CN1718592A - Fluorescence labeling hydrophobic modified chitin polymer, its preparation method and application - Google Patents
Fluorescence labeling hydrophobic modified chitin polymer, its preparation method and application Download PDFInfo
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Abstract
A fluorescently-labeled hydrophobic modified oligochitose polymer used for preparing target medicine or fluorescent signal amplifier carrier is prepared through reaction between aqueous solution of oligochitose, fatty acid and cross-linking coupler, and reacting on isothiocyano fluorescent larbel in ice bath, darc and magnetic stirring conditions to obtain said polymer, and ultrasonically dispersing it in water to obtain its colloidal microparticles.
Description
Technical field
The invention belongs to compounds process for production thereof, relate to the preparation method of fluorescence labeling hydrophobic modified chitin polymer and as the application of nano level micelle particulate carrier in life science.
Background technology
In recent years, particulate carrier has obtained increasing concern and application in the drug targeting field.As study more liposome, nanoparticle, microballoon and polymeric micellar etc.These particulate carriers since its particle diameter little, can grow circulation, good biocompatibility in vivo and, be considered to a kind of good targeting drug delivery system through characteristics such as microbial films.Having unique advantage aspect macromolecular drug and the gene drug delivery, be the focus in current research field especially.
Drug targeting therapeutics is divided into three developmental stage with the drug targeting treatment.Early stage is the histoorgan target, as the small particle size passive target that utilizes particulate gathers tumor tissues etc. to tissues such as livers by " enhanced sees through and retention effect " (enhanced permeability and retention effect).Subsequently, the drug targeting treatment develops into cell-targeting.Recently, there is the scholar to begin to propose organoid target notion, is mainly used in the disease that the organoid pathology causes, as mitochondriopathy etc.
At present, there has been the investigator to carry out the cellular uptake research of particulate carrier.People such as Lee have studied the cellular uptake process of polyhistidyl-polyoxyethylene glycol (polyHis-b-PEG) particulate.But, be still waiting further research for the cellular uptake mechanism of particulate, in the intracellular transport approach and content such as how to realize discharging in the cell of entrained medicine.
Chitosan is a kind of natural polycation polysaccharide with biocompatibility, biodegradable, low toxicity.In decades, chitosan is widely used in excipient substance, hemostatic material, tissue engineering bracket etc.There is molecular weight big (hundreds thousand of) in commercially available chitosan, and the viscosity height such as can't dissolve at shortcoming under physiological pH (7.4) condition.In recent years, the people's physico-chemical property and application thereof of solubility oligochitosan (being low molecular chitosan) that begin one's study.
The chitosan structure study on the modification that has carried out relates generally to the wetting ability of polymer slightly water-soluble chitosan structure is modified, as the N-carboxymethylation, and grafting PEG molecule etc.; Hydrophilic and the hydrophobic grouping of grafting simultaneously on chitosan molecule, preparation has the material of amphipathic characteristic; And grafting functional groups such as lactose molecule etc. reach targeting.Along with carrying out of oligochitosan research, synthetic and the physico-chemical property research of amphipathic oligochitosan, and, begin to cause people's extensive attention in the application of aspects such as industry, biomedicine, agricultural.
Summary of the invention
An object of the present invention is to provide fluorescence labeling hydrophobic modified chitin polymer, have following general structure:
R wherein
1Be fluorophor, R
2Be alkyl chain, n is the polymerization degree of oligochitosan, and the molecular weight of oligochitosan is less than 200kDa, part free amino group on the oligochitosan chain is replaced by alkyl, and amino group substitution degree is 1~50%, has positive charge, the zeta current potential is at 5~80mV, and particle diameter is at 5~1000nm, the covalent attachment fluorescence molecule.
Second purpose of the present invention provides the preparation method of fluorescence labeling hydrophobic modified chitin polymer, realizes by following scheme:
(1) hydrophobic modified chitin preparation: getting commercially available molecular weight is the high-molecular weight chitosan (90~95% deacetylation) of 450kDa, stirring and dissolving under 55~60 ℃ and pH5.0 condition, add cellulose degraded in cellulase and chitosan ratio 0.5: 100 (w/w), remove by filter impurity, according to service requirements, respectively from molecular weight 0.10kDa, 10kDa, 50kDa, 100kDa, in the ultra-filtration membrane of 200kDa, select suitable ultra-filtration membrane ultrafiltration classification, the ultrafiltrated lyophilize, (preferred oligochitosan is 0.1~50kDa) less than 200kDa to get molecular weight, deacetylation is greater than 80% lower molecular weight oligochitosan, gel permeation chromatography molecular weight.Get the above-mentioned oligochitosan aqueous solution,, control 50 ℃~90 ℃, reacted 5~48 hours according to oligochitosan, lipid acid, crosslinked coupler carbodiimide mol ratio 1: 1~50: 1~50.End reaction liquid dialysis purifying, lyophilize obtains hydrophobic modified chitin; Lipid acid is selected from myristic acid, Palmiticacid, oleic acid, stearic acid, the mountain Yu acid etc. any.
(2) fluorescence labeling hydrophobic modified chitin preparation: get the hydrophobic modified chitin aqueous solution, according to hydrophobic modified chitin: isothiocyano group fluorescent marker mol ratio 1: 1~50, under ice bath, lucifuge and magnetic agitation condition, reacted 5~48 hours.End reaction liquid dialysis purifying, lyophilize obtains fluorescence labeling hydrophobic modified chitin.Isothiocyano group fluorescein is selected from fluorescein isothiocyanate (fitc) or TRITC.
(3) preparation of fluorescence labeling hydrophobic modified chitin polymer micelle: (mg/ml, ratio w/v) in water, prepare the polymeric micellar particulate with the fluorescence labeling hydrophobic modified chitin ultra-sonic dispersion to press 1~10000: 1000.
Because the molecular weight of chitosan itself is bigger, water-soluble for improving it, research in the past mainly realizes by grafting hydrophilic radical (as sulfonic group).This grafting method except can increasing the solubility property of material to a certain extent, can not bring the remarkable improvement on the material functions of use.Reduce the molecular weight of chitosan, obviously help improving chitosan originally in aqueous medium, particularly the solubility property under higher pH environment.In the present invention, at first select the lower molecular weight oligochitosan for use.Find in the research that molecular weight is less than the oligochitosan of 200kDa, possessed good water-soluble under higher pH environment, and cytotoxicity reduces significantly with the decline of molecular weight.Thereby enlarged the Application Areas of chitosan, can be as good drug carrier material.
Oligochitosan of the present invention is that molecular weight distribution is narrow, the metastable lower molecular weight oligochitosan of physico-chemical property.Because the high molecular oligochitosan is behind the process enzyme liberating, molecular weight distribution is than broad, and the present invention obtains the oligochitosan of different molecular weight section by the ultra-filtration membrane classification of PSPP, and wherein preferred oligochitosan molecular weight is 0.1~50kDa
Among the present invention, selected main raw material(s) oligochitosan comes from the nature Crustacean, has low toxicity, biodegradable characteristic.Grafting lipid acid is the important composition composition of the daily food of the mankind.The carbodiimide crosslinking is adopted in the grafting reaction, and this method is widely used in biochemical field, and the reaction conditions gentleness is easy to control, and its byproduct of reaction can conveniently be removed by dialysis method, can guarantee to generate the biocompatibility of product.
Among the present invention, the method for preparing the micelle carrier is simple.Because grafting itself has unique parents' structure, the amphiphilic polymer with suitable hydrophilic and oleophilic base ratio, spontaneous gathering forms polymeric micellar in water.The modification oligochitosan for preparing under the optimal conditions, the particle diameter that forms micelle in deionized water be less than 100nm, the surface zeta potential current potential be on the occasion of.
The 3rd purpose of the present invention provides the fluorescence labeling hydrophobic modified chitin polymer micelle as the application of organoid target particulate carrier in the preparation targeted drug.
A further object of the present invention provides the carrier that the fluorescence labeling hydrophobic modified chitin polymer micelle amplifies as fluorescent signal, uses in the fluorescent mark that substitutes traditional gene chip.
Usefulness of the present invention is: the fluorescence labeling hydrophobic modified chitin polymer micelle that provides is a kind of particulate carrier with good organoid target, can be applicable to life science and pharmacy field.Utilize this carrier, the target that can be used for this each organoid in cell of carrier distributes and studies; Explore approach and mechanism that macromolecular substance enters cell; For the treatment of developing organoid targeted drug, provide theory and technical foundation.
Among the present invention, adopt the fluorescent mark technology of micelle,, provide the necessary technology means for studying micelle at intracellular distribution and target.Micelle itself possesses unique nuclear-membrane structure, and has positive charge, will be the development of pharmaceutical carrier technology, particularly carries some biomacromolecules and enters cell and specific organoid, establishes more solid working foundation.Find in the research that after hatching altogether with cell, this particulate has the organoid target function fast by cellular uptake, the micelle that the present invention prepares gathers some organoid target.The fluorescent mark micelle can be by the positive charge of self, and portability protein or active biomolecule enter drug target; By fluorescent tracer technique, can be applicable to function and the mechanism important exploration means of this micelle as macromolecule medicament carrier.For cell traffic and the pathways metabolism thereof of studying macromolecular substance provides the good technical means, also provide technical foundation simultaneously for the treatment of developing organoid targeted drug.
The fluorescently-labeled micelle of synthetic of the present invention also can be used as the carrier that fluorescent signal amplifies, and purposes is very extensive in the field, forward position of life science.Can be used as the fluorescent signal carrier of gene chip, become the strong instrument of genomics research.Because there are a lot of shortcomings in the fluorescent mark means that adopt in original biochip technology, as a little less than the fluorescent signal, detection technique height, equipment price costliness and efficient is low etc.The present invention adopts fluorescently-labeled micelle suitable with the biomolecules yardstick, can be incorporated on the DNA, substitutes the fluorescent mark means of traditional gene chip, for its clinical application is paved the way.
The preparation method of this particulate carrier provided by the invention, method is simple.Selected main raw material(s) oligochitosan comes from the nature Crustacean, has low toxicity, biodegradable characteristic.
Description of drawings
Fig. 1 is for hatching A549 cellular uptake photo behind the 30min with the fluorescent mark micelle.
Fig. 2 is the variation of fluorescence intensity in the cell behind the A549 cellular uptake fluorescent mark micelle.
Embodiment
Example one:
1, the preparation of oligochitosan
Chitosan (molecular-weight average 450kDa) 6g adds in the aqueous hydrochloric acid of 200mL 1.25% (v/v), stirring and dissolving under 55~60 ℃ of conditions, transfer pH to 5.0 with weak ammonia or dilute hydrochloric acid, add cellulase in cellulase and chitosan ratio 0.5: 100 (w/w), after the controlling reaction time 8 hours, with reaction product 4000r * min
-1Centrifugal 10 minutes, supernatant liquor was with 0.45 μ m millipore filtration pre-treatment, and with the ultra-filtration membrane ultrafiltration classification of different molecular weight, the ultrafiltrated lyophilize obtains the oligochitosan of certain molecular weight.And be 50.6kDa by the gel permeation chromatography molecular-weight average.
2, the preparation of fluorescence labeling hydrophobic modified chitin
Get above-mentioned oligochitosan 5.0g, the accurate title, decide.After adding 40ml distilled water stirring and dissolving, add carbodiimide 30mg, stirring and dissolving.With the myristic acid of 0.35g, add in the 10ml methanol solution, behind the ultrasonic dissolution, add in the above-mentioned oligochitosan solution, at 400rmin
-1Under the magnetic agitation condition, 60 ℃ of controlled temperature are more than the reaction times 24h.End reaction liquid is put dialysis tubing, and distilled water dialysis 24h removes byproduct of reaction.The dialyzate lyophilize prepares hydrophobic modified chitin.
The amino group substitution degree of oligochitosan behind the hydrophobically modified adopts the trinitro-benzene-sulfonic acid method to measure.(0.5~9mg), the accurate title, decide, and is dissolved in respectively in the redistilled 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 4mg and be dissolved in the 2ml redistilled water, with the method operation, measure the absorption value at 344nm wavelength place, calculating its substitution value by typical curve is 4.8%.
Get 0.1g TRITC and 0.5g hydrophobic modified chitin, be dissolved in respectively in 60% ethanolic soln after accurate title is fixed.At ice bath, lucifuge and magnetic agitation (400rmin
-1) under the condition, with the hydrophobic modified chitin ethanolic soln, be added drop-wise in the rhodamine solution, the same terms continues reaction 10h down.End reaction liquid is put dialysis tubing, and redistilled water dialysis 24h removes the unreacted rhodamine.The dialyzate lyophilize gets fluorescence labeling hydrophobic modified chitin.
3, fluorescence labeling hydrophobic modified chitin micelle preparation
Get fluorescence labeling hydrophobic modified chitin 10mg, the accurate title, decide.Add the ultrasonic 10min of an amount of distilled water water-bath and disperse, be settled to 1000ml, get the fluorescence labeling hydrophobic modified chitin micelle solution.
Zetasizer 3000HS analysis-e/or determining particle diameter and surface potential.The median size of fluorescence labeling hydrophobic modified chitin micelle is 322.2nm; Surface potential is 40.9 ± 0.1mV.
Example two:
1, the preparation of oligochitosan
Chitosan (molecular-weight average 450kDa) 6g adds in the aqueous hydrochloric acid of 200mL 1.25% (v/v), stirring and dissolving under 55~60 ℃ of conditions, transfer pH to 5.0 with weak ammonia or dilute hydrochloric acid, add cellulase in cellulase and chitosan ratio 0.5: 100 (w/w), controlling reaction time 24 hours, after question response finishes, with reaction product 4000r * min
-1Centrifugal 10 minutes, supernatant liquor was with 0.45 μ m millipore filtration pre-treatment, and with the ultra-filtration membrane ultrafiltration classification of different molecular weight, the ultrafiltrated lyophilize obtains the oligochitosan of certain molecular weight.And be 10.1kDa by the gel permeation chromatography molecular-weight average.
2, the preparation of fluorescence labeling hydrophobic modified chitin
Get 0.5g chitosan (molecular-weight average 10.1kDa), the accurate title, decide.After adding 40ml distilled water stirring and dissolving, add carbodiimide 55mg, stirring and dissolving.With the stearic acid of 0.08g, add in the 10ml ethanolic soln, behind the ultrasonic dissolution, add in the above-mentioned oligochitosan solution, at 400rmin
-1Under the magnetic agitation condition, 60 ℃ of controlled temperature are more than the reaction times 24h.End reaction liquid is put dialysis tubing, and distilled water dialysis 24h removes byproduct of reaction.The dialyzate lyophilize prepares hydrophobic modified chitin, and the trinitro-benzene-sulfonic acid method of pressing in the example one is measured, and substitution value is 9.1%.
Get 40.0mg fluorescein isothiocyanate (fitc) and 0.5g hydrophobic modified chitin, be dissolved in respectively in 80% ethanolic soln after accurate title is fixed.At ice bath, lucifuge and magnetic agitation (400rmin
-1) under the condition, with the hydrophobic modified chitin ethanolic soln, be added drop-wise in the fluorescein isothiocyanate (fitc) solution, the same terms continues reaction 24h down.End reaction liquid is put dialysis tubing, and redistilled water dialysis 24h removes the unreacted fluorescein.The dialyzate lyophilize gets fluorescence labeling hydrophobic modified chitin.
3, fluorescence labeling hydrophobic modified chitin micelle preparation
Get fluorescence labeling hydrophobic modified chitin 20mg, the accurate title, decide.Add the ultrasonic 10min of an amount of distilled water water-bath and disperse, be settled to 100ml, get the fluorescence labeling hydrophobic modified chitin micelle solution.
Zetasizer 3000HS analysis-e/or determining particle diameter and surface potential.The median size of fluorescence labeling hydrophobic modified chitin micelle is 32.2nm, is unimodal distribution; Surface potential is 51.6 ± 0.2mV.
Example three:
1, the preparation of oligochitosan
Chitosan (molecular-weight average 450kDa) 6g adds in the aqueous hydrochloric acid of 200mL 1.25% (v/v), stirring and dissolving under 55~60 ℃ of conditions, transfer pH to 5.0 with weak ammonia or dilute hydrochloric acid, add cellulase in cellulase and chitosan ratio 0.5: 100 (w/w), controlling reaction time 20 hours, after question response finishes, with reaction product 4000r * min
-1Centrifugal 10 minutes, supernatant liquor was with 0.45 μ m millipore filtration pre-treatment, and with the ultra-filtration membrane ultrafiltration classification of different molecular weight, the ultrafiltrated lyophilize obtains the oligochitosan of different molecular weight.And be 18.8kDa by the gel permeation chromatography molecular-weight average.
2, the preparation of fluorescence labeling hydrophobic modified chitin
Get 0.5g oligochitosan (molecular-weight average 18.8kDa), the accurate title, decide.After adding 40ml distilled water stirring and dissolving, add carbodiimide 0.12mg, stirring and dissolving.With the stearic acid of 0.16g, add in the 10ml ethanolic soln, behind the ultrasonic dissolution, add in the above-mentioned oligochitosan solution, at 400rmin
-1Under the magnetic agitation condition, 80 ℃ of controlled temperature are more than the reaction times 24h.End reaction liquid is put dialysis tubing, and distilled water dialysis 24h removes byproduct of reaction.The dialyzate lyophilize prepares hydrophobic modified chitin, and the trinitro-benzene-sulfonic acid method of pressing in the example one is measured, and substitution value is 17.8%.
Get 16.0mg fluorescein isothiocyanate (fitc) and 0.5g hydrophobic modified chitin, be dissolved in respectively in 16ml and 20ml 50% ethanolic soln after accurate title is fixed.At ice bath, lucifuge and magnetic agitation (400rmin
-1) under the condition, with the hydrophobic modified chitin ethanolic soln, be added drop-wise in the fluorescein isothiocyanate (fitc) solution, the same terms continues reaction 24h down.End reaction liquid is put dialysis tubing, and redistilled water dialysis 24h removes the unreacted fluorescein isothiocyanate (fitc).The dialyzate lyophilize gets fluorescence labeling hydrophobic modified chitin, and is stand-by.
3, fluorescence labeling hydrophobic modified chitin micelle preparation
Get fluorescence labeling hydrophobic modified chitin 40mg, the accurate title, decide.Add the ultrasonic 10min of an amount of distilled water water-bath and disperse, be settled to 20ml, get the fluorescence labeling hydrophobic modified chitin micelle solution.
Zetasizer 3000HS analysis-e/or determining particle diameter and surface potential.The median size of fluorescence labeling hydrophobic modified chitin micelle is 28.9nm, is unimodal distribution; Surface potential is 50.9 ± 0.1mV.
With the A549 cell is model cell, at 37 ℃, and 5%CO
2Culturing cell after the centrifugal collection, is pressed every hole 1 * 10 to being the logarithmic growth after date under the condition
5The density of individual cell is inoculated 24 well culture plates, the pre-cultivation 24 hours in the incubator.Adding the fluorescence labeling hydrophobic modified chitin micelle solution in nutrient solution hatches.Micelle concentration is controlled to be 50~300 μ gml
-1After cultivating certain hour, observe, and collecting cell regularly with the fluorescence inverted microscope, after the lysis with the fluorescent value of fluorescent spectrophotometer measuring intracellular fluid.
Result of study shows, hatch 30min after, micelle begins to be distributed in the cell, and targeted cells nuclear, referring to Fig. 1, Fig. 2, Fig. 2 shows, when carrier concn is 50 μ gml
-1The time, fluorescence intensity level is 8.33 in the cell when hatching 15min; Behind the 2h, fluorescence intensity is increased to 11.83, and is corresponding, when micelle concentration is 300 μ gml
-1The time, intracellular fluorescence intensity is increased to 78.69 from 34.45, and the cellular uptake of visible micelle has time and concentration dependent, and along with the increase of incubation time and micelle concentration, cellular uptake presents the trend of increase.
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the embodiment of front is interpreted as only illustrating, but not limits the scope of the invention by any way.
Claims (9)
1, fluorescence labeling hydrophobic modified chitin polymer has following general structure:
The molecular weight of oligochitosan is less than 200kDa, and the part free amino group on the oligochitosan chain is replaced by alkyl, and amino group substitution degree is 1~50%, wherein R
1Be fluorophor, R
2Be alkyl chain, n is the polymerization degree of oligochitosan, has positive charge, and the zeta current potential is at 5~80mV, and particle diameter is at 5~1000nm, the covalent attachment fluorescence molecule.
2, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 1 is characterized in that realizing by following steps:
(1) hydrophobic modified chitin preparation: get molecular weight 450kDa, the oligochitosan of 90~95% deacetylations, stirring and dissolving under 55~60 ℃ and pH5.0 condition, by cellulase and chitosan weight ratio is 0.5: 100 adding cellulose degraded, remove by filter impurity, with ultra-filtration membrane ultrafiltration classification, the filtrate lyophilize, get molecular weight less than 200kDa, deacetylation is greater than 80% lower molecular weight oligochitosan, the gel permeation chromatography molecular weight, get the above-mentioned oligochitosan aqueous solution, according to oligochitosan, lipid acid, crosslinked coupler carbodiimide mol ratio 1: 1~50: 1~50 is controlled 50 ℃~90 ℃, reacts 5~48 hours, end reaction liquid dialysis purifying, lyophilize obtains hydrophobic modified chitin;
(2) fluorescence labeling hydrophobic modified chitin preparation: get the hydrophobic modified chitin aqueous solution, according to hydrophobic modified chitin, isothiocyano group fluorescent marker mol ratio 1: 1~50, under ice bath, lucifuge and magnetic agitation condition, reacted 5~48 hours, end reaction liquid dialysis purifying, lyophilize obtains fluorescence labeling hydrophobic modified chitin;
(3) fluorescence labeling hydrophobic modified chitin polymer micelle preparation: by 1~10000: 1000mg/ml, the ratio of w/v in water, prepares the polymeric micellar particulate with the fluorescence labeling hydrophobic modified chitin ultra-sonic dispersion.
3, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 2 is characterized in that: the preferred oligochitosan of step (1) is the lower molecular weight oligochitosan of 0.1~50kDa.
4, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 2 is characterized in that: step (1) lipid acid is selected from myristic acid, Palmiticacid, oleic acid, the stearic acid, docosoic etc. any.
5, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 2 is characterized in that: isothiocyano group fluorescent marker is isothiocyano group fluorescein or TRITC in the step (2).
6, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 2 is characterized in that: the mol ratio of oligochitosan, lipid acid and isothiocyano group fluorescent marker is 1: 1~50: 1~50 in the step (2).
7, the preparation method of fluorescence labeling hydrophobic modified chitin polymer according to claim 2 is characterized in that: fluorescence labeling hydrophobic modified chitin and water are 1~10000: 1000 according to by weight/volume in the step (3), disperse under ultrasound condition.
8, fluorescence labeling hydrophobic modified chitin polymer according to claim 1 is as the application of organoid target particulate carrier in the preparation targeted drug.
9, fluorescence labeling hydrophobic modified chitin polymer according to claim 1 is used in the fluorescent mark that substitutes traditional gene chip as the carrier that fluorescent signal amplifies.
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CN110104627A (en) * | 2019-04-02 | 2019-08-09 | 贵州大学 | A method of mesoporous carbon is prepared by carbon nitrogen source of chitosan oligosaccharide |
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