CN102440961A - Targeting polymer micelle containing acid-sensitive subsurface, and preparation method thereof - Google Patents
Targeting polymer micelle containing acid-sensitive subsurface, and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a targeting polymer micelle containing acid-sensitive subsurface, and a preparation method thereof. The polymer micelle is in a core-shell structure. Targeting ligand molecules are grafted on a hydrophilic segment terminal of the shell, and a hydrophobic anti-tumor medicine is loaded by a hydrophobic segment of the core. An acid-sensitive response subsurface exists between the core and the shell, wherein the acid-sensitive response subsurface can load negative electricity quantum dots. When a pH value is 5.0, the acid-sensitive layer is hydrophobic; when the pH value is 7.4, the acid-sensitive layer is hydrophobic. According to the invention, with the targeting molecules, the tumor targeting function of the micelle is realized; with the quantum dots loaded on the acid-sensitive subsurface, a visualizing function is realized; with low-pH-responsibility of the acid-sensitive subsurface, a controlled-release function can be realized, and the release of the medicine can be controlled. With the micelle system provided by the invention, the release behavior of the medicine is improved, and a premature-release problem of the micelle system in blood circulation is possible to be solved. With an internalization behavior mediated by the targeting molecules, the treatment effect of the medicine is enhanced. With the loaded quantum dots, monitoring during the treatment process can be realized.
Description
Technical field
The present invention relates to chemistry, pharmacy and biomedical engineering field, is a kind of the have micellar preparation of target polymer of acid-sensitive subsurface stratum and application aspect medicine sustained release and video picture specifically.
Background technology
Human beings'health in the tumor serious threat, and mortality rate is high.Oncotherapy has three kinds of main means: surgical operation, radiotherapy and chemotherapy (chemotherapy), wherein preceding two kinds are the locality treatment, when tumor diffusion takes place and shifts, take chemotherapy usually.And mostly chemotherapeutics commonly used clinically is the hydrophobic type medicine, and the dissolubility in water is very low, has limited their application.The micromolecule surfactants that adopt are to hydrophobic type chemotherapeutics emulsifying solubilising more during clinical use, but this dosage form blood stability is very poor, and the adjuvant toxic and side effects of interpolation is also very big, and release behavior is not easy control.Polymer micelle is a kind of novel medicament carrier, and it is that autonomous dress forms in water is that the kernel hydrophilic section is the nucleocapsid structure of shell with the hydrophobicity section by amphipathic copolymer, and the drug loading of hydrophobic type is in micellar nuclear.Polymer micelle can the load hydrophobic drug and the dissolubility of significantly increasing medicament, improve bioavailability of medicament.
Conventional micelle medicine carrying system exists two large problems, the one, medicine too early release in blood circulation, the 2nd, the tumor locus medicine assemble and release concentration not enough.Medicine too early release in blood circulation makes effective dose reduce, and the whole body toxic and side effects also can increase simultaneously, and medicine can make curative effect of medication reduce greatly in tumor locus gathering or release concentration inadequately.The release of medicine when therefore reducing body-internal-circulation, discharging medicine again behind the control medicine-carried system arrival tumor cell is a kind of effective means that reduces the whole body toxic and side effects, improves the therapeutic effect of medicine.
Avoid the too early release of medicine in blood, will improve the drug release behavior, this respect environmental response type micelle advantage is fairly obvious.Wherein acid-sensitive type micelle is crucial a kind of.The physiological environment pH value that has been found that blood in the medical research is about 7.4, and lysosomal pH value then is 4.5 ~ 5.0 in the cell.According to the difference of pH value under this two states, design acid-sensitive type micelle and can accomplish that micelle is stablized medicine in blood and do not discharge, and after arriving Cytolysosome, medicine just begins to discharge the performance curative effect.So just can reduce medicine and in blood, discharge the toxic and side effects of bringing, avoid the loss of medicine in blood circulation again.
Solve medicine adopts targeting modification inadequately usually at the aggregate concentration of tumor locus medicine transmission system.Introduce on polymer micelle surface tumor cell surface or tumor tissues blood vessel surface cross express its receptor part as targeted molecular (like folic acid, polypeptide or antibody etc.); Strengthen the endocytosis of tumor cell, thereby improve aggregate concentration and the burst size of medicine at tumor locus to medicine-carried system.
In addition, in oncotherapy, realize that real-time monitoring will have very significant meaning to tumor treatment.During the orthopaedic surgical operations treatment, monitoring in real time can make excision more thorough to accurate positioning tumor position.If can realize real-time monitoring in the chemotherapy, then make things convenient for chemotherapy effect is estimated.Many employing nuclear magnetic resonance, NMR video pictures or ultrasonoscopy are monitored in real time at present clinical.But nuclear magnetic resonance, NMR video picture or ultrasonoscopy are just estimated tissue; The appearance of fluorescence imaging technology can be studied people to tumor on cellular level, make things convenient for pair cell inner structure, cell-cell interaction, cellular signal transduction to carry out more deep understanding.The R and D of fluorescence imaging technology and fluorescent probe have two big difficulties: the one, and can't effectively overcome cell and cover what labelled molecule was signaled at the autofluorescence of visible region; The 2nd, the fluorescence organic dye molecule is prone to photobleaching takes place, and can't realize preferably the long-time fluorescent labeling of research molecule is observed.Quantum dot has unique optical property as novel fluorometric reagent: wide excitation wavelength range and emission wavelength is very narrow, fluorescence lifetime long, do not allow to be subject to other signal disturbs size that can be through controlling it by anti-photobleaching, emission wavelength and forms and adjust and its size characteristics also makes it be very suitable in bioluminescence forms images, doing fluorescent marker.
Summary of the invention
The objective of the invention is to overcome the too early release problem of medicine-carried system in blood circulation, a kind of target polymer micelle with acid-sensitive subsurface stratum is provided.
In order to overcome the deficiency of prior art, the present invention adopts following technical scheme:
Target polymer micelle with acid-sensitive subsurface stratum of the present invention is a nucleocapsid structure; The hydrophilic section tip of shell is connected to the targeting ligand molecular; The hydrophobic section load hydrophobic anticancer drug of kernel; But exist the acid-sensitive response subsurface stratum of one deck load negative electricity quantum dot between kernel and the shell, acid-sensitive layer is hydrophilic when pH 5.0, and acid-sensitive layer is hydrophobic when pH 7.4.
The used polymer of micelle is the triblock copolymer that the Polyethylene Glycol hydrophilic section of folic acid functionalization, acid-sensitive interlude and hydrophobic section are formed.When pH 5.0 with the hydrophobic type drug loading in micelle nuclear, acid-sensitive period of this moment is hydrophilic, not carrying medicament; PH was transferred to 7.4 o'clock, acid-sensitive section hydrophobic formation layer protective layer stops medicine when pH 7.4, to discharge, and plays the problem that prevents that medicine from discharging too early in blood circulation again.After medicine-carried system got into tumor cell through folate-mediated endocytosis, acid-sensitive layer was hydrophilic under the sour environment of the lysosome pH 4.5 ~ 5.0 of tumor cell, and medicine begins from micelle nuclear, to discharge, the performance therapeutical effect.
Described acid-sensitive interlude can be to gather aminoacyl diisopropyl ethylenediamine or polyacrylamide diisopropyl ethylenediamine, and hydrophobic section can be hydrophobic type molecules such as polyester or cholic acid.Adopt folic acid polyethylene glycol-Radix Asparagi acyl diisopropyl ethylenediamine-cholic acid triblock copolymer (FA-PEG-P (Asp-dip)-CA) in the embodiment of the invention.
Described hydrophobic type medicine can be paclitaxel hydrophobic type antitumor drug such as (PTX).
Target polymer micelle with acid-sensitive subsurface stratum of the present invention can provide real time monitoring function in chemotherapy, be the medicine-carried system that has the video picture function concurrently.The video picture function of medicine-carried system utilizes acid-sensitive intermediate layer this characteristic of positively charged when pH 5.0 to realize.In pH 5.0 medicine carryings, add electronegative quantum dot, through electrostatic interaction quantum dot is loaded to acid-sensitive layer, again pH is transferred to 7.4, acid-sensitive section hydrophobic skin at carrier micelle nuclear forms layer protective layer, and the negative electricity quantum dot is wrapped in protective layer.Acid-sensitive intermediate layer load negative electricity quantum in anti-release is too early lighted the function that can monitor fluorescence imaging in real time.
Described negative electricity quantum dot can be carboxylated CdSe/ZnS or other electronegative quantum dot.
Another object of the present invention provides the above-mentioned micellar method for preparing of target polymer with acid-sensitive subsurface stratum; Concrete steps are following: the end capped polyethylene glycol-Radix Asparagi of 10 weight portion folic acid acyl diisopropyl ethylenediamine-cholic acid triblock copolymer and 1 weight portion hydrophobic anticancer drug are dissolved in the oxolane; Under ultrasonication, slowly add in the acidic buffer, oxolane is vapored away naturally, filter the hydrophobic drug aggregation is removed; With centrifugal ultrafiltration pipe (MWCO=100 kD) centrifugal ultrafiltration 3 times; Add negative electricity quantum dot CdSe/ZnS (100 μ L, 8 μ moL/L), behind the mixing static 30 minutes; Regulate pH to 7.4, the centrifugal quantum dot of removing not load of 6000 r/min.The gained micelle shape is even, and it is 53.4 ± 0.8 nm that mean diameter is measured in light scattering, and it is about 40 nm that transmission electron microscope is measured particle diameter.
Compared with prior art, the present invention has following beneficial effect:
The target polymer micelle with acid-sensitive subsurface stratum of our design is to be that acid-sensitive section the triblock copolymer of folic acid functionalization self assembly under the condition of pH 5.0 forms through interlude.Utilize micelle nuclear load hydrophobic drug; Exist the intermediate layer of one section acid-sensitive sense between micelle nuclear and the shell; Under the condition of pH 5.0; Acid-sensitive section positively charged can pass through electrostatic interaction load elecrtonegativity quantum dot, and under the environment of pH 7.4, acid-sensitive section hydrophobic skin at micelle nuclear forms layer protective layer; The negative electricity quantum dot is wrapped in protective layer, and this layer can play the effect that prevents that medicine from discharging under the condition of pH 7.4 simultaneously.After micelle gets into tumor cell through folic acid targeting mediation, acid-sensitive section protonated hydrophilic that becomes under the condition of the low pH 5.0 of Cytolysosome, this moment, medicine can discharge the performance curative effect from micelle nuclear.This system is the initiatively too early Multifunction micelle medicine carrying system that discharges of targeting, video picture and anti-medicine of collection.Target polymer micelle with acid-sensitive subsurface stratum of the present invention can not only solve the too early release problem of medicine-carried system in blood, has improved aggregate concentration and the rate of release of medicine at tumor locus, can also realize real-time monitoring simultaneously.Oncotherapy and novel pharmaceutical formulation aspect there are very big researching value and application prospect.
Description of drawings
Fig. 1 is the dynamic light scattering scattergram of micelle particle diameter among the embodiment 3.The result shows PEG-P (Asp-dip)
17The micelle particle diameter that-CA forms is 53.4 ± 0.8 nm.
Fig. 2 is a micelle transmission electron microscope shape appearance figure among the embodiment 3.Quantum dot during pH 7.4 is evenly distributed on the skin of micelle kernel, and the micelle particle diameter is (40 nm) evenly, has explained that the result shows that quantum dot by the loading in the micelle sample of success, has also proved that we have acid-sensitive subsurface stratum by the micelle of design really simultaneously.
Fig. 3 is micelle vitro drug release figure among the embodiment 4; The acid-sensitive interlude ground number of repeat unit of used polymer is 17 among the embodiment; PEG-P (Asp-dip)-CA paclitaxel loaded back sample burst size in the buffer of pH 7.4 is very low, and in the buffer of pH 5.0, all shows the rapid release behavior.Medicine release in vitro result shows that the micelle volume that is designed has the function that prevents that medicine from discharging too early in pH 7.4.
Fig. 4 is the fluorescence release graphics of micelle when pH 5.0 of load FDA and QD among the embodiment 5.When the result is illustrated in pH 5.0, prolong in time, FDA discharges gradually, and its fluorescence intensity strengthens gradually; QD is slowly cancellation of fluorescence gradually in sour environment, and its fluorescence intensity weakens gradually.
Fig. 5 is the fluorescence release graphics of micelle when pH 7.4 of load FDA and QD among the embodiment 5.When the result is illustrated in pH 7.4, prolong in time, FDA and QD are stabilized in the micelle nuclear and do not discharge, and the fluorescence intensity of FDA and QD is constant basically.
Fig. 6 be among the embodiment 6 different micelle samples to the toxotest figure of Bel-7402 cell.Sample is respectively: FA-micelles, PTX/micelles, PTX/FA-micelles, PTX/FA-micelles/QD; Wherein FA-micelles is the hungry area bundle, and the experimental point series concentration that contains paclitaxel in back three's sample is (5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM), and incubation time is 36 h.The result shows that the cytotoxicity of hungry area bundle FA-micelles is minimum; The cytotoxicity of non-targeting group PTX/micelles takes second place; The more non-targeting group of the cytotoxicity of targeting group PTX/FA-micelles is high; Simultaneously the sample P TX/FA-micelles/QD of load P TX and QD is suitable basically with targeting group PTX/FA-micelles sample, and the toxicity that sample behind the introducing QD be described is less than increase.
The specific embodiment
Following examples have been done detailed description to the present invention.
Triblock copolymer FA-PEG-P (the Asp-dip)-CA's with acid-sensitive interlude of modified with folic acid is synthetic
(1) one end is Polyethylene Glycol (Allyl-PEG-OH) synthetic of hydroxyl for the pi-allyl other end
With in exsiccant reaction bulb, stirring 15 minutes behind the tetrahydrofuran solution of potassium naphthalide (4 mL) and propenyl (0.75 mL) mix homogeneously.The tetrahydrofuran solution (containing 1.5 g hexaoxacyclooctadecane-6-6 and 5 mL anhydrous tetrahydro furans) that under argon shield, adds anhydrous tetrahydro furan (20 mL) and hexaoxacyclooctadecane-6-6 then; Stirring after 15 minutes places cryosel to bathe cooling in mixture; And slowly feed exsiccant oxirane; Keep low temperature 24 hours so that polyreaction continues to carry out, continue reaction 72 hours under the room temperature.
(2) one ends are amino Polyethylene Glycol (Allyl-PEG-NH for the pi-allyl other end
2) synthetic
Take by weighing Allyl-PEG-OH (Mn=2000 Da; 20g) in exsiccant there-necked flask (250 mL), 80 ℃ of dry 4 h of evacuation add anhydrous methylene chloride (100 mL), pyridine (50 mL) and paratoluensulfonyl chloride (TsCl then; 5g), lucifuge is reacted 24 h under the room temperature.Reactant liquor is with HC1 (3 mol L-1) extracting and washing, and organic layer slowly is added drop-wise to solution in a large amount of absolute ethers with NaHCO3 (5g) washing, and vacuum drying gets white powder PEG
2k-OTS.
Above-mentioned white powder (10 g) and strong aqua ammonia (500 mL, 25%) are placed the 1000mL container, and reaction is 5 days under the room temperature.Use CH with volume
2C1
2After the extraction with its with volume NaOH solution (1.0 mol L
-1) fully mix and stir 2 h, the organic layer water is washed till neutrality, vacuum drying gets Allyl-PEG
2k-NH
2It is 80% that nuclear-magnetism calculates amino conversion ratio.
(3) benzyloxycarbonyl group aspartic acid anhydride (BLA-NCA) is synthetic
(250 mL) asparagine acid benzyl ester (25 g) of packing in exsiccant there-necked flask adds new ethyl acetate (100 mL) stirring and dissolving of steaming then.Be transferred in the exsiccant ground constant pressure funnel after in addition three surpalites (13.2948 g) being dissolved with new ethyl acetate (40 mL) of steaming.After the ethyl acetate solution temperature of asparagine acid benzyl ester risen to 40 ℃, stir and slowly drip the ethyl acetate solution of triphosgene, question response liquid changes transparent back and stirs ripening 1 hour, and logical argon bubbling is removed the hydrogen chloride that solution is produced.
Cross the unreacted asparagine acid benzyl ester of elimination, carry out reprecipitation new the steaming in the normal hexane, fully shake was put into refrigerator freezing at least 12 hours when crystal to be had is separated out.Freezing back is sucking filtration fast, and with the normal hexane washing for several times, vacuum drying obtains the BLA-NCA crystal.
(4) polyethylene glycol-aspartic acid copolymer (PEG-PBLA) is synthetic
With PEG
2k-NH
2(1.0 g) adds in the reaction bulb (50 mL), and 70 ℃ of vacuum drying 4 h with DMF (10 mL) dissolving, add DMF (2 mL) solution that contains 1.9 g BLA-NCA.React 24 h down in 35 ℃ behind the mix homogeneously.In cold diethyl ether, filtration is drained with solution precipitation, gets PEG
2k-PBLA
3.5k
(5) polyethylene glycol-aspartic acid-cholic acid (PEG-PBLA-CA) is synthetic
Take by weighing cholic acid (0.05 g), EDC (0.03 g) and NHS (0.02 g) add DMF/HCCl in reaction bulb (50 mL)
3After (V:V=1:1,4 mL) dissolving, stir 30min, add PEG2k-PBLA3.5k (0.55 g), room temperature reaction 24h.Solution precipitation in dehydrated alcohol, is filtered, washing, vacuum drying obtains PEG-PBLA-CA.
(6) (PEG-P's (Asp-dip)-CA) is synthetic for polyethylene glycol-Radix Asparagi acyl diisopropyl ethylenediamine-cholic acid
Take by weighing PEG-PBLA-CA (0.3 g) and add in the reaction bulb (50 mL),, add dip (4 mL), react 48h down in 35 ℃ of stirrings with DMF (1.0 mL) dissolving.Solution precipitation in absolute ether, is filtered, and vacuum drying gets PEG-P (Asp-dip)-CA.
(7) (FA-PEG-P's (Asp-dip)-CA) is synthetic for the polyethylene glycol-Radix Asparagi acyl diisopropyl ethylenediamine-cholic acid of folic acid functionalization
(0.3 g 1eq) places two mouthfuls of flasks, 70 ℃ of vacuum drying 1 h to take by weighing PEG-P (Asp-dip)-CA; Add DMF (5 mL) dissolving, (80 mg are 15eq.) with azodiisobutyronitrile (6 mg to add the mercaptoethylmaine hydrochlorate then; 0.8eq); At 70 ℃ of reaction 24 h, be deposited in the cold absolute ether, get NH
2-PEG-P (Asp-dip)-CA.
Folic acid (0.044 g) adds in the two-mouth bottle (50 mL), behind vacuum drying 4 h, adds exsiccant DMSO (15 mL); Add N-hydroxy-succinamide 0.023g (2eq) then successively; N, N'-dicyclohexylcarbodiimide 0.041g (2eq), lucifuge is reacted 12 h; The centrifugal DCU that removes generation, activatory then folic acid joins and is dissolved with NH
2In the 5 ml DMF solution of-PEG-P (Asp-dip)-CA (0.3 g); Be transferred to 8 to pH value of reaction system with triethylamine then; Lucifuge reaction 24h, with the bag filter of the MWCO=1k Da 48h that in distilled water, dialyses, lyophilization gets yellow powder shape product FA-PEG-P (Asp-dip)-CA.
The micellar preparation of target polymer with acid-sensitive subsurface stratum
(1) load QD micelle sample preparation: the mixture of FA-PEG-P (Asp-dip)-CA (2.0 mg) and PEG-P (Asp-dip)-CA (8.0 mg) is dissolved in the oxolane (THF, 1.0 mL), is added drop-wise to PBS (10 mL under the ultrasonic situation; PH=5.0) in; Stirred overnight vapors away THF naturally, and using the aperture is to add negative electricity quantum dot CdSe/ZnS (100 μ L behind the membrane filtration of 0.22 μ m; 8 μ moL/L); Behind the mixing static 30 minutes, regulate pH to 7.4, the centrifugal quantum dot of removing not load of 6000r/min.
(2) load QD and the sample preparation of paclitaxel micelle: mixture and the PTX (1.0 mg) of FA-PEG-P (Asp-dip)-CA (2.0 mg) and PEG-P (Asp-dip)-CA (8.0 mg) are dissolved in the oxolane (THF, 1.0 mL), are added drop-wise to PBS (10 mL under the ultrasonic situation; PH=5.0) in, stirred overnight vapors away THF naturally; The use aperture is that the membrane filtration of 0.45 μ m is removed the PTX aggregation; With centrifugal ultrafiltration pipe (MWCO=100 kD) centrifugal ultrafiltration 3 times, add negative electricity quantum dot CdSe/ZnS (100 μ L, 8 μ moL/L); Behind the mixing static 30 minutes, regulate pH to 7.4.The centrifugal quantum dot of removing not load of 6000r/min.
(3) load QD and fluorescein(e) diacetate (FDA) micelle sample preparation: mixture and the FDA (1.0 mg) of FA-PEG-P (Asp-dip)-CA (2.0 mg) and PEG-P (Asp-dip)-CA (8.0 mg) are dissolved in the oxolane (THF, 1.0 mL), are added drop-wise to PBS (10 mL under the ultrasonic situation; PH=5.0) in, stirred overnight vapors away THF naturally; The use aperture is that the membrane filtration of 0.45 μ m is removed the FDA aggregation; With centrifugal ultrafiltration pipe (MWCO=100 kD) centrifugal ultrafiltration 3 times, add negative electricity quantum dot CdSe/ZnS (100 μ L, 8 μ moL/L); Behind the mixing static 30 minutes, regulate pH to 7.4.The centrifugal quantum dot of removing not load of 6000r/min.
Embodiment 3Micellar particle diameter of target polymer and pattern testing experiment with acid-sensitive subsurface stratum:
The micellar size of load QD adopts the dynamic light scattering system to measure, and test result is seen Fig. 1; Its form is then observed definite through transmission electron microscope, test result is seen Fig. 2.
External release test with target polymer micelle (is example with paclitaxel loaded micelle) of acid-sensitive subsurface stratum
Make the extracorporeal releasing experiment of paclitaxel of the method for dialysis.Paclitaxel loaded PEG-P (Asp-dip)-CA micelle of newly doing is divided into two parts, and it is 5.0 that portion is kept its pH value, and another part transferred to 7.4.Again the sample of each pH value is divided into three parts (parallel laboratory tests) and installs in the bag filter that molecular cut off is 14000 Da, bag filter is put into the PBS buffer of the identical pH value of 100 mL.Place 37
℃Shaking table in, under the rotating speed of 75 r/min, the point in time sampling of setting is added the fresh buffer of equal volume afterwards.With the concentration of HPLC test sample, calculate the cumulative release amount of different time points.Wherein the used chromatographic column model of HPLC is the Ultimate of welch materials company
AQ-C18,5 μ m, 4.6 * 250mm, mobile phase is acetonitrile and water (V:V=50:50), flow velocity is 1.0 mL min
-1, column temperature is 40 ℃, the injected sample amount is 20 μ L.With time-the cumulative release amount is figure, obtains the curve that external PTX discharges from micelle, test result such as Fig. 3.
External fluorescence release test with target polymer micelle (micelle with load FDA and QD is an example) of acid-sensitive subsurface stratum
Get pH and be 7.4 load FDA and the 1.0 mg mL of QD
-1Micellar solution 1 mL, regulating pH is 5.0, is settled to 2 mL.With fluorescent spectrophotometer assay fluorescence intensity variation diagram in time, the excitation wavelength of fluorescent scanning is 430 nm, excites slit to be set to 10.0 nm, and the emission slit is set to 10.0 nm, and scanning speed is 500 nm min
-1Test result such as Fig. 4.
Using same parameter detecting pH is 7.4 load FDA and the 0.5 mg mL of QD
-1The fluorescence intensity of micellar solution is variation diagram in time, test result such as Fig. 5.
Embodiment 6In vitro toxicity test with polymer micelle of acid-sensitive subsurface stratum:
Detect of the influence of micelle sample with tetrazolium salts colorimetry (MTT) to the Bel-7402 ability of cell proliferation.All experimental result triplicates.With the Bel-7402 cell with 1 * 10
3The every hole of cell is inoculated in 96 orifice plates; Then with Bel-7402 cell 37 ℃ of cultivation 12h in 100 μ L RPMI 1640 (containing 10%FBS) culture medium; Then change the culture medium that does not contain FA and serum into; And add the different samples system and cultivate 36 h again, wherein sample sets has: 1) hungry area bundle (FA-PEG-P (Asp-dip)-CA) FA-micelles, 2) PEG-P (Asp-dip)-CA micelle paclitaxel loaded (PTX-micelles); 3) micelle of folic acid targeting paclitaxel loaded (PTX/FA-micelles), the 4) micelle of folic acid targeting paclitaxel loaded and quantum dot (PTX/FA-micelles/QD) simultaneously.At last, culture medium is changed into 150 μ L RPMI 1640 and adds 5 mg mL
-1MTT solution 50 μ L, 37 ℃ are continued to hatch 4 h.Stop cultivating, culture supernatant absorption in the hole is discarded, in every hole, add 150 μ L DMSO, shake 5 min, with the absorbance of each hole of enzyme-linked immunosorbent assay instrument monitoring at OD570 nm.With the blank group is benchmark, calculates cell proliferation rate.Test result such as Fig. 6.
Claims (9)
1. target polymer micelle with acid-sensitive subsurface stratum; Described nano-micelle structure is a nucleocapsid structure; The hydrophilic section tip that it is characterized in that shell is connected to the targeting ligand molecular, the hydrophobic section load hydrophobic anticancer drug of kernel, but exist the acid-sensitive response subsurface stratum of one deck load negative electricity quantum dot between kernel and the shell; Acid-sensitive layer is hydrophilic when pH 5.0, and acid-sensitive layer is hydrophobic when pH 7.4.
2. target polymer micelle as claimed in claim 1; It is characterized in that said micelle is made up of the Polyethylene Glycol-acid-sensitive section-hydrophobic section triblock copolymer of targeted molecular functionalization; Acid-sensitive section for gathering aminoacyl diisopropyl ethylenediamine or polyacrylamide diisopropyl ethylenediamine, and hydrophobic section is polyester or cholic acid.
3. target polymer micelle as claimed in claim 1 is characterized in that said targeting ligand molecular is a folic acid.
4. target polymer micelle as claimed in claim 1 is characterized in that described acid-sensitive section is the polymer of diisopropyl ethylenediamine functionalization, when pH 5.0 acid-sensitive section hydrophilic, when pH 7.4 acid-sensitive section hydrophobic.
5. target polymer micelle as claimed in claim 1 is characterized in that said nano-micelle for spherical, and diameter is 50 nm.
6. target polymer micelle as claimed in claim 1 is characterized in that said hydrophobic anticancer drug is a paclitaxel.
7. described micellar method for preparing of target polymer of claim 1 with acid-sensitive subsurface stratum; It is characterized in that comprising the steps: the end capped polyethylene glycol-Radix Asparagi of 10 weight portion folic acid acyl diisopropyl ethylenediamine-cholic acid triblock copolymer and 1 weight portion hydrophobic anticancer drug are dissolved in the oxolane; Under ultrasonication, slowly add in the acidic buffer, oxolane is vapored away naturally, filter the hydrophobic drug aggregation is removed; With centrifugal ultrafiltration pipe (MWCO=100 kD) centrifugal ultrafiltration 3 times; Add negative electricity quantum dot CdSe/ZnS (100 μ L, 8 μ moL/L), behind the mixing static 30 minutes; Regulate pH to 7.4, the centrifugal quantum dot of removing not load of 6000 r/min.
8. the micellar method for preparing of target polymer with acid-sensitive subsurface stratum as claimed in claim 7 is characterized in that said micelle will be at carrying medicament under the acid condition, and described acidic buffer is PBS (pH 5.0).
9. the micellar method for preparing of target polymer with acid-sensitive subsurface stratum as claimed in claim 7 is characterized in that, said filtration employing aperture is that the filter membrane of 0.45 μ m filters.
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| CN102796235A (en) * | 2012-06-07 | 2012-11-28 | 华东理工大学 | Copolymer based on environmental response and preparation method thereof |
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| CN106902078A (en) * | 2015-12-21 | 2017-06-30 | 复旦大学 | One kind contains taxol micelle nano cluster and preparation method thereof |
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| CN102796235B (en) * | 2012-06-07 | 2014-04-23 | 华东理工大学 | Copolymer based on environmental response and preparation method thereof |
| CN103059297A (en) * | 2012-12-28 | 2013-04-24 | 武汉大学 | Multifunctional degradable polyasparaginate modified polymer and preparation method thereof |
| CN106902078A (en) * | 2015-12-21 | 2017-06-30 | 复旦大学 | One kind contains taxol micelle nano cluster and preparation method thereof |
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