Porphyrin-functionalized amphiphilic diblock copolypeptides for photodynamic therapy

Article abstract of DOI:10.1002/pola.24451

A series of amphiphilic diblock copolypeptides (ADCs), 5-(4-aminophenyl)- 10,15,20-triphenyl-porphyrin (APP) conjugated poly(L-leucine)-block-polylysine (APP-L_nK_m) with different molar ratios of L-leucine unit and lysine unit were designed and synthesized. The optimized composition of the polypeptide was determined to be APP-L₁₀₉K₁₈₆, which has high fluorescence quantum yield and could self-assemble into micelles in an aqueous medium with mean particle size <30 nm. The in vitro study indicates that APP-L₁₀₉K₁₈₆ shows no significant dark cytotoxicity when the concentration is below 200 mg L^-1 for HepG2 and HeLa cells. In contrast, the polymer exhibits apparent phototoxicity with low IC ₅₀ values toward HepG2 and HeLa cells, implying that the potential high photodynamic therapy efficacy of the polymer. Copyright

Full text of DOI:10.1002/pola.24451

Porphyrin-Functionalized Amphiphilic Diblock
Copolypeptides for Photodynamic Therapy
ZE-YONG LI, HUI-YUAN WANG, CAO LI, XIAO-LU ZHANG, XIAO-JUN WU, SI-YONG QIN, XIAN-ZHENG ZHANG, REN-XI ZHUO
Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry,
Wuhan University, Wuhan 430072, China
Received 10 August 2010; accepted 19 October 2010
DOI: 10.1002/pola.24451
Published online 18 November 2010 in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: A series of amphiphilic diblock copolypeptides
ADCs), 5-(4-aminophenyl)-10,15,20-triphenyl-porphyrin (APP)
conjugated poly(L-leucine)-block-polylysine (APP-L with
no significant dark cytotoxicity when the concentration is
ꢀ
1
(
below 200 mg L for HepG2 and HeLa cells. In contrast, the poly-
mer exhibits apparent phototoxicity with low IC50 values toward
HepG2 and HeLa cells, implying that the potential high photody-
namic therapy efficacy of the polymer. VC 2010 Wiley Periodicals,
n m
K )
different molar ratios of L-leucine unit and lysine unit were
designed and synthesized. The optimized composition of the
polypeptide was determined to be APP-L109
K
186, which has
Inc. J Polym Sci Part A: Polym Chem 49: 286–292, 2011
high fluorescence quantum yield and could self-assemble into
micelles in an aqueous medium with mean particle size
KEYWORDS: amphiphiles; N-carboxyanhydride; drug delivery
<
30 nm. The in vitro study indicates that APP-L109
K
186 shows
systems; micelle; polypeptide; phototoxicity; self-organization
INTRODUCTION Photodynamic therapy (PDT) is a promising
particles, hydrophilic polymer, or amphiphilic polymer–PS
conjugates have been developed.
4
treatment for many types of tumors, including lung, esopha-
1
geal, cervical, bladder, and gastric tumors. PDT is based on
Synthesis and polymerizations of a-amino acid N-carboxyan-
the tumor-specific accumulation of a photosensitizer (PS), of-
ten a porphyrin derivative, followed by irradiation with visi-
ble light, which induces the generation of highly reactive ox-
ygen species (ROS) and then leads to irreversible destruction
hydrides (NCAs) were reported for the first time by Leuchs
9
in 1906. From then on, these cyclic and highly reactive
amino acid derivatives have been used for peptide synthesis
but mainly for the formation of polypeptides by ring opening
2
of the treated tissues. For early or localized disease, PDT
1
0
polymerizations (ROP).
The synthetic polypeptides pro-
can be a selective and curative therapy without damaging
surrounding healthy tissues. For PDT, PS is a key factor that
can absorb light with appropriate wavelength at the site of
the photodynamic reaction to produce ROS to mediate cellu-
lar toxicity. As the second generation of PS, porphyrin deriva-
duced from the NCAs are able to form highly ordered struc-
ture such as a-helix and b-sheet. The different self-assembly
morphologies could be observed by varying the polypeptide
compositions, sequences, and segments length. These poly-
peptides have good biocompatibility and biodegradability
because of the chemical and structural similarity to natural
biomolecules such as proteins.
3
tives are extensively studied because they are the effective
generators of singlet oxygen with favorable properties such
as selectivity for particular diseased tissues and relatively
4
fast elimination from the body, which minimizes side effects.
In aqueous medium, amphiphilic block copolymers possess
the ability to spontaneously self-assemble into nanosized
micelles with a typical core-shell structure with attractive
However, most of porphyrin derivatives are hydrophobic.
This lipophilic nature may be an important obstacle for the
preferential accumulation in cellular hydrophobic loci as
these molecules must be able to get into cells by crossing
properties as promising colloidal carriers for poorly water-
11,12
soluble and amphiphilic drugs as well as genes.
In par-
5
lipid membranes, and thus greatly limits the in vivo applica-
ticular, amphiphilic diblock copolypeptide (ADC) or protein
hybrid copolymers recently received much attention for the
fabrication of stimuli-responsive micelles and vesicles for
various biomedical applications, such as drug delivery and
tions. In addition, because of their chemical structure, most
PSs tend to aggregate in aqueous media as a result of the
propensity of the hydrophobic skeleton to avoid contact with
6
water molecules, which affect their efficacy in vivo due to
1
3–15
7
,8
gene therapy.
the decreased bioavailability and capacity to absorb light.
To overcome this problem, the delivery systems for porphy-
rin derivatives such as oil-dispersions, liposomes, polymeric
To effectively deliver the PS, the carrier must also be able to
incorporate the PS without loss or alteration of its activity.
Correspondence to: X.-Z. Zhang (E-mail: xz-zhang@whu.edu.cn)
Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 49, 286–292 (2011) V
2010 Wiley Periodicals, Inc.
C
286
WILEYONLINELIBRARY.COM/JOURNAL/JPOLA

ARTICLE
Given the high probability of repetitive dosing schedules, the
system must also be biodegradable and have little or no im-
for 3 days and distilled water for 4 days, and finally freeze-
dried.
1
6
munogenicity. Amphiphilic micelles based on polypeptide-
PS conjugates could satisfied these requirements. Herein, we
report a series of porphyrin-functionalized ADCs for PDT,
which have high fluorescence quantum yields. The polypep-
tides synthesized by ROP of NCAs have good biocompatibil-
ity and conspicuous PDT efficacy.
Synthesis of Poly(L-leucine)-block-polylysine (APP-L K )
n
m
The diblock copolymers were prepared by ROP of Z-Lys NCA
initiated by the macroinitiator APP-L in DMF with feed
n
ratios of 1/1 and 1/2 (Leu-NCA/Z-Lys-NCA molar ratio),
respectively. The reaction mixture was stirred under a N2
ꢁ
atmosphere for 4 days at 50 C. The products were purified
by dialysis against DMF for 3 days and distilled water for
EXPERIMENTAL
4
days. After freeze-drying, APP-Leucine109-b-Lysine(Z)107
Materials
(APP-L109K(Z)107) and APP-Leucine109-b-Lysine(Z)186 (APP-
N,N-Dimethylformamide (DMF; Shanghai Chemical Reagent)
was used after distillation under reduced pressure. Tetrahy-
drofuran (THF; Shanghai Chemical Reagent) was treated
with Na and then purified by vacuum distillation before use.
N(e)-Benzyloxycarbonyl-L-lysine (H-Lys(Z)-OH; 99%; Aldrich),
L-leucine (99%; Aldrich), n-hexane (Shanghai Chemical Rea-
gent), trifluoroacetic acid (TFA; 99%; Aldrich), hydrogen bro-
mide (33 wt % in glacial acetic acid; Sigma), and diethyl
ether (Shanghai Chemical Reagent) were used as received.
L
1
109K(Z)186) were obtained from the feed ratios of 1/1 and
/2 (Leu-NCA/Z-Lys-NCA molar ratio), respectively.
Subsequently, a 100-mL round-bottom flask was charged
with APP-L109K(Z)107 (155 mg) and TFA (8 mL) and was
placed in an ice bath. The mixture was stirred for 15 min.
HBr (3 mL of a 33 wt % solution in acetic acid) was then
added dropwise and the system was allowed to stir for 1 h.
Then diethyl ether (20 mL) was added to precipitate the
product. The mixture was centrifuged to isolate the green
solid precipitate. After drying, product was resuspended in
10 mL DMF, and the solution was placed in a dialysis bag
Measurements
1
The H NMR spectra of products were recorded on a Mer-
ꢀ
1
cury VX-300 spectrometer at 300 MHz (Varian) by using
TFA-d and dimethyl sulfoxide-d (DMSO-d ) as a solvent and
6 6
tetramethylsilane (TMS) as an internal standard.
(Molecular Weight Cut Off (MWCO) ¼ 3500 g mol ) and
dialyzed against distilled water for 4 days (water changed
every 8 h). After dialysis, the sample was lyophilized to give
the product (APP-L109
APP-L109K186 was obtained after the deprotection of 140 mg
APP-L109K(Z)186
K107) (60.7 mg). Similarly, 57.0 mg of
Fourier Transform Infrared (FT-IR) spectra were recorded on
an AVATAR 360 spectrometer. Samples were pressed into po-
tassium bromide (KBr) pellets.
.
Determination of Critical Micelle Concentration
Synthesis of 5-(4-Aminophenyl)-10,15,20-triphenyl-
porphyrin
Fluorescence spectra were recorded on a LS55 luminescence
spectrometer (PerkinElmer). Pyrene was used as a hydro-
5
-(4-Aminophenyl)-10,15,20-triphenyl-porphyrin (APP) was
2
1
1
7–19
phobic fluorescent probe. Aliquots of pyrene solutions (1.2
synthesized according to the literature procedure.
ꢀ
7
ꢀ1
ꢂ 10 mol mL in acetone, 50 lL) were added to contain-
ers, and the acetone was allowed to evaporate. One milliliter
aqueous solution of polymer with a particular concentration
was added to the container, which contained the pyrene resi-
due. The aqueous sample solutions containing pyrene resi-
Synthesis of Leu-NCA and Z-Lys-NCA
L-Leucine-N-carboxyanhydride (Leu-NCA) and N(e)-Benzylox-
ycarbonyl-L-lysine-N-carboxyanhydride (Z-Lys-NCA) were
2
0
synthesized according to a literature procedure. L-Leucine
ꢀ
6
(
(
2.62 g, 0.02 mol) was suspended in freshly distilled THF
90 mL) in a 250-mL three-necked flask. The system was
due at the same concentration of 6 ꢂ 10 M were kept at
room temperature for 24 h to reach the solubilization equi-
librium of pyrene in the aqueous phase. Emission wave-
length was carried out at 393 nm, and excitation spectra
were recorded ranging from 300 to 360 nm. Both excitation
and emission slit widths were 10 nm. From the pyrene exci-
tation spectra, the intensity ratio I338/I335 was analyzed as a
function of logarithm of the polymer concentration. A critical
micelle concentration (CMC) value was determined from
the intersection of the tangent to the curve at the inflection
with the horizontal tangent through the points at low
magnetically stirred under a steady flow of N , then triphos-
2
gene (5.94 g, 0.02 mol) in a THF solution (15 mL) was
added dropwise to the suspension. Subsequently, the reac-
ꢁ
tion mixture was heated to 50 C and stirred for 90 min to
yield a clear solution. The solution was condensed under
reduced pressure and then was poured into excess dried n-
hexane to obtain crude crystals of Leu-NCA, which were fur-
ther recrystallized from dried THF/n-hexane twice and dried
under vacuum. Z-Lys-NCA was synthesized according to a
similar procedure.
2
2
concentration.
Synthesis of APP-Ln
Micelle Formation
APP-poly(L-leucine) (APP-L ) was prepared by ROP of Leu-
NCA initiated by APP in DMF with a feed ratio of 1/80
The micelles of APP-L109K186 were prepared by a dialysis
method. Briefly, the polymer was dissolved in DMSO at an
n
ꢀ
1
(
APP/Leu-NCA molar ratio). The resultant solution of Leu-
initial concentration of 200 mg L
and then dialyzed
NCA and APP was stirred under a N atmosphere for 4 days
at 50 C. The product was purified by dialysis against DMF
against distilled water for 2 days using a dialysis bag
(MWCO ¼ 3500 g mol ).
2
ꢁ
ꢀ1
PORPHYRIN-FUNCTIONALIZED ADCs FOR PDT, LI ET AL.
287

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
SCHEME 1 Core-shell micelle
self-assembled from APP-L109
K
186
with phototoxicity toward cancer
cells.
Transmission Electron Microscopy
Dark Cytotoxicity of APP-L Km
n
Transmission electron microscopy (TEM) experiments were car-
ried out on a JEM-100CX II instrument operating at an accelera-
tion voltage of 80 KV. A drop of micelle suspension was placed
on a copper grid with formvar film and stained by a 0.2% (w/
v) solution of phosphotungstic acid before measurement.
In vitro cytotoxicity was evaluated using human hepatocellu-
lar liver carcinoma (HepG2) and human cervical carcinoma
(HeLa) cells. For dark cytotoxicity assay, 200 lL of cells in
Dulbecco’s modified eagle medium (DMEM) with a concen-
4
tration of 2.0 ꢂ 10 cells/mL was added to each well (4.0 ꢂ
3
1
0 cells/well) in a 96-well plate. After incubation for 24 h
Size Distribution Measurements
ꢁ
in incubator (37 C, 5% CO ), the culture medium in each
2
Zeta sizer Nano ZS (Malvern Instruments) was used to deter-
mine the size and size distribution of self-assembled micelles.
well was replaced by 200 lL of DMEM or 4-(2-hydroxy-
ethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, con-
taining the polymer with a particular concentration, and the
cells were further incubated for 48 h. Then, the medium was
replaced by fresh DMEM or HEPES and 20 lL of 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
ꢀ
1
The micelle-contained solution (ꢃ100 mg L ) was passed
through a 0.45-lm pore size filter before measurement.
Fluorescence Quantum Yields
Fluorescence emission spectra were recorded on a LS55 lu-
minescence spectrometer (PerkinElmer) spectrofluorimeter.
The excitation and emission slit widths were both 10 nm. All
absorption spectra were acquired using a Lambda Bio40 UV-
vis spectrometer (PerkinElmer). All the measurements were
performed within 3 h after preparation of the solutions. Stock
ꢀ
1
solution (5 mg mL ). After incubation for 4 h, 200 lL of
DMSO was added and shaken at room temperature. The opti-
cal density (OD) was measured at 570 nm with a Microplate
Reader Model550 (BIO-RAD). The viable rate was calculated
by the following equation: cell viability ¼ ODtreated/ODcontrol,
ꢀ
1
where OD
was obtained in the absence of polymer and
control
solutions of tetraphenylporphyrin (H TPP, 0.0001 mg mL in
2
ꢀ
1
ODtreated was obtained in the presence of polymer.
DMSO), APP-L109K107 (0.023 mg mL in phosphate buffered
ꢀ
1
saline (PBS) pH 7.4), and APP-L109K186 (0.047 mg mL in
PBS pH 7.4) were prepared. Each solution contains the same
concentration of porphyrin. The quantum yields were calcu-
Phototoxicity of APP-L Km
n
For phototoxicity assay, the HepG2 cells and HeLa cells were
cultured as described above for the dark cytotoxicity assay.
After the polymer-contained culture medium was added, the
plate with cells was then placed on ice and exposed to light
from a 150-W xenon lamp filtered through a 400–700-nm
long-pass filter for 20 min. The cells were returned to the in-
cubator overnight and assayed for viability. Other conditions
were the same as that for dark cytotoxicity assay.
2
3
lated using a secondary standard method. H TPP was used
2
as a secondary standard. According to this approach, the inte-
grated fluorescence intensity of the analyte (I) and standard
(
I
s
), the absorbencies of the analyte (A) and the standard (A
at excitation wavelength, the excitation wavelengths of analyte
k) and the standard (k ), and the refractive indexes of analyte
s
)
(
s
solution (n) and standard solution (n ) are related to the
s
quantum yield of the analyte (/) as follows:
RESULTS AND DISCUSSION
I As ks n2
Synthesis and Characterizations of APP-L K
n m
/
¼ /s
In this study, core-shell micelles self-assembled from poly-
mer APP-L109K186 with phototoxicity toward cancer cells
were prepared (Scheme 1). The polymer synthetic proce-
dures consist of four steps (Scheme 2): the synthesis of
Is A k n2
s
where, / is the quantum yield of the reference standard
s
2
4
(
0.11 for H TPP).
2
288
WILEYONLINELIBRARY.COM/JOURNAL/JPOLA

ARTICLE
SCHEME 2 Synthesis of Leu-
NCA, Z-Lys-NCA, and APP-L
n m
K .
1
Leu-NCA and Z-Lys-NCA, the synthesis of APP-L , the synthe-
was characterized by H NMR (in TFA-d) shown in Figure 2.
The characteristic signals at 0.94 (a), 1.63 (b), 1.73 (c), and
4.71 ppm (d) are assigned to the protons of leucine units.
The characteristic signals at 7–9 ppm (e) are assigned to the
porphyrin protons. The integration ratio of the two types of
signals indicates that the degree of polymerization is 109 (n
n
sis of APP-L K(Z) , and the deprotection of benzyloxycar-
n
m
n m
bonyl (Z) from APP-L K(Z) . The first step was to prepare
two monomers. Their chemical structures were confirmed by
1
H NMR (in Fig. 1). The second step was to prepare amino-
terminated APP-L , which was synthesized by ROP method
n
with APP as an initiator. The chemical structure of APP-Ln
¼ 109, M ¼ 13000). The third step was to polymerize the
n
1
1
FIGURE 1 H NMR spectra of (1) Leu-NCA and (2) Z-Lys-NCA.
FIGURE 2 H NMR spectrum of APP-L
n
.
PORPHYRIN-FUNCTIONALIZED ADCs FOR PDT, LI ET AL.
289

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
1
FIGURE 3 H NMR spectra of (1) APP-L109K(Z)107, (2) APP-L109K(Z)186, (3) APP-L109
K
107, and (4) APP-L109
K
186
.
second monomer (Z-Lys-NCA) with APP-L as a macroinitia-
was verified by the fluorescence technique using pyrene as a
probe. Concomitant with the increase in fluorescence inten-
sity, a red-shift from 335 to 338 nm takes place ascribed to
the micellization of the ADCs, because pyrene is preferen-
tially partitioned into the hydrophobic core of the micelles
with a change of the photophysical properties. From the plot
of fluorescence intensity ratio I338/I335 versus the logarithm
of polymer concentration in Figure 5, the CMC value is deter-
n
1
tor. The structure of APP-L K(Z)m was characterized by
H
n
NMR (in TFA-d). As shown in Figure 3(1) and (2), the
appearance of Z group signals (5.10 and 7.20 ppm) implies
that the two types of diblock copolypeptides with different
polylysine chain lengths are successfully synthesized. The
composition of the diblock copolypeptides are listed in Table
1. Finally, two types of ADCs were obtained after the depro-
ꢀ
1
tection of Z groups from APP-L K(Z) . As shown in Figure
mined to be 30.6 mg L
.
n
m
3
(3) and (4), the disappearance of Z group signals reveals
This micellar structure was further confirmed by TEM. As
shown in Figure 6, it is evident that the self-assembled
that the two types of polypeptides are successfully obtained.
The same conclusion is drawn from the FT-IR spectra. As
shown in Figure 4, the peaks at 1650 and 1550 cm are
attributed to the amide I band and amide II band in the
polypeptide chain, respectively.
ꢀ
1
Micelle Formation and Characterizations
The ADC APP-L109K186 could self-assemble into micelles in
an aqueous medium. The formation of APP-L109K186 micelles
TABLE 1 Feed Composition and Composition of APP-L
n m
K
a
b
APP/Leu /Lys
c
Molar Ratio in
Diblock
Diblock
APP/Leu-NCA/Z-Lys-NCA
Feed Molar Ratio
Copolypeptide
Copolypeptide
APP-L109
APP-L109
a
K
107
186
1/80/109
1/80/218
1/109/107
1/109/186
K
Leucine unit.
Lysine unit.
b
c
FIGURE 4 FT-IR spectra of (1) APP-L109K107 and (2) APP-
1
Calculated from H NMR spectra.
109 186
L K .
290
WILEYONLINELIBRARY.COM/JOURNAL/JPOLA

ARTICLE
TABLE 2 Fluorescence Quantum Yields of APP-L109
K107 and
APP-L109 186 in PBS (pH 5 7.4, Excitation at 420 nm, and
K
Emission at 650 nm)
Diblock Copolypeptide
/
APP-L109
K
K
107
186
0.24
0.25
APP-L109
polylysine, the aggregation pattern of APP-L109K107 becomes
elusive (data not shown). Therefore, we focused our study
on the APP-L109K186 micelles. From the dynamic light scat-
tering (DLS) shown in Figure 6, the mean size of APP-
L109K186 micelles is 120 nm. The difference in the sizes
determined by different methods is mainly attributed to the
fact that the size measured by the DLS is the hydrodynamic
diameter of micelles in aqueous solution, while the size
observed by TEM is the diameter of the freeze-dried
FIGURE 5 The intensity ratio I338/I335 in the excitation spectrum
as a function of logarithm of APP-L109 186 concentration. The
K
ꢀ
1
CMC is 30.6 mg L
.
2
5,26
micelles formed have a regular spherical shape and are well-
dispersed. From the TEM image, the sizes of micelles are
micelles. Similar conclusion was drawn in literature.
<
30 nm. Because of the shorter hydrophilic segments of
Fluorescence Quantum Yields
We know that conventional PSs tend to aggregate in aqueous
media as a result of their p–p interactions and lipophilic
FIGURE 6 TEM image of APP-L109
K
186 (A) micelles self-
FIGURE 7 Dark toxicity (square labelled line) and phototoxicity
(circle labelled line) of polymers for (A) HepG2 cells and (B)
HeLa cells.
assembled in aqueous medium and the sizes of micelles deter-
mined by DLS (B).
PORPHYRIN-FUNCTIONALIZED ADCs FOR PDT, LI ET AL.
291

JOURNAL OF POLYMER SCIENCE: PART A: POLYMER CHEMISTRY DOI 10.1002/POLA
nature, which affects their efficacy in vivo because of the
decreased bioavailability and capacity to absorb light, that is,
low tendency to aggregation in aqueous media is an impor-
tant requirement for the photodynamic agents. Herein, we
investigated the fluorescence quantum yields of APP-
L109K107 and APP-L109K186. As shown in Table 2, the porphy-
rin-functionalized diblock copolypeptides show high fluores-
cence quantum yields. In other words, there is no activity
loss or alteration occurred after the porphyrins being incor-
porated to the diblock copolypeptides.
3 Kreimer-Birnbaum, M. Semin Hematol 1989, 26, 157–173.
4
Konan, Y. N.; Gurny, R.; Allemann, E. J Photoch Photobio B
002, 66, 89–106.
2
5
Oenbrink, G.; Jurgenlimke, P.; Gabel, D. Photochem Photo-
biol 1988, 48, 451–456.
6
Rosenthal, I. Photochem Photobiol 1991, 53, 859–870.
7 Brasseur, N.; Ali, H.; Langlois, R.; Van Lier, J. E. Photochem
Photobiol 1988, 47, 705–711.
8
Isele, U.; Schieweck, K.; Van Hoogevest, P.; Capraro, H. G.;
Kessler, R. J Pharm Sci 1995, 84, 166–173.
In Vitro Cytotoxicity
As shown in Figure 7, no significant decrease in cell viability
occurs when the concentration of the APP-L109K186 is below
9 Leuchs, H. Ber Dtsch Chem Ges 1906, 39, 857–861.
1
0 Kricheldorf, H. R. Angew Chem Int Edit 2006, 45, 5752–5784.
ꢀ
1
200 mg L for HepG2 [Fig. 7(A)] and HeLa [Fig. 7(B)] cells.
1
1 Allen, C.; Maysinger, D.; Eisenberg, A. Colloid Surface B
Obviously, APP-L109K186 does not show apparent dark cyto-
toxicity. This is attributed to the self-assembly of the polyca-
1
999, 16, 3–27.
1
5
tionic segments, which greatly diminishes its cytotoxicity.
12 Gref, R.; Minamitake, Y.; Peracchia, M. T.; Trubetskoy, V.;
The phenomenon most likely is resulted from chain assem-
bly preventing free diffusion of the polycations to cell
surfaces.
Torchilin, V.; Langer, R. Science 1994, 263, 1600–1603.
1
1
1
3 Bae, Y.; Kataoka, K. Adv Drug Deliver Rev 2009, 61, 768–784.
4 Osada, K.; Kataoka, K. Adv Polym Sci 2006, 202, 113–153.
5 Holowka, E. P.; Sun, V. Z.; Kamei, D. T.; Deming, T. J. Nat
2
7
In contrast, the ADC exhibits visible phototoxicity toward
HepG2 cells and HeLa cells. APP-L109K186 shows slightly
higher phototoxicity in HepG2 cells than in HeLa cells, The
Mater 2007, 6, 52–57.
ꢀ
1
16 Poste, G.; Kirsh, R. Nat Biotechnol 1983, 1, 869–878.
IC50 values of APP-L109K186 are 60 and 100 mg L
HepG2 cells and HeLa cells, respectively.
for
17 Adler, A. D.; Longo, F. R.; Shergalis, W. J Am Chem Soc
1
964, 86, 3145–3149.
CONCLUSIONS
1
8 Kruper, W. J.; Chamberlin, T. A.; Kochanny, M. J Org Chem
In summary, a series of porphyrin-functionalized ADCs were
synthesized by ROP of Leu-NCA and Z-Lys-NCA, followed by
the deprotection of Z groups. DLS measurements and TEM
observation confirmed the micellar structure formed by the
self-assembly of the polymer synthesized. APP-L109K186
micelles exhibited no significant dark cytotoxicity and high
PDT efficacy against HepG2 and HeLa cells. These results
1989, 54, 2753–2756.
9 Wu, D. Q.; Li, Z. Y.; Li, C.; Fan, J. J.; Lu, B.; Chang, C.;
Cheng, S. X.; Zhang, X. Z.; Zhuo, R. X. Pharm Res 2010, 27,
1
1
87–199.
2
0 Daly, W. H.; Poche, D. Tetrahedron Lett 1988, 29, 5859–5862.
21 Hu, X. L.; Liu, S.; Chen, X. S.; Mo, G. J.; Xie, Z. G.; Jing, X.
B. Biomacromolecules 2008, 9, 553–560.
indicated APP-L109
cancer therapy.
K186 micelles could be a novel carrier for
2
2 Liu, Y. H.; Cao, X. H.; Luo, M. B.; Le, Z. G.; Xu, W. Y. J Col-
loid Interface Sci 2009, 329, 244–252.
This work was supported by the Ministry of Science and
Technology of China (2009CB930300), Trans (New)-Century
Training Programme Foundation for the Talents from the
Ministry of Education of China, and Natural Science Founda-
tion of Hubei Province, China (2009CDA024).
23 Fery-Forgues, S.; Lavabre, D.
J Chem Educ 1999, 76,
1260–1264.
2
4 Seybold, P. G.; Gouterman, M. J Mol Spectrosc 1969, 31,
1
–13.
2
5 Morita, T.; Horikiri, Y.; Suzuki, T.; Yoshino, H. Int J Pharm
2
001, 219, 127–137.
REFERENCES AND NOTES
2
6 Giacomelli, C.; Schmidt, V.; Borsali, R. Macromolecules
1
Levy, J. G.; Obochi, M. Photochem Photobiol 1996, 64, 737–
2007, 40, 2148–2157.
7 Pakstis, L. M.; Ozbas, B.; Hales, K. D.; Nowak, A. P.; Deming,
T. J.; Pochan, D. Biomacromolecules 2004, 5, 312–318.
739.
2
2
Dougherty, T. J. Photochem Photobiol 1987, 45, 879–889.
292
WILEYONLINELIBRARY.COM/JOURNAL/JPOLA