Intracellular Delivery of a Proapoptotic Peptide via Conjugation to a RAFT Synthesized Endosomolytic Polymer

• Published in: Molecular pharmaceutics 2010-04, Vol.7 (2), p.468-476
• Main Authors: Duvall, Craig L, Convertine, Anthony J, Benoit, Danielle S. W, Hoffman, Allan S, Stayton, Patrick S
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - chemical synthesis; Antineoplastic Agents - chemistry; Antineoplastic Agents - metabolism; Antineoplastic Agents - pharmacology; Cell Proliferation - drug effects; Flow Cytometry; HeLa Cells; Hemolysis - drug effects; Humans; Hydrogen-Ion Concentration; Membrane Potential, Mitochondrial - drug effects; Models, Theoretical; Peptides - chemistry; Polymers - chemical synthesis; Polymers - chemistry; Polymers - metabolism; Polymers - pharmacology
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/mp9002267

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 μM 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.