Clement H. Bamford

Interaction of a polymeric biguanide biocide with phospholipid membranes

Differential scanning calorimetry (DSC) and fluorescence polarization methods have been used to study the interactions between phospholipid membranes and a polymeric biocide, poly(hexamethylene biguanide hydrochloride) (PHMB). It was found that PHMB had very little effect on neutral lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), whereas it greatly reduced the phase transition temperature of phosphatidylglycerol (PG), an acidic lipid found in bacteria. Although the corresponding monomeric biocide had a similar effect on the PG bilayer, the behaviour towards mixed lipid bilayers of PC and PG has been shown to be completely different for the polymeric and monomeric biocides: viz. the former can induce isothermal phase separation into a PHMB-PG complex domain and a PC-enriched domain, whilst the latter cannot. This may account for the great difference in bactericidal activity between them. It is suggested that PHMB interacts primarily with negatively charged species in the membranes, inducing aggregation of acidic lipids in the vicinity of the adsorption site, where higher fluidity and higher permeability are expected. The results have shown that two factors might be crucial in the cidal activity of such types of cationic disinfectants as biguanides: phase separation and interaction with the hydrocarbon interior of the membranes. Polymeric biocides could be particularly effective by virtue of their ability to combine hydrophobic character and multiple charges within a single molecule.

Studies in polymer surface functionalization and grafting for biomedical and other applications

Abstract A simple and inexpensive treatment for surface functionalization and grafting carried out under mild aqueous conditions and suitable for prefabricated medical devices, has been developed. The two stages are performed separately; the first is hydroxylation by a peroxy compound, particularly a peroxydisulfate or peroxymonosulfate, in aqueous solution with strong nitrogen purging; and the second is grafting of a vinyl monomer by the conventional ceric ion technique. Types of polymer studied include polypropylene, polystyrene, polyacrylonitrile, polyurethanes, aliphatic polyesters, nylons, aromatic polyesters and polycarbonates. Surface properties of the substrate polymers may be greatly modified by this treatment. To illustrate possible applications for covalent coupling of bioactive molecules to polymers we have attached the blood-anticoagulants heparin and hirudin to the polyester Ecdel and to polystyrene, respectively. The products were highly bioactive.

Studies of a novel membrane for affinity separations: I. Functionalisation and protein coupling

Abstract A study is presented of the potentialities of an electrostatically-spun poly(ether—urethane—urea) as an affinity separation membrane. The spinning process produces a fibrous network with a high internal surface area. A variety of chemical methods has been used for functionalising and activating the membrane. Assessments of the capacities of the activated membranes for covalent coupling of protein A and human immunoglobulin G have given very encouraging data. Non-specific adsorption of both proteins by the inactivated polymer was negligible. Geometric considerations suggest that the limiting factor determining protein coupling is the accessible surface area rather than the number of coupling sites.

Non-classical free-radical polymerization: Degradative addition to monomer in the polymerization of 1-vinylimidazole

Abstract A study of the free-radical polymerization of 1-vinylimidazole at 70°C is described; most attention has been devoted to the (homogeneous) reaction in ethanol, but polymerizations in N , N -dimethylformamide (DMF), water and bulk monomer are also considered. The polymerization in ethanol is unusual in that the rate becomes effectively zero-order in monomer M at moderately high [M]. Kinetic results indicate the occurrence of a degradative reaction between propagating radicals and monomer (see below); the available molecular weight data suggest that bimolecular termination takes place by radical combination. Polymerization in DMF is generally similar although the kinetic treatment is rather less satisfactory. At high [M] there are indications of occlusion phenomena in DMF and these latter are marked in the (heterogeneous) bulk polymerization. In water, significant interactions between solvent and monomer, evidenced by viscosity-composition behaviour, affect the kinetics of polymerization, which, however, resemble those with the other solvents. Of considerable interest is the influence of pH on rates and degrees of polymerization, which change in the same sense and to nearly the same extent. We believe these findings imply suppression of the degradative reaction by protonation of the monomer. The degradative reaction between propagating chains and monomer is thought to be formation of a relatively unreactive radical by addition to position 2 of the monomer, rather than by chain transfer. Evidence for this is adduced from molecular weight data, which are not consistent with transfer, the existence of occlusion phenomena and the observations on the pH dependence in aqueous solution. Molecular weight distributions expected in polymerizations of this type are calculated in the Appendix. Reinitiation by the adduct radical, which probably occurs only to a limited extent under non-occlusion conditions, becomes important when occlusion is significant. A kinetic treatment of these phenomena is presented.

Studies in polymer surface modification and grafting for biomedical uses: 2. Application to arterial blood filters and oxygenators

The technique for polymer surface modification described in earlier papers has been applied to two components of an extracorporeal blood circulatory system used in open-heart surgery, with the object of improving their haemocompatibility. The devices were the arterial blood filter composed of nylon membranes and the blood oxygenator consisting of polypropylene microporous hollow fibres. Polyacrylamide and, in some cases, a mixture of polyacrylamide and poly(3-aminopropyl methacrylamide) have been covalently attached to the blood-contacting surfaces. The basic groups of the latter polymers provided a means of coupling heparin to the surface. The extent of heparin coupling was determined by radioactivity measurements with the aid of 35S-labelled heparin. The following determinations were made to assess haemocompatibilities: blood plasma clotting times (PTT); platelet adhesion to the nylon filters; concentrations of lymphocytes and neutrophils in whole blood after exposure to the surfaces. It is concluded that the overall process, which has the merits of cheapness and simplicity in application, has a beneficial effect on haemocompatibility.

Network formation III—Influence of organometallic initiator on network structure

The nature of the primary radicals arising from the interaction of halides and organometallic derivatives has been investigated with the aid of tracer and gelation techniques. Halides studied include carbon tetrachloride and esters of trichloracetic acid; with Mo(CO)6, Mn2(CO)10 and Pt(PPh3)4 these yield radicals derived from the halide by removal of chlorine atoms to give ‘attached’ primary radicals. The nickel derivatives Ni{P(OPh)3}4, Ni(CO)4, Ni(CO)2(PPh3)2 produce some radicals which do not contain an organic residue and are therefore ‘unattached’; these may be chlorine atoms or an adduct with monomer. Since unattached radicals promote branch formation, the use of the nickel derivatives increases the range of available network structures. Values for the ratio of combination to disproportionation in the termination reaction of methyl methacrylate polymerization are a byproduct of these investigations and are given for three temperatures.

Studies of the esterification of dextran: routes to bioactive polymers and graft copolymers

Abstract Problems associated with the esterification of dextran as a means of coupling bioactive molecules or introduction of functionality suitable for graft polymerization are considered. In particular, the importance of eliminating side-reactions which incorporate into dextran unwanted residues, e.g. groups containing nitrogen, is emphasized and practical techniques for minimizing this are described. We have developed a formamide-based solvent suitable for esterification with the aid of dicyclohexyl carbodiimide (DCC) and carbonyl di-imidazole (CDI) as coupling agents. The preferred catalyst is p -pyrrolidinopyridine. CDI has the advantage of enabling dimethylsulphoxide to be used as solvent for dextran and other hydroxylic polymers without inducing oxidation of hydroxyl groups. This coupling agent is flexible and offers a choice of two routes to esterification, each having its merits. We have optimized conditions for coupling by use of butyric acid as model. Esterification of dextran has been employed in the preparation of soluble bioactive macromolecules by coupling the anti-platelet agent (I) and also in the synthesis of graft copolymers via introduction of 2-bromopropionate groups. Crosslinking of dextran and the polymerization of dipyridamole have been effected by use of CDI.

Network properties: (1) Multiple glass transitions

Abstract Networks composed of polyvinyl trichloracetate ( Pvtca ) cross-linked by polymethyl methacrylate or polystyrene were prepared from mixtures of Pvtca and the appropriate monomer by thermal initiation with molybdenum carbonyl at 80°C or photoinitiation with manganese carbonyl at 25°C. From details of the syntheses the following properties of the networks were calculable: (1) the mean numbers of cross-linked units per weight average Pvtca chain, (2) the mean degree of polymerization of the cross-links and (3) the ratio (number of branches)/(number of cross-links). Dilatometric observations showed that normally the networks have two glass temperatures and are two-phase systems. The slopes of the volume-temperature curves between the transition points were higher than expected, markedly so with copolymers containing methyl methacrylate and to a much smaller extent with those containing styrene. It is concluded that in the former systems the Pvtca -rich phase contains considerable proportions of polymethyl methacrylate while in the latter there is only a small degree of incorporation of styrene into the Pvtca -rich phase. In agreement with these conclusions is the observation that for the polymethyl methacrylate systems the lower glass temperature is appreciably higher than the glass temperature of Pvtca . With the systems containing styrene this difference is not found. The influence of structural characteristics of the networks, particularly the branch/cross-link ratio and the length of the cross-links, on the compositions of the two phases is discussed.

Non-classical free-radical polymerization: 3. Diffusion-control in degradative addition☆

Abstract The different kinetic features associated with polymerizations in which retardation arises through degradative transfer and degradative addition processes are considered. Reasons are given for believing that in reactions retarded by degradative transfer neglect of the interaction between ‘inactive’ radicals is justifiable, while with degradative addition this is not so. A kinetic treatment of the latter is developed in which all three termination reactions between propagating and inactive radicals are assumed to be diffusion-controlled with a single rate coefficient and equations are derived which permit from experimental data on rates of polymerization estimation of the definitive kinetic parameters k p k′ − 1 2 t , k pm k′ − 1 2 t and k fm k′ − 1 2 t (kp, k′t, kpm, kfm are the rate coefficients of propagation, termination, reinitiation and degradative additions, respectively). The kinetic equations are satisfactorily consistent with ew experimental data on the polymerization of 1-vinylimidazole in ethanol solution at 70°C, for which there is strong evidence for the occurrence of degradative addition. A modified procedure for processing data for polymerizations with degradative transfer is put forward which is convenient for estimating the kinetic parameters and reveals in a simple manner the importance of re-initiation. It is suggested that this treatment could be generally useful in the early stages of the study of a retarded polymerization.

Network properties: 2. A broadline n.m.r. study of molecular motions in some multicomponent crosslinked polymers

Abstract A series of multicomponent polymers of known structure and consisting of poly(vinyl trichloroacetate) crosslinked with poly(methyl methacrylate) (PMMA) have been examined by broadline n.m.r. Both enhanced and retarded rotation of the α-methyl groups on the PMMA chains, compared with those in PMMA homopolymer, have been observed. A previous assessment of the morphologies of the same polymers, based on the results of dilatometric studies, has been extended. The modifications to the rotations of the α-methyl groups have been correlated with the morphologies of the multicomponent polymers and it is proposed that both effects arise as a result of changes in the distribution of conformations adopted by the PMMA chains in the multicomponent polymers compared with that in the pure homopolymer.