Using C24 Ceramide Nanoparticles to Help Skin Wounds Heal

Ceramide-based lipid nanoparticles were prepared via ethanol injection methods [17]. Briefly, distearoylphosphatidylcholine (DSPC, Croda, Seoul, Republic of Korea, 8.61 mg), cholesterol (Samchun Chemicals, Seoul, Republic of Korea, 12.31 mg), N-lignoceroyl-D-erythro-sphingosine (C24 ceramide, Avanti Polar Lipids, Alabaster, AL, USA, 23.96 mg), and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt (DPPG-Na, NOF, 3.12 mg) were dissolved in ethanol solution (3 mL) with a molar ratio of 13:38:44:5 and then added gently to deionized water (9 mL) with a volume ratio of 3:1. After agitation with continuous stirring at 500 rpm using a magnetic stirrer for 10 min, the mixture solution (12 mL) was treated to remove the ethanol via rotary evaporation (60 ± 5 °C). The production of the bare LNPs used a process almost similar to that used for producing C24-LNPs; the composition ratios are shown in Figure 1A. The final LNP solution (10 mg/mL) was characterized based on its particle size, polydispersity index, and zeta potential using a Zeta-sizer Nano ZS (Malvern, Seoul, Republic of Korea). Cryo-EM images of the C24-LNPs for microscopy were prepared by placing 5 μL of LNPs onto a 400-mesh lacey carbon-coated grid using single-sided blotting for 2 s; then, the sample grid was immediately immersed into nitrogen-cooled ethane. The C24-LNP morphology was then observed using a Krios G4 (Thermo Fisher Scientific, Seoul, Republic of Korea) at liquid nitrogen temperature and 300 kV.

Immortalized human skin keratinocytes, HaCaT purchased from the Korean Cell Line Bank (Seoul, Republic of Korea), and the cell were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (PS) at 37 °C under 5% CO₂. The cells were sub-cultured at 70–80% confluency and used with a passage below 30. The cells were tested negative for mycoplasma.

The cells were seeded in a 96-well cell culture plate (3 × 10⁴ cells) at 37 °C under 5% CO₂. The cells were treated with various concentrations of C24-LNP for 24 h at 37 °C, and cell viability was measured with a WST-8 cell counting kit (Dojindo, Kumamoto, Japan). The absorbance was measured at 450 nm using a microplate reader (BioTek, Seoul, Republic of Korea), and the negative control group’s relative survival rate (viability %) was calculated.

To assess the wound-healing effects of C24-LNP in vitro, HaCaT human skin keratinocyte cells were seeded (24-well culture plate, SPL) and cultured in reduced serum for 24 h. Then, the cells were scratched using a scratcher (Scar Scratcher, SPL). After 24 h, the cells were treated with different concentrations of C24-LNP for 48 h. The wound area was measured on days 0, 1, and 2 after the scratch. The human recombinant EGF protein (Thermo Fisher Scientific, Seoul, Republic of Korea, 10 ng/mL), bare LNP without C24 ceramide (100 μg/mL), or each LNP component (100 μg/mL) was treated via scratching and used as positive and negative controls. The recovered wound area was calculated using Image-J Software (Version 1.54m). The data were statistically evaluated from three independent experiments in three replicates.

The cells were treated with C24-LNP (500 µg/mL) for 24 h and then lysed in a RIPA buffer containing a protease inhibitor cocktail (Sigma-Aldrich, Seoul, Republic of Korea). A BCA assay kit was used to measure the total protein amounts (Thermo Fisher Scientific, Seoul, Republic of Korea). Samples containing equal amounts of protein were separated using SDS-PAGE on a 4–15% gradient gel (Bio-Rad, XT-Criterion, Seoul, Republic of Korea) and transferred onto polyvinylidene difluoride (PVDF) membranes. After the transfer, the membranes were blocked with 5% skim milk in TBS-T (Tris buffer saline-0.02% Tween) buffer for 1 h at room temperature. The following primary antibodies were used to determine the proliferation and their signal pathway: anti-Collagen I (Cell Signaling Technology, Danvers, MA, USA, rabbit-monoclonal, 1:1000), anti-Cytokeratin-14 (Santa Cruz Biotechnology, Heidelberg, Germany, mouse-monoclonal, 1:200), anti-Ki-67 (Santa Cruz Biotechnology, Heidelberg, Germany, mouse-monoclonal, 1:200), anti-PCNA (Santa Cruz, mouse-monoclonal, 1:200), anti-AKT (Cell Signaling Technology, Danvers, MA, USA, rabbit-polyclonal, 1:1000), anti-pAKT (Cell Signaling Technology, Danvers, MA, USA, rabbit-polyclonal, 1:1000), anti-ERK1/2 (Cell Signaling Technology, Danvers, MA, USA, rabbit-polyclonal, 1:1000), and anti-pERK1/2 (Cell Signaling Technology, Danvers, MA, USA, mono-polyclonal, 1:1000). Anti-β-actin (Santa Cruz Biotechnology, Heidelberg Germany, mouse-monoclonal, 1:10,000) was used as an internal loading control. Each primary antibody was incubated overnight at 4 °C. Following three washes with TBS-T buffer, a reaction with appropriate horseradish peroxidase (HRP)-conjugated mouse or rabbit secondary antibodies (Santa Cruz Biotechnology, Heidelberg, Germany) was carried out for 2 h at room temperature and finally detected using a Pierce ECL kit (Middleton, MA, USA). Bands were imaged via chemiluminescence imaging using an ImageQuant LAS500 (GE Healthcare, Chicago, IL, USA).

Male HR-1 hairless mice aged six weeks and weighing 18–20 g were purchased from Central Laboratory Animal Inc. (Seoul, Republic of Korea). All procedures followed the local institutional guidelines for animal care established by the Catholic University Animal Use and Care Committee (IACUC NO: 2024-0209-01). The mice were allowed to adapt to the environment for 1 week before the experiment. They were housed in cages with controlled temperatures (24–25 °C), normal light–dark cycles, and 60% humidity. In an in vivo animal model, full-thickness nude mouse skin wounds were induced on the back of the mice using a 6 mm biopsy punch (Mckesson, Irving, TX, USA) under isoflurane anesthesia (Hana Pharma Co., Ltd., Seoul, Republic of Korea) to determine the extent of wound healing. The mice were randomly assigned to the following four groups: non-treated negative control, EGF-protein-treated positive control, C24-LNP, and bare LNP without C24 ceramide.

To determine the therapeutic potential of C24-LNP for skin wounds, 10 µL of C24-LNP (1 mg/mL in DW, 4.4 µg C24) was applied topically daily for 10 days starting from the day of injury (day 0) according to previous in vivo wound-healing experiments [18]. The wound was covered with a transparent medical dressing (Tegaderm film, 3M). The wound size, as seen above, was measured on days 2, 4, 6, 8, and 10 after injury, and the recovery rate (%) was evaluated based on the degree of contraction of the wound area through photographic images measured using Fiji Image-J. Regeneration of the epithelium and dermis was assessed by histology and Western blot on the 4th and 10th days post-injury, respectively, compared with the control group. A Western blot analysis was performed using skin tissue lysates using the same procedure as a cell lysate analysis.

The excised tissues were fixed with 4% neutral paraformaldehyde and embedded in paraffin. The sections were cut at 6 µm thickness and deparaffinized according to the general protocol. The stained slides were observed using a Nikon Eclipse E200 microscope (Nikon, Tokyo, Japan).

MT staining was performed using paraffin sections to evaluate the collagen production effect of C24-LNP on skin dermal regeneration during wound healing. The protocol and histology analysis are similar to those of H&E. The amount of blue-stained collagen in the dermal layer was quantified using ImageJ software (Version 1.54m).

All values are expressed as mean ± SEM (standard error of the mean). The statistical analyses were performed using Graph Pad Prism9 (GraphPad Software Inc., La Jolla, CA, USA). In vitro cell proliferation, migration, and in vivo wound-healing rate (%) were assessed via an analysis of variance (ANOVA), followed by Tukey’s or Dunnett’s multiple comparison test with a significance level (α) of 0.05.

We synthesized C24-LNP with appropriate ratios of the neutral lipid, cholesterol, and anionic lipid compound (Figure 1A) [19]. To manufacture LNPs with high biocompatibility, we tried to exclude the use of cationic lipid compounds known to have cytotoxicity as much as possible. In addition, we attempted to maintain the cholesterol content as much as possible to maintain structural stability. We confirmed that a minimum of 5% of anionic lipid compounds, such as DPPG, must have aqueous solution stability. Finally, we have established the composition of the optimized C24-LNP. The nanoparticles composed of the optimal ratio exhibited very high aqueous stability. The analytical results showed that the average uniform size was 127 ± 5 nm, and their poly-diversity index (PDI) value was determined at 0.165 ± 0.007 via dynamic light scattering (DLS) analysis. The surface charge of the synthesized C24-LNP showed an anionic character of −51 ± 2 mV. From the cryo-EM analysis, we confirmed that the C24-LNPs were spherical, had a relatively uniform size distribution, and exhibited a bilayer structure (Figure 1B).

HaCat cells are immortalized human keratinocytes and have been extensively used to study epidermal homeostasis and its pathophysiology. Human keratinocytes treated with C24-LNP were subjected to in vitro assays. Cell viability was examined according to the treatment concentration of C24-LNP, and the cell growth rate increased rapidly compared with the control at concentrations above 50 µg/mL (Figure 2A). Cell growth reached saturation at 500 µg/mL, indicating that the effect depended on the treatment concentration of C24-LNP. We examined C24-LNP uptake into keratinocytes through energy-dependent endocytosis and whether the released C24 ceramide in the cytoplasm affected signaling related to cell proliferation (Figure S1). In addition, we confirmed the degree of effect by comparing it with the treatment using EGF protein, which is known to affect cell growth (Figure 2B and Figure S2A). When we treated the LNP, which consists of the absence of only the C24 ceramide component (called bare LNP), the effect on cell growth and biomarkers was small. These results suggest that C24 ceramide leakage into the cytoplasm affects cell growth. According to the Western blot analysis, the expression of biomarkers related to proliferation (Ki-67, PCNA, AKT, and ERK1/2) increased in the treatment group, and the expression levels were similar to or relatively higher than those of the positive group treated with epidermal growth factor (EGF) protein, which is known to promote cell growth. We observed that LNPs containing C24 ceramide can sufficiently increase the proliferation of cell lines in vitro without the biological drug EGF protein. EGF protein generally binds to EGFR in the cell membrane, affects RAS, and transmits signals to AKT and ERK to promote cell growth.

On the other hand, C24 ceramide directly affects ERK and AKT expression in the cytoplasm, regardless of the receptor on the cell membrane. We also investigated whether migration increases with cell growth stimulation. This experiment may indicate the extent to which wounds heal in the actual epidermis. Therefore, in vitro migration assays may indicate the effectiveness of wound-dressing therapies [20,21]. We investigated the extent of recovery via migration after culture by removing cells at 1500 µm intervals using a scratcher (Figure 2C and Figure S3A). The extent of recovery varied depending on the treatment concentration of C24-LNP, and a significant migration effect was observed compared with the control at concentrations above 500 µg/mL. In particular, the high-concentration C24-LNP treatment group of 1 mg/mL showed a recovery effect similar to that of the EGF protein drug treatment group. However, when each component of the non-LNP-formulated C24-LNP was treated in the cells, the migration effect was not enhanced (Figure S3B). Because the solubility of C24 ceramide is inherently low, the cellular uptake efficiency at the molecular level is considerably lower than that of the nanoparticle formulation. Furthermore, when treated with bare LNP without only C24 ceramide, a migration effect similar to that of the negative control was observed. These results demonstrate that nanoparticles containing C24 ceramide enhance both cell growth and migration effects related to cellular uptake.

The wound-healing effect of C24-LNP was evaluated using an animal model with a 6 mm biopsy punch-induced wound. The treatment groups received 10 μL of either C24-LNP or bare LNP (1 mg/mL). As a positive control, EGF (100 μg/mL) was applied at the same volume (10 μL). Wound recovery was monitored over time (Figure 3). On the fourth day, the C24-LNP and EGF groups showed significantly faster healing effects than the other groups. On the 10th day, while all treatment groups exhibited some level of recovery, the C24-LNP group achieved the highest recovery rate (86.9 ± 2.2%) compared with the control group (75 ± 1.2%), the EGF group (80.3 ± 0.4%), and the bare LNP group (74.2 ± 2.0%). (Mean ± SEM, n = 3, ** p < 0.05 vs. control or bare LNP.) This is thought to be observed because the dermal layer growth process is dominant until the fourth day, when the epidermal layer grows rapidly thereafter.

The healing effect on the skin was evaluated using a histological analysis of the treatment groups. An analysis of the H&E-stained sections confirmed that C24-LNP accelerated re-epithelialization on day 4 post-injury (Figure 4A,B). The epithelium of the C24-LNP and EGF-positive treatment groups recovered faster and more effectively to typical skin structure than the control group. In particular, the epithelium in the C24-LNP group showed significant thickness (37.0 ± 6.3 µm), and the dermal structure recovered rapidly. Keratinocytes and fibroblasts increased in the epidermis and dermis, which dominated cell flow or migration in the normal tissue area and induced proliferation.

In contrast, the control and bare LNP-treated groups showed slower recovery and were characterized by a thinner epithelial layer and an unclear dermal structure. Changes in the proliferation-related biomarkers in the tissue were confirmed by a Western blot analysis in an in vivo animal model (Figure 4C and Figure S2B). Similar to the in vitro results, the expression of most proliferation-related markers increased, with higher expression observed in the C24-LNP-treated group than in the EGF-treated group. The increase in proliferating cell nuclear antigen (PCNA), AKT, and ERK1/2 activation was prominent. PCNA mediates downstream cellular responses, including growth, survival, proliferation, migration, and angiogenesis, and the ERK1/2 pathway is essential for keratinocyte proliferation [22,23,24]. Increased pAKT activity further suggests that it promotes cell migration by enhancing the stability of cytoskeletal components or facilitating their remodeling. Studies have reported that the increased AKT expression promotes collagen formation and accelerates tissue regeneration in mouse wound models [25,26,27,28,29,30]. Collagen is important for maintaining skin elasticity, strength, and moisture, contributing to a firm and youthful appearance [31,32]. To investigate this further, collagen formation was analyzed in samples from an animal model [33]. H&E and collagen formation analyses of the treated animal models on day 4 showed that epithelial cell regeneration was evident and the number of fibroblasts in the dermal layer increased dramatically. Still, collagen formation had not yet occurred (Figure S4). However, in the tissues on day 10, the epithelial and dermal thicknesses increased to the level of normal skin. In addition, collagen expression and extracellular matrix (ECM) production, essential for skin regeneration, were significantly higher in the C24-LNP and EGF-positive treatment groups (Figure 5).

Data are contained within the article and. Supplementary Materials