**1. Introduction**

Multimodal cancer therapy involves the use of two or more treatment modalities with the aim to attack tumors on different sides by acting either on a single oncogenic pathway through different mechanisms or across parallel pathways without amplification of side effects [1–4]. In this frame, the combination of conventional chemotherapeutics with light-activated therapeutic treatments is a very appealing approach to potentiate the therapeutic outcome through anticancer synergistic/additive effects [5]. Light represents in fact as suitable and minimally invasive tool that permits the introduction of therapeutic species in biological environments with superb spatiotemporal control [6]. Among the light-activatable therapeutic treatments, photodynamic therapy (PDT) is so far one of the most promising to combat cancer diseases [7]. This treatment modality mainly exploits the cytotoxic effects of the highly reactive singlet oxygen (1O2), catalytically generated by energy transfer between the long-lived excited triplet state of a photosensitizer (PS) and the nearby molecular oxygen [8].

Another emerging light-activated approach is based on the photoregulated release of nitric oxide (NO) through the use of NO photodonors (NOPD) [9–11]. Although still confined to the research area, this NO-based PDT, namely NOPDT, has come to the limelight in recent years and holds very promising features in cancer treatment [12]. In fact, apart from playing multiple roles in the bioregulation of a broad array of physiological processes [13], NO has also proven to be an effective anticancer agent. If generated in an appropriate concentration range [14,15] this diatomic free radical can act not only as a cytotoxic agen<sup>t</sup> [16], but also as inhibitor of the ATP binding cassette (ABC) transporters responsible of the efflux of chemotherapeutics and of the induction of multidrug resistance (MDR) in cancer cells [17,18]. In contrast to the photocatalytic mechanism leading to 1O2 in PDT, the working principle of NOPDT exploits the excitation light to uncage NO leading, of course, to a consumption of the NOPD [9–11]. Noteworthy, both 1O2 and NO are multitarget species that have not a reduced efficacy in MDR cells due to their short lifetimes, and confine their action to short distances from the production site inside the cells (<20 nm for 1O2 and <200 μm for NO), reducing systemic toxicity issues common to many conventional drugs. Besides, since NO photorelease is independent of O2 availability, it may successfully complement PDT at the onset of hypoxic conditions, typical for some tumors, where PDT may fail [19].

The significant breakthroughs in nanomedicine permit nowadays the development of multifunctional platforms in which more therapeutic agents are entrapped in a single nanocarrier. This offers an unprecedented opportunity to devise a better scheme for precise and controlled delivery of multiple therapeutics to the same area of the body at a predefined extra/intracellular level [20–23]. In this context, nanoplatforms combining PDT and NOPDT are opening new horizons towards more effective and less invasive cancer treatments entirely based on "nonconventional" chemotherapeutics [24]. Besides, an increasing large number of nanoconstructs in which PDT has been combined with chemotherapy in the managemen<sup>t</sup> of several cancer types has been reported in literature [5]. In contrast, only recently NOPDT has been successfully used in combination with chemotherapeutics [25]. Based on this scenario, the achievement of a trimodal nanoplatform able to generate 1O2 and NO and, simultaneously, to deliver a conventional chemotherapeutic is a very challenging objective to pursue. Mesoporous silica nanoparticles (MSNs) are very suitable scaffolds to this end due to their high loading capacity, ease of surface functionalization, good biocompatibility, and satisfactory light transparency [26–30]. Moreover these inorganic scaffolds have been extensively used as excellent carrier for (i) conventional chemotherapeutics to reverse MDR [31], (ii) PDT agents alone and in combinations with chemotherapeutics [32–34], and (iii) spontaneous NO donors [35] and NOPD [36].

In this manuscript, we report the first example of engineered MSNs able to deliver "three bullets" for potential anticancer therapy. These MSNs covalently integrate in their molecular scaffold erythrosine and a nitroaniline derivative as suitable PS and NOPD, respectively, and encapsulate the chemotherapeutic doxorubicin (DOX) in a noncovalent fashion. We demonstrate that the photochemical properties of the PS and NOPD are preserved in an excellent manner in the MSNs containing all active components. As a result, the spontaneous release of DOX under physiological conditions can be combined with the photoregulated generation of 1O2, NO, or both by using light stimuli of appropriate energy in the visible light range.

#### **2. Materials and Methods**
