CN109253990A - Nano fluorescent probe for targeting pancreatic cancer circulating tumor cells - Google Patents
Nano fluorescent probe for targeting pancreatic cancer circulating tumor cells Download PDFInfo
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- CN109253990A CN109253990A CN201810265678.0A CN201810265678A CN109253990A CN 109253990 A CN109253990 A CN 109253990A CN 201810265678 A CN201810265678 A CN 201810265678A CN 109253990 A CN109253990 A CN 109253990A
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- 208000015486 malignant pancreatic neoplasm Diseases 0.000 title claims abstract description 51
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- C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- 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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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
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Abstract
The invention provides a nano fluorescent probe for targeting pancreatic cancer circulating tumor cells, which comprises H2O2Responsive fluorescent small molecules and polymers targeting pancreatic cancer surface markers, said H2O2Responsive fluorescent small molecules and targetsThe polymers of the surface markers for pancreatic cancer have opposite hydropathic and hydrophobic properties. According to the invention, through the specific combination of hyaluronic acid and CD44 antigen with high expression on the surface of pancreatic cancer tumor, the HA-NP-BE nano fluorescent probe can enter pancreatic cancer cells in a targeted manner to detect H in the cells2O2The imaging is carried out, and the kit has quick response, can effectively distinguish pancreatic cancer tumor cells from normal cells, detects CTC in pancreatic cancer tumor patients, and is helpful for early diagnosis of pancreatic cancer.
Description
Technical field
The invention belongs to biological fields, more particularly, are related to a kind of the intracellular of targeted pancreatic cancer circulating tumor cell
" Ratio-type " namo fluorescence probe of hydrogen peroxide.
Background technique
Circulating tumor cell (CTCs) detection at present mainly includes being enriched with and identifying two parts.Measured less since CTCs has,
The features such as heterogeneous and morphotropism, there are many detections that method can be used for CTCs at present.It is the with good grounds cell density of enrichment method, big
The physics method of the characteristics such as small, dielectricity, according to cell surface specific antigen include epithelial adherence molecule (EpCAM),
The immunological method of EGFR and the negative selection method for removing red blood cell, leucocyte；Identification method has flow cytometry, immunofluorescence
Method, fluorescence in situ hybridization, reverse transcription PCR and the sequencing of two generations.Wherein the method for negative selection and immunofluorescence technique is the most frequently used or effective.
Intracellular hydrogen peroxide is cell " second messenger ", related with the growth of cell, differentiation, migration and apoptosis.Carefully
Increasing for concentration of hydrogen peroxide intracellular can make intracellular DNA impaired, cause cell carcinogenesis.Existing research shows that circulation is swollen
Concentration of hydrogen peroxide obviously increases in oncocyte.
Chinese invention patent CN201710564408.5 disclose a kind of response of hydrogen peroxide ratiometer nano-probe and
It is applied, although having not by H2O2The internal standard fluorescent molecule rhodamine B that concentration influences is as reference, but rhodamine B and cumarin
Excitation wavelength is different, and the power of the laser in laser confocal microscope is difficult to be consistent, the transmitting effect of two kinds of fluorescence
Rate is also different, external H2O2Concentration standard curve is being applied to intracellular H2O2When detection, cellular environment and detection device
Disturbing factor it is too many, can only half-quantitative detection.
Hyaluronic acid can the specifically highly expressed CD44 in targets neoplastic cells surface.CD44 is in adhesion factor family
A member, be the film surface glycoprotein of adhesion between mediated cell and cell, cell and extracellular matrix, participate in the increasing of cell
It grows, break up, stick, migrate.CD44 is primarily involved in heterotypic adhesion, i.e., tumour cell and host cell and host matrix is viscous
Attached, heterotypic adhesion plays a driving role in tumor cell invasion transfer.
The enrichment method and identification method of circulating tumor cell used at present have limitation, as a result unsatisfactory.
Although failing to find epithelial cell, the benign disease and malignant neoplastic disease human peripheral of pancreas in the peripheral blood of normal person
In can find the equal nothings such as epithelial cell, and cellular morphology, including cell size, core size, nuclear morphology, cytoplasm/core ratio
Significant difference, therefore the enrichment based on physical method and identification method cannot be used for the detection of the circulating tumor cell of cancer of pancreas.
On the other hand, predict that most common tumor markers only have carbohydrate anti-for diagnosis of pancreatic cancer, curative effect monitoring and survival rates
Former (CA19-9) and carcinomebryonic antigen (CEA).But both tumor markers all there is a problem of one very big -- specificity is not high.
The patient of Lewis a and b- genotype blood group can not synthesize CA19-9.In addition, in nonmalignant disease such as cirrhosis, diabetes, slow
In property pancreatitis, cholangitis, obstructive jaundice and other alimentary tract cancers patient, CA19-9 is also possible to high expression.Even if
In resectable Pancreas cancer patients, up to 35% patient CA19-9 also not high expression.Simultaneously as the tumour of cancer of pancreas is thin
There is heterogeneity, in the epithelial-mesenchymal conversion process of cell (EMT), EpCAM and these epithelium marker expression poles CK19 in born of the same parents
It is likely to decrease.Therefore, capturing enrichment and identification CTCs respectively using EpCAM and CK19 antibody, there are limitations, in capture pancreas
Result is disappointing in the CTCs utilization of gland cancer.
Therefore, it is urgent to provide a kind of namo fluorescence probes of targeted pancreatic cancer circulating tumor cell, to solve the above problems.
Summary of the invention
The object of the present invention is to provide a kind of expression independent of cell surface EpCAM and CK19 antigen, targeting is swollen
The CD44 on oncocyte surface improves the detection efficiency of namo fluorescence probe, reduces the interference of leucocyte, and it is thin to reduce pancreatic tumour
False negative result and false positive results in born of the same parents' detection, the targeted pancreatic cancer circulating tumor cell with good biocompatibility
Namo fluorescence probe.
To achieve the goals above, the present invention provides a kind of spies of the nano fluorescent of targeted pancreatic cancer circulating tumor cell
Needle, which is characterized in that the probe includes H2O2The fluorescent small molecule of responsiveness and the polymerization of targeted pancreatic cancer surface marker
Object, the H2O2The fluorescent small molecule of responsiveness and the polymer of targeted pancreatic cancer surface marker have opposite hydrophilic and hydrophobic.
As a preferred embodiment, the H2O2The fluorescent small molecule of responsiveness includes naphthalimide-small point of borate fluorescence
One of sub- NP-BE, cumarin borate, fluorescein borate, carbazole-borate, two pyrroles of fluorine boron-borate.
As a preferred embodiment, the cancer of pancreas surface marker is CD44.
As a preferred embodiment, the polymer of the targeted pancreatic cancer surface marker is hyaluronic acid HA.
CD44 can be to intracellular H2O2Quick response has good biocompatibility, specifically targets neoplastic cells
And normal cell is since surface C D44 low expression is without absorbing namo fluorescence probe.The target of other targeted pancreatic cancer cells is also
Carcinomebryonic antigen (CEA), sugar antigen CA19-9 etc. choose the polymerization of suitable targeted pancreatic cancer surface marker according to marker
Object.
It is an advantage of the current invention that express this feature using tumor cell surface CD44 high, with hyaluronic acid to its into
Row targeting.Therefore, the hyaluronic acid of CD44 targeting is introduced as H2O2Responsiveness naphthalimide-borate fluorescent small molecule
Carrier, and wherein, naphthalimide-borate and H2O2Reaction front and back has blue and the two different colors of fluorescence of green,
It is a kind of fluorescent molecule of Ratio-type.Can with targeting it be entered based on this targeting Ratio-type namo fluorescence probe formed
In the highly expressed cancer of pancreas CTC of CD44 and to intracellular H2O2Imaging and semi-quantitative analysis, to finally identify CTC.By saturating
The highly expressed CD44 antigen specific binding of bright matter acid and pancreatic tumour surface, HA-NP-BE namo fluorescence probe can target
Property enter pancreatic cancer cell, detect intracellular H2O2And it is imaged, and there is quick responsiveness, it can be effectively regional
Divide human pancreatic carcinoma cell and normal cell, detects the CTC in pancreatic tumour patient, the early stage for facilitating cancer of pancreas examines
It is disconnected.
Detailed description of the invention
The synthesis of Fig. 1 hyaluronic acid-naphthalimide-borate graft polymers.
The nucleus magnetic hydrogen spectrum of Fig. 2 (a) HA and (b) HA-NP-BE graft copolymer.
(a) DLS curve and (b) TEM picture of Fig. 3 HA-NP-BE namo fluorescence probe.
200 μM of H is added in Fig. 42O2UV absorption of the HA-NP-BE nano-probe at (a) 450nm and at 350nm is strong afterwards
The ratio of degree and the fluorescence intensity at (b) 550nm are with the change curve in reaction time.
Fig. 5 HA-NP-BE namo fluorescence probe respectively with the H of various concentration2O2React the purple of (a) 30min and (b) 60min
Outer absorption spectrum.The H of HA-NP-BE namo fluorescence probe and various concentration2O2(c) is ultraviolet at 350nm and 450nm after reaction
Absorption intensity changes (d) and with H2O2The curve of concentration variation.
The H of Fig. 6 HA-NP-BE namo fluorescence probe and various concentration2O2Respectively in (a) 405nm after reaction 30min, (b)
The fluorescence spectrum (d) and fluorescence intensity and H excited under the excitation wavelength of 450nm and (c) 488nm2O2Linear pass between concentration
It is curve, the fluorescence intensity ratio and H when (e) 405nm excitation wavelength excites, at 550nm and 500nm2O2It is linear between concentration
Relation curve.
Cytotoxicity after Fig. 7 HA-NP-BE namo fluorescence probe and RAW 264.7 and PANC-1 incubation for 24 hours.
Fig. 8 HA-NP reference namo fluorescence probe and (a) RAW 264.7, (b) 293 HEK, (c) MIA-PaCa-2 and (d)
PANC-1 cell co-cultures the fluidic cell fluorogram after different time.
Fig. 9 HA-NP reference namo fluorescence probe and different cells co-culture the fluidic cell fluorescence intensity after different time.
The CD44 protein expression of Figure 10 immunoblotting assay difference cell surface.
The Zeta potential of Figure 11 HA-NP-BE namo fluorescence probe.
Figure 12 HA-NP-BE namo fluorescence probe and (a) RAW 264.7, (b) 293 HEK, (c) MIA-PaCa-2 and (d)
PANC-1 cell co-cultures the fluidic cell fluorogram after different time.
Figure 13 HA-NP-BE namo fluorescence probe and different cells co-culture the fluidic cell fluorescence intensity after different time.
Figure 14 normal cell (a) RAW 264.7 and (b) HEK 293 are co-cultured with HA-NP-BE namo fluorescence probe respectively
The laser confocal microscope picture of 0min, 30min, 60min and 120min, nucleus are dyed with Hoechst 33328.
Figure 15 cancer of pancreas (a) MIA-PaCa-2 and (b) PANC-1 cell are incubated for HA-NP-BE namo fluorescence probe respectively
The laser confocal microscope picture of 0min, 30min, 60min and 120min, nucleus are dyed with Hoechst 33328.
The laser confocal microscope picture of Figure 16 Patients with Pancreatic Cancer 1007CTC.CTC successively uses HA-NP-BE nano fluorescent
Probe and CK19-Alexa Fluor 647 are dyed.
The fluorescence intensity in each channel CTC, rate value and the H of calculating in Figure 17 Patients with Pancreatic Cancer 10072O2Concentration.
The laser confocal microscope picture of Figure 18 normal health human leukocytes.Leucocyte successively use HA-NP-BE nanometers it is glimmering
Light probe and CK19-Alexa Fluor 647 are dyed.
Specific embodiment
Hereinafter, technology of the invention is described in detail in conjunction with specific embodiment.It is appreciated that the various in detail below
Embodiment is only used for helping skilled in the art to understand the present invention, rather than limitation of the present invention.
The synthesis of embodiment 1.CD44 targeted nanometer fluorescence probe and tumor-targeting detection
Fig. 1 is hyaluronic acid-naphthalene imines-borate graft polymers synthesis, by 160mg (0.4mmol) hyaluronic acid
Sodium is dissolved in 50mL ultrapure water, and being vigorously stirred 2h dissolves it sufficiently.Then, 117.8mg (0.4mmol) DMTMM is added will
Activated carboxylic 30min on hyaluronic acid.Finally, 43.9mg (0.12mmol) NP-BE being dissolved in DMSO is added dropwise
Into above-mentioned mixed liquor, it is protected from light at room temperature for 24 hours.After reaction, it is with molecular cut off by above-mentioned reaction mixture
The bag filter of 14kDa is in DMSO/H2The in the mixed solvent of O (1:4v/v) is dialysed two days, is dialysed two days in pure water.Dialysis purification
Aqueous solution afterwards is freeze-dried to obtain HA-NP-BE graft polymers.HA-NP-BE graft polymers have it is good amphipathic,
It can be self-assembly of the H of CD44 targeting in aqueous solution2O2Responsive nano fluorescence probe.By HA-NP-BE namo fluorescence probe
With excessive H2O2Reaction 3h is stirred at room temperature, makes the pinacol borate deprotection on HA-NP-BE graft polymers, obtains
To HA-NP graft polymers, is then dialysed in the bag filter that molecular cut off is 14kDa with ultrapure water, remove excessive H2O2
The boric acid and pinacol small molecule generated with reaction, and it is self-assembly of HA-NP reference namo fluorescence probe in water.Pass through core
Magnetic hydrogen spectrum characterizes the chemical structure of HA-NP-BE graft polymers, as shown in Fig. 2, in 7.0~9.0ppm of chemical shift
Place occurs belonging to the characteristic peak of naphthalimide phenyl ring hydrogen, it was demonstrated that successfully NP-BE fluorescent molecule is grafted on HA.
Contain hydrophilic hyaluronic acid backbone and hydrophobic NP-BE side chain in HA-NP-BE graft polymers, this two
Parent's property structure can be self-assembly of the HA-NP-BE namo fluorescence probe of CD44 targeting in water.It is characterized with DLS and TEM
The self assembly behavior of HA-NP-BE graft polymers.DLS the result shows that, the average grain diameter of targeted nanometer fluorescence probe is 60nm
Left and right (Fig. 3 a).At the same time, the ruler for the nano spherical particle to be formed is assembled to HA-NP-BE graft polymers by tem observation
Very little very uniform, the test result of about 50nm or so (Fig. 3 b) and DLS is consistent.
In order to determine HA-NP-BE namo fluorescence probe and H2O2The optimum reacting time of reaction, respectively with UV absorption light
Spectrum and sepectrophotofluorometer have carried out dynamics research to it.It is added in the HA-NP-BE namo fluorescence probe of 200 μ g/mL
200 μM of H2O2, with the ratio of the ultraviolet absorption peak at ultraviolet tracking 450nm and at 350nm with HA-NP-BE namo fluorescence probe
And H2O2Reaction time variation tendency, excited at 450nm with sepectrophotofluorometer, record 550nm at fluorescence intensity
With the variation tendency in reaction time.Ultraviolet and fluorescence dynamics research show as shown in figure 4, and H2O2The 4000s of reaction
Interior, ultraviolet spectra shows I450/I350UV absorption ratio with HA-NP-BE namo fluorescence probe and H2O2Reaction time
Increase and rapidly increase, and good linearly increasing trend is presented, fluorescence spectrum shows that HA-NP-BE namo fluorescence probe exists
Preferable linear relationship is presented with the increase in reaction time in fluorescence intensity at 550nm in 2000s, both carries out in reaction
It tends to balance when to 7000s.Based on ultraviolet and fluorescence dynamic experiment as a result, glimmering by HA-NP-BE nanometers in subsequent experiment
Light probe and H2O2Reaction time be uniformly set as 30min.
In vitro in PBS environment, HA-NP-BE nano fluorescent is had rated with ultra-violet absorption spectrum and fluorescence spectrum respectively and is visited
For H2O2Responsiveness.As shown in figure 5, the maximal ultraviolet absorption peak of HA-NP-BE namo fluorescence probe is at 350nm, once
And H2O2After reaction, then 450nm has been arrived in red shift at maximal ultraviolet absorption peak, and with H2O2Concentration is continuously increased, at 350nm
Absorption peak constantly reduce, the absorption peak at 450nm constantly enhances.HA-NP-BE namo fluorescence probe and H2O2The time of reaction
Longer, which is more obvious (Fig. 5 c).In addition, UV absorption peak intensity and H at 450nm and at 350nm2O2Concentration
Change (Fig. 5 d) in a linear relationship.
Meanwhile the H of HA-NP-BE namo fluorescence probe and various concentration is also had rated with sepectrophotofluorometer2O2Reaction
The fluorescence intensity change of 30min.As shown in fig. 6, the green fluorescence intensity of HA-NP-BE namo fluorescence probe is with H2O2Concentration (0
~200 μM) increase and constantly enhance.To the increased fluorescence intensity of HA-NP-BE namo fluorescence probe and H2O2Concentration carries out line
Property fitting, both discoveries have a good linear relationship (Fig. 6 d), and the slope of a curve of fitting is that 0.91 (slope is said closer to 1
Bright curve it is linear better), R2It is 0.9988.From ultraviolet spectra it is found that HA-NP-BE namo fluorescence probe and H2O2After reaction
Maximal ultraviolet absorption is in 450nm, that is to say, that the maximum excitation wavelength of nano-probe is in 450nm.But in subsequent biologic applications
In, the either laser of flow cytometry or laser confocal microscope all without 450nm excitation wavelength.Therefore, glimmering
On light spectrophotometer, in addition to measuring the fluorescence intensity of HA-NP-BE namo fluorescence probe under the excitation wavelength of 450nm with H2O2
The variation of concentration, while also determining glimmering under conditions of common optical maser wavelength 405nm and 488nm in the instrument of biological detection
Intensity variation.Compare the fluorescence intensity discovery that three optical maser wavelengths excite lower HA-NP-BE namo fluorescence probe, 450nm excitation
Under fluorescence intensity it is most strong (Fig. 6 b), 405nm excitation will appear two fluorescence emission peak (respectively experiments of 500nm and 550nm
Under the conditions of in system not and H2O2The fluorescence probe of reaction and and H2O2The emission peak of the fluorescence probe of fully reacting), and
With H2O2Concentration increase, the fluorescence emission peak at 500nm constantly weakens, and the fluorescence emission peak at 550nm then can be continuous
Enhance (Fig. 6 a), H can be quantitatively detected by the ratio of the fluorescence intensity of the two2O2Concentration.As shown in fig 6e, by 550nm
With the fluorescence intensity ratio (I at 500nm550/I500) and H2O2Concentration carry out linear fit, obtained standard curve be y=
0.0341x+0.5361, R2It is 0.99988.The H2O2Concentration standard curve, due to being that the same excitation is same
Fluorescent molecule and the fluorescence for emitting two kinds of different wave lengths simultaneously, the influence of the ratio of fluorescence intensity not stimulated luminescence power,
Not by the Different Effects of the fluorescent emission efficiency of different fluorescent molecules, only and H2O2Concentration it is related, in actual biologic applications
In, disturbing factor is smaller, H that can more truly in reacting cells2O2It is horizontal.In addition, 488nm can equally excite HA-NP-BE
The fluorescence (Fig. 6 c) of namo fluorescence probe, but fluorescence intensity ratio 450nm and 405nm excitation are weak.It is green in order to more intuitively observe
Color fluorescence intensity changes from scratch, and the HA- in the laser activated cell of 488nm is selected in subsequent Bioexperiment
The fluorescence signal of NP-BE namo fluorescence probe, and then excited with 405nm in circulating tumor cell detection, pass through two kinds of fluorescence
The variation half-quantitative detection H of color2O2Concentration.
In order to which preferably HA-NP-BE namo fluorescence probe is applied in cancer of pancreas detection, nano-probe is had rated first
Biocompatibility.The inherent cytotoxicity of HA-NP-BE nano-probe is had detected with the mtt assay of standard, the cell strain of selection is
Normal cell RAW 264.7 and pancreatic cancer cell PANC-1.By RAW 264.7 and PANC-1 cell respectively with various concentration
HA-NP-BE namo fluorescence probe, which is incubated for, detects absorbance value with microplate reader afterwards for 24 hours, and calculates the survival rate of cell.Such as Fig. 7 institute
Show, after being incubated for for 24 hours, even if HA-NP-BE namo fluorescence probe at concentrations up to 1000 μ g/mL, RAW 264.7 and PANC-1
Also there is 80% cell survival rate.MTT experiment illustrates that HA-NP-BE namo fluorescence probe has good biocompatibility, can make
For the ideal material of biological detection.
The intracellular of the HA-NP-BE namo fluorescence probe of CD44 targeting, which is had rated, with flow cytometry takes the photograph ability.In vitro
Ultraviolet and fluorescence experiments all show HA-NP-BE namo fluorescence probe have good H2O2Responsiveness can not directly use nanometer
Fluorescence probe itself takes the photograph ability to evaluate into the cell.For this purpose, in advance by HA-NP-BE namo fluorescence probe and H2O2Sufficiently reaction,
It is stripped of to H2O2HA-NP reference namo fluorescence probe, fluorescence intensity is prepared in the pinacol borate group of responsiveness
No longer by intracellular H2O2The influence of concentration can effectively evaluate the ability of cellular uptake namo fluorescence probe.HA-NP is joined
Than namo fluorescence probe respectively with HEK 293, RAW 264.7, PANC-1 and MIA-PaCa-2 cell incubation 30min, 60min and
120min has found every kind with fluorescence intensity of the flow cytometry analysis reference nano-probe in tumour cell and normal cell
Cell is to the intake ability of namo fluorescence probe all different (Fig. 8), in the fluidic cell fluorogram of measure of cell intake
Fluorescence intensity it is for statistical analysis, the size of the intake ability of four kinds of cells is MIA-PaCa-2 > PANC-1 > RAW 264.7
The intake of reference namo fluorescence probe is presented with altogether in ≈ HEK 293 (Fig. 9), pancreatic cancer cell MIA-PaCa-2 and PANC-1
The time of culture increases and increased trend, even and if normal cell RAW 264.7 and HEK 293 and the incubation time of probe prolong
To two hours, intracellular fluorescence intensity does not also change significantly length.Above-mentioned experimental result can illustrate nano fluorescent
It is absorbed by human pancreatic carcinoma cell to the probe property of can choose, and cannot be introduced into normal cell.
It is above-mentioned the experimental results showed that, have very much can for the difference of normal cell and human pancreatic carcinoma cell intake namo fluorescence probe
Can be due to caused by the CD44 of cell surface, CD44 is a member in cell adhesion molecules family, mediated cell and cell,
The film surface glycoprotein of adhesion between cell and extracellular matrix, participates in the proliferation of cell, breaks up, sticks, migrating.CD44 master
The heterotypic adhesion of tumour cell Yu host cell and host matrix is participated in, and heterotypic adhesion is shifted in tumor cell invasion
In play a driving role.Therefore, the very possible high expression of the CD44 antigen on human pancreatic carcinoma cell surface, and the CD44 of normal cell
Then low expression.In order to verify this experimental phenomena, with Western Blot experimental analysis MIA-PaCa-2, PANC-1, RAW
The expression quantity of 264.7 and HEK, 293 4 kinds of cell surface CD44 albumen simultaneously demonstrates this imagination.As shown in Figure 10 a, MIA-
The band color of the CD44 of PaCa-2 is most deep, PANC-1 secondly, RAW 264.7 is compared with HEK 293 with internal reference ACTIN band,
Color is all shallower, and the band color of CD44 is deeper in Western Blot experiment illustrates that CD44 expressing quantity is more.Take CD44
The ratio of the gray value of the sum of the grayscale values internal reference band of protein band is further analyzed, as shown in fig. lob, CD44 expressing quantity
MIA-PaCa-2 > PANC-1 > RAW264.7 ≈ HEK 293, the rule of this and four kinds of cellular uptake HA-NP reference namo fluorescence probes
Rule is consistent, effectively demonstrates the imagination of experimental result.In addition, hyaluronic acid is that each structural unit contains on a kind of main chain
The natural biological macromolecular of carboxyl is self-assembly of nanometer as the water-solubility carrier of hydrophobic NP-BE fluorescent molecule in water
Particle, surface can have a large amount of negative electrical charge, and Zeta potential is -29.1mV (Figure 11).If nanoparticle does not have targeting,
Cell membrane itself is negatively charged, since like charges are mutually exclusive, are difficult to gulp down effect into cell by packet, demonstrate from side
HA-NP-BE namo fluorescence probe has targeting to human pancreatic carcinoma cell.
HA-NP-BE namo fluorescence probe is had rated to endogenous cellular H with flow cytometry2O2Responsiveness.Firstly, with
Normal cell RAW 264.7 and 293 cell of HEK are as negative control group, in this experiment, in order in more preferable simulation blood
Normal cell, specially selected the macrophage of mouse, macrophage is a kind of parting of leucocyte, and leucocyte is human body
A large amount of existing normal cells in blood.Streaming with respective blank control cell the experimental results showed that compare, HA-NP-BE receives
Even if the incubation time of rice fluorescence probe and 293 cell of RAW 264.7 and HEK extends to 2h, into the cell also almost without green
Color fluorescence signal (Figure 12 a and b).On the one hand, the CD44 expression on normal cell surface is lower, is difficult to absorb electronegative HA-NP-
BE namo fluorescence probe；On the other hand, the endogenous H in normal cell2O2It is horizontal lower in itself, HA-NP-BE nanometers can not be made
The green fluorescence of fluorescence probe has apparent enhancing, the two factors result in the intensity of cellular fluorescence of normal cell very jointly
It is low.On the contrary, using human pancreatic carcinoma cell MIA-PaCa-2 and PANC-1 as positive test group, as shown in Figure 12 c and d, carefully
Green florescent signal intracellular is remarkably reinforced, and illustrates the endogenous H in tumour cell2O2It is horizontal higher, it can be effectively by naphthalene Asia
Pinacol borate deprotection on amine-borate group, and enhance green fluorescence.Intracellular fluorescence intensity is carried out
It analyzes (Figure 13), it is different in conjunction with intake of two kinds of human pancreatic carcinoma cells to HA-NP-BE namo fluorescence probe, although
Its intracellular fluorescence intensity is almost the same after MIA-PaCa-2 and PANC-1 is incubated for 30min with nano-probe jointly, but nanometer
Probe is much higher in the intracellular concentration ratio PANC-1 of MIA-PaCa-2, although and the concentration of the intracellular nano-probe of PANC-1
It is lower, but its enhancing fluorescence intensity is but equally matched with MIA-PaCa-2, shows actually intracellular H2O2Horizontal PANC-1
It is more much higher than MIA-PaCa-2.
With laser confocal microscope to HA-NP-BE namo fluorescence probe to intracellular endogenous H2O2Level carries out glimmering
Light imaging analysis.As shown in figure 14, normal cell RAW 264.7 and HEK 293 is hardly visible the green fluorescence of naphthalene imines, only
Blue-fluorescence after thering is nucleus to be dyed by Hoechst 33328, and it is with HA-NP-BE namo fluorescence probe and normally thin
The extension of born of the same parents' incubation time, also without apparent green florescent signal.And to cancer of pancreas MIA-PaCa-2 and PANC-1 cell into
When row fluorescence imaging, in addition to the blue-fluorescence of nucleus, moreover it is possible to observe naphthalene imines-borate and into the cell interior in cytoplasm
Source property H2O2The green fluorescence issued after reaction removing pinacol borate protection, and with the extension of incubation time, green is glimmering
Luminous intensity is enhanced (Figure 15).Above-mentioned laser confocal microscope is the result shows that HA-NP-BE namo fluorescence probe can be effective
Ground enters cell, and to intracellular H2O2It is imaged, green fluorescence is weaker in normal cell, H2O2Concentration is lower, and pancreas cancerous swelling
Fluorescence in oncocyte is stronger, H2O2Concentration is higher, can efficiently differentiate normal cell and pancreatic tumour, realizes tumor target
Tropism detection.
The identification of 2. Patients with Pancreatic Cancer circulating tumor cell of embodiment
HA-NP-BE namo fluorescence probe can with targeting detect the endogenous H in pancreatic cancer cell2O2, and have fast
The responsiveness of speed, can efficiently differentiate human pancreatic carcinoma cell and normal cell, be applied to cancer of pancreas in adjuvant clinical
The detection of the circulating tumor cell of patient.Ethics Committee of Shanghai Communications University has approved this experiment.Collect pancreas clinically
Gland cancer patient's peripheral blood passes through the method enrichment cycles tumour cell of negative selection.Firstly, being removed using erythrocyte cracked liquid red thin
Born of the same parents using most leucocyte in the magnetic bead removal blood of CD45 antibody label, then successively use the CTC being enriched to respectively
HA-NP-BE namo fluorescence probe and CK19-Alexa Fluor 647 are dyed, and are finally seen under laser confocal microscope
It examines and counts.Fluorescence imaging analysis is carried out to the CTC captured, as shown in figure 16, although the CTC observed under CLSM is each
The green fluorescence intensity of cell is all not quite identical, but can observe bright green fluorescence, shows following for Patients with Pancreatic Cancer
H in ring tumour cell2O2It is horizontal very high.In addition, the CK19 marked by Alexa Fluor 647 red fluorescence is to detecting
Doubtful circulating tumor cell has carried out total verifying, and it is glimmering that the doubtful CTC of most of fluoresced green can be caught stronger red
Light, and red fluorescence and green fluorescence are not fully overlapped, that is, CK19 positive, H2O2The positive, but have also discovered CK19 yin
Property, H2O2The positive, that is, only stronger green fluorescence and the very weak cell of red fluorescence, this is likely to be because of epithelium
The low expression of CK19 caused by interstitial transition effects or caused by not expressing, as number is that three of 8,9 and 12 doubt in Figure 16
Like CTC.Thus, H2O2Positive cell is exactly circulating tumor cell, although due to the height heterogeneity of tumour, in tumour cell
H2O2Expression, which also has height, H that is low, but designing2O2The namo fluorescence probe of responsiveness is to H2O2With very high sensitivity,
Intracellular endogenous H can be effectively detected out by the enhancing of fluorescence intensity2O2, compared to by detecting cell surface
Specific proteins such as CK19 is sensitiveer.Pass through the H in detection tumour cell2O2To identify CTC, only H2O2The nanometer of responsiveness
Fluorescence probe, which enters, just can be effectively carried out detection in tumour cell living, and dead CTC can not absorb nano fluorescent spy
Needle, intracellular H2O2Small molecule also can be with the death of cell, and permeability of cell membranes increases or diffuses out cell or because of itself
It is unstable and decompose.And the specific proteins CK19 detection CTC for detecting cell surface is easy to because CK19 antibody and cell are incubated
The time educated has been grown or has entered cell by cell endocytic or nonspecific absorption or even apoptosis or death occurs in cell surface
The surface of circulating tumor cell also have CK19, ultimately cause nonspecific dyeing and generate false positive results.Therefore, pass through
Detect the H in CTC2O2To identify that CTC is more specific.Currently, clinically special tumour this to cancer of pancreas, due to EpCAM
Cause the verification and measurement ratio of CTC very low with the low expression of CK19, false negative result is very high.It is above-mentioned the experimental results showed that HA-NP-BE receives
Rice fluorescence probe is by detecting intracellular H2O2The CTC in pancreatic tumour patient can be effectively detected out in level, facilitate
The early diagnosis of cancer of pancreas.
Since HA-NP-BE namo fluorescence probe can emit simultaneously two kinds of different wave lengths under the excitation of 405nm excitation wavelength
Fluorescence, can use the ratio of blue-fluorescence and green fluorescence, according to Standard in vitro curve y=0.0341x+0.5361 calculate
H in circulating tumor cell2O2Level, as shown in figure 17, the H in Patients with Pancreatic Cancer CTC being calculated2O2Level for 10~
It is 30 μM, more consistent with concentration range reported in the literature.Although the standard curve is that fitting obtains in an in vitro environment, but same
A fluorescent molecule is excited by the same excitation wavelength, and the power of exciting light is identical, and the emission effciency of fluorescence is also identical, even if
H also can be accurately measured in complicated biological environment2O2It is horizontal.
In addition, using the leucocyte of normal healthy people as the negative control experiment group of CTC, it is glimmering to verify HA-NP-BE nanometers
The specificity that light probe detects Patients with Pancreatic Cancer circulating tumor cell.Successively use HA-NP-BE namo fluorescence probe and CK19-
Alexa Fluor 647 dyes the leucocyte of normal healthy people, as shown in the laser confocal microscope picture of Figure 18,
The leucocyte volume very little (< 10 μm) of normal person, without HA-NP-BE namo fluorescence probe and H2O2Green fluorescence letter after reaction
Number, show the H in normal human leukocytes2O2Horizontal very low, also the not no red fluorescent of CK19, shows leucocyte CK19 not
Expression or low expression.It is above-mentioned the experimental results showed that the HA-NP-BE namo fluorescence probe of CD44 targeting can be effectively reduced it is outer
The interference of leucocyte in all blood, improves the detection efficiency of probe.
This feature is expressed using tumor cell surface CD44 high, it is targeted with hyaluronic acid.Therefore, it introduces
The hyaluronic acid of CD44 targeting is as H2O2Responsiveness naphthalene imines-borate fluorescent small molecule carrier, and wherein, naphthalene imines-
Borate and H2O2Reaction front and back has blue and the two different colors of fluorescence of green, is a kind of fluorescence from ratiometer point
Son.The highly expressed cancer of pancreas of CD44 can be entered with targeting from ratiometer namo fluorescence probe based on this targeting formed
In CTC and to intracellular H2O2Imaging and semi-quantitative analysis, to finally identify CTC.Pass through targets neoplastic cells surface
The detection efficiency of namo fluorescence probe can be improved in CD44, reduces the interference of leucocyte, reduces in human pancreatic carcinoma cell detection
False negative result and false positive results.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (4)
1. a kind of namo fluorescence probe of targeted pancreatic cancer circulating tumor cell, which is characterized in that the probe includes H2O2Response
The fluorescent small molecule of property and the polymer of targeted pancreatic cancer surface marker, the H2O2The fluorescent small molecule and targeting of responsiveness
The polymer of cancer of pancreas surface marker has opposite hydrophilic and hydrophobic.
2. a kind of namo fluorescence probe of targeted pancreatic cancer circulating tumor cell according to claim 1, which is characterized in that
The H2O2The fluorescent small molecule of responsiveness includes naphthalimide-borate fluorescent small molecule NP-BE, cumarin borate, fluorescence
One of plain borate, carbazole-borate, two pyrroles of fluorine boron-borate.
3. a kind of namo fluorescence probe of targeted pancreatic cancer circulating tumor cell according to claim 1, which is characterized in that
The cancer of pancreas surface marker is CD44.
4. a kind of namo fluorescence probe of targeted pancreatic cancer circulating tumor cell according to claim 3, which is characterized in that
The polymer of the targeted pancreatic cancer surface marker is hyaluronic acid HA.
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| CN111704682A (en) * | 2020-07-03 | 2020-09-25 | 广东工业大学 | Compound and nano micelle |
| CN111892668A (en) * | 2020-07-03 | 2020-11-06 | 广东工业大学 | Compound, preparation method thereof, fluorescent probe and antitumor drug |
| CN115791727A (en) * | 2022-11-26 | 2023-03-14 | 吉林大学 | Application of fat-soluble fluorescent probe in neuronal death detection and detection method |
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| CN108844935B (en) * | 2018-07-06 | 2020-07-24 | 山西大同大学 | Preparation method and application of boron-nitrogen co-doped carbon dots |
| CN110044861A (en) * | 2019-05-09 | 2019-07-23 | 南宁师范大学 | The detection method of concentration of hydrogen peroxide |
| CN112924610A (en) * | 2021-02-01 | 2021-06-08 | 上海交通大学 | Mass spectrum-based ROS absolute quantification method in living cells and application thereof |
| CN113624727B (en) * | 2021-07-02 | 2024-06-18 | 湖北文理学院 | Method for detecting hydrazine concentration |
| CN113984729B (en) * | 2021-10-29 | 2022-10-28 | 中国科学院自动化研究所 | Synthesis method and application of hypoxic response type ratiometric probe |
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| CN115791727A (en) * | 2022-11-26 | 2023-03-14 | 吉林大学 | Application of fat-soluble fluorescent probe in neuronal death detection and detection method |
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| CN109251743B (en) | 2021-09-07 |
| CN109251743A (en) | 2019-01-22 |
| CN107543808A (en) | 2018-01-05 |
| CN109253990B (en) | 2021-10-15 |
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