Nanoscopic micelle delivery improves the photophysical properties and efficacy of
photodynamic therapy of protoporphyrin IX
Huiying Dinga, Baran D. Sumerc, Chase W. Kessingera, Ying Donga,b, Gang Huanga,
David A. Boothmana,b, Jinming Gaoa,⁎
aDepartment of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, United States
bLaboratory of Molecular Stress Responses, Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard,
Dallas, TX 75390, United States
cDepartment of Otolaryngology, Head and Neck Surgery, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, United States
a b s t r a c ta r t i c l ei n f o
Received 30 October 2010
Accepted 3 January 2011
Available online 10 January 2011
Nanodelivery systems have shown considerable promise in increasing the solubility and delivery efficiency of
hydrophobic photosensitizers for photodynamic therapy (PDT) applications. In this study, we report the
preparation and characterization of polymeric micelles that incorporate protoporphyrin IX (PpIX), a potent
photosensitizer, using non-covalent encapsulation and covalent conjugation methods. Depending on the
incorporation method and PpIX loading percentage, PpIX existed as a monomer, dimer or aggregate in the
micelle core. The PpIX state directly affected the fluorescence intensity and1O2generation efficiency of the
resulting micelles in aqueous solution. Micelles with lower PpIX loading density (e.g. 0.2%) showed brighter
fluorescence and higher1O2yield than those with higher PpIX loading density (e.g. 4%) in solution. However,
PDT efficacy in H2009 lung cancer cells showed an opposite trend. In particular, 4% PpIX-conjugated micelles
demonstrated the largest PDT therapeutic window, as indicated by the highest phototoxicity and relatively
low dark toxicity. Results from this study contribute to the fundamental understanding of nanoscopic
structure–property relationships of micelle-delivered PpIX and establish a viable micelle formulation (i.e. 4%
PpIX-conjugated micelles) for in vivo evaluation of antitumor efficacy.
Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Recently, photodynamic therapy (PDT) has received considerable
cancer and other diseases [1–3]. During PDT, a photosensitizer (PS)
drug is first intravenously administered into the patient. Upon light
activation at the targeted tissues, singlet oxygen (1O2) and other
[4,5]. Compared to other therapeutic modalities, PDT is much less
invasive than surgery, and is more tumor-selective than chemother-
apy and radiotherapy with minimal toxicity to normal tissues.
Currently, Photofrin®(porfimer sodium), Visudyne®(verteporfin),
Levulan®(5-aminolevulinic acid, 5-ALA) and Metvixia®(methyl
aminolevulinic acid) have been approved as PDT drugs by the Food
approved to treat head and neck cancers in the European Union .
Except for 5-ALA, all the other clinically approved PDT drugs are
porphyrin-based molecules, which are hydrophobic and have intrin-
sically low water solubilities. Excipient molecules such as lipid
mixtures (e.g. in Visudyne®) and ethanol/poly(propylene glycol)
(e.g. in Foscan®) are used to solubilize these agents for intravenous
injections. 5-ALA is a pro-drug that can be converted into protopor-
phyrin IX (PpIX) in rapidly proliferating tumor cells compared to
normal tissues leading to selective accumulation of PpIX in tumors
[6,7]. The endogenously produced PpIX in turn acts as a potent PS
allowing selective destruction of cancer cells. A major disadvantage of
because of its polarity. Moreover, it is unstable in aqueous solution
from the neutral to basic pH range. In the clinical setting, high doses of
5-ALA must be administered to reach clinically efficacious levels of
PpIX. So far, free PpIX cannot be directly injected intravenously due to
its low water solubility (~1 μg/mL). Moreover, dark toxicity as well as
singlet oxygen further limits the direct clinical use of PpIX [8,9].
Nanoparticles have been developed as an effective PDT drug
delivery platform in recent years , and extensive research efforts
have been devoted to the development of effective nanocarriers for
PDT agents including liposomes [11,12], dendrimers [13,14], polymer
nanoparticles [15,16] and polymeric micelles [17,18]. Among these,
polymeric micelles provide an intriguing option due to their unique
core–shell structures in aqueous environment. The hydrophobic core
of the micelles provides a naturally compatible environment for
Journal of Controlled Release 151 (2011) 271–277
⁎ Corresponding author.
E-mail address: email@example.com (J. Gao).
0168-3659/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
loading hydrophobic PS molecules, while the hydrophilic shells
stabilize the nanoparticles for increased water solubility and prolong
blood circulation. In addition to the solubility enhancement of
hydrophobic agents into aqueous solution, micelle encapsulation
can also greatly improve the photophysical and photochemical
properties of PS in aqueous solution. Singlet oxygen is believed to
be the main cytotoxic agent with PDT, and the properties of singlet
oxygen are sensitive to the microenvironment surrounding the PS.
The quantum yield, lifetime and diffusion distance of singlet oxygen
will vary depending on whether it is generated by free PS in aqueous
solution or PS encapsulated in micelles. Studies have shown longer
1O2lifetimes and improved1O2generation when porphyrin-based PS
is incorporated into micelle carriers , and that the produced1O2in
the micelle core can escape out of the core efficiently . Moreover,
the micelle formulation may protect PpIX from photobleaching by
ROS and reacting with biomolecules directly. Another advantage of
incorporating porphyrin-based PS agents into micelles includes the
ability to prevent aggregation, which can interfere with PS efficacy
and decrease the fluorescence of the PS dye. Previously, our lab
reported the use of polymeric micelles for the delivery of imaging
agents [21,22], and/or hydrophobic drugs [23,24] for cancer diagnos-
tic or therapeutic applications. In this study, we describe the use of
micelle carriers for the delivery of PpIX using a biocompatible and
biodegradable block copolymer, poly(ethylene glycol)-b-poly(D,L-
lactide) (PEG-PLA). A unique correlation of structure and photo-
physical properties of PpIX/PEG-PLA micelles was discovered. The
fluorescence efficiency,1O2quantum yield and PDT efficacy in cancer
cells were found to be highly dependent on the mode (i.e. physical
encapsulation vs. covalent conjugation) and PpIX loading density.
These results contribute to the fundamental understanding of the
nanocarrier effect on the photophysical properties of PpIX-containing
micelles, which will facilitate the implementation of these photosen-
sitive nanoparticles for PDT treatment of cancer and other diseases.
2.1. Syntheses of PEG-PLA and PpIX-conjugated PEG-PLA
The PEG-PLA (molecular weights of PEG and PLA blocks are 5 kDa)
copolymer was synthesized by ring-opening polymerization of
D,L-lactide (Alfa Aesar, Ward Hill, MA) in dry toluene at 115 °C for 24 h
using MeO-PEG-OH as a macroinitiator and Sn(Oct)2 as a catalyst
following a published procedure . For PEG-PLA–PpIX synthesis,
PpIX (0.1 mmol, Sigma-Aldrich) was first dissolved in 20 mL dry
THF. Then PEG-PLA (1.2 g), dicyclohexylcarbodiimide (0.1 mmol) and
N, N-dimethylaminopyridine were added under dry nitrogen and
stirred at room temperature for 48 h. After reaction, white precipitate
of dicyclohexylurea was filtered and the filtrate was concentrated
by rotavap. The residual mixture was dialyzed in distilled water and
lyophilized. UV–vis analysis showed that 75% of PEG-PLA-OH end
groups were conjugated with PpIX.
2.2. Micelle production and characterization
PpIX/PEG-PLA micelles were produced by a solvent evaporation
method as previously reported . Briefly, a proper amount of PEG-
PLA and free PpIX or PEG-PLA–PpIX were first dissolved in THF with
PpIX weight ratio varying from 0.04% to 4%. The mixture was added
dropwise to distilled water under sonication and then allowed to
evaporate overnight to remove THF. The resulting PpIX-micelles were
purified by centrifugation dialysis (MW cutoff 100 kDa) to remove
The PpIX-micelles were characterized by TEM for particle
morphology and zeta sizer (Malvern NanoZS) for zeta potential and
hydrodynamic diameters. The PpIX loading density was measured by
dissolving a solid micelle sample in THF and quantified by UV–vis
analysis using a previously established calibration curve. The UV–vis
absorption spectra of PpIX-micelles were recorded at room temper-
ature using a Shimadzu UV spectrophotometer (UV-1800), and the
emission spectra were obtained using a Hitachi fluorescence spectro-
2.3. Singlet oxygen detection
The amount of
absorbance or emission  of ADPA in the aqueous PpIX-micelle
solutions. Briefly, a series of air-saturated PpIX-micelle solutions
(1 mL) were prepared with anthracene-9,10-dipropionic acid (ADPA,
10 μM) and fresh PpIX micelles. The solution was illuminated under a
laser light (λ=532 nm, power density=10 mW/cm2). The consump-
tions of ADPA (A0–At, A0and Atare the absorption of ADPA at 378 nm
before and after irradiation, respectively) were followed by monitor-
ing its absorption decrease at 378 nm over time. Relative1O2yields
(ΦΔ) were calculated by the slopes of the ADPA conversion and
normalized to 0.2% PpIX-conjugated micelles.
1O2was measured by following the loss of UV
2.4. PDT efficacy of PpIX-micelles in H2009 cells
H2009 human lung cancer cells were cultured in RPMI 1640
medium supplemented with 5% fetal bovine serum and antibiotics
(Penicillin–Streptomycin) at 37 °C in a 10% CO2humidified incubator.
One day before the PDT treatments, H2009 cells were trypsinized
using 0.05% trypsin–EDTA and seeded (10,000 cells/well) into 48-well
plates. Cell culture media were then replaced by media containing
predetermined doses of PpIX micelles and incubated for 24 h. For the
PDT study, cells were illuminated with laser (λ=532 nm, power
density=20 mW/cm2) for 10 min. After irradiation, H2009 cells were
allowed to grow for an additional 5 days in fresh media. Relative cell
survival was measured by a DNA assay using Hoechst dye 33258 
and data were graphed as means of treated/control (T/C)±SE×100%
from three independent experiments performed in sextuplicate. Dark
toxicity was assessed from H2009 cells with PpIX-micelle incubation
but without laser light exposure. Student's t-test was performed for
statistical analysis (p value less than 0.05 was considered significant).
2.5. Confocal laser scanning microscopy of micelle uptake in H2009 cells
Confocal imaging studies were performed on a Nikon TE2000E
confocal laser scanning microscope. H2009 cells were seeded in a
glass-bottomed culture dish and allowed to attach overnight. The
media were replaced with fresh media containing different PpIX-
micelle samples at 100 μg/mL. H2009 cells were examined at different
time points with a 600× total magnification (λex=405 nm,
λem=605±35 nm). ImageJ software (National Institutes of Health)
was utilized to quantify the mean fluorescence intensity (MFI) of the
PpIX-micelles in vitro.
3.1. Production and physical characterization of PpIX-micelles
Two series of PpIX/PEG-PLA micelles were generated: one through
a non-covalent hydrophobic encapsulation strategy and another via
covalent conjugation of PpIX to the hydroxyl group of PEG-PLA
through ester linkage (Fig. 1A). In both series, PpIX loading density
(weight percentage of PpIX over micelles) was varied from 0.04% to
4%. Transmission electron microcopy (TEM) illustrated that PpIX-
containing micelles had a spherical morphology (Fig. 1B). Dynamic
light scattering (DLS) analysis showed that the micelle diameters
were approximately 30 nm (Fig. 1C), with slight although statistically
insignificant increase of micelle size with an increase in PpIX loading
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277
density (Table 1). Zeta potential (ξ) measurements showed that the
micelle surface became more negatively charged with increased
loading of PpIX in both series. For example, the ξ values decreased
from −6.9±1.5 to −13.3±1.7 and −19.6±1.3 mV for 0% (blank),
0.2% and 4% PpIX-encapsulated micelles, respectively (Table 1). A
similar trend was also observed for PpIX-conjugated micelles. We
attribute the decrease in ξ potential to the increased surface density of
the negatively charged carboxylate (–COO−) groups in higher PpIX-
3.2. UV–vis and fluorescence properties of PpIX micelles
The UV–vis absorption spectra of free PpIX monomer, dimer and
aggregates are shown in Fig. 2A. In ethyl acetate (EA), PpIX stayed as a
monomer with a sharp Soret band at 404 nm. At pH 11 in phosphate
buffer, PpIX existed as a dimer and the Soret band blue shifted to
380 nm. At pH 7 in phosphate buffer, a greatly broadened split Soret
whichcorrelateswiththeformationofextended aggregates insolution.
In ethyl acetate, PpIX monomer showed bright fluorescence at 632 nm
(λex=404 nm, Fig. 2B). For the PpIX dimer in an aqueous environment
(pH 11), the fluorescence intensity decreased with a blue shift of
emission peak (λem=620 nm, λex=404 nm). For PpIX aggregates at
pH 7, the fluorescence intensity became very weak (Fig. 2B).
The absorption spectra of PpIX-encapsulated micelles were highly
dependent on the loading percentage of PpIX (Fig. 2C). Between 0.04%
and 0.2% loading, PpIX existed mostly as a monomer in the micelle
core as indicated by the Soret band at 404 nm. At 0.5%, a mixture of
PpIX monomers and dimers was present. Above 1% in PpIX loading,
PpIX formed aggregates in the micelle core as shown by the broadly
split Soret bands. Fluorescence emission of PpIX-encapsulated
micelles reached a maximum (λem=630 nm) with 0.2% loading
density. At higher loading density (N1%), the fluorescence intensity
decreased dramatically (Fig. 2D), consistent with the formation of
PpIX aggregates inside the micelle core. For PpIX-conjugated micelles,
the absorption spectra showed that PpIX existed mostly as monomers
between 0.04 and 0.2% PpIX loading density (Fig. 2E). At 0.2%, PpIX-
conjugated micelles had a slightly higher fraction of PpIX in the
monomeric state as indicated by the sharper Soret band (half-peak
width was 63 nm) than 0.2% PpIX-encapsulated micelles (70 nm). At
higher PpIX loading (i.e. 2% or 4%), PpIX existed mostly in the dimeric
state as indicated by the blue-shifted Soret band. Similar to PpIX-
encapsulated micelles, the fluorescence intensity of PpIX-conjugated
micelles reached a maximum (λem=630 nm) at 0.2% PpIX loading
density and decreased significantly at higher PpIX loading (Fig. 2F).
For both PpIX-conjugated and PpIX-encapsulated micelles, we chose
0.2% and 4% loading densities for the subsequent photophysical and
PDT efficacy studies.
Fig. 1. (A) Schematic illustration of two different types of PpIX/PEG-PLA micelles from non-covalent encapsulation and covalent conjugation methods. At low loading density (i.e.
0.2%), PpIX exists as monomers in both micelle formulations; at high loading density (i.e. 4%), PpIX is present as aggregates or dimers in encapsulated and conjugated micelles,
respectively. (B)Arepresentativetransmissionelectronmicroscopy(TEM)imageof4%PpIX-conjugatedmicellescounter-stained with2%PTA.Thescalebaris100 nm.(C)Histogram
depicting hydrodynamic size distribution of 4% PpIX-conjugated micelles by dynamic light scattering (DLS).
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277
3.3. Efficiency of1O2generation
The formation of1O2was measured by the bleaching of ADPA dye,
which was monitored by following the net loss of ADPA absorption at
378 nm over time (Fig. 3) [28,29]. The relative quantum yields (ΦΔ) of
1O2generation from different micelle formulations were measured
and listed in Table 1. The ΦΔvalues were normalized to 0.2% PpIX-
conjugated micelles. At 0.2% PpIX loading density, PpIX-encapsulated
micelles showed a smaller ΦΔ(0.82) compared to PpIX-conjugated
micelles (1.0). At 4% PpIX loading density, PpIX-conjugated micelles
showed a much higher relative ΦΔvalue (0.48) than PpIX-encapsu-
inside the micelle cores (i.e. ΦΔ, monomerNΦΔ, dimerNNΦΔ, aggregates).
3.4. PDT efficacy in H2009 lung cancer cells
Fig. 4 shows the phototoxicity and dark toxicity of different PpIX-
micelles in H2009 lung cancer cells after 24 h incubation. For
phototoxicity measurement, the cells were irradiated with laser light
(λ=532 nm, power density=20 mW/cm2) for 10 min. Cell cytotoxi-
city was measured as the percentage of the viable cells over the
untreated cell control (i.e. without micelle incubation and laser
exposure). For blank PEG-PLA micelles, the H2009 cells did not show
any observable phototoxicity or dark toxicity (data not shown). For
PpIX-micelles, several major observations can be made from the PDT
data. First, the phototoxicities of 0.2% PpIX-micelles (either encapsu-
lated or conjugated formulation) were significantly lower than those
of 4% PpIX-micelles at the same micelle dose. For example, the IC50
(micelle concentration causing 50% toxicity) values were 51±4 and
21±2 μg/mL (p=0.002) for 0.2% and 4% PpIX-encapsulated micelles,
micelles,asillustratedby58±3 and3±2 μg/mL(p=0.0003) for0.2%
and 4% loading density, respectively. These data are contradictory to
observed for 0.2% PpIX-micelles. Second, 4% PpIX-encapsulated
Comparison of the physical properties of free PpIX and PpIX/PEG-PLA micelles.
PpIX samplesPpIX loading (wt.%)a
ξ (mV) Soret band (nm)Q-bands (nm) Emission (nm)
535, 562, 589, 642
506, 538, 574, 629
542, 565, 593, 640
506, 538, 575, 630
510, 542, 581, 635
aPpIX loading density was measured as the weight of PpIX over that of total micelles.
bBy dynamic light scattering.
cRelative1O2yields that were normalized to that of 0.2% PpIX-conjugated micelles.
dPpIX at 2 μM in phosphate buffer (PB) at pH 7 (aggregated state).
Fig. 2. UV–vis absorption (A,C,E) and fluorescence (B,D,F, λex=404 nm) spectra of free
PpIX, PpIX-encapsulated micelles, and PpIX-conjugated micelles, respectively. Free
PpIX (A,B) existed as monomers in ethyl acetate (EA), dimers in phosphate buffer (PB)
at pH=11, and aggregates in phosphate buffer (PB) at pH=7 ([PpIX]=8 μM in all
samples). PpIX-encapsulated micelles (C,D) show PpIX existed as monomers at low
loading density (b0.2%) and as aggregates at high loading density (N1%). In comparison,
PpIX-conjugated micelles (E,F) showed that PpIX existed as monomers at low loading
density (b0.2%) and as dimers at high loading density (N2%). All the micelles were
measured at 2 mg/mL concentration except for those indicated by a dilution factor in C
and E for higher loading PpIX-micelles.
Fig. 3. (A) Exemplar UV–vis spectra of ADPA in the presence of 0.2% PpIX-conjugated
micelles as a function of laser irradiation time (λ=532 nm, power density=10 mW/
cm2). Inset figure shows the reaction of ADPA with1O2. (B) ADPA consumption over
time in solutions containing different PpIX-micelles with (solid symbols) and without
laser irradiation (open symbols). A0and Atare absorption of ADPA at 378 nm before
and after irradiation, respectively.
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277
micelles showed significantly higher dark toxicity than the other
micelle formulations. For example, an 80% relative survival was
observed at a micelle dose of 8±3 μg/mL; in contrast, none of the
other PpIX-micelles showed similar dark toxicity even at the highest
micelle dose of 80 μg/mL. Third, 4% PpIX-conjugated micelles
demonstrated the largest PDT therapeutic window between the
phototoxicity (Fig. 4B, red open circles) and dark toxicity (Fig. 4B,
black solid circles). Their phototoxicity was the highest among all
micelles while the dark toxicity was comparable to 0.2% PpIX-micelles
and much lower than 4% PpIX-encapsulated micelles (Fig. 4).
3.5. Intracellular fluorescence of PpIX-micelles
Confocal laser scanning microscopy was used to examine micelle
uptake and PpIX release from different PpIX-micelles in H2009 cells
over time. Both 0.2% PpIX-micelle formulations had similar fluores-
cence data; therefore only 0.2% PpIX-encapsulated micelles were
shown (Fig. 5A). At 1 h, the 0.2% PpIX-micelles showed similar
intracellular fluorescence over the other two 4% PpIX-micelles. In
addition, the fluorescence intensity from the 0.2% PpIX-micelles did
not change considerably over the time course from 1 to 24 h (Fig. 5A
and D black bars). In contrast, the 4% PpIX-encapsulated micelles
(Fig. 5B and D red bars) showed a dramatic increase in fluorescence
intensity in H2009 cells from 1 to 4 h (e.g. MFI values were 3.6±1.1
and 20.7±3.7 at 1 h and 4 h, respectively; pb0.001). The MFI values
(20.7±3.7 and 22.4±3.8 at 4 and 24 h, respectively) remained
relatively the same from 4 to 24 h (p=0.52). Unlike the 4% PpIX-
encapsulated micelles, the intracellular fluorescence intensity of
H2009 cells incubated with 4% PpIX-conjugated micelles (Fig. 5C
and D blue bars) showed a pattern of steady increase from 2.8±0.4 at
1 h to 15.8±1.6 at 24 h (pb0.001).
The objective of the current study was to develop PpIX-micelles
and investigate their photophysical properties and PDT efficacy in
cancer cells. PpIX is a potent photosensitizer and although its
precursor, 5-ALA (Levulan®) has been clinically approved by the
FDA, poor membrane permeability was a major limiting factor to
achieve the desired clinical efficacy . To overcome this limitation,
we aimed to establish a micellar nanocarrier using a biocompatible
and biodegradable PEG-PLA copolymer to directly deliver PpIX to
cancer cells. Currently, Genexol™, a PEG-PLA micelle formulation for
the delivery of paclitaxel, has already been clinically approved for
cancer treatment in S. Korea .
PpIX is planar molecule with four conjugated pyrrole rings (Fig. 1)
. PpIX can easily aggregate in aqueous solution and its aggregation
behavior has been extensively studied as a function of pH and ionic
Fig. 4. Phototoxicity (red open symbols) and dark-toxicity (black solid symbols) of (A)
0.2% PpIX-micelles and (B) 4% PpIX-micelles as a function of micelle/PpIX dose after
24 h incubation. The relative survival was normalized to the control cells without light
nor PpIX. Both PpIX-encapsulated and PpIX-conjugated formulations were shown in
each figure. Laser irradiation conditions were: λ=532 nm, power density=20 mW/
cm2, total light dose=12 J/cm2.
Fig. 5. Confocal laser scanning microscopy images of H2009 lung cancer cells incubated
with PpIX-micelles over time. (A) 0.2% PpIX-encapsulated micelles; (B) 4% PpIX-
encapsulated micelles; and (C) 4% PpIX-conjugated micelles. Red fluorescence is from
structural moieties containing PpIX. All micelle concentrations were maintained at
100 μg/mL. The scale bars are 20 μm in A–C. (D) The mean fluorescence intensity from
10 cells as a function of incubation time for different PpIX-micelles. The p values were
calculated using the Student's t-test and indicated in D between paired groups of
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277
strength [32,33]. In thepH range of 0–3, PpIX stays asa monomer with
a sharp Soret band at 404 nm and four Q-bands; at pHN8, PpIX exists
as a dimer with a sharp Soret band at 388 nm and four weak Q-bands;
and in the pH range 3–7, PpIX forms extended aggregates with two
splitting weak Soret bands centered at 350 and 460 nm and four weak
Q bands. In these larger aggregates, porphyrins preferentially interact
axially through π–π interactions and laterally by edge-to-edge
hydrophobic interactions. Formation of intermolecular hydrogen
bonds between the carboxylic acids was further hypothesized to
contribute to the stabilization of the aggregated structures . At
physiological pH (7.4), PpIX has a very low aqueous solubility (~1 μg/
mL) and cannot be directly administered intravenously.
Polymeric micelles provide an attractive nanocarrier option for the
delivery of PpIX. In this study, we prepared PpIX-micelles by two
different strategies: non-covalent encapsulation of PpIX in the
hydrophobic cores of micelles and covalent conjugation of PpIX to
the core-forming block of the PEG-PLA copolymer (Fig. 1A). We
systematically examined the UV–vis absorption, fluorescence emis-
sion,1O2yield and PDT efficacy of different PpIX-micelles. At low PpIX
loading density (i.e., b0.2%), PpIX mostly stayed in the monomeric
state as indicated by the sharp Soret band at 404 nm, strong
fluorescence intensity, and high1O2yield regardless of incorporation
strategies. At high PpIX loading density (i.e., 4%), however, the
incorporation strategy had a dramatic effect on the PpIX state in the
micelles. In PpIX-encapsulated micelles, aggregated forms of PpIX
were observed as indicated by the split Soret bands and complete
quenching of fluorescence and
comparison, the 4% PpIX-conjugated micelles showed that PpIX
were present as dimers with retaining of considerable1O2generation
capacity (ΦΔ=0.48). These results are consistent with the previous
reports that only PpIX monomers and dimers can be photoexcited to
triplet state thus allowing for the generation of1O2[34,35].
micelles did not correlate with their phototoxicity in H2009 lung
micelles showed significantly lower phototoxicities compared to the
4% PpIX-micelles at the same micelle dose (Fig. 4). To investigate this
discrepancy, we used confocal laser scanning microscopy to examine
the micelle uptake and PpIX release in H2009 cells. For pegylated
micelle nanoparticles, cell uptake occurs through fluidic phase
endocytosis where nanoparticles are internalized via endosomes and
distributed to other intracellular organelles (e.g. lysosome, Golgi)
[36,37]. At 4% drug loading, both micelle formulations had very low
fluorescence emissions in cell culture media due to PpIX quenching
within the micelle core (Fig. 1). With 4% PpIX-encapsulated micelles,
rapid increasesin fluorescence in H2009 cells was observedin the first
4 h (Fig. 5). We attribute this increase to the release of free PpIX from
intact micelles as well as from dissociation of micelles into PEG-PLA
unimersandfree PpIX, whichmay furtherbindto cytosolicproteins or
membrane structures leading to further elevated fluorescence. For 4%
PpIX-conjugated micelles, higher intracellular fluorescence intensity
of some micelles into PEG-PLA–PpIX unimers. Similar micelle
dissociation events were also observed following exposure to
fluorogenic PEG-b-poly(ε-caprolactone) micelles under biological
conditions [38,39]. In comparison, 4% conjugated micelles were
more stable and had slower micelle dissociation kinetics compared
to 4% encapsulated micelles in cell culture medium (Supplementary
Fig. S1), which is consistent with the fast equilibrium for 4% PpIX-
encapsulated micelles and steady increase for 4% conjugated micelles
in intracellular fluorescence measurements.
Our data show that 4% PpIX-conjugated micelles provided the
largest PDT therapeutic index compared to the other micelle
formulations tested (Fig. 4). Several factors may contribute to this
enhanced performance. First, conjugation of PpIX to the PLA segment
prevented the formation of highly aggregated structures as in the 4%
1O2 generation (ΦΔ=0.06). In
1O2 generation efficiency from different PpIX-
PpIX-encapsulated micelles. Consequently, the intact 4% PpIX-
conjugated micelles had much higher1O2yield (ΦΔ=0.48) than 4%
PpIX-encapsulated micelles (ΦΔ=0.06). Second, it is known that
high concentrations of free PpIX in cells can induce dark toxicity due
to the binding of PpIX to mitochondrial membranes [40–42]. As such,
PpIX conjugation to the PEG-PLA copolymer can effectively prevent
the release of free PpIX and avoid dark toxicity. Third, 4% PpIX-
conjugated micelles showed pattern of steady increase in intracellu-
lar fluorescence, which suggest uptake and the slow release of
conjugated PpIX inside the cells for improved phototoxicity over
time. The prolonged PpIX accumulation may reflect the slower
micelle dissociation kinetics to release PEG-PLA–PpIX. In contrast, in
4% PpIX-encapsulated micelles, after an initial increase no changes in
fluorescence intensity were observed from 4 to 24 h, which indicates
the reaching of equilibrium between PpIX-micelle uptake, PpIX
release, and the subsequent clearance of released free PpIX. Although
4% PpIX-encapsulated micelles had higher intracellular fluorescence
over 4% PpIX-conjugated micelles in confocal studies, chemical
extraction of PpIX from cells showed that PpIX-conjugated micelles
had higher intracellular PpIX accumulation than that of encapsulated
micelles (Supplementary Fig. S2). These results further verified the
protection of PpIX and its photosensitive properties in conjugated
micelle formulations, making 4% PpIX-conjugated micelles the best
formulation for PDT efficacy. It is interesting to note that for
anticancer drug delivery, it is imperative that intact drug molecules
be released from nanocarriers. In this study, release of free PpIX from
4% PpIX-encapsulated micelles caused undesirable dark toxicity,
while 4% PpIX-conjugated micelles allowed for an effective dose
accumulation and higher phototoxicity with reduced dark toxicity in
This study systematically investigated several PEG-PLA micelle
formulations for the delivery of PpIX, a precursor of heme biosynthe-
sis and a potent PDT agent. Non-covalent encapsulation and covalent
conjugation strategies were employed to incorporate PpIX into PEG-
PLA micelle nanocarriers, which yielded monomeric, dimeric and
aggregated forms of PpIX in the micelle core. Although 0.2% PpIX-
micelles had higher fluorescence intensity and more efficient
generation in aqueous solution, 4% PpIX-micelles resulted in brighter
fluorescence and higher PDT efficacy in cancer cells as a result of PpIX
release and micelle dissociation. Among all formulations, 4% PpIX-
conjugated micelles provided the highest PDT efficacy with relatively
low dark toxicity. These results contribute to the mechanistic
understanding of the structure–property relationships of PpIX-
micelles and establish an optimal micelle formulation (e.g. 4% PpIX-
conjugated micelles) for subsequent in vivo evaluation in animals.
This research is supported by the National Cancer Institute
(R01CA122994 and R01CA129011 to JG), (R01CA102792 to DAB) and
a Susan G. Komen Foundation postdoctoral fellowship (PDF0707216).
This is manuscript CSCN058 from the program in Cell Stress and Cancer
Nanomedicine in the Simmons Comprehensive Cancer Center at the
University of Texas Southwestern Medical Center at Dallas.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277
 D.E. Dolmans, D. Fukumura, R.K. Jain, Photodynamic therapy for cancer, Nat. Rev.
Cancer 3 (2003) 380–387.
 A.P. Castano, P. Mroz, M.R. Hamblin, Photodynamic therapy and anti-tumour
immunity, Nat. Rev. Cancer 6 (2006) 535–545.
 J.P. Celli, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue, T.
Hasan, Imaging and photodynamic therapy: mechanisms, monitoring, and
optimization, Chem. Rev. 110 (2010) 2795–2838.
 J.W. Snyder, E. Skovsen, J.D. Lambert, P.R. Ogilby, Subcellular, time-resolved
studies of singlet oxygen in single cells, J. Am. Chem. Soc. 127 (2005)
 F. Ricchelli, S. Gobbo, G. Moreno, C. Salet, L. Brancaleon, A. Mazzini, Photophysical
properties of porphyrin planar aggregates in liposomes, Eur. J. Biochem. 253
 J.C. Kennedy, R.H. Pottier, D.C. Pross, Photodynamic therapy with endogenous
protoporphyrin IX: basic principles and present clinical experience, J. Photochem.
Photobiol. B 6 (1990) 143–148.
 P. Uehlinger, M. Zellweger, G. Wagnieres, L. Juillerat-Jeanneret, H. van den Bergh,
N. Lange, 5-Aminolevulinic acid and its derivatives: physical chemical properties
and protoporphyrin IX formation in cultured cells, J. Photochem. Photobiol. B 54
 S. Eleouet, N. Rousset, J. Carre, L. Bourre, V. Vonarx, Y. Lajat, G.M. Beijersbergen van
Henegouwen, T. Patrice, In vitro fluorescence, toxicity and phototoxicity induced
by delta-aminolevulinic acid (ALA) or ALA-esters, Photochem. Photobiol. 71
 J.M. Fernandez, M.D. Bilgin, L.I. Grossweiner, Singlet oxygen generation by
photodynamic agents, J. Photochem. Photobiol., B 37 (1997) 131–140.
 W. Chen, J. Zhang, Using nanoparticles to enable simultaneous radiation and
photodynamic therapies for cancer treatment, J. Nanosci. Nanotechnol. 6 (2006)
 H.P. Lassalle, D. Dumas, S. Gräfe, M.A. D'Hallewin, F. Guillemin, L. Bezdetnaya,
Correlation between in vivo pharmacokinetics, intratumoral distribution and
photodynamic efficiency of liposomal mTHPC, J. Control. Release 134 (2009)
 A. Johansson, J. Svensson, N. Bendsoe, K. Svanberg, E. Alexandratou, M. Kyriazi, D.
Yova, S. Grafe, T. Trebst, S. Andersson-Engels, Fluorescence and absorption
assessment of a lipid mTHPC formulation following topical application in a non-
melanotic skin tumor model, J. Biomed. Opt. 12 (2007) 034026.
 S. Battah, S. Balaratnam, A. Casas, S. O'Neill, C. Edwards, A. Batlle, P. Dobbin, A.J.
MacRobert, Macromolecular delivery of 5-aminolaevulinic acid for photodynamic
therapy using dendrimer conjugates, Mol. Cancer Ther. 6 (2007) 876–885.
 Y. Li, W.-D. Jang, N. Nishiyama, A. Kishimura, S. Kawauchi, Y. Morimoto, S. Miake,
T. Yamashita, M. Kikuchi, T. Aida, K. Kataoka, Dendrimer generation effects on
photodynamic efficacy of dendrimer porphyrins and dendrimer-loaded supra-
molecular nanocarriers, Chem. Mater. 19 (2007) 5557–5562.
 L.M. Rossi, P.R. Silva, L.L.R. Vono, A.U. Fernandes, D.B. Tada, M.c.S. Baptista,
Protoporphyrin IX nanoparticle carrier: preparation, optical properties, and
singlet oxygen generation, Langmuir 24 (2008) 12534–12538.
 A. Khdair, B. Gerard, H. Handa, G. Mao, M.P. Shekhar, J. Panyam, Surfactant-
polymer nanoparticles enhance the effectiveness of anticancer photodynamic
therapy, Mol. Pharm. 5 (2008) 795–807.
 C.J. Rijcken, J.W. Hofman, F. van Zeeland, W.E. Hennink, C.F. van Nostrum,
Photosensitiser-loaded biodegradable polymeric micelles: preparation, charac-
terisation and in vitro PDT efficacy, J. Control. Release 124 (2007) 144–153.
 A.M. Master, M.E. Rodriguez, M.E. Kenney, N.L. Oleinick, A.S. Gupta, Delivery of the
photosensitizer Pc 4 in PEG-PCL micelles for in vitro PDT studies, J. Pharm. Sci. 99
 B. Li, E.H. Moriyama, F. Li, M.T. Jarvi, C. Allen, B.C. Wilson, Diblock copolymer
micelles deliver hydrophobic protoporphyrin IX for photodynamic therapy,
Photochem. Photobiol. 83 (2007) 1505–1512.
 B.A. Lindig, M.A.J. Rodgers, A.P. Schaap, Determination of the lifetime of singlet
oxygen in D2O using 9, 10-anthracenedipropionic acid, a water-soluble probe,
J. Am. Chem. Soc. 102 (1980) 5590–5593.
 H. Ai, C. Flask, B. Weinberg, X. Shuai, M.D. Pagel, D. Farrell, J. Duerk, J.M. Gao,
Magnetite-loaded polymeric micelles as ultrasensitive magnetic-resonance
probes, Adv. Mater. 17 (2005) 1949–1952.
 C. Khemtong, C.W. Kessinger, J.M. Ren, E.A. Bey, S.G. Yang, J.S. Guthi, D.A.
Boothman, A.D. Sherry, J.M. Gao, In vivo off-resonance saturation magnetic
resonance imaging of alpha(v)beta(3)-targeted superparamagnetic nanoparti-
cles, Cancer Res. 69 (2009) 1651–1658.
 N. Nasongkla, E. Bey, J.M. Ren, H. Ai, C. Khemtong, J.S. Guthi, S.F. Chin, A.D. Sherry,
D.A. Boothman, J.M. Gao, Multifunctional polymeric micelles as cancer-targeted,
MRI-ultrasensitive drug delivery systems, Nano Lett. 6 (2006) 2427–2430.
 N. Nasongkla, X. Shuai, H. Ai, B.D. Weinberg, J. Pink, D.A. Boothman, J.M. Gao,
cRGD-functionalized polymer micelles for targeted doxorubicin delivery, Angew.
Chem. Int. Ed. 43 (2004) 6323–6327.
 X. Shuai, H. Ai, N. Nasongkla, S. Kim, J. Gao, Micellar carriers based on block
copolymers of poly(epsilon-caprolactone) and poly(ethylene glycol) for doxoru-
bicin delivery, J. Control. Release 98 (2004) 415–426.
 Y.F. Liu, W. Chen, S.P. Wang, A.G. Joly, Investigation of water-soluble x-ray
luminescence nanoparticles for photodynamic activation, Appl. Phys. Lett. 92
 C. Labarca, K. Paigen, A simple, rapid, and sensitive DNA assay procedure, Anal.
Biochem. 102 (1980) 344–352.
 M.A. Oar, J.A. Serin, W.R. Dichtel, J.M.J. Frechet, Photosensitization of singlet
oxygen via two-photon-excited fluorescence resonance energy transfer in a
water-soluble dendrimer, Chem. Mater. 17 (2005) 2267–2275.
 H.Y. Ding, X.S. Wang, L.Q. Song, J.R. Chen, J.H. Yu, Chao-Li, B.W. Zhang, Aryl-
modified ruthenium bis(terpyridine) complexes: quantum yield of1O2genera-
tion and photocleavage on DNA, J. Photochem. Photobiol. A 177 (2006) 286–294.
 D.W. Kim, S.Y. Kim, H.K. Kim, S.W. Kim, S.W. Shin, J.S. Kim, K. Park, M.Y. Lee, D.S.
Heo, Multicenter phase II trial of Genexol-PM, a novel Cremophor-free, polymeric
micelle formulation of paclitaxel, with cisplatin in patients with advanced non-
small-cell lung cancer, Ann. Oncol. 18 (2007) 2009–2014.
 W.S. Caughey, J.A. Ibers, Crystal and molecular structure of the free base
porphyrin, protoporphyrin IX dimethyl ester, J. Am. Chem. Soc. 99 (1977)
 L.M. Scolaro, M. Castriciano, A. Romeo, S. Patane, E. Cefali, M. Allegrini,
Aggregation behavior of protoporphyrin IX in aqueous solutions: clear evidence
of vesicle formation, J. Phys. Chem. B 106 (2002) 2453–2459.
 N.C. Maiti, S. Mazumdar, N. Periasamy, Dynamics of porphyrin molecules in
micelles—picosecond time-resolved fluorescence anisotropy studies, J. Phys.
Chem. 99 (1995) 10708–10715.
 M. Craw, R. Redmond, T.G. Truscott, Laser flash-photolysis of hematoporphyrins in
some homogeneous and heterogeneous environments, J. Chem. Soc. Faraday T 1
(80) (1984) 2293–2299.
 F. Ricchelli, Photophysical properties of porphyrins in biological membranes, J.
Photochem. Photobiol. B 29 (1995) 109–118.
 C. Allen, Y. Yu, A. Eisenberg, D. Maysinger, Cellular internalization of PCL20-b-
PEO44block copolymer micelles, Biochim. Biophys. Acta 1421 (1999) 32–38.
 S. Mukherjee, R.N. Ghosh, F.R. Maxfield, Endocytosis, Physiol. Rev. 77 (1997)
 H. Chen, S. Kim, W. He, H. Wang, P.S. Low, K. Park, J.X. Cheng, Fast release of
lipophilic agents from circulating PEG-PDLLA micelles revealed by in vivo forster
resonance energy transfer imaging, Langmuir 24 (2008) 5213–5217.
 R. Savic, T. Azzam, A. Eisenberg, D. Maysinger, Assessment of the integrity of poly
(caprolactone)-b-poly(ethylene oxide) micelles under biological conditions: a
fluorogenic-based approach, Langmuir 22 (2006) 3570–3578.
 S. Braverman, C. Helson, L. Helson, Hemin toxicity in a human epithelioid sarcoma
cell line, Anticancer Res. 15 (1995) 1963–1967.
 L. Goldstein, Z.P. Teng, E. Zeserson, M. Patel, R.F. Regan, Hemin induces an iron-
dependent, oxidative injury to human neuron-like cells, J. Neurosci. Res. 73
 S. Sandberg, Protoporphyrin-induced photodamage to mitochondria and lyso-
somes from rat liver, Clin. Chim. Acta 111 (1981) 55–60.
H. Ding et al. / Journal of Controlled Release 151 (2011) 271–277