Lycopene-Carrier Solid Dispersion loaded Lipid Liquid Crystal Nanoparticle: in vitro Evaluation and in vivo Wound Healing Effects (2025)

1. Introduction

Lycopene, a red carotenoid pigment primarily found in tomatoes and various other fruits and vegetables, has attracted considerable attention for its therapeutic potential, especially in wound healing. This natural compound possesses powerful antioxidant properties, effectively neutralizing harmful free radicals that can hinder the wound healing process [1-3]. Over 85% of dietary lycopene intake is derived from tomatoes, highlighting their significance as a primary source of this beneficial compound [4]. The therapeutic role of lycopene extends beyond its antioxidant functions. Research has shown its potential to promote wound healing through multiple mechanisms, including the reduction of oxidative stress, which protects cells and tissues from damage [5]. The modulation of the inflammatory response to prevent excessive inflammation that can disrupt healing and stimulate collagen synthesis, essential for tissue repair [6]. Additionally, lycopene enhances angiogenesis [7] and improves blood flow to the wound site, hence ensuring an adequate supply of oxygen and nutrients necessary for healing [8].

Moreover, lycopene has been recognized for its antimicrobial activity against pathogenic bacteria commonly associated with wound infections [9]. By inhibiting bacterial growth, lycopene helps create a more favorable environment for healing. Studies also indicate that lycopene promotes wound contraction, an important step in minimizing wound size and expediting recovery [10].

Despite the significant promise shown by lycopene as a wound healing agent across various bioassay systems, its clinical application is limited by challenges such as water insolubility and low bioavailability [3,11,12]. To harness the potential of lycopene as a therapeutic agent, substantial efforts must be made to enhance its bioavailability and facilitate targeted delivery to specific sites. This requires well-designed preclinical studies using animal models that closely replicate human conditions, thereby establishing the therapeutic efficacy of lycopene for wound healing.

One effective strategy to address these limitations is the incorporation of lycopene into nanoformulations, which can protect it from degradation and enhance its solubility in aqueous environments [13,14]. Nanotechnology provides innovative drug delivery systems that tackle the shortcomings of conventional methods, such as nonspecific biodistribution, inadequate targeting, poor oral bioavailability, and low therapeutic indices. Various nanocarriers have been explored for the delivery of lycopene, including vesicular nanocarriers, solid lipid nanocarriers, and lipid nanocarriers. These advanced delivery systems have shown promise in improving the bioavailability and therapeutic properties of lycopene when used in nanoformulations [15, 16].

Solid dispersion systems are advanced drug delivery formulations designed to enhance the dissolution rate and bioavailability of poorly water-soluble drugs. These systems consist of a solid carrier matrix with uniformly dispersed drug particles, such as eutectic mixtures like sulfathiazole-urea [17]. Solid dispersion systems were introduced by Obi and Sekiguchi in 1961, then it was further developed by Levy and Kani, along with Goldberg's work on solid solutions, underscored the influence of carrier composition on dissolution rates [17]. Recent studies have emphasized innovative carriers and preparation techniques aimed at improving bioavailability. Solid lipid nanoparticles and nano-dispersed lipid liquid crystals (LLC) have attracted significant attention from researchers for their potential in transdermal drug application [18-20]. Liquid crystals exist in a mesophase state, bridging the characteristics of solids and liquids. Their internal structures can adopt various geometrical conformations that are easily interchangeable, potentially facilitating the release of encapsulated drug substances during structural transitions. Liquid crystals exhibit distinctive physical properties that lie between the crystalline and liquid phases. Unlike the rigid, repetitive molecular arrangement found in crystals or the random mobility of liquids, liquid crystals in the mesophase display a unique state where molecules are both freely dispersed, similar to a liquid, and partially oriented, similar to a solid. This balance of order and mobility allows for dynamic shifts in their structures while maintaining characteristics of both solidity and fluidity, suggesting that encapsulated drugs may be released as the molecular assembly undergoes conformational changes [21, 22]. Highly stable liquid crystals composed of monoolein/oleic acid and phytol have been developed and applied to skin and mucous membranes [23]. These dispersed LLCs have been utilized to modulate skin permeability for various compounds, including [24], cyclosporine [25], naproxen [26], vitamin K [27] and propranolol [28].

The aim of this study was to develop lycopene-loaded lipid liquid crystal nanoparticles (LLCs) for application in wound healing by enhancing the solubility and bioavailability of lycopene. It was hypothesized that: (a) the incorporation of lycopene into solid dispersion systems with polymers would enhance its aqueous solubility, and (b) the integration of lycopene solid dispersions into LLCs matrices would facilitate controlled release, improve lycopene permeation across the skin, and stimulate wound healing more effectively than free lycopene. To test these hypotheses, specific objectives were established, including extracting lycopene from tomatoes and preparing solid dispersions to enhance solubility, characterizing the physicochemical properties of the lycopene solid dispersions, preparing lycopene-loaded LLCs, evaluating their drug loading, release, stability properties, and assessing the ex vivo skin permeation and wound healing efficacy of the formulations using cell-based and animal models.

2. Methods and Materials

Pure lycopene standard powder was obtained from BOC Sciences Inc. USA, while Polyvinylpyrrolidone-K30 (PVP-K30), Poloxamer 188 (Plx), Pluronic F127 (poloxamer 407), hydroxypropyl methylcellulose (HPMC) and glycerol monooleate were sourced from Sigma-Aldrich. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and ethanol were acquired from a local supplier (Dr. Mojallali, Iran).

2.1. Lycopene extraction using Soxhlet method

Lycopene was extracted from dried tomato paste powder and tomato paste production residue using the Soxhlet extraction method. Samples weighing 5 g were placed in a Soxhlet apparatus and extracted with a 1:1 mixture of acetone and ethyl acetate (total volume of 250 mL) for 12 hours. The solvent volume was adjusted to account for evaporation. The extract was then concentrated using a rotary evaporator and analyzed further. UV-Vis spectrophotometry was employed to quantify lycopene at the wavelengths corresponding to its peaks (470 nm).

2.2. Lycopene-carrier solid dispersion

Solid dispersion systems are a class of solid-state drug delivery platforms designed to enhance the dissolution rate and oral bioavailability of poorly water-soluble drugs. These systems consist of a solid polymeric carrier material where drug particles are uniformly dispersed at a molecular level. In this study, solid dispersions containing lycopene were prepared using polyvinylpyrrolidone (PVP) K30 and Poloxamer (Plx) at various weight ratios of carrier to lycopene (5:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:5). Lycopene-PVP K30 and Lycopene-Plx solid dispersions were prepared using the solvent evaporation method and the melting method, respectively.

For the solvent evaporation method, the specified ratios of PVP K30 and lycopene were placed in a beaker with a minimal amount of ethanol (for example, 1 g of the total solid in 100 mL of ethanol) and stirred. After two days under stirring conditions at room temperature, the solvent completely evaporated, resulting in the formation of glassy solids. These glassy solid clumps were then ground using a mortar and pestle, and the resulting particles were stored in sealed containers placed inside a desiccator for further physicochemical tests.

For the melting method, the specified ratios of Plx and lycopene were heated above their melting points (80 °C) and then allowed to cool at room temperature after stirring with a glass stirrer. The mortar and pestle used for grinding had been previously stored in the freezer to facilitate the grinding of the samples, as there was a risk of softening the Plx-containing samples during the grinding process. The ground materials were then passed through a 60-mesh sieve (with a pore diameter of 250 µm), and the obtained particles were kept in sealed containers inside the desiccator until their physicochemical properties were evaluated.

2.2.1. Physicochemical properties of lycopene

2.2.1.1. Saturation solubility

The saturated solubility of lycopene in extracted powder form (Lyc), as well as in physical mixtures (PM) and solid dispersion systems with Poloxamer 188 (Plx) and PVP K30, was determined in distilled water at 25 °C in a shaking water bath (Memmert, Germany) set at 200 rpm for 48 hours. The resulting mixtures were filtered through 0.22 μm syringe filters, and absorbance was measured using a UV spectrophotometer at 470 nm [29].

2.2.1.2. Fourier transform infrared spectroscopy

Apart from X-ray and DSC tests, FTIR was also employed to test the obtained samples. This test was conducted to evaluate the potential for drug degradation or the formation of hydrogen bonds with the carriers in the formulation. Medicinal powders were mixed with KBr powder, pressed into glass disks under high pressure, and analyzed using an FTIR-Perkin-Elmer Spectrum Two device for spectral analysis in the frequency range of 4000-400 cm⁻¹ with 32 scans [30].

2.3. Lipid Liquid Crystal Nanoparticles (LLCs)

Liquid crystalline formulations of lycopene were developed using glycerol monooleate (GMO) as the lipid phase and Pluronic F127 as a stabilizer. For the gel system, 6.4 g of GMO was melted at 55 °C and mixed with 0.6 g of ethanol for 12 hours. Subsequently, 425 mg of the lycopene-PVP solid dispersion was added. In the LLC system, 7 g of melted GMO was mixed with 3 g of water for 3 days. To 1.5 g of this gel, 300 mg of lycopene-PVP was added and mixed with 3 g of Pluronic F127 (1% w/w). This mixture was homogenized for 10 minutes and then subjected to probe sonication for an additional 10 minutes at 14 W to obtain a stable dispersion. The encapsulation efficiency (EE %) and drug loading (DL %) were calculated using the following equations:(Eq. 1) $$\text{EE\%}=\mathrm{}\frac{\text{Amountoftotaldrug}-\text{Amountoffreedrug}}{\text{Totaldrug}}\times 100$$(Eq. 1) (Eq. 2) $$\text{DL\%}=\mathrm{}\frac{\text{Amountofdruginnanoparticles}}{\text{Amountofdrug}+\text{Amountofnanoparticles}}\times 100$$(Eq. 2)

To determine the amount of unencapsulated drug, 500 µL of the LLC formulation containing 1% lycopene was transferred to a 15 mL Falcon Amicon® tube (centrifugal filters Amicon Ultra-4 with a 100 kDa molecular weight cut-off, Millipore, Germany). The sample was centrifuged at 5000 rpm for 20 minutes. The resulting filtrate was diluted to 10 mL with methanol using a volumetric flask. The absorbance of the solution was then measured at 470 nm using a spectrophotometer [31]. Each formulation was tested in triplicate, and the mean and standard deviation were calculated.

Given that the LLC formulation is intended for wound treatment and must possess appropriate retention on the wound, it was necessary for it to have relatively high viscosity. To achieve this, 10% w/w HPMC was added after preparing the LLC. The mixture was stirred until the HPMC was completely dissolved. Once dissolved at room temperature, it became viscous and suitable for application on wounds.

2.3.1. In vitro release

The in vitro release of lycopene from the formulations (LLC and LLC-HPMC) was studied using a dialysis bag method. A volume of the formulation was placed within a 10 kDa cut-off dialysis bag and immersed in a 0.2% w/w sodium dodecyl sulfate PBS solution at pH 7.4 while stirring at 37 °C. At predetermined time points up to 96 hours, samples were withdrawn from outside the bag and replaced with fresh buffer to maintain sink conditions and also keep the final volume constant. The concentration of lycopene in the samples was determined by measuring absorbance at its λ_max (470 nm).

2.3.2. Ex vivo Skin Permeability Assessment

Ex vivo permeation studies of lycopene-loaded LLC and LLC-HPMC formulations were conducted using static Franz diffusion cells and excised full-thickness mouse abdominal skin. Healthy skin was obtained from four mice, with hair removed 24 hours prior to the experiment. The mice were euthanized through spinal anesthesia, and the skin was cleaned and mounted in the diffusion cells. Subsequently, 1 mL of the lycopene-loaded LLC formulation was applied to the skin, which was equilibrated at 34 °C for 1 h. Samples were collected from the receptor fluid (RF) at 0.5, 1, 2, 4, 6, 18, 24, and 48 h. Each 2 mL sample was mixed with an equal volume of 0.2% w/w sodium dodecyl sulfate PBS and analyzed for lycopene content using UV-spectrophotometry (λ_max 470 nm). After the experiment, to calculate the remaining product (%RF), the skin was scraped to recover residual lycopene, which was dissolved in dichloromethane, centrifuged (11,000 rpm for 10 minutes), filtered, and analyzed. For % skin (the lycopene% penetrated the skin but did not pass into the release medium), skin pieces were soaked in dichloromethane for 3 days, centrifuged (5,000 rpm for 20 minutes), and analyzed [32].

Drug transport across the membrane was investigated using Fick's diffusion model. Permeability coefficients were derived from the following equations:(Eq. 3) $$\mathrm{P}\mathrm{}=\mathrm{}\text{slope}\mathrm{}/\mathrm{}\left(\mathrm{S}\mathrm{}\times {\mathrm{}\mathrm{C}}_{\mathrm{d}}\right),\mathrm{}\text{Slope}\mathrm{}=\text{dM}/\text{dt}$$(Eq. 3)

In these equations, the 'slope' is obtained by plotting the amount of drug permeated (dM) over time (dt). S represents the membrane surface area (cm²) and C_d denotes the drug concentration in the donor compartment.

All animal experimental protocols were approved by the Ethical Committee of Mashhad University of Medical Sciences (IR.MUMS.PHARMACY.REC.1399.038).

2.3.3. Morphology of LLC

The morphology of the LLC and LLC-HPMC was examined using polarized light microscopy under cross-polarized conditions. Formulations were placed on slides, covered with water, and visualized [33].

2.3.4. Rheology Testing

The viscosity of the LLC and LLC-HPMC formulations was measured using a Brookfield viscometer (KU-3 Viscometer, AMETEK®, USA) equipped with a CC3-14 spindle. Shear stress-shear rate profiles were obtained at 25 °C using Rheo3000 software to characterize the flow behavior of the formulations.

2.3.5. Stability Testing of Lipid Nanoparticles

The stability of the lycopene-loaded LLC and LLC-HPMC formulations was evaluated according to the International Conference on Harmonization (ICH) guidelines [34]. Samples were stored at 75 ± 5% humidity and 40 ± 2 °C for 6 months. At 0, 1, 3, and 6 months, particle size, polydispersity index (PDI), zeta potential, crystallinity, drug content, color, and pH were assessed.

2.4. MTT Cytotoxicity Assay

Human fibroblast cells (HDF) were cultured in T25 flasks until they reached 80% confluency. The cells were maintained in DMEM High Glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin-streptomycin) in a 5% CO₂ atmosphere with 80-90% humidity at 37 °C. To evaluate cell viability, HDF cells were seeded in 96-well plates at a concentration of 1 × 10⁴ cells per well and treated with various formulations, including lycopene derived from PVP using the solid dispersion method, LLC, LLC-HPMC, and formulations without lycopene, alongside a control group. The cells were incubated at 37 °C with 5% CO₂ for 24, 48, and 72 hours. After incubation, the medium was removed, and the cells were washed with PBS. Fresh medium containing 10 µL of MTT solution (5 mg/mL) was added, and the cells were allowed to incubate for an additional 4 hours. Following this, the medium was removed, and formazan crystals were solubilized in DMSO. Absorbance was measured at 570 nm and 630 nm using an ELISA reader. Cell viability was calculated using the formula:(Eq.4) $$\mathit{Cell\; Viability}\mathrm{}\left(\mathrm{\%}\right)=\frac{\mathit{Average\; absorbance\; of\; control\; samples}}{\text{Average absorbance of treated samples}}\times 100$$(Eq.4)

The test compared the cell viability treated with the aforementioned formulations to the control group at 24, 48, and 72 h.

2.5. Scratch Assay for Cell Migration

Cell migration was assessed in groups treated with LLC and LLC-HPMC formulations containing lycopene derived from the solid dispersion method, as well as in groups that contained only the lycopene solid dispersion. Human fibroblast cells were cultured in 12-well plates using high-glucose DMEM medium supplemented with 10% FBS and 1% antibiotic (penicillin/streptomycin) at 37 °C in a 5% CO₂ environment with 90% humidity. When the cells reached 90% confluence, a scratch was made across the cell monolayer to establish the initial time point (T0). The medium was then replaced with fresh medium, and optimized concentrations of the test compounds were added to the wells in triplicate. Cell migration was monitored by observing changes in cell shape within the scratch area at 2, 6, 12, 24, and 48 hours post-treatment, with control wells included for comparison. Migration was documented using an inverted microscope (Olympus IX53).

2.6. Animal Studies for Wound Healing Assessment

2.6.1. Animal Care and Grouping

Thirty male mice (25 ± 5 g) were housed under controlled conditions (24 ± 2 °C, 12-hour light/dark cycle) with unrestricted access to food and water. The mice were randomly divided into five groups (n = 6 per group), each receiving different treatments: the LLC-HPMC group, the drug-free LLC-HPMC group, the lycopene solid dispersion group, the positive control group (silver sulfadiazine), and the negative control group (no treatment) immediately after wounding.

2.6.2. Wounding Procedure

Deep open wounds were created on the dorsal area of the mice using 6 mm biopsy punches, removing all skin layers down to the panniculus carnosus. This model allowed for the examination of inflammation, granulation tissue formation, re-epithelialization, angiogenesis, and tissue regeneration. The mice were anesthetized with ketamine (10%) and xylazine (2%) cocktail (200 μL per mouse). Back Hair of mice was shaved, and wounds were created using the biopsy punch. The size of the wounds was measured with a ruler.

2.6.3. Histological Studies

Wound tissues were harvested on days 3, 7, 10, and 14 post-wounding and fixed in 10% formalin. Histological analysis included counting fibroblasts and assessing inflammation, angiogenesis, and re-epithelialization using hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) staining, and Masson's Trichrome (MT) staining. Connective tissue, particularly collagen, was visualized using Masson's Trichrome staining. A scoring system was employed, ranging from 0 (unhealed) to 12 (completely healed), assessing parameters such as re-epithelialization, epithelial thickness, keratinization, granulation tissue, scar formation, and tissue regeneration. Wound healing progression was documented through photographs taken at consistent intervals on days 3, 7, 10, and 14. Wound area measurements were analyzed using ImageJ software, calculating the percentage of wound closure with the following formula:(Eq.5) $$\mathit{Wound\; closure}\mathrm{}\left(\mathrm{\%}\right)=\frac{(\mathit{A}\mathrm{ₒ}-\mathit{A}\mathrm{x})}{\mathit{A}\mathrm{ₒ}}\mathrm{}\times 100$$(Eq.5)

Where Aₒ​ is the initial wound area and A_x is the wound area at a given time point.

2.7. Statistical Analysis

All data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test, with a significance level set at p < 0.05, was used for statistical analysis. Additionally, the Kruskal-Wallis test was used to asses histopathological studies. Graphs were generated, and analyses were performed using Excel 2021 (Microsoft, USA) and Prism 9.1 (GraphPad, USA).

3. Results

3.1. Lycopene extract and solid dispersion of lycopene-carriers

The final powder of the raw material was obtained using the Soxhlet extraction method. The lycopene product extracted from tomatoes is quantified using UV spectroscopy with standard lycopene purchased from BOC Sciences Inc., USA. The average yield of lycopene per kg of raw material was measured to be 407.0 ± 23.1 mg. The purity of the extracted lycopene is 87.5 ± 2.3%, and it is normal that the peaks obtained from XRD and DSC for extracted lycopene are not sharp. Therefore, the results obtained from the solid-dispersion method from the extracted lycopene with PVP cannot be interpreted using XRD and DSC, and focusing solely on FTIR and solubility measurement.

Lycopene is insoluble in water and the results of the saturation solubility tests for lycopene with PVP and Poloxamer (Plx) carriers in various proportions are presented in Figure 1. A statistically significant difference in lycopene solubility was noted between the solid dispersions incorporating Poloxamer compared to those with PVP carriers. PVP demonstrated greater efficacy in enhancing the aqueous solubility of lycopene than Poloxamer. When the ratio of PVP to lycopene was 2:1, the greatest effect on enhancing the solubility of lycopene was observed and this ratio was selected for further analysis.

FTIR spectroscopic analysis was performed to investigate the interactions between lycopene and PVP-K30 in various formulations. The FTIR spectra of lycopene (Lyc), PVP-K30, a physical mixture (PM), and solid dispersion systems of lycopene and PVP-K30 are displayed in Figure 2. A broad peak in the 3400 cm⁻¹ region and a bifurcated peak around 3000 cm⁻¹, corresponding to O-H and C-H stretching vibrations, are revealed in the lycopene spectrum. Similar bands are observed in the spectrum of PVP-K30, with characteristic amine group bands appearing around 3460 cm⁻¹. The FTIR spectra of both the physical mixture and the solid dispersion system exhibit comparable features, indicating similar interactions between lycopene and PVP-K30 in these systems.

3.2. In vitro evaluation of LLC and LLC-HPMC

Lycopene is a deep orange close to red, but it becomes a lighter orange when it is placed in a solid dispersion with PVP as the carrier. Ultimately, when it is incorporated into the LLC, the formulation appears white, indicating that lycopene is well-encapsulated within the liquid crystal nanoparticles. The percentage of lycopene encapsulation within the liquid crystal formulation was evaluated using an indirect method. The EE% and DL% were obtained to be 71.57 ± 4.08 and 4.42 ± 0.12, respectively.

The release profiles of LLC and LLC-HPMC systems containing lycopene were evaluated using a 10 kDa dialysis bag in a PBS solution at pH 7.4 (Figure 3A). Significant differences in release rates were not observed between the LLC and LLC-HPMC at all points. Moreover, the similarity factor (f₂) was calculated to be 74.06 ± 4.28%, while the difference factor (f₁) was determined to be 4.23 ± 1.28. This indicates that the addition of HPMC has no effect on the drug release process and this was proved by the similarity test. The reason for this phenomenon is that HPMC is hydrophilic and easily dissolves in an aqueous release environment, creating very little resistance to mass transfer for the lipid particles of the liquid crystals in releasing lycopene.

The morphology of LLC (Figure 3D) and LLC-HPMC (Figure 3E) was observed to take the form of a cubosome, with a dark field and a few visible particles, indicating an isotropic structure that does not respond to polarized light. In contrast, a reaction to polarized light would produce a texture-like or multi-colored birefringent field, exhibiting a hexosome (anisotropic) structure.

The obtained drug release data were fitted to various models such as zero-order, first-order, Higuchi, Weibull, and Korsmeyer-Peppas. The results showed that the best model that fits well with the release data is the Weibull release model (Figure 3C-D). This model showed a higher regression coefficient compared to the other models (Table 1). Additionally, fitting the release data to the Korsmeyer-Peppas model showed that n is less than 0.45, indicating that the drug release mechanism is diffusion. This suggests that the drug migrates from a concentrated area to a diluted area, while the degradation of the drug delivery system does not contribute to the drug release process.

3.3. Ex vivo release and Permeability

The release kinetics of lycopene from various formulations were assessed using the Franz diffusion cell method, with the findings illustrated in Figure 4A. The concentration of lycopene in the receptor compartment of the Franz diffusion cell was detectable one hour after the experiment commenced. The release profiles of the two formulations in Franz cells with mouse skin were found to be similar to the in vitro release observed in dialysis bags. The permeability coefficients for LLC and LLC-HPMC in mouse skin were calculated to be 7.56 cm/min and 7.48 cm/min, respectively, showing no significant difference between the two. There were no significant differences in retention product (% RP), % skin, and receptor fluid (% RF) between the LLC and LLC-HPMC formulations, indicating that the lipid crystal formulation, when incorporated into an HPMC gel matrix, does not exhibit substantial changes in skin permeation in mice (Figure 4B).

Rheological Analysis

The stress-strain results indicate that the LLC-HPMC follows a pseudoplastic Bingham model, as evidenced by the non-linear relationship observed in the rheological data (Figure 5).

3.4. Stability study

The stability study of lycopene-loaded LLC and LLC-HPMC formulations was conducted over 6 months at 75 ± 5% humidity and 40 ± 2 °C. Parameters such as particle size, polydispersity index (PDI), zeta potential, phase homogeneity, color, and drug content were analyzed periodically. One-way ANOVA results obtained for 0, 1, 3, and 6 months revealed that both LLC and LLC-HPMC formulations remained completely stable, with no significant differences observed at the specified time points (Table 2).

An important point in the stability of nanoparticles is that the zeta potential must be negative and less than -25 mV. Our findings indicate that the zeta potential of the formulations has continuously remained below -25 mV, demonstrating long-term stability and the absence of aggregation. Overall, the negative zeta potential indicates a stable dispersion, as like charges repel, helping to prevent aggregation of the lipid particles [35]. The zeta potential of lipid liquid crystals, such as those based on glycerol monooleate (or other glycerol derivatives) can be negative for several reasons namely surface charge, ionization of functional groups, interactions with counterions, dipole moments and hydration shell.

Considering the chemical composition of the system manufactured in the current study, glycerol monooleate can contribute to a net negative charge on the surface of the lipid particles. Certain functional groups within the lipid molecules can ionize, leading to the generation of negative charges. This is especially true if the pH of the environment allows for deprotonation of acidic groups. Furthermore, in the liquid crystal phase, the arrangement of lipid molecules can lead to the exclusion of certain cations, enhancing the relative negative charge on the surface. The molecular structure of glycerol monooleate may also create a dipole effect that could influence the overall charge distribution, contributing to a negative zeta potential. Finally, the formation of a hydration shell around the lipid particles can also influence the surface charge. The orientation of water molecules can affect the effective charge seen by surrounding ions [36, 37].

3.5. Cytotoxicity Assessment (MTT Assay)

To ensure the safety of the LLC and LLC-HPMC formulations, a cytotoxicity assessment was deemed essential. The cytotoxic effects of lycopene incorporated in LLC on fibroblast cells were evaluated through a cell viability assay using the MTT method. The viability of fibroblast cells was assessed at 24, 48 and 72 hours post-treatment with various formulations, including lycopene combined with PVP, LLC, and LLC-HPMC, both with and without lycopene. As illustrated in Figure 6, cell viability across all groups was compared to the control group at the specified time intervals.

At 24 hours, no significant changes in cell viability were observed in cells treated with lycopene and PVP compared to the control group. However, by 48 hours, the cell viability was 96.6%, and by 72 hours, it decreased slightly to 93.8%. These results indicate that lycopene does not exert cytotoxic effects on human fibroblast cells, as the treated cells maintained normal metabolic activity throughout the assessment period. Importantly, the survival rate in all experimental groups remained above 80%, which is generally considered a favorable outcome. Specifically, the group containing lycopene showed a survival rate comparable to that of the control group, with a p-value greater than 0.05, indicating a statistically insignificant difference. Survival rates observed at 24 hours were also statistically indistinguishable from the control.

In groups treated with LLC and LLC-HPMC containing lycopene, survival rates at 72 hours were recorded at 90% and 91.81%, respectively. Conversely, survival rates in the groups utilizing LLC and LLC-HPMC devoid of lycopene were slightly lower, at 87.06% and 86.62%, respectively. Overall, the findings suggest that lycopene, when incorporated into LLC and LLC-HPMC, exhibits no significant cytotoxicity towards human fibroblast cells over the assessed time points, supporting its potential for safe application in biomedical contexts.

3.6. Cell migration test results

As illustrated in the accompanying Figure 7, significant cell migration toward the artificial wound (scratch) was observed in the group treated with the LLC formulation containing lycopene at the specified time intervals. Notably, this group exhibited a more rapid filling of the distance between cells compared to the control group, indicating a stimulatory effect of lycopene on fibroblast cell migration and proliferation.

3.7. Animal Study Results

3.7.1. Macroscopic Examination of the Wound Healing Process

As mentioned in section 2.3, the LLC formulation alone is unsuitable for topical application due to the runny state of the LLC, they cannot remain on the skin for an extended period. To address this issue LLC must be incorporated into an HPMC gel matrix. Therefore, in animal study and the evaluation of the wound healing process, the LLC-HPMC formulation was utilized. To assess the wound healing rate, photographs of the wound sites were captured at prescribed intervals (days 0, 5, 7, 14, and 21), ensuring a standardized distance for imaging (Figure 8). The wound area was quantified using ImageJ software, and the percentage of wound closure was calculated. Results indicated significantly faster closure with the LLC from day 5 onward compared to other groups, correlating with enhanced healing effects. On day 10, a significant difference was observed between the lycopene-containing LLC-HPMC formulations and both the positive control group (silver sulfadiazine) and the negative control group, with a p-value of less than 0.0001 (Figure 9A).

3.7.2. Histological Results

Histological analysis was conducted to evaluate five key wound healing parameters (epithelial thickness, keratin production, granulation tissue formation, scar reconstruction, and re-epithelialization), with each parameter scored individually on a scale of 0-3. The LLC-HPMC formulation received the highest total score of 12, indicating superior wound healing effects for these two groups compared to the other treatment groups. Notably, the scores reflected improved re-epithelialization, granulation tissue development, and scar reconstruction with the LLC-HPMC relative to the controls (Figure 9B). The data obtained for LLC-HPMC was compared against the control group using the Kruskal-Wallis non-parametric test.

The histopathology of excised wound tissues from different treatment groups was examined (Figures 10 and 11). In the LLC-HPMC group (Group A), complete re-epithelialization and intact dermal structures were noted, characterized by a thin epidermal layer and normal dermal architecture. Masson’s Trichrome staining revealed a normal distribution of collagen fibers. The LLC-HPMC base (Group B) displayed partial re-epithelialization and granulation tissue formation beneath residual scabs. The Lyc-PVP solid dispersion (Group C) exhibited full re-epithelialization. In contrast, silver sulfadiazine (Group D) induced necrotic debris with no re-epithelialization and minimal collagen formation in granulation tissue. The control group (Group E) resembled Groups B and D. Histopathological assessments indicated that liquid crystal formulations, particularly LLC-HPMC, enhanced re-epithelialization and collagen deposition compared to standard treatments like silver sulfadiazine, demonstrating superior wound healing efficacy.

4. Discussion

The results indicated that the lycopene-loaded lipid liquid crystal nanoparticles system significantly improved lycopene solubility, skin permeation, and bioavailability compared to the controls. Moreover, it enhanced wound healing by accelerating closure and promoting re-epithelialization and collagen deposition. Lycopene has been shown to possess several medicinal properties that make it a promising agent for wound healing applications [38]. Previous studies have demonstrated lycopene's anti-angiogenic effects through the inhibition of microvascular cell proliferation and migration in vitro [39]. It also exhibits potent anti-inflammatory actions via the downregulation of pro-inflammatory cytokines and mediators, as evidenced in both in vitro and in vivo models of inflammation [40, 41]. These anti-inflammatory properties have led to the clinical use of lycopene for the treatment of arthritis. Additionally, lycopene has been found to mitigate spinal cord ischemic injuries, indicating its neuroprotective capabilities. Notably, lycopene shares similar pharmacological activities with curcumin, another natural compound extensively researched for its wound healing properties. Given its multifaceted effects targeting angiogenesis, inflammation, and tissue damage—all key processes in wound repair—lycopene represents a viable candidate for development as a wound treatment.

Comparing the results obtained for Plx and PVP, the results showed that PVP exhibited to be a better carrier for increasing the solubility of hydrophobic lycopene. At a ratio of 2:1 of PVP to lycopene, it achieved a significant increase in saturation solubility (Figure 1). Therefore, in the subsequent study, we examined the physicochemical properties of PVP with lycopene in a solid dispersion.

The studies examined various formats of lycopene formulations, emphasizing that the choice of solubilizer can greatly influence solubility outcomes. Interactions between lycopene and excipients such as PVP and PEG 6000 were highlighted throughout the investigations. It was noted that these interactions might include hydrogen bonding and van der Waals forces, which not only stabilize the amorphous form of lycopene but also enhance its solubility properties [42-45]. The importance of these interactions cannot be overstated, as they play a crucial role in stabilizing lycopene, preventing crystallization during storage, and improving its stability over time. The ability of PVP K30 to maintain lycopene's crystalline state while providing a solid matrix presents an intriguing approach for enhancing the stability and potential bioavailability of lycopene. In contrast, findings from previous research [46] indicate that lycopene may predominantly exist in an amorphous state when formulated as part of commercial products (e.g., Lycovit®), where characteristic diffraction peaks for lycopene were absent. The extensive interactions between lycopene and its solubilizers, such as PVP K30, may prevent crystallization or encourage partial amorphization without completely disrupting the integrity of the crystalline structure of lycopene. This adaptability is particularly beneficial for pharmaceutical formulations, as retaining crystalline properties can be crucial for stability while simultaneously promoting enhanced dissolution profiles associated with amorphous forms.

The FTIR results from this study indicate that lycopene is effectively dispersed within a PVP K30 solid dispersion system, with no significant chemical interaction between the two components detected (Figure 2C). This lack of strong interaction suggests that the stability of lycopene in the formulation is enhanced, potentially preventing the formation of unwanted by-products. It is suggested that the differences in chemical structures and polarities between lycopene and PVP K30 contribute to this behavior. The characterization of lycopene via FTIR, as reported in a study by Mirahmadi etal. in 2024, illustrated the presence of specific vibrational peaks corresponding to its chemical structure, including the characteristic C = C stretching at 1629.97 cm⁻¹ and bending vibrations at 1375.09 cm⁻¹. While some interactions between lycopene and solubilizers were noted, particularly with the FTIR spectra of the lycopene-PVP K30 combination exhibited only minimal shifts in peak positions and intensities [42,47]. This observation indicates that the chemical environment of lycopene is not significantly altered by PVP K30. In accordance with a study by Zardini etal. in 2018, the absence of new peaks in the FTIR spectra of lycopene-loaded formulations reinforces the notion that no chemical reactions, such as hydrolysis or esterification, are occurring between lycopene and the other components in the nanoparticle matrix. The observation that lycopene is predominantly dissolved rather than chemically modified suggests that the properties of lycopene are successfully retained in the formulation while its solubility is enhanced through physical means, rather than through the creation of novel chemical entities [43]. Additionally, the distinct peaks observed for PVP K30, particularly in the carbonyl and C–H stretching regions, coupled with the characteristic peaks of lycopene, affirm the compatibility of lycopene within the PVP K30 matrix. The FTIR analysis from studies [43,44] supports these conclusions, as no significant spectral changes were detected post-formulation, indicating that the integrity of lycopene is maintained in the solid dispersion.

This high percentage of encapsulation underscored the effectiveness of the method employed and the stability of the formulation. The physical interactions between lycopene and the lipid matrix appear to play a crucial role in this high encapsulation efficiency, aligning with findings from a study, which reported encapsulation efficiencies (EE) ranging from 64.79% to 78.89% in various nanocarrier formulations [43]. This similarity underscores the potential advantages of lipid-based systems for encapsulating lipophilic compounds like lycopene due to their low solubility in aqueous environments.

The release of lycopene-carrier from LLC and LLC-HPMC occurred through a diffusion mechanism, and the data fitting results indicated that the degradation of the formulation did not play a role in the release of lycopene (Figure 3A). The release in the in vitro environment reached a plateau after 60 minutes, which aligns well with the structure of cubosomes, showing a faster release compared to hexosomes. The enhanced permeability of the formulation, attributed to its lower viscosity and larger surface area, facilitates improved contact with the surrounding medium. This observation aligns with findings from previous studies, which identified a biphasic release pattern in lipid nanoparticles characterized by an initial rapid release phase followed by a slower release over time. The distribution of lycopene within the lipid matrix significantly affects this release profile, as the initial phase is influenced by the lipid surrounding lycopene, promoting higher mass transfer rates. The rapid release during the initial stage can also be attributed to the high surface area of the nanoparticles and the low viscosity of the matrix, which enhances diffusivity [48]. It should be noted that the release data from the experiment may not fully represent the actual release profile. This discrepancy is due to the use of a membrane in the experimental setup, which could potentially introduce artifacts into the dissolution data, as pointed out by Boyd in 2003 [49]. Despite this potential limitation, the data remains valuable for comparative analysis. The results can still be utilized for meaningful comparisons as the experiment was meticulously conducted under uniform conditions across all formulations.

The findings from the rheological evaluation suggest that the formulated system exhibits pseudoplastic Bingham fluid behavior, highlighting the complex interactions between the lycopene particles and the polymers within the formulation (Figure 5). This behavior implies that the formulation possesses both a yield stress, characteristic of Bingham fluids, and shear-thinning properties typical of pseudoplastic fluids.

The results demonstrate that the LLC formulation containing lycopene exhibits no significant cytotoxicity towards human fibroblast cells, indicating its safety for topical applications (Figure 6). Notably, at certain concentrations, lycopene may even exert a protective effect, supporting its potential as a beneficial ingredient in dermatological formulations. This aligns with findings from a study conducted by Cefali etal., which utilized resazurin staining to evaluate cytotoxicity and confirmed that lycopene extract promoted cell viability in macrophage and fibroblast lineages. The presence of live cells in wells treated with lycopene, even at high concentrations (200 µg/mL), further validates the safety profile of lycopene-rich formulations [50]. These findings collectively suggest that lipid-based systems containing lycopene not only enhance delivery but also maintain cellular integrity, reinforcing their utility for skin applications.

The observation of Cell migration further supports the hypothesis that these compounds positively influence cell growth and migration. Consistent with expectations, both formulations containing growth-promoting substances demonstrated a significantly faster migration rate relative to the control group, corroborating findings from previous animal model studies. The evaluation of cell behavior was based on morphological changes observed across the different treatment groups in comparison to the control.

The results of this study illustrate that LLC-HPMC containing lycopene significantly enhances fibroblast cell migration, indicating its potential effectiveness in wound healing (Figure 7). This finding aligns with recent research by Salunke etal. (2024), which demonstrated that lycopene emulgel notably improved wound closure and epithelialization in diabetic rats, achieving a reduction in wound size by 95.3% and 88.9%, respectively [51]. Such findings underscore the therapeutic promise of lycopene, particularly in applications for chronic wounds that often experience impaired healing due to underlying conditions like diabetes. The ability of lycopene to promote cellular migration and proliferation is likely attributed to its potent antioxidant properties, which mitigate oxidative stress, a critical factor in the wound healing process [52]. Support for this notion is provided by research conducted by Bavarsad etal., which demonstrated the effectiveness of lycopene in a skin-lightening cream, where its antioxidant capabilities contributed to improved skin conditions, indirectly suggesting similar mechanisms could be involved in wound healing [53]. Furthermore, a study by Wawrzyniak etal. (2023) highlighted lycopene’s role as a powerful scavenger of reactive oxygen species (ROS) and its involvement in skin aging processes. By reducing ROS levels, lycopene may enhance fibroblast viability and activity, both essential for wound healing. The synergy between lipid liquid crystalline formulations and lycopene could further amplify these effects, providing a stable and effective delivery system that promotes skin repair. Additionally, the mechanisms by which lycopene regulates insulin resistance pathways and enhances microvascular health are highly relevant. Improvement in vascular function can facilitate better nutrient and oxygen delivery to wound sites, thus accelerating the healing process [54]. In contrast, it has been shown by Honda etal. that the Z-isomers of lycopene exhibit even greater biological activities compared to its all-E-isomers, suggesting that the stability and efficacy of lycopene in the current formulations might be enhanced through specific isomeric configurations [55]. This raises interesting questions regarding the formulation processes employed and the potential to optimize lycopene’s efficacy based on its isomeric state. In conclusion, lipid liquid crystal formulations containing lycopene not only enhance fibroblast migration and promote wound healing but also represent a versatile approach to skincare that addresses both cosmetic and therapeutic needs. The positive outcomes observed in the present study, supported by a growing body of literature, indicate the potential for leveraging lycopene in clinical settings for effective wound management strategies. Future research should focus on optimizing these formulations to maximize their therapeutic effects and explore their applications in various dermatological conditions.

5. Conclusion

The incorporation of lycopene into lipid liquid crystal formulations significantly enhances its solubility, permeation, and therapeutic efficacy in wound healing applications. The results demonstrated that these innovative formulations promote fibroblast migration and proliferation. By addressing major challenges related to lycopene’s bioavailability and stability, this study establishes a solid foundation for the further development of lycopene-based therapies. The positive outcomes provide compelling evidence for the potential of lipid liquid crystal systems as effective tools for wound management and highlight lycopene's multifaceted role as a therapeutic agent. Future clinical studies will be essential for validating these findings and exploring the practical applications of these formulations in various dermatological conditions.

CRediT authorship contribution statement

Elham Khodaverdi and Jebrail Movaffagh: Conceptualization, Data curation, Investigation, Methodology, and Writing – original draft. Soheil Tafazzoli Mehrjardi: Investigation, Validation, Software, Writing original draft, Farhad Shahverdi and Hossein Kamali: Validation, Methodology, Funding acquisition, Supervision, Project administration, Writing- review & editing; Ali Nokhodchi: Formal analysis, Writing- review & editing

Declaration of Competing Interest

The authors declare that they have no conflict of interest.

Acknowledgments

The authors are grateful for the financial support provided by Mashhad University of Medical Sciences with 981639 number.

Funding

The author(s) reported there is no funding associated with the work featured in this article.