Research Article

Bioavailability Enhancement of Aripiprazole Via Silicosan Particles:Preparation, Characterization and In vivo Evaluation

Azza A. Mahmoud,1,2 Alaa H. Salama,2,3 Rehab N. Shamma,4,6 and Faten Farouk5

Received 13 June 2018; accepted 6 August 2018

Abstract. The aim of this study was to design a novel carrier for enhancing the bioavailabilityof the poorly water-soluble drug, aripiprazole (ARP). Silicosan, the applied carrier, wasobtained by chemical interaction between tetraethyl orthosilicate (TEOS) and chitosan HCl.Different ARP-loaded silicosan particles were successfully prepared in absence and presenceof one of the following surfactants; Tween 80, Poloxamer 407 and cetyltrimethylammoniumbromide (CTAB). The prepared ARP-loaded silicosan particles were thoroughly investigatedfor their structures using FTIR, XRD, and DSC analysis as well as their particle size, zetapotential, flowability, drug content, and in vitro drug release efficiencies. The prepared ARP-loaded silicosan particles were characterized by amorphous structure, high drug entrapmentefficiency and a remarkable improvement in the release of aripiprazole in simulated gastricfluid. SEM and EDX revealed that the morphology and silica atom content in theprepared ARP-loaded silicosan particles were affected by the used surfactant in theirformulations. The selected ARP-loaded silicosan particles were subjected to in vivo studyusing rabbits. The obtained pharmacokinetic results showed that the relative bioavailabilityfor orally administered ARP-loaded silicosan particles (SC-2-CTAB) was 66% higher relativeto the oral suspension (AUC0-10h was 16.38 ± 3.21 and 27.23 ± 2.35 ng.h/mL for drug powderand SC-2-CTAB formulation, respectively). The obtained results suggested the unique-structured silicosan particles to be used as successful vehicle for ARP.

KEY WORDS: aripiprazole; tetraethyl orthosilicate; surfactant; dissolution; bioavailability.

INTRODUCTION

Aripiprazole (ARP) is an antipsychotic drug with proventherapeutic effect against schizophrenia (1), and approved bythe FDA for the treatment of acute manic and mixed episodesassociated with bipolar disorder. ARP is a partial D2, and 5-HT1A receptor agonist, and like the other atypical antipsy-chotics, it is a 5-HT2A receptor agonist (2). ARP is a poorlywater-soluble drug and classified as class II drug according to

BCS (3). Previous attempts to increase ARP solubility wereperformed through complexation with cyclodextrins (4, 5),application of nanomilling and coprecipitation (6), formationof nanosuspensions (7), and through formation of 3D printedorodispersible films (8). Incorporation of ARP into nanoparti-cles like solid lipid nanoparticles (9) and into poly(caprolactone) nanoparticles (10) has also been evaluated.

In the field of pharmaceutical technology, a drug carriersystem should be simple, easily prepared, having high entrap-ment efficiency and a controlled drug release in the body.Recently, silica particles have been exploited as matrices forenhancing the entrapment and dissolution rate of candidateactive moieties. Silica particles present an attractive host to theactive agent owing to its low toxicity, excellent biocompatibility,particle morphology, and chemical character of the silica surface(11). In 2011, nonporous silica nanoparticles for cancer imagingwere approved for the first in-human clinical trial (12,13).Therefore, the polymer-silica composites represents a promisingclass of materials for drug delivery.

Drug loading within silica carrier can be carried out usingvarious novel loading approaches, viz, microwave irradiation,solventmethods, and supercritical fluid (14–17).Drugmoleculescan be stabilized either on the silica surface or within silica poresin an amorphous state, which enhances the dissolution rate of

1 Department of Pharmaceutics and Pharmaceutical Technology,Faculty of Pharmaceutical Sciences and Pharmaceutical Industries,Future University in Egypt, Cairo, Egypt.

2 Department of Pharmaceutical Technology, Pharmaceutical andDrug Industries Research Division, National Research Centre,Dokki, Cairo, Egypt.

3 Department of Pharmaceutics, Faculty of Pharmacy, Ahram Cana-dian University, 6th of October City, Cairo, Egypt.

4 Department of Pharmaceutics and Industrial Pharmacy, Faculty ofPharmacy, Cairo University, Kasr El-Aini Street, Cairo, 11562,Egypt.

5 Department of Pharmaceutical Chemistry, Faculty of Pharmacy,Ahram Canadian University, 6th of October City, Cairo, Egypt.

6 To whom correspondence should be addressed. (e–mail:rehab.shamma@pharma.cu.edu.eg)

AAPS PharmSciTech (# 2018)DOI: 10.1208/s12249-018-1145-6

1530-9932/18/0000-0001/0 # 2018 American Association of Pharmaceutical Scientists

the drug (18,19). Yilmaz and Bengisu (20) studied the potentialof drug loading onto silica microspheres through a single stepsol–gel process (20).

In this study, tetraethyl orothosilicate (TEOS) has been usedas a carrier for the preparation of ARP-loaded silicosan particles.Different volume ratios between TEOS and chitosanHCl solution(0.2% w/v) have been tested and their effect on the particle sizeand zeta potential of the prepared ARP-loaded silicosan particleswere evaluated. The optimum formulation was further modifiedthrough the incorporation of different surfactants. FT-IR studieshave been used to evaluate possible interactions between the drugand the polymers and surfactants used. Morphological examina-tion was done using scanning electron microscopy imagingcoupled with energy dispersive spectrometry. Furthermore, solid-state characterization using differential scanning calorimetry andX-ray diffraction have been used to evaluate the physical state ofthe drug in the prepared ARP-loaded silicosan particles. Theselected formulations were lyophilized and evaluated for in vitrodrug release. Finally, the in vivo performance of the optimizedformulations have been evaluated using male Albino rabbits, andthe pharmacokinetic parameters were calculated and comparedwith that of the drug suspension.

MATERIALS AND METHODS

Materials

Aripiprazole (ARP) was supplied from Alembic Pharmaceu-ticals Limited, Karakhadi, India. Tetraethyl orthosilicate (98%;TEOS),Poloxamer407,dialysis tubingcellulosemembrane(molec-ular weight cut of 14,000 g/mol), dichloromethane, and methanol(HPLCgrade)werepurchasedfromSigma-Aldrich,USA.Chitosanhydrochloride (CS-HCl) was gifted from Zhejiang ChemicalsImport andExportCorporation,China.Tween80andethanolwerepurchased from El-Nasr Pharmaceutical Chemicals Company,Egypt. Cetyltrimethylammonium bromide (CTAB) was obtainedfromBioBasic Inc., Canada. Isopropyl alcoholwas purchased fromSDS, France. All other reagents were of analytical grade and wereused as received.All water usedwas deionized, bi-distilledwater.

Preparation of ARP-Loaded Silicosan Particles

For the preparation of ARP-loaded silicosan particles, thedrug was dissolved in isopropyl alcohol (0.5 mL) and then it wasmixed with 1 mLTEOS using a vortex mixer (Reax Top VortexMixer, Heidolph, Germany). Then, the mixture was added tothe CS-HCl aqueous solution (0.2% w/v). The preparation wasthen left over night at room temperature until the formation of agel structure. The compositions for the prepared ARP-loadedsilicosan particles are listed in Table I.

For the preparation of surfactant containing ARP-loadedsilicosan particles, accurately weighed amount of the surfactant(1%w/vCTAB, Tween 80, or Poloxamer 407) was first dissolvedin the CS-HCl solution, then the same procedure conducted forthe preparation of ARP-loaded silicosan particles was applied.

Finally, the resulting gel structures were placed in widemouth bottles, frozen at − 80°C for 24 h, then lyophilized in aChrist freeze dryer (ALPHA 2-4 LD plus, Germany) under atemperature of − 80°C and vacuum of 7 × 10−2 mbar for 24 h.The obtained powders from the lyophilized samples werestored in desiccators until use.

Evaluation of the Prepared ARP-Loaded Silicosan Particles

Fourier Transform Infrared Spectroscopy (FTIR)

The FTIR spectra were recorded for ARP, CS-HCl,TEOS, Tween 80, Poloxomer 407, CTAB, lyophilizedARP-free, and ARP-loaded silicosan particles usingBruker FTIR spectrophotometer (Model 22, Bruker, UK)using the KBr disk technique. The FTIR measurementswere performed in the scanning range of 4000–400 cm−1

at ambient temperature.

Scanning Electron Microscope (SEM) Imaging Coupled withEnergy Dispersive X-ray Analyzer (EDX)

Morphology of selected lyophilized ARP-loaded silicosanparticles were observed using a SEM (Jeol JSM-6400; JEOL Ltd.,Tokyo, Japan). The samples were fixed using double-sidedadhesive tape on a brass stub and then it was subjected tosputter-coated with a thin layer of gold. Imaging of the sampleswas operated using SEM at an excitation voltage of 20 kV.Quantitative chemical analysis on particle surfaces was performedby energy dispersive X-ray analyzer (EDX).

Entrapment Efficiency

Fixed amount of the prepared ARP-loaded silicosanparticles was mixed with 1 mL ethanol to dissolve the non-entrapped drug crystals, followed by centrifugation for 15 minat 20,000 rpm (Sigma 3-30 K, Spincontrol Comfort, Ger-many). The residue was then separated and dissolved indichloromethane and assessed for entrapment efficiencyspectrophotometrically at 368.5 nm (UV-1800 UV-VIS Spec-trophotometer, Shimadzu, Japan).

The entrapment efficiency was calculated using thefollowing equation:

Entrapment efficiency %ð Þ

¼ amount of drug in the silicosan particlesamount of added drug

� 100

Evaluation of the Lyophilized ARP-Loaded SilicosanParticles

Determination of Freeze-Drying Process Yield

The obtained silicosan powder after the freeze-dryingprocess was weighed and the process yield was calculated bythe following equation:

%yield ¼ recovered massmass entered in the freeze dryer

� 100

Determination of Particle Size and Zeta Potential

The particle size and zeta potential of the preparedlyophilized ARP-loaded silicosan particles were measured

Mahmoud et al.

using Mastersizer 2000 and ZetaSizer Nano ZS, respectively(Malvern Instruments, UK). The lyophilized powder of ARP-loaded silicosan particles was dispersed in distilled waterprior to measurement.

In vitro Drug Release

The in vitro ARP release from selected ARP-loadedsilicosan particles was evaluated at both gastric and intestinalpH using USP II dissolution apparatus (Hanson SR8-Plus,USA) following a pH change method. Accurately weighedamount of lyophilized ARP-loaded silicosan particles (equiv-alent to 10 mg ARP) were dispersed into 300 mL of 0.1 NHCl (pH 1.2). After 2 h, 100 mL of trisodium phosphate(0.26 M) were added to each vessel to achieve a pH of 6.8 andthe experiment was run for further 6 h (21). The temperatureof the medium was maintained at 37 ± 0.5°C while the speedof the paddle was adjusted at 50 rpm throughout theexperiment. At definite time intervals, 5 mL samples werewithdrawn and filtered using a dissolution filter (10 μmUHMW polyethylene Cannula dissolution filter, Hanson,USA) and replaced with fresh medium in order to maintainsink conditions. The in vitro release of 2 mL ARP aqueoussuspension (prepared by direct dispersion of 10 mg ARP in2 mL distilled water) was also done for comparison using thesame procedure mentioned above. Samples were subjected toHPLC analysis using a sensitive, selective, and accurateHPLC method that was developed and validated before thestudy for determination of ARP concentrations in collectedsamples. All chemicals and reagents were of analytical grade,solvents used were of HPLC grade. HPLC system consistingof isocratic pump LC-10 AD and a UV/VIS detector SPD-10A was connected to a SCL-10A integrator; Knaueradvanced scientific UV 2250 (Berlin, Germany). The analyt-ical column was Eurosphere C18 column, 250 mm × 4.6 I.D.mm, particle size 5 μm (Berlin, Germany). The isocraticmobile phase comprised methanol:water (90:10, v/v). Themobile phase was filtered and degassed. The flow rate was setat 1.2 mL/min and the column effluent was monitoredcontinuously using UV detection at 254 nm, the injectionvolume was 20 μL.

X-ray Diffractometry (XRD)

X-ray diffraction pattern was recorded using Philips-PW-1050 diffractometer equipped with filter Ni, Cu Kα radiation.The tube was operated at a voltage of 40 kV and a current of20 mA. The XRD patterns of the plain ARP, CS-HCl, CTAB,Poloxamer 407, ARP-free, and ARP-loaded silicosan parti-cles were recorded.

Differential Scanning Calorimetry (DSC)

The thermal behavior of the pure ARP, CS-HCl,Poloxamer 407, CTAB, ARP-free, and ARP-loaded silicosanparticles were traced using Shimadzu differential scanningcalorimeter (DSC-50, Shimadzu, Japan). For each scan, 2 mgof each sample were weighed and placed in a hermeticallysealed aluminum pan. The samples were then heated in atemperature range of 25–350°C at a constant heating rate of10°C/min under a nitrogen atmosphere.

TableI.

Com

position

andCha

racterizationforSilicosan

Particles

Formulation

code

Com

position

Entrapm

ent

efficien

cy(%

)Cha

racterizationof

lyop

hilized

aripiprazole-lo

aded

silicosan

Surfactant

TEOSto

Chitosan

HCl*

ratio(V

/V)

Process

yield(%

)Particlesize

(μm)

Zetapo

tential(mV)

Ang

leof

repo

se(θ)

Hau

sner’ratio

Carr’inde

xRE

(%)**

SC-1

–1:1

10.135

±5.17

––

––

––

SC-2

–1:2

58.330

±6.71

69.42±5.12

14.859

±0.106

−40.2±1.55

22.3±1.41

1.07

±0.10

6.66

±0.24

82.67±3.01

SC-3

–1:6

37.720

±1.05

––

––

––

SC-2-2

–1:2

24.335

±1.82

––

––

––

SC-2-TW

Twee

n80

1:2

48.840

±1.11

62.97±2.24

24.009

±0.515

−14.05

±0.63

32.8±1.62

1.28

±0.11

22.22±0.55

81.79±2.49

SC-2-Plx

Polox

amer

407

1:2

68.855

±2.41

64.97±1.54

33.773

±2.106

10.98±1.01

26.7±1.48

1.16

±0.10

14.28±0.56

62.65±0.82

SC-2-C

TAB

CTAB

1:2

65.050

±6.21

86.59±0.15

48.033

±3.876

33.1±3.11

24.2±1.61

1.10

±0.03

9.52

±0.72

95.72±0.74

Alltheform

ulations

wereprep

ared

using5-mgdrug

except

forSC

-2-2,w

here

10-m

gdrug

was

used

initsprep

aration.

CTA

B,c

etyltrim

ethy

lammon

ium

brom

ide;

RE,release

efficien

cy*C

hitosanHClsolution

was

prep

ared

inconcen

trationof

0.2%

w/v

**The

releaseefficien

cyfordrug

suspen

sion

was

18.96±2.96%

Aripiprazole-Loaded Silicosan Particles

Flow Properties of Lyophilized ARP-Loaded SilicosanParticles

Two methods were used for powder flowability measure-ment. The first method (Hausner’s ratio and Carr’s index)was used to calculate the bulk density and tapped density ofthe powder blends (22). The lower the Hausner ratio and theCarr index; the better is the powder flowability. The secondmethod was the characterization of flowability by measuringthe angle of repose according to the fixed funnel andfree standing cone method (23). This angle represents themaximum possible angle between the surface of a powder pileand the horizontal plane.

In vivo Studies in Rabbits

Study Design. The in vivo studies were carried out tocompare the bioavailability and pharmacokinetic parametersof 10 mg ARP from two different treatments in white NewZealand male Albino rabbits (2.5–3 kg) using a non-blind,cross-over design. Three rabbits were randomly assigned toeach treatment group. Food was withdrawn 10 h prior to thestudy with water ad libitum. In the early morning, theassigned treatments were administered. In treatment A,rabbits received ARP aqueous suspension (prepared bydirect dispersion of ARP in distilled water). In treatment B,rabbits received ARP-loaded silicosan particles preparedusing CTAB (SC-2-CTAB). Both treatments were dispersedin 2-mL distilled water to achieve an amount of ARPequivalent to 10 mg ARP per kg body weight of the rabbit,administered through an intragastric tube. Blood samples(2 mL) were withdrawn retro-orbitally at 0 (predose), 0.5, 1,1.5, 2, 3, 4, 6, 8, and 10 h after treatement administration.Blood samples were collected into heparinized tubes andplasma was obtained by centrifugation at 4000 rpm for15 min. The plasma was pipetted into glass tubes and frozenat − 20°C until analysis. All animal experiments wereapproved by the Research Ethics Committee (REC) forAnimal Subject Research at the Faculty of Pharmacy, CairoUniversity, Egypt and operated according to the NationalInstitutes of Health guide for the care and use of Laboratoryanimals (NIH Publications No. 8023, revised 1978).

Chromatographic Conditions. Chromatographic separa-tion was performed on a JASCO HPLC system equippedwith a PU-20180 Plus pump and a DG-2090-54 degasser.The elution was achieved on an Inertsil RP-HPLC ODScolumn (4.6 × 250 mm, 5 μm) using isocratic elution. Themobile phase was composed of methanol and water(90:10 v/v) with a detection wavelength of 254 nm. ARPwas eluted at 16.5 min while the internal standard(aceclofenac) was eluted at 11.5 min. The quantitationanalysis was performed based on peak area ratio betweenARP and the internal standard. The analytical methodwas validated according to ICH guidelines and applied forthe determination of ARP in samples.

Standard Calibration Curve Construction and AssayValidation. All assays were performed in independent tripli-cates. The lower limit of quantification was 0.1 ng/mL and alinear response (r2 = 0.992) was obtained.

Plasma Analysis. Sample preparation was performedusing a modified protein precipitation method in which300 μL of the plasma were mixed with 10 μL of theinternal standard (to give a concentration of 10 ng/mL)and 700 μL of acetone. The samples were left to stand for15 min with vortex mixing. Then, sample-precipitatedproteins were separated by centrifugation at 6000 rpm.The supernatant (600 μL) was withdrawn and evaporatedto dryness. Samples were then reconstituted in the mobilephase (30 μL) prior to injection.

All frozen plasma samples obtained from the rabbitsafter receiving treatments A and B were thawed at ambienttemperature and assayed as described above without theaddition of ARP.

Pharmacokinetic Analysis. Data from plasma analysiswere analyzed for each rabbit using WinNonlin®(version1.5, Scientific consulting, Inc., Cary, NC). Non-compartmental analysis was pursued and the pharmaco-kinetic variables; Cmax (maximal drug concentration; ng/mL), Tmax (time for maximal drug concentration; h) andAUC(0-t) (area under the curve; ng.h/mL) were generated.

The elimination half-life (t1/2) was calculated as t1/2 =Ln2/k. The relative bioavailability (frel) was calculated forARP-loaded silicosan particles (SC-2-CTAB) relative to ARPaqueous suspension as AUCSC-2-CTAB/AUCsusp. All resultswere expressed as mean n = 3 ± SD.

Statistical Analysis

Statistical analysis of the in vivo studies were based onuntransformed values for Cmax and AUC variables andobserved values for t1/2. The nonparametric Signed-RankTest (Mann-Whitney’s test) was used to compare Tmax

between the two treatment groups.

RESULTS AND DISCUSSION

Preparation and Characterization of ARP-Loaded SilicosanParticles

In our study, isopropyl alcohol was used to dissolvethe drug, as it is miscible with TEOS unlike methanol andethanol. Preliminary studies were conducted in order todetermine the feasibility of preparing ARP-loadedsilicosan particles with an acceptable particle size andentrapment efficiency-values, with the aid of CS-HCl.Successful formulations were obtained at room tempera-ture without the need of elevated temperature or anyother harsh conditions that may affect sensitive drugs.Three different TEOS:CS-HCl volume ratios (1:1, 1:2, and1:6 v/v) were prepared containing fixed ARP concentra-tion for the prepared ARP-loaded silicosan particles.Furthermore, ARP-loaded silicosan particles were pre-pared with the incorporation of Tween 80, Poloxamer407, or CTAB.

Mahmoud et al.

Evaluation of the Prepared ARP-Loaded Silicosan Particles

Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra were assessed to demonstrate theinteraction between the components in the formulation(Fig. 1a). The FTIR spectrum of TEOS showed absorp-tion bands at 2976, 2927, and 2893 cm−1 due to aliphaticC–H stretching. The band located at 1086 cm−1 is relatedto the stretching vibrations of C–O groups. The CS-HClshowed a broad peak in its FTIR spectrum at 3439 cm−1

contributing to the stretching of NH2 and OH groups. Thestretching vibrations of C-O group for the CS-HClappeared at 1091 cm−1 and at 1024 cm−1. The bendingvibrations of aliphatic C–H stretching were located at2888, 2849, and 2767 cm−1.

The FT-IR spectrum of Tween 80 showed intense bandsat 2922 and 2869 cm−1 due to the aliphatic C–H stretching.The O–H group showed absorption band at 3423 cm−1. Theband at 1735 cm−1 can be attributed to the O–C=O groupwhile the band at 1643 cm−1 can be attributed to the

stretching of the C=C group. The band at 1107 cm−1 isassigned to asymmetric C–O stretching vibrations. Poloxamer407 showed a broad band at 3442 cm−1 corresponding to theO–H group. The absorption bands being noticed at 2968 and2888 cm−1 are due to aliphatic C–H stretching. The peakappearing at 1109 cm−1 is assigned to C–O stretchingvibrations. The FTIR spectrum of CTAB revealed strongabsorption C–H stretching bands at 2918 and 2849 cm−1. Theband at 1473 cm−1 is due to C–N stretching mode.

The interaction between TEOS and CS-HCl can bedetected from the evolution of new IR band for Si–N in thefinal material. This band can be attributed to the newcovalent bond formed between the Si–O–CH2–CH3 groupsof TEOS and C–NH2HCl groups of CS-HCl. The presence ofthe band for the Si–N group at 953, 955, 955, and 961 cm−1 forformulation prepared without surfactant, with Tween 80, withPoloxamer 407, and with CTAB, respectively, clearly confirmsthe interaction between TEOS and CS-HCl to form thesilicosan particles, as shown in Fig. 1a.

The FTIR spectra for formulations prepared usingPoloxamer 407 or CTAB confirm the presence of the bands

500 1000 1500 2000 2500 3000 3500 4000

13

Wavenumber (cm-1)

1

2

3

4

5

6

7

8

9

10

11

12

14

`O

OHOH

OO

NH2HClNH2HCl

OH

CH2OH CH2OHCH2OH

OO OH

NH

OH

4 SiO O

O

O

OHNH2HCl NH2HCl

OH OH OH

OH

OH

OH

OH

CH2OH CH2OH CH2OH

OO

OO

N4

O

O

OO

O

O

a

b c

Fig. 1. a FTIR spectra for (1) chitosan HCl (CS-HCl), (2) TEOS, (3) Tween 80, (4) Poloxamer 407, (5)CTAB, drug-free formulations: (6) SC-2, (7) SC-2-TW, (8) SC-2-Plx, and (9) SC-2-CTAB, ARP-loadedformulations: (10) SC-2, (11) SC-2-TW, (12) SC-2-Plx, (13) SC-2-CTAB, and (14) aripiprazole (ARP).Chemical structure for the formulated silicosan particles: b prepared without surfactant or using Poloxamer407 or CTAB and c prepared using Tween 80

Aripiprazole-Loaded Silicosan Particles

for the active groups of the surfactants. Such results confirmthat Poloxamer 407 or CTAB did not form chemicalinteraction with TEOS or CS-HCl and gives silicosanstructure demonstrated in Fig. 1b. On the other hand, withthe data obtained, it was found that Tween 80 was involved inthe interaction between TEOS and CS-HCl. The disappear-ance of the O–C=O band for Tween 80 at 1735 cm−1 gaveevidence for chemical interaction between this group and theC–NH2HCl group of CS-HCl resulting in formation of theC=N group at 1541 cm−1. This result reveals that thecomposition was changed due to introduction of Tween 80to give the silicosan structure demonstrated in Fig. 1c.

The FTIR spectrum of ARP (Fig. 1a) shows many sharpabsorption bands representing the different functional groups inthe drugmolecule. The absorbance bands at 3437 and 3348 cm−1

are corresponding to O–H and N–H stretching, respectively.The absorption band at 3112 cm−1 represents aromatic C–Hstretching while the bands at 2978 and 2930 cm−1 are due toaliphatic C–H stretching. The N–C=O group showed intensepeak at 1674 cm−1. The bands due to stretching of C–NandC–Clgroups appeared at 1161 and 792 cm−1, respectively. Thearomatic C=C stretching appeared at 1626, 1492, and 1454 cm−1.

ARP retained its characteristic peaks in the preparedformulations confirming that it was not involved in theinteraction between TEOS and CS-HCl (Fig. 1a). Thecharacteristic amide group (N–C=O) for ARP located at1674 cm−1 was shifted to lower frequencies, to 1634, 1636,1634, and 1633 cm−1 for formulation prepared withoutsurfactant, with Tween 80, with Poloxamer 407 and withCTAB, respectively. The shift of the amide group for theARP can be related to the hydrogen bond formation between

this group and the O–H group in CS-HCl and/or terminalsilanol groups in the silicosan particles, which confirm bindingbetween CS-HCl and ARP.

These results conveyed a clear picture about interac-tion between TEOS and CS-HCl and binding the newprepared particles to ARP, which is specifically importantfor orally administered silica-based drug delivery system.These changes suggested that the formation of hydrogenbonds between ARP and the silicosan particles. Thissituation may be due to the establishment of differenttypes of host–guest interactions (H-bond) between ARPand silicosan particles (24).

Scanning Electron Microscope Imaging

The morphology of ARP-loaded silicosan particleswas investigated by SEM (Fig. 2). It could be clearly seenthat the different structures of silicosan network areformed upon changing the surfactant type. Fig. 2a showsthat preparing ARP-loaded silicosan particles in theabsence of any surfactant cause the silicosan network toappear as interconnected mesh with no well-definedparticles. On the other hand, upon using Tween 80, thesilicosan network appeared as agglomerated porous struc-tures covered with some connected particles formingramified long chains (Fig. 2b). Figure 2c clearly showsthat ARP-loaded silicosan particles prepared usingpoloxamer consists of tiny particles that seems to beconnected in the shape of randomly orientedcoagglomerates. Using CTAB as a surfactant to prepareARP-loaded silicosan particles produced spherical

a b

c d

20 µm 30 µm

50 µm 50 µm

Fig. 2. SEM images and EDX figures (intensity in counts versus energy in keV) for a SC-2, b SC-2-TW, c SC-2-Plx, and d SC-2-CTAB

Mahmoud et al.

particles aggregated in semi-spherical agglomerates inter-connected with short chains (Fig. 2d).

The presence of silica atoms,ARP, CS-HCl, and surfactantsin different ARP-loaded silicosan particles were further con-firmed by the elemental analysis (EDX) (Fig. 2). The EDXfigures indicate the elemental analysis of the ARP-loadedsilicosan particles. It is obvious in Fig. 2 that the oxygen, carbon,nitrogen, and silica atoms exist in all the investigated silicosanparticles as expected. The elemental analysis also yielded theatomic percentage of Si element which were equal to 1.76% forSC-2 (ARP-loaded silicosan particles with no surfactant), whilethe approximate Si percentages were 20.93, 17.74, and 34.98%for SC-2-TW, SC-2-PLX, and SC-2-CTAB, respectively,confirming successful preparation of the fabricated silicosanparticles. EDX data showed increase in the intensity of silicaatompeak upon including surfactants intoARP-loaded silicosanparticles (SC-2-TW, SC-2-PLX, and SC-2-CTAB). This couldconclude that the interaction between the TEOS and CS-HCl islargely increased when surfactants were included in thefabrication media. Furthermore, SC-2-CTAB formulation pre-pared using CTAB as surfactant had the highest content of silica(34.98%). This may be attributed to the positive charge ofCTAB which may prefer to attract and emulsify the negativelycharged TEOS (source of silica atoms) resulting in theformation of silicosan particles with high percentage of silicaatoms.

Entrapment Efficiency of ARP-Loaded Silicosan Particles

Results of the entrapment efficiency for the preparedARP-loaded silicosan particles are presented in Table I.Results showed that increasing the TEOS:CS-HCl volumeratio from 1:1 (SC-1) to 1:2 (SC-2) or 1:6 (SC-3) resulted in asignificant increase in entrapment efficiency value from10.1% for SC-1 to 58.3 and 37.7% for SC-2 and SC-3,respectively (p < 0.05). This may be attributed to the increasein the amount of CS-HCl which interacts with TEOS toproduce more silicosan particles that will entrap the drug.Increasing the amount of CS-HCl would result in less numberof TEOS molecules that will react with single chitosanmolecule.

Further increase in TEOS:CS-HCl volume ratio from 1:2(SC-2) to 1:6 (SC-3) was not coupled with an increase in theentrapment efficiency values of the ARP-loaded silicosanparticles, on the contrary, the entrapment efficiency de-creased. Such significant decrease in entrapment efficiency(p < 0.05) was attributed to the large volume of the aqueousphase containing CS-HCl added to the TEOS which resultedin dilution of the drug and its precipitation (this was visuallyinspected during the preparation).

In an attempt to improve the entrapment efficiency valueof the prepared formulation, ARP-loaded silicosan particles(SC-2) was modified by increasing the drug loading duringpreparation from 5 to 10 mg (formulation SC-2-2). Unfortu-nately, increasing drug-loading concentration did not improvethe entrapment efficiency of the drug, but on the contrary, agreat detraction of drug entrapment value was produced. Thismay be due to the saturation of the isopropyl alcohol with thedrug that was rapidly precipitated upon contact with theaqueous solution of the CS-HCl.

Among the tested formulations, ARP-loaded silicosanparticles (SC-2) with TEOS:CS-HCl volume ratio of 1:2showed the highest entrapment efficiency value. Therefore,this formulation was selected for further incorporation ofsurfactants, namely, Tween 80, Poloxamer 407, or CTAB,seeking improvement of the tested parameters.

The entrapment efficiency-values of the obtainedsilicosan particles prepared in presence of surfactants wereenhanced upon using Poloxamer 407 or CTAB during theformulation (p < 0.05) due to their solubilization effect on thedrug (25). The highest value for entrapment efficiency(68.855 ± 2.403%) was recorded for SC-2-Plx. Previous stud-ies had proven the superiority of Poloxamer 407 in increasingthe drug entrapment and loading capacities as it providesgreat stabilization during particle formation (26). On theother hand, using Tween 80 caused a significant decrement inthe entrapment efficiency of the obtained formulation(p < 0.05) due to its consumption in the formation of thenew chemical structure of silicosan particles (Fig. 2c).

A noticeable remark to be documented is that upon theuse of Tween 80 or Poloxamer 407 as a surfactant inpreparing the silicosan particles, faster gel formation (lesstime for gel formation; less than 24 h) was observed.Silicosan particles containing Tween 80 showed faster gelformation than that containing Poloxamer 407. On the otherhand, ARP-loaded silicosan particles containing CTAB didnot affect the gelling time compared to formulation lackingsurfactant. A possible explanation is the high HLB-values ofTween 80 and Poloxamer 407 relative to CTAB, favoringgreater mixing of the phases (organic TEOS phase andaqueous CS-HCl phase). HLB-values for Tween 80,Poloxamer 407, and CTAB are 15, 18–23, and 10, respec-tively. Although Poloxamer 407 has higher HLB value thanthat of Tween 80 and was expected to show faster silicosanparticles formation than that for formulation containingTween 80, it showed longer gelling time. This may beattributed to the higher viscosity of the Poloxamer 407solution that might have caused retardation of phases mixing.Further characterization studies (DSC and XRD) wereperformed in order to thoroughly investigate the nature andcomposition of the obtained ARP-loaded silicosan particles.

The promising preliminary study results for SC-2 formu-lation and its modified surfactant-formulations habilitatedthem for further subjection to further studies.

Determination of Process Yield, Particle Size, and ZetaPotential

Considering the importance of yield value as a significantparameter for scaling-up process, yield values for theprepared formulations were investigated in the current study(Table I). It can be clearly demonstrated in the table thepromising high values for this parameter which indicates thesuccess of the applied ARP-loaded silicosan particles prepa-ration method. Yield values ranged from 62.971 ± 2.245 to86.586 ± 0.145% with the maximum value attended for CTABcontaining formulation. The high process yield value for SC-2-CTAB may be due to the high melting point of CTAB (249–253°C) (27) compared to those for Tween 80 (− 20.56°C) (28)and Poloxamer 407 (53–57°C) (29) which resulted in a lesssticky product.

Aripiprazole-Loaded Silicosan Particles

It was found that silicosan formulation with higher silicaatom content (SC-2-CTAB) had the higher particle size valueand silicosan particles with lower silica atom content (SC-2)had the lowest particle size value. This could be attributed tothe molecular volume for the formed silicosan whereincreasing the number of reacted TEOS with the chitosanmolecule would result in more bulky molecule that would bebacked in large volume particles.

The zeta potential value for the lyophilized SC-2formulation exhibited a negative value due the predominanceof silanol group having a larger electron cloud, due to theunshared lone pairs, giving negative charge for the formula-tion. Such negative zeta potential value for the lyophilizedSC-2 was decreased upon using Tween 80 in the formulationdue to the interaction of Tween 80 with TEOS. On the otherhand, using Poloxamer 407 during the preparation of SC-2-Plx resulted in the formation of ARP-loaded silicosanparticles with positive zeta potential value which indicatethe presence of CS-HCl that was not involved in the reaction.The SC-2-CTAB preparation possessed a high positive zetapotential value of 33.1 mV due to the presence of thepositively charged surfactant, CTAB.

Determination of Physical State of the Drug Within thePrepared ARP-Loaded Silicosan

Solubility/dissolution-limited bioavailability can be over-come through the preparation of amorphous drug forms (30).However, the amorphous state can change back to thecrystalline state during processing or storage. Stabilization of

amorphous drugs through the adsorption in/on materials isgaining much interest (31).

In order to get the benefit from high solubility anddissolution, the challenge of poor stability of amorphouscompounds has to be addressed seriously. The interaction ofARP with silicosan particles, either via adsorption and/orintegration within the matrix, could change its physical form;especially in the presence of the unique structure of theprepared ARP-loaded silicosan particles represented by theircharacteristic high surface area. The well-known importantrole of drug’s crystallinity on its dissolution rate andconsequently its increased bioavailability has driven us toexplore it. Thus, DSC and XRD were used to determine thecrystallinity/amorphous state of the entrapped ARP withinthe prepared ARP-loaded silicosan particles.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetrical analysis wasemployed to evaluate the molecular state of ARP in theprepared formula. DSC thermograms of ARP, CS-HCl,Poloxamer 407, CTAB, ARP-free and ARP-loaded silicosanparticles are shown in Fig. 3a. As illustrated in Fig. 3a, thefree ARP was characterized by a single, sharp meltingendothermic peak at 183.9°C, corresponding to its meltingpoint, revealing a crystalline anhydrous substance typicalbehavior. CS-HCl thermogram showed two endothermicpeaks; one at 91.73°C related to water loss associated withhydrogen-bonding to the hydrophilic groups of CS-HCl, andthe other at 221.2°C, in addition to an exothermic peak at

50 100 150 200 250 300 350 400 450

1

Temperature (°C)

g/W

wolf

ta

eH

2

3

4

5

6

7

8

9

10

11

12

5 10 15 20 25 30 35 40 45 50 55 60 65 70

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5

6

7

8

9

10

11

12

)st

nu

oc

(yti

sn

etnI

Angle (2-Theta°)

a b

Fig. 3. a DSC curves and b X-ray diffraction for (1) aripiprazole, (2) chitosan HCl (CSHCl), (3) poloxamer 407, and (4)CTAB, drug-free formulations: (5) SC-2, (6) SC-2-TW, (7) SC-2-Plx, and (8) SC-2-CTAB formulations, ARP-loadedformulations: (9) SC-2, (10) SC-2-TW, (11) SC-2-Plx, and (12) SC-2-CTAB

Mahmoud et al.

218.1°C, indicating its degradation (32). Poloxamer 407 showsan endothermic peak at 58.01°C corresponding to its meltingpoint. The DSC thermogram of CTAB demonstrates twoendothermic peaks at 61.58 and 433.3°C. Interestingly, all thepeaks of the tested excipients disappeared in the preparedARP-free silicosan particles, suggesting complete transforma-tion of the excipients to the amorphous state.

All the evaluated ARP-loaded silicosan particles re-vealed the disappearance of the melting endothermic peakof ARP crystals, thus provided indirect evidence for theeffective adsorption of ARP in the internal network ofsilicosan particles. The DSC thermograms for the medicatedformulations were similar to their counterpart non-medicatedformulations. The absence of the characteristic peaks of ARPwithin the medicated formulations confirms its transformationto its amorphous form and its complete entrapment within theprepared silicosan formulations (33). These results supportedthat the ARP crystals changed to the amorphous (34) ormolecular state (35) during the loading procedure.

X-ray Diffractometry (XRD)

Figure 3b shows XRD diffractograms of ARP, CS-HCl,Poloxamer 407, CTAB, ARP-free, and ARP-loaded silicosanparticles. XRD data confirm the obtained results from DSCanalysis. The XRD diffractograms for the medicated and non-medicated formulations showed amorphous structure. ARPlost its crystallinity and fully integrated within the preparedARP-loaded silicosan particles.

The diffraction pattern of ARP powder revealed princi-ple intense peak at 11.07° and intense peaks at 6.9°, 6.3°, and3.6°, suggesting that the drug existed as crystalline material.The powder XRD of ARP-loaded silicosan particles showedthe typical halo pattern due to amorphous nature of thesilicosan particles in the range 4–80° and the peaks attribut-able to the crystalline ARP was not available. This typicaldiffuse pattern indicated the entirely amorphous nature ofARP in the system. According to Williams et al., lack ofcrystallinity is an added evidence for the formation ofinclusion complex (36). This proves that silicosan particleskept their structure after loading with ARP and the includedARP was not arranged in a crystalline form.

Flow Properties of the Prepared ARP-Loaded SilicosanFormulations

Powder flow properties are important aspects in processingoperations, such as flow from hoppers, mixing, and compression.A uniform flow from the hoppers into the die cavity ensuresuniform tablet weight and content uniformity. Poor powder flowpresents amajor problem to the pharmaceutical industry (37). Theeffect of the three surfactants on the flow and dissolutionproperties of the prepared ARP-loaded silicosan particles werestudied. The flowability of the powder blends was determinedusingHausner’s ratio, Carr’s index, and angle of repose.Hausner’sratio (HR)was related to the inter particle friction; powders with alow inter particle friction had a ratio of approximately 1.25indicating a good flow. Generally, powders with Carr’s indexbelow 25% have very good flow properties (37). Powders withangle of repose above 50° have unsatisfactory difficult flowproperties, while those with values of 25–40° represent reasonable

flowpotential, whereasminimumangles close to 25° correspond tovery good flow properties (38). From the results presented inTable I, all the prepared ARP-loaded silicosan particles showedacceptable values for Hausner’s ratio, Carr’s index, and angle ofrepose (Table I). Such free-flowing powder properties preventsparticles from aggregation, ensures great surface area allowing theparticles to be rapidly diffused and easily dispersed upon contactwith the desired vehicle (39). Such results would refer to theefficacy of the resulted lyophilized powder to be filled into hardgelatin capsule shell.

In vitro Drug Release

The ARP release profiles from different silicosan hostswere compared with that of pure ARP and shown in Fig. 4.Great enhancement of ARP release fromARP-loaded silicosanparticles compared with ARP alone was observed. The greatenhancement of ARP release from ARP-loaded silicosanparticles could be attributed to both the high specific surfacearea that cause both high drug adsorption/loading and for highdrug release rate (40) and the presence of free unreacted silanolpresent on the surface groups on the silicosan surface whichrenders the particles’ surface hydrophilic providing goodwettability of the particles. Silanol groups can interact withproton donor/acceptor groups of the molecules hosted in thesilicosan through hydrogen bonding (as confirmed in the FTIRresults) which are easily broken upon hydrating in the releasemedium. Water enters into the silicosan structure and disloca-tion of the guest molecules occurs towards the externalenvironment by diffusion (41). This dissolution behavior canalso be attributed to the following: first, the increase in surfacearea of ARP by its adsorption on the surface of silicosanparticles that provided larger effective dissolution area. Second,the existence of ARP in an amorphous state (confirmed byDSCand powder XRD studies), as the witnessed dissolutionimprovement of ARP was associated with alteration in thesolid-state properties of the drug (42). In general, the amor-phous structure of any hydrophobic compound has higherdissolution rate than the crystalline form, in aqueous media(43). Third, the wettability of the hydrophilic silicosans, wheresilica particles have a hydrophilic surface owing to the presenceof the silanol groups; therefore, it has good wettability foraqueous media.

All in vitro release data ofARP from different ARP-loadedsilicosan particles were evaluated, and the RE% was calculatedfrom the area under the release curve at time t. It is expressed asthe percentage of the area under the release curve to therectangle corresponding to 100% drug release, for the sametotal time, according to the following equation (44):

RE% ¼ ∫t0y� dty100 � t

� 100

where, RE is the release efficiency, y is considered thepercentage drug released at certain time t and y100 is the100% drug release at time t.

The release profiles of ARP from surfactant enrichedARP-loaded silicosan particles show that CTAB containingformulations had an improved drug release profile comparedto the corresponding formulation lacking surfactant. This

Aripiprazole-Loaded Silicosan Particles

could be attributed to the high surface charge of the CTABcontaining particles (+ 33.1 ± 3.11 mV) as well as the solubi-lization effect for CTAB, which resulted in greater dispersionin the particles within the medium with greater surface areaexposed to the release medium and greater, hence, enhanceddrug solubilization and release. On the other hand, Tween 80containing formulations had a similar drug release profilewith that of the corresponding formulation lacking surfactant.This could be attributed to the involvement of Tween 80 inthe interaction between TEOS and CS-HCl as previouslyproven from FTIR analysis (Fig. 1). Moreover, Poloxamer407 containing formulations had a slower drug release profilecompared to that of the corresponding formulation lackingsurfactant. This can be attributed to the hydrophilic nature ofPoloxamer 407. Upon contact with release medium at 37°C,excessive hydrogen bonding between water molecule andethereal oxygen of Poloxamer 407 occurs forming a highlyviscous gel layer resulting in drug release retardation (45).

Release efficiency values of the surfactant enriched ARP-loaded silicosan particles were calculated to be 62.65 ± 0.82,81.79 ± 2.49, and 95.72 ± 0.74%, for Poloxamer 407, Tween 80,and CTAB containing formulations, compared to 82.67 ± 3.01for the surfactant-free formulation (SC-2). Although slowerrelease was expected from Tween 80 containing formulationsowing to the greater particle size and subsequent smaller area ofthe particles compared to SC-2, no significant difference wasobserved between the release efficiency values of SC-2 and SC-2-TW (p > 0.05). This suggests that the increase in particle sizewas compensated by hydrophilic nature of Tween 80 that causeddrug solubilization. As expected, the RE for ARP plain powderwas very low (18.96 ± 2.96%), due to its hydrophobic nature andpoor wettability. SC-2-CTAB slicosan particles with the largestvalue for release efficiency was chosen for in vivo study.

In vivo Study

To assess the impact of loading ARP into the silicosanparticles (SC-2-CTAB) on the pharmacokinetic parameters

and the in vivo bioavailability of ARP, rabbits were admin-istered a dose of 10 mg/kg of SC-2-CTAB through the oralroute and results were compared to an equal dose of ARPoral suspension (Fig. 5). The obtained pharmacokineticresults showed that the relative bioavailability for ARP-loaded silicosan particles (SC-2-CTAB) was 66% higherrelative to the oral suspension (AUC0-10h was 16.38 ± 3.21and 27.23 ± 2.35 ng.h/mL for ARP powder and SC-2-CTABformulation, respectively). The maximum ARP plasma con-centration reached following the administration of thesilicosan particles was 1.7-fold higher compared to that ofthe ARP suspension (7.69 ± 1.93 and 4.53 ± 1.40 ng/mL,respectively), reached within the same time (tmax = 1.5 h forboth treatments). The significantly higher values of the extentparameters (Cmax and AUC0-10h) were observed following theadministration of ARP-loaded silicosan particles (SC-2-CTAB) is attributed to the enhanced solubility and dissolu-tion of ARP in the silicosan particles (p < 0.05). No significantdifference was observed in the values of t1/2 following bothtreatments (8.94 ± 7.70 and 8.83 ± 6.27 h for drug powder and

0

20

40

60

80

100

120

140

0 2 4 6 8

Cum

ulat

ive

drug

rel

ease

(%)

Time (h)

SC-2 SC-TW SC-Plx SC-CTAB Drug suspension

Fig. 4. In vitro aripiprazole release from different silicosan particles in comparison withdrug suspension (the dotted line presents the difference between the release done inpH 1.2 (first 2 h), and pH 6.8 (next 6 h))

0

1

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8

9

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0 2 4 6 8 10

)L

m/gn(noitartnecnoc

amsalp

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A

Time (h)

Drug suspension SC-2-CTAB particles

Fig. 5. Mean ARP concentration in rabbit plasma after oraladministration of ARP suspension and ARP-loaded silicosan parti-cles (SC-2-CTAB)

Mahmoud et al.

SC-2-CTAB formulation, respectively; p > 005). This is con-sistent with the pharmacokinetic theory, where the increase inabsorption should not affect drug elimination (46).

CONCLUSION

Inthepresentwork, silicosanparticles,noveldeliverycarriers,for enhancement of the oral performance of aripiprazole (ARP)weredesigned.TheFT-IRstudyconfirmedtheinteractionbetweentetraethyl orthosilicate (TEOS) and chitosan-HCl in order to formthe silicosan particles. Furthermore, among the used surfactants,only Tween 80 was found to interact with silicosan formulations.The inclusionofARPinsilicosanparticles improvedtremendouslyits dissolution in simulated gastricfluid.Moreover, investigation ofARP state in these novel carriers revealed that the loaded drugappear in a molecular amorphous state. ARP-loaded silicosanparticlespreparedusingCTABwithhighestsilicaatomcontenthadthe highest enhancement effect on the drug dissolution. Theprepared ARP-loaded silicosan particles succeeded to enhancethe ARP bioavailability after oral administration to rabbits incomparison with that of plain drug suspension. The association ofthe unique-structure of silicosan and the molecular state of ARPpresents a potential candidate for delivering the poorly watersoluble,ARP, with enhanced oral bioavailability.

COMPLIANCE WITH ETHICAL STANDARDS

All animal experiments were approved by the ResearchEthics Committee (REC) for Animal Subject Research at theFaculty of Pharmacy, Cairo University, Egypt and operatedaccording to the National Institutes of Health guide for thecare and use of Laboratory animals (NIH Publications No.8023, revised 1978).

Conflict of Interest The authors declare that there is noconflict of interest.

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