2 research outputs found
Stabilization of Ostwald Ripening in Low Molecular Weight Amino Lipid Nanoparticles for Systemic Delivery of siRNA Therapeutics
Lipid
nanoparticles (LNPs) represent the most clinically advanced
technology for the systemic delivery of therapeutic siRNA in vivo.
Toward this end, a novel class of LNPs comprising low molecular weight
(MW) ionizable amino lipids having asymmetric architecture was recently
reported. LNPs of these amino lipids,
termed asymmetric LNPs, were shown to be highly efficacious and well
tolerated in vivo; advances were enabled by improved endosomal escape,
coupled with enhanced amino lipid metabolism and clearance. In this
work, we show that, in contrast to their desirable pharmacological
performance, asymmetric LNPs present a significant pharmaceutical
developability challenge, namely physical instability limiting extended
shelf life. Using orthogonal characterization methods, we identify
the mechanism of LNP instability as Ostwald ripening and establish
it to be driven predominantly by the asymmetric amino lipid component.
Through rational optimization of LNP physical and macromolecular properties,
we are able to significantly attenuate or entirely eliminate the Ostwald
ripening instability. Modulation of LNP size, for example, effectively
halts particle growth. Similarly, optimization of LNP macromolecular
packing through deliberate selection of structurally matched colipids
significantly diminishes the rate of ripening. This later experimental
observation is substantiated by molecular dynamics simulations of
LNP self-assembly, which establish a quantitative dependence of LNP
macromolecular order on colipid structure. In totality, the experimental
and molecular dynamics outcomes of this work support the rational
design of LNP physical and chemical properties leading to effective
Ostwald ripening stabilization and enable the advance of asymmetric
LNPs as a clinic-ready platform for siRNA therapeutics
Assessing the Heterogeneity Level in Lipid Nanoparticles for siRNA Delivery: Size-Based Separation, Compositional Heterogeneity, and Impact on Bioperformance
A primary consideration when developing lipid nanoparticle
(LNP)
based small interfering RNA (siRNA) therapeutics is formulation polydispersity
or heterogeneity. The level of heterogeneity of physicochemical properties
within a pharmaceutical batch could greatly affect the bioperformance,
quality, and ability of a manufacturer to consistently control and
reproduce the formulations. This article studied the heterogeneity
in the size, composition, and <i>in vitro</i> performance
of siRNA containing LNPs, by conducting preparative scale fractionation
using a sephacryl S-1000 based size-exclusion chromatography (SEC)
method. Eight LNPs with size in the range of 60–190 nm were
first evaluated by the SEC method for size polydispersity characterization,
and it was found that LNPs in the range of 60–150 nm could
be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties
were fractionated, and fractions were studied in-depth for potential
presence of polydispersity in size, composition, and <i>in vitro</i> silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse
following results of a semipreparative SEC fractionation that showed
similar size, chemical composition, <i>in vitro</i> silencing
activity, and cytotoxicity across the fractions. Therefore, LNP A
represents a relatively homogeneous formulation and offers less of
a challenge in its pharmaceutical development. In contrast, LNP B
fractions were shown to be significantly more polydisperse in size
distribution. Interestingly, LNP B SEC fractions also exhibited profound
compositional variations (e.g., 5 fold difference in N/P ratio and
3 fold difference in lipid composition) along with up to 40 fold differences
in the <i>in vitro</i> silencing activity. The impact of
LNP size and formulation composition on <i>in vitro</i> performance
is also discussed. The present results demonstrate the complexity
and potential for presence of heterogeneity in LNP-based siRNA drug
products. This underscores the need for tools that yield a detailed
characterization of LNP formulations. This capability in tandem with
the pursuit of improved formulation and process design can lead to
more facile development of LNP-based siRNA pharmaceuticals of higher
quality