Impact of hydrophobic chain composition on amphiphilic macromolecule antiatherogenic bioactivity

Article abstract of DOI:10.1021/bm500809f

Amphiphilic macromolecules (AMs) composed of sugar backbones modified with branched aliphatic chains and a poly(ethylene glycol) (PEG) tail can inhibit macrophage uptake of oxidized low-density lipoproteins (oxLDL), a major event underlying atherosclerosis development. Previous studies indicate that AM hydrophobic domains influence this bioactivity through interacting with macrophage scavenger receptors, which can contain basic and/or hydrophobic residues within their binding pockets. In this study, we compare two classes of AMs to investigate their ability to promote athero-protective potency via hydrogen-bonding or hydrophobic interactions with scavenger receptors. A series of ether-AMs, containing methoxy-terminated aliphatic arms capable of hydrogen-bonding, was synthesized. Compared to analogous AMs containing no ether moieties (alkyl-AMs), ether-AMs showed improved cytotoxicity profiles. Increasing AM hydrophobicity via incorporation of longer and/or alkyl-terminated hydrophobic chains yielded macromolecules with enhanced oxLDL uptake inhibition. These findings indicate that hydrophobic interactions and the length of AM aliphatic arms more significantly influence AM bioactivity than hydrogen-bonding.

Full text of DOI:10.1021/bm500809f

Article
pubs.acs.org/Biomac
Open Access on 07/28/2015
Impact of Hydrophobic Chain Composition on Amphiphilic
Macromolecule Antiatherogenic Bioactivity
Allison Faig,† Latrisha K. Petersen,‡ Prabhas V. Moghe,‡,§ and Kathryn E. Uhrich*,†,‡
‡
§
†Department of Chemistry and Chemical Biology, Department of Biomedical Engineering, and Department of Chemical and
Biochemical Engineering, Rutgers University, Piscataway, New Jersey 08854, United States
S
* Supporting Information
ABSTRACT: Amphiphilic macromolecules (AMs) composed of
sugar backbones modified with branched aliphatic chains and a
poly(ethylene glycol) (PEG) tail can inhibit macrophage uptake of
oxidized low-density lipoproteins (oxLDL), a major event
underlying atherosclerosis development. Previous studies indicate
that AM hydrophobic domains influence this bioactivity through
interacting with macrophage scavenger receptors, which can
contain basic and/or hydrophobic residues within their binding
pockets. In this study, we compare two classes of AMs to
investigate their ability to promote athero-protective potency via
hydrogen-bonding or hydrophobic interactions with scavenger
receptors. A series of ether-AMs, containing methoxy-terminated
aliphatic arms capable of hydrogen-bonding, was synthesized.
Compared to analogous AMs containing no ether moieties (alkyl-AMs), ether-AMs showed improved cytotoxicity profiles.
Increasing AM hydrophobicity via incorporation of longer and/or alkyl-terminated hydrophobic chains yielded macromolecules
with enhanced oxLDL uptake inhibition. These findings indicate that hydrophobic interactions and the length of AM aliphatic
arms more significantly influence AM bioactivity than hydrogen-bonding.
conditions), and as a result of their systemic administration and
mechanism of action statins do not directly treat atherogenic
sites in the arteries.10−12 When statins are not tolerated by
patients or when patients are genetically predisposed to
increased LDL levels as in familial hypercholesterolemia, lipid
apheresis therapies can be used to extracorporeally remove
plasma lipoproteins (i.e., LDL) from the blood.13−17 Apheresis
methods often utilize adsorbents, which contain ligands that
interact with and retain LDL, including dextran sulfate,
polyacrylate, heparin, and phosphates, and carriers such as
PVA microspheres, cellulose beads, nonwoven fabrics, and other
polymer systems.13,14,16−19 While these therapies lower LDL
levels and improve atherosclerosis outcomes, problems remain:
long-term, expensive treatments ($40 000−100 000 USD
annually) are required to maintain efficacy, treatment access is
limited, and many current adsorbents have low LDL selectivity
and poor mechanical properties.14−17,19,20 Consequently,
researchers are currently targeting various steps in the
atherosclerotic cascade described above, including monocyte
recruitment, macrophage-mediated cholesterol metabolism, and
plaque regression to impede the inflammatory progression and
improve treatment efficacy.6
1. INTRODUCTION
Atherosclerosis, a major cause of mortality worldwide, is an
inflammatory disease characterized by arterial plaque develop-
ment.1−5 During the early stages of atherosclerosis, low-density
lipoprotein (LDL) accumulates in the subendothelial space
where various cells catalyze its oxidative modification.4,6−8 This
oxidized LDL (oxLDL) initiates an inflammatory response, in
which monocytes are recruited to sites of endothelial
dysfunction, migrate into the subendothelial space, and
subsequently differentiate into macrophages.1,3,4,6,7,9 Macro-
phages then internalize oxLDL primarily through scavenger
receptors A (SRA) and B (CD36), resulting in unregulated
modified lipid accumulation and foam cell formation.2,3,5−7,9
Foam cells promote the inflammatory process and lead to
atherosclerotic plaque formation, narrowing the artery, and
cardiovascular events, including hypertension, stroke, and
myocardial infarction.2,3,6,9 In this work, we seek to mitigate
atherogenesis via new designs of macromolecules that interfere
with oxLDL uptake, and thus de-escalate the atherosclerotic
development.
Statins are the most well-known and widely prescribed
therapeutic for treating coronary artery disease.10,11 They slow
the atherosclerotic cascade through inhibiting hepatic cholesterol
biosynthesis and subsequently increase the expression of hepatic
LDL receptors to lower serum LDL levels. However, statins can
have undesirable side effects, including muscle toxicity, cognitive
problems, and metabolic issues (e.g., liver toxicity or thyroid
Received: June 2, 2014
Revised: July 24, 2014
Published: July 28, 2014
© 2014 American Chemical Society
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As an alternative strategy to treat atherosclerosis, researchers
are investigating means to abrogate the atherosclerotic cascade
by preventing oxLDL trafficking and uptake within the blood
vessel walls.21,22 In vivo studies have indicated that apolipopro-
tein E-null mice deficient in certain scavenger receptors (e.g.,
SRA or CD36) result in significantly smaller atherosclerotic
lesions and a decreased uptake of modified LDL (e.g.,
oxLDL).21−23 Given that oxLDL uptake can lead to foam cell
formation and atherosclerotic plaque development, inhibiting
oxLDL uptake could impede atherogenesis. Previously, our lab
demonstrated that amphiphilic macromolecules (AMs) inhibit
scavenger receptor-mediated oxLDL uptake, particularly through
competitive inhibition of SRA and CD36.24 These sugar-based,
PEGylated AMs are comprised of a sugar backbone that is
acylated with aliphatic chains and conjugated to a poly(ethylene)
glycol (PEG) tail.25 Given their amphiphilicity, AMs self-
assemble into nanoscale micelles in aqueous environments25
with a PEG shell that may shield uptake by the reticuloendo-
thelial system, potentially prolonging in vivo blood circulation
times.26 Upon discovering AMs’ antiatherosclerotic activity,
various studies were conducted to elucidate their bioactive
mechanism. Dynamic light scattering (DLS) studies indicated
that AMs containing an anionic charge (e.g., carboxylate moiety)
within their hydrophobic domain complex with unmodified
LDL, but do not complex with oxLDL, likely due to charge
repulsion resulting from oxLDL’s increased net negative
charge.27 As these AMs did not interact with oxLDL yet
prevented its accumulation in macrophages, further immunoloc-
alization and antibody blocking assays were conducted and
demonstrated that AMs interact with macrophage scavenger
receptors and subsequently prevent oxLDL uptake through these
receptors.24,28−30 A library of AMs was generated by systemati-
cally modifying AM structural elements, and quantitative
structure−activity relationship (QSAR) models were developed
to determine the most prominent athero-protective AM
features.31−34 The hydrophobic domain plays a key role; the
presentation of the aliphatic arms influences AM athero-
protective bioactivity.33−35
Though structure−activity relationships provided significant
insights regarding AM efficacy, a more rational approach for
developing bioactive AMs with increased potency would be
inspired by the physicochemical attributes of the scavenger
receptor binding pockets. While certain scavenger receptors
contain basic residues in their oxLDL binding domains,36,37
others contain hydrophobic residues near their oxLDL binding
sites.38 Increasing the AM hydrophobicity through extending
alkyl chain lengths or decreasing PEG tail lengths could increase
AM interactions with hydrophobic receptor pockets, whereas the
addition of heteroatoms into the hydrophobic domain could
enable hydrogen-bonding interactions with basic residues,
ultimately reducing oxLDL uptake by mimicking scavenger
receptor interactions with hydrophobic oxidized lipids.39−41 To
decipher which interactions more effectively influence athero-
protective bioactivity through repressing oxLDL uptake, a series
of novel ether-containing AMs (ether-AMs) capable of hydro-
gen-bonding was synthesized based on a linear tartaric acid (TA)
backbone and compared to analogous AMs containing no ether
moieties (alkyl-AMs) that would exhibit stronger hydrophobic
interactions (Figure 1). The relative hydrophobicity of all AMs
was varied by altering both the aliphatic chain and PEG tail
lengths to determine whether more lipophilic AMs would mimic
the interactions of hydrophobic, oxidized lipids with scavenger
Figure 1. Chemical structures of ether- and alkyl-AMs.
receptors and thereby exhibit increased athero-protective
bioactivity.
2. MATERIALS AND METHODS
2.1. Materials. All reagents and solvents were purchased from
Sigma-Aldrich (Milwaukee, WI) and used as received unless otherwise
noted. Hydrochloric acid (HCl, 1 N), dibenzyl tartrate (DBT), and
polytetrafluoroethylene (PTFE) syringe filters were purchased from
Fisher Scientific (Fair Lawn, NJ). Silica gel was purchased from VWR
(Radnor, PA). Monomethoxy-poly(ethylene glycol)-amine (mPEG-
amine) was purchased from Laysan Bio (Arab, AL) and azeotropically
distilled with toluene prior to use. Reagents for cell culture, toxicity
studies, and oxLDL uptake studies include human buffy coats purchased
from The Blood Center of New Jersey (East Orange, NJ), Ficoll-Paque
premium 1.077 g/mL purchased from GE Healthcare (Fairfield, CT),
RPMI-1640 purchased from ATCC (Manassas, VA), macrophage
colony stimulating factor purchased from PeproTech (Rocky Hill, NJ),
penicillin/streptomycin purchased from Lonza (Basel, Switzerland),
alamarBlue assay, fetal bovine serum, and Hoechst 33342 purchased
from Life Technologies (Carlsbad, CA), unlabeled oxLDL purchased
from Biomedical Technologies Inc. (Ward Hill, MA), and 3,3′-
dioctadecyloxacarbocyanine (DiO) labeled oxLDL purchased from
Kalen Biomedical (Montgomery Village, MD).
2.2. Characterization. Proton (1H) and carbon (13C) nuclear
magnetic resonance (NMR) spectra were obtained using a Varian 400 or
500 MHz spectrophotometer. Samples were dissolved in deuterated
chloroform (CDCl3), and a few drops of dimethyl sulfoxide (DMSO-d6)
added, if necessary; trimethylsilane was used as an internal reference.
Fourier transform infrared (FT-IR) spectra were recorded on a Thermo
Scientific Nicolet iS10 spectrophotometer using OMNIC software with
an average of 32 scans. FT-IR samples were either pressed into
potassium bromide (KBr) discs (1 wt % sample) or solvent-cast onto
sodium chloride plates.
AM precursor molecular weights were determined using a
ThermoQuest Finnigan LCQ-DUO system equipped with a syringe
pump, an optional divert/inject valve, an atmospheric pressure
ionization (API) source, a mass spectrometer (MS) detector, and the
Xcalibur data system. Samples were prepared at a concentration of 10
μg/mL in methanol (MeOH) or dichloromethane (DCM) using 1%
acetic acid or 1% ammonia for positive or negative ion modes,
respectively. AM weight-averaged molecular weight (Mw) and
polydispersity index (PDI) data were determined by gel permeation
chromatography (GPC) using a Waters LC system (Milford, MA),
equipped with a 2414 refractive index detector, 1515 isocratic HPLC
pump, 717plus autosampler, and a Jordi divinylbenzene mixed-bed GPC
column (7.8 × 300 mm, Alltech Associates, Deerfield, IL). Samples were
prepared at 10 mg/mL in DCM and filtered with 0.45 μm PTFE syringe
filters prior to autoinjection. DCM was used as the eluent at a flow rate of
1 mL/min. An IBM ThinkCentre computer with WaterBreeze version
3.20 software was used for data collection and processing, with Mw
calibrated against broad PEG standards (Waters, Milford, MA).
2.3. Synthesis. 2.3.1. Synthesis of n-Methoxyalkanoic Acid
Chains (2). The preparation of 8-methoxyoctanoic acid (2a) is
presented as an example. According to modified literature procedures,42
anhydrous MeOH (8 mL) was cooled to 0 °C, potassium hydroxide
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(KOH, 13.80 mmol) added, and the solution stirred for 30 min. A
solution of 8-bromooctanoic acid (1a, 4.60 mmol) in anhydrous MeOH
(7 mL) was then added via syringe. The reaction mixture was heated to
reflux temperatures and stirred overnight. After cooling to room
temperature, MeOH was removed in vacuo and the resulting crude
mixture reconstituted in 1 N HCl (25 mL) and diethyl ether (5 mL).
The crude product was extracted using diethyl ether (4 × 30 mL), the
combined organic layers were washed with 50:50 brine/H2O (30 mL)
and dried over magnesium sulfate (MgSO4), and then the solvent
removed in vacuo. 2a was then purified on silica gel via column
chromatography using a hexanes/acetic acid/ethyl acetate gradient
(99.8:0.2:0 to 98:1:1).
8-Methoxyoctanoic Acid (2a). Yield: 0.70 g, 87% (pale yellow oil).
1H NMR (500 MHz, CDCl3): δ 3.32 (t, 2H, OCH2), 3.27 (s, 3H,
OCH3), 2.26 (t, 2H, CH2CO), 1.53 (m, 4H, CH2), 1.27 (b, 6H, CH2).
13C NMR (500 MHz, CDCl3): δ 179.57, 72.95, 58.51, 34.16, 29.53,
29.21, 29.14, 26.03, 24.80. IR (cm−1, thin film from diethyl ether):
3600−3100 (OH, COOH), 1709 (CO, COOH). ESI-MS m/z: 173.3
(M − 1).
(t, 4H, OCH2), 3.37 (s, 6H, OCH3), 2.45 (t, 4H, CH2CO), 1.62 (m, 8H,
CH2), 1.31 (b, 20H, CH2). 13C NMR (500 MHz, CDCl3): δ 172.90,
168.51, 73.26, 70.74, 58.35, 33.79, 29.12, 28.87, 28.74, 28.59, 28.39,
25.76, 24.72. IR (cm−1, KBr): 3650−3300 (OH, COOH), 1760 (CO,
ester), 1736 (CO, COOH). ESI-MS m/z: 517.3 (M − 1).
2,3-Bis(12-methoxydodecanoyl) TA (4c). Two-step yield: 0.17 g,
99% (white powder). 1H NMR (500 MHz, CDCl3 with DMSO): δ 5.69
(s, 2H, CH), 3.36 (t, 4H, OCH2), 3.33 (s, 6H, OCH3), 2.42 (m, 4H,
CH2CO), 1.59 (m, 8H, CH2), 1.26 (b, 28H, CH2). 13C NMR (500
MHz, CDCl3): δ 171.89, 167.42, 72.23, 70.22, 57.85, 33.15, 29.00, 28.93,
28.89, 28.84, 28.79, 28.62, 28.36, 25.50, 24.11. IR (cm−1, KBr): 3650−
3300 (OH, COOH), 1761 (CO, ester), 1737 (CO, COOH). ESI-
MS: m/z: 573.9 (M − 1).
2.3.4. Synthesis of Ether-AMs (5). The synthesis of ether-AMs is
presented as an example (5a2). Following a modified literature
procedure,25 4a (0.45 mmol) and catalytic 4-(dimethylamino)-
pyridinium 4-toluenesulfonate (DPTS, 0.15 mmol) were dissolved in
a mixture of anhydrous DCM (10 mL) and anhydrous dimethylforma-
mide (DMF, 3 mL). This solution was added to 2 kDa mPEG-amine
(0.15 mmol). Upon complete dissolution of PEG, dicyclohexylcarbo-
diimide (DCC, 1 M in DCM, 0.48 mmol) was added dropwise via
syringe and the reaction stirred for 48 h at room temperature under
argon. The reaction mixture was cooled to −20 °C, and the white solid
precipitate (dicyclohexylurea) removed via vacuum filtration. The
filtrate was then diluted with DCM (25 mL) and washed with 0.1 N HCl
(1 × 40 mL) and brine (2 × 40 mL). The organic layer was dried over
MgSO4 and concentrated in vacuo. Ether-AM (5a2) was then
precipitated from diethyl ether (50 mL) and isolated via centrifugation
(Hettich EBA 12, Beverly, MA; 3500 rpm, 5 min), and the diethyl ether
decanted. The product was washed with diethyl ether (50 mL × 4) and
isolated with centrifugation and decanting, as above. The PEG Mw used
to synthesize the ether-AMs will be denoted numerically in kilodaltons
as a subscript (e.g., 5a2).
10-Methoxydecanoic Acid (2b). Yield: 1.93 g, 84% (light orange
solid). 1H NMR (400 MHz, CDCl3): δ 3.35 (t, 2H, OCH2), 3.32 (s, 3H,
OCH3), 2.32 (t, 2H, CH2CO), 1.57 (m, 4H, CH2), 1.28 (b, 10H, CH2).
13C NMR (500 MHz, CDCl3): δ 179.53, 73.11, 58.53, 34.19, 29.64,
29.55, 29.52, 29.34, 29.21, 26.24, 24.94. IR (cm−1, thin film from DCM):
3600−3100 (OH, COOH), 1709 (CO, COOH). ESI-MS m/z: 201.3
(M − 1).
12-Methoxydodecanoic Acid (2c). Yield: 0.70 g, 80% (white solid).
1H NMR (400 MHz, CDCl3): δ 3.38 (t, 2H, OCH2), 3.34 (s, 3H,
OCH3), 2.34 (t, 2H, CH2CO), 1.60 (m, 4H, CH2), 1.27 (b, 14H, CH2).
13C NMR (500 MHz, CDCl3): δ 180.08, 73.12, 58.61, 34.32, 29.77,
29.73, 29.68, 29.66, 29.59, 29.42, 29.25, 26.30, 24.90. IR (cm−1, KBr):
3600−3100 (OH, COOH), 1731 (CO, COOH). ESI-MS m/z: 229.3
(M − 1).
1
5a2. Yield: 0.28 g, 78% (beige waxy solid). H NMR (400 MHz,
2.3.2. Synthesis of 2,3-Bis(n-methoxyalkanoyl) Dibenzyl Tartrate
(DBT) (3). The synthesis of 2,3-bis(8-methoxyoctanoyl) DBT (3a) is
presented as an example. DBT (0.67 mmol), 2a (1.40 mmol), and
catalytic dimethylaminopyridine (DMAP, 0.13 mmol) were dissolved in
anhydrous DCM (10 mL) under argon. Upon complete dissolution, 1-
ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI, 2.80 mmol)
was added as a coupling reagent and the reaction was stirred overnight
under argon. The reaction mixture was diluted with DCM (25 mL) and
washed with aqueous solutions of 10% potassium bisulfite (3 × 40 mL)
and saturated sodium bicarbonate (3 × 40 mL) to remove the EDCI
urea byproduct and unreacted 2a, respectively. The organic layer was
then washed with brine (40 mL) and dried over MgSO4, and the product
(3) isolated in vacuo. As the product appeared as a viscous liquid for the
shorter aliphatic chain lengths, yield was not calculated. Instead, a two-
step yield was calculated following the next synthetic step. The detailed
characterization of 3a−c can be found in the Supporting Information.
2.3.3. Synthesis of 2,3-Bis(n-methoxyalkanoyl) TA (4). The
synthesis of 2,3-bis(8-methoxyoctanoyl) TA (4a) is presented as an
example. 3a (0.67 mmol, theoretical) was deprotected following
modified literature procedures,34,43 using H2(g) and a 10% w/w
palladium on carbon (Pd/C) catalyst in a 1:1 DCM/MeOH solvent
system (HPLC grade, 6 mL total). The reaction mixture was passed
through a Celite filter using 1:1 DCM/MeOH (HPLC grade, 300 mL
total) to remove the catalyst, and the filtrate concentrated in vacuo. Pure
product was precipitated from (4a) or triturated in (4b and 4c) hexanes
and isolated via vacuum filtration.
CDCl3): δ 6.79 (b, 1H, CONH), 5.62 (dd, 2H, CH), 3.63 (m, ∼200H,
CH2CH2O), 3.35 (m, ∼13H, 2OCH3, OCH2), 2.41 (m, 4H, CH2CO),
1.59 (m, 8H, CH2), 1.32 (b, 12H, CH2). Mw, 2.5 kDa; PDI, 1.1.
1
5a5. Yield: 1.34 g, 85% (off-white powder). H NMR (400 MHz,
CDCl3): δ 6.66 (b, 1H, CONH), 5.62 (dd, 2H, CH), 3.64 (m, ∼500H,
CH2CH2O), 3.35 (m, ∼13H, 2OCH3, OCH2), 2.42 (m, 4H, CH2CO),
1.60 (m, 8H, CH2), 1.33 (b, 12H, CH2). Mw, 6.2 kDa; PDI, 1.1.
1
5b2. Yield: 0.22 g, 63% (beige waxy solid). H NMR (500 MHz,
CDCl3): δ 6.62 (b, 1H, CONH), 5.58 (dd, 2H, CH), 3.65 (m, ∼200H,
CH2CH2O), 3.34 (m, ∼13H, 2OCH3, OCH2), 2.44 (m, 4H, CH2CO),
1.60 (m, 8H, CH2), 1.30 (b, 20H, CH2). Mw, 2.3 kDa; PDI, 1.1.
5b5. Yield: 0.34 g, 98% (off-white solid). 1H NMR (400 MHz,
CDCl3): δ 6.70 (b, 1H, CONH), 5.58 (dd, 2H, CH), 3.64 (m, ∼500H,
CH2CH2O), 3.34 (m, ∼13H, 2OCH3, OCH2), 2.41 (m, 4H, CH2CO),
1.59 (m, 8H, CH2), 1.29 (b, 20H, CH2). Mw, 6.3 kDa; PDI, 1.1.
1
5c2. Yield: 0.27 g, 55% (beige waxy solid). H NMR (500 MHz,
CDCl3): δ 6.90 (b, 1H, CONH), 5.56 (dd, 2H, CH), 3.65 (m, ∼200H,
CH2CH2O), 3.38 (m, ∼13H, 2OCH3, OCH2), 2.40 (m, 4H, CH2CO),
1.58 (m, 8H, CH2), 1.27 (b, 28H, CH2). Mw, 2.6 kDa; PDI, 1.1.
1
5c5. . Yield: 0.13 g, 78% (off-white solid). H NMR (500 MHz,
CDCl3): δ 6.67 (b, 1H, NH), 5.54 (dd, 2H, CH), 3.65 (m, ∼500H,
CH2CH2O), 3.38 (m, ∼13H, 2OCH3, OCH2), 2.41 (m, 4H, CH2CO),
1.60 (m, 8H, CH2), 1.27 (b, 28H, CH2). Mw, 6.4 kDa; PDI, 1.1.
2.3.5. Synthesis of Aliphatic TA Derivatives (6). The synthesis of
aliphatic TA derivatives is presented as an example (6a). Aliphatic TA
derivatives were synthesized following a modified literature procedure.44
In brief, L-TA (7.00 mmol) and zinc chloride (2.20 mmol) were
suspended neat in decanoyl chloride (52.50 mmol) and heated to 95 °C.
After stirring 24 h, the reaction mixture was cooled to room temperature
and quenched with H2O (30 mL) and diethyl ether (100 mL), then
vigorously stirred for 30 min. This solution was washed with H2O (5 ×
100 mL), and the organic layer concentrated in vacuo to yield a viscous
brown liquid. Pure product (6) was precipitated from 1 L of stirring
hexanes and isolated via vacuum filtration. The length of the product’s
aliphatic chains will be indicated by the lettering a−c, with a given TA
derivative (e.g., 6a) having aliphatic chains of analogous length to the
2,3-Bis(8-methoxyoctanoyl) TA (4a). Two-step yield: 0.24 g, 77%
(off-white solid). 1H NMR (400 MHz, CDCl3): δ 5.72 (s, 2H, CH), 3.57
(m, 2H, OCH2), 3.49 (m, 2H, OCH2), 3.40 (s, 6H, OCH3), 2.59 (quin,
2H, CH2CO), 2.39 (quin, 2H, CH2CO), 1.66 (m, 8H, CH2), 1.33 (m,
12H, CH2). 13C NMR (500 MHz, CDCl3): δ 172.86, 168.10, 73.07,
70.90, 58.34, 34.06, 28.39, 28.20, 28.18, 25.55, 24.85. IR (cm−1, KBr):
3650−3300 (OH, COOH), 1762 (CO, ester), 1736 (CO,
COOH). ESI-MS m/z: 461.1 (M − 1).
2,3-Bis(10-methoxydecanoyl) TA (4b). Two-step yield: 0.53 g, 85%
(white powder). 1H NMR (500 MHz, CDCl3): δ 5.76 (s, 2H, CH), 3.46
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a
Scheme 1. Synthetic Scheme for Ether-AMs
a
The PEG Mw used will be denoted numerically in kilodaltons as a subscript with x = 45 yielding 2 kDa PEG and x = 113 yielding 5 kDa PEG (e.g.,
5a2 and 5a5 signify an ether-AM with a 2 kDa and 5 kDa PEG tail, respectively).
previously discussed n-methoxyalkanoyl derivatives (e.g., 4a). The
detailed characterization of 6a−c can be found in the Supporting
Information.
emission shifts from 332 to 334.5 nm. The ratio of absorption of pyrene
in micelles (334.5 nm) to pyrene alone (332 nm) was thus plotted
against the logarithm of AM concentration, and the inflection point of
this curve was taken as the CMC.25
2.3.6. Synthesis of Alkyl-AMs (7). The synthesis of alkyl-AMs is
presented as an example (7a2). Alkyl-AMs were prepared in the same
manner as were the previously discussed ether-AMs, using 6a (0.50
mmol), DPTS (0.17 mmol), mPEG-amine (0.17 mmol), and DCC
(0.53 mmol). Additional anhydrous DMF was used if necessary to fully
solubilize 6 prior to adding it to mPEG-amine. The PEG Mw used to
synthesize the alkyl-AM will also be denoted numerically in kilodaltons
as a subscript (e.g., 7a2).
2.5. Dynamic Light Scattering (DLS) Measurements. DLS
analysis was performed on Zetasizer Nano ZS90 instrument (Malvern
Instruments, Southboro, MA) in triplicate with a 90° scattering angle.
AM samples were dissolved in HPLC grade H2O (10 mg/mL) and
equilibrated for 24 h at 37 °C with gentle agitation (60 rpm). Solutions
were passed through 0.45 μm PTFE syringe filters prior to size
measurements, and Z-average sizes were collected and analyzed.
2.6. Cell Culture. Peripheral blood mononuclear cells (PBMCs)
were isolated from human buffy coats (Blood Center of New Jersey; East
Orange, NJ) by centrifugation through Ficoll-Paque density gradient
(GE Healthcare). PBMCs were plated into T-175 flasks, and monocytes
selected via adherence after 24 h. Monocytes were cultured for 7 days in
RPMI 1640 (ATCC) supplemented with 10% fetal bovine serum (FBS),
1% penicillin/streptomycin, and 50 ng/mL macrophage colony-
stimulating factor (M-CSF) for differentiation into human monocyte-
derived macrophages (HMDMs).30,35
2.7. Cell Viability Studies. To screen cellular toxicity of the AMs,
the alamarBlue assay was carried out according to manufacturer’s
protocol. In brief, HMDMs were plated in a 96-well plate at 150 000
cells/mL in basal media (RPMI 1640 supplemented with 10% FBS and
1% penicillin/streptomycin) and allowed to rest for 24 h. Cells were
then treated with the desired concentration of AM (10−5 - 10−3 M)
diluted in basal media for 24 h. Following incubation, the treatment
(media containing specific AM concentrations) was removed and
alamarBlue (diluted 1 to 10 in basal media) was added to each well and
cells incubated for 24 h. The supernatant was then transferred to a new
plate and absorbance read on a spectrophotometer (Infinite 200 Pro,
7a2. Yield: 0.25 g, 60% (off-white waxy solid). 1H NMR (400 MHz,
CDCl3): δ 6.97 (b, 1H, CONH), 5.55 (dd, 2H, CH), 3.65 (m, ∼200H,
OCH2), 3.39 (s, 3H, OCH3), 2.40 (m, 4H, CH2CO), 1.62 (m, 4H,
CH2), 1.26 (b, 24H, CH2), 0.88 (t, 6H, CH3). Mw, 1.9 kDa; PDI, 1.1.
7a5. Yield: 0.40 g, quantitative (off-white solid). 1H NMR (400 MHz,
CDCl3): δ 6.90 (b, 1H, CONH), 5.53 (dd, 2H, CH), 3.66 (m, ∼500H,
OCH2), 3.38 (s, 3H, OCH3), 2.39 (m, 4H, CH2CO), 1.63 (m, 4H,
CH2), 1.26 (b, 24H, CH2), 0.88 (t, 6H, CH3). Mw, 5.3 kDa; PDI, 1.1.
7b2. Yield: 0.30 g, 60% (off-white waxy solid). 1H NMR (400 MHz,
CDCl3): δ 6.84 (b, 1H, CONH), 5.54 (dd, 2H, CH), 3.66 (m, ∼200H,
OCH2), 3.38 (s, 3H, OCH3), 2.40 (m, 4H, CH2CO), 1.62 (m, 4H,
CH2), 1.25 (b, 32H, CH2), 0.88 (t, 6H, CH3). Mw, 2.1 kDa; PDI, 1.1.
7b5. Yield: 0.36 g, quantitative (off-white solid). 1H NMR (400 MHz,
CDCl3): δ 6.85 (b, 1H, CONH), 5.56 (dd, 2H, CH), 3.65 (m, ∼500H,
OCH2), 3.38 (s, 3H, OCH3), 2.41 (m, 4H, CH2CO), 1.61 (m, 4H,
CH2), 1.25 (b, 32H, CH2), 0.89 (t, 6H, CH3). Mw, 5.4 kDa; PDI, 1.1.
7c2. Yield: 0.28 g, 55% (off-white waxy solid). 1H NMR (500 MHz,
CDCl3): δ 6.91 (b, 1N, CONH), 5.55 (dd, 2H, CH), 3.66 (m, ∼200H,
OCH2), 3.38 (s, 3H, OCH3), 2.41 (m, 4H, CH2CO), 1.62 (m, 4H,
CH2), 1.26 (b, 40H, CH2), 0.88 (t, 6H, CH3). Mw:, 2.1 kDa; PDI, 1.1.
7c5. Yield: 0.50 g, quantitative (off-white solid). 1H NMR (400 MHz,
CDCl3): δ 6.92 (b, 1H, CONH), 5.53 (dd, 2H, CH), 3.64 (m, ∼500H,
OCH2), 3.38 (s, 3H, OCH3), 2.40 (m, 4H, CH2CO), 1.62 (m, 4H,
CH2), 1.25 (b, 40H, CH2), 0.88 (t, 6H, CH3). Mw, 5.8 kDa; PDI, 1.1.
2.4. Critical Micelle Concentration (CMC) Measurements. AMs
were dissolved in HPLC grade H2O and diluted to a series of
concentrations ranging from 1 × 10−3 to 1 × 10−10 M. Separately, a stock
solution of pyrene was prepared in HPLC grade acetone (5 × 10−6 M)
and 0.5 mL of this solution was added to a series of vials. Acetone was
removed in vacuo, and AM solutions (5 mL) were added. AM-pyrene
solutions were incubated for 48 h at 37 °C with gentle agitation (60
rpm) to allow pyrene to partition into the AM micelles. Fluorescence
studies were then conducted on a RF-5301PC spectrofluorometer
(Shimadzu Scientific Instruments, Columbia, MD), using pyrene as the
fluorescent probe. Emission was measured from 300−360 nm with a 390
nm excitation wavelength. Upon micelle formation, pyrene partitions
into the micelle hydrophobic core and the maximum wavelength
Tecan, Mannedorf, Switzerland) at 570 and 600 nm.
̈
2.8. OxLDL Uptake by Macrophages. HMDMs were cocultured
with 3,3′-dioctadecyloxacarbocyanine- (DiO-)labeled oxLDL (1 μg/
mL) and unlabeled oxLDL (4 μg/mL) with or without different AM
concentrations, ranging from 10−5 to 10−3 M, in basal media (RPMI
1640 supplemented with 10% FBS and 1% penicillin/streptomycin) for
24 h. Treatments were then removed and replaced with cold phosphate
buffered saline (PBS) and placed on ice packs. HMDMs were removed
from wells by vigorous pipetting and transferred to 5 mL tubes,
centrifuged at 1000 rpm for 10 min, and fixed in 1% paraformaldehyde
(150 μL). The oxLDL fluorescence associated with HMDMs was
quantified on a FACScalibur (Beckton Dickenson, Franklin Lakes, NJ)
flow cytometer, collecting 10 000 events/sample, and analyzed with
Flow Jo software (Treestar, Ashland, OR). This study included a
minimum of three experimental replicates. Data is presented as percent
of oxLDL uptake as determined by the following equation:
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Figure 2. 1H NMR spectra for 5a2 ether-AM synthesis as an example: 8-methoxyoctanoic acid 2a (A), 2,3-bis(8-methoxyoctanoyl) DBT 3a (B), 2,3-
bis(8-methoxyoctanoyl) TA 4a (C), and ether-AM 5a2 (D).
DiO oxLDL MFI of treatment sample
DiO oxLDL MFI of oxLDL only control sample
were attempted in which methoxy-terminated alkanoyl chlorides
%oxLDL uptake = 100 ×
were prepared from thionyl chloride and reacted with L-TA in
the presence of a Lewis acid catalyst.25 These conditions,
however, required an excess of 2 and resulted in incomplete
acylation. As an alternative, 2 was coupled to a protected TA
backbone (DBT, Scheme 1) through EDCI coupling, resulting in
complete acylation while using near stoichiometric amounts of 2.
The acylated DBT derivative (3) was subsequently deprotected
via hydrogenolysis, using a 10% w/w Pd/C catalyst, to give the
acylated TA product (4). To finally generate the ether-AMs (5),
4 was coupled to mPEG-NH2 using DCC coupling with a DPTS
catalyst. A stoichiometric excess of 4 and DCC ensured that PEG
coupled to only one of 4’s two carboxylic acids. Ether-AM
precursors’ chemical structures were confirmed via NMR and
FT-IR spectroscopies and MS, while ether-AM synthesis was
verified by 1H NMR spectroscopy and GPC.
2.9. Statistical Analysis. OxLDL uptake studies were conducted in
experimental triplicate. The results were then evaluated using Student’s t
test, with significance criteria assuming a 95% confidence level (P <
0.05). Standard error of the mean is reported in the form of error bars on
the graphs of the final data.
3. RESULTS AND DISCUSSION
3.1. AM Synthesis and Characterization. Ether-AMs were
synthesized to assess whether incorporating heteroatoms,
specifically ethers, into the terminal-end of alkyl chains of the
hydrophobic domain would enhance AM bioactivity by
promoting hydrogen-bonding interactions with scavenger
receptor binding pockets, potentially reducing oxLDL uptake
even further. To this end, a series of methoxy-terminated long-
chain carboxylic acids was first synthesized to serve as the AMs’
hydrophobic arms. Using the Williamson ether synthesis,
potassium methoxide, generated from MeOH and KOH, was
reacted with bromo-terminated alkanoic acids (1) to yield
methoxy-terminated alkanoic acids (2) via an SN2 reaction.45 To
acylate the TA backbone with 2, typical AM synthetic methods
1H NMR spectroscopy was critical in affirming successful
synthesis of ether-AMs and their precursors. Figure 2 presents
the NMR spectra obtained during the synthesis of 5a2, as an
example. Successful 8-methoxyoctanoic acid (2a) synthesis was
confirmed by the appearance of a triplet and singlet at 3.32 and
3.27 ppm (d in Figure 2A), corresponding to the methylene and
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a
Scheme 2. Synthetic Scheme for Alkyl-AMs
a
The PEG Mw used will be denoted numerically in kilodaltons as a subscript with x = 45 yielding 2 kDa PEG and x = 113 yielding 5 kDa PEG (e.g.,
7a2 and 7a5 signify an alkyl-AM with a 2 kDa and 5 kDa PEG tail, respectively).
methyl protons adjacent to the methoxy oxygen atom. The
relative integration of DBT’s methine singlet (g in Figure 2B) to
signals associated with the 8-methoxyoctanoyl arms demon-
strated complete acylation to form 3a, with two aliphatic arms
present per DBT backbone. Disappearance of the aromatic and
benzyl proton signals (e and f in Figure 2B) illustrated the
complete deprotection of 3a to produce 4a (Figure 2C). Finally,
successful PEGylation to yield 5a2 was confirmed by the
appearance of a large ∼200 proton PEG multiplet (i in Figure
2D). The 1:2 ratio of the amide proton signal (h in Figure 2D) to
the methine proton signal of the TA backbone further indicated
that PEG was only conjugated to one side of the TA derivative.
In addition to synthesizing ether-AMs, a series of analogous
alkyl-AMs (Scheme 2) was prepared to compare the influence of
hydrogen-bonding and hydrophobic interactions on AM
physicochemical and biological properties. These alkyl-AMs (7,
Figure 1) only differed from ether-AMs in that the methoxy
oxygen atom was replaced with a methylene group, yielding AMs
with saturated aliphatic arms of analogous lengths to the ether-
AMs. To synthesize these analogues, previously reported
methods were used in which L-TA was reacted with an acyl
chloride to generate a modified TA hydrophobe (6) that was
subsequently coupled to mPEG-amine using DCC. AM and AM
precursor chemical structures were confirmed via the afore-
mentioned methods.
most methylenes within the hydrophobic domain, these AMs
were likely less soluble in water as compared to 5a and 5b,
resulting in lower CMC values. Furthermore, as micellization is
entropically driven by the displacement of water molecules from
the hydrophobic domain, these results likely stem from 5c AMs
having more water molecules associated with their hydrophobic
domains prior to micellization, resulting in a larger entropic
increase and thus a greater free energy decrease upon
micellization.46 Although 5c AMs’ CMC values were lower
than those of other ether-AMs, all ether-AMs exhibited CMCs
near 10−4 M. In comparing ether-AMs to their respective alkyl-
AM analogues (e.g., 5a2 vs 7a2), all alkyl-AMs exhibited CMC
values lower than the analogous ether-AMs, ranging approx-
imately from 10−6 to 10−4 M (Table 1). Similar to the trends
among the ether-AMs (5), the analogues’ (7) lower CMC values
likely result from the increased hydrophobicity of 7 as compared
to 5. Given that lower CMCs can provide greater stability against
dilution, the alkyl-AMs would be more likely to remain in
micellar assemblies when diluted under physiological con-
ditions.47
To determine whether AM micelles exhibited nanoscale sizes
suitable for biomedical applications, the micelles were next
measured using DLS. Prior to DLS measurements, AMs were
incubated in water for 24 h at 37 °C to mimic physiological
conditions. In comparing the ether-AMs, 5a and 5b exhibited
sizes near 100 nm while 5c exhibited smaller sizes, regardless of
PEG Mw (Table 1). The smaller sizes of 5c AMs may result from
enhanced hydrophobic interactions upon micellization due to
their larger hydrophobic domains,48 which could overcome
potential repulsion caused by the methoxy moieties. Further-
more, other investigators have reported that different micelle
morphologies (e.g., spindle-like, rod-like, or bowl-like) result
when increasing the length of block copolymers’ hydrophobic
domains.49−52 Although the DLS method employed assumes
Brownian motion of sphere-shape particles, it is plausible that
alternate micelle morphologies are present, giving rise to the
smaller particle sizes. Despite the range in sizes (12−119 nm), all
ether-AMs remained within a size range (10−200 nm)
considered optimal for enhanced stability in vivo.47 In contrast
to ether-AMs, all alkyl-AMs exhibited smaller sizes, ranging from
8 to 15 nm. As these analogues are more hydrophobic than the
ether-AMs, these results correlate well with the ether-AM size
trends, suggesting larger hydrophobic domains yield stronger
hydrophobic interactions and smaller micelle sizes. Finally, all
2000 Mw AMs exhibited smaller sizes than their 5000 Mw
counterparts (e.g., 5a2 vs 5a5). This phenomenon likely resulted
from 2000 Mw AMs’ smaller PEG size, as seen in previous
Once the synthesis of all ether- and alkyl-AMs was confirmed,
their physicochemical properties were evaluated. When AM
concentrations in aqueous environments exceed a CMC, they
self-assemble into micelles; this transition was measured using an
established fluorimetry assay.25 In evaluating ether-AMs alone, it
was observed that, while keeping the PEG Mw constant, 5c AMs
exhibited slightly lower CMC values than those of 5a and 5b,
which were comparable (Table 1). As 5c AMs contained the
Table 1. Physicochemical Properties of Ether-AMs (5) and
Alkyl-AMs (7), with 2000 Mw Compounds Shown on the Left
and 5000 Mw Compounds Shown on the Right
2000 Mw
CMC (M)
5000 Mw
CMC (M)
AM
size (nm)
AM
size (nm)
5a2
7a2
5b2
7b2
5c2
7c2
1.97 × 10−4
9.79 × 10−5
1.99 × 10−4
3.43 × 10−5
1.24 × 10−4
6.94 × 10−6
102.3 0.3
8.0 0.1
93.5 1.4
7.3 0.1
12.5 0.0
8.1 0.1
5a5
7a5
5b5
7b5
5c5
7c5
2.19 × 10−4
5.79 × 10−5
2.02 × 10−4
3.20 × 10−5
9.01 × 10−5
7.49 × 10−6
111.4 0.8
15.3 0.1
119.8 2.0
11.2 0.1
28.7 0.5
10.6 0.2
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Figure 3. Effect of administering varying concentrations of 5000 Mw (A) and 2000 Mw (B) ether-AMs (dark gray) and alkyl-AM analogues (light gray)
on percent of oxLDL uptake in HMDMs. AMs of specific alkyl lengths are grouped between the dashed lines, and the AM treatments not investigated
due to cytotoxicity issues are indicated as text on the graph. Significant deviations from the oxLDL positive control (black) are denoted by asterisks (∗)
on the graph.
literature.53,54 Despite variations in sizes, ether-AMs exhibited
suitable sizes for drug delivery applications, while some alkyl-
AMs displayed sizes slightly smaller than the desirable size range.
3.2. AM Biological Properties. Prior to assessing ether- and
alkyl-AMs’ antiatherogenic potential, cytotoxicity was screened
vitro studies were conducted in the presence of serum proteins to
mimic physiological conditions; however, serum proteins are
capable of disrupting micelle integrity30,58−60 and may impact
AM bioactivity. Previous studies have indicated that in
comparison to serum-free conditions AMs’ efficacy decreases
in the presence of serum proteins, which may result from serum
protein interactions with AMs.32 The presence of serum
proteins, therefore, allows for a more realistic understanding of
ether- and alkyl-AM bioactivity.
in HMDMs at concentrations ranging from 10−5 to 10−3
M
(Supporting Information Figures S1 and 2). Treatments that
resulted in 70% or more viable cells were considered nontoxic.
While the most hydrophobic ether-AM, 5c2, was cytotoxic only
at the highest concentration administered (10−3 M), all other
ether-AMs were nontoxic at all concentrations tested. Of the
alkyl-AMs, 7b2 and 7c5 were cytotoxic at 10−3 M and 7c2
exhibited cytotoxicity at both 10−3 and 10−4 M. In agreement
with previously published results on nanoscale systems,55−57
these results suggest that as AM hydrophobicity is increased, the
macromolecules become more cytotoxic. Conversely, AMs with
larger PEG tails showed improved cell viability over AMs with
smaller molecular weight PEG tails (i.e., 5000 vs 2000 Da,
respectively). Furthermore, ether-AMs, containing two addi-
tional, ethereal oxygen atoms within their hydrophobic domain,
are better tolerated by HMDMs than alkyl-AMs when
administered at higher concentrations.
To assess the impact of the hydrophobic chain architecture on
AM antiatherogenic bioactivity, HMDMs were coincubated with
AMs at concentrations ranging from 10−5 to 10−3 M and
fluorescently labeled oxLDL. This broad range of concentrations
(e.g., above and below CMC values) was investigated to
determine the influence of concentration and the presence of
micelles (or unimers) on AM bioactivity. Furthermore, all in
As shown in Figure 3A, the hydrophobic chain composition
and AM concentration play an integral role in 5000 Mw PEG tail
AM antiatherogenic bioactivity. As the ether-AMs’ aliphatic chain
length increases, their ability to inhibit oxLDL uptake increases,
such that 5c5 > 5b5 > 5a5, with 5a5 exhibiting no bioactivity. This
phenomenon is concentration-dependent, with higher ether-AM
concentrations resulting in more oxLDL uptake inhibition,
except for 5a5. For example, while HMDMs treated with 10−4
5c5 exhibit 73% oxLDL uptake, those cells incubated with 10−3
M
M
5c5 significantly repress oxLDL uptake to less than 2% (Figure
3A). Furthermore, 10−3 M 5c5 reduced the amount of oxLDL
internalized by cells by such a significant magnitude that only
5.3% of the cells had any oxLDL in them at all (Figure 4A). Given
that 5b5 administered at 10−3 M significantly inhibited oxLDL
uptake (Figure 3A) despite its larger-sized micelles, it appears
that micelle size alone does not dictate AMs’ antiatherogenic
potential. Further, as 5a5 exhibited no bioactivity at 10−3
M
despite its micellar assembly, it is likely that the chemical
composition of the AM more strongly influences bioactivity than
the corresponding micellar configuration. Although AM micellar
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Figure 4. Percent of HMDMs positive for oxLDL after incubation with varying concentrations of 5000 Mw (A) and 2000 Mw (B) ether-AMs (dark gray)
and alkyl-AM analogues (light gray). AMs of specific alkyl lengths are grouped between the dashed lines, and the AM treatments not investigated due to
cytotoxicity are indicated as text on the graph. Significant deviations from the oxLDL positive control (black) are denoted by asterisks (∗) on the graph.
structure does not demonstrate a pronounced effect on AM
bioactivity, the increased size and PEG shielding provided by the
micellar assembly would likely increase the AMs biological
stability when administered in a clinical setting.26,47
bioactivity,34 it is plausible that the longer aliphatic chains
arrange more favorably within scavenger receptor binding
pockets through the aforementioned hydrophobic interactions.
In comparing the 2000 Mw ether-AMs to the corresponding
alkyl-AMs (Figure 3B), similar trends were apparent with AMs
showing reduced oxLDL uptake as their concentration, hydro-
phobicity, and aliphatic chain length increases. Only 7a2
significantly reduced the number of oxLDL positive HMDMs
(1.4%, Figure 4B) when administered at 10−3 M; fewer AM
concentrations were investigated for these studies, however, due
to toxicity. This data suggests that the amphiphilic balance
provided by the higher molecular weight PEG chains is critical for
minimizing cellular toxicity and highly hydrophobic domains are
detrimental to cellular viability. Although AMs containing 2000
Mw PEG are relatively more hydrophobic than their 5000 Mw
counterparts, PEG Mw did not have a pronounced effect on AM
bioactivity. These results agree with previously published results,
suggesting that while PEG size can modulate cytotoxicity, the
AM hydrophobic domain dominates antiatherogenic bioactiv-
ity.32 While relatively high micromolar concentrations are
required to achieve significant oxLDL uptake inhibition (i.e.,
10−3−10−4 M) and may pose biocompatibility concerns,
previous in vivo studies demonstrated that a previously
synthesized AM containing a larger hydrophobic domain with
a 5000 Mw PEG tail exhibits no significant toxicity in mice when
administered via intraperitoneal injection at approximately 4500
μM (2000 mg/kg).62 As the tested in vivo concentration is higher
than in vitro concentrations used in this work, the most potent
Alkyl-AMs with 5000 Mw PEG tails (Figure 3A, light gray)
show similar trends to the aforementioned ether-AMs (Figure
3A, dark gray), exhibiting reduced oxLDL uptake as AM
concentration and/or alkyl chain length are increased; however,
they were much more efficacious in preventing oxLDL uptake
than the ether-AMs. When administered at 10−4 M, for example,
7a5 (29%), 7b5 (4.7%), and 7c5 (1.5%) showed significantly
lower oxLDL uptake than their corresponding ether-AMs 5a5
(112%), 5b5 (108%), and 5c5 (73%). In fact, the 10−3
M
concentrations of the 5000 Mw alkyl-AMs were so potent that less
than 7% of HMDMs were positive for any oxLDL at all (Figure
4A). As alkyl-AMs repressed more oxLDL uptake than analogous
ether-AMs and AM potency increased with increasing aliphatic
arm length, these results indicate that hydrophobicity and the
length of AMs’ aliphatic arms play a more significant role than
hydrogen-bonding in modulating athero-protective bioactivity.
Given that macrophage scavenger receptors (e.g., CD36) contain
hydrophobic residues near their oxLDL binding pockets,38 it is
plausible that AMs primarily interact with scavenger receptors
through hydrophobic interactions, resulting in reduced oxLDL
uptake. Furthermore, previous literature suggests that the
scavenger receptor ligands of different lengths exhibit varying
activity, likely resulting from how the ligands arrange within the
receptor pocket.61 As previous research demonstrated that
increasing AM hydrophobicity does not always improve
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Figure 5. Dose response of most efficacious AM treatments.
ether- and alkyl-AMs could serve as viable atherosclerosis
treatments.
significance of hydrophobicity and aliphatic chain length in
modulating bioactivity, critical for the design of next-generation
antiatherogenic AMs.
Collectively, these studies demonstrate that increasing hydro-
phobicity through longer aliphatic chains and minimal
heteroatoms improves bioactivity. Although certain AM treat-
ments, such as 10−4 M 7a5, exhibited a significant reduction in
oxLDL uptake (29%, Figure 3A), the majority of HMDMs (98%,
Figure 4A) were still positive for oxLDL. While this treatment
reduced oxLDL uptake, oxLDL was still able to accumulate in
macrophages that could, over time, elicit the atherosclerotic
cascade. In comparing the dose response of the most efficacious
AMs tested (Figure 5), 7c5 is the most promising candidate for
antiatherosclerotic therapies: it has the lowest CMC value,
effectively inhibits both oxLDL uptake and accumulation in
macrophages at a lower concentration (10−4 M) than the other
AMs tested, and was not cytotoxic at this concentration. Studies
assessing this candidate’s in vivo bioactivity and biocompatibility
are the focus of future work, aiming to identify an appropriate
administration route that maximizes antiatherosclerotic efficacy
with minimal adverse effects.
ASSOCIATED CONTENT
* Supporting Information
■
S
Characterization measurements for 2,3-bis(n-methoxyalkanoyl)
dibenzyl tartrate intermediates and alkyl-AM precursors are
included, as well as results from cell viability screenings. This
material is available free of charge via the Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Mailing address: Department of Chemistry and Chemical
Biology, Rutgers University, 610 Taylor Road, Piscataway, New
Jersey 08854, USA. Telephone: 848-45-0361. Fax: 732-445-
7036. E-mail: keuhrich@rutgers.edu.
Notes
The authors declare no competing financial interest.
4. CONCLUSIONS
ACKNOWLEDGMENTS
■
Ether- and alkyl-AMs were synthesized to assess the relative
contributions of hydrogen-bonding and hydrophobic interac-
tions in oxLDL uptake inhibition in primary human macro-
phages. Ether-AMs displayed higher CMCs and larger hydro-
dynamic diameters than corresponding alkyl-AM analogues,
likely due to their decreased hydrophobicity and the presence of
hydrophilic moieties in the hydrophobic domain, respectively.
Hydrophobicity and aliphatic chain length also played a critical
role in the antiatherogenic potential of the AMs to inhibit oxLDL
accumulation, with more hydrophobic AMs (i.e., alkyl-AMs and/
or AMs with longer aliphatic arms) showing a greater reduction
in oxLDL uptake. However, the amphiphilic balance provided by
the higher Mw PEG tails and ether moieties were beneficial for
minimizing cellular toxicity. Therefore, AMs with larger PEG
components (i.e., 5000 Mw PEG tails) and larger hydrophobic
domains (i.e., longer alkyl chains and/or no ether moieties) were
the lead candidates due to their combined biocompatibility and
high oxLDL inhibition potential. These findings reinforce the
This study was supported by the National Institutes of Health
(NIH R21093753 to P.V.M. and NIH R01 HL107913 to P.V.M.
and K.E.U.) and the U.S. Department of Education fellowship for
Graduate Assistance in Areas of National Need (GAANN, A.F.).
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