Background
In recent years, with the progress of cardiac surgery, the proportion of secondary operations has been gradually increased, and adhesion has become an important factor affecting the effectiveness of the secondary operations. As in coronary artery bypass grafting, adhesions can lead to a significant increase in the incidence of bleeding-related serious complications.
Currently, anti-blocking materials can be divided into two broad categories, non-absorbable materials and bioabsorbable materials. Non-resorbable materials include synthetic membranes and xenograft membranes. Wherein the synthetic membrane comprises silicone rubber, an expanded polytetrafluoroethylene membrane, a polyethylene membrane, a terylene net and the like. Bioabsorbable materials include polylactic acid, carboxymethyl cellulose, hyaluronic acid films, and the like.
The non-absorbable materials currently used as anti-adhesion materials as permanent foreign materials in the body may cause inflammatory reactions in the body. The single membrane for preventing heart adhesion only plays a role in isolation, and has no good effects in promoting tissue healing, reducing myocardial fibrosis and inflammatory reaction and improving heart function recovery. In addition, the current gel or film for preventing postoperative adhesion cannot be cut and attached as required, and once the film and the heart are adhered, the film and the heart are difficult to tear off, so that secondary injury of tissue contusion can be caused.
Chinese patent CN201711425189 discloses an absorbable anti-adhesion material used after cardiac surgery and a membrane prepared from the absorbable anti-adhesion material, wherein an isolation membrane prepared from chitosan, gelatin, glycerol, collagen and silk fibroin has good biocompatibility, can be absorbed and degraded, can prevent postoperative adhesion between the heart and surrounding tissues and protect heart functions, and can only play a role in physical adhesion release and does not have anti-inflammatory and anti-oxidation capabilities.
Chinese patent CN201310214708.2 discloses an anti-adhesion liquid for wound irrigation and surgical operation, a preparation method and application thereof, which uses sodium carboxymethyl cellulose, brown alginate oligosaccharides, hydroxyethyl starch, polyethylene glycol and sodium chloride, and prepares membranous substances or liquid by mixing with injection water in a stirring or crosslinking mode, has various functions of hemostasis, antibiosis, anti-adhesion and healing promotion, can not be flushed at specific parts as required, and can possibly influence the external area of an operation area, and has no anti-inflammatory and anti-oxidation capabilities.
In view of this, the present invention has been made.
Disclosure of Invention
In order to solve the technical problems, the invention provides an artificial pericardium capable of preventing postoperative tissue adhesion and relieving heart fibrosis and inflammatory reaction.
Specifically, the technical scheme of the invention is as follows:
In a first aspect, the invention provides a drug-loaded artificial pericardium, which is prepared from the following raw materials, by mass, 0.575-0.625 parts of polyethylene oxide (PEO polyethylene oxide), 0.09-0.11 parts of polyethylene glycol (PEG), 0.9-1.1 parts of alpha-cyclodextrin (alpha-CD), 0.160-0.240 parts of sodium carboxymethylcellulose (NaCMC) and 0.005-0.015 parts of colchicine.
The drug-carrying artificial pericardium provided by the invention is made of absorbable materials with good biocompatibility, is not easy to cause inflammatory reaction in vivo, can be degraded and absorbed, can play a certain role in promoting recovery of heart through slow release, has good effects in promoting tissue healing, reducing myocardial fibrosis and inflammatory reaction and improving heart function recovery, can be cut as required, can attach specific parts of heart, can completely wrap the heart after being wetted by water, and can not cause contusion to tissues.
Preferably, the drug-loaded artificial pericardium is prepared from the following raw materials, by mass, 0.600 part of polyethylene oxide, 0.1 part of polyethylene glycol, 1.0 part of alpha-cyclodextrin, 0.200 part of sodium carboxymethyl cellulose and 0.01 part of colchicine.
Preferably, the molecular weight of the polyethylene oxide is 1X 106-3×106.
Preferably, the polyethylene glycol has a molecular weight of 1500-2500.
The invention provides a preparation method of the drug-loaded artificial pericardium according to the first aspect, which comprises the following steps of adding alpha-cyclodextrin into 50-70 ℃ polyethylene glycol aqueous solution, mixing, adding polyethylene oxide aqueous solution and colchicine sequentially, mixing, drying at 50-70 ℃ for 12-24 hours, adding sodium carboxymethyl cellulose aqueous solution on the surface of the mixture, and continuously drying at 50-70 ℃ for 12-24 hours.
The invention uses alpha-cyclodextrin, polyethylene glycol and polyethylene oxide as main materials for preparing the medicine for preventing postoperative cardiac adhesion, has good water reactivity and complete tissue wrapping property, can be cut as required, uses colchicine as the medicine loaded by the artificial pericardium, can slowly release medicine effect through a film, and reduces cardiac fibrosis degree and inflammatory reaction.
Preferably, the mass fraction of the aqueous polyethylene oxide solution is 3.5% -4.5%.
Preferably, the mass fraction of the polyethylene glycol aqueous solution is 1.5% -2.5%.
Preferably, the mass fraction of the sodium carboxymethyl cellulose aqueous solution is 1.6% -2.4%.
More preferably, the preparation method comprises the steps of dissolving 0.575-0.625 parts by mass of polyethylene oxide with molecular weight of 1X106-3×106 in 14-16 parts by volume of ultrapure water to prepare a polyethylene oxide aqueous solution with mass fraction of 3.5% -4.5%, dissolving 0.09-0.11 parts by mass of polyethylene glycol with molecular weight of 1500-2500 in 4-6 parts by volume of ultrapure water, slowly adding 0.9-1.1 parts by mass of alpha-cyclodextrin in a 55-65 ℃ water bath after vortex mixing, dissolving in 55-65 ℃ water bath for 50-70 seconds to obtain a mixture of polyethylene oxide and alpha-cyclodextrin, adding 14-16 parts by volume of polyethylene oxide aqueous solution with mass fraction of 3.5% -4.5%, preparing a mixed solution of alpha-cyclodextrin, polyethylene oxide and polyethylene glycol, adding 0.005-0.015 parts by mass of colchicine, stirring for 1.5-2.5 minutes by rotating centrifugal stirring by using a mixer 1500-2500, repeatedly adding the mixture into a carboxyl glass oven with mass of the mixture at 55-65 ℃ for 50-70 seconds to obtain a mixture with mass fraction of 3.5% -4.5%, and removing air bubbles in an oven with dry condition, and standing overnight, and removing the mixture in an oven with dry condition at the temperature of 1-65 ℃ to obtain a dry condition, wherein the mixture is subjected to a dry condition, and the mixture is subjected to a dry condition, and air bubble condition is removed, and the air condition is prepared.
The preparation method provided by the invention is simple, and the prepared drug-loaded artificial pericardium has good biocompatibility, can be absorbed and degraded, is convenient to implement (water reactivity and pruning as required), and can reduce heart fibrosis and inflammatory response.
The beneficial effects are that:
the invention provides a drug-loaded artificial pericardium and a preparation method thereof. The drug-loaded artificial pericardium provided by the invention can be used for relieving adhesion between the heart and surrounding tissues after cardiac surgery and relieving inflammatory reaction and fibrosis degree after surgery. The experimental group and the control group are compared after the myocardial infarction operation by animal experiments to find that the membrane has the effect of relieving heart adhesion, and the biological safety and the anti-adhesion layer anti-fibroblast adhesion capability of the membrane are proved according to in vitro cell experiments. In addition, according to the detection of the drug concentration, the film has the effect of drug slow release, can maintain the relative drug concentration for a period of time after operation, and lightens the inflammatory reaction and the fibrosis degree. The membrane has good water reactivity and biodegradability, can be completely attached by intermolecular acting force and heart tissue after wetting, does not depend on viscosity and heart adhesion, avoids secondary damage to the tissue caused by tearing again, and can be completely degraded in a certain time of implantation in a living body. In addition, compared with the traditional anti-adhesion flushing liquid, the film can be cut as required and attached to the required part, so that the influence of excessive flushing on other tissues is avoided.
Detailed Description
The invention provides an artificial pericardium with good biocompatibility, absorbability and degradability, convenient implementation (water reactivity and pruning as required), heart fibrosis and inflammatory reaction alleviation and other functions for preventing and treating tissue adhesion after heart operation.
The invention adopts the following technical scheme:
An artificial pericardium for preventing postoperative tissue adhesion and relieving heart fibrosis and inflammatory reaction is prepared from alpha-cyclodextrin (alpha-CD), polyethylene glycol (PEG), polyethylene oxide (PEO), sodium carboxymethylcellulose (NaCMC) and colchicine. The materials are all absorbable and degradable materials, have good biocompatibility and can not generate autologous inflammatory reaction.
The artificial pericardium provided by the invention uses alpha-CD, PEG, PEO as a main material for preparing the inner layer of the film, wherein a-CD and PEG are used as skeleton parts of the film, PEO is used as a crosslinking part of the film, the film can be cut as required after the preparation is finished, meanwhile, the artificial pericardium has good water reactivity, the film can shrink after being wetted by water, the film can be completely wrapped with attached tissues, and the tissue can not be contused after the film is uncovered.
The artificial pericardium provided by the invention uses NaCMC as a material for preparing the outer layer of the film, the NaCMC can provide a physical isolation layer for tissues, the fibrin generated by surrounding tissues is prevented from being adhered to the heart, and the occurrence of postoperative heart adhesion can be effectively prevented. And NaCMC has good biocompatibility with tissues, and does not cause self-inflammatory reaction.
In one of the more specific and preferred embodiments provided herein, the preparation method comprises dissolving 0.600g PEO with a molecular weight of 2X 106 in 15mL ultrapure water to prepare 15mL PEO solution with a mass fraction of 4%. Dissolving 0.1g of PEG with molecular weight of 2000 in 5mL of ultrapure water, slowly adding 1g of alpha-CD into a water bath with the temperature of 60 ℃ for dissolution after vortex mixing, carrying out ultrasonic water bath for 1 minute in the water with the temperature of 60 ℃ to obtain a PEG-alpha-CD mixture, adding 15mL of PEO solution with the mass fraction of 4% to prepare an alpha-CD/PEG/PEO solution, adding 10mg of colchicine, carrying out centrifugal stirring for 2 minutes by using a mixing deaerator 2000, and repeating for 2 times. The resulting thick mixture was added to a glass bottom petri dish, sealed in a 60 ℃ oven overnight (12 h) to remove air bubbles from the mixture. Adding 10mL of 2% NaCMC solution on the surface of the mixture, and drying in a 60 ℃ oven for over night (12 h) to obtain the final drug-loaded artificial pericardium product
The artificial pericardium provided by the invention uses colchicine as a medicine loaded on the inner layer of the film, so that the degree and the area of heart fibrosis after operation or after myocardial infarction can be effectively reduced, the recovery of heart functions is facilitated, and simultaneously, the reduction of heart inflammatory reaction and the adhesion between the heart and surrounding tissues after operation are facilitated.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
No endpoints of the ranges and any values disclosed in the specification are limited to the precise range or value, and such range or value should be understood to encompass values approaching those range or value. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "detailed description," or "some embodiments," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the embodiments. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
In the examples provided in this specification, no particular technique or condition is identified, either as described in the literature in this field or as a product specification. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
Example 1
The embodiment provides a drug-loaded artificial pericardium capable of preventing postoperative tissue adhesion and relieving heart fibrosis and inflammatory reaction, which is prepared by the following steps:
0.600g of PEO having a molecular weight of 2X 106 was dissolved in 15mL of ultrapure water to prepare 15mL of a PEO solution having a mass fraction of 4%. Dissolving 0.1g of PEG with molecular weight of 2000 in 5mL of ultrapure water, slowly adding 1g of alpha-CD into a water bath with the temperature of 60 ℃ for dissolution after vortex mixing, carrying out ultrasonic water bath for 1 minute in the water with the temperature of 60 ℃ to obtain a PEG-alpha-CD mixture, adding 15mL of PEO solution with the mass fraction of 4% to prepare an alpha-CD/PEG/PEO solution, adding 10mg of colchicine, carrying out centrifugal stirring for 2 minutes by using a mixing deaerator 2000, and repeating for 2 times. The resulting thick mixture was added to a glass bottom petri dish, sealed in a 60 ℃ oven overnight (12 h) to remove air bubbles from the mixture. And adding 10mL of 2% NaCMC solution on the surface of the mixture, and putting the mixture into a 60 ℃ oven for drying overnight (12 h) to obtain the final drug-loaded artificial pericardium finished product.
Example 2
The embodiment provides a drug-loaded artificial pericardium capable of preventing postoperative tissue adhesion and relieving heart fibrosis and inflammatory reaction, which is prepared by the following steps:
0.625g of PEO having a molecular weight of 3X 106 was dissolved in 16mL of ultrapure water to prepare 16mL of a PEO solution having a mass fraction of 3.9%. Dissolving 0.11g of PEG with molecular weight of 2000 in 5.5mL of ultrapure water, slowly adding 1.1g of alpha-CD into 65 ℃ water bath for dissolution after vortex mixing, carrying out ultrasonic water bath for 1 minute in 65 ℃ water to obtain a PEG-alpha-CD mixture, adding 16mL of PEO solution with mass fraction of 3.9%, preparing the alpha-CD/PEG/PEO solution, adding 10mg of colchicine, centrifuging and stirring for 2 minutes by using a mixing deaerator 2000 revolutions, and repeating for 3 times. The resulting thick mixture was added to a glass bottom petri dish, sealed in a 65 ℃ oven overnight (12 h) to remove air bubbles from the mixture. And adding 10mL of 2% NaCMC solution on the surface of the mixture, and putting the mixture into a 65 ℃ oven for drying overnight (12 h) to obtain the final drug-loaded artificial pericardium finished product.
Example 3
The embodiment provides a drug-loaded artificial pericardium capable of preventing postoperative tissue adhesion and relieving heart fibrosis and inflammatory reaction, which is prepared by the following steps:
0.575g of PEO having a molecular weight of 2X 106 was dissolved in 14mL of ultrapure water to prepare 14mL of a PEO solution having a mass fraction of 4.1%. Dissolving 0.09g of PEG with molecular weight of 2000 in 4.5mL of ultrapure water, slowly adding 0.9g of alpha-CD into a water bath with the temperature of 60 ℃ for dissolving after vortex mixing, carrying out ultrasonic water bath for 1 minute in 55 ℃ to obtain a PEG-alpha-CD mixture, adding 14mL of PEO solution with the mass fraction of 4.1 percent to prepare the alpha-CD/PEG/PEO solution, adding 10mg of colchicine, and carrying out centrifugal stirring for 2 minutes by using a mixing deaerator for 2000 revolutions, and repeating for 3 times. The resulting thick mixture was added to a glass bottom petri dish, sealed in a 55 ℃ oven overnight (12 h) to remove air bubbles from the mixture. And adding 10mL of 2% NaCMC solution on the surface of the mixture, and drying the mixture in a 55 ℃ oven overnight (12 h) to obtain the final drug-loaded artificial pericardium finished product.
Experimental example 1
The experimental example uses the drug-loaded artificial pericardium prepared in example 1 as experimental material to detect the anti-blocking capability, drug release concentration, microstructure, water reaction characteristic, biosafety and degradability.
The specific detection method comprises the following steps:
and (3) anti-adhesion capability, namely ligating anterior descending branches of coronary arteries of a heart of a mouse to construct an myocardial infarction model, implanting a cut artificial pericardium patch in a ligature area, performing photographing comparison between the implanted artificial pericardium patch and a non-implanted group after 28 days, and observing adhesion effect of the heart and surrounding tissues.
Drug release concentration the artificial pericardium is placed in a 24-well plate Transwell chamber with the drug carrying layer facing downwards to simulate the artificial pericardium to release drug on the surface of the heart. Ultrapure water was added to the well plate at the lower layer of the cell, and the absorbance of the solution in the well plate was measured every 1 day, 2 days, 3 days, and 4 days. Preparing a colchicine standard concentration reagent, measuring the absorbance of the reagent to obtain a colchicine absorbance-concentration standard curve, substituting the measured absorbance to obtain the drug release concentration on different days, and drawing a drug release curve.
The microstructure is that an artificial pericardium with the size of 1mmX and 1mm is stuck on a sample carrying table by a conductive adhesive tape, and a scanning electron microscope is used for observing the microstructure of the microstructure double-layer film.
The water reaction characteristic is that the prepared artificial pericardium film is cut into a rectangle with the length of 1cm X0.5cm, and is placed in a culture dish filled with ultrapure water, and the film reacts after meeting water in one eye of time.
Biosafety primary mouse fibroblasts are respectively transplanted on 6-hole plates containing artificial pericardium and no film to form an experimental group and a control group, and F-actin and DAPI are used for carrying out immunofluorescence staining on the cells to observe the growth state of the cells.
Degradability-the artificial pericardium is implanted on the surface of the heart of the mouse, and the degradation condition of the artificial pericardium is observed after 7 days by opening the chest.
The detection results are shown in FIGS. 1-6.
As can be seen from FIG. 1, the heart was less adherent to surrounding tissue than the untreated control group.
From figure 2, it can be seen that the drug-loaded artificial pericardium gradually reaches equilibrium in 4 days, indicating the drug-releasing effect of the film.
As can be seen from fig. 3, the artificial pericardium has a double-layer structure under a scanning electron microscope, and the medicine carrying layer structure is loose, so that the medicine release is facilitated, the anti-adhesion layer structure is compact, the medicine release can be prevented, and the directional release of colchicine is ensured to a certain extent.
As can be seen from FIG. 4, the artificial pericardium reacts rapidly with water, wets and shrinks within 2 seconds, indicating the rapid water-reactivity of the film, which can rapidly conform to tissue on the wetted heart surface.
As can be seen from FIG. 5, cells grew well on the artificial pericardium and cardiac fibroblast adhesion on the artificial pericardium was less than in the control group, and the anti-fibroblast adhesion ability of the film on the surface reacted with its anti-blocking properties.
As can be seen from FIG. 6, the film was mostly degraded 7 days after implantation of the artificial pericardium on the surface of the heart, demonstrating its degradability.
It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to the above-mentioned embodiments, it should be understood by those skilled in the art that the technical solution described in the above-mentioned embodiments may be modified or some technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the spirit and scope of the technical solution of the embodiments of the present invention.