Luciferase Reporter Cell Line - 5637

For patients with bladder cancer (BCa), intravesical chemotherapy is strongly recommended following transurethral resection of bladder tumors. However, this form of local adjuvant therapy presents drawbacks, namely causing non-specific tissue damage and exhibiting poor penetration of the bladder mucosa. Here, researchers synthesized fluorinated polylysine micelles (PLLF) by reacting polylysine (PLL) with heptafluorobutyric anhydride. Due to the modification with fluorinated chains, PLLF is capable of self-assembling into nanoparticles (NPs) and efficiently condensing plasmids, demonstrating excellent gene transfection efficiency in vitro. When loaded with a CRISPR-Cas9 system designed to target the DAD1 gene (Cas9-sgDAD1), the resulting PLLF/Cas9-sgDAD1 NPs effectively suppressed DAD1 expression in BCa cells and induced BCa cell apoptosis via the MAPK signaling pathway. Furthermore, in vivo studies demonstrated that intravesical administration of PLLF/Cas9-sgDAD1 NPs achieved significant therapeutic efficacy without eliciting systemic toxicity.

Based on positive results obtained from in vitro experiments-specifically, the confirmation that PLLF/Cas9-sgDAD1 nanoparticles (NPs) can specifically inhibit bladder tumor growth while exhibiting excellent mucosal penetration capabilities-researchers conducted intravesical therapeutic experiments using an orthotopic bladder tumor mouse model. To establish this model, researchers inoculated 5 × 10⁵ 5637 cells stably expressing firefly luciferase (designated as 5637Luc) into the bladder walls of female BALB/c nude mice (6–8 weeks old). On Day 3, the successful establishment of tumors was confirmed by detecting bioluminescence signals; subsequently, the tumor-bearing mice were randomly divided into three groups (5 mice per group) to receive treatment with different formulations: PBS, PLL/Cas9-sgDAD1, and PLLF/Cas9-sgDAD1. As illustrated in Figure 1A, intravesical instillation treatments were administered every three days for a total of four doses; on Day 17, tumor growth was monitored again via bioluminescence imaging using an IVIS Lumina system. Throughout the entire treatment regimen, the body weights of all nude mice were continuously monitored. The body weight change curves presented in Figure 1B demonstrate an acceptable range of fluctuation, indicating that the intravesical administration of PLL/Cas9-sgDAD1 and PLLF/Cas9-sgDAD1 induces negligible systemic toxicity. By comparing the bioluminescence intensities of the orthotopic bladder tumors at the beginning and end of the treatment period (i.e., Day 3 and Day 17), the significant anti-tumor efficacy of the PLLF/Cas9-sgDAD1 nanoparticles was conclusively demonstrated (Figure 1C).

Figure 1. Intravesical anticancer evaluation of PLLF/Cas9-sgDAD1 NPs. (Tang, Dongdong, et al., 2024)