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Chitosan possesses many characteristics of an ideal gene delivery system. However, the transfection efficiency of conventional chitosans is generally found to be low. In this study, we investigated the self-branching of chitosans as a strategy to improve its gene transfer properties without compromising its safety profile. Self-branched (SB) and self-branched trisaccharide-substituted (SBTCO) chitosans with molecular weights of 11-71 kDa were synthesized, characterized, and compared with their linear counterparts with respect to transfection efficiency, cellular uptake, formulation stability, and cytotoxicity. Our studies show that in contrast with unmodified linear chitosans that were unable to transfect HeLa cells, self-branched chitosans mediated high transfection efficiencies. The most efficient chitosan, SBTCO30, yielded gene expression levels two and five times higher than those of Lipofectamine and Exgen, respectively, and was nontoxic to cells. Nanoparticles formed with SBTCO chitosans exhibited a higher colloidal stability of formulation, efficient internalization without excessive cell surface binding, and low cytotoxicity.

The blood-brain barrier (BBB), composed of tightly organized endothelial cells, limits the availability of drugs to therapeutic targets in the central nervous system. The barrier is maintained by membrane bound efflux pumps efficiently transporting specific xenobiotics back into the blood. The efflux pump P-glycoprotein (P-gp), expressed at high levels in brain endothelial cells, has several drug substrates. Consequently, siRNA mediated silencing of the P-gp gene is one possible strategy how to improve the delivery of drugs to the brain. Herein, we investigated the potential of siRNA-chitosan nanoparticles in silencing P-gp in a BBB model. We show that the transfection of rat brain endothelial cells mediated effective knockdown of P-gp with subsequent decrease in P-gp substrate efflux. This resulted in increased cellular delivery and efficacy of the model drug doxorubicin.

Human metapneumovirus (hMPV) causes severe airway infection in children that may be caused by an unfavorable immune response. The nature of the innate immune response to hMPV in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. We examined nasopharynx aspirates and blood samples from hMPV-infected children without detectable co-infections. The expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mRNA quantification while blood plasma proteins were determined by a multiplex immunoassay. Several genes were significantly up-regulated at mRNA and protein level in the hMPV infected children. Most apparent was the expression of the chemokine IP-10, the pro-inflammatory cytokine IL-18 in addition to the interferon inducible gene ISG54. Interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes IL-1β and NLRP3. Overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hMPV mediated lung disease and the antiviral response in children with severe infection. Our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hMPV-infected children.