CN110123748A - Double drug-carrying polymer micelles of a kind of folate-mediated targeting and its preparation method and application - Google Patents
Double drug-carrying polymer micelles of a kind of folate-mediated targeting and its preparation method and application Download PDFInfo
- Publication number
- CN110123748A CN110123748A CN201910324103.6A CN201910324103A CN110123748A CN 110123748 A CN110123748 A CN 110123748A CN 201910324103 A CN201910324103 A CN 201910324103A CN 110123748 A CN110123748 A CN 110123748A
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- pga
- targeting
- pll
- double drug
- carrying polymer
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Classifications
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- 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
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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- 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
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- A—HUMAN NECESSITIES
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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
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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
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Molecular Biology (AREA)
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- Polymers & Plastics (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Dispersion Chemistry (AREA)
- Biophysics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses double drug-carrying polymer micelles of a kind of folate-mediated targeting and its preparation method and application, the polymer micelle is to be formed by nanoparticle by being grafted taxol, gemcitabine and the polyglutamic acid of folic acid respectively, then with polylysine electrostatic self-assembled.Compare the prior art, the double drug-carrying polymer micelles of targeting of the invention, it is combined by taxol and two medicine of gemcitabine, it can reach better antitumous effect, and solve the problems, such as that taxol soluble is poor, drug effect is improved by targeting, reduces toxicity, provides possibility for the targeting low toxicity treatment of cancer.
Description
Technical field
The present invention relates to double drug-carrying polymer micelles of a kind of folate-mediated targeting and its preparation method and application, belong to load
Medicine polymer micelle technical field.
Background technique
Nano medication is as a kind of emerging technology, for the accurate positioning and early diagnosis of tumour, targeting, long-acting and combine and control
Treatment provides important research/development platform, for the bottleneck for overcoming the targeting of conventional medicament non-specificity and non-selective damage body tissue
Problem provides possibility.A large number of researchers are based on polymer micelle, dendrimer, quantum dot, nanogold, nanometer Jie in recent years
The design of the nano-medicament carriers such as hole silicon has synthesized the Nano medication that largely can be used for tumour diagnosis and treatment, mainly passes through " core-shell structure copolymer " structure
The methods of design, surface modification improve Nano medication performance.Compared with the excessive method of the toxic side effects such as traditional physics and chemotherapy,
Nano medication is more accurate, and curative effect is more excellent.
However, chemotherapy of tumors effect is still undesirable, on the one hand, such as water-soluble the characteristics of due to drug itself physicochemical property
Official post drug is not effectively achieve the tumor tissues of special construction；On the other hand, multi-drug resistance of the tumor is also chemotherapy failure
Major reason；In another aspect, chemotherapeutics lacks targeting, causing the serious toxic side effect of patient is also clinically Chemo-Therapy
The major obstacle for the treatment of.The targeting of angle research reversing tumor MDR and raising drug from drug delivery system are that a kind of raising is anti-swollen
Tumor medicine curative effect reduces dosage simultaneously and reduces the new approaches of toxic side effect, has become the hot spot of antitumor research.
Summary of the invention
Goal of the invention: in order to solve the above technical problems, the present invention provides a kind of double medicines that carry of folate-mediated targeting to polymerize
Object micella and its preparation method and application, the polymer micelle are able to solve the problem of taxol soluble difference, and use folic acid
Targeting is conducive to improve anticancer effect.
Technical solution: to achieve the above object of the invention, the invention adopts the following technical scheme:
A kind of double drug-carrying polymer micelles of folate-mediated targeting, which is by being grafted taxol, Ji respectively
The polyglutamic acid of western his shore and folic acid, then nanoparticle is formed by with polylysine electrostatic self-assembled.
The taxol, gemcitabine and folic acid are connected on polyglutamic acid by ester bond on molecule.
Amino on the polyglutamic acid on remaining carboxyl and polylysine is able to carry out electrostatic self-assembled and forms nanometer
Particle.
The preparation method of the double drug-carrying polymer micelles of the targeting, comprising the following steps:
1) Polylysine Polymers macromolecular PLL is prepared；
2) polyglutamic acid polymer macromolecular PGA is prepared；
3) taxol PTX and gemcitabine GEM are grafted on PGA molecule respectively, by the carboxyl on folic acid FA by repairing
It is grafted on PGA molecule after decorations hydroxyl, is respectively formed graft PGA-PTX, PGA-GEM and PGA-FA；
4) by three kinds of grafts obtained by step 3), electrostatic self-assembled is carried out in buffer solution with PLL and forms nanoparticle
FA-PMDNPs targets double drug-carrying polymer micelles to get described.
As preferred:
In step 1), the method for preparing Polylysine Polymers macromolecular PLL is as follows:
Tetrahydrofuran (THF) makes triphosgene decompose generation phosgene as nucleopilic reagent, and Lys (Z) is obtained under the action of phosgene
It with n-hexylamine is that initiator causes its ring-opening polymerisation and obtains PLL (Z) to Lys (Z)-NCA, Lys (Z)-NCA, PLL (Z) is in room temperature
Under the conditions of (preferably 2h) is reacted with HBr, slough benzyloxycarbonyl group (Cbz), obtain macromolecule polyalcohol PLL.
In step 2), the method for preparing polyglutamic acid polymer macromolecular PGA is as follows:
Tetrahydrofuran (THF) makes triphosgene decompose generation phosgene as nucleopilic reagent, and Glu (Z) is obtained under the action of phosgene
It with n-hexylamine is that initiator causes its ring-opening polymerisation and obtains PBLG to Glu (Z)-NCA, Glu (Z)-NCA, PBLG is in alkaline condition
Under slough benzyl, obtain macromolecule polyalcohol PGA.
In step 3):
The PGA is connect by ester bond with PTX, GEM respectively under DMAP/EDCI catalysis, obtains PGA-PTX and PGA-
GEM；
The FA end group activated carboxylic is simultaneously reacted with 3- aminopropanol, obtains the compound FA-OH, PGA that end group is hydroxyl
With FA-OH under DMAP/EDCI catalysis, PGA-FA is obtained.
Nanoparticle (FA-PMDNPs) in step 4) the preparation method is as follows:
PGA-GEM, PGA-PTX and PGA-FA are dissolved in PBS buffer solution respectively, and mixed with PLL stock solution, it will
Mixture be incubated at 4 DEG C 10-14 hours to get.
The present invention finally provides the double drug-carrying polymer micelle application in preparations of anti-tumor drugs of the targeting.
Since tumour cell fast breeding needs a large amount of nucleic acids, tumor cell surface will be overexpressed folic acid by
Body, and it is then highly conserved on normal cell surface.The specificity of the specificity and combination that are identified using folacin receptor with its ligand,
It can achieve the accurate targeting to tumour, increase drug in the enrichment of lesions position, improve curative effect, reduce dosage, reduce
The toxic side effect of normal tissue.
Polylysine and polyglutamic acid are a kind of poltpeptides, and good biocompatibility is biodegradable.Pass through ester bond
After carrying medicament can slow release drug in the cell, reach slow release effect.
Folic acid is a kind of vitamin, the further synthetic DNA of the synthesis by participating in purine and thymidine and RNA.By
A large amount of nucleic acids are needed in tumour cell fast breeding, therefore tumor cell surface will be overexpressed folacin receptor, and just
Normal cell surface is then highly conserved.FA and FR binding force are strong, have high selectivity to tumour.Known using folacin receptor and its ligand
The specificity of other specificity and combination, can achieve the accurate targeting to tumour, increases drug in the enrichment of lesions position, mentions
High curative effect reduces dosage, reduces normal tissue toxic side effect.
Taxol is by interfering cytoskeleton component part --- micro-pipe normal function is thin to realize inhibition and kill cancer
Born of the same parents；Gemcitabine is a kind of Difluoronucleosides class antimetabolite anticarcinogen for destroying cellular replication.The total transmission of both drugs is controlled
Treatment can achieve synergistic effect, realize lower dosage and better therapeutic effect.
Therefore, the present invention loads to taxol on polyglutamic acid by ester bond, can be with as outer layer using polyglutamic acid-folic acid
The folic acid is set to be exposed to nanoparticle surface well to realizing targeting, as the FA and tumor cell surface on nanoparticle
After FR is combined, lysosome is reached after cell endocytic, reaches slow release effect.Utilize the taxol antitumor machine different with gemcitabine
System, two medicines combination, can achieve better antitumous effect.
Technical effect: compare the prior art, and the double drug-carrying polymer micelles of targeting of the invention pass through taxol and Ji Xi
He is combined two medicine of shore, can reach better antitumous effect, and solve the problems, such as that taxol soluble is poor, is mentioned by targeting
High-drug-effect reduces toxicity, provides possibility for the targeting low toxicity treatment of cancer.
Detailed description of the invention
Fig. 1: PLL nuclear-magnetism characterization；
Fig. 2: PLL IR Characterization；
Fig. 3: PGA nuclear-magnetism characterization；
Fig. 4: PGA IR Characterization；
Fig. 5: the standard curve of taxol, gemcitabine and folic acid；
The IR Characterization that Fig. 6: (A) is PGA-PTX, (B) is PGA-GEM, (C) is PGA-FA；
The nuclear-magnetism characterization that Fig. 7: (A) is PGA-PTX, (B) is PGA-GEM, (C) is PGA-FA；
Fig. 8: FA-PMDNP characterization, wherein (A) is the relationship of PGA and PLL mass ratio and hydration partial size；(B) for PGA with
The relationship of PLL mass ratio and Zeta potential；(C) partial size for being optimum quality ratio FA-PMDNP；It (D) is tem observation best in quality
The FA-PMDNP pattern of ratio；
Fig. 9: the release profiles of taxol and gemcitabine in pH 5.5 and pH 7.4；
Figure 10: the relation curve of the hemolysis rate of various concentration FA-PMDNPs and PMDNPs.
Specific embodiment
Technical solution of the present invention is further illustrated below by specific embodiment.
Embodiment 1
1) Polylysine Polymers macromolecular (PLL) is prepared:
Step 1: formula Lys (Z) (5g, 17.85mmol) is dissolved in 65 milliliters of anhydrous tetrahydro furans, stirs 30min
Afterwards, phosgene (2.12g, 7.15mmol) is added, 50 DEG C are reacted 3 hours in the case where being filled with condition of nitrogen gas.Be added big head for precooling just oneself
Alkane stirs 10min, and suction filtration obtains compound L ys (Z)-NCA.1H NMR(400MHz,DMSO-d6): δ 9.11 (1H, α-NH),
7.34(5H,Ph),5.00(2H,-CH2Ph),4.42(1H,-CH),2.98(2H,-CH2),1.69(2H,-CH2),1.4(2H,-
CH2),1.28(2H,-CH2).FT-IR(cm-1): 943 (the stretching vibration absorption band NCA of O=C-O-C=O), 1688 (C=O
In the Cbz group), 1774,1812 (C=O in the NCA), 1250 (C-O in the Cbz group).
Step 2: compound L ys (Z)-NCA (5.10g, 16.65mmol) obtained by upper step is dissolved under the action of nitrogen
It is added in reaction in 25mLDMF, then by the hexylamine (55.65 μ g, 0.55mmol) for being dissolved into 1ml DMF, is heated to 35 DEG C instead
It answers 3 days.The methyl tertiary butyl ether(MTBE) stirring 10min of big head for precooling is added, it is compound PLL (Z) that suction filtration, which obtains white powder,.Purifying
PLL (Z) afterwards is dried at room temperature for.1H NMR(400MHz,DMSO-d6):7.32(ArH in the Cbz group),4.99
(-CH2 in the Cbz group),and 0.82(-CH3 in hexylamine).FT-IR(cm-1): 1692 (C=O in
the Cbz group),1627 and 1537(-NHCO-,amide bond of the PLL(Z)repeating unit),
697(Ph in the Cbz group),1250(C-O in the Cbz group).
Step 3: PLL (Z) (3.6g, 0.45mmol) is dissolved in HBr (13.25g, 33wt%, acetic acid in), is stirred at room temperature
2h precipitates PLL with the methyl tertiary butyl ether(MTBE) of pre-cooling, repeats Purification by filtration.Filter cake after purification is dried at room temperature for.1H NMR
(400MHz,D2O):4.27(-CH),1.13-1.84(-CH2 in PLL and hexylamine)0.82(-CH3 in
hexylamine).FT-IR(cm-1):1627 and 1537(-NHCO-,amide bond of the PLL(Z)repeating
unit).Wherein, polymerization degree n is selected from 45-70, preferably n=50.(its nuclear-magnetism and IR Characterization are shown in Fig. 1 and Fig. 2 respectively).
2) polyglutamic acid polymer macromolecular (PGA) is prepared:
Step 1: formula Glu (5g, 21.07mmol) is dissolved in 65 milliliters of anhydrous tetrahydro furans, after stirring 30min, is added
Enter phosgene (2.5g, 8.43mmol), 50 DEG C are filled with nitrogen and react 3 hours.The n-hexane stirring 10min of big head for precooling is added, takes out
Filter obtains compound Glu (Z)-NCA.1H NMR(400MHz,DMSO-d6)δ9.12(1H,α-NH),7.48–7.23(5H,Ph),
5.10(2H,CH2), Ph 4.45 (1H ,-CH), 2.53 (2H ,-CH2), 1.99 (2H ,-CH2) .FT-IR (cm-1): 928 (O=C-
The stretching vibration absorption band of O-C=O), 1703 (C=O in the Cbz group), 1780 (C=O in the NCA).
Step 2: compound Glu (Z)-NCA (4.5g, 17.16mmol) obtained by upper step is dissolved in 25ml's under a nitrogen
DMF, then the hexylamine (31.56mg, 0.31mmol) for being dissolved in 1mlDMF is added in reaction, it is heated to 35 DEG C and reacts 3 days.It is added big
The methyl tertiary butyl ether(MTBE) of head for precooling stirs 10min, and it is compound PBLG that suction filtration, which obtains white powder,.1H NMR(400MHz,
DMSO-d6)δ7.24(Ph in the Cbz),5.03(-CH2 in the Cbz),3.93(-CH),0.81(-CH3 in
Hexylamine) .FT-IR (cm-1): where polymerization degree n is selected from 45-70, preferably n=53.
Step 3: it takes compound PBLG (3.4g, 0.31mmol) obtained by step to be dissolved in 29mL methanol, hydrogen-oxygen is then added
Change in sodium (5mL, 12.4M) solution, normal-temperature reaction 3h, repeats filtering purification.10mL ethyl alcohol is added in post-processing, in revolving speed
It is centrifuged 10min under 9000rmp to be precipitated, is washed repeatedly with ethyl alcohol precipitating 3-4 times, adjusts pH to 7-8.By the precipitating after centrifugation
5mL hydrochloric acid EA is added and stirs 10min, is centrifuged again, precipitating is dried to obtain polyglutamic acid (PGA) with vacuum oven.1H
NMR(400MHz,D2O)δ12.13(-COOH),4.24(-CH),0.84(-CH3 in hexylamine).FT-IR(cm-1):
Wherein, polymerization degree n is selected from 45-70, preferably n=53.(its nuclear-magnetism and IR Characterization are shown in Fig. 3 and Fig. 4 respectively).
3) preparation of PGA-PTX, PGA-GEM and PGA-FA:
One, PGA-PTX is prepared
Compound PGA (400mg, 3.3mmol) is dissolved in 4mL dimethyl sulfoxide, be added EDCI (632mg, 3.3mmol) and
PTX (80mg, 0.0937mmol) is added after stirring 30min in DMAP (200mg, 1.64mmol), after room temperature reaction for 24 hours, reaction solution
PGA-PTX is obtained with bag filter (MWCO=1000D) dialysis 48h.(its infrared and nuclear-magnetism characterization is shown in Fig. 6 and Fig. 7 respectively).
Two, PGA-GEM is prepared
Compound PGA (400mg, 3.3mmol) is dissolved in 4mL dimethyl sulfoxide, be added EDCI (632mg, 3.3mmol) and
GEM (80mg, 0.3mmol) is added after stirring 30min in DMAP (200mg, 1.64mmol), and after room temperature reaction for 24 hours, reaction solution is used
Bag filter (MWCO=1000D) dialysis 48h obtains PGA-GEM.(its infrared and nuclear-magnetism characterization is shown in Fig. 6 and Fig. 7 respectively).
Three, PGA-FA is prepared
(1) FA (200mg, 0.45mmol) is weighed to be dissolved in 5mL dimethyl sulfoxide, addition NHS (54.09mg,
0.47mmol), it after 1h is stirred at room temperature in EDCI (129.26mg, 0.67mmol), is added Et3N (35.3mg, 0.47mmol), is protected from light
Stir 36h.Post-processing, which is added, is pre-chilled methyl tertiary butyl ether(MTBE): acetone=7:3 stirring 20min, by filter cake 100mL methyl after suction filtration
Tertbutyl ether: it washes for acetone=7:3 more times.It filters, filter cake is dried in vacuo to obtain compound FA-NHS.
(2) by compound FA-NHS (200mg, 0.372mmol) obtained by upper step and 3- aminopropanol (27.9mg,
It 0.372mmol) is dissolved in 5mL dimethyl sulfoxide, is stirred at room temperature for 24 hours.The a large amount of methyl tertiary butyl ether(MTBE)s of post-processing addition: n-hexane=
7:3 (v/v) stirs 10min, filters, filtration cakes torrefaction is obtained pale yellow powder FA-OH.
(3) by compound PGA (200mg, 1.65mmol), EDCI (316.86mg, 1.65mmol), DMAP (201.95mg,
It 1.65mmol) is dissolved in 4mL dimethyl sulfoxide, stirs 30min.FA-OH (66mg, 0.13mmol) is added to stir 24 hours.It will
Reaction solution obtains product PGA-FA with bag filter (MWCO=1000D) dialysis 48h.(its infrared and nuclear-magnetism characterization see respectively Fig. 6 and
Fig. 7).
3) nanoparticle (FA-PMDNPs) is prepared
By PGA-GEM (1mg/mL, 4mL), PGA-PTX (1mg/ml, 4ml) and PGA-FA (1mg/ml, 2ml) are dissolved in
In 1mL PBS buffer solution (10mM, pH7.4), and mix with the PLL stock solution of various amounts (0.01mg/ml) generate it is various
PGA-GEM/PTX/FA and PLL mass ratio.Mixture is incubated for 3 hours at 4 DEG C.Use Malvern Zeta SizerNano
The average grain diameter (Fig. 8) and zeta current potential (Fig. 8) of the FA-PMDNC of Series Measurement different quality ratio.Use transmission electron microscope
Observe the pattern (Fig. 8) of the FA-PMDNP of optimum quality ratio.
Embodiment 2
(1) measurement of sample solid content:
Weighing vial 5 times simultaneously calculates average value, takes 1mL sample (such as PGA-PTX) to be put into vial, is being dried in vacuo
It dries in case to constant weight.Then it weighs bottle weight 5 times and calculates average value, obtain sample solid content.
(2) drafting of standard curve:
FA, PTX and the GEM solution that various concentration is prepared with methanol measure absorbance using ultraviolet specrophotometer.Folic acid
Detection wavelength is 283nm, and taxol Detection wavelength is 230nm, and gemcitabine Detection wavelength is 269nm, corresponding according to each concentration
UV absorption draw standard curve, as shown in Figure 5.The concentration of drug in sample is calculated according to standard curve.
(3) calculation formula of drugloading rate and grafting rate:
Drugloading rate=sample drug concentration/sample solid content；Grafting rate=drug molar concentration/polymer mole is dense
Degree.
Drug grafting rate and mass percent, as shown in Table 1:
Embodiment 3: the performance test of the double drug-carrying polymer micelles of folate-mediated targeting of the present invention
(1) nanometer containing folic acid FA-PMDNPs preparation:
Preferred mass ratio is 20:1: taking PGA-PTX and PGA-GEM with liquid-transfering gun, it is water-soluble that 50 μ LPLL are added in PGA-FA
Liquid obtains clear transparent solutions.By attracting each other for positive and negative charge, stands overnight and be self-assembled into nanoparticle.
(2) preparation of folic acid nanoparticle PTX-GEM NPs is free of:
Preferred mass ratio is 20:1: taking each 300 μ L of PGA-PTX and PGA-GEM with liquid-transfering gun, it is water-soluble that 400 μ LPLL are added
Liquid obtains the clear transparent solutions of 1mL.By attracting each other for positive and negative charge, stands overnight and be self-assembled into nanoparticle.
(3) stability of FA-PMDNPs and PMDNPs
Nano particle (0.5mg/mL) is kept at room temperature.In subsequent 24,48,72,96h, pass through DLS (Ma Er
Literary dynamic light scattering) monitoring sample.All stability experiments carry out in triplicate.The experimental results showed that obtained nanoparticle
Son is since in four days, change of size is little, so nano particle is relatively stable
(4) vitro drug release
FA-PMDNPs and PMDNPs dispersion solution (PH=7.4,2mL, PTX=GEM=0.18mg/mL) is placed in
It analyses in bag (MWCO=3kDa, Spectrum Laboratories).By bag filter be placed in 28mL acetate buffer (10mM,
PH5.5 contains 0.5% (w) Tween80) or PBS buffer solution (10mM, pH7.4, containing in 0.5% (w) Tween80).At 37 DEG C
It is stirred with 400rpm.In each particular point in time (0,1,2,4,8,24,48 and 72 hour), periodically takes out 1mL sample and use phase
The fresh buffer of same volume is replaced.Then by the sample of taking-up by UPLC (Themo) measure different time points PTX and
GEM concentration.(PTX:0.93min, y=0.2191x-0.2455R2=0.998, GEM:5.94min, y=0.2883x-
0.0252R2=0.998).For each sample, all drug release experiments are carried out in triplicate.As a result Fig. 9 is seen, by the test
It is more to measure the amount of amount that PTX, GEM discharge in acid condition obviously than discharging under weak basic condition, this is because PTX, GEM with
PGA is connected by ester bond.
(5) hemolytic test
New blood from healthy BABL/c is collected into the centrifuge tube containing heparin.By the anticoagulated whole blood of acquisition with
1000g is centrifuged 10 minutes.Then it three times and is resuspended in PBS with PBS Washed Red Blood Cells (RBC), obtains 4% suspension (v/v).
Into each centrifuge tube be added 0.5mL red blood cell suspension, then with the NPs (4,8,12,16,20mg/ of 0.5mL various concentration
ML it) mixes.Distilled water and PBS are incubated with the red blood cell suspension of same volume respectively, as positive and negative control.
After 37 DEG C incubate 1 hour, mixture is centrifuged 10 minutes at 1000g.Existed by using hemoglobin detection kit
The hemoglobin concentration in supernatant is detected at 510nm.The hemolysis rate of RBC: hemolysis rate=(OD is calculated using following formulaSample-
ODPBS)/(ODDistilled water-ODPBS) × 100%.
The hemolysis rate of FA-PMDNPs and PMDNPs is measured below 3% by the test.Various concentration FA-PMDNPs and
The relation curve of the hemolysis rate of PMDNPs, is shown in Figure 10.
Claims (9)
1. a kind of double drug-carrying polymer micelles of folate-mediated targeting, which is characterized in that the polymer micelle is by being grafted respectively
The polyglutamic acid of taxol, gemcitabine and folic acid, then nanoparticle is formed by with polylysine electrostatic self-assembled.
2. the double drug-carrying polymer micelles of folate-mediated targeting according to claim 1, which is characterized in that the Japanese yew
Alcohol, gemcitabine and folic acid are connected on polyglutamic acid by ester bond on molecule.
3. the double drug-carrying polymer micelles of folate-mediated targeting according to claim 1, which is characterized in that the polyglutamic
Amino on acid on remaining carboxyl and polylysine is able to carry out electrostatic self-assembled and forms nanoparticle.
4. the described in any item preparation methods for targeting double drug-carrying polymer micelles of claim 1-3, which is characterized in that including with
Lower step:
1) Polylysine Polymers macromolecular PLL is prepared；
2) polyglutamic acid polymer macromolecular PGA is prepared；
3) taxol PTX and gemcitabine GEM are grafted on PGA molecule respectively, the carboxyl on folic acid FA is passed through into modification hydroxyl
It is grafted to after base on PGA molecule, is respectively formed graft PGA-PTX, PGA-GEM and PGA-FA；
4) by three kinds of grafts obtained by step 3), electrostatic self-assembled is carried out in buffer solution with PLL and forms nanoparticle FA-
PMDNPs targets double drug-carrying polymer micelles to get described.
5. the preparation method according to claim 4 for targeting double drug-carrying polymer micelles, which is characterized in that in step 1),
The method for preparing Polylysine Polymers macromolecular PLL is as follows:
Tetrahydrofuran (THF) makes triphosgene decompose generation phosgene as nucleopilic reagent, and Lys (Z) obtains Lys under the action of phosgene
(Z)-NCA, Lys (Z)-NCA are that initiator causes its ring-opening polymerisation and obtains PLL (Z) with n-hexylamine, and PLL (Z) is at room temperature
It is reacted with HBr, sloughs benzyloxycarbonyl group (Cbz), obtain macromolecule polyalcohol PLL.
6. the preparation method according to claim 4 for targeting double drug-carrying polymer micelles, which is characterized in that in step 2),
The method for preparing polyglutamic acid polymer macromolecular PGA is as follows:
Tetrahydrofuran (THF) makes triphosgene decompose generation phosgene as nucleopilic reagent, and Glu (Z) obtains Glu under the action of phosgene
(Z)-NCA, Glu (Z)-NCA are that initiator causes its ring-opening polymerisation and obtains PBLG with n-hexylamine, and PBLG is sloughed under alkaline condition
Benzyl obtains macromolecule polyalcohol PGA.
7. the preparation method according to claim 4 for targeting double drug-carrying polymer micelles, which is characterized in that in step 3):
The PGA is connect by ester bond with PTX, GEM respectively under DMAP/EDCI catalysis, obtains PGA-PTX and PGA-GEM；
The FA end group activated carboxylic is simultaneously reacted with 3- aminopropanol, obtains compound FA-OH, PGA and the FA- that end group is hydroxyl
OH obtains PGA-FA under DMAP/EDCI catalysis.
8. the preparation method according to claim 4 for targeting double drug-carrying polymer micelles, which is characterized in that received in step 4)
Rice corpuscles FA-PMDNPs's the preparation method is as follows:
PGA-GEM, PGA-PTX and PGA-FA are dissolved in PBS buffer solution respectively, and mixed with PLL stock solution, will be mixed
Object be incubated at 4 DEG C 10-14 hours to get.
9. claim 1-3 is described in any item to target double drug-carrying polymer micelle application in preparations of anti-tumor drugs.
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