Bakuchiol-loaded amphiphilic polymeric nanoparticles: enhancing stability and multifunctionality through encapsulation

Abstract

Bakuchiol (BK), a natural meroterpene isolated from Psoralea corylifolia, has gained significant attention as a functional cosmetic ingredient due to its antioxidant, anti-inflammatory, and antibacterial properties. However, its high hydrophobicity and chemical instability limit its practical application. In this study, we developed a robust and efficient delivery system for BK using polyglyceryl-10 laurate (PG10-L), an amphiphilic polymer, via a simple and scalable nanoprecipitation strategy. This approach helps address an important challenge in achieving stable long-term aqueous formulations of BK without relying on complex synthetic processes. The resulting BK-loaded nanoparticles (BK@NPs) exhibited a small particle size (< 200 nm), high encapsulation efficiency (≈ 99%), and remarkable colloidal stability for over 12 weeks. Notably, BK@NPs significantly enhanced biological efficacy compared to free BK, resulting in markedly improved antioxidant and antibacterial performance. Furthermore, the formulation demonstrated excellent cytocompatibility in NIH-3T3 murine fibroblasts along with effective suppression of intracellular reactive oxygen species (ROS). These findings highlight that the PG10-L-based nanoprecipitation strategy provides a robust and green platform to enhance the multifunctional potential of bakuchiol for advanced dermo-cosmetic applications.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13036-026-00644-x.

Background

Bakuchiol (BK) is extracted from the seeds of the Babchi plant (Psoralea corylifolia) and is used in traditional medicine in India and China [1, 2]. It is a terpene-based phenol that exhibits strong antibacterial, antioxidant, anti-inflammatory, antiosteoporotic, and antitumor activities [3]. BK acts as a natural preservative in various formulations because of its excellent antibacterial properties. Therefore, it is a safe alternative to synthetic preservatives since it is both biocompatible and environmentally friendly [4, 5]. While extending the shelf life of products, its high biodegradability minimizes negative impacts on consumers and the environment; hence, it provides fresher and safer options [6]. Unlike many synthetic additives, BK is highly biocompatible as a naturally derived bioactive substance with relatively few reported side effects. In particular, its strong antioxidant ability helps neutralize free radicals, preventing oxidative stress and subsequent cell damage, thereby alleviating aging [7–9]. Furthermore, the anti-inflammatory properties of BK effectively manage inflammation by reducing redness and swelling [10–12].

However, the practical efficacy of BK is limited without appropriate formulation strategies [13]. Various studies have attempted to address these issues by processing BK using polymeric, lipid-based, or hybrid nanocarrier systems to improve its stability and bioactivity in aqueous environments [14–18]. Nevertheless, significant problems such as stability and shelf life issues, variability in effects, decreased absorption rates, and bioirritation remain [19]. In particular, BK is unstable in external environments such as in the presence of heat and oxygen and may become discolored or produce an unpleasant odor owing to oxidation and deterioration, significantly reducing the efficacy of the active ingredient [20]. This can cause adverse effects and allergic reactions. In addition, the fat-soluble characteristics of BK (log P ≈ 6.13) make it difficult to effectively penetrate biological tissues [21, 22]. In aqueous environments, free BK molecules readily undergo hydrophobic aggregation and phase separation [23, 24], which prevents the exposure of its functional groups to reactive species. Consequently, its activity is weakened in aqueous environments, making it challenging to achieve the expected effects [25]. Furthermore, the intensity of the stimulation applied to the body varies owing to an imbalance in concentration, leading to inconsistent effects [19].

Recent advances in nanomedicine have demonstrated that such delivery strategies can significantly enhance the delivery efficiency of hydrophobic phytochemicals, leading to improved bioavailability and favorable biological responses [26, 27]. Notably, biomimetic polymer-based systems have emerged as innovative platforms capable of modulating the cellular microenvironment and promoting regenerative processes [28, 29].

Despite these efforts, conventional BK delivery systems often suffer from low loading efficiency and insufficient long-term stability in aqueous media [16–19]. Many reported systems rely on complex multi-step fabrication procedures or high-energy processing, which complicates manufacturing and makes it difficult to preserve the biological activity of oxidation-sensitive BK [18–20]. Therefore, there is a pressing need for the development of a robust stabilization platform that can maintain BK bioactivity over extended periods without oxidation or phase separation, while simultaneously enabling a simplified manufacturing process with minimal organic solvent usage [18, 20].

In this study, we propose a nanoprecipitation-based formulation strategy, building upon innovative delivery technologies [30–33], by employing polyglyceryl-10 laurate (PG10-L), a highly biocompatible amphiphilic emulsifier [34, 35]. Owing to its amphiphilic molecular structure, PG10-L can effectively solubilize hydrophobic compounds without the need for high-energy processing, making it particularly suitable for BK encapsulation [36]. Compared with other amphiphilic carriers that often rely on complex block copolymers or multi-component surfactant systems, PG10-L enables a simplified single-component formulation, thereby reducing formulation complexity while remaining compatible with solvent-minimized nanoprecipitation processes. The nanoprecipitation approach adopted in this work enables rapid, reproducible, and environmentally benign “green synthesis,” thereby minimizing ecological impact while maximizing nanoparticle safety and biocompatibility [37–39]. The overall concept of this study—including the preparation of BK-loaded nanoparticles (BK@NPs; Fig. 1A) and the investigation of their antibacterial (Fig. 1B), antioxidant (Fig. 1C), and anti-inflammatory (Fig. 1D) properties—is summarized in Fig. 1. This plant extract–based nanoparticle platform is expected to maximize the functional potential of BK while offering broad applicability as an eco-friendly formulation strategy [40–42].

Fig. 1.

Schematic of (A) the manufacturing process for BK@NPs, (B) improved antibacterial effect of BK@NPs, (C) improved antioxidant effect of BK@NPs, and (D) NO production and reduction using Griess reagent after the treatment of LPS and BK@NPs for 24 h in RAW264.7 macrophage cells

Materials and methods

Materials

PG10-L was purchased from Nikko Chemicals (Tokyo, Japan). BK (≥ 95% purity, CAS No. 10309-37-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA; Product No. SMB00604). Deionized water (DIW) was purchased from HyClone (Logan, UT, USA). Methylene blue and DPPH were purchased from Sigma Aldrich. Staphylococcus aureus (American Type Culture Collection (ATCC) 6538) was used for the antibacterial analysis. Luria-Bertani (LB) agar was purchased from BD Difco (Franklin Lakes, NJ, USA). Murine fibroblast NIH 3T3 cells and RAW 264.7 murine macrophages (ATCC, MD, USA) were used for the in vitro assays. Dulbecco’s modified Eagle’s medium (DMEM) and penicillin-streptomycin (PS) were used for the in vitro cell culture experiments; fetal bovine serum (FBS) was obtained from Gibco (Grand Island, NY, USA). As reagents used for the in vitro MTT assay, dimethyl sulfoxide (DMSO, 99.8%) was obtained from Sigma-Aldrich, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was sourced from Invitrogen (Carlsbad, CA, USA). For the in vitro reactive oxygen species (ROS) scavenging assay, hydrogen peroxide (H₂O₂, 30%) was purchased from Junsei Chemical Co. (Tokyo, Japan), and 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) was obtained from Invitrogen. Lipopolysaccharide (LPS) and the Griess reagent used for the in vitro anti-inflammatory assays were purchased from Sigma-Aldrich. All the solvents were used in their original unpurified states.

Preparation of BK@NPs

BK NPs were prepared by the self-assembly of hydrophobic BK with amphiphilic PG10-L in an aqueous phase. PG10-L consists of a hydrophobic hydrocarbon chain and a hydrophilic carboxyl group. First, BK (1, 2, 3, 4, or 8 mg) and PG10-L (20 mg) were dissolved in 1 mL of EtOH in a 4 mL vial. The mixture was then stirred on a rotary shaker for 2 h at room temperature. Subsequently, the EtOH solution (1 mL) containing BK and PG10-L was delivered into 5 mL of DIW using a syringe pump (LEGATO 100, KD Scientific) under stirring at 530 rpm. The mixture was stirred at 530 rpm for 1 h to obtain the BK@NPs. EtOH was then removed by vacuum evaporation (approximately 2 h), and the dispersion was adjusted with DIW to a final volume of 5 mL. The NPs were prepared by incorporating BK loadings of 5, 10, 15, 20, and 40 wt% relative to PG10-L, and the loadings were carefully measured. The resulting products, designated as BK@NP × wt% (× wt% represents the BK loading of PG10-L), were prepared using a procedure identical to that used to produce the BK-free NPs of PG10-L, designated as BK@NP 0 wt%. For clarity, unless otherwise stated, the BK@NP concentrations are reported as NP mass. The BK-equivalent concentration (BK_eq) represents the mass concentration of BK within the NPs and was calculated as BK_eq = (NP mass) × (loading), and free BK was administered at BK_eq values matched to BK@NPs (Table 1).

Table 1.

Formulation composition and preparation conditions of BK@NPs

Sample	BK (mg)	PG10-L (mg)	BK loading (wt% vs. PG10-L)	EtOH volume (mL)	DIW volume (mL)	Final NP concentration (mg/mL)*
BK@NPs 0 wt%	0	20	0	1	5	4
BK@NPs 5 wt%	1	20	5	1	5	4
BK@NPs 10 wt%	2	20	10	1	5	4
BK@NPs 15 wt%	3	20	15	1	5	4
BK@NPs 20 wt%	4	20	20	1	5	4
BK@NPs 40 wt%	8	20	40	1	5	4

* Final NPs concentration is reported as total nanoparticle mass per volume after ethanol removal and volume adjustment to 5 mL with DIW

Characterization of BK@NPs

The physical and chemical properties of BK@NPs were characterized. The surface charge, hydrodynamic diameter, and polydispersity index (PDI) of the BK@NPs were measured using electrophoretic light-scattering spectroscopy (ELS-Z2; Otsuka Electronics Co., Tokyo, Japan) in combination with dynamic light scattering (DLS) analysis. In addition, the morphologies of the BK@NPs were examined at 25 °C using transmission electron microscopy (TEM, JEM-2100PlusHR, JEOL, Tokyo, Japan). TEM observations were performed by dropping the NP solution onto a carbon-coated copper grid without staining, followed by drying for 3 d at room temperature. The chemical structures and encapsulation status of the nanoparticles were analyzed using Fourier transform infrared spectroscopy (FT-IR). The spectra of pure BK, PG10-L, and freeze-dried BK@NPs were acquired using a PerkinElmer Frontier spectrometer (PerkinElmer, Waltham, MA, USA) in the scanning range of 400–4,000 cm⁻¹ with a resolution of 4 cm⁻¹ using the KBr pellet technique. To visually confirm the encapsulation, stability, and transparency of the dispersed phase, a dye solubility test using methylene blue was employed. A methylene blue solution prepared in distilled water was mixed with the BK@NP suspension (1:1 v/v) and stirred thoroughly. The mixture was then visually observed to determine the dispersion state and check for any precipitation [43]. After purifying the unloaded BK using Amicon Ultra-15 centrifugal filters with a molecular weight cutoff of 100 kDa, the loading efficiency and BK content of the NPs were evaluated using high-performance liquid chromatography (HPLC). Ultrafiltration was performed for 3 min at 1000 rpm. The following HPLC conditions were utilized in this experiment: a wavelength of 262 nm and an injection volume of 20 µL were employed to detect the sample, while a mobile phase comprising water and acetonitrile served as the eluent. The eluent was delivered at a constant rate of 1 mL/min under a controlled temperature of 35 °C. The gradient program consisted of the following steps: From 0 to 8 min, the percentage of water gradually increased from 0% to 36% in acetonitrile. From 8 to 20 min, the percentage of water gradually increased from 36% to 100% in acetonitrile. Finally, from 20 min to 23 min, the percentage of water remained constant at 100%. The retention time of BK was 11.473 min. The loading content and efficiency were determined as follows [44, 45]:

Where weight of fed BK is the initial mass of bakuchiol added during preparation, weight of unloaded BK is the mass of free bakuchiol measured in the filtrate after ultrafiltration, and weight of NPs is the total mass of the obtained nanoparticles.

Long-term stability of BK@NPs

The long-term stability of the optimized BK@NPs (15 wt%) was evaluated by monitoring the changes in hydrodynamic diameter and PDI. The nanoparticles were incubated in phosphate-buffered saline (PBS, pH 7.4) and 10% fetal bovine serum (FBS) at 25 °C and 37 °C, respectively. The measurements were conducted at predetermined time intervals (0, 1, 3, 5, 7, and 14 days) using DLS to assess their structural integrity under storage and physiological conditions.

In vitro drug release assay of BK@NPs

The in vitro release profile of BK from BK@NPs was evaluated using the dialysis bag diffusion technique. Briefly, 2 mL of the BK@NP dispersion (15 wt%) was placed into a ready-to-use dialysis device (Spectra-Por^® Float-A-Lyzer^® G2, MWCO 100 kDa; Spectrum Labs, USA). The device was immersed in 20 mL of phosphate-buffered saline (PBS). The release study was conducted at 37 ℃ at pH 5.0 and pH 7.4 under constant stirring. At predetermined time intervals (0, 0.5, 1, 2, 4, 8, 24, and 48 h), 1 mL of the release medium was withdrawn and replaced with an equal volume of fresh medium. The amount of released BK was quantified using HPLC.

Intracellular cargo release of BK@NPs

To visualize the cellular uptake and subsequent intracellular release, Nile Red (NR) was encapsulated into the NPs as a hydrophobic fluorescent model drug. NIH-3T3 cells were seeded in 24-well plates and incubated for 24 h. The cells were then treated with NR-loaded NPs and incubated for various time points (0, 0.5, 1, 2, 4, 8, 24, and 48 h). After incubation, the cells were washed three times with PBS to remove unbound NPs. The intracellular fluorescence of NR was observed using an inverted fluorescence microscope (Leica DMI8; Leica Microsystems, Germany) equipped with a Leica DFC7000 GT camera. Image acquisition and processing were performed using the LAS X software (Leica Microsystems).

In vitro cytotoxicity of BK@NPs

The biocompatibility of BK@NPs was assessed using NIH-3T3 murine fibroblast cells cultured in DMEM supplemented with 1% penicillin–streptomycin and 10% FBS. After the addition of BK@NPs, the cell viability was measured after 24 h using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Initially, cells were grown in 96-well plates, with each well containing 10,000 cells. After a 24-hour incubation period, cells were exposed to different concentrations of BK@NPs (from 1 to 100 µg/mL) and incubated at 37 °C for an additional 24 h. Afterwards, a medium containing 1 mg/mL MTT solution was added to each well, and the plates were incubated for 3 h. After incubation, the medium was removed, and the crystalline purple formazan dye was dissolved in dimethyl sulfoxide (DMSO). The optical density of the formazan produced by viable cells was measured at 570 nm using a microplate reader (BioTek, Winooski, VT, USA). The percentage of viable cells was determined using the following formula [46, 47]:

Here, ∆Α570 of test group represents the optical density (absorbance) of the cells treated with BK@NPs, and ∆Α570 of control group represents the absorbance of the untreated control cells.

To visually validate the cytotoxicity results, a Live/Dead assay was performed using a commercial kit (Live/Dead™ Viability/Cytotoxicity Kit, Invitrogen). NIH-3T3 cells were treated with various formulations of BK@NPs as described in the MTT assay section. After 24 h of incubation, the cells were stained with calcein-AM (2 µM) and ethidium homodimer-1 (4 µM) for 30 min at room temperature. The stained cells were imaged using a fluorescence microscope (Leica DMI8, Germany) to distinguish between live (green fluorescence) and dead (red fluorescence) cells.

Antibacterial activity of BK@NPs

The antibacterial activity of BK@NPs was evaluated against Staphylococcus aureus (S. aureus) (ATCC 6538, Gram-positive) and Escherichia coli (E. coli) (Gram-negative) using the broth microdilution method to determine the minimum inhibitory concentration (MIC), in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [48]. Bacteria were cultured in LB broth at 37 °C until reaching an optical density (OD600) of 0.1. The bacterial suspension was then treated with BK@NPs at various concentrations (31.25–4000 µg/mL) in 96-well plates and incubated for 24 h. The bacterial growth was monitored by measuring the absorbance at 600 nm using a microplate reader. To account for the turbidity interference caused by high concentrations of nanoparticles, the minimum bactericidal concentration (MBC) was determined by plating aliquots from the MIC wells onto agar plates. After incubation at 37 °C for 24 h, the plates were inspected for colony formation to distinguish between bacterial growth and nanoparticle turbidity.

DPPH radical scavenging activity of BK@NPs

The DPPH assay was used to assess the antioxidant activity of BK@NPs. Control groups were prepared using solutions with different concentrations (0–15 wt%) of BK in DIW. BK@NPs were prepared using four different BK concentrations (0, 5, 10, and 15 wt%). A 0.2-mM DPPH solution was diluted with ethanol, stirred for approximately 1 h, and then placed in a dark environment at 4 °C. Each BK@NP colloidal suspension was then combined with 50 µL of the prepared DPPH solution, resulting in a total volume of 200 µL. The control group consisted of a 50-µL aliquot of the DPPH solution mixed with 150 µL of DIW, which exhibited minimal antioxidant activity. Ascorbic acid (AA) was used as a positive control, whereas BK in DIW was used as a negative control for direct comparison with BK@NPs. The mixtures were incubated in the absence of light at a temperature of 25 °C for a period of 24 h. The absorbance of each mixture was measured at 515 nm by using a microplate reader (VICTOR X5; PerkinElmer, Singapore). The antioxidant activity was assessed using the following Eqs [49, 50]:

Where ∆Α515 of control is the absorbance of the DPPH solution without nanoparticles, and ∆Α515 of sample is the absorbance of the reaction mixture containing BK@NPs.

In vitro antioxidant activity of BK@NPs

To demonstrate the cellular antioxidant effects of the BK@NPs containing 15 wt% BK, NIH-3T3 mouse embryonic fibroblasts were cultured in 96-well plates at a density of 10,000 cells/well for 24 h. Oxidative stress was induced in fibroblasts using H₂O₂. After applying the samples to the cells, the subsequent changes in the ROS levels were quantified using the following methodology: We exposed fibroblasts to various concentrations of BK@NPs (1 to 1000 nM), along with 10 µM H₂O₂, and cultured them for 8 h. For this purpose, a negative control group without H₂O₂ and a positive control group with H₂O₂ were included. Subsequently, the NIH-3T3 cells were rinsed with PBS (200 µL per well) to remove any residual sample solution. Following this, the cells were incubated with a 10 µM H2DCFDA solution (100 µL per well) for 30 min in a dark environment. This solution served as a fluorescent probe for ROS detection. Finally, the antioxidant activity of the samples was quantified by measuring the fluorescence, in particular, the intensity of oxidized dichlorofluorescein (DCF), using a microplate reader with excitation and emission wavelengths of 485 and 535 nm, respectively [51, 52].

In vitro anti-inflammatory activity of BK@NPs

To assess the anti-inflammatory properties of BK@NPs within the cells, 15,000 RAW 264.7 cells were placed into each well (200 µL) of a 96-well plate and incubated for 24 h. LPS (20 ng/mL) was used to trigger an inflammatory response in RAW 264.7 cells, resulting in the production of nitric oxide (NO). Initially, the cells were treated with varying concentrations of BK@NPs (10 to 100 µg/mL) in the presence of LPS (20 ng/mL) and incubated for another 24 h. The negative control group was incubated without LPS or BK@NPs (and an LPS-only group served as the positive control for NO induction) for comparison with the experimental groups. Subsequently, the supernatant was carefully transferred to a fresh 96-well plate. Griess reagent was added to each well, and the plate was incubated for 10 min at room temperature. Finally, a microplate reader (VICTOR X5, PerkinElmer, Singapore) was used to measure the absorbance at 560 nm. The concentration of NO in the samples was quantified to evaluate the in vitro anti-inflammatory properties of BK@NPs [53, 54].

Statistical analysis

The experimental data were presented as mean values ± standard deviation, with each experiment conducted in triplicate. To compare the variations between the experimental groups, we used Student’s t-test. Statistical significance was confirmed for all assessments, with a p-value < 0.05.

Results and discussion

Characterization of BK@NPs

We formulated PG10-L NPs containing different concentrations of BK ranging from 0 to 40 wt%. PG10-L was chosen because of its excellent biocompatibility, safety profile, and amphiphilic structure, which facilitated self-assembly into stable NPs with a hydrophilic outer layer and a hydrophobic inner core in water, making it highly suitable for encapsulating hydrophobic compounds such as BK. As the BK concentration increased from 0 to 40 wt%, the size (Fig. 2A) and PDI (Fig. 2B) of the BK@NPs increased, indicating poor NP formation at 40 wt%. Moreover, the zeta potential of BK@NPs remained in a highly negative range with an increase in the BK concentration, reaching − 33.45 ± 0.6 mV at 40 wt%. This can be attributed to the presence of unencapsulated BK or changes in surface charge distribution (Fig. 2C). The state of the 15 wt% BK@NPs was visualized using TEM (Fig. 2D). The encapsulation of BK was visually confirmed using a methylene blue solubility test. BK dispersed in water appeared cloudy, indicating fine particle dispersion (Fig. 2E).

Fig. 2.

(A) Initial hydrodynamic diameter, (B) polydispersity index (PDI), and (C) zeta potential of BK@NPs with different BK loadings in the range of 5–40 wt%. (D) TEM images of 15wt% BK@NPs. (E) Digital images of the methylene-blue staining test. Data are presented as mean ± SD (n = 3)

When more BK was incorporated into the BK@NPs, the particles became blurry, whereas lower loading rates resulted in more distinct and well-defined particles. The addition of methylene blue resulted in a transparent suspension of BK@NPs containing up to 15 wt% BK, demonstrating the successful encapsulation of BK within the NPs at this concentration threshold. However, when the BK concentration reached 20 wt%, the suspension was not transparent, indicating the presence of unencapsulated BK. To further verify the encapsulation at the molecular level, FT-IR analysis was performed (Fig. S1). The spectrum of pure BK exhibited characteristic peaks, including O-H stretching at ~ 3350 cm^− 1 and C = C stretching vibrations at 1500–1650 cm^− 1. However, in the BK@NPs spectrum, these specific peaks of BK were largely masked by the broad signals of the PG10-L matrix. The absence of distinct BK peaks in the nanoparticle spectrum indicates that the drug was successfully entrapped within the hydrophobic core of the PG10-L micelles via hydrophobic interactions, rather than being adsorbed on the surface. To determine the encapsulation efficiency, the BK loading content (L.C.) and loading efficiency (L.E.) were evaluated using HPLC. The nanoparticles exhibited excellent encapsulation capability with L.E. values exceeding 99% for formulations with 5, 10, and 15 wt% BK loadings. Specifically, the 15 wt% BK@NPs showed a high L.E. of 99.87% and an L.C. of 14.98%, while maintaining a uniform particle size and narrow size distribution (PDI = 0.21). However, increasing the loading to 20 wt% resulted in a significant drop in L.E. to approximately 69.8% and led to particle instability. Based on these results, the 15 wt% formulation was determined to be the optimal condition and was used for subsequent experiments.

Long-term stability of BK@NPs

First, the long-term storage stability of BK@NPs was evaluated using DLS to determine the hydrodynamic diameters, PDI, and zeta potential changes under aqueous solution (DIW) conditions at 25 °C for 12 weeks (Fig. 3). The hydrodynamic diameters of the BK@NPs were stable without noticeable changes over a long period (up to 12 weeks) at BK concentrations ranging from 5 to 15 wt%. Moreover, the hydrodynamic diameters of all samples were less than 200 nm. In terms of size distribution, the optimized formulations (5–15 wt%) exhibited PDI values ranging from 0.18 to 0.22 (Fig. 2B) [55], indicating a relatively narrow and uniform distribution close to the ideal range for monodisperse systems. Although higher drug loadings (e.g., 40 wt%) resulted in increased heterogeneity, the selected 15 wt% BK@NPs maintained a low PDI (< 0.25) consistently over the 12–week monitoring period (Fig. 3B). This stability confirms that the nanoparticles are thermodynamically stable and resistant to aggregation in aqueous media. Additionally, as shown in Fig. 3C, the zeta potential of the BK@NPs remained stable in the range of -25 to -35 mV without significant fluctuations over the 12-week period, further demonstrating the surface charge consistency which contributes to electrostatic repulsion and colloidal stability. To further evaluate the robustness of the optimized nanoparticles under physiological conditions, we monitored the stability in phosphate-buffered saline (PBS, pH 7.4) and 10% fetal bovine serum (FBS) for 14 days (Fig. S2). Consistent with the DIW results, the nanoparticles exhibited excellent stability in PBS, maintaining their particle size (~ 150 nm) and PDI (< 0.2) throughout the 14-day period. This confirms structural integrity in saline environments. In the presence of 10% FBS, which mimics the protein-rich biological environment, the nanoparticles maintained their initial size and dispersity for up to 3 days. However, a gradual increase in particle size (to ~ 350 nm) and PDI was observed starting from day 5, likely due to the formation of a protein corona or non-specific interactions with serum proteins over extended periods. Although this instability might limit applications requiring prolonged systemic circulation, it presents no significant drawback for the primary intended use of BK@NPs in topical or dermo-cosmetic applications. Notably, the 3-day (72 h) stability window in serum fully covers the functional timeframe of the nanoparticles, as our release (Fig. 4A) and uptake (Fig. 4B) studies confirmed that BK delivery and internalization are largely completed within 48 h. Thus, the observed stability is considered sufficient for the intended biological application. Collectively, these results indicate that the fabricated BK@NPs have remarkable stability in both storage and biological environments, making them a reliable platform for sustained drug delivery.

Fig. 3.

Long-term stability of BK@NPs at BK concentration ranging from 5 to 15wt%. Variations in (A) hydrodynamic diameters, (B) polydispersity index (PDI), and (C) zeta potential of BK@NPs over 12 weeks. Data are presented as mean ± SD (n = 3)

Fig. 4.

(A) In vitro cumulative release profiles of BK from BK@NPs in PBS at pH 5.0 and pH 7.4 over 48 h at 37 °C. Measurements were taken at predetermined time intervals of 0.5, 1, 2, 4, 8, 24, and 48 h. (B) Fluorescence microscopy images of NIH-3T3 cells treated with Nile Red-loaded NPs for different time intervals (0, 0.5, 1, 2, 4, 8, 24, and 48 h), demonstrating time-dependent cellular uptake and intracellular cargo release. Data are presented as mean ± SD (n = 3)

In vitro release and intracellular behavior of BK@NPs

The in vitro cumulative release profiles of BK from BK@NPs in PBS (pH 7.4 and pH 5.0) are presented in Fig. 4A. At physiological pH (7.4), BK@NPs exhibited a biphasic release pattern characterized by an initial burst (~ 20% within 2 h), likely due to drug molecules adsorbed near the particle surface, followed by a gradual release phase reaching ≈ 90% after 48 h. This sustained release profile suggests that the PG10-L encapsulation effectively controls the diffusion of the hydrophobic cargo over an extended period, consistent with typical controlled-release nanocarrier systems [56]. Interestingly, the release was notably suppressed under acidic conditions (pH 5.0), reaching only ≈ 47% after 48 h. This significant difference compared to the release at pH 7.4 indicates that the PG10-L nanoparticles maintain superior structural integrity in acidic environments. This pH-dependent stability is advantageous for preventing premature drug leakage in acidic biological fluids or storage conditions, while ensuring effective release at physiological pH. To corroborate these findings in a biological system, the intracellular behavior was monitored in NIH-3T3 cells using NR-loaded NPs (Fig. 4B). A time-dependent increase in red fluorescence was observed from 0 to 48 h. While only weak fluorescence was detected initially (0–2 h), the intensity significantly increased after 4 h and reached its maximum level at 48 h within the monitored period. This trend mirrors the in vitro sustained release behavior at physiological pH, confirming that the nanoparticles effectively internalize and gradually release their hydrophobic payload within the cellular environment.

In vitro cytotoxicity activity of BK@NPs

To determine the optimal formulation range and safety profile, the cytotoxicity of BK@NPs was evaluated in two stages using the MTT assay and further validated by Live/Dead staining. The overall experimental scheme and the results of the formulation optimization are presented in Fig. 5. First, we assessed the viability of NIH-3T3 cells treated with BK@NPs loaded with varying amounts of BK (0, 5, 10, 15, and 20 wt%) at a fixed nanoparticle concentration of 100 µg/mL (Fig. 5A). This concentration represents the upper limit of the tested concentration range in this study. Formulations with up to 15 wt% BK loading maintained high cell viability (> 90%), comparable to the untreated control (CTL), even at this concentration. However, the 20 wt% BK@NPs group showed a significant reduction in viability (~ 65%), likely due to the instability of the nanoparticles and the presence of unencapsulated free BK. This toxicity at 20 wt% was visually corroborated by Live/Dead staining (Fig. 5B), where a substantial number of dead cells (red fluorescence) were observed, whereas the 0–15 wt% groups showed predominantly live cells (green fluorescence). Based on these results, 15 wt% was selected as the optimal loading content.

Fig. 5.

In vitro cytotoxicity of BK@NPs in NIH-3T3 cells. (A) Cell viability of NIH-3T3 cells treated with BK@NPs with varying BK loading contents (0–20 wt%) at a fixed nanoparticle concentration of 100 µg/mL. (B) Representative Live/Dead fluorescence images corresponding to the treatments in (A). Live cells are stained green (calcein-AM), and dead cells are stained red (ethidium homodimer-1). The 20 wt% group exhibits significant toxicity at this dose. The scale bar represents 500 μm. Data are presented as mean ± SD (n = 3)

To further confirm the safety of the optimized formulation, we evaluated the dose-dependent cytotoxicity of 15 wt% BK@NPs at concentrations ranging from 1 to 100 µg/mL (Fig. S3). The cells retained high viability (> 90%) across all concentrations, including the highest dose of 100 µg/mL (Fig. S3A). The Live/Dead assay images were consistent with the quantitative MTT data, showing a dense population of viable green cells with negligible red staining across all tested concentrations (Fig. S3B). Collectively, these results confirm that the optimized 15 wt% BK@NPs possess excellent biocompatibility. This favorable safety profile is closely correlated with the drug release kinetics shown in Fig. 4. The sustained release of bakuchiol from the PG10-L matrix prevents sudden exposure to high concentrations of the drug (dose dumping), thereby mitigating potential cellular stress and cytotoxicity while maintaining therapeutic efficacy.

Antibacterial activity of BK@NPs

To comprehensively evaluate the antibacterial efficacy, we conducted both a colony counting assay at a fixed dose and a standard MIC determination assay. First, the bactericidal effect was confirmed against S. aureus. At a fixed concentration of 600 µg/mL (BK-eq), BK@NPs significantly inhibited bacterial survival compared to free BK (Fig. 6A). Subsequently, to determine the effective concentration range and selectivity, a broth microdilution assay was performed against Gram-positive S. aureus and Gram-negative E. coli (Fig. 6B). BK@NPs exhibited potent inhibitory activity against S. aureus, suppressing bacterial growth to near-baseline levels starting from concentrations as low as 31.25 µg/mL. Interestingly, at high concentrations (> 1000 µg/mL), an apparent increase in OD600 was observed. However, this was attributed to the inherent turbidity of the high-concentration nanoparticle suspension rather than bacterial growth. This was confirmed by the MBC assay (Fig. 6C), where plating of the suspensions from 1000, 2000, and 4000 µg/mL wells resulted in no colony formation, proving complete bacterial eradication. In contrast, no significant inhibition was observed against E. coli across all tested concentrations, with growth levels comparable to the growth control. This indicates that BK@NPs exhibit selective antibacterial activity specific to Gram-positive bacteria, which aligns with previous reports on the spectrum of bakuchiol [2]. Furthermore, this potent antibacterial efficacy can be correlated with the release kinetics of the nanoparticles (Fig. 4). The nanoparticles act as a drug reservoir, ensuring that the bacteria are continuously exposed to effective concentrations of bakuchiol above the MIC level over the 24 h incubation period, which facilitates complete bacterial eradication as evidenced by the MBC results.

Fig. 6.

Antibacterial activity of BK@NPs. (A) Bacterial viability of S. aureus treated with free BK and BK@NPs at a fixed dose (600 μg/mL BK-eq), assessed by colony counting. (B) MIC determination using broth microdilution against S. aureus (Gram-positive) and E. coli (Gram-negative). The dashed red line indicates the growth control, and the dashed grey line indicates the sterility control. Note that the increased OD at high concentrations (>1000 µg/mL) in S. aureus is due to nanoparticle turbidity. (C) MBC confirmation plates for S. aureus treated with various concentrations of BK@NPs. The absence of colonies at 1000–4000 µg/mL confirms the bactericidal effect despite the high OD readings. Data are presented as mean ± SD (n = 3). (*p < 0.05)

In vitro antioxidant activity of BK@NPs

To assess the antioxidant activity of BK@NPs, DPPH and in vitro radical-scavenging activity assays were conducted. First, the antioxidant ability of the BK@NPs was evaluated using the DPPH radical-scavenging method. BK in DIW exhibited very low DPPH radical-scavenging activity, while BK@NPs showed significantly higher scavenging activity than free BK. This remarkable difference stems from the high lipophilicity of bakuchiol (reported log P ≈ 6.13), which leads to its immediate aggregation and phase separation in aqueous environments, thereby shielding its active phenolic groups from reactive species [21, 23]. This enhancement can be attributed to the improved aqueous dispersion of bakuchiol and its protection within the nanoparticle core, which helps preserve antioxidant functionality during the assay. The antioxidant effect of BK@NPs increased with an increase in the BK concentration from 5 to 15 wt% (Fig. 7A). In the case of 0 wt% BK@NPs (blank control without BK), DPPH radical scavenging was negligible, indicating that PG10-L (a solubilizer) had little effect on the antioxidant activity. These results indicate that nanoencapsulation is essential for preserving the antioxidant functionality of BK in aqueous environments. Compared with previously reported lipid- or polymer-based bakuchiol carriers, which often involve complex multi-step fabrication or multiple surfactants, the present BK@NPs offer a distinct advantage by employing a streamlined one-step nanoprecipitation strategy using a single amphiphilic solubilizer (PG10-L). This approach not only simplifies the manufacturing process but also achieves high encapsulation efficiency and long-term colloidal stability, thereby helping to mitigate typical trade-offs between formulation simplicity and delivery performance. In addition to improved aqueous dispersibility, the enhanced antioxidant activity of BK@NPs can be attributed to a combination of sustained release behavior and improved cellular accessibility. The controlled release of BK from the nanoparticle core is expected to maintain the temporal availability of bioactive BK, facilitating more effective interactions with reactive oxygen species (ROS) over time [57, 58]. Furthermore, as observed in the cellular uptake study (Fig. 4B), BK@NPs with sizes in the range of 100–200 nm were efficiently internalized, while the amphiphilic nature of the PG10-L carrier may promote interactions with the cellular membrane [59]. Together, these factors are expected to increase intracellular delivery efficiency compared with free BK [60], thereby contributing to the pronounced antioxidant activity observed in both chemical and cellular assays.

Fig. 7.

Antibacterial activity of BK@NPs. (A) Bacterial viability of S. aureus treated with free BK and BK@NPs at a fixed dose (600 µg/mL BK-eq), assessed by colony counting. (B) MIC determination using broth microdilution against S. aureus (Gram-positive) and E. coli (Gram-negative). The dashed red line indicates the growth control, and the dashed grey line indicates the sterility control. Note that the increased OD at high concentrations (> 1000 µg/mL) in S. aureus is due to nanoparticle turbidity. (C) MBC confirmation plates for S. aureus treated with various concentrations of BK@NPs. The absence of colonies at 1000–4000 µg/mL confirms the bactericidal effect despite the high OD readings. Data are presented as mean ± SD (n = 3). (*p < 0.05)

Subsequently, the intracellular ROS scavenging activity was evaluated in H₂O₂-stimulated NIH-3T3 fibroblasts using the H2DCFDA assay. With increasing BK@NPs concentration (1→1000 nM), the cellular ROS levels in the 15 wt% BK@NPs group decreased to ≈ 75%, ≈ 45%, ≈ 35%, and ≈ 15% of the H2O2-treated control (ROS group), respectively (corresponding to approximately 25%, 55%, 65%, and 85% reductions, respectively; Fig. 8B). In comparison, the ROS level of BK in DIW remained similar to that of the control at 1–10 nM and decreased to approximately 80% and 60% at 100 and 1000 nM, respectively. On the other hand, the ROS level of 0 wt% BK@NPs remained similar to that of the control at 1–10 nM, showed a transient decrease to approximately 55% at 100 nM, and returned to approximately 90%–100% at 1000 nM. Therefore, these two samples may have low in vitro ROS-scavenging activities. These results further emphasize the fact that the encapsulation of BK improves its antioxidant activity, thereby improving its activity in the aqueous phase. Similar studies using lipid- or polymer-based BK nanocarriers have reported moderate increases in antioxidant activity [61]. In contrast, our PG10-L encapsulation approach resulted in a significant ROS reduction of > 90%, which underscores its excellent radical-scavenging performance. This sustained antioxidant protection is also supported by the release profile, where the continuous release of BK allows for prolonged scavenging of intracellular ROS.

Fig. 8.

(A) Antioxidant activity of BK@NPs (0–15 wt%) compared to BK in DIW, assessed using the DPPH radical-scavenging assay. AA served as the positive control, and 0 wt% BK@NPs denotes the blank NP control (NP mass-matched). Values are expressed as % inhibition relative to the vehicle. (B) Intracellular ROS scavenging activity assessed using the H2DCFDA assay. NIH-3T3 cells were treated with 15 wt% BK@NPs at concentrations ranging from 1 to 1000 nM. Values are expressed as a percentage of the H₂O₂-treated control (ROS group = 100%). The ROS group represents the oxidative stress condition (highest ROS level), while the CTL group represents the untreated negative control (lowest ROS level). Data are presented as mean ± SD (n = 3). (*p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.0001)

In vitro anti-inflammatory activity of BK@NPs

The anti-inflammatory potential of BK@NPs was assessed by quantifying the inhibition of NO production in LPS-stimulated RAW 264.7 cells. When RAW 264.7 cells were treated with 15 wt% BK@NPs, NO production was significantly reduced, showing a dose-dependent reduction compared to the LPS-treated group (Fig. 8). In particular, at a concentration of 100 µg/mL, the NO levels were reduced by more than 96%. In addition, NO production was reduced by up to 20% and 8% in DIW and 0 wt% BK@NPs, respectively. These results indicate that PG10-L has little anti-inflammatory effect, and BK@NPs demonstrate an excellent inhibitory effect on the LPS-induced increase in NO concentration. Other BK encapsulation approaches have demonstrated partial NO suppression, but the PG10-L BK@NPs system developed in this study achieved nearly complete inhibition (> 96%), highlighting its potential advantage in anti-inflammatory applications.

Conclusion

In this study, we formulated BK-loaded NPs (BK@NPs) via a nanoprecipitation approach using PG10-L as a simple and scalable strategy to overcome the intrinsic solubility and stability limitations of bakuchiol in aqueous environments. The proposed nanoencapsulation platform effectively preserved the functional performance of bakuchiol, demonstrating significant antioxidant, anti-inflammatory, and antibacterial activities, while maintaining colloidal stability and biocompatibility. Blank particles (0 wt% BK) served as particle-only controls, allowing the contribution of the BK payload to be distinguished from carrier effects. Compared with previously reported self-assembling BK nanocarriers [62], the present formulation showed comparable or improved encapsulation efficiency and sustained colloidal stability under the examined conditions, although direct comparisons under identical protocols remain necessary. Overall, our findings support the feasibility of BK@NPs as a versatile bakuchiol delivery platform capable of enabling consistent bioactivity across multiple functional assays. Given that the current evidence is limited to in vitro and microbiological evaluations, future studies should investigate release kinetics in complex media, batch-to-batch consistency, scale-up reproducibility, comprehensive inflammatory cytokine profiling, and in vivo safety and efficacy. Such studies will be essential for translating this platform toward pharmaceutical, cosmetic, and functional product applications.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AA

Ascorbic acid

ATCC

American Type Culture Collection

BK

Bakuchiol

BK_eq

Bakuchiol-equivalent

BK@NPs

Bakuchiol-loaded nanoparticles

CLSI

Clinical and Laboratory Standards Institute

CTL

Control

DCF

Dichlorofluorescein

DIW

Deionized water

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified Eagle’s medium

DMSO

Dimethyl sulfoxide

DPPH

2,2-diphenyl-1-picrylhydrazyl

EtOH

Ethanol

FBS

Fetal bovine serum

FT-IR

Fourier transform infrared spectroscopy

H2DCFDA

2,7-dichlorodihydrofluorescein diacetate

HPLC

High-performance liquid chromatography

LB

Luria-Bertani

L.C.

Loading content

L.E.

Loading efficiency

LPS

Lipopolysaccharide

MBC

Minimum bactericidal concentration

MIC

Minimum inhibitory concentration

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

MWCO

Molecular weight cut-off

NO

Nitric oxide

NPs

Nanoparticles

NR

Nile Red

OD

Optical density

PBS

Phosphate-buffered saline

PDI

Polydispersity index

PG10-L

Polyglyceryl-10 laurate

PS

Penicillin-streptomycin

ROS

Reactive oxygen species

SD

Standard deviation

TEM

Transmission electron microscopy

Author contributions

Dongseong Seo: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Sohyeon Yu: Writing – original draft, Validation, Methodology, Formal analysis, Data curation. Taeho Kim: Software, Methodology, Investigation, Data curation. Jonghyun Park: Visualization, Validation. Hyungjun Kim: Validation, Resources, Funding acquisition, Conceptualization. Daekyung Sung: Writing – review & editing, Supervision, Resources, Methodology, Conceptualization.

Funding

This research was financially supported by the Ministry of Small- and Medium-Sized Enterprises (SMEs) and Startups (MSS), Korea, under the “Regional Specialized Industry Development Plus Program (R&D, S3369610)” supervised by the Korea Technology and Information Promotion Agency for SMEs (TIPA). This work was supported by the National Research Foundation of Korea (NRF) [grant number 2021M3C1C3097647] funded by the Korean Government (MSIT) and by the Korea Institute of Ceramic Engineering and Technology (KICET) [grant number 1415181794].

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.