Structure-property optimization of Ezetimibe nanocrystals by computationally guided bottom-up engineering for enhanced bioavailability

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Pathade, Shubhangi A. Thool, Varsha B. Pokharkar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7488181/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in BioNanoScience → Version 1 posted 9 You are reading this latest preprint version Abstract Ezetimibe (EZT), a BCS class II drug, is a selective cholesterol absorption inhibitor used to treat high blood cholesterol. However, its clinical efficacy is limited by poor solubility and bioavailability. This study aims to address the low solubility and bioavailability of EZT. Aiming to improve the solubility, dissolution, and bioavailability of this hydrophobic drug by formulating EZT nanocrystals (EZT-NCs) using an innovative antisolvent precipitation-ultrasonication method. This bottom-up approach of optimizing structure and properties through particle size reduction, followed by lyophilisation, holds promises for enhanced therapeutic performance and effectiveness. Optimization of variables, including solvent: antisolvent ratio, poloxamer188 (P188) concentration, and ultrasonication amplitude, was achieved using Box-Behnken Design (BBD) as a computational tool, to produce uniform nanosized crystals with good dispersibility. Optimized EZT-NCs showeda particle size of 340±12.00 nm, PDI of 0.12±0.05, and zeta potential of -46±0.15 mV. DSC and pXRD confirmed reduced crystallinity. Scanning Electron microscopy (SEM) confirmed a nanometric size range, and in vitro dissolution revealed 85.17% release for EZT-NCs within 1 hour, a 1.87-fold increase over pure EZT. The everted gut sac model showed EZT-NCs had 5.23 times higher permeability than pure EZT, due to their nanometric size and P-gp inhibition by P188. Furthermore, EZT-NCs achieved a C max of 8.22μg/mL, with an AUC 0-48 that was 2.15 times higher than pure EZT. EZT-NCs demonstrated improved aqueous solubility, dissolution range, and bioavailability, suggesting their potential for an enhanced oral delivery approach. Ezetimibe Nanocrystals Antisolvent precipitation-ultrasonication Box-Behnken Design Oral bioavailability Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Ezetimibe (EZT), 1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one, is a selective inhibitor of cholesterol absorption.It effectively prevents the intestinal uptake of both dietary and biliary cholesterol without interfering with the absorption of fat-soluble vitamins and nutrients by blocking the Niemann-Pick C1-like l (NPC1L1) protein[ 1 , 2 ]It belongs to the BCS class II drug identified by high permeability and poor water solubility, with an oral bioavailability of approximately 35%, and is highly lipophilic, having a log p of 4.5[ 3 ]. Its low water solubility of 0.00846 mg/mL and the following slow dissolution rate are the causes of its varied bioavailability. Moreover, it has a significant efflux by phosphoglycoprotein (P-gp) [ 4 , 5 ]. As solubility is a crucial factor in effective drug therapy across all routes of delivery, pharmaceutical technologists face a significant obstacle when developing new pharmaceutical products, due to the high lipophilicity and low solubility of nearly 50% of active pharmaceutical ingredients (API) discovered by high-throughput screening[ 6 , 7 ]. Approximately 40% of drugs are poorly soluble, causing inadequate absorption and higher doses for therapeutic effects, potentially leading to toxicities and increasing drug development costs[ 8 ]. Over the years, numerous approaches have been developed to increase the oral bioavailability of these APIs-like solid dispersions [ 9 ], co-crystals [ 10 , 11 ] cyclodextrin inclusion complexes [ 12 ]. Self-microemulsifying drug delivery system (SMEDDS)[ 13 ], Self-nanoemulsifying drug delivery system (SNEDDS)[ 14 ]. The preparation of these formulations for therapies was laborious and time-consuming due to the high quantity of surfactants and co-surfactants, causing toxicological concerns. Challenges included restricted drug loading capacity, premature drug release, biocompatibility issues, and manufacturing complexity. Whereas, nanotechnology-based formulations showed significant potential with fewer drawbacks[ 15 ]. Nanocrystals have garnered significant interest as a nanotechnology-based strategy for the reduction of the size of drugs, to improve the solubility and bioavailability of poorly soluble drugs [ 16 ]. Nanocrystals are nanosized formulations that comprise nano-scaled drug particles stabilized by an appropriate stabilizer or surfactant. The average particle sizes of these formulations are less than 1 µm, ideally between 200 nm and 500 nm. Nanocrystals can exhibit various states, including fully crystalline, partially crystalline, or entirely amorphous.[ 17 – 19 ]. Nanocrystals gained widespread acceptance due to their distinguishing features, they are carrier-free, consisting entirely of the drug itself, and are easily scalable. Their significant advantages are the large specific surface area, under the equation of Noyes-Whitney, resulting in rapid dissolution, and by the Freundlich-Ostwald relation, to higher saturation concentration[ 20 – 22 ]. This promoted their use in formulation development, along with a high drug loading and nanoscale distribution. Nanocrystals are synthesized using two methods: top-down and bottom-up. Top-down methods use high-energy processes to break down the drug particles, while bottom-up methods use precipitation from a supersaturated drug solution. Top-down is more widely applicable but has limitations in particle size reduction efficiency, high energy input, and concerns about solid-state changes, chemical degradation, and residual metal content[ 23 ]. Therefore, we aimed to enhance the solubility and oral bioavailability of EZT by optimizing its structure-properties through the formulation of nanocrystals using the antisolvent precipitation ultrasonication method, a bottom-up approach designed to overcome the limitations of top-down techniques. The antisolvent precipitation-ultrasonication method is highly efficient, scalable, and environmentally friendly for pharmaceutical applications. This method offers solvent-free, stable nanocrystals with better particle size reduction without high energy input, making it a reliable and eco-friendly alternative to ball milling and the evaporative precipitation into the aqueous solution method, respectively[ 24 , 25 ]. Hence, for optimization, we have screened the different stabilizers to identify the most suitable contenders for inhibiting Ostwald ripening. Then, successfully implemented a Box Behnken Design (BBD) as a computational tool to identify the critical variables, following the characterization of optimized EZT nanocrystals (EZT-NCs). The pharmacokinetic parameters of EZT-NCs were tested in male Wistar rats to assess in vivo performance, and an everted gut sac model was employed to study the influence of P-gp efflux inhibitors on improving EZT bioavailability. We hypothesize that prepared EZT-NCs show significantly enhanced solubility, dissolution rate, and bioavailability compared to pure EZT. 2. Materials and Methods 2.1 Materials Ezetimibe was received from MSN Laboratories Pvt Ltd (Telangana, India). Soluplus®, Kolliphor EL, Vitamin E-TPGS, and Tween® 80 were kindly supplied as gift samples by BASF Corporation (Navi Mumbai, India). Sodium lauryl sulfate (SLS) and Poloxamer 188 (P188) were purchased from Sigma-Aldrich (Mumbai, India). PVP K-30 and HPMC E5 were purchased from LobaChemiePvt Ltd, India. Mannitol, Lactose, and Trehalose were received as gift samples from Signet Excipients Pvt Ltd (Mumbai, India). Merck (Mumbai, India) provided HPLC-grade methanol and acetonitrile. In all formulations, Milli Q water was used. All the remaining chemicals and reagents were of analytical grade. 2.2 Methods 2.2.1 Screening of Stabilizers 2.2.1.1 Screening using saturation solubility Saturation solubility studies within an aqueous stabilizer solution were used to screen suitable stabilizers. Anionic, polymeric, and non-ionic stabilizers from various categories were assessed utilizing a miniature shake flask method to ascertain the solubility of EZT in stabilizer solutions[ 26 ].EZT solubility was assessed in 1%w/v concentration aqueous solutions of Soluplus®, P188, Tween 80®, SLS, Vitamin E-TPGS, Kolliphor-EL, PVP K-30, and HPMC E5. Vials containing 5 mL of aqueous stabilizer solution were filled with an excess of the drug (10 mg), and the vials were continuously shaken with a mechanical shaker at 37°Cand 150 rpm for 48 hours. The resulting suspensions were separated and filtered through a 0.22 µm syringe filter(Whatman Inc., Clifton, NJ, USA) after being centrifuged for 10 minutes at 20,000 rpm (Allegra 64R centrifuge, Beckman-Coulter ™, USA). The absorbance of the filters at 232 nm was measured with a UV-visible spectrophotometer (Jasco V-730 Spectrophotometer), and the filtrate contents were examined for EZT. The solubility studies were carried out in triplicate, and data were expressed as mean value ± SD. 2.2.1.2 Screening using particle size The stabilizers were screened and selected for further studies on the basis of the EZT saturation solubility study results in the above-mentioned various stabilizer solutions. The EZT nanocrystals were prepared from these stabilizers and characterized for particle size and PDI using the dynamic light scattering technique (Nano ZS90, Malvern Instruments Pvt. Ltd., Malvern, Worcestershire, England, UK). All experiments were conducted in triplicate. 2.2.2 Preparation of EZT nanocrystals The EZT-NCs dispersions were prepared by a bottom-up approach, specifically the anti-solvent precipitation-ultrasonication method. In brief, the solvent phase consisted of 10 mg of the drug dissolved in 1 mL of ethanol. Later on, the solvent phase was introduced into the antisolvent phase, consisting of a P188 solution prepared using 15 mL of Milli Q water at a rate of 1 mL/min. A high-intensity ultrasonicator(Vibra-Cell, Sonics and Materials, Inc., Newtown, CT)was used to apply continuous sonication throughout the solvent phase addition for 10 min at 40% amplitude with a 10:05 on: off cycle, to balance effective cavitation with heat management, avoiding thermal degradation of heat-sensitive ezetimib with consistent particle size reduction and uniformity. Then, ethanol from the formulation was evaporated by stirring the formulation on a magnetic stirrer for 1 to 2 h. 2.2.3 Lyophilization of EZT-NCs suspension The nanocrystalline suspension was powdered by lyophilizing the EZT-NCs. The suspension was frozen for 24 hours at -80˚C using an ultra-low freezer (U101, Innova, New Brunswick Scientific) and lyophilized over 72 hours using a lyophilizer (INLABCO, model no INFD-3P) at − 40°C condenser temperature with a 0.45 mbar vacuum. This study tested lactose, mannitol, and trehalose at 1% w/v for their appearance and aggregation, determining the suitable cryoprotectant using the re-dispersibility index (RDI)[ 27 ]. The resulting lyophilized powder was used for further characterization. RDI can be calculated using Eq. 1: RDI = [D0/D] Eq. 1 Where D0 and D represent the mean particle size before lyophilization and after lyophilization, respectively. 2.2.4 Optimization of EZT-NCs with Design of Experiments (DoE) A three-factor, three-level BBD was applied for the optimization of formulation factors and the investigation of key effects, interaction effects, and quadratic effects on the characteristics of the EZT nanocrystals. By preliminary experiments Solvent: antisolvent ratio (A), the Concentration of stabilizer (B), and Ultrasonication amplitude (C) were investigated as independent variables at three levels of low (-1), medium (0), and high (+ 1). The particle size (nm) and PDI were considered as response (dependent) variables listed in Table 1 . Analysis of variance (ANOVA) was applied to statistically analyze the model, whereas equations were constructed using Design-Expert 13® software. The experiments were conducted randomly according to the recommendation of the software, with a batch prepared to calculate deviations between theoretical and practical results, and the relative error and three-dimensional (3D) response surface plots were created to illustrate variable effects[ 28 – 30 ]. Table 1 List of dependent and independent variables in BBD for EZT-NCs Independent Variables Levels Low -1 Medium 0 High + 1 1. Solvent: antisolvent ratio (ml) (A) 1:10 1:15 1:20 2. Concentration of P188 (mg) (B) 10 15 20 3. Ultasonication amplitude (%) (C) 30 40 50 Dependent variables Constraints 1. Particle Size (nm) (X) Minimum 2. PDI (Y) Minimum Minimum 3. Characterization of EZT-NCs 3.1 Saturation Solubility Studies The saturation solubility of pure EZT, PM of EZT with poloxamer 188, and lyophilized EZT-NCs were evaluated in different media, such as distilled water, 0.1N HCL pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8, as well as 0.05 M acetate buffer pH 4.5 with 0.45% SLS. The solubility determination followed the aforementioned method. Solubility tests were carried out in triplicate, and results were provided as mean ± SD. 3.2 Particle size, PDI, and zeta potential The prepared EZT-NCs were characterized for particle size (z-average), PDI, and zeta potential by the dynamic light scattering technique (Nano ZS90, Malvern Instruments Pvt. Ltd., Malvern, Worcestershire, England, UK) at 25°C. Before measurement, the samples were diluted byMilli-Q®water (Millipore, France) and analyzed in triplicate. 3.3 Fourier transform infrared spectrometry analysis (FT-IR) The FTIR spectra of pure EZT, mannitol, PM of EZT with P188, and lyophilized EZT-NCs were obtained using FTIR spectroscopy (Jasco4100, Japan). The potassium bromide (KBr) was heated in a hot air oven for 15–20 minutes to activate it. Each sample was then combined with KBr in a 3:1 ratio and placed into the cavity.FTIR spectra were acquired with 4 cm − 1 resolution, scanning from 4000 to 400 cm − 1 for 50 scans. 3.4 Differential Scanning Colorimetry (DSC) Thermal analysis of pure EZT, PM of EZT with P188, and lyophilized EZT-NCs was conducted using the DSC-60 Plus Calorimeter (DSC 60; Shimadzu Corporation). Samples of 3 mg each were sealed in aluminum pans and were heated from 30°C to 350°C at 10°C/min under a dry nitrogen purge of 30 mL/min to produce the thermograms. 3.5 Powder X-ray diffraction (pXRD) Powder X-ray diffraction patterns of pure EZT, mannitol, PM of EZT with P188, and lyophilized EZT-NCs were recorded in an X-ray diffractometer (Xpert Pro MPD, Panalytical, Netherlands) using CuKα radiation as an X-ray source. Samples were scanned for a 2θ range of 0° to 60° at a scanning speed of 2°/minute at an operating voltage of 40 kV and current of 30 mA. 3.6 Scanning Electron Microscopy (SEM) The morphology of pure EZT and EZT-NCs was examined under a scanning electron microscope (FEI, Quanta 200, Netherlands). Samples were clanged on a double adhesive tape and were gold plated by a sputter coater mounted on aluminum plates and observed at an acceleration voltage of -30 kV. 3.7 Drug content The lyophilized EZT-NCs, weighing 10 mg of EZT, were precisely measured and then dissolved in 10 mL of methanol. After vortexing the solution for 5 minutes, it was filtered with a 0.22 µm PTFE syringe filter (Whatman Inc., Clifton, NJ, USA), further diluted with methanol, and assayed for EZT by a UV-visible spectrophotometer at 232 nm. All the samples were assayed in triplicate, and mean values ± SD were reported for the results. Drug content determination was derived using the mathematical Eq. 2 Drug content (%) = ((Observed drug content)/(Theoretical drug content))×100 Eq. 2 3.8 In vitro drug release studies The in vitro dissolution studies of EZT-NCs were conducted and compared with pure EZT. Pure EZT and EZT-NCs (equivalent to 10 mg of EZT) were filled into gelatine capsules of capsule size #1, and dissolution was performed in USP Type I apparatus (Electrolab Dissolution tester, India) using a 500 mL 0.05M acetate buffer of pH 4.5 with 0.45% SLS at 37°C ± 0.5°C and with stirring at 50 rpm (USFDA recommended dissolution media for EZT)[ 31 ]. An aliquot of 5 mL was withdrawn at different time intervals. A fresh, equivalent volume of dissolution medium was immediately substituted. The collected aliquots were filtered and assayed spectro-photometrically at 232 nm. The percent cumulative drug release was plotted against the time profile. 3.9 Stability Study The stability of the optimized EZT-NCs was evaluated following ICH guidelines. Samples were stored at 25 ± 2°C and 60 ± 5% RH for three months. Particle size, PDI, and zeta potential were determined at 1, 2, and 3 months after re-dispersing the lyophilized EZT-NCs, along with visual inspections for physical stability. 3.10 Ex vivo Permeability Study The inhibitory effects of P-gp by the pure EZT and EZT-NCs were assessed using an everted rat gut sac model. The study involved cleaning a 4 cm segment of the small intestine of a male Wistar rat with Krebs-Ringer Buffer (KRB) and aerating with an electric aerator. The segment was then filled with the drug suspension and EZT-NCs (1mg/mL). The tissue was then placed in 100 mL KRB solution, aerated with atmospheric air, kept at 37 ± 0.5°C, and stirred at 50 rpm. Samples were removed and replaced with fresh KRB solution. Permeability was studied for 60 minutes, and drug permeation was measured at 232 nm using a UV spectrophotometer[ 32 ]. The permeation flux (J, µg min⁻¹) was estimated based on the slope of the linear regression analysis, and the apparent permeability coefficient was obtained using Eq. 3. P app =J/(A× C 0 ) Eq. 3 Where J is the permeation flux, A is the intestinal surface area, and C 0 is the initial drug concentration. 3.11 Invivo pharmacokinetic study 3.10.1 Animals and dose administration Male Wistar rats were procured from Global Bioresearch Solutions, Pune, India, weighing 200–250 g, all animal experiments received prior approval from the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Bharati Vidyapeeth Deemed University (PCP/IAEC/2024/2–18), which is registered with the committee for Control and Supervision of Experiments on Animals, Government of India. Animals were housed in standard laboratory conditions with pelletized feed and filtered water, and abstained from 12 hours before the study, with daily health monitoring. The study involved two groups of nine rats each, each group receiving pure EZT and EZT-NCs, respectively, at a dosage of 3 mg/kg orally in a 0.75% w/v sodium CMC solution[ 33 – 35 ].0.5 mL of blood samples were collected from the retro-orbital plexus at specific intervals (1, 2, 4, 8, 10, 12, 24, 36, 48 hrs). The plasma was separated by centrifuging the samples in heparinized Eppendorf tubes at 8000 rpm for 15 minutes and then stored at -20 ◦C. 3.10.2 Preparation of plasma sample and data analysis Plasma samples were deproteinized using a methanol precipitation method, followed by centrifugation and injection into the HPLC system. Chromatography was conducted using a Jasco PU 1580 system and Borwin software, with separations on a Thermo C-18 column using 50:50 acetonitrile: water as the mobile phase, with slight modifications to the previously reported method[ 35 ]. Compounds were detected with a flow rate of 1 mL/min and an injection volume of 20µL at 232 nm using a UV detector. Pharmacokinetic parameters were analyzed using PKSolver (version 2.0) with a non-compartmental model and linear trapezoidal approach, presented as mean ± S.D.Estimated parameters included the time to reach maximum concentration (T max ), maximum drug concentration (C max ), area under the curve from zero to 48 hours (AUC 0 − 48 ), area under the curve from zero to infinity (AUC 0−∞ ), and half-life (t 1/2 ). 3.12 Statistical analysis The results for the in vivo pharmacokinetic studies were statistically analyzed using GraphPad Prism® version 5. Two-way ANOVA was used, followed by a Bonferroni post-hoc test. The findings were presented as means ± SD, and a p-value < 0.05 indicated significance. 4. Results and Discussion 4.1 Screening of stabilizers In the nanosizing process, stabilizers are initially screened based on their drug solubilising ability, which helps reduce surface free energy and prevent particle agglomeration, thereby ensuring nanosuspension stability [ 37 ]. It is postulated in the Lifshitz-Slyozov-Wagner (LSW) theory that the drug concentration in the dispersed phase directly affects Ostwald ripening. The drug solubility in the stabilizer solution creates a concentration gradient, causing smaller solubilized particles to migrate towards larger particles, resulting in particle growth, agglomeration, and crystallization. Therefore, for nano-colloidal systems, stabilizers should minimally affect drug solubility[ 38 – 40 ]. Stabilizers were screened according to their saturation solubility in aqueous solutions. Figure 3 (a) illustrates that the intrinsic aqueous solubility of EZT was minimally impacted by 1% w/v P188, HPMC E5, PVP K30, and SLS by providing critical steric and electrostatic stabilization, while Soluplus®, Kolliphor-EL, Tween 80® , and Vitamin E-TPGShad a significant effect on it due to their surfactant properties. As a result, P188, HPMC E5, PVP K30, and SLS were selected for further studies because they contribute to stability without significantly altering solubility, ensure narrow particle size distribution and robust physical stability, essential for consistent in-vivo performance and enhanced bioavailability. The initial experimental batches were prepared using the four stabilizers that had been screened earlier (Table 2 ). Following nanosuspension with P188 (USFDA-approved as a GRAS) showed the smallest particle size and PDI, along with the highest zeta potential. It is an amphiphilic block copolymer consisting of a hydrophobic polypropylene oxide segment that attaches to the drug surface and its hydrophilic polyethylene oxide segment, forming steric hindrance to suppress particle agglomeration and promote dispersibility and colloidal system stability. Hence, P188 was selected as the single steric stabilizer due to its ability to prevent submicron particle agglomeration [ 41 , 42 ]. Table 2 Experimental batches with screened stabilizers EZT Stabilizer Particle size (nm) PDI Zeta potential (mV) 10 mg 10 mg P188 605 ± 7.57 0.26 ± 0.17 − 45 ± 0.05 10 mg 10 mg HPMC E5 1021 ± 10.15 0.22 ± 0.21 -22 ± 0.07 10 mg 10 mg PVP K30 807 ± 21.00 0.35 ± 0.12 -43 ± 0.24 10 mg 10 mg SLS 988 ± 9.04 0.26 ± 0.11 -66 ± 0.12 4.2 Lyophilization of EZT-NCs suspension Lyophilization improves stability and extends shelf life by keeping the product in a dry form. Cryoprotectants prevent the destabilization of the colloidal dispersion of nanocrystals caused by the stress of freeze-drying and dehydration during lyophilisation [ 43 ]. Various cryoprotectants, including mannitol, lactose, and trehalose, were screened and analyzed based on product appearance (Table 3 ). From the above results, mannitol was found to be the most effective in forming a fluffy, free-flowing, and well-formed dried powder. This is attributed to the high collapse temperature of mannitol, which retained the porous structure with a cake-like appearance for the product[ 44 ]. Therefore, mannitol was selected as a suitable cryoprotectant and further screened for concentrations ranging from 0.5 to 1.5% (w/v). Table 3 Screening of cryoprotectants Cryoprotectant Concentration of cryoprotectant (%w/v) Appearance Mannitol 1 Fluffy and free-flowing Lactose 1 Aggregated and Sticky Trehalose 1 Aggregated The particle size and RDI of the reconstituted lyophilized aqueous dispersion of nanocrystals were measured (Table 4 ). Increasing the mannitol concentration beyond 1% (w/v) results in a linear increase in nanocrystal size, while insufficient cryoprotectant concentration of 0.5% (w/v) leads to incomplete coating and particle aggregation, both contributing to larger particle sizes. The addition of 1% (w/v) mannitol into the lyophilized aqueous dispersion of nanocrystals effectively prevents aggregation, resulting in an average particle size of 402 ± 23.25 nm. An RDI value close to 1 indicates that the nanoaggregates are fully re-dispersible. Therefore, 1% mannitol was determined to be the optimal concentration based on particle size and RDI [ 45 ]. Table 4 Effect of varying cryoprotectant concentrations on particle size and RDI Mannitol concentration (%w/v) Particle Size (nm) RDI 0.5 541 ± 10.82 1.12 1.0 402 ± 23.25 1.06 1.5 446 ± 36.38 1.14 4.3 Optimization of EZT-NCs with Design of Experiments (DoE) BBD was selected for the current study in order to examine both the main and interaction impacts of independent variables on the dependent variables. When there are three factors or more than three factors, BBD is highly effective in minimizing the number of runs compared to the central composite design. This approach evaluated the impact of Solvent: antisolvent ratio (A), P188 concentration (B), and Ultrasonication amplitude (C) on particle size (X) and PDI (Y). Seventeen runs were generated with five center points(Table 5 ). Multiple regression analysis was performed using Design-Expert 13® software to generate polynomial models, including linear, two-factor interaction, and quadratic. The model with the highest R², adjusted R², and predicted R² values were selected for optimal fit Table 6 . ANOVA and 3D plots were used to evaluate the impact of independent variables on dependent variables. Table 5 Box Behnken Design with observed responses Run Solvent: antisolvent ratio (ml) Concentration of P188 (mg) Ultrasonication amplitude (%) Particle Size (nm) PDI Zeta potential (mV) 1 1:20 15 50 531 ± 11.55 0.65 ± 0.03 -42 ± 0.15 2 1:15 20 50 825 ± 16.57 1.08 ± 0.05 -41 ± 0.25 3 1:10 15 30 455 ± 09.55 0.5 ± 0.06 -36 ± 0.25 4 1:15 15 40 340 ± 12.00 0.12 ± 0.05 -46 ± 0.15 5 1:10 15 50 642 ± 02.57 0.74 ± 0.02 -47 ± 1.15 6 1:15 15 40 392 ± 27.08 0.12 ± 0.01 -46 ± 0.60 7 1:20 20 40 750 ± 14.51 0.69 ± 0.05 -47 ± 0.50 8 1:20 10 40 761 ± 13.15 0.44 ± 0.06 -45 ± 0.50 9 1:15 10 50 600 ± 13.28 0.98 ± 0.03 -46 ± 0.17 10 1:15 15 40 342 ± 19.57 0.15 ± 0.01 -46 ± 0.15 11 1:15 15 40 382 ± 10.00 0.14 ± 0.01 -45 ± 0.28 12 1:15 15 40 388 ± 18.57 0.13 ± 0.01 -47 ± 0.05 13 1:10 20 40 750 ± 11.73 0.36 ± 0.06 -41 ± 0.23 14 1:10 10 40 640 ± 28.15 0.83 ± 0.02 -39 ± 0.05 15 1:15 10 30 731 ± 38.73 0.94 ± 0.02 -37 ± 0.23 16 1:20 15 30 640 ± 11.52 0.45 ± 0.05 -42 ± 0.17 17 1:15 20 30 631 ± 17.02 0.72 ± 0.06 -41 ± 0.52 Table 6 Model Fit Summary statistics for Particle size and PDI Response Model R² Adjusted R² Predicted R² Particle Size Quadratic 0.9930 0.9840 0.9760 PDI Quadratic 0.9983 0.9961 0.9785 Table 7 Optimized parameters along with predicted and observed values of responses Factors Predicted value Observed value Solvent: antisolvent ratio (ml) Concentration of P188 (mg) Ultrasonication amplitude(%) Particle Size (nm) PDI Particle Size (nm) PDI 1:15 15 40 368 0.12 340 ± 12.00 0.12 ± 0.05 4.3.1 Assessment of the design of the experiment The significance of each coefficient for the main effects and interaction terms in this BBD was evaluated using appropriate polynomial model equations and p-values. A lower p-value (p < 0.05) indicates a more significant coefficient, highlighting the substantial effect of the corresponding independent variables. In the regression equation, a negative value indicates an antagonistic influence on the dependent variable, whereas a positive value indicates a synergistic effect. 4.3.2 Effect of independent variables on particle size One important factor influencing the solubility of nanocrystals is particle size, which in turn improves the drug's bioavailability. The particle size of all 17 batches was analyzed, and it was found to be in the range of 340 ± 12.00 to 825 ± 16.57 nm (Table 5 ). The quadratic model suggested by BBD to describe the effect on particle size is significant with P < 0.05, while the lack of fit is not significant. The relationship of coded factors with particle size was evaluated by equations 4 and 3D plots. Particlesize = 368.8 + 24.375A-28B + 17.625C -30.25AB-74AC + 81.25BC + 113.35A 2 + 243.1B 2 + 84.85C 2 Eq. 4 In this polynomial Eq. 4, the model terms A, B, C, AB, AC, BC, A², B², and C² are significant (P < 0.05). Synergistic and antagonistic effects on the observed responses are represented by the positive and negative signs in the equation. According to the equation, it can be seen that the concentration of P188 (B) has an antagonistic effect that causes the particle size to decrease as factor B increases, while the solvent: antisolvent ratio (A) and ultrasonication amplitude (C) have an antagonistic effect on the particle size that causes the particle size to increase as factors A and B increase. However, these two factors, A and B, at certain levels may interact with other factors in a way that counteracts their positive influence. This complexity highlights the importance of considering interaction effects and not relying solely on the main effects. The 3D plots shown in Fig. 1 illustrate the effect of independent variables on particle size. As the solvent: antisolvent ratio increases from lower to higher levels, particle size initially increases, then decreases at an intermediate level, and finally increases again at higher levels. Similarly, as the concentration of P188 increases, particle size follows the same trend. This behavior is attributed to a lower solvent: antisolvent ratio, which reduces supersaturation, leading to less efficient mixing and slower nucleation, promoting particle growth. However, as the solvent-to-antisolvent ratio increases, the degree of supersaturation also increases, enhancing the nucleation rate and leading to the formation of smaller particles. Beyond the optimum level, further increases in the solvent: antisolvent ratio result in more nuclei, reducing the diffusion of particles and increasing collisions, which ultimately leads to larger particle sizes[ 44 , 47 ]. Lower concentrations of P188 result in inadequate stabilization due to insufficient adsorption and incomplete coverage of newly formed nanosized particles, leading to crystal growth and increased particle size. At higher concentrations, P188 forms a thicker adsorbed layer on the drug surface, resulting in slightly larger particles [ 48 ]. Similarly, the amplitude of ultrasonication is crucial for the efficient mixing of solvent and antisolvent. This is because the process generates cavitation bubbles through cycles of compression and rarefaction in the liquid dispersion medium. The collapse of these bubbles creates intense shock waves that impart high velocities to the suspended particles. Increasing the amplitude induces the solvent and antisolvent to mix more effectively with a steady reduction of particle size, leading to greater supersaturation and nucleation, and thereby resulting in colloidal particles. However, if the amplitude intensity is increased beyond an optimal point, particle size may slightly increase due to greater hindrance among newly formed particles, resulting in agglomeration [ 49 ]. Overall, the optimized conditions for particle size reduction directly contribute to nanocrystal stability by minimizing aggregation during storage and maintaining uniformity. Adequate P188 concentration and optimal ultrasonication amplitude prevent particle growth and agglomeration, thereby ensuring long-term stability and efficient drug delivery, which collectively enhances in vivo performance and therapeutic efficacy. 4.3.3 Effect of independent variables on PDI As a dimensionless parameter, PDI measures the distribution of particle sizes, ranging from 0 to 1. Values greater than 0.5 indicate a broad particle distribution, leading to Ostwald ripening. The developed formulations exhibited PDI values ranging between 0.12 ± 0.05 to 1.08 ± 0.05. BBD suggested that the model describing the effect on PDI is quadratic; P < 0.05 indicates that the model is significant, whereas the lack of fit is not significant. The relationship of coded factors to PDI was assessed by Eqs. 5 and 3D plots. PDI = 0.1320- 0.0250A − 0.0425B + 0.1050C + 0.1800AB- 0.0100AC + 0.0800BC + 0.0515A 2 + 0.3965B 2 + 0.4015C 2 Eq. 5 The model terms A, B, C, AB, BC, A², B², and C² had significant (P < 0.05) effects on the response. According to the response surface plots in Fig. 2 , the solvent: antisolvent ratio, P188 concentration, and ultrasonication amplitude significantly affected PDI, with notable interactive effects. As the solvent: antisolvent ratio increased, slight changes in PDI were noted. At the optimal ratio, high supersaturation led to uniform nuclei distribution and lower crystal growth rates, resulting in a narrower size distribution[ 50 ].PDI was greatly influenced by P188 levels and ultrasonication amplitude. Higher P188 concentrations decreased particle size and PDI by inhibiting steric hindrance, as lower stabilizer levels slowed stabilizer migration, causing insufficient surface stabilization and larger PDI. Also, higher P188 levels provided a thicker nanoparticle coating, enhancing stability. However, as the ultrasonication amplitude increased, monodispersity was observed up to some extent, but later particles were subjected to higher energies, and due to greater attrition, they lost their repulsive forces and agglomerated, resulting in higher PDI[ 51 ]. Where lower PDI ensures minimal aggregation risks, enhanced solubility, and dissolution rate. 4.3.4 Selection of Optimized Formulation The analysis of variables of formulation showed that there is a strong relationship between solvent: antisolvent ratio, P188 concentration, ultrasonication amplitude, particle size, and PDI. 3D response surface diagrams were plotted to emphasize the effects of the solvent: antisolvent ratio, P188 concentration, and ultrasonication amplitude interactions on particle size and PDI. Finally, the optimal formulation was determined through the point prediction approach in the softwareTable 7. The optimized EZT-NCs dispersion has shown a particle size of 340 ± 12.00 and PDI of 0.12 ± 0.05 using the composition solvent: antisolvent ratio (1:15), P188 concentration (15mg), ultrasonication amplitude (40%), and was used for further study. 4.3.5 Determination of Zeta Potential Zeta potential is a key indicator of nanocrystal dispersion stability. Values greater than + 30 mV or less than − 30 mV indicate stability, as higher zeta potential values signify stronger repulsive forces among the particles, which in turn contribute to the stability of the suspension by preventing aggregation. The zeta potential of optimized EZT-NCs was − 46 ± 0.15 mV. All formulations showed a zeta potential ranging from − 36 ± 0.25 mV to -47 ± 1.15 mV (Table 5 ). These values implied that P188 provided sufficient coverage on the nanocrystal surfaces, contributing to a stable dispersion. 5. Characterization of EZT nanocrystals 5.1 Saturation solubility Studying solubility at different pH levels and in distilled water provides crucial insights into drug behavior, aiding in the absorption prediction and the selection of an appropriate dissolution medium. The study compared the saturation solubility of EZT-NCs to pure EZT and PM of EZT with P188 in five different media: distilled water, 0.1N HCL pH 1.2, acetate buffer pH 4.5, phosphate buffer pH 6.8, and 0.05 M acetate buffer pH 4.5, finding that the solubility was 1.2–2.5 and 1.8–2.2 fold to that of EZT and PM of EZT with P188 in different media, respectively. The enhanced solubility of EZT-NCs is attributed to their smaller particle sizes. Nanoinization increases saturation solubility, as per the Ostwald-Freundlich and Noyes-Whitney equations[ 52 ]. Kelvin’s equation also explains that the strong curvature of nanosized particles increases dissolution pressure[ 53 ]. Thus, results highlight the critical role of particle size reduction to enhance the aqueous solubility and oral bioavailability[ 54 , 55 ]. 5.2 Fourier transform infrared spectrometry analysis FT-IR spectra of pure EZT, P188, PM of EZT with P188, and lyophilized EZT-NCs are illustrated in Fig. 1 (b). The FTIR analysis of pure EZT showed characteristic peaks of O − H stretching in alcohols at 3287.07 cm − 1 , C = O stretching of β-lactam ring at 1717.3 cm − 1 , C − H stretching in alkanes at 2916.42 cm − 1 , and p-substituted benzene ring vibration at 830.20 cm − 1, aligning with the literature data[ 56 ]. Mannitol exhibited O-H stretching vibrations with intermolecular H-bonds and C-H stretching vibrations. PM indicated characteristic peaks of P188, O-H stretching, and C-O stretching, respectively[ 57 , 58 ]. No spectral shift was noted in PM, suggesting no drug-stabilizer interaction. However, the interaction between drugs and stabilizers, especially through hydrogen bonding, significantly impacts characteristic peaks. The IR spectrum of the EZT-NCs formulation showed a shift in the carboxylic – OH peak, and a less prominent C = O stretching band at 1717 cm − 1 , likely due to hydrogen bonding between the C = O group of EZT and the -OH group of P188. This H-bonding was confirmed by stabilizer adsorption onto the drug crystal surface. The EZT-NCs showed characteristic peaks without any additional peaks, showing the chemical stability of the drug and stabilizer. 5.3 Differential Scanning Calorimetry Thermograms of pure EZT, mannitol, PM of EZT and P188, and EZT-NCs were depicted in Fig. 3 (c). The DSC thermogram of EZT indicated a characteristic sharp endothermic peak at 164.5°C, indicating the crystalline nature of pure EZT. The PM displayed distinct endothermic peaks at 51.5°C and 163.9°C, representing the presence of P188 and EZT, respectively. Mannitol exhibited an endothermic peak at 170.4°C, also reflecting its crystalline nature. However, the DSC analysis revealed an endothermic peak at 169.3°C for EZT-NCs, resembling the peak for mannitol. The significant amount of mannitol used as a cryoprotectant caused a dilution effect, masking the endothermic peak of the free drug. This disappearance of the EZT peak indicates complete drug encapsulation within the stabilizer matrix, suggesting that the stabilizer effectively coats and incorporates the drug molecules[ 59 ]. 5.4 Powder X-ray Diffractogram The pXRD pattern of the pure EZT, mannitol, PM of EZT with P188, and EZT-NCis is illustrated in Fig. 3 (d). Pure EZT displayed distinct high-energy diffraction peaks with 2θ values ranging from 8.04° to 32.56°, confirming its crystalline nature. The PM also showed characteristic peaks of EZT and P188 at 8.20° to 31.22°, 19.35°, and 23.48°, respectively, but with lower intensity. Whereas EZT-NCs exhibited diffraction peaks from 9.95° to 44.13°. The only variation noted between the pure EZT, PM of EZT with P188, and EZT-NCs is in the peak intensities. The variations in the relative peak intensities may be caused by the smaller particle size after size reduction, indicating the transformation of EZT into nanocrystals with reduced crystallinity, which is consistent with the DSC data. The decrease in peak intensity suggests amorphization[ 60 ]. The pXRD diffractograms also revealed that mannitol, which was employed in the lyophilization process, exhibited high-energy diffraction peaks within 2θ values ranging from 10.85° to 45.46°. These peaks obscured the characteristic diffraction peaks of EZT in the lyophilized formulation. This distinct diffraction pattern suggests that mannitol exists in a crystalline form rather than in an amorphous form, important for the long-term stability of nanocrystals[ 61 ]. 5.5 Scanning Electron Microscopy The surface morphology and shape were studied using SEM for pure EZT and its nanocrystals. Accordingly, Fig. 3 (e. A)depicts the pure EZT as an irregular, cylindrical, and rod-shaped crystalline structure. In contrast, the EZT-NCs exhibited a slightly spherical structure with smoother edges, Fig. 3 (e. B). This slight spherical shape of nanocrystals is potentially associated with the reduction in size to the nanoscale and the adsorption of P188 on hydrophobic drug particles. The slightly smoother surface and altered morphology of EZT-NCs result from stabilizer surface coating and lyophilization. Mannitol and P188 on the nanocrystal surface contribute to this effect, with P188 acting as a steric barrier to prevent particle agglomeration[ 62 ]. 5.6 Determination of Drug Content The analysis of the drug content in the optimized EZT-NCs showed 96.27 ± 0.96% of EZT content in the EZT-NCs formulation. The high drug content of nanocrystals is an enormous advantage, as the delivery system is free of carrier systems. 5.7 In vitro drug release study The dissolution profiles of pure EZT and EZT-NCs are depicted in Fig. 4 (a). Dissolution profiles revealed that pure EZT exhibited a release of 45.55% within 1 hour, whereas EZT-NCs achieved an 85.17% release within the same timeframe of 1 hr, indicating a 1.869-fold increase in drug release as compared to the pure drug. The increased dissolution rate is attributed to the reduced size of EZT-NCs, resulting in a larger surface area and thinner diffusion layer, which further reduces nanocrystal agglomeration and improves wetting and dispersibility[ 63 ]. According to the Prandtl equation, smaller particles have larger curvature, reducing diffusion distance and increasing dissolution velocity. As particle size decreases below 1 µm, the dissolution pressure and saturation solubility significantly increase[ 64 – 66 ]. Additionally, the existence of P188 at the drug-aqueous phase interface further aids dissolution by reducing surface tension through hydrogen bonding with water molecules, enhancing drug release[ 67 ]. 5.8 Stability Studies The optimized EZT-NCs, stored at room temperature (25 ± 2°C and 60 ± 5% relative humidity) for three months, were evaluated for their physical and chemical stability to determine the limits of their stability with storage recommendations. Table 8 illustrates the findings from the stability study of samples maintained at room temperature, indicating no significant alterations in particle size, PDI, and zeta potential. Table 8 Stability studies of EZT-NCs Sr. no Storage condition Storage time (months) Particle size (nm ± SD) PDI Zeta potential (mV ± SD) 1 Roomtemperature (25 ± 2°C/60 ± 5% RH) 0 379 ± 15.24 0.14 ± 0.05 -46 ± 0.15 1 385 ± 10.12 0.19 ± 0.08 -47 ± 0.21 2 391 ± 12.05 0.21 ± 0.04 -46 ± 0.32 3 394 ± 12.23 0.22 ± 0.03 -45 ± 0.24 5.9 Ex vivo Permeability Study An everted gut permeability study was performed to measure the intestinal permeation of EZT from pure EZT dispersion and EZT-NCs suspension. The ex vivo gut sac model predicts intestinal transport accurately due to its enzyme activity and transporter expression closely mirroring the human intestine. In the intestine, large amounts of P-gp efflux transporter are present; hence, to check the P-gp inhibitor activity of P188, this study was performed. In this study, the exsorption of EZT dispersion and EZT-NCs from serosal medium to mucus medium was evaluated. This study concluded that a greater amount of drug permeated into the mucous medium than the pure EZT dispersion shown in Fig. 4 (b), indicating that permeation of P188 incorporated EZT-NCs was more than that of pure EZT dispersion because of P-gp inhibition caused by P188. The apparent permeability coefficient (P app ) of EZT-NCs(5.73×10 − 5 cm/min) was approximately 5.23-fold higher than compared that of the pure EZT dispersion (1.09×10 − 5 cm/min). The increased permeability of EZT-NC can be ascribed to their nanometric size and the inhibitory effects of P188 on P-gp [ 68 – 70 ]. 5.10 In vivo pharmacokinetic study The in vivo pharmacokinetic study was performed to assess in vivo bioavailability. After oral administration of pure EZT and EZT-NCs, plasma concentrations were obtained and graphically illustrated in Fig. 5 . Also, the pharmacokinetic parameters determined from non-compartmental analysis are presented in Table 9 . The oral administration of pure EZT and EZT-NCs resulted in a C max of 3.10 ± 0.46µg/mL and 8.22 ± 0.50 µg/mL after 2 hours (T max ), respectively. The AUC 0 − 48 values for pure EZT and EZT-NCs were 95.65 (µg/mL*h) and 205.8 (µg/mL*h), respectively. Table 9 Pharmacokinetic parameters of EZT and EZT-NCs Sample Pharmacokinetic parameters C max (µg/ml) T max (h) AUC 0 − 48 (µg/ml*h) AUC 0−∞ (µg/ml*h) Dispersion of EZT 3.10 ± 0.46 2 95.65 ± 9.43 293.10 ± 23.98 EZT-NCs 8.22 ± 0.50 2 205.80 ± 27.65 299.47 ± 33.76 This suggests that the AUC 0 − 48 of EZT-NCs was 2.15 times higher than that of pure EZT when administered orally (p < 0.001). Thus, C max and AUC of the EZT-NCs demonstrated increased EZT bioavailability using the EZT-NCs formulation. This increased bioavailability could be related to the reduction of particle size to nanocrystals, significantly enhancing the dissolution rate. This improvement is attributed to a larger surface area, entanglement with the mucus layer, greater saturation solubility, and a higher concentration gradient between the GI lumen and blood[ 71 ] Furthermore, the potential of poloxamers to increase permeability by modifying the micro-viscosity of the cellular membrane contributed to improved bioavailability[ 72 ] These factors collectively lead to faster and more complete drug absorption, resulting in higher C max and AUC values. Conclusion The study successfully prepared EZT-NCs using the antisolvent precipitation–ultrasonication method, a bottom-up approach for size reduction. P188 was identified as an effective stabilizer for nanonizing EZT. Computational BBD design pinpointed significant parameters affecting response variables. The EZT-NCs demonstrated higher saturation solubility and dissolution rate than pure EZT. Everted gut sac results and in vivo study showed a considerable enhancement over those of the pure EZT, hence bioavailability can be effectively achieved using nanocrystals. In the future, it is crucial to carry out pharmacodynamics studies to validate additional therapeutic evidence and evaluate the potential enhancement of current clinical effectiveness using EZT-NCs. Declarations Acknowledgments The authors would like to thank BVDU, Poona College of Pharmacy, Pune, India, for providing research facilities. Funding This work does not involve any funding sources. Conflict of Interest The author(s) declare(s) that they have no conflicts of interest to disclose. Financial interests The authors declare they have no financial interests. Author Contributions Pratiksha Pathade: Writing original draft, drawing figures, and validation. Shubhangi Thool: Methodology, data curation, review, and editing. Varsha Pokharkar: Supervision, editing, resources, and review. 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Ultrasonication of chitosan nanoparticle suspension: Influence on particle size. Colloids Surf APhysicochem Eng Asp . 462:75-81. https://doi.org/10.1016/j.colsurfa.2014.08.028 Müller RH, Peters K. (1992). Nanosuspensions for the formulation of poorly soluble drugs: I. Preparation by a size-reduction technique. Int J pharm . 82(3):R7-10. https://doi.org/10.1016/0378-5173(92)90184-4 Buckton G, Beezer AE. (1992). The relationship between particle size and solubility. Int J pharm . 82(3):R7-10. https://doi.org/10.1016/0378-5173(92)90184-4 Gao L, Zhang D, Chen M, Zheng T, Wang S. (2007). Preparation and characterization of an oridonin nanosuspension for solubility and dissolution velocity enhancement. Drug Dev Ind Pharm .33(12):1332-9. https://doi.org/10.1080/03639040701741810 Cheng M, Yuan F, Liu J, Liu W, Feng J, Jin Y, Tu L. (2020). Fabrication of fine puerarin nanocrystals by Box–Behnken Design to enhance intestinal absorption. Aaps Pharmscitech.21:1-2. https://doi.org/10.1208/s12249-019-1616-4 Górniak A, Czapor-Irzabek H, Złocińska A, Karolewicz B. (2021). Physicochemical and dissolution properties of ezetimibe–aspirin binary system in development of fixed-dose combinations. J Therm Anal Calorim. 144(4):1219-27. https://doi.org/10.1007/s10973-020-09543-9 Pardhi VP, Jain K. (2021). Impact of binary/ternary solid dispersion utilizing poloxamer 188 and TPGS to improve pharmaceutical attributes of bedaquiline fumarate. J Drug Deliv Sci Technol. . 62:102349. https://doi.org/10.1016/j.jddst.2021.102349 Parmar KR, Shah SR, Sheth NR. (2011). Preparation, characterization, and in vitro evaluation of ezetimibe binary solid dispersions with poloxamer 407 and PVP K30. J Pharm Innov . 107-14.https://doi.org/10.1007/s12247-011-9104-8 Feng J, Zhang Y, McManus SA, Qian R, Ristroph KD, Ramachandruni H, Gong K, White CE, Rawal A, Prud'homme RK. (2019). Amorphous nanoparticles by self-assembly: processing for controlled release of hydrophobic molecules. Soft Matter. 15(11):2400-10. https://doi.org/10.1039/C8SM02418A Dizaj SM, Lotfipour F, Barzegar-Jalali M, Zarrintan MH, Adibkia K. (2015). Box-Behnken experimental design for preparation and optimization of ciprofloxacin hydrochloride-loaded CaCO3 nanoparticles. J Drug Deliv Sci Technol. 29:125-31. https://doi.org/10.1016/j.jddst.2015.06.015 Al Hazzaa SA, Rajab NA. (2023). Cilnidipine nanocrystals, formulation and evaluation for optimization of solubility and dissolution rate. Iraqi Journal of Pharmaceutical Sciences. 32(Suppl.):127-35. https://doi.org/10.31351/vol32issSuppl.pp127-135 Torge A, Grützmacher P, Mücklich F, Schneider M. (2017). The influence of mannitol on morphology and disintegration of spray-dried nano-embedded microparticles. Eur. J. Pharm. Sci . 104:171-9. https://doi.org/10.1016/j.ejps.2017.04.003 Sun, J., Wang, F., Sui, Y., She, Z., Zhai, W., Wang, C., & Deng, Y. (2012). Effect of particle size on solubility, dissolution rate, and oral bioavailability: evaluation using coenzyme Q 10 as naked nanocrystals. Int. J. Nanomedicine , 7 , 5733–5744. https://doi.org/10.2147/IJN.S34365 Salehi N, Al-Gousous J, Mudie DM, Amidon GL, Ziff RM, Amidon GE. (2020). Hierarchical mass transfer analysis of drug particle dissolution, highlighting the hydrodynamics, pH, particle size, and buffer effects for the dissolution of ionizable and nonionizable drugs in a compendial dissolution vessel. Mol. Pharm . 4;17(10):3870-84. https://doi.org/10.1021/acs.molpharmaceut.0c00614 Müller RH, Peters K. (1998). Nanosuspensions for the formulation of poorly soluble drugs: I. Preparation by a size-reduction technique. Int J pharm. 160(2):229-37. https://doi.org/10.1016/S0378-5173(97)00311-6 Fülöp V, Jakab G, Tóth B, Balogh E, Antal I. Study on optimization of wet milling process for the development of albendazole containing nanosuspension with improved dissolution. Period. Polytech. Chem. Eng .https://doi.org/10.3311/PPch.15569 Pawar VK, Gupta S, Singh Y, Meher JG, Sharma K, Singh P, Gupta A, Bora HK, Chaurasia M, Chourasia MK. (2015). Pluronic F-127 stabilised docetaxel nanocrystals improve apoptosis by mitochondrial depolarization in breast cancer cells: pharmacokinetics and toxicity assessment. J Biomed Nanotechnol .11(10):1747-63. https://doi.org/10.1166/jbn.2015.2158 Gao W. Precision Nanometrology. London: Springer London; 2010. Nguyen TT, Duong VA, Maeng HJ. (2021). Pharmaceutical formulations with P-glycoprotein inhibitory effect as promising approaches for enhancing oral drug absorption and bioavailability. Pharmaceutics . 13(7):1103. https://doi.org/10.3390/pharmaceutics13071103 Lotfy NS, Borg TM, Mohamed EA. (2021). The promising role of Chitosan–poloxamer 188 nanocrystals in improving diosmin dissolution and therapeutic efficacy against ferrous sulfate-induced hepatic injury in rats. Pharmaceutics . 13(12):2087. https://doi.org/10.3390/pharmaceutics13122087 Thadkala K, Nanam PK, Rambabu B, Sailu C, Aukunuru J. (2014). Preparation and characterization of amorphous ezetimibe nanosuspensions intended for enhancement of oral bioavailability. Int J Pharm Investig . 4(3):131. https://doi.org/10.4103/2230-973X.138344 Li M, Si L, Pan H, Rabba AK, Yan F, Qiu J, Li G. (2011). Excipients enhance intestinal absorption of ganciclovir by P-gp inhibition: assessed in vitro by everted gut sac and in situ by improved intestinal perfusion. Int J Pharm . 403(1-2):37-45. https://doi.org/10.1016/j.ijpharm.2010.10.017 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in BioNanoScience → Version 1 posted Editorial decision: Revision requested 16 Oct, 2025 Reviews received at journal 24 Sep, 2025 Reviews received at journal 17 Sep, 2025 Reviewers agreed at journal 09 Sep, 2025 Reviewers agreed at journal 06 Sep, 2025 Reviewers invited by journal 06 Sep, 2025 Editor assigned by journal 03 Sep, 2025 Submission checks completed at journal 02 Sep, 2025 First submitted to journal 29 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7488181","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512661931,"identity":"8bb3435e-1367-4fcf-b585-141e57baa6c2","order_by":0,"name":"Pratiksha R. Pathade","email":"","orcid":"","institution":"Poona College of Pharmacy, Bharati Vidyapeeth (Deemed University)","correspondingAuthor":false,"prefix":"","firstName":"Pratiksha","middleName":"R.","lastName":"Pathade","suffix":""},{"id":512661932,"identity":"3d196b13-57e1-40de-8258-2c0d4201cb85","order_by":1,"name":"Shubhangi A. Thool","email":"","orcid":"","institution":"Poona College of Pharmacy, Bharati Vidyapeeth (Deemed University)","correspondingAuthor":false,"prefix":"","firstName":"Shubhangi","middleName":"A.","lastName":"Thool","suffix":""},{"id":512661933,"identity":"4c99de2b-8f4d-42d5-ba5b-c42e730faee5","order_by":2,"name":"Varsha B. Pokharkar","email":"data:image/png;base64,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","orcid":"","institution":"Poona College of Pharmacy, Bharati Vidyapeeth (Deemed University)","correspondingAuthor":true,"prefix":"","firstName":"Varsha","middleName":"B.","lastName":"Pokharkar","suffix":""}],"badges":[],"createdAt":"2025-08-29 11:23:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7488181/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7488181/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12668-025-02358-8","type":"published","date":"2026-01-16T16:29:09+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91166594,"identity":"dfdca86a-5a3d-457f-8d53-36e14dbb38be","added_by":"auto","created_at":"2025-09-12 10:39:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119927,"visible":true,"origin":"","legend":"\u003cp\u003e3D response surface plot showing the effect of an independent variable on the particle size\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/b026fa1dd112df2061561537.png"},{"id":91166597,"identity":"d776a257-befb-41c9-acd2-ef2b31a75573","added_by":"auto","created_at":"2025-09-12 10:39:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120476,"visible":true,"origin":"","legend":"\u003cp\u003e3D response surface plot showing the effect of an independent variable on the PDI\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/995307c33373f0c5a09f4828.png"},{"id":91166602,"identity":"6ff15fd8-ca5a-4158-8f05-467e54751737","added_by":"auto","created_at":"2025-09-12 10:39:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":223750,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Saturation solubility of EZT in different stabilizers, (b) FTIR spectra of A) EZT, B) Mannitol, C) PM of EZT and P188 and D) EZT-NCs (c) DSC thermograms of A) EZT, B) Mannitol, C) PM of EZT and P188 and D) EZT-NCs (d) pXRD diffractograms of A) EZT B) Mannitol C) PM of EZT and P188 and D) EZT-NCs (e) SEM images of A) pure EZT and B) EZT-NCs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/aa41948702e23247044c63d4.png"},{"id":91166595,"identity":"b477c013-e230-4d3c-8251-e414a2f43e5b","added_by":"auto","created_at":"2025-09-12 10:39:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39037,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Dissolution profiles of pure EZT and EZT-NCs, (b) Permeability of EZT and EZT-\u003cstrong\u003eNCs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/0fe33d7c7cdec4845e342c79.png"},{"id":91166890,"identity":"de744068-5420-4c98-ad64-5e9f05cb8f04","added_by":"auto","created_at":"2025-09-12 10:47:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39814,"visible":true,"origin":"","legend":"\u003cp\u003ePharmacokinetic profile of pure EZT and EZT-NCs\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/0292bce966989f507e750c32.png"},{"id":100617518,"identity":"a67f9416-9bc2-42b0-a15f-91c08f76cafb","added_by":"auto","created_at":"2026-01-19 17:54:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2181376,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7488181/v1/02cd5a0d-77de-4b77-ad29-bdd8e9c1ef32.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Structure-property optimization of Ezetimibe nanocrystals by computationally guided bottom-up engineering for enhanced bioavailability","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEzetimibe (EZT), 1-(4-fluorophenyl)-3-[(3S)-3-(4-fluorophenyl)-3-hydroxypropyl]-4-(4-hydroxyphenyl) azetidin-2-one, is a selective inhibitor of cholesterol absorption.It effectively prevents the intestinal uptake of both dietary and biliary cholesterol without interfering with the absorption of fat-soluble vitamins and nutrients by blocking the Niemann-Pick C1-like l (NPC1L1) protein[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]It belongs to the BCS class II drug identified by high permeability and poor water solubility, with an oral bioavailability of approximately 35%, and is highly lipophilic, having a log p of 4.5[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Its low water solubility of 0.00846 mg/mL and the following slow dissolution rate are the causes of its varied bioavailability. Moreover, it has a significant efflux by phosphoglycoprotein (P-gp) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs solubility is a crucial factor in effective drug therapy across all routes of delivery, pharmaceutical technologists face a significant obstacle when developing new pharmaceutical products, due to the high lipophilicity and low solubility of nearly 50% of active pharmaceutical ingredients (API) discovered by high-throughput screening[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Approximately 40% of drugs are poorly soluble, causing inadequate absorption and higher doses for therapeutic effects, potentially leading to toxicities and increasing drug development costs[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Over the years, numerous approaches have been developed to increase the oral bioavailability of these APIs-like solid dispersions [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], co-crystals [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] cyclodextrin inclusion complexes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Self-microemulsifying drug delivery system (SMEDDS)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], Self-nanoemulsifying drug delivery system (SNEDDS)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The preparation of these formulations for therapies was laborious and time-consuming due to the high quantity of surfactants and co-surfactants, causing toxicological concerns. Challenges included restricted drug loading capacity, premature drug release, biocompatibility issues, and manufacturing complexity. Whereas, nanotechnology-based formulations showed significant potential with fewer drawbacks[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNanocrystals have garnered significant interest as a nanotechnology-based strategy for the reduction of the size of drugs, to improve the solubility and bioavailability of poorly soluble drugs [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Nanocrystals are nanosized formulations that comprise nano-scaled drug particles stabilized by an appropriate stabilizer or surfactant. The average particle sizes of these formulations are less than 1 \u0026micro;m, ideally between 200 nm and 500 nm. Nanocrystals can exhibit various states, including fully crystalline, partially crystalline, or entirely amorphous.[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Nanocrystals gained widespread acceptance due to their distinguishing features, they are carrier-free, consisting entirely of the drug itself, and are easily scalable. Their significant advantages are the large specific surface area, under the equation of Noyes-Whitney, resulting in rapid dissolution, and by the Freundlich-Ostwald relation, to higher saturation concentration[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This promoted their use in formulation development, along with a high drug loading and nanoscale distribution. Nanocrystals are synthesized using two methods: top-down and bottom-up. Top-down methods use high-energy processes to break down the drug particles, while bottom-up methods use precipitation from a supersaturated drug solution. Top-down is more widely applicable but has limitations in particle size reduction efficiency, high energy input, and concerns about solid-state changes, chemical degradation, and residual metal content[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTherefore, we aimed to enhance the solubility and oral bioavailability of EZT by optimizing its structure-properties through the formulation of nanocrystals using the antisolvent precipitation ultrasonication method, a bottom-up approach designed to overcome the limitations of top-down techniques. The antisolvent precipitation-ultrasonication method is highly efficient, scalable, and environmentally friendly for pharmaceutical applications. This method offers solvent-free, stable nanocrystals with better particle size reduction without high energy input, making it a reliable and eco-friendly alternative to ball milling and the evaporative precipitation into the aqueous solution method, respectively[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Hence, for optimization, we have screened the different stabilizers to identify the most suitable contenders for inhibiting Ostwald ripening. Then, successfully implemented a Box Behnken Design (BBD) as a computational tool to identify the critical variables, following the characterization of optimized EZT nanocrystals (EZT-NCs). The pharmacokinetic parameters of EZT-NCs were tested in male Wistar rats to assess \u003cem\u003ein vivo\u003c/em\u003e performance, and an everted gut sac model was employed to study the influence of P-gp efflux inhibitors on improving EZT bioavailability. We hypothesize that prepared EZT-NCs show significantly enhanced solubility, dissolution rate, and bioavailability compared to pure EZT.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eEzetimibe was received from MSN Laboratories Pvt Ltd (Telangana, India). Soluplus\u0026reg;, Kolliphor EL, Vitamin E-TPGS, and Tween\u0026reg; 80 were kindly supplied as gift samples by BASF Corporation (Navi Mumbai, India). Sodium lauryl sulfate (SLS) and Poloxamer 188 (P188) were purchased from Sigma-Aldrich (Mumbai, India). PVP K-30 and HPMC E5 were purchased from LobaChemiePvt Ltd, India. Mannitol, Lactose, and Trehalose were received as gift samples from Signet Excipients Pvt Ltd (Mumbai, India). Merck (Mumbai, India) provided HPLC-grade methanol and acetonitrile. In all formulations, Milli Q water was used. All the remaining chemicals and reagents were of analytical grade.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Methods\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Screening of Stabilizers\u003c/h2\u003e\u003cdiv id=\"Sec6\" class=\"Section4\"\u003e\u003ch2\u003e2.2.1.1 Screening using saturation solubility\u003c/h2\u003e\u003cp\u003eSaturation solubility studies within an aqueous stabilizer solution were used to screen suitable stabilizers. Anionic, polymeric, and non-ionic stabilizers from various categories were assessed utilizing a miniature shake flask method to ascertain the solubility of EZT in stabilizer solutions[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].EZT solubility was assessed in 1%w/v concentration aqueous solutions of Soluplus\u0026reg;, P188, Tween 80\u0026reg;, SLS, Vitamin E-TPGS, Kolliphor-EL, PVP K-30, and HPMC E5. Vials containing 5 mL of aqueous stabilizer solution were filled with an excess of the drug (10 mg), and the vials were continuously shaken with a mechanical shaker at 37\u0026deg;Cand 150 rpm for 48 hours. The resulting suspensions were separated and filtered through a 0.22 \u0026micro;m syringe filter(Whatman Inc., Clifton, NJ, USA) after being centrifuged for 10 minutes at 20,000 rpm (Allegra 64R centrifuge, Beckman-Coulter \u0026trade;, USA). The absorbance of the filters at 232 nm was measured with a UV-visible spectrophotometer (Jasco V-730 Spectrophotometer), and the filtrate contents were examined for EZT. The solubility studies were carried out in triplicate, and data were expressed as mean value\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section4\"\u003e\u003ch2\u003e2.2.1.2 Screening using particle size\u003c/h2\u003e\u003cp\u003eThe stabilizers were screened and selected for further studies on the basis of the EZT saturation solubility study results in the above-mentioned various stabilizer solutions. The EZT nanocrystals were prepared from these stabilizers and characterized for particle size and PDI using the dynamic light scattering technique (Nano ZS90, Malvern Instruments Pvt. Ltd., Malvern, Worcestershire, England, UK). All experiments were conducted in triplicate.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Preparation of EZT nanocrystals\u003c/h2\u003e\u003cp\u003eThe EZT-NCs dispersions were prepared by a bottom-up approach, specifically the anti-solvent precipitation-ultrasonication method. In brief, the solvent phase consisted of 10 mg of the drug dissolved in 1 mL of ethanol. Later on, the solvent phase was introduced into the antisolvent phase, consisting of a P188 solution prepared using 15 mL of Milli Q water at a rate of 1 mL/min. A high-intensity ultrasonicator(Vibra-Cell, Sonics and Materials, Inc., Newtown, CT)was used to apply continuous sonication throughout the solvent phase addition for 10 min at 40% amplitude with a 10:05 on: off cycle, to balance effective cavitation with heat management, avoiding thermal degradation of heat-sensitive ezetimib with consistent particle size reduction and uniformity. Then, ethanol from the formulation was evaporated by stirring the formulation on a magnetic stirrer for 1 to 2 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Lyophilization of EZT-NCs suspension\u003c/h2\u003e\u003cp\u003eThe nanocrystalline suspension was powdered by lyophilizing the EZT-NCs. The suspension was frozen for 24 hours at -80˚C using an ultra-low freezer (U101, Innova, New Brunswick Scientific) and lyophilized over 72 hours using a lyophilizer (INLABCO, model no INFD-3P) at \u0026minus;\u0026thinsp;40\u0026deg;C condenser temperature with a 0.45 mbar vacuum. This study tested lactose, mannitol, and trehalose at 1% w/v for their appearance and aggregation, determining the suitable cryoprotectant using the re-dispersibility index (RDI)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The resulting lyophilized powder was used for further characterization. RDI can be calculated using Eq.\u0026nbsp;1:\u003c/p\u003e\u003cp\u003eRDI = [D0/D] Eq.\u0026nbsp;1\u003c/p\u003e\u003cp\u003eWhere D0 and D represent the mean particle size before lyophilization and after lyophilization, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Optimization of EZT-NCs with Design of Experiments (DoE)\u003c/h2\u003e\u003cp\u003eA three-factor, three-level BBD was applied for the optimization of formulation factors and the investigation of key effects, interaction effects, and quadratic effects on the characteristics of the EZT nanocrystals. By preliminary experiments Solvent: antisolvent ratio (A), the Concentration of stabilizer (B), and Ultrasonication amplitude (C) were investigated as independent variables at three levels of low (-1), medium (0), and high (+\u0026thinsp;1). The particle size (nm) and PDI were considered as response (dependent) variables listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Analysis of variance (ANOVA) was applied to statistically analyze the model, whereas equations were constructed using Design-Expert 13\u0026reg; software. The experiments were conducted randomly according to the recommendation of the software, with a batch prepared to calculate deviations between theoretical and practical results, and the relative error and three-dimensional (3D) response surface plots were created to illustrate variable effects[\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of dependent and independent variables in BBD for EZT-NCs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eIndependent Variables\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eLevels\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLow\u003c/p\u003e\u003cp\u003e-1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMedium\u003c/p\u003e\u003cp\u003e0\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eHigh\u003c/p\u003e\u003cp\u003e+\u0026thinsp;1\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1. Solvent: antisolvent ratio (ml) (A)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2. Concentration of P188 (mg) (B)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3. Ultasonication amplitude (%) (C)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eDependent variables\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003e\u003cb\u003eConstraints\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1. Particle Size (nm) (X)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMinimum\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2. PDI (Y) Minimum\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e\u003cp\u003eMinimum\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Characterization of EZT-NCs","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Saturation Solubility Studies\u003c/h2\u003e\u003cp\u003eThe saturation solubility of pure EZT, PM of EZT with poloxamer 188, and lyophilized EZT-NCs were evaluated in different media, such as distilled water, 0.1N HCL pH 1.2, acetate buffer pH 4.5, and phosphate buffer pH 6.8, as well as 0.05 M acetate buffer pH 4.5 with 0.45% SLS. The solubility determination followed the aforementioned method. Solubility tests were carried out in triplicate, and results were provided as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Particle size, PDI, and zeta potential\u003c/h2\u003e\u003cp\u003eThe prepared EZT-NCs were characterized for particle size (z-average), PDI, and zeta potential by the dynamic light scattering technique (Nano ZS90, Malvern Instruments Pvt. Ltd., Malvern, Worcestershire, England, UK) at 25\u0026deg;C. Before measurement, the samples were diluted byMilli-Q\u0026reg;water (Millipore, France) and analyzed in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Fourier transform infrared spectrometry analysis (FT-IR)\u003c/h2\u003e\u003cp\u003eThe FTIR spectra of pure EZT, mannitol, PM of EZT with P188, and lyophilized EZT-NCs were obtained using FTIR spectroscopy (Jasco4100, Japan). The potassium bromide (KBr) was heated in a hot air oven for 15\u0026ndash;20 minutes to activate it. Each sample was then combined with KBr in a 3:1 ratio and placed into the cavity.FTIR spectra were acquired with 4 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e resolution, scanning from 4000 to 400 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 50 scans.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Differential Scanning Colorimetry (DSC)\u003c/h2\u003e\u003cp\u003eThermal analysis of pure EZT, PM of EZT with P188, and lyophilized EZT-NCs was conducted using the DSC-60 Plus Calorimeter (DSC 60; Shimadzu Corporation). Samples of 3 mg each were sealed in aluminum pans and were heated from 30\u0026deg;C to 350\u0026deg;C at 10\u0026deg;C/min under a dry nitrogen purge of 30 mL/min to produce the thermograms.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Powder X-ray diffraction (pXRD)\u003c/h2\u003e\u003cp\u003ePowder X-ray diffraction patterns of pure EZT, mannitol, PM of EZT with P188, and lyophilized EZT-NCs were recorded in an X-ray diffractometer (Xpert Pro MPD, Panalytical, Netherlands) using CuKα radiation as an X-ray source. Samples were scanned for a 2θ range of 0\u0026deg; to 60\u0026deg; at a scanning speed of 2\u0026deg;/minute at an operating voltage of 40 kV and current of 30 mA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Scanning Electron Microscopy (SEM)\u003c/h2\u003e\u003cp\u003eThe morphology of pure EZT and EZT-NCs was examined under a scanning electron microscope (FEI, Quanta 200, Netherlands). Samples were clanged on a double adhesive tape and were gold plated by a sputter coater mounted on aluminum plates and observed at an acceleration voltage of -30 kV.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Drug content\u003c/h2\u003e\u003cp\u003eThe lyophilized EZT-NCs, weighing 10 mg of EZT, were precisely measured and then dissolved in 10 mL of methanol. After vortexing the solution for 5 minutes, it was filtered with a 0.22 \u0026micro;m PTFE syringe filter (Whatman Inc., Clifton, NJ, USA), further diluted with methanol, and assayed for EZT by a UV-visible spectrophotometer at 232 nm. All the samples were assayed in triplicate, and mean values\u0026thinsp;\u0026plusmn;\u0026thinsp;SD were reported for the results. Drug content determination was derived using the mathematical Eq.\u0026nbsp;2\u003c/p\u003e\u003cp\u003eDrug content (%) = ((Observed drug content)/(Theoretical drug content))\u0026times;100 Eq.\u0026nbsp;2\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.8 \u003cem\u003eIn vitro\u003c/em\u003e drug release studies\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vitro\u003c/em\u003e dissolution studies of EZT-NCs were conducted and compared with pure EZT. Pure EZT and EZT-NCs (equivalent to 10 mg of EZT) were filled into gelatine capsules of capsule size #1, and dissolution was performed in USP Type I apparatus (Electrolab Dissolution tester, India) using a 500 mL 0.05M acetate buffer of pH 4.5 with 0.45% SLS at 37\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C and with stirring at 50 rpm (USFDA recommended dissolution media for EZT)[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. An aliquot of 5 mL was withdrawn at different time intervals. A fresh, equivalent volume of dissolution medium was immediately substituted. The collected aliquots were filtered and assayed spectro-photometrically at 232 nm. The percent cumulative drug release was plotted against the time profile.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Stability Study\u003c/h2\u003e\u003cp\u003e The stability of the optimized EZT-NCs was evaluated following ICH guidelines. Samples were stored at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH for three months. Particle size, PDI, and zeta potential were determined at 1, 2, and 3 months after re-dispersing the lyophilized EZT-NCs, along with visual inspections for physical stability.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.10 \u003cem\u003eEx vivo\u003c/em\u003e Permeability Study\u003c/h2\u003e\u003cp\u003eThe inhibitory effects of P-gp by the pure EZT and EZT-NCs were assessed using an everted rat gut sac model. The study involved cleaning a 4 cm segment of the small intestine of a male Wistar rat with Krebs-Ringer Buffer (KRB) and aerating with an electric aerator. The segment was then filled with the drug suspension and EZT-NCs (1mg/mL). The tissue was then placed in 100 mL KRB solution, aerated with atmospheric air, kept at 37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C, and stirred at 50 rpm. Samples were removed and replaced with fresh KRB solution. Permeability was studied for 60 minutes, and drug permeation was measured at 232 nm using a UV spectrophotometer[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The permeation flux (J, \u0026micro;g min⁻\u0026sup1;) was estimated based on the slope of the linear regression analysis, and the apparent permeability coefficient was obtained using Eq.\u0026nbsp;3.\u003c/p\u003e\u003cp\u003eP\u003csub\u003eapp\u003c/sub\u003e=J/(A\u0026times; C\u003csub\u003e0\u003c/sub\u003e) Eq.\u0026nbsp;3\u003c/p\u003e\u003cp\u003eWhere J is the permeation flux, A is the intestinal surface area, and C\u003csub\u003e0\u003c/sub\u003eis the initial drug concentration.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.11 \u003cem\u003eInvivo\u003c/em\u003e pharmacokinetic study\u003c/h2\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003e3.10.1 Animals and dose administration\u003c/h2\u003e\u003cp\u003e Male Wistar rats were procured from Global Bioresearch Solutions, Pune, India, weighing 200\u0026ndash;250 g, all animal experiments received prior approval from the Institutional Animal Ethics Committee (IAEC) of Poona College of Pharmacy, Bharati Vidyapeeth Deemed University (PCP/IAEC/2024/2\u0026ndash;18), which is registered with the committee for Control and Supervision of Experiments on Animals, Government of India. Animals were housed in standard laboratory conditions with pelletized feed and filtered water, and abstained from 12 hours before the study, with daily health monitoring. The study involved two groups of nine rats each, each group receiving pure EZT and EZT-NCs, respectively, at a dosage of 3 mg/kg orally in a 0.75% w/v sodium CMC solution[\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].0.5 mL of blood samples were collected from the retro-orbital plexus at specific intervals (1, 2, 4, 8, 10, 12, 24, 36, 48 hrs). The plasma was separated by centrifuging the samples in heparinized Eppendorf tubes at 8000 rpm for 15 minutes and then stored at -20 ◦C.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section3\"\u003e\u003ch2\u003e3.10.2 Preparation of plasma sample and data analysis\u003c/h2\u003e\u003cp\u003ePlasma samples were deproteinized using a methanol precipitation method, followed by centrifugation and injection into the HPLC system. Chromatography was conducted using a Jasco PU 1580 system and Borwin software, with separations on a Thermo C-18 column using 50:50 acetonitrile: water as the mobile phase, with slight modifications to the previously reported method[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Compounds were detected with a flow rate of 1 mL/min and an injection volume of 20\u0026micro;L at 232 nm using a UV detector. Pharmacokinetic parameters were analyzed using PKSolver (version 2.0) with a non-compartmental model and linear trapezoidal approach, presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.Estimated parameters included the time to reach maximum concentration (T\u003csub\u003emax\u003c/sub\u003e), maximum drug concentration (C\u003csub\u003emax\u003c/sub\u003e), area under the curve from zero to 48 hours (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;48\u003c/sub\u003e), area under the curve from zero to infinity (AUC\u003csub\u003e0\u0026minus;\u0026infin;\u003c/sub\u003e), and half-life (t\u003csub\u003e1/2\u003c/sub\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.12 Statistical analysis\u003c/h2\u003e\u003cp\u003eThe results for the \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetic studies were statistically analyzed using GraphPad Prism\u0026reg; version 5. Two-way ANOVA was used, followed by a Bonferroni post-hoc test. The findings were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicated significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Results and Discussion","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e4.1 Screening of stabilizers\u003c/h2\u003e\u003cp\u003eIn the nanosizing process, stabilizers are initially screened based on their drug solubilising ability, which helps reduce surface free energy and prevent particle agglomeration, thereby ensuring nanosuspension stability [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is postulated in the Lifshitz-Slyozov-Wagner (LSW) theory that the drug concentration in the dispersed phase directly affects Ostwald ripening. The drug solubility in the stabilizer solution creates a concentration gradient, causing smaller solubilized particles to migrate towards larger particles, resulting in particle growth, agglomeration, and crystallization. Therefore, for nano-colloidal systems, stabilizers should minimally affect drug solubility[\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Stabilizers were screened according to their saturation solubility in aqueous solutions. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) illustrates that the intrinsic aqueous solubility of EZT was minimally impacted by 1% w/v P188, HPMC E5, PVP K30, and SLS by providing critical steric and electrostatic stabilization, while Soluplus\u0026reg;, Kolliphor-EL, Tween 80\u0026reg;\u003csup\u003e,\u003c/sup\u003e and Vitamin E-TPGShad a significant effect on it due to their surfactant properties. As a result, P188, HPMC E5, PVP K30, and SLS were selected for further studies because they contribute to stability without significantly altering solubility, ensure narrow particle size distribution and robust physical stability, essential for consistent in-vivo performance and enhanced bioavailability.\u003c/p\u003e\u003cp\u003eThe initial experimental batches were prepared using the four stabilizers that had been screened earlier (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Following nanosuspension with P188 (USFDA-approved as a GRAS) showed the smallest particle size and PDI, along with the highest zeta potential. It is an amphiphilic block copolymer consisting of a hydrophobic polypropylene oxide segment that attaches to the drug surface and its hydrophilic polyethylene oxide segment, forming steric hindrance to suppress particle agglomeration and promote dispersibility and colloidal system stability. Hence, P188 was selected as the single steric stabilizer due to its ability to prevent submicron particle agglomeration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eExperimental batches with screened stabilizers\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEZT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStabilizer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eParticle size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eZeta potential\u003c/p\u003e\u003cp\u003e(mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 mg P188\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e605\u0026thinsp;\u0026plusmn;\u0026thinsp;7.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026minus;\u0026thinsp;45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 mg HPMC E5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1021\u0026thinsp;\u0026plusmn;\u0026thinsp;10.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 mg PVP K30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e807\u0026thinsp;\u0026plusmn;\u0026thinsp;21.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10 mg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 mg SLS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e988\u0026thinsp;\u0026plusmn;\u0026thinsp;9.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003e4.2 Lyophilization of EZT-NCs suspension\u003c/h2\u003e\u003cp\u003eLyophilization improves stability and extends shelf life by keeping the product in a dry form. Cryoprotectants prevent the destabilization of the colloidal dispersion of nanocrystals caused by the stress of freeze-drying and dehydration during lyophilisation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Various cryoprotectants, including mannitol, lactose, and trehalose, were screened and analyzed based on product appearance (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). From the above results, mannitol was found to be the most effective in forming a fluffy, free-flowing, and well-formed dried powder. This is attributed to the high collapse temperature of mannitol, which retained the porous structure with a cake-like appearance for the product[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Therefore, mannitol was selected as a suitable cryoprotectant and further screened for concentrations ranging from 0.5 to 1.5% (w/v).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eScreening of cryoprotectants\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCryoprotectant\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration of cryoprotectant (%w/v)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAppearance\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMannitol\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFluffy and free-flowing\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLactose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAggregated and Sticky\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTrehalose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAggregated\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe particle size and RDI of the reconstituted lyophilized aqueous dispersion of nanocrystals were measured (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Increasing the mannitol concentration beyond 1% (w/v) results in a linear increase in nanocrystal size, while insufficient cryoprotectant concentration of 0.5% (w/v) leads to incomplete coating and particle aggregation, both contributing to larger particle sizes. The addition of 1% (w/v) mannitol into the lyophilized aqueous dispersion of nanocrystals effectively prevents aggregation, resulting in an average particle size of 402\u0026thinsp;\u0026plusmn;\u0026thinsp;23.25 nm. An RDI value close to 1 indicates that the nanoaggregates are fully re-dispersible. Therefore, 1% mannitol was determined to be the optimal concentration based on particle size and RDI [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of varying cryoprotectant concentrations on particle size and RDI\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMannitol concentration (%w/v)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRDI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e541\u0026thinsp;\u0026plusmn;\u0026thinsp;10.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e402\u0026thinsp;\u0026plusmn;\u0026thinsp;23.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e446\u0026thinsp;\u0026plusmn;\u0026thinsp;36.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003e4.3 Optimization of EZT-NCs with Design of Experiments (DoE)\u003c/h2\u003e\u003cp\u003eBBD was selected for the current study in order to examine both the main and interaction impacts of independent variables on the dependent variables. When there are three factors or more than three factors, BBD is highly effective in minimizing the number of runs compared to the central composite design. This approach evaluated the impact of Solvent: antisolvent ratio (A), P188 concentration (B), and Ultrasonication amplitude (C) on particle size (X) and PDI (Y). Seventeen runs were generated with five center points(Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Multiple regression analysis was performed using Design-Expert 13\u0026reg; software to generate polynomial models, including linear, two-factor interaction, and quadratic. The model with the highest R\u0026sup2;, adjusted R\u0026sup2;, and predicted R\u0026sup2; values were selected for optimal fit Table\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. ANOVA and 3D plots were used to evaluate the impact of independent variables on dependent variables.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBox Behnken Design with observed responses\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRun\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSolvent: antisolvent ratio (ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConcentration of P188 (mg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUltrasonication amplitude (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eZeta potential (mV)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e531\u0026thinsp;\u0026plusmn;\u0026thinsp;11.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e825\u0026thinsp;\u0026plusmn;\u0026thinsp;16.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e455\u0026thinsp;\u0026plusmn;\u0026thinsp;09.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e340\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e642\u0026thinsp;\u0026plusmn;\u0026thinsp;02.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e392\u0026thinsp;\u0026plusmn;\u0026thinsp;27.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e750\u0026thinsp;\u0026plusmn;\u0026thinsp;14.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e761\u0026thinsp;\u0026plusmn;\u0026thinsp;13.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e600\u0026thinsp;\u0026plusmn;\u0026thinsp;13.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e342\u0026thinsp;\u0026plusmn;\u0026thinsp;19.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e382\u0026thinsp;\u0026plusmn;\u0026thinsp;10.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e388\u0026thinsp;\u0026plusmn;\u0026thinsp;18.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e750\u0026thinsp;\u0026plusmn;\u0026thinsp;11.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e640\u0026thinsp;\u0026plusmn;\u0026thinsp;28.15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e731\u0026thinsp;\u0026plusmn;\u0026thinsp;38.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e640\u0026thinsp;\u0026plusmn;\u0026thinsp;11.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e631\u0026thinsp;\u0026plusmn;\u0026thinsp;17.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eModel Fit Summary statistics for Particle size and PDI\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eResponse\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModel\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eR\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAdjusted R\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePredicted R\u0026sup2;\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParticle Size\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.9930\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9840\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9760\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eQuadratic\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.9983\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.9961\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.9785\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eOptimized parameters along with predicted and observed values of responses\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eFactors\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003ePredicted value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003eObserved value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSolvent: antisolvent ratio (ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConcentration of P188 (mg)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUltrasonication amplitude(%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eParticle Size (nm)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1:15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e368\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e340\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec30\" class=\"Section3\"\u003e\u003ch2\u003e4.3.1 Assessment of the design of the experiment\u003c/h2\u003e\u003cp\u003eThe significance of each coefficient for the main effects and interaction terms in this BBD was evaluated using appropriate polynomial model equations and p-values. A lower p-value (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicates a more significant coefficient, highlighting the substantial effect of the corresponding independent variables. In the regression equation, a negative value indicates an antagonistic influence on the dependent variable, whereas a positive value indicates a synergistic effect.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec31\" class=\"Section3\"\u003e\u003ch2\u003e4.3.2 Effect of independent variables on particle size\u003c/h2\u003e\u003cp\u003eOne important factor influencing the solubility of nanocrystals is particle size, which in turn improves the drug's bioavailability. The particle size of all 17 batches was analyzed, and it was found to be in the range of 340\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00 to 825\u0026thinsp;\u0026plusmn;\u0026thinsp;16.57 nm (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The quadratic model suggested by BBD to describe the effect on particle size is significant with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, while the lack of fit is not significant. The relationship of coded factors with particle size was evaluated by equations 4 and 3D plots.\u003c/p\u003e\u003cp\u003eParticlesize\u0026thinsp;=\u0026thinsp;368.8\u0026thinsp;+\u0026thinsp;24.375A-28B\u0026thinsp;+\u0026thinsp;17.625C -30.25AB-74AC\u0026thinsp;+\u0026thinsp;81.25BC\u0026thinsp;+\u0026thinsp;113.35A\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;243.1B\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;84.85C\u003csup\u003e2\u003c/sup\u003eEq.\u0026nbsp;4\u003c/p\u003e\u003cp\u003eIn this polynomial Eq.\u0026nbsp;4, the model terms A, B, C, AB, AC, BC, A\u0026sup2;, B\u0026sup2;, and C\u0026sup2; are significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Synergistic and antagonistic effects on the observed responses are represented by the positive and negative signs in the equation. According to the equation, it can be seen that the concentration of P188 (B) has an antagonistic effect that causes the particle size to decrease as factor B increases, while the solvent: antisolvent ratio (A) and ultrasonication amplitude (C) have an antagonistic effect on the particle size that causes the particle size to increase as factors A and B increase. However, these two factors, A and B, at certain levels may interact with other factors in a way that counteracts their positive influence. This complexity highlights the importance of considering interaction effects and not relying solely on the main effects.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe 3D plots shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e illustrate the effect of independent variables on particle size. As the solvent: antisolvent ratio increases from lower to higher levels, particle size initially increases, then decreases at an intermediate level, and finally increases again at higher levels. Similarly, as the concentration of P188 increases, particle size follows the same trend. This behavior is attributed to a lower solvent: antisolvent ratio, which reduces supersaturation, leading to less efficient mixing and slower nucleation, promoting particle growth. However, as the solvent-to-antisolvent ratio increases, the degree of supersaturation also increases, enhancing the nucleation rate and leading to the formation of smaller particles. Beyond the optimum level, further increases in the solvent: antisolvent ratio result in more nuclei, reducing the diffusion of particles and increasing collisions, which ultimately leads to larger particle sizes[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Lower concentrations of P188 result in inadequate stabilization due to insufficient adsorption and incomplete coverage of newly formed nanosized particles, leading to crystal growth and increased particle size. At higher concentrations, P188 forms a thicker adsorbed layer on the drug surface, resulting in slightly larger particles [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSimilarly, the amplitude of ultrasonication is crucial for the efficient mixing of solvent and antisolvent. This is because the process generates cavitation bubbles through cycles of compression and rarefaction in the liquid dispersion medium. The collapse of these bubbles creates intense shock waves that impart high velocities to the suspended particles. Increasing the amplitude induces the solvent and antisolvent to mix more effectively with a steady reduction of particle size, leading to greater supersaturation and nucleation, and thereby resulting in colloidal particles. However, if the amplitude intensity is increased beyond an optimal point, particle size may slightly increase due to greater hindrance among newly formed particles, resulting in agglomeration [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOverall, the optimized conditions for particle size reduction directly contribute to nanocrystal stability by minimizing aggregation during storage and maintaining uniformity. Adequate P188 concentration and optimal ultrasonication amplitude prevent particle growth and agglomeration, thereby ensuring long-term stability and efficient drug delivery, which collectively enhances in vivo performance and therapeutic efficacy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec32\" class=\"Section3\"\u003e\u003ch2\u003e4.3.3 Effect of independent variables on PDI\u003c/h2\u003e\u003cp\u003eAs a dimensionless parameter, PDI measures the distribution of particle sizes, ranging from 0 to 1. Values greater than 0.5 indicate a broad particle distribution, leading to Ostwald ripening. The developed formulations exhibited PDI values ranging between 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 to 1.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05. BBD suggested that the model describing the effect on PDI is quadratic; P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicates that the model is significant, whereas the lack of fit is not significant. The relationship of coded factors to PDI was assessed by Eqs.\u0026nbsp;5 and 3D plots.\u003c/p\u003e\u003cp\u003ePDI\u0026thinsp;=\u0026thinsp;0.1320- 0.0250A \u0026minus;\u0026thinsp;0.0425B\u0026thinsp;+\u0026thinsp;0.1050C\u0026thinsp;+\u0026thinsp;0.1800AB- 0.0100AC\u0026thinsp;+\u0026thinsp;0.0800BC\u0026thinsp;+\u0026thinsp;0.0515A\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.3965B\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;+\u0026thinsp;0.4015C\u003csup\u003e2\u003c/sup\u003e Eq.\u0026nbsp;5\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe model terms A, B, C, AB, BC, A\u0026sup2;, B\u0026sup2;, and C\u0026sup2; had significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) effects on the response. According to the response surface plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the solvent: antisolvent ratio, P188 concentration, and ultrasonication amplitude significantly affected PDI, with notable interactive effects. As the solvent: antisolvent ratio increased, slight changes in PDI were noted. At the optimal ratio, high supersaturation led to uniform nuclei distribution and lower crystal growth rates, resulting in a narrower size distribution[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].PDI was greatly influenced by P188 levels and ultrasonication amplitude. Higher P188 concentrations decreased particle size and PDI by inhibiting steric hindrance, as lower stabilizer levels slowed stabilizer migration, causing insufficient surface stabilization and larger PDI. Also, higher P188 levels provided a thicker nanoparticle coating, enhancing stability. However, as the ultrasonication amplitude increased, monodispersity was observed up to some extent, but later particles were subjected to higher energies, and due to greater attrition, they lost their repulsive forces and agglomerated, resulting in higher PDI[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Where lower PDI ensures minimal aggregation risks, enhanced solubility, and dissolution rate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec33\" class=\"Section3\"\u003e\u003ch2\u003e4.3.4 Selection of Optimized Formulation\u003c/h2\u003e\u003cp\u003eThe analysis of variables of formulation showed that there is a strong relationship between solvent: antisolvent ratio, P188 concentration, ultrasonication amplitude, particle size, and PDI. 3D response surface diagrams were plotted to emphasize the effects of the solvent: antisolvent ratio, P188 concentration, and ultrasonication amplitude interactions on particle size and PDI. Finally, the optimal formulation was determined through the point prediction approach in the softwareTable 7. The optimized EZT-NCs dispersion has shown a particle size of 340\u0026thinsp;\u0026plusmn;\u0026thinsp;12.00 and PDI of 0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 using the composition solvent: antisolvent ratio (1:15), P188 concentration (15mg), ultrasonication amplitude (40%), and was used for further study.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec34\" class=\"Section3\"\u003e\u003ch2\u003e4.3.5 Determination of Zeta Potential\u003c/h2\u003e\u003cp\u003eZeta potential is a key indicator of nanocrystal dispersion stability. Values greater than +\u0026thinsp;30 mV or less than \u0026minus;\u0026thinsp;30 mV indicate stability, as higher zeta potential values signify stronger repulsive forces among the particles, which in turn contribute to the stability of the suspension by preventing aggregation. The zeta potential of optimized EZT-NCs was \u0026minus;\u0026thinsp;46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 mV. All formulations showed a zeta potential ranging from \u0026minus;\u0026thinsp;36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25 mV to -47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 mV (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These values implied that P188 provided sufficient coverage on the nanocrystal surfaces, contributing to a stable dispersion.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"5. Characterization of EZT nanocrystals","content":"\u003cdiv id=\"Sec36\" class=\"Section2\"\u003e\u003ch2\u003e5.1 Saturation solubility\u003c/h2\u003e\u003cp\u003eStudying solubility at different pH levels and in distilled water provides crucial insights into drug behavior, aiding in the absorption prediction and the selection of an appropriate dissolution medium. The study compared the saturation solubility of EZT-NCs to pure EZT and PM of EZT with P188 in five different media: distilled water, 0.1N HCL pH 1.2, acetate buffer pH 4.5, phosphate buffer pH 6.8, and 0.05 M acetate buffer pH 4.5, finding that the solubility was 1.2\u0026ndash;2.5 and 1.8\u0026ndash;2.2 fold to that of EZT and PM of EZT with P188 in different media, respectively.\u003c/p\u003e\u003cp\u003eThe enhanced solubility of EZT-NCs is attributed to their smaller particle sizes. Nanoinization increases saturation solubility, as per the Ostwald-Freundlich and Noyes-Whitney equations[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Kelvin\u0026rsquo;s equation also explains that the strong curvature of nanosized particles increases dissolution pressure[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Thus, results highlight the critical role of particle size reduction to enhance the aqueous solubility and oral bioavailability[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e\u003ch2\u003e5.2 Fourier transform infrared spectrometry analysis\u003c/h2\u003e\u003cp\u003eFT-IR spectra of pure EZT, P188, PM of EZT with P188, and lyophilized EZT-NCs are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e (b). The FTIR analysis of pure EZT showed characteristic peaks of O\u0026thinsp;\u0026minus;\u0026thinsp;H stretching in alcohols at 3287.07 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;O stretching of β-lactam ring at 1717.3 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;\u0026minus;\u0026thinsp;H stretching in alkanes at 2916.42 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, and p-substituted benzene ring vibration at 830.20 cm\u003csup\u003e\u0026minus;\u0026thinsp;1,\u003c/sup\u003e aligning with the literature data[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Mannitol exhibited O-H stretching vibrations with intermolecular H-bonds and C-H stretching vibrations. PM indicated characteristic peaks of P188, O-H stretching, and C-O stretching, respectively[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. No spectral shift was noted in PM, suggesting no drug-stabilizer interaction. However, the interaction between drugs and stabilizers, especially through hydrogen bonding, significantly impacts characteristic peaks. The IR spectrum of the EZT-NCs formulation showed a shift in the carboxylic \u0026ndash; OH peak, and a less prominent C\u0026thinsp;=\u0026thinsp;O stretching band at 1717 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, likely due to hydrogen bonding between the C\u0026thinsp;=\u0026thinsp;O group of EZT and the -OH group of P188. This H-bonding was confirmed by stabilizer adsorption onto the drug crystal surface. The EZT-NCs showed characteristic peaks without any additional peaks, showing the chemical stability of the drug and stabilizer.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec38\" class=\"Section2\"\u003e\u003ch2\u003e5.3 Differential Scanning Calorimetry\u003c/h2\u003e\u003cp\u003eThermograms of pure EZT, mannitol, PM of EZT and P188, and EZT-NCs were depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (c). The DSC thermogram of EZT indicated a characteristic sharp endothermic peak at 164.5\u0026deg;C, indicating the crystalline nature of pure EZT. The PM displayed distinct endothermic peaks at 51.5\u0026deg;C and 163.9\u0026deg;C, representing the presence of P188 and EZT, respectively. Mannitol exhibited an endothermic peak at 170.4\u0026deg;C, also reflecting its crystalline nature. However, the DSC analysis revealed an endothermic peak at 169.3\u0026deg;C for EZT-NCs, resembling the peak for mannitol. The significant amount of mannitol used as a cryoprotectant caused a dilution effect, masking the endothermic peak of the free drug. This disappearance of the EZT peak indicates complete drug encapsulation within the stabilizer matrix, suggesting that the stabilizer effectively coats and incorporates the drug molecules[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003e5.4 Powder X-ray Diffractogram\u003c/h2\u003e\u003cp\u003eThe pXRD pattern of the pure EZT, mannitol, PM of EZT with P188, and EZT-NCis is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (d). Pure EZT displayed distinct high-energy diffraction peaks with 2θ values ranging from 8.04\u0026deg; to 32.56\u0026deg;, confirming its crystalline nature. The PM also showed characteristic peaks of EZT and P188 at 8.20\u0026deg; to 31.22\u0026deg;, 19.35\u0026deg;, and 23.48\u0026deg;, respectively, but with lower intensity. Whereas EZT-NCs exhibited diffraction peaks from 9.95\u0026deg; to 44.13\u0026deg;. The only variation noted between the pure EZT, PM of EZT with P188, and EZT-NCs is in the peak intensities.\u003c/p\u003e\u003cp\u003eThe variations in the relative peak intensities may be caused by the smaller particle size after size reduction, indicating the transformation of EZT into nanocrystals with reduced crystallinity, which is consistent with the DSC data. The decrease in peak intensity suggests amorphization[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The pXRD diffractograms also revealed that mannitol, which was employed in the lyophilization process, exhibited high-energy diffraction peaks within 2θ values ranging from 10.85\u0026deg; to 45.46\u0026deg;. These peaks obscured the characteristic diffraction peaks of EZT in the lyophilized formulation. This distinct diffraction pattern suggests that mannitol exists in a crystalline form rather than in an amorphous form, important for the long-term stability of nanocrystals[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec40\" class=\"Section2\"\u003e\u003ch2\u003e5.5 Scanning Electron Microscopy\u003c/h2\u003e\u003cp\u003eThe surface morphology and shape were studied using SEM for pure EZT and its nanocrystals. Accordingly, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (e. A)depicts the pure EZT as an irregular, cylindrical, and rod-shaped crystalline structure. In contrast, the EZT-NCs exhibited a slightly spherical structure with smoother edges, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e (e. B). This slight spherical shape of nanocrystals is potentially associated with the reduction in size to the nanoscale and the adsorption of P188 on hydrophobic drug particles. The slightly smoother surface and altered morphology of EZT-NCs result from stabilizer surface coating and lyophilization. Mannitol and P188 on the nanocrystal surface contribute to this effect, with P188 acting as a steric barrier to prevent particle agglomeration[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec41\" class=\"Section2\"\u003e\u003ch2\u003e5.6 Determination of Drug Content\u003c/h2\u003e\u003cp\u003eThe analysis of the drug content in the optimized EZT-NCs showed 96.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96% of EZT content in the EZT-NCs formulation. The high drug content of nanocrystals is an enormous advantage, as the delivery system is free of carrier systems.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec42\" class=\"Section2\"\u003e\u003ch2\u003e5.7 In vitro drug release study\u003c/h2\u003e\u003cp\u003eThe dissolution profiles of pure EZT and EZT-NCs are depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a). Dissolution profiles revealed that pure EZT exhibited a release of 45.55% within 1 hour, whereas EZT-NCs achieved an 85.17% release within the same timeframe of 1 hr, indicating a 1.869-fold increase in drug release as compared to the pure drug. The increased dissolution rate is attributed to the reduced size of EZT-NCs, resulting in a larger surface area and thinner diffusion layer, which further reduces nanocrystal agglomeration and improves wetting and dispersibility[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. According to the Prandtl equation, smaller particles have larger curvature, reducing diffusion distance and increasing dissolution velocity. As particle size decreases below 1 \u0026micro;m, the dissolution pressure and saturation solubility significantly increase[\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Additionally, the existence of P188 at the drug-aqueous phase interface further aids dissolution by reducing surface tension through hydrogen bonding with water molecules, enhancing drug release[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec43\" class=\"Section2\"\u003e\u003ch2\u003e5.8 Stability Studies\u003c/h2\u003e\u003cp\u003eThe optimized EZT-NCs, stored at room temperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity) for three months, were evaluated for their physical and chemical stability to determine the limits of their stability with storage recommendations. Table\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e illustrates the findings from the stability study of samples maintained at room temperature, indicating no significant alterations in particle size, PDI, and zeta potential.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eStability studies of EZT-NCs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSr. no\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eStorage condition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eStorage time (months)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eParticle size (nm\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePDI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eZeta potential (mV\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eRoomtemperature (25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C/60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e379\u0026thinsp;\u0026plusmn;\u0026thinsp;15.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e385\u0026thinsp;\u0026plusmn;\u0026thinsp;10.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e391\u0026thinsp;\u0026plusmn;\u0026thinsp;12.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e394\u0026thinsp;\u0026plusmn;\u0026thinsp;12.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e-45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec44\" class=\"Section2\"\u003e\u003ch2\u003e5.9 \u003cem\u003eEx vivo\u003c/em\u003e Permeability Study\u003c/h2\u003e\u003cp\u003eAn everted gut permeability study was performed to measure the intestinal permeation of EZT from pure EZT dispersion and EZT-NCs suspension. The \u003cem\u003eex vivo\u003c/em\u003e gut sac model predicts intestinal transport accurately due to its enzyme activity and transporter expression closely mirroring the human intestine. In the intestine, large amounts of P-gp efflux transporter are present; hence, to check the P-gp inhibitor activity of P188, this study was performed. In this study, the exsorption of EZT dispersion and EZT-NCs from serosal medium to mucus medium was evaluated. This study concluded that a greater amount of drug permeated into the mucous medium than the pure EZT dispersion shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (b), indicating that permeation of P188 incorporated EZT-NCs was more than that of pure EZT dispersion because of P-gp inhibition caused by P188. The apparent permeability coefficient (P\u003csub\u003eapp\u003c/sub\u003e) of EZT-NCs(5.73\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e cm/min) was approximately 5.23-fold higher than compared that of the pure EZT dispersion (1.09\u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003ecm/min). The increased permeability of EZT-NC can be ascribed to their nanometric size and the inhibitory effects of P188 on P-gp [\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec45\" class=\"Section2\"\u003e\u003ch2\u003e5.10 \u003cem\u003eIn vivo\u003c/em\u003e pharmacokinetic study\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003ein vivo\u003c/em\u003e pharmacokinetic study was performed to assess \u003cem\u003ein vivo\u003c/em\u003e bioavailability. After oral administration of pure EZT and EZT-NCs, plasma concentrations were obtained and graphically illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Also, the pharmacokinetic parameters determined from non-compartmental analysis are presented in Table\u0026nbsp;\u003cspan refid=\"Tab9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. The oral administration of pure EZT and EZT-NCs resulted in a C\u003csub\u003emax\u003c/sub\u003e of 3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u0026micro;g/mL and 8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 \u0026micro;g/mL after 2 hours (T\u003csub\u003emax\u003c/sub\u003e), respectively. The AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;48\u003c/sub\u003e values for pure EZT and EZT-NCs were 95.65 (\u0026micro;g/mL*h) and 205.8 (\u0026micro;g/mL*h), respectively.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab9\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 9\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePharmacokinetic parameters of EZT and EZT-NCs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e\u003cp\u003ePharmacokinetic parameters\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;48\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/ml*h)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAUC\u003csub\u003e0\u0026minus;\u0026infin;\u003c/sub\u003e\u003c/p\u003e\u003cp\u003e(\u0026micro;g/ml*h)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDispersion of EZT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e95.65\u0026thinsp;\u0026plusmn;\u0026thinsp;9.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e293.10\u0026thinsp;\u0026plusmn;\u0026thinsp;23.98\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEZT-NCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e205.80\u0026thinsp;\u0026plusmn;\u0026thinsp;27.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e299.47\u0026thinsp;\u0026plusmn;\u0026thinsp;33.76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThis suggests that the AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;48\u003c/sub\u003e of EZT-NCs was 2.15 times higher than that of pure EZT when administered orally (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Thus, C\u003csub\u003emax\u003c/sub\u003e and AUC of the EZT-NCs demonstrated increased EZT bioavailability using the EZT-NCs formulation. This increased bioavailability could be related to the reduction of particle size to nanocrystals, significantly enhancing the dissolution rate. This improvement is attributed to a larger surface area, entanglement with the mucus layer, greater saturation solubility, and a higher concentration gradient between the GI lumen and blood[\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e] Furthermore, the potential of poloxamers to increase permeability by modifying the micro-viscosity of the cellular membrane contributed to improved bioavailability[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] These factors collectively lead to faster and more complete drug absorption, resulting in higher C\u003csub\u003emax\u003c/sub\u003e and AUC values.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe study successfully prepared EZT-NCs using the antisolvent precipitation\u0026ndash;ultrasonication method, a bottom-up approach for size reduction. P188 was identified as an effective stabilizer for nanonizing EZT. Computational BBD design pinpointed significant parameters affecting response variables. The EZT-NCs demonstrated higher saturation solubility and dissolution rate than pure EZT. Everted gut sac results and \u003cem\u003ein vivo\u003c/em\u003e study showed a considerable enhancement over those of the pure EZT, hence bioavailability can be effectively achieved using nanocrystals. In the future, it is crucial to carry out pharmacodynamics studies to validate additional therapeutic evidence and evaluate the potential enhancement of current clinical effectiveness using EZT-NCs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank BVDU, Poona College of Pharmacy, Pune, India, for providing research facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work does not involve any funding sources.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare(s) that they have no conflicts of interest to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePratiksha Pathade: Writing original draft, drawing figures, and validation. Shubhangi Thool: Methodology, data curation, review, and editing. Varsha Pokharkar: Supervision, editing, resources, and review.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData can be made available on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXu Q, Deng Y, Xiao J, Liu X, Zhou M, Ren Z, Peng J, Tang Y, Jiang Z, Tang Z, Liu L. (2021). Three musketeers for lowering cholesterol: statins, ezetimibe and evolocumab. \u003cem\u003eCurr Med Chem. \u003c/em\u003e28(5):1025-41. https://doi.org/10.2174/0929867327666200505091738\u003c/li\u003e\n\u003cli\u003eShukr MH, Ismail S, Ahmed SM. (2019). Development and optimization of ezetimibe nanoparticles with improved antihyperlipidemic activity. \u003cem\u003eJ Drug Del Sci Technol.\u003c/em\u003e. 49:383-95. https://doi.org/10.1016/j.jddst.2018.12.001\u003c/li\u003e\n\u003cli\u003eShevalkar G, Vavia P. (2019). 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Excipients enhance intestinal absorption of ganciclovir by P-gp inhibition: assessed in vitro by everted gut sac and in situ by improved intestinal perfusion.\u003cem\u003eInt J Pharm\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e403(1-2):37-45. https://doi.org/10.1016/j.ijpharm.2010.10.017\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Ezetimibe, Nanocrystals, Antisolvent precipitation-ultrasonication, Box-Behnken Design, Oral bioavailability","lastPublishedDoi":"10.21203/rs.3.rs-7488181/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7488181/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEzetimibe (EZT), a BCS class II drug, is a selective cholesterol absorption inhibitor used to treat high blood cholesterol. However, its clinical efficacy is limited by poor solubility and bioavailability. This study aims to address the low solubility and bioavailability of EZT. Aiming to improve the solubility, dissolution, and bioavailability of this hydrophobic drug by formulating EZT nanocrystals (EZT-NCs) using an innovative antisolvent precipitation-ultrasonication method. This bottom-up approach of optimizing structure and properties through particle size reduction, followed by lyophilisation, holds promises for enhanced therapeutic performance and effectiveness. Optimization of variables, including solvent: antisolvent ratio, poloxamer188 (P188) concentration, and ultrasonication amplitude, was achieved using Box-Behnken Design (BBD) as a computational tool, to produce uniform nanosized crystals with good dispersibility. Optimized EZT-NCs showeda particle size of 340±12.00 nm, PDI of 0.12±0.05, and zeta potential of -46±0.15 mV. DSC and pXRD confirmed reduced crystallinity. Scanning Electron microscopy (SEM) confirmed a nanometric size range, and \u003cem\u003ein vitro\u003c/em\u003e dissolution revealed 85.17% release for EZT-NCs within 1 hour, a 1.87-fold increase over pure EZT. The everted gut sac model showed EZT-NCs had 5.23 times higher permeability than pure EZT, due to their nanometric size and P-gp inhibition by P188. Furthermore, EZT-NCs achieved a C max of 8.22μg/mL, with an AUC \u003csub\u003e0-48\u003c/sub\u003e that was 2.15 times higher than pure EZT. EZT-NCs demonstrated improved aqueous solubility, dissolution range, and bioavailability, suggesting their potential for an enhanced oral delivery approach.\u003c/p\u003e","manuscriptTitle":"Structure-property optimization of Ezetimibe nanocrystals by computationally guided bottom-up engineering for enhanced bioavailability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 10:39:02","doi":"10.21203/rs.3.rs-7488181/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-16T15:06:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-24T15:37:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-18T03:42:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178037612776832356169934610971519646777","date":"2025-09-09T16:53:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"291541878798777248033096269830397386495","date":"2025-09-07T00:47:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-06T22:28:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-03T12:42:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-02T12:16:08+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioNanoScience","date":"2025-08-29T11:20:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bionanoscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bnsc","sideBox":"Learn more about [BioNanoScience](http://link.springer.com/journal/12668)","snPcode":"12668","submissionUrl":"https://submission.nature.com/new-submission/12668/3","title":"BioNanoScience","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"97b92925-d159-4f77-817b-8448542ec96d","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:22:21+00:00","versionOfRecord":{"articleIdentity":"rs-7488181","link":"https://doi.org/10.1007/s12668-025-02358-8","journal":{"identity":"bionanoscience","isVorOnly":false,"title":"BioNanoScience"},"publishedOn":"2026-01-16 16:29:09","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-09-12 10:39:02","video":"","vorDoi":"10.1007/s12668-025-02358-8","vorDoiUrl":"https://doi.org/10.1007/s12668-025-02358-8","workflowStages":[]},"version":"v1","identity":"rs-7488181","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7488181","identity":"rs-7488181","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}