CN104906075B - Nano-carrier and preparation method thereof is delivered altogether - Google Patents
Nano-carrier and preparation method thereof is delivered altogether Download PDFInfo
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Abstract
The invention discloses a kind of nano-carrier of delivering medicine and gene and preparation method thereof altogether;It is specifically related to a kind of while the common delivering nano-carrier PLGA pSPE PEG Ligand of load medicine and genomic medicine.This delivery system is by spermine(spermine)With polyethyleneglycol diacrylate(PEGDA)The poly- spermine pSPE of two ends spermine is synthesized by Michael addition reaction, pSPE is with gathering(Y)Copolymer p LGA generates amphipathic PLGA pSPE by the condensation reaction of amino and carboxyl, and PLGA pSPE are connected the polyethylene glycol PEG of target head with an end carboxyl other end(HOOC‑PEG‑Ligand)PLGA pSPE PEG Ligand are generated by the condensation reaction of amino and carboxyl, it is self-assembly of nanoparticle, kernel PLGA can load insoluble drug well as hydrophobic material, pSPE contains substantial amounts of secondary amino group, lotus positive electricity, the gene of negative electrical charge, the stealth material PEG of graft strip target head can be carried well, the transmission of more preferable targeted drug can be realized under PEG and target head collective effect, is realized medicine and gene delivery to same target cell.
Description
Technical field
The present invention relates to a kind of nano-carrier of delivering medicine and gene altogether, and in particular to a kind of load gene simultaneously and controls
The targeting drug delivery system of medicine is treated, delivery system of the present invention can target target cell, and delivery of gene and medicine reach intracellular and made
With site, the purpose of Synergistic treatment is reached, belongs to technical field of medicine.
Background technology
Medicine is contained jointly with gene by high molecular nanometer carrier, target cell is delivered to jointly, is on the one hand slowly released
Medicine is put, another aspect expression treatment gene reaches synergistic effect, achievable drug therapy is cooperateed with gene therapy
Effect, is the malignant disease Perfected process such as treatment tumour so as to increase curative effect.
Small molecule polyamines is the excellent carrier of gene delivery, but its molecular weight is small, compresses the limited in one's ability of nucleic acid, therefore
It is necessary its synthetic polymer.Spermine (spermine, SPE) is the Typical Representative of polyamines small molecule.It is present in all
In eukaryotic, certain effect is played in being metabolized in the cell, is the material of good biocompatibility.And SPE has
Four amidos, are two secondary amine and two primary amine respectively.These amidos can pass through Michael addition reaction with acrylate-based
Poly- spermine (polyspermine, pSPE) of the generation with high buffer capacity.In addition, Poly(D,L-lactide-co-glycolide
(PLGA) it is, that biodegradable one birdss of the same feather flock together compound, with good biocompatibility, the U.S. has been obtained as pharmaceutic adjuvant
FDA ratifies.It is modified by the carboxyl to the polymer ends, the poly- spermine of grafting is prepared into amphipathic nature polyalcohol pSPE-
PLGA, wherein pSPE contain substantial amounts of secondary amino group, and lotus positive electricity can carry the gene of negative electrical charge well, kernel PLGA is as thin
Water-based material can load insoluble drug well, and have the advantages that sustained release, particle size are controllable.Poly- spermine pSPE is in vivo
Also biodegradable, the metabolite is the endogenous material largely existed in vivo.Therefore, pSPE-PLGA has good raw
Thing compatibility, is the ideal carrier that a kind of nontoxic, achievable gene and medicine are transmitted altogether.In pSPE-PLGA graft strip target heads
PEG, can realize more preferable targeted drug transmission under stealth material PEG and target head collective effect.
The content of the invention
Purpose:The present invention provides a kind of nano-carrier of delivering medicine and gene altogether, and the delivery system is to can be used for cooperateing with
Treatment malignant tumour or the medicine and gene of other malignant diseases such as liver fibrosis, hepatic sclerosis etc. deliver nanometer administration system altogether
System, is related to a kind of nanometer for being self-assembly of common load medicine and gene delivery system, self-assembled nanometer delivering system of the invention altogether
Medicine and gene can be delivered in target cell by system simultaneously, and gene and medicine are discharged in target cell cytoplasm.
Technical scheme:In order to solve the above technical problems, the technical solution adopted by the present invention is:
One kind delivers nano-carrier altogether, by the polymer support PLGA-pSPE-PEG-Ligand self assemblies of load medicine
Nanoparticle and therapeutic gene formation compound, form delivering medicine altogether and gene nano carrier;
PLGA-pSPE-PEG-Ligand is a kind of non-viral amphipathic cationic polymer, by amphipathic PLGA-
The Poly-cation of three sections of compositions of pSPE and the polyethylene glycol PEG (HOOC-PEG-Ligand) of end carboxyl one end target head, gathers
Compound carrier PLGA-pSPE-PEG-Ligand chemical structural formula is as follows:
Wherein, PLGA molecular weight is 2-100k;PSPE molecular weight is 2-100k;PEG molecular weight is 1-10k;Ligand is
Target head, selected from Man-6-P (M6P), galactolipin (Gal), vitamin A (VA), folic acid (FA), hyaluronic acid (HA).
Ligand is target head, and polyethylene glycol PEG, pSPE are the poly- spermine of polyspermine, poly lactic-co-glycolic acid copolymerization
Thing (PLGA).
Described common delivering nano-carrier, preparation method comprises the following steps:Using anti-phase solvent method, by medicine with
PLGA-pSPE-PEG-Ligand is codissolved in solvent, is added drop-wise to dropwise in certain volume deionized water, is used molecular cut off
500-14000 dialysis band dialysed at 4 DEG C 2d remove solvent, the polymer support PLGA-pSPE- of medicine must be encapsulated
The nanoparticle of PEG-Ligand self assemblies;Then therapeutic gene solution is added under vortex and isometric is loaded with treatment
In the PLGA-pSPE-PEG-Ligand nano-sized colloidal solutions of medicine, vortex 30s is stored at room temperature 30min, produced.It is applicable to
It is injected intravenously administration, subcutaneous administration, mucosa delivery etc..
The solvent is organic solvent, the one or more in anhydrous DMSO (dimethyl sulfoxide (DMSO)), methanol, ethanol;
And/or, the medicine is adriamycin, taxol, camptothecine, legalon.
The therapeutic gene is DNA or siRNA;Wherein, DNA is p53, HGF, Akt, PDCD;SiRNA be siBcl-2,
siAkt、siTGF-β1、siHSP47、siTIMP-1。
Preferably, described common delivering nano-carrier, it is characterised in that:If described for treating liver fibrosis
DNA is HGF, and siRNA is siTGF- β 1, siHSP47, siTIMP-1;The medicine is legalon;The target head is sweet
Reveal sugar -6- phosphoric acid (M6P), vitamin A (VA).If for treating liver cancer, the DNA is p53, Akt, PDCD, and siRNA is
siBcl-2;The medicine is adriamycin, taxol, camptothecine etc..The target head is galactolipin (Gal), hyaluronic acid
(HA) etc..
The present invention provides a kind of polymer support PLGA-pSPE-PEG-Ligand, and PLGA-pSPE-PEG-Ligand is one
Non-viral amphipathic cationic polymer is planted, by amphipathic PLGA-pSPE and the polyethylene glycol PEG of end carboxyl one end target head
(HOOC-PEG-Ligand) Poly-cation of three sections of compositions, polymer support PLGA-pSPE-PEG-Ligand chemistry
Structural formula is as follows:
Wherein, PLGA molecular weight is 2-100k;PSPE molecular weight is 2-100k;PEG molecular weight is 1-10k;Ligand is
Target head, selected from Man-6-P (M6P), galactolipin (Gal), vitamin A (VA), folic acid (FA), hyaluronic acid (HA).
Described polymer support PLGA-pSPE-PEG-Ligand, by spermine (spermine) and the propylene of polyethylene glycol two
Acid esters (PEGDA) synthesizes the poly- spermine pSPE of two ends spermine by Michael addition reaction, and pSPE is common with poly- (lactic acid-ethanol)
Polymers PLGA passes through the condensation reaction generation PLGA-pSPE, PLGA-pSPE of amino and carboxyl and gathering for end carboxyl one end target head
Ethylene glycol PEG (HOOC-PEG-Ligand) generates PLGA-pSPE-PEG-Ligand by the condensation reaction of amino and carboxyl;It is poly-
Compound carrier PLGA-pSPE-PEG-Ligand synthetic method is as follows:
Wherein, n, x, y, z are positive integer.
Polymer support PLGA-pSPE-PEG-Ligand, its preparation method specifically includes following steps:
1) pSPE synthesis:Using polyethyleneglycol diacrylate (PEGDA) and polyamine molecule spermine as construction unit,
Alternating connects into polymer pSPE, spermine (SPE) is dissolved in the solvent that 10-20 times of w/v is measured, polyethylene glycol two
Acrylate (PEGDA) is dissolved in the solvent that 10-20 times of w/v is measured, and SPE and PEGDA molar ratio are 1:1-
1.5:1, then PEGDA solution is slowly added drop-wise in SPE solution, then 12-48h is stirred at 4-100 DEG C;Products therefrom is molten
Solution is in 4 DEG C of deionized water, and then dialysed 2d with molecular cut off 2000-14000 dialysis band at 4 DEG C;Finally will production
Thing is freezed, and is produced pSPE, is stored in standby at -20 DEG C;
2) PLGA-pSPE is synthesized:Take appropriate PLGA to be dissolved in solvent, add DCC and NHS, at room temperature stirring reaction 0.5-
24h, is centrifuged off precipitation, and supernatant is mixed with the anhydrous DMSO solution containing pSPE, and pSPE and PLGA molar ratio are 1:1-
1.5:1, at room temperature stirring reaction 12-48h dialysed with molecular weight cut-off value 2000-14000 bag filter at 4 DEG C, obtain PLGA-
pSPE;
3) PLGA-pSPE-PEG-Ligand is synthesized:Appropriate HOOC-PEG-Ligand is taken, solvent dissolving adds DCC (two
Carbodicyclo hexylimide) and NHS, at room temperature stirring reaction 0.5-24h, be centrifuged off precipitation, supernatant with containing PLGA-pSPE's
Anhydrous DMSO solution is mixed, at room temperature stirring reaction 12-48h, with molecular weight cut-off value 2000-14000 bag filter at 4 DEG C
Dialysis, obtains PLGA-pSPE-PEG-Ligand.The solvent is organic solvent, one kind in anhydrous DMSO, methanol, ethanol
Or it is several.
A kind of above-mentioned HOOC-PEG-Ligand, its structural formula is as follows:
Wherein, PEG molecular weight is 1k-10k;Connecting key is ester bond or amido link.
Described HOOC-PEG-Ligand, when Ligand is Man-6-P (M6P), i.e. HOOC-PEG-M6P is closed
It is as follows into route
The preparation method of the HOOC-PEG-M6P specifically includes following steps:Appropriate HOOC-PEG-COOH is taken, first with nothing
Water DMSO dissolves, and then adds DCC and NHS, at room temperature stirring reaction 6-24h, is centrifuged off precipitation, supernatant and target containing M6P
The molar ratio of head Ligand anhydrous DMSO solution mixing, HOOC-PEG-COOH and target head is 1:1-1:1.5, stir at room temperature
Reaction 12-48h is mixed, is dialysed with the bag filter of molecular weight cut-off value 2000-5000 at 4 DEG C, obtains PLGA-pSPE-PEG;It is described
Solvent is organic solvent, the one or more in anhydrous DMSO dimethyl sulfoxide (DMSO)s, methanol, ethanol.
Beneficial effect:Prepared by the present invention contains substantial amounts of parahelium based on amphipathic nature polyalcohol pSPE-PLGA, wherein pSPE
Base, lotus positive electricity can carry the gene of negative electrical charge well, and kernel PLGA can load slightly solubility medicine well as hydrophobic material
Thing, and have the advantages that sustained release, particle size are controllable.Poly- spermine biodegradable in vivo, the metabolite is a large amount of in vivo
The endogenous material of presence;Poly(D,L-lactide-co-glycolide (PLGA), is that biodegradable one birdss of the same feather flock together compound, with good
Good biocompatibility, U.S. FDA approval is obtained as pharmaceutic adjuvant the sixth of the twelve Earthly Branches.Therefore, pSPE-PLGA has good bio-compatible
Property, it is the ideal carrier that a kind of nontoxic, achievable gene and medicine are transmitted altogether.In the PEG of pSPE-PLGA graft strip target heads,
More preferable targeted drug transmission can be realized under stealth material PEG and target head collective effect.And with very low cytotoxicity
Significantly transfection efficiency, delivers field with medicine in gene and has broad application prospects altogether
Brief description of the drawings
Fig. 1 is that pSPE, PLGA-pSPE, PLGA-pSPE-PEG-Ligand that the present invention is prepared according to embodiment 2,4,6 gather
The sign of compound:(A) hydrogen spectrogram, δ=1.4-1.6ppm (- CH2-, SPE), δ=3.8ppm (- CH2-, PEGDA);δ=1.5 (-
CH3-, PLGA), δ=4.9 (- CH-, PLGA), δ=5.2 (- CH2-, PLGA);δ=3.5 (- CH2-, PEG) (B) infrared spectrum
Figure, 3500~3300cm-1For secondary amine (- NH-) stretching vibration (pSPE);1750cm-1Left and right is carbonyl (C in carboxyl (- COOH)
=O) stretching vibration (PLGA);3500~3300cm-1For secondary amine (- NH-) stretching vibration, 1750cm-1Left and right be amido link (-
CONH- C=O stretching vibrations (PLGA-pSPE) in);3500~3300cm-1For secondary amine (- NH-) stretching vibration, 1750cm-1It is left
The right side is C=O stretching vibrations, 1270~1010cm in amido link (- CONH-)-1Left and right is C-O stretching vibrations (PLGA- in ehter bond
PSPE-PEG-Ligand) (C) gpc analysis.
Fig. 2 is the sign for the PLGA-pSPE-PEG-Ligand/DNA compounds that the present invention is prepared according to embodiment 9:(A)
Compound gel electrophoresis figures of the PLGA-pSPE-PEG-Ligand/DNA with different quality than preparation, (B) DNA protection and release
Evaluate.
Fig. 3 is that the PLGA-pSPE-PEG-Ligand polymer that the present invention is prepared according to embodiment 10 is different in various concentrations
The cytotoxicity at cell 24h, 72h time point:(A) L02 cells, (B) SMCC-7721 cells, (C) HepG2 cells, (mean ±
SD, n=3).
Fig. 4 is that the present invention is tested according to the in-vitro transfection of embodiment 11.
Embodiment
The present invention is further described below in conjunction with the accompanying drawings.
The present invention is realized by following technical scheme, is comprised the following steps that:
Spermine (spermine) synthesizes two ends essence with polyethyleneglycol diacrylate (PEGDA) by Michael addition reaction
The poly- spermine pSPE of amine, pSPE and poly- (lactic acid-ethanol) copolymer p LGA are generated by the condensation reaction of amino and carboxyl
PLGA-pSPE, PLGA-pSPE and the polyethylene glycol PEG (HOOC-PEG-Ligand) of end carboxyl one end target head by amino with
The condensation reaction generation PLGA-pSPE-PEG-Ligand of carboxyl.
Delivery system is fresh preparation to self-assembled nanometer altogether, prepares scheme specific as follows:
Using anti-phase solvent method, medicine and PLGA-pSPE-PEG-Ligand are codissolved in dimethyl sulfoxide (DMSO) DMSO, by
It is added drop-wise in certain volume deionized water, 2d removings of being dialysed with molecular cut off 500-14000 dialysis band at 4 DEG C
DMSO, must encapsulate the polymer support PLGA-pSPE-PEG-Ligand nanoparticles of medicine.
DNA solution is added to the PLGA-pSPE-PEG-Ligand solution of isometric encapsulating medicine under vortex
In, vortex 30s is stored at room temperature 30min, produced.
Self assembly prepared by above-mentioned preparation method delivers the nano-carrier of medicine and gene altogether, determines its particle diameter and electricity
Gesture.
Application of the nano-carrier of above-mentioned delivering medicine and gene altogether in treatment liver fibrosis, liver cancer.
Embodiment 1
PSPE synthetic schemes is specific as follows:In the absolute methanol that spermine (SPE) 1.2mmol is dissolved to 10mL, polyethylene glycol
In diacrylate (PEGDA) 1mmol dissolving 10mL absolute methanols, then PEGDA solution is slowly added drop-wise in SPE solution,
Then 36h is stirred at 40 DEG C.Products therefrom is dissolved in 4 DEG C of deionized water, then with the dialysis of molecular cut off 5000
Band is dialysed 2d at 4 DEG C.Finally product is freezed, is stored at -20 DEG C, produces pSPE.
Embodiment 2
The Structural Identification and molecular weight characterization of pSPE polymer
PSPE polymer is detected by proton magnetic and infrared identification structure, molecular weight by gel blocking chromatogram.
Embodiment 3
PLGA-pSPE is synthesized:Appropriate PLGA-COOH (molecular weight 10k) 1mmol is taken, is dissolved, added with the anhydrous DMSO of 10mL
DCC and NHS, stirring reaction 0.5-24h, is centrifuged off precipitation at room temperature, and the 10mL of supernatant and the pSPE containing 1.2mmol is anhydrous
DMSO solution is mixed, at room temperature stirring reaction 12h, and dialysed 2d with the bag filter of molecular weight cut-off value 10000 at 4 DEG C, is obtained
PLGA-pSPE。
Embodiment 4
The Structural Identification and molecular weight characterization of PLGA-pSPE polymer
PLGA-pSPE polymer is detected by proton magnetic and infrared identification structure, molecular weight by gel blocking chromatogram.
Embodiment 5
PLGA-pSPE-PEG is synthesized:Appropriate 1mmol HOOC-PEG-Ligand are taken, the anhydrous DMSO dissolvings of 10mL are added
DCC and NHS, stirring reaction 12h, is centrifuged off precipitation at room temperature, and the 10mL of supernatant and the PLGA-pSPE containing 1mmol is anhydrous
DMSO solution is mixed, at room temperature stirring reaction 24h, and dialysed to obtain PLGA- with the bag filter of molecular weight cut-off value 10000 at 4 DEG C
pSPE-PEG。
Embodiment 6
The Structural Identification and molecular weight characterization of PLGA-pSPE-PEG polymer
PLGA-pSPE-PEG polymer is examined by proton magnetic and infrared identification structure, molecular weight by gel blocking chromatogram
Survey.
Embodiment 7
The preparation of PLGA-pSPE-PEG self assembly blank nanoparticles
Using anti-phase solvent method, PLGA-pSPE-PEG is dissolved in 1mL dimethyl sulfoxide (DMSO)s DMSO, 10mL is added drop-wise to dropwise
In deionized water, dialysed 24h removing DMSO with the dialysis band of molecular cut off 1000 at 4 DEG C, obtains PLGA-pSPE-PEG nanometers
Grain.
Embodiment 8
The sign of PLGA-pSPE-PEG blank nanoparticles
The size and surface charge of PLGA-pSPE-PEG nanoparticles are determined using dynamic light scattering.Particle diameter about 70nm, Zeta
Potential about+25mV.
Embodiment 9
PLGA-pSPE-PEG self assembly blank nanoparticles are investigated to DNA binding abilities
DNA solution is added under vortex in isometric PLGA-pSPE-PEG blank nanoparticle solution, vortex 30s,
30min is stored at room temperature, common delivering nano-carrier is produced
DNA is compressed nanoparticle and protective capability is characterized by electrophoresis.PLGA-pSPE-PEG blank nanoparticle and DNA with
After different mass ratioes is combined, loading pigment is added, last volume is 10 μ L;In order to evaluate protection energy of the polymer to DNA
Power, DNaseI is used as digestive enzyme.It is added in 1% Ago-Gel, GelRed dyeing is used as electrolyte, 50V with TAE buffer solutions
Lower race 40min.
Embodiment 10
Cytotoxicity
Influence using MTS method analysis margin nanoparticles to L02, SMCC-7721, HepG2 cell inhibitory effect.Grow shape
The good cell of state is with 1 × 104Cells/well is added in 96 orifice plates, after incubated overnight, is discarded culture medium, is separately added into containing PLGA-
The culture medium of pSPE-PEG blank nanoparticle and PEI 25k, each sample at least 3 multiple holes, with normal cell as a control group, only
Plus the cell of culture medium is blank group.To make each above-mentioned treatment group have comparativity, cultivate after 24h and 72h, added per hole respectively
20 μ L MTS solution, 37 DEG C of shaking 4h, ELIASA 490nm detection absorbances (A) of lucifuge.
Embodiment 11
Transfection
At 37 DEG C, in 5%CO2Tieed up in the DMEM culture mediums containing 10%FBS of incubator (Thermo Scientific)
Hold cell.For transfection experiment, by L02 cells 10 × 104The amount of individual cells/well is seeded in 24 hole flat undersides, is incubated 18h
Afterwards, replaced with blank nanoparticle/pGL3 compounds in serum-free or culture medium containing 10% serum, be then incubated 4h.Use afterwards
Fresh culture medium is replaced, and 24h is incubated at 37 DEG C.Uciferase activity determines the method according to manufacturer.BCA albumen
Test kit is used to extract albumen, its concentration Chemiluminometer (Autolumat, LB953;EG&G
Berthold, Germany) determine.
Embodiment 12
DNA is HGF, and siRNA is siTGF- β 1, siHSP47, siTIMP-1;Medicine is legalon;Target head is sweet
Reveal sugar -6- phosphoric acid (M6P), vitamin A (VA), this materials application is in treating liver fibrosis.
Embodiment 13
DNA is p53, Akt, PDCD, and siRNA is siBcl-2;Medicine is adriamycin, taxol, camptothecine etc.;Target
Head is galactolipin (Gal), hyaluronic acid (HA), and this materials application is in liver cancer treatment.
The above is only the preferred embodiment of the present invention, it should be pointed out that:For the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. one kind delivers nano-carrier altogether, by the polymer support PLGA-pSPE-PEG-Ligand self assemblies of load medicine
Into nanoparticle and therapeutic gene formation compound, form delivering medicine altogether and gene nano carrier;
PLGA-pSPE-PEG-Ligand be a kind of non-viral amphipathic cationic polymer, by amphipathic PLGA-pSPE with
The Poly-cation of three sections of compositions of polyethylene glycol PEG (HOOC-PEG-Ligand) of one end carboxyl one end target head, it is polymer supported
Body PLGA-pSPE-PEG-Ligand chemical structural formula is as follows:
Wherein, PLGA molecular weight is 2-100k;PSPE molecular weight is 2-100k;PEG molecular weight is 1-10k;Ligand is target head,
Selected from Man-6-P (M6P), galactolipin (Gal), vitamin A (VA), folic acid (FA), hyaluronic acid (HA).
2. according to claim 1 deliver nano-carrier altogether, preparation method comprises the following steps:Using anti-phase solvent method,
Medicine and PLGA-pSPE-PEG-Ligand are codissolved in solvent, are added drop-wise to dropwise in certain volume deionized water, is used
Molecular cut off 500-14000 bag filter dialysed at 4 DEG C 2d remove solvent, the polymer support of medicine must be encapsulated
The nanoparticle of PLGA-pSPE-PEG-Ligand self assemblies;Then therapeutic gene solution is added in equal volume under vortex
Be loaded with the PLGA-pSPE-PEG-Ligand nano-sized colloidal solutions of medicine, vortex 30s is stored at room temperature 30min, i.e.,
.
3. according to claim 2 deliver nano-carrier altogether, it is characterised in that:The solvent is organic solvent, selected from nothing
One or more in water DMSO, methanol, ethanol;And/or, the medicine is adriamycin, taxol, camptothecine, fine grinding
Ji guest.
4. according to claim 1 or 2 deliver nano-carrier altogether, it is characterised in that:The therapeutic gene be DNA or
siRNA;Wherein, DNA is p53, HGF, Akt, PDCD;SiRNA be siBcl-2, siAkt, siTGF- β 1, siHSP47,
siTIMP-1。
5. a kind of polymer support PLGA-pSPE-PEG-Ligand, it is characterised in that:PLGA-pSPE-PEG-Ligand is one
Non-viral amphipathic cationic polymer is planted, by amphipathic PLGA-pSPE and the polyethylene glycol PEG of end carboxyl one end target head
(HOOC-PEG-Ligand) Poly-cation of three sections of compositions, polymer support PLGA-pSPE-PEG-Ligand chemistry
Structural formula is as follows:
Wherein, PLGA molecular weight is 2-100k;PSPE molecular weight is 2-100k;PEG molecular weight is 1-10k;Ligand is target head,
Selected from Man-6-P (M6P), galactolipin (Gal), vitamin A (VA), folic acid (FA), hyaluronic acid (HA).
6. polymer support PLGA-pSPE-PEG-Ligand according to claim 5, by spermine (spermine) with gathering
Glycol diacrylate (PEGDA) synthesizes the poly- spermine pSPE of two ends spermine, pSPE and poly- (breast by Michael addition reaction
Acid-glycolic) copolymer p LGA generates PLGA-pSPE, PLGA-pSPE and an end carboxyl by the condensation reaction of amino and carboxyl
The polyethylene glycol PEG (HOOC-PEG-Ligand) of one end target head generates PLGA-pSPE- by the condensation reaction of amino and carboxyl
PEG-Ligand;The synthetic method of the polymer support PLGA-pSPE-PEG-Ligand is as follows:
Wherein, n, x, y, z are positive integer.
7. polymer support PLGA-pSPE-PEG-Ligand according to claim 5, its preparation method specifically include with
Lower step:
1) pSPE synthesis:Using polyethyleneglycol diacrylate (PEGDA) and polyamine molecule spermine as construction unit, alternately
Polymer pSPE is connected into, spermine (SPE) is dissolved in the solvent that 10-20 times of w/v is measured, the propylene of polyethylene glycol two
Acid esters (PEGDA) is dissolved in the solvent that 10-20 times of w/v is measured, and SPE and PEGDA molar ratio are 1:1-1.5:1,
Then PEGDA solution is slowly added drop-wise in SPE solution, then stirs 12-48h at 4-100 DEG C;Products therefrom is dissolved in 4
DEG C deionized water in, then dialysed 2d with molecular cut off 2000-14000 bag filter at 4 DEG C;Finally product is frozen
It is dry, pSPE is produced, is stored in standby at -20 DEG C;
2) PLGA-pSPE is synthesized:Take appropriate PLGA to be dissolved in solvent, add DCC and NHS, at room temperature stirring reaction 0.5-24h, from
The heart removes precipitation, and supernatant is mixed with the anhydrous DMSO solution containing pSPE, and pSPE and PLGA molar ratio are 1:1-1.5:1,
Stirring reaction 12-48h is dialysed with molecular weight cut-off value 2000-14000 bag filter at 4 DEG C at room temperature, obtains PLGA-pSPE;
3) PLGA-pSPE-PEG-Ligand is synthesized:Appropriate HOOC-PEG-Ligand is taken, solvent dissolving adds DCC (two hexamethylenes
Base carbodiimide) and NHS, at room temperature stirring reaction 0.5-24h, be centrifuged off precipitation, supernatant with containing the anhydrous of PLGA-pSPE
DMSO solution is mixed, stirring reaction 12-48h at room temperature, with molecular weight cut-off value 2000-14000 bag filter at 4 DEG C thoroughly
Analysis, obtains PLGA-pSPE-PEG-Ligand.
8. polymer support PLGA-pSPE-PEG-Ligand according to claim 7, it is characterised in that:The solvent is
Organic solvent, the one or more in anhydrous DMSO, methanol, ethanol.
9. a kind of HOOC-PEG-Ligand as described in claim 1 or 5, its structural formula is as follows:
Wherein, PEG molecular weight is 1-10k;Connecting key is ester bond or amido link.
10. HOOC-PEG-Ligand according to claim 9, when Ligand is Man-6-P (M6P), i.e.,
HOOC-PEG-M6P, synthetic route is as follows
Specific preparation method step is as follows:Take appropriate HOOC-PEG-COOH, first with anhydrous DMSO dissolve, then add DCC and
NHS, stirring reaction 6-24h, is centrifuged off precipitation at room temperature, and the anhydrous DMSO solution of supernatant and the Ligand of target head containing M6P is mixed
Close, the molar ratio of HOOC-PEG-COOH and target head is 1:1-1:1.5, stirring reaction 12-48h, is retained with molecular weight at room temperature
The bag filter of value 2000-5000 is dialysed at 4 DEG C, obtains PLGA-pSPE-PEG;The solvent is organic solvent, selected from anhydrous
One or more in DMSO, methanol, ethanol.
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