Biodegradable polymers, produced from renewable sources, are alternatives to conventional synthetic polymers with their competitive mechanical properties, biocompatibility, processability, thermal stability, low-cost and environmentally-friendly properties [1
]. Polylactic acid (PLA) is one of the most widely used biodegradable polymers in biomedical applications, such as surgical plates, suture yards, and screws [2
]. Like every other biomaterial used in living tissue as an implant, PLA surface is also open to endogenous or exogenous bacterial contamination. Such contamination may cause nosocomial infection during hospitalization, followed by patient discomfort, extended hospitalization time, external drug load to recover, post-operation to remove the implant, and even morbidity [3
]. Biofilm formation on a biomaterial surface is a multistep process, which begins by bacterial contamination, mediated by physicochemical interaction on the surface, followed by bacterial adhesion through hydrogen bonds and proliferation by multilayering and clustering. Composed bacterial strain secretes an extracellular matrix (consist of polysaccharides, nucleic acids and proteins) to cover the colonies and creates a biofilm [11
]. The biofilm ruptures after reaching the critical amount of bacteria and releases to the surrounding tissue, resulting in potentially serious infections. Removing the existing biofilm is challenging by drug treatment and in most cases, the solution is the removal of the implant. Therefore, bacterial contamination needs to be inhibited at the first stage of adhesion. Such bacterial adhesion and biofilm formation depend on surface charge and density, the chemical composition of the surface, its topology (roughness) and hydrophilicity [4
]. Since only the biomaterial’s surface is in contact with the living tissue and environment during the implantation, creating an antibacterial surface to prevent bacterial adhesion is a valid and convenient approach, instead of blending the bulk material with antibacterial agents. In this way, antibacterial drug loading can be lowered to avoid the patient from the side effects of antibiotics as well as reduce the material cost and its release to the human body is controlled by covalent immobilization. One of the most commonly used ways to fabricate an antibacterial surface to prevent bacterial adhesion is using broad-spectrum antibacterial agents to immobilize on the biomaterials surface. Chlorhexidine (CHx) is a broad-spectrum bactericidal cationic compound belonging to the biguanide family and is toxic to both Gram-negative and Gram-positive bacteria [14
]. It is widely used as an ingredient in household disinfectants, skin/hand antisepsis, hospital disinfectants, dental cleaning products, and cosmetics [17
]. The action mechanism of the CHx targets bacterial cell membrane damage by electrostatic interactions of cationic CHx with anionic groups in the bacterial lipid layer to reduce cell viability or even finalize by cell death [19
Immobilization of the CHx onto an inert surface such as the poly(lactic) acid (PLA) surface is challenging due to its high hydrophilic nature and lack of free bonding groups. Antibacterial surface coating by the plasma mediated multistep physicochemical method is a promising technique to overcome this drawback. Plasma treatment is a non-thermal, fast and effective process without using any chemicals or toxins. Since most of the polymer surfaces lack active functional groups for further chemical bonding and they are mostly hydrophilic, plasma treatment can be used to functionalize the polymer surface by plasma particles to create oxygen-containing functional groups (such as hydroxyl, carbonyl, carboxyl) and also increase the surface wettability/hydrophilicity by plasma etching [22
]. The bulk material properties are not influenced by plasma treatment therefore its interaction is limited to the nanoscale [32
]. In addition to antibacterial activity, the cytotoxicity behavior of the antibacterial containing biomaterials is crucial because most of the antibacterial agents are toxic to cells, therefore their usage needs to be moderated to allow cell growth on the biomaterials after implantation.
In this work, a plasma mediated multistep physicochemical method was used to immobilize CHx onto the PLA surface. Apart from plasma functionalization, carbodiimide chemistry was applied using the coupling reagents of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC), and N-Hydroxysuccinimide (NHs) to increase the anchoring of CHx onto the surface. Surface hydrophilicity analysis was carried out using a water contact angle test, chemical analysis to observe elemental changes was performed by x-ray photoelectron spectroscopy, and surface morphology was investigated with a scanning electron microscope. Antibacterial performance of the samples was tested against Staphylococcus aureus (CCM 4516) as Gram-positive and Escherichia coli (CCM 4517) as Gram-negative representatives. Finally, in vitro cytocompatibility of the samples was studied using primary mouse embryonic fibroblast cells.