CN100571781C - Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof - Google Patents
Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof Download PDFInfo
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- CN100571781C CN100571781C CN 200710057173 CN200710057173A CN100571781C CN 100571781 C CN100571781 C CN 100571781C CN 200710057173 CN200710057173 CN 200710057173 CN 200710057173 A CN200710057173 A CN 200710057173A CN 100571781 C CN100571781 C CN 100571781C
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Abstract
The present invention relates to a kind of Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof.Biotinylated pulullan polysaccharide nanometer grains be with biotinylation pulullan polysaccharide derivant at organic solvent dissolution, add again and generate suspension in the entry, suspension is dialysed then, ultrafiltration and concentration, lyophilization gets final product, mean diameter is the 100-300 nanometer.Only need to add in the preparation process even not add any surfactant on a small quantity.Mild condition, safety.The nano-particle surface of preparation is smooth, regular.The Biotinylated pulullan polysaccharide nanometer grains of the present invention's preparation has the ability of specific bond tumor cell, is a kind of tumor-targeted carrier material that better has.
Description
Technical field
The present invention relates to field of medicaments, particularly a kind of Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof.
Technical background
Pulullan polysaccharide (Pullulan) Chinese also is translated into pullulan, pullulan, general poly-polysaccharide or pulullan.It is the extracellular polysaccharide of the mould generation of falx of sprouting, and with α-1, it is main that the 6-glycosidic bond constitutes the homotype polysaccharide in conjunction with maltose, be glucose by α-1, the 4-glycosidic bond is combined into maltotriose, two ends are again with α-1, the 6-glycosidic bond is with other maltotriose combination, and macromolecule polysaccharide so repeatedly is formed by connecting.α-1,4-glycosidic bond same α-1, the ratio of 6-glycosidic bond is 2: 1, the degree of polymerization (D.P) is 100-5000.Commodity pulullan mean molecule quantity 2 * 10
5, approximately form by 480 maltotrioses.This material is nontoxic, no mutagenic action, and colorless and odorless, edible, have excellent biological compatibility, can be degraded by the body endo-amylase, be a kind of excellent drug carrier material.Its aqueous solution viscosity is starkly lower than other polysaccharide solution, makes it more help practical application.Research shows that also pulullan polysaccharide can combine with the galactosylated acceptor of surface of hepatocytes, can be the medicine liver target new material is provided.But, because the water solublity of pulullan polysaccharide is extremely strong, so want in aqueous solution, to prepare can medicine carrying microgranule, must in preparation process, add cross-linking agent, as glutaraldehyde etc., these deleterious cross-linking agent are difficult to thoroughly remove at last handling process, will cause final product to have certain toxicity.In recent years, synthetic hydrophobically modified pulullan polysaccharide derivant has been widely used in the delivery system of number of chemical medicine, protein drug and genomic medicine as pharmaceutical carrier.
Biotin (biotin) claims biotin again, molecular formula C
10H
16N
2O
3S, chemical structural formula:
Biotin is a kind of water soluble vitamins of needed by human, but human body can not self be synthetic, must replenish by extraneous.Studies show that further the nearly all cell surface of human body all has biotin receptor expression, especially tumor cell.Tumor cell increases fast needs a large amount of trophic factors, and the biological example element is so cell surface meeting overexpression can form one like this and more normally organize the higher relatively zone in conjunction with biotin in conjunction with the receptor of biotin around tumor cell.Utilize these characteristics the target spot of biotin receptor as a kind of specific recognition can be had the tumor cell targeting by make material in conjunction with biotin.
Recent study shows that the pulullan polysaccharide derivant of hydrophobically modified can be self-assembled into nanoparticle by hydrophobic interaction in aqueous solution, and has been widely used in various chemicalses, the research of protein drug and genomic medicine.Nanoparticle can increase medicine in the lesions position time of staying as pharmaceutical carrier, reaches the purpose of slow release and controllable release; Method by chemical bonding is attached to nanoparticle surface to the functional groups that some have targeting or control drug release simultaneously, can realize the purpose of targeting and control sustained-release administration.The material that can be used for preparing nanoparticle must have good biocompatibility, biodegradable, and preparation method is easy.
Summary of the invention
The present invention aims to provide a kind of Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof.It is the modification to the pulullan polysaccharide carrier material, characteristics in conjunction with pulullan polysaccharide and biotin, the biotinylation pulullan polysaccharide derivant of the present invention's preparation, not only be easy in water solublity, form nanoparticle, particle size distribution range is narrow, and smooth surface is regular, and have the ability of specific bond tumor cell, be a kind of tumor-targeted carrier material that better has.
The advantage of preparation method of the present invention is that nanoparticle can directly form in aqueous solution, the organic reagent of introducing can eliminate by the method for rotary evaporation or dialysis, only needs to add in the preparation process even not add any surfactant, mild condition, safety on a small quantity.
Biotinylated pulullan polysaccharide nanometer grains provided by the invention be with biotinylation pulullan polysaccharide derivant at organic solvent dissolution, add again and generate suspension in the aqueous solution, suspension is dialysed then, ultrafiltration and concentration, lyophilization gets final product.Mean diameter is the 100-300 nanometer, coefficient of dispersion 0.002-0.250.
The step of Biotinylated pulullan polysaccharide nanometer grains preparation method of the present invention is as follows:
1) at first be in organic solvent, biotin, dicyclohexylcarbodiimide and 4-dimethylamino pyridine and pulullan polysaccharide reaction, through the biotinylation pulullan polysaccharide derivant that dialysis prepares, the biotin substitution value is 20-85%.
2) biotinylation pulullan polysaccharide derivant is dissolved in (organic facies) in the organic solvent fully, stirs down and is added to (water) in the aqueous solution again, forms the nanometer suspension; Water can be water or water and ethanol, water and acetone mixed solution.Water: ethanol (volume ratio)=1: 0.5～4, water: acetone (volume ratio)=1: 0.5～4; Biotinylation pulullan polysaccharide derivant quality concentration is 0.5-10% in the organic solvent;
3) with 2) in the nanometer suspension in 1-10L water, dialysed 3-12 hour;
4) nanometer suspension ultrafiltration and concentration adds the lyophilization of 1-5% freeze drying protectant.Ultrafiltration pipe molecular cut off 30,000, revolution 1000-1800rpm, time 10-20 minute.
Organic solvent of the present invention is dimethyl sulfoxide (DMSO); Can contain surface active agent polyvinyl alcohol (PVA molecular weight 30,000-70,000) in the aqueous solution, PVA concentration: 0.1-4%; Freeze drying protectant is: mannose, sorbitol, glucose, lactose or sucrose.Organic facies: water=1: 1～20 (volume ratio).
The Biotinylated pulullan polysaccharide nanometer grains of the present invention's preparation has the ability of specific bond tumor cell, is a kind of tumor-targeted carrier material that better has.
The advantage of Biotinylated pulullan polysaccharide nanometer grains of the present invention and preparation method thereof is only to use in the preparation process mechanical agitation of low speed, nanoparticle can directly form in aqueous solution, the organic reagent of introducing can eliminate by the method for rotary evaporation or dialysis, only needs to add in the preparation process even not add any surfactant on a small quantity.Mild condition, safety.The nano-particle surface of preparation is smooth, regular.Mean diameter is the 100-300 nanometer, and particle size distribution range is narrow.
Description of drawings:
Fig. 1, Biotinylated pulullan polysaccharide nanometer grains transmission electron microscope (TEM) photo.
Fig. 2, Biotinylated pulullan polysaccharide nanometer grains particle size distribution figure.
The specific embodiment
Embodiment 1:
Get biotin 2.44g, dicyclohexylcarbodiimide (DCC) 2.06g and 4-dimethylamino pyridine (DMAP) 611mg add dissolving fully in the 50ml anhydrous dimethyl sulphoxide (DMSO), and react 1 hour.Subsequently, the 244mg pulullan polysaccharide is joined in the above-mentioned reactant liquor reaction 6 days, be reflected under the magnetic agitation and carry out.After finishing, reaction, removes the sediment D CU in the reactant liquor with reactant liquor buchner funnel sucking filtration three times.The every 25ml of filtrate is put into a bag filter, again bag filter is put into 3L deionized water dialysis 6 days, changed water once in per 30 minutes.After dialysis finishes the white depositions in the bag filter is taken out, use the buchner funnel sucking filtration 3 times, the white solid on the funnel is collected, with 200ml washed with dichloromethane 3 times, reuse deionized water wash 3 times is after the drying, obtain white solid matter, the biotin substitution value is 81%.
The substitution value assay method is as follows: accurately take by weighing product 50mg, add dissolving with hydrochloric acid and be settled to 20ml.Hydrochloric acid solution is measured the S constituent content by inductively-coupled plasma spectrometer (IPC), again according to formula:
Substitution value %=S element (molal quantity)/anhydrous grape saccharide residue (molal quantity) * 100
Calculate the biotin substitution value.
Embodiment 2:
The biotinylation pulullan polysaccharide 10mg that gets embodiment 1 preparation is dissolved among the 2mlDMSO fully, under agitation 2mlDMSO solution is joined in the 4ml deionized water, forms rapidly to have light blue opalescent nanometer suspension.Pour the nanometer suspension in the bag filter (molecular cut off 8,000-14,000), dialysis is 8 hours in the 3L deionized water.It is 30,000 ultrafiltration pipes that nanometer suspension after the dialysis is used molecular cut off, carries out ultrafiltration and concentration under 1200rpm in centrifugal 15 minutes.In ultrafiltrate, add 5% mannitol and carry out lyophilization, obtain the white powder Biotinylated pulullan polysaccharide nanometer grains.Get a certain amount of white powder and be dispersed in again in the certain volume deionized water, get scattered nanometer suspension and put into the laser particle size analyzer sample cell and carry out granularmetric analysis.Recording mean diameter is 123.6 ± 0.7 nanometers, the coefficient of dispersion 0.094.Fig. 1, Biotinylated pulullan polysaccharide nanometer grains transmission electron microscope (TEM) photo.Fig. 2, Biotinylated pulullan polysaccharide nanometer grains particle size distribution figure.
Embodiment 3:
Get biotinylation pulullan polysaccharide 10mg and be dissolved in fully among the 2mlDMSO, under the low speed magnetic agitation 2mlDMSO drips of solution is added to the aqueous phase that 5ml deionized water and 5ml ethanol mixture form, formation has light blue opalescent nanometer suspension rapidly.Pour the nanometer suspension in the bag filter (molecular cut off 8,000-14,000), dialysis is 8 hours in the 3L deionized water.It is 30,000 ultrafiltration pipes that nanometer suspension after the dialysis is used molecular cut off, carries out ultrafiltration and concentration under 1200rpm in centrifugal 15 minutes.In ultrafiltrate, add 5% mannitol and carry out lyophilization, obtain the white powder Biotinylated pulullan polysaccharide nanometer grains.Mean diameter is 170.7 ± 0.5 nanometers, the coefficient of dispersion 0.065.
Claims (2)
1, a kind of preparation method of Biotinylated pulullan polysaccharide nanometer grains is characterized in that comprising the steps:
1) at first be in the organic solvent dimethyl sulfoxide, biotin, dicyclohexylcarbodiimide and 4-dimethylamino pyridine and pulullan polysaccharide reaction are through the biotinylation pulullan polysaccharide of dialysing and preparing;
2) the biotinylation pulullan polysaccharide is dissolved in fully and forms organic facies in the dimethyl sulfoxide, stirs to be added to down again and mixes in the aqueous phase solution, forms the nanometer suspension; The mass concentration of biotinylation pulullan polysaccharide is 0.5-10% in the described dimethyl sulfoxide; Described water is the water and the dehydrated alcohol of 1: 1 (v/v);
3) with step 2) in the nanometer suspension in 1-10L water, dialysed 3-12 hour;
4) nanometer suspension ultrafiltration and concentration adds the lyophilization of 1-5% freeze drying protectant mannitol; Ultrafiltration pipe molecular cut off 30,000 in the described ultrafiltration and concentration, revolution 1000-1800rpm, time 10-20 minute;
The mean diameter of the nanoparticle that obtains is the 123-170 nanometer, coefficient of dispersion 0.065-0.094.
2, preparation method according to claim 1 is characterized in that: step 2) to contain the surfactant molecule amount in the aqueous phase solution be 30,000-70,000 polyvinyl alcohol, concentration: 0.1-4%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104857560A (en) * | 2015-05-26 | 2015-08-26 | 西北大学 | Injectable hydrogel prepared from carboxylated pullulan and preparation method of injectable hydrogel |
| CN109984365B (en) * | 2019-05-13 | 2021-07-09 | 云南中烟工业有限责任公司 | Application of biotinylation pullulan polysaccharide derivative in improving biotin transfer rate of cigarette cut tobacco |
| CN110692962A (en) * | 2019-10-12 | 2020-01-17 | 山东省食品发酵工业研究设计院 | Preparation method of winter jujube composite powder containing DHA algae oil nanoparticles |
| CN111494326A (en) * | 2020-04-11 | 2020-08-07 | 南京正大天晴制药有限公司 | Apixaban tablet and preparation method thereof |
| CN111557928A (en) * | 2020-05-15 | 2020-08-21 | 南通大学 | Codonopsis pilosula acetylene glycoside-loaded pullulan nano microspheres for treating spinal cord injury |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10568839B2 (en) | 2011-01-11 | 2020-02-25 | Capsugel Belgium Nv | Hard capsules |
| US11319566B2 (en) | 2017-04-14 | 2022-05-03 | Capsugel Belgium Nv | Process for making pullulan |
| US11576870B2 (en) | 2017-04-14 | 2023-02-14 | Capsugel Belgium Nv | Pullulan capsules |
| US11878079B2 (en) | 2017-04-14 | 2024-01-23 | Capsugel Belgium Nv | Pullulan capsules |
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