Synthesis of poly(PEGA) Bioconjugates using RAFT Polymerization

Protein-polymer conjugates are widely used in biotechnology and medicine, and non-toxic polymers for bioconjugation would be advantageous for these applications. Herein, we reported the toxicity of polymers synthesized by two commonly employed controlled radical polymerizations: reversible addition-fragmentation chain transfer (RAFT) poly-merization and atom transfer radical polymerization (ATRP). Poly(polyethylene glycolacrylate) (poly(PEGA)) synthesized by RAFT polymerization using a trithiocarbonate chain transfer agent (CTA) was found to provide superior cell viability compared with corresponding polymers prepared by ATRP or RAFT, and therefore was selected as a promising polymer for bioconjugation. Poly (PEGA) was synthesized with different molecular weights and functionalized at the chain end with maleimide, which is known to bea specific reactive group with free thiols on the protein surface. We also attempted to synthesize bis-functionalized maleimide-poly(PEGA) to mimick dimeric structures of proteins frequently found in nature. Polymers were characterized by 1H NMR spectroscopy and size exclusion chromatography (SEC). The kinetics of the RAFT polymerizations were investigated and a linear evolution of molecular weight with monomer conversion was observed. The SEC (IR) analyses showed no detectable side reactions during thepolymerization. However, for the bis-functionalized maleimide-poly (PEGA), a loss of the maleimide end groups was observed during the deprotection procedure. Two modelproteins, V131C T4 lysozyme (T4L) and VEGF165, were expressed, purified, and subsequently conjugated to maleimide-poly (PEGA)s. The resultant conjugates were monitored and purified by SEC (UV), and further analyses were carried out by gel electrophoresis. Lytic activities of T4L conjugates and T4L were monitored by UV-Vis, and bioactivity was not observe

Baseline plasma fatty acids profile and incident cardiovascular events in the SU.FOL.OM3 trial: the evidence revisited.

OBJECTIVE: We aimed to investigate the association between baseline plasma fatty acids profile and the risk of future major cardiovascular events in patients with a history of ischaemic heart disease or ischemic stroke. METHODS: Baseline plasma fatty acids as well as established cardiovascular risk factors were measured in 2,263 patients enrolled in the SUpplementation with FOLate, vitamins B-6 and B-12 and/or OMega-3 fatty acids randomized controlled trial. Incident major cardiovascular, cardiac and cerebrovascular events were ascertained during the 4.7 years of follow up. Hazard ratios were obtained from Cox proportional hazards models after adjustment for cardiovascular risk factors. RESULTS: During the follow-up, 154, 379 and 84 patients had major cardiovascular, cardiac and cerebrovascular events respectively. Upon adjustment for gender, initial event, baseline age and BMI, the risk of developing a major cardiovascular event decreased significantly in successive quartiles of arachidonic acid (P trend<0.002), total omega 3 polyunsaturated fatty acids (P trend<0.03), docosapentaenoic acid (P trend<0.019), docosahexaenoic acid (P trend<0.004), eicosapentaenoic acid + docosahexaenoic acid (P trend<0.03) and eicosapentaenoic acid + docosapentaenoic acid + docosahexaenoic acid (P trend<0.02). This inverse association was borderline significant with increased quartiles of stearidonic acid (P trend<0.06). In the full model, only stearidonic acid remained inversely associated with the risk of developing a major cardiovascular event (P trend<0.035), a cardiac event (P trend<0.016) or a cerebrovascular event (P trend<0.014), while arachidonic acid was inversely associated with the risk a cerebrovascular event (P trend<0.033). CONCLUSION: The inverse association of long chain omega 3 polyunsaturated fatty acids with recurrence of Cardiovascular diseases was mainly driven by well-known cardiovascular risk factors. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN41926726