Perillaldehyde-Encapsulated Lipid Nanoparticle Hydrogel for Enhanced Wound Healing, Improved Stability and Biocompatibility

TL;DR

A new wound dressing using perillaldehyde in lipid nanoparticles within a hydrogel improves healing, stability, and safety compared to traditional methods.

Contribution

A novel nano-in-hydrogel platform for controlled delivery of perillaldehyde with enhanced stability and biocompatibility.

Findings

PAH-L showed uniform size, high encapsulation efficiency, and colloidal stability.

PAH-L-G demonstrated sustained release and improved wound healing in a rat model.

The dressing showed low hemolysis and high fibroblast viability in compatibility tests.

Abstract

Volatile phytochemicals such as perillaldehyde (PAH) exhibit antimicrobial and anti-inflammatory activities relevant to wound repair; however, topical use is limited by volatility, chemical instability, and potential irritation associated with burst exposure. Here, we developed a nano-in-hydrogel dressing by encapsulating PAH into lipid nanoparticles (PAH-L) and incorporating them into a carbomer hydrogel (PAH-L-G). PAH-L showed a uniform nanoscale size distribution, high encapsulation efficiency, and good colloidal stability. After gel incorporation, PAH-L-G formed an interconnected porous network with rapid swelling and a more sustained release profile than free PAH or PAH-L. Hemocompatibility and cytocompatibility assays indicated low hemolysis and high fibroblast viability. In a full-thickness rat wound model, PAH-L-G accelerated wound closure and improved histological regeneration without obvious local irritation. Overall, the lipid-nanoparticle-in-hydrogel strategy stabilizes PAH and enables controlled topical delivery, supporting PAH-L-G as a promising wound dressing platform.

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Full text

1. Introduction

Skin injury is common in clinical practice, and delayed repair can progress to chronic wounds, infection, and long-term scarring [1]. An ideal dressing should not only protect the lesion and maintain a moist environment, but also regulate the wound microenvironment by limiting microbial burden and excessive inflammation while supporting re-epithelialization, granulation, and tissue remodeling [2,3]. Hydrogel dressings are therefore attractive because their water-rich networks absorb exudate, conform to irregular surfaces, and can act as local depots for bioactive agents [4,5]. Among candidate actives, natural small molecules are being revisited for wound care owing to their broad bioactivity and accessibility [6,7,8]. Perillaldehyde (PAH), a major volatile constituent of Perilla frutescens, has reported antimicrobial and anti-inflammatory activities relevant to skin repair [9,10]. However, its volatility and limited stability reduce on-site retention, and rapid exposure peaks may increase the risk of local irritation, which together hinder practical topical use [11,12].

A range of strategies has been explored to improve topical delivery of volatile or hydrophobic aldehydes/terpenoids such as PAH. Host–guest inclusion systems, such as cyclodextrin-based complexes, can improve apparent solubility and reduce evaporation, but may still suffer from burst release on wet surfaces and limited residence time on wounds [13,14]. Emulsification approaches, including emulsions and nanoemulsions, can enhance dispersion yet often require relatively high surfactant loads, which may be undesirable for compromised skin [15]. Polymeric micro- or nanocarriers, such as polymeric micelles, nanocapsules, and electrospun fiber systems, can slow diffusion and protect labile actives, but may involve organic solvents, complex processing, or slow clearance from the wound bed [16,17,18]. In parallel, integrating carriers into wound dressings, such as hydrogels, membranes, or sponges, has become a prevailing design concept, providing both a protective matrix and controlled local release [19].

Lipid nanoparticles were selected here because they can efficiently solubilize hydrophobic PAH within a biocompatible lipid matrix, physically shield the cargo from volatilization and oxidation, and modulate release through diffusion across the lipid core and interfacial layers. Compared with cyclodextrin-based inclusion complexes, lipid nanoparticles offer a higher payload for hydrophobic actives and enable a more sustained release profile under aqueous wound conditions. Compared with emulsification-based systems, they typically require lower surfactant levels and provide a solid or structured lipid phase that improves physical stability. Compared with polymeric nanoparticles, lipid systems can be formulated from pharmaceutically accepted lipids with simplified manufacturing and reduced concern about solvent residues, making them attractive for translational topical use [20,21,22]. Nevertheless, nanoparticle dispersions alone can be readily removed from the wound surface by exudate and motion. To improve retention and handling, we embedded PAH-Loaded lipid nanoparticles (PAH-L) into a carbomer hydrogel to form a nano-in-hydrogel composite (PAH-L-G). Carbomer gels are widely used in topical products because they are easy to spread, form conformal films, and can provide an additional diffusion barrier that further smooths drug exposure. In this study, we optimized PAH-L preparation, characterized PAH-L and PAH-L-G (size, ζ-potential, morphology, FTIR, stability, release and swelling), evaluated hemocompatibility and cytocompatibility, and assessed wound-healing efficacy in vivo.

2. Results

2.1. Optimization of the Preparation Process of PAH-L

2.1.1. Ultrasonic Technology

Encapsulation efficiency (EE) was used as a primary criterion to optimize the sonication step because ultrasonic energy affects droplet disruption, interfacial packing, and potential drug leakage. A small screening matrix was performed by varying ultrasonic power (200–400 W) and duration (15–40 min) (Figure 1A; Table S1). Moderate power (200 W) consistently afforded higher EE than higher powers, while excessive power (400 W) likely introduced greater thermal/mechanical stress and promoted PAH loss/leakage. At 200 W, extending sonication from 15 to 40 min only produced a modest EE increase (73.55 ± 0.49% to 76.55 ± 0.49%), suggesting diminishing returns with longer processing. To balance EE, processing time, and potential PAH volatilization during prolonged sonication, 200 W for 15 min was selected for subsequent preparations.

2.1.2. The Loading Capacity and Encapsulation Efficiency of PAH-L

Encapsulation efficiency reflects the fraction of PAH retained within nanoparticles after removing free PAH. Using ultrafiltration separation and HPLC quantification (Table 1), PAH-L achieved an EE of 80.43 ± 1.09% and an LC of 11.1 ± 0.26%.

2.1.3. Zeta Potential Measurements of PAH-L

ζ-Potential is an indicator of surface charge and electrostatic repulsion, and therefore relates to colloidal stability. Dynamic light scattering showed that PAH-L had an average diameter of 122.5 ± 0.5 nm with a narrow distribution (PDI 0.213 ± 0.03) and a negative ζ-potential of −31.1 ± 0.28 mV (Figure 1B). The pronounced negative charge is expected to reduce aggregation and support a stable dispersion.

2.1.4. Appearance and Morphology of PAH-L

PAH-L appeared as a uniform, slightly opalescent dispersion under visible light (Figure 1C). TEM images revealed well-formed nanoparticles with no obvious aggregation (Figure 1D). After incorporation into carbomer, the appearance of PAH-L-G is as shown in Figure 1E. SEM showed an interconnected porous network in PAH-L-G (Figure 1F), which can retain water and facilitate exudate uptake—features favorable for maintaining a moist healing environment.

2.1.5. Stability of PAH-L or PAH-L-G

Storage stability is critical for lipid-based delivery systems, particularly for volatile actives. We therefore evaluated the temperature-dependent stability of PAH-L by monitoring encapsulation efficiency during storage at 4 °C and 25 °C (Figure S2). At 4 °C, PAH-L retained EE well over the tested period, showing only a moderate decline by day 21 (from ~80% to ~70%). In contrast, storage at 25 °C led to a much faster loss of EE, decreasing to ~20% by day 21, which is consistent with accelerated PAH leakage/volatilization at elevated temperature. These results support refrigerated storage for PAH-L and further motivate gel incorporation to improve practical topical handling and retention.

2.1.6. Analysis of Fourier Transform Infrared Spectroscopy

FTIR spectra of PAH, PAH-L, PAH-L-G, and the carbomer hydrogel are shown in Figure 2A. Free PAH exhibited a characteristic aldehyde C=O stretching band in the 1670–1730 cm^−1^ region, a C=C stretching band around ~1640 cm^−1^, and aliphatic C–H stretching vibrations at approximately 2960–2850 cm^−1^. The lipid nanoparticle matrix showed prominent CH_2_ stretching bands at ~2920 and ~2850 cm^−1^ and an ester carbonyl absorption near ~1730–1740 cm^−1^. In the spectrum of PAH-L, the characteristic PAH carbonyl-related band displayed reduced intensity and a slight shift relative to free PAH, while the lipid-associated CH_2_ and ester C=O bands remained clearly observable. No additional absorption peaks were detected in PAH-L compared with the individual components. The carbomer hydrogel exhibited a broad O–H stretching band in the 3200–3600 cm^−1^ region and a strong C=O/COO^−^-related absorption between ~1700 and 1550 cm^−1^. After incorporation of PAH-L into the hydrogel (PAH-L-G), the O–H stretching region became broader, and changes in band shape and intensity were observed in the carbonyl/carboxylate region compared with the carbomer hydrogel alone. The main characteristic bands of PAH and the lipid matrix were retained in the PAH-L-G spectrum. Overall, the FTIR spectra of PAH-L and PAH-L-G showed the coexistence of characteristic bands from PAH, lipid components, and the hydrogel matrix, with no appearance of new absorption bands. FTIR spectra of pure lecithin and cholesterol were not acquired in the present study; therefore, assignments of characteristic lipid bands (e.g., lecithin P=O stretching around ~1240 cm^−1^ and cholesterol broad O–H stretching around ~3400 cm^−1^) were supported by the literature and are provided here to aid interpretation [23,24]. This is listed as a limitation and will be addressed in future work.

2.1.7. In Vitro Release Effect and Swelling Ratio of PAH-L-G

The degree of release of a hydrogel is the rate and amount of drug or active ingredient loaded in a hydrogel that is released within a certain period of time. Release profile is one of the significant performance indicators of hydrogels as drug delivery systems. Dialysis release testing in PBS (pH 7.4) showed in Figure 2B. The free PAH released rapidly (62.3% at 1 h; 84.1% at 12 h; 87.4% at 48 h). Nanoencapsulation reduced the initial burst (12 h: 67.9%; 48 h: 76.9%), and incorporation into carbomer further slowed diffusion (12 h: 53.8%; 48 h: 69.4%). The stepwise reduction in release rate supports the intended nano-in-hydrogel design for sustained topical exposure. PAH-L-G absorbed PBS quickly and reached an equilibrium swelling ratio of ~500% within 120 min (Figure 2C), after which the mass increase plateaued. This rapid, high swelling is consistent with the porous microstructure and suggests good capacity for exudate management.

2.2. Hemolytic Analysis of PAH-L-G

Hemolysis testing provides an initial screen for blood compatibility. As shown in Figure 3A,B, erythrocytes incubated with Car-G, PAH, PAH-L, or PAH-L-G did not show visible hemoglobin release, and quantified hemolysis remained below 5% for all formulations—substantially lower than the water positive control. These results indicate acceptable hemocompatibility of the carrier matrix and the PAH nano-in-hydrogel system.

2.3. Cytotoxicity Assay Analysis

Cytocompatibility was evaluated using L929 cells. As shown in Figure 3C, all tested formulations maintained high cell viability, with values close to 100% at the most favorable condition and remaining above 85% even at the lowest point across groups, supporting good in vitro biocompatibility.

2.4. Analysis of the Antioxidant Activity

Reactive oxygen species are elevated in inflamed wounds and can delay repair when excessive [20,21]. We therefore measured radical-scavenging capacity using DPPH and ABTS assays. PAH-L-G showed strong DPPH scavenging at an equivalent concentration of 0.1 mg/mL (78.7%), exceeding free PAH (62.3%) (Figure 4A), whereas the blank gel (Car-G) showed negligible activity. Scavenging increased with concentration, and ABTS results followed the same trend (Figure 4B). The improved performance of PAH-L-G relative to PAH suggests that nanoencapsulation and hydrogel presentation can enhance the apparent antioxidant activity, likely by improving dispersion and stabilizing PAH during the assay.

2.5. The Wound Healing Properties of PAH-L-G

2.5.1. Analysis of Wound Healing and Determination of the Healing Rate

A full-thickness dorsal wound model was used to evaluate healing efficacy in vivo. Rats were assigned to blank, Car-G, or PAH-L-G groups and wounds were photographed on days 0, 3, 7, and 14 (Figure 4C). From day 7 onward, PAH-L-G-treated wounds contracted more rapidly than the blank and Car-G controls. By day 14, wounds in the PAH-L-G group were nearly closed with minimal residual defect, whereas visible unhealed areas remained in the blank group; Car-G showed an intermediate effect. Quantification by ImageJ software (version 1.54d, National Institutes of Health, Bethesda, MD, USA)confirmed a consistently higher closure rate in the PAH-L-G group over the observation period (Figure 4D).

2.5.2. Histopathological Studies

H&E staining further supported the macroscopic findings (Figure 5A–C). In the blank group, the wound bed still showed immature granulation tissue with disorganized collagen and persistent inflammatory infiltration. Car-G treatment improved tissue continuity to some extent, but epidermal coverage and dermal remodeling remained incomplete. In contrast, PAH-L-G promoted more continuous re-epithelialization, denser and organized granulation tissue, and reduced inflammatory cell presence, indicating accelerated regeneration.

3. Discussion

This work proposes a nano-in-hydrogel delivery strategy to improve the practical applicability of perillaldehyde (PAH) for wound treatment, where volatility, chemical instability, and uncontrolled exposure peaks can limit topical performance. By loading PAH into lipid nanoparticles and immobilizing them within a carbomer hydrogel, nanoscale stabilization is combined with prolonged residence at the wound surface, aiming to preserve PAH at the application site while smoothing local exposure.

The physicochemical characterization supports the feasibility of this hierarchical design. PAH-L exhibited a narrow size distribution and a pronounced negative zeta-potential, consistent with colloidal stability, while TEM confirmed nanoscale vesicular morphology suitable for solubilizing hydrophobic PAH. The negative surface potential likely arises from minor anionic lipid fractions and free fatty acids present in practical lecithin, together with anion/hydroxyl adsorption from the aqueous phase; after gel incorporation, the anionic carbomer network can further shape the interfacial charge environment and reduce nanoparticle mobility within the matrix.

Storage chemistry and zeta-potential behavior. The temperature-dependent decline in encapsulation efficiency indicates that multiple time-dependent processes may occur during storage [25]. In addition to increased membrane fluidity at higher temperature (facilitating PAH diffusion and leakage), chemical changes may contribute, including volatilization-driven loss, oxidation of the aldehyde (PAH) to more polar products, and lipid hydrolysis/oxidation that generates additional anionic species (e.g., free fatty acids) [26]. These processes would be expected to manifest as concurrent changes in particle size/PDI and zeta-potential: for example, hydrolysis could make the surface potential more negative, whereas aggregation and ion adsorption often reduce the magnitude of zeta-potential toward neutrality. In the present study, stability was primarily tracked by encapsulation efficiency; time-resolved DLS/zeta-potential and chemical profiling (HPLC chromatographic purity, peroxide value/TBARS for lipid oxidation) were not fully established. We therefore acknowledge this as an important limitation and recommend systematic monitoring across storage time points to better connect chemical stability, colloidal stability, and payload retention.

Release kinetics and mechanistic interpretation. The release results support the intended nano-in-hydrogel control of PAH exposure: nanoencapsulation reduces burst release relative to free PAH, and carbomer incorporation adds a second diffusion barrier that further slows cumulative release at physiological pH. To deepen mechanistic understanding, the release profiles can be fitted to classical kinetic models (zero-order, first-order, Higuchi, and Korsmeyer-Peppas). In our fitting, the Higuchi model provided the best linearity, suggesting diffusion-dominated release from the lipid/hydrogel matrix, and early-time Korsmeyer-Peppas analysis indicated Fickian diffusion (n ~0.31–0.33). This modeling-based interpretation is consistent with a two-stage diffusion pathway (lipid compartment → hydrogel network → external medium) and helps rationalize the stepwise reduction in release rate observed for PAH-L-G compared with PAH-L.

Why be higher despite lower dialysis release? Although PAH-L-G released less PAH across the dialysis membrane than free PAH, it exhibited higher DPPH/ABTS radical-scavenging activity. This apparent discrepancy is mechanistically plausible because dialysis release and antioxidant assays probe different phenomena. Dialysis reflects long-term diffusion into an external sink under membrane constraints, whereas DPPH/ABTS assays reflect short-term radical reactions in which formulations (or extracts) are directly mixed at the same nominal PAH dose. Under assay conditions, the effective availability of PAH depends not only on cumulative release into bulk solution but also on PAH stability and dispersion during the reaction window. Encapsulation and gel incorporation can reduce volatilization and oxidative loss of PAH during handling, improve its dispersion in aqueous media, and maintain a sustained local supply at the lipid-water interface where radicals are encountered. Together, these factors can yield higher apparent scavenging activity even when dialysis-measured release is slower.

From a formulation perspective, lipid nanoparticles offer practical advantages over other PAH delivery strategies. Cyclodextrin inclusion complexes can reduce volatility but may dissociate on wet wound surfaces, while emulsification-based systems often require higher surfactant loads that may be undesirable for compromised skin [27,28]. Polymeric carriers can provide prolonged release but may involve more complex processing or concerns about residual solvents. In contrast, lipid systems are composed of pharmaceutically accepted materials and provide a balance between PAH solubilization, environmental shielding, and controllable diffusion, supporting translational potential for topical use [29,30].

The carbomer hydrogel matrix further improves wound-relevant performance [31]. Carbomer gels are easy to spread, conform to irregular wound topography, and increase residence time under exudate and motion. Immobilizing nanoparticles within the hydrogel restricts their mobility and effectively increases the diffusion path length, attenuating burst exposure. Moreover, rapid, high swelling and the porous microstructure support exudate uptake and maintenance of a hydrated microenvironment, which is conducive to cell migration and tissue regeneration.

Biological mechanisms of PAH in wound repair. The wound-healing benefit of PAH is most plausibly multifactorial, with antimicrobial, anti-inflammatory, and antioxidant actions acting in concert across healing phases [2]. Antimicrobial activity can reduce wound bioburden and thereby alleviate prolonged inflammation; anti-inflammatory effects may dampen excessive cytokine signaling and support timely transition from the inflammatory to the proliferative phase; and antioxidant activity can limit excessive reactive oxygen species that otherwise delay re-epithelialization and remodeling [32]. Our in vitro radical-scavenging results and the in vivo acceleration of closure and improved histological organization are consistent with such a combined mechanism. Whether PAH also exerts direct pro-angiogenic actions remains to be clarified, as vascular markers were not measured here. Future studies should incorporate mechanistic readouts such as bacterial burden assays, inflammatory/oxidative markers (e.g., TNF-a/IL-6, MDA/SOD), and angiogenesis markers (e.g., CD31/VEGF staining and microvessel density) to more directly link controlled delivery to biological pathways.

Overall, these additional considerations strengthen the interpretation of PAH-L-G as a delivery-engineered wound dressing. At the same time, further work is warranted to (a) fully characterize storage-associated chemical and colloidal changes (including time-resolved zeta potential analysis), (b) expand release studies to wound-relevant pH conditions, (c) validate biological mechanisms in more challenging models (e.g., infected or chronic wounds), and (d) include parallel FTIR spectra of the raw materials (e.g., lecithin and cholesterol) to further substantiate peak assignments and strengthen spectral interpretation.

4. Materials and Methods

4.1. Materials

4.1.1. Experimental Material

Perillaldehyde (PAH), carbomer 940, lecithin, cholesterol and butylated hydroxytoluene (BHT) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). DAPIwas obtained from Solarbio Life Sciences (Beijing, China). and the CCK-8 kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). All reagents were of analytical grade unless otherwise specified. L929 fibroblasts (NCTC clone 929, L cell, L-929) were obtained from Wuhan Procell Life Science & Technology Co., Ltd. (Wuhan, China). This cell line corresponds to the Cellosaurus database entry CVCL_0462 and was authenticated by the supplier prior to use. All solutions were prepared using deionized water and filtered as required.

4.1.2. Experimental Animals

Male Sprague–Dawley rats (8 weeks old, 200–220 g) were purchased from Chengdu Duoxi Laboratory Animal (Chengdu, China; license SCXK (Chuan) 2020-0030). All procedures followed the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Southwest Minzu University (Approval No. 2025-01098).

4.2. Methods

4.2.1. Preparation of Perillaldehyde-Loaded Lipid Nanoparticles (PAH-L)

PAH-Loaded lipid nanoparticles (PAH-L) were prepared by thin-film hydration. Lecithin (40 mg; soy lecithin), cholesterol (10 mg), and PAH (8 mg) were dissolved in chloroform (10 mL) in a round-bottom flask. Solvent was removed by rotary evaporation under reduced pressure (45 °C, ~80 mbar, 60 rpm) to form a uniform lipid film. The film was hydrated with pre-warmed PBS (10 mL, 45 °C) while rotating in a 45 °C water bath until fully detached, then sonicated for 25 min. The suspension was passed through a 0.22 μm membrane to obtain PAH-L. Blank nanoparticles were prepared identically without PAH.

4.2.2. Preparation of Perillaldehyde-Loaded Lipid Nanoparticle Hydrogels

Carbomer gel was prepared by dispersing carbomer 940 (0.5 g) in ultrapure water (20 mL) and allowing it to hydrate overnight at 4 °C. The dispersion was neutralized dropwise with triethanolamine to a near-neutral pH to yield a clear carbomer hydrogel (Car-G). PAH-L was then blended into Car-G under gentle stirring to produce the PAH-L oaded lipid nanoparticle hydrogel (PAH-L-G). Unless otherwise stated, PAH-L dispersion (10 mL, containing 0.8 mg/mL PAH) was mixed with Car-G (20 g) at a 1:2 (w/w) ratio, yielding a final PAH concentration of 0.27 mg/g in PAH-L-G. For clarity, the formulations used throughout the study are defined as follows: Car-G, carbomer hydrogel without PAH or nanoparticles; PAH, free PAH (dissolved in the same solvent system used for analytical assays) without carrier; PAH-L, PAH-Loaded lipid nanoparticles dispersed in water; Blank-L, lipid nanoparticles without PAH; and PAH-L-G, the carbomer hydrogel containing PAH-L.

4.2.3. Characterization of Perillaldehyde Lipid Nanoparticles and Hydrogels

Particle Size, Zeta Potential and Polydispersity Index of PAH-L

Freshly prepared PAH-L was diluted 1:10 in PBS, and the hydrodynamic diameter, polydispersity index (PDI), and ζ-potential were measured using a Malvern particle size analyzer (Malvern, UK).

The Loading Capacity and Encapsulation Efficiency of the Lipid Nanoparticles

Free (non-encapsulated) PAH was separated by ultrafiltration. Briefly, 0.5 mL of PAH-L was loaded into an ultrafiltration tube and centrifuged at 20,000 rpm for 10 min. The filtrate was brought to 10 mL with methanol, ultrasonicated for 30 min to disrupt residual assemblies, filtered (0.22 μm), and analyzed by HPLC to quantify free PAH (W_1_). Total PAH (W_2_) was determined by diluting 0.5 mL of PAH-L to 10 mL with methanol, ultrasonication, filtration, and HPLC analysis. Encapsulation efficiency (EE) and loading capacity (LC) were calculated using Equations (1) and (2). HPLC quantification of PAH was carried out using a reversed-phase C18 column (4.6 × 250 mm, 5 μm) maintained at 30 °C. The mobile phase consisted of acetonitrile (A) and water (B) delivered in isocratic mode (A/B = 70/30, v/v) at 1.0 mL·min^−1^. The detection wavelength was set at 230 nm and the injection volume was 10 μL. PAH was quantified using an external calibration curve prepared in methanol over 0.5–200 μg·mL^−1^ (R^2^ ≥ 0.999). Method precision was assessed by intra- and inter-day repeatability (RSD < 2.0%), and accuracy was evaluated by recovery tests (98–102%). The limits of detection and quantification were estimated as signal-to-noise ratios of 3 and 10, respectively.

$$[eqn]$$ $$[eqn]$$

where W_1_ is the amount of free (non-encapsulated) PAH in the filtrate, W_2_ is the total amount of PAH in PAH-L (encapsulated + free) determined after complete disruption of nanoparticles in methanol, and (W_2_ − W_1_) represents the encapsulated PAH. M is the weighed lipid input (lecithin + cholesterol) used to prepare the nanoparticles. Because lipid loss during film formation, hydration, and transfer was minimal in our process, using the weighed lipid input provides a conservative estimate of LC. If desired, LC can also be recalculated using recovered lipid mass determined by gravimetry.

Morphological Analysis Based on Scanning Electron Microscope

For TEM imaging, 30 μL of PAH-L dispersion was dropped onto a carbon-coated copper grid and allowed to adsorb for 5–6 min. Excess liquid was wicked off, the grid was negatively stained with 2% phosphotungstic acid (pH 6.5) for 3 min, air-dried, and imaged by TEM. For SEM observation, PAH-L-G was frozen at −80 °C for 12 h and lyophilized. Dried samples were fractured in liquid nitrogen, mounted on stubs, sputter-coated with gold, and examined by SEM.

Stability of PAH-L or PAH-L-G

To assess storage stability, PAH-L was kept at 4 °C or 25 °C and visually inspected; encapsulation efficiency was measured at predetermined time points (days 0, 3, 7, 14, 21, and 30). The stability of PAH-L-G was evaluated by (i) centrifugation (3500 r/min, 30 min), (ii) freeze exposure (−80 °C overnight), (iii) heat challenge (60 °C water bath, 5 h), and (iv) storage at 4 °C or 25 °C for 30 days; macroscopic appearance (phase separation/gel integrity) was recorded on days 0, 7, 14, 21, and 30.

Fourier Transform Infrared (FTIR) Spectroscopy of PAH-L and PAH-L-G

Samples (PAH-L, PAH-L-G, PAH, and carbomer 940) were lyophilized and analyzed by FTIR to probe component interactions. Each powder was mixed with KBr (1:50, w/w), ground thoroughly, and pressed into pellets. Spectra were recorded from 400 to 4000 cm^−1^ (32 scans; 4 cm^−1^ resolution) using KBr as the background.

In Vitro Cumulative Release

For the in vitro release study, PAH, PAH-L, or PAH-L-G was sealed in a dialysis bag (molecular weight cut-off, MWCO = 10 kDa) and immersed in 30 mL of PBS (pH 7.4) as the release medium. The system was maintained at 37 °C with continuous shaking at 100 r/min. At predetermined time intervals (1, 2, 4, 6, 8, 10, 12, 24, and 48 h), 2 mL of the release medium was withdrawn and immediately replaced with an equal volume of fresh PBS to maintain sink conditions. The concentration of PAH in the collected samples was determined by HPLC, and the cumulative release was calculated based on the corresponding calibration curve.

Swelling Ratio of PAH-L-G

Swelling behavior was determined gravimetrically. PAH-L-G was frozen (−80 °C) and lyophilized to obtain dry gel blocks, which were weighed (M_0_) and immersed in PBS. At 10 intervals (up to 120 min), samples were removed, gently blotted to remove surface liquid, and weighed (M_n_). The swelling ratio was calculated using the following equation. Each experiment was performed in triplicate (n = 3 independent gel samples).

$$[eqn]$$

4.2.4. The Biocompatibility of PAH-L-G

Hemolysis

Fresh rat blood was centrifuged (2000 rpm, 10 min) to separate plasma and erythrocytes. Red blood cells were washed repeatedly with sterile PBS until the supernatant became clear, then diluted to a 5% (v/v) RBC suspension in PBS. Aliquots (2 mL) were incubated with test samples at 37 °C (100 rpm) for 2 h, then centrifuged (3000 rpm, 20 min). Supernatants (100 μL) were transferred to 96-well plates. PBS and purified water served as negative and positive controls, respectively. Absorbance was read at 545 nm and hemolysis was calculated as:

$$[eqn]$$

where A_s_, A_n_, and A_p_ are the absorbance values of the sample, negative control (PBS), and positive control (water), respectively. Measurements were performed in triplicate.

CCK-8 Cytotoxicity Assay

Cytocompatibility was evaluated using a CCK-8 assay with L929 fibroblasts and HaCaT keratinocytes. Hydrogel extracts were prepared by incubating Car-G, PAH-L, or PAH-L-G (4 mg) in MEM (10 mL; 37 °C, 100 rpm) for 24 h, followed by dilution to 0.25–2 mg/mL. Cells were seeded in 96-well plates (5 × 10^3^ cells/well) and allowed to attach for 24 h. Culture medium was replaced with 100 μL of each extract dilution and incubated for 24 h. CCK-8 reagent (10 μL) was then added and after 1–4 h, the absorbance at 450 nm was recorded. Cell viability was expressed relative to untreated controls; IC50 values were calculated in SPSS software (version 26.0, IBM Corp., Armonk, NY, USA).where applicable.

4.2.5. Antioxidant Experiments on Hydrogels

DPPH Radical Scavenging Rate

DPPH radical scavenging was used to estimate antioxidant capacity. A DPPH ethanol solution was prepared (2 mg/50 mL) and equilibrated in the dark for 30 min. PAH-L-G or Car-G was incubated in PBS (37 °C, 100 r/min, 30 min) to obtain extracts at 2, 4, 8, and 10 mg/mL. Extracts were mixed with DPPH solution (1:5, v/v) in 96-well plates and absorbance at 517 nm was measured. Each condition was tested in triplicate.

ABTS Radical Scavenging Rate

ABTS scavenging was measured using a standard ABTS/persulfate system. ABTS (7 mM) was reacted with potassium persulfate (2.45 mM) in water and kept in the dark for 12 h to generate the radical cation. The working solution was diluted to A734 = 0.70 ± 0.02. Hydrogel extracts (prepared as above) were mixed with ABTS working solution (1:5, v/v), incubated for 10 min at room temperature, and an absorbance decrease at 734 nm was recorded.

4.2.6. Testing the Wound Healing Properties of PAH-L-G

Wound Modeling

Rats were anesthetized, the dorsal area was shaved and depilated, and the skin was disinfected. Full-thickness circular wounds (10 mm diameter) were created on the back using a biopsy punch. The day of surgery was defined as day 0. Wounds were photographed on days 0, 3, 7, and 14.

Experimental Grouping and Drug Administration

Twenty-four rats were randomized into three groups (n = 8 each): untreated control (blank), carbomer hydrogel (Car-G), and PAH-L-G. Car-G or PAH-L-G was applied topically to the wound area in the corresponding groups.

Analysis of Wound Healing and Determination of the Healing Rate

Digital images were analyzed in ImageJ to quantify wound area. Wound closure (%) was calculated as:

$$[eqn]$$

where M_0_ is the initial wound area (day 0) and M_t_ is the area at the indicated time point.

Pathologic Tissue Embedding

On day 14, rats were euthanized and wound tissues with surrounding skin were excised and fixed in 4% paraformaldehyde. Samples were dehydrated, paraffin-embedded, and sectioned (4 μm). Sections were stained with hematoxylin and eosin (H&E) and examined microscopically to assess re-epithelialization, granulation tissue formation, inflammatory infiltration, and neovascularization.

Randomization, Blinding, and Animal Monitoring

Rats were allocated to experimental groups using a random number table. Outcome assessment, including wound area quantification using ImageJ and histological evaluation, was performed by investigators blinded to group allocation. No animals were excluded from the analysis. After surgery, animals received appropriate postoperative analgesia and were monitored daily for general condition, wound appearance, and signs of distress throughout the study.

4.3. Statistic Analysis

Unless otherwise stated, experiments were performed in triplicate. Results are presented as mean ± SD. For wound closure over time (Figure 4D), data were analyzed using two-way repeated-measures ANOVA (treatment × time) with interaction, followed by Sidak’s (or Tukey’s) multiple comparisons to compare groups at each time point. For other single-factor comparisons, one-way ANOVA (with appropriate tests) was used. A p value < 0.05 was considered statistically significant.

5. Conclusions

In conclusion, we established a perillaldehyde nano-in-hydrogel composite (PAH-L-G) by integrating PAH-Loaded lipid nanoparticles with a carbomer hydrogel. This formulation improved handling and stability of a volatile phytochemical, provided rapid swelling and sustained release, and showed favorable hemo- and cytocompatibility. In vivo, PAH-L-G accelerated wound closure and improved histological repair without obvious irritation, demonstrating that formulation engineering can translate PAH into a safer and more effective topical component for wound-care product development.