Analytical Characterization of Heterogeneities in mRNA-Lipid Nanoparticles Using Sucrose Density Gradient Ultracentrifugation.

Rational design and robust formulation processes are critical for optimal delivery of mRNA by lipid nanoparticles (LNPs). Varying degrees of heterogeneity in mRNA-LNPs can affect their biophysical and functional properties. Given the profound complexity of mRNA-LNPs, it is critical to develop comprehensive and orthogonal analytical techniques for a better understanding of these formulations. To this end, we developed a robust ultracentrifugation method for density-based separation of subpopulations of mRNA-LNPs. Four LNP formulations encapsulating human erythropoietin (hEPO) with varying functionalities were synthesized using two ionizable lipids, A and B, and two helper lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), along with cholesterol and DMG-PEG-2K. Upon ultracentrifugation on a sucrose gradient, a distinct pattern of "fractions" was observed across the gradient, from the less dense topmost fraction to the increasingly denser bottom fractions, which were harvested for comprehensive analyses. Parent LNPs, A-DOPE and B-DOPE, were resolved into three density-based fractions, each differing significantly in the hEPO expression following intravenous and intramuscular routes of administration. Parent B-DEPE LNPs resolved into two density-based fractions, with most of the payload and lipid content being attributed to the topmost fraction compared to the lower one, indicating some degree of heterogeneity, while parent A-DEPE LNPs showed remarkable homogeneity, as indicated by comparable in vivo potency, lipid numbers, and particle count among the three density-based fractions. This study is the first to demonstrate the application of density gradient-based ultracentrifugation (DGC) for a head-to-head comparison of heterogeneity as a function of biological performance and biophysical characteristics of parent mRNA-LNPs and their subpopulations.

The experiences of nurses who insert central venous access devices.

There has been much role expansion in nursing in the last two decades, with advanced nurses now performing minimally invasive surgery such as central venous access device insertion. However, there is a lack of research exploring the perceptions of nurses performing these procedures. This study explored the lived experiences of nurses who perform minimally invasive surgery, namely central venous access device insertion. Three key themes emerged from this analysis:stress associated with the unpredictable nature of the procedure,coping with responsibility and a patient-focused approach. Although the practitioners experience a degree of stress in the role, they also experience job satisfaction and feel that their roles have a positive impact on the patients in their care. The study findings provide information to managers and nurses performing these roles, and suggest how stress and burnout can be prevented.