CN101693179A - Method for preparing biodegradable nano-microspheres - Google Patents
Method for preparing biodegradable nano-microspheres Download PDFInfo
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- CN101693179A CN101693179A CN200910309385A CN200910309385A CN101693179A CN 101693179 A CN101693179 A CN 101693179A CN 200910309385 A CN200910309385 A CN 200910309385A CN 200910309385 A CN200910309385 A CN 200910309385A CN 101693179 A CN101693179 A CN 101693179A
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Abstract
A method for preparing biodegradable nano-microspheres belongs to the technical field of medicinal nano material, and comprises dissolving hydrophilic quantum dots and polypeptide or protein or antibodies in phosphate buffered solution to get an aqueous phase mixture, ultrasonically dissolving poly (lacticoglycolic acid) copolymer in an organic solvent, adding the organic solvent into the aqueous phase mixture to mix thoroughly, getting water-in-oil colostrums after treatment of ultrasonic emulsification, adding the water-in-oil colostrums into polyvinyl alcohol, and obtaining the biodegradable nano-microspheres after normal temperature mixing, and centrifugal filtering and washing. The method for preparing the biodegradable nano-microspheres takes PLGA for biological degradation as the load material, combines the fluorescent characteristic of the quantum dots and the pesticide effects of the polypeptide, the protein and the antibodies to prepare the biodegradable nano-microspheres with marking effect and treatment effect by a multiple emulsion method, and the nano-microspheres are biodegradable and have the effect of slow release.
Description
Technical field
What the present invention relates to is the product in a kind of medicinal nano material technology field and preparation method thereof, specifically is a kind of preparation method with biodegradable nano-microspheres of mark and treatment function.
Background technology
Degradable poly lactic acid/ethanol copolymer (poly lactide-co-glycolide with excellent biological compatibility, PLGA) nanoparticle was used to prepare Biodegradable slowly-releasing or targeted delivery of drugs system after deliberation nearly 30 years, was the focus of research both at home and abroad.PLGA has obtained drugs approved by FDA at present as operation suture thread, angiocarpy bracket controlled release drug coating, and the material of injection micro-capsule, microballoon, implants etc.According to character, method of administration and the drug release time of medicine, select the lactic acid copolymerization of different molecular weight, different optical activity for use, the polymerization ratio of lactide not of the same race and glycolide adopts corresponding preparation technology to control medicine and reaches different release models; Can also pass through the induction system of PLGA preferred agents, thereby obtain the novel form of medicine.Nanoparticle can make the drug concentration of administration part maintain treatment level for a long time, reduces administration number of times and dosage, thereby prevents on a plurality of aspects or alleviate toxic and side effect.The end product of PLA hydrolysis is water and carbon dioxide, in ask that product lactic acid also is normal glycometabolism product in the body, so nontoxic, the nonirritant of this polymer and has good Bc.
Along with the high speed development of biotechnology, medicines such as polypeptide, protein, antibody continue to bring out, existing multiple at present critical treatment medicine listing.The basic formulation of biotech drug is freeze-dried, although its curative effect of conventional formulation is early confirmed by clinical, but because the half-life is short, very unstable again under the room temperature situation, need long-term frequent drug administration by injection, from patients ' psychological and financial burden angle, these all difficult acceptance, in addition, the medicine that has must surpass certain hour and could effectively play a role.So, be carrier material with PLGA, comprise that medicines such as polypeptide, protein, antibody make microballoon, can reach slowly-releasing, long-acting purpose in vivo, be the frontier that nearly various countries scholar during the last ten years studies energetically.
To the evaluation of drug effect and intuitively observe the residing position of medicine, need carry out certain mark to medicine, mark commonly used at present is isotope labeling and fluorochrome label, but isotope labeling exists radiation and pollution, and the dye marker existence needs certain wavelengths and the high light source activation of energy, radiative spectral width, and red light district has hangover, defectives such as photostability is poor, detects the background height, and sensitivity is low.Quantum dot-labeled fluorescence analysis principle is similar to existing fluorescence analysis with detection technique, its significant advantage is to have good photostability, under the lasting light irradiation, can keep several hrs, fluorescence lifetime is long, the fluorescence that suppresses natural materials self by the control time disturbs, and is highly sensitive.And for slow releasing pharmaceutical cyclic process in vivo to monitor be a long-term complicated process, its mark is needed to detect for a long time, quantum dot can address that need just, overcome the defective that fluorescent dye is easy to quencher, and directly observe medicine and the residing position of carrier thereof by fluorescence imaging, the Real Time Observation curative effect of medication is played a very important role.
Find through literature search prior art, Chinese patent literature CN101327189A, put down in writing a kind of " have mark and treatment bifunctional method for preparing nano alcohol liposome material ", this technology is with 5-FU and the dissolving of hydrophilic quantum dot and be dispersed in the PBS.As carrier, preparation contains the nano alcohol liposome of 5-FU and quantum dot with phosphatide.But this technology exists entrapment efficiency low, and easy shortcoming such as leakage, thereby causes the early stage release rate height of medicine and quantum dot, thereby can not reach the purpose of slowly-releasing.
Summary of the invention
The present invention is directed to the prior art above shortcomings, a kind of preparation method of biodegradable nano-microspheres is provided, with biodegradation material PLGA as carrier material, adopt the method for emulsion, drug effect in conjunction with medicines such as the fluorescent characteristic of quantum dot and polypeptide, protein, antibody, be prepared into biodegradable nano-microspheres, have the effect of slowly-releasing with mark and result of treatment.
The present invention is achieved by the following technical solutions: the medicine that will have the quantum dot of fluorescent characteristic and have a treatment function be wrapped in can biodegradable polymer P LGA in, and pass through the emulsion technology, obtain Nano microsphere.
The present invention specifically may further comprise the steps:
The first step, a kind of being dissolved in the PBS with in hydrophily quantum dot and polypeptide, protein or the antibody obtains aqueous mixture.
Described hydrophily quantum dot is selected CdTe, CdSe, ZnS, ZnSe or CdSe/ZnS core/shell type semiconductor-quantum-point for use, its fluorescent emission wavelength from 488nm to 605nm between.
In the described aqueous mixture: the concentration of hydrophily quantum dot is that the concentration of 0.005～0.03g/ μ L, polypeptide, protein or antibody is 0.1～0.5g/ μ L.
The pH of described PBS is 7.4.
Second step joined the PLGA ultrasonic dissolution in the aqueous mixture behind organic solvent and fully stirring, after ultrasonic emulsification is handled, obtained the Water-In-Oil colostrum then.
The concentration of described PLGA is 1～5g/ μ L, and wherein the mol ratio of DL-LA: GA is 90: 10,80: 20,75: 25,60: 40 or 50: 50.
Described organic solvent is: a kind of or its combination in chloroform, carrene, acetone, ethanol or the ethyl acetate.
Described ultrasonic emulsification is handled and is meant: setting ultrasonic power 40～100W carries out ultrasonic dispersion and continues to mix 1 hour after 1～5 minute under the 700r/min condition.
The 3rd step joined the Water-In-Oil colostrum in the polyvinyl alcohol, and after the normal temperature stir process, the centrifugal filtration washing obtains biodegradable nano-microspheres.
Described temperature stir process is meant that the rotating speed with 700～1000rpm/min stirred 4～16 hours under room temperature environment.
The concentration of described polyvinyl alcohol is 0.5～10g/ μ L.
Described centrifugal filtration washing is meant centrifugal speed between 6000～12000rpm/min, and centrifugation time 5～30 minutes adopts deionized water washing 3 times after filtering.
The present invention is wrapped in water miscible quantum dot and the medicine with therapeutic action among the Biodegradable material PLGA simultaneously, and is prepared into Nano microsphere by the method for emulsion.Utilize the fluorescence labeling effect of quantum dot, the therapeutic action of medicine, the slow releasing function of Biodegradable vehicles, and the preparation method of emulsion obtain having mark and the bifunctional biodegradable nano-microspheres of treatment.
The specific embodiment
Below embodiments of the invention are elaborated, present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment 1
The first step with 0.005% emission wavelength be the CdTe quantum dot that disperses in the 488nm water and 0.01% thymic peptide to be dissolved in pH be in 7.4 PBSs, obtain aqueous mixture.
Second step with 1% PLGA (DL-LA: the GA mol ratio is 90: 10) ultrasonic dissolution in chloroform.Under the 700r/min stirring condition, join in the aqueous mixture of the first step, the auxiliary emulsification of 40W power ultrasonic 3 minutes continues under the room temperature to stir 1 hour, obtains Water-In-Oil (W/O) colostrum.
The W/O colostrum that the 3rd step obtained for second step under the churned mechanically condition of 700r/min joins in the polyvinyl alcohol (PVA) of 10 milliliters of 0.5g/ μ L, room temperature, the lasting stirring 10 hours, chloroform volatilizees fully, emulsion droplet begins the hardening balling-up, filter and wash 3 times, obtain having the Biodegradable nanometer material of mark and treatment dual-use function.Vacuum freeze-drying gets freeze-dried powder, and sealing is placed on 4 ℃ of preservations.
The biodegradable nano-microspheres that present embodiment prepares, microballoon is by PLGA parcel quantum dot and thymic peptide, through fluorescent spectrophotometer assay, has the fluorescence of emission wavelength at 480～490nm, the envelop rate of thymic peptide is between 40～70%, measure through laser particle analyzer, its average grain diameter is at 8～10 microns.
This enforcement preparation gained biodegradable nano-microspheres, release is 78% after 12 days.Experiment shows outside the prepared fluorescence medicament nano spheroid can continue release, has obvious slow release effect, and keeps good fluorescence property.
Embodiment 2
The first step, with 0.01% fluorescent emission wavelength be the water-soluble quantum dot CdTe@CdSe of 530nm and 0.3% EGF (EGF) to be dissolved in pH be in 7.4 the PBS, obtain aqueous mixture.
In second step, the PLGA with 3% (DL-LA: the GA mol ratio is 75: 25) ultrasonic dissolution is in acetone.Under the 700r/min stirring condition, join in the aqueous mixture of the first step, the ultrasonic auxiliary emulsification of 60W 3 minutes continues under the room temperature to stir 1 hour afterwards, obtains Water-In-Oil (W/O) colostrum.
The 3rd step, the W/O colostrum that under churned mechanically condition second step was obtained joins in the polyvinyl alcohol (PVA) of certain volume, suitable concentration, room temperature, 800rpm/min stirred 4 hours, acetone volatilizees fully, emulsion droplet begins the hardening balling-up, centrifugal and wash 3 times, can make biodegradable nano-microspheres with fluorescence labeling and treatment dual-use function.Vacuum freeze-drying gets freeze-dried powder, and sealing is placed on 4 ℃ of preservations.
The biodegradable nano-microspheres that present embodiment prepares, microballoon is by PLGA parcel quantum dot and EGF, through fluorescent spectrophotometer assay, have the fluorescence of emission wavelength between 520～540nm, the envelop rate of EGF is between 30～50%.Measure through laser particle analyzer, its average grain diameter is between 500～800 nanometers.
This enforcement preparation gained biodegradable nano-microspheres, the vitro drug release experiment shows, the 9th day cumulative release degree 51.8%, and keep good fluorescence property, have obvious slow release effect and fluorescence property.
Embodiment 3
The first step, with 0.03% fluorescent emission wavelength be the water-soluble quantum dot CdSe@ZnS of 605nm and 0.005% monoclonal antibody drug to be dissolved in pH be in 7.4 the PBS, obtain aqueous mixture.
In second step, the PLGA with 5% (DL-LA: the GA mol ratio is 50: 50) ultrasonic dissolution is in ethyl acetate.Under the 700r/min stirring condition, join in the aqueous mixture of the first step, ultrasonic auxiliary emulsification 5 minutes continues under the room temperature to stir 1 hour afterwards, obtains Water-In-Oil (W/O) colostrum.
The 3rd step, under churned mechanically condition, the second W/O colostrum that obtain of step joined in 10 milliliters of the 10g/ μ L polyvinyl alcohol (PVA), under room temperature, 1000rpm/min, stirred 20 hours, ethyl acetate volatilizees fully, emulsion droplet begins the hardening balling-up, centrifugal and wash 3 times, can obtain having the biodegradable nano-microspheres of mark and treatment dual-use function.Vacuum freeze-drying gets freeze-dried powder, and sealing is placed on 4 ℃ of preservations.
The biodegradable nano-microspheres that present embodiment prepares, microballoon is by PLGA parcel quantum dot and monoclonal antibody drug, through fluorescent spectrophotometer assay, has the fluorescence of emission wavelength between 520～540nm, the envelop rate of monoclonal antibody drug is between 30～50%, measure through laser particle analyzer, its average grain diameter is between 100～500 nanometers.
This enforcement preparation gained biodegradable nano-microspheres, the vitro drug release experiment shows that microballoon can continue to discharge BSA and reach more than the 35d, and keeps good fluorescence property, has obvious slow release effect and fluorescence property.
Claims (9)
1. the preparation method of a biodegradable nano-microspheres is characterized in that, may further comprise the steps:
The first step, a kind of being dissolved in the PBS with in hydrophily quantum dot and polypeptide, protein or the antibody obtains aqueous mixture;
Second step joined the PLGA ultrasonic dissolution in the aqueous mixture behind organic solvent and fully stirring, after ultrasonic emulsification is handled, obtained the Water-In-Oil colostrum then;
The 3rd step joined the Water-In-Oil colostrum in the polyvinyl alcohol, and after the normal temperature stir process, the centrifugal filtration washing obtains biodegradable nano-microspheres.
2. the preparation method of biodegradable nano-microspheres according to claim 1, it is characterized in that, described hydrophily quantum dot is selected the hud typed semiconductor-quantum-point of CdTe, CdSe, ZnS, ZnSe or CdSe-ZnS for use, its fluorescent emission wavelength from 488nm to 605nm between.
3. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, in the described aqueous mixture: the concentration of hydrophily quantum dot is that the concentration of 0.005～0.03g/ μ L, polypeptide, protein or antibody is 0.1～0.5g/ μ L.
4. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, the pH of described PBS is 7.4.
5. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, the concentration of described PLGA is 1～5g/ μ L, and wherein the mol ratio of DL-LA: GA is 90: 10,80: 20,75: 25,60: 40 or 50: 50.
6. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, described organic solvent is: a kind of or its combination in chloroform, carrene, acetone, ethanol or the ethyl acetate.
7. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, described ultrasonic emulsification is handled and is meant: setting ultrasonic power 40～100W carries out ultrasonic dispersion and continues to mix 1 hour after 1～5 minute under the 700r/min condition.
8. the preparation method of biodegradable nano-microspheres according to claim 1 is characterized in that, described temperature stir process is meant that the rotating speed with 700～1000rpm/min stirred 4～16 hours under room temperature environment.
9. the preparation method of biodegradable nano-microspheres according to claim 1, it is characterized in that, described centrifugal filtration washing is meant centrifugal speed between 6000～12000rpm/min, and centrifugation time 5～30 minutes adopts deionized water washing 3 times after filtering.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008454A (en) * | 2010-12-03 | 2011-04-13 | 上海交通大学 | Daidzein-entrapped PLGA nanoparticles and preparation method thereof |
| CN103239408A (en) * | 2013-04-26 | 2013-08-14 | 天津大学 | Preparation method of submicron order core/shell structure PLGA (Poly(Lactic-co-Glycolic) microsphere with uniform particle size |
| CN108160013A (en) * | 2017-12-15 | 2018-06-15 | 华南理工大学 | A kind of Water-soluble carbon quantum dot slow-release microcapsule and preparation method and application |
| CN110709067A (en) * | 2017-11-30 | 2020-01-17 | G2G生物公司 | Method for preparing biodegradable microspheres with improved stability and storage stability |
| CN111773394A (en) * | 2019-04-04 | 2020-10-16 | 复旦大学 | Beta-galactosidase fluorescent probe nano-microsphere and preparation method and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1132758A (en) * | 1995-04-06 | 1996-10-09 | 中国科学院成都有机化学研究所 | High-molecular microglobe and its prepn and use |
| US6962716B1 (en) * | 2000-09-27 | 2005-11-08 | Board Of Regents, The University Of Texas System | Compositions and methods for biodegradable microspheres as carriers of bioactive substances |
-
2009
- 2009-11-06 CN CN2009103093859A patent/CN101693179B/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102008454A (en) * | 2010-12-03 | 2011-04-13 | 上海交通大学 | Daidzein-entrapped PLGA nanoparticles and preparation method thereof |
| CN102008454B (en) * | 2010-12-03 | 2012-07-04 | 上海交通大学 | Daidzein-entrapped PLGA nanoparticles and preparation method thereof |
| CN103239408A (en) * | 2013-04-26 | 2013-08-14 | 天津大学 | Preparation method of submicron order core/shell structure PLGA (Poly(Lactic-co-Glycolic) microsphere with uniform particle size |
| CN103239408B (en) * | 2013-04-26 | 2014-09-17 | 天津大学 | Preparation method of submicron order core/shell structure PLGA (Poly(Lactic-co-Glycolic) microsphere with uniform particle size |
| CN110709067A (en) * | 2017-11-30 | 2020-01-17 | G2G生物公司 | Method for preparing biodegradable microspheres with improved stability and storage stability |
| US11311854B2 (en) | 2017-11-30 | 2022-04-26 | G2Gbio, Inc. | Method for preparing biodegradable microspheres having improved stability and storage stability |
| CN110709067B (en) * | 2017-11-30 | 2022-10-14 | G2G生物公司 | Method for preparing biodegradable microspheres with improved stability and storage stability |
| CN108160013A (en) * | 2017-12-15 | 2018-06-15 | 华南理工大学 | A kind of Water-soluble carbon quantum dot slow-release microcapsule and preparation method and application |
| CN108160013B (en) * | 2017-12-15 | 2020-08-18 | 华南理工大学 | Water-soluble carbon quantum dot sustained-release microcapsule and preparation method and application thereof |
| CN111773394A (en) * | 2019-04-04 | 2020-10-16 | 复旦大学 | Beta-galactosidase fluorescent probe nano-microsphere and preparation method and application thereof |
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| CN101693179B (en) | 2012-02-15 |
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