and triblock polymers

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Anticoagulant properties of poly(sodium 2-(acrylamido)-2methylpropanesulfonate)-based di- and triblock polymers Bartlomiej Kalaska, Kamil Kami#ski, Joanna Miklosz, Keita Nakai, Shin-ichi Yusa, Dariusz Pawlak, Maria Nowakowska, Andrzej Mogielnicki, and Krzysztof Szczubia#ka Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00691 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Anticoagulant properties of poly(sodium 2(acrylamido)-2-methylpropanesulfonate)-based diand triblock polymers Bartlomiej Kalaska1, Kamil Kamiński2, Joanna Miklosz1, Keita Nakai3, Shin-Ichi Yusa3, Dariusz Pawlak1, Maria Nowakowska2, Andrzej Mogielnicki1*, Krzysztof Szczubiałka2*

1

Department of Pharmacodynamics, Medical University of Bialystok, Mickiewicza 2c,

15-089 Bialystok, Poland 2

Faculty of Chemistry, Jagiellonian University, Gronostajowa 2, 30-387 Kraków, Poland

3

Department of Applied Chemistry, Graduate School of Engineering, University of Hyogo,

Himeji, Hyogo, Japan

Keywords Anticoagulant, block copolymer, PAMPS, PEG, PMPC, antiplatelet

1 ACS Paragon Plus Environment

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Abstract

Di- and triblock copolymers with low dispersity of molecular weight were synthesized using radical addition-fragmentation chain transfer polymerization. The copolymers contained anionic poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS) block as an anticoagulant component. The block added to lower the toxicity was either poly(ethylene glycol) (PEG) or poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC). The polymers prolonged clotting times both in vitro and in vivo. The influence of the polymer architecture and composition on the efficacy of anticoagulation and safety parameters was evaluated. The polymer with the optimal safety/efficacy profile was PEG47-b-PAMPS108, i.e., a block copolymer with the degree of polymerization of PEG and PAMPS blocks equal to 47 and 108, respectively. The anticoagulant action of copolymers is probably mediated by antithrombin, but it differs from that of unfractionated heparin. PEG47-b-PAMPS108 also inhibited platelet aggregation in vitro and increased the prostacyclin production, but had no antiplatelet properties in vivo. PEG47-b-PAMPS108 anticoagulant activity can be efficiently reversed with a copolymer of PEG and poly(3-(methacryloylamino)propyl trimethylammonium chloride) (PMAPTAC) (PEG41-b-PMAPTAC53, HBC), which may be attributed to the formation of polyelectrolyte complexes with PEG shells without anticoagulant properties.

Introduction As a beneficial consequence of the progress in medical sciences, the 20th century witnessed an unprecedented expansion of human life expectancy. This was accompanied with the shift in morbidity and mortality from infectious to noninfectious diseases1. Among the latter, the pathologies, whose common underlying mechanism is thrombosis, are nowadays the leading cause of deaths worldwide. The family of thromboembolic diseases include deep vein thrombosis and pulmonary embolism (collectively known as venous thromboembolism), 2 ACS Paragon Plus Environment

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myocardial infarction, acute coronary syndrome, ischemic stroke, to mention only the most common. Just two of them, ischemic heart disease and ischemic stroke, account for one in four deaths worldwide2. Therefore, antithrombotic therapy is one of the most frequently applied forms of medical treatment and, consequently, antithrombotic drugs, i.e., antiplatelet drugs, anticoagulants and fibrinolytic drugs, belong to the most important pharmaceuticals nowadays3. Antiplatelet drugs prevent the aggregation of platelets and formation of platelet clots4, while anticoagulants are the drugs which prevent formation of fibrin from fibrinogen and formation of fibrin5 clots during primary and secondary hemostasis, respectively. Among the latter, unfractionated heparin (UFH) is the oldest known anticoagulant (in continuous use for 70 years) and, together with other heparinoids and Vitamin K antagonists (VKAs), it is still the leading drug in the treatment of thromboembolic disorders. Most of the anticoagulants available on the market may cause severe adverse effects including hemorrhage, anemia, insomnia, hypotension, osteoporosis, fetal abnormalities, skin necrosis, etc.6. Paradoxically, anticoagulants may also be prothrombotic, like in the case of “coumarin necrosis”7 or heparin-induced thrombocytopenia (HIT)/thrombosis syndrome8. An important factor determining the safety of an anticoagulant is the availability of its effective, selective and fast-acting antidote. Unfortunately, many of the currently applied antiplatelet drugs or anticoagulants do not have an antidote (e.g. aspirin, fondaparinux9), or have an antidote with a delayed onset of action (e.g., Vitamin K for VKAs10), or have only partially effective antidote (e.g. protamine for low-molecular-weight heparins (LMWHs)11), or have an antidote showing a high incidence of severe adverse effects (e.g. protamine for UFH12). Therefore, many new pharmaceuticals have been developed with superior properties compared to the old generation anticoagulants. For example, LMWHs, which drew attention in the mid-1970s and early 1980s13, were found to be generally safer than UFH, e.g., the risk



of hemorrhage or HIT for LMWHs is lower14. Therefore LMWHs have replaced UFH in many indications15. Moreover, LMWHs can be administered subcutaneously simplifying the anticoagulant therapy, although this way of administration is still much less comfortable for patients than the oral one, like in the case of direct oral anticoagulants (DOACs)16. Some novel anticoagulants can be administered once daily without monitoring (for example dabigatran etexilate, a direct oral thrombin (fIIa) inhibitor, and rivaroxaban, a direct oral factor Xa (fXa) inhibitor)15. In spite of the effort made to develop improved drugs, the new anticoagulants still have their shortcomings. Their activity may be difficult to measure, e.g., LMWHs, fondaparinux and idraparinux have little effect on routine coagulation tests such as activated partial thromboplastin time (aPTT) and activated clotting time (ACT)17. Many of prospective anticoagulants were withdrawn from the clinical tests at different stages due to unacceptable adverse effects, e.g. ximelagatran, an oral thrombin inhibitor, was withdrawn because of potential hepatic toxicity18. Therefore, the quest for novel, efficient, easy to administer, safer and cost-effective anticoagulants is still continued. When trying to predict anticoagulant activity during the drug development one may look for common molecular patterns in this group of pharmaceuticals. A coumarin moiety is an example of a molecular unit responsible for the activity of the class of low molecular weight anticoagulants such as VKAs. In polymers the presence of sulfate and/or sulfonate groups was found to induce the anticoagulant properties. Examples of such polymers include naturally19,20 and synthetically21,22 sulfated polysaccharides. The anticoagulant activity of sulfate/sulfonate-containing polymers could be related to the degree of sulfation/sulfonation. For example, fucoidan requires a minimal charge density of 0.5 sulfate groups per glucose unit and a minimum chain length of 70 glucose units23. Other


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important structural factors of the sulfated/sulfonated polysaccharides influencing their anticoagulant activity involve molecular weight and the pattern of sulfation/sulfonation24. The inherent drawbacks of the natural polymers such as high dispersity of the molecular weight, batch-to-batch and source-to-source variability, and difficult purification procedures directed the research on polymeric anticoagulants towards the synthetic polymers such as sulfonated polyisoprene (SPIP)25, sulfonated polyurethanes (PUs)26, sulfonated poly(ethylene oxide)27, poly(sodium styrene sulfonate) (PSSS)28, poly(sulfohexyl methacrylate)29, poly(γglutamic

acid)sulfonate30,

and

poly(sodium

2-acrylamido-2-methylpropanesulfonate)

(PAMPS)31. To our knowledge, all the synthetic anticoagulant polymers studied so far were, however, obtained using conventional radical polymerization technique which yields polymers with a poor control of the molecular weight and quite high dispersity of the molecular weight. To obtain polymers with well-defined and controllable molecular weight and low dispersity index one of the controlled radical polymerization (CRP) methods should be used instead32. Among them the reversible addition-fragmentation chain transfer (RAFT) polymerization method33 is of special interest in the context of polymers designed as drugs candidates. It allows carrying out polymerization reactions of a wide range of monomers in a variety of solvents (including water), does not require toxic metal catalysts, is tolerant to the presence of many functional groups, allows synthesis of end-functionalized polymer chains and various polymeric architectures (block, graft, gradient, star, dendritic, etc.), and can be performed in bulk, solution, emulsion, and dispersion34. The above mentioned advantages of RAFT have encouraged us to use this technique to obtain well-defined polymers with anticoagulant properties. We have selected PAMPS as the main component of the prospective polymers responsible for their anticoagulant properties35. Copolymers of PAMPS and poly(acrylic acid) (PAA) have been already shown to possess



anticoagulant activity in vitro as measured using prothrombin time (PT), aPTT, and thrombin time (TT). This activity was stronger at higher concentrations and at higher content of 2(acrylamido)-2-methylpropanesulfonic acid (AMPS) in the copolymers36. However, our approach was to focus not only on the anticoagulant activity, but concomitantly on the biocompatibility and safety. PAMPS was found to be nontoxic for a developing chick embryo37, however, the intravenous administration of 20 mg/kg b.w. of PAMPS in rats resulted in a high mortality rate38. To limit the potential toxicity of PAMPS in intravenous applications and improve its biocompatibility we block-copolymerized PAMPS with neutral poly(ethylene glycol) (PEG) and zwitterionic poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC). Conjugation of a substance (e.g. protein) with the former polymer, called pegylation, is a well-known method of improving its solubility, thermal and physical stability, preventing aggregation in vivo and during storage, reducing toxicity, immunogenicity, antigenicity, hemolysis and erythrocyte aggregation, extending circulation time in blood, protection from enzyme digestion, and decreasing kidney filtration39. Recently, pegylation was also applied to improve the biocompatibility of a dendrimeric polycation showing heparin reversal activity40, i.e., opposite to that of PAMPS. Conjugation with PMPC was also reported to improve the polymer biocompatibility41, reduce thrombogenicity (platelet aggregation42 and fibrin formation43), and reduce protein adsorption44. These properties were ascribed to the uniquely weak interaction of the polymer with surrounding water molecules which retain the structure similar to that of free water45. Interestingly, the copolymers of PAMPS and PEG described in this paper form spherical polyion complexes (PICs) with the cationic block copolymers of PEG and poly(3(methacryloylamino)propyl trimethylammonium chloride) (PMAPTAC)46. The PICs have cores formed by the charged blocks surrounded by a corona of PEG blocks. As we have


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recently shown, the cationic block polymer PEG-b-PMAPTAC, termed as Heparin Binding Copolymer (HBC), is a strong inhibitor of UFH with efficacy and safety parameters superior to those of protamine47. Significantly, HBC reversed also enoxaparin, an LMWH, which is inhibited by protamine only partially, and fondaparinux, for which there is no antidote currently. Thus, we found out that PEG-b-PAMPS anticoagulants presented in this paper and HBC-type polymers form a well-matching pair of a pegylated polyanionic anticoagulant and a pegylated polycationic antidote in human blood, the approach that has been never applied before for anticoagulants and their antidotes. Our in vitro aPTT assay showed promising results of complete neutralization of PEG-b-PAMPs by HBC in an appropriate mass ratio. Their full interaction in living organism will be further investigated by our group.

Experimental section

Materials The RAFT chain transfer agents, 4-cyanopentanoic acid dithiobenzoate (CPD) and poly(ethylene glycol)-based chain transfer agent (PEG41-CTA) were synthesized according to the methods reported by McCormick and coworkers48 and modified method of Chong and coworkers46, respectively. α,ω-Bis-hydroxy poly(ethylene glycol) (HO-PEG-OH, numberaverage molecular weight Mn=9.40×103 Da, degree of polymerization DP=227, molecular weight distribution Mw/Mn=1.06, Aldrich), 2-(methacryloyloxy)ethyl phosphorylcholine (MPC, 96%, NOF Corp.), 4,4’-azobis(4-cyanopentanoic acid) (V-501, 98%, Wako), 2(acrylamido)-2-methylpropanesulfonic acid (AMPS, 95%, Wako), 4-hydroxy-2,2,6,6tetramethylpiperidinyl-1-oxy (HTEMPO, free radical, 98%, Aldrich), sodium hydrogen sulfite (NaHSO3, Fluka, solution for synthesis, 38–40% in water), potassium persulfate (K2S2O8,



Aldrich, 99.99%), dimethylsulfoxide (DMSO, HPLC grade, POCH Gliwice), Griess reagent (1% w/v sulfanilic acid/0.1% w/v N-(1-naphtyl) ethylenediamine-dihydrochloride in 2.5% v/v H3PO4, Sigma), trisodium citrate (≥99%), calcium sulfate (≥99.99%) (Sigma-Aldrich, Germany), aPTT and PT reagents (Bio-Ksel, Poland), anti-factor Xa (anti-fXa) and anti-factor IIa (anti-fIIa) assay kits (Sekisui Diagnostics, USA), AT, thrombin/AT (TAT) complex, heparin cofactor II (HCII) assay kits (Cloud-Clone Corp., USA), 6-Keto-PGF1α assay kit (Cayman Chemical Company, USA), unfractionated heparin (Polfa Warszawa, Poland), pentobarbital (Biovet, Poland), collagen (Chrono-log, USA), diagnostic kit for determination of calcium concentration (Cormay, Poland) were used as received. Water was purified using a Millipore Milli-Q system. (3-(Methacryloylamino)propyl)trimethylammonium chloride (MAPTAC) (50 wt% in water) from Aldrich was passed through an inhibitor-remover column.

Synthesis of polymers

Preparation of PAMPS275 homopolymer Poly(sodium 2-acrylamido-2-methyl-1-propanesulfonate) with the degree of polymerization of 275 (PAMPS275), was synthesized according to the method developed in our laboratories and described in an earlier publication49 using nitroxide-mediated polymerization (NMP). AMPS (0.07 mol) was neutralized quantitatively with Na2CO3. Then, the redox initiator (K2S2O8:NaHSO3, 1:1 molar ratio, 1 mol%) and HTEMPO (1.5 mol%) were added. The polymerization was carried out in DMSO:water mixture (8:1 v/v, 50 mL) at 125°C. The reaction was completed after 6.5 h and the resulting polymer was precipitated with diethyl ether and dried in a vacuum oven at 40°C. The polymer was then dissolved in water, dialyzed


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for a week (Fisher cellulose tubing, cutoff molecular weight 12,000–14,000 g/mol) and freeze-dried.

Preparation of PAMPS-containing Block Copolymers

Synthesis of PMPC20-b-PAMPS198 PMPC macro-chain transfer agent was synthesized according to the previously reported method50. Mn(NMR), DP estimated from 1H NMR, and Mw/Mn for the obtained polymer (PMPC20 block) estimated from gel-permeation chromatography (GPC), were 6.21×103 Da, 20, and 1.03, respectively. A predetermined amount of AMPS (2.50 mg, 12.1 mmol) was neutralized with the proper amount of NaOH in water (12.1 mL). PMPC20 (375 mg, 0.0603 mmol, Mn(NMR) = 6.21×103 Da, Mw/Mn = 1.03) and V-501 (6.73 mg, 0.0240 mmol) were added to the solution. The solution was deoxygenated by purging with Ar gas for 30 min. Block copolymerization was carried out at 70°C for 4 h. The diblock copolymer (PMPC20-bPAMPS198) was purified by dialysis against pure water for two days and recovered by a freeze-drying technique (2.88 g, 91.2%). Mn(NMR) for PMPC20-b-PAMPS198 and DP for the PAMPS block estimated from 1H NMR were 4.72×104 Da and 198, respectively (Figure S1 in Supplementary Information). Mw/Mn estimated from GPC was 1.25 (Figure S2 in Supplementary Information).

Synthesis of PAMPS48-b-PEG227-b-PAMPS48 Poly(ethylene glycol)-based bifunctional chain transfer agent (CPD-PEG-CPD) was synthesized according to the literature51. Mn and Mw/Mn were estimated by GPC to be 1.00×104 Da and 1.18, respectively. DP was 227 estimated from 1H NMR. A predetermined



amount of AMPS (3.13 g, 15.1 mmol) was neutralized with the proper amount of NaOH in water (30.6 mL). V-501 (15.5 mg, 0.0552 mmol) and CPD-PEG-CPD (1.60 g, 0.151 mmol, Mn(GPC) = 1.00×104 Da, Mw/Mn = 1.18) were added to the solution. The solution was deoxygenated by purging with Ar gas for 30 min. Polymerization was carried out at 70°C for 16 h. After polymerization, the mixture was poured into a large excess of THF to precipitate the resulting polymer, which was then dissolved in water and dialyzed against pure water for one day. The triblock copolymer (PAMPS48-b-PEG227-b-PAMPS48, AEA) was recovered by a freeze-drying technique (4.05 g, 85.8%). Mn(NMR) (Figure S3 in Supplementary Information) and Mw/Mn values determined by GPC (Figure S4 in Supplementary Information) were 3.26×104 Da and 1.42, respectively. DP of both PAMPS blocks was 48, as estimated by 1H NMR.

Synthesis of PEG47-b-PAMPS108 and PEG47-b-PAMPS29 Preparation method and full characterization data for PEG47-b-PAMPS108 and PEG47-bPAMPS29 were described in our previous report46. The Mn(NMR) values for PEG47-bPAMPS108 and PEG47-b-PAMPS29 were 2.71×104 Da and 9.00×103 Da, respectively. The Mw/Mn values for PEG47-b-PAMPS108 and PEG47-b-PAMPS29 estimated from GPC were 1.17 and 1.15, respectively.

Synthesis of PEG41-b-PMAPTAC53 The synthesis of PEGx-b-PMAPTACy block copolymers has been described previously46. Briefly, the PEG41-PMAPTACn diblock polymers were prepared by dissolving of MAPTAC (5.53 g, 25.0 mmol), V-501 (69.2 mg, 0.247 mmol), and PEG41-CTA (1.13 g, 0.501 mmol) in water (41.0 mL). The mixture was degassed by purging with Ar gas for 30 min. Polymerization was carried out at 70°C for 5 h. The polymerization mixture was dialyzed


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against pure water for two days. PEG41-PMAPTACn was recovered using a freeze-drying technique (5.58 g, 83.8%).

Characterization of polymers GPC measurements were performed using a Tosoh RI-8020 refractive index detector equipped with Shodex 7.0 µm bead size GF-7M HQ column (exclusion limit ~107) working at 40°C under a flow rate of 0.6 mL/min. A phosphate buffer (50 mM, pH 9) containing 10 vol% acetonitrile was used as eluent. The values of Mn and Mw/Mn were determined based on calibration carried out using standard poly(sodium styrenesulfonate) samples. 1H Proton NMR spectra were obtained on a Bruker DRX-500 spectrometer operating at 500.13 MHz using deuterium lock. Malvern Nano ZS light scattering apparatus working at a scattering angle of 173° was used for dynamic light scattering (DLS) determination of hydrodynamic size as well as for static light scattering (SLS) determination of molecular weight measurements. The DLS measurements for the diblock copolymers were carried out in 0.1 M NaCl while the SLS measurements for PAMPS275 were performed in 1 M NaCl aqueous solutions applying the measured refractive index increment (dn/dc = 0.143 mL/g). The size of complexes formed by HBC and PEG47-b-PAMPS108 was studied in PBS. PEG47-b-PAMPS108 was dissolved at 3 mg/mL. To that mixture, the HBC solution in PBS buffer was added (12 mg/ml), and the size of the complexes formed was monitored. Data analysis was carried out with software provided by Malvern. Viscometric measurements were performed for PAMPS275 using Ubbelohde viscometer (SI Analytics, type 530 13/Ic) equipped with ViscoClock. The measurements were performed in 1 M NaCl aqueous solutions and the molecular weight of PAMPS275 was calculated using Mark-Houwink equation applying previously reported values of constants52.



Isothermal titration calorimetry (ITC) measurements ITC technique was used to study the interactions between PEG47-b-PAMPS108 polymer and thrombin, AT, albumin, HBC, and platelet factor 4 (PF4). The ITC measurements were performed using Malvern MICROCAL PEAQ-ITC instrument. All compounds were dissolved in PBS. Concentrations of thrombin, AT, albumin, HBC, PEG47-b-PAMPS108 and PF4 were 0.000210748, 0.0000344, 0.000210748, 0.001078, 0.00005 or 0.0001 mol/L, and 0.003 mol/L, respectively. The measurement temperature was 25°C and the mixing speed was 750 rpm. The measuring chamber contained 270 µl of PEG47-b-PAMPS108 solution to which 2 µL aliquots of the respective compound solution were added.

Measurements of calcium concentration Calcium binding properties of PAMPS-based polymers were evaluated using saturated solution of calcium sulfate. Four hundred and fifty µL of calcium sulfate saturated solution was incubated for 10 min with 50 µL of polymers solution in the final concentration of 0.1-1 mg/mL. Concentration of free calcium ions was measured in the supernatant after 20 min of centrifugation (3500×g at 4°C) using automated clinical biochemical analyzer (Mindray BS 120, Germany).

Animals Male Wistar rats were obtained from the Centre of Experimental Medicine in Medical University of Bialystok, bred in a 12-hour light/dark cycle in temperature and humiditycontrolled room, grouped cages as appropriate, and allowed to have ad libitum access to sterilized tap water and standard chow in specific pathogen-free conditions. All the procedures involving animals were approved by Local Ethical Committee (Permit Numbers 28/2012, 108/2015, and 2/2018) and conducted in accordance with directive 2010/63/EU of


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the European Parliament and of the Council on the protection of animals used for scientific purposes, ARRIVE guidelines53, and the national laws. All animals were euthanized by exsanguination at the end of experiments.

In vitro efficacy and safety experiments Forty male Wistar rats were anesthetized by intraperitoneal injection of pentobarbital (45 mg/kg). The blood samples were collected from heart and drawn into 3.13% trisodium citrate. One part of anticoagulated blood samples was used to measure platelet aggregation; second part was used to count the blood cells; third part was centrifuged and pooled plasma was used to measure aPTT, PT, anti-fXa activity, and anti-fIIa activity. If the anti-fIIa activity was more than 1.2 U/mg, a value of 1.2 U/mg was ascribed for the sake of statistical analysis. Sodium citrate anticoagulated blood sample or citrate pooled plasma was incubated with studied polymers or UFH solution in the final concentration of 0.005-1 mg/mL. The control samples were incubated with the PBS solution (vehicle). The results of in vitro experiments were expressed as a percentage of the control samples.

In vivo efficacy and safety experiments Sixty-eight male Wistar rats weighing 178.0±12.8 g were randomly divided into 7 groups, anesthetized by an intraperitoneal injection of pentobarbital (45 mg/kg) and placed in a supine position on a heated operation table. The PAMPS-based polymers or UFH (6 mg/kg, 1 mL/kg) were administered into the right femoral vein. Vehicle (PBS, 1 mL/kg) treated animals served as a control group. Heart rate (HR), tissue perfusion of rat’s paw, blood oxygen saturation and respiratory rate (RR) were measured using the PhysioSuite Physiological Monitoring Modular System (Kent Scientific Corporation) for 30 min after PAMPS-based polymers administration. The blood samples were taken from the heart at the



end of the experiment for measurement of aPTT, PT, anti-fXa activity, anti-fIIa activity, AT concentration, TAT concentration, HCII concentration, prostacyclin metabolite (6-Keto PGF1α) concentration, platelet aggregation, and blood cell count. If the anti-fIIa activity was more than 1.2 U/mg, a value of 1.2 U/mg was ascribed for the sake of statistical analysis.

In vitro neutralization of the lead polymer by HBC The neutralization of our lead polymer by HBC was assessed by measuring aPTT, as described previously47,54,55. In brief, 10 µL of the PEG47-b-PAMPS108 (0.1 mg/mL of plasma) was mixed with 200 µL of plasma. After 2 min and 30 s of incubation, 10 µL of a solution containing increasing concentrations of HBC (0.025–0.1 mg/mL of plasma) were added. After 2 min and 30 s of incubation, the aPTT values were automatically determined with an optical method.

The measurement of coagulation parameters, platelet aggregation, and blood cell count aPTT and PT were automatically determined by an optical method (Coag Chrom 4000, BioKsel, Poland) adding routine laboratory reagents (Bio-Ksel, Poland). Anti-fXa and anti-fIIa was analyzed with the chromogenic assay in 96-well plate using a microplate reader (Dynex Tech., USA) according to the kit manufacturer instructions (Sekisui Diagnostics, USA). AT, TAT complex, and HCII concentration was analyzed with the enzyme-linked immunosorbent assay (ELISA) in 96-well plate using a microplate reader (Dynex Tech., USA) at 450 nm according to the instructions of kits manufacturer (Cloud-Clone Corp., USA). 6-ketoPGF1α concentration was measured with ELISA kit using a microplate reader (Dynex Tech., USA) at 405 nm according to the instructions of the kit’s manufacturer (Cayman Chemical, USA). Platelet aggregation was measured after incubation of blood (500 µL) and PAMPS-based


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polymers, UFH solution (500 µL) in the final concentration of 0.005-1 mg/mL or 0.9% NaCl solution (500 µL) for 20 min at 25°C, and then for 15 min at 37°C. The changes in impedance were registered during 6 min after collagen addition (7.5 µg/mL) using Chrono-log aggregometer (Chrono-log Corp., USA). The aggregation curve was described by maximal extension (MaxA), the slope of platelet aggregation (Slp), lag phase (Lag), and area under the curve (AUC). The blood cells were counted using the Animal Blood Counter (ABC Vet, Horiba, France).

Statistical analysis Shapiro-Wilk’s test of normality was used for data distribution analysis. The normally distributed data were shown as mean±SD and analyzed using unpaired Student t-test. The non-Gaussian data were presented as median with lower and upper limits and analyzed using the non-parametric Mann-Whitney test. P values less than 0.05 were considered significant. The data were analyzed with GraphPad Prism 6 software.

Results and discussion

Characterization of the studied polymers We have synthesized three diblock and one triblock copolymers, all containing PAMPS blocks as the active component (i.e., responsible for their anticoagulant activity) (Figure 1). The other block was either neutral PEG block or zwitterionic PMPC block yielding an overall strongly negative charge of the polymers. As discussed in the Introduction section, the role of PEG and PMPC blocks was to decrease the toxicity of the polymers. The polymers were denoted as PAMPSx, PMPCx-b-PAMPSy, PEGx-b-PAMPSy, and PAMPSx-b-PEGy-b-



PAMPSx, where x and y were the degrees of polymerization of relevant blocks. The only polymer containing PMPC block was PMPC20-b-PAMPS198 with quite short PMPC block and much longer PAMPS block. Two PEG-b-PAMPS diblock copolymers, PEG47-bPAMPS27 and PEG47-b-PAMPS108, contained PEG blocks of the same length while the length of their PAMPS blocks differed significantly. In the only triblock copolymer studied, PEG constituted long central block with much shorter PAMPS end blocks. PAMPS275 homopolymer was used as a reference to evaluate the influence of the PEG and PMPC blocks on the toxicity and safety parameters of the polymers. Molecular weight of the PEG blocks in all the polymers was greater than 400 Da which is considered a minimum value for PEG to be resistant to toxic oxidative degradation by alcohol dehydrogenase and aldehyde dehydrogenase56. On the other hand, total molecular weight of the polymers did not exceed 40-60 kDa, which is a limiting value for a nondegradable polymer to undergo renal clearance57.

Figure 1. The structures of the studied block polymers and the relative lengths of their blocks


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Biomacromolecules

The results of NMR and GPC analyses and the structural parameters (molecular weights and their dispersities) of the studied polymers are given in the Supplementary Information (Tables S1-S2, Figures S1-S4). Thanks to the application of RAFT polymerization technique for the synthesis of the block copolymers, the dispersities (given by the

ெೢ ெ೙

ratio) of the polymer molecular weights were

very low, ranging from 1.17 to 1.25 for the diblock copolymers and 1.42 for the triblock copolymer. These values can be translated into the standard deviations of the number average molecular weights (ܵ‫ܦ‬ெ೙ ) using the following formula58:

ܵ‫ܦ‬ெ೙ = ‫ܯ‬௡ ට

ெೢ ெ೙

−1

(1)

The respective values of ܵ‫ܦ‬ெ೙ and

ௌ஽ಾ೙ ெ೙

are given in Table 1. The above equation points

out the importance of achieving possibly low

ெೢ ெ೙

of polymeric materials of biomedical

interest. For example, the value of ܵ‫ܦ‬ெ೙ for a polymer with a polymer with

ெೢ ெ೙

ெೢ ெ೙

= 1.1 is about half of that for

= 1.4, which can also be considered as reasonably low value.

Since an important parameter of polyelectrolytic anticoagulants and their antidotes is the charge of the polymeric chain (both absolute and per kDa), the respective values were calculated and compared with those of UFH and protamine sulfate (PS) (Table 1). Compared to that of UFH, the total charge of the block polymer chains was much lower for PEG47-bPAMPS27, comparable for PEG47-b-PAMPS108 and PAMPS48-b-PEG227-b-PAMPS48 and much greater for PMPC20-b-PAMPS198.

Table 1. Characteristics of the studied polymers



Polymers

‫ܯ‬௡ (kDa)

PAMPS275

‫ܯ‬௡ ‫ܯ‬௪

ܵ‫ܦ‬ெ೙ (kDa)

ܵ‫ܦ‬ெ೙ ‫ܯ‬௡

Page 18 of 49

Charge/chain

Charge/kDa

-275

-4.8

1.18

PEG47-b-PAMPS27

9.0

1.15

3.5

0.39

-27

-3.0

PEG47-b-PAMPS108

27.1

1.17

11.1

0.41

-108

-3.99

PAMPS48-b-PEG227-b-PAMPS48

32.6

1.42

21.1

0.65

-96

-2.94

PMPC20-b-PAMPS198

47.2

1.25

23.6

0.50

-198 a

-4.19 b

UFH

-73 to -75

PS

+21 to +25 c

-5.00 b +4.54 to +5.40 d

a

net charge of the chain, there are additionally 20 positive and 20 negative charges in the zwitterionic PMPC block b

based on 40

c

based on 59

d

assuming Mw of 4626 Da 60

The anticoagulant properties of PAMPS-based polymers The anticoagulant activity of the block polymers was tested by measuring the influence of the polymers on the hemostatic parameters in vitro (Figure 2 and Figure 3) and in vivo (Figure 4 and Figure 5).


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Biomacromolecules

Figure 2. Effects of PAMPS-based polymers and unfractionated heparin (UFH) on activated partial thromboplastin time (aPTT, A) and prothrombin time (PT, B). **p

and triblock polymers

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