CN103251955B - A kind of macromolecule target medicine carrier for tumor of bladder perfusion therapy and preparation method thereof - Google Patents
A kind of macromolecule target medicine carrier for tumor of bladder perfusion therapy and preparation method thereof Download PDFInfo
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- CN103251955B CN103251955B CN201210037859.0A CN201210037859A CN103251955B CN 103251955 B CN103251955 B CN 103251955B CN 201210037859 A CN201210037859 A CN 201210037859A CN 103251955 B CN103251955 B CN 103251955B
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Abstract
The invention discloses a kind of targeting macromolecule medicine-carried system for tumor of bladder perfusion therapy and preparation method thereof.Described targeting macromolecule medicine-carried system, be with aliphatic polyester be hydrophobic phase, the poly(ethylene oxide) of end-functionalization is aqueous-favoring block copolymer micelle medicine-carried system, antitumor drug is wrapped in the hydrophobic inner core of micelle or with the modifiable functional group in hydrophilic segment and carries out chemical bonding.By carrying out the hydrophilic segment end group of copolymer modifying the macromolecule medicine-carried system that can obtain having target function and fluorescent tracing effect.This medicine-carried system has significant targeting to bladder cancer cells, significantly can suppress the growth of bladder cancer cells, and realizes stable and drug releasing rate that is that continue, shows its important application prospect in tumor of bladder perfusion therapy.
Description
Technical field
The present invention relates to a kind of macromolecule target medicine carrier for tumor of bladder perfusion therapy and preparation method thereof.
Background technology
Tumor of bladder is disease common in Urology Surgery, at present, the whole world has the tumor of bladder case newly-increased more than 1,200 ten thousand every year, wherein 5,400,000 occurs in developed country, 6700000 cases are at developing country (MartinePloeg, KatjaK.H.Aben, LambertusA.Kiemeney, Thepresentandfutureburdenofurinarybladdercancerintheworl d, WorldJournalofUrology, 2009,27 (3), 289-293).The tumor of bladder tumor disease that sickness rate is very high in China is also urinary system and male genital.In bladder cancer, superficial bladder cancer (superficialbladdercancer) accounts for 60-80%.After traditional therapeutic modality is mainly ocal resection, ancillary drug is treated, due to the physiological structure that bladder is special, and the general drug toxicity adopting intravesical drug conveying (intravesicaldrugdelivery) i.e. perfusion therapy to avoid whole body.But because the complex biological scholarship and moral conduct of superficial bladder tumor is, the postoperative easy recurrence of 50-70% superficial tumor, wherein the recurrence of 30-40% causes the enhancing of increasing of malignancy or wetting capacity, makes the difficulty for the treatment of further sharply increase.Therefore, the therapeutic effect how improving superficial bladder tumor is most important for the treatment level improving bladder cancer.
At present, more common in bladder instillation to treat chemicals has thiophene to replace to send (Thiotepa), mitomycin (MitomycinC), amycin (Doxorubicin), epirubicin (Epirubicin) etc.Although these small-molecule drugs show certain therapeutic effect in the perfusion therapy of tumor of bladder, but, because small-molecule drug is easily got rid of with urine, short in the intravesical time of staying, thus need repeatedly to pour into, not only expense improves, and side effect also can be caused to increase, more can cause the Drug resistance of tumor, bring difficulty to follow-up treatment.Meanwhile, due to the physiological characteristics that bladder is special, can small-molecule drug be caused through bladder by systemic Absorption, produce toxic and side effects.
Macromolecule medicine-carried system is in widespread attention due to the advantage of its uniqueness, the infiltration at tumor tissues position with enhancing that the nanometer size effect that macromolecule carrier has uniqueness causes and stop effect (enhancedpermeationandretention is called for short EPR effect).Simultaneously, due to the multiformity of chemical constitution, macromolecule can connect the part (as antibody, polypeptide, saccharide, somatomedin etc.) with tumor cell surface receptor with high affinity, the specific binding of receptor and part is utilized to realize the active targeting of macromolecule medicine-carried system to tumor, thus increase therapeutic effect, reduce toxic and side effects.Therefore, macromolecule medicine-carried system has become one of focus studied in drug delivery system.
The applied research of macromolecule medicine-carried system in tumor of bladder perfusion therapy, bibliographical information is little.Some researchs mainly concentrate on (the ShelleyMDetal.Intravesicaltherapyforsuperficialbladderca ncer:Asystematicreviewofrandomisedtrialsandmeta-analyses .CancerTreatmentReviews.2010 such as selection, action time, dosage of medicine, 36,195-205).Have report to utilize chitosan to apply PCL, PLL prepared the nanoparticle of embedding mitomycin and achieved bladder instillation to treat and achieve certain effect (EremBilensoya, CanSarisozena, Gunesetal.Intravesicalcationicnanoparticlesofchitosanand polycaprolactoneforthedeliveryofMitomycinCtobladdertumor s, InternationalJournalofPharmaceutics.2009,371,170-176).Also report is had to be applied in the treatment of tumor of bladder by various EGF-R ELISA, but its lower responsiveness limits its application (PeterC.Black, PiyushK.Agarwaletal, Targetedtherapiesinbladdercancer-anupdate, UrologicOncology:SeminarsandOriginalInvestigations, 2007,25,433-438).Also have report that CS-PAA magnetic polymer microsphere is carried out zoopery and obtain certain effect, but need externally-applied magnetic field, bring certain trouble to treatment.
Summary of the invention
The invention provides target medicine carrier of a kind of tumor of bladder perfusion therapy and preparation method thereof.
The target medicine carrier of tumor of bladder perfusion therapy provided by the present invention is the micelle of amphipathic nature block polymer, described micelle has nucleocapsid structure, its shell is made up of the hydrophilic segment in described amphipathic nature block polymer, and kernel is made up of the hydrophobic segment in described amphipathic nature block polymer; Described hydrophilic segment is the poly(ethylene oxide) with function end group, described function end group is following 1) or 2): the biologically functional molecule 1) bladder cancer cells to targeting, 2) bladder cancer cells is had to biologically functional molecule and the fluorescence molecule of targeting; Described hydrophobic segment is biodegradable aliphatic polyester.
Wherein, described micelle is of a size of 10 ~ 500nm.
The number-average molecular weight of described amphipathic nature block polymer is 1,500 ~ 600,000, wherein aliphatic polyester segment is 0.01 ~ 1,000 with the ratio of the mean molecule quantity of poly(ethylene oxide) segment, and the number-average molecular weight of aliphatic polyester segment is 1,000 ~ 500,000, the number-average molecular weight of poly(ethylene oxide) segment is 500 ~ 100,000.
Described aliphatic polyester specifically can be selected from the copolymer of homopolymer that any one monomer following formed and the formation of at least two kinds of monomers one or more: caprolactone (CL), lactide (LA) and Acetic acid, hydroxy-, bimol. cyclic ester (GA).Described lactide comprises meso-lactide, levorotatory lactide and dextrorotation lactide.
The hydrophilic outer shell of above-mentioned micelle is connected to various functional molecular by chemical modification, and hydrophobic inner core then can the various antitumor drug of physically trapping.Micellar surface connects various targeted molecular, there is double targeting, comprise (1) active targeting, the specific receptor etc. of tumor cell surface is utilized to design micellar surface and make itself and tumor cell have specific binding and reach the targeting to tumor cell, make pharmaceutical carrier be enriched in tumor locus and enter tumor cell so that realize drug release containment tumor cell growth, realize the targeted therapy of tumor; (2) passive target, prepared micelle size, between 20 ~ 200nm, is easy to enter tumor locus and rests on tumor locus, i.e. the effect of EPR passive target.In addition, connect the fluorescence molecule such as Fluorescein isothiocyanate, RB 200 by the functional group at micellar surface hydrophilic segment thus make macromolecule medicine-carried system have fluorescent tracing effect, conveniently observing it at intracellular distribution, quantitatively characterizing and evaluating drug effect.
Described targeted molecular and the growth factor receptorses etc. such as folic acid, linear polypeptide (as RGD) or ring type polypeptide are comprised to the biologically functional molecule that bladder cancer cells has a targeting, to make micellar carrier, there is active targeting function to bladder cancer cells.
Described fluorescence molecule comprises the fluorescence signaling molecule such as Fluorescein isothiocyanate (FITC), RB 200 (RBITC), has fluorescent tracing function to make micellar carrier.
When with the micelle of above-mentioned amphipathic nature block polymer for the medicine-carried system of carrier for the preparation of tumor of bladder perfusion therapy time, the antitumor drug of institute's load is hydrophobic drug, and it is embedded in the hydrophobic inner core of micelle by physical action in preparation process; The antitumor drug of institute's load is hydrophilic medicament, and it is by changing its hydrophilic and make its hydrophobic then physically trapping or by carrying out chemical bonding and load with the modifiable functional group in hydrophilic segment end group.Described antitumor drug specifically can be selected from amycin (Doxrubin), ametycin (MitomycinC), paclitaxel (Paclitaxel), pirarubicin (pirarubicin), hydroxy camptothecin (Hydroxycamptothecin), thio-tepa (Thiotepa), bacillus calmette-guerin vaccine (Calmette-Guerinvaccine), mitoxantrone (MitoxantroneHcl), second AY-62013 (Ethoglucid), gemzar (Gemcitabin), gemcitabine (Gemcitabine), in cisplatin (Cisplatin) one or more etc.
Macromolecule targeted drug micellar carrier of the present invention, the biodegradable aliphatic polyester using FDA to ratify to use safely in human body and poly(ethylene oxide) (PEO) are raw material, synthesizing amphipathic block copolymer.
With aliphatic polyester as polycaprolactone (PCL), polylactide (PLA) and water-soluble polyethylene oxide (PEO) synthesizing amphipathic block copolymer PEO-b-PCL/PLA are example, and the preparation process as the Amphiphilic Block Copolymer Micelles of pharmaceutical carrier is as follows:
1, open loop anionic polymerisation synthesizing amphipathic block copolymer
The synthetic route of poly(ethylene oxide) and aliphatic polyester (comprising Poly-L-lactic acid, dextrorotation polylactic acid, PDLLA and polycaprolactone) block copolymer (PEO-b-PCL/PDLA/PLLA/PLA) is as follows:
With two (trimethyl silicane) nitrogen potassium ([(CH
3)
3si]
2nK) be initiator, under argon shield, by oxirane monomers (EO) and aliphatic ester monomer: caprolactone (CL) or lactide (LA), levorotatory lactide (DLA), one or several in dextrorotation lactide (LLA) carry out the block copolymer that anionic ring-opening polymerization obtains aliphatic ester and oxirane, as poly-(oxirane-b-caprolactone) block copolymer (PEO-b-PCL), poly-(oxirane-b-lactic acid) block copolymer (PEO-b-PLA), poly-(oxirane-b-caprolactone/Poly-L-lactic acid/dextrorotation polylactic acid/PDLLA) block copolymer (PEO-b-PCL/PDLA/PLLA/PDLLA).
The molecular structural formula of polymer used in the present invention is as follows:
2, the synthesis of the Amphipathilic block polymer of targeted molecular modification
Utilize the functional group of hydrophilic end in Amphipathilic block polymer as amino N H
2, carboxy CO OH, hydroxyl OH, methoxyl group CH
3o etc. carry out association reaction with targeted molecular folic acid (FA), polypeptide (as linear RGD or ring type polypeptide C (RGDfk)) etc., obtain the target product that targeted molecular is modified.
3, the synthesis of the amphipathic nature block polymer of fluorescence molecule modification
Utilize the functional group of hydrophilic end in Amphipathilic block polymer as amino N H
2, carboxy CO OH, hydroxyl OH, methoxyl group CH
3the fluorescence molecules such as O etc. and Fluorescein isothiocyanate (FITC), RB 200 (RBITC) carry out chemical bond reaction, obtain the target product that fluorescence molecule is modified.
4, the micelle of fluorescence and/or targeting modification is prepared
The amphipathic nature block polymer that the targeted molecular obtained in step 2 or step 2 and 3 and/or fluorescence molecule are modified is dissolved in organic solvent and forms oil phase, above-mentioned oil phase is slowly added drop-wise in aqueous phase under agitation, stirs dialysis removing organic solvent after 2 ~ 24 hours and obtain the micellar solution of fluorescence and/or targeting modification.
5, macromolecule medicine-carried system is prepared
By step 2) amphipathic nature block polymer modified of the targeted molecular that obtains or step 2) and 3) amphipathic nature block polymer of the fluorescence that obtains and targeted molecular modification is dissolved in organic solvent; Hydrophobic anticancer drug or the hydrophilic anti-tumor medicine after hydrophobic treatment being dissolved in described organic solvent and forming oil phase, or by hydrophilic anti-tumor medicine and step 1) modified functional group in the amphipathic nature block polymer prepared is dissolved in described organic solvent after carrying out chemical bonding and forms oil phase; Described oil phase is added drop-wise under agitation in excessive aqueous phase, stirs dialysis removing organic solvent after 2 ~ 24 hours and obtain polypeptide drug-loaded micelle solution.
Described targeted molecular and the growth factor receptorses etc. such as folic acid, linear polypeptide (as RGD) or ring type polypeptide are comprised to the biologically functional molecule that bladder cancer cells has a targeting.
Described fluorescence molecule comprises the fluorescence signaling molecule such as Fluorescein isothiocyanate (FITC), RB 200 (RBITC).
Targeting macromolecule medicine-carried system of the present invention is mainly used in the perfusion therapy of tumor of bladder, to bladder cancer cells, there is special targeting and to be enriched in around bladder cancer cells thus to enter tumor cell and discharge medicine, playing the therapeutical effect that it is good.The macromolecule medicine-carried system built by amphipathic nature block polymer that the present invention proposes, preparation method is simple, drug loading is high, to bladder cancer cells T24 cell, there is good targeting, and can significantly inhibition tumor cell growth so that kill tumor cell, and stable and drug releasing rate that is that continue can be realized, and this macromolecule medicine-carried system does not exist by problems such as protein adsorption in course of conveying, show its important application prospect in tumor of bladder perfusion therapy.
Accompanying drawing explanation
Fig. 1 is NH
2the synthetic route of-PEO-b-PCL.
Fig. 2 is the synthetic route of FITC-PEO-b-PCL.
Fig. 3 is the synthetic route of FA-PEO-b-PCL.
Fig. 4 be carrier micelle prepare schematic diagram.
Fig. 5 is the endocytosis of confocal laser scanning microscope cell to micelle; Green to be fluoresced by FITC fluorescence micelle; Wherein a is the result of FA-PEO-b-PCL and FITC-PEO-b-PCL mixed micelle and cell interaction, and b is OCH
3-PEO-b-PCL and the mixed micelle of FITC-PEO-b-PCL and the result of cell interaction.
Fig. 6 is the quantitative study result of micelle to T24 tumor cell targeting.
Fig. 7 is the toxicity test (MTT) of carrier micelle to T24 tumor cell.
Detailed description of the invention
Below by specific embodiment, the present invention will be described, but the present invention is not limited thereto.
Experimental technique described in following embodiment, if no special instructions, is conventional method; Described reagent and material, if no special instructions, all can obtain from commercial channels.
Embodiment 1: the synthesis of Amphipathilic block polymer
1) synthesis of PEO-b-PCL
Synthetic route as shown in Figure 1.Dewater oxirane monomers (EO) and oxolane (THF) solvent deoxygenation, reaction unit pump drainage three times, and applying argon gas is protected, and is placed in ice-water bath.The EO monomer transfer of cool drying in reaction tube, is experimentally designed molecular weight, under argon shield, by THF and two (trimethyl silicane) nitrogen potassium [(CH by the two prong of utilization
3)
3si]
2nK initiator joins in reaction tube successively.React after 2-5 days, under argon shield, add the THF solution of certain volume caprolactone monomer (CL).Continue reaction after 0.5-3 hour, add acetic acid cessation reaction, polymerizate is precipitated in excessive cold diethyl ether the amphipathic nature block polymer NH obtaining having Amino End Group
2-PEO-b-PCL.The structure of polymerizate and composition can pass through gel permeation chromatography (GPC) and proton nmr spectra (
1hNMR) jointly confirm.
Take the succinic anhydride of 0.020g, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) of 0.042g, 4-(the N of 0.0266g, N-dimethylamino) pyridine (DMAP) is dissolved in 15mlTHF, stirred at ambient temperature 0.5-8 hour.Pelleting centrifugation removing reaction produced, then adds the NH of 1.000g
2-PEO-b-PCL, continues reaction after 2-5 days, polymerizate is precipitated in excessive cold diethyl ether the amphipathic nature block polymer COOH-PEO-b-PCL obtaining end carboxyl.The structure of polymerizate and composition can by GPC and
1hNMR confirms.
2) synthesis of PEG-b-PLA
At stannous octoate (Sn (Oct)
2) under catalysis, carry out the ring-opening polymerisation of meso-lactide (DLLA) with terminal hydroxy group PEG for initiator, obtain amphipathic nature block polymer PEG
114-b-PDLLA
350.
Exemplary steps is as follows: the PEG-OH adding 0.100g in the reaction bulb of drying, the DLLA of 0.5041g, after deaeration in condenser dewaters, be filled with argon, adds 2.55ul, Sn (Oct) that concentration is 0.982mol/L
2toluene solution and solvent toluene 5ml.48h is reacted in the oil bath of putting into uniform temperature after sealing system.Polymerizate is precipitated among excessive cold diethyl ether/methanol mixed solvent, obtains product P EG
114-b-PDLLA
350.The structure of polymerizate and composition can by GPC and
1hNMR confirms.Respectively with levorotatory lactide (LLA) and dextrorotation lactide (DLA) for monomer, adopt same synthetic method and step, can PEG be obtained
114-b-PLLA
350and PEG
114-b-PDLA
350.
3) synthesis of PEG-b-P (CL-co-LA)
According in embodiment 1 2) method prepare PEG-b-P (CL-co-LA).In the reaction bulb of drying, add the PEG-OH of 0.100g, the LA (racemization, left-handed or dextrorotation) of 0.5041g and the CL of 0.2450g, after deaeration in condenser dewaters, be filled with argon, add 2.55ul, Sn (Oct) that concentration is 0.982mol/L
2toluene solution and solvent toluene 10ml.48h is reacted in the oil bath of putting into uniform temperature after sealing.Polymerizate is precipitated in excessive cold diethyl ether/methanol mixed solvent, obtains product P EG
114-b-P (CL
107-co-LA
350).The structure of this product and composition can by GPC and
1hNMR confirms.
4) synthesis of PEG-b-P (GA-co-LA)
According in embodiment 1 2) method prepare PEG-b-P (GA-co-LA).The PEG-OH of 0.100g is added, the Isosorbide-5-Nitrae-dioxane-2 of LA and 0.3270g of 0.5041g in the reaction bulb of drying, 5-acetyl butyryl (Acetic acid, hydroxy-, bimol. cyclic ester, GA), after deaeration in condenser dewaters, be filled with argon, add 2.55ul, Sn (Oct) that concentration is 0.982mol/L
2toluene solution and solvent toluene 10ml.48h is reacted in the oil bath of putting into uniform temperature after sealing.Polymerizate is precipitated in the mixed solvent of excessive cold diethyl ether/methanol, obtains product P EG
114-b-P (GA
270-co-LA
350).The structure of this product and composition can by GPC and
1hNMR confirms jointly.
5) synthesis of PEG-b-P (GA-co-LA-co-CL)
According in embodiment 1 2) method prepare PEG-b-P (GA-co-LA-co-CL).In the reaction bulb of drying, add the PEG-OH of 0.100g, the GA of CL and 0.3270g of LA, 0.2450g of 0.5041g, after deaeration in condenser dewaters, be filled with argon, add 2.55ul, Sn (Oct) that concentration is 0.982mol/L
2toluene solution and solvent toluene 10ml.48h is reacted in the oil bath of putting into uniform temperature after sealing.Polymerizate is precipitated in excessive cold diethyl ether/methanol mixed solvent, obtains product P EG
114-b-P (GA
270-co-LA
350-co-CL
107).The structure of this product and composition can by GPC and
1hNMR confirms jointly.
Embodiment 2: the synthesis of the polymer that fluorescence molecule and targeted molecular are modified
1) synthesis of FITC-PEO-b-PCL
Synthetic route as shown in Figure 2.Take 0.0780g Fluorescein isothiocyanate (FITC) to be dissolved in 5ml dimethyl sulfoxide (DMSO), join the NH that 1.0g is housed
2-PEO
5k-b-PCL
5kreaction tube in, in the oil bath of 20-100 DEG C react 72 hours, after secondary of dialysing in distilled water, centrifugal removing supernatant, obtains product FITC-PEO after lower sediment lyophilization
5k-b-PCL
5k.The structure of this product and composition can by GPC and
1hNMR confirms jointly.Adopt and use the same method, replace FITC with tetramethylrhodamine (RBITC), can RBITC-PEO-b-PCL be obtained.
2) synthesis of FA-PEO-b-PCL
Synthetic route as shown in Figure 3.By 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 0.0396g), N-hydroxy-succinamide (NHS, 0.0160g) be dissolved in the DMSO of 5mL with folic acid (FA, 0.0609g), activated carboxylic 2 hours.Then NH is added
2-PEO
5k-b-PCL
5k(0.5g), react 72 hours.After dialysing three days in intermediate water, centrifugal removing supernatant, again dissolves and removes insoluble matter in acetone, revolves steaming and obtains faint yellow product.The structure of this product and composition can by GPC and
1hNMR confirms jointly.
3) synthesis of GRGDY-PEO-b-PCL
First take the GRGDY (glycine-arginine-glycine-aspartic acid-tyrosine sequence) of 0.1132g, the N of 0.0486g, N '-carbonyl dimidazoles (CDI) is dissolved in the DMSO of 5ml, reacts after 0.5-4 hour, adds the NH of 1g
2-PEO
5k-b-PCL
5k, continue reaction 2-5 days.After in intermediate water, dialysis removes unreacted micromolecule in three days, lyophilization obtains product GRGDY-PEO
5k-b-PCL
5k.The structure of this product and composition can by GPC and
1hNMR confirms jointly.
4) synthesis of C (RGDfk)-PEO-b-PCL
First the COOH-PEO of 1g is taken
5k-b-PCL
5k, the NHS of the EDC of 0.2000g, 0.2300g is dissolved in the DMF of 10ml (DMF), stirs 2-12 hour with activated carboxyl under room temperature.Add the C (RGDfk) of 0.115mg, react 72 hours in the oil bath of 20-100 DEG C, after secondary of dialysing in distilled water, centrifugal removing supernatant, obtains product C (RGDfk)-PEO after lower sediment lyophilization
5k-b-PCL
5k.The structure of this product and composition can by GPC and
1hNMR confirms jointly.
Implementation column 3: the preparation of micelle
1) preparation of common micelle
Take the PEO that 10mg end group is amino, carboxyl or methoxyl group
5k-b-PCL
5kbe dissolved in the DMSO of 1mL, stir and make it fully dissolve in 0.5-12 hour.Get the above-mentioned solution of 100 μ l with microsyringe slowly to drip under the stirring of gentleness in the intermediate water of 10mL, continue to stir dialysis after 2-24 hour and obtain micellar solution to remove organic solvent in three days, be placed in 4 DEG C of refrigerators and save backup.
2) preparation of targeting micelle
Preparation process and condition the same, but polymer used is the FA-PEO of modified with folic acid
5k-b-PCL
5kor the GRGDY-PEO that line style is peptide modified
5k-b-PCL
5kor C (the RGDfk)-PEO that ring type polypeptide is modified
5k-b-PCL
5k.
3) preparation of fluorescence micelle
Preparation process and condition the same, but polymer used be 5mg fluorescence molecule modify FITC-PEO
5k-b-PCL
5kor the CH of RBITC-PEO-b-PCL and 5mg unmodified
3o-PEO
5k-b-PCL
5kmixture.
4) there is the preparation of the micelle of targeting and fluorescent dual effect
Preparation process and condition the same, but polymer used be 5mg fluorescence molecule modify FITC-PEO
5k-b-PCL
5kor RBITC-PEO
5k-b-PCL
5kthe FA-PEO modified with 5mg targeted molecular
5k-b-PCL
5k, GRGDY-PEO
5k-b-PCL
5kor C (RGDfk)-PEO
5k-b-PCL
5kin a kind of mixture of polymer.
5) preparation of carrier micelle
Preparation process and condition are with 4), but add antitumor drug in polymer solution as amycin, the mass ratio of polymer and amycin is 2: 1.Preparation process as shown in Figure 4.
The micelle obtained utilizes light scattering technique (DLS), and ultraviolet spectrophotometer and spectrofluorophotometer measure its particle diameter, drug loading and critical micelle concentration (CMC) respectively, and acquired results is in table 1.
The particle diameter of table 1 micelle, envelop rate, drug loading and CMC result
Embodiment 4: micelle is to the targeting adsorption test of T24 cell
1) cell culture
Select human bladder tumor cell T24 cell to carry out experiment in vitro, cell culture (adds penicillin, the streptomycin of 10% hyclone and 1%) in 37 DEG C and 5%CO in F-12 culture medium
2hatch in incubator.During experiment, get the good cell of growth conditions with 1 × 10
5the density kind in individual/hole is in 24 orifice plates.When cell is spread to 70%, old culture medium is abandoned in suction, after rinsing three times by phosphate buffered solution (PBS), add fresh culture medium, add the CH3O-PEO5k-b-PCL5k fluorescence micellar solution of the unmodified of a certain amount of variable concentrations (0.15mg/ml, 0.3mg/ml, 0.45mg/ml) simultaneously respectively or there is the FA-PEO of targeting and fluorescent dual effect
5k-b-PCL
5kmicellar solution continues to cultivate.
2) confocal laser scanning microscope
After cell culture certain hour, inhale and abandon micellar solution and old culture medium, PBS rinses three times to remove extracellular micelle.Add the glycerol mounting of 70%.Laser confocal microscope (CLSM) is utilized to observe micelle to the targeting absorption of T24 cell and the process entering cell.Acquired results is shown in Fig. 5.Fig. 5 Green is fluoresced by the intrafascicular FITC of fluorescent glue, wherein a is the result of FA-PEO-b-PCL targeting fluorescence micellar solution and cell interaction, from Fig. 5 a, micelle successfully enters cell interior and shows stronger fluorescence intensity, illustrates that more micelle successfully enters cell interior; B is CH
3the result of O-PEO-b-PCL fluorescence micelle and cell interaction, from Fig. 5 b, intracellular fluorescence intensity is starkly lower than Fig. 5 a, and the quantity and the speed that enter the micelle of cell are starkly lower than Fig. 5 a, illustrates that the targeting of the micelle prepared by us to bladder cancer cells is obvious.
3) quantitative study
After cell culture certain hour, inhale and abandon micellar solution and old culture medium, PBS rinses three times to remove extracellular micelle.96 orifice plates of black are transferred to after adding certain density NaOH solution dissolved cell.The fluorescent value that selective exitation wavelength is 480nm, emission wavelength is 535nm, test cell lysate.According to following formulae discovery micelle to the adsorption rate of cell:
Acquired results is shown in Fig. 6.As shown in Figure 6, along with the time increases, intracellular fluorescence intensity increased along with the time; With the intracellular fluorescence intensity of the targeting micelle effect fluorescence intensity apparently higher than the cell interior of common micelle effect, micelle targeting is obvious.
4) Study of cytotoxicity
Get the good cell of growth conditions with 1 × 10
5the density kind in individual/hole is in 96 orifice plates.When cell is spread to 70%, inhale and abandon old culture medium, add fresh culture medium, add respectively simultaneously a certain amount of variable concentrations (0.1mg/ml, 0.2mg/ml) by the CH of non-medicine carrying
3o-PEO
5k-b-PCL
5kthe FA-PEO that micellar solution or targeted molecular are modified
5k-b-PCL
5k, GRGDY-PEO
5k-b-PCL
5k, C (RGDfk)-PEO
5k-b-PCL
5kblank micelle in the carrier micelle CH of one or several and load amycin
3o-PEO
5k-b-PCL
5k/ DOX, FA-PEO
5k-b-PCL
5k/ DOX, GRGDY-PEO
5k-b-PCL
5k/ DOX, C (RGDfk)-PEO
5k-b-PCL
5kin one or several continue cultivate.After 72 hours, every hole adds the 3-(4 that 20ul concentration is 5mg/ml, 5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) solution, act on after 0.5-4 hour, select 490nm wavelength, enzyme linked immunological monitor measures each hole absorbance value, and result as shown in Figure 7.
Above test cell line result shows, the obstacle that prepared micelle successfully can cross cell membrane enters cell interior, as shown in Figure 5.The experimental group adding targeting micelle is stronger relative to the experimental group fluorescence of common micelle, illustrate that folic acid and peptide modified micelle can be enriched in cell membrane more, faster and enter cell, show the targeting good to tumor cell, as shown in Figure 5, Figure 6.Carrier micelle and after tumor cell effect a period of time, reveals obvious inhibitory action to its growth table, and the carrier micelle that particularly targeted molecular is modified shows higher cytotoxicity, and result as shown in Figure 7.
Claims (2)
1. target medicine carrier is for the preparation of the application in the targeting macromolecule medicine-carried system of tumor of bladder perfusion therapy;
Described target medicine carrier, be the micelle of amphipathic nature block polymer, described micelle has nucleocapsid structure, and its shell is made up of the hydrophilic segment in described amphipathic nature block polymer, and kernel is made up of the hydrophobic segment in described amphipathic nature block polymer; Described hydrophilic segment is the poly(ethylene oxide) with function end group, described function end group is following 1) or 2): the biologically functional molecule 1) bladder cancer cells to targeting, 2) bladder cancer cells is had to biologically functional molecule and the fluorescence molecule of targeting; Described hydrophobic segment is biodegradable aliphatic polyester;
Described micelle is of a size of 10 ~ 500nm;
The number-average molecular weight of described amphipathic nature block polymer is 1,500 ~ 600,000, wherein aliphatic polyester segment is 0.01 ~ 1,000 with the ratio of the mean molecule quantity of poly(ethylene oxide) segment, and the number-average molecular weight of aliphatic polyester segment is 1,000 ~ 500,000, the number-average molecular weight of poly(ethylene oxide) segment is 500 ~ 100,000;
Described aliphatic polyester be selected from the copolymer of homopolymer that any one monomer following formed and the formation of at least two kinds of monomers one or more: caprolactone, lactide and Acetic acid, hydroxy-, bimol. cyclic ester;
The described biologically functional molecule having a targeting to bladder cancer cells is folic acid or RGD.
2. application according to claim 1, is characterized in that: described fluorescence molecule is selected from Fluorescein isothiocyanate and/or RB 200.
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