Green approach for fabrication of bacterial cellulose-chitosan composites in the solutions of carbonic acid under high pressure CO₂

Highlights

• Bacterial cellulose-chitosan films were obtained in “green” carbonic acid solutions.

• The chitosan amount in the composite was tripled.

• Chitosan nanoaggregates covered the microfibrils of the bacterial cellulose.

• Chitosan impregnation increased the specific surface area of the material.

Abstract

The functionalization of the bacterial cellulose (BC) surface with a chitosan biopolymer to expand the areas of possible applications of the modified BC is an important scientific task. The creation of such composites in the carbonic acid solutions that were performed in this work has several advantages in terms of being biocompatible and eco-friendly. Quantitative analysis of chitosan content in the composite was conducted by tritium-labeled chitosan radioactivity detection method and this showed three times increased chitosan loading. Different physicochemical methods showed successful incorporation of chitosan into the BC matrix and interaction with it through hydrogen bonds. Microscopy results showed that the chitosan coating with a thickness of around 10 nm was formed in the bulk of BC, covering each microfibril. It was found that the inner specific surface area increased 1.5 times on deposition of chitosan from the solutions in carbonic acid.

Introduction

One of the important areas of the development of materials science in terms of environmental management is the creation of biocomposites from renewable natural materials with advanced functional properties. One of the promising natural material is bacterial cellulose (BC). It can be produced by certain types of bacteria, including Gluconacetobacter (sometimes called Acetobacter). BC has the same chemical formula as the well-known plant cellulose, but differs from it in physical, mechanical and chemical properties. First, BC has a developed ultrathin nanofibrillar structure with a fiber diameter from units to tens of nanometers (thickness may vary depending on the synthesis method), while in plant microfibrils they are assembled into bundles and clusters with diameters an order of magnitude larger (Iguchi, Yamanaka, & Budhiono, 2000; Shoda & Sugano, 2005). Secondly, BC is a chemically purer material than plant cellulose, which contains several impurities, such as lignin or hemicelluloses. Such unique properties of BC as biocompatibility, slow biodegradability, and high water absorption capacity make it a material of choice in biomedicine (Rajwade, Paknikar, & Kumbhar, 2015). Slow biodegradability makes BC suitable as a carrier matrix for long-term use. BC is used in medicine and bioengineering in applications such as wound and burn-healing dressings, artificial skin and artificial blood vessels, scaffolds for tissue engineering, and many others (Bäckdahl, Risberg, & Gatenholm, 2011; Fu, Zhang, & Yang, 2013; Lin, Lien, Yeh, Yu, & Hsu, 2013).

Another example of the well-known natural polymer is chitosan, a derivative of a widespread, renewable natural resource chitin, which is found in the exoskeleton of crustaceans and the cell walls of fungi. Chitosan is also known for its possible applications in the field of biomedical technologies, since it has many properties that are advantageous for this area, for example, biocompatibility, low toxicity, antimicrobial activity against certain strains, biodegradability, hemostatic properties, and the ability to stabilize metal nanoparticles (Bakshi, Selvakumar, Kadirvelu, & Kumar, 2020).

Chitosan and BC biopolymers are similar in structure, and both are obtained by simple procedures (deacetylation and purification, respectively) from natural renewable resources. Each of these biogenous polymers has its own unique and promising properties, and their combination can lead to significant benefits. Indeed, chitosan modification of the BC surface leads to the formation of new functional groups in it, which can expand the scope of materials from BC. Bacterial cellulose itself has no antimicrobial activity to prevent wound infection. Chitosan can introduce this property into a material made from BC, forming a film on the surface of the BC fibrils through the formation of hydrogen bonds. It is also known that BC has little antioxidant capacity against reactive oxygen species (Schönfelder et al., 2005), while functionalization of BC with chitosan followed by adsorption and stabilization of gold nanoparticles could solve this problem. Another advantage of using BC and chitosan composites for tissue regeneration is that during biodegradation chitosan breaks down into compounds similar to glycosaminoglycans, which provide better fibroblast proliferation and ordered collagen deposition (Nge, Nogi, Yano, & Sugiyama, 2010).

Several methods are known for creating composites from BC and chitosan: by impregnating the BC in a solution of chitosan in acetic acid (Li et al., 2017; Lin et al., 2013; Ul-Islam, Shah, Ha, & Park, 2011), grinding the BC into powder and dispersing it in a solution of chitosan in acetic acid, while the resulting mixture is then cross-linked with glutaric aldehyde (Wahid et al., 2019), adding chitosan initially to the culture medium before growing BC films (Jia et al., 2017; Phisalaphong & Jatupaiboon, 2008), mechanically mixing two polymers by electrospinning (Ardila et al., 2016). Each method has its benefits as well as drawbacks, for example, the presence of cytotoxic or allergenic residues in the final product, the heterogeneity of the composite, as well as technological difficulties. Therefore, it seems important to develop a new, biocompatible and environmentally friendly approach to create such composite materials.

Carbonic acid can be used as a replacement for traditional acidic solvents with improved properties for dissolving biopolymers (Pigaleva et al., 2014), forming nanostructures (Pigaleva, Bulat et al., 2015) and creating composites (Novikov et al., 2018). The carbonic acid solution is water saturated with CO₂ under pressure. Carbon dioxide molecules dissolve in water and then react to form carbonic acid, which then dissociates into protons and hydrogen carbonate anions (Pigaleva, Elmanovich, Kononevich, Gallyamov, & Muzafarov, 2015). Chitosan is soluble in acidic media; therefore, according to traditional methods, it is dissolved and applied to various biomaterials, including cellulose, from acetic acid solutions. The effect of acetic acid on the biomaterial and its residual presence in the biomaterial in some cases can provoke an allergic reaction in humans when the product is used for medical purposes (Wuethrich, 2011). Thus, for the use of polymer composites with chitosan in medical applications, in which such material is in contact with the human body, there is a need to select a different solvent. This solvent must meet the criteria for non-toxicity, biocompatibility, and be potentially non-allergenic. These conditions are met by a carbonic acid solution, which, when the pressure is released, turns into water (Pigaleva et al., 2014). At high pressures of hundreds of bars, the pH of the aqueous phase of a biphasic system H₂O/CO₂ decreases to values less than 3, which makes it suitable for dissolving chitosan. Accordingly, in such an environment, the surface modification of bacterial cellulose with chitosan can be carried out. In addition to the obvious advantages of biocompatibility, environmental friendliness and the absence of solvents undesirable for biomedical applications in carbonic acid solution, the use of high pressures can increase the amount of chitosan applied to bacterial cellulose compared to soaking methods in traditional solvents. Composites based on collagen and chitosan with enhanced antimicrobial properties, biocompatibility, and resistance to calcification and bioresorption was formed in a carbonic acid medium are reported in the scientific literature (Gallyamov et al., 2014, 2018). Moreover, there is data on the improvement in the mechanical properties of the biomaterials themselves in carbonic acid solutions (Gallyamov et al., 2014; Pigaleva et al., 2019). Recently, the ability of carbonic acid under high pressure of CO₂ to extract bacteria from bacterial cellulose was investigated (Pigaleva et al., 2019). However, the synthesis of composites from bacterial cellulose and chitosan in this promising medium has not yet been performed. Thus, it is important to investigate the possibility of creating such composites in a carbonic acid solution, which is new to this field, and to identify the main advantages of the resulting material. Of course, it is necessary to take into account the problem of carbon dioxide emission during the abovementioned high-pressure process. Indeed, especially in the scaling up to industrial production, it would be extremely promising to use a continuous flow process in which the released carbon dioxide is collected and recycled (Han & Poliakoff, 2012). This will solve the problem both in terms of environmental friendliness of the manufacture and of people’s emission of carbon dioxide during their work shift.

In this article, we propose to use water saturated with CO₂ under high pressure (i.e. carbonic acid solution) for BC-based composite synthesis for two purposes. On one hand, we combine two naturally occurring polymers in one composite with enhanced functionality in a biocompatible, eco-friendly and self-neutralising solution of carbonic acid. On the other hand, we utilize the high-pressure medium for more effective penetration of dissolved chitosan into BC matrix. It can be expected that the new material will be of increased purity, with a higher loading of chitosan into the matrix, which can be extremely useful in some biomedical applications. The present work aims to study the structure and main properties of the modified BC tissue and to compare the results with those obtained by conventional synthesis methods of the composites containing bacterial cellulose and chitosan.