We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.
Special (lipid) delivery: The role of the ionizable lipid pKa in the in vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pKa value and silencing of the mouse FVII gene (FVII ED50) was found, with an optimal pKa range of 6.2–6.5 (see graph). The most potent cationic lipid from this study has ED50 levels around 0.005 mg kg−1 in mice and less than 0.03 mg kg−1 in non‐human primates.
Lipid nanoparticles (LNP) containing ionizable cationic
lipids
are the leading systems for enabling therapeutic applications of siRNA;
however, the structure of these systems has not been defined. Here
we examine the structure of LNP siRNA systems containing DLinKC2-DMA(an
ionizable cationic lipid), phospholipid, cholesterol and a polyethylene
glycol (PEG) lipid formed using a rapid microfluidic mixing process.
Techniques employed include cryo-transmission electron microscopy, 31P NMR, membrane fusion assays, density measurements, and
molecular modeling. The experimental results indicate that these LNP
siRNA systems have an interior lipid core containing siRNA duplexes
complexed to cationic lipid and that the interior core also contains
phospholipid and cholesterol. Consistent with experimental observations,
molecular modeling calculations indicate that the interior of LNP
siRNA systems exhibits a periodic structure of aqueous compartments,
where some compartments contain siRNA. It is concluded that LNP siRNA
systems formulated by rapid mixing of an ethanol solution of lipid
with an aqueous medium containing siRNA exhibit a nanostructured core.
The results give insight into the mechanism whereby LNP siRNA systems
are formed, providing an understanding of the high encapsulation efficiencies
that can be achieved and information on methods of constructing more
sophisticated LNP systems.
Limit size systems are defined as the smallest achievable aggregates compatible with the packing of the molecular constituents in a defined and energetically stable structure. Here we report the use of rapid microfluidic mixing for the controlled synthesis of two types of limit size lipid nanoparticle (LNP) systems, having either polar or nonpolar cores. Specifically, limit size LNP consisting of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC), cholesterol and the triglyceride triolein were synthesized by mixing a stream of ethanol containing dissolved lipid with an aqueous stream, employing a staggered herringbone micromixer. Millisecond mixing of aqueous and ethanol streams at high flow rate ratios (FRR) was used to rapidly increase the polarity of the medium, driving bottom-up synthesis of limit size LNP systems by spontaneous assembly. For POPC/triolein systems the limit size structures consisted of a hydrophobic core of triolein surrounded by a monolayer of POPC where the diameter could be rationally engineered over the range 20-80 nm by varying the POPC/triolein ratio. In the case of POPC and POPC/cholesterol (55/45; mol/mol) the limit size systems achieved were bilayer vesicles of approximately 20 and 40 nm diameter, respectively. We further show that doxorubicin, a representative weak base drug, can be efficiently loaded and retained in limit size POPC LNP, establishing potential utility as drug delivery systems. To our knowledge this is the first report of stable triglyceride emulsions in the 20-50 nm size range, and the first time vesicular systems in the 20-50 nm size range have been generated by a scalable manufacturing method. These results establish microfluidic mixing as a powerful and general approach to access novel LNP systems, with both polar or nonpolar core structures, in the sub-100 nm size range.
Der Einfluss des pKS‐Werts von ionisierbaren Lipiden auf den siRNA‐Transport durch Lipidnanopartikel in vivo wurde anhand zahlreicher Modifikationen der Lipid‐Kopfgruppen studiert. Dabei wurde ein Zusammenhang zwischen pKS‐Wert und Stummschaltung des Maus‐FVII‐Gens (FVII ED50) gefunden: pKS‐Werte von 6.2–6.5 erwiesen sich als optimal (siehe Diagramm). Für das wirksamste kationische Lipid betrug ED50 etwa 0.005 mg kg−1 in Mäusen und <0.03 mg kg−1 in Primaten (außer Menschen).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.