Synthesis and cellular studies of PPIX-homing peptide conjugates

Article abstract of DOI:10.1142/S1088424612500599

Five amphiphilic protoporphyrin IX-peptide conjugates bearing the sequences ATWLPPR, AAhexPQRRSARLSA and cERGDPhe conjugated via the propionic side chains, were synthesized and evaluated in vitro using two cell lines: human carcinoma HEp2 and human leukem

Full text of DOI:10.1142/S1088424612500599

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2012; 16: 603–615
DOI: 10.1142/S1088424612500599
Published at http://www.worldscinet.com/jpp/
Synthesis and cellular studies of PPIX-homing peptide
conjugates
Martha Sibrian-Vazquez, Xiaoke Hu,Timothy J. Jensen and M. Graça H. Vicente*◊
Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, USA
Dedicated to Professor Emanuel Vogel in memoriam
Received 9 December 2011
Accepted 10 February 2012
ABSTRACT: Five amphiphilic protoporphyrin IX-peptide conjugates bearing the sequences
ATWLPPR, AAhexPQRRSARLSA and cERGDPhe conjugated via the propionic side chains, were
synthesized and evaluated in vitro using two cell lines: human carcinoma HEp2 and human leukemia
HL-60. All conjugates were found to have low dark- and photo-toxicities in both cell lines, and 3 to
10-fold higher accumulation was observed within HL-60 vs. HEp2 cells, depending on the nature of the
peptide sequence. The preferential subcellular sites of localization for all conjugates were found to be
the lysosomes in HEp2 cells, and the mitochondria in HL-60 cells, suggesting different mechanisms of
cellular internalization.
KEYWORDS: porphyrin, peptide, PDT, toxicity, cell uptake.
Targeted therapies are useful for the specific delivery
INTRODUCTION
of drugs into tumor cells, and have been shown to
Photodynamic therapy (PDT) is a form of photo-
improve the biodistribution and biological efficacy of
chemotherapy (PCT) that involves light activation of a
photosensitizers [3]. The general strategy involves the
tumor-localized photosensitizer, which produces reactive
non-covalent or covalent attachment of drugs to targeting
oxygen species, such as 1O2, that lead to selective photo-
moieties or vectors that specifically bind tumor antigens
oxidativedestructionoftumorcells[1]. Twoprotoporphyrin
or over-expressed enzymes or receptors on cancer
and VisudyneTM

IX (PPIX) derivatives, Photofrin
cells. Examples of targeting moieties are monoclonal
antibodies, proteins, peptides, oligonucleotides, and
glycosides [4]. In particular, the use of homing peptides
to target integrins, a family of cell surface proteins
responsible for cell–cell and cell–matrix adhesion with
a major role in the survival of endothelial cells, has been
used for the specific delivery of cytotoxic drugs [5].
Moreover, studies have shown that the tumor vasculature
can be targeted by homing peptides containing an RGD
(Arg–Gly–Asp) sequence, which bind to b and v3ab
integrins on angiogenic endothelial cells [6]. Both linear
and cyclic peptides containing the RGD sequence have
been designed or isolated from phage display experiments
as ligands for the v3 integrin. Cyclic peptides containing
the RGD sequence have revealed higher specificity for
integrins and increased stability towards enzymatic
hydrolysis compared with the linear counterparts [7].
In vitro studies have shown that photosensitizer-homing
(Fig. 1), are FDA-approved for the PDT treatment of
various cancers, including lung, melanoma, bladder and
Barrett’s esophagus, and in the latter case, for the “wet-
form” of age-related macular degeneration. Since both
drugs are mixtures of compounds and not tumor-targeted,
a considerable amount of research in the last decade
has centered on the investigation of targeted sensitizers
of defined structure, and with improved selectivity for
tumor tissues. On the other hand, the aminolevulinic acid
(5-ALA)-induced intracellular accumulation of PPIX,
as a consequence of ferrochelatase’s limited activity, is
also used for the PDT treatment of keratoses, basal cell
carcinoma and malignant gliomas [2].
SPP full member in good standing
*Correspondence to: M. Graça H. Vicente, email: vicente@lsu.
edu, tel: +1 225-578-7442
Copyright © 2012 World Scientific Publishing Company

604
M. SIBRIAN-VAZQUEZ ET AL.
higher-order
chromatin
structure
O
Me
Me
Me
MeO2C
MeO2C
and to facilitate the transcriptional
activation of mammalian genes [11].
The 31 amino acid synthetic peptide
(KDEPQRRSARLSAKPAPPKPEPKPK-
KAPAKK) from the HMGN2 fragment
containing the PQRRSARLSA sequence
was shown to be readily internalized
by human leukemia HL-60 and breast
cancer MDA-MB-435 cells and to
localize in the nuclei, both in vitro and
in vivo, following intravenous injection
into tumor-bearing nude mice [11].
O
Me
Me
N
N
NH
N
Me
NH
N
Me
HN
HN
Me
Me
Me
CO2H
CO2R2
CO2H
n = 1-9
CO2R1
n
R1 = H; R2 = Me
R1 = Me; R2 = H
Fig. 1. Structures of Photofrin (left) and VisudyneTM (right)

peptide conjugates are selectively delivered within cell
lines over-expressing avb3 integrins, heterodimeric
glycoproteins, and they retain their PDT efficacy [8].
Several PPIX conjugates to homing peptides and
other targeting molecules including folic acid [9] have
been reported to date [4]. A PPIX-cyclic RGDfk peptide
conjugate was found to have higher tumor accumulation
and retention than free PPIX, both in vitro using integrin
positive SiHa cells, and in vivo in a mouse CaNT
tumor model [10]. Herein we report the synthesis and
characterization of a series of conjugates of PPIX to
homing peptides and a vascular endothelial growth
factor (VGEF). Homing peptide sequences include
the cyclic ERGDPhe sequence, the VEGF sequence
ATWLPPR and the PQRRSARLSA sequence. The
last sequence is found in the HMGN2 protein, a highly
conserved nucleosomal protein involved in unfolding
RESULTS AND DISCUSSION
Synthesis
The PPIX-peptide conjugates 4–9 were synthesized as
shown in Schemes 1 and 2, from commercially available
PPIX (1). The targeting peptide sequences conjugated to
thepropionicacidchainsofPPIXareATWLPPR,AAhex-
PQRRSARLSA and the cyclic peptide cERGDPhe.
Our previous studies on meso-tetraphenylporphyrin
(TPP)-peptide conjugates suggest that the incorporation
of a low molecular weight polyethylene glycol (PEG)
linker reduces intramolecular interactions between the
porphyrin and the peptide moiety, favoring extended
conformations for the conjugates [12, 13]. In addition,
one or two PEG groups generally increase the tumor cell
uptake and water-solubility of porphyrin macrocycles
Me
Me
Me
Me
1. EDCI/DMAP/ p-benzyloxybenzylalcohol
Wang resin 0.87 mmol/g in DMF
N
N
NH
N
NH
N
HN
HN
2. EDCI/DMAP/MeOH
3. CH2Cl2:TFA 1:1
Me
Me
Me
Me
CO2H
CO2H
CO2H
1: PPIX
CO2Me
2
Me
t
1. NH2(CH2)2(OCH2CH2)5OCH2CO2 Bu
HBTU/Et3N/ CH2Cl2
2. CH2Cl2:TFA 1:1 (95%)
3. peptidyl resin/PyBOP/DIEA/DMF
4. phenol/TFA/TIS/H2O (58-62%)
Me
N
NH
N
HN
Me
Me
O
R
O
CO2Me
O
N
H
O
5
3: R = OH
4: R = ATWLPPR
5: R = AAhexPQRRSARLSA
Scheme 1. Synthesis of PPIX-peptide conjugates 4 and 5
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SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
605
Me
Me
1. NH2(CH2)2(OCH2CH2)6(CH2)2NH-Boc
PyBOP/Et3N/ CH2Cl2
2. CH2Cl2:TFA 1:1 (73%)
Me
Me
Me
N
N
NH
N
NH
N
HN
HN
3. protected cERGDPhe/PyBOP/DIEA/DMF
4. phenol/TFA/TIS/H2O (27%)
Me
Me
Me
H
H
N
CO2H
O
N
CO2H
1: PPIX
O
O
N
H
O
R
R
O
N
H
O
6
6
6: R = H
9: R = cERGDPhe
1. peptidyl resin/PyBOP/DIEA/DMF
2. phenol/TFA/TIS/H2O (17-55%)
Me
O
Me
O
O
N
NH
N
CO2H
HN
NH
NH
HN
HN
O
Me
Me
H
H
N
N
O
HN
NH2
O
CO2R2
CO2R1
cERGDPhe
7: R1 = OH, R2 = AAhexPQRRSARLSA
8: R1 = R2 = AAhexPQRRSARLSA
Scheme 2. Synthesis of PPIX-peptide conjugates 7, 8 and 9
[14]. In this series of PPIX conjugates, the peptides were
either linked directly to the propionic acid side chains
of PPIX (in 7 and 8) or via a short PEG linker (in 4, 5
and 9). The mono and di-peptide PPIX conjugates were
synthesized in solution- or solid-phase using standard
conjugation methodologies [15].
respectively, after deprotection and cleavage from
the solid support, followed by reversed-phase HPLC
purification. The PPIX-di-peptide conjugate 9 containing
the cERGDPhe cyclic peptide sequence, was synthesized
from di-pegylated PPIX 6 containing terminal amino
groups. Porphyrin 6 was prepared in 73% overall yield,
by conjugation of both the propionic acid chains of PPIX
with commercially available monoamino-protected
As shown in Scheme 1, the PPIX-mono-peptide
conjugates 4 and 5 were synthesized by selective
esterification of PPIX in solid phase support [16] following
by conjugation of the remaining propionic acid group, after
cleavage from the solid support under acidic conditions.
The free carboxylic group of PPIX mono-methyl ester 2 (as
regioisomeric mixture), was conjugated to a low molecular
weight PEG linker via amide bond formation using HBTU
and Et3N [13]. Acid hydrolysis of the terminal tert-butyl
ester using a 1:1 mixture of CH2Cl2/TFA provided the free
carboxylic acid 3 in 95% yield. Porphyrin 3 was activated
as the benzotriazole ester with PyBOP and conjugated on
solid support to the corresponding peptide sequence via
amide bond formation. After deprotection and cleavage
from the solid support, PPIX-mono-peptide conjugates 4
and 5 were purified by reversed HPLC and isolated in 58
and 62% yields, respectively.
NH2CH2CH2(OCH2CH2)6CH2CH2NH-Boc,
following
by cleavage of the Boc group using CH2Cl2/TFA 1:1.
Reaction of the activated d-benzotriazole ester of the
glutamic acid residue on the protected ERGDPhe cyclic
peptide sequence, in solution phase, with porphyrin 6,
followed by deprotection of the arginine and aspartic acid
residues using a 5:88:2:5 mixture of phenol/TFA/TIS/
H2O afforded the corresponding PPIX-di-cERGDPhe
conjugate 9 in 27% yield.
All PPIX conjugates were characterized by NMR, MS
and UV-vis spectrophotometry (see Supporting information).
Cell studies
Timedependentcellularuptake. Thecellularuptakeof
PPIX-peptide conjugates 4, 5, 7, 8 and 9 was investigated
in a time dependent manner at a concentration of 10 mM in
both human squamous cell carcinoma HEp2 and human
myeloid leukemia HL-60 cells (Fig. 2). The conjugation
The conjugation of activated PPIX benzotriazole
ester to the peptide sequence AAhexPQRRSARLSA on
solid support (Scheme 2) gave a mixture of the mono-
and di-peptide conjugates 7 and 8 in 17 and 55% yields,
Copyright © 2012 World Scientific Publishing Company
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606
M. SIBRIAN-VAZQUEZ ET AL.
Fig. 2. Time-dependent uptake of PPIX-peptide conjugates 4 (blue), 5 (purple), 7 (black), 8 (red), and 9 (green) at 10 mM by
(a) HEp2 cells and (b) HL-60 cells
of the peptide sequences via the propionic acid chains of
PPIX produced amphiphilic conjugates that are believed
to show enhanced affinity for lipid/aqueous interfaces
and increased cellular uptake compared with hydrophilic
molecules, which generally show decreased interactions
with the lipid bilayer cell membrane [15, 17].
peptide, accumulated the most of all conjugates in both
cell lines, while its corresponding di-peptide derivative,
conjugate 8, accumulated to a lesser extent, maybe due to its
higher hydrophilicity. All conjugates showed similar uptake
kinetics in both cell lines, accumulating more rapidly in the
first hour after exposure to cells, and reaching a plateau after
2 h in HEp2 cells, and 4–8 h in HL-60 cells.
In human HEp2 cells higher uptake was observed for
conjugates 5, 7, and 8 containing the more hydrophilic
AAhexPQRRSARLSA peptide sequence bearing three
arginine residues, than for conjugates 4and 9that contain the
ATWLPPRandcERGDPhesequences,respectively,bearing
only one arginine residue. We have previously observed
that the cellular uptake of porphyrin conjugates bearing
short peptide sequences, increases with increasing number
of arginine residues [12]. Although pegylated porphyrin
conjugates tend to accumulate to a larger extent within cells
than the non-pegylated derivatives [13, 14], conjugates 4,
5, and 9 bearing a PEG linker exhibited relatively lower
uptake compared with 7 and 8 bearing a three carbon spacer,
due to the nature of the peptide sequence. Interestingly,
the pegylated PPIX-peptide conjugate 9 containing two
zwitterionic cERGDPhe and two PEG linkers was the least
accumulated within HEp2 cells.As expected, in HL-60 cells
a significantly higher cellular uptake was observed for all
PPIX conjugates compared with
Cytotoxicity. The dark cytotoxicity and phototoxicity
(1 J/cm2) of PPIX-peptide conjugates 4, 5, 7, 8 and 9
were evaluated in both HEp2 and HL-60 cells exposed
to increasing concentrations of each conjugate for 24 h,
and the results obtained are summarized in Table 1
(the IC50 values were calculated from dose-response
curves). All conjugates were found to have low
cytotoxicity in the dark in both cell lines (IC50 > 100 mM),
with the exception of conjugate 8 which was moderately
toxic to HEp2 cells, with determined IC50 = 74 mM. Upon
exposure to low light dose (1 J/cm2), conjugate 7 was the
most toxic to the HL-60 cells, with IC50 = 7 mM. Conjugate
7 bearing the tri-cationic PQRRSARLSA sequence also
had the highest cellular uptake in both cell lines. Both the
amount of photosensitizer accumulated within cells and
its subcellular distribution affect its phototoxicity, and
consequently determine its biological efficacy [18].
Table 1. Cytotoxicity (Cell Titer Blue assay, light dose ~1 J/cm2) in human HEp2 and HL-60
that in HEp2 cells. The highest
increase in uptake was observed
for conjugate 9 (about 10-fold)
followed by conjugate 4 (about
5-fold); all other conjugates
showed about a 3-fold increase
in uptake in HL-60 vs. HEp2
cells. Therefore, the differences
observed in the cellular uptake
for these PPIX conjugates are
mostly attributed to the nature
of the peptide sequence. It is
interesting to note that PPIX
conjugate 7, containing only
cells
Compound
Human carcinoma HEp2 cells
Human leukemia HL-60 cells
Dark
Phototoxicity Ratio
Dark
Phototoxicity Ratio
toxicity
(IC50, mM)
(IC50, mM)
toxicity
(IC50, mM)
(IC50, mM)
4
5
7
8
9
>100
>100
>100
>73.8
>100
>10
>10
>10
>10
>10
>10
>10
>10
>7.4
>10
>100
>100
>100
>100
>100
>20
>20
>20
>20
7.1
>5
>5
>5
>5
>14
one
AAhexPQRRSARLSA
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SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
607
Intracellular localization. The subcellular localization
3.9 × 150 mm (Waters, USA) column and a stepwise
gradient.
of all PPIX-peptide conjugates was investigated in
both HEp2 and HL-60 cells at a 10 mM concentration
using fluorescence microscopy. Figures 3–7 show the
fluorescent pattern observed for all PPIX conjugates in
HEp2 cells and its overlay with the organelle specific
fluorescent probes LysoSensor Green (lysosomes),
Mitotracker Green (mitochondria), DiOC6 (ER), and
BODIPY FL C5-ceramide (Golgi complex). Similar
figures were obtained using HL-60 cells (see Supporting
information). In HEp2 cells all conjugates collected in
vesicles, as shown by the punctuate pattern observed
in Figs 3–7, some of which correlated with the cell
lysosomes. When Cremophor EL was used as a nonionic
emulsifier in the incubation media, conjugates 4 and 9
were also found in the ER. Although it has been reported
that the PQRRSARLSA sequence has the ability to target
and accumulate in the nuclei of tumor endothelial cells
[11], no nuclear localization was observed for conjugates
5, 7, and 8 containing this peptide sequence. This could be
a result of an endocytic mechanism of cellular uptake for
these conjugates, thus favoring their localization within
endosomal vesicles [15]. On the other hand, in HL-60 cells
all PPIX-peptide conjugates were found in mitochondria
(see Figs S14–S18 of the Supporting information),
suggesting a different mechanism of cellular uptake in this
cell line. In addition, conjugates 4 and 5 were also found
in lysosomes, and conjugate 7 in the ER of HL-60 cells.
Synthesis
Peptide synthesis. Peptide sequences were prepared
on an automated peptide synthesizer (Applied Biosystems
Pioneer, Peptide Synthesis System, USA) in a 0.2 mmol
scale, using the Fmoc strategy of solid-phase peptide
synthesis. A 4-fold excess of the Fmoc-protected amino
acids were coupled to the PAL-PEG-PS resin using
PyBOP as the activating agent. The peptide sequences
prepared using this methodology were ATWLPPR and
AAhexPQRRSARLSA. After the final coupling reaction
and the successive removal of the Fmoc group, the
resin was washed with DMF and isopropyl alcohol, and
then dried under vacuum. The dried resin containing
the protected amino acid sequences were used in the
coupling reaction to the PPIX derivatives. Protected
cyclic peptide cERGDPhe was synthesized following a
protocol described in the literature [19]. The PPIX mono-
methyl ester 2 was synthesized in solid support using a
NovaPEG Wang resin (0.87 mmol/g) and as described in
the literature [16]. The spectroscopic characterization for
this compound is in agreement with that reported in the
literature [16].
Porphyrin 3. Porphyrin 2 (50 mg, 0.086 mmol) was
dissolved in 50 mL of CH2Cl2. To this solution Et3N
(26 mg, 0.258 mmol) and HBTU (32.6 mg, 0.086 mmol)
were added. The mixture was stirred at room temperature
for 10 min, then NH2CH2CH2(OCH2CH2)5OCH2CO2tBu
(37.7 mg, 0.095 mmol) was added [13]. The reaction
mixture was stirred at room temperature for 24 h. The
solvent was evaporated under vacuum and the target
porphyrin was isolated by flash column chromatography
on silica gel using CHCl3/MeOH for elution. Yield 58.8
mg, 72%. Protected porphyrin 2 (50 mg) was dissolved in
4 mL of a mixture of CH2Cl2/TFA 1:1. The mixture was
stirred at room temperature for 4 h, the solvent evaporated
under vacuum and the residue triturated with ethyl ether.
The green residue was dried under vacuum. Yield 44.7
EXPERIMENTAL
General
Unless otherwise indicated, all commercially
available starting materials were used directly without
further purification. Reactions under anhydrous
conditions were performed in dried and distilled
solvents under an argon atmosphere. All reactions
were monitored by TLC using Sorbent Technologies
0.25 mm silica gel plates with or without UV indicator
(60F–254). Silica gel Sorbent Technologies 32–63
1
mg (95%). H NMR (300 MHz; CDCl3): dH, ppm -4.74
1
(2H, s, pyrrole-NH), 1.27–2.03 (4H, m, CH2CONH),
3.37–3.61 (36H, m, OCH2-PEG, CH2NH-PEG, CH3-
pyrrol), 3.81–4.02 (4H, m, CH2-pyrrol, COOCH3), 4.24
(2H, s CH2CO), 6.11–6.17 (2H, m, CH2 vinyl), 6.24–6.33
(2H, m, CH2 vinyl), 8.02–8.17 (2H, m, CH vinyl), 9.54
(1H, s, CH ar pyrrol), 9.59 (1H, s, CH ar pyrrol), 9.66
(1H, s, CH ar pyrrol), 9.74 (1H, s, CH ar pyrrol). MS (HR
ESI): m/z 897.4498 (calcd. for [M + H]+ 897.4523).
General procedure for syntheses of PPIX-peptide
conjugates on solid support. Peptidyl resin (0.0165
mmol) was introduced into a glass synthesizer, swelled in
DMFfor1h,andthenwashedwithDMF(2×5mL).Tothe
peptidyl resin was added 500 mL of a solution containing
0.033 mmol of pegylated PPIX 3, 0.099 mmol of DIEA
and 0.033 mmol of PyBOP. The reaction mixture was
mm was used for flash column chromatography. H
and 13C NMR were obtained on a ARX-300 Bruker
spectrometer. DQCOSY, NOESY and TOCSY spectra
were obtained on a Varian Inova-500 spectrometer.
Chemical shifts (d) are given in ppm relative to CDCl3
1
(7.26 ppm, H) unless otherwise indicated. Electronic
absorption spectra were measured on a Perkin Elmer
Lambda 35 UV-vis spectrophotometer. Mass spectra
were obtained at the LSU Mass Spectra Facility. HPLC
separation and analysis were carried out on a Dionex
system including a P680 pump and a UVD340U,
detector. Semi-preparative HPLC was carried out using
a Luna C18 100 Å, 5 mm, 10 × 250 mm (Phenomenex,
USA) column and a stepwise gradient; analytical HPLC
was carried out using a Delta Pak C18 300 Å, 5 mm,
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M. SIBRIAN-VAZQUEZ ET AL.
Fig. 3. Subcellular localization of PPIX-peptide conjugate 4 in HEp2 cells at 10 mM for 24 h. (a) Phase contrast, (b) overlay
of conjugate 4 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence,
(g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h) and (j) overlays of organelle tracers with conjugate 4
fluorescence. Scale bar: 10 mm
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SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
609
Fig. 4. Subcellular localization of PPIX-peptide conjugate 5 in HEp2 cells at 10 mM for 24 h. (a) Phase contrast, (b) overlay
of conjugate 5 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence, (g)
MitoTracker Green fluorescence, (d), (f), and (h) overlays of organelle tracers with conjugate 5 fluorescence. Scale bar: 10 mm
Copyright © 2012 World Scientific Publishing Company
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M. SIBRIAN-VAZQUEZ ET AL.
Fig. 5. Subcellular localization of PPIX-peptide conjugate 7 in HEp2 cells at 10 mM for 24 h. (a) Phase contrast, (b) overlay
of conjugate 7 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence,
(g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h) and (j) overlays of organelle tracers with conjugate 7
fluorescence. Scale bar: 10 mm
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SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
611
Fig. 6. Subcellular localization of PPIX-peptide conjugate 8 in HEp2 cells at 10 mM for 24 h. (a) Phase contrast, (b) overlay
of conjugate 8 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence,
(g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h) and (j) overlays of organelle tracers with conjugate
8 fluorescence. Scale bar: 10 mm
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M. SIBRIAN-VAZQUEZ ET AL.
Fig. 7. Subcellular localization of PPIX-peptide conjugate 9 in HEp2 cells at 10 mM for 24 h. (a) Phase contrast, (b) overlay
of conjugate 9 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence,
(g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h) and (j) overlays of organelle tracers with conjugate
9 fluorescence. Scale bar: 10 mm
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SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
613
shaken overnight at room temperature and then filtered
filtered and the solvent evaporated under vacuum.
The target compound was isolated by flash column
chromatography on alumina Grade V using chloroform/
methanol 9:1 for elution. Yield 162 mg (74%). The Boc-
protected pegylated PPIX (0.15 g, 0.1047 mmol) was
dissolved in 8 mL of a mixture of CH2Cl2/TFA 1:1 and
stirred at room temperature for 4 h. The solvent was
evaporated under vacuum and the residue triturated
with ethyl ether, then dried under vacuum. Yield 130 mg
(98%). 1H NMR (300 MHz; CDCl3): dH, ppm 1.25–1.27
(4H, J = 1.25 NH2), 2.92–3.72 (80H, m, OCH2-PEG,
CH2NH-PEG,CH2NHCO-PEG,CH2CONH,CH3pyrrole),
6.32–6.42 (4H, m, CH2 vinyl), 7.2 (2H, broad s, NHCO-
PEG), 7.61 (2H, broad s, CH vinyl), 8.14–8.25 (2H, m,
CH vinyl), 10.40 (2H, s, CH ar pyrrole), 10.46 (1H, s, CH
ar pyrrole), 10.72 (1H, s, CH ar pyrrole). MS (HR ESI):
m/z 1263.7496 (calcd. for [M + H]+ 1263.7486).
to give a dark purple resin. The resin was washed to
remove un-reacted pegylated PPIX, first with DMF until
the filtrate was colorless, then with dichloromethane and
methanol, before being dried under vacuum. Cleavage
and deprotection was carried out by treatment of the
dried resin with 2 mL of a mixture of TFA/Phenol/TIS/
H2O, 88/5/2/5 at room temperature for 4 h. The resin was
filtered, washed with TFA (3 × 2 mL), and the filtrates
combined and evaporated under vacuum to give a green
residue. Addition of cold diethyl ether yielded a green
precipitate, which was washed repeatedly with diethyl
ether and dried under vacuum. The purification of the
PPIX-peptide conjugates was carried out by reversed-
phase HPLC on a Luna C18 semi-preparative column (10 ×
250 mm, 5 mm) (Phenomenex, USA) using a mixture of
water/acetonitrile both containing 0.1% TFA, with a
stepwise gradient. The fraction containing the conjugate
was collected and lyophilized to yield pure conjugate.
The purity of the peptides was >95% as obtained by
HPLC on an analytical Delta Pak C18 (3.9 × 150 mm,
5 mm) column.
PPIX conjugate 7. Yield 8 mg (17%). UV-vis
(methanol): lmax, nm (e, M-1.cm-1) 404.75 (61050), 508
(9 230), 546 (7 050), 580 (5 120), 638 (3 285). MS (HR
ESI): m/z 1796.9993 (calcd. for [M + H]+ 1796.9950).
PPIX conjugate 8. Yield 44 mg (55%). UV-vis
(methanol): lmax, nm (e, M-1.cm-1) 408 (84 600), 510 (8
PPIX conjugate 4. Yield 26.8 mg (58%). UV-vis
(methanol): lmax, nm (e, M-1.cm-1) 430 (136 932), 510 (7
750), 545 (7 188), 582 (4 902), 638 (3 702). H NMR
1
1
926), 545 (7 174), 581 (5 522), 637 (3 434). H NMR
(500 MHz, d6-DMSO): dH, ppm 0.97 (6H, s, CH3 Leu),
1.14 (6H, s, CH3 Leu), 1.30 (6H, s, CH3 Ala), 1.34 (6H,
s, CH3 Ala), 1.38–1.49 (4H, m, CH2-CH2-CH2 AAhex),
1.68 (24H, s, b CH2 Arg, g CH2 Arg), 2.15 (12H, s, b CH2
Gln, b CH2 Pro, g CH2 Pro), 2.29 (4H, s, CH2-CONH
AAhex), 2.56 (4H, s, CH2-pyrrole), 2.96 (4H, s, CH2-
NHCO AAhex), 3.34 (3H, s, CH3 pyrrole), 3.54 (9H, s,
CH3 pyrrole), 3.95–4.74 (36H, m, a CH Pro, a CH Gln,
a CH Arg, a CH Arg, a CH Ser, a CH Ala, a CH Arg,
a CH Leu, a CH Ser, a CH Ala, b CH2 Ser), 6.69–6.72
(2H, m, CH2 vinyl), 6.93–6.96 (2H, m, CH2 vinyl), 7.25–
7.86 (21H, m, NH AAhex, NH Pro, NH Gln, NH Arg,
NH Arg, NH Ser, NH Ala, NH Arg, NH Leu, NH Ser,
NH Ala, e CH Arg), 8.01–8.02 (1H, m, CH vinyl), 8.94–
9.02 (1H, m, CH vinyl), 10.97 (3H, s, CH ar pyrrole),
10.84 (1H, CH ar pyrrole). MS (HR ESI): m/z 3031.7564
(calcd. for [M]+ 3031.7320).
(500 MHz, d6-DMSO): dH, ppm 0.08 (3H, s, CH3-Leu),
0.25 (3H, s, CH3-Leu), 0.78–0.98 (4H, m, d CH2 Thr, b
CH2 Leu), 1.09–1.14 (1H, m, d CH Leu), 1.40 (3H, m,
b CH3 Ala), 1.71–1.91 (8H, m, b CH2 Arg, d CH2 Arg,
b CH2 Pro, d CH2 Pro), 2.04–2.12 (6H, m, d CH2 Pro,
CH2CONH), 2.16–2.18 (2H, m, b CH2 Pro), 2.32–3.0
(2H, m, dCH2 Arg), 3.18–3.64 (51H, m, d CH2 Pro, d
CH2 Pro, b CH2 Trp, CH3 pyrrole, COOCH3 pyrrole,
OCH2-PEG, CH2NH-PEG, CH2CO), 5.30–5.35 (2H, m,
CH2 vinyl), 5.52–5.59 (2H, m, CH2 vinyl), 6.01–7.17
(9H, m, CH ar Trp, NH Arg, NH Trp, NH Thr, e CH
Arg), 7.59–7.62 (1H, m, CH vinyl), 7.77–7.79 (1H, m,
CH vinyl), 9.37 (1H, s, CH ar pyrrole), 9.43 (1H, s, CH
ar pyrrole), 9.89 (1H, s, CH ar pyrrole), 9.96 (1H, s, CH
ar pyrrole). MS (HR ESI): m/z 859.9697 (calcd. for [M +
H]2+ 859.9688).
PPIX conjugate 5. Yield 35.4 mg (62%). UV-vis
(methanol): lmax, nm (e, M-1.cm-1) 418 (221 712), 510
(8 706), 545 (7 184), 582 (5 176), 638 (3 654). MS (HR
ESI): m/z 711.7398 (calcd. for [M]3+ 711.7371).
PPIX conjugate 9. Protected cyclic peptide
cERGDPhe was dissolved in 2 mL of anhydrous DMF, to
this solution was added DIEA (30.7 mg, 0.2374 mmol),
followed by the addition of PyBOP (51.4 mg, 0.0989
mmol). The mixture was stirred at room temperature
5 min, then porphyrin 6 (50 mg, 0.0396 mmol) was
added in one portion and the reaction mixture stirred
for an additional 24 h at room temperature. The PPIX-
cERGDPhe protected conjugate was isolated by flash
column chromatography on silica gel using CH2Cl2/
methanol 4:1 for elution. Deprotection was carried out
using a mixture TFA/Phenol/TIS/H2O, 88/5/2/5 at room
temperature for 4 h. The solvent was evaporated under
vacuum and the residue triturated with ethyl ether,
then dried under vacuum. Yield 23 mg (27%). UV-vis
(methanol): lmax, nm (e, M-1.cm-1) 406 (78 648), 510 (10
Porphyrin 6. Under an argon atmosphere, PPIX
di-hydrochloride salt (0.1 g, 0.1563 mmol) was
suspended in 100 mL of anhydrous CH2Cl2. To this
suspension was added Et3N (0.095 g, 0.938 mmol)
and the mixture was heated at 35 °C until PPIX was
completely dissolved. To the solution was added PyBOP
(0.3438 mmol), the mixture was stirred at 35 °C for
5 min, then NH2CH2CH2(OCH2CH2)6CH2CH2NH-Boc
(0.141 g, 0.3126 mmol) was added in one portion. The
mixture was stirred at 35 °C for an additional 20 min,
then at room temperature for 12 h. The organic phase
was washed with water (3 × 100 mL), dried over Na2SO4,
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 613–615

614
M. SIBRIAN-VAZQUEZ ET AL.
088), 544 (8312), 582 (6520) 638 (4580). 1H NMR (500
MHz, d6-DMSO): dH, ppm 0.4–1.32 (6H, m, b-H-Glu,
b,g-H-Arg), 1.90–2.02 (2H m, b-H-Glu), 2.33–2.69 (6H,
m, b-H-Asp, CH2-pyrrole), 2.79–2.95 (4H, m, b-CH-
Phe), 3.06–3.10 (4H, m, d-CH-Arg), 3.15–3.19 (4H, m,
CH2NHCO-PEG), 3.27–3.22 (8H, m, CH2NHCO-PEG,
a-CH Gly), 3.37–3.61 (56 H, m, OCH2-PEG, 12H;
CH3-pyrrole), 3.96–4.00 (2H, m, a-CHGlu), 4.05 (2H,
dd, J = 15.2/7.7 Hz, a-NHGly), 4.12–4.16 (2H, m,
a-CH-Arg), 4.45 (2H, q, J = 7.2 Hz, a-CH-Phe), 4.60–
4.65 (2H, m, a-CH-Asp), 5.32 (2H, m, CH2 vinyl), 5.65
(2H, m, CH2 vinylic), 6.77 (4H, s, NH2-Arg), 7.13–7.26
(10H, m, CH -Phe ar), 7.42–7.45 (2H, m, e-NHArg),
7.60 (1H, m, CH vinyl), 7.62–7.65 (3H, m, NH-PEG, CH
vinyl), 7.98–8.02 (4H, m, NH-Phe, NH-Asp), 8.05 (1H, d,
J = 7.2 Hz, NH-Glu), 8.30 (1H, dd, J = 3.8/7.4 Hz,
NH-Gly), 9.33 (1H, s, CH ar pyrrole), 9.37 (1H, s, CH ar
pyrrole), 9.48 (1H, s, CH ar pyrrole), 9.75 (1H, s, CH ar
pyrrole), 9.86 (1H, s, CH ar pyrrole). MS (HR ESI): m/z
812.7564 (calcd. for [M]3+ 812.7544).
cytotoxicity experiment. For the suspension cells HL-60,
the plates were centrifuged before the loading medium or
PBS buffer was removed.
Time-dependent cellular uptake. Both HL-60 and
HEp2 cells were plated at 10000 per well in a Costar
96 well plate and allowed to grow overnight. Conjugate
stocks were prepared in water at a concentration of
10 mM and then diluted into medium to final working
concentrations. The cells were exposed to 10 mM of each
conjugate for 0, 1, 2, 4, 8, and 24 h. At the end of the
incubation time the loading medium was removed and
the cells were washed with PBS. For the suspension cells
HL-60, the plates were centrifuged before the loading
medium or PBS buffer was removed. The cells were
solubilized upon addition of 100 mL of 0.25% Triton
X-100 (Calbiochem) in PBS. To determine the conjugate
concentration, fluorescence emission was read at 410/
650 nm (excitation/emission) using a BMG FLUOstar
plate reader. The cell numbers were quantified using
the CyQuant reagent (Molecular Probes).
Microscopy. The HEp2 cells were plated on LabTek
2 chamber coverslips and incubated overnight, before
being exposed to 10 mM of conjugate for either 1 or
18 h. For the co-localization experiments the cells were
incubated for 18 h concurrently with conjugate and one
of the following organelle tracers (Molecular Probes), for
30 min: MitoTracker Green 250 nM, LysoSensor Green
50 nM, DiOC6 5 mg/mL, and BODIPY FL C5 ceramide
1 mM. The slides were washed three times with growth
medium and new medium containing 50 mM HEPES pH
7.4 was added. Fluorescent microscopy was performed
using a Zeiss Labovert 200M inverted fluorescent
microscope fitted with standard FITC and Texas Red filter
sets (Chroma). The images were aquired with a Zeiss
Axiocam MRM CCD camera fitted to the microscope.
For the HL-60 cells, 10,000/100 ml cells were seeded
in each vial in a 96 Costar microplate and allow to grow
for 24 h. The cells were then exposed to 10 mM of each
compound for 18 h. Organelle tracers were obtained from
Invitrogen and used at the following concentrations:
LysoSensor Green 50 nM, MitoTracker Green 250
nM, ER Tracker Green 100 nM, and BODIPY FL C5
ceramide 1 mM. The organelle tracers were diluted into
medium and the cells were incubated concurrently with
PPIX conjugate and tracers for 30 min. The cells were
collected, centrifuged and washed with PBS. Fifty ml
of washed cells were added to slide glass and gently
covered by coverslips. The images were acquired using
a Leica DMRXA microscope and DAPI, GFP and Cy5
filter cubes (Chroma Technologies).
Cell studies
All tissue culture media and reagents were obtained
from Invitrogen. Human HEp2 cells were obtained
from ATCC and maintained in a 50:50 mixture of
DMEM:Advanced MEM containing 5% FBS. HL-60
cells were obtained from ATCC and maintained in
the ATCC-formulated Iscove’s Modified Dulbecco’s
Medium (IMDM) supplemented with 1% Penicillin-
Streptomycin and 20% FBS. Both of the cells were
sub-cultured biweekly to maintain sub-confluent stocks.
Cytotoxicity.TheHL-60andtheHEp2cellswereplated
and allowed 36–48 h to attach. The cells were exposed
to increasing concentrations of PPIX-conjugate up to
100 mM and incubated overnight. The loading medium
was then removed and the cells fed medium containing
Cell Titer Blue (Promega) as per manufacturer’s
instructions. Cell viability was then measured by reading
the fluorescence at 520/584 nm using a BMG FLUOstar
plate reader. The signal was normalized to 100% viable
(untreated) cells and 0% viable (treated with 0.2%
saponin from Sigma) cells. For the suspension cells
HL-60, the plates were centrifuged before the loading
mediumorPBSbufferwasremoved. Forthephototoxicity
experiments, the HL-60 and HEp2 cells were prepared
as described above for the dark cytotoxicity assay and
treated with conjugate concentrations of 0, 0.625, 1.25,
2.5, 5, and 10 mM. After compound loading, the medium
was removed and replaced with medium containing 50
mM HEPES pH 7.4. The cells were then placed on ice
and exposed to light from a 100 W halogen lamp filtered
through a 610 nm long pass filter (Chroma) for 10 min.
An inverted plate lid filled with water to a depth of 5 mm
actedasanIRfilter.Thetotallightdosewasapproximately
1 J/cm2. The cells were returned to the incubator overnight
and assayed for viability as described above for the dark
CONCLUSION
Five new PPIX-peptide conjugates bearing both linear
and cyclic homing peptides linked either directly or via
a low molecular weight PEG group to the propionic side
chains of PPIX, were synthesized in moderate yields
Copyright © 2012 World Scientific Publishing Company
J. Porphyrins Phthalocyanines 2012; 16: 614–615

SYNTHESIS AND CELLULAR STUDIES OF PPIX-HOMING PEPTIDE CONJUGATES
615
using solution- and solid-phase techniques. The peptides
and Plaetzer K. Photochem. Photobiol. Sci. 2008;
7: 283–289.
consisted of a VEGF sequence (ATWLPPR), a sequence
fromtheHMGN2nucleosomalprotein(PQRRSARLSA),
and a cyclic RGD-containing sequence (cERGDPhe)
for targeting avb3 integrins, which are heterodimeric
transmembrane glycoprotein receptors over-expressed in
actively dividing endothelial cells. The PPIX conjugates
containing the PQRRSARLSA peptide efficiently
accumulated within human carcinoma HEp2 cells,
probably due to the three arginine residues. In human
leukemia HL-60 cells, 3 to 10-fold higher accumulation
within cells was found for all conjugates. The most
efficiently taken-up by both cell lines was conjugate 7,
bearing only one AAhexPQRRSARLSA sequence linked
directly to one of the PPIX propionic side groups. The
main sites of subcellular localization for these conjugates
were found to be the lysosomes in HEp2 cells, possibly
as a result from endocytosis, and the mitochondria in the
HL-60 cells, suggesting different uptake mechanisms in
these two cell lines. All PPIX conjugates showed low
dark- and photo-toxicities towards both cell lines. The
most photo toxic conjugate was 7(IC50 = 7mM, 1 J/cm2)
to HL-60 cells.
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Acknowledgements
The authors thank Martha Juban for syntheses of
peptide sequences on solid support. The work described
was supported by the US National Institutes of Health,
grant number R21 CA139385.
Supporting information
NMR of selected compounds, HPLC traces and
microscopy images for conjugates (Figs S1–S18) are
given in the supplementary material. This material is
available free of charge via the Internet at http://www.
worldscinet.com/jpp/jpp.shtml.
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J. Porphyrins Phthalocyanines 2012; 16: 615–615