CN105854027A - Amphiphilic self-assembled nanomicelle based on nano Low-generation PAMAM (polyamidoamine) dendrimer and application thereof - Google Patents
Amphiphilic self-assembled nanomicelle based on nano Low-generation PAMAM (polyamidoamine) dendrimer and application thereof Download PDFInfo
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Abstract
The invention relates to an amphiphilic self-assembled nanomicelle based on nano Low-generation PAMAM (polyamidoamine) dendrimer and application thereof, particularly obtained conjugating low-generation PAMAM dendrimer and a hydrophobic agent and self-assembling in water via ultrasonic vibration. The nanomicelle has active molecules or a prodrug with drug loading capacity; the chemical conjugation of the hydrophobic agent and the low-generation PAMAM dendrimer helps improve the defects of poor solubility of the hydrophobic drug and low bioavailability, and a new idea is provided to solve the problem that traditional clinical chemotherapeutics are high in toxicity and low in safety coefficient; the nanomicelle can also function as a nanocarrier to carry other hydrophobic drugs, enabling joint use of drugs and enhancing therapeutic effect.
Description
Technical field
The present invention relates to a kind of amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer and application thereof.
Background technology
Molecular self-assembling (molecular self-assembly) refers in equilibrium conditions, and molecule is by intermolecular non-
Covalent bond weak interaction, such as Van der Waals force, hydrogen bond, hydrophobic interaction, electrostatic interaction, π-π interaction etc., spontaneous combination is formed
There is the stable aggregation of certain 26S Proteasome Structure and Function or supramolecular process.The aggregation of molecular self-assembling formation or supermolecule
It is generally of unimolecule or the more unexistent properties of low levels of molecular aggregation, such as optical, electrical, biological nature etc..General next
Saying, the self assembly of molecule has the minor alteration of structure enlarge-effect, i.e. molecular structure can cause the topological structure of whole molecule
Occur great variety, not only so molecule self assembly will not because of structure change and affect its characteristic, impart on the contrary
The assembling that self-assembly some more novel characteristics, such as pattern and structure are more enriched, special self assembly is machine-processed, special
Body performance and the feature such as easy functionalization and intelligent response, these performances make the great development prospect of molecular self-assembling.
Dendrimer (dendrimer) owing to having nanoscale, highly-water-soluble, monodispersity and adjustability of structure,
Can be formed the nanoparticle of suitable size by molecular self-assembling, the conveying that therefore can be used as cancer therapy drug and imaging medicine carries
Body.Compared to liposome, the covalent bond in dendrimer support provides more stable carrier structure, enables to withstand life
The change of reason condition, and drug molecule can be protected not decomposed rapidly, it is ensured that its specific medicinal property, promote drug molecule
Certain drug effect concentration is kept in tumor cell.Research finds that nano-carrier system based on dendrimer is poly-compared to linear
Compound carrier (such as PEG) has more superior pharmaceutical carrier performance, and this is the nano-scale and in the solution due to dendrimer
Closely caused by spherical structure.Polyamide-amide (PAMAM) Shi Yileiyou center dissipates to external symmetry and highly branched new
The tree-shaped macromolecular compound of type nanometer, has regular, fine structure, in monodispersity, has good dissolubility, 0 ~ 10 generation grain
Footpath scope is 10～130nm.Nano level particle diameter makes PAMAM be easier to through the biological barrier such as blood vessel wall, cell membrane, to its core
The heart and surface are modified, can with linker because of materials such as, antibody and vaccine, internal layer cavity can packaging medicine molecule, as medicine
The carrier of thing orientation transport.PAMAM dendrimer, as a new generation's pharmaceutical carrier, can pass through electrostatic interaction, hydrophobic interaction
Or hydrogen bond action is drug encapsulation portion in the inner, it is possible to by electrostatic, covalently bind in its surface formation complex, thus increase
Drug solubility, improves drug bioavailability.The research of PAMAM dendrimer and application have obtained continuous development, are subject to
The concern that numerous areas is increasingly extensive such as arrive from material science to biological medicine.
Application to PAMAM dendrimer at present focus primarily ones the aspects such as the carrier of medicine and gene, such as patent
Disclosing derivant and the application thereof of a kind of novel PAMAM in CN201010142282, its described derivant is phenylalanine
The PAMAM dendrimer that coupling is modified, for transfection and the internal transfection of gene of siRNA, it is to utilize PAMAM tree
On shape molecular surface, the positive charge of primary amine group reaches to deliver purpose to electronegative DNA or RNA adsorption, but
Electrostatic adsorption power is compared on the weak side with covalent effect power, less stable.CN201310404541 discloses a kind of based on
The self-assembled nanometer genophore complex of PAMAM, it is by mutual electrostatic interaction self assembly group by HA, PAMAM and gene
The compound system become, but Electrostatic Absorption belongs to more weak interaction, utilizes this mode carrying medicament, and drug loading can be led to by ratio
Cross chemical coupling little with physically trapping two ways, and poor stability, slow release effect is inconspicuous.Public affairs in CN201410271351
A kind of folic acid-PAMAM-ursolic acid the Nano medication with cancer target of cloth, the original material that this Nano medication is used is
Highly toxic G3 and G5 PAMAM type dendrimer (PAMAM), need to first pass through the chemical reaction ammonia by PAMAM surface
Base is replaced as hydroxyl to reduce the toxicity of material itself, then by folic acid and ursolic acid and the hydroxyl reaction on PAMAM surface, finally
Synthesis target product.Therefore this course of reaction is complicated, and production cost is higher, and this limits its application to a certain extent.Combine
Upper described, the studies above all uses the PAMAM of high algebraically as object of study (PAMAM algebraically was 3 ~ 6 generations), and height is for PAMAM
Synthesis step is loaded down with trivial details, causes high cost；Additionally, height carries a large amount of amino for PAMAM surface, easy and organism is interior with negative electricity
Various biomembranes interact, therefore there is higher cytotoxicity, it is easier to occur haemolysis.
For making up the deficiencies in the prior art, the present invention uses low generation PAMAM dendrimer as object of study.Relative to
High for PAMAM, the amino quantity of low generation pamam dendrimer molecular surface greatly reduces, and the most therefore toxicity be substantially reduced.And low generation
PAMAM synthesis step is simple, with low cost, is more suitable for large-scale application.Additionally, the space steric effect of low generation PAMAM is far away
Less than high for PAMAM, this just end modified provides convenience for it.But low generation PAMAM self can not become nano-micelle, because of
This, utilize hydrophobic drug and its coupling so that it is have amphipathic nature, and be self-assembled into nano-micelle further.It is thus possible to
Enough under aqueous environments, form stable nano-micelle structure.This nano-micelle not only can be independently as bioactive molecule or precursor medicine
For treating, being alternatively arranged as pharmaceutical carrier, bag carries other dewatering medicaments further, it is achieved the therapeutic alliance to medicine, to strengthen
The therapeutic effect of nano-micelle.
Different pharmaceutical drug combination can increase target spot, the multiple target spots on disabling signal Signal Transduction Pathways, it is possible to reduces swollen
The drug resistance of tumor.The present invention based on low generation PAMAM, by the coupling with dewatering medicament, not only improves medicine molten first
The defect that Xie Du is poor, bioavailability is relatively low, it is often more important that construct amphiphilic nano carrier systems, gives its bag medicine carrying
The potentiality of thing, to realize the use in conjunction of medicine, have not been reported this kind of application method of low generation PAMAM, have greatly
Novelty, has the biggest application prospect.
Summary of the invention
It is an object of the invention to provide a kind of amphipathic self-assembled micelle based on low generation PAMAM dendrimer and answer
With, including its preparation method and the application in fields such as pharmacy thereof.This nano-micelle is based on low generation PAMAM(G0Such as formula III, G1As
Formula IV) dendrimer, bioactive molecule or the precursor medicine possessing Drug loading capacity can be obtained by chemical modification.
III
IV
For reaching above-mentioned purpose, the present invention provides a kind of amphipathic self-assembled micelle based on low generation PAMAM dendrimer, and it is special
Levy and be by chemical method at low generation PAMAM dendrimer G0Generation (formula III), G1Hydrophobic drug is introduced on generation (formula IV),
Increase dendrimer hydrophobicity, give it amphipathic so that it is can self assembly be stable nano-micelle in an aqueous medium；
Described low generation PAMAM dendrimer, for polyamidoamine dendrimer, refers mainly to G0、G1Generation, but it is not limited to this, meet
Above-mentioned principle；Described hydrophobic drug, based on carboxylic acids medicine, can carry out idol by amido link and PAMAM dendrimer
Connection, but it is not limited to this, chemical coupling can be carried out with PAMAM dendrimer so that it is become amphipathic dendrimer, described
Hydrophobic drug includes but not limited to: antitumor drug includes but not limited to this: ursolic acid, methotrexate, aminopterin-induced syndrome, Qi Dun
Fruit acid, gamlogic acid；Cardiovascular drugs includes but not limited to: atorvastatin, fluvastatin, Pitavastatin, linoleic acid；Anti-inflammatory agent
Thing includes but not limited to: aspirin at interior salicylic acid, ibuprofen at interior aryl alkanoic acid class, meclofenamic acid interior
That acids fragrant；Drug for liver disease includes but not limited to: ursodesoxycholic acid.
Concrete, 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；Described amphipathic conjugate is as shown in Formulas I or Formula II；
I
II
Wherein in Formulas I, Ra is dewatering medicament；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；
Wherein in Formula II, Ra is dewatering medicament；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；Rd is hydrogen or hydrophobic medicine
Thing；Re is hydrogen or dewatering medicament；Rf is hydrogen or dewatering medicament；Rg is hydrogen or dewatering medicament；
Described dewatering medicament is the medicine containing carboxyl.
Described dewatering medicament be ursolic acid, methotrexate, aminopterin-induced syndrome, pemetrexed, oleanolic acid, tetrachloro fragrant that
Acid, chlorambucil, gamlogic acid, Raltitrexed, linoleic acid, enalapril, fosinopril, Candesartan, gemfibrozil, department's handkerchief
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, ibuprofen, meclofenamic acid, that acid fragrant
Or ursodesoxycholic acid.
It is preferably ursolic acid or aspirin.
The mode of its self assembly is ultrasonic vibration.
It is in preparing antitumor drug and as pharmaceutical carrier application in preparing antitumor drug.
The preparation process of described 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, adds catalyst a, catalyst b, obtain reactive intermediates；
(2) synthesis of the amphipathic conjugate of PAMAM-hydrophobic drug
Low generation PAMAM is dissolved in organic solvent, adds the reactive intermediates obtained in catalyst c, with above-mentioned steps and carry out
Reaction further, controls temperature to reaction completely.Then reactant liquor is dialysed in organic solvent, distilled water successively, finally
Lyophilizing i.e. can get amphipathic conjugate.
(3) described synthetic route is as follows:
Wherein catalyst a, catalyst b include but not limited to: dicyclohexylcarbodiimide (DCC)/N-hydroxysuccinimide
(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 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
Taking amphipathic conjugate soluble in water, supersound process is i.e. available based on low generation PAMAM dendrimer amphipathic from group
Dress nano-micelle.Particle size analyzer, scanning electron microscope etc. is used to measure self-assembled composite in aqueous after dilution
Particle diameter distribution and CHARGE DISTRIBUTION characterize.
3. using PAMAM-hydrophobic drug conjugate as carrier, the preparation nano-micelle complex containing insoluble drug
Take the amphipathic conjugate of PAMAM-hydrophobic drug and other dewatering medicaments are codissolved in organic solvent, N2Dry up, add
Water, the amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer that i.e. can obtain bag medicine carrying thing after supersound process is multiple
Compound.After dilution, particle size analyzer, scanning electron microscope etc. is used to measure self-assembled composite particle diameter in aqueous
Distribution and CHARGE DISTRIBUTION characterize.
Preparation method is:
(1) hydrophobic drug is dissolved in organic solvent, adds catalyst a, catalyst b, obtain reactive intermediates；
(2) low generation PAMAM is dissolved in organic solvent, adds the reactive intermediates obtained in catalyst c, with above-mentioned steps and enter
Traveling single step reaction, after process, both must be based on the amphipathic conjugate of low generation PAMAM dendrimer, it can be in an aqueous medium
It is self-assembled into as the amphipathic self-assembled nano micelle based on low generation PAMAM dendrimer described in stable.
Described 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；
Described catalyst c:4-(N, N-dimethylamino) pyridine or 4-(N, N-diallyl amino) pyridine；
Concrete:
(1) dewatering medicament is dissolved in oxolane with NHS, DCC, the dewatering medicament-NHS finally obtained by stirring, remove impurity；
(2) taking dewatering medicament-NHS and add in methanol, after stirring, in methanol, after dialysis, again in water, dialysis is last
It is dried available dewatering medicament-low generation PAMAM；
(3) self assembly of amphiphilic nano micelle: take dewatering medicament-low generation PAMAM and be dissolved in distilled water, after ultrasonic vibration, both
Amphipathic self-assembled micelle based on low generation PAMAM dendrimer described in.
More specifically:
(1) dewatering medicament is dissolved in oxolane with NHS, DCC it is dissolved according to the ratio of mol ratio 1:1:1 ~ 1:10:10, logical
Dewatering medicament-the NHS obtained after crossing stirring 24 ~ 48h, filtering and impurity removing, removing solvent chromatography；
(2) take dewatering medicament-NHS and add in methanol, with low generation PAMAM dendrimer according to 1:2 ratio at room temperature,
Magnetic agitation reaction 48h, the rear 12 ~ 24h that first dialyses in methanol, then dialyse in water 24 ~ 48h, and last lyophilization can obtain
Dewatering medicament-low generation PAMAM；Bag filter used by dialysis selects specification as required, and specification is MWCO=500-2000；
(3) take dewatering medicament-low generation PAMAM and be dissolved in distilled water with the ratio of 1mg/mL, after ultrasonic vibration, both described based on
The amphipathic self-assembled micelle of low generation PAMAM dendrimer.
The self-assembled nano micelle of its low generation PAMAM dendrimer is alternatively arranged as carrier bag and carries other dewatering medicaments.
Other dewatering medicaments are: Erlotinib, Suo Fei replace Buddhist nun, methotrexate, amycin, paclitaxel, rheum emodin, meter Fei Si
The medicine such as ketone, metapristone.
Preparation method is: be codissolved in organic by the amphipathic self-assembled micelle of low generation PAMAM dendrimer with dewatering medicament
In solvent, by volatilization or N2Dry up film forming, add water in the water quality ratio than 1 ~ 100:1000, by ultrasonic be
Bag can be obtained and carry the nano-micelle complex of other dewatering medicaments.
Described amphipathic self-assembled micelle based on low generation PAMAM dendrimer, not only can be separately as prodrug
Or bioactive molecule plays a role, it is possible to as nano-carrier, bag carries other hydrophobic drugs and plays a role, it is achieved the associating of medicine
Treatment.
The method have the advantages that
1. the present invention is based on low generation PAMAM, relative to and height for PAMAM, low generation PAMAM synthesis step is simple, with low cost,
It is more suitable for large-scale application.And, the space steric effect of low generation PAMAM is far smaller than high for PAMAM, and this just repaiies for its end
Decorations are provided convenience.Additionally, height carries a large amount of amino for PAMAM surface, the interior various biomembranes with negative electricity of easy and organism
Interact, therefore there is higher cytotoxicity, it is easier to haemolysis occurs.
2. hydrophobic drug and the chemical coupling of low generation PAMAM dendrimer, can improve hydrophobic drug dissolubility
, there is, for solving clinical classic chemotherapy medicine, the problem offer that toxicity is big, safety coefficient is low in the defect that difference, bioavailability are relatively low
New approaches.
The most synthetically prepared nano-micelle acts not only as bioactive molecule or precursor medicine is independently used for disease treatment, also
Can wrap and carry other dewatering medicaments, it is achieved the drug combination of medicine as nano-carrier, heighten the effect of a treatment.Reasonably drug combination is not
Only can reduce toxicity, also can reduce drug resistance, improve curative effect.
Accompanying drawing explanation
Fig. 1 ursolic acid modify based on G0 The amphipathic self-assembled nano micelle synthetic route chart of PAMAM dendrimer；
Fig. 2 ursolic acid modify based on G0 The amphipathic self-assembled nano micelle synthetic route chart of PAMAM dendrimer；
Prepared by Fig. 3 based on ursolic acid, G0 The particle diameter distribution of the amphipathic self-assembled nano micelle of PAMAM dendrimer
Figure；
Fig. 4 G0、G1、G5PAMAM dendrimer acts on the MTT experiment result (4h) of breast carcinoma cancerous cell (4T1)；
Prepared by Fig. 5 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on hepatocarcinoma
The MTT experiment result (4h) of cell (HepG2)；
Prepared by Fig. 6 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on hepatocarcinoma
The MTT experiment result (24h) of cell (HepG2)；
Prepared by Fig. 7 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on melanin
The MTT experiment result (4h) of oncocyte (B16F10)；
Prepared by Fig. 8 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on melanin
The MTT experiment result (24h) of oncocyte (B16F10)；
Prepared by Fig. 9 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on skin squama
The MTT experiment result (4h) of shape cancerous cell (SCL)；
Prepared by Figure 10 based on ursolic acid, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on skin
The MTT experiment result (24h) of squamous cancer cell (SCL)；
Prepared by Figure 11 based on ursolic acid, G1 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on mammary gland
The MTT experiment result (4h) of cancerous cell (MCF-7)；
Prepared by Figure 12 based on ursolic acid, G1 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on mammary gland
The MTT experiment result (24h) of cancerous cell (MCF-7)；
Prepared by Figure 13 based on ursolic acid, G0 The amphipathic conjugate bag of PAMAM dendrimer carries Erlotinib self assembly
Nano-micelle acts on the MTT experiment result (24h) of lung carcinoma cell (A549)；
Prepared by Figure 14 based on aspirin, G0 The amphipathic self-assembled nano micelle of PAMAM dendrimer acts on lung
The MTT experiment result (24h) of cancerous cell (A549)；
Prepared by Figure 15 based on aspirin, G0 The amphipathic conjugate bag of PAMAM dendrimer carries Erlotinib from group
Dress nano-micelle acts on the MTT experiment result (24h) of lung carcinoma cell (A549)；
Prepared by Figure 16 based on ursolic acid, the amphipathic conjugate self assembly of PAMAM dendrimer and bag carry dewatering medicament mistake
Journey.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
The synthesis of the amphipathic dendrimer conjugate that embodiment 1 ursolic acid is modified
(1) based on G0Synthesis (the G of the amphipathic conjugate of PAMAM dendrimer0-UA2As a example by)
1. the synthesis of UA-NHS
Take 2.28g(5mmol) ursolic acid (be called for short: UA, lower with) in 150mL round-bottomed flask, add 50mL oxolane, treat UA
After dissolving, add the 1.15g(10mmol dissolved with 10mL acetonitrile) NHS, then at 10 ~ 15 DEG C, dropwise 25mL tetrahydrochysene is used in dropping
The 2.06g(10mmol that furan dissolves) DCC.Magnetic agitation 24h under room temperature.After reaction terminates, it is filtered to remove precipitate, then uses
Rotary Evaporators removes oxolane, obtains white powder crude material.The UA-NHS that silica gel column chromatography isolated is pure, washes
De-agent is dichloromethane: methanol (v/v)=50:1.Its high resolution mass spectrum is shown in Fig. 1.
② GO-UA2Synthesis
Taking 0.44g(0.8mmol) UA-NHS is in 50mL round-bottomed flask, and addition 15mL methanol, ice bath drips 0.21g
(0.4mmol) G0 PAMAM solution.Under room temperature, lucifuge magnetic agitation 48h.After reaction terminates, reactant liquor is dialysed in methanol
(MWCO=1000) 24h, dialyse in water 48h the most again.After dialysis terminates, carry out lyophilization and can arrive white powder, i.e. G0-
UA2Product.Its high resolution mass spectrum is shown in Fig. 2.
(2) based on G1Synthesis (the G of your amphipathic conjugate of PAMAM dendrimer1-UA1As a example by)
1. the synthesis of UA-NHS
Take 2.28g(5mmol) ursolic acid (be called for short: UA, lower with) in 150mL round-bottomed flask, add 50mL oxolane, treat UA
After dissolving, add the 1.15g(10mmol dissolved with 10mL acetonitrile) NHS, then at 10 ~ 15 DEG C, dropwise 25mL tetrahydrochysene is used in dropping
The 2.06g(10mmol that furan dissolves) DCC.Magnetic agitation 24h under room temperature.After reaction terminates, it is filtered to remove precipitate, then uses
Rotary Evaporators removes oxolane, obtains white powder crude material.The UA-NHS that silica gel column chromatography isolated is pure, washes
De-agent is dichloromethane: methanol (v/v)=50:1.Its high resolution mass spectrum is shown in Fig. 1.
② G1-UA1Synthesis
Take 0.22g(0.4mmol) UA-NHS in 50mL round-bottomed flask, add 15mL methanol, be added drop-wise to 0.21g(0.4mmol)
G1In PAMAM ice bath solution.Under room temperature, lucifuge magnetic agitation 48h.After reaction terminates, reactant liquor is dialysed in methanol
(MWCO=1500) 24h, dialyse in water 48h the most again.After dialysis terminates, carry out lyophilization and can arrive white powder, i.e. G1-
UA1Product.
The synthesis of the amphipathic dendrimer conjugate that embodiment 2 methotrexate is modified is (based on G1PAMAM dendrimer, G1-
Me 1As a example by)
Take 0.182g(0.4mmol) methotrexate (Methotrexate be called for short: Me, lower with) in 150mL round-bottomed flask, add
After 50mL DMF dissolves, add the 0.115g(10mmol dissolved with 10mL acetonitrile) NHS, then 10 ~ 15
The 0.206g(10mmol that at DEG C, dropwise dropping is dissolved with 25mL oxolane) DCC.Magnetic agitation 24h under room temperature.Reaction terminates
After, it is filtered to remove precipitate, obtains the organic solution of reactive intermediates；Draw reactant liquor to be added drop-wise to dissolved with 0.571g
(0.4mmol) G1In PAMAM ice bath solution.Under room temperature, lucifuge magnetic agitation 48h.After reaction terminates, reactant liquor at N, N-
Dialyse in dimethylformamide (MWCO=1500) 1 ~ 3d, and then dialyse in water 3d, and lyophilization i.e. can get methotrexate and repaiies
The amphipathic dendrimer conjugate of decorations.
The synthesis of the amphipathic dendrimer conjugate that embodiment 3 aspirin is modified is (based on G0PAMAM dendrimer, G0-
ASP 1As a example by)
Take 72mg(0.4mmol) aspirin (Aspirin be called for short: ASP, lower with) in 150mL round-bottomed flask, add 50mL bis-
After chloromethanes dissolves, add the 0.115g(10mmol dissolved with 10mL acetonitrile) NHS, at 10 ~ 15 DEG C, then dropwise drip use
The 0.206g(10mmol that 25mL oxolane dissolves) DCC.Magnetic agitation 24h under room temperature.After reaction terminates, it is filtered to remove precipitation
Thing, obtains the organic solution of reactive intermediates；Draw reactant liquor to be added drop-wise to dissolved with 0.21g(0.4mmol) G0PAMAM ice bath
In solution.Under room temperature, lucifuge magnetic agitation 48h.After reaction terminates, reactant liquor is dialysed in DMF
(MWCO=500) 1 ~ 3d, then dialyse in water 3d, and lyophilization i.e. can get the amphipathic dendrimer that methotrexate is modified
Conjugate.
The synthesis of the amphipathic dendrimer nano-micelle that embodiment 4 ursolic acid is modified is (with G0-UA2As a example by)
Take 1mg G0-UA2Conjugate is dissolved in 1mL distilled water, ultrasonic vibration 15min ~ 1h.It is observed that molten in ultrasonic procedure
Liquid can be gradually become the phenomenon of transparent colloid by muddiness.After ultrasonic end, after taking 1uL dilution 1000 times, use Malvern laser
Particle size analyzer determination self-assembled composite particle diameter in aqueous.Measurement result shows, G0The amphipathic dendrimer of PAMAM
Defining the more uniform nanoparticle of granularity in aqueous, its size is as shown in Figure 3.
The bag that embodiment 5 ursolic acid is modified carries the synthesis of the amphipathic dendrimer nano-micelle of Erlotinib
Take 1mg G0-UA2, 1mg Erlotinib be codissolved in 1mL methanol, N2Dry up, add 1mL water, ultrasonic 10min.Ultrasonic knot
Bundle i.e. can obtain bag and carry the self-assembled nano micelle of Erlotinib cancer therapy drug.After taking 1uL dilution 1000 times, use granulometry
The mensuration self-assembled composite such as instrument, scanning electron microscope particle diameter in aqueous is distributed and CHARGE DISTRIBUTION characterizes.This
Under the conditions of to record envelop rate be 30%, particle diameter distribution shows, it is 420nm that bag carries the nano-micelle Average Particle Diameters of Erlotinib.
The bag that embodiment 6 aspirin is modified carries the synthesis of the amphipathic dendrimer nano-micelle of Erlotinib
Take 1mg G0-ASP1, 1mg Erlotinib be codissolved in 1mL methanol, N2Dry up, add 1mL water, ultrasonic 10min.Ultrasonic knot
Bundle i.e. can obtain bag and carry the self-assembled nano micelle of Erlotinib cancer therapy drug.After taking 1uL dilution 1000 times, use granulometry
The mensuration self-assembled composite such as instrument, scanning electron microscope particle diameter in aqueous is distributed and CHARGE DISTRIBUTION characterizes.
The amphiphilic nano micelle that embodiment 7 ursolic acid is modified is as the precursor medicine inhibitory action to tumor proliferation
Mtt assay detection medicine is to hepatocellular carcinoma H22, melanoma cell B16F10, cutaneous squamous cancerous cell SCL and breast carcinoma
Cell MCF-7,4T1, the inhibited proliferation of lung cell A549
1. take the tumor cell one bottle being in exponential phase state, after digestion, make 1 × 105The cell suspension of individual/ml.
2. cell suspension is moved into 96 orifice plates, every hole 100uL, puts 37 DEG C, 5% CO2Incubator is cultivated 24h.
3. remove culture medium, add self-assembled composite, every hole 100uL according to Concentraton gradient, separately set UA group and PAMAM
Group.Effect 24h after (or 4h), remove pastille culture medium, in every hole add serum-free, without phenol red medium 100uL, add
MTT solution 10uL, continues to hatch 4h.
4. discarding supernatant in plate, every hole adds 100uL DMSO, and vibrate 10min, uses microplate reader to detect each hole light and inhales
Receipts value (OD value), and calculate the proliferation inhibition rate of cell: cell survival rate (%)=medication group mean OD value ÷ blank group is put down
All OD value × 100%, carries out data process with GraphPad Prism software, and result is shown in 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 even particle size distribution, mean size is 222nm；As shown in Figure 4, G5
The toxicity of PAMAM is far above low generation PAMAM(G0、G1Generation)；Fig. 5 ~ 12,14 show, the G after ursolic acid, aspirin are modified0
PAMAM and G1PAMAM dendrimer nano-micelle active anticancer is greatly improved relative to having had for ursolic acid, and all presents one
Fixed dosage-concentration dependent, illustrates that the low generation dendrimer nano-micelle after ursolic acid modification can improve the curative effect of medicine.
Figure 13,15 it is the nano-micelle effect that proliferation of lung cancer cells is suppressed that bag carries Erlotinib, relative to being used alone nanometre glue
Bundle G0-UA2, Erlotinib, the drug effect after drug combination significantly improves, show preparation treatment tumor medicine in terms of there is pole
Big application prospect.
Claims (6)
1. an amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer, it is characterised in that it is by amphipathic idol
Connection thing in water self assembly and obtain；Described amphipathic conjugate is as shown in Formulas I or Formula II；
I
II
Wherein in Formulas I, Ra is dewatering medicament；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；
Wherein in Formula II, Ra is dewatering medicament；Rb is hydrogen or dewatering medicament；Rc is hydrogen or dewatering medicament；Rd is hydrogen or hydrophobic medicine
Thing；Re is hydrogen or dewatering medicament；Rf is hydrogen or dewatering medicament；Rg is hydrogen or dewatering medicament；
Described dewatering medicament is the medicine containing carboxyl.
2. amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer as claimed in claim 1, its feature exists
In, described dewatering medicament is ursolic acid, methotrexate, aminopterin-induced syndrome, pemetrexed, oleanolic acid, four clofenamic acides, benzene fourth
Acid chlormethine, gamlogic acid, Raltitrexed, linoleic acid, enalapril, fosinopril, Candesartan, gemfibrozil, Sparfloxacin, Ah
Atorvastatin, mefenamic acid, Raltitrexed, indomethacin, fluvastatin, edatrexate, Pitavastatin, edatrexate, sub-oil
Acid, all-trans-retinoic acid, 9-cis-retinoic acid, aspirin, salicylic acid, ibuprofen, meclofenamic acid, that acid fragrant or Bears deoxygenate
Cholic acid.
3. amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer as claimed in claim 1, its feature exists
In, described dewatering medicament is ursolic acid or aspirin.
4. amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer as claimed in claim 1, its feature exists
Mode in its self assembly is ultrasonic vibration.
5. amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer as claimed in claim 1 is anti-swollen in preparation
Application in tumor medicine.
6. amphiphilic nano self-assembled micelle based on low generation PAMAM dendrimer as claimed in claim 1 carries as medicine
Body application in preparing antitumor drug.
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| CN106581693A (en) * | 2016-11-03 | 2017-04-26 | 福州大学 | Application of ursolic acid conjugate having anticancer activity and serving as drug carrier or molecular probe carrier |
| CN107349429A (en) * | 2017-07-18 | 2017-11-17 | 福州大学 | A kind of conjugate carrier-free self-assembled nanometer grain of aptamer ursolic acid and its preparation and application |
| CN109456490A (en) * | 2018-10-23 | 2019-03-12 | 青岛大学 | A kind of preparation method and product of dendritic macromole hydrogel |
| CN111298132A (en) * | 2020-02-22 | 2020-06-19 | 新乡医学院 | Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof |
| CN113057939A (en) * | 2021-03-09 | 2021-07-02 | 湖北工业大学 | Preparation method and application of chemically modified gelatin micelle coated tripterine medicine |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106581693A (en) * | 2016-11-03 | 2017-04-26 | 福州大学 | Application of ursolic acid conjugate having anticancer activity and serving as drug carrier or molecular probe carrier |
| CN106581693B (en) * | 2016-11-03 | 2019-07-09 | 福州大学 | A kind of application of the ursolic acid conjugate as pharmaceutical carrier or molecular probe carrier with anticancer activity |
| CN107349429A (en) * | 2017-07-18 | 2017-11-17 | 福州大学 | A kind of conjugate carrier-free self-assembled nanometer grain of aptamer ursolic acid and its preparation and application |
| CN109456490A (en) * | 2018-10-23 | 2019-03-12 | 青岛大学 | A kind of preparation method and product of dendritic macromole hydrogel |
| CN111298132A (en) * | 2020-02-22 | 2020-06-19 | 新乡医学院 | Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof |
| CN111298132B (en) * | 2020-02-22 | 2022-06-24 | 新乡医学院 | Tree-shaped molecule gemcitabine self-assembled nano prodrug and preparation method and application thereof |
| CN113057939A (en) * | 2021-03-09 | 2021-07-02 | 湖北工业大学 | Preparation method and application of chemically modified gelatin micelle coated tripterine medicine |
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