# Microbots Gene Delivery System Based on Bifidobacteria in a Tumor Model
## 4 Discussion  
Current strategies to fight cancer usually present certain disadvantages since they are not selective for cancer cells and affect normal tissues. That is why we are looking for new alternatives [3; 15]. In cancer, the use of microbots based on anaerobic bacteria as specific transporters targeted to tumor tissues has been explored since they have more complex mechanisms that allow them to reach specific tissues, cross nonpermeable barriers easily, and modulate their microenvironment [7]. In this study, we developed microbots based on Bifidobacterium and fluorescent magnetic nanoparticles coated with chitosan and pHRE-Luc to be used as gene carriers. We showed the simultaneous use of Bifidobacterium with NCs (microbots) to deliver pDNA in an in vivo model and the transgene was highly expressed in tumor tissue compared with other organs. The microbot does not express the transgene (bioluminescent gene); rather, it acts as a delivery vector that transfects the tumor cells and transfers the transgene for expression by the mammalian cells. The bioluminescent gene is loaded on the NCs, which are carried on the bacterial surface. Herein, we take advantage of the anaerobic bacterial habit to combine with functional NPs and the hypoxic promoter, which induce high gene reporter expression in response to hypoxia and improve the efficiency of treatment in the tumor hypoxic environment. These microbots are biocompatible and comparatively safe. NCs that were a size of 200-300 nm were obtained (Figure 1A); this size is ideal for transfections in tumor therapy since they can extravasate to the tumor area [16]. Zeta potential of +16 mV was obtained (Figure 1B), and a positive potential represents an advantage in targeted delivery therapies and transfection efficiency since the cell membrane is negatively charged, so cationic NCs can have a strong electrostatic interaction with the cell, which results in rapid endocytosis and allows for efficient transfection [17]. An excessive positive charge can cause cytotoxicity, nonspecific interactions, and blood aggregation [18]. The magnetic NPs used for the elaboration of NCs in this study are composed of an SPIO core, which is a material used in clinical magnetic resonance diagnoses. The NPs shell was made of silica, a biocompatible material component similar to those used in daily supplements of calcium, and the fluorescence was given by a covalently bound organic fluorophore that has been approved by the Food and Drug Administration (FDA) [19]. To protect the pDNA, chitosan was used, which is a biocompatible and nontoxic biopolymer, whose use has been approved by the FDA and has already been used in various food and medical areas [20]. Figure 7: Microbot Biodistribution. For the determination of the shape and morphology of the microbots, AFM was used, and the results confirmed that the NCs uniformly adhered to the bacterium's surface (Figure 3). In addition, this result was validated by SEM, and microbots with a diameter between 1-2 \(\upmu\)m were observed (Figure 2). The in vivo distributions of microbots and reporter gene expression was determined in a tumor-bearing mouse model two days after IV administration by luminescence and fluorescence. Greater red fluorescence was obtained in the tumor tissue than in the other organs (spleen, heart, liver, lung, kidney), indicating the presence of microbots in the tumor area. In addition, when comparing the specific transport efficiency of the two systems, r the microbots were better than the NCs alone group (Figure 5). The fluorescence obtained from the tumor tissue does not compare with that of the other organs; however, minimum fluorescence was observed in the liver and kidney since they play an important role in the excretion of nanoparticles and substances [21; 22].