| Fluorescence imaging technology is one of the most powerful tools in the biomedical field.At present,more and more fluorescence probes have been developed to equip the"ammunition storehouse" for fluorescence imaging.Since the aggregation-induced emission(AIE)effect was discovered in 2001,it has broken through the limitation of traditional aggregation-caused emission(ACQ)fluorescent probes in the biological field.With the advantages of strong fluorescence signals,excellent photostability and excellent biocompatibility,AIE luminogens have been widely used in clinical medicine research fields,such as tumor imaging,brain vascular imaging,circulating tumor cells detection,etc.In this paper,the fluorescent probes with AIE property are used to explore their applications in urology,and to provide new strategies to solve practical clinical problems.This paper is divided into two parts:the first is the intraoperative fluorescence imaging of ureter;the second is the rapid differential diagnosis of urinary tract infection.Part I Near-Infrared-II Aggregation-Induced Emission(AIE)Dots for Intraoperative Identification of UreterObject:The fluorescent probe 2TT-oC6B dots with near-infrared-? region(NIR-?)and AIE property were prepared for intraoperative identification of ureter.The physicochemical properties and biological safety of 2TT-oC6B dots were evaluated,and the practical application value of 2TT-oC6B dots in ureter imaging was explored.Methods:(1)Synthesized AIE molecule:2TT-oC6B,and analyzed the product using nuclear magnetic resonance(NMR).(2)2TT-oC6B and 1,2-Distearoyl-Sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene Glycol)-2000](DSPE-PEG2000)were used to prepare NIR-? fluorescent probe:2TT-oC6B dots.The absorption spectrum,emission spectrum,fluorescence quantum yield(QY),morphology,and particle size of 2TT-oC6B dots were characterized by ultraviolet/visible spectrophotometer,fluorescence spectrophotometer,fluorescence quantum yield meter,transmission electron microscope(TEM)and dynamic light scattering(DLS),respectively.(3)AIE properties:the fluorescence signals of 2TT-oC6B dots and indocyanine green(ICG)with different concentrations in deionized water or urine were tested by NIR-? imaging system.(4)Chemical stability of 2TT-oC6B dots:the fluorescence signals of 2TT-oC6B dots and ICG with different pH from 3-11 in deionized water or urine were imaged by NIR-? imaging system.(5)Photostability of 2TT-oC6B dots:the fluorescence signals of 2TT-oC6B dots and ICG in deionized water or urine under continuous 808 nm laser irradiation(50 mW/cm2)for 25 min were captured by NIR-? imaging system.(6)After retrograde or antegrade injection of ureter with 2TT-oC6B dots,NIR-? fluorescence imaging of ureter under 808 nm laser irradiation was evaluated by NIR-? imaging system.(7)For comparing the differences between 2TT-oC6B dots and ICG in NIR-? imaging of ureter under 808 nm laser irradiation,NIR-? fluorescence intensity of ureter imaging with retrograde injection of 2TT-oC6B dots and ICG under 808 nm laser irradiation with different thickness of beef slices(0 mm,1.5 mm,3 mm,4.5 mm)was recorded by NIR-? fluorescence system.(8)The iatrogenic ureteral injury(IUI)models:complete/partial ligation,ligation,complete/partial disconnection of ureter;ureteral recanalization model;and ureteral diseases models:foreign body,stricture,abnormal course of ureter were prepared,and then the ureteral fields were imaged by NIR-? fluorescence system respectively.(9)For comparing the differences between 2TT-oC6B dots and ICG in NIR-? imaging of ureter under laparoscopic xenon light,after two ureters were injected with 2TT-oC6B dots and ICG respectively,two ureters were imaged with the NIR-? fluorescence imaging system when laparoscopic xenon light was 5 cm,10 cm and 15 cm away from the ureteral field.(10)Cytotoxicity evaluation of 2TT-oC6B dots:the cytotoxicity of 2TT-oC6B dots on human uroepithelial cells SV-HUC-1 and human bladder cancer cells T24 was evaluated by cell counting kit-8(CCK-8).(11)To assess the in vivo safety of 2TT-oC6B dots,the kidneys,ureters,and bladders of rabbits were collected,and then stained with hematoxylin and eosin(H&E).Results:(1)Both TEM and DLS manifested uniform spheres in shape with an average diameter of about 160 nm.The 2TT-oC6B dots exhibited maximal absorption and emission at 733 nm and 1030 nm,respectively.The QY of 2TT-oC6B dots was measured to be 11.0%in the NIR-? region.(2)The fluorescence intensity of the 2TT-oC6B dots in deionized water or urine proportionally increased with increasing concentrations.However,the fluorescence intensity of ICG was slightly increased at very low concentrations,but significantly quenched at high concentrations.(3)The fluorescence intensity of 2TT-oC6B dots was negligibly changed by varying the pH from 3-11 in deionized water and urine,respectively.In addition,the fluorescence intensity of 2TT-oC6B dots was not affected by urine,which was comparable with that in deionized water.Contrarily,ICG displayed a significant decrease in fluorescence intensity possibly due to the ionic interactions in urine.(4)Upon continuous 808 nm laser irradiation(50 mW/cm2),2TT-oC6B dots displayed good photostability with an ignorable variation in fluorescence intensity,while the intensity decreased by over 95%in ICG.(5)After 2TT-oC6B dots were injected into the lumen of the ureter anterogradely or retrogradely,the ureter was immediately lighted up under NIR-?fluorescence system,which revealed the clear course and peristaltic flow of the ureter.(6)The ureter bearing 2TT-oC6B dots was significantly brighter than that of ICG.The signal-to-background ratio(SBR)of 2TT-oC6B dots were about 2.4 times that of ICG on NIR-?fluorescence imaging.(7)NIR-? fluorescence imaging of ureter with AIE dots intraureteral injection was clearly visualized,as the ureteral region were covered with two beef slices,about 3 mm.Even as the ureteral region was covered with three beef slices,4.5 mm,slight fluorescence signals could still be distinguished.However,NIR-? fluorescence signals of the ureter with ICG intraureteral injection were hardly captured when the ureteral region was covered with only a 1.5 mm slice of beef.(8)Iatrogenic ureteral injuries(IUIs),ureteral common diseases and ureteral recanalization could be detected by NIR-? fluorescence imaging with 2TT-oC6B dots.(9)NIR-? fluorescence imaging of ureter with 2TT-oC6B dots under the laparoscopic xenon light was evaluated.As the laparoscopic xenon light source approached the ureteral region gradually,from 15 cm to 10 cm and then to 5 cm,NIR-? fluorescence imaging of ureter with retrograde AIE dots injection could be seen more and more clearly.However,the NIR-? fluorescence of ureter with ICG retrograde injection can hardly be captured by the sensitive InGaAs camera,even as the distance between laparoscopic xenon light source to the imaging platform was 5 cm.(10)When 0.10 mg/mL 2TT-oC6B dots were added to both SV-HUC-1 and T24 cells,almost no cytotoxicity occurred.Even if the concentration of 2TT-oC6B dots was as high as 0.20 mg/mL,the growth of both cells was not significantly affected.(11)The hematoxylin and eosin(H&E)staining showed that there was no obvious damage or inflammatory lesion in urinary organs of the rabbits.Conclusions:In summary,we proposed a strategy for intraoperative identification of ureter using NIR-? AIE dots in living rabbits.Noteworthy,2TT-oC6B dots exhibited maximal NIR-? emission at 1030 nm with a high fluorescence QY(11.0%),which was sufficient to enable intraoperative identification of ureter via NIR-? fluorescence imaging.Compared to ICG,2TT-oC6B dots embodied the advantages of high brightness,good chemical stability in urine,and superb photostability,which guaranteed the clarity and reliability of NIR-? fluorescence signal.In vivo NIR-? imaging with 2TT-oC6B dots via intraureteral injection enabled us to visualize the ureter with high SBR and deep fluorescence penetration depth.Furthermore,not only ureteral injuries,some common diseases but also ureteral recanalization could be detected using 2TT-oC6B dots.Interestingly,NIR-?imaging of the ureter could even be performed under conventional laparoscopic xenon light without 808 nm laser,due to the high fluorescence efficiency of 2TT-oC6B dots.Moreover,2TT-oC6B dots showed good biosafety and negligible biotoxicity.Thus,our work realized the intraoperative identification of ureter using highly stable and bright NIR-? AIE dots,providing a new platform for real-time fluorescence imaging during clinical operations.Part ? Fluorescent Probe Based on Aggregation-Induced Emission Mechanism for Rapid Differential Diagnosis of Urinary Tract InfectionObject:Based on aggregation-induced emission(AIE)mechanism,a fluorescent probe for rapid identification of pathogenic bacteria and fungi was synthesized,the physical and chemical characteristics of the fluorescent probe were evaluated,and its feasibility of rapid identification and diagnosis of urinary tract infection(UTI)was tested.Methods:(1)Synthesize AIE molecule:IQ-Cm.1H NMR,13C NMR,HRMS spectra,absorption spectrum,emission spectrum,single crystal structure,fluorescence quantum yield(QY),morphology,and particle size of IQ-Cm were characterized by nuclear magnetic resonance(NMR),high resolution mass spectrometer(HRMS),ultraviolet/visible spectrophotometer,fluorescence spectrophotometer,X-ray single crystal diffractometer,fluorescence quantum yield meter,transmission electron microscope(TEM)and dynamic light scattering(DLS),respectively.(2)The photostability of IQ-Cm was tested in time series mode by a confocal laser scanning microscope(CLSM).(3)Pathogen staining:IQ-Cm was added into pathogens PBS suspension with the final concentration of 10 ?M(G-bacteria(OD600=1.0),G+bacteria(OD600=1.0)or fungi(OD600=2.0)).The mixtures were incubated for about 10 min at room temperature.(4)Pathogen Imaging.(a)For naked-eye identification of pathogens,after incubating pathogens with IQ-Cm,the mixture was observed under 365 nm UV irradiation.(b)For microscope imaging,after incubating pathogens with IQ-Cm,the pathogen suspension was transferred to glass slide,slightly covered by a coverslip,left for 2 min for immobilization and then imaged.(5)Zeta potential measurements and assessment of antimicrobial activity of IQ-Cm.The zeta potential was measured by ZetaPALS potentiometer.The antimicrobial activity of IQ-Cm against three pathogens was evaluated by traditional surface plating method.(6)Co-staining experiment.Three pathogens were incubated with 10 ?M of IQ-Cm and 5 ?g/ml of propidium iodide(PI)in PBS solution for 10 min and then imaged on a fluorescence microscope.(7)Mechanism of IQ-Cm staining E.coli.75%alcohol was used for preparation of cell membrane-destroyed E.coli.Then the treated E.coli were incubated with 10 ?M of IQ-Cm and 5 ?g/ml of PI in PBS solution for 10 min and then imaged on a fluorescence microscope.ethylenediaminetetraacetate(EDTA)was used for preparation of E.coli with disrupted outer membrane but intact inner membrane.Then the treated E.coli were incubated with 10 ?M of IQ-Cm in PBS solution for 10 min and then imaged on a fluorescence microscope.(8)Mechanism of IQ-Cm staining C.albicans.C.albicans was incubated with 1 ?M of IQ-Cm and 100 nM of Mito-tracker Green for 10 min and then imaged on a fluorescence microscope.(9)Detection of urinary tract infection.The urinary tract infection model caused by single pathogen was prepared,and the staining steps and imaging conditions were the same as those of Methods(3)and Methods(4).The urinary tract infection model that evolved from bacterial infection to fungal infection was prepared.The staining steps and imaging conditions were the same as those of Methods(3)and Methods(4b).Results:(1)Characterization of IQ-Cm.The synthesized product was confirmed by 1H NMR,13C NMR,HRMS spectra.The single crystal structure demonstrated that IQ-Cm adopted a twisted 3D conformation with large torsional angles for the aromatic groups,i.e.72.4—77.8° torsion of phenyl groups from the isoquinolinium core and 23.5—31.8° torsion of coumarin and isoquinolinium moiety from the phenyl linker.The distance between the nearest two coumarin or two isoquinolinium planes was about 11 A.IQ-Cm crystal gave intensive orange-red emission at 620 nm with a fluorescence quantum yield of 5.7%.Under UV light irradiation,IQ-Cm presented a remarkable solvatochromism effect.With increasing the water content from 0 to 80%in DMSO/water mixtures,the emission intensity of IQ-Cm at 501 nm gradually decreased,along with a slight red-shift in the emission maxima,due to the enhanced twisted intramolecular charge transfer(TICT)effect by the more polar water in the surrounding environment.Further increasing the water content from 80%to 98%,a large red-shifted emission at 643 nm appeared and increased,showing an AIE phenomenon because of the formation of aggregates.DLS result and TEM image showed that IQ-Cm forms rod aggregates of about 1 ?m.(2)Photostability of IQ-Cm.After 120 consecutive scans,there was almost no loss of fluorescent signal in IQ-Cm.(3)Pathogen Imaging.(a)For naked-eye identification of pathogens,after incubating pathogens with IQ-Cm,the fluorescence emission of IQ-Cm was obviously lighted up with three distinguishable emission colors.The E.coli suspension gave the weak orange-yellow fluorescence,the S.aureus suspension presented orange-red fluorescence while C.albicans gave the bright yellow emission.(b)For microscope imaging,after incubating pathogens with IQ-Cm,the labeled three pathogens showed the different emission colors,i.e.,green and orange for E.coli,orange-red for S.aureus and yellow for C.albicans.(4)Zeta potential measurements and assessment of antimicrobial activity of IQ-Cm.After adding IQ-Cm,the surface potentials of S.aureus and C.albicans did not obviously change while that of E.coli became more positive.The killing rates of IQ-Cm to E.coli,S.aureus and C.albicans were 10%,100%and 25%,respectively.(5)Mechanism of IQ-Cm staining E.coli.In the case of G-E.coli with the barrier of outer membrane,IQ-Cm primarily targeted their negative surface,and thus a major of E.coli were almost nonemissive.Only a small portion of E.coli with compromised outer membrane or dead E.coli were lighted up by IQ-Cm.When IQ-Cm molecules mainly inserted into the cell membrane of E.coli with compromised outer membrane,green fluorescence was seen,or when IQ-Cm molecules mainly entered in the cell protoplasm of dead E.coli,orange fluorescence was presented.(6)Mechanism of IQ-Cm staining S.aureus.The strong interaction of IQ-Cm with S.aureus facilitated IQ-Cm to enter the cell cytoplasm of S.aureus.In large polar and glass-like microenvironment of cell cytoplasm,IQ-Cm gave a red-shifted emission of orange color.(7)Mechanism of IQ-Cm staining C.albicans.IQ-Cm mainly located in the mitochondria of C.albicans.In the mediate polar environment of mitochondrial membrane,an intermediate yellow emission was obtained.(8)Detection of urinary tract infection.The urinary tract infection model caused by single pathogen was readily detected by IQ-Cm in naked-eye identification and microscope imaging.The urinary tract infection model that evolved from a bacterial infection to a fungal infection could be detected by microscope imaging.Conclusions:Based on the AIE mechanism and different characteristics of the cell structure and microenvironment in pathogens,a fluorescent probe for rapid identification of G-bacteria,G+bacteria and fungi was designed:IQ-Cm.IQ-Cm was mainly localized in the cell membrane and cytoplasm of G-bacteria,cytoplasm of G+bacteria,and mitochondria of fungi.At the level of the naked eye,G-bacteria gave the weak orange-yellow fluorescence,G+bacteria presented orange-red fluorescence while fungi gave the bright yellow emission.At the cellular level,three pathogens showed the different emission colors,green and orange for G-bacteria,orange-red for G+bacteria and yellow for fungi.By providing discernible fluorescent emission colors,IQ-Cm could quickly identify G-bacteria,G+bacteria,and fungi.More importantly,IQ-Cm showed great potential for rapid diagnosis of urinary tract infections caused by single pathogen,and the evolution of urinary tract infections from bacterial infection to fungal infection could also be monitored by IQ-Cm in a timely and intuitive way.Therefore,our research provided a fast and easy platform for the diagnosis of pathogens,successfully transforming the information of pathogenic bacteria and fungi into different fluorescent colors,and providing a timely and reliable reference for the formulation of clinical treatment plans. |
PDF Full Text Request