# Microbots Gene Delivery System Based on Bifidobacteria in a Tumor Model
## 3 Results
### Fluorescence and Luminescence Analysis  
Two principles were evaluated: the specific transport efficiency, and the gene expression efficiency. In vivo microbot delivery was performed in a tumor-bearing mouse model that was established by inoculation of melanoma cancer cells subcutaneously. When the solid tumor reached approximately 1 \(\pm\) 0.5 mm in diameter, three gene delivery systems (microbots, NCs alone, and naked pDNA) were injected into the tail vein. The NCs loaded in the membrane of the microbot carry an organic fluorophore with which the location of the nanoparticles in the organs of the mice was determined. The highest fluorescent signal was observed in the tumor compared to the healthy spleen, kidney, liver, lung, and heart tissues (\(p\) < 0.001). These results indicated that microbots reach the deep tissue of solid tumors through the bloodstream, delivering NCs to hypoxic tumors. The system of the microbots with magnetic influence (5307 RFU/mg of protein) had a higher fluorescence signal compared to the NC system (3458 RFU/mg of protein) (\(p\) < 0.05) and the negative control (758 RFU/mg of protein) (\(p\) < 0.001). This result indicates that the microbots were more efficient for the delivery of NCs to the tumor area than the NCs alone group (Figure 5). Figure 4: Zeta potential of the microbots. (**A**) Hydrodynamic zeta potential of microbots with a potential of \(-\)28 mV with intensity = 100% and, (**B**) Hydrodynamic zeta potential of _Bifidobacterium breve_ wild-type with a potential of \(-\)30 mV with intensity = 100% and St Dev = 4.1. Results obtained were from three measurements. NCs loaded in the membrane of microbots carry pDNA (pHRE-Luc) which contains a gene that codes for luciferase, with which the site of transfection and expression in mice could be determined. The highest luminescence signal was determined in the tumor compared to the healthy tissues: the spleen, liver, lung, and heart (\(p<0.0001\)). The system of the microbots with magnet influence (7411 RLU/mg of protein) had a higher luminescence signal compared to the NC system (4412 RLU/mg of protein) and naked plasmid system (2824 RLU/mg of protein). This result indicates that the microbots were more efficient for the specific delivery of genes to the tumor area (Figure 6). Figure 5: Fluorescence analysis in organs and tumor tissue. Notes: Relative fluorescence intensities plotted as a bar graph displaying the signal for different organs of tumor-bearing mice 48 h after IV of microbots. The RFU/mg protein was normalized to milligrams of protein. The data represent the mean \(\pm\) the standard error of the mean of three repetitions. Significant differences are shown (* \(p<0.05\) and *** \(p<0.001\)). Abbreviation: Negative control represent phosphate buffered saline; M+M group, microbots with the influence of a magnetic field; NCs+M, nanocomplex with the influence of a magnetic field; IV, intravenous injection; RFU, relative fluorescence units. Figure 6: Luciferase gene expression in different organs of mice. Notes: Relative luminescence intensities plotted as a bar graph displaying the signal for different organs of tumor-bearing mice 48 h after IV of microbots. The RLU/mg protein was normalized to milligrams of protein. All treatments had the vector pHRE-Luc. The data represent the mean \(\pm\) the standard error of the mean of three repetitions. Significant differences are shown (* \(p<0.05\), *** \(p<0.001\) and *** \(p<0.0001\)). Abbreviation: Negative control represent phosphate-buffered saline; M+M group, microbots with the influence of a magnetic field; NCs+M, nanocomplex with the influence of a magnetic field; \(p\), naked plasmid; IV, intravenous injection; RLU, relative light units.