Maturation of Nanodiscs to HDL-like Particles via Lecithin-

Cholesterol Acyl Transferase

Chinonso C. Opara

Abstract
High Density Lipoprotein (HDL) particles are spherical
structures, which play a fundamental role in the reverse cholesterol
transport (RCT) pathway, where cholesterol is carried to the liver for
excretion. Apolipoprotein (Apo) A-1 is the primary protein component of
HDL and plays a critical role in the RCT pathway. As lipid and
cholesterol are transported from cells, they are encircled by Apo A-1,
forming discoidal pre--HDL particles, which subsequently undergo
cholesterol-dependent maturation via lecithin-cholesterol acyl
transferase (LCAT), forming spherical HDL. Man-made HDL have many
applications in nanotechnology, including intracellular delivery of drug
cargo. However, the large size distribution of HDL made from full length
Apo A-1 impedes drug delivery precision. We use a shorter construct of
Apo A-1, as a membrane scaffold protein for nanodiscs which resemble
pre--HDL particles, but differ in their more uniform size range. We
hypothesize that treatment of nanodiscs containing cholesterol with
LCAT will result in spherical HDL-like particles with a more narrow size
window. Current data suggests that LCAT-mediated maturation of
cholesterol-containing nanodiscs yields HDL-like particles with a
narrow size distribution. The insight gained from this study serves to
establish a baseline for using matured nanodiscs as drug delivery
vehicles with a uniform size range.

Introduction
High density lipoprotein (HDL) particles are known as the
good cholesterol because they aid in the excretion of cholesterol
from the body via the reverse cholesterol transport (RCT) pathway.
The RCT pathway begins with the cellular expulsion of cholesterol
and fatty acids, which are subsequently encircled by apolipoprotein
(Apo) A-1 the primary protein component of HDL. This forms
discoidal pre--HDL particles, which mature into spherical HDL.
This is accomplished by the enzyme, lecithin-cholesterol acyl
transferase (LCAT), which esterifies cholesterol to fatty acids
within the discoidal pre--HDL particles, resulting in a more
hydrophobic lipid assembly, leading to sphere formation.
Subsequently, mature HDL are taken up by the liver, resulting in
cholesterol excretion by the bile
1
.
While HDL is utilized in the body for transporting
cholesterol, researchers are currently developing them as a drug
delivery platform by exploiting the hydrophobic core for
encapsulating drug cargo
2,3
. However, man-made HDL constructed
from full length Apo A-1 results in a population of particles with a
large size window
4
. This is problematic for drug delivery, as it
generates uncertainty in dosage (Figure 1).


Figure 1. HDL particles of different sizes carrying varying amounts of drug cargo.

Conversely, by using a shorter construct (MSP1D1) of Apo A-1
as a membrane scaffold protein (MSP), nanodiscs which resemble
discoidal pre--HDL particles can be made, via a procedure that
mimics the first stage of the RCT pathway (Figure 2). This results in a
solution of particles with a narrow size distribution
5
, compared with
nascent and man-made discoidal pre--HDL made from full length Apo
A-1
6,7
.

1
Groen et al., The Ins and Outs of Reverse Cholesterol Transport.
2
Murakami et al., Intracellular Drug Delivery by Genetically Engineered High-
Density Lipoprotein Nanoparticles.
3
Zhang and Chen, Recombinant High Density Lipoprotein Reconstituted with
Apolipoprotein AI Cysteine Mutants as Delivery Vehicles for 10-
Hydroxycamptothecin.
4
Shahzad et al., Targeted Delivery of Small Interfering RNA Using
Reconstituted High-Density Lipoprotein Nanoparticles.
5
Denisov et al., Directed Self-Assembly of Monodisperse Phospholipid
Bilayer Nanodiscs with Controlled Size.
6
Vedhachalam et al., Influence of Apolipoprotein (Apo) A-I Structure on
Nascent High Density Lipoprotein (HDL) Particle Size Distribution.
7
Scanu et al., Degradation and Reassembly of a Human Serum High-Density
Lipoprotein. Evidence for Differences in Lipid Affinity among Three Classes of
Polypeptide Chains.

Figure 2. Nanodisc preparation: membrane scaffold protein combines with lipids
dissolved in detergent to form mixed micelles. Nanodiscs are formed upon removal of the
detergent.

Subsequent maturation of cholesterol-containing nanodiscs has
shown to yield spherical HDL-like particles, with three or more
nanodiscs combining to form two or more HDL-like particles
8
.


Figure 3. HDL-like particle preparation: LCAT esterifies cholesterol to fatty acids in the
nanodiscs. Three or more nanodiscs combine to form two or more HDL-like particles.

Nanodisc maturation may also result in HDL-like particles with
a uniform size range. In addition, with further manipulation of the MSP
and lipid, the HDL-like particles could be tuned to locate specific cell
types for targeted drug delivery.


8
Silva et al., Structure of Apolipoprotein A-I in Spherical High Density
Lipoproteins of Different Sizes.
Results and Discussion
Effect of Cholesterol and LCAT Concentration on Formation of HDL-
like Particles
Nanodisc formulations containing varying amounts of
cholesterol (0%, 5%, 7.5%, 10%, or 12.5% - mole) were incubated
without or with LCAT (2.5 mg/mL or 5 mg/mL). Size exclusion
chromatography (SEC) of the incubation mixtures suggests HDL-like
particle formation is proportional to nanodisc cholesterol content and
LCAT concentration (Figure 4). The peaks around 19.5 minutes are
residual lipid aggregates resulting from formation of the nanodiscs,
which elute around 25 minutes. The peaks around 23 minutes become
more pronounced as nanodisc cholesterol content and LCAT
concentration are increased. The biggest difference in broadness of the
23 minute peak is between the incubation mixture containing 0%
cholesterol nanodiscs and no LCAT (top left panel) and the incubation
mixture containing 12.5% cholesterol nanodiscs and 5 mg/mL LCAT
(bottom right panel). This indicates that the HDL-like particles elute
around 23 minutes and that their formation is augmented with increasing
nanodisc cholesterol content and LCAT concentration.


Figure 4. SEC traces of the incubation mixtures. Nanodiscs elute around 25 minutes.
HDL-like particles are represented by the shoulder around 23 minutes, directly before
the nanodiscs. HDL-like particle formation increases with increasing LCAT
concentration and nanodisc cholesterol content, as is shown by the broadening of the 23
minute peak.

However, HPLC traces containing no LCAT also possess a slight
peak around 23 minutes. Rather than being HDL-like particles, these
peaks are potentially lipid aggregates with diameters close to that of the
HDL-like particles. The former would counter the mechanism of
nanodisc maturation, where LCAT esterifies cholesterol to fatty acids
within the nanodiscs. Yet, the lipid aggregates eluting around 19.8
minutes do not increase in the presence of LCAT. This proposes that the
lipid aggregates eluting around 23 minutes also do not intensify when
LCAT is added, suggesting that the broadening of the 23 minute peak is
accomplished by way of maturation of the nanodiscs to HDL-like
particles via LCAT.

Resulting Size Distributions from LCAT Incubation Mixtures
In order to assess the size of the nanodiscs, peak III
corresponding to the nanodiscs from the incubation mixture containing
the 0% cholesterol nanodisc formulation and no LCAT was collected via
SEC and subjected to static light scattering (SLS) (Figure 5). The
nanodiscs displayed a polydispersity of 8.6% around 8.5 nm, which is
similar to the literature value of ~9.7 nm
9
.


Figure 5. Static light scattering size distribution histogram of peak III from the
incubation mixture containing the 0% cholesterol nanodisc formulation and no LCAT.
The nanodiscs display a polydispersity of 8.6% around 8.5 nm.

9
Denisov et al., Directed Self-Assembly of Monodisperse Phospholipid
Bilayer Nanodiscs with Controlled Size.
From the incubation mixture containing the 12.5% cholesterol
nanodisc formulation and 5 mg/mL LCAT, peaks II and III
corresponding to the nanodiscs and HDL-like particles, respectively
were collected through SEC and analyzed by SLS (Figure 6). Because
the representative peaks of nanodiscs and HDL-like particles were
collected, in the histogram below, two size distributions were expected: a
population of nanodiscs around 8.5 nm and a population of HDL-like
particles with a slightly larger diameter than the nanodiscs. Instead size
distributions around 75 nm and 260 nm were observed. This is
potentially due to confounding of the data by larger particles in the
solution. In the future, this analysis should be repeated for the purpose of
obtaining an accurate diameter for the HDL-like particles.


Figure 6. Static light scattering size distribution histogram of peaks II and III from the
incubation mixture containing the 12.5% cholesterol nanodisc formulation and 5 mg/mL
LCAT. Two distributions, one for nanodiscs and another for HDL-like particles, around
10 nm were expected. Rather, distributions around 75 nm and 260 nm were observed,
possibly due to confounding of the data by larger particles in solution.

Visual Comparison of Nanodiscs and HDL-Like Particles
Electron microscopy (EM) was used to visualize the nanodiscs
and HDL-like particles. Peak I from the incubation mixture containing
the 0% cholesterol nanodisc formulation and no LCAT representing
nanodiscs (Figure 7) and peaks II and III from the incubation mixture
containing the 12.5% cholesterol formulation and 5 mg/mL LCAT
(Figure 8) representing both nanodiscs and HDL-like particles,
respectively were selected for EM analysis.
When the nanodiscs are viewed from their sides, they look like
two adjacent rods with a dark band (MSP) in between them. This is
consistent with their structure, as two MSPs are wrapped around the
hydrophobic core. When viewed from the top, the dark band disappears,
while the nanodiscs remain rod-like in appearance.


Figure 7. Electron microscopy image of peak III (purified for nanodiscs) from the
incubation mixture containing the 0% cholesterol nanodisc formulation and no LCAT.
When viewed from their sides (within red circle), the nanodiscs appear rod-like with a
dark band, representing the membrane scaffold protein, through their middle. When
viewed from the top (within blue circle), the dark bands are no longer visible and the
nanodiscs still appear rod-like. Size bar = 100 nm.

From the EM, the HDL-like particles appear to have a narrow
size distribution, which will have to be confirmed by SLS. They also
appear more circular than the nanodiscs, which is also consistent with
their spherical shape. However, from the 2D image, it is not possible to
fully discern the HDL-like particles from the nanodiscs. A 3D
reconstruction of the EM image will be needed in order to provide more
spatial detail in regards to the structural differences between the two
particles.


Figure 8. Electron microscopy image of peaks II and III (purified for HDL-like particles
and nanodiscs) from the incubation mixture containing the 12.5% cholesterol
formulation and 5 mg/mL LCAT. The HDL-like particles (within yellow circle) are
similar in manifestation to the top view of the nanodiscs, but appear more circular, from
the 2D image, than the nanodiscs. Size bar = 100 nm.

The data presented from this work propose that discoidal
nanodiscs can be converted to spherical HDL-like particles with a narrow
size distribution via LCAT. HDL-like particle formation is proportional
to nanodisc cholesterol content and LCAT concentration. In the future,
adjustment of nanodisc cholesterol content will be needed in order to
further optimize HDL-like particle formation. This work establishes a
baseline for utilizing matured nanodiscs as a platform for intracellular
drug delivery.

Materials and Methods
Lipids
Lipids were purchased in powder form and dissolved in 100%
chloroform. 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) was
purchased from Avanti Polar Lipids and cholesterol was purchased from
Sigma. Lipid films were prepared by dispensing the desired amount of
lipid using a Hamilton glass syringe into glass culture tubes. Excess
solvent was removed via nitrogen stream followed by overnight vacuum
desiccation.
Lipids containing varying amounts of cholesterol (POPC +
cholesterol 0%, 5%, 7.5%, 10%, and 12.5% - mole) were made by
adding appropriate amounts of cholesterol dissolved in chloroform into
POPC dissolved in chloroform. The resulting lipid film was obtained, as
described above.

POPC Phosphate Assay
POPC concentration was assessed via phosphate assay. 1 and 2
L aliquots of POPC stock solution dissolved in chloroform were
dispensed at the bottom of glass culture tubes. Phosphate standards (0 to
228 mM) were treated likewise. Phosphate moieties were cleaved in a
fume hood above 200
O
C in 8.9 N sulfuric acid. Subsequently, hydrogen
peroxide was added to completely oxidize the remaining organic
material. Samples were heated again above 200
O
C to remove remaining
peroxide. Next, the phosphorous-molybdate complex was formed
through addition of 2.5% ammonium molybdate and 10% ascorbic acid
and subsequent heating at 100
O
C to allow the blue hue to results. When
cooled to room temperature, the samples were transferred to a clear flat-
bottom microplate and the absorbance was read at 820 nm.

MSP1D1 Expression
The expression vectors were purchased from Addgene and the
protein was expressed in BL21 Star (DE3) cells (Invitrogen) using the
pET expression system. A starting culture was inoculated from freshly
streaked plates into 10 mL of Luria Broth (LB) with 30 mg/L kanamycin
and allowed to grow for 8-12 hours at 37
O
C and 220 rpm. The starting
cultures was then diluted into 100 mL of LB with kanamycin and
allowed to grow for another 8-12 hours. The cultures were subsequently
added to 900 mL LB with kanamycin in 2.8 L fernbach flasks. One liter
cultures were grown for 2-3 hours, or until the OD was 0.6. Then, the
cultures were induced with Isopropyl -D-1-thiogalactopyranoside
(IPTG, Sigma) at a final concentration of 1mM. Before being harvested
by centrifugation at 8000xg for 10 minutes, the cells were then allowed
to grow for 2.5-3 more hours. Prior to purification, cell pellets were
stored wet at -80
O
C.

MSP1D1 Purification
MSP1D1s were purified via the N-terminal poly-histidine
utilizing a 20 mL column of Ni-NTA fast-flow resin (Qiagen). Prior to
each round of purification, the column was regenerated, stripping the
nickel with 50 mM EDTA, washing with water and 20% ethanol, and
then recharging with 100 mM NiBr
2
. The cell pellet from 6 L was
thawed on ice prior to resuspension in 50 mL of 20 mM phosphate
buffer, 1 mM phenylmethlysulfonyl fluoride (PSMF) and 5 g/mL of
deoxyribonuclease I, pH 7.4. The cells were lysed by sonication 30
second bursts, followed by 30 seconds on ice, for a total of six minutes.
Subsequently, the lysate was clarified by centrifugation at 30,000xg for
30 minutes.
The clarified lysate was loaded on the column through gravity
and the flow-through was reloaded to obtain maximal binding. Next, the
column was washed using 5 column volumes each of wash 1 (40 mM
tris, 300 mM NaCl, 1% Triton X-100, pH 8.0), wash 2 (40 mM Tris, 300
mM NaCl, 50 mM Na cholate, 20 mM imidazole, pH 8.0) and wash 3
(40 mM tris, 300 mM NaCl, 50 mM imidazole, pH 8.0) before elution
with elution buffer (40 mM Tris, 300 mM NaCl, 400 mM imidazole, pH
8.0). Five column volumes (100 mL) were then collected in 10 mL
aliquots. The elution was pooled and dialyzed against DFB containing
0.5 mM EDTA. The concentration was ascertained via UV-vis
absorbance at 280 nm and the protein was concentrated to a final
concentration between 150-170 M, aliquoted, and stored at -20
O
C.
Purity was determined by SDS-PAGE.

Nanodisc Preparation
The nanodiscs were made by resuspending lipid films,
containing varying amounts of cholesterol (0%, 5%, 7.5%, 10%, and
12.5% - mole) in disc forming buffer (DFB, 20 mM Tris, 100 mM NaCl,
pH 7.4) and cholate. The solutions were heated at 37
O
C to assist
solvation, and then repeatedly sonicated and vortexed until they became
clear. MSP1D1 was added at a POPC:MSP1D1 ratio of 65:1.
The final concentration of cholate in the incubation was 20mM,
which is 2-fold greater than the critical micelle concentration, in order to
ensure complete solubilization of the components. The solution was then
allowed to incubate at 4
O
C with constant agitation for 1 hour. Detergent
removal was accomplished by addition of prewashed Amberlite XAD-2
at 0.5 g/mL with constant agitation at 4
O
C for 4 hours. The resulting
nanodisc solutions were removed from the Amberlite XAD-2 utilizing a
25 gauge needle stored at 20
O
C.

LCAT Preparation
The mammalian LCAT construct (CHO-hLCATH6) was
expressed and purified by Michael J. Dabrowski in our lab. The Chinese
Hamster Ovary cells were maintained in DMEM F12 media with 10%
heat-inactivated fetal bovine serum, 0.01% pen-strep, 2mM glutamine
and vitamins. Upon attaining 95% competency, the cells were washed in
Hanks solution and incubated for 72 hours in PFX-CHO serum-free
media with the above mentioned supplements. 10mM sodium butyrate
was added after 24 hours to boost expression. After 48 hours, the media
was collected and centrifuged to remove cell debris. The supernatant,
containing the expressed LCAT, was subsequently loaded onto a His60
column for purification.

LCAT Reactions
LCAT reactions were performed by incubating 2 M nanodiscs
containing varying amounts of cholesterol (0%, 5%, 7.5%, 10%, or
12.5% - mole), 5 mM -mercapthoethanol, and LCAT at two different
concentrations (2.5 mg/mL or 5 mg/mL). After 4 hours, the reaction
mixtures were stored at 4
O
C.

Size Exclusion Chromatography
Size exclusion chromatography (SEC) was performed on a
calibrated Superdex 200 10/300 column, at an isocratic flow rate of 0.5
mL/min using DFB as the mobile phase.

Static Light Scattering
Static light scattering was performed on a DynoPro NanoStar
dynamic light scattering instrument from Wyatt Technology. For each
sample 30 acquisitions lasting 10 seconds were taken, producing an
averaged spectrum. Samples were prepared by collecting fractions
eluting at 23.3-27.8 minutes (peak III from the 0% cholesterol nanodisc
formulation containing no LCAT) and 21.3-28.3 minutes (peaks II and
III from the 12.5% cholesterol nanodiscs formulation containing 5
mg/mL LCAT) from the SEC column and concentrated using micro spin-
columns.

Electron Microscopy
Electron microscopy was performed by James Williams and
Jamie Ebner at the University of Washington Electron Microscopy Suite
in the Department of Biochemistry. Samples were prepared by collecting
fractions eluting at 23.3-27.28 (peak III from the 0% cholesterol
nanodisc formulation containing no LCAT) minutes and 21.3-28.3
(peaks II and III from the 12.5% cholesterol nanodiscs formulation
containing 5 mg/mL LCAT) minutes from the SEC column and
concentrated using micro spin-columns. Samples were diluted 1 to 20
and then stored at 4
O
C prior to spotting on freshly glow-discharged
carbon grids. Subsequently, the grids were stained with Nano-W
(Nanoprobes, provided by Dr. Kelly Lee in the Department of Medicinal
Chemistry) and allowed to dry for at least 1 hour before EM analysis.




































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Acknowledgments
I would like to thank my faculty mentor, William Atkins in the
Department of Medicinal Chemistry, for his advising and for his
provision of supplies and equipment for the project. I would also like to
give thanks to Michael Dabrowski and Wynton McClary from the Atkins
lab for their guidance in making nanodiscs, performing necessary assays
and for providing the LCAT. I would in addition like to thank James
Williams and Jamie Ebner from the Kelly Lee lab, also in the
Department of Medicinal Chemistry, for their support in collecting the
electron microscopy images. Also, I would like to thank the University
of Washington Ronald E. McNair Program for allowing me the
opportunity to publish in this journal.

Chinonso C. Opara
Medicinal Chemistry, William Atkins
chinonsoopara@gmail.com

My research interests center around drug delivery. I am attending
Vanderbilt Medical School to receive an M.D.