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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Preparation of novel phospholipid-based sonocomplexes for improvedintestinal permeability of rosuvastatin: In vitro characterization, dynamicsimulation, Caco-2 cell line permeation and in vivo assessment studies

Wessam Hamdy Abd-Elsalam1, Sara Nageeb El-Helaly1, Mohammed Abdallah Ahmed1,Abdulaziz Mohsen Al-mahallawi⁎,1

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt

A R T I C L E I N F O

Keywords:SonocomplexesUltrasound irradiationRosuvastatinPhospholipid complexesCaco-2 cell line

A B S T R A C T

The study aimed to fabricate innovative drug-phospholipid complexes termed “sonocomplexes” adopting ul-trasound irradiation to increase the liposolubility and to enhance the intestinal absorption of rosuvastatin as amodel drug for BCS class III active pharmaceutical ingredients (APIs). A 22 full factorial design was fashioned toinvestigate the influence of phosphatidylcholine content in the phospholipid (∼30 and 60%) and molar ratio ofphospholipid to rosuvastatin (1:1 and 2:1) on physicochemical properties of sonocomplexes. In comparison topure drug, sonocomplexes showed a minimum of about 2 folds and a maximum of about 15 folds increase inlipophilicity (expressed in terms of partition coefficient, P). Results of molecular docking, dynamic simulations,Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) confirmed the stronginteractions between rosuvastatin and the phospholipid via hydrogen bonding interaction, van der Waals forcesand hydrophobic interaction. The complexation efficiency reached around 99% and transmission electron mi-croscopy (TEM) of the aqueous dispersion of the optimal sonocomplex showed spherical nanosized vesicles. Theoptimal sonocomplex showed significantly superior Caco-2 cells permeability and markedly better oral bioa-vailability compared to the pure drug. In summary, sonocomplexes can be considered as effective approach forenhancing the liposolubility and consequently the intestinal permeability of BCS class III drugs.

1. Introduction

Sonochemistry is situated as one of the most influential tools in thesynthesis of various chemical and biological compounds. The utilizationof high intensity ultrasonic irradiation delivers an extraordinary reac-tion conditions; represented by short duration of extremely high tem-peratures and pressures in liquids (Bang and Suslick, 2010).

The theory of sonochemistry is mainly based on the process ofacoustic cavitation (Manickam and Ashokkumar, 2014; Pokhrel et al.,2016). The propagating sound wave creates tiny acoustic bubbles; thatencompass an incredible amount of energy which can act as a spark forthe initiation of different chemical reactions. The use of ultrasound formaterial synthesis is a well-established concept. Compared to othermethods, sonochemistry is economical and highly effective methodaffording enough quantity of energy that is able to accomplish chemicalreactions under mild conditions of temperature and pressure (Suslick,1995).

Phospholipids are important cell membrane components, exertingthe actions of keeping cell membrane fluidity. Over the past few dec-ades, model phospholipids membranes and vesicles have provided aplatform for drug delivery (Allen and Cullis, 2013). In addition, phos-pholipids complexes have been studied with the purpose of augmentingthe therapeutic efficacy of drug molecules facing poor oral absorptionand diminished intestinal permeability. Hence, these complexes max-imize the affinity of the drugs to their targeted site of action by facil-itating their uptake through biological membranes (Cui et al., 2006).

Rosuvastatin (RS) is a fully synthetic lipid-lowering agent used toreduce circulating low-density lipoprotein cholesterol (LDL-C) levels;and consequently, decrease the risk of cardiovascular diseases. RS is aselective competitive inhibitor of 3-hydroxy-3-methylglutaryl coen-zyme A (HMG-CoA) reductase which is the rate controlling enzyme ofthe mevalonate pathway that produces cholesterol. RS belongs to classIII active pharmaceutical ingredients (APIs) according to the bio-pharmaceutical classification system (BCS). Thereby, the drug possesses

https://doi.org/10.1016/j.ijpharm.2018.07.005Received 10 February 2018; Received in revised form 30 June 2018; Accepted 1 July 2018

⁎ Corresponding author at: Faculty of Pharmacy, Cairo University, Kasr El-Ainy Street 11562, Egypt.

1 The authors contributed equally to this work.E-mail address: Abdulaziz.mohsen@pharma.cu.edu.eg (A.M. Al-mahallawi).

International Journal of Pharmaceutics 548 (2018) 375–384

Available online 03 July 20180378-5173/ © 2018 Elsevier B.V. All rights reserved.

T

high solubility but suffers from poor permeability; which in turn resultsin low absolute bioavailability (∼20%) (Bergman et al., 2010; Ponnurajet al., 2015). Therefore; the complexation between RS as a model drugfor BCS class III compounds and phospholipid could enhance the oralbioavailability and subsequently; the therapeutic efficacy of the parentmolecule.

The present work aimed to use the ultrasound energy to formulatephospholipids complexes of RS (sonocomplexes). The objectives of thisstudy are summarized as follows: (i) RS-phospholipid sonocomplexeswere prepared for the first time adopting the ultrasound energy, orwhat can be named as “sonocomplexation”. These sonocomplexes wereprepared using two types of phospholipids at two different molar ratios.(ii) The apparent solubility and the partition coefficient of the preparedsonocomplexes were explored in order to evaluate the alteration insolubility properties of sonocomplexes compared to that of the puredrug. Molecular docking together with Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) wereperformed to investigate the nature of RS-phospholipid interaction inthe sonocomplex. In addition, molecular dynamic simulation was donein order to study the behavior of sonocomplex molecule when dispersedin water. (iii) To verify the enhanced permeability of the prepared so-nocomplexes over the pure drug, Caco2 cell-line permeation and in vivoabsorption studies in rats were conducted for the aqueous dispersion ofoptimal sonocomplex in comparison to drug solution.

2. Materials and methods

2.1. Materials

Rosuvastatin (RS) was a kind gift from Marcyrl pharmaceutical in-dustries (Cairo, Egypt). L-α-phosphatidylcholine from soybean (TypeIV-S, ≥30%), L-α-phosphatidylcholine from egg yolk (∼60%), n-oc-tanol, tetrahydrofuran (THF) and methanol (HPLC grade) were pur-chased from Sigma–Aldrich Chemical Co. (St. Louis, Missouri, USA).Ortho-phosphoric acid (HPLC grade) was obtained from Merck(Darmstadt, Germany). Acetone was procured from El-NasrPharmaceutical Chemicals Co. (Cairo, Egypt). All other reagents were ofanalytical grade.

2.2. Preparation of RS–sonocomplexes

A 22 full factorial design was utilized for the forecasting and theinvestigation of the experimental trials to choose the optimal sono-complex (Table 1). Sonocomplexation was carried out using two typesof phospholipids; namely, phospholipid from soybean and phospholipidfrom egg yolk, to investigate the influence of phosphatidylcholinecontent in the phospholipid (∼30 and 60%, respectively) on the phy-sicochemical properties of the prepared sonocomplexes. In addition,two molar ratios of phospholipid to RS (1:1 and 2:1) were utilized toevaluate the effect of molar ratio on sonocomplexes properties. Thecompositions of the prepared sonocomplexes are listed in Table 2. In

sonocomplexation technique, specified weights of RS and phospholipidwere added to an accurate volume of tetrahydrofuran (THF), then themixture was sonicated in an ultrasonic water-bath sonicator (Crest Ul-trasonics Corp., NJ, USA) at room temperature for 30min. Followingthat, the organic solvent was removed under vacuum overnight and thedried residues were left in a desiccator for 24 h. The obtained sono-complexes were then reserved in an amber colored glass bottles andstored at room temperature.

In this study, Design-Expert software (Version 10, Stat-Ease Inc.,MN, USA) was used for the generation and evaluation of the statisticalexperimental design. Means were compared by ANOVA and sig-nificance level was set at P=0.05. Suitable regression models weredriven to enable navigation of the experimental space (Montgomery,2001). Numerical optimization was performed using the program ac-cording to the constraints set based on the utilization of desirabilityfunctions. All experiments were performed at least three times. All datawere presented as mean ± standard deviation.

2.3. Solid state characterization of the sonocomplexes

2.3.1. Differential scanning calorimetry (DSC)The thermograms of pure RS, phospholipid, RS-phospholipid phy-

sical mixture and the sonocomplex were recorded via DSC-60 differ-ential scanning calorimeter (Shimadzu, Kyoto, Japan) in order toevaluate the complex formation. Inspected samples were sealed in flat-bottomed aluminum pan and heated at a constant rate of 10 °C/minutefrom 20 to 200 °C in an atmosphere of nitrogen. The thermal behaviorwas integrated via an analyzer. The apparatus was calibrated withpurified indium (99.9%).

2.3.2. Fourier transform infrared (FT-IR) spectroscopyFT-IR spectra of pure RS, phospholipid, RS-phospholipid physical

mixture and the sonocomplex were recorded in potassium bromidediscs to confirm the formation of sonocomplex. The samples werescanned over the range 4000–400 cm−1 at ambient temperature; usingAffinity-1 FT-IR spectrophotometer (Shimadzu, Kyoto, Japan).Technical procedures of smoothing the spectra and the baseline corre-lation were applied.

2.4. Molecular docking

Molecular docking was used to predict the interactions between RSand phospholipid. All the molecular docking steps were done usingMOE version 2014.0901 (Chemical Computing Group Inc., Montreal,Canada). RS and dilinoleoylphosphatidylcholine (DLPC) were drawn,charges were assigned and energy was minimized with Amber10:EHTforce field to an energy gradient of 0.1 kcal/mol. RS was then dockedinto DLPC. The best docking type was chosen based on the bindingenergy scores and FTIR characterization results.

2.5. Molecular dynamic simulation

The molecular dynamic behavior of a monomer of RS-phospholipidsonocomplex in water system was investigated by molecular dynamicsimulations using MOE version 2014.0901. The most favorable RS/DLPC sonocomplex obtained from the docking step was used for mo-lecular dynamic simulation using Amber10:EHT force fields. A cubicbox of water molecules with periodic boundary conditions was set up asthe simulation system. The energy was minimized, and the system washeated from 0 °K to 310 °K by Nose-Poincare thermostat. A simulationwas then run for 1 nano second (ns).

2.6. High performance liquid chromatography (HPLC) analysis

The quantification of RS was performed using an isocratic reportedHPLC method with modification (Shah et al., 2011). The HPLC system

Table 122 Full factorial design used for the optimization of RS–phospholipid sono-complexes.

Factors (independent variables) Levels

Phospholipid type Soy bean Egg yolkPhospholipid: RS (molar ratio) 1:1 2:1

Responses (dependent variables) Desirability Constraints

Water solubility (mg/mL) MinimizePartition coefficient (P) MaximizeParticle size of the aqueuse dispersion of the

sonocomplex (nm)Minimize

Abbreviation: RS, rosuvastatin.

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consisted of a Zorbax Extend-C18 column (4.6 mm×250mm) con-taining 3.5 µm size adsorbent as stationary phase (Agilent technologies,Santa Clara, California, USA), LC-10AD pump, SPD-10A UV detectorand CR6A Chromatopac integrator (Shimadzu, Kyoto, Japan). Thecolumn was maintained at room temperature (25.0 ± 2.0 °C). Themobile phase consisted of mixture of methanol and double distilledwater (68:32, v/v, respectively) and the pH was adjusted to 3.0 usingortho-phosphoric acid. The elution was fixed at a flow rate of 1.0 mL/min. Effluents were monitored at 243.4 nm. The method was validatedfor linearity, accuracy, selectivity and precision.

2.7. Determination of RS content in sonocomplexes and calculation ofcomplexation efficiency (CE%)

The CE% of sonocomplexes was determined by dividing the differ-ence between the total content of RS in the complex and the free RS;that did not form a complex with phospholipid, by the total RS content(Song et al., 2008). Both total and free RS content in the sonocomplexwere determined by the above mentioned HPLC method. In order todetermine the total RS content in the formed complexes, approximately5mg of the complex were allowed to dissolve in 5mL of methanol.Then, the methanolic solution was evaporated to dryness under vacuumand the dried residue was reconstituted with the mobile phase to beanalyzed by HPLC.

According to the physicochemical properties of RS and phospho-lipid; both free and complexed RS dissolve in methanol. On the otherhand, only free RS can dissolve in acetone while phospholipid isacetone-insoluble, forming a colloidal dispersion. The free RS contentwas determined as follows: 5 mg of the complex were dispersed in 3mLacetone and vortexed for 2min. The dispersion was then centrifuged at15000 rpm for 30min. The clear supernatant was taken and evaporatedto dryness under vacuum then reconstituted with the mobile phase.Twenty microliters aliquot of the reconstituted solution were injectedinto the HPLC system.

2.8. Solubility studies and determination of n-octanol/water partitioncoefficient

2.8.1. Solubility studiesSolubility determinations of RS and sonocomplexes were carried out

by adding excess quantities of the tested sample to 10mL of water insealed glass containers. The containers were shaken for 24 h at 37 °Cand 100 rpm and then centrifuged at 15,000 rpm for 30min (Maitiet al., 2007; Ruan et al., 2010). After filtering the supernatant, 1 mL ofthe clear solution was withdrawn and mixed with the mobile phase.Aliquot of the resulting solution was injected into HPLC to determinethe concentration of RS.

2.8.2. Determination of n-octanol/water partition coefficient (P)N-octanol/water partition coefficient (P) measurements were con-

ducted to verify the liposolubility enhancement of RS after sonocom-plex formation. Accurately weighed amount of RS or sonocomplex wasadded to 10mL of water in sealed glass containers and agitated for 24 hat 37 °C (Yue et al., 2010). Thereafter; they were centrifuged at15,000 rpm, for 30min to remove excessive undissolved residues. Ten

mL of n-octanol were added to the aqueous solution and agitated for24 h at 37 °C. Separation of the water phase and n-octanol phase wereconceded by centrifugation at 4000 rpm for 15min, followed by fil-tration of both phases through a double 0.45 μm membrane (Morsiet al., 2017). The concentrations of the drug in both layers were de-termined using HPLC and the partition coefficient (P) was calculatedfrom the following equation:

=P Co Cw

Where Co is the concentration of the drug in n-octanol; Cw is theconcentration in water.

2.9. Particle size, polydispersity index and zeta potential measurements

The particle diameter (z-average) and the polydispersity index (PDI)of the appropriately diluted aqueous dispersions of sonocomplexes weredetermined by dynamic light scattering technique using Zetasizer NanoZS (Malvern Instrument Ltd., Worcestershire, UK). The zeta potential(ζ) was measured in distilled water using the same instrument by ob-serving the electrophoretic mobility of the particles in an electricalfield. All measurements were performed in triplicate and the meanvalues were calculated.

2.10. Transmission electron microscopy (TEM)

The inherent morphology of the vesicles formed upon dispersing theoptimal sonocomplex in water was visualized via TEM (Joel JEM 1230transmission electron microscope, Tokyo, Japan). One drop of the ap-propriately diluted sample was positioned onto carbon-coated coppergrid. The excess was drawn off with a filter paper. Subsequently, thesamples were dried for 5min at room temperature and then subjectedto TEM observation (Ruan et al., 2010).

2.11. Sonocomplex stability

In order to investigate physical stability, the optimal sonocomplexformulation was stored for 45 days in amber colored glass bottles andstored at room temperature. At the end of the storage period, the so-nocomplex was evaluated with respect to its appearance, residual drugcontent, CE%, particle size of the reconstituted sonocomplex, watersolubility and n-octanol/water partition coefficient (P). Statisticalanalysis of the obtained results was performed by Student’s t-test usingSPSS 17.01 software (SPSS Inc., Chicago, USA). Difference at P≤ 0.05was considered significant.

2.12. Transport studies

2.12.1. Caco-2 cells cultureThe human Caco-2 cells were obtained from the American Type

Culture Collection (Manassas, USA). The Caco-2 cell culture is con-sidered by the FDA, as a viable simulation of human intestinal ab-sorption. At the Egyptian National Cancer Institute (Cairo, Egypt), theCaco-2 cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM) supplemented with 10% fetal bovine serum (FBS), 1% L-glu-tamine and 1% penicillin/streptomycin. In order to allow maturation,

Table 2Composition and in-vitro characterization of the prepared RS–phospholipid sonocomplexes.

Formula Phospholipid type Molar ratio (PL: RS) Water solubilitya (mg/mL) Partition coefficienta Particle sizea (nm) PDIa Zeta potentiala (mV)

F1 Soy bean 1:1 1.401 ± 0.05 0.141 ± 0.03 800.2 ± 15 0.65 ± 0.03 −39.45 ± 0.35F2 Soy bean 2:1 0.325 ± 0.01 0.794 ± 0.36 545.2 ± 40 0.49 ± 0.13 −39.35 ± 0.49F3 Egg yolk 1:1 0.416 ± 0.05 0.906 ± 0.06 554.3 ± 57 0.34 ± 0.09 −16.3 ± 0.99F4 Egg yolk 2:1 0.376 ± 0.01 1.144 ± 0.16 477.55 ± 70 0.42 ± 0.23 −28.1 ± 0.71

Abbreviations: RS, rosuvastatin; PL, phospholipid; PDI, polydispersity index.a Data represented as mean ± standard deviation (n= 3).

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the cells were stored in an incubator at 37 °C, 5% CO2 and 95% relativehumidity.

2.12.2. Cell culture for transport studiesFor transport studies, the Caco-2 cells were seeded on polycarbonate

inserts in Transwell® 6-well plates (pore size; 0.4 µm, growth area;4.67 cm2) at a density of 50,000 cells/cm2. Culture medium was addedto apical (AP) (1.5 mL) and basolateral (BL) (2.6 mL) sides and wasreplaced every other day for the first week and daily thereafter. After18–21 days post-seeding, the cells were well-developed. The integrity ofthe monolayers was evaluated by the lucifer yellow (LY) permeabilityassay according to Piazzini et al. (2017). The critical maximum diffu-sion of LY to recognize leaky monolayers was estimated to be less than3% of initial concentration.

2.12.3. Transport experimentTransport studies were carried out according to the procedure de-

scribed by Hubatsch et al. (2007). Before conducting the transport ex-periment, the culture medium was removed and the Caco-2 cellmonolayer was washed three times with pre-warmed (37 °C) blankHank’s balanced salt solution (HBSS) supplemented with 25mM HEPES(pH 7.4) and then incubated at 37 °C with the new buffer. After themonolayer was equilibrated in blank HBSS for 30min, the AP chamberwas loaded with 1.5mL of a HBSS-based solution containing the drug ordispersed sonocomplex (160 μg/mL of RS), while the BL chamber wascharged with 2.6mL of the fresh HBSS buffer. Hundred μL samples weretaken from BL side (the receiver) at 0, 0.5, 1, 2, 3 and 4 h and they wereanalyzed for drug content by HPLC. This was followed by the transfer of100 µL of fresh buffer to the BL chamber to keep the volume invariable.The cumulative amount (μg) of RS permeated was plotted as a functionof time and the apparent permeability coefficient (Papp) was calculatedusing the following equation (Li et al., 2014):

= ×P dQdt C A

1app

0

where dQ/dt is the transport rate (µg/s), C0 is the initial drugconcentration on the AP side (µg/mL) and A is the surface area of themembrane (4.67 cm2). Statistical analysis of the obtained results wasperformed by Student’s t-test using SPSS 17.01 software. Difference atP≤ 0.05 was considered significant.

2.13. Preclinical pharmacokinetic studies in rats

2.13.1. Study designThe protocol of the in vivo studies was evaluated and approved by

the Research Ethics Committee in the Faculty of Pharmacy, CairoUniversity, Egypt. The in vivo absorption studies of the aqueous dis-persion of the best achieved RS sonocomplex, relative to an aqueousdrug solution, were carried out in male Wister rats (200 ± 20 g) toprovide a preliminary indication of the intrinsic impact of the phos-pholipid-based sonocomplex formation on drug absorption. The animalexperiments followed a two-treatment, non-blind, randomized, paralleldesign. The rats were cared for and handled according to the institu-tional regulations for experimental animals. They were housed in ananimal care facility under standardized conditions of controlled tem-perature (25 ± 0.5 °C), humidity (50–60%) and alternate 12 h light–-dark cycles. They were kept on standard pellet diet while water wasmade available ad libitum. These measures were taken to minimize thefactors that can impact the animal welfare and/or influence the re-producibility and validity of the derived preclinical data.

2.13.2. Oral administration of treatmentsThe in vivo pharmacokinetic studies following the oral administra-

tion of the treatments were evaluated in male Wistar rats. Ten adultmale Wistar rats were randomly assigned to one of two groups of equalnumber. On the study day, the overnight (12 h) fasted rats of Group I

were orally treated with an aqueous RS solution (treatment A)equivalent to 5mg/kg (Basu et al., 2013), while Group II rats wereorally treated with an aqueous dispersion containing predeterminedamount of the optimal sonocomplex (treatment B) equivalent to thesame RS dose of the first group. Blood samples were collected, undermild ether anesthesia, prior to and at intervals up to 72 h after oraladministration of treatments. The samples were immediately placedinto 1.5 mL micro-centrifuge heparinized tubes and centrifuged for5min at 5000g. The plasma samples were separated, kept in labeledglass tubes and frozen at −20 °C till analysis.

2.13.3. Sample processingThe extraction of RS was carried out according to the procedure as

described by Shah et al. (2011) with modification. In brief, 600 µL ofabsolute ethanol was added to 200 µL of plasma and vortexed (2min)for deproteination. Then, one mL of diethyl ether was added, vortexedfor 5min and centrifuged at 3500 rpm at 0 °C for 5min. The super-natant organic layer was separated in a test tube and evaporated tocomplete dryness using centrifugal vacuum concentrator (Eppendorf5301, Germany) at 40 °C. After drying, the residue was reconstituted in500 µL of mobile phase, vortexed for 2min and 20 µL sample was in-jected into HPLC system for analysis.

2.13.4. Pharmacokinetic and statistical analysisThe pharmacokinetic analysis of the data was performed according

to the non-compartmental analysis using WinNonlin® software. Themaximum concentration (Cmax) and time to reach maximum con-centration (Tmax) were directly obtained from the concentration–timecurves. The area under the curve from zero to 72 h (AUC(0–72), ng.h/mL) was estimated by the log-linear trapezoidal rule. The results wereexpressed as mean (± standard deviation) values of 5 rats. Statisticalanalysis of the determined pharmacokinetic parameters was performedusing the same software. A P-value of 0.05 was employed to assess thestatistical significance of the treatments on the derived pharmacoki-netic parameters.

3. Results & discussion

3.1. Preparation of RS-sonocomplexes

Ultrasound irradiation is an impressive tool, which can be used forthe synthesis of nanostructured materials. In this manuscript, the fac-tors selected and levels used were based on preliminary trials (data notshown) to determine the probable ranges of the independent variables.It is known that the two phospholipid types used in this study are freelysoluble in complexation solvent (THF) while RS is slightly soluble. So,the sonication time was set to be 30min which was the time needed forsonocomplex formation as indicated by obtaining a clear solution aftersonicating the drug-phospholipid mixture in THF for that period oftime.

3.2. Mechanism of sonocomplexation

Ultrasonic irradiation provides unusual reaction conditions; namely,a short duration of extremely high temperatures and pressures in li-quids. Remarkably, such extraordinary conditions are not derived di-rectly from ultrasound itself; in fact, ultrasound wavelengths are muchbigger than molecular dimensions. Thus, no molecular interaction be-tween ultrasound waves and the used chemicals took place (Bang andSuslick, 2010). Instead, when the ultrasound waves pass through THFsolution, it creates regions inside the solution with high and low pres-sure according to periodic compression and expansion (Plesset andProsperetti, 1977). This change in pressure precedes the process ofacoustic cavitation i.e. nucleation, growth and collapse of acousticbubble. Air molecules dissolved in the solution diffuse to form bubblesat the low-pressure cycle. On reaching the next cycle, the high external

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pressure compresses the bubble and the matter inside violently. Thisprocess of bubble growth and compression continues until the externalpressure dominates and the bubble collapses, as shown in Fig. 1. Re-ported theories states that pressure and temperature inside the bubblehas been calculated to rise to ∼1000 bar and ∼5000 °K during cavi-tation (Bang and Suslick, 2010; Xu et al., 2013).

The core region, known as the hotspot, features high-energy particle

collision that generates energy as high as 13 eV (Flannigan and Suslick,2005). Such drastic conditions can induce abnormal physical and che-mical changes and facilitate the complexation reaction between phos-pholipids and RS to produce RS-phospholipid complex, sonocomplex(Suslick et al., 1996; Mdleleni et al., 1998; Pokhrel et al., 2016). Thecomplexation phenomenon arises either from implosion due to ambientpressure or explosion due to boundary to produce microjets (Bang and

Fig. 1. The mechanism of sonocomplexation between phospholipid and rosuvastatin.

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Suslick, 2010). The imploding bubbles produce shock waves that ac-celerate RS and phospholipid molecules to several hundred meters persecond (Pokhrel et al., 2016). Collision at this speed results in drasticstructural transformation and formation of RS sonocomplexes.

3.3. Solid state characterization of the sonocomplexes

3.3.1. DSC studiesThe superimposed thermograms of RS, phospholipid, their physical

mixture and the sonocomplex, are illustrated in Fig. 2. RS displayed abroad endothermic peak at 145.77 °C, attributing to its crystallinecharacter, while phospholipid thermogram lacked any characteristicmelting behavior ensuring its amorphous nature. Unlike the physicalmixture thermogram, RS endothermic peak was almost nonexistent inthe sonocomplex thermogram. Actually, sonocomplex displayed anamorphous nature; that is completely different from that of the drug.These findings demonstrate the complex formation through creation ofhydrogen bond and van der Waal's hydrophobic interaction forces be-tween the drug and the phospholipid molecules (Jena et al., 2014).

3.3.2. FT-IR studiesFig. 3 demonstrates the FTIR spectra of RS, phospholipid, their

physical mixture and the sonocomplex. The FT-IR spectrum of RS re-vealed characteristic strong and broad band at 3390.86 cm−1 indicatingOeH stretching, while a sharp band of NeH stretching appeared at2962.66 cm−1. The band at 1739.79 cm−1 is assigned for C]Ostretching of carboxylic group. The other principle peaks are present at2916.37 cm−1 for olefinic ]CeH stretching, 1604.77 cm−1 for C]Nstretching, 1558.48 cm−1 for C]C stretching, 1508.33 cm−1 for NeHbending, and 1436.04 cm−1 and 1381.03 cm−1 for asymmetric andsymmetric bending vibration of CeH group, respectively. The band at1338.60 cm−1 allocates the asymmetric vibration for S]O, while thatat1157.29 cm−1 represents CeF stretching vibrations. The peaks at775.38 cm−1, 567.07 cm−1 and 520.78 cm−1 are the absorption peaksfor out of plane C]C of benzene ring, and that at 1230.58 cm−1 is thebending vibration for CeH. The FT-IR data of phospholipid showedcharacteristic bands of quaternary nitrogen at 3444.87 cm−1. The peakat 1739.79 cm−1 assigns for C]O stretching, while the bandat1234.44 cm−1 and 1068.56 cm−1 corresponds to P]O stretching andPeOeC stretching, respectively (Jena et al., 2014).

Comparative spectral analysis showed no shift in the absorptionpeaks of the drug or the phospholipid, when compared to the physicalmixture. On the other hand, the sonocomplex FT-IR chart showed sig-nificant changes in many peaks. The charts bared shift in the position ofOeH peak of the drug from 3390.86 cm−1 to 3333.14 cm−1, andquaternary nitrogen band of the phospholipid from 3444.87 cm−1 to

3383.14 cm−1. Furthermore, the OeH peak of the drug was obviouslysuppressed, while the quaternary nitrogen band of phospholipid wasrelatively narrower. These results set a great evidence of the creation ofintermolecular hydrogen bonding interactions between the two func-tional groups, indorsing the formation of drug phospholipid complexes.

3.4. Molecular docking and molecular dynamic simulations

Molecular docking was performed to find out the possible interac-tions between RS and the major component in the egg yolk phospho-lipid, DLPC (as indicated by the manufacturer’s certificate). Theminimum energy complex structure is shown in Fig. 4a. The dockingenergy was −5.72 kcal/mol. The interactions between RS and DLPCwere hydrogen bonding interaction, van der Waals forces and hydro-phobic interaction. Results of molecular docking conforms with FTIRresults which indicated a strong interaction between RS and at the polarhead of phospholipid.

A short time dynamic simulation was done to predict the config-uration of sonocomplex monomer when dispersed in water. Fig. 4bshows a snapshot of a sonocomplex monomer after 1 ns simulation. Thesimulation showed that RS interaction with polar head of phospholipidis stable after dispersion of sonocomplex in water.

3.5. Determination of complexation efficiency (CE%), water solubility andn-octanol/water partition coefficient (P)

Phospholipids are ampholytics surfactants; characterized by theirsignificant biocompatibility, ready availability and their ability tomagnify the liposolubility of water soluble drugs through a complexa-tion process. The complexation efficiency (CE%) was found to be about99% in the prepared sonocomplexes.

Fig. 5 shows the influence of the independent variables on theaqueous solubility and partition coefficient (P) of the prepared sono-complexes. The solubility of sonocomplexes ranged from 0.325 ± 0.01to 0.958 ± 0.05mg/mL as shown in Table 2. It is clear that there was amarked decrease in the water solubility of the prepared complexes incomparison to the measured pure drug solubility (10.68 ± 0.92mg/mL), suggesting the more lipophilic nature of sonocomplexes relative toRS. It is worth mentioning that for all the sonocomplexes, the maximumdose of RS (40mg) can still dissolve in less than 250mL which is thecriteria for highly soluble drugs. Statistical analysis revealed that thephospholipid type and the molar ratio of phospholipid to RS had sig-nificant effects on the sonocomplex solubility (P < 0.0001 for bothvariables). Regarding the phospholipid type, it was clear that the so-nocomplexes prepared with phospholipid from soybean source ex-hibited greater water solubility than those complexed with egg yolk

Fig. 2. DSC thermograms of pure rosuvastatin, egg yolk phospholipid, physical mixture, and the optimal sonocomplex (F4).

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phospholipid. This can be attributed to the fact that the degree of un-saturation of soybean phosphatidylcholine is greater than that of eggyolk phosphatidylcholine. The higher the degree of lipid unsaturation,the greater the water solubility is. On the other hand, increasing themolar ratio of phospholipid to RS resulted in enhancing the liposolu-bility of sonocomplexes and consequently led to a decrease in theirwater solubility. Complexing RS with phospholipid imparts the am-phiphilic surfactant nature of the phospholipid to the hydrophilic drugand accordingly modifies its lipophillic character (Pathan andBhandari, 2011).

In accordance with the water solubility measurements, the sono-complexes showed increased lipophilic behavior compared to the puredrug. The measured partition coefficient (P) of RS was found to be0.073 ± 0.002. As demonstrated in Table 2, the sonocomplexesshowed partition coefficient (P) values ranged from 0.141 ± 0.03 to1.144 ± 0.16. The sonocomplexes showed a minimum of about 2 foldsand a maximum of about 15 folds increase in P value relative to RS.This can be explained in the terms of the enhanced liposolubility of thesononcomplexes and the disguising of polar group of RS by phospho-lipids (Yue et al., 2010).

3.6. Particle size, polydispersity index and zeta potential

Particle size is an imperative parameter that influences the bio-distribution and the pharmacokinetics of the formed complexes. Whendispersed in water, the prepared sonocomplexes form vesicular (lipo-some-like) structures, since they are phospholipid-based systems. Theparticle size measurements of the aqueous dispersions of the sono-complexes are presented in Table 2. The particle size values of theaqueous dispersions of the prepared complexes were ranging from477.55 ± 70 to 800.2 ± 15 nm. Factorial analysis of variance showed

Fig. 3. FTIR spectra of (a) pure rosuvastatin, (b) egg yolk phospholipid, (c) physical mixture, and (d) the optimal sonocomplex (F4).

Fig. 4. (a) The minimum energy sonocomplex structure, (b) a snapshot of asonocomplex monomer after 1 ns simulation (water molecules are hidden).

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Fig. 5. The effects of factors of phospholipid type and phospholipid: RS molar ratio on the water solubility of sonocomplexes (a, b), partition coefficient of sono-complexes (c, d), and particle size of the aqueous dispersions of sonocomplexes (e, f).

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that phospholipid type possessed a significant effect on the particle size(P= 0.0251). It was noticed that sonocomplexes prepared with phos-pholipid from soybean source exhibited larger particle size than thosecomplexed with egg yolk phospholipid. It could be inferred that theparticle size is dependent on the percentage of phosphatidylcholinepresent in the phospholipid (∼30 and 60% for soybean and egg yolk,respectively). The quantity of phosphatidylcholine in the phospholipidis widely varied in accordance with the source of the phospholipid. Theincrease in the percentage of phosphatidylcholine would allow thedevelopment of markedly smaller vesicles (upon reconstituting the so-nocomplex with water); due to the higher emulsification power ofphosphatidylcholine. These results are, to some extent, in agreementwith the data obtained by Tadros and Al-mahallawi (2015), who de-monstrated that at a fixed bovine serum albumin concentration, theincrease in egg yolk L-α-lecithin concentration would allow the devel-opment of significantly smaller nanoparticles. In addition, statisticalanalysis revealed that increasing the phospholipid to RS molar ratioresulted in a significant decrease in particle size of the aqueous sono-complexes dispersions (P=0.0216). As previously mentioned in thediscussion of the effect of phospholipid type on the particles size of theaqueous dispersions of sonocomplexes, this could be explained in termsof the increased emulsification at higher phospholipid content.

Table 2 shows the values of polydispersity index (PDI) and zetapotential (ζ) of the aqueous dispersion of the prepared sonocomplexes.The PDI values are within the range of 0.34 ± 0.09 to 0.65 ± 0.03,indicating satisfactory degree of size uniformity. The zeta potential (ζ)is a pertinent parameter with regards to the stability of the dispersedsonocomplexes. It is clear that all of the aqueous dispersions of theprepared sonocomplexes exhibited acceptable ζ values, ranging from−16.3 ± 0.99 to −39.45 ± 0.35mV, which may indicate the ac-ceptable stability of the dispersed sonocomplexes (Shamma andElsayed, 2013).

3.7. Elucidation of the optimal sonocomplex

After generating the final equations relating the dependent and in-dependent variables, optimization was done for three studied re-sponses; namely, water solubility, partition coefficient (P) and particlesize. Numerical optimization searched the design space using the finalmodels created for each studied response. It aimed at finding factors’levels that meet the constraints set for different responses. The criteriaset for the optimum sonocomplex (achieving maximum values of watersolubility and partition coefficient (P), and minimum values for particlesize of the aqueous dispersion of the sonocomplex) were established insonocomplex F4, which is prepared using egg yolk phospholipid and RSat a molar ratio of 2:1. Therefore, F4 was selected as the optimum so-nocomplex for further investigations.

3.8. Morphological characterization

Morphological examination of the aqueous dispersion of the optimalsonocomplex (F4) by TEM showed perfectly spherical shaped non-ag-gregating vesicles with a narrow size distribution in nanometer rangethat confine with the particle size analyses results (Fig. 6).

3.9. Sonocomplex stability

The optimal sonocomplex (F4) was subjected to physical stabilitystudy. Following 45 days of storage at room temperature (25 °C), therewas no visible alteration in the appearance of F4. In addition, therewere no significant differences (P > 0.05) between the stored sono-complex and fresh one in the CE%, particle size, water solubility andpartition coefficient (P), demonstrating that the storage at the specifiedconditions did not affect the properties of the prepared sonocomplex.

3.10. Caco-2 cells transport studies

Caco-2 cells monolayer was utilized as a model to study the drugabsorption across the intestinal barrier. This cell line is very widelyused to investigate the trans-epithelial transport of drugs. (Piazziniet al., 2017). Fig. 7 demonstrates the results of RS permeation across theCaco-2 cell monolayer obtained from the aqueous dispersion of op-timum sonocomplex (F4) in comparison with the drug solution. It isevident that the permeation profile of the aqueous dispersion of sono-complex was superior when compared to the drug solution. The cal-culated apparent permeability coefficient (Papp) values of RS solutionand optimal sonocomplex were found to be 0.59 ± 0.12× 10−6 cm/sand 2.27 ± 0.29×10−6 cm/s, respectively. Based on the calculatedPapp values and permeation profiles, it can be concluded that the aqu-eous dispersions of the optimal sonocomplex (F4) showed a significantimprovement (P < 0.05) in the drug permeation across the Caco-2cells monolayer relative to the drug solution. The obtained resultsconfirmed that the optimal sonocomplex exhibited the Papp values ne-cessary for complete intestinal absorption in humans (Artursson andKarlsson, 1991). These findings can be interpreted in the light of theincreased lipophilicity of the fabricated phospholipid sononcomplexesas evident from their higher P values relative to the un-complexed drug(Artursson and Karlsson, 1991).

3.11. In vivo absorption studies in rats

RS pharmacokinetics following oral administration of an aqueousdrug solution (treatment A) and the aqueous dispersion of the optimalsonocomplex (F4, treatment B) were evaluated in rats and the plasmaconcentration–time curves of both treatments are shown in Fig. 8.Pharmacokinetic analysis showed that both formulae achieved max-imum drug concentration within 2 h indicating that there was no dif-ference between them in the rate of drug absorption. However, the

Fig. 6. Transmission electron micrograph (TEM) of the aqueous dispersions ofthe optimum sonocomplex (F4).

Fig. 7. The permeability studies of drug solution and aqueous dispersion ofoptimal sonocomplex (F4) via transport through monolayered membrane ofhuman Caco-2 cell seeded on polycarbonate inserts in Transwell® plate.

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obtained plasma concentration–time profiles revealed the superiority ofthe optimal sonocomplex relative to drug solution regarding the extentof RS availability in the systemic circulation. Statistical analysis de-monstrated that there was a significant difference (P < 0.0.5) betweenthe calculated values of maximum drug concentration (Cmax) of theoptimal sonocomplex and drug solution (86.21 ± 8.84 and76.01 ± 5.87 ng/mL, respectively). In addition, the calculatedAUC[ ]0

72for the optimal sonocomplex (F4) was significantly higher(P < 0.05) than that of the drug solution (1424.19 ± 106.64 and870.90 ± 81.63 ng.h/mL, respectively). In other words, the selectedsonocomplex achieved 1.13 folds increase in the Cmax and 1.63 foldsincrease in the estimated AUC[ ]0

72 compared to the drug solution. Theobtained results could be attributed to the enhanced lipophilicity of RSupon sonocomplexation with egg yolk phospholipid (as evident fromtheir higher P values relative to the un-complexed RS) which conse-quently led to better passive diffusion of the drug across gastrointestinalmembrane upon oral administration. Hence, it could be concluded thatphospholipid-based sonocomplexes effectively improved the oral bioa-vailability of RS, demonstrating the potential of these systems to im-prove the lipophilicity and consequently the absorption characteristicsof BCS class III drugs following oral administration.

4. Conclusions

In the present study, RS-phospholipid sonocomplexes were suc-cessfully prepared by ultrasound irradiation as an alternative techniqueto conventional thermal method. The CE% of the prepared sonocom-plexes was very high (around 99%). The fabricated sonocomplexesshowed different physicochemical attributes in comparison with pureRS, including higher partition coefficient and lower water solubility. Acomplete 22 factorial design was implemented aiming to prepare op-timal sonocomplex, which demonstrated about 15 folds increase in thepartition coefficient (P) value relative to RS. The transport studies viaCaco-2 models validated a substantial augmentation in the bio-pharmaceutical performance of RS. It revealed that the prepared so-nocomplexes were successful in enhancing RS permeation with respectto an aqueous drug solution. In addition, the in vivo pharmacokineticstudies in rats confirmed the superior oral bioavailability of the sono-complex relative to the drug solution. The increase of the Caco-2 cellspermeation and oral bioavailability of sonocomplex appear to be due toimproved lipophilicity of the complexed drug. Succinctly, the for-mulation of RS as phospholipid sonocomplex adopting a new technique“sonocomplexation” can be considered as an immense potential tool forcreation of phospholipid-drug complexes applying ultrasound irradia-tion as an energy source.

Acknowledgments

We would like to acknowledge Marcyrl pharmaceutical industriesfor gifting us free medical samples that aided in this work.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of this article.

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