Can targeted nanoparticles distinguish cancer metastasis from inflammation?

Introduction: Nanoparticles have potential in exploiting the leaky vasculature of primary tumors and facilitating enhanced intratumoral drug delivery, leading to its extensively research for cancer drug delivery. To enhance their selectivity and uptake by cancer cells, targeting ligands have been extensively employed to augment nanoparticle accumulation within tumors and their uptake by cancer cells. A few popular targets include fibronectin, αvβ3 integrin, and folate receptor. However, these ligands often target molecules upregulated not only in tumors but also in inflamed tissues.

Given that a significant portion of cancer patients exhibit comorbidities such as hypertension, chronic obstructive pulmonary disease (COPD), rheumatologic diseases, or diabetes, it is imperative to assess whether targeting ligands can effectively distinguish between the tumor microenvironment and inflammation. Fibronectin plays a crucial for cancer cell migration but is also elevated in inflamed tissues during the healing process. Similarly, αvβ3 integrin is involved in the mesenchymal phenotype of cancer cells promoting migration and metastasis, but also plays a role as adhesion molecules for leukocyte recruitment in inflammation. Moreover, targeting ligands may make nanoparticles highly immunogenic, leading to their rapid recognition and uptake by phagocytes in tumors or inflamed tissues. For instance, when folic acid is utilized as a ligand for nanoparticles, rapid recognition by phagocytes occurs.

In this study, we investigate the ability of targeted and untargeted nanoparticles to distinguish between sites of early metastasis and sites of inflammation.

Materials and

Methods: Using a 60-nm liposome as a representative nanoparticle, we generated three variants of targeted nanoparticles employing commonly used targeting ligands for fibronectin, αvβ3 integrin, or folate receptor. The deposition of these nanoparticles was compared with that of standard untargeted nanoparticles. We evaluated nanoparticle deposition in the lungs of mice representing three distinct biological landscapes: healthy lungs, lungs with aggressive metastasis, and lungs exhibiting general pulmonary inflammation.

For pulmonary metastases, we utilized the D2.A1 model of murine breast cancer, representing proliferating metastasis. For the lung inflammation model, we induced lung inflammation using bleomycin. Bleomycin is a chemotherapeutic drug known to induce severe lung inflammation in mice, ultimately leading to pulmonary fibrosis. This model was employed 5 days after bleomycin administration, corresponding to the pre-fibrotic inflammatory phase. Using fluorescently labeled nanoparticles and ex vivo fluorescence imaging of organs, we evaluated nanoparticle deposition in the lungs of mice 4 hours post systemic injection. Ex vivo fluorescence imaging of lungs was conducted using the IVIS Spectrum system. Finally, flow cytometry was performed to analyze the cellular uptake of nanoparticle variants by different immune cell subsets in the lungs.

Results, Conclusions, and Discussions: We generated three targeted nanoparticle variants—fibronectin-targeting, folate-targeting, and αvβ3 integrin-targeting—and compared their deposition in lungs with that of standard untargeted nanoparticles. Among the four nanoparticle types, fibronectin-targeting and untargeted nanoparticles exhibited the highest deposition in lungs containing aggressive metastasis, with a 2-fold higher deposition compared to folate-targeting nanoparticles.

While fibronectin-targeting and untargeted nanoparticles demonstrated superior performance in aggressive metastasis, it is critical to acknowledge that the common targets for cancer drug delivery are not exclusive to the tumor microenvironment but are also present in general inflamed tissues. Investigation of nanoparticle deposition in the bleomycin-induced pulmonary inflammation model revealed that only the untargeted nanoparticles exhibited higher deposition in metastases than in lungs with inflammation. All targeted nanoparticle variants in lungs with metastases closely resembled those in lungs with inflammation. (Fig 1.)

Additionally, flow cytometry analysis revealed predominant accumulation of all nanoparticle variants in immune cells rather than cancer cells. For instance, in the case of fibronectin-targeting nanoparticles, the number of nanoparticle-positive macrophages and dendritic cells was 16-fold higher than nanoparticle-positive cancer cells.

Our findings highlight the limitations of targeted nanoparticles in distinguishing between cancer metastasis and general inflammation, which could have significant clinical implications for nanoparticle-mediated drug delivery in cancer therapy. Inflamed tissues resulting from comorbidities may act as a sink, diverting cancer drugs away from tumor sites and reducing therapeutic efficacy. Moreover, nanoparticles were predominantly found in immune cells rather than cancer cells, suggesting limitations in conventional nanoparticle mediated anticancer drug delivery to cancer cells. However, this phenomenon may be advantageous for immunotherapy applications utilizing nanoparticles to deliver immunomodulatory agents to immune cells.

Acknowledgements (Optional): This work was supported by grants from the National Cancer Institute R01CA253627 (E.K.), R01CA278633 (E.K. ), P30CA043703 (E.K.), and the Angie Fowler AYA Cancer Research Fund (E.K.). T.J.M. was supported by the NSF graduate research fellowships program. A.B. was supported by a fellowship from the NIH Interdisciplinary Biomedical Imaging Training Program (T32EB007509).