Polyphosphate-based compositions

Abstract

The present disclosure provides a composition that includes: a biocompatible macromolecule, such as a polymer, comprising cationic functional groups and anionic functional groups, and polyphosphate and methods for the manufacture of said composition. The disclosure further discloses the use of said composition for application to an individual at a site of bleeding in order to control said bleeding.

Description

POLYPHOSPHATE-BASED COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of US Provisional Application No. 63 / 709,082, filed on October 18, 2024, the contents of which are hereby incorporated herein by reference in their entirety. FIELD

[0002] The present disclosure relates to compositions that include polyphosphate and a biocompatible macromolecule. BACKGROUND

[0003] The following paragraphs are not an admission that anything discussed in them is prior art or part of the knowledge of persons skilled in the art.

[0004] Polyphosphates are produced and released by platelets and accelerate blood clot formation. Some polyphosphates have been used as a procoagulating agent, and have been used to modulate blood coagulation and fibrinolysis. INTRODUCTION

[0005] The following introduction is intended to introduce the reader to this specification but not to define any invention. One or more inventions may reside in a combination or sub-combination of the apparatus elements or method steps described below or in other parts of this document. The inventors do not waive or disclaim their rights to any invention or inventions disclosed in this specification merely by not describing such other invention or inventions in the claims.

[0006] During the clotting process, different molecules, proteins, clotting factors and inorganic electrolytes such as calcium and magnesium act at different stages of the clotting pathways. Platelets also release linear polyphosphates (PPs) with degrees of polymerization, Dp, of 60-100 at final serum concentrations up to ~50 µM. Synthetic PPs with similar Dpvalues as those secreted by platelets have been shown to decrease clotting time of the blood by acting at different stages of the clotting cascade. Coacervates made from divalent cations and PPs with Dpvalues ranging from 160- 10,000, also showed promising hemostatic properties with decrease in blood clotting time of 30-60 %, mostly through their effect on the extrinsic pathway. Mechanistically, PPs play different roles in blood clotting: they promote activation of the intrinsic pathway byacting as assembly template surfaces for different proteins essential in the intrinsic pathway, play the role of a cofactor and accelerate factor XI back activation by thrombin, act as a cofactor and accelerate the activation of factor V by thrombin and by factor Xa. Activation of factor V protects factor Xa from the function of tissue factor pathway inhibitor (TFPI), so the presence of PP abrogates the anticoagulant function of TFPI. All these effects cause an earlier thrombin burst which causes a faster clotting. The earlier thrombin burst also causes an earlier activation of the thrombin-activatable fibrinolysis inhibitor, resulting in a delayed fibrinolysis and stability of the clot. A few hours after contacting the blood or release by platelets, PPs degrade via the activity of phosphatases and lose their procoagulant function, limiting the expansion of the clot and allowing the wound healing process. PPs with similar size as those secreted by platelets have been shown to also reverse the effect of different anticoagulant drugs, such as inhibitors of factor Xa including Rivaroxaban, and shorten the clotting time of the blood obtained from patients with hemophilia by up to 80%.

[0007] However, PP in its glassy solid powder form or dissolved in an aqueous solution lacks the mechanical properties required for acting as an initial physical barrier against bleeding. In addition, in some applications it is desirable for the PP to be site- specifically delivered at a controlled rate to the bleeding site, otherwise excess PP in the blood can scavenge multivalent cations that are required for blood clotting, potentially causing delayed clotting, or preventing it completely.

[0008] Polyphosphates may also be used to aid bone regeneration, such as by increasing the rate of apatite mineral formation and / or the amount of apatite mineral that is formed. PPs induce the osteogenic differentiation of stem cells and play a role in regulating the formation of hydroxyapatite, the main mineral component of bones, by acting as the reservoir of the required inorganic molecules. During bone mineralization, tissue non-specific alkaline phosphatase cleaves the polyphosphate chains, increasing the orthophosphate concentration which contributes to an increase in the rate of and / or in the amount of apatite mineral formation.

[0009] In one aspect, the present disclosure provides a composition that includes: (1) a biocompatible macromolecule, such as a polymer, having cationic functional groups and anionic functional groups, and (2) polyphosphate. Without wishing to be bound by theory, the authors of the present disclosure believe that the cationic functional groups electrostatically attract the polyphosphate, while the anionic functional groups electrostatically repel the polyphosphate as well as increase the dispersion of the composition in aqueous media and / or increase fluid absorption by the composition. Theauthors of the present disclosure believe that changing the ratio of the cationic to the anionic functional groups can change the overall attraction between the macromolecule and the polyphosphate, and therefore can change the release rate of the polyphosphate from the composition. In some examples, the molar ratio of the cationic functional groups to the anionic functional groups is from about 10:1 to about 0.1:1, such as from about 4:1 to about 0.9:1.

[0010] The biocompatible macromolecule may have a chitosan backbone whose monomers independently include, at the 2-position of the D-glucosamine:– NH(CO)CH2CH2CO2-; –NH3+; or –NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8, such as a physiological solution.

[0011] The polyphosphate may have a degree of polymerization (Dp) from about 20 to about 10,000, such as from about 50 to about 200.

[0012] The weight ratio of the polyphosphate to the biocompatible macromolecule may be from about 1:100,000 to about 1:20 (polyphosphate : biocompatible macromolecule), such as from about 1:20,000 to about 1:30.

[0013] In a specific example, the biocompatible macromolecule has a polymer backbone that includes chitosan; the chitosan backbone includes monomers that independently include, at the 2-position of the D-glucosamine: –NH(CO)CH2CH2COO-; – NH3+; or –NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8 the molar ratio of the –NH3+to the –NH(CO)CH2CH2COO- is from about 5:1 to about 0.8:1; the polyphosphate has a degree of polymerization from about 60 to about 1000; and the polyphosphate and the biocompatible macromolecule are present in a weight ratio of about 1:20,000 to about 1:50 (polyphosphate : biocompatible macromolecule).

[0014] Biocompatible macromolecules according to the present disclosure may be used for site-specific delivery of polyphosphate for clotting blood in a subject, such as during surgery or trauma care; site-specific delivery of polyphosphate for antimicrobial activity in a subject; or site-specific delivery of polyphosphate for bone regeneration in a subject.

[0015] In another aspect, the present disclosure provides a method that includes mixing a polyphosphate solution with a solution of a biocompatible macromolecule, such as a polymer, that comprises cationic functional groups and anionic functional groups; freezing the polyphosphate and biocompatible macromolecule mixture; and lyophilizing the frozen mixture to form a lyophilized composition comprising the polyphosphate andthe biocompatible macromolecule. The lyophilized composition may be applied to a bleeding site in a subject.

[0016] The biocompatible macromolecule and / or the polyphosphate may be as described above.

[0017] In yet another aspect, the present disclosure provides a method that includes reacting chitosan with succinic anhydride, where a portion of the amine groups in the chitosan are kept protonated during the reaction, to produce a carboxylated chitosan; and mixing a polyphosphate solution with a solution of the carboxylated chitosan, for example in a solution that has a pH from 6 to 8.

[0018] The method may additionally include freezing the polyphosphate and carboxylated chitosan mixture; and lyophilizing the frozen mixture to form a lyophilized composition comprising the polyphosphate and the carboxylated chitosan. The lyophilized composition may be applied to a bleeding site in a subject. BRIEF DESCRIPTION OF DRAWINGS

[0019] Examples according to the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0020] FIG.1 is a microfluidic system used to measure the clotting time for blood in contact with sponges according to the present disclosure under dynamic conditions when blood flows in capillaries.

[0021] FIG.2 shows images of the system of FIG.1 at different time points, illustrating clotting over time.

[0022] FIG.3 shows images of citrated, recalcified and PP-contained recalcified whole bovine blood 25 and 60 min into the clotting process. The photos show inverted vials containing the blood samples at these two time points. The sample with PP contained 500 µM PP.

[0023] FIG.4 is a graph illustrating the color intensity in channel 2 of the microfluidic system of FIG.1 when there is no sponge in the column versus when LCS sponges, with or without PP, are present.

[0024] FIG.5 is an image of a powder according to the present disclosure.

[0025] FIG.6 shows a powder duster spraying the powder of FIG.5.DETAILED DESCRIPTION

[0026] In one aspect, the present disclosure provides a composition that includes: (1) a biocompatible macromolecule, such as a polymer, that includes cationic functional groups and anionic functional groups, and (2) polyphosphate.

[0027] In the context of the present disclosure, “biocompatible macromolecule” should be understood to refer to a macromolecule that is non-toxic to a biological system, such as an animal patient, for the duration of time that the macromolecule would be in use.

[0028] The term “macromolecule” should be understood to refer to molecule with a high relative molecular mass, the structure of which essentially includes the multiple repetition of units derived, actually or conceptually, from molecules of low relative molecular mass. A molecule can be regarded as having a high relative molecular mass if the addition or removal of one or a few of the units has a negligible effect on the molecular properties.

[0029] Examples of macromolecules include polymers, such as polysaccharides; proteins; and polypeptides. Macromolecules may be linear or branched. Macromolecules may be synthetic or purified from a natural source.

[0030] The cationic functional groups may be cationic, and the anionic functional groups are anionic, when the biocompatible macromolecule is in a solution that has a pH from 6 to 8.

[0031] The molar ratio of the cationic functional groups to the anionic functional groups may be from about 10:1 to about 0.1:1, such as from about 4:1 to about 0.9:1. Increasing the relative amount of the cationic functional groups is believed to result in greater electrostatic attraction with the polyphosphate, and therefore a reduced release rate under physiological conditions. Increasing the relative amount of the anionic function groups is believed to result in a reduced electrostatic attraction with the polyphosphate, and therefore an increased release rate under physiological conditions. In the context of the present disclosure, a “high” proportion of anionic groups may correspond to a ratio of around 0.9:1 (cationic functional groups to anionic functional groups); a “medium” proportion of anionic groups may correspond to a ratio of around 1.5:1 (cationic functional groups to anionic functional groups); and a “low” proportion of anionic groups may correspond to a ratio of around 4:1 (cationic functional groups to anionic functional groups).

[0032] The cationic functional groups may include a protonated or quaternary nitrogen, such as: -NR3+, wherein each R is independently H, methyl, ethyl, propyl orbutyl; a heterocyclic nitrogen-based functional group, such as a triazolium, a pyridinium, or an imidazolium functional group; a cyclic nitrogen-based functional group, such as a piperidinium, a piperazinium, or a pyrrolidinium functional group; an amidinium ion; an alkylated thiourea; or a protonated urea.

[0033] An exemplary macromolecule with various exemplary cationic functional gr reN NN NH NH H N+2H2Scheme 1

[0034] The anionic functional groups may include: carboxylate, phosphate, sulfate, sulfonate, or hydroxyl.

[0035] An exemplary macromolecule with various anionic functional groups is illustrated below in Sche- - OSO3 OPO2HScheme 2

[0036] The biocompatible macromolecule may include zwitterionic functional groups, such as phosphatidylcholine functional groups, phosphatidylethanolamine functional groups, or serine-based or phosphatidylserine functional groups. An exemplary macromolecule with various zwitterionic functional groups is illustrated below in scheme 3.HN+(CH3)3NH + 3 Scheme 3

[0037] Macromolecules according to the present disclosure may include combinations of one or more different cationic functional groups, anionic functional groups, and / or zwitterionic functional groups.

[0038] The biocompatible macromolecule may include the cationic, anionic and / or zwitterionic functional groups. Alternatively, or additionally, the biocompatible macromolecule may be modified to include the cationic, anionic and / or zwitterionic functional groups. For example, an uncharged biocompatible macromolecule may be chemically modified to add cationic and anionic functional groups, or may be chemically modified to add zwitterionic functional groups. In another example, a biocompatible macromolecule may include cationic functional groups, such as an amine group that would be protonated under physiological conditions, and may be chemically modified to add a cationic group. In yet another example, the biocompatible macromolecule includes the cationic and anionic functional groups, and / or the zwitterionic functional groups, and is not chemically modified.

[0039] The biocompatible macromolecule may be or may have a backbone that includes: an oligo- or poly-saccharide, such as dextran, cyclodextrin, cellulose, etherified carboxymethyl cellulose, oxidized cellulose, or chitosan; a protein, such as collagen, or gelatin; a polypeptide, such as poly-lysine; or a synthetic polymer, such as polyaniline.

[0040] It should be understood that macromolecules according to the present disclosure may inherently include cationic and anionic functional groups. Accordingly, the macromolecule may not require chemical modification to introduce anionic or cationic functional groups. An example of a macromolecule that inherently includes cationic and anionic functional groups is collagen.

[0041] It should also be understood that macromolecules according to the present disclosure may be chemically modif nionic or cationic functional groups.Accordingly, the macromolecule may have a backbone to which the anionic and / or cationic functional groups are introduced. An example of such a macromolecule is chitosan onto which anionic groups are added.

[0042] Chitosan is a linear polysaccharide that includes β-(1→4)-linked D- glucosamine and N-acetyl-D-glucosamine units. Chitosan may be made by deacetylating a portion of the N-acetyl-D-glucosamine units in chitin, which is a long-chain polymer of N-acetylglucosamine, such as by treating the chitin with sodium hydroxide. Using chitosan as the biocompatible macromolecule may be beneficial when the composition is for use as a hemostatic agent as chitosan also has hemostatic properties.

[0043] Some of the amine functional groups in the chitosan can be protonated and act as the cationic functional groups of the biocompatible macromolecule. Other of the amine functional groups can be chemically modified to introduce an anionic functional group, such as by reacting the amine with a cyclic anhydride, such as succinic anhydride, glutaric anhydride, maleic anhydride, or phthalic anhydride. Amine groups in other biocompatible macromolecules could be similarly chemically modified.

[0044] In one example, the biocompatible macromolecule has a polymer backbone that includes chitosan, and the chitosan backbone includes monomers that independently include, at the 2-position of the D-glucosamine: –NH(CO)CH2CH2COO-; –NH3+; or – NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8. About 10% to about 15% of the monomers in the chitosan may include – NH(CO)CH3. The molar ratio of the –NH3+to the –NH(CO)CH2CH2COO- may be from about 5:1 to about 0.8:1.

[0045] The chitosan backbone may have a molecular weight of about 5 kDa to about 7000 kDa, such as at least about 300 kDa, for example about 1200 kDa to about 2500 kDa, or about 1800 kDa.

[0046] Collagen is a structural protein in the extracellular matrix of various connective tissues. The amino acid sequence of collagen includes both positively and negatively charged amino acid residues, such as lysine, arginine, and histidine; and aspartic acid and glutamic acid, respectively. Using collagen as the biocompatible macromolecule may be beneficial when the composition is for use as a hemostatic agent as collagen also has hemostatic properties.

[0047] Polyphosphate is a polymer formed from phosphate structural units linked by shared oxygen atoms. It should be understood that, in the context of the present disclosure, polyphosphate refers to linear polymers containing a few to several thousand residues of orthophosphate linked by phosphoanhydride bonds. The number ofphosphate units present in the polymer may be referred to as the “degree of polymerization” (Dp). The polyphosphate according to the present disclosure may have a degree of polymerization from about 20 to about 10,000, such as from about 30 to about 200. Polyphosphates with a Dpfrom about 60 to about 100 have been identified as being produced and released by activated platelets, and as activating blood coagulation factor XII. Polyphosphates with a Dpfrom about 30 to about 50 have been shown to stimulate osteogenesis and chondrogenesis, and promote the growth and differentiation of bone marrow-derived mesenchymal stem cells (MSCs). Without wishing to be bound by theory, the authors of the present disclosure believe that the site-specific amount of phosphate contributes to the biological effects, and so a lower concentration of polyphosphates that have a larger Dpmay provide a substantially equivalent biological effect as a higher concentration of polyphosphates with a smaller Dp. For example, a 0.01 wt% mixture of polyphosphates having an average Dp of about 1000 may provide a substantially equivalent biological effect as 0.1 wt% mixture of polyphosphates having an average Dpof about 100.

[0048] The weight ratio of the polyphosphate to the biocompatible macromolecule may be from about 1:100,000 to about 1:20 (polyphosphate : biocompatible macromolecule), such as from about 1:20,000 to about 1:30.

[0049] In a specific example according to the present disclosure, the biocompatible macromolecule has a polymer backbone that includes chitosan; the chitosan backbone includes monomers that independently include, at the 2-position of the D-glucosamine: –NH(CO)CH2CH2COO-; –NH3+; or –NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8; the molar ratio of the –NH3+to the – NH(CO)CH2CH2COO- is from about 5:1 to about 0.8:1; the polyphosphate has a degree of polymerization from about 60 to about 1000; and the polyphosphate and the biocompatible macromolecule are present in a weight ratio of about 1:20,000 to about 1:50 (polyphosphate : biocompatible macromolecule).

[0050] Compositions according to the present disclosure may include one or more additional components. For example, a composition according to the present disclosure may include: an antibiotic; an antimicrobial compound, such as silver; or a nanomaterial, such as peptide nanoparticles, liposomes, lipid nanoparticles, or metal or metal oxide nanoparticles, for example copper-, zinc oxide-, gold-, or silver-based nanoparticles.

[0051] A composition according to the present disclosure may be in the form of, or may be formulated in: a powder, a flake, a lyophilized foam or sponge, a macroscopic bead, a nanoparticle, a microparticle a hydrogel a nanofiber, or a sheet. For example thecomposition may be: coated on or impregnated in a gauze or sponge; or formulated for application as a spray or a multi-layered sheet. In a specific example, a composition that includes chitosan as the backbone of the biocompatible macromolecule may be lyophilized and formed into a sponge. In another example, the lyophilized material may be processed into flakes or a powder. A composition in the form of a powder may be prepared by milling the composition in the form of a lyophilized sponge using a ball-mill. The resulting powder may be sieved to collect a powder having a mean size from 1 µm to 1,000 µm. In particular examples, the power may have a mean size that is from 10 µm to 100 µm, such as from 20 µm to 60 µm, for example a mean size that is 30 + / - 5 µm. The power may be sieved using 63 µm and 20 µm sieves. A composition in the form of a powder may be sprayed, such as using a powder duster. In yet another example, the biocompatible macromolecule, such as a carboxylated chitosan, may be spun into a gauze or dressing, and the polyphosphate may be added to the gauze or dressing. Adding the polyphosphate to the gauze or dressing may include adding a solution of polyphosphate to the gauze or dressing, and allowing the gauze or dressing to dry.

[0052] In some examples according to the present disclosure, polyphosphate is released from the biocompatible macromolecule at a rate of about 0.1 µg to about 100 µg, such as from about 0.5 µg to about 1 µg, of polyphosphate per hour per milligram of composition.

[0053] Compositions according to the present disclosure may be used for site- specific delivery of polyphosphate for clotting blood in a subject, such as during surgery or trauma care; site-specific delivery of polyphosphate for antimicrobial activity in a subject; or site-specific delivery of polyphosphate for bone regeneration in a subject.

[0054] Compositions according to the present disclosure may be especially useful for site-specific delivery of polyphosphate for clotting blood in a subject who has an impaired ability to make blood clots, for example wherein the subject suffers from hemophilia or is taking an anticoagulant medication, such as rivaroxaban, apixaban, unfractionated heparin, or enoxaparin.

[0055] In another aspect, the present disclosure provides a method that includes: mixing a polyphosphate solution with a solution of a biocompatible macromolecule, such as a polymer, that includes cationic functional groups and anionic functional groups; freezing the polyphosphate and biocompatible macromolecule mixture; and lyophilizing the frozen mixture to form a lyophilized composition comprising the polyphosphate and the biocompatible macromolecule. The lyophilized composition may be applied to a bleeding site in a subject.

[0056] The biocompatible macromolecule and / or the polyphosphate may be as discussed above.

[0057] In another aspect, the present disclosure provides a method that includes: reacting chitosan with succinic anhydride, wherein a portion of the amine groups in the chitosan are kept protonated during the reaction, to produce a carboxylated chitosan; and mixing a polyphosphate solution with a solution of the carboxylated chitosan. The polyphosphate solution may be mixed with the solution of the carboxylated chitosan in a solution that has a pH from 6 to 8.

[0058] The method may additionally include freezing the polyphosphate and carboxylated chitosan mixture; and lyophilizing the frozen mixture to form a lyophilized composition that includes the polyphosphate and the carboxylated chitosan. The lyophilized composition may be applied to a bleeding site in a subject.

[0059] During the reaction of the chitosan with the succinic anhydride, about 10% to about 80%, such as from about 40% to about 70%, of the amine groups in the chitosan may be kept protonated.

[0060] The chitosan may have a molecular weight of about 1200 kDa to about 2500 kDa, such as about 1800 kDa.

[0061] About 10% to about 15% of the monomers in the chitosan may include - NH(CO)CH3.

[0062] The polyphosphate may be as discussed above, for example the polyphosphate may have a degree of polymerization from about 60 to 100.

[0063] Experimental – Materials and Methods

[0064] PP synthesis and Chitosan functionalization: Sodium polyphosphate (PP) with an average Dpvalue of 83 was synthesized as reported in S. Fanaee, M. J. Filiaggi, Mater Adv 2023, 4, 1678 and A. Momeni, M. J. Filiaggi, J Non Cryst Solids 2013, 382, 11.

[0065] Briefly, sodium phosphate monobasic monohydrate (NaH2PO4·H2O, Sigma Aldrich, Canada) and sodium carbonate (Na2CO3, Sigma, Canada) were mixed at a Na / P molar ratio of 1.015. The mixture was melted at 900 °C for 4 h, then quickly quenched on a clean copper plate to produce a glassy film. The glass was then broken to small pieces followed by milling (planetary micro mill, Fritsch, Germany) for 30 min to create a fine powder.

[0066] Carboxylated chitosan samples with three different degrees of carboxylation (DC), were prepared by modification of chitosan using succinic anhydride (SA, Sigma, Switzerland). A high molecular weight chitosan (1800 kDa) with a degree of deacetylation (DD) of 0.87 was used in this study (Glentham Life Sciences, UK). A 1 wt% dispersion ofchitosan in water was prepared by addition of 1 M hydrochloric acid (HCl, Sigma, Canada) to protonate all the available amine groups on chitosan and dissolve the chitosan. An equivalent volume of 0.5 M sodium bicarbonate (NaHCO₃ ,Sigma, Canada) solution was then added to deprotonate half of the amine groups to increase the pH to 6.4, the pKa of the amine groups on chitosan. Under this condition, chitosan polymer chains have half of their amine groups protonated for complete dissolution in the solvent and the other half deprotonated and available for reaction with SA. The amount of SA needed to achieve the intended DC was calculated and gradually added to the solution in solid form assuming a stoichiometric reaction between SA and deprotonated amine. Since addition of the SA to the solution lowers the pH and can cause protonation of amine groups thereby interrupting the carboxylation reaction, further 0.5 M sodium bicarbonate was gradually added at the same time with the SA to maintain the pH at 5.5- 6.0. After addition of all the SA powder, the pH was adjusted to 7.4 by addition of 1 M sodium hydroxide (NaOH, Sigma, Canada) to complete the reaction under rigorous mixing for 30 min. The polymer was then precipitated using 2-propanol (Fisher, Canada) and subsequently dissolved in water at a concentration of 1 w%. This purification step was repeated two more times. Eventually, the polymer was dried under vacuum by lyophilization (Labconco® FreeZone®, USA) and milled manually to achieve a powder.

[0067] Characterization of PP and carboxylated chitosan samples: The average Dpof PP and its polydispersity (PD) were determined using liquid31P NMR spectroscopy (Bruker AV 300 MHz) on a 10 wt% PP l ti i D O i th f ll i ti(1) where Q1 is the area under the peak at approximately -7 ppm, corresponding to the terminal P atoms, and Q2is the area under the peak at approximately -21 ppm, corresponding to the P atoms within the chains. The PD was determined by serial fractionation of the PP solution in water using acetone, measuring the average Dpof each fraction, and calculating the weight average (Mw), and number average (Mn) molecular weight of the PP according to PD = Mw / Mn.

[0068] Functionalized chitosan samples were analyzed using1H NMR spectroscopy. Briefly, 5 mg of each sample was dissolved in a mixture of 980 µL D2O and 20 µL DCl and analyzed under the following parameters: 70 °C temperature, number of scans of 65, relaxation delay of 6 s, and acquisition time of 2 s. The degree of carboxylation was calculated as follows:(2) where Hd, Hc, and Ha are areas under the peaks of protons related to deacetylated monomers, carboxyl groups and acetyl groups, at approximately 5.3, 3.0, and 2.4 ppm, respectively. Degree of acetylation (DA) was also calculated based on the following equation: (3)

[0069] Preparation of hemostatic sponges: Hemostatic sponges were prepared by mixing functionalized chitosan and PP solutions. Different amounts of a 5 wt% PP solution in water were gradually added to 1 wt% aqueous solutions of samples with: DC of 0.45 (referred to as “high carboxylation”, or HCS), 0.32 (referred to as “medium carboxylation”, or MCS), and 0.18 (referred to as “low carboxylation”, or LCS) under vigorous stirring for 30 min. Final mixtures containing 0, 0.005, 0.05, 0.5, 1.0 and 2.0 w% total PP were then frozen at -80 °C for 3 h and freeze-dried for 72 h, ultimately forming spongy products.

[0070] The three sponges had the following molar percentages of anionic, cationic, and L MHCS 45% 43% 13% Table 1

[0071] Swelling and PP release: Carboxylated chitosan samples containing 0.5 w% PP, HCS-0.5% PP, MCS-0.5% PP, and LCS-0.5% PP, were used for studying the swelling ratio and P release. Disks with diameters of 4 mm were prepared by weighing out 40 mg of each sample (n = 3) and compressing them using a manual disk maker. The disks were then placed in tissue culture plate inserts (Transwell™ with membrane pore size of 8 µm, VWR®) filled with 4 mL of 20 mM TBS, pH 7.4. TBS was chosen as it offers the same pH and salt concentration as the blood, while being free of phosphorous that may interfere with P release measurements. The disks were removed at 0.25, 0.5, 0.75,1, 1.5, 2, 3, 5, 8, 24, 48, and 72 h time points, placed on a paper filter to remove surface moisture and weighed. At each time point, the buffer in contact with the disks was collected to be used for release studies and replaced with an equivalent amount of fresh buffer. The swelling ratio was calculated based on the following equation: (4) where Wtis the weight of each disk at time t, and W0is the initial weight of the disks (40 mg). The collected buffers in contact with disk samples at different time points were spiked with 2% nitric acid and analyzed using ICP to quantify P content. The percentages of released P over time with respect to the initial P content of each disk were then used to determine PP release behaviour of the three functionalized chitosan samples.

[0072] Interaction of PP with chitosan: The effect of the degree of carboxylation on binding of PP with functionalized chitosan samples was investigated using ITC (Microcal VP-ITC, US). Here, a 0.1 w% PP solution was gradually injected into a 0.1 w% carboxylated chitosan dispersion while a constant temperature of 25 °C was maintained between the sample cell and the reference cell (filled with a buffer). Under these conditions, any heat absorbed or released by interaction between the macromolecules is compensated with the internal heating system to keep the cells at the same temperature, yielding raw data showing the power used for this compensation over time (µcal s−1), which is then converted to heat (kcal mol−1of titrant) to produce thermograms showing the ∆H of each injection against the molar ratio of the titrant to macromolecule. Twenty- nine 10-µL injections of 0.1 wt% PP were added to a sample cell filled with 0.1 wt% CC solutions with different carboxylation degrees at 5 min intervals. The experiments were conducted at 25 °C and the solutions were in PBS buffer with pH 7.4. The heat of dilution, obtained from titrating PP into the buffer, was subtracted from the heat thermograms.

[0073] CC polymers apparent electrical potential: The apparent ζ-potential values, assuming equivalent spherical particles for polymer chains, for CC polymer samples were measured using electrophoretic DLS by a Litesizer 500 instrument (Anton Paar, USA). The polymer solutions were injected into Omega cuvettes at a concentration of 0.2 w% in PBS buffer with pH = 7.4. The samples were allowed to equilibrate for 2 minutes at 25 °C before each measurement. The ζ-potential values were determined for each sample after 4 independent runs using Smoluchowski approximation.

[0074] Rheological characterization of blood clotting: The blood clotting capability of HCS, MCS, and LCS samples containing different amounts of PP was quantitativelyanalyzed by measuring the rheological properties of whole bovine blood (CL1700-500C, Cedarlane®). The HCS, MCS, and LCS sponge samples containing 0, 0.005, 0.05, 0.5,1.0, and 2.0 w% PP capable of releasing a maximum of 0, 5, 50, 500, 1000, and 2000 µM PP with the sponge to blood weight ratio of 1 to 100, respectively, were evaluated. The sponge samples were processed into flakes by exposing to liquid nitrogen (−196 °C) while mixing using a glass rod. The obtained flakes were then freeze-dried and stored at room temperature until use. The sponge flakes allowed for homogeneous distribution of samples in blood during the rheological characterizations.

[0075] Subsequently, citrated blood and a calcium chloride solution (CaCl2,75 mM) were placed in a water bath at 37 °C for 3 min. The CaCl2solution was then added to the blood to a final Ca2+concentration of 5 mM followed by brief vortex mixing. The recalcified blood was then dispensed on the rheometer plate. The sample flakes were dispersed on the blood (1 mg sample / 100 µL blood) and clotting proceeded with the sample sandwiched between the plates using a 40-mm parallel-plate geometry. The temperature was set at 37 °C and evaporation was minimized by covering the circumference of the geometry using silicon oil (45-55 mm² s−1viscosity, Thermo Scientific). To assess the impact of PP alone, PP solution was introduced to the blood before its deposition between the plates at final PP concentrations of 5, 50, 500, 1000, and 2000 µM, similar to the concentrations used with the sponges. Rheological characterizations of citrated blood and recalcified blood were also analyzed as controls.

[0076] All the rheological properties were measured under oscillatory shear using an HR10 rheometer (TA Instruments, USA). Coagulation was monitored over time at an angular frequency ω = 10 rad s−1and strain amplitude γ = 1% (in the linear viscoelastic regime). When the properties reached a steady-state (up to 6 h), a frequency sweep in the range ω = 0.1–100 rad s−1was undertaken at γ = 1%, followed by a strain sweep γ = 0.1%–300% at ω = 10 rad s−1.

[0077] Clotting evaluation under dynamic conditions using a microfluidic system: A microfluidic system was designed and fabricated (FIG.1) to measure the clotting time for blood in contact with the sponges under dynamic conditions when blood flows in capillaries. The design consisted of two inlets for citrated blood (inlet 1) and calcium solution (inlet 2) connected to two outlets through two spiral microchannels (300 µm × 200 µm). The outlets were connected to two conical columns (FINNTIP®1000, Fisher Scientific) containing 5 mg of identical sponge samples.

[0078] In channel 2, the citrated blood and calcium solution mix and flow toward column 2, where blood can clot upon contact with the sponge. Conversely, unmixedcitrated blood in channel 1, which lacks clotting capability, flows toward column 1. Initially, the channels exhibit equal hydraulic resistance, resulting in the complete filling of both, as shown in the top view images (FIG.2). As clotting begins in column 2, the hydraulic resistance in channel 2 increases, leading to a reduction in blood flow compared to channel 1. Upon complete clotting in column 2, blood flow in channel 2 ceases, causing it to transition from red to clear.

[0079] Throughout the experiments, the device was recorded from both top and side views. The top-view recordings were analyzed using a custom MATLAB code to track and report the red color intensity change in the channels. Briefly, the MATLAB code: • reads the video; • allows the user to draw two rectangular regions of interest (ROIs) in the displayed frame; • asks the user to input a time interval for analyzing the frames throughout the video; • for each frame at specific time intervals, extracts the intensity values of the red pixels in the defined ROIs; • generates plots showing the red intensity over time for each ROI; and • smooths red intensity signals and exports the smoothed data.

[0080] The clotting time was reported as the time required for the intensity to reach zero in channel 2 and confirmed by side-view recordings showing the cessation of blood flow in column 2. The clotting times were statistically compared using a student’s t-test (P < 0.05, N=3). The device provides a reliable approach that could be used for evaluation of haemostatic properties of various materials, from powders to sponges, reducing the number of in vivo animal tests required for efficacy evaluation.

[0081] The microfluidic device containing the discussed network of microchannels was fabricated from a polydimethylsiloxane (PDMS) layer, bonded to a 25 mm × 70 mm × 1 mm glass slide (Fisher Scientific). The PDMS layer was fabricated by replica molding of PDMS (10 : 1 ratio of elastomer and curing agent, SYLGARD™ 184, Dow®, USA) onto a 3D-printed (MARS 4 Ultra, Elegoo, China) plastic master mold. The PDMS pre-polymer was poured over the master mold and cured at 80 °C for 3 h. The PDMS layer and the glass slide were then bonded together by plasma bonding at 1 Torr and 50 W for 90 s (PDC-001-HP Harrick Plasma, USA). Before each experiment, the assembled device was washed with deionized water and then with pure ethanol followed by drying using compressed air. The inlets were fed with their relevant liquids from 10 mL plastic syringes (Fisher Scientific) connected to them by polyethylene tubing and glass capillaries (innerdiameter of 0.11 cm, Fisher Scientific). The syringe containing blood was placed in an injection pump (FUSION 200, Chemyx, US) with the injection rate set at 40 µL min−1while the syringe containing calcium stock solution (500 mM) was placed in another syringe pump (HEPHO, China) with its injection rate set at 5 µL min−1.

[0082] In vitro cell toxicity evaluations: Cytotoxicity of PP and the highly carboxylated chitosan (HCS) was studied using a lactate dehydrogenase (LDH) assay (LDH Assay Kit, Sigma) with the reaction being monitored by following the change in absorbance at 490 nm. NIH / 3T3 fibroblast cells (ATCC, USA) were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (vol / vol) fetal bovine serum, L-glutamine (4 mM), penicillin G (100 μg / mL), and streptomycin (100 μg / mL) at 37 °C in a humidified atmosphere with 5% CO2and 95% air. The NIH / 3T3 cells were seeded in a 96 well plate at a seeding density of 2,500 cells / well. The cells were incubated for 24 h to allow attachment and growth before the PP or HCS solutions were added to the media with final concentrations of 0 – 104µM or 0 – 1 w%, respectively. The NIH / 3T3 cells were incubated in the treated media for 24 h before assessing for cytotoxicity at a confluency of 75%.

[0083] In vitro bacterial growth evaluation: disks (20 mg) containing either HCS-0.5% PP, MCS-0.5% PP, LCS-0.5% PP, or Chitosam™(a hemostatic, non-woven chitosan dressing spun directly from chitosan) were prepared in triplicate and sterilized under UV for 60 min. The disks were placed in a 48-well plate followed by addition of 500 µL of Staphylococcus aureus suspensions in BHI broth (OD600=0.001) to each well. The well plates were covered and incubated for 5 h. Subsequently, 20 µL of media from each well was removed and mixed with 180 µL PBS for spot-plating. The spot plates were then incubated for 18 hr and read for CFU mL−1to evaluate their support of bacteria growth in the presence of the samples.

[0084] Preparation of a hemostatic powder: A powder format of the polyphosphate- carboxylated chitosan hemostatic material was prepared by milling sponges that were prepared as discussed above. The sponges were milled using a ball-mill for 10 minutes at medium power. The resulting powder was sieved using 63 µM and 20 µm sieves, and analyzed for shape and size (Morphophologi 4, Malvern). An exemplary image is shown in FIG.5. The powder had a mean size of 31.7 µM and circularity index of 0.8 (n=8400). The sprayability of the powder was tested and confirmed using a powder duster (illustrated in FIG.6). The powder material was demonstrated to have a high liquid absorption capacity (not shown), which is desirable since a high fluid absorption helps thelocal concentration of clotting factors and red blood cells, and this leads to improved hemostasis compared to hemostasis without the powder material.

[0085] Results

[0086] The synthesized PP in this study had an average Dpof 83 as determined using nuclear magnetic resonance spectroscopy (31P NMR), which is within the 60-100 range secreted by platelets and has been shown to have pro-hemostatic characteristics. The PP had a low polydispersity of Mw / Mn= 1.05. The synthesized PP was a brittle glassy solid with a glass transition temperature, Tg, of ~ 300 ºC and lacked the proper handling properties for physically blocking blood flow in hemostatic applications alone in this form.

[0087] As the carrier of PP, carboxylated chitosan (CC) samples that can have both attractive and repulsive interactions with PP were synthesized. A controlled number of negatively-charged carboxyl groups were added to the backbone of chitosan with final degrees of carboxylation (DC) in the range 0.09-0.48 as determined by1H NMR spectroscopy (defined as the ratio of monomers carrying a carboxyl group to all monomers in the chitosan polymer). These different numbers of negatively-charged carboxyl groups were introduced to chitosan to control the extent of bonding between chitosan and PP through competing electrostatic interactions between negatively-charged phosphate units on PP, positively-charged amine, and negatively-charged carboxyl groups on the CC samples, potentially allowing control over the PP release kinetics. Additionally, carboxylation of chitosan allows its dissolution in neutral pH and makes the final freeze-dried sponge greatly hydrophilic, which is favorable in hemostasis applications where a high capacity of blood and exudate absorption is required.

[0088] Samples with DC of 0.45 (referred to as “high carboxylation”, HCS), 0.32 (referred to as “medium carboxylation”, MCS), and 0.18 (referred to as “low carboxylation”, LCS) were used in this study. The results of the LDH assay on PP and the sa Ce a y . . . . . (%)100 ± 5.410.65 13.84 15.39 9.64 6.48 Ta Cy (%)100 ± 0.86. . . . 143 267 2.72 3.48Table 3

[0089] Concentrations of PP from 1 – 1000 µM PP, compared to control (0 µM), did not have any significant effect on the viability of the cells. The only significant effect (p < 0.05) was observed when the cells were treated with a highly concentrated, 10000 µM PP solution. However, the PP content of the samples in this study did not exceed 2 w%, which in similar conditions to rheology, PP release, and microfluidic studies, where 1 mg / 100 µL blood was used, and only in case of complete release would a concentration of 2000 µM occur. However, based on the release studies, complete release only happens at extended periods of time (> 48 h). Therefore, PP release from the samples in the desired application times (up to 24 h) is not cytotoxic as its concentration would be far below 10000 µM. It has been well established in the literature that chitosan is non- cytotoxic and safe to use. So, to evaluate the effect of chitosan carboxylation on the viability of the cells, the sample with the greatest carboxylation degree (i.e., HCS) was chosen and its effect was studied in various concentrations from 0.05 – 1 w%. None of the concentrations affected the viability of the cells significantly compared to the control group (0 w%). Therefore, it can be concluded that the hemostatic sponges containing PP and carboxylated chitosan prepared in this study are not cytotoxic.

[0090] The following table presents bacteria growth evaluation of the samples considering the effect of carboxylation degree. None of the samples exhibited a more favorable environment for bacterial growth 5 h post incubation, indicating that carboxylation degree is not a significant factor in bacterial growth compared to the ChitosamTMcontrHCS-0.5%PP 7.54E+08 1.98E+08 Table 4

[0091] Cell toxicity studies of HCS samples indicated no sign of reduced cell viability in the presence of these polymers, while PP was also found non-toxic to cells within the concentration range used in preparing all sponges in this study. In addition, the HCS, MCS and LCS samples showed antibacterial performance 5 h post incubation that was comparable to ChitosamTM, a 100% non-modified, commercially available chitosan-basedhemostatic sponge dressing, indicating that carboxylation degree did not negatively affect the natural antibacterial performance of chitosan as a hemostatic polymer.

[0092] Before testing the hemostatic properties of the PP-CC sponges, the effect of PP addition to blood was quantitatively studied using rheological analysis to evaluate the effective concentration range of PP for accelerated blood clotting. Citrated bovine blood lacks the ability to clot and shows a storage modulus G’ = 0.5 Pa, and a loss modulus G’’ = 0.2 Pa, which remain almost constant over time. However, recalcified (RC) blood clots over time, and its G’ increases following equation:where G’0 is the initial storage modulus before clotting, G’maxis the maximum storage modulus at long times, t is time, t1 / 2is the time when G’ = G’max / 2, and α is the relation coefficient. Similar sigmoidal models have been used previously for predicting mechanical properties and gelation kinetics of hydrogels as the gelation occurs (see, for example, V. Adibnia, R. J. Hill, J Rheol (N Y N Y) 2016, 60, 541). Addition of PP to the recalcified blood decreased the blood clotting time (FIG.3). Quantitatively, the table below shows that addition of 5, 50, 500, and 1000 µM PP to the blood, decreased t1 / 2by 40-70% compared to the recalcified blood with no PP.Table 5

[0093] This is consistent with the qualitative pro-hemostatic effect of PP reported in other studies. However, the effect of PP was dose-dependent such that addition of up to 50 µM of PP to the blood consistently decreased the blood clotting time, while further increases in PP concentration resulted in progressively longer blood clotting times. Notably, addition of 2000 µM PP completely inhibited the clotting of the blood. This effect could arise because PP has attractive interactions with Ca2+, so excess PP could result in scavenging the Ca2+required for blood clotting. Also, in the case of 1000 µM PP, the clot had a G’max of less than 40 Pa, suggesting that although PP decreased the clotting time, at such high concentrations, it negatively affects the mechanical properties of the blood clot. Lower concentrations of PP (5-500 µM), however, resulted in G’maxvalues of 100- 150 Pa, close to 100 Pa for the normal recalcified blood. This significant dose-dependenteffect of PP on the blood clotting kinetics highlights the importance of its controlled release for effective blood clotting. It should be noted that fibrin clots have been shown to have evident strain-stiffening behavior due to their fibrous structure that plays a significant role in the wound healing process. Importantly, addition of PP did not change the typical strain-stiffening behavior of the blood clot.

[0094] Based on the results obtained from the effect of PP concentration on the blood clotting, PP-CC sponges containing 0.5 w% PP were prepared from HCS, MCS and LCS by forming the lyophilized PP-CC compositions into disks, as discussed above.

[0095] Upon PP release from these sponges the PP concentration in a buffer with a weight 100-times larger than the weight of the sponge would be ≤ 500 µM at any time. These PP-CC sponges were highly hydrophilic and swelled in aqueous media. When the dried sponges underwent swelling analysis in Tris buffered saline (TBS) at pH = 7.4, it was shown that at different time points within 0 - 8 hrs, HCS and MCS had similar swelling behaviors. HCS reached its maximum swelling ratio of 17.5 at 8 h, remaining unchanged thereafter up to 24 h perhaps due to the disintegration of the gel and diffusion of highly hydrophilic polymers through the membrane as the sample swells by absorption of the buffer. In contrast, MCS continued swelling and reached its maximum swelling of 24-times its dry weight in 24 h but showed a mass loss afterwards similarly to the HCS hydrogel. In comparison, LCS swelled at a slower rate and reached its maximum of 7.5 after 8 h. This sample did not swell beyond this point nor exhibit any significant weight loss due to bulk degradation. Compared to the other commercially available chitosan- based hemostatic material, ChitosamTM, LCS showed a similar swelling profile, whereas MCS and HCS showed more than double the swelling ratios within the first 5 h of exposure to the buffer. Considering that hemostatic devices are typically used for less than 5 h on the wound, these data highlight a potentially significant increase in blood absorption for MCS and HCS in heavy bleeding conditions.

[0096] During the swelling stage, the buffer media were tested for phosphorous (P) release using inductively coupled plasma mass spectrometry (ICP). Between 0-8 h, considering that the bulk disintegration in all the samples was low and their swelling rate was high, the P release was dominated by a diffusion-based mechanism of PP out of the swelled sponge. This hypothesis was supported by fitting the data to the Higuchi model that describes the diffusional release of a substance from a homogenous matrix into the medium:where Qmis the amount of substance released at time t, D is the diffusion coefficient of the substance in the matrix, and c is a constant parameter. During 0-8 h for all the three samples, P concentration in the buffer changed linearly with √t with slopes of 20, 16.7, and 13.9 for HCS, MCS, and LCS, respectively, emphasizing that within this time frame, the release is primarily governed by PP diffusion out of the hydrogel. As the slope changed linearly with √D, it could be concluded that the rate of release (diffusion) from HCS-0.5% PP > MCS-0.5% PP > LCS-0.5% PP. This observation may suggest that the attractive interactions between PP and CC were stronger in LCS compared to MCS and HCS because of the higher number of amine groups on the polymer. All samples showed PP release within the concentration range of < 500 µM, which is the effective range of PP for blood clotting.

[0097] To investigate the molecular interactions between PP and CC, isothermal titration calorimetry (ITC) analyses were conducted. These interactions are expected to be primarily governed by electrostatic attraction between phosphate groups on PP and the protonated amine groups on CC, as well as repulsive interactions between phosphate groups on PP and carboxyl groups on CC. All measurements were conducted at pH 7.4, using phosphate buffer saline (PBS) solution as the solvent in both the reference and sample cells.

[0098] The variations of the total enthalpy of interactions, Q, with the ratio of phosphate units of PP to protonated amine groups of CCs, n, are shown in the table be N r / -4.35 -0.2 333 -045 2.65 -1.2211.13 -0.09 8.50 -0.23 6.78 -0.34 Table 6 and can be explained by two interconnected phenomena: (i) an endothermic CC unfolding event by addition of PP through repulsive interactions between phosphate groups on PP and carboxyl groups on CC, and (ii) an exothermic binding event between PP and CC through attractive interactions of phosphate groups on PP and protonated amine groups on CC. Although all three CC samples carry a net negative charge with apparent ζ-potential values of -21.8, -16 and -8.8 mV, for HCS, MCS, and LCS, respectively, they all carry both positively- and negatively-charged functional groups through protonated amine (with pka ~ 6.4) and carboxyl groups, as evident from the1H NMR spectra of the polymers. Therefore, the polymer chains are expected to have inter- chain electrostatic attractive interactions, with more frequency for LCS compared to MCS and HCS. Upon addition of PP to the HCS solution in the sample cell, an endothermic reaction was observed, likely due to the unfolding of HCS chains. This unfolding, however, resulted in more protonated amine being available for interaction with phosphate groups of PP. Therefore, by addition of more PP, the balance between the HCS chain unfolding and binding between phosphate and protonated amine caused a local minimum in the enthalpy curve at n* = dQ / dn = 0. Similar phenomena occurred with MCS and LCS, with the difference that the initial endothermic enthalpy of interaction was smaller while n* obtaining lower values of n 064, 2.19, and 2.50 for LCS, MCS, andHCS, respectively, due to the lower number of carboxyl groups on the MCS and LCS polymer chains. From this point, addition of more PP resulted in less heat being produced by binding phosphates to protonated amine groups because of the decrease in the number of available amine groups. Notably at this stage, the value of n** = d2Q / dn2= 0, which signifies the nominal stoichiometric value where the reactive sites are all occupied, increases from 4.54 to 6.00 to 7.85 for LCS, MCS, and HCS, respectively. This suggests that with the larger negative charge of HCS compared to MCS and LCS, more phosphate groups relative to amine groups are needed to occupy the binding sites as the electrostatic repulsion between PP and CC decreases the probability of phosphate / amine binding. These observations explain how the competing electrostatic interactions between protonated amine groups, phosphate groups, and carboxyl groups affect complexation of PP with CC and lead to the release kinetics of PP being significantly faster when embedded in CC polymer networks with higher amount of carboxylation of the chitosan chains as observed above.

[0099] Analysis of swelling and PP release indicated that LCS, MCS and HCS sponges may behave differently when exposed to blood due to the different intermolecular competing electrostatic interactions in these samples, which results in different release kinetics and fluid absorption capacity. To quantitatively evaluate blood clotting kinetics and mechanical properties of the blood clot in the presence of the sponges, rheological characterization of recalcified bovine blood in presence of LCS, MCS, and HCS containing 0 – 2 wt% PP was performed for 1 mg sponge / 100 µL blood at 37 ˚C. For these experiments, samples were made into fine flakes to achieve a homogenous distribution on the blood and more consistent and homogeneous blood clotting results.

[0100] The following tables shows representative G’ time-sweep data for the three samples with th T13.54 34.2169 50.3039 255.172103.60 93.7669 102.994 439.654177.89 92.1159 129.129 455.310.1 84.1199 8.14742 69.6636 5.25473 384.004 15.8451100 116.807 74.04 81.9711 52.7109 486.739 90.8495 Ta O 0 0 0 0 0 0 2.50991 105.37 2.50639 140.413 2.49904 1 7 2 6399.923 6 1 Table 9

[0101] The G’ time-sweep data were fitted with Equation 5, with the resulting t1 / 2, G’0, an LG’0(Pa)L L92.74 83.50 66.58 50.81 62.38 95.83 6 1 2 7 1 9 Table 10

[0102] All three samples showed rapid swelling in the blood, which resulted in the G’0values shown in Table 10. The values of G’0were significantly larger for LCS than MCS and HCS due to less swelling of LCS samples, which resulted in mechanically stiffer gels. The average G’0values for HCS, MCS, and LCS containing different PP amounts were 30 ± 15, 38 ± 18, and 243 ± 27 Pa, which are significantly higher than the near-zero G’0observed for RC blood treated with PP solutions. This initial swelling can play a significant role in the hemostatic ability of the sponges by acting as a physical barrier to the blood flow. Over time, the blood clotting process became dominant and the G’ value rose until it reached a plateau when a stable clot was formed. HCS, MCS, and LCS without added PP had t1 / 2values of 206, 168, and 93 min, respectively. The decrease in clotting time with the decrease in the degree of carboxylation could be due to the presence of more protonated amine groups in polymers with lower DC, since these functional groups attract negatively-charged red blood cells, helping to crosslink the clot and form a solid structure faster. By addition of 0 – 2 w% PP, the t1 / 2values decreased by up to 70 %, 60%, and 45%, for HCS, MCS, and LCS, respectively. This improvement in clotting time indicates that, through controlled-release of PP from the sponges, the PP concentration in the blood remained in the effective range for hemostasis of < 500 µM, which is consistent with the amount of PP release from the sponges after 3 h of exposure to fluids that was reported above. Table 10 shows the G’maxvalues for samples with different amounts of PP. The average G’maxvalues of the samples made from HCS, MCS, and LCS containing 0 – 2 w% PP were 114 ± 29, 109 ± 21, and 502 ± 69 Pa, respectively, indicating significantly stiffer clots formed in presence of LCS samples, which is consistent with lower swelling capacity of this polymer, while PP did not play any significant role inchanging the mechanical properties in these experiments. The frequency-independent G’ of clots with the three sponges indicated that they are all solid hydrogel structures with stable low-frequency plateaus. In addition, the sponges did not change the strain- stiffening behavior of blood clots regardless of PP concentration or degree of carboxylation of the chitosan, suggesting that the fibrillar structure of the fibrin clots are still dominant in the microstructure of the blood clots formed in the presence of the sponges.

[0103] The effect of the PP-doped sponges on clotting time was also studied using a microfluidic system. Unlike rheological characterization of the clotting, which occurs under static conditions, the microfluidic approach enabled evaluation of the clotting times in a more realistic setting under blood flow (FIG.1). The device was video recorded from a top view and the clotting time was determined by monitoring the red color intensity change in the channels and columns containing RC blood (FIG.2). In the absence of any sponges, the device can be used to evaluate blood clotting dynamics of RC blood in channel 2 compared to citrated blood in channel 1 under an initial flow rate of 40 µL min−1. Presence of LCS sponges with and without PP accelerated blood clotting of RC blood as shown in FIG.4 as evaluated from the change in color intensity in channel 2. The blood clotting tim S C. . . . . . . . Time 3.83 5.35 5.09 5.40 6.73 5.54 2.07 0.36 (min) Table 11

[0104] Chitosam™did not significantly reduce the clotting time of the blood compared to the control group (RC blood in the absence of any sponge or PP). Similar to the results obtained for the rheological characterization of blood clotting, the HCS sponge did not show any significant improvement in blood clotting rate. However, addition of 0.5 w% PP to this HCS sponge reduced the blood clotting time by 35%, whereas this number was ~60% under the static conditions. For MCS, however, there was no indication of hemostatic efficacy with blood flow compared to the recalcified blood control despite 16% faster blood clotting under static conditions in the absence of PP. Nevertheless, addition of 0.5 w% PP to the MCS sponge reduced blood clotting time by 39% under flow compared to ~60% under the static conditions. The LCS sponge, on the other hand, resulted in a 59% and 76% decrease in the blood clotting time under blood flow without and with inclusion of PP, respectively, as compared to 53% and 75% under staticconditions, indicating the most consistent and effective blood clotting under static and flow conditions among all tested samples.

[0105] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the examples. However, it will be apparent to one skilled in the art that these specific details are not required. Accordingly, what has been described is merely illustrative of the application of the described examples and numerous modifications and variations are possible in light of the above teachings.

[0106] Since the above description provides examples, it will be appreciated that modifications and variations can be effected to the particular examples by those of skill in the art. Accordingly, the scope of the claims should not be limited by the particular examples set forth herein, but should be construed in a manner consistent with the specification as a whole.

Claims

CLAIMS 1. A composition comprising: a biocompatible macromolecule, such as a polymer, comprising cationic functional groups and anionic functional groups, and polyphosphate.

2. The composition according to claim 1, wherein the cationic functional groups are cationic, and the anionic functional groups are anionic, when the biocompatible macromolecule is in a solution that has a pH from 6 to 8.

3. The composition according to claim 1 or 2, wherein the molar ratio of the cationic functional groups to the anionic functional groups is from about 10:1 to about 0.1:1, such as from about 4:1 to about 0.9:

1.

4. The composition according to any one of claims 1 to 3, wherein the cationic functional groups include a protonated or quaternary nitrogen, such as: -NR3+, wherein each R is independently H, methyl, ethyl, propyl or butyl; a heterocyclic nitrogen-based functional group, such as a triazolium, a pyridinium, or an imidazolium functional group; a cyclic nitrogen-based functional group, such as a piperidinium, a piperazinium, or a pyrrolidinium functional group; an amidinium ion; an alkylated thiourea; or a protonated urea; preferably wherein the cationic functional groups include -NH3+.

5. The composition according to any one of claims 1 to 4, wherein the anionic functional groups include: carboxylate, phosphate, sulfate, sulfonate, or hydroxyl.

6. The composition according to any one of claims 1 to 3, wherein the biocompatible macromolecule comprises zwitterionic functional groups, such as phosphatidylcholine functional groups, phosphatidylethanolamine functional groups, or phosphatidylserine functional groups.

7. The composition according to any one of claims 1 to 6, wherein the biocompatible macromolecule is, or has a backbone comprising: chitosan; an oligo- or poly-saccharide, such as dextran, cyclodextrin, cellulose, etherified carboxymethyl cellulose, or oxidized cellulose;a protein, such as collagen, or gelatin; a polypeptide, such as poly-lysine; or polyaniline.

8. The composition according to claim 7, wherein the biocompatible macromolecule has a polymer backbone comprising chitosan, and the chitosan backbone comprises monomers that independently include, at the 2-position of the D-glucosamine: – NH(CO)CH2CH2COO-; –NH3+; or –NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8.

9. The composition according to claim 7 or 8, wherein: about 10% to about 15% of the monomers in the chitosan comprise -NH(CO)CH3; and / or the chitosan backbone has a molecular weight of about 1200 kDa to about 2500 kDa, such as about 1800 kDa.

10. The composition according to claim 8 or 9, wherein the molar ratio of the –NH3+to the –NH(CO)CH2CH2COO- is from about 5:1 to about 0.8:

1.

11. The composition according to any one of claims 1 to 10, wherein the polyphosphate has a degree of polymerization (Dp) from about 20 to about 10,000, such as from about 30 to about 200.

12. The composition according to any one of claims 1 to 11, wherein the weight ratio of the polyphosphate to the biocompatible macromolecule is from about 1:100,000 to about 1:20 (polyphosphate : biocompatible macromolecule), such as from about 1:20,000 to about 1:

30.

13. The composition according to claim 1, wherein: the biocompatible macromolecule has a polymer backbone comprising chitosan; the chitosan backbone comprises monomers that independently include, at the 2- position of the D-glucosamine: –NH(CO)CH2CH2COO-; –NH3+; or –NH(CO)CH3when the biocompatible macromolecule is in a solution that has a pH from 6 to 8;the molar ratio of the –NH3+to the –NH(CO)CH2CH2COO- is from about 5:1 to about 0.8:1; the polyphosphate has a degree of polymerization from about 60 to about 1000; and the polyphosphate and the biocompatible macromolecule are present in a weight ratio of about 1:20,000 to about 1:50 (polyphosphate : biocompatible macromolecule).

14. The composition according to any one of claims 1 to 13, wherein the composition further comprises: an antibiotic; an antimicrobial compound, such as silver; or a nanomaterial, such as peptide nanoparticles, liposomes, lipid nanoparticles, or metal or metal oxide nanoparticles, for example copper-, zinc oxide-, gold-, or silver-based nanoparticles.

15. The composition according to any one of claims 1 to 14, wherein the composition is in the form of, or is formulated in: a powder, a flake, a lyophilized foam or sponge, a macroscopic bead, a nanoparticle, a microparticle, a hydrogel, a nanofiber, or a sheet; preferably wherein the powder has a mean size that is from 1 µm to 1,000 µm, such as from 10 µm to 100 µm, or from 20 µm to 60 µm, for example 30 + / - 5 µm.

16. The composition according to claim 15, wherein the composition is: coated on or impregnated in a gauze or sponge; or formulated for application as a spray or a multi-layered sheet.

17. The composition according to any one of claims 1 to 16, wherein the polyphosphate is released from the biocompatible macromolecule at a rate of about 0.1 µg to about 100 µg, such as from about 0.5 µg to about 1 µg, of polyphosphate per hour per milligram of composition.

18. The composition according to any one of claim 1 to 17 for: site-specific delivery of polyphosphate for clotting blood in a subject, such as during surgery or trauma care;site-specific delivery of polyphosphate for antimicrobial activity in a subject; or site-specific delivery of polyphosphate for bone regeneration in a subject.

19. The composition according to claim 18, wherein the subject has an impaired ability to make blood clots, for example wherein the subject suffers from hemophilia or is taking an anticoagulant medication, such as rivaroxaban, apixaban, unfractionated heparin, or enoxaparin.

20. A method comprising: mixing a polyphosphate solution with a solution of a biocompatible macromolecule, such as a polymer, that comprises cationic functional groups and anionic functional groups; freezing the polyphosphate and biocompatible macromolecule mixture; and lyophilizing the frozen mixture to form a lyophilized composition comprising the polyphosphate and the biocompatible macromolecule.

21. The method according to claim 20, further comprising applying the lyophilized composition to a bleeding site in a subject.

22. The method according to claim 20, further comprising milling the lyophilized composition to form a powder, optionally sieving the powder to collect a sieved powder having a mean size that is from 1 µm to 1,000 µm, such as from 10 µm to 100 µm, or from 20 µm to 60 µm, for example 30 + / - 5 µm.

23. The method of any one of claims 20 to 22, wherein the biocompatible macromolecule is as defined in any one of claims 1 to 10.

24. The method of any one of claims 20 to 23, wherein the polyphosphate is as defined in claims 11 or 12.

25. The method of claim 21 or 23, wherein the biocompatible macromolecule and the polyphosphate are as defined in claim 13.

26. A method comprising: reacting chitosan with succinic anhydride, wherein a portion of the amine groups in the chitosan are kept protonated during the reaction, to produce a carboxylated chitosan; and mixing a polyphosphate solution with a solution of the carboxylated chitosan.

27. The method according to claim 26, wherein the polyphosphate solution is mixed with the solution of the carboxylated chitosan in a solution that has a pH from 6 to 8.

28. The method according to claim 26 or 27, further comprising: freezing the polyphosphate and carboxylated chitosan mixture; and lyophilizing the frozen mixture to form a lyophilized composition comprising the polyphosphate and the carboxylated chitosan.

29. The method according to claim 27, further comprising applying the lyophilized composition to a bleeding site in a subject.

30. The method according to claim 28, further comprising milling the lyophilized composition to form a powder, optionally sieving the powder to collect a sieved powder having a mean size that is from 1 µm to 1,000 µm, such as from 10 µm to 100 µm, or from 20 µm to 60 µm, for example 30 + / - 5 µm.

31. The method according to any one of claims 26 to 30, wherein about 10% to about 80%, such as from about 40% to about 70%, of the amine groups in the chitosan are kept protonated during the reaction.

32. The method according to any one of claims 26 to 31, wherein about 10% to about 15% of the monomers in the chitosan comprise -NH(CO)CH3; and / or the chitosan has a molecular weight of about 1200 kDa to about 2500 kDa, such as about 1800 kDa.

33. The method according to any one of claims 26 to 32, wherein the polyphosphate has a degree of polymerization from about 60 to 100.

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