Bombesin receptor-targeted liposomes for enhanced delivery to lung cancer cells

Abstract

Background: Gastrin-releasing peptide is a member of the bombesin family of peptides. Its cognate receptor, gastrin releasing peptide receptor (GRPR), is widely expressed in cancers of the lung, pancreas and ovaries. Gastrin releasing peptide (GRP) is an autocrine growth factor in small cell lung cancer, which has very poor patient outcomes. High affinity antagonist peptides have been developed for in vivo cancer imaging. In this report we decorated pegylated liposomes with a GRPR antagonist peptide and studied its interaction with, and accumulation within, lung cancer cells. Results: An N-terminally cysteine modified GRPR antagonist (termed cystabn) was synthesised and shown to inhibit cell growth in vitro. Cystabn was used to prepare a targeted 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000) lipid conjugate that was formulated into liposomes. The liposomes displayed desirable colloidal properties and good stability under storage conditions. Flow cytometric and microscopic studies showed that fluorescently labelled cystabn-decorated liposomes accumulated more extensively in GRPR over-expressing cells than matched liposomes that contained no cystabn targeting motif. Conclusion: The use of GRPR antagonistic peptides for nanoparticle targeting has potential for enhancing drug accumulation in resistant cancer cells.

Keywords: GRPR; bombesin; liposome; lung cancer; targeting.

Figure 1

GRPR functionality in lung cancer cells. (A) Exemplar fluorescence trace from H345 cells loaded with Ca²⁺ reporter dye, Fura-2 AM before injection of GRPR agonist, Tyr⁴-Bn. Baseline fluorescence was monitored for 30 s before peptide injection. (B) Escalating concentrations of Tyr⁴-Bn were injected into H345 or H82 cells and the Fura-2 AM fluorescence emission monitored over 200 s. Peak Fura-2 ratios at 340/380 nm were plotted against agonist concentration. Data are mean ± SD. H345 cells were cultured over 5 days in selenite-insulin-transferrin (SIT) serum-free medium in the presence of escalating concentrations of Tyr⁴-Bn (C) or cystabn (D) before cell growth quantification by MTS assay. Similarly, (E, F) show MTS growth data for H82 cells in the presence of Tyr⁴-Bn (E) or cystabn (F). Error bars are SD.

Figure 2

Mass spectrometry characterisation of cystabn-lipid conjugate. MALDI–TOF mass spectra of crude DSPE-PEG₂₀₀₀-cystabn conjugate (A) and purified, post-dialysis, DSPE-PEG₂₀₀₀-cystabn conjugate (B).

Figure 3

Colloidal stability of liposomes. Control and target liposomes were exposed to PBS (A,C) or 10% FBS in PBS (B,D) at three temperatures for 24/72 h then analysed by DLS. Data shown are mean ± SD, n = 3.

Figure 4

GRPR targeting with cystabn increases cell accumulation of liposomes. (A) A549-GRPR cells were exposed to 1 μg/mL of control or target liposomes for 15–180 min before washing and analysis of liposomal 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE)-fluorescein intensity by flow cytometry. Fold-change median fluorescence intensity (MFI) was determined by correcting for the background MFI from cells exposed for matched time periods on ice. Data shown are mean ± SD, n = 3. A549-GRPR cells were exposed to 1 µg/mL of control (B) or target (C) liposomes (green) for 5 min, washed, fixed and nuclei labelled with Hoechst (blue).