Rapid and scale-independent microfluidic manufacture of liposomes entrapping protein incorporating in-line purification and at-line size monitoring

Within this paper we present work that has the ability to de-risk the translation of liposomes from bench to the clinic. We have used microfluidics for the rapid and scale-independent manufacture of liposomes and have incorporated in-line purification and at-line monitoring of particle size. Using this process, we have manufactured a range of neutral and anionic liposomes incorporating protein. Factors investigated include the microfluidics operating parameters (flow rate ratio (FRR) and total flow rate (TFR)) and the liposome formulation. From these studies, we demonstrate that FRR is a key factor influencing liposome size, protein loading and release profiles. The liposome formulations produced by microfluidics offer high protein loading (20–35%) compared to production by sonication or extrusion (<5%). This high loading achieved by microfluidics results from the manufacturing process and is independent of lipid selection and concentration across the range tested. Using in-line purification and at-line size monitoring, we outline the normal operating range for effective production of size controlled (60–100 nm), homogenous (PDI <0.2) high load liposomes. This easy microfluidic process provides a translational manufacturing pathway for liposomes in a wide-range of applications

Endotracheal Tube Cuff Pressures in Adult Patients Undergoing General Anaesthesia in Two Johannesburg Academic Hospitals

Background: Endotracheal tube (ETT) cuff pressure commonly exceeds the recommended range of 20–30 cm H₂O during anaesthesia. A set volume of air will not deliver the same cuff pressure in each patient and the pressure exerted by the ETT cuff can lead to complications, with either over- or under-inflated cuffs. These can include a sore throat and cough, aspiration, volume loss during positive pressure ventilation, nerve palsies, tracheomalacia and tracheal stenosis. No objective means of ETT cuff pressure monitoring is available in the operating theatres of Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and Chris Hani Baragwanath Academic Hospital (CHBAH). The ETT cuff pressure of patients undergoing general anaesthesia is therefore unknown.Method: ETT cuff pressure of 96 adult patients undergoing general anaesthesia without nitrous oxide at CMJAH and CHBAH was measured by one researcher. A RUSCH Endotest™ manometer was used to measure ETT cuff pressure in size 7.0 – 8.5 mm ETTs. The cuff inflation technique that was used by the anaesthetist was also documented.Results: The mean ETT cuff pressure recorded was 47.5 cm H₂O (range 10–120 cm H₂O). ETT cuff pressures exceeded 30 cm H₂O in 64.58% of patients. Only 18.75% of patients had ETT cuff pressures within the recommended range of 20–30 cm H₂O. There was no statistically significant difference between the ETT cuff pressures measured at the two hospitals. Minimal occlusive volume was the most frequent technique used to inflate the ETT cuff (37.5%); this was followed by inflating the ETT cuff with a predetermined volume of air in 31.25% of cases and palpation of the pilot balloon (27.08%). There was no statistically significant difference between the ETT cuff pressure measured and the inflation technique used by the anaesthetist.Conclusion: ETT cuff pressures of the majority of patients undergoing general anaesthesia at two academic hospitals were higher than the recommended range. ETT cuff pressure should routinely be measured using a manometer.Keywords: Adults, Endotracheal Tube Cuff Pressures, General Anaesthesia, Manomete

A phosphorylated light-chain component of myosin from skeletal muscle

1. The low-molecular-weight components of myosin from rabbit skeletal muscle migrated as four bands on polyacrylamide-gel electrophoresis in 8m-urea but only as three in systems containing sodium dodecyl sulphate. The two bands of intermediate mobility in 8m-urea (Ml2 and Ml3) had identical mobilities in sodium dodecyl sulphate. 2. The isolation of pure samples of all four low-molecular-weight components by DEAE-Sephadex chromatography is described. 3. The amino acid compositions of components Ml2 and Ml3 were identical. Further analyses showed the presence of 1 mol of phosphate/18500g of component Ml2 and less than 10% of this amount in component Ml3. Neither light component contained ribose. 4. Alkaline phosphatase from Escherichia coli converted component Ml2 into Ml3. Incubation with crude preparations of phosphorylase b kinase or protein kinase in the presence of ATP converted component Ml3 into Ml2. 5. Phosphorylation of component Ml3 with the kinases isolated from skeletal muscle and [γ-32P]ATP gave incorporation of 32P only into component Ml2 whether whole myosin or separated low-molecular-weight components were used. 6. High-voltage electrophoresis at pH6.5 and pH1.8 of a chymotryptic digest of 32P-labelled component Ml2 yielded one major radioactive peptide containing serine phosphate. 7. The amino acid sequence of this peptide was shown to be: Arg-Ala-Ala-Ala-Glu-Gly-Gly-(Ser,Ser(P))-Asn-Val-Phe. This sequence shows no obvious similarity to the site phosphorylated in the conversion of phosphorylase b into phosphorylase a by phosphorylase b kinase. 8. Evidence suggests that in vivo all the 18500-molecular-weight light chain is in the phosphorylated form. The extent of dephosphorylation that occurred during myosin extraction depended on the conditions employed.</jats:p

Silk nanoparticle manufacture in semi-batch format

Silk nanoparticles have demonstrated utility across a range of biomedical applications, especially as drug delivery vehicles. Their fabrication by bottom-up methods such as nanoprecipitation, rather than top-down manufacture, can improve critical nanoparticle quality attributes. Here, we establish a simple semi-batch method using drop-by-drop nanoprecipitation at the lab scale that reduces special-cause variation and improves mixing efficiency. The stirring rate was an important parameter affecting nanoparticle size and yield (400 < 200 < 0 rpm), while the initial dropping height (5.5 vs 7.5 cm) directly affected nanoparticle yield. Varying the nanoparticle standing time in the mother liquor between 0 and 24 h did not significantly affect nanoparticle physicochemical properties, indicating that steric and charge stabilizations result in high-energy barriers for nanoparticle growth. Manufacture across all tested formulations achieved nanoparticles between 104 and 134 nm in size with high β-sheet content, spherical morphology, and stability in aqueous media for over 1 month at 4 °C. This semi-automated drop-by-drop, semi-batch silk desolvation offers an accessible, higher-throughput platform for standardization of parameters that are difficult to control using manual methodologies

Volumetric scalability of microfluidic and semi-batch silk nanoprecipitation methods

Silk fibroin nanoprecipitation by organic desolvation in semi-batch and microfluidic formats provides promising bottom-up routes for manufacturing narrow polydispersity, spherical silk nanoparticles. The translation of silk nanoparticle production to pilot, clinical, and industrial scales can be aided through insight into the property drifts incited by nanoprecipitation scale-up and the identification of critical process parameters to maintain throughout scaling. Here, we report the reproducibility of silk nanoprecipitation on volumetric scale-up in low-shear, semi-batch systems and estimate the reproducibility of chip parallelization for volumetric scale-up in a high shear, staggered herringbone micromixer. We showed that silk precursor feeds processed in an unstirred semi-batch system (mixing time > 120 s) displayed significant changes in the nanoparticle physicochemical and crystalline properties following a 12-fold increase in volumetric scale between 1.8 and 21.9 mL while the physicochemical properties stayed constant following a further 6-fold increase in scale to 138 mL. The nanoparticle physicochemical properties showed greater reproducibility after a 6-fold volumetric scale-up when using lower mixing times of greater similarity (8.4 s and 29.4 s) with active stirring at 400 rpm, indicating that the bulk mixing time and average shear rate should be maintained during volumetric scale-up. Conversely, microfluidic manufacture showed high between-batch repeatability and between-chip reproducibility across four participants and microfluidic chips, thereby strengthening chip parallelization as a production strategy for silk nanoparticles at pilot, clinical, and industrial scales

Interbilayer-crosslinked multilamellar vesicles as synthetic vaccines for potent humoral and cellular immune responses

available in PMC 2011 September 1Vaccines based on recombinant proteins avoid the toxicity and antivector immunity associated with live vaccine (for example, viral) vectors, but their immunogenicity is poor, particularly for CD8+ T-cell responses. Synthetic particles carrying antigens and adjuvant molecules have been developed to enhance subunit vaccines, but in general these materials have failed to elicit CD8+ T-cell responses comparable to those for live vectors in preclinical animal models. Here, we describe interbilayer-crosslinked multilamellar vesicles formed by crosslinking headgroups of adjacent lipid bilayers within multilamellar vesicles. Interbilayer-crosslinked vesicles stably entrapped protein antigens in the vesicle core and lipid-based immunostimulatory molecules in the vesicle walls under extracellular conditions, but exhibited rapid release in the presence of endolysosomal lipases. We found that these antigen/adjuvant-carrying vesicles form an extremely potent whole-protein vaccine, eliciting endogenous T-cell and antibody responses comparable to those for the strongest vaccine vectors. These materials should enable a range of subunit vaccines and provide new possibilities for therapeutic protein delivery.Ragon Institute of MGH, MIT and HarvardBill & Melinda Gates FoundationUnited States. Dept. of Defense (contract W911NF-07-D-0004)National Institutes of Health (U.S.) (P41RR002250)National Institutes of Health (U.S.) (RC2GM092599

Correction : Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly

Correction for ‘Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly’ by Saphia A. L. Matthew et al., RSC Adv., 2022, 12, 7357–7373. https://doi.org/10.2039/D1RA07764C. The authors regret that there were sub-figure placement errors present in Fig. 4 and 5 of the main article. The sub-figure placement error in Fig. 4 was carried into Fig. S3, which shows additional statistical significances. The corrected figures are shown below

Genetic differentiation and admixture between sibling allopolyploids in the Dactylorhiza majalis complex

Allopolyploidization often happens recurrently, but the evolutionary significance of its iterative nature is not yet fully understood. Of particular interest are the gene flow dynamics and the mechanisms that allow young sibling polyploids to remain distinct while sharing the same ploidy, heritage and overlapping distribution areas. By using eight highly variable nuclear microsatellites, newly reported here, we investigate the patterns of divergence and gene flow between 386 polyploid and 42 diploid individuals, representing the sibling allopolyploids Dactylorhiza majalis s.s. and D. traunsteineri s.l. and their parents at localities across Europe. We make use in our inference of the distinct distribution ranges of the polyploids, including areas in which they are sympatric (that is, the Alps) or allopatric (for example, Pyrenees with D. majalis only and Britain with D. traunsteineri only). Our results show a phylogeographic signal, but no clear genetic differentiation between the allopolyploids, despite the visible phenotypic divergence between them. The results indicate that gene flow between sibling Dactylorhiza allopolyploids is frequent in sympatry, with potential implications for the genetic patterns across their entire distribution range. Limited interploidal introgression is also evidenced, in particular between D. incarnata and D. traunsteineri. Altogether the allopolyploid genomes appear to be porous for introgression from related diploids and polyploids. We conclude that the observed phenotypic divergence between D. majalis and D. traunsteineri is maintained by strong divergent selection on specific genomic areas with strong penetrance, but which are short enough to remain undetected by genotyping dispersed neutral markers.UE FWF; P22260UE: Y66

Mixing and flow-induced nanoprecipitation for morphology control of silk fibroin self-assembly

Tuning silk fibroin nanoparticle morphology using nanoprecipitation for bottom-up manufacture is an unexplored field that has the potential to improve particle performance characteristics. The aim of this work was to use both semi-batch bulk mixing and micro-mixing to modulate silk nanoparticle morphology by controlling the supersaturation and shear rate during nanoprecipitation. At flow rates where the shear rate was below the critical shear rate for silk, increasing the concentration of silk in both bulk and micro-mixing processes resulted in particle populations of increased sphericity, lower size, and lower polydispersity index. At high flow rates, where the critical shear rate was exceeded, the increased supersaturation with increasing concentration was counteracted by increased rates of shear-induced assembly. The morphology could be tuned from rod-like to spherical assemblies by increasing supersaturation of the high-shear micro-mixing process, thereby supporting a role for fast mixing in the production of narrow-polydispersity silk nanoparticles. This work provides new insight into the effects of shear during nanoprecipitation and provides a framework for scalable manufacture of spherical and rod-like silk nanoparticles

Analysis of surfactant-associated bacteria in the sea surface microlayer using deoxyribonucleic acid sequencing and synthetic aperture radar

The sea surface microlayer (SML) is the upper 1 mm of the ocean, where Earth’s biogeochemical processes occur between the ocean and atmosphere. It is physicochemically distinct from the water below and highly variable in space and time due to changing physical conditions. Some microorganisms influence the composition of the SML by producing surfactants for biological functions that accumulate on the surface, decrease surface tension, and create slicks. Slicks can be visible to the eye and in synthetic aperture radar (SAR) satellite imagery. This study focuses on surfactant-associated bacteria in the near-surface layer and their role in slick formation where oil is present