CN107022048A - A kind of temperature sensitive driver of macromolecular and its preparation method and application - Google Patents
A kind of temperature sensitive driver of macromolecular and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of temperature sensitive driver of macromolecular and its preparation method and application, the temperature sensitive driver of macromolecular includes the targeting module being made up of polypeptide ligand, the drive module being made up of temperature sensitive polymer and the trigger module being made up of photothermal deformation molecule, the trigger module is connected in targeting module, the targeting module is connected with drive module, the driver can be targeted to epicyte protein by polypeptide ligand, effective active targeting mode by molecular recognition is specific to be combined with memebrane protein, activating driver by local heating in cell membrane surface makes its aggregation of collapsing produce drag force, realize the assembly and regulation of different membrane receptor proteins, studied for signal path, immune cell activation and oncotherapy etc. provide new available strategy.
Description
Technical field
The invention belongs to technical field of polymer materials, it is related to temperature sensitive driver of a kind of macromolecular and preparation method thereof and answers
With.
Background technology
Cell-membrane receptor albumen can recognize, compound be generated with reference to single-minded biologically active ligand, so as to activate and start
A series of physical chemical change, and then cause the final biological effect of the material.These biological effects include cell propagation, cell
Differentiation, Apoptosis etc..The method of existing regulation and control membrane receptor protein oligomerization mainly has three kinds：1) matched somebody with somebody using bioactivity
Body；2) light science of heredity；3) magnetic tuning.But the drawbacks of every kind of method has respective.Biologically active ligand can not be realized to acceptor egg
Manipulation on time and space in vain.Light science of heredity transfects the albumen of photaesthesia dependent on transgenic technology in the cell, and process is multiple
It is miscellaneous, and application limitation, it is impossible to realize and the receptor protein of locomitivity difference is regulated and controled.Although magnetic tuning method can be realized to acceptor
Active control of the albumen on time and space, but this method is dependent on complicated instrument processing design.
CN106397577A discloses a kind of stimuli-responsive collagen polypeptide polymer, and the polymer is with glue
Former polypeptide is that the amino on main chain, its side chain passes through the bonded reactive grafting alkyloxy-ethers poplar bundles primitive side chain of dynamic so that
The collagen polypeptide polymer prepared has temperature and pH double-responses, but the material is not to be based on cell membrane table
Temperature and the pH response in face, it is impossible to be directly used in the regulation and control of cellular level.
Therefore, in this area, expect to obtain a kind of temperature sensing material regulated and controled with membrane receptor protein oligomerization.
The content of the invention
In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of temperature sensitive driver of macromolecular and its preparation side
Method and application.
To reach this goal of the invention, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of temperature sensitive driver of macromolecular, and the temperature sensitive driver of macromolecular is included by polypeptide
Targeting module, the drive module being made up of temperature sensitive polymer and the trigger module being made up of photothermal deformation molecule that part is constituted,
The trigger module is connected in targeting module, and the targeting module is connected with drive module.
In the present invention, the temperature sensitive driver of the macromolecular is referred to as macromolecular temperature sensing material.
The present invention temperature sensitive driver undergone phase transition using temperature-responsive polymeric material above and below specified temp, from relax
Exhibition state is transformed into the state of aggregation collapsed, and produces mechanical motive force.It has critically important in physical device structure and biodegradable delivery medicine
Application.
Polypeptide ligand is attached by the present invention by covalent bond with temperature sensitive polymer, and connection has photo-thermal in the molecule
The molecule of conversion character obtains the temperature sensitive actuator material of macromolecular as thermal source, and the driver can pass through polypeptide ligand target
To epicyte protein, activating driver by local heating in cell membrane surface makes its aggregation of collapsing produce drag force, utilizes
The drag force that its phase transformation is produced carries out simple, active to the receptor protein oligomerization of cell membrane surface and accurately manipulated.The temperature
Quick driver system, new material is provided for signal path research, and being that the offer such as immune cell activation and oncotherapy is new has
Effect strategy.
Preferably, the polypeptide ligand is can be with the protein bound arbitrary amino acid sequences polypeptide of cell membrane surface receptors
In any one or at least two combination；
Preferably, the amino acid sequence of the polypeptide ligand be MSRTMS, GYHWYGYTPQNVI, RGD or CNSCWSKD,
Further preferred MSRTMS.
In the present invention, the polypeptide ligand is the polypeptide with specific amino acid sequence, its corresponding specific knowledge
Other albumen, the corresponding identification albumen of such as MSRTMS is HER2 (human epidermal growth factor acceptor -2) albumen,
The corresponding identification albumen of GYHWYGYTPQNVI is HER1 (human epidermal growth factor acceptor -1) albumen, the corresponding identification eggs of RGD
It is integrin alpha v beta 3 in vain, the corresponding identification albumen of CNSCWSKD is DR4/5 (death receptor 4 or 5).
Preferably, the temperature sensitive polymer is poly-N-isopropyl acrylamide, poly- N, N- acrylamides, poly- N- hydroxyls
Methyl-propyl Methacrylamide, poly- N-2,2- dimethyl 1,3- dioxolanemethyls acrylamide, poly- N-2- methoxyl groups 1,3-
Dioxy second cyclohexyl methyl acrylamide, poly- N-2- ethyoxyls 1,3- dioxy second cyclohexyl methyls acrylamide, polymethylacrylic acid oligomerization second
In diol ester, poly N-vinyl isobutyramide, polymethyl vinyl ether, poly-N-vinylcaprolactam or poly- 2- ethyl oxazolines
Any one or at least two composition.
Preferably, the temperature sensitive polymer be NIPA and N-2 hydroxyl N-isopropylacrylamides copolymer,
The copolymer or NIPA and the copolymerization of acrylic acid of NIPA and N- hydroxyethyl acrylamides
In thing any one or at least two combination, preferably NIPA and/or N-2 hydroxyl N-isopropylacrylamides
Copolymer.
Preferably, when the temperature sensitive polymer is copolymer, its synthetic method is activity/controllable free-radical polymerisation, preferably
RAFT polymerize (reversible addion-fragmentation chain transfer polymerization).
Preferably, the chain transfer agents of the RAFT polymerizations are N, N'- dimethyl N, N'- bis- (4- pyridine radicals) thiuram two
Sulfide, 2- (dodecyl trithiocarbonic acid ester group) -2 Methylpropionic acid, double (dodecyl sulfanyl thiocarbonyl) curing
Thing, 2- cyano group -2- propyl group dodecyls trithiocarbonate, the sulphur of 2- cyano group -2- propyl group benzo two, 4- cyano group -4- [(dodecanes
Base sulfanyl thiocarbonyl group) sulfanyl] valeric acid, 4- cyano group -4- (phenyl formyl sulfenyl) valeric acid, cyanogen methyl dodecyl trithio
For carbonic ester, cyanogen methyl (phenyl) dithiocarbamates, the thio first of methyl -2- propionic acid methyl (4- pyridines) amino two
Appointing in acid esters, methyl -2- (dodecyl trithiocarbonate) -2 Methylpropionic acid ester or the sulphur of 2- phenyl -2- propyl group benzo two
Meaning it is a kind of or at least two combinations, further preferred 2- cyano group -2- propyl group dodecyl trithiocarbonates.
Preferably, the temperature sensitive polymer is the polymer with structure shown in formula I：
Wherein x=200-1200 (such as 200,230,250,280,300,350,380,400,440,480,500,530,
550th, 580,600,620,650,680,700,750,780,810,850,880,900,1000,1100 or 1200), y=1-60
(such as 1,3,5,8,10,15,18,20,25,30,35,40,45,50,55 or 60), and x/y=19-280 (such as 19,20,
23rd, 25,28,30,35,40,50,60,80,100,130,150,180,200,230,250 or 280), such as x=1168, y=
60 or x=1127, y=40 or x=1158, y=14 or x=1447, y=9 or x=624, y=34 or x=596, y=18 or x
=642, y=7 or x=698, y=4 or x=258, y=14 or x=252, y=11 or x=251, y=3 or x=280, y=
1, preferably x=642, y=7；
Preferably, the molecular weight of the temperature sensitive polymer be 20-80kD, such as 20kD, 25kD, 30kD, 35kD, 40kD,
45kD, 50kD, 55kD, 60kD, 65kD, 70kD, 75kD or 80kD, preferably 40kD.When the molecular weight of temperature sensitive polymer is less than
During 20kD, the response of the temperature sensitive driver of macromolecular of the present invention (or being the temperature sensitive driver of macromolecular) can be caused to become
Difference, and molecular weight is too small or too big, can cause material to increase the toxicity of cell.
Preferably, the polypeptide ligand as side chain graft on temperature sensitive polymer skeleton.On temperature sensitive polymer skeleton
Multiple polypeptide ligands can be grafted.
Preferably, the grafting is realized by Michael's addition method, i.e., matched somebody with somebody by the double bond in temperature sensitive polymer and polypeptide
Sulfydryl carries out Michael's addition in body, and polypeptide ligand sequence is grafted on the skeleton of temperature sensitive polymer so as to realize.
Preferably, polypeptide ligand connection ratio is 0.5-5%, such as 0.5%, 0.8%, 1%, 1.2%, 1.5%,
1.8%th, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.5%, 4.8% or 5%,
It is preferred that 1%.In the present invention, the polypeptide ligand connection ratio refers to that polypeptide ligand is connected on temperature sensitive polymer skeleton
Amount, particularly：Polypeptide ligand is with constituting the monomer or monomer mixture of temperature sensitive polymer (when polymer is by two or more monomers
Polymerization is when obtaining, and monomer mixture refers to the summation of two or more monomers) molar percentage.
Polymer backbone chain length and polypeptide ligand connection ratio control can be had in appropriate scope in the present invention
Beneficial to the response temperature range and response sensitivity for adjusting the temperature sensitive driver of macromolecular of the present invention.
Preferably, the photothermal deformation molecule is purpurin 18 (purpurin-18), burnt pheophytin, Cy5, Cy6
In Cy7 any one or at least two combination, preferred purpurin 18.
In the present invention, the molecule thermal source that functions as of photothermal deformation molecule promotes it to temperature sensitive polymer offer heat
It is changed into hydrophobic collapsed state from hydrophilic extended position.The advantage of molecule thermal source is can be locally to heat, controllability
Height, it is favourable for biologic applications.
Preferably, the photothermal deformation molecule is connected on polypeptide ligand by amido link.
On the other hand, the present invention provides the preparation method of the temperature sensitive driver of macromolecular as described above, the preparation method
For：By polypeptide solid-state reaction method synthesis polypeptide ligand sequence, the photothermal deformation molecule in connection on peptide sequence, and by stepping
The polypeptide ligand sequence for being connected with photothermal deformation molecule is grafted on by Ke Er additions obtains described on the skeleton of temperature sensitive polymer
The temperature sensitive driver of macromolecular.
In the present invention, the solid-phase synthesis and on peptide sequence connection on photothermal deformation molecule and Michael
Addition etc. can be realized using methods known in the art, and specific narration is no longer done here.
On the other hand, the present invention provides a kind of material of regulation and control membrane receptor protein aggregation, and the material includes as described above
The temperature sensitive driver of macromolecular.
The temperature sensitive driver of macromolecular of the present invention can be specific effectively to pass through the active targeting mode of molecular recognition
Combined with memebrane protein, temperature sensitive polymer skeleton drive membrane protein oligomerization in the case of to temperature, the memebrane protein of oligomerization
Have impact on downstream signaling pathway, and then influence the biological function of cell, thus realize from the immune cell activation of cellular level and
Oncotherapy etc..
On the other hand, the present invention provides a kind of drug delivery materials, and the drug delivery materials include as described above big
The temperature sensitive driver of molecule.
Relative to prior art, the invention has the advantages that：
The temperature sensitive driver of macromolecular of the present invention is constructed using temperature-responsive polymer-polypeptide hybrid material, the driving
Device can be targeted to epicyte protein by polypeptide ligand, effectively by the way that the active targeting mode of molecular recognition is specific and film
Protein binding, activating driver by local heating in cell membrane surface makes its aggregation of collapsing produce drag force, realizes different films
The assembly and regulation of receptor protein, is that signal path research, immune cell activation and oncotherapy etc. provide new available strategy.
Brief description of the drawings
The structural representation for the temperature sensitive driver of macromolecular that Fig. 1 is prepared for the present invention；
Fig. 2 is polypeptide ligand CK (P18) GGMSRTMS for being connected with purpurin 18 prepared by embodiment 1 MALDI-TOF
Spectrogram；
Fig. 3 is the nucleus magnetic hydrogen spectrum figure of the temperature sensitive driver of macromolecular prepared by embodiment 1；
Fig. 4 is polypeptide ligand CK (P18) GYHWYGYTPQNVI's for being connected with purpurin 18 prepared by embodiment 2
MALDI-TOF spectrograms；
Fig. 5 is the nucleus magnetic hydrogen spectrum figure of the temperature sensitive driver of macromolecular prepared by embodiment 2；
Fig. 6 is few in cell membrane surface regulation and control membrane receptor protein using the temperature sensitive driver of macromolecular prepared in embodiment 1
The schematic diagram of dimerization；
Fig. 7 is confocal microscope of the temperature sensitive driver of macromolecular for preparing of embodiment 1 in cell membrane surface
Confocal microscope figure when figure, wherein A figures are no laser irradiation, B figures are that fluorescence co-focusing when having laser irradiation shows
Micro mirror figure, its scale is 10 μm；
Fig. 8 is the temperature sensitive driver of macromolecular for preparing of embodiment 1 in the scanning electron microscope (SEM) photograph of cell membrane surface, wherein A
Figure is scanning electron microscope (SEM) photograph when no laser irradiates, and B figures are scanning electron microscope (SEM) photograph when whetheing there is laser irradiation, and its scale is 2 μm；
Fig. 9 is that the macromolecular that the embodiment 1 observed with the FRET passages of confocal microscope is prepared is temperature sensitive
The situation map for occurring fluorescence resonance transfer that driver is assembled in cell surface；
Figure 10 is the suppression situation curve that the temperature sensitive driver of macromolecular that embodiment 1 is prepared is bred to SKBR3 cells
Figure.
Embodiment
Technical scheme is further illustrated below by embodiment.Those skilled in the art should be bright
, the embodiment be only to aid in understand the present invention, be not construed as to the present invention concrete restriction.
Experimental method in following embodiments, is conventional method unless otherwise specified；Experiment material used, such as without
Specified otherwise, is available from commercially available from routine biochemistry chemical reagent work.
Embodiment 1
The structural representation of the temperature sensitive driver of macromolecular prepared in the present embodiment is as shown in figure 1, it is included by polypeptide
Targeting module, the drive module being made up of temperature sensitive polymer and the trigger module being made up of photothermal deformation molecule that part is constituted,
The trigger module is connected in targeting module, and the targeting module is connected with drive module.Wherein photothermal deformation molecule is purple
Red pigment -18, polypeptide ligand is that CKGGMSRTMS (in order to connect purpurin 18, with the addition of amino acid K；In order to by polypeptide and polymer
It is connected, with the addition of amino acid C.GG is flexible spacer), it is connected with polypeptide ligand CK (P18) GGMSRTMS of purpurin 18 point
Minor structure is as follows：
In the present embodiment, the synthetic method of the temperature sensitive driver of macromolecular is as follows：
1) synthesis of polypeptide ligand：
The Wang resins of 0.35mM load capacity are selected in synthesis, wherein the N-terminal of first amino acid (serine) is protected by Fmoc
Shield, C-terminal is fixed on resin.The Fmoc protections of N-terminal are sloughed with the DMF solution of 20% (v/v) hexahydropyridine, then with indenes three
Ketone method of testing detection deprotection result.Then by the carboxyl of next amino acid 0.4M N-methylmorpholine (NMM) and 10 times
In the DMF solution activation of BTA-N, N, N', N'- the tetramethylurea hexafluorophosphoric acid ester (HBTU) of amino acid, and it is added to
Take off in de-protected resin and react 1 hour.According to said method, remaining all amino acid are all got on by condensation reaction connection,
The connecting peptides for being fixed on resin are formed, wherein lysine uses Fmoc-Lys (Dde)-OH.
2) synthesis of purpurin 18 connecting peptides：
In step 1) complete after the synthesis of all amino acid, the Dde of lysine side-chain is removed with 2% hydrazine hydrate, then
The purpurin 18 molecule activated is repeated the above steps and is coupled at the N-terminal of lysine；Then use and contain 2.5% water and 2.5%
The trifluoroacetic acid solution of tri isopropyl silane removes synthetic polypeptide from resin, while the side chain for removing amino acid is protected
Shield；Trifluoroacetic acid is removed with rotary evaporation, then the crude product of polypeptide is precipitated with absolute ether, wash and dry；Finally
From anti-phase preparative liquid chromatography, by peptide purification.
The condition of purge process is：Mobile phase is the acetonitrile containing 0.1% trifluoroacetic acid and containing 0.1% trifluoroacetic acid
Distilled water；Parameter is gradient elution from the water of 5% acetonitrile/95% to 60% acetonitrile/40% water, flow velocity is 10ml/min, during processing
Between be 30min.The MALDI-TOF collection of illustrative plates of purpurin 18 connecting peptides obtained by the above method as shown in Fig. 2 as seen from the figure,
The molecular weight for being connected with the polypeptide ligand of purpurin 18 is 1604.8.
3) synthesis of temperature sensitive polymer skeleton：
NIPA and N-2 hydroxyls N-isopropylacrylamide are pressed into different mol ratio (99：1) it is added to Shrek
In bottle, 2- cyano group -2- propyl group dodecyl trithiocarbonates and azodiisobutyronitrile are then added, with N, N '-dimethyl first
Acid amides (DMF) dissolves, and after concentration is 1.5g/ml, stirring and dissolving, sealing system is passed through nitrogen 30 minutes, 70 DEG C of reactions 10 of constant temperature
Hour；Reacted solution is added in bag filter, dialysed 3 days, freeze-drying obtains pale yellow powder shape solid.
The above-mentioned polymer of also 14mmol amounts and 0.35g 4,4' methylene bis (2,6 di t butyl phenol) are added
Enter into reaction vessel, and dissolved with 35mL dry DMFs.By 30mmol acrylic acid isopropyl nitrile ethyl ester and 50mg tin dilaurate
Dibutyl tin is added in reaction vessel, second son stirring and dissolving.40 DEG C of constant temperature reacts 48 hours.By reacted solution ether
Precipitation is filtered and dialysed 1 day, and freeze-drying obtains the pale yellow powder shape solid of double bond modification.Pass through nuclear-magnetism and gel infiltration color
Spectrum determines polymer architecture and molecular weight.
4) synthesis of polymer-polypeptide attachment：
The polymers copolymers of 0.045mmol peptide molecules CK (P18) GGMSRTMS and 0.03mmol double bond amounts are dissolved in
In 1mL DMSO, it is placed in reaction vessel, adds 1 and drip triethylamine, stirring and dissolving, sealing system is passed through nitrogen 30 minutes, it is permanent
37 DEG C of temperature is reacted 3 days；Reacted solution is added in bag filter, dialyses 24 hours, obtains pure by the method for thermoprecipitation
Temperature sensitive polymer-polypeptide linker, freeze-drying obtains pulverulent solids.
The structure of the obtained temperature sensitive driver of macromolecular is as follows：
What is prepared in the present embodiment is connected with polypeptide ligand CK (P18) GGMSRTMS's of purpurin 18
MALDI-TOF spectrograms are as shown in Fig. 2 as seen from the figure, the molecular weight 1604.8 of polypeptide
Structural characterization is carried out to the obtained temperature sensitive driver of macromolecular using nucleus magnetic hydrogen spectrum, as a result as shown in figure 3, can by figure
To find out, chemical displacement value δ=3.43ppm and δ=3.18ppm correspond to-OCH respectively2- with-CH2-；δ=7.5-7.0, δ=
- NH- ,-CH- and-CH in 3.85ppm NIPAs corresponding with δ=1.05ppm3.δ=9.0-8.0 correspondences are more
- CONH- on peptide.
Embodiment 2
The step of using with embodiment 1 (1) identical polypeptide ligand synthetic method has synthesized polypeptide ligand CK (P18)
GYHWYGYTPQNVI, its structure is as follows：
Purpurin 18 is connected on the polypeptide ligand by the step of using with embodiment 1 (2) identical method, then
Progress step (3) same as Example 1 and (4) prepare the temperature sensitive driver of macromolecular, and its structure is as follows：
What is prepared in the present embodiment is connected with polypeptide ligand CK (P18) GYHWYGYTPQNVI's of purpurin 18
MALDI-TOF spectrograms are as shown in figure 4, as seen from the figure, and the molecular weight of polypeptide is 2375.0, and molecule adds the peak of sodium ion to be
2397.0。
Structural characterization is carried out to the obtained temperature sensitive driver of macromolecular using nucleus magnetic hydrogen spectrum, as a result as shown in figure 5, can by figure
To find out, chemical displacement value δ=3.43ppm and δ=3.18ppm correspond to-OCH respectively2- with-CH2-；δ=3.85ppm and δ=
- CH- and-CH in 1.05ppm correspondence NIPAs3.- CONH- on δ=9.0-8.0 correspondence polypeptides.
Embodiment 3
The temperature sensitive driver of macromolecular that embodiment 1 is prepared regulates and controls membrane receptor in mode shown in Fig. 6 in cell membrane surface
Protein oligomerization, pulls receptor protein oligomerization to verify that method is as follows driver：
The temperature sensitive driver molecule that FITC is marked with 3 μM of concentration in confocal wares with adherent SKBR3 cells
Half an hour is incubated altogether, molecule is combined with cell surface receptor protein；Laser confocal microscope imaging results as shown in fig. 7,
Scanning electron microscope (SEM) photograph when wherein A figures are no laser irradiation, B figures are scanning electron microscope (SEM) photograph when whetheing there is laser irradiation, are not giving laser
During irradiation, driver molecule does not almost enter born of the same parents, cell surface fluorescence due in extended configuration, spreading over cell surface
It is weaker；When under 655nm wavelength, using 4.77W cm-2Laser intensity irradiating sample, local temperature is increased to driver molecule
On phase transition temperature, molecule is caused to be collapsed aggregation, the aggregation fluorescence of formation becomes strong.Meanwhile, seen using thermal field ESEM
A figures and B in X rays topographs of the driver molecule after cell surface laser pre-irradiation, Fig. 8 is examined to scheme to be the temperature sensitive driving of macromolecular respectively
Scanning electron microscope (SEM) photograph of the device in cell membrane surface without laser and when having laser irradiation, it follows that driver molecule is really by laser
The state (A figures) of sprawling of pre-irradiation becomes the coherent condition of collapsing (B figures) after laser irradiation.To sum up, it is glimmering by laser co-focusing
The surface imaging of photoimaging and thermal field ESEM is common to prove that driver molecule occurs aggregation after laser irradiation and collapsed, this
Assemble the ability that the motive force for generation of collapsing has potential modulin aggregation.
Aggregation in order to verify driver molecule pulls and result in the oligomerization of receptor protein, by with FRET effects
It is intracellular that fusion protein ECFP-HER2 and EYFP-HER2 are transfected into HEK-293T.With the FRET passages of confocal microscope
The amount of fluorescence resonance transfer occurs for observation.As a result it is as shown in Figure 9, it can be seen that to contrast the cell irradiated without laser or blank
Control cell, the FRET levels of laser irradiation group are obviously improved, it was demonstrated that temperature sensitive driver pulls receptor oligomerization.
It can be verified using identical experimentation and obtain the temperature sensitive driver of macromolecular that embodiment 2 prepares to HER1
Receptor oligomerization has regulating and controlling effect.
Embodiment 4
The temperature sensitive driver of macromolecular prepared in the present embodiment to embodiment 1 carries out Proliferation Ability detection：
Above-mentioned temperature sensitive driver molecule is dissolved in phosphate buffer (PBS solution), driven with the evaluation of crystal violet method of testing
Dynamic suppression of the device to SKBR3 cell growths.By SKBR3 cells kind in 12 orifice plates, density is 20000 cells/wells.Cell
Amount can be quantified by the absorption of crystal violet.4% is used at different time points (0 day, 2 days, 4 days, 6 days, 8 days, 12 days)
Paraformaldehyde fix cell, and with 0.1% crystal violet solution to cell dyeing 15 minutes, PBS is washed after three times, uses 1mL
1% sodium dodecyl sulfate solution extractive crystallization is purple, and absorption value is tested under 570nm wavelength with ELIASA.Each experiment weight
It is multiple three times, average.
As a result temperature sensitive driver as shown in Figure 10, it can be seen that different disposal has an impact to the propagation of cell, of the invention
Under laser triggering, there is good inhibition to the propagation of SKBR3 cells, this is due to that the oligomerizations of HER2 acceptors is disturbed
Pernicious signal finally causes Proliferation Ability to the transmission of intracellular.
Applicant states that the present invention illustrates the temperature sensitive driver of macromolecular and its preparation of the present invention by above-described embodiment
Methods and applications, but the invention is not limited in above-described embodiment, that is, do not mean that the present invention has to rely on above-described embodiment
It can implement.Person of ordinary skill in the field is it will be clearly understood that any improvement in the present invention, to raw material selected by the present invention
The addition of equivalence replacement and auxiliary element, selection of concrete mode etc., all fall within protection scope of the present invention and open scope it
It is interior.
Claims (10)
1. a kind of temperature sensitive driver of macromolecular, it is characterised in that the temperature sensitive driver of macromolecular includes being made up of polypeptide ligand
Targeting module, the drive module being made up of temperature sensitive polymer and the trigger module being made up of photothermal deformation molecule, the triggering
Module is connected in targeting module, and the targeting module is connected with drive module.
2. the temperature sensitive driver of macromolecular according to claim 1, it is characterised in that the polypeptide ligand is can be with cell
Film surface receptor protein combine arbitrary amino acid sequences polypeptide in any one or at least two combination；
Preferably, the amino acid sequence of the polypeptide ligand is MSRTMS, GYHWYGYTPQNVI, RGD or CNSCWSKD, enters one
Walk preferred MSRTMS.
3. the temperature sensitive driver of macromolecular according to claim 1 or 2, it is characterised in that the temperature sensitive polymer is poly- N-
N-isopropylacrylamide, poly- N, N- acrylamides, poly- N- hydroxymethylpropyls Methacrylamide, poly- N-2,2- dimethyl
1,3- dioxolanemethyls acrylamide, poly- N-2- methoxyl groups 1,3- dioxy second cyclohexyl methyls acrylamide, poly- N-2- ethyoxyls 1,
3- dioxy second cyclohexyl methyls acrylamide, polymethylacrylic acid oligomeric ethylene glycol ester, poly N-vinyl isobutyramide, poly- ethylene methacrylic
Base ether, poly-N-vinylcaprolactam, poly- 2- ethyl oxazolines, N-2 hydroxyls N-isopropylacrylamide or N- hydroxyethyl acrylamides
In any one or at least two composition；
Preferably, the temperature sensitive polymer is different for copolymer, the N- of NIPA and N-2 hydroxyl N-isopropylacrylamides
In the copolymer of the copolymer or NIPA and acrylic acid of propylacrylamide and N- hydroxyethyl acrylamides
Any one or at least two combination, preferably NIPA and/or N-2 hydroxyl N-isopropylacrylamides copolymerization
Thing.
4. the temperature sensitive driver of macromolecular according to claim 3, it is characterised in that the temperature sensitive polymer is copolymer
When, its synthetic method is activity/controllable free-radical polymerisation, preferably RAFT polymerizations；
Preferably, the chain transfer agents of the RAFT polymerizations are N, N'- dimethyl N, N'- bis- (4- pyridine radicals) thiuram curing
Thing, 2- (dodecyl trithiocarbonic acid ester group) -2 Methylpropionic acid, double (dodecyl sulfanyl thiocarbonyl) disulphide,
2- cyano group -2- propyl group dodecyls trithiocarbonate, the sulphur of 2- cyano group -2- propyl group benzo two, 4- cyano group -4- [(dodecyl sulphur
Alkylthiono) sulfanyl] valeric acid, 4- cyano group -4- (phenyl formyl sulfenyl) valeric acid, the thio carbon of cyanogen methyl dodecyl three
Acid esters, cyanogen methyl (phenyl) dithiocarbamates, methyl -2- propionic acid methyl (4- pyridines) aminodithioformic acid
It is any in ester, methyl -2- (dodecyl trithiocarbonate) -2 Methylpropionic acid ester or the sulphur of 2- phenyl -2- propyl group benzo two
It is a kind of or at least two combination, further preferred 2- cyano group -2- propyl group dodecyl trithiocarbonates.
5. the temperature sensitive driver of macromolecular according to any one of claim 1-4, it is characterised in that the temperature sensitive polymer
For the polymer with structure shown in formula I：
Wherein x=200-1200, y=1-60, and x/y=19-280；It is preferred that x=642, y=7；
Preferably, the molecular weight of the temperature sensitive polymer is 20-80kD.
6. the temperature sensitive driver of macromolecular according to any one of claim 1-5, it is characterised in that the polypeptide ligand is made
It is side chain graft on temperature sensitive polymer skeleton；
Preferably, the grafting is realized by Michael's addition method；
Preferably, the polypeptide ligand connection ratio is 0.5-5%, preferably 1%.
7. the temperature sensitive driver of macromolecular according to any one of claim 1-6, it is characterised in that the photothermal deformation point
Son is purpurin 18, burnt pheophytin, in Cy5, Cy6 or Cy7 any one or at least two combination, it is preferably purplish red
Element -18.
8. the preparation method of the temperature sensitive driver of macromolecular according to any one of claim 1-7, it is characterised in that described
Preparation method is：By polypeptide solid-state reaction method synthesis polypeptide ligand sequence, the photothermal deformation molecule in connection on peptide sequence,
And the polypeptide ligand sequence for being connected with photothermal deformation molecule is grafted on the skeleton of temperature sensitive polymer by Michael's addition
To the described temperature sensitive driver of macromolecular.
9. a kind of material of regulation and control membrane receptor protein aggregation, it is characterised in that the material is included as any in claim 1-7
The temperature sensitive driver of macromolecular described in.
10. a kind of drug delivery materials, it is characterised in that the drug delivery materials are included such as any one of claim 1-7
The temperature sensitive driver of described macromolecular.
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