Bladder cancer (BC) is a prevalent and costly malignancy among urinary carcinomas globally, primarily due to the need for life-long surveillance and follow-up. Non-muscle invasive bladder cancer (NMIBC) accounts for approximately 75% of BC cases [1]. Despite transurethral resection of bladder tumors in NMIBC patients, recurrence rates remain high, with up to 78% of patients experiencing recurrence within 5 years [2]. The standard treatment for intermediate and high-risk NMIBC involves intravesical instillations of Bacillus Calmette-Guérin (BCG) with or without chemotherapy drugs, such as mitomycin C, gemcitabine, doxorubicin, and epirubicin, for a maintenance period of 1–3 years [3,4]. While this approach has shown significant success, recurrence still occurs in 60% of patients after 1 year [[5], [6], [7]]. Furthermore, the survival rates of BC have no improvement in the past three decades [8].

The limited therapeutic efficacy of current treatments for bladder cancer can be attributed to the formidable bladder penetrating barrier that impedes the drugs from exerting their effects. The urothelium layer, with its tight junctions and abundant glycosaminoglycan on the surface, significantly hinders the absorption of intravesical agents [9]. In addition, due to the special physiological structure of bladder, the efficacy of intravesical therapy is compromised by the frequent drug dilution, short retention time and poor pharmaceutical absorption [10]. Patients have to undergo frequent intravesical instillations with large doses of drugs, leading to serious side effects, decreased quality of life, and placing a heavy burden on their families [11]. For instance, patients with intermediate-risk NMIBC are recommended to accept 1 year of the full dose BCG (81 mg per instillation) intravesical therapy (induction plus 3, 6, 12 months, 3 times a week) or intravesical chemotherapy [12,13]. Adverse effects of BCG treatment are common, such as bacterial cystitis, malaise, skin rash, and even life-threatening sepsis [14]. Similarly, commonly used intravesical chemotherapeutic agent mitomycin C (40 mg per instillation) can cause side effects such as allergic skin reactions, irritative voiding symptoms, and dysuria [15,16]. Given these challenges, it is imperative to explore new strategies for BC treatment that offer enhanced therapeutic efficacy while minimizing systemic side effects.

Oxaliplatin (OXA) is a promising alternative to cisplatin in advanced bladder cancer chemotherapy due to its reduced nephrotoxicity [17]. OXA has shown potential in chemo-immunotherapy as it can induce immunogenic cell death (ICD) [18,19]. However, its systemic adverse effects and the development of drug tolerance have limited its application. To overcome these drawbacks, inert OXA prodrugs that can be converted to active OXA in tumor cells have been explored, and these prodrugs have been incorporated into nanoparticles to improve their targeting ability and safety [20,21].

Along with chemotherapy, BCG is the primary immunotherapy drug for NMIBC, while the mechanism of its action is still being elucidated. It has been posited that BCG interacts with innate immune cells and urothelial cells (including tumor cells) directly, induces antigen-presenting, activates innate and adaptive immune responses [14,22]. As previously mentioned, the clinical performance of BCG is hindered by the serious adverse effects and the need for large doses. The shortage of approved BCG manufacturers worldwide has also prompted investigations into low-dose BCG instillation [23,24]. Considering the advantages of oxaliplatin pro-drug nanoparticles and BCG, it is worth exploring whether these agents can be co-delivered intravesically to achieve a higher anti-tumor efficiency by activating tumor-specific immune responses with lower doses and fewer treatments. Such a co-delivery system may hold significant promise for improving bladder cancer therapy.

Furthermore, to address the challenge of prolonging the dwell time and enhancing drug permeability in the bladder, mucoadhesive polymers-based delivery systems for intravesical therapy have been extensively explored [25,26]. Among these, chitosan (CS), a natural polymer with abundant amino groups, has garnered significant attention due to its cationic properties, mucosal adhesion, and permeability enhancement capabilities. CS has been utilized in various drug delivery forms such as nanoparticles, films and hydrogels [27]. Herein, we employed CS solution as a carrier for drug delivery.

In our current study, we synthesized a glutathione (GSH)-responsive lipophilic oxaliplatin prodrug (octadecyl-OXA-carboxylic acid, OOC) and incorporated it into cationic liposomes (denoted as LRO) containing a stearyl cell-penetrating peptide C18-R8H3 [28]. The LRO formulation facilitated deeper penetration into bladder tumor tissues and released OXA by the reductant GSH in tumor cells. To further enhance therapeutic efficacy, LRO and BCG were co-delivered in viscous CS solution (LRO-BCG/CS) for treating murine orthotopic BC (Scheme 1). LRO-BCG/CS significantly prolonged the retention time of the cargos and enhanced their permeability across the bladder urothelium. Upon intravesical instillations, OXA induced ICD while the combination of BCG effectively remodeled the tumor immune microenvironment and activated systemic immune responses. This chemo-immunotherapy approach markedly inhibited tumor growth after only three instillations at very low doses of OXA and BCG compared with the doses used in previous researches. Overall, our study presents a novel and effective approach for BC therapy, offering the potential to improve intravesical treatments and patient outcomes while minimizing adverse effects and economic burden. LRO-BCG/CS holds great promise for translational potential in BC patients.