CN105348506B - The preparation of glutamic acid TPGS block copolymers and its application in targeted drug transmission - Google Patents

The preparation of glutamic acid TPGS block copolymers and its application in targeted drug transmission Download PDF

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CN105348506B
CN105348506B CN201510869800.1A CN201510869800A CN105348506B CN 105348506 B CN105348506 B CN 105348506B CN 201510869800 A CN201510869800 A CN 201510869800A CN 105348506 B CN105348506 B CN 105348506B
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tpgs
glutamic acid
liposome
block copolymers
modifications
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CN105348506A (en
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孙进
何仲贵
李琳
狄兴盛
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Shenyang Pharmaceutical University
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Abstract

The present invention relates to a kind of preparation of glutamic acid TPGS copolymers and the application of the amphipathic liposome for targetting material modification in disease target transmission.Described amphipathic targeting material, using glutamic acid as target head, polyethylene glycol increases the pliability of target head, and hydrophobic alpha tocopherol ester is the holdfast position with phosphatide.The liposome of targeting material modification can be used as the carrier of various antineoplastic targeted deliveries, and the glutamic acid of surface modification and the big neutral amino acid transporter 1 interaction of expression high on blood-brain barrier and tumor cell membrane can be passed through, effectively improve the ability and cellular uptake and antitumor activity across blood-brain barrier of liposome.The liposome stability is good, safe, and targeting is good, can be used to be injected intravenously, and has larger market application foreground.

Description

Glutamic acid-TPGS block copolymers preparation and its targeted drug transmission in Using
Technical field
The invention belongs to the new auxiliary material of pharmaceutical preparation and novel form field, it is related to the system of amphipathic nature polyalcohol glutamic acid-TPGS It is standby, and its application as targeting material in active targeting drug delivery system.
Background technology
The wettability growth of brain tumor and blood-brain barrier have had a strong impact on the clinical therapeutic efficacy of brain tumor.Chemotherapy is brain Knurl treatment conventional means, but most small-molecule drug and all macromolecular drugs across blood-brain barrier poor ability, perhaps Many activity antineoplastic high is just announced to fail due to across blood-brain barrier ability in exploitation early stage.Therefore, on blood-brain barrier Some acceptors and auxotype transporter of height expression turn into research focus, such as：Vitamin transporter, amino acid transporter, grape Sugar transporters.These indispensable auxotype transporters on tumor locus and blood-brain barrier can be developed as brain tumor actively The novel targets of targeted therapy.Nano-carrier is extensive due to can improve the curative effect of cancer therapy drug and reduce drug side-effect For targeted drug delivery system, the nano-carrier of PEGylation can more show some unique superiority, such as：Extension nanometer system The circulation time in vivo of agent, the pliability and targeting for increasing part etc..With nano-carrier as drug delivery machinery, with blood brain screen Expression transporter high is target spot simultaneously for barrier and tumour, is a kind of effectively brain targeted drug delivery system.
In recent years, more and more sight were transferred to the research of the nano particle preparations of tumour transporter target, and they pass through The surface modification of body substrate is transported through to nanometer formulation, substrate when transporter is touched, by transporter to its surface Substrate carries out high-affinity identification, combines, and then caves in, enters born of the same parents.Nanometer formulation can rely on expression high on tumor cell membrane Transporter reaches raising cellular uptake amount, increases the purpose of tumor killing effect.In addition, some auxotype transporters are simultaneously in blood Expression high on brain barrier, targeted nano preparation promotes across the blood brain screen of medicine by the transporter of expression high on blood-brain barrier first Barrier ability, then brain tumor is further targetted by identical target spot.Here, we are intended to exploitation targeting blood-brain barrier and brain The nanometer formulation of the neutral amino acid transporter body 1 (LAT1) of expression high, studies its across blood-brain barrier ability and brain glue in glioma The targeting ability of matter knurl.
The content of the invention
It is an object of the invention to provide one kind there is across blood-brain barrier ability high, tumor-targeting, extension medicine partly to decline Phase, not only micella can be self-assembly of with itself but also the amphipathic nature material-glutamic acid-TPGS of nanometer formulation dressing agent can have been made.
Second purpose of the invention is the preparation method for providing above-mentioned glutamic acid-TPGS block copolymers.
Third object of the present invention is to provide effect of the glutamic acid-TPGS block copolymers in targeted drug transmission.
The present invention is achieved through the following technical solutions above-mentioned purpose：
Using glutamic acid as target head, polyethylene glycol is water-wet side to glutamic acid-TPGS block copolymers, there is provided target head it is flexible Property, hydrophobic alpha-tocopherol esters are hydrophobic side, the holdfast position with other medicines carrier kernel.It is a kind of good stability, target The good active targeting material of tropism.
The structural formula formula of described glutamic acid-TPGS block copolymers is as follows：
Described targeting material, n is 11-110, and the molecular weight of polyethylene glycol is 500-5000, preferably 500-1000.
Its preparation process：By carboxyl and the glutamic acid of amido protecting, such as：N- benzyloxycarbonyl groups-Pidolidone -1- benzyl esters (Z- Glu-OBzl, I), it is dissolved in the organic good solvents such as q. s. methylene chloride, dimethyl sulfoxide (DMSO), in the presence of catalyst, such as：1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides (EDC) and DMAP (DMAP), lucifuge ice bath 1h-2h, then reacts 12h-48h with TPGS (II) under 30 DEG C of N2 protections, and separated purifying obtains the III of faint yellow solid.Ⅲ Compound takes off the blocking group of glutamic acid, then obtain final chemical combination by further isolating and purifying by palladium carbon reduction reaction Thing-glutamic acid-TPGS block copolymers (Glu-TPGS, IV).
The block copolymer is faint yellow solid, is soluble in dichloromethane, N, dinethylformamide, dimethyl sulfoxide (DMSO) etc. Organic solvent.
Described glutamic acid-TPGS block copolymers are the good active targeting materials of a kind of good stability, targeting.
Described glutamic acid-TPGS block copolymers can be used for modification contain taxanes, camptothecin, Anthraquinones resist Any material in tumour medicine or dihydropyridines, NSAIDs or derivatives thereof；Gene class medicine is DNA or SiRNA Liposome, its method of modifying can be prepared using film dispersion method, and use following step：By phosphatide, cholesterol and glutamic acid- TPGS block copolymers (30-40:1-2:2-4, w/w/w) it is dissolved completely in appropriate dichloromethane, at moderate temperatures, rotation Dry film forming, adds 2-5mL hydrated agent aquations, and after Probe Ultrasonic Searching, obtain the liposome with brain targeting.
The invention has the advantages that：Prepare a kind of strong amphipathic nature polyalcohol-glutamic acid of new targeting- TPGS block copolymers, carrier preparation process is gentle, easy to operate.The liposome of prepared glutamic acid-TPGS modifications, prepares letter Just, particle diameter is smaller and homogeneous, and envelop rate is high, and good stability, targeting is good.In vitro cell experiment and internal across blood-brain barrier ability Proving the liposome of glutamic acid-TPGS modifications of the invention has preferable across blood-brain barrier ability and tumor-targeting.
Brief description of the drawings
Fig. 1 is the glutamic acid-TPGS block copolymer structures of the embodiment of the present invention 11HNMR spectrograms
The dynamic optical of the docetaxel liposome (DTX-TGL) that Fig. 2 is modified for the glutamic acid-TPGS of the embodiment of the present invention 2 Scattering determines micella grain-size graph and perspective electron microscope
Fig. 3 be the glutamic acid-TPGS of the embodiment of the present invention 2 modification docetaxel liposome/and TPGS modification it is many west he The DSC figures of match liposome (DTX-TGL/DTX-TL)
A：Docetaxel, B：Docetaxel physical mixture C：The docetaxel liposome of TPGS modifications, D：TPGS-Glu The docetaxel liposome of modification
Fig. 4 for the embodiment of the present invention 2 glutamic acid-TPGS modify docetaxel liposome and TPGS modification it is many west he The plasma stability assay of match liposome (DTX-TGL/DTX-TL)
The docetaxel liposome change of size of DTX-TL TPGS modifications
The docetaxel liposome change of size of DTX-TGL TPGS-Glu modifications
The docetaxel liposome polydispersity index change of-DTX-TL PDI TPGS modifications
The docetaxel liposome polydispersity index change of-DTX-TGL PDI TPGS-Glu modifications
Fig. 5 for the embodiment of the present invention 2 glutamic acid-TPGS modify docetaxel liposome and TPGS modification it is many west he The dilution stability experiment of match liposome (DTX-TGL/DTX-TL)
The docetaxel liposome change of size of DTX-TL TPGS modifications
The docetaxel liposome change of size of DTX-TGL TPGS-Glu modifications
The docetaxel liposome polydispersity index change of-DTX-TL PDI TPGS modifications
The docetaxel liposome polydispersity index change of-DTX-TGL PDI TPGS-Glu modifications
Fig. 6 is the docetaxel liposome of the glutamic acid-TPGS of the embodiment of the present invention 2 modifications and the docetaxel of TPGS modifications The extracorporeal releasing test of liposome (DTX-TGL/DTX-TL)
Release of the docetaxel liposome of the TPGS of DTX-TL 7.4 modifications in pH7.4
Release of the docetaxel liposome of DTX-TGL 7.4TPGS-Glu modifications in pH7.4
Fig. 7 for the embodiment of the present invention 2 glutamic acid-TPGS modify docetaxel liposome and TPGS modification it is many west he Cell toxicant 72h of match liposome (DTX-TGL/DTX-TL) to brain glioblastoma cell C6
The docetaxel liposome of DTX-TL TPGS modifications
The docetaxel liposome of DTXTGL TPGS-Glu modifications
DTX-Sol docetaxel DMSO solution agent
Fig. 8 for the embodiment of the present invention 2 glutamic acid-TPGS modify docetaxel liposome and TPGS modification it is many west he Cell toxicant 96h of match liposome (DTX-TGL/DTX-TL) to brain glioblastoma cell C6
The docetaxel liposome of DTX-TL TPGS modifications
The docetaxel liposome of DTX-TGL TPGS-Glu modifications
OTX-Sol docetaxel DMSO solution agent
Fig. 9 is the docetaxel liposome of the glutamic acid-TPGS modifications of the embodiment of the present invention 2 and the cumarin of TPGS modifications The brain glioblastoma cell C6 intake situations of 6 liposomes (C6-TGL/C6-TL)
The coumarin 6 liposome of C6TL TPGS modifications
The coumarin 6 liposome of C6TGL TPGS-Glu modifications
The coumarin 6 liposome that Figure 10 is modified glutamic acid-TPGS for the glutamic acid-TPGS density of the embodiment of the present invention 2 Brain glioblastoma cell C6 intake situation
Figure 11 for the embodiment of the present invention 2 coumarin 6 liposome modify glutamic acid-TPGS of different aminoacids substrate and The influence of neutral big amino acid transporter 1 (LAT1) intake on glioma C 6 cell film
Figure 12 is across the blood-brain barrier ability of the DIR liposomes of the glutamic acid-TPGS/TPGS of the embodiment of the present invention 2 modifications
DIR:DIR solutions TGL：The DIR liposomes of TPGS-Glu modifications
TL：The DIR liposomes of TPGS modifications.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but therefore will not invent the implementation described in being limited in Among example scope.
Embodiment 1
The preparation of glutamic acid-TPGS block copolymers.
By carboxyl and the glutamic acid of amido protecting, such as：N- benzyloxycarbonyl groups-Pidolidone -1- benzyl esters (Z-Glu-OBzl, I), It is dissolved in q. s. methylene chloride, in 1- (3- dimethylaminopropyls) -3- ethyl-carbodiimide hydrochlorides (EDC) and 4- diformazan ammonia Then yl pyridines (DMAP), lucifuge ice bath 1h reacts 12h with TPGS (II) under 30 DEG C of N2 protections, and separated purifying obtains light The III of yellow solid.III compound takes off the blocking group of glutamic acid by palladium carbon reduction reaction, then pure by further separating Change obtains final compound-glutamic acid-TPGS block copolymers (Glu-TPGS, IV).Reaction equation is as follows：
The molecular weight of polyethylene glycol is 1000 in step, but be not restricted to that this, polyethylene glycol of the invention also can be one The polyethylene glycol for hydroxyl modified is held, but be not limited to that above material.The molecular weight of polyethylene glycol can be 500-5000 In the range of.
Using nuclear magnetic resonance measuring1H-NMR hydrogen is composed to determine the structure of targeting material in embodiment 1, and the solvent of selection is D-DMSO, as a result such as Fig. 1.4.2ppm is that the proton peak on glutamic acid between the H on-CH-, 3.52-3.75ppm is the H in PEG. Below 3.0ppm is the typical proton H peaks in VE-succinate.
Embodiment 2
Film dispersion method prepares liposome and the TPGS modifications of the glutamic acid-TPGS modifications for carrying docetaxel or coumarin 6 Liposome
1mg docetaxels or coumarin 6 are weighed, is dissolved in q. s. methylene chloride, the paddy ammonia for adding 2mg embodiments 1 to prepare Acid-TPGS or TPGS, and 30mg soybean lecithin and the cholesterol of 1mg.Film forming is spin-dried for, the deionized water aquation of 2mL is added 30min, Probe Ultrasonic Searching 300W, 5min, the medicine not wrapped up is removed using mini-column centrifugation.
Will in embodiment 2 prepare liposome by dynamic light scattering and perspective Electronic Speculum determine liposome particle size and Form.Result such as Fig. 2, the particle diameter of liposome is about 80nm, and particle diameter distribution is narrow；Perspective electron microscope shows that drug-loaded liposome is particle diameter Homogeneous is spherical.
Table 1 for the embodiment of the present invention 2 glutamic acid-TPGS modify docetaxel liposome and TPGS modification it is many west he Match liposome (DTX-TGL/DTX-TL) to brain glioblastoma cell C6 72h and 96h IC50Value.
The docetaxel solution of table 1, the docetaxel liposome of TPGS modifications and the docetaxel fat of TPGS-Glu modifications IC of the plastid to C6 glioma cells50Value.
Embodiment 3
The docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome (DTX-TGL/ of TPGS modifications DTX-TL DSC figures)
The docetaxel liposome of glutamic acid-TPGS/TPGS modifications is prepared according to example 2, and is to freeze guarantor with mannitol Shield agent is freezed, and by existences of the dsc analysis DTX in liposome, analysis sample includes：DTX bulk drugs, DTX is former Material medicine and blank liposome physical mixture, lyophilized DTX-TGL and DTX-TL.
Fig. 3 results show that DTX is wrapped in phospholipid bilayer with unformed or molecular state.
Embodiment 4
The docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome of TPGS modifications
(DTX-TGL/DTX-TL) stability test in blood plasma
The docetaxel lipid that the docetaxel liposome and TPGS for preparing glutamic acid-TPGS modifications according to example 2 are modified Body, liposome is placed in the sugared nutrient solutions high of the DMEM containing 10%FBS, and liposome is determined in 0h, 1h by dynamic light scattering, The particle diameter of 2h, 4h, 6h, 8h, 10h, 12h and 24h.
Fig. 4 results show that the docetaxel liposome of glutamic acid-TPGS/TPGS modifications has preferable plasma stability.
Embodiment 5
The docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome (DTX-TGL/ of TPGS modifications DTX-TL dilution stability)
The docetaxel lipid that the docetaxel liposome and TPGS for preparing glutamic acid-TPGS modifications according to example 2 are modified Body, different multiples are diluted by liposome with PBS, and the particle diameter of liposome is determined by dynamic light scattering.
Fig. 5 results show the docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome of TPGS modifications With preferable dilution stability.
Embodiment 6
The docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome (DTX-TGL/ of TPGS modifications DTX-TL) extracorporeal releasing test
It is many that the docetaxel liposome and TPGS for preparing glutamic acid-TPGS modifications using dialysis investigation example 2 are modified The tablets in vitro feature of Xi Tasai liposomes.The polypeptide drug-loaded micelle solution containing 150 μ g is pipetted in bag filter, bag filter two ends press from both sides Tightly, it is respectively placed in the conical flask of the dissolution medium containing 30mL pH 7.4PBS (containing 0.5%Tween 80), in 37 DEG C of constant temperature Vitro release investigation is carried out with 100r/min in oscillator.2mL is sampled in 1,2,4,6,8,10,12,24h and 48h respectively, together When supplement 2mL fresh dissolution mediums, through 0.45 μm of filtering with microporous membrane, take 20 μ L carries out HPLC measure to sample.
Fig. 6 results show the docetaxel liposome of glutamic acid-TPGS modifications and the docetaxel liposome of TPGS modifications Drug release is slow, is conducive to more medicines to reach tumor locus.
Embodiment 7
Cytotoxicity experiment
By the brain glioblastoma cell (C6) in exponential phase with 3.5 × 104The DMEM sugared nutrient solutions high of/hole/0.1mL It is embedded in 96 orifice plates, what the docetaxel liposome and TPGS that the glutamic acid-TPGS for preparing embodiment 2 after 24h is modified were modified Docetaxel liposome is separately added into each hole with various concentrations, adds 100 μ L to contain liposome solutions per hole, and each concentration 3 is put down Row hole, puts incubation in incubator.After culture 72h and 96h, 96 orifice plates are taken out, the 5mg/mL MTT of 20 μ L, incubator are added per hole 96 orifice plate back-offs are sufficiently absorbed through residual liquid by middle incubation 4h, knockout plate in filter paper, and 150 μ L DMSO are added per hole in oscillator Middle vibration 10min, ELIASA determines the absorbance at each hole 492nm.Calculate inhibiting rate：
Inhibiting rate (%)=(1-AMedicine feeding hole/AControl wells) × 100%
Mtt assay determines liposome cytotoxicity result such as Fig. 7 and Fig. 8, and various concentrations drug-loaded liposome acts on C6 cells After strain 72h and 96h, cell inhibitory rate increases and increases with drug concentration and brooding time, and to the inhibitory action of cell by Strengthen in the targeting of glutamic acid.The IC of each preparation that table 1 is calculated50Value also embodies glutamic acid modified liposome Advantage.
Embodiment 8
Cellular uptake is tested
By the C6 cells of LAT1 expression high with 2 × 105The RPMI-1640 and DMEM nutrient solutions of/hole/0.1mL are embedded in 96 holes In plate, coumarin 6 liposome and the coumarin 6 fat of TPGS modifications that the glutamic acid-TPGS for preparing embodiment 2 after 24h is modified Plastid, the concentration for diluting identical coumarin 6 by HBSS buffer solutions is added in each hole, and per the μ L of hole 100, incubator is put in parallel 3 hole Middle incubation 1h and 3h.Supernatant is abandoned, adds the PBS solution juxtaposition of 50 μ l 0.5%TrtionX-100 (NaOH containing 0.2N) to shake per hole 1h is acted in bed.Then, it is 458nm in excitation wavelength, launch wavelength is that 525nm determines intensity of cellular fluorescence, and to every hole Determining the protein quantity is carried out, and calculates intake.In order to screen optimum targeting density, while prepare that there are different paddy ammonia The coumarin 6 liposome of acid-TPGS density modification, cellular uptake investigation is carried out according to above-mentioned.
The result of cellular uptake is shown in Fig. 9, Figure 10, in LAT1 expression knurl strains high, the coumarin 6 of glutamic acid-TPGS modifications The cellular uptake amount of liposome is higher than the coumarin 6 liposome that TPGS is modified.And the increasing of density is modified with targeting material Plus, cellular uptake increases, and maximum is reached in 10% cellular uptake.
Embodiment 9
The influence reality of the coumarin 6 liposome intake of the glutamic acid-TPGS modifications that different aminoacids substrate is mediated to LAT1 Test
By the C6 cells of LAT1 expression high with 2 × 105The RPMI-1640 of/hole/0.1mL and it is embedded in 96 orifice plates, after 24h The coumarin 6 liposome of glutamic acid-TPGS modifications prepared by embodiment 2, after being diluted by HBSS buffer solutions, with different aminoacids After well mixed, add in each hole, per the μ L of hole 100, parallel 3 hole is incubated 3h in putting 37 DEG C.Supernatant is abandoned, 50 μ l are added per hole 1h is acted in the PBS solution juxtaposition shaking table of 0.5%TrtionX-100 (NaOH containing 0.2N).Then, it is in excitation wavelength 458nm, launch wavelength is that 525nm determines intensity of cellular fluorescence, and determining the protein quantity is carried out to every hole, and calculates intake Amount.
Intake influence experimental result is shown in Figure 11, as a result shows as LAT1 high affinity substrates, leucine and phenylalanine There is obvious inhibitory action to the cohesive process of liposome and LAT1, it was demonstrated that targeting lipids physical efficiency is recognized and combines LAT1, promotes Cellular uptake.
Embodiment 10
Across the blood-brain barrier ability of the DIR liposomes of glutamic acid-TPGS modifications and the DIR liposomes of TPGS modifications
DIR liposomes and DIR solutions that example 2 prepares glutamic acid-TPGS/TPGS modifications are investigated using living imaging method Across blood-brain barrier ability.The liposome that KM mouse are loaded with DIR is given with 2mg/kg tail veins, after 8h, by the heart of mouse, liver, Spleen, lung, kidney and brain are taken out, and the distribution situation of liposome is observed by living imaging instrument.
Across blood-brain barrier capacity experimental result is shown in Figure 12, as a result shows that the liposome modified as glutamic acid-TPGS has More preferable across blood-brain barrier ability.

Claims (9)

1. glutamic acid-TPGS block copolymers, it is characterised in that：With polyethylene glycol as water-wet side, alpha-tocopherol esters are hydrophobic side, Glutamic acid is target head, and general structure is as follows：
Wherein, n is 11-110, and the molecular weight of polyethylene glycol is 500-5000.
2. the preparation method of glutamic acid-TPGS block copolymers according to claim 1, it is characterised in that use following steps Prepare：
Carboxyl and the glutamic acid of amido protecting are dissolved in the organic good solvent of q. s. methylene chloride, dimethyl sulfoxide (DMSO), in catalysis In the presence of agent, then lucifuge ice bath 1h-2h reacts 12h-48h with TPGS under 30 DEG C of N2 protections, and separated purifying obtains light Yellow solid；
By this faint yellow solid by palladium carbon reduction reaction, the blocking group of glutamic acid is taken off, then by further isolating and purifying Obtain final compound-glutamic acid-TPGS block copolymers.
3. preparation method according to claim 2, it is characterised in that the glutamic acid of carboxyl and amido protecting is N- benzyloxy carbonyls Base-Pidolidone -1- benzyl esters.
4. preparation method according to claim 2, it is characterised in that described catalyst is 1- (3- dimethylaminos third Base) one or two in -3- ethyl-carbodiimide hydrochlorides or DMAP.
5. application of the glutamic acid-TPGS block copolymers described in claim 1 in drug delivery system.
6. glutamic acid-TPGS the block copolymers described in claim 1 are as pharmaceutical carrier or dressing agent is in blood-brain barrier and swells Application in the delivering of knurl active targeting.
7. glutamic acid-TPGS the block copolymers described in claim 1 are targetting neutral big ammonia as pharmaceutical carrier or dressing agent Application in base acid transporter 1.
8. a kind of drug-carrying nanometer particle, it is characterised in that with the glutamic acid-TPGS block copolymers described in claim 1 be modification Agent, with liposome as drug-reservoir.
9. drug-carrying nanometer particle according to claim 8, it is characterised in that：Medicine in liposome is hydrophobic drug, parent Aqueous medicine or gene class medicine, described hydrophobic drug are taxanes, camptothecin, Anthraquinones antineoplastic or dihydro pyrrole The derivative of any material or the material in pyridine class, NSAIDs；Hydrophilic medicament be adriamycin, HCPT, Cis-platinum class；Gene class medicine is DNA or SiRNA.
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