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Development of “Inside‐Out” PEGylated Crosslinked Hemoglobin Polymers: A Novel Hemoglobin‐Based Oxygen Carrier (HBOC)
Author(s) -
Webster Kyle D,
Dahhan Dana,
Frosti Cheyanne,
Dean William,
Chaires Jonathan B,
Olsen Kenneth W
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.825.3
Subject(s) - pegylation , peg ratio , chemistry , hemoglobin , blood substitute , tetramer , polyethylene glycol , azide , polymer chemistry , combinatorial chemistry , biophysics , biochemistry , organic chemistry , enzyme , finance , economics , biology
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. αXL‐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 αXL‐Hb was then reacted with an 8‐arm Azide‐PEG (40 kDa). Analytical Ultracentrifugation (AUC) suggests the major PEGylated species has 3–4 αXL‐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 αXLHb p50 values following PEGylation. Large multi αXL‐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. Support or Funding Information Loyola University Chicago, and the Arthur J. Schmitt Fellowship 1Proposed schematic of Inside‐out PEGylated cross linked hemoglobin. It is more probable that two arms will bind to a single tetramer if Hb is activated at 2 :1 DIBO to Hb than one arm binding to an individual tetramer. The major inside‐out PEGylated hemoglobin polymer species produced is depicted (4 Hb : 1 PEG)