CN114854415A - 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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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/77—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals
- C09K11/7766—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing rare earth metals containing two or more rare earth metals
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- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
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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 layer NaYF 4 :Yb 3+ Outer shell NaYF 4 :Nd 3+ And modifying the NaYF shell 4 :Nd 3+ Surface dibenzocyclooctyne. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ions (Er) are avoided 3+ Ions and Nd 3+ Ions) with a high bright NIR-IIb fluorescence emission; endowing the probe with bio-orthogonality by modifying a functional group-alkynyl on the surface of the probe; when the rare earth optical probe and the tool are usedThe tumor extracellular vesicles with homologous targeting can enhance the tumor enrichment rate and the retention time of the optical probe when efficient bioorthogonal binding occurs in a living body, and can realize long-time dynamic fluorescence imaging of the living body tumor.
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
The rare earth doped Nanoparticles (NPs) have excellent optical characteristics such as no photobleaching, no flicker, narrow emission bandwidth, long service life and the like, so that the NPs become an optical probe with great development prospect. More importantly, erbium ion (Er) is doped 3+ ) The down-conversion nanocrystals (DCNPs) have characteristic fluorescence emission peak at 1525nm, and are suitable for living body imaging of NIR-IIb.
At present, Er 3+ Ion-doped optical probes have been designed and used to achieve micron-scale resolution of whole-body and cerebral vascular imaging at millimeter depths. Although some progress has been made in the field of in vivo fluorescence imaging, the rare earth optical probe often shows poor targeting and short retention time at a tumor part only by utilizing the traditional Enhanced Permeability and Retention (EPR) effect, and the application of the rare earth optical probe in the aspects of in vivo tumor detection and treatment is greatly limited.
At present, in order to improve the tumor targeting ability, researchers modify a plurality of 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 tumor targeting enrichment ability of the rare earth optical probe. However, the expression of biological receptors on tumor cells is limited, and the efficient binding of the receptors to ligands remains an obstacle to the efficient enrichment of probes at tumor sites.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects 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 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 the efficient enrichment of the probe at a tumor part is hindered in the prior art.
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+ NaYbF grown in the inner core 4 :Er 3+ ,Ce 3+ A surface;
shell NaYF 4 :Nd 3+ Said housing NaYF 4 :Nd 3+ Growing on the NaYF shell 4 :Yb 3+ A surface;
a dibenzocyclooctyne for modifying the shell NaYF 4 :Nd 3+ Of (2) is provided.
The rare earth optical probe is characterized in that the shell layer NaYF 4 :Yb 3+ Medium Yb 3+ The doping concentration of the ions is 100%.
The rare earth optical probe is of a spherical structure with the size of 21.5-29.5 nm.
A method for preparing the rare earth optical probe comprises the following steps:
preparation of Er by coprecipitation method 3+ Ions and Ce 3+ Ion-doped NaYbF 4 Kernel to obtain kernel NaYbF 4 :Er 3+ ,Ce 3+ ；
In the kernel NaYbF 4 :Er 3+ ,Ce 3+ The surfaces are sequentially connected to grow NaYF through a thermal cracking method 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ A shell, preparing a precursor of the rare earth optical probe;
then washing and centrifugally purifying 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 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 following steps of preparing Er by adopting a coprecipitation method 3+ Ions and Ce 3+ Ion-doped NaYbF 4 Inner core toolThe body includes:
mixing 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 a rare earth salt precursor solution;
adding NaOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F solution;
mixing the NaOH solution with NH 4 Sequentially dropwise adding the solution F into the rare earth salt precursor solution to obtain a mixed solution, keeping the mixed solution at room temperature for 0.5 to 1 hour, heating the mixed solution, and cooling the mixed solution to room temperature;
then centrifuging and washing to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
The preparation method of the rare earth optical probe comprises the following steps of preparing a core NaYbF 4 :Er 3+ ,Ce 3+ The surfaces are sequentially connected to grow NaYF through a thermal cracking method 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 the mixture into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing to obtain the NaYbF inner core 4 :Er 3+ ,Ce 3+ Surface of (2) is covered with a NaYF shell 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Mixing Y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Yb NPs is added into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing are carried out to prepare the precursor of the rare earth optical probe.
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 applied to the short-wave near-infrared tumor targeted fluorescence imaging of a nano probe;
wherein, the rare earth optical probe is the rare earth optical probe.
The rare earth optical probe is applied to bioorthogonal combination with tumor extracellular vesicles with homologous targeting, and NIR-IIb tumor targeted fluorescence imaging is achieved.
The application of the rare earth optical probe is that the tumor extracellular vesicles are extracellular vesicles which are secreted by human colon cancer cells and express azide genes.
Has the advantages that: the invention provides 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 layer NaYF 4 :Yb 3+ Outer shell NaYF 4 :Nd 3+ And modifying the NaYF shell 4 :Nd 3+ Surface dibenzocyclooctyne; the shell layer NaYF 4 :Yb 3+ NaYbF grown in the inner core 4 :Er 3+ ,Ce 3+ A surface; the shell NaYF 4 :Nd 3+ Growing on the NaYF shell 4 :Yb 3+ A surface. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ions (Er) are avoided 3+ Ions and Nd 3+ Ions) with a high bright NIR-IIb fluorescence emission; endowing the probe with bio-orthogonality by modifying a functional group-alkynyl on the surface of the probe; NIR-IIb fluorescence emission is taken 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 vesicle with the homologous targeting property are subjected to efficient biological orthogonal combination in a living body, the tumor enrichment rate and the retention time of the optical probe can be enhanced, the long-time dynamic fluorescence imaging of the living tumor can be realized, and the rare earth optical probe has a good application prospect in the field of optical biological imaging.
Drawings
FIG. 1 is an energy transfer path diagram, a corresponding TEM image and an element distribution diagram of a three-layer structured rare earth optical probe synthesized in example 1 of the present invention;
FIG. 2 is a graph of NIR-IIb fluorescence emission intensity obtained by rare earth optical probes with different Yb doping concentrations under the excitation of 808nm laser in example 2 of the invention;
FIG. 3 is a TEM image, a hydraulic radius analysis image and an IR spectrum of the bioorthogonal rare earth optical probe after DBCO is connected in example 3 of the present invention;
FIG. 4 is a graph showing the performance characterization and tumor targeting ability in vitro according to example 4 of the present invention;
FIG. 5 is a NIR-IIb in vivo tumor dynamic fluorescence imaging graph, a tumor site fluorescence signal intensity analysis graph and a probe enrichment amount analysis graph at a tumor site in example 5 of the present invention.
Detailed Description
The invention provides a rare earth optical probe and a preparation method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Optical bioimaging plays a crucial role in many biomedical fields, from early disease diagnosis to monitoring disease progression to image-guided treatment of life-threatening diseases, and to assess treatment efficacy and prognosis. The permeability of light in biological tissue depends on the absorption and scattering ability of the tissue components to light, which also directly affects the biological imaging quality, including imaging intensity, penetration depth, signal-to-noise ratio, and imaging resolution.
Therefore, the fluorescence bioimaging quality obtained using visible light (400-. Compared with the prior art, due to the weak absorption capacity, low scattering coefficient and negligible autofluorescence of biological tissues, the window NIR-IIb (1500-.
And the rare earth doped Nano Particles (NPs) have excellent optical characteristics, so that the rare earth doped nano particles become an optical probe with development prospect. However, in order to improve the tumor targeting ability, researchers modify a plurality of active targets such as folic acid, nucleic acid, antibody, polypeptide and the like on the surface of the rare earth optical probe to enhance the tumor targeting enrichment ability of the rare earth optical probe.
However, the expression of biological receptors on tumor cells is limited, and the efficient binding of the receptors to ligands remains an obstacle to the efficient enrichment of probes at tumor sites.
Based on the rare earth optical probe, the invention provides the rare earth optical probe which is Y 3+ 、Yb 3+ 、Er 3+ 、Ce 3+ 、Nd 3+ Rare earth nanocrystalline (NaYbF) with three-layer structure formed by ions 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+ (ii) a Shell NaYF 4 :Yb 3+ The shell layer NaYF 4 :Yb 3+ NaYbF grown in the inner core 4 :Er 3+ ,Ce 3+ A surface; shell NaYF 4 :Nd 3+ Said housing NaYF 4 :Nd 3+ Growing on the NaYF shell 4 :Yb 3+ A surface; a dibenzocyclooctyne for modifying the shell NaYF 4 :Nd 3 + Of (2) is provided.
The rare earth optical probe is designed into a three-layer structure, and the three-layer structure is provided with an inner core NaYbF from inside to outside 4 :Er 3+ ,Ce 3+ Shell layer NaYF 4 :Yb 3+ And a housing NaYF 4 :Nd 3+ The rare earth optical probe with the three-layer structure can avoid unnecessary rare earth ions (Er) 3+ Ions and Nd 3+ Ions) so that the rare earth optical probe has high-brightness NIR-IIb fluorescence emission; in addition, the invention provides NaYF on the shell 4 :Nd 3+ The surface of (A) is modified with dibenzoThe rare earth optical probe is modified by utilizing dibenzocyclooctyne, 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 peptide is combined with tumor extracellular vesicles with homologous targeting to form a bioorthogonal platform, the bioorthogonal 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 the dynamic fluorescence imaging of the living tumor for a long time (48 h).
Specifically, the invention utilizes a rare earth optical probe with high-brightness NIR-IIb fluorescence emission, endows the probe with bio-orthogonality capacity 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 a living body, the rare earth optical probe for modifying the alkynyl group and the tumor extracellular vesicle for expressing the azide group are subjected to efficient bioorthogonal combination by utilizing a copper-free click reaction, so that the targeted enrichment and retention time of the rare earth optical probe at a tumor part are enhanced, and the NIR-IIb living body tumor targeted fluorescence imaging under the excitation of 808nm is realized.
In some embodiments, the shell layer is a NaYF 4 :Yb 3+ Medium Yb 3+ The doping concentration of the ions is 100%.
In some embodiments, the rare earth optical probe is a spherical structure with a size between 21.5nm and 29.5 nm. Preferably, the rare earth optical probe is 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+ Ions and Ce 3+ Ion-doped NaYbF 4 Kernel to obtain kernel NaYbF 4 :Er 3+ ,Ce 3+ ；
Step S20: in the kernel NaYbF 4 :Er 3+ ,Ce 3+ The surfaces are sequentially connected to grow NaYF through a thermal cracking method 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ A shell, preparing a precursor of the rare earth optical probe;
step S30: then washing and centrifugally purifying 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 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 a three-layer structure, and the surface of the rare earth optical probe is modified by dibenzooctyne, so that energy loss caused by cross relaxation among rare earth ions can be avoided, the rare earth optical probe can generate a high-brightness NIR-IIb (1525nm) fluorescence signal under the condition of 808nm laser excitation, and the rare earth optical probe can be endowed with bioorthogonal capability, so that the rare earth optical probe has higher targeting property on tumors, and the resolution and signal-to-noise ratio of living tumor imaging are enhanced.
In some embodiments, the step S10 specifically includes the steps of:
step S11: mixing 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 a rare earth salt precursor solution;
step S12: adding NaOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F solution;
step S13: mixing the NaOH solution with NH 4 Sequentially dropwise adding the solution F into the rare earth salt precursor solution to obtain a mixed solution, keeping the mixed solution at room temperature for 0.5 to 1 hour, heating the mixed solution, and cooling the mixed solution to room temperature;
step S14: then centrifuging and washing to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
Specifically, the heating process in step S13 includes a low-temperature heating process and a high-temperature heating process; the low-temperature heating treatment is divided into three stages: the first stage is heat preservation at 50 ℃ for 30min, the second stage is heat preservation at 70 ℃ for 20min, and the third stage is heat preservation at 100 ℃ for 30 min; the purpose of the low-temperature treatment is mainly to remove excessive formaldehyde and water in the solution. The high-temperature treatment is heat preservation for 40-60min at the temperature of 280-320 ℃, and the high-temperature treatment is mainly used for enabling the mixed solution to react to generate kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
In some embodiments, the centrifugation in step S14 is performed using an ethanol solvent at 6000 rpm; followed by at least two washes with a 1:6 hexane/ethanol mixture.
In some embodiments, the step S20 specifically includes the steps of:
step S21: mixing Yb (CF) 3 COO) 3 、CF 3 COONa and said inner core NaYbF 4 :Er 3+ ,Ce 3+ Adding the mixture into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing to obtain the NaYbF inner core 4 :Er 3+ ,Ce 3+ Surface of (2) is covered with a NaYF shell 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Step S22: mixing Y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Yb NPs is added into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing are carried out to prepare the precursor of the rare earth optical probe.
By way of illustration: firstly, adopting a coprecipitation method, and reacting NaOH and NH 4 Separate addition of F to produce smaller size NaYbF 4 2% Er and 2% Ce nanocrystalline core, and then growing NaYF on the surface of the core by adopting a thermal cracking method 4 Yb shell followed by NaYbF 4 :2％Er,2％Ce@NaYF 4 Yb is a nucleus on which NaYF grows 4 30% Nd surface layer to obtain the final NaYbF 4 :2％Er,2％Ce@NaYF 4 :Yb@NaYF 4 30% of Nd nano-crystal. During the growth of the shell layer, the thickness of the shell layer and the composition of rare earth elements of the shell layer are controlled by adding different types and proportions of trifluoroacetic acid rare earth salts and sodium trifluoroacetate.
Then, in NaYbF 4 :2％Er,2％Ce@NaYbF 4 @NaYF 4 30% Nd nanocrystalline surface modification DSPE-PEG-NH 2 . Subsequently, amide reaction of amino group with ester group is carried out in DSPE-PEG-NH 2 DBCO-NHS is connected 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 an application of the rare earth optical probe, wherein 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.
In some embodiments, the rare earth optical probe is used for carrying out bio-orthogonal combination with tumor extracellular vesicles with homologous targeting to realize NIR-IIb tumor targeted fluorescence imaging.
In some embodiments, the tumor extracellular vesicles are human colon cancer cells (HCT-116) secreting azide-expressing extracellular vesicles.
In some embodiments, the route of capture of the extracellular vesicles is as follows:
step A: incubating N-azidoacetylmannosamine-tetraacylation (Ac4Mannaz) and HCT-116 for 48h, and expressing an azide group on the cell membrane surface of the HCT-116 by using a metabolic marker;
and B: kit for extracting azide group-expressing extracellular vesicles (EVs-N) secreted by HCT-116 cells 3 )。
In this embodiment, the DBCO-modified NaYbF 4 :Er 3+ ,Ce 3+ @NaYF 4 :Yb 3+ @NaYF 4 :Nd 3+ NPs and the extracellular vesicles which are secreted by HCT-116 and express azide groups are in efficient bioorthogonal combination in a living body, and have high targeting property on tumors.
The present invention will be described in further detail with reference to examples. It is also to be understood that the following examples are illustrative of the present invention and are not to be construed as limiting the scope of the invention, and that certain insubstantial modifications and adaptations of the invention by those skilled in the art may be made in light of the above teachings.
Example 1
Preparation of three-layer structured rare earth optical probe
Firstly, NaYbF is prepared by a coprecipitation method 4 2 percent of Er and 2 percent of Ce kernel. 0.96mmol of 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 was cooled to 40 ℃ 2.5mmol NaOH and 4.0mmol NH 4 F was dissolved in 6ml and 8ml of methanol, respectively, and then NaOH solution and NH were added 4 And sequentially dripping the solution F into the rare earth salt precursor solution, keeping the room temperature for 30min, sequentially heating the mixed solution to 50 ℃, keeping the temperature for 30min, heating to 70 ℃, keeping the temperature for 20min, and finally keeping the temperature at 100 ℃ for 30min to remove the 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 procedures were carried out under nitrogen flow protection. Finally, the nanoparticles were finally dispersed in 10mL of hexane for further use by adding 20mL of ethanol to the solution, centrifuging for 5 minutes at 6000rpm, followed by washing twice with a 1:6 hexane/ethanol mixture.
Subsequently, NaYbF was coated by thermal cracking 4 And (5) shell layer. First 0.5mmol Yb (CF) 3 COO) 3 、1mmol CF 3 COONa and 0.25mmol (2.5mL) of inner core nanocrystal (NaYbF) 4 2% Er, 2% Ce NPs) was added to a mixed solution of 10mL oleic acid and 10mL octadecene, then the mixture was heated to 120 ℃ for 45min, and then heated to 300 ℃ for 30 min. Subsequently, the reaction mixture was allowed to cool naturally to room temperature, and the product was obtained by washing with ethanol and centrifugation.
Finally, coating the outermost layer by thermal crackingNaYF 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 is 0,10,30,50,100) NPs with 10mL of oleic acid and 10mL of octadecene at normal temperature, then heating to 120 ℃ for 45min, then heating to 300 ℃ for 45min, and finally washing the reactant cooled to room temperature by ethanol and centrifuging to obtain the final nanocrystal.
The energy transfer path diagram, the corresponding TEM image and the element distribution diagram of the NIR-IIb rare earth optical probe with the three-layer structure are shown in FIG. 1.
FIG. 1 (a) shows an energy transfer path schematic of a three-layer rare earth optical probe; FIGS. 1(b) and (c) are TEM images of rare earth nanocrystals with core and shell two-layer structure; (d) the HAADF-STEM diagrams of the three-layer rare earth optical probe are shown, and the element distribution diagrams of Er, Ce, Yb, Y and Nd in the three-layer rare earth optical probe are shown in the diagrams (e) - (j).
Example 2
Evaluation of NIR-IIb fluorescence emission from rare earth optical probes
The rare earth optical probes with different Yb doping concentrations were approximately the same as those in example 1, except that the amount of the trifluoroacetate material was changed when the second shell was coated by thermal cracking, as follows: NaYF 4 :x％Yb 3+ (x ═ 0%, 10%, 30%, 50%, 100%) of the shell layer. (1-x%)/2 mmol of prepared Y (CF) 3 COO) 3 X%/2 mmol of Yb (CF) prepared 3 COO) 3 、1mmol CF 3 COONa and 0.25mmol (2.5mL) of inner core nanocrystal (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 to 300 ℃ for 30 min. Subsequently, the reaction mixture was allowed to cool naturally to room temperature, and the product was obtained by washing with ethanol and centrifugation.
The NIR-IIb fluorescence emission intensity images obtained by rare earth optical probes with different Yb doping concentrations under the excitation of 808nm laser are shown in figure 2.
FIG. 2 (a) shows NIR-IIb fluorescence emission spectra obtained by rare earth optical probes with different Yb doping concentrations under the excitation of 808nm laser; (b) and (3) a statistical graph of the fluorescence emission intensity of the rare earth optical probes with different Yb doping concentrations at 1525 nm.
Example 3
Biorthogonal 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 concrete steps are as follows: 35mg of DSPE-PEG-NH 2 Mixed (in 2ml chloroform) with 10mg of DCNPs (in 1ml chloroform), stirred overnight at room temperature, then heated to 60 ℃ to completely evaporate the organic solvent. Then adding 2mL of water into the product, performing ultrasonic treatment for 10min, and collecting NPs @ DSPE-PEG-NH 2 Centrifuged at 10000rpm for 10min, washed 3 times with water and finally dispersed in 2mL of distilled water.
Subsequently, DBCO-NHS was reacted with NH on the surface of the optical probe by amidation 2 And (4) connecting. In detail, 1.0mg DBCO-NHS was dissolved in a solution containing 200. mu.L DMF and 50. mu.L TEA,1mL of prepared DCNPs @ NH2), stirred overnight at room temperature, centrifuged at 12000rpm for 15min, washed 3 times with water, and dispersed in 2mL PBS. The final DBCO-modified optical probe was stored at 4 ℃ for later use.
The TEM image, the hydraulic radius analysis image and the infrared spectrum of the bioorthogonal rare earth optical probe connected with DBCO are shown in FIG. 3.
FIG. 3 (a) is a diagram showing a hydraulic radius analysis of a bioorthogonal rare earth optical probe; (b) a TEM image showing a bio-orthogonal rare earth optical probe; (c) and (3) representing a Fourier infrared test spectrogram of the bioorthogonal rare earth optical probe.
Example 4
Evaluation of Exo-N 3 Targeting to tumor cells
HCT-116 cells were seeded in 150mm dishes (5X 106 cells/dish) and after 24h of incubation, the medium was removed and Ac dissolved in DMSO 4 Mannaz (10 mu M) is incubated for 48h, and the extracellular vesicles are separated and purified from the collected HCT-116 cell culture supernatant by using a cell supernatant extracellular vesicle extraction kit, and the performance of the extracellular vesicles is characterizedAnd in vitro tumor targeting ability is shown in figure 4.
FIG. 4 (a) is a confocal image showing the expression of azide groups on the cell surface of HCT-116; (b) EVs-N 3 A TEM image of (B); (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 The optical probe targets the fluorescence image of HCT-116 cells under the conditions of (1).
Example 5
Evaluation of bioorthogonal platform for NIR-IIb in vivo tumor imaging
Will be 1 × 10 6 A single HCT-116 cell was inoculated subcutaneously into Balb/c mice. When the tumor volume reaches about 80-120mm 3 Tumor-bearing mice were randomly assigned to two groups. The control group is directly injected with bioorthogonal rare earth optical probe (100 mu L,2mg/ml) in tail vein, the experimental group is injected with the extracellular vesicles expressing the azide groups and the bioorthogonal rare earth optical probe (100 mu L,2mg/ml) in sequence in a tail vein injection mode, and living tumor NIR-IIb fluorescence signals at different time points are obtained through an imaging system.
An NIR-IIb living tumor dynamic fluorescence imaging graph, a tumor part fluorescence signal intensity analysis graph and probe enrichment analysis at a tumor part are shown in figure 5.
FIG. 5 (a) shows NIR-IIb in vivo tumor dynamic fluorescence imaging within 0-48 h; (b) graphs showing the change in fluorescence intensity over time at the tumor sites of two groups of mice; (c) shows the enrichment rate of the fluorescent probe in the main organs and tumors 48h after injection.
As shown in fig. 5, in the group without the injection of extracellular vesicles, NIR-IIb fluorescence was collected at the tumor site of the mouse 4h after the injection of the fluorescent probe, but the fluorescence signal gradually decreased with the passage of time, and almost no fluorescence signal was collected until 48 h. However, in the mouse group injected with the extracellular vesicles for 4 hours, a brighter fluorescence signal is collected at the tumor site of the mouse after the fluorescent probe is injected, and the signal is not obviously weakened until 48 hours, at this time, the fluorescence 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 providesA rare earth optical probe, a method for preparing the same and applications thereof are provided, the rare earth optical probe comprising: kernel NaYbF 4 :Er 3+ ,Ce 3+ Shell layer NaYF 4 :Yb 3+ Outer shell NaYF 4 :Nd 3+ And modifying the NaYF shell 4 :Nd 3+ Surface dibenzocyclooctyne; the shell layer NaYF 4 :Yb 3+ NaYbF grown in the inner core 4 :Er 3+ ,Ce 3+ A surface; the shell NaYF 4 :Nd 3+ Growing on the NaYF shell 4 :Yb 3+ A surface. The rare earth optical probe has a three-layer structure, and unnecessary rare earth ions (Er) are avoided 3+ Ions and Nd 3+ Ions) with a high bright NIR-IIb fluorescence emission; endowing the probe with bio-orthogonality by modifying a functional group-alkynyl on the surface of the probe; NIR-IIb fluorescence emission is taken 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 vesicle with the homologous targeting property are subjected to efficient biological orthogonal combination in a living body, the tumor enrichment rate and the retention time of the optical probe can be enhanced, long-time dynamic fluorescence imaging of the living tumor can be realized, and the rare earth optical probe has a good application prospect in the field of optical biological imaging.
It will be understood that the invention is not limited to the examples described above, but that modifications and variations will occur to those skilled in the art in light of the above teachings, and that all such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.
Claims (10)
1. 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+ NaYbF grown in the inner core 4 :Er 3+ ,Ce 3+ A surface;
shell NaYF 4 :Nd 3+ Said housing NaYF 4 :Nd 3+ Growing on the NaYF shell 4 :Yb 3+ A surface;
a dibenzocyclooctyne for modifying the shell NaYF 4 :Nd 3+ Of (2) is provided.
2. The rare earth optical probe as claimed in claim 1, wherein the shell layer NaYF is a shell layer 4 :Yb 3+ Medium Yb 3+ The doping concentration of the ions is 100%.
3. The rare earth optical probe according to claim 1, wherein the rare earth optical probe is a spherical structure having a size between 21.5nm and 29.5 nm.
4. A method for preparing a rare earth optical probe according to any one of claims 1 to 3, comprising the steps of:
preparation of Er by coprecipitation method 3+ Ions and Ce 3+ Ion-doped NaYbF 4 Kernel to obtain kernel NaYbF 4 :Er 3+ ,Ce 3+ ；
In the kernel NaYbF 4 :Er 3+ ,Ce 3+ The surfaces are sequentially connected to grow NaYF through a thermal cracking method 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ A shell, preparing a precursor of the rare earth optical probe;
then washing and centrifugally purifying 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 connecting DBCO-NHS on the basis of the ligand to prepare the rare earth optical probe.
5. The method for preparing the rare-earth optical probe according to claim 4, wherein the Er is prepared by a coprecipitation method 3+ Ions and Ce 3+ Ion-doped NaYbF 4 The kernel specifically comprises:
mixing 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 a rare earth salt precursor solution;
adding NaOH and NH 4 F is respectively dissolved in methanol to prepare NaOH solution and NH 4 F solution;
mixing the NaOH solution with NH 4 Sequentially dropwise adding the solution F into the rare earth salt precursor solution to obtain a mixed solution, keeping the mixed solution at room temperature for 0.5 to 1 hour, heating the mixed solution, and cooling the mixed solution to room temperature;
then centrifuging and washing to obtain the kernel NaYbF 4 :Er 3+ ,Ce 3+ 。
6. The method for preparing a rare earth optical probe according to claim 4, wherein the step is carried out in a core NaYbF 4 :Er 3+ ,Ce 3+ The surfaces are sequentially connected to grow NaYF through a thermal cracking method 4 :Yb 3+ Shell and NaYF 4 :Nd 3+ The shell specifically includes:
mixing Yb (CF) 3 COO) 3 、CF 3 COONa and said inner core NaYbF 4 :Er 3+ ,Ce 3+ Adding the mixture into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing to obtain the NaYbF inner core 4 :Er 3+ ,Ce 3+ Surface of (2) is covered with a NaYF shell 4 :Yb 3+ Coating to obtain NaYbF 4 :Er,Ce@NaYF 4 :Yb NPs；
Mixing Y (CF) 3 COO) 3 、Nd(CF 3 COO) 3 、CF 3 COONa and said NaYbF 4 :Er,Ce@NaYF 4 Yb NPs is added into a mixed solution of oleic acid and octadecene, and then heating, centrifuging and washing are carried out to prepare the precursor of the rare earth optical probe.
7. The method for producing a rare earth optical probe according to claim 4, wherein the method for producing a rare earth optical probe is performed in a nitrogen atmosphere.
8. The application of the rare earth optical probe is characterized in that the rare earth optical probe is applied to nano probe short wave near infrared tumor targeted fluorescence imaging;
wherein the rare earth optical probe is the rare earth optical probe according to any one of claims 1 to 3.
9. The use of the rare earth optical probe according to claim 8, wherein the rare earth optical probe is used for bio-orthogonal binding with tumor extracellular vesicles with homologous targeting to realize NIR-IIb tumor targeted fluorescence imaging.
10. The use of the rare earth optical probe as claimed in claim 8, wherein the tumor extracellular vesicles are azide gene-expressing extracellular vesicles secreted by human colon cancer cells.
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| CONG CAO等: "Ln3+-doped nanoparticles with enhanced NIR-II luminescence for lighting up blood vessels in mice" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117363358A (en) * | 2022-12-29 | 2024-01-09 | 先进能源科学与技术广东省实验室 | Bionic film-coated rare earth doped inorganic nano fluorescent probe and preparation method and application thereof |
| CN117363358B (en) * | 2022-12-29 | 2024-04-23 | 先进能源科学与技术广东省实验室 | Bionic film-coated rare earth doped inorganic nano fluorescent probe and preparation method and application thereof |
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| CN114854415B (en) | 2023-10-03 |
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