Effect of PEGâPDMAEMA Block Copolymer Architecture on

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Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin Salla Välimäki, Alexey Khakalo, Ari Ora, Leena-Sisko Johansson, Orlando J. Rojas, and Mauri A. Kostiainen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00699 • Publication Date (Web): 30 Jul 2016 Downloaded from http://pubs.acs.org on July 31, 2016

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Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin Salla Välimäkiǂ, Alexey Khakaloɸ, Ari Oraǂ, Leena-Sisko Johanssonɸ, Orlando J. Rojasɸ, and Mauri A. Kostiainenǂ,*

ǂ

Biohybrid Materials, Department of Biotechnology and Chemical Technology, Aalto University,

FI-00076 Aalto, Finland

ɸ

Biobased Colloids and Materials, Department of Forest Products Technology, Aalto University,

P.O. FI-00076 Aalto, Finland

ABSTRACT Heparin is a naturally occurring polyelectrolyte consisting of a sulfated polysaccharide backbone. It is widely used as an anticoagulant during major surgical operations. However, the associated bleeding risks require rapid neutralization after the operation. The only clinically approved antidote for heparin is protamine sulfate, which is however ineffective against low molecular weight heparin and can cause severe adverse reactions in patients. In this study, the facile synthesis of cationic-neutral diblock copolymers and their effective heparin binding is

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presented. Poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PDMAEMA) block copolymers were synthetized in two steps via atom-transfer radical polymerization (ATRP) using PEG as a macroinitiator. Solution state binding between heparin and a range of PEG-PDMAEMA block copolymers and one homopolymer was studied with dynamic light scattering and methylene blue displacement assay. Also in vitro binding in plasma was studied by utilizing a chromogenic heparin anti-Xa assay. Additionally, quartz crystal microbalance and multi-parametric surface plasmon resonance were used to study the surface adsorption kinetics of the polymers on a heparin layer. It was shown that the block copolymers and heparin form electrostatically bound complexes with varying colloidal properties, where the block lengths play a key role in controlling the heparin binding affinity, polyelectrolyte complex size and surface charge. With the optimized polymers (PEG114PDMAEMA52 and PEG114PDMAEMA100), heparin could be neutralized in dose-dependent manner, and bound efficiently into small neutral complexes, with a hydrodynamic radius less than 100 nm. These complexes did not have an effect on cell viability. Based on these studies, our approach paves the way for the development of new polymeric heparin binding agents.

KEYWORDS: heparin, block copolymer, polyelectrolyte complex, electrostatic binding

INTRODUCTION Heparin is a highly negatively charged polyanion composed mainly of uronic acid and glucosamine subunits (Scheme 1b).1 It is an effective blood anticoagulant as it interacts with the thrombin inhibitor antithrombin-III and therefore disrupts the blood clotting cascade.2 Anticoagulant activity of heparin has been widely used for clinical purposes, for example in surgeries. However, after the operation anticoagulant effect of heparin needs to be neutralized to


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regain the pristine clotting properties of blood. This is in most cases accomplished with a small cationic protein, protamine sulfate, which binds heparin through electrostatic interactions.3 Unfortunately, protamine is not effective in neutralization of low molecular weight heparin4 and may cause adverse effects5–7, such as hypotension (low blood pressure) and anaphylaxis (a serious allergic reaction) through uncontrolled activation of the complement system.

There has recently been significant interest in developing cationic small molecule and macromolecule alternatives for protamine. Among first substances, methylene blue was studied as a replacement of protamine, but it did not show effective binding in biological media.8 Also other cationic small molecules, such as Delparantag9 and calix[8]arene10, have been investigated. Interestingly, dendritic polymers, for example polyamidoamine (PAMAM) dendrimers, have shown generation-dependent binding11–13 and the studies indicate that high positive charge as such does not give the optimal results12, but instead also the charge ratio should be taken into account to avoid high toxicity. In addition to these alternatives, highly efficient self-assembling multivalent binders have been presented.14,15 This system is based on small amphiphiles that form micellar structures to display multivalent ligand arrays on their surface, but can also disassemble through hydrolysis. Furthermore, macromolecules, including polybrene16, dextran derivatives17,18 and a variety of block copolymers 19–21, have been used for neutralization of anticoagulant activity of heparin.

However, many of the early stage studies rely on heparin neutralization with plain cationic compounds, which can bind heparin efficiently, yet they often form large polyelectrolyte complexes with poor colloidal stability. Furthermore, the complexes are not sterically stabilized with biologically inert moieties and may still be recognized by the immune system. Therefore,


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cationic moieties has recently been combined with poly(ethylene glycol) (PEG) or similar compounds to enhance colloidal properties and biocompatibility.19–21 Here, we report a series of designed diblock copolymers consisting of a poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) based cationic block and a biologically inert PEG block. The polymers presented here can be synthesized from commercially available starting materials (Scheme 1a) in two facile steps. Atom transfer radical polymerization (ATRP) technique was used in the polymerization to gain polymers with controlled and narrow dispersity of molar mass. The heparin-binding PDMAEMA block is a water-soluble polycation, where the pKa of the tertiary amine groups is approximately 7.5, indicating that they are protonated in physiological conditions.22,23 For this reason, PDMAEMA has been one of the most interesting polymers for pharmaceutical and medical applications, such as gene transfection and drug delivery.24–26 It has also been shown to be less cytotoxic than other polycations, such as polyethylenimine (PEI).27 As mentioned, the biocompatibility of cationic macromolecules can be further enhanced by pegylation.28,29 For example, coating DNA-PDMAEMA polyplexes with PEG has been shown to improve the pharmacokinetic properties by reducing their aggregation with blood components and extending the blood circulation time.30,31 Additionally, it has been shown that pegylated polymer-heparin complexes do not impair the proliferation of cells.19 Moreover, complex size can be reduced by adding a PEG block to the cationic polymer.19,20 As protamine associated toxicity is suggested to be related to the large size of the heparin-protamine complexes,32 pegylation gives the possibility to optimize the complex size and consequently the toxicity. Therefore, in this work PDMAEMA was block copolymerized with PEG in order to increase the biocompatibility, control the complex size and cover the heparin complexes with a biologically inert and colloidally stable corona.


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Scheme 1. (a) Synthesis of PEG-PDMAEMA block copolymers. Block copolymers with varying block lengths were synthetized, along with one PDMAEMA homopolymer. (b) Simplified heparin structure. (c) Schematic presentation of complexation between heparin and PEGPDMAEMA block copolymer.

EXPERIMENTAL SECTION

Materials.

All reagents were purchased from Sigma-Aldrich and all except for DMAEMA were used as received. DMAEMA monomer was purified by passing through alumina column to remove inhibitor. PEGs were purchased from Fluka and dried in oven prior to use. All organic solvents were purchased from VWR. Heparin solutions were filtered through 0.2 µm GHP membrane syringe filter before usage.


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Methods.

Nuclear magnetic resonance (NMR). 1H spectra were recorded with a Bruker Avance 400 instrument using deuterated chloroform CDCl3 as the solvent. Residual solvent peak was used as an internal standard for peak calibration.

Gel permeation chromatography (GPC). GPC was used to determine the molar mass (Mn, Mw) and dispersity (Mw/Mn) of the polymers. All measurements were performed with a setup consisting of a Waters 515 HPLC-pump, Waters Styragel-columns (columns HR6, HR4 and HR2, effective molecular weight range of the set 500-10 000000 Da) and Waters 2410 refractive index –detector. The measurements were run in DMF containing 1 % of LiBr at 30 °C. Measurement was calibrated with poly(methyl methacrylate) standards with molar mass between 2 200 and 903 000 Da.

Attenuated total reflectance infrared spectroscopy (ATR-FTIR). The infrared spectra were recorded with a Unicam Mattson 3000 FTIR spectrometer equipped with PIKE Technologies GladiATR. All spectra were obtained at the range from 400 to 4000 cm-1 with a total of 16 scans and resolution of 32 cm-1.

Methylene blue displacement assay (MB displacement assay). A solution of heparin and methylene blue complex was mixed with polymer solutions of different concentrations and absorption was measured with a Biotek Cytation 3 microplate reader. Heparin-MB solution was prepared by mixing the heparin (0.5 mg/ml) and methylene blue (0.064 mg/ml) solutions in 1:4 volume ratio. Polymer solutions were prepared by diluting 10 mg/ml stock solution of the polymer to concentrations of 2.5, 2.0, 1.5, 1.0, 0.75, 0.5, 0.4, 0.3, 0.2, 0.1 and 0.05 mg/ml.


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Samples were prepared by mixing 100 µl of heparin-MB solution with 20 µl of polymer solution. This gave MB concentration of 0.043 mg/ml, heparin concentration of 0.08 mg/ml and polymer concentration ranging between 0.42 and 0.01 mg/ml. All samples were prepared in 50 mM TrisHCl buffer at pH 7.3. Absorption spectra between 400 and 750 nm were measured from 96 well plate without lid at room temperature. Ratio of absorbance intensity at 664 nm and 568 nm (λ(664)/λ(568)) was used to determine the amount of polymer needed for full displacement of methylene blue i.e. complexation between heparin and polymer. Measurements were repeated three times and reported as average values.

Heparin anti-Xa assay. Heparin neutralization with the polymers and protamine was examined by using a commercially available two stage kit, Biophen Heparin Anti-Xa (221010). Samples were prepared by adding heparin in 150 mM saline to human plasma to which polymers in 150 mM saline in different concentration were added. Samples were diluted and other reagents were reconstructed according to the manufacturer’s instructions. Final concentration of heparin was 0.075 IU/ml and mass ratio of polymer-to-heparin ranged between 0 and 2. To run the calorimetric assay, 40 µl of the samples was added to wells of 96-well microplate followed by addition of 40 µl antithrombin and 2 minutes incubation. This was followed by addition of factor Xa (40 µl), 2 minutes incubation, addition of the chromogenic substrate (40 µl) and 2 minutes incubation. Reaction was stopped with 80 µl of 2 % citric acid and absorbance at 405 nm was measured with Biotek Cytation 3 microplate reader. Anticoagulant activity is inversely proportional to the absorption intensity and percentage of neutralization was determined with help of calibration curve of heparin performed according to the manufacturer’s instructions (concentration 0-0.1 IU/ml).


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Dynamic light scattering (DLS). The hydrodynamic diameter and ζ-potential were measured using Zetasizer Nano ZS90 device (Malvern Instruments) with a 4 mW He-Ne ion laser at the wavelength of 633 nm and an Avalanche photodiode detector at an angle of 90°. Zetasizer software (Malvern Instruments) was used to obtain the data. Cumulant analysis was used to give the intensity mean value of size i.e. hydrodynamic diameter and Smoluchowski model was used to calculate the ζ-potential. Experiments were carried out at 25 °C.

Samples for size measurements were prepared by diluting 10 mg/ml heparin solution to concentration of 0.1 mg/ml and volume of 0.8 ml. Heparin solutions were titrated with 2 µl of 4 or 8 mg/ml polymer or protamine solution yielding concentrations ranging between 0.02 mg/ml and 0.2 mg/ml. Measurements were carried out in 50 mM Tris-HCl buffer at pH 7.3 with 0 mM and 150 mM NaCl. After every addition, the samples were allowed to equilibrate for 2 minutes. Each titration was carried out three times and all titration points were measured two times. Results present the average of these measurements.

ζ-potential measurements were carried out by mixing solutions of heparin (5 mg/ml) and polymer (5 mg/ml) into 10 mM Tris-HCl buffer at pH 7.3. In all samples, heparin concentration was 0.1 mg/ml, polymer/protamine concentration 0.05, 0.1, 0.15, 0.2 or 0.3 mg/ml and final sample volume 0.8 ml. After adding heparin and the polymer to buffer media samples were allowed to equilibrate for 2 minutes. Samples were measured in PMMA macro cuvettes using a ZEN1002 dip cell. Each measurement was carried out three times and average values are presented.


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Quartz crystal microbalance with dissipation monitoring (QCM-D). Interactions between surface immobilized heparin and polymers were investigated by using gold-coated sensors and a QCMD unit (E4 instrument, Q-Sense AB, Sweden). The sensors were first cleaned with UV/ozone treatment for 15 min followed by PEI adsorption from 0.1 wt-% solution for 15 min. After thorough rinsing with MilliQ water all crystals were dried with nitrogen gas. Heparin modification of PEI-coated sensors was performed in situ to establish irreversible binding and full surface coverage before polymer injection. After reaching stable baseline with heparin solution, rinsing with buffer solution was applied to remove loosely bound molecules (Figure S6). Finally, the polymer solutions were applied and the shifts in dissipation and frequency were monitored. All polymers were dissolved in 50 mM Tris-HCl buffer at pH 7.3 with or without 150 mM NaCl to yield 0.5 or 0.1 mg/ml polymer concentration. All solutions were filtered using 0.45 µm filters and degassed prior to use. Experiments were performed at a constant flow rate of 100 µl/min and the temperature was maintained at 23 °C. Voigt protocol (Q-Tools software, version 2.1, Q-Sense, Västra Frölunda, Sweden) was used to estimate the adsorbed thickness and mass of polymer layer on the heparin surface by assuming a density of 1200 kg/m3 for the adsorbed layer.

Multi-parametric surface plasmon resonance (MP-SPR). MP-SPR sensor chip preparation and heparin coating of the chips were performed as described for QCM-D sensors. Polymer adsorption onto heparin and properties of adsorbed layers were investigated by using multiparametric surface plasmon resonance device (MP-SPR Model Navi 200, Oy BioNavis Ltd., Finland). Equation 1 was used to determine the thickness of the adsorbed layer:

∆

= ∙


(1)

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where ∆SPR angle is a change in the SPR angle, ld is a characteristic evanescent electromagnetic field decay length (240 nm), estimated as 0.37 of the light wavelength; m is a sensitivity factor for the sensor (109.94°/RIU) obtained after calibration of the MP-SPR, n0 is the refractive index of the bulk solution (1.333 RIU) and na is the refractive index of the adsorbed layer (assumed value of 1.513 RIU measured for PDMAEMA33). The surface excess concentration was calculated according to equation 2:

= ×

(2)

where d is the calculated thickness of the adsorbed layer and ν is the specific density of the layer (1200 kg/m3).

X-ray photoelectron spectroscopy (XPS). The surface chemical composition of dried heparinpolymer films on QCM-D crystals was examined with an AXIS Ultra photoelectron spectrometer by Kratos Analytical. An X-ray source of monochromatic Al Kα irradiation at 100 W under neutralization was used. High resolution spectra were recorded along with elemental wide-region data. The analysis area was 400 × 800 µm2 and XPS spectra were recorded on 2-4 locations for each sample. Gold, gold-PEI and gold-PEI-heparin films were used as references. XPS data was obtained for crystals coated with 0.5 mg/ml polymer solutions.

Atomic force microscopy (AFM). Surface topography of the dried heparin-polymer films on QCM-D crystals was analyzed with a Nanoscope IIIa multimode scanning probe microscope (Digital Instruments, Santa Barbara, CA). Gold-PEI and gold-PEI-heparin films were used as references. Data was obtained for crystals coated with both 0.5 and 0.1 mg/ml polymer solutions.


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Surface areas of 3 × 3 and 1 × 1 µm2 were investigated in air using tapping mode with silicon cantilevers. Images were processed (flattening) and analyzed with NanoScope Analysis software.

Cell culture and MTT assay. Adenocarcinomic human alveolar basal epithelial cells (A549) were obtained from the American Type Culture Collection. The cells were maintained at 37 °C in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The culture was maintained at 37 °C under an atmosphere of 5% CO2 and 95% air. Cells were splitted in 1:5 ratio and seeded into 24-well culture plates (approximately 40 cells/well) 48 hours before the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Before adding sample solutions, the medium was replaced with new DMEM (200 µl). Thereafter, 20 µl of sterile filtered sample solution containing polymer or protamine (0.1-100 µM) or heparin-polymer/protamine complex (1 µM heparin, 1:1 mass ratio) was added to the cell culture. The samples were incubated for 24, 48 and 72 hours. After this, 20 µl MTT solution (5.0 mg/ml) was added into each well. The plates were incubated at 37 °C in 5% CO2 and 95% air for four hours in the dark. Finally, the cell culture media was removed and 150 µl DMSO was added to the cells. After adding DMSO, the plates were stirred and the solubilization of formazan crystals was confirmed before reading. The absorbance was measured with a microplate reader (Cytation 3, Biotek) at wavelength of 540 nm. All samples were duplicates and average values are presented.

RESULTS AND DISCUSSION

A series of four block copolymers comprising PDMAEMA and PEG, along with a PDMAEMA homopolymer, were synthetized by using ATRP (Scheme 1, see the supporting information for


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full experimental details). The polymers with varying block sizes were analyzed by 1H NMR and GPC, and detailed information of molar mass and dispersity (Mw/Mn) is given in Table 1. Molar mass of the homopolymer was calculated based on conversion calculated from the disappearance of double bond signals at 6.1 ppm and appearance of methyl proton NMR signal around 1 ppm. For the block copolymers, both theoretic molar mass (Mnth) based on the conversion and molar mass based on end group analysis (MnNMR) was determined, and the values are in good agreement. Polymers are named based on the number of repeating units (Table 1). Molar mass distributions determined with GPC were low. Also, ATR-FTIR spectra were recorded (Figure S4).

Table 1. Summary of polymers synthetized

weight fraction

a

degree of polymerization b

c

sample code

PEG

PDMAEMA

PEG

PDMAEMA71

0

100

-

-

PEG25PDMAEMA87

7

93

25

PEG114PDMAEMA19

63

37

PEG114PDMAEMA52

38

PEG114PDMAEMA100

24

a

Conv. (%)

PDMAEMA PDMAEMA

Mnth

MnNMR d

e

MnGPC f

f

(Mw/Mn)

(equiv.)

(Da)

(Da)

71.8

11 100

-

6000

1.12

87

72.1

12 400

15 000

8700

1.15

114

19

25.9

9 100

8 000

12700

1.08

62

114

52

57.4

14 000

13 000

16500

1.11

76

114

100

79.7

17 500

21 000

20200

1.19

Weight fractions for block copolymers calculated based on degree of polymerization and mass

of repeating unit. b Degree of polymerization for PEG calculated based on molar mass provided by the manufacturer. c Degree of polymerization for PDMAEMA block calculated based on endgroup analysis by using 1H NMR d Theoretical molar mass equivalent to PMMA standards:


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M(PEG) + ([M]/[init] × conv. × M(M)) e M(PEG) + DP × M(M) f Determined based on the GPC measurements

Polymer binding efficiency towards heparin was studied with methylene blue displacement assay, and the results indicate that the efficiency is mainly dependent on the length of the cationic PDMAEMA block (Figure 1). The main conclusion is that smaller amounts of polymers with long PDMAEMA blocks is needed to fully release methylene blue i.e. bind all heparin. For example, compared with PEG114PDMAEMA52, twice the amount of PEG114PDMAEMA19 is needed to release all of the heparin-bound methylene blue. However, when the length of cationic block is further increased no noteworthy difference in the efficiency is observed, as seen when PEG114PDMAEMA100 is compared to PEG114PDMAEMA52. On the other hand, length of the neutral block does not significantly affect binding efficiency as only a slightly smaller amount of PEG25PDMAEMA87 is needed for displacement of the methylene blue compared to PEG114PDMAEMA52 and PEG114PDMAEMA100. Additionally, homopolymer PDMAEMA71 behaves similarly as the block copolymers. Based on these results, PEG114PDMAEMA52 of our polymers was considered the optimal choice for heparin binding as it has relatively short cationic block and long neutral block, which is according to previous studies favorable for low activation of the complement system34, but simultaneously it binds with heparin as efficiently as polymers with longer cationic blocks.


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b)

a)

MB MB-hep MB-hep-pol

2

PDMAEMA71 PEG25PDMAEMA87 PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100

1

0 0

1 2 3 4 polymer : heparin mass ratio

Absorbance (a.u.)

3

λ(664)/λ(568)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

664

568

400

500 600 700 wavelength (nm)

Figure 1. Methylene blue (MB) binding assay results showing changes in absorbance when polymer concentration is increased in heparin-MB solution. (a) Increase in λ(664)/λ(568) ratio indicates the release of MB and binding between heparin and polymer. The data consists of the average values measured from three independent titrations. (b) UV/vis spectra examples of free MB, heparin bound MB, and heparin-polymer-MB mixture (released MB). Red marks indicate wavelengths used to monitor change between heparin and MB binding.

Also in vitro binding efficiency of polymers in plasma was studied by using a chromogenic antiXa assay and the results were compared to neutralization efficiency of protamine. In Figure 2, it can be observed that neutralization efficiency of the polymer is dependent on the polymer concentration and structure. For example, by increasing the polymer concentration, essentially higher percentage of neutralization is achieved. Additionally, as observed also in the MB displacement assay, longer cationic block provides more effective binding. This behavior is most pronounced when PEG114PDMAEMA100 and PEG114PDMAEMA19 are compared. Moreover, including a neutral block in to the cationic polymer does not lower the binding efficiency as can be seen when comparing the binding affinity of PEG114PDMAEMA100 and PDMAEMA71. Based on these measurements long cationic block is favorable in heparin binding and neural block does not affect the effectiveness. The assay shows that the polymers are able to neutralize heparin in


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dose-dependent manner, however, protamine is more effective, which indicates that further optimization of the polymer structures and neutralization assay is required.20

PDMAEMA71 PEG25PDMAEMA87 PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100 protamine

100

%

80

Neutralization

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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60

40

20

0 0

0.5

1

1.5

2

polymer : heparin mass ratio

Figure 2. Neutralization of heparin (0.75 IU/ml) by protamine and the synthetized polymers measured in triplicates with a heparin anti-Xa assay. Electrophoretic mobility of the polymer-heparin complexes was measured in order to study how the ζ-potential of the complexes is dependents on block lengths (Figure 3) and allow tuning of the final surface charge of the complexes. In general, when the cationic block is short, ζ-potential increases only slightly even at high polymer-to-heparin mass ratios where complex formation was observed with the MB displacement assay. When length of the cationic block is increased, complex surface charges attain neutral values when the mass ratio of PEG114PDMAEMA52 or PEG114PDMAEMA100 and heparin reaches 1.5 which also corresponds to full complexation according to the MB displacement assay. Electroneutral values are retained with further polymer addition. Complexes with a clearly positive surface potential (∼10 mV) are achieved only for


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protamine and for polymers when the neutral PEG block is short or absent. For both PEG25PDMAEMA87 and PDMAEMA homopolymer, ζ-potential reaches positive values with mass ratios below 1.5 (Figure 3). These results indicate that a long enough PEG chain is needed for effective shielding and obtaining complexes with a neutral surface charge. Similar results have been observed for other related systems.35

Considering the ζ-potential measurements and the polymers presented here, PEG114PDMAEMA52 performs most optimally since the ζ-potential of its complex with heparin is close to neutral at concentrations where full binding is observed with the MB displacement assay. PEG114PDMAEMA100 has the same characteristics but as concluded from MB displacement assay results, increasing length of the cationic block does not give additional benefit to the complexation. In general, it is important to have neutral particles preferably with stabilizing hydrophilic outer layer to prevent aggregation with blood components.30,36

20 10

ζ-potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0 -10 -20 PDMAEMA71 PEG25PDMAEMA87 PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100 protamine

-30 -40 -50 0.5

1

1.5

2

2.5

3


Figure 3. Change in ζ-potential upon complexation between heparin and different polymers and protamine in varying mass ratios. The experiment was conducted in triplicates and average values are presented.


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Complex size as a function of polymer concentration was measured with dynamic light scattering, and the results indicate that the complex size may be varied mainly by changing the length of the neutral block as well as polymer concentration. Figure 4 shows distinctive changes in hydrodynamic diameter of the complexes when polymer solution is added to heparin in physiologically relevant media (50 mM Tris-HCl pH 7.3, 150 mM NaCl). Firstly, without neutral block (PDMAEMA71), complex diameters are large reaching values near 2000 nm. For this polymer, complex size increases rather linearly after polymer:heparin mass ratio of 0.8:1. Around 1:1 mass ratio, the complexes phase separate and form a white precipitate. Similar precipitation is observed also when protamine is titrated into the heparin solution. As seen in Figure 4b, also the count rate increases clearly upon polymer and protamine addition, indicating complex formation. Decrease of count rate after a system specific point is commonly observed behavior of polyelectrolyte complexes.37

For block copolymers the phase behavior is different. For example, when PEG25PDMAEMA87 is titrated to heparin solution, complexes reach diameters around 600 nm and also scattering intensity increases notably. However, these complexes do not show visible aggregation. Macroscopic phase separation is most probably prevented due to the hydrophilic PEG block which makes the complexes soluble to water. These kind of complexes are also known as complex coacervate core micelles (CCCMs).37,38 With both the homopolymer and PEG25PDMAEMA87 complex size and standard deviation clearly increases when polymer is added to heparin solution. On the contrary, when length of the neutral block is increased, complexes become significantly smaller, even down to 40 nm in diameter, which is significantly smaller than size of heparin-protamine complexes with hydrodynamic diameter of 200 nm. In addition, the size of the polymer-heparin complexes remains constant even when an excess of


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polymer is added and the size range is in good agreement with similar previously studied systems.19 Additionally, small hydrodynamic diameter throughout the measurement indicates that only a few heparin and polymer chains form the complex and the shielding effect of PEG is likely to prevent formation of larger complexes. This kind of behavior is common for CCCMs.38 However, with a long neutral block and a short cationic block (PEG114PDMAEMA19), the complexes are slightly larger compared to PEG114PDMAEMA52 and PEG114PDMAEMA100, but after 1:1 mass ratio also these complexes reach stable values below 100 nm. The larger amount of PEG114PDMAEMA19 needed for complexation can be explained with the short cationic block which is not as efficient in heparin binding as the longer PDMAEMA polymers, and therefore more of the PEG114PDMAEMA19 polymer is needed to bind the heparin, leading also to slightly larger complexes. Similar need of higher amounts of PEG114PDMAEMA19 for full complexation is observed also in the MB displacement assay data.

With PEG114PDMAEMA19, PEG114PDMAEMA52 and PEG114PDMAEMA100 the count rates were low (Figure 4c) and therefore the values for hydrodynamic diameter should be considered as estimates. On the other hand, small but increasing count rate upon polymer titration can be considered as further proof of the increasing number of particles with fixed size, instead of size increase of existing particles.38 We note also that size and count rate development upon polymer titration was not affected by addition of 150 mM NaCl (Figure S5).

It has been suggested that protamine toxicity is related to the large size of heparin-protamine complexes and therefore size regulation of the complexes is highly important.32 Interestingly, in our system complex size can be changed by tuning block length of the copolymer. Additionally, to prevent aggregation and phase separation a neutral PEG block is also needed. PEG covering of


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polyelectrolyte complexes is also known to improve pharmacokinetic properties as it prevents aggregation with blood components.30,31

b)

1500

Derived count rate (kcps)

2000

c) 80 4000


1000 500 0

PDMAEMA71 PEG25PDMAEMA87 protamine

Derived count rate (kcps)

a) 2500

Hydrodynamic diameter (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3000

2000

1000

0.5

1

1.5

2

0


60

40

20

0

0 0

PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100

0.5

1

1.5

2


0

0.5

1

1.5

2


Figure 4. DLS data showing change in hydrodynamic diameter and count rate upon titration of heparin solution with given polymer, as indicated (50 mM Tris-HCl pH 7.3, 150 mM NaCl). The experiments were conducted in triplicates and average values are presented. (a) Larger complexes are formed for the PDMAEMA71 homopolymer and block copolymer with short PEG block. (b) For the PDMAEMA71 homopolymer, copolymers with short PEG block and protamine, count rates are higher. (c) Count rates below 100 kcps were recorded for block copolymer with longer PEG block

QCM-D was used to investigate binding of polymers on heparin surface under constant flow. The QCM-D is an acoustic instrument that simultaneously measures shifts in the resonance frequency (∆f) and energy dissipation (∆D) of an oscillating piezoelectric quartz crystal upon changes in the mass adsorbed on the crystal surface.39,40 Dissipation refers to the frictional losses that lead to damping of the oscillation frequency depending on the viscoelastic properties of the


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material. The frequency and dissipation changes were measured at a fundamental resonance frequency and its overtones.

As can be observed from the adsorption curves in Figure 5, each polymer behaves in a distinct manner. Firstly, based on the sharp frequency change (Figure 5a), all copolymers adsorb rapidly within minutes on the heparin surface. However, the initial binding is followed by a partial decrease in -∆f3 until a plateau is reached. Such signal reduction was most significant for PEG114PDMAEMA100. Similar behavior has been observed also for other multilayers composed of weak and strong polyelectrolyte.41–45 This behavior can be explained by the removal of polyelectrolyte complexes,41 desorption of ions,42 rearrangement of the layer43 or combination of these, and it can depend on polyelectrolyte molar mass,42 pH41–45 and electrolyte concentration44. Additionally, it must be considered that the heparin surface is not solid but it most likely changes conformation and exchanges water and ions upon polymer addition.

Based on the QCM sensograms the highest adsorption for block copolymers is observed with long neutral block and short cationic block (PEG114PDMAEMA19). After rinsing, the largest positive shift in frequency is observed for the copolymer with a short neutral block (PEG25PDMAEMA87). With this polymer, also dissipation (Figure 5b) decreases upon rinsing indicating that the layer becomes more rigid, and therefore most probably water is removed. This assumption is supported also by MP-SPR measurements (Figure 6) as no noteworthy desorption was observed upon rinsing. Similar behavior has been detected also previously in analogous systems and dissipation reduction was observed to be due to the substantial removal of water.46 For other block copolymers rinsing does not significantly affect ∆D, but upon polymer addition small positive shift in the dissipation was observed, which indicates that the layer becomes less


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rigid. This kind of behavior has been observed also before for PEG containing polymers47 with increasing positive ∆D upon increasing polymer concentration and PEG segment length.

The PDMAEMA71 homopolymer, on the other hand, displays a different binding characteristic. With PDMAEMA71 a fast adsorption is observed immediately after its addition, but instead of desorption a linear increase in -∆f3 is observed. Similar behavior is detected in the dissipation curve although the changes are relatively small. This distinct behavior of the homopolymer can most likely be explained by steric freedom and lack of PEG segments leading to continuing layer formation within timescale of the measurement.48 Further measurements are needed to fully understand the behavior.

Figure 5. Adsorption of polymers (0.1 mg/ml solutions in 50 mM Tris-HCl buffer pH 7.3, 150 mM NaCl) on heparin measured with QCM-D. Frequency (-∆f) and dissipation (∆D) shifts at the third overtone (15 MHz) are presented. (a) Shift in frequency, -∆f3 and (b) shift in dissipation, ∆D3. Polymer addition is indicated with P and rinsing with buffer is marked with R. (c)


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Schematic illustration of suggested ways of polymer adsorption on heparin layer, depending on polymer architecture.

Additionally, adsorbed polymer thickness and adsorbed mass (Table 2) were calculated based on Voight modeling49, and the results are in line with the sensogram data. As can be assumed based on the QCM sensograms, homopolymer adsorbs as a thick layer and therefore produces a large change in the interfacial mass. For the block copolymers, the highest calculated mass is observed with short cationic block and long neutral block, PEG114PDMAEMA19. In general, positively charged PDMAEMA adsorbs on the heparin layer and PEG blocks are extended into the buffer media as presented schematically in Figure 5c. Similar behavior has been observed also for other systems where diblock copolymer has been adsorbed on substrate covered with cationic polyelectrolyte.50 On the other hand, the adsorbed mass for PEG114PDMAEMA100 is significantly lower which could be due to possible partial desorption.

Table 2. Calculated thicknesses (h) and masses (∆m) based on QCM-D and MP-SPR data for polymer adsorption on heparin (sample concentration: 0.1 mg/ml).

sample code

hQCM-D hMP-SPR ∆mQCM-D ∆mMP-SPR 2 2 (nm) (nm) (mg/m ) (mg/m )

PDMAEMA71

2.68

0.91

3.21

1.10

PEG25PDMAEMA87

2.25

0.61

2.70

0.73

PEG114PDMAEMA19 3.04

0.68

3.65

0.82


0.57

3.08

0.68


0.42

1.41

0.51

MP-SPR was utilized to further investigate the interaction between polymers and heparin. This technique can give additional information to QCM-D since the MP-SPR signal is not affected by


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changes in water coupling. The adsorption of most PEG-PDMAEMA block copolymers (0.1 mg/ml in 50 mM Tris-HCl, 150 mM NaCl) was very fast and adsorption plateau was reached within few minutes as seen in Figure 6. Additionally, rinsing with buffer did not significantly alter the amount of surface-bound polymer. This behavior indicates electrostatic binding between heparin and the block copolymers.51 However, PEG114PDMAEMA100 made an exception as plateau was reached only after partial desorption. Also, compared to the block copolymers, the DMAEMA71 homopolymer performed differently since it produced a relatively linear increase in the ∆SPR angle with time. These results are in good agreement with the QCM-D measurements, where similar behavior was observed.

The block copolymer adsorption in MP-SPR measurements seems to mainly depend on the block lengths since greater block copolymer adsorbed mass is gained with short cationic blocks as seen also in the QCM-D sensograms. Additionally, layer masses and thicknesses for the polymer layers were calculated based on the MP-SPR data (Table 2). For all of the polymers, the results are similar as observed for QCM-D calculations. However, MP-SPR values for layer thicknesses and masses are lower than for QCM-D data which indicates that polymer layers are hydrated as coupled water is observed in QCM-D measurements but not in MP-SPR measurements.


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Figure 6. Adsorption of polymers on heparin layer measured with a MP-SPR. The timepoint where the polymer is added is marked with P and rinsing with buffer is indicated with R. Polymers were added as 0.1 mg/ml solutions in Tris-HCl buffer (pH 7.3, 150 mM NaCl).

QCM-D and MP-SPR measurements were also carried out with a five-fold higher polymer concentration (0.5 mg/ml). Results were similar although a greater desorption was observed for PEG114PDMAEMA100 as well as for PEG25PDMAEMA87 (Figure S7). The results of calculated thickness and adsorbed mass (Table S1) indicate that with higher polymer concentration polymers showing most pronounce desorption have also the lowest calculated thickness and mass. Therefore, based on the sensograms and calculated values we can conclude that layer formation is also dependent on the polymer concentration. To further investigate possible desorption, X-ray photoelectron spectroscopy was used to study chemical composition of the airdried polyelectrolyte layers. XPS atomic concentrations for the samples (Table S2) reveal that the gold solid support used was clearly exposed in the films containing PEG114PDMAEMA100 and the gold signal in this film was even higher than in the reference film of heparin. This


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indicates that both heparin and the polymer have been partially removed from the sensor surface. Also atomic force microscopy was used to study adsorption of the polymers on heparin. From the images, it can be seen that PEG114PDMAEMA100 has slightly lower RMS roughness (Table S3) which could indicate the removal of polyelectrolyte complexes, but the differences alone are too small to confirm this.

Overall, on the grounds of QCM-D and MP-SPR measurements, layer formation of the polymers on heparin seems to depend on three factors: the presence of uncharged PEG block, polymer concentration and relative block lengths. Firstly, the homopolymer adsorption continues as long as polymer is supplied (within the time scale of our measurements), whereas with block copolymers PEG segment seems to prevent further attachment after surface saturation. With PEG114PDMAEMA100, also desorption was observed, and based on the XPS measurements desorption seems to be due to removal of polyelectrolyte complexes from the surface. Similar dissolution with polymer solutions with high salt concentrations has been observed also before.52 Also, with these previously reported systems, flushing with buffer containing salt does not alone cause significant dissolution but instead a mixture of free polymer and salt results in desorption. In our system, also the polymer concentration affects the dissolution as with higher block copolymer concentrations desorption of layers is more pronounced upon polymer addition. Moreover, block lengths of the block copolymers have an effect on the polymer layer mass. With short cationic blocks adsorption is observed to be more effective.

The possible toxicity of the polymers and the polymer-heparin complexes was studied utilizing a colorimetric MTT dye assay by incubating adenocarcinomic human alveolar basal epithelial cells with the polymers and complexes for 24, 48 and 72 hours. Same experiments were performed


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also for protamine and heparin-protamine complexes to compare the toxicity profiles. From Figure 7a, where the cell viabilities after 24 hours of incubation with the polymers are presented, it can be observed that cells retained their viability, especially at low concentrations, whereas at high concentrations viability is decreased. Protamine and heparin show insignificant toxicity at all concentrations even though in previous heparin toxicity determinations heparin has showed concentration dependent toxicity.53 Similar trend in cell viability was observed also with 48 (Figure S8) and 72 (Figure 7b) hours of incubation. Complexes, with one-to-one mass ratio of polymer and heparin and 1 µM heparin concentration, did not show any significant decrease in the number of viable cells even with long incubation times as seen in Figure 7c. Especially complexes with block copolymers having long PEG block perform well.


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a) 140

Cell viability (%)

120

24 h

100

PDMAEMA71 PEG25PDMAEMA87 PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100 protamine heparin

80 60 40 20 0 only cells 0.01

b)

Cell viability (%)

0.1

1

10

100

Polymer concentration (µM) 140 120

72 h

100

PDMAEMA71 PEG25PDMAEMA87 PEG114PDMAEMA19 PEG114PDMAEMA52 PEG114PDMAEMA100 protamine heparin

80 60 40 20 0 only cells 0.01

0.1

1

10

100

Polymer concentration (µM)

c) 140 120

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100


80 60 40 20 0 only cells

24 h 24 h 48 h 72 h polymer complex complex complex

Figure 7. MTT assay results of the polymers and the polymer-heparin complexes compared with protamine and protamine-heparin complexes. The experiment was conducted two times and average values are presented. (a) Cell viability after incubating adenocarcinomic human alveolar basal epithelial cells with different concentrations of the polymers (0.01-100 µM) for 24 h. (b) Viability after incubating cells with different concentrations of the polymers (0.01-100 µM) for 72 h. (c) Cell viability after 24, 48 and 72 h incubation with complex solution containing 1:1


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mass ratio of heparin and polymer with 1 µM heparin concentration compared to incubation with 1 µM polymer solution for 24 h.

CONCLUSIONS

Here, we have shown that electrostatic complexation between heparin and polymers can be varied by changing molecular architecture of the PDMAEMA polymers. By adding a neutral water soluble PEG block to the cationic PDMAEMA polymer, smaller, water soluble and electroneutral complexes are obtained. On the other hand, too short cationic block does not guarantee sufficient heparin binding with neutral surface charge of the complexes, and therefore length of the cationic block has been optimized to be as short as possible to avoid excess toxicity, but long enough to effectively bind heparin. Moreover, efficient binding was demonstrated in biologically relevant media (pH 7.3, 150 mM NaCl) and dose-dependent neutralization in plasma.

From the series of polymers studied here, PEG114PDMAEMA52 combines the advantageous properties and is identified as the optimal polymer for heparin binding. Compared to PEG25PDMAEMA19, a smaller amount of PEG114PDMAEMA52 is needed for complete binding, resulting also in smaller complexes as seen in the MB displacement assay and DLS measurements. On the other hand, PEG114PDMAEMA100 has a longer cationic block but similar characteristics in complexation, indicating that increasing the length of the cationic block does not yield additional beneficial properties, other than slightly more efficient neutralization in antiXa assay. Moreover, ζ-potential of heparin complexes with PEG114PDMAEMA52 is promising as


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it reaches electroneutral values around mass ratios that correspond complete heparin binding according to the methylene blue displacement assay. Adsorption studies of PEG114PDMAEMA52 on heparin layer indicate efficient electrostatic binding of surface bound heparin. Moreover, complexes of PEG114PDMAEMA52 with heparin in one-to-one mass ratio show negligible toxicity in the MTT assay. Binding was also studied in vitro in plasma and the results give positive signs of heparin neutralization. However, additional in vitro and in vivo studies as well as optimization of the polymer structures to achieve same neutralization levels as with protamine are needed.

Most of the previous work has introduced heparin binders consisting only from cationic moieties10,15,16 which are often considered toxic24,25. PEG is well-known to enhance the polyelectrolyte complex properties of cationic moieties19–21, and therefore also in our study we have utilized this approach to improve colloidal properties and reduce the toxicity. Additionally, we have thoroughly characterized the system to find optimal polymer for heparin binding in physiologically relevant media. Overall, our results indicate the presented polymers are potential candidates for further development as an alternative for protamine.

ASSOCIATED CONTENT Supporting Information. Synthesis of the polymers; NMR spectra of the polymers; ATR-FTIR spectra of the polymers; DLS count rates for samples containing 150 mM NaCl; DLS data for measurements without NaCl; in situ heparin coating in QCM-D and MP-SPR; QCM-D, MP-SPR


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data for 0.5 mg/ml polymer concentrations; MTT assay for 48 hours of incubation; XPS atomic concentrations; AFM RMS roughnesses.

AUTHOR INFORMATION Corresponding Author * Correspondence should be addressed to Mauri A. Kostiainen ([email protected]).

ACKNOWLEDGMENT Financial support from Academy of Finland (projects 263504, 267497, 273645, 286845), Biocentrum Helsinki and Emil Aaltonen Foundation is gratefully acknowledged. This work was carried out under the Academy of Finland’s Centers of Excellence Programme (2014–2019) and made use of Aalto University Bioeconomy Facilities. Authors wish to thank Seija Lemettinen from laboratory of Polymer Chemistry at University of Helsinki for GPC measurements, Dr Joseph M. Campbell and Ritva Kivelä from Department of Forest Products Technology at Aalto University for XPS measurements and AFM imaging, respectively.

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Biomacromolecules

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(46) Vuoriluoto, M.; Orelma, H.; Johansson, L.-S.; Zhu, B.; Poutanen, M.; Walther, A.; Laine, J.; Rojas, O. J. J. Phys. Chem. B 2015, 119, 15275–15286. (47) Teramura, Y.; Kuroyama, K.; Takai, M. Acta Biomater. 2016, 30, 135–143. (48) Kenausis, G. L.; Vörös, J.; Elbert, D. L.; Huang, N.; Hofer, R.; Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104, 3298–3309. (49) Voinova, M. V.; Rodahl, M.; Jonson, M.; Kasemo, B. Phys. Scr. 1999, 59, 391. (50) de Vos, W. M.; Kleijn, J. M.; de Keizer, A.; Cohen Stuart, M. A. Angew. Chem. Int. Ed. 2009, 48, 5369–5371. (51) Hoogeveen, N. G.; Stuart, M. A. C.; Fleer, G. J. J. Colloid Interface Sci. 1996, 182, 146– 157. (52) Kovacevic, D.; van der Burgh, S.; de Keizer, A.; Cohen Stuart, M. A. Langmuir 2002, 18, 5607–5612. (53) Gurbuz, H. A.; Durukan, A. B.; Sevim, H.; Ergin, E.; Gurpinar, A.; Yorgancioglu, C. Blood Coagul. Fibrinolysis 2013, 24, 742–745.

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