Vitamin D Nanoliposomes to Improve Solubility, Stability, and Uptake Across Intestinal Barrier

Abstract

Background/Objectives: Vitamin D (VD) is a fat-soluble vitamin essential for bone health, and calcium and phosphorus absorption. Recently, new interesting functions are reported such as neuroprotective activity, regulatory roles in the immune system, and protective effects in cancer patients. However, the lipophilic nature of VD represents a limitation, as it is associated with low solubility and poor absorption; additionally, VD exhibits poor stability. Methods: Two nanoliposomes containing VD, conventional (LP-VD) and conjugated with D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS, LPT-VD), were developed. The physical and chemical stability during the storage and gastrointestinal stability, the dissolution profile, the cytotoxicity and the Caco-2 cellular uptake were investigated. Nanoliposomes were fully characterized determining sizes, PdI, Zeta potential, encapsulation efficiency and recovery and they were lyophilized to improve stability. Subsequently, the freeze-dried liposomes were encapsulated in hard gelatin capsules to mimic an oral dosage form, and they were subjected to dissolution test. Results: LP-VD exhibited an average size of 85.50 ± 5.70 nm, a PdI of 0.24 ± 0.06, and a ZP of -20.90 ± 4.37 mV. LPT-VD showed an average size of 61.70 ± 3.90 nm, a PdI of 0.26 ± 0.02, and a ZP of -9.45 ± 2.99 mV. The EE% values were 95.76 ± 1.26% and 97.54 ± 3.24% for LP-VD and LPT-VD, respectively. Both nanoliposomes solubilized 2 mg/mL of VD and improved both its storage stability and stability in aqueous and gastrointestinal environment. The freeze-dried products guarantee constant chemical-physical parameters for 28 days at 25 °C. VD dissolution profile was improved. Conclusions: Nanoliposomes, in particular LPT-VD, showed the best results in terms of chemical stability, dissolution profile, and Caco-2 cellular uptake, confirming the stabilization, bioenhancer properties and P-gp inhibition capabilities of TPGS.

Keywords: Caco-2 uptake; TPGS; Vitamin D; cytotoxicity; dissolution; drug delivery; nanoliposomes.

Figure 1

VD recovery percentage (R%) in lyophilized LP-VD and LPT-VD, stored at +25 °C. Data are reported as mean ± SD of n = 3.

Figure 2

LPT-VD and LP-VD physical stability in SGF (2 h) and SIF (4 h). Data are reported as mean ± SD of n = 3.

Figure 3

Cellular uptake of LP-6C and LPT-6C by Caco-2 cells after 2 h incubation at 37 °C. Images of nuclei stained with DAPI (blue), 6-Coumarin (green) and their overlay. Scale bar: 50 µm.

Figure 4

Dissolution release profiles of VD from a VD solution, freeze-dried LP-VD and LPT-VD in water (0.3% w/v SDS) at 37 °C. Data are reported as mean ± SD of n = 3.

Figure 5

Caco-2 cell viability evaluated by MTT assay (A) and cytotoxicity by LDH assay (B) when exposed for 2 h to 6C (2.5 and 5 µg/mL) or LP-6C and LPT-6C (2.5 and 5 µg/mL). Data are expressed as percentage of control (CRL, medium) and triton-X (TX) which represent, respectively, the maximum cell viability and cell cytotoxicity. Values represent the mean ± SEM of at least three experiments performed in triplicate. ** p < 0.01 and *** p < 0.001 vs. CRL (one-way ANOVA followed by Dunnett’s test).

Figure 6

Caco-2 cell viability evaluated by MTT assay (A) and cytotoxicity by LDH assay (B) when exposed for 2 h to VD (10 and 20 µg/mL) or LP-VD and LPT-VD (10 and 20 µg/mL). Data are expressed as percentage of control (CRL, medium) and triton-X (TX) which represent, respectively, the maximum cell viability and cell cytotoxicity. Values represent the mean ± SEM of at least three experiments performed in triplicate. *** p < 0.001 vs. CRL (one-way ANOVA followed by Dunnett’s test).