Design and Development of Self-Emulsifying Drug Delivery System to Improve the Solubility and Bioavailability of Ciprofloxacin

Summary

This study presents and describes the formulation of Ciprofloxacin Self-Emulsifying Drug Delivery Systems.

Abstract

Ciprofloxacin, a potent fluoroquinolone antibiotic, is used to treat various bacterial infections. This drug has low aqueous solubility and limited oral bioavailability. To overcome these limitations, this study focused on developing a Self-Emulsifying Drug Delivery System (SEDDS) for Ciprofloxacin, aiming to enhance its solubility and bioavailability. The formulation process involved selecting silicone oil, Tween 80, propylene glycol (PG), and polyethylene glycol (PEG) as the core components based on solubility studies. The optimization of SEDDS formulations was guided by pseudo-ternary phase diagrams, which helped in identifying the effective self-emulsifying regions and determining the optimal ratios of surfactant and co-surfactant. Evaluation of the SEDDS formulations involved droplet size and zeta potential measurements alongside Fourier-transform infrared (FT-IR) spectroscopy, confirming drug-excipient compatibility and successful drug incorporation. The F2 and F5 formulations exhibited droplet sizes of 320 nm and 202 nm, respectively, with corresponding zeta potentials of -11.4 mV and -13.38 mV, indicative of stability. Release studies showed an initial rapid release, with 88.2% released from F5 within the first 2 h, followed by a sustained release, reaching 93.1% after 5 h. The current formulations significantly improve the drug's solubility and bioavailability.

Introduction

Ciprofloxacin is a fluorinated quinolone with significant activity against both Gram-negative and Gram-positive bacteria, largely due to the presence of a fluorine atom at position 6 of its naphthyridine ring structure. This modification enhances its spectrum of antibacterial activity^1,2. As a synthetic fluoroquinolone antibiotic, it targets bacterial DNA gyrase or topoisomerase II, making it an effective treatment for various bacterial infections. Despite its broad-spectrum efficacy and generally safe profile, it faces challenges with low aqueous solubility and oral bioavailability, hindering its clinical application^3,4.

To address these issues, Self-Emulsifying Drug Delivery Systems (SEDDS) have been utilized. This is composed of a mixture of natural and synthetic oils, non-ionic surfactants, and co-solvents/surfactants, which significantly enhance the solubility of drugs^5,6,7. These systems are designed to form fine oil-in-water (O/W) emulsions or microemulsions upon contact with gastrointestinal fluids, facilitated by the gastrointestinal motility. This mechanism not only improves drug solubility but also facilitates absorption through the intestinal lymphatic pathway, thereby bypassing hepatic first-pass metabolism and enhancing bioavailability^8,9.

The rising number of hydrophobic drugs due to advancements in combinatorial and medicinal chemistry poses a challenge to oral bioavailability due to poor GIT absorption. This situation shows the importance of innovative drug delivery systems like SEDDS to improve the solubility and bioavailability of such compounds^10,11.

This study focused on formulating a stable SEDDS with varied lipid, surfactant, and co-surfactant ratios to enhance its water solubility and oral bioavailability. Prior research demonstrated the potential of SEDDS to significantly increase the bioavailability of hydrophobic drugs, as seen with acyclovir¹². Furthermore, the anhydrous nature of SEDDS formulations facilitates their encapsulation in gelatin capsules, offering good patient compliance and the potential for sustained and rapid drug effects¹³.

It includes self-micro-emulsifying and self-nano-emulsifying systems, produce emulsions with varying globule sizes, offering physical stability and ease of manufacturing. These characteristics lead to improved dissolution rates and bioavailability, as evidenced by the commercial success of SEDDS formulations¹⁴. It was extensively characterized through stability studies, droplet size, and zeta potential analysis, aiming to improve its aqueous solubility and oral bioavailability¹⁵.

It offers several advantages over conventional solubility enhancement methods such as solid dispersions, liposomes, and cyclodextrin complexes. Unlike solid dispersions, which require complex manufacturing processes, these are easy to formulate and scale up. Compared to liposomes, SEDDS exhibit superior physical stability and spontaneous emulsification in gastrointestinal fluids, improving drug absorption. Additionally, it enhances lymphatic transport, bypassing hepatic metabolism and increasing bioavailability¹⁶. A previous study has demonstrated the effectiveness of SEDDS in enhancing the bioavailability of poorly soluble drugs like acyclovir, proving their potential in drug delivery applications¹⁶. Its clinical effectiveness is limited by its poor aqueous solubility and low oral bioavailability, leading to inconsistent absorption and reduced therapeutic efficacy. Conventional approaches, such as solid dispersions and cyclodextrin complexes, have limitations in scalability and stability¹⁷. It offers a promising alternative by forming fine oil-in-water emulsions upon contact with gastrointestinal fluids, enhancing drug solubilization and absorption. It bypasses hepatic first-pass metabolism through the intestinal lymphatic pathway, improving bioavailability¹⁸. This study develops an SEDDS formulation to overcome solubility challenges, ensuring better therapeutic outcomes with enhanced stability, dissolution, and patient compliance. The primary goal of this study is to enhance the solubility and oral bioavailability of Ciprofloxacin by developing an SEDDS. It has low aqueous solubility and limited gastrointestinal absorption, which restricts its therapeutic effectiveness¹⁹. By formulating an optimized SEDDS, the drug can be efficiently solubilized in gastrointestinal fluids, leading to improved dissolution, enhanced absorption through the intestinal lymphatic system, and ultimately, increased bioavailability. This study focuses on optimizing the lipid, surfactant, and co-surfactant composition of SEDDS to achieve a stable formulation with improved drug release characteristics.

Protocol

NOTE: The details of the reagents and equipment used in this study are listed in the Table of Materials.

1. Solubility studies

1. Measure and mix an excess amount of drug (Ciprofloxacin) with the following selected oil combination: oleic acid oil (2 mL), olive oil (2 mL), castor oil (2 mL), sunflower oil (2 mL), mygliol oil (2 mL), surfactant like T-80 (2 mL), T-20 (2 mL), and co-surfactant including PG (2 mL), PEG200 (2 mL), PEG400 (2 mL), and PEG600 (2 mL).
2. Vortex the mixture thoroughly using the vortex mixer. Incubate the mixed samples at a constant temperature of 25 ± 2 °C using the thermo mixer.
3. Check the samples at predetermined time points (2 h, 6 h, 12 h, 18 h, and 24 h) to determine when equilibrium solubility is reached.
4. Centrifuge the samples at 16,770 x g for 15 min at room temperature to separate the supernatant from undissolved particles. Filter the supernatant through a 0.22 µm membrane filter to remove any remaining particulate matter.
5. Measure the concentration in the samples using the UV-Vis spectrophotometer at 277 nm. Mix the sample in a 50:50 ratio with methanol before analysis to prevent interference from oil in the absorbance readings.

2. Pseudo-ternary phase diagram construction

1. Prepare a combination of the following oils: oleic acid oil (2 mL), olive oil (2 mL), castor oil (2 mL), sunflower oil (2 mL), mygliol oil (2 mL), surfactants like T-80 (2 mL), T-20 (2 mL), and co-surfactant including PG (2 mL), PEG200 (2 mL), PEG400 (2 mL), and PEG600 (2 mL) based on solubility parameters.
2. Construct a pseudo-ternary phase diagram. Formulate the phase diagram for all surfactant and co-surfactant mixtures.
   NOTE: A pseudo-ternary phase diagram is a triangular diagram used in formulation science, especially in the development of emulsions, microemulsions, or self-emulsifying drug delivery systems (SEDDS), to visually represent the phase behavior of mixtures containing three main components: typically, oil, water, and a mixture of surfactants and co-surfactants (Smix). The pseudo-ternary phase diagram helps identify regions where the formulation forms: Clear microemulsions, Turbid emulsions, or Phase separation (unstable mixtures). By plotting various combinations of the three components, you can determine which ratios will result in a stable and homogenous system, ideal for drug delivery.
3. Vary the oil and Smix proportions from 1/9 to 9/1 for the graph of the pseudo-ternary phase. Add distilled water drop by drop to the mixture. Observe the change from a clear to a turbid solution.
4. Measure the quantity of water added by weighing the oil and surfactant mix before and after adding water. Calculate the percentage composition of all components using related software to plot a pseudo-ternary phase diagram.
5. Identify the stable emulsification area for every Smix based on the phase diagram.
   1. For each mixture, observe visually whether it is clear and transparent (microemulsion-stable), slightly turbid or bluish (nanoemulsion-possibly stable), milky, or phase separation (unstable).
   2. Record results on the diagram using different markings or colors. Plot data on the phase diagram. For each Smix ratio, plot all tested compositions, and mark the ones that formed stable emulsions/microemulsions. The region where these stable mixtures cluster is the emulsification zone.
   3. Identify the stable emulsification area. The stable emulsification area is the region in the diagram where most of the formulations form clear or slightly bluish emulsions quickly, remain stable over time (no phase separation). This area shows the best oil:Smix: water ratios for that specific Smix.Typically, the larger the area, the more robust the Smix ratio is at forming emulsions.
   4. Compare different Smix diagrams. After plotting diagrams for different Smix ratios, compare the size and shape of the emulsification zones. The Smix ratio with the largest stable area is usually preferred, but droplet size, drug solubility, and stability should also be considered.

3. SEDDS preparation

1. Prepare formulations with surfactant and co-surfactant mixture concentrations between 30% and 60%. Prepare four formulations for every phase diagram within this range.
2. Add 25 mg of the drug to 100 mg of the oil phase. Mix the oil phase and drug well using a vortex mixer.
3. Add an adequate quantity of Smix, depending on the analysis done in steps 2.3-2.5, to the oil drug mixture for 10 min. Mix well using homogenization.
4. Keep the oil content unchanged in all eight formulations (F1, F2, F3, F4, F5, F6, F7, F8). Make the quantity of Smix different in each of the eight formulations.

4. Physical characterization

1. Thermodynamic studies
   1. Centrifuge the formulations for 15 min at 6,037 x g. Use the supernatant and select only the formulations without phase separation for the freeze-thaw stress test.
   2. Subject the selected formulations to three freeze-thaw sets at 40 °C, room temperature, and 20 °C. Retain the formulations for 48 h at each temperature during the freeze-thaw sets.
2. Identification of self-emulsification time
   1. Prepare the dissolution apparatus II (USP). Pour 500 mL of distilled water into each dissolution vessel. Start stirring the water at 50 rpm and maintain a temperature of 37 ± 2 °C.
   2. Add 100 µL of the formulation sample to the apparatus. Observe the time of emulsion formation. The observed time represents the self-emulsification time.
3. Robustness to dilution
   1. Mix each formulation with water, acid buffer pH 1.20, and phosphate buffer pH 6.80 in the ratio of 1,000:1, 100:1, and 50:1, respectively. Observe each formulation visually.
   2. Reject all formulations that show emulsion breakage.
4. Cloud point measurement
   1. Take 1 mL of all formulations and mix them with 200 mL of water. Place the mixture in a water bath and slowly increase the temperature from 0 °C --75 °C.
   2. Observe the mixture for turbidity and record the temperature at which it becomes turbid. Repeat the test for stable preparations in triplicate for accurate results. A cloud point is the temperature at which a clear emulsion becomes turbid.
5. Droplet size and zeta potential analysis
   1. Dilute the selected formulations with water 100x. Mix the diluted formulations well with an ultrasonic homogenizer for at least 1 min.
   2. Analyze the zeta potential and particle size distribution as described in²⁰.
   3. Take 0.1 mL of the freshly prepared emulsion in a 25 mL glass beaker and dilute it with 9.9 mL of distilled water. Take 2.5 mL of this diluted solution from stock solution into a 3 mL quartz-cuvette.
   4. Click the DTS desktop icon to launch the software. Transfer the prepared sample to the 3 mL quartz cuvette. Confirm that no air bubbles remain.
   5. Open the cover and place the cuvette inside the device, making sure the cuvette is oriented correctly with respect to the path of the light beam. Click the Measurement icon.
   6. Take out the cuvette. Retrieve the sample or properly dispose of it. Save the data as a PDF in a personal folder for further use.
6. Morphology studies
   1. Dilute the sample formulation 50x using deionized water to create a well-dispersed sample.
   2. Spread a thin film of the diluted sample on a glass slide, positioning the obtained sample on the glass slide to obtain tiny films.
   3. Dehydrate the thin film by freeze-drying. Place the thin film into the freezer and dry the slide using freeze-drying at ultra-low temperatures (typically -40 °C to -80 °C).
   4. Analyze the freeze-dried sample using a Scanning Electron Microscope (SEM; 31,653x) to perform superficial morphology studies.
7. Fourier Transform Infra-Red (FT-IR) studies
   1. Set the instrument wave numbers between 4,000 cm⁻¹ and 400 cm⁻¹ using ambient air as a background and a resolution of 1 cm⁻¹. See²¹.
   2. Record spectra of pure drug and optimized formulation separately. Place the samples on the sampling area. Adjust count to 16 scans, resolution 4, gauge force 80, and pressure arm with a flat tip.
   3. Verify that the Auto Increment remains set to the Blank option so that the spectrum is automatically stored in the desired folder.
   4. To begin the sample measurement, click the Sample button. After selecting Sample, since there is no waiting period, be sure to have the sample ready and the pressure clamp reduced. Analyze all the samples individually, one by one.
8. In vitro drug release
   1. Prepare a stock solution of pure drug (0.17 mg/mL), F2, and F5 (0.1 w/v) in a preheated 50 mM phosphate buffer pH 7.4 at 37 ± 2 °C.
   2. Fill 1.0 mL of the pure drug base stock solution and 1 mL of each formulation in dialysis tubes. Dialyze each sample at 300 rpm at 37 ± 2 °C in 25 mL of phosphate buffer using a thermo-mixer.
   3. Take 0.1 mL aliquots from the medium at intervals of 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h. Replace the aliquots with an equal volume of 50 mM pH 7.4 phosphate buffer.
   4. Use fluorescence spectroscopy to determine the amount of pure drug after background correction through excitation at a wavelength of 335 nm and emission at a wavelength of 420 nm.

Results

Solubility study and Pseudo-ternary phase diagram
The solubility of pure drug in the chosen components is highest in silicone oil at 85 mg/mL, followed by Tween 80 at 70 mg/mL, PG at 50 mg/mL, and PEG at 30 mg/mL. The derived pseudo-ternary phase diagram revealed a constant ratio of surfactants to co-surfactants (Smix). In the diagram, a system composed of PG/PEG 600 and T-80 at a ratio of 1:6 demonstrated a large area capable of emulsification featuring silicone oil. Figure 1

Discussion

Approximately 40% of new chemical entities exhibit poor solubility in water, which results in reduced oral bioavailability²². This challenge significantly hampers the clinical application of many potential therapeutic compounds. It is a wide-ranging synthetic fluoroquinolone antibiotic categorized under BCS IV, exemplifying such drugs with solubility issues³. To overcome these solubility challenges, it is employed in research as a novel approach⁵. It...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ciprofloxacin	Feroze-Son Pharma (Nowshera 24,110, Pakistan	NA	Standard drug
Mygliol oil,	Dow Chemical Ltd, Bangkok 10,110, Thailand	NA
Oleic acid oil, silicone oil, olive oil, castor oil, and sunflower oil	Gaziantep (Gunedogu Anadolu 34,810, Turkey)	NA	Oil phase
Polyethene oxide 200	Sigma Aldrich (Saint Louis, Missouri 63,101, United States	8.17001	Co-surfactant
Polyethene oxide 400	Sigma Aldrich (Saint Louis, Missouri 63,101, United States	182028	Co-surfactant
Polyethene oxide 600	Ahad International Pharmaceutical, Dera Ismail Khan, 29,050, Pakistan	NA	Co-surfactant
Polyethylene Glycol (PG)	Sigma Aldrich (Saint Louis, Missouri 63,101, United States	1546401	Co-surfactant
Tween 20	Sigma Aldrich (Saint Louis, Missouri 63,101, United States	11332465001
Tween 80	Sigma Aldrich (Saint Louis, Missouri 63,101, United States	P8074	Surfactant