| Avian leukemia subgroup J virus(ALV-J)clinically can seriously damage the immune system and cause a variety of tumor diseases and persistent viraemia,seriously affecting the egg production rate,meat-feed ratio and other production performance of chickens,causing incalculable losses to the world poultry industry.Therefore,the prevention and control of avian leukemia subgroup J virus is urgent.Studies have shown that zinc finger antiviral protein(ZAP)has a significant inhibitory effect on ALV-J replication by overexpression in a chicken embryonic fibroblast cell line(DF-1).However,ZAP is mainly administered by intravenous injection,which is prone to stressful effects in birds,and the transport pathway and mechanism of action of ZAP in vivo are not clear.Thus,in this paper,three carriers based on sodium alginate and plant polysaccharides were prepared: the sodium alginate-atractylodis macrocephalae polysaccharide system,the sodium alginate-platycodon grandiflorum polysaccharide system and sodium alginate-atractylodis macrocephalae polysaccharide@polydopamine nanomicrocapsules,and the loading performance of the carriers and fluorescent tracer labeling in DF-1 cells were investigated to provide technical support for the visualization of fluorescent tracing of ZAP in the clinical setting,which mainly includes the following three aspects:(1)Three carrier materials with mass ratios of 0.5,1 and 2 were prepared using sodium alginate and atractylodis macrocephalae polysaccharide as carriers,and loaded with BSA and ZAP proteins successively.The carrier materials were characterized and their loading performance was compared.The comparison shows that the carrier loaded with ZAP with the mass ratio of sodium alginate to atractylodis macrocephalae polysaccharide of 2 is a homogeneous spherical nanoparticle and has a high encapsulation efficiency(about 80%),which is the optimal loading system.The optimal loading system was subjected to cytotoxicity assay,release performance assay and fluorescent labeling investigation.The results showed that the ZAP-loaded sodium alginate-atractylodis macrocephalae polysaccharide carrier material could maintain the cell viability of DF-1 at more than 80% with low cytotoxicity.The carrier was able to achieve more than 32% release of ZAP,and was able to show excellent fluorescence performance with graphite-phase carbon nitride quantum dots(g-CNQDs)modification.This study provides an idea for finding excellent sodium alginate-polysaccharide-based carrier materials.(2)In order to investigate the differences between the sodium alginate-platycodon grandiflorum system and the sodium alginate-atractylodis macrocephalae system and to select the optimal loading system,three different mass ratios of sodium alginate-platycodon grandiflorum polysaccharide carriers loaded with BSA and ZAP were prepared successively.Through material characterization and comparison of loading performance,the carrier with the mass ratio of sodium alginate to platycodon grandiflorum polysaccharide of 2 was determined as the optimal loading system under this system,followed by cytotoxicity determination,release performance determination and fluorescence tracing investigation.Comparing the sodium alginate-atractylodis macrocephalae system and the sodium alginate-platycodon grandiflorum polysaccharide system,it can be seen that the various properties of the two systems do not differ significantly.However,the SEM results showed that the ZAP-loaded sodium alginate-atractylodis macrocephalae system had a homogeneous spherical nanostructure,which had more potential to become a nanocapsule.Therefore,the carrier with the mass ratio of sodium alginate to atractylodis macrocephalae of 2 was selected as the optimal loading system for ZAP loading and used in the subsequent research work.In this work,the optimal loading system for ZAP was found by comparison,which laid the foundation for the subsequent intracellular fluorescence tracing study.(3)The SA-AM-ZAP@PDA nanocapsule wrapped with polydopamine(PDA)was prepared based on the carrier material with the mass ratio of sodium alginate to Atractylodes macrocephala polysaccharide of 2,and it was surface modified with g-CNQDs.The results of cytotoxicity assay experiments of the nanocapsules showed that the nanocapsules were less toxic to DF-1 cells and could maintain the cell viability of DF-1 at more than 99%.The results of cell imaging showed that SA-AM-ZAP@PDA-QDs nanocapsules had excellent fluorescence properties in DF-1 cells and successfully achieved fluorescent labeling of ZAP,and the addition of PDA also improved the biocompatibility of nanocapsules and enhanced their fluorescence properties.SA-AM-ZAP@PDA-QDs nanocapsules exerted a variety of effects such as immune enhancement,cell internalization,fluorescent labeling,etc.This study provides theoretical support for clinical studies on the visualization of ZAP transport pathways in vivo. |
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