CN102319453A - Drug-loaded stent with ultrasonic intelligent controlled release - Google Patents
Drug-loaded stent with ultrasonic intelligent controlled release Download PDFInfo
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- CN102319453A CN102319453A CN201110235823A CN201110235823A CN102319453A CN 102319453 A CN102319453 A CN 102319453A CN 201110235823 A CN201110235823 A CN 201110235823A CN 201110235823 A CN201110235823 A CN 201110235823A CN 102319453 A CN102319453 A CN 102319453A
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- amycin
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Abstract
The invention relates to a stent coating with ultrasonic intelligent controlled release and a preparation method and an application thereof. The invention also provides a drug-loaded stent with ultrasonic intelligent controlled release and a preparation method and an application thereof. The advantages of the invention are that: a new stent coating with the effect of ultrasonic intelligent controlled release is successfully constructed and synthesized; ultrasound is first found and confirmed to have control effect on the temperature-sensitive release of loaded drugs; and a new method is provided for future drug intelligent controlled release; a drug-loaded biliary tract stent with ultrasonic intelligent drug controlled release is synthesized, and a possible and feasible treatment means is provided for the treatment of malignant tumors of hollow organs; ultrasound is first found and confirmed to have significant effect on promoting the release of drugs coated by a temperature-sensitive material by changing the spatial structure of the temperature-sensitive material. By using ultrasound, the effect of drug intelligent controlled release at a designated site is achieved; local anticancer effect is brought into play; the toxic and side effect of anticancer drugs on important human tissue is reduced; and high clinical application value is provided.
Description
Technical field
The present invention relates to a kind of support, specifically, is a kind of carried stent with ultrasonic intelligent controlled release of temperature sensitive drug coated coating.
Background technology
In the interventional therapy field, for the narrow positions of organs such as blood vessel, trachea, esophagus, urethra, biliary tract, implant frame is expanded narrow positions usually, and it is unimpeded effectively to keep tube chamber.Support be not imported in the tube chamber under the expansion state, and is positioned stenosis, expands and places here.People have invented bracket for eluting medicament on bracket basis subsequently; Bracket for eluting medicament also is referred to as drug releasing stent; Carry medicine through encapsulating, in support is inserted tube chamber, behind the diseased region, discharge medicine through type of elution in the medicine autohemagglutination compound coating in the polymer of metal support surface.Present bracket for eluting medicament more is directly with hydrophobic polymer such as polylactic acid, and polycaprolactone etc. are as the coating drug loading, and this coating is uncontrollable to drug release.
Chinese patent document CN101862477A discloses a kind of support and application thereof with drug temperature-sensitive controlled-release function; Described support is made up of passive support and the drug-carried coat that covers rack surface; Described drug-carried coat is to be coated to rack surface by the coating with drug temperature-sensitive controlled-release function with the mode of spin coating, dip-coating or spraying to form; Coating has good temperature sensitivity and drug controlled-releasing function, and carried stent can effectively kill and wound cancerous cell.The PDT (HMME-PDT) that Liu Jia etc. disclose the mediation of amycin (DOX) associating hematoporphyrin monomethyl ether (sees for details: Liu Jia the effect of human liver cancer cell (HepG2); Deng. HMME-PDT is to the symphyogenetic research of human liver cancer cell HepG2 [J] in the DOX associating. the Chinese medicine physics magazine; 2008,25 (5): 819-822.).But about a kind of carried stent of the temperature sensitive drug coated coating with ultrasonically controlled-release effect and its production and application also do not appear in the newspapers at present.
Summary of the invention
The object of the invention one is to deficiency of the prior art, and a kind of bracket coating of ultrasonic intelligent controlled release is provided.
The object of the invention two is that a kind of carried stent of ultrasonic intelligent controlled release is provided.
For realizing above-mentioned purpose; The technical scheme that the present invention takes is: a kind of bracket coating of ultrasonic intelligent controlled release; The amphiphilic block copolymer that described coating is formed by temperature sensitive polymer and degradable hydrophobic polymer, degradable polymer supported body material and the chemotherapeutics and the sound sensitiser that are embedded in the coating are formed.
Described amphiphilic block copolymer is P-(NIPAAm-co-NHMAAm)-S3-C12, and described degradable polymer supported body material is PLLA.
Described chemotherapeutics is selected from a kind of in amycin, kaempferol, vincristine, camptothecine, epipodophyllotoxin, paclitaxel, the 5-fluorouracil, and described sound sensitiser is selected from a kind of in blood porphyrin derivant, ATX-70, ATX-S10, porfimer sodium, piroxicam, dimethyl sulfoxide, cytosine arabinoside, formicester acid, the anthracycline compound.
Described chemotherapeutics is an amycin, and sound sensitiser is a hematoporphyrin monomethyl ether, and described hematoporphyrin monomethyl ether is 20:1 with the drug level ratio of amycin.
A kind of method for preparing of bracket coating of ultrasonic intelligent controlled release, described method for preparing may further comprise the steps:
A, doxorubicin hydrochloride is dissolved in the acetone soln, adds ethylenediamine water miscible amycin is become oil-soluble, then with the degeneration amycin be dissolved in dichloromethane and the acetone mixed solution in;
B, take by weighing in this solution of polylactic acid and hematoporphyrin monomethyl ether dissolving respectively, and then take by weighing PNIPAM-NMA three thioesters and be dissolved in this solution, promptly get described bracket coating.
The application of the bracket coating of described ultrasonic intelligent controlled release in support.
For realizing above-mentioned second purpose; The technical scheme that the present invention takes is: a kind of carried stent of ultrasonic intelligent controlled release; Constitute by passive support and the coating that covers rack surface; Described carried stent is to be coated to rack surface by the arbitrary described coating of claim 1-6 with the mode of spin coating, dip-coating or spraying to form, and described support is Esophageal Stent, gastrointestinal tract support, biliary tract prosthesis, bronchial stent, bladder support or ureter bracket.
Described support is a biliary tract prosthesis, and described carried stent is a nick-eltitanium alloy stent.
A kind of method for preparing of carried stent of ultrasonic intelligent controlled release, the method for preparing of described support are that the mode of the arbitrary said coating for preparing of claim 1-4 with spin coating, dip-coating or spraying is coated on the rack surface.
The application of described carried stent in the tract tumor disease.
The invention has the advantages that:
1, successfully makes up and synthesized a kind of new bracket coating, find and confirm the ultrasonic control action that temperature sensitive medicine carrying is discharged that has first, for the intelligent controlled release of medicine from now on provides a kind of new method with ultrasonic intelligent controlled-release function;
2, synthesized medicine carrying biliary tract rack with ultrasonic intelligent medicine controlled releasing; Propose and confirmed the new role of carried stent in the malignant biliary tumor aid treatment; Be that support is when providing the hollow organ supporting role; Utilize support to take medicine, have certain tumor aid treatment effect, for the hollow organ treating malignant tumor provides a kind of possible, feasible treatment means for carrier;
3, find and confirm the ultrasonic temperature sensing material space structure that passes through to change, the remarkable effect that promotion temperature sensing material drug coated discharges first.Utilize the ultrasonic effect that can play the medicine intelligence controlled release of appointed part, bring into play partial antitumaous effect, reduce the toxic and side effects of cancer therapy drug, have high clinical value the human body vital tissue.
Description of drawings
Accompanying drawing 1 is the infrared spectrum of the temperature sensitive copolymer of amphiphilic.
Accompanying drawing 2 is pure nickel titanium alloy infrared detection spectrograms.
Accompanying drawing 3 is the amphiphilic block copolymer molecular structural formula.
Accompanying drawing 4 is copolymer NMR scanning.
Accompanying drawing 5 is amphipathic copolymer LCST testing results.
Accompanying drawing 6 is each material contact angle detection.
Accompanying drawing 7 is respectively to organize material drug release front and back electronics sonomicroscope scintigram.
Accompanying drawing 8 is the forward and backward SEM scanning of each material group ultrasonication.
Accompanying drawing 9 is structural representations with carried stent of temperature sensitive drug coated coating of the present invention.
Accompanying drawing 10 is external QBC survival rates under the drug combination condition.
Accompanying drawing 11 is the influences in different time point external inhibition cholangiocarcinoma cell propagation under ultrasonic and non-ultransonic condition of three kinds of materials of B/C/D.
Accompanying drawing 13 is through two the cause apoptosis situation of various materials to the QBC cell that detect of dying of streaming.
Accompanying drawing 14 is that nude mice is carried out ultransonic sketch map.
Accompanying drawing 15 is nude mice tumor formation figure.
Accompanying drawing 16 is drug level of ultrasonic front and back HMME and DOX in the blood.
Accompanying drawing 17 is ultrasonic front and back HMME and DOX drug level in the tumor body.
Accompanying drawing 18 is the back 7 days tumor body difference of treatment, and left side figure be a tumor body difference, and right side figure treats back 7 days tumor weight and the medicine inhibition situation to tumor.
Accompanying drawing 19 is the back 14 days tumor body difference of treatment, and left side figure be a tumor body difference, and right side figure treats back 14 days tumor weight and the medicine inhibition situation to tumor.
The specific embodiment
Below in conjunction with accompanying drawing the specific embodiment provided by the invention is elaborated.
One, amphiphilic block copolymer is synthetic:
1. Raft chain-transferring agent S-1-dodecyl-S '-(α, α '-dimethyl-α ' '-acetic acid) three thioesters (DMP) is synthetic
0.25g 4 bromide (1.6 mmol), 8.1g lauryl mercaptan (0.04 mol) and 19.24g acetone (0.33 mol) mixture are stirred and are cooled to 0 ℃, the N2 protection down dropping 3.35 g concentration be the NaOH solution of 50wt%.After 20 min, in 20 min, drip the mixed liquor of forming by 3.04g Carbon bisulfide (0.04 mol) and 5.2mL acetone.After reaction system begins to redden, slowly add 7.13g CHCl
3(0.06mol), in 15min, dripping 16 g concentration then is the NaOH solution of 50wt%.This system is 0 ℃ of following stirred overnight.After reaction finishes, add 60mL H to system
2O adds the concentrated hydrochloric acid acidify then.After acetone is removed in stirring, thick product is dissolved in 150 mL isopropyl alcohols, removes by filter insoluble matter, the filtrating evaporate to dryness obtains yellow solid.This solid carries out recrystallization with normal hexane.Obtain 4.6 g yellow crystals.Productive rate: 32%.
2. the RAFT polymerization of PNIPAM
3.40g NIPAAM (30 mmol), 0.11g chain-transferring agent DMP (0.3 mmol) and 4.93mg azodiisobutyronitrile (AIBN) (0.03 mmol) are dissolved in the 8.7mL oxolane (THF); Through freezing, bleed, the dissolving program, remove the oxygen in the system.The question response system is stirred to room temperature, reacts at 70 ℃ then.Whole process keeps stirring, and at N
2Carry out under the protection.Take a sample by scheme of designing in advance, take out sample and promptly use liquid nitrogen freezing, a part of sample is used for the nuclear-magnetism test, to confirm monomeric conversion ratio; Another part adds a small amount of THF dilution, ether sedimentation, dissolution precipitation several times after, filter and obtain buff powder, 45 ℃ of vacuum dryings.
3. PNIPAM-b-HMAM block copolymerization
With PNIPAM three thioesters is the macromolecular chain transfer agent.N-Isopropylacrylamide (NIPAAM) macromolecular chain transfer agent is mixed with certain mass ratio with NMA (HMAAM); Be dissolved in the oxolane of new steaming; Add an amount of AIBN as initiator, remove the oxygen in the system through freezing, the dissolving program of bleeding.Under nitrogen protection, place 70 ℃ of oil bath reactions 7 hours then.Pour in the ether after reaction finishes and precipitate, the product that obtains is dissolved in the dichloromethane again, and behind the ether sedimentation, the product that obtains is a pale yellow powder, is dried to constant weight in 45 ℃ of following vacuum drying ovens.
Two, coating liquid preparation:
1. non-temperature sensitive solution (PLLA) configuration:
Doxorubicin hydrochloride is dissolved in the acetone soln, adds excessive ethylenediamine the water solublity amycin is become oil-soluble; Then 10mg degeneration amycin is dissolved in the acetone mixed solution of 20ml dichloromethane and 10ml; Take by weighing the 900mg polylactic acid respectively then and the 200mg hematoporphyrin monomethyl ether is dissolved in this solution, dispose non-temperature sensitive PLLA coating liquid.
2. temperature sensitive solution allocation:
Doxorubicin hydrochloride is dissolved in the acetone soln, adds excessive ethylenediamine water miscible amycin is become oil-soluble; Then 10mg degeneration amycin is dissolved in the acetone mixed solution of 20ml dichloromethane and 10ml; Take by weighing respectively then in this solution of 900mg polylactic acid and 200mg hematoporphyrin monomethyl ether dissolving, and then take by weighing 270mg PNIPAM-NMA three thioesters and be dissolved in this solution, dispose temperature sensitive PLLA coating liquid.
3. coating procedure:
Take out after Nitinol is put into salpeter solution heat treatment 30min, with a large amount of deionized water rinsings, drying for standby.Nitinol is dipped in the above-mentioned coating liquid and takes out behind the 1min after the acidify, and dry 30s is dipped in the coating liquid again and takes out drying for standby behind the 1min.
4. nanometer gold-temperature sensitive coating hydridization:
Dispose the auric chloride aqueous solution of 1mg/ml and the sodium borohydride aqueous solution of 1mg/ml respectively.The Nitinol of temperature sensitive coating is dipped into 30min in the chlorogold solution, takes out, clean with deionization, drying is dipped into 5min in the sodium borohydride aqueous solution then, takes out washed with de-ionized water, drying for standby.
The sign of embodiment 2 polymer and polymer nano-particle
Polymer architecture characterizes:
P-(NIPAAM-b-HMAAM) copolymer structure is with Fourier transformation infrared spectrometer (FTIR) (Bruker; Equinoxss/Hyperion2000), nuclear magnetic resonance chemical analyser (NMR) (Bruker, Anance400), polymer powder is directly tested with the ATR method in the FTIR test; CDCl is used in the NMR test
3As solvent; With GPC (waters) determining molecular weight and molecular weight distributing index.
The test of polymer temperature-sensing property:
Aqueous copolymers solution LCST adopts nephelometry to measure; 5mg P-(NIPAAM-b-HMAAM) copolymer is dissolved in the 10mL deionization, makes the aqueous solution of P-(NIPAAM-b-HMAAM).(U-3310 Hitachi) records at wavelength 450nm place the aqueous solutions of polymers light transmission rate with ultraviolet spectra.Sample cell is used temperature controller constant temperature.Sample cell is tested temperature constant temperature to be measured 10 minutes then, and the temperature when transmitance becomes initial transmitance 50% is LCST.
One, composition and the phenetic analysis of temperature sensitive amphiphilic block copolymer P-(NIPAAm-co-NHMAAm)
1. infrared spectrum (FTIR) analyser detects the amphipathic copolymer composition
Adopt the sample preparation of KBr pressed disc method, on the Bruker EQUINOX55 type Fourier transformation infrared spectrometer of configuration DTGS detector and KBr beam splitter, analyze.The resolution of instrument is superior to 0.5cm ~ 1, scanning times, and sweep limits is 400 ~ 4000cm
-1
2. the nuclear magnetic spectrum analyser detects the amphipathic copolymer composition
Adopt the DMX-500 type NMR of German Bruker company to analyze the composition of copolymer, CDCl
3Be solvent, 25 ℃ of temperature.
3. the detection of amphipathic copolymer lower critical solution temperature (LCST)
(1) takes by weighing a certain amount of P-(NIPAAm-co-NHMAAm)-S3-C12 sample and be dissolved in the distilled water, be mixed with the solution of 5mg/ml, insert temperature chamber constant temperature.
(2) ultraviolet spectrophotometer overlaps under the constant temperature in homemade recirculated water heated at constant temperature, constant temperature 20min under each temperature, the light transmittance of mensuration 450nm fixed wave length.
(3) according to temperature-light transmittance curve, light transmittance sudden change mid point deserved temperature is decided to be the lower critical solution temperature LCST of polymer.
4. each material group combines contact angle detection with Nitinol:
Contact angle: be meant and on a solid level plane, drip a drop, the solid-liquid on the surface of solids-gas three-phase point of interface place, the angle that its liquid-vapor interface is become when being clipped in wherein with solid-liquid interface two tangent lines to liquid phase; Can reflect the moistening situation of liquid to the surface of solids, contact angle is littler, and moistening gets better, and material and support adhesion are stronger.Adopt contact angle detection appearance (specifications and models: KRUSS DSA 10-MK2 contact angle tester, manufacturer: Kr ü ss company, Germany).
Two, result and analysis:
(1) infrared analysis
Please with reference to accompanying drawing 1, accompanying drawing 1 is the infrared spectrum of the temperature sensitive copolymer of amphiphilic.Show among the figure that pure PLLA medication coat is at 1750cm
-1Locate the stretching vibration that strong absworption peak should belong to the two keys of C=O, corresponding to the PLLA ester bond; And in temperature sensitive medication coat at 1544cm
-1, 1646cm
-1And 3500cm
-1Near new absworption peak of appearance should be the deformation vibration of N=H key, is the absworption peak of temperature sensitive copolymer.Pure nickel titanium alloy infrared detection is illustrated in does not have absworption peak on the infrared spectrum, see accompanying drawing 2, and accompanying drawing 2 is pure nickel titanium alloy infrared detection spectrograms.
From figure, can find out 1650 cm
-1The strong absworption peak at place should belong to the two keys of C=O typical stretching vibration (the C=O stretching vibration appears at 1900 ~ 1650 cm
-1, be very characteristic and the strongest often absorption in the infrared spectrum); At 1544 cm
-1The absworption peak at place should be the deformation vibration of N-H key, and (the N-H deformation vibration is equivalent to CH
2The scissoring vibration mode, its absorption band is at 1440 ~ 1560 cm
-1); And a wide strong absorption bag occurred at 3500 cm-1 places, belonged to O-H and the N-H stretching vibration peak among the NIPAAm among the HMAAm respectively.The result shows that infrared spectrum has further proved P (NIPAAm-
Co-HMAAm)-S3-
C12The existence of block copolymer, P-(NIPAAm-co-HMAAm)-S3-
C12Block copolymer is by successfully preparation.
(2) nmr analysis
Accompanying drawing 3 is the amphiphilic block copolymer molecular structural formula, wherein is denoted as a, b, c, d, e, f and partly distinguishes Ha, Hb, Hc, Hd, He, Hf in the respective figure 4.Accompanying drawing 4 is copolymer NMR scanning.
NMR scanning shows 3.9 corresponding to Hb, and 3.8 corresponding to Hf, and 2.0 corresponding to He; 1.05 corresponding to Ha; 1.6-1.8,, confirm the existence of P (NIPAAm-co-HMAAm)-S3-C12 amphiphilic block copolymer for the isopropyl of PNIPAAm and PHMAAm and the proton of methylol go out the peak corresponding to Hc Hd
(3) amphipathic copolymer LCST detects
Please with reference to accompanying drawing 5, accompanying drawing 5 is amphipathic copolymer LCST testing results.We can see P (NIPAAm)-S3-C12 prepolymer and P (NIPAAm-in the drawings
Co-HMAAm)-LCST between the S3-C12 block copolymer changes.By P (NIPAAm-
Co-HMAAm)-micellar LCST that the S3-C12 block copolymer forms is about 42 ℃, is higher than the LCST of P (NIPAAm)-S3-C12.Mainly be because the cooperative effect that the hydroxyl of prepolymer disappears in a large number and hydrophobic section adds behind the formation block copolymer.Show about LCST to 42 ℃ (S3 is an initiator) adopting more hydrophilic NMA and the copolymerization of N-Isopropylacrylamide can effectively improve temperature sensitive amphipathic copolymer, this temperature meets thermotherapy temperature range in the body.
(4) each material winding feeler detects:
Please with reference to accompanying drawing 6, accompanying drawing 6 is each material contact angle detection, and wherein 1-4 is respectively the contact angle detection of blank nickel titanium alloy, PLLA medication coat, temperature sensitive medication coat and hybridized nanometer gold plating.
Nickel-titanium alloy material shows low contact angle, and when with the PLLA coating, it is big that contact angle becomes, and surpasses 100 °, and when containing the temperature sensitive polymer coating, contact angle obviously dwindles, and becomes comparatively hydrophilic, helps the release of medicine; Behind the hybridized nanometer gold, contact angle further dwindles, and hydrophilic further improves, this maybe with nanometer gold and temperature sensing material hydridization after, the temperature sensing material hydrophilic section migrates to material surface and increases relevant.
The preparation of carried stent:
Nick-eltitanium alloy stent is soaked in the mixed solution that above-mentioned technological process prepares, adopts dipping process, utilize molecule and intermolecular Van der Waals force effect, we have accomplished the preparation of medicine carrying biliary tract rack, have reached the purpose of steady load medicine.
Field emission scanning electron microscope and sonomicroscope that embodiment 3 each material group drug release front and rear surfaces change detect
One, experimental apparatus:
Field emission scanning electron microscope: specifications and models: Quanta 200 FEG manufacturers: FEI Company, the U.S..
Electronics sonomicroscope imaging system (look and reach by section; HIROX-7700): be used for detection material and absorbing the be stimulated acoustical signal of back generation of the heat wave that produced behind the luminous energy and material; Can be used for observing the material surface conformation change, have the advantage of quick, harmless and dynamic analysis.
Two, experiment analysis results:
Please with reference to accompanying drawing 7, accompanying drawing 7 is respectively to organize material drug release front and back electronics sonomicroscope scintigram.The diagram be * 400 amplification conditions under scanning result.A. PLLA coating; B. temperature sensing material coating; C. nanometer gold-temperature sensitive hybrid coating.1. medicine does not discharge; 2. drug release is not ultrasonic; 3. drug release is ultrasonic.
Electronics sonomicroscope scanning result showed before and after each organized the material drug release: non-temperature sensitive (PLLA group) has certain variation at ultrasonic forward and backward material surface, but changes not remarkable; Temperature sensitive group has a large amount of cavity spline structures to occur at ultrasonic forward and backward visible material surface local, and shows the comparatively change of homogeneous; Nanometer gold+temperature sensing material group is in the certain variation of ultrasonic forward and backward existence, but show as damaged than bulk, this maybe with nanometer gold temperature sensing material hydridization after, coating weakens relevant with the rack surface adhesion under the ultrasonication.
Please with reference to accompanying drawing 8, accompanying drawing 8 is the forward and backward SEM scanning of each material group ultrasonication, and A is before the ultrasonication, B is a scanning result after the ultrasonication, temperature sensitive group of left figure right and wrong (PLLA group), and right figure is temperature sensitive group.Obvious heterogeneity cavity spline structure, non-homogeneous release appear by the ultrasonic back of the visible PLLA material group of left figure; Be able to even release fast by the visible temperature sensitive group of material medicine after ultrasonication of right figure; The SEM scanning result showed before and after each organized the material drug release: the ultrasonic forward and backward appearance of each material group changes with electronics sonomicroscope scanning result.
The structure of embodiment 4 carried stents is formed
The Reference numeral and the ingredient that relate in the accompanying drawing are as follows:
1. blend coating 2. supports
11.PLLA the C12 part of coating 12. copolymer tail ends
13. temperature sensitive part 14. sound sensitisers
15. chemotherapeutics
Please with reference to accompanying drawing 9, accompanying drawing 9 is structural representations with carried stent of temperature sensitive drug coated coating of the present invention.Described carried stent is made up of blend coating 1 and support 2, and described blend coating 1 is the blend coating of copolymer p-(NIPAAm-co-NHMAAm)-S3-C12 and poly-lactic acid ester (PLLA), and described blend coating 1 covers the outer surface of support 2.Described blend coating 1 comprises C12 part 12, temperature sensitive part 13, sound sensitiser 14 and the chemotherapeutics 15 of PLLA coating 11, copolymer tail end.Described sound sensitiser 14 is embedded in the blend coating 1 with chemotherapeutics 15.
Described sound sensitiser 14 is blood porphyrin derivants, and described chemotherapeutics 15 is selected from a kind of in amycin, kaempferol, vincristine, camptothecine, epipodophyllotoxin, paclitaxel, the 5-fluorouracil.
Need to prove that described sound sensitiser 14 is hematoporphyrin monomethyl ethers, described chemotherapeutics 15 is amycin.
Best cholangiocarcinoma cell is killed and wounded the hematoporphyrin monomethyl ether of usefulness, the affirmation of amycin drug combination condition under the embodiment 5 external supersonic conditions
1. experiment purpose: maximum is killed and wounded cholangiocarcinoma cell usefulness under the ultrasound condition HMME and DOX drug combination condition detect, and are the synthetic experimental basis that provides of subsequent material
2. experimental technique step:
(1) earlier with exponential phase cell 2 * 10
5Cell/ml is inoculated in 35mm single hole culture plate, and every hole 2ml cultivated 24 hours.
(2) experimental group (amycin group DOX, hematoporphyrin monomethyl ether low dose group HMME LOW, hematoporphyrin monomethyl ether high dose group HMME HIGH, hematoporphyrin monomethyl ether low dosage add amycin group HMME LOW+DOX, the hematoporphyrin monomethyl ether high dose adds amycin group HMME HIGH+DOX) adds the culture fluid that contains the variable concentrations medicine; Matched group is then used isopyknic culture fluid; Every group of parallel hole of establishing more than 3; Rearmounted 37 ℃ of ultrasonication, 5% CO
2And the incubator of saturated humidity is cultivated 3,6,12,24 h respectively.The hematoporphyrin monomethyl ether low dosage is 5 μ g/ml, and the hematoporphyrin monomethyl ether high dose is 10 μ g/ml.
(3) according to timing node, to inhale and abandon supernatant, every then hole adds the MTT that contains 5 mg/ml of 2ml, and 37 ℃, 5% CO2 gas incubator continues to cultivate 4 hours.
(4) the careful suction abandoned supernatant; Every hole adds the DMSO Rong Xie Jia Za of 2ml, and vibration 10min treats that the MTT reduzate dissolves fully; From every ware sucking-off 100 μ l to 96 hole culture dishs; On enzyme mark view, use wavelength to be 492nm, reference wavelength is that 630nm measures optical density (OD) value, calculates the growth inhibition ratio of cell.
(5) date processing: inhibitory rate of cell growth=
3. experimental apparatus, material:
Cell strain: QBC cell (people's cholangiocarcinoma cell)
Reagent: (3-(4 for tetrazolium bromide; 5-dimethylthiazol-2-yl)-2; 5-diphenyl tetrazolium bromide, MTT), RPMI 1640 culture medium, calf serum, DMSO (dimethyl sulfoxide), PBS, 0.25% pancreatin, hematoporphyrin monomethyl ether, amycin.
Experimental apparatus: Bio-Rad company ELIASA (550 type), Heraeus company, CO
2Incubator (BB 5060 types), 96,24 hole culture dishs, multifunction supersonic appearance.
4. experimental result:
Statistical analysis: data are represented with meansigma methods ± standard deviation.Adopt mathematical statistics software SPSS13.0 to carry out the ttest statistical analysis; Please with reference to accompanying drawing 10; Find out among the result that ultrasonic group of each time point HMME high dose+amycin is maximum with non-ultrasonic group significant difference; Especially 24 hours action effect is more notable, so this drug level is as the synthetic data foundation of subsequent material, and promptly the two concentration proportioning of hematoporphyrin monomethyl ether and amycin is 20:1.
Embodiment 6 carries out material coating on above-mentioned condition basis, and carries out external medicine survival rate experiment
1. experiment purpose: synthesizing to check and respectively organize the influence of material through material to the survival rate of QBC cell
2. experimental apparatus, material:
Cell strain: QBC cell (people's cholangiocarcinoma cell)
Reagent: tetrazolium bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT), RPMI 1640 culture medium, calf serum, DMSO (dimethyl sulfoxide), PBS, 0.25% pancreatin, B, C, D.
A: hematoporphyrin monomethyl ether+amycin
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol
Experimental apparatus: Bio-Rad company ELIASA (550 type), the CO of Heraeus company
2Incubator (BB 5060 types), 96,24 hole culture dishs, multifunction supersonic appearance.
3. experimental technique step:
(1) earlier with exponential phase cell 2 * 10
5Cell/ml is inoculated in 35mm single hole culture plate, and every hole 2ml cultivated 24 hours.
(2) experimental group (B, C, D) adds the medicine carrying (volume 2ml) that contains the variable concentrations medicine, and matched group is then used isopyknic culture fluid, every group of parallel hole of establishing more than 3, rearmounted 37 ℃ of ultrasonication, 5% CO
2And the incubator of saturated humidity is cultivated 0,6,12,24,36 h respectively.
(3) according to timing node, to inhale and abandon supernatant, every then hole adds the MTT that contains 5 mg/ml of 2ml, and 37 ℃, 5% CO2 gas incubator continues to cultivate 4 hours.
(4) the careful suction abandoned supernatant; Every hole adds the DMSO Rong Xie Jia Za of 2ml, and 10 min that vibrate treat that the MTT reduzate dissolves fully; From every ware sucking-off 100 μ l to 96 hole culture dishs; On enzyme mark view, use wavelength to be 492nm, reference wavelength is that 630 nm measure optical density (OD) value, calculates the growth inhibition ratio of cell.
(5) date processing: inhibitory rate of cell growth=
4. experimental result
On this basis, synthesized nano material: blank group (ultrasonic/non-ultrasonic)
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol (ultrasonic/non-ultrasonic)
Measured the influence of three kinds of materials of B/C/D in different time point survival rate of pair cell under ultrasonic and non-ultransonic condition respectively, the result is please with reference to accompanying drawing 11.Statistical analysis: data are represented with meansigma methods ± standard deviation.Adopt mathematical statistics software SPSS13.0 to carry out the ttest statistical analysis.Each is organized ultrasonic group and compares with non-ultrasonic group
p>0.05 there was no significant difference, *
p<0.05, * *
p<0.01, * * *
p<0.001 there were significant differences in expression.
Statistical analysis shows in each material group effect 6 hours that ultrasonic front and back respectively organize cell survival rate and all do not have significant difference, act on 12 hours B, C, D groups and significant difference all occurs, and wherein to organize difference more remarkable for C; Act on 24 hours and continued to show as identical effect trend in 36 hours; Each group all presents obvious time dependence, and the effect of C group cell killing also shows as significant ultrasonic dependency.
The experiment of embodiment 7 Nanoalloy material vitro drug release
1. experiment purpose: through this test to confirm in the synthetic material HMME the drug release situation.
2. experimental apparatus and reagent:
The multi-functional ELIASA of Synergy 2 SL high-performance (BioTek, USA), the Fluoscence spectrofluorophotometer (Cary Eclipse, USA), PBS, the multifunction supersonic appearance, RPMI 1640 culture medium, calf serum, other reagent are analytical pure.
3. experiment is divided into groups: A: blank group (ultrasonic/non-ultrasonic)
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol (ultrasonic/non-ultrasonic)
4. experimental technique step and result:
Take the method for 0h after the ultrasonication, 3h, 6h, 12h, five different time nodes samplings of 24h detection of drugs concentration; Consult the excitation wavelength 485nm of amycin through document; Emission wavelength 590nm, and the excitation wavelength of hematoporphyrin monomethyl ether is 395nm, emission wavelength 613nm.
Draw amycin and hematoporphyrin monomethyl ether standard curve.Detect the drug level of each time point amycin and hematoporphyrin monomethyl ether, each sample is all got 100 μ L and is measured also curve plotting.Please with reference to accompanying drawing 5.
Statistical analysis: the burst size of more ultrasonic front and back HMME and DOX, the difference d that employing SAS computed in software is every group carries out test of normality, and the difference accord with normal distribution adopts the ANOVA check.
Figure 12-1 (material B, HMME), P=0.1837, P>0.05, show that these 2 groups of result differences do not have the significance meaning.Figure 12-2 (material B, DOX), P=0.1393, P>0.05, show that these 2 groups of result differences do not have the significance meaning.B group medicine can slowly discharge ultrasonic front and back there was no significant difference.Figure 12-3 (material C, HMME), P=0.00382,0.001>P>0.05, show that these 2 groups of result differences have the significance meaning.Figure 12-4 (material C, DOX), P=0.01478,0.001>P>0.05, show that these 2 groups of result differences have the significance meaning.C group medicine can slowly discharge, and ultrasonic back drug release is significantly accelerated, and the difference multiple reaches more than 2 times.
The two detection apoptosis that dye of embodiment 8 Annexin V-FITC/PI
1. experiment purpose: through the two apoptosis situation that cause of various materials that detect of dying of streaming to the QBC cell
2. experimental principle: the two methods of dying of Annexin V-FITC/PI are that (fluoresein-5-isothiocyanate, FITC) labelling Annexin V are used in combination PI simultaneously and refuse the method for dying apoptotic cell is carried out two dyeing with Fluorescein isothiocyanate.
3. main experimental apparatus and material
The Becton Dickinson FACSCalibur of company flow cytometer, the Becton Dickinson WINMID of company software, Annexin V – FITC test kit (Millipore), CO
2Incubator and conventional cell culture articles for use one cover etc.
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol
4. experimental procedure
(1) will be in cell inoculation to the 35mm single hole orifice plate of exponential phase, in 37 ℃, 5% CO
2Cultivate 24 h in the incubator and treat adherently, wait to grow to 50%.
(2) the blank group is disregarded, and independent ultrasonic group gives supersound process, and experimental group (B, C, D) adds the medicine carrying (volume 0.4*0.8cm) that contains the variable concentrations medicine; Matched group is then used isopyknic culture fluid; Every group of parallel hole of establishing more than 3, rearmounted 37 ℃ of ultrasonication, 5% CO
2And the incubator of saturated humidity is cultivated 0h, 6h, 12h, 24h, 36h respectively.
(3) by each time point collecting cell, wash cell 2 times with the PBS buffer, centrifugal collection (1500 rpm, 5 min).Add binding Buffer 400 μ l, mixing.
(4) add Annexin V – FITC reagent 5 μ l, 4 ℃ of 15min.
(5) add PI reagent 10 μ l, 4 ℃ of 5min.
(6) use the FACSCalibur flow cytometer to detect.
Left lower quadrant (LL) shows living cells; Right upper quadrant (UR) shows non-viable apoptotic cell; And right lower quadrant (LR) is a viable apoptotic cell.Event is illustrated in the cell number among UL, UR, LL, the LR; %Gated representes that the cell among UL, UR, LL, the LR accounts for the ratio of an inner cell; %Total representes that the cell among UL, UR, LL, the LR accounts for the ratio of all cells.
Apoptosis rate=viable apoptotic cell (LR %Gated)+non-viable apoptotic cell (UR %Gated).
5. experimental result
Divide into groups: blank group (ultrasonic/non-ultrasonic)
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol (ultrasonic/non-ultrasonic)
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol (ultrasonic/non-ultrasonic)
Please with reference to accompanying drawing 13, the apoptosis rate of material C group processing is the highest at 36h finally, and each material group apoptosis situation of statistical analysis prompting shows killing and wounding through causing the apoptosis effect of each material group pair cell with the MTT change detected is consistent in earlier stage; Wherein the C group is more remarkable in ultrasonic forward and backward apoptotic cell difference, finds identical with the MTT detection.
One, experiment purpose: make up the external mice with tumor model of QBC; And give HMME-SDT+ amycin nickel-titanium alloy material treatment; Detect the variation of tumor body size, tumor histology; And carry out the mensuration of the concentration of hematoporphyrin monomethyl ether and amycin in nude mice blood, the tumor tissue, and experimental basis is provided for further research.
Two, experimental technique
1. sample packet: A: negative control group (connect tumor ultrasonic but not administration),
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol (0h, 3h, 6h, 7d, 14d)
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol (0h, 3h, 6h, 7d, 14d)
D: 8 every group of hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol (0h, 3h, 6h, 7d, 14d) amount to 16 groups.
2. making of tumor model animal and blood sample collection concrete steps:
Animal: immunodeficient mouse---nude mice (purchasing Bi Kai company) in Shanghai
Tumor cell line: QBC cell line (people's cancer of biliary duct tumor cell)
Structure of animal pattern and blood sample collection:
(1) tumor cell is with 1 * 10
7, PBS is diluted to 0.2 ml, and to be inoculated in before the nude mice axil subcutaneous.
(2) administration.Each group material Nitinol sheet metal is cut into 0.2*0.8cm
2Size, two of every nude mice embeddings directly are embedded in Subcutaneous tumor and organize both sides.
(3) behind the material embedding 12h, ultrasonic, and put to death the nude mice of respectively organizing 0h, 3h, 6h time point, the tumor body being peeled preserve and formalin solution, eye socket is stored in 4 ℃ with blood after getting blood, in order to the drug level of subsequent detection tumor body in blood.Ultrasonic sketch map is seen Figure 14.Figure 15 left side figure is that 7 days tumor forms, and right figure is 14 days tumor formation figure.
(4) for the nude mice of respectively organizing 7d, 14d after ultrasonic, weigh every day and record, observe the survival condition of nude mice.
(5) nude mice of each group 7d is put to death in the 7th day after ultrasonic, and the stripping tumor is measured the volume of relevant tumor body, weight, and be stored in formalin solution, in order to follow-up section and immunohistochemical study.
(6) nude mice of each group 14d is put to death in the 14th day after ultrasonic, and the stripping tumor is measured the volume of relevant tumor body, weight, and be stored in formalin solution, in order to follow-up section and immunohistochemical study.
(7) collection of sample and detection: the sample for each group 0h, 3h, 6h carries out the detection of blood drug level and tumor vivo medicine concentration; Sample to 7d, 14d; Collect tumor tissue, measure body weight, volume, also be used for researchs such as follow-up paraffin section and SABC after the preservation.
3. sample packet
A: negative control group (connecing tumor but not administration)
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol group
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol group
D: hematoporphyrin monomethyl ether+amycin+temperature sensing material+nanometer gold+Nitinol group
Give the numbering shown in the following table 1:
Table 1
4. detection sample process
Tumor tissue handles:
Take by weighing the tumor tissues of 0.2g; The normal saline that with the w/v is 1:5 is with abundant homogenate .9 000 r/min of homogenizer; Get supernatant homogenate 0.5 mL and 1.0 g ammonium sulfate behind centrifugal 10 min. behind vortex mixing 5 min; Add saturated sodium bicarbonate solution 0.5 mL and chloroform/methanol (4:1, V/V) extract 2 mL. vortex mixings 5 min.Centrifugal 15 min of 1600 r/min shift lower floor's organic facies to another test tube; Adding chloroform/methanol (4:1.V/V) extract 2 mL again, handle coextraction 3 times as stated above. lower floor's organic facies is merged, and 37 ℃ of water-baths volatilize.Residue is with 1ml PBS dissolving, and centrifugal 10 min of 13 000 r/min get supernatant, and classifying and numbering is deposited.
Serum sample is handled:
The about 0.2ml of sample serum, accurate methanol 0.5ml, vortex mixing 3min, centrifugal (13000rpm) 10min of adding; Discard deposition, carefully draw methanol layer to another EP pipe, add methanol 0.5ml again, vortex mixing 3min; Centrifugal (13000rpm) 10min draws methanol layer to another EP pipe, hold over night, centrifugal (13000rpm) 10min; Draw methanol layer to another EP pipe, volatilize in the fume hood, 1 ml PBS dissolving, classifying and numbering is deposited.
The reference substance sample process:
(concentration: 10 mg/ml), add an amount of PBS, be diluted to 1 mg/ml, behind the 0.22um microporous filter membrane, classifying and numbering is deposited excessively to get female medicine amycin.Hematoporphyrin monomethyl ether (concentration 200mg/ml) is handled with quadrat method, is diluted to 1 mg/ml classifying and numbering, and it is subsequent use to keep in Dark Place.
5. the detection of blood substance concentration and tumor vivo medicine concentration
Experimental apparatus and reagent:
The multi-functional ELIASA of Synergy 2 SL high-performance (BioTek, USA), the Fluoscence spectrofluorophotometer (Cary Eclipse, USA), PBS,
B: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+Nitinol
C: hematoporphyrin monomethyl ether+amycin+temperature sensing material+Nitinol
D: hematoporphyrin monomethyl ether+amycin+non-temperature sensing material+nanometer gold+Nitinol
Multifunction supersonic appearance, RPMI 1640 culture medium, calf serum
Other reagent are analytical pure.
Experimental technique:
Take the detection method of 0h after the ultrasonication, 3h, 6h three different time nodes blood sample and tumor vivo medicine concentration; Consult the excitation wavelength 485nm of amycin through document; Emission wavelength 590nm, and the excitation wavelength of hematoporphyrin monomethyl ether is 395nm, emission wavelength 613nm.Draw amycin and hematoporphyrin monomethyl ether standard curve.Detect the drug level of each time point amycin and hematoporphyrin monomethyl ether, and curve plotting.Each group sample is all got 100 μ L and 96 porocyte culture dishs carry out fluoroscopic examination in fluorescence microplate reader.
Figure 16 is the drug level of ultrasonic front and back HMME and DOX in the blood.Figure 17 is ultrasonic front and back HMME and a DOX drug level in the tumor body.Statistical analysis: data are represented with meansigma methods ± standard deviation.Adopt mathematical statistics software SPSS13.0 to carry out the ttest statistical analysis, with P>0.05 there was no significant difference, *
p<0.05, * *
p<0.01, * * *
p<0.001 there were significant differences in expression.Statistical results show: the ultrasonic back of each experimental group lotus tumor topical remedy discharges increase is all arranged, and this possibly be that lotus tumor local space is narrow and small, due to local vibration increases after the ultrasonication; Compare with matched group; Drug level significantly increases after ultrasonication in the temperature sensing material group lotus tumor; And be significantly higher than other each groups; The increase that temperature sensing material release amount of medicine under the ultrasonication is described is relevant with ultrasonic local vibration, but its factor that plays a decisive role is the change of temperature sensing material self space structure or performance under the ultrasonication.Peripheral blood serum drug level testing result statistical results show is in the increase of the simultaneous peripheral blood serum drug level of lotus tumor local drug concentration increase; But its increasing degree is remarkable not as increasing degree in the lotus tumor, explains that lotus tumor local application has the advantage of low whole body toxic and side effects.
6. the medicine carrying nickel-titanium alloy material is to the influence of nude mice Subcutaneous tumor
(1) the medicine carrying niti material is seen table 2 to the influence (evaluating toxic and side effect) of nude mice body weight
Table 2
| Divide into groups | Number of elements (only) | Body weight (g) before the treatment | The 7 days body weight (g) in ground, treatment back | The 14th day body weight (g) in treatment back |
| |
8 | 23.033±1.0764 | 20.6±1.366748 | 18.225±1.08233 |
| US B | 5 | 23.28±0.759605 | 20.76±0.879204 | 18.965±0.98769 |
| US C | 5 | 23.025±0.865544 | 22.66±0.482701* | 19.836±1.1582* |
| US D | 5 | 23.05±0.695222 | 21.08±0.511859 | 19.103±0.84221 |
| |
3 | 23.154±01.178 | 20.53±0.93241 | 18.8672±1.2318 |
| |
3 | 23.087±1.083 | 20.21±0.88723 | 18.2553±1.0832 |
| |
3 | 23.358±1.224 | 20.42±0.78932 | 18.6639±0.97531 |
Nude mice all survives, and the mental status is good, and inoculated tumour cell suspension all can be in the subcutaneous 80mm that touches of inoculation position after 10 days
3About the tumor piece, volume is consistent, tumor formation rate 100%.Removing few part nude mice tumor surface has incrustation place of festering, and surplus nude mice tumor surface does not have the ulcer incrustation, and tumor peplos is complete.
(2) the weight of animals situation of change before and after the Drug therapy
Can find out from table 2; The body weight difference and the no significant difference of each group before the treatment, and 7,14 days the data show in treatment back, the body weight of material C group lays particular stress on than B, D and negative control group significantly; Illustrative material C is less to the influence of nude mice body, and the whole body toxic and side effects is lower.
(3) tumor size situation of change before and after the drug effect is seen table 3
Table 3
| Divide into groups | Number of elements (only) | Tumor body volume (mm before the treatment 3) | The 7th day tumor body volume (mm in treatment back 3) | The low 14 days tumor body volume (mm in treatment back 3) |
| |
8 | 83.37±13.29 | 1125.64±118.74 | 2053.38±228.86 |
| US B | 5 | 81.52±9.65 | 847.75±77.92* | 1893.77±315.69* |
| US C | 5 | 79.29±17.83 | 418.33±86.21** | 1208.19±276.52*** |
| US D | 5 | 76.58±11.44 | 773.26±57.88* | 1786.53±300.81* |
| |
3 | 82.93±12.92 | 1038.83±96.52 | 1996.52±219.56 |
| |
3 | 80.66±15.28 | 1089.39±106.63 | 2014.39±308.69 |
| |
3 | 78.69±13.79 | 1097.28±89.19 | 1984.37±247.92 |
Can find out from table 3; Respectively organize the gross tumor volume no significant difference property of mice with tumor before the treatment; And the tumor body volume of treating back 7 days, 14 days shows material B, C, D organizes and matched group more all shows difference, is illustrated under the medicament slow release, and each is organized lotus tumor propagation and all receives inhibition to a certain degree; And each ultrasonic front and back of material group volume is relatively, ultrasonic group of material C and the not ultrasonic difference maximum of comparing, and lotus tumor volume obvious difference shows that greater than other two groups the ultrasonic back of material C group is the highest to lotus tumor inhibition of proliferation rate; This explains that simple ultrasonic energy has certain inhibitory action to tumor proliferation, but this effect obviously will be weaker than because of the ultrasonic release amount of medicine of temperature sensing material space conformation due to changing that cause and significantly increases the influence that tumor proliferation is suppressed.
(4) the medicine carrying niti material is to lotus tumor inhibition of proliferation situation
Please with reference to accompanying drawing 18, Figure 18 is the back 7 days tumor body difference of treatment, and left side figure be a tumor body difference, and right side figure treats back 7 days tumor weight and the medicine inhibition situation to tumor.Extract tumor after contrast is respectively organized 7 days, can find out that the tumor body of material C group (amycin+hematoporphyrin monomethyl ether+temperature sensing material+Nitinol) obviously dwindles than other each group.From the data result analysis, treat and show material B, C after 7 days, the D group more all shows difference with matched group, shows that respectively organizing the material medicine all can discharge, and has certain inhibitory action to tumor proliferation.See from the heavy suppression ratio of tumor; Material B, C organize ultrasonic front and back relatively; Its suppression ratio is 11.93% and 15.91%, and the ultrasonic back of material C group reaches 34.09% to lotus tumor inhibition of proliferation rate, and illustrative material C group is ultrasonic obviously to be better than other each groups in the effect that suppresses lotus tumor propagation; The effect of combination of ultrasound temperature sensing material can effectively increase medicine carrying niti material release amount of medicine, strengthens medicine to the effect of lotus tumor inhibition of proliferation.
Please with reference to accompanying drawing 19, Figure 19 is the back 14 days tumor body difference of treatment, and left side figure be a tumor body difference, and right side figure treats back 7 days tumor weight and the medicine inhibition situation to tumor.Extract tumor after contrast is respectively organized 14 days, can find out that the ultrasonic back of material C group nude mice tumor body obviously dwindles than other each group.Find out that from data result treat and show material B, C after 14 days, the D group more all shows difference with matched group, the material C group is compared the difference maximum with contrast, show that drug release all has inhibitory action to tumor proliferation, this inhibitory action is remarkable with the material C group.The heavy suppression ratio of tumor sees that material B, the ultrasonic front and back of C lotus tumor inhibition of proliferation rate are 16.02% and 20.70%, and the material C group is for reaching 44.53%, apparently higher than other each groups.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the inventive method; Can also make some improvement and replenish, these improvement and replenish and also should be regarded as protection scope of the present invention.
Claims (10)
1. the bracket coating of a ultrasonic intelligent controlled release; It is characterized in that; The amphiphilic block copolymer that described coating is formed by temperature sensitive polymer and degradable hydrophobic polymer, degradable polymer supported body material and the chemotherapeutics and the sound sensitiser that are embedded in the coating are formed.
2. coating according to claim 1 is characterized in that, described amphiphilic block copolymer is P-(NIPAAm-co-NHMAAm)-S3-C12, and described degradable polymer supported body material is PLLA.
3. coating according to claim 1; It is characterized in that; Described chemotherapeutics is selected from a kind of in amycin, kaempferol, vincristine, camptothecine, epipodophyllotoxin, paclitaxel, the 5-fluorouracil, and described sound sensitiser is selected from a kind of in blood porphyrin derivant, ATX-70, ATX-S10, porfimer sodium, piroxicam, dimethyl sulfoxide, cytosine arabinoside, formicester acid, the anthracycline compound.
4. coating according to claim 4 is characterized in that described chemotherapeutics is an amycin, and sound sensitiser is a hematoporphyrin monomethyl ether, and described hematoporphyrin monomethyl ether is 20:1 with the drug level ratio of amycin.
5. the method for preparing of the bracket coating of a ultrasonic intelligent controlled release is characterized in that, described method for preparing may further comprise the steps:
A, doxorubicin hydrochloride is dissolved in the acetone soln, adds ethylenediamine water miscible amycin is become oil-soluble, then with the degeneration amycin be dissolved in dichloromethane and the acetone mixed solution in;
B, take by weighing in this solution of polylactic acid and hematoporphyrin monomethyl ether dissolving respectively, and then take by weighing PNIPAM-NMA three thioesters and be dissolved in this solution, promptly get described bracket coating.
6. according to the application of bracket coating in support of the arbitrary described ultrasonic intelligent controlled release of claim 1-4.
7. the carried stent of a ultrasonic intelligent controlled release; Constitute by passive support and the coating that covers rack surface; It is characterized in that; Described carried stent is to be coated to rack surface by the arbitrary described coating of claim 1-6 with the mode of spin coating, dip-coating or spraying to form, and described support is Esophageal Stent, gastrointestinal tract support, biliary tract prosthesis, bronchial stent, bladder support or ureter bracket.
8. carried stent according to claim 7 is characterized in that described support is a biliary tract prosthesis, and described carried stent is a nick-eltitanium alloy stent.
9. the method for preparing of the carried stent of a ultrasonic intelligent controlled release is characterized in that, the method for preparing of described support is that the mode of the arbitrary said coating for preparing of claim 1-4 with spin coating, dip-coating or spraying is coated on the rack surface.
10. according to the application of the arbitrary described carried stent of claim 7-8 in the tract tumor disease.
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Application publication date: 20120118 |