Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood

Abstract

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only beta-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production, biocompatibility, biodegradation, physical properties and applications

“…In contrast to mcl-PHAs, scl-PHAs are not soluble in acetone, thus, enabling a separation of both PHA classes (159). Recovery of PHA from bacterial cells using organic solvents is often applied in industrial processes and in laboratory scale PHA extractions due to the recovery efficiency of the process, simplicity, rapidity, polymer purity obtained and the possible removal of endotoxins from the recovered polymer, which is important for medical applications (160)(161)(162). Solvents extract the polymer without degrading it by improving the cellular membrane permeability and subsequent solubilization of the PHA (162).…”

“…Biocompatibility of PHB has been demonstrated in vivo under subcutaneous implantation of PHB films (161). Tissue reaction to films from PHB of different Mw (300, 450, 1000 and 1500 kDa) implanted subcutaneously was relatively low and did not change from tissue reaction to control glass plate.…”

“…It was shown that PHB or P (HB-co-HV), when in contact with blood, did not affect platelet responses, nor did the polymer activate complement system (214). However, more involved polymer purification procedures had to be followed to significantly reduce the amount of bacterial cell wall material associated with the purified PHA (161).…”

“…Tissue reaction to films from PHB of different Mw (300, 450, 1000 and 1500 kDa) implanted subcutaneously was relatively low and did not change from tissue reaction to control glass plate. The low tissue reaction to implanted PHB films indicates the high biocompatibility of PHB in vivo (161,214). This high biocompatibility of PHB is due to the presence of natural PHB oligomers and HB, the intermediate product of PHB degradation, in animal tissues at normal conditions in comparison with chemically synthesized biodegradable polymers, for example, polylactides and polyglycolides.…”

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

“…However, microspheres, though containing a small amount of biomaterial, have a large surface area and, being injected subcutaneously or intramuscularly, come in contact with extensive area inside the host organism, which can cause stronger tissue reaction (Anderson and Shive, 1997;Vospaleniye, 1995). (Sevastianov et al, 2003). The trademark of the material is Bioplastotan (Trademark "BIOPLASTOTAN".…”