Synthesis, characterization, and biocompatibility of biodegradable hyperbranched polyglycerols from acid-cleavable ketal group functionalized initiators

Article abstract of DOI:10.1021/bm300959h

Herein we report the synthesis of biodegradable hyperbranched polyglycerols (BHPGs) having acid-cleavable core structure by anionic ring-opening multibranching polymerization (ROMBP) of glycidol using initiators bearing dimethyl and cyclohexyl ketal groups. Five different multifunctional initiators carrying one to four ketal groups and two to four hydroxyl groups per molecule were synthesized. The hydroxyl carrying initiators containing one ketal group per molecule were synthesized from ethylene glycol. An alkyne-azide click reaction was used for synthesizing initiators containing multiple cyclohexyl ketal linkages and hydroxyl groups. The synthesized BHPGs exhibited monomodal molecular weight distributions and polydispersity in the range of 1.2 to 1.6, indicating the controlled nature of the polymerizations. The polymers were relatively stable at physiological pH but degraded at acidic pH values. The polymer degradation was dependent on the type of ketal structure present in the BHPG; polymers with cyclohexyl ketal groups degraded at much slower rates than those with dimethyl ketal groups at a given pH. Good control of polymer degradation was achieved under mild acidic conditions by changing the structure of ketal linkages. A precise control of the molecular weight of the degraded HPG was achieved by controlling the number of ketal groups within the core, as revealed from the gel permeation chromatography (GPC) analyses. The decrease in the polymer molecular weights upon degradation was correlated well with the number of ketal groups in their core structure. Our data support the suggestion that glycidol was polymerized uniformly from all hydroxyl groups of the initiators. BHPGs and their degradation products were highly biocompatible, as measured by blood coagulation, complement activation, platelet activation, and cell viability assays. The controlled degradation profiles of these polymers together with their excellent biocompatibility make them suitable for drug delivery and bioconjugation applications.

Full text of DOI:10.1021/bm300959h

Article
pubs.acs.org/Biomac
Synthesis, Characterization, and Biocompatibility of Biodegradable
Hyperbranched Polyglycerols from Acid-Cleavable Ketal Group
Functionalized Initiators
Rajesh A. Shenoi,† Benjamin F. L. Lai,† and Jayachandran N. Kizhakkedathu*,†,‡
†Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver BC,
Canada V6T 1Z3
‡Department of Chemistry, University of British Columbia, Vancouver BC, Canada V6T 1Z3
S
* Supporting Information
ABSTRACT: Herein we report the synthesis of biodegradable hyperbranched polyglycerols (BHPGs) having acid-cleavable
core structure by anionic ring-opening multibranching polymerization (ROMBP) of glycidol using initiators bearing dimethyl
and cyclohexyl ketal groups. Five different multifunctional initiators carrying one to four ketal groups and two to four hydroxyl
groups per molecule were synthesized. The hydroxyl carrying initiators containing one ketal group per molecule were synthesized
from ethylene glycol. An alkyne−azide click reaction was used for synthesizing initiators containing multiple cyclohexyl ketal
linkages and hydroxyl groups. The synthesized BHPGs exhibited monomodal molecular weight distributions and polydispersity
in the range of 1.2 to 1.6, indicating the controlled nature of the polymerizations. The polymers were relatively stable at
physiological pH but degraded at acidic pH values. The polymer degradation was dependent on the type of ketal structure
present in the BHPG; polymers with cyclohexyl ketal groups degraded at much slower rates than those with dimethyl ketal
groups at a given pH. Good control of polymer degradation was achieved under mild acidic conditions by changing the structure
of ketal linkages. A precise control of the molecular weight of the degraded HPG was achieved by controlling the number of ketal
groups within the core, as revealed from the gel permeation chromatography (GPC) analyses. The decrease in the polymer
molecular weights upon degradation was correlated well with the number of ketal groups in their core structure. Our data
support the suggestion that glycidol was polymerized uniformly from all hydroxyl groups of the initiators. BHPGs and their
degradation products were highly biocompatible, as measured by blood coagulation, complement activation, platelet activation,
and cell viability assays. The controlled degradation profiles of these polymers together with their excellent biocompatibility make
them suitable for drug delivery and bioconjugation applications.
(TMP), which is partially deprotonated using potassium
methoxide.10 Slow addition of monomer to a partially
deprotonated multifunctional initiator results in polymers
with controlled molecular weights and narrow polydispersity.
Polymers of varying molecular weights are accessible through
the proper choice of the polymerization conditions such as the
use of 1,4-dioxane as an emulsifier for achieving very high
molecular weights (300−1000 kDa)11 or employing macro-
initiators for the synthesis of medium molecular weight HPGs
1. INTRODUCTION
In the past few years there has been considerable advancement
in the use of dendritic and hyperbranched polymers for a
variety of biomedical applications including drug delivery, tissue
engineering, protein delivery, and gene transfection.1−6 Among
these, hyperbranched polyglycerols (HPGs) have received
much attention recently due to their special features.7−9
HPGs possess dendrimer-like properties and could be
synthesized by a single-step process and have a compact
globular structure. The most common method employed for
the synthesis of HPGs involves the anionic ring-opening
multibranching polymerization (ROMBP) of glycidol using a
multifunctional initiator, 1,1,1-tris(hydroxymethyl)propane
Received: March 23, 2012
Revised: August 20, 2012
© XXXX American Chemical Society
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(6−25 kDa).12 Haag and coworkers recently reported the
synthesis of cross-linked HPG particles of size varying from a
few nanometers to several micrometers using miniemulsion and
microfluidic template technologies.13 HPGs are shown to be
highly biocompatible, and a large number of hydroxyl groups in
the polymers serve as potential sites for the conjugation of
drugs and bioactive molecules such as peptides and
carbohydrates.14−22 HPGs with hydrophobic alkyl groups in
the core have been designed for applications such as human
serum albumin (HSA) substitute23 and the encapsulation of
hydrophobic drugs and several guest molecules.24−28 Function-
alization of HPGs with cationic groups has shown potential for
the delivery of nucleic acids as they bind DNA and condense
them into smaller particles (<100 nm).29,30 There have also
been reports on the use of HPGs modified with anionic groups
as heparin analogues and as anti-inflammatory agents.31−33 It
has been demonstrated that grafting of red blood cells (RBCs)
to HPGs results in increased antigen protection on the RBC
surfaces.34−36 The use of aldehyde-functionalized HPGs as
soluble peptide capture agents in proteomic analysis has been
studied.37,38 There is also a recent report on the conjugation of
choline phosphate to HPG and its use as a universal
biomembrane adhesive.39 All of these studies demonstrate the
versatility of HPGs for a variety of applications in bio/
nanomedicine.
Another attractive feature that makes HPGs suitable for drug
delivery is that their circulation half-lives in vivo can be
controlled by varying the molecular weights. For example,
HPGs of molecular weight 100 kDa and 500 kDa have
circulation half-lives of 32 h and 57 h, respectively.40 The
conjugation of drugs to such high-molecular-weight polymers
may result in better bioavailability of the drugs due to the
longer circulation times that in turn lead to better therapeutic
efficiency. In addition to this, conjugation of proteins or
carbohydrates to high-molecular-weight HPGs leads to multi-
valent interactions with proteins and cells.21,22 Despite these
advantages, in vivo studies have revealed relatively higher levels
of accumulation for higher molecular weight HPGs in organs
such as liver,39 10−14% of injected dose was accumulated in
the liver. The organ accumulation decreased with decreasing
molecular weight.39 Also, the glomerular filtration limit for
polymers is in the range of 50−65 kDa. This necessitates the
need for designing biodegradable versions of HPGs for the
development of long circulating drug delivery vehicles. Also,
controlling the molecular weight of the degraded fragment will
facilitate the clearance of these polymers via kidney without
organ accumulation and without affecting their biocompatibility
profile. This can be achieved by incorporating defined
degradation points within the polymer. One such approach
towards synthesizing biodegradable HPG nanogels has been
reported by Haag and coworkers using disulfide as the
degradable linkage that can be cleaved under the reductive
environment within cells.41 Acid-catalyzed miniemulsion
polymerization of triglycidyl ether with disulfide containing
cross-linkers was used for the synthesis of these biodegradable
HPG nanogels. However, there are no reports on the synthesis
of soluble biodegradable HPGs without the use of cross-linkers.
Here we report the synthesis of biodegradable hyper-
branched polyglycerols (BHPGs) using novel multifunctional
initiators containing different numbers of acid degradable
dimethyl and cyclohexyl ketal linkages in their structure. This is
the first report of ketal-group-containing HPGs. We selected
acid-degradable ketal group as the degradable moiety due to the
broad range of applications of pH sensitive polymers as delivery
vehicles for antitumor agents, proteins and nucleic acids as well
as for the treatment of acute inflammatory diseases. pH-
sensitive polymers could be more efficient in tumor targeting
due to the acidic environment of most tumor cells. Protein and
nucleic acid delivery requires the endosomal degradation of the
polymeric carrier and release of the therapeutic molecule to the
cytosol.42−46 The ketal groups can undergo degradation under
the mild acid conditions of the cellular compartments such as
endosomes.47 In this article, we report the synthesis of five
different ketal-containing initiators. BHPGs were synthesized
by the anionic ROMBP of glycidol using these initiators. We
have investigated polymerization of glycidol from different
initiators and studied their degradation behavior. The control
over polymer degradation was achieved using different ketal
linkages in the polymer. We have also investigated the cell
compatibility and hemocompatibility of the degradable HPGs
and their degradation products.
2. EXPERIMENTAL SECTION
2.1. Materials. Ethylene glycol (anhydrous, 99.8%), trimethyl
orthoacetate (99%), p-toluene sulfonic acid monohydrate (ACS
reagent, ≥98.5%), pyridinium p-toluenesulfonate (98%), molecular
sieves 5 Å, 2-methoxy propene (97%), trimethyl orthoformate (99%),
cyclohexanone (99.5%), 2-bromoethanol (95%), glycerol (99.5%),
pentaerythritol (98%), diethylene glycol (≥99%), copper sulfate
pentahydrate (ACS reagent, ≥98%), sodium ascorbate (≥98%), and
potassium methoxide (25% solution in methanol) were all purchased
from Sigma-Aldrich, Ontario and were used without further
purification. Glycidol (96%) (Sigma-Aldrich) was distilled under
reduced pressure before use and stored over molecular sieves at 4 °C.
Dichloromethane, diethyl ether, tetrahydrofuran, methanol, hexane
and ethyl acetate were procured from Fisher Scientific, Canada.
Ethylene glycol monoacetate,48 2-azido ethanol,49 1-Methoxycyclohex-
ene,50 bis(2-propynoxyethyl)ether,51 tris(2-propynoxy)propane51 and
tetrakis(2-propynoxymethyl)methane51 were synthesized as per the
previously reported literature.
2.2. Characterization. High-resolution mass spectra of the
compounds were recorded on a Waters/Micromass LCT time-of-
flight mass spectrometer. NMR spectra of the initiators were recorded
in CDCl3 on a Bruker Avance 300 MHz NMR spectrometer. NMR
spectra of the polymers were recorded in D2O or CD3OD. Inverse-
gated (IG) 13C NMR spectra of the polymers were done in deuterated
methanol (CD3OD) with a relaxation delay of 6 s. Molecular weights
of the polymers were determined by gel permeation chromatography
(GPC) on a Waters 2695 separation module fitted with a DAWN EOS
multiangle laser light scattering (MALLS) detector coupled to an
Optilab DSP refractive index detector, both from Wyatt Technology.
Molecular weight and polydipsersity of the polymers were measured
using GPC-MALLS in 10 mM phosphate-buffered 0.1 M NaNO3 at
pH 8.5. The analysis was performed using Waters Ultrahydrogel
columns (guard, linear and 120) as the mobile phase. We measured
the dn/dc of BHPG-DM-1, and it was found to be 0.12, which is
similar to that of HPG. The dn/dc value of the polymers is expected to
be similar because the initiator content in the polymer is about 5−
12%. The degree of branching of the polymers was calculated
according to the previously reported literature.10 To study the pH-
dependent degradation, we prepared polymer solutions in buffer
solutions in D2O having pH 1.1−8.2. pH 1.1 buffer was prepared using
potassium chloride and HCl, and pH 4.1 buffer was prepared using
potassium hydrogen phthalate and sodium hydroxide. Potassium
dihydrogen phosphate and sodium hydroxide were used for the
preparation of buffer solutions of pH between 5.5 and 8.2. For the
polymer degradation studies using NMR analysis, 12−15 mg of the
polymer was dissolved in the respective buffer, and their NMR spectra
were recorded at various time intervals. The percentage of degradation
was calculated from the ratio of the intensities of the peaks
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corresponding to ketal group in the polymer and ketone groups
generated during the degradation.
Ethyleneglycol monoacetate (7.2 g, 0.069 mol) and azidoethanol (6 g,
0.069 mol) were dissolved in THF (200 mL). p-Toluene sulfonic acid
(1.3 g, 0.0069 mol) was added to this solution and stirred for 10 min. 5
Å molecular sieves (100 g) was then added and stirred for another 10
min. Finally, 1-methoxy cyclohexene (6.2 g, 0.0552 mol) was added,
and the reaction mixture was stirred at room temperature for 48 h.
Sodium bicarbonate (0.5 g) was added to the mixture, stirred for 15
min, and filtered through a pad of K2CO3 kept over Celite bed. The
filtrate was evaporated to obtain the crude product, which was purified
by column chromatography on silica gel using 10% ethyl acetate/
2.3. Synthesis of Functional Initiators. 2.3.1. Synthesis of 2,2′-
[Propane-2,2-diylbis(oxy)] Diethanol (DMKI-1). Ethylene glycol
monoacetate was synthesized as per reported procedure.48 Ethylene
glycol (30 mL, 0.537 mol), trimethyl orthoacetate (90 mL, 0.698 mol),
and p-toluene sulfonic acid (2 g) were dissolved in 600 mL of
dichloromethane and stirred at room temperature for 4 h. Water (12.6
mL, 0.698 mol) was added to the reaction mixture and stirred for an
additional 1 h. Dichloromethane was removed under reduced pressure,
and the product 2-hydroxyethylacetate (2) was purified by flash
chromatography on silica gel using chloroform/acetone (9:1). Yield:
70%. 1H NMR (CDCl3, 300 MHz, δ): 2.09 (s, 3H), 3.83 (t, 2H), 4.19
(t, 2 H). Compound 2 (22 g, 0.211 mol) was dissolved in 300 mL of
THF. Pyridinium p-toluenesulfonate (PPTS, 2.2 g, 0.00844 mol) was
added and stirred for 15 min, followed by the addition of molecular
sieves (5 Å) (100 g) and stirring for an additional 15 min. To this
mixture, 2-methoxypropene (8.4 mL, 0.0844 mol) was added, and the
reaction mixture was stirred at room temperature for 48 h. THF was
removed under reduced pressure to yield propane-2,2-diylbis-
(oxyethane-2,1-diyl)diacetate (3) as a pale-yellow liquid. Yield: 60%.
1H NMR (CDCl3, 300 MHz, δ): 1.38 (s, 6H) 2.07 (s, 6H), 3.64 (t,
1
hexane as the eluent. Yield: 40%. H NMR (CDCl3, 300 MHz, δ)
1.41−1.68 (m, 10H), 2.06 (s, 3H), 3.36 (t, 2H), 3.61 (m, 4 H), 4.21 (t,
2H). The azido acetate compound (0.5 g, 1.85 mmol) and bis(2-
propynoxyethyl)ether (0.112 g, 6 mmol) were dissolved in methanol.
To this mixture was added an aqueous solution of CuSO4·5H2O
(0.071 g, 3.6 mmol) and sodium ascorbate (89 mg, 3.6 mmol), and the
solution was stirred at room temperature for 48 h. Methanol was
removed under reduced pressure; the residue was dissolved in
dichloromethane and washed three times with brine. The organic layer
was dried over anhydrous sodium sulfate, and methanol was removed
under reduced pressure to yield a dark-brown residue. The compound
was further purified by column chromatography on silica gel using 5%
methanol/dichloromethane as the eluent. Deprotection of the acetyl
groups of the compound was done using potassium carbonate in
methanol/water mixture using a similar procedure as that described for
4H), 4.18 (t, 4 H).
Deprotection of acetyl group in compound 3 was carried out by
treating with sodium hydroxide in methanol/water at room temper-
ature. After the reaction, brine was added and the product was
extracted with dichloromethane. Evaporation of dichloromethane
under reduced pressure yielded 2,2′-[propane-2,2- diylbis(oxy)]
1
CHKI-1. Yield: 63%. H NMR (CDCl3, 300 MHz, δ): 7.73 (2H),
4.69(4H), 4.52(4H), 3.82(4H), 3.68(8H), 3.59 (4H), 3.18(4H),
1.30−1.65 (m, 20H). 13C NMR (CDCl3, 75 MHz, δ): 144.34, 123.81,
100.07, 70.13, 69.23, 64.14, 61.26, 60.80, 58.07, 50.21, 33.10, 24.98,
22.42. HRMS: Calculated for [C30H52N6O9+Na]: 663.3693; found
663.3683.
2.3.4. Synthesis of CHKI-3. CHKI-3 was synthesized by a similar
procedure as that for CHKI-2 using tris(2-propynoxy)propane51 and
azidoethanol49 (Scheme 2). Tris(2-propynoxy)propane was synthe-
sized as per reported procedure.51 1H NMR (CDCl3, 300 MHz, δ):
7.80 (1H), 7.75(2H), 4.78(2H), 4.64(4H), 4.54(6H), 3.84(6H),
3.61(11H), 3.21(6H), 1.30−1.65(30H). 13C NMR (CDCl3, 75 MHz,
δ): 144.7, 144.27, 123.88, 100.14, 69.94, 64.41, 63.86, 61.32, 60.86,
58.11, 53.31, 50.25, 33.15, 25.03, 22.49. HRMS: Calculated for
[C42H71N9O12+Na]: 916.5120; found 916.5115.
2.3.5. Synthesis of CHKI-4. CHKI-4 was synthesized by a similar
procedure as that for CHKI-2 using tetrakis(2-propynoxymethyl)
methane51 and 2-azido ethanol.49 1H NMR (CDCl3, 300 MHz, δ):
7.72(4H), 4.55(16H), 3.84(8H), 3.61(8H), 3.45(8H), 3.21(8H),
1.30−1.65(40H). 13C NMR (CDCl3, 75 MHz, δ): 144.77, 123.85,
100.24, 68.91, 64.66, 61.32, 61.47, 60.96, 58.19, 50.29, 33.25, 25.13,
22.59. HRMS: Calculated for [C57H96N12O16+Na]: 1227.6965; found
1227.6971.
2.4. General Procedure for the Synthesis of Biodegradable
Hyperbranched Polyglycerols. Polymerization of glycidol was
carried out under an argon atmosphere in a three-necked glass reactor
equipped with a mechanical stirrer and a syringe pump. The new
initiators were dissolved in methanol, and 15% of the hydroxyl groups
were deprotonated by stirring with potassium methoxide (25%
solution in methanol) for 30 min. Methanol was removed under
reduced pressure for 5 h. The temperature was increased to 95 °C, and
glycidol was added very slowly using a syringe pump over several
hours. After the complete addition of the monomer, the reaction
mixture was stirred for an additional 3 h. The polymer was dissolved in
methanol and precipitated twice from acetone and dried in vacuum.
2.5. Blood Compatibility Analysis. Blood from healthy
consented unmedicated donors was collected into either a 3.8%
sodium citrated tube with a blood/anticoagulant ratio of 9:1 or a
serum tube at Centre for Blood Research, University of British
Columbia. ’The protocol was approved by University clinical ethical
committee. Platelet-rich plasma (PRP) was prepared by centrifuging
citrated whole blood samples at 150g for 20 min in an Allegra X-22R
centrifuge (Beckman Coulter, Canada). Platelet-poor plasma (PPP)
was prepared by centrifuging citrated whole blood samples at 1200g
for 20 min. Serum was prepared by centrifuging whole blood samples
1
diethanol (4) as yellow viscous oil. Yield: 40%. H NMR (CDCl3,
300 MHz, δ): 3.71 (t, 4H), 3.58 (t, 4H), 1.38 (s, 6H). 13C NMR
(CDCl3, 75 MHz, δ): 99.42, 61.65, 60.93, 24.17. HRMS: Calculated
for [C7H16O4+Na]: 187.0946; found: 187.0945.
2.3.2. Synthesis of 2,2′-[Cyclohexane-2,2-diylbis(oxy)] Diethanol
(CHKI-1). 1-Methoxy cyclohexene was synthesized as per the literature
procedure.50 p-Toluene sulfonic acid (0.50 g) was added slowly to a
mixture of cyclohexanone (5, 20 mL, 0.193 mol) and trimethyl
orthoformate (30 mL, 0.274 mol) and stirred at r.t. (22 °C) overnight.
It was then connected to a fractional distillation unit with a long
vigreux column, and the reaction mixture was distilled at atmospheric
pressure. Methanol and methyl formate were removed as first fraction,
followed by unreacted trimethyl orthoformate. The fraction that
distilled at 125−138 °C was collected as pure 1-methoxy cyclohexene
(6). To a solution of ethylene glycol monoacetate (5.2 g, 0.05 mol) in
THF (75 mL) was added p-toluene sulfonic acid (0.50 g, 0.0026 mol)
and 5 Å molecular sieves (25 g) and stirred for 15 min. Compound 6
(2.2 g, 0.0196 mol) was added, and the reaction mixture was stirred at
r.t. for 48 h. Sodium bicarbonate (0.5 g) was added and stirred for 15
min, and the reaction mixture was filtered through a pad of K2CO3
kept over Celite bed. The filtrate was evaporated to obtain the crude
product, which was purified by column chromatography on silica gel
1
using 5% ethyl acetate in hexane as the eluent. Yield: 46%. H NMR
(300 MHz, CDCl3, δ): 4.2 (t, 4H), 3.6 (t, 4H), 2.1 (s, 6H), 1.66 (m,
4H), 1.59 (m, 4H), 1.51(m, 2H). 13C NMR (75 MHz, CDCl3, δ):
170.9, 100.3, 63.8, 57.8, 33.4, 22.7, 21.9.
To a stirred solution of the product obtained in the previous step in
methanol, potassium carbonate and water were added and stirred at r.t.
for 15 h. Reaction mixture was filtered through Celite bed, washed
with methanol, and concentrated. The residue obtained was triturated
with excess diethyl ether (3 × 50 mL), and ether was removed under
reduced pressure. The product obtained was triturated again with
hexane (2 × 10 mL), and the hexane layer was decanted. The viscous
product was then dissolved in diethylether, dried over anhydrous
sodium sulfate, filtered, and evaporated to afford pure product, CHKI-
1
1 (8) as a white solid. Yield: 90%. H NMR (CDCl3, 300 MHz, δ):
3.75 (t, 4H), 3.58 (t, 4H), 1.69 (t, 4H), 1.52 (m, 4H), 1.43(m, 2H).
13C NMR (CDCl3, 75 MHz, δ): 99.23, 60.96, 60.20, 32.73, 24.61,
21.99. HRMS: Calculated for [C10H20O4+Na]: 227.1259; found
227.1261.
2.3.3. Synthesis of CHKI-2. 2-Azidoethanol49and bis(2-
propynoxyethyl)ether51 were synthesized as per reported procedure.
C
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Figure 1. Chemical structures of the hydroxyl functionalized ketal initiators containing one, two, three, and four ketal groups per molecule.
collected in serum tubes at 1200g for 30 min. The polymer stock
solutions were made in HEPES buffer at a concentration of 10 mg/
mL. To make the degraded polymer solution, we prepared BHPG
stock solutions in HEPES buffer at 10 mg/mL and kept them at 37 °C
for 15 days. It was evident from NMR analysis that the polymers were
completely degraded during this time (Supporting Information; Figure
S19). The biocompatibility of the degradation products was studied
using the completely degraded polymer solutions.
2.5.1. Activated Partial Thromboplastin Time Assay. Sodium
citrate anticoagulated PPP was used for activated partial thromboplas-
tin time (APTT) analysis. The effect of BHPGs and their degradation
products on the coagulation cascade was examined by mixing PPP
with the polymer or degraded polymer solution (9:1 v/v; 1 mg/mL
final concentration) at 37 °C. Control experiments were performed
with identical volumes of HEPES buffer solution to PPP. The
coagulation reagent actin FSL was used for APTT analysis. Each
experiment was repeated in triplicates on the STart4 coagulometer
(Diagnostica Stago, France) with plasma from three separate donors,
and the average SD was reported.
2.5.2. Platelet Activation Analysis. The level of platelet activation
in PRP was quantified by flow cytometry. PRP (90 μL) was incubated
at 37 °C with 10 μL of BHPGs or degraded BHPGs in HEPES buffer
to get a final concentration of the polymer 1 mg/mL. After 1 h,
aliquots of the incubation mixtures were removed for assessment of
the platelet activation state. Postincubation BHPG/platelet mixture (5
μL), diluted in HEPES buffer, was incubated for 20 min in the dark
with 5 μL of monoclonal anti-CD62P-PE (Immunotech) and 5 μL of
anti-CD42-FITC. The samples were then fixed with 1 mL of formal
saline solution (37% formaldehyde in saline). The level of platelet
activation was analyzed in a BD FACSCanto II flow cytometer
(Becton Dickinson) by gating platelets specific events based on their
FITC-CD42 fluorescence and light scattering profile. Activation of
platelets was expressed as the percentage of platelet activation marker
CD62P-PE fluorescence detected in the 10 000 total events counted.
Bovine thrombin (1 U/mL, Sigma) was used as a positive control, and
FITC-conjugated and PE-conjugated goat antimouse IgG polyclonal
antibodies (Immunotech) were used as the nonspecific binding
control. PRP incubated with identical volumes of HEPES as that of the
polymer was used a normal control. Duplicate measurements were
performed using PRP from three separate donors, and the average
SD values are reported.
microliters each of the BHPG polymers was mixed with 90 μL of fresh
human serum for 1 h at 37 °C to get a final concentration of BHPG in
serum of 1 mg/mL. Heat-activated human IgG (1 mg/mL) and 5 mM
EDTA were positive and negative controls, respectively. HEPES buffer
added serum was used as normal control. The postincubation mixture
(60 μL) was diluted with 120 μL of GVB2+ (CompTech). GVB2+
diluted mixture (75 μL) was incubated for 1 h at 37 °C with 75 μL of
antibody-sensitized sheep erythrocytes (CompTech). The reaction
was stopped by the addition of 300 μL of cold GVB-EDTA to each
sample. The samples were centrifuged, and the optical density of
supernatant at 540 nm was measured. Distilled water incubated
antibody-sensitized sheep erythrocytes was used as 100% lysis control
for the calculation. The total complement consumed was measured,
and the value is reported as the percent of complement activation.
2.5.4. Cell Viability Analysis. Human umbilical vein endothelial cells
(HUVECs) were used to examine the cell viability in presence of
BHPGs and their degradation products. The polymers were dissolved
in the endothelial cell growth medium (EGM-2 BulletKit, Lonza)
media, filtered, and incubated with the cells for 48 h. Starting with an
initial polymer concentration of 5 mg/mL, serial dilutions of the
polymer solutions with the media were performed to achieve the lower
concentrations. Degraded polymer solutions were made by keeping
the stock polymer solution in the media for 15 days at 37 °C and then
serially diluting the solution with media to obtain different
concentrations. HUVECs incubated with the media only were used
as the normal control for the study. At the end of the 2-day incubation
period, viability was measured by adding 18 μL of the 3-[4,5-
dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-
2H-tetrazolium] (MTS) reagent to each of the wells. The optical
density was measured at two wavelengths, 490 and 600 nm. The
medium only MTS value was used as the baseline and was expressed as
100% viability. HUVEC viability after exposure to the polymer samples
was expressed as the percentage of the cells viable compared with
medium-treated cells.
3. RESULTS AND DISCUSSION
3.1. Synthesis of Multifunctional Initiators Containing
Ketal Groups. Five different heterofunctional initiators
containing dimethyl and cyclohexyl ketal groups in their
structure were designed and synthesized (Figure 1). The
hydroxyl initiator containing a dimethyl ketal group, DMKI-1,
was synthesized from ethylene glycol as the starting material
2.5.3. Complement Activation Analysis. The level of complement
activation was measured by CH50 sheep erythrocyte lysis assay. Ten
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a
Scheme 1. Synthetic Route for Multifunctional Initiators with One Ketal Group
a
(A) 2,2′-[propane-2,2-diylbis(oxy)] diethanol (DMKI-1); (B) 2,2′-[cyclohexane-2,2-diylbis(oxy)] diethanol (CHKI-1).
(Scheme 1A). Ethylene glycol was first converted to its
monoacetate 2 by a reported procedure,48 which was then
reacted with 2-methoxy propene to generate the ketalized
diacetate 3. Deprotection of the acetyl groups with potassium
carbonate in methanol/water mixture yielded the compound 4.
The formation of the compound was confirmed by NMR
spectroscopy (Supporting Information; Figures S1 and S2)
afford the ketalized azido acetate compound 11. The trialkyne
12 was synthesized by the reaction of glycerol with propargyl
bromide using a reported procedure.51 The azide 11 and alkyne
12 were reacted in the presence of copper sulfate/sodium
ascorbate in methanol/water mixture to yield the compound
13, which was purified by column chromatography. Depro-
tection of the acetyl groups of 13 using potassium carbonate in
methanol/water afforded the trihydroxy compound 14 with
three cyclohexyl ketal linkages. The compound was charac-
1
which showed a signal at 1.39 ppm in the H NMR spectrum
characteristic of the methyl resonance of the dimethyl ketal
group (Supporting Information; Figure S1). To synthesize the
initiator containing cyclohexyl ketal group, CHKI-1, we
transformed cyclohexanone into its enol ether 6 by reaction
with trimethyl orthoformate in the presence of p-toluene
sulfonic acid50 (Scheme 1B). The enol ether was then reacted
with ethylene glycol monoacetate to yield the cyclohexyl
ketalized diacetate 7, which upon treatment with potassium
carbonate in methanol/water afforded the compound 8. NMR
spectra of the compound 8 are given in the Supporting
Information. (See Figures S3 and S4.)
Attempts to synthesize compounds with multiple ketal
groups using a similar procedure were not successful. The
reaction of trimethylolpropane or pentaerythreitol and ethylene
glycol monoacetate with 2-methoxypropene resulted in intra-
molecular cyclization reactions. So we employed an alkyne−
azide click reaction for synthesizing initiators CHKI-2, CHKI-
3, and CHKI-4 with multiple ketal linkages. A representative
synthetic procedure for CHKI-3 is shown in Scheme 2. In the
first step, one equivalent each of ethylene glycol monoacetate
and azido ethanol was reacted with 1-methoxy cyclohexene to
1
terized by H NMR that showed signals in the region 7.75 to
7.80 ppm corresponding to the triazole ring proton and those
at 1.3 to 1.65 ppm characteristic of the cyclohexyl protons. (See
the Supporting Information, Figures S7 and S8.) CHKI-2 and
CHKI-4 were synthesized using a similar procedure. NMR
spectra of the compounds are shown in the Supporting
Information. (See Figures S5, S6, S9, and S10.)
3.2. Synthesis of Core-Functionalized BHPGs. All five
compounds DMKI-1, CHKI-1, CHKI-2, CHKI-3, and CHKI-
4 were investigated as initiators for the anionic ROMBP of
glycidol (Scheme 3). The compounds were partially deproto-
nated (15% of the hydroxyl groups) using potassium
methoxide, and polymerizations were carried out at 95 °C
with slow monomer addition (Table 1). Polymer yields were in
the range of 70−80% with all initiators and were comparable to
1
those reported with trimethylol propane as the initiator. H
NMR spectra of the polymers showed characteristic signals for
the ketal group, which confirmed that the polymer was growing
from the ketal-group-containing initiators. The polymer
BHPG-DM-1 obtained from DMKI-1 initiator showed a signal
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Scheme 2. Synthetic Route for Cyclohexyl Ketal Initiator CHKI-3 Using Azide−Alkyne Click Chemistry
at 1.39 ppm corresponding to the methyl protons of the
dimethyl ketal group (Figure 2A and Supporting Information
Figure S11). Signals at 1.3 to 1.7 ppm characteristic of the
cyclohexyl ketal groups were observed for the polymers BHPG-
CH-1, BHPG-CH-2, BHPG-CH-3, and BHPG-CH-4 ob-
tained from initiators CHKI-1, CHKI-2, CHKI-3, and CHKI-
4, respectively (Figure 2C and Supporting Information Figures
S12−S15). The polymers BHPG-CH-2, BHPG-CH-3, and
BHPG-CH-4 also showed signals at 8.07, 8.04, and 7.98 ppm,
initiator.10 The number of hydroxyl groups on the initiator
has no significant effect on the degree of branching. Because
ketal groups are known to hydrolyze under acidic conditions,
GPC analysis of the polymers was performed in 0.1 N NaNO3
at pH 8.5 to avoid degradation of the polymers during analysis.
All polymers exhibited monomodal molecular weight distribu-
tion, and the polydispersities were in the range of 1.26 to 1.57
(Table 1). A representative GPC profile for BHPG-CH-4 is
shown in Figure 4, and GPC chromatograms of other polymers
are given in the Supporting Information (Figure S16). Results
showed that the polymerizations were controlled and glycidol
was polymerized uniformly from the initiators.
1
respectively, in the H NMR spectra corresponding to the
proton of the triazole ring, which further confirms that the
growth of the polymer chains was occurring from the initiators.
The branched nature of these polymers was confirmed by
inverse gated 13C NMR spectrum (Figure 3); the degree of
branching was in the range of 0.54 to 0.58 which was closer to
the values reported for HPGs synthesized from TMP
3.3. Degradation of the Core-Functionalized BHPGs.
After synthesizing BHPGs with well-defined degradation points
in their core, we investigated their degradation behavior at
different pH values. All of the newly developed BHPGs were
F
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Scheme 3. Synthetic Route for Biodegradable Hyperbranched Polyglycerol (BHPG-CH-4) Containing Four Ketal Linkages
from the Initiator CHKI-4
highly soluble in water. The presence of ketal group in the core
structure of the polymers makes them susceptible to
degradation under acidic conditions, resulting in the formation
of low-molecular-weight polymer fragments and small molec-
ular weight ketones. Thus, it is anticipated that polymers
BHPG-DM-1 and BHPG-DM-2 with dimethylketal groups
upon degradation will give two HPG fragments and acetone.
The polymers BHPG-CH-1, BHPG-CH-2, BHPG-CH-3, and
BHPG-CH-4 with cyclohexyl ketal groups will degrade to HPG
fragments and cyclohexanone. To study the degradation, we
dissolved the polymers in deuterated buffers of pH 1.1−8.2,
and their NMR spectra were monitored at different time
intervals. Degradation of the polymers was evident from the
decrease in the intensity of the signal due to ketal group and the
appearance of new signals from the ketone released during the
degradation. For example, in the case of BHPG-DM-1, the
signal at 1.39 ppm characteristic of the dimethyl ketal group
gradually disappeared with time and a new signal at 2.18 ppm
appeared due to the formation of released acetone (Figures 2B
and 5). In the case of polymers with cyclohexyl ketal groups,
broad signals at 1.3 to 1.7 ppm corresponding to the cyclohexyl
groups were shifted to sharper signals at 1.7 and 1.82 ppm, and
a new signal was observed at 2.32 ppm due to the
cyclohexanone produced during degradation (Figure 2D and
G
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Table 1. Characteristics of Acid-Cleavable BHPGs Synthesized from Ketal Initiators
degradation half life at different pH
a
Mn (Mw/Mn)
ketal initiator no. of ketal groups theoretical molecular weight before degradation after degradation
values (h)
polymer
5.5
0.3
6.0
6.5
7.4
BHPG-DM-1
BHPG-DM-2
BHPG-CH-1
BHPG-CH-2
BHPG-CH-3
BHPG-CH-4
DMKI-1
DMKI-1
CHKI-1
CHKI-2
CHKI-3
CHKI-4
1
1
1
2
3
4
5000
25000
10000
5000
5200 (1.3)
2800
13 900
4700
1
2.3
32.8
n.d.
b
26000 (1.6)
9500 (1.26)
4500 (1.24)
7500 (1.57)
10800 (1.54)
n.d.
2
n.d.
4.7
n.d.
13.1
19.2
27.4
30.1
138.6
168
528
564
2500
1.8
4
6.6
10000
2300
15.4
18.5
10000
3400
4.3
a
Percent of degradation was calculated from 1H NMR spectra by monitoring the disappearance of peaks due to the ketal group in the polymer and
appearance of the peaks due to the ketone formed as a result of degradation. The degradation half lives were determined from the plotting ln(A/A0)
against time, where A0 is the initial amount of the polymer and A is the concentration of the polymer that is not degraded at a given time t.
b
n.d.: not determined.
1
Figure 2. Polymer degradation studied by proton NMR spectroscopy. Shown are H NMR spectra of BHPG-DM-1 and BHPG-CH-3 before and
after degradation. (A) BHPG-DM-1 before degradation. The signal at 1.39 ppm corresponds to the ketal group. (B) BHPG-DM-1 after degradation.
The signal at 1.39 ppm due to ketal group disappears completely, and the new signal at 2.18 ppm corresponds to the formation of degradation
product acetone. (C) BHPG-CH-3 before degradation. The signal at 1.3 to 1.6 ppm corresponds to the cyclohexyl protons of the cyclohexyl ketal
group. (D) BHPG-CH-3 after degradation. The signals due to the ketal group completely disappeared, and new sharp peaks appear at 2.32 and 1.65
to 1.85 ppm due to the cyclohexanone formed as a result of degradation.
Figure 3. Inverse-gated 13C NMR (CD3OD, 75 MHz) of BHPG-CH-3. The polymer consists of linear (L13 and L14), dendritic (D), and terminal
(T) structural units. The peak assignments were made according to the literature.10
also see the Supporting Information; Figure S17). The time-
dependent degradation profiles of the different BHPGs at
different pH values are shown in Figure 6. The polymers
showed remarkable pH-dependent degradation kinetics; at
strongly acidic pH values (1.1 and 4.1), all polymers were
completely degraded within the first time point of the NMR
experiment (30 min). Controlled degradation profiles were
observed in the pH range 5.5−8.2, with slower degradation
rates at higher pH values (Figure 6A). The degradation half-
lives of the polymers were determined from the plots of ln(A/
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compared with BHPG-CH-1 with one cyclohexyl ketal group
without triazole group and BHPG-CH-2 with two cyclohexyl
ketal and two triazole groups. A similar trend was observed for
other pH values greater than 5.5. Below pH 5.5, the rate of
degradation was much faster for all polymers. The differences in
the degradation rates between the BHPGs containing three and
more triazole rings in the initiator core may be due to the
increased hydrophobicity of the core structure by the triazole
groups, which could possibly change the penetration of water to
the core. It has been reported that the degradation of ketal
groups could be controlled by varying the hydrophobicity of
long chain alkyl groups attached to the ketal moiety.54 There
were no significant differences in the hydrolysis rates between
BHPG-CH-3 and BHPG-CH-4 containing three and four
triazole rings, respectively.
To study the degradation behavior of the BHPGs under in
vivo conditions and within cells, the degradation was also
studied at 37 °C for the polymers BHPG-CH-1 and BHPG-
CH-3 at pH 5.5 and 7.4. At both of these pH values the
degradation was found to be two to three times faster at 37 °C
compared with that at 25 °C (Supporting Information; Figures
S18 and S19).
Figure 4. GPC-MALLS chromatograms in 0.1 N NaNO3 at pH 8.5 of
BHPG-CH-4 before and after degradation. The second elution peak
for the degraded product could be from the core as the degraded
product was not separated before injection. The molecular weight of
the degraded product was calculated from the main peak.
The degradation of BHPG was also studied by gel
permeation chromatography. The molecular weights of the
BHPGs before and after degradation are given Table 1. The
decrease in the molecular weights of degraded BHPG was
correlating very well with the number of ketal groups present in
the initiator molecules. Therefore, for BHPG-DM-1, which
contains one ketal group in the polymer chain, the molecular
weight decreased from 5200 to 2800 g/mol upon degradation.
Similar observations were made for BHPG-DM-2 and BHPG-
CH-1, which contain one ketal group, and their molecular
weights after degradation were approximately half the original
molecular weight. The BHPG-CH-3 and BHPG-CH-4 also
degraded similarly to low-molecular-weight HPGs, and the
decrease in molecular weight was proportional to the number
of ketal groups within the polymer (Figure 4). These results
together with the low polydispersity and NMR analysis of the
BHPGs suggest that the growth of the polymer chains occurred
uniformly from the hydroxyl groups of the ketal initiators.
3.4. Biocompatibility Evaluation of BHPGs. 3.4.1. Blood
Compatibility of BHPGs. Hemocompatibility is an important
criterion for polymers to be used for vascular applications.55
Therefore, the newly synthesized BHPGs and their degradation
products were evaluated for their blood compatibility by
measuring the blood coagulation, complement activation, and
platelet activation in presence of polymers. All of the
experiments were performed by adding the polymer solutions
or degraded polymer solutions in buffer to human plasma/
serum.
Blood coagulation in the presence of the BHPGs and their
degradation products was measured to investigate their pro- or
anticoagulant nature.56,57 Blood clotting time was measured
using clinical clotting assays in the presence of BHPGs. The
polymer solutions were incubated with human PPP at 37 °C,
and the clotting time was monitored by APTT that measures
the time (in seconds) taken for a fibrin clot to form after the
addition of partial thromboplastin reagent (actin) and calcium
chloride. The APTT of the plasma in the presence of BHPGs
and their degradation products was comparable to that of the
buffer control (Figure 7A), which shows that these polymers do
not cause any adverse effect on blood coagulation. The BHPGs
and their degradation products showed a similar profile as that
1
Figure 5. Polymer degradation kinetics by NMR analysis. H NMR
spectra (D2O, 300 MHz) showing the degradation of BHPG-DM-1 at
pH 5.5. The intensity of the signal at 1.39 ppm due to the dimethyl
ketal group gradually decreases with time, and a new signal appears at
2.18 ppm due to the formation of acetone as the degradation product.
A0) against time, where A0 is the initial amount of the polymer
and A is the concentration of the polymer that is not degraded
at any given time. The degradation half-lives of the polymers
are given in Table 1. The polymer BHPG-DM-1 with dimethyl
ketal group showed degradation half-lives of 0.3, 1, 2.3, and
32.8 h at pH 5.5, 6.0, 6.5, and 7.4, respectively. The degradation
half-lives of the cyclohexyl ketal group containing BHPG-CH-1
at pH 5.5, 6.0, 6.5, and 7.4 were, respectively, 2, 4.7, 13.1, and
138.6 h, respectively. All of these data clearly demonstrate that
degradation rates of the polymers can be changed by changing
the pH and the structure of ketal groups.
We compared the degradation profiles of BHPGs containing
cyclohexyl ketal group and dimethyl ketal groups (Figure
6B,C). A comparison of the degradation of different ketal
groups containing HPGs at pH 5.5 is given in Figure 6D. The
data show that the degradation of the cyclohexyl ketal group
containing HPG was slower than that of the BHPG containing
dimethyl ketal groups. For example, the degradation half-lives
of BHPG-DM-1 and BHPG-CH-1 at pH 5.5 were 0.3 and 2 h,
respectively (Table 1). This can be attributed to the difference
in the torsional strain involved during the hydrolysis of the
cyclohexyl ketal groups in comparison with dimethyl ketal
groups.52,53 As shown in Figure 6D, among the HPGs with
different numbers of cyclohexyl ketal groups, BHPG-CH-3 and
BHPG-CH-4 with three and four cyclohexyl ketal and triazole
groups, respectively, degraded at slightly lower rates at pH 5.5
I
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Figure 6. Degradation of core functionalized BHPGs. (A) pH-dependent degradation profiles of BHPG-DM-1, (B) BHPG-CH-1, and (C) BHPG-
CH-3. pH values are shown in the Figure. (D) Comparison of the degradation of BHPGs at pH 5.5. The BHPGs degraded rapidly at the acidic pH
values (1.1 and 4.1), whereas the degradation was more controlled in the pH 5.5−8.2. BHPGs with cyclohexyl ketal group degraded at much slower
rates than those with dimethyl ketal group.
Figure 7. Blood compatibility analysis of BHPGs. (A) Activated partial thromboplastin time (APTT) in human plasma measured at 37 °C. HEPES
buffer was used as normal control. (B) Complement activation in human serum measured by CH50 assay using antibody-sensitized sheep RBCs. IgG
and HEPES buffer were used as positive and normal controls, respectively. (C) Platelet activation in human-platelet-rich plasma by measuring the
expression of activation marker CD62P as measured by flow cytometry. Bovine thrombin and HEPES buffer were used as positive and normal
controls, respectively. Final concentration of polymer was 1 mg/mL in all cases.
J
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Figure 8. Cell viability of BHPGs (A) and their degradation products (B) against human umbilical vein endothelial cells measured by MTS assay
after 48 h of polymer incubation.
of HPGs reported.58 The slight variation in the buffer controls
used for the BHPG and their degradation products was due to
the donor variation as these experiments were performed
independently at different time points and blood was collected
from the different donors. Such donor variation is normal. In
each case, a comparison was made between the respective
buffer control with the polymers or the degradation products.
Complement activation upon interaction with polymers is an
indication of inflammatory potential of the polymer and an
indication of blood incompatibility. Activation of the comple-
ment system components can lead to several cellular responses
such as histamine release and induction of inflammation.55 The
level of complement activation by BHPGs was measured using
antibody-sensitized sheep erythrocyte complement consump-
tion assay.59 The BHPG samples were incubated with human
serum at 37 °C for 1 h, and the antibody-sensitized sheep
erythrocytes were added to this solution. Lysis of the sensitized
sheep erythrocytes was taken as a measure of complement
consumption. Erythrocyte lysis was quantified by the amount of
hemoglobin released. The level of complement activation by
the polymers was compared with that of buffer control and a
positive control, immunoglobulin G (IgG) (Figure 7B). Results
showed that BHPGs and their degradation products did not
activate the complement system, as evident from their
insignificant difference in values compared to that of the buffer
control.
Platelet activation upon interaction with polymers can result
in adverse effects such as thrombotic complications and arterial
embolization upon administration of the polymers.55 Platelet
activation by BHPGs was quantified by flow cytometry analysis.
The polymer solutions (1 mg/mL, final concentration) were
incubated with PRP for 1 h, and platelet activation was
measured as expression of platelet activation marker CD62P
using monoclonal anti-CD62P-FITC antibody. Platelet activa-
tion was expressed as the percentage of platelets that are
positive for CD62P. The platelet activation by BHPGs was
compared with that of control buffer, and polyethyleneimine
(PEI) was used as the positive control. As is evident from
Figure 7C, the extent of platelet activation by BHPGs was very
similar to that of the buffer control, which indicates that
BHPGs and their degradation products did not induce any
platelet activation.
polymer for 48 h and measuring the number of viable cells
by MTS assay. The results are shown in Figure 8, which
demonstrates that the BHPGs as well as their degradation
products were nontoxic to the cells up to a polymer
concentration of 5 mg/mL. This shows the excellent cell
compatibility of BHPGs and their degradation products.
4. CONCLUSIONS
We have demonstrated the synthesis of a new class of
biodegradable polymers based on core-degradable HPGs by
anionic ring-opening polymerization of glycidol using initiators
containing acid cleavable ketal linkages. Five different multi-
functional initiators containing a varying number hydroxyl
groups and ketal groups were synthesized. Both cyclohexyl-
ketal- and dimethyl-ketal-group-containing initiators were
synthesized. The polymerizations were well-controlled, as
evident from the low polydispersities and the monomodal
molecular weight distributions of the polymers. The polymers
were relatively stable at physiological pH values but exhibited
excellent control of degradation at acidic pH values. The
degradation rates of these polymers were controlled by varying
the structure of ketal groups and number of ketal groups in the
initiator. Polymers with cyclohexyl ketal groups in their core
showed slower degradation rates than those with dimethyl ketal
groups. The BHPGs were degraded to low molecular weights
depending on the number of ketal groups present in the BHPG,
which indicates that polymerization was occurring uniformly
from the hydroxyl groups of the initiators. Blood and cell
compatibilities of the BHPGs and their degradation products
were excellent. The ability to tune the degradation of BHPGs
by varying the ketal group structure in the initiator together
with their excellent biocompatibility makes these polymers
useful for a variety of drug delivery and bioconjugation
applications.
ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectra of the initiators and polymers, GPC-MALLS
traces of the polymers, NMR spectra showing the time-
dependent degradation of the BHPGs, and the degradation
profiles of the polymers at 37 °C are given. This material is
available free of charge via the Internet at http://pubs.acs.org.
3.4.2. Cell Compatibility of BHPGs. Cell compatibility of
BHPGs and their degradation products was evaluated by
incubating HUVECs with different concentrations of the
K
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AUTHOR INFORMATION
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*E-mail: jay@pathology.ubc.ca.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was funded by Canadian Institutes of Health
Research (CIHR). The LMB Macromolecular Hub at the UBC
Centre for Blood Research was funded by Canada Foundation
for Innovation and Michael Smith Foundation of Health
Research. J.N.K. is a recipient of a CIHR/CBS new investigator
in Transfusion Science and Michael Smith Foundation of
Health Research Career Investigator Scholar Award.
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