분자모사를 통한 혈액투석막 형성에 관한 연구(Ⅱ)

김용완, 김재진, 김영하

한국과학기술연구원, 생체재료연구센터

A Molecular Simulation Approach of Hemodialysis Membrane Formation(Ⅱ)

Yong-Wan Kim, Jae-Jin Kim, Young Ha Kim

Biomaterials Research Center, KIST

Introduction

Blood-compatible membranes[1] are a critical functional element in a variety of bioartificial organs. A great deal of effort has been focused on the interaction of blood and artificial surfaces as applying this delicate control to biomaterials would lead to superior enhancement of the property of medical devices. Current understanding suggests that hydrophilic surfaces form a tolerable interface with blood by minimizing protein adsorption and the interaction of cellular elements with the surface. It has generally been accepted that biomaterials adsorbing the least amount of the plasma protein fibrinogen following exposure to blood will support less platelet adhesion and therefore exhibit less thrombogenicity. Several studies suggest, however, that the conformation or orientation of immobilized fibrinogen rather than the total amount of adsorbed plays an important role in determining the blood compatibility of biomaterials. Recently, it was noted that fewer proteins are adsorbed on blood-compatible polymer surfaces and their original conformation is not changed.

Blends include simple mixtures such as membranes prepared from mixture of PVP(polyvinylpyrrolidone), sulfonated-PEG1k/styrene copolymer and polysulfone. The copolymer having the hydrophobic styrene block is anchored to the polysulfone membrane by hydrophobic force and the sulfonated hydrophilic block is towards blood, and then modifying the membrane surface into having hydrophilicity. This concept is developed for surface modification of polysulfone hemodialysis semi- permeable membrane by phase inversion process, which is difficult for surface modification of the membrane by chemical or radiation-induced grafting. In the phase inversion process, the polymer solution immersed in a non-solvent goes through controlled precipitation into a semipermeable network. Then, the hydrophilic PEG blocks are driven to the membrane surface in the blood environment.

Despite of enormous interest that the mechanisms of blood-compatibility on the surface of biomaterials thus have fundamental importance and their macroscopic properties have been studied seeking for practical applications, yet have not been fully understood due to much more complicated biomedical interface phenomena. Having practical limitations of computer modeling methods, MD simulations are an important complement to get a valuable insight into surface chemistry in macroscopic experimental investigation. A few efforts of optimized computational model have been developed for MD simulation of the blood-compatible biomaterials. There, however, are few publications concerned with the structural and dynamical properties such as mean-square displacement, self-diffusion coefficient and radial distribution functions. Also, most of calculations concerned small molecules and it is difficult to generalize the models into real field. This study involves the complete magnitudes and descriptions of the mechanism of interaction of blood/biomedical interface.

The purpose of this molecular dynamics (MD) simulations study was to get an insight into the adhesion of fibrinogen and the formation of blood-compatible hemodialysis membrane by blending of sulfonated PEG diblock copolymer (Styrene- PEG1k-SO3) with polysulfone. In blending the mixture, the phase separation by phase inversion and the conformation of diblock copolymer on a hemodialysis membrane have also been studied. Also, the time-dependent conformational changes in fibrinogen adsorbed to the surface- modified polysulfone membranes are focused to investigate the effect of primary chemical processes leading to the adhesion on the conformational changes. The results provide the advantage of using diblock copolymer for the surface modification of hemodialysis polysulfone membrane and the new synthetic strategies of the blood-compatible biomedical devices.

Experimental

-membrane fabrication The underlying synthetic method for the preparation of styrene/PEG1K-SO3 copolymer has been described previously[2]. Concentrated polymer solutions for membrane fabrication were prepared by weighing the appropriate constituents in a glass container. The solutions were mixed until a homogeneous solution was obtained as judged by optical clarity. Flat sheet membranes were fabricated using mixtures of styrene/PEG1K-SO3, PVP, DMF and polysulfone. The mixtures was poured onto a glass plate and then evenly coating the plate by drawing a retractable doctor blade across it. The plate was then immersed quickly and smoothly into the coagulation bath. After 5min in the coagulation bath, the membrane was removed and placed in a water bath for 2h, then extracted for 16h in a  80℃ water bath. The membranes were subsequently dried in a convection oven at 65℃ for at least 12h. XPS analyses were performed on microporous flat sheet membranes.

-blending simulation study The computational results were obtained using software programs from Molecular Simulations, Inc. The program combines modified Flory-Huggins model and molecular simulation techniques. MD calculations were carried out with the Cerius2 program. In the first stage, the geometry of PSF, DMF and  diblock copolymer was minimized from 300K to 1000K and then 50K to 300K with 50K increment. In the second stage of blending, the two polymer were simulated to get energy distribution(E11, E12, E21, E22) and coordination number(Z11, Z12, Z21, Z22). This work focuses on the miscibility and phase separation of each polymer. The information obtained includes phase diagrams (binodal and spinodal curves), thermodynamic mixing variables, the temperature dependent interaction parameter □, binding energy component analysis, and the identification of favorable binding configurations between molecular pairs.

-conformation of diblock  copolymer The Cerius2 package was used for all the MD simulations in the preparation and data collection. All calculations were performed using O2 Workstation-R5000. The polysulfone surface model is created from Surface builder, containing 16 independent polysulfone monomer. A diblock copolymer comprising the PEG1K, (CH2-CH2-O)22 unit was from polymer builder. One side of copolymer is sulfonated and the other side is bonded with styrene. Hydrogen was added to terminate the copolymer chains. The unit cell for MD simulation is cubic lattice(i.e. a cubic box with periodic boundary conditions) containing psf surface, water, and diblock copolymer according to its surface density. The cell parameters are a,b=80Å, c=120Å and □, □, □=90o. Parameters for potentials involving biopolymer and copolymer were calculated using PCFF. The initial atomic coordinates for polymer were taken from the X-ray structural determination of PEG and polysulfone. A time step 1 of fs was employed for the simulation of the equation of motion. The system was allowed to relax during 200ps using NVT ensemble simulation, in which the temperature was kept constant as 310K using a Nose-Hoover thermostat with a relaxation time of 0.1ps. It was found to have relaxed sufficiently after 100ps. Numbers of atoms, volume and temperature were monitored throughout the simulation as a means of assessing the validity of the model used.

Results and Discussion

In the case of sulfonated PEG grafted polymer surfaces, the biocompatibility has been increased so that it could be applied many biomedical fields[3]. To extend this knowledge of these phenomena, especially, MD simulation study was undertaken to model the character of blood-compatibility for fabrication of hemodialysis membrane(Fig 1). It is readily admitted that the geometrical model used is somewhat artificial. However, it does include some intuitively essential features that the surface layer is able to assume a higher degree of conformational freedom than the bulk. The results provides an insight of the essential conformational- dependent structure/dynamical features of a blood compatible surface, and the procedures used can be applied to compute any molecular dimensions with reasonable agreement between theoretical and experimental output.

Molecular dynamics (MD) simulations were undertaken to gain an understanding of the interfacial interactions between styrene/PEG1K-SO3 diblock copolymer and polysulfone surfaces in a blood-compatible membranes. The structural and dynamical properties of the model were analyzed to probe interfacial properties at each copolymer density. Three different molecular systems were used. In the first one, blends properties were simulated in order to inspect the miscibility of different polymers, while in the second one, conformational characteristics of diblock copolymer and anchoring states were simulated, showing different coordination modes of water on the copolymer and energetically most favorable surface structure. And in the third one, fibrinogen adsorption on the surface was simulated to inspect if the surface-modified hemodialysis membranes are blood-compatible, suggesting the probable mechanism that no conformational changes are essential to enhance the blood-compatibilty. The computational results provide more sophisticated insight into the fabrication of blood-compatible biomedical devices.

Fibrinogen-fibrinogen and fibrinogen-polymer surface adhesion is a result of receptor-ligand interactions and depends on the physicochemical properties of the interacting surface molecules, such as charge and hydrophilicity. The current conformation character of both fibrinogen and surface-modified polymer are necessary for an understanding the nature of blood-compatibility as the chemical composition and the surface structure of the surface affect the interfacial interactions, possibly through fibrinogen receptor conformation. It is supposed that fibrinogen undergoes conformational changes upon adsorption to the surface by changing the molecule from a compact form to an extended form.

Physical and chemical surface properties significantly influence the behavior of cells interacting with the surface, that is, the dynamics of the fibrinogen adhesion process, shape and rate of spreading. After initial ligand binding, fibrinogen form changes to the fibrin and regulates signal transmission. Presumably, in adhesion, the most critical events take place within a few seconds of the interaction. It is supposed that fibrinogen is able to adhere to foreign surfaces within a fraction of a second and that a time of 0.01-0.2 sec may be long enough for the interaction of colliding fibrinogens in suspension.

It has been studied the interaction between fibrinogen and copolymer surfaces of modified hydrophilicity. The sulfonated surfaces are not adhesive for fibrinogen and other proteins whereas the nonsulfonated surfaces are adhesive for these proteins, that is, the number of proteins adhered to nonsulfonated surfaces is high and is not influenced by the surface character. Also, it is noticed that unmodified hydrophobic groups stimulate the adhesion of the proteins to surfaces. The rate and strength of the fibrinogen-surface interaction depend on the density of sulfonic groups present at the surface. It is possible that the sulfonic groups present at the surfaces change the conformation of the fibrinogen adsorbed to the surfaces.

REFERENCES

[1] J. D. Bronzino, The biomedical engineering handbook. CRC Press, 1995

[2] H. J. Chun, J. J. Kim, et al., Polym. J., 22, (1990) 347

[3] D. K. Han, K. D. Park, et al., J. Biomed. Mater. Res., 27, (1993) 1063

Figure 1