Chitosan-based Bioactive Nanofibrous Hemostatic Agent for Emergency Care

Abstract

Uncontrollable bleeding in major arteries has been reported to cause preventable 50% of battlefield casualties and 31% of mortalities in civilians worldwide. In India, road traffic injuries are a major concern, which causes 40% of deaths due to hemorrhage, and there is a rise of 2.4% every year. Commercially available hemostatic agents require at least 1-2 minutes for blood clotting, and most are either difficult to apply, expensive, or produce exothermic reaction upon contact with blood to cause adverse reactions. The objective of this study was therefore to investigate a novel self- assembly-based facile method to fabricate chitosan-casein/gelatin nanofibers through polyelectrolyte complex (PEC) formation for rapid hemostasis. The efficacy of hemostasis was improved by optimizing the process parameters and incorporating biologically active nanoparticles to nanofibrous PECs. The also investigated the effect of nanoparticles (e.g., ZnO-NP, AgNP) on improving bioactivity of the chitosan-based nanofibrous PEC under in vitro and in vivo conditions. FTIR spectroscopy revealed that amide group (1630 cm-1) of chitosan and phosphate group (910 cm-1) of casein could form nanofibrous PEC with electrostatic interaction at pH 8.2±0.2. The chitosan and casein in the ratio of 30:70 (CC30), 50:50 (CC50), and 70:30 (CC70) nanofibrous PECs allowed platelet adhesion and rapidly absorbed blood fluid to form rapid blood clots within 9±3, 16±3, and 30±4 s, respectively, which were better than commercially available Celox™ (90±3s). Increasing the concentration of chitosan from 10% to 90% in the CC formulations increased the productivity (r=0.99) of PECs but led to increased blood clotting time (r=0.90) due to an increase in zeta potential (r=0.98), fiber diameter (r=0.93), and decreased surface porosity (r=- 0.99), absorption capacity (r=-0.99). The pH also influenced zeta potential of PEC, with an optimized pH of 8.0±0.1 yielding clear nanofibers. Sonication improved the segregation of nanofibers by promoting water removal. The optimized PECs containing chitosan and casein in ratio of 30:70 (CC30) at a pH of 8.0 and dehydration under sonication could clot the blood within 9±2s in vitro and 9±2s in rat femoral artery puncture model with no evidence of rebleeding. The CC-based nanofibrous PEC were highly hemocompatible, biocompatible, non-toxic, and non- immunogenic. The chitosan-casein PECs could also be developed as microporous hemostatic sponge (CC30G) with porosity of 73.00±4.74%, pore diameter of 42.66±5.33 μm, and rapid water absorption capacity (1165±55%). The CC30G sponge showed bacteriostatic action against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. Upon incorporation of AgNP, the sponge (CC30GS) acquired the bactericidal property. The sponge could also be combined with adhesive tape to develop as a hemostatic band-aid for bleeding in skin lacerations, cuts, and topical wounds. Bioactivity of CC-based PECs (CC30Z) could be improved by incorporation of ZnO-NPs without compromising their hemostatic efficiency or biocompatibility. ZnO-NP incorporated PECs (CC30Z) were bioactive in terms of their bactericidal effect against Gram-positive S. aureus and Gram-negative E. coli, promoting cellular metabolic enzyme activity (alkaline phosphatase, glutamate dehydrogenase, lactate dehydrogenase, and malate dehydrogenase) for skin regeneration, and enhanced platelet aggregation and activation for rapid hemostasis. Results further showed that replacing casein with gelatin in the chitosan-based PECs could also increase the compressive elastic modulus of PEC-induced blood clots from 21±2 kPa to 68.6 ± 6.4 kPa, which was five-fold better than a commercially available CeloxTM-induced blood clot. Ag-NP (CG30S) and ZnO-NP (CG30Z) incorporation in chitosan-gelatin PEC did not affect the fiber diameter, surface porosity, hemocompatibility, and biocompatibility of the chitosan-gelatin PECs nanofibers but had bactericidal effect on both Gram-positive S. aureus and Gram-negative E. coli. Both CG30S and CG30Z could clot the blood within 10 s under in vitro conditions and rat femoral artery puncture model in vivo. Further, CG30Z had excellent bioactivity in promoting cellular/tissue metabolic enzymes involved in skin regeneration and could enhance platelet aggregation as well as activation. Taken together, chitosan-casein/gelatin nanofibrous PEC could rapidly clot the blood with 10 s under in vitro conditions by promoting platelet activation and aggregation, rapid absorption of plasma, and activation of extrinsic coagulation pathway. It could also clot blood within 10 s in rat femoral artery puncture model and within 25s in rabbit ear artery model. The PEC was bioactive, bactericidal, hemocompatible, biocompatible, non-toxic, non-immunogenic and safe for use in animal models. The chitosan-based PECs could also be developed as hemostatic sponge for skin cuts and lacerations. Further pre-clinical trials on large animals should be conducted under GLP conditions before clinical trials in humans for a successful market-ready product.