(T1130-05-29) Sodium Alginate-Based Bio-Adhesive Hydrogel: A Hemostatic Agent, Endoscopically Deliverable to the Gastrointestinal Resection Site

Purpose: Endoscopic resection (ER) methods like endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) are used as the primary approach for the elimination of early gastrointestinal (GI) neoplasia. Hemorrhage is a well-established complication of ER and can present a difficulty to the endoscopist both intraoperatively and postoperatively in the cases of delayed bleeding (DB). Hemorrhage following ER has the potential to result in adverse outcomes. Current strategies for achieving hemostasis during ER, such as electrocoagulation and surgical clip application, may elevate the likelihood of thermal damage or obstruct subsequent resections ¹. Although epinephrine injections serve as a beneficial supplement to hemostasis, their advantages are typically short-lived, necessitating definitive intervention. Generally, synthetic topical hemostatic agents have been utilized to manage intraprocedural bleeding (IPB) and forestall delayed bleeding (DB) post-ER ¹. In this context, we developed a hemostatic agent that is comprised of two components, namely, sodium alginate and CaCl₂ that form a bio-adhesive hydrogel barrier, in vivo, for management of IPB and DB, moreover, it could be delivered to the resection site endoscopically through a catheter designed specifically to administer the hemostatic agent. Further objectives are to achieve better or comparable rheological and bio-adhesiveness properties of Ca alginate gel compared to the commercially available PuraStat^{® 2} hydrogel, which is comprised of 2.5% w/v of a synthetic repeating peptide (acetyl-[arginyl-alanyl-aspartyl-alanyl]4-amide tetrahydrochloride) in sterile water for injection supplied in a prefilled syringe.

Methods: The sodium alginate (Pronova UP MVG (IFF N&H Norway AS)) 2.8% w/v was dissolved in deionized water (DW) using a magnetic stirrer for 24 h until clear solution was obtained. In addition, a solution of CaCl₂ (1.2 M) was formulated by dissolving CaCl₂ in DW. Following this, the Na alginate and CaCl₂ solutions were mixed in a 10:1 ratio to fabricate Ca alginate gel. The viscosity of sodium alginate solution was determined concerning shear rate and shear stress to identify their Newtonian or non-Newtonian attributes using the Rheometer (DHR-2, TA Instruments, New Castle DE). Furthermore, the gelation/cross-linking reaction between sodium alginate and CaCl₂ was assessed by time sweep rheology. Samples were analyzed for storage modulus (G′), loss modulus (G″), and complex viscosity (η*) at a frequency of 10 rad/s during a time sweep experiment over 5 min. Moreover, the adhesion force of the selected hydrogels was determined utilizing an oscillation frequency sweep at 25 °C and 37 °C, encompassing aspects of viscosity and bio-adhesiveness. Overall, a detailed analysis was performed on the rheological characteristics as well as bio-adhesiveness of both Na alginate solution and Ca alginate gel and finally compared in all aspects to the commercially available PuraStat^{® 2}.

Results: Alginate is a naturally occurring linear anionic polysaccharide which consists of α-L-guluronate (G) residues and β-D-mannuronate (M) residues linked through 1-4 glycosidic bonds. The G and M residues may be arranged as blocks of consecutive G residues, consecutive M residues, and alternating M and G residues ³. Calcium binds to the guluronate blocks of adjacent alginate chains, thus, forming ionic cross-links between polymer chains, which leads to the ridged gel structure. The rigidity of a calcium alginate gel strongly correlates with the length of the alginate G-blocks; therefore, alginates with a high G/M ratio exhibit stronger gel properties than alginates with a lower ratio ². The rheological characterization of hydrogels revealed that as the shear rate increased from 0 to 1000 1/s, sodium alginate showed shear thinning likewise PuraStat^® (Figure 1). Also, the time sweep rheology confirmed the gelation reaction between sodium alginate and CaCl₂ leading to Ca alginate hydrogel formation in approximately 12.4 seconds (Figure 2). Moreover, the obtained higher Tan δ (delta) values of Ca alginate hydrogel as compared to PuraStat^® using oscillation frequency sweep rheology represented significantly more bio-adhesiveness of alginate hydrogel (Figure 3).

Conclusion: An alginate-based hydrogel was successfully formulated through a reaction with CaCl₂ using ionotropic gelation. Extensive in vitro and ex vivo studies of alginate hydrogel cross-linked with CaCl₂ demonstrated outstanding rheological and bio-adhesive characteristics similar to those of the commercially accessible PuraStat^®.

References: 1. Subramaniam S, Kandiah K, Thayalasekaran S, Longcroft-Wheaton G, Bhandari P. Haemostasis and prevention of bleeding related to ER: The role of a novel self-assembling peptide. United European Gastroenterol J. 2019 Feb;7(1):155-162. doi: 10.1177/2050640618811504. Epub 2018 Nov 5. PMID: 30788128; PMCID: PMC6374844.
2. Gil, Eun Seok et al. Comparison of PuraStat self-assembling peptide hydrogel versus mineral-based Hemospray for endoscopic hemostasis of upper and lower gastrointestinal lesions in pigs. Frontiers in Gastroenterology (2022).
3. Lee KY, Mooney DJ. Alginate: properties and biomedical applications. Prog Polym Sci. 2012 Jan;37(1):106-126. doi: 10.1016/j.progpolymsci.2011.06.003. PMID: 22125349; PMCID: PMC3223967

Acknowledgements: We acknowledge Origin Endoscopy and Maryland Innovation Initiative (MII) for funding this project.


Figure 1: Graphs showing the rheological properties of sodium alginate (2.8% w/v Pronova UP MVG) and PuraStat® as a function of shear rate vs shear stress as evaluated using rheometer at [A] 25 °C and [B] 37 °C. For these graphs, the curvature indicated non-Newtonian behavior. Viscosity profile during upcurve and downcurve of sodium alginate (2.8% w/v Pronova UP MVG) and PuraStat® [C] 25 °C and [D] 37 °C. Both represent shear-thinning behavior.


Figure 2: Graphs represent time-sweep rheology at 37 °C of sodium alginate (2.8% w/v Pronova UP MVG) [A] without and [B] with CaCl2, [C] PuraStat®, here the Tan δ (delta) < 1 exhibit an already gel character and [D] time onset gelation of 2.8% w/v Pronova UP MVG upon reaction with CaCl2. The gelation reaction between sodium alginate and CaCl2 leading to Ca alginate hydrogel formation in approximately 12.4 seconds is known as the gel point where the storage modulus crosses over the loss modulus and at which Tan δ becomes equal to 1.


Figure 3: Graphs represent frequency-sweep rheology at 37 °C of [A] sodium alginate (2.8% w/v Pronova UP MVG) cross-linked with CaCl2 and [B] PuraStat® and [C] Tan δ (delta) values of sodium alginate (2.8% w/v) cross-linked with CaCl2 and PuraStat® at a frequency of 0.1 rad/s to 100 rad/s. Tan δ values of Ca alginate hydrogel can be directly related to significantly more bio-adhesiveness as compared to PuraStat®.