Effect Of Human Serum Albumin On Aminated Pullulan Nanoparticle Preparation And Its Effect On Nano-Agent Function

Objective:Aminated pullulan polymers with different degrees of substitution were synthesized to prepare nanoparticles with different amounts of charge.Aminated hydrophobic modified pullulan?CHAP?,carboxylated hydrophobic modified pullulan?CHSP?and hydrophobic modified pullulan?CHP?nanoparticles were prepared respectively.Then the interaction between nanoparticles and human serum albumin?HSA?was studied.The drug release behavior of CHAP,CHSP and CHP nanoparticles was studied,and the structural changes of HSA and CHAP nanoparticles with different degrees of substitution were studied.The purpose of this study is to provide an experimental and theoretical basis for the interaction between nano-targeted drug preparations and biomacromolecules in vivo.Methods:After the hydrophobic modification of pullulan,different amounts of ethylenediamine were used to modify,and then three kinds of nanoparticles with different charge amounts were obtained.The structure of the products was confirmed by infrared spectroscopy and nuclear magnetic resonance spectroscopy,and the nanoparticles were prepared by dialysis.Nanoparticles were characterized by dynamic light scattering?DLS?and transmission electron microscopy?TEM?.Hydrophobically modified pullulan was separately modified by amination and carboxylation,and the products were confirmed by infrared spectroscopy and nuclear magnetic resonance spectroscopy.The prepared nanoparticles were characterized by DLS and TEM.Isothermal titration calorimetry?ITC?,Fluorescence spectroscopy and circular dichroism spectroscopy?CD?were used to study the interaction of nanoparticles with HSA.The drug release of drug-loaded pullulan nanoparticles was determined by dialysis method.ITC was used to study the changes of thermodynamic parameters of HSA complexes before and after drug loading.Results:Three different degrees of substitution of aminated hydrophobically modified pullulan nanoparticles,the particle size of the nanoparticles is between 200 nm and 300 nm,the zeta potential and charges increased with the increase of amino substitution degree.The morphology of NPs is an incompletely regular spherical structure.Three kinds of nanoparticles with different electrical properties were prepared:CHAP NPs,CHSP NPs and CHP NPs.The zeta potentials of the nanoparticles were+12.9 mV,-15.4 mV and-0.698 mV,respectively;the particle size was 73.1 nm,116.9 nm and 156.9 nm.The thermodynamic changes of CHP,CHSP and CHAP nanoparticles were determined by ITC.The enthalpy and entropy showed that HSA interacted with nanoparticles.The binding constants of CHSP,CHP and CHAP were 1.41×10⁴ M^-1,27.7×10⁴ M^-1 and 412×10⁴ M^-1,respectively,indicating that the positively charged CHAP binds most strongly to HSA.Fluorescence spectroscopy and CD method were used to determine the changes in protein structure after complexation of nanoparticles with HSA.The results showed that CHP nanoparticles caused the greatest decrease in the?-helix of HSA.The drug release rate of free mitoxantrone,nanoparticles after loading drug and nanoparticle composite HSA was compared.After 48 hours of reaction,the drug release rate of CHSP-HSA was the highest,and CHP-HSA was the lowest.However,the release rate of the nanoparticles was slower than that of the free drug and the drug-loaded nanoparticles.Compared with the unloaded CHAP NPs,the HSA complex drug-loaded CHAP NPs had opposite changes in coverage and binding constant,indicating that drug loading had an effect on the release of nanomedicine.Conclusion:The size and charge amount of the CHAP nanoparticles increase as the degree of amino substitution increases.The size,surface charge and surface modification of nanoparticles affect the complexation of NPs with HSA.The adsorption of HSA on the surface of NPs retards the release function of CHAP nano-formulation.