Piezoelectric microneedle preparation for non-invasive and non-drug treatment of lymphedema as well as preparation method and application of piezoelectric microneedle preparation

Abstract

The invention provides a piezoelectric microneedle preparation for non-invasive and non-drug treatment of lymphedema as well as a preparation method and application of the piezoelectric microneedle preparation. The piezoelectric microneedle preparation comprises a substrate with piezoelectric performance and a microneedle array which is distributed on one side of the substrate and has conductive performance. Wherein the substrate is prepared from a piezoelectric material and is subjected to polarization treatment; the microneedle array is prepared from a degradable material or a conductive polymer material, the degradable microneedle array is loaded with or coated with a conductive material, and the conductive polymer material can generate local charges. The micro-needle is high in penetration strength and small in invasion, and the pain of a patient is reduced; meanwhile, complex wire and electrode connection is not needed, so that the infection problem caused by a traditional power supply system is avoided; therefore, the cost is low, and long-term storage is facilitated; and a larger wound is not caused, so that the pain of a patient is relieved.

Description

Technical Field

[0001] The present invention belongs to the field of medical device technology and relates to the field of microneedle technology, and specifically relates to a piezoelectric microneedle preparation for non-invasive and drug-free treatment of lymphedema and a preparation method and application thereof. Background Art

[0002] The lymphatic system is the second largest circulatory system in the human body. Its main function is to drain lymph and remove foreign matter such as bacteria and viruses from the body. Once the lymphatic system is damaged, it will lead to disordered lymphatic drainage, causing interstitial fluid accumulation and tissue swelling, which is called lymphedema. To date, the total number of lymphedema patients in the world exceeds 250 million, and the number of patients in my country exceeds 3.5 million. It ranks second among disabling diseases and has a risk of death.

[0003] Lymphedema brings serious motor dysfunction and psychological burden to patients. Despite its wide impact, traditional treatment methods including surgery and conservative treatment have limited effects. Surgical treatment methods (liposuction, lymphatic transplantation, vascularized lymph node transplantation) are difficult to operate and cause great trauma to patients; conservative treatment methods (drug therapy, comprehensive detumescence treatment, intermittent compression therapy) require lifelong treatment, which significantly reduces the quality of life of patients. It can be seen that the above treatment methods can only temporarily reduce edema and are uncertain about alleviating and improving the development of the disease. More and more evidence shows that chronic inflammation is an important pathological sign of the progression of lymphedema, which is characterized by significant infiltration and changes of immune cells. Macrophages are an important immune cell that have been observed to accumulate in large numbers at the site of lymphedema lesions and are highly polarized to the M1 phenotype. These macrophages secrete a large number of proinflammatory cytokines (TNF-α, IL-1β, iNOS), which trigger a strong inflammatory response. Sustained inflammatory response leads to more severe tissue damage and high fibrosis.

[0004] Since macrophages have great plasticity, their polarization will be affected by various environmental and chemical factors inside and outside the cells, such as microorganisms, cytokines, pH, oxygen, etc. Although some progress has been made in macrophage polarization methods based on bioactive agents, immunomodulators and chemokines, it is still a huge challenge to find a sustained and effective method to regulate the polarization of macrophages from M1 to M2. It is worth noting that in current studies, physical factors have shown significant regulatory effects on macrophages. Electrical stimulation, as a physical signal, can directly mediate cell behavior through electrogenetic mechanisms. Therefore, regulating macrophage polarization phenotypes and pathological inflammatory microenvironments through electrical stimulation is expected to provide a novel and effective clinical method for the treatment of lymphedema. However, traditional electrical stimulation methods involve implanting cells on conductive materials and supplying power through connected wires and electrodes. Due to the wide distribution of electric potential on the surface of the material, cells cannot effectively sense local electrical stimulation. In addition, another issue worth noting is that once lymphedema is formed, it will cause subcutaneous tissue hyperplasia, thickening and hardening of the skin, and traditional percutaneous treatment methods are limited.

[0005] Therefore, introducing local electrical signals and solving the difficulties of transdermal drug delivery are crucial for the treatment of lymphedema. Summary of the invention

[0006] The present invention is directed to the above-mentioned problems, and non-invasive electrical stimulation is used to excite piezoelectric microneedles to generate local electrical signals, thereby selectively regulating macrophage polarization and factor secretion, providing a new clinical direction for the treatment of lymphedema.

[0007] In view of this, the first purpose of the present invention is to provide a piezoelectric microneedle preparation; the second purpose is to provide a method for preparing the piezoelectric microneedle preparation; and the third purpose is to provide an application of the piezoelectric microneedle preparation as a non-invasive lymphedema treatment product.

[0008] In order to achieve the above object, the specific technical solution adopted by the present invention is as follows:

[0009] In a first aspect of the present invention, a piezoelectric microneedle preparation is provided, comprising a substrate having piezoelectric properties and a microneedle array having conductive properties distributed on one side of the substrate.

[0010] The substrate is made of piezoelectric material and polarized; the microneedle array is made of degradable material or conductive polymer material, the degradable microneedle array is loaded or coated with conductive material, and the conductive polymer material can generate local charges.

[0011] When treating lymphedema, the preparation is covered on the lymphedema area, and after the microneedle array is inserted into the skin, a vibration device is used to approach the piezoelectric substrate, and mechanical energy is converted into electrical energy through mechanical vibration or ultrasound, so that the charge converges at the tip of the microneedle preparation, and the effect of intervening in lymphedema is achieved by regulating macrophage polarization and the local immune microenvironment. In addition, the substrate can be pressed with fingers to generate electrical signals.

[0012] Preferably, the diameter of the microneedle array is 200-800 μm, and the length of the array is 500-1200 μm.

[0013] Preferably, in terms of the substrate, its thickness is 100-1000 μm, and the piezoelectric material is selected from β-phase polyvinylidene fluoride or polyurethane; the substrate is prepared by electrospinning, casting molding or hot pressing molding;

[0014] The polarization treatment method of the substrate is as follows: it is mainly divided into two polarization methods, namely thermal polarization and electric field polarization. Both methods require the substrate to be pre-evaporated with a layer of metal electrode film (Al, Ag, etc.). Among them, the polarization conditions of thermal polarization are polarization electric field strength of 100-150MV / m, polarization temperature of 80-90°C, and polarization time of 45-60min; the polarization conditions of electric field polarization are needle electrode voltage of 15-16KV, mesh grid voltage of 5-6KV, polarization temperature of 60-70°C, and polarization time of 3-5min.

[0015] Preferably, in the microneedle array, the degradable material is selected from modified oligomeric hyaluronic acid, chitosan, carboxymethyl cellulose or starch; the loaded or coated conductive material includes graphene oxide, carbon nanotubes or nanowires, precious metal powder or oxide nanoparticles, which impart conductive properties to the degradable material.

[0016] The degradable material and the conductive material are combined in any of the following ways: (1) the degradable material is first mixed with the conductive material (graphene oxide, carbon nanotubes or nanowires), and then a microneedle array is prepared; (2) the degradable material is first prepared into a microneedle array, and then precious metal powder or oxide nanoparticles are coated on the surface of the microneedle body by sputtering.

[0017] Further preferably, the degradable material is first mixed with the conductive material (graphene oxide, carbon nanotubes or nanowires) in a mass ratio of 1:1; the noble metal powder or oxide nanoparticles are selected from gold, silver, platinum or a mixture.

[0018] In a preferred embodiment of the present invention, modified oligomeric hyaluronic acid is used to prepare a degradable microneedle array. The improved method of oligomeric hyaluronic acid is as follows:

[0019] (1) Add hyaluronic acid or sodium hyaluronate to a calcium, zinc or copper salt solution, let stand at room temperature for 3-12 hours to obtain a hyaluronate precipitate, put the precipitate into a dialysis bag and immerse it in a 5% dilute hydrochloric acid solution for 12-24 hours, then wash it with PBS 2-5 times, and finally dry it in a vacuum dryer to obtain a hyaluronate precipitate powder. The mass ratio of hyaluronic acid or sodium hyaluronate to calcium, zinc or copper salt is 1:1-5;

[0020] (2) Mixing hyaluronate precipitate powder and sodium hyaluronate at a mass ratio of 2:1-0.1, adding deionized water, and stirring evenly to obtain a hyaluronic acid composite pre-solution with a mass concentration of 5-20wt%.

[0021] Preferably, the calcium, zinc or copper salt is selected from calcium sulfate, calcium chloride, zinc sulfate, zinc chloride, copper sulfate, copper chloride or copper citrate;

[0022] The mass ratio between the hyaluronate precipitate powder and sodium hyaluronate is preferably 2:1;

[0023] The mass concentration of the hyaluronic acid compound pre-solution is preferably 10wt%.

[0024] The above method can significantly improve the mechanical strength of hyaluronic acid without using a cross-linking agent, and make it have good biosafety. At the same time, by compounding hyaluronate precipitate powder and sodium hyaluronate in a specific ratio, the grooves of the microneedle array can be well filled, and the prepared microneedles have sufficient penetration strength and almost no bubbles are generated.

[0025] Preferably, the microneedle body can also be prepared from conductive polymer materials, such as polypyrrole, polyaniline, polythiophene and its derivatives or polyacetylene, to directly prepare the microneedle array without conductive modification.

[0026] In a second aspect, the present invention provides a method for preparing the above-mentioned piezoelectric microneedle preparation, using any of the following preparation methods:

[0027] Method 1:

[0028] The conductive material is dispersed in the pre-solution constituting the degradable microneedle array material by magnetic stirring to obtain a precursor mixed suspension, and the precursor mixed suspension is added to the microneedle array mold for semi-curing treatment; the substrate material is added to the above-mentioned microneedle array mold for curing treatment to combine the substrate material with the microneedle array to prepare a piezoelectric microneedle preparation;

[0029] Method 2

[0030] The precursor solution of the degradable material is directly added to the microneedle array mold, and semi-cured, and then the substrate material is added and cured to combine the substrate material with the microneedle array to prepare a microneedle preparation; and then the prepared piezoelectric microneedle preparation is subjected to a noble metal sputtering treatment to prepare a piezoelectric microneedle preparation;

[0031] Method 3

[0032] The precursor solution of the conductive polymer material is directly added to the microneedle array mold, semi-cured, and then the substrate material is added and cured to combine the substrate material with the microneedle array to prepare a microneedle preparation.

[0033] Preferably, in the first to third methods, the microneedle array mold is prepared by an existing method, such as using polydimethylsiloxane as a template material, and pre-fabricating microneedle array molds with different pinhole arrangements and aperture sizes according to actual needs.

[0034] When preparing the microneedle array, the semi-curing treatment method is selected from any of the following methods: room temperature drying, heating drying, heating concentration followed by UV light drying. These three methods are common operations when preparing the microneedle array, and are obtained by conventional operating conditions or through experimental exploration based on the array material.

[0035] The curing treatment method is heating and drying curing;

[0036] Further preferably, in method one, the mass ratio between the degradable microneedle array material and the conductive material is 1:1; in method two, the mass ratio between the degradable microneedle array material and the sputtered precious metal material is 1:0.5, the sputtering time is 5 to 10 minutes, and the sputtering working pressure is 0.1 to 10 Pa.

[0037] In the three preparation methods, the preparation method of the substrate is the same. In a specific embodiment of the present invention, the substrate is prepared by an electrospinning method, as follows: PVDF or polyurethane powder is dissolved in an appropriate solvent (DMF: acetone (w / w) 6-7: 4-3), and after a certain reaction time at 70-80°C, a spinning solution with a mass concentration of 10-20% is obtained; then an electrospinning device is used to prepare a PVDF or polyurethane substrate, wherein the voltage is 12-15KV direct current, and the spinning solution is distributed from a stainless steel needle tip at a flow rate of 1-2mL / h, and the relative humidity and temperature are maintained at ≈25±5% and 25±1°C, respectively; the substrate is polarized according to the above method before use.

[0038] In the specific implementation method of the present invention, modified oligomeric hyaluronic acid is used as a degradable microneedle array material, which is mixed with graphene oxide powder and dissolved to obtain a suspension solution, and the microneedle array needle body is prepared after vacuum removal of bubbles; then it is combined with a cut substrate and cured to obtain a piezoelectric microneedle preparation.

[0039] The piezoelectric performance test shows that the piezoelectric microneedle preparation exhibits electrical neutrality without external stimulation. When ultrasonic or vibration stimulation is applied, the piezoelectric microneedle preparation deforms under ultrasound and generates an internal electric field, which enables the piezoelectric microneedle preparation to generate electrical signals. However, the piezoelectric performance of the device is not positively correlated with the content of graphene oxide. When the content of graphene oxide is 0.25 mg / mL, the output performance reaches the maximum value, even exceeding the output performance of using PVDF membrane alone.

[0040] At the same time, the piezoelectric microneedle preparation prepared by the present invention also has good biocompatibility and negligible cytotoxicity. After application, it can regulate the polarization of macrophages from M1 to M2 and effectively improve the inflammatory environment; the results of animal model experiments show that the piezoelectric microneedle preparation can alleviate the inflammatory pathological environment by regulating macrophage polarization, thereby promoting lymphatic vascular remodeling and lymphatic system function repair, which is beneficial to the treatment of lymphedema.

[0041] The third aspect of the present invention provides the use of any one of the piezoelectric microneedle preparations in the preparation of a lymphedema treatment product.

[0042] Preferably, the lymphedema treatment product is selected from a transdermal preparation that penetrates the subcutaneous tissue.

[0043] In a fourth aspect, the present invention provides a non-invasive lymphedema treatment system, comprising a piezoelectric microneedle preparation and an ultrasonic stimulation or mechanical stimulation device for generating stimulation in vitro. Mechanical energy is converted into electrical energy through mechanical vibration or ultrasonic action, so that the charge converges at the tip of the microneedle preparation, and the effect of intervening lymphedema is achieved by regulating macrophage polarization and the local immune microenvironment.

[0044] Functions and Effects of the Invention

[0045] In terms of treatment operation, the microneedle of the present invention has strong penetration force and low invasiveness, which reduces the pain of patients. At the same time, there is no need for complicated wires and electrode connections, thus avoiding infection problems caused by traditional power supply systems. There is no need to load drugs, so the cost is low and it is convenient for long-term storage. It will not cause large wounds, thereby alleviating the pain of patients.

[0046] In terms of preparation, the present invention first prepares a degradable microneedle body with conductive properties through a two-step method, then prepares a substrate with piezoelectric properties, and finally solidifies the two. Among them, the microneedle base and the substrate can be prepared with different materials according to different needs, which can be a single material or two or more materials. It has the characteristics of easy availability of materials, simple process, and low cost, which is conducive to industrial promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 An improved two-step method for preparing piezoelectric microneedle preparations;

[0048] Figure 2 The morphology of the piezoelectric microneedle preparation is characterized, including: (A) the morphology of the piezoelectric microneedle preparation under a stereo microscope and (B) the super-depth digital microscope; (C) the morphology of the PVDF substrate under a scanning microscope;

[0049] Figure 3 The performance characterization of the piezoelectric microneedle preparation includes: (A) the amplitude-voltage butterfly curve and phase voltage hysteresis loop of the piezoelectric microneedle preparation; (B) the linear relationship between the PFM amplitude and the applied voltage; (C) the output voltage of the piezoelectric microneedle preparation under the stimulation of an ultrasonic device; (D) the voltage and (E) the current of the piezoelectric microneedle preparation under the stimulation of a mechanical device;

[0050] Figure 4 The biocompatibility of the piezoelectric microneedle preparation, including: (A) CCK-8 test results of RAW 264.7 macrophages and (B) Hacat cells; (C) live / dead staining results of RAW 264.7 macrophages and (D) Hacat cells (3D);

[0051] Figure 5 The results of routine blood tests at week 6;

[0052] Figure 6 This is the result of routine organ sectioning;

[0053] Figure 7 The morphology of macrophages after different treatments under bright field microscope;

[0054] Figure 8 The flow cytometry test results of macrophages after different treatments;

[0055] Fig. 9 Elisa tests the secretion levels of related anti-inflammatory / pro-inflammatory factors after different treatments;

[0056] Fig.10 Mouse tail pictures and volume statistics;

[0057] Fig.11 These are the images of mouse tail tissue sections with lymphedema at week 3, including: (A) H&E and (B) Masson staining images;

[0058] Fig.12 This is immunofluorescence staining of the tail tissue of a mouse with lymphedema at week 3;

[0059] Fig.13 These are the images of mouse tail tissue sections with lymphedema at week 6, including: (A) H&E and its magnified image; (B) Masson staining image;

[0060] Fig.14 This is a picture of mouse tail lymphangiography at week 6. DETAILED DESCRIPTION

[0061] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0062] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturers.

[0063] 1. Preparation of piezoelectric microneedle preparations

[0064] The obtained microneedle array mold with a predetermined size is used to prepare a microneedle array, such as Figure 1 As shown, the improved two-step method is used for the preparation. The microneedle array mold uses a mold such as silicone or resin, and the mold includes a groove cavity and a microneedle array mold hole connected to the groove cavity.

[0065] First, prepare the microneedle array, that is, disperse the conductive materials of different concentrations in the soluble polymer solution by magnetic stirring to obtain a precursor mixed suspension. Then pour the precursor mixed suspension into the microneedle array mold to completely fill the mold holes, and then scrape off the excess solution with a scraper. Finally, place the substrate material in the mold, and after curing, combine it with the microneedle array to form a piezoelectric microneedle preparation.

[0066] Alternatively, the pre-solution of the degradable material is directly poured into the microneedle array mold, and then the substrate material is placed in the mold, and after curing, it is combined with the microneedle array, and finally the conductive material is sputtered to obtain the piezoelectric microneedle preparation.

[0067] Alternatively, a precursor solution of a conductive polymer material is added to a microneedle array mold, semi-cured, and then a substrate material is added and cured to combine the substrate material with the microneedle array to prepare a microneedle preparation.

[0068] The preparation method of the substrate material is not limited to electrospinning, but also includes a casting molding method and a hot pressing molding method.

[0069] When using, the preparation is covered on the lymphedema area. After the microneedle preparation penetrates the skin, the probe of the ultrasonic instrument is placed close to the piezoelectric substrate, and the mechanical energy is converted into electrical energy through the ultrasonic action, so that the charge converges at the tip of the microneedle preparation, and the effect of intervening lymphedema is achieved by regulating the polarization of macrophages and the local immune microenvironment. In addition, the substrate can be pressed with fingers to generate electrical signals.

[0070] Embodiment 1:

[0071] A preparation of a piezoelectric microneedle preparation, the steps are as follows:

[0072] Step 1: Prepare a conductive solution. Use a modified oligomeric hyaluronic acid solution as a soluble material, add 10 mg of graphene oxide powder into 10 mL of the soluble material aqueous solution, stir magnetically to obtain a uniformly mixed suspension solution, remove bubbles by vacuum, and set aside for later use.

[0073] The preparation method of the modified oligomeric hyaluronic acid is as follows: 10 mg of sodium hyaluronate is added to a solution containing 10 mg-50 mg of calcium salt, the concentration of the calcium salt solution is in the range of 0.5%-5wt%, and the solution is allowed to stand at room temperature for 3-12 hours to obtain a hyaluronate salt, the precipitate is placed in a dialysis bag and immersed in a dilute hydrochloric acid solution for 12-24 hours, and then washed with PBS for 2-5 times, and finally dried in a vacuum dryer to obtain a calcium hyaluronate powder; 10 mg of calcium hyaluronate powder and 5 mg of sodium hyaluronate powder are weighed and stirred to obtain a hyaluronic acid composite pre-solution with a mass concentration of 10%;

[0074] Step 2, prepare the piezoelectric substrate. The piezoelectric substrate is prepared using electrospinning technology. First, the PVDF powder is dissolved in an appropriate solvent (DMF: acetone (w / w) 6-7: 4-3), and reacted at 70-80°C for 30 minutes to obtain a high concentration PVDF solution (10-20% (w / w)). Then, an electrospinning device is used to prepare the PVDF substrate, wherein the voltage is 12-15KV direct current, and the PVDF solution is distributed from the stainless steel needle tip at a flow rate of 1-2mL / h, and the relative humidity and temperature are maintained at ≈25±5% and 25±1°C, respectively. In addition, the PVDF substrate is polarized before use.

[0075] Step 3: Prepare piezoelectric microneedle preparations. Pour the mixed suspension solution into the microneedle array mold, centrifuge and evacuate it several times to completely fill the mold holes, scrape off the excess solution with a scraper, and semi-cured to form the microneedle body; finally, place the cut substrate material in the mold, and after curing, combine it with the microneedle array to form a piezoelectric microneedle preparation.

[0076] Embodiment 2:

[0077] A piezoelectric microneedle preparation was prepared by referring to the method of Example 1, except that in step 1, graphene oxide powder was added to an aqueous solution of a soluble material to obtain a 1 mg / mL mixed suspension solution, which was then diluted to 0.5 mg / mL.

[0078] Embodiment 3:

[0079] A piezoelectric microneedle preparation was prepared by referring to the method of Example 1, except that in step 1, the mixed suspension solution obtained by adding graphene oxide powder to an aqueous solution of a soluble material was diluted to 0.25 mg / mL.

[0080] Embodiment 4:

[0081] A piezoelectric microneedle preparation is prepared by referring to the method of Example 1, except that in step 1, the mixed suspension solution obtained by adding graphene oxide powder to an aqueous solution of a soluble material is diluted to 0.1 mg / mL.

[0082] Embodiment 5:

[0083] A piezoelectric microneedle preparation is prepared by referring to the method of Example 1, except that in step 1, a mixed suspension solution does not need to be prepared, but a conductive polymer solution is directly prepared.

[0084] Embodiment 6:

[0085] A preparation of a piezoelectric microneedle preparation, the steps are as follows:

[0086] Step 1, prepare the piezoelectric substrate. The piezoelectric substrate is prepared using electrospinning technology. First, the PVDF powder is dissolved in an appropriate solvent (DMF: acetone (w / w) 6-7: 4-3), and reacted at 70-80°C for 30 minutes to obtain a high concentration PVDF solution (10-20% (w / w)). Then, an electrospinning device is used to prepare the PVDF substrate, in which the voltage is 12-15KV direct current, and the PVDF solution is distributed from the stainless steel needle tip at a flow rate of 1-2mL / h, and the relative humidity and temperature are maintained at ≈25±5% and 25±1°C, respectively. In addition, the PVDF substrate is polarized before use.

[0087] Step 2: Prepare a microneedle preparation. Use the modified oligomeric hyaluronic acid solution as a soluble material, pour the oligomeric hyaluronic acid solution into the microneedle array mold, make it completely fill the mold holes, scrape off the excess solution with a scraper, and finally place the cut substrate material in the mold, and combine it with the microneedle array after curing to form a microneedle preparation.

[0088] Step 3: Prepare a piezoelectric microneedle preparation. Fix the microneedle preparation obtained above in a sputtering instrument, use gold as the sputtering material, and sputter for 5-10 minutes at a working pressure of 0.1-10 Pa to finally obtain a piezoelectric microneedle preparation.

[0089] Embodiment 7:

[0090] A piezoelectric microneedle preparation is prepared by referring to the method of Example 6, except that in step three, silver is used as the sputtering material.

[0091] Embodiment 8:

[0092] A piezoelectric microneedle preparation is prepared by referring to the method of Example 6, except that in step three, platinum is used as the sputtering material.

[0093] 2. Performance test of piezoelectric microneedle preparations

[0094] 1. Morphological characterization of piezoelectric microneedle preparations

[0095] The morphology of the piezoelectric microneedle preparation was studied using a stereo microscope, an ultra-depth digital microscope, and a scanning electron microscope. Under the stereo microscope and ultra-depth digital microscope, it can be seen that the needles of the piezoelectric microneedle preparation remain intact and have the same size and shape. The needles are composed of cones and quadrilaterals, arranged in a 15×15 array, and the height of each needle is about 1000μm ( Figure 2 A. Figure 2 B). In addition, it can be seen from the scanning electron microscope that the PVDF film of the substrate is flat and uniform ( Figure 2 C).

[0096] 2. Characterization of piezoelectric properties of piezoelectric microneedle preparations

[0097] The performance of the piezoelectric microneedle was characterized using a piezoresponse force microscope and an electrometer. When the piezoelectric microneedle preparation was characterized using a piezoresponse force microscope, a butterfly curve and a ferromagnetic hysteresis loop appeared ( Figure 3 A), which is a typical manifestation of piezoelectric properties. Considering that the local electric field polarization hysteresis characterization is a method to locally quantify its piezoelectric coefficient, a linear fit is performed on the piezoelectric response force microscope amplitude voltage curve, and the results show that its slope is d 33 value, i.e. 38.5pm / V( Figure 3 B). In order to accurately analyze the piezoelectric properties of the piezoelectric microneedle preparation, a sandwich-shaped piezoelectric nanogenerator was also made to simulate its application environment. When a linear motor test system applied a force of 30N, the output performance of the piezoelectric microneedle preparation based on different graphene oxide concentrations, including open circuit voltage and short circuit current density, was tested. The results showed that the device performance was not positively correlated with the content of graphene oxide. When the graphene oxide content was 0.25mg / mL, the output performance reached the maximum value, even exceeding the output performance of the PVDF membrane alone ( Figure 3 D, 3E). In addition, ultrasonic equipment was used for testing, and it was found that the piezoelectric microneedle preparation exhibited electrical neutrality without ultrasonic stimulation. However, when ultrasound was applied, the piezoelectric microneedle preparation was deformed under ultrasound and generated an internal electric field, which enabled the piezoelectric microneedle preparation to generate electrical signals, and the output performance was optimal when the graphene oxide content was 0.25 mg / mL ( Figure 3 C).

[0098] 3. Biocompatibility of piezoelectric microneedle preparations

[0099] The in vitro toxicity of piezoelectric microneedle preparations on HaCat cells and Raw 264.7 macrophages was evaluated. First, cell proliferation was detected using a cell counting kit-8 (CCK-8). The results showed that both HaCat cells and Raw 264.7 macrophages showed sustained proliferation after 24, 48, and 72 hours of treatment with piezoelectric microneedle preparations ( Figure 4 A, 4B). Then a live / dead staining test was performed, and it was found that most cells showed green fluorescence, and relatively few cells showed red fluorescence. In addition, a three-dimensional cell culture experiment was performed using HaCat cells to more realistically simulate the in vivo situation. It was observed that the edges of the cell spheroids showed a large amount of green fluorescence, while the center of the cell spheroids showed a relatively small amount of red fluorescence due to the hypoxic environment, indicating that the piezoelectric microneedle preparation has good biocompatibility ( Figure 4 C, 4D).

[0100] In order to further ensure the biological safety during the administration, in vivo experiments were also conducted. After 6 weeks of treatment, the results of the blood routine test showed that all blood parameters, including white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), neutrophils (NEUT), and lymphocytes (LYMPH), were within the normal range ( Figure 5 ). At the same time, hematoxylin-eosin (H&E) staining was used to evaluate the toxicity of the material in the lungs, kidneys, liver, spleen, and heart, and no inflammatory infiltration or pathological changes were observed in these organs. Based on these findings, the piezoelectric microneedle preparation has good biosafety, negligible cytotoxicity, and has the potential for long-term utilization ( Figure 6 ).

[0101] 4. Piezoelectric microneedle preparations for regulating macrophage polarization and related inflammatory environment

[0102] Chronic inflammation of the dermis and subcutaneous tissue is a histological hallmark of lymphedema, and is typically characterized by changes in the abundance of immune cells. Macrophages are one of the main immune cells and infiltrate in large numbers in lymphedema tissues. In order to study the effect of piezoelectric microneedle preparations on macrophage polarization, lipopolysaccharide (LPS) was used to create an inflammatory environment as a positive control group to promote the transformation of macrophages from the M0 phenotype to the M1 phenotype. As can be seen from the bright field images, the M0 macrophages in the control group were round, while the M1 macrophages stimulated by LPS were irregularly round with multiple radial filopodia, and after treatment with the piezoelectric microneedle preparation, the cells showed an elongated spindle shape, which is consistent with the morphology of M2 macrophages ( Figure 7 ).

[0103] Subsequently, flow cytometry was used to analyze the expression of M1 macrophage polarization markers CD86 and M2 macrophage polarization markers CD206. The results showed that LPS treatment significantly increased the proportion of M1 macrophages, while after treatment with the piezoelectric microneedle preparation, the proportion of M1 macrophages marked by CD86 decreased and the proportion of M2 macrophages increased ( Figure 8 In addition, enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion levels of pro-inflammatory factors (TNF-α, IL-1β and iNOS) and anti-inflammatory factors (IL-10, IL-4 and Arg-1). The results showed that the expression levels of TNF-α, IL-1β and iNOS were significantly reduced after treatment with the piezoelectric microneedle preparation, while the expression levels of IL-10, IL-4 and Arg-1 were increased ( Fig. 9 ). Therefore, the piezoelectric microneedle preparation can regulate the polarization of macrophages from M1 to M2 and effectively improve the inflammatory environment.

[0104] 3. Piezoelectric microneedle preparations for the treatment of lymphedema

[0105] Establishment of animal model: A 0.5 cm circumferential incision was made at about 2 cm from the mouse tail by microsurgery, and the mouse tail lymphedema model was established by injecting methylene blue for color development and cutting off the main lymphatic vessels under a microscope.

[0106] Experimental groups: The mice were randomly divided into four groups, with at least five mice in each group: (1) sham operation group (sham operation + PBS treatment), (2) untreated group (surgery + untreated), (3) piezoelectric microneedle preparation group (surgery + piezoelectric microneedle preparation and pressing treatment), (4) ultrasound-mediated piezoelectric microneedle preparation group (surgery + ultrasound + piezoelectric microneedle preparation treatment). Different treatments were performed at the end of the second week after surgery, and three measurement points were established at -1.0 cm, +0.5 cm and +1.0 cm away from the incision site.

[0107] Photos of the mouse tails showed that, except for the sham operation group, all other groups had obvious swelling after surgery, indicating that the lymphedema model was successfully established, and the mouse tail appeared curled and stiff during the development of lymphedema. Statistical analysis of the results showed that the diameter of the mouse tail did not change much at -1.0 cm distal to the incision, and there was no statistically significant difference between the groups. In the untreated group, the diameter and volume of the mouse tail increased rapidly with time, and showed a slight downward trend in the later period, especially near the incision, but the regression was limited. In contrast, the diameter and volume of the mouse tail increased in the first few weeks of edema formation, but decreased significantly after treatment in the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group ( Fig.10 ).

[0108] At week 3, the tail tissue of mice was obtained, and the cross-section of the tail near the incision was taken for H&E and Masson staining. H&E staining images showed that compared with the sham operation group, the dermis structure of the untreated group, the piezoelectric microneedle preparation group, and the ultrasound-mediated piezoelectric microneedle preparation group was disordered, and the thickness of the subcutaneous soft tissue was significantly thickened, which also indicated the formation of lymphedema. Masson staining further observed that the collagen of the subcutaneous tissue of the sham operation group was neatly arranged, while the collagen structure of the subcutaneous tissue of the untreated group, the piezoelectric microneedle preparation group, and the ultrasound-mediated piezoelectric microneedle preparation group was disordered, accompanied by progressive fibrosis ( Fig.11 ). In addition, inflammation-related indicators were evaluated at week 3. CD86 and CD206 immunofluorescence staining showed that the red fluorescence in the untreated group increased and the green fluorescence decreased, indicating that the macrophages in the lymphedema tissue were obviously polarized toward the M1 phenotype, promoting the development of subcutaneous inflammation. On the contrary, the red fluorescence decreased and the green fluorescence increased relatively in the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group after one week of treatment ( Fig.12 ).

[0109] At the 6th week after the end of treatment, mouse tail tissue samples were collected for H&E and Masson staining studies. H&E staining images showed that the thickness of the subcutaneous tissue of the rat tail was significantly reduced after treatment in the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group. The local magnified images of H&E staining showed that the inflammatory cell infiltration in the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group was reduced, and the subcutaneous tissue structure was relatively intact. In contrast, the soft tissue of the untreated group was still thicker than that of the sham operation group. The enlarged H&E staining image showed that the epidermis of the untreated group was thickened, hyperkeratinized, and the amount of inflammatory cell infiltration increased, indicating tissue inflammatory changes. Long-term inflammatory stimulation can lead to fibrosis, and Masson staining showed that collagen deposition in the untreated group increased. However, after treatment in the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group, the distribution of collagen fibers in the dermis was orderly, and the percentage of collagen decreased ( Fig.13 ). In addition, the effect of inflammation relief on lymphatic remodeling was evaluated by IGC lymphangiography. After contrast agent injection, lymphatic vessels were clearly visible in the sham group; in the untreated group, no lymphatic vessels were seen above the incision after contrast agent injection due to abnormal lymphatic circulation. However, after treatment with the piezoelectric microneedle preparation group and the ultrasound-mediated piezoelectric microneedle preparation group, contrast agent was observed to flow through the incision and show obvious lymphatic vessels ( Fig.14 ). These results indicate that piezoelectric microneedle preparations can alleviate the inflammatory pathological environment by regulating macrophage polarization, thereby promoting lymphatic vascular remodeling and lymphatic system function repair, which is beneficial to the treatment of lymphedema.

[0110] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A piezoelectric microneedle preparation, characterized in that: The invention comprises a substrate with piezoelectric properties and a microneedle array with conductive properties distributed on one side of the substrate. Wherein, the substrate is made of piezoelectric material and subjected to polarization treatment; The microneedle array is made of a degradable material or a conductive polymer material. The degradable microneedle array is loaded with or coated with a conductive material. The conductive polymer material can generate local charges.

2. The piezoelectric microneedle preparation according to claim 1, characterized in that: in, The diameter of the microneedle array is 200-800 μm, and the array length is 500-1200 μm; The thickness of the substrate is 100-1000 μm, and the piezoelectric material of the substrate is selected from β-phase polyvinylidene fluoride or polyurethane; The substrate is prepared by electrostatic spinning, casting or hot pressing; The polarization treatment method of the substrate is selected from thermal polarization or electric field polarization. Both methods require that a layer of Al or Ag metal electrode film be evaporated on the substrate in advance; wherein, the polarization conditions of thermal polarization are polarization electric field strength of 100-150MV / m, polarization temperature of 80-90°C, and polarization time of 45-60min; and the polarization conditions of electric field polarization are needle electrode voltage of 15-16KV, mesh grid voltage of 5-6KV, polarization temperature of 60-70°C, and polarization time of 3-5min.

3. The piezoelectric microneedle preparation according to claim 1, characterized in that: in, In the microneedle array, the degradable material is selected from modified oligomeric hyaluronic acid, chitosan, carboxymethyl cellulose or starch; the loaded or coated conductive material includes graphene oxide, carbon nanotubes or nanowires, noble metal powder or oxide nanoparticles; The conductive polymer material is selected from polypyrrole, polyaniline, polythiophene and derivatives thereof or polyacetylene.

4. The piezoelectric microneedle preparation according to claim 3, characterized in that: in, The preparation method of the modified oligomeric hyaluronic acid is as follows: (1) adding hyaluronic acid or sodium hyaluronate to a calcium, zinc or copper salt solution, standing at room temperature for 3-12 hours to obtain a hyaluronate precipitate, placing the precipitate in a dialysis bag and immersing it in a 5% dilute hydrochloric acid solution for 12-24 hours, washing it with PBS 2-5 times, and finally drying it in a vacuum dryer to obtain a hyaluronate precipitate powder; wherein the mass ratio of hyaluronic acid or sodium hyaluronate to calcium, zinc or copper salt is 1:1-5; (2) Mixing hyaluronate precipitate powder and sodium hyaluronate at a mass ratio of 2:1-0.1, adding deionized water, and stirring evenly to obtain a hyaluronic acid composite pre-solution, wherein the mass concentration of the hyaluronic acid composite pre-solution is 5-20wt%.

5. The piezoelectric microneedle preparation according to claim 4, characterized in that: in, The calcium, zinc or copper salt is selected from calcium sulfate, calcium chloride, zinc sulfate, zinc chloride, copper sulfate, copper chloride or copper citrate; The mass ratio between hyaluronate precipitate powder and sodium hyaluronate is 2:1; The mass concentration of the hyaluronic acid compound pre-solution is 10wt%.

6. The method for preparing the piezoelectric microneedle preparation according to any one of claims 1 to 5, characterized in that: Use any of the following preparation methods: Method 1: Dispersing the conductive material in the pre-solution constituting the degradable microneedle array material by magnetic stirring to obtain a precursor mixed suspension, and adding the precursor mixed suspension into the microneedle array mold for semi-curing treatment; Adding the substrate material into the microneedle array mold and performing a curing treatment to combine the substrate material with the microneedle array to prepare a piezoelectric microneedle preparation; Method 2 Directly adding a precursor solution of a degradable material into a microneedle array mold, performing a semi-curing treatment, and then adding a substrate material and performing a curing treatment to combine the substrate material with the microneedle array to prepare a microneedle preparation; Then the prepared piezoelectric microneedle preparation is subjected to a noble metal sputtering treatment to prepare a piezoelectric microneedle preparation; Method 3 The precursor solution of the conductive polymer material is directly added to the microneedle array mold, semi-cured, and then the substrate material is added and cured to combine the substrate material with the microneedle array to prepare a microneedle preparation.

7. The preparation method according to claim 6, characterized in that: in, In the first to third methods, the semi-curing treatment method is selected from any one of the following methods: room temperature drying, heating drying, heating concentration followed by UV light drying; The curing treatment method is heating and drying curing; In method 1, the mass ratio between the degradable microneedle array material and the conductive material is 1:1; in method 2, the mass ratio between the degradable microneedle array material and the sputtered precious metal material is 1:0.5, the sputtering time is 5 to 10 minutes, and the sputtering working pressure is 0.1 to 10 Pa; The preparation method of the substrate is as follows: PVDF or polyurethane powder is dissolved in an appropriate solvent, and after reacting at 70-80°C for a certain period of time, a spinning solution with a mass concentration of 10-20% is obtained; then an electrospinning device is used to prepare the PVDF or polyurethane substrate, wherein the voltage is 12-15KV direct current, the spinning solution is distributed from a stainless steel needle tip at a flow rate of 1-2mL / h, and the relative humidity and temperature are maintained at ≈25±5% and 25±1°C, respectively; the substrate is polarized before use.

8. Use of the piezoelectric microneedle preparation according to any one of claims 1 to 5 in the preparation of a product for treating lymphedema.

9. The use according to claim 8, characterized in that: The lymphedema treatment product is selected from transdermal preparations.

10. A non-invasive lymphedema treatment system, characterized in that: The invention comprises the piezoelectric microneedle preparation according to any one of claims 1 to 5 and an ultrasonic stimulation or mechanical stimulation device for generating stimulation in vitro.

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