CN104151509A - H-type pH-sensitive degradable block copolymer and preparation method and application thereof - Google Patents
H-type pH-sensitive degradable block copolymer and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an H-type pH-sensitive degradable block copolymer and a preparation method and application thereof. The copolymer consists of a pH-sensitive polymethylacrylic acid chain segment and a biodegradable polylactic acid chain segment. The preparation method comprises the following steps: firstly, preparing tetrachloropolylactic acid by hydroxyl-terminated polylactic acid and dichloroacetyl chloride; then, preparing an H-type tert-butyl methacrylate-lactic acid copolymer by virtue of atom transfer radical polymerization reaction of tert-butyl methacrylate; and carrying out acidic hydrolysis. The copolymer can be self-assembled to a spherical core-shell micelle with the clinical agglomeration concentration of 19.7-32.5mg/L and the hydrodynamic diameter being less than 200nm. The micelle shows pH sensitivity and biodegradability, and the degradation rate changes along with copolymer composition and environmental media. The micelle has a higher release rate on 10-hydroxycamptothecine and taxol in a buffer solution with the pH value of 5.6-7.4 than a buffer solution with the pH value of 1.4, and shows pH-dependent cytotoxicity. The micelle can be used as a carrier with a hydrophobic drug which is released in a targeted manner for targeted treatment of intestinal cancer.
Description
Technical field
The invention belongs to biological medical polymer material technical field, be specifically related to responsive degradable block copolymer micelle of a kind of H type pH and preparation method thereof and the application in drug release.
Technical background
Colorectal cancer, also referred to as colon and rectum carcinoma or intestinal cancer, is a kind of cancer that uncontrollable Growth of Cells forms in colon, rectum (part for large intestine) or appendix, and human health has been formed to huge threat.The medicine of at present, treating this disease has 10-hydroxycamptothecine (HCPT) and taxol (PTX) etc.These medicine poorly water-solubles, unstable and have a side effect.Therefore, be necessary to design suitable drug delivery system, to drug degradation or leakage are dropped to minimum, and medicine be improved in the accumulation of target area.
Due to self-assembly nucleocapsid structure, size of particles and the surface properties of Amphiphilic Block Copolymer Micelles uniqueness, be considered at present a kind of desirable drug conveying carrier.The performance of this uniqueness makes them can dissolve unstable and insoluble drugs, avoids the non-selective absorption that produced by reticuloendothelial system (RES), thereby realizes passive target by high permeability and retention effect (EPR).In fact, show to improve drug conveying to target site, weakening non-specific toxicity as the polymer micelle of drug conveying, finally improved therapeutic efficiency.In order to treat colorectal cancer, copolymer micelle pharmaceutical carrier under one's belt (pH=1.0～2.5) should keep enough stable, and can discharge rapidly coated medicine in the enteron aisle of pH=5.1～7.8.
Patent CN 201010192970.8 discloses a kind of degradable acid-sensitive amphipathic segmented copolymer and its preparation method and application, the close hydrophobic section of multipolymer is made up of polyoxyethylene glycol and acid-sensitive group-di-isopropyl tertiary amine groups respectively, and copolymer micelle is less than 15.97% to the Rate of average load of Zorubicin (DOX).[the Chen W. such as the Chen Wei of University Of Suzhou, et al.J.Control Rel.2010,142 (1): 40-46] prepared with polyoxyethylene glycol and poly-(2,4,6-trimethoxy-benzene methylal tetramethylolmethane carbonic ether) be the responsive degradable polymer vesica of pH and the micella of block, polymer vesicle only has 30.1%～37.7% to the medicine load-carrying efficiency of PTX, and polymer micelle reaches 61.4%～65.2% to the medicine load-carrying efficiency of PTX, under the condition of acidic pH of pH=4.0 and 5.0 gentlenesses, the rate of release of PTX is the rate of release under physiological pH environment faster than them.[the Lee S.C. such as the Lee SC of Inha University of Korea S, et al., J.Control Rel.2003,89 (3): 437-446] synthesized the carrier that poly-(2-ethyl-2-oxazoline-6-caprolactone) segmented copolymer discharges as taxol drug, the bearing capacity of PTX in micella only has 0.5～7.6wt%, depends on hydrophobic block content.[the Kim S. such as the Kim S of Purdue Univ-West Lafayette USA, et al., J.Control Rel.2008, 132 (3): 222-229] prepared based on vinylformic acid and 4-(2-vinyl benzyloxy base)-N, the copolymer micelle that N-(nikethamide) is comonomer, its micelle-forming concentration is 31～86mg/L, the bearing capacity of PTX and coating efficiency are subject to pH impact, pH is greater than at 5 o'clock, bearing capacity and coating efficiency decline greatly, in the time that vinylformic acid molar content is greater than 20%, in the intestinal juice that is released in simulation of PTX from micella, (pH=6.5) discharged completely through 12 hours, and in the gastric juice (pH=1.6) of simulation, there is serious degraded in the PTX discharging.
Summary of the invention
A technical problem to be solved by this invention is to overcome the defects such as the early stage explosive type drug release of low, the easy generation of poor stability, coating efficiency that existing micella medicine exists, provide good, the water-soluble height of a kind of chemical stability, toxicity low and and blood constitutent interact poly-(methacrylic acid-lactic acid-methacrylic acid) segmented copolymer of the biodegradable H type of pH responsive type low.
Another technical problem to be solved by this invention is to provide a kind of preparation method for the responsive degradable segmented copolymer of above-mentioned H type pH.
The structural formula that solves the problems of the technologies described above adopted technical scheme and be this segmented copolymer is as follows:
The integer that in formula, m is 16～33, the integer that n is 10～31.
Reaction process and the preparation method of the above-mentioned responsive degradable segmented copolymer of H type pH are as follows:
1, synthetic chloro poly(lactic acid)
Taking the tetrahydrofuran (THF) that is dried as solvent; under nitrogen protection; the hydroxyl-terminated polylactic acid that is 3000～6000 by number-average molecular weight and dichloroacetyl chloride are 1:2.5～4 normal temperature esterification in molar ratio; and add triethylamine to neutralize the HCl discharging; reaction mixture, through standing, filtration, concentrating under reduced pressure, cold diethyl ether precipitation, room temperature vacuum-drying, obtains chloro poly(lactic acid).
2, poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of synthetic H type
Under anhydrous and oxygen-free condition, by chloro poly(lactic acid), Tert-butyl Methacrylate (tBMA), 1, 1, 4, 7, 10, 10-hexamethyl Triethylenetetramine (TETA) (HMTETA), CuBr is in molar ratio for 1:200～600:8:7 adds in the mixed solvent that dimethyl sulfoxide (DMSO) (DMSO) and the volume ratio of Virahol (IPA) are 1:1, 90 DEG C are reacted 24 hours in confined conditions, quench, use tetrahydrofuran (THF) diluted reaction mixture, mixture after dilution is removed copper complex by 200～300 order neutral alumina chromatography columns, steam except tetrahydrofuran (THF), precipitate with cold diethyl ether, filter, vacuum-drying, obtain poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of H type.
3, the responsive degradable segmented copolymer of synthetic H type pH
Poly-H type (Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer is dissolved in to 1 completely, in 4-diox, the aqueous hydrochloric acid that the massfraction that adds 8～10 times of this block copolymerization amounts is 36.5% is hydrolyzed 24 hours at 90 DEG C, and underpressure distillation removes 1,4-diox, by normal hexane precipitation, filter room temperature vacuum-drying, obtain the responsive degradable segmented copolymer of H type pH, concrete reaction process is as follows:
The hydroxyl-terminated polylactic acid that above-mentioned number-average molecular weight is 3000～6000 is provided by Jinan Dai Gang biomaterial company limited, dichloroacetyl chloride likes that by ladder is uncommon (Shanghai) changes into industrial development company limited and provides, Tert-butyl Methacrylate is provided by Aladdin company of the U.S., 1, Isosorbide-5-Nitrae, 7,10,10-hexamethyl Triethylenetetramine (TETA) is provided by Sigma-Aldrich company of the U.S..
The responsive degradable segmented copolymer of H type pH of the present invention is in the purposes of preparing in cancer therapy drug 10-hydroxycamptothecine or paclitaxel carried medicine micella, and its concrete preparation method is as follows:
Responsive H type pH degradable segmented copolymer, 10-hydroxycamptothecine or taxol are dissolved in to N completely for 10:1 in mass ratio, in the mixed solvent of dinethylformamide and acetone, it is in 2000 dialysis tubing that gained solution packs molecular weight cut-off into, being placed in deionized water or pH and being 7.4 PBS buffered soln dialyses, not coated medicine is removed in centrifugation, medicament-carried polymer micelle lyophilize, obtains the carrier micelle of the coated 10-hydroxycamptothecine of the responsive degradable segmented copolymer of H type pH or taxol.
Compared with prior art, the present invention has following beneficial effect:
1, the copolymer micelle that segmented copolymer of the present invention forms in the aqueous solution has low CAC value, satisfactory stability, responsive pH responsiveness, and phase transition point is corresponding with the pKa value (approximately 5.6) of polymethyl acrylic acid.
2, the copolymer micelle biodegradable that segmented copolymer of the present invention forms changes along with biological microenvironment, is to have good biodegradability in 7.4 and 37～40 DEG C of environment at pH.
3, the copolymer micelle that segmented copolymer of the present invention forms has high dewatering medicament encapsulation efficiency, and its drug release behavior has pH target controllability, and does not produce early stage explosive type release, is a kind of preferred drug carrier material.
4, block copolymer micelle of the present invention itself is nontoxic, and with pH, noticeable change does not occur; And micella base pharmaceutical preparation in the small intestine environment of pH=5.6 and 7.4, have than acid microenvironment in high drug toxicity, be applicable to the treatment of the diseases such as carcinoma of small intestine.
Brief description of the drawings
Fig. 1 is the infrared spectrogram of the responsive degradable segmented copolymer of the H type pH for preparing of embodiment 1.
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the responsive degradable segmented copolymer of the H type pH for preparing of embodiment 1.
Fig. 3 is the transmission electron microscope photo of the responsive degradable block copolymer micelle of the H type pH for preparing of embodiment 1.
Fig. 4 is the dependency of copolymer micelle transmittance (wavelength 500nm) to pH that the segmented copolymer of embodiment 1～3 forms.
Fig. 5 is the responsive degradable segmented copolymer of the H type pH of embodiment 1 and 2 is quality loss in time in the simulation Physiological Medium of 1.4 (stomaches) and 7.4 (small intestines) at pH.
Fig. 6 be the responsive degradable block copolymer micelle carrying of the H type pH for preparing of embodiment 1 taxol transmission electron microscope photo.
Fig. 7 is the drug release in vitro performance that the carrier micelle of the coated 10-hydroxycamptothecine of the responsive degradable segmented copolymer of the H type pH for preparing of embodiment 2 is accumulated under 37 DEG C, different pH.
Fig. 8 is the drug release in vitro performance that the carrier micelle of the coated taxol of the responsive degradable segmented copolymer of the H type pH for preparing of embodiment 2 is accumulated under 37 DEG C, different pH.
Fig. 9 is the drug release in vitro performance that the carrier micelle of the coated taxol of the responsive degradable segmented copolymer of the H type pH for preparing of embodiment 2 is accumulated in 40 DEG C, the environment of pH=5.6.
Figure 10 is that the carrier micelle of the coated taxol of free PTX, embodiment 2 copolymer micelles and embodiment 2 multipolymers is cultivated after 48 hours the toxicity test result to L929 cell in different pH environment.
Figure 11 is the D of the carrier micelle storage different time of the coated taxol of embodiment 2 multipolymers
hand the variation of PTX content in core.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail, but protection scope of the present invention is not limited only to these embodiment.
Embodiment 1
To prepare the responsive degradable segmented copolymer of the following H type pH of structural formula as example, raw materials used and preparation method thereof be:
1, synthetic chloro poly(lactic acid)
The hydroxyl-terminated polylactic acid that is 3000 by 2.0g (0.67mmol) number-average molecular weight is dissolved in 15mL dry tetrahydrofuran, stir, under nitrogen protection, drip 0.20mL (2.08mmol) dichloroacetyl chloride being dissolved in advance in 5mL dry tetrahydrofuran, after dripping, continue to stir 30 minutes, then gained solution is cooled to 0 DEG C, under nitrogen protection, drip 0.29mL (2.08mmol) triethylamine being dissolved in advance in 1mL dry tetrahydrofuran, the HCl discharging with neutralization, after room temperature reaction 24 hours, reaction mixture sat 2 hours, filter, filtrate decompression is concentrated into about 10mL, by the cold diethyl ether precipitation of 10 times of volumes, room temperature vacuum-drying, obtain chloro poly(lactic acid), its productive rate is about 70%.
2, poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of synthetic H type
Under anhydrous and oxygen-free condition, chloro poly(lactic acid) prepared by 0.32g (0.1mmol) step 1, 0.22mL (0.8mmol) 1, 1, 4, 7, 10, 10-hexamethyl Triethylenetetramine (TETA), 10.21mL (60mmol) Tert-butyl Methacrylate, 0.10g (0.7mmol) CuBr joins in the Shrek bottle that fills the mixed solvent that the volume ratio of 5mL dimethyl sulfoxide (DMSO) and Virahol is 1:1, 90 DEG C are reacted 24 hours in confined conditions, with water quenching, use tetrahydrofuran (THF) diluted reaction mixture, mixture after dilution by 200～300 order neutral alumina chromatography columns to remove copper complex, rotary evaporation is removed tetrahydrofuran (THF), and by the cold diethyl ether precipitation of 10 times of its volumes, filter, room temperature vacuum-drying is to constant weight, obtain poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of H type, its productive rate is 33%.
3, the responsive degradable segmented copolymer of synthetic H type pH
Poly-0.5g H type (Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer is dissolved in to 35mL1 completely, in 4-diox, and to add 3.5mL massfraction be 36.5% aqueous hydrochloric acid, at 90 DEG C, be hydrolyzed 24 hours, after reaction finishes, underpressure distillation removes 1,4-diox, the normal hexane precipitation of 10 times of its volumes for resistates, filter, room temperature vacuum-drying, to constant weight, obtains the responsive degradable segmented copolymer of white powder H type pH, and its transformation efficiency is 88%.Adopt infrared spectra, NMR (Nuclear Magnetic Resonance) spectrum to carry out structural confirmation to synthetic product, the results are shown in Figure 1～2.Through the test of Waters-Breeze type gel permeation chromatograph, the number-average molecular weight of this segmented copolymer is about 13940, and polydispersity index is 1.16.
Embodiment 2
To prepare the responsive degradable segmented copolymer of the following H type pH of structural formula as example, raw materials used and preparation method thereof be:
In poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer step 2 of synthetic H type of embodiment 1, the consumption of Tert-butyl Methacrylate becomes 6.81mL (40mmol), other steps are identical with embodiment 1, be prepared into the responsive degradable segmented copolymer of the synthetic H type pH of white powder, its transformation efficiency is 89%, test through Waters-Breeze type gel permeation chromatograph, the number-average molecular weight of this segmented copolymer is about 11320, and polydispersity index is 1.23.
Embodiment 3
To prepare the responsive degradable segmented copolymer of the following H type pH of structural formula as example, raw materials used and preparation method thereof be:
In poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer step 2 of synthetic H type of embodiment 1, the consumption of Tert-butyl Methacrylate becomes 3.40mL (20mmol), other steps are identical with embodiment 1, be prepared into the responsive degradable segmented copolymer of the synthetic H type pH of white powder, its transformation efficiency is 87%, test through Waters-Breeze type gel permeation chromatograph, the number-average molecular weight of this segmented copolymer is about 7250, and polydispersity index is 1.10.
Embodiment 4
To prepare the responsive degradable segmented copolymer of the following H type pH of structural formula as example, raw materials used and preparation method thereof be:
In the synthetic chloro poly(lactic acid) step 1 of embodiment 2, number-average molecular weight used is that 3000 hydroxyl-terminated polylactic acid is with waiting hydroxyl-terminated polylactic acid that mole number average molecular weight is 6000 to replace, other steps are identical with embodiment 2, be prepared into the responsive degradable segmented copolymer of the synthetic H type pH of white powder, its transformation efficiency is 88%, through the test of Waters-Breeze type gel permeation chromatograph, the number-average molecular weight of this segmented copolymer is about 13230, and polydispersity index is 1.11.
Contriver is dissolved in responsive the H type pH of 40mg embodiment 1～4 preparation degradable segmented copolymer in 4mL DMF respectively, under agitation condition, drip deionized water until there is oyster white, form copolymer micelle, continue to stir 12 hours, then with 1000mL deionized water room temperature dialysis 48 hours, dialysis tubing molecular weight cut-off is 2000, start deionized water of replacing per hour in 3 hours, then within every 8 hours, change a deionized water, to remove DMF.Adopt the pattern of the JEOLJEM-1210 type transmission electron microscope observation copolymer micelle of NEC company production; Adopt the critical reunion concentration (CAC) of Perkin Elmer LS55 type spectrophotofluorometer (excite with emission wavelength and be respectively 339 and 394nm, slit width is 10nm) test copolymer micelle; The BI-90Plus type dynamic light scattering that adopts the U.S. to be equipped with Argon ion laser is measured the at room temperature hydromeehanics diameter (D of polymer micelle
h) (wavelength 660 nm, 90 ° of deflection angles, output rating 15mW); Adopt the U.S. Delsa Nano S of Beckman Coulter Inc. type laser particle size analysis Zeta potential analyser to analyze micellar surface electric charge.Test result is in table 1 and Fig. 3.
The physical and chemical performance of the copolymer micelle of table 1 embodiment 1～4
| Sample | Pattern | CAC(mg/L) | D h(nm，DLS) a | Zeta potential (mV) a |
| Embodiment 1 | Spherical | 32.5 | 188.1±2.74 | -18.8 |
| Embodiment 2 | Spherical | 30.0 | 163.3±8.17 | -17.5 |
| Embodiment 3 | Spherical | 19.7 | 139.5±3.62 | -15.3 |
| Embodiment 4 | Spherical | 22.0 | 146.7±3.05 | -15.5 |
Note: in table, a represents that the concentration in PBS buffered soln that copolymer micelle is 7.4 at pH is 250mg/L.
The transmittance of the copolymer micelle that employing ultraviolet-visible pectrophotometer forms the segmented copolymer of embodiment 1～3 in the PBS of different pH values buffered soln tested, and test result is shown in Fig. 4.As can be seen from Figure 4, the transmittance of copolymer micelle is along with pH occurs significantly to change mutually, and wherein the pH phase transition point of the copolymer micelle of embodiment 1～3 is respectively 5.4,5.77,5.92, illustrates that copolymer micelle shows pH responsiveness.
It is in 1.4 and 7.4 PBS buffered soln that responsive 30mg H type pH degradable segmented copolymer is dissolved in respectively to 12mL pH, packing molecular weight cut-off into is in 3000 dialysis tubings, cultivate 3,7,14,21,30 days with 600mL PBS buffered soln at 37 DEG C, re-use molecular weight cut-off and be 1000 dialysis membrane by degradation solution 1000mL deionized water dialysis 24 hours, by dialyzate freeze-drying, weigh, be calculated as follows the biological degradation rate of segmented copolymer:
Biological degradation rate=[(W
0-W
a)/W
0] × 100%
W in formula
0and W
abe respectively the quality of segmented copolymer degraded front and back.As seen from Figure 5, the segmented copolymer of embodiment 1 and embodiment 2 is cultivated and is had respectively 49.6% and 45.3% mass loss in 30 days in the PBS buffered soln of 37 DEG C of pH=7.4, temperature, illustrates that the biological degradation rate of segmented copolymer increases with the increase of polymethyl acrylic acid block chain length.But in the PBS of pH=1.4 buffered soln, the biological degradation rate of segmented copolymer declines greatly, the Block copolymer rate of embodiment 2 only has 8%～10%.Therefore, segmented copolymer of the present invention is conducive to medicine in enteral release, and after degraded, can discharge by metabolism easily in small intestine.
Embodiment 5
The responsive degradable segmented copolymer of H type pH of embodiment 1～4 preparation is in the purposes of preparing in cancer therapy drug 10-hydroxycamptothecine carrier micelle, and concrete using method is as follows:
By responsive 40mg H type pH degradable segmented copolymer, 4mg 10-hydroxycamptothecine (HCPT) is dissolved in the mixed solvent that the volume ratio of 10mL DMF and acetone is 9:1 completely, it is in 2000 dialysis tubing that gained solution packs molecular weight cut-off into, being placed in 500mL pH and being 7.4 PBS buffered soln dialyses 48 hours, start deionized water of replacing per hour in 3 hours, then within every 8 hours, change a deionized water, then not coated medicine is removed in centrifugation, by medicament-carried copolymer micelle lyophilize, obtain the carrier micelle of the coated 10-hydroxycamptothecine of the responsive degradable segmented copolymer of H type pH.
Embodiment 6
The responsive degradable segmented copolymer of H type pH of embodiment 1～4 preparation is in the purposes of preparing in anti-cancer medicine paclitaxel carrier micelle, and concrete using method is as follows:
In embodiment 5,10-hydroxycamptothecine used such as uses to replace at the taxol (PTX) of quality, other steps are identical with embodiment 5, obtain the carrier micelle of the coated taxol of the responsive degradable segmented copolymer of H type pH, see Fig. 6, carrier micelle is spherical, and pattern is not subject to the impact of small-molecule drug carrying, the about 131nm of median size, medicine has entered in the core of micella.
The coated carrier micelle of 10-hydroxycamptothecine of the responsive degradable segmented copolymer of H type pH and the carrier micelle of coated taxol that embodiment 5 and 6 is obtained carry out stability, drug encapsulation and vitro drug release and vitro cytotoxicity test, and concrete test method and test result are as follows:
1, drug encapsulation test
The coated carrier micelle of 10-hydroxycamptothecine of the responsive degradable segmented copolymer of H type pH that embodiment 5 and 6 is obtained and the carrier micelle of coated taxol are dissolved in the mixed solvent that the volume ratio of DMF and acetone is 9:1 completely, adopt the ultraviolet-visual spectrometer that HIT's model is U-3900/3900H to measure their absorbancys under fixing absorbing wavelength 391nm (HCPT) and 228nm (PTX), calculate medicine loading capacity (LC) and encapsulation efficiency (EE) by following formula:
LC (wt.%)=(quality of the micella of the quality of micella Chinese traditional medicine/medicament-carried) × 100%
EE (wt.%)=(quality of micella Chinese traditional medicine/add drug quality) × 100%
Working curve is drawn by the free drug that uses different concns in DMF and acetone mixed solvent.Test-results is in table 2.
The coating efficiency of the copolymer micelle of table 2 embodiment 1～4 to HCPT and PTX
From table 2, along with the increase of polymethyl acrylic acid block chain length in segmented copolymer, its LC and EE improve gradually, and wherein PTX has the LC higher than HCPT and EE.Therefore, H type segmented copolymer of the present invention is more suitable for the carrier as PTX drug release.
2, vitro drug release experiment
The dialysis tubing sealing that is 2000 with molecular weight cut-off by the carrier micelle of the coated 10-hydroxycamptothecine of responsive 20mg H type pH degradable segmented copolymer and coated taxol, then immerse respectively 400mL pH value and be in 7.4,6.4 and 5.6 PBS buffered soln, and in 37 DEG C (normal body temperatures) and 40 DEG C (temperature of tumor tissues) lower stirring, to discharge medicine.At predetermined time interval, get 4mL PBS buffered soln and carry out quantitative analysis, meanwhile add the fresh PBS of 4mL to supplement to delivery systme.The HCPT of accumulative total and PTX burst size are measured by the ultraviolet absorptivity at 391nm and 228nm place, and are calculated as follows:
Accumulation drug release %=M
t/ M
0× 100%
M in formula
tfor the release amount of medicine of time t, M
0represent the medication amount of carrying in copolymer micelle.
From Fig. 7～8, the vitro drug release of HCPT and PTX is that pH relies on, and does not have initial stage explosive type to discharge.In the time of pH=1.4, only had 26% drug release through 96 hours, thereby can reduce medicine leakage under one's belt.Under the high pH condition that is not less than 5.6, drug release rate and burst size are along with pH increases and improves.Be 5.6,6.4 and 7.4 o'clock at pH, through 96 hours, HCPT cumulative release amount was respectively 64%, 78% and 84%, and pH=5.6 and 7.4 o'clock, through 96 hours, the PTX release amount of medicine of accumulation was respectively 60% and 77%.Bound drug is sealed LC and EE analysis in test, and segmented copolymer of the present invention is suitable for carrying and the release of PTX and HCPT.
The therapeutic efficiency that is coated the carrier micelle of taxol in order to understand the responsive degradable segmented copolymer of H type pH, to PTX, under tumour cell environment, the release behavior of the accumulation of (40 DEG C of pH=5.6, temperature) is studied, and the results are shown in Figure 9.As seen from Figure 9, total the burst size of PTX or speed improve with the raising of polymethyl acrylic acid block length.24 hours time, the Cumulative release amount of PTX from embodiment 1, embodiment 2 and embodiment 3 copolymer micelles is respectively 40%, 33% and 29%; Within 96 hours, be respectively 72%, 67% and the release of 61%, PTX there is the distinguishing feature of sustained release.Therefore, have enough PTX remaining for sustained release to guarantee long-term treatment effect.
3, vitro cytotoxicity test
Utilize the cytotoxicity of the free PTX of MTT chemical examination research, the embodiment 2 blank micellas of multipolymers formation and the carrier micelle of coated taxol.In the dish of 96 wells, by L929 mouse embryo fibroblasts at 37 DEG C, 5% CO
2in moistening atmosphere, with every well 1 × 10
4density cultivate one in DMEM medium 24 hours completely, supplement 10% foetal calf serum (DMEM in high glucose).Then the carrier micelle of the free PTX of different concns, blank micella and coated taxol is dissolved in cell culture medium, replaces the substratum in every well with solution prepared by 100 μ L.The sample of each concentration repeats three tests.Culture plate is at 37 DEG C, 5%CO
2hatch after 48 hours, add growth medium that 20 μ L MTT solution (5mg/mL) and 100 μ L are fresh to replace the sample solution of each well.Culture plate is at 37 DEG C, 5% CO
2in continue to maintain 4 hours, discard supernatant liquid.Then, the DMSO of 150 μ L is joined in each well to 10 minutes blue first a ceremonial jade-ladle, used in libation crystal with dissolving viable cell internalization of room temperature vibration.As a reference, under identical condition, cell kind is grown in fresh culture (negative control).Measure in each well first a ceremonial jade-ladle, used in libation crystal in the absorbancy of 490 nm by the general microplate reader that Bio-Rad laboratory equipment company limited of Britain model is 680, and optical density represents.Cell viability is with containing absorbancy (OD in the well of micelle suspension cultured cells
sample) to only having the absorbancy (OD of cell
contrast) percentage represent, be specifically calculated as follows:
Comparative survival rate of cells %=(OD
sample/ OD
contrast) × 100%
As seen from Figure 10, at pH=1.4 (gastric environment), under 5.6 and 7.4 (small intestine) three kinds different pH environment, blank micella is almost non-toxic within the scope of concentration 1～500mg/L, cultivate cell survival rate after 48 hours and exceed 95%, illustrate that the carrier that segmented copolymer of the present invention discharges as medicine control is that low toxicity is even nontoxic, the carrier micelle cytotoxicity of the coated taxol of multipolymer changes along with pH environment, when pH=1.4 (stomach), carrier micelle is low cytotoxicity, there is no significant difference (although cell viability is slightly lower than blank micella) with blank micellar phase ratio.But when pH=5.6 and 7.4 (small intestine), carrier micelle cytotoxicity improves, cell viability drops to 47.1% from 99.5%.In this different pH medium, cytotoxicity difference discharges consistent with the PTX of pH sensitivity.In the enteron aisle of pH=5.6 and 7.4, because discharging fast, PTX causes the toxicity high to cancer cells, the result for the treatment of obtaining.But because PTX still less leaks, this carrier micelle does not damage acid stomach.The specific selectivity of this target and pH susceptibility can provide a kind of synergistic effect.Presentation of results, the responsive degradable segmented copolymer of H type pH of the present invention is conducive to drug release, shows the cytotoxicity that pH relies on, and can effectively kill carcinoma of small intestine cell and can not cause damage to stomach.
4, storage stability test
By the carrier micelle of the coated taxol of embodiment 2 multipolymers, 9 DEG C of storages, as seen from Figure 11, the carrier micelle of coated taxol shows good physical stability, store after 35 days, the loss of the carrier micelle of coated taxol is lower than 6%, and do not observe PTX and reunite or sedimentation, D
hdrop to 142.9nm by 150.2nm, 4.7% the dimensional change of only having an appointment, illustrates the carrier that multipolymer of the present invention can discharge as medicine control.
Claims (4)
1. the responsive degradable segmented copolymer of H type pH, is characterized in that the structural formula of this segmented copolymer is as follows:
The integer that in formula, m is 16～33, the integer that n is 10～31.
2. the preparation method of the responsive degradable segmented copolymer of the H type pH of claim 1, is characterized in that it is made up of following step:
(1) synthetic chloro poly(lactic acid)
Taking the tetrahydrofuran (THF) that is dried as solvent, under nitrogen protection, the hydroxyl-terminated polylactic acid that is 3000～6000 by number-average molecular weight and dichloroacetyl chloride are 1:2.5～4 normal temperature esterification 24 hours in molar ratio, obtain chloro poly(lactic acid);
(2) poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of synthetic H type
Under anhydrous and oxygen-free condition, by chloro poly(lactic acid), Tert-butyl Methacrylate, 1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA), CuBr add in the mixed solution that the volume ratio of dimethyl sulfoxide (DMSO) and Virahol is 1:1 for 1:200～600:8:7 in molar ratio, 90 DEG C are reacted 24 hours, and separation and purification product obtains poly-(Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer of H type;
(3) the responsive degradable segmented copolymer of synthetic H type pH
Poly-H type (Tert-butyl Methacrylate-lactic acid-Tert-butyl Methacrylate) segmented copolymer is dissolved in to 1 completely, in 4-diox, the aqueous hydrochloric acid that the massfraction that adds 8～10 times of this block copolymerization amounts is 36.5%, at 90 DEG C, be hydrolyzed 24 hours, obtain the responsive degradable segmented copolymer of H type pH.
3. the responsive degradable segmented copolymer of the H type pH of claim 1 is in the purposes of preparing in cancer therapy drug 10-hydroxycamptothecine carrier micelle.
4. the responsive degradable segmented copolymer of the H type pH of claim 1 is in the purposes of preparing in anti-cancer medicine paclitaxel carrier micelle.
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