Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain

Source:  https://www.researchgate.net/publication/8690647_Specific_role_of_polysorbate_80_coating_on_the_targeting_of_nanoparticles_to_the_brain

Article (PDF Available)inBiomaterials 25(15):3065-71 · August 2004with68 Reads

DOI: 10.1016/j.biomaterials.2003.09.087 · Source: PubMed

Abstract
It was reported that nanoparticles with polysorbate 80 (Tween 80, T-80) coating represented tools used for delivering drugs to brain. Nevertheless, disputations were once aroused for some complications. Aimed to have a better understanding of the specific role of T-80 coating on nanoparticles and simplify the problem, the direct observation of brain targeting combined with in vivo experiments was carried out in this work using the model nanoparticles (MNPs). The presence of a complex composed by the model loading, T-80 and nanoparticles was found in the preparation of MNPs. The result was further supported by some surface properties of MNPs. Being bound to nanoparticles that were overcoated by T-80 later, was necessary for the loading to be delivered to brain. Partial coverage was enough for T-80 coating to play a specific role in brain targeting. It seemed that brain targeting of nanoparticles was concerned with the interaction between T-80 coating and brain micro-vessel endothelial cells. Therefore, the specific role of T-80 coating on nanoparticles in brain targeting was confirmed.

Biomaterials 25 (2004) 3065–3071
Specific role of polysorbate 80 coating on the targeting of
nanoparticles to the brain
Wangqiang Sun
a,b
, Changsheng Xie
a,b,
*, Huafang Wang
c
,YuHu
c
a
Nano Pharmaceutical Research Center, Huazhong University of Science and Technology, Wuhan 430074, China
b
Department of Material Science and Engineering, Huazhong University of Science and Technology, Luo-Yu Road 1037, Wuhan, Hubei 430074, China
c
Institute of Hematology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
Received 6 August 2003; accepted 22 September 2003
Abstract
It was reported that nanoparticles with polysorbate 80 (Tween 80, T-80) coating represented tools used for delivering drugs to
brain. Nevertheless, disputations were once aroused for some complications. Aimed to have a better understanding of the specific
role of T-80 coating on nanoparticles and simplify the problem, the direct observation of brain targeting combined with in vivo
experiments was carried out in this work using the model nanoparticles (MNPs). The presence of a complex composed by the model
loading, T-80 and nanoparticles was found in the preparation of MNPs. The result was further supported by some surface properties
of MNPs. Being bound to nanoparticles that were overcoated by T-80 later, was necessary for the loading to be delivered to brain.
Partial coverage was enough for T-80 coating to play a specific role in brain targeting. It seemed that brain targeting of nanoparticles
was concerned with the interaction between T-80 coating and brain micro-vessel endothelial cells. Therefore, the specific role of T-80
coating on nanoparticles in brain targeting was confirmed.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Polylactic acid (PLA); Nanoparticles; Polysorbate 80 (Tween 80,T-80); Brain targeting; Brain micro-vessel endothelial cells (BMECs);
Fluorescein isothiocyanate-dextran (FITC-dextran)
1. Introduction
Featured with tight continuous circumferential junc-
tions between them, brain micro-vessel endothelial cells
(BMECs) mainly build up the blood–brain barrier
(BBB) [1–5], which hinders water-soluble molecules
and those with molecular weight above 500, such as
the therapeutic peptides, proteins, genes and antibiotics,
from the circular system to the brain. The applicability
of medicines in brain diseases is thus limited.
In pharmaceutics, nanoparticles [4,6] are polymeric
particles with a size ranging from 10 to 1000 nm,
employed to carry drugs through incorporation or
absorption. Loaded by nanoparticles, drugs will be
released at right rate and dose at specific sites in body
during a certain time to realize the accurate delivery,
which will enhance the therapeutic efficacy and reduce
the toxicity and the side effect. It was reported that
nanoparticles overcoated by polysorbates (especially
polysorbate 80 (Tween 80, T-80)) were capable of
transporting the loaded drugs across BBB after admin-
istration, which supplied tools delivering drugs to brain
[4,7–10]. Nevertheless, disputations were once aroused
for some complications [11]. Up to now, most works
[4,7–11] were focused on nanoparticles of poly(alkylcya-
noacrylate) (PACA), a polymer that is not authorized to
application in human [12]. Brain targeting was char-
acterized mainly through physiological or pharmacolo-
gical reactions of testing animals that had administrated
model drugs mediated by nanoparticles [4,7–11]. PACA
will be rapidly degraded by esterases presented in
biological fluid and some toxic products will stimulate
or damage the central nervous system (CNS) [11].
Furthermore, possibly due to competitive adsorption,
complete desorption of the pre-loaded model drug after
surface modification by T-80 was reported [11]. Hence,
the doubt on the specific role of T-80 coating on
nanoparticles was put forward and a mechanism of a
ARTICLE IN PRESS
*Corresponding author. Department of Material Science and
Engineering, Huazhong University of Science and Technology, Luo-
Yu Road 1037, Wuhan, Hubei 430074, China. Tel.: +86-027-
87543840; fax: +86-027-87543776.
E-mail address: csxie@mail.hust.edu.cn (C. Xie).
0142-9612/$ – see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2003.09.087
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non-specific role of the permeabilization of BBB
concerned with the toxicity of PACA was surmised
[11]. Meanwhile pungent rebutment was also aroused
for the design and operation of the experiments
supporting the doubt [4]. These disputations reflected
that there lied some limitations in the works on
nanoparticles in brain targeting.
Aimed to have a better understanding of the specific
role of T-80 coating on nanoparticles in an easy way, we
tried to introduce new idea to investigate those problems
in this elementary work. A new kind of model
nanoparticle (MNP) was designed based on the surfac-
tant-free polylactic acid (PLA) nanoparticles. As a
typical biodegradable polyester currently employed in
clinic approved by Food and Drug Agency of US, PLA
[12–15] was employed instead of PACA to avoid the
toxic effects induced by the matrix material. Since the
suspensions of surfactant-free nanoparticles (SFNPs) of
biodegradable polyesters were relatively stable during
storage [16], the suspension of SFNPs of PLA was
adopted as a reference system to physically characterize
MNPs. The preparation of MNPs was formed through
incubation of the model loading with SFNPs, followed
by the treatment of overcoating with T-80. Fluorescence
microscopy is a physical method, once employed to
support some findings in brain targeting of nanoparti-
cles [7,8]. To further avoid the potential toxicity possibly
induced by some compositions of the drug carriers,
which had led to complications in experiments related to
physiological or pharmacological reactions of testing
animals [11], the direct observation of brain targeting
combined with in vivo experiments was carried out using
fluorescence microscopy. Having been widely applied in
in vivo or in vitro experiments observed with fluores-
cence microscopy [7,8,17], fluorescein isothiocyanate-
dextran (FITC-dextran, dextran labelled by fluorescein
isothiocyanate) was used as the model loading to probe
the distribution of MNPs in brain. Vascular perfusion
fixation was performed instead of immersion fixation
once used in the experiments of PACA nanoparticles [7],
because it is the best method for the study of CNS
morphology with light or electron microscopy in the
small laboratory animal, which can maintain the tissue
in as near a life-like state as possible, both morpholo-
gically and chemically [18].
2. Experiments
2.1. Materials, reagents and animals
PLA (racemic, 5000 in molecular weight) and FITC-
dextran (77,000 in molecular weight) were purchased
from Shandong Province Institute of Medical Equip-
ments, China and Sigma, respectively. Reagents were
analysis-grade chemicals made in China mainly includ-
ing acetonitrile, anhydrous ethanol, T-80, cobalt nitrate
hexahydrate (Co(NO
3
)
2
6H
2
O), ammonium thiocya-
nate (NH
4
SCN), chloroform, acetone, sodium chloride
(NaCl) and paraformaldehyde. These materials and
chemicals were applied as obtained without further
treatment. Experimental animals were inbred Kunming
mice (20–30 g in weight) supplied by the Experimental
Animal Center of Tongji Medical College, Huazhong
University of Science and Technology, China.
2.2. Preparation of nanoparticles
SFNPs were firstly prepared by a nanoprecipitation
method [15,19] with some modifications. In brief, PLA
was dissolved in acetonitrile and slowly added into 50%
(v/v) ethanol aqueous solution. The pre-formed suspen-
sion was slowly added to distilled water with shaking
and then treated in a rotative vacuum evaporator to
remove organic solvents and surplus water. Finally, the
suspension with a concentration of 1% (w/v) was
produced.
Based on the suspension of SFNPs as described
above, MNPs were formed at 20–25
C as below. FITC-
dextran was added into the preparation of SFNPs and
incubated for 24 h. Thereafter T-80 was added and
stored for another 24 h. The ratio among nanoparticles,
FITC-dextran and T-80 in weight was 10:1:10. Free
FITC-dextran and T-80 in the final preparation were
not further treated as those free model drugs and T-80 in
the preparations of model drug loaded nanoparticles of
PACA [7–11].
Two controls, T-80 coated nanoparticles (TNPs) and
FITC-dextran loaded nanoparticles (FNPs), were pre-
pared similar as MNPs, respectively, only without
adding FITC-dextran or T-80.
These suspensions of different nanoparticles (listed in
Table 1) were used in characterization of nanoparticles
and preparation of some injections administrated to the
testing animals thereinafter. When necessary, some
chemicals would be added and dissolved in the suspen-
sions therein.
2.3. Characterization of nanoparticles
The morphology of SFNPs was observed in a JEM-
10CX II transmission electron microscope (TEM)
ARTICLE IN PRESS
Table 1
Components added in different suspensions
Type of suspensions Components added in suspensions (w/v)
SFNPs SFNPs (1%)
TNPs SFNPs (1%)+T-80 (1%)
FNPs SFNPs (1%)+FITC-dextran (0.1%)
MNPs SFNPs (1%)+FITC-dextran
(0.1%)+T-80 (1%)
W. Sun et al. / Biomaterials 25 (2004) 3065–30713066
(JEOL, Japan) at 80 kV using the sample taken from the
suspension of SFNPs by C-coated Cu grips.
The amount of T-80 coating on TNPs or MNPs was
determined based on a quantitative test for poly(ethy-
lene oxide) with ammonium cobaltothiocyanate
(NH
4
[Co(SCN)
3
]) [20]. Briefly each suspension with a
known volume was centrifuged at 16,850g at 4
C for
30 min in a TLL-C table centrifuge (Beijing Sihuan
Instrument Plant, China), respectively. The supernatant
in each sample was discarded to eliminate unadsorbed
T-80. The pellets were rinsed with distilled water twice
and the washing solution was eliminated by another
centrifugation as described above. The nanoparticles
thus purified were resuspended in NH
4
[Co(SCN)
3
]
solution (dissolving Co(NO
3
)
2
6H
2
O (30 g/l) and
NH
4
SCN (200 g/l) in distilled water) with vigorous
shaking. The complex between T-80 coating on nano-
particles and NH
4
[Co(SCN)
3
] that might be produced
was then extracted into chloroform. The adsorption of
the chloroform solution was determined in a UV-
2102PC spectrophotometer (Unico (Shanghai) Instru-
ment Co., Ltd., China) at a wavelength of 318.5 nm,
respectively. The amount of T-80 coating in each sample
was calculated according to the determination.
To estimate the amount of FITC-dextran absorbed on
FNPs or MNPs, high-speed centrifugation and rinse as
described above were carried out to eliminate unad-
sorbed FITC-dextran in the suspensions with a known
volume. Each sample thus purified was lyophilized in a
LGJ table lyophilizer (Beijing Sihuan Instrument Plant,
China) and acetone was added then. To further
eliminate dissolved PLA, another high-speed centrifuga-
tion was performed and washed twice with acetone. The
washing solution was discarded by centrifugation again
as described above. The final precipitations were then
dissolved in distilled water and measured in an RF-540
fluorescence spectrophotometer (Shimadzu, Japan) at
excitation and emission wavelengths of 495 and 520 nm,
respectively. The amount of FITC-dextran absorbed on
FNPs or MNPs was estimated according those determi-
nations at last.
To test the size and the zeta potential (z) of those
nanoparticles therein before, samples from those sus-
pensions were diluted with distilled water by 50 folds,
respectively, and then each triply measured at 15
Cina
Zeta Pals zetasizer (Brookhaven Instruments, US).
Using SFNPs as a reference system, the hydrodynamic
thickness (d) of the layer adsorbed on different
nanoparticles was calculated as follows [20]:
d ¼
d
a
d
o
2
;
where d
a
is the diameter of nanoparticles with adsorbed
FITC-dextran and/or T-80 and d
o
is the diameter of
SFNPs.
2.4. In vivo experiments and fluorescence microscopy
analysis
Healthy mice were divided into seven groups (three
mice per group) in in vivo experiments. In each group
each mouse was intravenously injected with the same
injection at a dose of 0.2 ml in tail vein, respectively.
Those injections were suspensions or solutions contain-
ing some components of the preparation of MNPs and
0.9% (w/v) NaCl, a component of physiological saline
(see Table 2). In Group 1, the injection contained
MNPs. The other groups were controls, just prepared
before injection, including a mixture of FNPs and T-80,
a mixture of TNPs and FITC-dextran, a suspension of
FNPs, a mixture of SFNPs, FITC-dextran and T-80, a
mixture of FITC-dextran and T-80, as well as a solution
of FITC-dextran.
The vascular perfusion fixation with 4% paraformal-
dehyde solution was performed in those experimental
animals after intravenous injection for 45 min. Before
the perfusate was infused, vessels were rinsed to
eliminate residual blood. The prefixed brain tissues were
ARTICLE IN PRESS
Table 2
Main components in injections
Group Type of injections Main components in injections
1 A suspension of MNPs in saline MNPs suspension (FITC-dextran, 0.1% (m/v), 8 mg/kg; T-80, 1%
(m/v), 80 mg/kg)+NaCl (0.9% (m/v), 72 mg/kg)
2 A mixture of FNPs and T-80 in saline FNPs suspension (FITC-dextran, 0.1% (m/v), 8 mg/kg)+T-80 (1%
(m/v), 80 mg/kg)+NaCl (0.9% (m/v), 72 mg/kg)
3 A mixture of TNPs and FITC-dextran in saline TNPs suspension (T-80, 1% (m/v), 80 mg/kg)+FITC-dextran (0.1%
(m/v), 8 mg/kg)+NaCl (0.9% (m/v), 72 mg/kg)
4 A suspension of FNPs in saline FNPs suspension (FITC-dextran, 0.1% (m/v), 8 mg/kg)+NaCl
(0.9% (m/v), 72 mg/kg)
5 A mixture of SFNPs, FITC-dextran and T-80 in saline SFNPs suspension+FITC-dextran (0.1% (m/v), 8 mg/kg)+T-80
(1% (m/v), 80 mg/kg)+NaCl (0.9% (m/v), 72 mg/kg)
6 A mixture of FITC-dextran and T-80 in saline FITC-dextran (0.1% (m/v), 8 mg/kg)+T-80 (1% (m/v), 80 mg/
kg)+NaCl (0.9% (m/v), 72 mg/kg)
7 A solution of FITC-dextran in saline FITC-dextran (0.1% (m/v), 8 mg/kg)+NaCl (0.9% (m/v), 72 mg/kg)
W. Sun et al. / Biomaterials 25 (2004) 3065–3071 3067
immersed in the same perfusate, further fixed at 4
Cina
refrigerator overnight and cut into sections by a
vibratome thereafter. Those sections were observed in
an FM fluorescence microscope (Nikon, Japan) at
excitation and emission wavelengths of 495 and
520 nm, respectively.
3. Results and discussion
3.1. Characterization of model nanoparticles
As a reference system, the properties of SFNPs were
investigated at first in this work. As shown in Fig. 1,
SFNPs were solid and nearly spherical particles. From
Fig. 2, the effective diameter was 162.1 nm with a
narrow polydispersity index of 0.108 at the confidence
level of 95%.
From Table 3, both FITC-dextran and T-80 were
located on the surface of MNPs. The result suggested
that the preparation of MNPs was not just a mixture of
FITC-dextran, T-80 and nanoparticles and a complex
composed by FITC-dextran, T-80 and nanoparticles
was presented indeed. Compared with the two controls
the total amount of the layer adsorbed on MNPs was
the highest, while both FITC-dextran and T-80 bound
on MNPs were each lower than the situation of FITC-
dextran or T-80 lonely adsorbed on nanoparticles,
respectively. The dextran employed in this work is a
typical water-soluble polymer with high molecular
weight. Because of the large size of polymer chains, it
is not easy to displace adsorbed high molecular weight
molecules [22]. Hence, desorption of FITC-dextran pre-
adsorbed on nanoparticles due to competitive adsorp-
tion was unobvious. Since FITC-dextran was previously
bound to MNPs, the steric effect [22] could not be
ignored. The adsorption of T-80 on MNPs was
undoubtedly influenced. Consequently, T-80 only par-
tially covered MNPs.
Since SFNPs were uniform and nearly spherical, d of
the adsorbed layer on different nanoparticles could be
estimated. The results listed in Table 4 demonstrated
that the adsorbed layer on MNPs was thicker than those
of the controls, which was in good agreement with the
results therein before (Table 3). These results gave a hint
to support that a complex composed by FITC-dextran,
T-80 and nanoparticles was presented indeed in the
preparation of MNPs. As the surface become more
crowded when more surfactants or polymers are
adsorbed, the adsorbed are probably more laterally
compressed and trend to stretch in the direction
perpendicular to the surface, which will lead to d of
adsorbed layer increased [21,22].
The extent of positive shift in z of different
nanoparticles was listed in Table 5. The results were
content with those of d of the adsorbed layer (Table 4)
as described above, which further supported the
presence of a complex composed by FITC-dextran, T-
80 and nanoparticles. As a very important parameter on
the electrical properties of surface, z is defined as the
potential at the shear plane in the Stern model. It is
generally assumed in tests of double-layer theory that z
and the Stern potential (c
s
) are the same except some
situations, e.g. in the presence of absorbed non-ionic
species or polymers that force the shear plane further
away from the surface, reducing z relative to c
s
[22].
PLA carries the negative charge for its end group of
carboxylic acid [23], while FITC-dextran is a non-ionic
surfactant and T-80 is a kind of non-ionic polymer.
Therefore, the more the adsorbed, the thicker the
adsorbed layer and the more positive shift in z:
3.2. Direct observation of brain targeting
In vivo experiments were carried out to investigate the
specific role of T-80 coating in brain targeting. Contrary
to the data of PACA nanoparticles [11], Table 6 showed
nocaseofmortalitywasfoundineachgroup,which
ARTICLE IN PRESS
Fig. 1. TEM image of SFNPs.
100
0
20
40
60
80
100
0
10
20
30
40
50
60
70
80
90
100
Relative cumulative numbers (%)
Relative intensity of scattered light (%)
Diameter (nm)
60 70
80
9050
500
400300200
162.1
Fig. 2. Size distribution of SFNPs: density distribution and ––
cumulative distribution.
W. Sun et al. / Biomaterials 25 (2004) 3065–30713068
demonstrated the security of the compositions of the
preparation of MNPs. In body scissions of PLA will
take place and lactic acid will be produced by further
biodegradation. The degraded product, lactic acid,
enters the tricarboxylic acid cycle and is metabolized
as CO
2
and H
2
O at last [13]. It seemed that the effects of
toxicity of the carrier on CNS might be ignored in this
work.
From Table 6, fluorescence only appeared in Group 1
that injections contained MNPs. These indicated that
MNPs owned the property of brain targeting. Fluores-
cence was neither found in groups (Groups 4 and 7, also
see Table 2) without T-80 in injections nor observed in
Group 2 (also see Table 2) without T-80 coating on
nanoparticles. Such discriminations from Group 1
illustrated that brain targeting corresponded to the T-
80 coating on nanoparticles. The experiments of Oliver
et al. [11] suggested that the preparation of T-80 coated
nanoparticles was actually a simple mixture of model
drug, T-80 and nanoparticles. In groups (Groups 3, 5
and 6, also see Table 2) that FITC-dextran was just
mixed with T-80, however, no fluorescence was found.
The results contradicted those of Oliver et al. and
illustrated that brain targeting was related to the
complex composed by FITC-dextran, T-80 and nano-
particles. These data supported the findings of Kreuter
et al. [4,7–10], i.e. the coating of T-80 plays a specific
role in brain targeting. The experiments also suggested
that partial coverage was enough for T-80 coating to
play a specific role in brain targeting taken together with
the results therein before (Table 3).
Comparing with (a) and (b) in Fig. 3, one can further
find that fluorescence mainly located at the wall of brain
micro-vessels. Although it was very hard to discriminate
whether MNPs were adhered to the lining of the
BMECs or taken up by BMECs from fluorescence
microscopy, one thing seemed to be confirmed that
brain targeting of nanoparticles was concerned with the
interaction between T-80 coating and BMECs.
According to literatures, several mechanisms of
nanoparticle-mediated drugs across BBB had been
proposed [4,7,8,11]. Among them the mechanism of
endocytosis [4,7,8] was supported by many experiments.
It is very possible that polysorbates on the surface of
nanoparticles anchor apolipoprotein E (apo E) that
plays an important role in the transport of the low-
density lipoprotein (LDL) into brain. After apo E being
bound to the surface, nanoparticles mimic LDL-
particles to interact with LDL receptors on BMECs,
which makes them up-taken by BMECs [4].Onthe
contrary some workers suggested that in the situation of
PACA nanoparticles a non-specific opening of the tight
junctions between BMECs was first induced by the
carrier matrix and the unloaded model drug was then
penetrate into the CNS with the help of T-80 [11].From
this work the result illustrated that being bound to
nanoparticles that were overcoated by T-80 later, was
necessary for the loading to be delivered to brain.
Therefore, it seemed that the mechanism of endocytosis
was more reasonable than the latter.
4. Conclusions
A complex composed by the loading, T-80 and
nanoparticles was found in the preparation of MNPs.
ARTICLE IN PRESS
Table 4
d of the layer adsorbed on different nanoparticles
Nanoparticles Diameter (nm) d (nm)
SFNPs 162.1 0
TNPs 174.8 6.4
FNPs 194.2 16.1
MNPs 202.6 20.3
Table 5
z of different nanoparticles
Nanoparticles z (mV) Positive shift in z
(mV)
Relative positive
shift in z (%)
SFNPs 29.52 0 0
TNPs 26.32 3.20 10.8
FNPs 13.40 16.12 54.6
MNPs 10.17 19.35 65.5
Table 3
Amount of T-80 or FITC-dextran adsorbed on different nanoparticles
Nanoparticles Amount of T-80
(mg/mg)
Relative amount
of T-80 (%)
a
Amount of FITC-
dextran (mg/mg)
Relative amount of
FITC-dextran (%)
b
Total amount of the adsorbed
layer (mg/mg)
c
TNPs 30.7 100 0
d
0 30.7
FNPs 0
e
0 15.4 100 15.4
MNPs 25.6 83.4 14.0 90.9 39.6
a
Taking TNPs as the standard.
b
Taking FNPs as the standard.
c
Calculated by summing both the amount of T-80 and FITC-dextran.
d
Since TNPs were not treated with FITC-dextran, the value was supposed as zero.
e
Since FNPs were not treated with T-80, the value was supposed as zero.
W. Sun et al. / Biomaterials 25 (2004) 3065–3071 3069
The result was further supported by surface properties
of MNPs. Being bound to nanoparticles that were
overcoated by T-80 later, was necessary for the loading
to be delivered to brain. Partial coverage was enough for
T-80 coating to play a specific role in brain targeting. It
seemed that brain targeting of nanoparticles was related
to the interaction between the T-80 coating and BMECs.
The mechanism of endocytosis was more reasonable for
nanoparticle-mediated drugs across BBB. The specific
role of T-80 coating on nanoparticles in brain targeting
was thus confirmed.
Acknowledgements
This work was supported by the National Natural
Science Foundation of China (Grant Nos. 30170392 and
50271029).
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ARTICLE IN PRESS
Table 6
Comparison of fluorescence microscopy analysis on brain tissues
Group Type of injections Complex presented in
injections
Mortality before
vascular perfusion
Fluorescence in
brain tissues
1 A suspension of MNPs in saline FITC-dextran-T-80-
nanoparticles
None +
2 A mixture of FNPs and T-80 in saline FITC-dextran-
nanoparticles
None
3 A mixture of TNPs and FITC-dextran in saline T-80-nanoparticles None
4 A suspension of FNPs in saline FITC-dextran-
nanoparticles
None
5 A mixture of SFNPs, FITC-dextran and T-80 in saline None None
6 A mixture of FITC-dextran and T-80 in saline None None
7 A solution of FITC-dextran in saline None None
Note: +, green fluorescence appeared in the view under a fluorescence microscope; , no fluorescence was observed, completely dark in the view
under a fluorescence microscope.
Fig. 3. Fluorescence distributed in the brain tissue of the mouse from Group 1 after 45 min intravenous injection: (a) viewed under a fluorescence
microscope and (b) viewed under a photo microscope.
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