Preparation of nanoparticles. LENK-SQ NPs were prepared using the nanoprecipitation methodology. Briefly, the LENK-SQ bioconjugate (i.e., LENK-SQ-Diox, LENK-SQ-Dig, or LENK-SQ-Am) was dissolved in EtOH (8 mg/ml) and added dropwise under stirring (500 rpm) into a 5% aqueous dextrose solution (EtOH/dextrose solution volume ratio, 1:4). The solution became spontaneously turbid with a Tyndall effect, indicating the formation of the nanoparticles. EtOH was then completely evaporated using a Rotavapor (80 rpm, 30°C, 30 mbar) to obtain an aqueous suspension of pure LENK-SQ NPs (final concentration, 2 mg/ml). Blank SQ NPs (LENK-free NPs) were prepared by the same method as described above by adding dropwise an ethanolic solution of squalenic acid into 5% aqueous dextrose solution. Fluorescently labeled LENK-SQ NPs were also obtained by the same procedure, except that the fluorescent probe DiD was solubilized in the ethanolic phase together with the LENK-SQ-Am bioconjugate (DiD/LENK-SQ-Am ratio was 4 wt %) before addition to the dextrose solution. Fluorescence quencher LENK-SQ NPs were also prepared by the same way using DiR as a fluorescent probe (DiD/DiR/LENK-SQ-Am ratio was 2:2:100 wt). The peptide drug loadings into the NPs were expressed as percentage (%), calculated from the ratio between LENK peptide Mw and LENK-SQ bioconjugate Mw. The LENK-SQ NPs were regularly observed by cryo-TEM. All the NPs were freshly prepared and used within 2 hours (conservation at 4°C) before in vivo experiments.

DLS measurements. The mean particle size, polydispersity index (PDI), and zeta potential were primarily evaluated by DLS (Nano ZS, Malvern; 173° scattering angle at 25°C). The measurements were performed in triplicate following appropriate dilution of the NPs in water (DLS) or in 0.1 mM KCl (zeta potential). The results represent the mean and SD of three repeated sample preparations or more.

Cryo-TEM. The morphology of the LENK-SQ NPs was investigated by cryo-TEM. NPs were vitrified using a chamber designed and set up in the laboratory where both humidity and temperature could be controlled. Four microliters of solution of LENK-SQ NPs (4 mg/ml in Milli-Q water) was deposited onto a perforated carbon film mounted on a 200-mesh electron microscopy grid. The homemade carbon film hole dimensions were about 2 mm in diameter. Most of the drop was removed with a blotting filter paper, and the residual thin films remaining within the holes were quick-frozen by plunging them into liquid ethane cooled with liquid N2. The specimen was then transferred, using liquid N2, to a cryo-specimen holder and observed using a JEOL FEG-2010 electron microscope. Micrographs were recorded at 200 kV under low-dose conditions at a magnification of ×40,000 on SO-163 Kodak films. Micrographs were digitized using a film scanner (Super Coolscan 8000 ED, Nikon), and analyses were made using the ImageJ software.

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