Comparative study of photosensitizer loaded and conjugated glycol chitosan nanoparticles for cancer therapy.

Abstract

This study reports that tumor-targeting glycol chitosan nanoparticles with physically loaded and chemically conjugated photosensitizers can be used in photodynamic therapy (PDT). First, the hydrophobic photosensitizer, chlorin e6 (Ce6), was physically loaded onto the hydrophobically-modified glycol chitosan nanoparticles (HGC), which were prepared by self-assembling amphiphilic glycol chitosan-5β-cholanic acid conjugates under aqueous conditions. Second, the Ce6s were chemically conjugated to the glycol chitosan polymers, resulting in amphiphilic glycol chitosan-Ce6 conjugates that formed self-assembled nanoparticles in aqueous condition. Both Ce6-loaded glycol chitosan nanoparticles (HGC-Ce6) and Ce6-conjugated chitosan nanoparticles (GC-Ce6) had similar average diameters of 300 to 350 nm, a similar in vitro singlet oxygen generation efficacy under buffer conditions, and a rapid cellular uptake profile in the cell culture system. However, compared to GC-Ce6, HGC-Ce6 showed a burst of drug release in vitro, whereby 65% of physically loaded drugs were rapidly released from the particles within 6.5h in the buffer condition. When injected through the tail vein into tumor bearing mice, HGC-Ce6 did not accumulate efficiently in tumor tissue, reflecting the burst in the release of the physically loaded drug, while GC-Ce6 showed a prolonged circulation profile and a more efficient tumor accumulation, which resulted in high therapeutic efficacy. These comparative studies with drug-loaded and drug-conjugated nanoparticles showed that the photosensitizer-conjugated glycol chitosan nanoparticles with excellent tumor targeting properties have potential for PDT in cancer treatment.

Full text

Page 1

Comparative study of photosensitizer loaded and conjugated glycol chitosan
nanoparticles for cancer therapy
So Jin Leea,b,1, Heebeom Kooa,1, Hayoung Jeonga,c, Myung Sook Huha,d, Yongseok Choib, Seo Young Jeongc,
Youngro Byune, Kuiwon Choia, Kwangmeyung Kima,⁎, Ick Chan Kwona,⁎⁎
aBiomedical Research Center, Korea Institute of Science and Technology, 39-1 Haweolgog-Dong, Sungbook-Gu, Seoul 136-791, South Korea
bSchool of Life Sciences and Biotechnology, Korea University, Seoul 136-713, South Korea
cDepartment of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul, 130-701, South Korea
dResearch Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Korea
eDepartment of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and College of Pharmacy, Seoul National University,
599 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Korea
a b s t r a c ta r t i c l ei n f o
Article history:
Received 9 December 2010
Accepted 24 March 2011
Available online 30 March 2011
Keywords:
Glycol chitosan
Photodynamic therapy
Photosensitizer
Drug-loaded nanoparticle
Drug-conjugated nanoparticle
This study reports that tumor-targeting glycol chitosan nanoparticles with physically loaded and chemically
conjugated photosensitizers can be used in photodynamic therapy (PDT). First, the hydrophobic
photosensitizer, chlorin e6 (Ce6), was physically loaded onto the hydrophobically-modified glycol chitosan
nanoparticles (HGC), which were prepared by self-assembling amphiphilic glycol chitosan-5β-cholanic acid
conjugates under aqueous conditions. Second, the Ce6s were chemically conjugated to the glycol chitosan
polymers, resulting in amphiphilic glycol chitosan-Ce6 conjugates that formed self-assembled nanoparticles
in aqueous condition. Both Ce6-loaded glycol chitosan nanoparticles (HGC-Ce6) and Ce6-conjugated chitosan
nanoparticles (GC-Ce6) had similar average diameters of 300 to 350 nm, a similar in vitro singlet oxygen
generation efficacy under buffer conditions, and a rapid cellular uptake profile in the cell culture system.
However, compared to GC-Ce6, HGC-Ce6 showed a burst of drug release in vitro, whereby 65% of physically
loaded drugs were rapidly released from the particles within 6.5 h in the buffer condition. When injected
through the tail vein into tumor bearing mice, HGC-Ce6 did not accumulate efficiently in tumor tissue,
reflecting the burst in the release of the physically loaded drug, while GC-Ce6 showed a prolonged circulation
profile and a more efficient tumor accumulation, which resulted in high therapeutic efficacy. These
comparative studies with drug-loaded and drug-conjugated nanoparticles showed that the photosensitizer-
conjugated glycol chitosan nanoparticles with excellent tumor targeting properties have potential for PDT in
cancer treatment.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Chemicaldrugsaregenerallyconsideredtobeoneofthemostefficient
forms of cancer therapy. To achieve maximum therapeutic efficacy with
minimal side effects, specific delivery of anticancer drugs to the target
tumor site is highly desirable in cancer treatment [1]. For this purpose,
many research groups have developed various nano-size drug carriers,
suchasliposomes,polymerconjugates,inorganicparticles,andpolymeric
nanoparticles[2–5].Amongthese,polymericnanoparticleshavereceived
much attention from biomedical researchers because they possess many
useful properties and can be easily modified for clinical purposes [6,7].
Moreover, nanoparticles provide prolonged circulation in the blood and
accumulation to high levels in tumor tissue by avoiding rapid renal
clearance when injected intravenously [8].
A large number of chemical drugs for cancer therapy are
hydrophobic, and two methods are used to introduce hydrophobic
drugs into polymeric nanoparticles: 1) physically loading onto
polymeric nanoparticles and 2) chemical conjugation to polymeric
nanoparticles [9]. The physical loading technique has been widely
used with amphiphilic nanoparticles, but problems, such as the
instability of nanoparticles during blood circulation causing a burst of
release and loss of the loaded drugs, have been encountered [10]. The
chemical conjugation technique allows the conjugation of hydropho-
bic drugs to hydrophilic polymers, which can then self-assemble to
form spherical nanoparticles in aqueous conditions [11]. Importantly,
chemically conjugated drugs in nanoparticles demonstrate increased
stability, and unintended release is less frequent than with loaded
drugs. Even with similar polymers and drugs, the in vitro and in vivo
characteristics of nanoparticles can be largely changed by the method
employed to introduce the drugs into the nanoparticles. However, to
Journal of Controlled Release 152 (2011) 21–29
⁎ Corresponding author. Tel.: +82 2 958 5916; fax: +82 2 958 5909.
⁎⁎ Corresponding author. Tel.: +82 2 958 5912; fax: +82 2 958 5909.
E-mail addresses: kim@kist.re.kr (K. Kim), ikwon@kist.re.kr (I.C. Kwon).
1These authors contributed equally to this paper.
0168-3659/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2011.03.027
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
NANOMEDICINE

Page 2

the best of our knowledge, few studies have been performed for
analyzing these changes. Therefore, a comparative study of nanopar-
ticles physically loaded with or chemically conjugated to similar
polymers and drugs is expected to be helpful to researchers who
develop or use nanoparticles for drug delivery in cancer treatment.
Photodynamic therapy (PDT) with photosensitizers has emerged
as an effective therapeutic option for various tumors and other
diseases [12,13]. When the proper wavelength of light irradiates
photosensitizers, highly reactive singlet oxygen is generated, causing
damage to tumor tissues [14]. Along with singlet oxygen, photosen-
sitizers can emit fluorescence, which enables easy detection and
trackingof the photosensitizers during both in vitro and in vivo studies
[15]. Especially under in vivo conditions, this fluorescence is beneficial
for the imaging and quantification of photosensitizers in target tumor
tissues or other organs [16]. Moreover, even if they are conjugated to
other molecules like polymers, photosensitizers remain therapeutic
unlike most other drugs [17]. Because of these characteristics,
photosensitizers are suitable as model drugs for a comparative study
of loaded and conjugated polymeric nanoparticles under both in vitro
and in vivo conditions.
Chitosan is a natural polymer and a form of deacetylated chitin.
Chitosan is both biodegradable and biocompatible. Consequently,
chitosan has been widely used in various biomedical and pharmaceu-
ticalformulations[18].Glycolchitosan(GC)isachitosanderivativewith
ethyleneglycolgroupsonits backbone,and itswatersolubilityishighly
enhanced by these glycol groups. In previous papers, we developed
tumor-homing glycol chitosan nanoparticles and applied them as drug
carriers for cancer therapy [5]. When hydrophobic molecules, such as
5β-cholanic acid or protophorphyrin IX, were conjugated to a GC
polymer, the resulting amphiphilic conjugates formed self-assembled
hydrophobic GC nanoparticles (HGCs) with hydrophilic GC shells and
hydrophobic cores under aqueous conditions [19,20]. These HGCs
harboredvariousanticancerdrugsintheirhydrophobicinnercores,and
showedprolongedcirculationinthebloodandspecificdeliveryofdrugs
to tumors for cancer therapy [21,22].
Herein, we synthesized two kinds of tumor targeting nanoparticles
containing photosensitizers for PDT. We selected chlorin e6 (Ce6) as
the photosensitizer because of its hydrophobicity, its activation by
near infrared wavelengths, allowing it to act in deep tissue layers, and
high singlet oxygen generation efficiency [23]. We obtained Ce6-
loaded glycol chitosan nanoparticles (HGC-Ce6) and Ce6-conjugated
chitosannanoparticles(GC-Ce6),andcomparedtheinvitroandinvivo
characteristics of these two nanoparticles for PDT in cancer therapy.
We developed new photosensitizer-containing glycol chitosan nano-
particles and demonstrated their potential for efficient PDT of tumors.
Furthermore, this comparative study provides valuable information
about the in vivo behaviors of nanoparticles for drug delivery.
2. Materials and methods
2.1. Materials
Glycol chitosan (average molecular weight=250 kDa; degree of
deacetylation=82.7%), chlorin e6(Ce6), N-hydroxysuccinimide
(NHS), 5β-colanic acid, 1-ethyl-3-(3-dimethylaminopropyl)-carbo-
diimide hydrochloride (EDC), fluorescein isothiocyanate (FITC), and
p-nitroso-N,N′-dimethylaniline (RNO) were purchased from Sigma
(St. Louis, MO). Anhydrous methanol and dimethyl sulfoxide (DMSO)
were purchased from Merck (Darmstadt, Germany). All other
chemicals and solvents were analytical grade and were used without
further purification.
2.2. Preparation of HGC-Ce6 and GC-Ce6
Firstly, hydrophilic glycol chitosan (0.25 g, 1 μmol) was chemically
conjugated with hydrophobic 5β-cholanic acids (0.01 g, 28 μmol) in
1:1 (v/v) methanol/distilled water solution, and 42 μmol of NHS and
42 μmol of EDC were added as catalysts to the solution as previously
reported [5]. After stirring at room temperature for 1 day, the solution
wasdialyzedfor3 daysagainstmethanolandwater,andthenlyophilized
togiveawhitesolidpowder.TheresultingHGCwasoptimizedtocontain
160 molecules of 5β-cholanic acids per glycol chitosan polymer and
it formed a spherical nanoparticle (size=250 nm) in aqueous condi-
tion [5,24]. To prepare Ce6-loaded glycol chitosan nanoparticles (HGC-
Ce6), the photosensitizer, Ce6, was physically loaded onto HGCs by a
simpledialysismethod[25].Inbrief,50 mgofHGCin25 mlofDMSOand
Ce6solution(2.5,5or10 mgin6.25 mlofDMSO)weremixed,vigorously
stirred for 12 h at room temperature, and dialyzed for 3 days against
distilled water. The reaction mixture was then filtered through a 0.8um
syringefilterandlyophilizedtogiveagreenpowdercontainingHGC-Ce6.
Secondly, Ce6-conjugated glycol chitosan nanoparticles (GC-Ce6)
were prepared by in 1:9 (v/v) DMSO/distilled water containing glycol
chitosan and Ce6. Glycol chitosan polymer (50 mg, 200 nmol) was
dissolved in distilled water (10 ml) and Ce6 (2.5, 5 or 10 mg) was
dissolvedinDMSO(2 ml).Then,3.45 mg(30 μmol)ofNHSand5.73 mg
(30 μmol) of EDC were added to the solution, it was gently stirred at
room temperature. After 1 day, the reaction mixture was dialyzed for
3 days against methanol and water to remove free Ce6 molecules.
Finally, the product was finally filtered through a 0.8 μm syringe filter
and lyophilized to give a green powder containing GC-Ce6.
2.3. Characterization of HGC-Ce6 and GC-Ce6
The physical loading and chemical conjugation efficiency of Ce6 in
the nanoparticles were quantified using a Lambda UV–vis 7
spectrophotometer (Perkin-Elmer, CT). One percent (v/v) DMSO/
water solution was added to each nanoparticle (1 mg/ml) to make a
clear solution. The Ce6 concentration was determined was measuring
absorbance at 405 nm and referring to a standard curve of free Ce6
concentrations in the same DMSO/water solution. The size distribu-
tions of HGC-Ce6 and Ce6-GC were determined in PBS (1 mg/ml,
37 °C, pH 7.4) using dynamic light scattering (DLS) (Spectra Physics,
Mountain View, CA) and a digital autocorrelator (BI-9000AT,
Brookhaven, NY, USA) at 633 nm. The morphological images of the
nanoparticles were confirmed by transmission electron microscopy
(TEM) (CM30 electron microscope, Philips, CA). For this, each sample
was prepared on a 300-mesh copper grid coated with carbon at a
concentration of 1 mg/ml in distilled water. After drying the sample,
negative staining was performed with 2 wt.% uranyl acetate solution.
The chemical structures of HGC and GC-Ce6 were confirmed by
performing 1 H NMR (UnityPlus 300, Varian, CA, USA) on samples
prepared in 1:1 (v/v) D2O/CD3OD solution (data not shown).
2.4. Singlet oxygen generation and release profile of HGC-Ce6 and
GC-Ce6
The generation of singlet oxygen was determined using p-nitroso-
N,N′-dimethylaniline (RNO) as an indicator for photo-induced singlet
oxygen with histidine as the singlet oxygen trap [26]. One-hundred μl
of RNO (100 μl in distilled water, or 250 μM) and 300 μl of histidine in
distilled water (0.03 M) were dissolved in a quartz cuvette. Free Ce6,
HGC-Ce6 and GC-Ce6 (1 μg of Ce6) were dissolved in 1% (v/v) DMSO/
distilled water. To quantify singlet oxygen generation, 1.0 ml of each
solution was added to the RNO solution. Before measuring, each
sample solution was bubbled with oxygen for 10 min and then
irradiated with a 671 nm laser (220 mW/cm2). The optical density at
440 nm (λmaxof RNO) was monitored every 2 min using a spectro-
photometer (ISS, Champaign, IL).
ToobtainthedrugreleaseprofilesofHGC-Ce6andGC-Ce6,1.0 mgof
each sample was dissolved in 1.0 ml of PBS (pH 7.4) and sonicated at
90 W for 4 min. The solution was placed in a dialysis tube (molecular
weight cutoff=12 to 14 kDa, Spectrum®), and the tube was fully
22
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 3

immersed in20 mlof PBS andgentlyshakenat 100 rpmin a water bath
at 37 °C. At the proper time interval, samples (1.0 ml) were collected
fromtheoutersolutionandtheconcentrationofCe6wasdeterminedby
UV–vis spectroscopy (Lambda Vis 7 Spectrometer, Perkin-Elmer, CT).
2.5. Cellular imaging of HGC-Ce6 and GC-Ce6
To determine the cellular uptake characteristic of nanoparticles,
GC polymers of each nanoparticle were labeled with 0.1 wt.% FITC in
DMSO. FITC-labeled nanoparticles emitted strong green fluorescence,
as previously reported [15]. After FITC labeling, the physicochemical
properties, such as average particle size and drug content, were
unchanged compared to unlabeled nanoparticles. Squamous cell
carcinoma (SCC-7) cells were originally obtained from the American
Type Culture Collection (Rockville, MD) and cultured in RPMI 1640
(Gibco, Grand Island, NY) containing 10% FBS (Gibco, Grand Island,
NY). They were seeded onto 6-well plates (2×105cells/well) and
incubated for 1 day. Then, the medium was replaced with 2 ml of
serum-free RPMI medium containing free Ce6 (2.5 μg/ml), HGC-Ce6,
or GC-Ce6 (2.5 μg/ml of Ce6) and then incubated for 1 h. The cells
were then washed twice with PBS (with Ca2+, Mg2+) and fixed with
4 wt.% paraformaldehyde. The intracellular localization of FITC labeled
nanoparticles and Ce6 were determined using a fluorescence
microscope (IX81, Olympus). The fluorescence images were obtained
with the FITC filter set (Ex=488 nm, Em=500–550 nm) for FITC-
labeled nanoparticles and with the Cy5.5 filter set (Ex=670 nm,
Em=720 nm) for Ce6, respectively.
2.6. In vitro phototoxicity of HGC-Ce6 and GC-Ce6
SCC-7 cells were seeded onto 96-well cell culture plates (1×104
cells/well) and incubated for 1 day. Then the medium was replaced
with 200 μl of serum-free medium containing free Ce6, HGC-Ce6, or
GC-Ce6 (0.5, 1, 2, 4, 8 μg of Ce6, respectively). After 1 h incubation, the
cells were washed three times with PBS and fresh medium was added.
After these processes, cells were illuminated with a LED lamp (670–
690 nm, 100 mW/cm2) for 20 min. Cells were then incubated at 37 °C
for 1 day, and the cell viability was measured by performing MTT
assays.Cellviabilitywas represented asthe percentage of live cellsper
total none-treated cells. Using an Annexin V-FITC fluorescence
microscope kits (BD bioscience, San Jose, CA, USA), apoptotic cells
were observed in green fluorescence images after 12 h incubation.
2.7. In vivo imaging of HGC-Ce6 and GC-Ce6
Athymic nude mice (5 weeks old, Institute of Medical Science,
Tokyo) were used for comparing in vivo biodistribution and tumor
targeting efficacy of free Ce6, HGC-Ce6 and GC-Ce6. For tumor tissue
generation,HT-29humancolonadenocalcinomacells(5.0×106)were
injected into the left flanks of mice. When the tumor volume grew to
approximately150±30 mm3,asalinesolutionoffreeCe6,HGC-Ce6or
GC-Ce6 containing 2.5 mg/kg of Ce6 were injected into the tumor-
bearing mice via tail vein. Then, the in vivo biodistribution and tumor
targetingefficacyofeachsamplewereevaluatedbyputtingmiceonan
animal plate at 37 °C inside the eXplore Optix system (Advanced
Research Technologies Inc., Montreal, Canada) [27,28]. During non-
invasive whole-body imaging, laser power and detection time were
adjusted to20 μW and 0.3 s per point, respectively. The excitation and
emission spots were evaluated by raster-scanning in 1 mm steps over
the region of interest (ROI). To excite Ce6 molecules, a 670 nm pulsed
diode laser was used. Near infrared fluorescence emission (650 to
700 nm) was detected with a time-correlated single photon counting
system (Becker and Hickl GmbH, Berlin, Germany) and a fast
photomultiplier tube (Hamamatsu, Japan). To compare ex vivo organ
biodistributions of Ce6 in the nanoparticles, mice were sacrificed at
2 day after i.v. injection, and the excised organs (livers, lungs, spleens,
kidneys, hearts, and the tumors) were analyzed with a 12-bit CCD
camera (Image Station 4000 mm; Kodak, New Haven, CT) equipped
with a special C-mount lens and a near infrared emission filter (600–
700 nm; Omega Optical). The quantification data of fluorescence
intensity in all images was calculated as total photons per centimeter
squared per steradian (p/s/cm2/sr) (n=5) [16].
2.8. Therapeutic efficacy of HGC-Ce6 and GC-Ce6 in HT-29
tumor-bearing mice
The HT-29 tumor-bearing mice models for PDT were made by the
same methods as described above for the imaging studies. When
tumor volume grew to approximately 150±30 mm3, saline, free Ce6,
HGC-Ce6, and GC-Ce6 containing 2.5 mg/kg of Ce6 were injected into
the tail vein (n=5). At 4 h and 12 h post-injection, mice were
irradiated twice with a red laser (671 nm, 220 mW/cm2) for 30 min
each at the tumor site. Then, the therapeutic efficacy of treated mice
was evaluated by monitoring the tumor volumes calculated as
width×length×height×1/2 for 25 days. Differences between exper-
imental and control groups were determined using one-way ANOVA
and deemed statistically significant (indicated by an asterisk (*) in
figure) if pb0.01.
3. Results and discussion
3.1. Physicochemical properties of HGC-Ce6 and GC-Ce6
Hydrophobically modified glycol chitosan nanoparticles (HGC)
were prepared as nano-size drug carriers for cancer treatment
A
B
Hydrophobically modified
glycol chitosan (HGC)
Hydrophobic photosensitizer
conjugated glycol chitosan (GC-Ce6)
C
= 5 -cholanic acid
β
= Glycol chitosan
= chlorin e6
HGC-Ce6
(Drug loaded nanoparticle)
GC-Ce6
(Drug conjugated nanoparticle)
Fig. 1. Preparation of HGC-Ce6 and GC-Ce6. Chemical structure of (A) glycol chitosan-
5β-cholanic acid conjugate (HGC) and (B) glycol chitosan-chlorin e6 conjugate (GC-
Ce6). (C) Schematic illustration of HGC-Ce6 (drug-loaded nanoparticle) and GC-Ce6
(drug-conjugated nanoparticle).
23
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 4

(Fig. 1A) [5]. HGC containing no drugs formed stable self-assembled
nanoparticle with an average diameter of 250 nm in aqueous
condition. Many hydrophobic anticancer drugs can be successfully
loaded into the hydrophobic inner-cores of HGC by a simple dialysis
method[15].Tomakephotosensitizer-loadednanoparticles,bothHGC
and Ce6 were dissolved in DMSO and then dialyzed against distilled
water to produce Ce6-loaded HGC (HGC-Ce6) (Fig. 1A and C). When
5 wt.%, 10 wt.% and 20 wt.% of Ce6 were loaded into the HGC
nanoparticles, the physical loading efficiencies were approximately
96%, 90% and 81%, respectively (Table 1). On the other hand, the
photosensitizer-glycolchitosanconjugates wereproduced bythedirect
chemical conjugation of Ce6 to glycol chitosan polymer in the presence
ofNHSandEDC(Fig.1BandC).WhenthefeedratioofCe6waslessthan
10 wt.%inthereactionmixture,thechemicalconjugationefficiencywas
above 85%, but it dramatically decreased to about 50% when the feed
ratio of Ce6 was above 20 wt.% compared with the glycol chitosan
polymer in the reaction (Table 1). To compare the in vitro and in vivo
characteristics of HGC-Ce6 and GC-Ce6 for cancer treatment, we
optimized the feed ratio of Ce6 to 10 wt.%, wherein the content of Ce6
was about 8.7 to 9.0 wt.% in both types of nanoparticles. Therefore, we
usedthesetwonanoparticles(10 wt.%HGC-Ce6and10 wt.%GC-Ce6)in
the following studies unless otherwise noted.
In aqueous condition, both HGC-Ce6 and GC-Ce6 formed stable
self-assembled nanoparticle structures and their average size was
about 300 to 350 nm,as measuredby dynamic lightscattering(Fig. 2).
TEM images also showed a spherical morphology, because the
hydrophilic glycol chitosan molecules are located outside and the
physically loaded or chemically conjugated Ce6s are located inside
due to their hydrophobic intermolecular interactions.
3.2. In vitro singlet oxygen generation and release profile of HGC-Ce6
and GC-Ce6
We next measured the singlet oxygen generation of free Ce6 and
both types of nanoparticles using p-nitroso-N,N′-dimethylaniline
(RNO) as an indicator (Fig. 3A) [26]. Free Ce6 showed a very sharp
downward curve at OD 440 nm, indicating rapid generation of singlet
oxygen upon light exposure. The optical density of both HGC-Ce6 and
GC-Ce6 also decreased when irradiated, but the rate was slower than
that of free Ce6, due to the self-quenching properties of the
nanoparticles [11]. In particular, the amounts of singlet oxygen
generation of HGC-Ce6 and GC-Ce6 were very similar despite their
Table 1
Characteristics of HGC-Ce6 and GC-Ce6 (n=3).
SampleCe6 amount (%)Loading & conjugation
efficiency (%)
5 wt.%aHGC-Ce6
10 wt.% HGC-Ce6
20 wt.% HGC-Ce6
5 wt.% GC-Ce6
10 wt.% GC-Ce6
20 wt.% GC-Ce6
4.79±0.8
9.02±0.9
16.29±1.2
4.63±0.8
8.73±0.6
10.1±0.9
96.23±4.8
90.18±9.0
81.45±6.0
92.57±4.1
87.34±6.0
50.27±4.9
aThe weight feed ratio of Ce6 to HGC and GC nanoparticles.
Fig. 2. Particle sizes and morphological shapes of (A) HGC-Ce6 and (B) GC-Ce6. Size
distributions were determined by dynamic light scattering, and inserted images were
TEM images of each type of nanoparticle.
5 101520 25
10
20
30
40
50
60
70
80
90
100
Cumulative release (%)
Time (hr)
HGC-Ce6
GC-Ce6
B
0510 15202530
0
20
40
60
80
100
RNO Concentration (%)
Time (min)
Free Ce6
GC-Ce6
HGC-Ce6
0
0
A
Fig. 3. Singlet oxygen generation and release profile of HGC-Ce6 and GC-Ce6. (A) Singlet
oxygen generation determined using RNO as a sensor according to irradiation time.
Results represent the means±s.e. (n=3). (B) In vitro release profile of HGC-Ce6 and
GC-Ce6 measured with UV–vis spectroscopy in PBS at 37 °C. Results represent means±
s.e. (n=3).
24
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 5

different nanoparticle structures, suggesting that the singlet oxygen
generation of Ce6 was not significantly changed by the type of
nanoparticle under these conditions.
To evaluate the in vitro drug release profile, HGC-Ce6 and GC-Ce6
were immersed at 37 °C in PBS (pH 7.4) to mimic in vivo conditions
and time-dependent changes in drug concentration were measured in
the buffer system [15]. Ce6 was released rapidly from HGC-Ce6
(Fig. 3B), demonstrating that the hydrophobic interactions of Ce6s in
HGC were relatively weaker than those of covalent conjugation. Over
the 5 h incubation period, 58% of the loaded Ce6 was rapidly released
from the HGC. Hence HGC-Ce6 may lose a large amount of physically
loaded drugs into the blood stream under in vivo conditions. However,
as expected, GC-Ce6did not releasethe drug evenup to 1 day afterthe
start of incubation because of the robust nature of the covalent bond
betweenGCandCe6.Thus,chemicallyconjugateddrugsareexpectedto
show the same biodistribution of nano-sized drug carriers without any
loss of conjugated drugs in the blood stream. However, based on the
drug release study, the drug release profiles of HGC-Ce6 and GC-Ce6
showed substantial differences under aqueous conditions.
3.3. Cellular uptake and in vitro phototoxicity of HGC-Ce6 and GC-Ce6
To assay cellular uptake of nanoparticles in the SCC7 tumor cell
culture system, both nanoparticles were labeled with FITC and
observed through an FITC filter set (green color). Also, the precise
location of Ce6s after cellular uptake was observed by using a Cy5.5
filter set (red color). After 1 h of incubation, free Ce6 (2.5 μg/ml)-
treated cells presented a strong red color in the cytoplasm, indicating
that a large amount of Ce6 had been transported to and diffused
into the cytoplasm (Fig. 4). Tumor cells incubated with HGC-Ce6 or
GC-Ce6 nanoparticles (2.5 μg/ml of Ce6 in each case) showed green
FITC spots in the cytoplasm, indicating efficient cellular uptake of both
nanoparticles. In the case of HGC-Ce6 treated cells, most of the FITC
fluorescence was localized to the cytoplasm, and physically loaded
Ce6s werediffused throughout the cytoplasm,indicating thatthe Ce6s
were rapidly released from HGC after cellular uptake. These results
confirmed that HGC could deliver Ce6s into the cytoplasm of tumor
cells almost as efficiently as free Ce6 within one hour. These results
are in accordance with a previous study demonstrating that HGC
shows fast cellular uptake by several nondestructive endocytic
pathways, such as caveolae-mediated endocytosis, clathrin-mediated
endocytosis, and macropinocytosis [25,29]. However, in the case of
GC-Ce6-treated cells, the red fluorescentspots congregated morethan
those of HGC-Ce6. Furthermore, these large red spots mainly co-
localized with the green spots in the fluorescence images, indicating
that Ce6 of GC-Ce6 was chemically conjugated to the GC polymers and
could not be released from the nanoparticles. The different intracel-
lular behaviors of HGC-Ce6 and GC-Ce6 might substantially affect
their therapeutic efficacy against cancer.
Next, we studied the in vitro phototoxicity of HGC-Ce6 and GC-Ce6
in a tumor cell culture system. As shown in Fig. 5A, tumor cells were
incubated with free Ce6, HGC-Ce6, or GC-Ce6 (0.5, 1, 2, 4, 8 μg of Ce6,
respectively) for 2 h and then irradiated using an LED lamp at 671 nm
for 20 min. At 1 day post-irradiation, the phototoxicity of each sample
was determined by the MTT assay. The cell viability decreased as a
function of the increase in the concentration of Ce6, reflecting Ce6
phototoxicity. When the Ce6 concentration of free Ce6, HGC-Ce6, or
GC-Ce6 was less than 4 μg/ml, tumor cells treated with free Ce6
showed the greatest loss of cell viability. Importantly, HGC-Ce6-treated
cells showed higher phototoxicity than GC-Ce6-treated cells, whereas
both cell treatments showed similar in vitro singlet oxygen generation
efficiencies.This isconsistentwiththeideathatrapidlyreleasedCe6sin
the HGC-Ce6 can generate singlet oxygen at target cellular organelles
such as mitochondria, compared with congregated photosensitizers of
GC-Ce6 [30]. At a higher Ce6 concentration of 8 μg/ml, all tumor cells
treatedwithfreeCe6,HGC-Ce6,orGC-Ce6showedseverephototoxicity
Fig. 4. Fluorescence microscopy images of SCC-7 cells incubated with free Ce6, FITC labeled-HGC-Ce6 or FITC-labeled GC-Ce6.
25
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 6

at 1 day post-irradiation, which was confirmed by FITC-labeled
annexin-V (green apoptotic cells) (Fig. 5B). This result indicated that
all Ce6 molecules in HGC-Ce6 and GC-Ce6 would be therapeutically
efficient for PDT in cancer treatment.
3.4. In vivo biodistribution of HGC-Ce6 and GC-Ce6
To compare the in vivo biodistribution of physically loaded and
chemically conjugated Ce6s in nanoparticles, saline, free Ce6, HGC-Ce6,
or GC-Ce6 (2.5 mg/kg of Ce6) was injected intravenously into HT-29
human colon adenocarcinoma tumor-bearing mice. The in vivo
biodistribution of Ce6 molecules in each sample could be directly
monitored by non-invasive and real-time fluorescence imaging of the
whole body, because Ce6 can emit strong near infrared (NIR)
fluorescence for efficient tracking [31]. Therefore, it is possible to
image the in vivo localization of physically loaded or chemically
conjugated Ce6 molecules in in vivo systems without using additional
fluorescence dyes.
At 3 h post-injection, free Ce6-treated mice showed whole-body
fluorescence that was not seen in saline-injected mice. A strong signal
was detected in the liver tissue (Fig. 6A). Thus, we did not observe
tumor tissue specificity for free Ce6 in these mice. In addition, whole-
body fluorescence decreased rapidly, due to the rapid excretion of Ce6
[32,33]. In the case of HGC-Ce6, intense whole-body fluorescence was
observed at 3 h post-injection, indicating that physically loaded Ce6
circulates for a longer time than free Ce6. Moreover, there was a slight
increase in Ce6 tumor target specificity. However, the fluorescence of
the whole body and the tumor tissue decreased similarly and rapidly,
as observed for free Ce6 , indicating that physically loaded Ce6 was
rapidly released from HGC, as predicted by the in vitro release data in
Fig. 3B. However, GC-Ce6-treated mice displayed much higher tumor
targetspecificityat 1 h post-injection thaneither freeCe6or HGC-Ce6,
and the subcutaneous tumor tissues could be easily distinguished
from the surrounding tissue. Furthermore, the fluorescence of GC-Ce6
in tumor tissue was maintained for up to 2 days, indicating that the
chemically conjugated Ce6 molecules were not subject to rapid
excretion from the body. The total photon counts of GC-Ce6 in tumor
tissue were about 6–7 fold higher than that of free Ce6 and HGC-Ce6
(Fig. 6B).
Ex vivo fluorescence images also confirmed higher fluorescence in
the tumor tissue of GC-Ce6-treated mice, indicating superior in vivo
tumor targeting of GC-Ce6 compared to that of free Ce6 or HGC-Ce6
(Fig.6C).Theseinvivoandexvivoresultsdemonstratedthatdrugloaded
nanoparticleshadgreatertumortargetingspecificitythanthefreedrug,
although drug accumulation in the tumor tissue from HGC-Ce6 was
inefficient because of the burst in the release of the drug into the blood
stream. In contrast, the chemically conjugated Ce6 in GC-Ce6 nano-
particles was unaffected by the burst in drug release, and consequently
the GC-Ce6 nanoparticle displayed a more prolonged blood circulation
time and more efficient accumulation in the tumor tissue.
3.5. In vivo therapeutic efficacy of HGC-Ce6 and GC-Ce6
The in vivo therapeutic efficiencies of HGC-Ce6 and GC-Ce6 were
evaluated by measuring tumor growth rates in HT-29 tumor-bearing
mice with laser irradiation for PDT. When the inoculated tumor
size grew to 150±30 mm3, saline, free Ce6, HGC-Ce6 or GC-Ce6
(2.5 mg/kg of Ce6) was intravenously injected into the mice. After
post-injection periods of 4 h and 12 h, the tumor tissues were
irradiated with a red laser (670 nm, 250 mW/cm2) for 30 min and
thetumorsizewasmonitoredupto20 days.After7 daypost-injection,
only tumor tissue containing GC-Ce6 and irradiated with the laser
showed severe necrosis, indicating excellent phototoxicity in cancer
treatment, whereas free Ce6- and HGC-Ce6-treated mice failed to
show noticeable phototoxicity in tumor tissue, indicating their lower
therapeutic efficacy in cancer treatment (Fig. 7A).
The tumor growth rate of individual mice clearly showed the
excellent therapeutic results of GC-Ce6 in cancer therapy. As shown in
Fig. 7B, free Ce6-treated mice did not show an effective decrease in
tumor size comparedto the saline-treated mice. This is perhaps due to
lower tumor target specificity. HGC-Ce6-treated mice had slower
tumor growth rates than free Ce6-treated mice, but tumor suppres-
sion was incomplete in these mice. However, GC-Ce6-treated mice
showed a dramatic decrease in tumor volume. The final tumor
volumes in these mice were approximately 160 mm3at 20 day post-
injection, which was significantly smaller than that of free Ce6 or
HGC-Ce6-treated mice (about 760 mm3and 560 mm3, respectively).
This suggested that GC-Ce6 has potential as an effective Ce6-delivery
system for cancer treatment, most likely because of the robust nature
of the chemically conjugated Ce6 molecules in the glycol chitosan
nanoparticles. Taken together, the results demonstrate that there are
significant differences between drug-loaded and conjugated nano-
particles with respect in vivo biodistribution, tumor target specificity,
and cancer treatment.
02468
0
20
40
60
80
100
Cell Viability (%)
Concentration of Ce6 (μ μg/ml)
Free Ce6
HGC-Ce6
GC-Ce6
A
B
DIC
DAPIAnnexin V
No treat
HGC-Ce6
GC-Ce6
Free Ce6
50μ μm
Fig. 5. In vitro phototoxicity of HGC-Ce6 and GC-Ce6. (A) MTT assay data and
(B) Annexin V treated images of SCC-7 cells treated with free Ce6, HGC-Ce6, or GC-Ce6
and laser irradiation.
26
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 7

4. Conclusions
In this study, we have shown that drug-loaded and conjugated
nanoparticles perform differently during both in vitro and in vivo
experiments. We selected Ce6 and GC as a photosensitizer and a
biocompatible polymer, respectively, and developed drug-loaded
and drug-conjugated nanoparticles as model drug delivery systems
in cancer treatment. Both nanoparticles were well dispersed in the
aqueous system and formed stable nano-structures. HGC-Ce6
showed time dependant release of Ce6 and more efficient photo-
dynamic therapy than GC-Ce6 in the cell culture system. However
HGC-Ce6 did not accumulate efficiently in tumor tissue because of
the burst in drug release, even though it showed enhanced tumor
targeting compared with free drug. On the other hand, GC-Ce6 had a
prolonged circulation time and efficiently accumulated in the
tumor, which resulted in excellent therapeutic efficacy in tumor-
bearing mice. The results suggest that GC-Ce6 has great potential
in in vivo PDT for tumors, and provide valuable information about
in vivo burst release of drugs and nanoparticle stabilities for cancer
therapy.
Fig. 6. In vivo biodistribution of HGC-Ce6 and GC-Ce6. (A) In vivo time-dependant whole body imaging of athymic nude mice bearing HT-29 tumors after i.v. injection of saline, free
Ce6, HGC-Ce6, or GC-Ce6 (2.5 mg/kg of Ce6). (B) Quantification of in vivo tumor target specificity recorded as total photon counts per centimeter squared per steradian (p/s/cm²/sr)
of each tumor. (C) Ex vivo images of normal organs (liver, lung, spleen, kidney, and heart) and tumors excised at 2 days post-injection of saline, free Ce6, HGC-Ce6, or GC-Ce6.
27
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 8

Acknowledgements
This work was financially supported by the Real-Time Molecular
Imaging Project, the Global Research Laboratory Project, Fusion
Technology Project (2009-0081876) of MEST, National R&D Program
for Cancer Control of Ministry for Health and Welfare from Republic of
Korea (1020260), and the Intramural Research Program of KIST.
References
[1] M.A. Phillips, M.L. Gran, N.A. Peppas, Targeted nanodelivery of drugs and
diagnostics, Nano Today 5 (2010) 143–159.
[2] X. Li, L. Ding, Y. Xu, Y. Wang, Q. Ping, Targeted delivery of doxorubicin using
stealth liposomes modified with transferrin, Int. J. Pharm. 373 (2009) 116–123.
[3] E. Lee, J. Lee, I.-H. Lee, M. Yu, H. Kim, S.Y. Chae, S. Jon, Conjugated chitosan as a
novel platform for oral delivery of paclitaxel, J. Med. Chem. 51 (2008) 6442–6449.
[4] T.Y.Ohulchanskyy,I.Roy,L.N.Goswami,Y.Chen,E.J.Bergey,R.K.Pandey,A.R.Oseroff,
P.N. Prasad, Organically modified silica nanoparticles with covalently incorporated
photosensitizerforphotodynamictherapyofcancer,NanoLett.7(2007)2835–2842.
[5] K. Kim, J.H. Kim, H. Park, Y.-S. Kim, K. Park, H. Nam, S. Lee, J.H. Park, R.-W. Park, I.-S.
Kim, K. Choi, S.Y. Kim, K. Park, I.C. Kwon, Tumor-homing multifunctional
nanoparticles for cancer theragnosis: simultaneous diagnosis, drug delivery, and
therapeutic monitoring, J. Control. Release 146 (2010) 219–227.
[6] M.E. Fox, F.C. Szoka, J.M.J. Fréchet, Soluble polymer carriers for the treatment of
cancer: the importance of molecular architecture, Acc. Chem. Res. 42 (2009)
1141–1151.
[7] Z. Liu, Y. Jiao, Y. Wang, C. Zhou, Z. Zhang, Polysaccharides-based nanoparticles as
drug delivery systems, Adv. Drug Deliv. Rev. 60 (2008) 1650–1662.
[8] H.Maeda,J.Wu,T.Sawa,Y.Matsumura,K.Hori,Tumorvascularpermeabilityandthe
EPR effect in macromolecular therapeutics: a review, J. Control. Release 65 (2000)
271–284.
[9] K.Y.Choi,H.Chung,K.H.Min,H.Y.Yoon,K.Kim,J.H.Park,I.C.Kwon, S.Y.Jeong,Self-
assembled hyaluronic acid nanoparticles for active tumor targeting, Biomaterials
31 (2010) 106–114.
[10] A.S. Hasan, M. Socha, A. Lamprecht, F.E. Ghazouani, A. Sapin, M. Hoffman, P.
Maincent, N. Ubrich, Effect of the microencapsulation of nanoparticles on the
reduction of burst release, Int. J. Pharm. 344 (2007) 53–61.
[11] B.-c. Bae, K. Na, Self-quenching polysaccharide-based nanogels of pullulan/folate-
photosensitizer conjugates for photodynamic therapy, Biomaterials 31 (2010)
6325–6335.
[12] D.E.J.G.J. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for cancer,
Nat. Rev. Cancer 3 (2003) 380–387.
[13] C.M. Moore, D. Pendse, M. Emberton, Photodynamic therapy for prostate cancer-a
review of current status and future promise, Nat. Clin. Pract. Urol. 6 (2009) 18–30.
[14] L.K. Folkes, P. Wardman, Enhancing the efficacy of photodynamic cancer therapy by
radicals from plant auxin (indole-3-acetic acid), Cancer Res. 63 (2003) 776–779.
[15] S.J. Lee, K. Park, Y.-K. Oh, S.-H. Kwon, S. Her, I.-S. Kim, K. Choi, S.J. Lee, H. Kim, S.G.
Lee, K. Kim, I.C. Kwon, Tumor specificity and therapeutic efficacy of photosen-
sitizer-encapsulated glycol chitosan-based nanoparticles in tumor-bearing mice,
Biomaterials 30 (2009) 2929–2939.
[16] H. Koo, H. Lee, S. Lee, K.H. Min, M.S. Kim, D.S. Lee, Y. Choi, I.C. Kwon, K. Kim, S.Y.
Jeong, In vivo tumor diagnosis and photodynamic therapy via tumoral pH-
responsive polymeric micelles, Chem. Commun. 46 (2010) 5668–5670.
[17] N. Nishiyama, Y. Morimoto, W.-D. Jang, K. Kataoka, Design and development of
dendrimer photosensitizer-incorporated polymeric micelles for enhanced photo-
dynamic therapy, Adv. Drug Deliv. Rev. 61 (2009) 327–338.
[18] J.H. Park, G. Saravanakumar, K. Kim, I.C. Kwon, Targeted delivery of low molecular
drugs using chitosan and its derivatives, Adv. Drug Deliv. Rev. 62 (2010) 28–41.
[19] B.S. Lee, K. Park, S. Park, G.C. Kim, H.J. Kim, S. Lee, H. Kil, S.J. Oh, D. Chi, K. Kim, K.
Choi, I.C. Kwon, S.Y. Kim, Tumor targeting efficiency of bare nanoparticles does not
mean the efficacy of loaded anticancer drugs: Importance of radionuclide imaging
for optimization of highly selective tumor targeting polymeric nanoparticles with
or without drug, J. Control. Release 147 (2010) 253–260.
[20] S.J. Lee, H. Koo, D.-E. Lee, S. Min, S. Lee, X. Chen, Y. Choi, J.F. Leary, K. Park, S.Y.
Jeong, I.C. Kwon, K. Kim, K. Choi, Tumor-homing photosensitizer-conjugated
glycol chitosan nanoparticles for synchronous photodynamic imaging and
therapy based on cellular on/off system, Biomaterials 32 (2011) 4021–4029.
[21] K.H. Min, K. Park, Y.-S. Kim, S.M. Bae, S. Lee, H.G. Jo, R.-W. Park, I.-S. Kim, S.Y. Jeong,
K. Kim, I.C. Kwon, Hydrophobically modified glycol chitosan nanoparticles-
encapsulated camptothecin enhance the drug stability and tumor targeting in
cancer therapy, J. Control. Release 127 (2008) 208–218.
[22] J.-H. Kim, Y.-S. Kim, K. Park, S. Lee, H.Y. Nam, K.H. Min, H.G. Jo, J.H. Park, K. Choi, S.Y.
Jeong, R.-W. Park, I.-S. Kim, K. Kim, I.C. Kwon, Antitumor efficacy of cisplatin-loaded
glycol chitosan nanoparticles in tumor-bearing mice, J. Control. Release 127 (2008)
41–49.
[23] W.W. Chin, P.W. Heng, P.S. Thong, R. Bhuvaneswari, W. Hirt, S. Kuenzel, K.C. Soo,
M. Olivo, Improved formulation of photosensitizer chlorin e6 polyvinylpyrroli-
done for fluorescence diagnostic imaging and photodynamic therapy of human
cancer, Eur. J. Pharm. Biopharm. 69 (2008) 1083–1093.
[24] H.-Y. Hwang, I.-S. Kim, I.C. Kwon, Y.-H. Kim, Tumor targetability and antitumor
effect of docetaxel-loaded hydrophobically modified glycol chitosan nanoparti-
cles, J. Control. Release 128 (2008) 23–31.
[25] S. Park, S.J. Lee, H. Chung, S. Her, Y. Choi, K. Kim, K. Choi, I.C. Kwon, Cellular uptake
pathway and drug release characteristics of drug-encapsulated glycol chitosan
nanoparticles in live cells, Microsc. Res. Tech. 73 (2010) 857–865.
[26] R. Bachor, C.R. Shea, R. Gillies, T. Hasan, Photosensitized destruction of human
bladder carcinoma cells treated with chlorin e6-conjugated microspheres, Proc.
Natl. Acad. Sci. U. S. A. 88 (1991) 1580–1584.
[27] X.L. Wu, J.H. Kim, H. Koo, S.M. Bae, H. Shin, M.S. Kim, B.-H. Lee, R.-W. Park, I.-S.
Kim, K. Choi, I.C. Kwon, K. Kim, D.S. Lee, Tumor-targeting peptide conjugated pH-
responsive micelles as a potential drug carrier for cancer therapy, Bioconjug.
Chem. 21 (2010) 208–213.
[28] S. Lee, K.Y. Choi, H.Chung, J.H. Ryu, A. Lee, H. Koo,I.-C. Youn, J.H. Park, I.-S.Kim, S.Y.
Kim, X. Chen, S.Y. Jeong, I.C. Kwon, K. Kim, K. Choi, Real time, high resolution video
imaging of apoptosis in single cells with a polymeric nanoprobe, Bioconjug. Chem.
22 (2011) 125–131.
[29] H.Y. Nam, S.M. Kwon, H. Chung, S.-Y. Lee, S.-H. Kwon, H. Jeon, Y. Kim, J.H. Park, J.
Kim, S. Her, Y.-K. Oh, I.C. Kwon, K. Kim, S.Y. Jeong, Cellular uptake mechanism and
intracellular fate of hydrophobically modified glycol chitosan nanoparticles,
J. Control. Release 135 (2009) 259–267.
[30] J. Morgan, A.R. Oseroff, Mitochondria-based photodynamic anti-cancer therapy,
Adv. Drug Deliv. Rev. 49 (2001) 71–86.
Fig. 7. In vivo therapeutic efficacy of HGC-Ce6 and GC-Ce6. (A) Tumor images and
(B) tumor growth data after photodynamic therapy with free Ce6, HGC-Ce6, or GC-Ce6
(5 mg/kg of Ce6) in HT-29 tumor-bearing mice (*=pb0.01).
28
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE

Page 9

[31] S.Y. Park, H.J. Baik, Y.T. Oh, K.T. Oh, Y.S. Youn, E.S. Lee, A smart polysaccharide/
drug conjugate for photodynamic therapy, Angew. Chem. Int. Ed. 50 (2011)
1644–1647.
[32] G.P. Gurinovich, T.E. Zorina, S.B. Melnov, N.I. Melnova, I.F. Gurinovich, L.A.
Grubina, M.V. Sarzhevskaya, S.N. Cherenkevich, Photodynamic activity of chlorin
e6 and chlorin e6 ethylenediamide in vitro and in vivo, J. Photochem. Photobiol., B
13 (1992) 51–57.
[33] G.A.Kostenich,I.N.Zhuravkin,E.A.Zhavrid,Experimentalgroundsforusingchlorine6in
the photodynamic therapy of malignant tumors, J. Photochem. Photobiol., B 22 (1994)
211–217.
29
S.J. Lee et al. / Journal of Controlled Release 152 (2011) 21–29
NANOMEDICINE