CN114854415B - Rare earth optical probe and preparation method and application thereof - Google Patents
Rare earth optical probe and preparation method and application thereof Download PDFInfo
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- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
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Abstract
The invention discloses a rare earth optical probe and a preparation method and application thereof, wherein the rare earth optical probe comprises: kernel NaYbF 4 :Er 3+ ,Ce 3+ Shell NaYF 4 :Yb 3+ NaYF shell 4 :Nd 3+ And modifying the shell NaYF 4 :Nd 3+ Surface dibenzocyclooctyne. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ion (Er 3+ Ions and Nd 3+ Ions) with a high-intensity NIR-IIb fluorescence emission; the biological orthogonality is provided by modifying the functional group-alkynyl on the surface of the probe; when the rare earth optical probe and the tumor extracellular vesicles with homologous targeting are subjected to high-efficiency biological orthogonal combination in a living body, the tumor enrichment rate and the residence time of the optical probe can be enhanced, and long-time dynamic fluorescence imaging of living tumors can be realized.
Description
Technical Field
The invention relates to the technical field of optical biological imaging, in particular to a rare earth optical probe and a preparation method and application thereof.
Background
Rare earth doped Nanoparticles (NPs) have excellent optical properties such as no light bleaching, no flicker, narrow emission bandwidth, long lifetime, etc., making them a very promising class of optical probes. More importantly, erbium ion doped (Er 3+ ) The down-conversion nanocrystals (DCNPs) have characteristic fluorescence emission peaks at 1525nm, which are suitable for in vivo imaging of NIR-IIb.
Currently, er 3+ Ion doped optical probes have been designed and used to achieve millimeter depthWhole body and cerebral vessels imaging with micron-scale resolution. Although rare earth optical probes have made some progress in the field of in vivo fluorescence imaging, rare earth optical probes tend to exhibit poor targeting and short residence time at tumor sites, using only the traditional Enhanced Permeability and Retention (EPR) effects, greatly limiting their application in vivo tumor detection and therapy.
Currently, in order to improve the targeting ability of tumors, researchers modify various active targets such as folic acid, nucleic acid, antibodies, polypeptides and the like on the surface of a rare earth optical probe to enhance the targeting enrichment ability of the rare earth optical probe to tumors. However, the presence of such problems as limited expression of biological receptors on tumor cells, low binding efficiency of the receptors to the ligands, etc. still prevents efficient enrichment of the probe at the tumor site.
Accordingly, the prior art is still in need of improvement and development.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide a rare earth optical probe and a preparation method and application thereof, and aims to solve the problems that in the prior art, the binding efficiency of a receptor on the surface of the rare earth optical probe and a ligand on the surface of a modified rare earth optical probe is low, and efficient enrichment of the probe at a tumor part is hindered.
The technical scheme of the invention is as follows:
a rare earth optical probe comprising:
kernel NaYbF 4 :Er 3+ ,Ce 3+ ；
Shell NaYF 4 :Yb 3+ The shell layer NaYF 4 :Yb 3+ Grown on the inner core NaYbF 4 :Er 3+ ,Ce 3+ A surface;
shell NaYF 4 :Nd 3+ The shell NaYF 4 :Nd 3+ Grown on the shell layer NaYF 4 :Yb 3+ A surface;
dibenzocyclooctyne for modifying the shell NaYF 4 :Nd 3+ Is a surface of the substrate.
The rare earth optical probe comprisesIn the shell layer NaYF 4 :Yb 3+ Middle Yb 3+ The doping concentration of the ions was 100%.
The rare earth optical probe has a spherical structure with the size of 21.5 nm-29.5 nm.
The preparation method of the rare earth optical probe comprises the following steps:
preparation of Er by coprecipitation method 3+ Ion and Ce 3+ Ion doped NaYbF 4 Kernel, kernel NaYbF is prepared 4 :Er 3+ ,Ce 3+ ；
At the inner core NaYbF 4 :Er 3+ ,Ce 3+ The surface is sequentially connected and grown with NaYF by thermal cracking 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ A shell, a rare earth optical probe precursor is prepared;
then washing and centrifuging the rare earth optical probe precursor by using ethanol;
modifying amphiphilic DSPE-PEG-NH on the surface of the rare earth optical probe precursor 2 ；
Finally, in DSPE-PEG-NH 2 And (3) connecting DBCO-NHS on the basis of the ligand to prepare the rare earth optical probe.
The preparation method of the rare earth optical probe comprises the steps of preparing Er by adopting a coprecipitation method 3+ Ion and Ce 3+ Ion doped NaYbF 4 The kernel specifically comprises:
YbCl 3 ·6H 2 O、ErCl 3 ·6H 2 O、CeCl 3 ·7H 2 Adding the mixture of O, oleic acid and octadecene into a container, stirring and heating to 150 ℃, preserving heat for 1-2 hours, and cooling to 35-45 ℃ to prepare rare earth salt precursor solution;
NaOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F, solution;
the NaOH solution and NH 4 F, sequentially dripping the solution F into the rare earth salt precursor solution to obtain a mixed solution, and after the mixed solution is kept at room temperature for 0.5-1 hour, adding the mixed solutionCooling to room temperature after heat treatment;
then centrifugal and washing are carried out to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
The preparation method of the rare earth optical probe comprises the steps of 4 :Er 3+ ,Ce 3+ The surface is sequentially connected and grown with NaYF by thermal cracking 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ The shell specifically includes:
yb (CF) 3 COO) 3 、CF 3 COONa and said inner core NaYbF 4 :Er 3+ ,Ce 3+ Adding into oleic acid and octadecene mixed solution, heating, centrifuging, and washing to obtain NaYbF core 4 :Er 3+ ,Ce 3+ Surface-coated NaYF of (C) 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Yb NPs are added into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing are carried out to prepare the rare earth optical probe precursor.
The preparation method of the rare earth optical probe is carried out in a nitrogen atmosphere.
The application of the rare earth optical probe is that the rare earth optical probe is applied to the short wave near infrared tumor targeted fluorescence imaging of the nano probe;
wherein the rare earth optical probe is the rare earth optical probe.
The application of the rare earth optical probe is characterized in that the rare earth optical probe is used for generating biological orthogonal combination with tumor extracellular vesicles with homology targeting, so as to realize NIR-IIb tumor targeted fluorescence imaging.
The application of the rare earth optical probe, wherein the tumor extracellular vesicles are extracellular vesicles secreted by human colon cancer cells and expressing azide genes.
The beneficial effects are that: the invention provides a rare earth optical probe, a preparation method and application thereof, wherein the rare earth optical probe comprises: kernel NaYbF 4 :Er 3+ ,Ce 3+ Shell NaYF 4 :Yb 3+ NaYF shell 4 :Nd 3+ And modifying the shell NaYF 4 :Nd 3+ Dibenzocyclooctyne on the surface; the shell layer NaYF 4 :Yb 3+ Grown on the inner core NaYbF 4 :Er 3+ ,Ce 3+ A surface; the shell NaYF 4 :Nd 3+ Grown on the shell layer NaYF 4 :Yb 3+ A surface. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ion (Er 3+ Ions and Nd 3+ Ions) with a high-intensity NIR-IIb fluorescence emission; the biological orthogonality is provided by modifying the functional group-alkynyl on the surface of the probe; the NIR-IIb fluorescence emission is used as a receiving wave band of living body imaging, so that the resolution and the signal-to-noise ratio of living body tumor imaging are enhanced; when the rare earth optical probe and the tumor extracellular vesicles with homologous targeting are subjected to high-efficiency biological orthogonal combination in a living body, the tumor enrichment rate and the residence time of the optical probe can be enhanced, long-time dynamic fluorescence imaging of living tumors can be realized, and the optical probe has good application prospect in the field of optical biological imaging.
Drawings
FIG. 1 is an energy transmission path diagram, a corresponding TEM diagram and an element distribution diagram of a three-layer rare earth optical probe synthesized in example 1 of the present invention;
FIG. 2 is a graph showing the fluorescence emission intensity of NIR-IIb obtained by exciting rare earth optical probes with different Yb doping concentrations with 808nm laser in example 2 of the present invention;
FIG. 3 is a TEM image, a hydraulic radius analysis image and an infrared spectrum of the bio-orthogonal rare earth optical probe after DBCO connection in example 3 of the present invention;
FIG. 4 is a performance characterization and in vitro tumor targeting capability display of example 4 of the present invention;
FIG. 5 is a dynamic fluorescence imaging image of NIR-IIb living tumor, a fluorescence signal intensity analysis image of tumor sites, and an enrichment analysis image of probes at tumor sites in example 5 of the present invention.
Detailed Description
The invention provides a rare earth optical probe, a preparation method and application thereof, and the invention is further described in detail below in order to make the purposes, technical schemes and effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Optical biological imaging plays a vital role in many biomedical fields, from early disease diagnosis to monitoring disease progression to image-guided treatment of life threatening diseases, and to evaluate therapeutic efficacy and prognosis. The permeability of light in biological tissue depends on the absorption and scattering capabilities of the tissue components for light, which also directly affects biological imaging quality, including imaging intensity, penetration depth, signal-to-noise ratio, and imaging resolution.
Therefore, fluorescent biological imaging obtained with visible light (400-700 nm) and conventional near infrared (NIR-I; 700-950 nm) fluorescence wavelengths is poor in quality due to light absorption, light scattering and autofluorescence of biological tissue. In contrast, the window of NIR-IIb (1500-1700 nm) can realize deeper tissue penetration depth, high signal-to-noise ratio and excellent space-time resolution, and provides a new opportunity for diagnosis and treatment of deep focus diseases, due to the weak absorption capacity, low scattering coefficient and negligible autofluorescence of biological tissues.
Rare earth doped Nano Particles (NPs) have excellent optical characteristics, so that the nano particles become an optical probe with very good development prospect. However, in order to improve the targeting ability of tumors, researchers modify various active targets such as folic acid, nucleic acid, antibody, polypeptide and the like on the surface of a rare earth optical probe to enhance the targeting enrichment ability of the rare earth optical probe to tumors.
However, the presence of such problems as limited expression of biological receptors on tumor cells, low binding efficiency of the receptors to the ligands, etc. still prevents efficient enrichment of the probe at the tumor site.
Based on the above, the invention provides a rare earth optical probe, wherein the rare earth optical probe is Y 3+ 、Yb 3+ 、Er 3+ 、Ce 3+ 、Nd 3+ Rare earth nanocrystalline of three-layer structure composed of ions (NaYbF 4 :Er 3+ ,Ce 3+ @NaYF 4 :Yb 3+ @NaYF 4 :Nd 3+ NPs)。
Specifically, the rare earth optical probe includes: kernel NaYbF 4 :Er 3+ ,Ce 3+ The method comprises the steps of carrying out a first treatment on the surface of the Shell NaYF 4 :Yb 3+ The shell layer NaYF 4 :Yb 3+ Grown on the inner core NaYbF 4 :Er 3+ ,Ce 3+ A surface; shell NaYF 4 :Nd 3+ The shell NaYF 4 :Nd 3+ Grown on the shell layer NaYF 4 :Yb 3+ A surface; dibenzocyclooctyne for modifying the shell NaYF 4 :Nd 3 + Is a surface of the substrate.
The rare earth optical probe is designed into a three-layer structure, and the inner cores NaYbF are respectively arranged from inside to outside 4 :Er 3+ ,Ce 3+ Shell NaYF 4 :Yb 3+ Outer shell NaYF 4 :Nd 3+ The three-layer rare earth optical probe can avoid unnecessary rare earth ions (Er) 3+ Ions and Nd 3+ Ions) such that the rare earth optical probe has a highlighted NIR-IIb fluorescence emission; in addition, the invention is arranged on the shell NaYF 4 :Nd 3+ The surface of the rare earth optical probe is modified with Dibenzocyclooctyne (DBCO), so that the rare earth optical probe is endowed with biological orthogonality, and the dibenzocyclooctyne is utilized to modify the rare earth optical probe, so that the binding efficiency of a receptor on the surface of the rare earth optical probe and a ligand dibenzocyclooctyne can be improved, and further, the efficient enrichment of tumor parts is realized. When the tumor cell vesicle with homologous targeting is combined with the tumor cell vesicle to form a biorthogonal platform, the biorthogonal platform has the advantages of high tumor targeting rate, long enrichment time, high imaging resolution and low imaging signal-to-noise ratio, and has better biocompatibility and lower immunogenicity; moreover, the bioorthogonal platform can realize long-time (48 h) dynamic fluorescence imaging of living tumors.
Specifically, the invention utilizes a rare earth optical probe with high-brightness NIR-IIb fluorescence emission, endows the probe with bioorthogonal capability by modifying a functional group-alkynyl (-C≡C-) on the surface of the probe, and enables tumor Extracellular Vesicles (EVs) with homologous targeting to successfully express an azide group (-N) through metabolic labeling 3 ). In living bodies, the copper-free click reaction is utilized to enable the rare earth optical probe modified with alkynyl groups and the tumor extracellular vesicles expressing azide groups to be subjected to high-efficiency biological orthogonal combination, so that the targeted enrichment and residence time of the rare earth optical probe at tumor positions are enhanced, and the NIR-IIb living body tumor targeted fluorescence imaging under 808nm excitation is realized.
In some embodiments, the shell layer NaYF 4 :Yb 3+ Middle Yb 3+ The doping concentration of the ions was 100%.
In some embodiments, the rare earth optical probe is a spherical structure having a size between 21.5nm and 29.5 nm. Preferably, the rare earth optical probe has a uniform spherical structure with a size of 26.5 nm.
In addition, the invention also provides a preparation method of the rare earth optical probe, which comprises the following steps:
step S10: preparation of Er by coprecipitation method 3+ Ion and Ce 3+ Ion doped NaYbF 4 Kernel, kernel NaYbF is prepared 4 :Er 3+ ,Ce 3+ ；
Step S20: at the inner core NaYbF 4 :Er 3+ ,Ce 3+ The surface is sequentially connected and grown with NaYF by thermal cracking 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ A shell, a rare earth optical probe precursor is prepared;
step S30: then washing and centrifuging the rare earth optical probe precursor by using ethanol;
step S40: modifying amphiphilic DSPE-PEG-NH on the surface of the rare earth optical probe precursor 2 ；
Step S50: finally, in DSPE-PEG-NH 2 And (3) connecting DBCO-NHS on the basis of the ligand to prepare the rare earth optical probe.
According to the invention, the rare earth optical probe is designed to be of a three-layer structure and is modified by dibenzooctyne on the surface of the rare earth optical probe, so that energy loss caused by cross relaxation among rare earth ions can be avoided, a high-brightness NIR-IIb (1525 nm) fluorescent signal can be generated under the condition of 808nm laser excitation, the biological orthogonality of the rare earth optical probe can be endowed, the rare earth optical probe has higher targeting to tumors, and the resolution and the signal-to-noise ratio of living tumor imaging are enhanced.
In some embodiments, the step S10 specifically includes the steps of:
step S11: ybCl 3 ·6H 2 O、ErCl 3 ·6H 2 O、CeCl 3 ·7H 2 Adding the mixture of O, oleic acid and octadecene into a container, stirring and heating to 150 ℃, preserving heat for 1-2 hours, and cooling to 35-45 ℃ to prepare rare earth salt precursor solution;
step S12: naOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F, solution;
step S13: the NaOH solution and NH 4 Dropwise adding the solution F into the rare earth salt precursor solution in sequence to obtain a mixed solution, keeping the mixed solution at room temperature for 0.5-1 hour, and cooling the mixed solution to room temperature after heating treatment;
step S14: then centrifugal and washing are carried out to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
Specifically, the heating treatment in step S13 includes a low-temperature heating treatment and a high-temperature heating treatment; the low-temperature heating treatment is divided into three stages: the first stage is to keep the temperature at 50 ℃ for 30min, the second stage is to keep the temperature at 70 ℃ for 20min, and the third stage is to keep the temperature at 100 ℃ for 30min; the purpose of the low temperature treatment is mainly to remove excess formaldehyde and water from the solution. The high temperature treatment is to keep the temperature at 280-320 ℃ for 40-60min, and the high temperature treatment is mainly used for reacting the mixed solution to generate the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
In some embodiments, the centrifugation in step S14 is performed at 6000rpm using an ethanol solvent; followed by washing with a 1:6 hexane/ethanol mixture at least twice.
In some embodiments, the step S20 specifically includes the steps of:
step S21: yb (CF) 3 COO) 3 、CF 3 COONa and said inner core NaYbF 4 :Er 3+ ,Ce 3+ Adding into oleic acid and octadecene mixed solution, heating, centrifuging, and washing to obtain NaYbF core 4 :Er 3+ ,Ce 3+ Surface-coated NaYF of (C) 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Step S22: y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Yb NPs are added into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing are carried out to prepare the rare earth optical probe precursor.
By way of illustration: first, a coprecipitation method is adopted by mixing NaOH and NH 4 F separate addition of NaYbF to smaller size 4 2% Er,2% Ce nanocrystalline core, then, thermal cracking method is adopted to grow NaYF on the surface of the core 4 Yb shell layer followed by NaYbF 4 :2％Er,2％Ce@NaYF 4 Yb is taken as a nucleus, and NaYF is grown on the surface of the Yb 4 30% Nd surface layer, finally obtaining NaYbF 4 :2％Er,2％Ce@NaYF 4 :Yb@NaYF 4 30% of Nd nanocrystalline. In the shell growth process, the thickness of the shell and the composition of the rare earth elements of the shell are controlled by adding rare earth trifluoroacetate and sodium trifluoroacetate in different types and proportions.
Then, at NaYbF 4 :2％Er,2％Ce@NaYbF 4 @NaYF 4 30% Nd nanocrystalline surface modification DSPE-PEG-NH 2 . Subsequently, an amide reaction of the amino group with the ester group is used in DSPE-PEG-NH 2 The DBCO-NHS is linked on the basis of the ligand.
In some embodiments, the method of preparing the rare earth optical probe is performed under a nitrogen atmosphere.
In addition, the invention also provides application of the rare earth optical probe, which is applied to the nano probe shortwave near infrared tumor targeted fluorescence imaging; wherein the rare earth optical probe is the rare earth optical probe.
In some embodiments, the rare earth optical probe is used to bio-orthogonally bind to tumor extracellular vesicles with cognate targeting to achieve NIR-IIb tumor targeted fluorescence imaging.
In some embodiments, the tumor extracellular vesicles are extracellular vesicles secreted by human colon cancer cells (HCT-116) that express an azide gene.
In some embodiments, the route of acquisition of the extracellular vesicles is as follows:
step A: incubating N-azidoacetylmannosamine-tetraacylation (Ac 4 ManNAz) with HCT-116 for 48h, and using metabolic markers to make HCT-116 cell membrane surface express azido groups;
and (B) step (B): extraction of azide-expressing extracellular vesicles (EVs-N) secreted by HCT-116 cells using a kit 3 )。
In this embodiment, DBCO modified NaYbF 4 :Er 3+ ,Ce 3+ @NaYF 4 :Yb 3+ @NaYF 4 :Nd 3+ NPs and extracellular vesicles secreted by HCT-116 and expressing azide groups are combined in a biological mode in a living body in a high-efficiency biological orthogonal mode, and the targeting effect on tumors is high.
The following examples are further illustrative of the invention. It is also to be understood that the following examples are given solely for the purpose of illustration and are not to be construed as limitations upon the scope of the invention, since numerous insubstantial modifications and variations will now occur to those skilled in the art in light of the foregoing disclosure.
Example 1
Rare earth optical probe for preparing three-layer structure
First, naYbF was prepared by the coprecipitation method 4 2% Er and 2% Ce kernel. Will 0.96mmol YbCl 3 ·6H 2 O、0.02mmol ErCl 3 ·6H 2 O and 0.02mmol CeCl 3 ·7H 2 A mixture of O with 6mL oleic acid and 15mL octadecene was added to a 250mL flask, the solution was stirred and heated to 150℃for 60 minutes. Subsequently, after the solution had cooled to 40℃2.5mmol NaOH and 4.0mmol NH were added 4 F is dissolved in 6ml and 8ml of methanol respectively, and then NaOH solution and NH are added 4 And F, sequentially dripping the solution into the rare earth salt precursor solution, keeping the room temperature for 30min, sequentially heating the mixed solution to 50 ℃ and preserving heat for 30min, heating to 70 ℃ and preserving heat for 20min, and finally preserving heat at 100 ℃ for 30min to remove redundant methanol and water. Then the solution is quickly heated to 300 ℃ and kept for 50min, the heating reaction is stopped, and the solution is cooled to room temperature. All processes were performed under nitrogen flow protection. Finally, the nanoparticles were dispersed in 10mL of hexane for further use by adding 20mL of ethanol to the solution, centrifuging at 6000rpm for 5 minutes, followed by washing twice with a 1:6 hexane/ethanol mixture.
Subsequently, naYbF was coated by thermal cracking 4 A shell layer. First, 0.5mmol Yb (CF) 3 COO) 3 、1mmol CF 3 COONa and 0.25mmol (2.5 mL) of inner core nanocrystalline (NaYbF) 4 2% Er,2% Ce NPs) was added to a mixed solution of 10mL oleic acid and 10mL octadecene, and the mixture was then heated to 120℃for 45min and then heated to 300℃for 30min. Subsequently, the reaction mixture was allowed to cool naturally to room temperature, and the product was obtained by washing with ethanol and centrifuging.
Finally, coating the outermost layer NaYF by thermal cracking 4 30% Nd shell, 0.175mmol Y (CF) 3 COO) 3 ,0.075mmol Nd(CF 3 COO) 3 ,0.5mmol CF 3 COONa and 5ml NaYbF 4 :2％Er,2％Ce@NaYF 4 Mixing x% Yb (x= 0,10,30,50,100) NPs with 10mL of oleic acid and 10mL of octadecene at normal temperature, then heating to 120 ℃ for 45min, heating to 300 ℃ for 45min, and finally washing and centrifuging reactants cooled to room temperature by ethanol to obtain the final nanocrystalline.
The energy transfer path diagram, the corresponding TEM diagram and the element distribution diagram of the synthesized three-layer structure NIR-IIb rare earth optical probe are shown in figure 1.
Fig. 1 (a) shows a schematic energy transmission path of a three-layer rare earth optical probe; FIGS. 1 (b) and (c) show TEM images of rare earth nanocrystals having a core/shell two-layer structure; (d) HAADF-STEM diagrams showing three-layer rare earth optical probes, and diagrams (e) - (j) show the element distribution patterns of Er, ce, yb, Y, nd in the three-layer rare earth optical probes.
Example 2
Evaluation of NIR-IIb fluorescence emission of rare earth optical probes
The rare earth optical probes with different Yb doping concentrations were almost the same as in example 1, except that the amount of trifluoroacetate raw material was changed when the second shell layer was coated by thermal cracking, as follows: naYF 4 :x％Yb 3+ (x=0%, 10%,30%,50%, 100%) shell layer. (1-x%)/2 mmol of prepared Y (CF) 3 COO) 3 Yb (CF) prepared in x%/2mmol 3 COO) 3 、1mmol CF 3 COONa and 0.25mmol (2.5 mL) of inner core nanocrystalline (NaYbF) 4 2% Er,2% Ce NPs) was first added to a mixed solution of 10mL oleic acid and 10mL octadecene. The mixture was then heated to 120℃for 45min and then heated to 300℃for 30min. Subsequently, the reaction mixture was allowed to cool naturally to room temperature, and the product was obtained by washing with ethanol and centrifuging.
The NIR-IIb fluorescence emission intensity diagrams obtained by the rare earth optical probes with different Yb doping concentrations under 808nm laser excitation are shown in FIG. 2.
FIG. 2 (a) shows NIR-IIb fluorescence emission spectra obtained by using rare earth optical probes with different Yb doping concentrations under 808nm laser excitation; (b) A statistical plot of fluorescence emission intensity at 1525nm for rare earth optical probes of different Yb doping concentrations is shown.
Example 3
Biological orthogonal NIR-IIb rare earth optical probe
First, in NaYbF 4 :2％Er,2％Ce@NaYbF 4 @NaYF 4 30% Nd surface modified DSPE-PEG-NH 2 NPs, the specific steps are as follows: 35mg of DSPE-PEG-NH 2 Mix with 10mg DCNPs (in 1ml chloroform) and stir overnight at room temperature, then heat to 60℃to allow complete evaporation of the organic solvent. Then at the aboveAdding 2mL of water into the product, performing ultrasonic treatment for 10min, and collecting NPs@DSPE-PEG-NH 2 Centrifuge at 10000rpm for 10min, wash with water 3 times, finally disperse in 2mL distilled water.
Subsequently, using NH on the surface of DBCO-NHS and optical probe by amidation reaction 2 And (5) connection. In detail, 1.0mg of DBCO-NHS was dissolved in a solution containing 200. Mu.L of DMF and 50. Mu.L of TEA,1mL of prepared DCNPs@NH2), stirred at room temperature overnight, centrifuged at 12000rpm for 15min, washed 3 times with water, and dispersed in 2mL of PBS. The final DBCO modified optical probe was stored at 4 ℃ for later use.
TEM image, hydraulic radius analysis image and infrared spectrum of biological orthogonal rare earth optical probe connected with DBCO are shown in figure 3.
FIG. 3 (a) is a diagram showing the hydraulic radius analysis of a bioorthogonal rare earth optical probe; (b) a TEM image of a bioorthogonal rare earth optical probe; (c) And the Fourier infrared test spectrogram of the bioorthogonal rare earth optical probe is shown.
Example 4
Evaluation of Exo-N 3 Targeting tumor cells
HCT-116 cells were seeded in 150mm dishes (5X 106 cells/dish), and after incubation for 24h, the medium was removed, and Ac dissolved in DMSO 4 ManNAz (10. Mu.M) was incubated for 48h, and extracellular vesicles were isolated and purified from the collected HCT-116 cell culture supernatant using a cell supernatant extracellular vesicle extraction kit, the performance characterization and in vitro tumor targeting ability of which are shown in FIG. 4.
FIG. 4 (a) is a confocal imaging of HCT-116 cell surface azide expression; (b) EVs-N 3 A TEM image of (a); (c) a western plot of EVs versus EVs-N3; (d) Representing EVs-N 3 Targeting of HCT-116 cells at different time points; (e) Indicating the presence or absence of EVs-N 3 Fluorescent imaging of HCT-116 cells targeted by the optical probe.
Example 5
Evaluation of bioorthogonal platform for NIR-IIb in vivo tumor imaging
Will be 1X 10 6 The individual HCT-116 cells were inoculated subcutaneously into Balb/c mice. When the tumor isUp to about 80-120mm in volume 3 At this time, tumor-bearing mice were randomly assigned to two groups. The control group is directly injected into the biological orthogonal rare earth optical probe (100 mu L,2 mg/ml) by tail vein, the experimental group sequentially injects extracellular vesicles expressing azide groups and the biological orthogonal rare earth optical probe (100 mu L,2 mg/ml) by tail vein injection, and living tumor NIR-IIb fluorescent signals at different time points are obtained through an imaging system.
The dynamic fluorescence imaging diagram of the NIR-IIb living tumor and the fluorescence signal intensity analysis diagram of the tumor part and the enrichment analysis of the probe at the tumor part are shown in FIG. 5.
FIG. 5 (a) shows a dynamic fluorescence imaging of NIR-IIb living tumors over a period of 0-48 hours; (b) Graphs showing fluorescence intensity of tumor sites of two groups of mice over time; (c) The enrichment rate of the fluorescent probe in the main organ and the tumor after 48 hours of injection is shown.
As shown in FIG. 5, in the group without injecting extracellular vesicles, after 4 hours of injection of the fluorescent probe, NIR-IIb fluorescence was collected at the tumor site of the mouse, but the fluorescence signal was gradually reduced with the lapse of time until 48 hours, and almost no fluorescence signal was collected. However, in the group of mice injected with extracellular vesicles, after 4 hours of injection of fluorescent probes, a larger range and brighter fluorescent signal is collected at the tumor part of the mice, and the signal is not obviously weakened until 48 hours, at the moment, the fluorescent intensity is 8 times that of the control group, and the tumor enrichment rate is 5 times that of the control group.
In summary, the present invention provides a rare earth optical probe, a preparation method and an application thereof, where the rare earth optical probe includes: kernel NaYbF 4 :Er 3+ ,Ce 3+ Shell NaYF 4 :Yb 3+ NaYF shell 4 :Nd 3+ And modifying the shell NaYF 4 :Nd 3+ Dibenzocyclooctyne on the surface; the shell layer NaYF 4 :Yb 3+ Grown on the inner core NaYbF 4 :Er 3+ ,Ce 3+ A surface; the shell NaYF 4 :Nd 3+ Grown on the shell layer NaYF 4 :Yb 3+ A surface. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ion (Er 3+ Ions and Nd 3+ Ions) with a high-intensity NIR-IIb fluorescence emission; the biological orthogonality is provided by modifying the functional group-alkynyl on the surface of the probe; the NIR-IIb fluorescence emission is used as a receiving wave band of living body imaging, so that the resolution and the signal-to-noise ratio of living body tumor imaging are enhanced; when the rare earth optical probe and the tumor extracellular vesicles with homologous targeting are subjected to high-efficiency biological orthogonal combination in a living body, the tumor enrichment rate and the residence time of the optical probe can be enhanced, long-time dynamic fluorescence imaging of living tumors can be realized, and the optical probe has good application prospect in the field of optical biological imaging.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (5)
1. The preparation method of the rare earth optical probe is characterized by comprising the following steps:
YbCl 3 ·6H 2 O、ErCl 3 ·6H 2 O、CeCl 3 ·7H 2 Adding the mixture of O, oleic acid and octadecene into a container, stirring and heating to 150 ℃, preserving heat for 1-2 hours, and cooling to 35-45 ℃ to prepare rare earth salt precursor solution;
NaOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F, solution;
the NaOH solution and NH 4 Dropwise adding the solution F into the rare earth salt precursor solution in sequence to obtain a mixed solution, keeping the mixed solution at room temperature for 0.5-1 hour, and cooling the mixed solution to room temperature after heating treatment;
then centrifugal and washing are carried out to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ ；
Yb (CF) 3 COO) 3 、CF 3 COONa and said inner core NaYbF 4 :Er 3+ ,Ce 3+ Adding into oleic acid and octadecene mixed solution, heating, and centrifugingWashing, so that the inner core NaYbF 4 :Er 3+ ,Ce 3+ Surface-coated NaYF of (C) 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Adding Yb NPs into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing to prepare the rare earth optical probe precursor;
then washing and centrifuging the rare earth optical probe precursor by using ethanol;
modifying amphiphilic DSPE-PEG-NH on the surface of the rare earth optical probe precursor 2 ；
Finally, in DSPE-PEG-NH 2 The DBCO-NHS is connected on the basis of the ligand to prepare the rare earth optical probe;
the shell layer NaYF 4 :Yb 3+ Middle Yb 3+ The doping concentration of the ions is 100%; the rare earth optical probe is of a spherical structure with the size of 21.5 nm-29.5 nm;
the heating treatment includes a low-temperature heating treatment and a high-temperature heating treatment; the low-temperature heating treatment is divided into three stages: the first stage is to keep the temperature at 50 ℃ for 30min, the second stage is to keep the temperature at 70 ℃ for 20min, and the third stage is to keep the temperature at 100 ℃ for 30min; the high temperature heating treatment is to keep the temperature at 280-320 ℃ for 40-60min.
2. The method for preparing a rare earth optical probe according to claim 1, wherein the method for preparing a rare earth optical probe is performed under a nitrogen atmosphere.
3. Use of a rare earth optical probe prepared by the method for preparing a rare earth optical probe according to any one of claims 1-2 in preparing a short wave near infrared tumor targeted fluorescence imaging optical probe.
4. The use of a rare earth optical probe according to claim 3, wherein the rare earth optical probe is used for bio-orthogonal binding to tumor extracellular vesicles with homology targeting, and for achieving NIR-IIb tumor targeted fluorescence imaging.
5. The use of a rare earth optical probe according to claim 4, wherein the tumor extracellular vesicles are extracellular vesicles secreted by human colon cancer cells and expressing azide genes.
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