Construction Of Carboxylesterase-responsive Multifunctional Probes And Their Applications In The Diagnosis And Treatment Of Cervical Cancer

Objective:Cervical cancer is one of the most common cancers in women.In recent years,the incidence and mortality of cervical cancer are both high.Difficulties in early non-invasive diagnosis and lack of effective treatment means mainly account for the poor prognosis.Therefore,developing new precision diagnosis and treatment methods for cervical cancer is of great significance for reducing mortality and improving patients’quality of life.The theranostic probe integrating imaging and treatment modules,which can realize the imaging-guided accurate and personalized treatment.Near-infrared fluorescence imaging has been widely concerned because of its high sensitivity and low tissue damage.In terms of treatment,due to the high heterogeneity of malignant tumors and the inherent shortcomings of each treatment mode,the combination of multiple treatment methods can achieve better anti-tumor effects.Carboxylesterase（CES）is a class of hydrolytic enzymes,which is overexpressed in various malignant tumors including cervical cancer and breast cancer.It is one of the tumor markers of cell.Considering the above points,CES-activated multifunctional probes are proposed in this thesis the applications of the probes in the near-infrared fluorescence imaging and synergistic therapy of cervical cancer are studied,aiming at providing new technical means for the accurate diagnosis and treatment of cervical cancer.Method:1.Using the near-infrared hemicyanine dye HCH and hexaacetylaminocaproic acid（AcAH）as raw materials,small molecule phototheranostic probe HCH-AcAH was synthesized through esterification reaction.HCH-AcAH was characterized by high resolution mass spectrum,¹H and ¹³C nuclear magnetic resonance spectra.The response of HCH-AcAH to CES and the photodynamic performance of the reaction system were investigated by UV-vis and fluorescence spectra assays.Laser confocal microscope was used to observe the uptake of probe in cells.The level of intracellular ROS was studied using the commercial ROS probe DCFH-DA.MTT method and Calcein-AM/PI staining were used to study the cellular killing effect of the probe.2.Using HCH and phenylbutyrate nitrogen mustard（a chemotherapeutic drug）as raw materials,a small molecular probe HC was synthesized by esterification reaction.Furthermore,a multifunctional nanoprobe PLGA-PEG@HC was prepared by encapsulating HC with PLGA-PEG via microemulsion method.HC was characterized by high resolution mass spectrum,¹H and ¹³C nuclear magnetic resonance spectra.The morphology and particle size of PLGA-PEG@HC were characterized by TEM and DLS assays.The response of PLGA-PEG@HC to CES was studied by UV-Vis and fluorescence spectra.The photodynamic and sonodynamic properties of the reaction systems were investigated by commercial probes DCFH-DA and DPBF.The imaging behavior of PLGA-PEG@HC in different cells was observed by laser confocal microscope.MTT method and calcein-AM/PI staining were used to study the killing effect of PLGA-PEG@HC to different cells.In vivo small animal fluorescence imaging was used to evaluate the potential of PLGA-PEG@HC for tumor imaging in vivo.The therapeutic effect of PLGA-PEG@HC in vivo was evaluated by tumor growth curve,tumor size and quality of tumor-bearing nude mice,and the biological safety of PLGA-PEG@HC was assessed by body weight changes,main biochemical indexes tests and pathological sections staining of major organs.3.In order to further improve the targeting ability and overall performance of the probe,the amphiphilic molecule HA-TS was obtained by covalently incorporating D-α-tocopherol succinic acid（TS）to hyaluronic acid（HA）,then the multifunctional nanoprobe HA-TS@HC with active targeting ability was obtained by encapsulating HC with HA-TS.The structure and properties of HA-TS@HC were investigated with the similar means mentioned in the second part.Result:1.The small molecule phototheranostic probe HCH-AcAH showed highly sensitive and selective fluorescence enhancement response to CES,with the detection limit as low as 0.712 U/L.ROS were generated in the reaction system of HCH-AcAH and CES under the irradiation of 660 nm laser.HCH-AcAH can be used for CES imaging in He La cells.Moreover,HCH-AcAH exhibited significant killing effects to He La cells under 660 nm laser irradiation,accompanied with low cytotoxicity in dark.2.The multifunctional nanoprobe PLGA-PEG@HC were spherical,with a uniform size of 95 nm.PLGA-PEG@HC showed highly sensitive and selective fluorescence enhancement to CES.ROS were produced in the reaction system of PLGA-PEG@HC and CES under 660 nm laser or ultrasonic stimulation.Studies at the cellular level showed that PLGA-PEG@HC could kill tumor cells through combining chemotherapy,photodynamic and sonodynamic therapy,with low toxicity to normal cells.In vivo studies demonstrated that PLGA-PEG@HC could accumulate at tumor sites and the fluorescence was activated.Moreover,PLGA-PEG@HC exhibited significant antitumor effects through the combination of chemotherapy,photodynamic and sonodynamic therapy,with high biosafety.3.The multifunctional nanoprobe HA-TS@HC were uniformly spherical and well dispersed,with a particle size of about 52 nm.HA-TS@HC displayed intense fluorescence-enhancement to CES,and HAase could promote the reaction between HA-TS@HC and CES.ROS were generated in the reaction system of HA-TS@HC and CES under 660 nm laser or ultrasonic stimulation.Studies at the cellular level showed that HA-TS@HC was selectively taken up by CD44-overexpressed tumor cells,followed by the activation of fluorescence signals.However,HA-TS@HC was barely taken up by normal cells.HA-TS@HC could kill tumor cells through combining chemotherapy,photodynamic and sonodynamic therapy,while exhibited low toxicity to normal cells.In vivo studies suggested that HA-TS@HC can be used for the fluorescence imaging of tumor in vivo.In addition,HA-TS@HC could dramatically suppress the growth of tumor through synergistic chemotherapy,photodynamic and sonodynamic therapy,with negligible side effects on normal tissues.Compared with PLGA-PEG@HC,the antitumor performance of HA-TS@HC was improved,and more effective tumor inhibition could be achieved with fewer treatment times and lower laser and ultrasonic power.Conclusion:In this thesis,three CES-activated multifunctional probes with near-infrared fluorescence imaging and synergistic therapy abilities are constructed,which can be used for the effective treatment of cervical cancer guided by fluorescence imaging.These three probes showed the maximum emission at about 710 nm and could achieve NIR fluorescence imaging of CES,possessing the merits of low background interference,deep tissue penetration,and little damage to tissues.In addition,the combinational chemotherapy,photodynamic and sonodynamic therapy showed better antitumor effect compared with the single therapy.The developed multifunctional probes are simple,with definite composition and good biocompatibility,which may have potential application value in precision diagnosis and efficient treatment of cervical cancer and related diseases.