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. 2012 Aug 28;6(8):6922-9.
doi: 10.1021/nn301922x. Epub 2012 Jul 6.

In vitro-in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery

Kathryn A Whitehead  1 , Jonathan Matthews, Philip H Chang, Farnaz Niroui, J Robert Dorkin, Mariano Severgnini, Daniel G Anderson
Affiliations

In vitro-in vivo translation of lipid nanoparticles for hepatocellular siRNA delivery

Kathryn A Whitehead et al. ACS Nano. .

Abstract

A significant challenge in the development of clinically viable siRNA delivery systems is a lack of in vitro-in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time associated with generating in vitro-in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examination of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple experimental systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro experiments was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clinically viable siRNA delivery materials and have the potential to reduce experimental costs while improving the translation of materials into animals.

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Figures

Figure 1
Figure 1
Lipidoid formulation procedure can significantly impact in vitro – in vivo correlations. a) In vivo – in vitro data points can be classified as either correlating (dashed line), false positives (blue area), or false negatives (red area). b) In vitro data generated via lipidoid complexes did not correlate with in vivo Factor VII silencing data. c) In vitro data generated via lipidoid nanoparticles (LNPs) was more predictive, with a reduction in the quantity and severity of false negatives. d) The transfection ability of LNPs when formulated as complexes or nanoparticles varied significantly. Error bars in each panel represent standard deviation (n = 3 and 4 for FVII and luciferase activity data, respectively).
Figure 2
Figure 2
The 100-lipidoids tested in this study were structurally diverse. Lipidoids were synthesized via the conjugate addition of alkyl-amines to alkyl-acrylamides, alkyl-acrylates, or alkyl-epoxides where X = 9 and 12, Y = 10-14 and Z = 12.
Figure 3
Figure 3
The use of an immortalized hepatocellular line, Hepa1-6, for in vitro transfection experiments did not improve the trend observed between in vivo and in vitro data as compared to the data shown for HeLa cells in Figure 1c. Error bars represent standard deviation (n = 3 and 4 for Y and X error bars, respectively).
Figure 4
Figure 4
Primary hepatocytes, which are morphologically most similar to mouse hepatocytes, offer the best correlation between in vitro and in vivo transfection data. A) Transfection results for freshly-isolated primary hepatocytes were dose-dependent for 6 LNPs. A dose of 10nM was determined to be most appropriate for comparison with in vivo data. B) For the 6 lipidoids studied in A), primary cells transfected with LNPs offered the best correlation with in vivo activity, followed by HeLas transfected with LNPs. HeLa cells transfected with lipidoid complexes and Hepa1-6 cells transfected with LNPs did not correlate well with in vivo activity. Error bars represent standard deviation (n = 3).
Figure 5
Figure 5
The surface charge (a) and size (when less than 300 nm) (b, c) of lipidoid nanoparticles did not have any discernable effect on siRNA delivery to hepatocellular targets in vivo. Each data point in panels (a) and (b) represents a distinct lipidoid formulation. Panel (c) shows an unchanged silencing effect for the lipidoid C12-200 formulated into nanoparticles with identical chemical composition but varying diameter. Error bars in each panel represent standard deviation (n = 3).
Figure 6
Figure 6
While entrapment did not correlate with efficacy at high siRNA doses of 5 mg/kg (a), low dose data revealed that LNPs possessing entrapment efficiencies of ~75% perform the best in vivo (b). In vivo data collected at 0.5, 0.02, and 0.01 mg/kg siRNA doses are denoted in (b) by black circles, hatched squares, and white triangles, respectively. Error bars represent standard deviation (n = 3).
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