Nanospermidine in Combination with Nanofenretinide Induces Cell Death in Neuroblastoma Cell Lines

Abstract

A new strategy to cause cell death in tumors might be the increase of intracellular polyamines at concentrations above their physiological values to trigger the production of oxidation metabolites at levels exceeding cell tolerance. To test this hypothesis, we prepared nanospermidine as a carrier for spermidine penetration into the cells, able to escape the polyamine transport system that strictly regulates intracellular polyamine levels. Nanospermidine was prepared by spermidine encapsulation in nanomicelles and was characterized by size, zeta potential, loading, dimensional stability to dilution, and stability to spermidine leakage. Antitumor activity, ROS production, and cell penetration ability were evaluated in vitro in two neuroblastoma cell lines (NLF and BR6). Nanospermidine was tested as a single agent and in combination with nanofenretinide. Free spermidine was also tested as a comparison. The results indicated that the nanomicelles successfully transported spermidine into the cells inducing cell death in a concentration range (150–200 μM) tenfold lower than that required to provide similar cytotoxicity with free spermidine (1500–2000 μM). Nanofenretinide provided a cytostatic effect in combination with the lowest nanospermidine concentrations evaluated and slightly improved nanospermidine cytotoxicity at the highest concentrations. These data suggest that nanospermidine has the potential to become a new approach in cancer treatment. At the cellular level, in fact, it exploits polyamine catabolism by means of biocompatible doses of spermidine and, in vivo settings, it can exploit the selective accumulation of nanomedicines at the tumor site. Nanofenretinide combination further improves its efficacy. Furthermore, the proven ability of spermidine to activate macrophages and lymphocytes suggests that nanospermidine could inhibit immunosuppression in the tumor environment.

Spermine oxidase induces DNA damage and sensitizes fusion negative rhabdomyosarcoma cells to irradiation

“…We have also observed that forced expression of SMOX promotes cell cycle arrest and apoptosis of FN-RMS cells, with a concomitant reduction of their tumorigenic features, such as anchorage-independent growth. These results are in agreement with the previous finding that an increase in SPD intracellular levels induces reactive oxygen species (ROS) production (i.e., H 2 O 2 ), this leading to the death of neuroblastoma, breast or lung cancer cells ( McCloskey et al, 1996 ; Pledgie et al, 2005 ; Lodeserto et al, 2022 ). In this regard, it has to be considered an increase in SPD intracellular levels can cause cancer cell death also in a ROS-independent manner through Bim activation ( Guo et al, 2020 ).…”

“…Nevertheless however, search for efficient and selective SMOX or PAOX inhibitors of is still ongoing ( Di Paolo et al, 2019 ). Recently, nanoSPD have been used to carry SPM into neuroblastoma cells, impairing cell growth and potentiating the effects of nanofenretidine in vitro ( Lodeserto et al, 2022 ).…”

Naxitamab Activity in Neuroblastoma Cells Is Enhanced by Nanofenretinide and Nanospermidine

“…The dry product was reconstituted with water to 50 mg/mL NF and stored at −22 °C until use. Spermidine nanomicelles were prepared as previously reported [ 22 ] by mixing soy phosphatidylcholine (4 mmoles), glyceryl tributyrate (2 mmoles), 2-hydroxypropyl beta cyclodextrin (0.4 mmoles), KOH 10 N (400 µL, 4 mmoles), and spermidine (2 mmoles) to homogeneity to obtain a semisolid phase that was dispersed in PBS pH 7.4 to 50 mg/mL. The coarse nanomicelle suspension obtained was filtered through 0.2 µm filters, dialyzed for 72 h (dialysis membrane Mw cutoff 10 KD) against PBS pH 7.4, and then, the dialyzed phase was lyophilized.…”

“…Nanofenretinide is a nanoformulation based on phospholipid nanomicelles containing fenretinide, which demonstrated activity in a wide range of tumor cell lines and xenograft models [ 18 , 19 , 20 , 21 , 22 ]. The ability of Nanofenretinide to penetrate tumor cells and transport high drug amounts has been proven in NB cells that showed higher intracellular drug concentrations with respect to free fenretinide [ 21 ].…”

“…Nanospermidine is a nanoformulation made of phospholipid nanomicelles containing spermidine. It had been previously evaluated for its ability to transport supraphysiological concentrations of spermidine into NB cells inducing an ROS increase [ 22 ]. The activation of stress-associated pathways, triggered by the ROS increase, is expected to improve the cell sensitivity to naxitamab.…”