Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications.
A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 degrees C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein-co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85+/-3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.
Salmaso
Stefano
S
Pharmaceutical Sciences Department, University of Padua, Via F. Marzolo 5, 35131 Padua, Italy.
Bersani
Sara
S
Pennadam
Sivanand S
SS
Alexander
Cameron
C
Caliceti
Paolo
P
D000180:Acrylic Resins / Q000737:chemistry* / Q000493:pharmacokinetics; D000818:Animals; D000911:Antibodies, Monoclonal; D001360:Avidin / Q000097:blood / Q000737:chemistry* / Q000493:pharmacokinetics; D001710:Biotin / Q000276:immunology / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004337:Drug Carriers; D008099:Liver / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008956:Models, Chemical; D015394:Molecular Structure; D008970:Molecular Weight; D009391:Nephelometry and Turbidimetry; D044367:Phase Transition; D017785:Photons; D017434:Protein Structure, Tertiary; D012995:Solubility; D013057:Spectrum Analysis / Q000379:methods; D013696:Temperature; D014018:Tissue Distribution
D000180:Acrylic Resins; D000911:Antibodies, Monoclonal; D004337:Drug Carriers; C469412:poly(N-isopropylacrylamide-co-acrylamide); D001360:Avidin; D001710:Biotin