CN113633822A - Polymer nanofiber/microparticle photosolder composite microsphere and preparation method and application thereof - Google Patents
Polymer nanofiber/microparticle photosolder composite microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation and application of nanofiber materials, and relates to a polymer nanofiber/microparticle light-welding composite microsphere, a preparation method and application thereof, wherein the composite microsphere is a medical degradable nanofiber microsphere with microparticles loaded on the surface, the fiber microsphere consists of oriented nanofibers or nano yarns, the microparticles are degradable polymer microparticles loaded with bioactive substances, and are deposited and welded on the surface of the fiber microsphere; preparing an oriented nanofiber membrane or nano yarn by using a medical degradable polymer and a near-infrared dye as raw materials; welding the contact parts of the fibers and the particles by laser irradiation at different time; then preparing the short fibers into a short fiber homogeneous suspension and preparing the short fibers into the short fiber homogeneous suspension by an electrostatic spraying technology; the raw materials are biodegradable polymers, are non-toxic and have good histocompatibility, and can be gradually degraded along with the regeneration and reconstruction of tissues; the slow release factor material can be prepared according to the requirements, and has good application prospect in cell and tissue engineering.
Description
The technical field is as follows:
the invention belongs to the technical field of preparation and application of nanofiber materials, and particularly relates to a preparation method and application of a polymer nanofiber/particle photosolder composite microsphere.
Background art:
microspheres have long been used in drug delivery applications due to their controlled release capabilities. They are increasingly becoming essential building blocks for the fabrication of regenerative engineering scaffolds, due to their ability to provide porous networks, to provide high resolution control of spatial tissue, and to provide growth factors/drugs and/or nanophase materials. Microspheres are ideal tools for engineering complex tissues and biological interfaces because they provide a physicochemical gradient through the spatiotemporal release of bioactive factors and nanoparticles. Different methods for preparing microspheres affect the performance of micro and macro scaffolds. As a common scaffold preparation technology, electrostatic spinning is widely applied to tissue engineering. Microspheres prepared based on electrospinning provide good initial mechanical properties and allow for the controlled release of bioactive molecules to facilitate tissue regeneration. Electrostatic spray technology can produce particles with a diameter of 1-1000 μm, which can encapsulate drugs or bioactive molecules. They are widely used for drug delivery or targeting applications, largely because they can enhance the efficacy of drugs by providing a large surface area to volume ratio and provide spatial and temporal control over the release of drugs. In addition to being an excellent controlled release carrier, the shape of the microspheres is rigid and can be assembled together, alone or in combination with other materials, to create a porous three-dimensional (3D) structure that can serve as a scaffold for tissue engineering.
Microsphere design is generally considered in terms of composition, structure and function. In terms of composition, the microsphere material has good biocompatibility and degradability, and is beneficial to the growth of tissue cells; structurally, the microspheres provide controlled release drug carriers, cells, tissue culture scaffolds; functionally, the microspheres provide (1) control over protein or drug release; (2) delivering bioactive molecules in response to environmental stimuli, such as temperature and pH; (3) acting as a micro bioreactor embedded in a surrounding matrix, creating a growing environment for intrinsically complex tissue regeneration; (4) as a cellular transporter; (5) forming mesh pores inside the scaffold to promote cell growth and accelerate scaffold absorption; (6) the mechanical support is impregnated into the otherwise weaker scaffold matrix.
In order to prepare an ideal composite microsphere with injectability and functional sites for bioactive substances, drug delivery or cell delivery, the electrostatic spinning technology and the coaxial electrostatic spraying technology are widely applied. The electrostatic spinning and electrostatic spraying technology has the advantages of simple operation, strong repeatability, mass production, cheap and easily obtained raw materials. At present, most of synthetic macromolecules, natural proteins, degradable polymers or their blend compounds can be used in electrostatic spinning and electrostatic spraying technologies, and nano-scale fibers and micro/nano-scale particles can be prepared.
At present, no report exists on a preparation method of the oriented structure and the drug-loaded particle photosynthesized composite microsphere for tissue engineering.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provide a preparation method of polymer nanofiber and particle photosolder composite microspheres, and the composite microspheres prepared by the method simulate the change of cell niches and biological signals and have good biomechanical property, degradation property and cell compatibility.
In order to achieve the above object, the present invention provides a polymer nanofiber/microparticle photosolder composite microsphere, which has a structure of a medical degradable nanofiber microsphere with microparticles loaded on the surface, wherein the fiber microsphere is composed of oriented nanofibers or nano yarns, and the microparticles are biodegradable polymer microparticles loaded with bioactive substances, and are deposited and welded on the surface of the fiber microsphere.
The preparation of the composite microsphere is divided into two parts, wherein the first part is a welded nanofiber or yarn and a microparticle carrier loaded with bioactive substances, and the bioactive substances can be controlled to release; and in the second part, the material obtained in the first part is frozen and shredded to obtain short fiber/particle composite homogeneous suspension to prepare fiber microspheres, and the nano fiber/particle composite microspheres can be used for delivering bioactive substances and cells.
The invention also provides a preparation method of the polymer nanofiber/microparticle photosolder composite microsphere, the composite microsphere is a medical degradable nanofiber microsphere loaded with microparticles, and the microparticles are degradable polymer microparticles loaded with bioactive substances. Preparing an oriented nanofiber membrane or nano yarn by using a medical degradable polymer and a near-infrared dye as raw materials; welding the contact part of the fiber and the surface particles by laser irradiation at different time; then preparing the mixture into a short fiber homogeneous suspension and preparing the polymer microspheres by an electrostatic spraying technology, wherein the method comprises the following specific steps:
(1) preparing polymer nano-fibers: blending medical degradable polymer and near-infrared dye in a solvent to be uniformly dissolved to obtain a spinning solution, and then preparing a uniaxial orientation polymer nanofiber membrane by single-nozzle electrostatic spinning, or obtaining nano yarn by double-nozzle nano yarn preparation equipment;
(2) depositing carrier particles: flatly paving the uniaxial orientation nanofiber membrane or the nanometer yarn obtained in the step (1) on a receiving flat plate to serve as a receiving device, spraying carrier particles to the surface of the nanofiber membrane or the nanometer yarn by a coaxial electrostatic spraying device, and depositing the carrier particles on the surface of the nanofiber membrane or the nanofiber;
(3) photosolder polymeric nanofibers and microparticles: irradiating the nanofiber membrane or the nanometer yarn deposited with the carrier particles by using near-infrared laser to perform 'photosolder', and welding the contact part of the surfaces of the fibers or the yarns and the particles by laser irradiation at different time;
(4) preparing the polymer nano-fiber/particle photosolder composite microsphere: and (4) cutting the material welded in the step (3) into short fibers by a freezing microtome, dispersing the short fibers into a gelatin water solution to obtain a short fiber suspension, performing electrostatic spraying, receiving liquid nitrogen, performing freeze drying, and crosslinking glutaraldehyde to obtain the polymer nanofiber/particle photostable composite microspheres.
The carrier particle is prepared by spraying a shell layer solution and a core layer solution through coaxial electrostatic spraying, wherein the shell layer solution is a mixed solution containing a medical degradable polymer and a near-infrared dye, and the core layer solution is a solution containing a bioactive substance.
The medical degradable polymer is at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-caprolactone copolymer.
The near-infrared dye is at least one of indocyanine green and heptamethine cyanine micromolecule dyes.
The mass ratio of the traditional Chinese medicine degradable polymer to the near-infrared dye in the step (1) is 100 (0.5-1); the volume ratio of the total mass of the medical degradable polymer and the near-infrared dye to the solvent is (5-15) g: 10 mL.
The electrostatic spinning equipment in the step (1) comprises a propelling device, a needle cylinder, a high-voltage electrostatic generator and a high-speed rotary cylinder.
The nano yarn equipment in the step (1) comprises a propelling device, a needle cylinder, a high-voltage electrostatic generator, a collecting stick and a winding stick.
The parameters of the electrostatic spinning equipment in the step (1) are as follows: the flow rate of the medical degradable polymer and the near-infrared dye blended spinning solution is 1-5mL/h, the voltage of a spray head is 10-20kV, the distance between the spray head and the rotary drum is 12-15cm through a ground wire of the rotary drum, and the rotating speed of the rotary drum is 2500 plus 3000 rpm.
The nano yarn equipment parameters in the step (1) are as follows: the flow rate of the medical degradable polymer and the near-infrared dye blended spinning solution is 0.0010-0.0020mL/min, the voltage of a nozzle is 5-15kV, the rotating speed of a collecting roller is 350-600rpm, and the rotating speed of a winding roller is 25-50 rpm.
The solvent in the step (1) is one of hexafluoroisopropanol, dichloromethane, trifluoroethanol and a dichloromethane/N, N-dimethylformamide mixed solvent.
The shell layer solution of the carrier particle is a chloroform solution of degradable polymer and near-infrared dye, the bioactive substances in the core layer solution are bioactive protein, growth factors or small molecules and the like, and the solvent is deionized water or phosphate buffer solution and the like.
The preparation parameters of the carrier particles are as follows: the flow rate of the shell layer solution is 1-5mL/h, the flow rate of the core layer solution is 0.1-1.0mL/h, the voltage of a spray head is 15-20kV, a flat plate grounding wire is received, and the distance between the spray head and the flat plate is 12-15 cm.
The laser irradiation intensity in the step (3) is determined according to the heat released by the near-infrared dye absorbing the laser energy; the laser irradiation time is adjusted according to the time for the selected medical degradable polymer to reach the melting point.
In the step (4), the concentration of the short fiber suspension is 10-20mg/mL, the electrostatic spraying voltage is 0-15kV, the suspension flow rate is 1-10mL/h, the receiving distance is 5-15cm, the receiving time is adjusted according to the required amount, and the size of the receiving microspheres can be changed by adjusting parameters (such as voltage and flow rate).
On the whole, the composite microsphere material simulates a cell growth three-dimensional space and has good mechanical property, biocompatibility, controllable release property and degradation property; in detail, the medical degradable polymer has good biocompatibility and biodegradability, can be used as a cell growth support material, and can adjust the degradation time by selecting different polymers or adjusting the mixing ratio of the different polymers so as to adapt to the cell growth rate. Therefore, the polymer nano-fiber/particle photosynthesized composite microsphere prepared by the method is not only suitable for delivering bioactive substances and cells, but also has good application prospect in tissue repair and regeneration.
Compared with the prior art, the invention has the following beneficial effects:
(1) the inventor firstly welds carrier particles loaded with bioactive substances and nano fibers or nano yarns to prepare the composite microspheres with a fiber structure, the composite microspheres have injectability, the pores of the composite microspheres can meet the transfer of nutrient substances required by a cell growth environment, the composite microspheres also have a three-dimensional topological structure of a bionic natural extracellular matrix, the surface roughness and the fiber structure of the composite microspheres can promote cell adhesion, the slow release behavior of the particles loaded with the bioactive substances can be controlled, the microenvironment is adjusted, the adhesion, proliferation and growth of cells are facilitated, and the differentiation of stem cells can be induced in situ.
(2) The polymer nano fiber/particle composite microsphere prepared by the invention has adjustable size, the loaded bioactive substances can be diversified, and the degradation performance of the microsphere and the slow release curve of the load can be adjusted by selecting different degradable polymers, so that the material has wider adjustable range of performance and wide application.
(3) The raw materials of the polymer nanofiber and particle photosolder composite microsphere prepared by the invention are biodegradable high molecules, are nontoxic and have good histocompatibility, and can be gradually degraded along with the regeneration and reconstruction of tissues; meanwhile, the slow release factor materials at different time can be prepared according to the requirements, and the preparation method has good application prospect in cell and tissue engineering.
Description of the drawings:
FIG. 1 is a schematic diagram of the principle of the preparation method of the polymer nanofiber/microparticle photosolder composite microsphere according to the present invention.
FIG. 2 is an optical image and a Scanning Electron Microscope (SEM) image of bioactive substance-loaded microparticles according to the present invention, wherein A is an optical microscope bright field image and A is an SEM image1FITC fluorescence map, A2The two are superposed; b is the SEM image of the particle morphology, and C is the SEM image of the morphology after the nano-fiber and the particle are subjected to the optical welding.
FIG. 3 is a digital photo of the composite microsphere and SEM image of the microsphere, wherein A and B are both digital photos of the composite microsphere, C is a local SEM image of the microsphere, D is a local SEM image of the whole microsphere, E is a local SEM image of the microsphere, and F is a SEM image of the whole microsphere.
Fig. 4 is an immunofluorescence staining picture of polymer nanofiber/microparticle photosynthesized composite microsphere loaded adipose-derived stem cells and tendon-derived stem cells, wherein a is the loaded adipose-derived stem cells, B is the loaded tendon-derived stem cells, green is actin (F-actin), and blue is cell nuclei.
FIG. 5 is a slow release curve of the bioactive substance loaded in the polymer nanofiber/microparticle photosynthesized composite microsphere related to the invention, wherein electrostatic spraying voltage parameters of the A-C microsphere preparation are respectively as follows: 0kV, 3kV and 10 kV.
The specific implementation mode is as follows:
the invention is further illustrated by the following specific examples in combination with the accompanying drawings.
Example 1:
the embodiment relates to a preparation method of a polymer nanofiber/particle photosolder composite microsphere, wherein the raw materials for preparing the polymer nanofiber are polycaprolactone and green indole-phthalocyanine, the bioactive substances loaded on the particle are Bovine Serum Albumin (BSA) and fibronectin, and the preparation method specifically comprises the following steps:
(1) preparing polymer nano-fibers: accurately weighing 0.5g of Polycaprolactone (PCL) and 0.005g of indocyanine green (ICG) by using an electronic balance, dissolving the Polycaprolactone (PCL) and the indocyanine green (ICG) in 5mL of hexafluoroisopropanol, stirring the mixture overnight by using a magnetic stirrer, and uniformly stirring the mixture to obtain a PCL/ICG spinning solution containing 0.1 wt% of ICG; the PCL/ICG uniaxial orientation nanofiber membrane is prepared by adopting electrostatic spinning equipment in the prior art, and the spinning parameters are as follows: the flow rates of the spinning solutions are respectively 1mL/h, the distance between a spray head and a roller is 15cm, the voltage of the spray head is 15kV, the rotating drum is grounded, the rotating speed of the roller is 2500rpm, and a PCL/ICG uniaxial orientation nanofiber membrane is obtained after 2 hours;
(2) depositing carrier particles: cutting the PCL/ICG uniaxial orientation nanofiber membrane obtained in the step (1) into a size of 5cm multiplied by 5cm, fixing the PCL/ICG uniaxial orientation nanofiber membrane on a receiving flat plate, receiving a grounding wire of the flat plate, wherein the distance between the receiving flat plate and a spray head of a coaxial electrostatic spraying device is 15cm, the coaxial electrostatic spraying device sprays carrier particles to the surface of the nanofiber membrane, and the deposition time of the carrier particles on the surface of the nanofiber membrane is 30 min; the carrier particle is prepared by spraying a shell layer solution and a core layer solution through coaxial electrostatic spraying, wherein the shell layer solution is a chloroform solution containing PCL and ICG, and the core layer solution is an aqueous solution containing Bovine Serum Albumin (BSA) and fibronectin; the parameters of the coaxial electrostatic spraying device are as follows: the flow rate of the shell layer solution is 2mL/h, the flow rate of the core layer solution is 0.3mL/h, and the voltage of the spray head is 15 kV;
the preparation method of the shell solution comprises the following steps: accurately weighing 0.15g of Polycaprolactone (PCL) and 0.0015g of indocyanine green (ICG) by using an electronic balance, dissolving the Polycaprolactone (PCL) and the indocyanine green (ICG) in 5mL of chloroform, stirring the mixture for 5 hours by using a magnetic stirrer, and uniformly stirring the mixture to obtain a chloroform solution containing the ICG and the PCL;
the preparation method of the core layer solution comprises the following steps: dissolving Bovine Serum Albumin (BSA) and fibronectin in deionized water, wherein the concentration of BSA is 1mg/mL, and the concentration of fibronectin is 20 μ g/mL;
(3) photosolder nanofiber films and microparticles: irradiating the nanofiber membrane deposited with the carrier particles by using near-infrared laser to perform 'photosolder', wherein the near-infrared laser irradiation can excite ICG (ion-coupled plasma) to generate heat to reach the melting point of the nanofiber and the particles, and guiding the contact parts of the nanofiber and the particles to be welded together; the wavelength of the near infrared laser is 808nm, and the intensity of the laser irradiation is 4W/cm2The laser irradiation time is 3s, the laser irradiation distance is 2-3cm, and the temperature of the irradiated part observed by a thermal imager reaches 32 ℃;
the Scanning Electron Microscope (SEM) observes and photographs the morphology of the contact portion between the nanofiber membrane and the fine particles after welding, and the result is shown in fig. 2C.
The results show that: the contact part of the nano fiber and the particle is well welded.
(4) Preparing the polymer nano-fiber/particle photosolder composite microsphere: intercepting the nano fiber/particle obtained in the step (3) after the optical welding with the size of 1.5cm multiplied by 1.5cm, embedding with OCT frozen section embedding medium, freezing to a solid state in a refrigerator at minus 80 ℃, and cutting into short fibers with uniform length by a frozen section technology, wherein the length of the fibers is set to be 50 mu m; uniformly dispersing short fibers in an aqueous solution containing 5% of gelatin, wherein the mass volume concentration of the short fibers is 20mg/mL, preparing micro/nano fiber microspheres by using a container containing liquid nitrogen as a receiving device through an electrostatic spraying technology, wherein the flow rate is 4mL/h, the voltage is 0, 3, 6, 8 and 10kV respectively, the receiving distance is 10cm, and the receiving time is 10 min; and then freeze-drying for 12h, and crosslinking glutaraldehyde to obtain the polymer nanofiber/particle photosolder composite microspheres.
The digital camera photographs the finished composite microspheres prepared at different voltages and the composite microspheres were formed after freeze-drying, the results are shown in fig. 3A and 3B.
The results show that: under the condition that other parameters are not changed, the diameters of the composite microspheres prepared by different voltages are different, and the diameters of the composite microspheres are reduced along with the increase of the voltages; and freeze-drying to obtain more stable composite microspheres with different diameters.
The overall and local morphology of the freeze-dried finished product of the composite microspheres loaded with no carrier particles was observed and photographed by a Scanning Electron Microscope (SEM), and the results are shown in fig. 3D and 3C.
The results show that: the freeze-dried composite microspheres without carrier particles are spherical, and the fibers can be well connected by partially enlarging.
The preparation method of the composite microsphere without the carrier particles is the step (1) and the step (4) of the embodiment.
The overall and local morphology of the freeze-dried finished product of the composite microspheres loaded with carrier particles was observed and photographed by a Scanning Electron Microscope (SEM), and the results are shown in fig. 3F and 3E.
The results show that: the freeze-dried composite microspheres loaded with carrier particles are spherical, and fine connection among fibers and particles loaded with bioactive substances can be observed in a partial enlarged view.
Example 2:
this example is an experiment of the effect of the polymer nanofiber/microparticle photosynthesized composite microspheres prepared in example 1 on the morphology of adipose-derived stem cells. The adipose-derived stem cells are inoculated into a 24-well plate containing the composite microsphere material prepared in example 1 at the concentration of 10000 cells/well, and after 2 days of culture, F-actin/DAPI staining is carried out to observe the cell morphology, and the specific method comprises the following steps:
1. after 2 days of complete cell culture (volume percentage composition: 89% low sugar Dulbecco's Modified Eagle Medium (DMEM)) + 10% fetal bovine serum + 1% cyan/streptomycin), the old culture was removed and then washed 3 times with Phosphate Buffered Saline (PBS);
2. fixing cells with 1mL of tissue cell fixing solution with the mass percentage concentration of 4% at room temperature for 10min, removing the tissue cell fixing solution, and washing with PBS for 3 times;
3. penetrating with 1 mL/hole of 0.1% polyethylene glycol octyl phenyl ether (Triton X-100) water solution for 5min at room temperature, removing the Triton X-100 solution, and washing with PBS for 3 times;
4. blocking with 1 mL/hole of BSA (bovine serum albumin) aqueous solution with the mass percentage concentration of 1% for 30min-60min, removing the BSA solution and washing with PBS for 3 times;
5. f-actin (fibrous actin green fluorescent dye solution) and BSA (bovine serum albumin) aqueous solution with the mass percentage concentration of 1% are mixed according to the volume ratio of 1: 1000, adding 200 mu L of the mixed solution into each hole, and dyeing for 40min at room temperature in a dark place;
6. washing with PBS for 5min for 3 times;
7. the slides were mounted with mounting medium containing DAPI (phenylindole), observed under a laser scanning confocal microscope and photographed, and the results are shown in FIG. 4A.
The results show that: cells grow on the composite microspheres in an adhesion manner, and the axon stretching is good, which indicates that the polymer nanofiber/microparticle photosolder composite microspheres can promote the growth of the cells.
Example 3:
the embodiment relates to a preparation method of a polymer nanofiber/microparticle photosolder composite microsphere material, wherein the preparation raw materials of the polymer nanofiber are polycaprolactone and green indole phthalocyanine, the microparticle-loaded bioactive substance is insulin-like growth factor (IGF), and the preparation method comprises the following specific steps:
(1) preparing polymer nano-fibers: accurately weighing 0.96g of Polycaprolactone (PCL) and 0.008g of indocyanine green (ICG) by using an electronic balance, dissolving the Polycaprolactone (PCL) and the indocyanine green (ICG) in 8mL of hexafluoroisopropanol, stirring the mixture overnight by using a magnetic stirrer, and uniformly stirring the mixture to obtain a PCL/ICG spinning solution; the PCL/ICG nano yarn is prepared by adopting the electrostatic nano yarn equipment in the prior art, and the parameters are as follows: the flow rate of the spinning solution is 0.0015mL/min, the voltage of a nozzle is 9kV, the rotating speed of a collecting roller is 450rpm, and the rotating speed of a winding roller is 35rpm, so that PCL/ICG nano yarn is obtained;
(2) depositing carrier particles: orderly arranging the PCL/ICG nano yarns obtained in the step (1), fixing the PCL/ICG nano yarns on a receiving flat plate, receiving a grounding wire of the flat plate, wherein the distance between the receiving flat plate and a spray head of a coaxial electrostatic spraying device is 15cm, the coaxial electrostatic spraying device sprays carrier particles to the surface of the nano yarns, and the deposition time of the carrier particles on the surface of the nano yarns is 30 min; the carrier particle is prepared by spraying a shell layer solution and a core layer solution through coaxial electrostatic spraying, wherein the shell layer solution is a chloroform solution containing PCL and ICG, and the core layer solution is an aqueous solution containing insulin-like growth factor (IGF); the parameters of the coaxial electrostatic spraying device are as follows: the flow rate of the shell layer solution is 2mL/h, the flow rate of the core layer solution is 0.3mL/h, and the voltage of the spray head is 15 kV;
the preparation method of the shell solution comprises the following steps: accurately weighing 0.15g of Polycaprolactone (PCL) and 0.0015g of indocyanine green (ICG) by using an electronic balance, dissolving the Polycaprolactone (PCL) and the indocyanine green (ICG) in 5mL of chloroform, stirring the mixture for 5 hours by using a magnetic stirrer, and uniformly stirring the mixture to obtain a chloroform solution containing the ICG and the PCL;
the preparation method of the core layer solution comprises the following steps: dissolving insulin-like growth factor (IGF) in deionized water to obtain a solution, wherein the concentration of insulin-like growth factor (IGF) is 1 mg/mL;
(3) photosolder of nano-yarns and microparticles: irradiating the nano-linear deposited carrier particles by using near-infrared laser to perform 'photosolder', wherein the near-infrared laser irradiation can excite ICG to generate heat to reach the melting point of nano-yarn and particles, and guiding the contact parts of the nano-yarn and the particles to be welded together; the laser irradiation parameters were: the wavelength of the near infrared laser is 808nm, and the intensity of the laser irradiation is 4W/cm2The laser irradiation time is 3s, the laser irradiation distance is 2-3cm, and the temperature of the irradiated part observed by a thermal imager reaches 32 ℃;
(4) preparing the polymer nano-fiber/particle photosolder composite microsphere: intercepting the nano yarn obtained in the step (3) after the optical welding to obtain the size of 1.5cm multiplied by 1.5cm, embedding the nano yarn by an OCT embedding medium, freezing the nano yarn to be solid at the temperature of minus 80 ℃ in a refrigerator, cutting the nano yarn into short yarns with uniform length by a freezing slicing technology, and setting the fiber length to be 50 mu m; uniformly dispersing short fibers in an aqueous solution containing 5% of gelatin, wherein the mass volume concentration of the short fibers is 20mg/mL, preparing micro/nano fiber microspheres by using a container containing liquid nitrogen as a receiving device through an electrostatic spraying technology, wherein the voltage is 7kV, the receiving distance is 10cm, and the receiving time is 10 min; and then freeze-drying for 12h, and crosslinking glutaraldehyde to obtain the polymer nanofiber/particle photosolder composite microspheres.
Example 4:
this example is an experiment of the effect of the polymer nanofiber/microparticle photosynthesized composite microsphere material prepared in example 2 on tendon stem cell morphology. Cells are inoculated into a 24-well plate containing the composite microsphere material prepared in example 2 at the concentration of 10000 cells/well, and after 2 days of culture, F-actin/DAPI staining is carried out to observe the cell morphology, and the specific method comprises the following steps:
1. after culturing the cells in complete culture medium (89% low sugar Dulbecco's Modified Eagle Medium (DMEM) + 10% fetal bovine serum + 1% cyan/streptomycin by volume) for 2 days, removing the old culture medium, and then washing with Phosphate Buffered Saline (PBS) for 3 times;
2. fixing cells with 1mL of tissue cell fixing solution with the mass percentage concentration of 4% at room temperature for 10min, removing the tissue cell fixing solution, and washing with PBS for 3 times;
3. penetrating with 1 mL/hole of 0.1% polyethylene glycol octyl phenyl ether (Triton X-100) water solution for 5min at room temperature, removing the Triton X-100 solution, and washing with PBS for 3 times;
4. blocking with 1 mL/hole of BSA (bovine serum albumin) aqueous solution with the mass percentage concentration of 1% for 30min-60min, removing the BSA solution and washing with PBS for 3 times;
5. f-actin (fibrous actin green fluorescent dye solution) and BSA (bovine serum albumin) aqueous solution with the mass percentage concentration of 1% are mixed according to the volume ratio of 1: 1000, adding 200 mu L of the mixed solution into each hole, and dyeing for 40min at room temperature in a dark place;
6. washing with PBS for 5min for 3 times;
7. the slides were mounted with mounting medium containing DAPI (phenylindole), observed under a laser scanning confocal microscope and photographed, and the results are shown in FIG. 4B.
The results show that: cells grow on the composite microspheres in an adhesion manner, and the axon stretching is good, which indicates that the polymer nanofiber/microparticle photosolder composite microspheres can promote the growth of the cells.
Example 5:
this example is a slow release experiment of the polymer nanofiber/microparticle photosynthesized composite microsphere material prepared in example 1. Selecting three composite microspheres with preparation parameters of 0kV, 3kV and 10kV respectively for electrostatic spraying voltage in the step (4), respectively placing the three composite microspheres in 5mL Phosphate Buffer Solution (PBS), continuously oscillating a constant-temperature oscillating table, regularly measuring optical density (OD value) by a protein quantification kit (BCA method), and calculating cumulative release amount, wherein the specific method comprises the following steps:
1. weighing 3 composite microspheres with the same mass, placing the microspheres in a 15mL centrifuge tube, and adding 5mL Phosphate Buffered Saline (PBS) into each tube;
2. setting parameters of a constant-temperature shaking table to be 37 ℃, setting the rotating speed to be 80r/min, and placing 3 tubes of centrifuge tubes in the shaking table for continuous shaking;
3. at 5 time points of 1, 2, 3, 5, 7 days, 1mL of each of the 3 tube samples was sampled, and the optical density (OD value) was measured by a protein quantification kit (BCA method);
4. adding 2 μ L of sample and 98 μ L of working solution into each well of a 96-well plate, mixing well, incubating at 37 deg.C for 30min, and reading OD value at 562nm with an enzyme-labeling instrument;
6. calculating the protein concentration of the sample to be detected at each time point according to a standard curve equation, and calculating the cumulative release amount and the variance of the sample at each time point;
7. the cumulative release amount and variance of the samples obtained at each time point were plotted as a sustained release curve, and the results are shown in FIG. 5.
The results show that: the accumulated release amount of the polymer nanofiber/particle photosolder composite microsphere material is in an increasing trend along with the time lapse of the release protein amount.
Claims (10)
1. The polymer nanofiber/microparticle photosolder composite microsphere is characterized in that the structure of the composite microsphere is a medical degradable nanofiber microsphere with microparticles loaded on the surface, the fiber microsphere is composed of oriented nanofibers or nano yarns, the microparticles are degradable polymer microparticles loaded with bioactive substances, and the degradable polymer microparticles are deposited and welded on the surface of the fiber microsphere.
2. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microsphere as defined in claim 1, wherein the medical degradable polymer and the near-infrared dye are used as raw materials to prepare an oriented nanofiber membrane or a nano yarn; welding the contact parts of the fibers and the particles by laser irradiation at different time; then preparing the mixture into a short fiber homogeneous suspension and preparing the polymer microspheres by an electrostatic spraying technology, wherein the method comprises the following specific steps:
(1) preparing polymer nano-fibers: blending medical degradable polymer and near-infrared dye in a solvent to be uniformly dissolved to obtain a spinning solution, and then preparing a uniaxial orientation polymer nanofiber membrane by single-nozzle electrostatic spinning, or obtaining nano yarn by double-nozzle nano yarn preparation equipment;
(2) depositing carrier particles: flatly paving the uniaxial orientation nanofiber membrane or the nanometer yarn obtained in the step (1) on a receiving flat plate to serve as a receiving device, spraying carrier particles to the surface of the nanofiber membrane or the nanometer yarn by a coaxial electrostatic spraying device, and depositing the carrier particles on the surface of the nanofiber membrane or the nanofiber;
(3) photosolder polymeric nanofibers and microparticles: irradiating the nanofiber membrane or the nanometer yarn deposited with the carrier particles by using near-infrared laser to perform 'photosolder', and welding the contact part of the surfaces of the fibers or the yarns and the particles by laser irradiation at different time;
(4) preparing the polymer nano-fiber/particle photosolder composite microsphere: and (4) cutting the material welded in the step (3) into short fibers by a freezing microtome, dispersing the short fibers into a gelatin water solution to obtain a short fiber suspension, performing electrostatic spraying, receiving liquid nitrogen, performing freeze drying, and performing glutaraldehyde crosslinking to obtain the polymer nanofiber/particle photosolder composite microspheres.
3. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microsphere as claimed in claim 2, wherein the carrier microparticle is prepared by coaxially spraying a shell layer solution and a core layer solution through electrostatic spraying, wherein the shell layer solution is a mixed solution containing a medical degradable polymer and a near infrared dye, and the core layer solution is a solution containing a bioactive substance.
4. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microsphere as recited in claim 2, wherein said medical degradable polymer is at least one of polylactic acid, polycaprolactone, polylactic acid-glycolic acid copolymer and polylactic acid-caprolactone copolymer; the near-infrared dye is at least one of indocyanine green and heptamethine cyanine micromolecule dyes.
5. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microsphere in the step (1), wherein the mass ratio of the traditional Chinese medicine degradable polymer to the near-infrared dye in the step (1) is 100 (0.5-1); the volume ratio of the total mass of the medical degradable polymer and the near-infrared dye to the solvent is (5-15) g: 10 mL.
6. The method for preparing the polymeric nanofiber/microparticle photosynthesized composite microsphere as defined in claim 2, wherein the solvent in step (1) is one of hexafluoroisopropanol, dichloromethane, trifluoroethanol and a dichloromethane/N, N-dimethylformamide mixed solvent.
7. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microsphere as claimed in claim 3, wherein the shell solution of the carrier microparticle is a chloroform solution of degradable polymer and near infrared dye, and the bioactive substance in the core solution is bioactive protein, growth factor or small molecule.
8. The method for preparing the polymeric nanofiber/microparticle photosynthesized composite microsphere as defined in claim 2, wherein the laser irradiation intensity in the step (3) is determined according to the heat quantity released by the near-infrared dye absorbing the laser energy; the laser irradiation time is adjusted according to the time for the selected medical degradable polymer to reach the melting point.
9. The method for preparing the polymer nanofiber/microparticle photosynthesized composite microspheres as claimed in claim 2, wherein in the step (4), the concentration of the short fiber suspension is 10-20mg/mL, the electrostatic spraying voltage is 0-15kV, the suspension flow rate is 1-10mL/h, the receiving distance is 5-15cm, the receiving time is adjusted according to the demand, and the size of the received microspheres can be changed by adjusting the voltage and the flow rate.
10. Use of the polymeric nanofiber/microparticle photosynthesized composite microsphere as defined in claim 1 in tissue engineering for the delivery of bioactive substances and cells.
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