Anna Regiel-Futyra

Development of Noncytotoxic Chitosan–Gold Nanocomposites as Efficient Antibacterial Materials

This work describes the synthesis and characterization of noncytotoxic nanocomposites either colloidal or as films exhibiting high antibacterial activity. The biocompatible and biodegradable polymer chitosan was used as reducing and stabilizing agent for the synthesis of gold nanoparticles embedded in it. Herein, for the first time, three different chitosan grades varying in the average molecular weight and deacetylation degree (DD) were used with an optimized gold precursor concentration. Several factors were analyzed in order to obtain antimicrobial but not cytotoxic nanocomposite materials. Films based on chitosan with medium molecular weight and the highest DD exhibited the highest antibacterial activity against biofilm forming strains of Staphylococcus aureus and Pseudomonas aeruginosa. The resulting nanocomposites did not show any cytotoxicity against mammalian somatic and tumoral cells. They produced a disruptive effect on the bacteria wall while their internalization was hindered on the eukaryotic cells. This selectivity and safety make them potentially applicable as antimicrobial coatings in the biomedical field.

Bactericidal Effect of Gold–Chitosan Nanocomposites in Coculture Models of Pathogenic Bacteria and Human Macrophages

The ability of pathogenic bacteria to develop resistance mechanisms to avoid the antimicrobial potential of antibiotics has become an increasing problem for the healthcare system. The search for more effective and selective antimicrobial materials, though not harmful to mammalian cells, seems imperative. Herein we propose the use of gold-chitosan nanocomposites as effective bactericidal materials avoiding damage to human cells. Nanocomposites were obtained by taking advantage of the reductive and stabilizing action of chitosan solutions on two different gold precursor concentrations. The resulting nanocomposites were added at different final concentrations to a coculture model formed by Gram-positive (Staphylococcus aureus) or Gram-negative (Escherichia coli) bacteria and human macrophages. Gold-chitosan colloids exhibited superior bactericidal ability against both bacterial models without showing cytotoxicity on human cells at the concentrations tested. Morphological and in vitro viability studies supported the feasibility of the infection model here described to test novel bactericidal nanomaterials. Flow cytometry and scanning electron microscopy analyses pointed to the disruption of the bacterial wall as the lethal mechanism. Data obtained in the present study suggest that gold-chitosan nanocomposites are powerful and promising nanomaterials for reducing bacteria-associated infections, respecting the integrity of mammalian cells, and displaying high selectivity against the studied bacteria.

Study on inhibitory activity of chitosan-based materials against biofilm producing Pseudomonas aeruginosa strains.

Six antibiotic-resistant Pseudomonas aeruginosa strains, isolated from chronic diabetic foot infections, were chosen for studying the influence of different chitosan-based materials: chitosan solution and chitosan submicroparticles in both planktonic and 24 h-old biofilm-forming models. Chitosan solution occurred to be more effective in the reduction of bacterial populations than chitosan submicroparticles for both planktonic and biofilm-related Pseudomonas cells. It seems that the antimicrobial activity of the tested chitosan preparations depends on the individual bacterial strain susceptibility probably related to differences in the phenotypes and natural antioxidant abilities of Pseudomonas aeruginosa strains.

The quenching effect of chitosan crosslinking on ZnO nanoparticles photocatalytic activity

Zinc oxide (ZnO), the main component of several suntan lotions, generates highly oxidizing, cytotoxic and genotoxic reactive oxygen species (ROS) upon UV light absorption. In order to increase safety combined with ZnO use as a sunscreen, its photocatalytic activity should be efficiently quenched. In our studies commercial samples of zinc oxide nanoparticles were hybridized by ionotropic gelation with a natural biopolymer, chitosan (CS). The chemical crosslinking of the polymer in the presence of ZnO nanoparticles was performed. Significantly, in contrast to several CS–ZnO hybrid materials described in the literature, the obtained composites maintained the UV light absorption ability, while the photocatalytic activity towards chemical and biological substrates was totally quenched. Furthermore, a complete lack of photoelectrochemical response observed for the chitosan modified semiconductors confirmed the total inhibition of photoinduced interfacial electron transfer processes. Additionally, antibacterial activity against selected bacterial strains, Staphylococcus aureus and Escherichia coli was observed, although there was no cytotoxic effect against human keratinocytes. The nanocomposites resolve the problem of the risk associated with using semiconductor nanoparticles as ingredients of suntan lotions, cosmetics and dermatological formulations. The transparent polymeric coating allows the absorption of UV irradiation by ZnO particles and simultaneously blocks photogeneration of reactive radicals and oxygen species.

Chitosan-based nanocomposites for the repair of bone defects

Chitosan scaffolds of different deacetylation degrees, average molecular weights and concentrations reinforced with silica nanoparticles were prepared for bone tissue regeneration. The resulting nanocomposites showed similar pore sizes (<300 μm) regardless the deacetylation degree and concentration used in their formulation. Their mechanical compression resistance was increased by a 30% with the addition of silica nanoparticles as nanofillers. The biocompatibility of the three-dimensional chitosan scaffolds was confirmed by the Alamar Blue assay in human primary osteoblasts as well as the formation of cell spheroids indicative of their great potential for bone regeneration. In vivo implantation of the scaffolds in a mice calvaria defect model provided substantial evidences of the suitability of these nanocomposites for bone tissue engineering showing a mature and dense collagenous tissue with small foci of mineralization, vascularized areas and the infiltration of osteoblasts and osteoclasts. Nevertheless, mature bone tissue formation was not observed after eight weeks of implantation.

Chitosan-based coatings in the prevention of intravascular catheter-associated infections

Central venous access devices play an important role in patients with prolonged intravenous administration requirements. In the last years, the coating of these devices with bactericidal compounds has emerged as a potential tool to prevent bacterial colonization. Our study describes the modification of 3D-printed reservoirs and silicone-based catheters, mimicking central venous access devices, through different approaches including their coating with the well known biocompatible and bactericidal polymer chitosan, with the anionic polysaccharide alginate; also, plasma treated surfaces were included in the study to promote polymer adhesion. The evaluation of the antimicrobial action of those surface modifications compared to that exerted by a model antibiotic (ciprofloxacin) adsorbed on the surface of the devices was carried out. Surface characterization was developed by different methodologies and the bactericidal effects of the different coatings were assayed in an in vitro model of Staphylococcus aureus infection. Our results showed a significant reduction in the reservoir roughness (≤73%) after coating though no changes were observed for coated catheters which was also confirmed by scanning electron microscopy, pointing to the importance of the surface device topography for the successful attachment of the coating and for the subsequent development of bactericidal effects. Furthermore, the single presence of chitosan on the reservoirs was enough to fully inhibit bacterial growth exerting the same efficiency as that showed by the model antibiotic. Importantly, chitosan coating showed low cytotoxicity against human keratinocytes, human lung adenocarcinoma epithelial cells, and murine colon carcinoma cells displaying viability percentages in the range of the control samples (>95%). Chitosan-based coatings are proposed as an effective and promising solution in the prevention of microbial infections associated to medical devices.

Preparation and characterization of alginate/chitosan formulations for ciprofloxacin-controlled delivery:

In this work, alginate beads loaded with ciprofloxacin (AL_CP) and alginate beads loaded with ciprofloxacin and covered by chitosan (AL_CP_CS) were prepared by emulsification technique in combination with internal gelation method. Physicochemical characterization of the resulting formulations revealed the hydrodynamic diameter and Zeta potential ca. 160 nm and ca. −32 mV in the case of AL_CP and ca. 240 nm and ca. +14 mV in the case of AL_CP_CS (pH = 6.0), respectively. Kinetics of ciprofloxacin release from alginate/chitosan formulations was studied in different media (pH = 1.2, 6.8, and 7.4). Covering alginate core with a polycation such as chitosan moderates the drug release, resulting in a pH-sensitive hybrid controlled-release system. Herein, alginate beads with encapsulated ciprofloxacin covered with chitosan are proposed as an effective oral delivery system since the drug release from alginate core is limited in low pH solution (gastric conditions).

Generation of hydroxyl radicals and singlet oxygen by particulate matter and its inorganic components

Particulate matter (PM) can strongly affect redox biochemistry and therefore induce the response of the immune system and aggravate the course of autoimmune diseases. Nanoparticles containing transition metal compounds possessing semiconductor properties (TiO2, ZnO) may act as photocatalysts and accelerate the generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In this study, the NIST standard reference material, SRM 1648a, has been analyzed in terms of this consideration. Organic compounds present in SRM 1648a were removed by cold oxygen plasma treatment. Samples of SRM 1648a with removed organic content (<2% of organic carbon, <1% of nitrogen) were obtained within 2 h of this treatment. The treatment did not affect the morphology of the powder. The reference material and PM2.5 collected in Kraków are composed of smaller particles and nanoparticles forming aggregates. The efficiency of (photo)generation of hydroxyl radicals and singlet oxygen was compared for original and organics-free samples. The analyzed samples showed the highest activity towards ROS generation when exposed to UV-vis-NIR light, moderate under UV irradiation, and the lowest in dark. Data collected in the present study suggest that the organic fraction is mostly responsible for singlet oxygen generation, as almost twice higher efficiency of 1O2 generation was observed for the original NIST sample compared to the material without the organic fraction. However, particulate matter collected in Kraków was found to have a five times higher activity in singlet oxygen generation (compared for original NIST and Kraków dust samples).