Probiotic Escherichia coli Nissle 1917 propelled micro-robot with pH sensitivity for hypoxia targeted intestinal tumor therapy

Poor drug penetration in hypoxia area of solid tumor is a big challenge for intestinal tumor therapy and thus it is crucial to develop an effective strategy to overcome this challenge. Compared with other bacteria used for construction of hypoxia targeted bacteria micro-robot, the Escherichia coli Nissle 1917 (EcN) bacteria are nonpathogenic Gram-negative probiotic and can especially target and identify the signal molecules in the hypoxic region of tumor, and thus, in this study, we choose EcN to construct a bacteria propelled micro-robot for targeting intestinal tumor therapy. Firstly, the [email protected] with average diameter of 200 nm were synthesized and conjugated with EcN bacteria using EDC/NHS chemical crosslinking method to construct a EcN propelled micro-robot. The motility of micro-robot was then evaluated and the motion velocity of [email protected] was 3.78 µm/s. Compared with [email protected] without EcN driven, EcN bacteria propelled micro-robot transported much more [email protected] into the inner of HCT-116 3D multicellular tumor spheroids. However, the EcN bacteria are non-intracelluar bacteria which lead to the micro-robot can not directly enter into tumor cells. Therefore, we utilized acid-labile linkers of cis-aconitic amido bone to link EcN with [email protected] nanoparticles to achieve the pH sensitive separation of EcN with [email protected] from the micro-robot. At 4 h of incubation, the isolated [email protected] began to enter into the tumor cells through CLSM observation. In vitro live/dead staining results show that [email protected] induced much more cell death than [email protected] at 24 and 48 h of incubation with HCT-116 tumor cells in acid culture media (pH 5.3). For the validation of the therapeutic efficacy of the micro-robot for intestinal tumor, we established the HCT-116 subcutaneous transplantation tumor model. After 28 days of treatment, [email protected] dramatically inhibit tumor growth with tumor volume was around 689 mm3, induce much more tumor tissues necrosis and apoptosis. Finally, the toxicity of this micro-robot was investigated by pathological analysis the liver and heart tissues. We expect that the pH sensitive EcN propelled micro-robot here we constructed may be a safe and feasible strategy for intestinal tumor therapy.