Blood Components Interactions to Ionic and Nonionic Glyconanogels

Aug 17, 2015 - Department of Pathology and Centre for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada ... The data revea...
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Blood Components Interactions to Ionic and Nonionic Glyconanogels Ravin Narain,*,† Yinan Wang,† Marya Ahmed,† Benjamin F.L. Lai,‡ and Jayachandran N. Kizhakkedathu‡,§ †

Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 2G6, Canada Department of Pathology and Centre for Blood Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada § Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada ‡

S Supporting Information *

ABSTRACT: Nanogels are prominent examples of “smart” nanomaterials, which are designed to incorporate biologically relevant (macro)molecules for systemic delivery. Although these systems are carefully engineered, only a handful of studies discuss the blood compatibility of nanogels, and no systematic studies are available on how the presence of net or surface charges impacts the hemocompatibility of these nanomaterials. Therefore, in this study, temperature responsive, galactose based nanogels bearing net positive, negative, or neutral charges, either in the core or shell of nanogels, are prepared and are subsequently evaluated for their blood compatibility profiles. The nanogels containing neutral core and shell, cationic core with neutral shell, anionic core with neutral shell, neutral core with cationic shell, and neutral core with anionic shell are prepared by reversible addition− fragmentation chain transfer (RAFT) polymerization approach. The evaluation of complement activation, blood clot formation, platelet activation, red blood cells aggregation, and hemolysis provides a detailed analysis of structure activity relationship of blood compatibility profile of these nanogels. The data reveal that the physical and biological (blood compatibility) properties can be carefully tuned by embedding the charges in the core of temperature-responsive nanomaterials, protected by neutral carbohydrate based shells.

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INTRODUCTION Nanogels are carefully engineered nanosized systems composed of hydrophilic, hydrophobic, or amphiphilic polymers chains for the encapsulation and release of proteins, drugs, genes, and other biologically active molecules. Because of their high stability in aqueous dispersion, high loading capacity, and their responsiveness to pH, temperature, and ionic strength, they are extremely valuable as systemic delivery carriers.1 Engineered “smart” nanogels that responds to environmental factors are optimized systems for the controlled delivery of cargo at the desired location under physiological conditions.2,3 The rapid development of public knowledge about diseases and the unmet need for personalized medicines promote research and development efforts to produce novel biomaterials with desired efficacies. For instance, the notable differences in pH and temperature of normal versus cancerous tissues have pushed for the design of more advanced pH and temperature responsive nanomaterials for targeted drug delivery.2 Polymers, the structural units of polymeric nanomaterials, are extensively studied for their hemocompatibility and toxicity profiles.4−7 The net charge of polymers is reported to alter their hemocompatibility profiles. For example, cationic materials are reported to induce changes in blood coagulation,7 protein aggregation,8−10 red blood cells agglomeration, platelet activation, and complement activation.8 In general, anionic © 2015 American Chemical Society

polymers and surfaces inhibit complement activation and blood coagulation.11 Examples include heparins, heparin-mimics, and polysulfates.12,13 However, certain anionic inorganic polymers, like polyphosphates, are known to activate blood coagulation.14 The incorporation of charged polymeric chains into responsive nanomaterials, such as nanogels, significantly alters their chemical and biological properties, including drug encapsulation and release profiles, and biocompatibility.4−6 A recent report describes thrombogenic properties of chitosan-modified surface.7 Pereira et al. reported that glycol chitosan (GC)-based nanogels with strong net positive surface charge can successfully evade the immune system. GCs are nonhemolytic, are not uptaken by macrophages, and do not induce complement activation.8 In contrast, polyethylene glycol (PEG)ylated and non-PEGylated siRNA loaded dextran nanogels of net positive charge caused significant aggregation of platelets, while their negatively charged counterparts did not induce platelet aggregation. Moreover, the dispersion of PEGylated and non-PEGylated negatively charged nanogelsiRNA complexes in human plasma showed agglomeration and release of 50% of complexed siRNA.15 Received: July 3, 2015 Revised: August 14, 2015 Published: August 17, 2015 2990

DOI: 10.1021/acs.biomac.5b00890 Biomacromolecules 2015, 16, 2990−2997

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Biomacromolecules In another report, zwitterionic nanogels composed of poly(vinylpyridine-co-sulfobetaine methacrylate) P(VP-coSBA) copolymers showed enhanced hemocompatibility. The introduction of zwitterionic component “SBA” into polyvinylpyrrolidone (PVP) greatly affected platelet adhesion profile of copolymers. The zwitterionic copolymers of PVP exhibited excellent biocompatibility at all studied monomer ratios.16 The above-mentioned handful of reports on blood compatibility of charged nanogels do not allow a detailed correlation between their chemical and biological properties. Herein, we describe the detailed synthesis of galactose-based, well-tailored cationic, neutral, and anionic glyco-nanogels by reversible addition− fragmentation chain transfer (RAFT) polymerization. The introduction of charged species (positive or negative) either in the core or in the shell of nanogels allows a structuredependent study of their blood compatibility.

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Scheme 1. Synthesis of (a) Poly(LAEMA-st-AEMA) MacroCTA and (b) Cationic Carbohydrate-Based Nanogel (NG4)

MATERIALS AND METHODS

Materials. The 2-lactobionamidoethyl methacrylamide (LAEMA), 2-aminoethyl methacrylamide hydrochloride (AEMA), and the chain transfer agent (CTA), cyanopentanoic acid dithiobenzoate (CTP), were synthesized in the laboratory according to previously reported protocols.16−19 The 4,4′-azobis(cyanovaleric acid) (ACVA), methacrylic acid (MA), di(ethylene glycol) methyl ethyl methacrylate (DEGMA), and the cross-linker, N,N′-methylenebis(acrylamide) (MBAm) were purchased from Sigma-Aldrich (Oakville, ON, Canada). The organic solvents were from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Methods. Synthesis of Macro-CTA Carrying Different Charges by RAFT Polymerization. Macro-CTAs carrying different charges [poly(LAEMA) (neutral), poly(LAEMA-st-AEMA) (positive charges), or poly(LAEMA-st-MA) (negative charges)] were synthesized in a similar way as previously reported.4,19,20 In a typical protocol for poly(LAEMA-st-AEMA) synthesis, LAEMA (0.79 g, 1.7 mmol) and AEMA (0.28 g, 1.7 mmol) were dissolved in 5 mL of DI water with 1 mL of CTP (10 mg, 36 μmol) and ACVA (5 mg, 18 μmol) DMF stock solution in a 25-mL reactor. The solution was degassed by purging nitrogen for 30 min and placed in an oil bath at 70 °C for 5 h. The reaction was then quenched in liquid nitrogen, and the polymer obtained was precipitated in acetone and washed with methanol to remove the residual monomers and RAFT agents. The molecular weight and polydispersity of the polymers were determined by an aqueous gel permeation chromatography (GPC) (Viscotek GPC system) at room temperature with a flow rate of 1.0 mL/min. The 0.5 M sodium acetate/0.5 M acetic acid buffer was used as eluent, and pullulan standards (Mw = 500−404 000 g/mol) were used for calibration. The compositions of the polymers were determined using Varian 500 1H NMR by using D2O as the solvent. Synthesis of Nanogels by RAFT Polymerization. The synthesis of the nanogels was conducted at 70 °C by employing ACVA as the initiator and poly(LAEMA), poly(LAEMA-st-AEMA), or poly(LAEMA-st-MA) as macro-CTA. A typical procedure for the synthesis of poly[(LAEMA-st-AEMA)-b-(DEGMA-st-MBAm-st-LAEMA)] nanogel is as follows. In a 25-mL reactor, poly(LAEMA19-stAEMA19) macroCTA (200 mg, 0.016 mmol) and LAEMA (248 mg, 0.53 mmol) were first dissolved in 8 mL of DI water. Then DEGMA (400 mg, 2.13 mmol), MBAm as cross-linker (41 mg, 0.27 mmol) (10 mol % with respect to total moles of DEGMA and LAEMA), and ACVA (2.3 mg, 0.008 mmol) were dissolved in 2 mL of 2-propanol and added in the above solution. The solution was degassed by purging with nitrogen for 30 min, and the reaction was carried out at 70 °C for 24 h (Scheme 1). The reaction was then quenched in liquid nitrogen, and the product was purified by dialysis against DI water for 3 days using dialysis membrane with a molecular weight cutoff of 6000. The nanogel was obtained as a white powder after freeze-drying overnight and was stored in a refrigerator (4 °C).5,20 Characterization of Nanogels. Dynamic Light Scattering (DLS) and Zeta Potential Analysis. The hydrodynamic diameter and charge

on nanogel surfaces were determined at 15 and 37 °C in DI water using a Brookhaven DLS and a zeta potential instrument. The volumetric swelling/shrinking ratios of the nanogels at different temperatures were estimated as follows: %volumetric changing ratio =

VT1 − VT 2 × 100 VT1 3

3

( D2 ) − ( D2 ) = 3 ( D2 ) T1

T2

× 100

T1

Transmission Electron Microscopy (TEM). Size and morphology of nanogels at room temperature were also analyzed by TEM on a Philips transmission electron microscope operated at 80 kV and fitted with a CCD camera. A droplet of aqueous solution of nanogel (0.1 mg/mL) was placed on the TEM carbon coated copper grid and allowed to airdry overnight; the samples were stained with phosphotungstate prior to observation under TEM. Biological Reagents. Whole blood from healthy consented donors was either collected into 3.8% sodium citrated tube with a blood/ anticoagulant ratio of 9:1 or empty nonanticoagulant serum tube at the Centre for Blood Research, University of British Columbia. The University of British Columbia Human Ethics Committee approved the protocol. Platelet-rich plasma (PRP) was prepared by centrifuging citrated whole blood samples at 150 × g for 10 min in an Allegra X22R centrifuge (Beckman Coulter, Canada). Platelet-poor plasma (PPP) was prepared by centrifuging citrated whole blood samples at 1200 × g for 20 min. Serum was prepared by allowing the nonanticoagulated whole blood samples to clot and then centrifuging at 1200 × g for 30 min. Reagent for conventional coagulation assay activated partial thromboplastin time (APTT), actin FSL, was purchased from Siemens Healthcare. Anti-CD62PE and goat antimouse PE antibodies were purchased from Immunotech. Thromboelastography (TEG) cup and pin was purchased from Hemonetics (Cedarlane). Drabkins’ reagent was purchased from Sigma-Aldrich. GVB2+ (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3 with 0.15 mM CaCl2 and 0.5 2991

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37 °C. After 1 h of incubation, the polymer-treated serum samples were diluted with GVB2+ buffer in ratio 1:2 (60 μL of treated serum with 120 μL of GVB2+ buffer). The diluted serum was then incubated in equal volume with the antibody-sensitized sheep RBC (EA cells) for 1 h at 37 °C to measure the amount of complement activity remaining in the serum. Heat-aggregated IgG (final concentration 5 mg/mL) and PBS buffer were also incubated with GVB2+-diluted human serum for 1 h at 37 °C as the positive and negative controls, respectively, for the experiment. The reactions were stopped by the addition of 0.3 mL of GVB-EDTA. Control tubes containing equal volumes of EA cells and GVB2+ buffer were added with GVB-EDTA (blank control) or dH2O (100% lysis control). Intact EA cells were spun down at 6000 × g for 3 min, and the supernatants were sampled and measured at a wavelength of 540 nm. Percentage EA lysis was calculated using average absorbance values as follows:

mM MgCl2, pH 7.3) and antibody-sensitized sheep erythrocytes (EA) were purchased from CompTech (Tyler, TX). Stock solutions of the nanogels were made out to 1 and 10 mg/mL in phosphate-buffered saline (10 mM phosphate buffer in 2.7 mM potassium chloride and 137 mM sodium chloride, pH 7.4, PBS). One in ten dilutions of the nanogel samples were made with plasma, whole blood, or serum to achieve final nanogel concentrations in the analysis media at 0.1 and 1 mg/mL. Plasma Clotting Assays Analysis: APTT Assay. Sodium citrate anticoagulated PPP was used for APTT analysis. The effect of nanogel on the coagulation cascade was examined by mixing 180 μL of PPP with 20 μL of nanogel stock solutions (9:1 v/v; 0.1 or 1 mg/mL final concentration) at 37 °C. Control experiments were performed with identical volumes of PBS solution to PPP. Two-hundred microliters of actin FSL was mixed with the plasma−nanogel mixture. Each measurement was repeated in triplicate (100 μL of the mixture) on the STart4 coagulometer (Diagnostica Stago, France). Twenty microliters of 0.025 M of calcium chloride was added to initiate the intrinsic coagulation cascade. The average value of the clotting times from at least three different donors was reported. Plasma Clotting Assays Analysis: Thromboelastography (TEG). Whole blood clotting was investigated using a Thromboelastograph Hemostasis System 5000 (Hemoscope Corporation). For TEG study, the blood samples were used within 5 min of blood collection. Forty microliters of stock nanogel solutions (final concentration 1 mg/mL) was incubated with 360 μL of citrated whole blood. Three-hundred and forty microliters of the nanogel-whole blood sample suspension was transferred to TEG cups; the coagulation analysis began when the anticoagulated whole blood was recalcified with 20 μL of 0.20 M CaCl2 solution. Phosphate-buffered saline mixed with whole blood served as the normal control for the study. All TEG experiments were performed at 37 °C and ended 2 h after the initiation of clotting. Platelet Activation Analysis: Flow Cytometry. The level of platelet activation was quantified by flow cytometry analysis. Ninety microliters of PRP was incubated at 37 °C with 10 μL of stock nanogel samples (final concentration is 0.1 or 1 mg/mL). After 1 h, aliquots of the incubation mixtures were removed for assessment of the platelet activation state. Five microliters of postincubation platelet/nanogel mixture, diluted in 45 μL HEPES buffer, was incubated for 20 min in the dark with 5 μL of monoclonal anti-CD62PE. The reaction was then stopped with 0.3 mL of phosphate-buffered saline solution. The level of platelet activation was analyzed in a BD FACSCanto II flow cytometer (Becton Dickinson) by gating platelets specific events based on their light scattering profiles. Activation of platelets was expressed as the percentage of platelet activation marker CD62P detected in the 10 000 total events counted. Duplicate measurements for each donor and the mean values from three donors are reported. Platelets incubated with human thrombin (Sigma) (1 U/mL) were used as a positive control for the flow cytometric analysis, and PE conjugated goat antimouse IgG polyclonal antibody was used as the nonspecific binding control. Red Blood Cell Lysis. Thirty microliters of each of the stock nanogel solutions was mixed with 270 μL of 10% hematocrit red blood cell (RBC) suspension for 1 h at 37 °C. The dH2O incubated RBCs were used as the positive control (100% lysis), and phosphate-buffered saline mixed with the RBC was used as the normal control. The percent of RBC lysis is measured by the cyanmethemoglobin based on Drabkin’s assay. Twenty microliters of the RBC/nanogel suspension without centrifugation was added to 1 mL of Drabkin’s solution, and the optical density (OD) was measured. The incubated RBC/nanogel mixture was centrifuged, and 200 μL of supernatant was added to 1 mL of Drabkin’s solution, and the OD was measured. The OD was measured by a light spectrophotometer at a wavelength of 540 nm. The percent of RBC lysis in sample was calculated from the OD of supernatant and the OD of RBC/nanogel suspension. Complement Activation Analysis: CH50 Assay. A modified hemolytic assay was performed to analyze the level of complement activation by nanogels. Two incubation steps were utilized. First, stock polymer solutions (final concentrations of 0.1 and 1 mg/mL) were incubated and reacted with 90 μL of GVB2+-diluted human serum at

%EA lysis = (OD540,test sample − OD540,blank ) /(OD540,100%lysis − OD540,blank ) × 100 Percentage of complement activated (consumed) by the polymeric nanoparticles was expressed as 100% − % EA lysis.

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RESULTS AND DISCUSSION Polymers and polymeric nanoscaffolds are the most studied systemic delivery systems. Engineered synthetic materials offer the flexibility to produce nanomaterials of desired biological properties, sizes, and shapes. Polymeric nanogels are nanometer sized, smart, stimuli-responsive nanoparticles, which are extensively studied as delivery systems in vitro and in vivo. However, only a limited number of approaches address the issue of hemocompatibility of these nanomaterials.8−16 In addition, there is only limited information available on the influence of surface properties of nanogels on their interaction with various blood components. The surface charge of glyconanogels is found to impact their interaction with blood components. Thus, a systematic study on well-defined nanogels, with fine-tuning of positive and negative charges within the core or shell of nanogels, is required. Galactosebased nanogels bearing cationic, anionic, or neutral charges were prepared as depicted in Scheme 1. The preparation of nanogels was done in two steps. In the first step, a macroCTA containing a carbohydrate polymer alone or different charged components was prepared. LAEMA was either homopolymerized or copolymerized with cationic monomer, 2-aminoethyl methacrylamide, AEMA, or an anionic monomer, methacrylic acid, MA, via RAFT polymerization. The macro-CTAs were about 12 kDa with the carbohydrate to ionic monomer ratio ∼1:1 (Table 1; Supporting Information, Figure S1). The macro-CTAs were further polymerized, in the presence of cross-linker, with either, DEGMA alone, or in the presence of AEMA or MA to yield well-defined cationic, neutral, or anionic nanogels (Table 2). The temperature-responsive properties of the nanogels were studied by determining their sizes and net charges using DLS, Table 1. Molecular Weight (Mn) and PDI (Mw/Mn) of Macro-CTAs Synthesized by RAFT Process

2992

polymer

charge

Mn (GPC, g/mol)

Mw/Mn

P(LAEMA25) P(LAEMA21-st-AEMA16) P(LAEMA19-st-MA25)

neutral cationic anionic

11 700 12 300 11 100

1.23 1.22 1.25

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Biomacromolecules Table 2. Characteristics of Nanogels with Varying Compositions hydrodynamic diameter (PDI) (nm) nanogel

compositionsa

NG1

poly[(LAEMA25)-b-(DEGMA-st-MBAmst-LAEMA)180] poly[(LAEMA25)-b-(DEGMA-st-MBAmst-AEMA)180] poly[(LAEMA25)-b-(DEGMA-st-MBAmst-MA)180] poly[(LAEMA21-st-AEMA16)-b-(DEGMAst-MBAm-st-LAEMA)180] poly[(LAEMA19-st-MA25)-b-(DEGMA-stMBAm-st-LAEMA)180]

NG2 NG3 NG4 NG5 a

idealized core−shell nature of nanogels core-neutral, shellneutral core-cationic, shellneutral core-anionic, shellneutral core-neutral, shellcationic core- neutral, shellanionic

15 °C 86 ± 0.3 (0.20)

37 °C

Zeta potential (mV) 15 °C

37 °C

77.4 ± 0.2 (0.18)

0.00 ± 0.00

0.00 ± 0.00

111 ± 3 (0.34)

59.4 ± 1.8 (0.33)

0.00 ± 0.00

0.00 ± 0.00

110 ± 0.7 (0.02)

95.9 ± 0.03 (0.02)

−0.15 ± 0.04

−0.08 ± 0.05

86.5 ± 0.5 (0.30)

71.7 ± 0.4 (0.27)

24.63 ± 1.15

28.60 ± 0.63

60.3 ± 1.9 (0.34)

53.6 ± 0.6 (0.30)

−10.77 ± 0.47

−10.93 ± 1.18

The degree of polymerization values (DP) of cores were calculated as ([DEGMA] + [monomer] + [cross-linker])/[macro-CTA].

temperature-responsive core showed strong positive zeta potential, while NG5 containing anionic shell and neutral temperature-responsive core showed net negative zeta potential values. Interestingly, although NG5 contains more charged units as compared to NG4, the latter showed a higher absolute zeta potential value (Table 2). However, the actual charges on the surface of the nanogels may be different, and hence this can account for the difference in the zeta potential values.21−23 Nanogels with different core−shell structure and temperature-sensitive properties were investigated for their interaction with various blood components. We studied the influence of surface charges and presence of temperature-sensitive component on blood coagulation, blood clot formation, platelet activation, complement activation, and RBC aggregation and hemolysis using various in vitro assays. The influence on intrinsic pathway of blood coagulation by nanogels at two different concentrations was measured by APTT analysis. Results are shown in Figure 2. The nanogels containing cationic shell and neutral core (NG4, zeta potential 28.6 ± 0.63 mV) showed inhibition of blood coagulation at both concentrations studied. It should be noted that nanogels containing neutral shell and core (NG1, zeta potential 0.00 mV), neutral shell and cationic core (NG2, zeta potential 0.00 mV), and neutral shell and anionic core (NG3, zeta potential −0.08 mV) showed no significant effect on blood coagulation

TEM, and zeta potential measurements, respectively (Table 2, Figure 1). According to the DLS results (Table 2), the nanogels

Figure 1. TEM images of NG1 (neutral core and neutral shell) (A), NG2 (cationic core and neutral shell (B), and NG3 (anionic core and neutral shell) (C) synthesized via RAFT process.

shrank 27−85% in volume when the temperature was increased from 15 to 37 °C. As revealed by zeta potential values, the charged moieties in the core of nanogels were successfully masked by neutral glycopolymer shell at both 15 °C and physiological temperatures (37 °C). NG1, NG2, and NG3, bearing neutral shell, and neutral, cationic, and anionic temperature-responsive cores, respectively, show neutral zeta potentials. However, the presence of charged monomers in the shell of nanogels showed strong net positive or negative values depending upon the type of ionic monomer introduced in the macro-CTA. NG4 containing cationic shell and neutral

Figure 2. APTT of the glycopolymer nanogels measured at varying concentrations compared to buffer. The values given are the average ± SD from three independent measurements. 2993

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Figure 3. Blood coagulation measured using TEG. Shown are TEG traces (a), reaction time (R) (b), and clot strength (MA) (c) of the glycopolymer nanogels and macroCTAs at 1 mg/mL concentration. The values given are the average ± SD from three independent measurements.

charges on nanogel surface may also be contributing to the observed behavior, which cannot be captured by doing experiments using soluble polymers. Further work is needed to establish the cause of unusual TEG profile in the presence of different temperature-sensitive nanogels. The cationic shell component poly(LAEMA19-st-AEMA19) is delaying the clot formation, however, not influencing the MA values or TEG profile. Thus, except for NG3 (neutral shell and anionic core), for all other nanogels, the temperature-responsive component poly(DEGMA) is influencing the clot formation resulting in unusual clot and TEG morphology. On the basis of these studies, the only nanogel that did not influence the blood coagulation was NG3 with anionic core and a shell containing galactose and stimuli-sensitive poly(DEGMA). These data confirm the importance of nanogel design in the generation of biocompatible materials. The platelet interaction with nanogels was investigated by measuring the platelet activation. The expression of activation marker CD62P selectin is measured using fluorescently labeled antibodies in the presence of nanogels at two different concentrations (Figure 4). The activation of platelets results

profile. The presence of net negative charge on polymers is reported to increase blood-clotting time. For instance, carboxyl24 and sulfate25 groups containing polymers are known to possess anticoagulant activities. Although our glyconanogel bearing neutral core and anionic shell (NG5, zeta potential −10.93 ± 1.18 mV) contains carboxyl groups,20 it was not found to exhibit anticoagulant properties. To further investigate the influence of nanogels on blood coagulation, we measured the kinetics of blood clot formation, clot stiffness, and clot lysis by TEG analysis. The time to form initial clot (reaction time (R)) and maximum amplitude (MA values), which reflect clot stiffness, are reported. Figure 3, panel A gives the whole blood TEG profiles in the presence of various nanogels. Except for NG3, all other nanogels gave uncharacteristic TEG profiles. The R times for nanogels NG1, NG2, NG3, and NG5 were similar to that of the control buffer (Figure 3B); however, the NG4 (zeta potential +28 mV) showed longer R time. The increase in R time indicates that nanogels with neutral core and cationic shell (NG4) impaired blood coagulation. The data are consistent with APTT values reported in Figure 2. It has been reported that cationic macromolecules could enhance the blood clotting time by inhibiting key coagulation factors.26 The MA values showed considerable decrease in the presence of nanogels, regardless of the net charge of the nanogels, as compared to the buffer control. NG3 (cationic core and neutral shell) showed MA values similar to that of the buffer control. The changes in MA values could be due to the alteration of clot structure formed in the presence of nanogels. To further understand the unusual TEG profile and clot stiffness produced by different nanogels, we measured the whole blood TEG profile in the presence of water-soluble shell components of the nanogels, namely poly(LAEMA25), poly(LAEMA19-st-AEMA19), poly(LAEMA19-st-MA24), containing neutral, cationic, and anionic charges (Figure 3A−C). The TEG profiles of shell component, regardless of net charges, at identical concentration as nanogels (1 mg/mL) appear to be normal, which suggests that the unusual TEG profile (in combination with low MA values) of some of the nanogels is originating from the temperature-responsive component poly(DEGMA). This argument is supported by a recent observation that water-soluble temperature-sensitive polymers change the blood coagulation by altering the fibrin polymerization, clot structure, morphology, and the way in which platelets integrate to the clot structure.27 In addition, the local concentration of

Figure 4. Platelet activation (as determined by %CD62P expression at 1 mg/mL concentration of nanogels) induced by glycopolymer nanogels at varying concentrations. The values given are the average ± SD from three independent measurements from different donors.

in release of coagulation factors, such as thrombin, which further promotes platelet aggregation resulting in thrombus formation.26 The nanogels with net cationic shell and neutral core (NG4) showed slight increase in CD62P expression (∼30%) at 1 mg/mL concentration. All the other nanogels did not show significant platelet activation compared to the buffer 2994

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prevent the activation of the complement. In addition, observation of the current galactose-containing nanogel is different from complement activating dextran particles34 and hydroxyl-containing nanoparticles previously reported in the literature. Since we have limited ability to measure galactose density on these nanogels, more work is needed to understand this behavior. However, the current observation is important and will open up new avenues for glycopolymer-based nanogels for practical applications. In addition, the core−shell design is particularly important as certain groups can be masked against unwanted biological interaction by placing it within core and are further protected using a layer of carbohydrate rich shell layer as in the case of NG2. In this case, the primary amine groups are protected against complement activation when placed within the core. RBCs make 45% of blood volume and are an important component determining the hemocompatibility of a material. The cationic polymers, such as polyethylenimine (PEI) and protamine, are well-known to induce RBC aggregation and lysis.26 None of the glycol-nanogels (neutral, negative, and positive) induced RBC aggregation and hemolysis under the conditions studied (Figures 6 and 7). These results are

control. These results are consistent with previous studies where PEGylated dextran based nanogels with strong net positive charge are reported to cause platelet aggregation.11 It is also reported that cationic polymers significantly activate platelets.28 The complement activation is another important aspect of the study of blood component interaction. We used a modified CH50 analysis to measure the complement consumption in the presence of nanogels using antibody sensitized sheep erythrocytes.29 A previous report describes that the surface decoration of hydrophobic nanoparticles with stealth layer such as PEG is required to decrease the complement activation.20 In addition, the presence of surface groups such as hydroxyl groups and primary amine groups on the nanoparticles is shown to increase the complement activation.29−32 Dextrancoated nanoparticles and surfaces are also shown to be complement activating.33 Thus, it is our intention to investigate whether the glycopolymer-based nanogels induce complement activation. In our structures, we have a hydroxyl-rich layer of galactose-based polymer and primary amines on certain nanogels (NG2 and NG4). As shown in Figure 5, the presence

Figure 6. RBC hemolysis in the presence of glycopolymer nanogels. PBS buffer is used as a normal control, and distilled water (H2O) is used as positive control. The values given are the average ± SD from three independent measurements.

Figure 5. Percent complement consumption (% complement activation) caused by glycopolymer nanogels at different concentrations in serum after incubation for 1 h. IgG is used as a positive control. The values given are the average ± SD from three independent measurements. The values from PBS buffer control were used for normalizing the values.

consistent with previous reports where dextran based siRNA loaded nanogels of both net positive and negative charges were studied for their RBC interactions.15 Although we obtained a general understanding of the blood interaction with respect to the glyco-nanogel design, more studies are needed to obtain detailed mechanism of interaction.

of hydroxyl groups on the nanogels did not induce complement activation unlike those grafted on solid surface or nanostructures. The presence of primary amine groups on the shell of NG4 is inducing complement activation. However, the primary amine groups present in the core of the nanogel (NG2) are not inducing any complement activation. The data are important in many folds; unlike solid surfaces, the presence of sugar residues on nanogel surface is not inducing complement activation. This might be due to the fact that conformation of the sugar containing polymer chains on the nanogel surface may be different from that of solid surfaces.29 We have shown previously that the density of sugar units on glycopolymer surface is a critical parameter that determines the complement binding and activation. Above a certain density of sugars units, the surface-grafted glycopolymers showed an exponential increase in complement activation, and below this limit, there was no or minimal activation observed. We assume that in the case of our nanogels, the galactose density is low enough to

Figure 7. Nanogels and RBC interactions. Optical micrographs showing RBC aggregation in the presence of 1 mg/mL of glycopolymer nanogels. A cationic polymer, PEI, is used as a positive control. 2995

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For instance, interaction of various blood proteins such as thrombin, antithrombin, fibrinogen, etc. with nanogels is critical to elucidate the possible reasons behind the changes in blood coagulation in the presence of nanogels. Evaluation of protein corona on the surface of the nanogel is another important parameter that needs to be addressed. In addition, the structure of fibrin clot, clot architecture, and platelet binding and integration to the clot is important to elucidate observed TEG profiles. Clustering of charges on nanogel is an important characteristic that needs to be investigated. Future studies in our laboratories will address these issues.

CONCLUSIONS Nonionic and ionic, thermoresponsive colloidal materials bearing net positive or negative charge, either in the core or shell of nanogels, are synthesized by RAFT polymerization. The analysis of their physiochemical properties revealed the presence of thermoresponsive nanogels of ∼50−100 nm in diameter. The zeta potential data indicate that glycoshells can completely mask the charges introduced in the core of the nanogels; however, the presence of ionic moieties in glycoshells yields strong net charges to these nanomaterials. In general, the anionic glyconanogels maintain high blood compatibility at all studied concentrations. The cationic glyconanogels, where cationic content is introduced in the shell of glyconanogels, do exhibit slight increase in blood coagulation time, fibrinolysis, and platelet activation. The detailed screening of all nanogel samples revealed that anionic nanogels (NG3) are nonthrombogenic, hemocompatible materials, which can be used for further studies as drug delivery vector under in vivo conditions. These studies will also help the design of biocompatible nanogels for various biomedical applications. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00890.

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REFERENCES

(1) Kabanov, A. V.; Vinogradov, S. V. Nanogels as pharmaceutical carriers: Finite networks of infinite capabilities. Angew. Chem., Int. Ed. 2009, 48, 5418−5429. (2) Alvarez-Lorenzo, C.; Concheiro, A. Smart drug delivery systems: from fundamentals to the clinic. Chem. Commun. 2014, 50, 7743− 7765. (3) Ballauff, M.; Lu, Y. "Smart" nanoparticles: preparation, characterization and applications. Polymer 2007, 48, 1815−1823. (4) Bhuchar, N.; Sunasee, R.; Ishihara, K.; Thundat, T.; Narain, R. Degradable Thermoresponsive Nanogels for Protein Encapsulation and Controlled Release. Bioconjugate Chem. 2012, 23, 75−83. (5) Ahmed, M.; Narain, R. Intracellular Delivery of DNA and Enzyme in Active Form Using Degradable Carbohydrate-Based Nanogels. Mol. Pharmaceutics 2012, 9, 3160−3170. (6) Ahmed, M.; Jawanda, M.; Ishihara, K.; Narain, R. Impact of the Nature, Size and Chain Topologies of Carbohydrate-Phosphorylcholine Polymeric Gene Delivery Systems. Biomaterials 2012, 33, 7858− 7870. (7) He, Q.; Gong, K.; Ao, Q.; Ma, T.; Yan, Y.; Gong, Y.; Zhang, X. Positive charge of chitosan retards blood coagulation on chitosan films. J. Biomater. Appl. 2013, 27, 1032−1045. (8) Pereira, P.; Pedrosa, S. S.; Correia, A.; Lima, C. F.; Olmedo, M. P.; Gonzalez-Fernandez, A.; Vilanova, M.; Gama, F. M. Biocompatibility of a self-assembled glycol chitosan nanogel. Toxicol. In Vitro 2015, 29, 638−646. (9) Jones, C. F.; Campbell, R. A.; Brooks, A. E.; et al. Cationic PAMAM dendrimers aggressively initiate blood clot formation. ACS Nano 2012, 6, 9900−9910. (10) Travers, R. J.; Shenoi, R. A.; Kalathottukaren, M. T.; Kizhakkedathu, J. N.; Morrissey, J. H. Nontoxic polyphosphate inbibitors reduce thrombosis while sparring hemostasis. Blood 2014, 124, 3183−90. (11) Amiji, M.; Park, K. Surface modification of polymeric biomaterials with poly(ethylene oxide), albumin, and heparin for reduced thrombogenicity. J. Biomater. Sci., Polym. Ed. 1993, 4, 217− 234. (12) Ran, F.; Nie, S.; Li, J.; Su, B.; Sun, S.; Zhao, C. Heparin-like macromolecules for the modification of anticoagulant biomaterials. Macromol. Biosci. 2012, 12, 116−125. (13) Türk, H.; Haag, R.; Alban, S. Dendritic polyglycerol sulfates as new heparin analogues and potent inhibitors of the complement system. Bioconjugate Chem. 2004, 15, 162−167. (14) Smith, S. A.; Mutch, N. J.; Baskar, D.; Rohloff, P.; Docampo, R.; Morrissey, J. H. Polyphosphate modulates blood coagulation and fibrinolysis. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 903−8. (15) Naeye, B.; Deschout, H.; Roding, M.; Rudemo, M.; Delanghe, J.; Devreese, K.; Demeester, J.; Braeckmans, K.; De Smedt, S.; Raemdonck, K. Hemocompatibility of siRNA loaded dextran nanogels. Biomaterials 2011, 32, 9120−9127. (16) Chou, Y.-N.; Chang, Y.; Wen, T.-C. Applying Thermo-settable Zwitterionic Copolymers as General Fouling-resistant and Thermaltolerant Biomaterial Interfaces. ACS Appl. Mater. Interfaces 2015, 7, 10096−10107. (17) Benaglia, M.; Rizzardo, E.; Alberti, A.; Guerra, M. Searching for More Effective Agents and Conditions for the RAFT Polymerization of MMA: Influence of Dithioester Substituents, Solvent, and Temperature. Macromolecules 2005, 38, 3129−3140. (18) Ahmed, M.; Jiang, X.; Deng, Z.; Narain, R. Cationic GlycoFunctionalized Single-Walled Carbon Nanotubes as Efficient Gene Delivery Vehicles. Bioconjugate Chem. 2009, 20, 2017−2022. (19) Deng, Z.; Bouchekif, H.; Babooram, K.; Housni, A.; Choytun, N.; Narain, R. Facile synthesis of controlled-structure primary aminebased methacrylamide polymers via the reversible addition-fragmentation chain transfer process. J. Polym. Sci., Part A: Polym. Chem. 2008, 46, 4984−4996. (20) Deng, Z.; Ahmed, M.; Narain, R. Novel well-defined glycopolymers synthesized via the reversible addition fragmentation

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NMR data for the nanogels (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 780 492 1736. Fax: 780 492 2881. Author Contributions

Y.W. and M.A. made equal contributions to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funding from the Natural Sciences and Engineering Research Council of Canada (Discovery grant program) and Canada Foundation for Innovation (CFI). J.N.K. acknowledges funding from Canadian Institutes of Health Research. 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 CFI and the Michael Smith Foundation for Health Research (MSFHR). J.N.K. holds a Career Investigator Scholar award from the MSFHR. 2996

DOI: 10.1021/acs.biomac.5b00890 Biomacromolecules 2015, 16, 2990−2997

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Biomacromolecules chain transfer process in aqueous media. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 614−627. (21) Methacrylic acid [MAK Value Documentation, 2010]. The MAK Collection for Occupational Health and Safety; Wiley-VCH Verlag GmbH & Co.: Weinheim, Germany, 2012; pp 216−227. (22) Alidedeoglu, A. H.; York, A. W.; McCormick, C. L.; Morgan, S. E. Aqueous RAFT polymerization of 2-aminoethyl methacrylate to produce well-defined, primary amine functional homo- and copolymers. J. Polym. Sci., Part A: Polym. Chem. 2009, 47, 5405−5415. (23) Sunasee, R.; Wattanaarsakit, P.; Ahmed, M.; Lollmahomed, F. B.; Narain, R. Biodegradable and Nontoxic Nanogels as Nonviral Gene Delivery Systems. Bioconjugate Chem. 2012, 23, 1925−1933. (24) Li, L. L.; Cheng, C.; Xiang, T.; Tang, M.; Zhao, W. F.; Sun, S. D.; Zhao, C. S. Modification of polyethersulfone hemodialysis membrane by blending citric acid grafted polyurethane and its anticoagulant activity. J. Membr. Sci. 2012, 405−406, 261−274. (25) Huang, Y.; Shaw, M. A.; Mullins, E. S.; Kirley, T. L.; Ayres, N. Synthesis and anticoagulant activity of polyureas containing sulfated carbohydrates. Biomacromolecules 2014, 15 (12), 4455−4466. (26) Xia, Y.; Cheng, C.; Wang, R.; Qin, H.; Zhang, Y.; Ma, L.; Tan, H.; Gu, Z.; Zhao, C. Surface-engineered nanogel assemblies with integrated blood compatibility, cell proliferation and antibacterial property: towards multifunctional biomedical membranes. Polym. Chem. 2014, 5 (20), 5906−5919. (27) Shenoi, R. J.; Kalathottukaren, M. T.; Travers, R. J.; Lai, B. F.; et al. Affinity-based design of a synthetic universal reversal agent for heparin anticoagulants. Sci. Transl. Med. 2014, 6, 260ra150. (28) Lai, B. F. L.; Zou, Y.; Yang, X.; Yu, X.; Kizhakkedathu, J. N. Abnormal blood clot formation induced by temperature responsive polymers by altered fibrin polymerization and platelet binding. Biomaterials 2014, 35, 2518−2528. (29) Rodrigues, S. N.; Goncalves, I. C.; Martins, M. C. L.; Barbosa, M. A.; Ratner, B. D. Fibrinogen adsorption, platelet adhesion and activation on mixed hydroxyl-/methyl-terminated self-assembled monolayers. Biomaterials 2006, 27, 5357−5367. (30) Yu, K.; Lai, B. F.; Foley, J. H.; Krisinger, M. J.; Conway, E. M.; Kizhakkedathu, J. N. Modulation of Complement Activation and Amplification on Nanoparticle Surfaces by Glycopolymer Conformation and Chemistry. ACS Nano 2014, 8, 7687−7703. (31) Moghimi, S.; Andersen, A. J.; Ahmadvand, D.; Wibroe, P. P.; Andresen, T. L.; Hunter, A. C. Material properties in complement activation. Adv. Drug Delivery Rev. 2011, 63, 1000−1007. (32) Hamad, I.; Al-Hanbali, O.; Hunter, C.; Rutt, K. J.; Andresen, T. L.; Moghimi, S. M. Distinct Polymer Architecture Mediates Switching of Complement Activation Pathways at the Nanosphere−Serum Interface: Implications for Stealth Nanoparticle Engineering. ACS Nano 2010, 4, 6629−6638. (33) Lemarchand, C.; Gref, R.; Passirani, C.; Garcion, E.; Petri, B.; Muller, R.; Costantini, D.; Couvreur, P. Influence of polysaccharide coating on the interactions of nanoparticles with biological systems. Biomaterials 2006, 27, 108−118. (34) Reddy, S. T.; Van der Vlies, A. J.; Simeoni, E.; Angeli, V.; Randolph, G. J.; O'Neil, C. P.; Lee, L. K.; Swartz, M. A.; Hubbell, J. A. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat. Biotechnol. 2007, 25, 1159−1164.

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DOI: 10.1021/acs.biomac.5b00890 Biomacromolecules 2015, 16, 2990−2997