Lipid Nanoparticles Help Deliver EGF mRNA to Improve Wound Healing

The spontaneously immortalized human keratinocyte line HaCaT (QuiCell-H439, Shanghai Kuisai Biological Technology Co., Ltd., Shanghai, China) was cultured in DMEM (Gibco, Shanghai, China) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO₂ and authenticated through Short Tandem Repeat. Antarctic phosphatase (M0289L, New England Biolabs, Beijing, China) and the FlowOrigin M reagent kit were purchased from Mingtai Pharmaceutical Equipment Co., Ltd. (Shanghai, China). eGFP mRNA (BN-mRNA-eGFP-100) was purchased from Beinu Biotech (Shanghai, China). The calcein/PI cell viability/cytotoxicity assay kit (C2015S), crystal violet staining solution (C0121), H&E Stain (G1005), and Masson’s trichrome stain (G1006) were obtained from Beyotime (Shanghai, China) and Servicebio (Wuhan, China), respectively. Primary antibodies against EGF (27141-1-AP), collagen I (66761-1-Ig), collagen III (22734-1-AP), and E-cadherin (20874-1-AP) were purchased from Proteintech (Wuhan, China).

mRNA synthesis was performed as previously described [22]. Briefly, a linearized DNA template—purified after restriction digestion—was transcribed in vitro with T7 RNA polymerase at 37 °C for 2 h. Following transcription, free nucleotides and short RNA fragments were removed using an Ambion MEGAclear kit (Shanghai, China) as per the manufacturer’s instructions. The eluted RNA was incubated at 37 °C for 30 min with Antarctic Phosphatase (New England Biolabs, Beijing, China) to remove residual 5′-phosphates and then repurified through the same MEGAclear column. The final product was quantified on a NanoDrop One spectrophotometer (Thermo Scientific, Shanghai, China), adjusted to 1 mg mL⁻¹ in RNase-free 10 mM Tris-HCl and 1 mM EDTA (pH 7.4), aliquoted, and stored at –80 °C until use.

The preparation of LNP-mRNA_EGF was based on a previously reported method with further optimizations [23]. Briefly, the aqueous phase was prepared by diluting 0.3 mL of an mRNA solution (1 mg/mL) to a final concentration of 0.17 mg/mL using a sodium citrate buffer (pH 4.0). The organic phase was formulated by mixing four lipid components in equal volumes: DLin-MC3-DMA (25 mmol/L), DOPC (5 mmol/L), cholesterol (19.25 mmol/L), and DMG-PEG2000 (0.75 mmol/L). For synthesis, the aqueous and organic phases were utilized at volumes of 1.65 mL and 0.55 mL, respectively. The diluted aqueous phase was loaded into syringes and secured at the inlets of a new chip. Mixing was then carried out via a Microflow S chip device, yielding LNP-mRNA_EGF. The LNP was fabricated following a similar protocol.

Immediately following the microfluidic process, a 100 µL aliquot of the freshly prepared LNP-mRNA_EGF suspension was diluted tenfold with 0.22 µm filtered, particle-free ultrapure water. The Z-average diameter and polydispersity index (PDI) were determined via dynamic light scattering. For further morphological examination, 5 µL of the undiluted LNP sample was applied onto a glow-discharged 200-mesh copper grid. After allowing 60 s for adsorption, the residual liquid was removed via Whatman No. 1 filter paper, and the grid was air-dried at room temperature for two minutes. A subsequent negative staining procedure was performed: the grid was placed on a 20 µL drop of 2% (w/v) phosphotungstic acid (pH 7.0) for 30 s, followed by blotting and complete drying. Micrographs were acquired at an accelerating voltage of 200 kV.

The mRNA concentrations obtained by disrupting the LNP structure with SDS and without disrupting the LNP structure are designated as total mRNA and free mRNA, respectively. The encapsulation efficiency is calculated based on these values. The formula for encapsulation efficiency is Encapsulation efficiency = (total RNA concentration − free RNA concentration)/total RNA concentration × 100%.

LNP-mRNA_EGF was labeled with the fluorescent dye 3,3′-dioctadecyloxacarbocyanine perchlorate and co-incubated with HaCaT cells cultured in media at different pH levels for 2 h at 37 °C. After fixation and nuclear staining with DAPI, cellular uptake efficiency was assessed and quantified using fluorescence microscopy.

HaCaT cells were seeded in 96-well plates at a density of 5 × 10³ cells per well and cultured overnight in a 37 °C incubator. After cell attachment, the cells were treated with various concentrations of LNP and LNP-mRNA_GFP, followed by further incubation. After treatment, the medium was removed and replaced with fresh medium containing CCK-8 reagent, and the cells were incubated at 37 °C for 1 h. Subsequently, the optical density of each well was measured at 450 nm using a microplate reader to assess cell viability.

HaCaT cells were seeded in a 24-well plate at a density of 5 × 10⁴ cells per well and cultured overnight in a 37 °C incubator. The following day, the cells were treated with varying concentrations of LNP-mRNA_GFP and cultured further. Subsequently, the cells were trypsinized, harvested, and analyzed by flow cytometry to determine GFP expression efficiency. Similarly, GFP fluorescence intensity was observed and measured at different time points using fluorescence microscopy.

The concentration of EGF in cell culture supernatants was quantitatively determined by ELISA. The assay was performed with a commercial Human EGF ELISA Kit (E-EL-H0059, Elabscience, Wuhan, China) strictly following the manufacturer’s instructions. Briefly, cell culture supernatants were collected and centrifuged at 1000× g for 20 min at 4 °C to remove cells and debris. The clarified supernatants were either used immediately or stored at –80 °C for subsequent analysis. A standard curve was prepared by serially diluting the EGF standard to concentrations ranging from 3.91 to 250 pg/mL. Aliquots (100 µL) of each standard or sample were added to a 96-well microplate pre-coated with an anti-human EGF antibody and incubated at 37 °C for 90 min. After washing, biotinylated detection antibody followed by horseradish peroxidase-labeled avidin were added sequentially, with incubation at 37 °C for 60 min and 30 min, respectively. Following another wash step, TMB substrate solution was added to each well and incubated at 37 °C in the dark for 15 min. The reaction was stopped by adding 50 µL of stop solution per well, and the absorbance was immediately measured at 450 nm using a microplate reader.

A CCK-8 kit (C0038, Beyotime, Shanghai, China) was used to evaluate cell proliferation. HaCat cells were seeded into 96-well plates at a density of 5 × 10³ cells per well. After 24 h, the culture medium was replaced with Opti-MEM I reduced-serum medium, and the cells were treated with either LNP-mRNA_EGF or recombinant human EGF (rhEGF) protein, which served as a positive control. At 24 and 72 h post-treatment, CCK-8 reagent was subsequently added to each well, and the absorbance at 450 nm was measured to monitor cell proliferation.

HaCaT cells were seeded in 12-well plates and cultured until reaching 80–90% confluency. A sterile pipette tip was used to create a uniform scratch in the cell monolayer across the center of each well. After gently washing the cells twice with PBS, the medium was replaced with serum-free medium, and the initial scratch images (0 h) were captured under a microscope. The cells were then treated with different formulations and cultured for an additional 48 h. After incubation, the same scratch areas were re-imaged, and cell migration ability was evaluated by measuring the change in scratch width.

HaCaT cells were seeded in the upper chamber of a Transwell insert (8 μm pore polycarbonate membrane, Corning, Shanghai, China) at an appropriate density. After cell attachment, the medium was replaced with serum-free medium, and different formulations were added to the upper chamber. The lower chamber was filled with medium containing the corresponding treatments. Following 24 h of incubation, the Transwell inserts were removed. Non-migrated cells on the upper surface of the membrane were gently wiped away with a cotton swab. The migrated cells on the lower surface were fixed with 4% paraformaldehyde for 10 min, stained with 0.1% crystal violet for 15 min, and gently rinsed with PBS. The membranes were then observed under a microscope, and the number of migrated cells was counted.

All animal procedures were conducted in accordance with the guidelines of an approved Institutional Animal Care and Use Committee protocol (IACUC-20251010) and followed standard humane care practices. To assess the therapeutic effect of LNP-mRNA_EGF on wound healing, 55 male C57BL/6 mice (obtained from Shanghai Jihui Laboratory Animal Care Co., Ltd., Shanghai, China) were anesthetized, and the hair on their dorsal skin was carefully removed. An 8 mm full-thickness wound was created on the dorsal skin via a high-frequency laser. The mice were then administered intradermal injections (using a BD insulin syringe) at four periwound sites with one of the following: Normal Saline, empty LNP, LNP-mRNA_EGF (40 µL of LNP formulation containing 20 µg mRNA per injection site) or rhEGF. Wound healing was monitored by capturing digital images on days 0, 3, 6, and 10, and the wound area was quantified via ImageJ 10.2 software to determine the rate of closure.

On day 10, the mice were euthanized, and 8 mm punch biopsies were collected from the wound sites. The tissue samples were fixed in 4% formalin, subjected to dehydration through a graded ethanol series, and subsequently embedded in paraffin. Sections were stained with H&E for histological evaluation and with Masson’s trichrome for collagen visualization. Finally, the stained sections were imaged via an microscope (CKX41, Olympus, Shanghai, China).

Dorsal wound tissue samples were fixed in 4% paraformaldehyde (PFA), cryoprotected by immersion in 30% sucrose, and subsequently embedded in OCT compound prior to snap freezing. Sections of 7 μm thickness were prepared, followed by antigen retrieval via citrate buffer and permeabilization with 0.1% Triton X-100. The sections were then blocked with goat serum for one hour and incubated with primary antibodies targeting CD31, COLI/II, and E-cadherin at 4 °C overnight. After washing, the sections were exposed to Alexa Fluor-conjugated secondary antibodies and incubated in the dark for one hour at room temperature. Finally, the nuclei were stained with DAPI, and the slides were coverslipped with mounting medium for confocal microscopy analysis.

Skin tissue samples were collected at predetermined time points and thoroughly homogenized to prepare tissue lysates. The content of human EGF in the tissue lysates was quantitatively measured by ELISA. The specific experimental procedures were consistent with the aforementioned in vitro ELISA detection method.

The data were processed, and graphical representations were generated via GraphPad Prism software (version 10.1). For comparisons between two groups, unpaired Student’s t tests were applied. Comparisons across multiple groups were conducted by one-way analysis of variance (ANOVA), followed by Tukey’s test for post hoc analysis. A probability (p) value of less than 0.05 was considered statistically significant.

LNP-mRNA_EGF was created using microfluidic technology. To directly evaluate mRNA entrapment, agarose gel electrophoresis was performed, comparing LNP-mRNA_EGF with its intact lipid structure to samples that had been treated with SDS for lipid disruption. In Figure S1, a noticeable mRNA band was present only in the SDS-treated sample, where the lipid structure was disrupted, while the intact LNP sample showed no band. This result demonstrates that the mRNA in the prepared LNP-mRNA_EGF formulation is effectively encapsulated within the lipid nanoparticles. Additionally, the content measurement within the LNP showed an encapsulation efficiency of 88 ± 2% (Figure S2).

Transmission electron microscopy (TEM) characterization showed that both blank LNP and LNP-mRNA_EGF had a typical spherical shape (Figure 1A). Dynamic light scattering (DLS) analysis showed that the average hydrodynamic diameter of the blank LNP was consistent with the TEM observations (Figure 1B). Both formulations displayed low polydispersity indices (PDI), indicating a relatively uniform particle size distribution (Figure 1C). Furthermore, at pH 6.5, the Z-average size and PDI of both LNP and LNP-mRNA_EGF remained largely comparable to those at pH 7.4 (Figure 1D and Figure S3). The analysis of zeta potential revealed a notable variation dependent on pH, due to the protonatable characteristics of the lipid components. Figure 1E illustrates their distinct zeta potentials at different pH values: a negative charge at physiological pH 7.4, which reduces nonspecific cellular uptake, and a slightly positive charge in acidic environments. This pH-dependent charge reversal is anticipated to enhance electrostatic interactions with negatively charged cell membranes, thereby potentially promoting nanoparticle uptake at the slightly acidic wound microenvironment. To confirm these results, we used the green fluorescent dye 3,3′-dioctadecyloxacarbocyanine per-chlorate to label LNP-mRNA_EGF and measured its internalization in HaCaT cells across different pH environments. Data presented in Figure 1F,G demonstrate that cellular uptake was markedly enhanced at pH 6.5 relative to pH 7.4 (p < 0.01).

Furthermore, the storage stability of the nanoparticles was evaluated over a 10-day period by monitoring changes in Z-average size. The hydrodynamic diameter increased by only 10 ± 3% for the blank LNP and 19 ± 1% for LNP-mRNA_EGF, indicating good stability under storage conditions (Figure S4). These physicochemical characteristics collectively support the potential of LNP-mRNA_EGF as a stable and efficient mRNA delivery platform.

To evaluate the biosafety of the LNP in a systematic manner, we started by assessing its cytotoxicity in HaCaT cells. As shown in Figure 2A, with an increase in LNP dose, cell viability gradually lessened; however, concentrations up to 4.875 µg mL⁻¹ did not show a statistically significant decrease, implying this concentration is non-toxic. Thus, taking into account cell viability and possible therapeutic benefits, a concentration of 4.875 µg mL⁻¹ might be appropriate for in vitro studies. Following this, we explored if mRNA loading impacts the cytocompatibility of LNP. LNPs with GFP mRNA (LNP-mRNA_GFP) were compared against empty LNPs. The viability curves overlapped almost perfectly (Figure 2B), and live/dead staining confirmed that mRNA encapsulation did not increase cytotoxicity (Figure 2C), indicating that the particles retained their excellent safety profile after loading.

With safety established, we quantified the transfection efficiency. HaCaT cells were incubated for 12 h with increasing doses of LNP-mRNA_GFP, and GFP expression was analyzed via flow cytometry. The percentage of transfected cells increased with increasing particle concentration and plateaued at 2.438 µg mL⁻¹ (Figure 2D,E and Figure S5). Fluorescence microscopy revealed expression for 72 h; the signal intensity continued to increase through the final time point (Figure 2F and Figure S6), demonstrating that a single dose of LNP-mRNA_GFP can sustain robust protein expression for at least three days. Furthermore, to evaluate the expression level of EGF in HaCaT cells, we employed LNP-mRNA_EGF at varying concentrations. The results demonstrated a dose-dependent increase in EGF expression across the tested concentration range, with the peak level observed at 4.875 µg·mL⁻¹, which exhibited the highest EGF secretion (Figure S7).

Taken together, these data show that the LNP platform is biocompatible at therapeutically relevant concentrations and mediates efficient, prolonged mRNA expression in human keratinocytes, providing a solid rationale for advancing to in vivo wound healing studies.

EGF is known to drive HaCaT keratinocyte proliferation by activating the EGF receptor and its downstream ERK/Akt axis [24]. We therefore hypothesized that delivering EGF-encoding mRNA via LNP (LNP-mRNA_EGF) would reproduce proliferation. As early as 24 h posttreatment, cell viability assays revealed a significant increase in metabolic activity (Figure 3A). Because our earlier work revealed that LNP- mRNA remains expressed for at least 72 h, we extended the observation window. By 72 h, the proliferation index increased from 117 ± 3% at 24 h to 136 ± 2% (Figure 3B), indicating that continuously synthesized EGF exerts a sustained and amplified bioactive response.

In addition to promoting proliferation, LNP-mRNAEGF has been shown in studies to guide the migration of HaCaT cells [25]. Figure 3C,D demonstrate that the closure rate of monolayers treated with LNP-mRNA_EGF was significantly quicker and comparable to the rate achieved with recombinant EGF protein. Transwell assays corroborated these findings: the number of cells that traversed the membrane was significantly greater in the LNP-mRNA_EGF group (Figure 3E and Figure S8), collectively demonstrating a robust promigratory capacity.

Taken together, these data confirm that LNP-mRNA_EGF expresses bioactive EGF in HaCaT cells and simultaneously amplifies both proliferation and migration—key cellular events in cutaneous repair—thereby providing experimental support for its potential use in skin-regenerative therapy.

To assess the wound healing effectiveness of LNP-mRNA_EGF in living organisms, a model with an 8 mm diameter circular full-thickness skin defect was created in mice using a fractional CO₂ laser (Figure 4A). Subsequently, the mice were randomly divided into four groups, receiving treatments of LNP, LNP-mRNA_EGF, and rhEGF, respectively, with an equivalent volume of normal saline serving as the blank control group. According to Figure 4B, the wound area in the LNP-mRNA_EGF group was significantly smaller than in the control and LNP groups by day 6 after treatment, as shown by the photos and healing simulation diagrams. The quantitative data (Figure 4C) reveal that, by day 6, the LNP-mRNA_EGF group had an average residual area of 31% of the original, compared to 56% in the control group and 47% in the LNP group (p < 0.001), confirming that mRNA treatment significantly accelerated healing. By day 10, the wounds in the LNP-mRNA_EGF group had almost completely closed, whereas those in the other two groups still had >20% of the area remaining unhealed. To further investigate the in vivo kinetics of EGF expression, mice were administered a single dose of LNP-mRNA_EGF, and skin tissue from the wound region was collected at set time points for ELISA analysis. As shown in Figure S9, the level of EGF in the wound tissue increased over time, peaked on day 5, and remained elevated on day 7. These results demonstrate that a single administration of LNP-mRNA_EGF enables the sustained, local expression of EGF at the wound site, supporting its role in promoting the healing process.

Re-epithelialization is the first critical step in skin wound healing and occurs before the dermis begins to mature [26]. The rapid formation of a temporary barrier during injury stops excessive water loss and prevents bacteria from escaping, safeguarding the underlying tissue [27]. To verify the regeneration of the epidermis, hematoxylin and eosin (H&E) staining and Masson’s trichrome staining were performed on the wounds after treatment. As shown in Figure 4D, the tissue in the LNP-mRNA_EGF treatment group exhibited obvious epithelialization, whereas the control and LNP groups presented almost no epithelial formation. Furthermore, the results of Masson’s trichrome staining (Figure 4E,F) showed a significant enhancement in collagen deposition within the LNP-mRNA_EGF group (p < 0.0001).

To elucidate the potential mechanisms by which LNP-mRNA_EGF promotes wound healing in wounds, immunofluorescence staining was performed on the wound tissues. In Figure 5A, it is shown that the skin EGF levels in the LNP-mRNAEGF group were notably higher than those in the control and LNP groups, with a clear expression advantage over rhEGF (p < 0.001), suggesting that mRNA therapy can stably and continuously express EGF, achieving long-term therapeutic effects.

Collagen remodeling is the “final stage” of wound healing and is also the core link that determines the strength of the recovery of skin function. Type I collagen, after being deposited, is cross-linked by lysyl oxidase into dense, bundle-like structures, which provide mechanical support and serve as a barrier for the newly formed tissue [28]. If type III collagen is continuously overexpressed and cross-linking is insufficient, the fiber arrangement becomes disordered, which can easily lead to hypertrophic scarring. In the advanced phases, an increase in the ratio of type I to type III collagen and more organized bundle-like structures result in the wound becoming softer and flatter, with its function almost returning to normal [29]. Collagen staining was performed on the skin of each group, and, as shown in Figure 5B, compared with the control group, the LNP-mRNA_EGF group presented a significantly greater ratio of type I to type III collagen, indicating that this treatment can promote wound healing and improve skin function.

E-cadherin is the most abundant calcium-dependent adhesion molecule in epidermal cells. It forms junctions that “lock” adjacent keratinocytes together, ensuring that the entire epidermis remains intact under continuous external force without tearing [30]. The expression level of E-cadherin directly determines the integrity of the barrier and the ability to suppress inflammation [31]. Figure 5C shows that the expression of E-cadherin in the LNP-mRNA_EGF group was significantly greater than that in the control group, indicating that this formulation can not only accelerate healing but also rebuild the cellular barrier and suppress inflammation, demonstrating great potential for application.

Finally, H&E staining of samples from the heart, liver, spleen, lung, and kidney after treatment indicated preserved tissue architecture without any signs of inflammatory infiltration, necrosis, fibrosis, or immune-related injury (Figure S10). Concurrently, blood biochemical analysis showed no significant abnormalities. Collectively, these results indicate that LNP-mRNA_EGF exhibited a favorable safety profile in vivo during the treatment period (Figure S11).

This study aimed to develop an LNP-mRNA_EGF delivery platform and validate its overall therapeutic efficacy. Accordingly, the experimental design focused on endpoint measures such as wound closure rate, histological morphology, and changes in key protein expression. Within this framework, systematic longitudinal quantitative data on cellular behaviors—such as the migration rate of keratinocytes at the re-epithelialization front, and the dynamic number and spatial distribution of specific cell subsets in the dermis—were not collected. This limitation restricts a more in-depth, single-cell-level analysis of the dynamic mechanisms through which this treatment accelerates wound healing. Furthermore, although a single dose of 20 μg LNP-mRNA_EGF used in this study demonstrated significant pro-healing effects, whether this represents the optimal dosage regimen requires further systematic investigation and validation.

The data that support the findings of this study are available from the corresponding author upon request.