Study Of Biological Characteristics And Peripheral Nerve Tracing Technique Based On Fluorescent Gold Nanoclusters

The physical disability resulted by peripheral nerve injuries are commonly seen in clinical practice.Even after successful surgical repair the outcome is suboptimal or in some cases fail to recover.Currently,clinically available methods for evaluation of peripheral nerve regeneration and surgical outcome are simple clinical tests such as evaluation of muscle power,sensory exam,Tinel's sign and electromyogram,which are all subjective tests or lack accuracy.Therefore,accurate assessment of peripheral nerve regeneration helps in predicting outcome of surgical repair.A very traditional technique of neuroanatomical tracing in the field of neuroscience can directly visualize the nerve.Traditionally available neural tracers such as horseradish peroxidase,bacterial toxins and fluorescent dyes possess several limitations of time-consuming steps of immunostaining,complex procedure,poor fluorescence signal and photobleaching.Currently,there are many research papers on use of fluorescent nanoparticles in field of bioimaging and biomedical applications.With our previous research experience on carbon dots(carbon nanodots,polymer dots,graphene dots),in this study we introduce gold nanoclusters(AuNCs)due to its superior fluorescence excitation/emission wavelength(Red fluorescence)that overcomes the tissue and cell autofluorescence,which is an important feature in field of cellular imaging and nerve tracing.The reason of interest in these nanoparticles lie in their characteristics of excellent optical properties,chemical stability,low toxicity,good solubility,resistant to photobleaching and easy surface modifications.Semiconductor quantum dots are currently used in biosensors and bioimaging field.However,their heavy metal toxicity remains the greater limitation in its use in clinical field.Thus,these nanoparticles when compared to semiconductor quantum dots can be promising prospect due to their biocompatibility properties.This paper focuses on the cellular behavior,in vivo toxicology and development of nano neural tracer based on gold nanoclusters.In this study,the detail cellular behavior of the AuNCs was studied in rat pheochromocytoma cells(PC 12).Investigation tools used to study the uptake kinetics and endocytic pathways of nanoparticles were confocal laser scanning microscopy(CLSM),fluorescence activated cell sorter(FACS).Then,for intracellular trafficking,sub cellular distribution and co-localizations,different endocytic markers and organelles specific dyes were observed with CLSM and directly visualized its presence in the cellular ultra structures through transmission electron microcopy(TEM).Our results showed that AuNCs exhibited low cytotoxicity and efficient internalization inside neural cells.The cellular uptake was dose,time and energy-dependent.They mostly dispersed in endo-lysosomal structures but not seen inside the nucleus.After in vitro and in vivo toxicity it was proved that AuNCs are low in toxicity,and based on their cellular behavior,a fluorescent bio-probe for neural tracing was developed with conjugation of cholera toxin B subunit.Cholera toxin B subunit,is a non-toxic traditional neural tracer.A synthesis of cholera toxin B-Au nanoclusters conjugate(CTB-AuNC)via EDC/NHS coupling reaction was carried out.Its application as a retrograde tracer showed that it could bc transported via retrograde route by neurons in the peripheral nervous system in rats.