LDLR-targeted orlistat therapeutic nanoparticles: Peptide selection, assembly, characterization, and cell-uptake in...

Many cancers overexpress low-density lipoprotein receptors (LDLR), facilitating cholesterol metabolism for tumour growth. Targeting LDLR offers a promising strategy for selective drug delivery. Orlistat, a fatty acid synthase (FAS) inhibitor, has shown anti-cancer potential, particularly in tumours with high FAS expression. This study introduces an LDLR-Orlistat Targeted Nanoparticles (LDLR-OTNs) to enhance cancer cell uptake via LDLR-mediated endocytosis. The objectives include synthesizing lipid-based orlistat nanoparticles, functionalizing them with an 11-mer LDLR-binding peptide, assessing uptake and cytotoxicity in three LDLR- and FAS-expressing breast cancer cell lines (BT-474, MDA MB 453, MCF-7), and comparing uptake kinetics with non-targeted nanoparticles.

Orlistat nanoparticles (ONs) were synthesised via rapid solvent exchange, producing uncoated ONs, POPC-coated ONs (POPC-ONs), and LDLR-targeted ONs (LDLR-OTNs). Targeting was achieved by conjugating an 11-mer binding peptide (RLTRKRGLKLA) to DSPE-PEG5000 maleimide via click chemistry, confirmed by Ellman's test. Nanoparticles were characterised using DLS and TEM. Cellular uptake over 24 hours was assessed using fluorescence-labelled POPC-ONs and LDLR-OTNs, and uptake kinetics were analysed. Suramin-blocking studies were used to confirm LDLR-mediated uptake. A 48-hour cytotoxicity assay quantified IC₅₀ values in the aforementioned cell lines.

TEM data showed that LDLR-OTNs (33 nm) were smaller than untargeted POPC-ONs (58 nm) and uncoated ONs (67 nm). Ellman's test confirmed > 99.2% peptide conjugation. Cellular uptake of LDLR-OTNs was rapid, with significant fluorescence by 1 hour and a kinetic plateau at 24-48 hours, with data fitting to a modified exponential model, while that of untargeted POPC-ONs had lower initial uptake, following a logistic model. Suramin blocking reduced LDLR-OTN uptake, confirming receptor-mediated entry. Cytotoxicity assays yielded IC₅₀ values of 23.8 µM (BT-474), 25.8 µM (MDA MB 453), and 8.2 µM (MCF-7), with maximal inhibition at 48 h.

LDLR-OTNs demonstrated receptor-mediated uptake and potent cytotoxicity in LDLR- and FAS- overexpressing breast cancer cells. These findings support LDLR-targeted nanoparticles as a promising approach for delivering FAS inhibitors to LDLR-rich tumours, meriting further investigation in targeted cancer therapy development.