CN104434792B - Polymer micelle and preparation method thereof and antineoplastic pharmaceutical compositions, preparation and preparation method thereof - Google Patents
Polymer micelle and preparation method thereof and antineoplastic pharmaceutical compositions, preparation and preparation method thereof Download PDFInfo
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- CN104434792B CN104434792B CN201310416459.5A CN201310416459A CN104434792B CN 104434792 B CN104434792 B CN 104434792B CN 201310416459 A CN201310416459 A CN 201310416459A CN 104434792 B CN104434792 B CN 104434792B
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Abstract
The present invention relates to a kind of polymer micelle and preparation method thereof and antineoplastic pharmaceutical compositions, preparation and preparation method thereof.The polymer micelle includes the polysaccharide as shell and the retinene as kernel, wherein, the retinene is grafted on the polysaccharide by hydrazone key.Hydrazone key has pH sensitiveness, only can be just broken under specific pH environment and realize the complete release of loaded medicine, will not destroy the active group of the medicine, therefore the polymer micelle has higher stability；Also, due to the characteristic of retinene and polysaccharide in itself so that the polymer micelle has higher biocompatibility.
Description
Technical field
The present invention relates to nanosecond medical science technical field, more particularly to a kind of polymer micelle and preparation method thereof and resists swollen
Tumor medicine, preparation and preparation method thereof.
Background technology
Cancer is one of current most commonly seen Etiological for endangering human health and causing death.In China, every year
The number of cancer mortality has been occupied first of all kinds of causes of disease.Chemotherapy of tumors is one of the most frequently used method of current treating cancer, but mostly
Number chemotherapeutics dissolubilities are poor, medicine non-selectivity and drug resistance easily produced in human body, so as to limit it in clinical cancer
Application in treatment.
In recent years, the fast development of nanometer technology caused the rise of nanosecond medical science.Nanosecond medical science is a kind of new doctor
Field, refer to transmit medicine, gene, protein and developer etc. as pharmaceutical carrier by the material of nano-scale.
Nano-sized materials as carrier mainly include nanogel, nanoparticle, microcapsules, liposome, polymer micelle etc..
Wherein, polymer micelle be by amphipathic polymer in an aqueous medium self assembly and formed, be total to by hydrophobic section and hydrophilic section
With composition, there is unique nucleocapsid structure, wherein hydrophobic cores are surrounded by hydrophily shell.
The development of this nano-medicament carrier of the rise of nanosecond medical science, especially polymer micelle, carried for the treatment of cancer
New hope is supplied.It is that it shows as drug delivery system that polymer micelle, which is widely used in antineoplastic transmission system,
Unique advantage, compared with the surfactant of low molecule amount, polymer micelle has relatively low critical micelle concentration, Ke Yi
Under this big diluted ambient of body fluid, the good stability of polymer drug-carried system is remained in that, is advantageous to the treatment of cancer.
However, current polymer micelle due to the potential toxicity of polymer, in vivo poor biocompatibility and compared with
Low stability and be dfficult to apply to clinic.
The content of the invention
Based on this, it is necessary to provide the polymer micelle and its preparation side that a kind of biocompatibility is higher, stability is higher
Method.
Further, there is provided a kind of antineoplastic pharmaceutical compositions.
Further, a kind of anti-tumor medicinal preparation and preparation method thereof is also provided.
A kind of polymer micelle, including the polysaccharide as shell and the retinene as kernel, wherein, the retinene leads to
Hydrazone key is crossed to graft on the polysaccharide.
In one of the embodiments, the mass ratio of the polysaccharide and retinene is 10~100:20~500.
In one of the embodiments, the polysaccharide is glucan, sodium alginate, hyaluronic acid, heparin, chondroitin sulfate
Element, amylopectin, pectin, amylose or cyclodextrin.
In one of the embodiments, the retinene is all-trans-retinal or 11-cis retinal.
A kind of preparation method of polymer micelle, comprises the following steps：
The polysaccharide that buzane obtains connecting buzane is connected on the hydroxyl of polysaccharide, by retinene and the polysaccharide for connecting buzane
It is dissolved in solvent, in normal-temperature reaction 24~48 hours, isolates and purifies and the polymer micelle is obtained after drying.
In one of the embodiments, it is described that the step of buzane obtains connecting the polysaccharide of buzane is being connected on the hydroxyl of polysaccharide
Including：
The polysaccharide is dissolved in the first solvent, then adds pyridine and p-nitrophenyl chloroformate ester, and adds catalyst,
Reacted 4~24 hours under condition of ice bath, isolate and purify and obtain intermediate product after drying；And
The intermediate product is dissolved in the second solvent, adds hydrazine hydrate, is reacted 24~48 hours, isolates and purifies and dries
The polysaccharide for connecting buzane is obtained afterwards.
In one of the embodiments, in described the step of isolating and purifying and obtaining the polymer micelle after drying, point
Method from purifying is specially：Reactant is dialysed 2~5 days in the solvent, then dialysed 2~5 days in ultra-pure water,
Vacuum freeze drying obtains the polymer micelle.
A kind of antineoplastic pharmaceutical compositions, including above-mentioned polymer micelle swell with resisting on the polymer micelle is carried on
Tumor medicine.
In one of the embodiments, the antineoplastic be adriamycin, taxol, Docetaxel, fluorouracil,
Vincristine, cis-platinum, carboplatin or retinoic acid.
In one of the embodiments, the quality of the antineoplastic is the quality of the antineoplastic pharmaceutical compositions
1%~12%.
A kind of anti-tumor medicinal preparation, including ultra-pure water and the above-mentioned antitumor combination that is dissolved in the ultra-pure water
Thing.
In one of the embodiments, the concentration of the antineoplastic pharmaceutical compositions is 1~10mg/L.
A kind of preparation method of anti-tumor medicinal preparation, comprises the following steps：
Above-mentioned polymer micelle is dissolved in organic solvent, antineoplastic is then added, stirs at room temperature, mixing is equal
Dialysed after even with ultra-pure water, the anti-tumor medicinal preparation is obtained after aseptic process.
The retinene of above-mentioned polymer micelle is grafted on polysaccharide by hydrazone key, and hydrazone key has pH sensitiveness, only specific
PH environment under can just be broken and realize the complete release of loaded medicine, the active group of the medicine will not be destroyed, therefore
The polymer micelle has higher stability；Also, due to the characteristic of retinene and polysaccharide in itself so that the polymer micelle
With higher biocompatibility.
Brief description of the drawings
Fig. 1 is the structural representation of the polymer micelle of an embodiment；
Fig. 2 is the schematic diagram of the preparation method of the polymer micelle of an embodiment；
Fig. 3 is the structural representation of the antineoplastic pharmaceutical compositions of an embodiment；
Fig. 4 is the schematic diagram of the preparation method of the anti-tumor medicinal preparation of an embodiment；
Fig. 5 is the scanning electron microscope (SEM) photograph of the polymer micelle of embodiment 1；
Fig. 6 is the adriamycin release profiles in the anti-tumor medicinal preparation of embodiment 1 under condition of different pH；
Fig. 7 is the anti-tumor medicinal preparation of doxorubicin formulations, polymer micelle preparation and embodiment 1 to human breast carcinoma MCF-
7 cytotoxicities detect structure chart；
Fig. 8 is for MCF-7 Human Breast Cancer Cells in different time points to doxorubicin formulations and the antineoplastic system of embodiment 1
The intake situation testing result figure of adriamycin in agent；
Fig. 9 is the antineoplastic of physiological saline, doxorubicin formulations, the polymer micelle preparation of embodiment 1 and embodiment 1
Testing result figure of the preparation in nude mice Anticancer effect in vivo.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, below in conjunction with the accompanying drawings to the present invention
Embodiment be described in detail.Many details are elaborated in the following description in order to fully understand this hair
It is bright.But the invention can be embodied in many other ways as described herein, those skilled in the art can be not
Similar improvement is done in the case of running counter to intension of the present invention, therefore the present invention is not limited to the specific embodiments disclosed below.
Referring to Fig. 1, the polymer micelle 100 of an embodiment, including be mostly the polysaccharide of shell 10 and be used as kernel 30
Retinene.Polysaccharide is hydrophily shell, and retinene is hydrophobic cores, and it is amphipathic that retinene and polysaccharide form hydrophobe-hydrophile
Nucleocapsid structure, shell 10 and kernel 30 are connected by hydrazone key 20.
Polysaccharide is natural polysaccharide.Natural polysaccharide has higher stability, safety, hypotoxicity, hydrophily, is easy to chemistry and repaiies
Decorations and the characteristic such as biodegradable.
Preferably, polysaccharide is glucan (Dextran), sodium alginate（Alginate）, hyaluronic acid (Hyaluronic
Acid), heparin (Heparin), chondroitin sulfate (Chondroitin sulphate), amylopectin（Pullulan）, pectin
(Pectin), amylose（Amylose）Or cyclodextrin（Cyclodextrin）.
It is highly preferred that polysaccharide is glucan.Glucose as a kind of water-soluble and homogeneous molecular weight polysaccharide, be to be formed it is poly-
One of optimal candidate material of compound micella.Glucan is the polysaccharide that glucose is formed by 1,6- glycosidic bonds, is had preferable
Biocompatibility, biodegradability and water solubility, and its catabolite is endogenous material so that the polymer micelle
100 have higher biocompatibility.
Also, glucan can overcome the drug resistance of tumor drug resistance cell, the absorption of haemocyanin and interior rete cutaneum can be avoided
The clean-up effect of shape system, so as to extend circulation time inside polymer micelle 100.Thus, by above-mentioned polymer micelle 100
When the pharmaceutical composition that load antineoplastic obtains is used for treating cancer, the action time of antineoplastic can be extended, carried
High curative effect.
Retinene is all-trans-retinal or 11-cis retinal.
Retinene is grafted on polysaccharide by hydrazone key.Hydrazone key has pH sensitiveness, under the weakly acidic condition of tumor by local, hydrazone
Key will be broken, and so as to discharge the medicine of parcel in hydrophobic core, the medicine can be split away off still from polymer micelle
The structure that energy keeps original is constant, will not destroy the active function groups of medicine.But this system is under the conditions of normal physiological pH
It is stable, it is thus possible to pinpoint and discharge in tumor locus.
Meanwhile under the weakly acidic condition of tumor by local, retinene intactly can be split away off from polysaccharide, and be turned in vivo
It is melted into retinoic acid.Retinoic acid is multi-functional cell function instrumentality, and its adjustable range is very extensive, including cell differentiation, thin
Intracellular growth, cell propagation and Apoptosis etc..The retinoic acid being transformed by retinene can further suppress tumour cell
Growth.
ATRA has inhibitory action, such as colon cancer, leukaemia, tumor in digestive tract, lung cancer etc. to kinds of tumors.
Also, ATRA can improve sensitiveness of the tumour cell to antineoplastic, be advantageous to improve curative effect.Therefore, depending on
Yellow aldehyde is preferably all-trans-retinal, to be converted into all-trans retinoic acid in vivo.
Preferably, the mass ratio of polysaccharide and retinene is 10~100:20~500, with preferably comprehensive polysaccharide and retinene
Effect, the polymer micelle 100 is being obtained higher biocompatibility and while higher stability, use can be extended
The action time of the medicine of the polymer micelle 100 load, and make all-trans retinoic acid and drug synergism, improve curative effect.
The retinene of above-mentioned polymer micelle 100 is grafted on polysaccharide by hydrazone key, and hydrazone key has pH sensitiveness, only in spy
It can just be broken under fixed pH environment and realize the complete release of loaded medicine, the active group of the medicine will not be destroyed, because
This polymer micelle 100 has higher stability；Also, due to the characteristic of retinene and polysaccharide in itself so that the polymerization
Thing micella 100 has higher biocompatibility.
Because the polymer micelle 100 has higher stability, its hydrazone key only can be just broken under specific pH environment,
Be advantageous to fixed point, the Targeting delivery of the medicine of the polymer micelle 100 load, can be imitated by the high-permeability and delay of solid tumor
Should（EPR effects）By the passive target tumour cell of polymer micelle 100, effective aggregation of the medicine in tumor locus is realized, is improved
To the lethality of tumor tissues, and reduce the infringement of normal tissue.
Therefore, above-mentioned polymer micelle 100 is a kind of very safe and efficient pharmaceutical carrier.
The polymer micelle 100 is spherical.A diameter of 90 nanometers~120 nanometers of the polymer micelle 100.The polymer
The particle diameter of micella 100 is smaller, and load capacity is strong, and carrying drug ratio is high.
The preparation method of one embodiment polymer micelle, comprises the following steps：
The polysaccharide that buzane obtains connecting buzane is connected on the hydroxyl of polysaccharide, retinene and the polysaccharide for connecting buzane are dissolved in
In solvent, in normal-temperature reaction 12~48 hours, isolate and purify and the polymer micelle is obtained after drying.
Comprise the steps S110 and step connecting the step of buzane obtains connecting the polysaccharide of buzane on the hydroxyl of polysaccharide
S120。
Step S110：Polysaccharide is dissolved in the first solvent, then adds pyridine and p-nitrophenyl chloroformate ester, and add and urge
Agent, reacted 4~24 hours under condition of ice bath, isolate and purify and obtain intermediate product after drying.
First solvent is the solvent that can dissolve polysaccharide, pyridine, p-nitrophenyl chloroformate ester and catalyst, preferably diformazan
Base sulfoxide.
Preferably, concentration of the polysaccharide in the first solvent is 50~100mg/mL.
The effect of pyridine is to provide the progress that alkaline environment is advantageous to reaction.Preferably, the volume of pyridine and the first solvent
It is equal.
P-nitrophenyl chloroformate ester is as reactant.Preferably, the concentration of p-nitrophenyl chloroformate ester is 30~60mg/
mL。
Catalyst is that can be catalyzed the catalyst that polysaccharide and p-nitrophenyl chloroformate ester react, preferably 4- (dimethylamino)
Pyridine.Preferably, the mass ratio of catalyst and polysaccharide is 37.2:1000.
Preferably, the method isolated and purified is specially：3~4 times are washed with ethanol precipitation reactant, then with ethanol, then
Washed 2 times with ether.
It is appreciated that the method not limited to this isolated and purified, it is any can be by the method for above-mentioned purifying intermediate products
With for purifying the intermediate product.
Step S120：Intermediate product is dissolved in the second solvent, adds hydrazine hydrate, reacts 12~48 hours, isolates and purifies
And obtain connecting the polysaccharide of buzane after drying.
Second solvent is the solvent that can dissolve above-mentioned intermediate product and hydrazine hydrate, preferably water.
Hydrazine hydrate is excessive, so that reaction is carried out.Preferably, the mass ratio of intermediate product and hydrazine hydrate is 5:3.
Preferably, the method isolated and purified is specially：3 are dialysed in ultra-pure water with the bag filter of 3500 molecular cut offs
My god.
It is appreciated that the method not limited to this isolated and purified, any side that the above-mentioned polysaccharide for connecting buzane can be purified
Method can use.
Drying uses vacuum freeze drying.
Retinene and the polysaccharide for connecting buzane being prepared are dissolved in solvent, solvent is preferably dimethyl trident maple, in
After reacting 12~48 hours at room temperature, isolate and purify to obtain polymer micelle.The polysaccharide and retinene for connecting buzane are reacted
The step of generating polymer micelle is as shown in Figure 2.
Preferably, the method isolated and purified is specially：Reactant is dialysed in a solvent 2~5 days, be then ultra-pure water
Middle dialysis 2~5 days, vacuum freeze drying obtains polymer micelle.
Reactant is dialysed in a solvent 2~5 days first, the solvent for dialysis is identical with reaction dissolvent, will be not anti-
The retinene answered removes, and then dialyse 2~5 days in ultra-pure water, other water-solubility impurities are removed, and obtains that purity is high to gather
Compound micella.
The preparation method technique of above-mentioned polymer micelle is simple, and reaction condition is gentle, and energy consumption is low, and it is low to prepare cost.
Referring to Fig. 3, the antineoplastic pharmaceutical compositions 200 of an embodiment, including above-mentioned polymer micelle 100 and load
Antineoplastic on polymer micelle 100.In Fig. 3,100 be above-mentioned polymer micelle, and 40 be antineoplastic.
Antineoplastic is hydrophobic drug, and preferably adriamycin, taxol, Docetaxel, fluorouracil, Changchun are new
Alkali, cis-platinum, carboplatin or retinoic acid.
Hydrophobic antineoplastic is wrapped up by the hydrophobic inner core of above-mentioned polymer micelle 100 and is supported on polymer micelle
On 100.
Preferably, the quality of antineoplastic is the 1%~12% of the quality of antineoplastic pharmaceutical compositions.In the quality proportioning
Under, the coordinative role of antineoplastic and the retinoic acid being transformed by retinene can be preferably played, improves curative effect.
Above-mentioned antineoplastic pharmaceutical compositions 200 use above-mentioned polymer micelle 100 to be used as carrier, the polymer micelle 100
Hydrazone key can be broken under the weakly acidic condition of tumor by local and discharge hydrophobic cores parcel antineoplastic, this is antitumor
The active group of medicine is not destroyed, and antineoplastic can be realized tumor tissues are quick, fixed point, Targeting delivery.And
And because above-mentioned polymer micelle 100 has higher biocompatibility and relatively low cytotoxicity so that the antineoplastic
Composition 200 is smaller to the toxicity of human body, can be widely applied for clinic.
The anti-tumor medicinal preparation of one embodiment, including ultra-pure water and the above-mentioned antineoplastic that is dissolved in ultra-pure water
Composition 200.Ultra-pure water and antineoplastic pharmaceutical compositions 200 form liquid injection.
Preferably, the antineoplastic pharmaceutical compositions 200 concentration in ultra-pure water is 1~10mg/L.
The Zeta potential size of the anti-tumor medicinal preparation is about -14mv.
Using this anti-tumor medicinal preparation treatment tumour, the antineoplastic in said preparation can be in the fast quick-release of target spot
Put, curative effect is preferable；, can be while effectively treatment tumour also, said preparation is smaller to the toxicity of human body, greatly reduction pair
Side effect caused by human body.
The anti-tumor medicinal preparation is mainly used in the treatment of malignant tumour.Using when, be administered using intravenous injection.
The preparation method of the anti-tumor medicinal preparation of one embodiment, comprises the following steps：
Above-mentioned polymer micelle 100 is dissolved in organic solvent, antineoplastic is then added, stirs at room temperature, is mixed
Dialysed after closing uniformly with ultra-pure water, obtain anti-tumor agent.
Antineoplastic is preferably adriamycin, taxol, Docetaxel, fluorouracil, vincristine, cis-platinum, carboplatin
Or retinoic acid.
Organic solvent is the solvent that can dissolve polymer micelle and antineoplastic, preferably dimethyl sulfoxide (DMSO).It is organic
The dosage of solvent, which should ensure that, can fully dissolve polymer micelle and antineoplastic.
Stir at room temperature, be well mixed polymer micelle and antineoplastic.Preferably, stir 30 hours.
Referring to Fig. 4, dialysed after polymer micelle 100 and antineoplastic are well mixed with ultra-pure water, polymer
The self assembly in ultra-pure water of micella 100 and antineoplastic, antineoplastic dredging by polymer micelle 100 by hydrophobic effect
Water kernel is wrapped up and is carried on polymer micelle 100, forms the antineoplastic pharmaceutical compositions being dissolved in ultra-pure water, then carry out nothing
Anti-tumor medicinal preparation is obtained after bacterium processing.
Preferably, the time of ultra-pure water dialysis is 24 hours.
The method of aseptic process is preferably to be filtered with the sterile filters of 220nm.This aseptic process method need not heat,
Heat is avoided to produce harmful effect to antineoplastic pharmaceutical compositions.
The preparation method preparation technology of above-mentioned anti-tumor medicinal preparation is simple, and polymer micelle and antineoplastic are in water
With regard to antineoplastic pharmaceutical compositions, easy to operation, popularization can be self-assembled into.
It is expanded on further below by way of specific embodiment.
Embodiment 1
Prepare anti-tumor medicinal preparation and its characteristic research
（1）Prepare polymer micelle
1g glucans are dissolved in 20ml dimethyl sulfoxide (DMSO)s, 20ml pyridines is added after abundant dissolving, adds 0.618g
P-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, use ethanol precipitation after being reacted 4 hours under condition of ice bath,
Washed 3 times with ethanol again, then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in water, added
Amount hydration hydrazine reaction 24 hours, is then dialysed 3 days using the bag filter of 3500 molecular cut offs in ultra-pure water solution, after freezing
Obtain connecting the polysaccharide of buzane, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 in dimethyl sulfoxide (DMSO), anti-under normal temperature
After answering 24 hours, first dialysed 2 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialyse 3 in ultra-pure water afterwards
My god, polymer micelle is obtained after vacuum freeze drying.
The scanning electron microscope (SEM) photograph of the polymer micelle is shown in Fig. 5.As seen from Figure 5, the particle diameter of the polymer micelle be 90 nanometers~
120 nanometers.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 10 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs Ah mould
Plain 1 mass parts are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, Ran Houyong
The sterile filter filterings of 220nm, obtain anti-tumor medicinal preparation, are expressed as DD.
（3）Test the influence of the release of the adriamycin of above-mentioned anti-tumor medicinal preparation
In order to test the adriamycin release conditions of the anti-tumor medicinal preparation, in preparation process, 24 are dialysed in ultra-pure water
After hour, it is divided into and waits three parts of different bag filters of loading, then bag filter is immersed in pH5.0, pH6.5 and pH7.4 tri- respectively
The phosphate buffer of individual condition of different pH（PBS）In, with 37 DEG C, 150rpm shaking table simulated in vivo environment, different time points take
Outer liquid surveys the concentration of adriamycin, while fills into the identical pH of same volume PBS, as a result as shown in Figure 6.Under the conditions of pH=7.4,
Adriamycin all hardly discharges within 24 hours, and in pH=6.5, also very slowly, but the release of adriamycin is non-in pH=5.0 for release
It is often rapid.Because pH value is about 5.0 in the lysosome of tumour cell, therefore, the anti-tumor medicinal preparation can realize that adriamycin exists
Targeting, quick release in tumour cell.
（4）Above-mentioned anti-tumor medicinal preparation is to MCF-7 Human Breast Cancer Cells toxicity detection
By MCF-7 Human Breast Cancer Cells with 3 × 105/ ml is seeded in 96 orifice plates, per the μ l of hole 100, after overnight incubation respectively
Add free doxorubicin formulations（Dox）, above-mentioned anti-tumor medicinal preparation（DD）With above-mentioned polymeric micelle preparation（DR）, Dox and
In DD, the dosage of adriamycin employs 0.1,0.25,0.5,1,2 and 4 μ g/mL, six different dosage successively, the polymerization in DR
The amount of thing micella is consistent with the amount of adriamycin in DD, and the volume in each hole is 100 μ l.In 37 DEG C, 5%CO2Under the conditions of culture it is 44 small
When, add MTT and continue after cultivating 4 hours, nutrient solution is suctioned out, 150 μ l dimethyl sulfoxide (DMSO)s, purple to be crystallized are added in each hole
Fully after dissolving, OD is read490Value, as a result as shown in Figure 7.It can be seen from Fig. 7 that DD has the work for killing tumour cell well
With illustrating that the adriamycin in DD by cellular uptake and can discharge well, DR has relatively low cytotoxicity.
The test result shows, polymer micelle is to the small toxicity of cell, and after wrapping up adriamycin with the polymer micelle,
It can well discharge, can effectively kill tumour cell.Simple doxorubicin formulations are larger to the toxicity of cell, with above-mentioned polymerization
After thing micella is loaded, smaller cytotoxicity.
（5）MCF-7 Human Breast Cancer Cells are in intake of the different time points to the adriamycin in above-mentioned anti-tumor medicinal preparation
Situation detects
By MCF-7 Human Breast Cancer Cells with 5 × 104/ ml is seeded in 24 orifice plates, per hole 1ml, is added respectively after overnight incubation
Enter free doxorubicin formulations（Dox）With above-mentioned anti-tumor medicinal preparation（DD）, the dosage using adriamycin is 1 μ g/ml, and 1 is small
When, 6 hours, 12 hours and 24 hours four different sample points, the positive of adriamycin is intake of using flow cytomery
Cell number, testing result are as shown in Figure 8.As seen from Figure 8, DD can be absorbed by MCF-7 Human Breast Cancer Cells well, and with
The passage of time, positive cell number is more.
（6）Research of the above-mentioned anti-tumor medicinal preparation in nude mice Anticancer effect in vivo
MCF-7 Human Breast Cancer Cells are inoculated with the nude mice stern dorsal scs of 4~8 weeks（The inoculum concentration of every nude mice be 2 ×
106Cell, cell are resuspended in the DMEM culture mediums of 100ul serum-frees）, 100mm is grown to etc. knurl3After start to treat, experiment is divided into
Physiological saline group（Saline）, free adriamycin group (Dox), above-mentioned polymer micelle group (DR) and above-mentioned anti-tumor medicinal preparation
Group (DD).Dox dosage is 1.5mg/Kg, and DR dosage is suitable with the dosage of the polysaccharide nano-micelle carrier in DD.As a result such as
Shown in Fig. 9.As seen from Figure 9, DD groups can suppress tumour growth, free adriamycin well（Dox）Although have in vitro very
The effect of good kill tumour cell, but antitumous effect is very poor in vivo, and the free adriamycin of this explanation can not enter well
Enter to tumour cell.And DD has the function that good passive target, effective aggregation of the medicine in tumor locus, and energy can be realized
Release is wrapped in the adriamycin in hydrophobic core well, so as to effectively kill tumour cell.And single polymer latex
Shu Zaiti also shows good antitumous effect, and this is embodied mainly due to the effect of retinene.
Embodiment 2
Prepare anti-tumor medicinal preparation
（1）Prepare polymer micelle
1g amyloses are dissolved in 15ml dimethyl sulfoxide (DMSO)s, 15ml pyridines is added after abundant dissolving, adds
0.618g p-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, second is used after being reacted 4 hours under condition of ice bath
Alcohol precipitates, then is washed 3 times with ethanol, is then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in into water
In, add excess hydrazine hydrate and react 4 hours, then dialyse 3 in ultra-pure water solution using the bag filter of 3500 molecular cut offs
My god, obtain connecting the polysaccharide of buzane after lyophilized, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 in dimethyl sulfoxide (DMSO)
After reaction 12 hours, first dialysed 2 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialyse 3 in ultra-pure water afterwards
My god, polymer micelle is obtained after vacuum freeze drying.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 100 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs Japanese yew
The mass parts of alcohol 5 are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, Ran Houyong
The sterile filter filterings of 220nm, obtain anti-tumor medicinal preparation.
Embodiment 3
Prepare anti-tumor medicinal preparation
（1）Prepare polymer micelle
1g sodium alginates are dissolved in 10ml dimethyl sulfoxide (DMSO)s, 10ml pyridines is added after abundant dissolving, adds 0.6g
P-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, sunk after being reacted 10 hours under condition of ice bath with ethanol
Form sediment, then washed 3 times with ethanol, then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in water, added
Enter excess hydrazine hydrate to react 10 hours, then dialysed using the bag filter of 3500 molecular cut offs in ultra-pure water solution 3 days, frozen
Obtain connecting the polysaccharide of buzane after dry, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 react in dimethyl sulfoxide (DMSO)
After 48 hours, first dialysed 5 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialysed 5 days in ultra-pure water afterwards,
Polymer micelle is obtained after vacuum freeze drying.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 100 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs polyenoid
The mass parts of taxol 12 are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water,
Then filtered with the sterile filters of 220nm, obtain anti-tumor medicinal preparation.
Embodiment 4
Prepare anti-tumor medicinal preparation
（1）Prepare polymer micelle
1g hyaluronic acids are dissolved in 20ml dimethyl sulfoxide (DMSO)s, 20ml pyridines is added after abundant dissolving, adds
0.618g p-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, second is used after being reacted 18 hours under condition of ice bath
Alcohol precipitates, then is washed 3 times with ethanol, is then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in into water
In, add excess hydrazine hydrate and react 18 hours, then dialyse 3 in ultra-pure water solution using the bag filter of 3500 molecular cut offs
My god, obtain connecting the polysaccharide of buzane after lyophilized, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 in dimethyl sulfoxide (DMSO)
After reaction 30 hours, first dialysed 4 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialyse 4 in ultra-pure water afterwards
My god, polymer micelle is obtained after vacuum freeze drying.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 99 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs polyenoid
The mass parts of taxol 1 are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, so
Filtered afterwards with the sterile filters of 220nm, obtain anti-tumor medicinal preparation.
Embodiment 5
（1）Prepare polymer micelle
1g hyaluronic acids are dissolved in 20ml dimethyl sulfoxide (DMSO)s, 20ml pyridines is added after abundant dissolving, adds
0.618g p-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, second is used after being reacted 15 hours under condition of ice bath
Alcohol precipitates, then is washed 3 times with ethanol, is then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in into water
In, add excess hydrazine hydrate and react 48 hours, then dialyse 3 in ultra-pure water solution using the bag filter of 3500 molecular cut offs
My god, obtain connecting the polysaccharide of buzane after lyophilized, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 in dimethyl sulfoxide (DMSO)
After reaction 15 hours, first dialysed 2 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialyse 2 in ultra-pure water afterwards
My god, polymer micelle is obtained after vacuum freeze drying.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 99 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs polyenoid
The mass parts of taxol 1 are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, so
Filtered afterwards with the sterile filters of 220nm, obtain anti-tumor medicinal preparation.
Embodiment 6
（1）Prepare polymer micelle
1g cyclodextrin is dissolved in 20ml dimethyl sulfoxide (DMSO)s, 20ml pyridines is added after abundant dissolving, adds 0.618g
P-nitrophenyl chloroformate ester and 37.2mg4- (dimethylamino) pyridine, sunk after being reacted 20 hours under condition of ice bath with ethanol
Form sediment, then washed 3 times with ethanol, then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in water, added
Enter excess hydrazine hydrate to react 32 hours, then dialysed using the bag filter of 3500 molecular cut offs in ultra-pure water solution 3 days, frozen
Obtain connecting the polysaccharide of buzane after dry, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 react in dimethyl sulfoxide (DMSO)
After 42 hours, first dialysed 4 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialysed 4 days in ultra-pure water afterwards,
Polymer micelle is obtained after vacuum freeze drying.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 99 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs Changchun
The new mass parts of alkali 1 are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, then
Filtered with the sterile filters of 220nm, obtain anti-tumor medicinal preparation.
Embodiment 7
Prepare anti-tumor medicinal preparation
（1）Prepare polymer micelle
1g pectin is dissolved in 10ml dimethyl sulfoxide (DMSO)s, 10ml pyridines is added after abundant dissolving, adds 0.6g chlorine
Formic acid p-nitrophenyl ester and 37.2mg4- (dimethylamino) pyridine, ethanol precipitation is used after being reacted 20 hours under condition of ice bath, then
Washed 3 times with ethanol, then washed 2 times with ether, the intermediate product obtained after drying is substantially dissolved in water, added excessive
It is hydrated hydrazine reaction 20 hours, is then dialysed using the bag filter of 3500 molecular cut offs in ultra-pure water solution 3 days, after freezing
To the polysaccharide for connecting buzane, it is 5 in mass ratio to connect the polysaccharide of buzane and retinene:3 react 36 hours in dimethyl sulfoxide (DMSO)
Afterwards, first dialysed 5 days in dimethyl sulfoxide (DMSO), get rid of unreacted retinene, dialysed 5 days in ultra-pure water afterwards, vacuum is cold
It is lyophilized it is dry after obtain polymer micelle.
（2）Prepare anti-tumor medicinal preparation
The above-mentioned mass parts of polymer micelle 100 are measured, is substantially dissolved in the dimethyl sulfoxide (DMSO) of certain volume, weighs cis-platinum
12 mass parts are added in above-mentioned solution, and 30min is stirred under room temperature condition, then are dialysed 24 hours in ultra-pure water, Ran Houyong
The sterile filter filterings of 220nm, obtain anti-tumor medicinal preparation.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (11)
- A kind of 1. polymer micelle, it is characterised in that including the polysaccharide as shell and the retinene as kernel, wherein, institute Retinene is stated to graft on by hydrazone key on the hydroxyl of the polysaccharide；The mass ratio of the polysaccharide and retinene is 10~100:20~500；The retinene is all-trans-retinal or 11-cis retinal；The polymer micelle is spherical；The polymer micelle is pharmaceutical carrier.
- 2. polymer micelle according to claim 1, it is characterised in that the polysaccharide is glucan, sodium alginate, transparent Matter acid, heparin, chondroitin sulfate, amylopectin, pectin, amylose or cyclodextrin.
- 3. a kind of preparation method of polymer micelle, comprises the following steps：The polysaccharide that buzane obtains connecting buzane is connected on the hydroxyl of polysaccharide, retinene and the polysaccharide for connecting buzane are dissolved in In solvent, in normal-temperature reaction 12~48 hours, isolate and purify and the polymer micelle is obtained after drying；Wherein, the mass ratio of the polysaccharide and retinene is 10~100:20~500, the retinene be all-trans-retinal or 11-cis retinal, the obtained polymer micelle are spherical, and the polymer micelle is pharmaceutical carrier.
- 4. the preparation method of polymer micelle according to claim 3, it is characterised in that described to connect on the hydroxyl of polysaccharide Upper buzane, which obtains connecting the step of polysaccharide of buzane, to be included：The polysaccharide is dissolved in the first solvent, then adds pyridine and p-nitrophenyl chloroformate ester, and adds catalyst, in ice Reacted 4~24 hours under the conditions of bath, isolate and purify and obtain intermediate product after drying；AndThe intermediate product is dissolved in the second solvent, adds hydrazine hydrate, is reacted 12~48 hours, after isolating and purifying and drying To the polysaccharide for connecting buzane.
- 5. the preparation method of polymer micelle according to claim 3, it is characterised in that it is described isolate and purify and dry after In the step of obtaining the polymer micelle, the method isolated and purified is specially：Reactant is dialysed 2~5 in the solvent My god, then dialysed 2~5 days in ultra-pure water, vacuum freeze drying obtains the polymer micelle.
- 6. a kind of antineoplastic pharmaceutical compositions, it is characterised in that including the polymer latex as described in any one of claim 1~2 Beam and the antineoplastic being carried on the polymer micelle.
- 7. antineoplastic pharmaceutical compositions according to claim 6, it is characterised in that the antineoplastic be adriamycin, Taxol, Docetaxel, fluorouracil, vincristine, cis-platinum, carboplatin or retinoic acid.
- 8. antineoplastic pharmaceutical compositions according to claim 6, it is characterised in that the quality of the antineoplastic is institute State the 1%~12% of the quality of antineoplastic pharmaceutical compositions.
- A kind of 9. anti-tumor medicinal preparation, it is characterised in that including ultra-pure water and be dissolved in the ultra-pure water as right will Seek the antineoplastic pharmaceutical compositions described in 6.
- 10. anti-tumor medicinal preparation according to claim 9, it is characterised in that the antineoplastic pharmaceutical compositions it is dense Spend for 1~10mg/L.
- 11. a kind of preparation method of anti-tumor medicinal preparation, comprises the following steps：Polymer micelle as described in any one of claim 1~2 is dissolved in organic solvent, then adds antineoplastic, Stir at room temperature, dialysed after well mixed with ultra-pure water, the anti-tumor medicinal preparation is obtained after aseptic process.
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| CN108478532B (en) * | 2018-04-23 | 2020-12-15 | 滨州医学院 | Preparation method of beta cyclodextrin-dipalmitoliposome and application of beta cyclodextrin-dipalmitoliposome as drug carrier |
| CN108851084B (en) * | 2018-06-06 | 2021-06-15 | 福建省农业科学院农业工程技术研究所 | Colon positioning micelle loaded with quercetin and preparation method thereof |
| CN110354095A (en) * | 2019-07-01 | 2019-10-22 | 东南大学 | Target the pH sensitive transparent matter acid nanoparticle and preparation method thereof of atherosclerosis |
| CN112121011A (en) * | 2020-09-17 | 2020-12-25 | 广西大学 | Adriamycin corn starch grafted polymer micelle and preparation method thereof |
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| CN102766262A (en) * | 2012-07-20 | 2012-11-07 | 上海交通大学 | Preparation method for difunctional nanoparticle carrier and preparation method for difunctional nanoparticle preparation |
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| CN102766262A (en) * | 2012-07-20 | 2012-11-07 | 上海交通大学 | Preparation method for difunctional nanoparticle carrier and preparation method for difunctional nanoparticle preparation |
| CN103110955A (en) * | 2012-12-24 | 2013-05-22 | 深圳先进技术研究院 | Nano-drug carrier, reduction response nano-drug granules, nano-drug granular preparation and preparation method thereof |
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