CN106967167A - Protein/polypeptide polymer conjugate with fluorescent emission property and its preparation method and application - Google Patents
Protein/polypeptide polymer conjugate with fluorescent emission property and its preparation method and application Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention provides a kind of protein/polypeptide polymer conjugate with fluorescent emission property, with structure shown in Formulas I.Described protein/polypeptide polymer conjugate bridges protein/polypeptide and polymer by bifunctionality fluorescence molecule, described bifunctionality fluorescence molecule does not have or only has weak fluorescent emission ability in itself, fluorescence molecule just has strong fluorescent emission only after protein/polypeptide polymeric conjugation, therefore the conjugation procedure of the protein/polypeptide polymer conjugate can be by change in fluorescence in-situ monitoring, and the protein/polypeptide polymer conjugate can be applied to treat the transmission of polypeptide and cancer therapy drug.
Description
Technical field
The present invention relates to organic bridge joint molecular engineering field, more particularly to a kind of protein with fluorescent emission property/
Polypeptide-polymer conjugate and its preparation method and application.
Background technology
The covalent functionalization of polypeptide, protein and antibody and synthetic polymer, medicine and image probe forms important
Can be applied to the bioconjugate of clinical treatment, and protein-polymer conjugate, antibody-drug conjugates together, as allusion quotation
The example of type.Protein-polymer conjugate can trace back to 1970, Davis, and Abuchowski and partner report poly- second
The coupling of glycol (PEG) and bovine serum albumin(BSA).This technology is now known as PEGylation and extends to many polymerizations
In species type, e.g., responsive polymer and amphoteric ion polymer.The polymer of synthesis is connected on protein such as PEGylation
Many advantages can be brought, include the dissolubility and stability of enhancing protein, immunogenicity is reduced, increase blood circulation partly declines
Phase, at least PEGylated protein is by FDA (FDA) certification at present.On the other hand, antibody drug
The combination of the monoclonal antibody of conjugate (ADCs) and the selectively targeted diseased region of energy can kill cancer cell.Two kinds of FDA certifications
ADCs, brentuximabvedotin (trade name, Adcetris) and trastuzumab (trade name, Kadcyla) are at present may be used
Commercially available, about 40 kinds of the ADCs clinically used at present.
Protein-polymer conjugate is by growing side chain (graft from), grafting side chain (graft to) and big list
It is prepared by the method for body copolymerized grafting (graft through).The preparation of antibody-drug conjugates and protein-polymer is all
Dependent on the suitable efficient coupling reaction of selection and connection primitive.For protein/antibody coupling matter, selection does not influence
Protein/pharmaceutical activity and antibody function are best.Typically, protein/antibody organizational project native protein can be passed through
The modification (oxidation of e.g., the reduction of disulfide bond, the modification of one of carbon tip, or polysaccharide) of matter specific position, and directly using specific
The specific reaction of amino acid is realized.Functional polymer/medicine of synthesis is covalently connected on protein/antibody and mainly utilized
Orthogonal click reactions, such as Staudinger reactions, nitrine-alkynes cycloaddition (CuAAC) of copper catalysis, it is folded that tension force promotes
The cycloaddition (SPAAC) of nitrogen-cycloalkyne, D-A additions, Michael's addition, and oxime/hydrazone is formed by aldehyde and ketone.New design principle
Introducing, e.g., modularized design, gentle synthesis mode, the ability of optics spike and multifunctional unit further promotes this
The development in field.
Even it is worth noting that, the protein-polymer conjugate of optimal design, protein function and activity is bright
Aobvious decrease is inevitable.A kind of solution is to prepare the bioconjugate that can be broken, and is increased over time in vivo
Discharge natural protein.During changing in the cell, ADCs should be able to effectively discharge its active drug loading, to open up
Reveal cytotoxicity.However, monitoring finds the coupling of protein-polymer conjugate and antibody-drug conjugates and then released
The degree for putting protein/medicine depends on traditional ex situ technology, e.g., and SDS-polyacrylamide acid amides coagulates
Gel electrophoresis (SDS-PAGE), mass spectrum (MS), high performance liquid chromatography (HPLC), and volume removing chromatogram (SEC).Which prevent to poly-
The real-time monitoring of compound/drug conjugates in vitro with the release process on cellular level.
The content of the invention
In view of this, the technical problem to be solved in the present invention be to provide a kind of protein with fluorescent emission property/
Polypeptide-polymer conjugate and its preparation method and application, can by the method in-situ monitoring conjugation procedure of fluorescence monitoring, and
Transmission applied to treatment polypeptide and cancer therapy drug.
The invention provides a kind of protein/polypeptide-polymer conjugate with fluorescent emission property, with Formulas I institute
Show structure：
Wherein, POI is protein or polypeptide；Polymer is polymer.
The invention provides a kind of protein-polymer conjugates with fluorescent emission property, with knot shown in Formulas I-a
Structure：
Wherein, the POI is bovine serum albumin or salmon calcitonin, and the Polymer is polyethylene glycol.
The invention provides the preparation method of above-mentioned protein-polymer conjugates, comprise the following steps：
Bovine serum albumin containing sulfydryl, the polyethylene glycol of nitrine end group and compound B carry out Michael reactions and clicked on anti-
Should, prepare the protein-polymer conjugates shown in Formulas I-a；
Wherein, POI is bovine serum albumin, and n is 23~445；
Or comprise the following steps：
Salmon calcitonin, the polyethylene glycol of nitrine end group and compound B containing sulfydryl carry out Michael reactions and clicked on anti-
Should, prepare the protein-polymer conjugates shown in Formulas I-a；
Wherein, POI is salmon calcitonin；N is 23~445.
The invention provides a kind of polypeptide-polymer conjugate with fluorescent emission property, with knot shown in Formulas I-b
Structure：
Wherein, the POI is that polypeptide can be cut off in matrix metalloproteinase, and the Polymer is PTMC.
The invention provides the preparation method of aforementioned polypeptides-polymer conjugate, comprise the following steps：
The PTMC of the nitrine end group shown in the cut-off polypeptide of matrix metalloproteinase, formula D shown in formula C
Michael reactions and click-reaction are carried out with compound B, the polypeptide-polymer conjugate shown in Formulas I-b is prepared；
Wherein, m is 10~55.
It is preferred that, pass through the coupling efficiency of fluorescent emission intensity in-situ monitoring conjugate.
The polypeptide-polymer prepared the invention provides aforementioned polypeptides-polymer conjugate or above-mentioned preparation method is conjugated
The polymer vesicle of thing composition.
It is preferred that, the polymer vesicle particle diameter is 60~150nm.
It is preferred that, the polymer vesicle has matrix metalloproteinase enzyme response characteristic.
Monitored in real time the invention provides application of the above-mentioned polymer vesicle as pharmaceutical carrier, or as fluorescence indicator
The application of insoluble drug release.
Compared with prior art, sew the invention provides a kind of protein/polypeptide-polymer with fluorescent emission property
Compound, with structure shown in Formulas I.Described protein/polypeptide-polymer conjugate bridges albumen by bifunctionality fluorescence molecule
Matter/polypeptide and polymer, described bifunctionality fluorescence molecule do not have or only have weak fluorescent emission ability in itself, only
Fluorescence molecule just has strong fluorescent emission, therefore the protein/polypeptide-polymerization after protein/polypeptide-polymeric conjugation
The conjugation procedure of thing conjugate can be by change in fluorescence in-situ monitoring, and the protein/polypeptide-polymer conjugate can
Transmission applied to treatment polypeptide and cancer therapy drug.
Brief description of the drawings
Fig. 1 is the conjugation procedure change in fluorescence conditional curve that embodiment 2 prepares bovine serum albumin-polyethylene glycol conjugate
Figure；
Fig. 2 is that embodiment 2 prepares relation between the change in fluorescence and coupling efficiency of bovine serum albumin-polyethylene glycol conjugate
Figure；
Fig. 3 is ten sodium disulfonates-polyacrylamide gel that embodiment 2 prepares bovine serum albumin-polyethylene glycol conjugate
Electrophoretogram；
Fig. 4 is the conjugation procedure change in fluorescence conditional curve that embodiment 3 prepares salmon calcitonin-polyethylene glycol conjugate
Figure；
Fig. 5 is the volume removing chromatogram figure that embodiment 3 prepares salmon calcitonin-polyethylene glycol conjugate；
Fig. 6 is ten sodium disulfonates-polyacrylamide gel that embodiment 3 prepares salmon calcitonin-polyethylene glycol conjugate
Electrophoretogram；
Fig. 7 is the transmission electron microscope picture that capsule prepared by embodiment 6 ducks in drink；
Fig. 8 is the medicine controlled releasing curve map of embodiment 7.
Embodiment
The invention provides a kind of protein/polypeptide-polymer conjugate with fluorescent emission property, with Formulas I institute
Show structure：
Wherein, POI is protein or polypeptide；Polymer is polymer.
Protein/polypeptide-the polymer conjugate bridges protein/polypeptide with polymerizeing by bifunctionality fluorescence molecule
Thing, described bifunctionality fluorescence molecule does not have or only has weak fluorescent emission ability in itself, only when protein/many
Fluorescence molecule is just with strong fluorescent emission after peptide-polymer is conjugated, therefore the protein/polypeptide-polymer conjugate
Conjugation procedure can be by change in fluorescence in-situ monitoring, and the protein/polypeptide-polymer conjugate can be applied to treatment
The transmission of polypeptide and cancer therapy drug.
In some embodiments of the invention, the POI is bovine serum albumin, and the Polymer is polyethylene glycol,
Its structure is as shown in Formulas I-a：
Wherein, n is 23~445.
Above-mentioned protein-polymer conjugates are preferably prepared in accordance with the following methods：
Bovine serum albumin containing sulfydryl, the polyethylene glycol of nitrine end group and compound B carry out Michael reactions and clicked on anti-
Should, prepare the protein-polymer conjugates shown in Formulas I-a；
The present invention is to the raw material proportioning of above-mentioned reaction, reaction condition and is not particularly limited, can be conventional for this area
Michael reacts the proportioning and condition with click-reaction.
It is currently preferred, pass through the conjugated effect of the above-mentioned protein-polymer conjugates of fluorescent emission intensity in-situ monitoring
Rate.
In the other specific embodiment of the present invention, the POI is salmon calcitonin, and the Polymer is poly- second
Glycol, its structure is as shown in Formulas I-a：
Wherein, n is 23~445.
Above-mentioned protein-polymer conjugates are preferably prepared in accordance with the following methods：
Salmon calcitonin, the polyethylene glycol of nitrine end group and compound B containing sulfydryl carry out Michael reactions and clicked on anti-
Should, prepare the protein-polymer conjugates shown in Formulas I-a.
The present invention is to the raw material proportioning of above-mentioned reaction, reaction condition and is not particularly limited, can be conventional for this area
Michael reacts the proportioning and condition with click-reaction.
It is currently preferred, pass through the conjugated effect of the above-mentioned protein-polymer conjugates of fluorescent emission intensity in-situ monitoring
Rate.
In the other specific embodiment of the present invention, the POI is that polypeptide, its sequence can be cut off in matrix metalloproteinase
β APVGLIG β AC-SH are classified as, wherein, SH is sulfydryl (sulfydryl is located at carbon teminal cysteine residues), and aforementioned polypeptides are strong purchased from Shanghai
Shine biotechnology company, and the Polymer is PTMC.Its structure is as shown in Formulas I-b：
Wherein, m is 10~55.
Its preparation method is preferably：
The PTMC of the nitrine end group shown in the cut-off polypeptide of matrix metalloproteinase, formula D shown in formula C
Michael reactions and click-reaction are carried out with compound B, the polypeptide-polymer conjugate shown in Formulas I-b is prepared；
The present invention is to the raw material proportioning of above-mentioned reaction, reaction condition and is not particularly limited, can be conventional for this area
Michael reacts the proportioning and condition with click-reaction.
It is currently preferred, pass through the conjugated effect of the above-mentioned protein-polymer conjugates of fluorescent emission intensity in-situ monitoring
Rate.
The invention also discloses a kind of vesica, by aforementioned polypeptides-polymer conjugate or above-mentioned preparation method prepare it is many
Peptide-polymer conjugates are constituted.
The present invention is to the preparation method of the vesica and is not particularly limited, and can be prepared to be well known to those skilled in the art
Method.It is currently preferred, aforementioned polypeptides-polymer conjugate is dissolved in DMSO, plus deionized water, then use deionization
Water is dialysed.
The above-mentioned polymer vesicle that the present invention is provided is uniformly dispersed, and particle diameter distribution is uniform, and its particle diameter distribution is 60~
150nm。
Above-mentioned polymer vesicle has matrix metalloproteinase enzyme response characteristic, therefore can be used as pharmaceutical carrier application, or conduct
Fluorescence indicator monitors insoluble drug release in real time.
In order to further illustrate the present invention, the egg with fluorescent emission property provided with reference to embodiment the present invention
White matter/polypeptide-polymer conjugate and its preparation method and application is described in detail.
Difunctionality fluorescence molecule B synthetic route is as follows：
Embodiment 1c1 synthesis
4- bromosalicylaldehydes (1；3.11g,15.4mmol),Pd(PPh3)2Cl2(0.22g,0.31mmol),PPh3(0.061g,
0.23mmol), anhydrous tetrahydro furan (50mL) is added in the reaction flask containing magnetic stir bar, is heaved with dry nitrogen
Bubble is to reaction system degassing 30min, the drying Et then newly steamed3N (3.05g, 30.0mmol) and trimethyl acetenyl silicon
(1.67g, 17.0mmol) is added under nitrogen atmosphere, and solution becomes orange.Stir co-catalyst CuI after 20min
(0.088g, 0.46mmol) is added to reaction system under nitrogen atmosphere, and solution becomes burgundy.It is stirred overnight at room temperature, so
Solvent is removed afterwards and obtains dark brown solid, is dissolved into pentane and is filtrated to get yellow solution.Rotation removes all solvents, most
Afterwards, yellow crystals 2 (2.96g, yield are recrystallized to give in n-hexane twice:88.2%,>95%purity by HPLC).
1H NMR(CDCl3,δ,ppm,TMS):11.0 (s, 1H, benzene-OH),9.87(s,1H,-CH), O 7.48 (d, J=
8.4Hz, 1H, fragrant hydrogen), 7.07 (m, 2H, fragrant hydrogen), 0.26 (s, 9H ,-Si (CH 3)3)。
2 (2.85g, 13.1mmol) are dissolved in dry THF (40mL), then add 20mL contain KOH (0.74g,
MeOH solution 13.2mmol), reaction system is stirred at room temperature overnight, and then rotation hair removes all solvents, and remnant is again
It is dispersed in water, adds 1.0mL acetic acid and extracted with 3x200mL chloroforms.Merge organic phase and dried with anhydrous magnesium sulfate, filtered
After remove all solvents and obtain recrystallizing in brown solid, n-hexane and obtain yellow solid 3 (1.02g, yield twice:53.2%,
>95%purity by HPLC).
1H NMR(CDCl3,δ,ppm,TMS):11.0 (s, 1H, benzene-OH),9.89(s,1H,-CH), O 7.52 (d, J=
8.4Hz, 1H, fragrant hydrogen), 7.12 (m, 2H, fragrant hydrogen), 3.29 (s, 1H ,-C ≡ CH)。
By benzene sulfonyl chloride (2.51g, 14.3mmol), Et3N (2.15g, 21.3mmol) and N- acetoglycocolls (0.88g,
7.5mmol) it is dissolved in THF and is stirred at room temperature overnight.Remove after insoluble salt, compound 3 (1.02g, 7.0mmol) is added to
10h is stirred in reaction system and at 80 DEG C, 0 DEG C is then cooled to, obtains precipitation 4 (1.09g, yield:68.5%,>95%HPLC
Purity).
1H NMR(CDCl3,δ,ppm,TMS):9.80(s,1H,-CO-NH-), 8.57 (s, 1H, fragrant hydrogen), 7.67 (d, J
=8.4Hz, 1H, fragrant hydrogen), 7.35 (m, 2H, fragrant hydrogen), 4.40 (s, 1H ,-C ≡ CH),2.14(s,3H,-COCH 3)。
Compound 4 (1.09g, 4.8mmol), DMAP (DMAP) (0.11g, 0.96mmol), (Boc)2O
(2.11g, 9.8mmol) is dissolved in 40mL THF, and reaction system stirs 4h at 70 DEG C, is subsequently cooled to room temperature, adds MeOH
(10mL) and NH2NH2·H2O (0.96g, 19.2mmol), reaction system is stirred for 4h.Add CH2Cl2, then reactant mixture
Washed with 1MHCl solution, collect organic phase and use anhydrous MgSO4Dry.It is removed by filtration insoluble MgSO4, all solvents all use
Rotary evaporation is removed, and remnant is dissolved into CH2Cl2NaHCO is used after (40mL) and TFA (10mL) in the mixed solvent, stirring 2h3
Solution is washed, organic phase MgSO4Dry, filter out MgSO4Afterwards, rotary evaporation removes solvent and obtains target product 5 (0.76g), stands
Carve for synthesizing C1, it is specific as follows：
Compound 5 (0.76g, 4.1mmol) and maleic anhydride (2.01g, 20.5mmol) are dissolved in 100mL acetone, are returned
Night is flowed through, 0 DEG C is cooled to and obtains yellow solid precipitate.Above-mentioned yellow solid precipitate (1.03g) and a hydration p-methyl benzenesulfonic acid
(154mg) is dissolved in backflow in 30mL methanol and stayed overnight, and is cooled to 0 DEG C and obtains crude product yellow mercury oxide, with EtOAc/DCM (v/v=
1/2) it is further purified as eluant, eluent by column chromatography, obtains yellow solid C1 (480mg, yield:39.4%,>95%HPLC
Purity).
1H NMR(d6-DMSO,δ,ppm,TMS):10.28(s,1H,-CONH-), 8.65 (s, 1H, fragrant hydrogen), 7.73 (d,
J=8.1Hz, 1H, fragrant hydrogen), 7.49 (s, 1H, fragrant hydrogen), 7.38 (s, 1H, fragrant hydrogen), 6.74 (d, J=9.3Hz, 1H ,-
NHCOCH=CH-), 6.51 (d, J=8.7Hz, 1H ,-CH=CH-COO-),4.43(s,1H,-C≡CH),3.66(s,3H,-
COO-CH 3)。
13C NMR(CDCl3,δ,ppm TMS):167.5,164.2,157.6,149.9,131.9,129.7,128.8,
128.6,125.3,124.0,123.1,120.7,119.2,83.8,83.0,52.1。
RP-HPLC is analyzed:4.4min (mobile phases:MeOH/H2O v/v 4/1)。
ESI-MS:m/z calc.for C16H12NO5:298.06[M+H]+；found:298.0705.
The BSA and PEG of the difunctionality fluorescence molecule c1 of embodiment 2 mediations227-N3Fluorophore conjugate
Being dissolved in phosphate buffer (PBS), (70mL, pH 6.5,0.1M, contain bovine serum albumin BSA (498mg, 7.5 μm of ol)
1mM EDTA) in, while three (2- chloroethyls) phosphate ester salt acidulants (TCEPHCl) (21.5mg, 75 μm of ol) are dissolved in
In PBS (2.5mL), then it is added dropwise in above-mentioned BSA solution, after 4h, solution is dialysed with deionized water, and (2.0kDa is cut 24h
Stay molecular weight), then freeze to obtain BSAred。
BSA or BSAred(4mg, 0.06 μm of ol) is dissolved in PBS (0.9mL, pH 7.0,50mM), and C1 (0.6 μm of ol, it is molten
Solution is in 0.1mL DMSO), PEG227-N3(6mg, 0.6 μm of ol) and CuSO4/ Na- ascorbates (1/5 mol ratio) are added to molten
In liquid, 25 DEG C of stirring different times, by the change detection of in-situ observation fluorescent emission intensity about 420nm at it is conjugated enter
Journey.Reach after given time, 80 μ L sample solution is removed, then with 2mL PBS dilutions, quick addition copper absorption tree
Fat (Marine Chemistry company of the U.S., 100mg), is removed after copper ion, concussion 5min, before further SDS-PAGE tests, on
Clear liquid is filtered out by 0.22 μm of sterile syringe filter.
In course of reaction, its change in fluorescence figure is as shown in figure 1, relation such as Fig. 2 between its change in fluorescence and coupling efficiency
It is shown.
Gel electrophoresis is tested：SDS-PAGE experiments are operated on gel-electrophoretic apparatus (Bio-Rad), conjugated containing BSA-PEG
The solution (80 μ L) of thing is mixed with 20 μ L SDS-PAGE sample loading buffers, and 15.0wt% polyacrylamides are used according to standard scheme
Amine gel, gel electrophoresis band (is dyed bright with coomassie in ultraviolet light (365nm) or the irradiation of the white light of UVP EC3 imaging systems
It is blue) it can observe directly.
Its gel electrophoresis figure is as shown in Figure 3.
The salmon calcitonin (sCT) and PEG of the difunctionality fluorescence molecule c1 of embodiment 3 mediations227-N3Fluorophore conjugate
TCEPHCl (14.2mg, 50 μm of ol) is dissolved in PBS (2mL, pH 7.0,50mM), takes the 40 above-mentioned solution of μ L
(containing 1 μm of olTCEPHCl) is added to containing salmon calcitonin (sCT, 1.7mg, 0.5 μm of ol PBS (9mL, pH 7.0,0.05M))
Bottle in, be stirred at room temperature, RP-HPLC analysis show Cys1-Cys7Disulfide bond is in~30min quantification reduction.
200 μ LC1 DMSO solution (containing 0.2 μm of olC1), PEG227-N3(10.0mg, 1.0 μm of ol) and CuSO4/ Na- is anti-bad
Hematic acid salt (1/5 mol ratio) is added in the sCT of reduction (0.1 μm of ol is dissolved in 1.8mL PBS cushioning liquid), is stirred at 25 DEG C
The different times are mixed, are detected by the change of in-situ observation fluorescent emission intensity at~420nm after conjugated process, 4h, quickly
Add copper absorption resin (Marine Chemistry company of the U.S., 100mg) to remove after copper ion, concussion 5min, supernatant passes through
0.22 μm of sterile syringe filter is filtered out.80 μ L solution are tested for SDS-PAGE, and remaining solution is before RP-HPLC experiments are done
With deionized water dialysis 24h.
In course of reaction, its change in fluorescence figure is as shown in Figure 4.
The protein-polymer conjugates molecular weight prepared, its volume removing chromatogram are determined using size exclusion chromatography
Figure is as shown in Figure 5.
Gel electrophoresis is tested：SDS-PAGE experiments are operated on gel-electrophoretic apparatus (Bio-Rad), conjugated containing sCT-PEG
The solution (80 μ L) of thing is mixed with 20 μ L SDS-PAGE sample loading buffers, and 15.0wt% polypropylene has been used according to standard scheme
Acrylamide gel, gel electrophoresis band is in ultraviolet light (365nm) or the white light of UVP EC3 imaging systems irradiation (dyeing use
CoomassieBrilliant Blue) it can observe directly.
Its gel electrophoresis figure is as shown in Figure 6.
Embodiment 4 synthesizes the PTMC (PTMC-N of azide functionalization3)
Trimethylene carbonate (TMC) monomer (500mg, 4.9mmol) of recrystallization is dissolved in dry CH2Cl2In (1mL),
Then 3- azido -1- propyl alcohol (24mg, 0.24mmol) and 1,3- dicyclohexylurea (DCU)s (DCU, 8mg, 0.05mmol) are added dropwise dropwise
In dry CH2Cl2In (1mL), reactant mixture stirs 12h under room temperature under nitrogen atmosphere, reaction, tetrahydrofuran is quenched with acetic acid
Dilution, is then precipitated three times with excessive cold methanol, puts vacuum drying chamber into and be dried to obtain white powder PTMC-N3(160mg,
Yield 30.5%), the PTMC actual degree of polymerization by1H NMR are calculated and obtained, and are 14, therefore be abbreviated as PTMC14-N3。
The PVGLIG polypeptides and PTMC of the difunctionality fluorescence molecule C1 of embodiment 5 mediations14-N3Fluorophore conjugate
PTMC14-N3(7.5mg, 5.0 μm of ol) and C1 (1.5mg, 5.1 μm of ol) are dissolved in DMSO (1.8mL), PVGLIG
(8.1mg, 10.1 μm of ol) and CuSO4/ ascorbic acid (1/5 mol ratio), which is dissolved in after 0.2mL deionized waters, to be added, and reaction system exists
25 DEG C of stirrings, conjugation procedure is detected by fluorescent in situ.After co-cultivation~3h, the quick addition copper absorption resin (U.S.
Marine Chemistry company, 100mg) remove after copper ion, concussion 5min, supernatant is filtered out by 0.22 μm of sterile syringe filter,
Filtrate is collected by centrifugation vacuum drying chamber after precipitation and is dried overnight with excessive cold acetonitrile precipitation.
Embodiment 6 prepares PVGLIG-C1-PTMC14Polymer vesicle
PVGLIG-C1-PTMC14(2mg) is dissolved in 1mL DMSO, is added to the small of 15mL containing magnetic stirring bar
In bottle, stir add 9mL deionized waters (~500rpm) under 3h, stirring condition in~20s at room temperature, continue to use after stirring 5h
Deionized water dialysis (molecular cut off 3.5kDa) 24h.
The transmission electron microscope picture that the capsule of preparation ducks in drink is as shown in Figure 7.
Embodiment 7PVGLIG-C1-PTMC14Polymer vesicle embeds doxorubicin hydrochloride
PVGLIG-C1-PTMC14(2mg) is dissolved in 1mL DMSO, is added to the small of 15mL containing magnetic stirring bar
In bottle, the deionized water (5g/L, 2mL) containing DOXHCl is first added under stirring condition, the remaining same speed of 7mL deionized waters adds
Enter, continue to stir (molecular cut off the is 3.5kDa) 24h that dialysed with deionized water after 5h.DOX loading content~8.0wt%.
Its medicine controlled releasing application curves figure is as shown in Figure 8.
From above-described embodiment, above-mentioned protein/polypeptide-polymer conjugate prepared by the present invention can be by fluorescence
Emissive porwer in-situ monitoring coupling efficiency, and applied to the transmission for the treatment of polypeptide and cancer therapy drug.
The explanation of above example is only intended to the method and its core concept for helping to understand the present invention.It should be pointed out that pair
, under the premise without departing from the principles of the invention, can also be to present invention progress for those skilled in the art
Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
Claims (10)
1. a kind of protein/polypeptide-polymer conjugate with fluorescent emission property, it is characterised in that with knot shown in Formulas I
Structure：
Wherein, POI is protein or polypeptide；Polymer is polymer.
2. a kind of protein-polymer conjugates with fluorescent emission property, it is characterised in that with structure shown in Formulas I-a：
Wherein, the POI is bovine serum albumin or salmon calcitonin, and the Polymer is polyethylene glycol.
3. the preparation method of the protein-polymer conjugates described in a kind of claim 2, it is characterised in that including following step
Suddenly：
Bovine serum albumin containing sulfydryl, the polyethylene glycol of nitrine end group and compound B carry out Michael reactions and click-reaction,
Prepare the protein-polymer conjugates shown in Formulas I-a；
Wherein, POI is bovine serum albumin, and n is 23~445；
Or comprise the following steps：
Salmon calcitonin, the polyethylene glycol of nitrine end group and compound B containing sulfydryl carry out Michael reactions and click-reaction,
Prepare the protein-polymer conjugates shown in Formulas I-a；
Wherein, POI is salmon calcitonin；N is 23~445.
4. a kind of polypeptide-polymer conjugate with fluorescent emission property, it is characterised in that with structure shown in Formulas I-b：
Wherein, the POI is that polypeptide can be cut off in matrix metalloproteinase, and the Polymer is PTMC.
5. the preparation method of the polypeptide-polymer conjugate described in claim 4, it is characterised in that comprise the following steps：
The PTMC of the nitrine end group shown in the cut-off polypeptide of matrix metalloproteinase, formula D shown in formula C and change
Compound B carries out Michael reactions and click-reaction, prepares the polypeptide-polymer conjugate shown in Formulas I-b；
β APVGLIG β AC-SH formulas C；
Wherein, m is 10~55.
6. the preparation method according to claim 3 or 5, it is characterised in that conjugated by fluorescent emission intensity in-situ monitoring
The coupling efficiency of thing.
7. polypeptide prepared by the preparation method described in the polypeptide-polymer conjugate or claim 4 described in claim 4-poly-
The polymer vesicle of compound conjugate composition.
8. polymer vesicle according to claim 7, it is characterised in that the polymer vesicle particle diameter is 60~150nm.
9. polymer vesicle according to claim 7, it is characterised in that the polymer vesicle has matrix metalloproteinase enzyme sound
Answer characteristic.
10. polymer vesicle described in any one of claim 7~9 is as the application of pharmaceutical carrier, or is used as fluorescence indicator
The application of monitoring insoluble drug release in real time.
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