Hemolytic Activity of pH-Responsive Polymer-Streptavidin Bioconjugates

Abstract

Drug delivery systems that increase the rate and/or quantity of drug release to the cytoplasm are needed to enhance cytosolic delivery and to circumvent nonproductive cell trafficking routes. We have previously demonstrated that poly(2-ethylacrylic acid) (PEAAc) has pH-dependent hemolytic properties, and more recently, we have found that poly(2-propylacrylic acid) (PPAAc) displays even greater pHresponsive hemolytic activity than PEAAc at the acidic pHs of the early endosome. Thus, these polymers could potentially serve as endosomal releasing agents in immunotoxin therapies. In this paper, we have investigated whether the pH-dependent membrane disruptive activity of PPAAc is retained after binding to a protein. We did this by measuring the hemolytic activity of PPAAc-streptavidin model complexes with different protein to polymer stoichiometries. Biotin was conjugated to amine-terminated PPAAc, which was subsequently bound to streptavidin by biotin complexation. The ability of these samples to disrupt red blood cell membranes was investigated for a range of polymer concentrations, a range of pH values, and two polymer-to-streptavidin ratios of 3:1 and 1:1. The results demonstrate that (a) the PPAAc-streptavidin complex retains the ability to lyse the RBC lipid bilayers at low pHs, such as those existing in endosomes, and (b) the hemolytic ability of the PPAAc-streptavidin complex is similar to that of the free PPAAc.

<em>Ex Vivo</em> Red Blood Cell Hemolysis Assay for the Evaluation of pH-responsive Endosomolytic Agents for Cytosolic Delivery of Biomacromolecular Drugs

“…[14][15][16][17] pH-dependent hemolysis has been used to screen the ability of carriers to mediate endosomal release of biomacromolecular therapeutics (e.g. peptides, siRNA, ODNs, proteins), and results of this assay can be predictive of performance as an intracellular drug delivery vehicle.…”

Development of pH–responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery

“…There are many ways of improving nucleic acid release from particles and acid-sensitive trigger remains a way to achieve it. Particularly, synthetic polymers like poly (methyl methacrylate), poly (acrylic acid) [53], poly (N,N-iso-propylacrylamide) have been used to overcome this barrier. In the present work, the synthetic pHsensitive polyanion MAA was shown to increase the activity of the siRNA directed against RhoA in L929 mouse fibroblast cells in vitro.…”

Polycation gene delivery systems: escape from endosomes to cytosol

“…Synthetic anionic polymers that induce pH-responsive membrane disruption have been developed (Lackey et al 1999;Murthy et al 1999;Cheung et al 2001;Kyriakides et al 2002). These polymers were also designed to mimic anionic amphiphilic peptides that induce the endosomal membrane disruption.…”