**4. Targeting integrins for fluorescence imaging and photochemical/ photothermal treatment in ovarian cancer**

Photodynamic therapy (PDT) is a photochemical treatment modality involving the activation of a photosensitive molecule, a photosensitizer (PS), with light of an appropriate wavelength leading to the generation of reactive molecular species at the site of PS localization [155–157]. PSs can be conjugated to proteins or peptides, or formulated in delivery systems, to enhance selectivity or to improve photochemical potency [158–161]. As discussed previously, integrins play an important role in ovarian cancer progression, but targeting integrins for selective drug delivery remains challenging. There are a limited number of studies that focus on integrin targeting in

photochemistry-based applications. This section serves as a comprehensive review of the studies that have evaluated the effects of photosensitization on integrins, as well as the studies that target integrins to improve selectivity for fluorescence imaging and PDT of ovarian cancer. One photothermal therapy (PTT) study is also discussed at the end of this section to cover light-based practices that target integrins in ovarian cancer [162].

The effect of PDT on integrin expression and reorganization has been studied in the context of ovarian cancer by Runnels *et al.* [163]. In this study, OVCAR3 cells were maintained in monolayer or injected intraperitoneally into nude mice. *In vitro* and *in vivo* PDT treatments were carried out using a 690 nm argon ion pumped dye laser at 0.5 J/cm2 energy density following a 3-hour incubation of the cells with 0.092 μmol/L benzoporphyrin derivative monoacid (BPD). Subsequently, the cells were harvested and re-seeded on surfaces coated with ECM proteins: collagen IV, fibronectin, laminin, and vitronectin. Low-dose PDT (~ 85% cell survival) was shown to decrease the adhesion of OVCAR3 cells to collagen IV, fibronectin, laminin, and vitronectincoated substrates *in vitro* and *in vivo*. The authors further reported that the binding of OVCAR3 cells to collagen IV and laminin, but not fibronectin, was inhibited by the presence of an anti-β1 antibody, suggesting that the β1 subunit plays a role in the adhesion of OVCAR3 cells to select ECM proteins. It was also noted that BPD localized in and around mitochondria and caused intracellular damage upon irradiation, mainly mediated by singlet oxygen rather than other reactive molecular species. In this study, BPD-PDT-mediated photodamage was shown to impact integrin function and the integrity of focal adhesion plaques.

A limited number of studies have explored integrins as targets for selective delivery of imaging agents and PSs. In a recent study, Li and colleagues linked an RGD-peptide and IRDye 700 DX (IR700) to human serum albumin [164]. Compared to the untargeted nanoconjugate, cell delivery of the targeted nanoconjugate (cRGD-PEG-HSA-IR700) increased by 121-fold in αvβ3-expressing TOV21G cells. Cells were also treated using a 660 nm LED light source at an irradiance of 3.5 mW/cm<sup>2</sup> for 20 minutes [a fluence of 4.2 J/cm2 , not specified in the report]. PDT effectively killed the αvβ3-expressing TOV21G cells but did not affect αvβ3-negative NIH/3 T3 cells. The nanoconjugates were also tested on spheroids of SKOV3 cells grown in ultra-low attachment wells. Confocal microscopy images and live/dead staining assays revealed that cRGD-PEG-HSA-IR700 successfully penetrated the spheroids, generated cell killing, and caused long-term tumor suppression. An RGD peptide with EtNBS as the PS and a 5 kDa polyethylene glycol (PEG) chain has also been explored in the context of ovarian cancer [165]. Using this construct, cellular uptake was increased in genetically modified, α5 integrin-overexpressing OVCAR5 cells relative to wild-type OVCAR5 cells. PEGylated constructs aggregated less and generated more reactive molecular species compared to their non-PEGylated analogs. Dai *et al.* synthesized a compound called TTB, which exhibits aggregation-induced near infrared (NIR) fluorescence and generates reactive oxygen species when excited by white light [166]. TTB was integrated into an amphiphilic polymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (MPD) and conjugated with RGD peptide to target αvβ3 integrins. Efficacy of the construct for PDT and fluorescence imaging was evaluated *in vitro* and in animal models of prostate, cervical, and ovarian cancer. The integrin-targeted construct was shown to selectively accumulate in tumors, leading to cancer cell death *in vitro* and reduction of tumor size in tumorbearing mice, compared to controls.
