Verteporfin-loaded poly(ethylene glycol)-poly(beta-amino ester)- poly(ethylene glycol) triblock micelles for cancer therapy

Purpose: Many anticancer small molecules have poor aqueous solubility, and verteporfin (VP) has mainly been used as a photodynamic therapy agent, which carries photosensitivity and delivery limitations. PBAE-based materials have been developed extensively for nucleic acid delivery but are less explored for non-genetic hydrophobic drug delivery. There is a need for biodegradable, pH-sensitive micelles that can encapsulate VP and potentially evade macrophage clearance.

Hypothesis: PEG-PBAE-PEG triblock copolymers can self-assemble into micelles that encapsulate and pH-dependently release VP. Tuning the hydrophobicity of the central PBAE block will shift micelle morphology from spherical to filamentous. Filamentous micelles will reduce macrophage uptake, while spherical micelles will show greater cancer-cell uptake and cytotoxicity.

Aims: Synthesize two PEG-PBAE-PEG triblock copolymers with different hydrophobic PBAE blocks and PEG molecular weights. - Formulate and characterize VP-loaded spherical and filamentous micelles. - Measure VP loading, pH-sensitive release, and colloidal stability. - Compare macrophage uptake and anticancer efficacy in triple-negative breast cancer and small cell lung cancer cells.

Delivery system: Polymer: PEG-PBAE-PEG triblock copolymer. - PP1: B4S8m (1,4-butanediol diacrylate + octylamine) with 2 kDa mPEG-thiol. - PP2: B6S10m (1,6-hexanediol diacrylate + decylamine) with 800 Da mPEG-thiol. - Synthesis: Michael addition to form hydrophobic PBAE base, followed by thiol-ene PEGylation. - Nanoparticle type: Self-assembled micelles formed by nanoprecipitation. - Payload: Verteporfin (VP), a hydrophobic small-molecule anticancer drug. - Targeting ligand: None. - Morphologies: Spherical VP-loaded micelles (sVPM) ~50 nm; filamentous VP-loaded micelles (fVPM) ~31 nm width, ~651 nm length, aspect ratio ~20. - Surface charge: sVPM ~−3 ± 4 mV; sBM ~3 ± 5 mV, indicating PEG shielding. - Loading: sVPM loading capacity 5.36%, loading efficiency 43.7%. - Release: pH-sensitive, fastest at pH 5.0, slowest at pH 6.5, intermediate at pH 7.4.

Approach: In vitro only. Cell lines: MDA-MB-231 human triple-negative breast cancer, H446 human small cell lung cancer, and RAW 264.7 murine macrophages. - Uptake: 1.5 h incubation with free VP, sVPM, or fVPM; flow cytometry; n = 4. - Cytotoxicity: 2 h incubation with free VP, sVPM, or fVPM at 2.5–20 µM VP, then MTS at 2, 6, 24, and 48 h; n = 4. - Controls: free VP in 0.4% DMSO, blank micelles (sBM, fBM), and vehicle controls. - No in vivo model.

Key methods: ¹H NMR for polymer structure and PEGylation confirmation. - Pyrene fluorescence for critical micelle concentration (CMC). - TEM for micelle morphology and aspect ratio. - DLS and zeta potential for size, PDI, stability, and surface charge. - Fluorescence spectroscopy for VP loading and release kinetics. - Flow cytometry for cellular uptake. - MTS assay for cell viability/cytotoxicity.

Key results: CMC: PP1 micelles formed at 0.056 mg/mL. - Macrophage uptake: fVPM showed an 89% drop in cellular uptake percentage and a 5.6-fold drop in normalized geometric mean uptake compared with sVPM in RAW 264.7 macrophages. - Cancer cell killing: In H446 lung cancer cells, sVPM and fVPM at 5 and 10 µM VP, respectively, resulted in 100% cell killing. - Breast cancer killing: In MDA-MB-231 cells, both micelle types required 20 µM VP to kill at levels of 80% (sVPM) and 50% (fVPM). - Stability: sVPM was stable in human serum/PBS for 31 h; fVPM had higher PDI and lower macrophage uptake. - pH release: VP release was fastest at pH 5.0 and slowest at pH 6.5, supporting endosomal/lysosomal release potential.

Interpretation: This work demonstrates the first anisotropic PBAE-based self-assembled drug delivery system and one of the first validations of VP as a direct anticancer nanomedicine without photodynamic activation. Spherical micelles were superior for cancer-cell uptake and killing, while filamentous micelles were superior for evading macrophage uptake, suggesting a shape-dependent trade-off relevant to systemic delivery. PEG-PBAE-PEG micelles are proposed as biodegradable, pH-sensitive carriers for hydrophobic anticancer drugs.

Limitations: In vitro only: no in vivo pharmacokinetics, biodistribution, tumor accumulation, or survival data. - No active targeting ligand: delivery relies on passive accumulation and shape effects. - Limited cell panel: only MDA-MB-231, H446, and RAW 264.7 cells. - Filamentous micelles: lower cancer-cell uptake and lower cytotoxicity than spherical micelles. - VP mechanism: dependence on YAP/TEAD and p53 status was discussed but not directly tested. - Photodynamic therapy: not evaluated; the study focuses on non-PDT activity. - Long-term safety and clearance: not assessed. - Loading capacity: relatively modest (5.36%), and no in vivo dose escalation or toxicity study.