Next Generation Hemostatic Materials Based on NHS-Ester Functionalized Poly(2-oxazoline)s

Abstract

In order to prevent hemorrhage during surgical procedures, a wide range of hemostatic agents have been developed. However, their efficacy is variable; hemostatic devices that use bioactive components to accelerate coagulation are dependent on natural sources, which limits reproducibility. Hybrid devices in which chain-end reactive poly(ethylene glycol) is employed as active component sometimes suffer from irregular cross-linking and dissolution of the polar PEG when blood flow is substantial. Herein, we describe a synthetic, nonbioactive hemostatic product by coating N-hydroxysuccinimide ester (NHS)-functional poly(2-oxazoline)s (POx-NHS) onto gelatin patches, which acts by formation of covalent cross-links between polymer, host blood proteins, gelatin and tissue to seal the wound site and prevent hemorrhage during surgery. We studied different process parameters (including polymer, carrier, and coating technique) in direct comparison with clinical products (Hemopatch and Tachosil) to obtain deeper understanding of this class of hemostatic products. In this work, we successfully prove the hemostatic efficacy of POx-NHS as polymer powders and coated patches both in vitro and in vivo against Hemopatch and Tachosil, demonstrating that POx-NHS are excellent candidate polymers for the development of next generation hemostatic patches.

Figure 1

Schematic overview of application method and mechanism of action of poly(2-oxazoline) coated hemostatic patches. (A) Preparation of hemostatic patch by spray-coating POx-NHS onto a gelatin sponge. (B) Application of the patch onto the wound site. (C) Hemostasis is obtained by covalent cross-linking between the gelatin sponge, POx-NHS, blood proteins, and tissue in order to create a gel which seals off the wound surface and stops the bleeding.

Scheme 1. Synthesis of NHS-Ester Functionalized Polymers (POx-NHS; P1–P7)

Reagents and conditions: (i) methyl tosylate, 140 °C, CH₃CN, (ii) 0.1 M NaOH, rt, (iii) NHS–OH, DIC, DCM, rt, (iv) 2-amino-ethanol, 60 °C, 300 mbar, (v) succinic anhydride, DMAP, DMF/DCM (v/v, 1:9, rt).

Figure 2

SEM images of POx-NHS coated patches (G1–G4) and Hemopatch (PEG). Scale bars correspond to 1 mm or 100 μm (bottom right picture).

Figure 3

(A, B) In vitro tests. (A) Blood uptake capacity as a function of coating density (**P < 0.001, *P < 0.01); (B) In vitro adhesion test: (i) blood was applied between the patches, (ii) the patches were allowed to cross-link for defined time points (t1, t5, t15 min), (iii) the samples were placed in a Zwick Roell tensile bench and a vertical force was applied until failure, (iv) results of the adhesion test (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

In vivo study on pig spleen. (A) Images of the different prototypes at selected time points (0, 1, and 5 min), including the success rate of hemostasis. (B) Bleeding scores after 5 min according to the scoring system