WO2016095811A1 - 一种用聚乙二醇制备丝素纳微米球的方法及其在药物控释中的应用 - Google Patents

一种用聚乙二醇制备丝素纳微米球的方法及其在药物控释中的应用 Download PDF

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WO2016095811A1
WO2016095811A1 PCT/CN2015/097464 CN2015097464W WO2016095811A1 WO 2016095811 A1 WO2016095811 A1 WO 2016095811A1 CN 2015097464 W CN2015097464 W CN 2015097464W WO 2016095811 A1 WO2016095811 A1 WO 2016095811A1
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silk fibroin
solution
polyethylene glycol
silk
drug
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PCT/CN2015/097464
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English (en)
French (fr)
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吴建兵
刘健
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苏州丝美特生物技术有限公司
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Priority to EP15869318.4A priority Critical patent/EP3235512A4/en
Priority to US15/537,236 priority patent/US20170340575A1/en
Publication of WO2016095811A1 publication Critical patent/WO2016095811A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1658—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin

Definitions

  • the invention belongs to the field of biomedicine or pharmacy, and particularly relates to a method for preparing silk fibroin microspheres from polyethylene glycol and its application in controlled release of drugs.
  • Silk fibroin a natural high-molecular fibrin extracted from silk, accounts for 70% to 80% of silk.
  • Silk fibroin has excellent physical and chemical properties, is non-toxic, non-irritating, biocompatible, and biodegradable, and can largely meet the needs of biomaterials.
  • Silk fibroin has excellent processing properties, and can be used for preparing film, gel, microsphere, porous scaffold, etc., and can be applied to a drug carrier.
  • Chinese Patent Application No. 201310203722.2 discloses a preparation method of an injectable silk fibroin in situ gel which is formed by mixing an aqueous solution of silk fibroin with a solution of polyethylene glycol or propylene glycol to form a gel due to gel particles.
  • the smaller particle size is not suitable for loading drugs, so it is necessary to develop a simple and feasible method for preparing silk fibroin microspheres as a drug carrier.
  • the methods for preparing silk fibroin microspheres mainly include emulsion-organic solvent evaporation/extraction method, phase separation method, self-assembly method, supercritical fluid rapid expansion method, liposome template preparation method, microfluidization method and spray drying. Law and so on.
  • the emulsification method, the self-assembly method, and the lipid template method all need to add an organic solvent, and the residue of the organic solvent directly limits the clinical application of the silk fibroin microsphere.
  • the supercritical fluid rapid expansion method, the microfluidic method and the spray drying method are relatively simple, the apparatus and preparation conditions are complicated.
  • the preparation process of the phase separation method is relatively simple and safe and non-toxic, the operation process is cumbersome.
  • silk fibroin has obvious advantages as a drug carrier: first, its source is abundant and low in cost; secondly, silk fibroin as a natural polymer has good biodegradability and high biosafety; The diversification of crystallization mode can achieve the purpose of controlling the drug release rate by changing the conformational composition of silk fibroin, so that the drug is released in the targeted area, thereby improving the therapeutic effect of the drug. Therefore, the silk fibroin with excellent biocompatibility is prepared into microspheres/particles, which can make the two synergistic effects, so that the silk fibroin microspheres/particles not only have the biocompatibility unmatched by other materials, but also have Higher bioavailability and biosafety.
  • methods for preparing drug-loaded silk micron microparticles/microspheres mainly include emulsion-organic solvent evaporation/extraction method, phase separation method, self-assembly method, supercritical fluid rapid expansion method, liposome template preparation method, microflow Control and spray drying methods, etc., among which:
  • Emulsion-organic solvent evaporation/extraction method Thanonchat disperses the silk fibroin solution in an organic solution of ethyl acetate, and then adds it to the oil phase containing a surfactant (such as Span80) for stirring or sonication to form (W/O).
  • a surfactant such as Span80
  • Type emulsion followed by chemical crosslinking agent (glutaraldehyde) for curing.
  • the method utilizes the characteristics that the amino group on the silk fibroin is polycondensed with the aldehyde group of other compounds, and the silk fibroin micron particles are obtained by crosslinking, and the silk fibroin micron particles in the emulsion can be evaporated or extracted by the organic solvent. Separated.
  • the phase separation method Wang directly blends the silk fibroin solution with the PVA (polyvinyl alcohol) solution, and after ultrasonication or stirring, the blended solution is placed in an oven at 60 ° C to be dried, dissolved, and washed by centrifugation.
  • the silk fibroin microparticles can be adjusted to adjust the particle size of the silk fibroin microparticles by changing the concentration of the silk fibroin solution and the power of the ultrasound.
  • Self-assembly method Cao directly mixes the silk fibroin solution with the ethanol solution, stirs at room temperature for 2 min, and then freezes in a refrigerator (-5 ° C ⁇ -40 ° C) for 24 h, then takes it out, thaws at room temperature, and can be washed by centrifugation.
  • the particle size of the silk fibroin microparticles can be regulated by the concentration of the silk fibroin solution and the volume ratio of the silk fibroin solution to ethanol.
  • Supercritical fluid rapid expansion method Wenk dissolves the drug-loaded silk fibroin solution in the superfluid, and in a short time, the super fluid is ejected from the nozzle of a certain aperture by the frequency generating vibration device, and the wire is made under high voltage conditions.
  • the protein solution rapidly forms a crystal nucleus and prompts the nucleus to rapidly expand in a short time.
  • the droplets are collected by a liquid nitrogen collecting device and rapidly frozen to obtain a silk fibroin-loaded microsphere.
  • the particle size of the silk fibroin microparticles can be directly controlled by the pore size of the nozzle.
  • Liposome template preparation Wang dissolved DOPC (dioleoylphosphatidylcholine) in chloroform organic solution, dried to form a membrane under nitrogen, added a volume of silk fibroin solution, and diluted by deionized water. After that, it was directly frozen under liquid nitrogen for 15 min, and then thawed at 37 ° C for 15 min, then the thawed solution was poured into a beaker, stirred rapidly, and the solution was lyophilized and stored in a refrigerator at 4 ° C. By treatment with methanol and sodium chloride solution, the liposome is degraded to induce a change in the structure of the silk fibroin to form silk fibroin micron particles.
  • DOPC dioleoylphosphatidylcholine
  • Microfluidic method uses the principle of phase separation, uses a microfluidic device to isolate silk fibroin and PVA (polyvinyl alcohol) solution, and then controls the particle size of silk fibroin microparticles by controlling the flow rate of solution mixing.
  • the prepared microspheres have a relatively uniform particle size.
  • Hino sprays the silk fibroin solution directly into the hot air through an atomizing device, and then produces silk fibroin microparticles during the drying process.
  • the above method for preparing silk fibroin microparticles still has deficiencies, and there are great limitations on the later drug loading and application.
  • the emulsion-organic solvent evaporation/extraction method, the self-assembly method, and the liposome stencil method all require the addition of an organic solvent, and the residue of the organic solvent affects the stability of the small molecule drug and the activity of the macromolecular protein drug, thereby Directly limiting the clinical application of drug-loaded silk fibroin microparticles.
  • the supercritical fluid rapid expansion method, microfluidic method and spray drying method are relatively simple, but the device and preparation conditions are relatively complicated.
  • the drug loading rate is low and the cost is relatively high, which is not conducive to the later industrial application. Therefore, how to develop a preparation method of silk fibroin-loaded nano-microparticles with good drug controlled release effect, no organic solvent residue, simple operation, low preparation cost and good embedding effect has become an urgent problem to be solved.
  • the object of the present invention is to overcome the residual organic solvent in the preparation process of the drug-loaded nano-particles, the complicated process, the long time-consuming, the high cost, and the unfavorable industrialization.
  • Disadvantages such as production and clinical application provide a preparation method with high efficiency, safety, quickness and convenience, good resource utilization, low production cost, good biocompatibility, and certain value for industrial production and clinical application.
  • the method not only realizes the controlled release of the drug by regulating the particle size and the secondary crystal structure of the silk fibroin particles, but also solves the problem that some clinical drugs are difficult to be directly fixed in the silk fibroin micron particles by physical embedding method.
  • the invention provides a method for preparing silk fibroin microspheres from polyethylene glycol, comprising the steps of: separating a silk fibroin solution having a mass percentage of 1 to 30% with a polyethylene glycol having a mass percentage of 10 to 60%.
  • the solution was placed at a temperature of 4 to 60 ° C for 30 min, and then the silk fibroin solution and the polyethylene glycol solution were uniformly mixed at a certain ratio, and after centrifugation for a certain period of time, the mixture was centrifuged to prepare a silk fibroin microsphere suspension.
  • the present invention provides the use of the above method for preparing silk fibroin microspheres from polyethylene glycol, for use in the preparation and controlled release of drug-loaded silk micron microparticles, comprising the steps of: dissolving the drug in the mass In a polyethylene glycol solution having a percentage of 10 to 60%, or dissolving the drug in a silk fibroin solution having a mass percentage of 1 to 30%, and mixing the polyethylene glycol solution with the silk fibroin solution.
  • the blend was uniformly prepared, and then the blend was incubated for a certain period of time and then washed by centrifugation to obtain a silk fibroin-loaded nano-microparticle for drug controlled release.
  • the hydrophobic drug when the drug is a hydrophobic drug, the hydrophobic drug is dissolved in a polyethylene glycol solution and then mixed with a silk fibroin solution to prepare the blend; the hydrophobic drug is further The drug is prepared by dissolving the other drug in a silk fibroin solution and then mixing with a polyethylene glycol solution.
  • the hydrophobic drug comprises curcumin
  • the other drugs include a hydrophilic drug, doxorubicin, a polypeptide drug octreotide, a protein drug, bovine serum albumin, and a macromolecular hydrophilic model drug.
  • Sugar or CdTe quantum dots with fluorescent labeling include gemfibrozil, doxorubicin, a polypeptide drug octreotide, a protein drug, bovine serum albumin, and a macromolecular hydrophilic model drug.
  • the mass ratio of the silk fibroin contained in the silk fibroin solution to the drug ranges from 1000/1 to 1/10.
  • polyethylene glycol has a molecular weight in the range of 2,000 to 20,000.
  • the polyethylene glycol has a molecular weight percentage of 30 to 60% when the concentration molecular weight is 4000 and 6000, a mass percentage of 20 to 50% when the molecular weight is 10,000, and a mass percentage when the molecular weight is 20,000. It is 20 to 40%.
  • the silk fibroin solution has a pH of 3.0 to 11.0.
  • the silk fibroin solution has a salt ion treatment concentration of from 1 M/L to 0.01 M/L.
  • the volume ratio of the silk fibroin solution and the polyethylene glycol solution ranges from 5:1 to 1:10.
  • the blend is incubated at room temperature to 60 ° C for 0.5 to 24 h.
  • the silk fibroin solution has a dilution range of 5 to 20% by mass.
  • the silk fibroin solution is prepared as follows: the degummed cooked silk is infiltrated in a 9.3 M LiBr solution and placed in an oven at 60 ° C for 4 h, and dissolved to obtain a silk fibroin solution; The silk fibroin solution was poured into a dialysis bag and dialyzed against deionized water for 3 days. After the dialysis, the silk fibroin solution was centrifuged to remove insoluble impurities, and then poured into a dialysis bag and dialyzed against a polyethylene glycol solution having a molecular weight of 20,000 and 15 wt% for 24 hours. A concentrated silk fibroin solution was obtained.
  • the present invention has at least the following advantages:
  • the pharmaceutical auxiliary agent of the invention is prepared by blending polyethylene glycol with silk fibroin solution to prepare silk fibroin microspheres, or selecting a pharmaceutical auxiliary polyethylene glycol and silk fibroin solution to prepare silk fibroin particles.
  • the above preparation process does not require complicated equipment, the operation flow is simple and easy, the time is short, the cost is low, and the organic solvent is not added, and the prepared silk fibroin particles have high biosafety and can be directly applied to the clinic to realize optimal utilization of resources. ;
  • the method of the invention directly mixes and incubates and rapidly prepares the silk fibroin particles, does not require complicated experimental devices, does not require drying and film re-dissolution, simplifies the operation process, shortens the preparation time, and is beneficial to industrial production;
  • the method of the invention can prepare silk fibroin micron particles under normal temperature conditions, and does not need to be placed in a refrigerator for freezing or drying in an oven, which not only saves energy, but also reduces production cost;
  • the particle size of the silk fibroin particles prepared by the invention can be controlled by the relative molecular weight and concentration of polyethylene glycol and the concentration of silk fibroin, thereby creating conditions for drug controlled release drug loading;
  • the silk fibroin particles prepared by the method of the invention are used for embedding sustained-release drugs, and the selection range is wide, and is suitable for the hydrophobic and hydrophobic small molecule drugs, the polypeptide drugs and the protein drugs, etc., wherein the hydrophobic neutral drugs can pass through the molecules with the silk proteins.
  • the specific hydrophobic region is bound by hydrophobic interaction and embedded in the silk fibroin particles, thereby increasing the drug loading rate of the hydrophobic neutral drug.
  • FIG. 1 is a scanning electron micrograph of silk fibroin microparticles provided in Examples 1-4 of the present invention.
  • FIG. 2 is a particle size distribution diagram of silk fibroin microspheres prepared by blending different molecular weight PEG solutions and 8 wt% silk fibroin solutions according to Examples 1-4 of the present invention
  • Figure 3 is a scanning electron micrograph of a silk fibroin microsphere of Example 1-6 of the present invention.
  • Example 4 is a graph showing the particle size distribution of the silk fibroin microparticles and the viscosity of the polyethylene glycol solution provided in Example 2 of the present invention
  • Figure 5 is a scanning electron micrograph of silk fibroin microparticles provided in Example 3 of the present invention.
  • Figure 6 is a graph showing the particle size distribution and polyethylene glycol viscosity of the silk fibroin microparticles provided in Example 3 of the present invention.
  • Figure 7 is a scanning electron micrograph of silk fibroin microparticles provided in Example 4 of the present invention.
  • Figure 8 is a particle size diagram of silk fibroin microparticles provided in Example 4 of the present invention.
  • Figure 9 is a scanning electron micrograph of silk fibroin microparticles provided in Example 5 of the present invention.
  • Figure 10 is a scanning electron micrograph of silk fibroin microparticles provided in Example 6 of the present invention.
  • Figure 11 is a scanning electron micrograph of silk fibroin microparticles provided in Example 6 of the present invention.
  • Figure 12 is a scanning electron micrograph of silk fibroin microparticles provided in Example 7 of the present invention.
  • Figure 13 is a particle size diagram of silk fibroin microparticles provided in Example 7 of the present invention.
  • Figure 14 is a scanning electron micrograph of silk fibroin microparticles provided in Example 8 of the present invention.
  • Figure 15 is a topographical view of a single silk fibroin microsphere provided in Example 8 of the present invention.
  • Figure 16 is a cross-sectional view of a single silk fibroin microsphere provided in Example 8 of the present invention.
  • Figure 17 is a particle size diagram of silk fibroin microparticles provided in Example 8 of the present invention.
  • Figure 18 is an electromotive force diagram of the silk fibroin microparticles provided in Example 8 of the present invention.
  • Figure 19 is a graph showing the yield of silk fibroin microparticles provided in Example 8 of the present invention.
  • Figure 20 is an infrared spectrum diagram of silk fibroin microparticles provided in Example 9 of the present invention.
  • Figure 21 is a diagram showing the secondary structure content of the silk fibroin microparticles and the control sample provided in Example 9 of the present invention.
  • Figure 22 is an infrared spectrum diagram of silk fibroin microparticles provided in Example 10 of the present invention.
  • Figure 23 is a diagram showing the secondary structure content of the silk fibroin microparticles and the control sample provided in Example 10 of the present invention.
  • Figure 24 is an infrared spectrum diagram of silk fibroin microparticles provided in Example 11 of the present invention.
  • Figure 25 is a diagram showing the secondary structure content of the silk fibroin microparticles and the control sample provided in Example 11 of the present invention.
  • Figure 26 is an infrared spectrum of silk fibroin nanoparticles (supernatant) provided in Example 12 of the present invention.
  • Figure 27 is an infrared spectrum of silk fibro microparticles (secondary standing) provided in Example 12 of the present invention.
  • Figure 28 is an infrared spectrum of silk fibroin nanoparticles (primary standing) provided in Example 12 of the present invention.
  • Figure 29 is a laser confocal diagram of curcumin-silk submicron particles provided in Example 13 of the present invention.
  • Figure 30 is a laser confocal diagram of TMR-dextran-silk submicron particles provided in Example 14 of the present invention.
  • Figure 31 is a fluorescence micrograph of a CdTe-silk-silm microparticle provided in Example 15 of the present invention.
  • Figure 32 is a scanning electron micrograph of curcumin-silk-snap microparticles provided in Example 16 of the present invention.
  • Figure 33 is a laser confocal diagram of curcumin-silk submicron particles provided in Example 16 of the present invention.
  • Figure 34 is a scanning electron micrograph of doxorubicin hydrochloride-silk submicron particles provided in Example 17 of the present invention.
  • Figure 35 is a laser confocal diagram of doxorubicin hydrochloride-silk submicron particles provided in Example 17 of the present invention.
  • Figure 36 is a graph showing the cumulative release rate of curcumin-silk submicron particles provided in Example 18 of the present invention.
  • Figure 37 is a graph showing the cumulative release rate of TMR-dextran-silk-silm microparticles provided in Example 19 of the present invention.
  • Figure 38 is a graph showing the cumulative release rate of CdTe-silk-silm microparticles provided in Example 20 of the present invention.
  • Figure 39 is a graph showing the cumulative release rate of curcumin-silk submicron particles provided in Example 21 of the present invention.
  • Figure 40 is a graph showing the cumulative release rate of doxorubicin hydrochloride-silk submicron particles provided in Example 22 of the present invention.
  • Figure 41 is a graph showing the cumulative release rate of doxorubicin hydrochloride-silk submicron particles provided in Example 23 of the present invention.
  • Figure 42 is a scanning electron micrograph of octreotide-silk submicron particles provided in Example 24 of the present invention.
  • Figure 43 is a scanning electron micrograph of TMR-bovine serum albumin-silk submicron particles provided in Example 25 of the present invention.
  • Figure 44 is a laser confocal diagram of TMR-bovine serum albumin-silk-snap microparticles provided in Example 25 of the present invention.
  • Figure 45 is a cumulative release diagram of TMR-bovine serum albumin-silk sult microparticles provided in Example 26 of the present invention.
  • Figure 46 is a scanning electron micrograph of doxorubicin hydrochloride-silk fibroin nanoparticles (supernatant) provided in Example 27 of the present invention.
  • Figure 47 is a scanning electron micrograph of doxorubicin hydrochloride-silk microparticles (primary standing) provided in Example 27 of the present invention.
  • Figure 48 is a particle size distribution diagram of doxorubicin hydrochloride-silk fibroin nanoparticles (supernatant) provided in Example 27 of the present invention.
  • Example 49 is an electromotive force diagram of doxorubicin hydrochloride-silk fibroin nanoparticles (supernatant) provided in Example 27 of the present invention.
  • Figure 50 is a chart showing the encapsulation ratio of doxorubicin hydrochloride hydrochloride of doxorubicin hydrochloride-silk-sodium microparticles provided in Example 27 of the present invention.
  • Figure 51 is a laser confocal image of doxorubicin-silk microparticles (primary standing) provided in Example 27 of the present invention.
  • the present invention is not limited to the scope of the present invention.
  • the embodiments of the present invention are not limited to the scope of the present invention. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without creative efforts are within the scope of the present invention.
  • the method for preparing silk fibroin-loaded nano-microparticles for drug controlled release needs to consider avoiding the use of organic solvents and violent formation in the preparation process in order to maintain the stability and molecular activity of drug delivery.
  • the pellets are in a spherical condition.
  • the particle size and morphology of the microspheres prepared by phase separation can be controlled by changing the molecular weight of PVA (polyvinyl alcohol) and the weight ratio of PVA to silk protein.
  • PVA polyvinyl alcohol
  • the method of the present invention and the existing phase separation method are different in principle, operation flow, and drug application range.
  • the principle is mainly reflected in the phase separation method is to mix the PVA solution and the silk protein solution, the silk fibroin molecules are fully dispersed in the PVA solution by ultrasonic or stirring, and then the dispersed solution is placed in the oven.
  • the film is dried, formed into a film, dissolved, and washed.
  • the invention adopts the emulsion polymerization method to directly add the silk protein solution into the polyethylene glycol solution, and utilizes the hydrophilicity of the polyethylene glycol to rapidly absorb the large water molecules on the surface of the silk protein, and promote the hydrophobic regions of the silk proteins to be close to each other.
  • the phase separation method needs to place the blend liquid in a 60 ° C oven to form a film, and then dissolve and wash, and the present invention only needs to be incubated at room temperature.
  • the scope of application of the drug is different: the silk fibroin particles prepared by the phase separation method have a low drug loading rate for the hydrophobic neutral drug, and the polyethylene glycol of the method is an amphiphilic pharmaceutical auxiliary agent, and the hydrophobic neutral drug can be used. Dissolved in polyethylene glycol solution Medium, and then blended with silk protein to form a ball, the silk fibroin particles have a higher drug loading rate.
  • the silk fibroin-loaded nano-microparticle of the invention satisfies the effect of quantitatively and uniformly releasing the drug at a constant rate in drug controlled release, and keeping the blood drug concentration constant.
  • Select small molecule drugs such as curcumin, doxorubicin hydrochloride, macromolecular drugs, such as TMR-glucan, peptide protein drugs, such as octreotide, TMR-bovine serum albumin, etc., by physical embedding method
  • the possibility of clinical application of the late silk fibroin microparticles as a drug carrier is provided.
  • the concentration of the silk fibroin solution is determined by weighing a certain amount of silk fibroin solution, drying it in an oven at 60 ° C, and weighing it, and the ratio of the two is a concentration value (mass percentage, wt%).
  • the concentration of the silk fibroin solution generally obtained is about 7 wt%.
  • the concentration process of the silk fibroin solution is to transfer 100 ml of the prepared silk fibroin solution into the same Slide-a-lyzer dialysis bag, and then place the dialysis bag in a polyethylene glycol solution of 15 wt%, molecular weight 20,000, and 800 ml. After continuous dialysis for 24 h, about 30% by weight of the concentrated protein solution was obtained and stored in a refrigerator at 4 ° C.
  • the polyethylene glycol solutions of different molecular weights are disposed at different concentrations, the concentration of polyethylene glycol having a molecular weight of less than 1000 is 40-100% by weight; the concentration of polyethylene glycol having a molecular weight of 1000-6000 is 10-60% by weight; and the molecular weight is 10,000.
  • the concentration is 10-50% by weight; the concentration of molecular weight 20000 is 10-40% by weight;
  • the polyethylene glycol-silk fibroin blend was incubated at room temperature for 30 min;
  • the polyethylene glycols of different molecular weights are configured to different concentrations, the concentration of polyethylene glycol having a molecular weight of 2000-6000 is 10-60% by weight; the concentration of molecular weight 10000 is 10-50% by weight; the concentration of molecular weight 20000 is 10-40% by weight;
  • the polyethylene glycol-silk fibroin blend was incubated at room temperature for 30 min;
  • the concentration of the polyethylene glycol solution having a molecular weight of 2000 is 40 wt%
  • the concentration of the polyethylene glycol solution of 4000 is 40 wt%
  • the concentration of the polyethylene glycol solution having a molecular weight of 6000 is 40 wt%
  • the polyethylene glycol having a molecular weight of 10,000 is 40% by weight
  • the concentration of the polyethylene glycol solution having a molecular weight of 20,000 is 40% by weight;
  • the polyethylene glycol-silk fibroin blend was incubated at room temperature for 12 h;
  • the centrifuge tube is placed in a centrifuge and centrifuged, conditions: room temperature, 10 min, 12000 rpm / min;
  • the suspension of the silk fibroin microspheres was placed in deionized water, placed in a refrigerator at 4 ° C or dried under vacuum vacuum.
  • the silk fibroin micro-nanoparticles obtained by blending a polyethylene glycol solution having a molecular weight of 10,000 and a concentration of 40 wt% with a 8% by weight silk fibroin solution have a larger particle size.
  • the particle size of each group of silk fibroin microsphere samples was measured by a laser particle size analyzer, and each group was measured three times, and an average value was calculated.
  • the silk fibroin micron sphere particles obtained by blending a polyethylene glycol solution having a dose of 10000 and a concentration of 40 wt% with a 8 wt% silk fibroin solution have a large particle size.
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 3 it is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment, which were incubated at 25 ° C, 45 ° C, and 60 ° C, respectively. As can be seen, the morphology of the silk fibroin microparticles is affected by the incubation temperature.
  • the 8 wt% concentration of the silk fibroin solution was configured according to the method of Example 1;
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 4 it is the particle size of the silk fibroin microparticles provided in this embodiment and the polyethylene glycol solution.
  • the viscosity map, the square icon indicates the particle size, and the triangle icon indicates the viscosity.
  • the particle size of the silk fibroin microparticles is affected by the viscosity of the polyethylene glycol solution.
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 5 it is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment. As can be seen, the morphology of the silk fibroin microparticles is affected by the concentration of the polyethylene glycol solution.
  • FIG. 6 it is a particle size distribution of the silk fibroin microparticles provided in this embodiment and a polyethylene glycol viscosity diagram, a square icon indicates the particle diameter, and a circular icon indicates the viscosity. As can be seen, the particle size of the silk fibroin microparticles is affected by the viscosity of the polyethylene glycol solution.
  • the concentration of polyethylene glycol having a molecular weight of 10,000 is 50% by weight
  • Example 2 According to the method of Example 1, respectively, a silk fibroin solution having a concentration of 1, 2%, 5%, 8%, 12%, and 20% by weight was disposed.
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 7 it is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment. As can be seen, the morphology of the silk fibroin microparticles is affected by the concentration of the silk fibroin solution.
  • FIG 8 it is a particle size diagram of the silk fibroin microparticles provided in this example. As can be seen, the particle size of the silk fibroin microparticles is affected by the concentration of the silk fibroin solution.
  • the concentration of polyethylene glycol having a molecular weight of 10,000 is 50% by weight
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 9 it is a scanning electron micrograph of the silk fibroin microsphere provided in this embodiment. As can be seen, the morphology of the silk fibroin microspheres is affected by the addition of a methanol solution.
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG. 10 is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment, pH value They are 3.6 and 7.0 respectively. As can be seen, the morphology of the silk fibroin microparticles is affected by the pH of the silk fibroin solution.
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • FIG 12 it is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment, pH 3.6, 7.0, 10.0 of 0.2, 1, 5, 10, 15 wt% SF and 50 wt% molecular weight 10000 poly Glycol solution. As can be seen, the morphology of the silk fibroin microparticles is affected by the pH of the silk fibroin solution.
  • FIG. 13 it is a particle size diagram of the silk fibroin microparticles provided in this embodiment.
  • the pH value is 3.6, 7.0, 10.0, 0.2, 1, 5, 10, 15 wt% SF is blended with 50 wt% molecular weight 10000 PEG solution. .
  • the particle size of the silk fibroin microparticles is affected by the pH of the silk fibroin solution and the concentration of the silk fibroin solution.
  • the molecular weight of 10,000 polyethylene glycol is set to 20% (indicating the molecular weight of polyethylene glycol 10000, the concentration of 20wt%);
  • FIG 14 it is a scanning electron micrograph of the silk fibroin microparticles provided in this embodiment.
  • the first row of electron micrographs is before standing, the second row of electron micrographs is the supernatant after standing, and the third row is electron micrograph. It is a second static setting, and the fourth row of electron micrographs is once static, under the conditions of no ion, sodium ion, potassium ion and magnesium ion treatment.
  • the morphology of the silk fibroin microparticles is affected by the addition of salt ions.
  • FIG 15 is a surface topography of the individual silk fibroin microspheres provided in this example, as can be seen, the surface topography of the silk fibroin microspheres is affected by the added ions.
  • FIG 16 is a cross-sectional top view of the individual silk fibroin microspheres provided in this example, as can be seen, the cross-sectional morphology of the silk fibroin microspheres is affected by the added ions.
  • FIG 17 is a particle size diagram of the silk fibroin microparticles provided in the present example, as shown, the average particle size of the silk fibroin nanoparticles (supernatant) is about 350 nm, and the silk fibroin microparticles (secondary The average particle diameter of the stationary film was about 10 ⁇ m, and the particle diameter of the silk fibroin microparticles (primary standing) was about 40 ⁇ m on average.
  • FIG 19 is a graph of the yield of the silk fibroin microparticles provided in this example, as can be seen, the yield of the silk fibroin microparticles is affected by different salt ion treatments.
  • FIG 20 which is an infrared spectrum of the silk fibroin microparticles provided in this embodiment
  • the infrared spectrum of the silk fibroin microparticles (5%, 8%, 12%, 20% (wt) SF and 50% (wt) molecular weight 10000 polyethylene glycol solution blending, 8% (wt) and 50% (wt) molecular weight 10000 polyethylene glycol, methanol solution blending, control.
  • the secondary of silk fibroin microspheres The structure is affected by the addition of polyethylene glycol and methanol.
  • FIG 21 it is a secondary structure content diagram of the silk fibroin microparticles and the control provided in this example. As can be seen, the secondary structure content of the silk fibroin microparticles is affected by the addition of polyethylene glycol and methanol.
  • FIG 22 it is an infrared spectrum of the silk fibroin microparticles provided in this embodiment.
  • the secondary structure of the silk fibroin microspheres is affected by the incubation temperature.
  • FIG 23 it is a graph of the secondary structure content of the silk fibroin microparticles and the control provided in this example. As can be seen, the secondary structure content of the silk fibroin microparticles is affected by the incubation temperature.
  • Example 8 1. The lyophilized dried silk fibroin microparticles in Example 8 were selected and weighed 0.1 mg;
  • FIG. 24 it is an infrared spectrum of the silk fibroin microparticles provided in this embodiment. As can be seen, the secondary structure of the silk fibroin microparticles is affected by the pH of the silk fibroin solution.
  • FIG 25 it is a graph of the secondary structure content of the silk fibroin microparticles and the control provided in this example. As can be seen, the secondary structure content of the silk fibroin microparticles is affected by the pH of the silk fibroin solution.
  • Example 9 The lyophilized dried silk fibroin microparticles in Example 9 were selected and weighed 0.1 mg;
  • test condition was total reflection on the surface, and the wave number range was 400-4000 cm-1, thereby testing the infrared structure of each sample.
  • FIG. 27 it is an infrared spectrum of the silk fibroin particles (secondary standing) provided in the present embodiment.
  • the secondary structure of the silk fibroin microparticles is affected by the salt ion treatment.
  • FIG 28 it is an infrared spectrum of the silk fibroin particles (primary standing) provided in the present embodiment.
  • the secondary structure of the silk fibroin microparticles is affected by the salt ion treatment.
  • curcumin-polyethylene glycol-silk fibroin blend was incubated at room temperature for 2 h;
  • the centrifuge tube is placed in a centrifuge and centrifuged at room temperature, 10 min, 12000 rpm/min;
  • the drug loading rate of curcumin-silk-snap microparticles prepared by 5% silk protein was 0.27% ⁇ 0.07%, and the drug loading rate of curcumin-silk-silm microparticles prepared by 9% silk protein was 0.51 ⁇ 0.15%. .
  • the above solution is equal volume blending, the mass ratio of silk fibroin to TMR-glucan is 100/1;
  • the silk fibroin and TMR glucan blend solution are blended with the polyethylene glycol solution in an equal volume ratio, and gently pushed up and down with a sniffer to make the blend liquid uniformly mixed;
  • TMR-dextran-polyethylene glycol-silk fibroin blend was incubated at room temperature for 2 h;
  • TMR-dextran-silk-silica microparticles prepared by 1mg 5%, 9wt% silk protein solution, and add 1ml of lithium bromide solution, each group is called three samples;
  • the drug loading rate of TMR-dextran-silk-silm microparticles prepared by 5% silk protein was 0.13%, and the drug loading rate of TMR-dextran-silk-silm microparticles prepared by 9% silk protein was 0.26. %.
  • TMR-dextran-silk submicron particles provided in this example.
  • TMR-glucan is mainly distributed on the surface of silk fibroin microparticles.
  • the concentration ratio of silk fibroin to CdTe quantum dots in deionized water is 0.017 nmol CdTe/1 mg SF, and the final concentration of silk fibroin solution is 5, 8, 20 wt%;
  • a polyethylene glycol solution having a concentration of 50 wt% and a molecular weight of 10,000 is disposed;
  • the silk fibroin and CdTe blending solution is further blended with the polyethylene glycol solution in an equal volume ratio, and gently pushed up and down with a sniffer to make the blended liquid uniformly mixed;
  • the centrifuge tube is placed in a centrifuge for centrifugation, the conditions are room temperature, 10min, 12000rpm / min;
  • the encapsulation ratio of CdTe-silk-silk micro-particles prepared by 5% silk protein is 98.47% ⁇ 0.17%
  • the encapsulation ratio of CdTe-silk-silm micro-particles prepared by 8% silk protein is 98.89% ⁇ 0.23%, 20%.
  • the encapsulation ratio of the silk protein-prepared CdTe-silk submicron particles was 96.82% ⁇ 0.27%.
  • FIG. 31 it is a fluorescence micrograph of the CdTe-silk Su Na microparticles provided in this example. As can be seen, CdTe is uniformly distributed on the silk fibroin microparticles.
  • curcumin-polyethylene glycol-silk fibroin blend was incubated at room temperature for 24 h;
  • the centrifuge tube is placed in a centrifuge and centrifuged at room temperature, 10 min, 12000 rpm/min;
  • the drug loading rate of curcumin-silk-snap microparticles prepared by 5% pH 3.6 silk fibroin was 1.18% ⁇ 0.08%, and the drug loading of curcumin-silk-silm microparticles prepared by 5% pH 7.0 silk fibroin was carried out.
  • the rate is 0.64 ⁇ 0.06%.
  • FIG 32 it is a scanning electron micrograph of the curcumin-silk submicron particles provided in this example. As can be seen, the morphology of curcumin-silkner microparticles is affected by the pH of the silk fibroin solution.
  • FIG. 33 it is a laser confocal image of the curcumin-silk submicron particles provided in this example. As can be seen, curcumin is uniformly distributed in the silk fibroin microparticles.
  • the silk fibroin solution having a concentration of 5% pH 3.6 and pH 7.0 is separately disposed;
  • the centrifuge tube is placed in a centrifuge for centrifugation at room temperature, 10 min, 12000 rpm/min;
  • the drug loading rate of doxorubicin-silk-silm microparticles prepared by 5% pH 3.6 silk fibroin was 0.80% ⁇ 0.06%, and the doxorubicin-silk-sodium micron prepared by 5% pH 7.0 silk fibroin The drug loading rate of the particles was 1.90 ⁇ 0.05%.
  • FIG. 34 it is a scanning electron micrograph of the doxorubicin hydrochloride-silk submicron particles provided in this embodiment. As can be seen, the morphology of the doxorubicin-silk-sodium microparticles received the effect of the pH of the silk fibroin solution.
  • FIG. 35 it is a laser confocal image of the doxorubicin hydrochloride-silk nanoparticle provided in this embodiment.
  • the doxorubicin hydrochloride distribution is relatively uniform in the silk fibroin micron particles.
  • FIG 36 it is a cumulative release rate graph of curcumin-silk-snap microparticles provided in this example. As can be seen, the release rates of curcumin in silk fibroin-curcumin microspheres prepared at different concentrations were significantly different.
  • TMR-glucan Take the supernatant and measure the fluorescence value of TMR-glucan at a excitation wavelength of 555 nm and an emission wavelength of 590 nm with a microplate reader. Calculate the TMR-glucose by comparing the fluorescence-concentration standard curve of TMR-dextran. The amount of glycans released in the silk fibroin microparticles was calculated and the cumulative release rate was calculated.
  • FIG 37 it is a cumulative release rate diagram of the TMR-dextran-silk-silm microparticles provided in this example. As can be seen, the release rates of TMR-dextran in TMR-dextran-silk-silm microspheres prepared at different concentrations were significantly different.
  • FIG 38 it is a cumulative release rate diagram of the CdTe-silk submicron particles provided in this example. As can be seen, the release rates of CdTe quantum dots in CdTe-silk-silm microparticles prepared at different concentrations are significantly different.
  • FIG 39 it is a cumulative release rate diagram of the curcumin-silk submicron particles provided in this example. As can be seen, the release rates of curcumin in silk fibroin-curcumin microspheres prepared at different concentrations were significantly different.
  • FIG 40 it is a cumulative release rate diagram of doxorubicin hydrochloride-silk submicron particles provided in this example. As can be seen, the release rates of doxorubicin in silk fibroin-doxorubicin hydrochloride microspheres prepared at different concentrations were significantly different.
  • FIG 41 it is a cumulative release rate diagram of doxorubicin hydrochloride-silk submicron particles provided in this example. As can be seen, the release rates of doxorubicin hydrochloride in doxorubicin-silk nanospheres prepared at different concentrations were significantly different.
  • the mass ratio of silk fibroin to octreotide is 5 / 1;
  • the centrifuge tube is placed in a centrifuge and centrifuged at room temperature, 10 min, 12000 rpm/min;
  • FIG. 42 it is a scanning electron micrograph of octreotide-silk submicron particles provided in this example.
  • the centrifuge tube is placed in a centrifuge and centrifuged at room temperature, 10 min, 12000 rpm/min;
  • FIG 43 it is a scanning electron micrograph of TMR-bovine serum albumin-silk submicron particles provided in this example.
  • FIG 44 it is a laser confocal image of the TMR-bovine serum albumin-silk submicron particle provided in this example.
  • TMR-bovine serum albumin Take the supernatant and measure the fluorescence value of TMR-bovine serum albumin at the excitation wavelength of 555 nm and the emission wavelength of 590 nm with a microplate reader. Calculate the TMR-bovine by comparing the fluorescence-concentration standard curve of TMR-bovine serum albumin. The amount of serum albumin released in the silk fibroin microparticles, and the cumulative release rate is calculated;
  • TMR-bovine serum albumin in TMR-bovine serum albumin-silk submicron particles had a significant sustained release effect.
  • the molecular weight of 10,000 polyethylene glycol is set to 20% (indicating the molecular weight of polyethylene glycol 10000, the concentration of 20wt%);
  • the doxorubicin hydrochloride dissolved in the above salt ion solution is diluted with the above silk fibroin solution in an equal volume ratio to finally obtain a 15% silk fibroin solution, the concentration of doxorubicin is 1.5 mg/ml, and the salt ion concentration 0.1 mol/L; (the ratio of doxorubicin to silk fibroin protein is 1/100)
  • FIG. 46 it is a scanning electron micrograph of the doxorubicin hydrochloride-silica nanoparticle (supernatant) provided in the present embodiment. As shown in the figure, the morphology of the doxorubicin hydrochloride-silk nanoparticle is added by salt. The effect of ions.
  • FIG. 47 it is a scanning electron micrograph of the doxorubicin hydrochloride-silk microparticles provided in the present embodiment. As shown in the figure, the morphology of doxorubicin-silk fibroin microparticles (primary standing) is added by salt. The effect of ions.
  • FIG. 48 it is a particle size diagram of the doxorubicin hydrochloride-silica nanoparticle (supernatant) provided in this embodiment (before lyophilization and after lyophilization), as shown in the figure, doxorubicin hydrochloride-
  • the particle size of the silk fibroin nanoparticles is affected by the addition of salt ions and ultrasonic dispersion.
  • FIG. 49 it is an electromotive force diagram of the doxorubicin hydrochloride-silica nanoparticle (supernatant) provided in the present embodiment. As shown in the figure, the electromotive force of the doxorubicin hydrochloride-silk fibroin nanoparticle is affected by the addition of salt ions. influences.
  • FIG. 50 it is a graph of the doxorubicin hydrochloride encapsulation rate of the doxorubicin hydrochloride-silk nanometer microparticles provided in this embodiment.
  • the doxorubicin hydrochloride-silk nanoparticle loaded with doxorubicin is shown.
  • the efficiency is very high, close to 100%, and different salt ions also have an effect on the encapsulation rate of doxorubicin.
  • FIG. 51 it is a laser confocal image of the doxorubicin-silk microparticles (single standing) provided in this embodiment. As shown in the figure, the doxorubicin-silk fibroin microparticles loaded with the mold The distribution of the elements is relatively uniform and the intensity is high.

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Abstract

一种用聚乙二醇制备丝素纳微米球的方法：先将质量百分比1-30%的丝素蛋白溶液与质量百分比10-60%的聚乙二醇溶液放置在4-60℃温度下30min，然后将丝素蛋白溶液和聚乙二醇溶液混合，经孵育、离心洗涤制成丝素纳微米球。

Description

一种用聚乙二醇制备丝素纳微米球的方法及其在药物控释中的应用 技术领域
本发明属于生物医学或药剂学领域，具体涉及一种用聚乙二醇制备丝素纳微米球的方法及其在药物控释中的应用。
背景技术
丝素蛋白，是从蚕丝中提取的天然高分子纤维蛋白，含量约占蚕丝的70％～80％。丝素蛋白具有优良的理化性能，其无毒、无刺激性、生物相容性好，同时可被生物降解，很大程度上可满足生物材料的需求。丝素蛋白同时具有优异的加工性能，可以制备成膜，凝胶，微球，多孔支架等，可应用于药物载体。
中国专利申请第201310203722.2号公开了一种可注射丝素蛋白原位凝胶的制备方法，该方法通过将丝素蛋白水溶液和聚乙二醇或丙二醇溶液混合后凝固形成凝胶，由于凝胶颗粒的粒径较小不适用于负载药物，因此有必要开发一种简单可行的方法来制备丝素纳微米球以作为药物载体。
目前制备丝素纳微米球的方法主要有乳剂-有机溶剂蒸发/抽提法、相分离法、自组装法、超临界流体快速膨胀法、脂质体模板制备法、微流控法和喷雾干燥法等。乳化法、自组装法、脂质模板法都需要添加有机溶剂，而有机溶剂的残留会直接限制丝素纳微米球在临床上的应用。超临界流体快速膨胀法、微流控法和喷雾干燥法虽然比较简单，但装置和制备条件都比较复杂。相分离法的制备过程虽然相对简单并安全无毒，但操作流程比较繁琐。
另一方面，丝素蛋白作为药物载体具有明显的优势：一是其来源丰富、成本低廉；二是丝素蛋白作为天然高分子，其生物降解性好，生物安全性高；三是丝蛋白具有结晶方式多样化的特点，可以通过改变丝素蛋白的构象组成来达到控制药物释放速率的目的，使得药物在靶向区域释放，从而提高药物的疗效。因此将具有优异生物相容性的丝素蛋白制备成微球/颗粒，可使二者发挥协同作用，使丝素蛋白微球/颗粒不仅具有其他材料无法比拟的生物相容性，同时具有 较高的生物利用度和生物安全性。
目前制备载药丝素纳微米颗粒/微球的方法主要有乳剂-有机溶剂蒸发/抽提法、相分离法、自组装法、超临界流体快速膨胀法、脂质体模版制备法、微流控法和喷雾干燥法等，其中：
乳剂-有机溶剂蒸发/抽提法，Thanonchat将丝素蛋白溶液分散于乙酸乙酯的有机溶液中，然后加入含表面活性剂(如Span80)的油相中搅拌或超声处理，形成(W/O型)乳剂，再添加化学交联剂(戊二醛)进行固化。该方法是利用丝素蛋白上氨基易和其他化合物的醛基发生缩聚反应的特点，交联制得丝素纳微米颗粒，在乳剂中的丝素纳微米颗粒可以通过有机溶剂的蒸发或抽提得以分离。
相分离法，Wang将丝素蛋白溶液与PVA(聚乙烯醇)溶液直接共混，通过超声或搅拌处理后，将共混液放置在60℃的烘箱中烘干后再溶解，经离心洗涤后制得丝素纳微米颗粒，可通过改变丝素蛋白溶液的浓度和超声的功率来调控丝素纳微米颗粒的粒径。
自组装法，Cao将丝素蛋白溶液与乙醇溶液直接混合，在室温条件下搅拌2min后放置在(-5℃～-40℃)冰箱中冷冻24h，然后取出，室温解冻，离心洗涤便可制得丝素纳微米颗粒。该丝素纳微米颗粒的粒径可以通过丝素蛋白溶液的浓度及丝蛋白溶液与乙醇的体积比来调控。
超临界流体快速膨胀法，Wenk将载药的丝素蛋白溶液溶解在超流体中，短时间内通过频率发生振动装置将超流体从一定孔径的喷嘴中喷出，在高电压条件下，使丝素蛋白溶液快速形成晶核，并在短时间内促使晶核快速膨胀，通过液氮收集装置收集液滴并快速冷冻制得丝素载药微球。该丝素纳微米颗粒的粒径可以直接通过喷嘴的孔径来调控。
脂质体模版制备法，Wang将DOPC(二油酰磷脂酰胆碱)溶解在氯仿有机溶液中，在通氮气的条件下干燥成膜，加入一定体积的丝素蛋白溶液，经过去离子水稀释后，直接在液氮下冷冻15min，然后再在37℃的条件下解冻15min，接着将解冻的溶液倒入到烧杯中，快速搅拌，再将溶液冻干放置到4℃冰箱中保存。通过甲醇和氯化钠溶液的处理，使脂质体发生降解而诱导丝素蛋白的结构发生改变而形成丝素纳微米颗粒。
微流控法，Mitropoulos利用相分离的原理，通过使用微流控装置来隔离丝素蛋白和PVA(聚乙烯醇)溶液，然后通过控制溶液混合的流速来调控丝素纳微米颗粒的粒径，制备的微球粒径比较均一。
喷雾干燥法，Hino将丝素蛋白溶液通过雾化装置直接喷洒在热的空气中，随后在干燥的过程中制得丝素纳微米颗粒。
上述制备丝素纳微米颗粒的方法仍存在不足，对后期的药物装载及应用有很大的限制。其中，乳剂-有机溶剂蒸发/抽提法、自组装法、脂质体模版法都需要添加有机溶剂，而该有机溶剂的残留会影响小分子药物的稳定性和大分子蛋白药物的活性，从而直接限制载药丝素纳微米颗粒在临床上的应用。而超临界流体快速膨胀法、微流控法、喷雾干燥法虽然方法比较简单，但装置和制备条件都比较复杂，同时药物的载药率低，成本比较高，不利于后期的产业化应用。因此，如何研发一种药物控释效果好、无有机溶剂残留、操作简单、制备成本低、包埋效果好的丝素-载药纳微米颗粒的制备方法成为亟待解决的问题。
发明内容
鉴于上述丝素纳微米球及药物控释方面存在的缺陷，本发明的目的在于克服载药丝素纳微米颗粒制备过程中的有机溶剂残留、工序繁杂、耗时长、成本高、不利于产业化生产和临床应用等缺点，提供一种高效安全、快速简便、资源利用度好、生产成本低、生物相容性好、对产业化生产和临床应用有一定价值的制备方法。该方法不仅实现了通过对丝素颗粒的粒径大小和二级晶体结构的调控实现药物的可控释放，同时也解决了部分临床药物难以通过物理包埋方法直接固定在在丝素纳微米颗粒中的技术难题。
本发明提供的一种用聚乙二醇制备丝素纳微米球的方法，包括以下步骤：将质量百分比为1～30％的丝素蛋白溶液与质量百分比为10～60％的聚乙二醇溶液放置在4～60℃温度条件下30min，然后按一定比例将所述丝素蛋白溶液和聚乙二醇溶液混合均匀，孵育一定时间后离心洗涤，配制成丝素纳微米球悬浮液。
另一方面，本发明提供上述用聚乙二醇制备丝素纳微米球的方法的应用，应用于载药丝素纳微米颗粒制备及可控释放，包括下述步骤：将药物溶解在质 量百分比10～60％的聚乙二醇溶液中，或是将药物溶解在质量百分比1～30％的丝素蛋白溶液中，再将所述聚乙二醇溶液与所述丝素蛋白溶液混合均匀制得共混液，然后将所述共混液孵育一定时间后离心洗涤，制得用于药物控释的丝素-载药纳微米颗粒。
本发明进一步地，当所述药物为疏水性药物时，将所述疏水性药物溶解在聚乙二醇溶液中后再与丝素蛋白溶液混合制得所述共混液；除疏水性药物的其他药物，先将所述其他药物溶解在丝素蛋白溶液后再与聚乙二醇溶液混合制得所述共混液。
本发明更进一步地，所述疏水性药物包括姜黄素，所述其他药物包括亲水性药物阿霉素、多肽类药物奥曲肽、蛋白类药物牛血清白蛋白、大分子亲水性模型药物葡聚糖或具有荧光标记的CdTe量子点。
本发明进一步地，所述丝素蛋白溶液中所含有的丝素蛋白与所述药物的质量比范围为1000/1～1/10。
本发明进一步地，所述聚乙二醇的分子量范围为2000～20000。
本发明进一步地，所述聚乙二醇的浓度分子量为4000、6000时其质量百分比为30～60％，浓度分子量为10000时其质量百分比为20～50％，浓度分子量为20000时其质量百分比为20～40％。
本发明进一步地，所述丝素蛋白溶液的pH值在3.0～11.0。
本发明进一步地，所述丝素蛋白溶液的盐离子处理浓度从1M/L到0.01M/L。
本发明进一步地，所述丝素蛋白溶液和所述聚乙二醇溶液的体积比范围是5:1～1:10。
本发明进一步地，所述共混液在室温至60℃条件下孵育0.5～24h。
本发明进一步地，所述丝素蛋白溶液的稀释范围是质量百分比为5～20％。
本发明进一步地，所述丝素蛋白溶液的制备步骤如下：将已脱胶的熟丝浸润在9.3M的LiBr溶液中并置于60℃烘箱中4h，溶解后得到丝素蛋白溶液；将所述丝素蛋白溶液倒入透析袋中用去离子水透析3d；透析结束后将丝素蛋白溶液离心去除不溶物杂质，然后倒入透析袋中用分子量20000、15wt％的聚乙二醇溶液透析24h，获得浓缩的丝素蛋白溶液。
借由上述方案，本发明至少具有以下优点：
①本发明药用助剂聚乙二醇与丝素蛋白溶液共混来制备丝素纳微米球、或是选择药用助剂聚乙二醇与丝素蛋白溶液共混来制备丝素颗粒，上述制备过程不需要复杂的装置，操作流程简单易行、耗时短、成本低，而且不添加有机溶剂，所制备的丝素颗粒生物安全性高，可直接应用于临床，实现资源的优化利用；
②本发明方法直接混合孵育快速制备丝素颗粒，不需要复杂的实验装置，不需要干燥成膜再溶解，简化了操作流程，缩短了制备时间，有利于产业化生产；
③本发明方法在常温条件下就可制备丝素纳微米颗粒，不需要放置在冰箱中冷冻或烘箱中干燥，不仅节能，同时也降低了生产成本；
④本发明所制备的丝素颗粒的粒径可以受聚乙二醇相对分子量和浓度、以及丝素蛋白浓度的调控，为药物控释载药创造条件；
⑤本发明方法制备的丝素颗粒用来包埋缓释药物，选择范围很广，适用于亲疏水性小分子药物，多肽药物和蛋白药物等，其中疏水性中性药物可以通过与丝蛋白分子间特异的疏水区凭借疏水作用力结合并包埋在丝素颗粒中，从而提高疏水性中性药物的载药率。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例说明如后。
附图说明
图1是本发明实施例1-4提供的丝素纳微米颗粒的扫描电镜图；
图2是本发明实施例1-4不同分子量的PEG溶液与8wt％丝素蛋白溶液共混所制备的丝素纳微米球的粒径分布图；
图3是本发明实施例1-6丝素纳微米球的扫描电镜图；。
图4是本发明实施例2提供的丝素纳微米颗粒的粒径分布与聚乙二醇溶液的粘度图；
图5是本发明实施例3提供的丝素纳微米颗粒的扫描电镜图；
图6是本发明实施例3提供的丝素纳微米颗粒的粒径分布与聚乙二醇粘度图；
图7是本发明实施例4提供的丝素纳微米颗粒的扫描电镜图；
图8是本发明实施例4提供的丝素纳微米颗粒的粒径图；
图9是本发明实施例5提供的丝素纳微米颗粒的扫描电镜图；
图10是本发明实施例6提供的丝素纳微米颗粒的扫描电镜图；
图11是本发明实施例6提供的丝素纳微米颗粒的扫描电镜图；
图12是本发明实施例7提供的丝素纳微米颗粒的扫描电镜图；
图13是本发明实施例7提供的丝素纳微米颗粒的粒径图；
图14是本发明实施例8提供的丝素纳微米颗粒的扫描电镜图；
图15是本发明实施例8提供的单个的丝素微米球的表面形貌图；
图16是本发明实施例8提供的单个的丝素微米球的截面形貌图；
图17是本发明实施例8提供的丝素纳微米颗粒的粒径图；
图18是本发明实施例8提供的丝素纳微米颗粒的电动势图；
图19是本发明实施例8提供的丝素纳微米颗粒的产率图；
图20是本发明实施例9提供的丝素纳微米颗粒的红外光谱图；
图21是本发明实施例9提供的丝素纳微米颗粒及对照样的二级结构含量图；
图22是本发明实施例10提供的丝素纳微米颗粒的红外光谱图；
图23是本发明实施例10提供的丝素纳微米颗粒及对照样的二级结构含量图；
图24是本发明实施例11提供的丝素纳微米颗粒的红外光谱图；
图25是本发明实施例11提供的丝素纳微米颗粒及对照样的二级结构含量图；
图26是本发明实施例12提供的丝素纳米颗粒(上清液)的红外光谱图。
图27是本发明实施例12提供的丝素微米颗粒(二次静置)的红外光谱图。
图28是本发明实施例12提供的丝素纳米颗粒(一次静置)的红外光谱图。
图29是本发明实施例13提供的姜黄素-丝素纳微米颗粒的激光共聚焦图；
图30是本发明实施例14提供的TMR-葡聚糖-丝素纳微米颗粒的激光共聚焦图；
图31是本发明实施例15提供的CdTe-丝素纳微米颗粒的荧光显微镜图；
图32是本发明实施例16提供的姜黄素-丝素纳微米颗粒的扫描电镜图；
图33是本发明实施例16提供的姜黄素-丝素纳微米颗粒的激光共聚焦图；
图34是本发明实施例17提供的盐酸阿霉素-丝素纳微米颗粒的扫描电镜图；
图35是本发明实施例17提供的盐酸阿霉素-丝素纳微米颗粒的激光共聚焦图；
图36是本发明实施例18提供的姜黄素-丝素纳微米颗粒的累积释放率图；
图37是本发明实施例19提供的TMR-葡聚糖-丝素纳微米颗粒的累积释放率图；
图38是本发明实施例20提供的CdTe-丝素纳微米颗粒的累积释放率图；
图39是本发明实施例21提供的姜黄素-丝素纳微米颗粒的累积释放率图；
图40是本发明实施例22提供的盐酸阿霉素-丝素纳微米颗粒的累积释放率图；
图41是本发明实施例23提供的盐酸阿霉素-丝素纳微米颗粒的累积释放率图；
图42是本发明实施例24提供的奥曲肽-丝素纳微米颗粒扫描电镜图；
图43是本发明实施例25提供的TMR-牛血清白蛋白-丝素纳微米颗粒扫描电镜图；
图44是本发明实施例25提供的TMR-牛血清白蛋白-丝素纳微米颗粒激光共聚焦图；
图45是本发明实施例26提供的TMR-牛血清白蛋白-丝素纳微米颗粒累积释放图；
图46是本发明实施例27提供的盐酸阿霉素-丝素纳米颗粒(上清液)的扫描电镜图；
图47是本发明实施例27提供的盐酸阿霉素-丝素微米颗粒(一次静置)的扫描电镜图；
图48是本发明实施例27提供的盐酸阿霉素-丝素纳米颗粒(上清液)的粒径分布图；
图49是本发明实施例27提供的盐酸阿霉素-丝素纳米颗粒(上清液)的电动势图；
图50是本发明实施例27提供的盐酸阿霉素-丝素纳微米颗粒的盐酸阿霉素的封装率图；
图51是本发明实施例27提供的盐酸阿霉素-丝素微米颗粒(一次静置)的激光共聚焦图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整的描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，不用来限制本发明的范围。基于本发明的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明所提供的用于药物控释的丝素-载药纳微米颗粒的制备方法，为了能够保持药物传递的稳定性和分子活性，需要考虑在制备过程中尽量避免使用有机溶剂和剧烈的成颗粒成球条件。与现有相分离法制备丝素纳微米颗粒比较，相分离法制得微球的粒径和形貌可以通过改变PVA(聚乙烯醇)分子量、PVA与丝蛋白的重量比来进行调控，相分离法制备过程虽然相对简单并安全无毒，但需要对丝素蛋白和PVA共混液进行成膜、溶解、洗涤等操作，流程比较繁琐。
本发明方法与现有相分离方法在原理、操作流程、药物适用范围上均有不同。原理不同主要体现在：相分离法是采用的是将PVA溶液和丝蛋白溶液混合，通过超声或搅拌的方式将丝素蛋白分子充分分散在PVA溶液中，然后再将分散后的溶液放置在烘箱中烘干成膜，溶解，洗涤制得。而本发明采用乳化聚合法，直接将丝蛋白溶液加入聚乙二醇溶液中，利用聚乙二醇的亲水性，能够快速吸收丝蛋白表面大的水分子，促使丝蛋白的疏水区相互靠近，诱发分子间聚集自组装成微球。操作流程不同体现在：相分离法需要将共混液放置在60℃烘箱中烘干成膜，然后再溶解、洗涤，而本发明只需要室温孵育即可。药物适用范围不同体现在：相分离法制备的丝素颗粒对于疏水中性药物的载药率低，而本方法的聚乙二醇是两亲性药用助剂，可以将疏水性中性药物溶解在聚乙二醇溶液 中，然后与丝蛋白共混孵育成球，丝素颗粒的载药率较高。
本发明丝素-载药纳微米颗粒在药物控释方面满足定量匀速的向外释放药物、使血药浓度保持恒定的效果。选择小分子的药物，如姜黄素、盐酸阿霉素，大分子药物，如TMR-葡聚糖，多肽蛋白类药物，如奥曲肽、TMR-牛血清白蛋白等通过物理包埋的方法装载到丝素纳微米颗粒上，为后期的丝素纳微米颗粒作为药物载体的临床应用提供了可能性。
【实施例1】
1-1.丝素蛋白溶液的制备
称取25g已脱胶的熟丝浸润在100ml的9.3M的LiBr溶液中，并置于60℃烘箱中4h，每隔1h搅拌一次直至完全溶解，得到20％(W/V)的丝素蛋白溶液。将该丝素蛋白溶液倒入截留分子量为3500的Slide-a-lyzer透析袋中，将透析袋放入盛有去离子水的透析槽中透析3天，期间换水10次。透析结束后将丝素蛋白溶液在离心机中以9000rpm/min、20min、4℃的条件离心，然后出去不容物杂质。丝素蛋白溶液浓度的测定是先称取一定质量的丝素蛋白溶液，放在60℃烘箱中烘干，之后称重，两者之比为浓度值(质量百分比，wt％)。通常所获得的丝素蛋白溶液的浓度约为7wt％。丝素蛋白溶液的浓缩过程是将制备好的丝素蛋白溶液100ml移入到同样的Slide-a-lyzer透析袋中，再将透析袋放置在15wt％、分子量20000、800ml的聚乙二醇溶液中连续透析24h，获得约30wt％的浓缩蛋白溶液，放置在4℃冰箱中保存。
1-2.聚乙二醇-丝素蛋白共混液的制备
将1-1.中30wt％丝素蛋白溶液稀释至1％～5wt％；
将不同分子量的聚乙二醇溶液配置成不同的浓度，分子量低于1000的聚乙二醇的浓度为40-100wt％；聚乙二醇分子量1000-6000的浓度为10-60wt％；分子量10000的浓度为10-50wt％；分子量20000的浓度为10-40wt％；
分别取等体积的1-5wt％丝素蛋白溶液至等体积的不同分子量及浓度的聚乙二醇溶液中，用吸枪上下来回轻轻推打，使共混液混合均匀；
将聚乙二醇-丝素蛋白共混液放置在室温条件下孵育30min；
观察孵育后共混溶液的状态，并记录。
1-3.聚乙二醇-丝素蛋白共混液的制备
将1-1.中的30wt％丝素蛋白溶液稀释至6-30wt％；
将不同分子量的聚乙二醇配置成不同的浓度，聚乙二醇分子量2000-6000的浓度为10-60wt％；分子量10000的浓度为10-50wt％；分子量20000的浓度为10-40wt％；
分别取等体积的6-30wt％丝素蛋白溶液至等体积的不同分子量及浓度的聚乙二醇溶液中，用吸枪上下来回轻轻推打，使共混液混合均匀；
将聚乙二醇-丝素蛋白共混液放置在室温条件下孵育30min；
观察孵育后共混溶液的状态，并记录。
通过表1总结1-1.与1-2.中的实验结果，从记录的数据可以看出，分子量高的聚乙二醇容易与丝素蛋白溶液共混形成颗粒；聚乙二醇的分子量及浓度和丝素蛋白溶液的浓度是形成颗粒的直接影响因素。
表1丝素蛋白与聚乙二醇溶液共混30min后的状态表
Figure PCTCN2015097464-appb-000001
Figure PCTCN2015097464-appb-000002
Figure PCTCN2015097464-appb-000003
1-4.丝素纳微米球的制备
分别配置分子量2000的聚乙二醇溶液的浓度为40wt％，4000的聚乙二醇溶液的浓度为40wt％，分子量6000的聚乙二醇溶液的浓度为40wt％，分子量10000的聚乙二醇溶液的浓度为40wt％，分子量20000的聚乙二醇溶液的浓度为40wt％；
将1-1.中的30wt％丝素蛋白溶液稀释至8wt％，取该溶液至等体积的上述浓度及分子量的聚乙二醇溶液中；
将聚乙二醇-丝素蛋白共混液放置在室温条件下孵育12h；
孵育结束后，将离心管放置在离心机中离心，条件：室温，10min，12000rpm/min；
加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
用去离子水配置丝素纳微米球的悬浮液，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图1，从图中可以看出，分子量为10000、浓度为40wt％的聚乙二醇溶液与8wt％的丝素蛋白溶液共混后得到的丝素微纳米颗粒的粒径较大。
1-5.丝素纳微米球粒径的检测
分别取1-4.中的悬浮丝素纳微米球溶液，移入到一次性比色皿中；
用激光粒度仪测试各组丝素纳微米球样品的粒径，每组测三次，计算平均值。
参见附图2所示，它是丝素纳微米球的粒径分布图，从图中可以看出，分 子量为10000、浓度为40wt％的聚乙二醇溶液与8wt％的丝素蛋白溶液共混后得到的丝素纳微米球颗粒粒径较大。
1-6.丝素纳微米球的制备
1、配置分子量4000聚乙二醇30％(表示聚乙二醇分子量4000，浓度30wt％)；
2、按照实施例1方法配置5％浓度的丝素蛋白溶液；
3、按照体积2/1混合丝素蛋白和聚乙二醇溶液，分别在25℃，45℃，60℃条件下孵育24h；
4、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
5、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
6、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图3，它是本实施例提供的丝素纳微米颗粒的扫描电镜图，分别在25℃，45℃，60℃条件下孵育。如图可知，丝素纳微米颗粒的形貌受孵育温度的影响。
【实施例2】
1、选择实施例1-4中的不同分子量相同浓度的聚乙二醇；
2、用流变仪分别测试上述聚乙二醇的粘度；
3、按照实施例1方法配置8wt％浓度的丝素蛋白溶液；
4、按照体积1/1共混丝素蛋白和聚乙二醇溶液，并轻轻振荡混合均匀后在室温条件下孵育12h；
5、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
6、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
7、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图4，它是本实施例提供的丝素纳微米颗粒的粒径与聚乙二醇溶液的 粘度图，正方形图标表示粒径，三角形图标表示粘度。如图可知，丝素纳微米颗粒的粒径受聚乙二醇溶液粘度的影响。
【实施例3】
1、配置分子量10000聚乙二醇50％(表示聚乙二醇分子量10000，浓度50wt％)分别稀释至20％，30％；
2、用流变仪分别测试以上不同浓度聚乙二醇的粘度；
3、按照实施例1方法配置8％浓度的丝素蛋白溶液；
4、按照体积1/1混合丝素蛋白和聚乙二醇溶液，在室温条件下孵育24h；
5、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
6、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
7、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图5，它是本实施例提供的丝素纳微米颗粒的扫描电镜图。如图可知丝素纳微米颗粒的形貌受聚乙二醇溶液浓度的影响。
参见附图6，它是本实施例提供的丝素纳微米颗粒的粒径分布与聚乙二醇粘度图，正方形图标表示粒径，圆形图标表示粘度。如图可知，丝素纳微米颗粒的粒径受聚乙二醇溶液粘度的影响。
【实施例4】
1、配置分子量10000的聚乙二醇浓度50wt％；
2、按照实施例1方法分别配置1，2％，5％，8％，12％，20wt％浓度的丝素蛋白溶液
3、按照体积1/1混合丝素蛋白和聚乙二醇溶液，在室温条件下孵育2h；
4、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
5、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
6、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图7，它是本实施例提供的丝素纳微米颗粒的扫描电镜图。如图可知，丝素纳微米颗粒的形貌受丝素蛋白溶液浓度的影响。
参见附图8，它是本实施例提供的丝素纳微米颗粒的粒径图。如图可知，丝素纳微米颗粒的粒径受丝素蛋白溶液浓度的影响。
【实施例5】
1、配置分子量10000的聚乙二醇浓度50wt％；
2、按照实施例1方法配置8wt％浓度的丝素蛋白溶液；
3、按照体积10/10/1(SF/聚乙二醇/甲醇溶液)混合丝素蛋白和聚乙二醇溶液，在室温条件下孵育2h；
4、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
5、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
6、悬浮丝素纳微球，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图9，它是本实施例提供的丝素微米球的扫描电镜图。如图可知，丝素微米球的形貌受添加甲醇溶液的影响。
【实施例6】
1、配置分子量4000聚乙二醇60％(表示聚乙二醇分子量4000，浓度60wt％)；
2、分别配置1，5％浓度的丝素蛋白溶液；
3、分别调节上述丝素蛋白溶液的pH值至3.6，7.0；
4、按照体积1/1混合丝素蛋白和聚乙二醇溶液，室温条件下孵育24h；
5、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
6、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
7、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图10、11，是本实施例提供的丝素纳微米颗粒的扫描电镜图，pH值 分别为3.6和7.0。如图可知，丝素纳微米颗粒的形貌受丝素蛋白溶液pH的影响。
【实施例7】
1、配置分子量10000聚乙二醇50％(表示聚乙二醇分子量10000，浓度50wt％)；
2、分别配置0.2，1，5，10，15％浓度的丝素蛋白溶液；
3、分别调节上述丝素蛋白溶液的pH值至3.6，7.0，10.0；
4、按照体积1/1混合丝素蛋白和聚乙二醇溶液，室温条件下孵育24h；
5、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
6、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
7、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图12，它是本实施例提供的丝素纳微米颗粒的扫描电镜图，pH值为3.6，7.0，10.0的0.2，1，5，10，15wt％的SF与50wt％分子量10000聚乙二醇溶液。如图可知，丝素纳微米颗粒的形貌受丝素蛋白溶液pH的影响。
参见附图13，它是本实施例提供的丝素纳微米颗粒的粒径图，pH值为3.6，7.0，10.0的0.2，1，5，10，15wt％SF与50wt％分子量10000PEG溶液共混。如图可知，丝素纳微米颗粒的粒径受丝素蛋白溶液的pH值和丝素蛋白溶液浓度的影响。
【实施例8】
1、配置分子量10000聚乙二醇20％(表示聚乙二醇分子量10000，浓度20wt％)；
2、分别配置浓度为1mol/L的氯化钠，氯化钾，氯化镁溶液；
3、用去离子水溶解冻干粉，终浓度为30％；
4、加入等体积溶液的上述盐离子溶液，使得丝素蛋白溶液的终浓度为15％，盐离子的终浓度为0.5M；
5、按照体积1/1混合丝素蛋白盐离子溶液和聚乙二醇溶液，轻轻振荡混合均匀；
6、快速加入无水乙醇溶液(V上述共混液/V乙醇＝1/4)，混合均匀后，快速加入培养皿中，并移至60℃烘箱中；
7、干燥后取出培养皿，加入去离子水悬浮；
8、用超声波细胞粉碎机超声2min，振幅30％；
9、离心：室温20min，12000rpm；
10、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
11、悬浮丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图14，它是本实施例提供的丝素纳微米颗粒的扫描电镜图，第一行电镜图是静置之前，第二行电镜图是静置后上清液，第三行电镜图是二次静置，第四行电镜图是一次静置，分别在无离子，钠离子，钾离子，镁离子处理条件下。如图可知，丝素纳微米颗粒的形貌受添加盐离子的影响。
参见附图15，它是本实例提供的单个的丝素微米球的表面形貌图，如图可知，丝素纳微米球的表面形貌受添加离子的影响。
参见附图16，它是本实例提供的单个的丝素微米球的截面形貌图，如图可知，丝素纳微米球的截面形貌受添加离子的影响。
参见附图17，它是本实例提供的丝素纳微米颗粒的粒径图，如图可知，丝素纳米颗粒(上清液)的粒径平均值为350nm左右，丝素微米颗粒(二次静置)的粒径平均值在10μm左右，丝素微米颗粒(一次静置)的粒径平均在40μm左右。
参见附图18，它是本实例提供的丝素纳米颗粒的电动势图，如图可知，丝素纳米颗粒的电动势受不同盐离子处理的影响。
参见附图19，它是本实例提供的丝素纳微米颗粒的产率图，如图可知，丝素纳微米颗粒的产率受到不同盐离子处理的影响。
【实施例9】
1、选择实施例4，5中的冻干后干燥的丝素纳微米颗粒，分别称取0.1mg；
2、取少量的溴化钾粉末混合均匀，并用研杵研磨均匀，然后用压片机压片；
3、用傅里叶红外光谱仪测试：测试条件表面全反射，波数范围400-4000cm-1，以此测试各样品的红外结构；
4、选择8％丝素蛋白溶液制备的丝素纳微米颗粒(有无添加甲醇)，纯丝素蛋白冻干粉(未加聚乙二醇和甲醇作为对照样)，然后用Peakfit软件对各样品的二级结构含量进行模拟分析。
参见附图20，它是本实施例提供的丝素纳微米颗粒的红外光谱图，丝素纳微米颗粒的红外光谱图(5％，8％，12％，20％(wt)SF与50％(wt)分子量10000聚乙二醇溶液共混，8％(wt)与50％(wt)分子量10000聚乙二醇，甲醇溶液共混，对照样。如图可知，丝素微米球的二级结构受添加聚乙二醇和甲醇的影响。
参见附图21，它是本实施例提供的丝素纳微米颗粒及对照样的二级结构含量图。如图可知，丝素纳微米颗粒的二级结构含量受到添加聚乙二醇和甲醇的影响。
【实施例10】
1、选择实施例1-6中的冻干后干燥的丝素纳微米颗粒，分别称取0.1mg；
2、取少量的溴化钾粉末混合均匀，并用研杵研磨均匀，然后用压片机压片；
3、用傅里叶红外光谱仪测试：测试条件表面全反射，波数范围400-4000cm-1，以此测试各样品的红外结构；
4、选择上述丝素纳微米颗粒的红外光谱图，然后利用Peakfit软件对各样品的二级结构含量进行模拟分析。
参见附图22，它是本实施例提供的丝素纳微米颗粒的红外光谱图。如图可知，丝素微米球的二级结构受孵育温度的影响。
参见附图23，它是本实施例提供的丝素纳微米颗粒及对照样的二级结构含量图。如图可知，丝素纳微米颗粒的二级结构含量受到孵育温度的影响。
【实施例11】
1、选择实施例8中的冻干后干燥的丝素纳微米颗粒，分别称取0.1mg；
2、取少量的溴化钾粉末混合均匀，并用研杵研磨均匀，然后用压片机压片；
3、用傅里叶红外光谱仪测试：测试条件表面全反射，波数范围400-4000cm-1，以此测试各样品的红外结构；
4、选择上述丝素纳微米颗粒的红外光谱图，然后利用Peakfit软件对各样品的二级结构含量进行模拟分析。
参见附图24，它是本实施例提供的丝素纳微米颗粒的红外光谱图。如图可知，丝素纳微米颗粒的二级结构受丝素蛋白溶液pH值的影响。
参见附图25，它是本实施例提供的丝素纳微米颗粒及对照样的二级结构含量图。如图可知，丝素纳微米颗粒的二级结构含量受到丝素蛋白溶液pH值的影响。
【实施例12】
1、选择实施例9中的冻干后干燥的丝素纳微米颗粒，分别称取0.1mg；
2、取少量的溴化钾粉末混合均匀，并用研杵研磨均匀，然后用压片机压片；
3、用傅里叶红外光谱仪测试：测试条件表面全反射，波数范围400-4000cm-1，以此测试各样品的红外结构。
参见附图26，它是本实施例提供的丝素纳米颗粒(上清液)的红外光谱图。如图可知，丝素纳微米颗粒的二级结构受盐离子处理的影响。
参见附图27，它是本实施例提供的丝素微米颗粒(二次静置)的红外光谱图。如图可知，丝素纳微米颗粒的二级结构受盐离子处理的影响。
参见附图28，它是本实施例提供的丝素微米颗粒(一次静置)的红外光谱图。如图可知，丝素纳微米颗粒的二级结构受盐离子处理的影响。
【实施例13】
姜黄素-丝素纳微米颗粒的制备
1、分别称取0.5mg，0.9mg的姜黄素；
2、分别取1ml的50wt％分子量10000的聚乙二醇溶液溶解姜黄素；
3、配置5％，9wt％的丝素蛋白溶液各1ml；
4、将丝素蛋白溶液加入到姜黄素-聚乙二醇溶液中，用吸枪上下来回轻轻推打，使共混液混合均匀；
5、将姜黄素-聚乙二醇-丝素蛋白共混液放置在室温条件下孵育2h；
6、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
7、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
8、悬浮姜黄素-丝素纳微米颗粒，经冷冻真空干燥后保存；
9、分别称取1mg 5％，9wt％的丝蛋白溶液制备的姜黄素-丝素纳微米颗粒，加入1ml甲醇溶液，每组各称三个样品；
10、将离心管放在振荡器上振荡10min，然后放置在静音混合器中旋转2h；
11、取出离心管，离心，条件是室温，10min，12000rpm/min；
12、取上清液，用酶标仪在425nm处测姜黄素的吸光度值，对照姜黄素的吸光度-浓度标准曲线计算姜黄素在丝素纳微米颗粒中的含量，计算出载药率；
13、5％丝蛋白制备的姜黄素-丝素纳微米颗粒的载药率为0.27％±0.07％，9％丝蛋白制备的姜黄素-丝素纳微米颗粒的载药率为0.51±0.15％。
参见附图29，它是本实施例提供的姜黄素-丝素纳微米颗粒的激光共聚焦图。如图可知，姜黄素在丝素纳微米颗粒中分布比较均匀。
【实施例14】
TMR-葡聚糖-丝素纳微米颗粒的制备
1、分别用水溶解1.0mg/ml，1.8mg/ml的TMR-葡聚糖溶液；
2、分别配置10％，18wt％的丝素蛋白溶液；
3、将上述溶液等体积共混，丝素蛋白与TMR-葡聚糖的质量比为100/1；
4、配置浓度为50wt％分子量为10000的聚乙二醇溶液；
5、将丝素蛋白和TMR葡聚糖共混溶液按等体积比再与聚乙二醇溶液共混，用吸枪上下来回轻轻推打，使共混液混合均匀；
6、将TMR-葡聚糖-聚乙二醇-丝素蛋白共混液放置在室温条件下孵育2h；
7、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min， 12000rpm/min；
8、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
9、悬浮TMR-葡聚糖-丝素纳微米颗粒，经冷冻真空干燥后保存；
10、分别称取1mg 5％，9wt％的丝蛋白溶液制备的TMR-葡聚糖-丝素纳微米颗粒，加入1ml的溴化锂溶液，每组各称三个样品；
11、将离心管放在振荡器上振荡10min，然后放置在静音混合器中旋转2h，避光操作；
12、取出离心管，离心，条件是室温，10min，12000rpm/min；
13、取上清液，用酶标仪在激发波长550nm，发射波长590nm处测TMR-葡聚糖的荧光度值，对照TMR-葡聚糖的荧光度-浓度标准曲线计算TMR-葡聚糖在丝素纳微米颗粒中的含量，计算出载药率；
14、5％丝蛋白制备的TMR-葡聚糖-丝素纳微米颗粒的载药率为0.13％，9％丝蛋白制备的TMR-葡聚糖-丝素纳微米颗粒的载药率为0.26％。
参见附图30，它是本实施例提供的TMR-葡聚糖-丝素纳微米颗粒的激光共聚焦图。如图可知，TMR-葡聚糖主要分布在丝素纳微米颗粒的表面。
【实施例15】
CdTe-丝素纳微米颗粒的制备
1、用去离子水配置丝素蛋白与CdTe量子点浓度比为0.017nmol CdTe/1mg SF，丝素蛋白溶液的终浓度为5，8，20wt％；
2、配置浓度为50wt％分子量为10000的聚乙二醇溶液；
3、将丝素蛋白和CdTe共混溶液再与聚乙二醇溶液按等体积比共混，用吸枪上下来回轻轻推打，使共混液混合均匀；
4、将CdTe-丝素蛋白-聚乙二醇共混液放置在室温条件下孵育2h；
5、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
6、加入去离子水洗涤，去除上清液，该上清液保留测试用，反复离心，洗涤操作三次；
7、取上清液，用酶标仪在激发波长380nm，发射波长596nm处测CdTe量子点的荧光度值，对照CdTe的荧光度-浓度标准曲线计算CdTe在上清液中的含量，计算出封装率；
8、悬浮CdTe-丝素纳微米颗粒，经冷冻真空干燥后保存；
9、5％丝蛋白制备的CdTe-丝素纳微米颗粒的封装率为98.47％±0.17％，8％丝蛋白制备的CdTe-丝素纳微米颗粒的封装率为98.89％±0.23％，20％丝蛋白制备的CdTe-丝素纳微米颗粒的封装率为96.82％±0.27％。
参见附图31，它是本实施例提供的CdTe-丝素纳微米颗粒的荧光显微镜图。如图可知，CdTe均匀分布在丝素纳微米颗粒上。
【实施例16】
姜黄素-丝素纳微米颗粒的制备
1、称取1mg的姜黄素；
2、取1ml的50wt％分子量10000的聚乙二醇溶液溶解姜黄素；
3、配置浓度为5wt％的丝素蛋白溶液并分别调节pH值至3.6和7.0；
4、分别将丝素蛋白溶液加入到姜黄素-聚乙二醇溶液中，用吸枪上下来回轻轻推打，使共混液混合均匀；
5、将姜黄素-聚乙二醇-丝素蛋白共混液放置在室温条件下孵育24h；
6、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
7、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
8、悬浮姜黄素-丝素纳微米颗粒，经冷冻真空干燥后保存；
9、分别称取1mg 5％pH3.6和7.0的丝素蛋白溶液制备的姜黄素-丝素纳微米颗粒，加入1ml甲醇溶液，每组各称三个样品，；
10、将离心管放在振荡器上振荡10min，然后放置在静音混合器中旋转2h；
11、取出离心管，离心，条件是室温，10min，12000rpm/min；
12、取上清液，用酶标仪在425nm处测姜黄素的吸光度值，对照姜黄素的吸光度-浓度标准曲线计算姜黄素在丝素纳微米颗粒中的含量，计算出载药率；
13、5％pH 3.6丝素蛋白制备的姜黄素-丝素纳微米颗粒的载药率为1.18％±0.08％，5％pH 7.0丝素蛋白制备的姜黄素-丝素纳微米颗粒的载药率为0.64±0.06％。
参见附图32，它是本实施例提供的姜黄素-丝素纳微米颗粒的扫描电镜图。如图可知，姜黄素-丝素纳微米颗粒的形貌收到丝素蛋白溶液的pH值的影响。
参见附图33，它是本实施例提供的姜黄素-丝素纳微米颗粒的激光共聚焦图。如图可知，姜黄素在丝素纳微米颗粒中分布比较均匀。
【实施例17】
盐酸阿霉素-丝素纳微米颗粒的制备
1、称取1mg的盐酸阿霉素；
2、分别配置浓度为5％pH 3.6，pH7.0的丝素蛋白溶液；
3、用上述的丝素蛋白溶液溶解盐酸阿霉素颗粒；
4、配置浓度为50wt％分子量为10000的聚乙二醇溶液；
5、分别将含有盐酸阿霉素的丝素蛋白溶液加入到聚乙二醇溶液中，用吸枪上下来回轻轻推打，使共混液混合均匀；
6、将盐酸阿霉素-丝素蛋白-聚乙二醇共混液放置在室温条件下孵育24h；
7、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
8、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
9、悬浮盐酸阿霉素-丝素纳微米颗粒，经冷冻真空干燥后保存；
10、分别称取1mg 5％pH3.6和7.0的丝素蛋白溶液制备的盐酸阿霉素-丝素纳微米颗粒，加入1ml溴化锂溶液，每组各称三个样品；
11、将离心管放在振荡器上振荡10min，然后放置在静音混合器中旋转2h；
12、取出离心管，离心，条件是室温，10min，12000rpm/min；
13、取上清液，用酶标仪在600nm处测盐酸阿霉素的吸光度值，对照盐酸阿霉素的吸光度-浓度标准曲线计算盐酸阿霉素在丝素纳微米颗粒中的含量，计算出载药率；
14、5％pH 3.6丝素蛋白制备的盐酸阿霉素-丝素纳微米颗粒的载药率为0.80％±0.06％，5％pH 7.0丝素蛋白制备的盐酸阿霉素-丝素纳微米颗粒的载药率为1.90±0.05％。
参见附图34，它是本实施例提供的盐酸阿霉素-丝素纳微米颗粒的扫描电镜图图。如图可知，盐酸阿霉素-丝素纳微米颗粒的形貌收到丝素蛋白溶液的pH值的影响。
参见附图35，它是本实施例提供的盐酸阿霉素-丝素纳微米颗粒的激光共聚焦图。如图可知，盐酸阿霉素在丝素纳微米颗粒中分布比较均匀。
【实施例18】
1、称取10mg实施例13中的姜黄素-丝素纳微米颗粒，加入离心管中；
2、配置摩尔浓度为0.01mol/L，pH＝7.4的磷酸盐缓冲液(PBS)然后再加入5％的吐温80和3％的甲醇配制成释放液；
3、每个样品中分别加入1ml的上述释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在425nm处测姜黄素的吸光度值，对照姜黄素的吸光度-浓度标准曲线计算姜黄素在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图36，它是本实施例提供的姜黄素-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的丝素-姜黄素微球中的姜黄素的释放速率有明显差异。
【实施例19】
1、称取10mg实施例14中的TMR-葡聚糖-丝素纳微米颗粒，加入离心管中；
2、配置摩尔浓度为0.01mol/L，pH＝7.4的磷酸盐缓冲液(PBS)；
3、每个样品中分别加入1ml的PBS释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在激发波长为555nm，发射波长为590nm处测TMR-葡聚糖的荧光度值，对照TMR-葡聚糖的荧光度-浓度标准曲线计算TMR-葡聚糖在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图37，它是本实施例提供的TMR-葡聚糖-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的TMR-葡聚糖-丝素纳微米球中的TMR-葡聚糖的释放速率有明显差异。
【实施例20】
1、称取10mg实施例15中的CdTe-丝素纳微米颗粒，加入离心管中；
2、每个样品中分别加入1ml的去离子水；
3、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
4、离心，转速12000rpm，10min，室温条件；
5、取上清液，用酶标仪在激发波长为380nm，发射波长为596nm处测CdTe量子点的荧光度值，对照CdTe量子点的荧光度-浓度标准曲线计算CdTe量子点在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图38，它是本实施例提供的CdTe-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的CdTe-丝素纳微米颗粒中的CdTe量子点的释放速率有明显差异。
【实施例21】
1、称取10mg实施例16中的姜黄素-丝素纳微米颗粒，加入离心管中；
2、配置摩尔浓度为0.01mol/L，pH＝7.4的磷酸盐缓冲液(PBS)然后加入5％的吐温80和3％的甲醇配制成释放液；
3、每个样品中分别加入1ml的上述的释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在425nm处测姜黄素的吸光度值，对照姜黄素的吸 光度-浓度标准曲线计算姜黄素在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图39，它是本实施例提供的姜黄素-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的丝素-姜黄素微球中的姜黄素的释放速率有明显差异。
【实施例22】
1、称取10mg实施例17中的盐酸阿霉素-丝素纳微米颗粒，加入离心管中；
2、配置摩尔浓度为0.01mol/L,pH＝4.0的磷酸盐缓冲液(PBS)；
3、每个样品中分别加入1ml的PBS释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在480nm处测盐酸阿霉素的吸光度值，对照盐酸阿霉素的吸光度-浓度标准曲线计算盐酸阿霉素在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图40，它是本实施例提供的盐酸阿霉素-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的丝素-盐酸阿霉素微球中的阿霉素的释放速率有明显差异。
【实施例23】
1、称取10mg实施例17中的盐酸阿霉素-丝素纳微米颗粒，加入离心管中；
2、配置摩尔浓度为0.01mol/L，pH＝7.4的磷酸盐缓冲液(PBS)；
3、每个样品中分别加入1ml的PBS释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在480nm处测盐酸阿霉素的吸光度值，对照盐酸阿霉素的吸光度-浓度标准曲线计算盐酸阿霉素在丝素纳微米颗粒中释放量，并计算出累积释放率。
参见附图41，它是本实施例提供的盐酸阿霉素-丝素纳微米颗粒的累积释放率图。如图可知，不同浓度制备的盐酸阿霉素-丝素纳微米球中的盐酸阿霉素的释放速率有明显差异。
【实施例24】
奥曲肽-丝素纳微米颗粒的制备
1、用去离子水配置奥曲肽溶液，浓度为20mg/ml；
2、用去离子水溶解丝素冻干粉，浓度为100mg/ml；
3、并各取上述2ml的溶液共混，丝素蛋白与奥曲肽的质量比为5/1；
4、取分子量10.0K浓度50％的聚乙二醇，体积4ml；
5、将上述三种溶液共混，室温孵育24h；
6、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
7、保留上清液，反复离心洗涤三次，悬浮冷冻干燥；
参见附图42，它是本实施例提供的奥曲肽-丝素纳微米颗粒扫描电镜图。
【实施例25】
TMR-牛血清白蛋白-丝素纳微米颗粒的制备
1、用去离子水配置TMR-牛血清白蛋白溶液，浓度为0.5mg/ml，1ml；
2、用去离子水溶解丝素冻干粉，浓度为60mg/ml，5ml；
3、将上述溶液共混，丝素蛋白与TMR-牛血清白蛋白的质量比为600/1；
4、取分子量10.0K浓度50％的聚乙二醇，体积6ml；
5、将上述三种溶液共混，室温孵育24h；
6、孵育结束后，将离心管放置在离心机中离心，条件是室温，10min，12000rpm/min；
7、保留上清液，反复离心洗涤三次，悬浮冷冻干燥；
8、测试上清液中TMR-牛血清白蛋白的含量，利用酶标仪在激发波长为555nm，发射波长为590nm处测试TMR-牛血清白蛋白的荧光度然后绘制荧光度 -浓度标准曲线，然后再测试样品的荧光度，进而计算出上清液中TMR-牛血清白蛋白的含量，经计算，牛血清白蛋白的封装率为40.4±4.2％。
参见附图43，它是本实施例提供的TMR-牛血清白蛋白-丝素纳微米颗粒扫描电镜图。
参见附图44，它是本实施例提供的TMR-牛血清白蛋白-丝素纳微米颗粒激光共聚焦图。
【实施例26】
1、取实施例25的TMR-牛血清白蛋白-丝素纳微米颗粒，称取5mg；
2、配置摩尔浓度为0.01mol/L，pH＝7.4的磷酸盐缓冲液(PBS)；
3、每个样品中分别加入1ml的PBS释放液；
4、放置在37℃的烘箱中振荡释放，并在指定的时间内取出；
5、离心，转速12000rpm，10min，室温条件；
6、取上清液，用酶标仪在激发波长555nm，发射波长590nm处测TMR-牛血清白蛋白的荧光度值，对照TMR-牛血清白蛋白的荧光度-浓度标准曲线计算TMR-牛血清白蛋白在丝素纳微米颗粒中释放量，并计算出累积释放率；
参见附图45可知TMR-牛血清白蛋白-丝素纳微米颗粒中的TMR-牛血清白蛋白有明显的缓释效果。
【实施例27】
1、配置分子量10000聚乙二醇20％(表示聚乙二醇分子量10000，浓度20wt％)；
2、分别配置浓度为0.2mol/L的氯化钠，氯化钾，氯化镁溶液；
3、用去离子水溶解冻干粉，终浓度为30％；
4、称取24mg的盐酸阿霉素，并用4ml的去离子水室温溶解，得到浓度为6mg/ml的盐酸阿霉素溶液；
5、将上述摩尔浓度的盐离子按等体积比加入到6mg/ml的盐酸阿霉素溶液中，得到终浓度为3mg/ml的盐酸阿霉素溶液；
6、再用上述盐离子溶液溶解的盐酸阿霉素按等体积比稀释上述的丝素蛋白溶液，最终得到15％的丝素蛋白溶液，阿霉素的浓度为1.5mg/ml，盐离子浓度为0.1mol/L；(阿霉素与丝素蛋白质量比为1/100)
7、按照体积1/1混合盐离子处理的阿霉素-丝素溶液和聚乙二醇溶液，轻轻振荡混合均匀；
8、快速加入无水乙醇溶液(V上述共混液/V乙醇＝1/4)，混合均匀后，快速加入培养皿中，并移至60℃烘箱中；
9、干燥后取出培养皿，加入去离子水悬浮；
10、用超声波细胞粉碎机超声2min，振幅30％；
11、离心：室温20min，12000rpm；
12、加入去离子水洗涤，去除上清液，反复离心，洗涤操作三次；
13、悬浮盐酸阿霉素-丝素纳微米颗粒，放置在4℃冰箱中或经冷冻真空干燥后保存。
参见附图46，它是本实施例提供的盐酸阿霉素-丝素纳米颗粒(上清液)的扫描电镜图，如图可知，盐酸阿霉素-丝素纳米颗粒的形貌受添加盐离子的影响。
参见附图47，它是本实施例提供的盐酸阿霉素-丝素微米颗粒的扫描电镜图，如图可知，盐酸阿霉素-丝素微米颗粒(一次静置)的形貌受添加盐离子的影响。
参见附图48，它是本实施例提供的盐酸阿霉素-丝素纳米颗粒(上清液)的粒径图(冻干前和冻干悬浮后)，如图可知，盐酸阿霉素-丝素纳米颗粒的粒径受添加盐离子和超声分散的的影响。
参见附图49，它是本实施例提供的盐酸阿霉素-丝素纳米颗粒(上清液)的电动势图，如图可知，盐酸阿霉素-丝素纳米颗粒的电动势受添加盐离子的影响。
参见附图50，它是本实施例提供的盐酸阿霉素-丝素纳微米颗粒的盐酸阿霉素封装率图，如图可知，盐酸阿霉素-丝素纳米颗粒的装载阿霉素的效率很高，接近100％，同时不同盐离子对阿霉素的封装率也有影响。
参见附图51，它是本实施例提供的盐酸阿霉素-丝素微米颗粒(一次静置)的激光共聚焦图，如图可知，盐酸阿霉素-丝素微米米颗粒的装载阿霉素分布比较均匀，强度很高。
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护。

Claims (13)

  1. 一种用聚乙二醇制备丝素纳微米球的方法，其特征在于包括以下步骤：
    将质量百分比为1～30％的丝素蛋白溶液与质量百分比为10～60％的聚乙二醇溶液放置在4～60℃温度条件下30min，然后按一定比例将所述丝素蛋白溶液和聚乙二醇溶液混合均匀，孵育一定时间后离心洗涤，配制成丝素纳微米球悬浮液。
  2. 一种用聚乙二醇制备丝素纳微米球的方法的应用，用于药物控释，其特征在于包括下述步骤：
    将药物溶解在质量百分比10～60％的聚乙二醇溶液中，或是将药物溶解在质量百分比1～30％的丝素蛋白溶液中，再将所述聚乙二醇溶液与所述丝素蛋白溶液混合均匀制得共混液，然后将所述共混液孵育一定时间后离心洗涤，制得用于药物控释的丝素-载药纳微米颗粒。
  3. 如权利要求2所述的方法，其特征在于：当所述药物为疏水性药物时，将所述疏水性药物溶解在聚乙二醇溶液中后再与丝素蛋白溶液混合制得所述共混液；除疏水性药物的其他药物，先将所述其他药物溶解在丝素蛋白溶液后再与聚乙二醇溶液混合制得所述共混液。
  4. 如权利要求3所述的方法，其特征在于：所述疏水性药物包括姜黄素，所述其他药物包括亲水性药物阿霉素、多肽类药物奥曲肽、蛋白类药物牛血清白蛋白、大分子亲水性模型药物葡聚糖或具有荧光标记的CdTe量子点。
  5. 如权利要求2所述的方法，其特征在于：所述丝素蛋白溶液中所含有的丝素蛋白与所述药物的质量比范围为1000/1～1/10。
  6. 如权利要求1或2所述的方法，其特征在于：所述聚乙二醇的分子量范围为2000～20000。
  7. 如权利要求6所述的方法，其特征在于：所述聚乙二醇的浓度分子量为4000、6000时其质量百分比为30～60％，浓度分子量为10000时其质量百分比为20～50％，浓度分子量为20000时其质量百分比为20～40％。
  8. 如权利要求1或2所述的方法，其特征在于：所述丝素蛋白溶液的pH 值在3.0～11.0。
  9. 如权利要求1或2所述的方法，其特征在于：所述丝素蛋白溶液的盐离子处理浓度从1M/L到0.01M/L。
  10. 如权利要求1或2所述的方法，其特征在于：所述丝素蛋白溶液和所述聚乙二醇溶液的体积比范围是5:1～1:10。
  11. 如权利要求1或2所述的方法，其特征在于：所述共混液在室温至60℃条件下孵育0.5～24h。
  12. 如权利要求1或2所述的方法，其特征在于：所述丝素蛋白溶液的稀释范围是质量百分比为5～20％。
  13. 如权利要求1或2所述的方法，其特征在于：所述丝素蛋白溶液的制备步骤如下：将已脱胶的熟丝浸润在9.3M的LiBr溶液中并置于60℃烘箱中4h，溶解后得到丝素蛋白溶液；将所述丝素蛋白溶液倒入透析袋中用去离子水透析3d；透析结束后将丝素蛋白溶液离心去除不溶物杂质，然后倒入透析袋中用分子量20000、15wt％的聚乙二醇溶液透析24h，获得浓缩的丝素蛋白溶液。
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Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104592375A (zh) * 2014-12-16 2015-05-06 苏州丝美特生物技术有限公司 一种用聚乙二醇制备丝素微球的方法
CN106913900B (zh) * 2017-02-20 2020-07-10 苏州丝美特生物技术有限公司 丝素蛋白止血材料及其制备方法
CN107320450A (zh) * 2017-05-14 2017-11-07 广州医科大学 一种基于超临界二氧化碳技术的纳米制剂体系方法
WO2019094702A1 (en) 2017-11-10 2019-05-16 Cocoon Biotech Inc. Ocular applications of silk-based products
CN108465104A (zh) * 2018-03-07 2018-08-31 佛山实瑞先导材料研究院（普通合伙） 一种生长激素控释包衣及其制备方法
CN108316018A (zh) * 2018-03-15 2018-07-24 南通纺织丝绸产业技术研究院 一种丝素蛋白/壳聚糖功能性溶液的制备方法
CN108273132A (zh) * 2018-03-15 2018-07-13 南通纺织丝绸产业技术研究院 一种丝素蛋白/角蛋白复合多孔材料及其制备方法
CN108651861B (zh) * 2018-05-17 2021-07-27 南京农业大学 一种抗氧化姜黄素鸭血豆腐及其制备方法
AU2020288624A1 (en) 2019-06-04 2022-02-03 Cocoon Biotech Inc. Silk-based products, formulations, and methods of use
CN110812335B (zh) * 2019-12-04 2021-08-31 苏州大学 装载疏水药物的丝素蛋白微纳米颗粒缓释制剂及制备方法
CN111228215A (zh) * 2020-03-09 2020-06-05 王岩松 一种自组装可成像丝素蛋白水凝胶的制备方法
CN111759799A (zh) * 2020-07-08 2020-10-13 合肥工业大学 一种可注射胶体凝胶的制备方法及可注射胶体凝胶
CN113663119A (zh) * 2021-07-07 2021-11-19 核工业总医院 具有透视可显影功能的碘化油丝素蛋白栓塞微球制备方法
CN115337358B (zh) * 2022-07-19 2023-06-23 南通纺织丝绸产业技术研究院 具有多级梯度缓释功能的六味地黄丝素蛋白微球及其制法
CN115737601A (zh) * 2022-11-18 2023-03-07 安徽医科大学 一种采用层层自组装技术制备药物缓释制剂的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244277A (zh) * 2008-02-14 2008-08-20 苏州大学 丝素载药微球及其制备方法
CN104592375A (zh) * 2014-12-16 2015-05-06 苏州丝美特生物技术有限公司 一种用聚乙二醇制备丝素微球的方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8178656B2 (en) * 2004-06-11 2012-05-15 Trustees Of Tufts College Silk-based drug delivery system
US20100028451A1 (en) * 2006-09-26 2010-02-04 Trustees Of Tufts College Silk microspheres for encapsulation and controlled release
US9427499B2 (en) * 2008-11-17 2016-08-30 Trustees Of Tufts College Surface modification of silk fibroin matrices with poly(ethylene glycol) useful as anti-adhesion barriers and anti-thrombotic materials
US9566365B2 (en) * 2010-09-01 2017-02-14 Trustees Of Tufts College Silk fibroin and polyethylene glycol-based biomaterials
WO2014021954A2 (en) * 2012-04-13 2014-02-06 Trustees Of Tufts College Magneto-sensitive silk fibroin-based materials
AU2013329077A1 (en) * 2012-10-11 2015-04-23 Tufts University Compositions and methods for sustained delivery of glucagon-like peptide (GLP-1) receptor agonist therapeutics
KR20230021768A (ko) * 2013-03-15 2023-02-14 트러스티즈 오브 터프츠 칼리지 저분자량 실크 조성물 및 안정화 실크 조성물
CN103289107B (zh) * 2013-05-28 2015-02-04 苏州丝美特生物技术有限公司 一种可注射丝素蛋白原位凝胶的制备方法及其应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101244277A (zh) * 2008-02-14 2008-08-20 苏州大学 丝素载药微球及其制备方法
CN104592375A (zh) * 2014-12-16 2015-05-06 苏州丝美特生物技术有限公司 一种用聚乙二醇制备丝素微球的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BI, DIANZHOU;, PHARMACY, 30 November 1999 (1999-11-30), pages 465 *
See also references of EP3235512A4 *

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US20170340575A1 (en) 2017-11-30
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CN104592375A (zh) 2015-05-06
CN105597104A (zh) 2016-05-25
EP3235512A4 (en) 2018-08-15

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