The absence of any significant toxicity associated with EVs in vitro and in vivo support the prospective use of EVs for therapeutic applications and for drug delivery.

We have investigated the relationship between O2 delivery (DO2) and O2 consumption (VO2) in hindlimb muscle of anaesthetised rats during progressive systemic hypoxia. Since muscle vasodilatation that occurs during hypoxia is nitric oxide (NO) dependent, we examined the effects of the NO synthase (NOS) inhibitor nitro‐l‐arginine methyl ester (l‐NAME). In control rats (n= 8), femoral vascular conductance (FVC) increased at each level of hypoxia. Hindlimb DO2 decreased with the severity of hypoxia, but muscle VO2 was maintained until the critical DO2 value (DO2,crit) was reached at 0.64 ± 0.06 ml O2 min−1 kg−1; below this VO2 declined linearly with DO2. This is a novel finding for the rat but is comparable to the biphasic relationship seen in the dog. In another group of rats (n= 6), l‐NAME caused hindlimb vasoconstriction and attenuated the hypoxia‐evoked increases in FVC. DO2 was so low after l‐NAME administration that VO2 was dependent on DO2 at all levels of hypoxia. In a further group (n= 8), femoral blood flow and DO2 were restored after l‐NAME by infusion of the NO donor sodium nitroprusside (20 μg kg−1 min−1. Thereafter, hypoxia‐evoked increases in FVC were fully restored. Nevertheless, DO2,crit was increased relative to control (0.96 ± 0.07 ml O2 min−1 kg−1, P < 0.01). As NOS inhibition limited the ability of muscle to maintain VO2 during hypoxia, we propose that hypoxia‐induced dilatation of terminal arterioles, which improves tissue O2 distribution, is mediated by NO. However, since the hypoxia‐evoked increase in FVC was blocked by l‐NAME but restored by the NO donor, we propose that the dilatation of proximal arterioles is dependent on tonic levels of NO, rather than mediated by NO.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Hypoxia‐evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O2 sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O2 for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O2 tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l‐NAME, but restored when baseline FVC is restored by infusion of NO donor. This ‘restored’ hypoxic response, like the control hypoxic response, is inhibited by the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l‐NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l‐NAME with 8‐bromo‐cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l‐NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8‐cyclopentyl‐1,3‐dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; l‐NAME attenuated only the former response. We propose that in vivo, shear‐released NO increases the apparent Km of endothelial cytochrome oxidase for O2, allowing the endothelium to act as an O2 sensor, releasing adenosine in response to moderate falls in O2.

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