In vivo optical imaging

As a tumor model, the MDA-MB-231-Luc breast cancer cell line was used, which expressed the luciferase gene (Luc). With the cell line, sites and sizes of xenografts were visualized by bioluminescent imaging in vivo, and the merging images of bioluminescence and fluorescence were used to estimate the tumor-targeting ability of liposomes. Tumors were established by subcutaneously injecting 5 × 10⁶ MDA-MB-231-Luc cells under the front right axilla of immunocompromised 10-week-old female Balb/c nude mice. The tumor size was monitored every other day, and the mice were used for in vivo experiments when the tumor diameter reached 0.8–1.0 cm (typically 2 weeks after inoculation). Xenograft-bearing mice were randomly divided into two groups (n = 3 per group) and injected with LSS670-labeled Lp or LSS670-labeled ILp. The labeling reactions were carried out according to the manufacturer’s protocol. The animals were anesthetized using 3.5% isoflurane and maintained unconscious using 2.0–2.5% isoflurane during injection and imaging.

Optical imaging was performed with the Kodak in vivo FXPro imaging system (Carestream Health Inc., New York, USA), which combines multispectral fluorescence, luminescence, and digital X-ray capabilities in a single system. The imaging system consisted of a dark chamber with gas anesthesia inlet and outlet ports. The excitation and emission filters were set at 650 and 700 nm, respectively. All images were acquired using the following parameters, which were optimized to improve the signal-to-noise ratio: 60,000 s exposure time; 2 × 2 binning; 60 s acquisition time; 120 × 120 mm field of view; and f 2.25 aperture stop.

The animals were carefully wiped with alcohol to remove any fluorescing contaminants, and baseline imaging was performed. Nanoparticles (100 μL) were injected through the tail vein and in vivo fluorescence scanning was performed at various time points after injection. For bioluminescent imaging, the light-sensitive substrate D-luciferin was administered by intraperitoneal injection of approximately 2.5 mg luciferin/kg body weight. The exposure time was 3 min, with 4 × 4 binning.

Four additional tumor-bearing mice were intravenously injected with LSS670-labeled Lp or LSS670-labeled ILp and euthanized at 12 h post injection (at peak tumor uptake, based on imaging results). Biodistribution studies were carried out to validate quantitative fluorescent dye uptake values derived from ex vivo fluorescent scans. Tumor and major organs/tissues were collected, and fluorescence was measured and analyzed by the imaging system. Tumor xenografts were also frozen for confocal microscopy analysis.