**1. Introduction**

Over the last decades, polyamine research has continuously progressed, providing insight into the anabolic pathways and transport processes of polyamines. The effects of polyamines on cancer cells have also been explored in relation to different oncogenes and signaling pathways involved [1–3]. It has been demonstrated that oncogenes can affect the metabolism and function of polyamines by interfering with the expression and translation of key enzymes. Polyamines can also influence the expression of oncogenes in various ways, thus regulating the physiological function of cancer cells [4,5]. This research has prompted new ideas for cancer treatment. In particular, it has been demonstrated that the use of 2-difluoromethylornithine (DMFO), an inhibitor of ornithine decarboxylase (ODC), interferes with the polyamine biosynthesis and slows down the development of cancer in high-risk groups but DFMO has no significant effect on preventing cancer recurrence [6]. Moreover, the association of DFMO with inhibitors of polyamine transporters strongly

**Citation:** Lodeserto, P.; Rossi, M.; Blasi, P.; Farruggia, G.; Orienti, I. Nanospermidine in Combination with Nanofenretinide Induces Cell Death in Neuroblastoma Cell Lines. *Pharmaceutics* **2022**, *14*, 1215. https://doi.org/10.3390/ pharmaceutics14061215

Academic Editors: James J. Moon and Carlo Irace

Received: 27 March 2022 Accepted: 6 June 2022 Published: 7 June 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

improved the effect of single DFMO treatment in different tumor models by inducing significant levels of polyamine depletion in cells.

More recent attention has been given to polyamine analogs that upregulate polyamine catabolism and generate toxic compounds, such as H2O2, as a means to induce cancer cell death [7,8]. H2O<sup>2</sup> is formed, with 3-acetoamidopropanal, by the acetylpolyamine oxidase (PAOX) catalyzed conversion of N-acetylspermine and N-acetylspermidine into spermidine and putrescine, respectively. It is also formed by the conversion of N-acetylspermine in N-acetylspermidine, catalyzed by the spermine oxidase (SMOX) (Figure 1). Polyamine analogs, such as N1,N11-diethylnorspermine (DENSpm), and N1-ethyl-N11-[(cyclopropyl) methyl]-4,8- diazaundecane (CPENSpm), have provided cytotoxic responses in several tumor types by their ability to upregulate SMOX and Spermidine/spermine N1-acetyltransferase (SSAT) with a consequent increase of H2O<sup>2</sup> [9,10]. However, these molecules showed some drawbacks, such as poor specificity for cancer cells and induction of epithelial-mesenchymal transition-dedifferentiation in non-cancerous cells [11]. Overall, they showed poor positive outcomes in Phases I and II clinical trials [12].

**Figure 1.** Schematic representation of the polyamine catabolic pathway and absorption mechanisms in cells. Spermidine/spermine N1-acetyltransferase (SSAT) catalyzes the acetyl-group transfer from acetyl-CoA to the aminopropyl end of spermidine or spermine, producing N1-acetylspermidine or N1-acetylspermine, respectively. These acetylated polyamines are either excreted from the cell or used as substrates for peroxisomal N1-acetylpolyamine oxidase (PAOX), producing H2O<sup>2</sup> , 3 acetoamidopropanal, and either putrescine or spermidine, depending on the starting substrate. Alternatively, N1-acetylspermine can be directly converted to spermidine by spermine oxidase (SMOX) while generating H2O<sup>2</sup> and 3-aminopropanal. Polyamine absorption takes place by specific import-export carriers. Other possible mechanisms: passive diffusion and penetration by nanomicelles.

A new strategy to cause cell death in tumors might be based on increasing intracellular polyamine concentrations above physiological values to induce the production of oxidation metabolites at levels exceeding cell tolerance (Figure 1). However, the increase in intracellular polyamine concentration cannot be easily achieved by exogenous administration because the intracellular concentration of polyamines is tightly regulated by specific transporters that import or export polyamines depending on the cell necessity [13]. In

addition, passive diffusion is hampered by the low concentration gradient outside-inside the cell and the massive protonation of polyamines in body fluids [14–16]. For this purpose, we prepared nanospermidine by spermidine encapsulation in nanomicelles and evaluated its ability to induce cell death in two neuroblastoma cell lines: NLF and BR6. Moreover, we evaluated the combination of nanospermidine with nanofenretinide to assess if the contribution of a drug able to increase intracellular ROS levels [17–20] could improve the cytotoxic effect of nanospermidine on tumor cells. Indeed, we had previously demonstrated that nanofenretinide was highly active in neuroblastoma and DIPG tumor models, and the effects were mediated by ROS increase [21–24].

To better understand the biological effect of this treatment, we employed a new powerful microscopic technique, the quantitative phase imaging (QPI), which employs various methods (e.g., holography, ptychography) to retrieve the phase shift of light waves passing through the cells. QPI techniques measure the extent of phase delay generated by the sample and record it as pixel values within the generated image. Pixel intensity is determined by the physical thickness and the refraction index of the cells, the latter depending on biomolecule composition and organization [25,26]. In this study, a Lifecyte microscope was employed to perform QPI based on ptychography. This microscope collects multiple diffraction patterns from spatially overlapping regions of the samples to form QPI images and estimate cell number, confluence, cell dry mass, cell morphology, and motility [27–30]. Furthermore, it is built within a cell CO<sup>2</sup> incubator that maintains the plate at 37 ◦C and 5% CO2, thus allowing measures for long time periods without cell damage.

We used spermidine, among the other polyamines, as it has been proved to suppress tumorigenesis in healthy tissues and, in tumors, it contributed to the modulation of cancerrelated functions, including immunoregulation, autophagy, and apoptosis [31]. In the tumor microenvironment, in particular, spermidine can induce the autophagy-dependent release of ATP, which, in turn, promotes immune surveillance [32,33]. Additionally, spermidine demonstrated the ability to alter macrophage immunometabolism and stimulate CD8+ T-cells [34] and memory B-cell responses [35–37].
