Supramolecular intracellular delivery of an anionic porphyrin by octaarginine-conjugated per-O-methyl-β-cyclodextrin

Article abstract of DOI:10.1039/c4cc09042j

A convenient and efficient method for intracellular delivery of a water-soluble anionic porphyrin has been developed by utilizing its supramolecular interaction with per-O-methyl-β-cyclodextrin bearing an octaarginine chain as a cell-penetrating peptide.

Full text of DOI:10.1039/c4cc09042j

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Supramolecular intracellular delivery of an
anionic porphyrin by octaarginine-conjugated
per-O-methyl-b-cyclodextrin†
Cite this: Chem. Commun., 2015,
1, 2421
5
Received 13th November 2014,
Accepted 2nd January 2015
a
a
b
b
a
Hiroaki Kitagishi,* Fumihiko Chai, Shigeru Negi, Yukio Sugiura and Koji Kano
DOI: 10.1039/c4cc09042j
www.rsc.org/chemcomm
A convenient and efficient method for intracellular delivery of a small peptides consisting of less than 30 amino acid residues with a
water-soluble anionic porphyrin has been developed by utilizing its high density of basic amino acids (arginine or lysine). CPPs are
supramolecular interaction with per-O-methyl-b-cyclodextrin capable of assisting in the intracellular delivery of various bio-
9
bearing an octaarginine chain as a cell-penetrating peptide.
molecules and synthetic compounds. In general, the target mole-
cule is covalently attached to the CPP for effective delivery. However,
7,8
Porphyrins and their related compounds have been utilized in covalent conjugation of PS with CPP is time-consuming. In this
mammalian cells as photosensitizers (PS) in photodynamic tumor study, we describe a simple methodology for non-covalent delivery
therapy (PDT), contrast agents for magnetic resonance imaging of PS into cells using a CPP-conjugated artificial host molecule,
1
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4
(
MRI) and luminescent sensors for metal ions or oxygen.
per-O-methylated b-cyclodextrin (CD), which forms a stable inclu-
A variety of synthetic tetrapyrrole derivatives have been prepared sion complex with 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin
1
–4
to achieve these functions.
studies is delivery of the synthetic compounds into the cell. Some serum proteins.
hydrophobic tetrapyrroles can penetrate cell membranes without We chose the arginine octamer (R8) as a CPP because of its
An inevitable problem in these (TPPS) in an aqueous culture medium even in the presence of
1b,d,4
10
any chemical modifications.
In these cases, however, an structural simplicity and well-characterized cell-penetrating ability.
organic solvent such as dimethylsulfoxide (DMSO) is required to The R8 peptide with a terminal alkyne was conjugated with mono-6-
1
1
3
prepare a stock solution. Although a small amount of DMSO does azido-6-deoxy-per-O-methyl-b-CD (per-OMe-6-N -b-CD) by a
not affect cell viability, this method occasionally causes low copper(I)-catalyzed azide–alkyne cycloaddition reaction (CuAAC)
cellular uptake efficiency and unexpected cytotoxicity due to the (Scheme 1). Using the tris(3-hydroxypropyltriazolylmethyl)amine
1
2
+
formation of self-aggregates when the hydrophobic compound is (TBTA) ligand, which can suppress unfavorable oxidation of Cu
2+
dispersed from the organic solvent into an aqueous medium. In to Cu , the CuAAC reaction was performed in an aqueous
addition, non-specific hydrophobic interactions with biological com- solution under aerobic conditions and at ambient temperature.
ponents, such as serum proteins, must be considered. In contrast, Pure R8-conjugated per-O-methylated b-CD (R8-CD) was obtained
water-soluble cationic porphyrins, such as 5,10,15,20-tetrakis- after purification by reverse-phase liquid chromatography in a
(
N-methyl-4-pyridinium)porphyrin (TMPyP), are known to be effi- 52% yield. R8-CD was confirmed by elemental analysis, analytical
4,5
ciently transferred into cells without the use of organic solvents.
Therefore, TMPyP has often been used as the PS, although it tends to
chromatography and mass spectroscopy (Fig. S1, ESI†).
The intermolecular interaction between R8-CD and TPPS in
be accumulated in the nucleic acids of cells. In addition, self- aqueous solution was studied by UV-vis and NMR spectroscopy
assembled nanoparticles, including micelles and liposomes, have and size exclusion chromatography (SEC). It is known that
6
also been used for intracellular delivery of PS molecules.
2,3,6-tri-O-methyl-b-cyclodextrin (TMe-b-CD) strongly interacts
The use of cell-penetrating peptides (CPP) can effectively with TPPS to form a trans-type 2 : 1 inclusion complex, where
7
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deliver a large amount of PS molecules into cells. CPPs are two TMe-b-CD molecules interact with the 4-sulfonatophenyl
13
groups at the 5- and 15-positions. In contrast to the simple
UV-vis spectral changes observed when TPPS was titrated with
a
Department of Molecular Chemistry and Biochemistry, Faculty of Science and
Engineering, Doshisha University, Kyotanabe, Kyoto 610-0321, Japan.
E-mail: hkitagis@mail.doshisha.ac.jp
1
3b
TMe-b-CD,
a biphasic spectral change was observed in the
TPPS–R8-CD complex formation (Fig. 1). In the first phase, tetra-
anionic TPPS forms a water-insoluble ion-pair complex with octa-
cationic R8-CD, resulting in a weakening in the absorption band.
Further addition of R8-CD caused dissociation of the ion-pair
b
Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts,
Kyotanabe, Kyoto 610-0395, Japan
†
Electronic supplementary information (ESI) available: Experimental details and
Fig. S1–S11. See DOI: 10.1039/c4cc09042j
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Scheme 1 Synthetic route of R8-CD.
Fig. 2 The overlaid fluorescence/phase-contrast images of the HeLa cells
À6
À6
after treatment with (a, d) TPPS (5.0 Â 10 M), (b, e) TPPS (5.0 Â 10 M)
À5
À6
plus TMe-b-CD (1.2 Â 10 M), and (c, f) TPPS (5.0 Â 10 M) plus R8-CD
À5
(
1.2 Â 10 M) in different culture media (a–c: D-10; d–f: OPTI). Luminescence
was shown in red (excited at 435 Æ 25 nm for porphyrin) and blue (excited at
60 Æ 40 nm for DAPI). The images were obtained using an oil-immersion
objective lens (100Â).
3
15
followed by monitoring with fluorescence microscopy (Fig. 2).
Unsurprisingly, no fluorescence due to the free-base porphyrin was
observed from the cells in the absence of R8-CD. In the presence of
R8-CD, clear fluorescence signals due to the free-base porphyrin
were detected in the cells. In both the serum-containing and
À6
Fig. 1 (a) UV-vis spectral changes in TPPS (1.3 Â 10 M) upon addition of
R8-CD in phosphate buffer saline (PBS) at pH 7.4 and 25 1C. (b) The serum-free media, fluorescence signals were detected in endo-
changes in absorbance at 416 nm of TPPS as a function of [R8-CD]/[TPPS].
somal structures mainly located at the peripheries of the cell
nuclei. In addition, clear cytosolic dispersion was also observed
for the sample incubated in the serum-free medium (Fig. 2f).
1
0d
complex, resulting in the formation of the 2 : 1 inclusion complex. Similar observations were reported by Futaki et al., who found
As shown in Fig. 1b, the titration curve was almost saturated after that the R12 and R16 peptides in the serum-free medium
the addition of 2 equiv. of R8-CD, and the final spectrum was nearly showed cytosolic dispersion through an ATP-independent, direct
identical to that of the TPPS–TMe-b-CD complex (Fig. S2, ESI†). transmembrane mechanism. The arginine oligomers strongly
Therefore, R8-CD forms a 2 : 1 inclusion complex with TPPS in the adhered to the cell surface, and could be directly translocated
same manner as TMe-b-CD. The structural similarity of the R8-CD– into the cytosol due to potential differences across the cell
1
6
TPPS complex to the TMe-b-CD–TPPS complex was also confirmed membrane (the cell interior is relatively negative). Indeed,
by the pH titration, NMR titration, 2D NMR and the SEC analysis the cytosolic dispersion was also observed when the cells were
(Fig. S3–S5, ESI†).
incubated with the TPPS–R8-CD complex at 4 1C in the serum-
The cellular uptake of the TPPS–R8-CD complex was studied free medium (Fig. S7, ESI†), which indicates that the porphyrin
using HeLa cells. The TPPS solution with or without CD (2.4 equiv. was dispersed from the serum-free medium into the cytosol as
of TMe-b-CD or R8-CD) in phosphate buffer saline (PBS) was added a result of the direct transmembrane mechanism because
10d,17
to cells soaked in the culture medium. To investigate the effect ATP-dependent endocytosis should be blocked at 4 1C.
of serum, medium containing 10% serum (D-10) and serum- Non-specific interactions of the oligoarginine chain with albumin,
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4
free medium (OPTI) were used as the culture media. After 2 h a major acidic protein in serum, seem to reduce the effective
incubation, the cells were washed several times with PBS, concentration of the peptide that participates in the direct
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transmembrane pathway. As a result, the endocytosis mechanism
is the major cellular uptake pathway in the serum-containing
medium.
To understand the inclusion state of the TPPS–R8-CD
complex in cells, the cells containing the complex were lysed
by a freeze-thaw cycle in water, and the cell lysate was analyzed
by fluorescence spectroscopy. In the sample incubated in D-10
and OPTI, the cell lysate showed a characteristic fluorescence
spectrum resulting from the complex of TPPS with two R8-CD
molecules (lem = 643 and 704 nm). The spectrum was quite
distinguishable from free TPPS (Fig. S8, ESI†). These results
indicate that the inclusion phenomenon is sustained, even in
the cell-interior. The fluorescence intensity of the lysate was
increased as a function of incubation time and reached a
plateau in incubation more than 120 min (Fig. S8, ESI†).
Flow cytometry was applied to quantify the amount of TPPS
taken up by the cells. After incubation with the TPPS–R8-CD
complex, the number of highly fluorescent cells increased, as a
result of internalization of TPPS in the cells (Fig. 3). Owing to
the direct transmembrane pathway that occurs in the serum-
free medium, the cells incubated with the complex in OPTI
showed a wider distribution in the high fluorescence region
than those in the serum-containing medium. However, as
8a,10d
À6
compared to the previous intracellular delivery systems,
serum-induced inhibition was remarkably weak, and serum (5.0 Â 10 M) complexed with TMe-b-CD (1.2 Â 10 M), and TPPS (5.0 Â
proteins did not substantially interfere with cellular uptake of
the TPPS–R8-CD complex. Flow cytometry also revealed that bare
TPPS weakly interacted with the cells in OPTI (green line in
Fig. 4 Photo-induced cytotoxic effects of TPPS (5.0 Â 10 M), TPPS
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À5
À6
À5
1
0
M) complexed with R8-CD (1.2 Â 10 M) in different culture media
(
blue: OPTI; red: D-10). HeLa cells cultured in a 96-well plate were treated
with filtered light at 310–510 nm for 15 min. The cell viability was measured
by the MTT assay. (a) Photograph of the 96-well plate after the MTT assay.
Fig. 3a), whereas complexation with TMe-b-CD suppressed the The purple color indicates the presence of living cells. (b) The histogram
interaction (red line in Fig. 3a). Because TPPS strongly interacts for the cell viability. Each bar represents the mean Æ SD of the data
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obtained from at least three experiments.
with serum proteins, primarily serum albumin, the interaction
of TPPS with the cells was inhibited in the serum-containing
medium (Fig. 3b). Competitive titration experiments indicated that
R8-CD interacts more strongly with TPPS than BSA (Fig. S9, ESI†).
On the basis of these results, it can be concluded that R8-CD can be
widely used for intracellular delivery of water-soluble tetraaryl-
porphyrins, such as TPPS, even in the presence of serum proteins.
We next studied the photo-induced cytotoxicity in this system.
The TPPS solution with or without CD (TMe-b-CD or R8-CD) was
added to cells in the culture medium (OPTI or D-10), and filtered
light (310–510 nm) was successively irradiated to the cell cultures for
1
5 min. After photo-irradiation, the cell viability was measured
by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay
Fig. 4). Corresponding to the flow cytometry data shown in Fig. 3,
(
TPPS without serum showed weak toxicity due to weak interaction
with the cells. The photo-induced cytotoxic function of TPPS was
drastically reduced in the serum-containing medium. The TPPS–
TMe-b-CD complex did not show any photo-induced cytotoxicity in
4
Fig. 3 Flow cytometry histograms of HeLa cells (10 cells per measure- both types of media. Meanwhile, the TPPS–R8-CD complex
À6
ment). The cells were pre-treated with TPPS (5.0 Â 10 M; green lines),
showed very high cell-killing ability, regardless of the presence
of serum. Therefore, R8-CD is usable as an effective delivery
tool for PDT, even in the presence of competitive biomolecules
in the medium. Because R8-CD itself and the TPPS–R8-CD
complex did not show any cytotoxicity without light irradiation
À6
À5
TPPS (5.0 Â 10 M) plus TMe-b-CD (1.2 Â 10 M; red lines), and TPPS
À6
À5
(
5.0 Â 10 M) plus R8-CD (1.2 Â 10 M; blue lines) in different culture
media (a: OPTI; b: D-10). Untreated control cells are represented by the
black lines. The cells were individually excited at 643 nm and lumines-
cence signals were detected at 667 nm.
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Fig. S10, ESI†), the photo-induced cytotoxic effect of the TPPS–
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R8-CD (Fig. S11, ESI†).
(
(
In conclusion, we have successfully synthesized an octaarginine-
conjugated per-O-methyl-b-cyclodextrin (R8-CD) for the delivery
of an anionic tetraarylporphyrin, TPPS, into mammalian cells.
R8-CD forms a very stable 2 : 1 inclusion complex with TPPS in
aqueous solution in the same manner as TMe-b-CD without the
peptide chain. Because of the higher binding affinity of R8-CD
for TPPS, compared to serum proteins, R8-CD can be used as
a powerful molecular tool for intracellular delivery of tetraaryl-
porphyrins in vivo.
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