Construction Of Fluorescence/Photoacoustic Dual-modal Probes And Its Application In Imaging Of Acute Liver And Lung Injury

Recently,activatable molecular probes have attracted extensive attention and been widely used in the field of cell biology,biomedicine and environmental monitoring,due to the advantages of high sensitivity,fa st response and non-invasive.It provides an effective way to understand the function of bi omolecules and a basis for prediagnosis,pathological development and post-treatment of diseases through presice monitoring and imaging of specific biomarkers.However,the effective information obtained from in vivo imaging is limited by the insufficient tissue penetration depth of light.Therefore,the traditional activatable near-infrared fluorescent probes cann’t further meet the challenges of imaging deep and the accurate detection of biomarkers in vivo.In contrast,photoacoustic imaging methods that combine the advantages of optical and ultrasonic imaging to reduce tissue light scattering could provide promising signal-to-noise ratio and high-resolution images at depth in vivo.In addition,ratiometric imaging method can significantly improve the accuracy of experimental results through effectively avoid the interference of probe concentration and instrument fluctuation.Therefore,in order to achieve sensitive,accurate detection and high-resolution imaging of specific biomarkers in de pth,the development of activated multifunctional probes（e.g.,fluorescence/photoacoustic（FL/PA））with complementary advantages is of great significance.In above,a series of activatable dual-mode probes with excellent performance have been designed and synthesized based on the molecular structure modification and performance regulation of NIR hemicyanine and aza-BODIPY dyes for the early diagnosis and monitoring of acute liver injury and lung injury.The details are as follows:1)Hypoxia is an important pathological phenomenon in the course of drug-induced liver injury（DILI）.Effective monitoring of liver hypoxia is crucial for warning and treatment of DILI.However,because the correlation between hepatic hypoxia and DILI remains elusive and challenging,there are currently few studies for assessing liver hypoxia in vivo.Here,a novel photoacoustic/fluorescent（FL/PA）dual-modal probe（NO₂-CS）was rationally developed to image isoniazid（INH）induced liver hypoxia through detecting nitroreductase.The probe（NO₂-CS）was combined with the specific recognition substrate of nitroreductase（p-nitrobenzyl）with stable near-infrared fluorescent dye（NH₂-CS）.When the recognition group was specifically responded by nitro reductase,the fluorescence and photoacoustic signals of dye molecules were restored.In vivo studies showed that the probe could be used for FL/PA imaging of hypoxic DILI induced by INH.In addition,the occurrence of liver hypoxia in mice pre-treated with INH is also determined by up-regulation of hypoxia-inducible factor（HIF-1α）and the over-expressed NTR by electrophoresis analysis.It is believed that this probe can serve as an efficient tool to reveal hypoxia-related pathological progression of other liver diseases.2)The short excitation and emission wavelength of semi-cyanine fluorescent dyes is not conducive to ratio imaging in vivo.To addrress this issue,we first synthesised a novel NIR dye NF with a long excitation/emission wavelength and optically tunable hydroxyl group based on the xanthene skeleton through introduction of strong electron-sucking group（1-methyl-2-methyl-benz[c,d]iodolium salt）.And then,the NIR dye NF was further applied in construction of FL/PA dual modal probe.In addition,nitroxyl（HNO）,as one of highly reactive oxygen species in biological system,plays distinct biological roles in physiological process and anti-oxidation of organisms.However,the biological functions of endogenous HNO in DILI process still remains unclear owing to lacking of specific probe to monitor HNO in vivo.In view of above,a novel FL/PA probe（FB）was developed by coupling a HNO-responsive moiety（diphenylphosphinobenzoyl group）with fluorophore NF via an ester linker for detecting exogenous and endogenous HNO in vivo.By virture of the good response of to HNO through FL/PA dual model image,probe（FB）was further used for the detection of HNO in INH induced liver injury.These findings demonstrate that HNO is an ideal biomarker for DILI diagnosis,and FB are ideal fluorescent probes to study in situ HNO changes in various physiological and biochemical processes.3)The in-situ imaging of biological indicators in vivo is imperative for pathological research by utilizing activatable photoacoustic（PA）probe.However,the high-fidelity imaging in actual applications is greatly hampered by the inevitable false-signal response during blood circulation.Herein,we propose a controllable self-assembly and disassembly strategy（CADS）that can effectively perform probe in situ activation for high-fidelity imaging,by masking the reactive site to be inactivated in circulation.An amphiphilic molecular probe（9）was rationally constructed as a proof of concept,which is composed of two parts:the recognition moiety of superoxide anion radical(O₂^·-)caged aza-BODIPY as a hydrophobic group,and the PEG oligomer-bridgedβ-galactose as a hydrophilic group.Notably,the self-assembly of 9 as a nanoprobe（AP）in aqueous solution was obtained,causing the recognition site to be masked as inactivated due to the tight embedding of the hydrophobic group.More importantly,the stable AP exerted the false-signal silence during blood circulation,while the polarity-driven disassembly of AP allowed for activation of the recognition site towards endogenous O₂^·-within cells.By virtue of the positive feedback and specific accumulation ability,AP was further applied to precisely assess isoniazid（INH）induced liver injury through the desired ratiometric PA signals and high-fidelity imaging results.This strategy may provide a new insight for in situ imaging and elimination of off-site response.4)Real-time in situ imaging of acute lung injury is imperative for diagnosis and interventional therapy of patients.However,most of reported near infrared probes have been limitted for in situ imaging owing to the limitted blood circulation to accumulate at lung tissues.In order to solve this problem,we proposed an approach for developing a novel hemicyanine dye（FJH）with an optically tunable hydroxyl group and solubility via the combination of dyes screening and rational design strategy.In view of its excellent accumulation capacity of FJH,we further applied it to dwvelop molecular probe（FJH-C）for imaging acute lung injury through detecting esterase,which is constructed by introducing propyl formyl group.Upon addition of esterase,spectrometric studies showed that the probe FJH-C could be used for selectively and sensitively detecting esterases.And the probe FJH-C was demonstrated to accurately detect endogenous esterase in living cells and acute lung injury mouse model with high signal-to-back ratio through fluorescence/photoacoustic dual-mode imaging.We anticipate that the novel near infrared dyes could serve as an effective platform to design different FL/PA dual-mode probe for imaging lung diseases in the future.