CN115282045A

CN115282045A - Hydrogel accurate delivery system based on acupuncture needle, preparation method and application

Info

Publication number: CN115282045A
Application number: CN202210308021.4A
Authority: CN
Inventors: 崔文国; 林峰; 吴隆熙; 刘玉璞
Original assignee: SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS
Current assignee: SHANGHAI INSTITUTE OF TRAUMATOLOGY AND ORTHOPEDICS
Priority date: 2022-03-26
Filing date: 2022-03-26
Publication date: 2022-11-04

Abstract

The invention provides an acupuncture needle-based hydrogel accurate delivery system, a preparation method and application, and belongs to the technical field of biomedical materials. The hydrogel accurate delivery system based on the acupuncture needle comprises the acupuncture needle and a hydrogel coating, wherein a thread groove is formed in the needle body of the acupuncture needle, and the hydrogel coating is adhered to the thread groove of the acupuncture needle; the hydrogel coating is prepared by carrying out photo-crosslinking reaction on N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide and methacrylated hyaluronic acid to obtain an adhesive hydrogel, wrapping a carrying liposome, loading the carrying liposome in a thread groove, and drying the carrying liposome to form the hydrogel coating. The hydrogel accurate delivery system provided by the invention can realize smooth delivery of drugs or biological materials to subchondral bone in a minimally invasive manner on an acupuncture needle, regulate and control abnormal secretion of TGF-beta 1, and construct a stem cell migration channel of the subchondral bone, so as to achieve accurate positioning treatment of the subchondral bone.

Description

Hydrogel accurate delivery system based on acupuncture needle, preparation method and application

Technical Field

The invention belongs to the technical field of biomedical materials, relates to drug delivery in precise treatment, and particularly relates to an acupuncture needle-based hydrogel precise delivery system, and a preparation method and application thereof.

Background

Precision therapy, which generally includes optimized disease control strategies and optimized iatrogenic damage control strategies, currently occupies an important position in the medical field. Compared with the common treatment means, the accurate treatment method has the advantages that the medicine can be accurately positioned at the focus, the enrichment effect is generated at the focus part, the clinical curative effect is greatly improved, and the risk of side effects possibly brought by the treatment means can be obviously reduced.

At present, with continuous development and application of functionalized biomaterials, localized and accurate treatment of various diseases, such as hydrogel microspheres, electrostatic spinning and other biomaterials, is realized. However, due to the special physiological structure of some tissues or organs (such as spinal cord, cartilage, etc.), a natural physical barrier is created to the external treatment means, which greatly hinders the implementation of precise treatment. Therefore, how to design a drug delivery system which can powerfully break through physical barriers of various tissues and organs and realize accurate focus positioning is a major medical problem which needs to be overcome at present.

A large number of clinical applications have shown that Chinese acupuncture is the most mature means of precise positioning treatment in traditional medicine. The clinical application of Chinese acupuncture has been for thousands of years, and the efficacy of the Chinese acupuncture is tested by time and is popularized all over the world. The principle of acupuncture is that the micro-needle with the needle point diameter of 200-300 microns and the length of 100-120 mm is utilized to penetrate the physical barriers of various tissues and organs strongly and reach the focus directly, so as to accurately stimulate the focus and change the microenvironment of the focus, thereby achieving the effect of relieving or even treating diseases.

The material commonly used in acupuncture is an acupuncture needle, which is a microneedle prepared from a metal material, has physical hardness which cannot be achieved by organic polymers and silicon-based materials, can prepare the microneedle with the length of hundreds of millimeters, easily reaches various deep tissues of a human body, has the capability of strongly penetrating various hard tissue physical barriers, easily realizes the effect of inserting the needle from the skin, and strongly breaks through the physical barriers of various tissues to reach deep focuses of the human body.

As the diameter of the acupuncture needle tip is in the micron order, the acupuncture needle has very good micro-invasiveness, a patient hardly feels pain during the acupuncture treatment process, and the acupuncture part hardly bleeds. After the acupuncture treatment is finished, the micron-sized wound left by the acupuncture needle can be quickly self-healed, and a patient can leave a treatment room to start normal life without medical treatment such as bandaging. Therefore, in China, the patient has very high acceptance of acupuncture treatment, the rejection psychology in the face of invasive treatment of Western medicine does not exist, the patient can generally accept the acupuncture treatment frequency of 3-5 times per week, and the high-frequency and non-invasive treatment means also ensures the remarkable curative effect of the acupuncture treatment.

The traditional acupuncture needle prepared from the metal material has single chemical component and lacks of chemical groups for chemical modification, so that the traditional acupuncture needle is difficult to be specifically functionally modified.

Currently, acupuncture therapy mainly intervenes and treats focus parts through single physical stimulation, different pathological microenvironments often lack pertinence in the face of different diseases, and further expansion of acupuncture in the field of precise positioning treatment is severely limited.

Therefore, if the acupuncture needle can be functionally modified to play the effect of accurate positioning treatment aiming at the microenvironment of different diseases, the application of acupuncture in the field of accurate positioning treatment can be further expanded.

On the basis of acupuncture therapy, microneedle patches have been developed. The microneedle patch has abundant functional designs, can be used for positioning and accurately treating microenvironment of various diseases, and is widely applied to the field of medical treatment. The micro-needle patch can effectively penetrate into various tissues and organs, can be used for drug delivery aiming at a specific part, and becomes an ideal biological material for precise positioning treatment. Currently, microneedle patches have been used in various solid tumors and organs to achieve drug release and precise localized therapy. But the existing microneedle patch material still has the following two defects when aiming at the precise treatment of deep tissues: firstly, the depth of penetration is not enough, and only can act on superficial tissues; secondly, the hard physical barriers of some tissues and organs such as cartilage, ligament and the like cannot be broken through due to the lack of enough hardness. When people select materials for manufacturing the microneedle patch, the materials mainly focus on organic polymers and silicon-based materials, so that the two defects are not solved all the time, and the application and the expansion of the microneedle patch in the deep and accurate positioning treatment field are seriously hindered.

In order to overcome the defects of the Chinese acupuncture and the microneedle patch in deep and accurate treatment and fully develop the advantages of the Chinese acupuncture and the microneedle patch, researchers try to combine the Chinese acupuncture and the microneedle patch technology to prepare a multifunctional drug delivery carrier material so as to enrich the application of the material in the field of accurate positioning treatment.

Patent document CN 108245482 a discloses a spiral acupuncture needle with a thread structure on the needle body, through the spiral structure on the needle body, it can extract human tissue or body fluid during acupuncture treatment, thereby performing disease detection or gene detection, however, the outer surface of the needle body of the spiral acupuncture needle is provided with threads, the threads are arranged convexly on the needle body, thereby facilitating extraction of tissue fluid, the acupuncture needle structure can not be applied in the field of precise treatment, the threads which are externally protruded are used for loading medicine, and easily cause medicine loss, and can not reach the focus precisely.

Patent document CN 108338917A discloses an acupuncture needle having a groove formed in a needle body, which can extract human tissues or body fluids by using the groove structure of the needle body to perform disease detection or genetic testing. However, the above patent documents only relate to the basic extraction of human tissues or body fluids for disease detection or genetic testing, and do not discuss the further application of the above acupuncture needles in the field of precise treatment. How to fully utilize the groove structure of the acupuncture needle to finish the accurate delivery of the medicine, break through the hard physical barrier of tissues and organs and realize the accurate treatment of diseases still needs to be further researched.

Patent document CN 106061455A discloses a porous acupuncture needle, which is configured such that a plurality of micron-sized recessed holes are formed in the surface of the acupuncture needle, and drugs are loaded in the recessed holes, thereby achieving effective delivery of the drugs into the body and improving the therapeutic effect of acupuncture. Although the method can realize basic delivery of the medicine by borrowing the acupuncture needle, the requirement on the acupuncture needle is higher, the strength of the needle body is lower due to the large number of concave holes on the surface of the needle body, the needle body is easy to break when entering the body, and the hole structure can load the medicine, but the effective load capacity of the medicine is lower, the medicine is not easy to dissolve out of the holes, and the medicine release performance is uncontrollable. When the acupuncture needle is taken out, a large amount of the drug remains in the hollow hole, resulting in low drug delivery efficiency and failure to achieve the intended precise therapeutic effect.

Journal literature "Improvement in anti-administration of a ground-embedded microneedle array, sensors and Actuators B137 (2009) 274-280" discloses a microneedle patch structure, which is designed to have a microneedle structure with a groove, and realizes precise treatment by loading a drug in the groove of a microneedle and delivering the drug into the body. The research on the antigen or protein loaded in the groove of the microneedle patch indicates that the microneedle structure can release the antigen or protein into the body and can be used for positioning and accurate treatment. However, the micro-needle array structure is only suitable for superficial tissues, cannot perform positioning accurate treatment on deep tissues, and cannot penetrate hard physical barriers such as spinal cords or cartilage.

For the above precise treatment means, on one hand, the defects of poor drug delivery effect, uncontrollable drug release and no sustained release effect of the traditional acupuncture method exist, and on the other hand, the defects that the existing microneedle patch is only suitable for superficial tissues and cannot perform precise positioning treatment on deep tissues exist, and the above problems are urgently to be solved.

The hydrogel is a multifunctional drug-loaded biomaterial with wide application prospect, has great functionalization potential due to rich chemical groups, and plays an important role in realizing the functionalization of the acupuncture needle. The hydrogel-acupuncture needle system with minimally invasive and accurate positioning functions is developed, and is a main breakthrough direction for expanding the application of acupuncture needles in the field of accurate positioning treatment of deep tissues.

At the early stage, researchers have combined hydrogel with microneedles to endow the microneedles with more physiological functions. For example, xinjie Yin et al combines a hydrogel with a syringe needle, leaving the hydrogel in the needle hole while the syringe is in use, to reduce bleeding. Tsai-Yu Chen et al combine acupuncture and drug-loaded hydrogel to promote healing of the wound of diabetic patients. However, the above cases are limited to the treatment of skin or superficial blood vessels, and when the treatment needs to be performed deeply into the human body, and the treatment needs to be performed through several human tissues such as skin, tendon, muscle, cartilage, etc., and finally reaches the focus of subchondral bone, etc., a hydrogel microneedle capable of achieving the precise treatment efficacy has not been developed. The great obstruction of these physiological structures will cause the hydrogel to easily fall off from the upper half way of the acupuncture needle and not reach the focus smoothly. Therefore, how to ensure that the hydrogel loaded on the needle tip can not be separated in the process of inserting the acupuncture needle into a human body is a great problem troubling researchers.

Osteoarthritis (OA) is the most common chronic degenerative disease of the joints worldwide. For the precise treatment of OA, a number of functional biomaterials have been developed. Although these biomaterials are able to precisely localize to knee cartilage, they are effective in ameliorating cartilage damage and degeneration. However, one of the most important early factors facing OA: in subchondral bone lesions, almost no biological material can break through the physical barrier of cartilage, and accurate treatment is performed on subchondral bone.

It has been found that abnormal secretion of TGF-beta 1, a cytokine of subchondral bone in OA, leads to subchondral bone lesions (including abnormal remodeling, hardening, and mineralization) and ultimately accelerates progression of OA. However, the regulation of TGF-beta 1 abnormal secretion by systemic administration will inevitably result in major side effects. Meanwhile, stem cells of subchondral bone are a key factor for repairing cartilage damage. In clinical practice, drilling treatment is usually performed at a cartilage injury site during knee joint surgery to promote stem cells of subchondral bone to migrate to a cartilage defect, so that cartilage is repaired. However, this treatment can only be carried out in surgical patients.

In conclusion, the accurate positioning treatment of the subchondral bone can improve the mechanical environment of the cartilage, alleviate cartilage damage from the root, and the mesenchymal stem cells of the subchondral bone play an important role in repairing the cartilage damage. Therefore, accurate positioning treatment of subchondral bone is an ideal method for treating osteoarthritis, and how to develop a corresponding drug accurate delivery material to realize accurate positioning treatment of subchondral bone becomes a technical problem to be solved urgently.

Disclosure of Invention

In order to solve the technical problems, the invention provides a hydrogel precision delivery system based on an acupuncture needle, a preparation method of the hydrogel precision delivery system and application of precision positioning treatment in deep tissues. The technical purpose of the invention is as follows: on one hand, the acupuncture needle is improved, and the problems that the traditional acupuncture needle is single in function and cannot be well used for accurately delivering medicaments are solved; on the other hand, the method solves the problems that the medicine or hydrogel easily falls off from the acupuncture needle, can not smoothly pass through the physical barrier of deep tissues and has low medicine delivery efficiency in the existing acupuncture needle improvement method.

In order to achieve the above technical objectives and solve the above technical problems, one aspect of the present invention is to provide an acupuncture needle-based hydrogel precision delivery system, another aspect of the present invention is to provide a method for preparing the acupuncture needle-based hydrogel precision delivery system, and a third aspect of the present invention is to provide an application of the acupuncture needle-based hydrogel precision delivery system in deep tissue precision positioning therapy.

The invention firstly provides an acupuncture needle-based hydrogel accurate delivery system, which adopts the following technical scheme:

an acupuncture needle-based hydrogel precision delivery system comprises an acupuncture needle and a hydrogel coating, wherein a thread groove is formed in the needle body of the acupuncture needle, and the hydrogel coating is adhered to the thread groove of the acupuncture needle; the hydrogel coating is prepared by carrying out photo-crosslinking reaction on N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide and methacrylated hyaluronic acid to prepare adhesive hydrogel, wrapping a loading liposome, loading the loading liposome in a thread groove, and drying the loading liposome.

The technical concept of the invention is that firstly, a Chinese acupuncture needle (CA-needle) is taken as a precise delivery material penetrating deep cartilage tissue, and a threaded acupuncture needle (ST-needle) with a threaded groove on the surface of a needle body is designed by utilizing the performance of the CA-needle for breaking through a physical barrier strongly and the functions of micro-wound and precise positioning. The ST-needle can reach deep focus in a minimally invasive manner, hydrogel with adhesiveness is loaded in the thread groove of the ST-needle, and the hydrogel is wrapped with carrier liposome, so that substances such as medicines or biological materials and the like are smoothly delivered to subchondral bone to regulate abnormal secretion of TGF-beta 1, a stem cell migration channel of the subchondral bone is constructed, and the purpose of performing accurate positioning treatment on the subchondral bone is achieved.

However, since the diameter of the needle is in the micrometer range, the thread groove volume of the ST-needle is also very small, resulting in very limited medicine to be carried. The applicant initially adopted a means of directly loading the drug in the thread groove, and as a result, found that the drug delivery to the subchondral bone in a large amount cannot be achieved, the drug delivery efficiency is low, and precise positioning treatment of the subchondral bone cannot be achieved. Therefore, a brand new drug loading system needs to be constructed, so that the enrichment and the slow release of the drugs in the acupuncture needle groove are realized, and the drugs are smoothly delivered to a focus and then separated, so as to better perform accurate positioning treatment on subchondral bone.

On the basis of the above, the inventor selects hydrogel as a drug delivery carrier to prepare a drug-loaded hydrogel system, however, when the inventor tries to combine the hydrogel with ST-needle, the hydrogel can be loaded in the thread groove of ST-needle, but during acupuncture treatment, the following two problems are difficult to solve: firstly, hydrogel on the acupuncture needle is easy to be separated in a half way, and when the hydrogel passes through tissues such as skin, tendon, muscle, cartilage and the like of a human body, the hydrogel is easy to fall off from a thread groove and cannot smoothly reach a deep subchondral bone focus, so that the drug delivery rate is low; secondly, when the acupuncture treatment is finished and the needle is taken out, the hydrogel can not be smoothly and automatically separated from the threaded groove and then stays in the body, most of the hydrogel is still tightly adhered to the deep part of the threaded groove of the acupuncture needle and is taken out of the body, so that the drug delivery amount is very low, and the accurate positioning treatment effect is poor.

In view of the above phenomena, the inventors have made extensive research work with reference to a large number of documents, but have encountered great difficulties in the research process and experienced several failures, some of which are shown in comparative examples 1 to 7 of the present invention, and the results of the extensive failure research show that: the hydrogel can not be easily combined with a threaded acupuncture needle to realize accurate treatment on subchondral bone as envisaged by the inventor, and the delivery efficiency of the hydrogel in the practical application process is very low and only reaches about 20-30%. Therefore, how to ensure that the hydrogel loaded in the thread groove of the threaded acupuncture needle cannot be separated in advance in the body in the process of inserting the threaded acupuncture needle into the human body and the hydrogel can be smoothly separated from the thread groove and remained in the body when the needle is taken out is a great problem troubling researchers.

Finally, the inventor unexpectedly obtains a successful scheme through continuous innovation, innovatively designs an accurate delivery system of the adhesive hydrogel coating @ threaded acupuncture needle, and simultaneously realizes good application effects of resisting huge obstruction and preventing falling and separating acupuncture and hydrogel at a focus in an extremely ingenious mode, so that the delivery efficiency of the hydrogel reaches over 85 percent.

When the inventor constructs a hydrogel system, the hydrogel capable of adhering metal is innovatively designed, and the liposome carrying the drug is wrapped in the hydrogel, so that the long-term slow release of the drug is realized. Besides the properties of common hydrogel, methacrylated hyaluronic acid (HAMA) has a certain protection effect on cartilage, and N- [2- (3, 4-dihydroxyphenyl) ethyl]-2-methacrylamide (denoted DMA, CAS number: 471915-89-6, molecular formula: C ₁₂ H ₁₅ NO ₃ ) The methacryl group can participate in the photocrosslinking reaction of HAMA to obtain HAMA modified by dopamine group, which is a novel HAMA hydrogel (recorded as DMA @ HAMA) with an adhesive high-molecular interface. And then, combining the liposome with the hydrogel, loading the synthesized lipo @ DMA @ HAMA hydrogel system in the thread groove of the acupuncture needle, and drying to form a hydrogel coating so as to obtain the acupuncture needle-based hydrogel accurate delivery system.

When acupuncture penetrates human tissues, the hydrogel coating is tightly attached to the deep part of the thread groove and protected by the thread groove, so that the hydrogel accurate delivery system can resist huge obstruction and prevent falling without strong adhesion force. When the hydrogel enters the focus along with the acupuncture needle, the characteristics of the thread and the swelling characteristic of the hydrogel are skillfully utilized, and after the hydrogel absorbs body fluid to swell, the volume of the hydrogel rapidly expands and protrudes out of the thread groove, so that the hydrogel is in close contact with and adheres to peripheral tissues. Then, the needle is withdrawn while rotating the acupuncture along the direction of the thread, and the hydrogel is successfully retained in the focus.

The inventor also tries a method for combining other hydrogel components with an acupuncture needle, wherein the method comprises the steps of anchoring the medicine on a hydrogel network in a covalent bond mode and preparing a hydrogel medicine-carrying system with stronger adhesiveness, so that the medicine cannot be precisely delivered to the subchondral bone focus, the delivery efficiency of the medicine-carrying hydrogel only reaches 40-65%, and the technical effect of the invention cannot be realized.

Therefore, the scheme of the invention well breaks through the defect that the precise positioning treatment of the subchondral bone cannot be realized by combining the conventional hydrogel with the acupuncture needle, provides an innovative hydrogel precise delivery system based on the acupuncture needle, can well enable drugs or biological materials loaded by the hydrogel to penetrate a hard physical barrier of the cartilage through the threaded acupuncture needle, and smoothly reach the focus of the subchondral bone, and realizes the precise positioning treatment of the subchondral bone.

The hydrogel accurate delivery system based on the acupuncture needle is an accurate treatment system with great potential. The drug delivery device can be used for accurately delivering drugs for deep tissues such as subchondral bones and the like, can also be used for accurately delivering treatment for other focal parts with hard physical barriers, and can also be used for accurately delivering various biological materials.

Furthermore, the mass ratio of the N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide to the methacrylated hyaluronic acid in the hydrogel is 1. The hydrogel prepared according to the proportion can well ensure that the hydrogel can accurately reach subchondral bone under the protection of the threaded acupuncture needle, and can be easily separated from the threaded groove and left in the body. The hydrogel coating can be well adhered to the thread groove and does not exceed the surface of the acupuncture needle body.

Further, the mass ratio of the adhesive hydrogel to the liposome is 4 to 6.

Further, the diameter of the needle body of the acupuncture needle is 0.2-0.5mm, the thread pitch of the thread groove is 0.5-1mm, and the depth of the thread groove is 0.05-0.085mm.

Further, the liposome is used for loading a drug or a biological material.

Further, the medicament comprises baicalein.

The invention further provides a preparation method of the hydrogel precision delivery system based on the acupuncture needle, which comprises the following steps:

(1) Carving a groove on the surface of a needle body of a common acupuncture needle, extending along the needle body to form a thread shape, and preparing a thread microneedle;

(2) Preparing liposome with uniform size by a film method, reacting the liposome with a mixed solution of N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide and methacrylated hyaluronic acid, and carrying out photocrosslinking to obtain a hydrogel solution;

(3) And (3) loading the hydrogel solution obtained in the step (2) into the threaded groove of the threaded microneedle obtained in the step (1), curing through photo-crosslinking, and drying to obtain the accurate delivery system.

Further, the preparation steps of the thin film method in the step (2) are as follows: dissolving lecithin and cholesterol in chloroform according to the mass ratio of 3.

In the above method for preparing liposome, the drug or biomaterial can be added to form liposome loaded with drug or biomaterial, and then combined with hydrogel.

The invention also provides an application of the hydrogel precision delivery system based on the acupuncture needle, which is to use the precision delivery system as a carrier of medicines or biological materials for precision delivery to a focus site. The precise delivery system is particularly used as a carrier for precisely delivering the medicament to the spinal cord or the cartilage tissue, and particularly used for precisely delivering subchondral bone.

The invention has the following beneficial effects:

(1) The invention provides an acupuncture needle-based hydrogel accurate delivery system, which can firmly adhere loaded hydrogel in a thread groove of an acupuncture needle and accurately feed the loaded hydrogel into a focus to be treated under the protection of the thread groove. Meanwhile, the hydrogel is extruded and adhered with surrounding tissues after absorbing body fluid and expanding, so that the hydrogel is remained in a focus and the sustained release of the medicine is realized.

(2) The hydrogel accurate delivery system based on the acupuncture needle can easily break through the cartilage physical barrier and enter subchondral bones to realize accurate treatment of the subchondral bones, has high drug delivery rate, and has 80-85% of hydrogel transmission efficiency which is obviously higher than that of the traditional acupuncture needle (only 25%).

(3) When the hydrogel accurate delivery system based on the acupuncture needle is used for carrying out accurate positioning treatment on the subchondral bone of an OA rat, abnormal remodeling of the subchondral bone can be effectively inhibited, so that the degeneration and degradation of the cartilage are relieved, and the OA state of an illness is improved.

Drawings

FIG. 1 is a schematic diagram of CA-needle and ST-needle; a) Clinical use of CA-needle; b) A morphological schematic of CA-needle; c) Taking 360-degree macro photography on the micro-needle by using a camera; d) 360 ° display of the CA-needle tip; e) 360 ° of the ST-needle tip.

FIG. 2 is a schematic representation of hydrogel modification of ST-needle; a) A schematic diagram of hydrogel synthesis with adhesive metal surface properties; b) Transmission electron microscopy of liposomes; c) Particle size distribution of liposomes in aqueous solution; d) Scanning electron microscope images of lipo @ DMA @ HAMA hydrogel; e) Schematic representation of hydrogel modified ST-needle; f) The CA-needle tip was shown 360 ° after loading the hydrogel; g) ST-needle tips were shown 360 ° after loading the hydrogel (hydrogel stained dark blue for easy visualization); h) In vitro release of BAI by Lipo-BAI and Lipo-BAI @ DMA @ HAMA (n = 3).

FIG. 3 shows the perforation experiments on pig articular cartilage using CA-needle and ST-needle; a) Three major articular cartilages in pigs; B-D) femoral, tibial, talar articular cartilage perforation experiment: i) Displaying the photo in full; ii) a partial display photograph; iii) CA-needle in cartilage; iv) endochondral ST-needle; f) The time (n = 3) for the penetration of CA-needle and ST-needle into cartilage (black dashed line is cartilage contour line, blue dashed line is CA-needle and ST-needle contour line; and NS: it has no meaning).

FIG. 4 is a schematic representation of hydrogel transport through ST-needle; a) Schematic representation of ST-needle transport hydrogel; wherein i) CA-needle is unable to transport the hydrogel into the tissue, while ST-needle is successful in transporting the hydrogel into the tissue; ii) the dried hydrogel absorbs fluid from the tissue, the fluid swells and comes into intimate contact with the surrounding tissue, the ST-needle rotates and is removed, leaving the hydrogel behind in the tissue (blue arrows indicate fluid absorbed by the hydrogel in the surrounding tissue); b) In-vitro experiments verify the effect of the ST-needle transport hydrogel; wherein i) the hydrogel loaded CA-needle was inserted into pig cartilage (black arrow indicates hydrogel blocked by cartilage); ii) histological sections of subchondral bone; iii) Inserting the hydrogel-loaded ST-needle into pig cartilage; iv) histological sections of subchondral bone (black arrows indicate successful transport of hydrogel to subchondral bone); c) Cy-7 labeled hydrogel was used to verify that ST-needle was able to transport more hydrogel into cartilage than CA-needle; d) Quantitative analysis of fluorescence intensity (n = 3) (. About.p < 0.001).

FIG. 5 shows the cellular and molecular mechanisms of ST-needle treatment of OA; a) Schematic representation of inhibition of cytokine synthesis by ST-needle promotes stem cell migration; b) X-ray shows that ST-needle accurately reaches subchondral bone; c) Western blot of 15-LOX-1 and TGF-beta 1; D-E) carrying out quantitative analysis on Western blot results of 15-LOX-1 and TGF-beta 1; F-G) PCR map of mRNA of 15-LOX-1 and TGF-. Beta.1; h) Experimental schematic, ST-needle promotes MSC migration; i) Representative fluorescence images show vital staining of MSCs migrating in cartilage; j) Viable cells were quantified (n = 3) (. P < 0.05,. P < 0.01,. P < 0.001).

FIG. 6 ST-needle treatment of OA rats; a) Cy-7 marks the hydrogel, and verifies that the hydrogel can be retained in a rat body for a long time and continuously releases the drug; b) Quantitative fluorescence intensity analysis (n = 3); c) Micro-CT showed subchondral bone in rats at 4 and 8 weeks; e) BMD, BV/TV, tb quantification, subchondral bone Th (n = 3); (ns: no significance,. P < 0.05,. P < 0.01,. P < 0.001).

FIG. 7 shows that treatment reduced cartilage degeneration in OA rats (6 per group); a) Representative images of H & E staining of articular cartilage for each group; b) OARSI score for each group of articular cartilage; c) Safflower O-fast green staining represents images showing histological changes in five groups of cartilage; d) Relative glycosaminoglycan content for each group; e) Apoptotic cells TUNEL stained representative sections; f) Quantification of TUNEL positive cells; g) Type II collagen immunofluorescent staining of representative sections; h) Quantifying type II collagen positive cells; i) MMP-13 immunofluorescence staining representative sections; j) Quantification of MMP-13 positive cells; (ns: no significance, P < 0.05, P < 0.01, P < 0.001).

Fig. 8 is a schematic view showing hydrogel delivery using a horizontal groove acupuncture needle according to comparative example 1.

FIG. 9 is a schematic view showing hydrogel delivery using a screw-grooved needle for acupuncture in comparative example 2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail with reference to the following embodiments, it should be noted that the following embodiments are only for explaining and illustrating the present invention and are not intended to limit the present invention. The invention is not limited to the embodiments described above, but rather, may be modified within the scope of the invention.

Example 1

Design of acupuncture needle with screw thread groove

Taking a conventional chinese acupuncture needle (CA-needle) made of stainless steel as an example, the acupuncture application is shown in fig. 1, part a, the appearance of CA-needle is shown in fig. 1, part B, the tip of CA-needle is photographed and observed with a macro lens (see part C in fig. 1), it can be seen that the tip of CA-needle is sharp and smooth, and there is no special structure to support hydrogel (see part D in fig. 1).

In order to obtain hydrogel-supporting ability of the CA-needle, a threaded needle (ST-needle) was obtained by designing a special thread structure at the tip of the needle. The groove width of the ST-needle was about 200. Mu.M as shown by the microphotograph, and the groove was threaded from the needle tip to the needle tail (see section E in FIG. 1).

It should be noted that the threaded needle of the present invention is merely an example, and those skilled in the art can continuously modify the threaded needle, including the width and depth of the groove, the density of the thread, the extending distance of the thread at the needle tip, etc., so that the threaded needle can be adapted to various hydrogels having different physical properties to meet the different therapeutic requirements for various diseases.

(II) preparation of hydrogel

In order to load hydrogel well into the thread groove of ST-needle to achieve accurate drug delivery, the inventors conducted a great deal of research on the design of hydrogel, the research process of which is described in the following comparative examples 1-7. However, as shown in comparative examples 1 to 7, the hydrogel and the threaded acupuncture needle have a problem that the hydrogel easily slips from the threaded groove and a problem that the hydrogel is not smoothly separated from the threaded groove when the needle is removed, resulting in a low hydrogel delivery rate of only 20 to 65%, and thus, the drug delivery efficiency is not high and the therapeutic effect is poor.

Finally, through a great deal of research and innovation, the inventor designs a hydrogel with a metal adhesion function, a drug-loaded liposome is constructed in the hydrogel, and the hydrogel is dried to form a coating, so that the effect of smoothly delivering the drug to subchondral bone and smoothly separating the drug from the thread groove is realized, the delivery efficiency of the hydrogel reaches 80-85%, the drug delivery efficiency is high, the curative effect is remarkable, and the sustained-release time of the drug is long (as part A in fig. 2).

The preparation method of the hydrogel system successfully constructed by the invention comprises the following steps:

firstly, a drug-loaded liposome with uniform size (taking the drug-loaded baicalein as an example) is prepared by using a film method, and the specific preparation method comprises the following steps: 60mg of lecithin, 20mg of cholesterol and 8.0mg of baicalein were dissolved in a round-bottomed flask containing 30mL of chloroform, and the mixture was heated at 35 ℃ for 1 hour to completely evaporate the organic solvent, thereby obtaining a lipid film adhered to the bottom of the flask. Then, 3mL of double distilled water was added to the flask, and sonication was performed at 25 ℃ for 20 minutes to completely dissolve the lipid membrane in water, thereby obtaining a bilayer liposome having a micron size. The diameter of the liposome was measured by instrument to be about 100nm (as shown in part B of FIG. 2), and the particle size distribution was as shown in part C of FIG. 2. A strong probe sonication solution (60 single pulses/min, 130W) can be used for 5 minutes to prepare drug-loaded liposomes of smaller particle size.

Secondly, preparing the adhesive hydrogel by the following specific steps: weighing a proper amount of HAMA (methacrylic acid hyaluronic acid) freeze-dried powder and DMA (N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide) powder, dissolving in distilled water, controlling the HAMA concentration to be 3% -5% and the DMA concentration to be 1%, fully dissolving and uniformly stirring, and then adding 1% of liposome into the mixed solution to prepare Lipo @ DMA @ HAMA. The hydrogel can be cured under the ultraviolet crosslinking of 350nm wavelength to form solid hydrogel which is loaded with liposome and has an adhesion function. By observing Lipo @ DMA @ HAMA through an electron microscope, it can be seen that liposome nanoparticles (see part D in FIG. 2) with a diameter of about 100nm are uniformly distributed in the porous hydrogel.

The prepared DMA @ HAMA hydrogel and the threaded acupuncture needle are subjected to adhesion performance test, and the method comprises the following steps:

approximately 0.5mL each of the prepared DMA @ HAMA solution and the same concentration of HAMA solution was placed in the bottom of the centrifuge tube. The needle tip of the ST-needle was inserted between the two hydrogels and irradiated with UV light to cure the two hydrogels. Subsequently, the ST-needle tail is held in hand and the ST-needle is lifted. As a result, the ST-needle can be easily pulled out of the HAMA hydrogel without the adhesion function, while the DMA @ HAMA hydrogel with the adhesion function can well adhere to the ST-needle, and the ST-needle cannot be separated from the hydrogel.

(III) construction of hydrogel precision delivery system based on acupuncture needle

In order to prepare an acupuncture needle-based hydrogel accurate delivery system, the lipo @ DMA @ HAMA hydrogel is combined with ST-needle, and the specific method comprises the following steps: a proper amount of the aqueous solution of Lipo @ DMA @ HAMA was aspirated by a 1mL syringe, and the solution was dropped onto the CA-needle tip and into the ST-needle threaded groove, respectively, while being cured by ultraviolet irradiation (see, e.g., FIG. 2).

The hydrogel was stained dark blue, and the hydrogel-loaded acupuncture needle was photographed at a macro distance. As a result, it was observed that the hydrogel on the tip of the CA-needle was directly exposed on the metal surface and was significantly higher than the contour of the acupuncture needle (see F in FIG. 2); whereas the hydrogel on ST-needle was contained within the thread recess and did not rise above the acupuncture profile (see section G in figure 2). Therefore, the thread groove structure of ST-needle can well protect the hydrogel loaded in the ST-needle during the process of puncturing human tissues, and the construction success of the hydrogel precision delivery system based on the acupuncture needle is shown.

(IV) hydrogel drug-loaded Release Performance test

The drug Baicalein (BAI) is loaded in liposome (Lipo) and Lipo-BAI @ DMA @ HAMA hydrogel respectively to obtain two biological materials of Lipo-BAI and Lipo-BAI @ DMA @ HAMA-BAI. The two biological materials are placed in a dialysis membrane, soaked in PBS at 37 ℃, and the content of the drugs in the PBS is periodically detected. The drug release within Lipo-BAI was found to be faster, having been released by about 80% by day 5; the Lipo-BAI @ DMA @ HAMA-BAI biomaterial has a better sustained-release effect, and only releases about 50% of the medicament on the 5 th day. Further testing showed that drug release could last for about 3 weeks (see fig. 2, part H). Therefore, the Lipo @ DMA @ HAMA hydrogel material has very ideal drug slow release performance.

Example 2

Capability verification of hydrogel precision delivery system based on acupuncture needle for penetrating cartilage and reaching subchondral bone

To verify the ability of ST-needle to penetrate cartilage to deliver hydrogels to subchondral bone, pigs were selected as model animals for ex vivo experiments, and the three articular cartilage surfaces that are most common clinically were selected: femoral condyles, tibial plateau, talus to verify that ST-needle can be widely used for precise treatment of joint diseases in a plurality of different locations (see part a in fig. 3). Meanwhile, a common acupuncture needle (CA-needle) was selected as a control group. CA-needle was used on the left side of each articular surface and ST-needle was used on the right side, and images were recorded using macro photography technique for the case where the microneedles penetrated the cartilage. After the microneedle penetration test was completed, the penetrated portion was dissected to see whether the microneedles could enter the subchondral bone, and the depth of penetration of the microneedles into the subchondral bone was measured (see portions B-D in fig. 3).

From the measurements, it can be seen that the depth of penetration of both microneedles exceeds the thickness of the cartilage (see E in fig. 3) in the cartilage of the femur, tibia and talus. Therefore, the Chinese acupuncture needle can effectively penetrate through the cartilage physical barrier to reach the subchondral bone, and the capacity of ST-needle breaking through the physical barrier is not weakened after the threaded groove modification is carried out on the tip of the acupuncture needle. Statistics were made on the time of insertion of the needle for each experiment (see section F in fig. 3), and although the cartilage of the talus is relatively hard and requires more time to insert the subchondral bone, the total time of insertion of the needle is within 1 minute.

In conclusion, ST-needle has excellent capability of penetrating cartilage into subchondral bone, and has good clinical application prospect.

(II) capability verification of hydrogel delivery system based on acupuncture needle for accurate hydrogel delivery

The hydrogel coating on the tip of a conventional acupuncture needle (CA-needle) is blocked on the cartilage surface by the cartilage while the hydrogel coating on ST-needle is protected by the thread groove as the needle reaches the subchondral bone (see section i in FIG. 4). After ST-needle reaches the subchondral bone, the loaded hydrogel absorbs water in body fluid and expands, so that the ST-needle firmly fits with the surrounding subchondral bone. As the ST-needle rotation recedes, the hydrogel was successfully left behind in the subchondral bone (as shown in FIG. 4, panel II, part A).

Due to the micrometer-sized size of the acupuncture needle, it is difficult to obtain a clear picture of the above-mentioned process. Therefore, a screw was chosen as the enlarged model of ST-needle. First, as can be seen from the photo of the real object, the hydrogel was successfully "hidden" in the thread groove after loading the hydrogel into the thread groove, photo-curing and proper air-drying (see section i in B of FIG. 4). Subsequently, a wooden plate was used to simulate cartilage and subchondral bone, a hydrogel-loaded screw was inserted into the hole of the wooden plate, and PBS simulated body fluid was dropped to expand the hydrogel by absorbing water (see section B ii in fig. 4). As the screw is rotated backward, the hydrogel is retained in the holes of the wood plate (see section B, section iii and section vi in FIG. 4) as can be clearly seen by the photograph.

In addition, hydrogel-loaded ST-needle with fluorescent label was made and cartilage from the isolated porcine femur was removed to verify the ability of ST-needle to deliver hydrogel (see panel C in fig. 4). From the fluorescence intensity, the fluorescence value of the tip of the CA-needle after the needle is inserted into the cartilage and pulled out is reduced by only 25% compared with the initial value, which shows that only a small amount of hydrogel is remained in the cartilage. While the fluorescence value of ST-needle decreased by 82% of the initial value, indicating that ST-needle was able to deliver better hydrogel to subchondral bone (see panels D and E in FIG. 4).

(III) hydrogel precision delivery system biocompatibility test based on acupuncture needle

To evaluate the potential clinical application of drug-loaded Lipo @ dma @ hama, the in vitro cytotoxicity of Lipo-BAI and Lipo-ga @ dma @ hama on MSC was studied. After 1, 3 and 5 days of co-cultivation with different percolates, live/dead tests and CCK-8 tests were carried out. Live/dead staining showed that three groups of cells were almost alive with only a few dead cells in 5 days of culture. The cell density increased with the time of culture. In addition, the CCK-8 experiment result shows that the proliferation activity of the three groups of cells has no significant difference at all time points. These results indicate that the biomaterial prepared by the present invention has good biocompatibility with chondrocytes.

(IV) acupuncture needle-based hydrogel precision delivery system clinical treatment cytotoxicity test

Sterile ST-needle was taken and co-cultured with MSC for 1h to simulate the in vivo condition of the acupuncture needle. When the cells were stained live and dead, it was found that all MSCs around ST-needle were stained green, and almost no dead cells were stained red. After ST-needle was removed, MSCs were cultured for an additional 24h and live-dead cell staining was performed. It can be seen that the cell density of MSCs was significantly increased compared to 24h and also there were few dead cells stained red.

In conclusion, when the ST-needle is used for puncturing a human body for precise treatment, the ST-needle has no toxicity to surrounding cell tissues, and toxic substances are not remained after the ST-needle is pulled out to influence the growth of cells. (V) cellular molecular mechanism verification of hydrogel delivery system based on acupuncture needle for delivering hydrogel to treat OA

The mechanism for the precise treatment of OA using ST-needle delivered hydrogels has two aspects: after ST-needle delivered the hydrogel to subchondral bone, the hydrogel was retained in subchondral bone, releasing BAI continuously, and regulating abnormal remodeling of subchondral bone. ST-needle after the cartilage was extracted after the treatment was completed, micro-scale pores were left in the cartilage to serve as channels for MSC migration, so that the MSCs in the subchondral bone could migrate to the cartilage surface to promote cartilage repair (see section A in FIG. 5).

The rat subchondral bone cells were extracted from rats by establishing a rat OA model, setting up a control group and a treatment group (treated with ST-eedle, as shown in fig. 5, section B), and after extracting proteins and RNAs, WB and PCR assays were performed (as shown in fig. 5, section C-G). As a result, it was found that mRNA and protein of 15-LOX-1 and TGF-. Beta.1 were significantly elevated in rat subchondral bone in the OA group as compared with the control group, demonstrating that OA did cause abnormal secretion of subchondral bone cytokines. After the ST-needle treatment carried with the BAI medicine, mRNA and protein of 15-LOX-1 and TGF-beta 1 are remarkably reduced compared with the OA group, and the fact that the ST-needle precision treatment can effectively reverse secretion of abnormal cytokines is proved.

To verify that the micropores left after ST-needle treatment can aid in MSC migration, a cell migration assay was designed (see fig. 5, section H). The cartilage after the treatment with ST-needle was simulated by pricking dozens of through-holes on the cartilage disc of knee joint cartilage disc of rabbit using ST-needle, and used as a Perforated cartilage group (Perforated cartilage). Meanwhile, intact cartilaginous discs not punctured by ST-needle were used as the intact Cartilage group (Cartilage). The two cartilages were placed in Transwell chambers separately and cells were stained in the Transwell chambers at 12h and 24h, respectively, by adding MSC and culture medium according to the Transwell routine protocol (see section I in fig. 5). It is evident that the number of migrating cells in the Cartilage group is significantly less than in the blank group due to the blockage of the Cartilage disc; the Performed Cartilage group can help MSC to cross Cartilage to block cell migration because ST-needle leaves micro-pores on Cartilage disc, so that the number of migrated cells is significantly higher than that of Cartilage group (see J part in FIG. 5). In conclusion, the micropores created using ST-needle treatment are effective in helping MSC migration from subchondral bone to the cartilage surface.

(VI) research on alleviating subchondral bone lesions by using hydrogel precision delivery system based on acupuncture needle

In order to study the effect of lipo @ DMA @ HAMA in long-term retention in subchondral bone, lipophilic membrane fluorescent dye DiI was used to label liposomes. Labeled lipo @ dma @ hama was delivered to subchondral bone by ST-needle and fluorescence imaging was performed continuously for 5 weeks using IVIS (as in part a of fig. 6). Articular cavity injection with the fluorescent dye DiI alone was used as a control.

The results of the study showed that the fluorescence intensity of the control group decreased sharply in the first two days, and almost no fluorescence signal was detected on day 2, indicating that the retention effect of the free drug in the joint cavity was weak. In contrast, the fluorescence intensity of the lipo @ DMA @ HAMA hydrogel decreases very slowly, and decreases by about 50% only in week 3, and the fluorescence signal can last for more than one month, indicating that the lipo @ DMA @ HAMA hydrogel has very excellent retention effect in subchondral bone, ensuring that BAI can be released continuously for a long time (as part B in FIG. 6).

To further verify the curative effect of the accurate treatment of ST-needle on OA, a rat OA model was established by injecting iodoacetic acid into the joint cavity, and a blank Control was established (Control group). After successful molding, OA rats were treated by joint cavity injection with PBS (PBS group) and BAI (BAI group), and ST-needle, respectively.

Micro CT is widely applied to measurement of bone tissues at present, and compared with uncertainty of bone tissue slices and limitation of two-dimensional images, micro CT can perform X-ray scanning and three-dimensional reconstruction on the bone tissues, and can observe and measure data such as bone density, bone morphology and bone trabecular structure more intuitively. Two sampling time points of 4 weeks and 8 weeks are selected, rat subchondral bones are scanned through micro CT to obtain a three-dimensional model of the knee joint of the rat (as shown in a part C-D in figure 6), and three-dimensional related parameters (as shown in a part E-G in figure 6) related to the rat subchondral bones are obtained.

The experimental results show that: compared with the Control group, the bone density (BMD), bone fractional volume (BV/TV) and trabecular bone thickness (Tb.Th) of the PBS group were all increased and were statistically significant. The bone density, bone fraction volume, trabecular thickness of the treatment group treated with BAI were all lower than those of the PBS group; meanwhile, the ST-needle group treated with the ST-needle precision had significantly lower bone density, bone fraction volume, trabecular thickness than the PBS group, and had statistical significance. In conclusion, the abnormal remodeling of subchondral bone can be significantly improved by the precise treatment of subchondral bone of OA rats using ST-needle.

(VII) ability of needle-based hydrogel precision delivery system for improving cartilage lesions

Taking the knee joint of a rat from which the animal experiment is carried out to the eighth week, carrying out paraffin-embedded section, and carrying out morphological staining and immunofluorescence detection. Hematoxylin eosin (H & E) staining (as in section A of FIG. 7) and Safranin O-fast green staining (as in section B of FIG. 7) showed typical osteoarthritis features. The PBS group had irregular surfaces and had the most pronounced erosive fissures, the BAI group was the second, and the ST-needle group was the least pronounced.

The results of the OARSI scoring are shown in section C of fig. 7. The decrease in OARSI scores was seen in the other treatment groups compared to the PBS group, with the ST-needle group being the best, a 53.50% decrease, followed by a 27.95% decrease in the BAI group. It was shown that ST-needle maintained the cartilage matrix group more effectively (see section D in FIG. 7). Subsequently, apoptosis of chondrocytes was detected using TUNEL staining (as in part E of fig. 7), which indicates that: the ST-needle group had the least apoptotic cells (green staining) compared to the control group, and the BAI group was inferior.

The positive staining cells were quantitatively detected (as shown in fig. 7, panel F), and the results showed that the ST-needle group had the lowest apoptosis rate, 66.48% lower and the BAI group 25.88% lower than that of the PBS group. After ST-needle precise treatment, the apoptosis rate of chondrocytes under oxidative stress is reduced from 38.36 +/-5.48 percent to 12.86 +/-4.27 percent, which is obviously superior to 28.43 +/-5.87 percent of BAI group.

Immunofluorescent staining was used to detect the expression of the major biomarkers Collagen II and MMP-13 in cartilage (see FIG. 7, section G-J). The results show that: the level of collagen type II (red staining) expression was decreased in each treatment group compared to the control group, with the ST-needle group having the least decrease followed by the BAI group. The number of positively stained cells is shown in section H of FIG. 7, and compared with the PBS group, the expression level of collagen II in the ST-needle group is highest and increased by 128%, and the expression level of collagen II in the BAI group is increased by 100%.

MMP-13 is the main enzyme targeting cartilage degradation, and can degrade not only type II collagen in cartilage, but also proteoglycan, type IV and IX collagen, osteonectin and perlecan in cartilage. Thus, MMP-13 plays an important role in OA. The experimental results showed that MMP-13 expression level in the PBS group was significantly increased, which also resulted in degradation of cartilage matrix and progression of disease in OA. Compared with the PBS group, the expression level of MMP-13 in the ST-needle group is reduced by 66.48%, and the expression level in the BAI group is reduced by 25.88% (see J part in FIG. 7).

The above results indicate that, compared to the treatment means of injecting BAI into the joint cavity, the use of ST-needle for precise treatment can significantly inhibit abnormal remodeling of subchondral bone, thereby alleviating damage and degeneration of cartilage. The methods of the invention provide an attractive strategy for the treatment of OA.

The following comparative examples examine the use of drug delivery systems constructed by different combinations of hydrogel and threaded needles for precise positioning treatment of subchondral bone, and the results show that: none of the following approaches provides a good and precise delivery of drugs to subchondral bone, and their drug delivery efficiency is low.

Comparative example 1

Referring to an improvement of the needle in patent document CN 108338917A, the inventor constructed a plurality of horizontal grooves on the needle body of the needle, prepared HAMA hydrogel and lipo @ dma @ HAMA hydrogel by following the protocol described in example 1, and loaded the hydrogel on the needle having the horizontal grooves to construct a hydrogel precision delivery system based on the needle (as shown in fig. 8). As a result, the acupuncture needle with the horizontal groove structure can protect hydrogel from falling off the needle body and can directly reach subchondral bone lesions. However, in subsequent experiments, it is found that the hydrogel loaded in the horizontal groove is also taken out while the acupuncture needle is pulled out, and the hydrogel cannot be retained in a lesion, and the test according to the method of example 1 shows that the reduction amount of the hydrogel is only 35%, which indicates that the hydrogel delivery efficiency of the hydrogel precision delivery system is not higher than 35%.

Comparative example 2

In order to solve the problems of comparative example 1, the inventors constructed a needle having a screw-grooved structure, and prepared a hydrogel with reference to the protocol described in example 1, and loaded a HAMA hydrogel through the needle having the screw-grooved structure, and constructed a hydrogel precision delivery system based on the needle, so as to achieve the purpose of detaining the hydrogel in the lesion while withdrawing the needle (as shown in fig. 9). However, in subsequent experiments, it was found that, since the thread groove is formed around the entire acupuncture needle body by one circle, in order to ensure the mechanical strength of the acupuncture needle, the depth of the thread groove is significantly shallower than that of the horizontal groove in comparative example 1, which results in that the protective force of the thread groove to the hydrogel is weaker than that of the horizontal groove when the acupuncture needle is inserted into the human body, a large amount of HAMA hydrogel is dropped from the thread groove during acupuncture treatment, the subchondral bone cannot be smoothly reached, and the transport efficiency of the hydrogel is lower than that of the horizontal groove, and the decrease amount of the hydrogel is only 30% as measured according to the method of example 1, which indicates that the hydrogel precision delivery system has a low hydrogel transport efficiency of not higher than 30% and a hydrogel transport efficiency of not lower than that of the horizontal groove.

Comparative example 3

In order to solve the problems of comparative example 2, the inventors attempted to develop a hydrogel having an adhesive function to improve the protective property of the hydrogel in the thread groove and to improve the transportation efficiency thereof. The inventors have tried various "hydrogel-adhering" solutions and found that there are many difficulties in adhering the hydrogel to the surface of a metal needle.

(1) ST-needle was prepared for loading the hydrogel according to the protocol of example 1, and with reference to the preparation of adhesive hydrogels reported by Yang Gao et al (A Universal Strategy for gauge addition of Hot Soft Material, DOI: 10.1002/adfm.202003207), polymer chains were penetrated into the hydrogel and the adherends, triggering the cross-linking of the polymer, forming new topological entanglements with the existing network, and achieving topological Adhesion. However, when the hydrogel prepared by adopting the topological adhesion manner is combined with the ST-needle, the chemical modification of the surface of the acupuncture needle is required, which further reduces the depth of the thread groove, is not favorable for the adhesion of the hydrogel, and cannot successfully realize the accurate delivery of the hydrogel to the subchondral bone, and the delivery rate of the hydrogel is still low, only 28%, when tested by the method of example 1.

(2) ST-needle was prepared for loading the hydrogel according to the protocol of example 1, and an adhesive hydrogel capable of exhibiting strong, reversible, highly reproducible adsorption on silicon wafers, glass was prepared with reference to an artificial biomimetic octopus sucker microstructure adhesive patch reversibly usable in wet/dry adhesive systems reported by Sangyul Baik et al (A wet-solar adhesive patch assisted by physical properties of cups of octopi, DOI:10.1038/nature 22382). However, this type of adhesion is only suitable for relatively flat surfaces and does not have high adhesion to irregular surfaces, which greatly limits its application. When the inventors tried to combine the adhesive hydrogel with a threaded needle, and found that it was not suitable for the adhesion of the threaded groove of the needle, the hydrogel was delivered only at 35% efficiency as tested in the method of example 1.

In summary, it is extremely difficult to find a suitable adhesion scheme due to the metal material of the acupuncture needle, the limited surface area and the uneven thread structure.

Comparative example 4

In order to solve the problems of comparative example 3, inspired by the catechol group of mussel mucus, the inventors obtained a hydrogel having a metal-adhesion function by modifying dopamine on a HAMA hydrogel. The scheme does not need to carry out chemical modification on the metal surface, has no special requirements on the physical condition of the adhesion surface, can ensure that the hydrogel is firmly adhered in the thread groove of the acupuncture needle, and ensures that the hydrogel cannot be separated from the thread groove. However, subsequent research finds that although hydrogel with a strong adhesion function is prepared, the hydrogel cannot be smoothly separated from the thread groove after reaching the focus, and sustained release of the drug left in the focus during needle withdrawal cannot be achieved. The hydrogel delivery system was found to have a delivery efficiency of 55% for the hydrogel when tested according to the method of example 1.

Comparative example 5

In order to solve the problems of comparative example 4, the inventors dried the hydrogel adhered to the grooves of the needle thread prepared in example 4, but since the adhesive hydrogel supported the drug by the aqueous solution in the inside, the hydrogel supported the drug after drying to form a coating layer was largely lost. The results show that although the hydrogel delivery system has a delivery efficiency of 62% to the hydrogel, the drug loading in the hydrogel is reduced by more than 60%, and the treatment method still cannot successfully realize accurate delivery of the drug in the hydrogel to the subchondral bone.

Comparative example 6

In order to solve the problems of comparative example 5, the inventors tried to covalently bond the drug and the adhesive hydrogel network prepared in comparative example 4 by covalently anchoring the drug within the hydrogel network so that the drug is not lost during the drying process. However, subsequent experiments found that the drug release was extremely difficult due to the relatively strong covalent bond, and a large amount of drug remained inside the hydrogel network structure, showing that the drug release amount was only 50% after 5 days of delivery.

Comparative example 7

Compared with the prior art, the hydrogel network prepared in example 4 is modified on the basis of comparative example 6, and ionic bonds with relatively weak binding capacity are used for connecting the drug and the hydrogel network, and the scheme has a good effect on water-soluble drugs, but for fat-soluble drugs, the drug loading amount in the hydrogel is very limited due to very low solubility in water, and the loading amount on the fat-soluble drugs is not more than 10%, so that the application of the hydrogel + acupuncture system in medical treatment is greatly limited.

The results of the above studies of comparative examples 1 to 7 show that: in order to combine the acupuncture needle and hydrogel to construct a drug delivery system of hydrogel and acupuncture needle, there is a great difficulty in practical application, and it is very difficult to develop an acupuncture needle structure capable of loading hydrogel well so as to be able to smoothly deliver hydrogel to deep tissue lesions such as subchondral bone and the like, and to ensure that the hydrogel is highly efficient in delivery during acupuncture, and the hydrogel can be smoothly separated to remain in the lesions during needle withdrawal.

Finally, the invention selects the construction of a liposome secondary structure in the hydrogel to form a liposome-hydrogel drug-loading system. Due to the unique structure of the liposome, not only can water-soluble medicines be loaded, but also a large amount of fat-soluble medicines can be loaded. Meanwhile, chemical combination such as ionic bonds and the like is formed between the liposome and the hydrogel network, so that the liposome can be stably combined with the hydrogel network, and a liposome-hydrogel drug-loading system which is stable in physical structure and can load various drugs is constructed. On the basis, the invention innovatively designs a hydrogel coating @ acupuncture system which can simultaneously realize 'resistance to huge obstruction and prevention of falling-off' and 'separation of acupuncture and hydrogel at a focus' in an extremely ingenious mode.

The invention firstly constructs a thread groove at the tip of the acupuncture needle, and prepares the DMA @ HAMA hydrogel with an adhesive high molecular interface by utilizing the photocrosslinking reaction of DMA and HAMA. Subsequently, a hydrogel coating was formed by loading the synthesized lipo @ dma @ hama hydrogel system into the threaded groove and drying it. When acupuncture penetrates human tissues, the hydrogel coating is tightly attached to the deep part of the thread groove and protected by the thread groove, so that great resistance to falling off can be realized without strong adhesion force. After entering the focus, the characteristics of the thread and the swelling characteristic of the hydrogel are ingeniously utilized. After the hydrogel absorbs body fluid to swell, the volume of the hydrogel rapidly expands and protrudes out of the thread groove, so that the hydrogel is in close contact with peripheral tissues and adheres to the peripheral tissues. When the acupuncture needle is rotated and withdrawn along the direction of the thread, the hydrogel is successfully retained in the focus, so that the hydrogel accurate delivery system based on the acupuncture needle is well constructed, and experiments prove that the hydrogel accurate delivery efficiency of the hydrogel accurate delivery system can reach 80-85 percent.

Claims

1. An accurate hydrogel delivery system based on an acupuncture needle is characterized by comprising the acupuncture needle and a hydrogel coating, wherein a threaded groove is formed in a needle body of the acupuncture needle, and the hydrogel coating is adhered to the interior of the threaded groove of the acupuncture needle; the hydrogel coating is prepared by carrying out photo-crosslinking reaction on N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide and methacrylated hyaluronic acid to prepare adhesive hydrogel, wrapping a loading liposome, loading the loading liposome in a thread groove, and drying the loading liposome.

2. The needle-based hydrogel precision delivery system according to claim 1, wherein the mass ratio of N- [2- (3, 4-dihydroxyphenyl) ethyl ] -2-methacrylamide to methacrylated hyaluronic acid is 1.

3. The needle-based hydrogel precision delivery system according to claim 1, wherein the mass ratio of the adhesive hydrogel to the liposome is 4 to 6.

4. The needle-based hydrogel precision delivery system according to claim 1, wherein the needle body of the needle has a diameter of 0.2 to 0.5mm, a pitch of the thread groove of 0.5 to 1mm, and a depth of the thread groove of 0.05 to 0.085mm.

5. The needle-based hydrogel precision delivery system according to claim 1, wherein the liposome is loaded with a drug or a biomaterial.

6. The needle-based hydrogel precision delivery system according to claim 5, wherein the drug comprises baicalein.

7. A process for preparing the needle-based hydrogel precision delivery system for acupuncture according to any one of claims 1 to 6, comprising the steps of:

(1) Carving a groove on the surface of a needle body of a common acupuncture needle, extending along the needle body to form a thread shape, and preparing into a thread micro-needle;

(3) And (3) loading the hydrogel solution obtained in the step (2) into the threaded groove of the threaded microneedle obtained in the step (1), curing through photo-crosslinking, and drying to form a hydrogel coating, so as to obtain the acupuncture needle-based hydrogel precision delivery system.

8. The production method according to claim 7, wherein the thin film process in the step (2) is produced by: dissolving lecithin and cholesterol in chloroform according to the mass ratio of 3.

9. Use of the needle-based hydrogel precision delivery system according to any one of claims 1 to 6 or the needle-based hydrogel precision delivery system prepared by the method according to any one of claims 7 to 8 as a carrier for drugs or biomaterials for precision delivery to the lesion site.

10. The use of claim 9, wherein the use comprises the precision delivery system as a vehicle for precision delivery of a drug to spinal cord or cartilage tissue.