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

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

Figure 1

Chemical structures of hydrophilic compounds ascorbic acid and dehydroascorbic acid and lipophilic derivative, ascorbyl palmitate.

Figure 2

The structure of solid lipid nanoparticle. SLNs have lipid core with an incorporated lipid compound of interest and a surfactant layer on the surface which enables solubility in hydrophilic solutions. Ionic co-surfactants provide electrical stability and prevent the possible aggregation of nanoparticles.

Figure 3

Synthesis of solid lipid nanoparticles.

Figure 4

TEM images of SLN. SLN suspensions were contrasted with uranyl acetate and washed in ultra-pure water before visualization. Scale bar = 200 nm (a) SLN-AP 3% Pluronic; (b) blank SLN 3% Pluronic; (c) SLN-AP 10% Pluronic; and (d) blank SLN 10% Pluronic.

Figure 5

Cellular uptake of SLN-coumarin-6 during 48 h in cell lines HEK 293 and U2OS. Cells were treated with SLN-coumarin-6 and photographed at time points 6 h, 24 h and 48 h. Green fluorescence represents coumarin-6 that entered the cells. Scale bar = 100 µm.

Figure 6

Cytotoxicity of SLN AP and blank SLN synthesized with either 3% (a) or 10% (b) Pluronic F-68, as well as AA and DHA in free form (c), on HEK 293 and U2OS cell lines was tested by MTT test during 24 h. All treatments were normalized to ascorbate composition. Data are expressed as percentage of negative control, mean ± sd, n = 3. Holm–Šídák multiple unpaired t-tests were performed. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01.

Figure 7

In vitro ROS analysis in cell line U2OS. Cells were treated with AA in free form, SLN-AP and blank SLN for 1 h (a) and 6 h (b). Fluorescence was measured at 490 nm/570 nm. All treatments were normalized to ascorbate composition. Data are expressed as percent of negative control, mean ± sd, n = 3. Two-way ANOVA was performed with Dunnet’s correction. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.