The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EV production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the PfVps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production

In Situ Gelling Liquid Crystalline System as Local siRNA Delivery System

An effective short interfering RNA (siRNA) delivery system protects the siRNA from degradation, facilitates its cellular uptake, and promotes its release into the cytoplasm. Local administration of siRNA presents advantages over systemic administration, such as the possibility to use lower doses and allow local and sustained release. In this context, in situ solidifying organogels based on monoglycerides (MO), polyethylenimine (PEI), propylene glycol (PG) and tris buffer are an attractive strategy for intratumoral delivery of siRNA. In this study, precursor fluid formulation (PFF) composed of MO/PEI/PG/tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) was used to deliver siRNA to tumor cells. The internal structure of the gel obtained from PFF was characterized using small angle X-ray scattering (SAXS). In addition, its ability to complex siRNA, protect it from degradation, and functionally deliver it to tumor cells was investigated. Moreover, in vivo gel formation following intratumoral injection was evaluated. The gel formed in excess water from PFF was found to comprise a mixture of hexagonal and cubic phases. The system was able to complex high amounts of siRNA, protect it from degradation, promote siRNA internalization, and induce gene silencing in vitro in a variety of tumor cell lines. Moreover, a gel formed in situ following intratumoral injection in a murine xenograft model. In conclusion, PFF is a potential delivery system for local and sustained delivery of siRNA to tumor tissue after intratumoral administration

<i>In Situ</i> Gelling Liquid Crystalline System as Local siRNA Delivery System

An effective short interfering RNA (siRNA) delivery system protects the siRNA from degradation, facilitates its cellular uptake, and promotes its release into the cytoplasm. Local administration of siRNA presents advantages over systemic administration, such as the possibility to use lower doses and allow local and sustained release. In this context, <i>in situ</i> solidifying organogels based on monoglycerides (MO), polyethylenimine (PEI), propylene glycol (PG) and tris buffer are an attractive strategy for intratumoral delivery of siRNA. In this study, precursor fluid formulation (PFF) composed of MO/PEI/PG/tris buffer at 7.85:0.65:76.5:15 (w/w/w/w) was used to deliver siRNA to tumor cells. The internal structure of the gel obtained from PFF was characterized using small angle X-ray scattering (SAXS). In addition, its ability to complex siRNA, protect it from degradation, and functionally deliver it to tumor cells was investigated. Moreover, <i>in vivo</i> gel formation following intratumoral injection was evaluated. The gel formed in excess water from PFF was found to comprise a mixture of hexagonal and cubic phases. The system was able to complex high amounts of siRNA, protect it from degradation, promote siRNA internalization, and induce gene silencing <i>in vitro</i> in a variety of tumor cell lines. Moreover, a gel formed <i>in situ</i> following intratumoral injection in a murine xenograft model. In conclusion, PFF is a potential delivery system for local and sustained delivery of siRNA to tumor tissue after intratumoral administration