CN101524326A - Chlorin e6 chitosan-stearic acid graft micelle - Google Patents
Chlorin e6 chitosan-stearic acid graft micelle Download PDFInfo
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- CN101524326A CN101524326A CN200910097053A CN200910097053A CN101524326A CN 101524326 A CN101524326 A CN 101524326A CN 200910097053 A CN200910097053 A CN 200910097053A CN 200910097053 A CN200910097053 A CN 200910097053A CN 101524326 A CN101524326 A CN 101524326A
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- stearic acid
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- 239000008117 stearic acid Substances 0.000 title claims abstract description 97
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Abstract
The invention provides a preparation of chlorin e6 chitosan-stearic acid graft micelle. The preparation comprises a chitosan-stearic acid graft and chlorin e6, wherein, the chlorin e6 accounts for 4.76-16.76% of the total weight; the chitosan-stearic acid graft is formed by chemically grafting low molecular weight chitosan with average molecular weight being 18.0 KDa and stearic acid; and the amino substituted ration of the chitosan-stearic acid graft accounts for 4.96%. The invention utilizes the chitosan-stearic acid graft micelle to effectively pack the photosencitizer of chlorin e6, so as to prepare the intracellular administration preparation of chlorin e6 chitosan-stearic acid graft micelle. Compared with the chlorin e6 solution, the preparation of the invention can significantly improve the drug concentration of chlorin e6 in tumour cells and provide probability for the follow-up realization of efficient and harmless photodynamic therapy of tumors.
Description
Technical field
The invention belongs to targeting preparation, be specifically related to a kind of chlorin e 6 chitosan-stearic acid grafting micellar preparation and application thereof.
Background technology
(photodynamict herapy is to rise early 1980s and in the method for the novel rapidly treatment tumor of developed recently PDT) to the anti-tumor photosensitizer photodynamic therapy.Photodynamic therapy has little, the fast characteristics of producing effects of toxicity.By the sensibilization of biological light power, promptly after the laser irradiation of specific wavelength, photosensitizer is excited, by ground state (S
0) through excited singlet (S
1) change excited triplet state (T into
1), then by excited triplet state (T
1) make molecular oxygen in the surrounding medium become singlet oxygen (
1O
2) the isoreactivity material, the oxidation-sensitive group effect in they and the biomolecule causes its oxidation inactivation, finally causes death of neoplastic cells and shows its therapeutical effect.
The present light power curing cancer drug of using clinically such as hematoporphyrin derivative (HpD), phytochrome II etc. the porphyrin mix preparation that is complicated component exists that little, the effective photodynamic effect of absorptance at red light district is low, phototoxic reaction reaches shortcomings such as chemical composition is indefinite greatly.And chlorin e 6 to be chlorophyll stablize gets in touch one of the widest composition with biological activity in the catabolite, and become the object of study that attracts tremendous attention in the research of light power cancer curing medicine.There is report to show that the chlorin e 6 absorbing wavelength is at red light district (664nm), singlet oxygen productive rate Φ
ΔHigher, the suppression ratio of tumor is much better than hematoporphyrin derivative, and (hematoporphyrin derivative is HpD) to the suppression ratio of tumor.
Observe in animal experiment, the characteristic distributions of dihydro leaf fen e6 is that hold-up is big and relative time is longer in the long and tumor tissues of blood drug level height, half-life.Shortcoming is that dihydro leaf fen e6 presents long-time skin phototoxicity, and effective dose is big.Because tumor tissues is that relative selectivity absorbs to photosensitizer, does not have specificity, so be distributed with a spot of photosensitizer in the normal structure inevitably, may produce the light toxic and side effects.When being difficult to that light source is confined to the target position tissue, during as abdominal cavity and thoracic cavity tumor, in order to reduce the toxic and side effects to normal surrounding tissue, the photosensitizer formulations of exploitation targeting type just seems particularly important.
In recent years, in order to improve the concentration of photosensitizer at tumor cell, many seminar load on second filial generation photosensitizer on the appropriate carriers, make liposome, nanoparticle etc., by normal physiological processes optionally transport, productive set is to target tissue, increases substantially the photosensitizer anti-tumor activity, this is to improving the Drug therapy effect, reduce toxic and side effects, have crucial meaning.Richter etc.] discover that benzoporphyrin derivative monocyclic acids A makes liposome, can mediate medicine and promptly enter tissue.Vargas etc. make the PLGA nanoparticle with meso-(para hydroxybenzene) porphyrin (p-THPP), can obviously strengthen the effect of medicine to breast cancer cell.Konan-Kouakou etc. [discover, Verteporfin is made lactic acid-hydroxyacetic acid copolymer (PLGA) nanoparticle, the drug-carrying nanometer particle of small particle diameter is bigger to the photo-cell toxicity of breast carcinoma EMT-6 cell than the DMSO/PBS solution of medicine, and the speed that enters tissue is obviously accelerated.
(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, load 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 and retention effect).
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.
Summary of the invention
The purpose of this invention is to provide chlorin e 6 chitosan-stearic acid grafting micellar preparation, compare,, can significantly improve the drug level in the tumor cell by chlorin e 6 chitosan-stearic acid grafting micelle administration with the chlorin e 6 solution.
Chlorin e 6 chitosan-stearic acid grafting micelle is made up of chitosan-stearic acid grafting and chlorin e 6, and wherein chlorin e 6 accounts for the 4.76-16.67% of gross weight; Chitosan-stearic acid grafting is by the low-molecular weight chitoglycan of mean molecule quantity 18.0KDa, form with the grafting of stearic acid chemistry, the amino group substitution degree of chitosan-stearic acid grafting is 4.96%, 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.
Described chlorin e 6 chitosan-stearic acid grafting micelle prepares by the following method and gets:
Get 40mg oligochitosan-stearic acid grafting, the accurate title, decide, put in the 50mL beaker, add 8-32mL distilled water (pH is 5.7), pop one's head in ultrasonic 20 times (500w, work 2s stops 3s), get the micellar solution of 5mg/mL, adding is the chlorin alcoholic solution of 0.25mg/mL with volumetric concentration, ultrasonic 30 times of probe (400W, work 2s stops 3s) under the condition of ice bath, with mixed liquor bag filter (the MWCO 3.5KDa that packs into, SpectrumLaboratories, Laguna Hills, CA), with the 24h that dialyses under the deionized water room temperature, remove ethanol,, get chlorin e 6 chitosan-stearic acid grafting micelle powder the dialysis solution lyophilization.
Another object of the present invention provides the application of chlorin e 6 chitosan-stearic acid grafting micelle in the photosensitizer formulations of preparation targeting type.
Intracellular chlorin e 6 provides a kind of target administration carrier material for molecular target is positioned in the present invention, is specially chitosan-stearic acid grafting micelle.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 effectively sealing to second filial generation photosensitizer chlorin e 6, overcome dihydro leaf fen e6 and presented long-time skin phototoxicity, and effective dose is big, may produce shortcomings such as light toxic and side effects to normal structure.Chlorin e 6 chitosan-stearic acid grafting the micelle of preparation, be drug-delivery preparation in the cell, can improve the cell drug level that molecular target is positioned at the chlorin e 6 of tumor cell greatly, minimizing is learned treatment for the photodynamic tumor of follow-up realization high-efficiency low-toxicity possibility is provided the toxic and side effects of normal surrounding tissue.The foundation of the photosensitizer formulations of exploitation targeting type is provided.
Description of drawings
Fig. 1: the chlorin e 6 chitosan-micellar chlorin e 6 release in vitro of stearic acid grafting behavior.
Fig. 2: in the tumor cell drug level through the time change.
The specific embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1:
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) the synthetic and physical and chemical property determining of chitosan-stearic acid grafting
Get above-mentioned chitosan 0.1g, precision weighing, add 20mL 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: 25 takes by weighing stearic acid and is dissolved in the 10mL 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 chitosan-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 chitosan.Add 2N hydrochloric acid 2mL, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned chitosan-stearic acid grafting 4mg and be dissolved in the 2mL distilled water, with the method operation, measuring amino group substitution degree is 4.96%.
Adopt pyrene fluorescence spectrometry chitosan-stearic critical micelle concentration.Preparation variable concentrations chitosan-each 10mL of 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 0.036mg/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 28.9nm and 39.1mV.
3) chlorin e 6 chitosan-preparation of stearic acid grafting micelle and physical and chemical property determining thereof
Get 40mg oligochitosan-stearic acid grafting, precision claims fixed, puts in the 50mL beaker, adds 8mL distilled water (pH is 5.7), pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), gets the micellar solution of 5mg/mL.The alcoholic solution 8mL that adds the 0.25mg/mL chlorin e 6, the ultrasonic 30 (400W of probe under the condition of ice bath, work 2s stops 3s), with mixed liquor pack into bag filter (MWCO 3.5KDa, SpectrumLaboratories, Laguna Hills, CA), with the 24h that dialyses under the deionized water room temperature, remove ethanol, with the dialysis solution lyophilization, get chlorin e 6 chitosan-stearic acid grafting micelle powder.
After measured, the chlorin e 6 chitosan of 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting is 302.6nm, and surface potential is 40.5mV.Entrapment efficiency is 100%.Medicament contg is 4.76%.
Chlorin e 6 chitosan-micellar release in vitro of stearic acid grafting is carried out under 37 ℃ of constant temperature, frequency of oscillation 60rpm condition.Get chlorin e 6 chitosan-stearic acid grafting micelle pressed powder, disperse with deionized water (pH is 5.7), getting drug level is chitosan-stearic acid grafting micellar solution of 32 μ g/mL, get 2.5mL (the MWCO 7.0KDa in the bag filter that packs into respectively, Spectrum Laboratories, Laguna Hills CA), places bag filter the releasing tube that contains PBS 7.4 (containing 0.2% Tween 80) 20mL.Timing sampling 1.0mL, HPLC measures drug level, calculates the cumulative release amount.Fig. 1 is seen in the chlorin e 6 chitosan-micellar chlorin e 6 release in vitro of stearic acid grafting behavior.
4) the chlorin e 6 chitosan-micellar cell giving drugs into nose of stearic acid grafting concentration profile is measured
The trophophase lung epithelial tumor cell A549 cell of taking the logarithm, adjusting cell density with culture medium after the trypsinization is 1.0 * 10
5/ mL is inoculated in 12 well culture plates by the 4.0mL/ hole, the pre-24h that cultivates in the incubator, add chlorin e 6 chitosan-stearic acid grafting micelle respectively, constant drug level is 12.5g/mL, and compares with the free drug with concentration, in 37 ℃ hatch 2,4,6,8,12 respectively, behind the 24h, wash cell three times with PBS (pH 7.4), use the 50L trypsinization, with 0.5mL PBS collecting cell, the ice bath ultrasonic 20 (400W that pop one's head in, work 2s stops 3s), get cell breakage liquid.Get an amount of cell breakage liquid, add isopyknic dehydrated alcohol water-bath ultrasonication micelle, dissolved substance, sample introduction behind the 0.22m microporous filter membrane is measured medicament contg excessively.
Get above-mentioned cell breakage liquid 20L synchronously, add in 96 well culture plates, add the BCA working solution of 200L, place more than the 30min for 37 ℃.Measure light absorption value at the 570nm place with enzyme connection detector.According to the bovine serum albumin of concentration known (bovine serum albumin, BSA) production standard curve, and with the protein content (M) in this curve calculation cytolysate.Defined parameters F, when F is different time points, the ratio of drug level and intracellular protein content in the cell.Investigate the ingestion of medicines ability of cell according to following formula at different incubation times.
T is percentage rate (%)=F (the T)/F (0) * 100% (2) of cellular uptake medicine constantly
In the formula, F (T)=T is the interior drug concentrations of cell/T moment intracellular protein content constantly, and F (0)=0 is the initial concentration of medicine/0 moment intracellular protein content constantly
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 2 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 2.86% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 20.46%; After hatching 24 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 6.61% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 26.85%.
Embodiment 2:
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) the synthetic and physical and chemical property determining of chitosan-stearic acid grafting
Get above-mentioned chitosan 0.1g, precision weighing, add 20mL 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: 25 takes by weighing stearic acid and is dissolved in the 10mL 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 chitosan-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 chitosan.Add 2N hydrochloric acid 2mL, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned chitosan-stearic acid grafting 4mg and be dissolved in the 2mL distilled water, with the method operation, measuring amino group substitution degree is 4.96%.
Adopt pyrene fluorescence spectrometry chitosan-stearic critical micelle concentration.Preparation variable concentrations chitosan-each 10mL of 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 0.036mg/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 28.9nm and 39.1mV.
3) chlorin e 6 chitosan-preparation of stearic acid grafting micelle and physical and chemical property determining thereof
Get 40mg oligochitosan-stearic acid grafting, precision claims fixed, puts in the 50mL beaker, adds 16mL distilled water (pH is 5.7), pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), gets the micellar solution of 2.5mg/mL.The alcoholic solution 16mL that adds the 0.25mg/mL chlorin e 6, the ultrasonic 30 (400W of probe under the condition of ice bath, work 2s stops 3s), with mixed liquor pack into bag filter (MWCO 3.5KDa, SpectrumLaboratories, Laguna Hills, CA), with the 24h that dialyses under the deionized water room temperature, remove ethanol, with the dialysis solution lyophilization, get chlorin e 6 chitosan-stearic acid grafting micelle powder.
After measured, the chlorin e 6 chitosan of 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting is 288.2nm, and surface potential is 36.5mV.Entrapment efficiency is 100%.Medicament contg is 9.09%.
Chlorin e 6 chitosan-micellar release in vitro of stearic acid grafting is carried out under 37 ℃ of constant temperature, frequency of oscillation 60rpm condition.Get chlorin e 6 chitosan-stearic acid grafting micelle pressed powder, disperse with deionized water (pH is 5.7), getting drug level is chitosan-stearic acid grafting micellar solution of 32 μ g/mL, get 2.5mL (the MWCO 7.0KDa in the bag filter that packs into respectively, Spectrum Laboratories, Laguna Hills CA), places bag filter the releasing tube that contains PBS 7.4 (containing 0.2% Tween 80) 20mL.Timing sampling 1.0mL, HPLC measures drug level, calculates the cumulative release amount.Fig. 1 is seen in the chlorin e 6 chitosan-micellar chlorin e 6 release in vitro of stearic acid grafting behavior.
4) the chlorin e 6 chitosan-micellar cell giving drugs into nose of stearic acid grafting concentration profile is measured
The trophophase lung epithelial tumor cell A549 cell of taking the logarithm, adjusting cell density with culture medium after the trypsinization is 1.0 * 10
5/ mL is inoculated in 12 well culture plates by the 4.0mL/ hole, the pre-24h that cultivates in the incubator, add chlorin e 6 chitosan-stearic acid grafting micelle respectively, constant drug level is 12.5g/mL, and compares with the free drug with concentration, in 37 ℃ hatch 2,4,6,8,12 respectively, behind the 24h, wash cell three times with PBS (pH 7.4), use the 50L trypsinization, with 0.5mL PBS collecting cell, the ice bath ultrasonic 20 (400W that pop one's head in, work 2s stops 3s), get cell breakage liquid.Get an amount of cell breakage liquid, add isopyknic dehydrated alcohol water-bath ultrasonication micelle, dissolved substance, sample introduction behind the 0.22m microporous filter membrane is measured medicament contg excessively.
Get above-mentioned cell breakage liquid 20L synchronously, add in 96 well culture plates, add the BCA working solution of 200L, place more than the 30min for 37 ℃.Measure light absorption value at the 570nm place with enzyme connection detector.According to the bovine serum albumin of concentration known (bovine serum albumin, BSA) production standard curve, and with the protein content (M) in this curve calculation cytolysate.Defined parameters F, when F is different time points, the ratio of drug level and intracellular protein content in the cell.Investigate the ingestion of medicines ability of cell according to following formula at different incubation times.
T is percentage rate (%)=F (the T)/F (0) * 100% (2) of cellular uptake medicine constantly
In the formula, F (T)=T is the interior drug concentrations of cell/T moment intracellular protein content constantly, and F (0)=0 is the initial concentration of medicine/0 moment intracellular protein content constantly
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 2 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 2.86% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 20.46%; After hatching 24 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 6.61% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 26.85%.
Embodiment 3:
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) the synthetic and physical and chemical property determining of chitosan-stearic acid grafting
Get above-mentioned chitosan 0.1g, precision weighing, add 20mL 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: 25 takes by weighing stearic acid and is dissolved in the 10mL 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 chitosan-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 chitosan.Add 2N hydrochloric acid 2mL, shake up, measure absorption value, preparation standard curve at 344nm wavelength place.Get above-mentioned chitosan-stearic acid grafting 4mg and be dissolved in the 2mL distilled water, with the method operation, measuring amino group substitution degree is 4.96%.
Adopt pyrene fluorescence spectrometry chitosan-stearic critical micelle concentration.Preparation variable concentrations chitosan-each 10mL of 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 0.036mg/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 28.9nm and 39.1mV.
3) chlorin e 6 chitosan-preparation of stearic acid grafting micelle and physical and chemical property determining thereof
Get 40mg oligochitosan-stearic acid grafting, precision claims fixed, puts in the 50mL beaker, adds 32mL distilled water (pH is 5.7), pops one's head in ultrasonic 20 times (500w, work 2s stops 3s), gets the micellar solution of 1.25mg/mL.The alcoholic solution 32mL that adds the 0.25mg/mL chlorin e 6, the ultrasonic 30 (400W of probe under the condition of ice bath, work 2s stops 3s), with mixed liquor pack into bag filter (MWCO 3.5KDa, SpectrumLaboratories, Laguna Hills, CA), with the 24h that dialyses under the deionized water room temperature, remove ethanol, with the dialysis solution lyophilization, get chlorin e 6 chitosan-stearic acid grafting micelle powder.
After measured, the chlorin e 6 chitosan of 1mg/mL chitosan-stearic acid grafting concentration-micellar particle diameter of stearic acid grafting is 271.9nm, and surface potential is 35.0mV.Entrapment efficiency is 100%.Medicament contg is 16.67%.
Chlorin e 6 chitosan-micellar release in vitro of stearic acid grafting is carried out under 37 ℃ of constant temperature, frequency of oscillation 60rpm condition.Get chlorin e 6 chitosan-stearic acid grafting micelle pressed powder, disperse with deionized water (pH is 5.7), getting drug level is chitosan-stearic acid grafting micellar solution of 32 μ g/mL, get 2.5mL (the MWCO 7.0KDa in the bag filter that packs into respectively, Spectrum Laboratories, Laguna Hills CA), places bag filter the releasing tube that contains PBS 7.4 (containing 0.2% Tween 80) 20mL.Timing sampling 1.0mL, HPLC measures drug level, calculates the cumulative release amount.Fig. 1 is seen in the chlorin e 6 chitosan-micellar chlorin e 6 release in vitro of stearic acid grafting behavior.
4) the chlorin e 6 chitosan-micellar cell giving drugs into nose of stearic acid grafting concentration profile is measured
The trophophase lung epithelial tumor cell A549 cell of taking the logarithm, adjusting cell density with culture medium after the trypsinization is 1.0 * 10
5/ mL is inoculated in 12 well culture plates by the 4.0mL/ hole, the pre-24h that cultivates in the incubator, add chlorin e 6 chitosan-stearic acid grafting micelle respectively, constant drug level is 12.5g/mL, and compares with the free drug with concentration, in 37 ℃ hatch 2,4,6,8,12 respectively, behind the 24h, wash cell three times with PBS (pH 7.4), use the 50L trypsinization, with 0.5mL PBS collecting cell, the ice bath ultrasonic 20 (400W that pop one's head in, work 2s stops 3s), get cell breakage liquid.Get an amount of cell breakage liquid, add isopyknic dehydrated alcohol water-bath ultrasonication micelle, dissolved substance, sample introduction behind the 0.22m microporous filter membrane is measured medicament contg excessively.
Get above-mentioned cell breakage liquid 20L synchronously, add in 96 well culture plates, add the BCA working solution of 200L, place more than the 30min for 37 ℃.Measure light absorption value at the 570nm place with enzyme connection detector.According to the bovine serum albumin of concentration known (bovine serum albumin, BSA) production standard curve, and with the protein content (M) in this curve calculation cytolysate.Defined parameters F, when F is different time points, the ratio of drug level and intracellular protein content in the cell.Investigate the ingestion of medicines ability of cell according to following formula at different incubation times.
T is percentage rate (%)=F (the T)/F (0) * 100% (2) of cellular uptake medicine constantly
In the formula, F (T)=T is the interior drug concentrations of cell/T moment intracellular protein content constantly, and F (0)=0 is the initial concentration of medicine/0 moment intracellular protein content constantly
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 2 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 2.86% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 20.46%; After hatching 24 hours altogether with tumor cell, with the free drug form administration, cellular uptake medicine percentage rate only is 6.61% of total medicine, and with the administration of grafting micelle form, cellular uptake medicine percentage rate can reach more than 26.85%.
Claims (3)
1. chlorin e 6 chitosan-stearic acid grafting micelle, chitosan-stearic acid grafting is by the low-molecular weight chitoglycan of mean molecule quantity 18.0KDa, form with the grafting of stearic acid chemistry, the amino group substitution degree of chitosan-stearic acid grafting is 4.96%, it is characterized in that: described micelle is made up of chlorin e 6 and chitosan-stearic acid grafting, and wherein chlorin e 6 accounts for the 4.76-16.67% of gross weight.
2. chlorin e 6 chitosan according to claim 1-stearic acid grafting micelle is characterized in that: prepare by following steps:
Get 40mg oligochitosan-stearic acid grafting, the accurate title, decide, and puts in the 50mL beaker, add the 8-32mL distilled water, pH is 5.7, pops one's head in ultrasonic 20 times, get the micellar solution of 5mg/mL, adding is the chlorin alcoholic solution of 0.25mg/mL with volumetric concentration, and probe is ultrasonic 30 times under the condition of ice bath, with the mixed liquor bag filter of packing into, with dialysis under the deionized water room temperature 24 hours, remove ethanol,, get chlorin e 6 chitosan-stearic acid grafting micelle powder the dialysis solution lyophilization.
3. the application of chlorin e 6 chitosan according to claim 1-stearic acid grafting micelle in preparation targeting type photosensitizer formulations.
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Cited By (6)
| 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 |
| CN103638527A (en) * | 2013-12-05 | 2014-03-19 | 浙江大学 | Hemopoietin drug-carried nanoparticles and application thereof |
| CN107115527A (en) * | 2017-05-05 | 2017-09-01 | 李斯文 | A kind of sensitising agent compound and its preparation method and application |
| CN107875399A (en) * | 2017-10-26 | 2018-04-06 | 浙江大学 | A kind of Telmisartan modified chitosan oligosaccharide fatty acid nanoparticle and preparation and application |
| CN109464676A (en) * | 2018-10-25 | 2019-03-15 | 浙江中医药大学 | A kind of preparation method and product of the photosensitive targeted nano granule of chitosan oligosaccharide |
| CN114621746A (en) * | 2022-02-14 | 2022-06-14 | 苏州大学 | Afterglow luminescent nano material and preparation method and application thereof |
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2009
- 2009-03-30 CN CN200910097053A patent/CN101524326A/en active Pending
Cited By (10)
| 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 |
| 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 |
| CN103638527A (en) * | 2013-12-05 | 2014-03-19 | 浙江大学 | Hemopoietin drug-carried nanoparticles and application thereof |
| CN107115527A (en) * | 2017-05-05 | 2017-09-01 | 李斯文 | A kind of sensitising agent compound and its preparation method and application |
| CN107115527B (en) * | 2017-05-05 | 2020-06-30 | 李斯文 | Photosensitizer compound and preparation method and application thereof |
| CN107875399A (en) * | 2017-10-26 | 2018-04-06 | 浙江大学 | A kind of Telmisartan modified chitosan oligosaccharide fatty acid nanoparticle and preparation and application |
| CN107875399B (en) * | 2017-10-26 | 2020-04-03 | 浙江大学 | Telmisartan modified chitosan oligosaccharide fatty acid nanoparticle and preparation and application thereof |
| CN109464676A (en) * | 2018-10-25 | 2019-03-15 | 浙江中医药大学 | A kind of preparation method and product of the photosensitive targeted nano granule of chitosan oligosaccharide |
| CN109464676B (en) * | 2018-10-25 | 2022-03-15 | 浙江中医药大学 | Preparation method and product of chitosan oligosaccharide photosensitive targeting nanoparticles |
| CN114621746A (en) * | 2022-02-14 | 2022-06-14 | 苏州大学 | Afterglow luminescent nano material and preparation method and application thereof |
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