Transfersomal delivery of Centella asiatica promotes efficient excision wound healing in rats

Abstract

This study presents the development and evaluation of Centella Asiatica (CA)-loaded transfersomes (CANP) as a novel nanocarrier for transdermal delivery. CANP were prepared using an oil-in-water emulsion method, producing nanoparticles with a size of 135.22 ± 4.80 nm, a polydispersity index of 0.22 ± 0.01, and a zeta potential of −26.13 ± 0.58 mV. Stability tests confirmed consistent physicochemical properties under various storage conditions, with encapsulation efficiencies above 68% for madecassoside and 89% for asiaticoside. Ex vivo permeation studies using porcine skin showed significantly improved skin penetration compared to liposomes and niosomes, attributed to the high deformability index (1.31 ± 0.21 mg/cm²). In vitro cytotoxicity assays indicated cell viability above 80% across concentrations. Functionally, CANP reduced nitric oxide production in LPS-stimulated RAW 264.7 cells, demonstrating superior anti-inflammatory effects over native CA. CANP also promoted fibroblast proliferation and collagen production by 91.9% and 213.3% at days 7 and 14, respectively, exceeding vitamin C. Wound healing assays confirmed enhanced fibroblast migration and closure rates similar to fibroblast growth factor. In vivo, CANP hydrogels accelerated healing, with early fibroblast activity and collagen deposition between days 7–14, supporting epithelial regeneration over 21 days. Compared to controls, they more effectively reduced inflammation and increased dermal growth factor expression. These findings support CANP as a promising transdermal nanocarrier with enhanced skin penetration, anti-inflammatory activity, and regenerative potential. Encapsulating CA into transfersomes boosts its therapeutic efficacy, making it a strong candidate for advanced dermal applications.

1. Introduction

Wound management is a critical medical procedure aimed at alleviating intense pain, promoting recovery, and preventing severe health complications. Despite advances in treatment, the healing process of skin wounds is often hindered by numerous underlying factors that can delay recovery or, in some cases, prevent complete healing altogether.

The wound healing process generally comprises three interrelated stages: hemostasis/inflammation, proliferation, and remodeling (Gonzalez et al. 2016). During wound healing, the process is governed by a complex signaling network that involves growth factors, cytokines, and chemokines (Fernández-Guarino et al. 2023). Coagulation addresses irreversible cells and tissue necrosis caused by injury. Platelet-released cytokines recruit neutrophils and monocyte-derived macrophages to the wound. Macrophages clear necrotic debris and release growth factors, e.g. transforming growth factor-beta (TGF-β), transitioning the process to fibroblast proliferation and extracellular matrix (ECM) synthesis. In the remodeling phase, apoptosis of myofibroblasts and macrophages, along with the activity of matrix metalloproteinases and their inhibitors, restores tissue texture and strength. Furthermore, key factors, including the EGF family, TGF-β family, fibroblast growth factor (FGF) family, and vascular endothelial growth factor (VEGF), support ECM synthesis, deposition, and organization, including collagen, elastin, and glycosaminoglycans. Inflammatory mediators like interleukin-6 (IL-6) and tumor necrosis factor-alpha are essential in the inflammatory phase, while re-epithelialization restores skin integrity (Wilkinson and Hardman 2020). Imbalances in these processes can result in complications such as scarring or keloid formation (Lin and Lai 2024).

As a result, studies in wound management have increasingly focused on developing interventions and pharmacological agents to accelerate the healing process. Prompt and efficient wound treatment reduces patient suffering, lowers hospitalization costs, and mitigates the risk of severe complications (Lee et al. 2024). However, conventional therapeutic agents frequently show limited efficacy in addressing certain types of wounds, underscoring the need for innovative and cost-effective alternatives.

Among these alternatives, medicinal herbs have gained significant attention for long-standing use in traditional medicine to treat various ailments, including wounds (Xu et al. 2023). The rising incidence of skin wound cases in hospitals highlights the need for effective and affordable treatments. Dependence on conventional antimicrobials can be costly and occasionally less effective, creating an urgent demand for alternative solutions (Chinemerem Nwobodo et al. 2022; Aghamohammad and Rohani 2023).

Centella asiatica (CA), a perennial plant from the Apiaceae family, is a promising candidate widely recognized in traditional medicine for its wound-healing and skin disease treatment properties (Gohil et al. 2010; Bylka et al. 2014). CA has been traditionally used for various skin conditions, including wounds, scars, eczema, and psoriasis, due to its ability to accelerate wound contraction by stimulating fibronectin and collagen synthesis (Diniz et al. 2023). Previous studies have highlighted the pharmacological efficacy of CA in wound healing, primarily due to its active triterpenoid compounds—asiatic acid, madecassic acid, asiaticoside, and madecassoside—which stimulate collagen synthesis and promote tissue repair (Sengupta et al. 2021; Diniz et al. 2023). While most studies focus on the effects of individual compounds or combinations of these active triterpenoids, this study incorporates all four compounds with an innovative drug delivery system to enhance therapeutic potential.

To enhance the wound-healing potential of CA, the extract can be encapsulated in transfersomes for enhanced transdermal delivery of nanoparticles (CANP). Transfersomes are lipid-based nanocarriers composed of phospholipids, nonionic surfactants, and edge activators, which confer superior deformability and stability compared to conventional liposomes and niosomes (Rai et al. 2017; Malathi et al. 2021). These carriers are capable of encapsulating both hydrophilic and lipophilic agents, enabling better skin absorption and penetration (Riccardi et al. 2024).

This study aimed to develop a gel formulation containing CA transfersomes (CANP-Gel) and evaluate its efficacy in promoting wound healing using in vitro and in vivo excision models. It was hypothesized that CANP and CANP-Gel would enhance wound healing, accelerating closure, improving tissue regeneration, and reducing inflammation. This delivery system would improve CA absorption, promoting faster and more effective healing and offering a promising strategy for improved wound management.

2. Materials and methods

2.1. Preparation of formulated Centella asiatica nanoparticle (CANP) and gel containing CANP (CANP-Gel)

Centella Asiatica was cultivated in Nakhon Pathom, Thailand. After harvesting, the leaves were air-dried and subjected to extraction. Fresh Centella asiatica was oven-dried, ground into powder, and extracted with methanol under controlled temperature and time conditions. The extract was filtered, diluted, and mixed with an internal standard, then purified via solid-phase extraction. After washing and elution, samples were analyzed by UHPLC, as shown in Supplementary Table S1. Key triterpenes—including asiaticoside, madecassoside, asiatic acid, and madecassic acid—were quantified by comparing peak areas with reference standards (Supplementary Figure S1). A high-purity Centella asiatica extract (88.25%) was obtained using an in-house solvent extraction and fractional recrystallization method. The purified bright yellow powder contained madecassoside (45.39%), asiaticoside (38.34%), madecassic acid (2.34%), and asiatic acid (2.18%), respectively.

Soybean lecithin was purchased from Berli Jucker Public Company Limited (BJC) (Bangkok, Thailand). Cholesterol was purchased from The Sun Chemical Co.,LTD. (Bangkok, Thailand). Span 80 and Tween 80 were purchased from Croda Singaproe Pte Ltd. (Seraya Avenue, Singapore). Tocopherol acetate was purchased from Namsiang Co., Ltd. (Bangkok, Thailand). Propylene glycol was purchased from S. Tong Chemicals Co., Ltd. (Nonthaburi, Thailand). Microcare PHC, a combination of phenoxyethanol and chlorophenesin, was sourced from Chemico Inter Corporation Co., Ltd. (Bangkok, Thailand).

The CANP was synthesized according to the specific conditions and chemical compositions as shown in Table 1, consisting of lecithin: cholesterol: nonionic surfactants: edge activator; 2:1:2:1.6 in weight ratio. Briefly, the oil phase and water phase were heated at 80 °C–85 °C and homogenized at 8,000–10,000 rpm for 5–10 min using a high-speed homogenizer (Heidolph Silent Crusher M, Kenilworth, NJ, USA). The mixture was cooled to 50 °C and homogenized with an edge activator and preservative for another 10 min. Liposome and niosome formulations encapsulating CA extract were also prepared as controls. The stability of the formulated CANPs was evaluated at 30 °C and 40 °C for 6 months, following ASEAN guidelines for safety and nano-specific regulations on the stability studies and shelf-life assessment of traditional medicines. Key parameters, including particle size, polydispersity index (PdI), and zeta potential, were analyzed using dynamic light scattering to assess the stability and performance of the formulations. The pH and encapsulation efficiency percentages for madecassoside and asiaticoside were also evaluated.

Table 1.

Chemical components of formulated transfersomes encapsulating Centella asiatica.

Components (weight ratio)	Transfersome	Liposome	Niosome
Part A: oil phase
Cholesterol	1	1	1
Sorbitan oleate	1		1
Phospholipid: soybean lecithin	2	2
Propylene glycol	4	4	4
Part B: water phase
Centella asiatica extract	0.8	0.8	0.8
Polysorbate 80	1		1
Part C: Edge activator
Tocopherol acetate	1.6
Part D: preservatives	1.2	1.2	1.2

To formulate a minimum gel base containing CANPs, 1.2 g of Ammonium Acryloyldimethyltaurate/VP copolymer (aristoflex avc) was dispersed in 96.8 mg of deionized water using a magnetic stirrer till the proper clear gel formation was observed. Then, formulated CANPs were added into the pre-formed gel at 2% of the final concentration. The gel base with Blank and 50 ng FGF was crucial for in vitro wound healing, as it promotes the proliferation and migration of fibroblasts—the primary cells responsible for new tissue formation during healing by ECM and collagen components (Ornitz and Itoh 2001; Li et al. 2003). These were formulated as negative and positive controls, respectively.

2.2. The percentage of encapsulation efficiency (%EE)

The encapsulation or packaging efficiency of CANP was measured by High-Performance Liquid Chromatography (HPLC) using asiaticoside and madecassoside as standard markers. The 5 mg/mL of CANP were dissolved in 70% ethanol and centrifuged at 80,000 rpm for 2 hours to separate unencapsulated CA. After that, total CA content (upper part) and unencapsulated CA content (lower part) were qualified using Ultra HPLC.

$$\text{\% EE}=\frac{\text{Total CA content}-\text{Unencapsulated CA content}}{\text{Total CA content}}\mathrm{\times }\mathrm{ }100$$

2.3. Deformability index

The deformability study was performed using the extrusion method based on an adapted protocol from Modi and Bharadia (2023). Briefly, transfersome encapsulating CA was extruded through the filter membrane (pore size 50 nm), using a stainless steel holder. Niosomes and liposomes encapsulating CA were used as controls. The applied pressure was at 2.5 bar and the amount of vesicle suspension extruded within 5 min was measured and the deformative index of particle was calculated using an equation:

$$E=J\left[\frac{r_v}{r_p}\right]$$ 2

where

E is the elasticity of the vesicle membrane.

J is an extruded suspension amount.r_v is vesicle size.r_p is pore membrane diameter.

2.4. Ex vivo skin permeation

The permeation of nanoparticles through porcine skin was investigated and analyzed using fluorescent dye tracking and fluorescence microscopy (Olympus, UK). Fresh porcine skin was procured postmortem from a local slaughterhouse, authorized by the Provincial Livestock Office in Nakhon Pathom, Thailand, as a byproduct of the meat industry. No controls were imposed on rearing conditions to capture natural biological variability, enhancing the relevance to human heterogeneity. The method of porcine skin procurement was similar to that described in previous reports by Kong and Bhargava (2011) and Supanakorn et al. (2022). Briefly, skin was excised from the abdominal region of pigs weighing 20–30 kg. A portion of the freshly harvested skin was cut into 5 × 5 mm² pieces and immediately frozen at −80 °C for analysis. Before examination, the samples were thawed at room temperature for 1 hour to ensure complete melting without altering skin composition. The CANP, liposome encapsulating CA (CALP), and niosome encapsulating CA (CANI) were stained with 100 µg/mL tetramethylindocarbocyanine perchlorate (DiI dye). Unbound dye was removed through ultracentrifugation at 80,000 rpm for 4 hours. The stained particles were washed twice with phosphate-buffered saline (PBS), and their concentration was adjusted to 1 mg/mL to obtain stock solutions. To evaluate nanoparticle skin permeation, porcine skins were cut into 1.5 × 1.5 cm² pieces. A 100-µL of 1 mg/mL of CANP, CALP, and CANI was thoroughly applied to the surface of the cut skin. The treated skin was then placed into a 12-well transwell chamber with 0.4-µm pores (Costar, NY, USA), submerged in 200 µL of PBS (pH 7.4), and incubated for 4 hours. Following incubation, the skin samples were fixed, cryosectioned using a cryostat (Leica, Germany), and mounted onto glass slides. The basolateral medium was collected to quantify fluorescent dye concentrations using a fluorescence microplate reader at an excitation wavelength of 540 nm and an emission wavelength of 590 nm (Biotek synergy H1, USA). Skin permeability was calculated as a percentage relative to the initial fluorescence intensity.

2.5. Cell cytotoxicity assay

Ten thousand human dermal fibroblast cells were seeded into each well of 96 well-plates and cultured for 24 hours. Then, cells were treated with Blank, CANP and CA extract at different concentrations (0–250 µg/mL) for 24 hours. After that, 100 µL of 1 mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution was added into each well and incubated for 4 hours. Dimethyl sulfoxide was then used to dissolve formazan products, and the absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage using the following equation:

$$\text{\% Cell viability}=\frac{\text{Absorbance of treated cells}}{\text{Absorbance of untreated cells}}\mathrm{\times }100$$

2.6. Anti-inflammation assay (nitric oxide production)

RAW 264.7 macrophages; (mouse, product code ATCC-TIB-71, American Type Culture Collection (ATCC) company, Virginia, USA) were seeded at a density of 5 x 10⁴ cells/well in 48-well plates for 24 hours. After reaching 70%–80% confluence, cells were pretreated with 100 µg/mL of tested samples, including Blank (transfersome without extracts), CANP, CA extract, and 100 ng/mL dexamethasone as a positive control for 24 hours, and then stimulated with 0.1 µg/mL lipopolysaccharide (LPS) and incubated for an additional 24 hours. After treatment, the cells were harvested, and the supernatant was collected to measure nitrite production. Nitrite levels were determined by passing a mixture of nitrogen oxides (NO) into aqueous sodium hydroxide, followed by the addition of Griess reagent (Invitrogen, Singapore), according to the manufacturer’s protocol. Briefly, 150 μL of the cell supernatant was incubated with 150 μL Griess reagent for 15 min in the dark at room temperature. The absorbance was measured at 546 nm using a UV/Vis microplate reader. The fold changes in nitrite concentrations were calculated relative to untreated control.

2.7. Collagen production assay

A collagen production assay was performed in the same protocol by Eaknai et al. (2022). Briefly, human dermal fibroblast cells were seeded into 48 well-plates at 2.5 × 10⁴ cells/well and cultured for 24 hours. Then, cells were treated with 50 µg/mL of tested samples including Blank, CANP and CA extract, and additionally cultured for 7 and 14 days. After treatment, cells were washed thoroughly with PBS and fixed with 100 µL of 4% paraformaldehyde for 10 min. Cells were then washed twice with PBS. Produced collagen was stained with 100 µL of 0.1% direct red 80 solutions for 10 min. The unbound dye was removed by excessively washing with 0.01 N hydrochloric acid in 70% ethanol. Stained collagen was dissolved with 100 µL of 0.5 N sodium hydroxide and absorbance was measured at 540 nm using microplate reader. The collagen content was calculated as a percentage using the following equation:

$$\text{\% Collagen content}=\frac{\text{Absorbance of treated cells}}{\text{Absorbance of untreated cells}}\mathrm{\times } 100$$

2.8. Scratch wound healing assay

In vitro wound healing assay was performed to observe the directional cell migration. Human dermal fibroblast (ATCC number PCS-201-010, American Type Culture Collection (ATCC) Company, Virginia, USA) was seeded at 3 × 10⁵ cells/mL onto each chamber of silicon culture-insert 4 wells in dish 35 mm (ibidi, Germany) and cultured for 24 hours. After cultivation, culture-insert was removed, and cells were washed with PBS. Cells were then treated with 50 µg/mL of tested samples including Blank, CANP, CA extract, and vitamin C as a positive control. Additionally, vitamin C at 50 μg/mL was selected based on prior study (Bhubhanil et al. 2021), which demonstrated its potent antioxidant and anti-inflammatory effects in in vitro models at this concentration. Wound closure was photographed at 0, 24 and 48 hours to examine cell migration. The gap filled was examined by measuring changes in the wound area using ImageJ. Gap filled can be calculated as a percentage using the following equation:

$$\text{\% Gap filled}=\frac{{\mathrm{A}}_{t=0h\mathrm{ }}-A_{t=\mathit{\text{nh}}}}{A_{t=0h}\mathrm{ }}\text{×}100$$

When

A_t=0h is the wound area measured immediately after wounding.

A_t=nh is the wound area measured after wounding at each time point.

2.9. Animal

Twelve adult male Wistar rats (8 weeks old, weighing 185–210 g) were obtained from Nomura Siam International Co., Ltd. (Company registration number: 0105556040621, Bangkok, Thailand) and housed in standard-sized 24 × 36 × 24 inch polyvinyl acrylic plastic cages (2 rats/cage) at the Laboratory Animal Center, Thammasat University, Pathum Thani, Thailand (Animal supplier license number: B.2559/00023.001, certified by the Institute of Animals for Scientific Purposes Development, Thailand). All rats were kept at control temperature with a 12 hours:12 hours light/dark cycle and were received standard chow and distilled water ad libitum during the study. Following a 7-day acclimation period, rats were randomly divided into groups of four per time point and subjected to excision wounds, receiving standard treatment with either Blank hydrogel or the developed hydrogel for 21 days. Since wound healing response was optimal within 21 days, at each sampling time point (days 7, 14, and 21), rats were anesthetized using 5% isoflurane inhalation. The depth of anesthesia was assessed by checking reflexes and responses to touch on the foot or tail. Ethical approval All institutional and national guidelines, including those of the Institutional Animal Care and Use Committee of Thammasat University and the Ethical Principles and Guidelines for the Use of Animals by the National Research Council of Thailand, were strictly followed for the care and use of laboratory animals. Animal ethics approval was obtained for both rats and rabbits; however, this study focused on skin wound healing in rats, while rabbit irritation testing will be conducted in a future study.

2.10. Excision wound procedures

Rats were anesthetized with 5% isoflurane in an anesthesia induction chamber and maintained on a 3 L/min O₂ flowmeter using a mini mask of the R530 portable small animal anesthesia machine (RWD Life Science Co., Ltd., Guangdong, China). Then their dorsal skin was shaved using a mini-pet trimmer. Two circular surgical wounds (diameter: 10 mm) were created 2 cm apart at the dorsal thoracic level using aseptic surgical instruments, following the procedure outlined in previous studies (Bhubhanil et al. 2021; Lapmanee et al. 2024). Tramadol (VESNON-V 100, Buymed Thailand, Samut Prakan, Thailand) in sterile 0.9% saline was administered intraperitoneally to alleviate pain and tension following excision. Thereafter, the rats were housed individually with enrichment. The wound area was observed and cleaned on post-excision days (days 0, 7, 14 and 21). Rats were treated with Blank hydrogel and CANP-Gel for 21 days.

2.11. Measurement of wound contraction

Skin wounds were observed on days 0, 7, 14, and 21 by following the changes in wound area. The size of the wound was photographed, and calculated the percentage of wound contraction, taking starting size of the wound, 100 mm² as 100%, using the following formula:

$$\text{\% Wound contraction}=\frac{\text{Staring day wound size}-\text{Observed day wound size}}{\text{Starting day wound size}}\times 100$$

2.12. Collection of skin specimens

On days 7, 14, and 21 post-excisions and during treatment, 4 rats per group were sacrificed with overdose of isoflurane. The wound was excised and divided into two pieces. One piece of specimen was weighed and stored in liquid nitrogen for gene expression profiling of wound healing studies while the second specimen was fixed in 4% paraformaldehyde before embedding in paraffin for histological studies.

2.13. Histological analysis

Skin specimens collected on days 7, 14, and 21 were processed, and paraffin blocks were prepared. These blocks were sectioned at a thickness of 5 μm using a rotary microtome (Leica, Wetzlar, Germany) and mounted on clean glass slides. Sections were stained with Hematoxylin and Eosin (H&E) and Masson’s Trichrome (Sigma-Aldrich, St. Louis, MO, USA), then examined under a light microscope equipped with a Nikon DXM 1200 digital camera (Tokyo, Japan). Hematoxylin (deep blue-purple) stains nucleic acids and cell nuclei, while eosin (pink) nonspecifically stains proteins, highlighting the cytoplasm and extracellular matrix. Masson’s Trichrome (blue-green) selectively stains collagenous connective tissue fibers. Collagen intensity was quantified using ImageJ software. To assess cellular and vascular changes, ten random high-power fields (HPFs) at 400× magnification were selected per section, and stained cells and blood vessels were manually counted. Inflammatory responses were evaluated by quantifying mononuclear and polymorphonuclear leukocytes, along with markers of angiogenesis. Collagen deposition was further analyzed in ImageJ by calculating the ratio of blue-stained areas (hue: 155–175; saturation: 60–255; brightness: 0–255) within each HPF. Re-epithelialization was calculated using the following formula: (length of the newly formed epidermal layer/length of the wound between wound edges) × 100%. All histological evaluations were independently performed by two pathologists. For each specimen, three sections were randomly selected for analysis (Bhubhanil et al. 2021; Lapmanee et al. 2024).

2.14. Quantitative real-time PCR (qPCR)

The RNA from the skin from the healing wound area was collected and extracted using TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer’s protocol. The RNA was treated with DNase I (Thermo Fisher Scientific Inc, USA) to eliminate contamination with genomic DNA. The cDNA synthesis was made by reverse transcription using the High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, USA) following the manufacturer’s instructions and analyzed the expression of wound healing-related genes (i.e. Il6, Egf, Fgf2, Tgfb1, Col1a1, and Col3a1) using SYBR Green-based qPCR. The primers have been validated for specificity and efficiency by conventional qPCR, as previously described (Bhubhanil et al. 2021). The details of the primers used in this study are presented in Table 2. The relative mRNA expression was conducted by the CFX Manager^™ software (Bio-Rad Laboratories, Hercules, CA, USA) by performing the comparative Ct method. Beta-actin (Actb) was included as the reference gene for normalizing the target genes and for compensation of inter-PCR variation between each qPCR experiment. The expression level of each targeted gene is presented as a fold change relative compared to the level in the control group.

Table 2.

Primers sequences of Rattus norvegicus used in the qPCR study.

Gene	Primer sequence	Product	Access number
β-actin	F: 5′-CCCTGGCTCCTAGCACCAT-3′	80 bp	NM031144
(Actb)	R: 5′-GATAGAGCCACCAATCCACACA-3′
IL-6	F: 5′AACCTGAACCTTCCAAAGATGG-3′	168 bp	NM012589
(Il6)	R: 5′-TCTGGCTTGTTCCTCACTACT-3′
EGF	F: 5′-CTCAGGCCTCTGACTCCGAA-3′	93 bp	NM012842
(Egf)	R: 5′-ATGCCGACGAGTCTGAGTTG-3′
FGF-2	F: 5′-GATCCCAAGCGGCTCTACTG-3′	105 bp	NM019305
(Fgf2)	R: 5′-TAGTTTGACGTGTGGGTCGC-3′
TGF-β1	F: 5′-GGGCTACCATGCCAACTTCTG-3′	82 bp	NM021578
(Tfgb1)	R: 5′-GAGGGCAAGGACCTTGCTGTA-3′
Collagen 1 (Col1a1)	F: 5′-CATGTTCAGCTTTGTGGACCT-3′	94 bp	NM053304
	R: 5′-GCAGCTGACTTCAGGGATGT-3′
Collagen 3 (Col3a1)	F: 5′-GGGATCCAATGAGGGAGAAT-3′	128 bp	NM032085
	R: 5′-CCTTGCGTGTTTGATATT-3′

EGF: Epidermal growth factor; TGF-β1: Transforming growth factor beta 1; IL-6: Interleukin-6; F: Forward; R: Reverse.

2.15. Statistical analysis

Statistical analysis was performed using GraphPad Prism 10.0 (GraphPad Software, Inc., San Diego, CA). The results were expressed as mean ± standard error of the mean (SEM). The comparisons between groups were conducted using student’s-t test and/or between and within groups using one-analysis of variance (ANOVA) with post-hoc Tukey’s Honest test. All statistical tests were set at a significance level α of 0.05 (P < 0.05).

3. Results

3.1. Physicochemical and stability characterizations, transdermal delivery, cytotoxicity, cell proliferation, collagen production, and in vitro wound healing of formulated CANP

The CANP was formulated using oil in water emulsion method via high-speed homogenizer. The obtained particles exhibited spherical shapes with an initial size of 135.22 ± 4.80 nm, a PdI of 0.22 ± 0.01, and a zeta potential of −26.13 ± 0.58 (Figure 1A and Table 3). Liposomal and niosomal formulation of CA; CALP and CANI were also formulated as controls and their physiochemical characterizations were reported in Supplementary Table S2 and the hybrid composition of the developed transfersome was verified through melting temperature analysis using DSC, which revealed the thermal properties of both liposomes and niosomes (Supplementary Figure S2). Following the experiment aligned with ASEAN Guidelines on stability study and shelf-life of traditional medicines, our study demonstrated (Table 3) that CANP exhibits physiochemical stability, although a slight increase in the particle sizes was observed when stored at 40 °C compared to 30 °C. The pH value remained neutral, ranging from 7.1 to 7.8 throughout the experimental period, which supports its suitability as an active ingredient in various applications. The %EE of CANP was measured and monitored using the UHPLC technique. Over the six-month experimental period, the levels of both standard markers, made cassoside and asiaticoside, remained stable, averaging approximately 70% and 90%, respectively (Table 3). Considering the high %EE along with the characteristics of transfersomes, this synergy is expected to enhance the efficacy of CANP.

Figure 1.

Effects of formulated transfersomes encapsulating Centella asiatica on transdermal delivery were evaluated by the permeation of nanoparticles across porcine skin using fluorescent dye tracking. (A) transmission electron microscopy (TEM) photographs show a spherical individual transfersome indicating a core-shell structure (a) and a group of transfersomes with a vesicular structure, typical of liposomes of encapsulating Centella asiatica, CANP) (b), (B) The photographs, stained with non-fluorescent dye (buffer, BF), tetramethylindocarbocyanine perchlorate (DiI dye), or hematoxylin and eosin (H&E), show transfersomes encapsulating Centella asiatica (CANP), niosomes encapsulating Centella asiatica (CANI) and liposomes encapsulating Centella asiatica (CALP). Bar scale = 200 µm. (C) Skin permeability is expressed as mean ± SEM (n = 6) and calculated as a percentage relative to the initial fluorescence intensity.

Table 3.

Physicochemical properties and stability of formulated transfersomes encapsulating Centella asiatica at various time points and temperatures.

Parameters	Initial	1 month		2 months		3 months		6 months
		30 °C	40 °C	30 °C	40 °C	30 °C	40 °C	30 °C	40 °C
Size (nm)	135.22 ± 4.80	124.84 ± 2.02	125.43 ± 1.74	127.54 ± 3.10	134.19 ± 2.87	124.19 ± 1.77	130.01 ± 1.44	129.51 ± 3.33	150.30 ± 2.45
PdI	0.22 ± 0.01	0.24 ± 0.01	0.27 ± 0.01	0.27 ± 0.01	0.37 ± 0.01	0.26 ± 0.01	0.26 ± 0.01	0.23 ± 0.01	0.26 ± 0.01
Zeta potential (mV)	−26.13 ± 0.58	−29.26 ± 0.95	−29.29 ± 0.39	−28.56 ± 0.74	−34.63 ± 0.70	−28.39 ± 0.44	−29.22 ± 0.27	−26.96 ± 0.89	−27.93 ± 0.22
pH	7.84 ± 0.02	7.76 ± 0.01	7.65 ± 0.03	7.64 ± 0.01	7.40 ± 0.01	7.56 ± 0.01	7.31 ± 0.01	7.42 ± 0.01	7.15 ± 0.01
%EE (Madecassoside)	71.67 ± 0.34	72.66 ± 0.23	71.98 ± 0.36	69.80 ± 0.23	71.46 ± 0.36	69.73 ± 0.40	71.31 ± 0.25	69.21 ± 0.76	68.64 ± 0.33
%EE (Asiaticoside)	90.83 ± 0.21	92.01 ± 0.28	90.99 ± 0.20	91.33 ± 0.32	92.31 ± 0.34	90.05 ± 0.23	91.72 ± 0.45	90.34 ± 0.22	89.68 ± 0.31

Data are expressed as mean ± SEM. The transfersomes encapsulating Centella Asiatica (CANPs) were characterized for physicochemical properties and stability at 30 °C and 40 °C over 1, 2, 3, and 6 months. %EE: The percentage of encapsulation efficiency; PdI: Polydispersity.

To examine the ability of CANP for transdermal delivery, skin penetration studies were performed using ex vivo porcine skin. Fluorescent dye tracking analysis was employed to quantify the cumulative amount of dye transported across the skin. The cryosectioning of the treated skin revealed that CANP penetrated deeper into the skin compared to CALP and CANI (Figure 1B). Consistent with the fluorescence quantification results, the level of permeated fluorescent dye was significantly higher in the CANP-treatment compared to the CALP and CANI-treated groups (Figure 1B). These findings suggest that encapsulating CA extract into transfersomes facilitates its skin absorption, thereby enhancing its transdermal delivery. Noteworthy, the enhancement is likely due to the unique properties of transfersomes, which are highly elastic and deformable. As, the deformability index (DI) of CANP was measured at 1.31 ± 0.21 mg/cm², significantly higher than those of CALP (0.80 ± 0.06 mg/cm²) and CANI (0.32 ± 0.02 mg/cm²). Together, these results indicate that formulation of CA into transfersomes represents a highly effective nanocarriers for improving transdermal delivery and enhancing skin penetration of CA extract. Therefore, CANP was then used for further investigation.

To assess the formulated CANP cytotoxicity, cells were incubated with 2-fold dilution in the range of 0–1000 µg/mL Blank, CANP and CA extracts for 24 hours. Cell viability was then measured using an MTT assay. CANP, Blank NP, and CA extracts were not toxic to dermal fibroblast cells in all tested concentrations, as the percent cell viability was mostly higher than 80% at 24 hours (Figure 2A). Therefore, the concentrations at 100 µg/mL of tested samples was used for further experiments.

Figure 2.

The effects of formulated transfersomes encapsulating Centella asiatica (CANP) on cytotoxicity and anti-inflammatory activity were evaluated by MTT assay. A. cell viability assay in dermal fibroblast cells and B. nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, compared to Centella Asiatica extract (CA), blank transfersomes, and dexamethasone (DEX). Data are expressed as mean ± SEM (n = 4). **P < 0.01 and ***P < 0.001 compared to the non-treated control; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the LPS-treated control.

The murine macrophage cell line Raw264.7 is widely used for screening anti-inflammatory agents. In this study, we examined the effects of CA and CANP on NO production in LPS-stimulated macrophages. Our findings revealed that CANP significantly reduced nitrite accumulation, showing effects similar to those of dexamethasone (Figure 2B). Notably, the reduction in NO production was more pronounced in CANP-treated cells compared to those treated with CA. Together, these results suggest that CA exhibits anti-inflammatory activity, and its nanoformulation could enhance this inhibitory effect.

The CANP effects on the proliferation of human fibroblast cells and their collagen production were then examined. Cells treated with 2 µg/mL of CA and 100 µg/mL of CANP (2 µg/mL of extract equivalence) for 7 days showed a notable increase in proliferation compared to the control group. This increase was comparable to that observed in the positive control group treated with ascorbic acid (Figure 3A). Notably, the treatment with CA and CANP resulted in a significant enhancement in cell proliferation relative to the control, while ascorbic acid significantly enhanced this proliferation after 14 days of incubation (Figure 3A).

Figure 3.

The effects of formulated transfersomes encapsulating Centella Asiatica (CANP) on fibroblast proliferation and collagen production were evaluated by MTT assay and collagen staining. (A) The percentage of cell proliferation; (B) The percentage of total stained collagen; and (C) representative photographs of collagen staining on day 7 and day 14 were analyzed to assess the extent of collagen deposition. The results were compared to vitamin C (vit. C), Centella asiatica extract (CA) and blank nanoparticles. Data are expressed as mean ± SEM (n = 4). ***P < 0.001 compared to control on day 7 or day 14.

The potency of CANP in inducing collagen production in human dermal fibroblast cells was then investigated. Fibroblast cells were subjected to the treatment of 2 µg/mL of extract, 100 µg/mL of CANP (2 µg/mL of extract equivalence), and Blank NP for 7 and 14 days. 50 µg/mL of Vitamin C was used as a positive control. As shown in Figure 3(B), the stained collagen was noticeably enhanced by 28.6% and 91.1% in CA-treated cells after 7 and 14 days, whereas there was a significant increase in the collagen production of CANP treatment. The levels of stained collagen were improved by 91.9% and 213.3% at 7 and 14 days, respectively. The enhancement was more potent than the treatment of vitamin C, suggesting that CA and CANP were able to induce collagen synthesis in human skin cells and the encapsulation into transfersome could help to potentiate their effect on collagen production.

In this study, we then investigated the functions of CA and CANP on the induction of wound closure. As shown in Figure 4, CANP significantly accelerated wound gap closure in a time-dependent manner. According to the results presented in Supplementary Figure S3, 0.01% CANP was the highest concentration that maintained cell viability above 80% while also providing resistance to oxidative stress induced by 50, 100, and 200 μg/mL hydrogen peroxide. Based on these findings from the cell proliferation and antioxidant stress studies, we selected 100 μg/mL for initial evaluation, as pre-optimization trials indicated that this concentration is biologically active and nontoxic. Treatments with 2 µg/mL CA, 100 µg/mL CANP (2 µg/mL of extract equivalence), alongside 50 ng/mL FGF, over a 24-hour period led to increases in cell migration of 19%, 28%, and 33%, respectively, compared to the control group. After 48 hours, the increases in cell migration were further enhanced to 52%, 75%, and 76%. Remarkably, the wound gap closure achieved with 100 µg/mL CANPs was comparable to that seen with the FGF-positive control. These findings indicate that the treatment with 100 µg/mL CANP exhibits wound healing properties similar to those of the FGF-positive control.

Figure 4.

The effects of formulated transfersomes encapsulating Centella Asiatica (CANP) on wound healing potency in dermal fibroblast cells were evaluated by scratch assay. A. Representative photographs of scratch wound closure, and B. Percentage of gap filled at 24 and 48 hours. The results were compared to Centella Asiatica extract (CA), blank nanoparticles, and fibroblast growth factor (FGF). Data are expressed as mean ± SEM (n = 4). ***P < 0.001 compared to the non-treated control.

3.2. Effects of formulated CANP-Gel on in vivo wound closure, histological features, and targeted molecular changes in wound healing

To assess the role of CANP-Gel in wound healing, an in vivo wound healing assay was conducted. As shown in Figure 5(A), the characteristics of the wounds were carefully observed throughout the study. At day 7, the wounds treated with CANP-Gel demonstrated reduced pus formation and a relatively dry surface, suggesting a decrease in inflammatory exudate and a shift toward a more controlled inflammatory response. By day 14, the treated wounds exhibited minimal exudate, with a dry, crusted appearance, indicative of ongoing tissue regeneration and epithelialization. By day 21, although the reduction in wound size was slightly delayed relative to the control group, the CANP-Gel–treated wounds exhibited more organized tissue architecture, nearly complete epithelial closure, and minimal scar formation, indicating its efficacy in enhancing tissue repair and regeneration. These findings suggest that CANP-Gel effectively enhances the wound healing process, potentially through its anti-inflammatory and regenerative properties. Therefore, the wound contraction was measured on days 7, 14, and 21 following CANP-Gel treatment, revealing a progressive and significant increase in contraction rates across the experimental timeline. Specifically, wound closure was observed at 58.08% on day 7, 93.48% on day 14, and 98.63% on day 21 (Figure 5B). These results indicate that CANP-Gel treatment promotes accelerated wound healing compared to the control groups.

Figure 5.

Effects of gel containing formulated transfersomes encapsulating Centella Asiatica (CANP-Gel) on wound healing of the skin excision wounds in rats. (A) Photographs showing changes in rat skin wound closure from post-operation to day 21 and (B) the percentage of wound contraction on days 7, 14, and 21 post-wound excision. Data were expressed as mean ± SEM (n = 4/time point). *P < 0.05 and ***P < 0.001 compared to blank (control). Scale bar 1 cm.

As shown in Figure 6(A), histomorphological analysis of CANP-Gel–treated wounds revealed a significantly higher presence of mononuclear and polymorphonuclear leukocytes, fibroblasts, and newly formed blood capillaries on both days 7 and 14 post-treatment. Additionally, mild inflammatory cell infiltration was observed in the CANP group on day 3, as shown in Supplementary Figure 6(C), which aligns with the expected inflammatory phase of normal wound healing. CANP-Gel treatment also appeared to accelerate the proliferative phase, as indicated by a marked reduction in inflammatory cells (Figures 6B–C) and blood capillaries (Figure 6E) by day 21, along with an increased number of fibroblasts (Figure 6D).

Figure 6.

Effects of gel containing formulated transfersomes encapsulating Centella asiatica (CANP-Gel) on histopathology of the skin excision wounds in rats as evaluated by hematoxylin and eosin staining. (A) Representative histological images showing leukocytic inflammatory cells (arrow), blood vessels (arrowhead) and fibroblasts (asterisk). (B) Quantitative analysis of mononuclear leukocytes, (C) polymorphonuclear leukocytes, (D) fibroblasts, and (E) capillaries (angiogenesis) on days 7, 14, and 21 post-wound excision counted in 10 high-power field (400× magnification). Data were expressed as mean ± SEM (n = 3/time point). *P < 0.05, **P < 0.01 and *P < 0.001 compared to blank (control). Scale bar = 40 μm.

In addition, CANP-Gel-treated wounds demonstrated faster epithelialization compared to the Blank-treated wounds (Figure 7A). Enhanced collagen deposition (blue/green staining) was also observed in the CANP-Gel-treated wounds on day 7, when compared to the Blank-treated controls (Figure 7B). These findings collectively suggest that CANP-Gel effectively promotes both the inflammatory response and the wound remodeling process, contributing to accelerated wound healing.

Figure 7.

Effects of gel containing formulated transfersomes encapsulating Centella asiatica (CANP-Gel) on wound epithelialization and collagen deposit of the skin excision wounds in rats as evaluated by Masson’s Trichrome staining. (A) Photographs of histological changes in the epithelium and (B) collagen (green color) on days 7, 14, and 21 post-wound excision. (C) The percentage of re-epithelialization was calculated (re-epithelialization length/wound length) in 10 high-power field (40× magnification). (D) The percentage of collagen deposition was measured in 10 high-power field (HPF) (400× magnification). Data were expressed as mean ± SEM (n = 3/time point). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to blank (control). Scale bar = 1 mm.

At the molecular level, CANP-Gel treatment significantly upregulated the mRNA expression of Ilb6 on day 7, which could contribute to the acceleration of wound healing. Additionally, upregulation of fgf2, tgfb1, Col1a1, and Col3a1 were observed in the CANP-Gel-treated wounds (Figure 8). These molecules are critical mediators of wound healing, with fgf2 promoting angiogenesis and cell migration, tgfb1 driving collagen synthesis and scar formation, and Col1a1 and Col3a1 being major components of the ECM that contribute to tissue remodeling. Together, these findings suggest that CANP-Gel not only promoted fibroblast proliferation and migration but also facilitated the formation of a robust extracellular matrix, which are critical processes in wound healing and tissue regeneration in rat skin.

Figure 8.

Effects of gel containing formulated transfersomes encapsulating Centella asiatica (CANP-Gel) on gene expression of wound healing of the skin excision wounds in rats as evaluated by qRT-PCR. (A) Relative gene expression levels of interleukin-6 (IL6), (B) Epidermal growth factor (egf), (C) Fibroblast growth factor 2 (fgf2), (D) Transforming growth factor beta 1 (tgfb1), (E) Collagen Type I (Col1a1) and (F) Collagen Type III (Col3a1) on days 7, 14, and 21 post-wound excisions were normalized using beta-actin as an internal control. Data were expressed as mean ± SEM (n = 3/time point). *P < 0.05 and **P < 0.01 compared to blank (control).

4. Discussion

Among herbal medicines, CA has garnered significant attention due to its long-standing use in traditional medicine and its well-documented wound-healing properties. CA extract exhibits potent antioxidant and anti-inflammatory activities and accelerates wound healing by promoting fibroblast proliferation and enhancing collagen production (Sh Ahmed et al. 2019; Arribas-López et al. 2022). It contains active compounds such as asiatic acid, madecassic acid, asiaticoside, and madecassoside, which are known to enhance collagen synthesis, promote angiogenesis, and reduce inflammation (Sengupta et al. 2021; Diniz et al. 2023; Xu et al. 2023). These properties make CA a promising candidate for developing innovative wound management solutions, particularly in scenarios where conventional treatments may fall short due to limited efficacy or the emergence of drug resistance.

However, the therapeutic potential of CA is constrained due to certain limitations, such as its low solubility and poor bioavailability (Bansal et al. 2024). These challenges are primarily attributed to its hydrophobic behaviors, which results in poor aqueous solubility and poses significant barriers to its incorporation into transdermal delivery systems. To mitigate these limitations, nanoencapsulation strategies have been employed. Previous studies have demonstrated that formulating CA extracts into nanoparticles, particularly those with particle sizes below 100 nm, can markedly enhance their bioactivity (Zhu et al. 2021; Ebau et al. 2023).

Interestingly, incorporating CA into novel delivery systems such as transfersomes presents promising potential for enhancing wound healing outcomes. These advanced nanocarriers enable targeted and efficient delivery of active compounds, thereby maximizing its therapeutic effects. Transfersomes improve the bioavailability and penetration of CA into deeper skin layers, facilitating processes for efficient wound healing (Rai et al. 2017; Ramadon et al. 2022). Therefore, to create a formulation with enhanced wound healing action, the present study developed CANP and performed in vitro and in vivo to examine the efficacy of CANP-Gel on wound healing application.

Our results demonstrated that encapsulating CA into transfersomes using soybean lecithin, sorbitan oleate, polysorbate 80, and tocopherol acetate resulted in homogeneously spherical particles with an average size of 135 ± 4.8 and a deformability index of 1.31 ± 0.21 mg/cm². Moreover, the improved skin permeation observed in the porcine skin model demonstrates the potential of this transfersome-based delivery system to facilitate the targeted delivery of CA bioactives. The small particle size and deformability likely contributed to overcoming the skin’s stratum corneum barrier, enabling efficient drug penetration (Ghasemiyeh and Mohammadi-Samani 2020).

These findings align with the previous study by Surini and Djajadisastra (2018), which reported that encapsulation of Gotu kola leaf extract into transfersomes composed of phospholipid 90 G and Tween 80 produced spherical vesicles with a deformability index of 1.12. Additionally, in vitro studies utilizing Franz diffusion cells revealed that the transfersome gel containing Gotu kola leaf extract significantly enhanced asiaticoside penetration compared to the control gel lacking transfersomes (Surini and Djajadisastra 2018). Similar results were reported by Opatha et al. (2022). Three different types of transfersomes, composed of lecithin combined with either span 80, tween 80, or sodium deoxycholate, were utilized for the encapsulation of asiatic acids. The formulated nanoparticles were subsequently incorporated into gels and evaluated for in vitro permeation using Franz diffusion cells with Strat-M synthetic membranes. The findings indicated that all transfersomal formulations significantly enhanced asiatic acid penetration compared to non-formulated asiatic acids. Among these, the formulation using tween 80 as an edge activator was identified as the optimal strategy for asiatic acid encapsulation. Thus, a transfersomal gel demonstrated effective in vitro dermal penetration of CA, suggesting its potential as a therapeutic strategy for various skin disorders.

Additionally, our in vitro investigation revealed that CANP increased fibroblast cell proliferation and subsequently enhanced collagen production. The CANP formulation also showed greater improvement in wound closure compared to the non-encapsulated form, with a function similar to that of fgf mRNA expression. Furthermore, CANP demonstrated an anti-inflammatory effect by significantly inhibiting the production of NO, a key marker of the inflammatory pathway. This finding suggests that the delivery of CA compounds to targeted cellular sites was effective, as reported by Sun et al. (2020). This targeted delivery mechanism likely enhanced the bioactivity of CA, contributing to its overall wound-healing efficacy. Therefore, CANP has the potential to be a therapeutic agent for wound management.

Wound closure is a complex process heavily influenced by the ECM and cytokine signaling, with fibroblasts playing a pivotal role. These cells are essential for wound healing as they migrate to the wound site, proliferate, and secrete ECM components. Collagen, a primary ECM protein, is a key determinant of wound strength and integrity (Gurtner et al. 2008; Demidova-Rice et al. 2012). Our findings demonstrated that CANP-Gel significantly accelerated in vivo wound closure and re-epithelialization by modulating inflammation and enhancing collagen deposition. Furthermore, CANP-Gel actively promoted the inflammatory response and upregulated genes associated with wound healing, supporting its efficacy in facilitating the repair process in rat skin (i.e. IL6, Col1a1, Col3a1, fgf2, and tgfb1). These results are consistent with previous studies regarding to asiatic acid extracted from CA hydrogel microneedles increasing the rate of wound closure (Ryall et al. 2022). Active compounds such as asiaticoside, extracted from CA improved microcirculatory flow, and higher expression levels of superoxide dismutase and VEGF and lower malondialdehyde and inflammatory markers (i.e. IL-6 and IL-1β) in McFarlane-type dorsal rat flap model (Feng et al. 2021). Moreover, CA with the filopodia formation induces cell migration and subsequently promotes wound healing activity in the keratinocyte cell line via the activation of FAK, Akt, and MAPK signaling cascades (Singkhorn et al. 2018). Interestingly, the combination of asiaticoside and NO can inhibit the growth of bacteria in the wound surface, alleviate the inflammatory reaction of wound, and increase the expression of VEGF, iNOS, eNOS, CD34, and Wnt/β-Catenin signaling pathway in diabetic cutaneous ulcers (Nie et al. 2020).

This study demonstrates that CANP-Gel promotes wound healing through a multifaceted mechanism involving enhanced inflammation modulation, fibroblast proliferation, collagen synthesis, and tissue remodeling. The upregulation of specific dermal growth factors, along with improved histopathological outcomes, supports the efficacy of CANP-Gel as a promising wound-healing agent. However, this study did not include an assessment of sub-chronic toxicity, such as potential hepatotoxicity and nephrotoxicity. Comprehensive evaluation of systemic safety including biochemical, histopathological, and functional analyses of liver and kidney tissues (Javed and Ilyas 2025), remains essential for future studies to ensure the long-term biocompatibility of the formulation.

To address interspecies variability in transdermal delivery, porcine skin was selected for the ex vivo penetration assay due to its close anatomical and physiological resemblance to human skin, particularly in stratum corneum structure and lipid composition—key determinants of percutaneous absorption. This model preserves epidermal and dermal integrity, retains enzymatic activity and hydration levels, and allows for realistic evaluation of drug diffusion, accumulation, and biotransformation (Kocsis et al. 2022). It also reduces ethical concerns and inter-individual variability while providing mechanistic insights into drug penetration and release kinetics (Whitson and Black 2023). Although porcine models are well-established for studying skin permeability, using porcine skin requires more complex housing and handling, especially in early-stage preclinical research. Therefore, further investigation in this area remains essential. Conversely, rats were selected for the in vivo studies due to several practical and scientific advantages. The relatively small size and ease of handling make rats cost-effective and feasible for conducting controlled experiments with larger sample sizes. Additionally, rats have well-characterized wound healing physiology that shares many similarities with humans, including comparable inflammatory responses, angiogenesis, and tissue regeneration mechanisms (Weber et al. 2019). Importantly, a broad range of validated experimental tools is available for rats, such as specific histological markers, immunohistochemistry protocols, and gene expression profiling techniques, enabling detailed and quantitative assessment of wound healing processes (Ojeh et al. 2025). The rat model is thus widely adopted in preclinical research to study key aspects of wound repair and regeneration, facilitating evaluation of therapeutic efficacy with reliable reproducibility (Ghanbari et al. 2024). Although comparisons with commercial antibacterial wound healing gels are well established in previous studies (Bhubhanil et al. 2021), this study did not include a commercial product as a reference group due to significant variations among these products. Differences in mechanisms of action, active ingredients, formulation compositions, and recommended application protocols can markedly influence efficacy and mode of wound repair (European Medicines Agency 2018). Such heterogeneity complicates direct comparison and interpretation of results, potentially confounding the assessment of the novel formulation’s specific effects. Furthermore, regulatory guidelines and standardization for commercial wound care products vary across regions, further limiting the feasibility of using a single commercial gel as a universal benchmark (Sen 2024). Therefore, this study focused on characterizing the therapeutic potential of the tested formulation against untreated controls to provide a clearer evaluation of its intrinsic wound healing properties.

Future studies will incorporate advanced 3D imaging techniques, such as multiphoton microscopy and optical coherence tomography, to enhance the precision and spatial resolution of wound healing and skin disease assessments (Chen et al. 2023). These technologies enable detailed visualization of tissue architecture, cellular interactions, and extracellular matrix remodeling in three dimensions, providing a more comprehensive understanding of healing dynamics beyond traditional two-dimensional histology. Regarding histological analysis, while Masson’s trichrome staining was employed in this study to visualize collagen deposition and general tissue structure, inclusion of Sirius Red staining in future study is recommended. Sirius Red selectively binds to collagen fibers and, under polarized light microscopy, allows differentiation between collagen types and assessment of fiber maturity and organization (Liu et al. 2021). Although Sirius Red staining has been reported to detect slightly lower collagen densities compared to Masson’s trichrome, it offers complementary and confirmatory insights into collagen quality and remodeling during wound healing (Sharun et al. 2023). Combining these histological approaches with advanced imaging will improve the accuracy and depth of tissue regeneration evaluations.

Moreover, while qPCR provides valuable quantitative data on gene expression, it does not directly measure protein levels or post-translational modifications, which ultimately determine biological function. Therefore, Western blot analysis of key proteins including TGF-β and COL1A1 is essential to validate and complement the qPCR results by confirming that changes in mRNA correspond to alterations at the protein level. Additionally, assessment of antioxidant markers like superoxide dismutase activity and malondialdehyde concentration will provide critical insights into oxidative stress status and the formulation potential to mitigate reactive oxygen species–mediated tissue damage. Since oxidative stress plays a significant role in inflammation and impaired wound healing, inclusion of these biochemical assays will enhance understanding of the underlying anti-inflammatory mechanisms. These comprehensive analyses are planned for the next phase of investigation to strengthen mechanistic evidence supporting therapeutic efficacy of the formulation.

5. Conclusions

This study developed CANP as a novel transdermal delivery system. CANP demonstrated excellent physicochemical properties, enhanced skin penetration, stability, and safety, with high encapsulation efficiency and anti-inflammatory effects. In vitro, CANP significantly improved fibroblast proliferation and collagen production, outperforming vitamin C. In vivo, CANP-loaded hydrogels accelerated wound healing, reduced inflammation, and promoted tissue regeneration. These findings suggest that CANP is a promising nanocarrier for clinical applications in wound healing and skin regeneration, especially in the early phase. Future clinical studies could explore CANP-loaded patches or gels for treating chronic wounds and skin aging, potentially offering enhanced therapeutic outcomes through sustained release and improved patient compliance.

Funding Statement

This work was supported by a grant from the Fundamental Fund (FF2567) of the National Science and Technology Development Agency (NSTDA), project code P2351510.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval statement

This study obtained ethics approval and is part of a larger research project that may result in multiple related publications under one overarching ethics approval. The Institutional Animal Care and Use Committee (IACUC) of Thammasat University approved the use of animals and all experimental procedures under approval number 021/2562, initially granted in October 2019 and renewed until October 2021 due to delays in securing research grant funding. The use of animals was justified based on the need to assess safety, biocompatibility, and wound-healing efficacy of the tested formulations in an in vivo setting, which cannot be adequately replicated in vitro. A total of 12 adult male Wistar rats were used. All rats were housed in standard animal laboratory conditions with a temperature of 22 °C–25 °C, humidity of 45%–55%, and a 12:12 h light/dark cycle. Rats had free access to standard rodent chow and distilled water. Environmental enrichment included nesting materials and shelters. All procedures were performed under anesthesia with isoflurane, and all efforts were made to minimize pain and distress. This study also utilized porcine skin samples obtained from licensed meat suppliers registered with the Department of Livestock Development, Thailand. As these samples were collected postmortem from animals slaughtered for commercial purposes, additional ethical approval from the IACUC was not required. All research activities were carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org). This study used Centella asiatica leaves obtained from cultivated sources, grown by a local farm with appropriate permission. The collection and use of plant materials were conducted in compliance with institutional and national ethical guidelines. A voucher specimen was deposited at the National Herbarium (BKF), Department of National Parks, Wildlife and Plant Conservation, Thailand, under the code BKF No. 134226.

Data availability statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.