Lowering the siRNA Delivery Barrier: Alginate Scaffolds and Immune Stimulation - Nanotechnology for the Delivery of Therapeutic Nucleic Acids - page 198

delivery due to their increased circulatory half-life compared to non-
PEGylated liposomes [36]. A modified SNALP method was utilised by
Morrissey and colleagues in the earliest demonstration of a systemic
anti-viral effect by RNAi using a clinically feasible method. This study
showed reduced replication of a hepatitis B virus in mice via IV low
pressure injection of chemically modified siRNAs incorporated
into SNALPs [37]. SNALPs were further utilised in a study of RNAi-
mediated gene silencing in non-human primates [38] and in a proof
of concept study by the same research group of post-exposure RNAi
therapy of non-human primates against a lethal Ebola virus (EBOV)
challenge [39]. Overall, the SNALP system for systemic intravenous
delivery is a promising new technique for RNAi therapies.

9.3 HFDM: A Novel Method for Formulating

Stable siRNA-Loaded Lipid Particles for
in vivo Use

One of the issues with liposomes for systemic delivery is the
need to complex them with PEG. Addition of PEG is important to
ensure liposome accumulation in tissues following intravenous
administration. This is due to PEG's ability to create a steric barrier
on the surface of liposomes, resulting in decreased interactions with
serum proteins. These so-called “stealth liposomes”, of which SNALPs
are the classic example, are the most widely tested liposomes in
clinical trials to date. However, the presence of PEG in the formulation
can interfere with the binding efficiency between cationic lipids
and anionic siRNA, leading to inefficient entrapment of siRNA in
liposomes [40, 41]. More complex procedures have therefore been
required to prepare these PEGylated systems compared to those
used for conventional lipoplexes. These include the incorporation
of repeated freeze-thawing cycles in the formulation procedure [42]
or the use of techniques which involve ethanol/detergent dialysis
[35, 43], reverse-phase evaporation [44], or post insertion of PEG
onto pre-formed lipoplexes [15]. These procedures are labour
intensive, time consuming and result in aqueous end products
which may not be suitable for long term storage. To address this, we
developed a rapid, scalable, and easy method called the hydration-of-
freeze-dried-matrix (HFDM) method to produce stealth liposomes
for in vivo use (Fig. 9.1).