The EE% of KPN in the prepared quatsomes was determined by separating the unentrapped drug through filtration using Whatman filter paper (grade No. 1, pore size 11 μm) (El Hassab et al., 2025). An aliquot of 1 mL from the filtrate was subsequently sonicated in methanol to ensure complete drug release, and the concentration of KPN was quantified spectrophotometrically at λ_max = 257 nm (R ² = 0.995). Measurements were conducted in duplicate, and the results were expressed as the mean ± standard deviation (SD). The EE% was calculated according to the following equation (El-Shahed et al., 2024; Taw et al., 2025): E E % = e n c a p s u l a t e d   a m o u n t   o f   K P N T o t a l   a m o u n t   o f   K P N × 100 (1)

The physicochemical characterization of the prepared KPN-QS was conducted to evaluate their colloidal performance and predict formulation stability and vaginal adhesion potential. Key parameters including particle size (PS), poly-dispersity index (PDI), and zeta potential (ZP) were analysed, as these serve as critical indicators of nanosystem homogeneity, electrostatic stability, and mucoadhesive potential (Ahmed et al., 2025d). Measurements were performed using a Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, United Kingdom) operated at 25 °C and a fixed backscattering angle of 173° (Ahmed et al., 2025e). Prior to analysis, freshly prepared formulations were diluted (1:10 v/v) with distilled water until a faint opalescence as obtained, ensuring suitable particle dispersion and minimizing multiple scattering or vesicle aggregation. Each diluted sample was gently vortexed to achieve homogeneity before measurement (Fahmy et al., 2025; Saher et al., 2019). The PDI values were employed to assess the uniformity of the vesicular population, while ZP values were determined through electrophoretic mobility analysis, reflecting the surface charge intensity and predicting colloidal stability (Ahmed et al., 2025f). All measurements were performed in triplicate, and the mean ± standard deviation (SD) was used express the results to ensure reproducibility and analytical reliability (Farag et al., 2023; Saher et al., 2023).

The release of KPN from KPN-OQ was evaluated using dialysis bag diffusion technique (Ahmed et al., 2025h). 0.5 mL of the formulation (equivalent to 2.5 mg KPN) was added to a dialysis bag (molecular weight cut-off: 12,000–14,000 Da) that had been pre-soaked overnight in release medium. Both ends of the bag were firmly sealed to prevent any leakage during the experiment (Ragheb et al., 2025). Each filled dialysis bag was then placed into a glass bottle containing 50 mL of phosphate buffer saline, pH 4.5 containing 5% methanol kept under continuous stirring set at 100 rpm and 37 °C ± 0.5 °C (Hot plate magnetic stirrer, WiseStir, Daihan Scientific C. o., Ltd., Korea) (Ahmed et al., 2025a). To maintain sink conditions, 3 mL samples were taken out at predefined intervals (0.5, 1, 2, 4, 6, and 8 h) and promptly replaced with an equivalent volume of fresh buffer (Ahmed and Badr-Eldin, 2019). The collected samples were analysed spectrophotometrically at 257 nm to determine the concentration of KPN. A release study of KPN-SUSP in a buffer was conducted under identical conditions to verify the appropriateness of the dialysis membrane used and to compare the release profile of the suspension with the prepared quatsomes. The cumulative percentage of KPN released was computed at each time point and plotted against time (Ahmed et al., 2020). To better understand the mechanism governing KPN release from the optimized formulation, the obtained release data were mathematically analyzed using multiple kinetic models, including zero-order, first-order, Higuchi diffusion, and Korsmeyer–Peppas equations. These models were applied to determine the predominant release model. The correlation coefficient (R ²) values for each model were calculated, and the model exhibiting the highest R ² was considered the most appropriate descriptor of the release behavior (Ahmed et al., 2024b; Abdelhakeem et al., 2025).

A sample of KPN-OQ was placed onto a copper grid coated with carbon after being suitably diluted with distilled water. After removing the excess dispersion with filter paper, the sample was allowed to air dry at room temperature for 10 minutes (Albash et al., 2025b). It was then visualized using transmission electron microscope (TEM) (Hitachi HF-2000, Tokyo, Japan) operated at an accelerating voltage of 80 kV to examine the morphology of the selected formula (Elmahboub et al., 2024; Ahmed et al., 2023).

Particle growth, drug leakage, and other possible alterations were tracked to evaluate the stability of KPN-OQS. KPN-OQS was refrigerated for 3 months, and then its PS, PDI, EE%, ZP, and drug release profile were compared to freshly prepared samples (Ahmed et al., 2022). With SPSS® software (version 22.0; IBM Corp., Armonk, NY, United States), statistical analysis was performed using the paired Student’s t-test, with p < 0.05 deemed significant (Fahmy et al., 2026).

The antifungal activity of KPN against C. albicans ATCC10231 was evaluated (Al-mahallawi et al., 2021). The microdilution method was used to estimate the minimum inhibitory concentration (MIC) in accordance with the Clinical and Laboratory Standards Institute’s recommendations as previously described (El-Hema et al., 2025; Ismail et al., 2021). Briefly, the tested KPN formulations were serially diluted in Sabouraud Dextrose Broth (SDB) to obtain a range of concentrations (12.5–0.0224 mg/mL). A standardized fungal inoculum (10⁵–10⁶ CFU/mL) was prepared and added to each well of sterile 96-well microtiter plates. Microplates were incubated at 30 °C for 24–48 h, and fungal growth was assessed visually and spectrophotometrically by measuring optical density at 600 nm using a microplate reader (Biotek Synergy 2, SLFA model, United States). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the tested formulation that resulted in complete inhibition of visible fungal growth compared to the growth control. All experiments were performed independently in triplicate, and results were expressed as mean ± standard deviation.

KPN-OQS was tested in vivo to validate its antifungal activity in a vaginal drug delivery system. Female Wistar rats (150 ± 7 gm) were used in a murine Candida vaginal colonization model as described before (Eltabeeb et al., 2024; Ali et al., 2023). Estradiol (0.5 mg) was intraperitoneal injected 24 h before starting the fungal colonization. Dexamethasone (0.4 mg) was also injected intraperitoneally once daily, starting 24 h before colonization and continuing for three consecutive days during the colonization process. Fungal colonization was established by vaginally inoculating the rats with a suspension of C. albicans ATCC10231 (2-5 × 108 CFU) over a period of four consecutive days. Rats were randomly distributed into three groups (n = 6) and treatments were administered inter-vaginally (250 µL of the corresponding treatment using a soft plastic tip) 24 h post the last inoculation. The group size of six rats per treatment group was determined based on ethical considerations aligned with the 3Rs (Replacement, Reduction, Refinement) principle, aiming to minimize animal use while ensuring scientific validity. One group served as the negative control group and did not receive any treatment. The second group was treated with the KPN-SUSP. The third group was treated with the KPN-OQS. The vaginal lumen samples were collected from each rat using a sterile swab at 24, 48, and 72 h after treatment). The recovered C. albicans in the swaps were serially diluted in PBS and spotted on Sabouraud dextrose agar for the viable count as described before (Eltabeeb et al., 2024). The results of the tested groups were analysed and compared.

Vaginal specimens were excised and promptly fixed in 10% neutral buffered formalin. The samples were then processed using standard histological techniques: trimming, dehydration in ascending grades of ethanol, clearing in xylene and embedding in paraffin wax (Ahmed et al., 2021). Sections of 4–6 μm thickness were obtained using a rotary microtome and subsequently stained with hematoxylin and eosin (H&E) for general histopathological examination, following the method described by Bancroft and Gamble (Ahmed et al., 2025a).

The entrapment efficiency (EE %) of the prepared quatsomes ranged from 24.4% ± 0.4% to 96.9% ± 0.6% (Table 2). Statistical analysis (p < 0.05) revealed that all examined factors significantly influenced EE %, as illustrated in Figure 1a. Achieving high entrapment efficiency is essential for maximizing drug loading within the vesicular matrix, thereby enhancing therapeutic efficacy, ensuring sustained drug release, and minimizing dosing frequency.

The higher entrapment efficiency (EE %) observed in CPC-based quatsomes compared to CTAB-based ones is primarily related to the structural differences between their quaternary ammonium head groups. CPC contains an aromatic pyridinium ring, while CTAB features an aliphatic trimethylammonium group (Talman et al., 2025; Nemr et al., 2025). The aromatic nature of CPC enhances hydrophobic interactions, π–π stacking, and van der Waals forces within the bilayer, resulting in tighter molecular packing, lower membrane permeability, and superior drug retention. These stronger intermolecular interactions between CPC, cholesterol, and the lipophilic drug collectively produce a more cohesive and stable vesicular structure (Frolov et al., 2025).

In contrast, increasing the total amount of vesicle-forming materials (Factor B) led to a reduction in EE%. When the concentration of cholesterol and quaternary ammonium surfactants exceeded the optimal level, the excess amphiphiles promoted the formation of additional vesicles and micelles. This not only diluted the available drug among a larger number of vesicles but also diverted part of the surfactant to micellar structures that solubilized the drug in the aqueous phase rather than incorporating it into the bilayer (Omolo et al., 2018; Lombardo and Kiselev, 2022).

Furthermore, overly high cholesterol content (Factor C) can disturb the bilayer’s optimal packing arrangement, decreasing its flexibility and limiting drug accommodation within the hydrophobic core. Consequently, both compositional imbalance and phase competition contribute to the observed decline in EE% at higher material concentrations. Also, it could be due to the possible competition between cholesterol and KPN (lipophilic drug) to be incorporated within the lipid membrane (Sun et al., 2010).

Regarding PS, PS of the prepared quatsomes ranged from 108.7 ± 2.5 to 328 ± 13.4 nm (Table 2). Statistical analysis of the independent factors revealed that all the independent factors affected the PS of the prepared quatsomes significantly (p < 0.05) (Daralnakhla et al., 2021).

Firstly, as demonstrated in Figure 1b, CTAB-based vesicles were larger than those formed with CPC, although both possess identical C16 alkyl chains. This difference could be attributed to variations in head-group structure and counter-ion properties. The flexible, bulky tri-methyl-ammonium head-group in CTAB creates steric hindrance, favouring lower curvature and larger vesicles, whereas the planar, rigid pyridinium ring in CPC allows tighter packing and higher curvature. Additionally, CTAB contains bromide ions, while CPC contains chloride ions. Bromide has a larger atomic radius (∼114 pm) than Cl (∼99 pm), which increases head-group spacing at the vesicle surface. Furthermore, bromide binds more strongly to the positively charged head-groups, reducing the electrostatic repulsion between them. Reduced repulsion allows the bilayer to adopt a lower curvature, producing larger vesicles. In contrast, chloride’s weaker binding maintains higher repulsion, resulting in tighter curvature and smaller vesicles (Kim and Martin, 2023; Helgason et al., 2009).

Regarding factor B, increasing the amount of vesicle-forming materials from 150 mg to 200 mg resulted in a noticeable increase in vesicle size. This behaviour could be attributed to the higher availability of cholesterol and quaternary ammonium surfactant (QAS) molecules, which enhance bilayer assembly and permit looser molecular packing, thereby promoting vesicle growth. However, a further increase to 250 mg led to a decline in particle size (PS), likely due to the preferential formation of micelles at elevated QAS concentrations rather than their incorporation into the vesicular bilayer. Such micellization may deplete surfactant molecules from the vesicle surface, resulting in the formation of more compact structures or fragmented smaller vesicles, as demonstrated in Figure 2. These findings align with previously reported results by Kassem et al. (Kassem et al., 2023), confirming that excessive surfactant content can induce structural rearrangement and vesicle size reduction (Omolo et al., 2018; Lombardo and Kiselev, 2022).

With respect to factor C, an increase in the cholesterol-to-QAS ratio was found to correlate with a progressive rise in particle size (PS), which can be explained by a sequence of interrelated structural phenomena. Cholesterol molecules integrate within the bilayer matrix, orienting their hydroxyl groups toward the polar head region while embedding their rigid sterol nuclei among the hydrophobic alkyl chains. This intercalation enhances membrane rigidity and reduces bilayer fluidity, thereby reinforcing vesicular stability (Kaddah et al., 2018; Emad Eldeeb et al., 2019). Concurrently, cholesterol partially disrupts the ordered packing of the lipid components, leading to expansion of the intra-vesicular aqueous domain and facilitating greater water accommodation within the vesicular interior (Adel et al., 2021; Karal et al., 2022). Additionally, cholesterol increased the membrane stiffness, restrict the bilayer’s capacity to achieve high curvature, favouring the formation of larger-diameter vesicles with more stable structural organization (AbouSamra and Salama, 2017).

The polydispersity index (PDI) serves as a critical indicator of the homogeneity and size distribution of nanoscale vesicles, directly influencing their physical stability, reproducibility, and biological performance. Lower PDI values signify a more uniform vesicular population, which is desirable for ensuring consistent and predictable activity. In general, PDI values below 0.5 denote a monodisperse system with acceptable uniformity for pharmaceutical applications (N et al., 2022). In the current study, the PDI values of the optimized KPN-QS ranged between 0.23 ± 0.01 and 0.42 ± 0.03, confirming the formation of vesicles with a narrow and well-controlled particle size distribution across all experimental runs. Such findings reflect the efficiency and reproducibility of the formulation process, suggesting minimal aggregation or vesicle fusion. Furthermore, ANOVA demonstrated that none of the investigated formulation variables significantly affected PDI (p > 0.05). This result suggests that the vesicular uniformity was not sensitive to compositional variations within the studied design space. Consequently, although PDI was excluded from the final optimization criteria, the consistently low values demonstrate the robustness of the nanosystem and its appropriateness for localized vaginal drug delivery (Ahmed et al., 2025a).

The zeta potential (ZP) is a critical physicochemical parameter that defines the surface charge density and electrostatic stability of nano-systems. It provides insight into the extent of inter-vesicular repulsion, which directly influences the dispersion uniformity, aggregation tendency, and colloidal stability. Typically, nanocarriers possessing ZP values greater than ±30 mV are considered electrostatically stable, as the strong repulsive forces between charged particles hinder coalescence and sedimentation, thereby ensuring sustained dispersion (El Taweel et al., 2023). In the present study, the developed quatsomal formulations exhibited ZP values ranging from 36.4 ± 0.6 mV to 79.4 ± 3.4 mV, denoting a distinctly high positive surface charge that favours stable colloidal characteristics. Statistical analysis of the independent factors revealed that all the independent factors affected the PS of the prepared quatsomes significantly (p < 0.05). Figure 1c illustrates the effect of studied factors.

Regarding factor A, The higher zeta potential observed in Cetyl pyridinium chloride (CPC)–based quatsomes compared with those formulated using Cetyl trimethylammonium bromide (CTAB) can be primarily attributed to intrinsic structural and electrochemical differences between the two surfactants. CPC contains an aromatic pyridinium ring that carries a delocalized positive charge, allowing the charge to be more diffusely distributed and more readily exposed at the vesicle surface. This delocalization enhances electrostatic interactions with the surrounding aqueous medium and contributes to a more pronounced positive surface potential (Talman et al., 2025). In contrast, the quaternary ammonium head group of CTAB features a localized trimethylammonium charge, which, though positively charged, is more shielded by the bulky methyl substituents, resulting in a relatively weaker surface charge manifestation (Taw et al., 2025). Furthermore, the planar geometry of the pyridinium moiety in CPC allows for tighter interfacial alignment and stronger electrostatic repulsion among adjacent head groups, stabilizing the vesicle surface and preventing aggregation. The superior zeta potential exhibited by CPC-based quatsomes, ultimately translating into enhanced colloidal stability and improved mucoadhesive potential (Kim and Martin, 2023; Helgason et al., 2009).

When the total vesicle-forming material increased from 150 mg to 200 mg, a notable reduction in ZP was observed, followed by a subsequent increase when raised further to 250 mg. This biphasic response can be attributed to the complex interplay between electrostatic charge screening, vesicle crowding, and molecular packing density. At lower solid concentrations (150 mg), the vesicles were well dispersed, allowing the cationic head groups of QACs to remain fully ionized and oriented toward the aqueous environment, resulting in pronounced positive surface potential. Increasing the total amount to 200 mg led to a denser dispersion, which promoted partial charge neutralization through increased counter-ion adsorption and inter-vesicular interactions, effectively reducing the measurable ZP. At a higher surfactant concentration (=250 mg), the formation of smaller, more dispersed vesicles with increased surface curvature led to greater exposure of cationic moieties at the aqueous interface, thereby enhancing the electrostatic repulsion between vesicles (Omolo et al., 2018; Lombardo and Kiselev, 2022).

Meanwhile, the cholesterol-to-QAC ratio (factor C) exhibited a positively significant effect on ZP, which may be attributed to cholesterol’s ability to modulate the molecular packing and charge orientation within the bilayer. Cholesterol intercalates between adjacent alkyl chains, aligning its hydroxyl group near the polar head region. This ordered arrangement stabilizes the bilayer and optimizes the orientation of cationic groups, reducing charge shielding and favouring more effective surface charge presentation. At higher cholesterol ratios, the improved bilayer rigidity restricts QAC mobility, minimizing head groups entanglement and promoting enhanced exposure of positively charged sites toward the surrounding aqueous phase. This results in higher measured ZP values and improved colloidal stability.

The in vitro release profile of KPN from KPN-OQS and the corresponding KPN-SUSP were evaluated in phosphate buffer saline (pH 4.5, to mimic vaginal acidic nature) containing 5% methanol to maintain sink conditions (Figure 3a) (Ahmed et al., 2025a). KPN-OQ exhibited a biphasic release pattern, characterized by an initial burst release in the first 2 hours (38.21% ± 4.37%), followed by a sustained release phase extending up to 8 h. The rapid initial release may be attributed to the immediate diffusion of the surface-attached or unentrapped drug molecules (Hosny et al., 2018). Subsequently, the release rate slowed, indicating a diffusion-controlled release of quatsomes from the lipid bilayer. At 4 h, the cumulative amount of the drug released reached 58.84% ± 3.39%, and then a plateau-like phase was observed with final release of 86.48% ± 1.17% at 8 h. This sustainment could be attributed to the release of the entrapped drug within the vesicles. In contrast, KPN-SUSP exhibited a significantly slower release profile with only 12.45% ± 0.13% released at 0.5 h, increasing gradually to 27.34% ± 0.03% at 8 h. This slow release is related to the poor aqueous solubility of KPN and the absence of vesicle-mediated transport mechanism in the suspension.

The significantly enhanced release from quatsomes can be attributed to the nanoscale size of the vesicles, which provides a larger surface area for drug diffusion (Ahmed et al., 2025c; Bazaz et al., 2021). In addition, the presence of quaternary ammonium surfactants (QAS) and cholesterol in the bilayer may improve wettability and facilitate drug partitioning into the aqueous medium. Additionally, the positive surface charge of quatsomes could promote electrostatic interactions with the negatively charged buffer components, further enhancing drug release and promote stability (Albash et al., 2025a). Kinetic modeling provided further insight into the release behavior, revealing that the drug release profile of the optimized KPN formulation exhibited the best fit to the Higuchi diffusion model, signifying that diffusion through the vesicular matrix governed the release process.

The TEM micrographs of KPN-OQS showed that the vesicles were distinct with spherical morphology (as shown in Figure 3b). Furthermore, PS measured by Zetasizer was in a good harmony with TEM results (Sayed et al., 2021; Sakr et al., 2023). The vesicles displayed no evidence of aggregation or deformation, indicating the successful incorporation of quaternary ammonium surfactants and cholesterol, which likely contributed to the mechanical stability and integrity of the bilayer structure. The electrostatic repulsion generated by the surface charge effectively prevented vesicle–vesicle aggregation. Furthermore, QAS imparted steric stabilization by forming a hydrated barrier around each nanoparticle, thereby reducing interfacial tension and maintaining colloidal uniformity (Taw et al., 2025; Nemr et al., 2025).

KPN-OQS retained its physical characteristics till the end of storage period. A statistical comparison was made between the stored KPN-OQS key physical attributes and those of the freshly prepared samples. PDI (0.41 ± 0.02), EE% (115.21% ± 0.72%), PS (132.51 ± 3.11 nm), ZP (67.25 ± 4.1), and drug release after 8 h (Q8 h: 78.12% ± 0.96%) did not differ significantly (p > 0.05). This is might be attributed to the presence of cholesterol and QAS in its constructs. Cholesterol enhances the packing density of the vesicular membrane and consequently prevents drug leakage during storage (Ahmed et al., 2024b). Furthermore, QAS imparts a strong positive surface charge, providing electrostatic repulsion that inhibits vesicle aggregation. The combined effect of these components contributed to maintaining the vesicle’s integrity and uniformity, thus ensuring the preservation of particle size, surface charge, and entrapment efficiency over the studied period (Taw et al., 2025).

KPN demonstrated promising antifungal activity against C. albicans ATCC10231 with MIC of 5 ± 0 mg/mL.

A murine model of fungal vaginal infection was used to examine the KPN’s in vivo antifungal properties. C. albicans ATCC10231 suspension was vaginally injected into three groups of six female Wistar rats. The application of either the KPN-OQS or KPN-SUSP significantly reduced the fungal load recovered from the infected vagina compared to that of the negative control (no treatment) at all tested time intervals (One-way ANOVA, Tukey’s post hoc test, p < 0.0001) (Figure 4). The in vivo antifungal activity of KPN was considerably increased by the quatsomes, and at all tested time intervals, the antifungal activity of KPN-OQS was significantly higher than that of KPN-SUSP (One-way ANOVA, Tukey’s post hoc test, p < 0.01) (Figure 4). The fungal count retrieved from the KPN-OQS on the last day of the experiment was 4.807 and 2.941 logs lower than that of the negative control and KPN-SUSP groups, respectively. Interestingly, by the final day of the trial (72 h after treatment), the KPN-OQS had totally removed the colonized fungus from the vagina of three rats (Figure 4).

As shown in Figure 5, the rat vaginal samples of the normal control group showed normal histological structure of the vaginal wall, including intact stratified epithelial layers and intact lamina propria (a). Positive control (infected samples not treated) showed atrophy of the vaginal epithelium (b). Considering SUSP-treated group (c), photomicrographs showed no marked pathological changes in vaginal epithelium and lamina propria (Hematoxylin and Eosin stain). The KPN-OQS treated group (d) showed organized morphological features of the vaginal wall with intact epithelium without any microscopic alteration. However, histopathological analysis may not detect subtle irritation, mild inflammation, or functional changes. Therefore, future studies are warranted to include in vitro cytotoxicity assays on vaginal epithelial cells, evaluation of local inflammatory markers, and monitoring of animal body weight and behavior to provide a more comprehensive assessment of local and systemic toxicity.