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Fig. 1. Hot Homogenization and Ultrasonication Method. Created with Biorender.com |
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Fig. 2. Representative HPLC chromatograms and calibration curve (a) Chromatogram of Vitamin D3 with a retention time of 4.02 min. (b) Chromatogram of Azulene with a retention time of 1.9 min. Both compounds were effectively separated using a validated method with a shared mobile phase. (c) Calibration curve of Vitamin D3 demonstrating a linear relationship between concentration (0.25–10 µg/mL) and peak area, with a correlation coefficient (R2) of 0.998 |
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Fig. 3. Optical microscopy images showing the compatibility study between Glyceryl Monostearate (GMS) and Tefose® 1500 (PEG). (a and b) Solid lipids Glyceryl Monostearate (Kolliwax® GMS II) (GMS) and (c and d) Tefose® 1500 (PRG-6 Stearate/PEG-32 Stearate) (PEG) images under the optical microscopy |
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Fig. 4. Optical microscopy images of lipid mixtures. (a and b) Glyceryl Monostearate and Caprylic/Capric Triglyceride (GMS-CCT) mixture, which shows a clear, homogeneous dispersion with minimal crystallization, indicating strong compatibility. In contrast, (c and d) Tefose® 1500 and Caprylic/Capric Triglyceride (PEG-CCT) mixture exhibits visible oil droplets and delayed solidification, as marked by arrows, suggesting lower compatibility at this ratio. The images illustrate the miscibility and physical compatibility of solid and liquid lipids at a 1:1 ratio, The images were taken shortly after cooling from just above the solid lipid’s melting point |
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Fig. 5. Optical microscopy images showing different ratios of lipid mixtures. (a) CCT-GMS at a 3:7 ratio, (b) CCT-GMS at a 7:3 ratio, (c) CCT-PEG at a 3:7 ratio, and (d) CCT-PEG at a 7:3 ratio. The images were captured to evaluate the effect of varying solid-to-liquid lipid ratios on miscibility and physical homogeneity. The GMS-based mixtures (a, b) display more uniform structures and better dispersion, while the PEG-based mixtures (c, d) exhibit visible oil droplets and phase separation, particularly at higher CCT content. Arrows indicate regions of phase separation or oil droplet formation in PEG-containing mixtures. Among the ratios tested, the 1:1 GMS-CCT mixture (see Fig. 4(a, b)) exhibited the highest degree of compatibility, suggesting it as the most suitable composition for subsequent formulation development |
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Fig. 6. The illustration demonstrates the size and PDI of the placebo LNP formulation |
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Fig. 7. The illustration demonstrates the size and PDI of optimized LNP formulations |
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Fig. 8. Characterization of Lipid Nanoparticles (LNP) loaded with Vitamin D3. (a) Dynamic Light Scattering (DLS) analysis showing particle size distribution and polydispersity index (PDI) of the Vit D3-LNP formulation, (b) Zeta potential distribution measured by DLS, (c) Transmission Electron Microscopy (TEM) image of LNP morphology at 12,000 × magnification, (d) TEM image at 30,000 × magnification, and (e) TEM image at 80,000 × magnification, highlighting nanoparticle structure and distribution |
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Fig. 9. (a) Differential Scanning Calorimetry for Vitamin D3, LNP, and for Vit-D3-LNP, (b) Thermogravimetric (TGA) of Vitamin D3, LNP, and Vit-D3-LNP. (a) Differential Scanning Calorimetry (DSC) thermograms of pure Vitamin D3, lipid nanoparticles (LNP), and Vitamin D3-loaded lipid nanoparticles (Vit-D3-LNP). The DSC analysis reveal a sharp endothermic peak for pure Vitamin D3 at 82–86 °C (corresponding to the Vit-D3 melting point), indicating its crystalline nature, while the absence or broadening of this peak in Vit-D3-LNP confirm successful encapsulation and interaction with formulation components, including Caprylic/Capric Triglyceride (CCT), Glyceryl Monostearate (GMS – Kolliwax® GMS II), and Polyvinylpyrrolidone K30 (PVP K30 – Kollidon® 30). (b) Thermogravimetric Analysis (TGA) of pure Vitamin D3, LNP, and Vit-D3-LNP. TGA results demonstrate enhanced thermal stability of the formulations, with LNP and Vit-D3-LNP exhibiting a more gradual degradation profile compared to pure Vitamin D3. These findings confirm the successful integration of Vitamin D3 into the lipid-PVP matrix, improving its stability and encapsulation efficiency for potential topical delivery |
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Fig. 10. HPLC Analysis Results for Vitamin D3 Stability Under Different Storage Conditions after 30 Days. (a) Vitamin D3 reference concentration (initially 10 µg/mL). (b) Vitamin D3 degradation in LNPs stored in the dark: minimal loss (6%), confirming the protective effect against light exposure. (c) Under continuous light exposure, degradation increased to 9%, demonstrating Vitamin D3’s photosensitivity and supporting the need for dark storage to avoid light-induced isomer formation. (d) Refrigerated storage at 4 °C resulted in a 13% reduction, indicating moderate improvement in stability. (e) Freezing at –20 °C resulted in a 15% reduction, indicating only a slight enhancement over refrigeration. (f) Elevated temperature storage at 40 °C caused the most significant degradation (40%), highlighting the temperature sensitivity of Vitamin D3 and the importance of avoiding heat during storage |
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Fig. 11. Rheological profiles of the base cream and LNP-loaded cream (LNP-Cream)at 25 °C. These profiles show shear-thinning (pseudoplastic) behavior (a) for the cream and (b) for the LNP-cream. Viscosity decreases with increasing shear rate, demonstrating reduced flow resistance under mechanical stress |
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Fig. 12. Texture profile analysis of the base cream and LNP-loaded cream (LNP-Cream). (a) and (b) show the force-distance curves obtained from double compression cycle tests. (c) Quantitative comparison of texture parameters including hardness, adhesiveness, cohesiveness, and springiness. The LNP-Cream exhibited significantly lower hardness (P < 0.05) compared to the base cream, indicating a softer texture. Adhesiveness, cohesiveness, and springiness showed slight, non-significant (NS) reductions, suggesting preserved structural integrity and elasticity |
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Fig. 13. In vitro permeation test using three different donors: (a) and (b) Mean Cumulative amount (µg/cm2—All time points. (a) and (b) Mean cumulative drug amount (µg/cm2) at all time points (0, 2, 4, 6, and 24 h) following the in vitro penetration test (IVPT). The results show a significant difference (P < 0.05) between free Vitamin D3 and the cream formulation. Free Vitamin D3 exhibited minimal permeation, with only 0.001–0.002 µg/cm2 detected at 24 h, whereas the cream formulation demonstrated significantly higher permeation, reaching 0.006–0.009 µg/cm2. This suggests that the lipid nanoparticle (LNP) cream formulation enhances Vitamin D3 delivery by facilitating diffusion through the stratum corneum |
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Fig. 14. In vitro permeation test using three different donors. Mean Donor Drug Amount (µg/cm.2). Mean Donor drug amount (µg/cm2) following the in vitro penetration test (IVPT). The results indicate a significantly higher residual drug amount in the donor phase for free Vitamin D3 (1.1–1.38 µg/cm2) compared to the cream formulation (0.18–0.67 µg/cm2) (P < 0.05). This suggests improved skin permeation and retention of Vitamin D3 when incorporated into the lipid nanoparticle (LNP) cream formulation |
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Fig. 15. Multiphoton imaging (MPM) penetration images. Multiphoton microscopy (MPM) was used to evaluate the permeation and distribution of Rhodamine B-labelled lipid nanoparticles (LNP) and a cream containing Azulene across skin layers. Rhodamine B-labelled LNP (0.5% w/w dye) and Azulene cream were applied to frozen skin samples with a diffusion area of 1.13 cm2, and imaging was performed at 0, 4, and 24 h using a DermaInspect® multiphoton system. Fluorescence channels: red indicates Rhodamine B-labeled LNP; blue shows skin autofluorescence. Excitation wavelengths of 740 nm, 840 nm, and 770 nm were used for autofluorescence, Rhodamine B, and the combined formulations, respectively. In comparison, FLIM measurements captured fluorescence decay curves for Azulene (430–450 nm) and Rhodamine B (560–580 nm). Results showed that Rhodamine B-labelled LNP penetrated all skin layers at 4 h, with reduced fluorescence in deeper layers at 24 h. In contrast, Azulene cream demonstrated consistent fluorescence across all layers at both time points. FLIM imaging further visualized fluorescence distribution, confirming effective delivery and retention of both formulations, with Azulene dominating fluorescence in the combined system, indicating enhanced permeation and retention properties |
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Fig. 16. In Vitro Tests of Cell Viability, Inflammation, and multiphoton microscopy (MPM) images for LNP. (a) Cell Viability of free Vitamin D3 and Vitamin D3-LNP was evaluated in HaCaT cells after 24 h of exposure at concentrations of 1, 10, and 100 µg/mL. Cell viability (%) was calculated using the formula Cell Viability (%) = (Treated cells-Media)/(untreated cells-Media)*100. Both formulations maintained high cell viability (80–100%) across all concentrations, indicating no significant cytotoxic effects. (b) Inflammation test showing ROS levels in HaCaT cells treated with hydrogen peroxide (H₂O₂, 200 µM) with or without free Vitamin D3 and Vitamin D3-LNP. Intracellular ROS was detected using the DCFH-DA probe (DCF). The unit for DCF fluorescence intensity is typically relative fluorescence units (RFU) or simply arbitrary units (AU). H₂O₂ treatment significantly increased ROS levels (~ 250 AU), while both free Vitamin D3 and Vitamin D3-LNP significantly reduced ROS levels (P < 0.001), indicating strong antioxidant activity. (c), (d) and (e) Multiphoton Fluorescence Lifetime Imaging Microscopy (MPM-FLIM) images were captured after 24 h for the first four groups. Pseudocolored images are scaled from 0 to 3000 picoseconds (ps), with blue indicating shorter τm and red indicating longer τm. For the H₂O₂ + Vit D3 and H₂O₂ + Vit D3-LNP groups, cells were treated with 200 µM H₂O₂ for 2 h, followed by the respective formulations and incubation for an additional 24 h. The FLIM images demonstrated that Vitamin D3-LNP provides greater protection against H₂O₂-induced oxidative stress by restoring normal cell morphology and redox homeostasis. The H₂O₂ group showed the highest τm (~ 2600 ± 71.67 ps) (red), and lowest redox ratio (1.10 ± 0.04), reflecting severe oxidative stress. Vitamin D3-LNP-H₂O₂ treatment significantly reduced τm (~ 1800 ± 17.23 ps) (exhibited a shift toward blue) and improved the redox ratio (3.10 ± 0.17), closer to control levels (blue) (3.63 ± 0.10), highlighting its enhanced therapeutic potential |
