Construction And Evaluation Of Esterase/GSH Responsive Photoactivated Nano-pesticide Delivery System

Photoactivated pesticides are a new type of green pesticide with great development potential,but its in vitro photo-instability limits its application.Environmentally responsive polymer nanoparticles have attracted more and more attention as a new carrier for pesticide delivery systems.According to the physiological characteristics of insect cells rich in esterase and glutathione（GSH）,the design of esterase/GSH-responsive polymer nano-pesticides brings new hope to achieve the controlled release of photo-activated pesticides and to improve their in vitro photostability.In this paper,an esterase/GSH responsive polymer propesticide based on phenolic ester bond was constructed by using SA as main material,glycine as connecting arm and PB as pesticide model.The propesticide could self-assemble into core-shell structure nanoparticles in aqueous solution.Then,fluorescein-loaded photo-activated pesticide nanoparticles were prepared using fluorescein as a synergist by in-situ drug loading method.The main research contents and conclusions are as follows:Ⅰ.Preparation and characterization of PB-GSA polymer propesticideFirst,the esterification of Boc-glycine with protonated PB was carried out.Then the Boc protecting group of the resulting ester was removed by treatment with trifluoroacetic acid to give（PB-G）.Finally,the carboxyl group of SA was amidated with the amino group of PB-G to prepare polymer prodrug（PB-GSA）.The structure and purity of PB-GSA were characterized by FT-IR,¹H-NMR,HPLC and UV-Vis spectra.Ⅱ.Preparation and characterization of esterase/GSH responsive PB-GSA nanoparticles（1）PB-GSA nanoparticles were prepared by ultrasound-assisted self-assembly.TEM images showed that PB-GSA₃ nanoparticles had a spherical core-shell structure with uniform size.The dynamic light scattering（DLS）instrument showed that the average particle size of PB-GSA₁ and PB-GSA₃ nanoparticles were about 220±5.32nm and 345±7.43nm,respectively.And it had good storage stability and dilution stability.（2）In blank phosphate buffer solution（PBS）,the particle size of nanoparticles hardly changed,while in incubation with esterase or GSH,the average particle size of nanoparticles obviously increased.However,when nanoparticles were co-cultured with esterase and GSH,the particle size increased more rapidly.（3）Only about 6.5%PB was released after the nanoparticles were incubated in the blank PBS for 70 h.While in incubation with esterase or GSH,33.5%and 82.6%PB were released,respectively.When the nanoparticles were co-cultured with esterase and GSH,the release rate of PB was as high as 95.5%.Ⅲ.Preparation and in vitro drug release study of fluorescein-loaded nanoparticles（1）Fluorescein-loaded photo-activated pesticide nanoparticles were prepared using the PB-GSA as carriers by in-situ drug loading method.Studies showed that the larger the feed ratio（PB-G:SA）,the higher the drug loading rate of the nanoparticles and the larger their particle size.（2）About 11%of fluorescein was released within 24 h in the blank PBS.At the same time,the cumulative release rate of fluorescein reached 46%and 55%in PBS containing esterase or GSH,respectively.Under the combined incubation of esterase and GSH,the cumulative release rate of fluorescein reached 96%.Ⅳ.Evaluation of in vitro cell phototoxicity,photoactivity and photostability of PB-GSA nanoparticles（1）The fluorescence emission intensity of nanoparticle solutions containing equal amounts of PB to be significantly lower than that of free PB solutions indicating that the nanoparticles have the aggregation-caused quenching（ACQ）effect.After 270 min of illumination,the former had significantly lower PB degradation rate than the latter,and its fluorescence intensity was enhanced after GSH incubation.After incubation with GSH,the fluorescence emission intensity of the nanoparticles was enhanced.（2）The tyrosine oxidation rate of nanoparticles after GSH and/or esterase incubation was much higher than that of nanoparticles in blank PBS,and the oxidation rate of tyrosine in drug-loaded nanoparticles was about 18%higher than that of blank nanoparticles.The above shows that the photoactivity of the photoactivated pesticide nanoparticles has significant esterase and GSH responsiveness,and the loaded fluorescein effectively increased the photoactivity of the nano-pesticide.（3）The nanoparticles showed higher photocytotoxicity than equivalent free PB.And after pretreatment with in vitro light,the photocytotoxicity of free PB was greatly reduced,while the photocytotoxicity of nanoparticles was unchanged.