Efficient delivery of antibacterial agents directly to sites of tissue injury faces challenges such as poor drug stability and fast degradation by biological mechanisms. Biocompatible nanocarrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, we report the application of tailored polyionic particles via ionic interactions between negatively charged heparin and positively charge chitosan for efficient encapsulation of polyhexamethylene biguanide (PHMB) antibiotic. Negative zeta potential was required to encapsulate the positively charged PHMB. We demonstrate that the ratio of heparin to chitosan can be employed to create a tuned surface charge and maximize the bonding of the drug of choice as well as appropriate particle distribution and uniform morphology. Different formulations were evaluated in terms of size, polydispersity, surface charge, and morphology. Out of all these formulations, the best, negatively charged formulation at four-parts heparin to one-part chitosan was successfully encapsulated with PHMB and showed a sustained and controlled release in vitro for around 10 days. Reduced toxic responses (around 48% reduction) were observed from PHMB-loaded particles in contact with human dermal fibroblasts as compared to the soluble form of PHMB. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition as well as the direct killing of both Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) bacterial strains. The minimum inhibitory concentrations required to inhibit bacterial growth were determined by microplate dilution and LIVE/DEAD bacterial evaluation.

Chitosan has emerged as a useful biomaterial employed in tissue engineering and drug delivery applications due to its tunable and interesting properties. However, chitosan is protonated at biological pH and thus carries positive charges, which renders chitosan incompatible with conventional methods of RNA extraction. RNA extraction is an important step in investigating cell responses and behavior through studying their gene expression transcriptional profile. While some researchers have tried different techniques to improve the yield and purity of RNA extracted from cells encapsulated in chitosan-based biomaterials, no single study has investigated the effects of manipulating pH of the homogenate during RNA extraction on the yield and quality of total RNA. This study confirms the release and binding of RNA from chitosan to be pH dependent while analyzing the impact of pH changes during the tissue disruption and homogenization step of extraction on the resulting yield and quality of isolated RNA. This concept was applied to three commonly used methods of RNA extraction, using adult neural stem cells (aNSPCs) encapsulated within methacrylamide chitosan (MAC) as model chitosan-based bioscaffold. High pH conditions resulted in high yields with good quality using both TRIzol and CTAB. pH of the homogenate did not affect RNeasy spin columns, which works best in neutral conditions with good quality, however, the overall yield was low. Results in total show that pH affects RNA interaction with a chitosan-based bioscaffold, and thus alters the concentration, purity, and integrity of isolated RNA, dependent on the method used.

Neutrophils are a first line of host defense against infection and utilize a series of oxygen-dependent processes to eliminate pathogens. Research suggests that oxygen availability can improve anti-infective mechanisms by promoting the formation of reactive oxygen species. Also, oxygen can synergistically upregulate the antibacterial properties of certain antibiotics against bacteria by altering their metabolism and causing an increase in the antibiotic uptake of bacteria. Therefore, understanding the effects of oxygen availability, as provided via a biomaterial treatment alone or along with potent antibacterial agents, on neutrophil functions can lead us to the development of new anti-inflammatory and anti-infective approaches. However, the study of neutrophil functions in vitro is often limited by their short life span and nonreproducibility, which suggests the need for cell line-based models as a substitute for primary neutrophils. Here, we took advantage of the differentiated human leukemia-60 cell line (HL-60), as an in vitro neutrophil model, to test the effects of local oxygen and antibacterial delivery by fluorinated methacrylamide chitosan (MACF) hydrogels incorporated with polyhexamethylene biguanide (PHMB) antibacterial agent. Considering the natural modes of neutrophil actions to combat bacteria, we studied the impact of our dual functioning oxygenating-antibacterial platforms on neutrophil phagocytosis and antibacterial properties as well as the formation of neutrophil extracellular traps (NETs) and reactive oxygen species (ROS). Our results demonstrated that supplemental oxygen and antibacterial delivery from MACF–PHMB hydrogel platforms upregulated neutrophil antibacterial properties and ROS production. NET formation by neutrophils upon treatment with MACF and PHMB varied when chemical and biological stimuli were used. Overall, this study presents a model to study immune responses in vitro and lays the foundation for future studies to investigate if similar responses also occur in vivo.