pH-Promoted Release of a Novel Anti-Tumour Peptide by “Stealth” Liposomes Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells

Purpose: PEGylated pH-sensitive liposomes may improve delivery of anti-cancer peptides, but PEGylation can reduce cellular uptake and endosomal escape (“PEG dilemma”), and it was unknown whether PEGylated liposome components interfere with the intracellular mechanism of action of the delivered drug.

Hypothesis: PEGylated pH-sensitive liposomes can stably encapsulate and pH-dependently release the hTS-inhibitory peptide [D-Gln⁴]LR, avoid rapid macrophage uptake, deliver it to cisplatin-resistant ovarian cancer cells, and preserve its intracellular anti-tumour activity without altering the proteomic profile associated with its mechanism of action.

Aims: Prepare and characterize PEGylated and non-PEGylated pH-sensitive liposomes loaded with [D-Gln⁴]LR peptide. - Assess size, surface charge, hydrophilicity, serum stability, macrophage internalization, drug loading, encapsulation efficiency, and pH-dependent release. - Evaluate cytotoxicity of peptide-loaded liposomes in cisplatin-resistant C13* ovarian cancer cells. - Determine whether PEGylated pH-sensitive liposomes modify the peptide’s intracellular mechanism by Western blot analysis of hTS, TRAP1, HSP90AA1, and DHFR.

Delivery system: Platform: pH-sensitive liposomes, PEGylated (PpHL) and non-PEGylated (nPpHL). - Lipid composition: DOPE:CHEMS:DSPE (22.8:15.2:2 mM) for nPpHL; DOPE:CHEMS:DSPE_PEG (22.8:15.2:2 mM) for PpHL. - Preparation: Reverse-phase evaporation followed by homogenization. - Payload: [D-Gln⁴]LR octapeptide (LSCqLYQR), a novel human thymidylate synthase (hTS) inhibitor. - Targeting ligand: None; passive targeting via EPR and pH-sensitive endosomal release. - Fluorescent label: Rhodamine-DOPE for macrophage uptake studies. - Controls: Unloaded liposomes, naked peptide, SAINT-PhD peptide delivery system, 5-fluorouracil (5-FU).

Approach: Model: In vitro only. - Cell lines: C13 cisplatin-resistant human ovarian carcinoma; J774 murine macrophage line. - Liposome characterization: DLS, AFM, Rose Bengal surface hydrophobicity, serum stability in 100% FBS up to 72 h. - Macrophage uptake: Flow cytometry and confocal microscopy at 0.25–24 h with rhodamine-labeled liposomes. - Drug loading/release: LC-MS/MS quantification; release in PBS pH 7.4; leakage at pH 7.4, 6.5, 5.5, and 4.0. - Cytotoxicity: MTT on C13 cells; 15 h incubation with 0.05–0.250 mg/mL liposomes, then 48 h culture. - Western blot: hTS, TRAP1, HSP90AA1, DHFR; β-actin normalization; treatments with 5-FU, peptide-loaded PpHL, and SAINT-PhD–peptide. - Statistics: One-way ANOVA; significance at p < 0.05.

Key methods: Dynamic light scattering for size, PDI, and zeta potential. - Atomic force microscopy for morphology. - Rose Bengal test for surface hydrophilicity. - Flow cytometry and confocal microscopy for macrophage internalization. - LC-MS/MS for peptide loading, encapsulation efficiency, and release. - MTT assay for cytotoxicity. - Western blot and densitometry for intracellular protein modulation.

Key results: Physicochemical properties: nPpHL: 282 ± 24 nm, PDI 0.376, ζ = −52.10 ± 9.38 mV. PpHL: 196 ± 43 nm, PDI 0.267, ζ = −14.54 ± 4.73 mV. Drug loading ~11.73–12.06 µg/mg; encapsulation efficiency 36–38%. - Surface/serum stability: PpHL had higher surface hydrophilicity (PQ slope 1.9 vs 2.6) and remained stable in serum for 72 h, whereas nPpHL doubled in size within minutes. - Macrophage uptake: nPpHL reached ~45% positive J774 cells at 8 h; PpHL showed only ~5% positive cells at 24 h. - pH-dependent release: At pH 7.4, initial release was 11–14% and stable. At pH 5.5, PpHL size increased to ~466 nm with marked drug leakage; leakage further increased at pH 4.0. - Cytotoxicity: Naked peptide was inactive. Unloaded liposomes showed >80% cell viability. Peptide-loaded nPpHL was cytotoxic from 0.05 mg/mL; peptide-loaded PpHL showed significant cytotoxicity from 0.075 mg/mL and was slightly less cytotoxic than nPpHL but comparable to SAINT-PhD delivery. - Western blot: 5-FU increased hTS by ~60%. PpHL-delivered peptide did not modify hTS levels (p > 0.05). No major changes were seen in hTS, HSP90AA1, or DHFR; TRAP1 showed a slight increase. The PEGylated liposome did not alter the peptide’s intracellular protein modulation pattern.

Interpretation: PEGylated pH-sensitive liposomes efficiently deliver the anti-cancer peptide, provide stealth behaviour, release the payload at acidic pH, and preserve its intracellular mechanism of action, supporting their potential as safe nanocarriers for resistant ovarian cancer; further in vivo xenograft studies are warranted.

Limitations: In vitro only; no in vivo pharmacokinetics, biodistribution, or efficacy data. - Single cisplatin-resistant ovarian cancer cell line. - PEGylation slightly reduced cytotoxicity compared with non-PEGylated liposomes. - Encapsulation efficiency was relatively low (36–38%), though the peptide is highly target-specific. - Western blot analysis was limited to four proteins. - No long-term stability, repeat-dose, or toxicity studies. - Authors note that in vivo rat xenograft studies would be useful to confirm potential.