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Drug Delivery
ISSN: 1071-7544 (Print) 1521-0464 (Online) Journal homepage: http://www.tandfonline.com/loi/idrd20
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:[email protected]
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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