Effect of Carboxymethyl Nanochitosan Loaded with Streptomycin on Staphylococcus aureus and Pseudomonas aeruginosa

   This study aimed to synthesise carboxymethylnanochitosn as nanochitosan derivative from nanochitosan synthesized from soluble chitosan (low molecular weight) and encapsulate streptomycin then characterize and bioassay them on pathogenic gram positive and gram negative bacteria and Rhabdomyosarcoma cell- line. Ionic gelation method was used to synthesize nanchitosan from soluble and insoluble chitosan (high molecular weight). Dissolved chitosan was found to be superior to non-soluble in the preparation of nanochitosan. The concentration of (2 mg/100 of 2% glacial acetic acid) chitosan was selected as the highest concentration to obtain the highest yield of nanochitosan with a constant ratio of Tripolyphosphate. Carboxymethylnanochitosan was synthesized from nanochitosan in an acid-base method with a yield of 420 mg / 3g nanochitosan. It has been appointed a maximum absorbance (λ max) for streptomycin, carboxymethylnanochitosan and carboxymethylnanochitosan encapsulate
streptomycin will be 275, 265 and 270 nm respectively. It has been appointed the standard curves for streptomycin, carboxymethylnanochitosan and carboxymethylnanochitosan encapsulate streptomycin  compounds and calculated the absorbency values (α) which were 0.002, 0.004 and 0.006 respectively. The loading efficiency of carboxymethylnanochitosan with streptomycin was 90%.FTIR spectroscopy of carboxymethylnanochitosan and carboxymethylnanochitosan with streptomycin showed that, absorption peaks at the same frequencies 3000 -3800 cm
-1 with the deep in stretching of the absorption percent for carboxymethylnanochitosan with streptomycin. Scanning electron Microscope (SEM) three-dimensional morphological structures of chitosan,
nanochitosan, carboxymethylnanochitosan and carboxymethylnanochitosan with streptomycin showed different forms. It was found the aggregation of carboxymethylnanochitosan spheres and entrapped the  streptomycin within carboxymethylnanochitosan. Results of Atomic force spectroscopy (AFM) three dimensions images illustrated that the concentration 2mg/100 ml of 2% glacial acetic acid of soluble chitosan gave 54.64 nm average diameter and 90% of nanoparticles with diameter 80.00 nm. Insoluble chitosan at concentration 2mg/100 ml of 2%glacial acetic acid gave average diameter 105.52 nm and 90% of nanoparticles with diameter 170 nm. According to the process of carboxymethylnanochitosan preparation, it was obtained nanoparticles size with average diameter 35nm and 90% of the 56nm diameter. After loading carboxymethylnanochitosan on streptomycin, the average size of the nanoparticles was increased to reach 37nm and 90% of particles diameter increased to 62nm. These results confirmed the loading process of carboxymethylnanochitosan with streptomycin. Results of particle size distribution showed the median particle size of carboxymethylnanochitosan was 770 nm and 526 nm for carboxymethylnanochitosan with streptomycin. Nanochitosan prepared from insoluble chitosan showed no inhibition zone on Staphylococcus aureus and Pseudomonas aeruginosa whereas, from soluble chitosan (2 and 20 mg),nanochitosan gave inhibition zone against Pseudomonas aeruginosa only in comparison with the results of sensitivity to streptomycin. It was found that carboxymethylnanochitosan was not affected on the response of isolated.Staphylococcus aureus was become sensitive against streptomycin starting from concentration 100 µg/mL while; Pseudomonas aeruginosa became sensitivity from concentration 300 µg/mL and was more resistant than Staphylococcus aureus.  The results of sensitivity carboxymethylnanochitosan with streptomycin were similar to streptomycin for each isolate. In vitro anticancer test efficacy of streptomycin, carboxymethylnanochitosan and carboxymethylnanochitosan with streptomycin against Rhabdomyosarcoma cell line was evaluated. It was found that 50 µg/ml of carboxymethylnanochitosan with streptomycin more effective than 100µg/ml of streptomycin and growth inhibition was 37.5%, 38.5% respectively after 24 hrs of incubation.