## 2 Materials and Methods  
### Molecular Docking Simulation  
Molecular docking is a powerful method for predicting protein-ligand interactions between a bimolecular receptor (human serum proteins) and ligand (PES and ZW-PES). Herein, we study the effect of dipole orientation of 2-methacryloyloxyethyl phosphorylcholine (MPC), 2-((2-(methacryloyloxy)ethyl)dimethylammonio)ethyl methyl phosphate (MMP), and butyl (2-((2-(methacryloyloxy)ethyl)dimethylammonio)ethyl) phosphate (MBP) zwitterionic structures, having opposite dipole orientation, on protein-ligand interactions and PES hemocompatibility. The 3D X-ray structures of HSA (PDB code: 1 AO6), FB (PDB code: 3 GHG), and TRF (PDB code: 1D3K) were downloaded from the RCSB protein data bank (PDB) and used in the docking study. The crystallography ligand, as well as the water molecules and other ions, were removed from the protein chains. The ligand structures were created using ChemDraw software (Version 8.0), and Chem3D Ultra was used for their energy minimization. For energy minimization, the initial ligand geometries were created based on ChemDraw drawings. The ligand structures were then assigned MM2 force field parameters, and the energy was minimized using the optimization algorithm in Chem3D until a convergence criterion was met. The optimized molecular structures (mol format) were converted to PDBQT formats using the AutoDock tools 4.2.6. Molecular docking between human serum proteins and the ligands was performed using AutoDockVina software version 4.0 (Developed by the Scripps Research Institute, La Jolla, CA, USA) to evaluate the optimal structural properties of inherent protein-ligand interactions through molecular prediction [38]. Figure 1 represents the dipole array along the zwitterion structures, which can either be directed toward the backbone of the ZW chain (as in MMP and MBP) or away from the backbone (as in MPC). In addition, the optimized and energy-minimized structures of these ligands and PES are presented in Figure 1. According to the recommended procedure for molecular docking [39], the steps listed below were performed [40]:  
1. Removing water molecules, ligands, ions, and metal atoms from human serum proteins, downloaded from PDB;
2. Adding Kollman charges;
3. Merging nonpolar hydrogens;
4. Using AutoDock 4 to convert to PDBQT format.  
In our molecular docking simulations, we made specific assumptions to ensure the accuracy of our findings. A docking box of size (40 \(\times\) 40 \(\times\) 40) A (illustrating active sites) [35; 41; 42] was placed around the crystallographic ligand. In order to optimize efficiency, protein residues with atom sizes larger than 7.0 A were eliminated from the docking box. Utilizing the Lamarckan Genetic Search Algorithm (LGA) [43], 15 computational runs were conducted to ensure the accuracy of the data. The forms of interactions between docked proteins and zwitterionic ligands were determined by measuring the intermolecular energy. To convert mol to PDB format, the PyMOL 2.5.5 (Designed by Schrodinger, LLC, New York, NY, USA) molecular graphics system was used as a potent molecular viewer with remarkable 3D capabilities. Additionally, all the minimized energy conformers were acquired from ChemDraw and presented with ChemDraw, PyMOL, and Discovery Studio Visualizer 21.1.0.20298. By measuring the intermolecular energy of the ligand-protein complex, types of interactions between the docked protein and the ligand were studied through 2D interaction diagrams and ball-stick figure interactions drawn using PyMOL.