Chitosan dressings containing inorganic additives and levofloxacin as potential wound care products with enhanced hemostatic properties

Highlights

• Chitosan based wound dressings with inorganic additives were prepared

• Wound dressings with additives exhibited enhanced swelling properties in comparison with chitosan

• Improved in vitro and in vivo hemostatic action was confirmed

Abstract

Despite the progress in the development of hemostatic products, efficient treatment solutions to control hemorrhage upon wounding are still necessary. Chitosan (CS) is a natural hydrogel-forming polysaccharide, easy to modify for specific applications. Inorganic compounds in turn possess documented hemostatic properties. In this study, innovative hemostatic products based on CS, containing the inorganic additives aluminum chloride, aluminum sulfate hydrate or iron(III) sulfate and the antibiotic Levofloxacin were prepared, and their potential use as hemostatic materials was investigated. Structural characteristics, physical state and drug loading/release properties were examined. Strong interactions developed between CS and the additives, the pore size in the resulting products was affected, swelling increased up to 2500% and the stability of the wound dressings improved. The crystallinity of Levofloxacin reduced, and its release was immediate. The materials showed biocompatibility upon contact with cultured keratinocytes, hemocompatibility and hemostatic efficacy in vitro and in vivo.

Introduction

Hemostasis is the first stage of the wound healing process activated upon injury, that results in the control of bleeding and the formation of a protective barrier [1]. The mechanism of hemostasis includes: 1) vasoconstriction, 2) formation of a platelet plug, and 3) blood coagulation [2].

In cases of severe wounds, the use of hemostatic products is necessary to compensate for the compromised first step of wound closure [3]. According to the United States' military statistics, nearly 50% of deaths on the battlefield are caused by excessive bleeding, whereas more than 15% of the casualties are completely preventable [4].

Ongoing research is focused on designing products that can stop bleeding quickly and without side effects. The characteristics of an ideal hemostatic material are: rapid arrest of arterial and/or venous bleeding with no thermal effect (a sharp rise in temperature in the wound) [5], non-toxicity, flexibility, adaptability to the wound's shape, easy to remove without leaving residues, and stability in extreme temperatures [6].

Recently, various hemostatic agents were added to the list of available treatments, with different structures, coagulant composition, and modes of action. Commercially available ones are based on kaolin (Combat Gauze®), methyl cellulose (Surgicel®, Oxycel®), fibrinogen and thrombin (Thachosil®, Evarrest®) and gelatin (Spongostan®) [[7], [8], [9], [10], [11]]. Although easy-to-use and non-invasive, most of them are moderately effective in stopping severe bleeding, expensive, or raise safety concerns [9,12].

Chitosan (CS) is as a naturally derived polymer that plays a leading role in the development of new hemostatic products [13]. CS is a cationic polysaccharide with bactericidal properties [14], renewable, nontoxic, biodegradable and hydrophilic with high reactivity, promotes coagulation, flocculation and biosorption. The hemostatic properties of chitosan are due to direct electrostatic interactions between negatively charged red blood cells and platelets and the positively charged CS. Researchers and pharmaceutical companies are focusing on the hemostatic properties of CS by formulating it into several hemostatic products. CS-based hemostatic products exist in the form of bandages as Chitoflex®, HemCon® and ChitoGauze®, powders as Chitodine® and Celox Granules®, gels as Chitoseal® and XStat®, which is a mini hemostatic sponge dressing that is applied on a wound cavity using a lightweight applicator [11]. Some of these hemostatic products have drawbacks that limit their use in excessive bleeding [15]. Specifically, Chitodine® which contains CS powder with adsorbed elemental iodine can induce hypersensitivity reactions such as urticaria, angioedema, cutaneous hemorrhage or purpura, fever, arthralgia, lymphadenopathy, and eosinophilia [16]. The reported hypersensitivity reactions are not caused by all the CS-containing hemostatic products, concurring that CS is not responsible for these side effects. Hemostatic gauzes and patches are difficult to apply on small wounds without cutting or tearing it to fit [17]. HemCon® is a stiff, brittle hemostatic gauze that was difficult to apply effectively on a wound, and due to its low efficiency it was replaced by Chitoflex® [7]. Chitoflex® is an antibacterial, biocompatible wound dressing which adheres strongly to tissue surfaces and seals the wound surface [16]. Another CS-based hemostatic dressing currently used throughout the world by civilian and military first responders in control of compressible hemorrhage is Celox Granules®. It is an effective CS-based hemostatic that promotes clot formation through adsorption, dehydration, and facilitates blood cell bonding [18]. ChitoGauze® is a hemostatic dressing composed of a polyester medical gauze that is coated with CS. It is used for the external, temporary control of severely bleeding wounds [19]. However, Celox Granules®, ChitoGauze® and Chitoflex® were characterized inappropriate hemostatic agents when the access to the bleeding vessel is limited [18,20]. XStat is the first hemostatic agent designed for use in deep-tract or narrow-entrance wounds and is a cellulose sponge coated with CS [16,20]. Among many commercially available hemostatic agents, most hemostatic dressings which are currently recommended for clinical use are based on CS.

Ongoing research is focusing on advanced hemostatic CS-based materials with good biocompatibility, rapid hemostatic ability and low manufacturing cost [21]. CS was combined with various synthetic polymers, such as poly (vinyl alcohol) (PVA). CS-modified PVA sponge was found to be a hemostatic compound with desirable chemical, mechanical and physical properties, such as fast swelling [6]. Hemostatic products were also designed with CS and natural polymers – collagen for instance. Collagen imparts outstanding properties to CS, owing its porous structure that accelerates the absorption of water from blood flow on the wound site, improving the concentration of clotting factors [3]. Inorganic additives such as nanobioglass, silica, ions of calcium, phosphate and zinc, were also used with CS to develop composite systems with improved blood clotting properties. Inorganic materials seem to play a major role in initiating the coagulation cascade [3,14,15] and have been traditionally used as agents for hemorrhage control [22]. The most common inorganic hemostatic agents are aluminum chloride (ACl), iron(III) sulfate (FS), and occasionally aluminum sulfate (AS). ACl (5–25 wt%) is one of the most used astringents and acts by constricting blood vessels and extracting fluid from tissues [23]. FS is widely used as a hemostatic agent [24], since when it comes in contact with blood, a ferric ion–protein complex is formed that acts as a seal on the vessel wall, accelerating hemostasis more when compared to ACl [25]. It coagulates blood so quickly (1–3 min) that it must be placed directly against the damaged tissue. AS is a biocompatible, effective hemostatic agent with low-grade inflammation effect [26].

Hemostatic products with the ability to create gels when in contact with water and/or blood without being dissolved exhibit unique advantages. The gel can form an active coating that protects the wounded area from germs, absorbs and retains water within its structure, while removing excess exudates from the wound surface, and allows proper gaseous exchange [27]. CS can form such hydrogels by ionic cross-linking.

In this study, we hypothesized that the addition of different inorganic compounds in CS could lead to a wound dressing with improved stability, compact structure and enhanced hemostatic activity. The inorganic compounds were expected to play a dual role; that of ionic crosslinkers of CS, and that of hemostasis accelerators. To do so, CS wound dressings with the inorganic compounds AS, FS or ACl in various concentrations and loaded with the antibiotic levofloxacin (LEV) were prepared. LEV is an antibacterial drug, active against both Gram-positive and Gram-negative bacteria [28] and was used as a model antibiotic. To our knowledge, the combination of CS with ACl, AS or FS for hemostatic wound dressings has not been explored yet. All materials were in powder form with the aim to allow application to wounds of any shape and depth, and to be able to suppress external hemorrhage that is not amenable to other bleeding control methods [29]. These additives were expected to enhance the hemostatic action and the quality of CS wound dressings, thus protecting the wound from external factors. The morphology, chemical structure, stability, swelling, drug loading and release rate were evaluated. Finally, the in vitro and in vivo hemostatic potential of the materials was examined.