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.

Figure 1. Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)] polymer design

Multifaceted carrier properties were incorporated via RAFT polymerization using pyr-ECT to form a diblock architecture designed to possess aqueous solubility and pH-dependent membrane disruptive properties. The monomer chemical functionalities highlighted were chosen in order to produce the desired properties for each polymer block. Importantly, module 3 was designed to be near charge neutrality at physiologic pH (approximately 50% DMAEMA protonation and 50% PAA deprotonation predicted) but to undergo a transition to a more hydrophobic and positively charged state in lower pH environments.

Figure 2. pH-dependent membrane disruption by poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]

Polymer hemolysis was quantified at concentrations ranging from 1-40 μg/mL relative to 1% v/v Triton X-100. This experiment was completed 2 times in triplicate, yielding similar results. The data shown represent a single experiment conducted in triplicate ± standard deviation.

Figure 3. SDS PAGE gel validating polymer-peptide conjugation via a reducible disulfide linkage

(A) Increasing molar ratio of polymer:peptide to 2 or greater resulted in disappearance of the free peptide band as determined by staining with coomassie blue. (B) Treatment with the reducing agent TCEP disrupted the disulfide linkage, resulting in visualization of free peptide on the gel.

Figure 4. Polymer enhanced intracellular peptide delivery

Representative images illustrating (A) punctate peptide staining (green) in the samples delivered peptide alone and (B) dispersed peptide fluorescence within the cytosol following delivery of peptide-polymer conjugate. Samples were treated for 15 minutes with 25 μM peptide and prepared for microscopic examination following DAPI nuclear staining (blue).

Figure 5. Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)]-Antp-BH3 conjugate induction of HeLa cell death

HeLas were delivered (A) 10 μM or (B) 25 μM peptide conjugated to the Poly[HPMA]-b-[(PAA)(BMA)(DMAEMA)], peptide alone, an equivalent amount of the polymer alone, or peptide conjugated to a non-pH responsive poly(HPMA). At 1, 2, 4, and 6 hours, cell lysate was collected and assayed for LDH content relative to samples receiving no treatment. Representative data ± standard deviation are shown from 1 of 3 independent studies done in quadruplicate. * indicates Conjugate treatment was significantly different (p<0.05) than Peptide, Polymer, and HPMA Conjugate groups.

Figure 6. Pro-apoptotic indicators peptide conjugate bioactivity

In each test, HeLas were given fresh media (NT), or media containing 10 μM Antp-BH3 (peptide), 10 μM Antp-BH3 polymer conjugate (conjugate), or an equivalent amount of the polymer alone (polymer). (A) The JC-1 dye was added following 2 hours of treatment, and flow cytometry was used to assess the percent of cells exhibiting loss of mitochondrial membrane integrity. Representative data ± standard error mean are shown from combined data from 2 independent studies done in triplicate. *indicates p<0.05 vs NT, Polymer, and Peptide treatments using Tukey's method for pairwise comparisons. (B) To measure caspase activation, after 30 minutes of incubation, a fluorescent caspase 3/7 substrate was added to the media, and fluorescent readings were taken after 1 hour. Caspase activity was expressed relative to samples receiving no treatment (NT). Representative data ± standard error mean are shown from combined data from 3 independent studies done in triplicate. *indicates p<0.05 vs NT and Polymer treatments using Tukey's method for pairwise comparisons.

Scheme 1. Synthesis of Pyridyl Disulfide CTA

Scheme 2. Polymer-peptide conjugation via thiol-disulfide exchange reaction