Construction Of Triphenylamine-based Aggregation-induced Emission Nanoparticles And Its Application In Targeted Theranostic Of Cervical Cancer

Objective:Cervical cancer was the third most common cancer of women,which was the one of the leading causes of cancer-related death in worldwide.The incidence of cervical cancer was increasing in recent years.However,there was lack of effective treatment of cervical cancer duo to the imprecise diagnosis and intervention.Hence,cancer cells targeted theranostic and emerging strategy such as photodynamic theropy were of great demand.With the development of materials science,functionalized nanomaterials resented one novel strategy of cervical cancer.Molecules with aggregation-induced luminescence（AIE）characteristics received more and more attention due to its advantages,such as excellent light stability,bright fluorescence,high contrast,and large Stokes shift.AIE molecules held great potential in biomedical fields.However,absence of targeting ability,poor biocompatibility,and poor stability in physiological body fluids limited its real application.In present study,we prepared nanomaterials with AIE properties,and functionalized them to endow with tumor cell targeting ability,good water solubility,biocompatibility.We also determined its application in the theranostics of cervical cancer in vitro and in vivo.The main work was as follows:1.The AIE molecule M1 was synthesized and characterized,and a novel nanoparticle FFM1 was prepared by grafting this molecule with folic acid-modified F127.It could specifically target He La cells by recognizing the highly expressed folate receptor（FR）on the surface of He La cells.FFM1 NPs could release reactive oxygen species and induce tumor cells apoptosis upon light irradiation.It had excellent targeted imaging and photodynamic killing of He La cells,and showed application potential in targeted diagnosis and photodynamic therapy of cervical cancer.2.Two AIE molecules,MTB-PCBZ and BTZA-PCBZ,were synthesized and characterized respectively,and two AIE nanoparticles were obtained by the nanoparticle precipitation method.Screening of AIE nanoparticles with better properties makes them suitable for photodynamic therapy of tumors under the guidance of targeted imaging,and better applied to the diagnosis and treatment of cervical cancer.Methods:1.Using 2-（4-（diphenylamino）phenyl）-acetonitrile and 2,5-dipropoxytereph-thalaldehyde as raw materials,AIE molecule M1 was obtained by one-step Knoevenagel reaction.The structure of M1 was characterized by means of ¹H NMR and ¹³C NMR.M1was encapsulated by folic acid-functionalized F127 to form nanoparticles（FFM1 NPs）,and the UV-Vis absorption spectrum and fluorescence spectroscopy was used to characterize luminous properties and in vitro photodynamic properties of M1 and its nanoparticles.The imaging ability of FFM1 NPs at the cellular level was studied by laser confocal imaging.Evaluation of biocompatibility of FR non/low/high expressing cells and ROS killing effect after light exposure using MTT and Calcein-AM/PI assays.The targeting imaging ability of FFM1 NPs was explored by in vivo imaging system;The therapeutic effect and biosafety of FFM1 NPs in vivo were evaluated by mouse body weight,tumor volume,blood biochemical indexes and HE staining for pathological analysis.2.Using(4-（bis（4-methoxyphenyl）aminophenyl）boronic acid or(4-（bis（4-phenyl）-aminophenyl）boronic acid and-bromo-4-aldobenzo[C][1,2,5]thiadiazole,tetrakis-（triphenylphosphine）palladium as raw materials,respectively,two AIE molecules MTB-PCBZ and BTZA-PCBZ were synthesized by Knoevenagel reaction.The structures of two AIE molecules were characterized by means of ¹H NMR.The optimal structures of the two molecules were obtained by theoretical calculation.AIE nanoparticles（FFM and FFB NPs）were obtained by nanoparticle precipitation.The luminous properties and photodynamic properties in vitro of AIE molecules and their nanoparticles were studied by UV-vis and fluorescence spectroscopy.The effect of FFM and FFB NPs on cell viability in the absence of light and the generation of reactive oxygen species after light was investigated by MTT method.Targeted imaging ability to He La cells was investigated using confocal laser imaging.In vivo targeted imaging ability of FFB NPs was investigated using small animal imaging system.The therapeutic effect and biosafety of FFB NPs in vivo were analyzed by mouse body weight,tumor volume,blood biochemical indexes and HE staining for pathological analysis.Results:1.The synthesized M1 exhibited good AIE performance,and FFM1 NPs had excellent photostability,high fluorescence quantum yield and reactive oxygen species release ability.Cell experiments showed that FFM1 NPs could target He La cells,showed a strong fluorescent signal,and could generate a large amount of ROS after xenon lamp irradiation to induced cells apoptosis of He La.In vivo imaging studies showed that FFM1 NPs could specifically target and image at the tumor sites of tumor nude mice.In vivo treatment experiments showed that FFM1 NPs could effectively inhibit tumor growth,with little effect on major tissues and organs,liver and kidney functions.2.The synthesized MTB-PCBZ and BTZA-PCBZ had good AIE properties,FFM and FFB NPs had good photostability and reactive oxygen species generation capacity;In vitro experiments showed that compared with FFM NPs,FFB NPs had higher fluorescence quantum yield.Cell imaging experiments showed that both nanoparticles could target He La cells with high FR expression for imaging.MTT experiments proved that the two nanoparticles had good biocompatibility,and the photodynamic killing effect of FFB NPs was obvious under xenon lamp irradiation.Finally,FFB NPs were selected for in vivo research,and the results showed that FFB NPs could be targeted to tumor sites for fluorescence imaging,and they had obvious inhibition effect on nude mice tumor under the guidance of fluorescence imaging,and had little effect on major tissues and organs,liver and kidney function.Conclusion:In this study,three triphenylamine structure based nanomaterials with AIE properties were Prepared.M1 nanoparticles encapsulated with folic acid-functionalized F127 by nanoparticle precipitation method.They had high quantum yield and good light-driven reactive oxygen species generation ability,which were successfully applied to in vivo targeted imaging and photodynamic therapy of cervical cancer.In addition,on the basis of M1,electron-withdrawing groups were introduced into the triphenylamine structure and synthesized MTB-PCBZ and BTZA-PCBZ,which got fluorescence emission within the near-infrared region.Encapsulation of F127 functionalized with folic acid enabled them to specifically target tumor cells with high FR expression.MTB-PCBZ and BTZA-PCBZ not only maintained the original AIE fluorescence properties,but also had good biocompatibility.Finally,they were well applied to cancer cell-targeted imaging and therapy in vivo.The as developed nanomaterials could be used as a potential clinical agent for tumor-targeted imaging,diagnosis and photodynamic therapy.