Maturation of Nanodiscs to High Density Lipoprotein-Like Particles

via Lecithin-Cholesterol Acyl Transferase

Chinonso Opara, Mike Dabrowski, Wynton McClary and William Atkins
University of Washington, Department of Medicinal Chemistry
Introduction Results
Evidence of Larger Particles with
Increasing LCAT and Nanodisc Lipid
Cholesterol Content
Acknowledgements
The authors would like to thank James Williams and Jamie Ebner from
the Kelly Lee lab, in the Department of Medicinal Chemistry, for their
generous support in collecting the electron microscopy images.
Nanodiscs and HDL-Like Particles Are
Similar in Shape When Viewed in 2D
High Density Lipoprotein (HDL) particles are spherical structures, which
play a fundamental role in down regulating the amount of cholesterol in
the body. Apolipoprotien (Apo) A-1 is the primary protein component of
HDL. Man-made HDL have many applications in nanotechnology,
including drug delivery. However, the large size distribution of HDL made
from full length Apo A-1 impedes the precision of drug delivery.
Nanodiscs Display Narrow Size
Distribution, While Shoulder Exhibits
Larger size Window
Conclusions
HDL-like particle formation is proportional to LCAT
concentration and nanodisc lipid cholesterol content.

Future work is needed to differentiate nanodiscs
from HDL-like particles.

This work establishes a baseline for using HDL-like
particles created from nanodiscs as a drug delivery
vector.
We use a shorter construct of Apo A-1 as a membrane scaffold protein
(MSP) for preparing nanodiscs, which are disc-shaped synthetic
membrane models that resemble premature HDL particles. Importantly
Nanodiscs are less polydispersed than the latter.
Premature HDL particles undergo a cholesterol-dependent maturation to
HDL particles through Lecithin-Cholesterol Acyl Transferease (LCAT). We
hypothesize that treatment of nanodiscs containing cholesterol with LCAT
will result in spherical HDL-like particles with a more narrow size window.
Membrane Scaffold Protein
Lipid plus Detergent
Mixed Micells
Detergent
Removal via
Amberlite
XAD-2
Nanodiscs
Nanodiscs Containing Cholesterol
HDL-like Particles
LCAT
HDL Particles of Different Sizes Containing Varying Amounts of Drug Cargo
Time (min)
A
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r
b
a
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2
8
0

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0 mg/mL
LCAT
2.5 mg/mL
LCAT
5 mg/mL
LCAT
Figure 1. SEC traces of the incubation mixtures. Nanodiscs elute at around 25
minutes. HDL-like particles are believed to be the shoulder at around 23 minutes
directly before the nanodiscs.
Nanodisc formulations containing varying amounts of lipid cholesterol
were incubated with or without LCAT. Size exclusion chromatography
(SEC) of the incubation mixtures suggests HDL-like particle formation is
proportional to nanodisc cholesterol content and LCAT concentration
Figure 2. SLS size
distribution historgram of
peak III from the 0%
cholesterol Nanodisc
formulation with no
LCAT. The nanodiscs
are approximately 10 nm
in diameter with a low
level of polydispersity.
Individual peaks were enriched, as illustrated, and scanned via static
light scattering (SLS) to assess their corresponding particle sizes.
Figure 3. SLS size
distribution histogram of
peak II from the 12.5%
cholesterol Nanodisc
formulation with 5 mg/mL
LCAT. Two distributions
around 10 nm were
expected, but larger
particles in the sample
may have confounded
the data.
Electron microscopy (EM) photographs were taken of the peaks enriched
for either nanodiscs or HDL-like particles. Nanodiscs appear rod-like,
while HDL-like particles seem more circular. From the 2D images, it is
not possible to fully discern nanodiscs from the latter.
Figure 4. a) EM images of the nanodisc-enriched peak. When viewed from their
sides, nanodiscs appear rod-like with a dark band (MSP) through their middle. When
viewed from the top, the dark bands are not visible. b) Em images of the HDL-like
particle-enriched peak.
a). b).