Abstract
Objective: The evolution of antimicrobial resistance is a universal obstacle that necessities the innovation of more effective and safe antimicrobial alternatives with synergistic properties. The purpose of this study was to investigate the possible improvement of cephalexin antimicrobial treatments by loading into chitosan-based nanoparticles, then evaluate their antibacterial and antibiofilm activities as well as determination of its cytotoxicity.
 Methods: Chitosan nanoparticles (CSNPs) were prepared by ionic gelation method. Parameters were studied to optimize the particle size of CSNPs including pH, stirring rate, homogenization and ultra-sonication time. Size was measured by transmission electron microscope (TEM) and Zeta sizer, morphology seen by scanning electron microscope (SEM). Entrapment efficiency, drug loading and drug content were calculated. Stability of both plain and loaded chitosan Nano-carriers, Drug release and Kinetics also compatibilities were studied. Antimicrobial activity of CSNPs and cephalexin loaded CSNPs were evaluated against 4 Gram-positive and 4 Gram-negative standard and clinical isolates by microdilution method, also assessment of antibiofilm activity of both formulas was investigated against two biofilm producers clinical isolates by tube assay in addition to determination of their cytotoxicity by MTT(3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
 Results: Chitosan nanoparticles and its loaded antibiotics proved compatible combination with small Zeta size, suitable Zeta potential, maximum EE% and drug-loading capacity, sustained controlled release properties followed diffusion kinetic model and six month stability studies. Cephalexin loaded CSNPs showed better antimicrobial activity than plain CSNPs. Synergistic effects were found against S. aureus (ATCC 25923), B. subtilis (ATCC 9372), S. epidermidis, E. faecalis, P. aeruginosa (ATCC 29853) in addition to two carbapenem resistant isolates k. pneumoniae and E. coli. Also cephalexin loaded CSNPs exhibited antibiofilm activity against E. faecalis clinical isolate. Even though, cephalexin loaded CSNPs exhibited significant antibacterial activity, it showed less toxicity against mammalian cells, it had IC50 equal to 231.893 and did not exhibit any cytotoxicity against the WI-38 fibroblast cells at concentration 23.4 µg/ml.
 Conclusion: Cephalexin loaded CSNPs possessed good stability and sustained release effect in addition to its antimicrobial, antibiofilm activities and reduced cytotoxicity.
Highlights
The evolution of microbial resistance against different classes of antibiotic has attracted more attention and forced researchers to formulate innovative antimicrobial agents that exhibit efficient antimicrobial activity against the frequently increasing multidrug resistant pathogens [1]
There are several mechanisms that explain the antibacterial activity of chitosan, the most acceptable one assumes that chitosan binds to the negatively charged bacterial surface disturbing the cell membrane and altering its permeability attach to DNA causing inhibition of DNA replication and cell death [6]
By studying the chitosan concentration effect (0.1, 0.15 and 0.2) % w/v, while constant other parameter such as concentration of sodium tri polyphosphate 1 mg/ml and pH 5, 1100 r/min stirrer rate, 22000 r/min homogenization for 3 min and ultra-sonication time 45 min., It was observed that with increase in the concentration of chitosan the appearance of the solution changed from clear viscous liquid to opalescent fluid and precipitated the solution became opalescent indicating the formation of Nano chitosan with smallest Nano size ranged (23-35 nm)
Summary
Plain chitosan nanoparticles and cephalexin loaded nanoparticles were prepared based on the modified ionotropic gelation with minor modification [25, 26]. Different concentrations of the antibiotic were used, cephalexinloaded CSNPs were formed spontaneously upon the dropwise addition of an aqueous solution of sodium TPP to 20 ml of a 0.2 % w/v chitosan solutions, containing different five cephalexin concentrations (0.05, 0.1, 0.15, 0.2 and 0.25% w/v), with constant stirring at high r/min, followed by homogenization and sonication. Different chitosan concentrations effect (0.1, 0.15 and 0.2) %w/v were studied at constant other parameter such as concentration of sodium tri polyphosphate 1 mg/ml and pH 5,stirring at 1100 r/min, homogenization at 22000 r/min for 3 min and ultra-sonication time 45 min at room temperature. The samples were withdrawn at different time intervals over a period of six month and they were observed visually and under optical microscope and examined morphologically by TEM for the change in consistency and appearance of drug crystals upon storage. The experiment was performed in triplicate, and the result is expressed as the mean±SD [38]
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