CN105943499B - It is a kind of can be in the drug-loading system and preparation method thereof of tumor locus specific drug release - Google Patents
It is a kind of can be in the drug-loading system and preparation method thereof of tumor locus specific drug release Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- 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
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- 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
- A61K47/02—Inorganic compounds
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
Abstract
The invention discloses one kind can be in the drug-loading system and preparation method thereof of tumor locus specific drug release.The drug-loading system, which refers to, has one layer of acid-sensitive inorganic salts nanometer shell in the water-wet side for the drug-carrying polymer micelle containing disulfide bond and water-wet side carboxylated for containing anti-tumor drug.Experiment shows: the dual responsiveness of reduction and acid can be achieved at the same time in drug-loading system of the present invention, only specific drug release is carried out in tumor locus, targeting is obvious, not only solve the dissolubility of anti-tumor drug, and significantly improve the bioavilability of anti-tumor drug, it is obvious and low to the toxic side effect of other normal tissue organs to the toxic effect of tumour cell, there is significant value to the treatment of tumor disease.
Description
Technical field
The present invention is to be related to one kind to belong to targeting in the drug-loading system and preparation method thereof of tumor locus specific drug release
Medicine-feeding technology field.
Background technique
Tumour has become most important one of the disease for threatening human life and health.Currently, the method master for the treatment of tumour
There are radiotherapy, surgical excision and chemotherapy.Wherein, chemotherapy is the treatment the most commonly used method of tumour, still
There is many deficiencies for traditional administration mode.Such as: the half-life short of most of anticancer drugs in vivo, in tumor locus
Drug concentration is insufficient, causes therapeutic effect not significant；Give reach therapeutic effect dosage can normal tissue organ generate it is tight
The toxic side effect of weight；Anticarcinogen has the characteristics that water-insoluble, and vascular drug delivery is difficult, and bioavilability is low.Therefore, anticancer drug exists
Use clinically is limited to very much, it is necessary to prepare effective delivering that suitable drug-loading system carries out anticancer drug.
Polymer micelle is a kind of currently used medicinal nano carrier, be by amphiphilic block copolymer in water from
The advantages that assembling is formed, and has partial size small, and drugloading rate is high, and poorly water soluble drugs solubility can be improved.But polymer micelle is through dilute
It releases that rear stability is poor, especially passes through the high dilution of blood, can release the drug in advance in body circulation, so that drug biological utilisation
It spends low.In order to solve micella stability problem, there is scholar to be chemically crosslinked on the surface of micella, although the method can increase glue
The stability of beam, but micella can be reduced in the drug release of tumor locus, cause tumour to lead to the problem of multidrug resistance to drug.
Additionally, it is well known that tumor locus group is woven with apparent pathological characters, and such as: tumor locus rich blood vessel, blood vessel
Wall gap is wider, poor structural integrity, lymphatic return missing etc., so that causing the selective high-permeability of nanometer particle
And anelasticity, this phenomenon are also referred to as the high-permeability and retention effect (EPR effect) of solid tumor mass；Meanwhile tumour portion
The blood oxygen wretched insufficiency of position, cell anaerobic respiration makes lactic acid and carbonic acid aggregation cause slightly acidic environment, in addition, anoxic meeting
Gathering for the reducing substances such as inside tumor glutathione is caused to generate stronger reproducibility microenvironment.Therefore, rationally using swollen
The specific microenvironment at tumor position, research and develop it is a kind of can solve polymer micelle stability in vivo and its with tumor locus drug
The drug-loading system of contradictory problems between release will be of great significance to effective delivering of anticancer drug.
Summary of the invention
In view of the above-mentioned problems existing in the prior art and demand, the object of the present invention is to provide one kind can be in tumor locus spy
The drug-loading system and preparation method thereof of opposite sex drug release, provides a kind of effective route of delivery for anticancer drug.
For achieving the above object, The technical solution adopted by the invention is as follows:
It is a kind of can in the drug-loading system of tumor locus specific drug release, refer to contain anti-tumor drug containing two sulphur
The water-wet side of key and the drug-carrying polymer micelle of water-wet side carboxylated has one layer of acid-sensitive inorganic salts nanometer shell.
The drug-carrying polymer micelle containing disulfide bond and water-wet side carboxylated is by containing disulfide bond and water-wet side
The polymer micelle of carboxylated is contained to obtain to anti-tumor drug；It is described containing disulfide bond and water-wet side carboxylated it is poly-
Closing object micella is to be self-assembly of to obtain in deionized water by the polymer containing disulfide bond and water-wet side carboxylated；Described
Polymer containing disulfide bond and water-wet side carboxylated be by with carboxyl end groups or can carboxylated modified end groups hydrophilic polymeric
Object and hydrophobic organic and the small molecule compound containing disulfide bond are copolymerized the graft copolymerization to be formed by amido bond or ester bond
Object.
The anti-tumor drug is hydrophobic anticancer drug, including but not limited to: taxanes, cyclosporine class, Huang
Ketone, camptothecin, vinca, Anthraquinones, berbine and Antitumor Drugs of Podophyllotoxins.
It is described with carboxyl end groups or can the hydrophilic polymers of carboxylated modified end groups be polysaccharide or amino acid polymer,
The polysaccharide includes but is not limited to: hyaluronic acid, heparin, low molecular weight heparin, desulfated heparin, chondroitin, chondroitin sulfate,
Alginic acid, glucose, chitosan and fungi polysaccharide；The amino acid polymer includes but is not limited to: polyhistidyl and poly- bad ammonia
Acid.
The hydrophobic organic includes but is not limited to: deoxycholic aicd, cholic acid, gallbladder alcohol, carboxylated gallbladder alcohol, contain 2~18
The fatty acid of a C, the alkylamine containing 2~18 C and the alkylol containing 2~18 C.
The small molecule compound containing disulfide bond includes but is not limited to: cystamine, 3,3 '-dithiodipropionic acids, 2,
2 '-two sulphur diethanols, N, bis- (propylene acyl) cystamines of N '-, 3,3 '-two thiobis (sulfonic acid Succinimidyl Propionate), 3- (2-
Pyridyidithio) propionic acid N-hydroxy-succinamide ester, 2- pyridyidithio ethylamine, Cystine acrylamide, N, N '-
Bis- (tertbutyloxycarbonyl)-l-cysteine, 3,3 '-two thio third imido dimethyl phthalate dihydrochlorides, bis- (2- metering system) ethoxies
Two sulphur of base and N- [2- (two sulphur of 2- pyridine)] ethylmethyl amide.
The acid-sensitive inorganic salts include but is not limited to: calcium phosphate and calcium carbonate.
Preferably, the polymer containing disulfide bond and water-wet side carboxylated is that deoxycholic aicd-cystamine-is saturating
Bright matter acid graft copolymer, octanoic acid-cystamine-hyaluronic acid graft copolymer, cholic acid-cystamine-hyaluronic acid graft copolymer, gallbladder
Sour -2,2 '-two sulphur diethanols-hyaluronic acid graft copolymer or deoxycholic aicd-cystamine-chondroitin sulfate graft copolymer.
Preferably, the acid-sensitive inorganic salts nanometer shell with a thickness of 5~15nm.
It is of the present invention to include the following steps: in the preparation method of the drug-loading system of tumor locus specific drug release
A) polymer micelle containing disulfide bond and water-wet side carboxylated is prepared；
B) preparation contains the drug-carrying polymer micelle containing disulfide bond and water-wet side carboxylated of anti-tumor drug；
C) pH value of the obtained drug-carrying polymer micelle of regulating step b) is to 7.0~7.4, then with the drug-carrying polymer
Micella is soft template, using interionic active force, is existed using sequence dripping method (sequential addition approach)
The water-wet side of the drug-carrying polymer micelle generates one layer of acid-sensitive inorganic salts nanometer shell.
Furtherly, the preparation of the step a) polymer micelle is to make the polymerization containing disulfide bond and water-wet side carboxylated
Object and deionized water are stirred at room temperature to formation micella.
Furtherly, the preparation of drug-carrying polymer micelle described in step b) is first by anti-tumor drug with can pharmaceutically connect
Dissolved by solvent, then with containing disulfide bond and water-wet side can the polymer micelle of carboxylated carry out ultrasonic mixing, then adopt
Organic solvent is removed with dialysis, is finally separating removing free drug.
Furtherly, sequence dripping method described in step c) includes the following steps:
1. according to the carboxyl mole on drug-carrying polymer micelle surface, be added dropwise in right amount with formed acid-sensitive inorganic salts sun from
Component；
2. the anion source in right amount to form acid-sensitive inorganic salts is added dropwise according to the mole for the cationic source being added dropwise；
3. being alternately and repeatedly added dropwise, to form the acid-sensitive inorganic salts nanometer shell of required thickness.
Preferably, the acid-sensitive inorganic salts are calcium phosphate or calcium carbonate, described to form acid-sensitive inorganic salts
Cationic source is calcium ion source, and the anion source for forming acid-sensitive inorganic salts is phosphate ion sources or carbanion
Source.
As further preferred scheme, the calcium ion source selects calcium nitrate, calcium chloride or calcium sulfate, the phosphate radical from
Component selects disodium hydrogen phosphate or diammonium hydrogen phosphate, and the carbanion source selects sodium carbonate.
Preferably, the number being alternately and repeatedly added dropwise is controlled at 6~18 times.
Experiment shows: after drug-loading system of the present invention enters in vivo, at normal physiological pH, keeping stablizing, not release
Drug is put, but after arrival tumor locus, the acid-sensitive inorganic salts nanometer shell of water-wet side and internal reduction sensitivity key (two sulphur
Key) drug can be caused to discharge from the drug-loading system by the faintly acid of tumor locus and high reproducibility environment Specific lytic
Tumor locus is acted on out, to realize the targeted therapy to tumour.
Compared with prior art, the present invention have following conspicuousness the utility model has the advantages that
The present invention pass through first construct containing disulfide bond and water-wet side can carboxylated polymer micelle, utilize polymer micelle
Anti-tumor drug is contained, is then generated outside one layer of acid-sensitive inorganic salts nanometer in the water-wet side of drug-carrying polymer micelle
Shell not only using the advantages that partial size possessed by polymer micelle is small, drugloading rate is high, but also can solve polymer micelle and be deposited
Stability it is poor, can release the drug in advance in body circulation so that defect problems such as drug bioavailability are low, especially can also be simultaneously
The dual responsiveness for realizing reduction and acid makes the drug-loading system only carry out specific drug release in tumor locus, and targeting is bright
It is aobvious, the dissolubility of anti-tumor drug is not only solved, and significantly improve the bioavilability of anti-tumor drug, it is thin to tumour
The toxic effect of born of the same parents is obvious and low to the toxic side effect of other normal tissue organs, has significant valence to the treatment of tumor disease
Value.
Detailed description of the invention
Fig. 1 is drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid prepared by embodiment 1
Micella) dynamic light scattering diagram；
Fig. 2 is drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid prepared by embodiment 1
Micella) transmission electron microscope picture (A, B) under different times mirrors；
Fig. 3 is transmission electron microscope picture (A, B) of the drug-loading system prepared by embodiment 1 under different times mirrors；
Fig. 4 is the energy spectrum diagram of drug-loading system prepared by embodiment 1；
Fig. 5 is bent with the variation of standing time for the partial size of drug-carrying polymer micelle prepared by embodiment 1 and drug-loading system
Line comparison diagram；
Fig. 6 is the light scatter intensity of drug-carrying polymer micelle prepared by embodiment 1 and drug-loading system with standing time
Change curve comparison diagram；
Fig. 7 is the drug release patterns in vitro of drug-loading system prepared by embodiment 1；
Fig. 8 is that drug-carrying polymer micelle prepared by embodiment 1 and drug-loading system are external anti-under different load concentrations
Function of tumor comparison diagram；
Fig. 9 is that drug-carrying polymer micelle prepared by embodiment 9 and drug-loading system are external anti-under different load concentrations
Function of tumor comparison diagram.
Specific embodiment
The present invention is further explained with attached drawing combined with specific embodiments below.It should be understood that these embodiments are merely to illustrate
The present invention rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to
Normal condition or according to the normal condition proposed by manufacturer.
Embodiment 1
1, the preparation of cystamine modified hyaluronic acid:
0.5mmol hyaluronic acid (HA) is dissolved in the phosphate buffer solution (PBS) of 50mL, 0.01M, pH 7.4, so
2mmol 1- ethyl-(3- dimethylaminopropyl) carbodiimide (EDC) and 0.4mmol n-hydroxysuccinimide are added afterwards
(NHS), it reacts 15 minutes at room temperature；5mmol cystamine (CYS) is added, the reaction was continued at room temperature 4 hours；By reaction solution
It is sealed in 3500 molecular cut off bag filters, dialyses respectively in the sodium chloride solution of 0.1M and deionized water 1 day and 2 days,
And cystamine modified hyaluronic acid intermediate is arrived by being freeze-dried.
2, the preparation of deoxycholic aicd Acibenzolar:
By 1mmol deoxycholic aicd, the dicyclohexyl carbodiimide (DDC) of 1.2mmol, 1.2mmol N- hydroxysuccinimidyl acyl
Imines (NHS) is dissolved in tetrahydrofuran (THF), is reacted under ice bath 30 minutes, is then reacted 12 hours at room temperature；It filters
Precipitating is removed, is precipitated with excessive ice n-hexane, is filtered to get deoxycholic aicd Acibenzolar.
3, deoxycholic aicd-cystamine-hyaluronic acid graft copolymer preparation:
It is that 0.08~0.25:1 feeds intake in formamide according to the molar ratio of deoxycholic aicd Acibenzolar and cystamine hyaluronic acid,
It reacts 24 hours at room temperature, reaction was completed, respectively in the mixed solution of ethyl alcohol and water that volume ratio is 1:1, deionized water
In respectively dialyse 1 day and 2 days, and by freeze-drying to get arrive deoxycholic aicd-cystamine-hyaluronic acid graft copolymer.
4, the preparation of drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella):
It takes 36mg deoxycholic aicd-cystamine-hyaluronic acid graft copolymer to be dissolved in 10mL deionized water, stirs at room temperature
It mixes 15 minutes, obtains polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella)；
Separately antitumor drug paclitaxel (PTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution with it is upper
It states polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella) to be mixed, ultrasound is carried out after being stirred at room temperature 30 minutes
Processing, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, are then with molecular cut off
3500 bag filter is dialysed 12 hours in deionized water, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm, after
0.45 μm of filter membrane is to remove free drug to get (taxol (PTX)-deoxycholic aicd-cystamine-is transparent to drug-carrying polymer micelle
Matter acid micella).
5, the preparation of drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts and carry
Then the pH value of medicine micella is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (calcium nitrate of 0.1M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Aqueous solution), phosphate ion sources are added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
(0.1M disodium hydrogen phosphate aqueous solution) continues stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid of the present invention is arrived
Micella).
Drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella) to above-mentioned preparation and
Drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid micella) performs the following performance tests:
A) measurement of drugloading rate and encapsulation rate:
Medicine assay, determination condition are carried out with HPLC method are as follows:
Mobile phase is acetonitrile: water=55:45 (v/v), and chromatographic column is kromasil C18 column, flow velocity 1mL/min, detection
Wavelength is 227nm, and column temperature is 30 DEG C, and sample volume is 20 μ L；Drugloading rate and encapsulation rate are calculated respectively with formula (1), (2):
Medication amount/drug-carrying polymer micelle total amount × 100 formula (1) in drugloading rate (%)=drug-carrying polymer micelle
Medication amount/dosage × 100 formula (2) in encapsulation rate (%)=drug-carrying polymer micelle
After measured: the encapsulation rate of prepared drug-carrying polymer micelle is 87.69%, and drugloading rate 30.15% illustrates this
The invention polymer micelle can be very good to contain hydrophobicity anti-tumor activity medicine, and it is antitumor can to dramatically increase hydrophobicity
The solubility of drug, and then be expected to improve its bioavilability.
B) particle size determination:
After drug-carrying polymer micelle (PTX- deoxycholic aicd-cystamine-hyaluronic acid micella) is diluted with water 5 times, use
Zetasizer Nanoseries (Malvern, UK) is this implementation in 633nm, 25 DEG C, He-Ne laser determination its partial size, Fig. 1
The dynamic light scattering diagram of drug-carrying polymer micelle (PTX- deoxycholic aicd-cystamine-hyaluronic acid micella) prepared by example, by Fig. 1
It can be seen that: the partial size of drug-carrying polymer micelle prepared by the present embodiment (PTX- deoxycholic aicd-cystamine-hyaluronic acid micella) exists
129 ± 0.86nm, PDI (polydispersity index) are as follows: 0.08 ± 0.014, PDI are smaller, show the partial size of drug-carrying polymer micelle
For Nano grade and it is evenly distributed；Drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid micella)
Zeta potential is -29.9 ± 0.42mV, is in electronegativity.
The partial size being measured in the same method and distribution, as the result is shown: (calcium phosphate nano shell-PTX- is gone prepared drug-loading system
Oxycholic acid-cystamine-hyaluronic acid micella) partial size be 134.8 ± 0.5nm, PDI be 0.11 ± 0.008, Zeta potential be-
16.9 ± 1.17mV, relative to drug-carrying polymer micelle (PTX- deoxycholic aicd-cystamine-hyaluronic acid micella), although partial size has
Increased, but still is Nano grade and is evenly distributed；In addition, according to the bright of the Zeta potential absolute value of prepared drug-loading system
Aobvious decline (being down to 16.9 by 29.9), illustrates that combination has occurred in calcium ion and the negative potential on drug-carrying polymer micelle surface.
C) transmission electron microscope observing:
It is tested with analytic type transmission electron microscope (JEM-2010, Japan), test condition are as follows: acceleration voltage 200kv, light source
La86, minimum beam spot 1.5nm, be put into multiplying power be 50~1,200,000, point resolution 0.25nm, sample tilt angle ± 40 °, energy
Spectral resolution 136ev.
Fig. 2 is drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyalomitome prepared by the present embodiment
Sour micella) transmission electron microscope picture (A, B) under different times mirrors, as seen from Figure 2: prepared drug-carrying polymer micelle is in rule
Spheroidal, and it is uniform in size, appearance structure is good；
Fig. 3 is transmission electron microscope picture (A, B) of the drug-loading system prepared by the present embodiment under different times mirrors, as seen from Figure 3:
Particle shape can also be clearly seen in the case where no negative staining, show to contain organic/inorganic substance on the surface of drug-carrying polymer micelle,
It further relates to generate inorganic salts (calcium phosphate) nanometer shell；
Fig. 4 is the energy spectrum diagram of drug-loading system prepared by the present embodiment, as seen from Figure 4；Amorphous on drug-loading system surface
The essential element of body inorganic matter is calcium and phosphorus, further relates to Surface Creation inorganic salts (calcium phosphate) in drug-carrying polymer micelle
Nanometer shell.
D) stability experiment:
Respectively by the drug-carrying polymer micelle of preparation (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella) and
Drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid micella) is placed in 10% serum solution, is set
In being shaken in 37 DEG C of water bath with thermostatic control shaking casees with 120rpm, respectively in 1,2,4,8,12,24,48,72,96,120h sampling, then
Partial size is measured by step b), observes the variation of the partial size and light scatter intensity of drug-carrying polymer micelle and drug-loading system.
Fig. 5 is bent with the variation of standing time for the partial size of drug-carrying polymer micelle prepared by the present embodiment and drug-loading system
Line comparison diagram；As seen from Figure 5: with the extension of standing time, the partial size of drug-carrying polymer micelle is increased to by 145.5nm
215.2nm (increases 32.4%), and the partial size of drug-loading system increases to 173.4nm (increasing only 10.5%) by 155.2nm,
Illustrate that drug-loading system of the present invention relative to drug-carrying polymer micelle, has apparent stability.
Fig. 6 is the light scatter intensity of drug-carrying polymer micelle prepared by the present embodiment and drug-loading system with standing time
Change curve comparison diagram；As seen from Figure 6: with the extension of standing time, the light scatter intensity of drug-carrying polymer micelle significantly drops
Low (decreasing ratio up to 64.2%), and the light scatter intensity of drug-loading system is not substantially reduced (decreasing ratio only has 15.6%),
This also illustrates that drug-loading system of the present invention has significant stability.
E) vitro drug release is tested:
1mL (0.2mg/mL) drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid is taken respectively
Micella) (molecular cut off 3500) is sealed in bag filter, it is then respectively placed in the following 4 kinds of dissolution mediums of 60mL: 1. also
Former dissolution medium: the PBS of the pH=7.4 containing 0.5% Tween-80 and 40mmol glutathione (GSH)；2. non-reduced release is situated between
Matter: the PBS of the pH=7.4 containing 0.5% Tween-80；3. sour dissolution medium: the PBS of the pH=5.0 containing 0.5% Tween-80；④
Reduction and sour dissolution medium: the PBS of the pH=5.0 containing 0.5% Tween-80 and 40mmol glutathione (GSH)；Again respectively 37
Under the conditions of DEG C, with 120rpm water bath with thermostatic control shaking, taken respectively in 1,2,4,8,12,24,48,72,96,120,144,168,216h
Sample measures the PTX content released, then calculates drug release ratio, draws respective drug release patterns.
Fig. 7 is the drug release patterns in vitro of drug-loading system prepared by the present embodiment, as seen from Figure 7: small by 216
When, in the PBS of pH=7.4, the burst size of the PTX in the drug-loading system is only 34.08%, is shown non-under physiological environment
Often stablize；And in the PBS of pH=5.0, at 24 hours, the burst size of the PTX in drug-loading system just reached 36.1%, 216
Hour in, cumulative release amount reaches 78.61%, show the drug-loading system have apparent sensitivity to acid drug release behavior, this be because
For under acidic environment, corrosion occurs for inorganic salts (calcium phosphate) nanometer shell, so that drug-carrying polymer micelle is exposed to outside, thus
Drug has occurred comparatively fast to discharge；In the PBS of the pH=7.4 of the GSH containing 40mmol, PTX's in 216 hours drug-loading systems
Cumulative release amount is about 67.7%, higher than the cumulative release amount for being free of GSH, shows the drug-loading system also and have significantly to restore quick
Perceptual drug release behavior；Also, under acid and reducing environment (the PBS+40mmol GSH of pH=5.0), the load medicine at 216 hours
For the cumulative release amount of PTX in system up to 91.9%, release drug is most, shows drug-loading system of the present invention while having acid
With reduction dual-sensitivity drug release behavior.
F) to the toxicity test of MDA-MB-231 breast cancer cell:
MDA-MB-231 breast cancer cell is seeded on 96 orifice plates, about 8000, every hole cell, using containing 10% tire ox
The drug-carrying polymer micelle and drug-loading system of prescribed concentration, every group of multiple 6 holes are added in the DMEM culture medium overnight incubation of serum.In
Culture 48 hours in incubator, discard pastille culture medium, and PBS cleaning adds MTS to continue to be incubated for 1 hour, measures the OD value of 490nm.
Cancer cell survival rate is calculated according to formula (3):
Cancer cell survival rate (%)=experimental group OD value/control group OD value × 100 formula (3)
Fig. 8 is that drug-carrying polymer micelle prepared by the present embodiment and drug-loading system are external anti-under different load concentrations
Function of tumor comparison diagram, as seen from Figure 8: under the same conditions, compared to drug-carrying polymer micelle, load medicine system of the present invention
System can be such that the survival rate of cancer cell is substantially reduced, and illustrate that anticancer effect is obvious.
Embodiment 2
1, the preparation of cystamine modified hyaluronic acid:
With described in embodiment 1.
2, the preparation of deoxycholic aicd Acibenzolar:
With described in embodiment 1.
3, deoxycholic aicd-cystamine-hyaluronic acid graft copolymer preparation:
With described in embodiment 1.
4, the preparation of drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella):
With described in embodiment 1.
5, the preparation of drug-loading system (calcium carbonate nano shell-PTX- deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts and carry
Then the pH value of medicine micella is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (nitric acid of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Calcium aqueous solution), carbanion source is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 1 hour
(0.045M aqueous sodium carbonate) continues stirring 1 hour；6~18 times are alternately and repeatedly added dropwise (according to the nanometer shell of required generation
Thickness controlled)；
Alternate dropwise addition terminates, and after stirring 10 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get another drug-loading system (calcium carbonate nano shell-PTX- deoxycholic aicd-cystamine-hyalomitome of the present invention is arrived
Sour micella).
Embodiment 3
1, the preparation of cystamine modified hyaluronic acid:
With described in embodiment 1.
2, octanoic acid-cystamine-hyaluronic acid graft copolymer preparation:
According to mole of the octanoic acid hyaluronic acid modified with 1- ethyl-(3- dimethylaminopropyl) carbodiimide, cystamine
Than feeding intake in formamide for 5:5:1, at room temperature react 24 hours, reaction was completed, respectively volume ratio be 1:1 ethyl alcohol with
It respectively dialyses in the mixed solution of water, in deionized water 1 day and 2 days, and arrives octanoic acid-cystamine-hyalomitome by being freeze-dried
Sour graft copolymer.
3, the preparation of drug-carrying polymer micelle (taxol (PTX)-octanoic acid-cystamine-hyaluronic acid micella):
It takes 60mg octanoic acid-cystamine-hyaluronic acid graft copolymer to be dissolved in 10mL deionized water, is stirred at room temperature 15
Minute, obtain polymer micelle (octanoic acid-cystamine-hyaluronic acid micella)；
Separately antitumor drug paclitaxel (PTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution with it is upper
It states polymer micelle (octanoic acid-cystamine-hyaluronic acid micella) to be mixed, be carried out at ultrasound after being stirred at room temperature 30 minutes
Reason, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, are then with molecular cut off
3500 bag filter is dialysed 12 hours in deionized water, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm, after
0.45 μm of filter membrane arrives drug-carrying polymer micelle (taxol (PTX)-octanoic acid-cystamine-hyaluronic acid to remove free drug
Micella).
4, the preparation of drug-loading system (calcium phosphate nano shell-PTX- octanoic acid-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-octanoic acid-cystamine-hyaluronic acid micella), adjusts and carry medicine glue
Then the pH value of beam is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by sad institute according to the mole of the micellar surface carboxyl
Substituted carboxyl mole), it is that (calcium nitrate of 0.1M is water-soluble for 1:2 dropwise addition calcium ion source according to the molar ratio of calcium ion and carboxyl
Liquid), phosphate ion sources (0.1M is added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
Disodium hydrogen phosphate aqueous solution), continue stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer shell of required generation
Thickness is controlled)；
Alternate dropwise addition terminates, and after stirring 1 hour with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get another drug-loading system (calcium phosphate nano shell-PTX- octanoic acid-cystamine-hyaluronic acid glue of the present invention is arrived
Beam).
Embodiment 4
1, the preparation of drug-carrying polymer micelle (taxol (PTX)-octanoic acid-cystamine-hyaluronic acid micella):
With described in embodiment 3.
2, the preparation of drug-loading system (calcium carbonate nano shell-PTX- octanoic acid-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-octanoic acid-cystamine-hyaluronic acid micella), adjusts and carry medicine glue
Then the pH value of beam is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by sad institute according to the mole of the micellar surface carboxyl
Substituted carboxyl mole), it is that calcium ion source (the calcium nitrate water of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Solution), carbanion source (0.045M is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 5 hours
Aqueous sodium carbonate), continue stirring 5 hours；6~18 times are alternately and repeatedly added dropwise (according to the thickness of the nanometer shell of required generation
It is controlled)；
Alternate dropwise addition terminates, and after stirring 10 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get another drug-loading system (calcium carbonate nano shell-PTX- octanoic acid-cystamine-hyaluronic acid glue of the present invention is arrived
Beam).
Embodiment 5
1, the preparation of 2,2 '-two sulphur diethanol modified hyaluronic acids:
0.5mmol hyaluronic acid (HA) is dissolved in the phosphate buffer solution (PBS) of 50mL, 0.01M, pH 7.4, so
2mmol 1- ethyl-(3- dimethylaminopropyl) carbodiimide (EDC) and 0.4mmol n-hydroxysuccinimide are added afterwards
(NHS), it reacts 15 minutes at room temperature；2,2 '-two sulphur diethanol of 5mmol is added, the reaction was continued at room temperature 24 hours；
Reaction was completed, is precipitated out 2,2 '-two sulphur diethanol modified hyaluronic acid intermediates with acetone, filters and is sealed in retention point
Son amount is freeze-dried the reaction solution dialysed to be dialysed 2 days in 3500 bag filter in deionized water to get to 2,2 '-
Two sulphur diethanol modified hyaluronic acid intermediates.
2,-two sulphur diethanol of cholic acid -2,2 '-hyaluronic acid graft copolymer preparation:
According to cholic acid and the modified hyalomitome of 1- ethyl-(3- dimethylaminopropyl) carbodiimide, 2,2 '-two sulphur diethanols
The molar ratio of acid is that 5:5:1 is dissolved in formamide, is reacted at room temperature 24 hours, reaction was completed, respectively in ethyl alcohol: water (volume ratio 1:
1) it respectively dialyses 1 day and 2 days in mixed solution and deionized water, freeze-drying is to get saturating to 2,2 '-two sulphur diethanol of cholic acid--
Bright matter acid graft copolymer.
3, the system of drug-carrying polymer micelle (- two sulphur diethanol of taxol (PTX)-cholic acid -2,2 '-hyaluronic acid micella)
It is standby:
Cholic acid -2 45mg are taken, 2 '-two sulphur diethanols-hyaluronic acid graft copolymer is dissolved in 10mL deionized water,
It stirs 15 minutes at room temperature, obtains polymer micelle (2,2 '-two sulphur diethanol of cholic acid-- hyaluronic acid micella)；
Separately antitumor drug paclitaxel (PTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution with it is upper
It states polymer micelle (2,2 '-two sulphur diethanol of cholic acid-- hyaluronic acid micella) to be mixed, after being stirred at room temperature 30 minutes
It is ultrasonically treated, ultrasound condition is intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, then with retention
The bag filter that molecular weight is 3500 is dialysed 12 hours in deionized water, to remove ethyl alcohol, and 10 points is centrifuged at 3500rpm
Clock arrives drug-carrying polymer micelle (taxol (PTX)-cholic acid -2,2 '-two after 0.45 μm of filter membrane to remove free drug
Sulphur diethanol-hyaluronic acid micella).
4, the system of drug-loading system (- two sulphur diethanol of calcium phosphate nano shell-PTX- cholic acid -2,2 '-hyaluronic acid micella)
It is standby:
Measure 10mL drug-carrying polymer micelle (- two sulphur diethanol of taxol (PTX)-cholic acid -2,2 '-hyaluronic acid glue
Beam), the pH value of carrier micelle is adjusted to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted by total carboxyl mole on hyaluronic acid used by cholic acid institute according to the mole of the micellar surface carboxyl
Substituted carboxyl mole), it is that (calcium nitrate of 0.1M is water-soluble for 1:2 dropwise addition calcium ion source according to the molar ratio of calcium ion and carboxyl
Liquid), phosphate ion sources (0.1M is added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
Disodium hydrogen phosphate aqueous solution), continue stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer shell of required generation
Thickness is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get another drug-loading system (cholic acid -2 calcium phosphate nano shell-PTX-, 2 '-two sulphur diethyls of the present invention are arrived
Alcohol-hyaluronic acid micella).
Embodiment 6
1, the system of drug-carrying polymer micelle (- two sulphur diethanol of taxol (PTX)-cholic acid -2,2 '-hyaluronic acid micella)
It is standby:
With described in embodiment 5.
2, the system of drug-loading system (- two sulphur diethanol of calcium carbonate nano shell-PTX- cholic acid -2,2 '-hyaluronic acid micella)
It is standby:
Measure 10mL drug-carrying polymer micelle (- two sulphur diethanol of taxol (PTX)-cholic acid -2,2 '-hyaluronic acid glue
Beam), the pH value of carrier micelle is adjusted to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted by total carboxyl mole on hyaluronic acid used by cholic acid institute according to the mole of the micellar surface carboxyl
Substituted carboxyl mole), it is that calcium ion source (the calcium nitrate water of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Solution), carbanion source (0.045M is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 5 hours
Aqueous sodium carbonate), continue stirring 5 hours；6~18 times are alternately and repeatedly added dropwise (according to the thickness of the nanometer shell of required generation
It is controlled)；
Alternate dropwise addition terminates, and after stirring 10 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get another drug-loading system (cholic acid -2 calcium carbonate nano shell-PTX-, 2 '-two sulphur diethyls of the present invention are arrived
Alcohol-hyaluronic acid micella).
Embodiment 7
1, the preparation of the modified chondroitin sulfate of cystamine:
0.171g chondroitin sulfate (CS) is dissolved in 150mL deionized water, 0.575g 1- ethyl-(3- bis- is then added
Dimethylaminopropyl) carbodiimide (EDC) and 0.07g n-hydroxysuccinimide, it reacts 30 minutes at room temperature, then
It is added 1.65g cystamine (CYS), the reaction was continued at room temperature 8 hours；Reaction solution is sealed in the dialysis that molecular cut off is 3500
It in bag, respectively dialyses in the sodium chloride solution of 0.1M and deionized water respectively 1 day and 2 days, and is freeze-dried and changes to get to cystamine
Property chondroitin sulfate intermediate.
2, the preparation of deoxycholic aicd Acibenzolar:
With described in embodiment 1.
3, deoxycholic aicd-cystamine-chondroitin sulfate graft copolymer preparation:
Molar ratio according to deoxycholic aicd Acibenzolar and the modified chondroitin sulfate of cystamine is that 0.08~0.25:1 feeds intake in first
In amide, at room temperature react 24 hours, reaction was completed, respectively volume ratio be 1:1 ethyl alcohol and water mixed solution in, go
It respectively dialyses in ionized water 1 day and 2 days, and arrives deoxycholic aicd-cystamine-chondroitin sulfate graft copolymerization by being freeze-dried
Object.
4, the preparation of drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella):
36mg deoxycholic aicd-cystamine-chondroitin sulfate graft copolymer is taken to be dissolved in 10mL deionized water, at room temperature
Stirring 15 minutes, obtains polymer micelle (deoxycholic aicd-cystamine-chondroitin sulfate micella)；
Separately antitumor drug paclitaxel (PTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution with it is upper
It states polymer micelle (deoxycholic aicd-cystamine-chondroitin sulfate micella) to be mixed, be surpassed after being stirred at room temperature 30 minutes
Sonication, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, then use molecular cut off
It dialyses 12 hours for 3500 bag filter, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm in deionized water, after
0.45 μm of filter membrane arrives drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-sulfuric acid to remove free drug
Chondroitin micella).
5, the preparation of drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-chondroitin sulfate micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella), adjusts
Then the pH value of carrier micelle is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted and is deoxygenated by total carboxyl mole on chondroitin sulfate used according to the mole of the micellar surface carboxyl
Carboxyl mole replaced cholic acid), it is that the calcium ion source (nitric acid of 0.1M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Calcium aqueous solution), phosphate ion sources are added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
(0.1M disodium hydrogen phosphate aqueous solution) continues stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium phosphate nano shell-PTX- deoxycholic aicd-cystamine-chondroitin sulfate of the present invention is arrived
Plain micella).
Embodiment 8
1, the preparation of drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella):
With described in embodiment 7.
2, the preparation of drug-loading system (calcium carbonate nano shell-PTX- deoxycholic aicd-cystamine-chondroitin sulfate micella):
It measures 10mL drug-carrying polymer micelle (taxol (PTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella), adjusts
Then the pH value of carrier micelle is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted and is deoxygenated by total carboxyl mole on chondroitin sulfate used according to the mole of the micellar surface carboxyl
Carboxyl mole replaced cholic acid), it is that the calcium ion source (nitre of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Sour calcium aqueous solution), carbanion is added dropwise for 1.9:1 according still further to the molar ratio of calcium ion and carbanion after stirring 3 hours
Stirring 5 hours is continued in source (0.045M aqueous sodium carbonate)；6~18 times are alternately and repeatedly added dropwise (according to outside the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 8 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium carbonate nano shell-PTX- deoxycholic aicd-cystamine-chondroitin sulfate of the present invention is arrived
Plain micella).
Embodiment 9
1, the preparation of cystamine modified hyaluronic acid:
With described in embodiment 1.
2, the preparation of deoxycholic aicd Acibenzolar:
With described in embodiment 1.
3, deoxycholic aicd-cystamine-hyaluronic acid graft copolymer preparation:
With described in embodiment 1.
4, the preparation of drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-hyaluronic acid micella):
It takes 36mg deoxycholic aicd-cystamine-hyaluronic acid graft copolymer to be dissolved in 10mL deionized water, stirs at room temperature
It mixes 15 minutes, obtains polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella)；
Separately anti-tumor drug Docetaxel (DTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution
It is mixed with above-mentioned polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella), is carried out after being stirred at room temperature 30 minutes
Ultrasonic treatment, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, then with retention molecule
Amount is dialysed 12 hours in deionized water for 3500 bag filter, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm, then
It crosses 0.45 μm of filter membrane and arrives drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-Guang to remove free drug
Amine-hyaluronic acid micella).
5, the preparation of drug-loading system (calcium phosphate nano shell-DTX- deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts
The pH value of carrier micelle is saved to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (calcium nitrate of 0.1M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Aqueous solution), phosphate ion sources are added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
(0.1M disodium hydrogen phosphate aqueous solution) continues stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium phosphate nano shell-DTX- deoxycholic aicd-cystamine-hyaluronic acid of the present invention is arrived
Micella).
6) toxicity test of drug-carrying polymer micelle and drug-loading system prepared by MDA-MB-231 breast cancer cell:
MDA-MB-231 breast cancer cell is seeded on 96 orifice plates, about 8000, every hole cell, using containing 10% tire ox
The drug-carrying polymer micelle and drug-loading system of prescribed concentration, every group of multiple 6 holes are added in the DMEM culture medium overnight incubation of serum.In
Culture 48 hours in incubator, discard pastille culture medium, and PBS cleaning adds MTS to continue to be incubated for 1 hour, measures the OD value of 490nm.
Cancer cell survival rate is calculated according to formula (3):
Cancer cell survival rate (%)=experimental group OD value/control group OD value × 100 formula (3)
Fig. 9 is that drug-carrying polymer micelle prepared by the present embodiment and drug-loading system are external anti-under different load concentrations
Function of tumor comparison diagram, as seen from Figure 9: under the same conditions, compared to drug-carrying polymer micelle, load medicine system of the present invention
System can be such that the survival rate of cancer cell is substantially reduced, and illustrate that anticancer effect is obvious.
Embodiment 10
1, the preparation of drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-hyaluronic acid micella):
With described in embodiment 9.
2, the preparation of drug-loading system (calcium carbonate nano shell-DTX- deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts
The pH value of carrier micelle is saved to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (nitric acid of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Calcium aqueous solution), carbanion source is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 5 hours
(aqueous sodium carbonate of 0.045M) continues stirring 5 hours；6~18 times are alternately and repeatedly added dropwise (according to outside the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 10 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium carbonate nano shell-DTX- deoxycholic aicd-cystamine-hyaluronic acid of the present invention is arrived
Micella).
Embodiment 11
1, deoxycholic aicd-cystamine-chondroitin sulfate graft copolymer preparation:
With described in embodiment 7.
2, the preparation of drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella):
36mg deoxycholic aicd-cystamine-chondroitin sulfate graft copolymer is taken to be dissolved in 10mL deionized water, at room temperature
Stirring 15 minutes, obtains polymer micelle (deoxycholic aicd-cystamine-chondroitin sulfate micella)；
Separately anti-tumor drug Docetaxel (DTX) 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution
Mixed with above-mentioned polymer micelle (deoxycholic aicd-cystamine-chondroitin sulfate micella), be stirred at room temperature 30 minutes it is laggard
Row ultrasonic treatment, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, then with retention point
Son amount is dialysed 12 hours in deionized water for 3500 bag filter, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm,
Drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-Guang is arrived after 0.45 μm of filter membrane to remove free drug
Amine-chondroitin sulfate micella).
3, the preparation of drug-loading system (calcium phosphate nano shell-DTX- deoxycholic aicd-cystamine-chondroitin sulfate micella):
It measures 10mL drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella),
The pH value of carrier micelle is adjusted to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted and is deoxygenated by total carboxyl mole on chondroitin sulfate used according to the mole of the micellar surface carboxyl
Carboxyl mole replaced cholic acid), it is that the calcium ion source (nitric acid of 0.1M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Calcium aqueous solution), phosphate ion sources are added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
(0.1M disodium hydrogen phosphate aqueous solution) continues stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium phosphate nano shell-DTX- deoxycholic aicd-cystamine-chondroitin sulfate of the present invention is arrived
Plain micella).
Embodiment 12
1, the preparation of drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella):
With described in embodiment 11.
2, the preparation of drug-loading system (calcium carbonate nano shell-DTX- deoxycholic aicd-cystamine-chondroitin sulfate micella):
It measures 10mL drug-carrying polymer micelle (Docetaxel (DTX)-deoxycholic aicd-cystamine-chondroitin sulfate micella),
The pH value of carrier micelle is adjusted to 7.0, is then stirred at room temperature with the revolving speed of 400rpm；
It (is subtracted and is deoxygenated by total carboxyl mole on chondroitin sulfate used according to the mole of the micellar surface carboxyl
Carboxyl mole replaced cholic acid), it is that the calcium ion source (nitre of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Sour calcium aqueous solution), carbanion source is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 3 hours
(aqueous sodium carbonate of 0.045M) continues stirring 3 hours；6~18 times are alternately and repeatedly added dropwise (according to outside the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 8 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
It filters to get a kind of drug-loading system (calcium carbonate nano shell-DTX- deoxycholic aicd-cystamine-chondroitin sulfate of the present invention is arrived
Plain micella).
Embodiment 13
1, deoxycholic aicd-cystamine-hyaluronic acid graft copolymer preparation:
With described in embodiment 1.
2, the preparation of drug-carrying polymer micelle (ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella):
It takes 36mg deoxycholic aicd-cystamine-hyaluronic acid graft copolymer to be dissolved in 10mL deionized water, stirs at room temperature
It mixes 15 minutes, obtains polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella)；
Separately anti-tumor drug ciclosporin A 20mg is taken to be dissolved in 600 μ L dehydrated alcohols, by gained drug solution with it is above-mentioned
Polymer micelle (deoxycholic aicd-cystamine-hyaluronic acid micella) is mixed, and is carried out at ultrasound after being stirred at room temperature 30 minutes
Reason, ultrasound condition are intermittent ultrasonic (ultrasonic 3s stops 2s) under ice bath, total ultrasound 30 minutes, are then with molecular cut off
3500 bag filter is dialysed 12 hours in deionized water, to remove ethyl alcohol, and is centrifuged 10 minutes at 3500rpm, after
0.45 μm of filter membrane arrives drug-carrying polymer micelle (ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid to remove free drug
Micella).
3, the preparation of drug-loading system (calcium phosphate nano shell-ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts and carry medicine glue
Then the pH value of beam is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (calcium nitrate of 0.1M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Aqueous solution), phosphate ion sources are added dropwise for 1.6:1 according still further to the molar ratio of calcium ion and phosphate anion after stirring 30 minutes
(0.1M disodium hydrogen phosphate aqueous solution) continues stirring 30 minutes；6~18 times are alternately and repeatedly added dropwise (according to the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 30 minutes with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
To get a kind of drug-loading system of the present invention is arrived, (calcium phosphate nano shell-ciclosporin A-deoxycholic aicd-cystamine-is transparent for filtering
Matter acid micella).
Embodiment 14
1, the preparation of drug-carrying polymer micelle (ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella):
With described in embodiment 13.
2, the preparation of drug-loading system (calcium carbonate nano shell-ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella):
It measures 10mL drug-carrying polymer micelle (ciclosporin A-deoxycholic aicd-cystamine-hyaluronic acid micella), adjusts and carry medicine glue
Then the pH value of beam is stirred at room temperature with the revolving speed of 400rpm to 7.0；
It (is subtracted by total carboxyl mole on hyaluronic acid used by deoxidation gallbladder according to the mole of the micellar surface carboxyl
Carboxyl mole replaced acid), it is that the calcium ion source (nitric acid of 0.045M is added dropwise in 1:2 according to the molar ratio of calcium ion and carboxyl
Calcium aqueous solution), carbanion source is added dropwise for 2:1 according still further to the molar ratio of calcium ion and carbanion after stirring 6 hours
(aqueous sodium carbonate of 0.045M) continues stirring 6 hours；6~18 times are alternately and repeatedly added dropwise (according to outside the nanometer of required generation
The thickness of shell is controlled)；
Alternate dropwise addition terminates, and after stirring 5 hours with the revolving speed of 400rpm, stands 2 hours at room temperature, then dialysed,
To get a kind of drug-loading system of the present invention is arrived, (calcium carbonate nano shell-ciclosporin A-deoxycholic aicd-cystamine-is transparent for filtering
Matter acid micella).
Finally need indicated herein be: the above is only part preferred embodiments of the invention, should not be understood as to this hair
The limitation of bright protection scope, those skilled in the art's above content according to the present invention make it is some it is nonessential improvement and
Adjustment all belongs to the scope of protection of the present invention.
Claims (5)
1. one kind can be in the preparation method of the drug-loading system of tumor locus specific drug release, which comprises the steps of:
A) polymer micelle containing disulfide bond and water-wet side carboxylated is prepared, it may be assumed that make containing disulfide bond and water-wet side carboxylated
Polymer and deionized water be stirred at room temperature to formed micella；
B) preparation contains the drug-carrying polymer micelle containing disulfide bond and water-wet side carboxylated of anti-tumor drug, it may be assumed that will first resist
Tumour medicine is dissolved with pharmaceutically acceptable solvent, then with containing disulfide bond and water-wet side can carboxylated polymer latex
Shu Jinhang ultrasonic mixing, then organic solvent is removed using dialysis, it is finally separating removing free drug；
C) pH value of the obtained drug-carrying polymer micelle of regulating step b) is to 7.0~7.4, then with the drug-carrying polymer micelle
For soft template, using interionic active force, the water-wet side using sequence dripping method in the drug-carrying polymer micelle generates one layer
Acid-sensitive inorganic salts nanometer shell, the sequence dripping method include the following steps:
1. the cation in right amount to form acid-sensitive inorganic salts is added dropwise according to the carboxyl mole on drug-carrying polymer micelle surface
Source；
2. the anion source in right amount to form acid-sensitive inorganic salts is added dropwise according to the mole for the cationic source being added dropwise；
3. being alternately and repeatedly added dropwise, to form the acid-sensitive inorganic salts nanometer shell of required thickness；
Wherein:
The polymer containing disulfide bond and water-wet side carboxylated is by with carboxyl end groups or can carboxylated modified end groups
Small molecule compound of the hydrophilic polymer with hydrophobic organic and containing disulfide bond be copolymerized and to be formed by amido bond or ester bond
Graft copolymer.
2. preparation method according to claim 1, it is characterised in that: it is described have carboxyl end groups or can carboxylated it is modified
The hydrophilic polymer of end group is hyaluronic acid or chondroitin sulfate.
3. preparation method according to claim 1, it is characterised in that: the hydrophobic organic is deoxycholic aicd, cholic acid
Or octanoic acid.
4. preparation method according to claim 1, it is characterised in that: the small molecule compound containing disulfide bond is
Cystamine or 2,2 '-two sulphur diethanols.
5. preparation method according to claim 1, it is characterised in that: the acid-sensitive inorganic salts are calcium phosphate or carbonic acid
Calcium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1859905A (en) * | 2003-10-15 | 2006-11-08 | 日本株式会社Ltt生物医药 | Method of controlling particle size of retinoic acid nanoparticles coated with polyvalent metal inorganic salt and nano particles obtained by the controlling method |
| CN103156811A (en) * | 2013-01-08 | 2013-06-19 | 北京工业大学 | Preparation method for amphiphilic polymer drug micelle with calcium phosphate shell |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1859905A (en) * | 2003-10-15 | 2006-11-08 | 日本株式会社Ltt生物医药 | Method of controlling particle size of retinoic acid nanoparticles coated with polyvalent metal inorganic salt and nano particles obtained by the controlling method |
| CN103156811A (en) * | 2013-01-08 | 2013-06-19 | 北京工业大学 | Preparation method for amphiphilic polymer drug micelle with calcium phosphate shell |
Non-Patent Citations (2)
| Title |
|---|
| Biological evaluation of redox-sensitive micelles based on hyaluronic acid-deoxycholic acid conjugates for tumor-specific delivery of paclitaxel;J.Li，et al;《International Journal of Pharmaceutics》;20150202;第38-48页 |
| Redox-sensitive micelles self-assembled from amphiphilic hyaluronic aciddeoxycholic;J.Li,et al;《Biomaterials》;20111212;第33卷;第2310-2320页 |
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