A Novel Tumor-targeting Nanoprobe Design and Its Biological Effects Study

Author:Yao Chen Zuo

Supervisor:wu ming hong wang yan li


Degree Year:2019





Graphene quantum dots(GQDs)have been received increasing attention owing to their excellent physicochemical properties,high biocompatibility and tunable optical properties,which makes GQDs suitable for a wide range of potential applications in biomedical research.Numerous studies have examined surface functionalization of GQDs for the application in cancer diagnosis and drug delivery.In this thesis,we synthesized a new graphene-based tumor cell nuclear targeting fluorescent nanoprobe(GTTN),negatively charged sulfonic-graphene quantum dots via a modified alkali-mediated hydrothermal molecular fusion method which show specific tumor cell nuclear targeting perfomance both in vitro and in vivo.GTTN is a graphene-like single-crystalline structure amphiphilic fluorescent probe with a periphery that is functionalized by sulfonic and hydroxyl groups.It has the characteristic of specific tumor cell targeting,as it can directly cross the cell membrane and specifically target to the tumor cell nuclei by the changed permeability of the tumor cell membranes in the tumor tissue.This probe itself need no bio-ligand modification for tumor cell targeting,and has no intervention in cells of normal tissues.The tumor cell nuclear targeting mechanism is studied in this paper.In vitro,GTTN is repelled out of the cell membrane because of the repulsive force between negatively charged GTTN and the cell membranes which contributes to the low distribution in normal tissues in vivo.However,the plasma membrane-crossing process can be activated by incubating cells in ultrathin film culture medium because of the attachment of GTTN on cell membranes.Molecular dynamics simulations demonstrated that,once transported across the plasma membrane,the negatively charged functional groups of these GQDs will leave the membrane with a self-cleaning function retaining a small enough size to achieve penetration through the nuclear membrane into the nucleus.Our study shows that the specific tumor cell nuclear targeting mechanism of GTTN is closely related to its small size,negatively charged surface,specific microenvironment of the tumor(less interstitial fluid,high IFP)and self-cleaning.It suggests that GTTN may be developed into novel tools for tumor-specific imaging and therapeutics.This fluorescence probe can also recognize tumor tissue at a very early stage.This achievement in efficient and accurate tumor cell targeting will speed up the arrival of a new era of tumor diagnosis and treatment.In addition,GTTN with good biocompatibility and tunable optical properties may has a wide range of potential applications in biomedical fields.In this work,we also comprehensively detected the behavior of negatively charged GTTN in vivo and in vitro,using positively charged Amino-GQDs with similar construction as a comparison.These materials both show good chemical and photo-stability and considerable differences in their in vitro and in vivo imaging properties and toxicological behavior.Positively charged Amino-GQDs could easily pass through cell membranes and localize in cell cytoplasm,while showed acute toxicity in vivo causing death of mice even after tail vein injection at very low concentration.Negatively charged GTTN could not pass through the cell membrane even after 48 h co-incubation under normal culture conditions because the repulsive force between cell membrane and GTTN.However,specific targeting of cell nuclei was found when cells were cultured in an ultrathin film with GTTN.Both materials shows good performance for both in vitro and in vivo imaging,however,the toxicity issues of Amino-GQDs may limit their in vivo applications.Negatively charged GTTN shows low toxicity both in vitro and in vivo,which indicates its good potential for clinical applications.Besides,nowadays,two photon fluorescence imaging(TPFI)is considered to be a more suitable and popular way for cellular and deep tissue imaging with minor autofluorescence background,deeper tissue penetration,efficient light detection,reduced photobleaching and phototoxicity.GTTN,as a bright fluorescence probe,also exhibits strong fluorescence under two photon excitations.Moreover,GTTN displays little photobleaching,low toxicity and can emit quite strong fluorescence over a wide range of pH values.In in vitro/in vivo and deep tumor tissue two photon imaging study,GTTN prefers to stay out of the cells in vitro because the negatively charged surface of GTTN and cell membrane.However,the cell nucleus targeting process can be turned on by mild heating or laser irradiation of cells cultured in an ultrathin film.Furthermore,GTTN shows an extraordinary capability for targeting the nuclei of cancer cells in vivo,for a wide range of cancer types while there is no uptake by cells in normal tissue.Tumor nuclei targeting GTTN also exhibits strong fluorescence signal in deep tumor tissues for 500 μm depth under two photon microscopy.Herein,the good biocompatibility and extraordinary photo-and chemical-stability support the potential application of GTTN in TPFI of biological tissues and tumor monitoring and detection.To sum up,GTTN with good biocompatibility,tunable optical properties and specific tumor cell targeting ability allows it a super bright future for biomedical application.