CN103784410A - Preparation method of isatis indigotica root polysaccharide methoxyl poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) polymer microspheres - Google Patents
Preparation method of isatis indigotica root polysaccharide methoxyl poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) polymer microspheres Download PDFInfo
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Abstract
The invention discloses a preparation method of isatis indigotica root polysaccharide methoxyl poly(ethylene glycol)-poly(lactic acid) (MPEG-PLA) polymer microspheres. A multiple emulsion method is adopted to prepare the isatis indigotica root polysaccharide MPEG-PLA block copolymer microspheres. The preparation method comprises the specific steps: adding a polysaccharide solution into an oil phase of a dichloromethane solution of MPEG-PLA, stirring at a high speed to obtain a milk white initial emulsion, adding the initial emulsion into a polyvinyl alcohol (PVA) aqueous solution, stirring at a high speed for 2-5 minutes, adding a multiple emulsion onto a magnetic stirrer, stirring at a rate of 50-200 rpm until an organic solvent completely volatilizes, finally, centrifuging the rest white emulsion at a speed of 3,000-5,000 rpm for 15 minutes, decanting supernatant, collecting precipitates, washing the precipitates with tri-distilled water for three times, and freeze-drying to obtain the isatis indigotica root polysaccharide MPEG-PLA microspheres. The drug carrying microspheres obtained by the method have a round shape according to the scanning electron microscope results, and are uniform in particle size, the average particle size is 874.3 nm, the dispersion coefficient is 0.749, the encapsulation efficiency is as high as 86%, the release rate is as high as 79.1%, the release time is long (26 days), the release is sustained and smooth, and the burst release rate is relatively low.
Description
Technical field
The present invention relates to a kind of method of preparing medicine carrying microballoons, particularly a kind of Banlangen Polysaccharide MPEG-PLA method for preparing polymer micro.
Background technology
Radix Isatidis is the dry root of cruciate flower plant Isatis indigotica Fort. (Isatisindigoticafort), is the main source of Radix Isatidis.Clinical conventional Chinese medicine, begins to be loaded in Shennong's Herbal, and bitter in the mouth is cold in nature, has heat-clearing and toxic substances removing, removing heat from blood sore-throat relieving function.For diseases such as purple dark, the mumps of maculae caused by violent heat pathogen, crimson tongue, scarlet fever, major part pestilence, erysipelas, carbuncle.
Recent study proves that Banlangen Polysaccharide is the class material that in Radix Isatidis extract, content is more, biological activity is stronger.Banlangen Polysaccharide has immunoloregulation function, and specific immunity and nonspecific immunity, humoral immunization or cellular immunization are had to certain facilitation.Xu Yimin etc. have confirmed that lumbar injection Banlangen Polysaccharide can significantly promote the immunologic function of mice.Report, Banlangen Polysaccharide can promote the growth of mouse thymus and the propagation of thymocyte cell simultaneously, indirectly maintains the microenvironment of thymus, promotes T lymphocyte, thymic epithelial cell to secrete thymosin and cytokine, thereby improves the immunity of body.But Banlangen Polysaccharide is showing certain difficulty aspect the practical applications such as animal immune, for example administration number of times is many, requires great effort consuming time, and repeatedly immunity causes distress to body, the problems such as immunoenhancement result is bad, therefore in the urgent need to delaying releasing mechanism research to polysaccharide.
Targeting drug delivery system (targetingdrugsystem, TDS), that carrier circulates medicine and optionally make medicine concentrate in target organ, target tissue, target cell by topical or systemic blood, and the drug-supplying system that curative effect is high, toxic and side effects is little, be the 4th generation pharmaceutical dosage form, be considered to the suitable dosage forms of anticarcinogen.This system used carrier normally some macromolecular materials, have plenty of naturally, have plenty of synthetic.PLA-PEG copolymer (MPEG-PLA, or PELA) is the one in conventional macromolecular material, and it is after series reaction, to react with Polyethylene Glycol (PEG) product obtaining by polylactic acid (PLA) or monomer whose.
Polylactic acid (PLA) has the features such as biological nontoxic, biodegradability and biocompatibility, is one of focus of pharmaceutical carrier research over nearly 40 years, has obtained U.S. FDA to ratify for human body, and has been widely used for the other industries such as organizational project.Internal metabolism research discovery, PLA middle catabolite is in vivo lactic acid, and the end-product of degrading after tricarboxylic acid cycle is CO2 and H2O, and these products are homergy products of human body and animal, can be discharged to external after lungs and kidney processing.But because PLA is the polymer that contains a large amount of ester bonds, and ester bond is hydrophobic group, therefore has very large difficulty for the embedding of hydrophilic medicament.In order to change the hydrophobic nature of PLA, can increase its hydrophilic by the electric charge that changes polymer surfaces on the one hand; Also can, by adopting hydroaropic substance (as Polyethylene Glycol) to carry out structural modification to PLA, can improve preferably the hydrophobicity of PLA on the other hand, increase its load to hydrophobic drug.Polyethylene Glycol (PEG) be a kind of polyether high molecular material except the good hydrophilic property of just having mentioned, also there is good biocompatibility.
Microsphere (microsphere) means medicine dissolution or is dispersed in the small spherical entity forming in adjuvant, that is matrix scaffold microgranule.The particle diameter of microsphere between 1-250 μ m, is often suspended in oil conventionally.After medicine is made microsphere, main feature is slow release long-acting and plays targeting.General microsphere is passive target, while being less than 7 μ m, is generally absorbed by macrophage in liver, spleen, and the microsphere that is greater than 7-10 μ m is held back in the mode of machinery filtration by the minimum capillary bed of lung conventionally, is entered in lung tissue or lung qi bubble by macrophage picked-up.In the motor process of gastrointestinal tract, monokaryon-mononuclear phagocyte system or other pipeline, may there is uneven distribution in microsphere, comprise 1. inhomogeneous rotation; 2. inhomogeneous release.Here it is causes the general prominent reason of releasing of drug bearing microsphere system, and in the time using MPEG-PLA copolymer as pharmaceutical carrier, can effectively reduce the prominent phenomenon of releasing of medicine.
In sum, MPEG-PLA block copolymer, with its good biodegradable, biocompatibility and amphipathic feature, has unique advantage in targeting drug delivery system and drug sustained release system.We can come telomerized polymer size and distribution by the molecular weight that changes molecular weight, content and the copolymer of PEG in copolymer, thereby further regulate the rate of release of hydrophobic drug or insoluble drug.Moreover to the virose medicine of internal organs, novel form is prepared in research for some, in reduction, it must play certain effect aspect the toxic and side effects of internal organs.In addition, also have scholar that the bags such as nucleic acid vaccine are downloaded in MPEG-PLA copolymer both at home and abroad, make some originally because very easily destroyed water-soluble medicine can direct oral cavity administration, and minimizing medicine is to body stimulation.But to make MPEG-PLA block copolymer be used widely, also must overcome the difficult problem in this polymer preparation process, at present the main preparation method adopting is ring-opening polymerisation method, but selecting etc. of for example purge process in the method step, catalyst is all problem demanding prompt solution.
Summary of the invention
The problem existing for solving above-mentioned prior art, the present invention proposes a kind of Banlangen Polysaccharide MPEG-PLA method for preparing polymer micro.
For achieving the above object, technical scheme of the present invention is:
A kind of Banlangen Polysaccharide MPEG-PLA method for preparing polymer micro, adopt multi-emulsion method to prepare Banlangen Polysaccharide MPEG-PLA block copolymer microsphere, it is characterized in that, concrete steps are: polysaccharide solution is joined in the oil phase of MPEG-PLA dichloromethane solution, under high-speed stirred, obtain milky colostric fluid, colostric fluid is joined in PVA aqueous solution, high-speed stirred 2--5min, the speed with 50--200rpm on magnetic stirring apparatus that subsequently double emulsion is placed on stirs until organic solvent is evaporated completely, finally, the centrifugal 15min of speed by last milky white liquid with 3000--5000rpm, abandon supernatant, get precipitation, precipitation is after tri-distilled water cyclic washing 3 times, lyophilization obtains Banlangen Polysaccharide MPEG-PLA microsphere.
Described MPEG-PLA block polymer adopts the MPEG-PLA that molecular weight is 1000-5000, and in MPEG-PLA dichloromethane solution, MPEG-PLA block copolymerization substrate concentration is 100ug/ml.
Described Banlangen Polysaccharide body concentration of aqueous solution is 0.06g/ml;
Described PVA concentration of aqueous solution is 0.5%.
Described MPEG-PLA dichloromethane solution: Banlangen Polysaccharide aqueous solution: PVA aqueous solution volume ratio is 1-10:1-10:1-10.
Described high-speed stirred speed is 10000--30000rpm.
With respect to prior art; beneficial effect of the present invention is: adopt preparing micro spheres by multiple emulsion process: polymer is dissolved in water mutually in insoluble organic solvent; pharmaceutical aqueous solution (water) is dispersed in to the w/o type emulsion that wherein forms water soluble drug; the aqueous solution that configuration contains stabilizing agent and protection glue, as outside water, forms W/O/W type emulsion in being distributed to outside water by making w/o type emulsion under the condition stirring.Through evaporation, filtration, the dry medicine carrying microballoons that obtains.The medicine carrying microballoons electron-microscope scanning result profile that the method obtains is round and smooth, even particle size, and mean diameter 874.3nm, the coefficient of dispersion is 0.749, and envelop rate is up to 86%, and release rate is up to 79.1%, and release time is longer, be 26 days, and slow release is mild, and dashing forward, it is lower to release rate.
Accompanying drawing explanation
Fig. 1 is Banlangen Polysaccharide MPEG-PLA microsphere cumulative release curve.
Fig. 2 a is MS2-3 microsphere Electronic Speculum in embodiment.
Fig. 2 b is the blank microsphere Electronic Speculum of MS2-3 in embodiment.
Fig. 2 c is MS2-5 microsphere Electronic Speculum in embodiment.
Fig. 2 d is the blank microsphere Electronic Speculum of MS2-5 in embodiment.
Fig. 2 e is MS6-3 microsphere Electronic Speculum in embodiment.
Fig. 2 f is the blank microsphere Electronic Speculum of MS6-3 in embodiment.
Fig. 2 g is MS6-5 microsphere Electronic Speculum in embodiment.
Fig. 2 h is the blank microsphere Electronic Speculum of MS6-5 in embodiment.
The specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Described raw material all can obtain from open commercial sources if no special instructions.
The preparation of test example 1 microsphere
1.1 preparing micro spheres by multiple emulsion process
This test adopts multi-emulsion method to prepare Banlangen Polysaccharide MPEG-PLA polymer microballoon: accurately take 1.2g and dry to the Banlangen Polysaccharide of constant weight and be dissolved in 20ml distilled water, fully stir it is dissolved completely, then getting certain volume polysaccharide solution joins in the oil phase of 20ml containing the MPEG-PLA dichloromethane solution of certain solubility, under high-speed stirred, obtain milky colostric fluid, colostric fluid is joined to 100ml containing in a certain proportion of PVA aqueous solution, high-speed stirred 3min, the speed with 100rpm on magnetic stirring apparatus that subsequently double emulsion is placed on stirs until organic solvent is evaporated completely, approximately need 12h.Finally, the centrifugal 15min of speed by last milky white liquid with 4500rpm, abandons supernatant, gets precipitation, and precipitation is after tri-distilled water cyclic washing 3 times, and lyophilization obtains Banlangen Polysaccharide MPEG-PLA microsphere.4 ℃ save backup.
1.2 orthogonal processing designs
The optimised process of preparing for obtaining Banlangen Polysaccharide MPEG-PLA polymer microballoon, this EXPERIMENTAL DESIGN MPEG-PLA (2000) polymer be coated with Banlangen Polysaccharide, the while tests according to the orthogonal table of four factor three levels, as table 1.
Table 1 is prepared Banlangen Polysaccharide MPEG-PLA polymer microballoon orthogonal processing
Note: MPEG-PLA-dichloromethane is fixed volume 20ml as oil phase; Polysaccharide concentration is 0.06g/ml;
The outer water volume of PVA is 100ml.
2 testing results
2.1 orthogonal processing gained are respectively organized particle diameter and the envelop rate of microsphere
Use multi-emulsion method to prepare MPEG-PLA (2000) and MPEG-PLA (6000) polymer microballoon by the orthogonal processing of setting, under optical microscope, there is certain difference with oily mirror observation particle diameter and envelop rate, although microspherulite diameter prepared by the each technique of the former with the latter is more or less the same, but the former is higher than the latter for envelop rate, show that MPEG-PLA (2000) polymer is more suitable for for coated Banlangen Polysaccharide.In addition, make above two indexs of microsphere reach optimum process condition to be: Banlangen Polysaccharide volume is that 10ml, concentration are 0.06g/ml, MPEG (2000)-PLA block copolymerization substrate concentration is 100ug/ml, PVA concentration be 0.5% and mixing speed be 16000rpm, wherein MPEG (2000)-PLA-dichloromethane is fixed volume 20ml as oil phase, and the outer water volume of PVA is 100ml.Be MS2-3, A1B3C3D3 in table 2 and
Table 3.
Table 2 Banlangen Polysaccharide MPEG-PLA (2000) microspheres
Note: " * " shows that interior water volume is larger to the grain diameter influence of microsphere; And mixing speed is larger on the envelop rate impact of microsphere.
Table 3 Banlangen Polysaccharide MPEG-PLA (6000) microspheres
Note: " * " show mixing speed to prepare microsphere envelop rate and grain diameter influence larger.
The drug loading of 2.2 microspheres
Choose respectively microsphere prepared by MS2-3, MS2-5, MS6-3 and tetra-techniques of MS6-5, the drug loading of measuring each group of microsphere through sulphuric acid anthrone method is in table 4.From table 2-4, the drug loading of MS2-3 technique group is minimum, only has 3%.
The microsphere drug loading of table 4 different process group
| ? | MS2-3 | MS2-5 | MS6-3 | MS6-5 |
| Drug loading (%) | 3 | 5 | 4 | 6 |
2.3 appearance morphosiss
As Fig. 2 a-2h chooses respectively microsphere prepared by MS2-3, MS2-5, MS6-3 and tetra-techniques of MS6-5, carry out scanning electron microscope scanning.Wherein, microsphere profile prepared by MS2-3 technique is round and smooth, and granular size is more even, and effect is best.
2.4 particle diameters and surface potential
Choose respectively blank microsphere and year polysaccharide microsphere prepared by MS2-3, MS2-5, MS6-3 and tetra-techniques of MS6-5, utilize laser particle size analyzer to carry out grain diameter measurement and potential determination.Wherein, MS2-5 technique group is prepared the particle diameter minimum of microsphere, and Average Particle Diameters is 353.1nm.The particle diameter of MS2-3 technique group thus obtained microsphere is greater than other technique groups, and size is 874.3nm, and the coefficient of dispersion is 0.749, higher than other technique groups, but has 41% particle size distribution in 0.883nm left and right, is significantly less than other technique groups.
2.5 release in vitro curves
Choose respectively microsphere prepared by MS2-3, MS2-5, MS6-3 and tetra-techniques of MS6-5, set up glucose standard curve, survey each group of different time and be discharged into the absorbance of polysaccharide in medium with visible-uv-spectrophotometric, obtain accordingly the accumulative total release rate of different time sections.Wherein, microsphere sustained-release prepared by MS2-3 technique group is mild, and prominent to release rate lower, and release rate is higher, reaches 79.1%, delays release time the longest, and slow-release time is 26 days.The results are shown in Figure 1.
Comprehensive above all key elements of considering about slow release, adopting MPEG-PLA block polymer to be coated with under the prerequisite of Banlangen Polysaccharide, optimum process condition is: polymer molecular weight should be selected MPEG (2000)-PLA block copolymer, MPEG (2000)-PLA block copolymerization substrate concentration is 100ug/ml, and MPEG (2000)-PLA-dichloromethane is fixed volume 20ml as oil phase; Banlangen Polysaccharide volume is 10ml, and concentration is 0.06g/ml; PVA concentration is that 0.5%, PVA is that outer water volume is 100ml; Mixing speed is 16000rpm.
Test example 2
The impact of Radix Isatidis crude polysaccharides MPEG (2000)-PLA polymer microballoon on mouse immunity
1 materials and methods
The preparation of 1.1 years Radix Isatidis crude polysaccharides MPEG (2000)-PLA polymer microballoons
Adopt multi-emulsion method to prepare Banlangen Polysaccharide MPEG (2000)-PLA polymer microballoon: accurately to take 1.2g and dry to the Banlangen Polysaccharide of constant weight and be dissolved in 20ml distilled water, fully stir it is dissolved completely, then getting 10ml polysaccharide solution joins in the oil phase of 20ml containing MPEG (2000)-PLA dichloromethane solution of 100ug/ml, under 16000rpm high-speed stirred, obtain milky colostric fluid, colostric fluid is joined to 100ml containing in the PVA aqueous solution of 2% ratio, high-speed stirred 3min, the speed with 100rpm on magnetic stirring apparatus that subsequently double emulsion is placed on stirs until organic solvent is evaporated completely, approximately need 12h.Finally, the centrifugal 15min of speed by last milky white liquid with 4500rpm, abandons supernatant, gets precipitation, and precipitation is after tri-distilled water cyclic washing 3 times, and lyophilization obtains Banlangen Polysaccharide MPEG-PLA microsphere.4 ℃ save backup.
1.2 laboratory animal
Body weight 25gSPF Kunming white mice, purchased from Henan Province's Experimental Animal Center.
1.3 medicine preparation and groupings
Banlangen Polysaccharide MPEG (2000)-PLA block copolymer microsphere 400mg, 200mg, 100mg and blank MPEG (2000)-PLA block copolymer microsphere 400mg, 200mg, 100mg that accurate weighing is prepared, add 2ml tri-distilled water standardize solution, be made into concentration and be followed successively by the administration concentration of 200mg/ml, 100mg/ml, 50mg/ml.Precision takes the Banlangen Polysaccharide 14mg of preparation in test one, adds 7ml tri-distilled water standardize solution, and being made into concentration is the test Drug level of 2mg/ml.Prepare again the normal saline of 1000ml.All adopt 121 ℃, high pressure 30min sterilizing.
1.4 intraperitoneal injection of drugs
32 Kunming female mices are divided into 8 groups at random, inject successively 0.2ml Banlangen Polysaccharide MPEG (2000)-PLA block copolymer microsphere and blank MPEG (2000)-PLA block copolymer microsphere high concentration group (200mg/ml), middle concentration group (100mg/ml), low concentration group (50mg/ml), Banlangen Polysaccharide group and normal saline group for the 1st group to the 8th group, wherein Banlangen Polysaccharide and normal saline group successive administration 3 days, carries sugared microsphere and a blank microsphere group administration 1 day.
Detection and the variation tendency of 1.5 mouse blood immunocyte indexes
Measure the numerical value of the haematogenic immunity cell index of the 7th day, 14 days two time points after each sample administration of each test group with Hematometer.
Total IgG horizontal detection and variation tendency in 1.6 mouse bloods
Operate the 7th day, the 14th day mice serum total IgG level that detect respectively administration by the requirement of test kit description.
2 results
2.1 each test group haematogenic immunity cell index results
Asepticly take each test group mice periphery anticoagulation, detect the wherein index such as leukocyte, total lymphocyte count with Hematometer, carry the total white blood cells of 3 concentration groups of sugared microsphere and Banlangen Polysaccharide group a little more than blank microsphere group and normal saline group, but it is not remarkable respectively to organize difference; The total lymphocyte count of carrying sugared microsphere 20mg group and Banlangen Polysaccharide group is significantly higher than other each group and normal saline matched groups (P ﹤ 0.05), and other are respectively organized and remarkable (P ﹥ 0.05) (the results are shown in Table 5) of total lymphocyte count difference of normal saline group.
Administration the 14th day, carry the total white blood cells of 3 concentration groups of sugared microsphere and Banlangen Polysaccharide group a little more than blank microsphere group and normal saline group, but it is not remarkable respectively to organize difference; The total lymphocyte count of carrying sugared microsphere 20mg group and Banlangen Polysaccharide group is significantly higher than other each group and normal saline matched groups (P ﹤ 0.05) (the results are shown in Table 6).
The 7th day each test group leukocyte of table 5 administration and lymphocyte average and diversity ratio are
Note: in same column, same letter represents that group difference is not remarkable, and different letter representation group differences are remarkable.
The 14th day each test group leukocyte of table 6 administration and lymphocyte average and diversity ratio are
Note: in same column, same letter represents that group difference is not remarkable, and different letter representation group differences are remarkable.
Total IgG horizontal detection result in 2.2 mouse bloods
As can be seen from Table 7, administration is after 7 days, and the total IgG level of carrying sugared microsphere 20mg group is higher, is only second to Banlangen Polysaccharide group, and higher than carrying sugared microsphere 40mg and 10mg group, and apparently higher than blank microsphere group and normal saline group.And administration the 14th day, the total IgG level of carrying sugared microsphere 40mg, 20mg and 10mg group is high compared with Radix Isatidis holosaccharide group, significant difference, and be significantly higher than blank microsphere and normal saline group, wherein the highest to carry sugared microsphere 20mg group; The total IgG level of blank 3 groups of microsphere and normal saline group is still lower, and 4 group differences are not remarkable; But on the whole, in mouse blood, total IgG level is reducing.
Relatively (unit: ug/ml) of the total IgG level error opposite sex in different time mouse blood after table 7 administration
Note: in same column, same letter represents that group difference is not remarkable, and different letter representation group differences are remarkable.
Test example 3
Radix Isatidis crude polysaccharides MPEG (2000)-PLA polymer microballoon is on the LT impact of In vitro culture chicken spleen
1 materials and methods
The preparation of 1.1 years Radix Isatidis crude polysaccharides MPEG (2000)-PLA polymer microballoons
Adopt multi-emulsion method to prepare Banlangen Polysaccharide MPEG (2000)-PLA polymer microballoon: accurately to take 1.2g and dry to the Banlangen Polysaccharide of constant weight and be dissolved in 20ml distilled water, fully stir it is dissolved completely, then getting 10ml polysaccharide solution joins in the oil phase of 20ml containing MPEG (2000)-PLA dichloromethane solution of 100ug/ml, under 16000rpm high-speed stirred, obtain milky colostric fluid, colostric fluid is joined to 100ml containing in the PVA aqueous solution of 2% ratio, high-speed stirred 3min, the speed with 100rpm on magnetic stirring apparatus that subsequently double emulsion is placed on stirs until organic solvent is evaporated completely, approximately need 12h.Finally, the centrifugal 15min of speed by last milky white liquid with 4500rpm, abandons supernatant, gets precipitation, and precipitation is after tri-distilled water cyclic washing 3 times, and lyophilization obtains Banlangen Polysaccharide MPEG-PLA microsphere.4 ℃ save backup.
1.2 laboratory animal
20 week age Sanhuang chicken, purchased from Agricultural University Of He'nan's herding station.
1.3 medicine preparation and groupings
Accurate weighing Banlangen Polysaccharide MPEG (2000)-PLA block copolymer microsphere 6mg, 3mg, 1.5mg and blank MPEG (2000)-PLA block copolymer microsphere 3mg, add tri-distilled water 10ml volumetric flask standardize solution, be made into concentration and be followed successively by the administration concentration of 600ug/ml, 300ug/ml, 150ug/ml and 300ug/ml.Precision takes the Banlangen Polysaccharide 3mg of preparation in test one, adds tri-distilled water and dissolves standardize solution in the volumetric flask of 10ml, and being made into concentration is the test Drug level of 300ug/ml.Precision takes concanavalin A, Con A (ConA) 1mg again, adds tri-distilled water and dissolves standardize solution in the volumetric flask of 100ml, and concentration is 10ug/ml.Prepare again the normal saline of 1000ml.Except ConA test group is with the degerming of 0.22um membrane filtration, other 6 groups all adopt 121 ℃, high pressure 30min degerming.Every group is repeated 5 holes.
1.4 chicken lymphocyte transformation tests (WST-8 method)
The aseptic chicken spleen of winning, be placed in the sterile petri dish that fills Hank`s liquid, wash the peplos of removing afterwards surface coverage for 2 times, spleen is shredded with shears, in impouring dismembyator, grind again, use again 8 layers of filtered through gauze, getting filtrate is added in the centrifuge tube that fills equal-volume lymphocyte separation medium lentamente, do not stir and make muddy, the centrifugal 10min of 2000rpm, abandon supernatant, observe sedimentation cell volume, add erythrocyte cracked liquid with 9:1 volume ratio, leave standstill 2min, 2000rpm recentrifuge 8min, abandon supernatant, add immediately the Hank`s liquid of 9ml, mix, again with the centrifugal 5min of 2000rpm, abandon supernatant, again with Hank`s liquid washing 2 times, last centrifugal front counting, after use RPMI1640 complete culture solution resuspended, be diluted to 5 × 106/ml.
Lymphocyte suspension obtained above is added on 96 porocyte culture plates, every hole 90ul, then every hole adds each test group medicine 100ul preparing in step 1.3, adds subsequently 10ul mitogen ConA, establishes ConA control wells, cell contrast and culture fluid zeroing hole simultaneously.Culture plate is put into the incubator of 37 ℃, saturated humidity and 5%CO2 and cultivated 48h, cultivate and finish the CCK-8 solution (including the test kit of WST-8) that the every hole of front 4h adds 10ul, then continue to cultivate 1h, measure absorbance at 450nm.
2 results
As shown in Table 8, the OD450 value of the Banlangen Polysaccharide microsphere group of Banlangen Polysaccharide and variable concentrations is all significantly higher than blank microsphere group and normal saline group, wherein Banlangen Polysaccharide group is significantly higher than Banlangen Polysaccharide microsphere group, and not remarkable with positive control ConA group difference.
Table 8 Banlangen Polysaccharide microsphere is on the LT impact of In vitro culture chicken spleen
Note: in same column, same letter represents that group difference is not remarkable, and different letter representation group differences are remarkable.
Above-mentioned is illustrative rather than determinate with reference to embodiment, therefore in the variation and the modification that do not depart under general plotting of the present invention, within should belonging to protection scope of the present invention.
The above, be only the specific embodiment of the present invention, but protection scope of the present invention is not limited to this, and any variation of expecting without creative work or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.
Claims (6)
1. a Banlangen Polysaccharide MPEG-PLA method for preparing polymer micro, adopt multi-emulsion method to prepare Banlangen Polysaccharide MPEG-PLA block copolymer microsphere, it is characterized in that, concrete steps are: polysaccharide solution is joined in the oil phase of MPEG-PLA dichloromethane solution, under high-speed stirred, obtain milky colostric fluid, colostric fluid is joined in PVA aqueous solution, high-speed stirred 2--5min, the speed with 50--200rpm on magnetic stirring apparatus that subsequently double emulsion is placed on stirs until organic solvent is evaporated completely, finally, the centrifugal 15min of speed by last milky white liquid with 3000--5000rpm, abandon supernatant, get precipitation, precipitation is after tri-distilled water cyclic washing 3 times, lyophilization obtains Banlangen Polysaccharide MPEG-PLA microsphere.
2. preparation method as claimed in claim 1, is characterized in that, described MPEG-PLA block polymer adopts the MPEG-PLA that molecular weight is 1000-5000, and in MPEG-PLA dichloromethane solution, MPEG-PLA block copolymerization substrate concentration is 100ug/ml.
3. preparation method as claimed in claim 1, is characterized in that, described Banlangen Polysaccharide body concentration of aqueous solution is 0.06g/ml.
4. preparation method as claimed in claim 1, is characterized in that, described PVA concentration of aqueous solution is 0.5%.
5. preparation method as claimed in claim 1, is characterized in that, described MPEG-PLA dichloromethane solution: Banlangen Polysaccharide aqueous solution: PVA aqueous solution volume ratio is 1-10:1-10:1-10.
6. preparation method as claimed in claim 1, is characterized in that, described high-speed stirred speed is 10000--30000rpm.
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| CN115534471A (en) * | 2022-10-10 | 2022-12-30 | 上海同新服材新材料科技有限公司 | Biodegradable express delivery bag containing plant polysaccharide and preparation method thereof |
| CN115534471B (en) * | 2022-10-10 | 2024-05-10 | 上海同新服材新材料科技有限公司 | Biodegradable express bag containing plant polysaccharide and preparation method thereof |
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