Bacterial ghosts (BGs) can be prepared by both genetic and chemical means. Genetic method include using lysis gene . Chemical method include incubation with numerous agents for a short time at their minimum inhibitory or minimum growth concentrations (MIC or MGC). The aim of this study is to prepare the BGs with a new protocol via exposing the bacterial cells to tween 80 for an extended period of time followed by sudden reduction of the surrounding pH. ATCC 13311 was used for this purpose. The cells were incubated in 7% / tween 80 solution in Muller-Hinton broth for 24 h at 37 °C then pH was decreased to 3.6 by adding lactic acid for one hour. The bacterial pellets were separated by high speed centrifugation, and then washed three times by half normal saline solution. High quality BGs were visualized by scanning electron microscopy (SEM) revealing punctured cells with intact outer shells and at least one intramembranous tunnel. The absence of vital cells was confirmed by subculturing. The release of respective amounts of proteins and DNA is another evidence of ghost's production. In addition, the integrity of cells was proved by visualization of Gram-stained cells using light microscopy. In conclusion, this new protocol is simple, economic and feasible for BGs preparation.

Background. The present study focused on the green synthesis of silver nanoparticles (AgNPs) using the Astragalus spinosus Forssk. aqueous extract. In addition, we evaluated the antibacterial activity of AgNPs as well as some cellular mechanisms against Actinomyces viscosus and Streptococcus mutans as the most causative agents of tooth decay. Methods. In this study, AgNPs were green synthesized by the precipitation method based on the reduction of silver ions (AgNO3) by A. spinosus extract. Antibacterial effects of the green synthesized AgNPs were performed by measuring the minimum inhibitory concentration (MIC) and minimum bactericidal concentrations (MBC) through micro broth dilution method. In addition, we evaluated the reactive oxygen species (ROS) production, nucleic acid leakage, and protein leakage as the main antibacterial mechanisms of the green synthesized AgNPs against A. viscosus and S. mutans. The cytotoxicity effects of AgNPs against on human normal (NOF18 cells) and oral cancer (SCC4 cells) cell lines were also evaluated using MTT assay. Results. The green synthesized AgNPs have a spherical shape and are relatively uniform in size in the range of 30-40 nm. The MIC values for S. mutans and A. viscosus of the green synthesized AgNPs were 10.6 and 13.3 μg/ml, respectively, whereas the MBC values for S. mutans and A. viscosus of the green synthesized AgNPs were 21.3 and 26.6 μg/ml. The findings exhibited that ROS production, nucleic acid leakage, and protein leakage were increased after treatment of A. viscosus and S. mutans by the green synthesized AgNPs. The results demonstrated that the 50% inhibitory concertation (IC50) values of AgNPs on NOF18 and SCC4 cells were 93.3 μg/ml and 41.2 μg/ml, respectively. Conclusion. Overall, the results of this study showed that A. spinosus extract has a good ability to produce silver nanoparticles. The AgNPs produced have significant antibacterial effects against some tooth decay bacteria. Our results also revealed that the green synthesized AgNPs are more cytotoxic against cancerous cell line than normal cell line. Further in vivo studies are required to investigate the side effects and to evaluate the effectiveness of these bacteria.

Cell- based targeted delivery is recently gain attention as a promising platform for delivery of anticancer drug in selective and efficient manner. As a new biotechnology platform, bacterial ghosts (BGs) have novel biomedical application as targeted drug delivery system (TDDS). In the current work, Salmonellas’ BGs was utilized for the first time as hepatocellular cancer (HCC) in-vitro targeted delivery system. Successful BGs loading and accurate analysis of doxorubicin (DOX) were necessary steps for testing the applicability of DOX loaded BGs in targeting the liver cancer cells. Loading capacity was maximized to reach 27.5 µg/mg (27.5% encapsulation efficiency), by incubation of 10 mg BGs with 1 mg DOX at pH 9 in constant temperature (25 °C) for 10 min. In-vitro release study of DOX loaded BGs showed a sustained release (182 h) obeying Higuchi sustained kinetic release model. The death rate (tested by MTT assay) of HepG2 reached to 64.5% by using of 4 μg/ml, while it was about 51% using the same concentration of the free DOX ( P value < 0.0001 One-way ANOVA analysis). The proliferative inhibitory concentration (IC50) of the DOX combined formula was 1.328 µg/ml that was about one third of the IC50 of the free DOX (3.374 μg/ml). Apoptosis analysis (tested by flow-cytometry) showed more accumulation in early apoptosis (8.3%) and late apoptosis/necrosis (91%) by applying 1 μg/ml BGs combined DOX, while 1 μg/ml free DOX showed 33.4% of cells in early apoptosis and 39.3% in late apoptosis/necrosis, ( P value ˃ 0.05: one-way ANOVA). In conclusion, DOX loaded Salmonellas’ BGs are successfully prepared and tested in vivo with promising potential as hepatocellular cancer (HCC) targeted delivery system.

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