Platelet distribution in rabbits following infusion of liposomes

This investigation determined the organ distribution of liposomes containing egg phosphatidylcholine and cholesterol with egg phosphatidylglycerol (PG liposomes) or without (PC liposomes) and the effect of each liposome on platelet distribution in rabbits. Eight minutes after 51chromium-labelled platelets were given intravenously, either saline (n = 7), iodinated PG liposomes (n = 5) or iodinated PC liposomes (n = 5) were infused. Two minutes later the organ distribution of 51Cr-platelets and 125I-liposomes were compared. The PG liposomes produced a 41 ± 5% reduction in circulating platelet counts while PC liposomes did not. The PG liposomes decreased circulating 51Cr-platelets by a factor of 2 and increased platelet recoveries in the liver and lungs. The increased platelet recovery in the liver was associated with a greater PG liposome recovery. When animals receiving PG liposomes were studied over 60 minutes, both the labelled and unlabelled platelet counts returned to control values by 30 minutes and the 51Cr-platelet distribution between organs was similar to control values. These data indicate that platelets and PG liposomes initially sequester together and that this platelet-liposome interaction is specific for PG liposomes. However, the platelet sequestration is transient and by 60 minutes the platelets were released and circulating

Anionic lipid nanoparticles preferentially deliver mRNA to the hepatic reticuloendothelial system

Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved.Supramolecular & Biomaterials Chemistr

Lipid nanoparticle-mediated hit-and-run approaches yield efficient and safe in situ gene editing in human skin

Despite exciting advances in gene editing, the efficient delivery of genetic tools to extrahepatic tissues remains challenging. This holds particularly true for the skin, which poses a highly restrictive delivery barrier. In this study, we ran a head-to-head comparison between Cas9 mRNA or ribonucleoprotein (RNP)-loaded lipid nanoparticles (LNPs) to deliver gene editing tools into epidermal layers of human skin, aiming for in situ gene editing. We observed distinct LNP composition and cell-specific effects such as an extended presence of RNP in slow-cycling epithelial cells for up to 72 h. While obtaining similar gene editing rates using Cas9 RNP and mRNA with MC3-based LNPs (10-16%), mRNA-loaded LNPs proved to be more cytotoxic. Interestingly, ionizable lipids with a pK(a) ∼ 7.1 yielded superior gene editing rates (55%-72%) in two-dimensional (2D) epithelial cells while no single guide RNA-dependent off-target effects were detectable. Unexpectedly, these high 2D editing efficacies did not translate to actual skin tissue where overall gene editing rates between 5%-12% were achieved after a single application and irrespective of the LNP composition. Finally, we successfully base-corrected a disease-causing mutation with an efficacy of ∼5% in autosomal recessive congenital ichthyosis patient cells, showcasing the potential of this strategy for the treatment of monogenic skin diseases. Taken together, this study demonstrates the feasibility of an in situ correction of disease-causing mutations in the skin that could provide effective treatment and potentially even a cure for rare, monogenic, and common skin diseases

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection

Modulation of membrane structure by Ca2+ and dibucaine as detected by 31P NMR

The polymorphic phase behaviour of model membrane systems consisting of 20 mol% bovine brain phosphatidylserine and 80 mol% egg yolk phosphatidylethanolamine has been examined employing 31P NMR techniques. It is shown that the addition of Ca2+ to such systems can trigger isothermal bilayer to hexagonal (HII) phase transitions, and that such effects can be reversed by the subsequent incorporation of the local anaesthetic dibucaine. These results are discussed in terms of a recent model for membrane fusion (Cullis, P.R. and Hope, M.J. (1978) Nature 271, 672–674) and mechanisms of anaesthesia

Acyl chain orientational order in large unilamellar vesicles: comparison with multilamellar liposomes: a 2H and 31P nuclear magnetic resonance study

Large unilamellar vesicles (LUVs) composed of 1-[2H31]palmitoyl-2-oleoyl phosphatidylcholine (POPC-d31), with diameters of approximately 117 +/- 31 and 180 +/- 44 nm, were prepared by extrusion through polycarbonate filters with pore sizes of 0.1 and 0.2 microns, respectively. The 2H nuclear magnetic resonance (NMR) spectra obtained at 21 degrees C contain two components: a broad component (approximately 17 kHz linewidth) corresponding to the methylene groups and a narrower component originating from the methyl groups. Spectra with increasing powder pattern characteristics were obtained by reducing the rate of phospholipid reorientations by addition of glycerol (to increase the solvent viscosity) and by lowering the temperature. Full powder spectra, characteristic of liquid-crystalline bilayers, were obtained for both LUV samples at 0 degrees C in the presence of 50 wt% glycerol. Individual quadrupolar splittings were not resolved in these spectra, due to broader linewidths in the LUVs, which have significantly shorter values for spin-spin relaxation time T2 measured from the decay of the quadrupolar echo (90 microseconds) than the multilmellar vesicles (MLVs; 540 microseconds). Smoothed order parameter profiles (OPPs) were obtained for these samples by integration of the dePaked spectra. The OPPs were very similar to the OPP of POPC-d31 MLVs in 50 wt% glycerol at the same temperature, indicating that orientational order in MLVs and LUVs with a diameter of > or = 100 nm is essentially the same. The presence of 80 wt% glycerol was found to have a disordering effect on the vesicles