Long-circulating Mechanism Of MePEG-PLGA Nanoparticles And The Properties Of TNF-α Blocking Peptide Loaded Nanoparticles

Recently,nano drug delivery systems (NDDS) that are made up of biodegradablematerials have been the hotspot research in modern pharmacy.However,to date,somemechanisms involved in the long-circulating NDDS have not been fully understood.Inthis study,a series of methoxypolyethyleneglycol modified poly (D,L-lactide-co-glycolide) nanoparticles (MePEG-PLGA-NPs) were prepared by the spontaneousemulsification solvent diffusion (SESD) method,and the correlations among the surfaceproperty and size of nanoparticles,opsonization by serum protein and phagocytic uptakewere studied to illustrate the long-circulating mechanisms for MePEG-PLGA-NPs.Tumornecrosis factor alpha blocking peptide (TNF-BP) was loaded on MePEG-PLGA-NPs byelectrostatic interaction,and TNF-BP loading capacity,bioactivity and the in vitro drugrelease were studied.The research was mainly concerned with the following aspects.(1) MePEG-PLGA copolymer was synthesized by ring-opening polymerization.Themolecular weight (MW) of the MePEG-PLGA copolymer was determined by GPCchromatograph.The structure of copolymer was confirmed by the Fourier transforminfrared spectrum (FT-IR) and ¹H-NMR spectrum.The results showed that the MW of thecopolymer was about 38000 Da.The blank MePEG-PLGA-NPs were prepared by theSESD method,and the NPs were characterized in terms of particle size,zeta potential andmorphology.The main preparation parameters were optimized in order to obtain theMePEG-PLGA-NPs with the desired characteristics.It obtained the optimal conditions forpreparation of nanoparticles that the emulsifier was Tween 80 (0.2%),the concentration ofcopolymer in the organic phase was 8.0 mg/ml,and the volume ratio of organic phase toaqueous phase was 1:6.Average size and zeta potential of optimized MePEG-PLGA-NPswere 80.2±7.7 nm and -24.55±3.50 mV,respectively.The morphology ofMePEG-PLGA-NPs was examined by transmission electron microscope (TEM) andatomic force microscope (AFM),and it appeared that the nanoparticles were spherical inshape with smooth surface and without any aggregation or adhesion.(2) A series of MePEG-PLGA-NPs were prepared.The influences of MePEG chain length,MePEG content and particle size on serum protein adsorption and complementactivation were investigated systematically,and their capacity for adsorbing albumin andthe serum total proteins was measured by a bicinchoninic acid (BCA) protein assay.Theadsorption of serum total IgG was investigated by enzyme-linked immunosorbent assay(ELISA).The results showed that the longer MePEG chains grafted on NPs surfaces,thelower the protein adsorption including albumin,immunoglobulin G (IgG) and the serumtotal protein adsorption.With the contents of MePEG on NPs surfaces increased from2.5% to 10.0%,a reduction of IgG adsorption was observed.BSA and serum total proteinadsorption were reduced with the increase of MePEG content on NPs surfaces up to 7.5%(w/w),but further increase led to a slight increase of adsorption.Complement activityperformed by hemolytic CH₅₀ test indicated that PLGA-NPs with MePEG content withinthe range of 5.0-7.5 % induces the lowest of complement consumption.Complementfragments (iC3b and C4d) in serum analyzed by ELISA demonstrated that the MePEGcoating could reduce the complement activation via both the classical and the alternativepathways.For the different size of MePEG-PLGA-NPs,protein adsorption andcomplement activation were increased along with the particle size increased from 73.6 to405.1 nm.However,no significant difference could be observed when particle size above405.1 nm.(3) To study the influence of MePEG modification,particle size and opsonization byserum proteins on uptake efficiency of MePEG-PLGA-NPs by phagocytes,nilered-labeledNPs were prepared by SESD method.The uptake efficiency of NPs by murine peritonealmacrophages (MPM) was measured by fluorescence spectrometer and fluorescentmicroscope.Phagocytic uptake of MePEG-PLGA-NPs by MPM involvedserum-independent and serum-dependent phagocytosis.Only serum-dependentphagocytosis was significantly prevented by MePEG modification on PLGA-NP surfaces,and this effect was mainly ascribed to the reduction of IgG adsorption and complementactivation.The surface modification of PLGA-NPs with higher amount and longer chainof MePEG can efficiently inhibit opsonization by serum and subsequently reduce theuptake by phagocytes.However,the inhibition of phagocytic uptake of NPs by MePEGchain was not obviously enhanced when MePEG₅₀₀₀ content above 7.5%.The particle sizeis another critical parameter for the uptake of NPs by phagocytes.Serum-independent phagocytosis decreased and serum-dependent phagocytosis increased with the increase ofparticle size,which contributed to the highest uptake for the MePEG-PLGA-NPs with sizeabout 400 nm.(4) Tumor necrosis factor alpha blocking peptide (TNF-BP) was loaded onMePEG-PLGA-NPs by electrostatic interaction,and the influences of some factors onentrapment efficiency and drug loading such as the type of emulsifiers,the emulsifierconcentration,the polymer concentration,TNF-BP concentration,the pH and ionicstrength of incubation medium were studied.The results showed that the average drugloading and entrapment efficiency for the optimized NPs were 1.36±0.05% and 54.40±1.56 %,respectively.In vitro release studies showed that the more TNF-BP loaded on NPs,the easier the peptides released,and the peptide release became slower for NPs in lowerionic strength medium.It was confirmed that most of TNF-BP remained active afterreleased in phosphate buffer solution by means of MTT.This study demonstrated that MePEG on PLGA-NPs surfaces only significantlyinhibit serum-dependent phagocytosis,which was mainly ascribed to the reduction of IgGadsorption and complement activation.Serum-independent phagocytosis was decreasedand serum-dependent phagocytosis was increased with the increase of particle size.ForTNF-BP,it was shown that MePEG-PLGA-NPs possessed good drug loading,controlledrelease and stability.In the present study,it illustrated the long-circulating mechanisms ofthe NDDS to some extent,and it can also provide new information for the design of moreefficient NDDS for peptides and proteins in medical applications.Based on the presentwork,it is necessary to investigate the pharmacokinetics and tissue distribution oflong-circulating NDDS and in vivo efficacy assessment in the future.