Rebamipide nanocrystal with improved physicomechanical properties and its assessment through bio-mimicking 3D intestinal permeability model

Abstract

This study investigated the formulation and characterization of rebamipide nanocrystals (REB-NCs) to enhance the solubility and permeability of rebamipide, an anti-ulcer medication known for its low aqueous solubility and permeability, classified as BCS class IV. Employing high-pressure homogenization and wet milling techniques, we successfully achieved nanonization of rebamipide, resulting in stable nanosuspensions that were subsequently freeze-dried to produce REB-NCs with an average particle size of 223 nm. Comprehensive characterization techniques, including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) confirmed the crystalline nature of the nanocrystals and their compatibility with the selected excipients. The saturation solubility study revealed a remarkable three-fold enhancement in PBS pH 7.4 compared to rebamipide API, indicating the effectiveness of the nanocrystal formulation in improving drug solubility. Furthermore, 3D in-vitro permeability assessments conducted on Caco-2 cell monolayers demonstrated an noticeable increase in the permeability of REB-NCs relative to the pure active pharmaceutical ingredient (API), highlighting the promise of this formulation to enhance drug absorption. The dissolution profile of the nanocrystal tablets exhibited immediate release characteristics, significantly outperforming conventional formulations in terms of the dissolution rate. This research underscores the potential of nanomilling as a scalable, environment-friendly, and less toxic approach to significantly enhance the bioavailability of rebamipide. By addressing the challenges associated with the solubility and permeability of poorly water-soluble drugs, our outcome offers insightful information into developing efficient nanomedicine strategies for enhancing therapeutic outcomes.

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Md Samim Sardar

Md Samim Sardar is an MS student in the Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India. His project work is focused on nanocrystal-based solid oral formulation development of poorly water-soluble drugs with improve the solubility and bioavailability.

Prof. (Dr.) Velayutham Ravichandiran

Dr. V. Ravichandiran is the current Director of the National Institute of Pharmaceutical Education and Research Kolkata, West Bengal, India since 2015 and Director Additional Charge of National Institute of Pharmaceutical Education and Research Hajipur, Bihar, India since 2020. He visited various countries viz., Germany, Australia, Malaysia, and Singapore. He received various awards and was elected member of various Central and state Universities and Pharmacy Councils. He currently contributes to various scientific and research committees as Chairman and Member. He has been awarded multiple accolades, including the Silver Medal from Citrex, University of Malaysia Pahang, in 2016. He was awarded the Best Teacher accolade by The TN Dr. MGR Medical University in Chennai in 2011, and the Best Pharmacist distinction by the Indian Pharmaceutical Association in 2010. He has been recognised as top 2% Scientist in global contest per Survey by the Stanford/Elsevier List 2024. He has published 190 publications with an H-index of 33, i10-index of 125, and citations of 5791 and 17 patents at his credit.

Dr Subhadeep Roy

Dr. Subhadeep Roy currently holds the position of Assistant Professor in the Department of Pharmacology and Toxicology at the National Institute of Pharmaceutical Research in Kolkata, India. He received his PhD from Babasaheb Bhimrao Ambedkar University, Lucknow, where he investigated the role of polyunsaturated fatty acids in estrogen receptor-positive breast cancer. He has also worked as a visiting senior research fellow at the ID3S laboratory, Translational Health Science and Technology Institute, Faridabad, in Additive manufacturing inspired targeted drug delivery and compartment trafficking in human pulmonary macrophages. He has completed his postdoctoral program at IIT, Delhi, and Pennstate College of Medicine, USA. He was involved in different biomaterial and regenerative medicine-related work in his post-doctoral study. He has authored numerous works in internationally recognized journals that undergo rigorous peer review. Dr. Roy's research area focuses on 3D microphysiological disease models, Tissue Engineering, and Bioinspired/Biomimetic cutting-edge translational pharmaceutical research.

Dr. Santanu Kaity

Dr. Santanu Kaity is an assistant professor at the Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India. Before this, he was associated with formulation research and development at Mylan Laboratories Limited, A Viatris Company, Hyderabad, India, and at the Pharmaceutical Technology Centre, Centre of Excellence, Cadila Healthcare Limited, Zydus Cadila, Ahmedabad, India. Dr. Kaity received his doctorate from the Birla Institute of Technology, Ranchi, India. Dr. Kaity's research areas are focused on drug delivery, formulation development, and translational research.

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1. Introduction

The quinolone derivative, [2-(4-chlorobenzoylamino)-3-[2(1H)-quinolinone-4-yl] propanoic acid], rebamipide (REB) is utilized to treat acute gastritis, chronic gastritis, and gastric mucosal lesions.^1,2 It is also used in conditions like dry eye.³ The potent antiulcer drug, REB, works primarily by enhancing endogenous prostaglandin synthesis, scavenging free radicals generated from oxygen, and reducing inflammation and neutrophil activity. According to available research, REB is safe and effective against NSAID-induced GI injuries caused by different types of NSAIDs.⁴ Oral formulations of REB are eagerly awaited as potential solutions for gastrointestinal damage caused by NSAIDs. However, the effectiveness of REB is limited owing to its low solubility and permeability, resulting in a bioavailability of less than 10% in humans. Hence, it is classified as a biopharmaceutical classification system IV (BCS IV). Previous investigations, including solubility tests, have indicated that REB is nearly insoluble in polar and nonpolar solvents.⁵ Modification of such molecules into nanocrystals could be an effective strategy for the enhancement of solubility and permeability. Nanocrystals are pure drug crystals with diameters within the nanometer range. These crystals are stabilized or enclosed by a thin layer of inactive excipients. Nanocrystals consist only of drugs and do not contain carrier materials.⁶ In the pharmaceutical sector, nanocrystals are often defined as having sizes ranging from a few nanometers to 1000 nm. Consequently, researchers have turned to innovative formulation techniques, such as producing nanocrystals of ultrafine drug particles stabilized by excipients that retain a high loading capacity.⁷ Compared to other nanoparticles, such as polymeric nanoparticles, the high drug loading of nanocrystals (NCs) guarantees the efficient transport of drugs to cells or tissues and maintains a powerful therapeutic concentration to provide the desired pharmacological effects.⁸ Previous studies have explored various strategies for enhancing bioavailability of REB, including the development of nano-based formulations. REB nanocrystal based solid oral formulation significantly improved bioavailability compared to conventional formulations.⁹ Additionally, REB nanocrystals incorporated into hydrogels enhanced therapeutic effects in a hamster model of oral mucositis.¹⁰ However, these studies focused on specific applications and did not extensively investigate the physicochemical properties and stability of the REB nanocrystals. Despite these advancements, the literature lacks a comprehensive exploration of REB nanocrystals, particularly concerning the techniques for their preparation, characterization, and resultant in vitro and in vivo performance. Specifically, while high-pressure homogenization has been successfully employed in nanoparticle fabrication for various compounds, systematic studies dedicated to optimizing these methods for REB remain scarce. Additionally, the way that formulation components affect the stability and bioavailability of REB nanocrystals has not been thoroughly investigated. As such, there is a critical need to bridge these research gaps by enhancing the solubility and permeability of rebamipide through nanocrystal formulations and establishing a clear correlation between physicochemical properties and therapeutic outcomes.

Microfluidization or milling was used to prepare the nanocrystal suspensions. The suspension was lyophilized to obtain a dried powder for tablet formulation development after mixing with other suitable excipients. The lyophilized powders were characterized using various techniques. An in vitro study was performed on Caco-2 cells single layers to access the permeability of the prepared REB-NCs. This study not only explores the synergistic effects of these methods on particle size reduction and solubility enhancement but also introduces a novel stabilization strategy using biocompatible surfactants to maintain the integrity of the nanocrystals. Furthermore, the comprehensive evaluation of the permeability profiles of nanocrystals through advanced in vitro models presents a significant advancement over traditional formulations, which often overlooks the impact of particle morphology on drug absorption. Moreover, our work includes a comparative analysis of the developed nanocrystal formulation against marketed formulations, thereby establishing the superiority of our approach in bioavailability and therapeutic outcomes. By addressing the limitations of previous studies and focusing on the specific challenges associated with rebamipide, our study contributes valuable knowledge to the field of nanomedicine. This also provides a feasible approach for improving the clinical outcomes of similar BCS class of therapeutic agents.

2. Materials and methods

2.1 Materials and reagents

The materials utilized in this study include various chemical reagents and laboratory equipment. Rebamipide was sourced from Dr Reddy's Laboratories, Hyderabad, India, having a molecular mass of 370.786 g mol⁻¹ and purity of >99%. Crystal growth inhibitors included D-mannitol (purity: 99%, molecular weight: 182.17 g mol⁻¹, Batch No: 9149161) and lactose monohydrate (purity: 98–99%, molecular weight: 360.31 g mol⁻¹, Batch No: 5546975), both obtained from Sisco Research Laboratories Pvt. Ltd (SRL), Taloja, Maharashtra. Tween 80, a surfactant with a molecular mass of 1310 g mol⁻¹ (Lot No: BCBW3163), was procured from Sigma-Aldrich Unilab, MO, USA. Solvents and buffers included acetonitrile and methanol (both gradient HPLC grade, CAS Nos: 75-05-8 and 67-57-1, respectively, from Advent Chembio), triethylamine (CAS No: 121-44-8), orthophosphoric acid (CAS No: 7664-58-2), sodium dihydrogen phosphate (CAS No: 7558-79-4), ammonium acetate (CAS No: 631-61-8), sodium hydroxide (CAS No: 1310-73-2), HPLC water (CAS No: 7732-18-5), disodium hydrogen phosphate (CAS No: 7558-79-4), from Merck Life Science, acetic acid (CAS No: 64-19-7, from Avera Synthesis), and sodium acetate (CAS No: 127-09-3, from Avera Synthesis). Cell culture media and supplements included DMEM (Catalog No: 11965-092), RPMI 1640 (Catalog No: 1875093), fetal bovine serum (Catalog No: 10082147), Anti–Anti (Catalog No: 15240096), and Geltrex (Catalog No: A1413302), all sourced from Gibco. The dyes and antibodies used were live/dead™ Viability/Cytotoxicity kit (Catalog No. L3224, Invitrogen), goat anti-mouse IgG2a Alexa Fluor™ 594 (Catalog No: S-21135), and Alexa Fluor™ 488 phalloidin (Catalog No. A12379). Other reagents included PBS buffer (Catalog No. 10010023, Gibco), Hank's balanced salt solution (HBSS) (Catalog No: 14025092, Gibco), Dulbecco's phosphate-buffered saline (Catalog No: TCL021, Gibco), HEPES buffer (Catalog No. TCL021, Himedia), and lucifer yellow (Catalog No. L453), dextran (40000) (Catalog No. D1845), and dimethyl sulfoxide (DMSO) (Catalog No: D12345). The laboratory equipment used was Corning Costar 6.5 mm/3 μm/12 inserts/24 healthy plates (Catalog No. 3415). All reagents were used as received with further purification where applicable, and Milli-Q grade water was utilized for all the study.

2.2 Methods

2.2.1 High-pressure homogenization. The high-pressure pump forced the pre-homogenized liquid through an interaction chamber. When it reached the interaction chamber, it consisted of ceramic microchannels that split the liquid into two streams. Then, at extremely high speeds, these streams are combined and mixed, resulting in solid shear, impact, and cavitation, which reduces the size of the droplets or particles in emulsions and suspensions. The efficiency of Homogenizers is influenced by many operational factors, including the temperature, pressure, valve number of passes, flow rate, and impingement design. Initially, an aqueous suspension of rebamipide and excipient was prepared to formulate the nanocrystals. Two suspensions were prepared: one with lactose monohydrate and the other with mannitol. Accurately weighed, 5 g lactose was solubilized in 25 mL of Milli-Q water, and at last, 1% (w/v) Tween 80 was mixed. Then this solution was sonicated for 15 min, and then 1 g of REB was added and mixed thoroughly. Similarly, another suspension containing 5 g mannitol was prepared. Then, these suspensions were individually microfluidised and subjected to high-pressure homogenization using a microfluidizer (MICROFLUIDISER LM 20, USA). The micro-fluidized suspensions were then dried for 24 h in a lyophilizer (Optics Technology, India) and stored in a tightly closed container at room temperature.^11,12

2.2.2 Media milling. Ball milling is a technique in which the particle size is reduced by impact and attrition, mainly using metallic balls that are often made of zirconium (ZrO₂) or steel. The dimensions of the nanostructure may be changed by modifying the number and various dimensions of the balls, the composition of the balls, the composition of the cylinder surface, the rotational velocity, and the material to be milled. Ball mills often combine and pulverize materials into small particles. A hollow cylindrical shell revolves around its axis in a ball mill. Rebamipide nanocrystals were prepared using Tween 80 as a surfactant and lactose and mannitol were used as crystal growth arresters. Media milling was done by slight modification of the earlier reported method.⁹ For this, initially, mannitol (5 grams) was added to 30 mL Milli-Q water. Subsequently, a 1% (w/v) solution of Tween 80 was prepared and subjected to sonication for 30 minutes until it became clear. Following this, 1 gram of REB was incorporated into the mixture. The ball-milling process was then carried out using a Retsch ball mill (Retsch, Germany) with varying durations. A zeta sizer (Malvern Panalytical, UK) was used to further assess the prepared suspension's particle size.^13,14

2.3 Physicochemical characterization of particles

2.3.1 Powder X-ray diffraction (PXRD). REB, lactose, mannitol, and nanocrystals stabilized with mannitol and lactose were examined using a PANalytical X-ray powder diffractometer (Netherlands). All the samples were run at 45 kV and 40 mA, covering a range of 5°–70° (2θ). The outcomes were examined employing the X'PERT High Score Plus program.^14,15

2.3.2 Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectra of REB, lactose, mannitol, and both nanocrystals were acquired using a UATR (Universal Attenuated Total Reflectance) accessory on a PerkinElmer (Frontier) spectrophotometer (4000–400 cm⁻¹). The FT-IR data of the pure drug and excipients were compared with those of the resulting nanocrystals. Data were evaluated using Spectrum IR software. All samples were scanned in the 400–4000 cm⁻¹ range of wavelength.¹⁶

2.3.3 Solubility of nanocrystals. REB API and its nanocrystal saturation solubility were determined by suspending an excess amount of the material in a phosphate buffer (pH 7.4). The solutions were agitated on an orbital shaker (Tarson, India) at 25 ± 2 °C for approximately 24 hours. Afterward, the solutions were filtered using a 0.2 μm syringe nylon filter, and their absorbance were determined with a UV spectrophotometer at 228 nm (Thermo Scientific, USA).¹⁷

2.3.4 Scanning electron microscope (SEM). The morphological properties of the REB, lactose, mannitol, and nanocrystal formulations were evaluated using SEM (Gemini SEM 360, Zeiss, UK). Before imaging, the samples underwent electric conduction sputter coating of gold via fine autocoater (Model: Q 150R S plus). The coated pieces were placed under a 30 kV acceleration voltage after being attached to double-sided adhesive carbon tape for imaging.¹⁶

2.3.5 Transmission electron microscope (TEM). The morphology of nanocrystals stabilized with mannitol, after dispersion in aqueous media, was investigated using TEM (FEI Tecnai G2F20, Netherlands). Prior to analysis, a small amount of the dispersed emulsion was placed on the copper grid coated with carbon and stain with 2% w/v phosphotungstic acid. It was then air-dried and subjected to microscopic scanning.¹⁸

2.3.6 Atomic force microscopy (AFM). This technique was utilized to analyze the morphology of Rebamipide nanocrystals with mannitol. The research used contact mode atomic force microscopy (AFM). Quadratic mica sheets (muscovite IV type) recently cleaved (obtained from Structure Probe Inc./SPI Supplies, Westchester, PA, USA) and measured 10 × 10 mm were affixed to metal pads. These sheets were utilized to apply a solution containing nanoparticles. The imaging was performed utilizing the Oxford instrument (Asylum Research) type MFP3D Origin. The scan speed was adjusted to 1.00 Hz, and the scan sizes were measured at 5.30 μm. The sample was diluted in double-distilled water at a 1 : 100 ratio. It was then carefully placed on a glass slide and allowed to dry under normal circumstances to prevent particle movement. Glass slide holds the mica and the sample. The data were shown using topographic and phase imaging techniques. AFM generates the topographic image by measuring the force between the tip and the surface. Meanwhile, phase imaging involves mapping the phase delay between the periodic signal that drives the cantilever and the cantilever's oscillations. Afterwards, the Igor Pro 6.38B01 software was used to flatten all the photos. Identical software was used for both section analysis and particle size determination.¹⁹

2.3.7 Differential scanning calorimetry(DSC). It is utilized to analyze REB, a placebo (mannitol), the formulated nanocrystal with mannitol, and their corresponding physical combinations. A DSC Q20 instrument from TA-Instruments Waters (LLC, USA) was used for the analysis. Calibration of the device was carried out using pure indium, which has 156.6 °C melting point and 25.45 j g⁻¹ het of fusion. Samples weighing between 1–2 mg were placed in a pan and sealed with a lid. The experiment was performed over a temperature range of 25–350 °C, with a heating rate of 10 °C min⁻¹, in a nitrogen atmosphere at a flow rate of 50 cc min⁻¹.²⁰

2.3.8 Thermogravimetric analysis (TGA). It was conducted on pure REB, mannitol, and nanocrystal samples containing mannitol using a Discovery HP TGAinstrument (TA Instruments, New Castle, DE, USA). The samples were kept at 25–500 °C in a nitrogen atmosphere (60 mL min⁻¹) at an increment of 10 °C min⁻¹ to obtain the TGA curves.²¹

2.3.9 Vapor sorption analysis. The water vapor sorption isotherms were recorded using an Anton Paar Autosorb 6100 FKM XR-AG instrument. The measurements were conducted in a specialized vapor sorption cell, that is, a type B short-cell small bulb without a filler rod. The temperature was maintained at 298.15 K throughout the analysis by using a recirculating Dewar. The data points were collected over a relative pressure (p/p₀) range of 0.05–0.98. Before the measurement, distilled water was placed in the vapor source and prepared for measurement by vacuuming the dissolved air and impurities. Manifold heating was used to keep the temperature of 50 °C during the analysis. Data analysis was performed using advanced software, Anton Paar Kaomi for Autosorb.²²

2.3.10 Storage stability study. The storage stability study of nanocrystals was determined by subjecting them nanocrystals to open exposure at 50 °C for 30 days. After the 15th and 30^th days, a small sample was withdrawn, and FT-IR and PXRD were performed to check its physical and chemical stability after the open exposure study.²³

2.4. In vitro studies

The primary human intestinal cell lines Caco-2, HT-29, and HCT-15 cell lines were procured from the National Center for Cell Science, Pune, India. Caco-2 (passage 18) and HT-29 (passage 12) cells were cultured in DMEM. HCT-15 (passage number 8) were cultured in RPMI media with 10% fetal bovine serum and 1% antibiotic–antimycotic at 37° in a 5% CO₂/95% air humidified incubator (Thermo HERA cell, USA).²⁴

2.4.1. Cell viability studies. The cellular viability of the developed REB-NCs was evaluated in Caco-2, HT-29, and HCT-15 cell lines, using the MTT assay. The cell was cultured in a 96-well plate (1 × 10⁵ cells per well) and incubated for approximately 24 h to ensure proper growth and adherence. The old media was withdrawn from the wells, and new media was introduced. Then, the cell was treated with different concentrations of REB, and REB-Man-NC for 24 hours. After that, the media was aspirated, and the cells were washed carefully with 1× PBS. Next, 10 μL MTT (5 mg mL⁻¹) was added to each sample well and incubated for approximately 3 h. After carefully aspirating all media, formazan aggregates were dissolved by adding 100 μL of DMSO into each well. Subsequently, the absorbance was recorded at 570 nm utilizing a plate reader (Multiskan™ Spectrophotometer, A51119600C) to determine cell viability.²⁵

2.4.2. Live/dead analysis. The live/dead assay employs the use of two fluorescent dyes, calcein AM and ethidium homodimer, for the simultaneous staining of live and dead cells. Briefly, Caco-2, HT-29, and HCT-15 cells were cultured in 6-well plates with coverslips (1 × 10⁵ cells per well) and kept in an incubator with 5% CO₂ at 37 °C (Thermo HERA cell, USA) for 24 h. The cells were treated with IC50 of the REB and REB-Man-NC for 24 h. After rinsing the cells twice with 1× PBS, calcein and ethidium homodimers were added, and the plate was incubated at 37 °C for 45 min. The solution was removed, and the cells were washed twice with 1× PBS. The cells on the coverslip were then fixed with fixative (Image-IT I28900) for 15 min, rinsed with 1× PBS, and incubated with Hoechst solution (H21492) for 10 min. Then, further extensive washing was done with 1× PBS, and the coverslips were monitored under a confocal microscope (Leica DMi8, STELLARIS 5).²⁶

2.5. Intestinal permeability assessment

2.5.1. Caco-2 monolayer development. The cells were harvested, resuspended in culture media, and seeded in the apical compartment of the Transwell® (80 000 cells per insert, with 3 μm pore size and 0.33 cm² surface area). The media was changed every three days for 21 days. Caco-2 monolayer integrity was measured by analyzing transepithelial electrical resistance (TEER) and the permeability of zero permeable markers.

2.5.2. Transepithelial electrical resistance (TEER) measurement. An EVOM2 Voltohmeter, having STX2 Chopstick probes, was used to measure TEER to maintain monolayer integrity during the 21 days of the experiment. The TEER value was determined using eqn (1):

2.5.3. Lucifer yellow (LY) permeability assay. To determine LY passage/rejection, we did a LY permeability assay using Caco-2 cell line cultured in a Transwell®. First, the TEER of Caco-2 monolayers was recorded, and then both the chambers of Transwell® were filled with HBSS, and the plate was incubated at 37 °C for 1 h. The HBSS was withdrawn, and then 100 μg mL⁻¹ of LY in HBSS was introduced into the donor (apical) compartment, while the receiver (basal) compartment contained 800 μL of HBSS and placed in an incubator at 37 °C. At specific time intervals, 100 μL of the sample was collected, and the same volume was replenished with HBSS in the basal compartment. The samples were subsequently examined using a multimode reader (VarioskanTM Lux Multimode, VLBL0TD2) with excited and emitted wavelengths of 430 nm/540 nm, respectively.²⁷

The percentage of LY passage was determined viaeqn (2):

where RFU represents relative fluorescent units.

2.5.4. Dextran-40000 permeability assay. The transport of dextran-40000 across the Caco-2 cell single layer was assessed using a dextran permeability assay. After the TEER measurement, the Transwell® chambers were filled with HBSS, and the plate was kept in incubator at 37 °C for 1 h. The HBSS was removed, and 100 μg mL⁻¹ of dextran-40000 in HBSS was introduced into the donor (apical) compartment and the basal compartment contains 800 μL of HBSS and is incubated at 37 °C. 100 μL of the sample was collected at specific time intervals (15, 30, 60, 90, 120, 150, and 180 minutes), and the same volume was replenished with HBSS in the basal chamber. The samples were subsequently examined via a multimode reader at excitation and emission wavelengths of 495 nm/520 nm, respectively.²⁸

2.5.5. In vitro permeability study. Permeability studies were performed using Transwell inserts (Corning, Corning, NY, USA). Transport studies were performed when TEER values reached 500 Ω cm², and TEER was monitored continuously during the experiment time to maintain membrane integrity. To determine the donor to receiver and receiver to donor directional transport, 200 μL of rebamipide (10 μM) and its formulation were kept in the apical compartment, and 800 μL of rebamipide (10 μM) and its formulation were kept in the basal compartment. The apical and basal compartments were used to collect samples at 15, 30, 60, 90, 120, 150, and 180 minutes. The samples were subsequently substituted with an equivalent volume of the new assay medium. All samples were kept at −20 °C until further analysis. The quantity of active pharmaceutical ingredients (API) present in the samples was quantified via HPLC.²⁹

Similarly, the transport of standard drugs metoprolol (2.99 μM), Acyclovir (2.66 μM), prazosin (10 μM), digoxin (10 μM), and verapamil (10 μM) across the Caco-2 monolayer was evaluated in both the apical to basal and basal to apical direction. The apical and basal compartments were used to collect samples at 15, 30, 60, 90, 120, 150, and 180 minutes. The samples were subsequently substituted with an equivalent volume of the new assay media. The quantity of drug present in the samples was quantified via HPLC.³⁰

The apparent permeability (Papp) of each model was calculated using the eqn (3):³¹

Papp = (V_r × dQ/dt) × (1/A × C₀) (3)

where V_r is the volume of the receiver compartment, dQ/dt is the rate of permeation (in millimoles per second), A is the membrane's surface area (cm²), and C₀ is the initial drug concentration (μM). The efflux ratio or transport ratio was determined by calculating the ratio of Papp in the basal to apical direction versus Papp in the apical to basal direction, as shown in the following equation:³²

Efflux ratio (ER) = Papp_{(B to A)}/Papp_{(A to B)}. (4)

2.5.6. HPLC measurement conditions. The amount of drug present in the samples collected from the apical and basolateral compartments was estimated using an Agilent HPLC (Agilent 1260 Infinity II) with a UV diode array (HS G7117C) detector. Standard calibration curves were prepared by diluting the drug solution assayed. An Infinity Lab C18 column (5 μm, 250 × 4.6 mm, Agilent, USA) was utilized to separate the drug from the samples. The details of the analytical methods for the standard medicines are listed in Table S3 (ESI).†

The mobile phase consists of 10 mM phosphate buffer (pH 4.5) and acetonitrile to analyze rebamipide and its formulations. For REB, a gradient mode was used at a 1 mL min⁻¹ flow rate with 10 μL of sample injection through a C18 column. The effluent was detected using an Agilent 1260 variable-wavelength diode-array detector. Rebamipide was measured at 228 °C with a typical retention time of 6.6 min.³³ The gradient elution procedure for rebamipide is presented in Table 1.

Table 1 Gradient elution procedure of rebamipide API

Run time (min)	Mobile phase-A: phosphate buffer pH 4.5 (%v/v)	Mobile phase-B: acetonitrile (%v/v)
0	80	20
1	80	20
3	70	30
4.8	60	40
7	80	20

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2.6. Manufacturing of final dosage form

2.6.1. Tabletability, compressibility, and compactibility study. Powder samples of pure REB and REB nanocrystals were consolidated using a hydraulic press (PCI analytics, model HP-15TM). The media for tablet formation was equipped with a flat-faced punch (Stainless-steel) and die approximately of 13 mm in diameter. A sample of the powder weighing approximately 100 mg was manually loaded into the die. The powder was then compressed at pressures ranging from 125 to 200 MPa with a hold time of 30 s. The resulting tablets were left at room temperature for a full day to ensure a total elastic rebound. Measurements of the mass and dimensions of the compact, taken 24 h post-compression, were utilized to determine the compact density, solid fraction, and porosity. In the tabletibility analysis, the force required to break the tablet along its diameter was measured using a texture analyzer (Brookfield Ametek, model CTX). The breaking force was the maximum force that caused the tablet to fracture. The tensile strength of the tablets was computed viaeqn (5).³⁴

σ = 2F/πTD (5)

The tablet tensile strength (σ) was measured in megapascals (MPa), tablet breaking force (F) was measured in newtons (N), and tablet diameter (D) and thickness (H) were measured in millimeters (mm). The highest compaction pressure was 200 MPa. A PXRD study was conducted to examine the stress-induced polymorphic transitions. The calculated tensile strength data were plotted against compaction forces. In the compressibility study, the porosity of the compacts was measured. Porosity was determined by utilizing the density of the tablet (ρ_tablet) and the powder's actual density (ρ_true) using eqn (6).³⁵

Porosity = 1 − (ρ_tablet/ρ_true) (6)

Tablet density and powder actual density were measured, and porosity was calculated from this data. The actual density of powders was measured using a helium pycnometer (AccuPyc II 1340, Micrometric, USA). Then, for the compressibility study, porosity vs. different compaction forces of compacts were plotted. Compactibility was observed by plotting the graph tensile strength of compacts vs. porosity.^36,37

2.6.2. Tablet preparation. The mass of each manufactured tablet was fixed to ensure the same amount of REB in each dosage unit. The lyophilized nanocrystal powder was mixed with tablet excipients and sifted multiple times to obtain a free-flowing, uniformly mixed blend to manufacture the tablets. The blend was lubricated with #40 ASTM sieve passed magnesium stearate. Tablets were formulated through direct compression employing a rotary tablet press (Proton, Gujarat, India).

2.6.3. In vitro dissolution study. A dissolution test was done via a USP II rotating paddle apparatus (Labindia DS 14000 Smart). The paddle speed was set to 100 rpm, and the temperature of the dissolution medium (phosphate buffer at pH 7.4) was kept constant at 37 ± 0.5 °C. The dissolution study utilized 900 mL of the medium. Aliquot samples 5 mL were taken from each dissolution chamber at 10, 20, 30, 40, 50, and 60 min intervals. An aliquot was taken from each vial, and the amount of dissolved REB was analyzed via a UV spectrophotometer. The dissolution behavior of the nanocrystal tablets was compared with that of the marketed tablet Rebagen (rebamipide) tablets 100 mg (Macleods Pharma).

3. Results and discussion

3.1 Nanocrystal by high-pressure homogenization

REB and other excipients were dispersed in Milli-Q water and sonicated for 15 min. Process optimization was performed by changing the pressure to 10 000 psi, 15 000 psi, and 20 000 psi and changing the number of cycles to 5, 10, 15, and 20 cycles. Each sample was evaluated using particle size analysis (ZS Xplorer software, Malvern Zeta Sizer). From the particle size estimation, it was found that the size of the F5 formulation was the minimum (345 nm). The particle size data for the other batches are presented in Table 2. Each microfluidised suspension was lyophilized (OPTICS Technology, India) for further use. Hence, the process parameters, such as a pressure of 15 000 psi and 15 cycles, were considered optimum for high-pressure homogenization.

Table 2 Process optimization parameters and their respective size of formulation with lactose and mannitol (high-pressure homogenization)

Formulation number	Pressure (Psi)	Number of cycles	Particle size (nm) (with lactose)	Particle size (nm) (with mannitol)
Pure API particle size: 2920 nm.
F1	10 000	10	509.6	—
F2	10 000	15	473.5	—
F3	10 000	20	445.5	—
F4	15 000	10	580.6	—
F5	15 000	15	356.9	—
F6	15 000	20	604.6	—
F7	20 000	10	536.9	—
F8	20 000	15	567.4	—
F9	20 000	20	506.4	—
F10	10 000	10	—	458.3
F11	10 000	15	—	471.5
F12	10 000	20	—	519.0
F13	15 000	10	—	575.4
F14	15 000	15	—	359.5
F15	15 000	20	—	467.0
F16	20 000	10	—	487.8
F17	20 000	15	—	508.6
F18	20 000	20	—	494.2

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Process optimization showed that the optimized processing conditions reduced the particle size to a similar extent for lactose and mannitol (356.9 nm and 359.5 nm, respectively). Furthermore, we varied the amount of lactose and mannitol at optimized pressures and cycles to identify whether the excipient level affects particle size during processing through microfluidization. The observations are presented in Table 3. For both lactose and mannitol, we observed a larger particle size when the amount was increased (7.5 g) or decreased (2.5 g) than the central point (5.0 g).

Table 3 Composition optimization of batches prepared by high-pressure homogenization

Formulation	Rebamipide (g)	Lactose (g)	Mannitol (g)	Tween 80 (1%)	Distilled water (mL)	Particle size (nm)
F19	1.0	2.5	—	1	25	586.8
F5	1.0	5.0	—	1	25	356.9
F20	1.0	7.5	—	1	25	654.7
F21	1.0	—	2.5	1	25	436.9
F14	1.0	—	5	1	25	359.5
F22	1.0	—	7.5	1	25	690.5

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3.2 Nanocrystal by media milling

Since we could not reduce the particle size beyond a certain level by microfluidization, and for both lactose and mannitol, the reduced particle size was almost identical, we explored another method, media milling by ball mail, in this study. The REB and other excipients were dispersed in Milli-Q water and sonicated for 15 min. Process optimization was performed by changing the milling duration from 30 to 60 and 90 min. The process optimization of each sample was performed by particle size analysis using the method mentioned earlier. Each microfluidised suspension was lyophilized for further use. Here, we observed that formulation B2 and B3 with mannitol resulted in a particle size of 223.5 nm and 222.7 nm, respectively (Table 4). Since the particle size did not differ significantly between 60 and 90 min ball-milled samples, we considered the milling condition of 60 min at a frequency of 30 s⁻¹ as optimized to obtain the lowest possible particle size. However, using the ball-milling technique, we could not achieve significant size reduction in the case of a drug-excipient mixture with lactose monohydrate.

Table 4 Process optimization batches and their respective particle size with lactose and mannitol (media milling)

Formulation number	Time (min)	Frequency (per s)	Particle size (nm) (with lactose)	Particle size (nm) (with mannitol)
B1	30	30	—	230.1
B2	60	30	—	223.5
B3	90	30	—	222.7
B4	30	30	762.6	—
B5	60	30	762.4	—
B6	90	30	854.6	—

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Furthermore, composition optimization (Table 5) was performed using varying amounts of mannitol and lactose. In the case of lactose, no notable differences were observed in particle size among the batches. In the case of mannitol-containing batches, the batch executed with 5 g of mannitol showed the lowest particle size among all optimization trial batches.

Table 5 Formulation optimization of nanocrystal prepared by media milling

Formulation	Rebamipide (g)	Mannitol (g)	Lactose (g)	Tween 80 (% w/v)	Distilled water (mL)	Particle size (nm)
B7	1.0	2.5	—	1	25	277.3
B2	1.0	5	—	1	25	223.5
B8	1.0	7.5	—	1	25	371.4
B9	1.0	—	2.5	1	25	758.0
B5	1.0	—	5	1	25	762.4
B10	1.0	—	7.5	1	25	767.0

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The composition was finalized based on the saturation solubility study of batches with minimum particle size, lactose monohydrate (356.9 nm), and mannitol (223.5 nm).

The energy input in ball milling can be relatively high, leading to a technique that reduces the particle to a significant mechanical stress on the particles.³⁸ The liquid reduces the friction between the grinding media and particles, thereby minimizing the heat generated. Excessive heat can lead to the aggregation of particles. Liquid-assisted grinding (LAG) helps prevent particle aggregation, a common issue in high-energy processes such as microfluidization. Without proper control, microfluidization can sometimes lead to the re-agglomeration of particles. In LAG, the grinding energy is distributed more uniformly in the presence of the liquid, which can result in a finer and more consistent particle size reduction. In microfluidization, the energy distribution is less uniform, leading to a broader particle size distribution. LAG allows for more precise control over grinding parameters (e.g., liquid type, amount, and grinding time), which can be optimized to achieve smaller particle sizes.³⁹ This could be why the base technique reduced the particle size to a lesser extent than microfluidization.

3.3 Physicochemical characterization of prepared nanocrystal powder

3.3.1 Powder X-ray diffraction (PXRD). It was conducted to ascertain the crystalline or amorphous nature of the samples. The diffraction spectra of rebamipide, lactose, and freeze-dried powdered rebamipide nanocrystals prepared by microfluidization are shown in Fig. 1(A). The diffraction spectra of rebamipide, mannitol, and freeze-dried powdered rebamipide nanocrystals prepared by media milling are shown in Fig. 1(B).

Fig. 1 PXRD and FTIR spectra of drug, excipient, and nanocrystals. (A) PXRD spectra of REB, lactose, and REB-Lac-NC (B) PXRD spectra of REB, mannitol, and REB-Man-NC (C) FTIR spectra of REB, lactose, and REB-Lac-NC (D) FTIR spectra of REB, mannitol, and REB-Man-NC.

Clear and distinct peaks at specific 2θ values were observed in the diffractograms of rebamipide, confirming its crystalline nature. The diffractograms of the formulations showed that the peak positions of pure rebamipide and lyophilized nanocrystals were similar, indicating no significant differences, indicating that the lyophilized nanosuspension was in the same crystalline state. However, some new peaks were identified owing to the high-energy input during microfluidization. This aligns with research on rebamipide formulations, in which an attempt was made to improve their bioavailability by preparing lipid nanoemulsions or solid lipid nanoparticles.^40,41

3.3.2 Fourier transform infrared spectroscopy (FT-IR). It is an advanced analytical method employed to detect functional groups, molecular structures, and chemical bonds within a sample by analyzing its infrared light absorption or transmission. FT-IR spectra of the optimized premix with lactose and mannitol are presented in Fig. 1(C) and (D). The FT-IR spectra of the premix with lactose and mannitol revealed that pure rebamipide peaks were retained in the nanocrystal premix, indicating that nanonization did not alter the chemical nature of rebamipide. This observation aligns with previous studies in which the integrity of drug molecules was preserved after nanonization.⁴² Specifically, the prominent peaks at 3269 cm⁻¹ (C–H stretching), 1723 cm⁻¹ (C=O ester group), and 1642 cm⁻¹ (CO–NH group) were consistent with the literature values for rebamipide.⁴³ Similar retention of the characteristic peaks has been reported in studies involving other drugs, where nanonization did not affect their chemical structure.⁴⁴ This consistency across different studies underscores the compatibility of rebamipide with the excipients used, as no new peaks or shifts were observed, indicating no chemical interaction between the drug and excipients. Tables 6 and 7 show the major band assignments for nanocrystals with lactose and mannitol, respectively. These results are aligned with previous data.⁴¹

Table 6 Major FT-IR bands in rebamipide and REB-Lac-NC prepared by micro fluidization

Peaks (API)	Functional group	Peaks (REB-Lac-NC)	Functional group
3269 cm^−1	Aliphatic C–H stretching	3275 cm^−1	Aliphatic C–H stretching
1723 cm^−1	C=O (ester)	1724 cm^−1	C=O (ester)
1640 cm^−1	CO–NH	1643 cm^−1	CO–NH
1538 cm^−1	N=O	885 cm^−1	=CH–H

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Table 7 Major FT-IR bands in rebamipide and REB-Man-NC prepared by media milling

Peaks (API)	Functional group	Peaks (REB-Man-NC)	Functional group
3269 cm^−1	Aliphatic C–H stretching	3277 cm^−1	Aliphatic C–H stretching
1723 cm^−1	C=O (ester)	2938 cm^−1	CHO
1640 cm^−1	CO–NH	1725 cm^−1	C=O (ester)
1538 cm^−1	N=O	1645 cm^−1	CO–NH
1337 cm^−1	R–NO2	883 cm^−1	=CH–H

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3.3.3 Saturation solubility study of nanocrystals. A saturation solubility study is essential for understanding the physical properties of nanocrystals. Particle size reduction by nanonization have the probability to increase the solubility and permeability of Rebamipide (BCS IV drug). It has been observed that REB, the Active Pharmaceutical Ingredient (API), exhibited a solubility of 0.0259 mg mL⁻¹ in PBS buffer of pH 7.4. Regarding the premix, the one combined with lactose (F5) showed a 1.65 mg mL⁻¹ solubility. In comparison, the one combined with mannitol (B2) demonstrated a higher solubility of 3.86 mg mL⁻¹ in the same media (Fig. 2A). These results align with earlier research demonstrating that nanonization effectively enhances the solubility of drugs with poor water solubility.⁹ From the solubility analysis, we observed that the nanocrystal premix with mannitol had the highest solubility. Nanocrystals with lactose showed a moderate solubility improvement, but not to the same extent as the premix prepared with mannitol. Similarly, the use of mannitol as a stabilizing agent has been documented to improve the solubility of various drugs, owing to its hydrophilic nature. The observed solubility enhancement in our study with mannitol aligns with these findings, indicating its superior performance over lactose in enhancing the solubility of REB nanocrystals.⁴⁵ In this study, the increased saturation solubility of the mannitol premix could be primarily attributed to two factors: the particle size of the premix suspension and the physicochemical properties of mannitol in its lyophilized powder form. It is well established that smaller particle sizes can result in higher solubility because of the higher surface area exposed to the media. As the premix with mannitol had the lowest particle size (B2), it showed higher solubility than the batch with lactose monohydrate (F5). Furthermore, mannitol can create a stable hyperosmotic formulation in a powder mix, which has the capability to enhance the solubility of the Active Pharmaceutical Ingredient (API). Mannitol, a sugar alcohol, exhibits enhanced solubility because of its unique chemical properties. It forms hydrogen bonds with water molecules through its six hydroxyl groups (OH), thereby enhancing water solubility. Its crystalline structure, characterized by large intermolecular spaces, allows water molecules to penetrate, leading to higher dissolution. In brief, the hydrogen bonding, crystalline structure, and osmotic properties of mannitol explain its excellent water solubility.⁴⁶ The crystalline nature of mannitol also helps form a stable matrix for nanocrystals, which can enhance the solubility of the encapsulated active ingredient. Lactose, especially in its crystalline form (alpha-lactose monohydrate), has a less stable crystalline structure than mannitol does. This can result in low solubility.⁴⁷ Furthermore, as a monohydrate, lactose molecules is already in interaction with water molecules. Hence, the tendency to interact further with water molecules will be lower in such conditions than in mannitol.

Fig. 2 Solubility and microscopic images. (A) Solubility graph of nanocrystals (REB-Lac-NC and REB-Man-NC) and REB API, (B) SEM image of rebamipide, (C) SEM image of mannitol, (D) SEM image of REB-Man-NC, (E) SEM image of magnified strand, (F) TEM image of REB-Man-NC, (G) surface AFM image of REB-Man-NC, (H) 3D AFM image of REB-Man-NC.

As the goal of the study was to improve the physicochemical and mechanical properties of REB for improving its solubility, permeability, and development of suitable dosage form, and as we observed that the optimized premix with mannitol (B2) showed the most considerable solubility improvement, we selected the REB-mannitol nanocrystal (REB-Man-NC) for further characterization and formulation development.

3.3.4 Scanning electron microscope. SEM images offer detailed insights into the surface morphology and microstructure of nanocrystal samples, capturing information at both the micro- and nanoscale levels. SEM produces a vastly magnified image using electrons to form an image.⁴⁸ SEM images of Pure Rebamipide, mannitol, and REB-Man-NC are given in Fig. 2(B, C, and D), respectively. Pure Rebamipide was observed as a small acicular-shaped homogeneous crystal (acicular crystals) with a smooth texture. Mannitol appeared as irregular-shaped globules. REB-Man-NC exhibited a regular needle-shaped structure. Upon further magnification, it was observed that the strands were covered with tiny particles on the surface (Fig. 2E). These findings align with earlier research indicating that rebamipide nanocrystals prepared by the bead mill method exhibited spherical particles with enhanced solubility and stability.⁴⁵ Additionally, mannitol, as a stabilizing agent, has been shown to influence the morphology of nanocrystals, demonstrating that mannitol can form needle-like structures when combined with certain APIs, which aligns with our findings.⁴⁹ However, the presence of API or mannitol fragments has not yet been confirmed. Further microscopic analysis was conducted to gather comprehensive data on the size, shape, and characteristics of the drug particles.

3.3.5 Transmission electron microscope. The morphology of REB-Man-NC was further evaluated using TEM to identify the morphological characteristics of REB-Man-NC. The TEM images of the nanocrystals show rod-shaped structures, as shown in Fig. 2(F). A cluster of high-aspect-ratio nanostructures that resembled rods was observed in the photograph. These structures vary in length and orientation, and are closely packed together. While some rods remain apart, others are gathered together. Similar to the SEM images of the premix, the TEM image also shows irregular tiny particles attached to the strands. However, the TEM image was also insufficient to visualize the shape and size of the drug nanocrystals in the premix. We further characterized the premix with atomic force microscopic study.⁵⁰

3.3.6 Atomic force microscopy. It utilizes two distinct modes of data acquisition during scanning: surface imaging and 3D imaging. Fig. 2(G) and (H) show the surface image and three-dimensional (3D) image of REB-Man-NC, respectively. The surface picture depicts topographical features using the probe to follow the surface, whereas the 3D image provides the differentiation between nanocrystal segments. From the AFM images, the size of the nanocrystals was found to be approximately 200 nm, which is similar to that observed in the particle size analysis of the REB nanosuspension after milling. Atomic Force Microscopy (AFM) images showed a surface populated with multiple spherical protrusions. The color scale in the image signifies height variations, with dark brown indicating higher regions and brighter indicating lower regions. The nanocrystals exhibited size variability and a predominantly near-spherical shape, and the height variations hint at the surface roughness. The AFM study could be a good indicator of the API nanocrystal size and shape, as the method of sample preparation involved dilution of the sample with distilled water. As mannitol is highly water soluble, after solubilization in distilled water, near-spherical insoluble drug particles were observed on the flat, uniform, dried bed of mannitol. Thus, the AFM study confirmed that REB particles were converted into nanocrystals upon media milling.

3.3.7 Differential scanning calorimetry. DSC thermograms for pure Rebamipide (REB), mannitol, and REB-Man-NC are given in Fig. 3(A). The thermogram of pure REB displayed a distinct, sharp endothermic peak at 308.36 °C, indicating its characteristic melting point.⁵¹ This suggests that all rebamipide crystals undergo a phase transition at this temperature, and no moisture absorption occurs owing to their pronounced hydrophobicity. Mannitol exhibited a distinct endothermic peak at 170.80 °C. In the REB-Man-NC thermogram, the drug peaks shifted towards a lower temperature range with diminished intensity, potentially owing to a baseline shift. Such shifts can be attributed to the weight of the sample or specific heat capacity alterations.⁵² This thermal analysis provided insights into the physical characteristics of the dispersed solid powder and its melting and crystallization patterns. The absence of new peaks in the formulation implied no physical incompatibility between the nanocrystal drug components and mannitol. REB-Man-NC shows a sharp endothermic curve at 168 °C and a minor endothermic curve at 315 °C, mirroring the peaks of mannitol and the pure active pharmaceutical ingredient (API), respectively. A single peak signifies that the drug sample was devoid of impurities. However, the characteristic melting endotherm of rebamipide appeared to be slightly displaced from its original position, accompanied by a decrease in peak intensity.

Fig. 3 (A) DSC thermogram of rebamipide, mannitol, and REB-Man-NC, (B) TGA of rebamipide, mannitol, and REB-Man-NC, (C) FT-IR spectra for initial sample of REB-Man-NC, 15 days and 30 days stored sample, and (D) PXRD spectra for initial REB-Man-NC sample, 15 days and 30 days stored sample.

3.3.8 Thermogravimetric analysis. It is a method used to check changes in mass relative to temperature or time within a controlled atmosphere. This technique is valuable for assessing the thermal stability, decomposition, phase transitions, and other thermal properties of nanocrystals. The high surface-to-volume ratio of nanocrystals may result in distinct thermal stability compared to that of bulk materials. TGA can reveal the onset temperature of decomposition or phase transitions. The loss of weight seen in the TGA indicates the loss of volatile components, desorption of surface species, or degradation of the nanocrystals. The size of the nanocrystals influences their thermal behavior. Smaller nanocrystals typically have a higher surface-area-to-volume ratio, leading to enhanced reactivity and thermal properties that differ from those of larger crystals. The TGA curves of REB, mannitol, and REB-Man-NC are shown in Fig. 3(B). No peak was observed before 100 °C in these curves, indicating the absence of moisture. The onset temperature for pure rebamipide starts at 307 °C, which is similar to its melting point. These results are in agreement with earlier research findings. For instance, Jeon and Sohn (2016) reported that rebamipide polymorphs exhibit similar thermal behavior, with endothermic peaks corresponding to their melting points.⁵³ Mannitol shows the onset of melting at 299.4 °C. In the REB-Man-NC, two-phase weight loss was identical to that of mannitol at 275 °C, which is very similar to the onset of mannitol, and further curves were observed near 307 °C, which is very similar to that of pure rebamipide; hence, we observed that the nanocrystal formulation retained both mannitol and pure API characteristics. The observed thermal behavior in our study suggests that the nanocrystal formulation retains the properties of both mannitol and pure rebamipide, indicating no chemical interaction between the API and excipients, which aligns with the outcomes of other studies.⁹

3.3.9 Vapor sorption analysis. Vapor adsorption and desorption isotherms were obtained using an advanced Anton Paar Kaomi for Autosorb v1.00 instrument (Anton Paar GmbH). The experiments were conducted at a temperature of 25 °C. The relative pressure was progressively raised from 0.5 to 1, in increments of 0.5 at each step. The nanocrystal sample was measured in one cycle at a relative partial pressure 0.5–1. At 1 Relative Pressure, the sample adsorbs by 13.22 cm³ STP per g owing to water vapor sorption, as shown in Fig. 4(A). During subsequent desorption, the sample was desorbed, the vapor amount was equal to the amount absorbed at the starting point, and the entire process was reversible. In this study, we observed that the nanocrystals were non-hygroscopic. BET isotherm analysis was performed using sorption desorption isotherm analysis. Fig. 4(B) shows the BET isotherm of the nanocrystals. The BET isotherms showed a regression coefficient of 0.998, which is the best fit. The surface area of the nanocrystals is 15.271 m² g⁻¹, as shown in Fig. 4(B). These results align with earlier research, confirming the effectiveness of nanonization in enhancing the solubility of poorly soluble drugs. This consistency with previous studies underscores the reliability and validity of our findings. For instance, one study reported that cellulose nanocrystals exhibit similar non-hygroscopic behavior and a high surface area, which is attributed to their nanoscale size and surface properties.⁵⁴ Additionally, another report demonstrated that nanoporous carbon materials showed reversible water vapor adsorption–desorption cycles, indicating stability and non-hygroscopicity.⁵⁵ Our study's observed BET surface area aligns with these findings, highlighting the stability and non-hygroscopic nature of REB-Man-NC even after its extreme size reduction in the nanoscale range and creating a premix with highly water-soluble mannitol.

Fig. 4 (A) Vapor sorption isotherm of REB-Man-NC, (B) BET isotherm of REB-Man-NC.

3.3.10 Storage stability study. The stability of nanocrystals during storage can be evaluated through several techniques, such as FT-IR and PXRD. FT-IR is useful for identifying the chemical composition and structural characteristics of nanocrystals. Additionally, PXRD can be employed to determine the crystalline or amorphous nature of the samples, providing insights into their physical stability over time. FT-IR can be used to assess storage stability and monitor changes in functional groups or chemical bonds in nanocrystals over time. PXRD can be employed to monitor changes in the crystal structure or phase transitions to assess storage stability.⁵⁶ The resulting diffraction pattern can be analyzed to identify any shifts in the peak positions, changes in the peak intensities, or the appearance of new peaks over time, indicating changes in the crystallinity or phase composition. The FT-IR spectra of the REB-Man-NC samples stored for 15 and 30 d are presented in Fig. 3(C). We observed that the stored samples were stable after 15 and 30 days of storage at 50 °C as all the signature peaks of the samples were identical to the initial data. Similarly, the PXRD spectra of the REB-Man-NC samples [Fig. 3(D)] showed characteristics similar to those of the initial sample of REB-Man-NC. Thus, it can be said that the samples were stable under open exposure conditions throughout the study period.

3.4 In vitro studies

3.4.1 Cell viability assay. To determine the effectiveness of the developed REB-NCs under the biological milieu, we first studied their toxicity on Caco-2, HT-29, and HCT-15 cell lines. The finding provide that at a concentration of 200 μg mL⁻¹, ∼35–45% cell viability was observed for REB, whereas the same was ∼40–52% for REB-Man-NC (Fig. 5A, B and C) in Caco-2, HT-29, and HCT-15. The MTT results indicated that REB and REB-Man-NC did not exhibit any toxic effects on different intestinal cells, with IC50 value of 156.6 μg mL⁻¹ for REB and IC50 value of 185.1 μg mL⁻¹ for REB-Man-NC in Caco-2 cells. The IC50 value against HT-29 cells was 116.9 μg mL⁻¹ for REB, and the IC50 value was 124.5 μg mL⁻¹ for REB-Man-NC. In addition, the IC50 values against HCT-15 were 126.4 μg mL⁻¹ for REB, and the IC50 value was 134.3 μg mL⁻¹ for REB-Man-NC. A high IC50 value suggests that REB-NC exhibits minimal toxicity in Caco-2, HT-29, and HCT-15 cells (Table 8).

Fig. 5 Effect on the cell viability after 24 h treatment with various concentrations of REB and REB-Man-NC on (A) Caco-2, (B) HT-29, and (C) HCT-15 cells.

Table 8 Cell viability assay detail of REB API and REB-Man-NC

S. no.	Formulation	Caco-2 (IC50)	HT-29 (IC50)	HCT-15 (IC50)
1.	REB	156.6 μg mL^−1	116.9 μg mL^−1	126.4 μg mL^−1
2.	REB-Man-NC	185.1 μg mL^−1	124.5 μg mL^−1	134.3 μg mL^−1

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3.4.2 Live/dead assay. The live-dead fluorescence assay measures the esterase activity within living cells using Calcein-AM and the loss of plasma membrane integrity in dead cells using ethidium homodimer-1. Confocal microscopy images of Caco-2, HT-29, and HCT-15 cells treated with REB and REB-Man-NC displayed (Fig. 6) normal morphology with high green fluorescence and negligible red fluorescence, confirming the cytocompatible and anti-apoptotic effects of REB-NCs.

Fig. 6 Live/dead analysis after 24 h treatment of REB and REB-Man-NC on (A, B and C). Caco-2, (D, E and F). HT-29, and (G, H and I). HCT-15 cells.

3.4.3 Intestinal permeability assessment.
3.4.3.1 TEER measurement. The integrity of the Caco-2 monolayer was evaluated by TEER measurements for 21 days. Caco-2 cells showed a TEER value of 458 ± 12.56 Ωcm² on day 3 and 710 ± 13.25 Ωcm² on day 21. Caco-2 cells showed a gradual increase in TEER values after day 15 compared with the initial days, as shown in Fig. 7(A, B and C). Higher TEER values suggested the formation of a tight junction between the cells, further confirming monolayer formation. Studies have revealed that TEER values of 500–1000 Ωcm² are indicative of fully formed tight junctions in differentiated Caco-2 monolayers.^57,58

Fig. 7 Caco-2 monolayer development in Transwell® (3 μm pore size) and TEER measurement (A, B and C). Permeability of paracellular markers, lucifer yellow (D), dextran-40000 (E). Apparent permeability assessment of standard drugs, metoprolol (F), Acyclovir (G), prazosin (H), verapamil (I), digoxin (J), and digoxin in combination with verapamil (K and L) and test compounds, REB and REB-Man-NC (M), in Caco-2 monolayer with % drug release (N). Three repetitions of the data were averaged, the standard deviation among repeats was represented by error bars, and one-way ANOVA was used to determine statistical significance before Bonferroni's multiple comparisons test was applied (***p < 0.001).

3.4.3.2 Permeability of paracellular markers.
3.4.3.2.1 Lucifer yellow. LY shows the potential to cross the intestinal epithelial barrier through three major paracellular pathways: pore, leak, and unrestricted pathways. In this research, the Caco-2 monolayer was grown for 21 days to establish a differentiated and functional intestinal barrier. The monolayer integrity was evaluated by measuring the LY passage. The result showed that the transport of LY through the Caco-2 monolayer was 0.67 ± 0.04% after 180 minutes (Fig. 7D), indicating the formation of a tight junction between Caco-2 cells. The low permeability of LY across Caco-2 cells is a strong indicator of the development of tight junctions between cells. Tight junctions serve as a seal between Caco-2 cells, establishing a robust barrier that significantly influences the paracellular permeability of LY.^59,60
3.4.3.2.2 Dextran-40000. The permeability of dextran-40000 was evaluated after 21 days using a fluorescence plate reader in the Caco-2 monolayer. The results suggested that the permeability of dextran-40000 after 180 min was 0.89 ± 0.05% (Fig. 7E), indicating almost negligible permeability of dextran-40000 in the Caco-2 monolayer. Studies have shown that a Caco-2 monolayer with well-developed tight junctions displayed low paracellular permeability of dextran-40000.^61,62
3.4.3.3 In vitro permeability assessment. The European Medicines Agency (EMA) and the Food and Drug Administration (FDA) emphasize the Caco-2 permeability assay as the “gold standard” for evaluating intestinal permeability under laboratory conditions.⁶³ Standardization of the Caco-2 monolayer comprises a permeability study for standard compounds having high, moderate, and low permeability that range in human intestinal absorption ability.⁶⁴ Caco-2 cells demonstrate elevated expression of P-glycoprotein (P-gp) transporters, which depends on the duration of culture, passage number, and culture conditions.^65,66 A previous study reported higher expression of P-gp after 17 days of monolayer development.⁶⁷ Therefore, standardization should include P-gp efflux substrates. In this study, five standard drugs with different BCS characteristics (metoprolol, prazosin, Acyclovir, digoxin, and verapamil) were used in the Caco-2 monolayer.⁶⁸ To assess active transport mediated by P-gp transporters, 10 μM digoxin or 10 μM digoxin with 10 μM verapamil as an inhibitor was utilized. For the BCRP transport studies, 10 μM prazosin was used. In this study, experimental permeability data for the standard drugs (metoprolol, prazosin, Acyclovir, digoxin, and verapamil) and the test compound (rebamipide and its formulation) across the Caco-2 monolayer were evaluated using apparent permeability (Papp). The result showed that metoprolol (2.99 μM dose) was highly permeable with a value of 5.94 × 10⁻⁶ in the apical-to-basal transport and 19.9 × 10⁻⁶ in basal-to-apical transport, which declined slowly over a period of time, as shown in Fig. 7(F). Similar observations have been previously reported.^69,70 The apparent permeability of Acyclovir (2.66 μM dose) was found to be 4.69 × 10⁻⁶ cm s⁻¹ in the apical-to-basal direction and 10.8 × 10⁻⁶ cm s⁻¹ in the basal-to-apical direction, which indicates less permeability than metoprolol. This suggests higher permeability of Acyclovir with a higher efflux ratio than that reported by Shah et al., with a Papp value of 0.352 × 10⁻⁶ cm s⁻¹ at a dose of 2.5 mM.⁷¹ The apparent permeability of prazosin (10 μM dose) was 57.7 × 10⁻⁶ cm per in⁻¹ the apical-to-basal transport and 1781 × 10⁻⁶ cm s⁻¹ in the basal-to-apical direction. Prazosin showed the highest permeability as compared to the values reported by Kumar et al., 3.7 × 10⁻⁶ cm s⁻¹ in the apical-to-basal transport and 38.2 × 10⁻⁶ cm s⁻¹ in the basal-to-apical transport at a dose of 500 μM with a three times higher efflux ratio.⁷² Of all the standard drugs utilized in this study and one that was reported, digoxin exhibited the highest permeability, as shown in Fig. 7(J). The apparent permeability of digoxin (10 μM dose) was found to be 1019 × 10⁻⁶ cm s⁻¹ in the apical-to-basal transport and 3569 × 10⁻⁶ cm s⁻¹ in the basal-to-apical transport, as compared to the reported digoxin value of 1.45 × 10⁻⁶ cm s⁻¹ in the apical-to-basal transport and 23.27 × 10⁻⁶ cm s⁻¹ in the basal-to-apical transport at a dose of 1 μM.⁷³ The apparent permeability of verapamil (10 μM dose) was found to be 37.4 × 10⁻⁶ cm s⁻¹ in the apical-to-basal transport and 98.4 × 10⁻⁶ cm s⁻¹ in the basal-to-apical transport as compared to the reported verapamil value, done by Jarc et al., 32.1 × 10⁻⁶ cm s⁻¹ in the apical-to-basal transport and 28.6 × 10⁻⁶ cm s⁻¹ in the basal-to-apical transport at a dose of 70 μM.⁷⁴ When digoxin was administered with verapamil, a potent P-gp inhibitor, the Papp value of the drug decreased by one-third, as shown in Fig. 7(K and L), indicating that the P-gp transporter is responsible for drug efflux.

Similarly, the apparent permeabilities of the test compounds (rebamipide and its formulation) were calculated. The Papp values of rebamipide and its formulations at different time points are shown in Table S2 (ESI).† Enhanced permeability of rebamipide was obtained in our Caco-2 monolayer model at a dose of 10 μM, with values of 13.02 × 10⁻⁴ cm s⁻¹ in the apical-to-basal transport and 1.66 × 10⁻⁴ cm s⁻¹ in the basal-to-apical transport, in contrast to the published values of 1.51 × 10⁻⁵ cmin⁻¹ the apical-to-basal transport and 2.50 × 10⁻⁵ cm s⁻¹ in the basal-to-apical transport.⁷⁵ The REB-Man-NC showed enhanced permeation with an eighteen-fold higher efflux ratio than the REB API over a 3 h period, as shown in Fig. 7(M) and Table 9. The increased permeability of REB-Man-NC may have been due to the presence of Tween-80 and mannitol in the formulation. The permeability of the majority of the low-permeability drugs was considerably enhanced by tween-80.⁷⁶ As such, the permeability of mannitol is low, but the addition of other substances, such as interleukin-6, enhances its permeation.⁷⁷ Thus, the permeability of the formulation was increased by the addition of mannitol and tween-80. In addition, the decreased particle size and increased solubility increase the concentration gradient across the membrane, thereby improving permeability.⁷⁸

Table 9 Caco-2 cell permeability of REB and REB-Man-NC

System	Papp × 10^−4 (cm s^−1)		Efflux ratio
	Apical to basal	Basal to apical
REB	13.02	1.66	0.12
REB-Man-NC	17.12	38.04	2.22

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The result showed the higher permeation of both the test drugs (i.e., rebamipide) and standard drugs (i.e., metoprolol, prazosin, Acyclovir, digoxin, and verapamil). The Papp values for metoprolol, prazosin, Acyclovir, digoxin, and verapamil at different time points are listed in Table S1 (ESI).†

3.5 Tabletability, compressibility, and compactibility study

Tabletability is the correlation between the tensile strength of a tablet and applied compression pressure. Tabletability, compressibility, and compactibility profiles were plotted to assess the tableting performance of REB and REB-Man-NC; these profiles are given in Fig. 8(A), (B), and (C), respectively. To create these plots, the physical dimensions and breaking force of each compact were measured at each pressure point as the compression pressure increased from 125 to 200 MPa, as shown in Fig. 8(A). The porosity of the powder compressibility profile was a function of the applied pressure. Under a given stress, more compressible materials have a higher solid fraction and lower final porosity, which is a prerequisite for a good tabletability profile. As shown in Fig. 8(B), REB-Man-NC also showed a good compressibility and tabletability profile. The tensile strength of the tablets is shown as a function of porosity in the compactibility profile. Therefore, powdered materials are considered to be more compact if they produce stronger compacts at a given porosity.

Fig. 8 (A) Tabletability profile, (B) compressibility profile, (C) compactibility profile plots of REB and REB-Man-NC. (D) Comparison of tabletability between API and premix, (E) images of final compressed tablets with REB-Man-NC, (F) in vitro dissolution study of pure API, REB REB-Man-NC tablets, and the marketed tablet in phosphate buffer of pH 7.4.

As shown in Fig. 8(C), REB-Man-NC has a lower bed porosity than pure REB, indicating the superior compressibility and compactable characteristics of nanocrystals. Finally, a tabletability profile was created by our analysis, and we noticed an upward trend in tensile strength as compression pressure increased, reaching a plateau at higher pressures. The data suggest that a compression force within the 175–200 MPa range was optimal for achieving the maximum tensile strength in the compact. Further increase in the compression force did not contribute to an enhancement in the tensile strength of the tablet. Typically, the strength of a tablet increases with increasing compression pressure, eventually stabilizing the compacted mass. However, an inverse relationship was observed when tablet strength diminished with increasing pressure. This phenomenon is indicative of over-compaction, which can lead to tablet flaws.⁷⁹ Upon examining the tabletability graph, it was discernible that the tablet's tensile strength increased with increasing compression pressure, but reached a saturation point at higher pressures. The tensile strength apex of the tablet was attained using a compression force of 200 MPa. From these plots, we can observe that the nanocrystals have better tabletability characteristics than the pure REB. Nanocrystal powders have far better tableting behavior than pure REB API powders, which showed very weak compaction properties. Without other excipients, complete tablets can be formed with the premix over the compaction pressure range. Furthermore, after 24 h of rest, there was no sign of delamination or capping of tablets.

3.6 Preparation of tablet formulation

Tablets containing the lyophilized nanocrystals were developed by mixing the lyophilized REB-Man-NC with other extra-granular materials. The exact final composition and batch size are presented in Table 10. The images of the compressed tablets are presented in Fig. 8(E). The tablets were compressed by direct compression method. Excipients like MCC 102 (diluent), magnesium stearate (lubricant), and polyplasdone XL (super disintegrant) were used for tablet manufacturing. The details of the tablet unit composition are presented in Table 10. These excipients have excellent flowability and compressibility, making them suitable for direct compression. A mix of lyophilized nanocrystals with the tablet excipients was subjected to direct compression by a rotary compression machine using a caplet shape “D” type 19 mm × 8 mm punch by direct compression method. Weight variation was found to be 700 ± 2.07 mg. The hardness range of the tablet was found to be 12.2 ± 2.7 kp. The friability of the tablet was found to be 0.144% w/w. The drug content uniformity was analyzed by UV-visible spectroscopy. The % drug content of the tablets was observed to be 97.52 ± 2.03%. The results were within the range, indicating uniformity of drug content. The tablet shows an average disintegration time of 11 minutes 42 s.

Table 10 Materials for the preparation of the tablet with REB-Man-NC premix

Sl no.	Components	Mg/unit	Gram/batch (50 units)
Intra-granular
1	REB-Man-NC premix	600.00	30.00
Extra-granular
2	Microcrystalline cellulose 102	64.00	3.20
3	Crospovidone XL	30.00	1.50
4	Magnesium stearate	6.00	0.30
Total weight of a tablet		700.00	35.000

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3.7 In vitro dissolution study

The dissolution tests for the manufactured tablets were conducted in triplicate using a USP type II (paddle) apparatus. The tests utilized 900 mL of phosphate buffer at pH 7.4 as the dissolution medium, maintained at a temperature of 37 ± 0.5 °C. The paddle speed was set to 100 RPM for the REB-Man-NC tablet dissolution study. The dissolution characteristics of the REB-Man-NC powder, in-house tablet formulation with REB-Man-NC, and marketed formulation are presented in Fig. 8(F). The dissolution velocity of the REB-Man-NC tablet was faster than that of the marketed 100 mg tablet formulation. Nearly 76% of the REB was dissolved in just ten minutes from the nanocrystal tablet. This was better than the marketed tablet, from which near about 50% of REB was dissolved under the same conditions. The API showed only 20% dissolution after ten minutes. After 15 min, the REB-Man-NC tablet showed nearly 81% drug release, which is close to official drug release criteria for immediate release tablets (should release ≥85% drug in 15 min). The faster dissolution in the REB-Man-NC tablet formulation may be attributed to its extreme size reduction to the nanoscale level and its association with highly water-soluble mannitol. From this study, it can be concluded that REB-Man-NC tablets have the potential to show more significant dissolution and intestinal permeability improvement.

4. Conclusion

In conclusion, this study successfully demonstrated that nanocrystalline rebamipide (REB) significantly enhances its solubility and permeability, addressing the critical limitations posed by its low bioavailability as a BCS class IV drug. By applying high-pressure homogenization and wet milling techniques, REB was converted into nanocrystals with an average particle size of 223 nm, leading to an impressive three-fold increase in its solubility in phosphate buffer at pH 7.4. Comprehensive characterization methods, including FT-IR, DSC, and PXRD, confirmed the crystalline stability and compatibility of the nanocrystals with the excipients. In vitro studies utilizing Caco-2 monolayers have validated an eighteen-fold enhancement in permeability compared to pure REB. Simultaneously, the dissolution profiles of the nanocrystal tablets indicated close to immediate drug release characteristics, further underscoring their therapeutic potential. These findings establish nano-milling as an effective and scalable platform technology to increase the rate of dissolution and solubility of drugs that are poorly soluble in water, such as rebamipide. Our research not only provides a promising approach for improving oral bioavailability but also contributes new insights into the formulation strategies necessary for maximizing the clinical efficacy of this important therapeutic agent.

Author contributions

S. K. and S. R. conceptualized the study and designed the studies and methodologies. V. R. helped with funding and manuscript review. M. S. S., K. P. K., S. K. S., and U. G. performed manufacturing and material characterization. M. K., K. A., S. K. S., and U. G. performed permeation-related experiments. M. S. S., K. P. K., S. K. S., and M. K. drafted the manuscript. All authors have reviewed the final version of the manuscript and approved its submission.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgements

S. K. would like to thank the Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Kolkata, for supporting this work. S. R. acknowledges the Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Kolkata, for supporting this work. The authors are thankful to the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for their generous funding support to the institute. We acknowledge the analytical support from Dr. Rekha Bhar (Anton Paar). The authors also acknowledge instrumental support from Shiv Nadar University, IoE, Department of Chemistry for AFM study. All graphics were developed using a licensed version of BioRender.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4nr03137g

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