CN105854027B - A kind of amphiphilic nano self-assembled micelle and its application based on low generation PAMAM dendrimer - Google Patents
A kind of amphiphilic nano self-assembled micelle and its application based on low generation PAMAM dendrimer Download PDFInfo
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Abstract
The present invention relates to a kind of amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer and its applications.Be specifically related to it is a kind of be coupled based on low generation PAMAM dendrimer and dewatering medicament after, pass through ultrasonic vibration self assembly in water.Its bioactive molecule for having Drug loading capacity or precursor medicine, the chemical coupling of hydrophobic drug and low generation PAMAM dendrimer, the lower defect of hydrophobic drug poor solubility, bioavailability can be improved, that there are toxicity to solve the problems, such as clinical classic chemotherapy drug is big, safety coefficient is low provides new approaches.It also can be used as nano-carrier simultaneously, contains other dewatering medicaments, realizes the drug combination of drug, heighten the effect of a treatment.
Description
Technical field
The present invention relates to a kind of amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer and its applications.
Background technique
Molecular self-assembling (molecular self-assembly) refers to that in equilibrium conditions molecule passes through intermolecular non-
Covalent bond weak interaction, such as Van der Waals force, hydrogen bond, hydrophobic effect, electrostatic interaction, π-π interaction, spontaneous combination are formed
The process of stable aggregation or supermolecule with certain structure and function.The aggregation or supermolecule that molecular self-assembling is formed
Usually there are unimolecule or the unexistent some properties of low levels of molecular aggregation, such as light, electricity, biological nature.It is general next
It says, the self assembly of molecule has structure enlarge-effect, i.e. the minor alteration of molecular structure will lead to the topological structure of entire molecule
Great variety occurs, so the self assembly of molecule is non-but not influences its characteristic because of the change of structure, imparts instead
The some more novel characteristics of self-assembly, for example pattern and structure are more abundant, special self assembly mechanism, special assembling
The features such as body performance and easy functionalization and intelligent response, these performances make the great development prospect of molecular self-assembling.
Dendrimer (dendrimer) due to nanoscale, highly-water-soluble, monodispersity and adjustability of structure,
The nanoparticle of suitable size can be formed by molecular self-assembling, therefore can be used as anticancer drug and the conveying load of drug is imaged
Body.Compared to liposome, the covalent bond in dendrimer bracket provides more stable carrier structure, enables to withstand life
The variation of manage bar part, and drug molecule can be protected not decomposed rapidly, guarantee its specific medicinal property, promotes drug molecule
Certain drug effect concentration is kept in tumour cell.Research finds that the nano-carrier system based on dendrimer is poly- compared to linear
Closing object carrier (such as PEG) has more superior pharmaceutical carrier performance, this is because the nano-scale of dendrimer and in the solution
Caused by close spherical structure.Polyamide-amide (PAMAM) be it is a kind of from center to external symmetry diverging and it is highly branched new
The tree-shaped macromolecular compound of type nanometer has regular, fine structure, is in monodispersity, there is good dissolubility, 0 ~ 10 generation grain
Diameter range is 10~130nm.Nanoscale partial size makes PAMAM be easier to penetrate the biological barriers such as vascular wall, cell membrane, to its core
The heart and surface are modified, and can connect the substances such as gene, antibody and vaccine, internal layer cavity can packaging medicine molecule, as medicine
The carrier of object orientation transport.PAMAM dendrimer can pass through electrostatic interaction, hydrophobic interaction as pharmaceutical carrier of new generation
Or drug encapsulation portion in the inner can also be formed on its surface compound by electrostatic, covalent bond, to increase by hydrogen bond action
Drug solubility improves drug bioavailability.The research and application of PAMAM dendrimer have obtained continuous development, by
The increasingly extensive concern of numerous areas such as arrive from material science to biological medicine.
The carrier etc. for focus primarily oning drug and gene to the application of PAMAM dendrimer at present, such as patent
Derivative and its application of a kind of novel PAMAM are disclosed in CN201010142282, the derivative is phenylalanine
It is coupled the PAMAM dendrimer of modification, for the transfection of siRNA and the internal transfection of gene, it is to utilize PAMAM tree
The positive charge of primary amine group reaches delivery purpose to negatively charged DNA or RNA suction-operated on shape molecular surface, but
Electrostatic adsorption power is compared on the weak side with covalent effect power, and stability is poor.One kind is disclosed in CN201310404541 to be based on
The self-assembled nanometer genophore compound of PAMAM is to pass through mutual electrostatic interaction self assembly group by HA, PAMAM and gene
At compound system, but Electrostatic Absorption belongs to weaker interaction, carrying medicament in the way of this, and drug loading can be than logical
It crosses chemical coupling and wants small with physically trapping two ways, and stability is poor, slow release effect is unobvious.It is public in CN201410271351
A kind of folic acid-PAMAM- ursolic acid the Nano medication with cancer target of cloth, original material used by the Nano medication are
Highly toxic G3 and G5 polyamide-amine type dendrimer (PAMAM) need to first pass through chemical reaction for the ammonia on the surface PAMAM
Base is replaced as hydroxyl to reduce the toxicity of material itself, then folic acid and ursolic acid is reacted with the hydroxyl on the surface PAMAM, finally
Synthesize target product.Therefore the reaction process is complicated, and production cost is higher, this limits its application to a certain extent.It is comprehensive
Upper described, the studies above is all made of the PAMAM of high algebra as research object (PAMAM algebra was 3 ~ 6 generations), and height is for PAMAM
Synthesis step is cumbersome, causes cost excessively high；In addition, high carry a large amount of amino for the surface PAMAM, easily with negative electricity is had in organism
Various biological membrane interactions, therefore there are higher cytotoxicities, it is easier to haemolysis occur.
To make up the deficiencies in the prior art, the present invention is using low generation PAMAM dendrimer as research object.Relative to
Height greatly reduces for PAMAM, the amino amount of low generation pamam dendrimer molecular surface, therefore toxicity also substantially reduces.And low generation
PAMAM synthesis step is simple, low in cost, is more suitable for large-scale application.In addition, the space steric effect of low generation PAMAM is much
Less than height for PAMAM, this is just that its end modified is provided convenience.But low generation PAMAM itself cannot at nano-micelle, because
This, is coupled with it using hydrophobic drug, makes it have amphipathic nature, and be further self-assembled into nano-micelle.So as to
It is enough that stable nano-micelle structure is formed under aqueous environments.The nano-micelle not only can independently be used as bioactive molecule or precursor medicine
For treating, it is alternatively arranged as pharmaceutical carrier, further contains other dewatering medicaments, realizes the combination therapy to drug, with enhancing
The therapeutic effect of nano-micelle.
Different pharmaceutical drug combination can increase target spot, and multiple target spots on disabling signal Signal Transduction Pathways can also reduce swollen
The drug resistance of tumor.Based on low generation PAMAM, by the coupling with dewatering medicament, it is molten to not only improve drug for the first time by the present invention
Xie Du is poor, the lower defect of bioavilability, it is often more important that constructs amphiphilic nano carrier systems, assigns it and contain medicine
The potentiality of object have not been reported such application method of low generation PAMAM with realizing the use in conjunction of drug, have great
Novelty has very big application prospect.
Summary of the invention
The purpose of the present invention is to provide a kind of amphipathic self-assembled micelle based on low generation PAMAM dendrimer and its answer
With application including preparation method and its in fields such as pharmacy.The nano-micelle is based on low generation PAMAM(G0Such as formula III, G1Such as
Formula IV) dendrimer, the bioactive molecule or precursor medicine that have Drug loading capacity can be obtained by chemical modification.
III
IV
In order to achieve the above object, the present invention provides a kind of amphipathic self-assembled micelle based on low generation PAMAM dendrimer,
It is characterized in that by chemical method in low generation PAMAM dendrimer G0Generation (formula III), G1Hydrophobicity medicine is introduced on generation (formula IV)
Object increases the hydrophobicity of dendrimer, assigns that its is amphipathic, makes it in an aqueous medium can the stable nanometre glue of self assembly
Beam；The low generation PAMAM dendrimer is polyamidoamine dendrimer, refers mainly to G0、G1Generation, but not limited to this, symbol
It closes and states principle；The hydrophobic drug can be carried out based on carboxylic acids drug by amido bond and PAMAM dendrimer
Coupling, but not limited to this, chemical coupling can be carried out with PAMAM dendrimer, become amphipathic dendrimer, institute
Stating hydrophobic drug includes but is not limited to: anti-tumor drug includes but is not limited to this: ursolic acid, methotrexate (MTX), aminopterin-induced syndrome, neat
Pier tartaric acid, gambogicacid；Cardiovascular drugs include but is not limited to: Atorvastatin, Fluvastatin, Pitavastatin, linoleic acid；It is anti-inflammatory
Drug includes but is not limited to: including the aryl alkanoic acid class, Meclofenamic Acid including the salicylic acid, brufen including aspirin
That fragrant acids；Drug for liver disease includes but is not limited to: ursodesoxycholic acid.
Specifically, a kind of amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer, it is characterised in that its
By amphipathic conjugate in water self assembly and obtain；The amphipathic conjugate is as shown in Formulas I or Formula II；
I
II
Wherein Ra is dewatering medicament in Formulas I；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；
Wherein Ra is dewatering medicament in Formula II；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；Rd is hydrogen or hydrophobic
Drug；Re is hydrogen or dewatering medicament；Rf is hydrogen or dewatering medicament；Rg is hydrogen or dewatering medicament；
The dewatering medicament is the drug containing carboxyl.
The dewatering medicament be ursolic acid, methotrexate (MTX), aminopterin-induced syndrome, pemetrexed, oleanolic acid, tetrachloro it is fragrant that
Acid, Chlorambucil, gambogicacid, Raltitrexed, linoleic acid, enalapril, fosinopril, Candesartan, Gemfibrozil, department's pa
Sha Xing, Atorvastatin, mefenamic acid, Raltitrexed, Indomethacin, Fluvastatin, Edatrexate, Pitavastatin, Yi Daqu
Sand, linoleic acid, all-trans retinoic acid, 9-cis-retinoic acid, aspirin, salicylic acid, brufen, Meclofenamic Acid, that fragrant acid
Or ursodesoxycholic acid.
Preferably ursolic acid or aspirin.
The mode of its self assembly is ultrasonic vibration.
It is in the preparation of antitumor drugs and as pharmaceutical carrier application in preparation of anti-tumor drugs.
The preparation step of the amphipathic self-assembled micelle based on low generation PAMAM dendrimer is as follows:
1. the preparation of the amphipathic conjugate of PAMAM- hydrophobic drug
(1) synthesis of hydrophobic drug reactive intermediates
Hydrophobic drug is dissolved in organic solvent, catalyst a, catalyst b is added, obtains reactive intermediates；
(2) synthesis of the amphipathic conjugate of PAMAM- hydrophobic drug
Low generation PAMAM is dissolved in organic solvent, catalyst c is added, with reactive intermediates obtained in above-mentioned steps
It is further reacted, control temperature to fully reacting.Then reaction solution is dialysed in organic solvent, distilled water successively,
Finally amphipathic conjugate can be obtained in freeze-drying.
(3) synthetic route is as follows:
Wherein catalyst a, catalyst b include but is not limited to: dicyclohexylcarbodiimide (DCC)/N- maloyl is sub-
Amine (NHS) chemical method, 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDC)/N- hydroxysuccinimide
(NHS) method, Avidin (avidin)/biotin (biotin) method and N- hydroxysuccinimide (NHS)/maleoyl-
Imines (maleimide) chemical method；Catalyst c includes but is not limited to: 4- (N, N- dimethylamino) pyridine (DMAP), 4-
(N, N- diallyl amino) pyridine (DAAP).
2. the preparation of the amphipathic self-assembled nano micelle of PAMAM- hydrophobic drug
Take amphipathic conjugate soluble in water, ultrasonic treatment can be obtained based on the amphipathic of low generation PAMAM dendrimer
Self-assembled nano micelle.Self-assembled composite is measured water-soluble using particle size analyzer, scanning electron microscope etc. after diluting
Particle diameter distribution and distribution of charges in liquid are characterized.
3. preparing the nano-micelle compound containing insoluble drug using PAMAM- hydrophobic drug conjugate as carrier
The amphipathic conjugate of PAMAM- hydrophobic drug and other dewatering medicaments is taken to be codissolved in organic solvent, N2Drying,
Water is added, the amphipathic self-assembled nanometer glue based on low generation PAMAM dendrimer for containing drug can be obtained after ultrasonic treatment
Beam compound.After diluting, in aqueous solution using the measurement such as particle size analyzer, scanning electron microscope self-assembled composite
Particle diameter distribution and distribution of charges are characterized.
The preparation method comprises the following steps:
(1) hydrophobic drug is dissolved in organic solvent, catalyst a, catalyst b is added, obtains reactive intermediates；
(2) low generation PAMAM is dissolved in organic solvent, catalyst c is added, produced among activity obtained in above-mentioned steps
Object is further reacted, and after processing, both obtains the amphipathic conjugate based on low generation PAMAM dendrimer, can be in aqueous Jie
It is self-assembled into matter as the stable amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer.
The catalyst a and catalyst b is dicyclohexylcarbodiimide, N- hydroxysuccinimide, 1- ethyl-(3-
Dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, N- hydroxysuccinimide, Avidin, biotin, N- hydroxysuccinimide
Or two kinds in maleimide；
Catalyst c:4- (N, the N- dimethylamino) pyridine or 4- (N, N- diallyl amino) pyridine；
It is specific:
(1) dewatering medicament and NHS, DCC are dissolved in tetrahydrofuran, pass through the dewatering medicament-finally obtained that stirs, cleans
NHS；
(2) it takes dewatering medicament-NHS to be added in methanol, after stirring, after dialysing in methyl alcohol, dialyses in water again
Finally the low generation PAMAM of dewatering medicament-can be obtained in drying；
(3) self assembly of amphiphilic nano micella: the low generation PAMAM of dewatering medicament-is taken to be dissolved in distilled water, ultrasonic vibration
Afterwards, the amphipathic self-assembled micelle based on low generation PAMAM dendrimer is both obtained.
It is more specific:
(1) that dewatering medicament and NHS, DCC are dissolved in the ratio in tetrahydrofuran according to molar ratio 1:1:1 ~ 1:10:10 is molten
In passing through 24 ~ 48h of stirring, filtering and impurity removing, remove solvent and chromatograph dewatering medicament-NHS obtained after separation；
(2) dewatering medicament-NHS is taken to be added in methanol, with low generation PAMAM dendrimer according to the ratio of 1:2 in room temperature
Under, magnetic agitation reaction 48h, rear first dialysis 12 in methyl alcohol ~ for 24 hours, then dialyse 24 ~ 48h in water, and being finally freeze-dried can
Obtain the low generation PAMAM of dewatering medicament-；Dialysis bag filter used selects specification as needed, and specification is MWCO=500-2000；
(3) the low generation PAMAM of dewatering medicament-is taken to be dissolved in distilled water with the ratio of 1mg/mL, after ultrasonic vibration, both it is described
Amphipathic self-assembled micelle based on low generation PAMAM dendrimer.
The self-assembled nano micelle of its low generation PAMAM dendrimer is alternatively arranged as carrier and contains other dewatering medicaments.
Other dewatering medicaments are as follows: Tarceva, Suo Fei replace Buddhist nun, methotrexate (MTX), adriamycin, taxol, rheum emodin, meter Fei Si
The drugs such as ketone, metapristone.
The preparation method comprises the following steps: the amphipathic self-assembled micelle of low generation PAMAM dendrimer is codissolved in dewatering medicament organic
In solvent, pass through volatilization or N2Drying film forming, adds water in the ratio with water quality than 1 ~ 100:1000, is by ultrasound
It can obtain containing the nano-micelle compound of other dewatering medicaments.
The amphipathic self-assembled micelle based on low generation PAMAM dendrimer, not only can be separately as pro-drug
Or bioactive molecule plays a role, and also can be used as nano-carrier, contains other hydrophobic drugs and plays a role, realizes the joint of drug
Treatment.
The invention has the following advantages:
1. the present invention is based on low generation PAMAM, relative to and height for PAMAM, low generation PAMAM synthesis step is simple, at low cost
It is honest and clean, it is more suitable for large-scale application.Moreover, the space steric effect of low generation PAMAM is far smaller than height for PAMAM, this is just its end
It is terminal modified to provide convenience.In addition, high carry a large amount of amino for the surface PAMAM, easily with various lifes that negative electricity is had in organism
Object membrane interaction, therefore there are higher cytotoxicities, it is easier to haemolysis occurs.
2. the chemical coupling of hydrophobic drug and low generation PAMAM dendrimer, can improve hydrophobic drug solubility
Difference, the lower defect of bioavailability, that there are toxicity to solve the problems, such as clinic classic chemotherapy drug is big, safety coefficient is low provides
New approaches.
3. the nano-micelle being synthetically prepared acts not only as bioactive molecule or precursor medicine is independently used for disease treatment, also
It can be used as nano-carrier, contain other dewatering medicaments, realize the drug combination of drug, heighten the effect of a treatment.Reasonable drug combination is not
Toxicity can be only reduced, drug resistance can be also reduced, improves curative effect.
Detailed description of the invention
Fig. 1 ursolic acid modification based on G0 The amphipathic self-assembled nano micelle synthetic route chart of PAMAM dendrimer；
Fig. 2 ursolic acid modification based on G0 The amphipathic self-assembled nano micelle synthetic route chart of PAMAM dendrimer；
Based on ursolic acid, G prepared by Fig. 30 The partial size of the amphipathic self-assembled nano micelle of PAMAM dendrimer point
Butut；
Fig. 4 G0、G1、G5PAMAM dendrimer acts on the MTT experiment result (4h) of breast cancer cancer cell (4T1)；
Based on ursolic acid, G prepared by Fig. 50 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on liver
The MTT experiment result (4h) of cancer cell (HepG2)；
Based on ursolic acid, G prepared by Fig. 60 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on liver
The MTT experiment result (for 24 hours) of cancer cell (HepG2)；
Based on ursolic acid, G prepared by Fig. 70 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on black
The MTT experiment result (4h) of pigment oncocyte (B16F10)；
Based on ursolic acid, G prepared by Fig. 80 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on black
The MTT experiment result (for 24 hours) of pigment oncocyte (B16F10)；
Based on ursolic acid, G prepared by Fig. 90 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on skin
The MTT experiment result (4h) of skin squamous cancer cell (SCL)；
Based on ursolic acid, G prepared by Figure 100 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on skin
The MTT experiment result (for 24 hours) of skin squamous cancer cell (SCL)；
Based on ursolic acid, G prepared by Figure 111 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on cream
The MTT experiment result (4h) of adenocarcinoma cell (MCF-7)；
Based on ursolic acid, G prepared by Figure 121 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on cream
The MTT experiment result (for 24 hours) of adenocarcinoma cell (MCF-7)；
Based on ursolic acid, G prepared by Figure 130 The amphipathic conjugate of PAMAM dendrimer contains Tarceva from group
Dress nano-micelle acts on the MTT experiment result (for 24 hours) of lung carcinoma cell (A549)；
Based on aspirin, G prepared by Figure 140 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on
In the MTT experiment result (for 24 hours) of lung carcinoma cell (A549)；
Based on aspirin, G prepared by Figure 150 The amphipathic conjugate of PAMAM dendrimer contains Tarceva certainly
Assemble nanometer micella acts on the MTT experiment result (for 24 hours) of lung carcinoma cell (A549)；
Prepared by Figure 16 based on ursolic acid, PAMAM dendrimer amphipathic conjugate self assembly and contain hydrophobic medicine
Object process.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention
Technical solution is described further, but the present invention is not limited only to this.
The synthesis of the amphipathic dendrimer conjugate of 1 ursolic acid of embodiment modification
(1) it is based on G0Synthesis (the G of the amphipathic conjugate of PAMAM dendrimer0-UA2For)
1. the synthesis of UA-NHS
Take 2.28g(5mmol) ursolic acid (referred to as: UA, similarly hereinafter) and in 150mL round-bottomed flask, 50mL tetrahydrofuran is added,
After UA dissolution, the 1.15g(10mmol dissolved with 10mL acetonitrile is added) NHS, it is then added dropwise dropwise at 10 ~ 15 DEG C and uses 25mL
The 2.06g(10mmol of tetrahydrofuran dissolution) DCC.Magnetic agitation is for 24 hours at room temperature.After reaction, it is filtered to remove sediment, so
Tetrahydrofuran is removed with Rotary Evaporators afterwards, obtains white powder crude material.The isolated pure UA- of silica gel column chromatography
NHS, eluant, eluent are methylene chloride: methanol (v/v)=50:1.Its high resolution mass spectrum is shown in Fig. 1.
② GO-UA2Synthesis
Taking 0.44g(0.8mmol) for UA-NHS in 50mL round-bottomed flask, 0.21g is added dropwise in addition 15mL methanol, ice bath
(0.4mmol) G0 PAMAM solution.At room temperature, it is protected from light magnetic agitation 48h.After reaction, reaction solution is dialysed in methyl alcohol
(MWCO=1000) for 24 hours, then dialyse 48h in water again.After dialysis, white powder, i.e. G can be arrived by carrying out freeze-drying0-
UA2Product.Its high resolution mass spectrum is shown in Fig. 2.
(2) it is based on G1Synthesis (the G of your amphipathic conjugate of PAMAM dendrimer1-UA1For)
1. the synthesis of UA-NHS
Take 2.28g(5mmol) ursolic acid (referred to as: UA, similarly hereinafter) and in 150mL round-bottomed flask, 50mL tetrahydrofuran is added,
After UA dissolution, the 1.15g(10mmol dissolved with 10mL acetonitrile is added) NHS, it is then added dropwise dropwise at 10 ~ 15 DEG C and uses 25mL
The 2.06g(10mmol of tetrahydrofuran dissolution) DCC.Magnetic agitation is for 24 hours at room temperature.After reaction, it is filtered to remove sediment, so
Tetrahydrofuran is removed with Rotary Evaporators afterwards, obtains white powder crude material.The isolated pure UA- of silica gel column chromatography
NHS, eluant, eluent are methylene chloride: methanol (v/v)=50:1.Its high resolution mass spectrum is shown in Fig. 1.
② G1-UA1Synthesis
Taking 0.22g(0.4mmol) for UA-NHS in 50mL round-bottomed flask, addition 15mL methanol is added drop-wise to 0.21g
(0.4mmol) G1In PAMAM ice bath solution.At room temperature, it is protected from light magnetic agitation 48h.After reaction, reaction solution in methanol
For 24 hours, then dialyse middle dialysis (MWCO=1500) 48h in water again.After dialysis, white powder can be arrived by carrying out freeze-drying,
That is G1-UA1Product.
The synthesis of the amphipathic dendrimer conjugate of 2 methotrexate (MTX) of embodiment modification (is based on G1Tree-shaped point of PAMAM
Son, G1- Me 1For)
Take 0.182g(0.4mmol) methotrexate (MTX) (Methotrexate abbreviation: Me, similarly hereinafter) in 150mL round-bottomed flask,
After the dissolution of 50mL n,N-Dimethylformamide is added, the 0.115g(10mmol dissolved with 10mL acetonitrile is added) NHS, then exist
The 0.206g(10mmol dissolved with 25mL tetrahydrofuran is added dropwise at 10 ~ 15 DEG C dropwise) DCC.Magnetic agitation is for 24 hours at room temperature.Reaction
After, it is filtered to remove sediment, obtains the organic solution of reactive intermediates；Reaction solution is drawn to be added drop-wise to dissolved with 0.571g
(0.4mmol) G1In PAMAM ice bath solution.At room temperature, it is protected from light magnetic agitation 48h.After reaction, reaction solution in N, N-
Dialyse (MWCO=1500) 1 ~ 3d in dimethylformamide, and then dialyse 3d in water, is freeze-dried to obtain methotrexate (MTX) and repairs
The amphipathic dendrimer conjugate of decorations.
The synthesis of the amphipathic dendrimer conjugate of 3 aspirin of embodiment modification (is based on G0Tree-shaped point of PAMAM
Son, G0- ASP 1For)
Take 72mg(0.4mmol) and aspirin (Aspirin abbreviation: ASP, similarly hereinafter) and in 150mL round-bottomed flask, it is added
After the dissolution of 50mL methylene chloride, the 0.115g(10mmol dissolved with 10mL acetonitrile is added) NHS, then at 10 ~ 15 DEG C dropwise
The 0.206g(10mmol dissolved with 25mL tetrahydrofuran is added dropwise) DCC.Magnetic agitation is for 24 hours at room temperature.After reaction, it crosses and filters out
Sediment is removed, the organic solution of reactive intermediates is obtained；Reaction solution is drawn to be added drop-wise to dissolved with 0.21g(0.4mmol) G0
In PAMAM ice bath solution.At room temperature, it is protected from light magnetic agitation 48h.After reaction, reaction solution in n,N-Dimethylformamide
Middle dialysis (MWCO=500) 1 ~ 3d, then dialyse 3d in water, is freeze-dried to obtain the amphipathic tree of methotrexate (MTX) modification
Shape molecule conjugate.
The synthesis of the amphipathic dendrimer nano-micelle of 4 ursolic acid of embodiment modification is (with G0-UA2For)
Take 1mg G0-UA2Conjugate is dissolved in 1mL distilled water, ultrasonic vibration 15min ~ 1h.It can be observed in ultrasonic procedure
The phenomenon that transparent colloid being gradually become by muddiness to solution.After ultrasound, after taking 1uL to dilute 1000 times, using Malvern
Laser particle analyzer measures the partial size of self-assembled composite in aqueous solution.Measurement result shows G0PAMAM is amphipathic tree-shaped big
Molecule forms the more uniform nanoparticle of granularity in aqueous solution, and particle size is as shown in Figure 3.
The synthesis of the amphipathic dendrimer nano-micelle for containing Tarceva of 5 ursolic acid of embodiment modification
Take 1mg G0-UA2, 1mg Tarceva be codissolved in 1mL methanol, N21mL water, ultrasonic 10min is added in drying.It is super
Sound terminates that the self-assembled nano micelle for containing Tarceva anticancer drug can be obtained.After taking 1uL to dilute 1000 times, using granularity
Analyzer, scanning electron microscope etc. measure self-assembled composite particle diameter distribution in aqueous solution and distribution of charges carries out table
Sign.Measuring encapsulation rate under this condition is 30%, and particle diameter distribution shows that the nano-micelle Average Particle Diameters for containing Tarceva are
420nm。
The synthesis of the amphipathic dendrimer nano-micelle for containing Tarceva of 6 aspirin of embodiment modification
Take 1mg G0-ASP1, 1mg Tarceva be codissolved in 1mL methanol, N21mL water, ultrasonic 10min is added in drying.It is super
Sound terminates that the self-assembled nano micelle for containing Tarceva anticancer drug can be obtained.After taking 1uL to dilute 1000 times, using granularity
Analyzer, scanning electron microscope etc. measure self-assembled composite particle diameter distribution in aqueous solution and distribution of charges carries out table
Sign.
The amphiphilic nano micella of 7 ursolic acid of embodiment modification is as precursor medicine to the inhibiting effect of tumor proliferation
Mtt assay detects drug to hepatocellular carcinoma H22, melanoma cells B16F10, cutaneous squamous cancer cell SCL and cream
The inhibited proliferation of adenocarcinoma cell MCF-7,4T1, lung cell A549
1. taking one bottle of tumour cell in logarithmic growth phase state, 1 × 10 is made after digestion5The cell of a/ml is outstanding
Liquid.
2. cell suspension is moved into 96 orifice plates, every hole 100uL sets 37 DEG C, 5% CO2It is cultivated for 24 hours in incubator.
3. removing culture medium, self-assembled composite is added according to concentration gradient, every hole 100uL separately sets UA group and PAMAM
Group.After effect for 24 hours (or 4h), pastille culture medium is removed, serum-free is added in every hole, without phenol red medium 100uL, adds
MTT solution 10uL continues to be incubated for 4h.
4. discarding supernatant in plate, 100uL DMSO is added in every hole, vibrates 10min, is detected each hole light using microplate reader and is inhaled
Receipts value (OD value), and calculate the proliferation inhibition rate of cell: cell survival rate (%)=medication group mean OD value ÷ blank control group is flat
Equal OD value × 100%, carries out data processing with GraphPad Prism software, as a result sees Fig. 4 ~ 12.
Fig. 1, Fig. 2 are that ursolic acid modifies low generation PAMAM, prepare the synthetic route of amphipathic conjugate.Fig. 3 shows preparation
G0-UA2Amphipathic dendrimer self-assembled nano micelle particle diameter distribution is uniform, mean size 222nm；As shown in Figure 4, G5
The toxicity of PAMAM is much higher than low generation PAMAM(G0、G1Generation)；Fig. 5 ~ 12,14 show the G after ursolic acid, aspirin modification0
PAMAM and G1PAMAM dendrimer nano-micelle anticancer activity is greatly improved with respect to having for ursolic acid, and is all presented one
The curative effect of drug can be improved in fixed dosage-concentration dependent, the low generation dendrimer nano-micelle after illustrating ursolic acid modification.
Figure 13,15 are the effect for containing the nano-micelle of Tarceva and inhibiting to proliferation of lung cancer cells, relative to exclusive use nanometre glue
Beam G0-UA2, Tarceva, the drug effect after drug combination significantly improves, and shows there is pole in terms of the drug of preparation treatment tumour
Big application prospect.
Claims (1)
1. a kind of amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer, it is characterised in that it is by amphipathic idol
Connection object in water self assembly and obtain；The amphipathic conjugate is as shown in Formulas I or Formula II；
I;
II
Wherein Ra is dewatering medicament in Formulas I；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；
Wherein Ra is dewatering medicament in Formula II；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；Rd is hydrogen or hydrophobic medicine
Object；Re is hydrogen or dewatering medicament；Rf is hydrogen or dewatering medicament；Rg is hydrogen or dewatering medicament；
The dewatering medicament is ursolic acid or aspirin.
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