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Drug Delivery

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Intranasal brain-targeted clonazepampolymeric micelles for immediate control ofstatus epilepticus: in vitro optimization, exvivo determination of cytotoxicity, in vivobiodistribution and pharmacodynamics studies

Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M.Rashed & Ahmed B. Ibrahim

To cite this article: Samia A. Nour, Nevine S. Abdelmalak, Marianne J. Naguib, Hassan M.Rashed & Ahmed B. Ibrahim (2016) Intranasal brain-targeted clonazepam polymeric micellesfor immediate control of status epilepticus: in vitro optimization, ex vivo determination ofcytotoxicity, in vivo biodistribution and pharmacodynamics studies, Drug Delivery, 23:9,3681-3695, DOI: 10.1080/10717544.2016.1223216

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Drug Deliv, 2016; 23(9): 3681–3695! 2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/10717544.2016.1223216

RESEARCH ARTICLE

Intranasal brain-targeted clonazepam polymeric micelles for immediatecontrol of status epilepticus: in vitro optimization, ex vivo determinationof cytotoxicity, in vivo biodistribution and pharmacodynamics studies

Samia A. Nour1, Nevine S. Abdelmalak1, Marianne J. Naguib1, Hassan M. Rashed2, and Ahmed B. Ibrahim2

1Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt and 2Labeled Compounds Department, Hot Lab. Center,

Egyptian Atomic Energy Authority, Cairo, Egypt

Abstract

Clonazepam (CZ) is an anti-epileptic drug used mainly in status epilepticus (SE). The drugbelongs to Class II according to BCS classification with very limited solubility and highpermeability and it suffers from extensive first-pass metabolism. The aim of the present studywas to develop CZ-loaded polymeric micelles (PM) for direct brain delivery allowing immediatecontrol of SE. PM were prepared via thin film hydration (TFH) technique adopting a centralcomposite face-centered design (CCFD). The seventeen developed formulae were evaluated interms of entrapment efficiency (EE), particle size (PS), polydispersity index (PDI), zeta potential(ZP), and in vitro release. For evaluating the in vivo behavior of the optimized formula, bothbiodistrbution using 99mTc-radiolabeled CZ and pharmacodynamics studies were done inaddition to ex vivo cytotoxicty. At a drug:Pluronic� P123:Pluronic� L121 ratio of 1:20:20 (PM7), ahigh EE, ZP, Q8h, and a low PDI was achieved. The biodistribution studies revealed that theoptimized formula had significantly higher drug targeting efficiency (DTE¼ 242.3%), drugtargeting index (DTI¼ 144.25), and nose-to-brain direct transport percentage (DTP¼ 99.30%)and a significant prolongation of protection from seizures in comparison to the intranasallyadministered solution with minor histopathological changes. The declared results reveal theability of the developed PM to be a strong potential candidate for the emergency treatmentof SE.

Keywords

Brain, micelles, clonazepam, central compos-ite, intranasal

History

Received 6 June 2016Revised 1 August 2016Accepted 8 August 2016

Introduction

Status epilepticus (SE) represents a medical emergency that is

associated with high morbidity and mortality (Manno, 2003).

It is defined as continuous or intermittent seizures lasting

more than 5 min, without full recovery of consciousness

between seizures (Chen & Wasterlain, 2006). It requires

immediate intervention (Brophy et al., 2012) as the longer the

seizures, the greater the risk of cerebral damage (Macri,

2010). Treatment involves intravenous administration of a

central nervous system (CNS) depressant, namely, of benzo-

diazepine (BDZ) class (Lockey, 2002).

Clonazepam (CZ) is a potent, long-acting nitrobenzodia-

zepine derivative with anticonvulsant, muscle-relaxant, and

anxiolytic properties. It increases the effects of d-aminobu-

tyric acid (GABA) via modulation of the GABA receptor

(Nardi et al., 2013). Furthermore, CZ offers advantages over

other BDZ due to longer duration of action (Rey et al., 1999).

Clinical studies also revealed that clinical symptoms resolved

more completely with CZ (Lockey, 2002).

Oral or intravenous administration of CZ releases the drug

directly into the peripheral circulation that results into both

limited uptake across the blood-brain barrier (BBB) (Vyas

et al., 2006) and distribution to non-targeted sites which leads

to a number of side effects including palpitation, hair loss and

anorexia (Roche, 2009).

In addition, oral or intravenous administration of the drug

to patients suffering acute SE might be impractical or

inconvenient. From one side, intravenous administration

requires a qualified personnel or a near hospital facility.

From the other side, SE may impair the ability of the patient

for swallowing tablets (Anon, 2015). Thus, intranasal drug

delivery would present a competitive pathway for drug

targeting. It protects the drug from first-pass elimination

(Illum, 2003), circumvents the obstacles of BBB via olfactory

region allowing direct delivery to the CNS (Pires et al., 2009).

Moreover, intranasal delivery is considered to be simple,

convenient, and cost-effective (Marx et al., 2015).

CZ has been previously formulated as intranasal mucoad-

hesive microemulsion for brain targeting (Vyas et al., 2006), it

has been formulated also as solid lipid nanoparticles for

parental administration. To our knowledge, CZ has not been

formulated as polymeric micelles (PM) nanocarriers for

intranasal administration. Thus, herein, mixed PM wereAddress for correspondence: Marianne J. Naguib. Email:marian_joseph@hotmail.com

developed and optimized as another potential system for brain

targeting of CZ.

PM are nanoscopic structure formed by amphiphilic block

copolymers composed of hydrophilic and hydrophobic chains

that self-assemble in water, above a certain concentration

named the critical micelle concentration (CMC) (Chiappetta

& Sosnik, 2007). They consist of an inner core of assembled

hydrophobic segments capable of solublizing lipophilic

substances and an outer hydrophilic corona serving as a

stabilizing interface between the hydrophobic core and the

external aqueous environment (Francis et al., 2004).

PM have the advantage of by-passing the P-glycoprotein

(P-gp) efflux since they are transported into the cells via

receptor-mediated endocytosis in contrast to the typical free

drug diffusion (Srivalli & Lakshmi, 2012). P-gp are drug

efflux protein that hinders distribution of many drugs to the

brain, intestine, and multidrug-resistant (MDR) tumors (Amin,

2013). However, such systems have the drawbacks of forma-

tion of aggregates with a large size, which falls outside the

apparent preferred size range for drug delivery systems using

nanoscale particles and lack of stability in aqueous dispersion

leading to phase separation (Oh et al., 2004).

So, the aim of the present study was to formulate and

optimize stable PM for rapid brain targeting of CZ. PM are

expected to provide rapid nose-to-brain delivery with greater

transport and resident of the drug in the brain. This can help

to increase drug efficacy, reduce side effects, and decrease the

dose and dosing frequency. The performance of the prepared

micelles was evaluated in vitro using different criteria, ex vivo

for cytotoxic properties and in vivo in mice using biodistribu-

tion of 99mTc-clonazepam and appropriate pharmacodynam-

ics models.

Materials and methods

Materials

CZ was a kind gift from Amoun Pharmaceuticals (Elabour

city, Egypt), poly(ethylene glycol)-block-poly(propylene

glycol)-block-poly(ethylene glycol) (Pluronic� L121,

Pluronic� P123), acetonitrile and pentylenetetrazole (PTZ)

were procured from Sigma Chemicals Company (St. Louis,

MO). Spectra/Pore� dialysis membrane (12 000–14 000

molecular weight cutoff) was purchased from Spectrum

Laboratories Inc. (Los Angeles, CA). Technetium-99m was

eluted as 99mTcO4� from 99Mo/99mTc generator, Monrol

Company, Kocaeli, Turkey. Ethanol, disodium hydrogen

phosphate, potassium dihydrogen phosphate, and sodium

chloride were from El-Nasr Chemical Co. (Cairo, Egypt).

Experimental design

A three-level three-factor central composite face-centered

design (CCFD) was applied. The independent variables were;

P123 concentration (X1), Pluronics�:drug ratio (X2), and

hydration volume (X3). The levels of each factor were

designated as (�1, 0, +1) and their corresponding actual

values are shown in Table 1. The composition of the 17

formulae of the 33 CCFD is shown in Table 2. Analysis of

variance (ANOVA) was carried out to estimate the signifi-

cance of model and terms. Probability p values (p50.05)

denoted significance.

Preparation of CZ-loaded PM

PM were prepared adopting thin film hydration (TFH)

technique (Dua et al., 2012). In brief, CZ (10 mg) and

mixture of Pluronics� (L121 and P123) – predetermined

weights – were accurately weighed and dissolved in aceto-

nitrile (10 ml) in a one liter round-bottomed flask.

Acetonitrile was slowly evaporated under vacuum at 50 �Cusing rotary evaporator (Heidolph VV 2000, Burladingen,

Germany) at 90 rpm such that a thin dry film of the

components was formed on the inner wall of the flask. The

dried thin film was hydrated with the designed amount of

distilled water (Table 2) by rotating the flask in water bath at

30 �C using rotary evaporator at 210 rpm for 1 h under normal

pressure. To increase the stability of the formed PM, the

obtained dispersion was sonicated for 1 min in a bath

sonicator (Crest Ultrasonic Corp., Trenton, NJ).

In vitro evaluation of the formulated PM

Determination of entrapment efficiency

Ethanol was selected as an appropriate solvent for the lysis of

the prepared PM (Ryu et al., 2000). Total drug content

(free + entrapped) of the prepared formulae was determined

Table 2. Factors’ levels for the 33 central composite face-centereddesign (CCFD) used to prepare CZ polymeric micelles.

Factors’ levels

Formula code X1 X2 X3

Factorial pointsPM1 50 20 5PM2 100 20 5PM3 50 40 5PM4 100 40 5PM5 50 20 10PM6 100 20 10PM7 50 40 10PM8 100 40 10

Axial pointsPM9 50 30 7.5PM10 100 30 7.5PM11 75 20 7.5PM12 75 40 7.5PM13 75 30 5PM14 75 30 10

Center pointsPM15 75 30 7.5PM16 75 30 7.5PM17 75 30 7.5

X1: P123 conc., X2: Pluronic�: drug ratio, and X3: hydration volume.

Table 1. Variables in 33 central composite face-centered design (CCFD)for CZa polymeric micelles.

Levels

Factors �1 0 1

X1: P123 conc. 50% 75% 100%X2: Pluronics:drug ratiob 20:1 30:1 40:1X3: Hydration volume 5 7.5 10

aWeight of CZ¼ 10 mg.bMixture of Pluronics� (P123 and L121) according to X1 levels.

3682 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

by dissolving PM (0.5 ml) in ethanol and then measuring the

UV absorbance using spectrophotometer (Shimadzu, model

UV-1601 PC, Kyoto, Japan) at the predetermined lmax of CZ

in ethanol (309 nm) (Patel et al., 2012). In order to measure

EE%, the PM suspension was filtered through 0.2 mm

millipore filter as to remove unentrapped drug (Wiens et al.,

2004). 0.5 ml of the separated PM were disrupted by

sonication with ethanol and the concentration of the entrapped

drug was measured spectrophotometrically at the same lmax.

The EE% was calculated using the following formula

(Equation 1):

EE % ¼ amount CZ entrapped ðmgÞtotal amount of CZ mgð Þ � 100 ð1Þ

The measurements were done in triplicates and the mean

values ± standard deviation (SD) were calculated.

Determination of particle size (PS), polydispersity index

(PDI), and zeta potential (ZP)

The mean PS, PDI, and ZP were determined by Zetasizer

Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) at

25 �C. For determining PS, zetasizer system measures the

Brownian motion of the particles in the sample using

dynamic light scattering (DLS). As for ZP, it is measured

using a combination of electrophoresis and laser Doppler

velocimetry techniques. These techniques measure how fast

a particle moves in a liquid when an electrical field is

applied – i.e. its velocity. The formulations were properly

diluted with distilled water to have a suitable scattering

intensity (Abdelbary & Tadros, 2013). The results were

recorded in triplicates.

In vitro release

The CZ release from the developed PM was assessed in

triplicates using the membrane diffusion technique (Samia

et al., 2012). Calculated volume of the filtered PM

containing 1 mg of the drug according to the predetermined

EE, was placed in a dialysis bag (soaked overnight). The

bag was then immersed in 50 ml of the release medium in

amber colored bottles (due to light sensitivity of the drug)

(Shaji & Aditi, n.d.). Because of very limited solubility of

the drug, the release medium consisted of ethanol/water

mixture in the ratio of 1:1 (Sharma et al., 2014). The

bottles were then placed in a thermostatically controlled

shaking water bath operating at 100 shake per minute and a

temperature of 37 ± 0.5 �C (Yang et al., 2013). About 3 ml

of the release medium were withdrawn at predetermined

time intervals (0.25, 0.5, 1, 2, 4, 6, 8 h) and immediately

replaced by an equal volume of fresh release medium.

Percentage of drug released was calculated and plotted

versus time. The release of the drug solution was done

simultaneously. The drug release profiles were fitted to

zero, first, and Higuchi diffusion models (Higuchi, 1963).

The model with the highest coefficient of determination

(R2) was considered the best fitting. The time required for

the release of 50% of the loaded drug (t50%) was

calculated and checked statistically.

Selection of the optimized PM formula

Desirability was calculated using Design-Expert� software

(Version 7, Stat-Ease Inc., Minneapolis, MN) and considered

to optimize the studied responses depending on the provided

results. The significant responses were taken into consider-

ations while the non-significant factors were not. The PM

formula with the highest desirability value (close to 1) was

taken for further investigation.

Differential scanning calorimetry (DSC)

Samples of pure drug, components (Pluronic� P123 and

Pluronic� L121) and drug-loaded PM (PM7) were heated in

an aluminum pan at a rate of 5 �C/min in an atmosphere of

nitrogen to 400 �C and the thermograms were recorded

(Salama et al., 2012).

Transmission electron microscopy (TEM)

One drop of the optimized formula (PM7) was placed on a

copper grid and the excess was removed using a filter paper

and left to dry at room temperature. Then, one drop of

phosphotungstic acid aqueous solution (2%, w/v, negative

staining) was added and the excess was similarly removed and

similarly dried. Finally, the grid was examined under a

transmission electron microscope (Jeol JEM 2100, Tokyo,

Japan).

Effect of storage

The investigated formula (PM7) was assessed following

storage at controlled room temperature (25 �C ± 2) over 4

weeks (Han et al., 2009). At the end of the storage period, the

PM were evaluated with respect to their appearance, EE%,

PS, and Q8 h. Statistical analysis of the obtained results was

performed by Student’s t-test using SPSS 17.0� software

(SPSS Inc., Chicago, IL). Difference at p50.05 was

considered significant. The release profile of the stored PM

was compared to that of the freshly prepared ones according

to the model independent mathematical approach of Moore &

Flanner (1996). The similarity factor (f2) was calculated

according to the following equation (Equation 2):

f2 ¼ 0 log 1 þ 1

n

� � Xn

t¼1

ðRt � TtÞ2" #�0:5

� 100

8<:

9=; ð2Þ

where n is the number of sampling points, Rt and Tt are the

mean percent released from reference (fresh) and from test

(stored) at time t. An f2 value � 50 indicates that the release

profiles are similar, whereas smaller values may imply

dissimilar release profiles.

Ex vivo assessment of nasal cytotoxicity

Histopathological analysis of isolated sheep mucosa treated

with CZ-loaded PM of choice was done to assess the possible

local cytotoxic effects of the developed formula.

Isolation of sheep nasal mucosa

The head of a 1.3-year-old sheep weighing 55 kg was obtained

from the local slaughter house (Cairo, Egypt), within 10 min

DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3683

of the sacrifice. The nasal cavity was exposed with a

longitudinal excision through the lateral wall of the nose

without damaging the septum (Vandekerckhove et al., 2009).

The mucous membrane was then carefully removed and

immediately washed and immersed in ice-cold Ringer’s

solution (Du et al., 2006).

Application of PM and controls

Three segments from each of the anterior and posterior

sections of the nasal mucosa were separated, each segment

was then excised into three pieces. The parts were randomly

distributed into three groups so that each group contains equal

number of anterior and posterior segments. Group 1 was

treated with pH 6.4 phosphate-buffered saline (PBS) (negative

control) (Jagtap et al., 2015), Group 2 received isopropyl

alcohol (positive control) (Kumar et al., 2009), and Group 3

was exposed to CZ-loaded PM. All the three groups received

equal volumes (2 ml) of the treatment. After 2 h, the pieces

were washed with distilled water and preserved in 10%

formalin in saline solution (Al-Saraj, 2010).

Histopathological studies

Sections of 5 mm were stained with hematoxylin and eosin and

then examined using light microscope (Chen et al., 2014)

(National model 138, China).

Biodistribution in mice

Radiolabelling of CZ

Direct labeling method was used to prepare 99mTc-CZ under

reductive conditions in the presence of sodium dithionite

(Na2S2O4) as a reducing agent (Geskovski et al., 2013). In a

10 ml amber colored penicillin vial, 1.2 ml of CZ solution in

absolute ethanol containing 0.3–3 mg of CZ was placed.

Then, 1 ml of freshly prepared Na2S2O4 solution in distilled

water (containing 10–100 mg of Na2S2O4) was added fol-

lowed by 100 ml of freshly eluted 99mTc (7.2 MBq) then the

pH was adjusted using different volumes of 0.1 M HCl and/or

0.1 M NaOH solutions. The reaction mixture was shaken by

electrical vortex and left at ambient temperature for

predetermined time intervals before calculating the radio-

chemical yield.

Different factors that affect the radiolabeling process

(sodium dithionite amount, CZ amount, reaction pH, tem-

perature, and time) were studied and optimized to obtain the

highest radiochemical yield. Experiments studying each factor

were done in triplicate. Differences in the data were evaluated

with one-way ANOVA test. The level of significance was set

at p50.05. Results for p are reported and all the results are

given as mean ± standard deviation.

Radiochemical yield assessment

The radiochemical yield and the in vitro stability of 99mTc-CZ

complex were assessed by paper chromatography (PC) and

thin layer chromatography (TLC).

Acetone was used as a mobile solvent to evaluate the

percent of free 99mTcO4� while the reduced hydrolyzed

99mTcO2 was determined using ethanol:water:ammonium

hydroxide mixture (2:5:1, v/v/v) (Sakr et al., 2013; Essa

et al. 2015).

Preparation of radiolabelled CZ-loaded PM

The radiolabeled CZ (99mTc-CZ) was used to prepare PM

formula of choice (radiolabeled PM7) via TFH technique

adopting the aforementioned procedures with slight modifi-

cation, namely, the radiolabeled drug (99mTc-CZ) was added

to the water of hydration. This is due to the interaction of the

organic solvent (acetonitrile) with the chemicals used in drug

radiolabeling.

Animal study

The protocol of the study (code: PI 1114) was reviewed and

approved by Research Ethics Committee-Faculty of

Pharmacy, Cairo University (REC-FOPCU) in Egypt.

The studies were carried out using male Swiss albino mice

(20–25 g). The animals were housed under constant environ-

mental (room temperature 25 ± 0.5 �C relative humidity; 65%

with a 12 h on/off light schedule) and nutritional (fed with

standard mice diet with free access to water) conditions

throughout the experimental period. On the study day, the

mice were divided into 3 groups (18 mice per group). The

conscious animals were administered intranasal (I.N) 99mTc-

CZ solution (Group A), intranasal (I.N) 99mTc-PM7 formula

(Group B), and intravenous (I.V) 99mTc-PM7 formula (Group

C) at a CZ dose equivalent to 6 mg/g body weight. For

intranasal administration, the mice were held from the back in

a slanted position. The preparations were administered at the

openings of the nostrils (Abd-Elal et al., 2016) using

micropipette (200ml) fixed with low density polyethylene

tube having 0.1 mm internal diameter at the delivery site. The

procedure was performed gently, allowing the animals to

inhale all the preparation (Salama et al., 2012). For I.V

administration, 99mTc-PM7 was injected through the tail vein

of Group C mice. At different time intervals (0.25, 0.5, 1, 2, 4,

8 h), 3 mice were sacrificed. To calculate the percentage

uptake, all mice tissues and organs are separated and counted

individually for their radioactivity uptake. Since its impos-

sible to separate all muscles, bones and blood of mice, a

sample of each of them is separated and a known factor for

each of them is used to calculate the whole muscle, bone, and

blood radioactivity level. Blood, bone, and muscles were

assumed to be 7, 10, and 40% of the total body weight,

respectively (Motaleb et al., 2012; Rashed et al., 2014).

Subsequently, the brain was dissected, washed with normal

saline, made free from adhering tissue/fluid, and weighed.

The weight of the individual tissue/organ was determined.

The radioactivity of each sample as well as the background

was counted in a well-type NaI (Tl) crystal coupled to SR-7

scaler ratemeter. Percent injected dose per gram (% ID/

gram ± SD) in a population of three mice for each time point

were reported.

The pharmacokinetics parameters of different CZ prepar-

ations were determined for each mice including: maximum

CZ radioactivity uptake % injected dose per gram tissue

(%ID/g) (blood or brain) (Cmax) and time to reach Cmax (Tmax).

The area under the concentration-time curves from zero to 8 h

(AUC0–8 h%ID/g) and area under the curve from zero to

3684 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

infinity (AUC0–1 h%ID/g) were estimated using Kinetica�

2000 software (Innaphase, Philadelphia, PA).

The relative bioavailability of intranasal PM prepared

using 99mTc-CZ in comparison to 99mTc-CZ solution was

calculated adopting the following formula (Serralheiro et al.,

2014) (Equation 3):

Relative bioavailability % ¼ AUCPM0�1ð Þ i:n

AUCsolution0�1ð Þ i:n� 100

ð3Þ

The ability of formula of choice (PM7) for brain targeting

following intranasal administration can be calculated in terms

of drug targeting efficiency (DTE) (Zhao et al., 2007), drug

targeting index (DTI) (Khan et al., 2009), and nose-to-brain

direct transport percentage (DTP) (Zhang et al., 2006).

DTE represents time average partitioning ratio of the drug

between brain and blood and can be calculated using the

following equation (Equation 4):

DTE % ¼ AUCbraini:n

AUCbloodi:n� 100 ð4Þ

DTI values of CZ formulations were obtained from the

following equation (Equation 5):

DTI % ¼ AUCbrain=AUCbloodð Þ i:n

AUCbrain=AUCbloodð Þ i:vð5Þ

where AUCbrain is the area under brain CZ concentration-time

curve from zero to 8 h and AUCblood is the area under blood

CZ concentration-time curve from zero to 8 h.

For the direct nose-to-brain transport (DTP) which repre-

sents the percentage of drug directly transported to the brain

through the olfactory and trigeminal neural pathway, the

following equation is used (Equation 6):

DTP % ¼ Bi:n � Bx

Bi:n� 100 ð6Þ

where Bi.n is the total brain AUC(0–8) following intranasal

administration and Bx is a fraction of the brain AUC(0–8)

contributed by the systemic circulation through the BBB

following the intranasal administration and was calculated

according to Equation (7):

Bx ¼Bi:v

Pi:v� Pi:n ð7Þ

where Bi.v is the brain AUC(0–8) following intravenous

administration, Pi.v is the blood AUC(0–8) following intraven-

ous administration and Pi.n is the blood AUC(0–8) following

intranasal administration.

Pharmacodynamics study

The pharmacodynamics study was conducted according to the

protocol described by Florence et al. (2011). Male Swiss

albino mice weighing from 25 to 35 g were allocated

randomly to four different groups (n¼ 60). Convulsions

were introduced by intraperitoneal injection of PTZ

(100mg/g of body weight) (Jelenkovic et al., 2008). Animals

were treated with normal saline administered intransally as

negative control and with different CZ preparations, namely,

CZ solution intranasally (CZSi.n), CZ solution intravenously

(CZSi.v), and CZ intranasal PM (PM7) in a dose of 4 mg/g of

body weight (Cote et al., 2013). CZ treatments were

administered 15, 30, and 45 min before i.p injection of PTZ.

The time required for the onset of seizures from the time of

injection of PTZ was recorded and taken as the evaluation

parameter. Statistical analysis of the obtained results was

performed by ANOVA followed by post-hoc test using SPSS

17.0� software (Chicago, IL).

Results and discussion

Presence of Pluronics� with different hydrophilic–lipophilic

balance (HLB) could help in achieving the optimum thermo-

dynamic and kinetic stabilities for the formed micelles. It was

assumed that low HLB Pluronics� would increase the

thermodynamic stability of the micelles due to the tight

hydrophobic interactions with propylene oxide blocks (Dutra

et al., 2015). On the other hand, the high HLB Pluronics�

would increase the kinetic stability of the micelles due to the

steric hindrance that minimize micelle aggregation (Lee et al.,

2011)

The 17 developed formulae were successfully prepared

using TFH technique adopting a CCFD. This design requires

much fewer experiments than a full-factorial design.

Generation and evaluation of the experimental design was

carried out using the Design-Expert� software. The design

consisted of 8 factorial points, 6 axial points, and 3 center

points, giving a total of 17 formulae. The factorial points help

in estimating the linear terms and two factor interactions, the

axial points help in estimating the quadratic terms, and the

center points were repeated three times to estimate the pure

experimental uncertainty at the factor levels (Aboelwafa &

Makhlouf, 2012).

The results of the measured responses are given under the

following headings:

In vitro evaluation

Entrapment efficiency

The entrapment efficiency ranged from 12.7% (PM11) to

85.62% (PM7) (Table 3). The statistical analysis revealed that

the three investigated factors can significantly (p50.0001)

affect the ability of the drug to be incorporated in the PM

formed .The reduced equation, after omitting the non-

significant model terms, in terms of coded variables, was as

follows:

EE% ¼ 21:96� 14:52X1 þ 17:26X2 þ 5:12X3

� 5:14X1X2 þ 2:85X2X3 þ 11:89X21 þ 6:25X2

2

For X1 (P123 conc.) it was found that increasing P123

conc. would lead to a decrease in the entrapment ability of the

drug due to its low lipophilicity (HLB ¼ 8) compared to L121

(HLB¼ 1). This can be explained as follows, the increase in

L121 conc. was associated with the decrease of P123 conc.

Abundance of L121 provides higher lipophilicity due to an

HLB value of 1 (Batrakova et al., 2003) which provides a

favorable medium for the incorporation of the water insoluble

molecule of CZ (El-Dahmy et al., 2014). Similar results were

obtained by Xu et al. (2012) who observed the increase in

DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3685

folate loading after incorporation of L121 in the formulated

mixed micelles. As for X2 (Pluronics�:drug ratio), it was

observed that elevation of Pluronic� amount resulted in

higher drug incorporation. This can be explained on the basis

that increasing Pluronics� ratio would result in subsequent

increase in the presentation of the triblock copolymer L121

amount having longer hydrophobic segments favoring drug

interaction. All this in addition to the possible hydrogen bond

formed between the drug molecule and the Pluronics� that

increases with the increase of their amount (Kim et al., 2010).

Regarding X3 (hydration volume): increasing water

volume was found to significantly increase entrapment.

Enough water molecules must exist in the Pluronic� –

water mixture to bind all EO segments. Increasing the water

content higher than the amount of water needed to bind EO

segments will swell only the shrunk-bulky EO blocks (the PO

blocks remain anhydrous) (Kunieda et al., 2001). Swelling of

the EO blocks gives an increased interface area per EO block

which would alter the interface curvature (Svensson et al.,

2000), so that the Pluronic� micelle aggregates shapes

generally appear as spheres entrapping more drug. Adequate

precision was calculated by the Design-Expert� software to

demonstrate the signal to noise ratio to ensure that the model

could be applied to navigate the design space, whereas a ratio

greater than 4 is desirable (de Lima et al., 2011). Also,

predicted R2 was calculated as a measure of how good the

model could predict a response value by comparing

the calculated value with the adjusted R2 (Annadurai et al.,

2008). Adequate precision was 35.398 with reasonable

difference between the predicted R2 (0.9384) and the adjusted

R2 (0.9817).

Particle size (PS), polydispersity index (PDI), and zeta

potential (ZP)

The mean PS of the prepared PM formulae ranged from

83.77 nm (PM3) to 132.7 nm (PM6) (Table 3). Polynomial

analysis using quadratic model revealed the absence of

statistical significant between the studied variables. This is

expected in case of PS analysis due to separation of the

formed PM using 0.2 mm millipore filter extruding all

particles above 200 nm. Also it is worth noticing that the

difference between the highest PS and the lowest PS is only

48.93 nm.

Concerning PDI, the values obtained ranged between 0.19

and 0.45 (Table 3) which could be within the acceptable range

(Cho et al., 2014). Interestingly, polynomial statistical ana-

lysis using quadratic model revealed that two of the

investigated factors (X1 and X3) can significantly affect PDI

values.

The reduced equation, after omitting the non-significant

model terms, in terms of coded variables, was as follows:

PDI ¼ 0:29þ 0:056X1 � 0:046X3

Knowing that the Mwt of P123 is 5800 (Sang & Coppens,

2011) and that of L121 is 4400 (BASF, 2004), increasing

P123 conc. (X1) was associated with increase in the average

Mwt of Pluronics� mixture resulting in less kinetically

restricted encapsulation process of the drug on Pluronic�

surface so a less uniform distribution of PS (higher PDI) was

obtained (Abdelbary et al., 2015). As for hydration volume

(X3), increased levels of phase volume ratio and water volume

decreased the PDI. This might be explained by the formation

of more nucleation sites per unit volume of the antisolvent.

Hence, less drug molecules precipitated per nucleation site

and a more uniform distribution for the PS was obtained

resulting in lower PDI (Aghajani et al., 2012).

ZP can be considered as an important indicator of physical

stability of nanodispersions (Heurtault et al., 2003). A higher

electric charge on the surface of the nanoparticles will prevent

aggregation because of the strong repellent forces among

particles giving more stable dispersions (Honary & Zahir,

2013). Generally, ZP values above 20 mV indicate that

nanosuspensions are well dispersed with considerable stabil-

ity (Hornig et al., 2009). Results of ZP are compiled in Table

3, it ranged from �7.12 mV (PM2) to �28 mV (PM15)

indicating that some formulae had better stability (higher than

20 mV) than others. Quadratic model analysis of the measured

values showed that none of the studied variables (p40.05)

could significantly affect the ZP of PM.

Table 3. The measured responses of the central composite face-centered design (CCFD) of CZ polymeric micelles(mean ± SD, n¼ 3).

Formula code Y1 ¼ EE (%) PS (nm) Y2 ¼ PDI ZP (mV) Y3 ¼ Q8h (%) Y4 ¼ t50% (h)

PM1 25.40 ± 2.39 113.25 ± 1.13 0.20 ± 0.02 �20.90 ± 3.15 96.82 ± 3.80 3.14 ± 0.18PM2 13.21 ± 3.28 87.34 ± 2.31 0.34 ± 0.01 �7.12 ± 2.21 67.12 ± 1.75 5.58 ± 0.27PM3 66.87 ± 1.36 83.77 ± 2.18 0.28 ± 0.05 �23.30 ± 2.08 80.90 ± 1.98 4.24 ± 0.06PM4 31.01 ± 4.03 95.40 ± 0.19 0.41 ± 0.03 �15.70 ± 1.28 64.53 ± 4.06 5.75 ± 0.80PM5 35.87 ± 1.82 88.67 ± 1.14 0.20 ± 0.01 �20.55 ± 2.56 74.28 ± 3.13 3.78 ± 0.20PM6 13.46 ± 1.92 132.70 ± 4.23 0.19 ± 0.07 �18.25 ± 3.15 70.97 ± 0.37 3.72 ± 0.03PM7 85.62 ± 0.63 124.15 ± 5.56 0.19 ± 0.05 �24.60 ± 4.25 97.21 ± 2.04 2.21 ± 0.22PM8 45.75 ± 0.27 95.75 ± 4.56 0.30 ± 0.02 �19.85 ± 1.22 86.54 ± 1.70 3.71 ± 1.09PM9 51.42 ± 4.25 102.40 ± 2.22 0.26 ± 0.01 �18.95 ± 0.98 86.09 ± 1.34 3.70 ± 0.99PM10 16.55 ± 1.84 125 ± 5.18 0.45 ± 0.07 �15.70 ± 1.28 70.90 ± 0.28 6.35 ± 0.08PM11 12.70 ± 2.56 106.07 ± 3.47 0.23 ± 0.05 �17.95 ± 0.67 72.90 ± 0.93 4.58 ± 0.04PM12 43.99 ± 3.47 91.24 ± 4.5 0.26 ± 0.01 �15.45 ± 1.15 89.85 ± 0.35 2.81 ± 0.90PM13 18.19 ± 1.02 93.74 ± 5.13 0.31 ± 0.08 �17.80 ± 3.55 89.34 ± 2.06 2.85 ± 0.09PM14 25.1 ± 2.73 102.69 ± 5.33 0.19 ± 0.04 �19.25 ± 1.98 68.11 ± 0.46 5.10 ± 1.37PM15 22.2 ± 1.15 112.70 ± 2.23 0.25 ± 0.03 �28.00 ± 1.08 73.13 ± 0.86 4.28 ± 0.18PM16 18.8 ± 2.34 119.90 ± 1.23 0.36 ± 0.02 �25.80 ± 2.66 74.99 ± 1.39 4.81 ± 0.44PM17 24.2 ± 2.18 116.30 ± 4.56 0.28 ± 0.02 �26.90 ± 2.50 73.69 ± 0.92 4.50 ± 0.41

3686 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

In vitro release

Release of CZ from PM was done in ethanol in water (1:1).

This is due to the very limited solubility of CZ in water,

(saturated solubility in water &0.00522 mg/ml) (Patel &

Purohit, 2009). In vitro cumulative release profiles of the drug

from different formulations are shown in Figure 1. CZ release

from drug solution was investigated as control. It reached

&100% within 3 h, this suggested that the drug could freely

diffuse through dialysis membrane (Wei et al., 2009).

Regarding ANOVA analysis of the amount released after 8 h

(Q8h) and time required for release of 50% of the drug

(t50%), two factors interaction model was adopted. The

results revealed that X1 ¼ P123 conc. had a statistical

Figure 1. In vitro CZ release profiles frominvestigated polymeric micelle and the drugsolution in ethanol:water (1:1) at 37 ± 0.5 �C,mean ± SD, n¼ 3.

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8

Cum

ula�

ve c

lona

zepa

m re

leas

d (m

g %

)

Time (hr)

Drug PM1 PM2 PM3 PM4 PM5 PM6

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8

Cum

ula�

ve c

lona

zepa

m re

leas

d (m

g %

)

Time (hr)

Drug PM7 PM8 PM9 PM10 PM11 PM12

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7 8

Cum

ula�

ve c

lona

zepa

m re

leas

d (m

g %

)

Time (hr)

Drug PM13 PM14 PM15 PM16 PM17

DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3687

significant change on the measured variables. The reduced

equations, after omitting the non-significant model terms, in

terms of coded variables, for Q8h and t50% were: Q8h ¼78.71–7.52X1 and t50% ¼ 4.18 + 0.8X1, respectively.

Increasing P123 conc. lead to decrease in release rate of

the drug. This could be explained on the basis that P123 has a

higher molecular weight in comparison to L121 which means

more abundance of O and OH points that enhance attachment

to the drug molecule via hydrogen bonds leading to slower

release rate (Tang et al., 2012). It worth noticing that the PM

exhibited biphasic release. This included an initial burst

release of the drug located in the shell or at the core–shell

interface, followed by a slow release phase corresponding to

the diffusion of the drug from the core (Torchilin & Amiji,

2010). This indicates that the PM could not only solubilize the

poorly soluble drug (CZ), but also sustained its release.

The in vitro drug release profiles of the investigated PM

could be best fitted to Higuchi-diffusion model (highest R2,

Table 4). This is in line with the results reported earlier by

Gaber et al. (2006) who formulated beclomethasone dipro-

pionate as PM intended for pulmonary delivery.

Selection of the optimized PM formula

Desirability was estimated to predict the composition of the

formula of choice by maximizing EE and Q8h and

minimizing PDI and t50%. PS and ZP were not taken into

consideration as the results showed statistical insignificant

differences (Nour et al., 2015). The highest desirability value

obtained was 0.921 and it was associated with the independ-

ent variables, namely, X1¼50%, X2¼40, and X3¼10

corresponding to formula PM7. Consequently, this formula

was selected for further investigation.

Differential scanning calorimetry

The DSC study was done for CZ, Pluronics� (P123, L121)

and for CZ-loaded polymeric micelle candidate formula

(PM7).

Figure 2 shows the DSC thermogram of CZ with a sharp

characteristic endothermic peak at 238 �C (Roni et al., 2011)

indicating its crystalline state. Concerning thermograms of

P123 and L121, small endothermic peaks were detected at

39.4 �C and 120.35 �C, respectively, indicating their boiling

points. Regarding the DSC thermogram of CZ-loaded

formula, a very small peak was observed at 182.47 �Cindicating a micellization endotherm. This is in accordance

with Juggernauth et al. (2011) working on encapsulation of

laponite in nanoparticles containing Pluronic� F127. On the

other hand, the disappearance of the characteristic endother-

mic peak of CZ indicates the entrapment of the drug in the

developed PM (Leyva-Gomez et al., 2014).

Transmission electron microscopy (TEM)

Photomicrographs of CZ-loaded PM (PM7) are illustrated in

Figure 3. It is clear that the developed micelles were fairly

dispersed in aqueous medium (Figure 3a) and formed

homogeneous small-sized spherical micellar structures with

a smooth surface. A closer look on the photomicrograph

(Figure 3b) would show a perfect spherical shape of the

formed PM.

Characteristics of stored PM

There was no observed aggregation or change in the

appearance of CZ PM (PM7) after storage at controlled

Figure 2. DSC thermograms of CZ,Pluronic� P123, Pluronic� L121, and theoptimized polymeric micelle (PM7).

−6

−4

−2

0

2

4

6

8

10

0 50 100 150 200 250 300 350 400

mW

Temperature (°C)

CZ P123 L121 PM7

Table 4. Fitting CZ release to zero, first, and Higuchi diffusion models.

Correlation coefficient

Formulacode

Zeroorder

Firstorder

Higuchidiffusion

Best fittingmodel

PM1 0.832 0.733 0.941 Higuchi diffusionPM2 0.885 0.792 0.968PM3 0.723 0.584 0.862PM4 0.904 0.771 0.977PM5 0.946 0.820 0.994PM6 0.907 0.773 0.981PM7 0.799 0.634 0.920PM8 0.684 0.560 0.831PM9 0.796 0.650 0.903PM10 0.879 0.753 0.961PM11 0.873 0.711 0.956PM12 0.823 0.684 0.929PM13 0.790 0.657 0.914PM14 0.872 0.757 0.960PM15 0.913 0.758 0.981PM16 0.928 0.636 0.937PM17 0.926 0.789 0.988

3688 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

room temperature for 4 weeks. Such findings are in harmony

with that obtained by Oh et al. (2004) who found that

Pluronics� L121/F127 mixtures (in ratio, 1:1 w/w) formed

stable dispersions with small PS. In the present study, the

recorded EE, PS, and Q8h for the stored PM7 formula were

82.66% ± 2.18, 131.5 nm ± 5.78, and 98.6% ± 0.15, respect-

ively. The respective values for the freshly prepared PM7

were 85.62% ± 0.63, 124.15 nm ± 5.56 ,and 97.21% ± 2.04.

Statistical analysis revealed that there was no significant

difference (p40.05) in the measured variables of the stored

PM when compared to the freshly prepared ones. Calculating

similarity factor produced a value of 66.70 indicating that the

storage at the specified conditions had no marked effect on

the release of the drug (Han et al., 2009).

Nasal toxicity

The local toxicity effect of the candidate PM was examined

on sheep nasal mucosa in both anterior and posterior regions

in comparison to pH 6.4 PBS (negative control) and isopropyl

alcohol (positive control). The results are illustrated in

Figures 4 and 5.

As depicted in Figure 4(a), nasal mucosa treated with PBS,

revealed no change in the histological structures with normal

stratified squamous epithelium and intact underlying con-

nective tissue containing sebaceous glands and hair follicles.

Upon exposure to isopropyl alcohol (Figure 4b), sloughing of

the epidermal lining with disfiguration of the underlying

tissue was observed. Applying formula PM7 to the anterior

part of the nasal mucosa showed no change with normal

epidermis, dermis, and connective tissue (Figure 4c).

Examining the posterior part, treated with pH 6.4 PBS as a

negative control, revealed normal pseudostratified columnar

epithelium with submucosa, submucosal glands, and cartil-

aginous layer (Figure 5a). On exposure to isopropyl alcohol,

sloughing of the epithelium was noticed with complete

distortion of the submucosal layer (Figure 5b). On the other

hand, with PM7 minor thinning of the epithelium was noticed

(Figure 5c). This results are in line with Kolsure & Rajkapoor

(2012) who formulated zolmitriptan in nanomicellare carrier

using Pluronic� F127 and pluronic� F68, histopathological

studies revealed the absence of significant effect on the

microscopic structure of mucosa as the surface epithelium

lining and the granular cellular structure of the nasal mucosa

were totally intact.

Radiolabeling of CZ

The highest radiochemical yield of 99mTc-CZ was

94.3 ± 0.25%. Such maximum yield was obtained using

2 mg CZ and 50 mg sodium dithionite. Radiolabeling reaction

was done at ambient temperature (27 ± 3 �C) for 30-min

reaction time at pH 5 (Figure 6a–e). 99mTc-CZ complex

showed good in vitro stability up to 24 h.

Biodistribution study

The ability and extent of an intranasal formula to deliver the

drug to the brain can be mathematically calculated using

different parameters, namely, (i) relative bioavailability, (ii)

DTE (Zhao et al., 2007), (iii) DTI (Khan et al., 2009), and (iv)

DTP percentage (Zhang et al., 2006).

In the current study, radiolabeled preparations were

administered to mice as follows: (i) intranasal 99mTc-CZ

solution, (ii) intranasal 99mTc-PM7, and (iii) intravenous99mTc-PM7. The radioactivity was determined in blood and

brain at different time intervals up to 8 h.

Figure 7 reveals that CZ conc. in brain of mice receiving

intranasal 99mTc-PM7 was higher than both intranasal 99mTc-

CZ solution and intravenous 99mTc-PM7 (p50.05).

Concerning blood results (Figure 8), intravenous 99mTc-PM7

showed the highest blood accumulation of the drug due to

direct delivery of the drug to the blood, followed by intranasal99mTc-CZ solution and then intranasal 99mTc-PM7. These

differences were proved to be statistically significant

(p50.05).

Brain/blood ratios computed for different radiolabeled

preparations (Table 5) were obtained by dividing brain

reading by blood reading for each mouse at each time

interval. Statistically higher brain/blood ratios of intranasal99mTc-PM7 (p50.05), in comparison to the intranasally

administered solution and to the intravenously administered

PM7 formula, indicates the brain targeting ability of the

optimized PM formula.

The pharmacokinetic behavior of the three administered

preparations were mathematically evaluated by the calcula-

tion of Cmax, Tmax, and AUC0–1 for brain and blood (Table 6).

For the brain, the values were (0.24, 4.28, 0.29) %ID/g, (0.25,

0.25, 0.5) h and (0.32, 2.68, 0.80) h%ID/g for intranasal99mTc-CZ solution, intranasal 99mTc-PM7 and intravenous99mTc-PM7, respectively (Table 6). The significantly higher

Figure 3. TEM photomicrographs of mixedPluronic� L121/P123 polymeric micelles(PM7).

DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3689

Cmax and AUC0–1 values of the intranasal 99mTc-PM7

confirm direct delivery of the drug to the brain in comparison

to the other two administered radiolabeled preparations.

This is further proved by the relative bioavailability (Table 6)

which was found to be 812.96% and 11.83% for brain and

blood, respectively.

Drug administered intranasally can reach brain using

mainly two different pathways: (i) either through reaching

Figure 4. Photomicrographs of the anterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positivecontrol, b), and CZ-loaded polymeric micelles (c) (100�).

Figure 5. Photomicrographs of the posterior segments of sheep nasal mucosa treated with pH 6.4 PBS (negative control, a), isopropyl alcohol (positivecontrol, b), and CZ-loaded polymeric micelles (c) (100�).

3690 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

the systemic circulation then crossing BBB into the brain or

(ii) direct nose-to-brain transport from the nasal mucosa

through the olfactory region and the trigeminal nerve

bypassing the BBB (Illum, 2003). Based on the results

of the biodistribution study, DTE, DTI, and DTP values

were calculated for both intranasal 99mTc-CZ solution and

intranasal 99mTc-PM7 (Table 7). DTE% represents time

average partitioning of drug between brain and blood

(Haque et al., 2014), while DTI is a measure of the

differential targeting between intranasal and intravenous

delivery (Taylor et al., 2010) and DTP% represents the

percent of drug directly transported to the brain by the

olfactory and trigeminal neural pathway (Haque et al., 2014).

Their values were 242.39%, 5.78%, 144.25, 3.46, and 99.3,

70.07 for intranasal 99mTc-PM7 and intranasal 99mTc-CZ

solution, respectively.

Figure 6. Variation of the radiochemical yield of 99mTc-clonazepam as a function of clonazepam amount (a), Na2S2O4 amount (b), pH (c), reactiontemperature, (d) and time (e).

DOI: 10.1080/10717544.2016.1223216 Intranasal brain-targeted CZ polymeric micelles for immediate control of status epilepticus 3691

These results are in accordance with Jain et al. (2010) and

Kanazawa et al. (2011) who found that intranasal PM have a

very high potential for brain targeting of zolmitriptan and

coumarin, respectively.

Pharmacodynamic studies

The ability of the preparations to protect mice from PTZ-

induced seizures after intravenous and intranasal administra-

tions was evaluated to compare the preparations and their

delivery routes (Florence et al., 2011). PTZ was administered

after predefined intervals of 15-, 30-, and 45-min posttreat-

ment with CZ preparations. The onset of seizures in animals

treated with different preparations and routes is shown in

Table 8. The saline-treated control group produced

convulsions with an onset of 58 secs, on average, at the

three time intervals. CZ solution (CZS) was administered

intravenously 15, 30, and 45 min prior to PTZ challenge. It

offered protection against PTZ-induced convulsions by

delaying the onset significantly (p50.05) for 30

(160.6 ± 7.02 s) and 45 min (139.0 ± 8.18 s) treatment group

in comparison with the control group (less than 60 s).

However, intravenous CZS failed to induce significant

protection after 15 min (p40.05).

Although intranasal CZ solution offered prolongation of

the onset of PTZ-induced seizures at all time intervals in

comparison to control groups (Table 8), these differences

were found to be statistically insignificant. This may be due to

the limited ability of the i.n. solution to deliver the drug in

adequate conc. to the brain. On the other hand, the offered

Table 5. Brain/blood distribution of CZ administration as intranasal 99mTc solution, intranasal 99mTc-PM7, and intravenous 99mTc-PM7 in male Swissalbino mice (mean ± SD, n¼ 3).

Time

Formulation/route of administration Organ or tissue 0.25 0.5 1 2 4 8

99mTc-CZ solution/intranasal Brain 0.24 ± 0.01 0.16 ± 0.01 0.12 ± 0.01 0.02 ± 0.00 0.02 ± 0.00 0.00 ± 0.00Blood 1.03 ± 0.27 1.37 ± 0.43 0.95 ± 0.09 0.83 ± 0.08 0.67 ± 0.15 0.44 ± 0.15Brain/blood 0.24 ± 0.05 0.13 ± 0.05 0.13 ± 0.03 0.02 ± 0.00 0.02 ± 0.00 0.00 ± 0.00

99mTc-PM7/intranasal Brain 4.28 ± 0.69 1.35 ± 0.24 0.43 ± 0.11 0.16 ± 0.03 0.13 ± 0.00 0.02 ± 0.00Blood 0.85 ± 0.20 0.63 ± 0.12 0.32 ± 0.05 0.10 ± 0.02 0.06 ± 0.02 0.01 ± 0.01Brain/blood 5.10 ± 0.45 2.14 ± 0.18 1.31 ± 0.13 1.72 ± 0.37 2.04 ± 0.15 1.5 ± 0.45

99mTc-PM7/intravenous Brain 0.24 ± 0.05 0.29 ± 0.08 0.14 ± 0.01 0.10 ± 0.02 0.08 ± 0.01 0.01 ± 0.02Blood 9.62 ± 1.30 8.72 ± 1.29 7.48 ± 0.97 6.42 ± 0.86 5.25 ± 1.33 2.76 ± 0.85Brain/blood 0.02 ± 0.00 0.03 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.00 ± 0.00

Figure 7. CZ concentration in brain atdifferent time intervals following adminis-tration of intranasal 99mTc-CZ solution,intranasal 99mTc-PM7 and intravenous 99mTc-PM7, mean ± SD, n¼ 3, in male Swiss albinomice.

0

1

2

3

4

5

0 1 2 3 4 5 6 7 8

CZ

Con

c. in

Bra

in (

%/g

)

Time (hr)

99mTc-CZ(i.n) 99mTc- PM7 (i.n) 99mTc-PM7(i.v)

Figure 8. CZ concentration in blood atdifferent time intervals following adminis-tration of intranasal 99mTc-CZ solution,intranasal 99mTc-PM7 and intravenous 99mTc-PM7, mean ± SD, n¼ 3, in male Swiss albinomice.

0

2

4

6

8

10

0 1 2 3 4 5 6 7 8

CZ

Con

c. in

Blo

od (

%/g

)

Time (hr)

99mTc-CZ(i.n) 99mTc- PM7 (i.n) 99mTc-PM7(i.v)

3692 S. A. Nour et al. Drug Deliv, 2016; 23(9): 3681–3695

protection produced by intranasal PM7 is significantly higher

(p50.05) than all treatment groups and the control at all time

intervals. It reached 424.33 ± 31.5, 332.33 ± 41.1, and

314.66 ± 24.58 after 15, 30, and 45 min, respectively. This

confirms the ability of the PM to directly deliver the drug to

the brain in high concentration depending on the ability of the

Pluronics� to overcome the P-gp efflux mechanism, in

addition to offering a solubilized form of the drug that

allows its immediate uptake and improved efficacy.

Conclusion

Kinetically and thermodynamically stable PM were success-

fully developed using TFH technique. The ability of the

optimized polymeric micelle formula (PM7) with an accept-

able PS range and ZP, the lowest PDI and the highest EE for

incorporation of the drug was confirmed by TEM and DSC

results. PM7 produced minor histopathological changes

without affecting the integrity of the sheep nasal mucosa. In

addition, the biodistribution and pharmacodynamics studies

demonstrated the rapid and effective brain uptake of CZ in

mice following intranasal administration of the suggested

formula. This may represent an alternative to intravenous

administration in the management of acute SE especially

when oral administration is not feasible or it is clinically not

possible to treat the patient before hospitalization. However,

clinical benefits to risk ratio of the developed formulation

have to be established for its appropriateness in the clinical

practice.

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of this article.

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Table 6. Pharmacokinetics parameters for CZ administration as intranasal 99mTc solution, intranasal 99mTc-PM7, and intravenous 99mTc-PM7 in maleSwiss albino mice (mean ± SD, n¼ 3).

Time

Formulation/route of administration Organ or tissue Cmax (%ID/g) Tmax (h) AUC0–1 (h%ID/g) AUC0–8 (h%ID/g) Relative bioavailabilitya

99mTc CZ solution/intranasal Brain 0.24 ± 0.01 0.25 0.32 ± 0.01 0.31 ± 0.01Blood 1.37 ± 0.43 0.5 10.18 ± 4.05 5.65 ± 1.20

99mTc PM7/intranasal Brain 4.28 ± 0.69 0.25 2.68 ± 0.51 2.62 ± 0.47 812.96 ± 154.6Blood 0.85 ± 0.20 0.25 1.12 ± 0.22 1.08 ± 0.22 11.83 ± 2.88

99mTc PM7/intravenous Brain 0.29 ± 0.08 0.5 0.80 ± 0.33 0.71 ± 0.19Blood 9.62 ± 1.30 0.25 61.15 ± 17.10 42.23 ± 8.48

aRelative bioavailability (RBA) in comparison to I.N CZ solution.

Table 8. Time (s) for the development of seizures in male Swiss albino mice (mean ± SD, n¼ 5).

Formula administered Administration after 15 min Administration after 30 min Administration after 45 min

Control group 59.33 ± 3.6 56 ± 6.02 59.66 ± 2.08CZS(i.n) 98 ± 10.5 84.33 ± 10.53 71.33 ± 9.07CZS(i.v) 62 ± 6.55 160.6 ± 7.02 139 ± 8.18PM7(i.n) 424.33 ± 31.5 332.33 ± 41.10 314.66 ± 24.58

EE: entrapment efficiency; PS: particle size; PDI: polydispersity index; ZP: zeta potential Q8h: amount releasedafter 8 h and t50%: amount for the release of 50% of the drug.

Table 7. The DTE%, DTI%, and DTP% of intranasal 99mTc-CZ solution and intranasal 99mTc-PM7 polymericmicelles relative to the intravenous 99mTc-PM7 in male Swiss albino mice (mean ± SD, n¼ 3).

Formulation/route of administration DTE% DTI DTP%

99mTc-CZ solution/intranasal 5.78 ± 1.01 3.46 ± 0.81 70.07 ± 7.0499mTc-PM7/intranasal 242.39 ± 10.30 144.25 ± 10.22 99.30 ± 0.04

DTE: drug targeting efficiency; DTI: drug targeting index; DTP: nose-to-brain direct transport percentage.

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