Journal of Biotechnology 145 (2010) 4753

Contents lists available at ScienceDirect

Journal of Biotechnology
j our nal homepage: www. el sevi er . com/ l ocat e/ j bi ot ec
Preparation and characterization of folate-poly(ethylene
glycol)-grafted-trimethylchitosan for intracellular transport of protein through
folate receptor-mediated endocytosis
Yu Zheng
a,b,c
, Xiangrong Song
d
, Michael Darby
e
, Yufeng Liang
f
, Ling He
f
, Zheng Cai
g
, Qiuhong Chen
h
,
Yueqi Bi
i
, Xiaojuan Yang
c
, Jiapeng Xu
j
, Yuanbo Li
k
, Yiyi Sun
l
, Robert J. Lee
c
, Shixiang Hou
a,b,
a
Division of Pharmaceutics, College of Pharmacy, Sichuan University, Renmin Nan Road 3rd section 17, Chengdu 610041, China
b
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education of PR China, Chengdu 610041, China
c
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
d
State Key Laboratory of Biotherapy, West China Hospital, Chengdu, China
e
Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
f
Division of Medicinal Chemistry, College of Pharmacy, Sichuan University, Renmin Nan Road 3rd section 17, Chengdu 610041, China
g
College of Pharmaceutical Science, Southern Medical University, GuangZhou, China
h
College of Materials and Bioengineering, Chengdu University of Technology, Chengdu, China
i
Academy of Chinese Medicine Sciences, Renmin Nan Road 4th section 51, Chengdu, Sichuan, China
j
The Department of Chemical and Biomolecular Engineering, The Ohio State University, USA
k
Yangtze River Pharmaceutical Group Sichuan Hairong Pharmaceutical Co., Ltd., Du Jiangyan, China
l
College of Pharmacy, Chengdu University of Medical Science, Chengdu, China
a r t i c l e i n f o
Article history:
Received 11 March 2009
Received in revised form 30 August 2009
Accepted 5 September 2009
Keywords:
Folate-PEG-g-TMC conjugate
Intracellular protein delivery
Folate
Targeting
a b s t r a c t
To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins
to specic tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was
synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and u-
orescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable
weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers
andthe negativelychargedFITC-BSA. Intracellular uptake of FITC-BSAwas specicallyenhancedinSKOV3
cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared
with A549 cells (folate receptor decient cell line). Folate-PEG-g-TMC shows promise for intracellular
transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.
2009 Elsevier B.V. All rights reserved.
1. Introduction
Although therapeutic proteins are attractive candidates for the
treatment of various human diseases, their therapeutic effects are
still hampered by rapid enzymatic degradation, low target site
specicity and low permeability of cell membranes (Torchilin and
Lukyanov, 2003). Many approaches including polymer conjuga-
tion carrier, microreservoir delivery system, receptor-mediated
endocytosis and transduction using protein transduction domains
(PTDs) have been used to solve these problems. Despite that
transduction via PTDs is very efcient in intracellular delivery of
proteins, it also lacks specicity (Kim et al., 2005). Alternatively,

Corresponding author at: Division of Pharmaceutics, College of Pharmacy,

Sichuan University, Renmin Nan Road 3rd section 17, Chengdu 610041, China.
Tel.: +86 28 85502809; fax: +86 28 85502809.
E-mail address: housix@263.net (S. Hou).
the approach based on the receptor-mediated endocytosis process
possesses both specicity and efciency (Rihova, 1998; Lee et al.,
2005).
Folate receptors are vastly over-expressed in a wide variety of
human tumors, including ovarian, endometrial, colorectal, breast,
lung, renal cell carcinomas, brain metastases derived from epithe-
lial cancers and neuroendocrine carcinomas, but rarely are found
on normal cell surface (Sudimack and Lee, 2000). Folate conjugates
canbeinternalizedbycells throughfolatereceptor-mediatedendo-
cytic mechanism (Lee and Low, 1995). They are not substrates for
the reduced-folate carrier which is the main pathway for cellular
uptake of folate in mammalian cells. Though some folate receptors
areexpressedonthesurfaceof normal cells, theyarelocalizedtothe
surface of polarized epithelia facing lumen and cannot be reached
by intravenously administered folate conjugates (Low, 2004). Thus,
folate conjugates would not be toxic to normal cells. In previous
studies, folate has been conjugated to many polymers, such as
poly(l-lysine) (Kimet al., 2005), PEI (Liang et al., 2008) andchitosan
0168-1656/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2009.09.007
48 Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753
(Chan et al., 2007), etc. The delivery systems made by these folate
conjugates all increased either the intracellular uptake of proteins
or the transfectionrate of DNAcomparedwithunmodiedpolymer
delivery systems.
Chitosan is a cationic polyelectrolyte with biocompatible (Felt
et al., 1998) and biodegradable (Onishi and Machida, 1999) proper-
ties. N, N, N-Trimethylchitosan (TMC) is a permanently quaternized
chitosan derivative and possesses the merits of chitosan men-
tioned above. It can effectively condense proteins at physiological
pH and protects them against enzymatic degradation (Onishi and
Machida, 1999), and has been used to prepare delivery systems for
proteins (Onishi and Machida, 1999; Chen et al., 2007). Polyethy-
lene glycol-grafted-trimethyl chitosan(PEG-g-TMC) copolymer has
been synthesized and copolymer/insulin complex has been pre-
pared for peroral delivery of insulin (Jintapattanakit et al., 2007).
This complex better protects insulin against enzyme degrada-
tion than TMC/insulin complex because PEG segments hinder the
enzyme access to insulin and have a shielding effect on it.
Inthis study, folate was conjugatedto TMCthrougha PEGlinker.
Besides the shielding effect on protein, there are some other rea-
sons to use this linker. First, PEGylation decreases the toxicity of
TMC (Mao et al., 2005); secondly, the PEG linker increases spatial
freedom of the folate molecule and consequently enables the ef-
cient binding of folate to folate receptor (Ghaghada et al., 2005).
Folate-PEG-g-TMC/FITC-BSA complex was prepared through ionic
interaction between positively charged copolymers and negatively
charged protein. The intracellular uptake of the complex was mea-
sured and compared using two different cell lines, SKOV3 (a folate
receptor over-expressing cell line) and A549 (a folate receptor de-
cient cell line).
2. Materials and methods
2.1. Materials and reagents
NH
2
PEG3000NH
2
, 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDC), folate, N-hydroxysuccinimide,
succinic anhydride, FITC-BSA and CelLytic Mcell lysis reagent were
all obtained from Sigma, methoxypolyethylene glycol succinate
N-hydroxysuccinimide (mPEG-NHS, MW 3000) was purchased
from NOF Cooperation (Tokyo, Japan), chitosan (MW 50kDa,
degree of deacetylation (DD) 95%) was purchased from Haidebei
Marine Bioengineering Co. Ltd. (China), the other chemicals and
solvent were purchased from Kermel Co. Ltd. (China). Fetal bovine
serum (FBS), RPMI 1640 and folate-free RPMI 1640 media were
obtained from Gibco BRL (Grand Island, NY). Bio-Rad kit was
purchased from Bio-Rad Laboratories, Inc. Cells were provided by
the Cell Bank of the Chinese Academy of Science (Shanghai, China).
Protease inhibitor cocktail was purchased from Roche (USA).
Mov18 was purchased from Alexis Biochemicals (San Diego, USA).
SuperSignal West Dura trial kit was purchased from Pierce (USA).
2.2. Synthesis of mPEG-g-TMC
TMC was synthesized by the method of Polnok et al. (2004).
Degree of quaternization (DQ) is calculated using Eq. (1) as
described in the method of Polnok et al. and the tertiary amine
number as a percentage of total nitrogen number (DT) is calculated
using Eq. (2):
DQ(%) =
[(CH
3
)
3
]
9 [H]
100 (1)
DT(%) =
[(CH
3
)
2
]
6 [H]
100 (2)
[(CH
3
)
3
] and [(CH
3
)
2
] are the integrals of the quaternary amino
group peak at 3.3ppm and tertiary amino group peak at 2.5ppm,
respectively, and [H] is the integral of the
1
H peaks from 4.7 to
5.7ppm.
mPEG-NHS was conjugated to TMC. In detail, 5ml of an aque-
ous TMC (0.117g) solution was added to 50ml of a mPEG-NHS
(0.36g, 0.12mmol)/DMSO solution and stirred for 24h at room
temperature. The reaction mixture was dialyzed against water
(Spectra/Por

, MW cutoff 10kDa) to remove unreacted mPEG-

NHS. The product was freeze-dried. Yield: 89%.
1
H NMR (D
2
O,
in ppm): 2(CH
3
CO, 45H, t), 2.5(N(CH
3
)
2
, 402H, s), 3.3(N
+
(CH
3
)
3
,
1098H, s), 4.8(CH, 30H, m), 5.2(CH, 150H, m), 5.4(CH, 126H, m),
3.8(PEG, 5506H, bs). The number of PEG chain as a percentage of
total nitrogen number per mPEG-g-TMC molecule (graft ratio) was
calculated using Eq. (3):
Graft ratio(%) =
[PEG]
248 [H]
100 (3)
[PEG] is the integral of proton peak of PEG block at 3.8ppm
(OCH
2
). The number of the protonper PEGchainis 248 according
to the molecular mass of mPEG-NHS given by the supplier. [H] is
the integral of the
1
H peaks from 4.7 to 5.7ppm.
2.3. Synthesis of folate-PEG conjugate
The polyoxyethylene3000bis(amine) was convertedtoaninter-
mediate with one carboxyl terminal group by acylation with
succinic anhydrideas showninScheme1. Indetail, 5ml of asuccinic
anhydride (0.0084g, 0.084mmol)/methanol solution was added
dropwise to 50ml of a polyoxyethylene3000 bis(amine) (0.45g,
0.15mmol)/methanol solution and stirred for an additional 1h
at room temperature. The solvent was evaporated under reduced
pressure and the product was isolated via silica gel chromatogra-
phy using 88:11:4 CHCl
3
/MeOH/NH
4
OHas aneluent. The off-white
powder was determined to be pure by
1
H NMR (>95%). Yield: 30%.
1
HNMR (CDCl
3
, in ppm): 2.5(CH
2
COOH, 2H, t), 2.6(CH
2
CH
2
COOH,
2H, t), 2.9(CH
2
NH, 2H, t), 3.1(CH
2
NH
2
, 2H, t), 3.7(PEG, 242H, bs),
4.2(OCH
2
CH
2
NH
(2)
, 4H, t).
Folate was conjugatedto this intermediate via anamide linkage.
In detail, folic acid (0.295g, 0.67mmol) was activated by reacting
withEDC(1.29g, 6.73mmol) andNHS (0.093g, 0.81mmol) in20ml
anhydrous DMSO at room temperature for 3h. 2-Mercaptoethanol
(4.63ml, 65mmol) was added to quench the EDC. This solution was
then added dropwise with stirring to a 10ml anhydrous DMSO
solution of the intermediate (0.41g, 0.134mmol) and allowed to
react for 16h. The reaction mixture was dialyzed against DMSO
(Spectra/Por

, MW cutoff 1kDa) to remove unreacted folate, and

then the dialysis medium was changed to deionized water to
remove DMSO. Dialyzed product was freeze-dried. Yield: 92%.
1
H
NMR (DMSO-d6, in ppm): 1.9(CH, 1H, sextet), 2.0(CH, 1H, sextet),
4.4(CH, 1H, q), 4.5(CH
2
, 2H, d), 6.6(CH, 2H, d), 7.7(CH, 2H, d), 8.6(CH,
1H, s) and 3.5(PEG, 242H, bs).
2.4. Synthesis of folate-PEG-g-TMC
A 5ml of aqueous solution of TMC (0.117g) was added to a
50ml of folate-PEG conjugate (0.424g, 0.12mmol)/DMSO solution
containing EDC (0.460g, 2.4mmol) and NHS (0.033g, 0.288mmol)
and stirred for 24h at room temperature. The reaction mixture
was dialyzed against DMSO (Spectra/Por

, MW cutoff 10kDa) to
remove unreacted folate-PEG conjugate, and then dialyzed against
deionized water to remove DMSO. The product was freeze-dried.
Yield: 85%. The number of folate-PEG chain as a percentage of total
nitrogen number per folate-PEG-g-TMC molecule (graft ratio) was
Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753 49
Scheme 1. Synthetic route of folate-PEG-g-TMC. (A) Acylation of polyoxyethylene3000 bis(amine) using succinic anhydride; (B) preparation of folate-PEG conjugate; (C)
preparation of folate-PEG-g-TMC.
calculated using Eq. (4):
Graft ratio(%) =
[PEG]
266 [H]
100 (4)
[PEG] is theintegral of protonpeakof PEGat 3.8ppm(OCH
2
).
Thenumber of protonper PEGchainis 266accordingtothemolecu-
lar mass of polyoxyethylene3000 bis(amine) given by the supplier.
[H] is the integral of the
1
H peaks from 4.7 to 5.7ppm.
2.5. Characterization of copolymers
Nuclear magnetic resonance (NMR) spectra of both copolymers
dissolved in D
2
O were recorded on a Varia Unity Inova-400MHz
spectrometer (USA) at room temperature. Gel permeation chro-
matography (GPC) of both copolymers was carried out on Waters
Styragel HR columns (USA) using dimethylformamide as the eluent
(0.5ml/min). Fifteen microliter samples were injected each time.
The samples were analyzed with a Waters 2414 differential refrac-
tive index (RI) detector.
2.6. MTT assay
Amouse connective tissue broblast cell line, L929, was selected
to evaluate cytotoxicity following a previous method (Mao et al.,
2005). Relative cell viability at 3h compared to control cells con-
taining cell culture medium without copolymer was calculated
using Eq. (5):
Relative cell viability(%) =
[A]
test
[A]
control
100% (5)
IC
50
values, which represent concentration of the copolymers
resulting in 50% inhibition of cell growth, were calculated.
2.7. Preparation of complexes between copolymers and FITC-BSA
The appropriate amount of mPEG-g-TMC or folate-PEG-g-TMC
(0320g) serially diluted in 50l phosphate buffered saline at
pH7.2 (PBS) was mixed with 40g FITC-BSA dissolved in 50l PBS
and left at room temperature for 20min before use.
2.8. Characterization of complexes
The particle sizes and -potentials of two complexes with dif-
ferent weight ratios of copolymer to FITC-BSA were determined
at room temperature using a Zetasizer Nano ZS 90 (Malvern
Instruments Ltd., Malvern, UK). Morphology of the folate-PEG-g-
TMC/FITC-BSA complex at a weight ratio of 6 was analyzed by
atomic force microscopy (AFM). Freshly peeled mica was used as
sample support. Sample was analyzed within 2h after preparation.
Microscopy was performed on a Dimension 3100 (Veeco Instru-
ments, San Jose, USA) using commercial silicon tips attached to
an Itype cantilever with a length of 125m. The scan size was
2.5m2.5m.
2.9. Cell and cell culture conditions
SKOV3 cells (a human ovarian cancer cell line) and A549 cells
(a human lung carcinoma cell line) were maintained in standard
RPMI 1640 media supplemented with 10% (v/v) fetal bovine serum.
The cells were cultured as a monolayer in a humidied atmosphere
containing 5% CO
2
at 37

C.
50 Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753
2.10. Immunoblotting
The method of Stella et al. (2007) was used with some modica-
tions. Briey, SKOV3 and A549 cells were harvested and lysed with
lysis buffer containing a protease inhibitor cocktail. 30g of cel-
lular protein was applied to immunoblotting. Folate receptor blots
were incubated with a monoclonal antibody to folate receptor
(Mov18). -Actin was used as an internal control and blots were
incubated with indicated antibody. Signals were developed with a
SuperSignal West Dura trial kit.
2.11. Intracellular transport of the complexes
SKOV3 cells (folate receptor over-expressing cell line) and A549
cells (folate receptor decient cell line) were used for intracellu-
lar transport of complexes. The medium for culturing SKOV3 cells
was changed to folate-free RPMI 1640 one week before this exper-
iment. The cells were seeded onto a 24-well tissue culture plate at
a density of 3.310
4
cells/well and allowed to growfor 24h before
the treatment. Some cells were incubated with serumfree medium
containing 40g/ml Mov18 for 1h before the treatment. The cells
were then incubated in 1ml of serum free medium with or with-
out folate (1mM) containing100gFITC-BSAor various complexes
containing equivalent amount of FITC-BSA at 37

C for 3h. After

that, the media were aspirated and the cells were washed six times
with 0.9% NaCl (pH3 adjusted with acetic acid) to remove the com-
plexes present inthe medium. 200l lysis buffer was addedto each
well for half an hour and then the cell lysates were centrifuged at
14,500g for 10min at 4

C. The intracellular uptake of the FITC-

BSAinthe cell lysates was determinedby spectrouorophotometry
(Hitachi f-4600, Japan). Excitation and emission wavelengths were
494 and 520nm, respectively. The protein concentration in the cell
lysates was measured using a Bio-Rad kit.
2.12. Fluorescent microscopy
SKOV3 cells were seeded onto a 24-well tissue culture plate at a
density of 3.310
4
cells/well and allowed to grow for 24h before
the treatment. The cells were then incubated in 1ml of serum free
mediumwithor without folate (1mM) containing 100g FITC-BSA
or various complexes containing equivalent amount of FITC-BSA at
37

C for 3h. The cells were washed six times with 0.9% NaCl (pH
3 adjusted with acetic acid) to remove the complexes present in
the medium. The cells were imaged by uorescence microscopy
(Olympus FV1000, Japan) using a 488nm excitation wavelength.
3. Results and discussions
3.1. Characterization of the copolymers
The DQ and DT of synthesized TMC were 39% and 22%, respec-
tively.
1
H NMR spectra of the two copolymers are shown in Fig. 1.
Both copolymers showsignal at 3.8ppm([CH
2
O]
n
) for PEG and
signals at 2.5 (N(CH
3
)
2
), 3.3 (N
+
(CH
3
)
3
) and 4.75.7 (CHO) ppm
for TMC. Folate-PEG-g-TMC show signals at 6.6 (b), 7.7 (c), 8.5
(a) ppm for folate (Fig. 1A). These results are consistent with the
expectedchemical structureof thecopolymers. Themolecular mass
of TMC was calculated as around 75.2kDa based on the DQ, DT
and molecular mass of chitosan. According to the molecular mass
and DD of chitosan given by the supplier, there are 308 saccharide
Fig. 1.
1
H NMR spectra of copolymers (A) folate-PEG-g-TMC; (B) mPEG-g-TMC.
Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753 51
Fig. 2. Characterization of complexes: (A) Particle size and -potential variations of the two complexes as a function of copolymer/FITC-BSA weight ratio; (B) AFM image of
folate-PEG-g-TMC/FITC-BSA complex at a weight ratio of 6.
residues per chitosan molecule. Thus the number of quaternary
amine is 120. The composition of folate in the folate-PEGconjugate
is 88.04.3% (mol%) based on
1
H NMR spectrum of the folate-PEG
conjugate (data not shown). The graft ratio of PEG in mPEG-g-
TMC is 7.20.8% and that of folate-PEG in folate-PEG-g-TMC is
6.60.4%, indicating that the lowmolecular mass folate (441.4Da)
does not affect the conjugation of folate-PEG to TMC. The molecu-
lar mass of folate-PEG-g-TMC was calculated as around 132.8kDa.
Both mPEG-g-TMC and folate-PEG-g-TMC show unimodal molec-
ular mass distribution in the GPC eluograms (data not shown)
suggesting there are negligible intermediate prepolymers present
in the copolymer products.
The IC
50
values of TMC, mPEG-g-TMC and folate-PEG-g-TMC are
80, 720 and 680g/ml, respectively, which is consistent with pre-
vious work (Mao et al., 2005) and suggests that the copolymers are
less toxic than TMC and that folate conjugation does not increase
the toxicity of folate-PEG-g-TMC.
3.2. Characterization of the complexes
According to previous work, the pK
a
values of mono- and di-
methylated amines are around 6 (Chen et al., 2007), which means
they will not be ionized at the pH at which the complexes were
prepared. Only tri-methylated amines can react with negatively
charged FITC-BSA with a pI value of 4.8. Fig. 2A shows the par-
ticle sizes of the two complexes with different weight ratios of
copolymer to protein. There is no signicant difference between
the particle sizes of the two complexes at the same weight ratio
(p>0.05). The change in particle size of the mPEG-g-TMC/FITC-BSA
complex as a function of the weight ratio of copolymer/FITC-BSA
is similar to that of the folate-PEG-g-TMC/FITC-BSA complex. The
particle sizes of both complexes sharply increased up to a weight
ratio of 1.6, then decreased to less than 200nm thereafter which
indicates the formation of polyelectrolyte complex by ionic inter-
action. Similar phenomenon was also found in previous research
(Lee and Low, 1995). Theoretically, one molecule of folate-PEG-g-
TMC with a mass/charge ratio around 1107 (the molecular mass
to the number of tri-methylated amine) can bind 1.2 FITC-BSA
molecules giving a weight ratio of folate-PEG-g-TMC/FITC-BSA of
1.7 on the assumption that all negatively charged Asp and Glu
residues of FITC-BSA (99 per molecule) fully interact with all
quaternary amines of folate-PEG-g-TMC. This suggests that the
weight ratio of copolymer/FITC-BSA for charge neutralization is
1.7. As a result, the increase of particle sizes of the complexes
with weight ratios at and below 1.6 is likely due to aggregation
of the complexes caused by decreased repellent forces among
particulates. The assumption above can also be conrmed by the
change of -potentials of the complexes. Neutral complexes were
formed when copolymer/FITC-BSA weight ratio are between 0.8
and1.6 whereas negatively andpositively chargedcomplexes were
detected at copolymer/FITC-BSA ratios belowand above that range
(Fig. 2A). The complexes with the weight ratios above 1.6 in this
study are comparable in size to complexes made of poly(ethylene
glycol)-graft-trimethyl chitosan and insulin (Mao et al., 2005). The
AFM image of folate-PEG-g-TMC/FITC-BSA complex is shown in
Fig. 2B and conrms the formation of spherical and uniform com-
plexes.
3.3. Immunoblotting
By immunoblotting with the specic monoclonal antibody
Mov18 for human folate receptor , folate receptor expression
in SKOV3 cells and A549 cells was determined. The immunoblots
showed a broad band at 3840kDa for the SKOV3 cell line and just
a thin band for the A549 cell line (Fig. 3). The molecular weight
of folate receptor is consistent with previous report (Stella et al.,
2007). It can be concluded that folate receptor has high expression
in the SKOV3 cell line and very low expression in the A549 cell
line. Therefore, the SKOV3 cell line was used in the cellular uptake
experiment of the complexes, and the A549 cell line was used as a
negative control.
3.4. Intracellular transport of the complexes
Intracellular uptake of folate-PEG-g-TMC/FITC-BSA complex by
SKOV3 cells is greatly enhanced in a dose-dependent manner at
weight ratios of folate-PEG-g-TMC/FITC-BSA above 2.8 while that
of mPEG-g-TMC/FITC-BSA complex is only slightly enhanced with
the increase of weight ratio of mPEG-g-TMC/FITC-BSA (Fig. 4). Thus
there is a signicant difference betweenthe intracellular uptakes of
the two complexes when the weight ratio is above 2.8 (Fig. 4A). By
pretreating cells with medium containing Mov18, the intracellular
52 Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753
Fig. 3. Immunoblottinganalysis of folatereceptor in(A) A549cell lineand(B) SKOV3
cell line.
uptake of folate-PEG-g-TMC/FITC-BSA complex at a weight ratio of
6 was blocked, while that of mPEG-g-TMC/FITC-BSA complex at a
weight ratio of 6 was not obviously affected. The inuence of excess
free folate in media on the intracellular uptake of the complexes
was also compared. Because the free folate in the medium (1mM)
will competitively bind to folate receptor (Lee and Low, 1995),
the intracellular uptake of folate-PEG-g-TMC/FITC-BSA complex is
inhibited by 79%. The intracellular uptake of mPEG-g-TMC/FITC-
BSA complex at a weight ratio of 6 is minimally inuenced by the
addition of folate to the medium (Fig. 4B).
Inorder tofurther verifytheeffect of folatereceptor ontheintra-
cellular uptakes of the complexes, A549 cells decient in folate
receptors were used for the intracellular uptake experiment. The
intracellular uptake of folate-PEG-g-TMC complex by A549 cells is
not greatly enhanced when the weight ratio of copolymer/FITC-
BSA is above 2.8 and there is no signicant difference between
the intracellular uptakes of both complexes. The slight increase
in intracellular uptakes of both complexes by A549 cells when
the complex/FITC-BSA weight ratio increases can be explained by
the increased nonspecic effects of the complexes on intracellular
uptakes (Kimet al., 2005; HaradaandKataoka, 1998) (Fig. 4C). Addi-
tionally, neither the pretreatment with Mov18 nor the addition of
excess free folate in the medium (1mM) affected the intracellular
uptake of either complex (Fig. 4D). These phenomena indicate that
the folate-PEG-g-TMC/FITC-BSA complex can specically enhance
the intracellular uptake of FITC-BSA in SKOV3 cells through folate
receptor-mediated endocytosis but not in A549 cells.
Fig. 4. (A) Intracellular uptakes of the two complexes by SKOV3 cells cultivated in folate-free medium as a function of copolymer/FITC-BSA weight ratio; (B) Intracellular
uptakes of FITC-BSA and two complexes at a weight ratio of 6 by SKOV3 cells cultivated in medium with 1mM folate (FOL+) or without folate (FOL) or pretreated with
MOv18 (Mov18); (C) Intracellular uptakes of two complexes by A549 cells cultivated in folate-free mediumas a function of copolymer/FITC-BSAweight ratio; (D) Intracellular
uptakes of FITC-BSA and two complexes at a weight ratio of 6 by A549 cells cultivated in mediumwith 1mMfolate (FOL+) or without folate (FOL) or pretreated with Mov18.
Results were the mean of three independent experiments and statistically analyzed using one-way ANOVA. Two asterisks: p<0.01; one asterisk: p<0.05.
Y. Zheng et al. / Journal of Biotechnology 145 (2010) 4753 53
Fig. 5. Fluorescent microscopy images of SKOV3 cells cultivated in folate-free medium following incubation for 3h with (A) FITC-BSA; (B) folate-PEG-g-TMC/FITC-BSA
complex at a weight ratio of 6; (C) mPEG-g-TMC/FITC-BSA at a weight ratio of 6. An equivalent amount of FITC-BSA was used.
3.5. Fluorescent microscopy
The intracellular uptake of FITC-BSAbythe SKOV3cells is almost
negligible (Fig. 5A). The intracellular transport of folate-PEG-g-
TMC/FITC-BSA complex is very efcient (Fig. 5B), however, that of
mPEG-g-TMC/FITC-BSA complex is less efcient (Fig. 5C), which is
consistent with Fig. 4.
4. Conclusions
Folate-PEG-g-TMC was successfully synthesized. Nano-scaled
spherical folate-PEG-g-TMC/FITC-BSA complex was prepared and
employedas anewintracellular proteindeliverysystem. Intracellu-
lar uptake of FITC-BSA was greatly enhanced using this complex in
cells over-expressingfolate receptor, whichwas attributedtofolate
receptor-mediated endocytosis. This intracellular protein delivery
systemcan also be extended to other negatively charged therapeu-
tic proteins.
Acknowledgements
This study was nancially supported by National Science Foun-
dation of China (NSFC) (30772668).
References
Chan, P., Kurisawa, M., Chung, J.E., Yang, Y.Y., 2007. Synthesis and characterizationof
chitosan-g-poly(ethyleneglycol)-folateas a non-viral carrier for tumor-targeted
gene delivery. Biomaterials 28, 540549.
Chen, F., Zhang, Z.R., Huang, Y., 2007. Evaluation and modication of N-trimethyl
chitosan chloride nanoparticles as protein carriers. Int. J. Pharm. 336, 166173.
Felt, O., Buri, P., Gurny, R., 1998. Chitosan: a unique polysaccharide for drug delivery.
Drug Dev. Ind. Pharm. 24, 979993.
Ghaghada, K.B., Saul, J., Natarajan, J.V., 2005. Folate targeting of drug carriers: a
mathematical model. J. Controlled Release 104, 113128.
Harada, A., Kataoka, K., 1998. Novel polyion complex micelles entrapping enzyme
molecules in the core: preparation of narrowly-distributed micelles from
lysozyme and poly(ethylene glycol)poly(aspartic acid) block copolymer in
aqueous medium. Macromolecules 31, 288294.
Jintapattanakit, A., Junyaprasert, V.B., Mao, S.R., Sitterberg, J., Bakowsky, U., Kissel,
T., 2007. Peroral delivery of insulin using chitosan derivatives: a comparative
study of polyelectrolyte nanoxomplexes and nanoparticles. Int. J. Pharm. 342,
240249.
Kim, S.H., Jeong, J.H., Joe, C.O., Park, T.G., 2005. Folate receptor mediated intracel-
lular protein delivery using PLLPEGFOL conjugate. J. Controlled Release 103,
625634.
Lee, E.S., Na, K., Bae, Y.H., 2005. Doxorubicin loaded pH-sensitive polymeric
micelles for reversal of resistant MCF-7 tumor. J. Controlled Release 103, 405
418.
Lee, R.J., Low, P.S., 1995. Folate-mediatedtumor cell targetingof liposome-entrapped
doxorubicin in vitro. Biochim. Biophys. Acta 1233, 134144.
Liang, B., He, M.L., Xiao, Z.P., 2008. Synthesis and characterization of folate-
PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery. Biochem.
Biophys. Res. Commun. 367, 874880.
Low, P.S., 2004. Folatereceptor-targeteddrugs for cancer andinammatorydiseases.
Adv. Drug Deliv. Rev. 56, 10551058.
Mao, S.R., Shuai, X.T., Unger, F., Wittmar, M., Xie, X.L., Kissel, T., 2005. Synthesis, char-
acterization and cytotoxicity of poly(ethylene glycol)-graft-trimethyl chitosan
block copolymers. Biomaterials 26, 63436356.
Onishi, H., Machida, Y., 1999. Biodegradation and distribution of water-soluble chi-
tosan in mice. Biomaterials 20, 175182.
Polnok, A., Borchard, G., Verhoef, J.C., Sarisut, N., Junginger, H.E., 2004. Inuence of
methylation process on the degree of quaternization of N-trimethyl chitosan
chloride. Eur. J. Pharm. Biopharm. 57, 7783.
Rihova, B., 1998. Receptor-mediatedtargeteddrugor toxindelivery. Adv. DrugDeliv.
Rev. 29, 273289.
Stella, B., Marsaud, V., Arpicco, S., Geraud, G., Cattel, L., Couvreur, P., Renoir, J.M., 2007.
Biological characterization of folic acid-conjugated poly(H
2
NPEGCA-co-HDCA)
nanoparticles in cellular models. J. Drug Target. 15 (2), 146153.
Sudimack, J., Lee, R.J., 2000. Targeted drug delivery via the folate receptor. Adv. Drug
Deliv. Rev. 41, 147162.
Torchilin, V.P., Lukyanov, A.N., 2003. Peptide and protein drug delivery to
and into tumors: challenges and solutions. Drug Discov. Today 8, 259
266.