Hybrid polyglycerols with long blood circulation: synthesis, biocompatibility, and biodistribution.

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Hybrid Polyglycerols with Long Blood Circulation: Synthesis, Biocompatibility, and Biodistributiona Muhammad Imran ul-haq, Benjamin F. L. Lai, Jayachandran N. Kizhakkedathu*

HO

O

HO O

O

O

OH

O

OH OH

OH

OH OH

HO O

O

O

OH

O

OH OH O OH O OH

OH OH OH OH O O OH O OH OH O O O O OH O O O OH O O OH OH O O O HO O OH OH O O O O O OH OH O O HO O OH HO OH OH O HO OH O O OH O OH O O O O OH HO HO OH O O OH OH O O OH HO O O OH O HO OH OH OH O O OH OH HO OH O

%age of dose injected / ml plasma

Multifunctional polymers with defined structure and biocompatibility are critical to the development of drug delivery systems and bioconjugates. In this article, the synthesis, in vitro blood compatibility, cell viability, in vivo circulation, biodistribution, and clearance of hybrid copolymers based on linear and branched polyglycerol are reported. Hybrid polyglycerols (M n 100 kDa) are synthesized with different compositions (15–80 mol% linear polyglycerol). Relatively small hydrodynamic size and radius of gyration of the hybrid polyglycerols suggest that they are highly compact functional nanostructures. The hybrid polyglycerols show excellent blood compatibility as determined by measuring their effects on blood coagulation, red blood cell aggregation, hemolysis, platelet, and complement activation. The cell viability in presence of hybrid polyglycerols is excellent up to 10 mg mL1 concentration and is similar to both dextran and polyvinyl alcohol. Furthermore, tritium labeled hybrid polyglycerol shows long blood circulation (t1/2b ¼ 34 h) with 100 minimal organ accumulation in mice. 80 Multifunctionality, compact nature, LPG-HPG-50 biocompatibility, and the long blood (t β = 34.17± 6.22 h) 60 circulation make these polymers attrac40 tive for the development of bioconju20 gates and drug delivery systems. 0 1/2,

0

25

50

75

100

125

150

Time (h)

1. Introduction Biocompatible polymers with defined pharmacokinetics and multifuctionality have significant potential in the Dr. M. Imran ul-haq, B. F. L. Lai, Prof. J. N. Kizhakkedathu Centre for Blood Research, Department of Pathology and Laboratory Medicine, 2350 Health Sciences Mall, Vancouver BC V6T 1Z3, Canada Dr. M. Imran ul-haq, B. F. L. Lai, Prof. J. N. Kizhakkedathu Department of Chemistry, The University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver BC V6T 1Z3, Canada E-mail: [email protected] a Supporting Information is available online from the Wiley Online Library or from the author.

Macromol. Biosci. 2014, 14, 1469–1482 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

development of drug delivery vehicles and bioconjugates for the treatment of various diseases.[1–8] Conjugation of drugs to the polymers is an effective method to prolong their circulation half-life as well as to give favorable tissue distribution.[9–11] Polyglycerols (PGs), a class of highly hydrophilic polyether polymers, available in linear and branched form have attracted significant attention in the recent years due to their excellent biocompatibility profile compared to PEG and multifunctionality.[5,12–14] The ease of synthesis with good control of molecular weight properties of this class of polymers is another advantage.[15] The in vivo blood circulation of PGs can be fine tuned with the molecular weight of the polymers; both linear and hyperbranched PGs showed long circulation in mice when the molecular weights are high.[16,17] Due to their long blood

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DOI: 10.1002/mabi.201400152

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M. I. ul-haq, B. F. L. Lai, J. N. Kizhakkedathu www.mbs-journal.de

circulation and biocompatibility, PGs have attracted considerable attention in the development of long acting therapeutics, cancer imaging, and other potential biomedical applications.[18–29] The introduction of branched/linear units within the structure is a well-known strategy to tailor the properties of polymers.[30,31] Linear-dendritic diblock copolymers, in which a dendron is attached to a linear polymer chain, have received great attention in recent years because of their unique structure, novel properties, and potential applications in catalysis and as biomaterials.[32–35] Initial work in this field was from Frechet group on poly(benzyl ether) dendrimers[36] and Hammond and co-workers[37] on poly(amido amine) dendrimers attached to a linear polymer chain. Frey and coworkers[38–40] synthesized several types of linear-hyperbranched diblock copolymers with defined molecular properties. However, there is limited information available on the biocompatibility and in vivo properties of such hybrid structures in the current literature. It is anticipated that the hybrid copolymers of linear and branched polymers may have different biological properties and pharmacokinetics profile compared to their respective linear or branched counterparts due to the differences in architecture or topology as highlighted by Szoka and co-workers.[41] Thus in the present work we investigated the synthesis, biocompatibility, biodistribution, and blood circulation of high molecular weight hybrid copolymers consists of different contents of linear and hyperbranched polyglycerols. The biocompatibility of the linear polyglycerols and hyperbranched polyglycerols were reported individually.[14,16] However, there is no information currently available on the synthesis, biocompatibility, circulation time, and biodistribution of high molecular weight hybrid linear-branched polyglcerols. In the case of hybrid polyglycerols, the difference in the functional end groups may affect their interactions with biological macromolecules and cells. It is anticipated that hybrid polyglycerols with different proportions of primary and secondary hydroxyl groups may have potential advantages in bioconjugation applications.

2. Experimental Section 2.1. Materials and Methods Glycidol (96%) (Sigma–Aldrich) was distilled under reduced pressure before use and stored over molecular sieves at 4 8C. Trimethylolpropane (TMP) was obtained from Fluka (ON, Canada). Tritiated methyl iodide (C3H3I) was obtained from American Radiolabeled Chemicals (St. Louis, MO) as a solution in toluene and used after dilution in dimethylsulfoxide (DMSO). Cellulose ester dialysis membranes (MWCO 1000 and 10 000) were obtained from Spectra/Por Biotech (CA, USA). Deuterated solvents were obtained from Cambridge Isotope Laboratories, 99.8%D. All other chemicals and reagents were purchased from Sigma–Aldrich Canada Ltd (Oakville, ON).

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2.2. Techniques 1

H NMR spectra were recorded on a Bruker Avance 300 and 400 MHz NMR spectrometer using deuterated solvents (Cambridge Isotope Laboratories, 99.8% D) with the solvent peak as a reference. The absolute 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, Inc., Santa Barbara CA; the details have been described previously.[42]

2.3. Synthesis of Hybrid Linear-Branched Polyglycerols Ethoxy ethyl glycidyl ether (EEGE) monomer was synthesized by a procedure reported previously.[43] The hybrid copolymers of linear and hyperbranched polyglycerol (h(LPG-HPG)) were synthesized using a modified procedure reported.[16,43] For a typical polymerization the following conditions were used. Initially TMP was added to the flask under argon atmosphere followed by 0.11 mL of potassium methylate solution in methanol (20 wt%). Reaction mixture was stirred using a stirrer bar for 15 min and excess methanol was removed under vacuum. To this mixture anhydrous 1,4-dioxane was added under stirring. The flask was kept in an oil bath at 95 8C and a solution of pre-mixed glycidol and EEGE was added dropwise over a period of 12 h using a syringe pump. After the completion of monomer addition, the polymerization mixture was stirred for additional 5 h. The product was dissolved in methanol, neutralized by passing three times through a column containing cation-exchange resin (Amberlite IRC-150). The polymer was then precipitated into excess of acetone and diethyl ether mixture depending on the mole ratio of the EEGE component; hybrid polymer with more than 50% EEGE content was precipitated using diethyl ether. The composition of EEGE and glycidol in the hybrid polymer was determined by 1H NMR spectroscopy. The polymer was stirred with 25 mL of 35% HCl for deprotection of EEGE component. After the acid treatment, the polymer was purified by dialysis against water using a regenerated cellulose membrane (MWCO 1000) and recovered by freeze drying. The hybrid polymers were characterized for their molecular weight properties, composition, and molecular size. Five different compositions of linearhyperbranched hybrid polyglycerols ranging from 80 to 15 mol% linear component were synthesized. The HPG component present in the hybrid polymer was taken as the branched portion of the polymer and was used for calculating the branching density.

2.4. In Vitro Blood and Cell Viability Analyses 2.4.1. Techniques Platelet activation analysis was performed on a BD Biosciences FACS Canto II flow cytometer. The activated partial thromboplastin time (APTT) and prothrombin time (PT) were determined by a coagulation analyzer using mechanical end point determination (ST4, Diagnostica Stago). Optical microscopy was performed on a Zeiss Axioskop 2plus microscope and images were obtained with


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microscope-mounted black and white CCD camera (Qimaging Retiga 1300; exposure times less than 500 ms) was used. Cellomics TM Arrayscan VTI automated fluorescence imager for Propidium Iodide live cell staining.

2.4.4. Blood Compatibility Analysis Blood compatibility of different hybrid polyglycerols (LPG-HPG-80 to LPG-HPG-15 (5 different compositions) (Table 1) was determined by measuring their influence on RBC aggregation and hemolysis, blood coagulation (APTT, PT), platelet activation and complement activation by various analyses reported previously from our laboratory. Detailed procedures are given in the published reports.[12,16,26,45,46] We also compared the blood compatibility of the hybrid polyglycerols with polyvinyl alcohol (PVA) and dextran (hydroxyl rich polymers) having similar molecular weights.

2.4.2. Reagents APTT and PT reagents were purchased from Dade Behring. For platelet activation analysis, anti-CD62pPE, and goat anti-mouse PE antibodies were purchased from Immunotech. GVB2þ (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3 with 0.15 mM CaCl2, and 0.5 mM MgCl2, pH 7.3), antibody-sensitized sheep erythrocytes (EA) were purchased from CompTech (Tyler, TX, USA). Human umbilical vein endothelial cells (HUVECs) were obtained from Lorus Pharmaceuticals (Allendale, NJ), Endothelium Growth Medium-2 (EGM-2) and BulletKit (CC-3162) media were purchased from Lonza, Inc. Hoechst 33342 stain was purchased from Invitrogen. Propidium iodide solution was purchased from Sigma–Aldrich (ON, Canada).

2.4.5. Cell Viability Analysis 2.4.5.1. MTT Assay Cell viability of hybrid polyglycerols against HUVECs after incubation at different concentrations (1.25–10.0 mg mL1) was studied by MTT assay. Cells were plated at approximately 3000 cells per well in a 96 well plate and left to grow overnight. Polymer stock solution was prepared in EGM-2 medium and filtered through 0.2 mm filter. Final polymer concentrations were 1.25, 2.5, 5.0, and 10 mg mL1 and incubated for 48 h. Cell viability was determined using an MTT assay kit (CellTiter 96) Aqueous One Solution Cell Proliferation Assay (Promega, Madison WI) following the manufacture’s protocol. The untreated cells and medium alone were used as positive and negative controls, respectively. The percent cell viability was calculated: % cell viability ¼ [(treated cells – negative control)/(untreated cells negative control)] 100.

2.4.3. Blood Collection Blood was drawn from healthy unmedicated consented donors at the Centre for Blood Research by an approved protocol by the University of British Columbia clinical ethics committee. Blood was collected in 3.8% sodium citrated tube with a blood/ anticoagulant ratio of 9:1 (BD Vacutainer buffered citrate sodium (0.105 M; 9:1)) or EDTA (Fisher Scientific, New Jersey) or in serum tubes. Platelet-rich plasma (PRP) was prepared by centrifuging the citrated whole blood samples at 150 g for 10 min in an Allegra X-22R centrifuge (Beckman Coulter, Canada). Platelet-poor plasma (PPP) was prepared by centrifuging the citrated whole blood samples at 1200 g for 20 min. Serum was prepared by centrifuging whole blood containing serum tube at 1200 g for 30 min. All the centrifugation procedures were performed at room temperature (22 8C). Red blood cell (RBC) suspension was prepared by washing packed RBCs with phosphate buffered saline four times.

2.4.5.2. Propidium Iodide Cell Staining Roughly, 3000 HUVECs per well were seeded in a 96-well Plate 1 d before treatment with polymers and controls. Polymer stock solution was prepared in EGM-2 medium and filtered through 0.2 mm filter. Final polymer concentrations were 1.25, 2.5, 5.0, and 10 mg mL1 and incubated for 48 h. Media and 50% DMSO were used as normal and positive control, respectively. At the end of

Table 1. The composition and molecular characteristics of hybrid polyglycerols.

LPG [mol%]

HPG [mol%]

Mw [kDa]

Mn [kDa]

Mw/Mn [PDI]

Rg [nm]a)

Rh[nm]b) (TDA)

Degree of branchingc)

LPG-HPG-80

80

20

106

94.5

(1.12)

5.81

4.45

0.11

LPG-HPG-70

70

30

118

102

(1.15)

5.94

4.55

0.17

LPG-HPG-50

55

45

114

99.0

(1.15)

5.96

4.57

0.25

LPG-HPG-30

32

68

119

100

(1.18)

6.07

4.66

0.38

LPG-HPG-15

16

84

114

104

(1.10)

6.23

4.78

0.47

Dextran-100

–

–

110

–

–

–

–

PVA-100

–

–

125

–

–

–

–

Polymer

The molar content of LPG and HPG component in the hybrid copolymer was calculated using integral data from 1H NMR spectroscopy (D2O, DMSO-d6 and CDCl3) of the hybrid polymers before deprotection; a)Radius of gyration determined by Viscotek triple detector; b) Hydrodynamic radius determined by Viscotek triple detector; c)Degree of branching of the hybrid polymer were calculated theoretically from the composition and from the experimental determined branching density of HPG (0.56).



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incubation period, 100 mL of staining solution (1 mL of Hoechst, stock 20 mg mL1, and 100 mL propidium iodide (PI), stock 50 mg mL1 in PBS, in a 10 mL pre-warmed medium) was added directly to the wells without aspirating the treatment medium (to prevent loss of cells due to aspiration). The final concentration of PI was 0.5 mg mL1 and that of Hoechst in each well was 0.5 mg mL1. The staining solution was incubated with the cells for 30 min at 37 8C. The 96-well plate was scanned in a Cellomics TM Arrayscan VTI automated fluorescence imager. The cells were imaged with a 20 objective. The untreated wells (media) were identified as reference wells during the scan and the data output %. Responder indicated as a percentage of cells with average intensity in cytoplasm greater than the average intensity in the untreated cells. Hoechst 33342 is a blue fluorescent stain that labels all cell nuclei. Propidium iodide is a red fluorescent nuclear and chromosomal counterstain that does not penetrate the live cells and therefore live cells will not be stained. The dead cells with compromised cell membranes will take up the PI and show up as bright fluorescent objects than the live cells. The orange colored cells at the edges were the cell that are not completely located within the field of view. Absolute values were reported along with the media control and positive control (50% DMSO).

2.5. Biodistribution and Blood Circulation in Mice 2.5.1. Radio-Labelling of Hybrid Polyglycerol (h(LPG-HPG-50)) Tritium labelling of h(LPG-HPG-50) was performed by the partial conversion of hydroxyl groups to methyl ether using tritiated methyl iodide. We followed our previously published protocol for radio-labelling experiments.[16,17,22] Briefly, 0.3 g of polymer was dissolved in 3 mL of 1-methyl-2-pyrrolidinone (NMP) and approximately 5% of the hydroxyl groups were deprotonated by a reaction with sodium hydride (10 mg). To this reaction mixture, tritiated methyl iodide (toluene solution) dissolved in DMSO was added to methylate approximately 1% of hydroxyl groups; the reaction mixture was stirred at room temperature (22 8C) for 15 h and the labelled polymer was purified by dialysis against water using dialysis membrane of MWCO 1000 until the dialysate had low amounts of radioactivity (this normally take 48 h). The polymer solution was then filtered through a syringe filter (0.2 mm) and the total polymer weight was determined from the total volume, and the dry weight of a known volume (1 mL) of solution after freeze drying. The polymer solution was then concentrated by evaporating off the water in a fume hood and the final concentration in 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.

2.5.2. Animal Study Protocol The animal studies were performed at the Experimental Therapeutics laboratory at the B.C. Cancer Research Centre, Vancouver, BC, Canada. The protocol was reviewed and approved by the Institutional Animal Care Committee (IACC) at UBC. Female Balb/C mice (6–8 weeks) were injected intravenously (bolus) via

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lateral tail vein (three mice per group) at a prescribed dose of 50 mg kg1. The injected volume was 200 mL/20 g mice. Blood was collected at various time intervals (0.5, 1, 2, 4, 8, 24, 48, 103, and 144 h) by cardiac puncture after termination by CO2 inhalation. Plasma was separated by centrifuging samples at 2 000 rpm for 10 min. Urine and feces were collected by placing the animals in a metabolic cage and were analyzed for radioactivity by scintillation counting. After termination, major organs such as liver, kidney, spleen, lungs, and heart were removed, weighed, and processed for scintillation counting. Livers were made into a 30% homogenate in water using a tissue homogenizer. Aliquots (in triplicate) of 200 mL homogenate were transferred to scintillation vials. All other organs were dissolved in 500 mL Solvable. Vials were incubated at 50 8C overnight, then cooled prior to addition of 50 mL 200 mM EDTA, 25 mL 10 M HCl and 200 mL 30% H2O2. This mixture was incubated at room temperature for 1 h prior to addition of 5 mL scintillation cocktail. Samples were analyzed for radioactivity by scintillation counting.

2.6. Statistical Analysis Statistical analysis (Student’s t-test) was performed with MS Excel using two samples with two equal variances method. Paired comparisons were considered significant if p < 0.05. All the data are presented as mean standard deviation unless otherwise mentioned.

3. Results and Discussion 3.1. Synthesis and Characterization of Hybrid Polyglycerols Hybrid copolymers of linear and hyperbranched polyglycerols with different molar ratios of linear and branched structure with an average molecular weight around 100 kDa were synthesized by anionic ring opening multibranching polymerization (ROMBP). The characteristics of the polymers are given in Table 1. Copolymerization of EEGE and glycidol monomers followed by the deprotection with HCl afforded the hybrid polymers (Figure 1).[14,16,43] The composition of the hybrid polyglycerols was calculated from the 1H NMR spectra of the polymers before deprotection; the calculated composition was similar to the feed ratio of the monomers. The hybrid polymers were named in the following convention. A polymer with 80:20 linear/branched content was named as h(LPG-HPG-80) and ‘‘80’’ stands for the molar content of linear polyglycerol component and ‘‘20’’ for the hyperbranched polyglycerol component in copolymer. Similar convention was followed for other polymers. The use of 1,4-dioxane as polymerization medium generated high molecular weight polymers with low PDI. The absolute molecular weights of the polymers were measured by multiangle light scattering coupled with gel permeation chromatography.




Figure 1. Synthesis scheme for hybrid polymers based on linear-hyperbranched polyglycerol. The hybrid polyglycerols were synthesized by anionic ring opening polymerization of EEGE and glycidol monomers in 1,4-dioxane at 95 8C.

The 1H NMR spectra of the polymers before and after the deprotection of hydroxyl groups confirm the structure of the hybrid polymers (Figure 2); the characteristic peaks of both EEGE and glycidol were found.[16] The presence of signals from protecting group (Ha, Hb, and Hc) demonstrated that EEGE was present as a component in the hybrid polymer as expected. The Hc proton of the protecting group of EEGE and OH group of the hyperbranched component were contributing to the peak at 4.66 ppm. Therefore NMR experiments were carried out at 70 8C to provide better analysis of this overlapping peak at 4.66 ppm. The change in peak position of the hydroxyl groups (4.66–4.33 ppm) in the 1 H NMR at 70 8C in comparison to that collected at 25 8C with respect to the peaks from EEGE protecting group (Hc ¼ 4.66 ppm) confirmed the presence of hyperbranched component in the final hybrid copolymer.[16] Absence of characteristic peaks of the hydroxyl protecting groups (Ha, Hb, and Hc) in the 1H NMR of final deprotected hybrid polyglycerol confirmed its complete removal. The peaks at 3.44, 3.56, and 3.70 ppm were due to —CH— and —CH2— groups of the polymer, which is coming from EEGE and hyperbranched component; the branching density is theoretically calculated (Table 1). Peaks in NMR spectra at 3.13–3.15 ppm were due to the moisture present in DMSO-d6 solvent. The HPG component present in the hybrid polymer was taken as the branched portion of the polymer. Representative GPC chromatographs of hybrid polyglycerols are given in Figure S1 (Supporting Information). The compact structure of the hybrid polyglycerols was evidenced by their small hydrodynamic size measured (Table 1) which is consistent with the data reported for linear and hyperbranched polyglycerols.[16] The compactness parameter, Rg/Rh ratio,[42] also did not change with changes in the composition of the polymer and it remained around 1.30.


3.2. In Vitro Biocompatibility Analysis of Hybrid Polyglycerols The biocompatibility of high molecular weight hybrid polyglycerols was analyzed and compared with PVA (PVA100) and dextran (dextran-100) having similar molecular weights (100 kDa). Dextran and PVA were selected due to the presence of large number of hydroxyl groups in these polymers and these polymers were extensively used in many biomedical and biotechnological applications. Dextran is a branched polysaccharide made of many glucose molecules and PVA is a linear polymer. This comparison is anticipated to provide a benchmark for the performance hybrid polyglycerols for their use in various potential applications. 3.2.1. Hemocompatibility Analysis Blood compatibility is an important criterion for materials intended for intravenous administration.[47] Therefore all the synthesized hybrid polyglycerols (Table 1) were tested for their influence on RBC aggregation and hemolysis, blood coagulation, platelet activation, and complement activation at 37 8C and the results were compared with hydroxyl rich dextran-100 and PVA-100. 3.2.1.1. RBC Aggregation and Hemolysis Irreversible RBC aggregation upon interaction with polymers is detrimental to the cardiovascular system or organs upon their intravenous administration.[48] Under normal conditions, RBC forms reversible aggregates called ‘‘rouleaux’’ in the presence of plasma proteins. However, irreversible RBC aggregates that cannot be broken down easily by shear forces due to the blood flow can increase the


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Figure 2. 1H NMR spectra of hybrid polymers in DMSO-d6 at 70 8C.

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blood viscosity and is harmful to the vital organs. Such irreversible RBC aggregation can also change the deformability of RBCs. The changes in RBC aggregation and morphology in whole blood in the presence of different hybrid polyglycerols (LPG-HPG-80, 50, 30, 15), dextran-100 and PVA-100 at various concentrations (1–10 mg mL1) were measured. Optical micrographs shown in Figure 3 illustrate the differences between different polymers. The hybrid polyglycerols did not show any changes in RBC aggregation or shape at all the polymer concentrations in comparison to the buffer control. The shape of the RBCs

appeared to be normal. In contrast, the PVA-100 showed RBC aggregation at 1.0 and 8.0 mg mL1 concentration. Dextran-100 at 10 mg mL1 also showed some aggregation of RBC. These results clearly demonstrate the superiority of hybrid polyglycerols and provide evidence that these structures do not interact strongly with RBCs. RBC lysis in presence of the LPG-HPG-80, 50, 30, 15, dextran-100, and PVA-100 was also measured using washed RBCs (10% hematocrit). Polymers were incubated with RBC suspension at 1:9 v/v for 1 h at 37 8C and centrifuged. The lysis was measured and compared with the buffer control. Although, there was considerable RBC aggregation with PVA-100, there was no significant hemolysis compared to the buffer control (2–3%). Hybrid polyglycerols and dextran-100 also showed similar levels of hemolysis as that of the buffer control (Figure 4) which suggested that there was not much membrane damage upon interaction with these polymers. 3.2.1.2. Blood Coagulation Analysis Blood coagulation in the presence of the polymers was measured to investigate their pro- or anticoagulant nature.[46,49] For this purpose, the prothrombin time (PT) and APTT were determined. PT measures the extrinsic coagulation pathway (as well as the common pathway) and APPT is used to evaluate the intrinsic coagulation pathways. Polymer samples at various final concentrations (1, 5, and 10 mg mL1) were incubated with sodium citrate anticoagulated blood plasma at a ratio of 1:9 v/v at 37 8C.[44,45] Results are shown in Figure 5A and B; the

Figure 3. Effect of polymer concentration on RBC aggregation in whole blood. Optical micrographs of human RBCs incubated with hybrid polyglycerols at different concentrations (1 and 10 mg mL1). Data for dextran-100 and PVA-100 are also shown. HEPES buffer was used as normal control. RBC aggregation by PVA-100 was performed at 1 and 8 mg mL1 due to the poor solubility and technical difficulties.


Figure 4. Effect of polymer concentration on hemolysis. HEPES buffer and H2O were used as normal and positive (þve) controls, respectively. The hemolysis in presence of PVA-100 was performed at 1 and 8 mg mL1 due to the poor solubility and technical difficulties. Insert picture (10 mg mL1) the visual appearance of polymers tested for RBC lysis.


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Figure 5. A)-Effect of hybrid polyglycerols, dextran-100 and PVA-100 on APTT and B)-prothrombin time (PT) measured in human plasma.

values reported are averages of two different donors done in triplicates. The clotting times in presence of hybrid polyglycerols were comparable to that of the buffer control (Figure 5A and B) suggested that these polymers did not cause any adverse effect on blood coagulation. Similarly, dextran-100 did not show any significant change in the blood coagulation compared to the buffer control at all the concentrations studied. PVA-100 showed a slight increase in APTT values at the highest concentration studied (5 mg mL1). It was not possible to make measurements at 10 mg mL1 for PVA-100. 3.2.1.3. Platelet and Complement Activation Analyses Platelet activation upon interaction with polymers can result in adverse effects such as thrombotic complications

and arterial embolization upon intravenous administration of the polymers.[47] Platelets activation upon interaction with polymers was measured from the expression of CD62P on the surface of activated platelets by flow cytometry analysis. Polymers were incubated with platelet rich plasma (PRP) at different concentrations (1, 5, and 10 mg mL1 final concentration) at a ratio of 1:9 v/v at 37 8C. Results were compared with the buffer control and positive control (thrombin) (Figure 6A). Different hybrid polyglycerols, dextran-100, and PVA-100 at all concentrations did not show any significant platelet activation compared to the buffer control. The high platelet activation in presence of thrombin (96%) indicated that platelets used in the study have normal physiological function. The platelet activation data were also supported with scattered plots given in Figure S2 (Supporting Information).

Figure 6. A) Effect of hybrid polyglycerols, dextran-100 and PVA-100 on platelet activation estimated from CD62P expression. Polymers were incubated in PRP at 37 8C for 1 h and the platelet activation was measured using by flow cytometry. Thrombin was used as control (þ) and buffer was used as control (). B) Effect of hybrid polyglycerols, dextran-100, and PVA-100 on complement activation measured by CH50 analysis. Sensitized sheep erythrocytes were used for measuring the amount of complement proteins consumed during the incubation of polymers in serum. IgG was used as control (þ), EDTA as control () and complement activation was presented as a function of % activation.

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The complement system is an integral part of our innate immune system. Complement activation upon interaction with a synthetic material can provide some important information regarding its inflammatory potential. Activation of the complement can also lead to several cellular responses such as histamine release and induction of inflammation.[47] It has been previously reported that hydroxyl containing polymers such as dextan and PVA activate the complement when attached to the surfaces.[50] Complement activation in presence of polymers was measured by a CH50 analysis using antibody sensitized sheep erythrocytes in human serum. Hybrid polyglycerols (Figure 6B) showed similar complement consumption compared to the buffer control suggested that none of the hybrid polyglycerols activated complement system in human serum at all the polymer concentrations. Similarly soluble PVA-100 and dextran did not activated the complement system. All these data supported the fact that hybrid polyglycerols are blood compatible. 3.3. Cell Viability Analyses The viability of the HUVECs in presence of hybrid polyglycerols were studied by two different methods. Results from MTT assay are shown in Figure 7A. Cell viability results showed that hybrid polyglycerols (LPGHPG-80, 50, and 15) were non-toxic to HUVECs at various concentrations (1.25–10.0 mg mL1). There was no decrease in cell viability even at high concentrations suggested that hybrid polyglycerols are non-toxic. The greater than 100% viability of the cells might be due to the experimental conditions used as these polymers may not increase the growth of the cells. This argument is based on the results given in Figure 7B where we measured the absolute number of viable cells. The cell viability measured using propidium iodide live cell staining assay is given in Figure 7B and C. This method gave absolute number of viable cells and more accurate representation of the toxicity to cells.[46] In this assay, Hoechst blue stain labels cell nuclei and propidium iodide as a red fluorescent nuclear and chromosomal counterstain. As propidium iodide will not penetrate into the live cells, only dead cells get stained. Dead cells with compromised cell membranes will take up the PI stain and will show up as bright fluorescent objects than live cells. The cell viability was >90% in presence of hybrid polyglycerols (LPG-HPG-80, 50, 30, and 15) and were similar to the media control suggesting that hybrid polyglycerols are non-toxic to HUVECs at the polymer concentrations studied. Significant cell death was observed in presence of DMSO-50% (DMSO50% þ Media-50%) and only less than 5% cells survived after 48 h. Images of HUVECs with propidium iodide and Hoechst-33258 staining also indicated the percentage of


live cells with hybrid polyglycerols and dead cells with DMSO-50%. 3.4. In Vivo Studies of High Molecular Weight Hybrid Polyglycerol (LPG-HPG-50) 3.4.1. Blood Circulation, Biodistribution, and Clearance in Mice After determining the blood compatibility and cell compatibility of hybrid polyglycerols, their blood circulation, biodistribution, and clearance in mice were investigated. Since all the polymers showed similar biocompatibility profile, the hybrid copolymer LPG-HPG-50 was selected for this study. The polymer was tritium labeled by modifying a portion of the hydroxyl groups to methoxy groups using tritiated methyl iodide. We have shown previously that tritiated methoxy groups are stable in vivo.[16,17] The labeled LPG-HPG-50 was injected intravenously via tail vein in normal mice (Balb/C) at a dose of 50 mg kg1, and the radioactivity in plasma and organs was monitored at different time intervals (0.5, 1, 2, 4, 8, 24, 48, 103, and 144 h). We compared our results with linear polyglycerol (LPG-100), hyperbranched polyglycerol (HPG100) and PVA having similar molecular weights reported in the literature.[16,51,52] Figure 8 shows the plasma level of LPG-HPG-50; both the percentage of injected dose (%ID) and amount of polymer in plasma as a function of time. There was a gradual decrease in the amount of polymer present in plasma with time for LPG-HPG-50. The data was analyzed using a standard two-compartment open model.[53] The system consists of a central compartment (C) that is mainly plasma, and the tissue compartment (T). The elimination process is represented by compartment E. The time course of the blood concentration of the molecules after their i.v administration showed the following two-phase decrement pattern; a rapid decrease in the early stage after injection (a-phase) and the subsequent slow decrease (b-phase). The time course of polymer concentration in blood is described by the Equation 1 and the parameters A, B, a, and b in the equation are determined from the time profile of blood concentration by curve fitting. CðtÞ ¼ A:eat þ B:ebt

ð1Þ

The circulation half lives, t1/2a and t1/2b, were calculated as (ln2)/a, (ln2)/b and the complete data set is given in Table 2. The circulation half life (t1/2b) of LPG-HPG-50 was 34.17 6.22 h, which was in between the LPG and HPG of similar molecular weights (LPG-100–31.89 4.12 h) and HPG-100–39.24 8.89 h). This data showed that hybrid LPG-HPG-50 polymer retained more in vasculature than linear polyglycerol and less than the hyperbranched


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Figure 7. In vitro cell viability of hybrid polyglycerols and dextran-100 at varying concentrations against HUVECs after 48 h of incubation at 37 8C using A) cell viability determined by MTT assay. Media and DMSO (50%) was used as normal and positive control, respectively. B) Cell viability determined by propidium Iodide cell staining assay. DMSO (50%) was used as a positive control. C) Images of HUVEC cells with propidium and Hoechst-33342 staining. Hoechst blue stain labels cells nuclei. Propidium iodide is a red fluorescent nuclear and chromosomal counterstain. Arrows denote nuclei within images that are positive for propidium iodide (dead cells). Labeled nuclei within white circles denote cells negative for propidium iodide (live cells).

polyglycerol.[16] We attribute the higher circulation half life of LPG-HPG-50 compared to LPG was due to the increased branching of the polymer and its compact nature (Table 1). Although the hybrid LPG-HPG-50 gave lower circulation time than hyperbranched polyglycerol and the value was considerably higher than that of other linear water soluble

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polymeric structures such as PVA, dextran and PEG with similar molecular weights in mice as reported previously by other investigators.[52,54] For example, the circulation half life of PVA-125K (M w -125 KDa) was 20.2 h[52] and circulation half life of PEG (M w -125 KDa) was 21.5 h.[54] The obtained blood circulation half life of the hybrid LPG-HPG-




Figure 8. Pharmacokinetic profile of hybrid polyglycerol h(LPG-HPG-50) in Balb/C mice. A) A plot of percentage of injected dose of the polymer in plasma with time and B) the amount of polymer in plasma with time. Each data point represents the average of three mice and error bars are based on standard deviations. Dotted lines show the fitting of the data. The values for LPG-100 kDa, HPG-100 kDa were taken from previously published data given in ref.[16]

Table 2. Pharmacokinetic parameters and renal clearance data for hybrid LPG-HPG-50 in Balb/C mice.

P0

Urinary excretion

Vc

[mg mL1

[mL

a

b

t1/2

k12

k2

k21

AUC0-/

Polymer

plasma]

plasma]

[h1]

[h1]

[h]

[h1]

[h1]

[h1]

[mg mL1 h1]

data 8h

48 h 144 h

LPG-HPG-50

0.561

0.077

14.24

0.35

2.88

0.046

0.006

0.313 0.020 34.17 0.133 0.039 0.161 0.308

0.003

6.22

0.08

0.01

3.72

0.007

P0, Initial polymer dose; Vc, Volume of the central compartment; k21, tissue to blood; k12, blood to tissue; k2, Elimination; AUC0-1, values indicate the exposure of the animals for the polymer material.

50 was higher than any linear polymer of similar molecular weight.[16,51] We recently reported the unusual compact structure of the linear polyglycerols[16] compared to other linear polymers and was attributed to its long circulation in mice. Our current data on hybrid polyglycerols support the fact that along with compactness, the branching of the polymers is also influencing its vascular residence time. The AUC0-1 value of hybrid LPG-HPG-50 was 14.24 which is higher than LPG-100 (12.67) and lower than HPG-100 (19.13 mg mL1 h1), indicating the lower exposure of polymers to the animals.[16] The elimination constant value (k2) also showed the similar trend as AUC0-1 indicating that LPG-HPG-50 was eliminated faster than HPG and slower than LPG from circulation and it supports the half-life of hybrid LPG-HPG-50 lies between linear and branched polymers. 3.4.2. Biodistribution and Clearance of Hybrid LPG-HPG-50 We also measured the biodistribution profiles of LPG-HPG50 by analyzing the residual radioactivity by scintillation


counting in harvested organs (liver, spleen, heart, lungs, and kidneys) at different time intervals (24, 48, and 103 h). The percentage of injected dose (ID) per gram (%ID/g) of the tissue in different organs are shown in Figure 9. For comparison, we also inserted the published data on linear and hyperbranched polyglycerol of similar molecular weights from Imran ul-haq et al.[16] There was no considerable accumulation of LPG-HPG-50 in vital organs suggesting the absence of non-specific elimination/excretion pathways. In the liver up to 4% ID/g was present and the accumulation level of LPG-HPG-50 of was lower than LPG-100 at all the time points and difference was not significant. In the spleen up to 6% ID/g of LPG-HPG-50 was present and the accumulation was lower than LPG-100 at all-time points. For other organs studied (heart, kidney, and lungs), the level of accumulation of hybrid LPG-HPG-50 was similar to LPG-100 but lower than that of HPG-100 at all the time points which suggest that the hyperbranched structure was interacting differently with the cells in these organs compared to the linear or hybrid architecture. The difference in the accumulation was higher at the initial time points. The reason for this behavior is unclear at this


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Figure 9. Percentage of ID/g of liver, spleen, kidney, heart, and lung at different time points for hybrid polyglycerol h(LPG-HPG-50) in mice. The values for LPG-100 kDa, HPG-100 kDa were taken from previously published data given elsewhere [16] and were given for comparison. Each data point represents the average of three mice. Significant results are compared between three polymers. LPG-100 & LPG-HPG-50. LPG-HPG-50 & HPG-100. LPG-100 & HPG-100.

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time, however the differences in the circulation time of the polymers, differences in the types of cells present in these organs (e.g., residual macrophages), perfusion of the organs, and so on, may be involved which warrant further investigation. Tissue accumulation was found to decrease with time in the case of kidney, heart, and lungs. Very low urinary clearance was observed for LPG-HPG-50. The experimentally determined values for the clearance of the hybrid polyglycerols through the feces indicate that polymer was mainly cleared through the liver. Approximately 5.6% injected dose of h(LPG-HPG-50) was eliminated within first 24 h and almost 10.5% after 144 h (total).

4. Conclusion High molecular weight hybrid polyglycerols with different molar contents of linear and branched component were synthesized. Structural and chemical characterizations of the hybrid polyglycerols confirmed their highly compact nature. The biocompatibility analyses demonstrated that the hybrid polyglycerols are highly biocompatible and have similar properties as that of linear and hyperbranched polyglycerols. The hybrid polyglycerols showed distinct behavior compared to PVA and dextran in certain blood compatibility analysis. The hybrid polyglycerol with 50% linear and branched component showed long circulation half-life in mice. The circulation half-life of hybrid polyglycerol (LPGHPG-50) was between the linear polyglycerol and hyperbranched polyglycerol. The differences in the vascular residence time may be due to the differences in the branching of the polymers. The accumulation of hybrid polyglycerol in vital organs was minimal and was comparable to the high molecular weight PEG suggesting the absence of non-specific elimination/excretion pathways for this polymer. The excellent biocompatibility, simple synthesis, multifunctionality, and long circulation make hybrid polyglycerol as potential candidates for the development of long circulating drug delivery systems and conjugates.

Acknowledgements: The authors acknowledge the funding from the Canadian Institutes of Health Research (CIHR). The authors thank the LMB Macromolecular Hub at the UBC Center for Blood Research for the use of their research facilities. These facilities are supported in part by grants from the Canada Foundation for Innovation, British Columbia Knowledge Development Fund and the Michael Smith Foundation for Health Research (MSFHR). Authors also thank Drs. Marcel Bally and Nancy Dos Santos for their help with animal studies at IDP Centre of British Columbia Cancer Research Centre. JNK holds a Career Investigator Scholar award from MSFHR. Supporting Information:Additional characterization data is given in the supporting information


Received: March 27, 2014; Revised: May 28, 2014; Published online: July 11, 2014; DOI: 10.1002/mabi.201400152 Keywords: biodistribution; blood circulation; blood compatibility; cell viability; linear-branched hybrid polyglycerol

[1] S. Svenson, D. A. Tomalia, Adv. Drug. Delivery Rev. 2005, 57, 2106. [2] V. P. Torchilin, Adv. Drug. Delivery Rev. 2006, 58, 1532. [3] R. Duncan, Nat. Rev. Cancer. 2006, 6, 688. [4] J. Khandare, T. Minko, Prog. Polym. Sci. 2006, 31, 359. [5] R. Haag, F. Kratz, Angew. Chem. Int. Ed. 2006, 5, 1198. [6] C. C. Lee, J. A. Mackay, J. M. J. Frechet, F. C. Szoka, Nat. Biotechnol. 2005, 23, 1517. [7] E. R. Gillies, J. M. J. Frechet, Drug Discovery Today 2005, 10, 35. [8] J. Kopecek, Polym. Med. 1977, 7, 191. [9] S. Sadekar, A. Ray, M. Janat-Amsbury, C. M. Peterson, H. Ghandehari, Biomacromolecules 2011, 12, 88. [10] J. Fang, H. Nakamura, H. Maeda, Adv. Drug. Delivery Rev. 2011, 63, 136. [11] S. Svenson, T. D. Tomalia, Adv. Drug. Delivery Rev. 2005, 57, 2106. [12] R. K. Kainthan, S. R. Hester, E. Levin, D. V. Devine, D. E. Brooks, Biomaterials 2007, 28, 4581. [13] H. Frey, R. Haag, Rev. Mol. Biotechnol. 2002, 90, 257. [14] R. K. Kainthan, J. Janzen, E. Levin, D. E. Devine, D. E. Brooks, Biomacromolecules 2006, 7, 703. [15] D. Wilms, S. Stiriba, H. Frey, Acc. Chem. Res. 2010, 43, 129. [16] M. Imran ul-haq, B. L. Lai, R. Chapanian, J. N. Kizhakkedathu, Biomaterials 2012, 33, 9135. [17] R. K. Kainthan, D. E. Brooks, Biomaterials 2007, 28, 4779. [18] R. K. Kainthan, J. Janzen, J. N. Kizhakkedathu, D. V. Devine, D. E. Brooks, Biomaterials 2008, 29, 1693. [19] O. Kleifeld, A. Doucet, U. Keller, A. Prudova, O. Schilling, R. K. Kainthan, A. E. Starr, L. J. Foster, J. N. Kizhakkedathu, C. M. Overall, Nat. Biotechnol. 2010, 28, 281. [20] X. Yu, Z. Liu, J. Janzen, I. Chafeeva, S. Horte, W. Chen, R. K. Kainthan, J. N. Kizhakkedathu, D. E. Brooks, Nat. Mater. 2012, 11, 468. [21] J. Derneddea, A. Rauschb, M. Weinhart, S. Endersa, R. Taubera, € gelb, A. Boninb, R. Haag, Proc. K. Lichad, M. Schirnerd, U. Zu Natl. Acad. Sci. U.S.A. 2010, 107, 19679. [22] R. Chapanian, I. Constantinescu, D. E. Brooks, M. D. Scott, J. N. Kizhakkedathu, Biomaterials 2012, 33, 3047. [23] M. Calderon, M. A. Quadir, S. K. Sharma, R. Haag, Adv. Mater. 2010, 22, 190. [24] C. Mugabe, B. A. Hadaschik, R. K. Kainthan, D. E. Brooks, A. I. So, M. E. Gleave, H. M. Burt, B. J. U. Int. 2008, 103, 978. [25] C. Mugabe, P. A. Raven, L. Fazli, J. H. E. Baker, J. K. Jackson, R. T. Liggins, A. I. So, M. E. Gleave, A. I. Minchinton, D. E. Brooks, H. M. Burt, Biomaterials 2012, 33, 692. [26] S. Lee, K. Saito, H. R. Lee, M. J. Lee, Y. Shibasaki, Y. Oishi, B. S. Kim, Biomacromolecules 2012, 13, 1190. [27] Y. Oikawa, S. Lee, D. H. Kim, D. H. Kang, B. S. Kim, K. Saito, S. Sasaki, Y. Oishi, Y. Shibasaki, Biomacromolecules 2013, 14, 2171. [28] M. Imran ul-haq, J. L. Hamilton, B. L. Lai, R. A. Shenoi, S. Horte, I. Constantinescu, H. A. Leitch, J. N. Kizhakkedathu, ACS Nano 2013, 7, 10704. [29] K. Saatchi, P. Soema, N. Gelder, R. Misri, K. McPhee, J. H. E. €feli, Bioconjugate Baker, S. A. Reinsberg, D. E. Brooks, U. O. Ha Chem. 2012, 23, 372.


1481


[30] a) M. Jikei, M. Kakimoto, Prog. Polym. Sci. 2001, 26, 1233; b) C. Gao, D. Yan, Prog. Polym. Sci. 2004, 29, 183. €m, Adv. Polym. Sci. 1999, [31] A. Hult, M. Johansson, E. Malmstro 143, 1–34. [32] I. Gitsov, J. Polym. Sci. Part A: Polym. Chem. 2008, 46, 5295. [33] C. R. DeMattei, B. H. Huang, D. A. Tomalia, Nano Lett. 2004, 4, 771. [34] L. Y. Zhu, X. F. Tong, M. Z. Li, E. J. Wang, J. Phys. Chem. B 2001, 105, 2461. [35] L. Y. Zhu, X. F. Tong, M. Z. Li, E. Wang, J. Polym. Sci. Part A: Polym. Chem. 2000, 38, 4282. [36] I. Gitsov, K. L. Wooley, J. M. J. Frechet, Angew. Chem. Int. Ed. 1992, 31, 1200. [37] J. Iyer, K. Fleming, P. T. Hammond, Macromolecules 1998, 31, 8757. [38] A. M. Fischer, H. Frey, Macromolecules 2010, 43, 8539. [39] E. Barriau, M. A. Garcıa, H. Kautz, H. Frey, Macromol. Rapid. Commun. 2005, 26, 862. [40] F. Wurm, U. Kemmer-Jonas, H. Frey, Polym. Int. 2009, 58, 989. [41] M. E. Fox, F. C. Szoka, J. M. J. Frechet, Acc. Chem. Res. 2009, 42, 1141. [42] R. K. Kainthan, E. B. Muliawan, S. G. Hatzikiriakos, D. E. Brooks, Macromolecules 2006, 39, 7708.

1482

[43] S. Stiriba, H. Kautz, H. Frey, J. Am. Chem. Soc. 2002, 124, 9698. [44] R. K. Kainthan, M. Gnanamani, M. Ganguli, T. Ghosh, D. E. Brooks, S. Maiti, J. N. Kizhakkedathu, Biomaterials 2006, 27, 5377. [45] N. A. A. Rossi, I. Mustafa, J. K. Jackson, H. M. Burt, S. A. Horte, M. D. Scott, J. N. Kizhakkedathu, Biomaterials 2009, 30, 638. [46] J. M. Breier, N. M. Radio, W. R. Mundy, T. J. Shafer, Toxicology 2008, 105, 119. [47] M. B. Gobert, M. V. Sefton, Biomaterials 2004, 25, 5681. [48] (Eds: S. Chien, D. M. Surgenor), ‘‘Red Blood Cell’’, Academic Press, New York 1975, pp 1032–1121. [49] K. M. Hansson, S. Tosatti, J. Isaksson, J. Wettero, M. Texter, T. L. Lindahl, Biomaterials 2005, 26, 861. [50] A. Yusuke, K. Masako, T. Mitsuaki, I. Hiroo, ACS Appl. Mater. Interfaces 2009, 1, 2400. [51] Y. Tabata, Y. Murakami, Y. Ikada, J. Controlled Release 1998, 50, 123. [52] T. Yamaoka, Y. Tabata, Y. Ikada, J. Pharm. Pharmacol. 1995, 47, 479. [53] R. E. Notari, ‘‘An Introduction’’, Marcel Dekker, Inc., New York 1980. [54] T. Yamaoka, Y. Tabata, Y. Ikada, J. Pharm. Sci. 1994, 83, 601.



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