Leishmaniasis is a neglected tropical disease that mainly affects socially vulnerable populations. It is caused by various Leishmania species, and manifests primarily as cutaneous (CL) or visceral leishmaniasis (VL). AmBisome, a commercial formulation of amphotericin B (AmB) with conventional liposomes, is the most effective for VL treatment, but its efficacy is limited in some CL cases and immunocompromised patients. This work evaluated a new PEGylated liposomal formulation (LAmB-RA) coencapsulating AmB and retinoic acid (RA), as an immunomodulator, for CL treatment. LAmB-RA displayed a mean size of 125 nm, a polydispersity index <0.2, and high encapsulation efficiencies for AmB (97.1%) and RA (94.7%). Although SAXS analysis indicates that RA did not induce major structural rearrangements in the liposomal bilayer, comparison of circular dichroism spectra between LAmB-RA and the liposomal AmB formulation without RA (LAmB) revealed slightly different aggregated states of AmB. In addition, LAmB-RA showed a significantly reduced hemolytic activity, compared to LAmB. Pharmacokinetic analysis of the PEGylated formulations showed higher C max and prolonged plasma exposure of AmB compared to AmBisome. In Leishmania major-infected mice, LAmB-RA significantly reduced lesion size and parasite burden (99%) versus untreated control. In the Leishmania amazonensis model, it markedly inhibited lesion progression and was the only treatment to significantly reduce parasite load in the spleen (by 60%). Additionally, LAmB-RA promoted an increase in the IFN-γ/IL-10 ratio in antigen-stimulated splenocytes compared to LAmB, indicating a Th1-skewed immune response. These results support LAmB-RA as a promising therapeutic strategy for CL, combining prolonged circulation and enhanced efficacy.

Macrophages play a vital role in controlling tissue homeostasis, yet their dysregulation can drive the pathogenesis of various diseases. In this context, P2Y2, a purinergic receptor that binds ATP, could be involved in several biological mechanisms in inflammatory, infectious, or cancer diseases. Lipid nanoparticles (LNPs) varying by the difference of lipid moiety of the polyethyleneglycol-lipid (PEG-lipid) conjugate entering their composition were prepared by microfluidic mixing technology and loaded with a siRNA targeting the P2y2 mRNA. Three PEG-lipids with varying saturated alkyl chain lengths - DMG-PEG (C14), DPPE-PEG (C16), and DSPE-PEG (C18) - were evaluated for formulation optimization. The results showed that all siRNA-LNPs were efficiently taken up by unpolarized (M0) and M2-polarized bone marrow-derived macrophages (BMDMs). Despite comparable cellular uptake across formulations, their performance in gene silencing differed significantly. While (DMG-PEG)-LNPs demonstrated the highest efficacy in both cellular models, (DSPE-PEG)-LNPs remained ineffective in knocking down the target gene. Our findings further revealed that gene silencing is more efficient in M2 BMDMs than in M0 BMDMs, emphasizing the influence of macrophage polarization. Additionally, the PEG-lipid type notably influenced the intracellular distribution patterns of lipids and siRNA, with DPPE-PEG and DSPE-PEG formulations leading to higher siRNA compartmentalization. In contrast, the DMG-PEG formulation enabled more effective siRNA delivery to the cytoplasm. This research provides valuable insights into the impact of different PEG-lipid anchors on the performance of siRNA-LNPs targeting macrophages in vitro, paving the way for developing new macrophage-based immunotherapies.

This study investigates the potential of pulmonary delivery of siRNA as an emergency therapy for acute lung injury (ALI). To obtain a quick anti-inflammatory response, TNF-α, a critical pro-inflammatory cytokine, is knocked down for its early involvement in inflammatory responses. Therefore, TNF-α siRNA-lipid nanoparticles (LNPs) are designed and characterized for cellular uptake in lipopolysaccharide (LPS)-activated RAW264.7 murine macrophages, primary alveolar macrophages, and neutrophils from a murine ALI model. Intracellular trafficking and siRNA cytoplasmic release are evaluated in untreated and LPS-activated RAW264.7 cells. LPS-activated cells exhibit a fast and strong uptake of LNPs, including in primary cells. In RAW264.7 cells, significant endosomal escape and siRNA cytoplasmic release are observed after 16 h. In vitro efficacy studies reveal consistent TNF-α inhibition across pre-, co-, and post-incubation protocols, confirming the versatility of siRNA-LNPs in preventive or curative conditions. After intratracheal administration of TNF-α siRNA-LNPs in a murine ALI model, the distribution of LNPs demonstrates an accumulation in immune cells, including macrophages and neutrophils, reducing TNF-α and IL-6 levels, indicating a rapid anti-inflammatory effect. This work underscores the efficacy of TNF-α siRNA-LNPs in treating lung inflammatory diseases like ALI and highlights the importance of optimizing LNP distribution and delivery timing to enhance therapeutic outcomes.

The beta-adrenergic system is a potent stimulus for enhancing cardiac output that may become deleterious when energy metabolism is compromised as in heart failure. We thus examined whether the AMP-activated protein kinase (AMPK) that is activated in response to energy depletion may control the beta-adrenergic pathway. We studied the cardiac response to beta-adrenergic stimulation of AMPKα2-/- mice or to pharmacological AMPK activation on contractile function, calcium current, cAMP content and expression of adenylyl cyclase 5 (AC5), a rate limiting step of the beta-adrenergic pathway. In AMPKα2-/- mice the expression of AC5 (+50%), the dose response curve of left ventricular developed pressure to isoprenaline (p<0.001) or the response to forskolin, an activator of AC (+25%), were significantly increased compared to WT heart. Similarly, the response of L-type calcium current to 3-isobutyl-l-methylxanthine (IBMX), a phosphodiesterase inhibitor was significantly higher in KO (+98%, p<0.01) than WT (+57%) isolated cardiomyocytes. Conversely, pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) induced a 45% decrease in AC5 expression (p<0.001) and a 40% decrease of cAMP content (P<0.001) as measured by fluorescence resonance energy transfer (FRET) compared to unstimulated rat cardiomyocytes. Finally, in experimental pressure overload-induced cardiac dysfunction, AMPK activation was associated with a decreased expression of AC5 that was blunted in AMPKα2-/- mice. The results show that AMPK activation down-regulates AC5 expression and blunts the beta-adrenergic cascade. This crosstalk between AMPK and beta-adrenergic pathways may participate in a compensatory energy sparing mechanism in dysfunctional myocardium.

We investigated the mechanisms underlying the effects of the antidepressant fluoxetine on behavior and adult hippocampal neurogenesis (AHN). After confirming our earlier report that the signaling molecule β-arrestin-2 (β-Arr2) is required for the antidepressant-like effects of fluoxetine, we found that the effects of fluoxetine on proliferation of neural progenitors and survival of adult-born granule cells are absent in the β-Arr2 knockout (KO) mice. To our surprise, fluoxetine induced a dramatic upregulation of the number of doublecortin (DCX)-expressing cells in the β-Arr2 KO mice, indicating that this marker can be increased even though AHN is not. We discovered two other conditions where a complex relationship occurs between the number of DCX-expressing cells compared to levels of AHN: a chronic antidepressant model where DCX is upregulated and an inflammation model where DCX is downregulated. We concluded that assessing the number of DCX-expressing cells alone to quantify levels of AHN can be complex and that caution should be applied when label retention techniques are unavailable.