RESUMEN
Lipid nanoparticles (LNPs) are advanced core-shell particles for messenger RNA (mRNA) based therapies that are made of polyethylene glycol (PEG) lipid, distearoylphosphatidylcholine (DSPC), cationic ionizable lipid (CIL), cholesterol (chol), and mRNA. Yet the mechanism of pH-dependent response that is believed to cause endosomal release of LNPs is not well understood. Here, we show that eGFP (enhanced green fluorescent protein) protein expression in the mouse liver mediated by the ionizable lipids DLin-MC3-DMA (MC3), DLin-KC2-DMA (KC2), and DLinDMA (DD) ranks MC3 ≥ KC2 > DD despite similar delivery of mRNA per cell in all cell fractions isolated. We hypothesize that the three CIL-LNPs react differently to pH changes and hence study the structure of CIL/chol bulk phases in water. Using synchrotron X-ray scattering a sequence of ordered CIL/chol mesophases with lowering pH values are observed. These phases show isotropic inverse micellar, cubic Fd3m inverse micellar, inverse hexagonal [Formula: see text] and bicontinuous cubic Pn3m symmetry. If polyadenylic acid, as mRNA surrogate, is added to CIL/chol, excess lipid coexists with a condensed nucleic acid lipid [Formula: see text] phase. The next-neighbor distance in the excess phase shows a discontinuity at the Fd3m inverse micellar to inverse hexagonal [Formula: see text] transition occurring at pH 6 with distinctly larger spacing and hydration for DD vs. MC3 and KC2. In mRNA LNPs, DD showed larger internal spacing, as well as retarded onset and reduced level of DD-LNP-mediated eGFP expression in vitro compared to MC3 and KC2. Our data suggest that the pH-driven Fd3m-[Formula: see text] transition in bulk phases is a hallmark of CIL-specific differences in mRNA LNP efficacy.
Asunto(s)
Liposomas , Nanopartículas , Animales , Ratones , Nanopartículas/química , Micelas , Concentración de Iones de Hidrógeno , ARN Mensajero/genética , ARN Mensajero/química , ARN Interferente Pequeño/genéticaRESUMEN
BACKGROUND: Darbepoetin-alpha is applicable at longer injection intervals. Our early experience in children on peritoneal dialysis suggested increased injection pain compared to epoetin-beta, possibly due to technical differences or patient anxiety. METHODS: To verify a possible difference in the painfulness of the injected fluids per se, we performed a prospective, randomized, double-blind trial in 13 paediatric end-stage renal disease patients. They received three injections of equivalent doses of darbepoetin-alpha or epoetin-beta in 0.6 ml saline, using neutral syringes and 27G needles, at 4 week intervals. Pain perception was recorded immediately and after 30 min on a visual analogue scale (VAS, 0 = no pain, 10 = maximal pain; complemented by 5 faces for young children). RESULTS: The patients perceived more intense immediate injection pain with darbepoetin-alpha than with epoetin-beta (5.4 +/- 1 vs 2.3 +/- 0.6, P < 0.05). This was confirmed by the impression of the parents (5.3 +/- 1 vs 2.0 +/- 0.9, P < 0.05) and the nurses (4.4 +/- 1 vs 2.2 +/- 0.6, P < 0.05). General injection pain was inversely related to patient age (R = -0.77, P = 0.001). Six patients perceived no or a mild difference in injection pain, whereas 7 subjects reported a markedly higher pain score (> or =4 VAS points) with darbepoetin-alpha. After 30 min, the injection site was largely painless with both drugs. No significant local reactions occurred with either medication (0.3 +/- 0.1 vs 0.3 +/- 0.1 on a 5-score scale). CONCLUSIONS: Subcutaneous injections of darbepoetin-alpha are more painful than those of epoetin-beta in the majority of paediatric patients. The observed difference in painfulness is related to the nature of the injected compounds and may limit the subcutaneous applicability of darbepoetin-alpha in children.