CN108653741B - Metal organic coordination polymer coated natural sericin microsphere and preparation method and application thereof - Google Patents
Metal organic coordination polymer coated natural sericin microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a sericin microsphere and a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) extracting sericin from the silkworm cocoons by a high-temperature alkali extraction method to prepare a pure sericin solution; (2) adding the sericin solution into the corn oil, stirring and dispersing, and dropwise adding a cross-linking agent to obtain sericin microspheres; (3) adding acetone into the mixed emulsion, and solidifying the sericin microspheres; (4) washing and drying to obtain sericin microsphere solid; (5) incubating sericin microspheres with a medicament to obtain medicament-loaded sericin microspheres; (6) adding phenolic substances and metal ions under a neutral or alkalescent condition to obtain the drug-loaded sericin microsphere coated by the metal-organic coordination polymer. The sericin microsphere prepared by the method has a stable structure, uniform particle size and good dispersion in water, and retains the fluorescence characteristic of sericin. The drug-loaded sericin microspheres coated by the metal organic coordination polymer have the characteristics of high drug loading rate, pH sensitivity and the like.
Description
Technical Field
The invention relates to the field of biomedical composite materials, in particular to a natural sericin microsphere, a drug-loaded sericin microsphere wrapped by a metal organic coordination polymer, a preparation method and an application thereof.
Background
Sericin (Silk Sericin) is a natural high-molecular viscous protein wrapped on the surface of Silk fibroin in silkworm cocoons, accounts for about 20-30% of the content of the whole silkworm cocoons, mainly consists of polypeptides with molecular weight of 24-400 kDa, and comprises 18 amino acids such as serine, aspartic acid, glycine and the like. The shortage of people to recognize sericin for a long time leads the sericin to be treated as waste in the silk weaving industry, wastes a large amount of precious resources and causes environmental pollution. In recent years, people find that sericin has prominent performances in the aspects of moisture retention, antibiosis, antioxidation, anticoagulation, promotion of cell proliferation and adhesion and the like, and meanwhile, the sericin has wide attention in the field of biomedical materials due to good hydrophilicity, biodegradability and biocompatibility. At present, researchers dope sericin with other high molecular materials to prepare a composite hydrogel scaffold or a composite membrane, and apply the composite hydrogel scaffold or the composite membrane to tissue engineering repair and drug delivery systems. In addition, researchers have used lithium bromide extraction methods to obtain undegraded sericin, and have prepared a variety of injectable sericin hydrogels with simple chemical crosslinking, which have good biocompatibility and degradability, are capable of supporting cell adhesion and proliferation, and as a scaffold material for tissue engineering can promote repair of a variety of soft tissue injuries. Sericin is a sustainable and easily-obtained natural polymer material, has good gelling property, biocompatibility, biodegradability and natural cell adhesion performance, is concerned in tissue engineering repair and research of drug carriers, and has great development prospect.
Microspheres (microspheres) are spherical particles with a particle size of 1-100 μm prepared from natural or synthetic polymer materials. Because many medicines can not be directly used or the effect of direct use is not ideal, people use high molecular materials to embed medicines to prepare the medicine carrying microspheres, thereby achieving the effect of controlling the medicine administration. The polymer microsphere not only has the advantages of easy separation and extraction and the like of the solid phase carrier, but also has the advantages of low price, large specific surface area, good dispersibility, easy functionalization, adjustable biocompatibility and the like. With the deepening of understanding of sericin, researchers pay more and more attention to and search for sericin microspheres. The current methods for preparing sericin microspheres mainly comprise a coagulation precipitation method, a spray drying method and a spray freeze drying crosslinking method, but the methods have the following defects: 1) the preparation process is complex and is not easy to implement, for example, a spray freeze-drying method needs to spray a sericin mixed solution into a space with the temperature of minus 80 ℃ for coagulation and collection, and sericin microspheres can be obtained after freeze-drying; 2) the particle size of the prepared microsphere is difficult to accurately control, for example, the appearance and the size of the sericin microsphere prepared by a coagulation precipitation method are difficult to control; 3) the release rate of the carried medicine is not easy to control, for example, the medicine carrying sericin microsphere medicine prepared by adopting a coagulation precipitation method or a spray freeze drying method has too high release speed.
Based on the above, the sericin microspheres with regular appearance, uniform particle size and good dispersibility are successfully prepared by combining a water-in-oil emulsification method with a chemical crosslinking method for the first time. The method is simple and easy to implement, high in yield, free of toxic by-products and free of toxicity and harm in the preparation process; the obtained sericin microsphere has smooth surface, controllable size (5-100 μm), and fluorescence property; the microsphere can effectively carry out the carrying of medicines and cells based on the good biocompatibility and cell adhesion of sericin. Furthermore, the surface of the drug-loaded microsphere is coated with a plurality of layers of metal-organic coordination polymers by a layer-by-layer self-assembly method, the release speed of the drug can be controlled by changing the number of layers of the polymers coated on the surface of the microsphere, and the drug can be intelligently released in a responsive manner according to the pH value of the environment. The natural sericin microspheres prepared by the emulsification method and the sericin microspheres wrapped by the metal organic coordination polymer have wide application prospects in the research fields of drug carriers, cell carriers, tumor diagnosis and treatment, fluorescent tracing, tissue engineering and the like.
Disclosure of Invention
The invention aims to overcome the defects of the traditional microsphere preparation method and provide a preparation method and application of sericin microspheres and drug-loaded sericin microspheres wrapped by metal-organic coordination polymers, which have the advantages of simple process, high yield and good biocompatibility.
In order to achieve the purpose, the invention provides sericin microspheres, drug-loaded sericin microspheres coated by a metal-organic coordination polymer and a preparation method thereof, wherein the preparation method comprises the following steps:
(1) weighing a certain amount of silkworm cocoons, extracting sericin by a high-temperature alkali extraction method, and centrifuging, dialyzing and freeze-drying to obtain natural sericin solid;
(2) dissolving the sericin solid obtained in the step (1) in water to prepare a sericin solution with the concentration of 10 wt%;
(3) dropwise adding the sericin solution obtained in the step (2) into corn oil (containing a certain amount of emulsifier Tween-20), and stirring to fully emulsify the corn oil;
(4) dropwise adding a cross-linking agent into the emulsion obtained in the step (3), and stirring to fully cross-link the emulsion;
(5) dropwise adding acetone into the emulsion obtained in the step (4), and stirring to fully solidify the emulsion;
(6) centrifuging the mixed solution obtained in the step (5) to obtain the solidified sericin microspheres, washing the solidified sericin microspheres for three times by acetone, and performing vacuum drying to obtain the pure natural sericin microsphere solid.
(7) And (4) mixing the natural sericin microspheres obtained in the step (6) with a medicament for reaction to obtain the medicament-loaded sericin microspheres.
(8) Reacting the drug-loaded sericin microspheres obtained in the step (7) with phenolic substances such as tannic acid and metal ions such as iron ion solution in buffer solution to obtain drug-loaded sericin microspheres coated by metal organic coordination polymer; or reacting the natural sericin microsphere buffer solution obtained in the step (6) with phenolic substances such as tannic acid and metal ions such as iron ions to obtain the natural sericin microsphere coated by the metal-organic coordination polymer.
Further, the silkworm cocoon selected in the step (1) is a normal silkworm cocoon variety (white jade, Haoyue, etc.), and the silkworm fibroin deletion mutant variety (185Nd-s，140Nd-sEtc.) purchased from the institute for silkworm industry, academy of agricultural sciences, china.
Further, the specific process for extracting sericin comprises the following steps:
1) weighing a certain amount of silkworm cocoons, shearing the silkworm cocoons, washing the silkworm cocoons with water, and extruding the silkworm cocoons to remove water;
2) soaking the silkworm cocoon fragments obtained in the step 1) in Na with the concentration of 0.01-0.20 mol/L2CO3Heating and stirring at 600 rpm at 80-100 deg.C in water solution for 0.5-1 hr, wherein 20-30 mL Na is added into per gram of silkworm cocoon2CO3An aqueous solution;
3) centrifuging the solution in the step 2) at 3500 rpm for 5-10 min, and removing insoluble precipitates to obtain a clear solution;
4) dialyzing the solution obtained in the step 3) in deionized water for 2-3 days by a dialysis bag with MW of 3500 Da, and changing water once every 6 hours;
5) centrifuging the solution obtained in the step 4) at 8000 rpm for 10-15 min, and removing insoluble precipitate to obtain a clear solution;
6) quickly freezing the solution obtained in the step 5) in liquid nitrogen, placing in a vacuum freeze dryer for 2-3 days until completely freeze-drying to obtain pure sericin solid, and placing in a refrigerator at-20 ℃ for sealed storage.
Further, the dosage of the sericin solution and the corn oil in the step (3) is 1:5-20, namely 1 mL: 5-20 mL; the using amount of the emulsifier Tween-20 in the corn oil is 1-10%, namely 10 mL of the corn oil contains 1000 mu L of Tween-20100-.
Further, in the step (4), the concentration of the cross-linking agent glutaraldehyde is 25%, the addition amount is 5-20% of the reaction system, namely 0.5-2.0 mL of 25% glutaraldehyde is added into 10 mL of the reaction system; the stirring speed was 800 rpm and the crosslinking time was 1-4 hours.
Further, the amount of acetone added in the step (5) is 5-10 times of that of the reaction system, namely 50-100 mL of acetone is added into 10 mL of the reaction system; the stirring speed was 800 rpm and the curing time was 20-60 minutes.
Further, the drug in the step (7) is an anti-tumor drug, a hormone or an antibiotic.
Further, in the step (8), the concentration of the Tris buffer solution with the pH value of 7.0-9.0 is 10-20 mM, the concentration of the added tannic acid solution and the concentration of the added ferric chloride hexahydrate solution are 20-40 mg/mL and 10-20 mg/mL respectively, and the adding amount is 0.5-1.0%, namely 5-10 mu L of each tannic acid and ferric chloride hexahydrate solution are added into 1 mL system.
Further, the tannic acid used in the step (8) may be replaced by other common natural polyphenols such as anthocyanins, catechins, gallic acid, arbutin, and the like, and catechol-modified polymers.
Further, the iron ion solution used in the step (8) may be replaced by other metal ion solutions such as aluminum ions, copper ions, magnesium ions, and the like.
The invention provides a preparation method of a metal organic coordination polymer-wrapped natural sericin microsphere carrier.
The invention can control the size and the shape of the microsphere by adjusting the concentration of sericin, the concentration of an emulsifier, the water-oil ratio and the like, and can control the release speed of the medicament by changing the layer number of the metal organic coordination polymer coated on the surface, thereby being applicable to different biomedical applications.
The scheme of the invention has the following advantages:
(1) the method has the advantages of simple operation, low cost, high yield, universal applicability, no need of special complex instruments and the like; (2) the prepared sericin microspheres have good stability, biocompatibility and bioactivity, and are regular in appearance, controllable in size, uniform in particle size and good in dispersity; (3) the release speed of the medicine can be controlled by increasing or decreasing the number of layers of the metal-organic coordination polymer on the surface of the microsphere. Has wide application prospect in the fields of tissue engineering, tumor diagnosis and treatment, fluorescent tracing, drug carriers, cell carriers and the like.
Drawings
FIG. 1 is a shape diagram of a common microscope with sericin microspheres dispersed in water according to the embodiment of the invention, wherein the sericin solution concentration is 10 wt%, the emulsifier concentration is 2%, the water-oil ratio is 1:10, and the emulsification time is 15 min.
FIG. 2 is a shape diagram of a fluorescent microscope with sericin microspheres prepared by dispersing sericin solution with 10 wt% of concentration, 2% of concentration of emulsifier, 1:10 of water-oil ratio and 15 min of emulsification in water according to an embodiment of the invention.
FIG. 3 is a shape diagram of sericin microspheres prepared in an embodiment of the invention under a common microscope in a dry state with a sericin solution concentration of 10 wt%, an emulsifier concentration of 2%, a water-oil ratio of 1:10, and an emulsification time of 15 min.
FIG. 4 is a shape diagram of sericin microspheres prepared in an embodiment of the invention under a fluorescence microscope in a dry state with a sericin solution concentration of 10 wt%, an emulsifier concentration of 2%, a water-oil ratio of 1:10, and an emulsification time of 15 min.
FIG. 5 is a scanning electron microscope topography of sericin microspheres prepared in the embodiment of the invention with a sericin solution concentration of 10 wt%, an emulsifier concentration of 2%, a water-oil ratio of 1:10, and an emulsification time of 15 min.
FIG. 6 is a shape diagram of a common microscope with sericin microspheres dispersed in water according to the embodiment of the invention, wherein the sericin solution concentration is 15 wt%, the emulsifier concentration is 2%, the water-oil ratio is 1:10, and the emulsification time is 15 min.
FIG. 7 is a shape diagram of a fluorescent microscope with sericin microspheres prepared by dispersing sericin solution with a concentration of 15 wt%, an emulsifier concentration of 2%, a water-oil ratio of 1:10 and an emulsification time of 15 min in water according to an embodiment of the invention.
FIG. 8 is a shape diagram of a common microscope with sericin microspheres dispersed in water according to the embodiment of the invention, wherein the sericin solution concentration is 10 wt%, the emulsifier concentration is 6%, the water-oil ratio is 1:10, and the emulsification time is 15 min.
FIG. 9 is a shape diagram of a fluorescent microscope with sericin microspheres prepared by dispersing sericin solution with 10 wt% of concentration, 6% of concentration of emulsifier, 1:10 of water-oil ratio and 15 min of emulsification in water according to an embodiment of the invention.
FIG. 10 is a shape diagram of a normal microscope with sericin microspheres prepared by dispersing sericin solution with 10 wt% concentration, 2% concentration of emulsifier, 1:20 water-oil ratio and 15 min emulsification time in water according to an embodiment of the invention.
FIG. 11 is a shape diagram of a fluorescent microscope with sericin microspheres prepared by dispersing sericin solution with 10 wt% of concentration, 2% of concentration of emulsifier, 1:20 of water-oil ratio and 15 min of emulsification in water according to an embodiment of the invention.
FIG. 12 is a general appearance diagram of natural sericin microspheres coated with different numbers of layers of tannic acid-iron ion coordination polymers.
Fig. 13 is a general appearance diagram of drug-loaded sericin microspheres coated with different layers of tannic acid-iron ion coordination polymers.
Fig. 14 is a drug release curve diagram of drug-loaded sericin microspheres coated with different layers of tannic acid-iron ion coordination polymers under different pH conditions.
FIG. 15 is a graph showing the effect of sericin microspheres coated with 2 layers of a tannin-iron ion coordination polymer on red blood cells in blood.
FIG. 16 is a graph showing the effect of sericin microspheres coated with 2 layers of tannin-iron ion coordination polymers on the viability of tumor cells.
FIG. 17 shows the effect of drug-loaded sericin microspheres coated with 2 layers of tannic acid-iron ion coordination polymer and free drug on the viability of tumor cells.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting. In the following detailed description of the present invention, certain specific details are set forth in order to provide a better understanding of the present invention.
The invention is further illustrated by the following examples:
the silkworm cocoon used in the present invention is silkworm cocoon (white jade, Haoyue, etc.) or silk fibroin-deficient cocoon (185Nd-s, 140Nd-sEtc.); the corn oil is edible non-transgenic corn germ oil; the rest of the reagents were analytically pure.
Example 1: synthesis of natural sericin microspheres
(1) Washing 20 g of silkworm cocoon, draining, soaking in 400 mL of 0.02 mol/L Na2CO3Stirring and heating the solution at 100 ℃ and 600 rpm for reaction for 1 hour, cooling, centrifuging at 3500 rpm for 5 minutes to remove insoluble precipitates, dialyzing the upper clear solution in deionized water by a 3500 Da dialysis bag for 2 to 3 days, centrifuging the solution at 8000 rpm for 10 minutes to remove the insoluble precipitates, quickly freezing the upper clear solution in liquid nitrogen, and completely lyophilizing in a vacuum lyophilizer to obtain pure sericin solid;
(2) preparing 1 mL of 10 wt% sericin solution, namely dissolving 100 mg sericin in 1 mL of deionized water, dropwise adding the solution into corn oil containing 2% emulsifier Tween-20 at a water-oil ratio of 1:10, namely emulsifying the corn oil at 800 rpm at room temperature for 15 minutes, wherein 10 mL of the corn oil (containing emulsifier Tween-20200 mu L) is used;
(3) adding a cross-linking agent glutaraldehyde into the mixed emulsion, wherein the concentration is 25%, the addition amount is 10% of the reaction system, namely, 1 mL of 25% glutaraldehyde is added, and crosslinking is carried out for 2 hours at room temperature at 800 rpm;
(4) adding acetone into the mixed emulsion, wherein the addition amount of the acetone is 4-5 times of that of the reaction system, namely adding 50 mL of acetone, and curing for 40 minutes at the room temperature of 800 rpm;
(5) centrifuging the mixed emulsion at 3500 rpm for 8 minutes, and removing the supernatant to obtain sericin protein microsphere particles;
(6) washing the sericin microspheres with acetone for three times, centrifuging at 3500 rpm for 8 minutes each time, and vacuum-drying the final precipitate to obtain the sericin microspheres.
The result is shown in fig. 1-5, the sericin microsphere prepared by the emulsification method is a sphere with regular morphology, uniform particle size, good dispersibility in water, and natural fluorescence property, and the dried microsphere can still keep regular morphology and can see obvious fluorescence.
Example 2: effect of different concentrations of sericin on the synthesis of sericin microspheres
(1) Washing 20 g of silkworm cocoon, draining, soaking in 400 mL of 0.02 mol/L Na2CO3Stirring and heating the solution at 100 ℃ and 600 rpm for reaction for 1 hour, cooling, centrifuging at 3500 rpm for 5 minutes to remove insoluble precipitates, dialyzing the upper clear solution in deionized water by a 3500 Da dialysis bag for 2 to 3 days, centrifuging the solution at 8000 rpm for 10 minutes to remove the insoluble precipitates, quickly freezing the upper clear solution in liquid nitrogen, and completely lyophilizing in a vacuum lyophilizer to obtain pure sericin solid;
(2) preparing 100 mu L of 15 wt% sericin solution, namely dissolving 15 mg sericin in 100 mu L of deionized water, dropwise adding the solution into corn oil containing 2% emulsifier Tween-20, wherein the water-oil ratio is 1:10, namely 1 mL of the used corn oil (containing emulsifier Tween-2020 mu L), and emulsifying at 800 rpm for 15 minutes at room temperature;
(3) adding a cross-linking agent glutaraldehyde into the mixed emulsion, wherein the concentration is 25%, the addition amount is 10% of the reaction system, namely adding 100 mu L of 25% glutaraldehyde, and crosslinking for 2 hours at room temperature at 800 rpm;
(4) adding acetone into the mixed emulsion, wherein the addition amount of the acetone is 4-5 times of that of the reaction system, namely adding 5 mL of acetone, and curing for 40 minutes at the room temperature of 800 rpm;
(5) centrifuging the mixed emulsion at 3500 rpm for 8 minutes, and removing the supernatant to obtain sericin protein microsphere particles;
(6) washing the sericin microspheres with acetone for three times, centrifuging at 3500 rpm for 8 minutes each time, and vacuum-drying the final precipitate to obtain the sericin microspheres.
As shown in FIGS. 6 to 7, the particle size of the microspheres gradually increased with increasing concentration of the sericin solution, and the fluorescence characteristics were not changed.
Example 3: effect of different dosages of emulsifier on sericin microsphere Synthesis
(1) Washing 20 g of silkworm cocoon, draining, soaking in 400 mL of 0.02 mol/L Na2CO3Stirring and heating the solution at 100 ℃ and 600 rpm for reaction for 1 hour, cooling, centrifuging at 3500 rpm for 5 minutes to remove insoluble precipitates, dialyzing the upper clear solution in deionized water by a 3500 Da dialysis bag for 2 to 3 days, centrifuging the solution at 8000 rpm for 10 minutes to remove the insoluble precipitates, quickly freezing the upper clear solution in liquid nitrogen, and completely lyophilizing in a vacuum lyophilizer to obtain pure sericin solid;
(2) preparing 100 mu L of 10 wt% sericin solution, namely dissolving 10 mg sericin in 100 mu L of deionized water, dropwise adding the solution into corn oil containing 6% emulsifier Tween-20, wherein the water-oil ratio is 1:10, namely 1 mL of the used corn oil (containing emulsifier Tween-2060 mu L), and emulsifying at 800 rpm for 15 minutes at room temperature;
(3) adding a cross-linking agent glutaraldehyde into the mixed emulsion, wherein the concentration is 25%, the addition amount is 10% of the reaction system, namely adding 100 mu L of 25% glutaraldehyde, and crosslinking for 2 hours at room temperature at 800 rpm;
(4) adding acetone into the mixed emulsion, wherein the addition amount of the acetone is 4-5 times of that of the reaction system, namely adding 5 mL of acetone, and curing for 40 minutes at the room temperature of 800 rpm;
(5) centrifuging the mixed emulsion at 3500 rpm for 8 minutes, and removing the supernatant to obtain sericin protein microsphere particles;
(6) washing the sericin microspheres with acetone for three times, centrifuging at 3500 rpm for 8 minutes each time, and vacuum-drying the final precipitate to obtain the sericin microspheres.
As shown in FIGS. 8-9, the particle size of the microspheres increased significantly with increasing emulsifier dosage, the fluorescence characteristics did not change, but the morphology tended to be irregular, so 2% emulsifier was selected as the optimum dosage.
Example 4: effect of different Water-oil ratios on the Synthesis of sericin microspheres
(1) Washing 20 g of silkworm cocoon, draining, soaking in 400 mL of 0.02 mol/L Na2CO3Heating the solution at 100 deg.C and 600 rpm under stirring for 1 hr, cooling, centrifuging at 3500 rpm for 5 min to remove insoluble precipitate, dialyzing the upper clear solution in 3500 Da dialysis bag in deionized water for 2-3 days, and centrifuging at 8000 rpm for 10 min to remove insoluble precipitateInsoluble precipitate, quickly freezing the upper clear solution in liquid nitrogen, and completely lyophilizing in a vacuum lyophilizer to obtain pure sericin solid;
(2) preparing 100 mu L of 10 wt% sericin solution, namely dissolving 10 mg sericin in 100 mu L of deionized water, dropwise adding the solution into corn oil containing 2% emulsifier Tween-20, wherein the water-oil ratio is 1:20, namely 2 mL of corn oil (containing emulsifier Tween-2040 mu L) is used, and emulsifying at 800 rpm for 15 minutes at room temperature;
(3) adding a cross-linking agent glutaraldehyde into the mixed emulsion, wherein the concentration is 25%, the addition amount is 10% of the reaction system, namely adding 100 mu L of 25% glutaraldehyde, and crosslinking for 2 hours at room temperature at 800 rpm;
(4) adding acetone into the mixed emulsion, wherein the addition amount of the acetone is 4-5 times of that of the reaction system, namely adding 10 mL of acetone, and curing for 40 minutes at the room temperature of 800 rpm;
(5) centrifuging the mixed emulsion at 3500 rpm for 8 minutes, and removing the supernatant to obtain sericin protein microsphere particles;
(6) washing the sericin microspheres with acetone for three times, centrifuging at 3500 rpm for 8 minutes each time, and vacuum-drying the final precipitate to obtain the sericin microspheres.
As shown in FIGS. 10 to 11, the particle size of the microspheres gradually increased with increasing water-oil ratio, and the fluorescence characteristics were not changed.
By combining the above examples and figures, it can be seen that sericin microspheres with different particle sizes and morphologies can be prepared by controlling and changing the concentration of the sericin solution, the content of the emulsifier and the water-oil ratio, so that the sericin microspheres have wider application.
Example 5: synthesis of sericin microspheres wrapped by metal organic coordination polymers with different layers
(1) The phenolic substance used in this study was tannic acid and the metal ion was iron ion. 0.80 g of tannic acid solid was dissolved in 20 mL of deionized water to prepare a tannic acid solution of 40 mg/mL. 0.20 g of ferric chloride hexahydrate solid is dissolved in 20 mL of deionized water to prepare 10 mg/mL of iron ion solution.
(2) Dispersing 5 mg sericin microspheres in 0.5 mL deionized water, adding 5 μ L iron ion solution after ultrasonic dispersion, adding 5 μ L tannic acid solution after vortex for 1 minute, adding 20 mM Tris buffer solution with the pH value of 8.0 for 0.5 mL after vortex for 1 minute, centrifuging at 6000 rpm for 4 minutes to obtain sericin microspheres encapsulated by the iron tannate ion coordination polymer, and washing with deionized water for 3 times.
(3) And (3) repeating the step (2) to obtain sericin microspheres wrapped by the metal organic coordination polymers with different layers.
As shown in FIG. 12, from left to right, the sericin microspheres which are not coated and coated with 1, 2, 3 and 5 layers of the tannin-iron ion coordination polymer are seen, the sericin microspheres are dispersed in water and are milk white, the sericin microspheres coated with the metal organic coordination polymer are grey black, and the color is increased with the increase of the number of coating layers.
Example 6: synthesis of drug-loaded sericin microspheres wrapped by metal-organic coordination polymers with different layers
(1) The phenolic substance used in this study was tannic acid and the metal ion was iron ion. The drug-loaded microspheres used in the study were sericin microspheres loaded with doxorubicin. 0.80 g of tannic acid solid was dissolved in 20 mL of deionized water to prepare a tannic acid solution of 40 mg/mL. 0.20 g of ferric chloride hexahydrate solid is dissolved in 20 mL of deionized water to prepare 10 mg/mL of iron ion solution.
(2) Dispersing 5 mg drug-loaded sericin microspheres in 0.5 mL deionized water, adding 5 muL iron ion solution after ultrasonic dispersion, adding 5 muL tannic acid solution after 1 minute of vortex, adding 0.5 mL 20 mM Tris buffer solution with the pH value of 8.0 after 1 minute of vortex, centrifuging at 6000 rpm for 4 minutes to obtain the drug-loaded sericin microspheres encapsulated by the iron tannate ion coordination polymer, and washing for 3 times by deionized water.
(3) And (3) repeating the step (2) to obtain the drug-loaded sericin microspheres wrapped by the metal-organic coordination polymers with different layers.
As shown in fig. 13, the drug-loaded sericin microspheres which are not coated and coated with 1, 2, 3 and 5 layers of the tannin-iron ion coordination polymer are sequentially arranged from left to right, and it can be seen that the sericin microspheres of doxorubicin are dispersed in water to be in red, the drug-loaded sericin microspheres coated with the metal organic coordination polymer are in deep red, and the color is increased with the increase of the number of the coating layers.
Example 7: drug release of drug-loaded sericin microspheres encapsulated by metal organic coordination polymer under different pH conditions
(1) The study used the broad-spectrum anti-tumor drug doxorubicin hydrochloride. Dissolving 100 mg of doxorubicin hydrochloride in 200 mL of PBS (pH 8.5), adding 500 mg of sericin microspheres, stirring at room temperature in a dark place for 24 hours, washing with deionized water for 3 times, and completely freeze-drying to obtain drug-loaded sericin microspheres;
(2) dispersing 5 mg of drug-loaded sericin microspheres in 0.5 mL of deionized water, adding 5 muL of ferric chloride solution (10 mg/mL) after ultrasonic dispersion, adding 5 muL of tannic acid solution (40 mg/mL) after 1 minute of vortex, adding 0.5 mL of 20 mM Tris buffer solution with the pH of 8.0 after 1 minute of vortex, centrifuging at 6000 rpm for 4 minutes to obtain the drug-loaded sericin microspheres encapsulated by tannic acid, and washing with deionized water for 3 times to repeatedly encapsulate to obtain the drug-loaded sericin microspheres encapsulated by tannic acid with different layers;
(3) 3 mg of tannin-encapsulated drug-loaded sericin microspheres were dispensed into 4 2 mL EP tubes (1 mg per tube), and 1 mL of buffers of pH 5.0, 6.5 and 7.4 were added thereto at 37oAnd C, incubation in a dark place in an incubator in a slow oscillation mode, taking the supernatant at different time points, detecting the absorbance at 494 nm by using an ultraviolet spectrophotometer, calculating the amount of the adriamycin released at different time points according to an adriamycin standard curve, and simultaneously replacing different buffer solutions for continuous incubation.
The results are shown in fig. 14, which illustrates that the drug release rate of the drug-loaded sericin microspheres gradually decreases with the increase of the number of tannin encapsulating layers, and meanwhile, the drug-loaded sericin microspheres have a pH sensitive property, i.e., acidic conditions promote the release of drugs, and thus are suitable for the release of drugs in cellular endosomes.
Example 8: blood compatibility of native sericin microspheres and metallo-organic coordination polymer encapsulated sericin microspheres
(1) Two layers of tannin-iron ion coordination polymers are encapsulated on the surface of the natural sericin microsphere, the encapsulated sericin microsphere and the unencapsulated sericin microsphere are disinfected by alcohol to prepare solutions with different concentration gradients, and in addition, PBS buffer solution is used as a negative control, and 0.2% Triton X100 solution is used as a positive control.
(2) Selecting a conventional normal anticoagulation blood specimen, centrifuging at 800 rpm for 5 minutes, discarding the supernatant, washing red blood cells for 2-3 times by using a PBS buffer solution, collecting lower layer red blood cells, and preparing into a 2% red blood cell suspension.
(3) 100 μ L of microsphere suspension and 900 μ L of erythrocyte suspension were mixed, incubated at 37 ℃ for 4 hours in a shaker, centrifuged at 8000 rpm for 10 minutes, and the supernatant was collected.
(4) The collected supernatant was measured for absorbance at 545 nm using a microplate reader.
The results are shown in fig. 15, and it can be seen that the addition of sericin microspheres or metal-organic coordination polymer-coated sericin microspheres does not cause rupture of erythrocytes, i.e., the hemocompatibility is good.
Example 9: cell compatibility of native sericin microspheres and metallo-organic coordination polymer encapsulated sericin microspheres
(1) Encapsulating two layers of tannin-iron ion coordination polymers on the surface of the natural sericin microsphere, and preparing solutions with different concentration gradients by sterilizing the encapsulated and unencapsulated sericin microspheres with alcohol;
(2) natural sericin microspheres and sericin microspheres encapsulating two layers of tannin-iron ion coordination polymers are incubated with 4T1 cells for 24 hours and 48 hours, and then the activity of 4T1 cells is detected by an MTT method.
The results are shown in fig. 16, which are the results after 24 and 48 hours of incubation of the cells and the microspheres, respectively, and indicate that the activity of the 4T1 cells is not significantly affected by the natural sericin microspheres no matter whether the microspheres are encapsulated by the tannin-iron ion coordination polymer, which indicates that the biocompatibility of the sericin microspheres is good.
Example 10: killing effect of drug-loaded sericin microspheres encapsulating two layers of tannin-iron ion coordination polymers on tumor cells
(1) Encapsulating two layers of tannin-iron ion coordination polymers on the surfaces of doxorubicin-loaded sericin microspheres, and sterilizing the encapsulated and non-encapsulated drug-loaded sericin microspheres with alcohol to prepare solutions with different concentration gradients;
(2) free adriamycin, drug-loaded sericin microspheres and drug-loaded sericin microspheres encapsulating two layers of tannin-iron ion coordination polymers are incubated with 4T1 cells for 24 hours and 48 hours, and then the MTT method is adopted to detect the activity of 4T1 cells.
The results are shown in fig. 17, which are the results after 24 and 48 hours of co-incubation of the cells and the drug-loaded microspheres respectively, and show that the drug-loaded sericin microspheres encapsulating two layers of tannin-iron ion coordination polymers have a certain killing effect on 4T1 cells and can effectively exert the anti-tumor effect.
Finally, it should be noted that the above-mentioned embodiments are only specific examples of the present invention, and the present invention is not limited to the above-mentioned embodiments, and many modifications are possible, so that all modifications directly or conceivably made from the disclosure of the present invention should be considered as the protection scope of the present invention.
Claims (9)
1. The preparation method of the natural sericin microspheres, which are porous microspheres made of natural sericin, comprises the following steps:
(1) weighing silkworm cocoon, extracting sericin by a high-temperature alkali extraction method, centrifuging, dialyzing and freeze-drying to obtain pure natural sericin solid;
(2) dissolving the sericin solid obtained in the step (1) in water to prepare a sericin solution with the concentration of 10 wt%;
(3) dropwise adding the sericin solution obtained in the step (2) into corn oil containing emulsifier Tween-20, and stirring to fully emulsify the sericin solution to obtain emulsion; the volume ratio of the sericin solution to the corn oil containing emulsifier Tween-20 is 1: 5-100; the volume ratio of the dosage of the emulsifier Tween-20 to the dosage of the corn oil in the corn oil containing the emulsifier Tween-20 is 0.1-10: 100, the stirring speed is 800 rpm, and the emulsifying time is 5-30 minutes;
(4) dropwise adding a chemical cross-linking agent into the emulsion obtained in the step (3), and stirring to fully cross-link the emulsion to obtain cross-linked emulsion;
(5) dropwise adding acetone into the crosslinked emulsion obtained in the step (4), and stirring to fully solidify the crosslinked emulsion;
(6) centrifuging the mixed solution obtained in the step (5) to obtain the solidified sericin microspheres, washing the solidified sericin microspheres for three times by acetone, and performing vacuum pumping to obtain the pure natural sericin microspheres.
2. The method of claim 1, wherein: the extraction method of the sericin solid in the step (1) comprises the following steps:
1) weighing common silkworm cocoon or fibroin-deficient mutant silkworm cocoon, shearing, washing with deionized water, and squeezing to remove excessive water to obtain silkworm cocoon fragment;
2) soaking the silkworm cocoon fragments obtained in the step 1) in Na with the concentration of 0.01-0.2 mol/L2CO3Heating and stirring the solution at 80-100 deg.C and 600 rpm for 0.5-1 hr, wherein each gram of silkworm cocoon is dispersed in 10-50 mL of Na2CO3A solution;
3) centrifuging the solution obtained in the step 2) at 3500 rpm for 5 min, and removing insoluble precipitates to obtain a clear solution I;
4) dialyzing the clear solution I obtained in the step 3) in deionized water for 2-3 days, and changing water once every 6 hours; obtaining a dialysis solution;
5) centrifuging the dialyzed solution obtained in the step 4) at 8000 rpm for 10 min, and removing insoluble precipitates to obtain a clear solution II;
6) quickly freezing the clear solution II obtained in the step 5) in liquid nitrogen, putting the clear solution in a vacuum freeze dryer for 2-3 days until the clear solution is completely freeze-dried to obtain pure sericin solid, and putting the pure sericin solid in a refrigerator at the temperature of-20 ℃ for sealed storage.
3. The method of claim 1, wherein:
in the step (4), the chemical cross-linking agent is glutaraldehyde or genipin, and the addition amount of the chemical cross-linking agent is 5-50% of the volume of the emulsion; the stirring speed is 800 rpm, and the crosslinking time is 1-3 hours;
the amount of acetone added in the step (5) is 5-20 times of the volume of the cross-linked emulsion; the stirring speed was 800 rpm and the curing time was 40 minutes.
4. Natural sericin microspheres produced by the method according to any one of claims 1 to 3.
5. Drug-loaded sericin microspheres obtained by physically mixing the natural sericin microspheres of claim 4 with a drug.
6. The drug-loaded sericin microsphere according to claim 5, wherein: the medicine is an anti-tumor medicine, hormone or antibiotic.
7. A drug-loaded sericin microsphere coated by a metal-organic coordination polymer, wherein the drug-loaded sericin microsphere of claim 5 reacts with a phenolic substance and a metal ion solution under a neutral or alkalescent condition to obtain the drug-loaded sericin microsphere coated by the metal-organic coordination polymer; the phenolic substance comprises tannin, anthocyanin, catechin, gallic acid, arbutin or catechol group modified polymer; the metal ions comprise one or more of iron ions, aluminum ions, copper ions and magnesium ions.
8. The metal-organic coordination polymer coated natural sericin microsphere, which is prepared by reacting the natural sericin microsphere as claimed in claim 4 with a phenolic substance and a metal ion solution under neutral or weakly alkaline conditions to obtain the metal-organic coordination polymer coated natural sericin microsphere.
9. The use of the natural sericin microspheres as defined in claim 4, the drug-loaded sericin microspheres coated with a metal-organic coordination polymer as defined in claim 7, or the natural sericin microspheres coated with a metal-organic coordination polymer as defined in claim 8 in the preparation of a tumor diagnosis and treatment material, a drug delivery material, a cell carrier material, a fluorescent tracer material, or a tissue engineering material.
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