CN103980498A - Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof - Google Patents
Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof Download PDFInfo
- Publication number
- CN103980498A CN103980498A CN201410227934.9A CN201410227934A CN103980498A CN 103980498 A CN103980498 A CN 103980498A CN 201410227934 A CN201410227934 A CN 201410227934A CN 103980498 A CN103980498 A CN 103980498A
- Authority
- CN
- China
- Prior art keywords
- pla
- tpgs
- preparation
- dendroid
- catalyzer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention discloses a dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as a preparation method and an application thereof. The dendritic amphiphilic block copolymer H-PLA-b-TPGS has the general structural formula shown as the formula I, wherein in the formula I, m is between 16 and 42; n is between 16 and 64; and H represents a hyperbranched polyester H20, H30 or H40. The method for preparing the dendritic amphiphilic block copolymer H-PLA-b-TPGS is simple and free of pollution. The obtained copolymer is good in biocompatibility and biodegradability, and is taken as an adjuvant medicine material in applications of preparing medicine-carried nanoparticles, medicine-carried microspheres or composite tissue engineering scaffolds.
Description
Technical field
The present invention relates to a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS and preparation method thereof and application.
Background technology
Due to it, good biocompatibility and degradability are widely used in target slow-release, controlled release research as drug administration system carrier to Biodegradable polymer material in vivo.Dendritic macromole is the novel synthetic polymer of a class occurring in recent years, they have highly branched, highly symmetrical, single dispersion and a lot of unique character such as high surface functional group density, present the incomparable advantage of other material, therefore in a lot of Application Areass, especially in biological medicine and nanosecond medical science field, be subject to paying close attention to extremely widely, become a new study hotspot now.Dendritic macromole has following distinguishing feature compared with traditional linear macromolecule: (1) dendritic macromole has clear and definite molecular weight and molecular dimension, compound with regular structure, molecular volume, shape and function base all can accurately be controlled on molecular level; (2) dendritic macromole is generally set out by core, constantly outside branch, when algebraically is lower, be generally open molecular configuration, with the increase of algebraically and the continuation of branching, since the 4th generation, molecule is changed into the spherical three-dimensional structure of outer tight interior pine by the loose condition (of surface) of opening wide, intramolecule has wide cavity, and molecular surface has high functional group densities; (3) dendritic macromole has good reactive behavior and containing ability, can import a large amount of reactivities or functional groups at point subcenter and molecular end, as macromolecular material [the Cheng Y with specific function, et al., Chem.Soc.Rev., 40 (2011) 2673 and Carlmark A, et al., Chem.Soc.Rev., 42 (2013) 5858].Fig. 1 is the structural representation of polyester dendrimer.
Because having wide cavity, dendritic macromole inside there is high functional group densities with surface, therefore be widely used in drug delivery field, for example internal cavities can packaging medicine molecule, and end group can connect the target parts such as polymkeric substance, folic acid and aptamer by modifying.Dendritic macromole mainly concentrates on pharmaceutical carrier, genophore, immunological reagent, boron neutron capture therapy technology (BNCT), medical science contrast medium etc. in the application of biomedical sector at present.
Poly(lactic acid) (PLA) is to get permission in the world the main carriers material of the slow controlled release medicament of clinical application at present, there is good biocompatibility, biodegradability and tensile strength, nontoxic at human body, without accumulation, therefore it has very tempting application prospect and high commercial value at biomedical sector.Vitamin E TPGS (TPGS) is the abbreviation of polyethylene glycol 1000 vitamin E succinic acid ester (D-α-tocopherol polyethylene glycol1000succinate), it is the soluble derivative of vitamin-E, carboxyl and cetomacrogol 1000 (PEG1000) esterification by VE-succinate (TOS) form, relative molecular weight is about 1513, molecular structure is as follows, loaded " American Pharmacopeia ".TPGS has been widely used in pharmaceutical preparation, foods and cosmetics research, TPGS is flaxen waxy solid, be close to tasteless, it is a kind of amphiphile, amphiphilic molecule, there are a hydrophilic polar head and a hydrophobic aliphatic carbon chain afterbody molecule, can be water-soluble, also can be dissolved in most of polar organic solvents.
be Bai Situo series product, use polyvalent alcohol as core, react and form with alcohol acid.Its dendriform structure is polymerized by special core and dimethylol propionic acid (Bis-MPA).The basic product obtaining is the dendriform polyester with hydroxy functional group.
series product, for example H20, H30 and H40, owing to thering is biodegradability, biocompatibility, ball-like structure and a lot of terminal functionality, be widely used in designing unimolecular micelle [Tu C, et al., Polymer, 54 (2013) 2020 and Zeng X, et al., J.Polym.Sci.Part A:Polym.Chem., 50 (2012) 280].
These materials are nontoxic at human body, without accumulation, and all used by U.S. FDA approval, and therefore it has very tempting application prospect and high commercial value at biomedical sector.
Than single linear polymeric, the star-like improvement that the high molecular polymer of design shape has brought the performances such as many physics, chemistry, biology in advance such as grade.Compared with the linear polymeric of same molecular amount, star-like polymer has less hydrodynamic volume, viscosity in solution is little, while forming micella, there is lower micelle-forming concentration (CMC), can greatly improve like this medicine-carried nano particles stability in vivo, medicine is had to higher encapsulation rate and drug loading [Prabaharan M, et al., Biomaterials, 30 (2009) 3009].
Summary of the invention
The object of this invention is to provide a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS.
Second object of the present invention is to provide the preparation method of a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS.
The 3rd object of the present invention is to provide the application of a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS.
Technical scheme of the present invention is summarized as follows:
A kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS, its general structure is suc as formula shown in I:
In formula I: m=16-42, n=16-64; H represents hyper-branched polyester
h20, H30 or H40;
TPGS-is group shown in formula II:
In formula II: p=23.
The preparation method of above-mentioned a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS, comprise the steps: the preparation of (1) H-PLA dendroid multipolymer: by mole%, with lactide monomer and the 0.1%-10% hyper-branched polyester of 90%-99.9%
h20, H30 or H40 are raw material, and at the first catalyzer, under oxygen free condition, at 120-180 DEG C, polyreaction 8-12 hour, obtains number-average molecular weight M
nfor the H-PLA dendroid multipolymer of 51080-73600;
(2) preparation of carboxylated branch-shape polymer H-PLA-COOH: in organic solvent, by mole%, taking the second catalyzer of the Succinic anhydried of the H-PLA dendroid multipolymer of 5%-30%, 40%-90% or maleic anhydride, 5%-30% as raw material, at 10-40 DEG C of reaction 10-48 hour, obtain reaction solution, precipitate with precipitation agent, filter, to precipitate and be dried, obtain carboxylated branch-shape polymer H-PLA-COOH;
(3) in organic solvent, adding mol ratio is H-PLA-COOH and the TPGS of 1:20-80, add dehydrating condensation agent, under the effect of the third catalyzer, at 10-40 DEG C of reaction 10-48 hour, filter, filtrate is precipitated with precipitation agent, filter, will precipitate and be dried, obtaining molecular weight is the dendroid amphipathic nature block polymer H-PLA-b-TPGS of 69320-135800.
The first catalyzer is preferably stannous octoate, stannous iso caprylate, organic guanidine, metallic zinc, tributyltin chloride, ferric acetyl acetonade, zinc lactate, nano zine oxide, taurine, ethanol iron, n-propyl alcohol iron, Virahol iron or propyl carbinol iron, and the molar weight of described the first catalyzer is the 0.1%-0.5% of lactide monomer molar weight.
Can also be that the H-PLA dendroid multipolymer that step (1) is obtained carries out purifying, purification step is: H-PLA dendroid multipolymer is dissolved in methylene dichloride, ethyl acetate or dioxane, add methyl alcohol, ether or sherwood oil to make described multipolymer precipitation, filter, to precipitate vacuum-drying, obtain the H-PLA dendroid multipolymer of purifying.
The second catalyzer preferably from following a) and b) any one or be a) and b) mixture of 1-10 composition according to mol ratio: a) be pyridine, 2-picoline, 4-picoline or DMAP; B) be triethylamine, quadrol, triethylene diamine or tetrem alkene triamine.
The third catalyzer is pyridine, 2-picoline, 4-picoline or DMAP, and the add-on of the third catalyzer is 3-10 times of described H-PLA-COOH molar weight.
Organic solvent be selected from following at least one: dioxane, methylene dichloride, acetonitrile, toluene, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) and DMF.
The preferred ether of precipitation agent, normal heptane, sherwood oil, methyl alcohol and ethanol at least one.
Dehydrating condensation agent is preferably N, N-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, and the consumption of described dehydrating condensation agent is 1-60 times of described H-PLA-COOH molar weight.
Dendroid amphipathic nature block polymer H-PLA-b-TPGS as pharmaceutical excipient in the application of preparing in drug-carrying nanometer particle, medicine carrying microballoons or composite tissue engineering support.
The method that the present invention prepares H-PLA-b-TPGS dendroid multipolymer is simple, pollution-free.The multipolymer obtaining has good biocompatibility, biodegradability, as pharmaceutical excipient in the application of preparing in drug-carrying nanometer particle, medicine carrying microballoons or composite tissue engineering support.It is a kind of up-and-coming material.
Brief description of the drawings
Fig. 1 is the synthetic schematic diagram of dendroid amphipathic nature block polymer H-PLA-b-TPGS.
Fig. 2 be multipolymer H40-PLA nucleus magnetic resonance figure (
1hNMR).
Fig. 3 be H40-PLA-COOH nucleus magnetic resonance figure (
1hNMR).
Fig. 4 be H40-PLA-b-TPGS nucleus magnetic resonance figure (
1hNMR).
Fig. 5 is field emission scanning electron microscope collection of illustrative plates (FESEM) collection of illustrative plates of carrying docetaxel H20-PLA-b-TPGS nanoparticle.
Fig. 6 is particle diameter and the size distribution result that laser particle analyzer detects carrying docetaxel H20-PLA-b-TPGS nanoparticle.
Fig. 7 is the result that zeta potential instrument is measured medicine carrying H20-PLA-b-TPGS nanoparticle.
Fig. 8 is the vitro drug release curve of carrying docetaxel PLA-b-TPGS and carrying docetaxel H20-PLA-b-TPGS nanoparticle.
Fig. 9 be carrying docetaxel H20-PLA-b-TPGS nanoparticle, blank H20-PLA-b-TPGS nanoparticle (nanoparticle suspension concentration identical with drug-carrying nanometer particle) and
cytotoxicity experiment result to MCF-7 cell at 24h.
Figure 10 be carrying docetaxel H20-PLA-b-TPGS nanoparticle, blank H20-PLA-b-TPGS nanoparticle (nanoparticle suspension concentration identical with drug-carrying nanometer particle) and
cytotoxicity experiment result to MCF-7 cell at 48h.
Figure 11 be carrying docetaxel H20-PLA-b-TPGS nanoparticle, blank H20-PLA-b-TPGS nanoparticle (nanoparticle suspension concentration identical with drug-carrying nanometer particle) and
cytotoxicity experiment result to MCF-7 cell at 72h.
Figure 12 is that the Coumarin-6 nanoparticle that carries prepared by laser confocal scanning electron microscope observation H30-PLA-b-TPGS copolymer material is hatched the MCF-7 cell of 2h at 37 DEG C.Nucleus is dyed redness with PI, and it is green carrying Coumarin-6 nano particle, respectively by EGFP passage and I passage observation of cell picked-up situation: figure A is the situation (green) of observing by EGFP passage; Figure B is the situation (redness) of observing by PI passage; Figure C is the result after the doubling of the image of observing by EGFP passage with by PI passage.
Figure 13 is for carrying taxol H40-PLA-b-TPGS nanoparticle transmission electron microscope TEM figure
Figure 14 is the Electronic Speculum figure that carries Ibuprofen BP/EP H40-PLA-b-TPGS microball preparation.
Figure 15 is the scanning electron microscope (SEM) photograph that carries gelatin/calcium phosphate bone cement composite tissue engineering support of Ibuprofen BP/EP microballoon.
Embodiment
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 ordinary method; Described reagent and material, if no special instructions, all can obtain from commercial channels.
The preparation method of embodiment 1 dendroid amphipathic nature block polymer H20-PLA-b-TPGS, comprises the steps:
(1) preparation of H20-PLA dendroid multipolymer: by mole%, 98.5% lactide monomer and 1.5% hyper-branched polyester got
h20 initiator is raw material, puts into polymerizing pipe, adds the first catalyzer tributyltin chloride of lactide monomer molar weight 0.5%, vacuumizes, inflated with nitrogen, in triplicate, when vacuum tightness polymerizing pipe tube sealing during higher than 70pa, 120 DEG C of heating, polyreaction 12 hours, obtains number-average molecular weight M
nit is the H20-PLA dendroid multipolymer of 51080 (m=42, n=16);
H20-PLA dendroid multipolymer is dissolved in methylene dichloride, adds methyl alcohol to make multipolymer precipitation, filter, will precipitate vacuum-drying, obtain the H20-PLA dendroid multipolymer of purifying;
Experimental results show that the tributyltin chloride that substitutes this step with stannous octoate, organic guanidine, metallic zinc, ferric acetyl acetonade, zinc lactate, taurine, ethanol iron, n-propyl alcohol iron, Virahol iron or propyl carbinol iron, other can catalysis obtain H20-PLA dendroid multipolymer with this step;
(2) preparation of carboxylated branch-shape polymer H20-PLA-COOH: in organic solvent acetonitrile, by mole%, the H20-PLA dendroid multipolymer that step (1) taking 30% obtains, 40% maleic anhydride, the second catalyzer of 30% are raw material, and at 10 DEG C, reaction is 48 hours, obtain reaction solution, add precipitation agent and precipitate, make precipitation completely, filter, to be deposited in 40 DEG C of vacuum-dryings, obtain carboxylated branch-shape polymer H20-PLA-COOH; The quality of dioxane is 10 times of raw materials quality; DMAP (DMAP) and triethylamine that the second catalyzer is 1:1 by mol ratio form; Ether and methyl alcohol that precipitation agent is 1:1 by volume ratio form;
2-picoline and triethylene diamine that to experimental results show that by mol ratio be 1:1, or the mol ratio 4-picoline that is 1:1 and triethylamine substitute DMAP (DMAP) and the triethylamine that the mol ratio of this step is 1:1 and make catalyzer, can catalysis obtain carboxylated branch-shape polymer H20-PLA-COOH;
(3) in methylene dichloride, adding mol ratio is H20-PLA-COOH and the TPGS of 1:20, add the dehydrating condensation agent N of 16 times of suitable H20-PLA-COOH molar weights, N-dicyclohexylcarbodiimide (DCC), under the effect of the third catalyzer pyridine of 3 times of suitable H20-PLA-COOH molar weights, at 10 DEG C, reaction is 28 hours, filter, filtrate is precipitated with precipitation agent, filter, to be deposited in 40 DEG C of vacuum-dryings, can obtain molecular weight is 69320 (m=42, n=16, p=23) dendroid amphipathic nature block polymer H20-PLA-b-TPGS.Precipitation agent is that volume ratio is methyl alcohol and the ether of 1:2.
Experimental results show that the pyridine that is substituted this step by 4-picoline, can catalysis obtain dendroid amphipathic nature block polymer H20-PLA-b-TPGS.See Fig. 1.
The preparation method of embodiment 2, dendroid amphipathic nature block polymer H30-PLA-b-TPGS, comprises the steps:
(1) preparation of H30-PLA dendroid multipolymer: by mole%, 90% lactide monomer and 10% hyper-branched polyester got
h30 initiator is raw material, puts into polymerizing pipe, adds the first catalyzer stannous iso caprylate of lactide monomer molar weight 0.1%, vacuumizes, inflated with nitrogen, in triplicate, when vacuum tightness polymerizing pipe tube sealing during higher than 70pa, 180 DEG C of heating, polyreaction 8 hours, obtains number-average molecular weight M
nit is the multipolymer H30-PLA dendroid multipolymer of 68370 (m=27, n=32);
H30-PLA dendroid multipolymer is dissolved in ethyl acetate, adds ether to make multipolymer precipitation, filter, will precipitate vacuum-drying, obtain the H30-PLA dendroid multipolymer of purifying.
(2) preparation of carboxylated branch-shape polymer H30-PLA-COOH: in organic solvent dioxane, by mole%, the H30-PLA dendroid multipolymer that step (1) taking 5% obtains, 90% Succinic anhydried, the second catalyzer of 5% are raw material, and at 40 DEG C, reaction is 10 hours, obtain reaction solution, add precipitation agent and precipitate, make precipitation completely, filter, to be deposited in 40 DEG C of vacuum-dryings, obtain carboxylated branch-shape polymer H30-PLA-COOH; The quality of dioxane is 6 times of raw materials quality; Pyridine and quadrol that the second catalyzer is 10:1 by mol ratio form; Normal heptane and ethanol that precipitation agent is 2:1 by volume ratio form;
(3) in toluene, adding mol ratio is H30-PLA-COOH and the TPGS of 1:80, add the dehydrating condensation agent N of 1 times of suitable H30-PLA-COOH molar weight, N-dicyclohexylcarbodiimide (DCC), under the effect of the third catalyzer 2-picoline of 10 times of suitable H30-PLA-COOH molar weights, at 40 DEG C, reaction is 24 hours, filter, filtrate is precipitated with precipitation agent, filter, to be deposited in 40 DEG C of vacuum-drying 10h, can obtain number-average molecular weight is 93500 (m=27, n=32, p=23) dendroid amphipathic nature block polymer H30-PLA-b-TPGS.Precipitation agent is that volume ratio is normal heptane and the ethanol of 1:2.
The structural confirmation data of this embodiment products therefrom and embodiment 3, without substantive difference, repeat no more herein.
The preparation method of embodiment 3, dendroid amphipathic nature block polymer H40-PLA-b-TPGS, comprises the steps:
(1) preparation of H40-PLA dendroid multipolymer: get 99.9% lactide monomer and 0.1% hyper-branched polyester by molar content
h40 is raw material, put into polymerizing pipe, the catalyst nano zinc oxide that adds lactide monomer molar weight 0.1%, vacuumizes inflated with nitrogen, repeat 3 times, tube sealing under vacuum state, polyreaction 10 hours at 150 DEG C, obtaining number-average molecular weight is 73600 (m=16, n=64) H40-PLA dendroid multipolymer, is shown in Fig. 2.
H40-PLA dendroid multipolymer is dissolved in dioxane, adds sherwood oil to make multipolymer precipitation, filter, will precipitate vacuum-drying, obtain the H40-PLA dendroid multipolymer of purifying.
(2) preparation of carboxylated branch-shape polymer H40-PLA-COOH: in tetrahydrofuran (THF), by mole%, the H40-PLA dendroid multipolymer that step (1) taking 5% obtains, 75% Succinic anhydried, the second catalyzer of 20% are raw material, 30 DEG C of reactions 24 hours, obtain reaction solution, precipitate with precipitation agent sherwood oil, make precipitation completely, filter, and precipitate by methanol wash, to precipitate 40 DEG C of vacuum-dryings, obtain carboxylated branch-shape polymer H40-PLA-COOH, see Fig. 3; The quality of dioxane is 10 times of raw materials quality; The DMAP that the second catalyzer is 1:1 by mol ratio and tetrem alkene triamine form;
(3) at N, in dinethylformamide, adding mol ratio is H40-PLA-COOH and the TPGS of 1:65, add dehydrating condensation agent 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide of 60 times of suitable H40-PLA-COOH molar weights, under the effect of the third catalyzer DMAP of 5 times of suitable H40-PLA-COOH molar weights, at 25 DEG C, reaction is 24 hours, filter, filtrate is precipitated with precipitation agent, filter, to be deposited in 40 DEG C of vacuum-drying 10h, can obtain number-average molecular weight is 135800 (m=16, n=64, p=23) dendroid amphipathic nature block polymer H40-PLA-b-TPGS, see Fig. 4.Precipitation agent is that volume ratio is sherwood oil and the methyl alcohol of 1:1.
Experiment showed, with the DMF of the alternative the present embodiment of dimethyl sulfoxide (DMSO) and make organic solvent, can complete the reaction of this step.
Proton nmr spectra (
1hNMR) and the dendritic amphipathic nature block polymer H40-PLA-b-TPGS of gel permeation chromatography (GPC) result proof tree synthesized successfully.
Wherein, Fig. 2 is that polymkeric substance H40-PLA, Fig. 3 are the hydrogen nuclear magnetic resonance spectrogram of H40-PLA-COOH and Fig. 4 H40-PLA-b-TPGS.In the hydrogen nuclear magnetic resonance of H40-PLA, a peak (δ=5.18ppm) belongs to methyne in rac-Lactide (CH), and b peak (δ=1.56ppm) belongs to methyl (CH in rac-Lactide
3), c peak (δ=4.35ppm) and d peak (δ=1.44ppm) belong to respectively PLA segment end be connected hydroxyl methyne-CH-and-CH
3peak, illustrates successful ring-opening polymerization under the condition that rac-Lactide is initiator at H40.δ=1.23, peak and 4.20ppm distribute and belong to
h40 methyl and methylene peak.The 1HNMR of H40-PLA shows that dendroid poly(lactic acid) is successfully synthetic.By integration b peak (δ=1.56ppm) and d peak (δ=1.44ppm), can calculate molecular weight and the polymerization degree of PLA on every chain of H40-PLA.In H40-PLA, the molecular weight Mn of every chain and polymerization degree DP are respectively 1152 and 16.
The H40-PLA-COOH's of Fig. 3
1in HNMR, the new peak e of appearance, f (δ=2.65-2.72ppm) is the methylene peak of Succinic anhydried in carboxylated H40-PLA, illustrates that H40-PLA is by successful carboxylation reaction.
In the end in the linked reaction of a step H40-PLA-COOH and TPGS, products therefrom H40-PLA-b-TPGS.From H40-PLA-b-TPGS's
1in HNMR spectrogram (Fig. 4), can find out, g peak (δ=3.65ppm) belongs to the methylene peak-CH in TPGS
2-CH
2o.Nucleus magnetic resonance figure (
1hNMR) the dendritic amphipathic nature block polymer H40-PLA-b-TPGS of specification tree is successfully synthesized.The molecular weight of polymkeric substance can be respectively by gel permeation chromatography figure (GPC) and nmr spectrum (
1hNMR) calculate.The molecular weight of dendroid multipolymer is as shown in table 1.
The molecular weight of table 1 dendroid multipolymer
Embodiment 4, prepare the H20-PLA-b-TPGS nanoparticle of carrying docetaxel (Docetaxel)
Utilize ultrasonic emulsification/solvent evaporation method to prepare carrying docetaxel (Docetaxel) H20-PLA-b-TPGS nanoparticle.Preparation method is as follows: accurately take H20-PLA-b-TPGS multipolymer and 10mg Docetaxel powder prepared by 100mg embodiment 1, be dissolved in 10ml methylene dichloride.Under agitation condition, the massfraction that this solution is joined to 120ml is in the 0.03%TPGS aqueous solution.Under condition of ice bath, disperse 120s with 25w power ultrasonic, form emulsion oil-in-water, organic solvent is removed in decompression volatilization.The centrifugal 15min of 20000rpm, uses deionized water wash three times, to remove TPGS and free Docetaxel medicine.Gained precipitation is resuspended in 10ml deionized water, and lyophilize obtains carrying docetaxel H20-PLA-b-TPGS nanoparticle product.Utilize same method to prepare carrying docetaxel PLA-b-TPGS nanoparticle as a control group.
The scanning electron microscope that this embodiment prepares gained carrying docetaxel H20-PLA-b-TPGS nanoparticle the results are shown in Figure 5, and Nanoparticle Size is compared with homogeneous, and smooth in appearance is spherical in shape, and particle diameter is greatly about 150nm left and right.
As shown in Figure 6, nanoparticle narrower particle size distribution, particle diameter, greatly about 150nm left and right, has further confirmed the observations of scanning electron microscope.
As shown in Figure 7, the Zeta potential of nanoparticle is in-10mV left and right, and the absolute value of surface charge is higher, and between particle, repulsive interaction is stronger, thereby stable at disperse phase camber, and current potential is at the nanoparticle distributional stability in vivo of-10mV left and right.
As shown in table 2, using 0.03%TPGS to prepare drug loading as emulsifying agent is 10% polyene-containing taxol nanoparticle, can make encapsulation rate (HPLC method mensuration) reach and approach 100%.
HPLC testing conditions is as follows:
Chromatographic column is that (5 μ m) for YMC, 150mm*4.6mm for C-18 post
Moving phase: acetonitrile: water (50:50, V/V)
Flow velocity: 1.0ml/min
Column temperature: 30 DEG C
Ultraviolet detection wavelength is 227nm
Docetaxel typical curve linear equation is:
A=21.59*C (R
2=0.9998) wherein A is color atlas peak area; C is sample concentration.
As shown in Figure 8, dialysis method is measured the medicament slow release curve of nanoparticle, and this embodiment gained drug-carrying nanometer particle of 10mg is scattered in 5ml release medium PBST solution (by 8.5g NaCl, 2.2g Na
2hPO
4, 0.3g NaH
2pO
4, 1.0g tween-80 and deionized water 1000ml composition, and obtain through autoclaving.) in, form suspension.Nano particle suspension is placed in to dialysis tubing, seals sack.Airtight dialysis tubing is put into 50ml centrifuge tube, adds 15mlPBST, is placed in water bath with thermostatic control shaking table in 37 DEG C, 120rpm vibration.In a certain time interval, from centrifuge tube, take out 10ml solution for analyzing, supplement the fresh PBST of equivalent simultaneously in centrifuge tube.In the sample of collecting, add 2ml dichloromethane extraction, aqueous phase discarded.The sample that extraction obtains is through being dissolved in 5ml moving phase acetonitrile: water (50/50, V/V), passes into N
2make methylene dichloride volatilization, until solution becomes clarification, filter filters, and then adds moving phase, is settled to 10ml.Moving phase is used front through 0.45 μ m membrane filtration, and supersound process.Each sample introduction 20 μ l, the peak area of HPLC working sample, when test condition and survey encapsulation rate, the condition of HPLC used is identical.In conjunction with the typical curve (A=21.59*C) of Docetaxel, calculate the amount of each Docetaxel discharging, draw the outer release profiles of medicament-carried nano granule according to data, acquired results is shown in Fig. 8.
As shown in Figure 8, carrying docetaxel PLA-b-TPGS has similar release profiles with carrying docetaxel H20-PLA-b-TPGS nanoparticle, is two-phase release characteristic and follows initial " burst effect ".H20-PLA-b-TPGS nanoparticle release rate is faster, easily meets clinical requirement.
As shown in Fig. 9-11, adopt MMT method to measure the cytotoxicity of this nanoparticle: by MCF-7 cell (ATCC, Rockville, MD) be inoculated in 96 porocyte culture plates, after cell cultures 24h is adherent, discard outmoded substratum, rinse once with PBS, add testing sample, positive control, negative control to cultivate respectively 24h, 48h, 72h.Behind specific time interval, discard outmoded substratum, rinse once with PBS, every hole adds the cell culture medium of 100 μ l containing MTT1mg/ml, hatches after 4h, discards MTT for 37 DEG C, every hole adds the dimethyl sulfoxide (DMSO) (DMSO) of 100 μ l, cultivate 2h for dark 37 DEG C, vibration 10min, by the absorbancy of microplate reader mensuration 570nm wavelength.Result shows, the blank H20-PLA-b-TPGS nanoparticle of medicine carrying does not have good biocompatibility, because it does not all have obvious toxicity to MCF-7 cell under different nanoparticle suspension concentration; And carrying docetaxel H20-PLA-b-TPGS nanoparticle has obvious cytotoxicity, and cytotoxicity is greater than commercial Docetaxel preparation docetaxel
in addition, MTT experimental result explanation carrying docetaxel H20-PLA-b-TPGS nanoparticle has time and concentration dependent to the toxicity of MCF-7 cell.
Figure 10 be carrying docetaxel H20-PLA-b-TPGS nanoparticle, blank H20-PLA-b-TPGS nanoparticle (nanoparticle suspension concentration identical with drug-carrying nanometer particle) and
cytotoxicity experiment result to MCF-7 cell at 48h.
Figure 11 be carrying docetaxel H20-PLA-b-TPGS nanoparticle, blank H20-PLA-b-TPGS nanoparticle (nanoparticle suspension concentration identical with drug-carrying nanometer particle) and
cytotoxicity experiment result to MCF-7 cell at 72h.
Table 2. is prepared in polyene-containing taxol nanoparticle process with H20-PLA-b-TPGS copolymer material, the impact of drug loading and emulsifying agent particle diameter, Zeta potential and the encapsulation rate on nanoparticle
The H30-PLA-b-TPGS nanoparticle of Coumarin-6 is carried in embodiment 5, preparation
Utilize the preparation of ultrasonic emulsification/solvent evaporation method to carry the H30-PLA-b-TPGS nanoparticle of Coumarin-6.Preparation method is as follows: accurately take H30-PLA-b-TPGS dendroid multipolymer prepared by 50mg embodiment 2 and the Coumarin-6 of 1mg, be dissolved in 6ml methylene dichloride.Under agitation condition, this solution is joined in the 0.03%TPGS aqueous solution of 80ml.Under condition of ice bath, disperse 120s with 25w power ultrasonic, form emulsion oil-in-water, organic solvent is removed in decompression volatilization.The centrifugal 15min of 20000rpm, uses deionized water wash three times, to remove de-emulsifier TPGS and free Coumarin-6.Gained precipitation is resuspended in 8ml deionized water, and lyophilize must be carried the H30-PLA-b-TPGS nanoparticle product of Coumarin-6.Nanoparticle particle diameter is in 200nm left and right.
MCF-7 cell suspension is evenly inoculated in 6 porocyte culture plates, then adds 1ml substratum, 37 DEG C, 5%CO
2in incubator, cultivate 24h.In MCF-7 cell, add year Coumarin-6 nano particle of 250mg/l, continue to cultivate 2h.With ice-cold PBS flushing three times, add methyl alcohol fixed cell 20min, discard methyl alcohol, add propidium iodide (PI) dye liquor to hatch 5min, rinse three times with PBS again, can in cellular uptake experiment, carry the position of Coumarin-6 nanoparticle in cell by nuclear location is determined.Figure 12 is to the picked-up result of carrying Coumarin-6 nano particle of being prepared by H30-PLA-b-TPGS with laser confocal scanning electron microscope observation MCF-7 cell.As can be seen from the figure, only hatching after 2h with cell, nanoparticle is just absorbed by cell.Merge with figure B the figure C obtaining from figure A and can know to such an extent that see, greeny nanoparticle great majority are arranged in tenuigenin, tightly surround the nucleus taking on a red color.
The H40-PLA-b-TPGS nano particle of taxol (Paclitaxel, PTX) is carried in embodiment 6, preparation
Utilize the nanometer precipitator method to prepare the H40-PLA-b-TPGS nano particle of year taxol (Paclitaxel, PTX).Preparation method is as follows: accurately take H40-PLA-b-TPGS multipolymer and a certain amount of taxol powder prepared by 100mg embodiment 3, be dissolved in the acetone solvent of 8ml.Under agitation condition, this solution is joined in the TPGS aqueous solution that the massfraction of 120ml is 0.03%, stirring spends the night to volatilize removes organic solvent.The centrifugal 15min of 20000rpm, uses deionized water wash three times, to remove TPGS and free taxol drug.Gained precipitation is resuspended in 5ml deionized water, and lyophilize must be carried the H40-PLA-b-TPGS nanoparticle product of taxol PTX.This embodiment prepares the transmission electron microscope results that gained carries taxol H40-PLA-b-TPGS nanoparticle product and sees Figure 13, and as seen from the figure, this nano particle diameter is 60nm left and right, shape particle spherical in shape.
Many Ibuprofen BP/EPs H40-PLA-b-TPGS microballoon is carried in embodiment 7, preparation
Utilize solvent evaporation method preparation to carry Ibuprofen BP/EP H40-PLA-b-TPGS microballoon.Preparation method is as follows: accurately take H40-PLA-b-TPGS multipolymer prepared by 200mg embodiment 3 and the Ibuprofen BP/EP powder of 40mg, be dissolved in 20ml methylene dichloride, under agitation condition, this solution is joined in polyvinyl alcohol (PVA) aqueous solution that the mass concentration of 500ml is 0.5%.Under 600rpm rotating speed, stir two hours, form emulsion oil-in-water, under 500rpm rotating speed, volatilization is spent the night.The centrifugal 15min of 2000rpm, uses deionized water wash three times, to remove de-emulsifier PVA and free ibuprofen pharmaceutical.Gained precipitation is resuspended in 10ml deionized water, and lyophilize must be carried Ibuprofen BP/EP H40-PLA-b-TPGS microball preparation.As shown in figure 14, carry Ibuprofen BP/EP H40-PLA-b-TPGS microsphere features smooth surface, particle diameter is in 3 μ m left and right.The drug loading that carries Ibuprofen BP/EP H40-PLA-b-TPGS microballoon is about 15%, and encapsulation rate is more than 75%.
Embodiment 8, the preparation of carrying gelatin/calcium carbonate bone cement composite tissue engineering support of Ibuprofen BP/EP microballoon
Microballoon prepared by 0.9g embodiment 7 is dispersed in 100g deionized water, adds 15 gelatin (Qinghai gelatin limited-liability company), is warmed to 37 DEG C gelatin is dissolved completely.10g calcium phosphate cement bracket (Usbiomaterials Corp.) is immersed in above-mentioned solution, in 37 DEG C of vacuum drying ovens, vacuumize 1 hour.Take out support, surface is wiped dry with filter paper, and dry 3 days of lower 37 DEG C of vacuum obtains carrying gelatin/calcium phosphate bone cement composite tissue engineering support of Ibuprofen BP/EP microballoon.Figure 15 is the scanning electron microscope (SEM) photograph that carries gelatin/calcium phosphate bone cement composite tissue engineering support of Ibuprofen BP/EP microballoon, as can be seen from the figure, carrying Ibuprofen BP/EP microballoon is evenly distributed in the gelatin matrix on calcium phosphate bone cement hole wall, and the inside also has a lot of holes, these holes can continue to maintain the function of material as support, can grow into space is provided for cell adhesion and new organization, accelerated material degraded, accelerates skeleton reparation.
Claims (10)
1. a dendroid amphipathic nature block polymer H-PLA-b-TPGS, its general structure is suc as formula shown in I:
In formula I: m=16-42, n=16-64; H represents hyper-branched polyester
h20, H30 or H40;
TPGS-is group shown in formula II:
In formula II: p=23.
2. the preparation method of a kind of dendroid amphipathic nature block polymer H-PLA-b-TPGS of claim 1, is characterized in that comprising the steps:
(1) preparation of H-PLA dendroid multipolymer: by mole%, with lactide monomer and the 0.1%-10% hyper-branched polyester of 90%-99.9%
h20, H30 or H40 are raw material, and at the first catalyzer, under oxygen free condition, at 120-180 DEG C, polyreaction 8-12 hour, obtains number-average molecular weight M
nfor the H-PLA dendroid multipolymer of 51080-73600;
(2) preparation of carboxylated branch-shape polymer H-PLA-COOH: in organic solvent, by mole%, taking the second catalyzer of the Succinic anhydried of the H-PLA dendroid multipolymer of 5%-30%, 40%-90% or maleic anhydride, 5%-30% as raw material, at 10-40 DEG C of reaction 10-48 hour, obtain reaction solution, precipitate with precipitation agent, filter, to precipitate and be dried, obtain carboxylated branch-shape polymer H-PLA-COOH;
(3) in organic solvent, adding mol ratio is H-PLA-COOH and the TPGS of 1:20-80, add dehydrating condensation agent, under the effect of the third catalyzer, at 10-40 DEG C of reaction 10-48 hour, filter, filtrate is precipitated with precipitation agent, filter, will precipitate and be dried, obtaining molecular weight is the dendroid amphipathic nature block polymer H-PLA-b-TPGS of 69320-135800.
3. preparation method according to claim 2; it is characterized in that described the first catalyzer is stannous octoate, stannous iso caprylate, organic guanidine, metallic zinc, tributyltin chloride, ferric acetyl acetonade, zinc lactate, nano zine oxide, taurine, ethanol iron, n-propyl alcohol iron, Virahol iron or propyl carbinol iron, the molar weight of described the first catalyzer is the 0.1%-0.5% of lactide monomer molar weight.
4. according to the preparation method described in claim 2 or 3, it is characterized in that the H-PLA dendroid multipolymer that step (1) is obtained carries out purifying, purification step is: H-PLA dendroid multipolymer is dissolved in methylene dichloride, ethyl acetate or dioxane, add methyl alcohol, ether or sherwood oil to make described multipolymer precipitation, filter, to precipitate vacuum-drying, obtain the H-PLA dendroid multipolymer of purifying.
5. preparation method according to claim 2, it is characterized in that described the second catalyzer be selected from following a) and b) any one or be a) and b) mixture of 1-10 composition according to mol ratio: a) be pyridine, 2-picoline, 4-picoline or DMAP; B) be triethylamine, quadrol, triethylene diamine or tetrem alkene triamine.
6. preparation method according to claim 2, is characterized in that described the third catalyzer is pyridine, 2-picoline, 4-picoline or DMAP, and the add-on of the third catalyzer is 3-10 times of described H-PLA-COOH molar weight.
7. preparation method according to claim 2, it is characterized in that described organic solvent be selected from following at least one: dioxane, methylene dichloride, acetonitrile, toluene, dimethyl sulfoxide (DMSO), tetrahydrofuran (THF) and DMF.
8. preparation method according to claim 2, it is characterized in that described precipitation agent be ether, normal heptane, sherwood oil, methyl alcohol and ethanol at least one.
9. preparation method according to claim 2, it is characterized in that described dehydrating condensation agent is N, N-dicyclohexylcarbodiimide or 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide, the consumption of described dehydrating condensation agent is 1-60 times of described H-PLA-COOH molar weight.
Dendroid amphipathic nature block polymer H-PLA-b-TPGS claimed in claim 1 as pharmaceutical excipient in the application of preparing in drug-carrying nanometer particle, medicine carrying microballoons or composite tissue engineering support.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410227934.9A CN103980498A (en) | 2014-05-27 | 2014-05-27 | Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410227934.9A CN103980498A (en) | 2014-05-27 | 2014-05-27 | Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103980498A true CN103980498A (en) | 2014-08-13 |
Family
ID=51272682
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410227934.9A Pending CN103980498A (en) | 2014-05-27 | 2014-05-27 | Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103980498A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188550A (en) * | 2016-07-08 | 2016-12-07 | 清华大学深圳研究生院 | Dendritic block copolymer p AM PGlu b TPGS and preparation method and application |
| CN109096453A (en) * | 2018-06-21 | 2018-12-28 | 济南大学 | A kind of pectination amphipathic vinylidene group block copolymer and its application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880381A (en) * | 2010-06-30 | 2010-11-10 | 清华大学深圳研究生院 | Segmented copolymer modified by polyethylene glycol 1000 vitamin E succinic acid ester, preparation method and applications thereof |
| US20120328706A1 (en) * | 2000-08-30 | 2012-12-27 | Medivas, Llc | Polymer particle delivery compositions and methods of use |
| CN103159959A (en) * | 2013-03-07 | 2013-06-19 | 清华大学深圳研究生院 | M-PLGA-TPGS star type amphiphilic copolymer and preparation method and application |
-
2014
- 2014-05-27 CN CN201410227934.9A patent/CN103980498A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120328706A1 (en) * | 2000-08-30 | 2012-12-27 | Medivas, Llc | Polymer particle delivery compositions and methods of use |
| CN101880381A (en) * | 2010-06-30 | 2010-11-10 | 清华大学深圳研究生院 | Segmented copolymer modified by polyethylene glycol 1000 vitamin E succinic acid ester, preparation method and applications thereof |
| CN103159959A (en) * | 2013-03-07 | 2013-06-19 | 清华大学深圳研究生院 | M-PLGA-TPGS star type amphiphilic copolymer and preparation method and application |
Non-Patent Citations (3)
| Title |
|---|
| SIE HUEY LEE ET AL.: "Nanoparticles of poly(lactide)-tocopheryl polyethylene glycol succinate (PLA-TPGS) copolymers for protein drug delivery", 《BIOMATERIALS》, vol. 28, 23 January 2007 (2007-01-23) * |
| XIAOLONG TANG ET AL.: "Paclitaxel-loaded nanoparticles of star-shaped cholic acid-core PLA-TPGS copolymer for breast cancer treatment", 《NANOSCALE RESEARCH LETTERS》, vol. 8, no. 420, 17 October 2013 (2013-10-17) * |
| ZHIPING ZHANG ET AL.: "In Vitro and In Vivo Investigation on PLA-TPGS Nanoparticles for Controlled and Sustained Small <olecule Chemotherapy", 《PHARMACEUTICAL RESEARCH》, vol. 25, no. 8, 29 May 2008 (2008-05-29), XP 019613177 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106188550A (en) * | 2016-07-08 | 2016-12-07 | 清华大学深圳研究生院 | Dendritic block copolymer p AM PGlu b TPGS and preparation method and application |
| CN106188550B (en) * | 2016-07-08 | 2019-03-12 | 清华大学深圳研究生院 | Dendritic block copolymer p AM-PGlu-b-TPGS and the preparation method and application thereof |
| CN109096453A (en) * | 2018-06-21 | 2018-12-28 | 济南大学 | A kind of pectination amphipathic vinylidene group block copolymer and its application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103073730B (en) | CA-(PLA-ran-PLC-b-PEC) periodic copolymer and preparation method and application thereof | |
| CN104288833B (en) | A kind of containing can the composite reactive bone cement and preparation method thereof of slow release drug-loading nanoparticles | |
| CN109010846B (en) | Polyethylene glycol-chitosan-curcumin polymer, drug-loaded nanoparticles thereof and preparation method | |
| Zhang et al. | Synthesis and characterization of a new multifunctional polymeric prodrug paclitaxel–polyphosphoester–folic acid for targeted drug delivery | |
| Tao et al. | Synthesis of cholic acid-core poly (ε-caprolactone-ran-lactide)-b-poly (ethylene glycol) 1000 random copolymer as a chemotherapeutic nanocarrier for liver cancer treatment | |
| CN110698718B (en) | Hollow polymer microsphere coated with nano hydroxyapatite and preparation method and application thereof | |
| Gao et al. | Facile preparation of shell crosslinked micelles for redox-responsive anticancer drug release | |
| US20190110987A1 (en) | A method for preparation of hybrid amphiphilic star copolymer nano micelles | |
| Dai et al. | Biomimetic star-shaped porphyrin-cored poly (l-lactide)-b-glycopolymer block copolymers for targeted photodynamic therapy | |
| Yu et al. | Self-assembly of pH-responsive biodegradable mixed micelles based on anionic and cationic polycarbonates for doxorubicin delivery | |
| Chang et al. | Methoxy poly (ethylene glycol)-b-poly (valerolactone) diblock polymeric micelles for enhanced encapsulation and protection of camptothecin | |
| Zheng et al. | Novel micelles from graft polyphosphazenes as potential anti-cancer drug delivery systems: drug encapsulation and in vitro evaluation | |
| Zhou et al. | A twin-tailed tadpole-shaped amphiphilic copolymer of poly (ethylene glycol) and cyclic poly (ε-caprolactone): synthesis, self-assembly and biomedical applications | |
| Li et al. | Synthesis and characterization of amphiphilic block polymer poly (ethylene glycol)-poly (propylene carbonate)-poly (ethylene glycol) for drug delivery | |
| Lv et al. | Biodegradable depsipeptide–PDO–PEG-based block copolymer micelles as nanocarriers for controlled release of doxorubicin | |
| CN103159959B (en) | Star-like amphipathic multipolymer of a kind of M-PLGA-TPGS and preparation method thereof and application | |
| Sun et al. | Self‐assembled micelles prepared from poly (ɛ‐caprolactone)–poly (ethylene glycol) and poly (ɛ‐caprolactone/glycolide)–poly (ethylene glycol) block copolymers for sustained drug delivery | |
| Yang et al. | Synthesis and characterization of amphiphilic block copolymer of polyphosphoester and poly (l‐lactic acid) | |
| CN103980498A (en) | Dendritic amphiphilic block copolymer H-PLA-b-TPGS as well as preparation method and application thereof | |
| Zhang et al. | Preparation of core cross-linked PCL-PEG-PCL micelles for doxorubicin delivery in vitro | |
| Xu et al. | Comparative study of paclitaxel physically encapsulated in and chemically conjugated with PEG‐PLA | |
| CN102807677B (en) | CA-PLGA-TPGS (Cholic Acid-Poly-Lactic-co-Glycolic Acid copolymer-Tocopherol Polyethylene Glycol Succinate) star copolymer as well as preparation method and application thereof | |
| Wang et al. | Preparation of pH-sensitive dextran nanoparticle for doxorubicin delivery | |
| CN101717495B (en) | PCL-PLA-TPGS copolymer as well as preparation method and application thereof | |
| KR102176982B1 (en) | Reactive oxygen-sensitive ferrocene-based nano particles and method for preparing the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| AD01 | Patent right deemed abandoned | ||
| AD01 | Patent right deemed abandoned |
Effective date of abandoning: 20170412 |