Imparting mineral affinity to fetuin by bisphosphonate conjugation: a comparison of three bisphosphonate conjugation schemes.
Protein conjugation to bisphosphonic acids (BPs), such as 1-amino-1,1-diphosphonate methane (aminoBP) and 3,5-di(ethylamino-2,2-bisphosphono)benzoic acid (diBP), was proposed as a foundation for bone-specific delivery of protein therapeutics. This study was performed to directly compare the mineral affinity of protein-BP conjugates prepared by three different approaches. Fetuin, serving as a model protein, was derivatized with BPs by the following approaches: (i) by attaching the aminoBPs onto protein lysines using succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC); (ii) by attaching the aminoBPs onto protein carbohydrates using 4-(maleimidomethyl)-cyclohexane-1-carboxyl-hydrazide (MMCCH), and (iii) by conjugating diBP to protein lysines using the carbodiimide chemistry. The results indicated that conjugation of aminoBP and diBP to fetuin by all three means unequivocally enhanced the protein's affinity for hydroxyapatite in vitro. Similarly, conjugation of aminoBP and diBP onto fetuin increased the protein's retention in a mineral-containing matrix (Pro-Osteon) when the proteins were implanted in a rat subcutaneous model. Upon parenteral administration, however, no discernible differences were found between the SMCC- or MMCCH-linked conjugates and unmodified fetuin to target to bony tissues. DiBP-fetuin conjugates, however, led to successful bone targeting after intravenous injection in rats. We conclude that all three conjugation schemes were equally effective in imparting an affinity to the proteins toward mineral-containing matrices. Bone targeting, however, was achieved only with diBP conjugation to fetuin, supportive of the superior ability of this BP with a higher density of bisphosphonic acid groups.
Gittens
Sébastien A
SA
Department of Chemical and Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, AB, Canada.
Bansal
Geeti
G
Kucharski
Cezary
C
Borden
Mark
M
Uludag
Hasan
H
D000818:Animals; D001842:Bone and Bones / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005260:Female; D006882:Hydroxyapatites / Q000737:chemistry / Q000378:metabolism; D020007:Implants, Experimental; D008903:Minerals / Q000737:chemistry*; D015394:Molecular Structure; D063065:Organophosphonates / Q000737:chemistry*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000494:pharmacology
D006882:Hydroxyapatites; D008903:Minerals; D063065:Organophosphonates; D000509:alpha-Fetoproteins