Enzyme-Cleavable Polymeric Micelles for the Intracellular Delivery of Proapoptotic Peptides

Abstract

Peptides derived from the third Bcl-2 homology domain (BH3) renormalize apoptotic signaling by antagonizing prosurvival Bcl-2 family members. These potential peptide drugs exhibit therapeutic activities but are limited by barriers including short circulation half-lives and poor penetration into cells. A diblock polymeric micelle carrier for the BIM BH3 peptide was recently described that demonstrated antitumor activity in a B-cell lymphoma xenograft model [Berguig et al., Mol. Ther. 2015, 23, 907–917]. However, the disulfide linkage used to conjugate the BIM peptide was shown to have nonoptimal blood stability. Here we describe a peptide macromonomer composed of BIM capped with a four amino acid cathepsin B substrate (FKFL) that possesses high blood stability and is cleaved to release the drug inside of target cells. Employing RAFT polymerization, the peptide macromonomer was directly integrated into a multifunctional diblock copolymer tailored for peptide delivery. The first polymer block was made as a macro-chain transfer agent (CTA) and composed of a pH-responsive endosomolytic formulation of N,N-diethylaminoethyl methacrylate (DEAEMA) and butyl methacrylate (BMA). The second polymer block was a copolymer of the peptide and polyethylene glycol methacrylate (PEGMA). PEGMA monomers of two sizes were investigated (300 Da and 950 Da). Protein gel analysis, high performance liquid chromatography, and coupled mass spectrometry (MS) showed that incubation with cathepsin B specifically cleaved the FKFL linker and released active BIM peptide with PEGMA300 but not with PEGMA950. MALDI-TOF MS showed that incubation of the peptide monomers alone in human serum resulted in partial cleavage at the FKFL linker after 12 h. However, formulation of the peptides into polymers protected against serum-mediated peptide degradation. Dynamic light scattering (DLS) demonstrated pH-dependent micelle disassembly (25 nm polymer micelles at pH 7.4 versus 6 nm unimers at pH 6.6), and a red blood cell lysis assay showed a corresponding increase in membrane destabilizing activity (<1% lysis at pH 7.4 versus 95% lysis at pH 6.6). The full carrier–drug system successfully induced apoptosis in SKOV3 ovarian cancer cells in a dose-dependent manner, in comparison to a control polymer containing a scrambled BIM peptide sequence. Mechanistic analysis verified target-dependent activation of caspase 3/7 activity (8.1-fold increase), and positive annexin V staining (72% increase). The increased blood stability of this enzyme-cleavable peptide polymer design, together with the direct polymerization approach that eliminated postsynthetic conjugation steps, suggests that this new carrier design could provide important benefits for intracellular peptide drug delivery.

Stimuli-Responsive Polymeric Nanocarriers for Drug Delivery, Imaging, and Theragnosis

“…In another study, a peptide macromonomer was designed from BIM and cathepsin B substrate, which was then incorporated into copolymeric deblock system of DEAEMA/BMA and DEAEMA. The system was successful in the intracellular delivery of peptides [ 161 ].…”

Spherical Nucleic Acid Nanoparticles: Therapeutic Potential

“…Many strategies for intracellular protein delivery have been examined, including covalent [79–81] or non-covalent conjugation to polymers and cell penetrating peptides [82, 83], or delivery via nanocarriers such as liposomes [84], micelles [85, 86], and lipid nanoparticles [87, 88]. Brodin and colleagues hypothesized that the unique features of SNAs could be exploited to enable superior intracellular protein delivery.…”

“…Intracellular protein delivery has widespread biomedical applications [75–77], but proteins are very challenging to deliver intracellularly due to their large size, charged surface, low stability in biological fluids, and degradation during systemic circulation [78]. Many strategies for intracellular protein delivery have been examined, including covalent [79–81] or non-covalent conjugation to polymers and cell penetrating peptides [82, 83], or delivery via nanocarriers such as liposomes [84], micelles [85, 86], and lipid nanoparticles [87, 88]. Brodin and colleagues hypothesized that the unique features of SNAs could be exploited to enable superior intracellular protein delivery.…”

Extremely long tumor retention, multi-responsive boronate crosslinked micelles with superior therapeutic efficacy for ovarian cancer

“…Polymeric micelles demonstrated their superior potential in drug delivery for cancer therapy in several aspects, such as superior capability to encapsulate water insoluble chemotherapeutic drugs, prolonged in vivo circulation time and preferential accumulation at tumor site via the enhanced permeability and retention (EPR) effect due to their relatively smaller particle size (<100 nm) [11, 16–21]. However, there are some challenges that have hampered the clinical translation of this type of nanoparticles.…”