Acupuncture needle loaded with hydrogel drugs, and preparation method therefor and application thereof

Abstract

Disclosed is an acupuncture needle loaded with hydrogel drugs, which is used for treating osteoarthritis. Accurate treatment for osteoarthritis pathological environments is achieved by combining a spiral groove structure with specific hydrogel loaded drugs. Non-invasive dynamic assessment of disease sub-typing is achieved by means of responsive degradation of three hydrogels to specific biomarkers of osteoarthritis at different pathological stages, in combination with fluorescence signal changes. After the acupuncture needle is inserted into an osteoarthritis lesion site, specific enzymes or factors in different pathological environments trigger degradation of the corresponding hydrogels, thereby releasing the loaded drugs and achieving targeted treatment.

Description

An acupuncture needle loaded with hydrogel drug, its preparation method and application Technical Field

[0001] This invention relates to the field of medical device technology, specifically to an acupuncture needle loaded with hydrogel drugs, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a common degenerative joint disease, mainly characterized by synovitis, articular cartilage degeneration, and fibrosis. Traditional treatments include medication, physical therapy, and surgery, but these have drawbacks such as systemic side effects and limited therapeutic efficacy. Acupuncture, as a traditional Chinese medicine therapy, has been shown to be effective in relieving osteoarthritis symptoms; however, traditional acupuncture needles have limited functionality and cannot achieve precise treatment targeting the underlying pathological conditions.

[0003] In recent years, the integration of controlled drug release technology with medical devices has become a research hotspot. However, existing technologies mostly employ simple coatings or porous structures, making it difficult to achieve specific drug release targeting different pathological environments. Patent CN115282045A discloses a hydrogel precision delivery system based on acupuncture needles. This system firmly adheres the hydrogel carrier to the threaded grooves of the acupuncture needle, and under the protection of these grooves, it is precisely delivered into the lesion requiring treatment, achieving precise treatment of the subchondral bone, alleviating cartilage degeneration and degradation, and improving the condition of osteoarthritis. However, osteoarthritis pathogenesis includes three main stages: inflammation, matrix degradation, and fibrosis. Patent CN115282045A only achieves drug delivery to the subchondral bone and cannot achieve precise treatment targeting the specific pathological environment.

[0004] In recent years, the concept of molecular subtyping of osteoarthritis (OA) has been proposed. Based on the heterogeneity of disease-driving mechanisms, OA patients are classified into different subtypes through molecular biomarker profiling. Each subtype corresponds to unique pathological features, disease progression trajectories, and therapeutic targets, thereby achieving precise diagnosis and targeted therapy. Therefore, we selected three main stages of OA pathogenesis (inflammatory stage, matrix degradation, and fibrosis) and three main targets (IL-1β, MMP13, and TGF-β) to develop an acupuncture needle capable of targeted therapy against different pathological environments (inflammation, matrix degradation, and fibrosis) in OA. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to solve the above-mentioned technical problems and provide an acupuncture needle loaded with hydrogel drugs to achieve targeted drug release against the pathological environment of osteoarthritis, thereby improving the therapeutic effect.

[0006] Technical solution: The present invention provides an acupuncture needle loaded with hydrogel drugs. The acupuncture needle has a spiral groove on its body, and the hydrogel drugs are filled in the spiral groove of the acupuncture needle. The spiral groove is divided into a first segment, a second segment and a third segment from the tip of the acupuncture needle upward to the middle, and each segment is filled with a hydrogel drug.

[0007] Furthermore, the first segment is filled with GelMA hydrogel, which is specifically designed for the matrix degradation environment in osteoarthritis. The GelMA hydrogel is loaded with Batimastat drug, which specifically responds to MMP13.

[0008] The second segment is filled with boronic ester bond-modified GelMA hydrogel, which is specifically designed for inflammatory environments. The boronic ester bond-modified GelMA hydrogel is loaded with Anakinra drug, which specifically responds to IL-1β.

[0009] The third segment is filled with CYKC (Cys-Lys-Tyr-Cys tetrapeptide) modified GelMA hydrogel, which is specifically designed for fibrotic environments. The CYKC modified GelMA hydrogel is loaded with docetaxel, which specifically responds to TGF-β.

[0010] Furthermore, the acupuncture needle has a total length of 70-80mm, a spiral groove total length of 35-40mm, a pitch of 0.5mm, and a groove depth of 0.1-0.14mm. It is made of medical-grade stainless steel, and the spiral groove is created on the needle body using precision machining technology.

[0011] The present invention also provides a method for preparing an acupuncture needle loaded with a hydrogel drug, comprising the following steps:

[0012] 1) On ordinary acupuncture needles, make spiral grooves 35-40mm above the needle tip, with a pitch of 0.5mm and a groove depth of 0.1-0.14mm;

[0013] 2) Preparation of GelMA hydrogels, boronic ester bond modified GelMA hydrogels, and CYKC modified GelMA hydrogels;

[0014] The method for preparing the boronic ester bond modified GelMA hydrogel is as follows: add phenylboronic acid to the GelMA solution and oscillate it fully in an ultrasonic reactor for 30 minutes.

[0015] The preparation method of the CYKC modified GelMA hydrogel is as follows: CYKC is added to the GelMA solution and the solution is fully vibrated for 30 minutes using an ultrasonic reactor.

[0016] 3) Drug loading was performed on the three hydrogels in step 2). Specifically, Anakinra drug was mixed with GelMA hydrogel, Batimastat drug was mixed with boronic ester bond modified GelMA hydrogel, and docetaxel drug was mixed with CYKC modified GelMA hydrogel, and then cured by photocrosslinking.

[0017] 4) The three hydrogels obtained in step 3) are sequentially filled onto the acupuncture needles prepared in step 1). The amount of Anakinra, Batimastat, and docetaxel added is 0.8 mg each. Specifically, the first segment of the spiral grooved needle is inserted into the Batimastat-GelMA hydrogel prepared in Example 2, irradiated with a 405 nm light source for 20-30 seconds to gel, then the needle is withdrawn, and the excess gel is scraped off with a blade. Then, the filled portion is wrapped with aluminum foil, and the second segment of the spiral grooved needle is inserted into the Anakinra-boron ester bond-GelMA hydrogel, irradiated with a 405 nm light source for 20-30 seconds to gel, then the needle is withdrawn, and the excess gel is scraped off with a blade. Then, the filled portion is wrapped with aluminum foil, and the third segment of the spiral grooved needle is inserted into the docetaxel-CYKC-GelMA hydrogel, irradiated with a 405 nm light source for 20-30 seconds to gel, then the needle is withdrawn, and the excess gel is scraped off with a blade. This creates a three-section hydrogel-filled acupuncture needle.

[0018] The present invention also provides the application of acupuncture needles loaded with hydrogel drugs in the preparation of drug delivery systems for treating osteoarthritis.

[0019] Beneficial effects:

[0020] This invention provides a multifunctional spiral groove acupuncture needle for treating osteoarthritis. By combining a spiral groove structure with a drug-loaded specific hydrogel, it achieves precise treatment targeting the pathological environment of osteoarthritis. Through the degradation rate of the specific hydrogel, it enables the diagnosis of molecular subtypes of osteoarthritis, realizing integrated diagnosis and treatment, and has significant clinical application value.

[0021] This invention utilizes the responsive degradation of specific biomarkers for different pathological stages of osteoarthritis (OA) by three types of hydrogels, combined with changes in fluorescence signals, to achieve non-invasive dynamic identification of disease subtyping. When an acupuncture needle is inserted into the lesion site of osteoarthritis, specific enzymes or factors (MMP13, IL-1β, TGF-β) in different pathological environments trigger the degradation of the corresponding hydrogels, thereby releasing the loaded drugs for targeted therapy. Attached Figure Description

[0022] Figure 1 is a schematic diagram of the structure of the acupuncture needle of the present invention.

[0023] Figure 2 is a schematic diagram of hydrogel filling.

[0024] Figure 3 shows representative protein blot bands of MMP13, ADAMTS5, and type I collagen (Col I) and their corresponding quantitative statistical analysis.

[0025] Figure 4 is an example of the rat open field experiment in Example 7. From left to right, the groups are the control group, the DMM group, the acupuncture treatment group, the acupuncture-hydrogel group, and the acupuncture-hydrogel-drug group.

[0026] Figure 5 shows the experimental data of activity level, activity time, walking distance, and average speed of the five groups of rats in Example 7. Detailed Implementation

[0027] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0028] The Anakinra drug mentioned in this invention, also known as Anakina, was purchased from Aladdin, catalog number rp173459; the Batimastat drug, also known as Batimastat, was purchased from Aladdin, catalog number B125646; the Docetaxel drug was purchased from Aladdin, catalog number D107320; the GelMA hydrogel was purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.; and the CYKC (Cys-Lys-Tyr-Cys tetrapeptide) was purchased from Nanjing Genscript Biotech Co., Ltd.

[0029] Example 1: Preparation of GelMA hydrogel

[0030] (1) Gelatin dissolution: Dissolve the gelatin powder in phosphate buffer (PBS) preheated to 50-60℃ to prepare a 10% (w / v) gelatin solution and stir until completely dissolved.

[0031] (2) Methacrylation reaction: Cool the gelatin solution to 40-50℃, slowly add methacrylic anhydride (MA), the amount of MA added is 5-10% of the mass of gelatin, and stir the reaction for 2-4 hours under nitrogen protection.

[0032] (3) Dialysis purification: The reaction solution is placed in a dialysis bag and dialyzed with deionized water for 3-5 days to remove unreacted methacrylic anhydride and byproducts. After dialysis, the solution is freeze-dried to obtain solid GelMA.

[0033] (4) Hydrogel preparation: Dissolve solid GelMA in PBS to prepare a 5-20% (w / v) GelMA solution, add photoinitiator Irgacure 2959 (final concentration 0.5-1% w / v), stir evenly and inject into the mold, irradiate under ultraviolet light (wavelength 365 nm) for 30-60 seconds to complete photocrosslinking and curing.

[0034] Example 2: Preparation of Batimastat-GelMA hydrogel

[0035] 0.8 mg of Batimastat was mixed with 2 ml of 5% (w / v) GelMA hydrogel prepared in Example 1, and cured by photocrosslinking. The mixture was then irradiated with a 405 nm light source for 20-30 seconds to induce gelation, resulting in Batimastat-GelMA hydrogel.

[0036] Example 3: Preparation of Anakinra-boron ester bond-GelMA hydrogel

[0037] 0.5 g of phenylboronic acid was added to 2 ml of 5% (w / v) GelMA solution prepared in Example 1, and the solution was thoroughly agitated in an ultrasonic reactor for 30 minutes to allow the boron ester bonds of phenylboronic acid to modify the hydroxyl groups of GelMA, resulting in a boron ester bond modified GelMA hydrogel. Then, 0.8 mg of Anakinra drug was mixed with 2 ml of boron ester bond modified GelMA hydrogel, and the mixture was cured by photocrosslinking. The gel was then irradiated with a 405 nm light source for 20-30 seconds to obtain Anakinra-boron ester bond-GelMA hydrogel.

[0038] Example 4: Preparation of docetaxel-CYKC-GelMA hydrogel

[0039] 5 mg of CYKC was added to 2 ml of 5% (w / v) GelMA solution prepared in Example 1, and the mixture was thoroughly agitated in an ultrasonic reactor for 30 minutes to covalently modify the CYKC peptide onto the GelMA molecular chain, resulting in CYKC-modified GelMA hydrogel. Then, 0.8 mg of docetaxel was mixed with 2 ml of CYKC-modified GelMA hydrogel, and the mixture was cured by photocrosslinking. The gel was then irradiated with a 405 nm light source for 20-30 seconds to obtain docetaxel-CYKC-GelMA hydrogel.

[0040] Example 5: Preparation of acupuncture needles loaded with hydrogel drugs

[0041] 1) On ordinary acupuncture needles, a spiral groove is made on the needle body through precision machining technology. The spiral groove is made 40mm from the needle tip upwards, with a pitch of 0.5mm and a groove depth of 0.1mm, as shown in Figure 1.

[0042] 2) The three hydrogels obtained in Examples 2, 3, and 4 are sequentially filled into the acupuncture needles prepared in step 1). Specifically, the first segment of the spiral grooved needle is inserted into the Batimastat-GelMA hydrogel prepared in Example 2, irradiated with a 405nm light source for 20-30 seconds to gel, then the needle is withdrawn, and excess gel is scraped off with a blade. Then, the filled portion is wrapped with aluminum foil, and the second segment of the spiral grooved needle is inserted into the Anakinra-boron ester bond-GelMA hydrogel, irradiated with a 405nm light source for 20-30 seconds to gel, then the needle is withdrawn, and excess gel is scraped off with a blade. Then, the filled portion is wrapped with aluminum foil, and the third segment of the spiral grooved needle is inserted into the docetaxel-CYKC-GelMA hydrogel, irradiated with a 405nm light source for 20-30 seconds to gel, then the needle is withdrawn, and excess gel is scraped off with a blade. This forms an acupuncture needle filled with three hydrogel segments. As shown in Figure 2, different colors represent different hydrogels.

[0043] Example 6: Preparation of hydrogel acupuncture needles

[0044] The preparation method is the same as in Example 5, except that the filled hydrogel does not contain the drug, but the GelMA hydrogel prepared in Example 1 is filled into the three-segment spiral groove.

[0045] Example 7: Quantitative analysis of expression changes of key pathological biomarkers using Western blotting technology

[0046] To clarify the specific inhibitory effects of the three therapeutic agents on their respective targets, we established an IL-1β-induced osteoarthritis inflammation model at the cellular level and quantitatively analyzed the expression changes of key pathological markers using Western blotting. The figure below shows representative protein blot bands of MMP13, ADAMTS5, and type I collagen (Col I) and the corresponding quantitative statistical analysis. The control group represents normal bone and joints, and the IL-1 group represents the IL-1β-induced osteoarthritis inflammation model.

[0047] (1) ADAMTS5 expression and the regulatory role of Anakinra

[0048] Figure 3-A shows the expression of ADAMTS5. IL-1β stimulation significantly upregulated ADAMTS5 expression (p<0.05), indicating that our cell model successfully mimicked the pathological features of enhanced inflammatory factors in OA. After the addition of Anakinra (IL-1+Anakinra group), ADAMTS5 expression was significantly reduced (p<0.05 compared to the IL-1β group). This result not only demonstrates the anti-inflammatory effect of Anakinra, but more importantly, reveals the molecular mechanism by which it delays cartilage matrix degradation by specifically inhibiting ADAMTS5.

[0049] (2) MMP13 expression and the inhibitory effect of Batimastat

[0050] As shown in Figure 3-B, compared with the control group, IL-1β stimulation significantly increased the protein expression level of MMP13 (p<0.01), indicating that our cell model successfully simulated the pathological features of enhanced matrix degradation in OA. However, after treatment with Batimastat (IL-1+Batimastat group), the expression of MMP13 was significantly reduced (p<0.01 compared with the IL-1β group). This demonstrates that Batimastat can almost completely reverse IL-1β-induced high expression of MMP13 and specifically inhibit the matrix degradation pathway.

[0051] (3) Col I expression and the inhibitory effect of docetaxel

[0052] In Figure 3-C, we observed that IL-1β stimulation significantly promoted Col I expression (p<0.01), indicating that the inflammatory microenvironment can induce the expression of fibrosis-related proteins. After treatment with Docetaxel (IL-1+Docetaxel group), Col I expression was significantly reduced (p<0.01 compared to the IL-1β group). This finding confirms that Docetaxel can specifically inhibit the fibrosis process and prevent abnormal collagen deposition in the progression of OA pathology.

[0053] Example 8: Fractal Mechanism

[0054] (1) Diagnosis of inflammatory OA

[0055] Hydrogel: Boron ester bond modified GelMA (IL-1β responsive)

[0056] Fluorescent label: Cy5 (red)

[0057] Judgment criteria: Increased IL-1β levels in an inflammatory environment → triggering the breaking of boronic ester bonds → rapid decay of Cy5 fluorescence → decay rate is positively correlated with inflammatory activity.

[0058] (2) Diagnosis of matrix-degradable OA

[0059] Hydrogel: Natural GelMA (responsive to MMP13)

[0060] Fluorescent label: FITC (green)

[0061] Judgment criteria: MMP13 overexpression during matrix degradation → hydrolysis of GelMA collagen network → decrease in FITC fluorescence intensity → degradation rate reflects the degree of cartilage destruction.

[0062] (3) Diagnosis of fibrotic OA

[0063] Hydrogel: CYKC peptide-modified GelMA (responsive to TGF-β)

[0064] Fluorescent label: DAPI (blue)

[0065] Judgment criteria: In the fibrotic microenvironment, TGF-β activates cathepsins, which then cleave CYKC peptide chains, leading to the gradual disappearance of DAPI fluorescence. The slope of signal attenuation indicates the progression of fibrosis.

[0066] By comparing the relative decay rates of the three-color fluorescence, the dominant pathological process can be determined:

[0067] The fastest decline in Cy5 indicates an inflammation-dominant type (early OA); the fastest decline in FITC indicates a matrix degradation type (mid-stage OA); and the fastest decline in DAPI indicates a fibrotic type (late-stage OA).

[0068] Example 9: Therapeutic Effect

[0069] Set up a group control group (blank control, normal rats that did not receive any treatment, represented by sham in Figure 5).

[0070] DMM group (rats underwent DMM surgery (medial meniscus instability surgery) to induce osteoarthritis, but no treatment was given; DMM is used to represent this group in Figure 5).

[0071] The simple acupuncture treatment group (rats underwent DMM surgery to induce osteoarthritis, and were treated with simple acupuncture, represented by needle in Figure 4).

[0072] Acupuncture-hydrogel group (Rats underwent DMM surgery to induce osteoarthritis, and were treated with acupuncture + hydrogel, i.e., the hydrogel acupuncture needles prepared in Example 6, represented by hydrol in Figure 5).

[0073] Acupuncture-hydrogel-drug group (Rats underwent DMM surgery to induce osteoarthritis, and were treated with acupuncture + hydrogel + drug, i.e., the hydrogel drug acupuncture needle prepared in Example 5, represented by drug in Figure 5)

[0074] To test the performance of the acupuncture needles loaded with hydrogel drugs of the present invention, osteoarthritis models were first established in rats, and the rats were treated with the aforementioned acupuncture needles. Then, an open field test was conducted (the open field test involves allowing rats to move in an open area for 3 minutes, recording their movement trajectory throughout (automatically recorded by an instrument), and the instrument automatically generates the rat's activity level, activity time, walking distance, and average speed to assess the rat's behavior). It was found that after treatment, the rats' behavioral characteristics were significantly more active than before treatment. Figure 4 shows examples of the open field test results for five groups of rats, from left to right: control group, DMM group, acupuncture treatment group alone, acupuncture-hydrogel group, and acupuncture-hydrogel-drug group. The open field test figures in Figure 5, from left to right, compare the activity level, activity time, walking distance, and average speed of the five groups of rats. As shown in the figure, after acupuncture-hydrogel-drug treatment, the activity level of the rats was significantly improved compared with the DMM group (statistically significant), the activity time of the rats was significantly improved compared with the DMM group (statistically significant), and the movement distance and average speed of the rats were significantly improved (statistically significant).

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An acupuncture needle loaded with a hydrogel drug, characterized in that, The acupuncture needle has a spiral groove on its body, and the hydrogel drug is filled in the spiral groove of the acupuncture needle. The spiral groove is divided into a first section, a second section and a third section from the tip of the acupuncture needle upward to the middle, and each section is filled with a hydrogel drug.

2. The acupuncture needle loaded with hydrogel drug according to claim 1, characterized in that, The first segment is filled with GelMA hydrogel, which is loaded with Batimastat drug; the second segment is filled with boronic ester-modified GelMA hydrogel, which is loaded with Anakinra drug; and the third segment is filled with CYKC-modified GelMA hydrogel, which is loaded with docetaxel drug.

3. The acupuncture needle loaded with hydrogel drug according to claim 2, characterized in that, The acupuncture needle has a total length of 70-80mm, a total spiral groove length of 35-40mm, a pitch of 0.5mm, and a groove depth of 0.1-0.14mm.

4. A method for preparing an acupuncture needle loaded with a hydrogel drug as described in any one of claims 2-3, characterized in that, Includes the following steps: 1) On ordinary acupuncture needles, make spiral grooves 35-40mm above the needle tip, with a pitch of 0.5mm and a groove depth of 0.1-0.14mm; 2) Preparation of GelMA hydrogels, boronic ester bond modified GelMA hydrogels, and CYKC modified GelMA hydrogels; 3) Drug loading was applied to the three types of hydrogels from step 2); 4) The three types of hydrogels obtained in step 3) are sequentially filled onto the acupuncture needles prepared in step 1).

5. The method for preparing an acupuncture needle loaded with a hydrogel drug according to claim 4, characterized in that, The method for preparing the boronic ester bond modified GelMA hydrogel is as follows: add phenylboronic acid to the GelMA solution and oscillate it fully in an ultrasonic reactor for 30 minutes.

6. The method for preparing an acupuncture needle loaded with a hydrogel drug according to claim 4, characterized in that, The preparation method of the CYKC modified GelMA hydrogel is as follows: CYKC is added to the GelMA solution and the solution is fully vibrated for 30 minutes using an ultrasonic reactor.

7. The method for preparing an acupuncture needle loaded with a hydrogel drug according to claim 4, characterized in that, Step 3) specifically involves mixing Anakinra drug with GelMA hydrogel, Batimastat drug with boronic ester bond modified GelMA hydrogel, and docetaxel drug with CYKC modified GelMA hydrogel, and then curing them by photocrosslinking.

8. The method for preparing an acupuncture needle loaded with a hydrogel drug according to claim 7, characterized in that, The dosage of Anakinra, Batimastat, and docetaxel was 0.8 mg each.

9. The use of the acupuncture needle loaded with hydrogel drug according to any one of claims 1-8 in the preparation of a drug delivery system for treating osteoarthritis.

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