Objective: Colon adenocarcinoma is characterized by the downregulation of the retinoic acid receptor, making natural retinoids such as all-trans retinoic acid, 9-cis retinoic acid and 13-cis retinoic acid effective in treatment and chemoprevention due to their ability to increase RARβ expression. However, major limitations to their use include tolerability and acquired resistance. In this study, we evaluated fenretinide, a semisynthetic derivative of all-trans retinoic acid, in an HT-29 cell line. Fenretinide was evaluated both as a free drug and encapsulated in self-assembling phosphatidylcholine nanosystems with the aim of increasing the aqueous solubility and cell availability of the drug. Methods: Fenretinide was encapsulated in lipid nanosystems obtained in water by the dispersion of an amphiphilic mixture of phospholipids, glyceryl tributyrate and polysorbate 80. The physico-chemical characterization of the nanosystems was carried out by dynamic light scattering and spectrophotometry. The biological activity was evaluated by quantitative phase imaging microscopy, MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Results: Fenretinide in phosphatidylcholine nanosystems was more active than free fenretinide in inhibiting HT-29 cells’ proliferation, as indicated by quantitative phase imaging data. Indeed, encapsulated fenretinide increased duplication time, decreased dry mass and decreased the rate of cell growth more efficiently than fenretinide. Moreover, encapsulated fenretinide effectively decreased the motility of the cells that survived the treatment. Conclusions: The results indicate that the proposed nanosystems can be considered a valuable alternative to natural retinoids in the chemoprevention and treatment of colorectal cancer. This is due to the favorable pharmacologic characteristics of fenretinide in colorectal cancer and the improved drug activity provided by nanoencapsulation.

Background: Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. At present, multi-agent chemotherapy and surgery provide only limited effects and the prognosis for patients with recurrent or metastatic disease remains poor, with 5-year survival rates below 30%. These challenges highlight the need for innovative therapeutic approaches targeting osteosarcoma more effectively. Fenretinide, a synthetic derivative of all-trans retinoic acid, has shown significant antitumor activity in various cancers. In a recent high-throughput drug screening study, fenretinide emerged as the most active molecule against diffuse midline glioma over more than 3500 compounds. Fenretinide also demonstrated cytotoxic activity against osteosarcoma cell lines in vitro and in preclinical models and is endowed with a favorable safety and toxicity profile. However, its poor water solubility and limited bioavailability have hindered its clinical translation. To improve fenretinide bioavailability and enhance tumor exposure, different nanotechnology-based drug delivery systems have been proposed. Here we propose a tertiary complex made of fenretinide, bovine serum albumin, and hydroxypropyl-betacyclodextrin, indicated as BSAF. Methods: BSAF was evaluated for the main physico-chemical parameters such as hydrodynamic size, zeta potential, stability to drug leakage, and the biological effect on the osteosarcoma cell line MG63. Results: BSAF showed hydrodynamic size at the nanoscale, enhanced drug solubilization, high drug loading and size stability to dilution, characteristics that make this complex useful for targeted therapy. When tested on the MG63 osteosarcoma cell line, BSAF demonstrated significantly enhanced cytotoxicity, with half-maximal inhibitory concentration (IC₅₀) values ~50% lower than free fenretinide. The complex was more efficient than free fenretinide in inhibiting cell migration as demonstrated by wound healing assay. Live-cell imaging analyses revealed a cytostatic effect at sub-cytotoxic concentrations. Specifically, treatment with concentrations below the IC₅₀ resulted in significantly prolonged cell doubling time, decreased cell divisions, increased cellular sphericity and thickness, and decreased cell area. These morphological changes are more consistent with cell cycle arrest rather than apoptosis. These findings were corroborated by stable dry mass measurements, an indication of a cytostatic state rather than progressive cell death. In addition, cell motility parameters (e.g., instantaneous velocity, track speed, and displacement) at the single-cell and population level were markedly reduced at sub-IC₅₀ concentrations, further supporting a cytostatic phenotype. Conclusions: Collectively, the new BSAF complex showed promise as a potential therapeutic agent for treating osteosarcoma cancer, due to the favorable physico-chemical characteristics and the cytotoxic/cytostatic effects on MG63 cells. BSAF effects may be therapeutically valuable, particularly in preventing tumor recurrence by suppressing the proliferative and migratory potential of residual drug-resistant clones. Unlike conventional anticancer agents that mainly rely on cell death, fenretinide, when complexed, demonstrates a dual capacity to induce both cytotoxic and cytostatic responses, depending on concentrations, potentially overcoming multiple resistance mechanisms that are generally associated with tumor exposure to drug sub-cytotoxic concentrations.