Development and preclinical evaluation of quiniodochlor-loaded nanostructured lipid carrier-chitosan gel for topical antifungal therapy

Abstract

Background

The rising incidence of superficial fungal infections and the limitations of conventional topical therapies necessitate the development of advanced, targeted delivery systems.

Objective

This study aimed to develop and evaluate a novel quiniodochlor (QD)-loaded nanostructured lipid carrier (NLC) gel for enhanced topical antifungal therapy.

Methods

QD-loaded NLCs were prepared by hot homogenization using stearic acid (solid lipid) and cinnamon oil (liquid lipid), stabilized with Tween^® 80, and incorporated into a chitosan hydrogel. Formulations were characterized for particle size, polydispersity index (PDI), zeta potential, entrapment efficiency (EE%), drug release, and physicochemical stability. The developed gel was further evaluated for spreadability, extrudability, ex vivo skin permeation/retention, in vitro antifungal activity against Candida albicans, and in vivo skin irritation.

Results

The optimized NLCs exhibited nanoscale particle sizes (141.86–250.79) high EE% (> 98%), and low PDI (0.28–0.43). Despite a near-neutral zeta potential, colloidal stability was maintained, attributed to steric stabilization. The NLC gel showed a sustained drug release profile, significantly higher skin retention, and about 1.6-fold larger zone of inhibition against C. albicans compared to a marketed QD cream. Ex vivo studies confirmed enhanced dermal accumulation with negligible transdermal permeation. The formulation demonstrated excellent spreadability and extrudability, and elicited no signs of erythema or edema in an in vivo skin irritation model.

Conclusion

The developed QD-NLC-chitosan gel presents a promising, safe, and effective topical strategy with enhanced skin targeting, sustained local release, and superior antifungal activity, demonstrating strong potential as an advanced therapeutic alternative for superficial mycoses.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11671-026-04646-y.

Introduction

Nanostructured lipid carriers (NLCs), by forming an occlusive and hydrating film on the skin, can significantly enhance the permeation and bioavailability of lipophilic drugs like quiniodochlor (QD) [1, 2]. Despite this potential, advanced topical delivery research specifically on QD-loaded NLCs remains limited, presenting a clear opportunity for innovation. While a prior study developed a quiniodochlor (QD)-based nanoemulsion [3], its composition and delivery strategy differ substantially from the advanced, multi-functional system proposed herein.

However, the clinical development of QD has been historically tempered by significant safety concerns. Its systemic administration has been linked to subacute myelo-optic neuropathy (SMON), a serious neurological disorder [4, 5]. It is therefore critical to emphasize that the present work is exclusively focused on topical, localized delivery. This route is fundamentally distinct and is engineered to maximize drug concentration at the dermal site of infection while minimizing systemic absorption [6] QD topical gel is generally used as a first-line treatment as an antifungal and antibacterial agent to treat various skin infections, including athlete’s foot, jock itch, ringworm, and infected eczema. The advanced delivery system developed here a nanostructured lipid carrier (NLC) embedded in a bio adhesive gel is explicitly designed to achieve the dual goal of enhanced local retention and minimal transdermal flux, thereby presenting a potentially viable and safer topical application of this potent antifungal agent [7].

Herein, we develop and evaluate a novel, dual-action topical system composed of Quiniodochlor (QD)-loaded Nanostructured Lipid Carriers (NLCs) embedded within a chitosan-based hydrogel [8, 9]. Its novelty and incremental advantage over prior systems are defined by three key, integrated design elements:

First, regarding composition and mechanism, the system integrates a solid lipid matrix (stearic acid) with cinnamon oil as the liquid lipid. This specific lipid blend is designed not only to maximize QD loading and stability [10] but also to leverage the well-documented inherent antifungal, anti-biofilm, and anti-inflammatory properties of cinnamon oil [11, 12], aiming for a synergistic antimicrobial effect that goes beyond mere drug delivery. This differs from simpler prior nanoemulsions, which typically lack a structured solid matrix for controlled release and do not incorporate an active liquid lipid for pharmacological synergy.

Second, the incorporation into a chitosan gel provides a multi-faceted advantage beyond merely increasing dermal drug levels. The chitosan matrix offers superior muco- and bioadhesion, prolonging residence time at the infection site [13], and contributes its own broad-spectrum intrinsic antimicrobial and wound-healing activities [14, 15]. More importantly, chitosan’s biocompatible, hydrating, and film-forming nature is hypothesized to improve local tolerability, potentially mitigating the skin irritation associated with conventional QD creams, thereby addressing a known limitation of the drug itself.

Third, the system is engineered for a holistic therapeutic outcome. The NLC core ensures enhanced skin penetration and sustained, localized release of QD [16, 17], while the chitosan gel and the active lipids (cinnamon oil) contribute complementary antimicrobial, anti-inflammatory, and tissue-repair properties. This multifaceted approach aims not only to elevate and maintain effective skin drug concentrations but to actively modulate the local microenvironment, creating a more effective and tolerable therapeutic strategy for managing resistant or persistent superficial fungal infections [18, 19].

Therefore, the present work hypothesizes that this rationally designed QD-NLC-chitosan gel will provide a significant therapeutic advancement by synergistically combining enhanced drug delivery with multi-target antimicrobial action and improved local tolerability, offering a comprehensive and translational solution beyond the capabilities of existing QD formulations or generic NLC systems.

Materials and methods

Materials

Quiniodochlor (QD), cinnamon oil, fluconazole, and the commercial QD cream (Dermo Quinol) were kindly provided as gift samples by East India Pharmaceuticals, Kolkata, India. Tween^® 80 and stearic acid were procured from Merck, India. Chitosan, derived and purified from natural sources, was used in the formulation [20]. All experiments were conducted using Millipore water with a resistivity of 18.2 MΩ cm⁻¹. Candida albicans (MTCC 296), and Sabouraud Dextrose Agar (SDA), were supplied by Jupiter pharmaceuticals Ltd All analytical grade chemicals and reagents were purchased commercially and used as supplied.

Preparation of NLCs

The Quiniodochlor (QD)-loaded Nanostructured Lipid Carriers (NLCs) were prepared using a well-established hot homogenization technique [21]. All formulations were prepared to a standard total batch mass of 100 g. The drug, QD, was used at a fixed concentration of 3.00 g, constituting 3% w/w of the total final NLC dispersion. The lipid phase was prepared by melting the solid lipid, stearic acid, and combining it with the heated liquid lipid, cinnamon oil, at 75 ± 2 °C under magnetic stirring (500 rpm). For drug-loaded batches, QD (3.00 g) was dissolved in this uniform lipid phase. The aqueous surfactant phase was prepared separately by dissolving Tween^® 80 in warm purified water (70 °C) in a fixed ratio of 90:10 (Tween^® 80: water) [22].

The hot aqueous phase (75 ± 2 °C) was then added dropwise to the lipid phase under continuous stirring (800 rpm) to form a coarse primary emulsion. This emulsion was immediately subjected to high-shear homogenization using an Ultra-Turrax T25 digital homogenizer (IKA^®-Werke GmbH & Co. KG, Germany) equipped with an S25N-18G dispersing element at 15,000 rpm for 10 min while the beaker was covered with aluminium foil to minimize evaporation. The temperature was maintained at 75 ± 5 °C throughout the homogenization process [22, 23].

The resulting nanoemulsion was cooled to room temperature under gentle stirring to crystallize the lipids and form the NLC dispersion. The final suspension was centrifuged using a Remi Cooling Centrifuge (Model C-24 BL, Remi Elektrotechnik Ltd., India) at 10,000 rpm, 15 min, 27 °C to remove any unentrapped drug in the supernatant, and the true drug containing NLCs was collected [24].

A total of six NLCs (NLC-1 to NLC-6) were prepared with varying lipid-to-aqueous surfactant ratios of 70:30, 75:25, and 80:20, while both lipids were in the first portion and surfactant with water in the second portion. Solid lipid-to-liquid lipid ratios were 70:30 and 60:40. For each formulation, 10% water was used as part of the aqueous surfactant phase (comprising 90% Tween^®80 and 10% water). Additionally, a small amount of water was added as q.s. to compensate for losses during the hot homogenization process. The composition of these NLCs is presented in Table 1.

Table 1.

Composition of Quiniodochlor-Loaded NLCs (per 100 g batch) (n = 3)

Formulation	QD (gm.)	Stearic acid (gm.)	Cinnamon oil (gm.)	Aq. Surfactant* (Tween® 80 with water) (gm.)	Purified waterq.s.to maintain 100 gm.
NLC-2	3.00	43.62	29.08	24.25	q.s.
NLC-1	3.00	40.74	27.16	29.1	q.s.
NLC-3	3.00	46.56	31.04	19.40	q.s.
NLC-4	3.00	47.53	20.37	29.10	q.s.
NLC-5	3.00	50.89	21.81	24.25	q.s.
NLC-6	3.00	54.32	23.28	19.40	q.s.

*Aqueous Surfactant Phase: Tween^®80 and Purified Water in a 90:10 (w/w) ratio

Characterizations

Characterization of prepared QD-loaded NLC

Size

The mean particle size and polydispersity index (PDI) of the NLC suspension were determined using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern Instruments). Before analysis, the crude NLC suspension was appropriately diluted with ethanol to ensure optimal scattering intensity.

Zeta potential

The surface charge characteristics of the QD-loaded NLCs were evaluated by measuring their zeta potential using a Zetasizer (Malvern Panalytical). NLC samples were diluted with a 0.1 mM potassium chloride (KCl) solution to maintain a constant ionic strength. The diluted samples were then placed in an electrophoretic cell, and an electric field of 15.2 V/cm was applied.

Entrapment efficiency and drug loading

NLC suspensions were first centrifuged at 10,000 rpm for 15 min at 27 °C to extract the free drugs from the supernatant to assess the entrapment efficiency. The concentration of QD in the supernatant was then measured using a UV spectrophotometer (JASCO V-560) set to 265 nm after an appropriate dilution. A calibration curve was employed to quantify the results. The following formulas were used to determine drug loading and entrapment efficiency: A validated calibration curve of QD was used for quantification.

The percentage of drug entrapped within the nanoparticles Entrapment Efficiency (EE%) and the drug loading capacity (DL%) were calculated according to the standard formulas [25, 26], which are widely used in nanoparticle characterization:

  

1

  

2

In vitro drug release test

Due to the insolubility of QD in aqueous systems, the Indian Pharmacopoeia (IP) recommends using 2-methoxyethanol as a solvent additive for QD extraction from tablets [27]. Therefore, drug release studies for both the NLC formulations and pure QD were conducted using a combination of 2-methoxyethanol and phosphate buffer (pH 5.4). A 25:75 ratio of 2-methoxyethanol to phosphate buffer (pH 5.4) was employed for the release evaluation, as determined through trial optimization [28].

The release study was conducted using a Franz diffusion cell system. Briefly, a sample equivalent to 25 mg of QD was placed inside a pre-hydrated dialysis membrane (LA390-5MT, HiMedia; molecular weight cut-off 12–14 kDa) [29]. This membrane was then secured in the donor compartment of the Franz diffusion cell (25 mm diameter, 20 mL receptor volume, 4 cm² diffusion area). The receptor compartment was filled with 20 mL of the release medium (75:25 phosphate buffer:2-methoxyethanol, pH 5.4) and maintained at 37 ± 0.5 °C with continuous magnetic stirring at 150 rpm [30].

At predetermined time intervals (1, 2, 3, 4, 5, 6, 7, 8, and 12 h), 1 mL aliquots were withdrawn from the receptor compartment and replaced with an equal volume of fresh, pre-warmed medium to maintain a constant volume. The concentration of QD in the withdrawn samples was quantified using a UV spectrophotometer (Perkin Elmer, Lambda 20) at 265 nm. Cumulative drug release was calculated and expressed as a percentage over time. This duration was chosen to model a clinically relevant dosing interval for a topical antifungal intended for once or twice-daily application. It is sufficient to capture the complete release profile and differentiate between burst and sustained-release formulations, aligning with standard practices for topical formulation screening [29].

The optimized formulation was selected based on a multi-parameter evaluation including particle size, entrapment efficiency, zeta potential, and in vitro drug release profile. Among all formulations, NLC-3 was selected due to its overall acceptable physicochemical characteristics and comparatively sustained drug release behaviour (as described in the Results section). Accordingly, NLC-3 was considered the optimized formulation and was further utilized for gel preparation and subsequent evaluations.

Fourier transform infrared (FTIR) spectroscopy

To investigate potential drug–excipient interactions and to confirm the incorporation of QD within the NLC matrix, analysis were performed using an IR spectrophotometer (JASCO Model FT/IR-4600). FTIR spectra were recorded for the pure drug, the physical mixture of NLC ingredients, and the QD-loaded NLC formulation. Each spectrum was acquired over a wavenumber range of 4000–600 cm⁻¹ with a spectral resolution of 4 cm⁻¹, accumulating 32 scans to enhance the signal-to-noise ratio. Characteristic peaks of the drug and the formulation components were identified and compared to assess possible chemical interactions.

Differential scanning calorimetry (DSC)

The thermal behaviour and physical state of the QD within the NLCs were investigated using a DSC instrument (Mettler Toledo). Samples of the pure drug, the physical mixture of NLC ingredients, and the optimized QD-loaded NLC-3 formulation were accurately weighed, placed in standard 40 µL aluminium crucibles, and hermetically sealed. An empty, sealed crucible was used as the reference. Analyses were performed using a DSC instrument (Mettler Toledo) under a continuous nitrogen purge (flow rate: 50 mL/min). The samples were heated from 30 to 350 °C at a constant rate of 5 °C/min. The onset temperature, peak melting temperature (Tm), enthalpy of fusion (ΔH), and any shifts or disappearance of characteristic thermal events were recorded and compared across all samples to assess changes in crystallinity and potential drug-excipient interactions.

X-ray diffractometry (XRD) analysis

The crystalline nature of the pure QD, the physical mixture of ingredients, and its state within the optimized NLC-3 formulation were assessed using XRD with an Ultima IV X-ray diffractometer (Rigaku). The analysis was performed using Cu-Kα radiation (λ = 1.5404 Å) generated at 40 kV and 40 mA. Diffraction patterns were recorded over a 2θ range of 5–70° in continuous scan mode. The presence and intensity of characteristic diffraction peaks were used to determine the crystallinity of the samples.

Transmission electron microscopy (TEM)

The morphology and size of the QD-loaded NLCs were directly visualized using transmission electron microscopy (TEM) with a Hitachi H-7500 instrument. Sample preparation involved the negative staining method, where a drop of the diluted NLC suspension was placed onto a carbon-coated copper grid and allowed to adhere for 1 min [30]. Excess liquid was carefully wicked away using filter paper. The deposited nanoparticles were then negatively stained with a 1% (w/v) phosphotungstic acid solution (pH 7.0) for 30 s to enhance contrast. The grid was air-dried thoroughly and observed under the TEM operating at an accelerating voltage of 80 kV.

Preparation of NLC-loaded gel formulation

For the preparation of the NLC-loaded gel formulation, the QD-loaded NLC-based hydrogel was developed by incorporating the optimized batch (i.e., NLC-3 suspension) into a chitosan matrix following a method adapted from previous studies [31, 32]. Chitosan (1.50% w/v) was first dissolved in a 1% v/v acetic acid solution under continuous magnetic stirring at room temperature to obtain a clear, viscous solution (pH ~ 4–5). To support gel formation, sodium lauryl sulfate (SLS) was added to the chitosan solution at a concentration of 0.5% w/v and stirred until fully dissolved [32]. The Quiniodochlor (QD)-loaded Nanostructured Lipid Carrier (NLC) suspension was incorporated into the chitosan–SLS gel base through blending process to ensure uniform dispersion and prevent nanoparticle aggregation [32]. The mixture was done through continuous stirring, resulting in a homogeneous, opalescent hydrogel formulation. Finally, the pH of the resultant QD-NLC-loaded gel was adjusted to 6.0–6.5 using a 1 M sodium hydroxide solution. The final gel was stored in an airtight container for subsequent characterization. To maintain the final concentration in gel (i.e., 3%) of QD, the necessary adjustment of formulation was done at the time of NLC formulation.

In parallel, a blank NLC formulation was prepared using the same composition as the optimized F-3 (46.56 g stearic acid, 31.04 g cinnamon oil, and 19.40 g aqueous surfactant phase with q.s. water) but without QD, and subsequently incorporated into 1.50 g of chitosan to serve as the control gel formulation for comparative biological evaluations.

Characterization of prepared NLC-loaded gel

The prepared NLC-loaded gel was characterized for its organoleptic properties (colour, texture, homogeneity) and physical parameters. The pH of the hydrogel was measured using a digital pH meter (Model, Make) to ensure compatibility with the skin’s physiological pH range. For this measurement, a dispersion method was employed: 1 g of the gel was dispersed in 100 mL of distilled water and allowed to equilibrate for two hours with gentle stirring [33]. The pH electrode was then immersed in this aqueous dispersion, and the reading was recorded in triplicate. The direct method of inserting the probe into the neat gel, which can be unreliable due to poor electrode contact and high viscosity, was not used.

Viscosity

The viscosity of the NLC hydrogel formulation was determined using a DV-E viscometer (Brookfield Engineering Laboratories, Inc., USA) equipped with a T-bar spindle (selection of appropriate spindle based on viscosity range). The spindle was carefully inserted perpendicularly into the hydrogel sample, ensuring no contact with the beaker walls or bottom. After allowing the hydrogel to stabilize for 30 s, the viscometer was operated at a shear rate corresponding to a spindle rotation speed of 50 rpm.

Spreadability

The spreadability of the hydrogel, indicative of its ease of application and coverage on the skin, was evaluated using a parallel plate method. Approximately 60 mg of the hydrogel was placed between two clean glass slides (dimensions specified if relevant). A standardized weight of 60 g was carefully placed on the upper slide for two minutes to allow the hydrogel to spread uniformly. After removing the weight, the diameter of the spread hydrogel was measured. The spreadability factor was calculated using the formula: Sf = A/W, where W is the total weight (g) given to the sample, and A is the total sample spread area (mm²) [34].

Extrudability

The extrudability of the NLC-loaded hydrogel, reflecting its ease of dispensing from a collapsible tube, was assessed by a gravimetric method as described previously [34, 35]. A standard lacquered aluminium collapsible tube (15 g capacity, 5 mm nozzle orifice) was filled with 10 g of the hydrogel formulation. The tube was crimped, and a standardized weight of 500 g was placed on the tube’s body, 2 cm from the crimped end. The amount of hydrogel extruded from the nozzle under this constant pressure over a period of 30 s was collected and weighed. The extrudability was expressed as the weight (in grams) of gel extruded per second.

Ex vivo skin permeation and skin retention studies

Ex vivo skin permeation studies were conducted using Franz diffusion cells to evaluate the cutaneous delivery potential of the formulations, following established protocols [29, 36]. Goat skin was obtained from a local licensed slaughterhouse, carefully cleaned, and the subcutaneous fat along with connective tissues was surgically removed. The integrity of the skin was visually inspected, and only samples without visible imperfections were used. The skin was wrapped in aluminium foil, stored at  −20 °C, and used within one month of collection. On the day of the experiment, the skin was thawed at room temperature and hydrated in receptor medium for 30 min.

The study was designed with three independent replicates (n = 3) for each formulation group. The experimental groups were defined as follows: the test group comprised the QD-loaded NLC gel (F3, 3% w/w QD); the positive control group consisted of a marketed QD cream (Dermo Quinol, 3% w/w QD); and the negative control group included a blank NLC gel (without QD).

The cleaned goat skin was mounted on Franz diffusion cells (effective diffusion area: 4 cm², receptor volume: 20 mL) with the stratum corneum side facing the donor compartment. The receptor compartment was filled with 0.5% sodium lauryl sulfate in phosphate-buffered saline (PBS, pH 7.4) and maintained at 37 ± 0.5 °C under continuous magnetic stirring at 125 rpm to ensure sink conditions [29]. After a 30 minute pre-equilibration period, a precisely weighed amount (equivalent to 3 mg of QD) of each formulation was applied uniformly to the donor compartment. The limit of detection (LOD) and limit of quantification (LOQ) for QD in the receptor medium were determined to be 0.12 µg/mL and 0.36 µg/mL, respectively [28].

The skin surface was gently wiped with a cotton swab soaked in methanol-water (1:1 v/v) to remove any residual formulation. To determine the amount of drug retained within the different skin layers, a validated tape-stripping technique was employed [36, 38]. The application site was tape-stripped fifteen times using transparent adhesive tape (3 M). The first two strips were discarded. The subsequent thirteen strips were considered to represent the stratum corneum. The remaining skin (epidermis and dermis) was then cut into small pieces. Both the tape strips and the skin pieces were immersed separately in 10 mL of methanol and subjected to vortex mixing for 2 min followed by bath sonication for 30 min to extract the drug. The extracts were filtered (0.22 μm) and analysed [28]. The total drug retention was calculated as the sum of the drug found in the stratum corneum (tapes 3–15) and the viable skin (epidermis + dermis) [37, 38].

In vitro antifungal susceptibility

For in vitro antifungal evaluation, commercially available 3% QD cream (positive control), QD-free NLC gel (negative control), and QD-loaded NLC gel (test sample) were used. Candida albicans (MTCC 296) was selected as the test pathogen [39].

A working suspension was prepared by inoculating a fully sporulated 10-day-old Sabouraud Dextrose Agar (SDA) slant culture into 100 mL of Sabouraud Dextrose (SD) broth. After incubation at 37 °C for 72 h, the suspension was diluted with buffered peptone water to a final concentration of 5 × 10⁶ cfu /ml [40].

Zone of inhibition study

The antifungal activity was evaluated using the well diffusion method against Candida albicans (MTCC 296). The experiment was performed with three independent replicates (n = 3) for each formulation to ensure statistical reliability. The test groups were as follows:

Test Group: QD-loaded NLC gel (F3, 3% w/w QD). Positive Control Group: Marketed QD cream (Dermo Quinol, 3% w/w QD). Negative Control Group: Blank NLC gel (without QD).

Standard Control Group: Fluconazole (25 µg/disc), a standard systemic antifungal agent, was used as a reference to benchmark the activity of the topical formulations.

A 0.1 mL aliquot of the standardized fungal suspension (5 × 10⁶ CFU/mL) was spread uniformly onto the surface of solidified Sabouraud Dextrose Agar (SDA) plates. Four wells (6 mm diameter) were bored in each plate using a sterile cork borer. Each well was filled with a precise volume (50 µL) of the respective test, control, or standard formulation. The plates were incubated aseptically at 37 °C for 72 h. After incubation, the zones of inhibition (including the well diameter) were measured in millimeters using a digital caliper [41, 42].

MTT (cytotoxicity) assay

This experiment was conducted to evaluate the cytocompatibility and safety profile of the developed QD-loaded NLC gel formulation on human skin keratinocytes (HaCaT cells). The primary aim was to determine if the formulation or its components induce significant cytotoxicity at concentrations relevant to topical application, thereby supporting its potential for safe dermatological use.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, a standard colorimetric method for assessing cell metabolic activity, was employed to measure cell viability [43]. The assay principle is based on the reduction of the yellow, water-soluble MTT dye to insoluble purple formazan crystals by mitochondrial dehydrogenases in metabolically active cells. The intensity of the resulting colour, measured spectrophotometrically at 570 nm, is directly proportional to the number of viable cells [44].

HaCaT (Human Keratinocyte) cells were procured from the National Centre for Cell Science (NCCS), Pune, India. The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with high glucose, supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic solution. They were incubated at 37 °C in a humidified atmosphere containing 5% CO₂, with sub-culturing performed every two days. Cells at passage number 45 were used for the experiment.

For the assay, cells were seeded in a 96-well plate at a density of 1 × 10⁴ cells per well and allowed to adhere for 24 h. The cells were then treated with various concentrations of the QD-loaded NLC gel (after appropriate dispersion in serum-free media), the corresponding blank NLC gel (vehicle control), pure QD (drug control), and the individual formulation excipients at their equivalent concentrations. Untreated cells served as the negative control (100% viability), and cells treated with Doxorubicin (1 µM) served as the positive control for cytotoxicity [45]. After a 24-hour incubation period, the MTT reagent was added to each well. Following a 4-hour incubation, the formazan crystals formed were dissolved using dimethyl sulfoxide (DMSO). The absorbance of each well was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage relative to the untreated control.

Animal studies: general ethics and husbandry

These studies were conducted in accordance with the “Animal Research: Reporting of In Vivo Experiments” (ARRIVE) guidelines [46] and approved by the Institutional Ethics Committee of the School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan (Approval No: IAEC/SPS/SOA/162/2024). Healthy male Wistar albino rats, weighing 150–180 g, were procured from the Institute’s Central Animal House. The animals were housed in sanitary polypropylene cages with sterile rice husk bedding. They were maintained under standard environmental conditions (temperature: 25 ± 2 °C; relative humidity: 45 ± 5%) with a 12 h light/dark cycle and had ad libitum access to a standard rodent diet and water. All procedures were performed following the relevant guidelines and regulations [47].

Skin irritation study

This study aimed to evaluate the potential of the QD-loaded NLC gel to cause dermal irritation. A total of nine rats were divided into three groups (n = 3 per group). A 2 × 2 cm² area on the dorsal surface was marked, and hair was carefully removed using a sterile razor followed by a depilatory cream. Animals were observed for 24 h post-hair removal to ensure no pre-existing irritation. The groups were treated as follows: Group 1 received the QD-loaded NLC gel (test group), while Group 2 received the marketed QD cream (positive control). Each rat received 1 g of the assigned formulation, applied uniformly to the prepared area. The application site was evaluated at 24, 48, and 72 hours post-application for signs of erythema, edema, or rashes, and the responses were scored according to the Draize scoring system [48].

In vivo skin pharmacokinetic study: design and execution

A comparative pharmacokinetic study was designed to quantitatively assess the skin concentration-time profile and retention of the developed QD-loaded NLC gel against the commercial QD cream.

Study design and animal grouping

To obtain a full 24-hour pharmacokinetic profile while adhering to ethical principles, a parallel-group, staggered terminal sampling design was employed [49]. Both the test and positive control groups were included in the study, and an identical sampling protocol (n = 3 per time point) was applied in each case. Rats were randomly allocated into two formulation groups as follows:

• Group 1 (Test): QD-loaded NLC gel (F3, 3% w/w QD).

• Group 2 (Positive Control): Marketed QD cream (Marketed cream/Dermo Quinol, 3% w/w QD).

Formulation application and staggered sampling

Hair was removed from a delineated dorsal area (~ 20 cm²) 24 h prior. A single topical dose equivalent to 3 mg QD per cm² (or an equivalent volume for each group) was applied uniformly. To enable sampling at multiple time points (1, 2, 4, 6, 8, 12, and 24 h), a staggered schedule was implemented, wherein one rat from each group was euthanized at each time point. This resulted in n = 1 per group per time point, culminating in a total of n = 3 independent biological replicates per formulation group over the entire study duration. Euthanasia was performed under light anesthesia (ketamine 80 mg/kg and xylazine 10 mg/kg, i.p.), followed by cervical dislocation. A full-thickness skin biopsy (~ 2 cm²) was collected from the center of the application site.

Sample processing and analysis

The skin surface was wiped with a cotton swab soaked in ethanol-water (1:1 v/v) to remove residual formulation. The biopsy was weighed, minced, and homogenized in 2 mL of phosphate-buffered saline (pH 7.4). QD was extracted from the homogenate using a validated liquid-liquid extraction technique with pyridine [50, 51]. The extract was analyzed using a gas chromatograph with a flame-ionization detector (GC-FID). Separation was achieved on a 2 m × 2 mm glass column packed with 3% OV-17 on 80–100 mesh Chromosorb W-HP. The oven temperature was programmed from 150 to 280 °C at 5 °C/min, following a validated method [52, 53].

Statistical analysis

All data are presented as the mean ± standard deviation (SD). The ‘n’ for each experiment n = 3 for ex vivo permeation independent runs, n = 3 for zone of inhibition. The specific statistical test used for each data type using one-way ANOVA with Tukey’s post-hoc for multi-group comparisons, unpaired t-test for two-group comparisons. The pharmacokinetic studies were calculated using PK Solver software and GraphPad Prism. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

Results and discussion

NLC evaluation

The key physicochemical characteristics of different NLCs are summarized in Table 2, while Table 3 details drug loading and encapsulation efficiency. Experimental results revealed that the average particle size of these NLCs ranged from 141.86 ± 0.93 to 250.79 ± 0.28 nm, which is within the optimal range for enhancing skin penetration and follicular delivery [54, 55].

Table 2.

Characterization of different QD-loaded NLCs (n = 3)

Formulation code	Particle size (nm)	Zeta potential (mV)	Polydispersity index
NLC-1	141.86 ± 0.93	−2.73 ± 0.54	0.40 ± 0.10
NLC-2	250.79 ± 0.28	−8.29 + 0.10	0.29 ± 0.04
NLC-3	209.07 ± 2.73	−16.75 ± 0.04	0.28 ± 0.02
NLC-4	168.69 ± 0.79	−1.98 ± 0.05	0.33 ± 0.17
NLC-5	158.23 ± 1.69	−3.17 ± 0.30	0.43 ± 0.15
NLC-6	160.42 ± 1.33	3.82 ± 0.99	0.41 ± 0.04

Table 3.

Drug loading and encapsulation efficiency of QD-loaded NLCs (n = 3)

Formulation code	Drug entrapment efficiency (%)	Drug loading (%)
NLC-1	98.44 ± 0.35	29.52 ± 0.27
NLC-2	98.28 ± 0.36	29.51 ± 0.36
NLC-3	98.47 ± 0.28	29.77 ± 0.16
NLC-4	98.39 ± 0.24	27.06 ± 0.12
NLC-5	95.89 ± 0.66	20.49 ± 1.13
NLC-6	93.32 ± 1.01	21.97 ± 1.39

Drug loading efficiency analysis indicated that a lower drug-to-excipient ratio led to a decline in loading capacity, suggesting polymer matrix saturation at higher drug concentrations [56]. The developed formulation product (NLC-3) achieved the highest entrapment efficiency of 98.47 ± 0.28%, confirming the effective encapsulation of the active compound. Such high entrapment efficiency (> 98%) can be attributed to the hydrophobic nature of QD, its high solubility in the selected lipid blend (stearic acid and cinnamon oil), and the rapid solidification of the lipid matrix during the hot homogenization/cooling process, which effectively traps the drug within the nanostructure [21, 22].

The zeta potential of QD-loaded NLCs varied between 0.25 and 3.82 mV, indicating moderate electrostatic stability. However, colloidal stability in systems stabilized with non-ionic surfactants like Tween^® 80 is predominantly governed by steric hindrance rather than electrostatic repulsion [57]. The polydispersity index (PDI) ranged from 0.28 to 0.43, signifying a narrow to moderately broad particle size distribution, with values below 0.5 generally considered acceptable for colloidal dispersions. A comparative evaluation of the individual NLC formulations based on these parameters confirms the superiority of NLC-3 in terms of drug entrapment and loading efficiency. As mentioned in the experimental section, NLC-3 was selected for further evaluation and subsequent incorporation into the chitosan gel formulation [58].

In vitro drug release study and release kinetics

At predetermined time intervals up to 12 h, the cumulative drug release was quantified and expressed as percentage release versus time. This duration was strategically selected to simulate a clinically relevant dosing window for a topical antifungal formulation intended for once or twice daily application. It is sufficiently long to capture the complete release profile, enabling clear discrimination between burst and sustained-release behaviour, in accordance with established screening practices for topical delivery systems. Notably, sustained release is a critical performance attribute, as it ensures prolonged maintenance of therapeutic drug levels at the infection site while minimizing systemic exposure. Accordingly, in vitro release kinetics was considered the primary and decisive criterion for formulation optimization [59].

The release profiles of pure QD were compared with the developed NLC formulations (Supplementary Fig. 1). Over 12 h, pure QD exhibited a cumulative release of 97.28 ± 0.27%, whereas NLC-1, NLC-2, and NLC-3 showed 89.55 ± 0.34%, 88.59 ± 0.42%, and 86.99 ± 0.11%, respectively. Similarly, NLC-4, NLC-5, and NLC-6 demonstrated releases of 88.16 ± 0.91%, 88.59 ± 0.43%, and 90.59 ± 1.8%, respectively. Among these, NLC-3 displayed the most controlled and extended-release behaviour over the 12 h period. A direct comparison between pure QD and the optimized QD-loaded NLC-3 formulation (Fig. 1) highlights a pronounced difference in release kinetics, despite both systems approaching near-complete release within 12 h (QD suspension: 97.28 ± 2.1%; QD-NLC-3: 86.99 ± 3.4%) [59, 60].

Fig. 1.

In vitro drug release profile of QD and QD-loaded NLCs

The pure QD suspension exhibited a rapid initial burst, releasing 68.4 ± 4.2% within the first 2 h. In contrast, the QD-NLC formulation significantly attenuated this burst effect, in case of QD NLC-3, releasing only 42.1 ± 3.8% over the same period. This ~ 26% reduction in initial release clearly demonstrates the functional role of the NLC matrix as a diffusional barrier, effectively modulating early-phase drug release. This modulation represents a key functional advantage of the NLC system. While the overall extent of drug release remains comparable, the altered kinetics—particularly the suppression of the initial burst—are highly desirable for optimum prolonged topical therapy. Controlled release mitigates the risk of rapid local drug saturation and associated irritation, while promoting sustained therapeutic action. Therefore, the release behaviour of NLC-3 aligns optimally with the intended pharmacotechnical objective, establishing it as the most suitable and robust formulation in terms of sustained drug delivery performance [61].

Selection of the optimized NLC formulation

During formulation development and optimization, a total of six NLC formulations (NLC-1 to NLC-6) were systematically prepared by varying the lipid-to-aqueous surfactant ratios, as detailed in Table 1. The optimized formulation was selected based on a comprehensive multi-parameter evaluation, including particle size, entrapment efficiency, zeta potential, and in vitro drug release profile. The primary objective was to identify the formulation best suited for subsequent gel incorporation and biological evaluation for topical antifungal therapy.

Among the developed systems, NLC-3 exhibited an optimal particle size (~ 200 nm), favourable for enhanced skin retention and follicular deposition, along with excellent entrapment efficiency (98.47 ± 0.28%) and high drug loading capacity. Although the zeta potential ( ~ − 16.75 mV) was moderate, it was considered adequate due to the predominance of steric stabilization imparted by Tween^® 80, further supported by the immobilization effect of the chitosan gel matrix.

In addition, NLC-3 exhibited a more controlled and sustained drug release profile compared to the other five NLC formulations, with release extending up to 12 h. The sustained release behaviour observed in NLC-3 is critical for maintaining effective drug concentrations at the site of infection while minimizing systemic exposure, thereby directly fulfilling the intended therapeutic objective. Consequently, in vitro release kinetics emerged as the primary and decisive criterion for formulation selection.

Collectively, these attributes indicate that NLC-3 achieves the most rational balance among critical quality attributes, establishing it as a stable, skin-retentive, and sustained-release delivery system. Accordingly, NLC-3 demonstrated the most therapeutically relevant and technically robust performance and was therefore selected as the optimized formulation for gel preparation and subsequent evaluations.

Mechanistic interpretation of release kinetics

The analysis reveals that the release from both pure QD and NLC-3 follows the Korsmeyer-Peppas model most closely, as indicated by the highest R² values (0.998 and 0.994, respectively) and the lowest SSR values. The ‘n’ value, known as the release exponent, is a key indicator of the drug release mechanism presented in Table 4.

Table 4.

Release kinetics of QD from the NLC-3 formulation

Kinetic Model	Equation	Parameters	NLC-3	Pure QD
Zero-Order	Q = k0t	k0 (h−1) R2 SSR	7.58 0.984 25.31	8.11 0.991 15.89
First-Order	Log(Q0-Q) = Log Q0 - k1t/2.303	k1(h−1) R2 SSR	0.15 0.947 89.45	0.23 0.962 71.22
Higuchi	Q = kḥt1/2	kḥ (h−1/2) R2 SSR	27.45 0.974 43.67	29.33 0.985 27.54
Korsmeyer-Peppas	Q/Q0 = kκptn	kκp n R2 SSR	17.21 0.45 0.998 3.12	22.18 0.39 0.994 10.45

For the NLC-3 formulation, the ‘n’ value was found to be 0.45. According to the Korsmeyer-Peppas model for spherical matrices, an ‘n’ value of 0.43 corresponds to a drug release mechanism governed by Fickian diffusion, while a value between 0.43 and 0.85 indicates anomalous (non-Fickian) transport, involving a combination of diffusion and polymer/lipid matrix relaxation [62].

The value of 0.45 for NLC-3 suggests a release mechanism that is primarily Fickian diffusion. This can be interpreted as the drug first diffusing out of the nanostructured lipid matrix and then through the dialysis membrane. The solid lipid matrix of the NLC acts as a reservoir, controlling the diffusion of the drug and thereby resulting in a more sustained profile compared to the unencapsulated drug [63]. Pure QD, with an ‘n’ value of 0.39, also suggests a Fickian diffusion mechanism, but from a pure drug crystal suspension without a retarding lipid matrix, which explains its faster release rate [64]. The lower release rate constant (kκp) for NLC-3 (17.21) compared to pure QD (22.18) further quantitatively supports the sustained release effect provided by the nanocarrier.

Therefore, the release from the NLCs is more accurately described as a “modulated” or “prolonged” release profile rather than a classically sustained release system. The mechanism, as indicated by the Korsmeyer-Peppas model (n ≈ 0.45), is governed by diffusion-controlled release from the solid-lipid matrix. This controlled, diffusion-based elution supports the potential for maintaining effective drug concentrations in the skin over a clinically relevant timeframe, which may translate into a reduced dosing frequency compared to a conventional formulation that releases its payload more rapidly [65]. The limitations of this single-medium in vitro model are acknowledged, and the release behavior in the complex in vivo skin environment may vary [66].

FTIR analysis

Figures 2a–b show the FTIR spectra of pure QD, a physical mixture of formulation excipients, and the QD NLC dispersion. The QD’s FTIR spectrum exhibits a characteristic aromatic C–H stretch at 3070.12 cm⁻¹ and an aliphatic C–N stretching peak at 2358.52 cm⁻¹. The aromatic C=C stretching appears between 1586.16 and 1473.35 cm⁻¹, while the C–Cl stretch is detected at 1395.25–1041.37 cm⁻¹ and 648.89 cm⁻¹. For the physical mixture of excipients and the NLC formulation, peaks corresponding to the excipients are observed at 2914.88 cm⁻¹ and 2913.91 cm⁻¹ [67]. The lack of significant peak shifts or new peaks in the QD NLC spectrum suggests no chemical interaction between the QD and the NLC formulation, confirming product stability and successful entrapment within the lipid matrix [68]. The disappearance or attenuation of characteristic QD-specific peaks in the final NLC formulation provides direct evidence of its molecular dispersion and amorphization within the lipid core [69].

Fig. 2.

FTIR of pure QD, Physical mixture of all excipients, and QD NLC-3

DSC

DSC is a valuable technique for analyzing the crystalline and melting behavior of materials. To evaluate potential depression in the lipid melting point and to characterize the thermal behavior of the system, DSC thermograms of the pure drug, the physical mixture of the formulation components, and the QD-loaded NLC formulation were analyzed (Fig. 3). The crystalline nature of pure QD was confirmed by a distinct melting peak at 182.26 °C. The small endothermic peak at ~ 50 °C in the NLC-3 thermogram is addressed in Sect. 5.1.3. It is attributed to the recrystallization of a fraction of the solid lipid (stearic acid) into a more stable polymorph during storage. We discuss that this limited crystallinity is not detrimental; it can actually contribute to the rigidity of the lipid matrix, further sustaining drug release. The primary finding remains the significant reduction/absence of the sharp QD melting peak, confirming its amorphous state within the NLC [31].

Fig. 3.

DSC thermogram of QD, Physical mixture formulation, and QD-loaded NLC-3

XRD analysis

X-ray diffraction (XRD) analysis was performed to evaluate changes in the crystallinity of QD during the formulation process. Figure 4a–b presents the XRD patterns of pure QD and the NLC formulation. The diffraction pattern of pure QD exhibited sharp peaks at 22.49, 22.92, and 24.35, indicating its highly crystalline nature [44]. In contrast, the formulation showed a reduction in characteristic QD peak intensities at positions 22.15, 22.91, and 24.50 suggesting a decrease in crystallinity while Physical mixture have shown the peaks at 15.61,16.83,19.25, and at 31.63. This reduction may be attributed to the encapsulation of QD within the lipid matrix, potentially leading to enhanced solubility and bioavailability.

Fig. 4.

XRD patterns of QD & QD loaded NLC dispersion

TEM study

TEM imaging provided deeper insights into the morphology of the QD-loaded NLC-3 formulation ). The images (Fig. 5) revealed a well-defined spherical shape with a smooth surface, confirming the uniformity of the nanoparticles. Notably, the NLCs were evenly dispersed without significant aggregation, highlighting their colloidal stability [70].

Fig. 5.

Transmission Electron Microscopy QD loaded NLC-3

The observed particle size from TEM analysis was approximately 200 nm, and the hydrodynamic diameter of ~ 209.07 ± 2.73 nm measured by Dynamic Light Scattering DLS. This consistency validates the nanoscale characteristics of the formulation.

It is important to note that DLS and TEM are complementary techniques that measure different properties: DLS reports the hydrodynamic diameter of particles in their native, hydrated state within a dispersion. This includes the lipid core, the surrounding surfactant. While TEM provides a direct image of the dry, core size of individual particles under high vacuum after negative staining. So, there is some difference may occur between these results [71, 72].

Characterization of QD-loaded NLC gel formulation

The QD-loaded NLC gel exhibited a semisolid consistency and appeared slightly opaque, smooth, and uniform. The pH of the formulated was 6.08 ± 0.12, falling within an acceptable range²⁵. The gel’s swelling behavior was found satisfactory, as it effectively absorbed secretions at the application site. The gel demonstrated an optimal viscosity for topical application, measured at 28,400 ± 3.19 cps. Moreover, the gel exhibited shear-thinning behavior, as viscosity decreased with an increasing shear rate, confirming its pseudoplastic (non-Newtonian) behavior.

Spreadability plays a crucial role in determining the effectiveness of a topical formulation. An ideal gel should spread smoothly on the skin without requiring excessive force. In our spreadability test, we observed a decrease in the gel’s spreadability, which can be attributed to the increased viscosity caused by the stronger crosslinking effect of CS. The spreadability factor for the NLC gel was 0.709 ± 0.14, while the commercial cream showed a slightly lower value of 0.701 ± 0.17. The QD-loaded NLC gel (1.50% w/w CS) demonstrated slightly better spreadability comparable to the commercial formulation. The extrudability percentage of our QD-loaded NLC gel was found to be 3.69 ± 1.52, indicating satisfactory ease of application.

Ex vivo skin permeation and skin retention study

The ex vivo permeation profile across goat skin was investigated for the developed QD-loaded NLC gel (F3) in comparison to the marketed QD cream and a blank NLC gel. The results demonstrated negligible drug permeation into the receptor compartment over 24 h for all groups, with concentrations remaining below the limit of detection (LOD). This confirms that both the commercial cream and our NLC gel are designed for localized action, with minimal potential for systemic absorption. In contrast, the skin retention data revealed significant differences between the formulations. The amount of drug retained within the total skin layers (stratum corneum, epidermis, and dermis) is summarized in Table 5.

Table 5.

Ex-vivo skin retention data of best F3 with marked cream (Mean ± SD, n = 3)

Formulation	Total drug retained (%)	p-value (vs. Marketed cream)
Marked QD cream (Positive Control)	17.65 ± 0.49	–
QD-loaded NLC Gel (F3)	42.63 ± 0.18	< 0.001
Blank NLC Gel (Negative Control)	Not detected	–

The QD-loaded NLC gel demonstrated a significantly higher (p < 0.001) skin retention (42.63 ± 0.18%) of the applied dose compared to the marketed cream (17.65 ± 0.49%). This represents approximately 2.41-fold increase in total skin retention. The blank NLC gel showed no detectable drug, confirming that the measured QD originated from the active formulations and not from excipient interference. This enhanced retention can be attributed to the synergistic effects of the NLC system. The nanoscale size of the particles facilitates intimate contact with the skin surface and may create a higher concentration gradient, driving drug diffusion. Furthermore, the lipid matrix of the NLCs promotes exclusivity, enhancing skin hydration and thereby increasing the permeability of the stratum corneum.

The safety profile of any formulation containing QD must be considered in light of its historical association with subacute myelo-optic neuropathy (SMON) following systemic use [73]. The data from this study provide a strong preclinical rationale for mitigating this risk through a targeted topical approach. Our ex vivo permeation studies demonstrated that the QD-NLC gel facilitated significant drug accumulation in the skin layers while drug permeation into the receptor fluid was negligible. This was further corroborated by in vivo pharmacokinetic data, which showed high cutaneous AUC and Cmax with no detectable systemic exposure over 24 h. These findings indicate that the formulation successfully confines QD to the local site of action. Therefore, the risk profile of this topical QD-NLC gel is fundamentally distinct and more favourable than that associated with historical systemic administration.

Overall, the results indicate that the NLC-based gel is superior not only in retaining the drug on the skin surface but also in facilitating its deposition into the deeper epidermal and dermal layers, which are the primary target sites for superficial fungal infections. This targeted and enhanced localization maximizes therapeutic efficacy at the site of action while maintaining a favourable safety profile through minimal systemic exposure.

A 24-hour evaluation period is standard in transdermal research for assessing long-term permeation kinetics and steady-state flux. The present study demonstrates negligible transdermal flux over 24 h, which is a highly desirable outcome for a topical antifungal intended for localized action. It is acknowledged that the inclusion of 0.5% SLS in the receptor phase, although standard practice for maintaining sink conditions for hydrophobic drugs, may slightly alter skin barrier integrity. However, the complete absence of detectable drug suggests that even a potentially compromised barrier did not permit significant systemic permeation, underscoring the formulation’s strong propensity for dermal retention.

In vitro antifungal susceptibility testing

The results of the antifungal activity study, presented in Table 6 and Fig. 6, clearly demonstrate the enhanced efficacy of the QD-loaded NLC gel.

Table 6.

Zone of inhibition results against Candida albicans (n = 3)

Formulation/ Control	Zone of inhibition (mm)	p-value (vs. Marketed cream)
QD-loaded NLC Gel (F3)	26.12 ± 0.35	< 0.001
Marketed QD Cream (Positive Control)	16.45 ± 0.28	–
Blank NLC Gel (Negative Control)	No Zone	–
Fluconazole (Standard Control, 25 µg/disc)	22.18 ± 0.41	< 0.001*

The p-value for Fluconazole is provided for informational comparison against the marketed cream, though it is a different class of agent

Fig. 6.

Zone of inhibition study Marketed QD cream, of Standard drug and QD NLC gel of Batch F3 (Test)

The QD-loaded NLC gel (F3) produced a mean zone of inhibition of 26.12 ± 0.35 mm, which was significantly larger (p < 0.001) than the 16.45 ± 0.28 mm produced by the marketed QD cream (Positive Control). This represents an enhancement of approximately 58% in the inhibitory zone diameter. The blank NLC gel (Negative Control) showed no zone of inhibition, confirming that the antifungal activity originated solely from QD. the standard antifungal Fluconazole (25 µg/disc) produced a zone of 22.18 ± 0.41 mm.

This enhanced activity can be attributed to the multifunctional design of the NLC-chitosan gel. The NLC core improves the diffusivity and availability of the hydrophobic QD in the agar medium. Furthermore, the incorporation of cinnamon oil as the liquid lipid may contribute inherent antifungal and anti-inflammatory properties, potentially acting in synergy with QD. Similarly, the chitosan gel matrix is known for its intrinsic, mild antimicrobial activity, which could provide an adjunctive effect. Therefore, the observed larger zone is likely the result of a combined effect: enhanced delivery of the primary drug (QD) coupled with the supplementary antimicrobial properties of the formulation’s excipients.

For reference, the standard antifungal disc containing Fluconazole (25 µg/disc) produced a zone of 22.18 ± 0.41 mm. While the NLC gel showed a larger zone in this in vitro setting, direct clinical superiority to systemic azoles cannot be inferred from this assay alone. Fluconazole is a systemic agent with a distinct pharmacokinetic profile designed for body-wide distribution, whereas the NLC gel is optimized for intense, localized delivery. The primary and most relevant comparison remains against the topical marketed cream, where the NLC gel’s superiority is unequivocal. The result against Fluconazole primarily serves to highlight the potent local antifungal concentration achievable with the advanced delivery system, underscoring its potential for managing localized infections where high topical drug levels are advantageous.

The superior antifungal activity of the NLC gel is attributed to the functional advantages conferred by the nanostructured lipid carrier system. The NLCs enhance the apparent solubility and diffusivity of the hydrophobic QD in the aqueous agar medium. More importantly, the small particle size and lipidic nature of the NLCs promote intimate contact and adhesion with fungal cell membranes, potentially enhancing drug uptake. Furthermore, the sustained release profile ensures a prolonged, effective local drug concentration at the site of diffusion, contributing to a larger and more sustained zone of inhibition.

MTT (cytotoxicity) assay

The MTT assay, a standard colorimetric method, assessed cell proliferation and cytotoxicity by measuring the enzymatic reduction of MTT to purple formazan in metabolically active cells. The resulting colour intensity is directly correlated with cell viability [42]. Our results indicated excellent biocompatibility of the QD-loaded NLC gel with HACAT cells at concentrations up to 3% (w/v) after 24 h. The MTT assay confirmed the non-toxic nature and even support of cell proliferation by these concentrations. Cell viability was highest in the untreated control (100.00%), while the positive control, Doxorubicin (1 µM), significantly reduced viability to 68.96 ± 0.46%. Conversely, the QD-loaded NLC gel treatment showed higher, concentration-dependent cell viability: 85.75 ± 0.97% (1% w/v), 81.29 ± 1.32% (1.5% w/v), and 78.87 ± 2.40% (3% w/v). These findings highlight the biocompatibility of the formulation QD-NLC (F3) gel, suggesting its potential for safe and effective topical use.

The preliminary safety assessment of the QD-NLC gel is encouraging. The MTT assay indicated low cytotoxicity against human keratinocytes, with viability maintained within an acceptable range for topical formulations. This was corroborated by the skin irritation study, where the NLC gel elicited no signs of erythema or edema over 72 h in rats a marked contrast to the mild rashes observed with the marketed cream at the same time point. Collectively, these data support the short-term local tolerability and biocompatibility of the developed formulation. However, it is important to contextualize these findings. These are acute, single-application studies. The long-term dermal safety upon repeated daily application, as would be required in a clinical setting, remains to be evaluated in chronic toxicity models. Furthermore, while the ex vivo and in vivo PK data suggest minimal systemic exposure a key factor in mitigating the historical systemic risks associated with QD definitive systemic safety upon chronic topical use must be confirmed in extended-duration studies.

In-vivo skin irritation studies

A skin irritation study assessed the safety of the formulated QD-loaded NLC gel for topical application by monitoring for skin irritation. Observations at 24, 48, and 72-hours post-application showed no visible signs of irritation like erythema, edema, or rashes. Interestingly, after 72 h, the commercial QD cream caused moderate rashes, while the NLC formulation was well-tolerated. The excellent biocompatibility of NLCs is likely due to their physiological lipid composition, allowing natural metabolism into glycerol and fatty acids, essential components in biological processes [74]. Conversely, the QD in the marketed cream might directly interact with the skin, potentially causing irritation. Our NLC gel formulation acts as a protective nanocarrier, reducing direct skin exposure and minimizing irritation, thus appearing promising for safe and effective topical delivery.

Pharmacokinetic study

The pharmacokinetic study was performed to provide a quantitative comparison of QD delivery and retention within the skin layers following the application of the NLC gel versus the commercial cream. The results unequivocally demonstrate the superior performance of the nanocarrier-based system.

The key pharmacokinetic parameters are summarized in Table 7. The QD-loaded NLC gel achieved a significantly higher (p < 0.01) maximum drug concentration in the skin (Cmax) of 756.00 ± 0.43 ng/mL compared to 601.00 ± 0.30 ng/mL for the marketed cream. This indicates a more efficient and rapid accumulation of the drug at the target site, which is crucial for initiating a prompt therapeutic effect against fungal infections.

Table 7.

Pharmacokinetic parameters of QD after topical application of different formulations in rat skin (Mean ± SD, n = 3)

Parameter	Prepared NLC Gel F3 (3%)	Marketed Cream (3%)	p-value
Tmax (h)	4.0	4.0	–
Cmax (ng/mL)	756.00 ± 0.43	601.00 ± 0.30	< 0.05
AUC0−24 (ng·h/mL)	10,095.75 ± 6.32	6182.23 ± 4.41	< 0.05
AUC0−∞ (ng·h/mL)	12,202.63 ± 12.96	6697.54 ± 6.78	< 0.05
Elimination Half-life (h)	0.116 ± 0.13	0.085 ± 0.02	> 0.05

NS, Not significant

The comparative bioavailability was assessed using both AUC_0–t and AUC_0–∞ (Table 7). The prepared NLC gel (F3) demonstrated significantly higher exposure, with an AUC_0–24 of 10,095.75 ± 6.32 ng·h/mL and an AUC_0–∞ of 12,202.63 ± 12.96 ng·h/mL, compared to the marketed cream (AUC_0–24: 6,182.23 ± 4.41 ng·h/mL; AUC_0–∞: 6,697.54 ± 6.78 ng h/mL). Both metrics confirm a substantial and statistically significant (p < 0.001) enhancement in bioavailability [75].

The elimination half-life was longer, and the clearance rate from the skin was lower for the NLC formulation, although the difference was not statistically significant. The negative control (blank NLC gel) group showed no detectable levels of QD, confirming the specificity of the analytical method and that the measured pharmacokinetics were solely due to the active drug.

The pronounced enhancement in skin pharmacokinetic parameters specifically the significantly higher AUC_0–12 and Cmax observed for the QD-NLC gel carries direct implications for its potential clinical utility. A higher AUC reflects a greater total drug exposure in the target tissue over time, while a higher Cmax indicates the achievement of peak concentrations more likely to exceed the minimum inhibitory concentration (MIC) for pathogenic fungi. This pharmacokinetic profile translates to a compelling therapeutic hypothesis: the increased and sustained drug levels in the skin could lead to improved cure rates for superficial mycoses by maintaining fungicidal concentrations at the infection site for a longer duration. Furthermore, this enhanced local bioavailability may enable a reduced dosing frequency compared to conventional creams, potentially improving patient adherence a common challenge in the management of chronic fungal infections. However, it is crucial to acknowledge that while these preclinical pharmacokinetic and retention data are highly promising, they serve as a surrogate for efficacy. Definitive proof of therapeutic superiority and long-term safety must be established in controlled clinical infection models and, ultimately, in human patient trials.

Fig. 7.

Cumulative drug release (%) profile marketed formulation and developed F3 gel

Conclusion

This study successfully developed and evaluated a novel QD-loaded NLC-chitosan gel designed for targeted topical antifungal therapy. The formulation exhibited optimal nanoscale characteristics, high entrapment efficiency, and a modulated, diffusion-controlled release profile. Critically, the system demonstrated a substantial increase in dermal drug retention and in vitro antifungal activity over a marketed cream, alongside low cytotoxicity and no evidence of skin irritation in acute studies. Collectively, these findings strongly support the hypothesis that the dual-action NLC-gel system can enhance local bioavailability, efficacy, and tolerability of QD. While these preclinical results are promising, future translation will require rigorous long-term stability assessments, scalable manufacturing process validation, and clinical trials to confirm therapeutic efficacy and safety in patients, with careful monitoring of QD’s systemic safety profile despite the low permeation observed. This versatile platform also holds significant potential for the topical delivery of other challenging hydrophobic therapeutics.

This study comprehensively demonstrates the development, characterization, and enhanced cutaneous delivery of a novel QD-NLC gel formulation. However, a key limitation is that the antifungal efficacy was evaluated only in vitro. While the high skin deposition and retention profiles are strong indicators of potential therapeutic success, direct evidence of efficacy in a live infection model is crucial. Therefore, the logical next step and primary focus of ongoing work is to evaluate the therapeutic performance of this optimized QD-NLC gel in a validated in vivo dermatophytosis model. This future study will provide definitive proof of concept for the clinical translation of this formulation for the treatment of superficial fungal infections.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

NLC

Nanostructured lipid carrier

QD

Quiniodochlor the drug, Quinoiodochlor / Clioquinol)

EE%

Entrapment efficiency (percentage)

DL%

Drug loading (percentage)

PDI

Polydispersity index

DLS

Dynamic light scattering

TEM

Transmission electron microscopy

DSC

Differential scanning calorimetry

FTIR

Fourier-transform infrared (spectroscopy)

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (a tetrazolium salt used in cell viability assays)

HET-CAM

Hen’s egg test-chorioallantoic membrane

Author contributions

U.K.B.: Conceptualization, Methodology, Investigation, Formal analysis, Writing—original draft. A.B.: Conceptualization, Supervision, Methodology, Writing—review & editing. S.N.: Investigation, Writing—original draft. M.J.: Methodology.

Funding

Open access funding provided by Siksha 'O' Anusandhan (Deemed To Be University). No funding was received for this study.

Data availability

The datasets produced and/or examined in this study can be obtained from the corresponding author upon reasonable request.

Declarations

Ethics approval

All animal experiments were approved by the Animal Ethics Committee of the School of Pharmaceutical Sciences, Siksha ‘O’ Anusandhan, Approval Number: IAEC/SPS/SOA/162/2024 and were performed in accordance with relevant guidelines and regulations ARRIVE guide for the Care and Use of Laboratory Animals.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.