Vitamin D deficiency is a widespread health concern with implications ranging from bone disorders to immune system dysregulation. While oral supplementation is common, topical application offers a promising alternative, particularly for dermatological conditions like vitiligo, which is characterized by a loss of skin pigmentation. Conventional vitiligo treatments often fall short of achieving satisfactory repigmentation, but topical Vitamin D has shown potential in promoting melanocyte proliferation and differentiation, offering a targeted therapeutic strategy. However, highly lipophilic active substances such as Vitamin D encounter significant challenges in topical administration, including their inability to effectively penetrate the stratum corneum barrier and a tendency to deposit on the skin surface when formulated in creams.
To overcome these limitations, nanotechnology-based drug delivery systems, specifically Lipid Nanoparticles (LNPs), have emerged as a potential solution to enhance the therapeutic efficacy of drugs. LNPs are composed of biocompatible and biodegradable lipids, capable of encapsulating active pharmaceutical ingredients and protecting them from degradation. They offer advantages such as improved drug stability, bioavailability, targeted delivery, minimal skin irritation, high drug-loading capacity, and controlled and sustained release, facilitating drug retention in targeted skin layers. Furthermore, the integration of azulene, a compound known for its anti-inflammatory and skin-soothing effects, can complement Vitamin D’s action by supporting skin regeneration and reducing local oxidative stress, making this a promising approach for advanced skin treatments like vitiligo.
Methods
This study involved the preparation of Vitamin D3-loaded lipid nanoparticles (Vit D3-LNP) using a hot homogenization and ultrasonication method. A comprehensive optimization process was undertaken for formulation parameters (including surfactant and oil phase concentrations, and surfactant type) and process parameters (such as stirring speed and ultrasound time/amplitude), employing Central Composite Design and Box-Behnken Design statistical approaches. The optimized LNPs were then incorporated into an azulene cream vehicle. Physical and thermal characterizations were performed using Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), Differential Scanning Calorimetry (DSC), and Thermogravimetric Analysis (TGA). Chemical analysis, including encapsulation efficiency and stability testing, was conducted using High-Performance Liquid Chromatography (HPLC). In vitro assessments included cell viability and anti-inflammatory tests on HaCaT cells, skin permeation studies using Franz diffusion cells, and detailed penetration analysis with two-photon microscopy.
Key Findings
The research yielded several significant findings:
• Optimized LNP Characteristics: The developed Vit D3-LNP displayed favorable physical properties, with a particle size of 153.9 nm, a high zeta potential of -54.3 mV, and a low polydispersity index (PDI) of 0.216, all confirmed by TEM. These characteristics indicate a uniform and stable formulation.
• High Encapsulation Efficiency: The LNPs achieved a remarkable 96.98% encapsulation efficiency for Vitamin D3, demonstrating successful incorporation of the vitamin within the lipid matrix.
• Enhanced Thermal Stability: DSC and TGA analyses revealed that Vitamin D3 was successfully integrated into the lipid-polymer matrix, which improved its thermal stability compared to pure Vitamin D3, indicating protection from degradation.
• Impact of Storage Conditions on Stability: Stability studies highlighted the photosensitivity and temperature sensitivity of Vitamin D3. Light exposure resulted in a 9% reduction in Vitamin D3 content compared to 6% in dark storage at room temperature. Elevated temperatures (40°C) caused significant degradation (40% reduction), emphasizing the critical need for dark and room-temperature storage conditions to minimize degradation.
• Improved Cream Rheology and Texture: The LNP-loaded cream exhibited non-Newtonian, shear-thinning (pseudoplastic) behavior, which is ideal for topical application as it provides high viscosity at rest for stability but reduces resistance to flow during spreading. The cream also demonstrated a reduced hardness (12.97 g compared to 17.97 g for the control cream), suggesting a softer texture that could enhance spreadability and user comfort.
• Superior Skin Permeation and Retention: In vitro skin penetration tests showed that the cream formulation significantly enhanced Vitamin D3 permeation (0.006–0.009 µg/cm² at 24h) compared to free Vitamin D3 (0.001–0.002 µg/cm²). It also resulted in significantly higher drug retention in the stratum corneum and deeper skin layers (0.76–1.35 µg/cm² in SC for cream vs. 0.006–0.085 µg/cm² for free Vitamin D3), crucial for localized therapeutic effects.
• Effective Deep Skin Penetration: Two-photon microscopy confirmed that Rhodamine B-labelled LNPs, serving as a model for Vitamin D3, penetrated all skin layers (stratum corneum, stratum granulosum, and stratum spinosum) within 4 hours, showcasing their ability to transport encapsulated compounds across skin barriers.
• High Biocompatibility and Potent Antioxidant Effects: Both free Vitamin D3 and the Vit D3-LNP formulation demonstrated high cell viability (~80–100%) in HaCaT cells, affirming their biocompatibility. In an inflammation model, Vit D3-LNP treatment significantly reduced intracellular reactive oxygen species (ROS) levels, indicating strong antioxidant effects that were slightly more pronounced than with free Vitamin D3, and restored normal cellular morphology and redox homeostasis.
This study successfully developed and optimized a novel Vitamin D3-loaded lipid nanoparticle (LNP) system integrated into an azulene cream, effectively addressing the critical challenges of Vitamin D3 solubility, stability, and skin penetration for topical delivery. The research highlights the enhanced protective effects of the LNP formulation against ROS-induced stress and improved bioavailability of Vitamin D3, as evidenced by Fluorescence Lifetime Imaging Microscopy (FLIM) measurements. This comprehensive approach not only ensures high encapsulation efficiency and favorable biocompatibility but also enables sustained and targeted delivery to deeper skin layers, offering a promising therapeutic strategy for managing skin conditions such as vitiligo by promoting melanocyte proliferation and differentiation and reducing local oxidative stress.
The findings underscore the significant potential of this LNP-based azulene cream as an advanced topical delivery platform. Future research will concentrate on in vivo evaluation of skin permeation, bioavailability, and therapeutic efficacy, followed by clinical studies to assess long-term patient acceptability. Additionally, further investigations aim to refine deposition techniques and explore alternative biodegradable lipid carriers to continually enhance the formulation’s performance and stability.
Link to the study: https://link.springer.com/article/10.1007/s13346-025-01946-1
