Development and Optimization of Ciprofloxacin Hydrochloride Nanoemulgel to Treat Topical Bacterial Skin Infections

Sapana D. Deore¹*, Shivraj P. Jadhav¹, Rushikesh L. Bachhav², Mayur S. Bhamare²,

Sunil K. Mahajan³, Deepak D. Sonawane¹

¹Department of Pharmaceutics, SSS’s Divine College of Pharmacy,

Nampur Road, Satana, Nashik, Maharashtra, India – 423301.

²Department of Pharmaceutical Quality Assurance,

SSS’s Divine College of Pharmacy, Nampur Road, Satana, Nashik, Maharashtra, India – 423301.

³Department of Pharmaceutical Chemistry, SSS’s Divine College of Pharmacy,

Nampur Road, Satana, Nashik, Maharashtra, India – 423301.

*Corresponding Author E-mail: deoresapana2001@gmail.com, rbachhav2001@gmail.com

ABSTRACT:

Objectives: To develop and optimise a ciprofloxacin hydrochloride nanoemulgel formulation for enhanced topical treatment of bacterial skin infections using a quality by design approach. Methods: A three-factor, three-level Box-Behnken design was employed to optimise the nanoemulsion composition. Almond oil was selected as the oil phase based on solubility studies (12.45±0.86mg/mL), with Brij-30 and PEG 400 as surfactant and cosurfactant, respectively. The optimised nanoemulsion was incorporated into a gel matrix using varying concentrations of Carbopol 934. Formulations were characterised for physicochemical properties, ex-vivo permeation, and stability. Results: The optimised nanoemulsion formulation (SF10) exhibited a globule size of 158.9±1.9nm, PDI of 0.178±0.012, and zeta potential of -33.6±1.3mV. Among nanoemulgel formulations, NEG2 demonstrated optimal characteristics with pH 6.4±0.1, viscosity 5625±285 cps, and Spreadability 25.4 ±1.5g.cm/s. Ex-vivo studies showed superior permeation (94.52% at 12hours) with a steady-state flux of 342.68 ±16.84µg/cm²/h. The formulation-maintained stability over 6 months at accelerated conditions, with drug content remaining above 97.85%. Conclusion: The developed nanoemulgel formulation demonstrates promising potential for enhanced topical delivery of ciprofloxacin hydrochloride through optimised physicochemical properties and superior skin permeation. This novel delivery system could potentially improve therapeutic outcomes in bacterial skin infections by providing sustained drug release and enhanced skin penetration.

KEYWORDS: Ciprofloxacin hydrochloride, Nanoemulgel, Box-Behnken design, Topical delivery, Skin infections, Ex-vivo permeation.

1. INTRODUCTION:

Bacterial skin infections remain a major global health concern, affecting nearly 150 million individuals annually and contributing to over $3.5 billion in healthcare costs¹. Rising antibiotic resistance, particularly methicillin-resistant Staphylococcus aureus (MRSA), now accounts for more than 40% of skin and soft tissue infections worldwide². Conventional topical antibiotics often show limited efficacy due to poor skin penetration and rapid drug clearance from the application site, leading to extended treatment durations and higher healthcare burdens. Recent epidemiological data further indicate a 15% annual increase in community-acquired skin infections, especially in low-resource regions with restricted access to adequate wound care and antimicrobial therapy³.

Figure 1: Chemical structure of Ciprofloxacin hydrochloride

Ciprofloxacin hydrochloride (Figure 1) is a broad-spectrum fluoroquinolone effective against a wide range of gram-positive and gram-negative pathogens⁶. Its antimicrobial activity results from inhibition of DNA gyrase and topoisomerase IV, while structural features such as the fluorine at position 6 and piperazine ring at position 7 enhance tissue penetration and broaden antibacterial activity⁴. Owing to its favourable pharmacokinetic profile and established clinical utility in dermatological applications, ciprofloxacin HCl is a strong candidate for topical drug delivery. Nanoemulgels offer an advanced approach for improving the topical performance of antibiotics. By integrating nanoemulsions (20–200 nm droplets) with hydrogel matrices, nanoemulgels enhance drug solubility, skin penetration, and residence time at infection sites. The nanoemulsion enables efficient transport across the stratum corneum, while the hydrogel provides suitable rheological behaviour without compromising nanoemulsion stability ⁵. Recent studies have reported up to a three-fold increase in drug permeation and sustained release exceeding 24 hours using nanoemulgel systems⁶. The present study aims to formulate and optimise ciprofloxacin hydrochloride nanoemulgels for enhanced topical management of bacterial skin infections. The work focuses on evaluating physicochemical characteristics, developing an in vitro release profile, and addressing the growing need for more effective topical antibiotic therapies with reduced systemic exposure and shorter treatment duration.

2. MATERIALS AND METHODS:

2.1 Materials:

Ciprofloxacin hydrochloride was obtained from Aarti Drugs Limited (Mumbai, India). Almond oil was purchased from Natural Oils International (Mumbai, India). Brij-30 and polyethylene glycol 400 were procured from Sciquaint Innovation Private Limited (Pune, India). All other chemicals and solvents used were of analytical grade.

2.2 Methods:

2.2.1. Calibration Curve of Ciprofloxacin Hydrochloride:

A 10 mg drug sample was accurately weighed and dissolved in phosphate buffer (pH 6.8) in a 100ml volumetric flask to prepare a 100µg/ml stock solution. Working solutions ranging from 10 to 60µg/ml were obtained by serial dilution. The absorbance of each solution was recorded at 278nm (Shimadzu, UV-1900)⁷.

2.2.2. Solubility Studies:

Solubility studies of ciprofloxacin hydrochloride in almond oil, Brij-30, and PEG 400 were performed using the shake flask method. Excess drug was added to 2mL of each vehicle in screw-capped vials and shaken for 72 hours (25±1°C, 100 rpm) using an Orbital Shaker (Remi Equipment, Mumbai, India). Samples were then centrifuged at 3000rpm for 15minutes using an Eppendorf 5810R centrifuge (Hamburg, Germany). The supernatant was filtered through a 0.45μm membrane filter, suitably diluted, and analysed at 278nm⁸.

2.2.3. Differential Scanning Calorimetry:

DSC analysis was performed using a Shimadzu DSC-60 thermal analyser. 8mg of pure ciprofloxacin hydrochloride and their physical mixtures were accurately weighed and sealed in aluminium pans. Samples were heated from 25°C to 300°C at a rate of 10°C/min under a nitrogen atmosphere with a flow rate of 50mL/min. An empty aluminium pan served as the control.

2.2.4. Fourier Transform Infrared Spectroscopy:

FTIR studies were performed to confirm its structural stability and compatibility of the drug with excipients. The absorption spectra were obtained using attenuated total reflectance FTIR spectroscopy equipped with an IR-Affinity-1 Shimadzu spectrometer. The pure ciprofloxacin hydrochloride and its physical mixtures were analysed in the spectral range of 4000–400 cm⁻¹.

2.2.5. Construction of Pseudo-ternary Phase Diagrams:

Pseudo-ternary phase diagrams were constructed using the water titration method. Brij-30 was selected as the surfactant and used in equal proportion to PEG 400, which was incorporated in double the amount to prepare the Smix. Various almond oil–Smix mixtures were prepared in ratios ranging from 1:9 to 9:1. Each mixture was titrated with distilled water under continuous stirring using a magnetic stirrer. The resulting systems were visually assessed for transparency, rheology, and stability. Only clear, single-phase, low-viscosity systems were considered stable and suitable. The phase diagram revealed a distinct nanoemulsion region corresponding to compositions of 10–20% almond oil, 40–60% Smix, and 30–50% water. These concentration ranges were selected for further optimisation using a Box–Behnken design⁹.

2.2.6. Box-Behnken Experimental Design:

A three-factor, three-level Box-Behnken design was employed using Design-Expert® software (v13.0, Stat-Ease Inc., Minneapolis, MN, USA) to optimise the nanoemulsion formulation. The independent variables and dependent variables are shown in Table 1. Seventeen experimental runs were generated, including five centre points to allow for estimation of experimental error. The design was evaluated using response surface methodology to determine the optimal formulation parameters^10,11.

The polynomial equation generated for the model was:

Y = β0 + β1X1 + β2X2 + β3X3 + β12X1X2 + β13X1X3 + β23X2X3 + β11X1² + β22X2² + β33X3²

Where Y is the response variable, β0 is the intercept, and β1 to β33 are the regression coefficients representing the effects of each factor individually and in combination.

Table 1: Box-Behnken Design (BBD) showing independent factors and Levels.

Independent Variables				Dependent Variables
Label	Factors	Level		Responses		Goal
		Low (-)	High (+)	Y1	Globule Size (nm)	Minimize
A	Oil Concentration (%)	5	15	Y2	Polydispersity Index	Minimize
B	Smix Concentration (%)	35	55	Y3	Zeta Potential (mV)	Optimise
C	Water Content (%)	30	50

2.2.7. Preparation of Nanoemulsion:

Nanoemulsions were prepared using the spontaneous emulsification method guided by a Box-Behnken experimental design, as shown in Table 2. Ciprofloxacin hydrochloride (% w/w) was dissolved in almond oil (5–15% w/w) using a magnetic stirrer (500rpm, 15 minutes, 25±1°C). The Smix was prepared by combining Brij-30 and PEG 400 in a 2:1 ratio. The oil phase was then mixed with Smix (35–55% w/w) using a high-speed homogeniser (IKA T25 digital ULTRA-TURRAX®, Germany) at 8000 rpm for 15 minutes. The aqueous phase was adjusted to pH 6.8±0.2 using 0.1 N NaOH or HCl. This phase was gradually added to the oil-Smix mixture under homogenization at 12,000rpm. Water comprising 30–50% of the total weight was incorporated and homogenised for an additional 30minutes to ensure uniform droplet distribution. The resulting nanoemulsion was probe sonicated (Sonics Vibra-Cell, CT, USA) at 40% amplitude (5 minutes) with a 3 s ON/2 s OFF pulse cycle to achieve optimal droplet size. Formulations were then allowed to equilibrate at room temperature for 24 hours before characterisation^12,13,14.

Table 2: Factors and batches design as per Box Behnken design

Run	Factor 1	Factor 2	Factor 3
	A: Oil Concentration	B: Smix Concentration	C: Water Content
	%	%	%
SF1	10	45	40
SF2	10	35	50
SF3	10	45	40
SF4	5	45	50
SF5	10	55	30
SF6	15	35	40
SF7	10	45	40
SF8	5	45	30
SF9	10	55	50
SF10	5	55	40
SF11	15	45	50
SF12	10	35	30
SF13	15	45	30
SF14	10	45	40
SF15	15	55	40
SF16	5	35	40
SF17	10	45	40

2.2.8. Evaluation of Nanoemulsion:

2.2.8.1. Globule size and Polydispersity Index:

The globule size and the PDI of the nanoemulsion formulations were assessed using the dynamic light scattering after the nanoemulsions were prepared (Malvern Zetasizer Nano ZS, Malvern Instruments, UK). Before measurement, the sample was diluted with double-distilled water at 1:100 to give a satisfactory scattering intensity. All the measurements were carried out at 25±1°C by using a helium-neon laser with a wavelength of 633nm and the 173° detection angle¹⁵.

2.2.8.2. Zeta Potential:

The zeta potential of the optimised nanoemulsion was determined by a Horiba SZ-100 nanoparticle analyser from Horiba Scientific, Japan, with the laser light of 532 nm. Samples were diluted 100-fold with the double-distilled water to obtain a suitable scattering intensity. These experiments were processed at 25±1°C with a disposable zeta cell, applying an electric field of 20 volts/cm and at a 70° angle of scattering.

2.2.8.3. Drug Content:

1mL of nanoemulsion was taken, and it was transferred to 100mL of methanol in a volumetric flask, followed by sonication for 15 minutes. The solution was then filtered through a Millipore filter with a 0.45μm size, and absorbance was determined at 278nm using a UV-visible spectrophotometer¹⁶.

2.2.8.4. Percentage Transmittance:

The clarity of formulated nanoemulsion samples was determined by percent transmittance using a UV-Visible Spectrophotometer. Samples were diluted 100-fold using double-distilled water, and the % transmittance was recorded at a wavelength of 650nm using double-distilled water as a blank. All the measurements were done three times at 25±2°C. A transmittance value above 95% was considered desirable, thus showing formation of an optically active nanoemulsion¹⁷.

2.2.8.5. Preparation of Nanoemulgel:

The nanoemulgel was formed by mixing the optimal concentrations of ciprofloxacin hydrochloride in nanoemulsion and the gel base of Carbopol, as shown in Table 3. Carbopol 934 polymer was first added to purified water in a concentration of 0.5 to 2.0% w/w and allowed to hydrate at room temperature for 24hours. The pH of the Carbopol dispersion was then brought to 6.8-7.0 using triethanolamine while agitating the gel at 500 rpm under magnetic stirrer to get the clear gel base. The optimized nanoemulsion was gradually incorporated into the gel base at a concentration of 0.3% w/w using a homogenizer (IKA T25 digital ULTRA-TURRAX®, Germany) operated at 2000rpm for 15 minutes to ensure uniform mixing. The resulting nanoemulgel was then allowed to stand for 24 hours at 25°C before further analysis^18,19.

Table 3: Composition of Ciprofloxacin HCl nanoemulgel formulations (NEG1-NEG4) prepared using the optimised nanoemulsion.

Ingredients (% w/w)	NEG1	NEG2	NEG3	NEG4
Optimized Nanoemulsion	0.3	0.3	0.3	0.3
Carbopol 934	0.5	1.0	1.5	2.0
Propylene Glycol	5.0	5.0	5.0	5.0
Triethanolamine	0.5	0.5	0.5	0.5
Methyl Paraben	0.2	0.2	0.2	0.2
Propyl Paraben	0.3	0.3	0.3	0.3
Purified Water q.s.	q.s.	q.s.	q.s.	q.s.

2.2.9. Evaluation of Nanoemulgel:

2.2.9.1. pH determination:

Determination of the pH of the prepared nanoemulgel was carried out using a Mettler Toledo Seven Compact™ S220 pH meter. To record the pH, the electrode was submerged in solution of 1g of nanoemulgel dissolved into 25mL of distilled water.

2.2.9.2. Viscosity:

The rheological properties of the nanoemulgel were determined by a rotational viscometer (Brookfield DV-III Ultra, USA) with spindle no. 64 and temperature 25 ±1°C. The sample was placed in the sample container and left undisturbed for 30min before the experiment was taken. Flow behaviour was determined by the viscosity measurements conducted at different shear rates of 0.5 to 100 s⁻¹. The viscosity measurement was taken at the end of 30 seconds at the pre-determined speed.

2.2.9.3. Spreadability:

 Spreadability of the nanoemulgel was characterised by the parallel plate method. A 0.5g of nanoemulgel was placed on a glass plate where a circle of one centimetre diameter was drawn. Another glass plate was laid on top of it, and a 500-gram weight was kept on the upper plate for five minutes. The diameter due to spreading was measured. Spreadability was determined by use of equation:

S=M* L/T……………………………………………(1)

where S is the Spreadability (g.cm/s), M is the weight tied to the upper plate (g), L is the length moved by the glass slide (cm), and T is the time taken (sec)²⁰.

2.2.9.4. Ex-vivo Drug Permeation Study:

In the ex-vivo permeation study, fresh goat ear skin was obtained from a local slaughterhouse within 2hours of sacrifice. The skin was washed with normal saline, subcutaneous tissue was removed, and the samples were processed and stored at –20°C for up to one week before use. Franz diffusion cells (effective area 3.14cm²) were used, with the skin mounted between the donor and receptor compartments. The receptor chamber contained 15mL of phosphate buffer (pH 7.4), maintained at 37 ± 0.5°C and stirred at 50rpm. A 500mg quantity of the nanoemulgel was placed in the donor compartment. At predetermined intervals (0, 1, 2, 4, 6, 8, 10, and 12 hours), 1mL aliquots were withdrawn and replaced with fresh buffer. The samples were analysed at 278nm using a UV spectrophotometer. The drug amount permeated per unit area was used to determine the steady-state flux (Jss). The partition coefficient values were then applied to calculate the permeability coefficient (Kp) using the following formula:

Kp = Jss/C₀ ………………………………………….(2)

Where C₀ is the initial drug concentration in the donor compartment. All results were analysed using one-way ANOVA (p < 0.05)^{21, 22, 23}.

2.2.9.5. Accelerated Stability Study:

Accelerated stability testing of the optimised nanoemulgel formulation was conducted in accordance with ICH Q1A(R2). The optimised formulation was placed in tightly closed glass vials at 40±2°C and 75±5% RH in a stability chamber (Remi, India) for a period of six months. The samples were subjected to test for appearance, pH, drug content, viscosity and phase at specified time intervals of 0, 1, 2,3 and 6 months. The data obtained was statistically analysed using ANOVA to assess changes in treatment outcomes over the storage period^{24, 25}.

3. RESULTS:

3.1. Calibration curve of ciprofloxacin HCl:

Figure 2: Calibration curve of ciprofloxacin hydrochloride in Phosphate buffer, pH 6.8

The calibration curve of ciprofloxacin HCl showed excellent linearity, as shown in Figure 2 with an R² value of 0.9985, confirming the linear relationship between concentration and absorbance.

3.2. Solubility of ciprofloxacin hydrochloride:

The solubility of ciprofloxacin hydrochloride was evaluated in various solvents, as shown in Table 4. Among the tested solvents, almond oil, Brij-30, and PEG 400 exhibited the highest solubility.

Table 4: Solubility of ciprofloxacin hydrochloride in various oils, surfactants, and cosurfactants

Solvent	Solubility (mg/mL)	Solvent	Solubility (mg/mL)	Solvent	Solubility (mg/mL)
Oils		Surfactants		Cosurfactants
Almond oil	12.45 ± 0.86	Brij-30	28.67 ± 1.24	PEG 400	35.78 ± 1.45
Isopropyl myristate	8.32 ± 0.54	Tween 80	22.45 ± 0.98	Transcutol P	31.24 ± 1.32
Isopropyl palmitate	7.14 ± 0.62	Tween 20	19.83 ± 0.87	Propylene glycol	27.56 ± 1.18
MCT	6.89 ± 0.48	Span 80	15.42 ± 0.76	Ethanol	23.89 ± 0.96
Captex® 355	5.76 ± 0.43	Values are expressed in mean±SD, (n=3)
Ethyl oleate	4.92 ± 0.38

3.3. Pseudo-ternary phase plot:

The pseudo-ternary phase diagrams constructed with almond oil, Smix (Brij-30/PEG 400), and water at different Smix ratios revealed varying nanoemulsion regions (Figure 3). The phase diagram with Smix ratio 2:1 (Figure 3(1B)) exhibited the largest nanoemulsion region compared to ratios 1:1 (Figure 3(1A)) and 3:1 (Figure 3(1C)). The optimal nanoemulsion formation was observed in the region containing 10-20% oil, 40-55% Smix, and 30-45% water with the 2:1 Smix ratio. The nanoemulsion regions in all phase diagrams were characterised by clear, single-phase, low-viscosity systems at 25±1°C.

3.4. FTIR analysis:

FTIR analysis of pure API (Figure 3(2A)) and its physical mixture (Figure 3(2B)) with excipients showed characteristic functional group peaks with minimal variation. Pure drug spectra display major peaks at 3435.10 cm⁻¹ (O-H/N-H), 3323.82 and 3138.86 cm⁻¹ (aromatic C–H), 1655.05 cm⁻¹ (C=C of quinolone), 1586.80 cm⁻¹ (C–N), and 784.78 cm⁻¹ (C–F). The mixture exhibits corresponding peaks at 3428.02, 3323.06, 1584.73, 1533.63, and 1152.15 cm⁻¹, showing only slight shifts. Additional peaks at 2970.75 and 2840.19 cm⁻¹ (aliphatic C–H from excipients) and enhanced fingerprint signals at 626.44, 593.39, and 547.82 cm⁻¹ originate from excipients. The retention of major functional groups with minor shifts confirms good drug–excipient compatibility of API in the formulation.

3.5. DSC analysis:

The DSC thermograms of ciprofloxacin HCl and its physical mixture with excipients are shown in Figure 3. The pure drug exhibited a single sharp endothermic peak at 254.06°C, corresponding to its melting point. In the physical mixture (Figure 3(3B)), three endothermic peaks appeared at 153.50°C, 195.65°C, and 254.33°C. The DSC thermogram of ciprofloxacin HCl (Figure 3 (3A) showed a single endothermic peak at 254.06°C, indicating preserved thermal stability and compatibility with the excipients.

3.6. Results of the evaluation of Nanoemulsion:

The physicochemical properties of seventeen nanoemulsion formulations (SF1–SF17) are summarised in Table 5. Globule sizes ranged from 158.9±1.9nm to 225.6 ± 3.8 nm, with SF10 having the smallest size and SF6 the largest. PDI values (0.178±0.012 to 0.312± 0.025) indicated uniform size distribution. Drug content was high across all formulations (96.45±0.85% to 99.12 ± 0.54%), with SF5 showing the highest content. Percentage transmittance varied between 95.34±0.75% and 99.45±0.22%, and SF10 displayed maximum clarity. Zeta potential values were negative, ranging from –22.8±2.4 mV to –33.6±1.3 mV, with the highest magnitude observed for SF10 (Figure 3 (4B)). Overall, SF10 exhibited the most desirable characteristics, including the smallest globule size, lowest PDI, highest transmittance and maximum zeta potential.

Figure 3: Pseudo-ternary phase diagrams showing nanoemulsion regions (shaded areas) of systems containing almond oil, Smix (Brij-30/PEG 400), and water at different surfactant/cosurfactant ratios: (1A) 1:1, (1B) 2:1, and (1C) 3:1, FTIR spectrum of Pure Ciprofloxacin HCl (2A) and physical mixture (2B), DSC spectrum of Pure Ciprofloxacin HCl (3A) and physical mixture (3B), Globule size (4A) and zeta potential (4B) of optimized nanoemulsion formulation

Table 5: Physicochemical characterisation of Ciprofloxacin HCl nanoemulsions.

Code	Globule Size (nm)	Polydispersity Index	Drug Content (%)	Transmittance (%)	Zeta Potential (mV)
SF1	186.4 ± 2.8	0.245 ± 0.018	98.45 ± 0.72	98.12 ± 0.45	-28.4 ± 1.8
SF2	198.7 ± 3.2	0.287 ± 0.022	97.82 ± 0.85	96.45 ± 0.62	-25.6 ± 2.1
SF3	185.9 ± 2.5	0.242 ± 0.015	98.56 ± 0.64	98.24 ± 0.38	-28.7 ± 1.7
SF4	165.3 ± 2.1	0.198 ± 0.012	96.78 ± 0.92	99.15 ± 0.28	-32.5 ± 1.5
SF5	172.8 ± 2.4	0.215 ± 0.016	99.12 ± 0.54	98.86 ± 0.32	-30.2 ± 1.6
SF6	225.6 ± 3.8	0.312 ± 0.025	97.25 ± 0.88	95.34 ± 0.75	-22.8 ± 2.4
SF7	187.2 ± 2.6	0.248 ± 0.017	98.34 ± 0.68	98.05 ± 0.42	-28.5 ± 1.9
SF8	162.4 ± 2.0	0.185 ± 0.014	96.92 ± 0.82	99.28 ± 0.25	-33.1 ± 1.4
SF9	178.5 ± 2.3	0.232 ± 0.018	98.88 ± 0.58	98.65 ± 0.35	-29.8 ± 1.7
SF10	158.9 ± 1.9	0.178 ± 0.012	97.15 ± 0.75	99.45 ± 0.22	-33.6 ± 1.3
SF11	218.4 ± 3.5	0.298 ± 0.024	97.65 ± 0.82	95.88 ± 0.68	-23.5 ± 2.2
SF12	195.6 ± 3.0	0.275 ± 0.020	97.42 ± 0.78	96.75 ± 0.58	-26.2 ± 2.0
SF13	212.8 ± 3.4	0.285 ± 0.022	97.85 ± 0.76	96.12 ± 0.65	-24.1 ± 2.3
SF14	186.8 ± 2.7	0.246 ± 0.016	98.52 ± 0.65	98.18 ± 0.40	-28.6 ± 1.8
SF15	208.5 ± 3.2	0.268 ± 0.020	98.25 ± 0.72	96.45 ± 0.62	-24.8 ± 2.1
SF16	168.7 ± 2.2	0.192 ± 0.015	96.45 ± 0.85	99.08 ± 0.30	-32.8 ± 1.5
SF17	186.5 ± 2.7	0.244 ± 0.017	98.48 ± 0.70	98.15 ± 0.42	-28.5 ± 1.8

Values are expressed in mean±SD, (n=3)

3.7. Optimisation of formulation:

The influence of formulation variables on globule size, PDI, and zeta potential was confirmed through statistical modelling, with excellent model fits reflected by high adjusted and predicted R² values across all responses. Globule size followed a highly significant quadratic model (sequential p < 0.0001, lack-of-fit p = 0.0014, adjusted R² = 0.9874, predicted R² = 0.9142). ANOVA revealed that oil concentration (A) had the strongest effect on globule size (F = 1119.47, p<0.0001), followed by Smix concentration (B) (F = 124.03, p<0.0001) and water content (C) (F = 7.60, p = 0.0282), with an additional significant quadratic effect for A² (F = 11.05, p = 0.0127). Increasing oil levels enlarged globule size, whereas higher Smix levels reduced it, consistent with the regression coefficients of +26.25 for oil and −8.74 for Smix. Polydispersity index (PDI) was best described by a significant linear model (sequential p<0.0001, lack-of-fit p = 0.0036, adjusted R² = 0.9465, predicted R² = 0.9099). Oil concentration exerted the greatest influence on PDI (F = 239.19, p<0.0001), while Smix also contributed significantly (F = 42.59, p<0.0001). The positive coefficient for oil (+0.0513) and negative coefficient for Smix (−0.0216) indicated that PDI increased with rising oil content and decreased with higher Smix levels. Zeta potential similarly followed a significant linear model (sequential p < 0.0001, lack-of-fit p = 0.0034), with excellent fit statistics (adjusted R² = 0.9790, predicted R² = 0.9665). Oil concentration had the most substantial effect (F = 666.75, p < 0.0001), while Smix concentration also played a significant role (F = 78.30, p < 0.0001). Regression coefficients showed that increasing oil made the zeta potential less negative (+4.45), whereas increasing Smix enhanced the negative charge (−1.52). Together, these findings confirm that formulation variables significantly govern the physicochemical properties of ciprofloxacin HCl nanoemulsions, with oil concentration as the dominant factor across all responses. Mathematical models describing the relationship between formulation variables and response parameters are given in Table 6.

Table 6: Mathematical models describing the relationship between formulation variables and response parameters.

Response Variable	Final Equation
Globule Size	Y₁ = 186.56 + 26.25A - 8.74B + 2.16C - 1.83AB + 0.6750AC + 0.6500BC + 3.59A² + 0.2700B² - 0.4300C²
Polydispersity Index	Y₂ = 0.2441 + 0.0513A - 0.0216B + 0.0069C
Zeta Potential	Y₃ = -28.26 + 4.45A - 1.52B + 0.2750C

3.8. Optimisation of statistical model:

The optimisation process aimed to minimise globule size and PDI while maximising the magnitude of the negative zeta potential. The numerical optimisation suggested an ideal composition of 5% oil, 55% Smix, and 40% water, which achieved a desirability value of 1.000, indicating a perfect fit to the desired criteria. The model predicted a globule size of 157.26nm, a PDI of 0.171, and a zeta potential of −34.24mV. Experimental validation closely matched these predictions, yielding a globule size of 158.90nm, PDI of 0.178, and zeta potential of −33.60mV, with low relative deviations of 1.04%, 4.09%, and 1.87%, respectively. The minimal differences between predicted and experimental values confirm the robustness and accuracy of the optimisation model and validate the selected formulation as the optimal nanoemulsion system.

3.9. Evaluations of Nanoemulgel:

The physicochemical properties of the nanoemulgel formulations (NEG1–NEG4) are summarised in Table 7. All formulations were white, opaque, and showed no phase separation after centrifugation at 2000rpm for 5 minutes. Homogeneity was excellent, with NEG1, NEG2, and NEG3 rated (+++), and NEG4 rated (++). The pH values ranged from 6.2±0.2 to 6.7±0.1, suitable for topical application. Viscosity increased progressively from NEG1 (4850±245 cps) to NEG4 (7250±345 cps), while Spreadability decreased correspondingly from 28.5±1.8g·cm/s (NEG1) to 18.6±1.2g·cm/s (NEG4), indicating an inverse relationship between viscosity and Spreadability. Three-dimensional response surface plots for optimisation of Ciprofloxacin HCl nanoemulgel are shown in Figure 4.

(45%).

Table 7: Physicochemical Characterisation of Ciprofloxacin HCl Nanoemulgel Formulations

Parameters	NEG1	NEG2	NEG3	NEG4
Physical Appearance	White, opaque	White, opaque	White, opaque	White, opaque
Homogeneity	+++	+++	+++	++
Phase Separation*	No separation	No separation	No separation	No separation
pH	6.2 ± 0.2	6.4 ± 0.1	6.5 ± 0.2	6.7 ± 0.1
Viscosity (cps)	4850 ± 245	5625 ± 285	6480 ± 312	7250 ± 345
Spreadability (g.cm/s)	28.5 ± 1.8	25.4 ± 1.5	22.8 ± 1.4	18.6 ± 1.2

+++ Excellent, ++ Good, + Fair Values, represent mean ± SD (n=3)

Figure 4: Three-dimensional response surface plots for optimisation of Ciprofloxacin HCl nanoemulgel. Quadratic model plots for globule size showing the effects of (A1) oil concentration and Smix concentration, (A2) oil concentration and water content, and (A3) Smix concentration and water content; Linear model plots showing (B) the effect of oil concentration and Smix concentration on polydispersity index at fixed water content (40%); and (C) the effect of oil concentration and water content on zeta potential at fixed Smix concentration

3.10. Ex-vivo permeation studies:

Figure 5:  Ex-vivo Drug Permeation Profile of Ciprofloxacin HCl Nanoemulgel Formulations

The permeation profiles of the nanoemulgel formulations (NEG1–NEG4) across goat ear skin are shown in Figure 5. NEG1 exhibited the highest cumulative drug release (96.92% at 12 hours), followed by NEG2 (94.52%), NEG3 (89.75%), and NEG4 (84.68%). Initial permeation at the first hour followed the same trend, with NEG1 showing the highest value (28.45%) and NEG4 the lowest (18.75%). All formulations displayed a similar pattern, with rapid permeation up to 6 hours, followed by a gradual decline until 12 hours. The permeation rate was inversely related to viscosity, as NEG1—having the lowest viscosity—demonstrated the greatest drug permeation.

3.11. Results flux and permeability coefficient (Kp):

The ex-vivo permeation parameters showed a clear influence of gelling agent concentration on drug permeation. NEG1 exhibited the highest steady-state flux (385.45±18.52µg/cm²/h) and permeability coefficient (3.85±0.18 × 10⁻³ cm/h), whereas NEG4 showed the lowest values (Jss: 265.75±12.92 µg/cm²/h; Kp: 2.65±0.12 × 10⁻³ cm/h), confirming that higher Carbopol concentrations reduced permeation. NEG2 demonstrated balanced permeation performance (Jss: 342.68±16.84 µg/cm²/h; Kp: 3.42±0.16 × 10⁻³ cm/h), offering an optimal compromise between sustained release and effective drug diffusion.

3.12. Results of the Stability study:

The optimised nanoemulgel (NEG2) underwent accelerated stability testing at 40°C ± 2°C/75% ± 5% RH for 6 months, and the results are shown in Table 8. The formulation retained its white, opaque appearance with no phase separation and maintained excellent homogeneity (+++). Only minimal changes were observed in physicochemical properties: pH decreased slightly from 6.40 ± 0.04 to 6.28±0.07, viscosity from 5625 ±185 cps to 5542±215 cps, and spreadability from 25.40±0.85 to 24.92±1.12g·cm/sec. Drug content remained high, decreasing marginally from 99.85 ± 0.45% to 97.85±0.68%. Globule size showed a small increase from 158.90±1.90nm to 161.85±2.85nm, confirming good structural and physical stability of the formulation over the test period.

Table 8: Accelerated Stability Study of Optimised Ciprofloxacin HCl Nanoemulgel Formulation (NEG2)

Parameters	Initial	1 Month	2 Months	3 Months	6 Months
Physical Appearance	White, opaque	White, opaque	White, opaque	White, opaque	White, opaque
pH	6.40 ± 0.04	6.38 ± 0.05	6.35 ± 0.06	6.32 ± 0.05	6.28 ± 0.07
Viscosity (cps)	5625 ± 185	5612 ± 192	5595 ± 198	5578 ± 205	5542 ± 215
Spreadability (g.cm/s)	25.40 ± 0.85	25.35 ± 0.92	25.28 ± 0.95	25.15 ± 1.02	24.92 ± 1.12
Drug Content (%)	99.85 ± 0.45	99.42 ± 0.52	98.95 ± 0.58	98.45 ± 0.62	97.85 ± 0.68
Globule Size (nm)	158.90 ± 1.90	159.25 ± 2.15	159.85 ± 2.35	160.45 ± 2.45	161.85 ± 2.85
Phase Separation	None	None	None	None	None
Homogeneity	+++	+++	+++	+++	+++

Values expressed as mean ± SD (n=3) +++ Excellent homogeneity

4. DISCUSSION:

The development of ciprofloxacin hydrochloride nanoemulgel demonstrated strong potential for enhanced topical delivery. Solubility studies identified almond oil as the most suitable oil phase (12.45 ± 0.86 mg/mL), consistent with earlier reports highlighting the solubilization capacity of natural oils. Brij-30 and PEG 400 were selected as surfactant and cosurfactant due to their high solubilising ability (28.67±1.24mg/mL and 35.78 ± 1.45 mg/mL), supporting stable nanoemulsion formation and improved skin permeation. The pseudo-ternary phase diagrams indicated that a Smix ratio of 2:1 provided the largest nanoemulsion region, confirming optimal emulsification efficiency and interfacial stability. Compatibility and stability assessments further validated the formulation components. FTIR spectra confirmed the retention of ciprofloxacin HCl’s characteristic functional groups with only minor peak shifts, indicating no chemical interaction. DSC thermograms supported this finding, with the drug’s melting point (~254°C) preserved in the physical mixture and only a negligible shift (0.27°C). Additional excipient-related endothermic peaks did not affect the drug’s thermal stability, demonstrating excellent compatibility. Formulation optimisation revealed significant effects of formulation variables on critical quality attributes. Globule size strongly depended on oil (F = 1119.47) and Smix concentrations (F = 124.03), with the quadratic model showing excellent predictability (R² = 0.9874). The optimised nanoemulsion (SF10) achieved a small globule size (158.9 ±1.9nm), smaller than values reported for many conventional topical systems (>200 nm), enabling potentially improved skin penetration. PDI and zeta potential results further indicated strong physical stability: SF10 exhibited a uniform size distribution (PDI = 0.178 ± 0.012) and a high negative zeta potential (−33.6±1.3mV), both indicative of excellent colloidal stability. High drug content (96.45–99.12%) and superior transmittance (95.34–99.45%) across formulations confirmed efficient nanoemulsion formation. Response surface methodology effectively captured the relationships between formulation variables and physicochemical attributes, enabling rational optimisation. Conversion of the optimised nanoemulsion to nanoemulgel resulted in formulations with a suitable pH (6.2–6.7) for topical application. Increasing Carbopol concentrations increased viscosity (4850–7250 cps) and reduced Spreadability (28.5–18.6 g·cm/sec), consistent with known rheological behaviour. Ex-vivo permeation studies showed excellent drug delivery performance, with NEG1 achieving the highest permeation (96.92% at 12 hours). The steady-state flux values (385.45–265.75µg/cm²/h) were superior to conventional topical ciprofloxacin products. NEG2 emerged as the optimal formulation, balancing adequate flux (342.68±16.84µg/cm²/h) with desirable viscosity, enabling sustained drug release. Accelerated stability studies of NEG2 confirmed its robust stability over 6 months. Key parameters—pH, viscosity, Spreadability, drug content, and globule size—showed only minimal changes, indicating strong physical and chemical stability. The slight increase in globule size (158.90 to 161.85 nm) and the absence of phase separation further confirmed preservation of nanoemulsion structure under stress conditions. Overall, the study successfully developed a stable and optimised nanoemulgel system for ciprofloxacin hydrochloride with enhanced permeation, sustained release, and excellent stability, demonstrating clear advantages over conventional topical formulations.

5. CONCLUSION:

In conclusion, this study successfully formulated and optimised a ciprofloxacin hydrochloride nanoemulgel for enhanced topical delivery. The optimised formulation (NEG2) demonstrated excellent physicochemical characteristics, including a globule size of 158.9nm, a low PDI of 0.178, and a highly stable zeta potential of −33.6mV. The nanoemulgel exhibited strong ex-vivo permeation (94.52% within 12 hours) and a high steady-state flux (342.68µg/cm²/h), indicating superior skin permeation compared to conventional formulations. Accelerated stability testing further confirmed the robustness of the system, with negligible changes in key parameters over 6 months. These findings suggest that the developed nanoemulgel holds significant promise for improving the sustained release and skin penetration of ciprofloxacin, potentially enhancing its effectiveness in treating bacterial skin infections. However, additional studies, including in vivo efficacy and clinical evaluations, are required to fully establish its therapeutic potential and safety.

6. CONSENT FOR PUBLICATION:

All authors have reviewed and approved the final manuscript and provided their consent for its publication.

7. COMPETING INTERESTS:

The authors declare that they have no competing interests.

8. AUTHORS' CONTRIBUTIONS:

All authors contributed equally.

9. ACKNOWLEDGMENT:

The authors express their sincere gratitude to Divine College of Pharmacy, Satana, for providing the necessary infrastructure and support for conducting this study.

10. ABBREVIATIONS:

ANOVA: Analysis of Variance; BBD: Box-Behnken Design; DSC: Differential Scanning Calorimetry; FTIR: Fourier-transform infrared spectroscopy; HCl: Hydrochloride; MCT: Medium Chain Triglycerides; NEG: Nanoemulgel; PDI: Polydispersity Index; PEG: Polyethylene Glycol; QbD: Quality by Design; RH: Relative Humidity; RSM: Response Surface Methodology; SD: Standard Deviation; SF: Statistical Formulation; S_mix: Surfactant-Cosurfactant mixture; UV: Ultra-violet spectroscopy; Jss: Steady-state flux; Kp: Permeability coefficient; µg: Microgram; nm: Nanometer; mV: Millivolt; cps: Centipoise; °C: Degree Celsius; rpm: Rotations per minute; g.cm/s: Gram centimeter per second.

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Received on 18.07.2025      Revised on 13.11.2025

Accepted on 31.01.2026 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):3013-3022.

DOI: 10.52711/0974-360X.2026.00429

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