Polyethylene glycol (PEG) has been utilized in the development of pharmacological and therapeutic compounds. PEG increases hydrodynamic volume and circulation time of conjugated products and decreases immunogenicity. Previous PEG Hemoglobin-Based Oxygen Carriers (HBOCs) have coated the exterior of the Hb tetramer with multiple PEG chains. “Inside-out” PEGylation employs the reverse methodology, covering a functionalized 8-arm PEG backbone with multiple Hb tetramers. A new PEG-HBOC has been made by this method. The HBOC was made by copper free strain-promoted azide-alkyne cycloaddition click chemistry. aXL-Hb was made by crosslinking bovine Hb under deoxy conditions with dibromosalicyl fumarate (DBSF) followed by the activation of the crosslinked Hb with dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DIBO-NHS) to produce exposed alkynes on Lys residues. Activated aXL-Hb was then reacted with an 8-arm Azide-PEG (40 kDa). Analytical Ultracentrifugation (AUC) suggests the major PEGylated species has 3-4 aXL-Hb : 1 PEG. While the inside-out PEGylation methodology produced heterogeneous samples, it was reproducible in its heterogeneity. CD studies demonstrated that crosslinking and subsequent PEGylation did not alter the secondary structure. In addition, oxygen binding studies showed negligible changes in the aXLHb p50 values following PEGylation. Large multi aXL-Hb PEG complexes were successfully produced whilst maintaining the secondary structure and oxygen binding capabilities. Preliminary studies suggest that “Inside-Out” PEGylated Hb compounds will be efficacious as a HBOC.

Development of “inside-out" PEGylated crosslinked hemoglobin polymers: A novel hemoglobin-based oxygen carrier (HBOC)

251st National Meeting (2016)

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