Conventional treatment modalities for cancer include chemotherapy, radiotherapy, and surgery and their implementation depends on the primary origin, stage, and metastasis state. The treatments are associated with various complications, including morbidity, adverse systemic effects, toxicity, and multidrug resistance. Alternative and experimental approaches such as cell therapy, gene therapy, immunotherapy, and targeted therapy have yielded promising results [[1], [2], [3], [4], [5]] but also come with drawbacks that include high costs, toxicity, low efficacy in non-indicated cancer types, and the risk of failure in advanced clinical trials. It is therefore important to keep developing novel interventions that are non-to-minimally invasive, tumor-specific, non-toxic, and affordable for patients regardless of economic class. These experimental therapies could be deployed in a substitutive or complementary manner to currently used modalities.

Photodynamic therapy (PDT, Fig. 1) is such a treatment strategy that is explored for numerous types of solid tumors that arise in internal organs and organ structures [6], including non-resectable extrahepatic cholangiocarcinoma [[7], [8], [9], [10], [11], [12]]. Non-resectable extrahepatic cholangiocarcinoma is currently incurable and responds poorly to chemotherapy [13]. PDT has shown promise for this malignancy [[14], [15], [16], [17], [18]] but widespread clinical implementation has stalled in part due to the following reasons. Clinical caveats of PDT with approved PSs entail long-term skin photosensitivity that could lead to (severe) photoallergic reactions [12] and the corollary ethical concerns over indoors-ridden patients who only have a few months to live [19,20]. Moreover, there are noncircumventable photophysical restrictions that dictate outcome. Insufficient optical penetration of laser light precludes efficient photokilling across the entire tumor volume, especially in bulkier tumors [21]. Consequently, distally located tumor cells are sublethally afflicted by PDT [9] and in response activate survival mechanisms to remedy the hyperoxidative stress and cell damage [11,[21], [22], [23]], which enables the tumor cells to cope and ultimately accounts for tumor regrowth [24,25]. Nonetheless, PDT has several advantages over aforementioned treatments that warrant continued research, namely (1) the doubly selective nature of the treatment [12]; (2) general lack of systemic toxicity of photosensitizers [20]; (3) no development of drug resistance by tumor cells (applies to some PSs) [26,27]; (4) abscopal removal of residually viable tumor cells and metastases as secondary treatment effect [28,29], and (5) relatively low cost [20].

Our group has instituted measures to counter these PDT-specific clinical bottlenecks through the use of third-generation [20] and fourth-generation metallated phthalocyanine-based PSs [8,10,30] that are targeted to pharmacologically relevant locations in the tumor: the tumor microenvironment [7,9,12,19], the tumor endothelium [8,10,11,30,31], and the tumor parenchyma [32]. Liposomes are used to solubilize the lipophilic phthalocyanines and to encapsulate the PS molecules so as to sterically deter PS extravasation through the cutaneous microcirculation and minimize skin photosensitization. The simplest formulation – the interstitially targeted liposomes (ITLs) – encapsulating the PS zinc phthalocyanine (ZnPC) at a 0.003 PS:lipid ratio was shown to be taken up by all relevant cell types in vitro, including tumor cells (A431 [19], SK-ChA-1 [12]), endothelial cells (HUVECs) [12], fibroblasts (NIH-3 T3) [12], and macrophages (RAW 264.7) [12], despite the PEGylation. The extensive cell photosensitization yielded a 50% lethal concentration (LC50) range from 0.16 μM (SK-ChA-1 cells) to 2.03 μM (fibroblasts) 24 h after PDT of cell monolayers [12]. This formulation, consisting of DPPC and DSPE-PEG at a 96:4 M ratio, did not confer notable dark toxicity in multiple species (human and murine cell lines, zebrafish, chicken embryos, and mice) and was associated with moderate skin phototoxicity in vivo under exaggerated light conditions [12]. In mice bearing human triple negative breast cancer (MDA-MB-231) xenografts, the ZnPC-ITLs caused a 4-day (33%) delay in tumors reaching the human endpoint (tumor volume of ≥1800 mm3) after a single PDT session (starting tumor volume of 100–200 mm3, single i.v. bolus of 6 nmol ZnPC/animal, 24-h drug-light interval, 671-nm diode laser, cumulative radiant exposure of 200 J/cm2) compared to untreated tumor controls. At an administered PS dose that was 2 orders of magnitude lower than used in other comparable studies [[33], [34], [35]], the therapeutic efficacy in breast cancer xenografts was non-inferior in terms of tumor volume reduction [12].

Although the in vivo results with the ZnPC-ITLs provided satisfactory proof-of-concept at non-optimized conditions [12], a moderate degree of skin phototoxicity and semi-synthetic make-up of PEGylated liposomes are unacceptable and clinically risk-laden, respectively. In terms of the latter, liposomes can activate the complement system [36,37] and PEGylated liposomes can trigger accelerated blood clearance [38] due to a humoral immune response against PEG (anti-PEG IgM) after the first injection [39] and hepatic and splenic clearance of PEGylated particles in subsequent exposures [38,40]. This issue should not be underestimated in the wake of the recently developed mRNA vaccines, which contain both phospholipids and PEG [41]. Liposomes may also be cleared from the circulation by cells of the reticuloendothelial system (RES), reducing the effective dosage at the target site to therapeutically moot levels [42,43]. The accumulation of liposomes in macrophages, especially at higher doses, can influence the phagocytic activity, leading to immune suppression and hampered pathogen clearance [42,44,45] as well as reduced anti-tumor immune responsiveness [46]. Furthermore, the liposome preparation process requires organic solvents and chemicals [[47], [48], [49], [50]] that may not be biocompatible or GMP-compliant, and the production process itself may not be scalable to desired output levels.

Notwithstanding the availability of immunocompetent polymers for nanoparticle surface modification aimed at prolonging circulation time [51], there may be more effective and more practical routes to (targeted) PS delivery that are clinically feasible. In this study, bionanovesicles were prepared from tumor cells by simple sonication and loaded with ZnPC for PDT of cultured human extrahepatic cholangiocarcinoma (TFK-1) cells. The bionanovesicles were termed cellular vesicles (CVs; vesicles prepared by sonication of tumor cells without prior cell content removal) and cell membrane vesicles (CMVs; vesicles prepared by sonication of tumor cells with prior cell content removal). The CVs and CMVs are fundamentally distinct from secreted or actively formed extracellular vesicles, which entail exosomes, microvesicles, and apoptotic bodies that have been widely studied for purposes of drug delivery [52] and to some degree for PS delivery [53]. In contrast, the bionanovesicles were prepared by physically perturbing cells and structurally rearranging the cellular constituents, yielding nanovesicles with different intravesicular content, bilayer composition, and membrane surface architecture. This approach has not been investigated in the context of PS delivery and PDT. The CVs and CMVs were therefore studied in terms of ZnPC encapsulation efficacy, photosensitization potential of TFK-1 cells, dark toxicity, and photodynamic potency in TFK-1 cell monolayers. The main objectives of this first-in-its-class proof-of-concept study were to rule out cytotoxicity of the PS-encapsulating bionanovesicles and assess the level of phototoxicity in comparison to liposomal photonanomedicines.