Ivis lumina 3

Six-week old male NOD SCID mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., China, #406) weighing between 19 g to 22 g were used in our animal study. HCT116 cells (3 × 10⁶ cells) transduced with lentivirus were tail vein injected into the mice (n = 5 per group). Tumor metastasis were monitored weekly by an in vivo imaging system (Perkin Elmer IVIS Lumina III) from the third week after injection. Mice were intraperitoneally injected with 150 μg D-luciferin (Solarbio, China, #D8390) solution per gram body weight for 10 min before subjected to bioluminescent imaging. Tumor size and metastasis were quantified using Living Image software (Perkin Elmer IVIS Lumina III).

A muscle infection model was used to evaluate the in vivo targeting ability of the LNP@HMVs upon tail vein injection. The mice (n = 3) were shaved and then injected intramuscularly with E. coli (1 × 10⁸ CFU/20 g, 50 μl) on the right thigh, namely, the infected site. After 2 hours of infection, the mice were injected with LNPs, LNP@NMVs, LNP@OMVs, or LNP@HMVs via tail vein injection and imaged at predetermined time points (3, 6, 9, and 24 hours) using a small animal living imaging system IVIS (IVIS Lumina III, PerkinElmer, USA; λex/λem = 750/790 nm).
A lung infection model was also used to evaluate the in vivo targeting ability of the LNP@HMVs upon intratracheally administration. The mice (n = 3) were intratracheally inoculated with K. pneumoniae (1 × 10⁵ CFU/20 g, 50 μl). After 2 hours of infection, the mice were intratracheally administered with LNPs or LNP@HMVs and euthanized at predetermined time points (3, 6, 9, and 24 hours). The lungs were collected for analysis using the IVIS (IVIS Lumina III, PerkinElmer, USA; λex/λem = 750/790 nm).

The SK-ATF-Luc cells were used to assess the activity of ATF element after different treatments in vitro. The signal of Luc2 and hRLuc were acquired and measured in the same well using IVIS (Lumina Ⅲ, PerkinElmer). The cultured cells were treated with ViviRen (a final concentration of 60 μM) and hRLuc signals were acquired 3 min later. After another 30 min, cells were treated with D-luciferin (150 μg/mL) and Luc2 signals were acquired 5 min later. The relative activity of the ATF element was a ratio of the total photon from Luc2 to hRluc. hRluc was used as an endogenous control of cell numbers.

The green fluorescence protein (GFP) expression in the Hdac6-OE mouse was observed using the small animal in vivo optical two-dimensional imaging system (IVIS Lumina Ⅲ, perkinelmer, USA) according to the manufacturer's instructions.

Plasma extravasation was evaluated using the near-infrared fluorophore IR-676 (0.5 mg/kg, Spectrum-Info Ltd., Kyiv, Ukraine) dissolved in 5% (v/v) aqueous solution of Kolliphor HS 15 (polyethylene-glycol-15-hydroxystearate; Sigma-Aldrich, St. Louis, MO, USA), yielding a micellar contrast agent accumulating in inflamed areas due to increased vascular permeability [40 (link)]. The formula was injected intravenously (iv.) into anesthetized mice through the retroorbital veins with a 0.5 mL insulin needle, the bevel oriented downwards as described earlier [41 (link)] 5 h after the K/BxN serum transfer and on day 2. Short-term anesthesia of multiple subjects has been done by administration of 120/6 mg/kg ketamine-xylazine ip. Fluorescence imaging of both ankle joints was performed in pairs 20 min post-injection using the IVIS Lumina III (PerkinElmer, Waltham, MA, USA) in vivo imaging system with the following settings: auto acquisition time, Binning = 2, F/stop = 2, excitation/emission filter: 660/710 nm). Data were analyzed and regions of interests (ROI) were drawn around the ankle joints. Fluorescence was expressed as total radiant efficiency ([photons/s/cm²/sr]/[μW/cm²]).