In this article, we describe the development of a nanosized-glutathione peroxidase (GPx) mimic based on liposomes of which the amphiphilic selenenylsulfide derivative (R-Se-S-R') was incorporated into a lipid membrane. A lipid membrane-compatible selenenylsulfide derivative, 1-oxo-headecyl-seleno-l-cysteine-methyl-Se-yl-S-l-penicillamine methyl ester (OHSeP), was synthesized. X-ray photoelectron spectroscopy revealed that the sulfur and selenium atoms of the OHSeP molecule formed a selenenylsulfide linkage. The use of OHSeP easily allowed the introduction of the seleno-l-cysteine (SeCys) moiety into the liposomal membranes by mixing with the phosphatidylcholines (PCs), which gave rise to the GPx-like catalytic activity because of the selenium atom in the SeCys moiety. The penicillamine moiety of the OHSeP molecule incorporated into the OHSeP/PC liposomes was thought to orient toward the outer water phase. The OHSeP/PC liposomes generated the GPx-like catalytic activity, which was ascribed to the SeCys moiety that was introduced into the PC-based liposomes. Consequently, the lipid/water interface of the liposomal membranes could possibly provide an effective colloidal platform for the development of water-soluble nanosized GPx mimics.
In this article, we describe the development of a nanosized-glutathione peroxidase (GPx) mimic based on liposomes of which the amphiphilic selenenylsulfide derivative (R-Se-S-R') was incorporated into a lipid membrane. A lipid membrane-compatible selenenylsulfide derivative, 1-oxo-headecyl-seleno-l-cysteine-methyl-Se-yl-S-l-penicillamine methyl ester (OHSeP), was synthesized. X-ray photoelectron spectroscopy revealed that the sulfur and selenium atoms of the OHSeP molecule formed a selenenylsulfide linkage. The use of OHSeP easily allowed the introduction of the seleno-l-cysteine (SeCys) moiety into the liposomal membranes by mixing with the phosphatidylcholines (PCs), which gave rise to the GPx-like catalytic activity because of the selenium atom in the SeCys moiety. The penicillamine moiety of the OHSeP molecule incorporated into the OHSeP/PC liposomes was thought to orient toward the outer water phase. The OHSeP/PC liposomes generated the GPx-like catalytic activity, which was ascribed to the SeCys moiety that was introduced into the PC-based liposomes. Consequently, the lipid/water interface of the liposomal membranes could possibly provide an effective colloidal platform for the development of water-soluble nanosized GPx mimics.
In the periodic table, selenium is a member
of the chalcogen group
that shares similar chemical properties with oxygen, sulfur, and tellurium.
This element is an essential trace nutrient for mammals, birds, and
fish. Interest in exploring the biochemical propensity of selenium
has increased after the discovery of its biological role in the mid
1950s.[1] Selenium is now known in its multifarious
chemical forms as a trace element in living systems.[2] In particular, it is in the form of the 21st proteinogenic
amino acid “selenocysteine” (SeCys, Sec, or U).[3] The insertion of SeCys into polypeptide chains
is genetically guided by the uracil-guanine-adenine codon that acts
as the stop codon in a normal way,
and the resulting SeCys-inserted proteins (selenoproteins) broadly
occur in the entire body.[4,5] In humans, SeCys has
catalytic and/or structural roles in the 25 selenoproteins that were
deduced from a selenoproteome analysis.[6] The best-known selenoproteins are the glutathione peroxidases (GPxs)
that control the redox state in the living systems by catalyzing the
reduction of H2O2 and organic hydroperoxides
(R–OOH) to water and the corresponding alcohols (R–OH)
using glutathione in the reduced form (GSH) as the reducing substrate
(R–OOH + 2GSH → R–OH + GSSG + H2O).[7] Theseselenium-dependent GPxs are the major antioxidant
defense systems in mammals, birds, and fish. Among the five selenium-dependent
GPxs, phospholipid hydroperoxideGPx (GPx-4) plays a unique and vital
antioxidant role; it is the only enzyme that can directly catalyze
the reduction of phospholipid hydroperoxides.[8] A systemic knockout of the entire GPx-4 gene is lethal, becauseGPx-4 plays critical roles as a component of the mammalian antioxidant
defense system.[9]A catalytic cycle
for natural GPxs was first proposed by Ganther
and Kraus, in which a selenol (−SeH) in SeCysserves to reduce
the peroxide species.[10] Afterward, the
resulting selenenic acid (−SeOH) is recycled back to the selenol
through a sequential reaction with two molecules of GSH via the selenenylglutathione
(−Se–SG)-forming oxidized GSH (GSSG) and water (Figure ).[7,8,11] The selenenylsulfides (−Se–S−)
are the crucial intermediates in the GPx catalytic cycle. A recent
structural study of the natural GPxs elucidated a conserved structure
of functionally significant amino acid residues (asparagine, glutamine,
and tryptophan) in the vicinity of the SeCys (catalytic tetrad).[12] The catalytic activity of the natural GPxs requires
the supporting roles of several amino acid residues within their polypeptide
chains, which hardly seems to be achieved with low-molecular-mass
organoselenium compounds. The biological (therapeutic) effect of such
organoselenium compounds is mainly attributed to their peroxidase
catalytic activity, and the activity appears to depend on the reduction
of the selenenic acid to the selenol by thiols. Therefore, macromolecular
scaffolds with certain higher-order structures could possibly provide
a means for obtaining a better chemical stability of the selenium
atom and thus an improvement of the GPx-like catalytic activity. On
the basis of such an idea, we have synthesized polymer and/or polymeric
GPx mimics using biocompatible polymers and biopolymers. We have reported
a nanostructured GPx mimic using the pullulan conjugated with seleno-l-cystine (SeCyst), which improves the chemical stability of
the selenium atoms and hence increases the GPx-like catalytic activity.[13] A polypeptide material with a peculiar three-dimensional
structure is one of the potential scaffold candidates for such a purpose.
We have also developed albumin-supported GPx mimics with selenenylsulfide
as a functional element.[14] Many characteristics
of our macromolecular system were found to be different from the low-molecular-mass
GPx mimics: (i) the ability to improve the solubility of water and
stability of the chemically liable selenium compounds to function
as the active site; (ii) the capability of forming a hydrophobic environment
around the selenium compound; and (iii) the ability to concentrate
the local selenium compound in polymers.
Figure 1
Catalytic mechanism and
X-ray structure of glutathione peroxidase.
PDB ID: 1GP1.[11] GSH, glutathione in the reduced form;
GSSG, glutathione in the oxidized form; ROOH, peroxides.
Catalytic mechanism and
X-ray structure of glutathione peroxidase.
PDB ID: 1GP1.[11] GSH, glutathione in the reduced form;
GSSG, glutathione in the oxidized form; ROOH, peroxides.In the present study, we have developed a nanosized
GPx mimic based
on the molecular assembly of which an amphiphilic selenenylsulfide
derivative was incorporated into the liposomal membrane with a certain
molecular orientation. Liposomes are closed vesicles consisting of
lipid bilayer membranes. The lipid/water interface of the liposomal
membranes could provide a unique colloidal platform for various chemical
reactions.[15−18] A lipid bilayer membrane-compatible selenenylsulfide derivative,
1-oxo-hexadecyl-seleno-l-cysteine-methyl-Se-yl-S-l-penicillamine methyl ester (OHSeP),
was synthesized, and the nanosized structure of the liposomal membrane
was used as an alternative potential scaffold.
Experimental Section
Materials
SeCyst and l-penicillamine (Pen)
were obtained from Sigma Co., Ltd. (St. Louis, MO) and Tokyo Chemical
Industry Co., Ltd. (Tokyo, Japan), respectively. l-Cystine
(Cyst), n-hexadecanoic acid, butyric acid, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU) were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). 2,3-Diaminonaphthalene
(DAN) and glutathione in reduced form (GSH) were obtained from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). Egg yolk phosphatidylcholine
(PC, hydrogenated) was obtained from the NOF Corporation (Tokyo, Japan).
GSH reductase and nicotinamide adenine dinucleotide phosphate in the
reduced form (NADPH) were obtained from Wako Pure Chemical Ind., Ltd.
(Osaka, Japan). H2O2 used as the substrate for
the GPx-like catalytic activity measurements was purchased from Nacalai
Tesque, Inc. A Milli-Q Biocel system (Millipore Corp., Billerica,
MA) was utilized to generate water (>18 MΩ·cm), which
is
used throughout this study. All other chemicals were of commercial
reagent or special grades and used as received.
Syntheses of
OHSeP and Related Compounds
The SeCyst
methyl ester (H-SeCyst-OMe) was synthesized according
to the procedures reported by Bondanszky.[19] Briefly, a mixture of SeCyst (66.7 mg) and p-toluenesulfonic
acid (190 mg) dissolved in 10 mL of absolute CH3OH was
refluxed for 40 h. After the removal of the alcohol, the resulting
precipitate was left for 24 h in C2H5OC2H5. The obtained yellow solid material was washed
several times with C2H5OC2H5 and then dried under reduced pressure (yield 91.7%). H-SeCyst-OMe: 1H NMR (300 MHz, CD3OD) δ
2.37 (s, 3H), 3.29–3.31 (m, 2H), 3.85 (s, 3H), 4.42 (t, 1H, J = 12.6 Hz), 7.24 (d, 2H, J = 8.1 Hz),
7.71 (d, 2H, J = 8.4 Hz); MALDI TOF-MS [matrix: 2,5-dihydrobenzoic
acid (DHB), positive ion mode] calcd for C8H16N2O480Se2m/z 364.2, found 364.2.A mixture of the H-SeCyst-OMe (70.7 mg) and tert-butoxycarbonyl
(Boc)-Pen methyl ester (Boc-Pen-OMe, 52.6 mg) dissolved in 5 mL of
CH3CN containing N(C2H5)3 (30 μL) was stirred for 48 h at room temperature. After the
removal of the solvent, the resulting solid material was sequentially
washed with 10 w/v % citric acid and saturated NaHCO3,
and then dissolved in an appropriate volume of CH3COOC2H5 with anhydrous Na2SO4.
After the addition of n-hexadecanoic acid (15.5 mg),
the mixture was dissolved in 5 mL of CH3CN containing N(C2H5)3 (30 μL), and then stirred
at room temperature for another 24 h in the presence of TBTU (32.1
mg) (yield 61.8%). After the removal of the solvent, the resulting
solid was sequentially washed with 10 w/v % citric acid and saturated
NaHCO3. The Boc adduct was further exposed to 2 mL of CF3COOH to remove the Boc group. After the removal of the CF3COOH by evaporation, the resulting mixture was left in a 1:6
mixture of C2H5OC2H5 and
CH3(CH2)4CH3 for 24 h
to precipitate the solid materials and then separated by filtration.
Finally, the solid materials were dissolved in Milli-Q water, followed
by lyophilization, and then a pale yellow powder was obtained. OHSeP: 1H NMR (400 MHz, CD3OD) δ 0.89–0.91
(t, 3H, J = 6.8 Hz), 1.24–1.40 (m, 24H), 1.49–1.53
(d, 6H, J = 13.6 Hz), 1.58–1.65 (m, 2H), 2.22–2.26
(t, 2H, J = 7.4 Hz), 3.13–3.18 (dd, 1H, J = 9.0, 12.7 Hz), 3.39–3.18 (dd, 1H, J = 5.1 and 12.7 Hz), 3.74 (s, 3H), 3.87 (s, 3H), 4.11 (s, 3H), 4.75–4.78
(dd, 1H, J = 5.1 and 9.1 Hz); FAB-MS (matrix: glycerol)
calcd for C26H50N2O5S80Se m/z 583.3 [M + H]+, found 583.3.
PC and OHSeP at the
indicated molar ratio were mixed in a 100-mL round-bottom flask and
dissolved by adding 5 mL of CHCl3. The CHCl3 was gently removed. After the addition of 50 mL of 10 mM Tris/HCl
buffer (pH 7.4) to the flask, the mixture was vortexed for 1 h. Subsequently,
the resulting suspension was sonicated using a Sonifier 250D probe-type
sonicator (Branson, Danbury, CT) at 80 W for 15 min in an ice-water
bath. Opaque liposomal suspensions were then centrifuged at 40 000
rpm for 1 h. The supernatant was used in subsequent experiments as
the liposomal OHSeP solution.
Determination of Particle
Diameter and Zeta Potential of Liposomes
The particle diameter
and distribution of the liposomes were measured
by a dynamic light scattering (DLS) technique using a Zetasizer Nano
ZS (Malvern Instruments, Ltd., Worcestershire, UK) at 90° to
the incident beam (a 633 nm beam from a 4 mW He–Ne laser tube)
in a 1-cm pass-length quartz cell at 25 °C. Data fitting was
carried out using a multimodal algorithm supplied by Malvern Instruments.
The obtained correlograms were fitted to diffusion coefficients and
converted to a hydrodynamic diameter using the Einstein–Stokes
equation. The zeta potential of the liposomes was determined by a
laser Doppler method using an ELS-7500 (Otsuka Electronics Co., Ltd.,
Osaka, Japan) in a DTS1070 capillary cell.
Determination of Phosphorus
Concentration
After acid
digestion of the lipid vesicle suspensions, using a 4:1 mixture by
volume of HNO3 and HClO4, the phosphorus content
was determined based on vanadate, which can generate a yellow complex
with the phosphate ion.[20] The color produced
by the complexation was monitored at 440 nm. The amount of phosphorus
in liposomes was calculated by the difference in the phosphorus concentration
of the liposomal solution after centrifugation.
Determination
of Selenium Concentration
The selenium
concentrations in the specimens were determined by fluorometry using
DAN after acid digestion using a 4:1 mixture by volume of HNO3 and HClO4.[21] The selenium
standard solution [1000 ppm as selenium(IV) dioxide in 0.1 M HNO3] for the fluorometry was obtained from Kanto Chemical Co.,
Inc.
X-ray Photoelectron Spectroscopy
According to the procedures
reported previously,[22] the X-ray photoelectron
spectroscopy (XPS) was performed using an AXIS-ULTRA instrument fitted
with a delay-line detector and a monochromated Al Kα line (0.8339
nm, 1.486 keV) operating at 10 kV and 13 mA (Shimadzu Corp., Kyoto,
Japan).
Determination of GPx-like Catalytic Activity
The GPx-like
catalytic activity was determined according to the procedures reported
previously.[13,14] The liposome solution (final
selenium concentration, 5 μM) was sequentially combined with
ethylenediaminetetraacetic acid (1 mM), NaN3 (1 mM), GSH
reductase (1 unit), a reduced GSH (1 mM), and NADPH (0.25 mM) in 10
mM Tris/HCl buffer (pH 7.4). The reaction was initiated by the addition
of H2O2 solution (0.5 mM). Optical density (OD)
at 340 nm due to the NADPH was recorded every 10 s just after mixing
by inversion. The GPx-like catalytic activity was calculated using eq as micromoles of NADPH
oxidized per minute.where ΔODSMP is the decrease
in OD at 340 nm of the sample solutions between 10 and 70 s after
the addition of H2O2, ΔODBLK is the decrease in OD at 340 nm per minute of the solutions without
the liposome, εmM is the extinction coefficient for
1 mM NADPH solution [6.22/(mM·cm)], and c is
the final selenium concentration in μM.[23,24]
Results and Discussion
The catalytically active selenol
reduces peroxides to form a selenenic
acid, which further reacts with thiols to regenerate the selenenylsulfides.
The selenenylsulfide is the crucial intermediate in the catalytic
cycle of the natural GPxs (Figure ). Generally, the nucleophilic thiol attack at the
selenium atom is both kinetically and thermodynamically more favorable
than that at the sulfur atom.[25] A nucleophilic
attack of thiol at the selenium atom would lead to a thiol exchange
that hinders the regeneration of the catalytically active selenol.
An analysis of the catalytic site features of the natural GPx elucidates
that the sulfur atom in the selenenylsulfide intermediate is involved
in a weak interaction with the amidonitrogen atom of the threonine-54
residue, lowering the energy barrier to increase the possibility of
a nucleophilic attack of the negatively charged thiolate at the sulfur
atom in the selenenylsulfide.[26,27] The selenenylsulfide
intermediate can be converted to the corresponding selenol by high
concentrations of glutathione (GSH) in the living cell cytosol. Thus,
the thiol exchange that takes place at the selenium atom in the selenenylsulfide
is thought to result in poor GPx-like catalytic activity. In low-molecular-mass
selenium compounds, both the thiol exchange at the selenium atom and
the low reactivity of the selenol toward oxidation may result in poor
catalytic activity of the selenenylsulfide derivative. A key factor
for achieving a better GPx-like catalytic activity is to promote the
desired thiol exchange at the sulfur atom in the selenenylsulfide
and to generate the catalytically active selenol and disulfide. For
this purpose, we have attempted to use the lipid/water interface of
the liposomal membranes for mimicking the GPx-like catalytic activity
in water. A selenenylsulfide derivative with a lipid membrane-compatible n-hexadecanoyl chain was designed to orient the outer water
phase, for promoting the desired thiol exchange of the thiols (Figure ).
Figure 2
Schematic drawing of
a liposomal glutathione peroxidase mimic with
a selenenylsulfide-bearing lipid and its activation by a thiol exchange.
R1–SH, a thiol that can be attacked on the selenenylsulfide;
R2–SH, a thiol that can be released from the selenenylsulfide.
Schematic drawing of
a liposomal glutathione peroxidase mimic with
a selenenylsulfide-bearing lipid and its activation by a thiol exchange.
R1–SH, a thiol that can be attacked on the selenenylsulfide;
R2–SH, a thiol that can be released from the selenenylsulfide.In the present study, seleno-l-cystine (SeCys)
was used as the source material for the introduction of SeCys at the
lipid/water interface of the liposomes. The α-carboxyl groups
of SeCyst were esterified with methanol, and the H-SeCyst-OMe was allowed to react with the Boc-l-penicillamine
methyl ester (Boc-Pen-OMe) as a thiol exchange reagent with the diselenide
to form a selenenylsulfide between SeCys and Pen. Subsequently, the
selenenylsulfide derivative was then coupled with n-hexadecanoic acid to allow coupling with the membrane-compatible
moiety through its α-amino groups (OHSeP, Scheme ). The Boc protection at the α-carboxyl
group of Pen was removed by treatment with CF3COOH, followed
by mixing with phospholipid molecules. A selenenylsulfide intermediate
in the natural GPx catalytic cycle can be cleaved by the cosubstrate
GSH.[28] The selenenylsulfide linkage is
intrinsically less stable than the diselenide ones.[29−31] However, the
substitution of Cys by Pen notably improves the chemical stability
of labile selenium species, such as R–S–Se–S–R′,
and NO because of steric constraints by the two methyl groups at the
β carbon atom.[32]
p-TSA, p-toluenesulfonic acid; TEA, triethlyamine; TBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate; TFA, trifluoloacetic acid.To obtain information about the orientation of OHSeP molecules
in the liposomal membranes, its analogue, 1-butyryl-SeCys-methyl-Se-yl-S-l-penicillamine methyl
ester (BSeP), which possesses the n-butyryl chain
in place of the n-hexadecanoyl one, was synthesized
by similar synthetic procedures using n-butyric acid.
Although acetyl chain-substituted analogue of OHSeP was synthesized,
it is easily decomposed to produce elemental selenium as the terminal
product within a week at room temperature in a desiccator. Meanwhile,
OHSeP was fairly stable without developing red even when it was maintained
for several months under the same conditions, indicating that the
addition of a long acyl chain also resulted in improvement of the
chemical stability of selenenylsulfide derivatives. We expected that
the lipid/water interface could further stabilize selenenylsulfide
derivatives through interfering with any intermolecular interaction
among OHSeP molecules. As another control material, the cysteine analogue
(1-oxo-hexadecyl-l-cysteine-methyl-S-yl-S-l-penicillamine methyl ester, OHSP), which is
substituted by sulfur in place of selenium in the OHSeP molecule,
was similarly synthesized using Cyst as the starting material.Selenium has six naturally occurring isotopes and their abundances
are 8.73 for 82Se, 49.61 for 80Se, 23.77 for 78Se, 7.63 for 77Se, 9.37 for 76Se, and
0.89 for 74Se in at %. Thus, one selenium atom-containing
compound is supposed to show the characteristic mass spectral pattern,
as reflected by its natural isotope abundance. When the OHSeP and
BSeP samples were subjected to mass spectrometric analyses, the characteristic
selenium isotopic pattern involving one selenium atom in a molecular
ion was observed, and the 80Se-containing molecular ion
peaks of OHSeP and BSeP were at m/z 583.3 [M + H]+ and 414.4, respectively (Figure A,B). On the contrary, such
a distinctive feature of the isotopic pattern did not appear in the
mass spectrum of OHSP bearing a disulfide (Figure C).
Figure 3
Characteristic mass spectra of OHSeP (A), BSeP
(B), and OHSP (C).
Peaks containing naturally occurring isotopes are indicated with asterisks.
The natural abundances of the selenium and sulfur isotopes are 8.73
for 82Se, 49.61 for 80Se, 23.77 for 78Se, 7.63 for 77Se, 9.37 for 76Se, 0.89 for 74Se, 4.25 for 34S, 0.75 for 33S, and
94.99 for 32S (in at %).
Characteristic mass spectra of OHSeP (A), BSeP
(B), and OHSP (C).
Peaks containing naturally occurring isotopes are indicated with asterisks.
The natural abundances of the selenium and sulfur isotopes are 8.73
for 82Se, 49.61 for 80Se, 23.77 for 78Se, 7.63 for 77Se, 9.37 for 76Se, 0.89 for 74Se, 4.25 for 34S, 0.75 for 33S, and
94.99 for 32S (in at %).To characterize the oxidation and chemical bond state of
the sulfur
and selenium atoms, OHSeP was analyzed by XPS. The XPS measures the
kinetic energy of electrons emitted from sample surfaces under X-ray
radiation. Knowing the energy of the incoming X-ray beam and measuring
the kinetic energy of electrons leaving the surface determines the
binding energy obtained by the spectrometer. The binding energy is
specific to both the elements and electronic orbitals from which electrons
originate. The obtained absorption spectra and estimated values for
the binding energy are listed in Figure and Table , respectively. The sulfur 2p electrons from Cyst gave
an absorption peak at 161.4 and a shoulder at 162.4 eV (Figure A). The absorption peak shape
of OHSP (Figure C)
was almost identical to that of Cyst (Figure A). The values for the binding energy of
the sulfur 2p electrons in OHSP were 161.9 and 163.0 eV. The selenium
3p electrons from SeCyst gave two absorption peaks at 159.8 and 165.3
eV (Figure B). Meanwhile,
the sulfur 2p and selenium 3p electrons in OHSeP with the selenenylsulfide
linkage (Figure D)
provided distinctive absorption peaks at 164–169 and 157–164
eV. The two absorption peaks from OHSeP were separated into the sulfur
2p and selenium 3p components. The peak envelope of the sulfur 2p
and selenium 3p components from OHSeP (orange dotted line in Figure D) was mostly identical
to the observed spectrum (black solid lines in Figure D). In addition, the values for the binding
energy of the selenium 3p components of OHSeP (160.2 and 166.1 eV)
were nearly equal to those of SeCyst (159.8 and 165.3 eV). The values
for the binding energy of the sulfur 2p components of OHSeP (162.6
and 163.8 eV) were also close to those of Cyst and OHSP (161.9 and
163.0 eV) (Table ).
Therefore, the sulfur and selenium atoms in the OHSeP molecule were
thought to form the selenenylsulfide linkage and to mostly exist in
the oxidation state (number) of −1. A similar trend was also
observed in the XPS of BSeP.
Figure 4
X-ray photoelectron spectroscopy binding energies
of the selenium
3p and sulfur 2p electrons in Cyst (A), SeCyst (B), OHSP (C), and
OHSeP (D). Black solid line: found, brown solid line: selenium 3p3/2, green solid line: selenium 3p1/2, purple solid
line: sulfur 2p3/2, blue solid line: sulfur 2p1/2, orange dotted line: envelope.
Table 1
X-ray Photoelectron Spectroscopy Binding
Energies of Selenium 3p and Sulfur 2p Electrons in l-Cystine,
Seleno-l-Cystine, OHSP, and OHSeP
binding
energy (eV)
atom level
l-cystine
seleno-l-cystine
OHSP
OHSeP
selenium 3p3/2
none
159.8
none
160.2
selenium 3p1/2
none
165.3
none
166.1
sulfur 2p3/2
161.4
none
161.9
162.6
sulfur 2p1/2
162.4
none
163.0
163.8
X-ray photoelectron spectroscopy binding energies
of the selenium
3p and sulfur 2p electrons in Cyst (A), SeCyst (B), OHSP (C), and
OHSeP (D). Black solid line: found, brown solid line: selenium 3p3/2, green solid line: selenium 3p1/2, purple solid
line: sulfur 2p3/2, blue solid line: sulfur 2p1/2, orange dotted line: envelope.Small unilamellar liposomes
were prepared from the mixture of OHSeP
and egg yolk PC (hydrogenated) by the conventional Bangham method.[33] The particle diameters and zeta potentials of
the OHSeP/PC liposomes were determined by the dynamic light scattering
techniques (Tables and 3). The particle diameter of the PC (100)
liposome used in this study was 80.8 ± 1.0 nm. The particle diameter
of the OHSP (20)/PC (80) liposome, 85.3 ± 3.0 nm, was nearly
equal to that of the PC (100) liposome. The particle diameters of
the OHSeP/PC liposomes were slightly larger than those of the PC (100)
and OHSP (20)/PC (80) liposomes. The addition of a certain amount
of detergent, such as Triton X-100, to the liposome solutions is known
to break up the lipid bilayer structure to form the mixed micelles.
During the preparation of the liposomes, formation of the mixed micelles
was not observed even at 20 mol % of OHSeP, BSeP, and OHSP in the
mixtures with PC. Actually, remarkable decreases in the particle diameter
accompanying the addition of OHSeP, BSeP, and OHSP were also not observed
for the OHSeP (20)/PC (80) and OHSP (20)/PC (80) liposomes. The value
for the zeta potential of the PC (100) was almost zero because of
the electrically neutral head groups of PC and the BSeP/PC liposome.
The value for the zeta potential of the OHSeP/PC liposomes slightly
increased with an increase in the OHSeP content, which was seemingly
because of the head groups of the OHSeP embedded in the outer leaflet
of the PC bilayer membrane. A similar trend in the zeta potential
was also observed from the PC-free OHSeP solution and the OHSP/PC
liposome.
Table 2
Particle Diameter and Polydispersity
Index of Liposomes
itema
particle diameterb (nm)
polydispersity index
OHSeP (05)/PC (95)
113.7 ± 3.9
0.200
OHSeP (10)/PC (90)
114.4 ± 3.5
0.390
OHSeP (20)/PC (80)
102.2 ± 1.9
0.356
BSeP (20)/PC (80)
80.5 ± 1.2
0.190
OHSP (20)/PC (80)
85.3 ± 3.0
0.195
PC (100)
80.8 ± 1.0
0.205
OHSeP solution
173.5 ± 2.5
0.433
Values in parentheses
represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.
Mean ± standard deviation.
Table 3
Zeta Potential of
OHSeP/PC Liposomes
itema
zeta potentialb (mV)
OHSeP (05)/PC (95)
–3.51 ± 1.03
OHSeP (10)/PC (90)
–6.77 ± 2.81
OHSeP (20)/PC (80)
–12.40 ± 0.27
BSeP (20)/PC (80)
–2.25 ± 0.95
OHSP (20)/PC (80)
–10.20 ± 0.51
PC (100)
–1.56 ± 0.18
OHSeP solution
–12.00 ± 2.76
Values in parentheses represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.
Mean ± standard deviation.
Values in parentheses
represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.Mean ± standard deviation.Values in parentheses represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.Mean ± standard deviation.The OHSeP and PC composition
of the OHSeP/PC liposomes was actually
estimated from the determination of the selenium and phosphorus concentrations
in the liposome solutions (Table ). The actual lipid compositions of the OHSeP/PC liposomes
were almost in good agreement with those used in the preparation of
the respective liposomes, which demonstrated that OHSeP molecules
were mostly incorporated into the liposomal membrane structure.
Table 4
Lipid Composition Analysis of OHSeP/PC
Liposomes
compositionb (mol %)
itema
OHSeP or BSeP
PC
OHSeP (05)/PC (95)
5.6 ± 1.0
94.4 ± 0.8
OHSeP (10)/PC (90)
10.1 ± 1.2
89.9 ± 2.1
OHSeP (20)/PC (80)
22.1 ± 0.8
77.9 ± 3.8
BSeP (20)/PC (80)
87.1 ± 3.8
12.9 ± 1.3
PC (100)
0.0
100.0
Values in parentheses
represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.
Summation of OHSeP or BSeP
and PC
contents was defined as 100 mol %.
Values in parentheses
represent
mol % of OHSeP, BSeP, OHSP, and PC used in the preparation of liposomes.Summation of OHSeP or BSeP
and PC
contents was defined as 100 mol %.The orientation of OHSeP molecules in the liposomal
membranes is
a key factor affecting the expression of the GPx-like catalytic activity.
When the selenenylsulfide linkage of the OHSeP molecule was buried
in the hydrophobic interior of the liposomal membranes, the GPx-like
catalytic activity would hardly be found because the functional element
of OHSeP could not be accessible to the substrate peroxide species
and the coenzyme GSH. Liposomes were prepared from a mixture of BSeP
and PC (described as “BSeP/PC liposome” in tables and
figures), and the lipid composition of the obtained liposomes was
analyzed. If BSeP molecules could penetrate into the PC liposomal
membranes, the analytical results of the lipid composition of the
obtained liposomes were supposed to be nearly the same as those used
in the preparation of the liposomes and in the case of the OHSeP/PC
liposomes.
When the BSeP/PC liposomes were attempted to prepare at a 20:80 molar
ratio of BSeP to PC, the molar ratio of BSeP to PC in the obtained
liposome solution was estimated as 87.1 ± 3.8:12.9 ± 1.3
from the results of the selenium and phosphorus analyses. BSeP in
the obtained liposome solution was thought to be mostly free, not
incorporated into the liposomal membranes, that is, the Pen moiety
of BSeP was not thought to penetrate toward the hydrophobic interior
of the PC liposomal membranes. The obtained particle diameter of the
BSeP/PC liposomes was 80.5 ± 1.2 nm, which was almost identical
to that of the PC (100) liposome (Table ). From these results, BSeP bearing a short
acyl chain was thought to be much less preferable to the liposomal
membranes than OHSeP. Taken these results together, the Pen moiety
of the OHSeP molecule incorporated into the OHSeP/PC liposomes was
thought to orient toward the inner and outer water phases. The OHSP
molecules in the OHSP/PC liposomes were also thought to take a similar
orientation in the PC-bilayer membrane structure.The GPx-like
catalytic activity of the OHSeP/PC liposomes was further
evaluated by the NADPH method using H2O2 as
a substrate (Figure ). Both the PC (100) and the OHSP (20)/PC (80) liposomes did not
show any GPx-like catalytic activity. The BSeP (20)/PC (80) liposomes
showed a quite low value for the GPx-like catalytic activity, which
is thought to result from the free BSeP molecules in this liposome
solution. A PC-free OHSeP solution gave an even lower value. To improve
the solubility (dispersity) of OHSeP molecules, Triton X-100 and sodium
dodecyl sulfate at above respective critical micelle concentrations
and albumin were added to this PC-free OHSeP solution; however, no
remarkable changes in the values of its catalytic activity were observed.
On the other hand, the OHSeP/PC liposomes generated the GPx-like catalytic
activity, which is apparently ascribed to the SeCys moiety that was
introduced in PC-based liposomes. Such an activity of the OHSeP/PC
liposomes was higher than that of a solution of H-SeCyst-OMe with diselenide as a functional element. The observed
activity of the OHSeP/PC liposomes was much higher than that of ebselen,
a standard for comparing the GPx-like catalytic activity of selenium
compounds, for H2O2 (0.99 μmol Se–1 min–1)[34] and that of H-SeCyst-OMe for H2O2 (0.51 μmol Se–1 min–1).[34] The three OHSeP/PC liposomes with
different OHSeP contents (5, 10, and 20 mol %) demonstrated similar
values for the GPx-like catalytic activity. The OHSeP molecules in
the OHSeP/PC liposomes were thought to disperse favorably in the liposomal
membranes up to 20 mol % without forming structurally isolated aggregate.
The structures of the liposomal membrane also appear to be one of
the factors governing the GPx-like catalytic activity of the OHSeP
molecule. Thus, in addition to PC, other phospholipids that possess
various polar head groups may be effective for further improvement
of the GPx-like catalytic activity.
Figure 5
GPx-like catalytic activity of OHSeP/PC
liposomes for H2O2. n.d.: not detected.
GPx-like catalytic activity of OHSeP/PC
liposomes for H2O2. n.d.: not detected.In conclusion, we synthesized
a lipid membrane-compatible selenenylsulfide
derivative, OHSeP. SeCyst-bearing diselenide was substituted with
Pen to form the selenenylsulfide linkage. Then, the selenenylsulfide
(SeCys–Se–S–Pen) was conjugated with the lipid
membrane-compatible n-hexadecanoyl chain through
the α-amino group of the SeCys in OHSeP. The use of OHSeP easily
allowed the introduction of the SeCys moiety into the liposomal membranes
by mixing with phospholipids, which gave rise to the GPx-like catalytic
activity because of the selenium atom in the SeCys moiety. Overall,
the lipid/water interface of the liposomal membranes could possibly
provide an effective colloidal platform for the development of water-soluble
nanosized GPx mimics. Hence, our procedure using the selenenylsulfide
derivative would be extendable to the design of novel nanosized materials
with an antioxidant GPx-like catalytic activity. We will apply these
vesicular assemblies to medicine as a nanoparticulate antioxidant
material for the treatment of oxidative stress-related diseases.
Authors: Levi J Yant; Qitao Ran; Lin Rao; Holly Van Remmen; Toru Shibatani; Jason G Belter; Lucia Motta; Arlan Richardson; Tomas A Prolla Journal: Free Radic Biol Med Date: 2003-02-15 Impact factor: 7.376
Figure 1
Figure 2
Scheme 1
Figure 3
Figure 4
Figure 5