Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

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Colloids and Surfaces B: Biointerfaces

journal homepage: www.elsevier.com/locate/colsurfb

Erythrocytes load of low molecular weight chitosan nanoparticles as a potential

vascular drug delivery system
Wen Fan a,b,1 , Wei Yan b,1 , Zushun Xu b , Hong Ni a,
a
b

Faculty of Life Science, Hubei University, Wuhan 430062, China

Ministry of Education Key Laboratory for the Green Preparation and Application of Functional Materials, Hubei University, Wuhan 430062, China

a r t i c l e

i n f o

Article history:
Received 19 February 2012
Received in revised form 7 March 2012
Accepted 12 March 2012
Available online 20 March 2012
Keywords:
Low molecular weight chitosan
Nanoparticles
Erythrocyte carrier
Vascular drug delivery
Fluorescent labeling

a b s t r a c t
Low molecular weight (LMW) chitosan nanoparticles have attracted considerable attention as colloidal
drug carriers, but when applied to intravascular drug delivery, they are easy to be removed from
circulation by the reticuloendothelial system, which limits their applications as long-circulating or targetspecic carriers. Erythrocytes have a long circulation time in the blood, but they are sometimes not
suitable for loading and releasing of drug directly. The combination of LMW chitosan nanoparticles and
erythrocytes that complement each other is a desirable strategy to develop a multifunctional drug carrier. In this study, monodisperse, LMW chitosan nanoparticles were prepared by ionic gelation technique
and these nanoparticles were investigated with regard to their erythrocyte compatibility. Then the interactions between erythrocytes and uorescence-labeled LMW chitosan nanoparticles were studied by
confocal microscopy. The results of this study indicate that LMW chitosan nanoparticles show good compatibility with erythrocytes and they can be easily attached to the surface of erythrocyte membrane,
suggesting that erythrocytes load of LMW chitosan nanoparticles can be served as a potential vascular
drug delivery system.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Polymeric nanoparticles have been extensively investigated as
drug delivery carriers because of their multiple advantages, such as
the ability of protecting of drug from degradation, improving the
efciency of drug utilization and controlling the drug release rate
[1]. However, the major problem encountered in the application
of these polymeric nanoparticles in blood circulation is their short
circulation time due to the rapid clearance from circulation by the
bodys immune system, mainly by the reticuloendothelial system
(RES), causing the drug failed to maintain sustained therapeutic
blood concentration or to be delivered to non-RES target tissues or
cells [2]. Thus, development of long-circulating nanoparticles has
been a focus of many groups during the past decade. Currently,
production of nanoparticles with surface modications is the main
strategy to avoid rapid recognition of nanoparticles by RES [2,3].
Erythrocytes, as the autologous cells of the host body, have a
remarkably long life span of about 120 days and a widespread
circulation throughout the body. Application of erythrocytes as
slow drug release or site-targeted delivery system has been

Corresponding author. Tel.: +86 27 88661237; fax: +86 27 88661571.

E-mail address: nihongcs@gmail.com (H. Ni).
1
These two authors contributed equally to this work.
0927-7765/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2012.03.006

extensively studied [4]. Generally, transiently membrane defects

of 2050 nm will appear in erythrocyte membrane during hypotonic hemolysis [5]. Thus, extracellular drugs including peptides,
therapeutic enzymes and nucleic acids can be encapsulated into
erythrocytes through these resealable pores. However, one major
problem encountered is that the rapid leakage of certain encapsulated drugs from the loaded erythrocytes due to simple diffusion
[4,6]. The treatment of carrier erythrocytes with membrane stabilizing agents, such as glutaraldehyde can increase their osmotic
resistance, thus resulting in a decrease in the drug release rate. But
this will also cause a decrease in erythrocyte deformability, which
makes the erythrocytes more recognizable by RES organs, mainly
the liver and spleen. Moreover, structural changes in erythrocytes
may occur during the drug loading procedure, and this can also lead
to the recognition of erythrocytes by macrophages. Although these
expand the capability of erythrocytes targeting to RES, seriously
limit the advantage of erythrocytes as long-circulating carriers, and
in some cases, may pose toxicological problems [4,7].
In nature, some mammalian pathogens can bind to the exterior surface of erythrocyte and remain in circulation for several
weeks without showing apparent clinical symptoms. According to
this strategy, Chambers and Mitragotri reported a novel method
of prolonging intravascular particle circulation by anchoring
nanoparticles to the surface of erythrocytes [8,9]. They found that
polystyrene nanoparticles could adhere to erythrocytes through

W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

electrostatic and hydrophobic interactions in the absence of plasma

proteins. The erythrocytes-bound nanoparticles could remain in
circulation for several hours, up to a hundredfold greater than
that of free nanoparticles, then detach passively from erythrocytes
due to shear forces and cellcell interactions and eventually be
removed from the circulation without affecting the circulation of
erythrocytes themselves. Their studies also indicate that the binding strength between the nanoparticles and erythrocytes primarily
determines the circulation lifetime of nanoparticles.
In recent years, low molecular weight (LMW) chitosan, as a
kind of water-soluble chitosan, has shown remarkable advantages as colloidal drug carrier due to its high water solubility,
non-toxicity, biocompatibility, biodegradability, bioadhesive and
absorption enhancing properties. Moreover, the potential biological activities of LMW chitosan, such as antioxidant and antitumour
activities, also make it an ideal candidate for biomedical applications [10,11]. In a previous study, we developed a novel method
which allows a highly repeatability of producing monodisperse,
LMW chitosan nanoparticles through ionic gelation technique [12].
In this study, we expect that by anchoring these LMW chitosan
nanoparticles to the surface of erythrocytes, we could prolong the
circulation time of LMW chitosan nanoparticles in bloodstream
and thus expand their applications in vascular drug delivery, as
well as avoid the disadvantages of erythrocytes in drug encapsulation. To explore this possibility, LMW chitosan nanoparticles were
investigated with regard to their erythrocyte compatibility. Then
uorescence-labeled LMW chitosan nanoparticles were prepared
in order to allow the visualization of the interaction between LMW
chitosan nanoparticles and erythrocytes.

2. Materials and methods

2.1. Materials
Low molecular weight (LMW) water-soluble chitosan (viscosity 35 cps, deacetylation degree 91.5%) derived from crab shell, was
purchased from Jinhu Crust Product Co., Ltd, China. Fluorescein-5isothiocyanate (FITC) was purchased from Sigma Chemical Co., Ltd.
Sodium tripolyphosphate (TPP), glacial acetic acid, sodium hydroxide and all other chemicals were analytical grade, purchased from
Sinopharm Chemical Reagent Co., Ltd, China. Ultrapure water was
used throughout this study. Sodium chloride solution and isotonic
PBS buffer (pH 7.4) were passed through a 0.22 m syringe lter
(Millipore, USA) respectively before being used.

2.2. Preparation and characterization of LMW chitosan

nanoparticles
Monodisperse, LMW chitosan nanoparticles were prepared
according to our previous study, based on the ionic gelation of chitosan with TPP anions [12,13]. Briey, LMW chitosan was dissolved
in aqueous acetic acid (0.2 mg/mL) at a concentration of 0.5 mg/mL.
The chitosan solution was stirred overnight at room temperature
and the pH of the resulting solution was adjusted to 4.74.8 by
using 20 wt% sodium hydroxide solution. The chitosan solution was
then passed through a 0.45 m syringe lter to remove residues
of insoluble particles. TPP was dissolved in ultrapure water at a
concentration of 0.5 mg/mL and passed through a 0.22 m syringe
lter. To prepare chitosan nanoparticles, a magnetic stirrer was
placed in a chest freezer, in which the ambient temperature was
controlled at 24 C. Ten milliliters of chitosan solution in a 25 mL
round-bottom ask was preheated in a water bath at 60 C for
10 min, the ask was then placed on the magnetic stirrer stirring
at 700 rpm, 3.3 mL of 24 C TPP solution was quickly added to the

259

chitosan solution. The reaction was carried out for 10 min and the
resulting suspension was subjected to further analysis.
To investigate the aggregation behavior of LMW chitosan
nanoparticles in normal saline, the original suspension (0.5 mg/mL)
was mixed with an equal volume of 1.8% w/v sodium chloride solution, to produce a normal saline suspension (0.25 mg/mL). Then
after incubation at room temperature for different periods of time,
the z-average particle size, particle size distribution and polydispersity index (PDI) of the nanoparticles were measured at 25 C by
dynamic light scattering (DLS) on a high performance particle sizer
(HPPS-5001, Malvern, UK). Mean values were obtained from the
analysis of three different batches, each of them measured three
times.
2.3. Erythrocyte compatibility
Erythrocyte compatibility of the LMW chitosan nanoparticles
was evaluated in terms of hemolysis and morphological changes in
erythrocytes.
The hemolysis test was conducted as described by Bock and
Mller [14]. Whole blood was obtained from an adult rabbit, the
heparinized blood was centrifuged at 1000 g for 5 min to generate
packed red blood cells (RBC). The RBC were washed three times
with normal saline before being diluted with normal saline to
prepare an erythrocyte stock dispersion with a xed concentration of hemoglobin (3:11 centrifuged erythrocytes: normal saline).
The washing step was repeated in order to remove debris and
serum protein. One hundred microliters of the freshly prepared erythrocyte stock dispersion was added to 1 mL of freshly prepared
0.25 mg/mL chitosan nanoparticles suspended in normal saline.
After incubation under shaking at 37 C for 2 h, debris and intact
erythrocytes were removed by centrifugation at 750 g for 3 min.
Then 100 L of the resulting supernatant was dissolved in 2.0 mL
of an ethanol/HCl mixture [1 part HCl (37%, w/v) + 39 parts ethanol
(99%, v/v)]. This mixture dissolved all components and avoided the
precipitation of hemoglobin, with the exception of chitosan. Thus
an additional centrifugation was necessary (750 g for 3 min) to separate the non-soluble chitosan [15]. The absorption of the resulting
supernatant was determined at 398 nm by an UV spectrophotometer (UV-2000, Unico, China) against a blank sample. Normal saline
was used as a negative control (0% lysis), and ultrapure water was
used as a positive control (100% lysis). The trial was repeated and
the mean value of three measurements using different samples was
recorded.
For the study of morphological changes in erythrocytes induced
by an excessive amount of LMW chitosan nanoparticles, 50 L
of washed erythrocytes with a hematocrit of 70%, suspended in
normal saline, were incubated with 1 mL of 0.25 mg/mL chitosan
nanoparticles suspended in normal saline. The incubation was carried out at 37 C for 1 h under gentle shaking. Then 5 L of the
sample was mounted on a glass slide, viewed under an optical
microscope.
2.4. Erythrocyte sedimentation and agglutination
Fifty microliters of washed erythrocytes with a hematocrit of
70%, suspended in normal saline, were incubated with increasing
volumes of 0.25 mg/mL LMW chitosan nanoparticles suspended in
normal saline, respectively (Table 1). The incubations were carried
out at room temperature for 1 h. Ten microliters of the resulting
sedimentary erythrocytes were diluted to a hematocrit of 2% with
normal saline. To observe the behavior of erythrocyte sedimentation, the 2% erythrocyte suspension was mixed by gentle pipetting
and was left standing for 1 h at room temperature. For further
observation, the resulting sample was mixed by inversion and 5 L

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

Table 1
Washed erythrocytes (70% hematocrit) were incubated with increasing volumes of LMW chitosan nanoparticles (0.25 mg/mL).
Sample

10

11

12

Erythrocytes (L)
Nanoparticles (L)

50
0

50
5

50
10

50
20

50
30

50
40

50
50

50
60

50
70

50
80

50
90

50
100

of the sample was mounted on a glass slide, viewed under an optical

microscope.

2.5. Preparation and characterization of FITC-chitosan

nanoparticles
FITC-labeled chitosan was synthesized by the reaction between
the isothiocyanate group of FITC and the primary amino group
of chitosan [1618]. LMW chitosan was dissolved in 0.1 M aqueous acetic acid to prepare a 1 wt% chitosan solution. After stirring
for several hours at room temperature, the chitosan solution
was passed through a 0.45 m syringe lter. Then 100 mL of
dehydrated methanol followed by 50 mL of FITC in methanol
(0.5 mg/mL) was added to chitosan solution under stirring. The
mixture was stirred for 3 h in the dark at room temperature and the labeled chitosan was precipitated by adjusting
the pH of the mixture to 10 with 0.2 M NaOH. The precipitate was centrifuged and washed with a methanol/water mixture
(70:30, v/v) repeatedly until no uorescence was detected in the
supernatant (RF-540 spectrophotouorometer, Shimadzu, Japan,
exc = 490 nm, emi = 520 nm). The labeled chitosan was redissolved in 20 mL of 0.1 M acetic acid and dialyzed in the dark
against 3 L of ultrapure water for 3 days. The water being replaced
with fresh water every 6 h. Finally, the labled chitosan was
freeze-dried.

FITC-chitosan nanoparticles were prepared following the procedure described in Section 2.2. The morphological characteristics
of FITC-chitosan nanoparticles were examined using a high resolution transmission electron microscope (TEM, Tecnai G20, FEI,
Netherlands). A droplet of suspension was placed on a carbon lmcovered copper grid (200 mesh), 5 min later, the excess liquid was
removed by touching the edge of the copper grid with a piece of
lter paper. Then, a droplet of 2 wt% phosphotungstic acid solution
was placed on the copper grid, and the excess liquid was removed
again 5 min later. The sample was then air-dried before observation
by TEM.
The aggregation behaviors of FITC-chitosan nanoparticles in different saline solutions were studied with a laser scanning confocal
microscope (LSCM, Leica TCS SP2, Germany). The suspension of
FITC-chitosan nanoparticles was mixed with an equal volume of
1.8% w/v sodium chloride solution or isotonic PBS buffer. After incubation at room temperature for 5 h, the samples were mounted on
a glass slide and observed by LSCM, respectively.

2.6. Adhesion of FITC-chitosan nanoparticles onto erythrocyte

membrane
Fifty microliters of washed erythrocytes with a hematocrit of
70%, suspended in normal saline, were mixed and incubated with
50 L of 0.25 mg/mL FITC-chitosan nanoparticles suspended in

Fig. 1. (A) FITC-chitosan solution and (B) FITC-chitosan nanoparticles suspension under incandescent light; (C) FITC-chitosan solution and (D) FITC-chitosan nanoparticles
suspension under ultraviolet light.

W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

261

normal saline. The incubation was carried out at room temperature

for 1 h in the dark. The resulting erythrocytes were centrifuged and
washed twice with isotonic PBS buffer. The packed erythrocytes
were diluted with a PBS/glycerol mixture (9:1, v/v) and 5 L
of the sample was mounted on a glass slide and observed by
LSCM.

3. Results and discussion

3.1. Characterizations of LMW chitosan nanoparticles and
FITC-chitosan nanoparticles
In a previous study, we reported an effective method to prepare
LMW chitosan nanoparticles with high degree of monodispersity
and stability. As uorescent labeling provides a convenient and
sensitive approach for tracking the location of labeled nanoparticles, thus it is necessary to investigate whether the labeling
procedure will affect the monodispersity and stability of the
nanoparticles.
When observed under ultraviolet light, the FITC-chitosan solution exhibited a characteristic bright green uorescence of FITC,
while the FITC-chitosan nanoparticles exhibited a translucent
opalescent due to the scattered light from the nanoparticles (Fig. 1).
The chitosan nanoparticles had a mean particle size of 133 nm
and a PDI value of 0.029, while the FITC-chitosan nanoparticles
had a mean particle size of 152 nm and a PDI value of 0.034.
After stored at room temperature for 20 days, neither chitosan
nanoparticles nor FITC-chitosan nanoparticles showed any statistically signicant change in particle size and size distribution
(DLS data not shown). These results indicate that although the
FITC-labeling procedure leads to an increase in particle size, it has

Fig. 2. TEM image of FITC-chitosan nanoparticles stained with phosphotungstic

acid.

no apparent inuence on the monodispersity and stability of the

nanoparticles.
The typical morphology of the FITC-chitosan nanoparticles is
shown in Fig. 2. The nanoparticles exhibited a spherical shape and
had a narrow particle size distribution. TEM images of chitosan
nanoparticles were not presented since they appeared similar to
the FITC-chitosan nanoparticles.

Fig. 3. Aggregation morphologies of FITC-chitosan nanoparticles after 5 h of incubation in (A) isotonic sodium chloride solution and (B) diluted PBS buffer; (C) Particle size
distribution of LMW chitosan nanoparticles after different periods of incubation in isotonic sodium chloride solution.

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

3.2. Aggregation behaviors of LMW chitosan nanoparticles in

different saline solutions
Before incubation of chitosan nanoparticles with erythrocytes, the nanoparticles should be suspended in isotonic medium
(300 mOsmol/kg) which is necessary to maintain the normal morphology of erythrocytes [19]. We found that freeze-dried LMW
chitosan nanoparticles tended to aggregate and could not be
well redispersed, which is believed due to the strong inter-and
intramolecular hydrogen bonding [20]. Thus we prefer to mix the
original suspension of chitosan nanoparticles with an equal volume of twice isotonic medium. In further analysis by DLS, it was
found that even if the original suspension was mixed with an equal
volume of isotonic PBS buffer, the nanoparticles showed a rapid
aggregation and micro-particles were formed after 5 h of incubation (data not shown). This was also conrmed by LSCM in the
case of FITC-chitosan nanoparticles (Fig. 3B). However, when the
original suspension was mixed with an equal volume of twice isotonic sodium chloride solution, the nanoparticles underwent a slow
aggregation within 24 h (Fig. 3C). LSCM observations also demonstrated that FITC-chitosan nanoparticles were well dispersed after
5 h of incubation in isotonic sodium chloride solution (Fig. 3A).
As chitosan is a weak polyelectrolyte with a pKa around 6.5,
thus both the ionic strength and pH of the solution can inuence
the properties of chitosan particles [21]. The original suspension
provides an environment of low ionic strength and weak acid (pH
around 5.5), in which the protonated amino groups remaining on
the surface of chitosan nanoparticles are adequate to maintain the
colloidal stability due to the electrostatic repulsion between particles. However, when the nanoparticles are suspended in diluted
PBS buffer, not only the counter-ions will screen the protonated
amine groups, but also the protonated amino groups start to be
deprotonated under weak alkaline conditions, leading to a rapid
loss of electrostatic repulsion between particles, thus the particles
tend to aggregate rapidly. When the nanoparticles are suspended in
sodium chloride solution, only the shielding effect of counter-ions
is present and the aggregation of chitosan nanoparticles becomes
much slower. Therefore, in order to achieve good contact between
chitosan nanoparticles and erythrocytes, the nanoparticles were
suspended in isotonic sodium chloride solution before incubation
with erythrocytes.
3.3. Erythrocyte compatibility
Interestingly, it was observed that the hemolysis was negative at a concentration of 0.25 mg/mL LMW chitosan nanoparticles.
This is most likely due to that the nanoparticles caused negligible
damage to erythrocyte membrane, and moreover, the negatively
charged free hemoglobins were readily adsorbed by the positively
charged nanoparticles, causing the hemolysis was even lower than
the negative control (normal saline). Fig. 4 illustrates the morphology of erythrocytes after exposure to excessive amount of LMW
chitosan nanoparticles. It was found that although serious aggregation of erythrocytes occurred, no apparent changes in erythrocytes
morphology were observed, indicating that in the studied concentration range, the LMW chitosan nanoparticles had no deleterious
effects on erythrocytes morphology [22].
3.4. Erythrocyte sedimentation and agglutination
Fig. 5 shows the sedimentation morphology of erythrocytes
after incubation with increasing volumes of LMW chitosan
nanoparticles. It can be observed that compared to sample 1 (normal saline control), there were more erythrocytes deposited at the
bottom of the centrifuge tube in samples 28. However, in the
case of samples 912, erythrocytes became increasingly attached

Fig. 4. Morphology of erythrocytes after exposure to excessive amounts of LMW

chitosan nanoparticles (original magnication 1600).

to the tube wall. In further studies by using an optical microscope,

it demonstrates that erythrocytes were not agglutinated in samples 18, while increased agglutination of erythrocytes occurred in
samples 912 (Fig. 6). The results indicate that there exists a critical ratio between chitosan nanoparticles and erythrocytes, above
which erythrocytes start to be agglutinated. Thus, in the following
LSCM study, the mixing ratio of sample 7 was chosen to achieve
a balance between the carrying capacity of erythrocytes and the
avoidance of erythrocytes agglutination.
3.5. Adhesion of FITC-chitosan nanoparticles onto erythrocyte
membrane
As shown in Fig. 7, after incubation, FITC-chitosan nanoparticles were found attached to the surface of erythrocyte membrane.
The results indicate that after binding of chitosan nanoparticles to
erythrocyte membrane in normal saline, the PBS washing did not
lead to the separation of chitosan nanoparticles from erythrocyte
membrane. Thus, the anchoring of chitosan nanoparticles to erythrocyte membrane is expected to solve the problem that chitosan
nanoparticles tend to lose their stability in the bloodstream, which
has a high ionic strength and a pH buffer capacity, similar to that of
PBS. Instead, once the aggregation problem of chitosan nanoparticles has been solved, the slightly alkaline environment is benecial
to some extent, as it would lead to the deprotonation of chitosan,
which not only is expected to decrease the adsorption of plasma
proteins onto nanoparticles surface, but also should cause a contraction of chitosan nanoparticles and thus favors the slow release
of encapsulated drug [2327].
Pathogens in nature provide excellent examples of utilizing erythrocytes to evade the immune system [28,29]. The strategies that
combine the advantages of both nanoparticles and erythrocytes
including attaching nanoparticles to the surface of erythrocytes
or encapsulating nanoparticles into erythrocytes. Up to now, the
encapsulating strategy is limited to magnetic iron oxide nanoparticles. This is not only because the major challenge for many iron
oxide nanoparticle applications in medicine is to avoid RES clearance in order to maintain a longer blood circulation half-life, but
also because iron oxide nanoparticles usually have a hydrodynamic
diameter of less than 50 nm, which favors their encapsulation [3033]. Recently, Hamidi et al. reported that they had

W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

263

Fig. 5. (A) Sedimentation morphology of erythrocytes after incubation with increasing volumes of LMW chitosan nanoparticles; (B) contrast enhancement of (A).

Fig. 6. Aggregation morphology of erythrocytes after incubation with increasing volumes of LMW chitosan nanoparticles: (A) sample 1; (B) sample 7; (C) sample 9; (D)
sample 10 (original magnication 640).

Fig. 7. LSCM images of erythrocytes after incubation with FITC-chitosan nanoparticles: (A) uorescence image; (B) merged image.

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W. Fan et al. / Colloids and Surfaces B: Biointerfaces 95 (2012) 258265

encapsulated valproate-loaded chitosan nanoparticles into erythrocytes by hypotonic dialysis and the carrier demonstrated a
prolonged drug release behavior over 3 weeks in vitro [34]. However, they showed no evidence that chitosan nanoparticles were
encapsulated into erythrocytes. In addition to their anticipated
result that chitosan nanoparticles were encapsulated into erythrocytes, the naoparticles could also be attached to the surface
of erythrocytes, or in both cases. The ideal characteristics for a
substance to be encapsulated in erythrocytes include a small hydrodynamic diameter (usually smaller than 60 nm) and the lack of
interactions with erythrocyte membrane or the other cell constituents [4,32]. However, it is difcult for chitosan nanoparticles
to meet these constraints, since chitosan nanoparticles tend to
swell in aqueous media, and as found in this study, they can be
easily attached to the surface of erythrocyte membrane. Although
the strategy that encapsulating chitosan nanoparticles into erythrocytes may offers a better protection for the nanoparticles,
there still lack of effective and harmless method to encapsulate
relatively large nanoparticles [35]. Instead, by attaching chitosan
nanoparticles to the surface of erythrocytes, perhaps there would
be some factors that should be considered, such as different arrays
of opsonins as well as other plasma proteins, but it also offers the
opportunity of particle-target interactions, especially after attachment of specic ligands onto nanoparticles, such as peptides and
antibodies [2,25,36,37]. This could be exploited in targeted therapy, such as intravascular clotting in tumor blood vessels [38]. Thus,
the attaching strategy seems to be more appropriate to construct a
chitosan nanoparticles-erythrocytes based drug delivery system.
Erythrocytes are coated with a variety of proteins extending
from the membrane surface and many of these proteins are highly
glycosylated with sialic acid residues, whose carboxyl group (pKa
2.6) is mainly responsible for the negative surface charge of erythrocytes [8,39]. Thus, the electrostatic attraction between the
surface ionized groups of erythrocytes and chitosan nanoparticles
may play a role in maintaining the adhesion of chitosan nanoparticles to erythrocyte membrane. This analysis also implies that there
should be a decrease of the surface charge density of erythrocytes
due to a partial neutralization of sialic acid residues by chitosan
nanoparticles, which helps explain the results in Section 3.4 [40,41].
The sialic acid content of circulating erythrocytes decreases with
the aging of erythrocytes [42]. This is not only because the friction
of erythrocytes with themselves or endothelial cells during blood
circulation, but also related to the oxidative damage to erythrocyte membrane [43,44]. Removal of the terminal sialic acid residues
from membrane glycoproteins results in the exposure of the penultimate galactose residues which are the signal for removal of aged
erythrocytes from circulation by RES organs, mainly the liver [42].
In addition, several properties of erythrocytes are correlated with
their lifespan, including membrane phospholipid composition, surface antigens, integrin-associated protein CD47, shape as well as
their extent of deformability [36,45]. Thus, whether the interactions between LMW chitosan nanoparticles and erythrocytes will
lead to an exposure of galactose residues, or result in other changes
in erythrocytes that affect their circulation, require further study.

4. Conclusions
In this study, it was found that the uorescent labeling procedure did not affect the monodispersity and stability of the LMW
chitosan nanoparticles, and it was suggested that suspension of
LMW chitosan nanoparticles in sodium chloride solution instead
of PBS buffer could achieve a good dispersion of nanoparticles.
In the studied concentration range, the LMW chitosan nanoparticles caused negligible damage to erythrocyte membrane and
had no deleterious effects on erythrocytes morphology. Moreover,

by decreasing the volume of LMW chitosan nanoparticles mixed

with erythrocytes, it could avoid the agglutination of erythrocytes.
Owing to the good biocompatibility and natural bioadhesive properties of LMW chitosan, the LMW chitosan nanoparticles could
be served as a suitable drug carrier binding to erythrocytes. The
composite drug delivery system contains no harmful components
and is completely biodegradable. The possibility of combining the
advantages of both LMW chitosan nanoparticles and erythrocytes
as RES-evading drug carrier is attractive and requires more investigations in the future.

Acknowledgment
The authors appreciate Qiwei Yang and Ling Wang for their
assistance in this work.

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