Accepted Manuscript

Title: IN SITU NIOSOME FORMING MALTODEXTRIN

PRONIOSOMES OF CANDESARTAN CILEXETIL: IN
VITRO AND IN VIVO EVALUATIONS

Author: Nilufer Yuksel Zerrin Sezgin Bayindir Elif Aksakal

A. Tanju Ozcelikay

PII: S0141-8130(15)30024-6
DOI: http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.10.019
Reference: BIOMAC 5433

To appear in: International Journal of Biological Macromolecules

Received date: 11-7-2015

Revised date: 5-10-2015
Accepted date: 6-10-2015

Please cite this article as: N. Yuksel, Z.S. Bayindir, E. Aksakal, A.T. Ozcelikay, IN SITU
NIOSOME FORMING MALTODEXTRIN PRONIOSOMES OF CANDESARTAN
CILEXETIL: IN VITRO AND IN VIVO EVALUATIONS, International Journal of
Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.10.019

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 IN SITU NIOSOME FORMING MALTODEXTRIN PRONIOSOMES OF

CANDESARTAN CILEXETIL: IN VITRO AND IN VIVO EVALUATIONS IN

t
VITRO AND IN VIVO EVALUATION OF IN SITU NIOSOME FORMING

ip
PRONIOSOMES CONTAINING CANDESARTAN CILEXETIL

cr
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Nilufer Yuksel1*, Zerrin Sezgin Bayindir1, Elif Aksakal1, A. Tanju Ozcelikay2

1
Department of Pharmaceutical Technology, Faculty of Pharmacy, Ankara University, 06100
an
Tandogan, Ankara, Turkey

2
Department of Pharmacology, Faculty of Pharmacy, Ankara University, 06100 Tandogan,
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Ankara, Turkey
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*: Corresponding Author
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Prof. Nilufer Yuksel

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Ankara University
School of Pharmacy
Department of Pharmaceutical Technology
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06100 Tandogan ANKARA

TURKEY
Phone number: 00 90 312 203 3155
E-mail: nyuksel@pharmacy.ankara.edu.tr

Key words: Proniosomes, surfactants, particle size, dissolution, pharmacokinetics,

bioavailability.

1
Page 1 of 45
1. INTRODUCTION

The most of the recently investigated or commercially available drugs have low aqueous

solubility which results in variable bioavailabilities. For drug administration, oral route is

deemed as the most advantageous way. However, most of the available or recently

discovered drugs typically cause bioavailability problems due to poor solubility, unpredicted

t
absorption, intra- and interindividual variables and dose disproportion [1,2]. There are several

ip
approaches to enhance the solubility of poorly water soluble drugs such as: formation of drug

cr
complexes, drug derivatization, tailoring the solid state properties, addition of surfactants,

us
enhancement of surface area by micronization or nanonization, spray drying and drug

entrapment in micro- or nanoscale drug delivery systems [3].

an
Among the nanoscaled drug delivery systems, liposomes and niosomes are vesicular systems
M
with unique properties. Basically vesicular systems are spherical colloids formed of mono or

multiple lipid bilayers surrounding an aqueous phase. These systems come up with numerous
ed

advantages over the conventional dosage forms such as: enhancement on the solubility of

poorly water soluble drugs so as to provide drug delivery at much higher doses than the
pt

solubility of the drug; modification of drug release patterns; targeting of intended sites in the
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body and providing drug action in these regions; enhancement of drug bioavailability while

decreasing its toxicity [4-6]. Despite these advantages, both the physical and chemical
Ac

stability problems related to colloidal structure of the vesicular drug delivery systems are

inevitable. The main physical problems are swelling, fusion, sedimentation, aggregation, and

drug leakage during storage. The lipid components of vesicular systems are susceptible to

oxidation and hydrolysis which lead chemical instability. At that point, a clear advantage of

niosomes appears, due to the lack of easily hydrolysable phospholipids in their structure,

niosomes are deemed as more stable in chemical respect. Provesicular systems which are the

2
Page 2 of 45
dry powder or gel forms of the vesicular systems have been introduced as novel systems for

improving the vesicle physical stability while preserving their compositions and properties

[5,7,8].

Solid granular types of proniosomes are free flowing powders formed by coating the surface

t
of a water soluble carrier such as glucose, fructose, lactose, sorbitol, or maltodextrin by a

ip
lipid or surfactant solution in organic solvent. At the end of the coating process a dry

cr
formulation is obtained in which each water-soluble particle is coated with a thin surfactant

us
film [8-10].

an
Niosomes in dry powder form can be administered via oral, nasal, intravenous or other routes

after hydration. Especially their feature of conversion into solid dosage forms such as tablets,
M
capsules or beads generates a high potential for these systems. By this means, drug never

contacts with liquids until it reaches to patient. Proniosomes can form niosomal dispersions
ed

as they contact with body liquids. When required, certain amounts of proniosomes can be

measured and dehydrated with hot water by rapidly shaking for a few minutes prior to the
pt

administration [11,12]. The niosomes formed by the hydration of the solid powder possess
ce

the same structure with the niosomes prepared by the conventional techniques and also yield

more uniform size properties. These benefits of proniosomes make them a potential,
Ac

multipurpose carrier system for a wide range of active agents [13,14].

In the present study Candesartan cilexetil (CC), a prodrug and selective AT1 subtype

angiotensin II receptor antagonist, was selected as a model drug. CC is commonly used as

monotherapy or combined with diuretics and other antihypertensive agents. Following oral

administration it gets hydrolyzed during the absorption period and forms biologically active

3
Page 3 of 45
candesartan. The pKa value for CC is 6.3 and it is practically insoluble in water (<0.05

g/mL). CC is highly lipophilic and its partition coefficient (C octanol/Caqueous) at pHs of 1.1, 6.9

and 8.9 is >1000. CC is partly absorbed in gastrointestinal system due to its low solubility

under physiological pH interval and the absolute bioavailability of CC is at 15 % levels.

Depending on its poor solubility and low absorption characteristics, CC is classified as a

t
Class I II drug in Biopharmaceutics Classification System [15-17]. As an approach to

ip
improve the solubility and thereby the bioavailability of CC, preparation of its proniosomal

cr
formulations was planned in this study.

us
The aims of this research can be expressed as following: (i) technologically providing the
an
optimization estimating the proper parameters of the proniosome preparation process by

evaluation of different carrier, rotation speed of rotavapor, temperature, and membrane

M
charge states), (ii) characterization of the obtained proniosomes, selection of the CC loaded

proniosome formulation with the most appropriate properties, (iii) compression of the
ed

selected proniosomes as tablets, and conducting the characterization studies on them, and (iv)

evaluation of the in vivo behaviors of the proniosome tablets upon oral administration.
pt
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2. MATERIALS AND METHODS

2.1. Materials
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Candesartan cilexetil and candesartan were kind gifts from Abdi brahim Pharmaceuticals

(Istanbul, Turkey). Irbesartan, cholesterol, dicetyl phosphate, stearylamine, Span 60 (sorbitan

monostearate), hydroxypropyl methylcellulose (viscosity 80120 cP, 2% in H2O),

maltodextrin and heparin were purchased from Sigma (St. Louis, MO, USA); Tween 20

(polyoxyethylene sorbitan monolaurate) and 80 (polyoxyethylene sorbitan monooleate) were

purchased from Merck (Darmstadt, Germany); sorbitol was purchased from BioShop

4
Page 4 of 45
(Burlington, ON, Canada). Granulac 70, 200 and 230 was from Meggle (Wasserburg,

Germany), Kollidon CL was from BASF (Ludwigshafen, Germany) and Avicel PH 101 was

from FMC BioPolymer (PA, USA). All other reactive agents were of analytical grade.

2.2. Preparation of proniosomes and determination of the critical process parameters

t
ip
Slurry method was used to prepare the formulations given in Table 1 [9]. According to this

method, the calculated amounts of CC, Span 60, cholesterol and dicetyl phosphate (DCP) or

cr
stearylamine (SA) were transferred into a round bottom flask. Chloroform (10 ml) was added

us
to this mixture and sonicated in an ultrasonic bath. The accurately weighed carrier agents,

sorbitol or maltodextrin sieved to a particle size of 0.25-0.50 mm or lactose originated

an
Granulac carriers with different particle sizes was added to the obtained lipid solution. In

order to evaporate the organic solvent in the slurry mixture vacuum was applied in a
M
rotavapor for 10 min. At the end of this period, the obtained dry powder proniosomes were
ed

further incubated in a lyophilizer overnight to remove the solvent residues and the bulk was

completely dried. The process conditions and formulation variables are given in Table 1. The
pt

proniosomes were stored in tightly closed containers in desiccator at room temperature.

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2.3. Characterization of proniosomes

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2.3.1. Determination of product recovery and drug loading efficiency

The percent recovery is the ratio of the total obtained amount of proniosome powder to the

total solid content used for the preparation of proniosomes multiplied by 100. Ultracentrifuge

method was used to determine the entrapment efficiency [3,11,18]. In order to hydrate 1 g of

proniosomes, accurately weighed powder was made up to 10 ml with distilled water at 60C

and vortex mixed for 2 min. Afterwards, the samples were ultracentrifuged at 45 000 rpm,

4C for an hour (Beckman Coulter Optima XL-100K, Krefeld, Germany). The supernatant

5
Page 5 of 45
containing free drug was mixed with four parts methanol to dissolve the unloaded CC and

filtered through 0.45 m cellulose acetate filter (Sartorius AG, Goettingen, Germany) to

separate undissolved maltodextrin. Unentrapped CC concentration was determined by a

validated spectrophotometrical method at max =254 nm (wavelength of maximum

absorbance) (Schimadzu UV 1202/UV-VIS, Tokyo, Japan). Drug content in niosomes was

t
calculated by substracting the amount of the unentrapped drug from the theoretical drug

ip
amount in 1 g of proniosomes. The percentage drug loading was obtained as follows

cr
(Equation 1):

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Equation 1
an
M
2.3.2. Particle size and zeta potential measurements on niosomes derived from proniosomes

The niosome sample to be used for the analysis was prepared by mixing 100 mg proniosome
ed

in 1 ml water (60C) via 2 min vortexing thus providing the dissolution of the carrier and

diluting 50 l of this sample with 4 ml distilled water. Samples were filtered through
pt

Whatman no: 42 ashless filter paper. Formation of niosomes from hydrated proniosomes was
ce

demonstrated by using an optical microscope (Leica DM 4000B, Wetzlar, Germany).

Ac

In order to determine the particle size of the niosomal formulations dynamic light scattering

(DLS) technique was used. The results were obtained with a zetasizer (Malvern Zetasizer

Nano ZS, Malvern Instruments, Worcestershire, UK) utilizing HeNe Laser (633 nm) at

scattering angle of 175 at 25C (n=6). The same apparatus was used to measure the zeta

potential of the same niosomal samples [19].

6
Page 6 of 45
2.3.3. In vitro dissolution tests on proniosomes

The dissolution protocol given for the commercial tablet dosage forms containing

8 mg - CC in FDA Recommended Dissolution Methods was followed. Accordingly, the

dissolution test was performed by USP Apparatus II-Paddle method (USP Apparatus II,

t
Sotax AT7 Smart, Sotax AG, Basel, Switzerland) in 900 ml phosphate buffer containing 0.35

ip
% Tween 20, at 37 C and 50 rpm. After adding the weighed amount of proniosomal powder

cr
equivalent to 8 mg CC to the dissolution medium, at predetermined time intervals (10, 30,

us
60, 120, and 240 min) 5 ml of samples were withdrawn from the medium and the withdrawn

amount was replaced by fresh medium addition. The samples were then filtered through 0.45
an
m cellulose acetate filter and centrifuged at 45 000 rpm, 4C for an hour to separate the

dispersed niosomal vesicles. The CC concentration on the supernatant was determined by a

M
validated spectrophotometric method at max =258.5 nm.
ed

2.4. Selection of the final proniosome formulation and development of its proniosomal

tablet
pt

After the evaluation of proniosome formulations (F1-F8) prepared by using different process
ce

parameters and formulation variables, in terms of the drug loading percent, zeta potential,

particle size and distribution and dissolution properties, F7 proniosomes prepared with
Ac

maltodextrin as carrier was selected. In order to compress the tablets of this proniosomal

formulation, it was produced in several batches and the products were pooled for further

characterization in terms of their morphology and above mentioned properties (drug loading

percent, zeta potential, particle size and distribution and dissolution). This formulation was

coded as FP (pooled F7).

7
Page 7 of 45
In order to determine the formulation excipients, the tablet disintegration time was chosen as

the primary criterion. The concentration of disintegrating agent was determined by

compressing placebo tablets containing plain maltodextrin instead of proniosomal powder.

The final proniosomal tablet formulation (FT) was designed as follows:

t
FP (equivalent to 8 mg CC) 241 mg (76.99% w/w)

ip
Kollidon 24 mg (7.67% w/w)

cr
Avicel PH101 8 mg (15.34% w/w)

us
The following powder characterization tests were comparatively performed on maltodextrin,

proniosomes and proniosomal tablet mass.

an
M
2.4.1. Consolidation properties of the powders

The powder to be tested for its consolidation was filled into a 10 ml tarred graduated cylinder
ed

up to 10 ml mark. The graduated cylinder was weighted and the bulk density was calculated.

The graduated cylinder was further tapped with a free fall height of 2.5 cm at certain tapping
pt

number (5, 10, 20, 30, 50, 75, 100, 125, 200, 300, and 500) onto a wooden surface. The
ce

tapped density (TD) was calculated from the volume reduction by considering the powder

volume. By using these results, the relative density change (A) (Equation 2) and Hausner
Ac

index (HI) (Equation 3) were calculated [20,21]:

=
(Equation 2)

= (Equation 3)

8
Page 8 of 45
Linear correlation was established between the logarithm of relative density change and the

natural logarithm of tapping number, thus the slope and determination coefficients were

calculated.

2.4.2. Determination of the flow rate of powders

t
The flow rate was determined using a glass funnel with an orifice 10 mm in diameter by

ip
weighing the same quantity (10 g) of powders (n=5) each time. The angles of repose of

cr
powders were determined by the dynamic angle of repose method by using 30 mL of the

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granules (n=5) [22].

an
2.4.3. Determination of compressibility behavior of powders

A manual hydraulic press (Ucler, Istanbul, Turkey) with 10-mm flat face punch was used for
M
the determination of powder compressibility. Granules at appropriate amounts were pressed

at 43.33, 86.65, 173.31, 259.96, 346.62, 433.27 and 519.92 MPa. As the system reached to
ed

the desired pressure, it was preserved for 20 sec. The volume of the compact was calculated

by accurately measuring the compact height (n=3). Heckel equation was used for calculations
pt

(Equation 4) [23,24]:
ce

0
= + (Equation 4)
0
Ac

where vp is the volume of the obtained compact at each pressure applied, v is the true

volume of the compact (without pores), v0 is the initial granule volume, P is the applied

pressure. K is the slope of this linear equation and the intercept corresponds to ln v0/v0-v.

9
Page 9 of 45
2.5. Physical characteristics of proniosomal tablets

The final proniosomal tablet mass (FT) was weighed as 273 mg per tablet and compressed at

adequate pressure to obtain tablets with a hardness of approximately 75 N. A hand-operated

hydraulic press with 10-mm flat face punch was used. The hardness, thickness and

disintegration tests were conducted on proniosomal tablets. The tablet hardness was measured

t
with a Monsanto tester. The thicknesses of the tablets were accurately measured with a

ip
caliper. For the disintegration test, the given protocol in European Pharmacopeia 6 was

cr
followed [21]. The disintegration apparatus (Disintegration tester, Aymes, Istanbul, Turkey)

us
with six cells was operated with 900 ml of disintegration medium at 37C and following the

placement of the tablets in the medium the disintegration time was measured.
an
2.5.1. Zeta potential and particle size measurements on the niosomes derived from
M
proniosomal tablets

Following the hydration of 100 mg of the broken proniosomal tablet pieces in distilled water
ed

at 60C by a two minute vortexing process, the obtained niosomes were evaluated for their

zeta potential and particle size as described for niosomes derived from proniosomes. The
pt

niosome formation from hydrated proniosomal tablets was demonstrated by using an optical
ce

microscope (Leica DM 4000B, Wetzlar, Germany).

Ac

2.5.2. Morphological investigations on proniosomes and proniosomal tablets

The surface morphology of proniosomes prepared by using the final formulation (FP), the

broken pieces of the proniosomal tablets compressed by using FT formulation and uncoated

carrier particles were investigated by scanning electron microscopy (SEM) (JSM 6400,

JEOL Ltd., Tokyo, Japan). After sticking the double sided adhesive carbon tape on the

aluminum cylinder, the samples were placed on the other side. These samples were coated

10
Page 10 of 45
with gold and palladium via spraying nozzle. The surfaces of the samples were visualized by

electron scanning method, under high vacuum at 30 keV energy.

2.5.3. In vitro dissolution test on proniosomal tablets

The in vitro dissolution test on proniosomal tablets was performed as previously described

t
for proniosomes. In order to compare the dissolution performance of the proniosomal tablets,

ip
media at different gastrointestinal physiological pHs were used with/without Tween 80, aside

cr
from the dissolution medium recommended by FDA (below, number 7). The dissolution tests

us
were also performed on pure drug for 8 hours. The dissolution media and the max values used

in validated spectrophotometric methods for dissolved drug assay were as follows:

an
1. pH 1.2 simulated gastric fluid [25]. (max =248 nm)
M
2. pH 1.2 simulated gastric fluid with Tween 80 0.2 % - w/v. (max =255 nm)

3. pH 4.5 acetate buffer [25]. (max =256 nm)

ed

4. pH 4.5 acetate buffer with Tween 80 0.2 % - w/v. (max =258 nm)

5. pH 6.8 simulated intestinal fluid [25]. (max =256.5 nm)

pt

6. pH 6.8 simulated intestinal fluid with Tween 80 0.2 % - w/v. (max =259 nm)
ce

7. pH 6.5 phosphate buffer with Tween 20 0.35 % - w/v. (max =258.5 nm)
Ac

The dissolution data were evaluated by fitting to Weibull distribution, thus related parameters

explicating the type of dissolution profiles and dissolution time were obtained [26].

1
= (Equation 5)
1

in which is time, d is time at which 63.2% of the drug is dissolved, and is the shape

parameter [27].

11
Page 11 of 45
2.5.4. Differential scanning calorimetry analysis

Thermal properties of CC, Span 60, maltodextrin, cholesterol, FP proniosomes and the

physical mixture of FP proniosomes components were analyzed with Shimadzu DSC-60

(Tokyo, Japan) differential scanning calorimetry (DSC). The physical mixture of FP

t
proniosomes was prepared by mixing the FP ingredients in a glass mortar. The samples were

ip
heated from 20 to 300C under nitrogen atmosphere at a rate of 20C/min by using indium

cr
as a reference for calibration.

us
2.6. In vivo evaluation of proniosomal tablets

2.6.1. Animals
an
In order to investigate the in vivo performance of the formulations, approval has been taken
M
from the Animals Ethics Committee of Ankara University (Date: 27 April 2011, No: 2011-

111-416). Male Sprague-Dawley rats weighing 280340 g were used. The maintenance of the
ed

animals was made in standard cages at the animal house of Ankara University, Faculty of

Pharmacy (light: 07:00-19:00 h, dark: 19:00-07:00 h, at room temperature 251C). The

pt

laboratory animal welfare guidelines were followed while handling the animals.
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2.6.2. Dosage schedule and blood sampling

Ac

In the in vivo experiments to evaluate the bioavailability of CC, the animals groups were

formed as seen below (n= 8 per group):

Group 1: Oral administration of the aqueous suspension of the pure drug (control group)

Group 2: Oral administration of the aqueous suspension of the broken proniosomal tablet

(compressed FT) pieces.

12
Page 12 of 45
Rats were fed with standard diet prepared in pellet form. Prior the drug administration rats

were fasted for 12 h and water was supplied ad. libitum. The suspension form of pure drug

and broken proniosomal tablet pieces were prepared by dispersing the powders in 0.45 w/v

hydroxypropyl methylcellulose aqueous solution. The suspensions were administered to the

rats via gastric gavage at a CC dose of 10 mg/kg. At predetermined time points (0.25, 0.5, 1,

t
2, 4, 6, 8, 10, and 24 hour) 300 l of blood samples were collected from the tail vein of the

ip
animals into Eppendorf tubes containing 7 U heparin. After centrifuging the samples at 15000

cr
rpm for 5 min, 100 l of the plasma was withdrawn. At the end of the 6 th hour, animals were

us
allowed free access to food. The plasma samples were kept at -80C. Drug was extracted

from the samples and a validated HPLC method was used to determine the candesartan

concentration.
an
M
2.6.3. Sample preparation procedures and HPLC analysis

In order to extract candesartan from plasma samples, 100 l plasma was mixed with 50 l
ed

internal standard (irbesartan 2 g/ml) solution in methanol and 50 l methanol. The plasma

proteins were precipitated by adding 0.1 mg ZnSO4, 0.1 mg MgSO4, and 100 l
pt

acetonitrile/methanol (3:1) and vortexed for 10 min. Following the centrifugation at 15 000
ce

rpm for 5 min, the candesartan containing clear supernatant was directly injected to HPLC

[28,29].
Ac

The HPLC analysis was performed at 35C by using a DAD detector at a UV wavelength of

252 nm and a C8 reversed-phase column (250 mm x 4.6 mm x 5m) (Phenomenex Luna C8,

Torrence, CA, USA). The mobile phase was composed of acetonitrile:methanol:10 mM

KH2PO4 buffer (pH 3) (26:26:48). The injection volume and flow rate were 35 l and 1.0

ml/min, respectively. Candesartan concentrations were plotted against the peak area ratios of

13
Page 13 of 45
candesartan to irbesartan and the calibration curve was obtained. The linearity range of the

method was 83.31666.7 ng/ml (determination coefficient of 0.9960). Detection and

quantification limits of the method were 51.22 ng/ml and 155.20 ng/ml, respectively. The

precision during the same day (intraday assay) and different days (interday assay) at low,

medium and high concentrations of candesartan was evaluated. The relative standard

t
deviations for the types of precision were always within the acceptable limits (less than 5%)

ip
at all concentrations tested.

cr
us
2.6.4. Pharmacokinetic analysis

The pharmacokinetics of candesartan in free and proniosomal tablet forms were evaluated by
an
Kinetica software (Thermo Kinetika ver. 5.0, Thermo Fisher Scientific, Waltham, MA, USA)

using a noncompartmental model (Ritschel et al., 1999). The area under the concentration vs.
M
time curve from time zero to 24 h (AUC0t) and to infinity (AUC0) was computed by

loglineer trapezoidal method. The maximum plasma concentrations (C max) and the required
ed

time (Tmax) were calculated on the plasma concentration-time curves. The relative

bioavailability (% RB) was calculated by Equation 6 [30]:

pt
ce

AUC 0 (test )
% RB x100
AUC 0 (ref )
(Equation 6)
Ac

AUC0 (test) : AUC value of pure drug.

AUC0 (ref) : AUC value of proniosomal tablets.

2.7. Statistical evaluation

One way ANOVA and t-tests were used for the statistical comparison of the results by using

SPSS 9.0 for Windows (SPSS, Chicago, IL).

14
Page 14 of 45
3. RESULTS AND DISCUSSION

3.1. Preparation and characterization of proniosomes

The main component of niosomes is nonionic surfactants. Cholesterol is added to provide

membrane stabilization of niosomes. The typical cholesterol:surfactant molar ratio to obtain

niosomes with adequate physical stability is 1:1. It is possible to add charge inducing agents

t
(such as dicetylphosphate-DCP, stearylamine-SA) at a molar ratio of 0.05 for aggregation

ip
prevention and stabilization of the colloidal niosome dispersions [19]. As a critical parameter,

cr
the gel to liquid phase transition temperature (T c) of niosome forming surfactants, plays an

us
important role on enhancing the entrapment efficiency. Below this temperature the

permeability of the lipid bilayers is very low and so is the drug leakage. Span 60 has a T c of
an
50C and it is among the most commonly used surfactants that provide high entrapment

efficiency [6-31]. In the present study Span 60 was selected as the nonionic surfactant to
M
benefit from its mentioned advantage. The drug: sufactant: cholesterol: DCP or SA

concentrations were 11.4:11.4:11.4:1.2 mM (Table 1).

ed

The carriers used in proniosome formulations provide a wide surface for the niosome
pt

components, thus provide enhanced drug loading efficiency. The carriers must be safe,
ce

nontoxic and possess adequate flow properties as a powder mass. The carrier should be

poorly soluble or practically insoluble in the organic solvent used in the preparation of the
Ac

loading mixture and in order to easily get hydrated its solubility in water should be very good

[12-32]. In this study, carriers with different properties were investigated for their efficacy on

the preparation of proniosomes with optimum the most suitable properties. Sorbitol,

maltodextrin and lactose originated Granulac carriers with different particle sizes (70 d90

<230 m, 200 d90 <92 m, and 230 d90 <53 m) were used. Proniosomes were prepared

by slurry method. The ratio of the carrier to the surfactant: cholesterol: DSP or SA mixture

15
Page 15 of 45
was chosen as 20:1 and it is equal to 12 mM niosomal mixture per gram carrier. Usage of

low surfactant loading seems appropriate for obtaining thin and smooth film. The necessity of

smooth films for rapid formation of uniform multilamellar vesicles by hydration and shearing

has been reported [9].

t
Since Granulac containing formulations did not form in proniosomes, these formulations

ip
were not included in Table 1. After the organic solvent evaporation, a viscous slurry was

cr
formed and stuck on the walls of the round bottom flask. This was attributed to partial

us
solubilization of originally insoluble lactose in chloroform due the presence of surfactant in

the formulation.
an
3.1.1. Product recovery and drug loading efficiency of proniosomes
M
The product recoveries of the formulations were 60.90-93.80 % (Table 2). Sorbitol

containing F1 formulation was prepared with a yield of 80.13 %. As sorbitol was dissolved
ed

in organic solvent, formation of a viscous slurry was observed and since the evaporation of
pt

the solvent was slow, partial sticking was seen on the wall of the flask. The relatively low

product recovery might be explained by this. It was mentioned that sorbitol is a more
ce

appropriate carrier for spraying method, in which solvent is added in portions by spraying

onto the carriers [7,14]. As another carrier, maltodextrin was used for formulation studies.
Ac

The initially investigated critical process parameter was the rotation speed of rotavapor

during solvent evaporation step. While preparing F2, F3, and F4 formulations the rotation

speeds of the ratovapor were 60, 30 and 90 rpm during organic solvent evaporation. The

product recoveries of these formulations were 92.85 %, 90.62 % and 60.90 % respectively

(Tablo 2). While preparing F3 and F4 formulations, the product was stuck on the wall of the

flask and proniosomes were formed as aggregates. At low rotational speed, the mixture in the

16
Page 16 of 45
round bottom flask preserved its surface area and the cohesive forces between maltodextrin

particles became dominant resulting in the formation of aggregates. As the speed of solvent

evaporation decreased, the viscosity of the slurry increased and thus, hindered the

formation of individual proniosomal particles. was increased due to slow solvent evaporation.

and result in aggregate formation. Conversely, at high rotational speed, the surface area of

t
mixture increased due to compatible movement of the mixture with the rotating flask based

ip
on the centrifugal force. This force and rapid solvent evaporation led the mass to strongly

cr
adhere to the wall of the flask.the absence of homogenous slurry spreading onto the wall of

us
flask, resulted in aggregate formation. As a result, theThe resulted mass on in the flask after

the evaporation of organic solvent was low in amount, had poor flowability and formed
an
aggregates. The optimum rotation speed which gave the highest product recovery was

estimated as 60 rpm. which gave the highest product recovery.

M

The solvent evaporation was provided both by vacuum and heat application at rotavapor and
ed

then, incubation of the proniosomes in lyophilizer. In order to observe the effect of

temperature on solvent evaporation, different formulations were prepared at 60 C and two

pt

different temperatures below 60 C (45C, 30C, and 60C for F2, F5 and F6 proniosomes,
ce

respectively). Temperature did not affect the product recovery significantly but the highest

recovery (93.80 %) was obtained with F6 prepared at 60C (Table 2). Hence, it was chosen as
Ac

the most appropriate evaporation temperature. Also, 60C was above T c value of Span 60

and at this temperature the improved coating efficiency of the carrier particles with surfactant

film might be expected. At this stage F7 formulation prepared without negative charge

inducer dicetyl phosphate which is commonly used for enhancing the stability of niosomes

derived from proniosomes and F8 formulation with a positive charge inducer, stearylamine,

were prepared. The product recoveries of both formulations were very high.

17
Page 17 of 45
The drug loading efficiencies of the niosomes obtained by the hydration of proniosomes were

within the range of 97.28 - 99.80 % (Table 2). Drug loading was not influenced by the

changes on the parameters such as temperature and rotation speed during solvent evaporation.

Besides, both the carrier and charge inducing agents did not change the loading efficiency.

t
ip
Since CC is a lipophilic active agent, it was successfully incorporated into proniosome

formulations prepared with Span 60 which has a low HLB value of 4.70.

cr
us
3.1.2. Particle size and zeta potential measurements on niosomes derived from proniosomes

The particle size of the niosomes derived by the hydration of proniosomes was at nanoscale
an
and varied between 185-375 nm, PDI values were between 0.307 0.519 (Table 2). While

the temperature change did not affect the product recovery, the particle size of niosomes
M
formed from the proniosomes by hydratation was smaller with the increasing process

temperature. This result shows that the temperature clearly affected the spreading of the
ed

surfactant film onto the carrier particles. The particle size of the niosomes derived from the
pt

proniosomes prepared above Tc value of Span 60 at 60C were smaller (F6 and F7

formulations). Low PDI values demonstrated the homogeneity of the particle size
ce

distribution. When compared with the classical niosomes, during the preparation of

proniosomes the surfactant film is coated on the surface of each particle instead of the inner
Ac

surface of the round bottom flask. Thence, a thinner film layer onto a much wider surface is

obtained by coating. By easy hydration of this film layer, smaller niosomal vesicles are

formed with high drug loading capacity. Abd-Elbary et al. have compared the properties of

the niosomes prepared by reverse phase evaporation with those derived from proniosomes

prepared by slurry method [32]. The conventionally prepared niosomes were shown to be

significantly larger and heterogeneous than those derived from proniosomes.

18
Page 18 of 45
DCP was used as charge inducer in F1 and F6 formulations and the measured zeta potential

values were between -32.930.44 mV and -49.000.87 mV (Table 2). Although DCP was not

added to F7 formulation, it demonstrated a comparable negative charge with other

formulations (-43.650.54 mV). This was attributed to the negative charge of CC. Beside the

t
ester structure assembled with the carboxyl group, the free electron couples on nitrogen and

ip
oxygen atoms on CC molecule induce this negative charge. The zeta potential of SA

cr
containing F8 formulation (-6.170.18 mV) was very lower than the required value for good

us
stabilization. For this reason aggregation and sedimentation was observed on the niosomal

dispersions derived from proniosomes. As all zeta potential measurements were compared,
an
dicetyl phosphate usage does not seem to be necessary to provide stabilization of CC loaded

niosomes.
M

In order to estimate the formation of niosomes from hydrated proniosomes, the aqueous
ed

dispersions were evaluated under optic microscope. Spherical niosome formation was

observed in all formulations. The images of several samples were given in Fig. 1.
pt

Please Insert Figure 1

ce

3.1.3. In vitro dissolution tests on proniosomes

Ac

According to the data obtained from the dissolution test performed in phosphate buffer

containing 0.35 % (w/v) Tween 20 at pH 6.5, drug release from F4 and F5 formulations were

respectively 71.9 and 71.3 % at the end of four hour and these were lower than the drug

release obtained in other formulations (Fig. 2). F4 was prepared by using the fastest rotation

speed (90 rpm) and heating the system up to 45C during the solvent evoporation step. As it

was mentioned before, at this high rotation speed, instead of individual free flowing powder

19
Page 19 of 45
particles, aggregates were formed. For F5 formulation in which, rotation speed and

temperature were 60 rpm and 30C, a powder with good flowability was not obtained due to

insufficient organic solvent removal and the formulation was resulted in a rigid aggregate

after lyophilization. It is thought that, in both formulations homogenous surfactant film did

not form and hydration was slowed down to form niosomes. The highest drug release was

t
observed for F2 (91.9 %) and F7 (94.7 %) (Fig. 2). Out of the obtained results, it was

ip
concluded that among the investigated parameters solvent evaporation temperature and

cr
rotation speed were effective on in vitro drug release profiles.

us
Please Insert Figure 2

an
3.2. Selection of the final proniosome formulation and development of its proniosomal

tablet
M
Due to higher product recovery and drug loading percent, smaller particle size, and the

highest drug dissolution, maltodextrin based F7 formulation was selected. In further studies,
ed

this formulation was produced in series and pooled (FP formulation). FP formulation has

shown similar characteristics with the original formulation F7 (Table 2, Fig. 1 and 2).
pt
ce

Compared with niosomes, the advantage of proniosomes for oral administration is that they

can be filled into hard gelatin capsules or compressed in tablets. One of the aims of this study
Ac

was to prepare tablet formulations of proniosomes and investigate the possible changes on

their properties. Apart from the proniosomes, cross-linked polyvinyl pyrrolidone (Kollidon-

CL) as super disintegrant which enables disintegration by swelling and capillary actions were

added to the tablet formulation (FT). Microcrystalline cellulose (Avicel PH 101), a direct

tableting agent, was used to benefit from its disintegrant property from capillary action. Thus,

it was intended to provide the release of proniosomes by rapid tablet disintegration.

20
Page 20 of 45
3.2.1. Characterization of the proniosome powder and proniosomal tablet formulation:

Flow properties, consolidation and compressibility

The flowability and consolidation of the powder are important for homogenous filling of the

compression mass into the die cavity during tablet compression, homogenous density

t
distribution within the tablet and the mechanical properties of the tablets.

ip
cr
The flowability of powders is determined by the angle of repose and flow rate. The angle of

us
repose for maltodextrin was 36.22 and considered as fair from powder flowability respect

while the flowability of proniosomal powder (FP) was qualified as excellent with an angle of
an
respose of 29.85 and that of proniosomal compression mass (FT) was considered as good

with a value of 31.44 (Table 3) [21]. Likewise, the fastest flow was observed for
M
proniosomes and then for proniosomal compression mass (Table 3). Another indicator of

powder flowability is Hausner index (HI) values. For maltodextrin HI was 1.512 which
ed

corresponds to very poor flowability. On the other hand the estimated HI values of 1.220 and

1.203 for FP and FT were classified as fair from flowability respect (Table 3) [21]. Other
pt

studies also confirmed that compared to the carriers, dry vesicular systems were formed as
ce

non-sticky, free flowing powders [33,34]. The flow property of bulk material results from the

cohesive forces acting on individual particles such as van der Waals, electrostatic, surface
Ac

tension, interlocking, and friction [35]. These results revealed that, the flowability of

maltodextrin was improved by the surfactant film coating on the surface of maltodextrin and

the presence of other excipients which reduce the interactions between maltodextrin particles.

The initial step in tablet formation is the settlement of the powder particles close to each other

for compression and reduction of the bulk density which means they should consolidate [20].

21
Page 21 of 45
Afterwards, tablets are formed by plastic deformation and fragmentation. The slope values

(m) of the relative density change vs. the number of taps, i.e., packing rates for maltodextrin,

proniosomes (FP), and proniosomes containing tablet formulation (FT) are given in Table 3.

Accordingly, the lowest relative density change was observed in maltodextrin. An increase

was observed in the relative density change for proniosomal compression mass (FT). This is

t
compatible with the HI results.

ip
cr
The Heckel equation describes the relationship of the compact density with the applied

us
pressure. The rate of density increase with applied pressure is proportional to the volume

fraction of pores [23]. In this study, volume reduction of the compacts was used instead of
an
density increase under the applied pressure in Heckel equation. The reciprocal value of the

slope in Equation 4 (Py=1/K) represents the mean yield pressure by which a substance resists
M
the deformation process. Py, indicates the plasticity of the particles and it can be used for the

classification of materials from very soft to hard. The value of intercept (I) is related to the
ed

die filling and particles slipping over each other during rearrangement before deformation

and bonding of the separate particles at the beginning of the compression [23,24,36]. The
pt

parameters for Heckel equation are given in Table 3. Among the I values for maltodextrin
ce

(0.878) and FP (0.986) FT (1.248), a significant difference was observed at 10% level

between maltodextrin and FT (p<0.10). The Py values for maltodextrin and FP were 64.97
Ac

MPa and 70.75 MPa, respectively. Against the smallest slope (0.0106) of the FT, the highest

Py value (96.55 MPa) was calculated and this was significantly different from the Py of

maltodextrin (p<0.10). The presence of tablet excipients in the proniosomal tablet

compression mass reveals the need of higher pressure to provide plastic deformation and

beyond fragmentation during tablet compression.

22
Page 22 of 45
3.3. Physical characteristics of proniosomal tablets

In order to not to delay the tablet disintegration, the approximate tablet hardness was set to 75

N by adjusting the applied pressure during tablet compression. Considering the properties

given in Table 3, the hardness of the proniosomal tablets was 74.78 N and the disintegration

time was approximately 8 min.

t
ip
The particle size, zeta potential and optic images of the niosomes obtained from proniosomal

cr
tablets were investigated to see whether the initial proniosome properties were changed after

us
tablet compression or not (FT formulation; Table 2 and Fig. 1). As the obtained results were

compared with the ones from proniosome derived niosomes, it was confirmed that the
an
compression pressure during tablet formation did not change the niosome properties.
M
3.3.1. Morphological analysis

During the proniosome formation, the surface of maltodextrin carrier particles (0.25-0.50
ed

m) was coated with non-ionic surfactant solution in organic solvent (Table 1). SEM analysis

was performed to observe the morphological changes on the surface during this process and
pt

after tablet compression. As seen in Fig. 3, uncoated maltodextrin particles demonstrated

ce

irregular shape and there were some foldings and pores on their surface.

Please Insert Figure 3

Ac

As the surfactant coating on the smooth surfaces is homogenous as a thin film, the foldings

and pores might be filled with the surfactant solution, thus lead the formation of thick coating

layer. Therefore, the surface properties and surface area of the carrier are effective on the

formation of a smooth surfactant coating [9].

23
Page 23 of 45
However, the images of proniosomes in Fig. 3 were not significantly different from that of

the uncoated carrier, indicating the formation of homogeneous film layer of surfactant onto

the surface of maltodextrin during proniosome preparation. The proniosomal tablets were

directly split and visualized by SEM without powdering. On the broken surface of the tablets,

densification and fractures were observed due to the pressure applied during tablet

t
compression.

ip
cr
3.3.2. In vitro dissolution test on proniosomal tablets

us
The in vitro dissolution tests were conducted on proniosomal tablets (compressed FT) and

pure drug in dissolution media simulating various gastrointestinal pHs with/without

an
surfactant which lowers the surface tension to provide wetting (Fig. 4a and 4b, respectively).

The in vitro dissolution tests were conducted on proniosomal tablets (compressed FT) and
M
pure drug in dissolution media simulating the physiological gastrointestinal tract conditions

(pH and surface tension). The surface tensions of the dissolution media are very effective on
ed

the dissolution kinetics of active agents. The surface tension of human gastrointestinal fluids

are found to be 35-50 mN/m independent of the secretion rate and pH. Dissolution media
pt

with surface tension in this range are considered as more ideal for in vitro release testing [37].
ce

Therefore, Tween 80 was added at a ratio of 0.2 % - w/v to mimic the surface tension of

gastrointestinal fluids and provide the wetting of the drug.

Ac

Dissolution profiles of CC from pure form and proniosomal tablet are shown in Fig. 4. Drug

dissolution was not observed from both the pure drug a proniosomal tablet form in simulated

gastric fluid (pH 1.2) with/without Tween. In acetate buffer (pH 4.5) without Tween, pure

drug did not get dissolved but 10.47 % of the drug was dissolved from the proniosomal

tablets by the end of four hour. The CC amounts dissolved in pH 4.5 medium with Tween 80

24
Page 24 of 45
at the end of four hour were 56.14 % and 12.49 % for proniosomal tablets and pure drug,

respectively. The dissolution percents for CC in pH 6.8 medium without surfactant were

45.95 for proniosomal tablets, and 16.47 % for the pure drug. The highest drug release (96.97

% for proniosome tablets and 70.13% for pure drug) was observed in phosphate buffer pH

6.5 with 0.35 w/v Tween 20 which is the FDA recommended medium which meets the sink

t
condition and the subsequent highest release was obtained (78.23% for proniosomal tablets

ip
and 56.44% for pure drug) in phosphate buffer pH 6.8 with 0.2% w/v Tween 20.

cr
Please Insert Figure 4

us
Dissolution of CC in higher pHs is expected due to its pKa at 6.3. The pH-dependent

dissolution behavior of CC was stated; at pH 1.2 and pH 6.8 its solubility is 0.23 g/ml and
an
1.40 g/ml respectively [16]. Despite of these low solubility values, the high dissolution

percents obtained in this study revealed that the solubility of CC was enhanced via niosomes
M
derived from proniosomal tablets and CC was released from niosomes in molecular form.

However, the highest drug release percents obtained in phosphate buffer pH 6.5 containing
ed

0.35 w/v Tween 20 was not observed in other dissolution media, probably due to low

surfactant concentration (0.2 % w/v) in their contents. In a research on the effect of Tween
pt

20 on CC solubility, it was stated that addition of Tween 20 (0.35 % 0.70 % w/w) to 0.05
ce

mol/L of phosphate buffer pH 6.5 can dramatically increase the apparent solubility of CC

from 0.8 g/ml even to 353 g/ml. They also indicated that Tween 20 effect in lower pH or in
Ac

water was much smaller (20 g/ml in pH 4.5) [16]. Therefore, since the sink condition was

not achieved in other dissolution mediums, precipitation of the dissolved drug might be an

expected result [17].

The analysis of the dissolution data with Weibull distribution has confirmed this result.

Dissolution data from pure drug and the tablets containing proniosomes was compared

25
Page 25 of 45
through Weibull distribution with its parameters describing the types of dissolution profiles

and dissolution time. The shape parameter, , characterizes the profile as either exponential

(=1), S-shaped with upward curvature followed by a turning point (>1), or as one with

steeper initial slope than consistent with the exponential (<1) [27]. If a comparison was

made for the d values which represent the time for 63.2% drug release, d value of

t
proniosomal tablets was lowest at pH 6.5 medium containing 0.35% Tween 20 (29.85 min)

ip
among the other dissolution media. Pure drug also gave the lowest d value at pH 6.5 medium

cr
containing 0.35% Tween 20 and then drug dissolution was stopped. The shape parameter was

us
generally less than one indicating a rapid initial release followed by a slow release.

3.3.3. Differential scanning calorimetry analysis

an
Drug-excipient interactions in proniosomes were investigated by performing thermal

analysis. Thermograms of pure drug and formulation components were presented in Fig. 5a-
M
d. The melting peaks of cholesterol and Span 60 were observed at 148.67C and 66.08C

indicating their crystalline form (Fig. 5a and 5b) . The second peak at 193.35C in the
ed

thermogram of Span 60 showed the flash point (Fig. 5b). A sharp endothermic peak

corresponding to the melting of crystalline drug was found at 168.64C and immediately
pt

afterwards an exothermic peak was observed which might indicate the drug degradation (Fig.
ce

5c) [38, 39]. In the thermogram of maltodextrin an endothermic peak corresponding to its

melting point was present at 173.84C and then, decomposition was seen at around 250C
Ac

(Fig. 5d). The peaks in the thermogram of the physical mixture of excipients in FP

formulation at 160.74C, 172.88C, and 191.03C were considered as comparatively shifted

peak of cholesterol and peaks of CC and Span 60 (Fig.5e). There is not any apparent peak

indicating the crystalline state of CC within FP formulation; the characteristic peak of CC at

168C was disappeared (Fig. 5f) demonstrating the incorporation of CC in proniosomes and

formation of its amorphous form after incorporation. This amorphous form can explain the

26
Page 26 of 45
enhanced drug dissolution. Disappearance of the CC peak can be also attributed to the high

interactions between CC and proniosome components. Thus, the formation of endothermic

peak at 205.14C can be due to this interaction (Fig. 5f) .

Please Insert Figure 5

t
ip
3.4. In vivo evaluation of proniosomal tablets

cr
Candesartan is the therapeutically active form of CC and it is formed by the ester hydrolysis

us
of the drug. Fig. 5 6 shows the plot of plasma concentration of candesartan vs. time for the

suspension forms of pure drug and broken proniosomal tablets (compressed FT). The
an
obtained pharmacokinetic parameters are given in Table 4. Candesartan could be detected in

blood samples up to 10-hour after oral administration and drug concentration of the collected
M
samples at 24th hour was below the detection limits of HPLC. The experimental results

indicated that the plasma concentration of candesartan in rats was higher than that of pure
ed

CC. Cmax values were 382.39 56.558 ng/ml and 1053.1110.32 ng/ml for pure drug and

proniosomal tablet (p<0.0001). The estimated Tmax was 1.190.0.19 h for proniosomes tablet
pt

and this was significantly shorter than the T max (3.50.63 h) for pure drug (p<0.01). These
ce

results were compatible with the drug dissolution results in Table 3. The AUC0t and

AUC0values for proniosome tablets were significantly higher compared to the calculated
Ac

values for pure drug (p<0.01). The relative bioavailability of proniosomal tablets vs. pure

drug was 185.88 % upon oral administration of these two forms (Equation 6). The increase in

the rate and extent of absorption for the proniosomal tablet could be attributed to the increase

in the rate and extent of drug dissolution in GI tract. The more rapid absorption of drug from

proniosomal tablets was reported to be beneficial in the treatment of hypertension or heart

failure, particularly in case of emergency [3739].

27
Page 27 of 45
Please Insert Figure 56

The in vivo pharmacokinetic behavior demonstrated that the proniosomal tablets could

improve the oral bioavailability of CC which was attributed to the niosomes formed from

t
proniosomes. Due to its hydrophobic nature CC is not located in the aqueous core of the

ip
niosome but in the lipid bilayer that surrounds this core. The enhancement on the

cr
bioavailability can be associated with the following factors: (i) solubilization of the active

us
agent in the gastrointestinal system thus make it available for intestinal absorption, (ii)

contribution of the nanometric size of niosomes that increase the accessibility to enterocyte's
an
surface and improve the permeability across the intestinal membrane, and (iii) the possible

uptake of niosomes by transcytosis of M cells of Peyers patches present in intestine [19,

M
3739].
ed

4. CONCLUSION

Conversion of the vesicular systems such as liposomes and niosomes into commercial
pt

products is quite limited due to the critical physical stability problems. Formulation of
ce

vesicular systems as more stable provesicular systems by preserving their superior properties

may substantially provide the introduction of these systems to patient usage. Especially the
Ac

dry granular forms of proniosomes can be formulated as capsules, tablets or solutions to

prepare just before usage for oral administration and these are the most preferred forms in

terms of providing patients compliance and usage. Besides, proniosomes may provide

flexibility on large scale production. Proniosome formulations were found to be promising

systems that may enhance the oral bioavailability of other lipophilic drugs that cannot be

28
Page 28 of 45
formulated due to their low solubility and limited oral bioavailability (such as anticancer

agents).

DECLARATION OF INTEREST

t
The authors report no declarations of interest.

ip
cr
ACKNOWLEDGEMENTS

us
This study has been supported by TUBITAK (The Scientific and Technological Research

Council of Turkey) under grant 111S283.

an
M
ed
pt
ce
Ac

29
Page 29 of 45
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ip
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ip
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pt

[37] M. Efentakis, J.B. Dressman, Gastric juice as a dissolution medium: surface

tension and pH, Eur. J. Drug Metab. Pharmacokinet. 23(1998) 97-102.

ce

[36][38] C. Detroja, S. Chavhan, K. Sawant, Enhanced antihypertensive activity

Ac

of candesartan cilexetil nanosuspension: formulation, characterization and

pharmacodynamic study, Sci. Pharm. 79 (2011) 635651.

[37][39] Z. Zhang, F. Gao, H. Bu, J. Xiao, Y. Li, Solid lipid nanoparticles

loading candesartan cilexetil enhance oral bioavailability: in vitro characteristics

and absorption mechanism in rats, Nanomed-Nanotechnol. 8 (2012) 740-747.

34
Page 34 of 45
 Table 1: The composition of proniosome formulations and process parameters.

Ingredients F1 F2 F3 F4 F5 F6 F7 F8

Candesartan cilexetil (mg) 69.61 69.61 69.61 69.61 69.61 69.61 69.61 69.61
Cholesterol (mg) 44.23 44.23 44.23 44.23 44.23 44.23 44.23 44.23

t
Span 60 (mg) 49.28 49.28 49.28 49.28 49.28 49.28 49.28 49.28

ip
Dicetyl phosphate (mg) 6.49 6.49 6.49 6.49 6.49 6.49 - -
Stearylamine (mg) - - - - - - - 3.23

cr
Chloroform (mL) 10 10 10 10 10 10 10 10
Sorbitol (g) 2 - - - - - - -
Maltodextrin (g) - 2 2 2 2 2 2 2

us
Process parameters
Rpm 60 60 30 90 60 60 60 60
Temperature (C) 45 45 45 45 30 60 60 60
Time (min) 10 10 10 an 10 10 10 10 10
M
ed
pt
ce
Ac

35
Page 35 of 45
Table 2: The product recovery of proniosome formulations and the properties of niosomes
derived by hydration of the formulations.

Product % Drug Particle PDIbSE Zeta Potential

t
Formula recovery Loading Size (mV) SE

ip
(%) SEa (nm)SE

cr
F1 80.13 98.65 0.09 375 7 0.458 0.027 -42.35 0.71
F2 92.85 97.34 0.07 323 4 0.519 0.006 -43.08 0.06
F3 90.62 98.60 0.02 334 1 0.464 0.012 -32.93 0.44

us
F4 60.90 97.28 0.09 264 5 0.471 0.031 -43.50 0.10
F5 90.62 99.25 0.04 212 3 0.503 0.0003 -42.13 0.22
F6 93.80 99.80 0.02 185 1 0.392 0.002 -49.00 0.87
F7
F8c
92.90
93.60
99.09 0.04
99.19 0.08
204 2
-
an 0.307 0.004
-
-43.65 0.54
-6.17 0.18
FPd 93.20 99.20 0.02 217 2 0.408 0.015 -45.75 0.42
M
FTe - - 222 2 0.364 0.003 -45.60 0.35
a
Standard error; bPolydispersity index; cF8 The niosomes obtained by the hydration of F8
proniosomes were in aggregate form and their particle size was not measurable; dFP:Pooled F7
formulation;eFT:Compressed FT formulation containing FP proniosomes.
ed
pt
ce
Ac

36
Page 36 of 45
Table 3: Characterization of the carrier and proniosomal powders before and after tableting.

Powders

t
ip
Characteristics Maltodextrin Proniosomes (FP) Tablet formulation
( standard error) containing
proniosomes (FT)

cr
Angle of repose () 36.22 29.85 31.44
Flow rate (g/sec) 1.306 0.068 3.124 0.071 2.94 0.042

us
Tapped density (g/ml) 0.372 0.360 0.373
Bulk density (g/ml) 0.245 0.295 0.310
HI 1.512 1.220 1.203
Consolidation
m 0.129
an0.136 0.215
R2 0.8854 0.9715 0.9182
M
Compressibility
K 0.0158 0.0017 0.0143 0.0009 0.0106 0.0010
I 0.878 0.133 0.986 0.093 1.248 0.0060
ed

Py(1/K; Mpa) 64.97 8.16 70.75 4.97 96.55 10.05

Proniosomal tablets (compressed FT)
Thickness (cm) Hardness (N) Disintegration time (min)
pt

0.3 0 74.78 1.23 8.46 0.0085

Comparative dissolution behavior of tablets containing proniosomes in different
dissolution media by Weibull distribution model parameters
ce

Dissolution media Pure drug Tablets containing proniosomes

d (min) d (min)
18.92x105
Ac

pH 4.5 - - 0.2447
pH 4.5 with Tween 80 0.1093 19.62x109 0.5206 263.2
9
pH 6.8 0.0795 49.73x10 0.3373 1042
pH 6.8 with Tween 80 0.2096 805.4 0.4923 93.84
pH 6.5 with Tween 20 0.0664 29.85 0.6484 43.28

37
Page 37 of 45
 Table 4: The bioavailability parameters obtained from pure drug and proniosomal tablets
containing proniosomes (compressed FT).

Cmax (ng/ml) t max (min) AUC (0t) AUC(0)

(ng/mL/min) (ng/mL/min)

t
Tablet Pure Tablet Pure Tablet Pure Tablet Pure

ip
drug drug drug drug
1 619.64 312.51 1 6 1775.3 2616.2 2373.2 6565.8
2 1485.9 337.32 0.5 2 4511.6 1571.7 4936.9 1807.6

cr
3 984.83 411.33 1 4 5125.3 2206.8 9312.2 2523.5
4 1159.9 746.57 1 2 5224.0 2800.3 6070.0 2931.6

us
5 743.44 303.08 2 6 3762.7 1888.9 4904.7 2858.3
6 1407.0 329.13 1 2 5925.3 7080.6 6412.8 2123.7
7 830.70 407.14 1 2 3227.4 2015.5 5263.6 3769.8
8 1193.3 212.07 2 4 6213.7 1276.6 6589.7 2093.6

Mean 1053.1 382.39 1.19 3.5

an
4470.6 2682.1 5732.9 3084.2
SEa 110.32 56.558 0.19 0.63 525.49 652.91 693.71 543.02
95% CIb
M
LBc 752.23 248.66 0.74 2.02 3228.1 1138.2 4092.5 1800.3
UBd 1314.0 516.13 1.63 4.98 5713.3 4226.0 7373.2 4368.3
e
Sig
ed

p 0.00009** 0.003* 0.0051* 0.009*

a b c d
*p<0.01; **p<0.0001. Standard error. 95% confidence interval. Lower bound of CI. Upper bound
of CI.e Significance.
pt
ce
Ac

38
Page 38 of 45
Figure List:

Figure 1: The optic microscope images of niosome formulations derived by the hydration of

F7, FP proniosomes and proniosomal tablets (compressed FT).

t
Figure 2: Comparison of dissolution data for proniosome formulations in phosphate buffer

ip
containing 0.35 % Tween 20 at pH 6.5 (n=4).

cr
Figure 3: Surface morphologies of maltodextrin, maltodextrin based proniosomes (FP) and

us
their tablet (compressed FT) taken by SEM.

Figure 4: CC dissolution profiles from proniosomal tablets (compressed FT) (A) and pure

drug (B) at different dissolution media (n=4).

an
Figure 5: DSC thermograms of (a) cholesterol, (b)Span 60, (c) CC, (d) maltodextrin, (e)
M
physical mixture of CC +Span 60+maltodextrin+cholesterol, (f) FP formulation.

Figure 6: Comparison of the candesartan-plasma profiles for proniosomal tablet

ed

(compressed FT) and pure drug (C).

pt
ce
Ac

39
Page 39 of 45
Figure1

5m 5m 2m

F7 FP FT

t
ip
cr
us
an
M
ed
pt
ce
Ac

Page 40 of 45
Figure2

Figure 2

100

t
90

ip
80
Drug dissolved (%)

cr
70

us
60 F1
50 F2
F3
40 an F4
F5
30
F6
M
20 F7
10 F8
FP
FS
ed

0
0 30 60 90 120 150 180 210 240
Time (min)
pt
ce
Ac

Page 41 of 45
 Figure3

Proniosomal tablet Proniosome Maltodextrin Figure 3

Ac X1000

ce
pt
ed
M
X5000

an
us
cr
ip
t

Page 42 of 45
Figure4

Figure 4

100
Drug dissolved (%)

80

60

t
40

ip
20

cr
0
0 60 120 180 240 300 360 420 480
Time (min)

us
pH 6,8_Tween
pH 6.8_Tween pH 4,5_Tween
pH 4.5_Tween pH 1,2
pH 1.2_Tween
_ Tween pH 6,5
pH 6.5_Tween
_Tween
pH 6,8
pH 6.8 pH 4,5
pH 4.5 pH 1,2
pH 1.2

B
an
M
100
Drug dissolved (%)

80
ed

60

40
pt

20
ce

0
0 60 120 180 240 300 360 420 480
Time (min)
Ac

pH6,8
pH 6.8 pH 4,5
pH 4.5 pH1,2
pH 1.2HCl pH6,5_Tween
pH 6.5_Tween
pH6,8_Tween
pH 6.8_Tween pH 4,5_Tween
pH 4.5_Tween pH 1.2_Tween
pH1,2_Tween

Page 43 of 45
Figure5

Figure 5

DSC
mW
a

t
c

ip
d

cr
e

us
f

50 100 150
an 200 250 300
Temp [C]
M
ed
pt
ce
Ac

Page 44 of 45
Figure6

Figure 6

t
ip
cr
us
an
M
ed
pt
ce
Ac

Page 45 of 45