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Unimolecular Micelles based on Hydrophobically Derivatized Hyperbranched Polyglycerols: Biodistribution Studies Rajesh Kumar Kainthan† and Donald E. Brooks*,†,‡ Department of Pathology and Laboratory Medicine, Centre for Blood Research, and Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5. Received March 6, 2008; Revised Manuscript Received September 9, 2008
We recently reported the synthesis and testing of a new class of unimolecular micelles based on hyperbranched polyglycerols as second generation synthetic plasma expanders and as general drug delivery vehicles. A detailed biodistribution study of two derivatized hyperbranched polyglycerols of different molecular weights derivatized with hydrophobic groups and short poly(ethylene glycol) chains is reported in this article. In mice, these materials are nontoxic with circulation half-lives as high as 31 h, controllable by manipulating the molecular weight and the degree of PEG derivatization. Organ accumulation is low, presumably due to the “pegylation” effect. Thermal degradation and hydrolysis data suggest that these polymers are highly stable with a long shelf life, a major advantage for a pharmaceutical product. Degradation under acidic conditions has been observed for these polymers.
INTRODUCTION There is a huge clinical demand for human serum albumin (HSA), with the world market being in the ∼$1.5B/yr range (1). Because of supply and contamination problems with the human material, a number of alternatives have been developed as plasma expanders, but they all suffer from significant failings compared to the native protein (2, 3). While they can reproduce the osmotic function of HSA, none provide one of the most significant physiological functions of HSA, namely, the binding and transport in the circulation of a wide variety of small molecules including fatty acids, steroids, metal ions, and bilirubin, as well as hydrophobic drugs. Moreover, since most are linear polysaccharides, they increase plasma viscosity significantly, typically cause deleterious red cell aggregation, and exhibit a variety of complex physiological effects (4-7). Our overall goal is to develop a second-generation synthetic substitute for HSA consisting of a dendritic biocompatible polymer that not only has superior volume replacement properties compared to other plasma expanders, due to its low intrinsic viscosity, but also will closely mimic the compatibility, binding, and transport properties of the natural material. Toward this end, we recently reported the synthesis and preliminary testing of several derivatized hyperbranched polyglycerols (dHPGs) functionalized with octadecyl chains and monomethyl poly(ethylene glycol) (MPEG-400) oligomers (8). A simple one-pot synthetic strategy has been developed with the aim of reducing the cost of synthesis in an industrial setting. These dHPGs were shown to be highly biocompatible using various in Vitro hemocompatibility studies owing to the presence of PEG oligomers which protect the alkyl chains from interacting with cell membranes and plasma proteins. Being highly branched, these polymers have low intrinsic viscosity values and therefore are expected to increase blood viscosity only slightly. Moreover, they behave * Corresponding author. Centre for Blood Research, Life Sciences Centre, 2350 Health Sciences Mall, University of British Columbia, Vancouver, BC V6T 1Z3, Canada. Fax: (604) 822-7742, E-mail address:
[email protected] (D. E. Brooks). † Department of Pathology and Laboratory Medicine, Centre for Blood Research. ‡ Department of Chemistry.
as unimolecular micelles and have been shown to bind fatty acids and model hydrophobic drugs, implying that these polymers can also be applied as general drug carriers (8, 9). Due to the presence of functionalizable hydroxyl groups, they can also be decorated with targeting groups for directed drug therapy or bioactive materials that could be active intracellularly. An ideal synthetic plasma expander should have a reasonably long plasma circulation half-life compared to that of HSA (∼15 days) and should be metabolized or excreted as soon as it leaves the blood compartment. The circulation half-life of a polymer generally increases with increase in molecular weight and size; however, organ accumulation and uptake by the reticuloendothelial system (RES) also increases. Therefore, a balance is often sought for optimum performance. Long-term tissue deposits are a potential problem and still remain one of the drawbacks of the currently used plasma expanders (10). A detailed study of the biodistribution of two dHPGs therefore was carried out in mice, the results of which are the subject of the current manuscript.
EXPERIMENTAL PROCEDURES Materials. All chemicals were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON) and used without further purification except as noted. Glycidol (96%) was purified by vacuum distillation and stored over molecular sieves in a refrigerator (2-4 °C). 1,2-epoxyoctadecane was synthesized by the peroxidation of octadecene with m-chloroperbenzoic acid. R-epoxy,ωmethoxy poly(ethylene glycol) 350 (MPEG epoxide) was synthesized from a reaction of MPEG 350, sodium hydroxide, and epichlorohydrin. Two dHPGs of molecular weight 40 kDa (dHPG-40) and 83 kDa (dHPG-83) were prepared as previously reported (8). Polymer Characterization. NMR spectra were recorded on a Bruker Avance 400 MHz NMR spectrometer using deuterated solvents (Cambridge Isotope Laboratories, 99.8% D) with the solvent peak as a reference. Molecular weights and polydispersities of the polymers were determined by gel permeation chromatography (GPC) connected to a multiangle laser light scattering (MALLS) detector (GPC-MALLS). The GPC system used consists of a Waters 2690 separation module fitted with a DAWN EOS MALLS detector from Wyatt Technology Corp.
10.1021/bc800090v CCC: $40.75 2008 American Chemical Society Published on Web 10/11/2008
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Figure 1. Structure of the derivatized hyperbranched polyglycerol (dHPG). Table 1. Physical Characteristics of the dHPG Copolymers polymer
C18 mol %
MPEG mol %
glycidol mol %
no. of C18a
Mn × 10-4
Mw/Mn
[η] (mL/g)
Rh nm (QELS)
dHPG- 40 dHPG-83
1.6 1.6
20.4 29.4
78 69
4.4 7.6
4.0 8.3
1.5 1.8
7.2 7.9
6.5 7.7
a Number of C18 chains per molecule; Mn number average molecular weight; Mw/Mn polydispersity; [η] intrinsic viscosity; QELS quasi elastic light scattering; Rh average hydrodynamic radius.
with 18 detectors placed at different angles (laser wavelength ) 690 nm) and a refractive index detector (Optilab DSP from Wyatt Technology Corp.). An Ultrahydrogel linear column with bead size 6-13 µm (elution range 103 to 5 × 106 Da) and an Ultrahydrogel 120 with bead size 6 µm (elution range 150 to 5 × 103 Da) from Waters were used. An aqueous 0.1 N NaNO3 solution was used as the mobile phase at a flow rate of 0.8 mL/ min. The dn/dc value for polyglycerol was determined to be 0.12 mL/g in aqueous 0.1 N NaNO3 solutions and was used for molecular weight calculations. The data were processed using Astra software provided by Wyatt Technology Corp. All thermogravimetric analyses, to study the thermal stability of the polymers, were performed on a TGA Q500 (TA Instruments, USA). A constant ramping temperature program (20.0 °C/min to 800 °C) was used in the experiments with a balance gas flow of 40 mL/min (Nitrogen) and a sample gas flow of 60 mL/min (Nitrogen). The real-time weight percentage and TGA chamber temperature were recorded. Analysis of the data was performed using TA Universal Analysis 2000 software (version 4.2E, TA Instruments, USA) to find the onset points. In Vitro Aqueous Degradability. The polymer dHPG-83 was dissolved in buffer solutions of pH 5 (30 mM acetate buffer with 70 mM NaNO3) and 7.4 (30 mM phosphate buffer with
70 mM NaNO3) at a concentration of 10 mg/mL and incubated at 37 °C. Samples were withdrawn at 1, 2, and 4 week intervals and analyzed by size exclusion chromatography for molecular weight characteristics. Radiolabeling of dHPGs. Radiolabeling was done by partial conversion of hydroxyl groups to methoxides using tritiated methyl iodide. Briefly, 1 g of polymer was dissolved in 10 mL dimethylsulfoxide (DMSO) and approximately 5% of the hydroxyl groups were converted to potassium alkoxides by reaction with potassium hydride (35 mg). To this, 20 µL of tritiated methyl iodide (toluene solution) dissolved in 1 mL DMSO was added and the reaction mixture stirred at room temperature for 15 h; then, 5 mL water was added and the reaction mixture was acidified with dilute HCl. The polymer was purified by dialysis against water using a dialysis membrane of MWCO 1000 until the dialyzate contained low amounts of radioactivity; this normally took 24 h. The polymer solutions were then filtered through a syringe filter (0.2 µm) and the total polymer weight was determined from the total volume and the dry weight of a known volume (100 µL) of solution after freeze-drying (measured in duplicate). The polymer solution was then concentrated by evaporating off the water in a fume hood, and the final concentration of 100 mg/mL in 150 mM
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Figure 2. Time-dependent GPC chromatograms of dHPG-83 after incubation in buffer solutions of pH 7.4 and 5.0 at 37 °C.
Figure 3. Thermal decomposition profiles of the dHPG polymers.
saline solution was made by appropriate dilution with an aqueous NaCl solution. This allowed the specific activity to be determined by counting an aliquot of solution. Animal Studies. All animal experiment protocols were reviewed and approved by the Institutional Animal Care
Figure 4. GPC chromatograms of dHPG-83 before and after autoclaving.
Committee (IACC) at UBC prior to conducting the studies. The care, housing, and use of animals were performed in accordance with the Canadian Council on Animal Care Guidelines. The studies were conducted at the British Columbia Cancer
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dHPG- 83 every 2 weeks for 8 weeks. One week following the final injection, all mice received an i.v. injection of the radiolabeled dHPG-83, and blood was collected at different time points. Plasma was collected and assessed for polymer levels by scintillation counting.
RESULTS AND DISCUSSION
Figure 5. Change in body weight after administration of the dHPG polymers.
Research Centre under the supervision of Dr. Dawn Waterhouse and Prof. Marcel Bally. Female Balb/C mice (7-11 weeks) were heated under a lamp for 1-2 min to increase blood circulation, and gently placed into a mouse restrainer for injection. The tail was wiped down with 70% isopropanol to clean the injection site and to aid in increasing circulation. Mice were injected intravenously (bolus) via lateral tail vein with a polymer solution of concentration 100 mg/mL (four mice per group) to a prescribed dose of 1 g/Kg. The injected volume was 200 µL/20 g mouse. Mice were then returned to their cages. All animals were observed post-administration, at least once a day, and more if deemed necessary, during the pretreatment and treatment periods for mortality and morbidity. In particular, signs of clinical ill health were based on body weight loss, change in food consumption, and behavioral changes such as altered gait, lethargy, and gross manifestations of stress. Blood was collected at various time intervals (30 min, 2, 4, 8, 24, and 48 h, and 7, 14, and 30 days) by cardiac puncture after termination by CO2 inhalation. Plasma was separated by centrifuging samples at 2500 rpm for 15 min. Aliquots of plasma were placed in scintillation vials and analyzed with a Packard 1900 scintillation counter after adding 5 mL scintillation cocktail. Mice in one group (time point 48 h) were housed in a metabolic cage. Urine and stools were collected as pooled samples at 1, 4, 8, 24, and 48 h post-injection. Aliquots of urine were analyzed by scintillation counting. Stool samples were pooled and made into a 30% homogenate in a known amount of water prior to obtaining aliquots for scintillation counting. Upon termination, liver, spleen, kidney, lungs, and heart were removed from all the mice, weighed, and processed for scintillation counting. Livers (one lobe) were made into a 30% homogenate in a known amount of water using a polytron tissue homogenizer. Aliquots (in triplicate) of 200 µL homogenate were transferred to scintillation vials. All other organs were dissolved in 500 µL SOLVABLE. Vials were incubated at 50 °C overnight, then cooled prior to addition of 50 µL 200 mM EDTA, 25 µL 10 M HCl, and 200 µL 30% H2O2. This mixture was incubated at room temperature for 1 h prior to addition of 5 mL scintillation cocktail. Samples were analyzed by scintillation counting. Evaluation of Antibody Mediated Clearance. Female Balb/c mice (total of 16) were individually weighed and divided into groups of 4. Mice in groups 3 and 4 received injections of
Synthesis and Characterization. The synthetic method we have employed for the dHPGs is a simple and efficient one-pot approach based on anionic ring-opening polymerization of epoxides. The structure of the polymer is given in Figure 1; the details of the synthesis have been described in an earlier manuscript (8). Briefly, an HPG of molecular weight 7000 g/mol was prepared by anionic ring-opening multibranching polymerization of glycidol from partially deprotonated trimethylolpropane using potassium methylate (11). HPG has numerous hydroxyl end groups, with the number per molecule being roughly equal to the degree of polymerization. Some of these were modified with C18 alkyl chains (2-5%) and MPEG-350 chains (mol wt 350) (20-40%) by sequential addition of 1,2epoxyoctadecane and MPEG-epoxide. The unreacted alkyl epoxide was removed from the methanolic polymer solution by extraction with hexane. The final polymer product was purified by dialysis against water to remove any unreacted PEG chains and then freeze-dried. Two dHPGs of molecular weight 40 kDa and 83 kDa were prepared for biodistribution studies; their molecular characteristics are given in Table 1. The polymers were purified by dialysis. The molecular weight distributions of the polymer are narrow, in the range 1.5-1.8. In Vitro Aqueous Degradability. The dHPG polymer backbones contain -C-C- and -C-O-C- bonds only. Since these are not substrates for common enzymes in the circulation, no rapid enzymatic breakdown in blood is anticipated. We tested the stability of dHPG-83 in buffer solutions of pH 5 (lysosomal pH) and 7.4 (physiological pH) incubated at 37 °C by monitoring its molecular weight distribution profile over time by GPC. The chromatograms are shown in Figure 2. The RI traces indicated only a small decrease in peak molecular weight and the appearance of material in the 20 000-40 000 range after 14 and 30 days when the polymers were incubated at pH 7.4 (Figure 2A,B). However, the polymers degraded more noticeably at pH 5.0 and a much more prevalent lower molecular weight fraction, in the range 10 000-40 000, could be seen in the RI traces for the 15 and 30 day samples with an obvious reduction in the peak molecular weight (Figure 2C,D). The average molecular weight decreased from 83 to 20 K. Since the HPG core was reported earlier to be stable under similar conditions (12), the decrease observed might be due to degradation of the PEG chains, with the degradation of PEG under acidic conditions having been reported (13). However, the occurrence of relatively high molecular weight PEG chains would be expected to be relatively rare at the monomer concentrations used in the synthesis, so this is unlikely to be the whole explanation. A more detailed study of the degradation reaction is under way. Thermal Degradation. A desirable property of dHPGs would be sufficient thermal stability to allow them to be autoclaved for sterilization. The core HPGs are thermally quite stable with onset of thermal decomposition in the range 360-390 °C (14, 15). The thermograms obtained for dHPG-40 and dHPG-83 are shown in Figure 3. It is evident from the figure that the derivatized HPGs are also highly stable up to a temperature of 300 °C with no indication of thermal decomposition. The onset of decomposition is around 350 °C. Aqueous solutions of dHPG at two different concentrations (50 mg/mL and 100 mg/mL) were autoclaved under standard conditions for sterilization, and the GPC chromatograms before and after the sterilization
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Figure 6. Plot of polymer concentration of dHPG polymers in plasma with respect to time. Table 2. Rate Constants Determined from Pharmacokinetic Analysis of Polymer Concentrations in Plasma over Timea polymer dHPG-40 dHPG-83 a
P0 Vc (mg/mL plasma) (mL plasma) 6.8 ( 1.4 11.7 ( 0.4
2.94 ( 0.62 1.7 ( 0.06
R (h-1)
β (h-1)
t1/2 (h)
k12 (h-1)
k21 (h-1)
k2 (h-1)
0.028 ( 0.007 0.20 ( 0.07 24.9 ( 6.2 0.060 ( 0.089 0.12 ( 0.05 0.046 ( 0.027 0.022 ( 0.002 0.61 ( 0.11 31.0 ( 2.9 0.249 ( 0.130 0.34 ( 0.06 0.040 ( 0.011
AUC0-∞mg.mL-1.h 148.4 ( 44.9 295.4 ( 29.9
k12 and k21 represent the rate constants for transfer between the plasma and tissue compartments; k2 is the rate constant for elimination.
procedure are shown in Figure 4. The chromatograms are indistinguishable with no change in molecular weight. These results demonstrate that aqueous solutions of dHPG polymers are sufficiently stable to allow them to be autoclaved. Animal Studies. Mouse Toxicity. The derivatized HPGs dissolved in isotonic saline were injected i.v. into Balb/c mice at 0.5 g/Kg and 1 g/Kg, two mice per concentration per compound, and followed for 30 days for signs of toxicity and body weight loss. No untoward indicators were found, and all the animals grew normally with no signs of distress, as was found for underivatized HPGs (16). No signs of toxicity (e.g., lethargy, dry eyes, change in food consumption, altered gait, or scruffy coats) were observed. The animals were followed for 30 days for weight changes, and no weight loss was observed during this period (Figure 5). On autopsy, liver, gall bladder, spleen, lung, kidney, heart, intestine, lymph nodes, and bladder were examined macroscopically for gross pathology; all the organs appeared normal with no unusual finding observed. Biodistribution. The circulating levels of both polymers are shown in Figure 6 as a function of time, expressed as milligrams polymer per milliliter of plasma. The data were analyzed by a standard two-compartment open pharmacokinetic model (12, 17). The rate constants (k12 - plasma to tissue, k21 - tissue back to plasma, and k2 - elimination) calculated from the graphs are given in Table 2. The plasma half-life for dHPG-40 was ∼25 h, while that of dHPG-83 was ∼31 h. The value for the area under the plasma concentration curve (AUC0-∞) was ∼2 times that for the higher molecular weight polymer, implying that this polymer’s tissue exposure was twice that of the lower molecular weight material. The results show that it is possible to adjust the retention time in blood and tissue exposure by manipulating the molecular weight through changes in the PEG content of the polymer, since both the dHPGs had similar HPG contents (16 000 and 24 000 for the low and high molecular weights, respectively, whereas the PEG contents were around 22 500 and 56 000). The core HPG molecular weight can also be varied with related effects as we have shown recently with higher molecular weight
non-pegylated material (12). The plasma half-lives obtained for these polymers are consistent with the periods required for postoperative support in many cases (18). Lower molecular weight polymers, which would be excreted faster, can be made by reducing the molecular weight of HPG or degree of substitution with PEG; in some surgical settings, faster excretion is preferred. Even though the molecular weight of the dHPG83 is double that of dHPG-40, the plasma half-life did not change dramatically. This might be due to the plasma half-life’s dependence on many factors, one of which is the molecular size, which increases only weakly with molecular weight for hyperbranched polymers. The plasma half-lives observed for these polymers are higher than those for linear polymers of similar molecular weights, presumably due to their globular shape and relative rigidity. For example, poly-N-(2-hydroxylpropyl)methacrylamide (HPMA), which has been extensively studied for anticancer drug delivery, has a circulation half-life of only 25 h for a molecular weight of 556 000 (19). The hydrodynamic radii of dHPG polymers are well above the glomerular filtration threshold, which is about 3.7-6.0 nm pore radius (20). Perhaps more relevant is the inability of dHPGs to pass through pores via reptation, an option available for linear polymers (21, 22). A related observation is that longer circulation times have been reported for pegylated polyester bow-tie dendrimers, which likewise cannot reptate (13). The values for the distribution rate constants were calculated from the graphs and are summarized in Table 2. While the two polymers were eliminated from the system at essentially the same rate, the transfer of the higher molecular weight polymer dHPG-83 from blood to tissue (k12) was about 4 times faster and the reverse process (k21) almost 3 times faster than the rates for dHPG-40, consistent with the higher tissue accumulation observed for the higher molecular weight polymer as discussed below. At termination, liver, spleen, lungs, and heart were collected and processed for scintillation counting. Levels of polymer in the organs were analyzed. The data was not corrected for the
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Figure 7. Levels of polymer accumulated over time in liver (A), spleen (B), kidney (C), lungs (D), and heart (E).
plasma present in these organs, so the high levels of radioactivity up to 48 h are largely due to the plasma present in them. Both polymers accumulate in liver and spleen to a small extent with values below 5% ID/g tissue (ID ) initial dose) over the 30 day study period (Figure 7A,B). The low levels observed for these polymers compared to HPGs and other polymers reported
in the literature evidently are due to the presence of PEG chains, supporting the concept that PEG grafting reduces uptake by the RES (23, 24). The levels in the organs were higher for the higher molecular weight polymer, which remained almost constant in liver and increased in spleen during the study period. A similar dependence of tissue accumulation on molecular weight has
Unimolecular Micelles based on Hyperbranched Polyglycerols Table 3. Polymer Levels in Urine and Feces at the Times Indicateda dHPG-83
dHPG-40
time (h)
% dose in feces
% dose in urine
% dose in feces
% dose in urine
1 4 8 24 48
0.02 0.05 0.25 0.03 0.07
not collected 1.47 2.33 13.44 0.61
0.03 0.97 11.56 9.25 2.91
not collected not collected 9.8 17.1 0.75
a Error bars are not shown as the data are obtained from pooled samples.
Figure 8. Plot of polymer concentration of dHPG-83 in plasma of animals preinjected with the polymer with respect to time.
been reported for other polymers, likely because the total tissue exposure was higher as indicated by the greater AUC0-∞value for dHPG-83 (Table 2) (25, 26). The levels of the lower molecular weight polymer in kidney, lungs, and heart were below 1% ID/g tissue (Figures 7C,D,E). The levels of the higher molecular weight polymer were around 1% in kidney and lungs and ∼2% in heart. Similar organ accumulations have been reported for hetastarch, which is widely used clinically (25, 27). These levels of apparent accumulation were accompanied by no gross effects on organ histology, however; the animals remained healthy throughout the observation period. The fate of the residual polymer in these organs after 30 days is unknown, and long-term toxicity studies are needed to assess the effect. However, the in Vitro degradation data suggest that the polymer might degrade in the acidic compartments of the cells following uptake, resulting eventually in slow excretion from the body. Enzymatic degradation of polyethers has been reported earlier (28-30). A metabolic study with a group of mice (n ) 4) injected with the polymers was performed by collecting urine and feces as pooled samples at 1, 4, 8, 24, and 48 h. The values of polymer clearance into urine and feces are given in Table 3. Approximately 50% of dHPG-40 was excreted from the body within 24 h, with 27% through urine and 22% through feces. The dHPG-83 is mostly eliminated through urine with 17% eliminated within 24 h. Even though the amount of polymer present per unit mass of feces was similar for each, the total amount of dGHPG-83 excreted was only ∼1% due to significantly lower amounts of feces excreted within 48 h for this group of animals. Most urinary excretion was over within 24 h for both polymers. EValuation of Antibody Mediated Clearance. We have not developed an enzyme-linked immunosorbent assay (ELISA) for
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the anti-dHPG polymer family due to the lack of a positive control. However, we have tested for evidence of antibody mediated clearance by attempting to sensitize mice with a sequence of prior injections before measuring the plasma depletion curve. If these dHPG polymers are immunogenic, the plasma half-lives of the polymer injected subsequently should be reduced due to the antibody mediated clearance mechanism (31-33). In order to study this, 16 mice were divided into groups of 4. Mice in groups 3 and 4 were injected with non-radiolabeled dHPG-83 every 2 weeks for 8 weeks at dose of 1 g/kg. One week following the final injection, all mice received an i.v. injection of the radiolabeled dHPG-83 at a dose of 1 g/kg, and blood was collected at different time points. Plasma was collected and assessed for polymer levels by scintillation counting. All the animals grew normally during the study period, and no signs of toxicity or weight loss were observed (data not shown). On autopsy, all organs appeared normal with no gross pathology. The levels of dHPG-83 in the plasma for both the groups of animals, determined by scintillation counting, are shown as a function of time in Figure 8. It can be seen that the polymer levels are very similar with no significant change in plasma halflife, indicating that there is no significant antibody mediated clearance of the polymer. The dHPG polymers, with potential applications as an albumin substitute or a general drug delivery vehicle, are highly biocompatible as evidenced by the in ViVo results described above. The results also suggest that these polymers are nonimmunogenic, as they can be administered in repeated doses without change in plasma half-lives. The polymers were welltolerated by mice at doses as high as 1 g/kg. The plasma halflives exceeded 24 h and can be controlled by varying the molecular weight through changing the polyglycerol core or the PEG content, as in the present case. The tested polymers are of sufficient size to not pass out immediately into the urine. The overall data considering the plasma half-life and the organ accumulation suggests that a derivatized HPG polymer with a number-average molecular weight of 40 000 is well-suited to an application as a plasma expander. The presence of radioactivity over relatively long periods of time suggests that these structures do not break down appreciably in the circulation over the time courses utilized. However, the in Vitro degradation studies suggest that these PEG-containing polymers could degrade slowly, particularly under the acidic conditions of an intracellular environment.
ACKNOWLEDGMENT This work was funded by grants from The Bayer Canadian Blood Services Hema-Quebec Partnership Fund (1614230, 1664725, 1721923) and a Canadian Institutes of Health Research Proof of Principle grant. We thank Canadian Institutes of Health Research, Natural Science and Engineering Research Council of Canada, the Canada Foundation for Innovation, Canadian Blood Services, Michael Smith Foundation for Health Research and BC Knowledge Development Fund for infrastructure support especially the LMB Macromolecular Hub at the UBC Centre for Blood Research. We thank Prof. Marcel Bally and Dr. Dawn Waterhouse for the animal experiments and Dr. Johan Janzen and Mrs. Irina Chafeeva for the help with the radiolabeling experiments.
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