Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery.
Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.
Yi
Xiang
X
Department of Pharmaceutical Sciences and Center for Drug Delivery and Nanomedicine, College of Pharmacy, University of Nebraska Medical Center, Omaha, Nebraska 68198-5830, USA.
Batrakova
Elena
E
Banks
William A
WA
Vinogradov
Serguei
S
Kabanov
Alexander V
AV
D000818:Animals; D002417:Cattle; D002460:Cell Line; D004285:Dogs; D016503:Drug Delivery Systems; D042783:Endothelial Cells / Q000187:drug effects*; D006735:Horseradish Peroxidase / Q000737:chemistry; D015394:Molecular Structure; D011060:Poloxalene / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D020442:Poloxamer / Q000138:chemical synthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D013997:Time Factors; D014018:Tissue Distribution
C422648:pluronic block copolymer p85; D020442:Poloxamer; C464484:pluronic block copolymer P123; D011060:Poloxalene; D006735:Horseradish Peroxidase