CN115554249A - Microfluidic preparation method of self-assembled polypeptide microspheres - Google Patents
Microfluidic preparation method of self-assembled polypeptide microspheres Download PDFInfo
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Abstract
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps: step (1), respectively preparing a water phase solution and an oil phase solution; step (2), respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a chip pipeline to obtain self-assembled polypeptide microspheres; the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel and photoinitiator; the oil phase solution contains span 80 and mineral oil. The microfluidic preparation method can stably prepare the self-assembled polypeptide microspheres for a long time to obtain microspheres with uniform shapes. The self-assembled polypeptide microsphere has self-assembly characteristics, can be self-assembled in a microfluidic chip to form a microsphere, and can realize long-acting slow release of a therapeutic functional drug.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a microfluidic preparation method of self-assembled polypeptide microspheres.
Background
Self-assembling polypeptide is a short peptide fragment with hydrophilic and hydrophobic amino acids alternately appearing, and is commonly used as a functional drug delivery material at present. The self-assembly polypeptide can rapidly and spontaneously form cross-linked nano-fibers in an electrolyte solution with the pH value close to the isoelectric point of the self-assembly polypeptide, and then the cross-linked nano-fibers are converted into the three-dimensional fiber network hydrogel. Based on the characteristics, the controlled release material with enzyme reactivity and tumor targeting can be formed by inserting the enzymolysis substrate sequence and the tumor targeting sequence into the self-assembly polypeptide sequence. In the form of drug-loaded material administration, injectable microspheres are more convenient and minimally invasive than bulk gels or scaffolds that require surgical implantation. In the existing preparation method of the microspheres, the traditional emulsion chemical crosslinking method for curing the microspheres has the defects of agglomeration and uneven size, and easily causes the inactivation of bioactive functional drugs.
The micro-fluidic technology can conveniently control the generation of micron-sized liquid drops, and is an effective method for efficiently preparing hydrogel microspheres with uniform sizes. The editable self-assembly polypeptide is combined with the microfluidic technology, so that an intelligent functional drug delivery platform is formed. However, spontaneous cross-linking of self-assembled polypeptides has determined that it is not possible to prepare microspheres for a long time using microfluidic technology. Because it blocks the microfluidic channel after crosslinking into a gel and it is difficult to maintain the shape of the microspheres for a long time.
Therefore, it is necessary to provide a microfluidic preparation method of self-assembled polypeptide microspheres to overcome the deficiencies of the prior art.
Disclosure of Invention
The invention aims to avoid the defects of the prior art and provides a microfluidic preparation method of self-assembled polypeptide microspheres. The microfluidic preparation method of the self-assembled polypeptide microspheres can stably prepare the self-assembled polypeptide microspheres for a long time to obtain microspheres with uniform shapes.
The above object of the present invention is achieved by the following technical measures:
provides a microfluidic preparation method of self-assembled polypeptide microspheres, which comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
The aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine.
The oil phase solution contains span 80 and mineral oil.
Preferably, the preparation steps of the aqueous solution comprise the following steps:
step A1, respectively adding self-assembled polypeptide, photo-crosslinking hydrogel and photoinitiator into deionized water, and stirring to obtain a gel-forming precursor aqueous solution;
and step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering, and standing to obtain an aqueous phase solution.
Preferably, the oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing to obtain the oil phase solution.
Preferably, the step (2) is to inject the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip respectively, apply ultraviolet light to the tail end of a pipeline of the chip, adjust the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, extrude the aqueous phase solution and the oil solution together into a pore channel of the microfluidic chip through air pressure, and form a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
Preferably, the step A1 is to add the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 50-70 ℃ respectively, and stir for 10-50 min to obtain the gel-forming precursor aqueous solution.
Preferably, the step A2 is to mix the functional drug and the gel-forming precursor aqueous solution, filter the mixture through a filter membrane, and stand the mixture for 1 to 2 hours to obtain an aqueous phase solution.
Preferably, the oil phase solution is prepared by adding span 80 into mineral oil, mixing, and standing for 0.5-2 h to obtain the oil phase solution.
In the gel-forming precursor aqueous solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.25-2.00.
The concentration of the functional medicine in the gel-forming precursor water solution is 20 mg/ml-500 mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 1-2.
Preferably, the ratio of the relative pressure values of the aqueous phase solution and the oil phase solution is 20 to 100:70 to 140.
Preferably, the filter is a 0.22 μm filter.
Preferably, the microfluidic chip is a flow focusing pipeline microfluidic chip.
Preferably, the self-assembling polypeptide is at least one of a matrix metalloproteinase 1-sensitive self-assembling polypeptide, a self-assembling KLDL-12 peptide, a self-assembling RADA-16 peptide, a self-assembling IEIK-13 peptide, a self-assembling KLDL-12 peptide derivative, a self-assembling RADA-16 peptide derivative, or a self-assembling IEIK-13 peptide derivative.
Preferably, the photo-crosslinking hydrogel is at least one of methacrylic acidylated gelatin freeze-dried powder, methacrylic acidylated hyaluronic acid freeze-dried powder, methacrylic acidylated fibroin freeze-dried powder, methacrylic acidylated chondroitin sulfate freeze-dried powder, methacrylic acidylated sericin freeze-dried powder or polyethylene glycol diacrylate freeze-dried powder.
Preferably, the functional drug is at least one of exosome, drug-loaded liposome or high molecular functional drug.
Preferably, the photoinitiator is a LAP photoinitiator.
Preferably, the diameter of the self-assembled polypeptide microspheres is 50-200 μm.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps: step (1), respectively preparing a water phase solution and an oil phase solution; step (2), respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a chip pipeline to obtain self-assembled polypeptide microspheres; the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel and photoinitiator; the oil phase solution contains span 80 and mineral oil. The microfluidic preparation method can stably prepare the self-assembled polypeptide microspheres for a long time to obtain microspheres with uniform shapes. The self-assembled polypeptide microsphere has self-assembly characteristics, can be self-assembled in a microfluidic chip to form a microsphere, and can realize long-acting slow release of a therapeutic functional drug.
Drawings
The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.
FIG. 1 is a preparation diagram of a microfluidic preparation method of self-assembled polypeptide microspheres of the present invention.
FIG. 2 is a gel point curve for self-assembling polypeptides.
FIG. 3 is a shear-thinning curve for a self-assembling polypeptide.
FIG. 4 is a graph showing the distribution of the particle sizes of the self-assembled polypeptide microspheres of examples 4 to 9.
FIG. 5 is a scanning electron micrograph of the self-assembled polypeptide microspheres of example 4.
FIG. 6 is a graph of rheological profiles of self-assembled polypeptide microspheres from example 4.
FIG. 7 is a photograph of the in vitro release of self-assembled polypeptide microspheres of example 4.
Fig. 8 is a picture of mature osteoblasts using self-assembled polypeptide microspheres of example 4.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials, reagent materials and the like used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical operation companies unless otherwise specified. The matrix metalloproteinase 1 sensitive self-assembly polypeptide, the RADA-16 peptide, the RADA-12 peptide, the KLDL-12 peptide derivative and the IEIK-13 peptide derivative are purchased from Shanghai Betay Biotech, inc., and the matrix metalloproteinase 1 (MMP 1 enzyme for short) is purchased from Sigma. Span 80 and mineral oil were purchased from Sigma. Methacrylic anhydrified gelatin is available from Shanghai Allantin Biotech Co., ltd. The methacryloylated hyaluronic acid lyophilized powder, the methacryloylated fibroin lyophilized powder, the methacryloylated chondroitin sulfate lyophilized powder, the methacryloylated sericin lyophilized powder and the polyethylene glycol diacrylate lyophilized powder are all purchased from Suzhou Yongqin spring Intelligent Equipment Co.
Example 1.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, respectively adding self-assembled polypeptide, photo-crosslinking hydrogel and photoinitiator into deionized water, and stirring to obtain a gel-forming precursor aqueous solution;
and step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering, and standing to obtain an aqueous phase solution.
More specifically, step A1 is to add the self-assembled polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 50 ℃ to 70 ℃ respectively, and stir for 10min to 50min to obtain a gel-forming precursor aqueous solution.
More specifically, the step A2 is to mix the functional medicine and the gel-forming precursor aqueous solution, filter the mixture through a filter membrane, and stand the mixture for 1 to 2 hours to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing and standing to obtain the oil phase solution.
More specifically, the oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 0.5-2 h to obtain the oil phase solution.
And (3) specifically, the water-phase solution and the oil-phase solution obtained in the step (1) are respectively injected into corresponding pipeline ports of the microfluidic chip, ultraviolet light is applied to the tail end of a chip pipeline, the relative pressure of the water-phase solution and the relative pressure of the oil-phase solution are adjusted, then the water-phase solution and the oil-phase solution are extruded into a pore channel of the microfluidic chip together through air pressure, and a water-in-oil structure is formed after the water-in-oil structure is converged, so that the self-assembled polypeptide microspheres are obtained.
In the gel-forming precursor aqueous solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.25-2.00.
The concentration of the functional medicine in the gel-forming precursor water solution is 20 mg/ml-500 mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 1-2. The relative pressure value ratio of the water phase solution to the oil phase solution is 20-100: 70 to 140.
Wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
The self-assembly polypeptide is at least one of matrix metalloproteinase 1 sensitive self-assembly polypeptide, self-assembly KLDL-12 peptide, self-assembly RADA-16 peptide, self-assembly IEIK-13 peptide, self-assembly KLDL-12 peptide derivative, self-assembly RADA-16 peptide derivative or self-assembly IEIK-13 peptide derivative. The photo-crosslinking hydrogel is at least one of methacrylic acid acylated gelatin freeze-dried powder, methacrylic acid acylated hyaluronic acid freeze-dried powder, methacrylic acid acylated fibroin freeze-dried powder, methacrylic acid acylated chondroitin sulfate freeze-dried powder, methacrylic acid acylated sericin freeze-dried powder or polyethylene glycol diacrylate freeze-dried powder. The functional drug is at least one of exosome, drug-loaded liposome or macromolecule functional drug. The photoinitiator is a LAP photoinitiator.
The self-assembled polypeptide microsphere has self-assembling characteristic, so that the self-assembled polypeptide microsphere can be self-assembled in a micro-fluidic chip to form a skeleton structure of the microsphere, and a medicament with a treatment function can be slowly released for a long time.
Example 2.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 60 ℃ respectively, and stirring for 30min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine and the gelling precursor aqueous solution, filtering through a filter membrane, and standing for 1.5 hours to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing, and standing for 1 hour to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the gel-forming precursor aqueous solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.50-1.00.
The concentration of the functional medicine in the gel-forming precursor water solution is 30 mg/ml-300 mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 1. The relative pressure value ratio of the water phase solution to the oil phase solution is 40-80: 90 to 120.
Wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
The self-assembly polypeptide is at least one of matrix metalloproteinase 1 sensitive self-assembly polypeptide, self-assembly KLDL-12 peptide, self-assembly RADA-16 peptide, self-assembly IEIK-13 peptide, self-assembly KLDL-12 peptide derivative, self-assembly RADA-16 peptide derivative or self-assembly IEIK-13 peptide derivative. The photo-crosslinking hydrogel is at least one of methacrylic acid acylated gelatin freeze-dried powder, methacrylic acid acylated hyaluronic acid freeze-dried powder, methacrylic acid acylated fibroin freeze-dried powder, methacrylic acid acylated chondroitin sulfate freeze-dried powder, methacrylic acid acylated sericin freeze-dried powder or polyethylene glycol diacrylate freeze-dried powder. The functional drug is at least one of exosome, drug-loaded liposome or macromolecule functional drug. The photoinitiator is a LAP photoinitiator.
The self-assembly polypeptide microsphere of the invention endows the microsphere with complex functions including but not limited to enzyme responsiveness, temperature sensitivity and tumor targeting by editing the self-assembly polypeptide structure, can be used for tissue engineering and cancer treatment, and effectively promotes the tissue repair capability.
The microfluidic preparation method can stably prepare the self-assembled polypeptide microspheres for a long time to obtain microspheres with uniform shapes. The self-assembled polypeptide microsphere has self-assembly characteristics, can be self-assembled in a microfluidic chip to form a microsphere, and can realize long-acting slow release of a therapeutic functional drug.
Example 3.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 60 ℃ respectively, and stirring for 30min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1.5 hours to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing, and standing for 1 hour to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.50-1.00.
The concentration of the functional medicine in the gel-forming precursor water solution is 50 mg/ml-200 mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 1. The relative pressure value ratio of the water phase solution to the oil phase solution is 40-80: 90 to 120.
Wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
The self-assembly polypeptide is at least one of matrix metalloproteinase 1 sensitive self-assembly polypeptide, self-assembly KLDL-12 peptide, self-assembly RADA-16 peptide, self-assembly IEIK-13 peptide, self-assembly KLDL-12 peptide derivative, self-assembly RADA-16 peptide derivative or self-assembly IEIK-13 peptide derivative. The photo-crosslinking hydrogel is at least one of methacrylic acid acylated gelatin freeze-dried powder, methacrylic acid acylated hyaluronic acid freeze-dried powder, methacrylic acid acylated fibroin freeze-dried powder, methacrylic acid acylated chondroitin sulfate freeze-dried powder, methacrylic acid acylated sericin freeze-dried powder or polyethylene glycol diacrylate freeze-dried powder. The functional drug is at least one of exosome, drug-loaded liposome or macromolecule functional drug. The photoinitiator is a LAP photoinitiator.
The self-assembled KLDL-12 peptide derivative and the self-assembled RADA-16 peptide derivative of the present invention the self-assembled IEIK-13 peptide derivative refers to a series of substances containing the corresponding functional peptide.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 4.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 50 ℃ respectively, and stirring for 50min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine and the gelling precursor aqueous solution, filtering through a filter membrane, and standing for 2 hours to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing, and standing for 1 hour to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinked hydrogel and the photoinitiator are 0.25.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 0.1. The relative pressure value ratio of the water phase solution to the oil phase solution is 20:70.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
The self-assembly polypeptide is matrix metalloproteinase 1 sensitive self-assembly polypeptide. The photo-crosslinking hydrogel is methacrylic acid acylated gelatin freeze-dried powder. The functional medicine is a medicine-carrying liposome. The photoinitiator was a LAP photoinitiator.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 5.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 70 ℃, and stirring for 10min to obtain a gel precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1h to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing, and standing for 1 hour to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 2.00.
The concentration of the functional medicine in the gel-forming precursor water solution is 50 mg/ml-200 mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. The relative pressure value ratio of the water phase solution to the oil phase solution is 100:140.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
The self-assembly polypeptide is matrix metalloproteinase 1 sensitive self-assembly polypeptide, and the amino acid sequence of the self-assembly polypeptide is KLDLVPMSMRGGKLDL. The photo-crosslinking hydrogel is methacrylic acid acylated gelatin freeze-dried powder. The functional medicine is polymer functional medicine. The photoinitiator is a LAP photoinitiator.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 6.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (3) specifically, the water-phase solution and the oil-phase solution obtained in the step (1) are respectively injected into corresponding pipeline ports of the microfluidic chip, ultraviolet light is applied to the tail end of a chip pipeline, the relative pressure of the water-phase solution and the relative pressure of the oil-phase solution are adjusted, then the water-phase solution and the oil-phase solution are extruded into a pore channel of the microfluidic chip together through air pressure, and a water-in-oil structure is formed after the water-in-oil structure is converged, so that the self-assembled polypeptide microspheres are obtained.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.50.
The concentration of the functional drug in the gel-forming precursor aqueous solution is 50mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is a self-assembly KLDL-12 peptide derivative. The photo-crosslinking hydrogel is methacrylic acid acylated gelatin freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
It should be noted that the methacrylated gelatin (GelMA) of the present invention is a photosensitive biomaterial, and has biocompatibility, degradability, hardness, and the ability to promote cell attachment and growth. The GelMA structure contains a methylacryloylation substituent group, which causes the GelMA structure to initiate free radical polymerization crosslinking reaction under the conditions of a photoinitiator and ultraviolet light to form a complex hydrogel three-dimensional network.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic component induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 7.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (3) specifically, the water-phase solution and the oil-phase solution obtained in the step (1) are respectively injected into corresponding pipeline ports of the microfluidic chip, ultraviolet light is applied to the tail end of a chip pipeline, the relative pressure of the water-phase solution and the relative pressure of the oil-phase solution are adjusted, then the water-phase solution and the oil-phase solution are extruded into a pore channel of the microfluidic chip together through air pressure, and a water-in-oil structure is formed after the water-in-oil structure is converged, so that the self-assembled polypeptide microspheres are obtained.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 1.00.
The concentration of the functional drug in the gel-forming precursor aqueous solution is 100mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is a KLDL-12 peptide derivative. The photo-crosslinking hydrogel is methacrylated hyaluronic acid freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic induction mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 8.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (3) specifically, the water-phase solution and the oil-phase solution obtained in the step (1) are respectively injected into corresponding pipeline ports of the microfluidic chip, ultraviolet light is applied to the tail end of a chip pipeline, the relative pressure of the water-phase solution and the relative pressure of the oil-phase solution are adjusted, then the water-phase solution and the oil-phase solution are extruded into a pore channel of the microfluidic chip together through air pressure, and a water-in-oil structure is formed after the water-in-oil structure is converged, so that the self-assembled polypeptide microspheres are obtained.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.50.
The concentration of the functional drug in the gel-forming precursor aqueous solution is 150mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is a KLDL-12 peptide derivative. The photo-crosslinking hydrogel is methacrylated silk fibroin freeze-dried powder. The functional medicine is osteogenic induction bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic component induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 9.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinked hydrogel and the photoinitiator are 0.75.
The concentration of the functional drug in the gel-forming precursor aqueous solution is 200mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is RADA-16 peptide. The photo-crosslinking hydrogel is methacrylated sericin freeze-dried powder. The functional medicine is osteogenic induction bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic component induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 10.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.75.
The concentration of the functional drug in the gel-forming precursor water solution is 250mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is RADA-12 peptide derivative. The photo-crosslinking hydrogel is methacrylic acylated sericin freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 11.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a pipeline of the chip to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 65 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine and the gelling precursor aqueous solution, filtering through a filter membrane, and standing for 1,5h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.75.
The concentration of the functional drug in the gel-forming precursor aqueous solution is 300mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 2. Relative pressure value ratio of the aqueous phase solution to the oil phase solution of 70:100.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is IEIK-13 peptide derivative. The photo-crosslinking hydrogel is polyethylene glycol diacrylate freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microspheres can be used for preparing the self-assembled polypeptide microspheres well and stably for a long time in the range of the embodiment 1, and the obtained microspheres with more uniform shapes are obtained.
Example 12.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a chip pipeline to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 63 ℃ respectively, and stirring for 45min to obtain a gel-forming precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1.0h to obtain an aqueous phase solution.
The oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 1.5h to obtain the oil phase solution.
And (3) specifically, the water-phase solution and the oil-phase solution obtained in the step (1) are respectively injected into corresponding pipeline ports of the microfluidic chip, ultraviolet light is applied to the tail end of a chip pipeline, the relative pressure of the water-phase solution and the relative pressure of the oil-phase solution are adjusted, then the water-phase solution and the oil-phase solution are extruded into a pore channel of the microfluidic chip together through air pressure, and a water-in-oil structure is formed after the water-in-oil structure is converged, so that the self-assembled polypeptide microspheres are obtained.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.25.
The concentration of the functional drug in the gel-forming precursor water solution is 30mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 0.5. The relative pressure value ratio of the water phase solution to the oil phase solution is 50:140.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembly polypeptide is self-assembly KLDL-12 peptide. The photo-crosslinking hydrogel is polyethylene glycol diacrylate freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microsphere can prepare the self-assembled polypeptide microsphere in the range of the embodiment 1 well and stably for a long time, and the obtained microsphere with a more uniform shape is obtained.
Example 13.
A microfluidic preparation method of self-assembled polypeptide microspheres comprises the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
and (2) respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a chip pipeline to obtain the self-assembled polypeptide microspheres.
Wherein the aqueous solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine. The oil phase solution contains span 80 and mineral oil.
The preparation steps of the aqueous phase solution of the invention comprise the following steps:
step A1, adding the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator into deionized water preheated to 63 ℃ respectively, and stirring for 45min to obtain a gel precursor aqueous solution.
And step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering through a filter membrane, and standing for 1.0h to obtain an aqueous phase solution.
The preparation method of the oil phase solution comprises the steps of adding span 80 into mineral oil, mixing, and standing for 1.5 hours to obtain the oil phase solution.
And (2) respectively injecting the aqueous phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, applying ultraviolet light to the tail end of a chip pipeline, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil phase solution into a pore channel of the microfluidic chip together through air pressure, and forming a water-in-oil structure after convergence to obtain the self-assembled polypeptide microspheres.
In the aqueous phase solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 1.25.
The concentration of the functional drug in the gel-forming precursor water solution is 500mg/ml.
In the oil phase solution, the volume ratio of span 80 to mineral oil is 0.1. The relative pressure value ratio of the water phase solution to the oil phase solution is 100:80.
wherein the filter membrane is a 0.22 μm filter membrane; the micro-fluidic chip is a flow focusing pipeline micro-fluidic chip.
Wherein the self-assembling polypeptide is a self-assembling RADA-16 peptide derivative. The photo-crosslinking hydrogel is polyethylene glycol diacrylate freeze-dried powder. The functional medicine is osteogenesis inducing bone marrow mesenchymal stem cell exosome. The photoinitiator is LAP blue light initiator.
The extraction of the osteogenic component induced mesenchymal stem cell exosome solution comprises the following steps: selecting mesenchymal stem cells in logarithmic growth phase, then carrying out osteogenic differentiation induction on the mesenchymal stem cells for 7-14 days, using a culture medium containing 10% (w/t) of exosome-free serum all the time in the induction process, collecting cell culture supernatant after the induction is finished, and extracting osteogenic induction mesenchymal stem cell exosome solution by adopting a differential centrifugation method.
Experiments prove that the microfluidic preparation method of the self-assembled polypeptide microsphere can prepare the self-assembled polypeptide microsphere in the range of the embodiment 1 well and stably for a long time, and the obtained microsphere with a more uniform shape is obtained.
And (5) performing experiments and verification.
1. Aqueous solution Performance test
Selecting matrix metalloproteinase 1 sensitive self-assembly polypeptide (KLDL-MMP 1), dissolving 200 mu g of KLDL-MMP1 in 20 mu l of deionized water to prepare stock solution of 10mg/ml, and adding 20 mu l of stock solution into a 1.5ml EP tube; adding 180 mul of sterile distilled water and total 200 mul of solution, and uniformly blowing, beating and mixing to obtain the polypeptide hydrogel.
The above polypeptide hydrogel was dropped on 20 mm parallel plates, and gel point formation and shear thinning properties were measured by a rheometer. The gelation time of the polypeptide hydrogel was monitored at 25 ℃ room temperature at a frequency of 1Hz and a strain of 1% for 1 hour, and the intersection of the storage modulus (G') and loss modulus (G ") represents the gel point, as shown in fig. 2, where the gel point of the polypeptide hydrogel is 25-30 minutes.
Also at room temperature, from 0.1 to 100s using a rheometer -1 The viscosity and shear stress of the above polypeptide hydrogel were measured, as shown in fig. 3.
Experimental results show that the self-assembly polypeptide can meet the requirement that a micro-fluidic chip runs for at least 30 minutes, so that the self-assembly polypeptide microspheres can be stably prepared for a long time. The polypeptide hydrogel has the shear thinning characteristic, and can meet the requirement that a micro-fluidic chip extrudes a water phase into a pore channel.
2. Particle size experiment of self-assembled polypeptide microspheres
The self-assembled polypeptide microspheres prepared in examples 6 to 11 were observed under a microscope, and the particle size of each microsphere was counted, as shown in fig. 4.
In FIG. 4, it can be seen that the diameters of the self-assembled polypeptide microspheres prepared in examples 6-11 are all in the range of 100 μm to 200. Mu.m.
3. Microscopic morphology experiment of self-assembled polypeptide microspheres
The self-assembled polypeptide microspheres prepared in example 6 were air-dried at room temperature overnight, and then gold foil was sputtered to a thickness of 10 nm on the surface of the self-assembled polypeptide microspheres using a gold plating apparatus. The microscopic morphology of the microspheres was then observed with a scanning electron microscope, as shown in FIG. 5.
In FIG. 5, it can be seen that the self-assembled polypeptide microspheres prepared in example 6 have uniform size, smooth surface and stable processed form, and thus are good drug carriers.
4. Rheological property experiment of self-assembled polypeptide microspheres
The self-assembled polypeptide microspheres prepared in examples 6 to 11 were subjected to rheological property detection by dynamic sweep frequency test of a rheometer at room temperature, and the test frequency range was 0.1rad/s to 10rad/s.
The strain amplitude used was in the linear viscoelastic region, determined by dynamic amplitude sweep testing of hydrogel microsphere samples, with a frequency of 6.28rad/s and a shear amplitude range of 0.1% to 10%, yielding figure 6.
Therefore, the self-assembled polypeptide microspheres prepared by the invention have stable mechanical properties and are not easy to fluctuate, so the self-assembled polypeptide microspheres are good drug carriers.
5. In vitro release experiment of self-assembled polypeptide microspheres
The PKH 67-labeled exosomes were mixed in an aqueous phase and self-assembled polypeptide fluorescent microspheres were prepared using the method of example 6. Mu.l of microspheres were added to 2ml of simulated body fluid or simulated body fluid containing 0.7mg/ml MMP enzyme and individual microspheres were observed.
At 0d, 3d, 6d, 9d, 12d and 15d, observing and tracking the fluorescence of each group by using a confocal laser scanning microscope, as shown in fig. 7, the release speed of the exosomes wrapped in the slow-release microspheres is slower until 15 days, the exosomes are still released, and the self-assembled polypeptide fluorescent microspheres in the simulated body fluid containing the mmp enzyme are rapidly degraded, so that the enzyme-responsive drug release is realized, and the clinical application potential is extremely high.
As can be seen from FIG. 7, the self-assembled polypeptide fluorescent microsphere prepared by the invention can effectively slow down the release of exosome in vitro, has the effect of prolonging the half-life period of exosome, and can endow a material delivery system with more functions by changing the sequence of self-assembled polypeptide.
6. Self-assembled polypeptide microsphere therapeutic application verification experiment
In order to evaluate the therapeutic potential of the self-assembled polypeptide microspheres, the SD rat skull defect model was treated with the self-assembled polypeptide microspheres prepared in example 6, specifically:
SD rats were divided into a sham operation group, an exosome treatment group, and a general microsphere group, and the self-assembled polypeptide microsphere group prepared in example 6 was administered with corresponding drug treatments except for the sham operation group. At 7 days, 14 days after dosing, half of the rats were sacrificed and rat cranial samples were collected. After decalcification, the samples were paraffin-embedded and sectioned. The slices are deparaffinized, hydrated, antigen repaired and sealed. Then using anti-osteocalcin antibody as primary antibody to incubate overnight, using DAB to develop color after secondary antibody is incubated for 1 hour, finally counterstaining with hematoxylin, dehydrating, clearing and mounting after washing, and finally using osteocalcin to recognize mature osteoblast in new bone formation, as shown in figure 8.
Wherein the sham operation group means that only the surface skin of the cranium part is cut, and bone defect is not made; the exosome treatment group was injected with only the exosomes carried in example 6; the common microsphere set was the example 6 microspheres without exosomes. The preparation method of the general microspheres is the same as that of example 6, except that no exosome is added to the water phase.
Experimental results show that the exosome derived from the stem cells which is wrapped by the microspheres and slowly released can better promote the repair of bone tissues, and more mature bone cells exist, namely the self-assembled polypeptide microspheres obtained by the invention can effectively promote the bone repair.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (10)
1. A microfluidic preparation method of self-assembled polypeptide microspheres is characterized by comprising the following steps:
step (1), respectively preparing a water phase solution and an oil phase solution;
step (2), respectively injecting the water phase solution and the oil phase solution obtained in the step (1) into corresponding pipeline ports of the microfluidic chip, and applying ultraviolet light to the tail end of a chip pipeline to obtain self-assembled polypeptide microspheres;
the aqueous phase solution contains self-assembly polypeptide, photo-crosslinking hydrogel, photoinitiator and functional medicine;
the oil phase solution contains span 80 and mineral oil.
2. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 1, wherein the preparation step of the aqueous phase solution comprises the following steps:
step A1, adding self-assembly polypeptide, photo-crosslinking hydrogel and photoinitiator into deionized water respectively, and stirring to obtain a gel-forming precursor aqueous solution;
and step A2, mixing the functional medicine with the colloid-forming precursor aqueous solution, filtering, and standing to obtain an aqueous phase solution.
3. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 2, wherein: the oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing to obtain the oil phase solution.
4. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 3, wherein the step (2) comprises injecting the aqueous phase solution and the oil phase solution of step (1) into corresponding pipeline ports of the microfluidic chip, respectively, applying ultraviolet light to the end of the pipeline of the chip, adjusting the relative pressure of the aqueous phase solution and the relative pressure of the oil phase solution, then extruding the aqueous phase solution and the oil solution together into the pore channels of the microfluidic chip by air pressure, and forming a water-in-oil structure after the aggregation, thereby obtaining the self-assembled polypeptide microspheres.
5. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 4, wherein: the step A1 is specifically that the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are respectively added into deionized water preheated to 50-70 ℃, and stirred for 10-50 min to obtain a gel-forming precursor aqueous solution;
and the step A2 is to mix the functional medicine and the gelling precursor aqueous solution, filter the mixture through a filter membrane, and stand the mixture for 1 to 2 hours to obtain an aqueous phase solution.
6. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 5, wherein: the oil phase solution is prepared by adding span 80 into mineral oil, mixing and standing for 0.5-2 h to obtain the oil phase solution.
7. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 4, wherein: in the gel-forming precursor aqueous solution, the mass percentages of the self-assembly polypeptide, the photo-crosslinking hydrogel and the photoinitiator are 0.25-2.00;
in the oil phase solution, the volume ratio of span 80 to mineral oil is 1-2;
the concentration of the functional medicine in the gel-forming precursor water solution is 20 mg/ml-500 mg/ml;
the relative pressure value ratio of the water phase solution to the oil phase solution is 20-100: 70 to 140.
8. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 7, wherein: the filter membrane is a 0.22 mu m filter membrane;
the microfluidic chip is a flow focusing pipeline microfluidic chip.
9. The microfluidic preparation method of self-assembled polypeptide microspheres according to claim 1, wherein: the self-assembly polypeptide is at least one of matrix metalloproteinase 1 sensitive self-assembly polypeptide, self-assembly KLDL-12 peptide, self-assembly RADA-16 peptide, self-assembly IEIK-13 peptide, self-assembly KLDL-12 peptide derivative, self-assembly RADA-16 peptide derivative or self-assembly IEIK-13 peptide derivative;
the photo-crosslinking hydrogel is at least one of methacrylic acidylated gelatin freeze-dried powder, methacrylic acidylated hyaluronic acid freeze-dried powder, methacrylic acidylated fibroin freeze-dried powder, methacrylic acidylated chondroitin sulfate freeze-dried powder, methacrylic acidylated sericin freeze-dried powder or polyethylene glycol diacrylate freeze-dried powder;
the functional drug is at least one of exosome, drug-loaded liposome or high molecular functional drug;
the photoinitiator is a LAP photoinitiator.
10. The microfluidic preparation method of self-assembled polypeptide microspheres of claim 1, wherein: the diameter is 50-200 μm.
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| WO2013189282A1 (en) * | 2012-06-19 | 2013-12-27 | 广州帝奇医药技术有限公司 | Polypeptide-medicine-slow-releasing microsphere preparation and preparation method therefor |
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| CN113368062A (en) * | 2021-05-28 | 2021-09-10 | 南方医科大学 | Microfluidic preparation method of exosome sustained-release microspheres for promoting cartilage repair, sustained-release microspheres and application thereof |
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| CN113368062A (en) * | 2021-05-28 | 2021-09-10 | 南方医科大学 | Microfluidic preparation method of exosome sustained-release microspheres for promoting cartilage repair, sustained-release microspheres and application thereof |
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