CN117442544A

CN117442544A - Pirfenidone sustained-release microneedle patch for resisting myocardial fibrosis and preparation method thereof

Info

Publication number: CN117442544A
Application number: CN202311151842.2A
Authority: CN
Inventors: 王嵘; 侯骉
Original assignee: 6th Medical Center of PLA General Hospital
Current assignee: 6th Medical Center of PLA General Hospital
Priority date: 2023-09-07
Filing date: 2023-09-07
Publication date: 2024-01-26

Abstract

The invention provides a Pirfenidone sustained-release microneedle patch for resisting myocardial fibrosis and a preparation method thereof, wherein the patch comprises a substrate and a microneedle array, the substrate is made of uncured GelMA/PVP (methacryloylated gelatin/polyvinylpyrrolidone) composite material, the microneedle array fixed on the substrate is made of cured GelMA/PVP composite material, and the microneedle material is internally added with medicine Pirfenidone (PFD). The micro-needle patch provided by the invention has good biocompatibility by using GelMA/PVP, the PFD and the GelMA/PVP are mixed to prepare the PFD slow-release micro-needle patch, the micro-needle patch is fixed at the myocardial infarction part of the heart in the coronary artery bypass operation, the micro-needle with a specific structural design and the excellent biological safety and adhesion characteristics of the GelMA/PVP are utilized, the micro-needle patch can be stably fixed in an infarcted area for a long time, the characteristic of slow-release PFD is achieved by natural degradation of a base material, the retention rate of the PFD in the infarcted area is increased, and the PFD can continuously play the roles of anti-inflammation and anti-fibrosis.

Description

Pirfenidone sustained-release microneedle patch for resisting myocardial fibrosis and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a Pirfenidone (PFD) sustained-release microneedle patch for resisting myocardial fibrosis and a preparation method thereof.

Background

Ischemic Heart Failure (IHF) is a worldwide health problem, mainly caused by Acute Myocardial Infarction (AMI). Myocardial fibrosis and subsequent left ventricular remodeling following acute myocardial infarction can lead to a range of pathological disorders such as left ventricular wall tumors, ischemic mitral insufficiency, etc., which inevitably develop IHF. Thus, in addition to timely revascularization (within 6 hours after AMI), early inhibition of myocardial fibrosis and left ventricular remodeling is another key to the integrated therapeutic strategy of AMI. Previous studies have shown that angiotensin converting enzyme inhibitors/angiotensin receptor blockers (ACEI/ARB) are effective in preventing left ventricular remodeling and have been strongly recommended by guidelines. However, these drugs are often administered orally and cannot be targeted to the infarcted area, thereby diminishing their effect of inhibiting left ventricular remodeling and improving left ventricular function. Furthermore, they are not always suitable for patients with early hemodynamic instability of AMI.

Coronary Artery Bypass Grafting (CABG) is a complete revascularization technique and has long-term efficacy, and has proven to be a reliable means of treating ischemic heart disease. However, for the treatment of acute myocardial infarction, CABG has long been a two-line treatment scheme for thrombolytic therapy and Percutaneous Coronary Intervention (PCI), and the main reason is that, besides the high risk of AMI early surgery, the failure of CABG alone to reverse AMI post-left ventricular remodeling is a very important reason. In recent years, with the development of surgical techniques and perioperative management, CABG can be safely performed in the first week after AMI. Therefore, it has become extremely urgent and necessary to develop an anti-myocardial fibrosis treatment method that can be used contemporaneously in CABG surgery.

Pirfenidone is an oral polypyridine small molecule compound with the ability to inhibit fibrosis, inflammatory response and oxidative stress. Based on these characteristics, it has been widely used in the treatment of fibrotic diseases including lung, kidney, liver and bone marrow. In recent years, research has found that pulmonary and cardiac diseases have similar pro-fibrotic pathways, and PFDs are therefore considered potential therapeutic agents for inhibiting myocardial fibrosis. However, pirfenidone is only orally administered, and its half-life is short of about 2.5 hours, so that high-dose administration is generally required to achieve the desired therapeutic effect, however, the resulting impairment of liver and kidney functions is the biggest obstacle restricting oral administration thereof.

Disclosure of Invention

Aiming at the problem that the current CABG operation lacks in the synchronous period to inhibit/reverse fibrosis of myocardial infarction areas, the invention provides a PFD slow-release microneedle patch (MNP) for resisting myocardial fibrosis and a preparation method thereof, and the PFD slow-release microneedle patch is prepared by mixing PFD and GelMA/PVP by utilizing good biocompatibility of GelMA/PVP (methacryloylated gelatin/polyvinylpyrrolidone), and is fixed in myocardial infarction areas in the CABG operation, and the PFD slow-release microneedle patch can be stably fixed in the infarction areas for a long time by utilizing excellent biological safety and adhesion characteristics of microneedles and GelMA/PVP with specific structural design, so that the characteristics of slow-release PFD can be achieved by natural degradation of a base material, the retention rate of the PFD in the infarction areas can be increased, and the PFD can continuously play the roles of resisting inflammation and fibrosis.

The technical scheme provided by the invention is as follows:

in a first aspect, a pirfenidone slow release microneedle patch for resisting myocardial fibrosis comprises a substrate and a microneedle array, wherein the substrate is made of uncured GelMA/PVP composite material; the microneedle array fixed on the substrate is made of a solidified GelMA/PVP composite material, and a medicine pirfenidone PFD is added in the microneedle material.

Further, the pirfenidone slow release microneedle patch for resisting myocardial fibrosis is prepared by the following steps:

mixing and uniformly stirring GelMA, PVP and deionized water containing a photoinitiator to obtain a GelMA/PVP substrate;

adding PFD into GelMA/PVP substrate to obtain micro-needle matrix containing PFD;

adding the microneedle matrix containing the PFD into a mould, removing bubbles in vacuum, removing redundant microneedle matrix on the surface of the mould, drying in an oven at 40-50 ℃, repeatedly adding the microneedle matrix containing the PFD into the mould, drying for multiple times, irradiating ultraviolet rays of the dried microneedle array, and photo-curing to form;

and coating the GelMA/PVP substrate on a mould under the light-shielding condition, covering the microneedle array, vacuum drying to form a substrate, and continuously drying the GelMA/PVP-MNP-PFD system obtained after drying at 40-50 ℃ until the GelMA/PVP-MNP-PFD system is successfully separated from the mould.

Further, in the step of uniformly mixing and stirring GelMA, PVP and deionized water containing a photoinitiator to obtain the GelMA/PVP substrate, the mass ratio of the GelMA to the PVP is 4 (0.9-1.1).

Further, in the step of uniformly mixing and stirring GelMA, PVP and deionized water containing a photoinitiator to obtain the GelMA/PVP substrate, the content of the photoinitiator in the deionized water containing the photoinitiator is 0.02-0.05 wt%, and the mass ratio of GelMA to the deionized water containing the photoinitiator is 4 (40-60).

Still further, in the step of preparing the PFD-containing microneedle substrate, the PFD is added to the GelMA/PVP substrate as an aqueous PFD ethanol solution.

Still further, in the step of preparing the PFD-containing microneedle substrate, the PFD content in the PFD-containing microneedle substrate is 8 to 10mg/mL.

And further, in the step of irradiating the dried microneedle array with ultraviolet rays and photo-curing and forming, the microneedle array is irradiated with ultraviolet rays for 30-40 seconds and photo-curing and forming is performed.

Furthermore, in the step of coating the GelMA/PVP substrate on the mould and covering the microneedle array under the light-shielding condition, the GelMA/PVP substrate for preparing the microneedle substrate is selected, or the GelMA/PVP substrate without the photoinitiator is selected to be coated on the mould and cover the microneedle array.

Further, the diameter of the tip of the micro needle is 10-20 mu m, the diameter of the root is 280-320 mu m, the total length is 1/4-1/2 of the thickness of cardiac muscle, and the length of the front end of the micro needle, which is smaller than 50 mu m, is 1/5-1/4 of the total length of the micro needle.

In a second aspect, a method for preparing a PFD sustained release microneedle patch for preventing myocardial fibrosis comprises the following steps:

According to the pirfenidone slow-release microneedle patch for resisting myocardial fibrosis and the preparation method thereof, the pirfenidone slow-release microneedle patch has the following beneficial effects:

(1) According to the pirfenidone slow-release microneedle patch for resisting myocardial fibrosis and the preparation method thereof, provided by the invention, the drug carrier material needs to have excellent biocompatibility and biodegradability in consideration of biosafety factors, so that GelMA is selected as a matrix material. Compared with connective tissue, the resistance generated during penetration of myocardial tissue is higher, and the mechanical property of GelMA serving as an independent matrix material is difficult to meet the strength requirement of a microneedle array on myocardial penetration. According to the invention, by researching and considering the requirements of mechanical strength and biological safety, the medicine carrier is determined to be made of a GelMA/PVP composite material, and PVP has excellent biocompatibility and biodegradability, and the mechanical strength of GelMA can be regulated and controlled, so that the myocardial puncture requirement is met. The mass ratio of GelMA to PVP is 4 (0.9-1.1). At this ratio, the required gel state of GelMA hydrogel will not change due to the addition of PVP in large amounts, nor will the difficulty of use increase due to too little PVP addition to reduce the mechanical strength of the microneedle.

(2) According to the pirfenidone slow-release microneedle patch for resisting myocardial fibrosis and the preparation method thereof, PFD is added into GelMA/PVP base material in the form of PFD ethanol aqueous solution, and ethanol can be removed by heating during subsequent microneedle drying and molding; through the addition and removal of the ethanol at different stages, the full mixing of the PFD and the water phase is realized, and the stability of the drug effect of the microneedle patch and the industrial production are facilitated.

(3) According to the pirfenidone slow-release microneedle patch for resisting myocardial fibrosis and the preparation method thereof, provided by the invention, the mobility of far beyond other organs of the heart is considered, the microneedles in the microneedle array are designed to be the bee-like needles, and the fixation effect of the microneedle patch on the heart is improved by improving the microneedle form, so that the efficacy is beneficial to playing.

Drawings

FIG. 1 is a flow chart of the preparation of a PFD sustained release microneedle patch against myocardial fibrosis;

FIG. 2 is a view of a micropin of example 1 in a fluorescence microscope;

FIG. 3 is a Scanning Electron Microscope (SEM) image of the microneedle of example 1;

FIG. 4 shows cardiac function and myocardial fibrosis levels 7 days after treatment of rats in example 1;

FIG. 5 shows cardiac function and myocardial fibrosis levels 28 days after treatment of the rats in example 1;

FIG. 6 is a graph showing myocardial apoptosis levels in rats using a laser scanning confocal microscope in example 1;

FIG. 7 shows the levels of gap junction proteins in the MI rat model, from top to bottom, for the peripheral Infarct zone, the Infarct zone, and the Remote zone, respectively, for CX-43 staining in example 1;

FIG. 8 is a graph of microneedle stress, left graph showing that the mechanical properties of the microneedles do not change when the PFD is added, and right graph showing that individual microneedles bend or break when the pressure reaches 0.4N;

FIG. 9 is a statistical graph of in vitro sustained release rate of the microneedle patch of example 1;

FIG. 10 shows the results of PFD and GelMA/PVP matrix material biotoxicity tests;

FIG. 11 shows myocardial fibrosis levels of rats treated with the microneedle patches of example 1 and comparative example 1.

Detailed Description

The features and advantages of the present invention will become more apparent and clear from the following detailed description of the invention.

The word "exemplary" is used herein to mean "serving as an example, embodiment, or illustration. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments. Although various aspects of the embodiments are illustrated in the accompanying drawings, the drawings are not necessarily drawn to scale unless specifically indicated.

To increase the retention rate of the PFD in the infarcted area, the inventors developed a PFD-rich microneedle patch. The GelMA/PVP has good biocompatibility, the PFD and the GelMA/PVP can be mixed to prepare a PFD slow-release microneedle patch, the microneedle array on the microneedle patch is used for penetrating into cardiac muscle in the implementation of coronary artery bypass surgery, the microneedle with specific structural design and the excellent biological safety and adhesion characteristic of the GelMA/PVP are used, the PFD slow-release microneedle patch can be stably fixed in an infarcted area for a long time, and the characteristic of slow-release PFD is achieved through natural degradation of a base material, so that the retention rate of the PFD in the infarcted area is increased, and the PFD can continuously play the anti-inflammatory and anti-fibrosis roles.

The invention provides a PFD slow-release microneedle patch for resisting myocardial fibrosis, which comprises a substrate and a microneedle array, wherein the substrate is made of uncured GelMA/PVP composite material; the microneedle array fixed on the substrate is made of a solidified GelMA/PVP composite material, and a medicine PFD is added in the microneedle material.

The PFD slow release microneedle patch for resisting myocardial fibrosis is prepared by the following steps:

(1) GelMA (M) _W = 200,000 ～ 300,000), PVP and deionized water containing 0.02-0.05 wt% of photoinitiator are mixed and stirred uniformly according to the mass ratio of 4 (0.9-1.1) (40-60), and the GelMA/PVP substrate is obtained. Wherein the photoinitiator includes, but is not limited to, irgacure2959;

(2) Adding PFD into GelMA/PVP base material according to the proportion of 8-10 mg/mL to prepare a microneedle matrix containing PFD;

(3) Adding the microneedle matrix containing the PFD into a PDMS (polydimethylsiloxane prepolymer, poly (dimethylsiloxalle)) mould, removing bubbles in vacuum for 5min, removing redundant microneedle matrix on the surface of the mould, placing the mould in an oven at 40-50 ℃, and drying for 30-40 min; the PDMS mold is used as a female mold for molding the microneedles, and is provided with microneedle grooves which are arranged corresponding to the microneedle arrays; drying the microneedle matrix, and repeatedly adding the microneedle matrix containing the PFD into the mold, and drying to improve the loading capacity of the PFD in the microneedle patch MNP; irradiating the dried microneedle array with ultraviolet rays for 30-40 s, and performing photo-curing molding; the photo-curing molding has certain requirements on photo-curing time, and the strength of the micro-needle is weak when the photo-curing time is too short; too long a photo-curing time increases the brittleness of the microneedle body, and at the same time increases the crosslinking degree and excessively prolongs the degradation time in vivo.

(4) Coating the GelMA/PVP substrate on a mould under the condition of avoiding light, covering the microneedle array, vacuum drying for 5min to form a substrate, and continuously drying the GelMA/PVP-MNP-PFD system obtained after drying at 40-50 ℃ until the GelMA/PVP-MNP-PFD system is successfully separated from the mould. Wherein, the GelMA/PVP substrate of the forming substrate can be selected from the GelMA/PVP substrate of the microneedle substrate prepared in the step (1), or the GelMA/PVP substrate without the photoinitiator is prepared again.

The PFD slow release microneedle patch for resisting myocardial fibrosis is an in-situ patch, namely is directly used for targeting organ tissues, and the drug carrier material must have excellent biocompatibility and biodegradability in consideration of biosafety factors, so GelMA is selected as a matrix material. Compared with connective tissue, the resistance generated during penetration of myocardial tissue is higher, gelMA is used as an independent matrix material, and the mechanical property of the GelMA is difficult to meet the strength requirement of the microneedle array on myocardial penetration. The inventor researches and considers the requirements of mechanical strength and biological safety to determine the GelMA/PVP composite material, and PVP has excellent biocompatibility and biodegradability as well as can regulate and control the mechanical strength of GelMA so as to meet the myocardial puncture requirement. The mass ratio of GelMA to PVP is 4 (0.9-1.1). At this ratio, the required gel state of GelMA hydrogel will not change due to the addition of PVP in large amounts, nor will the difficulty of use increase due to too little PVP addition to reduce the mechanical strength of the microneedle.

The inventor finds that aggregation phenomenon exists when PFD is dispersed in water or GelMA/PVP base material in the development process, and a microneedle matrix with uniformly dispersed active ingredients cannot be obtained. The inventors determined that: dissolving PFD in ethanol, and adding deionized water to obtain PFD ethanol water solution, wherein the mass ratio of ethanol to water is 1:9-2:8; and adding the PFD into the GelMA/PVP substrate in the form of PFD ethanol aqueous solution, and heating to remove ethanol during subsequent microneedle drying and molding. By adding and removing ethanol at different stages, the PFD and the water phase are fully mixed.

In the present invention, the microneedle patch acts on the tissue of the internal organ of the living body to achieve in-situ administration, which is significantly different from the conventional microneedle patch attached to the skin. On the basis of considering whether the product is degraded or not, it is necessary to further consider the difficulty level of the product degradation. The substrate of the microneedle patch is free of drugs and serves to support the microneedle array and adhere to the organ tissue, but is expected to break away from the organ tissue and degrade as soon as possible at the end of administration. For this purpose, the invention creatively designs the substrate and the micro-needle array forming process, wherein the substrate is made of uncured GelMA/PVP composite material, and the micro-needle array is made of cured GelMA/PVP composite material. .

The target organ of the microneedle patch is a heart, the heart is contracted 60 to 100 times per minute by means of cardiac muscle to drive blood into aorta, the heart has more active beating property unlike other organs, and the heart is a dynamic organ, so that higher requirements are provided for the embedding capability of the microneedle patch, and in particular, under the condition that the substrate is an uncured composite material, the substrate is degraded faster than the microneedles, and the adhesion performance to the heart is reduced, the microneedle structure is required to be designed so that the microneedles stay in the cardiac muscle stably in the administration period.

The inventor designs the micro-needle in the micro-needle array in a similar cone shape, the diameter of the tip of the micro-needle is 10-20 mu m, the diameter of the root is 280-320 mu m, the total length is 1/4-1/2 of the thickness of cardiac muscle, and the length of the front end diameter of the micro-needle smaller than 50 mu m is 1/5-1/4 of the total length of the micro-needle.

The micro-needle has main medicine carrying function, and after penetrating into cardiac muscle, it swells with water and the pressure of volume swelling to cardiac muscle is used to promote the micro-needle to stay in cardiac muscle. The diameter of the front end of the micro needle is less than 50 mu m, and the length is 1/5-1/4 of the total length of the micro needle, so that the micro needle is deep in cardiac muscle and swells more quickly when meeting water, and the effect of firm embedding is achieved in a longer range of deep cardiac muscle.

The total length of the microneedle is a specific choice taking into account the thickness of the myocardium and the exertion of the drug effect; if the height is too low and is lower than the minimum value of the range, the length of the lower part of the micro needle is small to ensure the embedding effect of the front end of the micro needle, so that the drug loading amount is small, the penetration resistance is increased under the condition that the diameter of the root part is unchanged, and the repulsive force of the cardiac muscle to the micro needle is also increased; if the height is too high and above the maximum value of the above range, the myocardial contractile function may be affected.

The length of the diameter of the front end of the micro needle is less than 50 mu m and is 1/5-1/4 of the total length of the micro needle, so that the micro needle can smoothly penetrate deep into cardiac muscle like an elongated bee needle, and the long length and the small diameter meet the effect of rapid swelling and anchoring, thereby being beneficial to embedding into organ tissues. If the length of the microneedle front end diameter smaller than 50 μm is shorter than 1/5 of the total length of the microneedle, the residence time of the entire microneedle in the cardiac muscle is shortened.

Examples

Example 1 preparation of microneedle patches

The preparation flow of the microneedle patch is shown in fig. 1, and comprises the following steps:

(1) Mixing liquid polydimethylsiloxane prepolymer PDMS and a curing agent according to a mass ratio of 10:1, uniformly stirring, vacuumizing to remove bubbles in the mixture, pouring the mixture into a horizontally placed container, and heating the container in a baking oven at 60 ℃ for 10 hours to obtain a polydimethylsiloxane sheet with uniform thickness; and then taking the sheet out of the container, putting the sheet into a working bin of a laser engraving machine, adjusting laser parameters according to the required solid microneedle geometry, and carrying out micro-nano processing on the polydimethylsiloxane sheet so as to prepare the microneedle template.

(2) 100mg of PFD is dissolved in 1mL of ethanol, 10mL of deionized water containing 0.025% of the photoinitiator Irgacure2959 is added to obtain an aqueous solution of PFD ethanol, and the aqueous solution of PFD containing 0.025% of the photoinitiator Irgacure2959 is obtained by stirring at a temperature of 40-45 ℃.

(3) GelMA (M) _W = 200,000 ～ 300,000), PVP, 0.025% photoinitiator Irgacure2959 in a mass ratio of 4:1:50, and stirring for 4h at 50 ℃ after mixing to obtain a GelMA/PVP substrate.

(4) The PFD microneedle-based material was composed of 0.025% aqueous Irgacure2959 photoinitiator, gelMA, PVP without PFD.

(5) Dropping the microneedle matrix containing the PFD into a PDMS mold, removing bubbles in vacuum for 5min, removing redundant microneedle matrix on the surface of the mold, placing the PDMS mold into a baking oven at 45 ℃, drying for 30min, repeatedly adding the microneedle matrix containing the PFD into the mold, and drying, consuming about 500 mu L of the microneedle matrix containing the PFD, and improving the loading capacity of the PFD in the microneedle patch; irradiating the dried microneedle array with ultraviolet rays for 30s, and performing photo-curing molding;

coating a GelMA/PVP substrate on a mould under a light-proof condition to cover a microneedle array, vacuum drying for 5min to form a substrate, and drying the GelMA/PVP-MNP-PFD system obtained after drying at 45 ℃ overnight until the GelMA/PVP-MNP-PFD system is successfully separated from the mould, so as to obtain the PFD slow-release microneedle patch for resisting myocardial fibrosis.

To verify the formability of the microneedle patch, a microneedle patch for adults was prepared, which had a microneedle size of 15×15 array, a base thickness of 800 to 1000 μm, a microneedle tip diameter of 10 to 20 μm, a root diameter of about 1900 μm, a total length of about 5000 μm (1/2 of the thickness of the myocardium of an adult), a spacing between adjacent microneedles of about 600 μm between rows or columns, and a length of 1200 μm with a diameter of the tip of the microneedle of less than 50 μm of about 1/4 of the total length of the microneedles.

For in vivo test development, a microneedle patch for rats was prepared, which had a microneedle size of 15×15 array, a base thickness of 100 to 150 μm, a microneedle tip diameter of 10 to 20 μm, a root diameter of about 300 μm, a total length of about 800 μm (1/2 of the myocardial thickness of the rat), a spacing between adjacent microneedles between rows or columns of about 600 μm, and a length of 200 μm with a diameter of the tip of less than 50 μm of 1/4 of the total length of the microneedles. In the microneedle patch for rats, a microneedle stereofluorescent microscope image is shown in fig. 2, and a microneedle scanning electron microscope image is shown in fig. 3.

Experimental example 1

And constructing a rat myocardial infarction injury model. Myocardial infarction model was prepared by ligating the anterior descending branch of the left coronary artery of the rat. SD rats of 6-8 weeks old were taken and water was prohibited for 6 hours prior to surgery. SD rats were weighed by electronic scale, then placed in a pre-anesthesia tank for anesthesia, and when there was no reflex in the hind limbs of the rat jaw, they were fixed on the rat operating table, supine was placed, and 20-30 μg/kg of atropine was injected intraperitoneally to reduce airway secretions. And the electrode of the MP150 data acquisition system detector is connected to observe the change of the rat operation center electrogram. The neck and chest of the rat are removed. The neck skin was cut longitudinally about 1-2 cm and the subcutaneous tissue and muscle separated, the trachea and thyroid glands were fully exposed, the trachea was blunt separated and the posterior band passed. Lifting the wire, cutting the trachea half-cycle parallel trachea cannula at the position of a trachea ring by using ophthalmic scissors, connecting the trachea half-cycle parallel trachea cannula with an inhalation anesthesia machine, adjusting parameters of the anesthesia machine, and controlling the oxygen flow to be 0.6L/min and the concentration of isoflurane to be 3%. The skin was cut longitudinally about 2-3 cm at the left edge of the sternum, and the subcutaneous tissue, pectoral major muscle and anterior saw muscle were blunt separated. The spreader fully exposes the rib and intercostal muscles after spreading. Intercostal muscles are passively separated between the third and fourth intercostals, exposing the pleura. The cotton swab is used for piercing the pleura and entering the thoracic cavity when in expiration. The small animal spreader spreads the ribs, tears the pericardium, and reveals the heart. If necessary, the left lung can be isolated by padding cotton balls on the left outer side of the heart. After probing the left atrial appendage position, the ligation site was determined (2 mm below the boundary of the pulmonary artery with the left atrial appendage). The anterior descending left coronary artery was ligated using a 6-0 (13 mm needle) prolene wire. After no obvious bleeding is detected, the chest is closed. And one drainage tube is placed in the chest cavity. The intercostal muscles were sutured and then the surgical field muscles and skin were disinfected with iodine. The pectoral large muscle and the anterior saw muscle were closed and the skin was sutured. Residual intrathoracic fluid and fluid are aspirated through the drain tube with a 10mL syringe, and the drain tube is removed. After the rats wake up, the tracheal cannula was removed, airway blood clots and secretions were removed, and the tracheotomy was sutured with a 6-0 (13 mm needle) prolene line. The surgical field was sterilized with iodophor and the neck incision was closed. Rats were observed for 30 minutes and placed back into the cage without abnormalities.

Grouping animals: sham group (Sham operation group), AMI group (positive control group), AMI-MNP group (this group transplanted MNP contained no PFD), AMI-PFD-MNP group (microneedle patch for rat, microneedle array 15×15 in example 1), treatment mode: transplanting in situ in the infarction area.

And (3) observing curative effect: cardiac ultrasonography and Masson staining were performed at day 7 and day 28 after molding, and cardiac function and fibrosis levels were evaluated, and the results are shown in fig. 4 and 5. The WGA and CX-43 staining was performed at 28 days to evaluate the level of myocardial hypertrophy and the level of myocardial electrical signal transduction from a molecular perspective, and the results are shown in fig. 6 and 7.

According to fig. 4, masson trichromatic staining showed a 3-fold decrease in infarct size and a 2-fold increase in left ventricular wall thickness at day 7 in the AMI-PFD-MNP group compared to the AMI and AMI-MNP groups.

According to fig. 5, masson trichromatic staining showed a 3-fold decrease in infarct size and a 3-fold increase in wall thickness for the AMI-PFD-MNP group compared to the AMI and AMI-MNP groups at day 28.

According to fig. 6, wheat germ lectin staining found that the cross-sectional area of Cardiomyocytes (CMs) was significantly reduced in the AMI-PFD-MNP group compared to the other groups, while there was no significant difference between the AMI-MNP group and the AMI group, suggesting that PFD could reduce the occurrence of local cardiomyocyte hypertrophy by delaying fibrosis, and consequently avoid the occurrence of cardiomyocyte apoptosis.

According to FIG. 7, we assessed the protective effect of PFD on myocardial cell electrical and biomolecular signaling in the infarcted area (Infarct zone) by detecting the expression level of CX-43 protein on day 28. In addition to having anti-fibrotic effects, PFD has been found to reduce the loss of CX-43 protein in the infarct zone. In the AMI-PFD-MNP group, the expression level of the infarct zone CX-43 was increased 2.5-fold. These results fully demonstrate that AMI-PFD-MNP can promote intercellular signaling in infarct zone, express specific cardiac markers to maintain the integrity of electrical signal network, reduce the occurrence of arrhythmia after acute myocardial infarction.

In order to evaluate whether the mechanical properties of GelMA/PVP-MNP are affected by the addition of the PFD, the stress of the microneedles on the GelMA/PVP-MNP and the GelMA/PVP-MNP-PFD is analyzed, and particularly, the influence of the drug encapsulation on the mechanical properties of the GelMA/PVP-MNP is evaluated by measuring a force-displacement curve. The results are shown on the left side of fig. 8, and the slopes of the GelMA/PVP-MNP and GelMA/PVP-MNP-PFD curves are not significantly different, which indicates that the mechanical properties of GelMA/PVP-MNP are not affected by adding a certain dose of medicine. In the force-displacement curve of a single Microneedle (MN), as shown on the right in fig. 8, there is a distinct inflection point at a displacement of 400 μm, indicating that MN will bend or fracture only when the pressure exceeds 0.4N.

To describe the drug release kinetics, gelMA/PVP-MNP-PFD was immersed in 0.1M Phosphate Buffered Saline (PBS) and an in vitro release experiment was performed. The soaking solution was subjected to centrifugal pretreatment with a 1000MW ultrafiltration centrifuge tube to remove GelMA and PVP. PFD concentration was determined by 318 column reverse phase high performance liquid chromatography at 310nm (super ODS2;5 μm,4.6 mm. Times.150 mm). In high performance liquid chromatography testing, we found that GelMA/PVP-MNP-PFD could release the PFD continuously in vitro for up to 28 days, as shown in FIG. 9.

Experimental example 2

To assess the toxicity of PFD and GelMA/PVP matrix materials, we used H9c2 cells for in vitro assessment. Briefly, H9c2 cells were grown at 5X 10 ⁴ The density of individual cells/well was inoculated into 96-well plates and cultured in cell culture media (1% penicillin-streptomycin-gentamicin mixed solution, 10% fetal bovine serum and 89% Dulbeck's Modified Eagle Medium (DMEM). Cells were incubated with DMEM containing GelMA/PVP-MNP and GelMA/PVP-MNP-PFD dilutions extracts and PFD, respectively, for 72 hours.

In the cell assay, CCK-8 assay showed that none of the three materials affected cell proliferation and no significant difference in cell viability between the 72h groups (FIG. 10), indicating that these three materials did not negatively affect cell viability and proliferation.

Comparative example 1

The preparation method of the microneedle patch was the same as in example 1, however, the microneedle size was 15×15 array, the microneedle tip diameter was 10 to 20 μm, the root diameter was about 300 μm, the microneedle height was about 600 μm, the distance between adjacent microneedles between rows or columns was 600 μm, and the length of the microneedle tip diameter smaller than 50 μm was 100 μm.

The substrates of the microneedle patches prepared in example 1 and comparative example 1 were processed to 100 to 150 μm to reduce the influence of the substrates on the investigation of the microneedle anchoring properties.

Two microneedle patches are adopted to carry out in-situ transplantation in the infarct area of the rat myocardial infarction injury model, the materials are obtained after 7 days of investigation, and the rat heart is subjected to Masson dyeing.

Heart function and fibrosis levels were assessed by performing a cardiac ultrasound examination and Masson staining on both groups of rats and the results are shown in figure 11. It is clear that the area of myocardial fibrosis in the rats in example 1 is significantly smaller than that in comparative example 1, due to the insufficient anchoring ability of the microneedles in the myocardium in comparative examples 1-2, rendering the PFD in the microneedles incapable of exerting an anti-fibrotic effect.

The invention has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

What is not described in detail in the present specification is a well known technology to those skilled in the art.

Claims

1. The pirfenidone slow-release microneedle patch for resisting myocardial fibrosis is characterized by comprising a substrate and a microneedle array, wherein the substrate is made of uncured GelMA/PVP composite material; the microneedle array fixed on the substrate is made of a solidified GelMA/PVP composite material, and a medicine pirfenidone PFD is added in the microneedle material.

2. The pirfenidone slow release microneedle patch for preventing myocardial fibrosis according to claim 1, wherein the patch is prepared by:

3. The pirfenidone slow release microneedle patch for preventing myocardial fibrosis according to claim 2, wherein in the step of mixing and stirring GelMA, PVP and deionized water containing a photoinitiator uniformly to obtain a GelMA/PVP substrate, the mass ratio of GelMA to PVP is 4 (0.9-1.1).

4. The pirfenidone slow release microneedle patch for preventing myocardial fibrosis according to claim 2, wherein in the step of mixing and stirring GelMA, PVP and deionized water containing a photoinitiator uniformly to obtain a GelMA/PVP substrate, the content of the photoinitiator in the deionized water containing the photoinitiator is 0.02-0.05wt%, and the mass ratio of GelMA to the deionized water containing the photoinitiator is 4 (40-60).

5. The sustained release pirfenidone microneedle patch for preventing myocardial fibrosis according to claim 2, wherein the PFD is added to the GelMA/PVP substrate as an aqueous PFD ethanol solution in the step of preparing the microneedle substrate containing the PFD.

6. The sustained release pirfenidone microneedle patch for preventing myocardial fibrosis according to claim 2, wherein the PFD content in the PFD-containing microneedle matrix in the step of preparing the PFD-containing microneedle matrix is 8-10 mg/mL.

7. The sustained-release pirfenidone microneedle patch for preventing myocardial fibrosis according to claim 2, wherein in the step of ultraviolet irradiation and photo-curing and molding of the dried microneedle array, the microneedle array is subjected to ultraviolet irradiation for 30-40 s and photo-curing and molding.

8. The PFD sustained release microneedle patch of claim 2, wherein in the step of coating the mold with the GelMA/PVP substrate and covering the microneedle array under the dark condition, the GelMA/PVP substrate for preparing the microneedle substrate is selected, or the GelMA/PVP substrate without the photoinitiator is selected to be coated on the mold and covering the microneedle array.

9. The pirfenidone slow release microneedle patch for preventing myocardial fibrosis according to claim 1, wherein the tip diameter of the microneedle is 10-20 μm, the root diameter is 280-320 μm, the total length is 1/4-1/2 of the myocardial thickness, and the length of the diameter of the front end of the microneedle less than 50 μm is 1/5-1/4 of the total length of the microneedle.

10. The preparation method of the pirfenidone slow-release microneedle patch for resisting myocardial fibrosis is characterized by comprising the following steps of: