CN108727598A - Containing bonded graft copolymer of β-Carboxylamide and its preparation method and application - Google Patents
Containing bonded graft copolymer of β-Carboxylamide and its preparation method and application Download PDFInfo
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- 0 CCC(C)(C)C(C)CC(*C(C)(C)C(*1CC1)[N+])=O Chemical compound CCC(C)(C)C(C)CC(*C(C)(C)C(*1CC1)[N+])=O 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G81/00—Macromolecular compounds obtained by interreacting polymers in the absence of monomers, e.g. block polymers
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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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
Abstract
The invention discloses a kind of graft copolymer poly (ae-Asp-g-mPEG-g-Hap) containing β-Carboxylamide key, shown in its structure such as formula (I), it for main chain, and is grafted hexahydrophthalic anhydride and mPEG-2000 with poly- aminoethyl aspartic acid (poly (ae-Asp)).The invention also discloses the preparation methods of the graft copolymer, including the phosphoric acid catalyzed polycondensation of (1) L-Aspartic acid synthesizes PSI；(2) the complete open loop PSI synthesis poly (ae-Asp) of ethylenediamine；(3) mPEG with to nitro chloro-carbonic acid benzene fat NPC synthesizing polyethylene glycol monomethyl ether activity fat (mPEG-NP)；(4) mPEG-NP is grafted to the-NH of poly (ae-Asp)2, synthesis poly (ae-Asp-g-mPEG)；(5) in-NH2On continue be grafted hexahydrophthalic anhydride, synthesize the formula (I) graft copolymer.Formula (I) graft copolymer of the present invention, which can be used for constructing, carries adriamycin nano micella (poly (Asp-g-mPEG-g-Hap) Dox), it has preferable drug release property in acid condition, lower cytotoxicity and good cell are phagocytic, are with a wide range of applications.
Description
Technical field
The invention belongs to biological medicine technology, nanometer medicine and field of new materials, and in particular to containing β-carboxylic acyloxy amine key
The synthesis and its assembling of graft copolymer and the intelligent nano to tumour cell lysosome applied to preparation with acid triggering release
The purposes of micellar carrier.
Background technology
Malignant tumour seriously threatens the physical and mental health of the mankind, and clinical treatment is still based on small-molecule drug chemotherapy at present.
Small molecule chemotherapeutic drug achieves immense success in the early stage, but then will produce serious toxic side effect, and has low selectivity,
The defects of easy drug resistance, forces people to be badly in need of improving it.Scientific workers propose the medicine for tumor microenvironment
The concept of object transmission system, the i.e. rapid growth of tumor tissues cause it to be different from the microenvironment of normal structure, such as weary oxygen,
Acidity enhancing, temperature increase, and growth factor and hydrolysising protease secretion increase.Wherein, pH sensitive medicaments transmission system obtains
Research extensively, is designed according to the difference of normal human tissue and tumor microenvironment pH, and the pH of normal human blood is 7.35-
7.45, and it is 6.6-6.8 that the pH of tumor tissues is slightly lower, the pH of tumour cell endosome is 6.0-6.5, and the pH of lysosome is lower to be
5.0-5.5.According to this feature, make drug-loading system in normal blood circulation can holding structure relatively stablize, drug is transported
It is sent to after tumor locus the fast degradation under the low pH effects of tumor environment, drug is discharged, to which the poison for reducing normal tissue is secondary
Effect improves curative effect.
The unstable amido bond of acid is one of the research hotspot of pH sensitive medicaments transmission systems, such as β-carboxylic acid amide, cis- crow
Head amido bond.Amino is reacted with acid anhydrides (such as hexahydrophthalic anhydride, 2,3- dimethyl maleic anhydrides, citraconic anhydride) can form β-carboxyl
Amido bond.β-Carboxylamide key is stablized relatively in alkalinity and neutral environment, and is cracked in acidic environment, it was confirmed that β-carboxyl
Amido bond is highly suitable as the nano-medicament carrier construction unit of acid-sensitive.Zhang Jianhua project has been combined into the poly- (second of methoxyl group
Allyl diglycol)-b- poly- (6-caprolactone-co- γ-dimethylmaleimide -6-caprolactone), polyester segment is with acid-sensitive
β-carboxylic acyloxy amine key, can be assembled into micella.Under the conditions of 6.0 pH, β-carboxylic acid amide hydrolysis generates cationic primary amine, leads to electricity
Lotus reversion is turned negative number to positive number, and then makes drug quick release (Biomacromolecules 2014,15,4281-4292).
Adriamycin (Doxorubicin, Dox) is a kind of clinically widely applied cell cycle nonspecific agent (CCNSA), right
Kinds cancer has significant curative effect.But in Long-term clinical in use, finding that Dox has strong toxic side effect, especially heart
Toxicity and bone marrow suppression toxicity, secondly for the tumour cell of multidrug resistance, Dox is not especially sensitive.
Invention content
In view of the drawbacks of the prior art, the present invention provides a kind of Nano medication transmission systems of delivery Dox, using nanometer
Transmission system can deliver Dox and reach specific position, reduce the toxicity of normal tissue, and discharge rapidly and bear in privileged site
The drug Dox of load.The present invention is assembled into the load adriamycin nano micella poly (ae- of nano-scale with graft copolymer and Dox
Asp-g-mPEG-g-Hap) Dox can realize long-acting cycle in blood, be enriched in tumor locus, absorbed through tumour cell
Afterwards, can be under conditions of the acid condition pH 5.0 of lysosome, as amido bond is broken, micella decomposes, and successfully releases Dox.
The present invention proposes a kind of graft copolymer poly (ae-Asp-g-mPEG-g- of β containing acid-sensitive-Carboxylamide key
Hap), the graft copolymer poly (ae-Asp-g-mPEG-g-Hap) is poly- (aminoethyl aspartic acid-grafting-polyethylene glycol
Monomethyl ether-grafting-hexahydro benzamide), wherein the β-Carboxylamide key is pH responsive type amido bonds；
Shown in the structure such as formula (I) of the graft copolymer poly (ae-Asp-g-mPEG-g-Hap)；
In formula (I), x=1-8, y=1-41, m=11-109.
Preferably, x=2-4；Y=9-18；M=40-46.
In the graft copolymer poly (ae-Asp-g-mPEG-g-Hap)：The grafting rate DG of mPEGmPEG=17.4%,
The grafting rate DG of hexahydrophthalic anhydrideHap=76.9%.Wherein, grafting rate be according to the nuclear magnetic resonance spectroscopy of formula (1) (1H-NMR it) integrates
Than the average value of calculating, DGmPEGUnit to be grafted mPEG accounts for the percentage of main chain total monomer, DGHapFor grafting hexahydrophthalic anhydride
Unit accounts for the percentage of the total unit of main chain.
Wherein, the main chain of the graft copolymer poly (ae-Asp-g-mPEG-g-Hap) is poly (ae-Asp), knot
Shown in structure such as formula (3)
In formula (3), n=12-23.
The invention also provides a kind of graft copolymer poly (ae-Asp-g- of β containing acid-sensitive-Carboxylamide key branch
MPEG-g-Hap preparation method), includes the following steps：
(1) in a solvent, under the catalytic action of acid, polycondensation reaction occurs for L-Aspartic acid shown in formula (1), obtains formula
(2) polysuccinimide shown in (PSI)；
(2) in a solvent, ring-opening reaction occurs under the action of ethylenediamine for polysuccinimide (PSI), and complete open loop obtains
To poly- aminoethyl aspartic acid shown in formula (3) (poly (ae-Asp))；
(3) in a solvent, under the action of acid binding agent, with to nitro chloro-carbonic acid benzene fat NPC esterification occurs for mPEG, obtains
To poly glycol monomethyl ether activation fat (mPEG-NP) shown in formula (4)；
(4) in a solvent, poly glycol monomethyl ether activation fat (mPEG-NP) is grafted to-NH of poly (ae-Asp)2On,
It obtains (aminoethyl aspartic acid-grafting-poly glycol monomethyl ether) (poly (ae-Asp-g-mPEG)) poly- shown in formula (5)；
(5) in the first solvent, under the effect of the catalyst, hexahydrophthalic anhydride Hap shown in formula (6) and N- hydroxysuccinimidyl acyls
Imine reaction generates hexahydrophthalic anhydride NHS activity fat；
In the second solvent, in-the NH of poly (ae-Asp-g-mPEG)2On continue be grafted hexahydrophthalic anhydride NHS activity fat,
Poly- (the poly- second of aminoethyl aspartic acid-grafting-two of the graft copolymer that obtains β containing acid-sensitive-Carboxylamide key shown in formula (I)
Alcohol monomethyl ether-grafting-hexahydro benzamide) (poly (ae-Asp-g-mPEG-g-Hap))；Shown in reaction process such as route (1)：
In a specific embodiment, the step of preparing formula (I) specifically includes：Hexahydrophthalic anhydride Hap is weighed to stir to anhydrous DMF
Dissolving is mixed, n-hydroxysuccinimide 1.3g (NHS) is weighed and is added in reaction bulb, the DMAP of the 9mg of catalytic amount, room temperature is added
After reacting 8h, hexahydrophthalic anhydride NHS activity fat is obtained, mixture is concentrated, is added to (ae-Asp-g-mPEG) containing 1.32gpoly and (presses
PEG grafting rates 17.4% are calculated, and there are about-the NH of 2.1mmol2) aqueous solution in, reaction for 24 hours, with the dialysis of molecular cut off 3500
Bag dialysis mixed liquor 3d, is lyophilized to obtain product graft copolymer poly (ae-Asp-g-mPEG-g-Hap), yield 76%.
In route (1), n=4-50, x=1-8, y=1-41, m=11-109.
Preferably, n=12-23, x=2-4；Y=9-18；M=40-46.
In step (1), the acid is selected from phosphoric acid, sulfuric acid or p-methyl benzenesulfonic acid；Preferably, it is phosphoric acid.
In step (1), the solvent of the polycondensation reaction is selected from sulfolane (sulfolane), 1,3,5- trimethylbenzenes, DMF；It is excellent
Selection of land is sulfolane (sulfolane).
In step (1), the L-Aspartic acid, phosphoric acid molar ratio be (5-15)：1；Preferably, it is 10：1.
In step (1), the temperature of the polycondensation reaction is 150-180 DEG C；Preferably, it is 170 DEG C.
In step (1), the time of the polycondensation reaction is 8-15h；Preferably, it is 12h.
In step (1), preferably carry out under nitrogen protection.
In step (2), the solvent of the ring-opening reaction is selected from DMF, DMSO, DCM；Preferably, it is DMF；Further preferably
Ground is anhydrous DMF.
In step (2), the PSI, ethylenediamine molar ratio be 1：(2-4)；Preferably, it is 1：3.6.
In step (2), the temperature of the ring-opening reaction is 0 DEG C -50 DEG C；Preferably, it is 25 DEG C of room temperature.
In step (2), the time of the ring-opening reaction is 4-12h；Preferably, it is 8h.
In step (2), the ethylenediamine act as open loop PSI rings, provides-NH2For graft reaction.
In step (2), preferably carry out under nitrogen protection.
In step (3), the solvent of the esterification is selected from acetonitrile, DCM, DMF；Preferably, it is acetonitrile.
In step (3), the acid binding agent is selected from triethylamine, n,N-diisopropylethylamine, pyridine；Preferably, it is triethylamine；
The acid binding agent act as promoting esterification progress.
It is the mPEG, 1 to the molar ratio of nitro chloro-carbonic acid benzene fat NPC in step (3)：(1-3)；Preferably, it is 1：2.
In step (3), the temperature of the esterification is -5-25 DEG C；Preferably, it is 0 DEG C.
In step (3), the time of the esterification is 12-36h；Preferably, it is for 24 hours.
In step (4), the solvent of the graft reaction is selected from water, methanol, ethyl alcohol；Preferably, it is water, further preferably
Ground is deionized water.
In step (4) ,-NH in the poly (ae-Asp)2, mPEG-NP molar ratio be (4-15)：1；Preferably, it is
8：1.
In step (4), the temperature of the graft reaction is 0-50 DEG C；Preferably, it is 25 DEG C of room temperature.
In step (4), the time of the graft reaction is 12-50h；Preferably, it is 36h.
In step (5), first solvent is selected from DMSO, DCM, DMF；Preferably, it is DMF；It is further preferred that being nothing
Water DMF.
In step (5), the catalyst is selected from DMAP, pyridine；Preferably, it is DMAP.The catalyst act as urging
Change forms hexahydrophthalic anhydride NHS activation fat.
In step (5), the n-hydroxysuccinimide act as activation hexahydrophthalic anhydride, forms hexahydrophthalic anhydride NHS and lives
Change fat.
In step (5), second solvent is selected from water, methanol, ethyl alcohol；Preferably, it is deionized water.
In step (5), the poly (ae-Asp-g-mPEG), raw material hexahydrophthalic anhydride molar ratio be 1：(2-10)；It is preferred that
Ground is 1：5.
In step (5), the temperature of the graft reaction is 0-50 DEG C；Preferably, it is 25 DEG C.
In step (5), the time of the graft reaction is 12-40h；Preferably, it is 36h.
In the specific embodiment of the present invention, the graft copolymer (poly (ae-Asp-g-mPEG-g- are prepared
Hap route)) is as follows：
The invention also provides the graft copolymer poly containing β-Carboxylamide key being prepared according to the above method
(ae-Asp-g-mPEG-g-Hap), shown in structural formula such as formula (I).
The invention also provides by the graft copolymer poly of β containing acid-sensitive-Carboxylamide key shown in the formula (I)
(ae-Asp-g-mPEG-g-Hap) it is used to prepare the application of pharmaceutical carrier (blank micella)；DLS measures blank micella poly (ae-
Asp-g-mPEG-g-Hap) average grain diameter is 98.1nm, narrow distribution, the good (PDI of monodispersity:0.111).
Wherein, the drug is fat-soluble medicine；Preferably, the drug is fat-soluble medicine adriamycin.
Wherein, the carrier is nano-micelle carrier；Preferably, the nano-micelle carrier is acid-sensitive type.
Wherein, the nano-micelle carrier has acid response in tumour cell lysosome.
Wherein, the tumour includes breast cancer, lung cancer, liver cancer etc., and such as tumour cell can be lung carcinoma cell.
Wherein, the structure of the nano-micelle carrier is as shown in Figure 1 C.
The invention also provides a kind of systems carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox
Preparation Method the described method comprises the following steps：
(1) 20mg poly (ae-Asp-g-mPEG-g-Hap) copolymers and 4mg DoxHCl is taken to be dissolved in 0.5mL's
4 μ L triethylamines are added in DMSO, 2h are stirred, until being completely dissolved.(2) mixed solution is added dropwise to the 5mL that mixing speed is 500rpm
It in ultra-pure water, is then charged into the bag filter of molecular cut off 3500, PBS dialysis 48h removes the drug and DMSO being not loaded with.Its
Reaction process is as shown in Figure 1B.
The invention also provides a kind of load adriamycin nano micella poly (ae-Asp-g- being prepared by the above method
mPEG-g-Hap)·Dox；It is 77.5nm, narrow distribution, the good (PdI of monodispersity that DLS, which measures its average grain diameter,：0.102),
Zeta potential is -12.34mV.Meet the requirement of passive target, transmission electron microscope (TEM) confirms spherical micelle pattern.Carry adriamycin
The extracorporeal releasing experiment of nano-micelle poly (ae-Asp-g-mPEG-g-Hap) Dox is the results show that under conditions of 7.4 pH
Relatively stable, cumulative release amount is 25% in 34h, and cumulative release amount is 62% under the conditions of 5.0 pH, it was demonstrated that carrier micelle
With acid-sensitive Release Performance.Opposite normal incubation medium, carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap)
NHs of the Dox in the weak base containing lysosome4A549 cytotoxicities are obviously reduced in the culture medium of Cl, it was demonstrated that after neutralizing lysosomal pH,
The drug release process of carrier micelle is suppressed, it follows that conclusion carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-
Hap) Dox has lysosomal pH dependence in the drug release of cellular level.Flow cytomteric experiments are the results show that with free Dox
(DoxHCl) it compares, the phagocytosis amount for carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox increases.
The invention also provides load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox to make
Application in the drug of standby treating cancer；The cancer includes breast cancer, lung cancer, liver cancer.
The passive target of load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox prepared by the present invention
Behavior and quick drug release process specifically can be described as：Carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap)
Dox is enriched in tumor tissues, after being absorbed by tumour cell, in the acidity of lysosome after long-acting cycle fully realizes EPR effects
Under conditions of condition pH 5.0, as amido bond is broken, micella decomposes, and successfully releases Dox.
The structure and load adriamycin nano micella of graft copolymer according to the present invention containing β-Carboxylamide key branch
As shown in figure iD, implementation steps are as follows for poly (ae-Asp-g-mPEG-g-Hap) Dox schematic diagrames：
The first step：The synthesis of graft copolymer poly (ae-Asp-g-mPEG-g-Hap) and characterization
(1) polycondensation of L-Aspartic acid phosphoric acid catalyzed synthesizes PSI；(2) the complete open loop PSI synthesis poly (ae- of ethylenediamine
Asp)；(3) mPEG with to nitro chloro-carbonic acid benzene fat NPC synthesizing polyethylene glycol monomethyl ether activity fat mPEG-NP；(4) polyethylene glycol
Monomethyl ether activity fat mPEG-NP is grafted to the-NH of poly (ae-Asp)2, synthesis poly (ae-Asp-g-mPEG)；(5) in-NH2
Continue to be grafted hexahydrophthalic anhydride, synthesizes the graft copolymer poly (ae-Asp-g-mPEG-g-Hap) containing β-Carboxylamide key.
Second step：The degradation of graft copolymer poly (ae-Asp-g-mPEG-g-Hap) and its nano-micelle construct with
Characterization
With fluorescamine method to graft copolymer poly (ae-Asp-g-mPEG-g-Hap) different acid ph values (pH 6.0,
PH 5.5, pH 5.0) and normal blood pH value (pH 7.4) buffer solution in amino content be monitored, reflect polymer
Degradation situation in real time carries out dynamic analysis to degradation curve.Poly (ae-Asp-g-mPEG-g-Hap) glue is prepared with dialysis
Beam measures its critical micelle concentration, and particle size and distribution are measured with dynamic light scattering (DLS).
The structural schematic diagram of poly (ae-Asp-g-mPEG-g-Hap) micella is as shown in Figure 1 C.
Third walks：Carry assembling and the Release Performance of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox
Research
It is prepared with dialysis and carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and prepare gelation
Dry powder measures the encapsulation rate and drugloading rate of carrier micelle with ultraviolet absorption method.Carrier micelle shape is observed with transmission electron microscope (TEM)
Looks measure particle size and distribution, at different acid ph values (pH 6.0, pH 5.5, pH 5.0) with dynamic light scattering (DLS)
With carry out carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox in normal blood pH value (pH 7.4)
Release Performance research.
4th step：Carry the Study of cytotoxicity of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox.
Tetrazolium bromide (MTT) colorimetric determination carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox,
DoxHCl carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and in normal incubation medium and contains respectively
20,30,40,50mM NH4Cytotoxicity of the test to A549 in Cl culture mediums.
5th step：Carry the cell phagocytosis of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox.
Phagocytosis behaviors of the MCF-7 to poly (ae-Asp-g-mPEG-g-Hap) Dox micellas is studied, is done with DoxHCl
Control, cultivate respectively 2,4 and for 24 hours after, the culture medium of cell surface drug containing is washed away with PBS, with measured by flow cytometry cell
Swallow the fluorescent value of carrier micelle and DoxHCl.
The beneficial effects of the present invention are graft copolymer poly (ae-Asp-g- of the present invention containing β-Carboxylamide key
MPEG-g-Hap poly- to the open-loop products of polysuccinimide (PSI) with ethylenediamine) using acid-sensitive β-carboxylic acyloxy amine key as branch
Aminoethyl aspartic acid (poly (ae-Asp)) is main chain, is grafted hexahydrophthalic anhydride and mPEG-2000.The invention also discloses preparations
The method of the graft copolymer, including the phosphoric acid catalyzed polycondensation of (1) L-Aspartic acid synthesize PSI；(2) the complete open loop of ethylenediamine
PSI synthesizes poly (ae-Asp)；(3) mPEG activates fat mPEG- with to nitro chloro-carbonic acid benzene fat NPC synthesizing polyethylene glycol monomethyl ethers
NP；(4) poly glycol monomethyl ether activation fat mPEG-NP is grafted to the-NH of poly (ae-Asp)2, synthesis poly (ae-Asp-g-
mPEG)；(5) in-NH2Continue to be grafted hexahydrophthalic anhydride, synthesizes the graft copolymer (poly (ae-Asp- containing hexahydrobenzene amido bond
g-mPEG-g-Hap).Graft copolymer prepared by the present invention is stablized in neutral conditions, is reduced with pH, amido bond crack velocity
Accelerate.The graft copolymer poly (ae-Asp-g-mPEG-g-Hap) of the present invention can be further used for structure load adriamycin and receive
Rice micellar carrier (poly (Asp-g-mPEG-g-Hap) Dox) has preferable drug release property in acid condition, lower
Cytotoxicity and good cell are phagocytic；The present invention utilizes β-response of the Carboxylamide key to tumour cell lysosomal pH 5.0
Property, selection grafting hexahydrophthalic anhydride wraps up hydrophobic drug adriamycin using its hydrophobicity hexatomic ring as kernel.And β-COOH
With-the NH of Dox2Between positive and negative charge attract improve drugloading rate, stablize carrier micelle.Select the mPEG of good biocompatibility as
Hydrophilic segment synthesizes poly- (aminoethyl aspartic acid-grafting-methoxy poly (ethylene glycol)-grafting-hexahydro benzamide) (poly (ae-
Asp-g-mPEG-g-Hap)), and then its load adriamycin micella poly (ae-Asp-g-mPEG-g-Hap) Dox is built.Research
Its in vitro toxicity and active anticancer.
Description of the drawings
Fig. 1：A is to carry the formation of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and in tumour
Micella disintegrates under Cytolysosome condition of acidic pH, discharges the process schematic of adriamycin；B is to carry adriamycin nano micella poly
(ae-Asp-g-mPEG-g-Hap) the preparation process schematic diagram of Dox；C is blank micella poly (ae-Asp-g-mPEG-g-
Hap structural schematic diagram)；D is the structural representation for carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox
Figure.
Fig. 2 is the graft copolymer poly (ae-Asp-g-mPEG-g-Hap) that is measured with fluorescamine method molten in acetate buffer
Liquid (pH5.5, pH 5.0,10mM) or phosphate buffer (pH 7.4, pH 6.0,10mM) acid degradation curve graph (mean ±
SD, n=3).
Fig. 3 is that graft copolymer poly (ae-Asp-g-mPEG-g-Hap) degrades song in the acetate buffer solution of pH 5.0
Line dynamic analysis, the linear regression graph of-ln (1-DP) and time t.
Fig. 4 is to be changed with concentration with the scattered light intensity of DLS measurement blank micella poly (ae-Asp-g-mPEG-g-Hap)
Curve, catastrophe point are CMC, are calculated as 0.166.
Fig. 5 is blank micella poly (ae-Asp-g-mPEG-g-Hap) and carries adriamycin nano micella poly (ae-Asp-
G-mPEG-g-Hap) the particle diameter distribution (DLS figures) of Dox.
Fig. 6 is to carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox transmission electron microscope shape appearance figures.
Fig. 7 adriamycins standard curve of ultraviolet absorption value to concentration at 480nm.
Fig. 8 carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox in different pH buffer solutions
Drug release behavior.In 7.4 media of pH, drug releasing rate is slower, and cumulative release amount is about 25% in 34h.With medium
PH reduces drug release amount and obviously increases, and in 5.0 media of pH, burst size is up to 62% in 34h, about 7.4 conditions of pH in the same time
Under 2.5 times.
Fig. 9 is blank micella poly (ae-Asp-g-mPEG-g-Hap), DoxHCl and load adriamycin nano micella poly
(ae-Asp-g-mPEG-g-Hap) Dox is under the conditions of normal incubation medium, and carries adriamycin nano micella poly (ae-Asp-
G-mPEG-g-Hap) Dox is in NH containing 40mM4Cytotoxicity under Cl culture medium conditions.
Figure 10 is DoxHCl and carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox in 2,4 Hes
When for 24 hours in MCF-7 fluorescence content average value statistical chart.
Specific implementation mode
In conjunction with following specific examples and attached drawing, the present invention is described in further detail.The process of the implementation present invention,
Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to the following content specially referred to
It is bright that content is not particularly limited.
The synthesis of 1 polysuccinimide PSI of embodiment
It weighs L-Aspartic acid 13.3g (0.1mmol) to evenly spread in the sulfolane of 35mL, 0.5mL phosphoric acid is added and makees
For catalyst, under nitrogen protection, filtrate is added drop-wise to the methanol of 300mL by 170 DEG C of reaction 12h dropwise after reaction solution filtering
In, filtration washing obtains faint yellow solid, 50 DEG C of vacuum drying 48h, yield 87% afterwards for several times.
Shown in the structure of the PSI such as formula (2 ')；
In formula (2 '), n=12-23.
The synthesis of 2 poly- aminoethyl aspartic acid poly (ae-Asp) of embodiment
PSI 5g (0.05mol monomers) prepared by Example 1 are added in the DMF containing 50mL Non-aqueous processings, nitrogen
It under protection, is slowly dropped in 12mL ethylenediamines (0.18mol), stirs 8h at room temperature, after reaction solution filtering, used after filtrate concentration
Methanol dissolves, and is added drop-wise in cold ether, reprecipitation 3 times obtains faint yellow solid, yield 79%.
Shown in the structural formula such as formula (3 ') of the poly (ae-Asp)；
In formula (3 '), n=12-23.
3 poly glycol monomethyl ether of embodiment activates the synthesis of fat (mPEG-NP)
Dry mPEG-20005g (2.5mmol) and triethylamine 0.7mL (5mmol) is weighed to be added containing 50mL acetonitriles
In three-neck flask, then weighs and 15mL acetonitriles is dissolved in nitro chloro-carbonic acid benzene fat NPC 1g (5mmol), be added dropwise in flask,
Mixture reacts for 24 hours at 0 DEG C, is then cooled to -20 DEG C, and 12h is settled out the salt of triethylamine, and filtrate is precipitated to obtain end with ether
Product mPEG-NP.
Shown in the structural formula of the mPEG-NP such as formula (4 ')；
In formula (4 '), m=40-46.
The conjunction of poly- (aminoethyl aspartic acid-grafting-poly glycol monomethyl ether) poly (ae-Asp-g-mPEG) of embodiment 4
At
Poly (ae-Asp) 0.63g (4mmol-NH prepared by Example 22) be dissolved in 15mL deionized waters, it is added
Into the round-bottomed flask of 50mL, the mPEG-NP 1.1g (0.5mmol NP) for weighing the preparation of embodiment 3 are added in flask, room temperature
36h is reacted, is transferred to after reaction in the bag filter of molecular cut off 3500, deionized water is changed once per 6h, after dialysing 3 days
Freeze-drying obtains poly (ae-Asp-g-mPEG) solid, yield 83%.The grafting rate of mPEG is 17.4%.
Shown in the structural formula such as formula (5 ') of the poly (ae-Asp-g-mPEG)；
In formula (5 '), x=2-4, y=10-19, m=40-46.
Poly- (aminoethyl aspartic acid-grafting-poly glycol monomethyl ether-grafting-hexahydro benzamide) poly (ae- of embodiment 5
Asp-g-mPEG-g-Hap synthesis)
In a solvent, in-the NH of poly (ae-Asp-g-mPEG)2On continue hexahydrophthalic anhydride shown in grafting formula (6)
(Hap), poly- (the aminoethyl aspartic acid-grafting-of graft copolymer of β containing acid-sensitive-Carboxylamide key shown in synthesis formula (I)
Poly glycol monomethyl ether-grafting-hexahydro benzamide) (poly (ae-Asp-g-mPEG-g-Hap))；Reaction process such as route (1)
It is shown.It weighs in hexahydrophthalic anhydride Hap 1.54g (10mmol) to 50ml round-bottomed flasks, 20ml anhydrous DMF stirring and dissolvings is added, claim
It takes n-hydroxysuccinimide 1.3g (NHS, 11.3mmol) to be added in reaction bulb, the DMAP of the 9mg of catalytic amount, room temperature is added
Reaction 8h after concentrate, be added to poly containing 1.32g (ae-Asp-g-mPEG) (by PEG grafting rates 17.4% calculation, there are about
- the NH of 2.1mmol2) aqueous solution in, reaction for 24 hours, is dialysed mixed liquor 3d with the bag filter of molecular cut off 3500, is lyophilized
Product graft copolymer poly (ae-Asp-g-mPEG-g-Hap), yield 76%.
Shown in the structural formula such as formula (I ') of the graft copolymer poly (ae-Asp-g-mPEG-g-Hap)；
In formula (I '), x=2-4, y=9-18, m=40-46.
The acid degradation performance of 6 graft copolymer poly (ae-Asp-g-mPEG-g-Hap) of embodiment.
- the NH of polymer is measured with fluorescamine method2Changes of contents amount, with the fracture of amido bond, amino group concentration increases, glimmering
Luminous intensity enhances, and then calculates the degradation rate of amido bond.The solution of 1mg/mL is made of deionized water dissolving for sample to be tested, takes
The hac buffer (pH 5.5, pH 5.0,10mM is free of NaCl) or phosphoric acid buffer of the sample solution of 100 μ L and 100 μ L
Liquid (pH 7.4, pH 6.0,10mM is free of NaCl) is hatched at 37 DEG C, and the pH of the 100 μ L of the solution of 10 μ L is then taken every 1h
9.1 borate buffer dilution, is added the DMF solution (2mg/mL) of 10 μ L fluorescamines, hatches at room temperature in the mixture
10min.In excitation wavelength it is 390nm with microplate reader, launch wavelength is that 465nm surveys its fluorescence intensity.By sample and 0.01M
The absorption value of HCL incubated overnights is as 100% degradation.
Experimental result as shown in Fig. 2, with pH 7.4, simulate normally respectively by the buffer solution of pH 6.0, pH 5.5, pH 5.0
Blood, outside tumour cell, endosome, the pH of lysosome, under the conditions of as a result showing pH 7.4, the degradation amount of 36h amido bonds is only
17.2%, as pH value reduces, acid stronger, the faster amido bond fracture the more thorough, under the conditions of 5.0 pH, degradation in the same time
Amount reaches 93.0%, the results showed that graft copolymer poly (ae-Asp-g-mPEG-g-Hap) has significant acid-sensitive characteristic.
Change the influence being broken to amido bond for clearer relatively pH, the present invention analyzes degradation curve, finds
It meets level-one degradation kinetics equation and is dropped according to level-one as shown in figure 3, by taking the degradation in the buffer solution in pH=5.0 as an example
Solution kinetics equation derives that-ln (1-DP)=kt, DP represent the degradation percentage of t moment, substitutes into data and makees graph discovery, closely
K=0.0706, correlation coefficient r are obtained by linear regression and correlation analysis like the straight line for crossing origin for one2=
0.9945.With same method respectively to graft copolymer poly (ae-Asp-g-mPEG-g-Hap) in pH=7.4, pH=6.0
And the degradation situation under the conditions of pH=5.5 is analyzed, and level-one degradation kinetics equation is met.Calculate its degradation half life
t1/2Respectively 142.86h (pH=7.4), 32.90h (pH=6.0), 11.85h (pH=5.5), 10.07h (pH=5.0).Into
One step illustrates that the graft copolymer of the present invention is stablized relatively in pH=7.4, is reduced with pH, amido bond fracture is rapider, has
Acid-sensitive characteristic.
The measurement of 7 critical micelle concentration of embodiment (CMC)
5 freshly prepared blank micella poly (ae-Asp-g-mPEG-g-Hap) solution of embodiment is diluted to 2 step by step,
1,0.5,0.2,0.1,0.05,0.02,0.01,0.005,0.002,0.001mg/mL ten concentration, from small to large according to concentration,
It is measured 3 times with DLS successively.Scattered light intensity is made with concentration curve figure, it is dense to obtain its critical micell with cross extrapolation
Degree.
Experimental result is as shown in figure 4, scattered light intensity is mainly related with concentration with the grain size of micella particle in solution.?
More than CMC concentration, the micella particle homogeneity in solution is good, and particle diameter is almost a definite value (100nm or so), at this time with
Micellar concentration increase, particle concentration increases, scattered light intensity enhancing.In CMC concentration hereinafter, measuring that grain size is big and quality is not inconsistent
Close test request, it was demonstrated that there is no micella particles to exist in solution, and scattered light intensity angle value at this time is minimum (50kcps or so).It is logical
It is 0.166mg/mL to cross cross extrapolation and the critical micelle concentration of poly of the present invention (ae-Asp-g-mPEG-g-Hap) can be obtained
The assembling of 8 blank micella poly (ae-Asp-g-mPEG-g-Hap) of embodiment and grain size test
Graft copolymer poly (ae-Asp-g-mPEG-g-Hap) prepared by 20mg embodiments 5 is taken to be dissolved in the DMSO of 0.5mL
In, it is sucked in 1mL injections after being completely dissolved, mixing speed is slowly injected into as the 5mL of 500rpm with the speed of 20 μ L/min
It in ultra-pure water, is fitted into the bag filter of molecular cut off 3500 after stablizing 2h, 4 DEG C of dialysis 48h remove DMSO up to blank micella
poly(ae-Asp-g-mPEG-g-Hap).It is placed in 4 DEG C of refrigerators to preserve, blank micella poly (ae- need to be made for a long time by preserving
Asp-g-mPEG-g-Hap) freeze-dried powder.
Using DLS to the average grain diameter of freshly prepared blank micella poly (ae-Asp-g-mPEG-g-Hap), grain size point
Cloth, Zeta potential and polydispersity index (PDI) are tested.
Experimental result is as shown in figure 5, DLS measures blank micella poly (ae-Asp-g-mPEG-g-Hap) average grain diameter is
98.1nm, narrow distribution, the good (PDI of monodispersity:0.111).Prove that the blank micella meets the requirement of passive target.Study table
Bright, surface easily occurs interaction with albumen in blood plasma for the particle of positive charge and is polymerized to, Zeta potential test result -9.28mV
The surface of (non-attached drawing), electronegativity can help long circulating in micellar carrier.
Embodiment 9 carries the preparation of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox, grain size test
And pattern research
Take graft copolymer poly (ae-Asp-g-mPEG-g-Hap) and 4mg DoxHCl prepared by 20mg embodiments 5
It is dissolved in the DMSO of 0.5mL, 4 μ L triethylamines are added, stirs 2h, mixed solution, which is added dropwise to the 5mL that mixing speed is 500rpm, to be surpassed
It in pure water, is then charged into the bag filter of molecular cut off 3500, PBS dialysis 48h removes the drug and DMSO being not loaded with, and pays attention to
It is protected from light, is freeze-dried, be made and carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox freeze-dried powders.
Freshly prepared load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox is put down using DLS
Equal grain size, particle diameter distribution, Zeta potential and polydispersity index (PDI) are tested.It is seen using transmission electron microscope (TEM)
Examine the pattern for carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox.
Experimental result carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) as shown in figure 5, DLS is measured
The average grain diameter of Dox is 77.5nm, narrow distribution, the good (PDI of monodispersity:0.102).Zeta potential test result be-
12.34mV.Carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and blank micella poly (ae-Asp-
G-mPEG-g-Hap it) compares, grain size has the shrinkage of 20nm, it may be possible to-the NH of Dox2With graft copolymer poly (ae-Asp-g-
MPEG-g-Hap side chain-COOH) generates positive and negative charge and attracts, and after being loaded into Dox, increases the hydrophobicity of micelle inner core, makes glue
Beam kernel becomes even closer, while the effect that this positive and negative charge attracts also increases drugloading rate.
As shown in fig. 6, being the transmission electron microscope for carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox
(TEM) shape appearance figure.The assembly is spherical micelle as can be seen from Figure, is calculated by scale and shows grain size about in 55nm on TEM.
It is slightly less than the particle size data of DLS, reason may be the micella grain size that DLS is measured in aqueous solution, and TEM is the glue after nature volatilizes
Beam grain size, so there is the difference of about 20nm.
Embodiment 10 carries drugloading rate and the encapsulating of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox
The measurement of rate
Load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) the Dox freeze-dried powders DMSO for taking 5mg to prepare
Dissolving surveys the solution in the absorption value of 480nm with ultraviolet specrophotometer, measures the suction of the DMSO solution of isoconcentration gradient Dox
Receipts value draws the standard curve of absorbance value and concentration, substitutes into and obtains the concentration of the Dox in DMSO solution, and then is calculated
The drugloading rate and encapsulation rate of carrier micelle.
Drugloading rate DLC (wt%)=(quality of drug quality/micella of loading) × 100%
Encapsulation rate DLE (%)=(drug quality of drug quality/input of loading) × 100%.
Experimental result substitutes into Ah mould as shown in fig. 7, by its absorbance value at 480nm of UV spectrophotometer measuring
The standard curve y=0.0199x+0.0164, R that plain light absorption value does concentration2In=0.99933, load adriamycin is calculated and receives
Drugloading rate DLC=8.83%, the encapsulation rate DLE=39.69% of rice glue beam poly (ae-Asp-g-mPEG-g-Hap) Dox, such as
Shown in table 1.Should statistics indicate that, due to-the NH of Dox2With the side chain-of graft copolymer poly (ae-Asp-g-mPEG-g-Hap)
COOH generates positive and negative charge and attracts, and can improve drugloading rate.And carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-
Hap) dissolubility is good in aqueous solution by Dox, improves the water solubility of Dox.
Table 1
| Theoretical drugloading rate (wt%) | Drugloading rate (wt%) | Encapsulation rate (%) |
| 20% | 8.83% | 39.69% |
Embodiment 11 carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox in without medium
Drug release behavior is studied
Freshly prepared load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox of 1mL are packed into retention
It in the bag filter of molecular weight 1000, and is put into the brown vial of 25mL, at 37 DEG C, is separately added into the hac buffer of 10mL
(pH 5.5, pH 5.0,10mM) or phosphate buffer (pH 7.4, pH 6.0,10mM), in preset time interval, taking-up is released
Corresponding fresh buffer is changed in tapping, and the absorbance of each group release liquid is measured with ultraviolet specrophotometer, bent with Dox standards
Line compares, and calculates each group cumulative release amount.
Experimental results are shown in figure 8, and under the conditions of pH 7.4, Dox cumulative releases amount is 25% in 34h, and with pH value
It reduces, carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and the burst size of Dox is gradually increased, and release
Medicine is getting faster；Under the conditions of pH 5.0, Dox cumulative releases amount is 62% in 34h, using β-amido bond as the acid-sensitive of connecting key
Drug release behaviors of adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox under the conditions of each pH is carried fully to show
Its application prospect in drug delivery system.
Embodiment 12 carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox to people's non-small cell lung
Cancer cell A549 cell toxicity tests
The present invention further studies with tetrazolium bromide (MTT) colorimetric method and carries adriamycin nano micella poly (ae-Asp-g-
MPEG-g-Hap) cytotoxicities of the Dox to Non-small cell lung carcinoma cell A549.To blank micella poly (ae-Asp-g-
MPEG-g-Hap adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and DoxHCl), are carried respectively just
Normal culture medium (containing 10%FBS, 1% is dual anti-, F12 culture mediums) and contain 20,30,40 and 50mM NH4It is carried out under Cl culture medium conditions
Non-small cell lung cancer cell A549 toxotests.Experimental method is：
Step 1. will cultivate 5 A549 cells more than generation according to the cell concentration in 5000/hole uniformly by 180 holes μ L/
It is inoculated into 96 orifice plates, is placed in 37 DEG C, contain 5%CO2Wet environment under cultivate for 24 hours after.
Step 2. sucks old culture medium, is separately added into fresh normal incubation medium and contains 20,30,40 and 50mM NH4Cl's
A series of blank micella poly (ae-Asp-g-mPEG- that 20 μ L contain various concentrations are added in normal incubation medium group in culture medium
(a concentration of 0.008,0.016,0.033,0.065,0.130,0.260,0.530,1.060mg/mL) of blank micella g-Hap；?
Normal incubation medium and contain NH420 μ L are not added and carry adriamycin nano micella poly (ae-Asp-g-mPEG- for the nutrient media components of Cl
G-Hap) Dox and 20 μ L DoxHCl, the concentration being added before culture medium with the content meter of Dox is respectively 0.8,1.6,
3.2,6.4,12.8,25.6,51.2 and 102.4 μ g/mL (after culture medium is added, concentration is respectively 0.08,0.16,0.32,
0.64,1.28,2.56,5.12 and 10.24 μ g/mL), each concentration sets 6 holes.Continue after cultivating 48h.
The PBS solution (5mg/mL) of 10 μ L/ hole MTT is added in step 3., and after acting on 4h, three liquid in 50 holes μ l/ are added
(+5% isobutanol+0.01mol/LHCl of 10% lauryl sodium sulfate), 37 DEG C overnight, abundant lytic cell.
Step 4. measures the absorbance OD values at wavelength 570nm with microplate reader, and calculates each group A549 according to following formula
The survival rate of cell：
Cell viability (100%)=[(ODsample-ODblank)/(ODcontrol-ODblank)] × 100%
ODsample：The OD values of experimental group
ODblank：The not celliferous pure culture bases of 180 μ L are added in the OD values of blank group, i.e. step 1, other operations are as above.
ODcontrol：The pure culture base of the not drug containing of 20 μ L is added in the OD values of control group, i.e. step 2, other operations are as above.
Experimental result as shown in figure 9, the cells survival rate of blank micella in 95% or more (test concentrations of blank micella
Survival rate when only providing maximum concentration 1.06mg/ml for 0.008-1.06mg/ml, Fig. 9 is 95.66 ± 14.88%, it was demonstrated that its
Hypotoxicity feature.For carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and DoxHCl, with Dox
Concentration increases, and the survival rate of A549 cells reduces, and shows the fragmentation effect of concentration dependent.
The present invention is containing 20,30,40 and 50mM NH4A549 cytotoxicity tests are carried out under Cl culture medium conditions.NH4Cl is
A kind of common lysosome weak base has document report to use and contains NH4The medium culture cell of Cl can significantly improve Cytolysosome
PH.Experimental result is as shown in Figure 9, wherein DoxHCl is in normal incubation medium group and contains NH4The culture medium group of Cl (is in figure
Do not show) cytotoxicity there is no notable difference, experimental data almost the same.Relative to normal incubation medium group, carries adriamycin and receive
Rice glue beam poly (ae-Asp-g-mPEG-g-Hap) Dox is containing NH4Cells survival rate in Cl culture mediums significantly improves,
Especially NH containing 40mM4The difference of Cl most obviously (only provides NH containing 40mM in Fig. 94The experimental result of Cl culture medium groups, 20,
30,40mM NH4Cl groups are in table 2 with IC50Value provides).As a concentration of 1.28mg/L of Dox, in 95% confidence interval,
It carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox and carries adriamycin nano micella poly (ae-Asp-
g-mPEG-g-Hap)·Dox+40mM NH4Cl groups compare, P<0.0001, there is significant difference.
As shown in table 2, be deeper into research carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap)
Dox and DoxHCl has calculated separately their median lethal dose to the cytotoxicity difference of Non-small cell lung carcinoma A549
(IC50) value.Carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox (IC50=1.426) and DoxHCl
(IC50=0.9575) it compares, slightly improves, probably due to DoxHCl enters nucleus by free diffusing, direct killing is thin
Born of the same parents.Carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox need to release by the cracking of lysosomal acid condition
It puts, nucleus could be entered, therefore, toxicity slightly reduces.Carry adriamycin nano micella poly (ae-Asp-g-mPEG-g-
Hap) NH is added in Dox4The experimental group IC of Cl50, for normal culture group, it is significantly increased, especially carries adriamycin
Nano-micelle poly (ae-Asp-g-mPEG-g-Hap) Dox+40mM NH4Cl groups (IC50=4.389) it is normal culture group
(IC50=1.426) 3 times, cytotoxicity of the carrier micelle to A549 is significantly reduced.Because of lysosome weak base NH4Cl is neutralized
The pH of lysosome, causes amido bond that cannot degrade, micella cannot rupture, and Dox cannot discharge, and then cells survival rate significantly carries
Height, IC50Value significantly increases.Experimental result, which further demonstrates, carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-
Hap) the characteristic of the drug release dependence of lysosomal pH 5.0 of Dox micellas.Amido bond has good pH responses, carries adriamycin
Nano-micelle poly (ae-Asp-g-mPEG-g-Hap) Dox has good application prospect.
Table 2
Embodiment 13 carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox cell phagocytosis tests
Using flow cytometer to the load adriamycin nano micella poly (ae-Asp-g-mPEG-g- of MCF-7 cells
Hap) the cell phagocytosis amount of Dox and DoxHCl is studied, cell culture processes：By MCF-7 cell inoculations in six orifice plates
Upper preculture for 24 hours (20 × 104A/hole, the holes 2mL/), it is separately added into the load adriamycin nano that 1mL contains 1 μ g/mL Dox per hole
Micella poly (ae-Asp-g-mPEG-g-Hap) Dox and DoxHCl, set drug treating time as 2, and 4 and for 24 hours, control
1mL normal incubation medium cultures are added in group.After effect, each group cell is digested with pancreatin, is transferred in centrifuge tube, with pH 7.4
PBS buffer solution clean 3 times, be finally configured to 300-800 cell/ μ L with PBS, be transferred in 96 orifice plates, adriamycin is one
Kind autofluorescence substance, transmitting photoluminescence peak can be used for detecting quantitative in 575nm or so.With flow cytometer to the glimmering of each group
Light quantity is measured and analyzes the fluorescence intensity of each group.
Experimental result is as shown in Figure 10, the control group of not dosing, almost without fluorescence, corresponding average fluorescent strength value
(MFI) there was only 2.44, belong to the fluorescence intensity of cell itself.When cultivating 2h and 4h, adriamycin nano micella poly (ae- are carried
Asp-g-mPEG-g-Hap) be all higher than DoxHCl, the especially 4h of the MFI of Dox is to become apparent from, according to embodiment 11 and in fact
The supposition of example 12 is applied, largely carries adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox at this time still in lysosome
In do not have degradable, most cells are still survived, therefore show strong Dox fluorescence.And small-molecule drug is thin by diffusing into
Intracellular is directly entered nucleus killing tumor cell, and when cells survival state is deteriorated, the adherent reduced capability of cell holds very much
The fluorescence intensity for easily in sample handling processes, being washed off, therefore detecting with PBS is less than the carrier micelle group under in the same time.
In addition, when cultivating for 24 hours, detect the MFI values of DoxHCl slightly larger than load adriamycin nano micella poly (ae-
Asp-g-mPEG-g-Hap the reason of) Dox, intracellular Dox doses are reduced relatively is as time went on, to be largely accumulated in molten
Load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox in enzyme body, amido bond fracture, a large amount of Dox quilts
It releases, and as lysosome ruptures, the fluorescence intensity for leading to cell mortality, therefore detecting is less than in the same time
DoxHCl groups.
The protection content of the present invention is not limited to above example.Without departing from the spirit and scope of the invention, originally
Field technology personnel it is conceivable that variation and advantage be all included in the present invention, and with appended claims be protect
Protect range.
Claims (13)
1. a kind of graft copolymer poly (ae-Asp-g-mPEG-g-Hap) of β containing acid-sensitive-Carboxylamide key, feature exists
In shown in structure such as formula (I)；
In formula (I)：X=1-8；Y=1-41；M=11-109.
2. the graft copolymer poly (ae-Asp-g-mPEG-g- of β containing acid-sensitive-Carboxylamide key as described in claim 1
Hap), which is characterized in that the grafting rate DG of mPEGmPEGThe grafting rate DG of=17.4%, HapHap=76.9%.
3. the preparation of the graft copolymer poly (ae-Asp-g-mPEG-g-Hap) of β containing acid-sensitive-Carboxylamide key branch a kind of
Method, which is characterized in that include the following steps：
(1) in a solvent, under the catalytic action of acid, polycondensation reaction occurs for L-Aspartic acid shown in formula (1), obtains formula (2)
Shown in polysuccinimide；
(2) in a solvent, under the action of ethylenediamine ring-opening reaction occurs for polysuccinimide, obtains poly- ammonia shown in formula (3)
Ethyl aspartic acid (poly (ae-Asp))；
(3) in a solvent, under the action of acid binding agent, with to nitro chloro-carbonic acid benzene fat NPC esterification occurs for mPEG, obtains formula
(4) poly glycol monomethyl ether activation fat mPEG-NP shown in；
(4) in a solvent, poly glycol monomethyl ether activation fat mPEG-NP is grafted to the-NH of poly (ae-Asp)2On, obtain formula
(5) (aminoethyl aspartic acid-grafting-poly glycol monomethyl ether) poly (ae-Asp-g-mPEG) poly- shown in；
(5) in the first solvent, under the effect of the catalyst, hexahydrophthalic anhydride Hap and n-hydroxysuccinimide shown in formula (6)
Reaction generates hexahydrophthalic anhydride NHS activity fat；
In the second solvent, in-the NH of poly (ae-Asp-g-mPEG)2Hexahydrobenzene in upper grafting hexahydrophthalic anhydride NHS activity fat
Acid anhydride (Hap) obtains the graft copolymer poly (ae-Asp-g-mPEG-g- of β containing acid-sensitive-Carboxylamide key shown in formula (I)
Hap)；Shown in reaction process such as route (1)：
In route (1)：N=4-50, x=1-8, y=1-41, m=11-109.
4. method as claimed in claim 3, which is characterized in that in step (1), the acid is selected from phosphoric acid, sulfuric acid or to toluene
Sulfonic acid；The solvent of the polycondensation reaction is selected from sulfolane, 1,3,5- trimethylbenzenes, DMF；And/or the L-Aspartic acid, phosphoric acid
Molar ratio be (5-15)：1；And/or the temperature of the polycondensation reaction is 150-180 DEG C.
5. method as claimed in claim 3, which is characterized in that in step (2), the solvent of the ring-opening reaction be selected from DMF,
DMSO,DCM；And/or the molar ratio of the polysuccinimide, ethylenediamine is 1：(2-4)；And/or the ring-opening reaction
Temperature is 0-50 DEG C.
6. method as claimed in claim 3, which is characterized in that in step (3), the solvent of the esterification be selected from acetonitrile,
DCM,DMF；And/or the acid binding agent is selected from triethylamine, n,N-diisopropylethylamine, pyridine；And/or the mPEG, to nitre
The molar ratio of base chloro-carbonic acid benzene fat NPC is 1：(1-3)；And/or the temperature of the esterification is -5-25 DEG C.
7. method as claimed in claim 3, which is characterized in that in step (4), the solvent of the graft reaction is selected from water, first
Alcohol, ethyl alcohol；And/or-NH in the poly (ae-Asp)2, mPEG-NP molar ratio be (4-15)：1；And/or the grafting
The temperature of reaction is 0-50 DEG C；The time of the graft reaction is 12-50h.
8. method as claimed in claim 3, which is characterized in that in step (5), first solvent be selected from DMSO, DCM,
DMF；And/or the catalyst is selected from DMAP, pyridine；And/or second solvent is selected from water, methanol, ethyl alcohol；And/or institute
State poly (ae-Asp-g-mPEG), the molar ratio of hexahydrophthalic anhydride is 1：(2-10)；And/or the temperature of the graft reaction is 0-
50℃。
9. a kind of graft copolymer of β containing acid-sensitive that method as claimed in claim 3 is prepared-Carboxylamide key branch
poly(ae-Asp-g-mPEG-g-Hap)。
10. by the graft copolymer poly (ae-Asp-g- of the β containing acid-sensitive described in claim 1 or 9-Carboxylamide key
MPEG-g-Hap) it is used to prepare the application of pharmaceutical carrier, which is characterized in that the drug is fat-soluble medicine；The nanometre glue
Shu Zaiti is acid-sensitive type nano-micelle carrier.
11. application as claimed in claim 10, which is characterized in that the average grain diameter of the pharmaceutical carrier is 98.1nm, distribution
It is relatively narrow, the good PDI of monodispersity:0.111.
12. a kind of preparation method carrying adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox, feature exist
In the described method comprises the following steps：(1) 20mg poly (ae-Asp-g-mPEG-g-Hap) copolymers and 4mg Dox are taken
HCl is dissolved in the DMSO of 0.5mL, and 4 μ L triethylamines are added, and 2h is stirred, until being completely dissolved；(2) mixed solution is added dropwise to stirring speed
Degree is to be then charged into the bag filter of molecular cut off 3500 in the 5mL ultra-pure waters of 500rpm, and PBS dialysis 48h removings are not loaded with
Drug and DMSO, obtain load adriamycin nano micella poly (ae-Asp-g-mPEG-g-Hap) Dox.
13. a kind of load adriamycin nano micella poly (ae-Asp-g-mPEG- being prepared by claim 12 the method
G-Hap) Dox, which is characterized in that the average grain diameter for carrying adriamycin nano micella is 77.5nm, narrow distribution, monodisperse
Good, the PdI of property：0.102, Zeta potential is -12.34mV.
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| CN111499875A (en) * | 2020-04-13 | 2020-08-07 | 华东师范大学 | Graft copolymer containing cell-penetrating peptide, dopa and reductive detachable PEG (polyethylene glycol) as well as synthetic method and application thereof |
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| CN1629207A (en) * | 2003-11-03 | 2005-06-22 | 福利亚有限公司 | Methods of synthesis of polysuccinimide, copolymers of polysuccinimide and derivatives thereof |
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