Metabolic sugar labeling followed by the use of reagent-free Click chemistry is a demonstrated technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Here we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4ManAz-loaded MBs showed great stability under physiological conditions but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4ManAz from MBs was able to label 4T1 tumor cells with azido group and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 via subsequent Click chemistry. We demonstrated for the first time that Ac4ManAz loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting.However,selective metabolic labeling of the target tissues in vivo remains ac hallenge to overcome, which has prohibited the use of this techniquef or targeted in vivo applications.H erein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl Nazidoacetylmannosamine (Ac 4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac 4 ManAz-loaded MBs showed great stability under physiological conditions,b ut rapidly collapsed in the presence of tumor-localized ultrasound pulses.T he released Ac 4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent clickc hemistry. We demonstrated for the first time that Ac 4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be asimple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

Untreated non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) lead to irreversible liver damage. We hypothesized that a low-fat diet (LFD) or a high-fat diet (HFD) with soy protein isolate (SPI) would be an effective intervention to halt or reverse NAFLD progression. To test these hypotheses, we conducted two studies. In the first study, we fed a HFD to 7-week-old C57BL/6J mice to induce NAFLD compared to a LFD (control). Hepatic steatosis was monitored by quantitative ultrasound (QUS) scans (in vivo and ex vivo). Animals were euthanized after 0, 2, 4 and 6 weeks of feeding. In the second study, 7-week-old mice were randomized onto a LFD or HFD with SPI intervention after 4 weeks of feeding HFD. Animals from each group were scanned with QUS and euthanized after 4, 9 and 12 weeks of feeding. Animals fed the HFD developed NAFLD (100%) and NASH (80%) characterized by increased liver weight, lipid accumulation, and histological scores for inflammation by 4 weeks in the first study. In the second study, the LFD ameliorated this NAFLD phenotype after 5 weeks of feeding, however the SPI intervention failed to significantly attenuate NAFLD. QUS parameters were significantly increased with the HFDs (p<0.05) and steatosis grade (p<0.05) and were positively correlated with hepatic lipid concentrations. In conclusion, dietary modification may be effective at reversing NAFLD and NASH at early stages. Furthermore, QUS may become a valuable tool to track hepatic steatosis. Additional studies are needed to further evaluate the effectiveness of these interventions.

BACKGROUNDNon‐alcoholic fatty liver disease (NAFLD) is the most common liver disease in the Western world, affecting 20% of the population. If left untreated, NAFLD may progress into non‐alcoholic steatohepatitis (NASH), end‐stage liver disease or hepatocellular carcinoma. Emerging evidence indicates that soy protein may protect the liver from steatosis, which may improve health outcomes. We hypothesized that a soy protein intervention would be effective in protecting against further steatosis and disease progression in mice fed a high fat diet.METHODSIn this study, 8‐week‐old male C57BL/6J mice (n=70) were randomized onto an AIN‐93G control diet (CON, n=20) or a high‐fat atherogenic diet with a casein protein source (CAS, n=50). After 4 weeks on the CAS diet when animals were expected to develop NAFLD, CAS animals were randomized onto one of three diets: an atherogenic diet with a soy protein source (SOY, n=15), CAS (n=20) or the control diet (CAS‐CON, n=15). For this study, we utilized the novel non‐invasive QUS method developed by our lab in order to monitor hepatic fat content at various stages of steatosis. Each animal received liver ultrasound scans at baseline (in vivo) and at euthanasia (in vivo and ex vivo). At 4, 9 and 12 weeks on study, animals from each group were euthanized. At euthanasia, serum was collected and the liver was harvested. Half of the liver's left lateral lobe underwent hematoxylin and eosin (H&E) staining (3‐μm section). The right cranial lateral liver lobe was removed for ex vivo QUS scanning. The remainder of each liver was frozen for further analysis.RESULTSLiver weight, as a percentage of body weight, was significantly increased with both atherogenic diets (CAS and SOY). CAS animals had significantly higher accumulation of lipids in the liver at each respective time point (p<0.05) compared to either dietary control group (CON or CAS‐CON). CAS also had significantly higher serum cholesterol by week 12 compared to CON and CAS‐CON. The QUS was also significantly associated with the quantity of lipid in the liver. Ex vivo (p<0.0001) and in vivo (p=0.047) ultrasound attenuation was positively correlated with liver lipids. Serum alanine aminotransferase (ALT) and histology will be analyzed to determine hepatic damage and disease status. QUS attenuation and backscatter will be further correlated to histological measures of steatosis, fibrosis and inflammation in the liver. Total hepatic cholesterol will also be measured.CONCLUSIONSAtherogenic diets were associated with an increase in liver weight, liver lipids and ultrasound attenuation. Replacing casein with soy as the protein source after four weeks of feeding failed to significantly reverse hepatic lipid accumulation or liver weight. QUS attenuation was significantly associated with hepatic lipid accumulation. Serum cholesterol increased over the course of 12 weeks in the CAS group. The development of a QUS method to identify early stages of NAFLD and NASH would provide the capability to noninvasively quantify and monitor liver status and evaluate methods for intervention.Support or Funding InformationThis work was supported by NIH R37EB002641 and by the USDA National Institute of Food and Agriculture, Hatch project 228706.

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