CN107233577A - A kind of pH responses and the double medicine-carried nano particles and preparation method of cancer target and application - Google Patents
A kind of pH responses and the double medicine-carried nano particles and preparation method of cancer target and application Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
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- 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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/51—Nanocapsules; Nanoparticles
- A61K9/5107—Excipients; Inactive ingredients
- A61K9/513—Organic macromolecular compounds; Dendrimers
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
Abstract
Double medicine-carried nano particles of cancer target for the pH responses modified the invention discloses a kind of folic acid and poly-dopamine and preparation method and application, are prepared as：(1) preparation of hydrophobic drug nano-particle is carried；(2) modification of poly-dopamine；(3) modification of targeting ligand folic acid；(4) double drug incorporations of pH responses.The inventive method is simple, and wide adaptability, prepared nano-particle has the targeting of good biocompatibility, biodegradability and kinds of tumors, and double load medicines and pH response characteristics make it have good therapeutic effect.
Description
Technical field
The present invention relates to a kind of pH responses and the double medicine-carried nano particles and preparation method of cancer target and application.
Background technology
In recent years, cancer turns into the clinically main fatal disease of a class, and general tumor therapy is mainly controlled including operation
Treatment, chemotherapy and radiotherapy etc..Although these therapies are still clinically more effective method, they some at present
Weak point is also worth noting：Invasive treatment means and various toxic side effects usually bring larger pain to patient；Pass
The administering mode of system causes small-molecule drug random distribution in vivo, it is impossible to be effectively enriched in tumor locus, and a large amount of medications
The resistance to the action of a drug can be brought；These treatment means cannot distinguish between normal cell and tumour cell so that the medicine of internal random distribution exists
Other normal cells are caused damage while killing tumor cell.
Medicine delivery based on targeted nano carrier is a kind of Critical policies for overcoming traditional remedies shortcoming, and it passes through connection
The antineoplastic that the nano-carrier of targeting ligand is loaded accurately is delivered to tumor tissues and realizes that the control of medicine is released
Put.Targeted nano medicine has the following advantages：The solubility of hydrophobic drug is effectively increased based on nano-carrier；Pass through hydrophily base
Group modifies to extend the circulation time of medicine in vivo；Improve the bio distribution of medicine in vivo by targeting ligand.Using
This strategy enables medicine to be enriched in tumor tissues and specifically killing tumor cell, is reduced while improving curative effect
Toxic side effect.Meanwhile, polymer nano-particle is a class biodegradable material, wide with good biocompatibility
It is general to be applied to pharmaceutical carrier.Therefore, target polymer nano pharmaceutical carrier has the prospect for improving curative effect and the possibility of realization
Property.
The targeted molecular modification of nanoparticle surface is key technology therein, directly nanoparticle surface modified and
The method of previously prepared polymer-targeted molecular copolymer has the disadvantages that：Coupling agent, catalyst of use etc. need to pass through
Complicated purge process is rejected；Complicated reaction mechanism considerably increases the difficulty of synthesis, reduces the versatility of method.
Poly-dopamine is that a kind of have good biocompatibility and can stick to the surface modifier of surfaces of various materials.It is based on
Michal addition or Schiff base reactions, the targeted molecular with amino or sulfydryl can be in alkalescence condition
Under be advantageously connected on the poly-dopamine molecule for stick to nanoparticle surface to realize the preparation of targeted nano-particle.This
The method of kind can realize the simple and general preparation technology of targeted nano-particle.
Folacin receptor mediated target administration passes through folic acid and tumor cell surface mistake mainly using folacin receptor as target spot
The folacin receptor specific binding of expression, and then pharmaceutical carrier endocytosis is entered into cell.Research shows that folacin receptor is including breast
Several Kinds of Malignancy surface including gland cancer, cervical carcinoma, adenocarcinoma of lung, carcinoma of endometrium and oophoroma etc. is overexpressed, and sometimes may be used
100~300 times are higher by than normal structure.Therefore, folacin receptor mediated Nano medication goes for including breast cancer and palace
The treatment of Several Kinds of Malignancy including neck cancer.
Catechol and boric acid can generate the structure being stably covalently attached under neutral or basic conditions, while the structure exists
Catechol and boric acid can be cracked under acid condition, with invertibity, this process is as shown below.Due to poly-dopamine molecule
Upper sufficient catechol group and the cancer therapy drug bortezomib (BTZ) with boric acid structure, can be designed as downloaded medicine plan
Slightly：The medicine-carried nano particles of poly-dopamine surface modification can load bortezomib under neutral or basic conditions, in neutrality
Poly-dopamine effectively inhibits the activity of medicine with bortezomib stable bond in blood circulation, is released in acid tumor tissues
Put medicine and killing tumor cell.The strategy can reach double effects for carrying medicine and pH responses simultaneously, can greatly improve curative effect.
The content of the invention
The present invention provides double medicine-carried nano particles of a kind of pH responses and cancer target and preparation method and application.
Specifically, one aspect of the invention provides the preparation side of double drug-carrying nanometer particles of a kind of pH responses and cancer target
Method, it comprises the following steps：
(1) polymer nanoparticle for carrying hydrophobic drug is prepared；
(2) polymer nanoparticle for carrying hydrophobic drug is modified with poly-dopamine；
(3) polymer nano-particle for modifying poly-dopamine load hydrophobic drug with active targeting part is modified,
Obtain the polymer nanoparticle of active targeting part and the load hydrophobic drug of poly-dopamine modification；
(4) the load hydrophobic drug for modifying the drug loading with boric acid structure in active targeting part and poly-dopamine
Polymer nanoparticle on, obtain pH response and cancer target double drug-carrying nanometer particles.
In the inventive solutions, in step 4) in, drug loading is in pH in 7.5-12 weak alkaline aqueous solution
Carry out, the medicine with boric acid structure is dissolved in organic solvent, add suspension active targeting part and poly-dopamine modification
It is complete to reacting in the weak alkaline aqueous solution for the polymer nanoparticle for carrying hydrophobic drug, double drug-carrying nanometer particles are obtained after separation；
It is highly preferred that the mass ratio of the nanoparticle and bortezomib of active targeting part and poly-dopamine modification is 100:1-
20, more preferably 100:8-16.
The organic solvent is selected from acetone, dioxane, dimethyl sulfoxide (DMSO), dichloromethane, acetonitrile, tetrahydrofuran or N, N-
Dimethylformamide.
In the inventive solutions, step 1) for polymer and hydrophobic drug are dissolved in organic solvent, and add
Enter in the TPGS aqueous solution, reaction is complete to organic solvent volatilization, precipitation and separation, obtain carrying hydrophobic
The polymer nano-particle of property medicine；
Wherein, the polymer be selected from PLA, it is polyglycolic acid, Poly(D,L-lactide-co-glycolide, polycaprolactone, poly-
Ethylene glycol-Poly(D,L-lactide-co-glycolide, cholic acid-Poly(D,L-lactide-co-glycolide, polyethylene glycol 1000 vitamin E amber
One or more in amber acid esters-PLA；
The hydrophobic drug is selected from taxol, Docetaxel, Coumarin-6, cis-platinum, brufen, 10- hydroxy-camptothecins
One or more in alkali；
The organic solvent is selected from acetone, dioxane, dimethyl sulfoxide (DMSO), dichloromethane, acetonitrile, tetrahydrofuran or N, N-
Dimethylformamide.
In the inventive solutions, step 2) it is that the polymer nanoparticle for carrying hydrophobic drug is suspended in into pH to be
In 7.5-12 weak alkaline aqueous solution, dopamine hydrochloride is added, is reacted to complete, precipitation and separation, poly-dopamine modification is obtained
Load hydrophobic drug polymer nanoparticle.
In the inventive solutions, step 3) it is the polymer nanoparticle for carrying hydrophobic drug that poly-dopamine is modified
It is suspended in the weak alkaline aqueous solution that pH is 7.5-12, active targeting part is added in above-mentioned solution, reaction divides to complete
From the polymer nanoparticle that precipitation obtains active targeting part and the load hydrophobic drug of poly-dopamine modification；
In the inventive solutions, active targeting part be selected from folic acid polyethylene glycol amino, galactosamine (Gal),
A kind of polypeptide (Arg-Gly-Asp, RGD), aptamer sulfydryl (Aptamer-SH).
In the inventive solutions, step 2,3) or 4) in pH being selected from for 7.5-12 weak alkaline aqueous solution independence
One kind in PBS cushioning liquid, Tris cushioning liquid, aqueous sodium carbonate, aqueous sodium bicarbonate or sodium hydrate aqueous solution；
Preferably, pH is 8-9, more preferably 8.5.
Another aspect of the present invention provides double drug-carrying nanometer particles prepared by foregoing preparation method.
Another aspect of the invention provides a kind of pair of drug-carrying nanometer particle, and it is included the nanoparticle formed with polymer and carried
Hydrophobic drug is loaded on body skeleton, nanoparticulate carriers skeleton, nanoparticulate carriers skeleton surface is covered with poly-dopamine, poly- DOPA
Sticking on amine has the catechol group in active targeting part, poly-dopamine to also incorporate the medicine with boric acid base group；It is described
Double drug-carrying nanometer particles are the nanoparticle that pH is responded, and medicine is discharged in acid condition.
In the inventive solutions, the polymer in double drug-carrying nanometer particles is selected from PLA, polyglycolic acid, gathered
Poly lactic coglycolic acid, polycaprolactone, polyethylene glycol-polylactic acid-co-glycolic acid, cholic acid-polylactic acid-glycolic base
One or more in acetate multipolymer, polyethylene glycol 1000 vitamin E succinic acid ester-PLA；
The hydrophobic drug is selected from taxol, Docetaxel, Coumarin-6, cis-platinum, brufen, 10- hydroxy-camptothecins
One or more in alkali；
Medicine with boric acid structure is selected from bortezomib；
Active targeting part be selected from folic acid polyethylene glycol amino, galactosamine (Gal), a kind of polypeptide (Arg-Gly-Asp,
RGD), aptamer sulfydryl (Aptamer-SH).
Another aspect of the invention is preparing the application of antineoplastic there is provided foregoing double medicine-carried nano particles.Institute
State tumour and be selected from breast cancer, cervical carcinoma, adenocarcinoma of lung, carcinoma of endometrium and oophoroma.
The concrete technical scheme of the present invention includes：
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification, including
Following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg polymer is weighed, 2-10mg hydrophobic drugs are dissolved in 8-
In 10mL organic solvents, under conditions of being stirred at room temperature, 100mL 0.03% (w/v) polyethylene glycol dimension life is added dropwise to
In the plain E succinates aqueous solution, continue to be stirred at room temperature 10-15 hours with volatile organic solvent, 15000-18000rpm centrifugations 15-
30 minutes, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain carrying the polymer nano-particle of hydrophobic drug, freezing is dry
It is dry rear standby；
(2) modification of poly-dopamine：In proportion, the pH that nano-particle prepared in 50mg (1) is resuspended in 50mL is weighed
In=7.5-12 weak alkaline aqueous solution, 10-50mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 15000-18000rpm is centrifuged 15-30 minutes after reacting 3-6 hours, abandons supernatant, and precipitation is washed with deionized 3
It is secondary, obtain the polymer nano-particle of the load hydrophobic drug of poly-dopamine modification, freeze-dried back；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
PH=7.5-12 weak alkaline aqueous solution in, 50-100mg cancer target part folic acid polyethylene glycol amino is weighed, in room temperature
It is added under conditions of stirring in above-mentioned solution, 15000-18000rpm is centrifuged 15-30 minutes after reacting 3-6 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the polymer nanoparticle of folic acid and the load hydrophobic drug of poly-dopamine modification
Son, freeze-dried back；
(4) double load medicines：In proportion, weigh nano-particle prepared in 50mg (3) and be resuspended in 50mL pH for 7.5-12
Weak alkaline aqueous solution in, weigh 4-8mg bortezomibs and be dissolved in 200-300 μ L organic solvents, under conditions of being stirred at room temperature,
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 10-15 hours with volatile organic solvent, 15000-
18000rpm is centrifuged 15-30 minute, abandons supernatant, and precipitation is washed with deionized 3 times, obtains folic acid and poly-dopamine modifies
The double medicine-carried nano particles of cancer target of pH responses, freeze-dried back.
Polymer is selected from PLA, polyglycolic acid, Poly(D,L-lactide-co-glycolide, polycaprolactone, polyethylene glycol
Poly lactic coglycolic acid, cholic acid-Poly(D,L-lactide-co-glycolide or polyethylene glycol 1000 vitamin E succinic acid ester-
PLA.
Hydrophobic drug is selected from taxol, Docetaxel, Coumarin-6, cis-platinum, brufen or 10-hydroxycamptothecine.
Organic solvent is selected from acetone, dioxane, dimethyl sulfoxide (DMSO), dichloromethane, acetonitrile, tetrahydrofuran or N, N- diformazan
Base formamide.
Weak base aqueous solution be selected from PBS cushioning liquid, Tris cushioning liquid, aqueous sodium carbonate, aqueous sodium bicarbonate or
Sodium hydrate aqueous solution.
The double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification prepared by the above method.
The double medicine-carried nano particles of cancer target of the pH responses of above-mentioned folic acid and poly-dopamine modification are preparing antineoplastic
The application of thing.
The inventive method is simple, and there is wide adaptability, prepared nano-particle good biocompatibility, biology can drop
The targeting of solution property and kinds of tumors, double load medicines and pH response characteristics make it have good therapeutic effect, it is demonstrated experimentally that
Breast cancer can be targetted, and has therapeutic action to breast cancer.
Brief description of the drawings
Fig. 1 is the cancer target nano-particle of folic acid prepared by the embodiment 1 that dynamic light scattering is surveyed and poly-dopamine modification
CA-PLGA@PDA-PEG-FA/NPs (carrying docetaxel) size distribution.
Fig. 2 is the cancer target nano-particle CA-PLGA@PDA- of folic acid prepared by embodiment 1 and poly-dopamine modification
PEG-FA/NPs (carrying docetaxel) transmission electron microscope (TEM) collection of illustrative plates.
Fig. 3 is four kinds of nano-particles (CA-PLGA/NPs, CA-PLGA@PDA/ of carrying docetaxel prepared by embodiment 1
NPs, CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs) x-ray photoelectron power spectrum (XPS)
Full spectrum scanning figure.
Fig. 4 be embodiment 1 prepare carrying docetaxel nano-particle (CA-PLGA/NPs, CA-PLGA@PDA/NPs,
CA-PLGA@PDA-PEG-FA/NPs) Docetaxel In-vitro release curves.
Fig. 5 is the double drug-carrying nanometer particles of cancer target of the pH responses of folic acid prepared by embodiment 1 and poly-dopamine modification
The In-vitro release curves of the bortezomib of sub- CA-PLGA@PDA-PEG-FA+BTZ/NPs (carrying docetaxel).
Fig. 6 is nano-particle (CA-PLGA@PDA-PEG/NPs, the CA-PLGA@of carrying docetaxel prepared by embodiment 1
PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs) received with not carrying the CA-PLGA@PDA-PEG-FA/NPs of medicine
Rice corpuscles (with medicine-carried nano particles same concentrations) tests knot in 24 hours and 48 hours to the cytoactive of MCF-7 cells
Really, clinical Docetaxel preparationContrast (t-test, * p<0.05,**p<0.01,***p<
0.001)。
Fig. 7 is the photo of each group tumour after treating 14 days.Each group injects the carrying docetaxel of the preparation of embodiment 1 respectively
CA-PLGA@PDA-PEG/NPs, CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs, clinic are used
Docetaxel preparationContrast, physiological saline makees blank control.
Fig. 8 is the volume of tumour with variation diagram (t-test, the * p for the treatment of time<0.05,**p<0.01,***p<
0.001)。
Fig. 9 is the body weight of nude mice with the variation diagram for the treatment of time.
Figure 10 is the major organs and the histotomy figure of tumour of each group nude mice after treating 14 days.1,2,3,4 and 5 generation respectively
Table physiological saline,, CA-PLGA@PDA-PEG/NPs (carrying docetaxel), CA-PLGA@PDA-PEG-FA/NPs
(carrying docetaxel) and CA-PLGA@PDA-PEG-FA+BTZ/NPs (carrying docetaxel).
Figure 11 is that the nano-particle of load Coumarin-6 prepared by embodiment 2 handles MCF-7 cells after 0.5 hour and 2 hours
Laser confocal microscope (CLSM) picture.1,2,3 and 4 represent respectively CA-PLGA@PDA-PEG/NPs (load Coumarin-6),
CA-PLGA@PDA-PEG-FA/NPs (load Coumarin-6), CA-PLGA@PDA-PEG-FA+BTZ/NPs (load Coumarin-6) and
CA-PLGA@PDA-PEG-FA/NPs (load Coumarin-6)+free folic acid.
Embodiment
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Institute
It is conventional method unless otherwise instructed to state method.The raw material can be obtained from open commercial sources unless otherwise instructed.
Cholic acid-Poly(D,L-lactide-co-glycolide (CA-PLGA)
PLA (PLA)
Polyethylene glycol amino (NH2-PEG)
Folic acid polyethylene glycol amino (NH2-PEG-FA)
Taxol (Paclitaxel, PTX)
Docetaxel (Docetaxel, DTX)
Bortezomib (Bortezomib, BTZ)
Coumarin-6 (Coumarin-6, C6)
Embodiment 1
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification and its
Application in breast cancer targeted therapy, comprises the following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg cholic acid-Poly(D,L-lactide-co-glycolide (CA- is weighed
PLGA), 10mg Docetaxels, are dissolved in 8mL acetone, under conditions of being stirred at room temperature, and are added dropwise to the 0.03% of 100mL
(w/v) in the TPGS aqueous solution, continue to be stirred at room temperature 12 hours with the acetone that volatilizees, 18000rpm
Centrifugation 15 minutes, abandons supernatant, and precipitation is washed with deionized 3 times, obtains receiving for carrying docetaxel (Docetaxel, DTX)
Rice corpuscles CA-PLGA/NPs, freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 25mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle CA-PLGA@PDA/NPs of the carrying docetaxel of bar amine (poly dopamine, PDA) modification, after freeze-drying
It is standby；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 50mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle CA- of folic acid and the carrying docetaxel of poly-dopamine modification
PLGA@PDA-PEG-FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 6mg bortezomibs and be dissolved in 200 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles CA-PLGA@PDA-PEG-FA+BTZ/NPs (carrying docetaxel), freeze-dried back.
By the NH in step (3)2- PEG-FA replaces with NH2- PEG can obtain the load of polyethylene glycol and poly-dopamine modification
The nano-particle CA-PLGA@PDA-PEG/NPs of Docetaxel.
Step (1), (2), (3), the hydration particle diameter (by dynamic light scattering measurement) of nano-particle in (4) are respectively
103.4nm, 160.8nm, 166.4nm, 168.7nm, between 100-200nm and be distributed than more uniform, be conducive to tumour thin
The intake of born of the same parents；In addition, the Zete current potentials of above-mentioned four kinds of nano-particles be respectively -22.5mV, -18.4mV, -11.7mV, -
11.2mV, absolute value than larger, is conducive to nano-particle to be stabilized in dispersed phase.Wherein, Fig. 1 surveys for dynamic light scattering
Nano-particle CA-PLGA@PDA-PEG-FA/NPs (carrying docetaxel) prepared by the embodiment 1 obtained grain size distribution, puts down
Equal particle diameter is about 168.7nm and is distributed than more uniform；Fig. 2 is nano-particle CA-PLGA@PDA-PEG- prepared by embodiment 1
FA/NPs (carrying docetaxel) transmission electron microscope results, nano-particle is the smooth spherical structure with core shell structure, is said
Bright surface have passed through the modification of poly-dopamine and folic acid, and the particle diameter thus estimated is about 85nm and size is homogeneous；Two methods are surveyed
Particle diameter gap it is larger the reason for be nano-particle contraction in the drying process and collapse.
Embodiment 1 prepare carrying docetaxel four kinds of nano-particles (CA-PLGA/NPs, CA-PLGA@PDA/NPs,
CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs) XPS to compose scanning result entirely as shown in Figure 3.
From the figure 3, it may be seen that there are N1s peaks in the spectrogram of rear three kinds of nano-particles, it was demonstrated that the modification of poly-dopamine；CA-PLGA@PDA-PEG-
FA/NPs N1s peak intensities are in CA-PLGA@PDA/NPs, it was demonstrated that the modification of folic acid；CA-PLGA@PDA-PEG-FA+BTZ/NPs go out
Existing B1s peaks, it was demonstrated that the load of bortezomib.
Embodiment 1 prepare carrying docetaxel four kinds of nano-particles (CA-PLGA/NPs, CA-PLGA@PDA/NPs,
CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs) Docetaxel drugloading rate be respectively
9.96%th, 9.78%, 9.67%, 9.01%, illustrate that surface modification influences little to the drugloading rate of nano-particle；In addition, CA-
The drugloading rate of PLGA@PDA-PEG-FA+BTZ/NPs bortezomib is 9.18%.
As shown in figure 4, dialysis determines the Docetaxel elution profiles of nano-particle.It is prepared by step (1), (2), (3)
Carrying docetaxel nano-particle CA-PLGA/NPs, CA-PLGA@PDA/NPs, CA-PLGA@PDA-PEG-FA/NPs point
Also known as take 5mg to be dissolved in 1mL dissolution medium PBST solution (PBS solution, the Tween-80 comprising 0.1%w/v, pH 7.4), turn
Move on in bag filter (MWCO=3500, Shanghai life work) and seal, then be immersed in equipped with 15mL dissolution medium PBST solution
In 50mL centrifuge tubes, finally it is placed on water bath with thermostatic control shaking table in 37 DEG C of concussions.Taken out in certain time interval from centrifuge tube
1mL solution is used to analyze, and is supplemented the fresh solution of equivalent.The extraction of 1mL diethyl ether is added in the 1mL solution of taking-up, water is discarded
Lead to nitrogen volatilization diethyl ether after phase, treat that diethyl ether volatilization is complete, add 1mL mobile phase acetonitriles：Water (50/50, V/V) is through ultrasound point
Dissipate and after 0.1 μm of membrane filtration, take 20 μ L to be added in HPLC and measure.Measuring condition is：Mobile phase is acetonitrile：Water
(50/50, V/V)；Flowing phase velocity is 1.0mL/min；Using anti-phase C-18 pillars (150 × 4.6mm, 5 μm, C18, An Jie
Human relations)；Detection wavelength is 227nm.Its release profiles such as figure for changing over time is gone out according to the Specification Curve of Increasing of Docetaxel
4：Three kinds of nano-particles show the prominent of identical release mode, i.e. first stage and release and (a few days ago discharge about 50%) and second
The sustained release (being finally released slowly to 80% or so) in individual stage.
As shown in figure 5, dialysis determines the nano-particle CA-PLGA@PDA- of carrying docetaxel prepared by embodiment 1
The elution profiles of PEG-FA+BTZ/NPs bortezomib.Detailed process is similar to above-mentioned dialysis, only replaces following condition：Point
Three pH value (pH 7.4, pH 6.5, pH5.0) determine its release profiles；Mobile phase replaces with acetonitrile：Water (80/20, V/V)；Inspection
Survey wavelength is 270nm.Obtained bortezomib release profiles under conditions of pH 7.4 by 12 hours gold as shown in figure 5, release
Put about 25%；Burst size increases on a small quantity under conditions of pH 6.5；Under conditions of pH 5.0, two hour bortezomib releases
More than 50%, it is about 80% to eventually pass through 12 hours burst sizes.Found by contrasting, the bortezomib under conditions of pH 5.0
Rate of release and burst size all greatly improve, it was demonstrated that the pH responses of bortezomib release.
As shown in fig. 6, determining nano-particle with mtt assay 24 hours and 48 hours to the thin of breast cancer cell MCF-7
Cellular toxicity.By MCF-7 cells with 1 × 104The density of cells/well is inoculated on 96 orifice plates, overnight incubation and it is adherent after, use respectively
Nano-particle CA-PLGA@PDA-PEG-FA/NPs (not carrying medicine),(A kind of Docetaxel faces
Bed medication), CA-PLGA@PDA-PEG/NPs (carrying docetaxel), CA-PLGA@PDA-PEG-FA/NPs (carry polyenoid Japanese yew
Alcohol) and CA-PLGA@PDA-PEG-FA+BTZ/NPs (carrying docetaxel) handle 24 hours and 48 hours, wherein load medicine it is each
The concentration gradient of group Docetaxel is respectively 0.25,2.5,12.5,25 μ g/mL, and the concentration gradient of the nano-particle of medicine is not carried
It is consistent with the concentration of medicine-carried nano particles.Afterwards, 96 orifice plates add the MTT solution that 20 μ L concentration are 5mg/ml per hole, after
Continuous culture discards nutrient solution after 4 hours, and 150 μ L dimethyl sulfoxide (DMSO)s are added per hole and cause Jia Za within 30 minutes in lucifuge concussion on shaking table
Crystal is completely dissolved, and finally determines the absorbance per hole 490nm with ELIASA.
As a result show：CA-PLGA@PDA-PEG-FA/NPs (not carrying medicine) do not show cell toxicant in each concentration
Property, eliminate the cytotoxicity of pharmaceutical carrier；Remaining Docetaxel formula shows cytotoxicity, wherein three kinds carry medicine and receive
The cytotoxicity of rice corpuscles is all higher than the Docetaxel preparation of clinic The cell toxicant of three kinds of medicine-carried nano particles
Sex expression goes out concentration and time dependence；CA-PLGA@PDA-PEG-FA+BTZ/NPs prepared by embodiment 1 (carry polyenoid Japanese yew
Alcohol) most strong cytotoxicity is shown, show that antitumous effect can be significantly improved by targetting double medicines and pH response mechanisms of carrying.
Fig. 7 is the photo of each group tumour after treating 14 days.Each group injects three kinds of load polyenoid Japanese yews of the preparation of embodiment 1 respectively
Nano-particle (CA-PLGA@PDA-PEG/NPs, CA-PLGA@PDA-PEG-FA/NPs, the CA-PLGA@PDA-PEG-FA+ of alcohol
BTZ/NPs), another set injecting normal saline (saline) is used as blank control, the clinical Docetaxel of one group of injection
PreparationIt is used as control.As shown in fig. 7, relative to blank control, four kinds of Docetaxel formulas
The growth of tumour is inhibited, the inhibition of three kinds of medicine-carried nano particles is better thanWherein carrying docetaxel
CA-PLGA@PDA-PEG-FA+BTZ/NPs show best inhibition, and gross tumor volume is minimum.
Fig. 8 is the volume of tumour with the variation diagram for the treatment of time.When the tumour for the nude mice that MCF-7 cells are subcutaneously injected is long
To 50mm3When, it is randomly divided into the CA-PLGA@PDA-PEG/ that carrying docetaxel is injected intraperitoneally in five groups (every group 5) respectively
NPs, CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs and physiological saline and
(dosage is 10mg/kg).Every injection in 4 days once, tumor size and nude mice quality are every other day measured, by nude mice after 14 days
It is dead and isolate transplantable tumor.As shown in figure 8, relative to the blank control group of injecting normal saline, It is many with three kinds of loads
The nano-particle of alkene taxol shows to suppress the effect of tumour growth, the suppression of the nano-particle of three kinds of carrying docetaxels
Effect is superior toThe CA-PLGA@PDA-PEG-FA+BTZ/NPs of wherein carrying docetaxel show optimal suppression
Effect processed, gross tumor volume does not almost become big in 14 days treatment cycles, shows to target and double carries medicines and pH response mechanisms can
Significantly improve antitumous effect.As shown in Figure 9, it is shown that the quality change situation of each group nude mice after injection corresponding preparations, relatively
In the blank control group of injecting normal saline, the nano-particle of three kinds of carrying docetaxels influences little to the quality of nude mice, and
InjectionNude mice quality occur in that obvious reduction.This explanation relative to, what is prepared in embodiment 1 is more
The nanometer formulation of alkene taxol substantially reduces toxic side effect.
As shown in Figure 10, it is shown that the major organs (heart, liver, spleen, lung, kidney) and tumour of the nude mice by treatment in 14 days
Histotomy figure.Shown in figureShown to tumour cell with the nano-particle of three kinds of carrying docetaxels
Lethal effect, simultaneouslyThe damage to lung and liver is also shown, and the nano-particle of three kinds of carrying docetaxels does not have
Show the damage to major organs.This explanation relative to The nanometer system of the Docetaxel prepared in embodiment 1
Agent substantially reduces toxic side effect.
Embodiment 2
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification and its
Application in breast cancer targeted therapy, comprises the following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg cholic acid-Poly(D,L-lactide-co-glycolide (CA- is weighed
PLGA), 2mg Coumarin-6s, are dissolved in 8mL acetone, under conditions of being stirred at room temperature, and are added dropwise to 100mL 0.03% (w/
V) in the TPGS aqueous solution, continue to be stirred at room temperature 12 hours with the acetone that volatilizees, 18000rpm centrifugations
15 minutes, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain carrying the nano-particle of Coumarin-6 (Coumarin-6, C6)
CA-PLGA/NPs, freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 25mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle CA-PLGA@PDA/NPs of the load Coumarin-6 of bar amine (poly dopamine, PDA) modification, are freeze-dried standby
With；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 50mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle CA-PLGA@of folic acid and the load Coumarin-6 of poly-dopamine modification
PDA-PEG-FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 6mg bortezomibs and be dissolved in 200 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles CA-PLGA@PDA-PEG-FA+BTZ/NPs (load Coumarin-6), freeze-dried back.
By the NH in step (3)2- PEG-FA replaces with NH2- PEG can obtain the load of polyethylene glycol and poly-dopamine modification
The nano-particle CA-PLGA@PDA-PEG/NPs of Coumarin-6.
Figure 11 is three kinds of nano-particle (CA-PLGA@to load Coumarin-6 with confocal microscopy MCF-7 cells
PDA-PEG/NPs, CA-PLGA@PDA-PEG-FA/NPs, CA-PLGA@PDA-PEG-FA+BTZ/NPs) intake result, in addition
Also one group adds CA-PLGA@PDA-PEG-FA/NPs (load Coumarin-6) and free folic acid and is used to prove that folic acid is situated between simultaneously
The endocytosis led.MCF-7 cells are inoculated into Glass bottom culture dish, overnight incubation and it is adherent after, be separately added into 250 μ
The nano-particle of g/mL load Coumarin-6, wherein validation group are addition CA-PLGA@PDA-PEG-FA/NPs (load Coumarin-6)
(125 μ g/mL) and free folic acid (125 μ g/mL).After culture 0.5 hour and 2 hours, cell is washed 3 times with PBS, with 4%
Paraformaldehyde solution fix 15 minutes, cell is washed 3 times with PBS, nucleus 10 minutes is contaminated with DAPI, with PBS by cell
Washing 3 times, afterwards with confocal microscopy cellular uptake situation.As shown in figure 11,0.5 hour cell of culture has just been opened
Begin intake nano-particle, the Fluorescence Increasing of the nano-particle of cellular uptake after cultivating 2 hours, and wherein cell is to carrying Coumarin-6
CA-PLGA@PDA-PEG-FA/NPs and CA-PLGA@PDA-PEG-FA+BTZ/NPs intake is more or less the same, and fluorescence intensity is equal
The CA-PLGA@PDA-PEG/NPs for carrying Coumarin-6 are significantly stronger than, this illustrates that folate-mediated endocytosis substantially increases
Intake of the cell to nano-particle, and load bortezomib this technique does not influence its active targeting to act on.Add in addition
The fluorescence for entering the validation group of free folic acid substantially weakens, and this further demonstrates folate-mediated active targeting effect.
Embodiment 3
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification, including
Following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg Poly(D,L-lactide-co-glycolides (PLGA) are weighed,
10mg taxols, are dissolved in 10mL tetrahydrofurans, under conditions of being stirred at room temperature, and are added dropwise to 100mL 0.03% (w/v)
The TPGS aqueous solution in, continue to be stirred at room temperature 12 hours with the tetrahydrofuran that volatilizees, 18000rpm from
The heart 15 minutes, abandons supernatant, and precipitation is washed with deionized 3 times, obtains carrying the nanoparticle of taxol (Paclitaxel, PTX)
Sub- PLGA/NPs, freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 10mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 6 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle PLGA@PDA/NPs of the load taxol of bar amine (poly dopamine, PDA) modification, freeze-dried back；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 50mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle PLGA@PDA- of folic acid and the load taxol of poly-dopamine modification
PEG-FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 4mg bortezomibs and be dissolved in 200 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles PLGA@PDA-PEG-FA+BTZ/NPs (load taxol), freeze-dried back.
It is demonstrated experimentally that with the cholic acid-polylactic acid-glycolic base second of Poly(D,L-lactide-co-glycolide (PLGA) alternate embodiment 1
The double medicine-carried nano particles of cancer target of the pH responses of acid copolymer (CA-PLGA), obtained folic acid and poly-dopamine modification
PLGA@PDA-PEG-FA+BTZ/NPs characterize data is with embodiment 1 without substantive difference.
Embodiment 4
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification, including
Following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg polyethylene glycol-polylactic acids-co-glycolic acid is weighed
(PEG-PLGA), 10mg taxols, are dissolved in 10mL tetrahydrofurans, under conditions of being stirred at room temperature, are added dropwise to 100mL's
In 0.03% (w/v) the TPGS aqueous solution, continue to be stirred at room temperature 12 hours with the tetrahydrochysene furan that volatilizees
Mutter, 18000rpm is centrifuged 15 minutes, abandons supernatant, precipitation is washed with deionized 3 times, obtain carrying taxol (Paclitaxel,
PTX nano-particle PEG-PLGA/NPs), freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 10mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 6 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle PEG-PLGA@PDA/NPs of the load taxol of bar amine (poly dopamine, PDA) modification, are freeze-dried standby
With；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 50mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle PEG-PLGA@of folic acid and the load taxol of poly-dopamine modification
PDA-PEG-FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 4mg bortezomibs and be dissolved in 200 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles PEG-PLGA@PDA-PEG-FA+BTZ/NPs (load taxol), freeze-dried back.
It is demonstrated experimentally that with the cholic acid of polyethylene glycol-polylactic acid-co-glycolic acid (PEG-PLGA) alternate embodiment 1-
The cancer target of the pH responses of Poly(D,L-lactide-co-glycolide (CA-PLGA), obtained folic acid and poly-dopamine modification is double to be carried
Medicine nano-particle PEG-PLGA@PDA-PEG-FA+BTZ/NPs characterize data is with embodiment 1 without substantive difference.
Embodiment 5
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification, including
Following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg PLAs (PLA) are weighed, 10mg cis-platinums are dissolved in 8mL bis-
In methyl sulfoxide, under conditions of being stirred at room temperature, 100mL 0.03% (w/v) polyethylene glycol vitamin E amber is added dropwise to
In the amber acid esters aqueous solution, continue to be stirred at room temperature 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm is centrifuged 15 minutes, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains carrying the nano-particle PLA/NPs of cis-platinum, freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 50mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle PLA@PDA/NPs of the load cis-platinum of bar amine (poly dopamine, PDA) modification, freeze-dried back；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 100mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle PLA@PDA-PEG- of folic acid and the load cis-platinum of poly-dopamine modification
FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 8mg bortezomibs and be dissolved in 300 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles PLA@PDA-PEG-FA+BTZ/NPs (load cis-platinum), freeze-dried back.
It is demonstrated experimentally that with the cholic acid-Poly(D,L-lactide-co-glycolide (CA- of PLA (PLA) alternate embodiment 1
PLGA), the double medicine-carried nano particles PLA@PDA-PEG-FA+ of cancer target of the pH responses of folic acid and the poly-dopamine modification obtained
BTZ/NPs characterize data is with embodiment 1 without substantive difference.
Embodiment 6
A kind of preparation method of the double medicine-carried nano particles of cancer target of the pH responses of folic acid and poly-dopamine modification, including
Following steps：
(1) preparation of medicine-carried nano particles：In proportion, 100mg polyethylene glycol 1000 vitamin E succinic acid esters-poly- are weighed
Lactic acid (TPGS-PLA), 10mg cis-platinums are dissolved in 8mL dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, are added dropwise to
In 100mL 0.03% (w/v) the TPGS aqueous solution, continue to be stirred at room temperature 12 hours to volatilize
Dimethyl sulfoxide (DMSO), 18000rpm is centrifuged 15 minutes, abandons supernatant, and precipitation is washed with deionized 3 times, obtains carrying the nanometer of cis-platinum
Particle TPGS-PLA/NPs, freeze-dried back；
(2) modification of poly-dopamine：In proportion, weigh nano-particle prepared in 50mg (1) and be resuspended in 50mL's
In 10mM, pH=8.5 Tris buffer solutions, 50mg dopamine hydrochlorides are weighed, are added under conditions of being stirred at room temperature above-mentioned
In solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant, and precipitation is washed with deionized 3 times, obtains poly- many
The nano-particle TPGS-PLA@PDA/NPs of the load cis-platinum of bar amine (poly dopamine, PDA) modification, freeze-dried back；
(3) modification of targeting ligand folic acid：In proportion, weigh nano-particle prepared in 50mg (2) and be resuspended in 50mL
10mM, in pH=8.5 Tris buffer solutions, weigh 100mg cancer target part folic acid polyethylene glycol amino (NH2-PEG-
FA), it is added under conditions of being stirred at room temperature in above-mentioned solution, 18000rpm is centrifuged 15 minutes after reacting 3 hours, abandons supernatant
Liquid, precipitation is washed with deionized 3 times, obtains the nano-particle TPGS-PLA@of folic acid and the load cis-platinum of poly-dopamine modification
PDA-PEG-FA/NPs, freeze-dried back；
(4) double load medicines：In proportion, 10mM, pH=that nano-particle prepared in 50mg (3) is resuspended in 50mL are weighed
In 8.5 Tris buffer solutions, weigh 8mg bortezomibs and be dissolved in 300 μ L dimethyl sulfoxide (DMSO)s, under conditions of being stirred at room temperature, by
In the suspension for being added dropwise to above-mentioned nano-particle, continue to stir 12 hours with the dimethyl sulfoxide (DMSO) that volatilizees, 18000rpm centrifugations 15
Minute, supernatant is abandoned, precipitation is washed with deionized 3 times, obtain the cancer target of the pH responses of folic acid and poly-dopamine modification
Double medicine-carried nano particles TPGS-PLA@PDA-PEG-FA+BTZ/NPs (load cis-platinum), freeze-dried back.
It is demonstrated experimentally that with polyethylene glycol 1000 vitamin E succinic acid ester-PLA (TPGS-PLA) alternate embodiment 1
The tumor target of the pH responses of cholic acid-Poly(D,L-lactide-co-glycolide (CA-PLGA), obtained folic acid and poly-dopamine modification
To double medicine-carried nano particles TPGS-PLA@PDA-PEG-FA+BTZ/NPs characterize data and embodiment 1 without substantive difference.
Claims (10)
1. a kind of preparation method of double drug-carrying nanometer particles of pH responses and cancer target, it comprises the following steps：
(1) polymer nanoparticle for carrying hydrophobic drug is prepared；
(2) polymer nanoparticle for carrying hydrophobic drug is modified with poly-dopamine；
(3) polymer nano-particle for modifying poly-dopamine load hydrophobic drug with active targeting part is modified, and is obtained
The polymer nanoparticle of active targeting part and the load hydrophobic drug of poly-dopamine modification；
(4) the load hydrophobic drug for modifying the drug loading with boric acid structure in active targeting part and poly-dopamine is poly-
On compound nanoparticle, double drug-carrying nanometer particles of pH responses and cancer target are obtained.
2. preparation method according to claim 1, it is characterised in that in step 4) in, drug loading is 7.5-12 in pH
Weak alkaline aqueous solution in carry out, the medicine with boric acid structure is dissolved in organic solvent, add suspension active targeting part
It is complete to reacting in the weak alkaline aqueous solution of the polymer nanoparticle for the load hydrophobic drug modified with poly-dopamine, after separation
Obtain double drug-carrying nanometer particles；
It is highly preferred that the mass ratio of active targeting part and the nanoparticle and the medicine with boric acid structure of poly-dopamine modification is
100:1-20, more preferably 100:8-16；
It is highly preferred that the medicine with boric acid structure is selected from bortezomib；
It is highly preferred that the organic solvent is selected from acetone, dioxane, dimethyl sulfoxide (DMSO), dichloromethane, acetonitrile, tetrahydrofuran
Or N,N-dimethylformamide.
3. the preparation method according to claim any one of 1-2, it is characterised in that step 1) it is by polymer and hydrophobicity
Medicine is dissolved in organic solvent, and is added in emulsifier aqueous solution, and reaction is complete to organic solvent volatilization, and precipitation and separation is obtained
Carry the polymer nano-particle of hydrophobic drug；
Wherein, the polymer is selected from PLA, polyglycolic acid, Poly(D,L-lactide-co-glycolide, polycaprolactone, poly- second two
Alcohol-Poly(D,L-lactide-co-glycolide, cholic acid-Poly(D,L-lactide-co-glycolide, polyethylene glycol 1000 vitamin E butanedioic acid
One or more in ester-PLA；
The hydrophobic drug is in taxol, Docetaxel, Coumarin-6, cis-platinum, brufen, 10-hydroxycamptothecine
One or more；
The organic solvent is selected from acetone, dioxane, dimethyl sulfoxide (DMSO), dichloromethane, acetonitrile, tetrahydrofuran or N, N- diformazan
Base formamide；
The emulsifying agent is selected from TPGS, Tween-20, Tween-80, polyvinyl alcohol.
4. the preparation method according to claim any one of 1-3, it is characterised in that step 2) it is that will carry hydrophobic drug
Polymer nanoparticle is suspended in the weak alkaline aqueous solution that pH is 7.5-12, adds dopamine hydrochloride, reaction to complete, separation
Precipitation, obtains the polymer nanoparticle of the load hydrophobic drug of poly-dopamine modification.
5. the preparation method according to claim any one of 1-4, it is characterised in that step 3) it is the load that poly-dopamine is modified
The polymer nanoparticle of hydrophobic drug is suspended in the weak alkaline aqueous solution that pH is 7.5-12, and active targeting part is added to
In above-mentioned solution, reaction is to complete, and precipitation and separation obtains active targeting part and the load hydrophobic drug of poly-dopamine modification
Polymer nanoparticle；
Wherein, active targeting part be selected from folic acid polyethylene glycol amino, galactosamine (Gal), a kind of polypeptide (Arg-Gly-Asp,
RGD), aptamer sulfydryl (Aptamer-SH).
6. the preparation method according to claim any one of 2-5, wherein, step 2,3) or 4) in pH be 7.5-12 weak base
Property aqueous solution independence be selected from PBS cushioning liquid, Tris cushioning liquid, aqueous sodium carbonate, aqueous sodium bicarbonate or hydrogen-oxygen
Change one kind in sodium water solution；
Preferably, pH is 8-9, more preferably 8.5.
7. double drug-carrying nanometer particles prepared by the preparation method according to claim any one of 1-6.
8. a kind of pair of drug-carrying nanometer particle, it is included and born on the nanoparticulate carriers skeleton formed with polymer, nanoparticulate carriers skeleton
Hydrophobic drug is carried, nanoparticulate carriers skeleton surface is covered with poly-dopamine, and sticking in poly-dopamine has active targeting part, gathers
Catechol group on dopamine also incorporates the medicine with boric acid base group；Described pair of drug-carrying nanometer particle is the nanometer that pH is responded
Grain, discharges medicine in acid condition.
9. according to claim 8 pair of drug-carrying nanometer particle, wherein, the polymer is selected from PLA, polyglycolic acid, poly- breast
Acid-co-glycolic acid, polycaprolactone, polyethylene glycol-polylactic acid-co-glycolic acid, cholic acid-polylactic acid-glycolic base second
One or more in acid copolymer, polyethylene glycol 1000 vitamin E succinic acid ester-PLA；
The hydrophobic drug is selected from taxol, Docetaxel, Coumarin-6, cis-platinum, brufen, 10- hydroxy-camptothecins
One or more in alkali；
Medicine with boric acid structure is selected from bortezomib；
Active targeting part be selected from folic acid polyethylene glycol amino, galactosamine (Gal), a kind of polypeptide (Arg-Gly-Asp, RGD),
Aptamer sulfydryl (Aptamer-SH).
10. double medicine-carried nano particles described in claim any one of 6-9 are preparing the application of antineoplastic.
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| CN112603910A (en) * | 2020-12-31 | 2021-04-06 | 淮阴工学院 | Mesoporous polydopamine-loaded anthocyanin nanoparticle and preparation method thereof |
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| CN114588170A (en) * | 2020-12-04 | 2022-06-07 | 上海中医药大学附属龙华医院 | Cinobufagin-loaded PDA nano-drug and preparation method and application thereof |
| CN115337397A (en) * | 2021-05-10 | 2022-11-15 | 上海中医药大学附属龙华医院 | PDA nano-drug loaded with siRNA/PEI, preparation method and application thereof |
| CN116925337A (en) * | 2023-09-19 | 2023-10-24 | 广东药科大学 | Method for constructing targeted nano-carrier of dopamine derivative |
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| CN109276721B (en) * | 2018-09-19 | 2022-02-01 | 中山大学 | Targeting mesoporous polydopamine multifunctional nano diagnosis and treatment agent and preparation method and application thereof |
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| CN112603910A (en) * | 2020-12-31 | 2021-04-06 | 淮阴工学院 | Mesoporous polydopamine-loaded anthocyanin nanoparticle and preparation method thereof |
| CN113069432A (en) * | 2021-04-14 | 2021-07-06 | 深圳市人民医院 | Nanometer preparation for targeted repair of cardiac muscle and preparation method thereof |
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| CN113769110A (en) * | 2021-09-17 | 2021-12-10 | 清华大学深圳国际研究生院 | Nanometer carrier for photodynamic combined calcium overload and preparation method and application thereof |
| CN116925337A (en) * | 2023-09-19 | 2023-10-24 | 广东药科大学 | Method for constructing targeted nano-carrier of dopamine derivative |
| CN116925337B (en) * | 2023-09-19 | 2023-12-01 | 广东药科大学 | Method for constructing targeted nano-carrier of dopamine derivative |
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