CN101081876A - Subcellular organelle target directional Chitosan oligosaccharide-aliphatic acid grafting matter and preparation and application thereof - Google Patents
Subcellular organelle target directional Chitosan oligosaccharide-aliphatic acid grafting matter and preparation and application thereof Download PDFInfo
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- CN101081876A CN101081876A CN 200710069117 CN200710069117A CN101081876A CN 101081876 A CN101081876 A CN 101081876A CN 200710069117 CN200710069117 CN 200710069117 CN 200710069117 A CN200710069117 A CN 200710069117A CN 101081876 A CN101081876 A CN 101081876A
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- oligochitosan
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- acid grafting
- aliphatic acid
- chitosan oligosaccharide
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Abstract
The present invention provides chitosan-fatty acid grafting in the structure as shown, and the chitosan has molecular weight of 1-100 kDa, deacetylating degree of 70-100 % and partial free amino radicals substituted by alkyl radical in the substitution degree of 1-90 %. Through one improved alkylating modification process, degraded chitosan or low molecular weight chitosan is alkylation modified to form the chitosan-fatty acid grafting in special structure. The chitosan-fatty acid grafting may form micelle capable of being used as particle carrier for subcellular organelle in relevant research and preparing target medicine.
Description
Technical field
The invention belongs to the synthetic method of compound, relate to the preparation of chitosan oligosaccharide-aliphatic acid grafting, and the preparation of many hydrophobic cores chitosan oligosaccharide-aliphatic acid grafting micelle and this grafting micelle are as the application of nano level micelle particulate carrier in life science.
Background technology
In recent years, polymeric micellar has caused that in pharmaceutical preparation, biomedicine and field of polymer technology people pay attention to widely.Polymeric micellar is that autohemagglutination in aqueous medium forms by amphipathic block or graft copolymer, has nucleocapsid structure.Its hydrophobic chain segment constitutes the kernel of micelle, and hydrophilic segment forms the shell of micelle.Hydrophobic core can be the effect that insoluble drug provides bank; The wetting ability adventitia keeps the stability of micelle in aqueous environments, and can carry out physico-chemical property and modify reaching specific purpose, as the active target of micelle etc.
Polymeric micellar has many advantages as a kind of drug delivery system: the solvability by adjusting material, pH value, Zeta potential etc. can be controlled medicine release in vivo; Because the micelle particle diameter is very little, so can pass hemato encephalic barrier, also can promote the good absorption of stomach mucous membrane etc., the position that arrival large size particle can't pass through reaches the purpose of passive target; The polymeric micellar skeleton can be avoided the decomposition of medicine to a certain extent to the protection and the shielding effect of medicine, keeps stability of drug, reduces the toxicity of medicine; Compare with liposome, the drug loading of polymeric micellar is higher; The diversity of polymer materials makes with the polymkeric substance to be the also variation of pharmaceutical preparation of carrier, can satisfy various user demands.
Polymeric micellar mainly is used as the carrier of insoluble drug, particularly antitumor drug.Because the particle diameter of polymeric micellar is generally less than 100nm, can be gathered in tumor locus by " enhanced sees through and retention effect " (enhancedpermeability and retention effect).
Chitosan is the cationic polymers that a class is made up of glucosamine, has good biocompatibility, hypotoxicity and biodegradable.Chitosan is widely used in excipient substance, hemostatic material, tissue engineering bracket etc.Though with the chitosan is the polymeric micellar of the prepared particulate of basic material, nanoparticle and some hydrophobically modified chitosans, be widely used in the research of drug carrier material, but because the hydrophilic structure of carrier surface chitosan itself, so existing be difficult to be absorbed by the carrier of essentially consist, thereby finally influence the targeted therapy effect of medicine by cell with the chitosan.Simultaneously because the high molecular of chitosan, high viscosity and under the physiological pH condition shortcoming such as insoluble, increased the preparation difficulty of chitosan particle and nanoparticle, and the application that has limited hydrophobically modified chitosan micelle.
Summary of the invention
One object of the present invention provides chitosan oligosaccharide-aliphatic acid grafting, and its representative general structure is:
Wherein R is an alkyl chain, and its carbochain number is 12～22; N, x, y are the polymerization degree; The molecular weight of oligochitosan is 1～100kDa, and deacetylation is 70-100%; Part free amino group on the oligochitosan chain is replaced by alkyl, and amino group substitution degree is 1～90%.
Second purpose of the present invention provides the synthetic method of chitosan oligosaccharide-aliphatic acid grafting, realizes by following scheme:
(1) preparation of low-molecular weight chitoglycan (oligochitosan)
Get commercially available chitosan (70～100% deacetylation), under 55 ℃ and pH5.0 condition, stirred 2 hours, make the abundant swelling of chitosan after, by cellulase and chitosan ratio (w/w) adding in 0.5: 100 cellulase, degrade chitosan.Palliating degradation degree with viscosimetry control chitosan.The degradation solution of gained chitosan, remove impurity after filtration, according to service requirements, respectively from molecular weight 3kDa, 10kDa, 30kDa, in the ultra-filtration membrane of 50kDa, select suitable ultra-filtration membrane ultrafiltration classification, the ultrafiltrated lyophilize, deacetylation is that (preferred oligochitosan molecular weight is 1～100kDa) for the low-molecular weight chitoglycan of 70-100%, different molecular weight.Molecular weight with the gel permeation chromatography oligochitosan.
(2) chitosan oligosaccharide-aliphatic acid grafting preparation
The present invention is a couplant with carbodiimide (EDC) and N-hydroxy-succinamide (NHS), by the chemical reaction between the carboxyl of amino on the oligochitosan and lipid acid, and the preparation chitosan oligosaccharide-aliphatic acid grafting.Concrete synthetic method is: get 0.001M oligochitosan aqueous solution 100ml, after adding N-hydroxy-succinamide (mol ratio of oligochitosan and N-hydroxy-succinamide is 1: 10～1: the 1000) dissolving, reaction is 1 hour under the condition of room temperature and magnetic agitation; Other weighs a certain amount of lipid acid (mol ratio of oligochitosan and lipid acid is 1: 2～1: 200), after being dissolved in the dehydrated alcohol of 20ml under 40 ℃ the condition, add a certain amount of carbodiimide (mol ratio of lipid acid and carbodiimide is 1: 5), reaction is 1 hour under the condition of 50 ℃ of magnetic agitation.After above-mentioned two reaction solutions (reaction solution of oligochitosan and N-hydroxy-succinamide reaction solution and lipid acid and carbodiimide) respectively react 1 hour, above-mentioned two reaction solutions are mixed, continue under the condition of room temperature, magnetic agitation, to react 48 hours.End reaction liquid through the dialysis after, lyophilize, desciccate obtains chitosan oligosaccharide-aliphatic acid grafting behind 40 ℃ absolute ethanol washing.Lipid acid is selected from capric acid, myristic acid, Palmiticacid, oleic acid, stearic acid, the mountain Yu acid etc. any.
The preparation-obtained chitosan oligosaccharide-aliphatic acid grafting of the present invention, has the characteristic that in aqueous medium, forms micelle by self aggregation, the chitosan oligosaccharide-aliphatic acid grafting aqueous solution of 1mg/ml, can form particle diameter is that 5～1000nm, zeta current potential are the micelle of 5～80mV.The critical micelle concentration of chitosan oligosaccharide-aliphatic acid grafting is starkly lower than the critical micelle concentration of general tensio-active agent between 0.01～0.1mg/ml.When micelle runs into body fluid when diluted, can continue to keep the structure of micelle, show that the suitable carrier as medicine of chitosan oligosaccharide-aliphatic acid grafting micelle uses.
The 3rd purpose of the present invention provides chitosan oligosaccharide-aliphatic acid grafting and has application in the chitosan oligosaccharide-aliphatic acid grafting micelle of quick cellular uptake and subcellular organelle target in preparation.
The 4th purpose of the present invention provide chitosan oligosaccharide-aliphatic acid grafting preparation have that cytoplasm is detained and nucleus transport function medicine in application.
Chitosan oligosaccharide-aliphatic acid grafting micelle of the present invention has the special space structure of a plurality of hydrophobic core, can be absorbed fast by cell.Different according to the composition of chitosan oligosaccharide-aliphatic acid grafting and hydrophobic core number can embody the function that cytoplasm is detained and nucleus is transported respectively.
Usefulness of the present invention is: the present invention is on the research work basis in early stage, utilize degraded product, the low-molecular weight chitoglycan (oligochitosan) of chitosan, carrying out alkylation modifies, by improvement, synthesized the chitosan oligosaccharide-aliphatic acid grafting that a class has special construction to alkylation modified chitosan oligosaccharide (chitosan oligosaccharide-aliphatic acid grafting) synthetic method.By the formed grafting micelle of the chitosan oligosaccharide-aliphatic acid grafting that provides,, can be applicable to life science and pharmacy field for a kind of particulate carrier with good subcellular organelle target.Utilize this carrier, the target that can be applicable to this each organoid in cell of carrier distributes and studies; Explore approach and mechanism that macromolecular substance enters cell; For the treatment of developing subcellular organelle targeted drug, provide theory and technical foundation.
Description of drawings
Fig. 1: the oligochitosan of 10.2% amino group substitution degree-stearic acid grafting micelle, hatch fluorescent microscope photo after 2 hours altogether with the A549 cell.
Fig. 2: the oligochitosan of 48.6% amino group substitution degree-stearic acid grafting micelle, hatch fluorescent microscope photo after 2 hours altogether with the A549 cell.
Embodiment
The present invention is further described with accompanying drawing in conjunction with the embodiments.
Example one:
1, low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 550kDa (70% deacetylation), under 55 ℃ and pH5.0 condition, stirred 2 hours, make the abundant swelling of chitosan after, by cellulase and chitosan ratio (w/w) adding in 0.5: 100 cellulase, degrade chitosan.Palliating degradation degree with viscosimetry control chitosan.The degradation solution of gained chitosan is removed impurity after filtration, uses molecular weight to carry out the ultrafiltration classification as the ultra-filtration membrane of 10kDa and 30kDa.Get the ultrafiltrated lyophilize of molecular weight between 10kDa and 30kDa, deacetylation be 70%, the oligochitosan of molecular weight 22.4 kDa.
2, chitosan oligosaccharide-aliphatic acid grafting preparation and physical and chemical property determining
Get above-mentioned 0.0001mol oligochitosan, be dissolved in the 100ml distilled water, the aqueous solution of preparation oligochitosan, after the dissolving of adding 0.01mol N-hydroxy-succinamide, reaction is 1 hour under the condition of room temperature and magnetic agitation; Take by weighing the lipid acid (capric acid, stearic acid and mountain Yu acid) of 0.002mol in addition respectively, under 40 ℃ condition, be dissolved in the dehydrated alcohol of 20ml, add the carbodiimide of 0.01mol, reaction is 1 hour under the condition of 50 ℃ of magnetic agitation.After above-mentioned two reaction solutions respectively reacted 1 hour, the reaction solution with oligochitosan mixed with the reaction solution of lipid acid respectively, continued to react 48 hours under the condition of room temperature, magnetic agitation.End reaction liquid after dialysis, lyophilize, desciccate obtains oligochitosan-capric acid grafting, oligochitosan-stearic acid grafting and oligochitosan-mountain Yu acid grafting respectively behind 40 ℃ absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method, measure the amino group substitution degree of gained chitosan oligosaccharide-aliphatic acid grafting.(0.5～9mg), the accurate title, decide, and is dissolved in respectively in the distilled water of 2ml, adds the trinitro-benzene-sulfonic acid 2ml of the sodium hydrogen carbonate solution 2ml and 0.1% (w/v) of 4% (w/v), hatches 2h for 37 ℃ to get the different oligochitosans of measuring.Add 2N hydrochloric acid 2ml, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned chitosan oligosaccharide-aliphatic acid grafting 4mg and be dissolved in 2ml distilled water,, measure the absorption value at 344nm wavelength place, press the amino group substitution degree that typical curve calculates chitosan oligosaccharide-aliphatic acid grafting with the method operation.
Adopt the pyrene fluorescence spectrometry, the critical micelle concentration of chitosan oligosaccharide-aliphatic acid grafting.Get chitosan oligosaccharide-aliphatic acid grafting 10mg, the accurate title, decide, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml.The chitosan oligosaccharide-aliphatic acid grafting solution dilution of 1mg/ml is become each 10mL of chitosan oligosaccharide-aliphatic acid grafting solution of different concns, and (final concentration of pyrene is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), water-bath ultrasonic (400w, 30min).The excitation spectrum and the emmission spectrum of scanning pyrene are measured fluorescence intensity, and calculate critical micelle concentration.
Adopt the fluorescence collection method of going out, measure the hydrophobic core number of chitosan oligosaccharide-aliphatic acid grafting.Give a tongue-lashing pyridine (1-Dodecylpyridinium chloride with the chlorination hexadecyl, DPC) be the agent of going out of the fluorescence of pyrene collection, in sealing the chitosan oligosaccharide-aliphatic acid grafting micelle solution of pyrene, add quantitative DPC solution, fluorescence intensity before and after fluorescence spectrophotometry DPC adds, with the fluorescence collection kinetic equation (formula (1)) that goes out, calculate the concentration of the required hydrophobic material in hydrophobic core district (lipid acid):
ln(I
0/I)＝[Q]/[M] (1)
I
0Be respectively the fluorescence collection fluorescent emission intensity of agent before and after adding of going out with I; [Q] is the go out concentration of agent of fluorescence collection; [M] is for forming the concentration of single hydrophobic core district's desired fats acid.
The hydrophobic core district quantity n of single oligochitosan-stearic acid grafting molecule calculates according to following formula:
N=[lipid acid]/[M] (2)
Other gets chitosan oligosaccharide-aliphatic acid grafting 10mg, and accurate the title decides, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml, the chitosan oligosaccharide-aliphatic acid grafting micelle solution of preparation 1mg/ml.The particle diameter and the Zeta potential of Zetasizer 3000HS analysis-e/or determining chitosan oligosaccharide-aliphatic acid grafting micelle.
After measured, the physico-chemical property of oligochitosan-capric acid grafting, oligochitosan-stearic acid grafting and oligochitosan-mountain Yu acid grafting sees Table 1.
The physico-chemical property of table 1 oligochitosan-capric acid grafting, oligochitosan-stearic acid grafting and oligochitosan-mountain Yu acid grafting
| Grafting | Amino group substitution degree (%) | Critical micelle concentration (mg/ml) | Hydrophobic core number (individual) | Particle diameter (nm) | Zeta potential (mV) |
| Oligochitosan-capric acid | 10.5 | 0.05 | 1.85 | 67.9 | 38.7 |
| Oligochitosan-stearic acid | 9.9 | 0.04 | 2.17 | 56.8 | 43.6 |
| Oligochitosan-mountain Yu acid | 7.2 | 0.03 | 2.46 | 48.5 | 49.0 |
Example two:
1, low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (90% deacetylation), under 55 ℃ and pH5.0 condition, stirred 2 hours, make the abundant swelling of chitosan after, by cellulase and chitosan ratio (w/w) adding in 0.5: 100 cellulase, degrade chitosan.Palliating degradation degree with viscosimetry control chitosan.The degradation solution of gained chitosan is removed impurity after filtration, uses molecular weight to be 3kDa, 10kDa, 30kDa, and the ultra-filtration membrane of 50kDa carries out the ultrafiltration classification.Ultrafiltrated lyophilize after the classification, deacetylation be 90%, molecular weight is respectively 1.0kDa, 22.2 kDa, 33.4kDa, the oligochitosan of 54.6kda and 101.2kDa.
2, oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned 0.0001mol oligochitosan respectively and be dissolved in the 100ml distilled water, the preparation oligochitosan aqueous solution, after the dissolving of adding 0.01mol N-hydroxy-succinamide, reaction is 1 hour under the condition of room temperature and magnetic agitation; Other weighs the stearic acid of 0.002mol, after being dissolved in the dehydrated alcohol of 20ml under 40 ℃ the condition, adds the carbodiimide of 0.01mol, and reaction is 1 hour under the condition of 50 ℃ of magnetic agitation.After above-mentioned two reaction solutions respectively reacted 1 hour, the reaction solution with oligochitosan mixed with the reaction solution of lipid acid respectively, continued to react 48 hours under the condition of room temperature, magnetic agitation.End reaction liquid through the dialysis after, lyophilize, desciccate obtains the oligochitosan-stearic acid grafting of different molecular weight oligochitosan respectively behind 40 ℃ absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method, measure the amino group substitution degree of gained oligochitosan-stearic acid grafting.(0.5～9mg), the accurate title, decide, and is dissolved in respectively in the distilled water of 2ml, adds the trinitro-benzene-sulfonic acid 2ml of the sodium hydrogen carbonate solution 2ml and 0.1% (w/v) of 4% (w/v), hatches 2h for 37 ℃ to get the different oligochitosans of measuring.Add 2N hydrochloric acid 2ml, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned oligochitosan-stearic acid grafting 4mg and be dissolved in 2ml distilled water,, measure the absorption value at 344nm wavelength place, press the amino group substitution degree that typical curve calculates oligochitosan-stearic acid grafting with the method operation.
Adopt the pyrene fluorescent method, measure the critical micelle concentration of oligochitosan-stearic acid grafting.Get oligochitosan-stearic acid grafting 10mg, the accurate title, decide, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml.With oligochitosan-stearic acid grafting solution of 1mg/ml, be diluted to oligochitosan-each 10mL of stearic acid grafting solution of different concns, (final concentration of pyrene is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), water-bath ultrasonic (400w, 30min).The excitation spectrum and the emmission spectrum of scanning pyrene are measured fluorescence intensity, and calculate critical micelle concentration.
Adopt the fluorescence collection method of going out, measure the hydrophobic core number of oligochitosan-stearic acid grafting.Give a tongue-lashing pyridine (1-Dodecylpyridinium chloride with the chlorination hexadecyl, DPC) be the agent of going out of the fluorescence of pyrene collection, in sealing the oligochitosan of pyrene-stearic acid grafting micelle solution, add quantitative DPC solution, fluorescence intensity before and after fluorescence spectrophotometry DPC adds, the utilization fluorescence collection kinetic equation (formula (1)) that goes out calculates the concentration of the required hydrophobic material in hydrophobic core district (stearic acid).The hydrophobic core district quantity n of single oligochitosan-stearic acid grafting molecule calculates according to (2) formula.
Other gets oligochitosan-stearic acid grafting 10mg, and accurate the title decides, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml, oligochitosan-stearic acid grafting micelle solution of preparation 1mg/ml.Zetasizer 3000HS analyser is measured the particle diameter and the Zeta potential of oligochitosan-stearic acid grafting micelle respectively.
After measured, the physico-chemical property of the oligochitosan of different oligochitosan molecular weight-stearic acid grafting sees Table 2.
The physico-chemical property of the oligochitosan of the different oligochitosan molecular weight of table 2-stearic acid grafting
| Oligochitosan molecular weight (kDa) | Amino group substitution degree (%) | Critical micelle concentration (mg/ml) | Hydrophobic core number (individual) | Particle diameter (nm) | Zeta potential (mV) |
| 1.0 | 89.5 | 0.01 | 1.60 | 5.2 | 59.6 |
| 22.2 | 10.2 | 0.04 | 2.32 | 53.5 | 42.8 |
| 33.4 | 7.2 | 0.03 | 3.35 | 108.5 | 49.0 |
| 54.6 | 5.6 | 0.07 | 3.68 | 132.4 | 46.6 |
| 101.2 | 1.1 | 0.10 | 1.20 | 198.4 | 35.3 |
Example three:
1, low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 750kDa (100% deacetylation), under 55 ℃ and pH5.0 condition, stirred 2 hours, make the abundant swelling of chitosan after, by cellulase and chitosan ratio (w/w) adding in 0.5: 100 cellulase, degrade chitosan.Palliating degradation degree with viscosimetry control chitosan.The degradation solution of gained chitosan is removed impurity after filtration, uses molecular weight to carry out the ultrafiltration classification as the ultra-filtration membrane of 10kDa and 30kDa.Ultrafiltrated lyophilize after the classification, deacetylation be 100%, molecular weight is the oligochitosan of 25.8kDa.
2, oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned 0.0001mol oligochitosan, be dissolved in the 100ml distilled water, the aqueous solution of preparation oligochitosan adds the 0.01mol N-hydroxy-succinamide, and after the dissolving, reaction is 1 hour under the condition of room temperature and magnetic agitation; Other takes by weighing the stearic acid of 0.002mol, after being dissolved in the dehydrated alcohol of 20ml under 40 ℃ the condition, adds the carbodiimide of 0.01mol, and reaction is 1 hour under the condition of 50 ℃ of magnetic agitation.After above-mentioned two reaction solutions respectively reacted 1 hour, the reaction solution with oligochitosan mixed with the reaction solution of lipid acid respectively, continued to react 48 hours under the condition of room temperature, magnetic agitation.End reaction liquid through the dialysis after, lyophilize, desciccate obtains the oligochitosan-stearic acid grafting of deacetylation behind 40 ℃ absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method, measure the amino group substitution degree of gained oligochitosan-stearic acid grafting.(0.5～9mg), the accurate title, decide, and is dissolved in respectively in the distilled water of 2ml, adds the trinitro-benzene-sulfonic acid 2ml of the sodium hydrogen carbonate solution 2ml and 0.1% (w/v) of 4% (w/v), hatches 2h for 37 ℃ to get different amount oligochitosans.Add 2N hydrochloric acid 2ml, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned oligochitosan-stearic acid grafting 4mg and be dissolved in the 2ml redistilled water,, measure the absorption value at 344nm wavelength place, press the amino group substitution degree that typical curve calculates oligochitosan-stearic acid grafting with the method operation.
Adopt the pyrene fluorescent method, measure the critical micelle concentration of oligochitosan-stearic acid grafting.Get oligochitosan-stearic acid grafting 10mg, the accurate title, decide, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml.With oligochitosan-stearic acid grafting solution of 1mg/ml, be diluted to oligochitosan-each 10mL of stearic acid grafting solution of different concns, (final concentration of pyrene is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), water-bath ultrasonic (400w, 30min).The excitation spectrum and the emmission spectrum of scanning pyrene are measured fluorescence intensity, and calculate critical micelle concentration.
Adopt the fluorescence collection method of going out, measure the hydrophobic core number of oligochitosan-stearic acid grafting.Give a tongue-lashing pyridine (1-Dodecylpyridinium chloride with the chlorination hexadecyl, DPC) be the agent of going out of the fluorescence of pyrene collection, in the oligochitosan-stearic acid grafting micelle solution of load pyrene, add quantitative DPC solution, fluorescence intensity before and after fluorescence spectrophotometry DPC adds, the utilization fluorescence collection kinetic equation (formula (1)) that goes out calculates the concentration of the required hydrophobic material in hydrophobic core district (stearic acid).The hydrophobic core district quantity n of single oligochitosan-stearic acid grafting molecule calculates according to (2) formula.
Other gets oligochitosan-stearic acid grafting 10mg, and accurate the title decides, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml, oligochitosan-stearic acid grafting micelle solution of preparation 1mg/ml.Zetasizer 3000HS analyser is measured the particle diameter and the Zeta potential of oligochitosan-stearic acid grafting micelle respectively.
After measured, deacetylation is that the physico-chemical property of oligochitosan-stearic acid grafting of 100% sees Table 3.
Table 3 deacetylation is the physico-chemical property of oligochitosan-stearic acid grafting of 100%
| Deacetylation (%) | Amino group substitution degree (%) | Critical micelle concentration (mg/ml) | Hydrophobic core number (individual) | Particle diameter (nm) | Zeta potential (mV) |
| 100 | 12.6 | 0.04 | 2.62 | 87.9 | 35.8 |
Example four:
1, low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (90% deacetylation), under 55 ℃ and pH5.0 condition, stirred 2 hours, make the abundant swelling of chitosan after, by cellulase and chitosan ratio (w/w) adding in 0.5: 100 cellulase, degrade chitosan.Palliating degradation degree with viscosimetry control chitosan.The degradation solution of gained chitosan is removed impurity after filtration, uses molecular weight to carry out the ultrafiltration classification as the ultra-filtration membrane of 10kDa and 30kDa.Get the ultrafiltrated lyophilize of molecular weight between 10kDa and 30kDa, deacetylation be 90%, the oligochitosan of molecular weight 19.2kDa.
2, oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Oligochitosan-stearic acid grafting prepares feed ratio and sees Table 4.
Table 4 oligochitosan-stearic acid grafting prepares feed ratio
| Sequence number | Oligochitosan (mol) | N-hydroxy-succinamide (mol) | Stearic acid (mol) | Carbodiimide (mol) |
| 1 | 0.0001 | 0.005 | 0.001 | 0.005 |
| 2 | 0.0001 | 0.01 | 0.002 | 0.01 |
| 3 | 0.0001 | 0.02 | 0.004 | 0.02 |
| 4 | 0.0001 | 0.05 | 0.01 | 0.05 |
Get above-mentioned quantitative oligochitosan, be dissolved in the 100ml distilled water, the aqueous solution of preparation oligochitosan adds quantitative N-hydroxy-succinamide, and after the dissolving, reaction is 1 hour under the condition of room temperature and magnetic agitation; Other takes by weighing quantitative stearic acid, after being dissolved in the dehydrated alcohol of 20ml respectively under 40 ℃ the condition, adds quantitative carbodiimide respectively, and reaction is 1 hour under the condition of 50 ℃ of magnetic agitation.After above-mentioned two reaction solutions respectively reacted 1 hour, the reaction solution with oligochitosan mixed with the reaction solution of lipid acid respectively, continued to react 48 hours under the condition of room temperature, magnetic agitation.End reaction liquid through the dialysis after, lyophilize, desciccate obtains the oligochitosan-stearic acid grafting of different amino group substitution degrees respectively behind 40 ℃ absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method, measure the amino group substitution degree of gained oligochitosan-stearic acid grafting.(0.5～9mg), the accurate title, decide, and is dissolved in respectively in the distilled water of 2ml, adds the trinitro-benzene-sulfonic acid 2ml of the sodium hydrogen carbonate solution 2ml and 0.1% (w/v) of 4% (w/v), hatches 2h for 37 ℃ to get different amount oligochitosans.Add 2N hydrochloric acid 2ml, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned oligochitosan-stearic acid grafting 4mg and be dissolved in the 2ml redistilled water,, measure the absorption value at 344nm wavelength place, press the amino group substitution degree that typical curve calculates oligochitosan-stearic acid grafting with the method operation.
Adopt the pyrene fluorescent method, measure the critical micelle concentration of oligochitosan-stearic acid grafting.Get oligochitosan-stearic acid grafting 10mg, the accurate title, decide, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml.With oligochitosan-stearic acid grafting solution of 1mg/ml, be diluted to oligochitosan-each 10mL of stearic acid grafting solution of different concns, (final concentration of pyrene is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), water-bath ultrasonic (400w, 30min).The excitation spectrum and the emmission spectrum of scanning pyrene are measured fluorescence intensity, and calculate critical micelle concentration.
Adopt the fluorescence collection method of going out, measure the hydrophobic core number of oligochitosan-stearic acid grafting.Give a tongue-lashing pyridine (1-Dodecylpyridinium chloride with the chlorination hexadecyl, DPC) be the agent of going out of the fluorescence of pyrene collection, in sealing the oligochitosan of pyrene-stearic acid grafting micelle solution, add quantitative DPC solution, fluorescence intensity before and after fluorescence spectrophotometry DPC adds, the utilization fluorescence collection kinetic equation (formula (1)) that goes out calculates the concentration of the required hydrophobic material in hydrophobic core district (stearic acid).The hydrophobic core district quantity n of single oligochitosan-stearic acid grafting molecule calculates according to (2) formula.
Other gets oligochitosan-stearic acid grafting 10mg, and accurate the title decides, and is dissolved in distilled water, pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is settled to 10ml, oligochitosan-stearic acid grafting micelle solution of preparation 1mg/ml.Zetasizer 3000HS analyser is measured the particle diameter and the Zeta potential of oligochitosan-stearic acid grafting micelle respectively.
After measured, the physico-chemical property of the oligochitosan of different stearic acid feed ratio-stearic acid grafting sees Table 5.
The physico-chemical property of the oligochitosan of the different stearic acid feed ratio of table 5-stearic acid grafting
| Stearic acid charging capacity (mol) | Amino group substitution degree (%) | Critical micelle concentration (mg/ml) | Hydrophobic core number (individual) | Particle diameter (nm) | Zeta potential (mV) |
| 0.001 | 5.5 | 0.05 | 2.02 | 101.4 | 41.8 |
| 0.002 | 10.2 | 0.03 | 2.63 | 65.3 | 43.3 |
| 0.004 | 20.1 | 0.02 | 5..75 | 43.2 | 39.7 |
| 0.01 | 48.6 | 0.01 | 8.65 | 23.1 | 37.5 |
3, oligochitosan-stearic acid grafting micelle cellular uptake
Get 0.1g fluorescein isothiocyanate (fitc) and 0.5g oligochitosan-stearic acid grafting, be dissolved in respectively in 60% ethanolic soln after accurate title is fixed.Under ice bath, lucifuge and magnetic agitation (400rpm) condition, with oligochitosan-stearic acid grafting ethanolic soln, be added drop-wise in the fluorescein isothiocyanate (fitc) solution, the same terms continues reaction 10h down.End reaction liquid is put dialysis tubing, and redistilled water dialysis 24h removes the unreacted fluorescein isothiocyanate (fitc).The dialyzate lyophilize gets fluorescein isothiocyanate (fitc) mark oligochitosan-stearic acid grafting.
Get fluorescein isothiocyanate (fitc) mark oligochitosan-stearic acid grafting 10mg, the accurate title, decide.Add an amount of redistilled water, the ultrasonic 10min of water-bath disperses, and is settled to 1000ml, gets fluorescein isothiocyanate (fitc) mark oligochitosan-stearic acid grafting micelle solution.
With people II type pulmonary epithelial cells A549 cell is model cell, at 37 ℃, and 5%CO
2Culturing cell is to being the logarithmic growth after date under the condition, and centrifugal collection is by every hole 1 * 10
5The density of individual cell is inoculated 24 well culture plates, the pre-cultivation 24 hours in the incubator.Adding fluorescein isothiocyanate (fitc) mark oligochitosan-stearic acid grafting micelle solution in nutrient solution hatches.Micelle concentration is controlled to be 100 μ g/ml.After cultivating certain hour, observe with the fluorescence inverted microscope.The fluorescence microscope result shows that oligochitosan-stearic acid grafting micelle can be absorbed by cell fast, and according to the difference of the amino group substitution degree of grafting, embodies the function that cytoplasm is detained and nucleus is transported respectively.It is oligochitosan-stearic acid grafting micelle of 10.2% and 48.6% that Fig. 1 and Fig. 2 are respectively amino group substitution degree, hatches fluorescent microscope photo after 2 hours altogether with the A549 cell.Referring to Fig. 1 and Fig. 2, amino group substitution degree is respectively after oligochitosan-stearic acid grafting of 10.2% and 48.6% and A549 cell hatch 2 hours altogether, embodies the function that cytoplasm is detained and nucleus is transported respectively.Fig. 1 is the oligochitosan-stearic acid grafting micelle of 10.2% amino group substitution degree, hatches fluorescent microscope photo after 2 hours altogether with the A549 cell, and wherein A figure is a fluorescein isothiocyanate (fitc) tagged polymers micelle fluorescence photo, and B figure is that nuclear dyes the contrast photo.Fig. 2 is the oligochitosan-stearic acid grafting micelle of 48.6% amino group substitution degree, hatches fluorescent microscope photo after 2 hours altogether with the A549 cell, and wherein A figure is a fluorescein isothiocyanate (fitc) tagged polymers micelle fluorescence photo, and B figure is that nuclear dyes the contrast photo.
Claims (6)
1, a kind of chitosan oligosaccharide-aliphatic acid grafting, its representative general structure is:
Wherein R is an alkyl chain, and its carbochain number is 12～22, n, and x, y are the polymerization degree, and the molecular weight of oligochitosan is 1～100kDa, and deacetylation is 70-100%, and the part free amino group on the oligochitosan chain is replaced by alkyl, and amino group substitution degree is 1～90%.
2, the synthetic method of chitosan oligosaccharide-aliphatic acid grafting according to claim 1 is characterized in that realizing by following steps:
(1) preparation of lower molecular weight oligochitosan
Get commercially available high-molecular weight chitosan, 70～100% deacetylations, under 55 ℃ and pH5.0 condition, stirred 2 hours, after making the abundant swelling of chitosan, cellulase and chitosan ratio added cellulase in 0.5: 100 by weight percentage, degrade chitosan, palliating degradation degree with viscosimetry control chitosan, the degradation solution of gained chitosan, remove impurity after filtration, according to service requirements, respectively from molecular weight 3kDa, 10kDa, 30kDa is in the ultra-filtration membrane of 50kDa, select suitable ultra-filtration membrane ultrafiltration classification, the ultrafiltrated lyophilize, selecting the oligochitosan molecular weight for use is 1～100kDa, deacetylation is the low-molecular weight chitoglycan of 70-100%, with the molecular weight of gel permeation chromatography oligochitosan;
(2) preparation of chitosan oligosaccharide-aliphatic acid grafting
Get above-mentioned 0.001 M oligochitosan aqueous solution 100ml, add N-hydroxy-succinamide, the mol ratio of oligochitosan and N-hydroxy-succinamide is 1: 10～1: 1000, and after the dissolving, reaction is 1 hour under the condition of room temperature and magnetic agitation; Other takes by weighing lipid acid, the mol ratio of oligochitosan and lipid acid is 1: 2～1: 200, after being dissolved in the dehydrated alcohol of 20ml under 40 ℃ the condition, adds carbodiimide, the mol ratio of lipid acid and carbodiimide is 1: 5, and reaction is 1 hour under the condition of 50 ℃ of magnetic agitation; After above-mentioned two reaction solutions respectively react 1 hour, above-mentioned two reaction solutions are mixed, continue under the condition of room temperature, magnetic agitation, to react 48 hours, after dialysis, lyophilize, desciccate obtains chitosan oligosaccharide-aliphatic acid grafting behind 40 ℃ absolute ethanol washing.
3, the synthetic method of chitosan oligosaccharide-aliphatic acid grafting according to claim 2, it is characterized in that: it is that 5～1000nm, zeta current potential are the micelle of 5～80mV that the chitosan oligosaccharide-aliphatic acid grafting aqueous solution of 1mg/ml forms particle diameter, and the critical micelle concentration of chitosan oligosaccharide-aliphatic acid grafting is between 0.01～0.1mg/ml.
4, the synthetic method of chitosan oligosaccharide-aliphatic acid grafting according to claim 2 is characterized in that: formed chitosan oligosaccharide-aliphatic acid grafting micelle has the special space structure of a plurality of hydrophobic core.
5, chitosan oligosaccharide-aliphatic acid grafting according to claim 1 has application in the chitosan oligosaccharide-aliphatic acid grafting micelle of quick cellular uptake and subcellular organelle target in preparation.
6. chitosan oligosaccharide-aliphatic acid grafting according to claim 1 preparation have that cytoplasm is detained and nucleus transport function medicine in application.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101791412A (en) * | 2010-04-09 | 2010-08-04 | 浙江大学 | Acyclovir-chitosan-stearic acid grafting, synthetic method and application thereof |
| CN104861078A (en) * | 2015-05-20 | 2015-08-26 | 齐鲁工业大学 | Cellulose based macromolecular cross-linking agent, preparation method thereof and application of cellulose based macromolecular cross-linking agent in preparation of modified gelatin |
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| CN101791412A (en) * | 2010-04-09 | 2010-08-04 | 浙江大学 | Acyclovir-chitosan-stearic acid grafting, synthetic method and application thereof |
| CN101791412B (en) * | 2010-04-09 | 2011-12-07 | 浙江大学 | Acyclovir-chitosan-stearic acid grafting, synthetic method and application thereof |
| CN104861078A (en) * | 2015-05-20 | 2015-08-26 | 齐鲁工业大学 | Cellulose based macromolecular cross-linking agent, preparation method thereof and application of cellulose based macromolecular cross-linking agent in preparation of modified gelatin |
| CN104861078B (en) * | 2015-05-20 | 2017-06-30 | 齐鲁工业大学 | Macromolecules cross-linking agent, its preparation method based on cellulose and its application in modified gelatin is made |
| CN105920612A (en) * | 2016-06-12 | 2016-09-07 | 浙江大学 | Glucosamine-behenic acid grafting substance and preparation method |
| CN107880152A (en) * | 2017-10-26 | 2018-04-06 | 浙江大学 | The reduction response type chitosan stearylamine grafting and preparation and application that angiopep 2 is modified |
| CN109394678A (en) * | 2018-10-15 | 2019-03-01 | 安徽徽科生物工程技术有限公司 | Tissue irrigating solutions and its application in the medical field |
| CN110452314A (en) * | 2019-08-07 | 2019-11-15 | 浙江大学 | Anoxic sensitivity response type chitosan nitroimidazole grafting and preparation and application |
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