Photochemical Bionanoreactor for Efficient Visible-Light-Driven in Vitro Drug Metabolism

Article abstract of DOI:10.1021/acs.analchem.7b00677

In light of the significance of cytochrome P450 (CYP) catalyzed drug metabolism for drug development and toxicity screening, it is very important to imitate natural metabolic pathways accurately and efficiently in vitro. Herein, a novel and simple photochemical bionanoreactor has been constructed for efficient visible-light-driven in vitro drug metabolism based on eosin-Y-functionalized macroporous ordered silica foams (MOSF-EY). Because of the unique transfer of photoinduced electrons from photosensitizers to CYP heme domain, CYP catalyzed drug metabolism can be in vitro driven by the MOSF-EY nanoreactor under the irradiation of visible light. In such a case, the utilization of expensive electron donors, such as NADPH, can be avoided. Meanwhile, the in vitro drug metabolism approach exhibits high efficiency because of the fast adsorption of both CYP and drug molecules from the bulk solution into the nanopores of MOSF-EY, where the enzyme and substrate are highly concentrated and confined in nanospace to achieve a high reaction rate. Taking advantage of these attractive merits, the first example of photochemical bionanoreactor has been successfully applied in in vitro metabolism of both purified drug molecules and real tablets. Not only excellent CYP-catalyzed drug metabolism but also enzyme inhibition assay has been performed with the MOSF-EY photochemical bionanoreactor.

Full text of DOI:10.1021/acs.analchem.7b00677

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Article
Photochemical Bionanoreactor for Efficient
Visible-Light-Driven in-vitro Drug Metabolism
Xiaoni Fang, Yaokai Duan, Yujie Liu, Gary Adkins, Weijun
Zang, Wenwan Zhong, Liang Qiao, and Baohong Liu
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017
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Analytical Chemistry
Photochemical Bionanoreactor for Efficient Visible-Light-
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Driven in-vitro Drug Metabolism
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Xiaoni Fang, Yaokai Duan, Yujie Liu, Gary Adkins, Weijun Zang, Wenwan Zhong, Liang Qiao, Baohong Liu
1
. Department of Chemistry, Institute of Biomedical Sciences and State Key Lab of Molecular Engineering of Poly-
mers, Fudan University, Shanghai 200433, China
. Shanghai Stomatological Hospital, Fudan University, Shanghai 200433, China
. Department of Chemistry, University of California, Riverside 92501, United States
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ABSTRACT: In light of the significance of cytochrome P450 (CYP) catalyzed drug metabolism forꢀdrug development and
toxicity screening, it is very important to in-vitro imitate natural metabolic pathways accurately and efficiently. Herein, a
novel and simple photochemical bionanoreactor has been constructed for efficient visible-light-driven in-vitro drug me-
tabolism based on eosin Y functionalized macroporous ordered silica foams (MOSF-EY). Due to the unique transfer of
photo-induced electrons from photosensitizers to CYP heme domain, CYP catalyzed drug metabolism can be in-vitro
driven by the MOSF-EY nanoreactor under the irradiation of visible light. In such a case, the utilization of expensive elec-
tron donors, such as NADPH, can be avoided. Meanwhile, the in-vitro drug metabolism approach exhibits high efficiency
due to the fast adsorption of both CYP and drug molecules from the bulk solution into the nanopores of MOSF-EY, where
the enzyme and substrate are highly concentrated and confined in nanospace to achieve a high reaction rate. Taking ad-
vantage of these attractive merits, the first example of photochemical bionanoreactor has been successfully applied in in-
vitro metabolism of both purified drug molecules and real tablets. Not only excellent CYP catalyzed drug metabolism, but
also enzyme inhibition assay has been performed with the MOSF-EY photochemical bionanoreactor.
As one of the major superfamily of multifunctional
monooxygenases, cytochrome P450 (CYP) is involved in a
variety of metabolic reactions of endogenous and exoge-
nous compounds in living organisms. Since CYP in-
volves in multi component electron transfer chains, effi-
cient and continuous supplementation of electrons to
CYP is required to maintain the catalytic cycle of CYP
during drug metabolism. The cycle features the delivery
of electrons from NADPH to cytochrome P450 reductase
Therefore, cumbersome procedures, such as the modifica-
tion of electrodes, are inevitable. Among all the alterna-
tive strategies, light driven CYP-mediated drug metabo-
lism has drawn significant attention during the last few
years. For instance, quantum dots, eosin Y and Ru(II)
photosensitizer have been used as electron suppliers for
1
-5
6, 20-22
drug metabolism.
However, the reported light-
driven drug metabolism methods exhibit slow kinetics in
bulk solution reaction, leading to inefficient metabolism
and identification of metabolites at low concentrations of
CYP or drugs. The aforementioned shortcomings should
be overcome in terms of the urgent need of highly effi-
cient in-vitro CYP catalyzed drug metabolism approaches.
(
CPR) and subsequently to CYP heme. Practical applica-
tion of CYP in in-vitro drug metabolism is still a challenge
because of the inherent low stability and activity of the
enzyme, as well as the dependence on expensive NADPH
6-9
and redox partners. However, in-vitro CYP reactions are
Nanoreactors have attracted great attentions and of-
23-27
of great importance for drug development and toxicity
fered distinctive advantages in enzymatic catalysis.
For
10, 11
screening.
To date, the average costs of developing a
example, Liu et al. have developed electrochemistry driv-
new drug is ~2.5 billion US dollar, and > 90% of clinical
en TiO nanotube arrays for the study of enzyme bio-
catalysis and drug metabolism. Recently, mesoporous
2
12-14
31
trials of drug candidates are failed.
Therefore, the con-
struction of a novel platform to in-vitro imitate the natu-
ral metabolic pathways accurately and efficiently at low
cost is urgently demanded.
materials, such as macroporous ordered silica foams
(MOSF), have been reported as excellent candidates of
28-30
enzymatic nanoreactors.
Benefiting from their rela-
Great efforts have been made to imitate in-vitro CYP
tively large pore sizes and excellent biocompability, mes-
oporous materials have been employed to pre-immobilize
high concentration of enzymes and substrates within the
catalyzed drug metabolism, e.g. by replacing expensive
15, 16
NADPH with NADPH recycling systems,
using elec-
17-19
trochemistry methods,
or employing light-activated
nanopores and confine enzymatic reactions in nanoscale
6
23,28
domains.
approaches. The use of NADPH recycling systems is of
scientific interests, but suffers from the low tolerance of
monooxygenases toward peroxides, which results in rapid
inactivation of the monooxygenases. Electrochemically-
driven CYP catalyzed drug metabolism relies on the direct
electron transfer between CYP and electrode, which has
turned out to be a cost-effective method. However, it is
difficult to achieve the direct electron transfer to the
deeply buried iron-heme centers of CYP. In addition, the
stability and activity of CYP are limited on bare electrodes.
With theoretical predictions and experi-
mental validations, we have previously demonstrated that
mesoporous materials can offer distinctive advantages in
hosting CYP catalyzed drug metabolism, including fast
reaction kinetics, minimal consumption of multiple en-
zymes, good stability, and rapid separation of products
24
from metabolic reaction systems. However, mesoporous
materials have not been extensively exploited for light-
driven CYP catalyzed drug metabolism. It’s then desirable
to design a photochemical nanoreactor, which can realize
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NADPH- and reductase-free, light-driven, and highly effi- drothermally treated at 100 °C for another 24 h after keep-
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cient CYP catalyzed in-vitro drug metabolism.
ing static for 24 h. The resulting white precipitates were
filtered, washed thoroughly by water to remove any salts,
and then dried at room temperature. The final MOSF
products were obtained after baking the white precipi-
tates at 550 °C for 5 h.
Herein, a novel and simple photochemical bionanore-
actor has been constructed by introducing eosin Y (EY)
modified macroporous ordered silica foams (MOSF-EY)
as hosts for visible-light-driven and CYP catalyzed drug
metabolism. By introducing the remarkable photosensi-
tizer EY in the macroporous frameworks of MOSF,
MOSF-EY not only serves as an electron supplier, but also
a bionanoreactor for drug metabolism. The bionanoreac-
tor, in fact, offers abundant electrons for metabolic reac-
tion and facilitates their access to CYP’s heme domain.
Therefore, the efficiency of in-vitro drug metabolism can
be significantly enhanced. As a proof-of-concept of the
approach, purified drug molecules and real tablets were
chosen as substrates. Both faster reaction kinetics and
higher conversion ratio were achieved compared to tradi-
tional in-vitro drug metabolism system by NADPH. Fur-
thermore, enzyme inhibition assay has also been realized
with the MOSF-EY photochemical bionanoreactor. All the
results demonstrate that such a photochemical bioreactor
can offer a clinically relevant approach with potential ap-
plication in efficient drug candidates screening and
thereby the development of new drugs.
MOSF was functionalized with amino groups. In a
standard modification process, APTES was used as cou-
3
3
pling agent. MOSF materials were firstly dried and de-
gassed at 110 °C, and then dispersed in dry toluene (0.1 g
MOSF in 30 g toluene). An excess of APTES (3 mL) was
added under stirring and the mixture was stirred and re-
fluxed for 24 h at 110 °C. The resulting solid was filtered
and washed sequentially by toluene, dichloromethane
and ethanol three times, respectively. The final NH2-
MOSF products were obtained after through drying at
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70 °C overnight. Finally, 10 μg EY and 20 μg NH -MOSF
2
were added in PB buffer with a final volume of 100 μL and
stirred for 10 min at room temperature, followed by cen-
trifugation and washing by deionized water thoroughly to
obtain the MOSF-EY materials.
Scanning electron microscope (SEM) images were ob-
tained by SUPERSCAN SSX-550 scanning electron micro-
scope (Shimadzu, Japan). Transmission electron micros-
copy (TEM) images were obtained with a JEOL 2011 mi-
croscope operated at 200 kV (JEOL, Tokyo, Japan). Sam-
ples for TEM measurements were suspended in ethanol
and supported on a carbon-coated copper grid. The infra-
red spectra were obtained using an FT-IR360 (Nicolet,
Thermo Scientific). Cyclic voltammetric experiments were
performed with a CHI 660C electrochemical analyzer (CH
Instruments) with a conventional three-electrode config-
uration, where modified glassy carbon electrode (GCE)
with the diameter of 3 mm was employed as working elec-
trode, Pt wire served as counter electrode, and a saturated
calomel electrode (SCE) was used as reference electrode.
A green lamp was used as the irradiation source to initiate
CYP catalyzed drug metabolism (λ: 534 ± 5 nm, P: <80
mW, Φ: 22Χ90 mm).
EXPERIMENTAL SECTION
Chemicals. Cytochrome P450 CYP3A4 expressed in E.
coli (human, recombinant), cytochrome P450 CYP3A4
isozyme microsomes expressed in baculovirus-infected
insect cells (human, recombinant) with cytochrome P450
reductase and cytochromeb5, cytochrome P450 reductase
(
human, recombinant) expressed in baculovirus infected
insect cells, reduced nicotinamide adenine dinucleotide
phosphate (NADPH), nifedipine (≥98%, powder), caf-
feine (≥99%, powder), tetramethyl orthosilicate (TMOS,
≥
99%), EO20PO70EO20
(
P123, where EO is
poly(ethylene oxide) and PO is poly(propylene oxide), dry
toluene, (3-aminopropyl) triethoxysilane (APTES), eosin Y
(≥90%) were purchased from Sigma Chemical Co. (St.
Visible-Light-Driven CYP Catalyzed Drug Metabo-
lism by the MOSF-EY Photochemical Bioreactor. The
visible-light-driven metabolism by the MOSF-EY bi-
onanoreactor was conducted in a microcentrifuge tube
Louis, MO, USA). Methanol, acetonitrile, and methylene
chloride were purchased from Dikma Technologies Inc.
(
Lake Forest, CA). Testosterone (≥98%), ketoconazole (
98%), sodium sulphate anhydrous, sodium acetate an-
≥
o
(1.5 mL) at 37 C. Quantitative reaction mixture consisted
hydrous, acetic acid were obtained from Sinopharm
Chemical Reagent Co. (Shanghai, China). The stock solu-
tions of drugs and caffeine were prepared in methanol.
NADPH was prepared in potassium phosphate (PB) buff-
er, 0.1 M, pH = 8.0. All reagents were used as received
without further purification. Deionized water (18.2 MΩ
cm) used for all experiments was obtained from a Milli-Q
system (Millipore, Bedford, MA).
of CYP (5 μL), MOSF-EY (20 μg), and nifedipine (4 μg) or
testosterone (2 μg) in 20 μL of potassium phosphate (PB,
0.1 M)-TEOA (0.1 M) buffer (pH ≈8.0). It is noteworthy
that TEOA serves as an electron donor in the light driven
CYP catalyzed drug metabolism. For metabolic reaction,
o
the mixture was pre-incubated at 37 C for 5 min for the
adsorption of substrates. Green lamp was employed to
initiate the oxidation reaction. After being incubated at 37
Synthesis and Characterization of MOSF-EY Mate-
rials. MOSF materials were synthesized according to a
o
C for a certain period, the reaction was quenched by the
32
addition of ice−methylene chloride. At the same time, the
drug molecules with their metabolites were extracted by
methylene chloride, the detailed process was introduced
in Supporting Information. At last, methylene chloride
previously reported method. Briefly, 1.00 g of P123 and
1
.7 g of Na2SO4 were dissolved in 30 mL of NaAc-HAc
buffer solution (0.02 M, pH=5.0) to form a homogenous
solution under stirring at 35 °C. And then 1.52 g of TMOS
was added to the solution under stirring. After 5 minutes,
the stirring was stopped. The resultant mixture was hy-
was dried under a N stream without heating, and the
2
dried samples were redissolved in methanol for further
2
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EY. These results gave strong evidence about the success-
analysis. We also carried out the same reaction for 10 min
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without EY, TEOA, or green light as control experiments.
When the metabolism of nifedipine sustained-release
tablets was studied, a similar method was used. 5 μL CYP,
ful covalent binding of EY to the amino modified MOSF.
20 μg MOSF-EY materials, and 10 μg nifedipine sustained-
release tablets were added in 20 μL of potassium phos-
phate (PB, 0.1 M)-TEOA (0.1 M) buffer (pH ≈8.0) and
then incubated at 37 °C for a certain period. Before
HPLC−MS analysis, the extracted samples were centri-
fuged to remove precipitates thoroughly.
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In-vitro metabolism of nifedipine assisted by NADPH
3
4
way was also performed. Briefly, 4 μg of nifedipine was
dissolved in PB buffer (0.1 M). After the addition of CYP (5
μL) and cytochrome P450 reductase (5 μL) of microsomes
(
10 μL), the mixture was pre-incubated at 37 °C for 5 min.
Then 60 μg of NADPH was added to the solution to initi-
ate the oxidation reaction for 10 minutes. Finally, the me-
tabolites were extracted, redispersed, and analyzed by
HPLC−MS.
Chemical Inhibition Study. Chemical inhibition stud-
ies were performed by adding ketoconazole to the incuba-
tion mixture of nifedipine or testosterone before the irra-
diation of green light.
LC−MS Analysis. For rapid screening of reactive me-
tabolites, the reaction mixture was analyzed by HPLC−MS.
Samples were first subjected to chromatographic separa-
tion with an Agilent series 1260 HPLC system (Agilent
Technologies, Palo Alto, CA) integrated with an au-
tosampler and a UV detector. 20 μL of sample solution
was injected into a VP-ODS C18 column (250 mm × 4.6
mm i.d., 5μm, Agilent) at the flow rate of 0.6 mL/min.
The detailed separation process is explained in the Sup-
porting Information. The fractions from LC were intro-
duced into a 6460 QQQ mass spectrometer (Agilent) that
was operated in positive ion mode under optimized pa-
rameter conditions (refer to the Supporting Information).
The structure information on reactive metabolites can be
deduced from tandem MS spectra. Additionally, for quan-
titative analysis of the metabolites, 20 μL of caffeine (0.1
μg/μL) was added as an internal standard before HPLC-
MS analysis.
Figure 1. FTIR spectra of (a) MOSF, (b) NH -MOSF and (c)
2
MOSF-EY.
The morphology and structure of MOSF-EY were fur-
ther validated by SEM and TEM. Figure 2a&b display the
SEM images of MOSF-EY. Compared to MOSF (Figure S1),
the foam-like structure can be well-preserved after sur-
face modification with EY, and the pore size is still ∼100
nm. The TEM images also reveal the foam structure of
MOSF-EY with hexagonal column assemblies (Figure 2c).
From both SEM and TEM, no large aggregates were ob-
served for MOSF-EY, suggesting the well-dispersed state
of EY species. As shown in Table S1, MOSF has a surface
2
3
area of 351 m /g and pore volume of 1.25 cm /g, whereas
the surface area and pore volume of MOSF-EY decrease to
2
3
2
98 m /g and 1.09 cm /g, respectively, which can be at-
tributed to the impregnation of EY on the surface of
MOSF. The ζ potentials of MOSF and NH -MOSF were
2
measured as −35.6 and +17.5 mV in 0.1 M PB buffer (pH ≈
8.0), respectively, indicating the successful modification
of MOSF by APTES. Thus, NH -MOSF was positively
2
charged that can be served as an adsorbent for anionic EY
through the charge interaction between NH -MOSF and
2
EY. After the modification of EY on NH -MOSF, the ζ
2
potential of MOSF-EY decreased to –5.1 mV due to the
modification of the negatively charged EY on the surface
RESULTS AND DISCUSSION
of NH -MOSF, further demonstrating the structure of
2
Construction and optimization of the EY-MOSF
photochemical bionanoreactor. The EY-MOSF bi-
onanoreactor was constructed and optimized for efficient
visible-light-driven CYP catalyzed in-vitro drug metabo-
lism. The chemical modification of MOSF with EY
(MOSF/EY ratio of 10:1 (w/w)) was validated by Fourier
transform infrared (FT-IR) spectroscopy. In Figure 1a, the
MOSF-EY.
−
1
absorption peak of MOSF at 1083 cm is ascribed to the
stretching vibration of −Si−O. In the case of amino modi-
fied MOSF (Figure 1b), the peaks around 1568 and 2931
–
1
cm can be attributed to the grafted -NH2 groups and -
CH - groups of silica coupling agent (APTS), respectively.
2
After modification with EY (Figure 1c), new bands at 1456
−
1
−1
cm and 1557 cm correspond to aromatic C=C stretching
−
1
of EY. In addition, FT-IR bands at 1351 and 1616 cm are
assigned to aliphatic C–O stretching and N-H bending of
Figure 2. (a, b) SEM and (c) TEM images of MOSF-EY.
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Scheme 1. Schematic illustration of the MOSF-EY photochemical bionanoreactor assisted light-driven efficient drug me-
tabolism.
Considering that the absorbance of MOSF-EY materials
to visible light is relevant to the amount of EY, a series of
MOSF-EY materials were fabricated by varying the
amount of EY in order to construct a photochemical bio-
reactor with excellent performance in CYP catalyzed drug
metabolism. The amount of EY ranging from 0 to 200 μg
was used to modify MOSF (1 mg). As shown in Figure S2,
the maximum absorbance of MOSF-EY to light with the
wavelength from 520 to 600 nm was observed when the
EY amount was 100 μg/ mg MOSF. Further increase of the
amount of EY could not lead to a better light harvesting.
Therefore, the MOSF-EY material with a final MOSF/EY
ratio of 10:1 (w/w) was applied to assist visible-light-
driven CYP catalyzed in-vitro drug metabolism.
change from red to yellow of MOSF-EY was observed
after the addition of CYP, corresponding to the red-shift
of the absorption peak of MOSF-EY at 537 nm, Figure 3a.
Benefiting from the interaction between MOSF-EY and
CYP and the outstanding absorbance of MOSF-EY to visi-
ble light, the reduction potential of CYP shifted signifi-
cantly from −0.68 V to −0.77 V (Figure 3b) under light
irradiation. The negative shift is a result of the co-
EY-MOSF as a photochemical bionanoreactor for
CYP catalyzed drug metabolism. As illustrated in
Scheme 1, MOSF-EY serves not only as an electron suppli-
er but also a bionanoreactor for highly efficient CYP cata-
lyzed drug metabolism. The process is a series of reac-
tions confined in nanospaces, including: (i) enrichment of
drug and CYP in the nanopores of MOSF-EY; (ii) excita-
tion of electrons from the highest occupied molecular
orbital (HOMO) to the lowest unoccupied molecular or-
bital (LUMO) of MOSF-EY under the irradiation of visible
light. MOSF-EY will obtain photons and produce single
excited state MOSF-EY1*, and subsequently triplet excited
state MOSF-EY3* are produced during the process; (iii)
transfer of the photoexcited electrons from MOSF-EY
LUMO to CYP heme to induce CYP catalyzed drug me-
tabolism; (iv) reduction of the oxidized MOSF-EY to its
ground state by triethanolamine (TEOA).
The interaction between MOSF-EY and CYP is essen-
tial for the electron transfer reactions during the light-
driven drug metabolism. Spectrophotometry and cyclic
voltammetry were performed to investigate the electron
transfer process between MOSF-EY and CYP heme. Sig-
nificant red-shift in spectrophotometry could be observed
Figure 3. (a) Spectrophotometric analysis of the interac-
tion between MOSF-EY and CYP. (b) Cyclic voltammetric
measurement of CYP modified electrode in the absence
and presence of MOSF-EY. SCE: saturated calomel elec-
trode.
3
5
when EY is bound to proteins. As expected, the color
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Figure 4. Chromatograms of the metabolic reaction products of (a) nifedipine and (b) testosterone from 10 min of CYP
catalyzed metabolism under various experimental conditions as indicated on the figures. Mass spectra of (c) nifedipine
and (d) testosterone metabolism with the assistance of MOSF-EY photochemical bionanoreactor. Tandem MS by CID of
(
e) the peak at m/z=345 on Figure 4(c), and (f) the peak at m/z=305 on Figure 4(d).
reduction of EY and CYP, confirming the presence of elec-
tron transfer from MOSF-EY to CYP. During the EC
measurement, CYP was immobilized on the electrode.
Therefore, only EY on the surface of MOSF may react with
the immobilized CYP. Nevertheless, the reaction between
CYP and EY under light irradiation was demonstrated.
Thus, MOSF-EY can be used as an excellent photochemi-
cal bionanoreactor towards CYP catalyzed drug metabo-
lism.
Metabolism of nifedipine and testosterone as-
sisted by the EY-MOSF photochemical bionanoreac-
tor. To demonstrate the feasibility of the proposed proto-
col, the MOSF-EY photochemical bionanoreactor was
used to assist the metabolism of nifedipine and testos-
terone catalyzed by CYP. For direct comparison, metabol-
ic reaction in bulk solution was carried out with the same
amount of CYP and substrates. The chromatography in
Figure 4 shows the results of nifedipine and testosterone
metabolism with and without the assistance of MOSF-EY
under different conditions. As illustrated on Figure 4a,
the peaks with retention time (RT) at 17 and 27 min can
be attributed to dehydronifedipine and nifedipine, re-
spectively. For testosterone, the major metabolite was
observed at 18 min. No metabolite of nifedipine or testos-
terone can be detected in the absence of EY, TEOA or
visible-light, indicating that the electron pairs and light
source are indispensable for light-driven CYP catalyzed
drug metabolism. Moreover, CYP catalyzed drug metabo-
lism can be performed more efficiently with the MOSF-EY
photochemical bionanoreactor. The amounts of generat-
ed metabolites were much larger than that obtained after
10 min of in-bulk-solution metabolism by EY, TEOA and
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visible-light irradiation. Figure 4c&d show the corre- As demonstrated in Figure S4 (Supporting Information),
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sponding mass spectra of nifedipine, testosterone and
their metabolic reaction products obtained by the MOSF-
EY photochemical bioreactor. The structure of the meta-
bolic products of nifedipine and testosterone were con-
firmed by tandem MS (Figure 4e&f). Both the molecular
weight and fragments indicated the generation of dehy-
dronifedipine and 6β-hydrotestosterone.
The reaction kinetics of drug metabolism assisted by
MOSF-EY is mainly dependent on the amount of immobi-
lized CYP, affinity between enzyme and substrate, the
pore size of MOSF-EY, and the enzymatic activity. Kinetic
data were obtained using the Michaelis-Menten model.
The detailed calculations of KM & Vmax values were illus-
trated in Supporting Information. Benefiting from the
large pore size and negatively charged surface, MOSF-EY
photochemical bionanoreactor shows highly fast adsorp-
tion kinetics and high loading capacity to the positively
charged CYP in potassium phosphate (PB) buffer, pH =8.0.
about 88% of the maximum adsorption amount of CYP
could be achieved within 2 min. The introduction of the
bionanoreactor in CYP catalyzed drug metabolism not
only dramatically in-creases the local concentration of
CYP, but also facilitates the access of substrates to CYP.
As shown in Figure 5 and Table S2 (Supporting Infor-
mation), the Km for nifedipine and testosterone metabo-
lism are 5.06 ± 0.45 μM and 53.37 ± 4.01 μM, respectively,
which are much lower than previously reported methods,
indicating that the immobilized CYP has good affinity to
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substrates.
Moreover, the apparent Vmax values for
nifedipine and testosterone metabolism assisted by the
MOSF-EY photochemical bionanoreactor are 47.09 ± 1.10
and 74.15 ± 5.92 nmol/min/nmol CYP, respectively, much
larger than those described in previous reported ap-
3
6,41
proaches.
The large Vmax values in our photochemical
system come from the enhanced activity of enzymes in
confined nanospace, demonstrating that the MOSF-EY
bionanoreactor can provide a suitable microenvironment
to maintain the stability and catalytic activity of enzymes.
All of these results indicate that CYP catalyzed drug me-
tabolism assisted by MOSF-EY is highly advantageous in
enhancing metabolic reaction kinetics.
To further confirm the efficiency of the MOSF-EY pho-
tochemical bionanoreactor in CYP catalyzed drug metab-
olism, the absolute amount of metabolites of nifedipine
and testosterone were determined and compared to other
in-vitro drug metabolism methods. As shown in Table 1, a
total of 48.60 ± 3.97 nmol dehydronifedipine and 26.09 ±
4.31 nmol 6β-dehydroxytestosterone were obtained based
on the MOSF-EY photochemical bioreactor, which are
much more than traditional in-bulk-solution metabolism
of the same amount of substrates relying on the con-
sumption of expensive NADPH and reductase. Even when
microsomes containing CYP and CPR were used to drive
the metabolism of nifedipine and testosterone in solution,
the amount of generated metabolites was still much less
than the MOSF-EY approach. Such a difference in drug
metabolism is likely due to the improved efficiency of
electron transfer in the MOSF-EY system compared to
bulk solution reactions, and the nanoconfinement effect
of enzymatic reaction in nanospaces. The improvement of
absolute amount of metabolites plays a key role in drug
screening, especially for complex and trace drugs. Such
advances will contribute to the development of drug with
desired safety and efficiency.
Figure 5. Michaelis-Menten plots for CYP catalyzed (a)
nifedipine (0-200 μM) and (b) testosterone (0-500 μM)
metabolism assisted by the MOSF-EY photochemical bi-
onanoreactor. Metabolic reactions were performed with 5
μL CYP in 0.1 mL potassium phosphate (PB, 0.1 M)-TEOA
Encouraged by the prominent features of the proposed
protocol, the MOSF-EY photochemical bionanoreactor
was further applied to the in-vitro metabolism of nifedi-
pine sustained release tablets. The UV−HPLC chromate-
(
0.1 M) buffer for 10 min. Error bar shows the standard
deviation (n=3).
Table 1. Summary of the Absolute Amount of Metabolites Obtained by Different Strategy (n=3)
metabolism strategy
MOSF-EY nanoreactor/CYP4A4/hv
EY/CYP3A4/hv
Dehydronifedipine(nmol)
48.60 ± 3.97
6β-dehydroxytestosterone (nmol)
26.09 ± 4.31
25.69 ± 5.28
21.35 ± 2.92
CYP3A4/CPR/NADPH
15.06 ± 4.26
24.02 ± 2.83
12.18 ± 3.50
18.84 ± 5.17
CYP3A4 microsome/NADPH
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Analytical Chemistry
whether the developed MOSF-EY photochemical bi-
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onanoreactor can be applied to enzyme inhibition analy-
sis, MOSF-EY assisted CYP catalyzed drug metabolism
was carried out in the presence of ketoconazole, a com-
monly used inhibitor of CYP mediated nifedipine metabo-
lism. As shown in Figure 7, there is not any dehydronife-
dipine determined after the addition of ketoconazole,
indicating that ketoconazole is still an active inhibitor of
CYP in the MOSF-EY photochemical bionanoreactor
based in-vitro metabolism system. All these superiorities
render the MOSF-EY photochemical bionanoreactor
based in-vitro metabolism as a novel clinically relevant
biotechnological approach with potential application in
efficient drug screening and the development of new
drugs.
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Figure 6. Chromatograms of 30 min nifedipine sustained-
release tablets oxidation activated by the (a) MOSF-EY
and (b) NADPH in solution system. Metabolic reactions
were performed with 5 μL CYP, 10 μg nifedipine sus-
tained-release tablets in 20 μL of potassium phosphate
CONCLUSIONS
In summary, we have demonstrated that CYP catalyzed
drug metabolism can be effectively carried out in the ab-
sence of expensive NADPH and redox partner based on
the MOSF-EY photochemical bionanoreactor under visi-
ble light illumination. MOSF-EY materials inherit the
virtues of photosensitizer and nanoreactor, thereby act as
both an electron supplier and a bionanoreactor. With the
MOSF-EY photochemical bionanoreactor, much faster in-
vitro metabolic reaction kinetics is obtained. It is antici-
pated that the novel and simple MOSF-EY photochemical
bionanoreactor for visible-light-driven drug metabolism
would lead to promising advance in new drug develop-
ment with desired safety and therapeutical efficiency.
(
PB, 0.1 M)-TEOA (0.1 M) buffer for 30 min with and
without the assistance of MOSF-EY photochemical biore-
actor.
grams in Figure 6 illustrate the results of nifedipine sus-
tained release tablets oxidation activated by NADPH and
the MOSF-EY photochemical system, respectively. More
than 2/3 nifedipine could be consumed after 30 min with
the MOSF-EY photochemical bioreactor, whereas only 1/3
nifedipine were consumed under the same condition by
the NADPH based in solution reaction system. The result
clearly indicates that MOSF-EY can be used as a simple
and efficient photochemical bioreactor for the in-vitro
metabolism of complex samples.
ASSOCIATED CONTENT
Supporting Information
Detailed descriptions of the LC−MS experiments, data for
the absorbance of MOSF-EY materials relevant to the
amount of EY, structure of MOSF, and adsorption kinetic
of CYP by MOSF-EY. This material is available free of
charge via internet at http://pubs.a.org/.
AUTHOR INFORMATION
Corresponding Author
*
*
E-mail: bhliu@fudan.edu.cn. Fax:(+86) 21-6564-1740
E-mail: liang_qiao@fudan.edu. Fax:(+86) 21-6564-1740
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Figure 7. UV-HPLC chromatograms of nifedipine metab-
olism assisted by the MOSF-EY photochemical bioreactor
(a) without and (b) with the presence of ketoconazole.
Metabolic reactions were performed with 5 μL CYP, 4 μg
nifedipine, 1 μM ketoconazole in 20 μL of potassium
phosphate (PB, 0.1 M)-TEOA (0.1 M) buffer for 30 min
with the assistance of MOSF-EY photochemical bionano-
reactor.
This work was supported by NSFC (21175028, 21375022 and
21105014). LQ would also like to thank the Thousand Tal-
ents Program and NSFC (81671849) for support.
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