title: Intracellular delivery of a proapoptotic peptide via conjugation to a RAFT synthesized endosomolytic polymer.
abstract: Peptides derived from the third B-cell lymphoma 2 (Bcl-2) homology domain (BH3) can heterodimerize with antiapoptotic Bcl-2 family members to block their activity and trigger apoptosis. Use of these peptides presents a viable anticancer approach, but delivery barriers limit the broad application of intracellular-acting peptides as clinical therapeutics. Here, a novel diblock copolymer carrier is described that confers desirable pharmaceutical properties to intracellular-acting therapeutic peptides through site-specific molecular conjugation. This polymer was prepared using reversible addition-fragmentation chain transfer (RAFT) to form a pyridyl disulfide end-functionalized, modular diblock copolymer with precisely controlled molecular weight (M(n)) and low polydispersity (PDI). The diblock polymer (M(n) 19,000 g/mol, PDI 1.27) was composed of an N-(2-hydroxypropyl) methacrylamide (HPMA) first block (M(n) 13,800 g/mol, PDI 1.13) intended to enhance water solubility and circulation time. The second polymer block was a pH-responsive composition designed to enhance endosomal escape and consisted of equimolar quantities of dimethylaminoethyl methacrylate (DMAEMA), propylacrylic acid (PAA), and butyl methacrylate (BMA). A hemolysis assay indicated that the diblock polymer undergoes a physiologically relevant pH-dependent switch from a membrane inert (1% hemolysis, pH 7.4) to a membrane disruptive (61% hemolysis, pH 5.8) conformation. Thiol-disulfide exchange reactions were found to efficiently produce reversible polymer conjugates (75 mol % peptide reactivity with polymer) with a cell-internalized proapoptotic peptide. Microscopy studies showed that peptide delivered via polymer conjugates effectively escaped endosomes and achieved diffusion into the cytosol. Peptide-polymer conjugates also produced significantly increased apoptotic activity over peptide alone in HeLa cervical carcinoma cells as found using flow cytometric measurements of mitochondrial membrane depolarization (2.5-fold increase) and cell viability tests that showed 50% cytotoxicity after 6 h of treatment with 10 muM peptide conjugate. These results indicate that this multifunctional carrier shows significant promise for proapoptotic peptide cancer therapeutics and also as a general platform for delivery of peptide drugs with intracellular targets.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle Washington 98195, USA.
authors.forename: Craig L
authors.initials: CL
authors.lastname: Duvall
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Convertine
authors.forename: Danielle S W
authors.initials: DS
authors.lastname: Benoit
authors.forename: Allan S
authors.initials: AS
authors.lastname: Hoffman
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Stayton
mesh_terms: D000970:Antineoplastic Agents / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D006367:HeLa Cells; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008962:Models, Theoretical; D010455:Peptides / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D010455:Peptides; D011108:Polymers