Abstract
Vitamin D3-loaded lipid nanoparticles (Vit D3-LNP), integrated into an azulene cream, were developed to enhance the topical delivery and stability of Vitamin D3. The LNP was formulated using a lipid mixture and hot homogenization-ultrasonication, with comprehensive characterization revealing a particle size of 153.9 nm, a high zeta potential (-54.3 mV), and a PDI of 0.216, which TEM confirmed. Encapsulation efficiency was high (96.98%), indicating successful incorporation of Vitamin D3 within the lipid matrix. Stability studies revealed the impact of light exposure on Vitamin D3 degradation. In vitro, release, and skin penetration studies using Franz diffusion cells and two-photon microscopy demonstrated enhanced drug permeation and retention in deeper skin layers with the cream formulation. Cell Viability test confirmed high cell viability (~ 80–100%) for both free Vitamin D3 and the LNP formulation; also inflammation test showed a significant reduction in ROS levels with Vitamin D3-LNP treatment. These findings highlight the therapeutic value of LNP in managing conditions like Vitiligo, providing insights into the design of stable, effective Vitamin D3 delivery systems for dermal applications, and offering a promising approach for advanced skin treatments.
Introduction
Vitamin D deficiency has become a prevalent health concern worldwide, with implications ranging from bone disorders to immune system dysregulation. While oral supplementation remains a common approach for addressing this deficiency, topical application offers a promising alternative, particularly in dermatological conditions such as vitiligo. Vitiligo, characterized by the loss of skin pigmentation, presents a unique challenge as conventional treatments often fall short of achieving satisfactory repigmentation. Topical Vitamin D application has shown potential in promoting melanocyte proliferation and differentiation, offering a targeted therapeutic strategy for Vitiligo management [1]. To enhance the efficacy of such topical treatments, additional agents with complementary properties may be incorporated. Azulene (sodium 1,4-dimethyl-7-isopropylazulene-3-sulfonate), known for its anti-inflammatory and skin-soothing effects, represents one such candidate. Derived from common yarrow, chamomile, and absinthe, it is a compound found in cosmetic products and has demonstrated potential in managing skin inflammation. In the context of vitiligo, its anti-inflammatory properties may support skin regeneration and reduce local oxidative stress, thereby complementing the action of Vitamin D. In Italy, it is available for topical use in a colloidal vehicle under the brand name Veralga azulene® gel [2].
The possibility of delivering vitamin D through the skin either to improve skin conditions or to reach internal organs represents the primary target. The inability of highly lipophilic active substances, such as Vitamin D, to pass through the stratum corneum barrier represents a challenging task in administering these compounds topically. Additionally, when administered in cream formulations, Vitamin D tends to deposit on the skin surface due to its affinity with the vehicle. To overcome this limitation and to promote Vitamin D permeation through the skin, despite its physicochemical characteristics, specialized transdermal delivery systems with penetration enhancement properties are required [3, 4].
Nanotechnology-based drug delivery systems have gained widespread attention for their ability to enhance the therapeutic efficacy of various drugs. Among them, nanoparticles, which are solid colloidal particles typically ranging in diameter from 1 to 100 nm, are widely explored due to their unique physicochemical properties. These include metal nanoparticles, polymeric micelles, polymeric nanoparticles, and lipid-based systems such as liposomes and solid lipid nanoparticles. Their capacity to improve drug stability, bioavailability, and targeted delivery makes them suitable not only for pharmaceuticals but also for applications in cosmetics and skincare [5,6,7]. Lipid nanoparticles (LNPs), introduced in the 1990 s, have emerged as particularly promising carriers in the field of nanomedicine. Composed of biocompatible and biodegradable lipids, LNPs encapsulate active pharmaceutical ingredients (APIs) and protect them from degradation processes such as oxidation [8]. These nanoparticles exhibit minimal skin irritation, high drug-loading capacity, and the ability to provide controlled and sustained release [9]. When applied topically, they form an occlusive film that enhances skin permeation and facilitates drug retention in the targeted skin layers, increasing therapeutic effectiveness [10, 11].
Several studies have indicated that formulations containing the same drug at the same concentration can exhibit varying drug delivery rates through the skin due to the physicochemical properties of the vehicle used. Moreover, formulations employing the same ingredients can also result in different drug delivery rates based on the droplet size of the emulsion. In vitro drug release tests provide valuable insights into evaluating the impact of formulation factors such as dose formulas, vehicle composition, and drug solubility in vehicles. These considerations are crucial when formulating topical preparations [12, 13].
Formulation components have two primary effects on skin permeation. Firstly, they can modify the lamellar structure of intercellular lipids in the stratum corneum. These components can potentially enhance or impede the permeation of the drug through the skin by altering the organization and packing of the intercellular lipids. This, in turn, influences the pathway and rate at which drug molecules can traverse the stratum corneum. Secondly, formulation components can impact the solubility properties of lipids. The solubility of both the drug and the vehicle in these lipids can affect drug permeation through the skin. The lipid composition of the stratum corneum is a significant determinant of skin permeability. The composition of lipids in the stratum corneum plays a vital role in determining skin permeability. Formulation components can modulate the solubility of the drug in these lipids, consequently influencing the drug’s ability to penetrate the skin [14,15,16].
The size of LNP plays a crucial role in the stability of the formulation, drug release rate, and its behavior in the body. Various factors influence particle size, including lipid and surfactant properties, production techniques, and processing conditions (such as time, temperature, pressure, and the number of cycles). In both high-pressure homogenization and high-shear homogenization, larger particle sizes are observed with higher melting point lipids, attributed to increased viscosity of the dispersed phase [17]. The lipid composition significantly impacts the quality of LNP dispersion, and variations in lipid composition from different suppliers and batches are noteworthy. A higher lipid content (> 5–10%) increases particle size and polydispersity due to enhanced viscosity, which affects homogenization efficiency and particle agglomeration. The surfactant’s properties and concentration also influence the size and efficacy of LNP. Smaller particle sizes result in an increased surface area, leading to thermodynamic instability and phase separation through Ostwald ripening. Adequate surfactant concentration is crucial to cover newly formed surfaces and prevent phase separation by reducing interfacial tension [18], which, in turn, can impact the overall efficacy of the LNP formulation. Surfactant/cosurfactant mixtures have been observed to enhance LNP stability and reduce particle size compared to formulations with surfactant alone. The type of surfactant used influences the homogenization parameter required for a specific formulation. For instance, LNP dispersions stabilized with ionic surfactants exhibit smaller particle sizes compared to those stabilized by nonanoic surfactants. Researchers emphasize the need to determine the optimum surfactant concentration for each formulation to ensure stability and efficacy [19].
We have undertaken the present study to develop a topical Vitamin D formulation optimized with lipid nanoparticles (LNPs) to enhance its effectiveness in treating skin-specific conditions, such as Vitiligo. Unlike oral supplementation, which is subject to systemic metabolism and distribution, a localized topical approach offers more targeted therapeutic action. Additionally, to address the limitations related to skin penetration and surface deposition of Vitamin D, the formulation utilizes the unique properties of LNPs, including enhanced penetration, physicochemical stability, and controlled release, to maximize its therapeutic potential in dermatological applications.
Download the full article as PDF here Vitamin D-loaded lipid nanoparticles
or read it here
Materials
Sigma-Aldrich, Australia, provided standard Vitamin D3 (Purity ≥ 99.0%, HPLC grade), Glyceryl Monostearate (Kolliwax® GMS II), and Polyvinylpyrrolidone (PVP) (Kollidon® 30). PEG-35 Castor Oil (CO) and Flaxseed Oil (FSO) were also obtained from Sigma-Aldrich, Australia. Propylene Glycol Monolaurate (Lauroglycol FCC) (PML) and Tefose® 1500 (PRG-6 Stearate/PEG-32 Stearate) (PEG) were gifted by Gattefosse Pharmaceuticals, Australia. Ethoxylated Oleyl Alcohol 20 OE (EOA – Chemonic OE20) was sourced from Croda Pharma.
Our lab sourced HPLC-grade acetonitrile (ACN), methanol, medium-chain triglycerides (Caprylic/Capric Triglyceride, CCT), ultra-pure water (Milli-Q, IONPURE, USA), Propylene Glycol (PG), Disodium EDTA, Poloxamer 407, Shea Butter Cetyl Esters (AGREEN W), Glyceryl Stearate, Cetearyl Alcohol (combined with Polysorbate 60), and Phenoxyethanol
AL-Smadi, K., Imran, M., Abdoh, A. et al. Vitamin D-loaded lipid nanoparticles: antioxidant properties, preparation, optimization, and in vitro characterization. Drug Deliv. and Transl. Res. (2025). https://doi.org/10.1007/s13346-025-01946-1
Read more on Vitamins & Minerals here:
