Evaluating the efficiency and tumor selectivity of phthalocyanine nanoemulsions using tumor infiltration models

Introduction: Chemo- and radiotherapies, while critical in treating cancer, often compromise ovarian function, putting fertility at risk. For prepubertal girls and women requiring immediate treatment, an option is to remove and cryopreserve ovarian tissue for later transplantation after remission. However, in leukemia patients, this approach carries a significant risk: reintroducing malignant cells through the transplanted tissue, potentially leading to a recurrence of the disease (1). One of the most straightforward solutions is the development of purging strategies that can effectively eliminate these malignant cells ex vivo from ovarian tissue (2,3). Yet, ovarian tissue samples from leukemia patients are both scarce and highly valuable for research, with no certainty that all specimens contain malignant cells. This scarcity presents a major challenge in advancing effective cancer therapies. To address this issue, we have developed an engineered tumor-infiltration mimicking model (ETIM) using PEGylated fibrin hydrogels. This model replicates the tumor microenvironment through its mechanical tunability and biological relevance, allowing for the co-culture of ovarian and cancer cells to simulate tumor invasion dynamics more accurately. After establishing the realistic 3D ETIM that closely mimics ovarian tissue infiltrated by cancer cells, these models will be used to evaluate the efficacy and tumor selectivity of phthalocyanine nanoemulsions. Our findings offer important insights into tumor behavior within ovarian environments and hold promise for advancing cancer research, as well as the development of more targeted therapeutic strategies. Materials and Methods: Acute myeloid leukemia cells (HL-60) and ovarian stromal cells (SCs) were co-cultured at a 1:100 ratio within a hydrogel matrix engineered to replicate extracellular conditions. The hydrogel’s structural properties and cytocompatibility were evaluated using scanning electron microscopy (SEM) and LIVE/DEAD viability assays. Immunofluorescence targeting CD43 and Ki-67 was employed to analyze cancer cell identification, proliferation, and density dynamics. Following the preparation of ETIMs, these models were utilized to assess the efficacy of our established photodynamic therapy strategies. These approaches involved nanoemulsions (NE) incorporating two phthalocyanine photosensitizers—aluminum (III) phthalocyanine (AlPc) and zinc (II) phthalocyanine (ZnPc)—which demonstrated selective eradication of leukemic cancer cells (2). The size and morphology of these nanoemulsions were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Finally, the efficiency of these approaches will be evaluated on developed ETIMs using immunofluorescence and tumor-specific gene expression by reverse transcription polymerase chain reaction (RT-PCR), verifying the persistence of malignant cells within the ETIMs (Fig. 1). Results: Fibrin-based hydrogels were used to create a 3D tumor microenvironment, providing mechanical support while regulating tumor behavior. The PEGylated fibrinogen-thrombin reaction formed a dense hydrogel with a compact morphology. SEM analysis revealed a tightly packed fibrous network with an average fiber diameter of 97 nm, indicating enhanced mechanical stability (Fig. 2). Cytocompatibility tests showed no toxicity toward SCs or HL-60 cells after 24 hours. CD43/Ki-67 immunofluorescence staining effectively detected healthy and malignant cells while tracking cell density changes. The ETIM model accurately simulated leukemia cell infiltration into ovarian tissue, with stable malignant cell activity and proliferation over five days. Discussion: Fibrin-based hydrogels provided a suitable environment for cell growth and infiltration, mimicking the extracellular matrix with strong mechanical stability and biocompatibility. The stable proliferation of malignant cells within ETIM suggests its potential as a reliable platform for studying tumor progression and drug response. This model effectively replicates leukemia cell behavior in ovarian tissue, supporting its use in therapeutic evaluations. Conclusion: The PEGylated fibrin hydrogel scaffold offers a stable, supportive platform for engineered tumor models, promoting cell viability and proliferation. Ongoing studies will assess AlPC-Ne and ZnPc-NE within this model to advance tissue engineering strategies for improved therapeutic applications.