title: Toxicity, Immunogenicity, Uptake, and Kinetics Methods for CPPs.
abstract: Cell-penetrating peptides (CPPs) have been utilized as delivery vectors for various payloads, both in vitro and in vivo. Similar issues as for any other drug delivery systems: cytotoxicity and the tendency to induce innate immune response may limit their applications in clinics. Therefore, assessment of cytotoxicity and immunogenicity is an important step toward characterization of applicability of these delivery vehicles. Studying internalization mechanisms and kinetics of CPPs provides important information for the development of novel and more efficient cellular delivery vectors. This chapter describes methods and protocols for investigation of cytotoxicity and immunogenic activities of CPPs in vitro and in vivo as well as methods for studying cellular uptake and internalization kinetics of CPPs. In the first section we describe methods for in vitro cell viability studies and ELISA assay, which allows to measure cytokine release in cell culture media and in blood serum in response to different CPP applications. This chapter also provides a protocol for assessing caspase-1 activity essential for inflammation. In the second section of this chapter, we describe a comprehensive method and protocol for determining the endocytosis mechanisms utilized in CPP uptake by using luciferin-CPP conjugates and endocytosis inhibitors.
authors.affiliation: Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Nooruse 1/517, Tartu, 50411, Estonia, julia.uusna@ut.ee.
authors.forename: Julia
authors.initials: J
authors.lastname: Uusna
authors.forename: Kent
authors.initials: K
authors.lastname: Langel
authors.forename: Ülo
authors.initials: Ü
authors.lastname: Langel
mesh_terms: D000818:Animals; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D057846:Cell-Penetrating Peptides / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity*; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D004705:Endocytosis; D057075:Enzyme Assays / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods
chemical_list: D057846:Cell-Penetrating Peptides; D016207:Cytokines; D020169:Caspases