Nanoscale amphiphilic macromolecules with variable lipophilicity and stereochemistry modulate inhibition of oxidized low-density lipoprotein uptake

Article abstract of DOI:10.1021/bm400537w

Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) and counteract foam cell formation, a key characteristic of early atherogenesis. To investigate the influence of lipophilicity and stereochemistry on the AMs' physicochemical and biological properties, mucic acid-based AMs bearing four aliphatic chains (2a) and tartaric acid-based AMs bearing two (2b and 2l) and four aliphatic chains (2g and 2k) were synthesized and evaluated. Solution aggregation studies suggested that both the number of hydrophobic arms and the length of the hydrophobic domain impact AM micelle sizes, whereas stereochemistry impacts micelle stability. 2l, the meso analogue of 2b, elicited the highest reported oxLDL uptake inhibition values (89%), highlighting the crucial effect of stereochemistry on biological properties. This study suggests that stereochemistry plays a critical role in modulating oxLDL uptake and must be considered when designing biomaterials for potential cardiovascular therapies.

Full text of DOI:10.1021/bm400537w

Communication
pubs.acs.org/Biomac
Nanoscale Amphiphilic Macromolecules with Variable Lipophilicity
and Stereochemistry Modulate Inhibition of Oxidized Low-Density
Lipoprotein Uptake
Dawanne E. Poree,† Kyle Zablocki,‡ Allison Faig,† Prabhas V. Moghe,‡,§ and Kathryn E. Uhrich*,†
‡
§
†Departments of Chemistry and Chemical Biology, Biomedical Engineering, and Chemical and Biochemical Engineering, Rutgers
University, Piscataway, New Jersey 08854, United States
ABSTRACT: Amphiphilic macromolecules (AMs) based on
carbohydrate domains functionalized with poly(ethylene glycol)
can inhibit the uptake of oxidized low density lipoprotein (oxLDL)
and counteract foam cell formation, a key characteristic of early
atherogenesis. To investigate the influence of lipophilicity and
stereochemistry on the AMs’ physicochemical and biological
properties, mucic acid-based AMs bearing four aliphatic chains
(2a) and tartaric acid-based AMs bearing two (2b and 2l) and four
aliphatic chains (2g and 2k) were synthesized and evaluated. Solution aggregation studies suggested that both the number of
hydrophobic arms and the length of the hydrophobic domain impact AM micelle sizes, whereas stereochemistry impacts micelle
stability. 2l, the meso analogue of 2b, elicited the highest reported oxLDL uptake inhibition values (89%), highlighting the crucial
effect of stereochemistry on biological properties. This study suggests that stereochemistry plays a critical role in modulating
oxLDL uptake and must be considered when designing biomaterials for potential cardiovascular therapies.
structure has been systematically varied to determine the role of
PEG chain length and architecture, carboxylic acid location,
type and number of anionic charges, and rotational motion of
the anionic group.14 The role that comparative hydrophobicity
and stereochemistry play in inhibiting oxLDL uptake, however,
has not been actively explored. Based on previous molecular
modeling and experimental studies, the hydrophobic domain of
these AMs appears to be actively involved in binding to
macrophage scavenger receptors.15 These previous studies
correlate well with literature that suggests that hydrophobic
interactions play a major role in protein−polymer complex-
ation.16−18
As a preliminary study to probe the effect of lipophilicity on
the polymer’s physicochemical and biological properties, our
research group compared 2a to an analogous AM comprised of
an L-tartaric acid (L-TA) backbone bearing only two aliphatic
chains (2b, Figure 1a). Investigating the physicochemical
properties of these two AMs showed that an increase in
lipophilicity rendered more stable micelles, as determined by
the critical micelle concentration (CMC, a measure of solution
stability), with larger hydrodynamic radii. To investigate the
impact of lipophilicity on their biological properties, these AMs
were tested for their ability to inhibit oxLDL uptake in
peripheral blood mononuclear cells (PBMCs) under serum-free
conditions. While both polymers inhibited oxLDL uptake, 2a
was more efficacious, inhibiting 52% of oxLDL uptake in
PBMCs compared to 35% inhibition achieved by 2b (Figure
I. INTRODUCTION
Atherosclerosis, a disease characterized by occlusion of the
arteries, is triggered by the build-up of oxidized low density
lipoprotein (oxLDL) in vascular intima.1 The oxLDL
accumulation generates an inflammatory response, resulting in
the recruitment of circulating monocytes, followed by their
differentiation into macrophages, resulting in the upregulation
of macrophage scavenger receptors.2 The uptake of oxLDL is
mediated by these scavenger receptors, namely scavenger
receptor A (SR-A) and cluster of differentiation 36 (CD36),3−5
leading to unregulated cholesterol accumulation and foam cell
formation, a key characteristic of the onset of atherogenesis.6,7
To date, cholesterol-lowering therapies (i.e., statins) are the
most common methods for management of the long-term
effects of atherosclerosis. These drugs indirectly ameliorate the
cascade of atherosclerosis by decreasing cholesterol synthesis;
however, the ultimate impact on the deposition of oxLDL in
the blood vessel walls has not been clearly established. A more
direct and promising approach in the treatment and prevention
of atherosclerosis involves designing functional inhibitors
against scavenger receptors to abrogate uncontrolled oxLDL
uptake.8−11 Our research group has previously prepared
nanoscale amphiphilic macromolecules (AMs) capable of
inhibiting oxLDL uptake through competitive inhibition of
SR-A and CD36 scavenger receptors in IC21 macrophage
cells.12 Comprised of a mucic acid backbone, four aliphatic
chains, and a poly(ethylene glycol) (PEG) tail, these
biocompatible AMs (2a, Figure 1a) form nanoscale micelles
in aqueous media at relatively low critical micelle concen-
trations (10−7 M).13 To determine the key structural
components critical for oxLDL uptake inhibition, this AM
Received: April 16, 2013
Revised: June 18, 2013
Published: June 24, 2013
© 2013 American Chemical Society
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Figure 1. (a) Chemical structures of nanoscale AMs bearing four and two aliphatic arms, respectively. (b) AM inhibition of oxLDL in PBMC
macrophages.
acid monohydrate was azeotropically distilled with toluene to remove
1b). Although these results suggest that lipophilicity impacts
physicochemical and biological properties, it must be noted that
the sugar backbones of 2a and 2b have different stereo-
chemistries: mucic acid is a chiral, optically inactive, meso
compound, and L-TA is chiral, but optically active. Studies
performed by our research group and others have demon-
strated that stereochemistry can greatly impact a polymer’s
physicochemical and biological properties.15b,19−21 Further-
more, because chirality influences numerous biological events/
processes, stereoselective interactions between chiral materials
and biological systems has been the topic of recent reviews.22 It
is, therefore, possible that this disparity in the properties of 2a
and 2b is a consequence of lipophilicity, stereochemistry, or
both. Investigation into the contribution each factor makes to
our AMs’ biological activities would, therefore, aid our
understanding of the scavenger receptor binding mechanism
and enable our ability to optimize polymer design for
atherosclerotic treatments.
Herein, we present the synthesis of novel nanoscale AMs
comprised of an L-TA backbone that bears four aliphatic
chains, with the goal of ascertaining the influence of
lipophilicity on polymer properties. Preparation of these AMs
is achieved in two manners: (1) growing dendrons from the
hydroxyl groups of L-TA, thus incorporating branching onto
the sugar backbone (i.e., dendronized) or (2) coupling two L-
TA backbones to each other, yielding an AM with a disugar
backbone (i.e., disugar). The physicochemical properties of
these polymers are assessed as well as their ability to inhibit
oxLDL uptake in PBMC macrophages. Additionally, a meso
analog of (2b) was prepared (called 2l) to determine the
influence of stereochemistry on the AM properties.
water (3 × 50 mL) and dried under high vacuum for 4 h.
1
2.2. Instrumentation. H NMR spectra were obtained using a
Varian 400 or 500 MHz spectrophotometer with TMS as the internal
reference. Samples were dissolved in CDCl3, or CDCl3 with a few
drops of DMSO-d6 if necessary. IR spectra were recorded on a
ThermoScientific Nicolet is10 series spectrophotometer using
OMNIC software by solvent-casting samples on a salt plate. Mass
spectrometry was done on ThermoQuest Finnigan LCQ-DUO system
that includes 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 HPLC-grade CH2Cl2. Molecular
weights (MW) were determined using size exclusion chromatography
(SEC) with respect to PEG standards (Sigma-Aldrich) on a Waters
Stryagel HR 3 THF column (7.8 × 300 mm). The Waters LC system
(Milford, MA) was equipped with a 2414 refractive index detector, a
1515 isocratic HPLC pump, and 717plus autosampler. Samples (10
mg/mL) were dissolved in THF and filtered using 0.45 μm pore size
nylon or PTFE syringe filters (Fisher Scientific). Dynamic light
scattering (DLS) analysis was carried out on a Zetasizer nanoseries
ZS90 (Malvern instruments) in triplicate. CMC studies were carried
out on a Spex fluoromax-3 spectrofluorometer (Jobin Yvon Horiba) at
25 °C in triplicate.
2.3. Synthesis. 2.3.1. Synthesis of 2c. 2c was prepared in the same
manner as the previously synthesized 0cM,25 using 2b (1.06 g, 0.19
mmol), N-hydroxysuccinimide (NHS) (0.09 g, 0.77 mmol), and N′-
dicyclohexylcarbodiimide (DCC) (1 M in DCM) (0.31 mL) to yield
1
2c as a white powder (0.92 g, 85%). H NMR (CDCl3): δ = 0.86 (t,
6), 1.26 (m, 32), 1.60 (b, 4), 2.39 (b, 4), 2.90 (s, 4), 3.41 (m, ∼400),
5.66 (s, 2); Mw = 5.5 kDa; PDI = 1.07.
2.3.2. Synthesis of 2d. 2d was prepared similar to the previously
prepared 1N,26 using 2c (0.51 g, 0.09 mmol), propylamine (48.7 μL,
0.73 mmol), and triethylamine (NEt3) (197.4 μL, 1.42 mmol) to yield
1
2d as a white powder (0.42 g, 82%). H NMR (CDCl3): δ = 0.85 (t,
6), 1.21 (m, 32), 1.58 (b, 4), 2.28 (b, 4), 3.38 (s, 2), 3.41 (m, ∼400),
4.42 (s, 2), 5.30 (s, 1), 5.74 (s, 1); Mw = 5.6 kDa; PDI = 1.06.
2.3.3. Synthesis of 2e. Lauryl-acylated tartaric acid23 (0.30 g, 0.59
mmol) and NHS (0.27 g, 2.36 mmol) were weighed into a round-
bottom flask and placed under Ar(g). Anhydrous dichloromethane
(DCM) and 6 mL of anhydrous dimethyl formamide (DMF) were
then added to the round-bottom flask to dissolve the reagents. 1.48
mL DCC (1 M in DCM) was added dropwise to the reaction flask
over 1 h via syringe pump. The reaction mixture was stirred at room
temperature under argon for 24 h, cooled, and the resulting white solid
precipitate (dicyclohexylurea) was removed by vacuum filtration. The
filtrate was washed with 0.1 N HCl (20 mL), followed by 50:50
2. MATERIALS AND METHODS
2.1. Materials. All reagents and solvents were purchased from
Sigma-Aldrich and used as received unless otherwise noted. High-
performance liquid chromatography (HPLC)-grade solvents were
used unless otherwise noted. Monomethoxy-poly(ethylene glycol)
(mPEG, Mn = 5000 Da) was azeotropically distilled with toluene prior
to use. The following compounds were prepared as previously
described: 2a,13 2b,23 and benzylidene-protected 2,2-bis-
(hydroxymethyl)propionic acid (BP-BMPA).24 (2l), a structural
analogue of 2b, was also prepared using the same procedure as 2b,
but using meso-tartaric acid monohydrate. Prior to use, meso-tartaric
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Figure 2. Synthetic scheme for linear disugar AM, 2g.
brine:water (2 × 20 mL), dried over MgSO4, and concentrated via
rotary evaporation. The product was precipitated from hexanes
yielding 2e as a white solid (0.42 g, 29%). IR (cm−1, thin film from
CHCl3): 1831, 1745. 1H NMR (CDCl3): δ = 0.87 (t, 6), 1.26 (m, 32),
1.65 (m, 4), 2.48 (t, 4), 2.83 (s, 8), 6.23 (s, 2). 13C NMR
(CDCl3):14.34, 22.91, 24.58, 25.73, 29.46, 29.57, 29.68, 29.85, 32.14,
33.50, 68.61, 161.75, 167.98, 172.18. [M + NH4]+theo = 726.9, GC-MS:
(0.67 g, 92%). IR (cm-1, thin film from CHCl2): 3458, 3328, 1736. 1H
NMR (CDCl3): δ = 1.01 (s, 6), 3.59 (dd, 4), 4.58 (dd, 4), 4.72 (d, 2),
5.02 (d, 2), 5.44 (s, 2), 5.84 (s, 2), 7.25 (m, 20). 13C NMR (CDCl3):
17.76, 42.91, 68.21, 71.42, 73.22, 73.73, 101.99, 126.54, 128.25,
128.30, 128.66, 134.85, 138.04, 165.45, 172.62.
2.3.7. Synthesis of 2i. (2i) was prepared using an established
literature procedure,24 using 2h (0.65 g) 10% w/w Pd/C, HPLC grade
DCM (15 mL), and HPLC grade methanol (15 mL), yielding 2i as
white crystals (0.31 g, 97%). IR (cm−1, thin film from THF): 3408
(br), 1742.1H NMR (CDCl3): δ = 1.01 (s, 6), 3.52 (m, 8), 5.39 (s, 2).
13C NMR (CDCl3): 17.76, 42.91, 68.21, 71.42, 73.22, 73.73, 165.45,
172.62.
[M + NH4]+ = 726.1.
calc
2.3.4. Synthesis of 2f. 2d (0.12 g, 0.02 mmol) was added to a
round-bottom flask and dissolved in 5 mL of anhydrous DCM and 5
mL of anhydrous DMF. After the addition of NEt3 (50 μL, 0.36
mmol), the reaction mixture was allowed to stir under Ar(g). 2e
(0.015 g, 0.02 mmol) was dissolved in DCM (5 mL) and added
dropwise to the reaction flask via syringe pump at a rate of 1 mL/h.
Upon complete 2e addition, the reaction was allowed to stir at room
temperature under argon for 24 h. The reaction was filtered to remove
insoluble triethylamine salts. The filtrate was washed with 0.1 N HCl
(20 mL), followed by 50:50 brine:water (2 × 20 mL), dried over
MgSO4, and concentrated via rotary evaporation. The product was
precipitated from diethyl ether yielding 2f as a white solid (0.097 g,
2.3.8. Synthesis of 2j. 2i (0.36 g, 0.95 mmol), lauroyl chloride (1.1
mL, 4.76 mmol), and zinc chloride (0.04 g, 0.30 mmol) were added to
a round-bottom flask. Anhydrous DCM (2 mL) was added, and the
reaction was stirred at room temperature under argon for 24 h. Water
(5 mL) and diethyl ether (10 mL) were added to quench the reaction.
After stirring for 1 h, the reaction mixture was diluted with diethyl
ether (20 mL) and washed with water (5 × 20 mL), dried over
MgSO4, and concentrated via rotary evaporation. The product was
precipitated from cold hexanes (refrigerated for 2 days) yielding 2j as
white crystals (0.34 g, 32%). IR (cm−1, thin film from CH2Cl2): 3514,
1
75%). H NMR (CDCl3): δ = 0.87 (t, 12), 1.21 (m, 64), 1.59 (b, 8),
2.38 (b, 8), 2.90 (s, 4), 3.41 (m, ∼400), 5.30 (s, 1), 5.74 (s, 1); Mw =
6.3 kDa; PDI = 1.07.
1
1746. H NMR (CDCl3): δ = 0.86 (t, 12), 1.26 (m, 70), 1.59 (b, 8),
2.29 (t, 8), 4.16 (m, 8), 5.62 (s, 2). 13C NMR (CDCl3): 13.08, 17.79,
21.67, 23.58, 23.68, 27.90, 28.05, 28.13, 28.16, 28.23, 28.32, 28.36,
28.38, 28.43, 28.55, 28.57, 30.89, 32.12, 32.31, 42.91, 68.21, 71.42,
73.22, 73.73, 165.45, 168.12. [M − 2H]−theo = 1109.1, GC−MS: [M −
2H]− = 1109.2.
2.3.5. Synthesis of 2g. Glycine (0.0015 g, 0.02 mmol) was added to
a round-bottom flask and dissolved in anhydrous DCM (5 mL) and
anhydrous DMF (5 mL). Upon addition of NEt3 (10 μL, 0.07 mmol),
the reaction mixture was allowed to stir under Ar(g). 2f (0.03 g, 0.005
mmol) was dissolved in 5 mL DCM and added dropwise to the
reaction flask via syringe pump at a rate of 1 mL/h. Upon complete 2e
addition, the reaction was allowed to stir at room temperature under
argon for 24 h. The reaction was filtered to remove insoluble
triethylamine salts The filtrate was washed with 0.1 N HCl (20 mL),
followed by 50:50 brine:water (2 × 20 mL), dried over MgSO4, and
concentrated via rotary evaporation. The product was precipitated
from diethyl ether yielding 2f as a white solid (0.01 g, 33%) 1H NMR
(CDCl3): δ = 0.87 (t, 12), 1.21 (m, 64), 1.59 (b, 8), 2.38 (b, 8), 3.41
(m, ∼400), 5.50 (s, 2); Mw = 6.3 kDa; PDI = 1.07.
2.3.9. Synthesis of 2k. 2k was prepared using an established
literature procedure,13 using 2j (0.20 g, 1.8 mmol), mPEG (0.28 g,
0.06 mmol), DCC (0.19 mL, 1.9 mmol), and 4-(dimethylamino)-
pyridinium p-toluene-sulfonate (DPTS) (0.02 g, 0.007 mmol) to yield
1
2k as a white powder (0.29 g, 85%). H NMR (CDCl3): δ = 0.88 (t,
12), 1.30 (m, 70), 1.61 (b, 8), 2.29 (t, 8), 3.63 (m, ∼400H), 4.18 (m,
8), 5.5 (s, 1), 5.7 (s, 1); Mw = 6.3 kDa; PDI = 1.15.
2.4. CMC Measurements. A solution of pyrene, a fluorescence
probe molecule, was made up to a concentration of 5 × 10−6 M in
acetone. Samples were prepared by adding 1 mL of pyrene solution to
a series of vials and allowing the acetone to evaporate. AMs were
dissolved in HPLC-grade water and diluted to a series of
concentrations from 1 × 10−3 M to 1 × 10−10 M. AM−pyrene
2.3.6. Synthesis of 2h. 2h was prepared using an established
literature procedure24 using dibenzyl-L-tartrate (0.33 g, 0.99 mmol),
BP-BMPA anhydride (1.05 g, 2.46 mmol), and 4-dimethylaminopyr-
idine (DMAP) (0.06 g, 0.49 mmol), yielding 2h as light yellow crystals
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Figure 3. Synthetic scheme for dendronized AM, 2k.
solutions (10 mL) were shaken overnight at 37 °C to allow partition of
the pyrene into the micelles. The concentration of pyrene in all
samples was 5 × 10−7 M. Emission was performed from 300 to 360
nm, with 390 nm as the excitation wavelength. The maximum
absorption of pyrene shifted from 332 to 334.5 nm on micelle
formation.27 The ratio of absorption of encapsulated pyrene (334.5
nm) to pyrene in water (332 nm) was plotted as the logarithm of
polymer concentrations. The inflection point of the curve was taken as
the CMC.
TE2000-S. oxLDL uptake was quantified using ImageJ and normalized
to conditions receiving no polymer treatment.
2.8. Statistical Analysis. Each in vitro experiment was performed
at least twice, and three replicate samples were investigated in each
experiment. Five images per well were captured and analyzed. The
results were then evaluated using analysis of variance (ANOVA).
Significance criteria assumed 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.
2.5. ClogP Calculations. ClogP values were derived using the
CambridgeSoft ChemDraw software. The calculated values were of the
AM hydrophobic domain as the PEG component was constant for all
polymers.
2.6. Cell Culture. 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 were selected via plastic
adherence by washing thrice with phosphate buffered saline (PBS)
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 M-CSF (macrophage colony-
stimulating factor) for differentiation into macrophages.
2.7. oxLDL Oxidation. PBMC-derived macrophages were
cocultured with 10 μg/mL of 3,3′-dioctadecyloxacarbocyanine (DiO)
labeled oxLDL (Kalen Biomedical) and nanolipoblocker (NLB)
micelles (10−5 to 10−7 M) for 24 h in serum-free RPMI 1640. Cells
were then fixed with 4% paraformaldehyde and counterstained with
Hoechst 33342 prior to epifluorescent imaging using a Nikon Eclipse
3. RESULTS AND DISCUSSION
Preparation of novel nanoscale AMs based on L-TA and
bearing four aliphatic chains was achieved via two synthetic
methods: (1) coupling two L-TA backbones, yielding an AM
with a linear backbone (referred to as “linear disugar” in this
paper); and (2) incorporating branch points by growing
dendrons from the L-TA hydroxyl groups (referred to as
“dendronized”). The linear disugar AM was prepared by
esterification of the previously synthesized 2b23 with NHS to
yield 2c. The NHS group was subsequently displaced by
ethylene diamine to form the amine-terminated AM, 2d.
Coupling of this polymer to a di-NHS, lauryl-acylated L-TA
(2e) yielded the NHS-capped linear disugar, 2f. Amidation
using glycine rendered the carboxylic acid-terminated disugar,
2g, as the final product (Figure 2). Polymers prepared at each
step in the synthesis were characterized via 1H NMR and SEC.
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The synthesis of the dendronized AM was based on a
divergent synthesis using an anhydride coupling developed by
Ihre et al.24 (Figure 3). Dibenzyl-L-tartrate was coupled with
the previously reported benzylidene-protected BP-BMPA
anhydride using DMAP as the acylating catalyst to afford 2h
at a 92% yield. The benzylidene protecting groups as well as the
benzyl esters were removed by catalytic hydrogenolysis using
H2(g) and 10% w/w Pd/C as catalyst. Upon removal of catalyst
by filtration, the deprotection rendered L-TA with four terminal
hydroxyl groups (2i) in near quantitative yields. Using the
dendronized L-TA, the corresponding AM was synthesized by
modifying a previously published method for the preparation of
2a, which has a mucic acid backbone.13 Briefly, the two-step
procedure involves acylating 2i with lauroyl groups followed by
coupling to PEG. During the initial acylation step, some
modifications were required when 2i was used in place of mucic
acid. For example, to achieve an acceptable yield (40%) of 2j,
the number of equivalents of acylating agent (lauroyl chloride)
was significantly reduced from 15 (with mucic acid) to 5 (with
dendronized L-TA), as isolation and purification proved
problematic with a large excess of lauroyl chloride. It was also
necessary that the reaction occur at room temperature and in
solvent (DCM). Coupling of the PEG and 2j using DCC as the
coupling agent and DPTS as the catalyst proceeded as reported,
yielding the dendritic AM, 2k, in 85% yield. The resultant
self-assembly, an observation also made by Makino et al. in
their study of the in vivo blood clearance of lactosomes.29
The new AMs were then assessed for their ability to inhibit
oxLDL internalization in PBMC macrophages (Figure 4). In
Figure 4. Role of AMs with varying hydrophobicity on the in vitro
inhibition of oxLDL uptake in PBMC macrophages.
1
polymer was characterized via SEC and H NMR.
vitro experiments were carried out by incubating the cells with
10−6 M polymers and fluorescently labeled oxLDL for 24 h at
37 °C. As a control, the basal uptake of oxLDL when no
polymer was present was evaluated. The previously synthesized
2a13 and 2b22 were compared to the newly synthesized
polymers. Based on the improved inhibition of oxLDL
internalization of 2a (52%) relative to 2b (35%), it was
anticipated that increasing the overall hydrophobicity of the L-
TA based polymers would result in decreased oxLDL
internalization. The converse, however, was observed: both
2g and 2k were far less efficacious in inhibiting oxLDL uptake
(11% and 27% inhibition, respectively). This result suggests
that just the extrinsic hydrophobicity of AMs does not uniquely
govern blockage of macrophage oxLDL uptake mechanisms,
but that other factors likely contribute to 2a’s improved efficacy
of oxLDL inhibition.
With these unique AMs, the impact of hydrophobicity on the
physicochemical properties, namely, hydrodynamic radius and
CMC, was evaluated. CMC values were measured using a
previously reported fluorimetry technique using pyrene as the
fluorescence probe.27a The linear disugar AM, 2g, formed
micelles of ∼117 nm in diameter while the dendronized AM,
2k, formed ∼17 nm micelles. The larger micelles formed by 2g
may be attributed to the increased length of the hydrophobic
core, a consequence of tethering two L-TA sugars. A similar
trend was observed by Zeng and Pitt28 who, when preparing
the amphiphilic copolymer poly(ethylene oxide)-b-poly(N-
isopropylacrylamide(NIPAAM)-co-2-hydroxylethyl methacry-
late-lactaten), observed that lengthening of the hydrophobic
poly(NIPAAM) block resulted in larger micelles. Both AMs
exhibited ClogP values (2g: 17.36, 2k: 21.00; see Table 1)
Because 2a and 2b differ not only in their overall
lipophilicity, but also in stereochemistry, we probed the
influence of stereochemistry on AM physicochemical and
biological properties. A new AM was prepared, 2l (Figure 5a),
to be structurally analogous to 2b while being stereochemically
analogous to 2a. Analysis of the solution behavior of 2l revealed
micelles that were similar in size (∼8 nm) to 2b, but more
stable (CMC values of 10−6 M as opposed to 10−5 M) under
physiological conditions. These findings correlate well with the
results above: the number of hydrophobic arms and the length
of the hydrophobic domain influence micelle size, while
stereochemistry influences the solution stability of micelles.
Recently, our research group performed a study comparing the
oxLDL inhibition of 2a to a structurally analogous, but
stereochemically different AM based on saccharic acid.15b
Although the AMs differed by only one stereocenter in the
hydrophobic domain, their ability to inhibit oxLDL internal-
ization was vastly different with 2a showing 60% inhibition
compared to 10% inhibition by the saccharic acid-based
polymer. Based on this earlier work, it was anticipated that
AMs based on L- and meso-tartaric acid (2b and 2l,
respectively) would also have markedly different biological
a
Table 1. Physicochemical Properties of AMs
b
polymer
size (nm)
CMC (M)
ClogP
2a
2b
2g
2k
2l
20
7
1.20 × 10−7
1.25 × 10−5
1.58 × 10−5
5.84 × 10−5
6.12 × 10−6
20.37
9.09
117
17
8
17.38
21.00
9.09
a
The hydrodynamic size and critical micelle concentrations were
experimentally measured; the hydrophobicity coefficient was estimated
for the non-PEG components of AMs. Z-average size.
b
similar to that of their four-arm, mucic acid-based analogue, 2a
(20.37). These results suggest that micelle size is influenced by
the number of hydrophobic arms as well as by the length of the
hydrophobic domain, i.e., overall lipophilicity. In regards to
micelle assembly, both 2g and 2k have CMC values on the
order of 10−5 M, similar to that of 2b. Each of these polymers
possess an L-TA backbone, which suggests that the stereo-
chemistry of the hydrophobic core plays a key role in micelle
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Figure 5. (a) Chemical structure of AM bearing two aliphatic arms (2b) and an equivalent AM with meso stereochemistry (2l). (b) Effect of
stereochemistry on the in vitro inhibition of oxLDL uptake in PBMC macrophages.
properties. Additionally, it was hypothesized that if stereo-
chemistry is a major contributor to polymer-scavenger receptor
binding, then the ability of 2l to inhibit oxLDL uptake should
be similar to the stereochemically analogous 2a. The results of
this current study confirmed that minute changes, such as
altering one stereocenter along the polymer’s sugar backbone,
greatly affects oxLDL uptake and also revealed 2l as a better
inhibitor to oxLDL uptake than the “gold standard”, 2a.
Although it has less overall lipophilicity relative to 2a, 2l
showed the highest degree of inhibition of oxLDL internal-
ization, 89% (Figure 5b). This result further demonstrates that
overall AM lipophilicity may not be the most critical factor in
governing oxLDL inhibition, but rather, stereochemistry of the
hydrophobic domain could dramatically influence the polymer-
blockage of oxLDL uptake.
The authors thank Li Gu for assistance with critical micelle
concentration measurements.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 732-445-0361; Fax: 732-445-7036; E-mail: keuhrich@
rutgers.edu.
■
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
This study was supported by NIH grants (R21093753 to
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