*Article* **Synthesis and In Vitro Characterization of Ascorbyl Palmitate-Loaded Solid Lipid Nanoparticles**

**Maja Ledinski <sup>1</sup> , Ivan Mari´c <sup>2</sup> , Petra Peharec Štefani´c <sup>1</sup> , Iva Ladan <sup>3</sup> , Katarina Caput Mihali´c <sup>1</sup> , Tanja Jurkin <sup>2</sup> , Marijan Goti´c <sup>4</sup> and Inga Urli´c 1,\***


**Abstract:** Antitumor applications of ascorbic acid (AA) and its oxidized form dehydroascorbic acid (DHA) can be quite challenging due to their instability and sensitivity to degradation in aqueous media. To overcome this obstacle, we have synthesized solid lipid nanoparticles loaded with ascorbyl palmitate (SLN-AP) with variations in proportions of the polymer Pluronic F-68. SLNs were synthesized using the hot homogenization method, characterized by measuring the particle size, polydispersity, zeta potential and visualized by TEM. To investigate the cellular uptake of the SLN, we have incorporated coumarin-6 into the same SLN formulation and followed their successful uptake for 48 h. We have tested the cytotoxicity of the SLN formulations and free ascorbate forms, AA and DHA, on HEK 293 and U2OS cell lines by MTT assay. The SLN-AP in both formulations have a cytotoxic effect at lower concentrations when compared to ascorbate applied the form of AA or DHA. Better selectivity for targeting tumor cell line was observed with 3% Pluronic F-68. The antioxidative effect of the SLN-AP was observed as early as 1 h after the treatment with a small dose of ascorbate applied (5 µM). SLN-AP formulation with 3% Pluronic F-68 needs to be further optimized as an ascorbate carrier due to its intrinsic cytotoxicity.

**Keywords:** ascorbate; ascorbyl palmitate; drug delivery; cellular uptake; nanoparticles; antitumor effect
