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Feng, J. Qi, W. Fan, Y. Ma, H. He, F. Xia, X. Dong, W. Zhao, Y. Lu and W. Wu, Nanoscale, 2016, DOI:
10.1039/C6NR07581A.

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DOI: 10.1039/C6NR07581A

Nanoscale

Paper

Evidence of nose-to-brain delivery of nanoemulsions: cargoes but

not vehicles
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Received 00th January 20xx,

Accepted 00th January 20xx Ejaj Ahmada, Yunhai Fengb, Jianping Qia, Wufa Fana, Yuhua Maa, Haisheng Hea, Fei Xiaa,
Xiaochun Donga, Weili Zhaoa,c, Yi Lua,†, Wei Wua,†

Nanoscale Accepted Manuscript

DOI: 10.1039/x0xx00000x

www.rsc.org/ Abstract
The nose-to-brain pathway is proved to be a shortcut for direct drug delivery to the brain.
However, whether and to what extent can nanoparticles be delivered through this passage
are still awaiting validation with evidence. In this study, nose-to-brain transportation of
nanoparticles is tracked by fluorescent bioimaging strategies using nanoemulsions (NEs) as
model carriers. Identification of NEs in biological tissues is based on on→off signal switching
of a new kind of environment-responsive dyes embedded, P2 and P4, whereas two
conventional probes, DiR and coumarin-6 (C6), are embedded to represent the cargoes.
Evidence on translocation of NEs is collected either by live imaging or ex vivo histological
examination in rats after nasal administration. Results suggest that NEs with a particle size of
about 100 nm, either naked or coated with chitosan, show longer retention duration in
nostrils and slower mucociliary clearance than larger ones. P2 signals, representing integral
NEs, can be found in mucosa and trigeminal nerve for all size groups, whereas only weak P2
signals are detected in olfactory bulb for chitosan-coated NEs of 100 nm. Confocal
microscopy further confirms translocation of integral 100-nm NEs in nasal mucosa and along
the trigeminal nerve in decremental intensity. Weak signals of P4 probe, also representing
integral NEs, can be detected in the olfactory bulb but few in the brain. NEs as big as 900 nm
cannot be transported to the olfactory bulb. However, the DiR or C6 signals that represent
the cargoes can be found in intense amount along the nose-to-brain pathway and finally
reach the brain. Evidence shows that integral NEs can be delivered to the olfactory bulb, but
few to the brain, whereas the cargoes can be released and permeate into the brain in more
amount.
is highly challenging owing to the presence of the
Introduction blood-brain barrier (BBB), which precludes entry of
more than 95% of available pharmaceuticals.1 To
The incidence of chronic brain disorders, especially date, various drug delivery strategies via the
age-related neuro-degenerative diseases, is intravenous route fail to achieve significant
increasing at an alarming rate throughout the world. improvement in overcoming BBB obstacles.2 Besides
Effective treatment of the central nervous system trying more efficient “penetrating” approaches such
diseases, such as the Alzheimer’s and the Parkinson’s, as nanoscale delivery systems, it is inspired to explore
alternative pathways. Recently, the nose-to-brain
route attracts great interest due to the short
a.
School of Pharmacy, Fudan University, Key Laboratory of Smart Drug Delivery of transportation passages from the nasal cavity to the
MOE and PLA, Shanghai 201203, China
b.
Department of Otorhinolaryngology Head & Neck Surgery, Dahua Hospital, brain via olfactory and trigeminal nerve,
c.
Shanghai 200237, China circumventing BBB and providing quick transport of
Key Laboratory for Special Functional Materials of the Ministry of Education,
Henan University, Kaifeng 475001, China pharmaceuticals from nose to brain.3 Moreover,
† Corresponding author. E-mail: wuwei@shmu.edu.cn; fd_luyi@fudan.edu.cn intranasal delivery is a non-invasive route with
Electronic Supplementary Information (ESI) available. See
DOI: 10.1039/x0xx00000x improved safety and patient compliance.4

This journal is © The Royal Society of Chemistry 20xx Nanoscale | 1

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Several approaches have been tried to increase
the influx of drugs from nose to brain by using various
nanocarriers such as nanoemulsions,5 liposomes,6
micelles,7 nanosuspensions,8 solid lipid nanoparticles9
and chitosan-coated nanoconstructs.10 All these
formulations, without exception, show high influx of
drugs to the brain in comparison with the solution
counterparts of the drugs. Underlying mechanisms
includes longer retention of the nanoformulations in
nasal mucosa, enhanced permeation through
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mucosal epithelia, prevention from enzymatic
degradation and inhibition of P-gp efflux pump.11,12 A
key issue in nose-to-brain delivery is whether and to
what extent these nanocarriers can be translocated
to the cerebral regions. Elucidation of this issue helps
to weigh the balance between efficacy and toxicity,
and to clarify some of the basic drug delivery
controversies, e.g. active targeting of nanocarriers to
brain tissues. To date, only paradoxical results have
been obtained. The exact mechanisms of facilitated
nose-to-brain delivery by nanocarriers remain as a
secret.
This difficult situation is partly attributed to the
lack of functional approaches to track the
transportation of the nanocarriers. Labeling with
radioactive or fluorescent probes represents one of
the expedient approaches to trace nanocarriers
during translocation from nose to brain. Technetium
(99mTc) has been used to label the encapsulated
drugs5,13 or the nanocarriers per se14,15 to study the in
vivo distribution by Gama scintigraphy. Fluorescent
probes, including DiR, coumarin 6 (C6) and rhodamine
B, were also physically incorporated or covalently
linked to either the drugs or the carriers to track the
translocation of nanocarriers inside the body by live
imaging.7,10,16 These studies provide certain important
information on the in vivo fate of nanocarriers both
spatially and temporally. However, these approaches
experience obvious defects because dissociated
probes still give off signals, which significantly
compromises the accuracy of detection of the
particles. Smart or environment-responsive probes
should be employed to discriminate the nanocarriers
from signals of free probes in vivo.
Recently, our laboratory developed a series of
near-infrared (NIR) fluorescent 4,4′-difluoro-4-bora-
3a,4a-diaza-s-indacene (BODIPY) and aza-BODIPY
dyes, which exhibit sensitive aggregation-caused
quenching (ACQ) effects responding to water.17-20 This
kind of ACQ probes are highly lipophilic and can be
tightly embedded into either lipid or polymeric matrix
nanoparticles. The dyes give off fluorescent signals in
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well dispersed state in the matrix, whereas the
fluorescence quenches immediately upon release
from nanoparticles due to degradation of the matrix.
This on→off signal switching provides accurate and
sensitive signaling on the integrity as well as
biodegradation kinetics of the nanocarriers. In
addition, these dyes bear some specific
characteristics of the BODIPY family, such as intense
absorption, high quantum yields, tunable emissive
wavelengths, superior stability and pH insensitivity,
which enable them to survive the harsh in vivo
environments. Thus, these ACQ probes have been
adopted to study the in vivo fate of nanoparticles in
Nanoscale Accepted Manuscript
the gastrointestinal tract.17-20 Since the stimulus here
is the ubiquitous water, it is theoretically correct that
this kind of ACQ probes can be used to track the
voyage of certain nanocarriers throughout the whole
body.
Nanoemulsions (NEs) are heterogeneous systems
that consist of ultrafine oil-in-water dispersions
stabilized by surfactant molecules.21 Due to the
superior solubilisation capacity, thermodynamic
stability and relatively easy preparation to other
nanocarriers, NEs have been widely explored to
enhance nose-to-brain drug delivery.8,22,23 Previous
results indicated that excipients contained in NEs
might facilitate permeation through nasal epithelia to
brain,24 whereas coating with chitosan (CS) extends
the nasal residence time of NEs, facilitating high influx
of drug from nose to brain.13,25 However, the exact
role that NE particles play in nose-to-brain delivery is
still not fully explored.
In this study, the ACQ probes, P2/P4, are used to
study the fate of NEs via intranasal delivery, while
two conventional probes, DiR/C6, are used to
represent the cargoes. Specific attention is paid to
collecting evidence on nose-to-brain translocation of
NEs. Various crucial factors that affect intranasal
resident time and permeation along the nose-to-brain
path such as particle size, mucoadhesive coating with
chitosan and in situ gelling using thermosensitive
polymers were also investigated simultaneously.
Experimental
Materials
Labrafac® CC (caprylic–capric acid triglycerides) was
purchased from Gattefossé S. A. (Saint-Priest, France).
Solutol® HS15 (a mixture of free polyethylene glycol
660 and polyethylene glycol 660 hydroxystearate)
was provided by BASF (Ludwigshafen, Germany).
Coumarin 6 (C6) was purchased from Sigma-Aldrich
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(St. Louis, USA). DiR iodine was from Fanbo
Biochemicals Co. (Beijing, China). P2 and P4 were
synthesized in our lab; refer to previous publications
for their physicochemical properties.17-20 See Table 1
for the basic properties of the dyes used in this study.
Deionized water was prepared using a Milli-Q
purification system (Millipore, Billerica, MA, USA).
Other chemicals and reagents were of analytical
grade and purchased locally.
In vivo or ex vivo experiments were carried out in
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male SD rats (200 ± 20 g, Shanghai Laboratory Animal
Centre, Shanghai, China). All experiments were
approved by the institutional ethical committee and
performed in complicance with the relevant laws and
institutional guidelines at School of Pharmacy, Fudan
University.
Preparation of NEs
NEs of different particle sizes Crude emulsions
were prepared by a high-shear homogenizer (Scientz
Biotechnology Co., Ltd., China). Briefly, appropriate
amount of dye dissolved in dichloromethane was
added into 5 g Labrafac® WL1349/Labrafac CC (Table
1S). After thorough mixing and evaporation of
dichloromethane, the mixture was dispersed in an
aqueous solution (35 mL) containing 8.5% (w/v)
Solutol® HS15. The crude emulsions were produced
after the mixture was sheared at 10,000 rpm for 30
sec. The crude emulsions were further homogenized
through a high-pressure homogenizer (AH 100 D; ATS
Engineering Inc, Brampton, ON, Canada) to obtain 80
nm NEs (NE-80) at 1000 bar for 3 min and 200 nm
(NE-200) NEs at 150 bar for 2 min, respectively. A
membrane-emulsification apparatus [Senhui
Microsphere Technology (Suzhou) Co., Ltd., China]
was used to prepare 500 nm (NE-500) and 900 nm
(NE-900) NEs. NE-500 were obtained by extruding the
crude emulsions through a membrane tube with a
pore size of 1.2 μm at 1.6 MPa for 4 cycles, followed
by another 0.8-μm one at 1.8 MPa for 3 cycles. As for
preparation of NE-900, the crude emulsions were
extruded through a 1.2-μm membrane tube at 1.2
MPa for 2 cycles.
Chitosan-coated NEs The ingredients and
techniques for the preparation of chitosan-coated
NEs (CS-NE) were almost the same as for NE-80.
However, in order to coat chitosan onto the surfaces
of NEs, sodium dodecyl sulphate (SDS) was added in
the mixture of Labrafac® CC and Solutol® HS15 when
preparing the coarse emulsions to introduce negative
charges onto the surfaces of the NEs (Table 2S). Then,
0.5% (w/v) chitosan solution was used as the aqueous
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phase instead of water to achieve a layer of coating
with chitosan.
A control formulation of NEs dispersed in
chitosan solution (CS+NE-80), but not coated with
chitosan, was prepared following the same
procedures as for CS-NE, except that SDS was not
used when preparing the coarse emulsions.
Thermosensitive in situ gel of NEs Nostril
temperature of rats was reported to be around 35°C.
Therefore, a mixture of poloxamer 407 (P407, 18%, w/v)
and poloxamer 188 (P188, 16%, w/v), with sol↔gel
transition temperature around 30°C,26 was used as the
Nanoscale Accepted Manuscript
thermosensitive in situ gel. NEs dispersed in
thermosensitive gel are encoded as P-NE.
Characterization of NEs
The average size and polydispersity index (PDI) of NEs
were measured at 25°C, in triplicate, using a Malvern
Zetasizer Nano® (Malvern Instruments, Malvern UK)
with a 4 mW He–Ne laser at 633 nm under ambient
temperature. For the measurement, the NEs were
diluted in Milli-Q water beforehand (0.2 mL NEs
dispersed into 0.8 mL water).
Morphology of the NEs was studied using TEM
(Jeol JEM 2100F, Japan). Ten microliter of NEs was
added into 990 µL distilled water (1:100), stained with
2% phosphotungstic acid and applied on carbon-
coated grid for TEM observation.
The fluorescence intensity of NEs was measured
using a well-plate quantification method by IVIS
Spectrum Live Imaging System (PerkinElmer, USA).
Briefly, NEs were diluted in Milli-Q water (0.2 mL
mixed with 1.4 mL water). Then 100 μL of the diluted
NEs was instilled into 96-well plates in triplicate and
was imaged at the maximum excitation/emission
wavelengths (P2: 710/760 nm; P4: 640/680 nm; DiR:
740/800 nm; C6: 460/510 nm). The average radiant
efficiency (ARE) of NEs was determined by a region of
interest (ROI) quantification method.
Administration of NE formulations
Male SD rats were divided into eight groups, three in
each group. The P2-loaded formulations include NE-
80, NE-200, NE-500, NE-900, CS-NE, P-NE and CS+NE.
In addition, DiR-loaded CS-NE was set as a control
group to highlight the transportation of the cargos.
Animals were anaesthetized by intraperitoneal
injection of 700 µL 10% (w/v) chloral hydrate aqueous
solution before intranasal administration. Then,
animals were placed in a supine position, and 10 µL of
the formulations was administered slowly and gently
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through micropipette into one nostril of each rat.
Administration was repeated for 10 times to
administer a total of 100 µL formulation at an interval
of 1 min for each rat.
Live imaging of intranasal retention
After administration, in vivo live imaging of the
animals from both ventral and dorsal side was
acquired at 0.5, 1, 2, 4, 8, 12 and 16 hr using IVIS
Spectrum Live Imaging System. During the whole
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imaging process, animals were narcotized by an on-
line gas anesthetizing system using isoflurane
(Shandong Keyuan Pharmaceutical Co., Ltd, China).
Biodistribution study
Animals in groups of NE-80, CS-NE-108 and NE-900
were sacrificed at 1 hr post administration. Blood was
collected into heparinized tubes. One hundred micro-
liter of blood was added into 96-well black plates for
determination of fluorescent intensity. Vital organs
like heart, liver, spleen, lung, kidney and pancreas
were removed, washed with saline, weighted and
placed into black plates for ex vivo imaging. ARE of
blood and total radiant efficiency (TRE) of organs was
determined by a ROI quantification method.
Ex vivo histological examination
After 1 hr post administration of NE-80, CS-NE-108
and NE-900, animals were sacrificed, and nasal
mucosa, brain and trigeminal nerve were excised.
Brain was removed carefully with olfactory bulb
remaining intact with the brain and avoiding
fluorescent interference due to contamination. Ex
vivo imaging of nasal mucosa, brain and trigeminal
nerves were performed. Furthermore, brain was
sliced into thin sections for ex vivo imaging.
Confocal laser scanning microscopy
Animals of P4/C6-loaded NE-80, CS-NE-108 and NE-
900 groups were sacrificed at 1 hr post administration
and nasal mucosa, brain and trigeminal nerve were
excised and fixed using 4% paraformaldehyde for 24
hr. After air-drying for 1-2 hr under ambient
conditions, the samples were treated with 30%
sucrose solution till they were settled down to the
bottom of tubes. Finally, samples were frozen at −80
°C and sliced into 10-µm sections by Leica CM3050 S
Cryostat. Sections were observed by Zeiss LSM 510
confocal laser scanning microscopy (CLSM) (Carl Zeiss
Inc., Germany). Image display and analysis were
performed using the software provided by the
supplier.
Results and discussion
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In order to elucidate the mechanisms underlying
nose-to-brain delivery of nanoparticles, it is important
to track both the nanoparticles and the payloads
simultaneously. Conventionally, the biodistribution of
drugs or payloads along the nose-to-brain passages
are usually monitored to acquire evidence of
enhanced cerebral drug delivery. However, the in vivo
behaviours of the nanoparticles per se have never
been clarified. Fortunately, by using novel
environment-responsive fluorescent probes
developed in our laboratory, the in vivo fate of
nanoparticles, mainly lipid-based ones, can be
accurately monitored.17-20 Fig. 1A is a schematic
Nanoscale Accepted Manuscript
depiction of the rationale employed in this study. Two
kinds of fluorescent probes are encapsulated
simultaneously into NEs. Signals of environment-
responsive probes, P2 and P4, are used to represent
the NE particles because the probes encapsulated in
NEs are emissive, and free or released probes quench
instantaneously and absolutely upon degradation of
NEs. Signals of conventional probes, C6 and DiR, are
used to represent the payloads or drugs because they
exhibit steady fluorescence whether they are
embedded or not. Based on their wavelengths, either
P2/DiR or P4/C6 pairs are employed in this study for
live imaging or ex vivo imaging, respectively.
Concurrent tracking of the transportation of both NEs
and the payloads provides very important
information on the underlying mechanisms of nose-
to-brain delivery.
The quantity of various dyes incorporated into NEs
is chosed on the basis of our previous works.17-20 The
primary purpose of choosing dye loading levels, as
specified in Table 1S, is to achieve sufficient
fluorescence intensity for both in vitro and in vivo
observations. Moreover, it is also meant to attain
similar levels of fluorescence in the initial
administered doses for parallel comparison in a row.
Yet it is hard to adjust the dye loadings to achieve the
same level of fluorescence. Therefore, we use relative
fluorescence, expressed as the percentage of
fluorescence measured at time points to the originally
administered dose, when carring out comparisons.
Fig. 1B schematically reveals the virtual pathways
of nose-to-brain delivery. In fact, the brain is well
protected from the nasal cavity by the cribriform
plate of ethmoid bone. There are two possible
passages from nose to brain, i.e. the olfactory nerves
that end up at the olfactory bulb and the maxillary
branch of the trigeminal nerve. The olfactory bulb is
adjacent to the front lobe of the brain, while the
trigeminal nerve leads to the brain stem. The goal of
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this study is to track the translocation of both the NE
droplets and the payloads along these nose-to-brain
pathways. Evidence collection is primarily based on
identification of either the NE particles or the
payloads in various tissue samples by live imaging or
ex vivo imaging.
Preparation and characterization of NEs
The size, PDI and zeta potential of the NEs are shown
in Table 3S. The CS-NEs used in this study are of size
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108.5 nm, PDI 0.165 and zeta potential +26 mV,
respectively. The presence of SDS affects zeta
potential, but have no significant effect on the size of
CS-NEs (Table 2S). The size, PDI and zeta potential of
P-NE is found to be 235 nm, 0.143 and -1.17 mV,
respectively. The bulk gelling potential of poloxamer
(P407/188) significantly increases the size from 80 nm
to 235 nm and PDI from 0.11 to 0.143. Surface
morphology of the CS-NEs is analyzed by TEM (Fig.
1S). However, no characteristic evidence of chitosan
coating is seen in the TEM image. The particle size
observed in TEM is in line with that measured by a
Zetasizer.
Intranasal retention
Fig. 2A shows the retention of NEs of different sizes in
the nasal mucosa of SD rats as visualized by IVIS
spectrum live imaging. NEs having a size > 200 nm
show less retention in the nasal mucosa compared to
smaller ones (Fig. 2B). Almost all NEs with a size > 200
nm are cleared by the nasal mucociliary clearance
mechanism after 4 hr post administration, while both
NE-80 and NE-200 show some retention even up to
16 and 12 hr, respectively (Fig. 2B). Interestingly, the
overall trend of NEs retention is found to be size-
dependent in the following order: NE-80 > NE-200 >
NE-500 ≈ NE-900. It is of note that there are big
standard errors for the mucoretention data due to
repeated administration and inter-individual
variations. However, the overall trend suggests that
significant numbers of NE-80 and NE-200 might
penetrate the mucus layer and enter deep into the
nasal mucosa. On the contrary, it seems that less
number of NE-500 and NE-900 are able to penetrate
the mucus layer and reach the surface of nasal
mucosa. Quick regeneration of mucus effectively
removes the adhered NEs from the nasal mucosa,
which thus creates obstacles for their free movement.
An additional constraint concerning nose-to-brain
drug delivery is the natural defense mechanism –
nasal mucociliary clearance. Therefore, it is essential
to localize the formulation on the mucosal layer to
prevent rapid nasal clearance.27 Here we take
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approaches of in situ gelling using thermosensitive
poloxamers or thickening with chitosan, a
bioadhesive polymer, to retain NEs in the nasal
mucosa for longer duration. Higher retention of P-NEs
in the nostril is due to gel formation, while higher
retention of CS-NEs is due to electrostatic interaction
between positively charged chitosan and negatively
charged biomembranes,28 as well as the high viscosity
of the dispersion.29 Fig. 2C,D shows the quantitative
analysis results of live imaging for surface-decorated
NEs. Live imaging shows higher percent retention of
P-NE in the first 2 hr post administration compared to
NE-80, CS+NE and CS-NE-108. However, after 2 hr
Nanoscale Accepted Manuscript
their retention is insignificant and completely
untraceable after 8 hr due to nasal mucociliary
clearance, which suggests that bulk poloxamer gelling
system is somehow effective in retention of NEs in
nostril. On the other hand, retention of NE-80, CS+NE
and CS-NE-108 is observed up to 16 hr. NE-80 show
significantly higher retention at the first 30 min post
administration compared to CS-NE-108 and CS+NE.
However, at 1, 2 and 4 hr post administration
retention of CS-NE-108 becomes significantly higher
than NE-80. Moreover, retention of NE-80 and CS-NE-
108 shows no significant difference after 8 hr post
administration. CS+NEs show lower retention at 30
min compared to NE-80; however, at 1, 2 and 4 hr
post administration CS+NE retention is either
comparable or a little bit higher compared to NE-80.
On the contrary, retention of CS-NE-108 is
significantly higher at all time points compared to
CS+NE. Our findings here comply with results
obtained by other researchers.10,30,31 The high
retention of NE-80 is due to their smaller size, while
even higher retention of CS-NE-108 in comparison
with NE-80 is due to their interaction with negatively
charged membranes. Besides, CS-NE-108 possibly
enters deep into the nasal mucosa and enter through
the paracellular route as well upon opening of the
tight junctions between cells by chitosan 32,33. The
longer retention of CS+NE in nasal mucosa than
naked NEs adds strength to the tight junction-opening
hypothesis.
Biodistribution of NEs
IVIS live imaging of the dorsal side of the brain show
the position of NE-80, CS-NE-108 and NE-900 at
different time points (Fig. 2). A small ring of intense
P2 signal is only prominent at 4 and 8 hr post
administration in NE-80 and CS-NE-108 groups, while
similar patterns, but of less intensity, are observed at
1 and 2 hr for the NE-900 group. Due to the
limitations of live imaging, it is difficult to locate the
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signals to a specific organ or tissue. Therefore, no
information on nose-to-brain delivery can be
obtained by live imaging.
Blood and different vital organs are also analyzed
by ex vivo imaging. No P2 signals are detected in
blood or any vital organ. However, some signals are
observed in the trachea attached to the lungs (images
not shown) in some animals, which might be due to
the direct movement of the particles along with
mucus from anterior to posterior part of the nasal
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cavity and finally to the trachea through the
nasopharynx, rather than absorption via systemic
circulation. There is possibility that some NEs might
enter into the systemic circulation through the blood
vessels that present in the respiratory region of nasal
mucosa; however, the amount must be too small to
be detected by ex vivo imaging.
Ex vivo analysis of translocation
Translocation of integral NEs to nasal mucosa and
trigeminal nerve Fig. 3 shows the ex vivo imaging of
retention of NE-80, CS-NE-108 and NE-900 in the
nasal mucosa and trigeminal nerve of SD rats at 1 hr
post administration. The intensity of P2 signals in the
nasal mucosa is in line with the live imaging results.
Quantification of ex vivo imaging of nasal mucosa
shows retention of NEs in the order of CS-NE-108 >
NE-80 > NE-900 with significant difference between
groups (P < 0.05). Moreover, quantification of P2
signal suggests size dependent translocation via
trigeminal nerves. The translocation of NE-80, CS-NE-
108 and NE-900 groups is found to be 4.4%, 3.5% and
2.7% of the total applied NEs, respectively.
Encapsulation of both P4 and C6 dyes together in
NEs enables simultaneous tracking of both NEs and
the cargoes by CLSM. Intense C6 signal (green) and P4
signal (red) are observed from both olfactory and
respiratory regions in the horizontal as well as vertical
sections of each of NE-80, CS-NE-108 and NE-900
groups (Fig. 4). This observation clearly suggests that
a large number of NEs present in intact form
throughout the nasal mucosa. A little bit higher levels
of both C6 and P4 signals was observed in the
respiratory regions at 1 hr post administration, which
might be ascribed to their direct contact with NEs
administered.
Fig. 5 reveals the presence of intense green C6
signals in NE-80, CS-NE-108 and NE-900 groups in all
three sections taken from the nostril, middle and
brain stem regions of the trigeminal nerve. Presence
of P4 signals in all sections of the trigeminal nerve for
NE-80, CS-NE-108 and NE-900 clearly reinforces ex
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vivo imaging of trigeminal nerve. However, relative
intense P4 signals are found in the trigeminal nerve in
the following order NE-80 > CS-NE-108 > NE-900 due
to their relatively high percentage of translocation
through this route (Fig. 5A). Presence of intense
green signal, irrespective of the groups, in the
trigeminal nerve suggests that C6 might first be
released in the nostril and then permeate into the
trigeminal nerves. Following the passive diffusion
path, released C6 can be quickly transported from the
nostril to the brain via the trigeminal nerve.
Previously, it was reported that intranasally
administered drugs could enter in high concentration
Nanoscale Accepted Manuscript
into the olfactory nerve and trigeminal nerve, and be
transported into the brain.34 Moreover, the
transportation of the drugs to the brain through
these nerves is very fast, within 10 min of
administration.35 However, the presence of very weak
P4 signal in all three sections in NE-80 group indicated
that small amount of NE-80 might be transported in
intact form through this route. While the presence of
relatively intense P4 signals in the trigeminal nerve of
CS-NE-108 group in the nostril and middle sections
ssuggests that some intact CS-NE-108 is also
transported via this route, however, presence of
relatively weak signal in the brain stem region
indicates that transportation of CS-NE-108 may be
relatively slow, possibly due to their larger size and
viscosity due to presence of chitosan as compared to
NE-80. On the other hand, presence of relatively high
levels of P4 signals only in the nostril region in
comparison to the middle and brain stem region in
NE-900 group suggest that due to large size their rate
of translocation from the trigeminal nerve was
significantly slow. This observation also reinforces ex-
vivo imaging of low percentage of NE-900 in
trigeminal nerve in comparison to CS-NE-108 and NE-
80.
Translocation of integral NEs to brain Recently,
several studies performed in rodent model suggested
that NEs enhanced direct nose-to-brain transport of
small molecular drug.10,13 However, no particles
either in the olfactory bulb or the brain could be
found after continuous instillation of chitosan-coated
latex up to 4 consecutive days.31 Further investigation
on polystyrene nanoparticles with particle sizes of 20,
100 and 200 nm, respectively, either naked or
surface-modified with polysorbate 80 or chitosan,
indicated no penetration across excised porcine nasal
mucosa mounted in a vertical Franz diffusion cell.36
To date, no concrete evidence has yet been provided
in support of transport of integral nanocarriers from
nose to brain.
This journal is © The Royal Society of Chemistry 20xx
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Nanoscale
In this study, brains of NE-80, CS-NE-108 and NE-
900 groups are excised for ex vivo imaging from both
dorsal and ventral side. Ex vivo imaging shows very
weak P2 signals mainly in both olfactory bulb and
ventral region for the CS-NE-108 group, while weak
P2 signals are only observed sporadically in the
ventral surface of the brain in some of the NE-80
group animals. Noticeably, no P2 signals are detected
in any part of the brain for the NE-900 group (Fig. 6).
This finding clearly suggests that only minute number
Published on 15 December 2016. Downloaded by Fudan University on 15/12/2016 16:09:50.
of intact NE-80 and CS-NE-108 enter into brain.
Earlier, Huang and Donovan37 studied the transport of
polysterene nanoparticles ranging from 10–500 nm
across excised rabbit nasal respiratory epithelia, and
found that both 10-nm carboxylated and 200-nm
amine-modified PS particles were transported
paracellularly and transcellularly, but in very minute
quantities. Chitosan has been widely documented to
affect the permeability of epithelial membrane
through its interaction with the junctional complexes
between epithelial cells. It can transiently open the
tight junctions and consequently allow a paracellular
passage of materials through the epithelial barrier.
32,33
Presence of CS-NE-108, however in minute
quantity in the olfactory bulb, suggests that a small
fraction of small-size CS-NE-108 might follow the
paracellular route to enter into the olfactory bulb.
Since the average diameter of olfactory axon is about
200 nm (for two-month-old rabbits), particles of
sufficiently small sizes may translocate from olfactory
epithelia to olfactory bulb or brain.31,38 It is possible
that some NE-80 and CS-NE-108 reach the olfactory
bulb through this axon. However, strong P2 signals
are detected mainly at cribriform plate, suggesting
that a good number of NE-80 and CS-NE-108 reach
the cribriform plate, but only a small fraction into the
olfactory bulb, which clearly contradicts earlier
findings of localization of NEs payloads to the
olfactory bulb. It is implied that the translocation is
not due to translocation of the nanocarriers
themselves, but the diffusion of the model drug/dye
after release from the nanocarrier matrices.13-15
Moreover, some of the released drugs/dyes may
enter into the brain via trigeminal nerves as well.34 In
general, only a very small percentage of applied NE-
80 and CS-NE-108 enter into olfactory bulb through
olfactory nerve in intact form. Slices of the brain
tissues are further scrutinized and only faint P2
signals are found in limited sections of the brain in
NE-80, CS-NE-108 and NE-900 group (Fig. 6).
To obtain more supportive data, CLSM of the brain
tissue is further performed to strengthen the
evidence of ex vivo imaging. The P4 signals provide
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DOI: 10.1039/C6NR07581A
clear evidence on whether intact NEs enter into the
brain, whereas green C6 signals speak the traces of
the cargoes. CLSM images show intense green signal
from sections taken from different brain regions of
NE-80 and CS-NE-108 groups, but weak signals for NE-
900 (Fig. 7). Very faint P4 signals are observed only in
NE-80 and CS-NE-108 groups in the olfactory bulb
section only (Fig. 7). It suggested that only NEs of very
small size can be translocated to the brain in intact
form though in extremely small amount.
Nose-to-brain transport of the payload Fig. 8
shows the presence of DiR dye signal in the brain at 1
hr post administration in CS-NE encapsulating DiR.
Nanoscale Accepted Manuscript
Fluorescence of DiR dye is observed on both dorsal
and ventral surfaces of the brain; however, intense
signals are mainly observed on the olfactory bulb.
Moreover, ventral surface shows relatively strong
signals in comparison with dorsal surfaces. The
ophthalmic and maxillary branches of the trigeminal
nerve are playing pivotal role for nose-to-brain
delivery of drugs. These neurons are forming
branches that pass directly through the nasal mucosa.
In fact, it has been established that these neurons
influx neurotrophic factor, IGF-1, to the brain stem
and spinal cord areas in an in vivo rat model.34
Therefore, entry of drug in the rostral part of the
brain is via olfactory as well as trigeminal nerve. On
the other hand, trigeminal nerve is shown to enhance
drug to the caudal areas of the brain. Presence of
model DiR signal in the caudal region suggests that
DiR is transported through the trigeminal nerve as
well.
Conclusion
By tracing the signals of various fluorescent probes
used to label NEs, the translocation profiles of
integral NEs, as well as the cargoes, along the
passages from nose to brain have been roughly
outlined with imaging evidence both in vivo and ex
vivo. Particle size, rather than surface coating with
chitosan or thickening with thermosensitive in situ gel,
plays the most important role in determining the in
vivo fate of NEs. NEs with particle size of about 100
nm or smaller show prolonged resident duration in
the nasal cavity, whereas NEs of larger sizes show
faster clearance. Integral NEs of 100 nm can be
transported through the trigeminal and the olfactory
nerve to olfactory bulb, following a decremental
intensity gradient along the nose-to-brain path, and
finally to the brain, but in minute quantity. NEs of 900
nm can also be transported along the nose-to-brain
passage but in slower pace, which apparently can be
attributed to the faster mucociliary clearance and
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higher diffusion resistance encountered. On the
contrary, the cargoes, as represented by either C6 or
DiR, can be transported in higher intensity through
the maxillary nerve to the trigeminal nerve as well as
olfactory nerve to the olfactory bulb and finally to the
brain. This study provides evidence indicating
transportation of the cargoes but few integral NEs
into the brain. However, the exact amount of nose-
to-brain delivery needs to be validated in future
studies.
Published on 15 December 2016. Downloaded by Fudan University on 15/12/2016 16:09:50.
Acknowledgements
This study was financially supported by National
Natural Science Foundation of China (81573363,
21372063) and Shanghai Commission of Science and
Technology (14JC1490300). We thank Prof. Qizhi
Zhang and Dr. Qingfeng Liu at School of Pharmacy,
Fudan University for help with animal experiments.
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Table 1 Basic properties of various dyes used in this study

Dye λabs λem Colour ACQ Tracing Hydrophobicity

Nanoscale Accepted Manuscript

effect target
P2 708 732 Red Yes Particle High
P4 651 662 Red Yes Particle High
C6 460 500 Green No Cargo High
DiR 748 780 Red No Cargo High

Fig. 1 Schematic depiction of the rationale of accurate detection of integral nanoparticles (A)
and virtual passages of nose-to-brain delivery (B)

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Fig. 2 Retention of NEs in the nasal mucosa of SD rats analysed by IVIS live imaging from ventral side (A,
B) and quantification of total fluorescence in nostrils as a function of time (C, D)

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Fig. 3 Ex vivo imaging (A) and quantification of fluorescence (B) in nasal mucosa

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Fig. 4 Histological examination of horizontal sections of nasal mucosa, vertical sections of

olfactory region and vertical sections of respiratory region in both C6 and P4 channels.

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Fig. 5 Histological examination of different sections taken from the nostril,

middle and brain stem regions of trigeminal nerve. Green and red colors
represent the signals of C6 and P4, respectively

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Fig. 6 Ex vivo imaging of P2 fluorescence signals in the brain of SD rats 1
hr post administration

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Fig. 7 Confocal laser scanning microscopy images of the brain tissues taken from different Nanoscale Accepted Manuscript
position at 1 hr post administration in SD rats.

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Fig. 8 Diffusion of DiR fluorescence in the brain of SD rat at 1 and 2 hr post
administration after nasal administration of chitosan-coated nanoemulsions
(CS-NE-108) in both dorsal and ventral side.

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