Preparation Of Gallic Polyphenols Nanospheres:From Mechanism To Therapeutic Applications

Object:The purpose of this research is to prepare spherical nanoparticles with regular morphology directly from the decoction of Turkish galls in traditional Chinese medicine,and explore their formation mechanism and potential applications in the fields of antibacterial,antioxidant,pH response,biobase materials and intracellular protein delivery.Methods:（1）Spherical nanoparticles（TG-NPs）were prepared from Turkish galls extract（TGE）by differential centrifugation,and characterization was carried out by scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,malvern laser particle size instrument,ultraviolet-visible spectrospectroscopy（UV）,and Fourier infrared spectroscopy（FT-IR）;（2）The antioxidant capacity of TG-NPs on DPPH and ABTS radical scavenging was evaluated,and pH-responsive degradation was studied by observing the degradation of TG-HP NPs in different pH buffer solutions and the changes of polyphenol content;（3）High performance liquid chromatography-mass spectrometry（LC-MS）technology and energy dispersive X-ray spectroscopy（EDX）were used to analyze the chemical composition and elemental distribution of TG-NPs,and the existence of proteins/amino acids was verified by ninhydrin experiments;（4）According to the formation mechanism,gallic polyphenols and 10 functional proteins were self-assembled in a simple way to obtain gallic polyphenol protein nanospheres TG-protein NPs,and their characterization and properties were investigated,including stability,pH responsiveness and potential as bioactive templates;（5）The inhibitory effect of TG-NPs and TG-LYZ NPs on E.coli and S.aureus and the removal effect of biofilms were detected by oxford cup method,plate counting method,SEM bacterial morphology observation method,live/dead bacteria staining method and crystal violet staining experiment;（6）The distribution of nanosphere TG-protein NPs in A549 cells was observed by biological transmission electron microscopy,and the process of fluorescently labeled TG-protein NPs entering cells was tracked by laser confocal microscopy（CLSM）.Changes in biological activity after protein entry into cells were detected byβ-galactosidase staining assay.Results:（1）Gallic polyphenol nanoparticles in TGE are regular spherical nanoparticles.TG-LP NPs have a diameter of 413.89±202.95 nm,TG-HP NPs have a diameter of 230.34±59.48 nm.（2）TG-LP NPTs and TG-HP NPS retained the antioxidant capacity of TGE;Nanospheres TG-HP NPs are prone to degradate at pH>7.0,and the content of gallic polyphenols is reduced.（3）The main chemical components of gallic polyphenol nanospheres TG-NPs are gallic polyphenols,containing three elements:C,O and N,and ninhydrin experiments confirmed that nanospheres contain proteins;The molding mechanism of nanospheres is formed by self-assembly of gallic polyphenolic compounds and a small amount of protein present in Turkish galls.（4）Gallic polyphenols can self-assemble with10 different functional proteins to form TG-protein NPs,which have pH responsiveness that is easily degradable under alkaline conditions;TG-HP NPs and TG-BSA-1 NPs had good stability in 14 days;TG-BSA NPs can be used as bioactive templates to modify Ag nanoparticles or TA-Fe^III coatings on their surfaces.（5）Gallic polyphenol nanospheres have good inhibitory effect and biofilm scavenging ability against E.coli and S.aureus.（6）Gallic polyphenol protein nanospheres can be used as an effective carrier for intracellular protein delivery and retain part of the biological activity of the protein.Conclusion:TG-NPs could be prepared from Turkish galls extracts by differential centrifugation method,and the particle size of nanoparticles could be adjusted by different centrifugal forces,which exhibit strong antioxidant capacity and pH responsiveness,and it was confirmed that TG-NPs mainly formed through the self-assembly of gallic polyphenols and proteins existing in galls itself.According to formation mechanism,gallic polyphenol compounds can be assembled with 10 functional proteins to obtain TG-protein NPs,which can be used as bioactive template materials,and support pH response,antibacterial therapy and protein delivery.