Towards Bimodal Optical Monitoring of Photodynamic Therapy with Targeted Nanoconstructs: A Phantom Study  
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Increase in the efficiency of PDT treatment requires the development of protocols for a PDT procedure employing aids for both controlling the delivery of a PS to the tumor area, and monitoring the photodynamic reaction during light irradiation.  
Optical imaging techniques demonstrate high potential for noninvasive monitoring of PS accumulation and photobleaching in the course of a PDT procedure [4]. Since PSs typically exhibit strong fluorescence, fluorescence imaging techniques are usually employed for non-invasive PS distribution monitoring [5; 6; 7; 8; 9], while decrease of fluorescence signal as a result of light irradiation indicates efficient photodynamic reaction [5; 10]. Benefits of fluorescence imaging (FL) include high sensitivity due to low far-red autofluorescence, and relative simplicity of hardware implementation. On the other hand, FL does not allow for depth-resolved visualization, while multiple scattering leads to image blurring [11]. In this respect, complementary application of an auxiliary depth-resolving imaging modality can provide additional information on drug localization.  
Targeted nanoconstructs are a modern type of PS carrier that combine the drug itself with additional complexes, ensuring targeted delivery, and contrasting agents, providing high sensitivity for monitoring techniques. Employment of benzoporphyrin derivative (BPD), a clinically approved photosensitizer, as a component of a nanoconstruct can significantly enhance the efficiency of photodynamic therapy against cancer cells, due to the red-shifted absorption peak (\(\lambda_{\text{ex}}=690\) nm), fast accumulation, and rapid excretion [12; 13; 14]. However, the excitation spectral band in the visible range and relatively low fluorescence quantum yield of BPD [15] limit the abilities of fluorescence imaging at large depths. This aspect requires the introduction of an additional contrasting agent as a component of a nanoconstruct. Infrared fluorescence marker IRDye800 is a potential candidate for complementary use with BPD in nanoconstructs, owing to its higher fluorescence brightness and the excitation spectrum shifted to the near-IR spectral range [16].  
Optoacoustic imaging [17] has high potential as a complementary technique for localizing PS accumulation due to the absorption-based contrast, the in-depth resolution, and relatively high imaging depth [18]. A spectral approach in optoacoustics, allowing for tuning of the probing wavelength to the absorption maxima of the employed contrast agent provides high imaging sensitivity. In the case that the nanoconstruct contains a contrast agent in addition to the PS, dual-wavelength optoacoustic imaging can provide an additional benefit of complementarily localizing both dyes. Moreover, as mentioned above, the absorption band of IRDye800 is shifted to the IR range with respect to the absorption band of BPD, which provides deeper probing of IRDye800-labeled objects compared to those labeled with BPD.  
A previous study demonstrated the efficacy of complementary application of FL and OA in monitoring targeted nanoconstructs in frames of numerical Monte Carlo simulations [19]. This paper presents the next step in the study of this bimodal monitoring approach, demonstrating its efficiency in a phantom study where the BPD and IRDye800-containing inclusions embedded in a biotissue mimicking phantom are imaged by both FL and OA modalities and the potentiality of joint use of these techniques is estimated.