INTRODUCTION:

Infectious diseases caused by fungi occur in Indonesia. The prevalence of the disease reaches 52%¹. There are four main chemical groups of antifungal drugs: imidazole, echinocandins, polyene, and flucytosin. The imidazole group is the most widely used group of antifungals because it has a broad spectrum of activity and exemplary safety. A common treatment for fungal infections is ketoconazole, an antifungal medication belonging to the imidazole group^1,2.

Ketoconazole is lipophilic, with a Log P value of 3.1 and very low solubility in water (0.05 mg/L at 25⁰C). It is included in the BCS class II group, which has low solubility and high permeability, so its bioavailability is low. The properties of ketoconazole cause the substance to not penetrate the skin, so the drug cannot work effectively because it cannot reach the target treatment^3,4,5.

Methods that can be done to improve the properties of ketoconazole to increase its bioavailability, make water- soluble complexes, use surfactant systems, nanoparticles, solid dispersion formulations, and the formation of vesicles (liposomes, niosomes, transfersomes and etosomes)^6,7,8,9. Niosomes are one of the methods that can be used to increase the bioavailability of drugs because they can improvement the solubility and penetrability of drugs. Niosomes are vesicle systems with structures such as unilameral, multilamellar and polyhedral spheres. Niosom has several advantages, including better stability, which makes it less likely to form aggregates and reduce leakage from the drug. The main components of niosomes are surfactants, membrane stabilizers and hydration mediums. Niosomes are made using non-ionic surfactants, which have the advantages of being non-toxic, safe, and free- flowing. They have good solubility in water, making it easy to hydrate^10,11,12.

The proper selection of surfactants can affect the drug's entrapment efficiency and the size of the drug vesicles. This significantly affects the HLB value and the surfactant's chemical properties. Surfactants with an HLB value ranging from 14 to 17 are unsuitable for liposomal, but surfactants with an HLB value of 8.6 can produce niosomes with good entrapment efficiency. The HLB value of surfactants that can form niosome vesicles ranges from 4 to 8^13,14.

Alkyl ether, alkyl esters, alkyl amides, and sucrose fatty acid esters are examples of common non-ionic surfactants¹⁵. One common surfactant that used in the production of niosomes is Span 60. Span 60 can increase the penetration of the fluconazole niosome¹⁶. Span 60 also increases the dissolution ability of the niosome of the cyclosporin A¹⁷. The fatty acid sucrose ester is also a non-ionic surfactant that can be used to manufacture niosomes. It has a sugar substituent, sucrose, as the polar head group and fatty acid as the non-polar group. One type of sucrose ester fatty acid is SEP which has an HLB value of 16¹⁸. SEP has advantages, including lowering the surface tension of oil and water, being non-toxic, and being biodegradable¹⁹. Previous research found that formulas with a total HLB of 7-7.5 had high entrapment efficiency²⁰.

Evaluation of the niosome system that needs to be carried out to ensure the quality of the niosome system formed is the size of the vesicles, polydispersity index, zeta potential, morphological analysis, Entrapment efficiency and drug release test. In addition, a stability test also needs to be done to see if the drug trapped in the vesicles can last for a long time. The purpose of stability testing is to determine the influence of various environmental factors such as temperature, humidity, and light on the quality of a drug substance or drug product over time and to establish a retest period for the drug ingredient or the shelf life of the drug product and the recommended storage conditions²¹.

Based on the description above, it is necessary to research the manufacture of Ketoconazole niosomes using a combination of S-60 and SEP as a surfactant so that an optimal and physically stable niosome system is obtained to ensure the quality of the niosomes and the ability of the drug to be absorbed in the niosomes for a long time, as well as increasing the solubility and penetration of the drug. It is hoped that increasing the solubility and penetration of the drug will increase the effectiveness of ketoconazole as an antifungal so that the treatment of diseases caused by fungi using ketoconazole can cause optimal treatment effects and reduce side effects.

MATERIALS AND METHODS:

Materials:

Ketoconazole (Aarti Drugs Limited), S-60 (Nitro Chemical), Sucrose Ester Palmitat (Compass Food), Cholesterol (Nippon Fine Chemical), Chloroform (Merck), Methanol (Merck), Phosphate Buffer Saline (PBS) (Solarbio), Aqua pi (Ikapharmindo Putramas), dialysis bag.

Tool:

Uv-Vis Spectrophotometer (Shimadzu), Rotary Evaporator, Fourier Transform Infra-Red (FTIR) (Agilent), Particle Size Analyzer (PSA) (Beckman Coulter), Climatic Chamber, magnetic stirrer and glassware.

Methods:

Ketoconazole Niosomal Formulation:

The composition of the niosome is a surfactant, membrane stabilizer and carrier. Previous research has been carried out to optimize the formula with a ratio of surfactant and membrane stabilizer of 1:0.2 with various variations in the combination comparison between S-60 and SEP HLB value of 7-7.5 are produced as the optimal formula because had high entrapment efficiency(20). The membrane stabilizer used is cholesterol, which serves to maintain the permeability of niosomes and make the size of niosomes vesicles larger, thereby increasing drug absorption in niosomes (22).

Method of Preparation Ketoconazole Niosomes:

The procedure begins with the addition of Ketoconazole, S-60, SEP, and cholesterol to 10mL of chloroform. After that, the mixture is transferred into a 100mL round pumpkin. The mixture is dried using a rotary evaporator at a temperature of 55-65ºC until it is completely dry. The film layer is hydrated using 20mL of phosphate pH

7.4 for 1hour on a rotary evaporator at a temperature of 55-65ºC. A niosome is formed and sonicated for 20 minutes (23).

Niosomes Evaluation Niosomal Organoleptic Test:

The organoleptic test carried out includes the physical appearance of the niosome system visually.

Determination of Vesicle Size, Polydispersity Index and Zeta Potential:

To evaluate the vesicle size, polydispersity index, and zeta potential, the sample was diluted with aqua distillate in a 1:10 ratio using the Particle Size Analyzer tool at the University of Muhammadiyah, Prof. DR. HAMKA(24,25). After that, the sample was placed into the cell to be read and examined. The test was carried out.

Functional Group Analysis using FTIR:

All components contained in niosomes and their functional groups were evaluated using FTIR. Testing using FTIR aims to see the interaction between the niosomes. The sample is placed on the sample tray, the FTIR machine is turned on, and the results obtained are analyzed^23,26.

Entrapment Efficiency (EE) Testing:

Niosome suspension 0.65mL was inserted into the Eppendorf tube and centrifuged at 1300rpm for 1hour. The deposits formed are put into a 10mL measuring flask, and then PBS pH 7.4 (containing 10% v/v methanol) is added. Each sample was screened using a 0.45μm membrane; then, ketoconazole levels were measured using Ultra High-Performance Liquid Chromatography-Ultra Violet (UHPLC-UV) at a wavelength of 205nm. The motion phase used is acetonitrile: water pH 6 with a ratio of 60:40. Flow rate of 1 ml/min using column C18. The per cent Entrapment efficiency is calculated using the formula^23,27:

Amount of drug on niosomes

% EE = ---------------------------------------------- × 100

Amount of drug on preparation

Morphological imaging:

The morphology of niosomes was observed and analysed using Transmission Electron Microscopy (TEM). Observation was carried out by diluting the sample using deionised water with a ratio of 1:1, then 10 μL of the diluted solution was put into the object glass and left to dry, and viewed^28,29.

In Vitro Drug Release Test:

The ketoconazole drug release test in the niosome system was carried out using a dialysis bag. The test was carried out using a paddle according to the method in USP, where the device was set to a speed of 100rpm at a temperature of 37±0.2^oC (imitating human body temperature) in 200mL PBS pH 7.4 (containing 10% v/v methanol) as the dissolved medium. A number of samples (niosomes system) containing 37.5mg of ketoconazole were weighed and put into a dialysis bag and then into a dissolved device. A 5mL sample was drawn at specific intervals, i.e., 0.25, 0.5, 1, 2, 4, 8, 12, and 24hours. The volume of the drawn sample is refilled with PBS pH 7.4. Each sample was then screened using a 0.45μm membrane before being analyzed using UHPLC-UV at a wavelength of 205nm. The mobile phase used is acetonitrile: water pH 6 with a ratio of 60:40. Flow rate of 1mL/min using column C18. The drug release curve is depicted from the graph between concentration vs time^23,27.

Ketoconazole Niossome Stability Test:

A ketoconazole niosome stability test was performed to assess the effect of temperature and humidity on the storage of ketoconazole in the niosomes system. Stability tests are carried out by the International Convention on Harmonization (ICH) guidelines. The optimal ketoconazole niosome system is stored at cold temperatures (4±2⁰C with RH 60±5%), room temperature (25±2⁰C with RH 60±5%) and high temperatures (40±2⁰C with RH 75±5%). Furthermore, evaluation was carried out in the form of organoleptics, vesicle size, polydispersion index, zeta potential, Entrapment efficiency, and in vitro drug release tests at weeks 0, 2, 4, 6, 8, 10 and 12^21,30.

Data Analysis:

The stability test results were analyzed using two-way ANOVA to determine the significant difference (p<0.05) and to see the effect of temperature and humidity on the physical and chemical properties of ketoconazole niosomes.

RESULT:

Organoleptic:

The ketoconazole niosome produced is a white solution. Visual observation results show stability in ketoconazole niosomes formed when stored at temperatures of 4, 25, and 40⁰C for 12 weeks. The physical appearance of niosomes did not change when stored at temperatures of 4, 25 and 40 for 12 weeks.

Table 2. Test results of particle size stability, polydispersity index and zeta potential at 25⁰C

Week To	Particle Size(nm)		Polydispersity Index		Zeta Potential (mV)
Week To	F1	F2	F1	F2	F1	F2
0	429.40±6.30	433.50±6.22	0.571±0.00	0.571±0.00	-62.58±4.99	-35.00±1.98
2	468.23±25.54	431.17±7.25	0.571±0.00	0.571±0.00	-66.46±1.56	-60.69±1.51
4	409.07±10.27	427.23±5.64	0.571±0.00	0.571±0.00	-70.76±1.63	-67.88±1.33
6	394.50±12.71	383.63±11.82	0.571±0.00	0.571±0.00	-58.50±2.96	-65.40±4.29
8	359.73±22.31	347.13±4.57	0.571±0.00	0.571±0.00	-63.55±3.69	-66.45±1.98
10	357.13±1.42	365.73±6.62	0.571±0.00	0.571±0.00	-52.55±3.70	-52.68±5.73
12	348.63±6.15	348.00±2.11	0.571±0.00	0.571±0.00	-52.06±4.32	-51.48±4.96

Table 3. Test results of particle size stability, polydispersity index and zeta potential at 4⁰C

Week To	Particle Size(nm)		Polydispersity Index		Zeta Potential (mV)
Week To	F1	F2	F1	F2	F1	F2
0	429.40±6.30	433.50±6.22	0.571±0.00	0.571±0.00	-62.58±4.99	-35.00±1.98
2	424.07±13.42	403.83±9.61	0.571±0.00	0.571±0.00	-65.14±4.84	-68.55±3.14
4	449.10±9.51	395.10±7.89	0.571±0.00	0.571±0.00	-54.70±4.48	-63.35±3.03
6	376.27±6.62	373.63±5.72	0.571±0.00	0.571±0.00	-57.16±3.95	-64.76±1.21
8	375.30±13.00	380.80±7.50	0.571±0.00	0.571±0.00	-52.85±2.05	-65.92±6.83
10	357.80±5.03	377.87±8.12	0.571±0.00	0.571±0.00	-52.57±3.81	-59.64±5.17
12	358.87±18.28	361.20±11.32	0.571±0.00	0.571±0.00	-58.40±6.33	-60.75±1.76

Table 4. Particle size stability test results, polydispersity index and zeta potential at 40⁰C

Week To	Particle Size(nm)		Polydispersity Index		Zeta Potential (mV)
Week To	F1	F2	F1	F2	F1	F2
0	429.40±6.30	433.50±6.22	0.571±0.00	0.571±0.00	-62.58±4.99	-35.00±1.98
2	340.97±5.57	425.43±3.57	0.571±0.00	0.571±0.00	-70.89±0.49	-75.96±5.04
4	322.20±2.17	415.73±9.09	0.571±0.00	0.571±0.00	-64.03±4.91	-62.14±3.87
6	349.10±7.98	411.07±1.31	0.571±0.00	0.571±0.00	-67.81±3.81	-67.12±7.37
8	321.40±2.31	414.57±3.03	0.571±0.00	0.571±0.00	-70.81±5.67	-63.34±14.49
10	305.57±7.61	404.00±11.98	0.571±0.00	0.571±0.00	-61.10±12.77	-66.16±9.65
12	287.40±7.46	388.63±19.88	0.571±0.00	0.571±0.00	-62.91±7.67	-67.50±1.53

The results of the niosome stability test shown in Table 1-3 show a change in the particle size and zeta potential of the niosome, but these changes are insignificant. The particle size of the niosome in formulas 1 and 2 changed from 429.40 nm and 433.5nm to 358.87nm and 361.20 nm at 12⁰C temperature storage. At storage at 25⁰C, the particle sizes of the formula 1 and 2 niosomes changed to 348.63nm and 348.00nm over 12 weeks. At niosome storage at 40⁰C, the particle size of the formula 1 and 2 niosome changes to 287.40nm and 388.63nm over 12 weeks. The particle size in formulas 1 and 2 changed to smaller from week 0 to week 12. This can be caused by Ostwald ripening events in the niosome, which cause the rupture of the formed globular to become smaller. The results of statistical analysis showed that temperatures of 4 and 25⁰C did not affect particle size but storage at 40ºC affected particle size (31).

The polydispersity index <0,5. The vesicle size homogeneity is shown by the polydispersity index. If the resultant vesicles have a size of 0.000, it means that they are very uniform, and if they have a size of 1.000, they are very non-uniform³². The polydispersivity index of ketoconazole niosomes did not change at 8 weeks of storage at temperatures of 4, 25 and 40⁰C, which was 0.571.

The zeta potential of the niosomes in formulas 1 and 2 changes from -62.58 mV and -35.00mV to -58.40mV and -60.75mV at 12⁰C temperature storage. At storage at 25⁰C, the zeta potentials of the niosome formulas 7 and 8 changed to -52.06mV and -51.48mV over 12 weeks. At niosome storage at 40⁰C, the zeta potentials of niosome formulas 1 and 2 changed to -62.91mV and -67.50mV over 12 weeks. The zeta potential in formulas 1 and 2 changes from week 0 to week 12, but the value is still above ±30mV. The electrical charge present in the vesicles, which influences the migration of particles to form aggregates, is shown by the zeta potential value. A good zeta potential is above ±30mV. The whole formula has a good zeta potential value so that the formed niosome system will be more stable and have less chance of aggregate formation (33,34). Statistical analysis results showed that temperatures of 4 and 25⁰C did not affect the zeta potential, but storage at 40ºC affected the particle size so it can be concluded that Ketoconazole niosomes are stable at storage temperatures of 4 and 25ºC.

Sample Structure analysis results by FTIR:

Figure 2. FTIR Structure of F 1-2 Niosomes

The presence of a hydroxyl group is indicated by the sharp and broad peak in the S-60 spectrum at wavelength 3390, followed by an aromatic group at wavelength 2916/cm and an ester bond C=O at wavelength 1731/cm. A broad and sharp peak at wavelength 2931/cm indicated the presence of an acetyl group, followed by 2901/cm, which indicated the presence of a CH=CH group, 3401/cm, which indicated the presence of a hydroxyl group, and 2866/cm, which indicated the existence of a symmetrical -CH3 group, according to the cholesterol spectrum. A sharp peak at 1720/cm in the spectra of sucrose ester palmitate indicates the existence of an ester group (C=O bond), while 2916/cm indicates the presence of a -CH3 group. The presence of a C=O bond was suggested by a high peak in the ketoconazole spectrum at a wavelength of 1645/cm, followed by a symmetrical aromatic C=C bond at 1584/cm and an asymmetrical aromatic C=C bond at 1509. FTIR testing proves that the niosome system does not form new compounds but only in physical interactions(30,31,32,34).

Drug release tests are performed to determine the amount of drug the niosome system can release through the semipermeable membrane. The drug release test of the niosome system can be carried out in 2 ways: using a dialysis bag inserted into a beaker containing PBS or a membrane placed between the donor and the recipient using Franz diffusion cells⁴². In this study, the drug release test was carried out using a dialysis bag inserted into a glass beaker containing PBS pH 7.4 and then run on a magnetic stirrer because the Ketoconazole niosome system can be administered topically.

The results of the in vitro drug release test in Table 5 show that the niosome system can increase the release of ketoconazole. The drug release value carried out for 24 hours showed an increase in the amount of ketoconazole in the system that was able to penetrate the membrane compared to ketoconazole, which was not made into the niosome system. Formula 2 results in higher drug release than Formula 1, presumably because Formula 2 has a higher Entrapment efficiency value than Formula 1. Formula 2 also contains more SEP than Formula 1. Sucrose Ester Palmirate is a surfactant with hydrophilic groups on the head and lipophilic fatty acids on the tail. This can aid in making hydrophilic medications more soluble so they can more readily pass through membranes18,43. The more drugs that are trapped in the niosome, the more drugs can also be released because the niosome contains non-ionic surfactants that contain hydrophilic and lipophilic groups that can help the trapped drugs to be able to penetrate the membrane and can increase the solubility of the drug^44,45.

The stability test results of drug release showed a decrease in drug release that occurred in F1 and F2 stored at temperatures of 4, 25 and 40⁰C. The most significant decrease occurred in niosomes stored at 40ºC. This is in line with a reduction of entrapment efficiency of niosomes stored at 40⁰C, causing the content of drugs trapped in the vesicles to decrease, causing a decrease in drug release. The more drugs that can be released from the niosome system the better the niosomes that are formed, so it can be concluded that the niosomes that are formed are more stable if they are stored at temperatures of 4 and 25ºC because there is no significant decrease in the levels of drugs released by the niosomal system for 12 weeks.

Drug Release Kinetics Results:

The results of Ketoconazole release are incorporated into the equation to determine the mechanism of ketoconazole release from the niosomal system. The drug release model is determined based on zero orde, first orde, Higuchi and Korsemeyer Peppas equations. Zero orde indicates a constant release of the active substance. First orde indicates that the release of the drug depends on the concentration of the drug. Higuchi's model illustrates that drug release is influenced by time. The Korsmeyer-Peppas model describes drug bending controlled by an erosion mechanism¹⁷.

The study's results in Table 6 show the release of ketoconazole in solution. Niosomes F1 and F2 follow the Higuchi model. This illustrates that the release of drugs from the three systems follows the law of diffusion, i.e., the drug will penetrate the membrane and slow down as the diffusion distance increases⁴⁶.

CONCLUSION:

The combination of S-60 and SEP as a surfactant produces ketoconazole niosomes with good physical properties and physical stability. The niosome system can increase the drug release of ketoconazole compared to ketoconazole, which is not made into the niosome system. Stability tests showed that the ketoconazole niosome system was stable at 4 and 25⁰C temperature storage.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This research was funded by Majelis Pendidikan Tinggi Penelitian dan Pengembangan PP Muhammadiyah (RisetMu), Contract Number 0258.702/I.3/D/2024.

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Received on 10.06.2025 Revised on 18.10.2025

Accepted on 29.12.2025 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2089-2096.

DOI: 10.52711/0974-360X.2026.00300

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formula	Entrapment Efficiency (%) Week 0	Entrapment Efficiency (%) Week 12
Formula	Entrapment Efficiency (%) Week 0	Temperature 4ºC	Temperature 25ºC	Temperature 40ºC
F1	84.55±0.15	84.83±0.64	85.02±0.14	83.48±0.50
F2	90.32±0.17	89.08±0.24	90.79±0.41	87.40±0.23

	Medication that is released for 24 hours (%) Week 0	Medication that is released for 24 hours (%) Week 12
		Temperature 4ºC	Temperature 25ºC	Temperature 40ºC
Ketoconazole	42.86±0.26	-	-	-
F1	92.38±0.13	91.92±0.08	88.96±0.08	83.76±0.13
F2	96.50±0.09	94.16±0.53	91.69±0.55	87.78±0.18

Formula	Order 0		Order 1		Higuchi		Krosmeyer Peppas
Formula	k0	R2	K1	R2	kh	R2	k	R2	n
Ketoconazole Solution	0.0022	0.9729	4x10^-7	0.8488	0.7699	0.9812	0.0318	0.9675	2.4574
F1	0.0010	0.8388	1x10^-9	0.6855	0.5594	0.9651	0.0026	0.8732	2.3106
F2	0.0007	0.7884	4x10^-9	0.6064	0.4990	0.9366	0.0026	0.8022	2.1919

Kori Yati^1,2, Septiana Tri Pamungkas¹, Fitria Nugrahaeni², Fith Khaira Nursal^1,2*

MATERIALS AND METHODS:

Materials:

Tool:

Methods:

Method of Preparation Ketoconazole Niosomes:

Niosomes Evaluation Niosomal Organoleptic Test:

Determination of Vesicle Size, Polydispersity Index and Zeta Potential:

Functional Group Analysis using FTIR:

Entrapment Efficiency (EE) Testing:

Morphological imaging:

Ketoconazole Niossome Stability Test:

Data Analysis:

RESULT:

Organoleptic:

Particle Size, Polydispersity Index and Zeta Potential

Sample Structure analysis results by FTIR:

Entrapment efficiency results:

Niosomal Morphological Imaging Results:

In vitro Drug release test results

Drug Release Kinetics Results:

CONCLUSION:

CONFLICT OF INTEREST:

ACKNOWLEDGMENTS:

REFERENCES:

Kori Yati1,2, Septiana Tri Pamungkas1, Fitria Nugrahaeni2, Fith Khaira Nursal1,2*

MATERIALS AND METHODS:

Materials:

Tool:

Methods:

Method of Preparation Ketoconazole Niosomes:

Niosomes Evaluation Niosomal Organoleptic Test:

Determination of Vesicle Size, Polydispersity Index and Zeta Potential:

Functional Group Analysis using FTIR:

Entrapment Efficiency (EE) Testing:

Morphological imaging:

Ketoconazole Niossome Stability Test:

Data Analysis:

RESULT:

Organoleptic:

Particle Size, Polydispersity Index and Zeta Potential

Sample Structure analysis results by FTIR:

Entrapment efficiency results:

Niosomal Morphological Imaging Results:

In vitro Drug release test results

Drug Release Kinetics Results:

CONCLUSION:

CONFLICT OF INTEREST:

ACKNOWLEDGMENTS:

REFERENCES:

Kori Yati^1,2, Septiana Tri Pamungkas¹, Fitria Nugrahaeni², Fith Khaira Nursal^1,2*