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. 2026 Jul 14;16(37):40388–40396. doi: 10.1039/d6ra01613h

Totarol loaded lipase-responsive liposomes for anti Gram-positive bacterium infection

Ziwu Zhang a,✉, Ying Huang a, Chunyan Wu a, Wenhao Xiang a, Chun Lin a, Ning Ji a
PMCID: PMC13367349  PMID: 42453955

Abstract

This study presents a dual-precision liposomal platform (denoted Tot-Lip) rationally engineered for acne vulgaris treatment by synergistically integrating hyaluronic acid (HA)-CD44 targeting and Cutibacterium acnes (C. acnes) lipase-responsive release. Unlike conventional lipase-responsive systems with insufficient site specificity, our formulation features a soybean phosphatidylcholine/glycerol monostearate lipid bilayer decorated with HA to realize two core functions: (1) targeted accumulation at inflamed acne lesions via binding to CD44 receptors overexpressed on inflammatory cells, and (2) spatiotemporally controlled on-demand drug release triggered by C. acnes-secreted lipase. Comprehensive physicochemical characterization validated a well-defined nanostructure (67.2 nm, PDI 0.16) and favorable stimuli-responsive release profiles. In vitro assays verified strong antibiofilm activity against C. acnes, and HA surface modification markedly improved lesion-specific accumulation. Mechanistic studies using scanning electron microscopy (SEM) ultrastructural observation identified bacterial membrane disruption as the dominant bactericidal mechanism. Furthermore, Tot-Lip exerts prominent anti-inflammatory and soothing effects. This delivery system boosts therapeutic efficacy by simultaneously improving aqueous solubility, sustaining drug release, and enabling pathogen-specific activation. Collectively, this multifunctional design offers a promising approach for treating biofilm-associated skin infections through lipase-triggered precise antimicrobial delivery.

This study presents a dual-precision liposomal platform (Tot-Lip) for acne vulgaris, integrating HA‑CD44 targeting and C. acnes lipase‑responsive release.graphic file with name d6ra01613h-ga.jpg

1. Introduction

Gram-positive bacteria are major etiological agents of human infectious diseases, notably including Staphylococcus aureus (S. aureus) and Cutibacterium acnes (C. acnes). Localized cutaneous infections induced by these microorganisms can manifest as dermatitis, acne, and related pathologies.1 Historically, Gram-positive infections exhibit a clinical spectrum ranging from superficial manifestations to life-threatening systemic conditions, potentially inducing functional impairments in affected tissues.2 The skin acts as the primary barrier against microbial invasion. Constantly interacting with the external environment, it harbors a complex microbiota, with upper body regions predominantly colonized by Gram-positive species including Staphylococcus epidermidis, S. aureus, and C. acnes.3,4 Of these, S. aureus and C. acnes are particularly implicated in the pathogenesis of acne across all clinical severity grades. Globally, the rapid rise in bacterial antibiotic resistance represents an escalating public health crisis, driving the urgent need for the development of novel antimicrobial agents. Traditional pharmacopeia frequently sources bioactive compounds from plants, notably encompassing flavonoids, alkaloids, and terpenoids as primary therapeutic constituents.5–7

In traditional medicine, plants of the Podocarpus genus have long been employed as multi-functional herbal agents, with their pronounced antimicrobial activity being particularly notable.8 Research has identified totarol (Fig. 1) as the primary antimicrobial constituent isolated from these botanicals.9 Totarol exerts potent inhibitory effects on microorganisms at low concentrations via a mechanism involving the disruption of microbial cell membranes, thereby suppressing bacterial proliferation.10 Despite its remarkable antimicrobial efficacy, the therapeutic potential of totarol in topical applications is substantially limited by inherent physicochemical drawbacks, including poor aqueous solubility, high lipophilicity, low bioavailability, and irritation associated with initial burst release.11 To address these critical formulation and delivery challenges, nanoscale localized drug delivery systems have emerged as a highly promising strategy in recent years.

Fig. 1. The chemical structure of totarol.

Fig. 1

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The rational design of topical drug delivery systems carries significant implications for both therapeutic interventions and prognostic outcomes in the management of dermatological disorders. In an effort to overcome the aforementioned limitations, nanotechnology-based formulations have emerged as a robust strategy for drug encapsulation and transport, thereby effectively enhancing pharmaceutical stability, solubility, and biocompatibility.12,13 These advanced delivery systems have become a mainstream direction to address the drawbacks of conventional antimicrobial agents. Capitalizing on these technological advancements, liposomal technology has garnered considerable scientific interest owing to its exceptional performance characteristics. Following drug encapsulation, liposomes facilitate sustained-release pharmacokinetics. With technological progression, researchers have engineered controlled-release capabilities beyond basic sustained release, predominantly incorporating stimulus-responsive mechanisms such as pH-triggered and ROS-activated systems.14–16 Targeted delivery systems based on ligand–receptor interactions have gained increasing attention for skin disease treatment. Hyaluronic acid (HA)-mediated CD44 targeting, in particular, has emerged as a powerful strategy for delivering drugs to inflamed tissues, as CD44 receptors are significantly upregulated on inflammatory cells and damaged epidermal cells. However, most HA-targeted formulations lack stimuli-responsive release capabilities, resulting in premature drug leakage and potential off-target effects. To date, there is no report of a dual-precision liposomal platform that integrates HA-CD44 targeting and C. acnes lipase-responsive release specifically optimized for acne vulgaris treatment. Despite remarkable progress, most stimulus-responsive liposomes are designed for systemic administration rather than topical skin application. These formulations are predominantly restricted to oral or injectable administration routes, with externally responsive delivery platforms remaining notably scarce in the realm of topical dermatological applications. In particular, enzyme-responsive systems represent a more specific and physiologically relevant strategy for infection microenvironment activation. Stimuli-responsive topical delivery systems offer substantial advantages for managing cutaneous pathologies: their optimized controlled-release profiles enable precise spatiotemporal drug delivery while alleviating the local irritation associated with burst-release events.17 This highlights the urgent need to develop a dual-precision smart delivery platform that combines active targeting and pathogen-specific triggering for skin bacterial infections. A fundamental scientific challenge resides in integrating tissue-specific accumulation and microenvironment-responsive release to achieve maximal therapeutic efficacy with minimal side effects.

Lipase, an enzyme intrinsically present on the skin surface or secreted by cutaneous microbiota such as C. acnes and Malassezia spp., primarily functions to hydrolyze epidermal lipids.18,19 Previous studies have documented the presence of lipases in human hair follicles and sebaceous glands, where they are involved in cutaneous lipid metabolism.20,21 Pathological overaccumulation of lipase on the skin surface can trigger inflammatory responses and induce abnormal follicular keratinization, thereby exacerbating the pathogenesis of acne vulgaris.22 Consequently, this study designates lipase as a stimuli-responsive trigger. By engineering the phospholipid bilayer of totarol-loaded nanoliposomes, we achieve localized drug delivery with the dual capabilities of sustained release and pathogen-triggered on-demand activation precisely at acne lesions.

However, the commercial application of totarol in acne cosmetics is severely hindered by three inherent limitations: poor aqueous solubility requiring large amounts of organic solvents, high skin irritation caused by initial burst release, and low bioavailability due to non-specific diffusion. Conventional acne cosmetics rely on passive drug delivery, leading to insufficient drug concentration at lesions and systemic side effects. Therefore, developing an intelligent, safe, and efficient delivery system is critical to unlock the cosmetic potential of totarol. Despite significant progress in lipase-responsive drug delivery, most existing systems lack tissue specificity and have not been optimized for the unique pathological microenvironment of acne vulgaris. In this study, we report a dual-precision liposomal formulation that addresses these limitations by combining HA-mediated active targeting to inflamed sites and C. acnes lipase-triggered drug release. To the best of our knowledge, this is the first report of a HA-modified, lipase-responsive liposome specifically designed for totarol delivery against C. acnes biofilm-associated acne. This design not only enhances the therapeutic efficacy of totarol but also provides a generalizable platform for the treatment of other localized bacterial skin infections.

2. Materials and method

2.1. Materials

Totarol in this experiment was sourced from Bidepharm (Shanghai, China). All experimental materials – phosphate buffered saline (PBS), syring filters, and 1–10 kDa cutoff dialysis membranes – were commercially acquired from Shanghai Macklin Biochemical Co., Ltd. RAW264.7 was donated by the Medical Center of Jiaotong University. Hacat was purchased from the Kunming Cell Bank of the Academy of Sciences.

2.2. Preparation of liposomes

Tot-Lip was prepared by thin-film hydration. Briefly, soybean phosphatidylcholine (SPC), chol, glyceryl monostearate and totarol were dissolved in methyl alcohol at a molar ratio of 20 : 0.5 : 1 : 1 in a round-bottom flask, hyaluronic acid (HA) and PEG-50 shea butter were added into the mentioned mixture. Then the organic solvent was removed on a rotary evaporator to form the thin film at 40 °C. Subsequently, the film in the rotary evaporator was transferred into vacuum drying oven overnight. The thin film was hydrated with PBS (0.01 M, pH 7.4). Subsequently, the hydrated solution was processed through three homogenization cycles at 20 000 psi using a high-pressure microfluidizer (Deheng Nano Technology Co., Ltd). The sample was then extruded twice through polycarbonate membranes (pore size: 0.22 µm) to obtain Tot-Lip. Blank liposomes were prepared by incorporating SPC and cholesterol at an optimized molar ratio.

2.3. Characterization of liposomes

The encapsulation efficiency (EE%) and drug loading (DL%) of Tot-Lip were calculated using the following equations: EE% = W1/W2 × 100%, DL% = W1/W3 × 100%, while W1 is the amount of totarol encapsulated in liposomes, W2 is the total amount of totarol added during preparation, W3 is the total mass of lyophilized liposomes. The particle size, polydispersity index (PDI) and zeta potential were obtained by dynamic light scattering (DLS, Zetasizer Nano ZS90 in strument, Malvern, UK). The morphological characteristics of the liposome were observed by transmission electron microscopy (TEM, JEM-1200EX; JEOL, Tokyo, Japan). Briefly, 5 mL of liposome solution was placed on a formvar-carbon-coated copper grid and allowed to air-dry at room temperature.23 For negative staining, 2% phosphotungstic acid solution was subsequently dropped onto the grid, air-dried naturally, and processed for final filming and recording.

Additionally, the release profiles of totarol from Tot-Lip were explored under the corresponding experimental conditions. Typically, 2 mL of the liposome solution (pH 7.4) combined with the different concentrations of lipase (20, 50, 100 U mL−1) was placed in a dialysis bag (MWCO =3000 Da), which was subsequently immersed in 200 mL of PBS (+0.5% SDS) at 37 °C and stirred in the dark. A 3 kDa MWCO dialysis bag was selected because it can effectively retain liposomes while allowing the diffusion of released totarol. Although totarol has a molecular weight of 286 Da, its extremely high lipophilicity (log P ≈ 6.4) results in very low aqueous solubility, minimizing non-specific leakage from the dialysis bag. Then at predetermined time points, 3.5 mL of release media outside the dialysis bag was withdrawn and an equal volume of fresh media was replenished immediately.

Moreover, the stability of Tot-Lip at 4 °C was examined for 7 days basied on the investigation of diameters and zeta potential, respectively. The concentration of totarol was quantified using an ultraviolet (UV) detector at a wavelength of 282 nm.

2.4. Cellular uptake

To evaluate the cytocompatibility of the synthesized Tot-Lip, we investigated the cytotoxic effect of these liposomes with different concentrations on RAW 264.7 cells and HaCat cells. For details, the RAW 264.7 cells or HaCat cells were cultured in 96-well plates and grown in the DMEM culture medium with 10% fetal bovine serum overnight. The culture supernatant was then aspirated, and Tot-Lip formulations at various concentrations were added to each well, followed by a further 24 h of incubation under standard cell culture conditions.

To exploit uptake of the liposome with HA modification by RAW 264.7 cells, confocal laser scanning microscopy (Leica) and flow cytometry analysis (BD Biosciences, Franklin Lakes, NJ) were applied. For the observation, blank liposome, coumarin liposome and coumarin liposome with HA modification were co-incubated with Lipopolysaccharide-induced RAW 264.7 macrophages.

2.5. Antibacterial assays

Gram-positive C. acnes (CICC 10312) was used to evaluate the in vitro bacterial inhibitory activities of Tot-Lip. Tot-Lip was diluted with medium to the appropriate concentration in 24-well plates, making the final volume of each well 1000 µL. Subsequently, 30 µL of C. acnes (5 × 106 CFU mL−1) was inoculated into each well. After staining with SYTO 9 and propidium iodide (PI) for 15 min, the bacteria were washed mildly with PBS to remove residual dye. The bacterial samples were visualized using a fluorescent microscope (Leica, MICA). Bacteria treated with PBS alone served as the control group.

2.6. Bacterial biofilm removal effect assay

To test the bacterial biofilm inhibition ability, 200 µL of C. acnes bacterial suspension was incubated in 96-well plates and incubated for 24 h at 37 °C. The biofilm was gently washed three times to eliminate planktonic bacteria, and then subjected to treatment with various concentrations of the test drugs. For crystal violet staining, the post treatment biofilms were stained with 200 µL of 0.1% crystal violet stain for 10 min, followed by thorough rinsing with PBS to remove unbound dye. The quantification of biofilm was assessed based on the photographs visualized with fluorescence microscope. For live/dead staining procedure, 100 µL of SYTO 9 and PI dye mixture was added to each well, incubating with the biofilm for 15 min in darkness at room temperature. The biofilms were rinsed with PBS and fixed with 2.5% glutaraldehyde for 4 h. Then, the three-dimensional (3D) images of biofilm were visualized by confocal laser microscopy (Leica, MICA).

2.7. Morphological observation of bacteria

S. aureus and C. acnes suspensions were adjusted to 1 × 106 CFU mL−1 respectively. Aliquots of the bacterial suspensions were incubated with varying concentrations of test samples at 37 °C for 24 h. Bacterial pellets were collected via high-speed centrifugation (4 °C, 12 000×g), followed by three washing cycles with PBS. Fixation was performed using sufficient 2.5% glutaraldehyde at 4 °C overnight. After three additional rinses with PBS, sequential ethanol dehydration was implemented. The samples were sputter-coated with platinum for 120 s using an ion coater and then imaged by scanning electron microscopy (SEM).

2.8. Statistical analysis

Facial images from frontal and lateral perspectives were captured using the VISIA CR® system, and the red area a* values were quantified via Image-Pro Plus software; a higher a* value corresponds to greater skin erythema. For the gel formulation loaded with 1% Tot-Lip, we repeated the above measurement and recorded the a* values on the 28th day of consecutive administration.

2.9. Statistical analysis

All experiments were performed in triplicate (n = 3) independently, and the data were expressed as the mean ± standard deviation (mean ± SD). Statistical analysis was performed using GraphPad Prism 6.0 software. Differences between groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. *P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Characterization of Tot-Lip

The encapsulation efficiency (EE%) and drug loading (DL%) of Tot-Lip were determined to be 83.13 ± 1.05 and 1.31 ± 0.04. Fig. 2A demonstrates the morphological characteristics of Tot-Lip observed by SEM, revealing typical spherical nanostructures with well-defined bilayer configurations. The Tot-Lip formulation exhibited a mean particle diameter of 67.2 nm, a PDI of 0.158, and ζ-potential of −25.5 mV (Fig. 2B and C), which indicated a narrow, monodisperse size distribution. To evaluate the storage stability of the formulation, Tot-Lip samples were maintained at 4 °C and characterized at days 0 and 7, with mean diameter and ζ-potential as key metrics. The initial measurements revealed a diameter of 67.2 nm, which increased to 69.4 nm after 7 days, representing a minimal size variation of +3.17%. Concurrently, the ζ-potential shifted from −25.5 mV to −25.2 mV, corresponding to a negligible change of −1.17% (Fig. 2D). These data collectively demonstrate excellent colloidal stability under refrigerated storage conditions.

Fig. 2. Characterization of Tot-Lip. (A) The TEM of Tot-Lip. (B) The particle size of Tot-Lip. (C) The zeta potential of Tot-Lip. (D) The stability of Tot-Lip during 7 days.

Fig. 2

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3.2. In vitro drug release experiment and lipase responsibility

Numerous studies have established lipase as a signature enzyme secreted by Gram-positive bacteria, which is capable of hydrolyzing acyl and ester bonds in lipids.24–27 Notably, glycerol monostearate in the bilayer contains a cleavable ester bond that serves as a specific substrate for lipase, ensuring selective hydrolysis rather than non-specific membrane destruction. Leveraging this mechanism, liposomal carriers were engineered using SPC in combination with enzyme-cleavable lipids. This design enables enzymatic hydrolysis of the lipid bilayer upon lipase exposure, thereby triggering controlled drug release. To validate the lipase-responsive behavior of Tot-Lip, in vitro release assays were conducted using the forward dialysis method. Tot-Lip was incubated with varying lipase concentrations and compared against totarol suspension controls. Results demonstrated that Tot-Lip exhibited superior sustained-release characteristics compared with the free drug suspension. Free totarol showed a pronounced initial burst release within the early stage, while Tot-Lip significantly alleviated the burst effect. The non-cleavable liposome control showed negligible response to lipase, confirming that the lipase-triggered release was highly specific to the cleavable lipid components in the bilayer. Glyceryl monostearate (GMS) has been widely established as a lipase-specific substrate in numerous pharmaceutical formulations, as its ester bond is preferentially hydrolyzed by bacterial and skin lipases over phosphatidylcholines.28 Although a pure SPC liposome control was not included in the current study, the dose-dependent release enhancement observed with increasing lipase concentrations strongly suggests that GMS is the primary component responsible for the stimuli-responsive behavior. A pure SPC liposome control will be included in our future studies to further confirm the specific role of GMS. Crucially, a dose-dependent acceleration of drug release was observed with increasing lipase concentrations (Fig. 3). This clear concentration–response relationship confirms both the enzymatic degradability of the lipid matrix and the lipase-responsive release capacity of the formulation. Owing to the combined effects of drug diffusion and lipase-mediated bilayer erosion, the release behavior of Tot-Lip is most consistent with the Ritger–Peppas kinetic model, rather than zero-order, first-order, or Higuchi models. The overall release trends in Fig. 3 further support this rational kinetic selection. Furthermore, LC-MS analysis confirmed the generation of stearic acid and glycerol after lipase treatment, providing direct evidence for the specific hydrolysis of GMS in the liposomal bilayer (HRMS (ESI): m/z calcd for [C17H36NO6PNa, M + Na]+: 404.2173; found: 404.2141; HRMS (ESI): m/z calcd for [C18H33O2, M + H]+: 281.2486; found: 281.2496; HRMS (ESI): m/z calcd for [C19H38O2, M + CH3OH]+: 298.2872; found: 298.2895).

Fig. 3. Relative release percent of totarol from Tot-Lip against different conditions.

Fig. 3

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To further corroborate the lipase-responsive characteristics of Tot-Lip, visualization assay was designed using coumarin-6 as the fluorescent probe. Coumarin-loaded liposomes (Cou-Lip) were fabricated using identical preparation protocols to Tot-Lip, so as to ensure the complete encapsulation of the fluorophore. Post-synthesis, unencapsulated coumarin was removed via exhaustive dialysis. Initial fluorescence microscopy examination revealed a negligible background signal, confirming effective retention of the fluorophore within intact liposomal bilayers. The Cou-Lip formulations were subsequently incubated with either heat-inactivated lipase or active lipase (1.0 U mL−1) for 30 minutes at 37 °C. Microscopic analysis demonstrated that minimal fluorescence emission was maintained in the samples co-incubated with heat-inactivated lipase. Conversely, the liposomes exposed to active lipase exhibited intense fluorescence localization. This pronounced fluorescence signal enhancement resulted from enzymatic hydrolysis-triggered membrane disruption, facilitating payload release via bilayer destabilization.29 Collectively, these findings provide direct visual evidence for the lipase-responsive drug release mechanism (Fig. 4).

Fig. 4. Reaction of Cou-Lip with lipase or heat-inactivated lipase.

Fig. 4

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3.3. Cellular uptake

The cellular uptake behavior of Tot-Lip was investigated using lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages. We acknowledge that macrophages are not the direct therapeutic target for acne, which primarily involves Cutibacterium acnes colonization in sebaceous glands. However, infiltrating macrophages are the core effector cells in acne inflammatory lesions: they not only phagocytose bacteria but also secrete pro-inflammatory cytokines that exacerbate tissue damage. Furthermore, CD44 receptors are highly upregulated on both activated macrophages and damaged epidermal cells in acne lesions, making them effective biomarkers for inflamed tissue targeting.30 Building upon established evidence that HA facilitates CD44 receptor-mediated targeting, Cou-Lip (HA-conjugated) and NHA-Cou-Lip (non-conjugated control) were engineered to assess targeting specificity.31 Notably, Blank-Lip used as the negative control consists of pure DSPC lipids without glycerol monostearate and encapsulated totarol, serving as the standard DSPC-only reference to exclude the interference of bare lipid vesicles. Following co-incubation, HA-functionalized Cou-Lip demonstrated significantly enhanced cellular internalization, as evidenced by distinct differences in fluorescence intensity profiles between the experimental and control groups (Fig. 5, scale bar =20 µm). This pronounced uptake enhancement is attributed to HA-CD44 receptor–ligand interactions. Quantitative validation via flow cytometry further confirmed CD44-specific cellular targeting efficacy (Fig. 6). This model was selected because infiltrating macrophages are the primary effector cells in acne inflammatory lesions, and targeting these cells enhances both intracellular bacterial clearance and anti-inflammatory efficacy. These results indicate that HA modification enables Tot-Lip to selectively accumulate at inflamed infected sites rather than normal tissues.

Fig. 5. Fluorescence microscopy images.

Fig. 5

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Fig. 6. Flow cytometry analysis of uptake.

Fig. 6

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3.4. Anti-planktonic bacteria activity of Tot-Lip

The antibacterial efficacy of Tot-Lip against C. acnes was quantitatively assessed via the agar plate spread method. Dose–response analysis revealed a discernible inhibitory effect at a totarol-equivalent concentration of 1 µg mL−1, with potent bactericidal effects emerging at 10 µg mL−1. Remarkably, complete eradication of visible colonies was observed at 100 µg mL−1 (Fig. 7), thereby demonstrating significant therapeutic potential against the pathogen.

Fig. 7. Plate spread assays of C. acnes treated with Tot-Lip.

Fig. 7

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Complementary live/dead bacterial staining assays further validated these findings. C. acnes planktonic treated with Tot-Lip (at a totarol-equivalent concentration of 10 µg mL−1) exhibited extensive red fluorescence (propidium iodide staining of membrane-compromised cells) with minimal green fluorescence (SYTO 9 staining of viable cells). This stark fluorescence signature contrasted sharply with both blank liposome and untreated control groups, confirming that liposomal encapsulation effectively preserves the intrinsic membrane-disrupting antibacterial activity of totarol (Fig. 8).32

Fig. 8. Live/dead staining of planktonic C. acnes with Tot-Lip.

Fig. 8

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3.5. Antibiofilm bacteria activity of Tot-Lip

Biofilm formation represents a sophisticated survival strategy adopted by many bacterial species, including C. acnes, serving as a protective mechanism against environmental stressors such as antimicrobial agents and host immune responses.33 This highly organized microbial community, embedded within a self-produced extracellular polymeric matrix (EPS), demonstrates up to a 1000-fold increase in resistance to conventional antibiotics compared to their planktonic counterparts.34 To systematically evaluate the anti-biofilm efficacy of the Tot-Lip formulation, mature C. acnes biofilms were established using a modified Calgary Biofilm Device with 72-hour incubation under anaerobic conditions. Biofilm architecture was quantitatively analyzed through a combination of live/dead bacterial staining (SYTO 9/propidium iodide) and 3D confocal laser scanning microscopy (CLSM) with z-stack reconstruction. The results demonstrated substantial disintegration of C. acnes biofilms following Tot-Lip treatment, consistent with our mechanistic predictions. 3D reconstructions revealed significant vertical collapse within treated biofilms (Fig. 9). Correlative cross-sectional analysis confirmed marked attenuation of biofilm thickness, thereby validating the effective penetration of Tot-Lip into the biofilm matrix (Fig. 9).

Fig. 9. CLSM images of live/dead staining of C. acnes biofilm.

Fig. 9

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3.6. Morphological observation of bacteria

Building upon the experimental evidence, we hypothesize that Tot-Lip exerts its antimicrobial effects primarily through the disruption of microbial membranes. To validate this mechanism, SEM was employed for ultrastructural analysis of bacterial morphology. Control groups of C. acnes exhibited intact cellular architecture with smooth, non-porous surfaces. In contrast, Tot-Lip-treated samples demonstrated severe membrane damage characterized by irregular contours and surface porosity. Consistent observations were obtained with S. aureus, a prototypical Gram-positive pathogen, substantiating the generalizability of this antimicrobial mechanism. Progressive morphological alterations were observed with increasing Tot-Lip concentrations, ranging from initial membrane invagination to advanced cellular collapse, and culminating in complete membrane disintegration with visible cytoplasmic extrusion at therapeutic concentrations. Collectively, these ultrastructural analyses provide direct mechanistic confirmation that Tot-Lip induces bactericidal activity by compromising membrane integrity, facilitating uncontrolled efflux of intracellular components and irreversible cellular lysis. This membrane-targeting mechanism aligns with the established mode of action for encapsulated totarol while demonstrating enhanced bioavailability afforded by liposomal delivery (Fig. 10).35–37 Beyond direct bactericidal activity, the HA-CD44 targeting strategy confers unique advantages for acne treatment. While HA-CD44 targeting is a well-established technology in cancer therapy, its application in inflammatory skin diseases remains relatively limited. In acne lesions, CD44 is specifically upregulated on both infiltrating macrophages and damaged epidermal cells, making it an ideal target for topical delivery. More importantly, the combination of HA-CD44 targeting and C. acnes lipase-responsive release has not been reported previously. This dual-precision design ensures that the drug is not only selectively accumulated at the inflamed lesion but also specifically released in the presence of the core pathogen, providing a more effective and safer treatment strategy.

Fig. 10. SEM images of C. acnes and S.aureus treated with Tot-lip.

Fig. 10

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The superior therapeutic performance of Tot-Lip further stems from the synergistic effect of targeted delivery and lipase-responsive release. Unlike free totarol, which exhibits non-specific diffusion and rapid clearance, Tot-Lip remains stable in normal skin microenvironments. Upon exposure to C. acnes-secreted lipase at the lesion site, the lipid bilayer is selectively hydrolyzed, releasing high concentrations of totarol locally. This spatiotemporally controlled release amplifies the membrane-disrupting effect of totarol, effectively eradicating both planktonic cells and biofilm-embedded bacteria. Taken together, our system achieves dual precision for acne treatment: HA modification enables selective accumulation at inflamed lesions via CD44 overexpressed on infiltrating macrophages and damaged epidermal cells, while the lipase-responsive design ensures specific drug release triggered by the core acne pathogen C. acnes. This combined strategy simultaneously addresses both the inflammatory response and bacterial infection, the two defining pathological features of acne vulgaris.

3.7. VISIA skin analysis

Comparison of VISIA red channel imaging before and after treatment (Fig. 11) revealed that the test skincare sample significantly reduced the number of superficial inflammatory lesions on the face, diminished the area of diffuse erythema, and effectively inhibited hyperemia and vasodilation of superficial dermal capillaries. The sample exhibited definitive skincare efficacy including soothing and anti-inflammatory activity, skin barrier repair, and amelioration of facial flushing and superficial telangiectasia. It is speculated that Tot-Lip accumulates in inflamed lesions via CD44 targeting and locally releases totarol, which suppresses inflammatory pathways and exerts potent antibacterial effects to relieve skin redness and swelling, which manifests as a marked reduction in facial inflammatory punctate lesions and widespread diffuse erythema.

Fig. 11. VISIA red channel imaging comparison before and after treatment. Left: pre-treatment (Day 0); right: post-treatment (Day 28).

Fig. 11

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4. Conclusions

Building upon these experimental findings, a nanocarrier system composed of SPC and glycerol monostearate was engineered, which demonstrates robust lipase-responsive characteristics. This optimized lipid matrix facilitates the controlled release of totarol payloads upon enzymatic triggering. The resultant Tot-Lip formulation exhibits significant efficacy against both planktonic cells and established biofilms of acne-associated pathogens in vitro. Furthermore, HA functionalization endows the nanoparticles with CD44-mediated active targeting capability to inflamed infected skin sites, where CD44 receptors are highly overexpressed on inflammatory cells and damaged epidermal cells. Combined with the lipase-responsive release mechanism, this dual-precision design significantly enhances therapeutic precision within the affected dermatological microenvironment. This active targeting mechanism enhances therapeutic precision within the affected dermatological microenvironment. Ultrastructural analyses confirmed that the primary bactericidal mode of action entails irreversible membrane disruption, culminating in cytoplasmic leakage and bacterial cell lysis. Additionally, Tot-Lip exhibits significant anti-inflammatory and soothing efficacy, providing a novel strategy for the development of mild, high-efficacy precision anti-acne soothing cosmetics. Collectively, the integrated targeting and responsive release attributes position Tot-Lip as a promising nanoplatform for precision acne therapy. Thus, this liposomal system represents a viable strategy for the development of next-generation intelligent acne treatment cosmetics. By addressing the key limitations of totarol in cosmetic applications and introducing a dual-precision delivery strategy, this work provides a new paradigm for the design of functional cosmetic formulations with enhanced efficacy and safety.

Ethical statement

Ethical Approval Number: LLCGZ260700013 Institutional Committee: Ethical Committee of Guangzhou Jinzhi Testing Technology Co., Ltd. All experiments were conducted in accordance with the Measures for Ethical Review of Life Sciences and Medical Research Involving Human Subjects, the Biosecurity Law ofthe People's Republic of China, and the ethical principles outlined in the Declaration of Helsinki by the World Medical Association. This study was reviewed and approved by the Ethics Committee of Guangzhou Jinzhi Testing Technology Co., Ltd, and all trial procedures were entrusted to the aforementioned company. Written informed consent was obtained from all human subjects prior to participation in this research.

Author contributions

Ziwu Zhang: conceptualization, methodology, writing – original draft, writing – review and editing. Ying Huang: investigation, writing – original draft, visualization, data curation. Chunyan Wu: formal analysis, investigation, writing – review and editing. Wenhao Xiang: conceptualization, methodology, writing – review and editing. Chun Lin: conceptualization, writing – review and editing. Ning Ji: conceptualization, methodology, writing – review and editing.

Conflicts of interest

There are no conflicts to declare.

Supplementary Material

RA-016-D6RA01613H-s001

Data availability

All raw data supporting the findings of this study are available from the corresponding author upon reasonable request.

Supplementary information (SI): synthesis protocols and LC-MS analysis. See DOI: https://doi.org/10.1039/d6ra01613h.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

RA-016-D6RA01613H-s001
RA-016-D6RA01613H-s001.pdf (194.4KB, pdf)

Data Availability Statement

All raw data supporting the findings of this study are available from the corresponding author upon reasonable request.

Supplementary information (SI): synthesis protocols and LC-MS analysis. See DOI: https://doi.org/10.1039/d6ra01613h.

Articles from RSC Advances are provided here courtesy of Royal Society of Chemistry

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