CN108017783B - Polymer and the preparation method and application thereof with high potency drugs load performance - Google Patents
Polymer and the preparation method and application thereof with high potency drugs load performance Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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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
- A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/641—Branched, dendritic or hypercomb peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- 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
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
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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
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/40—Polyamides containing oxygen in the form of ether groups
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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
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/48—Polymers modified by chemical after-treatment
Abstract
The present invention provides a kind of polymer and the preparation method and application thereof with high potency drugs load performance, the macromolecule is with poly(ethylene glycol)-bPoly- [(N- 2- ethoxy)-asparagine] based on, and it modifies to obtain by phenyl boric acid, macromolecule material of main part is the polyethylene glycol and polyaminoacid with good biocompatibility, the phenyl boric acid group of polyaminoacid side chain can reach efficient drug loading by Coordination interaction with the drug containing amino, to clinically common chemotherapeutics such as adriamycin, Epi-ADM, Irinotecan etc. have superelevation drugloading rate (be greater than 50%) and close to 100% encapsulation rate, and carrier micelle release has the responsiveness of hydrogen peroxide；The freeze-dried powder of carrier micelle has a good solubility in aqueous solution, and the particle diameter of formation is small and narrowly distributing；It prepares simultaneously simply, convenient for promoting, there is huge development prospect in bio-medical material especially drug delivery field.
Description
Technical field
The present invention relates to small-molecule drug loads and delivering field, and are related to amino acid technical field, more particularly to have
The polymer and the preparation method and application thereof of high potency drugs load performance.
Background technique
High molecular nanometer drug refers to using nanotechnology, and small-molecule drug is wrapped in the macromolecule carrier of self assembly
Portion, preparing scale is nanoscale delivery system.Macromolecule carrier is usually amphiphatic polymer, can be formed
The water solubility and internal stability of carrier micelle can be improved for wrapping up dewatering medicament, hydrophilic shell in hydrophobic inner core.High score
Sub- Nano medication can extend drug blood circulation time, and by tumor locus " infiltration of enhancing and retention effect ", (EPR is imitated
Answer) realize passive target, while can use the targeting conveying and control release of the design realization drug of carrier, finally improve medicine
The curative effect of object simultaneously reduces toxicity.Has the Nano medication listing based on the high molecular materials such as polyester or polyaminoacid at present
(Genexol-PM) it or is in clinical investigation phase (NK105, NC6004, CT-2103).And the country is about high molecular nanometer
The exploitation of drug also has a large amount of relevant report, for example, a kind of polyaminoacid material, can wrap up adriamycin and improve its stabilization
Property；A kind of macromolecule can be with compound small molecule cis-platinum, and obtained compound has good dissolubility and biocompatibility.
Although high molecular nanometer drug is quickly grown in recent years, successfully clinical conversion is still limited by a nanometer medicine for it
The various properties of object itself, such as drugloading rate, encapsulation rate, dissolubility and release performance.Currently, many high molecular nanometer drugs are all
It is to be prepared using self-assembling methods such as nanoprecipitations.However, high molecular material be generally difficult to obtain by these methods it is very high
Drugloading rate and encapsulation rate.Wherein, the aggregation that interaction too strong between drug is induced is a master for causing the above results
Want reason.The interaction reduced between drug molecule is designed by reasonable structure or is improved between drug and macromolecule carrier
Interaction potentially contribute to realize efficient drug loading.There is document report to reduce drug point by small molecule dimer
Rapid aggregation behavior between son, and the drug for realizing superelevation supports, however this method synthesis step is complicated, answers in clinic
There is very big difficulty in used time.
Summary of the invention
The present invention provides a kind of high molecular materials, and especially the high molecular material side chain contains phenyl boric acid group, can be with
Realize that there is hydrogen peroxide responsiveness to the structure efficiently supporting, and designing containing amino drug by coordination, it can be with
Realize the release of carrying medicament specificity.
The present invention adopts the following technical scheme:
A kind of polymer has structure shown in formula (I)；
In formula (I), R1Selected from hydrogen, alkyl or replace alkyl；
R2Selected from-NH- or-R5(CH2)rNH-, wherein R5For-O- ,-OCONH- ,-OCO- ,-NHCOO- or-NHCO-, 1≤
r≤10；
R3Selected from hydrogen or following group:
R3 `Selected from hydrogen or following group:
R4Selected from hydrogen or hydrophobic grouping；
20≤n≤500；5≤m≤200.
In above-mentioned technical proposal, the alkyl is the alkyl of C1 ~ C40；In the substitution alkyl, substituent group is sulfydryl, sugar
Residue, aldehyde radical, carboxyl, vinyl, alkynyl, succimide, maleimide, biotin, RGD class small peptide, LHRH class small peptide
Or folic acid；The hydrophobic grouping is alkyl, benzyl, cholesterol formoxyl, acetyl group, cholic acid group or the deoxycholic acid of C4 ~ C20
Base.
In high molecular material provided by the invention based on polyaminoacid, it is preferred that the poly- amino containing phenyl boric acid structure
Acid fragment ratio in total polyaminoacid is not less than 20%；It is furthermore preferred that the group ratio of phenyl boric acid structure 40%~60% it
Between；The ratio is too low or too high may will affect drug carrying ability or material solubility property.
Preferably, 40≤n≤250；M is the degree of polymerization of aspartic acid segment in main chain, it is preferred that 10≤m≤50.
In preferred technical solution, R1For methyl；R2For-NH-；R4For acetyl group, polymer is with formula (I-a) at this time
Structure:
R3Selected from hydrogen or following group:
R3 `Selected from hydrogen or following group:
。
The high polymer based on amino acid that the present invention designs is made by introducing phenyl boric acid group in the high molecular side group
It can enhance the stability of carrier and drug in self assembling process by coordination with the drug containing amino, reduce small
The aggregation of molecular drug, to realize efficient drug loading；The phenyl boric acid group particularly preferably designed has hydrogen peroxide responsiveness,
Medicine-carried system may be implemented to discharge in tumor locus specificity；Current high molecular material is solved during preparing Nano medication
The existing drug reunion problem low with drugloading rate.
The present invention also provides a kind of preparation methods of polymer with formula (I) structure, include the following steps:
(1) compound and shielded amino acid monomer with formula (II) or formula (III) structure are anti-in organic solvent
It answers, obtains the polymer with protecting group；The amino acid monomer is carboxylic acid anhydrides in γ-benzyl-L-aspartate ester-N-；
(2) polymer with protecting group is reacted with ethanol amine, obtains the copolymer with formula (IV) structure, is
Poly(ethylene glycol)-bPoly- [(N- 2- ethoxy)-asparagine] copolymer；
(3) it by the copolymer with formula (IV) structure and the small molecule reaction with formula (V) structure, obtains with formula
(VI) intermediate of structure；Finally, the intermediate with formula (VI) structure obtains the polymerization with formula (I) structure by dialysis
Object；The polymer that specially there is the intermediate of formula (VI) structure to hydrolyze the formula that finally obtains (I) structure in dialysis procedure.
The present invention also provides a kind of preparation methods of polymer lyophilized powder, include the following steps:
(1) compound and shielded amino acid monomer with formula (II) or formula (III) structure are anti-in organic solvent
It answers, obtains the polymer with protecting group；The amino acid monomer is carboxylic acid anhydrides in γ-benzyl-L-aspartate ester-N-；
(2) polymer with protecting group is reacted with ethanol amine, obtains the copolymer with formula (IV) structure, is
Poly(ethylene glycol)-bPoly- [(N- 2- ethoxy)-asparagine] copolymer；
(3) it by the copolymer with formula (IV) structure and the small molecule reaction with formula (V) structure, obtains with formula
(VI) intermediate of structure；Finally, it is polymer lyophilized that there is the intermediate of formula (VI) structure to obtain by dialysis, freeze-drying
Powder；The polymer that specially there is the intermediate of formula (VI) structure to hydrolyze the formula that finally obtains (I) structure in dialysis procedure.
The present invention also provides the preparation methods of intermediate, include the following steps:
(1) compound and shielded amino acid monomer with formula (II) or formula (III) structure are anti-in organic solvent
It answers, obtains the polymer with protecting group；The amino acid monomer is carboxylic acid anhydrides in γ-benzyl-L-aspartate ester-N-；
After the completion of polymerization reaction, the sealing end such as acetic anhydride, chloroacetic chloride, cholesterol formyl chloride (R is added4), preferably blocked with acetic anhydride；
(2) polymer with protecting group is reacted with ethanol amine, obtains the copolymer with formula (IV) structure, is
Poly(ethylene glycol)-bPoly- [(N- 2- ethoxy)-asparagine] copolymer；
(3) it by the copolymer with formula (IV) structure and the small molecule reaction with formula (V) structure, obtains with formula
(VI) intermediate of structure.
Compound structure of the present invention is as follows:
Formula (IV)
Formula (V)
R6For hydrogen or following group:
R6 `For hydrogen or following group:
In above-mentioned technical proposal, in step (1), the organic solvent is n,N-Dimethylformamide or N, N- dimethyl
Formamide and dichloromethane mixture；The reaction preferably carries out in anhydrous conditions.
In above-mentioned technical proposal, in step (3), the copolymer with formula (IV) structure and small point with formula (V) structure
Son reaction carries out in a solvent, and the solvent is n,N-Dimethylformamide and/or dimethyl sulfoxide；The reaction is preferably in nothing
It is carried out under the conditions of water；Preferably, the reaction carries out in the presence of organic base (such as 4-dimethylaminopyridine).
The present invention also provides a kind of load medicine complexes micelles, including polymer and active medicine with formula (I) structure, institute
Active medicine is stated preferably with the drug of amino, such as adriamycin, Epi-ADM, Irinotecan.
The present invention also provides the preparation methods of above-mentioned load medicine complexes micelle, are nanoprecipitation method, mainly include following step
Rapid: active medicine and the above-mentioned polymer with formula (I) structure are dissolved in N,N-dimethylformamide, dimethyl sulfoxide or tetrahydro
It in the organic solvents such as furans, then instills in aqueous medium, then dialysis removes organic solvent and ultimately forms load in aqueous solution
Medicine complexes micelle.Preferably, the mass ratio of the polymer and active medicine with formula (I) structure is (1~20): 1, this
The encapsulation rate of obtained carrier micelle is invented not less than 80%.
The present invention also provides the above-mentioned polymer application in preparations of anti-tumor drugs with formula (I) structure, or
Application as active drug carrier.
The present invention also provides the complexes micelle application in preparations of anti-tumor drugs of above-mentioned load medicine.
It is anti-in preparation that the present invention also provides the intermediates of the preparation method of above-mentioned intermediate preparation and the intermediate
Application in tumour medicine, or the application in preparation active drug carrier.
In conclusion the present invention provides a kind of high molecular material with excellent drug carrying ability, the macromolecule be with
The polymer of structure shown in formula (I) can be used for biomaterial especially drug delivery field.The macromolecule is with poly- (second two
Alcohol)-bPoly- [(N- 2- ethoxy)-asparagine] based on, preferably modify to obtain by phenyl boric acid.Macromolecule main body has good
The phenyl boric acid group of good biocompatibility, side chain can reach efficient drug by coordination with the drug containing amino
Load.Preliminary Results of the present invention show the material to clinically common chemotherapeutics such as adriamycin, Epi-ADM, Yi Li
For health etc. have superelevation drugloading rate (be greater than 50%) and close to 100% encapsulation rate, and drug release has tumor microenvironment
Responsiveness.Macromolecule preparation is simple, convenient for promoting, has huge application in biomaterial especially Nano medication field
Prospect.
Detailed description of the invention
Fig. 1 is the block copolymer mPEG with protecting group prepared by embodiment 1113-b-PBLA21With deuterated trifluoroacetic acid
Hydrogen nuclear magnetic resonance spectrogram when as solvent；
Fig. 2 is the block copolymer mPEG of formula (IV) structure prepared by embodiment 1113-b-PHEA21With deuterated trifluoroacetic acid
Hydrogen nuclear magnetic resonance spectrogram when with deuterated water as mixed solvent (v:v=1:9)；
The nuclear magnetic resonance spectroscopy when small molecule for the formula (V) structure that Fig. 3 is prepared for embodiment 2 is using deuterated chloroform as solvent
Figure；
The core when precursor material for the formula (VI) structure that Fig. 4 is prepared for embodiment 3 is using deuterated dimethyl sulfoxide as solvent
Magnetic resonance hydrogen spectrogram；
The nuclear-magnetism when macromolecule for the formula (I) structure that Fig. 5 is prepared for embodiment 3 is using deuterated dimethyl sulfoxide as solvent is total
Shake hydrogen spectrogram；
Fig. 6 is the drugloading rate and encapsulation rate of the macromolecule loading adriamycin micella of formula (I) structure in embodiment 4；
Fig. 7 is the micellar particle size distribution that drugloading rate prepared by embodiment 4 is 33.4%；
Fig. 8 be drugloading rate prepared by embodiment 4 be 33.4% micella under different hydrogen peroxide concentrations drug release it is bent
Line；
Fig. 9 is Toxicity test result figure of the macromolecule to NIH/3T3 cell of formula (I) structure prepared by embodiment 3；
Figure 10 be embodiment 4 prepare adriamycin drugloading rate be 33.4% micella to HeLa cell proliferation inhibitory effect.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below with reference to embodiment, these
Description is only further explanation the features and advantages of the present invention, rather than limiting to the claimed invention.
The method for producing polymer of formula (I) of the present invention is according to the following steps:
In anhydrous n,N-Dimethylformamide and methylene chloride in the mixed solvent (v:v 1:9), using with formula (II),
Primary amine groups in the polyethylene glycol of formula (III) cause carboxylic acid anhydrides polymerization in γ-benzyl-L-aspartate ester-N-, are reacting
Excessive acetic anhydride sealing end is added in Bi Houke, obtains the polymer with protecting group, then in n,N-Dimethylformamide and
Ethanol amine reaction, obtains the copolymer of formula (IV) structure.
In polymer process of the preparation with protecting group, the compound with formula (II) or formula (III) structure with
The molar ratio of carboxylic acid anhydride monomer is preferably 1:5 ~ 200 in the γ-benzyl-L-aspartate ester-N-, and more preferably 1:10 ~
50；The temperature of the reaction is preferably 20 DEG C ~ 30 DEG C；The time of the reaction is preferably 48h ~ 96h.
In the above-mentioned polymer with protecting group and ethanol amine reaction process, protecting group removes simultaneously under the action of organic base
It is further acted on ethanol amine, obtains the copolymer with formula (IV) structure.Wherein the temperature of the reaction be preferably 30 DEG C ~
40 DEG C, the time being stirred to react is preferably 12h ~ for 24 hours, and the solvent of the reaction is preferably n,N-Dimethylformamide.Instead
It after answering, is settled with excess diethyl ether, obtains the copolymer of formula (IV) structure after filtration, washing and drying.
The lyophilized co-polymer powder end of formula (IV) structure in order to obtain, above-mentioned sedimentation products n,N-Dimethylformamide is molten
Solution, dialyse for 24 hours ~ 72h in pure water, changes in dialysis procedure water 6 ~ 15 times, is freeze-dried, obtains the copolymerization with formula (IV) structure
Object freeze-dried powder.
The synthesis of the small molecule of formula (V) structure is carried out according to method as follows.Simple, by 4- methylol phenyl boric acid
Pinacol ester and N, N'- carbonyl dimidazoles are mixed in anhydrous methylene chloride, and abundant washing and drying, drains solvent after reaction
Obtain the small molecule of formula (V) structure.
The small molecule reaction of the copolymer of formula (IV) structure and (V) structure, obtains the intermediate of (VI) structure.Reaction is preferred
It is carried out in anhydrous n,N-Dimethylformamide, the catalyst of 4-dimethylaminopyridine (DMAP) as reaction is added.It is described to stir
The temperature for mixing reaction is preferably 40 DEG C ~ 60 DEG C.The time being stirred to react is preferably 48h ~ 96h.End of reaction obtains (VI)
The intermediate of structure.The intermediate does not need to be further purified, and can directly add distilled water dialysis, in the process pinacol quilt
Hydrolysis removing, obtains the macromolecule of formula (I) structure after freeze-drying.
The present invention is during preparation has the macromolecule of formula (I) structure, in γ-benzyl-L-aspartate ester-N-
Carboxylic acid anhydrides is raw material, is not particularly limited to carboxylic acid anhydrides source in this amino acid-N-, and following methods preparation is referred to:
L-Aspartic acid and benzyl alcohol react under the action of the concentrated sulfuric acid, post-treated to obtain γ-benzyl-L- days
Aspartic acid ester, the γ-benzyl-L-aspartate ester are reacted with bis- (trichloromethyl) carbonic esters (triphosgene), obtain γ-benzene
Carboxylic acid anhydrides in methyl-L-aspartate ester-N-.
The above-mentioned compound structure being related to is as follows:
Formula (II)
Formula (III)
Formula (IV)
Formula (V)
R6For hydrogen or following group:
R6 `For hydrogen or following group:
For a further understanding of the present invention, to polymer provided by the invention and its medicine compound is carried below with reference to embodiment
Micella preparation method is described in detail, but protection scope of the present invention is not limited by the following examples.
Embodiment 1
The polyethyleneglycol with formula (II) structure that 5.00g number-average molecular weight is 5000 is added into dry reaction flask
Methyl ether, with 80mL dry toluene at 130 DEG C after azeotropic water removing 3h, the remaining toluene of decompressing and extracting；Obtained solid is dissolved
In 20mL dry methylene chloride, the first solution is obtained；By carboxylic acid anhydrides in 5.50g γ-benzyl-L-aspartate ester-N-
It is dissolved in the in the mixed solvent of 5mL dry n,N-Dimethylformamide and 50mL methylene chloride, obtains the second solution；In nitrogen
In atmosphere, the first solution is mixed with the second solution, is stirred to react 48h under the conditions of room temperature, nitrogen protection；Then 10mL is added
The reaction was continued for acetic anhydride for 24 hours.After reaction, decompression pumps most of solvent, then is settled with ether, filters, after dry,
The block copolymer with protecting group is obtained, is γ-benzyl-L-aspartate ester, protecting group is benzyl.Obtained band is protected
The block copolymer for protecting base carries out nuclear magnetic resonance spectroscopy, as a result referring to Fig. 1, the results showed that γ-benzyl-L-aspartate ester
The degree of polymerization is 21, which is denoted as mPEG113-b-PBLA21, structure is as follows:
Take the mPEG that 5.00g is obtained113-b-PBLA21The dry N,N-dimethylformamide of 45mL is dissolved at 35 DEG C
In, 4mL ethanol amine is added, is stirred to react for 24 hours, product is settled with ether, N, N- dimethyl methyl are used after being filtered, washed, drying
Amide dissolves, and dialyse 72h in pure water, changes in dialysis procedure water 10 times, then freeze-drying obtains block copolymer.
Nuclear magnetic resonance spectroscopy is carried out to obtained block copolymer, Fig. 2 is the block copolymer of the preparation of embodiment 1 with deuterated
Hydrogen nuclear magnetic resonance spectrogram when trifluoroacetic acid and deuterated water are as solvent, the results showed that, the block copolymer tool that embodiment 1 obtains
There is the structure of formula (IV), wherein R1It is methyl；R2For-NH-；R3For hydrogen；R4It is acetyl group；The yield of the block copolymer is
70%, wherein n=113, m=21 are denoted as mPEG113-b-PHEA21, specific structure is as follows:
。
Embodiment 2
In a dry round-bottomed flask, 7.2g 4- methylol phenyl boric acid pinacol ester and two miaow of 10.3g carbonyl is added
Azoles is added the dissolution of 50mL anhydrous methylene chloride, reacts at room temperature 12h.It is dilute that 500mL ethyl acetate is added into system after reaction
It releases, successively uses distilled water and saturated common salt water washing organic layer, then by organic layer with anhydrous MgSO4It is dried overnight.Decompression removes
Organic solvent is gone to finally obtain the small molecule of 7.6g formula (V) structure.Nuclear magnetic resonance spectroscopy is carried out to obtained block copolymer,
Fig. 3 be embodiment 2 prepare small molecule using deuterated dimethyl sulfoxide as solvent when hydrogen nuclear magnetic resonance spectrogram, the results showed that success
The small molecule is synthesized, specific structure is as follows:
Formula (V).
Embodiment 3
The tool prepared in 2.00g embodiment 1 is added into dry reaction flask for the high molecular material of formula (I) in order to obtain
Have the block copolymer of formula (IV) structure, the small molecule and DMAP(412mg of 1.0g formula (V) structure), vacuumize 12h.Then plus
Enter the dry n,N-Dimethylformamide dissolution of 10mL, is stirred to react 12h under the conditions of 50 DEG C, nitrogen protection.After reaction,
It is settled, is washed with excessive ether, filtered, after dry, obtain intermediate.Nuclear-magnetism is carried out to obtained midbody polymer
Resonance analyzing, as a result referring to fig. 4, the results showed that, phenyl boric acid group is successfully grafted on macromolecule.Obtained intermediate has formula
(VI) structure, wherein R1It is methyl；R2For-NH-；R6For phenyl boric acid group, ratio shared by phenyl boric acid group is 48%；R4It is
Acetyl group；Obtained intermediate specific structure is as follows:
Above-mentioned intermediate is dissolved in water, dialyse 72h in rear pure water, changes water 10 times, is obtained with formula in dialysis procedure
(I-a) polymer of structure, finally freeze-drying obtains freeze-dried powder, carries out nuclear magnetic resonance spectroscopy (Fig. 5) to obtained material, knot
Fruit shows pinacol by complete hydrolysis.
R3For following group:
N=113, m=21.
Embodiment 4
By the polymer 20mg of formula obtained in embodiment 3 (I-a) structure and different quality (4,10,13.3 or 20mg)
Doxorubicin hydrochloride be dissolved in 0.5mL n,N-Dimethylformamide, in course of dissolution be added equivalent be 1.5 times of doxorubicin hydrochloride
Triethylamine, be sufficiently mixed 3 hours, be then slowly dropped into the ultrapure water quickly stirred (2mL) in 37 DEG C of shaking table, be added dropwise
After continue to stir 3h, dialyse in pure water 12h, changes in dialysis procedure water 5 times, obtains load Ah mould finally by freeze-drying
The composite micelle of element.
Using ultraviolet-visible spectrum, adriamycin contains in the carrier micelle that the absorption measurement embodiment 4 of 480 nm obtains
Amount, is calculated by the following formula the loading (DLC) and encapsulation rate (DLE) of the adriamycin in micella:
DLC=(quality/micella gross mass of drug in micella) × 100%
DLE=(quality of drug/investment drug quality in micella) × 100%
Fig. 6 is the drugloading rate of carrier micelle prepared for embodiment 4 and summarizing for encapsulation rate, the results show that formula (I) structure
Macromolecule almost can quantitatively load adriamycin, encapsulation rate is close to 100%.
The micella of different drugloading rates is redissolved in PBS(pH=7.4), concentration to 0.1mg/mL utilizes dynamic light scattering point
Analysis, measures the hydrodynamics partial size of micella, the load medicine glue for being as a result 33.4% referring to the drugloading rate that Fig. 7, Fig. 7 are the preparation of embodiment 4
The hydrodynamics distribution map of beam, the results showed that micellar particle size is evenly distributed between 18nm ~ 48nm.Other different drugloading rates
Micellar particle size is also in 100 nm hereinafter, and narrow distribution.
Embodiment 5
By the polymer 20mg of formula obtained in embodiment 3 (I-a) structure and different quality (4,10,13.3 or 20mg)
Farmorubine Hydrochloride be dissolved in 0.5mL n,N-Dimethylformamide, in course of dissolution be added equivalent be Farmorubine Hydrochloride
1.5 times of triethylamine is sufficiently mixed 3 hours in 37 DEG C of shaking table, is then slowly dropped into the ultrapure water quickly stirred
(2mL) continues to stir 3h after being added dropwise, and dialyse in pure water 12h, changes in dialysis procedure water 5 times, finally by being freeze-dried
To the composite micelle of load Epi-ADM.
Using ultraviolet-visible spectrum in the carrier micelle that the absorption measurement embodiment 5 of 480 nm obtains Epi-ADM
Content, drugloading rate and encapsulation rate calculation formula reference implementation example 4.The results show that the macromolecule of formula (I) structure can almost quantify
Load Epi-ADM, the encapsulation rate of drug is close to 100%.
The micella of different drugloading rates is redissolved in PBS(pH=7.4), concentration to 0.1mg/mL utilizes dynamic light scattering point
Analysis, the results showed that all carrier micelle hydrodynamics partial sizes are in 100 nm hereinafter, and narrow distribution.
Embodiment 6
By the polymer 20mg of formula obtained in embodiment 3 (I-a) structure and different quality (4,10,13.3 or 20mg)
Irinotecan hydrochloride be dissolved in 0.5mL n,N-Dimethylformamide, in course of dissolution be added equivalent be irinotecan hydrochloride
1.5 times of triethylamine is sufficiently mixed 3 hours in 37 DEG C of shaking table, is then slowly dropped into the ultrapure water quickly stirred
(2mL) continues to stir 3h after being added dropwise, and dialyse in pure water 12h, changes in dialysis procedure water 5 times, finally by being freeze-dried
To the composite micelle of load Irinotecan.
Using ultraviolet-visible spectrum in the carrier micelle that the absorption measurement embodiment 6 of 360 nm obtains Irinotecan
Content, drugloading rate and encapsulation rate calculation formula reference implementation example 4.The results show that the macromolecule of formula (I) structure can almost quantify
Irinotecan is loaded, the encapsulation rate of drug is greater than 90%.
The micella of different drugloading rates is redissolved in PBS(pH=7.4), concentration to 0.1mg/mL utilizes dynamic light scattering point
Analysis, the results showed that all carrier micelle hydrodynamics partial sizes are in 100 nm hereinafter, and narrow distribution.
Embodiment 7
It is 7.4 that the composite micelle that the drugloading rate for taking the embodiment 4 of 5mg to prepare is 33.4%, which is dissolved in the pH value of 5mL 0.01M,
Phosphate buffer solution in, wherein containing 0.5% Tween 80, be then transferred to the bag filter that molecular cut off is 3500, use
The corresponding buffer of 45mL is dialysed, and dialysis carries out in the constant temperature oscillation case that temperature is 37 DEG C, revolving speed is 100, Mei Gete
It fixes time and samples 4mL, and supplement the buffer of corresponding amount；Using ultraviolet-visible spectrum 480nm absorption measurement release liquid
Concentration obtains cumulative percentage release with time increased variation relation, and releasing result is as shown in Figure 8.Releasing result shows
Drug release is slow under normal physiological conditions for carrier micelle, and drug can be released quickly under the conditions of 100 μM of hydrogen peroxide
It puts.It is such the result shows that carrier micelle release tool since the content of tumor tissues and intracellular hydrogen peroxide is higher than normal tissue
There is the responsiveness of tumor tissues microenvironment, has better specificity when applying in vivo.
Embodiment 8
It is investigated using polymer material toxicity of the MTT cell toxicity test to preparation, the specific steps are as follows:
1, logarithmic phase NIH/3T3 cell is collected, is inoculated in 96 orifice plates, 100 L(~ 7000 μ are contained in every hole) cell；
At 37 DEG C, saturated humidity, 5% CO224 h are cultivated in cell incubator；
2, culture solution is discarded after 24 h, the polymer dilution of formula (I-a) structure for embodiment 3 being prepared with culture medium is not to
Same concentration is added in 96 orifice plates, and 200 μ L, 3 multiple holes of every kind of concentration are added in every hole；At 37 DEG C, saturated humidity, 5% CO2Cell
48 h are cultivated in incubator；
3, after 48h, the 3-(4 that 20 μ L concentration are 5mg/mL, 5- dimethylthiazole -2 is added in every hole) -2,5- dimethyl four
Nitrogen azoles bromide (MTT) solution continues to cultivate 4h；Culture is terminated, culture solution in hole is sucked, 200 μ L dimethyl sulfoxides are added in every hole,
Low-speed oscillation 10min detects absorption value of each hole at 492nm with microplate reader, the cell survival under each concentration is calculated
Rate.
Fig. 9 is that the polymer of formula (I-a) structure prepared by embodiment 3 investigates result figure to NIH/3T3 cytotoxicity, as a result
Show that cell survival rate is 80% or more at various concentrations, it was demonstrated that high molecular material prepared by the present invention has good safety
Property.
Embodiment 9
1, logarithmic phase HeLa human cervical carcinoma cell is collected, is inoculated in 96 orifice plates, 100 L(~ 7000 μ are contained in every hole)
Cell；At 37 DEG C, saturated humidity, 5% CO224 h are cultivated in cell incubator；
2, culture solution is discarded after 24 h, is diluted with the carrier micelle that the drugloading rate that culture medium prepares embodiment 4 is 33.4%
It to various concentration, is added in 96 orifice plates, 200 μ L, 3 multiple holes of every kind of concentration are added in every hole；At 37 DEG C, saturated humidity, 5% CO2
48 h are cultivated in cell incubator；
3, after 48h, the MTT solution that 20 μ L concentration are 5mg/mL is added in every hole, continues to cultivate 4h；Culture is terminated, hole is sucked
200 μ L dimethyl sulfoxides are added in interior culture solution, every hole, and low-speed oscillation 10min detects suction of each hole at 492nm with microplate reader
The high molecular cell survival rate of each concentration is calculated in receipts value.
Figure 10 is that the carrier micelle that drugloading rate prepared by embodiment 4 is 33.4% investigates result figure to HeLa cytotoxicity, right
It is the pure medicine of adriamycin according to group.The result shows that the carrier micelle of preparation has good Cytostatic to tumor cell effect.
The present invention by improving the interaction between drug and small molecule to high molecular rational modification, enhancing carrier and
Stability of the drug in self assembling process, reduces the aggregation of small-molecule drug, to realize efficient drug loading, especially this
The design of sample does not need complicated synthesis step, in practical advantageously.
The general principles defined herein can without departing from the spirit or scope of the present invention, in other realities
It applies in example and realizes.Therefore, the present invention will not be limited to the embodiments shown herein, and is to fit to public with institute herein
The consistent widest scope of the principle and features of novelty opened.
Claims (7)
1. a kind of polymer has structure shown in formula (I)；
In formula (I), R1Selected from hydrogen, alkyl or replace alkyl；
R2Selected from-NH- or-R5(CH2)rNH-, wherein R5For-O- ,-OCONH- ,-OCO- ,-NHCOO- or-NHCO-, 1≤r≤
10；
R3Selected from hydrogen or following group:
R3 `Selected from hydrogen or following group:
R4Selected from hydrogen or hydrophobic grouping；
20≤n≤500；5≤m≤200；
The group ratio of phenyl boric acid structure is not less than 20%.
2. polymer according to claim 1, which is characterized in that the alkyl is the alkyl of C1 ~ C40；The substitution alkyl
In, substituent group is sulfydryl, saccharide residue, aldehyde radical, carboxyl, vinyl, alkynyl, succimide base, dimaleoyl imino, biotin
Base, RGD small peptide base, LHRH small peptide base or folic acid base；The hydrophobic grouping is alkyl, benzyl, the cholesterol formyl of C4 ~ C20
Base, acetyl group, cholic acid group or deoxycholic acid base.
3. polymer according to claim 1, which is characterized in that the preparation method of the polymer includes the following steps:
(1) compound with formula (II) or formula (III) structure reacts in organic solvent with shielded amino acid monomer, obtains
To the polymer for having protecting group；The amino acid monomer is carboxylic acid anhydrides in γ-benzyl-L-aspartate ester-N-；
(2) polymer with protecting group is reacted with ethanol amine, obtains the copolymer with formula (IV) structure；
(3) it by the copolymer with formula (IV) structure and the small molecule reaction with formula (V) structure, obtains with formula (VI)
The intermediate of structure；Finally, the intermediate with formula (VI) structure obtains the polymer with formula (I) structure by dialysis；
The compound structure being related to is as follows:
R6For hydrogen or following group:
R6 `For hydrogen or following group:。
4. polymer according to claim 3, which is characterized in that in step (1), the organic solvent is N, N- dimethyl methyl
Amide or N,N-dimethylformamide and dichloromethane mixture；The reaction carries out in anhydrous conditions；In step (3), tool
There are the copolymer of formula (IV) structure and the small molecule reaction with formula (V) structure to carry out in a solvent, the reaction is in no water bar
It is carried out under part, the reaction carries out in the presence of an organic base.
5. a kind of load medicine complexes micelle includes polymer and active medicine described in claim 1.
6. polymer application in preparation of anti-tumor drugs described in claim 1, or as preparation active drug carrier
Using.
7. carrying medicine complexes micelle application in preparation of anti-tumor drugs described in claim 5.
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| CN106008964A (en) * | 2016-05-20 | 2016-10-12 | 中国科学院长春应用化学研究所 | Water-soluble amino acid segmented copolymer, and preparation method and application thereof |
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