Self-quenchable biofunctional nanoparticles of heparin-folate- photosensitizer conjugates for photodynamic therapy

Article abstract of DOI:10.1016/j.carbpol.2011.05.011

Novel amphiphilic polysaccharide/PS conjugates synthesized by chemical conjugation of heparin with a hydrophobic photosensitizer (PS), pheophorbide a (PhA), and a targeting ligand, folate, were investigated for their potential application in photodynamic therapy (PDT). The anticoagulant activity of heparin-PhA (HP) and folate-heparin-PhA (FHP) conjugates was significantly decreased compared to that of heparin, thereby potentially reducing the hemorrhagic side effects. The critical self-quenching concentrations of the conjugates were decreased as the content of PhA rose. HP and FHP conjugates formed nano-sized particles in aqueous medium through a self-assembling process, and the nanoparticles were 130-170 nm in size, with a unimodal pattern of size distribution. Photoactivity of HP and FHP nanoparticles was evaluated by measuring the generation of singlet oxygen in DMF and PBS. The nanoparticles displayed a self-photoquenching effect in PBS, while the generation of singlet oxygen dramatically increased in DMF. HP and FHP nanoparticles exhibited marked phototoxicity on HeLa cells and were minimally dark-toxic without light treatment.

Full text of DOI:10.1016/j.carbpol.2011.05.011

Carbohydrate Polymers 86 (2011) 708–715
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Self-quenchable biofunctional nanoparticles of heparin–folate-photosensitizer
conjugates for photodynamic therapy
Li Lia,1, Byoung-chan Baeb,1, Thanh Huyen Trana, Kwon Hyeok Yoona, Kun Nab,∗, Kang Moo Huha,∗
a Department of Polymer Science and Engineering, Chungnam National University, Daejeon 305-764, Republic of Korea
b Department of Biotechnology, The Catholic University of Korea, Bucheon 420-743, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel amphiphilic polysaccharide/PS conjugates synthesized by chemical conjugation of heparin with a
hydrophobic photosensitizer (PS), pheophorbide a (PhA), and a targeting ligand, folate, were investigated
for their potential application in photodynamic therapy (PDT). The anticoagulant activity of heparin-PhA
(HP) and folate–heparin–PhA (FHP) conjugates was significantly decreased compared to that of heparin,
thereby potentially reducing the hemorrhagic side effects. The critical self-quenching concentrations
of the conjugates were decreased as the content of PhA rose. HP and FHP conjugates formed nano-sized
particles in aqueous medium through a self-assembling process, and the nanoparticles were 130–170 nm
in size, with a unimodal pattern of size distribution. Photoactivity of HP and FHP nanoparticles was
evaluated by measuring the generation of singlet oxygen in DMF and PBS. The nanoparticles displayed a
self-photoquenching effect in PBS, while the generation of singlet oxygen dramatically increased in DMF.
HP and FHP nanoparticles exhibited marked phototoxicity on HeLa cells and were minimally dark-toxic
without light treatment.
Received 16 March 2011
Received in revised form 10 May 2011
Accepted 11 May 2011
Available online 19 May 2011
Keywords:
Heparin
Photosensitizer
Photodynamic therapy
Self-assembled nanoparticles
Drug delivery
© 2011 Elsevier Ltd. All rights reserved.
1. Introduction
limited solubility in aqueous media. Therefore, the formulation of
an injectable PhA product with satisfactory solubility in aqueous
Photodynamic therapy (PDT) is a protocol that uses photo-
sensitizers (PSs) and light in the presence of oxygen to cause
selective damage to a target tissue (Dolmans, Fukumura & Jain,
2003). Because PSs are typically harmless without light, tumor site
treatment can be precisely targeted by selective illumination, lim-
iting the damage to surrounding healthy tissues. Pheophorbide
a (PhA), a second generation PS, is an anionic porphyrin deriva-
tive that can be easily prepared from chlorophyll. Its antitumor
effect when used as a PDT agent on a number of human cancer
cell types, including colonic cancer cells (Hajri et al., 2002), uter-
ine carcinoma cells (Tang, Liu, Zhang, Fong & Fung, 2009), hepatic
carcinoma cells (Tang et al., 2006), and human lung cancer cells
(Yin, Zhou, Jie, Xing & Zhang, 2004) has been reported. It is known
that PhA has a high extinction coefficient in the red region of the
spectrum. This provides an advantage over porphyrins used in PDT,
where longer wavelength lasers are used for better penetration into
tissues. In addition, its relatively fast elimination from the body
may shorten side effects caused by long-lasting cutaneous photo-
sensitivity (Roder, Hanke, Oelckers, Hackbarth & Symietz, 2000).
However, the intravenous use of PhA is greatly hampered by its
solutions is of great interest for PDT.
The formulation of hydrophobic PS using a nanoparticulate
drug delivery system is an attractive strategy (Bechet et al.,
2008). Nano-drug carriers may not only enhance the solubility
of hydrophobic PS, but also enable the incorporated drugs to be
passively transported and targeted to the tumor site through the
enhanced permeability and retention (EPR) effect. Furthermore, the
presence of active targeting molecules on the nanoparticle sur-
face can further enhance the therapeutic efficacy and reduce the
side effects of hydrophobic PDT agents, while achieving desired
effects and a greater uptake of the nanoparticles by tumor cells
(Allison, Mota, Bagnato & Sibata, 2008; Konan, Gurny & Allemann,
2002).
Among various biomaterials, natural polysaccharides seem to be
the most promising materials for the preparation of nano-PS carri-
ers due to their outstanding merits (Liu, Jiao, Wang, Zhou & Zhang,
2008), such as high water solubility, good biocompatibility, a wide
range of molecular weights, and an abundance of sources. In recent
studies, polysaccharides have been popularly used for the formu-
lation of hydrophobic PSs, such as protoporphyrin IX encapsulated
glycol chitosan-based nanoparticles (Lee et al., 2009), acetylated
hyaluronic acid/PhA conjugates (Li, Bae, & Na, 2010; Li, Huh, Lee, &
Kim, 2010), and pullulan/folate-PhA conjugates (Bae & Na, 2010).
These PS formulations showed great potential for application in
PDT.
∗
Corresponding authors. Tel.: +82 42 821 6663; fax: +82 42 821 8910.
E-mail addresses: kna6997@catholic.ac.kr (K. Na), khuh@cnu.ac.kr (K.M. Huh).
1
These authors contributed equally to this work.
0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2011.05.011

L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
709
In this study, we developed
a
new amphiphilic polysac-
organic solvent, and unreacted compounds. The dark green powder
was obtained after ultracentrifugation and freeze-drying (Fig. 1b).
charide/PS conjugate by chemical conjugation of heparin with
hydrophobic PhA and a targeting ligand, folate. Heparin, a highly
sulfated polysaccharide belonging to the family of glycosaminogly-
cans, has numerous important biological activities associated with
its interaction with diverse proteins, such as anticoagulant activity
(Linhardt, 1991), and inhibition of VEGF- and bFGF-mediated tumor
angiogenesis (Mousa & Petersen, 2009), tumor metastasis (Bobek
& Kovarik, 2004), and proliferation of arterial smooth muscle cells
(Smorenburg & Van Noorden, 2001). Recently, conjugates of hep-
arin with hydrophobic anticancer agents or low molecular weight
molecules have been widely investigated as antitumor drugs and
drug carriers (Cho, Moon, Park, Jeon, Byun & Lee, 2008; Park et al.,
2006; Park, Kim, Tran, Huh & Lee, 2010; Park, Tran, et al.2010). How-
ever, the conjugation of PS with heparin for use in actively targeted
PDT has not been reported.
Here, we report the syntheses and characterizations of
heparin-PhA (HP), and folate–heparin–PhA (FHP) conjugates. The
anticoagulant activities and the critical self-quenching concen-
trations (CQCs) of conjugates were studied. Physicochemical
properties, such as particle size and zeta potential, were also exam-
ined. The photoactivity of HP and FHP nanoparticles, as well as their
in vitro phototoxicities on HeLa cells, was also investigated.
2.2.3. Synthesis of HP and FHP conjugates
HP was synthesized by coupling aminated PhA with heparin
through amide covalent bonds using EDC as a coupling agent. In
brief, heparin (50 mg) was dissolved in 5 mL of formamide by gentle
heating. EDC (24.8 mg, in excess) was added to the heparin solution,
followed by mixing with aminated PhA in DMF (5 mL). The feed
ratios of aminated PhA to heparin are listed in Table 1. The reaction
was carried out at room temperature for 36 h under nitrogen gas.
After the coupling reaction, the reacted solution was dialyzed for
3 days to remove low molecular agents. The crude products were
obtained after freeze-drying, and then washed with methanol to
remove uncoupled aminated PhA. The final products were collected
by centrifugation, and dried under vacuum. For the synthesis of FHP
conjugates, the heparin solution and aminated PhA solution were
prepared first, and then EDC and aminated folate were added to the
heparin solution, followed by mixing with aminated PhA. The one-
step reaction for FHP and the purification steps were conducted
as those used for HP conjugates (Fig. 1c). The synthesized HP and
FHP conjugates were analyzed by 1H NMR spectroscopy (Bruker,
Germany).
2.3. Characterization of HP and FHP conjugates
2. Experimental
The coupling ratios of PhA and folate on the heparin backbone
were determined by a colorimetric method. Briefly, the lyophilized
HP or FHP was dissolved in 10 mL formamide to obtain a clear solu-
tion. The absorbance at 290 nm and 669 nm was read with a UV-VIS
spectrophotometer (Mini-1024, SHIMADZU, Japan). The content of
PhA and folate conjugated to heparin was calculated based on a cal-
ibration curve of PhA (669 nm) and folate (290 nm) in formamide,
respectively.
2.1. Materials
Heparin (Fraxiparin®), with an average molecular weight of
6000 Da, was obtained from Mediplex Co. (Korea). Pheophorbide, a
(PhA), was purchased from Frontier Scientific Inc. (Logan, UT, USA).
Folate, ethylenediamine, N,Nꢀ-dicyclohexylcarbodiimide (DCC), N-
hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC), 9,10-dimethylanthracene
(DMA), and pyrene were purchased from Sigma–Aldrich Chemical
Co. (St. Louis, MO, USA). Spectra/Por membranes were purchased
from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA
and Canada). RPMI 1640 medium, Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin
and streptomycin), and Dulbecco’s phosphate buffered saline
(DPBS) were obtained from GibcoBRL (Invitrogen Corp., Carlsbad,
CA, USA). All chemicals were analytical grade and used as received
without further purification.
The anticoagulant activities of HP and FHP conjugates were
determined by the FXa chromogenic assay that was described in
detail in our previous report (Li et al., 2011).
2.4. Measurement of the critical self-quenching concentration
(CQC)
The CQCs of HP and FHP conjugates were determined using
pyrene fluorescence spectroscopy, as described in the literature
(Bae & Na, 2010) with minimal modification. Briefly, pyrene solu-
tion in tetrahydrofuran (THF) (0.5 mg/mL) was added to 2 L of
dionized water in such an amount that the final concentration of
pyrene in water was 6.0 × 10−7 M. THF was removed by distilla-
tion at 70 ◦C for 3 h with vigorous stirring. HP and FHP solutions
with various concentrations were prepared and mixed with pyrene
solution. The final concentrations of HP and FHP conjugates in the
mixture ranged from 0.05 mg/L to 200 mg/L, which included a trace
of pyrene (3.0 × 10−7 M) in each vial. Fluorescence measurements
were performed at room temperature using a Jasco Model FP6500
spectrofluorometer (Japan). The slit widths were fixed at 10 nm.
Fluorescence excitation spectra of pyrene were recorded using an
emission wavelength of 390.0 nm.
2.2. Synthesis of heparin–PhA (HP) and folate–heparin–PhA
(FHP) conjugates
2.2.1. Preparation of aminated folate
The synthetic method for aminated folate was previously
reported elsewhere (Li, Bae, et al., 2010; Li, Huh, et al., 2010).
Briefly, folate (441 mg) dissolved in 20 mL of dimethyl sulfox-
ide (DMSO) was activated with DCC (248 mg) and NHS (230 mg)
at room temperature for 12 h. The by-product, dicyclohexylurea
(DCU), was removed by filtration. The resulting activated folate-
NHS was reacted with ethylenediamine (0.67 mL) and pyridine
(100 ␮L) at room temperature for an additional 12 h. The aminated
folate was precipitated with an excess of acetonitrile and washed
three times with diethyl ether. The yellow powder was obtained
after drying under vacuum (Fig. 1a).
2.5. Preparation and characterization of self-assembled HP and
FHP nanoparticles
2.2.2. Preparation of aminated PhA
The HP and FHP nanoparticles were prepared by a dialysis
method. HP or FHP conjugates (5 mg) were dissolved in 5 mL of
formamide, followed by sonication for 10 min to obtain a clear
solution. The solution was dialyzed against deionized water for
2 days. After filtration with a 0.45 ␮m syringe filter, the average
particle size, size distribution, and zeta-potential of each sample
PhA (160 mg) was activated with EDC (61.6 mg) and NHS
(61.6 mg) in 10 mL of dimethylformamide (DMF) at room tem-
perature for 12 h, and then ethylenediamine (36 ␮L) and pyridine
(20 ␮L) were added. After a 12 h reaction, the solution was dia-
lyzed for 2 days against deionized water to remove the byproducts,

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L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
Fig. 1. Schematic diagram of the synthesis of (a) aminated folate, (b) aminated PhA, and (c) FHP.
were measured using a dynamic light scattering instrument (ELS-Z
series, Otsuka Electronics, Japan). The morphology of HP and FHP
nanoparticles was observed by a field emission scanning electron
microscope (FE-SEM; JSM-7000F, JEOL, Japan) at 15 kV.
method. The standard stock solution of DMA in DMF was stored
in the dark at 4 ◦C until use, at which time it was diluted to a final
concentration of 1.184 × 10−5 M with DMF or PBS buffer. HP and
FHP nanoparticles (PhA, 1.5 ␮g/mL) were also dissolved in DMF or
PBS buffer (pH 7.4) and added to DMA. The mixtures were irradi-
ated at a light intensity of 3 mW/cm2 using a 670 nm laser source
(Institute of Electronics, The Catholic University of Korea). A colli-
mated laser beam was directed at the sample cuvette through an
optical fiber. The decrease in fluorescence intensity of DMA (emis-
sion 380–550 nm, with excitation at 360 nm) as a result of singlet
oxygen generation was monitored using a spectrofluoro photome-
ter controlled by a PC. Singlet oxygen quantum yield was calculated
by the following equation (Hoebeke & Damoiseau, 2002):
2.6. Observation of self-quenching effects of HP and FHP
nanoparticles
To monitor changes of fluorescence in different solvents, HP and
FHP nanoparticles were dissolved in PBS and DMF in EP-tubes,
respectively. Fluorescence images were detected with a KODAK
Image Station after being dissolved in different solvents. Images
were obtained using a 12-bit CCD camera (Image Station 4000 MM;
Kodak, New Haven, CT, USA) equipped with a special C-mount lens
and a long wave emission filter (600–700 nm; Omega Optical, Brat-
tleboro, VT, USA).
kS
kR
AR
AS
˚
=
×
× ref.˚ꢀ
ꢀ
where A is absorbance at 670 nm (the excitation wave length), k
is the gradient of the absorbance curve, S refers to the sample,
and R to the reference. SOQ (˚ꢀ) of each sample was calcu-
lated from the reference ˚ꢀ value (PhA 0.52: ref. ˚ꢀ) (Fernandez,
Bilgin & Grossweiner, 1997; Roslaniec, Weitman, Freeman, Mazur
& Ehrenberg, 2000).
2.7. Determination of singlet oxygen generation
The generation of singlet oxygen can be measured by the
decrease of fluorescence intensity of 9,10-dimethylanthracene
(DMA), utilizing fluorescent spectroscopy as an independent
Table 1
Characterization of HP and FHP conjugates.
Sample
Feed molar ratio
(folate:heparin:PhA)
Coupling ratio
(folate:heparin:PhA)
CQCa (mg/L)
Size (nm)
Zeta-potential (mV)
Anticoagulant
SOQ in DMFc
activityb (IU/mg)
HP1
HP2
FHP1
FHP2
–:1:6
–:1:10
5:1:6
–:1:4.67
–:1:7.24
3.04:1:4.14
2.67:1:6.71
1.73
0.39
4.90
1.08
174.1 3.2
134.6 1.5
167.0 1.7
145.2 3.0
−33.59 0.34
−30.70 0.55
−20.62 1.02
−20.44 0.78
71.7
51.6
55.4
40.2
0.47
0.47
0.48
0.47
5:1:10
a
CQC (critical self-quenching concentration).
The anticoagulant activity of heparin is 101 IU/mg.
SOQ (singlet oxygen quantum yield (˚ꢀ)): In DMF (PhA, 0.52).
b
c

L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
711
2.8. Cell cytotoxicity of HP and FHP nanoparticles
anticoagulant activity decreased according to the increase in the
amount of chemically coupled PhA and folate moieties. The conju-
gation of PhA and folate to the heparin backbone resulted in lower
anticoagulant activity of heparin, because the carboxyl groups of
heparin, which play an important role in anticoagulant activity,
were modified by conjugation. However, HP and FHP conjugates
may retain their antitumor efficacy because the sulfate groups,
which are important for endothelial cell binding or inhibition of
VEGF- and bFGF-mediated angiogenesis, are left unaltered (Barzu
et al., 1986). The application of heparin as an anticancer agent is
limited by its high anticoagulant activity, because the administra-
tion of heparin may induce serious side effects, such as hemorrhage,
thrombocytopenia, and osteoporosis. Thus, HP and FHP conjugates
with reduced anticoagulant activity could be safe for long term
delivery of PhA during PDT.
HeLa cells (5 × 104 per well) were seeded on 96-well plates
and cultured in 200 ␮L culture medium for 1 day. After incuba-
tion for 12 h with different concentrations of HP, FHP nanoparticles
and free PhA (0.0015–1.5 ␮g/mL of Pheo-A), the medium was
replaced with fresh RPMI 1640 just prior to irradiation at 1.2 J/cm2
by a 670 nm laser source (Institute of Electronics). After 24 h
of incubation, phototoxicity and dark-toxicity were determined
using 3-[4,5-dim ethylthiazol-2-yl]-3,5-diphenyltetrazolium bro-
mide dye (MTT dye, 2 mg/mL) uptake at 570 nm on an ELISA
reader.
3. Results and discussion
3.1. Synthesis and characterization of HP and FHP conjugates
3.2. Critical self-quenching concentrations (CQCs) of HP and FHP
conjugates
To our knowledge, among a variety of polysaccharides, heparin
is the unique polymer that possesses anti-cancer activities. Sev-
eral preclinical studies have indicated that heparin has a marked
effect on VEGF- and bFGF-mediated tumor angiogenesis, as well
as an inhibitory effect on tumor metastasis through interference
with endothelial cell adhesion (Bobek & Kovarik, 2004; Mousa
& Petersen, 2009; Smorenburg & Van Noorden, 2001). PDT is a
local treatment and generally cannot be used to treat cancer that
has spread (metastasized) (MacDonald & Dougherty, 2001). Hep-
arin could be used as a nanocarrier in PDT, used in combination
with antiangiogenic therapy, to enhance the response to PDT dur-
ing cancer treatment (Dolmans et al., 2003; Ferrario et al., 2000).
Here, we synthesized amphiphilic HP conjugates by conjugating
hydrophobic PhA to hydrophilic heparin. Folate conjugated HP was
synthesized for binding the folate-receptor in tumor cells during
actively targeted PDT. Aminated folate and PhA were initially pre-
pared by covalent bonding of the carboxyl group of folate and
PhA with ethylenediamine using DCC/NHS and EDC/NHS as cou-
pling agents, respectively (Fig. 1a and b). HP conjugates, with or
without folate conjugation, were then synthesized by a simple
one-step reaction of the amino groups of PhA and folate with the
carboxylic groups of heparin (Fig. 1c). The chemical compositions of
HP and FHP conjugates were confirmed by 1H NMR measurements.
The characteristic chemical shifts of HP conjugates correspond-
ing to heparin (3.9, 4.0 ppm) (Fig. 2a), and aminated PhA (4.6, 5.9,
6.4–6.5, 8.9, 9.4, 9.7–9.8 ppm) (Fig. 2c) were observed in the 1H
NMR spectrum of HP (Fig. 2d). Besides the characteristic peaks
of heparin and PhA, the typical peaks of aminated folate were
observed at 4.5, 6.7, 6.9, and 8.6 ppm in the 1H NMR spectrum of
FHP (Fig. 2e).
HP conjugates with different PhA contents were prepared by
varying the feed molar ratios of PhA to heparin (Table 1). The cou-
pling ratios of PhA increased from 4.67 to 7.24, corresponding to
PhA feed molar ratios of 6 and 10, respectively. For FHP conjugates,
the feed molar ratio of folate/heparin was fixed at 5/1. The coupling
ratios of folate and PhA for FHP1 were 3.04 and 4.14, and for FHP2
they were 2.67 and 6.71, respectively. The coupling ratios of folate
in FHP1 and FHP2 were similar to each other, while the coupling
ratio of PhA to the heparin backbone increased with increasing
the PhA feed molar ratio, indicating that the coupling reaction
of PhA/heparin was not as significantly affected by the presence
of folate moieties. Therefore, the one-step reaction method for
preparing the FHP conjugates can be considered as a feasible and
simple way to introduce the folate ligands and PhA to the heparin
backbone.
In general, the critical micelle concentration (CMC) or criti-
cal aggregation concentration (CAC) of amphiphiles is determined
using the pyrene fluorescence probe method. At values below the
CMC or CAC, pyrene is soluble in polar aqueous solution. When
micelles are formed, pyrene may preferentially partition into the
hydrophobic domain formed by a micellar core. As a medium
of high polarity changes to a non-polar environment, numer-
ous changes in the pyrene fluorescence spectra can be observed,
including an increase in the fluorescence intensity and a red
shift of the (0,0) band in the excitation spectra (Kalyanasundaram
& Thomas, 1977). When HP and FHP conjugates spontaneously
form nanoparticles, pyrene also preferentially partitions toward
the hydrophobic PhA core. However, probably due to the strong
quenching effect of the hydrophobic interaction between PhA
and pyrene, known as the fluorescence resonance energy trans-
fer effect induced by the aggregation behavior of PhA and ␲–␲
stacking interactions, a decrease of pyrene fluorescence inten-
sity was observed (Fig. 3a). Based on this special change, Li, Bae,
et al. (2010) and Li, Huh, et al. (2010) proposed a new term
(CQC) to describe the threshold concentration of polysaccharide-PS
amphiphiles required to quench the fluorescence of pyrene. A sim-
ilar phenomenon was observed in the study by Bae and Na (2010)
where the pyrene fluorescence intensity at 335 nm decreased, with
an increase in the concentration of folate–pullulan–PhA conju-
gates, and a red shift of (0,0) was not observed following the
addition of the polymer. Fig. 3b shows a plot of the fluorescence
intensity of pyrene at 335 nm vs. the concentration of FHP2; a
major change in the slope indicates the onset of self-quenching.
As shown in Table 1, the CQCs of HP and FHP conjugates were
in the range of 0.39–4.90 mg/L, which decreased with increas-
ing content of PhA, indicating that a higher hydrophobic PhA
content enabled the conjugates to spontaneously form nanopar-
ticles at a lower concentration. Nevertheless, the presence of folate
ligands did not show a significant effect on CQCs of HP conju-
gates at a nearly similar PhA content. The CQCs of FHP1 and HP1
were 4.90 mg/L and 1.73 mg/L, respectively, when the coupling
ratios of PhA were 4.14 and 4.67, respectively, indicating that
the CQC values of HP and FHP conjugates were highly depen-
dent on the core forming component, PhA. The CQC values of
HP and FHP conjugates were significantly lower than those of
folate–pullulan–PhA conjugates (0.08–0.27 g/L) (Bae & Na, 2010),
and acetylated hyaluronic acid–PhA conjugates (0.26–2.10 M) (Li,
Bae, et al., 2010; Li, Huh, et al., 2010). Because the intravenous
injection of nanoparticle solutions is associated with extreme dilu-
tion by circulating blood, the low CQC values suggest that the
HP and FHP nanoparticles are more favorable for systemic PhA
delivery.
As shown in Table 1, the anticoagulant activities of HP and
FHP conjugates ranged from 40.2 to 71.7 IU/mg, which were sig-
nificantly lower than that of unmodified heparin (101 IU/mg). The

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L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
Fig. 2. 1H NMR spectra of (a) heparin, (b) aminated folate, (c) aminated PhA, (d) HP, and (e) FHP.
Fig. 3. CQC measurement of FHP: (a) excitation spectra of pyrene in FHP2 conjugate, and (b) the plot of the fluorescence intensity of pyrene at 335 nm against concentration
of FHP2.

L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
713
Fig. 4. Size distribution of HP and FHP, measured by DLS: (a) HP2 and (b) FHP2; and morphology of HP and FHP, measured by FE-SEM: (c) HP2 and (d) FHP2.
3.3. Characterization of HP and FHP self-assembled nanoparticles
negative carboxyl groups were chemically modified via the cou-
pling reaction. This negative surface charge may cause HP and FHP
nanoparticles to possess long-term colloidal stability in a physio-
logical environment.
HP and FHP conjugates readily self-assembled in aqueous solu-
tions to form nanoparticles by a dialysis process. The driving force
of their self-assembling behavior is due to the hydrophobic inter-
action between conjugated PhA moieties. The nanoparticles may
have a core–shell structure with a hydrophobic PhA inner core sur-
rounded by a hydrophilic outer shell of heparin or heparin–folate.
The average particle size of HP and FHP nanoparticles ranged from
134.6 to 174.1 nm (Table 1), which decreased with increasing PhA
coupling ratio, due to the formation of a more compact hydropho-
bic inner core. However, the conjugation of folate ligand did not
induce a significant change in the particle size of HP and FHP
nanoparticles. For example, the particle sizes of HP1 and FHP1
were 174.1 nm and 167.0 nm, respectively, at a similar coupling
ratio for PhA (4.67 for HP1 and 4.14 for FHP1, respectively). The
size distribution of HP and FHP nanoparticles, measured by DLS,
showed a unimodal pattern (Fig. 4a and b). The FE-SEM images
showed spherical HP2 and FHP2 nanoparticles with a diameter of
80–100 nm, which was ∼30–40 nm smaller than the size deter-
mined by DLS (Fig. 4c and d). The particle size determined by DLS
measurement reflected the hydrodynamic diameter of the micelle
structure, which was swollen in aqueous solution, while FE-SEM
observed the size of nanoparticles in the dried state. It is known
that the large gaps (400–800 nm) between the endothelial cells
in the microvasculature of solid tumors enable nanoparticles to
preferentially accumulate and reside in the tumor mass via the
EPR effect (Matsumura & Maeda, 1986). Therefore, HP and FHP
nanoparticles with small size (<200 nm) are considered to be suit-
able for the accumulation of PhA at the targeted tumor through
the EPR effect. The HP and FHP nanoparticles were negatively
charged at their surface, as reflected in the zeta-potential values
(−20.44–33.59 mV) (Table 1), indicating that negatively charged
heparin covers the self-assembled nanoparticles. FHP nanoparti-
cles showed fewer negative charges than HP nanoparticles, which
may be because conjugated folate molecules partially shielded the
surface areas of negatively charged heparin outer shells, and more
3.4. Photoactivity and determination of singlet oxygen
production of HP and FHP nanoparticles
The photoactivity of free PhA, HP and FHP nanoparticles was
determined by KODAK Image Station. Fig. 5 shows the near-infra
red fluorescence (NIRF) images of free PhA, HP1, and FHP1 nanopar-
ticles at a similar PhA concentration in DMF (red arrow) or PBS
(blue arrow). Due to the FRET effect induced by the aggregation
behavior of PhA via hydrophobic and ␲–␲ stacking interactions,
the NIRF emissions of free PhA, HP, and FHP nanoparticles were
obscure in lipophobic (PBS) solvent. Contrasting NIRF images
were observed when free PhA, HP, and FHP nanoparticles were
dissolved in lipophilic (DMF) solvent. The results implied the self-
photoquenching potential of the HP and FHP nanoparticles.
To further prove the self-quenching effect of HP and FHP
nanoparticles, the generation of singlet oxygen by HP and FHP
Fig. 5. Bright and NIRF image of HP, FHP nanoparticles and PhA (blue arrow: in
PBS, red arrow: in DMF). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of the article.)

714
L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
Fig. 7. Viability of HeLa cells determined by MTT assay after treat ment of HP1,
FHP1 nanoparticles and PhA for 12 h: dark-toxicity ((ꢁ) HP1, (ꢂ) FHP1, (ꢀ) PhA)
and phototoxicity ((᭹) HP1, (ꢁ) FHP1, (ꢂ) PhA).
the incorporated PS may also have a self-photoquenching poten-
tial, due to the aggregation of PS at the inner core. However, during
circulation in the blood, the physical-loading system cannot con-
trol phototoxicity caused by the inadvertent leakage of PS from
the system, which leads to damage of normal tissue and blood
cells. Therefore, the use of PS-conjugated nanoparticles with a
self-quenching effect is a preferable system to switch phototox-
icity during PDT: during circulation, the phototoxicity of PS can
be completely suppressed until it reaches the target site, where
the suppression can be rapidly reversed by environmental condi-
tions such as pH, temperature, and enzymatic activity. Thus, the
nanoparticles with self-quenchable photoactivity based on HP and
FHP conjugates not only enhance the solubility of the hydrophobic
PhA, but also minimize the unfavorable phototoxicity in PDT.
3.5. Phototoxicity of HP and FHP nanoparticles
To estimate the PDT efficacy of HP and FHP nanoparticles, the
in vitro phototoxicity of the HP1 and FHP1 nanoparticles, as well
as free PhA, on HeLa cells was compared after treatment with or
without light at a dose of 1.2 J/cm2 at 670 nm, as shown in Fig. 7.
Without light treatment, free PhA, HP1, and FHP1 nanoparticles dis-
played a slight dark-toxicity (cell viability: 87.1–99.3%) in the range
of 0.0015–1.5 ␮g/mL. However, following light treatment, both the
HP1, and FHP1 nanoparticles, and free PhA showed significantly
enhanced cytotoxicity, implying that the PhA containing nanopar-
ticles effectively generated the cytotoxic species and induced the
cell depletion. Although the photoactivity was suppressed in aque-
ous medium due to photoquenching, the phototoxicity of HP and
FHP nanoparticles on living cells was maintained, which was prob-
ably due to a loss of the quenching effect by enzymatic attack
within cellular compartments. When nanoparticles are internal-
ized in cancer cells, a series of biological activities could induce
the decomposition of the heparin backbone (Ernst, Langer, Cooney
& Sasisekharan, 1995; Thunberg, Backstrom, Wasteson, Robinson,
Ogren & Lindahl, 1982) and the cleavage of the amide bond between
PhA and heparin; these interactions are involved in dissociation of
aggregated PhA, and thus photoactivity could be restored due to
loss of the quenching effect. Importantly, the phototoxicity of HP1
and FHP1 nanoparticles was higher than that of free PhA (IC50;
HP: 0.15 ␮g/mL; FHP: 0.14 ␮g/mL; PhA: 0.4 ␮g/mL). The relatively
greater phototoxicity of HP and FHP nanoparticles compared to
free PhA may be related to not only the different cellular uptake
Fig. 6. Change in DMA fluorescence due to generation of singlet oxygen by HP, FHP
nanoparticles and free PhA (a) in DMF and (b) in PBS.
nanoparticles in DMF and PBS, which governs the phototoxicity,
was determined by calculating the singlet oxygen quantum yield,
using DMA as the singlet oxygen trap. As shown in Fig. 6a, all
samples produced a sharp decline in DMA fluorescent intensity,
indicating the rapid generation of singlet oxygen upon laser irradi-
ation when free PhA, HP, and FHP nanoparticles were dissolved in
DMF. Singlet oxygen quantum yields (˚ꢀ) of HP1, HP2, FHP1, and
FHP2 were calculated as 0.47, 0.47, 0.48, and 0.47, respectively, by
a comparison of the slope for conjugates with the corresponding
slope for free PhA (˚ = 0.52). However, singlet oxygen generation
was significantly changed in PBS (Fig. 6b). The generation of singlet
oxygen dramatically decreased in PBS, according to the aggregation
of PhA, showing the quenching effect. That result implied the HP
and FHP nanoparticles could not be activated into the triplet state
by mutual energy transfer, resulting in a decline of the ability to
generate 1O2.
The self-quenching effect of the PS-conjugated nanoparticles is
advantageous for improving the therapeutic efficiency and lower-
ing the side effects of PDT, such as skin photosensitivity. Physical
incorporation of PS into polymeric nanocarrier systems is a con-
ventional method to improve the solubility of hydrophobic PS, and

L. Li et al. / Carbohydrate Polymers 86 (2011) 708–715
715
and localization characteristics of nanoparticles that leads to more
efficient intracellular PhA localization via the endocytic pathway,
but also reduced photoactivity of free PhA by possible negative
aggregation behavior resulting in quenching effect in an aqueous
environment. Therefore, the HP and FHP nanoparticles could be
efficient carriers for the targeted delivery of PhA, with minimal side
effects.
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4. Conclusions
Self-assembled nanoparticles based on HP and FHP conjugates
were prepared as potential PS carriers for use in PDT. The con-
jugation of PhA and folate molecules significantly lowered the
anticoagulant activity of heparin. In aqueous media, HP and FHP
conjugates formed self-assembled nanoparticles with a diame-
ter <200 nm. The low CQC values (0.39–4.90 mg/L) suggest that
the HP and FHP nanoparticles are stable for systemic PhA deliv-
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singlet oxygen quantum yield, indicated the autoquenching prop-
erties of HP and FHP nanoparticles. The cytotoxicity of HP and FHP
nanoparticles was higher than that of free PhA with light treatment,
while the nanoparticles revealed low cytotoxicity against cancer
cells without light. These findings indicated that the HP and FHP
nanoparticles could be efficient carriers with practical applications
in PDT.
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