CN101797220A - Oxaliplatin chitosan-stearic acid grafting micelle and application - Google Patents
Oxaliplatin chitosan-stearic acid grafting micelle and application Download PDFInfo
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Abstract
The invention provides an oxaliplatin chitosan-stearic acid grafting micelle which comprises 0.5 percent of chitosan-stearic acid grafting, 0.009-0.02 percent of oxaliplatin, 0-0.2 percent of lecithin and the balance of water. The oxaliplatin chitosan-stearic acid grafting micelle is respectively prepared by using a probe ultrasonic method, a film dispersion method and a lecithin mediated film dispersion method. The oxaliplatin chitosan-stearic acid grafting micelle has rapid ingestion function of passive targeting and cancer cells in a polymer micelle, can greatly improve the intracellular medicine concentration of a molecular target in the oxaliplatin in the cancer cells by encapsulating the oxaliplatin chitosan-stearic acid grafting micelle on the oxaliplatin, improves the cytotoxicity and shifts the multidrug resistance, and enhances the clinical anti-tumor efficacy of the oxaliplatin. The oxaliplatin chitosan-stearic acid grafting micelle can be applied to preparing an anti-tumor medicine.
Description
Technical field
The invention belongs to the preparation and the application of oxaliplatin targeting preparation, be specifically related to oxaliplatin chitosan-micellar preparation of stearic acid grafting and application thereof.
Background technology
Chemotherapy is a kind of important means for the treatment of cancer at present.Yet most of chemotherapeutic agents all lack special bio distribution in the body, cause thus normal cell and organ are caused toxic and side effects, thereby their clinical practice is restricted.In addition, tumor cell also becomes a very important factor that causes the chemotherapy of tumors failure to the drug resistance and the multidrug resistance of medicine.
Platinum medicine is a kind of chemotherapeutics with spectrum active anticancer, is also referred to as the DNA alkylating agent.Oxaliplatin is a third generation platinum series antineoplastic medicament, and it is by making the DNA interconnection and suppressing the synthetic purpose that reaches kill cancer cell of DNA.Yet oxaliplatin itself lacks the targeting to tumor tissues, drug distribution amount in tumor tissues seldom, and and erythrocyte between high degree of isolation arranged, making its treatment render a service greatly reduces, also cause simultaneously the sensory nerve pathological changes of frequently-occurring medicine toxicity such as tip and thrombocytopenia etc., thereby limited the clinical practice of oxaliplatin.In addition, tumor tissues also becomes a main cause that causes its chemotherapy failure to the generation of oxaliplatin toleration in therapeutic process.Therefore, develop newtype drug transmission system and just seem particularly important with targeting.Had about the report as pharmaceutical carrier such as liposome, nano-particle in more than ten years in past, what deserves to be mentioned is the polymer micelle that new development is in recent years got up, be considered to a kind of new drug carrier with very big potentiality.
(polymeric micelles, PMs) by amphipathic polymer spontaneous formation in aqueous environments, as kernel, the hydrophilic block has unique nucleocapsid structure as shell to polymeric micellar with the hydrophobicity block for it.Its kernel can be insoluble drug bank is provided, and the hydrophilic shell can make its disperse well in aqueous environments, thereby and can carry out physicochemical property to it and modify and obtain our desired characteristic.Polymer micelle is because its hydrophobic surface and smaller particle size (10-100nm) can be escaped engulfing of mononuclear phagocyte.Polymeric micellar self can be that the EPR effect realizes passive target by " enhanced infiltration and retention effect " (enhanced permeabilityand retention effect), and medicine is gathered tumor tissues.Polymeric micellar can also be realized the active targeting to tumor tissues by ligand modified.At present, existing with the report of polymer micelle as the oxaliplatin pharmaceutical carrier.Cabral etc. have prepared the block copolymer [PEG-b-P (Glu)] of Polyethylene Glycol and polyglutamic acid, wrap up the medicine carrying micelle that oxaliplatin obtains with it, its anti-tumor in vivo is active result show, compare with free drug, this medicine carrying micelle has improved the anti-tumor activity of medicine greatly, and have good distribution to tumor tissues, its drug level in tumor tissues is compared with free drug and has been improved 20 times.Because oxaliplatin has certain water solublity, wrap up with polymer micelle and also have some difficulties, be not a lot of about the report of this respect.
But chitosan is a kind of cationic carrier material with low toxicity, high-biocompatibility vivo degradation.With stearic acid (stearic acid, SA) grafting to chitosan (chitosan, on main chain CSO), obtain amphipathic chitose-stearic acid grafting (stearic acid-grafted chitosan oligosaccharide, CSO-SA).Discover that chitosan-stearic acid grafting can form micelle by self aggregation in aqueous medium, and have the function of quick cellular uptake.The present invention adopts chitosan-stearic acid grafting micelle to seal third generation platinum series antineoplastic medicament oxaliplatin, drug-delivery preparation in preparation oxaliplatin chitosan-stearic acid grafting micelle cell, by improving the drug level in the tumor cell, improve cytotoxicity and reverse multidrug resistance, rendeing a service for the clinical antitumor that improves oxaliplatin provides possibility.
Summary of the invention
An object of the present invention is to provide a kind of oxaliplatin chitosan-stearic acid grafting micelle, be to be positioned at intracellular oxaliplatin for molecular target, a kind of target administration carrier material is provided, formed by chitosan-stearic acid grafting, oxaliplatin, lecithin and water, wherein chitosan-stearic acid grafting account for gross weight 0.5%, oxaliplatin accounts for 0.009-0.02%, the 0-0.2% that lecithin accounts for gross weight, the 99.27-99.49% that water accounts for gross weight of gross weight; Chitosan-stearic acid grafting is by the low-molecular weight chitoglycan of mean molecule quantity 1.5kD～51kD, with C
10～C
22The fatty acid chemistry grafting form, the amino group substitution degree of oligochitosan is 1%～50% in chitosan-stearic acid grafting.When chitosan-stearic acid grafting concentration surpasses critical micelle concentration, can spontaneous formation grafting micelle in aqueous medium, micelle possesses by the cell function of picked-up fast.The micellar composition of grafting of the present invention has been patent " surface-modified hydrophobically modified chitin polymer administration micelle and preparation method thereof the " (patent No.: ZL200610051601.0) contained.
Oxaliplatin chitosan of the present invention-stearic acid grafting micelle obtains by following preparation method:
At first adopt the synthetic chitosan of method-stearic acid grafting of the patent No.: ZL200610051601.0: get chitosan 0.5g, precision weighing, add 80 ℃ of following stirring and dissolving of 30mL deionized water, take by weighing stearic acid 0.825g and carbodiimide (EDC) 5.5g, stirring and dissolving is dissolved in the 17mL ethanol solution, is heated to 80 ℃.Under 400rpm magnetic agitation condition above-mentioned alcoholic solution is added in the chitosan solution, 80 ℃ of constant temperature stirring reactions 4 hours are cooled to room temperature (25 ℃), end reaction liquid are put in the bag filter distill water dialysis 3 days.After the dialysis solution lyophilization, remove residual stearic acid, get chitosan-stearic acid grafting with absolute ethanol washing.Prepare oxaliplatin chitosan-stearic acid grafting micelle respectively by following three kinds of methods then:
(1) probe ultrasonic method: get 25mg oligochitosan-stearic acid grafting, precision claims fixed, adds the 5mL deionized water, and ice bath is popped one's head in ultrasonic 10 times (400w, work 2s stops 3s), gets the micellar solution of 5mg/mL.Adding the 0.5mL drug level is the oxaliplatin aqueous solution of 5mg/mL.Ultrasonic 50 times of probe (400W, work 2s stops 3s) gets oxaliplatin chitosan-stearic acid grafting micellar solution under the condition of ice bath.
(2) film dispersion method: get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, and adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, ultrasonic (same a) the dissolving back adding 0.75mL ethanol solution of water-bath probe.45 ℃ of following rotary evaporations remove and desolvate, and obtain the carrier micelle thin film, and it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution.
(3) lecithin mediation film dispersion method: get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, and adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, and adding solubility behind the water-bath ultrasonic dissolution is the lecithin alcoholic solution 0.75mL of 10mg/mL.45 ℃ of following rotary evaporations remove and desolvate, and obtain the carrier micelle thin film, and it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution.
Another object of the present invention provides the application of oxaliplatin chitosan-stearic acid grafting micelle in the preparation antitumor drug.Especially in the application in preparation anti-breast cancer, colorectal cancer, hepatocarcinoma, ovarian cancer, leukemia medicament.Compare with the oxaliplatin solution agent,, can significantly improve the drug level in the tumor cell, improve cytotoxicity and reverse multidrug resistance by oxaliplatin chitosan-stearic acid grafting micelle administration.
Usefulness of the present invention is: chitosan-stearic acid grafting micelle has the quick capture functions of intravital passive target of polymer micelle and tumor cell, by chitosan-stearic acid grafting micelle sealing to oxaliplatin, can improve the cell drug level that molecular target is positioned at the oxaliplatin of tumor cell greatly, improve cytotoxicity and reverse multidrug resistance, rendeing a service for the clinical antitumor that improves oxaliplatin provides possibility.
The explanation of accompanying drawing table
Fig. 1 is the growth inhibited curve of oxaliplatin chitosan-stearic acid grafting micelle to different tumor cells.
Fig. 2 be responsive and the drug-resistant tumor cell in drug level through the time variation.
The specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment one
1) preparation of low-molecular weight chitoglycan
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 95%) 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.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 chitosan molecule amount, 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 chitosan molecule amount with this elution curve.The weight average molecular weight of gained chitosan is 18.0kDa after measured.
2) chitosan-stearic acid grafting is synthetic
Get above-mentioned chitosan 0.5g, precision weighing adds 80 ℃ of following stirring and dissolving of 30mL deionized water, takes by weighing stearic acid 0.825g and carbodiimide (EDC) 5.5g, and stirring and dissolving is dissolved in the 17mL ethanol solution, is heated to 80 ℃.Under 400rpm magnetic agitation condition above-mentioned alcoholic solution is added in the chitosan solution, 80 ℃ of constant temperature stirring reactions 4 hours are cooled to room temperature (25 ℃), end reaction liquid are put in the bag filter distill water dialysis 3 days.After the dialysis solution lyophilization, remove residual stearic acid, get chitosan-stearic acid grafting with absolute ethanol washing.
Adopt 2,4, the 6-trinitro-benzene-sulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS) method measure chitosan-stearic acid grafting amino group substitution degree (substitution degree, SD).Get chitosan 10mg with molecular weight, place the volumetric flask of 10ml, use the deionized water standardize solution, promptly obtain the chitosan storing solution of 1mg/ml, get 0,10,50,100,200,300,400,500 μ l respectively, be settled to 2.0ml with deionized water, add 4% sodium bicarbonate 2.0ml and 0.1% trinitro-benzene-sulfonic acid 2.0ml, hatch 2h in 37 ℃ of water-baths.Add 2.0mol/L hydrochloric acid 2ml, shake up, the ultrasonic bubble of driving away utilizes ultraviolet spectrophotometer to measure absorbance, preparation standard curve at the 344nm place.Grafting 10mg decided in accurate in addition title, places the 10ml volumetric flask, the deionized water standardize solution.Get 300 μ l, with the method operation, measure the absorbance at 344nm place, the amino group substitution degree that calculates polymer by standard curve is 6.89%
Adopt the critical micelle concentration of pyrene fluorescence spectrometry chitosan-stearic acid grafting.The acetone soln 0.5ml that gets the 0.0012mg/ml pyrene puts into the test tube of 10ml, volatilizes acetone under 50 ℃.The grafting solution of preparation 0.001mg/ml～1mg/ml variable concentrations is got 5ml respectively and is joined that (the pyrene final concentration is 7 * 10-7mol/L), the ultrasonic 30min of room-temperature water bath in the above-mentioned test tube.The excitation spectrum and the emission spectra of scanning pyrene are determined at 374nm and 385nm fluorescence intensity, and determine that with the unexpected variation of measuring the I375/I385 slope critical micelle concentration of this grafting is 0.119mg/mL.
Adopt particle size and surface potential analyser, measure chitosan-micellar particle diameter of stearic acid grafting and surface potential.After measured, the chitosan of 1mg/mL-micellar particle diameter of stearic acid grafting and surface potential are respectively 34.8nm and 50.8mV.
3) oxaliplatin chitosan-stearic acid grafting micelle preparation
Get 25mg oligochitosan-stearic acid grafting, precision claims fixed, adds the 5mL deionized water, and ice bath is popped one's head in ultrasonic 10 times (400w, work 2s stops 3s), gets the micellar solution of 5mg/mL.Adding the 0.5mL drug level is the oxaliplatin aqueous solution of 5mg/mL.Ultrasonic 50 (400W, work 2s stops 3s) of probe get oxaliplatin chitosan-stearic acid grafting micellar solution under the condition of ice bath.The oxaliplatin chitosan of the 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting, current potential and the entrapment efficiency and the drug loading that record by inductively coupled plasma mass spectrograph (ICP-MS) the results are shown in Table 1.Chitosan-micellar number average bead diameter of stearic acid grafting is 30.4nm, and current potential is 50.3mV, and entrapment efficiency is 18.1%, and the drug loading amount is 1.78%.
Embodiment two
1) preparation of low-molecular weight chitoglycan
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 95%) 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.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 chitosan molecule amount, 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 chitosan molecule amount with this elution curve.The weight average molecular weight of gained chitosan is 18.0kDa after measured.
2) chitosan-stearic acid grafting is synthetic
Get above-mentioned chitosan 0.5g, precision weighing adds 80 ℃ of following stirring and dissolving of 30mL deionized water, takes by weighing stearic acid 0.825g and carbodiimide (EDC) 5.5g, and stirring and dissolving is dissolved in the 17mL ethanol solution, is heated to 80 ℃.Under 400rpm magnetic agitation condition above-mentioned alcoholic solution is added in the chitosan solution, 80 ℃ of constant temperature stirring reactions 4 hours are cooled to room temperature (25 ℃), end reaction liquid are put in the bag filter distill water dialysis 3 days.After the dialysis solution lyophilization, remove residual stearic acid, get chitosan-stearic acid grafting with absolute ethanol washing.
Adopt 2,4, the 6-trinitro-benzene-sulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS) method measure chitosan-stearic acid grafting amino group substitution degree (substitution degree, SD).Get chitosan 10mg with molecular weight, place the volumetric flask of 10ml, use the deionized water standardize solution, promptly obtain the chitosan storing solution of 1mg/ml, get 0,10,50,100,200,300,400,500 μ l respectively, be settled to 2.0ml with deionized water, add 4% sodium bicarbonate 2.0ml and 0.1% trinitro-benzene-sulfonic acid 2.0ml, hatch 2h in 37 ℃ of water-baths.Add 2.0mol/L hydrochloric acid 2ml, shake up, the ultrasonic bubble of driving away utilizes ultraviolet spectrophotometer to measure absorbance, preparation standard curve at the 344nm place.Grafting 10mg decided in accurate in addition title, places the 10ml volumetric flask, the deionized water standardize solution.Get 300 μ l, with the method operation, measure the absorbance at 344nm place, the amino group substitution degree that calculates polymer by standard curve is 6.89%
Adopt the critical micelle concentration of pyrene fluorescence spectrometry chitosan-stearic acid grafting.The acetone soln 0.5ml that gets the 0.0012mg/ml pyrene puts into the test tube of 10ml, volatilizes acetone under 50 ℃.The grafting solution of preparation 0.001mg/ml～1mg/ml variable concentrations is got 5ml respectively and is joined that (the pyrene final concentration is 7 * 10-7mol/L), the ultrasonic 30min of room-temperature water bath in the above-mentioned test tube.The excitation spectrum and the emission spectra of scanning pyrene are determined at 374nm and 385nm fluorescence intensity, and determine that with the unexpected variation of measuring the I375/I385 slope critical micelle concentration of this grafting is 0.119mg/mL.
Adopt particle size and surface potential analyser, measure chitosan-micellar particle diameter of stearic acid grafting and surface potential.After measured, the chitosan of 1mg/mL-micellar particle diameter of stearic acid grafting and surface potential are respectively 34.8nm and 50.8mV.
3) oxaliplatin chitosan-stearic acid grafting micelle preparation
Get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, and adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, adds the 0.75mL ethanol solution behind the water-bath ultrasonic dissolution.45 ℃ of following rotary evaporations remove and desolvate, and obtain the carrier micelle thin film, and it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution.The oxaliplatin chitosan of the 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting, current potential and the entrapment efficiency and the drug loading that record by inductively coupled plasma mass spectrograph (ICP-MS) the results are shown in Table 1.Chitosan-micellar number average bead diameter of stearic acid grafting is 44.9nm, and current potential is 51.8mV, and entrapment efficiency is 41.3%, and the drug loading amount is 3.97%.
Embodiment three
1) preparation of low-molecular weight chitoglycan
Get the chitosan that commercially available molecular weight is 450kDa (deacetylation is 95%) 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.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 chitosan molecule amount, 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 chitosan molecule amount with this elution curve.The weight average molecular weight of gained chitosan is 18.0kDa after measured.
2) chitosan-stearic acid grafting is synthetic
Get above-mentioned chitosan 0.5g, precision weighing adds 80 ℃ of following stirring and dissolving of 30mL deionized water, takes by weighing stearic acid 0.825g and carbodiimide (EDC) 5.5g, and stirring and dissolving is dissolved in the 17mL ethanol solution, is heated to 80 ℃.Under 400rpm magnetic agitation condition above-mentioned alcoholic solution is added in the chitosan solution, 80 ℃ of constant temperature stirring reactions 4 hours are cooled to room temperature (25 ℃), end reaction liquid are put in the bag filter distill water dialysis 3 days.After the dialysis solution lyophilization, remove residual stearic acid, get chitosan-stearic acid grafting with absolute ethanol washing.
Adopt 2,4, the 6-trinitro-benzene-sulfonic acid (2,4,6-trinitrobenzenesulfonic acid, TNBS) method measure chitosan-stearic acid grafting amino group substitution degree (substitution degree, SD).Get chitosan 10mg with molecular weight, place the volumetric flask of 10ml, use the deionized water standardize solution, promptly obtain the chitosan storing solution of 1mg/ml, get 0,10,50,100,200,300,400,500 μ l respectively, be settled to 2.0ml with deionized water, add 4% sodium bicarbonate 2.0ml and 0.1% trinitro-benzene-sulfonic acid 2.0ml, hatch 2h in 37 ℃ of water-baths.Add 2.0mol/L hydrochloric acid 2ml, shake up, the ultrasonic bubble of driving away utilizes ultraviolet spectrophotometer to measure absorbance, preparation standard curve at the 344nm place.Grafting 10mg decided in accurate in addition title, places the 10ml volumetric flask, the deionized water standardize solution.Get 300 μ l, with the method operation, measure the absorbance at 344nm place, the amino group substitution degree that calculates polymer by standard curve is 6.89%
Adopt the critical micelle concentration of pyrene fluorescence spectrometry chitosan-stearic acid grafting.The acetone soln 0.5ml that gets the 0.0012mg/ml pyrene puts into the test tube of 10ml, volatilizes acetone under 50 ℃.The grafting solution of preparation 0.001mg/ml～1mg/ml variable concentrations is got 5ml respectively and is joined that (the pyrene final concentration is 7 * 10-7mol/L), the ultrasonic 30min of room-temperature water bath in the above-mentioned test tube.The excitation spectrum and the emission spectra of scanning pyrene are determined at 374nm and 385nm fluorescence intensity, and determine that with the unexpected variation of measuring the I375/I385 slope critical micelle concentration of this grafting is 0.119mg/mL.
Adopt particle size and surface potential analyser, measure chitosan-micellar particle diameter of stearic acid grafting and surface potential.After measured, the chitosan of 1mg/mL-micellar particle diameter of stearic acid grafting and surface potential are respectively 34.8nm and 50.8mV.
3) oxaliplatin chitosan-preparation of stearic acid grafting micelle and physical and chemical property determining thereof
Get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, and adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, and adding solubility behind the water-bath ultrasonic dissolution is the lecithin alcoholic solution 0.75mL of 10mg/mL.45 ℃ of following rotary evaporations remove and desolvate, and obtain the carrier micelle thin film, and it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution.The oxaliplatin chitosan of the 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting, current potential and the entrapment efficiency and the drug loading that record by inductively coupled plasma mass spectrograph (ICP-MS) the results are shown in Table 1.Chitosan-micellar number average bead diameter of stearic acid grafting is 95.7nm, and current potential is 51.8mV, and entrapment efficiency is 47.2%, and the drug loading amount is 3.50%.
4) the oxaliplatin chitosan-application of stearic acid grafting micelle in antineoplaston
Adopting colorectal cancer (SGC-7901) cell, ovary (SKOV3) cancerous cell, hepatocarcinoma (BEL-7402) cell, leukemia (K562) cell, breast carcinoma (MCF-7) cell and adriamycin-resistant breast carcinoma (MCF-7/Adr) cell is model cell, compare with free drug, investigate the cytotoxicity of oxaliplatin chitosan-stearic acid grafting micelle by tetrazolium salts colorimetry (mtt assay), estimate oxaliplatin chitosan-micellar anti-tumor activity of stearic acid grafting tumor cell.Cell is cultivated in culture bottle with the RPMI-1640 culture fluid that contains 10% new-born calf serum, place 37 ℃ of incubators, feed 5%CO2 (relative humidity is 95%), changed culture fluid in 2 days one time, and under inverted microscope the observation of cell growth conditions, when treating that cell grows to nearly fusion, use trypsinization, the density of cell by 10000/ hole is inoculated in 96 well culture plates, cultivate 24h, after treating the cell attachment growth, culture fluid is removed in suction, with PBS flushing one time, adds fresh culture fluid then, the oxaliplatin medicine carrying micelle and the free oxaliplatin solution that add variable concentrations simultaneously respectively, each concentration is established 3 parallel holes, and establishes negative control hole, after continuing to cultivate 48h, add 5mg/ml MTT aqueous solution 20 μ l in every hole, cultivate 4h again, inhale and remove culture fluid in the hole, every Kong Zhongzai adds DMSO100 μ l dissolve purple crystallized product, behind 37 ℃ of jolting 30min, measure absorbance at 570nm with microplate reader.Cytotoxicity the results are shown in Table 2 and Fig. 1.The result shows, can significantly increase the toxicity of oxaliplatin medicine to tumor cell by the mediation of grafting micelle, and can realize reversing drug resistance, and it reverses multiple and is about 5.72 times.Among Figure 1A, SGC-7901 cell: (◆) free drug and (◇) embodiment three carrier micelles; SKOV3 cell: (■) free drug and () embodiment three carrier micelles; BEL-7402 cell: (▲) free drug and (△) embodiment three carrier micelles; K562 cell: (●) free drug and (zero) embodiment three carrier micelles.Among Figure 1B, MCF-7 cell: (■) free drug and () embodiment three carrier micelles; MCF-7/Adr cell: (●) free drug and (zero) embodiment three carrier micelles.Log C represents the logarithm of drug level.
5) in oxaliplatin chitosan-micellar cell of stearic acid grafting through the time determination of drug concentration
When MCF-7 becomes logarithmic growth with the MCF-7/Adr cell, they are pressed 100, the density in 000/ hole is seeded in 24 well culture plates, continue to cultivate 24 hours, after treating the cell attachment growth, to wherein adding oxaliplatin chitosan-stearic acid grafting micelle micelle (constant drug level is 20 μ g/ml), and compare with free drug with concentration, in 37 ℃ hatch 2h, 4h, 6h, 8h, 12h respectively after, discard original fluid, with ice PBS (pH7.4) flushing 3 times, stop the picked-up of medicine and remove the medicine that sticks on the cell membrane.Add 50 μ l pancreatin in cell, the cell dissociation that sticks on the culture plate is got off, then to wherein adding 950 μ l PBS, the collecting cell suspension is measured the concentration of the interior oxaliplatin of each time point cell with inductively coupled plasma mass spectrograph (ICP-MS).
Protein concentration is proofreaied and correct in the cell suspension: get above-mentioned cell suspension 20uL, add in 96 well culture plates, add the BCA working solution of 200uL, place more than the 30min down for 37 ℃.Measure light absorption value at the 570nm place with enzyme connection detector.According to the bovine serum albumin production standard curve of concentration known, and with the protein concentration in this curve calculation cell suspension.
Intracellular drug level through the time measurement result see Fig. 2.The result shows, mediates the drug level that can significantly increase in the tumor cell by the grafting micelle; At short notice, drug level increases along with the prolongation of incubation time in the cell.After hatching 12h altogether with tumor cell, 4.57 times have been improved with drug level in the cell of grafting micelle situation administration with comparing in the MCF-7 cell with the free drug form administration; 4.77 times have been improved with drug level in the cell of grafting micelle situation administration with comparing in the MCF-7/Adr cell with the free drug form administration.
The oxaliplatin chitosan that table 1 distinct methods makes-stearic acid grafting micelle physicochemical property
PI represents the dispersion index of number average bead diameter
Table 2 oxaliplatin chitosan-stearic acid grafting micelle is to the IC of different tumor cells
50Value
IC
50: expression half cell-lethal concentration.
Claims (5)
1. oxaliplatin chitosan-stearic acid grafting micelle, it is characterized in that, formed by chitosan-stearic acid grafting, oxaliplatin, lecithin and water, wherein chitosan-stearic acid grafting account for gross weight 0.5%, oxaliplatin accounts for 0.009-0.02%, the 0-0.2% that lecithin accounts for gross weight, the 99.27-99.49% that water accounts for gross weight of gross weight; Chitosan-stearic acid grafting is by the low-molecular weight chitoglycan of mean molecule quantity 1.5kD～51kD, with C
10～C
22The fatty acid chemistry grafting form, the amino group substitution degree of oligochitosan is 1%～50% in chitosan-stearic acid grafting.
2. a kind of oxaliplatin chitosan according to claim 1-micellar preparation method of stearic acid grafting, it is characterized in that, disclosed method according to CN100417417C obtains chitosan-stearic acid grafting earlier, prepares oxaliplatin chitosan-stearic acid grafting micelle respectively by three kinds of methods again:
(1) chitosan-stearic acid grafting preparation: get chitosan 0.5g, precision weighing adds 80 ℃ of following stirring and dissolving of 30mL deionized water, takes by weighing stearic acid 0.825g and carbodiimide (EDC) 5.5g, and stirring and dissolving is dissolved in the 17mL ethanol solution, is heated to 80 ℃.Under 400rpm magnetic agitation condition above-mentioned alcoholic solution is added in the chitosan solution, 80 ℃ of constant temperature stirring reactions 4 hours are cooled to room temperature (25 ℃), end reaction liquid are put in the bag filter distill water dialysis 3 days.After the dialysis solution lyophilization, remove residual stearic acid, get chitosan-stearic acid grafting with absolute ethanol washing;
(2) oxaliplatin chitosan-stearic acid grafting micelle preparation:
A. the ultrasonic method of popping one's head in
Get 25mg oligochitosan-stearic acid grafting, add the 5mL deionized water, ultrasonic 10 times of ice bath probe 400w, get the micellar solution of 5mg/mL, adding the 0.5mL drug level is the oxaliplatin aqueous solution of 5mg/mL, probe 400W is ultrasonic 50 times under the condition of ice bath, gets oxaliplatin chitosan-stearic acid grafting micellar solution;
B. film dispersion method
Get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, add the 0.75mL ethanol solution behind the water-bath ultrasonic dissolution, 45 ℃ of following rotary evaporations remove and desolvate, obtain the carrier micelle thin film, it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution;
C. lecithin mediates film dispersion method
Get 25mg oligochitosan-stearic acid grafting, the accurate title, decide, adding the 0.5mL drug level is in the oxaliplatin aqueous solution of 5mg/mL, adding solubility behind the water-bath ultrasonic dissolution is the lecithin alcoholic solution 0.75mL of 10mg/mL, 45 ℃ of following rotary evaporations remove and desolvate, obtain the carrier micelle thin film, it is disperseed with the 5mL deionized water, get oxaliplatin chitosan-stearic acid grafting micellar solution.
3. a kind of oxaliplatin chitosan according to claim 2-micellar preparation method of stearic acid grafting is characterized in that, among step B and the C, and the same A of the ultransonic condition of water-bath.
4. the application of a kind of oxaliplatin chitosan according to claim 1-stearic acid grafting micelle in the preparation antitumor drug.
5. the application of a kind of oxaliplatin chitosan according to claim 4-stearic acid grafting micelle in the preparation antitumor drug is characterized in that, the application in preparation anti-breast cancer, colorectal cancer, hepatocarcinoma, ovarian cancer, leukemia medicament.
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| CN102133176A (en) * | 2011-03-22 | 2011-07-27 | 济南宏瑞创博医药科技开发有限公司 | Oxaliplatin micelle preparation, preparation method and application thereof |
| CN107115531A (en) * | 2017-05-05 | 2017-09-01 | 浙江大学 | A kind of glycolipid polymers modification nano structured lipid carrier and preparation method and application |
| CN109394692A (en) * | 2018-12-12 | 2019-03-01 | 中国药科大学 | Dasatinib Graft copolymer micella, its freeze drying powder injection and preparation method and application |
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| CN1883708A (en) * | 2006-05-24 | 2006-12-27 | 浙江大学 | Surface-modified hydrophobically modified drug-carried chitosan polymer micelle and method for preparing same |
| CN101066461A (en) * | 2007-05-29 | 2007-11-07 | 浙江大学 | Application of cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle |
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| CN1883708A (en) * | 2006-05-24 | 2006-12-27 | 浙江大学 | Surface-modified hydrophobically modified drug-carried chitosan polymer micelle and method for preparing same |
| CN101066461A (en) * | 2007-05-29 | 2007-11-07 | 浙江大学 | Application of cell nucleus targeting chitosan-fatty acid graft as medicine carrier micelle |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102133176A (en) * | 2011-03-22 | 2011-07-27 | 济南宏瑞创博医药科技开发有限公司 | Oxaliplatin micelle preparation, preparation method and application thereof |
| CN102133176B (en) * | 2011-03-22 | 2012-09-05 | 济南宏瑞创博医药科技开发有限公司 | Oxaliplatin micelle preparation, preparation method and application thereof |
| CN107115531A (en) * | 2017-05-05 | 2017-09-01 | 浙江大学 | A kind of glycolipid polymers modification nano structured lipid carrier and preparation method and application |
| CN109394692A (en) * | 2018-12-12 | 2019-03-01 | 中国药科大学 | Dasatinib Graft copolymer micella, its freeze drying powder injection and preparation method and application |
| CN109394692B (en) * | 2018-12-12 | 2020-09-25 | 中国药科大学 | Dasatinib grafted polymer micelle, freeze-dried powder injection thereof, preparation method and application |
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