Effect Of Site-specific PEGylation With Linear Or Branched Pegs On The Structure,Pharmacodynamics And Pharmacokinetics Of Human Serum Albumin

Background and Objectives:Human serum albumin(HSA)is the most abundant protein in plasma,which is responsible for 80%of the colloid osmotic pressure of plasma and the main modulator of fluid distribution between body compartments.At the same time,HSA serves asa depot and carrier for many endogenous compounds including fatty acids,heme,bilirubin and tryptophan,and affects the pharmacokinetics/pharmacodynamics of many drugs.The main clinical indications of HSA are traumatic shock,burns,hypoalbuminemia,ascites and acute respiratory distress syndrome.But in some pathological cases such as injury and shock,the endothelial integrity is often compromised,causing the leak of HSA into theinterstitial space,which exacerbates interstitial edema and worsens the situation.So,the views on the clinical efficacy of HSA as a volume expander are inconsistent.In addition,the clinical dosage of HSA is relatively high.Though the recombinant HSAhad been successfully producedby the genetic engineering,the production of recombinant HSA with high purity,low cost and industrial-scale HSA still faces many challenges.So the supply for HSA is still very tense.In order to solve the problems of interstitial edema of HSA in the clinical application and the relatively tight source of HSA itself,HSA has been modified with high molecular weight PEG in our previous study.PEG-HSA with precise modification site and high purity was obtained.In the previous experimental study,the circular dichroism demonstrated that PEG covalent modification did not affect the secondary structure of HSA.The dynamic laser scattering confirmed that the PEG with 20 kDa could significantly increase the hydrodynamic radius of HSA.The pharmacokinetics study of HSA and its PEG modified products by 125I radiolabelling confirmed that the retention time of PEG-HSA in vivo was significantly prolonged.The acute lung injury model of mice and rat sepsis model confirmed that PEG modification could effectively reduce the leakage of HSA from blood vessels,making HSA play the role of blood volume expansion more effectively.Based on the previous research of our laboratory,in this study,we studied the efficacy of HSAsmodified by 20 kDa linear or branched PEG as blood volume expanders in the hemorrhagic shock model,and investigated whetherbranched PEG-modified HSA had advantages on the pharmacodynamics.The spatial orientation of different Mr and different configurations of PEGon the protein surface and their effect on the structureof HSA were studied by using small angle scattering technique.Surface plasmon resonance(SPR)was used to study the effect of diffent PEG conjugationson the binding capacity of hematin binding site near Cys34.In addition,because radiolabelling is unstable and easy to fall off,it could not reflect the real drug concentration in pharmacokinetics study.The present study applied nanoliquid chromatography tandem mass chromatography(NanoLC-MS/MS)to detect PEG-HSA specific peptide fragmentproduced by enzymolysisto establish a new method for determining the concentration of PEG-HSA in rat plasma,which is highly sensitive and selective.The Main Contents,Methodsand Results:1.Experimental study on the efficacy of site-specific PEGylated HSA in resuscitation from hemorrhagic shockTo evaluatethe resuscitative efficacy and the effect on reperfusion injury of two site-specific PEGylated human serum albumins(PEG-HSAs)modified with linear or branched PEG20kDa,compared with saline,8%HSA and 25%HSA,in a hemorrhagic shock model.Rats were bled to hemorrhagic hypovolemic shockand resuscitated with different resuscitation fluids.The mean arterial pressure(MAP)and blood gas parameters were measured.Hemorheology analysis was performed to evaluate the influence of resuscitationon red blood cells and blood viscosity.The microvascular state was indirectly characterized in terms of monocyte chemotactic protein-1(MCP-1)and endothelial nitric oxide synthase(eNOS)that related to shear stress and vasodilationrespectively.Combined with haematoxylin and eosin(HE)staining,the levels of inflammation-related factors and apoptosis-related proteins were used to evaluate the reperfusion injury in lungs.The results showed that the mean arterial pressures of HSA modified with linear and branched 20 kDa PEG(L-PEG-HSA and Y-PEG-HSA)were(91.00±2.10)mmHg and(91.8311.17)mmHg respectively,significantly higher than 8%HSA(HSA8).group((73.17±5.95)mmHg),25%HSA(HSA25)group((83.00±2.53)mmHg)and the normal saline group((67.29±4.46)mmHg).The blood gas parameters of PaO2,pH,Hb and BE in PEG-HSAs groups were also significantly improved compared withother groups at the end of resuscitation.The result suggests that PEG-HSAs can play a more effective role in expanding blood volume in a relatively longer time than HSA,and can improve the mean arterial pressure and blood gas parameters of rats more effectively.In addition,PEG-HSAs could promote the expression of MCP-1 and eNOS in rats,and significantly improve low-shear whole blood viscosity and plasma viscosity.The results of Western blot and HE staining showed that HSA25,L-PEG-HSA and Y-PEG-HSA could significantly inhibit the inflammation related factors(TNF-?,NF-?B)and the expression of apoptosis-related protein(bax,bcl-2)in the lung tissue.In this paper,no therapeutic advantageswere foundfor branchedPEG-modified HSA.The K value of erythrocyte sedimentation rate of PEG-HSAsgroups(L-PEG-HSA,4.38±1.01;Y-PEG-HSA,5.06±0.36)increased,compared with saline group(3.51 ±0.55),HSA8(3.24±0.46)and HSA25(3.20±0.81),especially in the Y-PEG-HSA group.And compared with saline group(0.92±0.08),HSA(HSA8,1.04±0.08;HSA25,1.11 ±0.28)could increase the red blood cell deformability index,but PEG modification would weaken this function of HSA,especially Y-PEGmodification(L-PEG-HSA,1.05 ± 0.04;Y-PEG-HSA,0.95 ± 0.06).2.Study on the structure of site-specific PEGylated HSA in solution and the heme binding affinity of native HSA and PEGylated HSAs.To obtain detailed structural informationof HSAsite specifically modified by single polyethylene glycol(PEG)chains of differentrelative molecular mass(Mr)or configuration at Cys34,a smallangle neutron scattering(SANS)study was carried out.In addition,SPR analysis was applied to investigate the influence of PEG chains with different Mror configuration on the binding affinity of heme pocket,which is adjacent to Cys34.The results showed that the radii of gyration(Rg)of HSA,PEG5kD-HSA,PEG10kD-HSA,PEG20kD-HSA,Y-PEG20kD-HSA,PEG40kD-HSA and Y-PEG40kD-HSA were2.27±0.01,2.42±0.04,2.44±0.01,2.52±0.01,2.61±0.02,2.50±0.02 and2.60±0.04 nm repectively,and their maximum dimension(Dmax)were repectively 6.22±0.04,6.79 ± 0.1,6.97 ± 0.07,7.07 ± 0.04,7.36 ± 0.11,7.00 ± 0·00 and7.49 ± 0.23 nmrepectively.This indicates that after PEG modification,the diameterof HSA insolutionincreases.The Rg and Dmax of branched PEGmodified HSA is relatively greater than those oflinear PEG-modified HSA.The interference curve of Y-PEG40kD-HSA is the most obvious at the small q range.And the apparent Mrof Y-PEG40kD-HSA was significantly larger than that of PEG40kD-HSA,indicating that the spatial structure of the branched PEG had a greater steric hindrance and could endue protein with stronger intermolecular repulsion.The rvalues of the maximum peak M of the P(R)function of HSA and PEG-HSAs areall about 3 nm,indicating that PEG modification has little effect on the structure of HSA itself.The three-dimensional conformations ofHSA and PEG-HSAs in solution construted by DAMMIN with the data of the small angle scattering showed that the PEG chain conjugated to HSA formed a random coil covering the protein surface,and more likely to form a sphericallycomplex with HSA.Compared with linear PEG of the same Mr,the two strands ofbranched PEGconnected to the same terminal could form two independent mutually exclusive random clusters on the HSA surface.The equilibrium dissociation constant(KD)of HSA,PEG5kD-HSA,PEG10kD-HSA,PEG20kD-HSA,Y-PEG20kD-HSA,PEG40kD-HSA,Y-PEG40kD-HSA and heme were(5.10±0.70)×10-9,(46.83 ±3.70)×10?9,(7.75 ±2.03)×10-9,(9.04±0.42)×10-9,(7.18±1.88)×10-9,(84.43±0.74)x10-9,(26.53±2.87)×10-9mol/L respectively.The results demonstrated that linear PEG10kD,linear PEG4OkD and branched PEG4OkD significantly reduced the binding affinity of HSA and heme,while linear PEG10kD,linear PEG20kD and branched PEG20kD had no effect on HSA and heme binding.3.Establishment ofquantitation of PEGylated albuminin in rat plasma based onNanoLC-MS/MS.A selective,sensitive and accurate liquid chromatography-tandem mass spectrometric(NanoLC-MS/MS)method was developed and validated for the determination of PEG-HSA in rat plasma.The method was mainly based on the simple extraction of PEG-HSA from plasma via the precipitation of endogenous protein using acidic ethanol and the NanoLC-MS/MS detection of the surrogate peptide generated from trypin digestion.The PEGylated bovine serum albumin(PEG-BSA)was added before the sample extraction and used as the internal standard.The HPYFYAPELLFFAK of PEG-HSA and HPYFYAPELLYYANK of PEG-BSA were chosen as the quantitative peptide.The MS/MS transitions of these two peptides were m/z 581.6 ? 779.4 and m/z 936.0 ? 1491.9.The tryptic peptides of plasma sample were separated on a C18 column(long,150mm;diameter 75?m).The formic acid(.FA)and acetonitrile(ACN)were used as the mobile phase reagent,and 0.1%FA/H2O was used as mobile phase A and 0.1%FA/ACN was used as mobile phase B,and the flow rate was 300 nl/min.The separation gradients were from5 min 8%B,45 min 35%B.50 min 90%B to 60 min 90%B.The method was linear over the concentration range of 50?3000 ng/mLfor analyte.The lower limit of quantification(LLOQ)was 50 ng/mL in rat plasma with intra-and inter-day precision as relative standard deviation(RSD)<15%and accuracy as relative error(RE)within 15%.Novelties and Conclusions:1.The efficacy of 20 kDa linear and branched PEG-modified HSA as blood volume expanders in the rat hemorrhagic shock model were investigatedfor the first time.The advantages of high Mr PEG modified HSA as ablood volume expander were confirmed.The relationship of high viscosity,high high osmotic pressureof PEG to the increase of the perfusion of microvessels and the reduction of the ischemia-reperfusion injury was elucidated.The effects of branched PEG modification on the aggregation and deformability of erythrocytes were found,providing an important reference for PEG-HSA clinical application.2.Asmallangle neutron scattering(SANS)study was carried out for the first time to obtain detailed structural informationof HSA site specifically modified by single polyethylene glycol(PEG)chains of different Mr or configuration at Cys34.In addition,SPR analysis was applied for the first time to investigate the influence of different PEG chains on the binding affinity of heme pocket,which is adjacent to Cys34.The study could theoretically explain the advantages of PEG modification on protein drugs in vivo,such as increased molecular radius,prolonged half-life,reduced immunogenicity,etc.And this study provided a new way for the optimization of Mr,structure,modifcation sites of PEGylation.3.A NanoLC-MS/MS method using plasma protein precipitation with water-miscible organic solvents and subsequent trypsin digestion to generate surrogate peptides for detection was developed and validated for the determination of PEG-HSA in rat plasma for the first time.The method has good specificity,accuracy,precision,extraction recovery and stability,which is suitable for the detection of pharmacokinetic samples.