Role of Polymer Architecture on the Activity of Polymer-Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders

Article abstract of DOI:10.1021/acs.biomac.5b00623

Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG-polymer bioconjugates, employing reversible addition-fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a "grafting-to" approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG-polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG-polymer bioconjugate. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.biomac.5b00623

Article
pubs.acs.org/Biomac
Role of Polymer Architecture on the Activity of Polymer−Protein
Conjugates for the Treatment of Accelerated Bone Loss Disorders
†
,‡,§
,†
,⊥
,¶
Bryan S. Tucker,
Jon D. Stewart,* J. Ignacio Aguirre,* L. Shannon Holliday,*
C. Adrian Figg,†,‡,§ Jonathan G. Messer, and Brent S. Sumerlin*
⊥
,†,‡,§
†
‡
§
Department of Chemistry, George and Josephine Butler Polymer Research Laboratory, and Center for Macromolecular Science
and Engineering, University of Florida, Gainesville, Florida 32611, United States
⊥
¶
Department of Physiological Sciences, College of Veterinary Medicine and Department of Orthodontics, College of Dentistry,
University of Florida, Gainesville, Florida 32610, United States
*
S Supporting Information
ABSTRACT: Polymers of similar molecular weights and
chemical constitution but varying in their macromolecular
architectures were conjugated to osteoprotegerin (OPG) to
determine the effect of polymer topology on protein activity in
vitro and in vivo. OPG is a protein that inhibits bone resorption
by preventing the formation of mature osteoclasts from the
osteoclast precursor cell. Accelerated bone loss disorders, such
as osteoporosis, rheumatoid arthritis, and metastatic bone
disease, occur as a result of increased osteoclastogenesis, leading
to the severe weakening of the bone. OPG has shown promise
as a treatment in bone disorders; however, it is rapidly cleared
from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit.
We aimed to improve the effectiveness of OPG by creating OPG−polymer bioconjugates, employing reversible addition−
fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely
branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a “grafting-to” approach,
and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG−polymer
bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic.
Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone
mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in
bone mineral density after administration of the loosely branched OPG−polymer bioconjugate.
INTRODUCTION
the polymer to react with a specific functional group on the
■
protein, most often primary amines or thiols found in lysine or
The conjugation of synthetic polymers to proteins has provided
a viable route to alter the solubility, activity, and blood circula-
3,17,24,25,26
cysteine residues, respectively.
This method provides
1
−8
the ability to completely characterize preformed polymers
prior to reaction with a protein. The well-defined polymers
derived from RDRP techniques have allowed end group
control, either by postpolymerization modifications or through
the use of a functionalized initiator, leading to well-defined
bioconjugates with control over the site-specificity and
tion times of proteins.
In particular, attaching poly(ethylene
glycol) (PEG) to a protein (i.e., “PEGylation”) has proven
effective to improve the efficacy of a number of clinically approved
9
−13
therapeutic proteins.
This increase in therapeutic effective-
ness is most often attributed to an increased hydrodynamic
diameter of the protein after bioconjugation, thereby decreasing
renal filtration and prolonging blood circulation time.
Advances in reversible-deactivation radical polymerization
RDRP) methods have provided a useful toolbox to control
the molecular weights, molecular weight distributions, and
architectures of polymers used in bioconjugations. Atom transfer
3,24−29
13
multiplicity of the polymers.
The versatility of RDRP
techniques has also been further demonstrated in a rapidly
growing area of bioconjugations using a “grafting-from”
strategy, which uses a protein as a macroinitiator or macro-
chain transfer agent to grow polymers directly from the
(
2,7,14,30,31,32
7,14
protein.
The grafting-from method provides some
radical polymerization (ATRP), nitroxide-mediated polymeri-
1
5−17
advantages compared to the grafting-to method such as purifi-
cation (e.g., separation of unreacted monomers vs separation
zation (NMP),
and reversible addition−fragmentation
3,18−23
chain transfer (RAFT) polymerization
have all been
successfully utilized to immobilize well-defined polymers to a
variety of proteins.
Traditionally, polymer−protein bioconjugates are prepared
by a “grafting-to” method, which uses a reactive end group on
Received: May 10, 2015
Revised: June 11, 2015
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.biomac.5b00623
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Article
of unreacted polymers) and control over the number of
polymers per protein.
Isotope, 99.9%), and chloroform-d (CDCl
3
, Cambridge Isotope,
9
9.8%) were used as received. The 2,2′-azobisisobutronitrile (AIBN,
Sigma-Aldrich, 98%) was recrystallized from ethanol. Poly(ethylene
glycol)methyl ether methacrylate (PEGMA, Sigma-Aldrich, Mn
There have been a number of studies that use polymer−
=
peptide bioconjugates to treat degenerative bone diseases and
33−35
500 g/mol) was passed through a column of basic alumina prior to
use. The 4-(cyanopentanoic acid) dithiobenzoate (CDB) and 4-cyano-
elicit bone growth.
In the present research, we investigated
the effect of conjugating a variety of polymer architectures to
osteoprotegerin (OPG), a therapeutically viable protein used in
the treatment of osteoporosis and other degenerative bone
4
-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDTPA)
54
were synthesized according to a previously published procedure.
1
Instrumentation. H NMR spectra were recorded on a Varian
36−39
diseases caused by increased osteoclastic bone resorption.
Innova2 500 MHz or a Varian Mercury 300 MHz NMR spectrometer
using the residual solvent signal as a reference. Molecular weight and
molecular weight distributions were determined by gel permeation
chromatography (GPC) in DMAc with 50 mM LiCl at 50 °C and a
OPG is a naturally occurring soluble decoy receptor involved
in the regulation of bone resorption by binding to the receptor
activator of nuclear factor kappa B (RANK) ligand (L),
preventing RANKL from binding to its target receptor, RANK,
−1
flow rate of 1.0 mL min (Agilent isocratic pump, degasser, and
autosampler; columns, (i) PLgel 5 μm guard + two ViscoGel I-series
40−43
on the surface of premature osteoclasts.
By inhibiting the
3
G3078 mixed bed columns, molecular weight range 0−20 × 10 and
RANK/RANKL interaction, OPG prevents osteoclast differ-
4
−1
0
−100 × 10 g mol ; or (ii) PLgel 5 μm guard + two PLgel 5 μm
−1
44−49
entiation and activation, which reduces bone resorption.
Mixed-D columns, molecular weight range 200−400,000 g mol ).
Detection consisted of (i) Wyatt Optilab T-rEX refractive index
detector operating at 658 nm and a Wyatt miniDAWN Treos light
scattering detector operating at 659 nm or (ii) Wyatt OptilabDSP
interferometric refractometer operating at 690 nm and a Wyatt
DAWN EOS light scattering detector operating at 685 nm. Absolute
molecular weights and molecular weight distributions were calculated
using the Wyatt ASTRA software. UV−vis measurements were
obtained using a Varian Carey 500 Scan UV−vis-NIR (near-infrared)
spectrophotometer.
Treatment of degenerative bone disorders with OPG has been
promising, and a recent study indicated that OPG may also be
50
useful in the treatment of muscular dystrophy. However,
OPG has a very short in vivo half-life, and multiple doses are
required to achieve a therapeutic benefit. Previous studies
have shown that conjugating OPG with PEG can improve the
blood circulation time of OPG (i.e., the pharmacokinetics),
but binding with RANKL (i.e., the pharmacodynamics) was
5
1,52
reduced.
Further studies suggested that changing the
Synthesis. N-(2-hydroxypropyl)methacrylamide (HPMA). To a
three-neck, 2 L round-bottom flask equipped with a mechanical
stirring device, thermometer, and addition funnel was added 1-amino-
2-propanol (75 mL, 0.96 mol). The reagent was dissolved in
dichloromethane (1 L) and cooled to −5 °C in a salt-ice bath.
Methacryloyl chloride (46 mL, 0.47 mol) was then added dropwise via
addition funnel. The reaction was stirred for 30 min at 0 °C then
slowly warmed to room temperature and left to stir overnight. The
reaction was filtered to remove 1-amino-2-propanol hydrochloride,
and the filtrate was concentrated to 500 mL and placed in a −20 °C
freezer overnight to crystallize the product. The resultant HPMA
architecture of the polymer from linear to branched could
provide the increased pharmacokinetics without complete
53
elimination of the pharmacodynamics of the protein.
We sought to further investigate the effect of polymer
branching density on the activity of a series of OPG−polymer
bioconjugates. Three polymers of varying branching density
were synthesized, including linear PEG, loosely branched PEG,
and densely branched PEG, while the molecular weight of each
architecture was held relatively constant. Each of the unique
architectures was then conjugated to OPG via a grafting-to
approach using the activated ester, N-hydroxysuccinimide
was isolated by filtration and recrystallized from acetone at −20 °C
1
(
5
52 g, 76%). H NMR (300 MHz, D O) δ: 5.72 (1H, s, CH C),
2
2
(
NHS), in the polymer end group. Control over the multiplicity
.47 (1H, t, CH C), 3.96 (1H, m, CH CH(CH )OH), 3.30 (2H,
2
2
3
of polymers per protein was elicited by adjusting the pH of
m, CH CH(CH )OH), 1.95 (3H, s, CH CCH ), 1.18 (3H, d,
2 3 2 3
the solution to favor functionalization of the N-terminus of the
CH CH(CH )OH).
2 3
3
protein. Using a grafting-to method allowed complete polymer
Methoxypolyethylene Glycol (mPEG)-Succinic Acid (mPEG-
COOH). To a 40 mL scintillation vial with septum cap and stir bar
were added mPEG (1.93 g, 0.386 mmol), succinic anhydride (201 mg,
characterization and end group modifications prior to coupling
with a valuable therapeutic protein. The OPG−PEG bioconju-
gates were analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) to ensure successful conjuga-
tion, and the retention of protein activity was demonstrated
using an in vitro osteoclast inhibition assay, which showed that
each bioconjugate retained high activity against osteoclast for-
mation. Finally, preliminary in vivo studies using peripheral
quantitative computed tomography indicated improved bone
mineral density of the loosely branched bioconjugate relative to
other OPG−polymer architectures.
2
.01 mmol), and TEA (218 mg, 2.13 mmol). The reagents were
dissolved in dry dichloromethane (10 mL) and allowed to stir at room
temperature. The reaction was monitored by H NMR spectroscopy
1
to observe the appearance of methylene protons adjacent to the
forming ester at 4.2 ppm. The reaction was quenched at 4 h, and the
polymer was purified by precipitation into diethyl ether (3× , 200 mL).
mPEG-NHS Ester (P1). To a 20 mL scintillation vial with septum
cap and stir bar were added mPEG-COOH (500 mg, 0.1 mmol) and
NHS (13 mg, 0.12 mmol). The reagents were dissolved in dry
dichloromethane (5 mL) and purged with N . EDC·HCl (24 mg,
2
0
.13 mmol) was dissolved in dichloromethane (2 mL) and added to
the reaction with stirring. The reaction was allowed to stir at room
temperature overnight followed by precipitation into diethyl ether
(500 mL). The polymer was filtered and dried under vacuum. The
activated ester polymer was used without further purification.
Poly(polyethylene glycol methyl ether methacrylate) (PolyPEG-
MA11). PEGMA (7.86 g, 15.7 mmol), CDB (223 mg, 0.798 mmol),
and AIBN (25.6 mg, 0.156 mmol) were added to a 25 mL Schlenk
flask with magnetic stir bar. Trioxane (142 mg) was added as an
internal reference, and the reagents were dissolved in 1,4-dioxane
(9 mL). The flask was sealed with a rubber septum, and the bright red
solution was degassed via three freeze−pump−thaw cycles. The
mixture was placed in an oil bath at 70 °C with stirring, and the
reaction was monitored by GPC and quenched at 2 h. The polymer
MATERIALS AND METHODS
■
Materials. Methacryloyl chloride (Alfa Aesar, 97%), 1-amino-2-
propanol (Alfa Aesar, 94%), succinic anhydride (TCI America, >95%),
triethylamine (TEA, Alfa Aesar, 99%), hydrazine (Alfa Aesar, 98+%),
tris(2-carboxyethyl)phosphine·HCl (TCEP, Alfa Aesar, 98%), methox-
ypolyethylene glycol (mPEG, Fluka, M = 5000 g/mol), 1-ethyl-3-(3-
n
(
9
(
dimethylamino)propyl)carbodiimide·HCl (EDC·HCl, Sigma-Aldrich,
8%), N-hydroxysuccinimide (NHS, Sigma-Aldrich, 99%), trioxane
Acros Organics, 99.5%), dichloromethane (BDH, 99.5%), diethyl
ether (Fisher Chemicals), 1,4-dioxane (Fisher Chemicals, 99%),
N,N-dimethylformamide (DMF, EMD, 99.8%), N,N-dimethylacetamide
(
DMAc, Sigma-Aldrich, 99%), deuterium oxide (D O, Cambridge
2
B
DOI: 10.1021/acs.biomac.5b00623
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Article
was purified by dialysis against water using a 3500 MWCO dialysis
membrane, and a dark red oil was isolated after lyophilization. The
sample was then dissolved in DMF, and hydrazine was used to remove
the dithiobenzoate end group (Mn, GPC = 6040 g/mol, Đ = 1.08).
Fluorescein Conjugation to PolyPEGMA . PolyPEGMA
11
room temperature. Dichloromethane was removed in vacuo, and the
polymer was used without further purification.
Conjugation to OPG. Two vials of recombinant human TNFRSF
11B-487 (OPG, 1 mg protein, Creative Biomart, His-tagged, lot
#265155) were warmed to room temperature, and each was dissolved
in phosphate buffer (250 μL, 100 mM, pH 7.5). Both solutions were
combined and placed in a Millipore Amicon Ultra 0.5 10 000 Da
MWCO ultrafiltration unit and centrifuged at 14 000 rpm at room
temperature for 10 min to remove interfering excipients. The protein
solution was diluted with phosphate buffer (500 μL, 100 mM, pH 7.5)
and centrifuged as before. This process was repeated three more
times to desalt the OPG prior to conjugation. The final OPG solution
(ca. 90 μL) had a concentration of approximately 22 μg/mL and was
stored on ice.
1
1
−2
(
2
(
163 mg, 2.69 × 10 mmol) was dissolved in DMF (3 mL) in a
0 mL scintillation vial with septum cap and purged with N . TCEP
2
−2
9.5 mg, 3.3 × 10 mmol) dissolved in DMF/H O (9/1) was added
2
to the reaction, followed by the addition of TEA (4.9 mg, 3.5 ×
−
2
−4
1
0
mmol) and acryloyl(fluorescein) (0.12 mg, 3.1 × 10 mmol).
The reaction was left to stir at room temperature for 16 h, and
−2
1
-(2-hydroxyethyl)-1H-pyrrole-2,5-dione (4.9 mg, 3.5 × 10 mmol)
was added as a solution in DMF. The reaction was allowed to stir for
h and then dialyzed against water using a 3500 MWCO dialysis
2
membrane. The product was isolated by lyophilization (158 mg).
Activated polymers were dissolved in phosphate buffer (100 mM,
pH 7.5) at concentrations of 2.5 mM immediately prior to conjugation.
OPG conjugations were carried out in 50 μL total volumes by mixing
OPG stock (20 μL, 7.3 nmol), polymer stock (15 μL, 37 nmol), and
phosphate buffer (15 μL, 100 mM, pH 7.5). After incubating for 24 h
at room temperature, each reaction mixture was placed in a separate
Millipore Amicon Ultra 0.5 10 000 Da MWCO ultrafiltration unit,
diluted with phosphate buffer (450 μL, 100 mM, pH 7.5), and
centrifuged at 14 000 rpm for 10 min at room temperature. This
washing was repeated three additional times. The final concentrated
protein samples were stored at 4 °C prior to biological assays.
Mouse Marrow Culture. The 1,25-dihydroxyvitamin D (1,25D )-
NHS Activation of PolyPEGMA -Fluorescein (P2). PolyPEGMA -
1
1
11
−2
fluorescein (147 mg, 2.43 × 10 mmol) and NHS (4.1 mg, 3.6 ×
−
2
10
mmol) were dissolved in dry dichloromethane (2 mL). The
reaction was purged with N , and EDC·HCl (5.5 mg, 2.9 ×
2
−
2
10
mmol) was added as a solution in dichloromethane (0.5 mL).
The reaction was left to stir overnight at room temperature.
Dichloromethane was removed in vacuo, and the polymer was used
without further purification.
Copolymerizations of PEGMA and HPMA. RAFT copolymeriza-
tions were performed using CDTPA and AIBN as the CTA and
initiator, respectively, with [CTA]:[I] = 5:1. The monomer feed ratios
were varied according to Table S1 of the Supporting Information, and
the total monomer conversions were limited to ≤16%. The monomers,
CTA, initiator, and trioxane were added to a 20 mL scintillation vial
equipped with a magnetic stir bar and septum cap, dissolved in DMAc
3
3
stimulated mouse marrow, in which osteoblasts and osteoclasts
differentiate coordinately over a period of 6 days, was produced as
5
5
described previously. Femora and tibia from Swiss-Webster mice
(8−20 g) that had been killed by cervical dislocation were dissected
from adherent tissue, and marrow was removed by clipping both bone
ends, inserting a syringe with a 25 gauge needle, and flushing the
marrow using αMEM plus 10% fetal bovine serum (αMEM D10).
The marrow was washed twice with αMEM D10 and then plated at a
(
3 M), purged with N for 30 min, and added to a preheated reaction
2
block at 60 °C. The monomer conversions were determined from
1
H NMR spectroscopy by integrating the vinyl protons from each
monomer relative to the trioxane standard. The reactivity ratios were
6
2
calculated graphically using the Finemann−Ross method, where r is
1
density of 1 × 10 cells/cm on tissue culture plates for 6 days in
−8
given by the slope and r is given by the negative intercept of a plot of
2
αMEM D10 plus 10 M 1,25D . Cultures were fed on day 4 by
3
G versus H (Tables S1 and S2 and Figure S6, Supporting Information).
replacing half the media per plate and adding fresh 1,25D . OPG and
3
Poly(HPMA -co-PEGMA ). HPMA (1.00. g, 6.98 mmol), PEGMA
1.18 g, 2.36 mmol), CDTPA (92.4 mg, 0.229 mmol), AIBN (8.2 mg,
.050 mmol), and trioxane (100 mg) as an internal standard were
9
6
OPG conjugates were added on day 1 and refreshed on day 4. After 6
days in culture, osteoclasts were abundant in control cultures. Cells
were fixed with 2% paraformaldehyde in citrate buffer for 20 min,
permeabilized by treatment with 1% Triton X-100 for 10 min, washed
in citrate buffer, and osteoclasts were detected by staining for tartrate-
resistant acid phosphatase (TRAcP) activity, which is a specific marker
for mouse osteoclasts, using the Leukocyte Acid Phosphatase (TRAP)
kit from Sigma (St Louis, MO). Cells expressing TRAcP activity
(
0
added to a 20 mL scintillation vial equipped with a septum cap and
magnetic stir bar. The reagents were dissolved in DMAc (3 mL),
purged with N for 30 min, and added to a preheated heating block at
2
60 °C. The reaction was quenched after 4 h by exposing the contents
to oxygen, and the polymer was purified by dialysis against water using
a 3500 MWCO membrane and lyophilized to yield an amorphous
solid (Mn, GPC = 7020 g/mol, Đ = 1.07).
5
5
were documented as described previously. The University of Florida
Institutional Animal Care and Usage Committee approved this
protocol.
Poly(HPMA -co-PEGMA )-Fluorescein. Poly(HPMA -co-PEGMA )
9
6
9
6
−2
(
150 mg, 2.0 × 10 mmol) was dissolved in DMF (3 mL) with stirring
in a 20 mL scintillation vial with septum cap. Hydrazine (0.05 mL,
mmol) was added, and the reaction turned colorless immediately.
Cell Cytotoxicity Assay. RAW 264.7 cells were grown as
5
6
described previously4. Raw 264.7 cells (ATCC) were plated at a
density of 1.25 × 10 cells per well in 24-well plates and treated with
OPG or derivatives of OPG as indicated. Adherent cells from three
2
After 30 min, the mixture was placed in a 3500 MWCO dialysis
membrane and dialyzed against water followed by lyophilization to
2
random fields per well (250 μm ) were counted and then averaged for
isolate the polymer. The end-group removed Poly(HPMA -co-PEGMA )
9
6
each well. Live cells were counted on day 2. On day 4, the cells were
fixed with 2% formaldehyde prior to counting. No overt signs of
toxicity, excess nonadherent cells, or cell debris were noted on either
days 2 or 4.
Animals and Experimental Groups. A total of 20 jugular vein-
cannulated male Sprague−Dawley rats aged 8−10 weeks arrived to the
Animal Care Services, University of Florida from a commercial vendor
−2
(
118 mg, 1.68 × 10 mmol) was dissolved in DMF (3 mL) in a 20 mL
scintillation vial with septum cap and stir bar and purged with N for
2
−
2
3
0 min. TCEP (5.6 mg, 2.0 × 10 mmol) was then added as a solution
2
−
in water (0.5 mL), followed by TEA (2.64 mg, 2.61 × 10 mmol).
−4
Acryloyl(fluorescein) (0.1 mg, 2 × 10 mmol) was added, and the
reaction was allowed to stir at room temperature for 20 h. 1-(2-
hydroxyethyl)-1H-pyrrole-2,5-dione (3.2 mg, 2.3 × 10− mmol) in
DMF (0.5 mL) was added and stirred 2 h, and the mixture was dialyzed
against water using a 3500 MWCO dialysis membrane. The product
was isolated by lyophilization to give a yellow solid (100 mg).
NHS Activation of Poly(HPMA -co-PEGMA )-Fluorescein (P3).
2
(Charles River, Ltd.). After arrival, rats were housed individually in
ventilated cages. The housing room was maintained at 68−79 °F
with an average humidity of 30−70% and a 12:12 h light/dark cycle.
All animal procedures were approved by the Institutional Animal Care
and Use Committee at the University of Florida (Gainesville, FL).
After a week of acclimation, rats were randomized into the following
five experimental groups (n = 4):
9
6
−
2
Poly(HPMA -co-PEGMA )-fluorescein (89.1 mg, 1.27 × 10 mmol)
and NHS (1.8 mg, 1.6 × 10 mmol) were dissolved in dry
dichloromethane (2 mL). The reaction was briefly purged with N ,
and EDC·HCl (3.0 mg, 1.6 × 10 mmol) was added as a solution in
dichloromethane (0.5 mL). The reaction was left to stir overnight at
9
6
−
2
2
−
2
(1) Group of rats euthanized at the beginning of the experiment
and received no treatment (Baseline control).
C
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Article
(
(
(
(
2) Group of rats that received a single bolus intravenous (IV)
calculate r (monomer reactivity ratio for PEGMA) = 0.98 and
1
injection of OPG alone (0.4 mg/kg) diluted in phosphate buffer
r2 (monomer reactivity ratio for HPMA) = 0.52 (Tables S1 and
S2, Figure S6, Supporting Information). These values indicate
that the copolymerization of the monomers is not random, with
PEGMA being preferentially consumed during the reaction.
Therefore, we altered the monomer feed ratio to target approxi-
mately 50% of the number of PEGMA repeat units as that of
the densely branched polymer described previously. While the
polymer likely contains a gradient microstructure rather than a
random copolymer, we believe that the polymer contains the
lower branching density needed to study the effect of branching
density on protein activity. The RAFT group was removed by
aminolysis, fluorescein was conjugated through Michael addition
(
pH 7.2) (Unmodified OPG).
3) Group of rats that received a single bolus IV injection of the
linear PEG−OPG bioconjugate (0.4 mg/kg) diluted in phosphate
buffer (pH 7.2) (OPG1).
4) Group of rats that received a single bolus IV injection of the
densely branched PEG−OPG bioconjugate (0.4 mg/kg) diluted
in phosphate buffer (pH 7.2) (OPG2).
5) Group of rats that received a single bolus IV injection of the
loosely branched PEG−OPG bioconjugate (0.4 mg/kg) diluted
in phosphate buffer (pH 7.2) (OPG3). Only one of the four
assigned rats was injected in this group due to loss of the
bioconjugate sample during filtration.
Rats from the baseline group and those rats after completion of
the 7-day period were euthanized by CO inhalation followed by
thoracotomy. Left femurs were excised and stripped of musculature,
placed in 10% buffered formalin for 48 h, and transferred to 70%
ethanol.
Peripheral Quantitative Computed Tomography (pQCT). For
the pQCT analysis, left femurs from all rats were scanned using a
Stratec XCT Research M instrument (Norland Medical Systems; Fort
Atkinson, WI) with software version 5.40. Scans were performed at
distances of 2, 4, and 6 mm proximal to the distal femur epiphyseal
growth plate. At this location, corresponding to the primary and
secondary spongiosa, is where endochondral ossification takes place
and bone grows in length and undergoes remodeling. Trabecular (trab)
bone mineral content (BMC), trab bone mineral density (trab BMD)
(
Figure S7, Supporting Information), and the carboxylic acid
2
end group was converted to an NHS ester.
Protein Conjugations. OPG was modified with each of the
polymer architectures using a grafting-to approach, in which
the pH value of the reaction was controlled to increase the
probability of selectively deprotonating the amine-terminus of
the protein, while the primary amines of lysine residues were
expected to be protonated and therefore rendered less
60,61
nucleophilic (Scheme 4).
Since coupling reactions of macro-
molecular reagents can be less efficient than those of small
molecules, five molar equivalents of polymer were used for each
bioconjugation. A control experiment was also performed, in
which pure OPG in the absence of polymer was subjected to the
same reaction and purification conditions employed during the
bioconjugation reaction and work-up. The purified OPG−PEG
conjugates (OPG1, OPG2, and OPG3 for the linear PEG,
densely branched PEG, and loosely branched PEG architectures,
respectively) were analyzed by SDS-PAGE, which demonstrated
successful polymer conjugation to OPG in addition to some
remaining unmodified protein (Figure 1).
5
7
and trab bone area (trab BA) were determined as previously described.
RESULTS AND DISCUSSION
■
Polymer Synthesis. Linear PEG, P1, was synthesized by
modifying the end group of commercially available mPEG (M =
n
5
000 g/mol) to contain an NHS activated ester (Scheme 1),
Scheme 1. Ring Opening of Succinic Anhydride with mPEG
and Formation of an NHS Activated Ester To Give P1
In Vitro Osteoclast Inhibition Assay. The in vitro activity
of each bioconjugate was measured using an osteoclast inhibi-
tion assay with 1,25-dihydroxyvitamin D -stimulated mouse
3
marrow cells and bioconjugate concentrations of 2, 20, and
2
00 ng/mL administered on days 1 and 4. Cells were fixed at
62
day 6 and stained for TRAcP, a marker for osteoclast activity.
Cells were divided into mononuclear, multinuclear (2−10 nuclei),
and giant (>10 nuclei), with each cell population representing
increasingly mature osteoclast cells. The osteoclast levels were
counted, and the results indicated that while 2 ng/mL was too
dilute to inhibit osteoclast growth for the unmodified OPG or
OPG1 and OPG2, the osteoclast count was reduced for OPG3.
Furthermore, a concentration of 20 ng/mL resulted in decreased
osteoclast counts for all samples receiving OPG relative to the
control (no OPG). At a concentration of 200 ng/mL, osteoclasts
were completely inhibited for all of the conjugates (Figure 2).
Importantly, each bioconjugate of OPG retained antiosteoclast
activity at concentrations of 20 and 200 ng/mL. To provide
evidence that the decrease in osteoclast activity is due to the
inhibition of osteoclast maturation by the OPG−RANKL interac-
tion and not due to general cytotoxicity of the bioconjugates,
a cell cytotoxicity study was performed using cells that serve as a
model for macrophages (Figure 3). The cells were shown to have
near-quantitative viability at 2 and 4 days at the concentrations
used in the osteoclast inhibition assay described, which sug-
gested that each bioconjugate was nontoxic and supported the
observation that the decrease in osteoclast maturation was indeed
a result of OPG−RANKL binding.
rendering the linear polymer reactive toward amine residues in
the protein.
The densely branched PEG architecture, P2 (polyPEGMA ),
11
was synthesized using RAFT polymerization of PEGMA
Scheme 2; Figure S3, Supporting Information). Because we
(
aimed to study the effect of varying branching density on
conjugate activity, the molecular weight of the polymers was
held constant, targeting an M of 5000 g/mol. Reports have
n
5
8
suggested in vivo cytotoxicity of dithiobenzoate end groups;
therefore, the RAFT group was removed by aminolysis. The
resultant thiol, a useful handle for further functionalization,
59
was exploited using Michael addition to incorporate fluorescein
as a convenient marker for in vitro and in vivo monitoring. The
end group removal and fluorescein conjugations were confirmed
by UV−vis spectroscopy (Figure S4, Supporting Information),
and the carboxylic acid α-end group was then converted to an
NHS ester for bioconjugations.
The loosely branched PEG architecture, P3 (poly(HPMA9-
co-PEGMA ), was prepared by copolymerizing PEGMA with
6
HPMA via RAFT polymerization (Scheme 3; Figure S5,
Supporting Information). We first studied the copolymerization
kinetics of PEGMA and HPMA by determining the monomer
reactivity ratios. The Finemann−Ross method was used to
Conjugation of even a single polymer can greatly reduce
6
,63−66
the activity of many proteins.
Thus, these results were
D
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Scheme 2. (A) RAFT Polymerization of PEGMA To Afford a Densely Branched Polymer, PolyPEGMA , Followed by
1
1
Aminolysis To Remove the RAFT Group. (B) Michael Addition Using Acryloyl(fluorescein). (C) Formation of the NHS
Activated Ester To Give P2
Scheme 3. (A) RAFT Copolymerization of PEGMA and HPMA. (B) RAFT Group Removal and Michael Addition with
Acryloyl(fluorescein). (C) Formation of NHS Ester To Afford P3
Scheme 4. OPG Bioconjugations with (A) P1 To Form OPG1, (B) P2 To Form OPG2, and (C) P3 To Form OPG3. Protein
Structure from the PDB (3URF)
promising because the OPG bioconjugates retained their ability
to prevent osteoclast formation, which suggests that they may
also decrease bone resorption in vivo. Furthermore, the loosely
branched bioconjugate, OPG3, actually had higher in vitro
activity than either OPG1 or OPG2, as indicated in a lower
osteoclast count (Figure 2).
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Biomacromolecules
Article
reliable way to determine bone mass is by determining bone
mineral density (BMD), a surrogate of bone mass, using pQCT.
We tested the antiresorptive activity of each bioconjugate in vivo
by measuring trabecular (trab) BMD, trabecular bone mineral
content (trab BMC), and trabecular bone area (trab BA) at the
distal femoral metaphyses of Sprague−Dawley rats at different
distances from the growth plate. For this purpose, 20 cannulated
rats were divided into five groups. One group was sacrificed at
the beginning of the experiment and received no treatment to
serve as a baseline control. The other four groups received single
bolus intravenous injections of unmodified OPG, OPG1, OPG2,
or OPG3 and were euthanized 7 days post-treatment. Femurs
were collected post-mortem, and BMD was analyzed by pQCT
at distances of 2, 4, and 6 mm proximal to the distal epiphyseal
growth plate (Figure S8, Supporting Information).
Figure 1. SDS-PAGE analysis of OPG bioconjugates. Lane: (1) molecular
weight marker, (2) unmodified OPG, (3) OPG1, (4) OPG3, and (5)
OPG2.
The pQCT analyses showed increased femur trab BMD, trab
BMC, and trab BA at the distal metaphysis of rats treated with
OPG3 compared to rats from the baseline, unmodified OPG,
OPG1, and OPG2 groups (Figure 4; Figure S9, Supporting
Figure 2. Osteoclast inhibition assay using 1,25-dihydroxyvitamin
D -stimulated mouse marrow cells. The control is the osteoclast count
3
of untreated cells. Background shading indicates different OPG
samples with doses on days 1 and 4, each with three concentrations of
2
, 20, and 200 ng/mL. Three cell types were countedmononuclear,
multinuclear, and giantindicated by the different bar colors blue,
orange, and green, respectively.
Figure 4. (A) Bone mineral density, (B) bone mineral content, and
Figure 3. Cell cytotoxicity assay using RAW 264.7 cells shown as the
percentage of cell survival relative to a control, which was untreated
cells. Each bioconjugate, indicated by background shading, was
administered at concentrations of 20 and 200 ng/mL, and live cells
were counted at 2 d (blue) and fixed with formaldehyde and counted
at 4 d (orange). Each sample is the average of four replicates.
(C) bone mineral area of OPG bioconjugates as the average from
2
−6 mm from the growth plate. Statistical analysis was accomplished
with one-way analysis of variance (ANOVA) followed by post-
ANOVA: multiple comparison Tukey Test or nonparametric Kruskal−
Wallis test; a = different from baseline, b = different from unmodified
OPG, and c = different from OPG1.
In Vivo Skeletal Effect Study. We aimed to show that the
effects of osteoclast inhibition observed in vitro could also be
translated to in vivo effectiveness. Since OPG ultimately prevents
differentiation and activation of osteoclasts, we reasoned that
we could monitor the efficacy of our bioconjugates in vivo by
monitoring the bone mass and bone volume in rats. A facile and
Information). While the linear and densely branched protein
bioconjugates showed no increase in BMD from either
unmodified OPG or the baseline control, we believe that the
loosely branched bioconjugate has enhanced antiresorptive
activity, consistent with the in vitro data described above.
Furthermore, since even unmodified OPG had no significant
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bone effect as compared to the control, it is possible that higher
concentrations or multiple doses of the bioconjugate would
further enhance the bone growth profile. When combined
with the nontoxic characteristics shown in the in vitro studies,
these in vivo results indicate promise for these materials as
an effective therapy in bone degenerative disorders. Further
pharmacokinetic studies are necessary to show the enhanced
blood circulation half-life of the bioconjugates, but the increase
in BMD is an exciting preliminary result that gives hope to the
translation from in vitro to in vivo efficacy.
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CONCLUSIONS
■
(
1
(
In summary, three polymers with linear, loosely branched, and
densely branched architectures were conjugated to OPG using
a grafting-to strategy. Control over the polymer branching
density was elicited using RAFT copolymerization of a PEGMA
macromonomer with HPMA, a water-soluble monomer that
1
(
67
leads to vinyl polymers with good biocompatibility. End group
control allowed further functionalization to render the polymers
reactive toward primary amines on proteins. Importantly, each
bioconjugate was shown to be nontoxic and retained high
activity toward the inhibition of osteoclasts. Preliminary in vivo
studies further supported the nontoxic character of the OPG
bioconjugates, and initial results suggested an increase in the
bone mineral density of the loosely branched OPG
bioconjugate. A more robust pharmacokinetics study is needed
to unequivocally show the therapeutic benefit of OPG−polymer
bioconjugates, but this report demonstrates the feasibility of
using such a system to treat bone degenerative diseases.
(
180−181.
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ASSOCIATED CONTENT
Supporting Information
■
(22) Cobo, I.; Li, M.; Sumerlin, B. S.; Perrier, S. Nat. Mater. 2015, 14,
143−159.
*
S
Experimental procedures, GPC chromatograms, 1H NMR
spectra, UV−vis spectra, and bone mineral density data. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.biomac.5b00623.
(23) Falatach, R.; McGlone, C.; Al-Abdul-Wahid, M. S.; Averick, S.;
Page, R. C.; Berberich, J. A.; Konkolewicz, D. Chem. Commun. 2015,
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5
(
O.; Leszczyszyn, O. I.; Blindauer, C.; Brayden, D. J.; Haddleton, D. M.
Soft Matter 2009, 5, 3038−3046.
AUTHOR INFORMATION
Corresponding Author
E-mail: sumerlin@chem.ufl.edu.
E-mail: jds2@chem.ufl.edu.
E-mail: aguirrej@ufl.edu.
E-mail: SHOLLIDAY@dental.ufl.edu.
■
*
*
*
*
(25) Jones, M. W.; Strickland, R. A.; Schumacher, F. F.; Caddick, S.;
Baker, J. R.; Gibson, M. I.; Haddleton, D. M. J. Am. Chem. Soc. 2012,
134, 1847−1852.
(26) Pokorski, J. K.; Hovlid, M. L.; Finn, M. G. ChemBioChem 2011,
12, 2441−2447.
(27) Nguyen, T. H.; Kim, S.-H.; Decker, C. G.; Wong, D. Y.; Loo, J.
A.; Maynard, H. D. Nat. Chem. 2013, 5, 221−227.
Author Contributions
The manuscript was written through contributions of all authors.
(28) Grover, G. N.; Lee, J.; Matsumoto, N. M.; Maynard, H. D.
Macromolecules 2012, 45, 4958−4965.
Funding
(29) Bays, E.; Tao, L.; Chang, C.-W.; Maynard, H. D.
Biomacromolecules 2009, 10, 1777−1781.
The authors would like to acknowledge the University of
Florida Research Foundation and the UF Seed Opportunity
Fund for partial support of this work.
(30) Murata, H.; Cummings, C. S.; Koepsel, R. R.; Russell, A. J.
Biomacromolecules 2013, 14, 1919−1926.
31) Wallat, J. D.; Rose, K. A.; Pokorski, J. K. Polym. Chem. 2014, 5,
545−1558.
32) Pokorski, J. K.; Breitenkamp, K.; Liepold, L. O.; Qazi, S.; Finn,
(
1
(
Notes
The authors declare no competing financial interest.
M. G. J. Am. Chem. Soc. 2011, 133, 9242−9245.
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