CN109456250A - Hot activation delayed fluorescence (TADF) nano-probe and preparation method thereof and the application in bio-imaging - Google Patents
Hot activation delayed fluorescence (TADF) nano-probe and preparation method thereof and the application in bio-imaging Download PDFInfo
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- CN109456250A CN109456250A CN201811325113.3A CN201811325113A CN109456250A CN 109456250 A CN109456250 A CN 109456250A CN 201811325113 A CN201811325113 A CN 201811325113A CN 109456250 A CN109456250 A CN 109456250A
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- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
Application the invention discloses a series of hot activation delayed fluorescence (TADF) nano-probes and preparation method thereof and in bio-imaging.Hot activation delayed fluorescence (TADF) the nano-probe molecule has structure shown in formula I.Compared with reported image probe molecule, hot activation delayed fluorescence nano-probe prepared by the present invention has better fluorescence intensity and fluorescence efficiency, greatly improve the image sensitivity of such molecule, improve the photoluminescent property of probe, with good water solubility, strong brightness and stability, it is oxygen barrier, but also there are potential fluorescence long life characteristics, it is clear to show in terms of cell imaging, stablize, the strong imaging effect of fluorescence signal, fluorescence lifetime can reach 271.20 microseconds or more, advantageously reduce self absorption effect and inner filtering effect, reduce transmitting light and scatters the detection mistake between light, reduce the background noise of fluorescence imaging, improve imaging signal to noise ratio, and it can be used for super-resolution fluorescence imaging experiments.
Description
Technical field
The present invention relates to a kind of hot activation delayed fluorescence (TADF) nano-probes, further relate to the preparation method of the nano-probe
And its application in bio-imaging.The invention belongs to pharmaceutical technology fields.
Background technique
TADF (thermally activated delayed fluorescence) hot activation delayed fluorescence material is not
Singlet exciton and Triplet exciton can be utilized in the case where containing noble metal simultaneously, is known as after fluorescent material and phosphorescence
Third generation organic photoelectrical material after material, for the first time in 2012 by Adachi et al. (Uoyama H, Goushi K, Shizu
K,Nomura H,Adachi C.Highly efficient organic light-emitting diodes from
delayed fluorescence.Nature.2012；492 (2012): 234-238.) synthesis.And TADF material is due to its single line
Energy level difference is smaller between state-triplet state, and triplet excitons can return to singlet by reversed intersystem crossing, forms singlet and swashs
Son then radioluminescence, which greatly improves the radioluminescence efficiency of exciton (Lee DR, Kim BS, Lee CW, Im Y,
Yook KS, Hwang SH, Lee JY.Above 30%external quantum efficiency in green
delayed fluorescent organic light-emitting diodes.Acs Appl Mater Inter.2015；7
(18):9625-9629.).TADF realizes quantum efficiency and is up to 30%, while having both high color purity, long life and low
(Tan Jihua, Huo Yanping, Cai Ning, nationality lack quick, Li Zongzhi to the advantages that starting voltage, and tension Electron donor acceptor type thermal activation is prolonged
Slow fluorescence blue light material progress organic chemistry .2017；37:2457-2480.).Therefore, TADF material has been mainly used in
In machine LED device, at the same time, efficient TADF material is in light-emitting guest, light emitting host, sensitizer, bioluminescence
The every field such as imaging obtain extensive utilization.
Imaging-PAM is used widely in fields such as biophysics, biochemistry and clinical medicine diagnosis, will
After the technologies such as fluorescence lifetime imaging and Laser Scanning Confocal Microscope, Excited Fluorescence Combined are micro- combine, since it is with very high
Sensitivity and molecular specificity, fluorescence lifetime imaging technology can quantitatively obtain the functional information of biomolecule and microenvironment, it
The deep structure and state of sample can be detected in certain circumstances, can also its sharp chemical sensitivity carry out spectroscopic diagnostics ( M.The art of fluorescence imaging with chemical sensors.Angew Chem
Int Ed Engl.2012；51(15):3532-3554.).Currently, fluorescence imaging have become it is a kind of tracking and understanding organism
The inside and outside indispensable general utility tool of complex biological environment, compared with other imaging patterns, fluorescence imaging provide it is a kind of when
Between and spatially all have high resolution, without technology (Chan J, the Dodani SC, Chang for encroaching on, having no toxic side effect
CJ.Reaction-based small-molecule fluorescent probes for chemoselective
bioimaging.Nat Chem.2012；4 (12): 973-984.), but fluorescence imaging be easy stimulated luminescence intensity, quenching and
The influence of many factors such as dye strength, stability (raise, Chen Danni, Guo Baoping, the foolish stupid fluorescence of ox by Liu Lixin, Qu Junle, woods
Service life is imaged and its applies Shenzhen University journal science and engineering version .2005 in biomedicine；22(2):133-141.).In addition,
That there are fluorescence life times is extremely short for fluorescent chemicals at present, meets the disadvantages of oxygen is unstable, serious to limit fluorescence lifetime imaging technology
The application of biological sample dynamic Diversity is provided and is popularized.On the other hand, fluorescence imaging also encounters universal limitation, such as group
Knit cell autofluorescence phenomenon (Genin E, Gao Z, Varela JA, Daniel J, Bsaibess T, Gosse I, Groc
L,Cognet L,Blanchard-Desce M."Hyper-bright"near-infrared emitting fluorescent
organic nanoparticles for single particle tracking.Adv Mater.2014；26(14):
2258-2261.), a kind of solution is that targeting fluorescence signal is isolated from background autofluorescence, is mainly relied on using spy
Fluorescent emission (Yao J, Yang M, Duan Y.Chemistry, biology, and medicine of the needle at near-infrared wavelength
of fluorescent nanomaterials and related systems:new insights into
biosensing,bioimaging,genomics,diagnostics,and therapy.Chem Rev.2014；114(12):
6130-78.), but usually there is small Stokes shift in typical organic near-infrared probe, can reuptake transmitting
Photon out leads to undesirable weak transmitting and background interference (Gao YT, Feng GX, Jiang T, Goh CC, Ng LG, Liu
B,Li B,Yang L,Hua JL,Tian H.Biocompatible Nanoparticles Based on Diketo-
Pyrrolo-Pyrrole(DPP)with Aggregation-Induced Red/NIR Emission for In Vivo
Two-Photon Fluorescence Imaging.Adv.Funct.Mater.2015,25(19),2857-2866.).In order to
The interference of background fluorescence is reduced, considers that the selection longer fluorescence probe of fluorescence lifetime is imaged to be applied to time-resolved fluorescence, than
Delay time appropriate such as is introduced between the long-term durability luminous measurement of pulse excitation light and probe, can not only be eliminated of short duration
Background fluorescence, while signal-to-noise ratio can also be improved.
Most of long-life probe compounds, such as iridium, europium, all have potential cell toxicant mostly based on rare earth heavy metal
Property (Lv W, Yang T, Yu Q, Zhao Q, Zhang KY, Liang H, Liu S, Li F, Huang W.A
Phosphorescent Iridium(III)Complex-Modified Nanoprobe for Hypoxia Bioimaging
Via Time-Resolved Luminescence Microscopy.Adv Sci(Weinh).2015；2(10):
1500107.), although the service life long enough of phosphorescence, it can be achieved that time resolution detect, since phosphorescence intensity is relatively weak, instrument
Precision requirement is high, the disadvantages of to the latent lesion of surrounding cellular tissue, limit its in bio-imaging application (LiT,
Yang D,Zhai L,Wang S,Zhao B,Fu N,Wang L,Tao Y,HuangW.Thermally Activated
Delayed Fluorescence Organic Dots(TADF Odots)for Time-Resolved and Confocal
Fluorescence Imaging in Living Cells and In Vivo.Adv Sci(Weinh).2017；4(4):
1600166.).Therefore, it is necessary to develop with long fluorescence lifetime, good biocompatibility, nontoxic, targeting effect is good
Novel nano probe application is imaged in active somatic cell time-resolved fluorescence.
In order to solve this phenomenon, the novel fluorescence nano-probe that some conjugated polymers are prepared in conjunction with probe is come across
In bio-imaging application, especially anoxic bio-imaging, they have an important feature, such as high brightness, rate of fasting ejection, excellent
Photostability, flicker free, nontoxic etc., in addition, the fluorescence probe of polymer wrapped can be adjusted to tens nanometers from several nanometers, and
Their spectral characteristic is not influenced, can also enhance fluorescence probe intensity and imaging effect.It is easy to close in addition, nano-probe also has
At and purifying, biological compatibility and degradability are good, chemical optics stablize the advantages that, be suitble to the developmental research of Bio-clinical field.
Summary of the invention
The novel hot activation that technical problem to be solved by the invention is to provide a kind of conjugated polymers in conjunction with probe is prolonged
Slow fluorescent nano probe, preparation method and the application in terms of bio-imaging.
To solve technical problem of the invention, the invention provides the following technical scheme:
A kind of novel fluorescence nano-probe molecule of the invention is a kind of compound with structure shown in formula I or it can connect
The salt received:
Wherein:
R1It is expressed as
R2It is expressed as
L1It is expressed as phenyl ring or substituted phenyl ring, it is preferred that L1Independently it is expressed as
R3、L2、R4、L3、R5Existence or non-existence；
R3In the presence of, be independently expressed as methylene, ehter bond, amino, carbonyl, ester group, amide groups or
R4In the presence of, be independently expressed as methylene, ehter bond, amino, carbonyl, ester group, amide groups or
R5In the presence of, independently it is expressed as
L2In the presence of, independently it is expressed asThe linear or branched alkyl group of C1~9, the linear chain or branched chain alkenyl of C1~9
Or the straight chain of C1~9 and branch alkynyl, wherein n=1-9；
L3In the presence of, it is independently expressed as the straight chain of C1~9 and the straight chain of branched alkyl, C1~9 and branched-chain alkenyl or C1~9 is straight
Chain and attachment alkynyl.
Wherein, it is preferred that the compound is selected from one of following compound:
Further, the present invention also provides the preparation method of the compound or its acceptable salt, including it is following
Step::
A) synthesis of DF-1
(1) compound a and aniline make solvent with acetic acid, protect in argon gas, reaction obtains compound b under the conditions of 120 DEG C；
(2) compound b, carbazole and NaH make solvent with THF, and reaction obtains compound DF-1 at room temperature；
B) synthesis of DF-2
(1) compound a and equal amido phenenyl acid make solvent with acetic acid, and reaction obtains compound c under the conditions of 4 DEG C；
(2) compound c, carbazole and NaH make solvent with DMF, and reaction obtains compound d under the conditions of 0 DEG C；
(3) compound d, N- (2- amino-ethyl) morpholine, HATU and DIPEA, makees solvent with DMF, reacts under room temperature
Obtain compound DF-2；
C) synthesis of DF-3
(1) compound d, [2- (2- amino ethoxy) ethyl] t-butyl carbamate, HATU and DIPEA, are made molten with DMF
Agent, reaction obtains compound e under room temperature；
(4) compound e makees solvent in TFA, is reduced to obtain compound f under room temperature；
(5) compound f, 2- carboxyethyl tri-phenyl-phosphorus bromide, HATU and DIPEA make solvent with DMF, under room temperature instead
It should obtain compound DF-3.
Further, the invention also provides the compound or its acceptable salt in preparation hot activation delayed fluorescence
Purposes in nano-probe.
Wherein, it is preferred that the dosage form of the hot activation delayed fluorescence nano-probe is micella, liposome, inclusion compound.
A kind of hot activation delayed fluorescence nano-probe micella is provided in one particular embodiment of the present invention, by institute
The compound stated or its acceptable salt and copolymer DSPE-PEG are prepared, it is preferred that the DSPE-PEG includes
DSPE-PEG600、DSPE-PEG2000、DSPE-MPEG2000、DSPE-PEG3000。
A kind of fluorescent nano probe micelle method preparing the hot activation delay, comprising the following steps:
(1) compound or its acceptable salt and copolymer DSPE-PEG are dissolved in tetrahydrofuran respectively, rotation bottle is moved into, surpasses
The sound several seconds uniformly mixes, and installation rotation bottle rotates 5 minutes, it is seen that uniform faint yellow screw-type film；
(2) ultrasonic aquation 2 minutes, clear pale yellow color liquid is formed, merging, which is shaken, moves into EP pipe after case is incubated for 1-2 hours, from
The heart takes supernatant, crosses film, saves in 4 DEG C of conditions, as fluorescent nano probe micella.
Wherein, it is preferred that by the compound or its acceptable salt and copolymer DSPE-PEG2000 in step (1)
It is dissolved in tetrahydrofuran respectively, is configured to the solution of 1mg/ml, 1ml DSPE-PEG2000 and 90 μ l compound solutions are moved into rotation
Bottle, ultrasonic several seconds uniformly mix；Installation rotation bottle, debugs 36 DEG C of water-bath；Pressure 0.65mp rotates 5min, removes rotation bottle and is added
2ml tetrahydrofuran is spin-dried for once again, it is seen that uniform faint yellow screw-type film.
Wherein, it is preferred that hydration process includes: to remove rotation bottle in step (2), and 1-2ml distilled water is added and shakes up, ultrasonic water
Change 2min, it is seen that clear pale yellow color liquid；Case is shaken in immigration, 37 DEG C, 110rpm, is incubated for 1-2 hours immigration EP and manages, 20000rpm,
It is centrifuged 5min, takes supernatant, crosses 220 μm of films three times, crosses 100 μm of films three times, is saved in 4 DEG C of conditions.
Further, the present invention also provides the hot activation delayed fluorescence nano-probe micellas in cell or living body water
Flat detection and the application in imaging, it is preferred that the cell is HePG2 cell or Hela cell.
Compared to the prior art, the beneficial effects of the present invention are:
Compared with reported image probe molecule, nano-probe molecule synthesized by the present invention has better fluorescence intensity
And fluorescence efficiency, the image sensitivity of such molecule is greatly improved, the photoluminescent property of such molecule is improved.
The preparation process of DF-1, DF-2 and DF-3 hot activation delayed fluorescence (TADF) nano-probe micella of the present invention has behaviour
Make simply, at low cost, high income, reaction process does not need expensive set without using reagents, reaction monitorings such as severe toxicity, danger
It is standby, the features such as rapid and accurate is monitored, industrialized production is suitble to.TADF nano-probe is free of heavy metal, no cytotoxicity, by it
It is prepared into nano-probe, is had the following advantages that, good aqueous solubility, Qiang Liangdu, stability is oxygen barrier, but also has potential fluorescence
Long life characteristics can show clear, stable, the strong imaging effect of fluorescence signal, especially this hair in terms of cell imaging
The fluorescence lifetime of bright DF-1, DF-2 and DF-3 hot activation delayed fluorescence nano-probe micella can reach 271.20 microseconds or more,
There is very big Stokes shift simultaneously, thus be more advantageous to this probe molecule reduce self absorption effect and inner filtering effect,
Reduce transmitting light and scatter detecting wrong, to reduce fluorescence imaging background noise, improving imaging signal to noise ratio between light,
And it can be used for super-resolution fluorescence imaging experiments.
Detailed description of the invention
Fig. 1 shows nano-probe full wavelength scanner spectrograms (a) of the present invention to absorb and (b) fluorescence intensity；
Fig. 2 indicates the present invention for measuring nano-probe micellar particle size distribution map；
Fig. 3 indicates the present invention for measuring co-focusing imaging of the nano-probe in Hela, HepG2 cell；
Fig. 4 indicate the present invention for measure in solvent nano-probe fluorescence lifetime attenuation curve (485nm excite,
630nm monitoring)；
A:DF-1 (10.0 μM)；B:DF-2 (10.0 μM)；C:DF-3 (10.0 μM)；
Fig. 5 indicate the present invention for measure intracellular nano-probe fluorescence lifetime fluorogram (405nm excite,
460nm monitoring).
A: fluorescence intensity；B: fluorescence lifetime.
Specific embodiment
Further describe the present invention below with reference to specific example, the advantages and features of the present invention will be with description and more
It is clear.But these examples be only it is exemplary, it is not intended to limit the scope of the present invention in any way.Those skilled in the art answer
It should be appreciated that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form repair
Change or replace, but these modifications and replacement are fallen within the protection scope of the present invention.
The synthesis of 1 compound b of embodiment
Operating process:
Compound a (4,5- difluorophthalic anhydride) (300mg) is fed intake with aniline (186mg) by 1:1.2 mass ratio,
It is dissolved in 5ml acetic acid, in 120 DEG C of lower reaction 4h of argon gas protection, room temperature is down to, is put into refrigerator overnight, crystallization has solid precipitation, filters
White solid is obtained, is washed, column purification is crossed, obtains white powder compounds b, 204mg, yield 57.1%.1H NMR
(400MHz, CDCl3) δ 7.79-7.75 (t, J=8.0Hz, 2H ,-Ar), 7.54-7.50 (m, 2H ,-Ar), 7.44-7.40 (m,
3H,-Ar).13C NMR(100MHz,CDCl3)δ165.25,156.06,155.91,153.46,153.31,131.32,
129.25,128.60,128.45,126.41,113.74,113.67,113.59,113.52.
The synthesis of 2 compound DF-1 of embodiment
Operating process:
It by carbazole (130mg) and NaH (34mg), is dissolved in THF and stirs 30min at room temperature, be added compound b (100mg),
Continue to be stirred to react 2h, cross column purification, rotation is evaporated, and obtains yellow powder compound DF-1,51mg, yield 16.3%.1H
NMR (400MHz, Methanol-d4) δ 8.25 (d, J=6.4Hz, 2H ,-Ar), 8.17-8.15 (m, 4H ,-Ar), 7.97 (d, J
=8.0Hz, 2H ,-Ar), 7.57-7.53 (m, 4H ,-Ar), 7.49-7.48 (m, 4H ,-Ar), 7.47-7.43 (m, 8H ,-Ar),
7.39–7.35(m,4H,-Ar),7.28–7.27(m,3H,-Ar).13C NMR(100MHz,Methanol-d4)δ165.91,
139.47,138.85,131.57,131.00,129.34,128.50,126.59,125.97,125.89,124.06,121.02,
120.21,109.43.
The synthesis of 3 compound c of embodiment
Operating process:
Compound a (4,5- difluorophthalic anhydride) (1g) and equal amido phenenyl acid (1.12g) are added to acetic acid
It is mixed in (30mL), solution is cooled to 4 DEG C overnight by reflux 4h., and extraction filters out solid chemical compound c 1.32g, yield
80%.1H NMR (400MHz, (CD3) 2SO) δ 13.14 (s, 1H ,-COOH), 8.22 (t, J=7.6Hz, 2H ,-Ar), 8.11-
8.08(m,2H,-Ar),7.61–7.59(m,2H,-Ar).13C NMR(100MHz,(CD3)2SO)δ167.11,165.33,
155.04,154.94,153.33,153.23,135.98,130.33,129.40,127.35,114.34,114.30,114.24,
114.20.
The synthesis of 4 compound d of embodiment
Operating process:
Compound c (1g) is dissolved in DMF (10mL) and NaH (330mg) is added in 0 DEG C, one hour, and carbazole is then added
(1.65g) reacts 1h. and is extracted with ethyl acetate (2x50ml), in Na2SO4Upper drying.With silica gel column chromatography (petroleum ether: acetic acid second
Ester=3:1) yellow solid compound d 1.47g, yield 75.6% are obtained after purification.1H NMR(400MHz,CDCl3)δ8.48
(s, 2H ,-Ar), 8.33 (d, J=8Hz 2H ,-Ar), 7.80-7.78 (m, 4H ,-Ar), 7.74 (d, J=8Hz 2H ,-Ar),
7.15 (d, J=8Hz, 4H ,-Ar), 7.11-7.05 (m, 8H ,-Ar) .13C NMR (100MHz, Methanol-d4) δ 170.89,
165.35,139.68,138.73,136.47,131.25,130.60,128.66,126.13,126.07,125.97,125.88,
124.05,121.07,120.19,109.33,29.77,29.71,29.60,29.54,29.43,29.32,27.21.
The synthesis of 5 compound DF-2 of embodiment
Operating process:
Compound d (200mg), HATU (153mg), DIPEA (87mg) are dissolved in DMF (10mL), react at room temperature 30min, so
N- (2- amino-ethyl) morpholine (52mg) reaction 0.5h. is added afterwards to be extracted with ethyl (2x50ml), in Na2SO4Upper drying.
With silica gel column chromatography (dichloro；Methanol=60:1) purifying, obtain yellow solid chemical compound DF-2 184mg, yield 77.5%.1H
NMR (400MHz, Methanol-d4) δ 8.43 (s, 2H ,-Ar), 8.09-8.07 (d, J=8.4Hz, 2H ,-Ar), 7.83-7.79
(m, 4H ,-Ar), 7.76-7.74 (d, J=8.4Hz2H ,-Ar), 7.22-7.20 (m, 4H ,-Ar), 7.01-7.08 (m, 8H ,-
), Ar 4.13-4.08 (m, 2H ,-CH2), 3.83 (t, J=5.6Hz, 4H ,-CH2), 3.71 (t, J=8Hz, 2H ,-CH2), 3.45
(t, J=6Hz, 2H ,-CH2), 3.24 (t, J=6.8Hz, 2H ,-CH2) .13C NMR (100MHz, Methanol-d4) δ
170.56,170.01,168.81,167.06,162.00,161.50,140.72,140.36,136.91,133.91,132.75,
129.26,127.75,126.90,126.84,125.31,121.96,121.08,110.73,65.18,58.73,53.68,
35.79.
The synthesis of 6 compound e of embodiment
Operating process:
Compound d (200mg), HATU (153mg), DIPEA (87mg) are dissolved in DMF (6mL) and react under room temperature
Then 30min. is added [2- (2- amino ethoxy) ethyl] t-butyl carbamate (100mg) and reacts 0.5h. ethyl acetate
(2x50ml) is extracted, in Na2SO4Upper drying.It is solid that yellow is obtained after purification with silica gel column chromatography (petroleum ether: ethyl acetate=1:2)
Body compound e 223g, yield 75.6%.1H NMR (400MHz, Methanol-d4) δ 8.28 (t, J=12.8Hz, 2H ,-
), CH 7.53-7.51 (m, 2H ,-Ar), 7.46-7.42 (m, 2H ,-Ar), 7.33-7.27 (m, 4H ,-Ar), 6.65 (t, J=
12.4Hz, 1H ,-CH), 6.32 (d, J=13.6Hz, 2H ,-CH), 4.18 (dd, J1=14.4Hz, J2=7.2Hz, 4H ,-
), CH2 1.75 (s, 12H ,-CH3), 1.41 (t, J=7.2Hz, 6H ,-CH3) .13C NMR (100MHz, Methanol-d4) δ
174.34,155.67,143.12,142.81,129.81,126.54,126.28,123.48,111.80,103.97,50.61,
39.98,27.87,12.58.HRMS(m/z)(M+):calcd.for C29H35N2+411.2795,found 411.2799.
The synthesis of 7 compound DF-3 of embodiment
Operating process:
Compound e (100mg) is dissolved in TFA (1mL) and reacts 0.5h under room temperature, and then evaporation removes TFA and is spin-dried for obtaining
Compound f, 2- carboxyethyl tri-phenyl-phosphorus bromide (50mg), HATU (10mg), DIPEA (10mg) are dissolved in DMF (3mL) room temperature reaction
30min. mixture extracts 30min with ethyl acetate (2x50mL), in Na2SO4Upper drying.With silica gel column chromatography (petroleum ether: second
Acetoacetic ester=1:2) yellow solid compound DF-3 72mg, yield 80.5% are obtained after purification.
1H NMR (400MHz, Methanol-d4) δ 8.52 (d, J=13.2Hz, 2H ,-Ar), 8.27 (d, J=8.8Hz,
2H,-CH),8.08–8.01(m,4H,-Ar),7.69–7.66(m,4H,-Ar),7.54–7.51(m,2H,-Ar),6.54(d,J
=13.2Hz, 2H ,-CH), 4.39-4.30 (m, 4H ,-CH2), 2.35 (t, J=7.2Hz, 2H ,-CH2), 2.05 (s, 12H ,-
CH3),2.00–1.95(m,2H,-CH2),1.80–1.72(m,2H,-CH2),1.62–1.56(m,2H,-CH2),1.52(t,J
=7.2Hz, 3H ,-CH3) .13C NMR (100MHz, Methanol-d4) δ 175.86,175.57,148.70,148.54,
139.24,138.82,134.45,134.27,132.32,132.27,130.88,130.44,129.70,128.41,127.89,
127.44,125.04,122.01,115.57,110.83,110.64,101.81,101.48,65.19,51.45,51.38,
47.99,44.01,39.37,33.32,26.85,26.01,25.84,25.74,24.26,18.81,11.24.
The preparation of 8 hot activation delayed fluorescence nano-probe micella of embodiment
Operating process:
(1) it weighs: weighing appropriate compound DF-1, DF-2 or DF-3 and DSPE-MPEG2000, be dissolved in tetrahydro furan respectively
It mutters, is configured to the solution of 1mg/ml.The solution of 1ml DSPE-MPEG2000 and 90 μ l DF-1, DF-2 or DF-3 are moved into and revolved
Bottle, ultrasonic several seconds uniformly mix.
(2) form a film: installation rotation bottle debugs 36 DEG C of water-bath；Pressure 0.65mp rotates 5min.It removes rotation bottle and 2ml tetra- is added
Hydrogen furans is spin-dried for once again with method, it is seen that uniform faint yellow screw-type film.
(3) aquation: removing rotation bottle, and 1-2ml distilled water is added and shakes up, ultrasonic aquation 2min, it is seen that clear pale yellow color liquid.
Case is shaken in immigration, 37 DEG C, 110rpm, is incubated for 1-2 hours.
(4) it filters: moving into EP pipe, 20000rpm is centrifuged 5min, takes supernatant, crosses 220 μm of films three times, crosses 100 μm of films three
It is secondary.It is saved in 4 DEG C of conditions, respectively obtains DF-1, DF-2 or DF-3 hot activation delayed fluorescence nano-probe micella.Measure average grain
Diameter is 56.01nm, and encapsulation rate 85.54%, concentration is 69.52 μM.
Experimental example 1: the photoluminescent property of hot activation delayed fluorescence nano-probe micella
(1) postpone nano-probe micella mother liquor using DF-1, DF-2 or DF-3 hot activation that DMAO prepares 1mM, by nanometer
Compound in probe micella is diluted to 20 μM of final concentration with DMEM (or PBS or water)；
(2) recovery cell
Hela, HepG2 cell are taken out out of -80 DEG C refrigerators, are placed in 37 DEG C of water, in thawing completely in 1-2min,
It in aseptic operating platform, is transferred into the culture bottle containing 4mL DMEM culture solution, and at 37 DEG C, contains 5%CO2Incubator
Inside cultivated.
(3) observation-passage-counting
Cell growth status is observed under the microscope, and until Hela, HepG2 cell adherent growth, convergence degree reaches
80%, it can pass on, old culture solution is outwelled in aseptic working platform, be rinsed with 2mL PBS, 0.25% pancreas of 1mL is added
Protease, carries out digestion 7min by 37 DEG C.After cell size is rounded, patting culture bottle makes carefully for observation under the microscope at this time
Born of the same parents, which fall off and suitable culture solution is added immediately, prevents digestion, is blown and beaten vitellophag at cell suspension, cell with dropper
Suspension dilutes 10 times, and cell count, preparation is inoculated in the chamber slide in 8 holes, and the cell number in every hole is 4 × 104It is a, it is placed in
37 DEG C, contain 5%CO2Incubator in be incubated overnight, no less than 12 hours.
(4) next day observes 8 orifice plate inner cell upgrowth situations, and convergence degree 70% or so discards old culture medium, PBS drift
200 μ l DF-1, DF-2 or DF-3 hot activation delay nano-probe micellar solution are added after washing, is placed in 37 DEG C, contains 5%CO2Training
It supports and is incubated for 2h in case.Then it is measured to full wavelength scanner (250nm-750nm) using microplate reader；Simultaneously in corresponding excitation wave
Strong point carries out the measurement of launch wavelength, as a result such as Fig. 1.Confirm photoluminescent property of the nano-probe in conjunction with Hela, HepG2.
Experimental example 2: hot activation delayed fluorescence nano-probe micella co-focusing imaging in cell
(1) postpone nano-probe micella mother liquor using DF-1, DF-2 or DF-3 hot activation that DMAO prepares 1mM, by nanometer
DF-1, DF-2 or DF-3 compound in probe micella is diluted to 5 μM of final concentration with DMEM；
(2) recovery cell
Hela, HepG2 cell are taken out out of -80 DEG C refrigerators, are placed in 37 DEG C of water, in thawing completely in 1-2min,
It in aseptic operating platform, is transferred into the culture bottle containing 4mL DMEM culture solution, and at 37 DEG C, contains 5%CO2Incubator
Inside cultivated.
(3) observation-passage-counting
Cell growth status is observed under the microscope, and until Hela, HepG2 cell adherent growth, convergence degree reaches
80%, it can pass on, old culture solution is outwelled in aseptic working platform, be rinsed with 2mLPBS, 0.25% pancreas of 1mL is added
Protease, carries out digestion 7min by 37 DEG C.After cell size is rounded, patting culture bottle makes carefully for observation under the microscope at this time
Born of the same parents, which fall off and suitable culture solution is added immediately, prevents digestion, is blown and beaten vitellophag at cell suspension, cell with dropper
Suspension dilutes 10 times, and cell count, preparation is inoculated in the chamber slide in 8 holes, and the cell number in every hole is 4 × 104It is a, it is placed in
37 DEG C, contain 5%CO2Incubator in be incubated overnight, no less than 12 hours.
(4) hot activation postpones nano-probe co-focusing imaging in the cell
Next day observes 8 orifice plate inner cell upgrowth situations, and convergence degree 70% or so discards old culture medium, after PBS rinsing
200 μ l DF-1, DF-2 or DF-3 hot activation delayed fluorescence nano-probe micellar solution are added, are placed in 37 DEG C, contain 5%CO2Training
It supports and is incubated for 2h in case.Then fluorescence imaging is carried out to cell with laser confocal microscope.It as a result as shown in figure 3, can from figure
To obtain, hot activation delayed fluorescence nano-probe can be imaged in Hela, HepG2 cell.
Experimental example 3: hot activation delayed fluorescence nano-probe micella fluorescence lifetime in solvent and cell measures
1, fluorescence lifetime measures hot activation delayed fluorescence nano-probe micella in a solvent
DF-1, DF-2 or DF-3 nano-probe micella mother liquor that 1mM is prepared using DMAO, by the change in nano-probe micella
It closes object and is diluted to 20 μM of final concentration with DMEM (or PBS or water).Then it is measured respectively with the survey of fluorescence lifetime imaging Q2 analyzer
Nano-probe in a solvent in fluorescence decay curve.
2, hot activation delayed fluorescence nano-probe micella fluorescence lifetime in cell measures
The preparation process of cell sheet:
(1) 24 orifice plates are taken, are operated in Biohazard Safety Equipment, (10 μ l) culture solution is dripped in all holes, is put into round lid
Then 1ml Hela, HepG2 cell suspension, cell count (4 × 10 is added in slide4), it is put into 5%CO2In incubator.
(2) it behind a length of full 80% or more the visual field of cell, takes out, PBS is rinsed 2 times, and it is solid that 4% paraformaldehyde, 500 μ l is added
Fixed, the set time is 10 minutes, is protected from light and is put into 4 DEG C of refrigerators, for use.
(3) it takes out cell after fixing to be rinsed 3 times with PBS, 20 μM of DF-1, DF-2 or DF-3 nano-probe is added in oscillation
Micellar solution after being incubated for the corresponding time, siphons away liquid after PBS rinsing with rifle and takes out coverslip.
(4) the 1 drop mountant of drop on glass slide, coverslip is put on glass slide, notices that cell is buckled to, siphons away coverslip
The extra mountant of surrounding applies 4 drop transparent nail polish up and down, extra mountant is wiped with swab stick, in coverslip surrounding
Coat uniform nail polish.
Then it is glimmering in the cell measurement hot activation delayed fluorescence nano-probe respectively to be surveyed with fluorescence lifetime imaging Q2 analyzer
Optical attenuation curve.
3, result
As a result as shown in Fig. 4 and Fig. 5, it can be seen that DF-1 hot activation delayed fluorescence nano-probe of the invention from the result
Micella, the service life is 271.20 μ s in a solvent, and the service life is 170 μ s, DF-2 hot activation delayed fluorescence nano-probe micellas in cell,
DF-3 hot activation delayed fluorescence nano-probe micella is equivalent to common long-life probe (the 10-30 μ s) service life and also greatly improves, when
After cell autofluorescence dies out, probe is still had, and is conducive to cell imaging detection.
Claims (10)
1. one kind is such as I compound represented of formula or its acceptable salt:
Wherein:
R1It is expressed as
R2It is expressed as
L1It is expressed as phenyl ring or substituted phenyl ring, it is preferred that L1Independently it is expressed as
R3、L2、R4、L3、R5Existence or non-existence；
R3In the presence of, be independently expressed as methylene, ehter bond, amino, carbonyl, ester group, amide groups or
R4In the presence of, be independently expressed as methylene, ehter bond, amino, carbonyl, ester group, amide groups or
R5In the presence of, independently it is expressed as
L2In the presence of, independently it is expressed asThe linear or branched alkyl group of C1~9, the linear chain or branched chain of C1~9 alkenyl or
The straight chain of C1~9 and branch alkynyl, wherein n=1-9；
L3In the presence of, be independently expressed as the straight chain of C1~9 and the straight chain of branched alkyl, C1~9 and branched-chain alkenyl or the straight chain of C1~9 and
Branch alkynyl.
2. compound as described in claim 1 or its acceptable salt, which is characterized in that the compound is selected from followingization
Close one of object:
3. the preparation method of compound as claimed in claim 2 or its acceptable salt, which comprises the following steps::
A) synthesis of DF-1
(1) compound a and aniline make solvent with acetic acid, protect in argon gas, reaction obtains compound b under the conditions of 120 DEG C；
(2) compound b, carbazole and NaH make solvent with THF, and reaction obtains compound DF-1 at room temperature；
B) synthesis of DF-2
(1) compound a and equal amido phenenyl acid make solvent with acetic acid, and reaction obtains compound c under the conditions of 4 DEG C；
(2) compound c, carbazole and NaH make solvent with DMF, and reaction obtains compound d under the conditions of 0 DEG C；
(3) compound d, N- (2- amino-ethyl) morpholine, HATU and DIPEA, makees solvent with DMF, and reaction obtains under room temperature
Compound DF-2；
C) synthesis of DF-3
(1) compound d, [2- (2- amino ethoxy) ethyl] t-butyl carbamate, HATU and DIPEA, make solvent with DMF,
Reaction obtains compound e under room temperature；
(2) compound e makees solvent with TFA, is reduced to obtain compound f under room temperature；
(3) compound f, 2- carboxyethyl tri-phenyl-phosphorus bromide, HATU and DIPEA make solvent with DMF, react under room temperature
To compound DF-3.
4. compound of any of claims 1 or 2 or its acceptable salt are in preparation hot activation delayed fluorescence nano-probe
Purposes.
5. purposes as claimed in claim 4, which is characterized in that the dosage form of the hot activation delayed fluorescence nano-probe is glue
Beam, liposome or inclusion compound.
6. a kind of hot activation delayed fluorescence nano-probe micella, which is characterized in that by compound of any of claims 1 or 2 or
Its acceptable salt and copolymer DSPE-PEG are prepared, it is preferred that the DSPE-PEG include DSPE-PEG600,
DSPE-PEG2000、DSPE-MPEG2000、DSPE-PEG3000。
7. a kind of prepare hot activation delayed fluorescence nano-probe micelle method as claimed in claim 6, which is characterized in that including with
Lower step:
(1) compound of any of claims 1 or 2 or its acceptable salt and copolymer DSPE-PEG are dissolved in tetrahydro furan respectively
It mutters, moves into rotation bottle, the ultrasonic several seconds uniformly mixes, and installation rotation bottle rotates 5 minutes, it is seen that uniform faint yellow screw-type film；
(2) ultrasonic aquation 2 minutes, clear pale yellow color liquid is formed, merging, which is shaken, moves into EP pipe after case is incubated for 1-2 hours, centrifugation takes
Supernatant crosses film, saves in 4 DEG C of conditions, as hot activation delayed fluorescence nano-probe micella.
8. the method for claim 7, which is characterized in that in step (1) by compound of any of claims 1 or 2 or
Its acceptable salt and copolymer DSPE-PEG2000 are dissolved in tetrahydrofuran respectively, are configured to the solution of 1mg/ml, by 1ml
DSPE-PEG2000 and 90 μ l compound solutions move into rotation bottle, and the ultrasonic several seconds uniformly mixes；Installation rotation bottle, debugs water-bath 36
℃；Pressure 0.65mp rotates 5min, removes rotation bottle addition 2ml tetrahydrofuran and is spin-dried for again once, it is seen that uniform faint yellow screw-type
Film.
9. the method for claim 7, which is characterized in that hydration process includes: to remove rotation bottle in step (2), and 1- is added
2ml distilled water shakes up, ultrasonic aquation 2min, it is seen that clear pale yellow color liquid；Case is shaken in immigration, and 37 DEG C, 110rpm, it is small to be incubated for 1-2
When move into EP pipe, 20000rpm is centrifuged 5min, takes supernatant, crosses 220 μm of films three times, crosses 100 μm of films three times, protects in 4 DEG C of conditions
It deposits.
10. hot activation delayed fluorescence nano-probe micella as claimed in claim 6 is in cell or the detection of living body level and imaging
Application, it is preferred that the cell be HePG2 cell or Hela cell.
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