3D Tumor-Mimicking Phantom Models for Assessing NIR I/II Nanoparticles in Fluorescence-Guided Surgical Interventions

Introduction: : Fluorescence image-guided surgery (FIGS) offers high spatial resolution and real-time feedback but is limited by shallow tissue penetration and autofluorescence from current clinically approved fluorophores. The near-infrared (NIR) spectrum, specifically the NIR-I (700-900 nm) and NIR-II (950-1700 nm), addresses these limitations with deeper tissue penetration and improved signal-to-noise ratios. However, biological barriers and suboptimal optical performance under surgical conditions have hindered the clinical translation of NIR-I/II nanoprobes. In vivo mouse models have shown promise, but do not replicate the complex optical scenarios encountered during real-world surgeries. Existing tissue-mimicking phantoms used to evaluate NIR-I/II imaging systems are useful but fall short when assessing nanoprobes in surgical environments. These phantoms often fail to replicate the tumor microenvironment, limiting their predictive assessment. To overcome these challenges, we propose developing tumor-mimicking phantom models (TMPs) that integrate key tumor features, such as tunable tumor cell densities, in vivo-like nanoparticle concentrations, biologically relevant factors (pH, enzymes), replicate light absorption components (hemoglobin), and light scattering components (intralipid). These TMPs enable more clinically relevant NIR-I/II nanoprobes assessments, including optical tissue penetration profiling, tumor margin delineation, and ex vivo thoracic surgery on porcine lungs. The components of TMPs can be further modulated to closely match the optical profiles of in vivo and ex vivo tumors. Additionally, 3D bioprinting technology facilitates a high-throughput platform for screening nanoprobes under realistic conditions. This approach will identify high-performing NIR-I/II probes with superior surgical utility, bridging the gap between preclinical findings and clinical applications, and ensuring results extend beyond traditional in vivo mouse studies.

Materials and

Methods: : A standard phantom stock solution was formulated by combining 720 mg of type-A gelatin, 150 mg of human hemoglobin, 900 µL of 20% intralipid, and 17.1 mL of 1X PBS. This prepared stock served as the foundational base for all subsequent phantom formulations (mimicking the tumor microenvironment along with key tumor attributes such as pH, enzymes, and morphology). For synthesizing nanoparticles (SPNs), DSPE-PEG was used to coat and trap the semiconducting polymer dyes (PFODBT, PCPDTBT, PDFT-T) following a nanoprecipitation method in PBS 1X. All the characterization was done using DLS Lite Sizer, Fluorescence spectrometer, and UV-VIS absorbance in the Srivastava Lab at Texas Tech University. Transmission electron microscopy was conducted in the Experimental Science Building I of Texas Tech University. To prepare the TMP, 1.5 mL of phantom stock solution was mixed with 500 µL of SPNs incorporated with pH, enzyme, or oxygen level. The mixtures (2 mL total) were transferred to silicone mold wells and frozen for a minimum of 1 hour to solidify. Different porcine tissues (fat, muscle, skin) were purchased from local grocery shops, dry-preserved porcine lungs were purchased from Nasco Education, and were used to evaluate optical performance of the nanoprobes in the NIR-I/II window by simulating real-time surgical scenarios. The fluorescence images were collected at the IVIS imager at Texas Tech University Experimental Science Building II and the IR Vivo imager at TTU Health Science Center, Amarillo. The phantom solution, along with SPN solution, was used to fabricate tumor tumor-mimicking phantom using Allevi 3 bioprinter.

Results, Conclusions, and Discussions:: The comprehensive design of tumor-mimicking phantoms enables an accurate replication of the optical properties and microenvironment of tumors, providing a significant improvement over traditional tissue phantoms. With our synthesized TMP model, it becomes possible to perform rapid assessments of nanoprobe performance in critical surgical scenarios, including tumor margin delineation and tissue penetration, using porcine tissue and ex vivo models. Tissue permeation profiling, both in NIR-I and NIR-II windows, shows that our approach could be leveraged to evaluate the tissue penetration performance of NIR NPs candidates being developed and tested in preclinical in vivo models, offering insights into how they might perform in an intraoperative surgical setting. Tumor margin delineation experiment showed how PCPDTBT and PDFT-T NPs are better performing in precise tumor margin delineation than PFODBT NP and can be further evaluated in clinical studies (Figure 2). The ex vivo porcine lung surgery underscores the potential of our TMPs in NIR-I/NIR-II-guided surgery simulations, highlighting their relevance for evaluating pre-clinical NPs and their ability to enhance surgical precision and outcomes in the development of fluorescence-guided surgical procedures (Figure 3).
Moreover, incorporating 3D bioprinting technology introduces the potential for high-throughput development of TMPs with optical and mechanical reproducibility, facilitating enhanced preclinical screening and validation and enabling the identification of the most effective NIR-I/II nanoprobes for surgical applications.

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