Fig 2: The inflammation targeting of the ICG loaded SM-liposomes was studied ex vivo from dextran sodium sulfate (DSS)-treated animals (a). The mice were treated with 4% DSS in drinking water for five days, and then regular water was given until the endpoint. Animals were sacrificed on days four, seven, and eight. Healthy non-treated animals were used as a control. The liposomes (125 µL 20 mM total lipid in PBS) were added via tail vein injection to the animals (n = 4 per disease group and control group). The mice were sacrificed, and the ICG fluorescence was analyzed from the proximal colon (dark column) and distal colon (white column) ex vivo 24 h after tail vein injection using a Night Owl Imager. Regions of Interest (ROIs) were drawn around two colon segments (proximal and distal colons) and were analyzed with the indiGO software. Y-axes represent Relative Fluorescence Intensity (RFI). Bars represent mean ± SEM. The targeting of the ICG- loaded SM-liposomes was verified by analyzing the ICG fluorescence from histological slices (b). The proximal (blue) and distal (grey) colon samples were collected and cut into 10 µm thin section slices. The fluorometric scanner was used to measure the ICG fluorescence from the thin section slices. Bars represent mean ± STDEV. Correlation between the aSMase activity (x-axel) and targeted ICG fluorescence (y-axel) was analyzed (c). The ICG fluorescence was measured from proximal colon samples using a Night owl Imager; ROIs were drawn around proximal colon samples and were analyzed with the indiGO software. The homogenate samples were collected from the same proximal colon samples, and aSMase activity was analyzed using an Amplex Red acid sphingomyelinase assay kit. The Amplex fluorescence was standardized to the tissue weight (mg). All the tissue sample activities were analyzed simultaneously.

Fig 3: Magnetoenzymatic Sphingomyelin (SM) liposomes. (a) Schematic illustration of SM-liposomes containing Fe3O4-nanoparticles (dark gray) embedded in the lipid membrane and a payload of imaging and/or drug molecule (star and diamond) inside the liposome. (b) TEM microscopy of SM liposomes. Liposomes were untreated and iron-free (panel I), untreated and loaded with iron (panel II), or iron-containing and additionally exposed to Sphingomyelinase (SMase) (0.4 U/mL) alone (panel III) or in combination with an AMF (6 mT, 20 min) (panel IV). (c) Release of cisplatin from SM-liposomes when the increased dose of SMase was used and AMF treatment was kept constant (20 min, 2 mT, 100 kHz). The (¦) shows results for SM-liposomes loaded with cisplatin, containing ferric nanoparticles (DSPC:SM:DOTAP:chol (20:30:20:30) mol/mol), and treated with different concentrations of SMase from 0 to 2 U/mL and AMF. The (?) group shows results for SM-liposomes (DSPC:SM:DOTAP:chol (20:30:20:30) mol/mol) with the same 0–2 U/mL SMase pretreatment but without AMF. After the treatment, liposomal solution was collected and dialyzed overnight against PBS, and the leakage was calculated by measuring the cisplatin absorbance inside and outside of the dialysis bag. Each bar represents the mean ± SEM. Significance determined by t-test and indicated by *. (d) SAXS spectra of SM liposomes (DSPC:SM:DOTAP:chol, molar % 20:30:20:30, respectively) (?), solution of Fe3O4 nanoparticles (¦) (0.3 mg/mL), and SM liposomes with Fe3O4 nanoparticles (?) (0.3 mg/mL) in PBS. The solution temperature is 25 °C. (e) Temperature increase (?T) of the liposome containing buffer after AMF 10-min treatment at RT. With the ASMase treated magneto enzymatic SM liposomes compositions, a 0.70 °C (+/- 0.2 standard deviations) increase in temperature from 24.0 to 24.70 °C was observed from before alternating magnetic field (AMF) stimulation and after commencing 20 min of AMF treatment (t = 20 min). For the non-ASMase-treated MESL compositions, a 1.30 °C (+/- 0.15 standard deviations) increase in temperature from 24.0 to 25.30 °C was observed from before the AMF stimulation and after commencing 20 min of AMF treatment (t = 20 min).

Fig 4: Characterization in vivo. (a) Blood clearance of the MESL liposomal particles loaded with ICG. The image shows a plot of ICG signal from the blood represented as %ID/mL, as determined by the absorbance at 800 nm at 5 (n = 3), 15 (n = 4), 30 (n = 6), 60 (n = 6), 90 (n = 3), and 120 (n = 4) min after intravenous injection. Each bar represents mean ± SEM. (b) Dynamic blood kinetics determined using photoacoustic imaging of the mouse femoral artery. RI in y-axes represents relative intensity for fluorescence. The injected amount was the same as well as c max in blood. Both nontreated SM- liposomes (?) (DSPC:SM:DOTAP:chol (20:30:20:30) mol/mol, loaded with Fe NPs and ICG)), as a control, and SM-liposomes pretreated with SMase (0.4 U), and AMF (5 min 2 mT, 100 kHz) (¦) were injected in the mouse tail vein and followed by imaging femoral artery for 15 min (p-value 0.0001, t-test two-tailed unpaired). (c) Biodistribution of ICG filled MESL liposomal nanoparticles for the first 2 h after IV injection. Fluorescence images were acquired after 5 (n = 3), 15 (n = 4), 30 (n = 6), 60 (n = 6), 90 (n = 3), and 120 (n = 4) min. Each bar represents the mean ± SEM. (d) ASMase activities analyzed with Amplex Red reagent-based assay in human SCC-9 tumor xenografts in mice. Muscle tissue was analyzed as control tissue. The activity was assessed immediately after mouse decapitation and dissection and was normalized by the weight of the tissue sample. In the average columns, both bars represent mean ± SEM. ** represent t-test results smaller than 0.005. (e) Histological staining of ASMase from SCC tumor xenograft slices. (f) We compared biodistribution of the liposomal ICG to Free ICG, which shows the liposomal ICG to be in circulation for longer. This result was also observed in the blood kinetic studies.

Fig 5: Immunohistochemical staining of aSMase (left upper panel) and macrophages (CD68) (right upper panel) in colons of mice suffering from dextran sodium sulfate (DSS)-induced acute colitis (upper row) (a). Mice were sacrificed after receiving 4% DSS for five days in their drinking water to induce acute colitis. The control animals had plain water (lower raw), mice were sacrificed, and colons were collected for immunohistochemical aSMase staining. Representative images of the same crypt fold of the proximal colon are shown. Arrows indicate possible co-localization. Scale bar = 25 µm. The lower row presents representative proximal colon from healthy control animals stained with aSMase (left) and CD68 (right).The aSMase expression was quantified from the histological slides (b). Quantification was done by measuring the area in crypt folds positive for aSMase staining and referring the resulting values to the total crypt fold area in the proximal colon (b) and distal colon (c). Bars represent mean ± SEM. (n = 3 per condition). Sphingomyelinase appearance was also analyzed from a human patient sample (d). Immunohistochemical staining of aSMase (upper row) and macrophages (CD68) (lower row) in colon tissue from a patient who has Crohn’s disease. Representative images show areas covering several crypts and crypt folds. Colonic tissue from areas regarded as non-inflamed (left) and inflamed (right) was collected, sliced, and immunohistochemically stained. Scale bar = 100 µm.