CN110152016A - A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification - Google Patents
A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification Download PDFInfo
- Publication number
- CN110152016A CN110152016A CN201910573130.7A CN201910573130A CN110152016A CN 110152016 A CN110152016 A CN 110152016A CN 201910573130 A CN201910573130 A CN 201910573130A CN 110152016 A CN110152016 A CN 110152016A
- Authority
- CN
- China
- Prior art keywords
- acid
- oxidisability
- micelle
- polyethylene glycol
- polymer micelle
- 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
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nanotechnology (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses carry medicine targeted nanometer polymer micelle and the preparation method and application thereof.Its composition of the load medicine targeted nanometer polymer micelle includes the PEG-PLGA copolymer that oxidisability ascorbic acid is modified and the hydrophobic drug contained by the copolymer.The present invention helps nano-micelle targeting by oxidisability ascorbic acid (DHA), using amphiphilic polymer nano micelle as pharmaceutical carrier, and anti-cancer medicine paclitaxel is put into micella.In tumor microenvironment, transhipment of the drug to cancer cell is can be improved in the special construction of oxidisability ascorbic acid, improves the simple delivering effect for relying on enhancing osmotic effect, the special nature of nano-micelle solves the solubility problem of anti-cancer medicine paclitaxel at the same time；The treatment of tumour can be effectively performed in the drug.
Description
Technical field
The invention belongs to field of medicaments, and in particular to a kind of targeting of the load taxol of oxidisability ascorbic acid modification is received
Rice glue beam.
Background technique
Taxol (PTX) is the organic matter that the mankind extract from natural products Chinese yew, has good anticancer effect
Fruit, action principle are: it in conjunction with 'beta '-tubulin and can promote its polymerization, inhibit its depolymerization, allow microtubule fasolculus cannot with it is micro-
Tubing center is connected, and induced mitogenesis was blocked in the G2/M phase, so that cell be made Schizoid death occur.However, anti-swollen
The solubility of tumor medicine taxol (PTX) in water is low, clinical preparationUsed solubilizer Cremophor EL
Toxic side effect is big, influences its clinical therapeutic efficacy, albumin taxolThe market price is expensive.
Polymer micelle is widely paid close attention in recent years as a kind of nano-medicament carrier.Polymer micelle it is hydrophobic
Kernel can be used as the memory space of hydrophobic drug, and insoluble drug is contained in wherein；Micella can be improved in its hydrophily shell
Stability in aqueous solution, and micella can be protected, avoid it from being penetrated by reticuloendothelial system and quickly removed in vivo.It is poly-
Close the effect that object micellar particle size is smaller, can deliver by enhancing infiltration retention effect (EPR) raising anti-tumor drug to tumor locus
Fruit.
Summary of the invention
It is an object of the present invention to provide a kind of load medicine targeted nanometer polymerizations for having targeting and treating double effects
Object micella.
Load medicine targeted nanometer polymer micelle provided by the present invention, composition is including copolymer micelle and by described total
The hydrophobic drug that polymers micella contains.
The copolymer is polyethylene glycol-polylactic acid-co-glycolic acid (DHA- of oxidisability ascorbic acid modification
PEG-PLGA) or oxidisability ascorbic acid modification polyethylene glycol-polylactic acid-co-glycolic acid (DHA-PEG-PLGA)
With the mixture of polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA)；Wherein, DHA-PEG- in the mixture
The molar content of PLGA is not less than 15%, preferably not less than 20%, such as 20%.
The partial size for carrying medicine targeted nanometer polymer micelle is 50~200nm, preferably 162~166nm.
The drugloading rate 1~5% for carrying hydrophobic drug in medicine targeted nanometer polymer micelle, preferably 2.22%-
2.42%.
The load medicine targeted nanometer polymer micelle, it is regular spherical shape that surface is observed under transmission electron microscope.
Wherein, polyethylene glycol-polylactic acid-co-glycolic acid of the oxidisability ascorbic acid modification is by Formulas I institute
The oxidisability ascorbic acid and N of two propargylations shown3- PEG-PLGA is prepared by Click reaction.
The N3The weight average molecular weight of-PEG-PLGA is 9000Da, wherein the molecular weight of PEG chain segment is 5000Da；PLGA
The content of segment is 4500Da；
PLGA segment (poly lactic-co-glycolic acid segment) is formed by two kinds of monomer polymerizations of lactic acid and hydroxyacetic acid, wherein cream
The molar ratio of acid and two kinds of monomers of hydroxyacetic acid is 75:25.
The specific preparation method of polyethylene glycol-polylactic acid-co-glycolic acid of the oxidisability ascorbic acid modification,
Include the following steps:
1) the oxidisability ascorbic acid of two propargylations shown in preparation formula I:
A, it reacts L-AA with alkali, obtains oxidisability ascorbic acid shown in Formula II；
B, it reacts oxidisability ascorbic acid with propargyl bromide, obtains the oxidation of two propargylations shown in Formulas I
Property ascorbic acid；
2) in the presence of L-AA sodium, cupric sulfate pentahydrate, keep the oxidisability of two propargylations shown in Formulas I anti-bad
Hematic acid and N3- PEG-PLGA carry out Click reaction to get.
In above method step 2), the reaction carries out in a solvent, and the solvent can be molten for the mixing of DCM and the tert-butyl alcohol
Agent, the two volume ratio can be 1:1.
The condition of the reaction are as follows: carry out in non-oxidizing atmosphere (such as argon gas) and under conditions of being protected from light, stir at room temperature
Mix reaction 8~for 24 hours, preferably 12h.
The step 2) further include: it is described after reaction, in the reaction system be added EDTA solution to remove Cu1+, so
Residue EDTA in alkali neutralization system is added afterwards, until pH meter registration close to 7.00, then evaporates, removes DCM not soluble in water.
Further, the step 2) further include: remaining liquid after above-mentioned processing is placed in 8KD bag filter, and will
Bag filter is put into stirring dialysis 12h in beaker and pours out liquid in beaker after the completion of dialysing, and changes water and continue the 12h that dialyses, it
After be lyophilized and weigh, obtain targeting DHA-PEG-PLGA polymer micelle.
The hydrophobic drug is preferably hydrophobic anticancer drug, excellent such as taxol, adriamycin, camptothecine, curcumin
It is selected as taxol.
It is a further object to provide the preparation methods of above-mentioned load medicine targeted nanometer polymer micelle.
The preparation method provided by the present invention for carrying medicine targeted nanometer polymer micelle, includes the following steps:
Using oxidisability ascorbic acid modification polyethylene glycol-polylactic acid-co-glycolic acid micella as carrier or with
Polyethylene glycol-polylactic acid-the co-glycolic acid and polyethylene glycol-polylactic acid-hydroxyacetic acid of oxidisability ascorbic acid modification
The mixed micelle of copolymer is carrier, is prepared using film hydration method or dialysis and carries medicine targeted nanometer polymer latex
Beam.
Specific step is as follows for the film hydration method: the poly- second two that hydrophobic drug and oxidisability ascorbic acid are modified
Alcohol-poly lactide-glycolide acid micella is dissolved in organic solvent, is removed the organic solvent, is obtained drug and copolymerization
The film of object micella；Then be dissolved in water the film, and ultrasonic treatment obtains the load medicine targeted nanometer polymer micelle.
Organic solvent described in the film hydration method can be methanol.
It is also another object of the present invention to provide the applications for carrying medicine targeted nanometer polymer micelle.
Application provided by the present invention is that the load medicine targeted nanometer polymer micelle is preparing anti-tumor drugs targeting
In application.
Load medicine targeted nanometer polymer micelle prepared by the present invention can effectively increase target-oriented drug.It can be by tumour
Reproducibility environment destroys fracture, so that molecule is fallen off and loses the amphiphilic of original molecule, and then micella is made to disintegrate.Oxidisability Vitamin C
Acid transporter, which enters cancer cell, can be reduced into ascorbic acid, and obvious gradient is formed inside and outside cancer cell.
The problem of in order to solve PTX dissolubility, safety, targeting and drug cost, it is anti-that the present invention is prepared for oxidisability
Polyethylene glycol (PEG)-polylactic acid (PLGA) conjugate of bad hematic acid (DHA) modification；Using anticancer drug PTX as delivery model
Drug is prepared for carrying the nano-micelle (PTX/DHA nano-micelle) of the DHA modification of PTX.Meanwhile in order to improve delivery efficiency, send out
Bright people carries out DHA modification to nano-micelle, to realize the active targeting to tumour.There are transporters in cancer cell, are responsible for
Hydrophilic nutriment and other must small-molecule substance, such as the supply of glucose, lactate and creatine.
Using nuclear magnetic resonance spectroscopy (1H NMR) structural characterization is carried out to DHA-PEG-PLGA conjugate.Pass through dynamic laser
Particle instrument (DLS) characterizes the partial size of nano-micelle with form；Targeting is assessed by CCK-8 method and non-target carries PTX glue
Beam is to 231 cell (human breast cancer cell of breast cancer；MDA-MB-231 cytotoxicity).The results show that DHA modification targeting micella
With good targeting and safety；With the extension of the administration time, targeting micella shows to be better thanIt is external anti-
Tumor promotion；Meanwhile drug cost is lower than albumin taxol
Compared with prior art, the present invention has the advantage that
1, it includes polyethylene glycol, poly lactide-glycolide acid etc. that Nano medication of the invention, which constitutes raw material, in vivo
After degradation overt toxicity will not be generated to body.The nano-micelle tool that polyethylene glycol and poly lactide-glycolide acid are formed
Have amphipathic, solves the problems, such as that taxol drug dissolubility is poor.
2, compared with traditional micella class drug, the present invention in oxidation ascorbic acid, the master to cancer cell may be implemented
Moving-target enhances the targeting of enhancing infiltration retention effect and drug to the accumulation with drug in cell；Oxidisability ascorbic acid
Ascorbic acid can be reduced into cancer cell by transporting, and obvious gradient is formed inside and outside cancer cell.
3, drug molecule taxol of the invention in conjunction with 'beta '-tubulin and can promote its polymerization in the case where being released effectively,
Inhibit its depolymerization, microtubule fasolculus cannot be connected with microtublue organizing center (MTOC), induced mitogenesis was blocked in the G2/M phase, to make thin
There is Schizoid death in born of the same parents.
Detailed description of the invention
Fig. 1 is the synthetic reaction flow chart of polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA).
Fig. 2 isSynthetic reaction flow chart.
Fig. 3 is polyethylene glycol-polylactic acid-co-glycolic acid (DHA-PEG-PLGA) of oxidisability ascorbic acid modification
Synthetic reaction flow chart.
Fig. 4 is oxidisability ascorbic acidSynthesize hydrogen spectrogram.
Fig. 5 is that polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA) synthesizes hydrogen spectrogram.
Fig. 6 is polyethylene glycol-polylactic acid-co-glycolic acid (DHA-PEG-PLGA) of oxidisability ascorbic acid modification
Synthesize hydrogen spectrogram.
Fig. 7 is paclitaxel concentration and ultraviolet light absorption peak area corresponding relationship standard curve.
Fig. 8 is target polymer nano micelle grain size distribution.
Fig. 9 is non-targeted polymer nanocomposite partial size distribution map.
Figure 10 is the cell viability figure of CCK-8 method measurement.
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto, all of the invention
Any modifications, equivalent replacements, and improvements etc. done within spirit and principle, should all be included in the protection scope of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
The target polymer nano micelle for the load taxol that embodiment 1, preparation oxidisability ascorbic acid are modified
One, the synthesis of non-targeted PEG-PLGA polymer:
Synthetic reaction flow chart is shown in Fig. 1.
Weigh PLGA-COOH (Mw=9000Da, wherein the molecular weight of PEG chain segment is 5000Da respectively with balance；PLGA
The content of segment is 4500Da；Composition ratio LA/GA=75/25) 40mg (restoring to room temperature), (the N- maloyl Asia NHS
Amine) 2.3mg and EDC (1- ethyl-(3- dimethylaminopropyl) carbodiimide) 5.73mg is placed in a reaction flask, take DMF 1ml
It is added in bottle and dissolves for solvent.The balloon and reaction flask of appropriate nitrogen are housed with tee tube connection, and multi-purpose using circulating water type
Vacuum pump sufficiently removes air 3~4 times in bottle, and gas is nitrogen in guarantee system.By reactant magnetic agitation in reaction flask
Device stirs the activation intermediate product PLGA-NHS for obtaining PLGA-COOH for 24 hours at room temperature.Weigh 50mg PEG-NH2Reaction is added
In bottle (if dissolved not exclusively, a small amount of DMF can be added dropwise into bottle or ultrasound, heat treatment are carried out to promote to dissolve to solution), after
It is continuous to dialyse after solution stirring 2 hours.It takes out regenerated cellulose bag filter (8KD), removes water-in-bag point.Liquid-transfering gun or drop
The PEG-PLGA solution that synthesis finishes is added into bag filter for pipe, removes extra air, the fixed bag filter both ends of clip cross.
Bag filter is put into the beaker for filling 2L water, and with magnetic stirrer for 24 hours (in dialysis procedure, can every 4h reach balance
The water in beaker is replaced afterwards, is preferably minimized the impurity concentration in bag filter).PEG-PLGA solution is transferred to 15ml EP pipe
In, diaphragm seal, and aperture is left on film with syringe needle, it is put into -80 DEG C of refrigerators and carries out freezing 2h, after freeze-drying
Obtain PEG-PLGA freeze-dried powder (i.e. PEG-PLGA polymer micelle).
The nuclear magnetic resonance spectroscopy (1H NMR) of PEG-PLGA polymer micelle characterizes:
A small amount of PEG-PLGA conjugate powder is weighed, with 500 μ L deuterated chloroforms (CDCl3) for solvent, core is added after dissolution
In magnetic tube.With tetramethylsilane (TMS) for internal standard, the 1H NMR of sample is measured with nuclear magnetic resonance.The method can detect PEG with
Whether the amidation process of PLGA succeeds, and is used to speculate the structure of polymer micelle.As a result as shown in Figure 5.As shown in Figure 5,
The polymer of step synthesis is really target product.
Two,Synthesis
Synthetic reaction flow chart is shown in Fig. 2.
In order to be attached the PEG molecule of DHA molecule and azide functionalization subsequently through Click reaction, we are by DHA
Molecule carries out two propargylations.It weighs potassium hydroxide 0.952g (17mmol) and is placed in round-bottomed flask, 60ml deionized water is added
It makes it dissolve, N2Protection, with the air in vacuum pump displacement round-bottomed flask, to remove oxygen.Weigh L-AA (3g,
It 17mmol) is added in round-bottomed flask, excludes oxygen again, 1h is stirred at room temperature.3.03ml (2eq, 34mmol) propargyl bromide is molten
In 120ml propyl alcohol, 40 DEG C of reaction 48h into reaction flask are added.After reaction, it is evaporated propyl alcohol, ethyl acetate 100ml is added
3 aqueous phase extracted of x collects ethyl acetate layer, and saturated sodium chloride solution is added, and extraction collects ethyl acetate layer, anhydrous sulphur is added
Sour sodium water removal, is evaporated ethyl acetate, obtains brown yellow oil liquid.Crude product crosses 200 mesh~300 mesh silicagel column, eluant, eluent
It is isolated and purified for methylene chloride: acetone=5:1 (v/v), obtains target compoundThrough1H-NMR,13C-HMR
It is identified with mass spectrum.
Three, the synthesis of DHA-PEG-PLGA polymer micelle is targeted
This reaction is in N3It is carried out after the connection of-PEG and PLGA and the synthesis of DHA, specific synthesis step is shown in Fig. 3.This is anti-
Should be attached using DHA as target head with PEG-PLGA polymer, using Vc-Na reproducibility by the copper in cupric sulfate pentahydrate from
Son is restored to Cu1+Using the catalyst as reaction.Due to Cu1+For noxious material, dialyse after the reaction using EDTA solution
To remove the substance.Since EDTA is dissolved in water in acidity, product output will affect, so needing to neutralize it.
It weighs respectively50mg、N3- PEG-PLGA 90mg, VcNa 50mg, cupric sulfate pentahydrate (CuSO4·
5H2O) 5mg is added in reaction flask, and DCM and tert-butyl alcohol 2ml is respectively taken to be dissolved.Prepare argon gas ball and connected with tee tube to react
Bottle, to solution boiling, (main purpose is to remove the extremely strong O of oxidisability to air in circulating water type vacuum pump removing bottle2).Use aluminium foil
Encapsulation reaction bottle (is protected from light), and is stirred to react 12h at room temperature with magnetic stirring apparatus.To after reaction, weigh 7~8mg's
EDTA powder is added in the beaker for filling about 4L water and dissolves.The NaOH solution that 0.5N or 1N is added into beaker carries out EDTA
It neutralizes, until pH meter registration is close to 7.00.It takes out reaction flask and carries out rotary evaporation, remove DCM not soluble in water.
It takes out bag filter (8KD), liquid in reaction flask is sucked out with dropper, dialysis is added after cleaning flask 2~3 times in distilled water
In bag, and bag filter is put into stirring dialysis 12h in beaker.After the completion of dialysing, liquid in beaker is poured out, and changes water and continues thoroughly
Analyse 12h.It is lyophilized and weighs later, obtain targeting DHA-PEG-PLGA polymer micelle.
Four, the preparation of drug-carrying polymer nano-micelle
Two kinds of drug-carrying polymer micelles, target polymer micella (the i.e. targeting DHA- of respectively DHA modification are devised herein
PEG-PLGA polymer micelle) and without the non-targeted polymer micelle (PEG-PLGA polymer micelle) further modified.
In targeting preparation, the 20% of the total PEG-PLGA polymer support of target polymer carrier (DHA-PEG-PLGA) Zhan
(molar ratio)；In non-targeted preparation, polymer support is PEG-PLGA polymer.At the same time, in drug-carrying polymer micelle
In, the molar ratio of chemotherapeutic drug Paclitaxel (PTX) and polymer support is 1:9.Prepare 2 round-bottomed flasks, weighs respectively certain
PTX, PEG-PLGA and DHA-PEG-PLGA of ratio are simultaneously added in flask.Targeting and non-target are respectively obtained using film hydration method
To the polymer micelle of group: a certain amount of methanol is added into flask and is dissolved, after rotary evaporation 30min, with big filter cabin
Vacuum pumping valve removes methanol remaining in bottle, obtains thin film in inner wall.Add suitable quantity of water to be dissolved, flask is placed in ultrasound
30-45min is ultrasonically treated in wave washer, accelerate drug self assembly, obtains the targeting for carrying medicine and non-targeted polymer latex
Beam.
The measurement and calculating of drug-carrying polymer micelle drugloading rate and encapsulation rate
The calculating of drugloading rate:
Using the concentration of PTX in high performance liquid chromatography (HPLC) measurement carrier micelle, drug-carrying polymer is calculated and receives
The drugloading rate of rice glue beam；Its principle be the ultraviolet band absorption peak of PTX PeakArea and the concentration of taxol it is linearly related.
By preparing the PTX acetonitrile solution of various concentration, measures specific UV absorption PeakArea and remove fitting concentration and PeakArea
The standard curve of relationship.Two groups of standard items have been prepared to reduce experimental error.
In the configuration of first group of standard items, prepares 10 EP and manage and be respectively labeled as 1~10, to 2~No. 9 ep Guan Zhongfen
It Jia Ru not 5ml acetonitrile.It takes 0.73mg PTX sample to be placed in No. 1 EP pipe, the dissolution of 10ml acetonitrile is added, keeps sample molten with oscillator
Solution is uniform.5ml solution is taken from No. 1 pipe, is added in No. 2 pipes and sufficiently vibrates.5ml solution is taken from No. 2 pipes, is added in No. 3 pipes
Sufficiently oscillation, and so on.0.77mg PTX sample is taken, and prepares second group of standard items according to above method.Two groups of standard items
In the concentration difference of adjacent EP pipe be 1/2.With the UV absorption peak area in high performance liquid chromatograph measurement standard items, compare dense
Degree obtains standard curve parsing formula according to data processing is carried out.
Prepare 2 EP pipes, it is with non-targeted 100 μ L of nano micellar solution of polymer and female with dilution in acetonitrile to be separately added into targeting
Liquid is put into measurement UV absorption peak area in high performance liquid chromatograph, is updated in standard curve and calculates drug to 600 μ L
Drugloading rate and encapsulation rate.
As a result as follows:
1, the drugloading rate of the targeting polymer nano micelle of the load taxol measured with high performance liquid chromatography is
2.23%；Encapsulation rate is 43.35%；
The targeting polymer nano micelle of the load taxol of above-mentioned preparation, surface are observed under transmission electron microscope
For regular spherical shape；
It is 164 nanometers that laser particle analyzer, which measures and carries the mean particle size of the targeting polymer nano micelle of taxol,；
Its average Zeta potential is measured using laser particle analyzer to be negative 11.6 millivolts.
2, the drugloading rate of the non-target tropism polymer nano micelle of the load taxol measured with high performance liquid chromatography is
3.16%；Encapsulation rate is 60.4%；
The non-target tropism polymer nano micelle of the load taxol of above-mentioned preparation, table are observed under transmission electron microscope
Face is regular spherical shape；
It is 164 nanometers that laser particle analyzer, which measures and carries the mean particle size of the targeting polymer nano micelle of taxol,；
Its average Zeta potential is measured using laser particle analyzer to be negative 11.6 millivolts.
Embodiment 2, pharmacodynamic test
It is thin to triple negative breast cancer MDA-MB-231 that CCK-8 method assessment targeting and non-target carry PTX polymer nano micelle
The cytotoxicity of born of the same parents.
Specific test method is as follows:
1, cell culture
By triple negative breast cancer cell MDA-MB-231 be added containing complete medium (94% basal medium,
5%FBS, 1% is dual anti-), 37 DEG C are placed in, 5%CO2In incubator, liquid is changed every other day.Reach about when cell is adherent
80%-90%, when cell monolayer adherent growth, can carry out secondary culture.
2, plating cells
The cell of logarithmic growth phase, is observed with microscope, if the adherent degree of cell is intensive, can be planted
Plate.In Biohazard Safety Equipment, former culture medium is taken out with dropper, and cleaned with 5mL D-Hanks.2mL pancreatin is added to patch
Parietal cell is dissolved.Dissolution cell is put into constant incubator 3 minutes, then takes out and whether checks cell under the microscope
It completely falls off, next step experiment is carried out if completely falling off.With liquid-transfering gun plus 6mL D-Hanks solution, and blow and beat under ten.It takes out
EP Guan Bingyou balance trim is centrifuged 5 minutes in the case where 1000 rpms under low speed desk centrifuge.After centrifugation, cell
Precipitating occurs and is layered with solution, takes out supernatant liquor with disposable dropper, and a certain amount of complete medium is added, is blown
It beats.
A drop solution is taken out, is dripped on tally, puts the counting for carrying out cell under the microscope later.After counting, it is added
A certain amount of complete medium, adjustment concentration are every milliliter 5 × 103The suspension of a cell, and be inoculated in 96 orifice plates, each
100 μ L of cell suspending liquid is added in hole, and be incubated in incubator makes its adherent for 24 hours.
3, cell toxicity test
After cell incubation for 24 hours after, discard culture solution.Different grouping, including Taxol group, PTX/DHA nano-micelle are set
The non-targeted nano-micelle group of group, PTX and control group.200 μ L are respectively added by test solution into 96 orifice plates for above-mentioned each group, are contained respectively
PTX concentration be 4692 (ng/ml), 938.4,187.68,37.536,7.5072,1.5014,
The culture solution of 0.30029 and 0 (ng/ml) this 7 kinds of various concentrations, parallel 4 holes of each concentration, to be free of cell
Culture medium be blank group, do not given containing cell drug cell be normal group, 37 DEG C, 5%CO2It is incubated in incubator.In dosing
48h afterwards takes out culture plate, and 100 μ L of CCK-8 solution is added in every hole, continues to take out after being incubated for 4h in 37 DEG C of incubators.With enzyme mark
Instrument measures trap at 570nm.And the survival rate of cell is calculated according to the trap in each hole.
4, test result
Under various concentration respectively with carry the targeting of medicine, non-targeted preparation withTumor cell survival test knot
Fruit.The IC of the targeting, non-targeted polymer and PTX drug that are obtained by experimental calculation50Value respectively 0.7727ng/ml,
20.48ng/ml and 152.3ng/ml.The results show that each preparation increases the fragmentation effect of tumour cell with the increase of concentration
By force.Such as Figure 10, three kinds of preparations have good fragmentation effect, and killing of the targeting preparation for tumour cell to tumour cell
Ability is strongest in three.
Claims (10)
1. a kind of load medicine targeted nanometer polymer micelle, composition includes copolymer micelle and is contained by the copolymer micelle
Hydrophobic drug；
The copolymer is that polyethylene glycol-polylactic acid-co-glycolic acid of oxidisability ascorbic acid modification or oxidisability resist
Polyethylene glycol-polylactic acid-the co-glycolic acid and polyethylene glycol-polylactic acid-co-glycolic acid of bad hematic acid modification
Mixture；Wherein, polyethylene glycol-polylactic acid-co-glycolic acid that oxidisability ascorbic acid is modified in the mixture
Molar content is not less than 15%.
2. load medicine targeted nanometer polymer micelle according to claim 1, it is characterised in that: the load medicine targeting is received
The partial size of rice polymer micelle is 50~200nm, preferably 162~166nm；
The drugloading rate 1~5% for carrying hydrophobic drug in medicine targeted nanometer polymer micelle, preferably 2.22%-
2.42%.
3. load medicine targeted nanometer polymer micelle according to claim 1 or 2, it is characterised in that: the oxidisability is anti-
Polyethylene glycol-polylactic acid-co-glycolic acid of bad hematic acid modification is that the oxidisability of two propargylations as shown in Formulas I resists
Bad hematic acid and N3Polyethylene glycol-polylactic acid-co-glycolic acid is prepared by Click reaction；
4. load medicine targeted nanometer polymer micelle according to claim 3, it is characterised in that: the N3Polyethylene glycol-
Poly lactic-co-glycolic acid is abbreviated as N3- PEG-PLGA, weight average molecular weight 9000Da, wherein the molecular weight of PEG chain segment is
5000Da；The content of PLGA segment is 4500Da；
PLGA segment is formed by two kinds of monomer polymerizations of lactic acid and hydroxyacetic acid, wherein mole of two kinds of monomers of lactic acid and hydroxyacetic acid
Than for 75:25.
5. load medicine targeted nanometer polymer micelle according to claim 3 or 4, it is characterised in that: the oxidisability is anti-
Polyethylene glycol-polylactic acid-co-glycolic acid preparation method of bad hematic acid modification, includes the following steps:
1) the oxidisability ascorbic acid of two propargylations shown in preparation formula I:
A, it reacts L-AA with alkali, obtains oxidisability ascorbic acid；
B, react oxidisability ascorbic acid with propargyl bromide, the oxidisability for obtaining two propargylations shown in Formulas I is anti-
Bad hematic acid；
2) in the presence of L-AA sodium, cupric sulfate pentahydrate, make the oxidisability ascorbic acid of two propargylations shown in Formulas I
With N3- PEG-PLGA carry out Click reaction to get.
6. load medicine targeted nanometer polymer micelle according to claim 5, it is characterised in that: in the step 2), institute
It states reaction to carry out in a solvent, the solvent is the mixed solvent of DCM and the tert-butyl alcohol, and the two volume ratio is 1:1；
The condition of the reaction are as follows: it is carried out in non-oxidizing atmosphere and under conditions of being protected from light, it is stirred to react 8 at room temperature~for 24 hours,
It is preferred that 12h；
The step 2) further include: it is described after reaction, in the reaction system be added EDTA solution to remove Cu1+, then plus
The step of entering residue EDTA in alkali neutralization system, until pH meter registration close to 7.00, then evaporates, removes DCM not soluble in water；
Further, the step 2) further include: liquid remaining after processing is placed in 8KD bag filter, and bag filter is put
Enter stirring dialysis 12h in beaker and pour out liquid in beaker after the completion of dialysing, and change water and continue the 12h that dialyses, freezing is dry later
It is dry, obtain targeting DHA-PEG-PLGA polymer micelle.
7. load medicine targeted nanometer polymer micelle according to claim 1 to 6, it is characterised in that: described to dredge
Aqueous pharmaceutical is hydrophobic anticancer drug, including taxol, adriamycin, camptothecine, curcumin.
8. carrying the preparation method of medicine targeted nanometer polymer micelle described in any one of claim 1-7, include the following steps:
Resist using polyethylene glycol-polylactic acid-co-glycolic acid micella of oxidisability ascorbic acid modification as carrier or with oxidisability
Polyethylene glycol-polylactic acid-the co-glycolic acid and polyethylene glycol-polylactic acid-co-glycolic acid of bad hematic acid modification
Mixed micelle is carrier, is prepared using film hydration method or dialysis and carries medicine targeted nanometer polymer micelle.
9. preparation method according to claim 8, it is characterised in that: specific step is as follows for the film hydration method: will
Hydrophobic drug and polyethylene glycol-polylactic acid-co-glycolic acid micella of oxidisability ascorbic acid modification are dissolved in organic
In solvent, the organic solvent is removed, obtains the film of drug and copolymer micelle；Then be dissolved in water the film, ultrasound
Processing, obtains the load medicine targeted nanometer polymer micelle.
10. load medicine targeted nanometer polymer micelle of any of claims 1-7 is preparing anti-tumor drugs targeting
In application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910573130.7A CN110152016A (en) | 2019-06-28 | 2019-06-28 | A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910573130.7A CN110152016A (en) | 2019-06-28 | 2019-06-28 | A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110152016A true CN110152016A (en) | 2019-08-23 |
Family
ID=67637159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910573130.7A Pending CN110152016A (en) | 2019-06-28 | 2019-06-28 | A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110152016A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115844822A (en) * | 2022-11-23 | 2023-03-28 | 上海交通大学医学院附属第九人民医院 | Oral drug-loaded micelle composition and preparation method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006113602A2 (en) * | 2005-04-15 | 2006-10-26 | Board Of Trustees Of Michigan State University | Ascorbate binding peptides |
| CN104277047A (en) * | 2013-07-08 | 2015-01-14 | 复旦大学 | Targeting functional molecule with glucose transporter affinity |
| CN104274834A (en) * | 2013-07-08 | 2015-01-14 | 复旦大学 | Environment-sensitive tumor-targeting polymer micelle and preparation method thereof |
| CN105646887A (en) * | 2016-01-05 | 2016-06-08 | 湖北大学 | Amphipathic high-molecular polymer, and preparation method and application thereof |
| CN106137962A (en) * | 2016-07-27 | 2016-11-23 | 毕云科 | A kind of glioma target polymer micelle loading carmustine and preparation method thereof |
| CN106265508A (en) * | 2015-06-24 | 2017-01-04 | 复旦大学附属华山医院 | The load paclitaxel targeting cross linked polymer micelle of a kind of DHA modification and preparation method and application |
| CN108245483A (en) * | 2016-12-28 | 2018-07-06 | 中国人民解放军第二军医大学 | A kind of polymer nano micelle system for containing insoluble anti-tumor medicament |
| US20190029970A1 (en) * | 2017-07-31 | 2019-01-31 | The Chinese University Of Hong Kong | Fatty acid conjugated nanoparticles and uses thereof |
-
2019
- 2019-06-28 CN CN201910573130.7A patent/CN110152016A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006113602A2 (en) * | 2005-04-15 | 2006-10-26 | Board Of Trustees Of Michigan State University | Ascorbate binding peptides |
| CN104277047A (en) * | 2013-07-08 | 2015-01-14 | 复旦大学 | Targeting functional molecule with glucose transporter affinity |
| CN104274834A (en) * | 2013-07-08 | 2015-01-14 | 复旦大学 | Environment-sensitive tumor-targeting polymer micelle and preparation method thereof |
| CN106265508A (en) * | 2015-06-24 | 2017-01-04 | 复旦大学附属华山医院 | The load paclitaxel targeting cross linked polymer micelle of a kind of DHA modification and preparation method and application |
| CN105646887A (en) * | 2016-01-05 | 2016-06-08 | 湖北大学 | Amphipathic high-molecular polymer, and preparation method and application thereof |
| CN106137962A (en) * | 2016-07-27 | 2016-11-23 | 毕云科 | A kind of glioma target polymer micelle loading carmustine and preparation method thereof |
| CN108245483A (en) * | 2016-12-28 | 2018-07-06 | 中国人民解放军第二军医大学 | A kind of polymer nano micelle system for containing insoluble anti-tumor medicament |
| US20190029970A1 (en) * | 2017-07-31 | 2019-01-31 | The Chinese University Of Hong Kong | Fatty acid conjugated nanoparticles and uses thereof |
Non-Patent Citations (4)
| Title |
|---|
| MA HAOJUN等: "Dehydroascorbic Acid and pGPMA Dual Modified pH-Sensitive Polymeric Micelles for Target Treatment of Liver Cancer", 《JOURNAL OF PHARMACEUTICAL SCIENCES》 * |
| PEI XIAOYU等: "Dehydroascorbic Acids-modified Polymer Micelles Target Cancer Cells to Enhance Anti-tumor Efficacy of Paclitaxel", 《SCIENTIFIC REPORTS》 * |
| 王伟: "《肿瘤药剂学》", 30 November 2016, 江苏凤凰科学技术出版社 * |
| 苏博，等: "载多柔比星PEG-PLGA纳米胶束制备及其抗三阴性乳腺癌作用", 《中国新药与临床杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115844822A (en) * | 2022-11-23 | 2023-03-28 | 上海交通大学医学院附属第九人民医院 | Oral drug-loaded micelle composition and preparation method thereof |
| CN115844822B (en) * | 2022-11-23 | 2024-05-14 | 上海交通大学医学院附属第九人民医院 | Oral drug-loaded micelle composition and preparation method thereof |
| WO2024109964A1 (en) * | 2022-11-23 | 2024-05-30 | 上海交通大学医学院附属第九人民医院 | Oral drug-loaded micelle composition and preparation method therefor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105833284B (en) | The building of taxol-oleic acid small molecule prodrugs self-assembled nanometer grain | |
| Kim et al. | Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel | |
| CN104177624B (en) | Dual Sensitive amphipathic three block copolymer containing disulfide bond and acylhydrazone key and preparation method and application | |
| CN103705943B (en) | A kind of preparation reducing response type pegylated medicament nano-composition and application thereof | |
| CN105669964B (en) | Biodegradable amphiphilic polymers, polymer vesicle prepared therefrom and the application of oophoroma special target | |
| CN101679021A (en) | Particulate drug delivery | |
| CN108559040B (en) | Polymer drug carrier, carrier micelle with redox response and AIE effect and preparation method thereof | |
| CN105617394A (en) | Self-assembled nano-system of unsaturated fatty acid-anti-tumor drug conjugates as well as preparation method and application thereof | |
| Li et al. | Antitumor drug Paclitaxel-loaded pH-sensitive nanoparticles targeting tumor extracellular pH | |
| CN107149592A (en) | Biological self-assembly nano-crystalline injection and preparation method with lympha targeted function | |
| CN104224710B (en) | A kind of docetaxel nanometer micella, its preparation method and application | |
| CN110483785A (en) | A kind of triblock polymer, medicament-carried nano micelle, Nano medication and its preparation method and application | |
| CN106421808A (en) | Preparation and application of hydroxyethyl starch modified anti-tumor medicine conjugate and assembling nanometer system thereof | |
| CN108144067A (en) | Tetravalence platinum compounds-bicyclic double bond amphipathic nature polyalcohol prodrug, its nano-micelle and preparation method and application | |
| CN105859990B (en) | The polymer of side chain sulfur-bearing caprylyl, its preparation method and polymer vesicle prepared therefrom and its application | |
| CN112089845A (en) | Taxane drug-adriamycin prodrug self-assembly nanoparticles and application thereof | |
| CN106883404A (en) | TPGS derivative and its preparation method and application | |
| CN107266384A (en) | N carboxyl inner-acid anhydride monomers and polyaminoacid based on 2 aminohexadecanoic acids and preparation method thereof | |
| Saeedi et al. | Redox and pH dual sensitive folate-modified star-like amphiphilic copolymer based on castor oil for controlled doxorubicin delivery | |
| CN110152016A (en) | A kind of targeted nanometer micella of the load taxol of oxidisability ascorbic acid modification | |
| CN106176602A (en) | A kind of targeting is in the Docetaxel chitosan nano-micelle of stomach organization and preparation method and application | |
| CN109180835A (en) | For antileukemie GSH/pH responsive nano drug delivery system and preparation method thereof | |
| CN107693505A (en) | Double dissolubility ROS sensitive nanoparticles of a kind of new profit and preparation method thereof | |
| CN111592605A (en) | Hyaluronic acid-cystamine-oleic acid polymer and application thereof in drug delivery | |
| CN106265513B (en) | A kind of effect of nano-paclitaxel and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190823 |