Development and Evaluation of Amine-Functionalized Multi-Walled Carbon Nanotubes Based Gel for Enhanced Topical Antifungal Delivery of Voriconazole

Development and Evaluation of Amine-Functionalized Multi-Walled Carbon Nanotubes Based Gel for Enhanced Topical Antifungal Delivery of Voriconazole | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Development and Evaluation of Amine-Functionalized Multi-Walled Carbon Nanotubes Based Gel for Enhanced Topical Antifungal Delivery of Voriconazole Vaishnavi Shelke, Manoj Shinde, Varsha Mane, Priyanka More This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9064633/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract A potent antifungal drug, voriconazole (VRZ), whose clinical performance is limited by its poor solubility. The current research aimed to investigate the capability of anime-functionalized multi-walled carbon nanotubes (MWCNTs-NH₂) as a carrier for the topical VRZ delivery. The VRZ-CNTs formulation was optimized using a 3² full factorial design. An optimized VRZ-CNTs showed reasonable particle size (102.9 ± 1.02 nm), suitable zeta potential (-37.3 ± 1.01 mV), and maximum entrapment efficiency (85.32 ± 1.07%), indicates the reliability and stability of the formulation. FTIR study verifies the compatibility of the formulation constituents, while DSC and XRD results reveal transmission of drug’s nature from crystalline to amorphous. SEM results suggested nanotubular morphology of VRZ-CNTs. The VRZ was released from formulation in sustained manner and showed 85.66 ± 2.33% drug release at the end of 8 hours. The optimized VRZ-CNTS effectively incorporated into gel with the use of carbapol 934, which showed adequate pH (6.74 ± 0.21), drug content (97 ± 2.56%), spreadability (26.5 ± 1.21 g·cm/sec) and viscosity (8963 ± 117 mPa·s). The gel illustrated markedly higher Ex-vivo skin permeation and drug retention. Skin irritation study indicated that the gel formulation was non-irritant. Moreover, in vivo evaluations confirmed the enhanced antifungal efficacy and better histopathological recovery of the VRZ–CNTs gel compared to conventional gel. Thus, VRZ-CNTS gel represents a promising and efficient alternative to conventional topical antifungal treatment. Voriconazole Carbon nanotubes Antifungal activity Factorial design Topical delivery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 1 Introduction Fungal infections occur when fungi colonize and proliferate in the human body which adversely affects superficial and also deeper tissues [ 1 ]. These infections are widely predominant, especially in persons with compromised immunity, and can exhibit in varying degrees of intensity ranging from mild or moderate superficial infections of the skin and nails to systemic conditions that may become potentially fatal [ 2 ]. General types of fungi responsible for such infections comprise yeasts, dermatophytes, and molds. Dermatophytes are often associated with infections such as ringworm and athlete’s foot, while yeasts like Candida species are prominent to cause mucosal and systemic infections, for example candidiasis, which can become extremely serious in immunosuppressed patients [ 3 , 4 ]. In contrast to bacterial infections, fungal infections are more difficult to treat because of structural and metabolic resemblance among fungal and human cells, which diminishes the availability of specific target for antifungal treatment [ 5 ]. Existing management approaches mostly depend on agents such as azoles, polyenes, allylamines, and echinocandins. From these, azoles, specifically imidazoles and triazoles, exert their effect by inhibiting ergosterol synthesis, an vital component of fungal cell membranes, consequently impairing survival of cell [ 6 , 7 ]. Despite its effectiveness, the phamacological potential of azoles is restricted by their poor solubility, low bioavailability, and dose-responsive adverse events, which demand the assessment of advanced drug delivery systems proficient to improve their effectiveness [ 8 ]. Nanotechnology-based carriers like solid-lipid nanoparticles, nanostructured lipid carriers, liposomes, niosomes, microemulsions, nanogels, polymeric nanoparticles, microneedles, carbon nanotubes (CNTs) etc. have been extensively investigated in recent times to conquer these hurdles. Nano carrier ystems can enhance the solubility, permeability, and stability of antifungal agents, thus enhancing site-specific delivery, prolonged release, and alleviating systemic side effects [ 9 , 10 ]. Among these nanocarriers, CNTs have attained significant attention because of their distinctive physicochemical characteristics, such as a greater aspect ratio, good mechanical strength, adaptable surface chemistry, high drug loading capacity, and capability to penetrate biological barriers [ 11 – 13 ]. Furthermore, functionalization of CNTs improves their dispersibility, drug-binding efficiency, and biocompatibility, making them ideal for drug delivery [ 14 ]. A second-generation triazole antifungal agent, Voriconazole (VRZ) is extremely effective against a wide range of fungal pathogens, including Candida , Aspergillus , and emerging resistant strains. VRZ act by inhibition of cytochrome P450-dependent 14-α-demethylase, which obstructs ergosterol biosynthesis, resulting in altered membrane permeability and subsequently death of fungal cell. Although it’s broad spectrum antifungal potential, VRZ is poorly water-soluble drug, showing variable absorption and low bioavailability [ 15 , 16 ]. To achieve therapeutic concentration high doses are often required, which markedly elevate the risk of hepatotoxicity, neurotoxicity, and drug interactions [ 17 ]. Thus, developing a delivery system that enhances solubility, increases bioavailability, and minimizes toxicity of VRZ is of immense important. Topical route offers a promising approach for antifungal drug delivery as it facilitates localized treatment specifically at the site of infection, minimizes systemic exposure, and offers patient convenience [ 18 ]. Nevertheless, the efficiency of conventional topical formulations is often limited by poor solubility of the active drug, restricted drug permeation through the stratum corneum,, and inadequate retention in the skin layers [ 19 ]. Embedding drug into nanocarriers such as CNTs and further this system incorporated to topical gel formulation has the potential to tackle these problems by facilitating effective drug encapsulation, improving solubility, as well as fostering sustained release at the site of infection [ 20 ]. This ensuring higher therapeutic efficacy of VRZ at lower doses while decreasing its adverse effectss [ 21 ]. Hence, developing a CNTs-based gel for VRZ delivery manifests a novel approach for the treatment of superficial fungal infections. Recent research has highlighted that CNTs can not only improve drug delivery but also demonstrate intrinsic antimicrobial potential, further promoting to their capability in treating infections [ 22 ]. Thus, integrating VRZ with functionalized CNTs in a gel formulation can significantly advance antifungal therapy by enhancing drug solubility, improving localized action, reducing adverse effects, and promoting faster recovery. The development of a CNTs-based gel for VRZ therefore represents a rational and innovative strategy that addresses the limitations of conventional antifungal formulations and offers promising prospects for the management of fungal skin infections. 2 Materials and Methods 2.1 Materials Voriconazole, the primary antifungal drug, was obtained from Mac-Chem Products (India) Pvt. Ltd., Mumbai. Amine-functionalized Multi-walled Carbon Nanotubes, which served as the drug carrier, were procured from Ad-nano Technologies Pvt. Ltd. Karnataka, India. Carbopol 934, used as a pH-responsive gelling agent, along with glycerin (humectant), methyl paraben (used both as a preservative and solubilizer), ethylenediamine (a building block), methanol, and ethanol (both used as solvents), were all purchased from S.D. Fine Chem., Mumbai, india. Each ingredient played a specific role in ensuring the stability, effectiveness, and safety of the final formulation. All chemicals and reagents used were of analytical grade. 2.2 Methods 2.2.1 Characterization of Amine-functionalized Multi-walled carbon nanotubes (MWCNTs-NH 2 ) 2.2.1.1 Organoleptic properties The MWCNTs-NH 2 were evaluated for organoleptic characters such as colour, odour, and appearance to ensure suitability, safety, and acceptability for formulation use [ 23 ]. 2.2.1.2 FTIR Study FTIR spectrum of amine-functionalized MWCNTs was recorded using a Bruker alpha spectrophotometer in the range of 4000–400 cm⁻¹ to confirm functional group modifications [ 24 ]. 2.2.1.3 SEM Analysis SEM (JEOL JSM-6390) was used to observe particle morphology of amine-functionalized MWCNTs by depositing a drop of sample solution on a stub and drying before imaging [ 25 ]. 2.2.1.4 TEM Analysis TEM analysis was performed after dispersing samples in isopropyl alcohol, and images were recorded using a Philips CM12 TEM to measure internal and external diameters and structural uniformity [ 26 ]. 2.2.2 Drug- excipient compatibility by FTIR FTIR spectroscopy (Bruker alpha spectrophotometer) was used to study interactions between Voriconazole and excipients (amine-functionalized MWCNTs, Carbopol, HPMC, glycerin, preservatives, and solvents). This confirmed stability by checking for new bonds or chemical alterations that could affect drug release and performance [ 27 ]. 2.2.3 Formulation of voriconazole loaded Carbon nanotubes Voriconazole stock solution (1 mg/ml in ethanol-water, 2:1) was mixed with MWCNTs-NH 2 (Concentration as mentioned in Table 1 ). The dispersion was sonicated for predetermined time, and stirred for 16 hours at room temperature. The drug-loaded nanotubes were obtained by centrifuging the clear supernatant at 3000 rpm for 10 minutes [ 28 ]. 2.2.4 Optimization of VRZ-CNTs A 3² factorial design was applied with two independent factors Carbon nanotubes concentration (X1) and sonication time (X2) at three levels each. Their effects on particle size (Y1), zeta potential (Y2), and entrapment efficiency (Y3) were studied using Design-Expert. The optimized formulation was selected by considering minimum particle size, and maximum zeta potential as well as entrapment efficiency [ 29 ]. 2.2.5 Characterization of VRZ-CNTs 2.2.5.1 Particle size and PDI Particle size and polydispersity index were determined using a Zetasizer Nano ZS at 25 ± 0.1°C with triplicate measurements [ 30 , 31 ]. 2.2.5.2 Zeta potential Surface charge of VRZ-CNTs was analyzed using Dynamic light scattering method (Zetasizer Nano ZS90) at 0.05 mg/mL in PBS (pH 7.4), with mean values obtained from triplicate runs [ 30 ]. 2.2.5.3 Entrapment efficiency (%EE) To determine EE, VRZ-CNTs were centrifuged at 5000 rpm for 15 min., and supernatant was analyzed at 256 nm by UV spectroscopy. The %EE was calculated from Eq. 1 [ 30 ]. $$\:EE=\frac{Total\:drug-Free\:drug}{Total\:drug}\times\:100\:\:\:\left(1\right)$$ 2.2.5.4 FTIR study FTIR spectra (Bruker alpha spectrophotometer) of VRZ-loaded CNTs were recorded using the KBr disk method (5 mg sample/100 mg KBr) over 4000–400 cm⁻¹ to confirm drug loading and interactions [ 31 ]. 2.2.5.5 DSC DSC thermograms of pure VRZ and VRZ-loaded CNTs were recorded (30–300°C, 10°C/min, nitrogen atmosphere) to assess compatibility and thermal behaviour [ 32 ]. 2.2.5.6 XRD XRD patterns of plain VRZ and VRZ-CNTs were obtained on a Rigaku Dmax 2500 diffractometer (Cu Kα, λ = 1.5418 Å, 40 kV, 40 mA) scanning 2θ = 5°–80° to analyze crystalline nature changes [ 33 ]. 2.2.5.7 SEM Surface morphology of VRZ-CNTs was observed using SEM (JEOL, Japan) after gold coating, under high vacuum at 15 V with varying magnifications [ 34 ]. 2.2.5.8 TEM The aqueous dispersion of VRZ-CNTs was dropped on carbon film coated copper grid, after airs drying the images were taken using high resolution transmission electron microscope (Philips CM12) [ 35 ]. 2.2.5.9 In vitro drug release Drug release from VRZ-CNTs and pure VRZ was studied using the dialysis bag method in PBS (pH 7.4). VRZ-CNTs (equivalent to 20 mg of VRZ) and VRZ (20 mg) dispersed with 5 ml PBS was placed separately in dialysis bags and immersed in 50 ml of PBS. The temperature of the system was maintained at 37°C and agitated at 100 rpm. Samples were collected up to 5 h and analyzed by UV spectrophotometer at 256 nm. Sink condition was maintained after sample collection at each time interval using same volume of fresh PBS [ 33 ]. 2.2.6 Formulation of VRZ-CNTs based topical gel VRZ-CNTs based gel was prepared by dispersing 200 mg Carbopol-934 in 15 ml of purified water, left for 24 h to swell, and mixed with VRZ-CNTs (equivalent to 100 mg VRZ). Remaining quantity of purified water was added to adjust the final weight to 20 g. The dispersion was neutralized with triethanolamine to adjust pH, yielding a homogeneous gel [ 36 ]. 2.2.7 Evaluation of VRZ-CNTs based topical gel 2.2.7.1 Appearance A VRZ-CNTs based topical gel was visually inspected for colour, homogeneity and phase separation as appearance is crucial for topical acceptability [ 37 ]. 2.2.7.2 pH The pH of VRZ-CNTs based topical gel was measured using a digital pH meter to ensure stability and skin compatibility [ 37 ]. 2.2.7.3 Drug content Drug content was determined by dissolving 2 g of gel in methanol, filtering, and analyzing by UV spectrophotometer at 256 nm [ 37 , 38 ]. 2.2.7.4 Viscosity Viscosity of VRZ-CNTs based topical gel was measured using a Brookfield viscometer with spindle no. 64 at constant speed of 6 rpm, and results were reported as mean of triplicate readings [ 37 , 38 ]. 2.2.7.5 Spreadability Spreadability of prepared gel was tested using the glass slide method [ 37 , 38 ]. A glass slide was placed on a plane platform at 37 ± 1°C and 1 g of VRZ-CNTs based topical gel was added on it. Another glass slide was placed over the gel loaded slide, 100 g standard weight was placed on the upper slide for a period of 1 min. Then the diameter of spread VRZ-CNTs based topical gel was measured. The spreadability of gel was calculated Eq. 2. \(\:\text{S}={d}^{2}\times\:\:\frac{\pi\:}{4}\) (2) Where, S = spredability, and d 2 =diameter. 2.2.7.6 Ex-vivo skin permeation Ex-vivo permeability of VRZ from VRZ topical gel and VRZ-CNTs based topical gel was studied using Franz diffusion cells (receptor chamber volume = 20 ml and diffusion area = 0.78 cm²). An excised goat abdominal skin was used as a membrane for the study. Phosphate buffer pH 7.4 was used as a diffusion membrane. The study was performed at 37 ± 0.5ºC with constant stirring. The VRZ topical gel and VRZ-CNTs based topical gel were uniformly spread on the surface of membrane from the side of donor compartment. At the intervals of 0, 0.5, 1, 2, 4, 6, and 8 h. aliquots of samples (1 ml) were withdrawn and to quantify the permeated VRZ samples were analyzed using a UV spectroscope at 256 nm a wavelength [ 39 ]. 2.2.7.7 Ex-vivo Drug retention To determine the drug retention, the treated area of the goat skin used in the permeability study was carefully excised and rinsed with phosphate buffer pH 7.4o remove adhered drug to the outer surface. Then the skin was cut into smaller pieces, and homogenized with 1 ml of ethanol. The homogenate was centrifuged at 8000 rpm for 10 min. a supernatant was collected and analyzed using UV- spectrophotometer to determine retention of VRZ in the skin layers [ 40 ]. 2.2.7.8 In vitro antifungal activity: In vitro Antifungal activity of VRZ-CNTs based topical gel, and pure VRZ was assessed using agar well diffusion method against Candida albicans, with inhibition zone measurements. A 100 µL of Candida albicans suspension was spread onto sterilized Sabouraud Dextrose Agar plates and then wells were prepared using sterile cork borer. The test solutions containing VRZ-CNTs based topical gel (equivalent to VRZ 10 µg), a negative control (saline), and a positive control VRZ (10 µg) were carefully placed in specific well. The prepared plates were incubated at 30°C for 48 h accessed for the fungal growth and inhibition zones [ 42 ]. 2.2.7.9 In vivo skin irritation study In-vivo skin irritation and antifungal activities were performed after approval of Institutional animal ethics committee (SCOP/IAEC/2025/07) for laboratory animal use and care. For in vivo studies Wistar albino rats (Either Male or Female), weighing 200–250 g were used. The rats were kept in a room with controlled cycles of 12 hours of light and 12 hours of darkness. Animals were given standard food and distilled water. Wistar rats were divided into 3 groups each group having 6 rats. Group I will be considered the normal control group, and animals from this group will receive distilled water (0.5 ml) topically. Group II will be considered the positive control group, and animals from this group will receive formalin (1%) 0.5 ml to induce irritation on the first day. Group III will be considered Test group, and animals from this group will receive 0.5 gram dose of VRZ-CNTs gel topically. Each formulation will be applied to a 1 cm² hairless area of the rat’s skin. The rats were then be returned to their cages and monitored. The rats were observed at 24, 48, and 72 hours after the application, and the signs of skin irritation, such as redness (erythema) or swelling (edema) were checked [ 43 ]. 2.2.7.10 In vivo antifungal activity The study consisted four groups, with six rats (n = 6) in each group. The groups will be randomly divided as follows: Group I was negative control group, which will have no fungal infection (fungus-free); Group II was positive control group, which was infected with fungi but not received treatment (untreated); Group III was infected rats treated for 10 days with Voriconazole gel (Marketed Formulation) and Group IV was infected rats treated for 10 days with VRZ-CNTs topical gel. Rats except group-I received 5 mg/kg of prednisolone intravenously for three days, to cause an immunosuppressive effect. The rats' dorsal (back) skin was shaved (1 cm²) 4 hours before the test. Candida albicans was used as the fungal strain, which adjusted to concentration of 10 6 CFU/ml. Each rat except group-I rats received an intradermal injection of 0.3 mL of the Candida albicans suspension in the middle of a shaved area on its exposed skin. Any mild edema at the injection site was removed by rubbing the area vigorously. After 72 hours, signs of fungal infection were appeared in the injected area. To prevent the rats from licking the skin, they were kept separately in individual cages. Rats from Group III and Group IV treated with 0.5 g of voriconazole gel (Marketed formulation) and VRZ-CNTs topical gel respectively, and analysed for recovery from skin damage. The rats were monitored for clinical signs of fungal infection, such as rashes, red patches, white particles, scaling, maceration, erythema (redness), cracking, and pus-filled pimples. Also Histopathologic examinations and Total leucocyte count were determined [ 44 ]. 2.2.8 Stability study Accelerated stability testing was performed at 40 ± 2°C temperature and 75 ± 5% Relative humidity (RH) for 3 months as per ICH guidelines by evaluating appearance, pH, drug content and viscosity for 3 motnhs [ 41 ]. 2.2.9 Statistical Analysis Analysis of variance (ANOVA) was applied to check the significance between different components. All experimental trials were performed in triplicate, and the results are interpreted as mean ± SD. 3 Results and discussion 2.3.1 Organoleptic properties of MWCNTs-NH₂ The amine-functionalized multi-walled carbon nanotubes appeared as a black, fluffy, and very light powder suggests a high surface area and porous structure, which are desirable properties for drug loading and adsorption. 2.3.2 FTIR spectrum of MWCNTs-NH₂ The FTIR spectrum of amine-functionalized carbon nanotubes is shown in Fig. 1 a. This technique helps identify the different types of chemical bonds in a substance by measuring how it absorbs infrared light at different wavelengths. In this graph, several peaks can be seen at specific positions, each representing a unique bond or group of atoms. A broad peak near 3768 cm⁻¹ indicates the presence of N–H bonds, which is expected after amine functionalization. The peak around 2929 cm⁻¹ comes from C–H stretching vibrations, showing that some aliphatic groups are present, possibly from remaining organic molecules or excipients. A small peak at 2348 cm⁻¹ may result from carbon dioxide or overtones. A strong and sharp peak at 1698 cm⁻¹ is characteristic of C = O (carbonyl) stretching, which may come from oxidation. The peak at 1214 cm⁻¹ likely represents C–N or C–O stretching, confirming the presence of amine or ether groups. This FTIR analysis confirms the presence of amine-functionalized carbon nanotubes [ 45 , 46 ]. 2.3.3 Scanning Electron Microscopy (SEM) of MWCNTs-NH₂ The Fig. 1 b shows SEM image of the MWCNTs-NH 2 . From the image, it was observed that the MWCNTs-NH 2 were long, tube-like structures that were tangled and clumped together. This web-like structure was mainly due to van der Waals forces pulling the tubes toward each other. The surface of the tubes appears smooth, which suggests that they may have been successfully treated with amine groups. Most tubes have a similar thickness, though some look slightly thicker likely because a few tubes are stuck together. 2.3.4 Transmission Electron Microscopy (TEM) MWCNTs–NH 2 The Fig. 1 c shows Transmission Electron Microscopy (TEM) analysis of MWCNTs-NH 2 . TEM is a powerful imaging technique that allows seeing the structure of materials at the nanometer scale. Image display CNTs at two different magnifications, i.e. 500 nm to 50 nm, giving a detailed look at their shape, size, and surface features. In the image of 500 nm scale, the MWCNTs-NH 2 appear as long, tangled, and thread-like structures, showing how they are dispersed. The image of 50 nm scale shows small black dots along the nanotube surfaces, which likely represent the amine groups chemically bonded during the functionalization process. In addition with this individual nanotube more clearly seen in this image, where the hollow tubular structure becomes visible, suggest their multi-walled nature. This confirms that the surface of the carbon nanotubes has been modified effectively, which is important for improving their compatibility in drug delivery, polymer composites, or other applications [ 47 ]. 2.3.5 Optimization of VRZ-CNTs A 3² full factorial design using Design-Expert software studied CNTs concentration (X1) and Sonication time (X2) on particle size (Y1), zeta potential (Y2), and entrapment efficiency (Y3). Positive and negative polynomial coefficients indicated variable effects, with statistically significant responses determined by p-values < 0.05. Scatter plots showed good agreement between actual and predicted values, validating the model’s reliability for optimization [ 48 ]. The obtained responses for nine runs are detained in Table 1 . Table 1 3² full factorial design for optimization of VRZ-CNTs. Run Factor X1 CNTs concentration (mg) Factor X2 Sonication time (min) Response Y1 Particle size (nm) Response Y2 Zeta potential (mV) Response Y3 Entrapment efficiency (%) 1 20 5 103.15 ± 1.14 -27.7 ± 1.52 87.9 ± 1.24 2 20 10 102.19 ± 0.5 -36.58 ± 172 92.76 ± 1.21 3 20 15 100.25 ± 1.05 -44.31 ± 1.20 94.89 ± 1.31 4 30 5 140.49 ± 0.97 -36.39 ± 1.31 82.4 ± 1.42 5 30 10 142.24 ± 1.20 -42.53 ± 1.34 83.2 ± 0.92 6 30 15 140 ± 2.10 -47.82 ± 1.26 86.5 ± 1.63 7 40 5 183.82 ± 1.41 -34.82 ± 1.48 79.5 ± 2.12 8 40 10 178.86 ± 1.32 -40.22 ± 1.42 88.2 ± 1.70 9 40 15 178.9 ± 1.21 -46.55 ± 1.38 85.5 ± 1.54 Table 2 ANOVA results for responses in optimization of VRZ-CNTs. Source Sum of squares Df Mean square F- value P- value Particle size Model 9293.39 2 4646.69 2311.51 < 0.0001 X1- CNTs Conc. 9281.88 9281.88 9281.88 9281.88 9281.88 X2 – Sonication Time 11.51 1 11.51 5.73 0.0538 Residual 12.06 6 12.01 Cor Total 9305.45 8 Zeta Potential Model 348.04 5 69.61 147.55 0.0009 X1- CNTs Conc. 28.17 1 28.17 59.71 0.0045 X2– Sonication Time 293.44 1 293.44 622.03 0.0001 X1X2 5.95 1 5.95 12.62 0.0380 Residual 19.89 1 19.89 0.0074 Cor Total 0.5904 1 Entrapment Efficiency Model 174.37 2 87.19 7.08 0.0264 X1- CNTs Conc. 107.10 1 107.10 8.69 0.0257 X2 – Sonication Time 67.27 1 67.27 5.46 0.0581 Residual 73.92 6 12.32 Cor Total 248.29 8 2.3.6 Effect on particle size The particle size of the Voriconazole loaded CNTs was determined using a Photon correlation spectroscopy (PCS) method, performed in triplicate. The lowest particle size was observed in the F3 batch (100.25 ± 1.05), while the F7 batch had the highest particle size (183.82 ± 1.41) as shown in Table 1 . The effects of independent variables on dependent variables are expressed with Eq. 3. Particle size (Y1) = + 141.10 + 39.33X1–1.39X2 (3) From the Eq. 3 it can be confirmed that, as CNTs concentration increases the particle size also increased due to systems viscosity [ 49 ], while longer sonication (up to 15 min) reduced size by breaking CNTs and improving dispersion [ 50 ]. The linear model showed excellent fit (R² = 0.9987, Adjusted R² = 0.9983, Predicted R² = 0.9970) with significant results (F-value 2311.51, p < 0.0001) depicted in Table 2 . Predicted and actual values closely matched, confirming model reliability. A 3D response surface plot for effect of independent variables on particle size is shown in Fig. 2 a. 2.3.7 Effect on Zeta potential: A zeta potential of VRZ-CNTs ranged from − 27.7 mV (F1) to -47.82 mV (F6), which influenced by CNTs concentration and sonication time (Table 1 ). The relationship between independent and dependent variables is depicted with Eq. 4. Zeta potential (Y2) = − 42.37 -2.17X1–6.33 X2 + 1.22 X1X2 + 3.88 X1 2 + 0.18 X2 2 (4) As CNTs concentration increases initially then zeta potential also increases, it could be due to increased charge density per unit volume. At highest concentration level of CNTs there was decrement in zeta potential. Longer sonication increases zeta potential due to better dispersion and surface charge distribution [ 51 ]. High zeta potential values are important as they prevent particle aggregation, ensuring colloidal stability and improved drug delivery efficiency [ 52 ]. A quadratic model was the best fit model for the zeta potential (R² = 0.9960, Adjusted R² = 0.9892, Predicted R² = 0.9636) and ANOVA results (Table 2 ) confirms the significant impact of independent variables on responses (F-value 147.55, p-value 0.0009). A 3D response surface plot for effect of independent variables on zeta potential is shown in Fig. 2 b. 2.3.8 Effect on Entrapment efficiency (EE) Entrapment efficiency (EE) of VRZ-CNTs ranged from 79.5% (F7) to 97.89% (F3), influenced by both CNTs concentration and sonication time (Table 1 ). The impact of independent variables on dependent variables is expressed with Eq. 5. Entrapment efficiency (Y3) = + 87.09–4.23X1 + 3.35X2 (5) From the Eq. 5 it can be observed that, higher CNTs concentration (40 mg) reduced EE due to drug leakage or reduced encapsulation. Longer sonication time improves entrapment efficiency due to better emulsification and droplet dispersion [ 53 ]. Hence, lower CNTs concentration with longer sonication gave the highest EE. The linear model showed good fit (R² = 0.7023, Adj R² = 0.6031) and confirms the significant impact of independent variables on responses (F-value 7.08, p-value 0.0264). High EE is important as it ensures maximum drug loading, improved therapeutic effect, and efficient drug delivery [ 54 ]. A 3D response surface plot for effect of independent variables on entrapment efficiency is shown in Fig. 2 c. 2.3.9 Validation of responses The optimization process aimed to fine-tune the formulation of the VRZ-CNTs by setting specific targets for the key dependent variables. The optimization criteria include maximized Entrapment efficiency to ensure the drug entrapped within vesicles and retained in the non-polar chain that distributes the drug into deep skin layers, Minimized Particle to ensure that the better drug release and maximized Zeta potential to ensure the stability of VRZ-CNTs. Using these criteria, a design of experiments (DOE) approach was employed to generate multiple solutions. A total of 9 solutions were identified, each representing a different combination of the independent variables –CNTs concentration and Sonication time. These solutions were evaluated based on their desirability scores, which reflect how well they meet the optimization criteria. The solution with the highest desirability score of 0.899 was selected as the optimized batch. This specific combination of sonication time, CNTs concentration and was determined to provide the most favourable balance of maximum entrapment efficiency, minimum particle size and maximum zeta potential. Once the optimized batch was selected, the model's accuracy was validated by comparing the predicted values of the dependent variables to the experimentally observed values. An additional optimized batch of the VRZ-CNTs was made with suggested optimized values for CNTs concentration (20 mg) and sonication time (15 Min.) and tested for model validation. The predicted responses i.e. particle size, zeta potential, and entrapment efficiency were 100.38 nm, -43.98 mV and 93.33% respectively. Which were compared with that of the observed values i.e. particle size 102.92 ± 5.66 nm (% bias 2.53), zeta potential − 42.85 ± 2.6 mV (% bias 2.68) and entrapment efficiency 91.85 ± 3.68% (% bias 1.58) and found to be closer to the predicted values. A % Bias was for all three responses was less than 3% confirms the good correlation between predicted and observed values. 2.3.10 Characterization of VRZ-CNTS 2.3.10.1 Particle size and PDI The Fig. 3 a represented the particle size and size distribution of the optimized VRZ–CNTs. This consistency indicated that the formulation process had been well-controlled and reproducible. The analysis showed a mean particle size of 102.92 nm, with a Z-Average size of particle size 103.9 nm (± 1.02). The mode, representing the most frequently occurring particle size, was 98.6 nm, and the standard deviation was 28.1 nm, suggesting a reasonably tight distribution around the mean. The Polydispersity Index (PDI) was 0.327, which pointed to a moderately narrow distribution. While slightly above the ideal value of 0.3 for highly monodisperse systems, this PDI was still within the acceptable range for VRZ-CNTs formulations, as values below 0.5 were generally considered suitable [ 55 ]. The graph displayed a single, sharp, and narrow peak, indicating that the particle sizes were relatively uniform and there was no presence of multiple particle populations. The graph demonstrated that the batch had been well-optimized in terms of particle size and distribution, making it suitable for further use. 2.3.10.2 Zeta potential Figure 3 b shows the zeta potential of optimized VRZ – CNTs. The sharp peak at -42.85 ± 2.6 mV particle size indicates that the particles carry a strong negative charge, which helps to keep them evenly dispersed without clumping. This means formulation is physically stable. Since zeta potential value greater than ± 30 mV is considered as it is more stable for colloidal systems, CNT are unlikely to aggregate, the graph shows a single sharp peak, which confirms that the sample is uniform and has a consistent charge distribution [ 56 ]. 2.3.10.3 Entrapment efficiency Entrapment efficiency tells about how much of the Voriconazole got trapped inside the carbon nanotubes. The entrapment efficiency of VRZ-CNTs was observed to be 91.85 ± 3.68%. An EE% above 80% is considered very effective in topical drug delivery systems especially for antifungal treatments because it means most of the drug is retained inside the carrier system [ 57 ]. 2.3.10.4 FTIR (Fig. 5 a) presents the FTIR spectra of pure VRZ, carbon nanotubes (CNTs), and VRZ-CNTs. Pure VRZ shows distinct peaks corresponding to key functional groups, including O–H (3203 cm⁻¹), aromatic and aliphatic C–H (3121–2878 cm⁻¹), aromatic C–C (1615–1509 cm⁻¹), triazole ring vibrations (1451–1332 cm⁻¹), ether linkages (1229–1047 cm⁻¹), and C–F bonds (1120 cm⁻¹). CNTs display minimal peaks due to their graphitic nature, but functionalization introduces broad O–H or N–H peaks (3400–3200 cm⁻¹) and a C = C signal near 1600 cm⁻¹. In the VRZ–CNTs spectrum, some peak shifts and intensity changes are observed, indicating physical interactions—likely hydrogen bonding—between VRZ and the CNTs [ 45 , 46 ]. However, all major functional peaks of VRZ remain present, confirming the drug's chemical stability after loading. These interactions suggest successful incorporation of VRZ into the CNTs, improving its solubility, thermal stability, and bioavailability compared to its pure crystalline form [ 58 ]. 2.3.10.5 DSC (Fig. 4 b) shows DSC thermograms compares two samples pure Voriconazole and VRZ-CNTs. DSC is used to see how a substance behaves when heated, it helps to understand things like how easily it melts, how stable it is with heat, and whether it is in a crystal or non-crystal (amorphous) form. Voriconazole has a sharp, deep peak around 132°C; this sudden drop shows the melting point of the drug and tells that VRZ is in a crystalline state [ 59 ]. Crystalline drugs usually don’t dissolve easily in water, which means they can be absorbed slowly in the body and may not work as effectively. In the VRZ-CNTs looks very different. It does not have a sharp melting peak like the pure drug. Instead, the curve is flatter and broader, especially around 200°C. This tells us that the VRZ crystal structure has changed or disappeared when combined with carbon nanotubes [ 60 ]. In this case, Voriconazole may have turned into an amorphous form, which usually dissolves faster and is absorbs better in the body. Also, the shift in the curve to higher temperatures shows that the VRZ is more stable with heat when it is loaded onto CNTs, this graph shows that when Voriconazole is combined with carbon nanotubes, it becomes less crystalline and more heat-stable [ 61 ]. 2.3.10.6 XRD A Fig. 5 a shows XRD of pure voriconazole and VRZ-CNTs. In the Pure Voriconazole defractogram, several sharp and well-defined peaks appear between 10° and 35° (2θ), confirming a highly crystalline structure. Crystalline forms of voriconazole generally have slower dissolution rates and limited solubility, which can affect absorption and reduce therapeutic efficiency [ 62 ]. In contrast, the defractogram of VRZ-CNTs shows a broad, less intense peak around 25° (2θ), while the sharp crystalline peaks present in pure voriconazole were nearly absent. This change indicates a transformation of voriconazole into an amorphous or partially amorphous state after incorporation into the carbon nanotubes [ 63 ]. The broad peak also reflects the typical signal of carbon nanotubes due to their graphitic structure. A smaller peak near 43° (2θ) in the same graph suggests a degree of structural order within the nanotube material [ 64 ]. This shift from crystalline to amorphous form is beneficial because amorphous voriconazole generally dissolves faster and is more soluble, enhancing its bioavailability. The amine-functionalized carbon nanotubes used as carriers offer a large surface area and strong interactions through functional groups, disrupting the regular crystal structure of voriconazole [ 65 ]. As a result, the formulation improves the physical stability, 2.3.10.7 SEM The images shown in Fig. 5 b and 5 c are Scanning Electron Microscopy (SEM) images of VRZ-CNTs at 10000X magnification and 20000X magnification respectively. SEM is used to visualize the surface structure and morphology of materials at very high magnification. The image at 10,000x magnification shows a dense and interconnected network of carbon nanotubes that appear as long, entangled, thread-like structures. The image taken at 20,000x magnification provides a clearer view of these nanotubes and reveals the fine distribution of the drug particles on the surface of the nanotubes. The overall structure appears rough and sponge-like, indicating a good dispersion of voriconazole within the nanotube matrix. The absence of sharp edges and crystals suggests that voriconazole has been successfully loaded onto the carbon nanotubes and may have transitioned into a more amorphous or dispersed state. This transformation is important because the amorphous form of a voriconazole generally has better solubility and absorption properties than its crystalline form. The functionalized carbon nanotubes provide a large surface area and active binding sites (due to amine groups), allowing for strong interaction with voriconazole [ 66 ]. As a result, drug particles are better distributed, more stable, and capable of controlled release. This improved morphology and dispersion seen in the SEM images suggests that the voriconazole-loaded carbon nanotubes show better performance in drug delivery than pure voriconazole. They could enhance bioavailability, prolong release and improve overall therapeutic effect. 2.3.10.8 TEM The Fig. 6 shows TEM analysis of VRZ-CNTs. Images are taken at two different magnifications, i.e. first showing 500 nm scale and second showing 50 nm, giving a detailed look at their diamentsions and surface features after loading of VRZ. In the image of 500 nm scale, the VRZ-CNTs appear as thin, elongated, and Tubular structures, with rough morphology indicating dispersion of drug in the nanotubes. The image of 50 nm scale shows internal morphology of specific tubular structure small black dots like structure inside the tubes confirms the loading and dispersion of drug. 2.3.10.9 In vitro release of VRZ loaded CNTs: Table 3 In vitro drug release from VRZ-CNTs and Plain VRZ Time (Hours) % CDR from VRZ-CNTs % CDR from Plain VRZ 0 0 0 1 37.33 ± 2.33 19.66 ± 2.66 2 49.66 ± 2.66 27.66 ± 1.66 3 61.00 ± 2.00 34.00 ± 2.00 4 71.33 ± 1.33 39.66 ± 1.66 5 75.00 ± 1.00 44.00 ± 1.00 6 79.00 ± 2.00 46.33 ± 1.33 7 82.33 ± 1.33 49.00 ± 0.55 8 85.66 ± 2.33 50.00 ± 0.25 A drug release study was carried out to compare the release behaviour between plain VRZ and VRZ-CNTs. The results showed that VRZ-CNTs released the drug much faster and in higher amounts than plain VRZ (Table 3 ). After 1 hour, VRZ-CNTs released about 38% (± 1.2) of the drug, while plain VRZ released only 20% (± 0.59). Finally, by the 8th hour, VRZ-CNTs showed a release of 85% (± 0.12), while plain VRZ remained at 39% (± 0.17) as shown in Fig. 7 a. These results confirmed that loading Voriconazole into carbon nanotubes improved its solubility and release rate, which was likely due to the smaller particle size, larger surface area, and better dispersion of the drug when carried by the nanotubes. Furthermore, the drug's transition to a more amorphous form and its interaction with functional groups on the CNT surface contribute to better solubility and faster dissolution [ 67 ]. Together, these factors make CNTs an effective carrier system for enhancing the therapeutic performance of voriconazole. A release mechanism from VRZ-CNTs was studied using different kinetic models, which include the zero order, first order, Higuchi, Hixon-Crowell, and Koresmyer-Peppas kinetics. The results of release kinetics are shown in Fig. 7 b to Fig. 7 f. The best fit model was found to be Koresmyer-Peppas model highest R² value (09896) with n < 0.5, confirms the release follows Fickian diffusion. 2.3.11 Evaluation of VRZ-CNTs Gel 2.3.11.1 Organoleptic properties The prepared VRZ-CNTs gel was evaluated for colour, homogeneity, and the occurrence of phase separation. The colour of gel was black due to the colour of CNTs was blackish. The prepared gel was found to be homogeneous without separation of phases suggests the appropriateness of formulation. 2.3.11.2 pH The average pH of VRZ-CNTs gel was 6.74 ± 0.21, which is close to the skin’s pH range, making the formulation safe and non-irritating for skin use [ 68 ]. 2.3.11.3 Drug content The drug content analysis showed a consistent and uniform distribution of voriconazole in the VRZ-CNTs based topical gel, with an average content of 97 ± 2.56%. This indicates that the drug is well incorporated into the formulation, ensuring reliable dosing [ 68 ]. 2.3.11.4 Viscosity The VRZ-CNTs based topical gel’s viscosity ranged between 8850 and 9080 mPa·s, with an average of 8963 mPa·s. This relatively high viscosity suggests that the gel is thick and stable, suitable for prolonged contact with the skin, which helps in sustained drug delivery [ 69 ]. 2.3.11.5 Spreadability In terms of application, the spreadability of the gel was measured at 26.5 ± 1.21 g·cm/sec, falling within the ideal range for topical gels. This means the gel can be smoothly and evenly applied on the skin, enhancing user comfort and effectiveness [ 68 , 69 ]. 2.3.11.6 Ex-vivo skin permeation The Ex-vivo skin permeability study was performed using goat abdominal skin. After 8 h of the study, the cumulative amount of VRZ permeated from the plain VRZ topical gel and VRZ-CNTs based topical gel was 71.96 ± 3.59 µg/cm² (Flux 8.99 µg/cm²/h) and 292.16 ± 7.08 µg/cm² (Flux 36.52 µg/cm²/h) respectively as shown in Fig. 8 a. Statistical analysis confirms that permeation of VRZ from VRZ-CNTs based topical gel was significantly greater than that from VRZ topical gel (P < 0.001). The greater permeability of VRZ from the VRZ-CNTs based topical gel is due to increased solubility, surface area, and sustained release of drug from the CNTs matrix, which help to maintains high concentration gradient across the skin. I addition to this, the positively charged amine groups of amine-functionalized multi-walled carbon nanotubes interact with negatively charged skin components, causes temporary lipid disruption and increases follicular penetration and enhances drug diffusion compared to plain VRZ gel [ 70 ]. 2.3.11.7 Ex-vivo Drug retention After 8 h of permeation study, the treated skin thoroughly washed, excised and evaluated for drug retention in the deeper skin layers. The VRZ-CNTs based topical gel showed significantly higher VRZ retention (70.53 ± 4.12 µg/cm²) compared to BRH-HG (23.87 ± 3.25 µg/cm²), as illustrated in Fig. 8 b. The enhanced retention is primarily due to strong hydrophobic interactions between voriconazole and the amine-functionalized multi-walled carbon nanotubes surface, which enable deeper carrier-mediated deposition within epidermal and dermal layers. Furthermore, the positively charged amine groups of MWCNTs-NH 2 electrostatically interact with negatively charged skin components, improving adhesion and prolongs residence time in deeper layers of the skin. The higher follicular penetration ability of MWCNTs-NH 2 further facilitates accumulation of VRZ in deeper tissues compared to plain VRZ gel [ 70 ]. 2.3.11.8 In-vitro antifungal activity The VRZ-CNTs gel exhibited a zone of inhibition of 33 ± 0.58 mm (Fig. 9 ), which was significantly larger than the standard drug (Voriconazole, 18 ± 0.25 mm), indicating enhanced antifungal effect, the plain gel (Without drug) showed no inhibition, confirming that the activity is solely due to the active formulation. The VRZ-CNTs gel showed better antifungal activity compared to both the standard drug (Voriconazole) and the plain gel. This was likely because the combination of Voriconazole with MWCNTs-NH 2 improved the drug’s effectiveness. The positively charged amine groups on CNTs may interact with the negatively charged fungal cell membrane allowing it to penetrate more efficiently. They also provided a controlled and sustained release of the drug, which kept the drug active for a longer time at the infection site [ 70 , 71 ]. 2.3.11.9 In-vivo Skin irritation The in-vivo skin irritation study was conducted to evaluate the safety of the VRZ-CNTs gel when applied topically. Since pharmaceutical gels can sometimes cause adverse skin reactions like redness, itching, or swelling, it is essential to confirm that all ingredients are non-irritating. In this study, healthy female Wistar albino rats were used, and the skin was monitored for signs of erythema (redness) and edema (swelling) at 24, 48, and 72 hours after application. The severity of these reactions was rated on a scale from 0 to 4, where 0 represents no reaction and 4 indicate a severe reaction [ 72 ]. Three groups were included in the study: a normal control group (no treatment), a positive control group treated with formalin (a known irritant), and a test group treated with the VRZ-CNTs gel. The normal control group showed no signs of redness or swelling at any time point (Fig. 10 a). The formalin treated group (Positive control) exhibited slight redness and swelling, confirming the sensitivity of the test (Fig. 10 b). Most importantly, the group treated with the VRZ-CNTs gel showed no signs of irritation, scoring 0 for both erythema and edema at all time points (Fig. 10 c). An average score of less than 2 indicates a non-irritant formulation. Since the VRZ-CNTs gel consistently scored 0 across all animals, it can be concluded that the formulation is non-irritating and safe for use on the skin. These findings support its suitability for topical application. 2.3.11.10 In vivo antifungal activity The study evaluated the effectiveness of VRZ–CNTs gel in treating deep skin fungal infections caused by Candida albicans in immunosuppressed rats. Initially, all rats showed healthy, intact skin with no signs of irritation or damage. After intradermal injection with Candida albicans (10⁶ CFU/mL), infection symptoms such as mild redness and irritation appeared within 24 hours in the infected groups. By the third day, the condition worsened, with clear signs of erythema, scaling, and skin cracking, confirming successful infection. Treatment was then initiated using two formulations: VRZ–CNTs gel and plain Voriconazole gel (marketed formulation). By the fifth day of treatment, rats treated with the VRZ–CNTs gel began shedding the infected layer, revealing healthy pink skin underneath, indicating rapid healing. Meanwhile, the group treated with the plain VRZ gel showed moderate improvement, but still had visible redness and scaling. By the tenth day, the VRZ–CNTs gel group had fully recovered with completely healed skin and no visible signs of infection or scarring (Fig. 11 ). In contrast, the plain VRZ gel group had not achieved full recovery. These findings demonstrate that VRZ–CNTs gel is significantly more effective in treating fungal skin infections, likely due to its enhanced drug delivery and sustained release properties. The VRZ–CNTs gel showed better healing than the plain VRZ gel, which still had mild redness and scarring. The untreated group had severe infection signs like pus and peeling. The enhanced effect of the VRZ–CNTs gel is due to amine-functionalized multi-walled carbon nanotubes, which helped the drug penetrate deeper and stay longer at the infection site. Their small size and large surface area improved drug delivery and antifungal action. It also facilitates sustained drug release and follicular targeting, which helps to maintain therapeutic levels for a prolonged period of time. In addition to this, the positively charged amine groups on CNTs enhances the interactions with negatively charged fungal cell membranes, which improves membrane permeation and enhances antifungal efficacy of VRZ-CNTs compared to plain VRZ gel [ 70 , 71 ]. The histopathological analysis (Fig. 12 ) revealed that the fungal infection-induced group (Positive control) displayed the most severe skin abnormalities, including severe hyperkeratosis, cellular infiltration, and epidermal hyperplasia (+++). These results confirmed extensive skin damage due to the fungal infection. In contrast, the normal control group had completely healthy skin with no signs of hyperkeratosis, infiltration, or hyperplasia (00). Rats treated with plain Voriconazole gel showed clear signs of healing compared to the infected group. The severity of skin damage was reduced to moderate hyperkeratosis, cellular infiltration, and moderate epidermal hyperplasia (++). This indicates that the antifungal effect of Voriconazole helped reduce inflammation and tissue damage, although not completely reversing the changes caused by the infection. The group treated with VRZ-CNTs gel also showed minimal hyperkeratosis, infiltration, and hyperplasia (+) as depicted in Table 4 . However, microscopic examination and skin appearance indicated superior healing, with almost normal tissue architecture and minimal inflammation. This suggests enhanced recovery in VRZ-CNTS treated group due to improved drug delivery and retention due to MWCNTs-NH 2 [ 73 ]. Table 4 Signs of Hyperkeratosis, Cellular infiltration and Epidermal hyperplasia in different study groups Group Hyperkeratosis Cellular infiltration Epidermal hyperplasia Normal control 00 00 00 Positive control +++ +++ +++ Test I ++ ++ ++ Test II + + + The Total Leucocytic Count (TLC) study assessed white blood cell levels to evaluate infection and immune response. The fungal infection group (Positive control) showed the highest TLC (13.23 × 10³/µL), indicating severe inflammation. The normal control had the lowest TLC (8.15 × 10³/µL), reflecting a healthy state i.e. no infection or immune response. The VRZ gel treated group showed moderate improvement (10.27 × 10³/µL), while the VRZ–CNTs treated gel group had a significantly lower TLC (9.15 × 10³/µL), suggesting more effective infection control and reduced inflammation due to enhanced drug delivery[ 48 , 49 ]. The higher TLC in the fungal infection group reflects a strong immune response to fight the infection. VRZ gel reduced infection and inflammation, lowering TLC moderately. The VRZ-CNTs gel improved drug delivery and healing more effectively, resulting in even less inflammation and a further reduced TLC [ 74 ]. 2.3.11.11 Stability Study For evaluation of stability of VRZ-CNTs gel, a three-months study was conducted at accelerated conditions (40 ± 2°C, 75 ± 5% RH). The results are depicted in Table 5 which confirmed that the appearance, pH, drug content, viscosity and spreadability of the VRZ-CNTs gel did not significantly changed after three months of storage at accelerated conditions. Thus, the developed VRZ-CNTs gel was found to be stable. Table 5 Stability study results of VRZ-CNTs gel Time (Months) Appearance pH Drug Content (%) Viscosity (mPa·s) Spreadability (g·cm/sec) 0 Black Homogeneous 6.74 ± 0.21 97 ± 2.56 8963 ± 117 26.50 ± 1.21 1 Black Homogeneous 6.70 ± 0.32 96.89 ± 1.64 8940 ± 125 2672 ± 0.87 2 Black Homogeneous 6.68 ± 0.26 96.80 ± 2.42 8915 ± 136 26.89 ± 1.58 3 Black Homogeneous 6.65 ± 0.15 96.75 ± 1.89 8910 ± 129 26.92 ± 1.66 4 Conclusion The present study successfully developed MWCNTs-NH₂ based gel for topical voriconazole delivery. The optimized VRZ-CNTs formulation exhibited adequate particle size, good zeta potential and higher entrapment efficiency, confirming stability and reproducibility of formulation. FTIR, DSC, and XRD validated compatibility of formulation components and verified transition of VRZ from crystalline to amorphous form, which facilitates enhancement in solubility and controlled release of drug. Incorporation of VRZ-CNTs into Carbopol 934 gel demonstrated suitable pH, spreadability, viscosity, and consistent drug content, assuring superiority of gel. Moreover, the VRZ-CNTs gel exhibited notably improved ex-vivo skin permeation and deeper skin retention, and also found to be non-irritant. In-vitro and In-vivo antifungal evaluation further validated enhanced therapeutic efficiency and histopathological recovery as that of conventional VRZ gel. These findings suggests that the VRZ-CNTs gel will be a safer, more effective, and promising alternative for conventional topical antifungal therapies. Declarations Acknowledgements Authors are thankful to Satara College of Phar­macy, Satara-415004, Maharashtra State, India for providing best of facility to conduct this research work. Author Contributions VS: Performed experimental work, manuscript drafting MS: Supervision, Conceptualization, manuscript drafting, Visualization, Investigation, VM: Manuscript editing, guid­ance AB: Data generation, manuscript writing Funding The authors declare that no funds, grants, or other support were received for completion of this research work. Data Availability The data will be made available on reasonable request from corresponding author. Competing interests The authors declare no competing interests. Animal Ethics All animal experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the Satara College of Pharmacy, Satara, Maharashtra, India under approval number SCOP/IAEC/2025/07. References McCormick, T. S., & Ghannoum, M. (2024). 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A., Elgamal, A. M., & ELsayed, W. M. (2023). Chenopodium murale juice shows anti-fungal efficacy in experimental oral candidiasis in immunosuppressed rats in relation to its chemical profile. Molecules , 28 (11), 4304. https://doi.org/10.3390/molecules28114304 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 15 May, 2026 Reviews received at journal 15 May, 2026 Reviews received at journal 13 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviews received at journal 07 Apr, 2026 Reviewers agreed at journal 06 Apr, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers invited by journal 24 Mar, 2026 Editor assigned by journal 24 Mar, 2026 Submission checks completed at journal 23 Mar, 2026 First submitted to journal 08 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9064633","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":611593056,"identity":"e64a0ff2-7a71-4d4a-9815-e756af715dc5","order_by":0,"name":"Vaishnavi Shelke","email":"","orcid":"","institution":"Satara College of Pharmacy, Satara","correspondingAuthor":false,"prefix":"","firstName":"Vaishnavi","middleName":"","lastName":"Shelke","suffix":""},{"id":611593070,"identity":"4fb86ef5-3fd2-4bbb-8be3-d2217ae249a7","order_by":1,"name":"Manoj 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spectrum, (b):\u003csub\u003e \u003c/sub\u003eSEM image,\u003csub\u003e\u0026nbsp;\u0026nbsp; \u003c/sub\u003eand (c):\u003csub\u003e \u003c/sub\u003eTEM images at 500 nm and 50 nm scales of MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/c7c4d34872136a2e4026fa52.png"},{"id":105566743,"identity":"7deae783-fbd3-4331-9533-c2021c86d9d0","added_by":"auto","created_at":"2026-03-27 12:57:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":67792,"visible":true,"origin":"","legend":"\u003cp\u003e3D plot showing effect of CNTs\u0026nbsp; concentration X1\u003csub\u003e \u003c/sub\u003eand\u0026nbsp; Ssonication time X2\u003csub\u003e \u003c/sub\u003eon (a): Particle size,\u0026nbsp; (b): Zeta potential and (c): Entrapment 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4","display":"","copyAsset":false,"role":"figure","size":67342,"visible":true,"origin":"","legend":"\u003cp\u003e(a): FTIR of VRZ and VRZ-CNTs, (b): DSC of VRZ, CNTs and VRZ-CNTs\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/6f4ce5eca6dac8296db08a1e.jpeg"},{"id":105487394,"identity":"6768493f-065c-4053-adfc-a0a70883370f","added_by":"auto","created_at":"2026-03-26 14:52:26","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":56646,"visible":true,"origin":"","legend":"\u003cp\u003e(a): X-ray diffractograms of VRZ and VRZ-CNTs, (b): SEM of VRz-CNTs at 10000X magnification, (c): SEM of VRZ-CNTs at 20000X 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vitro drug release from VRZ-CNTs, (b): Zero order kinetics, (c): First order kinetics, (d): Higuchi kinetics, (e): Hixon Crowell’s kinetics, (f): Korsmeyer-Peppas kinetics\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/4fd69a3ac2b9c733d3f37c67.jpeg"},{"id":105487396,"identity":"0f1410ea-868f-4e13-869b-274c0263e21f","added_by":"auto","created_at":"2026-03-26 14:52:26","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":41932,"visible":true,"origin":"","legend":"\u003cp\u003e(a): Ex-vivo skin permeation and (b): Drug retention study of VRZ gel and VRZ-CNTs gel\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/4121ccb9a9b454c59cebd9b9.jpeg"},{"id":105487402,"identity":"2b7fb9d0-de16-40a3-8791-1367bdc9208a","added_by":"auto","created_at":"2026-03-26 14:52:26","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":853816,"visible":true,"origin":"","legend":"\u003cp\u003eIn-vitro antifungal activity of VRZ-CNTs, Standard drug (VRZ), and Plain gel\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/136a7b15501f1302ee3db2a2.png"},{"id":105487399,"identity":"880051fe-cbbe-4c70-988e-ef772299c136","added_by":"auto","created_at":"2026-03-26 14:52:26","extension":"jpeg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":24884,"visible":true,"origin":"","legend":"\u003cp\u003eImages of skin irritation study (a): Normal control, (b): Positive control and (c): Test group.\u003c/p\u003e","description":"","filename":"floatimage10.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/87a8432b0e1feea6534ed675.jpeg"},{"id":105487400,"identity":"06706919-1624-4b8d-ad43-e88b30313f4a","added_by":"auto","created_at":"2026-03-26 14:52:26","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":142211,"visible":true,"origin":"","legend":"\u003cp\u003eResult of in vivo antifungal activity in wistar albino rats\u003c/p\u003e","description":"","filename":"floatimage11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/52b5d80dc2a45166ccf28032.jpeg"},{"id":105566206,"identity":"6cbd6262-97d8-46c1-89dd-85d0ebd69edd","added_by":"auto","created_at":"2026-03-27 12:55:40","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":317225,"visible":true,"origin":"","legend":"\u003cp\u003eResult of histopathology study on wistar albino rats\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/26c151656b27f0a2e2f554a9.png"},{"id":105752504,"identity":"2177c1a1-9747-4afd-978a-26b5ceb2cf27","added_by":"auto","created_at":"2026-03-30 16:02:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4027205,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9064633/v1/4d4ecc9c-737f-4b8e-b2a1-84a73b7bac3e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development and Evaluation of Amine-Functionalized Multi-Walled Carbon Nanotubes Based Gel for Enhanced Topical Antifungal Delivery of Voriconazole","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eFungal infections occur when fungi colonize and proliferate in the human body which adversely affects superficial and also deeper tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. These infections are widely predominant, especially in persons with compromised immunity, and can exhibit in varying degrees of intensity ranging from mild or moderate superficial infections of the skin and nails to systemic conditions that may become potentially fatal [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. General types of fungi responsible for such infections comprise yeasts, dermatophytes, and molds. Dermatophytes are often associated with infections such as ringworm and athlete\u0026rsquo;s foot, while yeasts like \u003cem\u003eCandida\u003c/em\u003e species are prominent to cause mucosal and systemic infections, for example candidiasis, which can become extremely serious in immunosuppressed patients [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In contrast to bacterial infections, fungal infections are more difficult to treat because of structural and metabolic resemblance among fungal and human cells, which diminishes the availability of specific target for antifungal treatment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Existing management approaches mostly depend on agents such as azoles, polyenes, allylamines, and echinocandins. From these, azoles, specifically imidazoles and triazoles, exert their effect by inhibiting ergosterol synthesis, an vital component of fungal cell membranes, consequently impairing survival of cell [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite its effectiveness, the phamacological potential of azoles is restricted by their poor solubility, low bioavailability, and dose-responsive adverse events, which demand the assessment of advanced drug delivery systems proficient to improve their effectiveness [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Nanotechnology-based carriers like solid-lipid nanoparticles, nanostructured lipid carriers, liposomes, niosomes, microemulsions, nanogels, polymeric nanoparticles, microneedles, carbon nanotubes (CNTs) etc. have been extensively investigated in recent times to conquer these hurdles. Nano carrier ystems can enhance the solubility, permeability, and stability of antifungal agents, thus enhancing site-specific delivery, prolonged release, and alleviating systemic side effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Among these nanocarriers, CNTs have attained significant attention because of their distinctive physicochemical characteristics, such as a greater aspect ratio, good mechanical strength, adaptable surface chemistry, high drug loading capacity, and capability to penetrate biological barriers [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, functionalization of CNTs improves their dispersibility, drug-binding efficiency, and biocompatibility, making them ideal for drug delivery [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A second-generation triazole antifungal agent, Voriconazole (VRZ) is extremely effective against a wide range of fungal pathogens, including \u003cem\u003eCandida\u003c/em\u003e, \u003cem\u003eAspergillus\u003c/em\u003e, and emerging resistant strains. VRZ act by inhibition of cytochrome P450-dependent 14-α-demethylase, which obstructs ergosterol biosynthesis, resulting in altered membrane permeability and subsequently death of fungal cell. Although it\u0026rsquo;s broad spectrum antifungal potential, VRZ is poorly water-soluble drug, showing variable absorption and low bioavailability [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To achieve therapeutic concentration high doses are often required, which markedly elevate the risk of hepatotoxicity, neurotoxicity, and drug interactions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Thus, developing a delivery system that enhances solubility, increases bioavailability, and minimizes toxicity of VRZ is of immense important. Topical route offers a promising approach for antifungal drug delivery as it facilitates localized treatment specifically at the site of infection, minimizes systemic exposure, and offers patient convenience [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nevertheless, the efficiency of conventional topical formulations is often limited by poor solubility of the active drug, restricted drug permeation through the stratum corneum,, and inadequate retention in the skin layers [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Embedding drug into nanocarriers such as CNTs and further this system incorporated to topical gel formulation has the potential to tackle these problems by facilitating effective drug encapsulation, improving solubility, as well as fostering sustained release at the site of infection [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This ensuring higher therapeutic efficacy of VRZ at lower doses while decreasing its adverse effectss [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Hence, developing a CNTs-based gel for VRZ delivery manifests a novel approach for the treatment of superficial fungal infections. Recent research has highlighted that CNTs can not only improve drug delivery but also demonstrate intrinsic antimicrobial potential, further promoting to their capability in treating infections [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, integrating VRZ with functionalized CNTs in a gel formulation can significantly advance antifungal therapy by enhancing drug solubility, improving localized action, reducing adverse effects, and promoting faster recovery. The development of a CNTs-based gel for VRZ therefore represents a rational and innovative strategy that addresses the limitations of conventional antifungal formulations and offers promising prospects for the management of fungal skin infections.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Materials\u003c/h2\u003e \u003cp\u003eVoriconazole, the primary antifungal drug, was obtained from Mac-Chem Products (India) Pvt. Ltd., Mumbai. Amine-functionalized Multi-walled Carbon Nanotubes, which served as the drug carrier, were procured from Ad-nano Technologies Pvt. Ltd. Karnataka, India. Carbopol 934, used as a pH-responsive gelling agent, along with glycerin (humectant), methyl paraben (used both as a preservative and solubilizer), ethylenediamine (a building block), methanol, and ethanol (both used as solvents), were all purchased from S.D. Fine Chem., Mumbai, india. Each ingredient played a specific role in ensuring the stability, effectiveness, and safety of the final formulation. All chemicals and reagents used were of analytical grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Methods\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Characterization of Amine-functionalized Multi-walled carbon nanotubes (MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e)\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.1 Organoleptic properties\u003c/h2\u003e \u003cp\u003eThe MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e were evaluated for organoleptic characters such as colour, odour, and appearance to ensure suitability, safety, and acceptability for formulation use [\u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.2 FTIR Study\u003c/h2\u003e \u003cp\u003eFTIR spectrum of amine-functionalized MWCNTs was recorded using a Bruker alpha spectrophotometer in the range of 4000–400 cm⁻¹ to confirm functional group modifications [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.3 SEM Analysis\u003c/h2\u003e \u003cp\u003eSEM (JEOL JSM-6390) was used to observe particle morphology of amine-functionalized MWCNTs by depositing a drop of sample solution on a stub and drying before imaging [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section4\"\u003e \u003ch2\u003e2.2.1.4 TEM Analysis\u003c/h2\u003e \u003cp\u003eTEM analysis was performed after dispersing samples in isopropyl alcohol, and images were recorded using a Philips CM12 TEM to measure internal and external diameters and structural uniformity [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Drug- excipient compatibility by FTIR\u003c/h2\u003e \u003cp\u003eFTIR spectroscopy (Bruker alpha spectrophotometer) was used to study interactions between Voriconazole and excipients (amine-functionalized MWCNTs, Carbopol, HPMC, glycerin, preservatives, and solvents). This confirmed stability by checking for new bonds or chemical alterations that could affect drug release and performance [\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.2.3 Formulation of voriconazole loaded Carbon nanotubes\u003c/h2\u003e \u003cp\u003eVoriconazole stock solution (1 mg/ml in ethanol-water, 2:1) was mixed with MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e (Concentration as mentioned in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The dispersion was sonicated for predetermined time, and stirred for 16 hours at room temperature. The drug-loaded nanotubes were obtained by centrifuging the clear supernatant at 3000 rpm for 10 minutes [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.2.4 Optimization of VRZ-CNTs\u003c/h2\u003e \u003cp\u003eA 3² factorial design was applied with two independent factors Carbon nanotubes concentration (X1) and sonication time (X2) at three levels each. Their effects on particle size (Y1), zeta potential (Y2), and entrapment efficiency (Y3) were studied using Design-Expert. The optimized formulation was selected by considering minimum particle size, and maximum zeta potential as well as entrapment efficiency [\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.2.5 Characterization of VRZ-CNTs\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.1 Particle size and PDI\u003c/h2\u003e \u003cp\u003eParticle size and polydispersity index were determined using a Zetasizer Nano ZS at 25 ± 0.1°C with triplicate measurements [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.2 Zeta potential\u003c/h2\u003e \u003cp\u003eSurface charge of VRZ-CNTs was analyzed using Dynamic light scattering method (Zetasizer Nano ZS90) at 0.05 mg/mL in PBS (pH 7.4), with mean values obtained from triplicate runs [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.3 Entrapment efficiency (%EE)\u003c/h2\u003e \u003cp\u003eTo determine EE, VRZ-CNTs were centrifuged at 5000 rpm for 15 min., and supernatant was analyzed at 256 nm by UV spectroscopy. The %EE was calculated from Eq.\u0026nbsp;1 [\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:EE=\\frac{Total\\:drug-Free\\:drug}{Total\\:drug}\\times\\:100\\:\\:\\:\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.4 FTIR study\u003c/h2\u003e \u003cp\u003eFTIR spectra (Bruker alpha spectrophotometer) of VRZ-loaded CNTs were recorded using the KBr disk method (5 mg sample/100 mg KBr) over 4000–400 cm⁻¹ to confirm drug loading and interactions [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.5 DSC\u003c/h2\u003e \u003cp\u003eDSC thermograms of pure VRZ and VRZ-loaded CNTs were recorded (30–300°C, 10°C/min, nitrogen atmosphere) to assess compatibility and thermal behaviour [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.6 XRD\u003c/h2\u003e \u003cp\u003eXRD patterns of plain VRZ and VRZ-CNTs were obtained on a Rigaku Dmax 2500 diffractometer (Cu Kα, λ = 1.5418 Å, 40 kV, 40 mA) scanning 2θ = 5°–80° to analyze crystalline nature changes [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.7 SEM\u003c/h2\u003e \u003cp\u003eSurface morphology of VRZ-CNTs was observed using SEM (JEOL, Japan) after gold coating, under high vacuum at 15 V with varying magnifications [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.8 TEM\u003c/h2\u003e \u003cp\u003eThe aqueous dispersion of VRZ-CNTs was dropped on carbon film coated copper grid, after airs drying the images were taken using high resolution transmission electron microscope (Philips CM12) [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section4\"\u003e \u003ch2\u003e2.2.5.9 In vitro drug release\u003c/h2\u003e \u003cp\u003eDrug release from VRZ-CNTs and pure VRZ was studied using the dialysis bag method in PBS (pH 7.4). VRZ-CNTs (equivalent to 20 mg of VRZ) and VRZ (20 mg) dispersed with 5 ml PBS was placed separately in dialysis bags and immersed in 50 ml of PBS. The temperature of the system was maintained at 37°C and agitated at 100 rpm. Samples were collected up to 5 h and analyzed by UV spectrophotometer at 256 nm. Sink condition was maintained after sample collection at each time interval using same volume of fresh PBS [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e2.2.6 Formulation of VRZ-CNTs based topical gel\u003c/h2\u003e \u003cp\u003eVRZ-CNTs based gel was prepared by dispersing 200 mg Carbopol-934 in 15 ml of purified water, left for 24 h to swell, and mixed with VRZ-CNTs (equivalent to 100 mg VRZ). Remaining quantity of purified water was added to adjust the final weight to 20 g. The dispersion was neutralized with triethanolamine to adjust pH, yielding a homogeneous gel [\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e2.2.7 Evaluation of VRZ-CNTs based topical gel\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.1 Appearance\u003c/h2\u003e \u003cp\u003eA VRZ-CNTs based topical gel was visually inspected for colour, homogeneity and phase separation as appearance is crucial for topical acceptability [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.2 pH\u003c/h2\u003e \u003cp\u003eThe pH of VRZ-CNTs based topical gel was measured using a digital pH meter to ensure stability and skin compatibility [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.3 Drug content\u003c/h2\u003e \u003cp\u003eDrug content was determined by dissolving 2 g of gel in methanol, filtering, and analyzing by UV spectrophotometer at 256 nm [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.4 Viscosity\u003c/h2\u003e \u003cp\u003eViscosity of VRZ-CNTs based topical gel was measured using a Brookfield viscometer with spindle no. 64 at constant speed of 6 rpm, and results were reported as mean of triplicate readings [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.5 Spreadability\u003c/h2\u003e \u003cp\u003eSpreadability of prepared gel was tested using the glass slide method [\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e]. A glass slide was placed on a plane platform at 37 ± 1°C and 1 g of VRZ-CNTs based topical gel was added on it. Another glass slide was placed over the gel loaded slide, 100 g standard weight was placed on the upper slide for a period of 1 min. Then the diameter of spread VRZ-CNTs based topical gel was measured. The spreadability of gel was calculated Eq.\u0026nbsp;2.\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:\\text{S}={d}^{2}\\times\\:\\:\\frac{\\pi\\:}{4}\\)\u003c/span\u003e \u003c/span\u003e (2) Where, S = spredability, and d\u003csup\u003e2\u003c/sup\u003e =diameter.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.6 Ex-vivo skin permeation\u003c/h2\u003e \u003cp\u003eEx-vivo permeability of VRZ from VRZ topical gel and VRZ-CNTs based topical gel was studied using Franz diffusion cells (receptor chamber volume = 20 ml and diffusion area = 0.78 cm²). An excised goat abdominal skin was used as a membrane for the study. Phosphate buffer pH 7.4 was used as a diffusion membrane. The study was performed at 37 ± 0.5ºC with constant stirring. The VRZ topical gel and VRZ-CNTs based topical gel were uniformly spread on the surface of membrane from the side of donor compartment. At the intervals of 0, 0.5, 1, 2, 4, 6, and 8 h. aliquots of samples (1 ml) were withdrawn and to quantify the permeated VRZ samples were analyzed using a UV spectroscope at 256 nm a wavelength [\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.7 Ex-vivo Drug retention\u003c/h2\u003e \u003cp\u003eTo determine the drug retention, the treated area of the goat skin used in the permeability study was carefully excised and rinsed with phosphate buffer pH 7.4o remove adhered drug to the outer surface. Then the skin was cut into smaller pieces, and homogenized with 1 ml of ethanol. The homogenate was centrifuged at 8000 rpm for 10 min. a supernatant was collected and analyzed using UV- spectrophotometer to determine retention of VRZ in the skin layers [\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.8 In vitro antifungal activity:\u003c/h2\u003e \u003cp\u003eIn vitro Antifungal activity of VRZ-CNTs based topical gel, and pure VRZ was assessed using agar well diffusion method against Candida albicans, with inhibition zone measurements. A 100 µL of Candida albicans suspension was spread onto sterilized Sabouraud Dextrose Agar plates and then wells were prepared using sterile cork borer. The test solutions containing VRZ-CNTs based topical gel (equivalent to VRZ 10 µg), a negative control (saline), and a positive control VRZ (10 µg) were carefully placed in specific well. The prepared plates were incubated at 30°C for 48 h accessed for the fungal growth and inhibition zones [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.9 In vivo skin irritation study\u003c/h2\u003e \u003cp\u003e In-vivo skin irritation and antifungal activities were performed after approval of Institutional animal ethics committee (SCOP/IAEC/2025/07) for laboratory animal use and care. For in vivo studies Wistar albino rats (Either Male or Female), weighing 200–250 g were used. The rats were kept in a room with controlled cycles of 12 hours of light and 12 hours of darkness. Animals were given standard food and distilled water.\u003c/p\u003e \u003cp\u003eWistar rats were divided into 3 groups each group having 6 rats. Group I will be considered the normal control group, and animals from this group will receive distilled water (0.5 ml) topically. Group II will be considered the positive control group, and animals from this group will receive formalin (1%) 0.5 ml to induce irritation on the first day. Group III will be considered Test group, and animals from this group will receive 0.5 gram dose of VRZ-CNTs gel topically. Each formulation will be applied to a 1 cm² hairless area of the rat’s skin. The rats were then be returned to their cages and monitored. The rats were observed at 24, 48, and 72 hours after the application, and the signs of skin irritation, such as redness (erythema) or swelling (edema) were checked [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section4\"\u003e \u003ch2\u003e2.2.7.10 In vivo antifungal activity\u003c/h2\u003e \u003cp\u003eThe study consisted four groups, with six rats (n = 6) in each group. The groups will be randomly divided as follows: Group I was negative control group, which will have no fungal infection (fungus-free); Group II was positive control group, which was infected with fungi but not received treatment (untreated); Group III was infected rats treated for 10 days with Voriconazole gel (Marketed Formulation) and Group IV was infected rats treated for 10 days with VRZ-CNTs topical gel. Rats except group-I received 5 mg/kg of prednisolone intravenously for three days, to cause an immunosuppressive effect. The rats' dorsal (back) skin was shaved (1 cm²) 4 hours before the test. Candida albicans was used as the fungal strain, which adjusted to concentration of 10\u003csup\u003e6\u003c/sup\u003e CFU/ml. Each rat except group-I rats received an intradermal injection of 0.3 mL of the Candida albicans suspension in the middle of a shaved area on its exposed skin. Any mild edema at the injection site was removed by rubbing the area vigorously. After 72 hours, signs of fungal infection were appeared in the injected area. To prevent the rats from licking the skin, they were kept separately in individual cages. Rats from Group III and Group IV treated with 0.5 g of voriconazole gel (Marketed formulation) and VRZ-CNTs topical gel respectively, and analysed for recovery from skin damage. The rats were monitored for clinical signs of fungal infection, such as rashes, red patches, white particles, scaling, maceration, erythema (redness), cracking, and pus-filled pimples. Also Histopathologic examinations and Total leucocyte count were determined [\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec35\" class=\"Section3\"\u003e \u003ch2\u003e2.2.8 Stability study\u003c/h2\u003e \u003cp\u003eAccelerated stability testing was performed at 40 ± 2°C temperature and 75 ± 5% Relative humidity (RH) for 3 months as per ICH guidelines by evaluating appearance, pH, drug content and viscosity for 3 motnhs [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec36\" class=\"Section3\"\u003e \u003ch2\u003e2.2.9 Statistical Analysis\u003c/h2\u003e \u003cp\u003eAnalysis of variance (ANOVA) was applied to check the significance between different components. All experimental trials were performed in triplicate, and the results are interpreted as mean ± SD.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec40\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec41\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec42\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec43\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec44\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec45\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec46\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec47\" class=\"Section3\"\u003e \u003cdiv id=\"Sec48\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec49\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec50\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec51\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec52\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec53\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec54\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec55\" class=\"Section4\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec56\" class=\"Section4\"\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec57\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec58\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec59\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec60\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec61\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec62\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec63\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec64\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec65\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec66\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec67\" class=\"Section3\"\u003e \u003c/div\u003e \u003cdiv id=\"Sec68\" class=\"Section3\"\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003ch2\u003e2.3.1 Organoleptic properties of MWCNTs-NH₂\u003c/h2\u003e\u003cp\u003eThe amine-functionalized multi-walled carbon nanotubes appeared as a black, fluffy, and very light powder suggests a high surface area and porous structure, which are desirable properties for drug loading and adsorption.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.2 FTIR spectrum of MWCNTs-NH₂\u003c/h2\u003e\u003cp\u003eThe FTIR spectrum of amine-functionalized carbon nanotubes is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea. This technique helps identify the different types of chemical bonds in a substance by measuring how it absorbs infrared light at different wavelengths. In this graph, several peaks can be seen at specific positions, each representing a unique bond or group of atoms. A broad peak near 3768 cm⁻¹ indicates the presence of N–H bonds, which is expected after amine functionalization. The peak around 2929 cm⁻¹ comes from C–H stretching vibrations, showing that some aliphatic groups are present, possibly from remaining organic molecules or excipients. A small peak at 2348 cm⁻¹ may result from carbon dioxide or overtones. A strong and sharp peak at 1698 cm⁻¹ is characteristic of C = O (carbonyl) stretching, which may come from oxidation. The peak at 1214 cm⁻¹ likely represents C–N or C–O stretching, confirming the presence of amine or ether groups. This FTIR analysis confirms the presence of amine-functionalized carbon nanotubes [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.3 Scanning Electron Microscopy (SEM) of MWCNTs-NH₂\u003c/h2\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb shows SEM image of the MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e. From the image, it was observed that the MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e were long, tube-like structures that were tangled and clumped together. This web-like structure was mainly due to van der Waals forces pulling the tubes toward each other. The surface of the tubes appears smooth, which suggests that they may have been successfully treated with amine groups. Most tubes have a similar thickness, though some look slightly thicker likely because a few tubes are stuck together.\u003c/p\u003e\u003ch2\u003e2.3.4 Transmission Electron Microscopy (TEM) MWCNTs–NH\u003csub\u003e2\u003c/sub\u003e\u003c/h2\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec shows Transmission Electron Microscopy (TEM) analysis of MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e. TEM is a powerful imaging technique that allows seeing the structure of materials at the nanometer scale. Image display CNTs at two different magnifications, i.e. 500 nm to 50 nm, giving a detailed look at their shape, size, and surface features. In the image of 500 nm scale, the MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e appear as long, tangled, and thread-like structures, showing how they are dispersed. The image of 50 nm scale shows small black dots along the nanotube surfaces, which likely represent the amine groups chemically bonded during the functionalization process. In addition with this individual nanotube more clearly seen in this image, where the hollow tubular structure becomes visible, suggest their multi-walled nature. This confirms that the surface of the carbon nanotubes has been modified effectively, which is important for improving their compatibility in drug delivery, polymer composites, or other applications [\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.5 Optimization of VRZ-CNTs\u003c/h2\u003e\u003cp\u003eA 3² full factorial design using Design-Expert software studied CNTs concentration (X1) and Sonication time (X2) on particle size (Y1), zeta potential (Y2), and entrapment efficiency (Y3). Positive and negative polynomial coefficients indicated variable effects, with statistically significant responses determined by p-values \u0026lt; 0.05. Scatter plots showed good agreement between actual and predicted values, validating the model’s reliability for optimization [\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e]. The obtained responses for nine runs are detained in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab1\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e3² full factorial design for optimization of VRZ-CNTs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eRun\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eFactor X1 CNTs\u003c/p\u003e \u003cp\u003econcentration (mg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eFactor X2 Sonication time\u003c/p\u003e \u003cp\u003e(min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eResponse Y1 Particle size (nm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eResponse Y2 Zeta potential (mV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eResponse Y3 Entrapment efficiency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e103.15 ± 1.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-27.7 ± 1.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e87.9 ± 1.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e102.19 ± 0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-36.58 ± 172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e92.76 ± 1.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e100.25 ± 1.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-44.31 ± 1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e94.89 ± 1.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e140.49 ± 0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-36.39 ± 1.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e82.4 ± 1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e142.24 ± 1.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-42.53 ± 1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e83.2 ± 0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e140 ± 2.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-47.82 ± 1.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e86.5 ± 1.63\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e183.82 ± 1.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-34.82 ± 1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e79.5 ± 2.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e178.86 ± 1.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-40.22 ± 1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e88.2 ± 1.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e178.9 ± 1.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e-46.55 ± 1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e85.5 ± 1.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab2\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eANOVA results for responses in optimization of VRZ-CNTs.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eDf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eMean square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eF- value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eP- value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\"\u003e \u003cp\u003eParticle size\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9293.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e4646.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e2311.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX1- CNTs Conc.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9281.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9281.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9281.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9281.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9281.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX2 – Sonication\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTime\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e11.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e11.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0538\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eResidual\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e12.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e12.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eCor Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e9305.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\"\u003e \u003cp\u003e\u003cb\u003eZeta Potential\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e348.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e69.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e147.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0009\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX1- CNTs Conc.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e28.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e28.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e59.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0045\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX2– Sonication\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTime\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e293.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e293.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e622.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX1X2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e12.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0380\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eResidual\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e19.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e19.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0074\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eCor Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.5904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\"\u003e \u003cp\u003e\u003cb\u003eEntrapment Efficiency\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eModel\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e174.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e87.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e7.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0264\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX1- CNTs Conc.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e107.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e107.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0257\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eX2 – Sonication\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eTime\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e67.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e67.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0.0581\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eResidual\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e73.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e12.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eCor Total\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e248.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003ch2\u003e2.3.6 Effect on particle size\u003c/h2\u003e\u003cp\u003eThe particle size of the Voriconazole loaded CNTs was determined using a Photon correlation spectroscopy (PCS) method, performed in triplicate. The lowest particle size was observed in the F3 batch (100.25 ± 1.05), while the F7 batch had the highest particle size (183.82 ± 1.41) as shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The effects of independent variables on dependent variables are expressed with Eq.\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eParticle size (Y1) = + 141.10 + 39.33X1–1.39X2 (3)\u003c/p\u003e\u003cp\u003eFrom the Eq.\u0026nbsp;3 it can be confirmed that, as CNTs concentration increases the particle size also increased due to systems viscosity [\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e], while longer sonication (up to 15 min) reduced size by breaking CNTs and improving dispersion [\u003cspan class=\"CitationRef\"\u003e50\u003c/span\u003e]. The linear model showed excellent fit (R² = 0.9987, Adjusted R² = 0.9983, Predicted R² = 0.9970) with significant results (F-value 2311.51, p \u0026lt; 0.0001) depicted in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Predicted and actual values closely matched, confirming model reliability. A 3D response surface plot for effect of independent variables on particle size is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea.\u003c/p\u003e\u003ch2\u003e2.3.7 Effect on Zeta potential:\u003c/h2\u003e\u003cp\u003eA zeta potential of VRZ-CNTs ranged from − 27.7 mV (F1) to -47.82 mV (F6), which influenced by CNTs concentration and sonication time (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The relationship between independent and dependent variables is depicted with Eq.\u0026nbsp;4.\u003c/p\u003e\u003cp\u003eZeta potential (Y2) = − 42.37 -2.17X1–6.33 X2 + 1.22 X1X2 + 3.88 X1\u003csup\u003e2\u003c/sup\u003e + 0.18 X2\u003csup\u003e2\u003c/sup\u003e (4)\u003c/p\u003e\u003cp\u003eAs CNTs concentration increases initially then zeta potential also increases, it could be due to increased charge density per unit volume. At highest concentration level of CNTs there was decrement in zeta potential. Longer sonication increases zeta potential due to better dispersion and surface charge distribution [\u003cspan class=\"CitationRef\"\u003e51\u003c/span\u003e]. High zeta potential values are important as they prevent particle aggregation, ensuring colloidal stability and improved drug delivery efficiency [\u003cspan class=\"CitationRef\"\u003e52\u003c/span\u003e]. A quadratic model was the best fit model for the zeta potential (R² = 0.9960, Adjusted R² = 0.9892, Predicted R² = 0.9636) and ANOVA results (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) confirms the significant impact of independent variables on responses (F-value 147.55, p-value 0.0009). A 3D response surface plot for effect of independent variables on zeta potential is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb.\u003c/p\u003e\u003ch2\u003e2.3.8 Effect on Entrapment efficiency (EE)\u003c/h2\u003e\u003cp\u003eEntrapment efficiency (EE) of VRZ-CNTs ranged from 79.5% (F7) to 97.89% (F3), influenced by both CNTs concentration and sonication time (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The impact of independent variables on dependent variables is expressed with Eq.\u0026nbsp;5.\u003c/p\u003e\u003cp\u003eEntrapment efficiency (Y3) = + 87.09–4.23X1 + 3.35X2 (5)\u003c/p\u003e\u003cp\u003eFrom the Eq.\u0026nbsp;5 it can be observed that, higher CNTs concentration (40 mg) reduced EE due to drug leakage or reduced encapsulation. Longer sonication time improves entrapment efficiency due to better emulsification and droplet dispersion [\u003cspan class=\"CitationRef\"\u003e53\u003c/span\u003e]. Hence, lower CNTs concentration with longer sonication gave the highest EE. The linear model showed good fit (R² = 0.7023, Adj R² = 0.6031) and confirms the significant impact of independent variables on responses (F-value 7.08, p-value 0.0264). High EE is important as it ensures maximum drug loading, improved therapeutic effect, and efficient drug delivery [\u003cspan class=\"CitationRef\"\u003e54\u003c/span\u003e]. A 3D response surface plot for effect of independent variables on entrapment efficiency is shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec.\u003c/p\u003e\u003ch2\u003e2.3.9 Validation of responses\u003c/h2\u003e\u003cp\u003eThe optimization process aimed to fine-tune the formulation of the VRZ-CNTs by setting specific targets for the key dependent variables. The optimization criteria include maximized Entrapment efficiency to ensure the drug entrapped within vesicles and retained in the non-polar chain that distributes the drug into deep skin layers, Minimized Particle to ensure that the better drug release and maximized Zeta potential to ensure the stability of VRZ-CNTs. Using these criteria, a design of experiments (DOE) approach was employed to generate multiple solutions. A total of 9 solutions were identified, each representing a different combination of the independent variables –CNTs concentration and Sonication time. These solutions were evaluated based on their desirability scores, which reflect how well they meet the optimization criteria. The solution with the highest desirability score of 0.899 was selected as the optimized batch. This specific combination of sonication time, CNTs concentration and was determined to provide the most favourable balance of maximum entrapment efficiency, minimum particle size and maximum zeta potential. Once the optimized batch was selected, the model's accuracy was validated by comparing the predicted values of the dependent variables to the experimentally observed values.\u003c/p\u003e\u003cp\u003eAn additional optimized batch of the VRZ-CNTs was made with suggested optimized values for CNTs concentration (20 mg) and sonication time (15 Min.) and tested for model validation. The predicted responses i.e. particle size, zeta potential, and entrapment efficiency were 100.38 nm, -43.98 mV and 93.33% respectively. Which were compared with that of the observed values i.e. particle size 102.92 ± 5.66 nm (% bias 2.53), zeta potential − 42.85 ± 2.6 mV (% bias 2.68) and entrapment efficiency 91.85 ± 3.68% (% bias 1.58) and found to be closer to the predicted values. A % Bias was for all three responses was less than 3% confirms the good correlation between predicted and observed values.\u003c/p\u003e\u003ch2\u003e2.3.10 Characterization of VRZ-CNTS\u003c/h2\u003e\u003ch2\u003e2.3.10.1 Particle size and PDI\u003c/h2\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea represented the particle size and size distribution of the optimized VRZ–CNTs. This consistency indicated that the formulation process had been well-controlled and reproducible. The analysis showed a mean particle size of 102.92 nm, with a Z-Average size of particle size 103.9 nm (± 1.02). The mode, representing the most frequently occurring particle size, was 98.6 nm, and the standard deviation was 28.1 nm, suggesting a reasonably tight distribution around the mean. The Polydispersity Index (PDI) was 0.327, which pointed to a moderately narrow distribution. While slightly above the ideal value of 0.3 for highly monodisperse systems, this PDI was still within the acceptable range for VRZ-CNTs formulations, as values below 0.5 were generally considered suitable [\u003cspan class=\"CitationRef\"\u003e55\u003c/span\u003e]. The graph displayed a single, sharp, and narrow peak, indicating that the particle sizes were relatively uniform and there was no presence of multiple particle populations. The graph demonstrated that the batch had been well-optimized in terms of particle size and distribution, making it suitable for further use.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.10.2 Zeta potential\u003c/h2\u003e\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb shows the zeta potential of optimized VRZ – CNTs. The sharp peak at -42.85 ± 2.6 mV particle size indicates that the particles carry a strong negative charge, which helps to keep them evenly dispersed without clumping. This means formulation is physically stable. Since zeta potential value greater than ± 30 mV is considered as it is more stable for colloidal systems, CNT are unlikely to aggregate, the graph shows a single sharp peak, which confirms that the sample is uniform and has a consistent charge distribution [\u003cspan class=\"CitationRef\"\u003e56\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.10.3 Entrapment efficiency\u003c/h2\u003e\u003cp\u003eEntrapment efficiency tells about how much of the Voriconazole got trapped inside the carbon nanotubes. The entrapment efficiency of VRZ-CNTs was observed to be 91.85 ± 3.68%. An EE% above 80% is considered very effective in topical drug delivery systems especially for antifungal treatments because it means most of the drug is retained inside the carrier system [\u003cspan class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.10.4 FTIR\u003c/h2\u003e\u003cp\u003e(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea) presents the FTIR spectra of pure VRZ, carbon nanotubes (CNTs), and VRZ-CNTs. Pure VRZ shows distinct peaks corresponding to key functional groups, including O–H (3203 cm⁻¹), aromatic and aliphatic C–H (3121–2878 cm⁻¹), aromatic C–C (1615–1509 cm⁻¹), triazole ring vibrations (1451–1332 cm⁻¹), ether linkages (1229–1047 cm⁻¹), and C–F bonds (1120 cm⁻¹). CNTs display minimal peaks due to their graphitic nature, but functionalization introduces broad O–H or N–H peaks (3400–3200 cm⁻¹) and a C = C signal near 1600 cm⁻¹. In the VRZ–CNTs spectrum, some peak shifts and intensity changes are observed, indicating physical interactions—likely hydrogen bonding—between VRZ and the CNTs [\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, all major functional peaks of VRZ remain present, confirming the drug's chemical stability after loading. These interactions suggest successful incorporation of VRZ into the CNTs, improving its solubility, thermal stability, and bioavailability compared to its pure crystalline form [\u003cspan class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.10.5 DSC\u003c/h2\u003e\u003cp\u003e(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb) shows DSC thermograms compares two samples pure Voriconazole and VRZ-CNTs. DSC is used to see how a substance behaves when heated, it helps to understand things like how easily it melts, how stable it is with heat, and whether it is in a crystal or non-crystal (amorphous) form. Voriconazole has a sharp, deep peak around 132°C; this sudden drop shows the melting point of the drug and tells that VRZ is in a crystalline state [\u003cspan class=\"CitationRef\"\u003e59\u003c/span\u003e]. Crystalline drugs usually don’t dissolve easily in water, which means they can be absorbed slowly in the body and may not work as effectively. In the VRZ-CNTs looks very different. It does not have a sharp melting peak like the pure drug. Instead, the curve is flatter and broader, especially around 200°C. This tells us that the VRZ crystal structure has changed or disappeared when combined with carbon nanotubes [\u003cspan class=\"CitationRef\"\u003e60\u003c/span\u003e]. In this case, Voriconazole may have turned into an amorphous form, which usually dissolves faster and is absorbs better in the body. Also, the shift in the curve to higher temperatures shows that the VRZ is more stable with heat when it is loaded onto CNTs, this graph shows that when Voriconazole is combined with carbon nanotubes, it becomes less crystalline and more heat-stable [\u003cspan class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.10.6 XRD\u003c/h2\u003e\u003cp\u003eA Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea shows XRD of pure voriconazole and VRZ-CNTs. In the Pure Voriconazole defractogram, several sharp and well-defined peaks appear between 10° and 35° (2θ), confirming a highly crystalline structure. Crystalline forms of voriconazole generally have slower dissolution rates and limited solubility, which can affect absorption and reduce therapeutic efficiency [\u003cspan class=\"CitationRef\"\u003e62\u003c/span\u003e]. In contrast, the defractogram of VRZ-CNTs shows a broad, less intense peak around 25° (2θ), while the sharp crystalline peaks present in pure voriconazole were nearly absent. This change indicates a transformation of voriconazole into an amorphous or partially amorphous state after incorporation into the carbon nanotubes [\u003cspan class=\"CitationRef\"\u003e63\u003c/span\u003e]. The broad peak also reflects the typical signal of carbon nanotubes due to their graphitic structure. A smaller peak near 43° (2θ) in the same graph suggests a degree of structural order within the nanotube material [\u003cspan class=\"CitationRef\"\u003e64\u003c/span\u003e]. This shift from crystalline to amorphous form is beneficial because amorphous voriconazole generally dissolves faster and is more soluble, enhancing its bioavailability. The amine-functionalized carbon nanotubes used as carriers offer a large surface area and strong interactions through functional groups, disrupting the regular crystal structure of voriconazole [\u003cspan class=\"CitationRef\"\u003e65\u003c/span\u003e]. As a result, the formulation improves the physical stability,\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.10.7 SEM\u003c/h2\u003e\u003cp\u003eThe images shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec are Scanning Electron Microscopy (SEM) images of VRZ-CNTs at 10000X magnification and 20000X magnification respectively. SEM is used to visualize the surface structure and morphology of materials at very high magnification. The image at 10,000x magnification shows a dense and interconnected network of carbon nanotubes that appear as long, entangled, thread-like structures. The image taken at 20,000x magnification provides a clearer view of these nanotubes and reveals the fine distribution of the drug particles on the surface of the nanotubes. The overall structure appears rough and sponge-like, indicating a good dispersion of voriconazole within the nanotube matrix. The absence of sharp edges and crystals suggests that voriconazole has been successfully loaded onto the carbon nanotubes and may have transitioned into a more amorphous or dispersed state. This transformation is important because the amorphous form of a voriconazole generally has better solubility and absorption properties than its crystalline form. The functionalized carbon nanotubes provide a large surface area and active binding sites (due to amine groups), allowing for strong interaction with voriconazole [\u003cspan class=\"CitationRef\"\u003e66\u003c/span\u003e]. As a result, drug particles are better distributed, more stable, and capable of controlled release. This improved morphology and dispersion seen in the SEM images suggests that the voriconazole-loaded carbon nanotubes show better performance in drug delivery than pure voriconazole. They could enhance bioavailability, prolong release and improve overall therapeutic effect.\u003c/p\u003e\u003ch2\u003e2.3.10.8 TEM\u003c/h2\u003e\u003cp\u003eThe Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e shows TEM analysis of VRZ-CNTs. Images are taken at two different magnifications, i.e. first showing 500 nm scale and second showing 50 nm, giving a detailed look at their diamentsions and surface features after loading of VRZ. In the image of 500 nm scale, the VRZ-CNTs appear as thin, elongated, and Tubular structures, with rough morphology indicating dispersion of drug in the nanotubes. The image of 50 nm scale shows internal morphology of specific tubular structure small black dots like structure inside the tubes confirms the loading and dispersion of drug.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.10.9 In vitro release of VRZ loaded CNTs:\u003c/h2\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab3\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro drug release from VRZ-CNTs and Plain VRZ\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eTime (Hours)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003e% CDR from VRZ-CNTs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003e% CDR from Plain VRZ\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e37.33 ± 2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e19.66 ± 2.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e49.66 ± 2.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e27.66 ± 1.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e61.00 ± 2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e34.00 ± 2.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e71.33 ± 1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e39.66 ± 1.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e75.00 ± 1.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e44.00 ± 1.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e79.00 ± 2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e46.33 ± 1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e82.33 ± 1.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e49.00 ± 0.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e85.66 ± 2.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e50.00 ± 0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eA drug release study was carried out to compare the release behaviour between plain VRZ and VRZ-CNTs. The results showed that VRZ-CNTs released the drug much faster and in higher amounts than plain VRZ (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). After 1 hour, VRZ-CNTs released about 38% (± 1.2) of the drug, while plain VRZ released only 20% (± 0.59). Finally, by the 8th hour, VRZ-CNTs showed a release of 85% (± 0.12), while plain VRZ remained at 39% (± 0.17) as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea. These results confirmed that loading Voriconazole into carbon nanotubes improved its solubility and release rate, which was likely due to the smaller particle size, larger surface area, and better dispersion of the drug when carried by the nanotubes. Furthermore, the drug's transition to a more amorphous form and its interaction with functional groups on the CNT surface contribute to better solubility and faster dissolution [\u003cspan class=\"CitationRef\"\u003e67\u003c/span\u003e]. Together, these factors make CNTs an effective carrier system for enhancing the therapeutic performance of voriconazole.\u003c/p\u003e\u003cp\u003eA release mechanism from VRZ-CNTs was studied using different kinetic models, which include the zero order, first order, Higuchi, Hixon-Crowell, and Koresmyer-Peppas kinetics. The results of release kinetics are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb to Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef. The best fit model was found to be Koresmyer-Peppas model highest R² value (09896) with n \u0026lt; 0.5, confirms the release follows Fickian diffusion.\u003c/p\u003e\u003ch2\u003e2.3.11 Evaluation of VRZ-CNTs Gel\u003c/h2\u003e\u003ch2\u003e2.3.11.1 Organoleptic properties\u003c/h2\u003e\u003cp\u003eThe prepared VRZ-CNTs gel was evaluated for colour, homogeneity, and the occurrence of phase separation. The colour of gel was black due to the colour of CNTs was blackish. The prepared gel was found to be homogeneous without separation of phases suggests the appropriateness of formulation.\u003c/p\u003e\u003ch2\u003e2.3.11.2 pH\u003c/h2\u003e\u003cp\u003eThe average pH of VRZ-CNTs gel was 6.74 ± 0.21, which is close to the skin’s pH range, making the formulation safe and non-irritating for skin use [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.3 Drug content\u003c/h2\u003e\u003cp\u003eThe drug content analysis showed a consistent and uniform distribution of voriconazole in the VRZ-CNTs based topical gel, with an average content of 97 ± 2.56%. This indicates that the drug is well incorporated into the formulation, ensuring reliable dosing [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.4 Viscosity\u003c/h2\u003e\u003cp\u003eThe VRZ-CNTs based topical gel’s viscosity ranged between 8850 and 9080 mPa·s, with an average of 8963 mPa·s. This relatively high viscosity suggests that the gel is thick and stable, suitable for prolonged contact with the skin, which helps in sustained drug delivery [\u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.5 Spreadability\u003c/h2\u003e\u003cp\u003eIn terms of application, the spreadability of the gel was measured at 26.5 ± 1.21 g·cm/sec, falling within the ideal range for topical gels. This means the gel can be smoothly and evenly applied on the skin, enhancing user comfort and effectiveness [\u003cspan class=\"CitationRef\"\u003e68\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.6 Ex-vivo skin permeation\u003c/h2\u003e\u003cp\u003eThe Ex-vivo skin permeability study was performed using goat abdominal skin. After 8 h of the study, the cumulative amount of VRZ permeated from the plain VRZ topical gel and VRZ-CNTs based topical gel was 71.96 ± 3.59 µg/cm² (Flux 8.99 µg/cm²/h) and 292.16 ± 7.08 µg/cm² (Flux 36.52 µg/cm²/h) respectively as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea. Statistical analysis confirms that permeation of VRZ from VRZ-CNTs based topical gel was significantly greater than that from VRZ topical gel (P \u0026lt; 0.001). The greater permeability of VRZ from the VRZ-CNTs based topical gel is due to increased solubility, surface area, and sustained release of drug from the CNTs matrix, which help to maintains high concentration gradient across the skin. I addition to this, the positively charged amine groups of amine-functionalized multi-walled carbon nanotubes interact with negatively charged skin components, causes temporary lipid disruption and increases follicular penetration and enhances drug diffusion compared to plain VRZ gel [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.7 Ex-vivo Drug retention\u003c/h2\u003e\u003cp\u003eAfter 8 h of permeation study, the treated skin thoroughly washed, excised and evaluated for drug retention in the deeper skin layers. The VRZ-CNTs based topical gel showed significantly higher VRZ retention (70.53 ± 4.12 µg/cm²) compared to BRH-HG (23.87 ± 3.25 µg/cm²), as illustrated in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb. The enhanced retention is primarily due to strong hydrophobic interactions between voriconazole and the amine-functionalized multi-walled carbon nanotubes surface, which enable deeper carrier-mediated deposition within epidermal and dermal layers. Furthermore, the positively charged amine groups of MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e electrostatically interact with negatively charged skin components, improving adhesion and prolongs residence time in deeper layers of the skin. The higher follicular penetration ability of MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e further facilitates accumulation of VRZ in deeper tissues compared to plain VRZ gel [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.11.8 In-vitro antifungal activity\u003c/h2\u003e\u003cp\u003eThe VRZ-CNTs gel exhibited a zone of inhibition of 33 ± 0.58 mm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e), which was significantly larger than the standard drug (Voriconazole, 18 ± 0.25 mm), indicating enhanced antifungal effect, the plain gel (Without drug) showed no inhibition, confirming that the activity is solely due to the active formulation. The VRZ-CNTs gel showed better antifungal activity compared to both the standard drug (Voriconazole) and the plain gel. This was likely because the combination of Voriconazole with MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e improved the drug’s effectiveness. The positively charged amine groups on CNTs may interact with the negatively charged fungal cell membrane allowing it to penetrate more efficiently. They also provided a controlled and sustained release of the drug, which kept the drug active for a longer time at the infection site [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.11.9 In-vivo Skin irritation\u003c/h2\u003e\u003cp\u003eThe in-vivo skin irritation study was conducted to evaluate the safety of the VRZ-CNTs gel when applied topically. Since pharmaceutical gels can sometimes cause adverse skin reactions like redness, itching, or swelling, it is essential to confirm that all ingredients are non-irritating. In this study, healthy female Wistar albino rats were used, and the skin was monitored for signs of erythema (redness) and edema (swelling) at 24, 48, and 72 hours after application. The severity of these reactions was rated on a scale from 0 to 4, where 0 represents no reaction and 4 indicate a severe reaction [\u003cspan class=\"CitationRef\"\u003e72\u003c/span\u003e]. Three groups were included in the study: a normal control group (no treatment), a positive control group treated with formalin (a known irritant), and a test group treated with the VRZ-CNTs gel. The normal control group showed no signs of redness or swelling at any time point (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003ea). The formalin treated group (Positive control) exhibited slight redness and swelling, confirming the sensitivity of the test (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003eb). Most importantly, the group treated with the VRZ-CNTs gel showed no signs of irritation, scoring 0 for both erythema and edema at all time points (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003ec). An average score of less than 2 indicates a non-irritant formulation. Since the VRZ-CNTs gel consistently scored 0 across all animals, it can be concluded that the formulation is non-irritating and safe for use on the skin. These findings support its suitability for topical application.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003ch2\u003e2.3.11.10 In vivo antifungal activity\u003c/h2\u003e\u003cp\u003eThe study evaluated the effectiveness of VRZ–CNTs gel in treating deep skin fungal infections caused by \u003cem\u003eCandida albicans\u003c/em\u003e in immunosuppressed rats. Initially, all rats showed healthy, intact skin with no signs of irritation or damage. After intradermal injection with \u003cem\u003eCandida albicans\u003c/em\u003e (10⁶ CFU/mL), infection symptoms such as mild redness and irritation appeared within 24 hours in the infected groups. By the third day, the condition worsened, with clear signs of erythema, scaling, and skin cracking, confirming successful infection. Treatment was then initiated using two formulations: VRZ–CNTs gel and plain Voriconazole gel (marketed formulation). By the fifth day of treatment, rats treated with the VRZ–CNTs gel began shedding the infected layer, revealing healthy pink skin underneath, indicating rapid healing. Meanwhile, the group treated with the plain VRZ gel showed moderate improvement, but still had visible redness and scaling. By the tenth day, the VRZ–CNTs gel group had fully recovered with completely healed skin and no visible signs of infection or scarring (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). In contrast, the plain VRZ gel group had not achieved full recovery. These findings demonstrate that VRZ–CNTs gel is significantly more effective in treating fungal skin infections, likely due to its enhanced drug delivery and sustained release properties.\u003c/p\u003e\u003cp\u003eThe VRZ–CNTs gel showed better healing than the plain VRZ gel, which still had mild redness and scarring. The untreated group had severe infection signs like pus and peeling. The enhanced effect of the VRZ–CNTs gel is due to amine-functionalized multi-walled carbon nanotubes, which helped the drug penetrate deeper and stay longer at the infection site. Their small size and large surface area improved drug delivery and antifungal action. It also facilitates sustained drug release and follicular targeting, which helps to maintain therapeutic levels for a prolonged period of time. In addition to this, the positively charged amine groups on CNTs enhances the interactions with negatively charged fungal cell membranes, which improves membrane permeation and enhances antifungal efficacy of VRZ-CNTs compared to plain VRZ gel [\u003cspan class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eThe histopathological analysis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e) revealed that the fungal infection-induced group (Positive control) displayed the most severe skin abnormalities, including severe hyperkeratosis, cellular infiltration, and epidermal hyperplasia (+++). These results confirmed extensive skin damage due to the fungal infection. In contrast, the normal control group had completely healthy skin with no signs of hyperkeratosis, infiltration, or hyperplasia (00). Rats treated with plain Voriconazole gel showed clear signs of healing compared to the infected group. The severity of skin damage was reduced to moderate hyperkeratosis, cellular infiltration, and moderate epidermal hyperplasia (++). This indicates that the antifungal effect of Voriconazole helped reduce inflammation and tissue damage, although not completely reversing the changes caused by the infection. The group treated with VRZ-CNTs gel also showed minimal hyperkeratosis, infiltration, and hyperplasia (+) as depicted in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. However, microscopic examination and skin appearance indicated superior healing, with almost normal tissue architecture and minimal inflammation. This suggests enhanced recovery in VRZ-CNTS treated group due to improved drug delivery and retention due to MWCNTs-NH\u003csub\u003e2\u003c/sub\u003e [\u003cspan class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab4\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSigns of Hyperkeratosis, Cellular infiltration and Epidermal hyperplasia in different study groups\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eHyperkeratosis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eCellular infiltration\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eEpidermal hyperplasia\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eNormal control\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003ePositive control\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eTest I\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e++\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003eTest II\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cp\u003eThe Total Leucocytic Count (TLC) study assessed white blood cell levels to evaluate infection and immune response. The fungal infection group (Positive control) showed the highest TLC (13.23 × 10³/µL), indicating severe inflammation. The normal control had the lowest TLC (8.15 × 10³/µL), reflecting a healthy state i.e. no infection or immune response. The VRZ gel treated group showed moderate improvement (10.27 × 10³/µL), while the VRZ–CNTs treated gel group had a significantly lower TLC (9.15 × 10³/µL), suggesting more effective infection control and reduced inflammation due to enhanced drug delivery[\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e]. The higher TLC in the fungal infection group reflects a strong immune response to fight the infection. VRZ gel reduced infection and inflammation, lowering TLC moderately. The VRZ-CNTs gel improved drug delivery and healing more effectively, resulting in even less inflammation and a further reduced TLC [\u003cspan class=\"CitationRef\"\u003e74\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003e2.3.11.11 Stability Study\u003c/h2\u003e\u003cp\u003eFor evaluation of stability of VRZ-CNTs gel, a three-months study was conducted at accelerated conditions (40 ± 2°C, 75 ± 5% RH). The results are depicted in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e which confirmed that the appearance, pH, drug content, viscosity and spreadability of the VRZ-CNTs gel did not significantly changed after three months of storage at accelerated conditions. Thus, the developed VRZ-CNTs gel was found to be stable.\u003c/p\u003e\u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" class=\"colspec\"\u003e\u003c/div\u003e\u003ctable id=\"Tab5\" border=\"1\"\u003e \u003ccaption\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStability study results of VRZ-CNTs gel\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003c/colgroup\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\"\u003e \u003cp\u003eTime (Months)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eAppearance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eDrug Content (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eViscosity (mPa·s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\"\u003e \u003cp\u003eSpreadability (g·cm/sec)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBlack Homogeneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e6.74 ± 0.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e97 ± 2.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e8963 ± 117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e26.50 ± 1.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBlack Homogeneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e6.70 ± 0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e96.89 ± 1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e8940 ± 125\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e2672 ± 0.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBlack Homogeneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e6.68 ± 0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e96.80 ± 2.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e8915 ± 136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e26.89 ± 1.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\"\u003e \u003cp\u003eBlack Homogeneous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e6.65 ± 0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e96.75 ± 1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e8910 ± 129\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\"\u003e \u003cp\u003e26.92 ± 1.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/table\u003e\u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eThe present study successfully developed MWCNTs-NH₂ based gel for topical voriconazole delivery. The optimized VRZ-CNTs formulation exhibited adequate particle size, good zeta potential and higher entrapment efficiency, confirming stability and reproducibility of formulation. FTIR, DSC, and XRD validated compatibility of formulation components and verified transition of VRZ from crystalline to amorphous form, which facilitates enhancement in solubility and controlled release of drug. Incorporation of VRZ-CNTs into Carbopol 934 gel demonstrated suitable pH, spreadability, viscosity, and consistent drug content, assuring superiority of gel. Moreover, the VRZ-CNTs gel exhibited notably improved ex-vivo skin permeation and deeper skin retention, and also found to be non-irritant. In-vitro and In-vivo antifungal evaluation further validated enhanced therapeutic efficiency and histopathological recovery as that of conventional VRZ gel. These findings suggests that the VRZ-CNTs gel will be a safer, more effective, and promising alternative for conventional topical antifungal therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eAuthors are thankful to Satara College of Phar\u0026shy;macy, Satara-415004, Maharashtra State, India for providing best of facility to conduct this research work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eVS: Performed experimental work, manuscript drafting MS: Supervision, Conceptualization, manuscript drafting, Visualization, Investigation, VM: Manuscript editing, guid\u0026shy;ance AB: Data generation, manuscript writing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e The authors declare that no funds, grants, or other support were received for completion of this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003eThe data will be made available on reasonable request from corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal Ethics\u0026nbsp;\u003c/strong\u003eAll animal experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of the Satara College of Pharmacy, Satara, Maharashtra, India under approval number SCOP/IAEC/2025/07.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMcCormick, T. S., \u0026amp; Ghannoum, M. (2024). Time to think antifungal resistance increased antifungal resistance exacerbates the burden of fungal infections including resistant dermatomycoses. \u003cem\u003ePathogens and immunity\u003c/em\u003e, \u003cem\u003e8\u003c/em\u003e(2), 158. https://doi.org/10.20411/pai.v8i2.656 \u003c/li\u003e\n\u003cli\u003eCasalini, G., Giacomelli, A., \u0026amp; Antinori, S. (2024). 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Chenopodium murale juice shows anti-fungal efficacy in experimental oral candidiasis in immunosuppressed rats in relation to its chemical profile. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e28\u003c/em\u003e(11), 4304. https://doi.org/10.3390/molecules28114304 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Voriconazole, Carbon nanotubes, Antifungal activity, Factorial design, Topical delivery","lastPublishedDoi":"10.21203/rs.3.rs-9064633/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9064633/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA potent antifungal drug, voriconazole (VRZ), whose clinical performance is limited by its poor solubility. The current research aimed to investigate the capability of anime-functionalized multi-walled carbon nanotubes (MWCNTs-NH₂) as a carrier for the topical VRZ delivery. The VRZ-CNTs formulation was optimized using a 3\u0026sup2; full factorial design. An optimized VRZ-CNTs showed reasonable particle size (102.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 nm), suitable zeta potential (-37.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 mV), and maximum entrapment efficiency (85.32\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07%), indicates the reliability and stability of the formulation. FTIR study verifies the compatibility of the formulation constituents, while DSC and XRD results reveal transmission of drug\u0026rsquo;s nature from crystalline to amorphous. SEM results suggested nanotubular morphology of VRZ-CNTs. The VRZ was released from formulation in sustained manner and showed 85.66\u0026thinsp;\u0026plusmn;\u0026thinsp;2.33% drug release at the end of 8 hours. The optimized VRZ-CNTS effectively incorporated into gel with the use of carbapol 934, which showed adequate pH (6.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21), drug content (97\u0026thinsp;\u0026plusmn;\u0026thinsp;2.56%), spreadability (26.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1.21 g\u0026middot;cm/sec) and viscosity (8963\u0026thinsp;\u0026plusmn;\u0026thinsp;117 mPa\u0026middot;s). The gel illustrated markedly higher Ex-vivo skin permeation and drug retention. Skin irritation study indicated that the gel formulation was non-irritant. Moreover, in vivo evaluations confirmed the enhanced antifungal efficacy and better histopathological recovery of the VRZ\u0026ndash;CNTs gel compared to conventional gel. Thus, VRZ-CNTS gel represents a promising and efficient alternative to conventional topical antifungal treatment.\u003c/p\u003e","manuscriptTitle":"Development and Evaluation of Amine-Functionalized Multi-Walled Carbon Nanotubes Based Gel for Enhanced Topical Antifungal Delivery of Voriconazole","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-26 14:52:21","doi":"10.21203/rs.3.rs-9064633/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T09:11:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T05:39:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T09:05:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157933455293363683111009969659856148234","date":"2026-05-08T04:19:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"137094811005829432570486109171301283759","date":"2026-04-23T11:50:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25224718610616448366424384438197468446","date":"2026-04-22T05:50:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-07T23:10:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54087323793349942017959633099985226449","date":"2026-04-06T22:51:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150172208622877086842087896888755493659","date":"2026-03-26T09:45:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36533114122276094322851166956716344294","date":"2026-03-26T08:47:24+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-24T08:27:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-24T08:22:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-24T00:39:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2026-03-08T13:41:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f1a0719c-3cf0-4b5c-beb1-08e52c22d77a","owner":[],"postedDate":"March 26th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T09:11:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-15T05:39:10+00:00","index":59,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-13T09:05:54+00:00","index":58,"fulltext":""},{"type":"reviewerAgreed","content":"157933455293363683111009969659856148234","date":"2026-05-08T04:19:07+00:00","index":55,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T09:27:27+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-26 14:52:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9064633","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9064633","identity":"rs-9064633","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}