The ability to activate drugs only at desired locations avoiding systemic immunosuppression and other dose limiting toxicities is highly desirable. Here we present a new approach, named local drug activation, that uses bioorthogonal chemistry to concentrate and activate systemic small molecules at a location of choice. This method is independent of endogenous cellular or environmental markers and only depends on the presence of a preimplanted biomaterial near a desired site (e.g., tumor). We demonstrate the clear therapeutic benefit with minimal side effects of this approach in mice over systemic therapy using a doxorubicin pro-drug against xenograft tumors of a type of soft tissue sarcoma (HT1080).

Hepatitis C virus (HCV) assembles its replication complex on cytosolic membrane vesicles often clustered in a membranous web (MW). During infection, HCV NS5A protein activates PI4KIII␣ enzyme, causing massive production and redistribution of phosphatidylinositol 4-phosphate (PI4P) lipid to the replication complex. However, the role of PI4P in the HCV life cycle is not well understood. We postulated that PI4P recruits host effectors to modulate HCV genome replication or virus particle production. To test this hypothesis, we generated cell lines for doxycycline-inducible expression of short hairpin RNAs (shRNAs) targeting the PI4P effector, four-phosphate adaptor protein 2 (FAPP2). FAPP2 depletion attenuated HCV infectivity and impeded HCV RNA synthesis. Indeed, FAPP2 has two functional lipid-binding domains specific for PI4P and glycosphingolipids. While expression of the PI4P-binding mutant protein was expected to inhibit HCV replication, a marked drop in replication efficiency was observed unexpectedly with the glycosphingolipid-binding mutant protein. These data suggest that both domains are crucial for the role of FAPP2 in HCV genome replication. We also found that HCV significantly increases the level of some glycosphingolipids, whereas adding these lipids to FAPP2-depleted cells partially rescued replication, further arguing for the importance of glycosphingolipids in HCV RNA synthesis. Interestingly, FAPP2 is redistributed to the replication complex (RC) characterized by HCV NS5A, NS4B, or double-stranded RNA (dsRNA) foci. Additionally, FAPP2 depletion disrupts the RC and alters the colocalization of HCV replicase proteins. Altogether, our study implies that HCV coopts FAPP2 for virus genome replication via PI4P binding and glycosphingolipid transport to the HCV RC. IMPORTANCELike most viruses with a positive-sense RNA genome, HCV replicates its RNA on remodeled host membranes composed of lipids hijacked from various internal membrane compartments. During infection, HCV induces massive production and retargeting of the PI4P lipid to its replication complex. However, the role of PI4P in HCV replication is not well understood. In this study, we have shown that FAPP2, a PI4P effector and glycosphingolipid-binding protein, is recruited to the HCV replication complex and is required for HCV genome replication and replication complex formation. More importantly, this study demonstrates, for the first time, the crucial role of glycosphingolipids in the HCV life cycle and suggests a link between PI4P and glycosphingolipids in HCV genome replication.

Systemic administration of antibiotics can cause severe side-effects such as liver and kidney toxicity, destruction of healthy gut bacteria, as well as multidrug resistance. Here, we present a bio-orthogonal chemistry-based strategy toward local prodrug concentration and activation. The strategy is based on the inverse electron-demand Diels–Alder chemistry between trans-cyclooctene and tetrazine and involves a biomaterial that can concentrate and activate multiple doses of systemic antibiotic therapy prodrugs at a local site. We demonstrate that a biomaterial, consisting of alginate hydrogel modified with tetrazine, is efficient at activating multiple doses of prodrugs of vancomycin and daptomycin in vitro as well as in vivo. These results support a drug delivery process that is independent of endogenous environmental markers. This approach is expected to improve therapeutic efficacy with decreased side-effects of antibiotics against bacterial infections. The platform has a wide scope of possible applications such as wound healing, and cancer and immunotherapy.

In this study, we have coupled the DNA polymerization capability of hybridization chain reaction (HCR) with the plasmonic properties of gold nanoparticles to develop a reprogrammable and multiplexed detection of three circulating oncomiRs (miR-10b, miR-21 and miR-141) dysregulated in various disease states of breast cancer. We have demonstrated that by simply changing the initiator (label-free short single stranded DNA) content of the HCR, while keeping everything else unchanged, the same nanoparticle assembly can be reprogrammed for the detection of the target oncomiRs individually or simultaneously in all possible combinations. We have shown that as little as 20 femtomoles of each oncomiR can be detected visually without using any analytical instrument. Furthermore, we demonstrated that the target oncomiR can be detected in an RNA pool isolated from a liquid biopsy mimic of breast cancer.