CROSS REFERENCE TO RELATED APPLICATIONS

This application is the national phase entry of International Application No. PCT/CN2017/111139, filed on Nov. 15, 2017, which is based upon and claims priority to Chinese Application No. CN2017107031485, filed on Aug. 16, 2017, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the technical field of molecular imaging, and particularly to a pH-responsive ultrasensitive fluorescent nanoprobe, preparation and using method thereof.

BACKGROUND

Molecular imaging can be used to study the occurrence, development and metastasis of diseases or tumors in vivo because of its ability to realize real-time, non-invasive and dynamic imaging at the living body level. Optical imaging has the advantages of like no radiation, high sensitivity, and low light damage to biological tissues, showing great potential in the research field of in vivo tumor monitoring. Due to the rapid proliferation of cells, tumor tissue tends to cause rapid glucose metabolism, large oxygen consumption, and excessive accumulation of acidic metabolites (such as lactic acid) in the tumor microenvironment. Therefore, the tumor microenvironment is generally characterized by weak acidity, hypoxia, production of a plurality of cytokines and high expression of biological enzymes. Conventional organic small molecule fluorescent probes, because of the non-specific distribution in vivo, will cause organic dyes generally to have the disadvantages of poor bio-distribution specificity, high background signal, poor light stability and easy photobleaching, thus being limited in the application of in vivo imaging, and cannot be applied to the detection of tiny tumors. The use of bio-intelligent responsive nanomaterial-loaded small molecule fluorescent probes can not only improve the light stability of small molecule fluorescent probes, but also achieve the high-efficient and specific enrichment of probes and the pH regulated fluorescence enhancement in the tumor sites, which has become a popular research direction in the field of molecular imaging technology.

SUMMARY

In view of the problems existing in the prior art, the present invention provides a pH-responsive ultrasensitive fluorescent nanoprobe, preparation and using method thereof.

The present invention is achieved by a pH-responsive ultrasensitive fluorescent nanoprobe, the pH-responsive ultrasensitive fluorescent nanoprobe is composed of pH-responsive matrix materials and organic small molecule dyes having fluorescent;

the negatively charged pH-responsive matrix materials are calcium phosphate (abbreviated as CaP), calcium hydroxyphosphate, fluorapatite, and calcium carbonate.

Further, positively charged pH-responsive matrix materials are zeolite imidazole framework (ZIF) material series;

the fluorescent organic small molecule dyes are positively charged dyes or negatively charged dyes.

Further, the positively charged dyes are IR780, RhB, or IR800.

Further, the negatively charged dyes are Cy3, Cy5, Cy5.5, Cy7, ICG, ICG-Der-01, ICG-Der-02, ICG-Der-03, IR820, Alexa Fluor 750, Alexa Fluor 700, Alexa Fluor 680, Alexa Fluor 660, Alexa Fluor 647, Alexa Fluor 635, Alexa Fluor 633, Alexa Fluor 610, Alexa Fluor 594, Alexa Fluor 568, Alexa Fluor 555, Alexa Fluor 546, Alexa Fluor 532, Alexa Fluor 514, Alexa Fluor 500, Alexa Fluor 488, or FITC.

Another purpose of the present invention is to provide a method for preparing the pH-responsive ultrasensitive fluorescent nanoprobe, and the method for preparing the pH-responsive ultrasensitive fluorescent nanoprobe includes the following three methods.

The first method, a positively charged dye is coated with a negatively charged matrix material, the dye is adsorbed on the PAA (polyacrylic acid) molecular chain by electrostatic interaction between the polyacrylic acid and the positively charged dye, and the PAA after dye adsorption is self-assembled to form a template in isopropyl alcohol, while the fluorescence of the dye is quenched by aggregation; the exposed carboxyl on the PAA is coordinated with Ca2+, and the calcium phosphate is deposited on the surface of the PAA sphere by adjusting the pH and adding phosphate for mineralization to form a dye-coated nanoparticle.

The second method, a negatively charged dye is coated with a negatively charged matrix material, the dye is enriched on the polyetherimide (PEI) molecular chain through the absorption of the negatively charged dye by PEI; meanwhile, a negatively charged PAA is added, the PEI absorbs both dye and PAA to self-assembled into a PEI/PAA nanospheres; the exposed carboxyl on the PAA is coordinated with Ca2+, and the calcium phosphate is deposited on the surface of the PAA sphere by adjusting the pH and adding phosphate for mineralization to form a dye-coated nanoparticle.

The third method, a negatively charged dye is coated with a positively charged matrix material, the dye molecules are adsorbed on the ligand (methylimidazole) molecules by the mutual electrostatic adsorption between the methylimidazole and the coated dye, meanwhile, the aggregation of the dye is caused, and the metal ions are added for being coordinated with the ligand to form a stable three-dimensional structure, thus forming a dye-coated nanoparticle.

Further, in the first method, the negatively charged pH-responsive matrix materials are calcium phosphate, calcium carbonate, calcium hydroxyphosphate, and fluorapatite; the positively charged dyes are IR780, RhB, or IR800.

Further, in the second method, the negatively charged pH-responsive matrix materials are calcium phosphate, calcium carbonate, calcium hydroxyphosphate, and fluorapatite; the negatively charged dyes are ICG, IR820, Alexa Fluor series dyes, and Cy series dyes.

Further, in the third method, the pH-responsive matrix materials are the MOF materials of the ZIF series; the negatively charged dyes are ICG, IR820, Alexa Fluor series dyes, and Cy series dyes.

Another purpose of the present invention is to provide a method for using the pH-responsive ultrasensitive fluorescent nanoprobe, and the method for using the pH-responsive ultrasensitive fluorescent nanoprobe includes: The nanoparticle is injected into the mouse via tail vein, which reaches the tumor site through the enhanced permeability and retention (EPR) effect and is enriched in the tumor site.

In normal tissues and blood, the pH is neutral or weakly alkaline, and the fluorescent molecules in the nanoparticles are in the state of aggregation quenching. In the tumor site, due to its weak acidic conditions, the matrix material CaP is dissolved and releases the fluorescent molecules, and the fluorescent recovers after the IR780 recovers to the free molecular state, thereby realizing fluorescence enhancement.

Another purpose of the present invention is to provide a CaP/IR780 probe and a CaCO3/RhB probe prepared by the pH-responsive ultrasensitive fluorescent nanoprobe.

Another purpose of the present invention is to provide a CaP/ICG probe and a CaCO3/Cy5 probe prepared by the pH-responsive ultrasensitive fluorescent nanoprobe.

Another purpose of the present invention is to provide a ZIF-8/ICG probe prepared by the pH-responsive ultrasensitive fluorescent nanoprobe.

The advantages and positive effects of the present invention are: the high-efficiency specificity enrichment and controllable release of the small molecule fluorescent probe at the tumor site can be achieved through the response of matrix materials to the weakly acidic conditions of the tumor microenvironment, the fluorescence intensity of the probe after release is significantly enhanced, and the ultrasensitive detections of the primary lesions and metastatic lesions of tumors are realized; matrix nanomaterials have pH response characteristic, under neutral conditions, fluorescent molecules loaded in nanoparticles are subjected to fluorescence quenching due to being in aggregation state; under the weakly acidic conditions of the tumor microenvironment, the matrix material is dissolved, so that the fluorescent molecules loaded in the nanoparticles recover to the single molecule state, the fluorescence is recovered, the tumor microenvironment acidic-responsive fluorescence enhancement is realized, and accurate imaging and real-time monitoring of early tumors can be realized.

The present invention can realize the high-efficiency enrichment of nanomaterials at the tumor site through the enhanced permeability and retention (EPR) effect of the nanomaterial at the tumor site, and the tumor-specific fluorescence enhancement can be achieved by utilizing the characteristic of pH response to the tumor microenvironment. Before reaching the tumor site, fluorescence quenching of fluorescent molecules in the probe was caused by the aggregation of the fluorescent molecules loaded in the matrix material, and the fluorescence signal was in a closed (OFF) state; while the probe is in a weakly acidic condition of the tumor microenvironment, the organic fluorescent molecules are released from the matrix material to achieve a pH-responsive “deaggregation” to enable the fluorescent signal recovery (ON). The OFF-ON switch minimizes the background signal of the fluorescent probe and significantly increases the imaging signal-to-noise ratio by nearly three times. Compared with the traditional small molecule fluorescent dyes, the sensitivity and specificity of fluorescence imaging can be greatly improved, and ultrasensitive detection of tumor microenvironment response can be realized; the specificity response probe prepared by the unique properties of the tumor microenvironment has the advantages of high targeting efficacy, low background signal, and high signal-to-noise ratio, and the ultrasensitive detection of tiny tumors smaller than 1 mm can be realized in vivo.

