The Adhesion Ability Assess On Liver Cancer Cell After Treated With Pegylated Anti-adhesion Peptide Yigsr

Purpose:Polypeptide YIGSR's ability to block the HCC cell adhere to extracellular matrix ex vivo has been proved in our previous experiment, but its low molecular weight (MW) and short half life time (T1/2) made it hard to be used in clinical. We try to modify the YIGSR using PEG and make sure the pegylated YIGSR keeps the anti-adhesion function and gets rid of the disadvantages in MW and T1/2 through the tests in stability, degrade speed in rat serum and anti-adhesion ability. We also hope these experimental data can contribute to PEG-YIGSR's further clinical use.Methods:Liquid chromatography-mass spectrometry (LC-MS) has been used to measure the pegylation rate and PEG-YIGSR's stability in different condition including pH, temperature and PEG quantity. SD rat serum proteolysis has been used to measure the degrade speed of PEG-YIGSR ex vivo and the area under curve (AUC) of remaining free YIGSR. Ex vivo cell matrix adhesion has been used to detect the anti-adhesion rate between liver cancer cell MHCC97H and laminin when PEG-YIGSR is presented.Results:1. Modification rate is more than 99% when pH shifts from 4.0 to 6.0. Among these the maximum modification rate is 99.99% when pH is 5.0 and the minimum modification rate is 99.5% when pH is 4.0.2. Modification rate increases with mole ratio increases when the pH of pegylation keeps at 5.0. Modification rate is 62.5% when mole ratio is 1:1 and modification rate is 99.999% when mole ratio is 10:1.3. PEG-YIGSR's ex vivo degrade speed are far slower than that of YIGSR.(1) Add YIGSR and PEG-YIGSR to rat serum respectively, stop the proteolysis and measure the free YIGSR concentration at a serial of time point. The concentration of YIGSR in YIGSR group is less than 0.44mg/L at 15 min time point and negative after 20 min time point. The concentration of YIGSR in PEG-YIGSR group is 1.77mg/L at 15 min time point and 0.11 mg/L at 60 min time point. After that the samples of PEG- YIGSR group undergo depegylation which means up-regulating pH to 9 and incubating in 37℃for another 24h. After depegylation the concentration of YIGSR increased 1.04mg/L (1.75μmol/L) at 60 min time point. According to the previous depegylation experiment, we can roughly estimate the remaining PEG-YIGSR in serum may be more than 200μmol/L, which means pegylation prolong the serum remaining time of YIGSR.(2) The AUCs of YIGSR concentration in YIGSR group and PEG-YIGSR group are significantly different (1146±47mg-min/L,1249±68mg-min/L, P=0.035).4. Pegylated YIGSR maintains the cell-matrix anti-adhesion ability.(1) The MHCC97H cell-matrix anti-adhesion rates of PEG-YIGSR (336μmol/L) and YIGSR (336μ.mol/L) are 19.5±4.5%and 20.1±4.3%respectively (P>0.05). Pegylated YIGSR maintains the cell-matrix anti-adhesion ability.(2) Different concentration PEG-YIGSR (336umol/L,168umol/L,84umol/L,42umol/L) show different anti-adhesion rate (19.5%±4.5%,11.4%±4.1%,9.8%±2.7%, 8.4%±3.6%, p＜0.05). Different incubate time (1h,2h,3h) also leads to different anti-adhesion rate (5.2%±2.5%,10.5%±3.3%,19.5%±4.5%, P＜0.05). Anti-adhesion rate increases following the concentration and incubation time of PEG-YIGSR increasing.Conclusion:1. The procedure of pegylating YIGSR has the characters of easy doing, high modification rate, and high stability.2. Pegylated YIGSR maintains the tumor cell-matrix anti-adhesion ability.3. Pegylated YIGSR shows slower ex vivo serum degrade speed, and may prolong the function time of anti-adhesion in vivo.