Design,synthesis And In Vitro Activity Evaluation Of Fluorescent Probes Based On Bisphosphate Derivatives

In recent years,fluorescent probes have become a common detection method,widely used in biomedicine,analytical chemistry,environmental science and other fields.Among them,the small molecule fluorescent probe is one of the important fluorescent probes,which has the advantages of high sensitivity,fast response speed,high safety and low cost,and is often used in molecular detection and biological imaging.Bone metastasis of malignant tumors has become one of the most serious complications,with the characteristics of high incidence,short survival time,low cure rate,and many complications.Tumor cells escape from the primary tissue and invade distant tissues,marking the transition of the tumor from a potentially curable disease to a malignant tumor.Once metastasis is almost incurable,it can only be treated to relieve symptoms.The clinical manifestations of bone metastasis include bone pain,pathological fractures,hypercalcemia,and spinal cord compression.These symptoms may seriously damage the patient's quality of life.Therefore,the early diagnosis and treatment of bone metastasis has always been one of the research hot spots.The commonly used detection methods include the calibration of serum biochemical indicators,nuclear imaging,etc.,and clinical treatment generally uses surgical treatment,local radiotherapy and systemic anti-cancer treatment Wait.This paper hopes to apply the optical imaging technology of small molecule fluorescent probe to the early detection of malignant bone metastasis.Rhodamine B is a basic dye with xanthene as the main structure;when the spiro ring is closed,there is no fluorescence,and under the action of H⁺or metal ions,the spiro ring will have strong fluorescence,which is an ideal"On-off"type fluorophore.Bisphosphonate drugs are used in the treatment of many metabolic bone diseases?mainly characterized by excessive bone resorption?.They are one of the most commonly used targeted drugs for the treatment of bone metastases from malignant tumors.They have demonstrated excellent performance both in vivo and in vitro Anti-tumor effect;has a high affinity for hydroxyapatite,can specifically target and bind calcium phosphate minerals in bones,and can prevent osteoclast-mediated bone resorption.In this paper,Rhodamine B was selected as the fluorescent group and bisphosphonate as the recognition group.A small-molecule fluorescent probe was designed and synthesized.It was suspected that it could target the site of bone metastasis,and it would ring open under acidic conditions.So it can detect the location of malignant bone metastasis in vivo in real time.Because the process of bone metastasis of malignant tumors will be accompanied by increased numbers of osteoclasts,blocked apoptosis,and enhanced activity.When osteoclasts play a role,they are first adsorbed on the surface of the bone,generating and releasing H⁺to acidify the surrounding microenvironment,resulting in hydroxyphosphorus The limestone dissolves locally,and Ca²⁺and the probe chelated with Ca²⁺are subsequently released,and the ring opens and emits fluorescence under acidic conditions,so the location of bone metastasis can be visualized.This article focuses on the design,synthesis and in vitro activity evaluation of this small molecule fluorescent probe.We designed and synthesized two kinds of probes,and the difference is the length of the carbon chain of the connecting group.By measuring the fluorescence emission spectrum,ultraviolet absorption spectrum,p H value response curve,concentration response curve and anti-interference of the probe,the optical performance of G0 was found to be better,so G0 with a shorter carbon chain was selected for further study.Subsequently,a comprehensive in vitro activity evaluation of G0 was carried out,including reaction time,optical stability,and fluorescence quantum yield.The experimental results show that:G0 will have a Stokes shift of 135 nm in a system with a p H value of 4.0;the reaction is complete within 5 seconds,and the fluorescence can be stably produced for more than 20 h;without interference from anions and cations in the system,it can specific response to p H value changes.Afterwards,we proved that G0 binds to hydroxyapatite particles quickly in vitro,and can achieve a binding rate of more than 80%in 15 min,and maintain stable binding for more than 3 h.In addition,G0 is not only sensitive to p H value in the solution,but also combined with hydroxyapatite particles,it can still respond to changes in p H value in the solid state and emit fluorescence in an acidic environment.Cell experiments show that G0 can exhibit better anti-tumor proliferation activity and less cytotoxicity in vitro,similar to the positive control zoledronic acid.The structure can be further optimized in the future,which may provide important clues for the early detection of malignant tumor bone metastasis.