CN106075539A - The nanofiber membrane preparation method of the core/shell structure of load Chinese medicine asiaticoside and wound dressing application - Google Patents

Abstract

The invention discloses the preparation method and application of the nano fibrous membrane of a kind of core/shell structure loading Chinese medicine asiaticoside.Being shell by lactic acid/ethanol copolymer (PLGA), polycaprolactone (PCL) load asiaticoside is inner core, prepares core/shell structure medicament-carrying nano-fiber membrane by coaxial electrostatic spinning method.The inventive method has prepares that raw material is easily obtained, simple to operate, Modulatory character is strong, be prone to the feature of large-scale industrial production.The nano fibrous membrane of the core/shell structure of the load Chinese medicine asiaticoside that the present invention prepares is applied to wound dressing, has the advantage that the wound that drug loading is controlled, utilization ratio of drug is high and outstanding promotees more effect.

Description

Preparation method and injury of nanofibrous membrane with core/shell structure loaded with traditional Chinese medicine asiaticoside Mouth Dressing Application

technical field

The present invention relates to the preparation of a drug-loaded nanofiber membrane, in particular to a method for preparing a nanofiber membrane with a core/shell structure loaded with the traditional Chinese medicine asiaticoside, and a nanofiber membrane with a core/shell structure loaded with the traditional Chinese medicine asiaticoside. Fibrous membranes are used in medical dressings that promote wound healing.

Background technique

The skin is the largest organ in the human body and has a high-level organizational structure. Its functions are to prevent bacterial erosion and infection, protect the human body, regulate body temperature, and excrete body fluids. In addition to the destruction of the skin and tissues, large-scale skin damage may also cause a series of complex pathological and physiological changes in the whole body, such as increased metabolism, excessive loss of water and protein, immune system disorders, etc., and even life-threatening. Therefore, regardless of the time after injury, it is necessary to temporarily cover the exposed wound with wound dressings to provide an environment conducive to wound healing and tissue repair, repair skin defect wound as soon as possible, and restore its physiological function. Traditional medical dressings are mainly made of gauze, cotton and other materials for cleaning or protecting wounds. They are cheap and easy to make, but they have congenital defects that are difficult to overcome: they have no obvious promotion effect on wound healing; dressing changes can cause pain and are easy to Damage new tissue; when the dressing is soaked, pathogens can easily pass through; changing the dressing requires a lot of work. With the continuous understanding of wounds and wound healing process, as well as the continuous development of material science, many new polymer materials are also used in the production of medical dressings, and play an increasingly important role in clinical applications.

Electrospinning technology is an efficient method for preparing nanofibers. In addition to the advantages of high specific surface area and porosity of ordinary electrospun membranes, the superfine fiber membranes obtained by coaxial electrospinning also have a unique core/shell structure. structure. When the inner core contains biologically active drugs, it has the function of protecting the drugs, keeping them stable in harsh environments, preventing their decomposition and denaturation, and achieving the purpose of continuously delivering biologically active substances or drugs. A kind of wound dressing processing method with good market prospect.

Centella asiatica (Centella asiatica), also known as half money, is a plant of the genus Centella asiatica, which is widely distributed in the south of the Yangtze River Basin. It has a history of more than two thousand years. Asiaticoside is the main monomer component extracted from the traditional Chinese medicine Centella asiatica. Modern pharmacological studies have shown that it has the effect of inhibiting the proliferation of fibroblasts. Asiaticoside reduces the expression of TGF-β1 mRNA that promotes scar hyperplasia and increases the expression _of TGF _- β3 mRNA that inhibits scar hyperplasia, thereby causing a significant decrease in the expression of inhibitors of metal matrix proteases (TIMPS) and achieving the promotion of type I collagen. Degradation, reduce the effect of scar hyperplasia. Patent No. CN201510994598.5 "A Medical Dressing with the Effect of Promoting Wound Healing and its Preparation Method", Patent No. CN201510994602.8 "Medical Film and Preparation Method for Promoting Skin Wound Repair", Patent No. CN201510994598 .5 patent "a medical dressing with the effect of promoting wound healing and its preparation method", the patent number is CN201510994613.6 patent "dressing and preparation method for promoting skin wound repair", although asiaticoside is used as an active ingredient It is used in wound dressings, but they are all administered in the form of gels or spreads. Different dosage forms and different administration methods have a huge impact on pharmacokinetics and relative bioavailability. Considering that asiaticoside is unstable and easily inactivated, it can be used as the main drug to prepare drug-loaded electrospun fiber membranes with a core/shell structure by coaxial electrospinning technology. This method not only makes asiaticoside more widely used clinically, but also opens up a new way for the development of traditional Chinese medicine formulations.

Contents of the invention

The invention provides a preparation method of a core/shell structure nanofiber membrane loaded with madecassoside, and an application of the drug-loaded nanofiber membrane as a wound dressing. With good biocompatibility and biodegradability biomedical polymer material polylactic acid/glycolic acid copolymer (PLGA) as the shell, polycaprolactone (PCL) load with good spinning performance and excellent mechanical properties Asiaticoside is used as the inner core, and a core/shell structure drug-loaded nanofiber membrane is prepared by coaxial electrospinning. The prepared drug-loaded nanofiber membrane has outstanding advantages in promoting wound healing and can be used for medical wound dressings. The preparation raw material is easy to obtain, the price is cheap, the preparation process is simple, and it is easy for large-scale industrial production, and has a good market prospect.

A method for preparing a nanofibrous membrane with a core/shell structure loaded with the traditional Chinese medicine asiaticoside, comprising the following steps:

(1) Dissolve lactic acid/glycolic acid copolymer (PLGA) in an organic solvent to make a shell spinning solution with a concentration of 0.1-0.3 g/mL;

(2) Dissolving polycaprolactone (PCL) in organic solvent II to form a solution with a concentration of 0.13-0.27 g/mL, and adding 0.02-0.08 g/mL asiaticoside to prepare an inner core spinning solution;

(3) Add the above two solutions of shell spinning solution and inner core spinning solution into two syringes respectively, connect them to the coaxial electrospinning device through a coaxial needle, and carry out high-voltage electrospinning to obtain Centella asiatica loaded Glycoside core/shell structured nanofibrous membranes.

The high-voltage electrospinning conditions are as follows: the power supply voltage is 12 kV-21 kV, the distance between the needle head and the receiving substrate is 12 cm-16 cm, the feeding speed is 0.5 mL/h-1.3 mL/h for the outer layer, and the inner layer is 2.0 mL/h～3.5 mL/h.

The first organic solvent is a mixed solution of dichloromethane and N,N-dimethylformamide; wherein, the volume ratio of dichloromethane and N,N-dimethylformamide is 3-4:1.

The second organic solvent is dichloromethane.

The nanofiber membrane prepared by the preparation method is characterized in that: the morphology of the nanofiber membrane is a core/shell structure.

The application of the loaded nanofiber membrane prepared by the method for preparing the nanofiber membrane with the core/shell structure loaded with the traditional Chinese medicine asiaticoside in wound healing promoting dressings.

Lactic acid/glycolic acid copolymer (PLGA) is selected as the shell spinning solution of the present invention because PLGA has excellent biocompatibility and biodegradability. In organisms, it can be degraded into lactic acid metabolized by living cells, and finally can be completely degraded into carbon dioxide and water to ensure the safety of organisms.

The carrier material of the inner core liquid is polycaprolactone (PCL), because PCL has excellent electrospinning performance, low cytotoxicity and excellent dimensional stability.

The volume ratio of the mixed solution of dichloromethane/N,N-dimethylformamide is preferably 3-4:1, which is conducive to the complete dissolution of PLGA to form a uniform solution.

The drug is the active ingredient Asiaticoside in the traditional Chinese medicine Centella asiatica, which has important functions such as promoting collagen synthesis and cardiovascular formation in the body, stimulating granulation growth, thereby accelerating cell proliferation and promoting wound healing.

The electrospinning device can adopt an existing general device in the field, and the needle adopts a coaxial needle.

Because polycaprolactone (PCL) is insoluble in water, and its organic solution will undergo phase separation after contacting with the water phase, resulting in the precipitation of the solute and becoming a gel-like substance. In order to ensure the stability of electrospinning, water should be avoided as a solvent. Therefore, for the PLGA/PCL system, the mixed solution of dichloromethane/N,N-dimethylformamide is selected as the solvent of PLGA, and the solvent of PCL is dichloromethane. methane. This compatible system can effectively reduce the interfacial tension between the inner and outer solutions, making the electrospinning process more stable and less prone to defects such as droplets.

One of the decisive factors for the formation of core/shell fibers is that the solution in the inner and outer tubes has a suitable concentration and flow rate: if the concentration is too small, electrospinning is unstable, and more core/shell fibers cannot be obtained, and if the concentration is too high, electrospinning will not be possible; the flow rate of the outer tube If it is too small, the inner layer solution cannot be wrapped, and the flow rate of the outer tube is too large, the inner layer solution is discontinuous, and the effect of coaxial electrospinning is close to that of ordinary electrospinning. Therefore, the concentration of the shell spinning solution in the present invention is 0.1 ~ 0.3 g/mL; the concentration of the inner core spinning solution is 0.13 ~ 0.27 g/mL, and the feeding rate shell is 0.8 mL/h ~ 2.0mL/h. Layer is 0.5 mL/h ~ 1.6 mL/h.

The concentration of the drug asiaticoside in the present invention is preferably 0.02 ~ 0.08 g/mL, which is to obtain an electrospun nanofiber membrane with uniform diameter distribution and smooth surface and to make the content of the drug in the drug-loaded electrospun membrane comparable to that of commercially available finished products. The medicine is similar.

The present invention has the following advantages:

(1) The present invention uses lactic acid/glycolic acid copolymer (PLGA) as the shell spinning material, which has excellent biocompatibility and biodegradability. As the first layer in contact with the skin, it can effectively ensure the safety of organisms;

(2) In the present invention, polycaprolactone (PCL), which has excellent electrospinning performance, low cytotoxicity and excellent dimensional stability, is used as the inner core spinning material loaded with drugs, which has good compatibility with drugs and can be Realize the effective loading of drugs and maintain the integrity of the shape of the fiber when used;

(3) By adjusting the power supply voltage, the distance between the needle and the receiving substrate, the feeding speed, the concentration of the spinning solution, and the environmental parameters during the electrospinning process, nanofiber membranes with different shapes and fiber diameters can be obtained, thereby realizing fiber Controllable preparation of membranes;

(4) The core-shell structure nanofibrous membrane loaded with traditional Chinese medicine asiaticoside according to the present invention not only has high porosity and specific surface area, but also overcomes the shortcomings of asiaticoside's instability and easy inactivation , using the biocompatibility and degradability of drug-loaded polymer materials, while protecting the wound, the drug is slowly released with the degradation of the carrier, which plays the role of anti-inflammatory and antibacterial, and accelerates the healing speed of the wound;

(5) The core-shell structure nanofibrous membrane loaded with traditional Chinese medicine asiaticoside prepared by the present invention is applied to wound dressings. Compared with ordinary gel or coated membranes, it has controllable drug loading, controllable release rate, and reduces Burst release can maintain the effective drug concentration of the drug in the environment for a long time, greatly improving the utilization rate and effect of the drug and many other technical advantages;

(6) The core-shell structure nanofiber membrane loaded with the traditional Chinese medicine asiaticoside prepared by the present invention is applied to wound dressings. Compared with traditional gauze dressings, it can greatly overcome the pain caused by dressing changes and easily damage new tissues, and the dressing is soaked The disadvantages are that pathogens are easy to pass through and the workload of changing dressings is large;

(7) The core/shell structure nanofibrous membrane loaded with the traditional Chinese medicine asiaticoside prepared by the present invention is applied to wound dressings, avoiding the cumbersome processing procedures of traditional Chinese medicine and its compound in the process of processing, and can overcome some defects caused by traditional Chinese medicine. The shortcoming of unstable curative effect caused by pharmacokinetics is conducive to the standardized research, development, production and management of drugs;

(8) The preparation method of the present invention is simple and easy to operate. While electrospinning the carrier material, the drug loading can be realized, and the required drug loading can be adjusted according to the different needs of disease treatment, and then it can be conveniently Realize individualized drug release therapy.

Description of drawings

Fig. 1 is the scanning electron micrograph of the core/shell structure nanofibrous film loaded with asiaticoside prepared in Example 1;

Fig. 2 is the transmission electron micrograph of the core/shell structure nanofibrous film loaded with asiaticoside prepared in Example 1;

Fig. 3 is the in vitro drug release curve of the core/shell structure nanofibrous membrane loaded with asiaticoside prepared in Examples 1, 2, 3, and 4;

Fig. 4 (a) is the naked eye observation photo of wound modeling;

Fig. 4 (b) is the macroscopic observation photo of wound healing of the core/shell structure nanofibrous membrane loaded with asiaticoside prepared in Examples 1, 2, 3, 4 after 2 days;

Fig. 4 (c) is the macroscopic observation photo of the point wound healing of the core/shell structure nanofibrous membrane loaded with asiaticoside prepared in Examples 1, 2, 3, and 4 after wounding for 5 days;

Fig. 4 (d) is the macroscopic observation photo of the point wound healing of the core/shell structure nanofibrous membrane loaded with asiaticoside prepared in Examples 1, 2, 3, and 4 after wounding for 7 days;

5A to 5E are histological slices of asiaticoside-loaded core/shell structure nanofibrous membranes prepared in Examples 1, 2, 3, and 4 observed under a microscope for wound repair 7 days after injury.

detailed description

Example 1

Dissolve lactic acid/glycolic acid copolymer (PLGA) in a mixed solution of dichloromethane and N,N-dimethylformamide (the volume ratio of dichloromethane to N,N-dimethylformamide is 3:1) , to make a shell spinning solution with a concentration of 0.15 g/mL; dissolve polycaprolactone (PCL) in dichloromethane to form a solution with a concentration of 0.13 g/mL, and add 0.02 g/mL of Centella asiatica Glycosides are formulated as an inner core spinning solution. Add the shell spinning solution and the core spinning solution into two syringes respectively, and connect them to the coaxial electrospinning device through a coaxial needle. When the power supply voltage is 12 kV, the distance between the needle and the receiving substrate is Under the spinning conditions of 13 cm, feeding speed of 0.5 mL/h for the outer layer and 2.0 mL/h for the inner layer, a core/shell nanofiber membrane loaded with asiaticoside was obtained.

Example 2

Dissolve lactic acid/glycolic acid copolymer (PLGA) in a mixed solution of dichloromethane and N,N-dimethylformamide (the volume ratio of dichloromethane to N,N-dimethylformamide is 3.5:1) , to make a shell spinning solution with a concentration of 0.20 g/mL; dissolve polycaprolactone (PCL) in dichloromethane to form a solution with a concentration of 0.15 g/mL, and add 0.04 g/mL of Centella asiatica Glycosides are formulated as an inner core spinning solution. Add the shell spinning solution and the core spinning solution into two syringes respectively, and connect them to the coaxial electrospinning device through a coaxial needle. When the power supply voltage is 14 kV, the distance between the needle and the receiving substrate is Under the spinning conditions of 14 cm, feeding speed of 0.7 mL/h for the outer layer and 2.4 mL/h for the inner layer, a core/shell nanofiber membrane loaded with asiaticoside was obtained.

Example 3

Dissolve lactic acid/glycolic acid copolymer (PLGA) in a mixed solution of dichloromethane and N,N-dimethylformamide (the volume ratio of dichloromethane to N,N-dimethylformamide is 4:1) , to make a shell spinning solution with a concentration of 0.23 g/mL; dissolve polycaprolactone (PCL) in dichloromethane to form a solution with a concentration of 0.20 g/mL, and add 0.06 g/mL of Centella asiatica Glycosides are formulated as an inner core spinning solution. Add the shell spinning solution and the core spinning solution into two syringes respectively, and connect them to the coaxial electrospinning device through the coaxial needle. The distance between the needle and the receiving substrate is Under the spinning conditions of 15 cm, feeding speed of 0.9 mL/h for the outer layer and 2.8 mL/h for the inner layer, a core/shell nanofiber membrane loaded with asiaticoside was obtained.

Example 4

Dissolve lactic acid/glycolic acid copolymer (PLGA) in a mixed solution of dichloromethane and N,N-dimethylformamide (the volume ratio of dichloromethane to N,N-dimethylformamide is 4:1) , to make a shell spinning solution with a concentration of 0.27 g/mL; dissolve polycaprolactone (PCL) in dichloromethane to form a solution with a concentration of 0.25 g/mL, and add 0.08 g/mL of Centella asiatica Glycosides are formulated as an inner core spinning solution. Add the shell spinning solution and the core spinning solution into two syringes respectively, and connect them to the coaxial electrospinning device through a coaxial needle. The distance between the needle and the receiving substrate is Under the spinning conditions of 16 cm, feeding speed of 1.0 mL/h for the outer layer and 3.0 mL/h for the inner layer, a core/shell nanofiber membrane loaded with asiaticoside was obtained.

Application example 1

Animal experiment of core-shell structure nanofibrous membrane loaded with asiaticoside applied to wound dressing: This experiment used a rat full-thickness skin defect wound model to observe the natural process of wound healing and evaluate the efficacy of electrospun membrane and drugs. Cut out a circular skin wound with a diameter of 1 cm on each side of the back spine of the rat, remove the epidermis, dermis, and subcutaneous connective tissue, and go deep into the muscle layer, and make 5 wounds per rat (Figure 4 (a) ).

The drug-loaded nanofiber membranes prepared in Examples 1, 2, 3, and 4 were respectively applied on four wounds A, B, C, and D, and wound E was covered with gauze as a blank control. Materials were collected regularly, and wound tissue sections at different time points after injury were observed with the naked eye, the growth of wound granulation tissue was analyzed, and the healing status of the four wounds was evaluated. The results are shown in Fig. 4(b–d).

Two days after the trauma, the skin of the wound margins in each group shrank and was accompanied by scabbing, and there was a severe inflammatory reaction on the surface of the wound, and the wound was red after the material was removed. The wounds treated with the films of Example 1, Example 2, Example 3 and Example 4 had less inflammatory exudates, and the wounds were whitish in color. Blank group E (covered with ordinary gauze) had the most severe inflammatory response and the largest wound area (Fig. 4(b)).

Five days after the trauma, the scarring of the wound was more obvious, and the area of the wound was significantly reduced. At this time, the wound was ruddy and the inflammatory reaction was basically over, and bright red granulation tissue could be seen. However, compared with the blank group E (covered with ordinary gauze), the wounds treated with the films of Example 1, Example 2, Example 3, and Example 4 obviously had better healing effects, and the epidermis had been formed (Figure 4(c)) .

After 7 days of wounding, the peripheries of the wounds treated with the films of Example 1, Example 2, Example 3 and Example 4 were almost completely covered by new epidermis, and eschar rarely existed. But the wound healing of the blank group E was significantly worse (Fig. 4(d)).

It shows that the prepared drug-loaded nanofibrous membrane can play an effective role in promoting wound healing.

Application example 2

Animal experiment of core/shell structure nanofibrous membrane loaded with asiaticoside applied to wound dressing: wound healing is a complex physiological repair process involving a variety of cells and tissues, and fibroblasts play a very important role in wound repair effect. In this experiment, a rat full-thickness skin defect wound model was used to observe the natural process of wound healing and evaluate the efficacy of electrospun membranes and drugs. Cut out a circular skin wound with a diameter of 1 cm at one centimeter on both sides of the back spine of the rat, remove the epidermis, dermis and subcutaneous connective tissue, and go deep into the muscle layer, and make 5 wounds per rat.

The drug-loaded nanofiber membranes prepared in Examples 1, 2, 3, and 4 were respectively applied on four wounds A, B, C, and D, and wound E was covered with gauze as a blank control. 7 days after injury, the wound tissue sections were observed under a microscope. The specific method is to anesthetize the rat with 10% chloral hydrate, and use sterile surgical instruments to take the newborn skin of the wound and the surrounding normal skin, reaching deep into the deep fascia on the surface of the muscle layer (take more skin around the incision to prevent damage) Newborn skin, but also easy to fix and not easy to curl). Flatten the removed skin with tweezers, stick the skin side down in a perforated plastic fixing box, immediately put the box in 4% paraformaldehyde fixative solution, and fix it in a refrigerator at 4°C for 24 hours. Shake it from time to time during the period, so that the tissue can be fixed more completely. The fixed tissues were taken out and washed with PBS solution with pH 7.4 every 5 min, twice. After rinsing, place in 20% sucrose solution for dehydration, after sinking to the bottom, change to 30% sucrose solution to continue dehydration and sink to the bottom, then change to 35% sucrose solution for dehydration until the tissue block sinks to the bottom. Put the dehydrated tissue into a cryostat for OTC embedding, and make slices with a thickness of 10-20 μm. Rinse the slices with PBS every 5 min for 3 times. The prepared sections were washed with water for 1-2 s, and stained with hematoxylin (60°C) for 30-60 s. Wash away the hematoxylin semen with running water and rinse with 1% hydrochloric acid ethanol. The sections were rinsed with running water and stained with 0.5% eosin solution for 30-60 s. Rinse with running water, 80% ethanol, 95% ethanol, and anhydrous ethyl alcohol for 1-2 s to make slices. The slices were rinsed with phenolic acid xylene for 2-3 s, and then washed twice with xylene for 2-3 s, and sealed with resin to make transparent slides for observation. The growth of wound granulation tissue was analyzed, and the healing status of the five wounds was evaluated. The results are shown in Figure 5A, Figure 5B, Figure 5C, Figure 5D, Figure 5E.

The main component of the intercellular substance in the granulation tissue is collagen, which is continuously synthesized, secreted, restructured, and renewed to continuously improve the structure and strength of the repaired tissue. Fibroblasts are the main cells that synthesize collagen. In the later stage of wound repair, fibroblasts participate in the reconstruction of repaired tissues by secreting collagenase.

After 7 days of injury, the wounds covered by the drug-loaded nanofiber membranes prepared in Examples 1, 2, 3, and 4 all had relatively loose fibroblasts and abundant capillaries, indicating that there was late granulation tissue formation, and new epithelial tissue Tissue covers the advanced granulation tissue wound, small blood vessels and hair follicles are formed, and the wound enters the final stage of repair. Compared with the other four groups, group E (Fig. 5E) had fewer mature fibroblasts and a lower maturity of granulation tissue.

Claims (6)

1. load a nanofiber membrane preparation method for the core/shell structure of Chinese medicine asiaticoside, comprise the steps:

(1) lactic acid/ethanol copolymer (PLGA) is dissolved in organic solvent one, makes the shell that concentration is 0.1 ~ 0.3 g/mL and spin Silk solution；

(2) polycaprolactone (PCL) is dissolved in organic solvent two, forms the solution that concentration is 0.13 ~ 0.27 g/mL, and add The asiaticoside of 0.02 ~ 0.08 g/mL is configured to inner core spinning solution；

(3) above-mentioned shell spinning solution, two kinds of solution of inner core spinning solution are separately added in two syringes, by coaxial syringe needle Connect into coaxial electrostatic spinning silk device, carry out high-voltage electrostatic spinning, obtain loading the core/shell structure nanofiber of asiaticoside Film.

The nanofiber membrane preparation method of the core/shell structure of load Chinese medicine asiaticoside the most according to claim 1, it is special Levying and be, described high-voltage electrostatic spinning condition is: the distance between supply voltage 12 kV～21 kV, syringe needle and reception substrate is 12cm～16 cm, feeding speed outer layer are 0.5 mL/h～1.3 mL/h, internal layer are 2.0 mL/h～3.5 mL/h.

The nanofiber membrane preparation method of the core/shell structure of load Chinese medicine asiaticoside the most according to claim 1, it is special Levying and be, described organic solvent one is the mixed solution of dichloromethane, DMF；Wherein, dichloromethane, N, N- The volume ratio of dimethylformamide is 3～4:1.

The nanofiber membrane preparation method of the core/shell structure of load Chinese medicine asiaticoside the most according to claim 1, it is special Levying and be, described organic solvent two is dichloromethane.

5. the nano fibrous membrane that the preparation method as described in any one of Claims 1-4 prepares, it is characterised in that: described nanometer The pattern of fibrous membrane is core/shell structure.

6. prepared by the nanofiber membrane preparation method of the core/shell structure of load Chinese medicine asiaticoside as claimed in claim 5 Nano fibrous membrane application in promoting wound care dressings.