Intracellular Delivery of a Proapoptotic Peptide Via Conjugation to a RAFT Synthesized Endosomolytic Polymer
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 (Mn) and low polydispersity (PDI). The diblock polymer (Mn 19,000 g/mol, PDI 1.27) was composed of an N-(2-hydroxypropyl) methacrylamide (HPMA) first block (Mn 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.
C. L. Duvall et al., "Intracellular Delivery of a Proapoptotic Peptide Via Conjugation to a RAFT Synthesized Endosomolytic Polymer," Molecular Pharmaceutics, vol. 7, no. 2, pp. 468-476, American Chemical Society (ACS), Apr 2010.
The definitive version is available at https://doi.org/10.1021/mp9002267
Materials Science and Engineering
Keywords and Phrases
Apoptosis; Bioconjugation; Cancer; Endosome escape; Intracellular drug delivery; RAFT polymerization; PH-responsive polymer; Therapeutic peptide
International Standard Serial Number (ISSN)
Article - Journal
© 2010 American Chemical Society (ACS), All rights reserved.
01 Apr 2010