CN106727425A - A kind of preparation method of lysozyme PLGA microballoons - Google Patents

A kind of preparation method of lysozyme PLGA microballoons Download PDF

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Publication number
CN106727425A
CN106727425A CN201611096061.8A CN201611096061A CN106727425A CN 106727425 A CN106727425 A CN 106727425A CN 201611096061 A CN201611096061 A CN 201611096061A CN 106727425 A CN106727425 A CN 106727425A
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lysozyme
microballoon
preparation
plga microballoons
plga
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郝青
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Shaanxi Huanke Biotechnology Co Ltd
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Shaanxi Huanke Biotechnology Co Ltd
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    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
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  • Organic Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

A kind of preparation method of lysozyme PLGA microballoons, belongs to field of material preparation, it is characterised in that comprise the following steps：（1）Lysozyme is dissolved in PBS liquid, interior water phase is formed；（2）Take to be dissolved in dichloromethane and form organic phase；（3）Interior water is added in organic phase, solidified microsphere is emulsified to obtain at the beginning of being carried out under condition of ice bath with ultrasonic cell disintegration instrument；（4）With deionized water centrifuge washing 3 times, lysozyme PLGA microballoons are obtained final product.With lysozyme as model drug, prepare microballoon, compare influence of the formulation factors to microballoon property in preparation process, and physicochemical property, the internal compatibility of microballoon and degradation characteristic are carried out to obtained microballoon, the microballoon monodispersity that emulsification condition is gentle, preparation process is difficult to make medicine be denatured and prepare is preferable, and preparation method process is simple of the present invention, it is easy to operate, be suitable to popularization and application.

Description

A kind of preparation method of lysozyme PLGA microballoons
Technical field
The invention belongs to field of material preparation, more particularly to a kind of preparation method of lysozyme PLGA microballoons.
Background technology
As biotechnology develops, albumen, polypeptide drug are quickly presented, currently on existing various biotech drugs City.Freeze-dried powder class preparation is the conventional dosage forms of this kind of medicine, although its determined curative effect, is easily degraded in vivo, is partly declined Phase is short, needs frequent drug administration by injection.
SPG membrane emulsifications are a kind of novel emulsion methods, and its principle is to apply a certain size pressure on dispersed phase to make It is separated into the more uniform drop of particle diameter after being SPG films by the micropore glass film of uniform pore diameter, by the continuous of continuous flowing Mutually wash away, reached on critical pressure from the critical value i.e. pressure of film sur-face peeling when liquid-drop diameter reaches, one can be formed The emulsion droplet of uniform grading.Microballoon can be prepared in conjunction with solvent evaporation method afterwards, its particle diameter can be come by SPG membrane apertures Control.The microballoon monodispersity that this method emulsification condition is gentle, preparation process is difficult to make medicine be denatured and prepare is preferable.
The content of the invention
Present invention seek to address that above mentioned problem, there is provided a kind of preparation method of lysozyme PLGA microballoons.
The technical scheme is that：
A kind of preparation method of lysozyme PLGA microballoons, it is characterised in that comprise the following steps：
（1）40mg lysozymes are dissolved in the PBS liquid of 200 μ l, pH7.4, interior water phase is formed；
（2）Take during PLGA is dissolved in 5ml dichloromethane and form organic phase；
（3）Interior water is added in organic phase, just emulsification is carried out under condition of ice bath with ultrasonic cell disintegration instrument, colostric fluid is film Dispersed phase in emulsion process is pressed into the outer water phase of 1% polyvinyl alcohol under nitrogen pressure by SPG films, is stirred under 400rpm rotating speeds Mix, organic solvent is volatilized completely, obtain solidified microsphere；
（4）With deionized water centrifuge washing 3 times, lysozyme PLGA microballoons are obtained final product.
The preparation method of lysozyme PLGA microballoons of the present invention, the lysozyme microballoon drugloading rate is 11.84%.
The preparation method of lysozyme PLGA microballoons of the present invention, the envelop rate of the lysozyme microballoon is 72.4%.
The preparation method of lysozyme PLGA microballoons of the present invention, the particle diameter of the lysozyme microballoon is 63-65 μm. SPG membrane emulsifications prepare a big feature of microballoon, and preparation method is gentle, and the emulsion droplet collision of preparation process is less, therefore pattern is justified It is whole without adhesion.By grain size analysis, it was demonstrated that the average grain diameter of microballoon is 63.89 μm, and span is 0.675.
The preparation method of lysozyme PLGA microballoons of the present invention, the mixing time is 4-4.5h.
The technical effects of the invention are that：
The preparation method of lysozyme PLGA microballoons of the present invention, with lysozyme as model drug, prepares microballoon, compares preparation During influence of the formulation factors to microballoon property, and physicochemical property, the internal compatibility of microballoon are carried out to obtained microballoon And degradation characteristic, emulsification condition is gentle, preparation process is difficult the microballoon monodispersity for making medicine be denatured and prepare preferably, and this The described preparation method process is simple of invention, it is easy to operate, be suitable to popularization and application.
Specific embodiment
Embodiment 1
A kind of preparation method of lysozyme PLGA microballoons, it is characterised in that comprise the following steps：
（1）40mg lysozymes are dissolved in the PBS liquid of 200 μ l, pH7.4, interior water phase is formed；
（2）Take during PLGA is dissolved in 5ml dichloromethane and form organic phase；
（3）Interior water is added in organic phase, just emulsification is carried out under condition of ice bath with ultrasonic cell disintegration instrument, colostric fluid is film Dispersed phase in emulsion process is pressed into the outer water phase of 1% polyvinyl alcohol under nitrogen pressure by SPG films, is stirred under 400rpm rotating speeds Mix, organic solvent is volatilized completely, obtain solidified microsphere；
（4）With deionized water centrifuge washing 3 times, lysozyme PLGA microballoons are obtained final product.
The preparation method of lysozyme PLGA microballoons of the present invention, the lysozyme microballoon drugloading rate is 11.84%.
The preparation method of lysozyme PLGA microballoons of the present invention, the envelop rate of the lysozyme microballoon is 72.4%.
The preparation method of lysozyme PLGA microballoons of the present invention, the particle diameter of the lysozyme microballoon is 63-65 μm. SPG membrane emulsifications prepare a big feature of microballoon, and preparation method is gentle, and the emulsion droplet collision of preparation process is less, therefore pattern is justified It is whole without adhesion.By grain size analysis, it was demonstrated that the average grain diameter of microballoon is 63.89 μm, and span is 0.675.
The preparation method of lysozyme PLGA microballoons of the present invention, the mixing time is 4h.
The particle diameter and its degree distribution situation of microballoon are determined using laser particle size analyzer dry method.Using differential scanning calorimeter Investigate the phase transition temperature of microballoon.Lysozyme powder, blank microballoon, lysozyme powder and the blank microballoon of 5 mg are weighed respectively Physical mixture and carry lysozyme microballoon, in the range of 20 DEG C -200 DEG C, nitrogen buffer gas, with 10 DEG C of speed liter Temperature, determines each sample respectively.Lysozyme microballoon is characterized using FTIS.Scan respectively molten The physical mixture of bacterium enzyme, blank microballoon, lysozyme and blank microballoon, lysozyme drug bearing microsphere.
The Tg of blank microballoon is 52. 98 DEG C;Lysozyme Tg is 83. 33 DEG C, but peak type is not sharp, because working as energy Reached solid lysozyme two, the domain of tertiary structure can when, the thermal capacitance of the sample there occurs obvious change, solid lysozyme Heat absorption is gradually stretched and is denatured into peptide chain;Lysozyme occurs in that two Tg with the physical mixture of blank microballoon, wherein 53. 08 DEG C it is believed that the Tg of material, but another is 71. 36 DEG C, it may be that lysozyme is influenceed by blank microballoon, and Tg there occurs Nearly 12 DEG C of change;Drug bearing microsphere only occurs in that a Tg, is 56. 24 DEG C, differs nearly 4 DEG C with the Tg of blank microballoon, and The Tg of medicine disappears.Illustrate that lysozyme is strictly to be wrapped in microballoon the inside, and cause the peak that its phase in version occurs to disappear not See, also cause that the Tg of material is changed, increased 4 DEG C.
After lysozyme microballoon is injected into rat muscle, have no that muscle groups are woven with abnormal response, but have fraction of inflammation anti- Should.Particularly at first week, the inflammatory cell of blueness is filled with around microballoon.Inflammatory reaction mitigates many after 1 week, says The histocompatbility of bright microballoon is good.And microballoon degraded situation in vivo is:The particle diameter of microballoon from the greater particle size of first week, It is gradually reduced.Microballoon has been degraded to ball interior after 4th week, causes the intensity of microballoon to decline, and spherical holding is not very Completely, there is the trend of rupture.But the degraded trend of microballoon is not obvious, it may be that sampling makes the difference and rat constitution of sample Difference, it is also possible to be to form bladder to wrap microballoon, have impact on the degraded of microballoon, needs further research.
With the increase of PLGA viscositys, the drugloading rate and envelop rate of microballoon all increase.Because the viscosity of organic phase increases Greatly, the particle diameter of microballoon may increase, and Drug loading capacity increased with the increase of microspherulite diameter.Release experiment in vitro In, the prominent effect of releasing of microballoon is reduced to 4%, because the big material of viscosity, the microballoon prepared with the increase of viscosity from 40% Can be finer and close, insoluble drug release is slow.The consumption of PLGA determines the concentration and its viscosity of organic phase in emulsion process, and organic phase is dense Degree is bigger, and the microsphere surface of preparation is finer and close, and solvent volatilization is also faster in microballoon, increases the quickening of microballoon curing rate, envelop rate It is high.The prominent degree of releasing of release in vitro also can be lower.
Different materials are mutually added in interior water, one side protected protein class medicine is not damaged in colostrum emulsion process, separately On the one hand pore-foaming agent is may act as, the medicine inside microballoon is smoothly discharged, increase total volume.Test result indicate that All additives are bigger than being not added with microballoon prepared by any material to the envelop rate and drugloading rate of microballoon.Result of study:Egg White envelop rate increases with the increase of interior water phase viscosity.But release in vitro result shows different additive to microballoon Chinese medicine The releasing trend of thing does not have much affect, and mainly influence is that the prominent of medicine releases degree in microballoon.The prominent of wherein PVA is released most Secondly it is mannitol for serious.Microexamination discovery is carried out by microballoon, after Nei Shui is added to additive, microsphere surface There is more larger hole, this is also to dash forward to release an increased reason.
Dosage not only influences entrapment efficiency, and is the key factor for influenceing microballoon cumulative release curve.With molten The increase of bacterium enzyme dosage, envelop rate reduction, prominent the releasing from 5. 18% of In Vitro Dissolution increases to 13. 41%.When dosage is high Just contain lysozyme higher in emulsion drop, concentration gradient of the lysozyme in inside and outside water phase is increased, so as to increase lysozyme The amount and medicine for being diffused into outer water phase are accumulated in microsphere surface.And in drugloading rate higher, lysozyme is released initially prominent Afterwards, an and then release faster.
When the volume of interior water phase increases, and organic phase constancy of volume, entrapment efficiency decline, the burst effect of release in vitro 12% then is increased to by 5%, and lysozyme cumulative release is very fast after initially prominent releasing.Outer water phase PVA concentration is to lysozyme microballoon Drugloading rate, envelop rate influence it is little, and the release in vitro tool of microballoon is had a certain impact.When the increase of outer water phase PVA concentration When, viscosity increase, while emulsification strengthens, makes the microsphere volume prepared less than normal, specific surface area increase, therefore rate of releasing drug Accelerate.

Claims (5)

1. a kind of preparation method of lysozyme PLGA microballoons, it is characterised in that comprise the following steps：
（1）40mg lysozymes are dissolved in the PBS liquid of 200 μ l, pH7.4, interior water phase is formed；
（2）Take during PLGA is dissolved in 5ml dichloromethane and form organic phase；
（3）Interior water is added in organic phase, just emulsification is carried out under condition of ice bath with ultrasonic cell disintegration instrument, colostric fluid is film Dispersed phase in emulsion process is pressed into the outer water phase of 1% polyvinyl alcohol under nitrogen pressure by SPG films, is stirred under 400rpm rotating speeds Mix, organic solvent is volatilized completely, obtain solidified microsphere；
（4）With deionized water centrifuge washing 3 times, lysozyme PLGA microballoons are obtained final product.
2. the preparation method of lysozyme PLGA microballoons according to claim 1, it is characterised in that：The lysozyme microballoon is carried Dose is 11.84%.
3. the preparation method of lysozyme PLGA microballoons according to claim 1, it is characterised in that：The lysozyme microballoon Envelop rate is 72.4%.
4. the preparation method of lysozyme PLGA microballoons according to claim 1, it is characterised in that：The lysozyme microballoon Particle diameter is 63-65 μm.
5. the preparation method of lysozyme PLGA microballoons according to claim 1, it is characterised in that：The mixing time is 4- 4.5h。
CN201611096061.8A 2016-12-02 2016-12-02 A kind of preparation method of lysozyme PLGA microballoons Pending CN106727425A (en)

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Application Number Priority Date Filing Date Title
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