β-Cyclodextrin-Coated Fluconazole Nanosuspensions: Formulation and Evaluation for Enhanced Ocular Delivery

β-Cyclodextrin-Coated Fluconazole Nanosuspensions: Formulation and Evaluation for Enhanced Ocular Delivery | 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 β-Cyclodextrin-Coated Fluconazole Nanosuspensions: Formulation and Evaluation for Enhanced Ocular Delivery Malkiet Kaur, Paramjot Maman, Manju Nagpal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7601516/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : Fluconazole, a potent antifungal agent, faces challenges in ocular therapy due to its poor aqueous solubility and limited bioavailability. Nanosuspensions formulations coated with β-cyclodextrin (β-CD) have the potential to enhance the delivery and therapeutic efficacy of such poorly soluble drugs. The study aimed to developβ-CD coated fluconazole nanosuspensions and evaluate their physicochemical properties, cytotoxicity and potential for improved ocular drug delivery. Methods : Fluconazole nanosuspensions were prepared using solvent evaporation method. The formulations were characterised for particle size, zeta potential, drug encapsulation efficiency and in vitro drug release profile. Cytotoxicity studies were performed using human corneal cells (HCET). Results : The results demonstrated that β-CD coating significantly improved the drug solubility and stability of drug. The particle size of coated nanosuspensions was found to be 275.39± 20.24 nm. In vitro evaluations showed prolonged drug release (83.24 ± 5.31%) at ocular surface. Additionally, the prepared nanosuspensions showed minimal cytotoxic effects which indicate the good biocompatibility of the prepared formulation for ocular use. Conclusion : The findings suggest thatβ-CD coated fluconazole nanosuspensions are a promising candidate for enhancing the therapeutic efficacy of fluconazole in ocular infections and can be used as an alternative to conventional dosage forms. β-cyclodextrin nanosuspension ocular delivery antifungal sustained release Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Despite of multiple researches in ophthalmic, Ocular drug delivery systems still face several challenges because of unique anatomy and physiological characteristics of eye. Human eye has several protective layers/barriers, such as the cornea, conjunctiva, and tear film, which significantly reduce the penetration and retention of administered formulations (Ahmed S et al., 2023 ). Moreover, most of conventional dosage forms like eye drops, ointments etc, have reduced precorneal residence time which further reduces the bioavailability and therapeutic efficacy of administered formulations (Liu LC et al., 2023 ). Other than this, many ophthalmic drugs such as fluconazole, face limitations in ocular treatment due to poor aqueous solubility, which further reduces the ability of drug to achieve therapeutic concentrations at the site of administration (Tanzawa A et al., 2022 ). Therefore, there is a critical requirement for innovative drug delivery strategies which have the ability to enhance the solubility, stability, and bioavailability of such ocular drugs (Volkova TV et al., 2021 ). Fluconazole is one of the widely used antifungal agents which is found effective in treatment of various ophthalmic fungal infections such as keratitis and endophthalmitis (Yosrey E et al., 2022 ). While effective, fluconazole suffers from poor aqueous solubility, rapid drainage, short ocular retention time, and poor penetration at corneal barrier results in suboptimal concentration of drug at administered site when given in form of eye drops and eye ointments. To overcome these limitations associated with fluconazole conventional formulations, the development of novel drug delivery systems (NDDS) has been an area of currently active researches (Nagai N et al ., 2022). Among the various NDDS approaches, nanosuspensions have emerged as a promising strategy for improving the delivery of poorly soluble drugs. Nanosuspensions are colloidal dispersions of submicron-sized drug particles that provide an increased surface area, leading to enhanced dissolution rates, solubility of drug (khan S et al., 2024 ). By reducing the particle size of poorly soluble drugs like Fluconazole, to a nano-meter scale, nanosuspensions can significantly improve the rate of drug absorption, resulting in enhanced ocular bioavailability. While nanosuspensions offer numerous advantages, however, the stability of the nanosuspension, sedimentation and aggregation of nano-sized dispersion still remain critical concerns (Fathi-Karkan S et al., 2024 ). One strategy to enhance the stability and ocular performance of nanosuspensions is the use of polymeric coatings. Coating materials can help stabilize the nanosuspensions with its improved interactions with ocular tissues, and modify their drug release profile. Among the various coating materials, β-cyclodextrin (β-CD) has gained significant attention because of its ability to produce inclusion complexes with poorly soluble drugs (Wang J et al ., 2020). β-CD is a cyclic oligosaccharide which contains about seven glucose units. It has the unique ability to encapsulate hydrophobic drug molecules within its hydrophobic cavity. This characteristic makes β-CD as a promising candidate for improving the solubility of lipophilic drugs like fluconazole (Khalid FM et al., 2023 ). Coating of fluconazole nanosuspensions with β-CD provides multiple advantages (Sadeh P et al., 2024 ) such as; 1) Providing stability to nanosuspensions by preventing the particle aggregation 2) Improves the bioavailability of drug by promoting better interaction with ocular tissues 3) enhanced mucoadhesive properties resulting in prolonged drug retention on the ocular surface with better residence time and therapeutic effectiveness of drug 4) Improvement of drug's release profile, allowing for a sustained release of fluconazole over time, which is essential for treating chronic ocular infections. The current study aims to prepare and evaluate β-CD-coated fluconazole nanosuspensions for ocular delivery. The primary objective of the study is to enhance the solubility and ocular bioavailability of fluconazole, improve the stability, and drug release profile. The outcome of this research could provide a promising strategy for the improvement of the therapeutic management of ocular fungal infections. The study results will offer more effective and sustained treatment options for ocular fungal infections as compared to traditional marketed formulations like eye drops and ointments. Materials and Methods Materials Fluconazole (purity ≥ 98%) was purchased from Sigma-Aldrich (India). β-Cyclodextrin (β-CD) was obtained from Loba Chemie Pvt. Ltd. (India). The solvents used in the formulation process, including water, methanol, and acetone, were of analytical grade and were supplied by Loba Chemie Pvt. Ltd (India). Polyvinyl alcohol (PVA) was used as a stabilizer and was purchased from Sigma-Aldrich. All other chemicals and reagents used were of analytical grade unless specified otherwise. Methods Preparation of Fluconazole Nanosuspensions Fluconazole (FLZ) nanosuspensions were prepared by using solvent evaporation method. A precise amount of FLZ (100 mg) was dissolved in about6 mL of organic volatile solvent, dichloromethane (DCM). The prepared FLZ solution was added dropwise using a syringe into 20 mL of distilled water containing hydroxypropyl ethyl cellulose (HPMC) as stabiliser in different concentrations (0.5%, 1%, and 5% w/v) with uniform magnetic stirring of 1000 rpm. These stabilizers prevent particle aggregation and ensure stability of the nanosuspension (Aldosari BN et al., 2024 ). The stirring was continued for 6 hours to ensure complete evaporation of dichloromethane. This step resulted in supersaturation of FLZ in aqueous medium, which result in the formation of nanosized FLZ particles through nucleation and crystallization processes. Continuous magnetic stirring ensured uniform distribution of FLZ and stabilizer which result in formation of stable nanosuspension. The prepared FLZ nanosuspension were stored at 4°C to prevent its degradation and maintain its integrity by minimising Ostwald ripening and particle aggregation (Wang P et al., 2019 ). Please refer Table 1 for different prepared formulations and optimization of FLZ- nanosuspensions. β-Cyclodextrin Coating of Fluconazole Nanosuspensions After the preparation of fluconazole nanosuspensions, a predetermined amount of β-CD (200 mg) was dissolved in deionized water and added dropwise to the nanosuspension while stirring continuously at 500 rpm. The mixture was kept under magnetic stirring for additional 4 hours to allow the formation of β-CD-FLZ inclusion complexes on the surface of the nanoparticles (Pawar P et al., 2021 ). The prepared β-CD-FLZ nanosuspension was subjected to high-frequency ultrasound treatment of 20 kHz for 10 minutes to break up any aggregates further and get more uniform size distribution. The final prepared β-CD-FLZ nanosuspension was stored in air tight glass ambient container (Aldosari BN et al., 2024 ). Please refer Table 2 for different optimization of β-CD-FLZ-nanosuspensions. Characterization of β-CD-FLZ Nanosuspensions After the preparation of β-CD coated fluconazole nanosuspensions, several characterization techniques were employed to evaluate stability and suitability for ocular delivery. Particle size and zeta potential analysis The particle size and zeta potential of prepared β-CD-FLZ nanosuspensions were measured using Delsa Nano C zeta sizer (Beckman. Coulter Pvt. Ltd.). Zeta potential is a measure of the surface charge on nanosized drug particles and is critical for determining the stability of the formulation. High zeta potential values generally have better stability due to high electrostatic repulsion, which prevents aggregation in nanosuspension (Abdelbary AA et al., 2013 ). Transmission electron microscopy (TEM) Study The morphology of prepared β-CD-FLZ nanosuspensions was examined using transmission electron microscopy (TEM). Transmission electron microscopy (TEM) was employed to assess the morphological characteristics of β-CD-FLZ nanosuspensions. A small drop of prepared β-CD-FLZ nanosuspensions was placed onto carbon-coated copper grids, which was then examined using TEM microscopy (200kV Tecnai™ T20, Mohali, India). Images were captured and analysed using dedicated imaging software to evaluate morphology of β-CD-FLZ nanosuspensions (Pawar P et al., 2021 ). Drug encapsulation efficiency The drug encapsulation efficiency (EE) was determined by measuring the amount of FLZ incorporated into the prepared β-CD-FLZ nanosuspensions. About 5 mL of prepared formulation was centrifuged at 10,000 rpm for 30 minutes to separate the free drug from the nanosuspension. 16 The supernatant was collected, and the drug concentration was determined using UV spectrophotometry at λ max of 260 nm. The encapsulation efficiency was calculated using the formula: EE (%) = \(\:\frac{Amount\:of\:drug\:encapsulated}{Total\:amount\:of\:drug\:added}\) X 100 In Vitro d rug release studies The in vitro drug release profile of β-CD-FLZ nanosuspensions was performed using the Franz diffusion cell. The in-vitro drug release profile of the optimized β-CD-FLZ nanosuspensions was assessed through triplicate analysis in order to minimise the errors. A dialysis membrane with molecular weight cut off range from 12–14 kDa, pre-soaked overnight in phosphate-buffered saline (PBS) of pH 7.4, was placed on Franz diffusion cell (Gupta H et al., 2011 ). The acceptor compartment was filled with Simulated Tear Fluid (STF, pH 7.4) as the dissolution medium and maintained at a constant stirring rate of 100 rpm. About 1 mL suspension of β-CD-FLZ nanosuspension was introduced into the donor compartment and sealed with parafilm to prevent evaporation. Drug release was monitored at predetermined time intervals (0.5, 1.0, 2.0, 3.0, 6.0,12.0, 24.0 hours). At each interval, 1 mL of the release medium was withdrawn and replaced with an equal volume of prewarmed STF. The collected samples were diluted appropriately and analysed with UV Spectroscopy (Friedrich I et al., 2005 ). The same process was repeated for plain FLZ-nanosuspensions to compare the release pattern with prepared β-CD-FLZ nanosuspensions. Cell line study Human corneal epithelial cells (HCET) were cultured in appropriate growth medium Hams F-12 nutrient medium with 10% fetal bovine serum (FBS) in a humidified incubator at 37°C and 5% carbon dioxide. The cells were seeded at a density of 1 × 10⁴ cells per well in 6-well plates and allowed to grow until they reached 70–80% confluence (Yang S et al., 2016 ). Once the HCET cells were ready for treatment, they were exposed to prepared β-CD-FLZ nanosuspensions of different concentrations of 10, 50, and 100 µg/mL and examined after 24 and 48 hours. A control group with no nanosuspension treatment was also included (Friedrich I et al., 2005 ). Statistical analysis All experiments were performed in triplicate in order to minimise the errors, and the results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). A p-value of < 0.05 was considered as statistically significant. Results Optimization of prepared β-CD-FLZ nanosuspensions Different formulations of FLZ nanosuspensions were prepared, and the F5 formulation was selected based on its zeta particle size. Table 1 represents the optimization of FLZ nanosuspensions Formulation Code FLZ (mg) HPMC (%W/V) Stirring Speed (rpm) Zeta Size (nm) F1 100 0.5 500 547.45 ± 23.56 F2 100 1 500 632.35 ± 13.23 F3 100 2 500 589.54 ± 21.94 F4 100 0.5 1000 345.89 ± 21.56 F5 100 1 1000 184.52 ± 22.65 F6 100 2 1000 268.24 ± 19.51 F5 formulation was selected further for coating with β-CD which was further optimised for β-CD concentration. F5 (B) was selected because of its ideal particle size for ocular use. Table 2 represents optimization of β-CD concentration Formulation Code FLZ (mg) HPMC (%W/V) Stirring Speed (rpm) β-CD concentration (mg) Zeta Size (nm) F5 (A) 100 1 1000 100 mg 217.52 ± 13.65 F5 (B) 100 1 1000 200 mg 275.39 ± 20.24 F5 (C) 100 1 1000 300 mg 417.36 ± 38.62 Particle Size and Zeta Potential Analysis The average size of the plain fluconazole nanosuspensions was found to be approximately 192 ± 15 nm. After coating with β-CD, the particle size of prepared β-CD-FLZ nanosuspension was found to be slightly increased to 275.39 ± 20.24 nm. This increase is likely due to the coating of FLZ nanoparticles with β-CD, which is considered to form a thin shell around drug particles. The prepared β-CD-FLZ nanosuspensions was found to be stable as indicated by its zeta potential, which was found to be -7 ± 3 mV. This negative value suggests good colloidal stability of prepared β-CD-FLZ nanosuspensions due to electrostatic repulsion between the particles, which helps to prevent aggregation and ensures the long-term stability. The particle size and zeta potential of one optimised formulation is shown in Fig. 1 and Fig. 2 respectively. TEM Study The morphology of prepared β-CD-FLZ nanosuspensions was analysed by using TEM study. The TEM images revealed that the drug particles in β-CD-FLZ nanosuspensions were uniformly dispersed, with spherical shapes and smooth surfaces. The smooth surface of FLZ particles after β-CD coating indicates that the FLZ particles were successfully encapsulated in the cyclodextrin cavity, which could enhance the stability and solubility of FLZ. Figure 3 gives TEM images of prepared β-CD-FLZ nanosuspensions. Drug encapsulation efficiency The encapsulation efficiency (EE) of FLZ in prepared β-CD-FLZ nanosuspensions was determined by UV spectrophotometric analysis. The encapsulation efficiency of the plain fluconazole nanosuspensions was found to be 67.23 ± 6.42%. However, after coating with β-CD, the encapsulation efficiency of final formulation was found to be slightly increased to 80.24 ± 7.65%. This indicates that β-CD not only stabilizes the drug nanoparticles but also aids in better drug loading by forming inclusion complexes with FLZ. In Vitro drug release studies The in vitro release profile of prepared β-CD-FLZ nanosuspensions was determined using the Franz diffusion cell. The release data showed that β-CD-FLZ nanosuspensions exhibited a sustained release of fluconazole over a 24-hour period, with approximately 75.37 ± 7.65% of the drug release at end of study. On the other hand, the plain FLZ nanosuspensions exhibited faster release, with about 83.24 ± 5.31% of drug release within 10 hours. The sustained release of the β-CD coated formulation is attributed to the formation of inclusion complexes between fluconazole and β-CD, which slows down the diffusion of the drug from the nanoparticles. Figure 4 represents the comparison of drug release profile in plain FLZ nanosuspensions and β-CD-FLZ nanosuspensions. Cell line study Cell line study was conducted to evaluate the biocompatibility of prepared β-CD-FLZ nanosuspensions. The results indicated that the prepared β-CD-FLZ nanosuspensions do not cause any significant damage to ocular cells when used in concentrations of 10 and 50 µg/ml supporting their potential for safe use in ocular drug delivery. High concentration results in slight expansion of ocular cells diameter probably because of intake of nanosuspensions in cells. Figure 5 shows the HCET cells morphological structure after 12 and 24 hours of using β-CD-FLZ nanosuspensions at different concentrations. The cell lines images showed that after treatment with different concentrations of prepared β-CD-FLZ nanosuspensions, the cells do not show any significant changes in their morphology, when visually examined under a light microscope. Any morphological alterations are indicative of cytotoxicity or stress in cells, such as cell shrinkage, membrane blebbing, detachment from the surface, or irregular shape. Discussion The successful preparation of β-CD-FLZ nanosuspensions demonstrates a promising approach to enhance the ocular delivery of FLZ-like drugs, which have limited aqueous solubility and ocular bioavailability. The results obtained from the physicochemical characterization and in vitro studies indicate that the β-CD coating significantly improved the properties of FLZ. The particle size of the β-CD coated nanosuspensions was found to be in the ideal range for ocular delivery. Nanosuspensions with particle sizes in the range of 200–300 nm desirable because this particle size is considered to be small enough to penetrate the corneal barrier and large enough to avoid rapid elimination of drugs via ocular drainage (Gaafar PM et al., 2014 ; Maged A et al., 2016 ). The enhanced drug encapsulation efficiency β-CD-FLZ Nanosuspensions indicates that β-CD played an important role in stabilizing the FLZ and preventing its loss during the preparation process. The improved solubility of FLZ due to β-CD coating is in line with previously conducted studies [12], which confirmed that β-CD can significantly increase the solubility of hydrophobic drugs by forming inclusion complexes. The sustained drug release observed with the β-CD coating of FLZ as compared to plain FLZ nanosuspensions. The faster release of the plain FLZ nanosuspensions could result in short-lived drug concentrations, limiting its effectiveness in the treatment of chronic ocular infections. However, the prolonged release of β-CD-FLZ nanosuspensions gives additional benefits in the treatment of chronic infections. The HECT cell line studies confirmed the prepared β-CD-FLZ nanosuspensions to be safe for corneal cells with minimal cytotoxicity effects at different concentrations. Conclusion In summary, the prepared β-CD-FLZ nanosuspensions demonstrated significant improvements in the solubility, stability, drug release profile, and ocular permeability of FLZ. The sustained release of the drug, enhanced corneal penetration, prolonged ocular retention, and enhanced biocompatibility make the prepared β-CD-FLZ nanosuspensions a promising formulation for ocular drug delivery. These findings highlight the potential of β-CD-FLZ nanosuspensions as an effective strategy for improving the treatment of ocular fungal infections. Declarations Ethical Statement Not applicable Competing Interests The authors declare that they have no conflict of interest. Funding statement Not applicable Author Contribution Malkiet Kaur and Paramjot Maman performed the experimental work and contributed to writing the manuscript. Manju Napgal designed the study work. Acknowledgements Authors are thankful to Chitkara College of Pharmacy, Chitkara University for providing necessary facilities to carry out the research work. References Abdelbary, A. A., Li, X., El-Nabarawi, M., Elassasy, A., & Jasti, B. (2013). Effect of fixed aqueous layer thickness of polymeric stabilizers on zeta potential and stability of aripiprazole nanosuspensions. Pharmaceutical Development And Technology , 18 (3), 730–735. 10.3109/10837450.2012.727001 Ahmed, S., Amin, M. M., & Sayed, S. (2023). 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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-7601516","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":523883954,"identity":"f2922adb-5cc7-4dfd-ac2c-72bcfefdef92","order_by":0,"name":"Malkiet Kaur","email":"","orcid":"","institution":"Maharishi Markandeshwar University, Mullana","correspondingAuthor":false,"prefix":"","firstName":"Malkiet","middleName":"","lastName":"Kaur","suffix":""},{"id":523883955,"identity":"2bda447b-1d0b-45b5-87d5-430b6c789aa7","order_by":1,"name":"Paramjot 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1","display":"","copyAsset":false,"role":"figure","size":44861,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZeta potential of final optimised β-CD-FLZ nanosuspension\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/cf37a878fc18766b6cd1b863.jpg"},{"id":92732370,"identity":"efb2b481-8f6e-4f18-8f9b-8384ad3aa2e3","added_by":"auto","created_at":"2025-10-03 16:06:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81165,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eParticle size of final optimised β-CD-FLZ nanosuspension\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/1746998750288a6b1ba9f482.jpg"},{"id":92732366,"identity":"73eee178-8c7f-45ba-8b9f-c3fefaeba46b","added_by":"auto","created_at":"2025-10-03 16:06:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":71793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTEM image of prepared of β-CD-FLZ nanosuspension\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/bb8bedc3ffc6be9e8dac9a72.jpg"},{"id":92732952,"identity":"6db66c9b-f461-4cf9-8409-72dd9503822c","added_by":"auto","created_at":"2025-10-03 16:14:07","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":76230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e drug release profile of plain FLZ nanosuspensions and β-CD-FLZ nanosuspensions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/468aa4568cc45867368625f2.jpg"},{"id":92732368,"identity":"bbb1d16d-46bb-4033-81a0-cfd687919152","added_by":"auto","created_at":"2025-10-03 16:06:07","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46406,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShape of HECT cells after exposing with β-CD-FLZ nanosuspensions\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/47c7116abf80ae7cda9323eb.jpg"},{"id":93380879,"identity":"825cc3fd-b0f1-4a69-9ef8-260a30dfe347","added_by":"auto","created_at":"2025-10-13 08:47:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1269425,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7601516/v1/07492ac8-e2ea-4f45-9598-54bbc1c19959.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"β-Cyclodextrin-Coated Fluconazole Nanosuspensions: Formulation and Evaluation for Enhanced Ocular Delivery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDespite of multiple researches in ophthalmic, Ocular drug delivery systems still face several challenges because of unique anatomy and physiological characteristics of eye. Human eye has several protective layers/barriers, such as the cornea, conjunctiva, and tear film, which significantly reduce the penetration and retention of administered formulations (Ahmed S et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, most of conventional dosage forms like eye drops, ointments etc, have reduced precorneal residence time which further reduces the bioavailability and therapeutic efficacy of administered formulations (Liu LC et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Other than this, many ophthalmic drugs such as fluconazole, face limitations in ocular treatment due to poor aqueous solubility, which further reduces the ability of drug to achieve therapeutic concentrations at the site of administration (Tanzawa A et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, there is a critical requirement for innovative drug delivery strategies which have the ability to enhance the solubility, stability, and bioavailability of such ocular drugs (Volkova TV et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFluconazole is one of the widely used antifungal agents which is found effective in treatment of various ophthalmic fungal infections such as keratitis and endophthalmitis (Yosrey E et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). While effective, fluconazole suffers from poor aqueous solubility, rapid drainage, short ocular retention time, and poor penetration at corneal barrier results in suboptimal concentration of drug at administered site when given in form of eye drops and eye ointments. To overcome these limitations associated with fluconazole conventional formulations, the development of novel drug delivery systems (NDDS) has been an area of currently active researches (Nagai N \u003cem\u003eet al\u003c/em\u003e., 2022). Among the various NDDS approaches, nanosuspensions have emerged as a promising strategy for improving the delivery of poorly soluble drugs. Nanosuspensions are colloidal dispersions of submicron-sized drug particles that provide an increased surface area, leading to enhanced dissolution rates, solubility of drug (khan S et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). By reducing the particle size of poorly soluble drugs like Fluconazole, to a nano-meter scale, nanosuspensions can significantly improve the rate of drug absorption, resulting in enhanced ocular bioavailability. While nanosuspensions offer numerous advantages, however, the stability of the nanosuspension, sedimentation and aggregation of nano-sized dispersion still remain critical concerns (Fathi-Karkan S et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). One strategy to enhance the stability and ocular performance of nanosuspensions is the use of polymeric coatings. Coating materials can help stabilize the nanosuspensions with its improved interactions with ocular tissues, and modify their drug release profile. Among the various coating materials, β-cyclodextrin (β-CD) has gained significant attention because of its ability to produce inclusion complexes with poorly soluble drugs (Wang J \u003cem\u003eet al\u003c/em\u003e., 2020). β-CD is a cyclic oligosaccharide which contains about seven glucose units. It has the unique ability to encapsulate hydrophobic drug molecules within its hydrophobic cavity. This characteristic makes β-CD as a promising candidate for improving the solubility of lipophilic drugs like fluconazole (Khalid FM et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Coating of fluconazole nanosuspensions with β-CD provides multiple advantages (Sadeh P et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) such as; 1) Providing stability to nanosuspensions by preventing the particle aggregation 2) Improves the bioavailability of drug by promoting better interaction with ocular tissues 3) enhanced mucoadhesive properties resulting in prolonged drug retention on the ocular surface with better residence time and therapeutic effectiveness of drug 4) Improvement of drug's release profile, allowing for a sustained release of fluconazole over time, which is essential for treating chronic ocular infections. The current study aims to prepare and evaluate β-CD-coated fluconazole nanosuspensions for ocular delivery. The primary objective of the study is to enhance the solubility and ocular bioavailability of fluconazole, improve the stability, and drug release profile. The outcome of this research could provide a promising strategy for the improvement of the therapeutic management of ocular fungal infections. The study results will offer more effective and sustained treatment options for ocular fungal infections as compared to traditional marketed formulations like eye drops and ointments.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eFluconazole (purity\u0026thinsp;\u0026ge;\u0026thinsp;98%) was purchased from Sigma-Aldrich (India). β-Cyclodextrin (β-CD) was obtained from Loba Chemie Pvt. Ltd. (India). The solvents used in the formulation process, including water, methanol, and acetone, were of analytical grade and were supplied by Loba Chemie Pvt. Ltd (India). Polyvinyl alcohol (PVA) was used as a stabilizer and was purchased from Sigma-Aldrich. All other chemicals and reagents used were of analytical grade unless specified otherwise.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMethods\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of Fluconazole Nanosuspensions\u003c/h2\u003e\u003cp\u003eFluconazole (FLZ) nanosuspensions were prepared by using solvent evaporation method. A precise amount of FLZ (100 mg) was dissolved in about6 mL of organic volatile solvent, dichloromethane (DCM). The prepared FLZ solution was added dropwise using a syringe into 20 mL of distilled water containing hydroxypropyl ethyl cellulose (HPMC) as stabiliser in different concentrations (0.5%, 1%, and 5% w/v) with uniform magnetic stirring of 1000 rpm. These stabilizers prevent particle aggregation and ensure stability of the nanosuspension (Aldosari BN et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The stirring was continued for 6 hours to ensure complete evaporation of dichloromethane. This step resulted in supersaturation of FLZ in aqueous medium, which result in the formation of nanosized FLZ particles through nucleation and crystallization processes. Continuous magnetic stirring ensured uniform distribution of FLZ and stabilizer which result in formation of stable nanosuspension. The prepared FLZ nanosuspension were stored at 4\u0026deg;C to prevent its degradation and maintain its integrity by minimising Ostwald ripening and particle aggregation (Wang P et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Please refer Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for different prepared formulations and optimization of FLZ- nanosuspensions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eβ-Cyclodextrin Coating of Fluconazole Nanosuspensions\u003c/h3\u003e\n\u003cp\u003eAfter the preparation of fluconazole nanosuspensions, a predetermined amount of β-CD (200 mg) was dissolved in deionized water and added dropwise to the nanosuspension while stirring continuously at 500 rpm. The mixture was kept under magnetic stirring for additional 4 hours to allow the formation of β-CD-FLZ inclusion complexes on the surface of the nanoparticles (Pawar P et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The prepared β-CD-FLZ nanosuspension was subjected to high-frequency ultrasound treatment of 20 kHz for 10 minutes to break up any aggregates further and get more uniform size distribution. The final prepared β-CD-FLZ nanosuspension was stored in air tight glass ambient container (Aldosari BN et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Please refer Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e for different optimization of β-CD-FLZ-nanosuspensions.\u003c/p\u003e\n\u003ch3\u003eCharacterization of β-CD-FLZ Nanosuspensions\u003c/h3\u003e\n\u003cp\u003eAfter the preparation of β-CD coated fluconazole nanosuspensions, several characterization techniques were employed to evaluate stability and suitability for ocular delivery.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eParticle size and zeta potential analysis\u003c/h2\u003e\u003cp\u003eThe particle size and zeta potential of prepared β-CD-FLZ nanosuspensions were measured using Delsa Nano C zeta sizer (Beckman. Coulter Pvt. Ltd.). Zeta potential is a measure of the surface charge on nanosized drug particles and is critical for determining the stability of the formulation. High zeta potential values generally have better stability due to high electrostatic repulsion, which prevents aggregation in nanosuspension (Abdelbary AA et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTransmission electron microscopy (TEM) Study\u003c/h3\u003e\n\u003cp\u003eThe morphology of prepared β-CD-FLZ nanosuspensions was examined using transmission electron microscopy (TEM). Transmission electron microscopy (TEM) was employed to assess the morphological characteristics of β-CD-FLZ nanosuspensions. A small drop of prepared β-CD-FLZ nanosuspensions was placed onto carbon-coated copper grids, which was then examined using TEM microscopy (200kV Tecnai\u0026trade; T20, Mohali, India). Images were captured and analysed using dedicated imaging software to evaluate morphology of β-CD-FLZ nanosuspensions (Pawar P et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDrug encapsulation efficiency\u003c/h3\u003e\n\u003cp\u003eThe drug encapsulation efficiency (EE) was determined by measuring the amount of FLZ incorporated into the prepared β-CD-FLZ nanosuspensions. About 5 mL of prepared formulation was centrifuged at 10,000 rpm for 30 minutes to separate the free drug from the nanosuspension.\u003csup\u003e16\u003c/sup\u003e The supernatant was collected, and the drug concentration was determined using UV spectrophotometry at λ\u003csub\u003emax\u003c/sub\u003eof 260 nm. The encapsulation efficiency was calculated using the formula:\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEE (%) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{Amount\\:of\\:drug\\:encapsulated}{Total\\:amount\\:of\\:drug\\:added}\\)\u003c/span\u003e\u003c/span\u003e X 100\u003c/h2\u003e\u003cp\u003e\u003cb\u003eIn Vitro\u003c/b\u003e \u003cb\u003ed\u003c/b\u003e\u003cb\u003erug release studies\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe in vitro drug release profile of β-CD-FLZ nanosuspensions was performed using the Franz diffusion cell. The in-vitro drug release profile of the optimized β-CD-FLZ nanosuspensions was assessed through triplicate analysis in order to minimise the errors. A dialysis membrane with molecular weight cut off range from 12\u0026ndash;14 kDa, pre-soaked overnight in phosphate-buffered saline (PBS) of pH 7.4, was placed on Franz diffusion cell (Gupta H et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The acceptor compartment was filled with Simulated Tear Fluid (STF, pH 7.4) as the dissolution medium and maintained at a constant stirring rate of 100 rpm. About 1 mL suspension of β-CD-FLZ nanosuspension was introduced into the donor compartment and sealed with parafilm to prevent evaporation. Drug release was monitored at predetermined time intervals (0.5, 1.0, 2.0, 3.0, 6.0,12.0, 24.0 hours). At each interval, 1 mL of the release medium was withdrawn and replaced with an equal volume of prewarmed STF. The collected samples were diluted appropriately and analysed with UV Spectroscopy (Friedrich I et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The same process was repeated for plain FLZ-nanosuspensions to compare the release pattern with prepared β-CD-FLZ nanosuspensions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell line study\u003c/h2\u003e\u003cp\u003eHuman corneal epithelial cells (HCET) were cultured in appropriate growth medium Hams F-12 nutrient medium with 10% fetal bovine serum (FBS) in a humidified incubator at 37\u0026deg;C and 5% carbon dioxide. The cells were seeded at a density of 1 \u0026times; 10⁴ cells per well in 6-well plates and allowed to grow until they reached 70\u0026ndash;80% confluence (Yang S et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Once the HCET cells were ready for treatment, they were exposed to prepared β-CD-FLZ nanosuspensions of different concentrations of 10, 50, and 100 \u0026micro;g/mL and examined after 24 and 48 hours. A control group with no nanosuspension treatment was also included (Friedrich I et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll experiments were performed in triplicate in order to minimise the errors, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). A p-value of \u0026lt;\u0026thinsp;0.05 was considered as statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eOptimization of prepared β-CD-FLZ nanosuspensions\u003c/h2\u003e\u003cp\u003eDifferent formulations of FLZ nanosuspensions were prepared, and the F5 formulation was selected based on its zeta particle size.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003erepresents the optimization of FLZ nanosuspensions\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFormulation Code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFLZ (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHPMC (%W/V)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStirring Speed (rpm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZeta Size (nm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e547.45\u0026thinsp;\u0026plusmn;\u0026thinsp;23.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e632.35\u0026thinsp;\u0026plusmn;\u0026thinsp;13.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e500\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e589.54\u0026thinsp;\u0026plusmn;\u0026thinsp;21.94\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e345.89\u0026thinsp;\u0026plusmn;\u0026thinsp;21.56\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e184.52\u0026thinsp;\u0026plusmn;\u0026thinsp;22.65\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e\u003cp\u003e268.24\u0026thinsp;\u0026plusmn;\u0026thinsp;19.51\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eF5 formulation was selected further for coating with β-CD which was further optimised for β-CD concentration. F5 (B) was selected because of its ideal particle size for ocular use.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003erepresents optimization of β-CD concentration\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFormulation Code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFLZ (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHPMC (%W/V)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStirring Speed (rpm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eβ-CD concentration (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZeta Size (nm)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF5 (A)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e217.52\u0026thinsp;\u0026plusmn;\u0026thinsp;13.65\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF5 (B)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e100\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e200 mg\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e275.39\u0026thinsp;\u0026plusmn;\u0026thinsp;20.24\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF5 (C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e100\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e300 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e\u003cp\u003e417.36\u0026thinsp;\u0026plusmn;\u0026thinsp;38.62\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eParticle Size and Zeta Potential Analysis\u003c/h2\u003e\u003cp\u003eThe average size of the plain fluconazole nanosuspensions was found to be approximately 192\u0026thinsp;\u0026plusmn;\u0026thinsp;15 nm. After coating with β-CD, the particle size of prepared β-CD-FLZ nanosuspension was found to be slightly increased to 275.39\u0026thinsp;\u0026plusmn;\u0026thinsp;20.24 nm. This increase is likely due to the coating of FLZ nanoparticles with β-CD, which is considered to form a thin shell around drug particles. The prepared β-CD-FLZ nanosuspensions was found to be stable as indicated by its zeta potential, which was found to be -7\u0026thinsp;\u0026plusmn;\u0026thinsp;3 mV. This negative value suggests good colloidal stability of prepared β-CD-FLZ nanosuspensions due to electrostatic repulsion between the particles, which helps to prevent aggregation and ensures the long-term stability. The particle size and zeta potential of one optimised formulation is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eTEM Study\u003c/h2\u003e\u003cp\u003eThe morphology of prepared β-CD-FLZ nanosuspensions was analysed by using TEM study. The TEM images revealed that the drug particles in β-CD-FLZ nanosuspensions were uniformly dispersed, with spherical shapes and smooth surfaces. The smooth surface of FLZ particles after β-CD coating indicates that the FLZ particles were successfully encapsulated in the cyclodextrin cavity, which could enhance the stability and solubility of FLZ. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e gives TEM images of prepared β-CD-FLZ nanosuspensions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eDrug encapsulation efficiency\u003c/h2\u003e\u003cp\u003eThe encapsulation efficiency (EE) of FLZ in prepared β-CD-FLZ nanosuspensions was determined by UV spectrophotometric analysis. The encapsulation efficiency of the plain fluconazole nanosuspensions was found to be 67.23\u0026thinsp;\u0026plusmn;\u0026thinsp;6.42%. However, after coating with β-CD, the encapsulation efficiency of final formulation was found to be slightly increased to 80.24\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65%. This indicates that β-CD not only stabilizes the drug nanoparticles but also aids in better drug loading by forming inclusion complexes with FLZ.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIn Vitro drug release studies\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e release profile of prepared β-CD-FLZ nanosuspensions was determined using the Franz diffusion cell. The release data showed that β-CD-FLZ nanosuspensions exhibited a sustained release of fluconazole over a 24-hour period, with approximately 75.37\u0026thinsp;\u0026plusmn;\u0026thinsp;7.65% of the drug release at end of study. On the other hand, the plain FLZ nanosuspensions exhibited faster release, with about 83.24\u0026thinsp;\u0026plusmn;\u0026thinsp;5.31% of drug release within 10 hours. The sustained release of the β-CD coated formulation is attributed to the formation of inclusion complexes between fluconazole and β-CD, which slows down the diffusion of the drug from the nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e represents the comparison of drug release profile in plain FLZ nanosuspensions and β-CD-FLZ nanosuspensions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eCell line study\u003c/h2\u003e\u003cp\u003eCell line study was conducted to evaluate the biocompatibility of prepared β-CD-FLZ nanosuspensions. The results indicated that the prepared β-CD-FLZ nanosuspensions do not cause any significant damage to ocular cells when used in concentrations of 10 and 50 \u0026micro;g/ml supporting their potential for safe use in ocular drug delivery. High concentration results in slight expansion of ocular cells diameter probably because of intake of nanosuspensions in cells. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the HCET cells morphological structure after 12 and 24 hours of using β-CD-FLZ nanosuspensions at different concentrations. The cell lines images showed that after treatment with different concentrations of prepared β-CD-FLZ nanosuspensions, the cells do not show any significant changes in their morphology, when visually examined under a light microscope. Any morphological alterations are indicative of cytotoxicity or stress in cells, such as cell shrinkage, membrane blebbing, detachment from the surface, or irregular shape.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe successful preparation of β-CD-FLZ nanosuspensions demonstrates a promising approach to enhance the ocular delivery of FLZ-like drugs, which have limited aqueous solubility and ocular bioavailability. The results obtained from the physicochemical characterization and \u003cem\u003ein vitro\u003c/em\u003e studies indicate that the β-CD coating significantly improved the properties of FLZ. The particle size of the β-CD coated nanosuspensions was found to be in the ideal range for ocular delivery. Nanosuspensions with particle sizes in the range of 200\u0026ndash;300 nm desirable because this particle size is considered to be small enough to penetrate the corneal barrier and large enough to avoid rapid elimination of drugs via ocular drainage (Gaafar PM et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Maged A et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The enhanced drug encapsulation efficiency β-CD-FLZ Nanosuspensions indicates that β-CD played an important role in stabilizing the FLZ and preventing its loss during the preparation process. The improved solubility of FLZ due to β-CD coating is in line with previously conducted studies [12], which confirmed that β-CD can significantly increase the solubility of hydrophobic drugs by forming inclusion complexes. The sustained drug release observed with the β-CD coating of FLZ as compared to plain FLZ nanosuspensions. The faster release of the plain FLZ nanosuspensions could result in short-lived drug concentrations, limiting its effectiveness in the treatment of chronic ocular infections. However, the prolonged release of β-CD-FLZ nanosuspensions gives additional benefits in the treatment of chronic infections. The HECT cell line studies confirmed the prepared β-CD-FLZ nanosuspensions to be safe for corneal cells with minimal cytotoxicity effects at different concentrations.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, the prepared β-CD-FLZ nanosuspensions demonstrated significant improvements in the solubility, stability, drug release profile, and ocular permeability of FLZ. The sustained release of the drug, enhanced corneal penetration, prolonged ocular retention, and enhanced biocompatibility make the prepared β-CD-FLZ nanosuspensions a promising formulation for ocular drug delivery. These findings highlight the potential of β-CD-FLZ nanosuspensions as an effective strategy for improving the treatment of ocular fungal infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthical Statement\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding statement\u003c/h2\u003e\u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMalkiet Kaur and Paramjot Maman performed the experimental work and contributed to writing the manuscript. Manju Napgal designed the study work.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eAuthors are thankful to Chitkara College of Pharmacy, Chitkara University for providing necessary facilities to carry out the research work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbdelbary, A. A., Li, X., El-Nabarawi, M., Elassasy, A., \u0026amp; Jasti, B. (2013). 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A., \u0026amp; Metwally, M. E. (2022). Implementation of HILIC-UV technique for the determination of moxifloxacin and fluconazole in raw materials and pharmaceutical eye gel. \u003cem\u003eScientific Reports\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e(1), 13388. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-022-17064-8\u003c/span\u003e\u003cspan address=\"10.1038/s41598-022-17064-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"β-cyclodextrin, nanosuspension, ocular delivery, antifungal, sustained release","lastPublishedDoi":"10.21203/rs.3.rs-7601516/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7601516/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Fluconazole, a potent antifungal agent, faces challenges in ocular therapy due to its poor aqueous solubility and limited bioavailability. Nanosuspensions formulations coated with β-cyclodextrin (β-CD) have the potential to enhance the delivery and therapeutic efficacy of such poorly soluble drugs. The study aimed to developβ-CD coated fluconazole nanosuspensions and evaluate their physicochemical properties, cytotoxicity and potential for improved ocular drug delivery.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Fluconazole nanosuspensions were prepared using solvent evaporation method. The formulations were characterised for particle size, zeta potential, drug encapsulation efficiency and \u003cem\u003ein vitro\u003c/em\u003e drug release profile. Cytotoxicity studies were performed using human corneal cells (HCET).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: The results demonstrated that β-CD coating significantly improved the drug solubility and stability of drug. The particle size of coated nanosuspensions was found to be 275.39± 20.24 nm. \u003cem\u003eIn vitro\u003c/em\u003e evaluations showed prolonged drug release (83.24 ± 5.31%) at ocular surface. Additionally, the prepared nanosuspensions showed minimal cytotoxic effects which indicate the good biocompatibility of the prepared formulation for ocular use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: The findings suggest thatβ-CD coated fluconazole nanosuspensions are a promising candidate for enhancing the therapeutic efficacy of fluconazole in ocular infections and can be used as an alternative to conventional dosage forms.\u003c/p\u003e","manuscriptTitle":"β-Cyclodextrin-Coated Fluconazole Nanosuspensions: Formulation and Evaluation for Enhanced Ocular Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-03 16:06:02","doi":"10.21203/rs.3.rs-7601516/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2f1ae21-e4f0-481a-8342-f0e403151c36","owner":[],"postedDate":"October 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T08:39:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-03 16:06:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7601516","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7601516","identity":"rs-7601516","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}