FITC was fully released and evenly distributed in the muscle gap, which is consistent with the results of IVIS imaging. Compared with intramuscular injection of the same amount of FITC, the use of nano-needle delivery technology can make the dye more evenly distributed and delivered to the deep tissue.

[0079] In summary, the present invention completes the combination of a main molecule capable of loading drug molecules and a linker through a series of chemical syntheses, and irreversibly (permanently) combines the main molecule-linker complex on the surface of traditional medical acupuncture needles through auxiliary means such as electrochemical deposition, thereby giving the acupuncture needles the ability to load drugs and being able to exert drug delivery function in traditional acupuncture treatment.

[0080] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A nano needle, characterized in that: The nano needles include acupuncture needles with a host molecule modified on the surface; The host molecule includes a cyclic molecule capable of forming an inclusion compound structure.

2. The nanoneedle according to claim 1, characterized in that: The cyclic molecules capable of forming inclusion complex structures include any one of cyclodextrin, cyclodextrin derivatives, cucurbituril or cucurbituril derivatives, or a combination of at least two of them.

3. The nanoneedle according to claim 2, characterized in that: The cyclodextrin includes any one of α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin or a combination of at least two thereof; Preferably, the cyclodextrin derivative includes any one of SBE-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, β-cyclodextrin hydrate, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, acetyl-β-cyclodextrin, (2-hydroxyethyl)-β-cyclodextrin, triacetyl-γ-cyclodextrin, succinyl-α-cyclodextrin-β-cyclodextrin, carboxymethyl-β-cyclodextrin, heptyl (2,3,6-tri-O-methyl)-β-cyclodextrin or 6-monodeoxy-6-monoamino-β-cyclodextrin hydrochloride, or a combination of at least two thereof.

4. The nanoneedle according to claim 2 or 3, characterized in that: The cucurbituril derivatives include sugammadex sodium and / or hydrated cucurbituril.

5. The nanoneedle according to any one of claims 1 to 4, characterized in that: The main molecule is connected to the surface of the acupuncture needle through a chemical linker or a chemical bond; Preferably, the chemical linker comprises any one or a combination of at least two of phenol, phenolic derivatives or adhesion peptides; Preferably, the phenolic derivative comprises any one of methyl salicylate, ethyl salicylate, methyl anthranilate or methyl cinnamate, or a combination of at least two thereof.

6. A method for preparing the nanoneedle according to any one of claims 1 to 5, characterized in that: The preparation method comprises: The host molecule is connected to the surface of an acupuncture needle.

7. The method for preparing the nanoneedle according to claim 6, characterized in that: The connection method includes an electrochemical method or a spray method; Preferably, the preparation method further comprises the step of modifying the main molecule with a chemical linker; Preferably, the method for modifying the chemical linker specifically comprises: The solution of the main molecule is mixed with the chemical linker and reacted to obtain the main molecule modified with the chemical linker.

8. The method for preparing the nanoneedle according to claim 7, characterized in that: The preparation method comprises the following steps: (1) mixing a solution of a main molecule with a chemical linker, reacting the mixture, and obtaining a main molecule modified with a chemical linker; (2) Using a solution of a main molecule modified with a chemical linker as an electrolyte solution and an acupuncture needle as a working electrode, an electrochemical workstation is used to perform an electrochemical process to obtain the nanoneedle.

9. A nanoneedle delivery system, characterized in that: The nanoneedle delivery system comprises the nanoneedle according to any one of claims 1 to 5 and a functional molecule; The functional molecules are loaded on the main molecules of the nano needle.

10. The nanoneedle delivery system according to claim 9, characterized in that: The functional molecules include any one of drugs, tracer molecules, fluorescent marker molecules, small molecule proteins or drug combinations, or a combination of at least two of them.

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