Polyamine graft copolymers

A series of polymine-graft copolymers of styrene [92-95] and hydroxyethyl methacrylate [96-98] were found to form a microdomain structure and exhibit unique biomedical behavior at the interface with living cells, such as blood platelets and lymphocytes. The most intensive studies were made with poly(hydroxyethyl methacryIate)-0ra/t-polyamine copolymers (HA)  

HA copolymers were prepared by radical copolymerization of HEMA with polyamine macromonomer which had been synthesized by a self-polyaddition reaction of N, N -diethyl-AT-(vinylphenethyI)ethyIenediamine (EDAS) in the presence of lithium diisopropylamide [98-100] 

It is known that lympocytes are composed of two major subpopulations T-cell and B-cell, each of which plays an essential role in all of the immunological reactions in living systems. There are strong medical demands for the separation of T- and B-cells, which is essential for the therapy and diagnosis of autoimmune diseases and cancer, as well as for HLA typing in transplantation. 

It had been found previously [106] that cell adhesion on the surface of HA copolymers decreased with increasing ambient pH. With polyHEMA, on the other hand, no pH-dependency was observed. We considered the adhesion to be caused primarily by ionic interaction between the lymphocytes and the HA surfaces. The degree of protonation (a) of amino groups in HA copolymer was estimated by acid-base titration of an HCI solution of polyamine macromonomer with NaOH solution. In physiological conditions (pH 7.2-7.4) about 50% of the amino groups of the macromonomer are protonated. In this pH range, the polyamine macromonomer was found to be insoluble. 

The isoelectric points of B cells and T cells are reported to be 3.8 and 4.6, respectively. This may explain the higher adhesivity of the former to the HA surface. For this reason, adhesivity of T cells are assumed to be more susceptible than B cells to the decrease in ionic character of the HA surface. 

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A series of polyamine-graft copolymers (See Sect. 4.3) were found to form microdomain structure and to exhibit unique biomedical behavior at the interface with living cells, e.g. blood platelets or lymphocytes. Although a number of postulates were proposed to explain the unique behavior of microdomain-structured surface, mechanisms for the mode of interaction of living cells with any of the domain-structured materials have not been adequately explained. In Sect. 4, the author will review results on the biomedical behavior of SPUs, HEMA-STY, and polyamine-graft copolymers, and discuss their interfacial properties in terms of the random network concept of water molecules on the material s surface. 

As stated in Chapter 1, microdomain-structured surfaces are believed to play an important role in their interactions with cells, proteins, and other biological elements. In this Chapter, the author will discuss biomedical behavior of three types of microdomain-structured materials segmented polyurethanes, A-B-A block copolymers, and polyamine-graft copolymers. 

This paper reviews the present status of affinity separation of cells based on the biospecific interaction of cellular receptors with proteinaceous ligands immobilized on a solid-phase matrix. Special emphasis was placed on the development of new matrix materials for immuno-affinity chromatography of lymphocyte subpopulations. Our newly developed matrix of poly(2-hydroxyethyl methacrylate)/polyamine graft copolymer offered novel advantages in (1) elimination of non-specific adsorption of lymphocytes and (2) simple immobilization procedure of ligand protein through non-covalent adsorption. This matrix allowed a rapid separation of preparative quantities of pure and vital lymphocyte subpopulations (IgG-positive and -negative cells) in excellent yield. 

Summarizing the above description, the surface of polyamine graft copolymers with a definite amount of polyamine branches showed an extremely small quantity of non-specifically adsorbed lymphocytes. This advantageous characteristic of polyamine graft copolymers led us to utilize them as solid-phase matrices for cell affinity chromatography. 

As the separation of lymphocyte subpopulations based on the expression of immunoglobulin (Ig) molecules on their plasma membrane surfaces is of considerable practical interest at the present time (13), we have applied polyamine graft copolymers as solid-phase matrices for the separation of IgG-positive (IgG" ") and -negative (IgG ) lymphocytes. The solid-phase matrix was prepared by coating graft copolymers on glass beads of 48 - 60 mesh by solvent evaporation techniques. 

Figure 1 (left). Structural formula of polystyrene/polyamine graft copolymer (SA copolymer). x represents wt% of polyamine portion in the copolymer. 

We found also that the amino-content of the polyamine-styrene graft copolymer, SAX (x is the polyamine content in wt.%) correlates closely with the adsorption behavior of FN or VN as it also does with the adhesivity of bovine aortic endo-thelial cells to the SA copolymer surface, as will be discussed in Sect. 4.4. 

In the course of our study with HA copolymers, we frequently observed that some of HA copolymer surfaces which have a small number of polyamine graft chains became sufficiently inert against blood platelets - as well as against lymphocytes - to reject their attachment [97, 108-113]. 

The p-DVB-piperazine adducts can also undergo self condensation whereby the macromonomers formed exhibit rather low molecular weights, and are insoluble in the reaction mixture (benzene or THF). They dissolve only upon the addition of an acid or in hot chloroform. Free-radical copolymerization of this macromonomer with styrene was carried out in benzene in the presence of some acetic acid (to obtain a homogeneous reaction mixture) to yields of about 20% M). Here again separation of the unreacted macromonomer is possible, and the polyamine content of the graft copolymer is very close to the amount contained in the reaction mixture. 

These suppressive effects of SA copolymers on cellular adsorption and shape change were observed for rat lymphocytes as well as platelets (58,59). Figure 2 clearly demonstrates the elimination of adsorption of lymphocytes on albuminated surfaces of SA copolymers containing 9 wt% polyamine branches (SA9). A change in the back-bone structure of the graft copolymer from polystyrene to the more hydrophilic poly(2-hydroxyethyl methacrylate) (PHEMA)... 

Graft copolymers of natural rubber and poly(methyl methacrylate) in latex form have been produced on a small scale. The rubber latex is treated with methyl methacrylate in the presence of an initiator (e.g. hydroperoxide activated by a polyamine). Generally, equal weights of rubber (dry weight) and monomer are used and the product consists of about 80% graft copolymer, 10% polyisoprene and 10% poly(methyl methacrylate). The material gives rise to vulcanizates with excellent physical properties at high hardness levels. 

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