CN101066461A - Application of cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle - Google Patents
Application of cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle Download PDFInfo
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Abstract
The present invention is application of medicine carrying chitosan-fatty acid grafting micelle in preparing targeting antitumor medicine carrier, especially antitumor medicine carrier targeting to lung cancer cell nucleus, cervical carcinoma cell nucleus and mammary cancer cell nucleus. The chitosan-fatty acid grafting micelle of the present invention has grafting carrier with low toxicity, and may be ingested fast by cell nucleus to target to cell nucleus and to raise the curative effect of antitumor medicine by up to 80 times. It has cell nucleus targeting function of permeating cell membrane fast with less destruction by intracellular lysosome. The grafting micelle with hydrophobic kernel can envelop hydrophobic antitumor medicine targeting cell nucleus to form medicine carrying grafting micelle. The present invention provides nanometer subcellular organelle administration for high efficiency low toxicity tumor treatment.
Description
Technical field
The invention belongs to the application of cell nucleus targeting antitumor drug medicine carrying micelle, relate to the application of chitosan-fatty acid graft as medicine carrier micelle in the preparation antitumor drug of cell nucleus targeting.
Background technology
Tumor is the major disease that directly threatens human health always, and chemotherapy of tumors is because the molecular targeted property of medicine shortage itself, thereby great treatment problems such as cure rate is low, toxic and side effects is huge occur.By the suitable carriers technology, with the direct targeting pathological tissues of medicine (organ), cell and subcellular organelle one of important means that solves the low and toxic and side effects of cancer chemotherapy cure rate.Present scientist both domestic and external has obtained certain progress by the nano-carrier technology on the tissue (organ) of antitumor drug and cell-targeting, but the curative effect of making a breakthrough property not, its essence is that the molecular action target spot of most antitumor drug is positioned at cell.Therefore, medicine molecular action target spot (subcellular organelle) targeted nanometer carrier material Study on Technology exploitation in the tumor cell is the key that breaks through the cancer chemotherapy bottleneck.
(polymeric micelles is a class novel nano carrier that was developing in recent years PMs) to polymeric micellar, has solubilising, targeting, low toxicity and macrocyclic advantage.Polymeric micellar has unique nucleocapsid structure by amphipathic polymer spontaneous formation in aqueous environments.Its kernel can be insoluble drug bank is provided, and the hydrophilic shell then can carry out physicochemical property to be modified, and can seal polypeptide protein class medicine and genomic medicine, and reach that targeting distributes in the body, escape mononuclear phagocyte engulf, improve effect such as biomembrane transhipment.Polymeric micellar self can gather tumor tissues by " enhanced seeing through and retention effect " (enhanced permeability andretention effect).
The molecular action target of most of antitumor drug is positioned at nucleus, as: amycin, ametycin etc.Existing chemotherapy of tumors technology has been continued to use the non-targeting mode of administration of medicine substantially, has serious toxicity, and therefore a lot of patients stop treatment, and compliance is poor.Domestic and international research person is by the passive target and the active targeting technology of carrier, improved tumor cell accumulating of medicine on every side to a certain extent, but do not embody significant antitumor curative effect, its subject matter is that the molecular action target of most of antitumor drug is positioned at nucleus, and existing carrier technique does not have the nucleus transport function.
Chitosan is the cationic polymer 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 prepared microgranule of stock and nanoparticle, has been widely used in the research of drug carrier material, because the hydrophilic structure of chitosan itself makes it be difficult to can't be realized the subcellular organelle targeting by the cell huge uptake.
Summary of the invention
The purpose of this invention is to provide the application of chitosan-fatty acid graft as medicine carrier micelle in the targeting vector of preparation antitumor drug.
Another object of the present invention provides chitosan-fatty acid graft as medicine carrier micelle is arranged in the targeting vector of nuclear antitumor drug at the preparation molecular target application.
Chitosan-fatty acid graft as medicine carrier micelle provided by the invention is used at the targeting vector that the preparation molecular target is arranged in the antitumor drug of lung carcinoma cell nuclear.
Chitosan-fatty acid graft as medicine carrier micelle provided by the invention is used at the targeting vector that the preparation molecular target is arranged in the antitumor drug of cervical cancer cell nuclear.
Chitosan-fatty acid graft as medicine carrier micelle provided by the invention is used at the targeting vector that the preparation molecular target is arranged in the antitumor drug of breast cancer cell nuclear.
Chitosan oligosaccharide-aliphatic acid grafting micelle provided by the invention, when chitosan oligosaccharide-aliphatic acid grafting concentration surpasses critical micelle concentration, can spontaneous formation grafting micelle in aqueous medium, micelle possesses by cell and absorbs fast, and the function of targeted cells nuclear.The composition of grafting micelle of the present invention is contained by patent " surface-modified hydrophobically modified chitin polymer administration micelle and preparation method thereof " (number of patent application 200610051601.0) and patent " a kind of non-viral gene transfection carrier and preparation method and purposes " (number of patent application 200610050516.2).
Adopt graft as medicine carrier micelle provided by the invention since this grafting carrier toxicity low, have by cell and absorb fast, and the function of targeted cells nuclear, the therefore maximum antitumor drug curative effect that can improve more than 80 times.
The present invention is with carboxylic lyophobic dust (as fatty acid), with the amino grafting of the part in the chitosan molecule structure, can strengthen the hydrophobicity of chitosan molecule structure, chitosan-the aliphatic acid grafting for preparing, this grafting can form the grafting micelle by self aggregation in aqueous medium.The hydrophobic cores of grafting micelle, but the hydrophobic drug molecule of solubilising.By chitosan hydrolyzate control molecular weight, obtain oligochitosan; By the ingredient proportion in the control grafting building-up process.
Usefulness of the present invention is: the chitosan oligosaccharide-aliphatic acid grafting micelle of synthetic low cytotoxicity, have quick permeate through cell membranes, seldom by lysosome in the cell destroy, the function of cell nucleus targeting, can prepare and have specific amino group substitution degree chitosan oligosaccharide-aliphatic acid grafting.This grafting micelle possesses by cell and absorbs fast, and concentrates in nuclear carrier target function.And by self the hydrophobic cores characteristic, seal molecular target and be positioned at nuclear hydrophobic anticancer drug, form graft as medicine carrier micelle, be the antineoplaston of high-efficiency low-toxicity, novel nanometer subcellular organelle targeting drug delivery system is provided.This carrier material is applied to the transmission that molecular target is positioned at the nucleus antitumor drug, can increases substantially the antitumor curative effect of such medicine.
Description of drawings
Fig. 1: the A549 cellular uptake fluorescence microscope photo of oligochitosan-stearic acid grafting micelle.
The specific embodiment
The present invention is further described by embodiment and accompanying drawing.
Embodiment 1: the antineoplaston of chitosan-fatty acid graft as medicine carrier micelle on lung cancer A549 cell used
1) low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 92.5%) 90g, add in the aqueous hydrochloric acid solution of 3000mL1.25% (v/v), under 55～60 ℃ of temperature conditions, swelling is after 2 hours, the cellulase (w/w) of adding 5% is degraded under 55～60 ℃ of temperature conditions.Control reaction temperature and time, the son amount that makes low score chitosan (oligochitosan).Gained degradation reaction liquid, (Biomax-10, Millipore Co. USA) after the ultrafiltration classification, get the lyophilization of molecular weight 10～50Kda ultrafiltrate to use 50Kda and 10Kda ultrafilter membrane.Gel permeation chromatography oligochitosan molecular weight, adopt TSK-gel G3000SW chromatographic column, 0.1mol/L sodium acetate (pH6.0) is a mobile phase, respectively with molecular weight 0.73,5.9,11.8,47.3,212.0 the glucosan standard specimen of 788.0Kda prepares elution curve, calculates the oligochitosan molecular weight with this elution curve.After measured, the weight average molecular weight of gained oligochitosan is 18.4kDa.
2) oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned oligochitosan 0.4g, precision weighing, add 30mL distilled water stirring and dissolving after, add the carbodiimide (EDC) of proportional quantity (mol ratio of oligochitosan and carbodiimide is 1: 100), stirring and dissolving.According to oligochitosan: stearic acid (mol ratio) 1: 20 takes by weighing stearic acid and is dissolved in the 20mL ethanol solution.With the mixed liquor of above-mentioned two kinds of solution, under 400rpm magnetic agitation condition, 80 ℃ of isothermal reactions 5 hours are cooled to room temperature (25 ℃), continue to stir 6 hours.End reaction liquid is put in the bag filter distill water dialysis 48 hours.After the dialysis solution lyophilization, remove residual stearic acid, get oligochitosan-stearic acid grafting with absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method to measure the amino group substitution degree of 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 bicarbonate solution 2mL and 0.1% (w/v) of 4% (w/v), hatched 2 hours for 37 ℃ to get not commensurability oligochitosan.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 distilled water, with the method operation, measuring amino group substitution degree is 9.52%.
Adopt pyrene fluorescence spectrometry oligochitosan-stearic critical micelle concentration.Each 10mL of preparation variable concentrations chitosan oligosaccharide-aliphatic acid grafting solution, (the pyrene final concentration is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), the ultrasonic 30min of room-temperature water bath.The excitation spectrum and the emission spectra of scanning pyrene are determined at 375nm and 396nm fluorescence intensity, and to measure I
375/ I
396The unexpected variation of slope, the critical micelle concentration of determining this grafting is 0.04mg/mL.
Adopt particle size and surface potential analyser, measure the particle diameter and the surface potential of oligochitosan-stearic acid grafting micelle.After measured, the particle diameter and the surface potential of the oligochitosan of 1mg/mL-stearic acid grafting micelle are respectively 24.6nm and 30.1mV.
3) oligochitosan-stearic acid grafting micelle cellular uptake
Adopt oligochitosan-stearic acid grafting fluorescent labeling micelle to carry out cellular uptake research.With fluorescein isothiocyanate (fitc) (Fluorescein isothiocyanate, FITC) labelling oligochitosan-stearic acid grafting.Get oligochitosan-stearic acid grafting 20mg, be dissolved in 50% methanol solution of 2.4mL; Other gets the 5.0mg fluorescein isothiocyanate (fitc), is dissolved in 5.0mL ethanol.The alcoholic solution of getting 0.5mL FITC mixes with the 0.5mL distilled water, under ice bath, lucifuge and magnetic agitation condition, methanol solution with oligochitosan-stearic acid grafting, be added drop-wise in the FITC-alcoholic solution, behind the room temperature reaction 24 hours, with distill water dialysis (MWCO10,000) 24 hours, the final FITC labelling oligochitosan-stearic acid grafting that gets, it is standby to keep in Dark Place.
Get the A549 cell, in the RPMI RPMI-1640 that contains 10% calf serum of having an appointment, cultivate (5%CO
2, 37 ℃ of incubators).The trophophase cell of taking the logarithm after the PBS rinse of pH7.4, adds trypsinization, and with the culture fluid dilution, by every hole 1 * 10
5The density of individual cell is inoculated in 24 well culture plates, cultivates 24 hours in the incubator.After the 24 well culture plate inner cell adherent growth, discard old culture fluid, after the PBS rinse 2 times, add fresh medium 1mL and FITC labelling oligochitosan-stearic acid grafting micelle solution (final concentration: 200 μ g/mL) respectively.After hatching certain hour,, put the fluorescence inverted microscope and observe and take pictures with PBS flushing cell 2 times.After grafting and A549 cell were hatched 2 hours, the fluorescence microscope photo of grafting cellular uptake was seen Fig. 1, and wherein A figure is a FITC labelling micelle cellular uptake fluorescence photo, and B figure is that nuclear dyes the contrast photo.
4) oligochitosan-stearic acid graft as medicine carrier micelle preparation
Get 40mg oligochitosan-stearic acid grafting, precision claims fixed, puts in the 50mL beaker, adds 40mL distilled water (pH is 5.7), pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is transferred to measuring bottle, and distilled water is settled to 50mL, gets the micelle solution of 0.8mg/mL.Pipette this oligochitosan-stearic acid grafting micelle solution 20mL, add the 7mg ametycin, ultrasonic 30 times of probe (400W, work 2s stops 3s) gets oligochitosan-stearic acid graft as medicine carrier micelle solution under the condition of ice bath.
After measured, the particle diameter of oligochitosan-stearic acid graft as medicine carrier micelle is 48.6nm, and surface potential is 30.4mV.Entrapment efficiency is 64.14%.
5) oligochitosan-stearic acid graft as medicine carrier micelle antitumor curative effect
The present invention is with inhibition rate of tumor cell (IC
50Value), estimate the antitumor drug effect of chitosan-fatty acid graft as medicine carrier micelle.With human lung cancer cell A549's cell is model, and in 96 porocyte culture plates, every hole adds 100 μ L and contains 5 * 10
3The culture fluid of individual A549 cell is put 37 ℃, 5%CO
2Incubator was cultivated 24 hours, after treating that cell is adherent fully, adding oligochitosan-stearic acid grafting, the oligochitosan-stearic acid graft as medicine carrier micelle solution and the mitomycin c solution of variable concentrations in the cell hole respectively, is contrast with undressed blank cell, and multiple hole is established in every hole.Normal DMEM culture fluid continues to cultivate 48 hours, and every hole adds concentration 5mg/mL Thiazolyl blue (MTT) solution 20 μ L, places 37 ℃, 5%CO
2Incubator continues to cultivate after 4 hours, abandons supernatant, and every hole adds dimethyl sulfoxide 150 μ L, measures absorbance with multi-functional microplate reader, and calculates cell inhibitory rate.Cell inhibitory rate adopts following formula to calculate:
Cell inhibitory rate %=(blank group absorbance-experimental group absorbance)/blank group absorbance * 100%
As calculated, the IC of oligochitosan-stearic acid grafting
50(fatality rate of cell half) is 309.5 μ g/mL, the IC of mitomycin c solution
50Be 17.84 μ g/mL, and the IC of oligochitosan-stearic acid graft as medicine carrier micelle solution
50Be 0.31 μ g/mL.The result shows that oligochitosan-stearic acid grafting is the low cytotoxicity material, and ametycin on lung cancer A549 cell, can improve 57.5 times of antitumor curative effects through oligochitosan-stearic acid grafting micelle Bao Zaihou.
Embodiment 2: the antineoplaston of oligochitosan-stearic acid graft as medicine carrier micelle on cervical cancer cell Hela cell used
1) low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 92.5%) 90g, add in the aqueous hydrochloric acid solution of 3000mL1.25% (v/v), under 55～60 ℃ of temperature conditions, swelling is after 2 hours, the cellulase (w/w) of adding 5% is degraded under 55～60 ℃ of temperature conditions.Control reaction temperature and time, the son amount that makes low score chitosan (oligochitosan).Gained degradation reaction liquid, (Biomax-10, Millipore Co. USA) after the ultrafiltration classification, get the lyophilization of molecular weight 10～50Kda ultrafiltrate to use 50Kda and 10Kda ultrafilter membrane.Gel permeation chromatography oligochitosan molecular weight, adopt TSK-gel G3000SW chromatographic column, 0.1mol/L sodium acetate (pH6.0) is a mobile phase, respectively with molecular weight 0.73,5.9,11.8,47.3,212.0 the glucosan standard specimen of 788.0Kda prepares elution curve, calculates the oligochitosan molecular weight with this elution curve.The weight average molecular weight of gained oligochitosan is 18.4kDa after measured.
2) oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned oligochitosan 0.4g, precision weighing, add 30mL distilled water stirring and dissolving after, add the carbodiimide (EDC) of proportional quantity (mol ratio of oligochitosan and carbodiimide is 1: 100), stirring and dissolving.According to oligochitosan: stearic acid (mol ratio) 1: 20 takes by weighing stearic acid and is dissolved in the 20mL ethanol solution.With the mixed liquor of above-mentioned two kinds of solution, under 400rpm magnetic agitation condition, 80 ℃ of isothermal reactions 5 hours are cooled to room temperature (25 ℃), continue to stir 6 hours.End reaction liquid is put in the bag filter distill water dialysis 48 hours.After the dialysis solution lyophilization, remove residual stearic acid, get oligochitosan-stearic acid grafting with absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method to measure the amino group substitution degree of 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 bicarbonate solution 2mL and 0.1% (w/v) of 4% (w/v), hatched 2 hours for 37 ℃ to get not commensurability oligochitosan.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 distilled water, with the method operation, measuring amino group substitution degree is 9.52%.
Adopt pyrene fluorescence spectrometry oligochitosan-stearic critical micelle concentration.Each 10mL of preparation variable concentrations chitosan oligosaccharide-aliphatic acid grafting solution, (the pyrene final concentration is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), the ultrasonic 30min of room-temperature water bath.The excitation spectrum and the emission spectra of scanning pyrene are determined at 375nm and 396nm fluorescence intensity, and to measure I
375/ I
396The unexpected variation of slope determines that the critical micelle concentration of this grafting is 0.04mg/mL.
Adopt particle size and surface potential analyser, measure the particle diameter and the surface potential of oligochitosan-stearic acid grafting micelle.After measured, the particle diameter and the surface potential of the oligochitosan of 1mg/mL-stearic acid grafting micelle are respectively 24.6nm and 30.1mV.
3) oligochitosan-stearic acid grafting micelle cellular uptake
Adopt oligochitosan-stearic acid grafting fluorescent labeling micelle to carry out cellular uptake research.With fluorescein isothiocyanate (fitc) (Fluorescein isothiocyanate, FITC) labelling oligochitosan-stearic acid grafting.Get oligochitosan-stearic acid grafting 20mg, be dissolved in 50% methanol solution of 2.4mL; Other gets the 5.0mg fluorescein isothiocyanate (fitc), is dissolved in 5.0mL ethanol after accurate title is fixed.The alcoholic solution of getting 0.5mL FITC mixes with the 0.5mL distilled water, under ice bath, lucifuge and magnetic agitation condition, the methanol solution of oligochitosan-stearic acid grafting is added drop-wise in the FITC-alcoholic solution, behind the room temperature reaction 24 hours, (MWCO 10 with distill water dialysis, 000) 24 hours, the final FITC labelling oligochitosan-stearic acid grafting that gets, it is standby to keep in Dark Place.
Get the A549 cell, in the RPMI RPMI-1640 that contains 10% calf serum of having an appointment, cultivate (5%CO
2, 37 ℃ of incubators).The trophophase cell of taking the logarithm after the PH7.4 PBS rinse, adds trypsinization and with the culture fluid dilution, by every hole 1 * 10
5The density of individual cell is inoculated in 24 well culture plates, cultivates 24 hours in the incubator.After the 24 well culture plate inner cell adherent growth, discard old culture fluid, after the PBS rinse 2 times, add fresh medium 1mL and FITC labelling oligochitosan-stearic acid grafting micelle solution (final concentration: 200 μ g/mL) respectively.After hatching certain hour,, put the fluorescence inverted microscope and observe and take pictures with PBS flushing cell 2 times.After grafting and A549 cell were hatched 2 hours, the fluorescence microscope photo of grafting cellular uptake was seen Fig. 1.
4) oligochitosan-stearic acid graft as medicine carrier micelle preparation
Get 40mg oligochitosan-stearic acid grafting, precision claims fixed, puts in the 50mL beaker, adds 40mL distilled water (pH is 5.7), pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), is transferred to measuring bottle, and distilled water is settled to 50mL, gets the micelle solution of 0.8mg/mL.Pipette this oligochitosan-stearic acid grafting micelle solution 20mL, add the 7mg ametycin, ultrasonic 30 times of probe (400W, work 2s stops 3s) gets oligochitosan-stearic acid graft as medicine carrier micelle solution under the condition of ice bath.
After measured, the particle diameter of oligochitosan-stearic acid graft as medicine carrier micelle is 48.6nm, and surface potential is 30.4mV.Entrapment efficiency is 64.14%.
5) oligochitosan-stearic acid graft as medicine carrier micelle antitumor curative effect
The present invention is with inhibition rate of tumor cell (IC
50Value), estimate the antitumor drug effect of chitosan-fatty acid graft as medicine carrier micelle.With cervical cancer cell Hela cell is model, and in 96 porocyte culture plates, every hole adds and contains 5 * 10
3100 μ L culture fluid of individual Hela cell are put 37 ℃, 5%CO
2Incubator was cultivated 24 hours, after treating that cell is adherent fully, adding oligochitosan-stearic acid grafting, the oligochitosan-stearic acid graft as medicine carrier micelle solution and the mitomycin c solution of variable concentrations in the cell hole respectively, is contrast with undressed blank cell, and multiple hole is established in every hole.Normal DMEM culture fluid continues to cultivate 48 hours, and every hole adds concentration 5mg/mL Thiazolyl blue (MTT) solution 20 μ L, places 37 ℃, 5%CO
2Incubator continues to cultivate after 4 hours, abandons supernatant, and every hole adds dimethyl sulfoxide 150 μ L, measures absorbance with multi-functional microplate reader, and calculates cell inhibitory rate.Cell inhibitory rate adopts following formula to calculate:
Cell inhibitory rate %=(blank group absorbance-experimental group absorbance)/blank group absorbance * 100%
As calculated, the IC of oligochitosan-stearic acid grafting
50(fatality rate of cell half) is 383.6 μ g/mL, the IC of mitomycin c solution
50Be 31.72 μ g/mL, and the IC of oligochitosan-stearic acid graft as medicine carrier micelle solution
50Be 0.39 μ g/mL.The result shows that oligochitosan-stearic acid grafting is the hypotoxicity material, and ametycin can improve 81.3 times of antitumor curative effects through oligochitosan-stearic acid grafting micelle Bao Zaihou on cervical cancer cell Hela cell.
Embodiment 3: the antineoplaston of different amino group substitution degree chitosan-fatty acid graft as medicine carrier micelle on lung cancer A549 cell used
1) low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 92.5%) 90g, add in the aqueous hydrochloric acid solution of 3000mL1.25% (v/v), under 55～60 ℃ of temperature conditions, swelling is after 2 hours, the cellulase (w/w) of adding 5% is degraded under 55～60 ℃ of temperature conditions.Control reaction temperature and time, the son amount that makes low score chitosan (oligochitosan).Gained degradation reaction liquid, (Biomax-10, Millipore Co. USA) after the ultrafiltration classification, get the lyophilization of molecular weight 10～50Kda ultrafiltrate to use 50Kda and 10Kda ultrafilter membrane.Gel permeation chromatography oligochitosan molecular weight, adopt TSK-gel G3000SW chromatographic column, 0.1mol/L sodium acetate (pH6.0) is a mobile phase, with molecular weight 0.73,5.9,11.8,47.3,212.0 the glucosan standard specimen of 788.0Kda prepares elution curve, calculates the oligochitosan molecular weight with this elution curve.The weight average molecular weight of gained oligochitosan is 18.4kDa after measured.
2) oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned oligochitosan 0.4g, precision weighing, add 30mL distilled water stirring and dissolving after, add the carbodiimide (EDC) of proportional quantity (mol ratio of oligochitosan and carbodiimide is 1: 100), stirring and dissolving.According to oligochitosan: stearic acid (mol ratio) 1: 10 takes by weighing stearic acid and is dissolved in the 20mL ethanol solution.With the mixed liquor of above-mentioned two kinds of solution, under 400rpm magnetic agitation condition, 80 ℃ of isothermal reactions 5 hours are cooled to room temperature (25 ℃), continue to stir 6 hours.End reaction liquid is put in the bag filter distill water dialysis 48 hours.After the dialysis solution lyophilization, remove residual stearic acid, get oligochitosan-stearic acid grafting with absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method to measure the amino group substitution degree of 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 bicarbonate solution 2mL and 0.1% (w/v) of 4% (w/v), hatched 2 hours for 37 ℃ to get not commensurability oligochitosan.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 distilled water, with the method operation, measuring amino group substitution degree is 5.40%.
Adopt pyrene fluorescence spectrometry oligochitosan-stearic critical micelle concentration.Each 10mL of preparation variable concentrations chitosan oligosaccharide-aliphatic acid grafting solution, (the pyrene final concentration is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), the ultrasonic 30min of room-temperature water bath.The excitation spectrum and the emission spectra of scanning pyrene are determined at 375nm and 396nm fluorescence intensity, and to measure I
375/ I
396The unexpected variation of slope determines that the critical micelle concentration of this grafting is 0.05mg/mL.
Adopt particle size and surface potential analyser, measure the particle diameter and the surface potential of oligochitosan-stearic acid grafting micelle.After measured, the particle diameter and the surface potential of the oligochitosan of 1mg/mL-stearic acid grafting micelle are respectively 31.4nm and 33.4mV.
3) oligochitosan-stearic acid graft as medicine carrier micelle preparation
Get 40mg oligochitosan-stearic acid grafting, the accurate title, decide, and puts in the 50mL beaker, add 40mL distilled water (pH is 5.7), pop one's head in ultrasonic 20 times (500w, work 2s stops 3s), be transferred in the volumetric flask, be settled to 50mL, get the micelle solution of 0.8mg/mL with distilled water.Pipette this oligochitosan-stearic acid grafting micelle solution 20mL, add the 7mg ametycin, ultrasonic 30 times of probe (400W, work 2s stops 3s) gets oligochitosan-stearic acid graft as medicine carrier micelle solution under the condition of ice bath.
After measured, the particle diameter of oligochitosan-stearic acid graft as medicine carrier micelle is 43.5nm, and surface potential is 32.9mV.Entrapment efficiency is 58.14%.
4) oligochitosan-stearic acid graft as medicine carrier micelle antitumor curative effect
The present invention is with inhibition rate of tumor cell (IC
50Value), estimate the antitumor drug effect of chitosan-fatty acid graft as medicine carrier micelle.With human lung cancer cell A549's cell is model, and in 96 porocyte culture plates, every hole adds and contains 5 * 10
3100 μ L culture fluid of individual A549 cell are put 37 ℃, 5%CO
2Incubator was cultivated 24 hours, after treating that cell is adherent fully, adding oligochitosan-stearic acid grafting, the oligochitosan-stearic acid graft as medicine carrier micelle solution and the mitomycin c solution of variable concentrations in the cell hole respectively, is contrast with undressed blank cell, and multiple hole is established in every hole.Normal DMEM culture fluid continues to cultivate 48 hours, and every hole adds concentration 5mg/mL Thiazolyl blue (MTT) solution 20 μ L, places 37 ℃, 5%CO
2Incubator continues to cultivate after 4 hours, abandons supernatant, and every hole adds dimethyl sulfoxide 150 μ L, measures absorbance with multi-functional microplate reader, and calculates cell inhibitory rate.Cell inhibitory rate adopts following formula to calculate:
Cell inhibitory rate %=(blank group absorbance-experimental group absorbance)/blank group absorbance * 100%
As calculated, the IC of oligochitosan-stearic acid grafting
50(fatality rate of cell half) is 418.2 μ g/mL, the IC of mitomycin c solution
50Be 17.84 μ g/mL, and the IC of oligochitosan-stearic acid graft as medicine carrier micelle solution
50Be 0.58 μ g/mL.The result shows that oligochitosan-stearic acid grafting is the hypotoxicity material, and ametycin can improve 30.8 times of antitumor curative effects through oligochitosan-stearic acid grafting micelle Bao Zaihou on lung cancer A549 cell.
Embodiment 4: the antineoplaston of different amino group substitution degree chitosan-fatty acid graft as medicine carrier micelle on cervical cancer cell Hela cell used
1) low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 92.5%) 90g, add in the aqueous hydrochloric acid solution of 3000mL1.25% (v/v), under 55～60 ℃ of temperature conditions, swelling is after 2 hours, the cellulase (w/w) of adding 5% is degraded under 55～60 ℃ of temperature conditions.Control reaction temperature and time, the son amount that makes low score chitosan (oligochitosan).Gained degradation reaction liquid, (Biomax-10, Millipore Co. USA) after the ultrafiltration classification, get the lyophilization of molecular weight 10～50Kda ultrafiltrate to use 50Kda and 10Kda ultrafilter membrane.Gel permeation chromatography oligochitosan molecular weight, adopt TSK-gel G3000SW chromatographic column, 0.1mol/L sodium acetate (pH6.0) is a mobile phase, with molecular weight 0.73,5.9,11.8,47.3,212.0 the glucosan standard specimen of 788.0Kda prepares elution curve, calculates the oligochitosan molecular weight with this elution curve.The weight average molecular weight of gained oligochitosan is 18.4kDa after measured.
2) oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned oligochitosan 0.4g, precision weighing, add 30mL distilled water stirring and dissolving after, add the carbodiimide (EDC) of proportional quantity (mol ratio of oligochitosan and carbodiimide is 1: 100), stirring and dissolving.According to oligochitosan: stearic acid (mol ratio) 1: 10 takes by weighing stearic acid and is dissolved in the 20mL ethanol solution.With the mixed liquor of above-mentioned two kinds of solution, under 400rpm magnetic agitation condition, 80 ℃ of isothermal reactions 5 hours are cooled to room temperature (25 ℃), continue to stir 6 hours.End reaction liquid is put in the bag filter distill water dialysis 48 hours.After the dialysis solution lyophilization, remove residual stearic acid, get oligochitosan-stearic acid grafting with absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method to measure the amino group substitution degree of 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 bicarbonate solution 2mL and 0.1% (w/v) of 4% (w/v), hatched 2 hours for 37 ℃ to get not commensurability oligochitosan.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 distilled water, with the method operation, measuring amino group substitution degree is 5.40%.
Adopt pyrene fluorescence spectrometry oligochitosan-stearic critical micelle concentration.Each 10mL of preparation variable concentrations chitosan oligosaccharide-aliphatic acid grafting solution, (the pyrene final concentration is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), the ultrasonic 30min of room-temperature water bath.The excitation spectrum and the emission spectra of scanning pyrene are determined at 375nm and 396nm fluorescence intensity, and to measure I
375/ I
396The unexpected variation of slope determines that the critical micelle concentration of this grafting is 0.05mg/mL.
Adopt particle size and surface potential analyser, measure the particle diameter and the surface potential of oligochitosan-stearic acid grafting micelle.After measured, the particle diameter and the surface potential of the oligochitosan of 1mg/mL-stearic acid grafting micelle are respectively 31.4nm and 33.4mV.
3) oligochitosan-stearic acid graft as medicine carrier micelle preparation
Get 40mg oligochitosan-stearic acid grafting, the accurate title, decide, and puts in the 50mL beaker, add 40mL distilled water (pH is 5.7), pop one's head in ultrasonic 20 times (500w, work 2s stops 3s), be transferred in the volumetric flask, be settled to 50mL, get the micelle solution of 0.8mg/mL with distilled water.Pipette this oligochitosan-stearic acid grafting micelle solution 20mL, add the 7mg ametycin, ultrasonic 30 times of probe (400W, work 2s stops 3s) gets oligochitosan-stearic acid graft as medicine carrier micelle solution under the condition of ice bath.
After measured, the particle diameter of oligochitosan-stearic acid graft as medicine carrier micelle is 43.5nm, and surface potential is 32.9mV.Entrapment efficiency is 58.14%.
4) oligochitosan-stearic acid graft as medicine carrier micelle antitumor curative effect
The present invention estimates the antitumor drug effect of chitosan-fatty acid graft as medicine carrier micelle with inhibition rate of tumor cell.With cervical cancer cell Hela cell is model, and in 96 porocyte culture plates, every hole adds and contains 5 * 10
3100 μ L culture fluid of individual Hela cell are put 37 ℃, 5%CO
2Incubator was cultivated 24 hours, after treating that cell is adherent fully, adding oligochitosan-stearic acid grafting, the oligochitosan-stearic acid graft as medicine carrier micelle solution and the mitomycin c solution of variable concentrations in the cell hole respectively, is contrast with undressed blank cell, and multiple hole is established in every hole.Normal DMEM culture fluid continues to cultivate 48 hours, and every hole adds concentration 5mg/mL Thiazolyl blue (MTT) solution 20 μ L, places 37 ℃, 5%CO
2Incubator continues to cultivate after 4 hours, abandons supernatant, and every hole adds dimethyl sulfoxide 150 μ L, measures absorbance with multi-functional microplate reader, and calculates cell inhibitory rate.Cell inhibitory rate adopts following formula to calculate:
Cell inhibitory rate %=(blank group absorbance-experimental group absorbance)/blank group absorbance * 100%
As calculated, the IC of oligochitosan-stearic acid grafting
50(fatality rate of cell half) is 492.5 μ g/mL, the IC of mitomycin c solution
50Be 31.72 μ g/mL, and the IC of oligochitosan-stearic acid graft as medicine carrier micelle solution
50Be 0.98 μ g/mL.The result shows that oligochitosan-stearic acid grafting is the hypotoxicity material, and ametycin can improve 32.4 times of antitumor curative effects through oligochitosan-stearic acid grafting micelle Bao Zaihou on cervical cancer cell Hela cell.
Embodiment 5: the antineoplaston of chitosan-fatty acid graft as medicine carrier micelle on breast cancer cell MCF-7 cell used
1) low-molecular weight chitoglycan (oligochitosan) preparation
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 92.5%) 90g, add in the aqueous hydrochloric acid solution of 3000mL1.25% (v/v), under 55～60 ℃ of temperature conditions, swelling is after 2 hours, the cellulase (w/w) of adding 5% is degraded under 55～60 ℃ of temperature conditions.Control reaction temperature and time, the son amount that makes low score chitosan (oligochitosan).Gained degradation reaction liquid, (Biomax-10, Millipore Co. USA) after the ultrafiltration classification, get the lyophilization of molecular weight 10～50Kda ultrafiltrate to use 50Kda and 10Kda ultrafilter membrane.Gel permeation chromatography oligochitosan molecular weight, adopt TSK-gel G3000SW chromatographic column, 0.1mol/L sodium acetate (pH6.0) is a mobile phase, with molecular weight 0.73,5.9,11.8,47.3,212.0 the glucosan standard specimen of 788.0Kda prepares elution curve, calculates the oligochitosan molecular weight with this elution curve.After measured, the weight average molecular weight of gained oligochitosan is 18.4kDa.
2) oligochitosan-preparation of stearic acid grafting and physical and chemical property determining
Get above-mentioned oligochitosan 0.4g, precision weighing, add 30mL distilled water stirring and dissolving after, add the carbodiimide (EDC) of proportional quantity (mol ratio of oligochitosan and carbodiimide is 1: 100), stirring and dissolving.According to oligochitosan: stearic acid (mol ratio) 1: 20 takes by weighing stearic acid and is dissolved in the 20mL ethanol solution.With the mixed liquor of above-mentioned two kinds of solution, under 400rpm magnetic agitation condition, 80 ℃ of isothermal reactions 5 hours are cooled to room temperature (25 ℃), continue to stir 6 hours.End reaction liquid is put in the bag filter distill water dialysis 48 hours.After the dialysis solution lyophilization, remove residual stearic acid, get oligochitosan-stearic acid grafting with absolute ethanol washing.
Adopt the trinitro-benzene-sulfonic acid method to measure the amino group substitution degree of 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 bicarbonate solution 2mL and 0.1% (w/v) of 4% (w/v), hatched 2 hours for 37 ℃ to get oligochitosan.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 distilled water, with the method operation, measuring amino group substitution degree is 14.8%.
Adopt pyrene fluorescence spectrometry oligochitosan-stearic critical micelle concentration.Each 10mL of preparation variable concentrations chitosan oligosaccharide-aliphatic acid grafting solution, (the pyrene final concentration is 7 * 10 to add quantitative pyrene respectively
-7Mol/L), the ultrasonic 30min of room-temperature water bath.The excitation spectrum and the emission spectra of scanning pyrene are determined at 375nm and 396nm fluorescence intensity, and to measure I
375/ I
396The unexpected variation of slope determines that the critical micelle concentration of this grafting is 0.03mg/mL.
Adopt particle size and surface potential analyser, measure the particle diameter and the surface potential of oligochitosan-stearic acid grafting micelle.After measured, the particle diameter and the surface potential of the oligochitosan of 1mg/mL-stearic acid grafting micelle are respectively 57.6nm and 36.8mV.
3) oligochitosan-stearic acid graft as medicine carrier micelle preparation
Take by weighing oligochitosan-stearic acid grafting 10mg, add water 5ml dissolving.Under magnetic agitation, it is an amount of dropwise to splash into the amycin dimethyl sulfoxide solution, and the dosage that makes amycin is 1.5%.After the stirring at room 4 hours, be transferred in the bag filter, the foreign minister is a water, dialyses and removes dimethyl sulfoxine in 24 hours.Dialysis solution is added the suitable quantity of water standardize solution in the 10ml volumetric flask, make that the final concentration of oligochitosan-stearic acid grafting is 1mg/mL.Dialysis solution is continued to remove as yet the not amycin solid of solubilising with the centrifugal 10min of 4000rpm, get the medicine carrying micelle.It is an amount of to get dispersion liquid, with particle size and surface potential detection instrument, measures the particle diameter and the surface potential of medicine carrying micelle.
After measured, the particle diameter of oligochitosan-stearic acid graft as medicine carrier micelle is 76.1nm, and surface potential is 35.2mV.Entrapment efficiency is 56.5%.
4) oligochitosan-stearic acid graft as medicine carrier micelle antitumor curative effect
The present invention estimates the antitumor drug effect of chitosan-fatty acid graft as medicine carrier micelle with inhibition rate of tumor cell.With breast cancer cell MCF-7 cell is model, and in 96 porocyte culture plates, every hole adds and contains 5 * 10
3100 μ L culture fluid of individual MCF-7 cell are put 37 ℃, 5%CO
2Incubator was cultivated 24 hours, after treating that cell is adherent fully, adding oligochitosan-stearic acid grafting, oligochitosan-stearic acid graft as medicine carrier micelle solution and the amycin solution of variable concentrations in the cell hole respectively, is contrast with undressed blank cell, and multiple hole is established in every hole.Normal DMEM culture fluid continues to cultivate 48 hours, and every hole adds (5mg/mL) solution of 20 μ L Thiazolyl blues (MTT), places 37 ℃, 5%CO
2Incubator continues to cultivate after 4 hours, abandons supernatant, and every hole adds dimethyl sulfoxide 150 μ L, measures absorbance with multi-functional microplate reader, and calculates cell inhibitory rate.Cell inhibitory rate adopts following formula to calculate:
Cell inhibitory rate %=(blank group absorbance-experimental group absorbance)/blank group absorbance * 100%
As calculated, the IC of oligochitosan-stearic acid grafting
50(fatality rate of cell half) is 285.7 μ g/mL, the IC of amycin solution
50Be 0.49 μ g/mL, and the IC of oligochitosan-stearic acid graft as medicine carrier micelle solution
50Be 0.18 μ g/mL.The result shows that oligochitosan-stearic acid grafting is the hypotoxicity material, and amycin can improve 2.7 times of antitumor curative effects through oligochitosan-stearic acid grafting micelle Bao Zaihou on breast cancer cell MCF-7 cell.
Claims (5)
1, cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle is used in the targeting vector of preparation antitumor drug.
2. cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle according to claim 1 is used at the targeting vector that the preparation molecular target is arranged in nuclear antitumor drug.
3, cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle according to claim 2 prepares the targeting vector that molecular target is arranged in the antitumor drug of lung carcinoma cell nuclear and uses.
4, cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle according to claim 2 prepares the targeting vector that molecular target is arranged in the antitumor drug of cervical cancer cell nuclear and uses.
5, cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle according to claim 2 prepares the targeting vector that molecular target is arranged in the antitumor drug of breast cancer cell nuclear and uses.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101773466A (en) * | 2010-03-09 | 2010-07-14 | 沈阳药科大学 | Oral administration nanometer polymer micelle medicine carrying system and preparation method thereof |
| CN101791412A (en) * | 2010-04-09 | 2010-08-04 | 浙江大学 | Acyclovir-chitosan-stearic acid grafting, synthetic method and application thereof |
| CN101797220A (en) * | 2010-04-06 | 2010-08-11 | 浙江大学 | Oxaliplatin chitosan-stearic acid grafting micelle and application |
| CN101596169B (en) * | 2009-07-06 | 2011-05-04 | 浙江大学 | Chitosan nanoparticle for encapsulating adenosine triphosphate and preparation method thereof |
| CN102743336A (en) * | 2012-07-04 | 2012-10-24 | 浙江大学 | Preparation method and application of curcumin chitosan-stearic acid graft micelle |
| CN103638527A (en) * | 2013-12-05 | 2014-03-19 | 浙江大学 | Hemopoietin drug-carried nanoparticles and application thereof |
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- 2007-05-29 CN CNB2007100691156A patent/CN100562341C/en not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101596169B (en) * | 2009-07-06 | 2011-05-04 | 浙江大学 | Chitosan nanoparticle for encapsulating adenosine triphosphate and preparation method thereof |
| CN101773466A (en) * | 2010-03-09 | 2010-07-14 | 沈阳药科大学 | Oral administration nanometer polymer micelle medicine carrying system and preparation method thereof |
| CN101773466B (en) * | 2010-03-09 | 2014-07-30 | 沈阳药科大学 | Oral administration nanometer polymer micelle medicine carrying system and preparation method thereof |
| CN101797220A (en) * | 2010-04-06 | 2010-08-11 | 浙江大学 | Oxaliplatin chitosan-stearic acid grafting micelle and application |
| CN101797220B (en) * | 2010-04-06 | 2011-12-07 | 浙江大学 | Oxaliplatin chitosan-stearic acid grafting micelle and application |
| 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 |
| CN102743336A (en) * | 2012-07-04 | 2012-10-24 | 浙江大学 | Preparation method and application of curcumin chitosan-stearic acid graft micelle |
| CN103638527A (en) * | 2013-12-05 | 2014-03-19 | 浙江大学 | Hemopoietin drug-carried nanoparticles and application thereof |
| CN105920612A (en) * | 2016-06-12 | 2016-09-07 | 浙江大学 | Glucosamine-behenic acid grafting substance and preparation method |
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