CN1317035C - Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group - Google Patents
Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group Download PDFInfo
- Publication number
- CN1317035C CN1317035C CNB2005100155747A CN200510015574A CN1317035C CN 1317035 C CN1317035 C CN 1317035C CN B2005100155747 A CNB2005100155747 A CN B2005100155747A CN 200510015574 A CN200510015574 A CN 200510015574A CN 1317035 C CN1317035 C CN 1317035C
- Authority
- CN
- China
- Prior art keywords
- polysaccharide
- solution
- hydroformylation
- hydrazides
- dissolved
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present invention discloses a method for assembling a multi-biological functional factor on the surface of a particle based on a hydrazide group, which belongs to an assembling technique for a multi-biological functional factor. The method comprises the following steps: subjecting glucan, starch or pullulan to amphiphilic modification and hydrazidation, or further using the hydrazide group or biotin coupling to realize partial biotinylation; preparing drug-carrying microballoons from amphiphilic modified hydrazidated polysaccharides or part of the biotinylation derivative, liposolubilc drug and biological degradable polymers, and separating and dispersing microballoon aqueous solution to buffer solution so as to form microballoon solution; dripping the microballoon solution to the buffer solution dissolved with biological factors for hatching, and using deionized water for scouring so as to obtain a particle of which the surface is assembled with a multi-biological functional factor. The present invention has simple operation, the surface multi-biological particle has important application foreground in the aspects such as drug delivery, controlled release, slow release, etc.
Description
Technical field
The present invention relates to a kind of method for assembling multi-biological functional factor on micro-particle surface, belong to multi-biological functional factor assemble method based on hydrazide group.
Background technology
Nanoparticle or microsphere be because its smaller particle size, the long body-internal-circulation time, and medicine played the effect of protection and controlled release, slow release, be the ideal carrier of medicine.Yet ideal disease treatment as treatment of cancer, requires medicine only to act on pathological tissues or cell, and to not influence of normal structure, promptly the targeting of medicine discharges; Some therapy then requires carrier both to have stronger load capacity, has good targeting again, as the transportation of epidermal growth factor, endothelial cell growth factor (ECGF), nerve growth factor.Above-mentioned various functional factor need combine with carrier, though and Biodegradable materials such as polylactic acid are the particle or the microsphere of substrate has excellent biological compatibility, lack the reactable group on its structure, combine with carrier thereby limited functional factor.Be rich in the reactable group in natural macromoleculars such as glucosan, Pullulan, starch, hyaluronic acid and the modifier structure thereof, and have excellent biological compatibility, degradability, the surface has the effect in the extension body internal recycle cycle similar to Polyethylene Glycol with the nanoparticle of polyose modification and microsphere.
The design of pharmaceutical carrier and target function factor combination is very crucial to the successful implementation of targeting preparation.Most in recent years research is confined to single functionalization of nanoparticle and microsphere surface, promptly only connects a kind of functional factor at nanoparticle or microsphere surface.Connected mode commonly used comprises: covalency such as thioether bond, disulfide bond, ester bond, amido link, acylhydrazone key, schiff alkali are coupled to be realized being connected with the biotin-avidin physical action.Single functionalized particles that said method is realized can not well be dealt with problems in some cases, as the nanoparticle targeting effect in vivo that is used for treatment of cancer is subjected to various biological barriers (as blood brain barrier, BBB) influence, hinder them and arrived targeting moiety smoothly, even their surface is connected with targeting substance, a kind of effective solution is introduced the multiple functionalization factor at particle surface exactly, wherein certain factor guiding particle passes through such as biological barriers such as BBB, and other factor then guides particle to arrive targeting moiety smoothly; The transportation of the and for example above-mentioned epidermal growth factor of mentioning, endothelial cell growth factor (ECGF), nerve growth factor also requires targeting in load, also can solve by the functionalization of the multi-biological factor at carrier surface.
Summary of the invention
The purpose of this invention is to provide a kind of method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group.This method is the binding site of biological molecule with the hydrazide group of microparticle surfaces and based on the biotin of hydrazide group, by easy reaction of acylhydrazone key and biotin-avidin physical action multiple bioactive molecule is coupled to microparticle surfaces, and the targeted drug of guiding medicine carrying microgranule discharges.
The present invention is realized that by following technical scheme a kind of method for assembling multi-biological functional factor on micro-particle surface based on aldehyde radical is characterized in that comprising the steps:
1. the preparation of amphiphilic polysaccharide derivates
1) preparation of hydroformylation polysaccharide: under 4 ℃ of lucifuge conditions, in aqueous systems, press sodium metaperiodate (NaIO
4) with compound of polysaccharide in the mol ratio 1: 2～50 of the sugar unit reaction 0.5～8h that feeds intake, with reactant liquor with molecular cut off 10,00～40,000 semipermeable membrane to deionized water dialysis 10～144h, lyophilization gets solid product; Described polysaccharide is selected from relative molecular mass 10,000～1,000,000 glucosan or starch or Pullulan.
2) preparation of amphiphilic modification hydroformylation polysaccharide
A. the preparation of cholesterol modification hydroformylation polysaccharide: cholesterol and succinic anhydride 1: 1 in molar ratio～5 are fed intake, and 70 ℃ are reacted 3～8h and generate cholesterol-3-hemisuccinic acid (Chol-Succ), ethyl alcohol recrystallization 1～3 time, vacuum drying under pyridine catalysis; Cholesterol-3-hemisuccinic acid and thionyl chloride 1: 2 in molar ratio～10 are dissolved in the dry chloroform of 10～50mL, 70 ℃ of reaction 1～10h, generate cholesterol-3-hemisuccinic acid acyl chlorides (Chol-Succ acyl chlorides), vacuum steams liquid, redissolves in the dry chloroform of 20mL residual solid stand-by; The hydroformylation polysaccharide that above-mentioned lyophilizing is obtained is dissolved among the dry DMSO of 40mL; drip several triethylamines; by the mol ratio of acyl chlorides and sugar unit is 1: 2～50 to measure the Chol-Succ acyl chlorides and be added dropwise in the hydroformylation polysaccharide solution; nitrogen protection; 80 ℃ of reaction 3～8h; product is to deionized water dialysis 10～144h, and the dialysis solution lyophilization gets cholesterol modification hydroformylation polysaccharide.
B. the preparation of cholic acid modification hydroformylation polysaccharide: the 1g cholic acid is dissolved in 20～60mL ethanol, 40～78 ℃ form Ethyl cholate. under the catalysis of 1～5mL concentrated hydrochloric acid, the alcoholic solution of Ethyl cholate. is slowly being dripped in the alcoholic solution that is dissolved with 0.5～10mL hydrazine hydrate under 40～78 ℃, reaction 3～8h, cooling precipitate ethyl alcohol recrystallization gets cholic acid hydrazides (CAH); 100mg hydroformylation polysaccharide is dissolved in the 8mL water, and CAH is dissolved among the 16mL DMF, and the mixing of two solution obtains a homogeneous system, and the mol ratio of sugar unit and CAH is 1: 2～50 in the system; The pH value of system is adjusted to 4, and room temperature reaction 1～24h, reaction solution pour in a large amount of methanol and be settled out product, filters, and with the methanol thorough washing repeatedly, vacuum drying under the room temperature gets cholic acid modification hydroformylation polysaccharide.
C. the preparation of phenoxy group modification hydroformylation polysaccharide: with antodyne (1,2-epoxy-3-phenoxypropane): the mol ratio of hydroformylation polysaccharide sugar unit is 1: 2～50 to feed intake, room temperature reaction 10-48h in 1M sodium hydroxide (NaOH) solution, with product sedimentation in ethanol, again deionized water is dialysed, 10～144h, lyophilization gets phenoxy group modification hydroformylation polysaccharide.
3) preparation of amphiphilic modification hydrazides polysaccharide and further part biological elementization product
Get a certain amount of amphiphilic modification hydroformylation polysaccharide and be dissolved in N, in the dinethylformamide (DMF), slowly be added drop-wise to the sodium acetate (Na of the adipic dihydrazide of mass concentration 5%, pH=5
2AC) in the buffer solution, with methanol extraction, lyophilization gets amphiphilic modification hydrazides polysaccharide behind room temperature reaction 5～24h.
Press biotin (Biotin) and N-hydroxy-succinamide (NHS) and N, 1: 1.5: 1.5 ratio of N '-dicyclohexylcarbodiimide (DCC) mol ratio is dosed among the dry DMF, 4～25 ℃ of reaction 24h, filter, filtrate dropping to is dissolved with in drying two first class sulfoxide (DMSO) systems of amphiphilic modification hydrazides polysaccharide, reaction 5～24h, with reactant liquor to the deionized water 48h that dialyses, the dialysis solution lyophilization, the amphiphilic modification hydrazides polysaccharide of further part biological elementization.The substitution value of the proportioning control Biotin of control Biotin and amphiphilic modification hydrazides polysaccharide.
2. medicine carrying microballoons preparation:
1) dialysis
A. be 1: 0.5～100: 0.5～100 to be dissolved in the dimethyl sulfoxide (DMSO) or the N of 1～1000 parts by volume altogether by weight with fat-soluble medicine, biodegradable polymers and amphiphilic modification hydrazides polysaccharide, in the dinethylformamide (DMF); Described fat-soluble medicine comprises: carmustine, indometacin, cyclosporin A, Aclacnomycin A, rifampicin, ketoprofen, tamoxifen, erythromycin or albendazole; Described biodegradable polymers comprises: relative molecular mass 5,000～100,000 polylactic acid (PLA), polyglycolic acid (PGA), pla-pcl (PCL) or polylactic acid-polyglycolic acid copolymer (PLGA); Described amphiphilic modification hydrazides polysaccharide is selected from the relative molecular mass 10 through cholesterol, cholic acid or phenoxy group hydrophobically modified, 000～1,000, the hydrazides product of 000 hydroformylation glucosan or hydroformylation starch or hydroformylation Pullulan, or further utilize hydrazide group and biotin in the hydrazides product coupled, realize the product of its part biological elementization.
B. be to use the semipermeable membrane of molecular cut off 7,000～50,000 with above-mentioned solution altogether, obtain the medicine carrying biodegradable polymers microgranule that the surface contains hydrazide group or part biological element deionized water dialysis 1～144h;
2) emulsion method
A. be 1: 0.5～100 to be dissolved in 1～1 altogether by weight with fat-soluble medicine and polymer, form oil phase in the mixed solution of the acetone of 000 parts by volume, dichloromethane or said two devices, described fat-soluble medicine comprises: carmustine, indometacin, cyclosporin A, Aclacnomycin A, rifampicin, ketoprofen, tamoxifen, erythromycin or albendazole; Described biodegradable polymers comprises: relative molecular mass 5,000～200,000 poly 3-hydroxy butyrate, polystyrene, polymethyl methacrylate or nylon.
B. the weight ratio by fat-soluble medicine and amphiphilic modification hydrazides polysaccharide is 1: 0.5～100, amphiphilic modification hydrazides polysaccharide is dissolved in 1～1, in the sodium dihydrogen phosphate-sodium hydrogen phosphate of 000 parts by volume (PBS) buffer solution, get water, described amphiphilic modification hydrazides polysaccharide is selected from through cholesterol, cholic acid or phenoxy group hydrophobically modified relative molecular mass 10,000～1,000, the hydrazides product of 000 hydroformylation glucosan or hydroformylation starch or hydroformylation Pullulan, or further utilize hydrazide group and biotin in the hydrazides product coupled, realize the product of its part biological elementization.
C. the oil phase that step a is made also continues till being stirred to particles solidify under the magnetic force condition at the aqueous phase that quick implantation step b under the stirring of 500～1200 rev/mins of rotating speeds makes.
3. the microsphere aqueous solution that step 1 is made is with 10,000～16, and is centrifugal under the 000rpm, removes supernatant; Again will be from going out the microsphere ultra-sonic dispersion in different buffer solution with parts by volume, carry out coupling with different bioactie agent;
4.1) coupled if utilize carboxyl in free hydrazide group of microsphere surface and the biological function factor structure to carry out chemistry, then under the condition of ice bath, 1: 0.1 by volume～4.0 microspheres solution with pH=4.0～6.02-(N-morpholino) ethyl sulfonic acid (MES) buffer solution environment drop to and are dissolved with two or more biotic factor of 0.1～2.0mg/mL and 0.005～0.5g/mL
1-[3-(dimethylamino) propyl group]-pH=4.0～6.0MES buffer solution of 3-ethyl carbodiimide (EDC) in, hatching 1～24h; Described biotic factor comprises transferrins, rabbit igg, EGF-R ELISA EGFR, TfR CD71, monoclonal antibody OX26, endothelial cell growth factor (ECGF) and nerve growth factor.
2) if by the sugar unit in the biological function factor that has the glycogen structure in some structure is carried out oxidation, realize coupled with the free hydrazide group of microsphere surface again, under 4～25 ℃, 1: 0.1 by volume～4.0 is that the microspheres solution of 4.0～6.0 sodium hydrogen phosphates-sodium citrate buffer solution environment is that 4.0～6.0 sodium hydrogen phosphates-sodium citrate buffer solution drip to the pH that is dissolved with two or more biotic factor of 0.1～2.0mg/mL with pH, hatching 1～5h; Described biotic factor comprises oxidation transferrins, oxidation rabbit igg.
3) if above-mentioned amphiphilic polysaccharide adopts part biological elementization modifier, then 1: 0.1 by volume～4.0 is 7.4～9.18 Na with pH
2CO
3-NaHCO
3The microspheres solution of buffered environment is 7.4～9.18 Na to the pH that is dissolved with two or more Avidin biotic factor of 0.1～2.0mg/mL
2CO
3-NaHCO
3Buffer drips, 37 ℃ of hatching 1～5h; Described biotic factor comprises Avidin transferrins, Avidin rabbit igg, Avidin EGFR, Avidin CD71, Avidin 0X26, Avidin endothelial cell growth factor (ECGF), Avidin nerve growth factor.
With artemia hatching solution 10,000～16, centrifugal under the 000rpm, deionized water wash three times is scattered in the normal saline with parts by volume.
Among the present invention, it is coupled that multifunctional agents is at the assembling mode of microparticle surfaces that the hydrazide group that utilizes carboxyl in the free hydrazide group of microsphere surface and the biological function factor structure or glycogen oxidation to obtain carries out chemistry, or pass through behind the hydrazide group biotinylation and Avidin factor generation physical bond.Utilize among the present invention method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group and preparation method thereof, it is regular to make shape, particle size range 50-1, the microsphere or the granule of the surperficial multi-functional assembling of 000nm and narrow diameter distribution.The present invention is simple to operate, by preparing the microgranule that the surface contains reactive group-hydrazide group, control reaction condition and reactions steps, can assemble two or more the biological function factor at microparticle surfaces, to satisfy different application requirements, prepared surface biological multifunction microgranule has important application prospects at aspects such as drug delivery, controlled release fertilizers.
Description of drawings:
Fig. 1 is the transmission electron microscope photo of the difunctionalization microsphere of embodiment 2 preparations, and mean diameter is about about 250nm, and particle size distribution is narrower, and microsphere surface is extremely smooth;
Fig. 2 is the dynamic light scattering test picture of the difunctionalization microsphere of embodiment 2 preparations;
Fig. 3 is the fluorescence microscope photo of the difunctionalization microsphere of embodiment 2 preparations, and particle solution presents green fluorescence, has proved the combination of the transferrins (Tf) of Fluorescein isothiocyanate (FITC) labelling at particle surface;
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment and accompanying drawing:
Embodiment 1:
1. the preparation of cholesterol hydrophobically modified hydrazides glucosan (Chol-Dex-CHO): (1) is dissolved in 1g cholesterol and 1g succinic anhydride in the 30mL dry pyridine, 70 ℃ are reacted 5h down, ethyl alcohol recrystallization 2 times, vacuum drying 24h gets cholesterol-3-hemisuccinic acid (Chol-Succ); (2) 1g cholesterol-3-hemisuccinic acid is dissolved in the dry chloroform of 20mL, drip the 2mL thionyl chloride to system under 70 ℃, reaction 6h generates cholesterol-3-hemisuccinic acid acyl chlorides (Chol-Succ acyl chlorides), with the reactant liquor evaporate to dryness, add the dry chloroform of 20mL again and redissolve; Under (3) 4 ℃ of lucifuge conditions, the 2g glucosan is dissolved in the 20mL deionized water, presses sodium metaperiodate (NaIO
4) with the mol ratio of sugar unit be 1: 5 reaction 8h that feeds intake, with reactant liquor with molecular cut off 14,000 semipermeable membranes to the deionization 48h that dialyses, lyophilization gets the hydroformylation glucosan; (4) above-mentioned lyophilizing is obtained to such an extent that the hydroformylation polysaccharide is dissolved among the dry DMSO of 40mL, drip several triethylamines, by the mol ratio of acyl chlorides and sugar unit is to measure Chol-Succ acyl chlorides at 1: 10 to be added dropwise in the hydroformylation dextran solution, nitrogen protection, 80 ℃ of reaction 6h, product is to the deionized water 48h that dialyses, the dialysis solution lyophilization, cholesterol modification hydroformylation glucosan; (5) get 0.5g cholesterol modification hydroformylation glucosan and be dissolved in 100mLN, in the dinethylformamide (DMF), slowly be added drop-wise to the sodium acetate (Na that contains the 0.5g adipic dihydrazide of 100mL pH=5
2AC) in the buffer solution, with methanol extraction, lyophilization gets cholesterol modification hydrazides glucosan behind the room temperature reaction 24h.
2. microsphere preparation: (1) takes by weighing 4mg cholesterol hydrophobically modified hydrazides glucosan (Chol-Dex-CHO), and 8mgPLGA 50,000 and 4mg indometacin are dissolved among the 6mLDMSO altogether; (2) room temperature with above-mentioned solution with the semipermeable membrane of molecular cut off 7,000 to the deionized water 48h that dialyses, obtain having the microsphere water solution system of obvious turbid light; (3) with microsphere aqueous solution 16,000rpm is centrifugal, removes supernatant; (4) from going out the microsphere ultra-sonic dispersion in 4mL pH=5.4MES buffer;
3. functionalization: under (1) condition of ice bath, microspheres solution is added drop-wise in the pH=5.4MES buffer that 2mL is dissolved with 2mg EDC, 2mg IgG and 50ug endothelial cell growth factor (ECGF) hatching reaction 12h; Centrifugal and ultrasonic cleaning three times to be removing unconjugated biotic factor and EDC under (3) 16, the 000rpm, the glucan-modified PLGA microsphere of surperficial difunctionalization.
Embodiment 2:
1. the preparation of the cholesterol hydrophobically modified hydrazides glucosan of part biological elementization: (1) preparation cholesterol hydrophobically modified hydrazides glucosan (with example 1); (2) with biotin (Biotin) 0.01mmol and N-hydroxy-succinamide (NHS) 0.015mmol and N, N '-dicyclohexylcarbodiimide (DCC) 0.015mmol is dissolved in the dry N of 5mL, and in the dinethylformamide (DMF), ice bath reaction 24h filters; (3) getting 1mL filtrate drops to 20mL and is dissolved with in dry dimethyl sulfoxide (DMSO) system of 50mg cholesterol modification hydrazides glucosan, room temperature reaction 12h, with reactant liquor to the deionized water 48h that dialyses, the dialysis solution lyophilization, the cholesterol modification hydrazides glucosan of further part biological elementization.
2. microsphere preparation: (1) altogether is dissolved in 4mLDMSO with 8mg PLA 3,0000 and 3mg cyclosporin A the cholesterol hydrophobically modified hydrazides glucosan Chol-Dex-NH-NH-Biotin (containing the hydrazides group) of 4mg part biological elementization; (2) under the room temperature to pH=5.0Na
2AC buffer solution dialysis (cut-off=14000g/mol) 48h forms the stable solution with foul leukorrhea light; (3) solution centrifugal and ultra-sonic dispersion are in 5mL pH=5.0 sodium hydrogen phosphate-sodium citrate buffer solution;
3. functionalization: under (1) ice bath, the microspheres solution room temperature is dropped to 1mL contain 2mg NaIO
4Oxidation and in the pH=5.0 sodium hydrogen phosphate-sodium citrate buffer solution of the transferrins (Tf) of FITC labelling, room temperature hatching 12h, 16, the 000rpm high speed centrifugation, from go out microsphere with buffer ultrasonic cleaning three times to remove not in conjunction with Tf; (2) with the Na of microsphere ultra-sonic dispersion in pH=7.6
2CO
3-NaHCO
3Buffer solution adds the rabbit igg of Avidinization, 37 ℃ of hatching 1h, 16, under the 000rpm centrifugal and ultrasonic cleaning three times removing not in conjunction with rabbit igg, the glucan-modified PLA ultramicron of surperficial difunctionalization.
Embodiment 3:
1. phenoxy group hydrophobically modified hydrazides Pullulan (Ph-Pull-NH-NH
2) preparation: (1) is dissolved in 0.5g hydroformylation Pullulan (preparation technology is with the preparation of the hydroformylation glucosan in the example 1) in 20mL 1M sodium hydroxide (NaOH) solution, with antodyne (1,2-epoxy-3-phenoxypropane) by antodyne: the unitary mol ratio of hydroformylation pulullan is to feed intake at 1: 10, room temperature reaction 24h, with product sedimentation in ethanol, again deionized water is dialysed, 24h, lyophilization gets phenoxy group modification hydroformylation Pullulan; (2) get 0.5g phenoxy group modification hydroformylation Pullulan and be dissolved in 100mL N, in the dinethylformamide (DMF), slowly be added drop-wise to the sodium acetate (Na that contains the 0.5g adipic dihydrazide of 100mLpH=5
2AC) in the buffer solution, with methanol extraction, lyophilization gets phenoxy group modification hydrazides Pullulan behind the room temperature reaction 24h.
2. microsphere preparation: (1) takes by weighing 4mg Ph-Pull-NH-NH
2Be dissolved in altogether among the 4mL DMF with 8mg polyglycolic acid 50,000 and 1mg Aclacnomycin A; (2) under the condition of ice bath, to pH=5.0Na
2AC buffer solution dialysis 48h forms the stable solution with foul leukorrhea light; (3) solution centrifugal and ultra-sonic dispersion are in 5mL pH=5.4MES buffer;
3. functionalization: microspheres solution is dripped in 2mL is dissolved with the pH=5.4MES buffer of 100ug EGFR (EGF-R ELISA), 100ul CD71 and 0.01g EDC, ice bath hatching 12h, 16, behind the 000rpm high speed centrifugation from go out microsphere with buffer ultrasonic cleaning three times removing unconjugated biotic factor, the Pullulan of surperficial difunctionalization is modified the polyglycolic acid ultramicron.
Embodiment 4:
1. cholic acid hydrophobically modified hydrazides starch (Cholic-Sta-NH-NH
2) preparation: (1) is dissolved in the 1g cholic acid in the 30mL ethanol, 78 ℃ form Ethyl cholate. under the catalysis of 4mL concentrated hydrochloric acid, the alcoholic solution of Ethyl cholate. is slowly being dripped in the alcoholic solution that is dissolved with the 5mL hydrazine hydrate under 78 ℃, reaction 8h, cooling precipitate ethyl alcohol recrystallization gets cholic acid hydrazides (CAH); (2) 100mg hydroformylation starch (preparation technology is with the hydroformylation glucosan in the example 1) is dissolved in the 8mL water, CAH is dissolved among the 16mL DMF, and the mixing of two solution obtains a homogeneous system, and the mol ratio of sugar unit and CAH is 1: 2～50 in the system; The pH value of system is adjusted to 4, and room temperature reaction 8h, reaction solution pour in a large amount of methanol and be settled out product, filters, and with the methanol thorough washing repeatedly, vacuum drying under the room temperature gets cholic acid modification hydroformylation starch; (3) get 0.5g cholic acid modification hydroformylation starch and be dissolved in 100mLN, in the dinethylformamide (DMF), slowly be added drop-wise to the sodium acetate (Na that contains the 0.5g adipic dihydrazide of 100mL pH=5
2AC) in the buffer solution, with methanol extraction, lyophilization gets cholic acid modification hydrazides starch behind the room temperature reaction 24h.
2. microsphere preparation: (1) is dissolved in (v/v=1: 1) obtain oil phase in 15mL acetone and the dichloromethane mixed solution with the 40mg polystyrene; (2) with 35mg Cholic-Sta-NH-NH
2Be dissolved in the pH=7.4PBS buffer solution of 50mL, stir 6h under the magnetic force condition, 800 rev/mins of rotating speeds get water; (3) with oil phase inject fast aqueous phase under the stirring of 800 rev/mins of rotating speeds and continue be stirred under the magnetic force condition ultra-micro carrier solidify till; (4) the microsphere liquid that step (3) is obtained is 10,000rpm centrifugalize, washing; (5) solidified microsphere is scattered in the 10mL pH=5.4MES buffer;
3. functionalization: under (1) condition of ice bath, microspheres solution is added drop-wise in the pH=5.4MES buffer that 3mL is dissolved with 3mg EDC, 2mg IgG and 2mg Tf hatching reaction 8h; Centrifugal and ultrasonic cleaning three times to be removing unconjugated biotic factor and EDC under (7) 16, the 000rpm, the starch of surperficial difunctionalization is modified polystyrene microsphere.
Embodiment 5:
1. cholic acid hydrophobically modified hydrazides glucosan (Cholic-Dex-NH-NH
2) preparation: preparation technology is with cholic acid hydrophobically modified hydrazides starch (Cholic-Sta-NH-NH in the example 4
2) preparation, only polysaccharide is changed to glucosan.
2. microsphere preparation: (1) is dissolved in (v/v=1: 3) obtain oil phase in 20mL acetone and the dichloromethane mixed solution with the 40mg poly 3-hydroxy butyrate; (2) with 40mg Cholic-Dex-NH-NH
2Be dissolved in the pH=7.4PBS buffer solution of 50mL, stir 6h under the magnetic force condition, 800 rev/mins of rotating speeds get water; (3) with oil phase inject fast aqueous phase under the stirring of 800 rev/mins of rotating speeds and continue be stirred under the magnetic force condition microsphere solidify till; (4) the microsphere liquid that step (3) is obtained is 10,000rpm centrifugalize, washing; (5) solidified microsphere is scattered in the 10mL pH=5.4MES buffer;
3. functionalization: under (1) condition of ice bath, microspheres solution is added drop-wise in the pH=5.4MES buffer that 2mL is dissolved with 1mg EDC, 25u1 Ox26 and 2mg Tf hatching reaction 8h; Centrifugal and ultrasonic cleaning three times to be removing unconjugated biotic factor and EDC under (7) 16, the 000rpm, the glucan-modified poly 3-hydroxy butyrate microsphere of surperficial difunctionalization.
Claims (1)
1. method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group is characterized in that may further comprise the steps:
1) preparation of amphiphilic polysaccharide derivates
(1) preparation of hydroformylation polysaccharide: under 4 ℃ of lucifuge conditions, in aqueous systems by the mol ratio 1: 2～50 of sugar unit in sodium metaperiodate and the compound of polysaccharide reaction 0.5～8h that feeds intake, with reactant liquor molecular cut off 10,00～40,000 semipermeable membrane is to deionized water dialysis 10～144h, and lyophilization gets solid product; Described polysaccharide is selected from relative molecular mass 10,000～1,000,000 glucosan or starch or Pullulan;
(2) preparation of amphiphilic modification hydroformylation polysaccharide
A. the preparation of cholesterol modification hydroformylation polysaccharide: cholesterol and succinic anhydride 1: 1 in molar ratio～5 are fed intake, and 70 ℃ are reacted 3～8h and generate cholesterol-3-hemisuccinic acid, ethyl alcohol recrystallization 1～3 time, vacuum drying under pyridine catalysis; Cholesterol-3-hemisuccinic acid and thionyl chloride 1: 2 in molar ratio～10 are dissolved in the dry chloroform of 10～50mL, 70 ℃ of reaction 1～10h, generate cholesterol-3-hemisuccinic acid acyl chlorides, vacuum steams liquid, redissolves in the dry chloroform of 20mL residual solid stand-by; The hydroformylation polysaccharide that above-mentioned lyophilizing is obtained is dissolved in the exsiccant dimethyl sulfoxide of 40mL, drip several triethylamines, by the mol ratio of acyl chlorides and sugar unit is 1: 2～50 to measure cholesterol-3-hemisuccinic acid acyl chlorides and be added dropwise in the hydroformylation polysaccharide solution, nitrogen protection, 80 ℃ of reaction 3～8h, product is to deionized water dialysis 10～144h, and the dialysis solution lyophilization gets cholesterol modification hydroformylation polysaccharide;
B. the preparation of cholic acid modification hydroformylation polysaccharide: the 1g cholic acid is dissolved in 20～60mL ethanol, 40～78 ℃ form Ethyl cholate. under the catalysis of 1～5mL concentrated hydrochloric acid, the alcoholic solution of Ethyl cholate. is slowly being dripped in the alcoholic solution that is dissolved with 0.5～10mL hydrazine hydrate under 40～78 ℃, reaction 3～8h, cooling precipitate ethyl alcohol recrystallization gets the cholic acid hydrazides; 100mg hydroformylation polysaccharide is dissolved in the 8mL water, and the cholic acid hydrazides is dissolved in the N of 16mL, and in the dinethylformamide, the mixing of two solution obtains a homogeneous system, and the mol ratio of sugar unit and cholic acid hydrazides is 1: 2～50 in the system; The pH value of system is adjusted to 4, and room temperature reaction 1～24h, reaction solution pour in a large amount of methanol and be settled out product, filters, and with the methanol thorough washing repeatedly, vacuum drying under the room temperature gets cholic acid modification hydroformylation polysaccharide;
C. the preparation of phenoxy group modification hydroformylation polysaccharide: by the mol ratio of antodyne and hydroformylation polysaccharide sugar unit is 1: 2～50 to feed intake, room temperature reaction 10-48h in the 1M sodium hydroxide solution, with product sedimentation in ethanol, again deionized water is dialysed, 10～144h, lyophilization gets phenoxy group modification hydroformylation polysaccharide;
(3) preparation of amphiphilic modification hydrazides polysaccharide and further part biological elementization product
Get amphiphilic modification hydroformylation polysaccharide and be dissolved in N, in the dinethylformamide, slowly be added drop-wise to mass concentration 5%, pH and be in the sodium acetate buffer solution of 5 adipic dihydrazide, with methanol extraction, lyophilization gets amphiphilic modification hydrazides polysaccharide behind room temperature reaction 5～24h;
Press biotin and N-hydroxy-succinamide and N, 1: 1.5: 1.5 ratio of N-dicyclohexylcarbodiimide mol ratio is dosed to dry N, in the dinethylformamide, 4～25 ℃ of reaction 24h, filter, filtrate dropping to is dissolved with in the exsiccant two first class sulfoxide systems of amphiphilic modification hydrazides polysaccharide, reaction 5～24h, with reactant liquor to the deionized water 48h that dialyses, the dialysis solution lyophilization, get the amphiphilic modification hydrazides polysaccharide of further part biological elementization, with the proportioning of control biotin and amphiphilic modification hydrazides polysaccharide, the substitution value of control biotin;
2) medicine carrying microballoons preparation:
(1) dialysis
A. be 1: 0.5～100: 0.5～100 to be dissolved in the dimethyl sulfoxide or the N of 1～1,000 parts by volume altogether by weight with fat-soluble medicine, biodegradable polymers and amphiphilic modification hydrazides polysaccharide, in the dinethylformamide; Described fat-soluble medicine comprises: carmustine, indometacin, cyclosporin A, Aclacnomycin A, rifampicin, ketoprofen, tamoxifen, erythromycin or albendazole; Described biodegradable polymers comprises: relative molecular mass 5,000～100,000 polylactic acid, polyglycolic acid, polycaprolactone or polylactic acid-polyglycolic acid copolymer; Described amphiphilic modification hydrazides polysaccharide is selected from the relative molecular mass 10 through cholesterol, cholic acid or phenoxy group hydrophobically modified, 000～1,000, the hydrazides product of 000 hydroformylation glucosan or hydroformylation starch or hydroformylation Pullulan, or further utilize hydrazide group and biotin in the hydrazides product coupled, realize the product of its part biological elementization;
B. be to use the semipermeable membrane of molecular cut off 7,000～50,000 with above-mentioned solution altogether, obtain the medicine carrying biodegradable polymers microgranule that the surface contains hydrazide group or part biological element deionized water dialysis 1～144h;
(2) emulsion method
A. be 1: 0.5～100 to be dissolved in 1～1 altogether by weight with fat-soluble medicine and polymer, form oil phase in the mixed solution of the acetone of 000 parts by volume, dichloromethane or said two devices, described fat-soluble medicine comprises: carmustine, indometacin, cyclosporin A, Aclacnomycin A, rifampicin, ketoprofen, tamoxifen, erythromycin or albendazole; Described biodegradable polymers comprises: relative molecular mass 5,000～200,000 poly 3-hydroxy butyrate, polystyrene, polymethyl methacrylate or nylon;
B. the weight ratio by fat-soluble medicine and amphiphilic modification hydrazides polysaccharide is 1: 0.5～100, amphiphilic modification hydrazides polysaccharide is dissolved in 1～1, in the sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution of 000 parts by volume, get water, described amphiphilic modification hydrazides polysaccharide is selected from through cholesterol, cholic acid or phenoxy group hydrophobically modified relative molecular mass 10,000～1,000, the hydrazides product of 000 hydroformylation glucosan or hydroformylation starch or hydroformylation Pullulan, or further utilize hydrazide group and biotin in the hydrazides product coupled, realize the product of its part biological elementization;
C. the oil phase that step a is made also continues till being stirred to particles solidify under the magnetic force condition at the aqueous phase that quick implantation step b under the stirring of 500～1200 rev/mins of rotating speeds makes;
3) with step 2) the microsphere aqueous solution that makes is with 10,000～16, and is centrifugal under the 000rpm, removes supernatant; Again will be from going out the microsphere ultra-sonic dispersion in different buffer solution with parts by volume, carry out coupling with different bioactie agent;
4) a. is coupled if utilize carboxyl in free hydrazide group of microsphere surface and the biological function factor structure to carry out chemistry, then under the condition of ice bath, 1: 0.1 by volume～4.0 microspheres solution with pH=4.0～6.02-(N-morpholino) ethyl sulfonic acid buffer solution environment drop to and are dissolved with two or more biotic factor of 0.1～2.0mg/mL and 0.005～0.5g/mL 1-[3-(dimethylamino) propyl group]-pH=4.0～6.02-(N-morpholino) ethyl sulfonic acid buffer solution of 3-ethyl carbodiimide in, hatching 1～24h; Described biotic factor comprises transferrins, rabbit igg, EGF-R ELISA EGFR, TfR CD71, monoclonal antibody OX26, endothelial cell growth factor (ECGF) and nerve growth factor;
B. if by the sugar unit in the biological function factor that has the glycogen structure in some structure is carried out oxidation, realize coupled with the free hydrazide group of microsphere surface again, under 4～25 ℃, 1: 0.1 by volume～4.0 is that the microspheres solution of 4.0～6.0 sodium hydrogen phosphates-sodium citrate buffer solution environment is that 4.0～6.0 sodium hydrogen phosphates-sodium citrate buffer solution drip to the pH that is dissolved with two or more biotic factor of 0.1～2.0mg/mL with pH, hatching 1～5h; Described biotic factor comprises oxidation transferrins, oxidation rabbit igg;
C. if above-mentioned amphiphilic polysaccharide adopts part biological elementization modifier, then 1: 0.1 by volume～4.0 is 7.4～9.18 Na with pH
2CO
3-NaHCO
3The microspheres solution of buffered environment is 7.4～9.18 Na to the pH that is dissolved with two or more Avidin biotic factor of 0.1～2.0mg/mL
2CO
3-NaHCO
3Buffer drips, 37 ℃ of hatching 1～5h; Described biotic factor comprises Avidin transferrins, Avidin rabbit igg, Avidin EGFR, Avidin CD71, Avidin OX26, Avidin endothelial cell growth factor (ECGF), Avidin nerve growth factor;
5) with artemia hatching solution 10,000～16, centrifugal under the 000rpm, deionized water wash three times is scattered in the normal saline with parts by volume.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100155747A CN1317035C (en) | 2005-10-24 | 2005-10-24 | Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100155747A CN1317035C (en) | 2005-10-24 | 2005-10-24 | Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1768860A CN1768860A (en) | 2006-05-10 |
| CN1317035C true CN1317035C (en) | 2007-05-23 |
Family
ID=36750564
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100155747A Expired - Fee Related CN1317035C (en) | 2005-10-24 | 2005-10-24 | Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1317035C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100594938C (en) * | 2007-04-20 | 2010-03-24 | 中国医学科学院生物医学工程研究所 | Pulullan polysaccharide carrier material having the function of targeting therapy of cancer |
| JP5860480B2 (en) | 2011-01-11 | 2016-02-16 | キャプシュゲル・ベルジウム・エヌ・ヴィ | New hard capsule containing pullulan |
| JP7222911B2 (en) | 2017-04-14 | 2023-02-15 | カプスゲル・ベルギウム・ナムローゼ・フェンノートシャップ | How to make pullulan |
| CN110678170A (en) | 2017-04-14 | 2020-01-10 | 比利时胶囊公司 | Pullulan polysaccharide capsule |
| CN110624422A (en) * | 2019-08-19 | 2019-12-31 | 广西医科大学 | Dialysis membrane for removing carbonylation protein and preparation method thereof |
| CN111249475B (en) * | 2020-02-24 | 2024-05-10 | 爱尔眼科医院集团股份有限公司 | Medicine and gene dual delivery system, and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1466594A (en) * | 2000-09-22 | 2004-01-07 | ʥŵ��-�ϳ�ʵ���ҹ�˾ | Polysaccharides with antithrombotic activity comprising at least a covalent bono with biotin or a biotin derivative |
| WO2005054301A1 (en) * | 2003-11-14 | 2005-06-16 | Chugai Seiyaku Kabushiki Kaisha | Crosslinked polysaccharide microparticles and process for producing the same |
-
2005
- 2005-10-24 CN CNB2005100155747A patent/CN1317035C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1466594A (en) * | 2000-09-22 | 2004-01-07 | ʥŵ��-�ϳ�ʵ���ҹ�˾ | Polysaccharides with antithrombotic activity comprising at least a covalent bono with biotin or a biotin derivative |
| WO2005054301A1 (en) * | 2003-11-14 | 2005-06-16 | Chugai Seiyaku Kabushiki Kaisha | Crosslinked polysaccharide microparticles and process for producing the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1768860A (en) | 2006-05-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1768859A (en) | Method for assembling multi-biological functional factor on micro-particle surface based on aldehyde group | |
| CN1317035C (en) | Method for assembling multi-biological functional factor on micro-particle surface based on hydrazide group | |
| Gericke et al. | Nanoparticles based on hydrophobic polysaccharide derivatives—formation principles, characterization techniques, and biomedical applications | |
| US7786095B2 (en) | Amphiphilic macrocyclic derivatives and their analogues | |
| Van De Manakker et al. | Cyclodextrin-based polymeric materials: synthesis, properties, and pharmaceutical/biomedical applications | |
| CA2627287C (en) | Persistent luminescence nanoparticles used in the form of a diagnosis agent for in vivo optical imaging | |
| Hammer et al. | Leuko-polymersomes | |
| US11369570B2 (en) | Aptamer bioconjugate drug delivery device | |
| EP1285021A1 (en) | Material based on biodegradable polymers and method for preparing same | |
| CN100571781C (en) | Biotinylated pulullan polysaccharide nanometer grains and preparation method thereof | |
| CN102145173B (en) | Human serum albumin complex hydrophobically modified pullulan nanoparticles and preparation method thereof | |
| Sy et al. | Surface functionalization of polyketal microparticles with nitrilotriacetic acid–nickel complexes for efficient protein capture and delivery | |
| JP4262090B2 (en) | Anticancer agent-chitosan complex forming self-aggregate and method for producing the same | |
| CN101721710B (en) | Cholesteryl-carboxymethyl Curdlan nanometer particle and preparing method | |
| JP6772282B2 (en) | New nano-preparation of anti-cancer drug and its manufacturing method | |
| JP2005503476A (en) | Biodegradable polymer and cyclodextrin-containing material | |
| CN1745848A (en) | Chitin-sodium alginate packed pylorus helicobacterium protein microballs and their preparation | |
| CN1931370A (en) | Glycopeptide conjugate microsphere or microcapsule and its prepn process | |
| CN1733312A (en) | Fluorouracil-dextran and process for preparing the same | |
| CN1887273A (en) | Prepn process of polysaccharide vitreous particle | |
| CN101015698B (en) | Medicament using rare earth transferring complex compound as vector | |
| CN108503718B (en) | Hydroxyalkyl starch conjugate and preparation method and application thereof | |
| CN106496603A (en) | Preparation method based on the self-assembled nano micelle of cationic amphiphic polymer | |
| CN110496226A (en) | A kind of preparation method of the nucleocapsid structure dendrimer pharmaceutical carrier of pH response | |
| CN1683016A (en) | Process for preparing medicine-carrying particle containing surface transferrin for glioma target chemical therapy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |