High incidence and death rates define bladder cancer, a major worldwide health burden. Ranked ninth most common cancer and the thirteenth leading cause of cancer-related mortality, an estimated 614,298 new cases and 220,506 deaths were recorded globally in 2022 [[1], [2], [3]]. Men are especially disproportionately affected; in 2022, they account for 523,674 fresh cases, sixth most common cancer in this population. Recurrence and metastases account mostly for the bad prognosis in advanced bladder cancer [4]. Because they damage healthy tissues, conventional treatment modalities including surgery, chemotherapy, and radiation often show non-selective cytotoxicity, resulting in major side effects including alopecia, tiredness, anemia, and leukopenia [[5], [6], [7], [8]]. More selective and effective therapeutic approaches are therefore desperately needed.

Emerging as a promising minimally invasive alternative for many diseases, including cancer [9,10] is photodynamic treatment (PDT). PDT generates cytotoxic reactive oxygen species (ROS) by means of the synergistic action of a photosensitizer, light, and molecular oxygen, so causing cellular destruction [11,12]. The characteristics of the photosensitizer, especially its capacity to effectively generate ROS upon light irradiation, exhibit great biocompatibility, and selectively accumulate in target tissues [13,14], hence define the efficacy of PDT in great part. Although many organic and inorganic nanomaterials have been investigated as photosensitizers and/or delivery vehicles to improve PDT results, each has natural restrictions.

Traditional organic photosensitizers sometimes suffer from poor water solubility, photobleaching, and a tendency to aggregate under physiological conditions, thus delivery systems [15,16] become necessary. Because of their special optical characteristics and possibility for surface functionalization, inorganic nanomaterials including gold nanoparticles (AuNPs) and quantum dots (QDs) have attracted a lot of interest in PDT [17]. While QDs show high photoluminescence quantum yields and tuned emission wavelengths [19], AuNPs can increase ROS generation by surface plasmon resonance-mediated energy transfer [18]. But questions about the possible long-term toxicity of heavy metals linked with AuNPs and QDs as well as their low biodegradability call for the investigation of substitute, biocompatible NPs [20].

A naturally occurring calcium phosphate mineral that is the inorganic component of bone and teeth, hydroxyapatite (HA) has garnered interest as a potential biomaterial for biomedical applications due to its exceptional osteoconductive, biocompatible, and biodegradable properties [21]. A flexible method to customize the physicochemical properties and functions of the HA lattice is provided by the possibility to replace calcium ions inside it with alternative metal ions [22]. Incorporating elements like cerium (Ce) and zinc (Zn), the development of dual mineral substituted hydroxyapatite (DHA) NPs recently shows promise as a creative platform for many biological uses [23]. To improve DHA NPs even more, surface modification of them has been studied. Photoensitizers such as Riboflavin (RFA) can help to increase biocompatibility, stability, and stop particle agglomerating. Generally regarded as non-toxic and biocompatible, RFA is an FDA-approved, water-soluble vitamin having uses in several biological settings. Although the original text notes RFA increasing the sensitivity of cancer cells to anticancer medications, this is not the main emphasis of its application as a coating in this work for PDT.

The application of RFA-coated DHA NPs as photosensitizers for PDT is investigated in this work. Although preliminary studies show that DHA NPs can produce ROS upon light irradiation [24], this work specifically aims to emphasize the possible advantages of DHA NPs over conventional PDT NPs such as gold NPs and quantum dots. We hypothesize that the special mix of cerium and zinc inside the hydroxyapatite matrix may produce a synergistic effect, so overcoming some constraints related with conventional NPs used in PDT and possibly leading to enhanced ROS generation, improved biocompatibility, and inherent biodegradability. This work will describe the synthesis, characterizing, and evaluation of RFA-coated DHA NPs together with a comparative study with known PDT NPs.

A well-characterized and extensively used human transitional cell carcinoma cell line derived from a grade III papillary bladder tumor [25], T24 bladder cancer cells are a fundamental in vitro model for analyzing bladder cancer progression and evaluating novel therapeutic approaches including PDT. Their fast spread and invasive capacity as well as their unique genetic and molecular profiles make them a pertinent target for study aiming at creating focused treatments. The steady and strong expansion of T24 cells in laboratory environments enables repeatable studies on drug efficacy, cellular signalling pathways, and the effects of new approaches including the use of bare and RFA-coated DHA NPs as photosensitizers to induce cytotoxicity and cell death via ROS generation.