E-selectin liposomal and nanotube-targeted delivery of doxorubicin to circulating tumor cells

Purpose: Metastasis is the leading cause of cancer mortality, and circulating tumor cells (CTCs) are key to metastatic spread. Doxorubicin (DXR) is effective but systemically toxic and nonspecific. A targeted delivery system was needed to capture and kill CTCs under physiologically relevant flow while reducing off-target toxicity.

Hypothesis: E-selectin/PEG-functionalized liposomal doxorubicin (ES-PEG L-DXR) will adhere to CTCs via E-selectin–ligand interactions under shear flow, capture and kill these cells, and halloysite nanotube (HNT) surface coating will enhance capture and targeted killing while sparing non-target blood cells.

Aims: Prepare and characterize E-selectin-targeted PEGylated liposomal doxorubicin and empty liposomes. - Assess DXR encapsulation, leakage, cellular uptake, and static cytotoxicity in model CTCs. - Evaluate capture and killing of model CTCs under flow in a microtubule device, with and without HNT coating. - Test targeted liposome efficacy in dilute suspension under cone-and-plate shear and assess toxicity to red blood cells and mononuclear cells.

Delivery system: Platform: PEGylated liposomal doxorubicin (L-DXR) with E-selectin targeting. - Liposome preparation: Thin lipid film hydration with 125 mM ammonium sulfate, freeze-thaw, extrusion; remote loading of DXR at DXR-to-lipid ratio 0.2:1 (w/w); >95% loading efficiency. - Targeting ligand: Recombinant human E-selectin/Fc chimera conjugated to DSPE-PEG₂₀₀₀-maleimide via thiolation, then attached to liposomes. - Payload: Doxorubicin. - Surface coating for capture: Halloysite nanotubes (HNT) with poly-L-lysine on microrenathane tubes. - Controls: Naked L-DXR, soluble DXR, empty liposomes (EL), ES-PEG EL, BSA- or milk-blocked surfaces. - Targeting ligand: E-selectin (no antibody).

Approach: Model: In vitro only. - Cell lines: COLO 205 colorectal adenocarcinoma and KG-1a acute promyelocytic leukemia as model CTCs. - Safety cells: Human red blood cells (RBCs) and mononuclear cells (MNCs) from healthy donors. - Static treatment: 18 h incubation with 0.001–5 µL liposomes; viability assessed at day 4 by trypan blue. - Microtube capture: Cells perfused at 0.5 dyn/cm² for 15 min, then 2.5 dyn/cm² for 2 h; captured cells collected and cultured; viability at day 4. - Cone-and-plate shear: Dilute cell/liposome suspension sheared at 2 dyn/cm² for 2 h; viability at day 4. - Replication: Typically n = 3; data mean ± SEM.

Key methods: Dynamic light scattering and zeta potential for liposome size/surface charge. - Flow cytometry and confocal microscopy for liposome adhesion and internalization. - Fluorescence spectrophotometry for DXR encapsulation and leakage. - Trypan blue exclusion for cell viability. - Fluorescence microscopy for DXR intrinsic fluorescence and immobilized liposomes. - Cone-and-plate viscometer and microtubule perfusion for shear-based capture/killing.

Key results: Liposome characterization: L-DXR 106.3 ± 5.9 nm, ζ = −24.56 ± 6.34 mV; PEG L-DXR 111.5 ± 6.4 nm, ζ = −8.49 ± 5.85 mV; ES-PEG L-DXR 143.7 ± 11.2 nm, ζ = −13.12 ± 6.28 mV. DXR loading >95%; minimal leakage over 39 days. - Static cytotoxicity: Maximum efficacy at 0.5 µL; >95% killing of KG-1a and COLO 205 cells with L-DXR or ES-PEG L-DXR; empty liposomes had no effect. ES-PEG L-DXR was as effective as non-targeted L-DXR. - Adhesion: ES-PEG L-DXR showed ~99.9% adherence to COLO 205 and KG-1a cells; non-targeted liposomes showed negligible adhesion. - Microtube capture and killing: ES-PEG L-DXR reduced KG-1a viability by ~20% and COLO 205 by ~30% versus controls. ES-PEG EL had no significant effect. RBCs showed no significant adhesion; MNC viability decreased ~16%. - Cone-and-plate shear: ES-PEG L-DXR reduced KG-1a viability by ~75% versus EL/untreated and ~65% versus naked L-DXR. COLO 205 viability decreased ~90% versus untreated. - HNT-coated tubes: ES-PEG L-DXR reduced COLO 205 viability by >87% versus smooth tubes and ~96% versus control HNT; KG-1a viability decreased ~35% versus smooth and ~45% versus control HNT.

Interpretation: The authors conclude that E-selectin-targeted liposomal doxorubicin can capture and kill CTCs under flow, and HNT-coated microtubules significantly enhance this effect. The immobilized device may reduce systemic drug exposure and offers a potential strategy to reduce metastasis by targeting CTCs in circulation.

Limitations: In vitro only; no in vivo metastasis or pharmacokinetic/biodistribution studies. - Model CTCs are established cell lines, not patient-derived CTCs. - Simplified flow and device conditions compared with human vasculature. - Off-target MNC viability decreased ~16%, indicating potential nonspecific effects. - Liposomes stored only up to one week before use; long-term stability not fully assessed. - No targeting ligand optimization or comparison with other selectins/ligands.