An Injectable Hemostat Composed of a Polyphosphate-Conjugated

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An Injectable Hemostat Composed of a Polyphosphate-Conjugated Hyaluronan Hydrogel Megumu Sakoda, Makoto Kaneko, Seiichi Ohta, Pan Qi, Shigetoshi Ichimura, Yutaka Yatomi, and Taichi Ito Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00588 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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An Injectable Hemostat Composed of a Polyphosphate-Conjugated Hyaluronan Hydrogel Megumu Sakoda†, Makoto Kaneko††, Seiichi Ohta†††, Pan Qi†††, Shigetoshi Ichimura††††, Yutaka Yatomi††, Taichi Ito†,†††,*

†Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ††Department of Clinical Laboratory Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan †††Center for Disease Biology and Integrative Medicine, The University of Tokyo, Hongo 7-31, Bunkyo-ku, Tokyo 113-0033, Japan ††††Department of Applied Bioscience, Kanagawa Institute of Technology, 1030 Shimo-ogino, Atsugi, Kanagawa 243-0292, Japan KEYWORDS polyphosphate, hyaluronan, injectable hydrogel, hemostasis

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ABSTRACT

We have developed a new hydrogel hemostat composed of hyaluronan (HA) conjugated with inorganic polyphosphate (PolyP). A hemostatic hydrogel, HAX-PolyP, was formed rapidly by mixing aldehyde-modified HA and hydrazide-modified HA conjugated with PolyP (HA-PolyP). Although the gelation rate decreased with increasing PolyP content, the gelation time was below 5 min. In addition, the hydrogel swelling volume decreased with increasing PolyP content, but the degradation rate did not depend on PolyP content and the hydrogel underwent complete degradation through hydrolysis over 3 weeks in phosphate buffered saline. HAX-PolyP showed similar biocompatibility with the HA hydrogel without PolyP conjugation in vitro and in vivo. Intraperitoneal administration of HAX-PolyP did not induce any adhesion in the peritoneum and clot formation in the lungs. Finally, HA-PolyP accelerated the coagulation rate of human plasma ex vivo, and HAX-PolyP showed as strong a hemostatic effect as fibrin glue in a mouse liver bleeding model in vivo.


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1. Introduction Because of the requirement to stop bleeding during many surgical procedures, hemostats are widely used clinically.1-3 In addition to sheet-type hemostats, several injectable hemostats are utilized and have been studied in terms of their operability using dual-syringe systems or spray devices in laparoscopic surgeries. Fibrin sealants, which are two-component products that combine thrombin and fibrinogen, have been widely used as injectable hemostatic agents 4; however, the risk of viral infection cannot be ignored, because fibrinogen and thrombin are blood products. Thrombin gelatin matrix, Floseal® 5 or Surgiflo® 2, was recently approved; however, it persists for 6 to 8 weeks after its administration, and a risk of clotting for heparinized blood is pointed out because of its adverse effect on the anticoagulation effects of heparin.2 Although some chemically-synthesized hemostats, such as the two-component polyethylene glycol surgical sealant (CoSeal®)

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and a self-assembling peptide sealant 7, have been developed, novel

synthetic hemostats are required for safer surgeries and therefore some new hemostats based on a new concept have been recently reported.3, 8 As a new procoagulant agent, inorganic polyphosphate (PolyP), stored in dense granules in platelets 9, has been reported to contribute to hemostasis through four different mechanisms, which are: (i) the initiation of the contact pathway of the blood coagulation cascade enhancement of coagulation factor V activation

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, (iii) fibrin clot stabilization

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12, 13

, (ii) the

, and (iv)

promotion of factor XI feedback activation by thrombin.14 Interestingly, these physiological roles of PolyP greatly depend on its degree of polymerization (DP).14, 15 While the procoagulant effect of platelet PolyP at a DP of 60–100 is not strong, high molecular weight PolyP strongly promotes the intrinsic coagulation cascade.15 Although PolyP has been extensively studied in terms of hematology, to date, only one study has investigated its use in potential hemostatic


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materials. Shin-Yeu Ong et al. (2008) fabricated a chitosan hydrogel crosslinked using PolyP via a polyion-complex (PIC), and studied its hemostatic effect using a whole blood coagulation experiment.16 Though a stable PIC was formed for gelation, a high concentration of PolyP was utilized to crosslink the hydrogel sufficiently. In addition, the chitosan/PolyP complex was not implanted in vivo; therefore, its biocompatibility was not investigated. Based on the low biocompatibility of cationic chitosan as an implantable material 17, new PolyP-based biomaterials are expected as hemostats. Recently, Donovan et al. reported PolyP nanoparticles prepared via precipitation in aqueous solution containing divalent metal cations. The activation of contact pathway by PolyP nanoparticles was confirmed by in vitro clotting assay.18 They further fabricated artificial dense granules using PolyP nanoparticles encapsulated in liposome.19 The PolyP nanoparticles-encapsulated liposomes also accelerated the contact pathway through activation of factor XII, suggesting a potential as procoagulant materials. In addition to these hemostatic properties, PolyP had been reported to have a differentiationinducing effect on osteoblast cells.20, 21 However, PolyP itself form a hydrogel only when present with an extremely high concentration of iron(III) nitrate, calcium nitrate, or aluminum nitrate 22, 23

; therefore, to use PolyP in bone tissue engineering, it is necessary to conjugate it to other

matrix hydrogels. Injectable hyaluronan (HA)-based hydrogels are versatilely used in tissue engineering, drug delivery, and as anti-peritoneal adhesion barriers.24, 25 We, for the first time, successfully synthesized hyrdrozide-modified HA conjugated with PolyP (HA-PolyP) and prepared injectable HA-based hydrogel conjugated with PolyP (HAX-PolyP) by mixing it with aldehyde-modified HA (HA-CHO).26 Crosslinking mainly occurred between aldehyde group in HA-CHO and hydrazide group in HA-PolyP to form HAX-PolyP. HAX-PolyP successfully enhanced the osteogenic differentiation of MC-3T3-E1 cell lines in vitro. Based on the


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hemostatic effects of PolyP, HAX-PolyP is expected to show hemostatic properties. However, its hemostatic effect and biocompatibility in vivo have not been investigated. In the present study, we investigated both the hemostatic effect and biocompatibility of HAXPolyP in vitro and in vivo (Figure 1A). The influence of the degree of modification of HAXPolyP on its gelation time and degradation rate was investigated. In addition, the biocompatibility of HAX-PolyP was evaluated using a cell viability assay in three cell lines: macrophages, fibroblast cells and mesothelial cells. The biocompatibility of HAX-PolyP in mice was also evaluated through intraperitoneal and subcutaneous administration. Finally, the ex vivo hemostatic effect of HA-PolyP was measured using a Sonoclot coagulation analyzer. The in vivo hemostatic effect of HAX-PolyP was also evaluated by measuring blood loss in a mouse liver bleeding model.


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Figure 1. (A) Schematic illustration of HAX-PolyP as an injectable hemostat. (B) Synthetic route of the following precursor polymers: (i) HA-CHO, (ii) HA-ADH, and (iii) HA-PolyP.

2. Materials and methods 2-1. Materials Hyaluronan (HA, Mw 850 kDa and 2.0 MDa) was kindly gifted by Denka Co. Adipic dihydrazide

(ADH),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride


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(EDC·HCl), ethanol, sodium periodate, ethylene glycol, imidazole, Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin-amphotericin B suspension (PSA), fetal bovine serum (FBS), non-essential amino acids solution (NEAA), insulin, epidermal growth factor (EGF), and hydrocortisone were purchased from Wako Pure Chemical Ind. (Osaka, Japan). Sodium hexametaphosphate and Minimum Essential Medium, with Eagle’s salts (MEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). A dialysis membrane (Spectra/Por, MWCO = 6–8 kDa) was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). A double-barrel syringe was kindly gifted by Baxter (Deerfield, IL, USA). Normal saline was purchased from Otsuka Pharmaceutical (Tokyo, Japan). Medium 199, containing 685 µM Lglutamine, 26 mM sodium bicarbonate and 25 mM HEPES, was purchased from Thermo Fisher Scientific (cat# 12340-030; Waltham, MA, USA). Standard human plasma (Coagtrol N) was purchased from Sysmex (Hyogo, Japan).

2-2. Synthesis of polyphosphate-modified hyaluronan ADH-modified HA (HA-ADH) was synthesized through the conjugation of ADH to HA (Mw 850 kDa), as previously reported.25 Polyphosphate-modified HA (HA-PolyP) was subsequently synthesized through a conjugation of the terminal hydroxyl groups of PolyP to the hydrazide groups of HA-ADH using a previously reported reaction scheme, as shown in Figure 1B(ii).26 Briefly, 45 mM imidazole and 16 mM EDC·HCl were dissolved in distilled water and adjusted to pH 6.0 using 1 N HCl. Sodium hexametaphosphate was then added at a final concentration of 3.3 mM. After readjusting the pH to 6.0 using 1 N NaOH, the aqueous mixture was maintained at room temperature for 1.5 hours, and subsequently purified through ethanol precipitation. The


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product was lyophilized for 3 days, and imidazole-modified PolyP (PolyP-Im) was obtained as a white powder. Then, 143 mL of HA-ADH aqueous solution (3.5 mg/mL, containing 3.86 mM of ADH groups to its disaccharide unit of HA-ADH) was mixed with a 0.1- to 1.0-fold molar excess of PolyP-Im (Figure 1B(iii)). The solution was reacted at 50°C for 5 hours after adjusting the pH to 8.5 using 1 N NaOH, and was subsequently purified through dialysis (MWCO = 6–8 kDa) against distilled water at room temperature. The final product, HA-PolyP, was obtained as a white powder through lyophilization for 3 days, and stored at 4°C.

2-3. Synthesis of aldehyde-modified hyaluronan HA (Mw 2.0 MDa) was modified into aldehyde-modified HA (HA-CHO) using a previously reported method (Figure 1B(i)).25,

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Briefly, HA was reacted with an equimolar amount of

sodium periodate for 2 hours at room temperature in the dark. The reaction was terminated by adding an excess amount of ethylene glycol (~4.5 mL). The product was purified through dialysis (MWCO = 6–8 kDa) against distilled water at room temperature. HA-CHO was obtained as a white powder after lyophilization for 3 days, and was stored at 4°C before use.

2-4. Characterization of HA-PolyP and turbidity measurement of HA-PolyP The FT-IR spectra of PolyP-Im, HA-ADH, and HA-PolyP were obtained using the potassium bromide disk method via a FT-IR-4200ST spectrometer (JASCO, Tokyo, Japan). The 1H NMR spectra of HA–ADH and HA–PolyP were obtained using a JNM-A500 spectrometer (JEOL, Tokyo, Japan). HA–ADH and HA–PolyP (10 mg/mL for both) dissolved in D2O were used for the measurement. In addition, the

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P NMR spectra of PolyP-Im and HA–PolyP were obtained


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using a JNM-A500 spectrometer. PolyP-Im and HA–PolyP (31.6 mg/mL for both) dissolved in D2O were used for the measurement. The degree of modification of PolyP was determined using a molybdenum assay and a TNBS assay.26 For the molybdenum assay, 1 mg of HA-PolyP was dissolved in 1 mL of 0.5 M sulfuric acid and heated to 80°C for 2 hours. The hydrolyzed HA-PolyP was then diluted to 1.0 µg/mL using distilled water and reacted with the reaction mixture (2.5 M sulfuric acid, 4.11 M tartar emetic solution, 32.4 M ammonium molybdate, 100 mM L-ascorbic acid, and distilled water) at a 4:1 volume for 10 min at room temperature. The amount of monophosphate in the sample was quantified by comparing the absorbance at 883 nm with a calibration curve of phosphate standard solutions using a V-630BIO UV–vis spectrometer (JASCO, Tokyo, Japan). For the TNBS assay, a 0.5 mg/mL HA-PolyP solution and a 0.29 mM TNBS solution were prepared using 0.1 M NaHCO3 (pH 8.5) as a solvent. 0.5 mL of the HA-PolyP solution was reacted with 0.25 mL of the TNBS solution for 2 hours at 37°C. The reaction was terminated by adding 0.125 mL of 1 N HCl. The amount of amine in the sample was quantified by comparing the absorbance at 335 nm with a calibration curve of ADH solutions using a V-630BIO UV–vis spectrometer. The turbidity of a HA-PolyP solution in distilled water (1.0 mg/mL) at different CaCl2 concentrations (0–50 mM) was measured using a V-630BIO UV–vis spectrometer at 405 nm. An increase in turbidity, caused by insoluble salt formation, was used as an indicator of chelation between HA-PolyP and Ca2+. We conducted four independent experiments for each Ca2+ concentrations. Data are expressed as average ± standard deviation.


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2-5. Characterization of HAX-PolyP HAX-PolyP was prepared by mixing equal amounts of HA-PolyP solution (20 mg/mL) and HA-CHO solution (20 mg/mL) for measurement of gelation time, and examination on swelling behavior and degradation behavior. HA-PolyP with different degrees of PolyP modification was used. The amount of PolyP in the HAX-PolyP was also changed by mixing HA-PolyP with HAADH that was not conjugated with PolyP. The mixture ratio of HA-ADH : HA-PolyP was varied as follows - 10 : 0, 7 : 3, 4 : 6, and 1 : 9, while keeping the total polymer concentration at 20 mg/mL. Hereafter, the resultant hydrogels are termed as HAX, HAX-PolyP (A/P = 7/3), HAXPolyP (A/P = 4/6), and HAX-PolyP (A/P = 1/9), respectively. A/P indicates the weight ratio of HA-ADH to HA-PolyP. Gelation time was measured by adding 100 µL of aqueous HA–CHO solution (20 mg/mL) to 100 µL of HA-PolyP/HA-ADH mixture solution, which was stirred at 300 rpm on a petri dish using a stirrer (RS-1DR; AS ONE, Osaka, Japan). During the mixing, the sample solution was observed visually. We determined the gelation time as the time until the mixture became a globule. The size of a stir bar was 2 × 5 mm and the stirring speed was 300 rpm, respectively. We conducted four independent experiments for each condition. We also measured the time dependency of the storage modulus (G’) and loss modulus (G’’) of HAX-PolyP with different mixture ratios of HA-PolyP/HA-ADH using a rheometer (MCR302; Anton Paar, Graz, Austria). The strain value and the frequency were fixed at 5% and 1 Hz, respectively. For the examination of swelling and degradation behavior, HAX-PolyP was fabricated in a cylindrical shape using a rubber mold. Equal amounts of HA-ADH/HA-PolyP mixture solution (20 mg/mL in PBS) and HA–CHO solution (20 mg/mL in PBS) were injected into the rubber mold sandwiched between two glass slides, using a double-barreled syringe (Baxter, Deerfield, IL). The diameter and


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thickness of the prepared hydrogel was 1.3 cm and 3 mm, respectively. The time course of swelling and degradation of the gel disks in PBS at 37°C was measured gravimetrically. The weight of the hydrogel was measured at each time point. The measurements were continued until complete degradation of the hydrogels over 3 weeks. The obtained weight of hydrogel at each time point was normalized to the initial weight to evaluate the degree of swelling and degradation. We measured the dry mass of the hydrogels with different mixture ratios of HAPolyP/HA-ADH on day 7 and day 14 of the swelling and degradation experiments.

2-6. In vitro cell viability assay In vitro cell viability in the presence of HA-ADH and HA-PolyP was evaluated through an MTT assay (Cell Titer 96; Promega, WI, USA) using a human mesothelial cell line (MeT-5A, ATCC), mouse macrophage cell line (RAW264.7, RIKEN Cell Bank), and a mouse fibroblast cell line (NIH3T3, RIKEN Cell Bank). We chose these cells because they play important roles in the peritoneal cavity, which is one of the possible application sites of HAX-PolyP. The growth media used for the MeT-5A cells was Medium 199 supplemented with 3.3 nM EGF, 361 nM hydrocortisone, 870 nM insulin, 1% PSA and 10% FBS. MEM supplemented with 1 mM NEAA, 1% PSA and 10% FBS was used for RAW264.7 cells. DMEM supplemented with 1% PSA and 10% FBS was used for NIH3T3 cells. Each cell line was grown and maintained in culture media at 37°C in 5% CO2. 5×104 cells were placed in each well of a 24-well plate, and incubated at 37°C in 5% CO2 overnight, and then the medium were replaced using medium containing 0.01 to 1 mg/mL of HA-ADH and HA-PolyP. On the third day after adding those materials in the case of MeT-5A and NIH3T3 cells, or the second day in the case of RAW264.7 cells, MTT assays were performed. 100 µL of tetrazolium salt solution was added into each well and incubated at 37°C


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for 4 hours. The purple formazan produced by active mitochondria was solubilized using a detergent solution (1 mL/well), and then the absorbance at 570 nm was measured using a plate reader (2030 ARVO V3; PerkinElmer, Waltham, MA, USA). The absorbance values were normalized to wells where no test materials were added to the media, to determine the cell viability. Statistical analysis was performed using one-way ANOVA, followed by Turkey HSD analysis. An assessment of in vitro cell viability in the presence of the hydrogels, HAX and HAXPolyP, was also performed in a similar manner. 100 µL each of HAX and HAX-PolyP were molded into 4 mm diameter and 10 mm height cylinder by mixing normal saline containing 20 mg/mL HA-ADH or HA-PolyP with normal saline containing 20 mg/mL HA-CHO using the double-barreled syringe. HAX or HAX-PolyP was added to Transwell® cell culture inserts (cat# 3422; Corning, Inc., Corning, NY, USA). After 2 days incubation at 37°C in 5% CO2 for RAW264.7 cells, and 3 days for NIH3T3 and MeT-5A cells, cell viability was measured using a MTT assay, as described above.

2-7. Subcutaneous and intraperitoneal administration of HAX-PolyP The experiments were performed at the Animal Research Section, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, the University of Tokyo. The Animal Care Committee of the University of Tokyo approved all procedures in this experiment before it began. ICR mice (7 weeks old, male) weighing from 30 to 32 g were purchased from CLEA Japan, Inc. (Japan), and housed in groups in a 6 AM−6 PM, light−dark cycle. All the mice were acclimated for at least 1 week before the experiment. HA-PolyP and HA-CHO were sterilized using UV irradiation for 2 hours and then dissolved in normal saline at a 20 mg/mL


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concentration. Anesthesia was induced using pentobarbital (Kyoritsu Seiyaku Co., Tokyo, Japan). 0.5 mL each of HA-PolyP and HA-CHO solutions were injected intraperitoneally using a double-barreled syringe. A further 0.5 mL each of HA-PolyP and HA-CHO solutions were injected subcutaneously into the postero-dorsal wall using the double-barreled syringe. Four mice received the above treatments. The mice were sacrificed 1 week after the injections, and the presence of hydrogel residue and adhesions in the peritoneum was evaluated. Residual hydrogel fragments, along with surrounding tissues and lungs, were sampled, fixed in 10% formalin, and processed for histology (hematoxylin-eosin [HE] stained slides) using standard techniques. HE-stained slides were observed using a fluorescence microscope (BZ-X700, Keyence Co, Osaka, Japan).

2-8. Ex vivo hemostatic effects of HA-PolyP The ethics committee of the faculty of medicine, the University of Tokyo approved all procedures in this experiment before it began (#10412). The effect of HA-PolyP on clot formation was investigated using a Sonoclot coagulation and platelet function analyzer (Sienco Inc., Boulder, CO, USA).27-29 The Sonoclot analyzer records an elasticity change in clots as a clot signal using an oscillating probe. 120 µL of HA-ADH, PolyP or HA-PolyP (1.0 mg/mL) was mixed with standard human plasma and warmed to 37°C. Immediately after the addition of 120 µL of CaCl2 solution (25 mM), the clot signal was measured using the Sonoclot analyzer. 120 µL of normal saline was used as a control condition instead of the addition of material solutions such as HA-PolyP.


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2-9. Evaluation of in vivo hemostatic effects using a liver bleeding model To evaluate the in vivo hemostatic effect of HAX-PolyP, we used ICR mice (7 weeks old, male) in a liver bleeding model, with slight modification to a previously reported protocol.30, 31 The experiments were performed at the Animal Research Section, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, the University of Tokyo. The Animal Care Committee of the University of Tokyo approved all the procedures in this experiment prior to its beginning. All the mice were acclimated for at least 1 week before the operation. The polymers were sterilized through UV irradiation for 2 hours and then dissolved in normal saline at a 20 mg/mL concentration. HA-PolyP was dissolved in 0.15 mL of normal saline. HACHO was dissolved in 0.15 mL of normal saline containing 0.5 M CaCl2. Then, both the solutions were mixed using a double-barreled syringe, and 0.3 mL of HAX-PolyP was prepared before the hemostatic experiment. The mice were anesthetized using pentobarbital. After an abdominal incision, serous fluid around the mouse liver was gently removed, and a pre-weighted filter paper (φ9 cm, 5C; ADVANTEC Co., Tokyo, Japan) on a paraffin film (5 cm × 5 cm) was placed beneath the liver. A 5 mm-wide resection was made in center of the left lobe of the liver using a scalpel. Immediately after the resection, 0.3 mL of HAX-PolyP was placed on the bleeding point. After 3 min, at which point the bleeding had already stopped, the weight of blood absorbed on the filter paper was immediately measured by weighing. No material or 0.3 mL of HAX was applied for negative control groups, while fibrin glue (Bolheal®; Chemo-sero Therapeutic Institute, Kumamoto, Japan) was applied for a positive control group.


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3. Results 3-1. Synthesis of HA-PolyP with various degrees of modification of PolyP PolyP-Im was conjugated to HA-ADH to obtain HA-PolyP, in accordance with our previous report.26 Conjugation of the PolyP onto HA was confirmed by changes in the FT-IR spectra before and after the conjugation (Figure 2A and B). Modification of HA with ADH was indicated by the emergence of a new peak at 1700 cm-1, reflecting stretching of the C=O of the amide groups. The IR spectrum of PolyP-Im showed a peak at 1300 cm-1, which corresponds to the symmetric stretching of the P=O of the phosphate group. Synthesis of HA-PolyP was confirmed by a new peak at 1300 cm-1, which reflected asymmetric stretching of the P=O of the phosphate group. Emergence of a new peak at 1700 cm-1 was also observed, reflecting stretching of the C=O of the amide groups. These FT-IR spectra confirmed the proper conjugation of the polyphosphate groups onto HA-ADH. The conjugation was also confirmed by 1H NMR and 31P NMR spectra, as reported in our previous work (data are not shown in the current study). The relationship between the degree of modification of PolyP and the reaction conditions was investigated. The effect of the reacted amount of PolyP on the degree of modification to the hydrazide group of the HA-ADH was examined via a molybdenum blue and a TNBS assay. The molybdenum blue assay can determine modified phosphates by counting the phosphate ions after the degradation of the conjugated PolyP, while the TNBS assay can detect unmodified amines of the hydrazide groups in HA-ADH, from which we could estimate the amount of modified phosphates. Both the molybdenum blue assay and the TNBS assay showed that the degree of modification of PolyP increased with an increasing amount of added PolyP (Figure 2C). By changing the equivalent of PolyP-Im with respect to the hydrazide groups as 0.1, 0.3, 0.5, and 1.0, the degree of modification of the PolyP, as determined via the molybdenum blue assay,


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increased to 0.95%, 6.1%, 7.0%, and 12% per the hydrazide groups, respectively. These results are consistent with the results of the TNBS assay, in which the degree of modification increased to 0.82%, 5.1%, 5.8%, and 11%, respectively. In our previous research, the degree of modification of HA-PolyP was not controlled by the reaction conditions. The results of the present research indicate that the degree of PolyP modification can be controlled by the added amount of PolyP.


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Figure 2. Characterization of HA-PolyP. (A, B) FT-IR spectra of PolyP-Im, HA-ADH, and HAPolyP. Arrows indicate new peaks resulting from modifications. (A) Whole spectra between 4000 and 400 cm-1. (B) Magnified spectra between 2000 and 400 cm-1. The absorbance at 1700 cm-1 is assigned as C=O. The absorbance at 1300 cm-1 is assigned as P=O. (C) Degree of modification of HA-PolyP, as determined through molybdenum blue assay and TNBS assay.


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3-2. Chelating properties of HA-PolyP with calcium ions Phosphates are known to chelate calcium ions, which play important roles in coagulation. The interaction of conjugated PolyP with calcium ions was examined via turbidity measurements (Figure 3). HA-PolyP was mixed with CaCl2 at various concentrations, and then its turbidity was measured via UV-vis. Free PolyP, HA, and HA-ADH were also used for comparison. The turbidity of unmodified HA was almost zero, and did not increase by increasing the calcium concentration. In addition, the turbidity of HA-ADH also did not increase following the addition of Ca2+, although HA-ADH without Ca2+ showed a higher turbidity than unmodified HA, presumably because of the increased hydrophobicity caused by ADH conjugation. On the other hand, the turbidity of free PolyP and HA-PolyP was increased by the addition of Ca2+, suggesting the chelation of Ca2+. In both free PolyP and HA-PolyP, the turbidity increased with increasing Ca2+, and this gradually reached a plateau around 20 mM. HA-PolyP showed turbidity increase with lower Ca2+ concentration than PolyP, which was similar level with blood calcium. These results suggested that HA-PolyP and HAX-PolyP can chelate blood Ca2+, which might affect hemostasis. To compensate this chelation, 25 mM of CaCl2 was added to all polymer samples for the following ex vivo evaluation of hemostatic activity. These results indicate that conjugated PolyP retained Ca2+ chelation ability.


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Figure 3. Effect of different polymer solutions and CaCl2 concentrations on turbidity measurements. The absorbance of PolyP, HA, HA-ADH, and HA-PolyP solutions with different CaCl2 concentrations was measured at 405 nm, and plotted against CaCl2 concentration.

3-3 Rapid gelation of HA-PolyP and the degradation kinetics of HAX-PolyP The conjugation of PolyP and its degree of modification affected the gelation behavior and the properties of the hydrogel. By mixing HA-PolyP with HA-CHO, a hydrogel can be formed via Schiff’s base formation, thanks to the hydrazide groups of HA-PolyP that are available for crosslinking, even after PolyP conjugation to HA-ADH.26 The gelation time measured via mixing by the stirrer was longer as the degree of modification of PolyP increased, from 17.6 to 209.6 s, as shown in Table 1. Moreover, the gelation time can also be controlled by using the mixture of HA-ADH and HA-PolyP. The gelation time became longer with increasing amounts of HA-PolyP, from 9.3 to 216.8 s, as shown in Table 1. We also tried to measure the gelation time of HAX-PolyP using the rheometer. However, because the gelation time was too short, G’ was already larger than G’’ and remained constant from the beginning of the measurements


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(Figure S1). HAX-PolyP showed G' of 28 Pa (A/P = 10/0), 22 Pa (A/P = 7/3), 22 Pa (A/P = 4/6), and 21 Pa (A/P = 1/9). These G' values are similar with that of previously reported HAX.25 These results suggested that HA-PolyP has a sufficient gelation rate as an injectable hydrogel. The time course of swelling and degradation in PBS at 37°C of HAX-PolyP with different mixture ratios of HA-ADH and HA-PolyP was measured gravimetrically. The degree of swelling of HAX-PolyP increased with an increasing ratio of HA-PolyP (Figure 4A). On day 7, while HAX (A/P = 10/0) swelled prominently, HAX-PolyP (A/P = 1/9) retained its initial structure (Figure 4C). On the other hand, the time required for the completion of degradation was similar between all the examined samples (Figure 4A). It took approximately 3 weeks for degradation, regardless of the amount of HA-PolyP. Dry mass of HAX-PolyP was also measured on day 7 and day 14 (Figure 4B). There was little difference in the dry mass between each hydrogel on both day 7 and day 14 (Figure 4B).

Table 1. Gelation time of cross-linkable hydrogel. Modification degree per HA [ % ]

Gelation Time [ s ]

Mixture ratio of HA-ADH and HA-PolyP [ - ]*

0.11

0.35

2.27

2.62

4.28

A/P = 10/0

A/P = 7/3

A/P = 4/6

A/P = 1/9

17.6±2.6

16.3±1.3

84.3±13.6

195.6±28.4

209.5±1.0

9.3±1.7

27.0±8.8

57.6±13.0

216.8±30.4

* A/P indicates the weight ratio of HA-ADH to HA-PolyP


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Figure 4. (A) Swelling and degradation kinetics of the hydrogels in PBS at 37°C with different mixture ratios of HA-ADH to HA-PolyP. Relative mass of the hydrogel (%) is the ratio of the weight of the hydrogel at each time point to the initial weight, expressed as a percentage. Data are expressed as average ± standard deviation (n = 4). (B) Dry mass of the hydrogels during immersion in PBS at 37°C with different mixture ratios of HA-ADH to HA-PolyP. Relative dry mass of the hydrogel (%) is the ratio of the dry weight of the hydrogel at each time point to the initial dry weight, expressed as a percentage. Data are expressed as average ± standard deviation (n = 4). (C) Appearance of HAX and HAX-PolyP. (i) Initial hydrogel before incubation and (ii) 7 days after starting incubation. The diameter of the hydrogel in (i) is 1.3 cm.


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3-4 Excellent in vitro biocompatibility of HA-PolyP and HAX-PolyP The cytotoxicity of the synthesized HA-PolyP was examined via a cell viability assay using three cell lines; RAW264.7 mouse macrophages, NIH3T3 mouse fibroblasts, and MeT-5A human mesothelial cells. HA-ADH was also used for comparison. In the case of RAW264.7 cells (Figure 5A), while 80–90% of the cells were alive in 0.01 mg/mL of the polymers, cell viability decreased with increasing concentrations. The cell viability was significantly lower in HA-PolyP compared with in HA-ADH. In the case of NIH3T3 cells (Figure 5B), the cytotoxicity observed in the RAW264.7 cells was mitigated for both HA-PolyP and HA-ADH. While cell viability in HA-ADH was almost constant at approximately 80% within the examined concentration range, that in HA-PolyP gradually decreased from 0.1 mg/mL. The cytotoxicity produced by HA-PolyP was milder in the case of MeT-5A cells (Figure 5C). Although cell viability at 1 mg/mL was decreased to approximately 26%, cell viability in HA-PolyP below 0.2 mg/mL was almost the same as that in HA-ADH, at approximately 80%. The cytotoxicity of HAX-PolyP was also examined through a cell viability assay using the three cell lines. HAX-PolyP (20 mg/mL concentration) did not show severe toxicity, unlike HAPolyP (Figure 5D). No significant difference was observed in the viabilities of cells incubated with HAX-PolyP and HAX. The cell viability of the three cell lines incubated with HAX-PolyP was almost the same as those incubated with HAX. Figures S2-4 show pictures of the three cell lines 1 day after the application of materials. The change in cell morphology was not observed by the application of materials in all three cell lines.


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Figure 5. Effect of HA-ADH and HA-PolyP on cell viability, as measured using MTT assay. Data are expressed as average ± standard deviation (n = 4). (A) Mouse macrophages (RAW264.7) after a 2 days incubation with polymers, (B) mouse fibroblast cells (NIH3T3) and (C) human mesothelial cells (MeT-5A) after a 3 days incubation with polymers. (D) Effect of 20 mg/mL HAX and 20 mg/mL HAX−PolyP on the cell viability of RAW264.7, NIH3T3 and MeT-


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5A cells, as measured using MTT assay. Data are expressed as average ± standard deviation (n = 4). ** and * indicated p < 0.001 and p < 0.05, respectively.

3-5 Excellent in vivo biocompatibility of HAX-PolyP in the mouse peritoneum. The biocompatibility of HAX-PolyP was examined via intraperitoneal and subcutaneous transplantation of the hydrogel into mice. No mice developed peritoneal adhesion after intraperitoneal administration of HAX-PolyP, as shown in Table 2, despite trauma induced by the injection. The hydrogels remained in the peritoneum 1 week after the injection (Table 2 and Figure 6A). HE staining was conducted to evaluate the effect of HAX-PolyP on surrounding tissues. HE staining of the hydrogel residues and surrounding tissues showed no peritoneal adhesion and inflammation (Figure 6B). A mesothelial cell layer was also observed at the interface between the residual HAX-PolyP and surrounding tissue (Figure 6B). In addition, in the case of subcutaneous transplantation, HAX-PolyP also remained under the dermis after 1 week (Table 2 and Figure 6D). The HE staining of the hydrogel residues and surrounding tissues did not indicate severe inflammation (Figure 6E). No decrease in body weight was observed, and all of the mice appeared healthy (Table 2). HE staining of lung was conducted for both administration routes, because PolyP with a DP of 60–100 was reported to induce pulmonary embolization following intravenous administration.10 However, in the current study, pulmonary embolism was not observed following intraperitoneal and subcutaneous administration of HAXPolyP (Figure 6C and F). These results suggest good biocompatibility of HAX-PolyP in vivo.


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Table 2. Evaluation of adhesions following intraperitoneal injection of hydrogels in mice Postperative day [ days ]

Administration route

Body weight [g]

Material residue Peritoneal adhesion

0

Intraperitoneally

33.5±1.3

0

Subcutaneously

33.5±1.7

7

Intraperitoneally

37.5±1.9

4/4

7

Subcutaneously

37.0±0.8

4/4

0/4

Figure 6. Intraperitoneally and subcutaneously administered HAX-PolyP 7 days after injection. Residues of the intraperitoneally (A, B, and C) and subcutaneously (D, E, and F) injected HAXPolyP were found in four mice after 7 days (n = 4). (A) Residues of the intraperitoneally injected HAX-PolyP 7 days after injection. (B) Hematoxylin & eosin (HE) stained sections of the interface between the hydrogel and abdominal wall 7 days after intraperitoneal injection of HAX-PolyP, ×10. (C) HE stained sections of the lung 7 days after intraperitoneal injection of


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HAX-PolyP, ×20. (D) Residues of the subcutaneously injected HAX-PolyP 7 days after injection. (E) HE stained sections of muscle tissue, 7 days after subcutaneous injection of HAXPolyP, ×10. (F) HE stained sections of the lungs, 7 days after subcutaneous injection of HAXPolyP, ×20.

3-6 Enhancement of clot formation of human blood by HA-PolyP The effect of HA-PolyP on hemostasis was examined in terms of the kinetics of clot formation and the mechanical properties of formed clots (Figure 7). Figure 7A shows changes in the mechanical strength of plasma containing an additive polymer, over time. The initial rise in the clot signal corresponds to the onset of clotting, while its slope thereafter, called as ‘clot rate (CR)’, represents the rate of clot development, which usually corresponds to the fibrin polymerization rate. The onset of clotting was almost identical for all examined samples. However, the slope of the clot signal after the onset was steeper in plasma containing HA-PolyP and free PolyP, compared with the other samples. These differences were further analyzed using the CR value shown in Figure 7B. The PolyP and HA-PolyP showed a 1.5-times higher CR compared with normal saline and HA-ADH (Figure 7B). These results suggest that the conjugated PolyP on HA could promote fibrin gel formation.


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Figure 7. (A) The effect of saline, HA-ADH, PolyP, and HA-PolyP solution on the mechanical properties of plasma during the coagulation process, as measured using a Sonoclot coagulation analyzer. The clot signal indicates the mechanical strength of the plasma with different polymer solutions. (B) Comparison of the clot rate between saline, HA-ADH, PolyP, and HA-PolyP solutions. The clot rate is defined as the slope of the clot signal during coagulation. It represents the rate of clot development, which usually corresponds to the fibrin polymerization rate. Data are expressed as average ± standard deviation (n = 4).

3-7 In vivo hemostatic capability of HAX-PolyP in a liver bleeding model The hemostatic activity of HAX-PolyP was examined using a mouse liver bleeding model. The HAX-PolyP was applied to the cut surface of the liver and the blood loss was measured to evaluate hemostatic activity. Hemostasis time was within 3 min in all cases. The amount of bleeding was decreased by HAX-PolyP application (Figure 8). The amount of bleeding in mice


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that received HAX-PolyP was significantly lower than in the groups without treatment and mice who underwent HAX application. Furthermore, even compared with a commercial hemostatic material, fibrin glue, HAX-PolyP produced almost the same level of hemostasis. These results indicate that the hemostatic activity of HAX-PolyP is comparable to that of fibrin glue.

Figure 8. Weight of blood lost from a bleeding mouse liver treated using 20 mg/mL HAX, 20 mg/mL HAX-PolyP and fibrin glue. Mice without application of a material were used as a control. Data are expressed as average ± standard deviation (n = 14, 12 or 10).

4. Discussion HAX-PolyP shows potential as an injectable hemostat and has the same effect as fibrin glue without using human blood products. HA-PolyP demonstrates rapid gelation properties for in situ gelation. The degree of modification of PolyP could be controlled by the amount of PolyPIm added at the reaction (Figure 2C). HA-PolyP formed hydrogels when mixed with HA-CHO.


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The gelation time was prolonged as PolyP content was increased, through a change in both the PolyP modification rate and the mixture rate with HA-ADH. This resulted from a decrease in the crosslinking point, caused by PolyP conjugation to the hydrazide group of HA-ADH or a decrease in the amount of HA-ADH in the mixture. Moreover, the degradation kinetics of the hydrogels were also affected by the ratio of HA-ADH to HA-PolyP. The hydrazide group in HAADH and the PolyP group in HA-PolyP are considered to cause electrostatic interaction that leads to an increase in crosslinking points, resulting in a difference in the degree of swelling. However, because the main crosslinking point created by the Schiff base did not change considerably, there was little difference in the dry mass and the degradation rate. Although HA-PolyP showed cytotoxicity above a certain polymer concentration, HAX-PolyP showed good biocompatibility in the cell viability assay (Figure 5). This was likely caused by a decrease in the free PolyP concentration following gelation. HAX and dextran-based Schiff base hydrogels have also demonstrated decreased cytotoxicity following gelation.32,

33

The

biocompatibility of the HAX-PolyP was further confirmed through intraperitoneal and subcutaneous injection into mice. Some materials, such as chitosan and its derivatives, have been reported to induce inflammation and peritoneal adhesions.17 However, HAX-PolyP did not cause inflammation in the surrounding subcutaneous tissue or adhesions in the peritoneal cavity, despite the perseverance of some material residues in these tissues. One specific concern in terms of the safety of PolyP-related material is pulmonary embolism formation. PolyP with a DP of 60–100, which is similar to the DP of the PolyP found in platelets, formed a thrombus following intravenous administration, as detailed in a previous report.10 However, we confirmed that HAXPolyP produced no pulmonary embolism using two different routes of injection. We consider that this difference can be explained by differing injection routes and the molecular weight of


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PolyP. Peritoneally and subcutaneously administered HAX-PolyP results in a gradual release of PolyP for a long period. During this process, PolyP would be degraded by enzymes in the tissue 34

, such as alkaline phosphatase, and therefore its migration into the blood stream would be

prevented. In addition, the degree of polymerization of the PolyP used in the current study was lower than that in the previous works. It has been reported that the hemostatic effect of PolyP highly depends on its DP. Therefore, it is possible that PolyP with a smaller DP would not induce thrombus formation. Another concern on safety of PolyP-related materials might be potential bone formation at the applied site, due to their osteoinductive properties. Because PolyP is naturally present in platelets and contributes to hemostasis, we consider that such bone formation would not be a critical issue for their use as an injectable hemostat. Further research on the relationship between bone formation and hemostasis will be necessary. These results suggested safety of HAX-PolyP. The obtained HA-PolyP accelerated blood coagulation in an ex vivo experiment using standard human plasma (Figure 7). It was revealed that while HA-PolyP did not affect the onset of clotting (clotting time) significantly, it increased the mechanical strength of clots immediately after the onset, meaning that a dense fibrin network was formed in the presence of HA-PolyP. It was reported that PolyP was incorporated into clots during fibrin polymerization and therefore the resulting clots had increased turbidity, thicker fibrils, increased hardness, and higher resistance to fibrinolysis.12 Our results are consistent with these previous reports. In vivo experiments using the liver bleeding model demonstrated that HAX-PolyP produced significantly decreased bleeding compared with a non-treated control and HAX. When HAXPolyP was compared with fibrin glue, the amount of bleeding was almost the same, suggesting that HAX-PolyP produced effective hemostatic activity. We consider that the conjugated PolyP,


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especially on the hydrogel surface, contributed to the hemostatic activity of HAX-PolyP. When applied to wound sites, PolyP contained in HAX-PolyP would get contact with blood without significant degradation. At that time, hemostasis would be promoted by PolyP via several mechanisms; the initiation of the contact pathway of the blood coagulation cascade10, the enhancement of coagulation factor V activation11, fibrin clot stabilization12, 13, and promotion of factor XI feedback activation by thrombin.14 Further optimization of the design of HA-PolyP, such as varying the DP of PolyP or the amount of PolyP in HAX-PolyP, could be helpful in improving the hemostatic effect of HAX-PolyP.

Conclusion An in situ cross-linkable hydrogel hemostat composed of HAX-PolyP was developed. The gelation time and swelling of HAX-PolyP could be controlled by varying its PolyP content. The good biocompatibility of HAX-PolyP was confirmed through a cell viability assay in vitro and intraperitoneal and subcutaneous administration to mice in vivo. Moreover, the hydrogel showed enhanced clotting ex vivo and efficient hemostatic activity in a mouse liver bleeding model in vivo, which was comparable to that of fibrin glue.

Supporting Information. Figure S1: Time dependency of the storage modulus G’ and loss modulus G’’ of the HAXPolyP with different mixture ratios of HA-PolyP and HA-ADH after the addition of HA-CHO. Figure S2: Microscopic images of mouse macrophages (RAW264.7) 1 day after the application of materials.


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Figure S3: Microscope images of mouse fibroblast cells (NIH3T3) 1 day after the application of materials. Figure S4: Microscope images of human mesothelial cells (MeT-5A) 1 day after the application of materials.

AUTHOR INFORMATION *Corresponding author. E-mail address: [email protected] Tel: +81-3-5841-1425; Fax: +81-3-5841-1697

ACKNOWLEDGMENT We thank Denka Co. for supplying hyaluronan. We also thank N. Kanno for her help in demonstrating the Sonoclot measurement technique. We are grateful to Keyence for the use of the fluorescence microscope, BZ-X700. This research was supported by Tokyo Society of Medical Sciences, Terumo Foundation for Life Sciences and Arts, and Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (16H02637).


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Table of Contents Graphic


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An Injectable Hemostat Composed of a Polyphosphate-Conjugated

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