Efficient drug metabolism strategy based on microsome-mesoporous organosilica nanoreactors

Article abstract of DOI:10.1021/ac503024h

A rapid and accurate in vitro drug metabolism strategy has been proposed based on the design of a biomimetic nanoreactor composed of amino-functionalized periodic mesoporous organosilica (NH₂-PMO) and microsomes. The amphiphilic nature and posi

Full text of DOI:10.1021/ac503024h

Article
pubs.acs.org/ac
Efficient Drug Metabolism Strategy Based on Microsome−
Mesoporous Organosilica Nanoreactors
Xiaoni Fang,† Peng Zhang,† Liang Qiao,‡ Xiaoyan Feng,† Xiangmin Zhang,† Hubert H. Girault,‡
and Baohong Liu*,†
†Department of Chemistry, Institutes of Biomedical Sciences, and State Key Laboratory of Molecular Engineering of Polymers, Fudan
University, Shanghai 200433, China
‡Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fed
erale de Lausanne, CH-1015 Lausanne, Switzerland
́ ́
S
* Supporting Information
ABSTRACT: A rapid and accurate in vitro drug metabolism strategy has
been proposed based on the design of a biomimetic nanoreactor composed
of amino-functionalized periodic mesoporous organosilica (NH2-PMO) and
microsomes. The amphiphilic nature and positive charge of NH2-PMO
make it highly suited for the immobilization of hydrophobic and negatively
charged microsomes to form nanoreactors, which can in turn extract
substrates from solutions. Such nanoreactors provide a suitable environ-
ment to confine multiple enzymes and substrates with high local
concentrations, as well as to maintain their catalytic activities for rapid
and highly effective drug metabolic reactions. Coupled with high-
performance liquid chromatography−mass spectrometry analysis, the
metabolites of nifedipine and testosterone were quantitatively characterized,
and the reaction kinetics was evaluated. Both the metabolism conversion
and reaction rate were significantly improved with the NH2−PMO
nanoreactors compared to bulk reactions. This strategy is simple and cost-effective for promising advances in biomimetic
metabolism study.
n modern drug development, assessment of safety always
384-well plate to form a series of parallel assays for
Iconsists of designed bioassays for toxicology and pharma- simultaneous metabolism of many drug candidates. The
automation facilitated parallel sample processing, thus enabling
high-throughput drug metabolism screening. However, as a
result of the relatively low concentration of microsomes and
drugs, the in-solution metabolic reaction, e.g., in a well of the
384-well plate, exhibited slow kinetics.
cokinetics, animal toxicity studies, and finally human clinical
trials.1 Approximately 40% of drug candidates fail at the clinical
testing stages owing to unanticipated toxicity, which drives up
drug development cost significantly.2,3 Screening of toxicity at
early stages of the drug discovery process can minimize such
failures by selecting drug candidates with acceptable levels of
both bioactivity and toxicity.4,5 In the past decade, the number
of screenable drug targets increased dramatically with the rapid
development of combinatorial chemistry and advances in
bioinformatics, genomics, and proteomics.6,7 However, the
gap between the number of screenable drug candidates and the
approved new drugs is widening. Therefore, development of
low-cost and high-throughput toxicity screening methods,
which can address early-stage toxicology and mimic human
metabolism of drug candidates to predict the likelihood of drug
candidate toxicity, is the major challenge in drug discovery.
Human liver microsomes are enriched sources of metabolic
enzymes, such as cytochrome P450 (CYP450) and uridine
diphosphoglucuronosyl transferase (UGT), which together are
responsible for nearly 80% of the metabolism of currently
marketed drugs and are therefore widely used for in vitro
metabolism and toxicity studies.8 Carlson et al.9,10 developed a
384-well plate assay system for high-throughput metabolism,
where microsomal enzymes were dispersed in each well of the
To address this issue, microsome-immobilized bioreactors
have attracted wide interest and offered distinct advantages,
including fast reaction kinetics, minimal consumption of
microsomes, good stability, long storage time, and rapid
separation of products from microsomal enzymes. Rusling et
al.11,12 designed a silica microbead bioreactor coated with DNA
and enzymes to measure reactive metabolites and DNA adduct
formation rates relevant to genotoxicity screening. Although
this bioreactor can contribute to the efficient analysis of major
and minor DNA adducts as well as the metabolites in less than
5 min of reaction time, there is still a potential to achieve higher
metabolism efficiency by simultaneously concentrating enzymes
and drugs in a nanoreactor. It is then desirable to design a
nanoreactor with a good affinity for both multiple enzymes and
multiple drugs for their enrichment, while the motion of
Received: August 14, 2014
Accepted: October 14, 2014
Published: October 14, 2014
© 2014 American Chemical Society
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molecules and the contact between enzymes and drugs is not
restricted.13−15
(TMB), dry toluene, (3-aminopropyl)triethoxysilane
(APTES), potassium chloride, and ethanol were purchased
from Sigma Chemical Co. (St. Louis, MO). Methanol,
acetonitrile, and methylene chloride were obtained from
Dikma Technologies Inc. (Lake Forest, CA). Nifedipine
sustained-release tablets were obtained from the Yangtze
River Pharmaccutical Group (Jiangsu, China). The phosphate
buffer (PB) solution (0.1 M, pH ≈ 7.4) was prepared by
KH2PO4 and K2HPO4. The stock solutions of drugs and
caffeine were prepared in methanol. NADPH was prepared in
PB solution. All reagents were used as received without further
purification. Deionized water (18.4 MΩ/cm) used for all
experiments was obtained from a Milli-Q system (Millipore,
Bedford, MA).
Synthesis and Characterization of NH2-PMO Materi-
als. PMO materials were synthesized according to a previously
reported method.31 Briefly, 0.5 g of F127, 2.5 g of KCl, and 0.5
g of TMB were dissolved in 30 g of HCl (0.1 M). After the
mixture was stirred for 6 h at 0 °C, 2.0 g of BTME was added.
The resultant mixture was hydrothermally treated at 100 °C for
another 24 h after being stirred for 24 h at room temperature.
The resulting powders were filtered and washed thoroughly by
the mixture of 60 mL of ethanol and 5 mL of HCl (2 M) at 60
°C to remove the template. The final PMO products were
obtained by filtration and dried at room temperature in air. In a
standard modification process,32 PMO materials were first dried
and degassed at 110 °C and then dispersed in dry toluene (0.2
g of PMO in 30 mL). An excess of APTES (2 mL) was added
under stirring, and the mixture was stirred and refluxed for 24 h
at 110 °C. The resulting solid was filtered and washed by
toluene, dichloromethane, and ethanol three times. The final
NH2-PMO products were obtained through drying at 70 °C.
Transmission electron microscopy (TEM) images were
directly taken with a JEOL 2011 microscope operated at 200
kV by dispersing the samples on a Cu grid with carbon films.
The infrared spectra were obtained using an FT-IR360
manufactured by Nicolet. A ζ potential meter (Malvern
Zetasizer Nano) was used to measure the ζ potentials of
material dispersed in PB solution at 298 K. A V-550 UV/vis
spectrophotometer (JASCO Corp., Japan) was used for the
quantification of nifedipine, testosterone, and NADPH to
characterize the adsorption kinetics of the substrates by NH2-
PMO.
On the basis of these considerations, mesoporous materials
are considered as potential candidates to form such a
nanoreactor. Mesoporous materials have been reported as
excellent hosts for the immobilization of proteins and enzymes
due to the highly ordered mesoporous structure and sufficient
functional groups for enzyme immobilization.16−21Additionally,
with the surface modification of mesoporous materials, selective
enrichment of proteins, such as phosphorylated protein,22
glycosidoprotein,23 and membrane proteins, can be realized.24
Furthermore, since the local environment inside the meso-
porous materials is mainly regulated by the surface
modification, fast enzymatic reactions can occur despite the
bulk buffer conditions,25,26 making it possible to perform
mesoporous-material-assisted enzymatic reaction in a buffer
condition optimized for substrate extraction instead of
enzymatic reaction itself.
It is a challenge to design a host material for the
simultaneous enrichment of different enzymes and drugs with
varied physical and chemical properties to realize complex
multistep metabolic reactions. During the in vitro drug
metabolism studies, microsomes and NADPH are normally
used, where NADPH acts as an electron donor while the
microsomal enzymes act as the catalysts of oxidative
metabolites of drug molecules by oxygen. The catalytic cycle
of the CYP450 enzyme in microsomes features delivery of
electrons from NADPH to CYP450 reductase (CPR) for
subsequent delivery to the P450 heme. The reduced heme is
further involved in the oxidation of substrate molecules by
oxygen. The microsomes are hydrophobic, while NADPH is
hydrophilic.27,28 In addition, both microsomes and NADPH are
negatively charged in normal reaction solutions.29,30 Therefore,
an amphiphilic periodic mesoporous organosilica (PMO) with
a surface modification of amino groups to have positive surface
charges (NH2-PMO) is designed and synthesized in this work
as a suitable candidate for the coenrichment of microsomes,
NADPH, and drugs.
Highly efficient metabolic reactions are demonstrated with
the assistance of NH2-PMO to form a biomimetic nanoreactor.
Not only does it enrich multiple enzymes and substrates to
form high local concentrations, the NH2-PMO nanoreactor can
also provide a suitable microenvironment to maintain the
stability and catalytic activity of the enzymes. Both a high
conversion ratio and a large reaction rate constant were
achieved when using nifedipine and testosterone as target
drugs. The quantification and structural characterization of the
metabolites were well achieved by high-performance liquid
chromatography−mass spectrometry (HPLC−MS) analysis.
The NH2-PMO-based biomimetic nanoreactor strategy is
simple, cost-effective, rapid, and accurate for drug metabolism
analysis. It is expected to accelerate the drug screening and lead
to promising advances in drug discovery and development.
Drug Metabolism in NH2-PMO Nanoreactors. A 10 μL
volume of CYP3A4 microsomes was added to 100 μL of NH2-
PMO PB solution (1 mg/mL). The mixture was then agitated
at 4 °C for 30 min for complete adsorption of the microsomes
onto NH2-PMO/PMO. Then the NH2-PMO/PMO materials
were pelleted by centrifugation and resuspended in 50 μL of PB
solution containing nifedipine (4 μg) or testosterone (2 μg).
For the metabolic reaction, the mixture was preincubated at 37
°C for 5 min for the adsorption of substrates, and then 60 μg of
NADPH was added to the incubation solution to initiate the
oxidation reaction. After being incubated at 37 °C for a certain
number of minutes, the reaction was quenched, and the drug
molecules with their metabolites were extracted by 1 mL of
ice−methylene chloride. At last, the methylene chloride was
dried under a N2 stream without heating, and the dried samples
were redissolved in methanol for further analysis.
EXPERIMENTAL SECTION
■
Chemicals. Cytochrome P450 CYP3A4 isozyme micro-
somes expressed in baculovirus-infected insect cells (human,
recombinant) with cytochrome P450 reductase and cyto-
chrome b5, reduced nicotinamide adenine dinucleotide
phosphate (NADPH), nifedipine (≥98%, powder), testoster-
one (≥98%, powder), caffeine (≥99%, powder), a triblock
copolymer, EO106PO70EO106 (Pluronic F127), bis-
(trimethoxysilyl)ethane (BTME), 1,3,5-trimethylbenzene
When the metabolism of nifedipine sustained-release tablets
was studied, a similar method was used. A 20 μL volume of
CYP3A4 microsomes and 50 μg of nifedipine sustained-release
tablets were preloaded into the NH2-PMO and then incubated
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with 120 μg of NADPH. Before HPLC−MS analysis, the
extracted samples were centrifuged to remove the precipitates
thoroughly.
In-Solution Drug Metabolism. The control experiments
of microsomal dispersions were performed according to the
published protocols.33,34 The same amounts of enzymes and
drugs as those used during the NH2-PMO nanoreactor
catalyzed reaction were suspended in 200 μL of PB solution.
After incubation, extraction, and redispersion, the metabolites
were analyzed by HPLC−MS.
Figure 2. (a) FT-IR spectra of NH2-PMO and PMO. (b) ζ potential
LC−MS Analysis. For rapid screening of reactive
metabolites, the reaction mixture was analyzed by HPLC−
MS. Samples were first subjected to chromatographic
separation with an Agilent series 1260 HPLC system (Agilent
Technologies, Palo Alto, CA) integrated with an autosampler
and a UV detector. A 20 μL volume of the sample solution was
injected into a VP-ODS C18 column (250 mm × 4.6 mm i.d., 5
μm, Agilent) at a flow rate of 0.6 mL/min. The detailed
separation process is explained in the Supporting Information.
The fractions from LC were introduced into a 6460 QQQ mass
spectrometer (Agilent) that was operated in positive ion mode
under the optimized parameter conditions (refer to the
Supporting Information). The structure information on reactive
metabolites can be deduced from tandem MS spectra.
Additionally, for quantitative analysis of the metabolites, 20
μL of caffeine (0.1 μg/μL) was added as an internal standard
before HPLC−MS analysis.
distributions of NH2-PMO and PMO.
peak arose around 1570 cm−1. This peak may be attributed to
the grafted −NH2 groups. However, the stretching vibration of
−NH2 at 3100−3300 cm−1 was not distinguishable, which can
be overlapped by the vibration absorption from the large
amount of silanol groups generated during the ethanol
extraction process.35,36 The ζ potentials of PMO and NH2-
PMO were measured as −27.3 and +32.5 mV in a PB solution
(Figure 2b), respectively, which demonstrates the successful
modification of PMO by amino groups in NH2-PMO. All the
aforementioned results indicated that the nanoreactor is
designed as a good candidate for hosting target drugs and
enzymes. As shown in Scheme 1, hydrophobic and negative
Scheme 1. Schematic Illustration of the NH2-PMO
Nanoreactor for Efficient Drug Metabolic Reaction
RESULTS AND DISCUSSION
■
Characterization of the Nanoreactors. The low
conversion rate is one of the main limitations of in vitro drug
metabolism. Nanoconfined reactors with designed structures
and functions are proposed to realize enhanced drug
metabolism. Considering the hydrophobic nature and negative
charge of the microsomes and hydrophilic nature and negative
charge of NADPH, a positively charged amphiphilic nano-
porous material is desirable for simultaneous immobilization of
microsomes and NADPH. To realize this purpose, NH2-PMO,
with the existence of both −CH2 groups and Si−O−Si groups
in the framework, by surface modification through incorpo-
ration of amino groups has been prepared as a host for drug
metabolism. In addition to the surface properties, NH2-PMO
holds a periodic mesoporous 3D structure of arrays of pores
∼20 nm in diameter that would not restrict the mass transfer of
molecules inside the host, Figure 1.
microsomes were first immobilized in the NH2-PMO nano-
reactor through hydrophobic and electrostatic interaction.
Afterward, such NH2-PMO−microsome nanoreactors were
easily dispersed in PB solution containing substrates and then
in the solution of NADPH. More than 90% of nifedipine and
testosterone and NADPH can be adsorbed into the NH2-PMO
nanoreactor within 2 min (Figure S1, Supporting Information).
In this way, the target substrates and multiple enzymes are
rapidly captured and concentrated inside the mesoporous
material to realize fast kinetics of drug metabolism due to the
nanoconfinement and enrichment effect in the NH2-PMO
reactors.
The FT-IR spectrum of bare PMO is shown in Figure 2a.
The dominant multipeaks at 1000−1100 cm−1 are typically
assigned to the Si−O bonds and Si−OH groups of the bare
PMO material. For organic-bridged materials, the peaks located
in the region of 2920 cm−1 can be assigned to the −CH2−
groups. In the case of NH2-PMO, an obvious additional single
Drug Metabolism by the NH2-PMO−Microsome
Nanoreactor. To test the proposed protocol for highly
efficient in vitro drug metabolism, the performance of NH2-
PMO−microsome nanoreactors has been evaluated by HPLC−
MS to characterize the metabolites using nifedipine as a
substrate. Nifedipine is a prototype of the dihydropyridine class
of calcium channel blockers that is widely used in the treatment
Figure 1. TEM images of (a) bare PMO and (b) NH2-PMO. The
insets are partially zoomed images of the materials.
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of hypertension, Prinzmetal’s angina pectoris, and other
vascular disorders. For humans, the primary product of
CYP3A4-catalyzed oxidation of nifedipine is dehydronifedipine
(DNIF; Scheme 2a). Nifedipine oxidation is a prototypic
CYP3A4 reaction, where the products can be simply measured
by UV−HPLC.37,38
a retention time tR ≈ 16 min, and the substrate was seen at tR ≈
27 min. For direct comparison, the same amounts of enzymes
and drugs were used in the bulk solution reaction system as
those used in the nanoreactor metabolic reaction system. As
shown in Figure 3a, much more metabolite of nifedipine was
observed by adding NH2-PMO in the metabolism system for 2
min compared to the in-solution metabolism. More than 50%
nifedipine had already been consumed after 10 min with the
assistance of NH2-PMO, whereas still only a small amount of
the metabolite was observed in the bulk solution reaction
system (Figure 3b). After 30 min of incubation with the NH2-
PMO−microsome nanoreactor, the majority of nifedipine had
already been oxidized (Figure 3c). When the reaction lasted for
60 min, still a large amount of nifedipine was not consumed by
the bulk solution reaction, and the amount of generated
metabolite was even less than that obtained after 10 min of in
vitro metabolism based on the NH2-PMO−microsome nano-
reactor. The performance of such nanoreactors for a clinical
sample was also investigated with the nifedipine sustained-
release tablets as the test sample. As shown in Figure S2
(Supporting Information), more than half of nifedipine in the
tablets was metabolized within 10 min in the presence of the
nanoreactor, while the in-solution reaction was much less
efficient.
Scheme 2. Oxidative Metabolism of (a) Nifedipine and (b)
Testosterone
Both results show that the metabolic reaction with the
assistance of the NH2-PMO−microsome nanoreactor is much
more efficient than the in-solution reaction. Besides the
periodic mesoporous structure, the positively charged surface
is also an important factor to realize the fast in vitro
The UV−HPLC chromatograms in Figure 3 illustrate the
results of nifedipine oxidation activated by NADPH with and
without the assistance of NH2-PMO−microsome nanoreactors
at different times. The metabolite of nifedipine was observed at
Figure 3. UV−HPLC chromatograms of nifedipine oxidation catalyzed by human liver CYP3A4 microsomes with and without the assistance of the
NH2−PMO nanoreactor monitored at 254 nm at different times: (a) 2 min, (b) 10 min, (c) 30 min, (d) 60 min. The nifedipine substrate was
observed at tR ≈ 27 min. The metabolite of nifedipine was observed at tR ≈ 16 min.
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metabolism. Figure S3 (Supporting Information) shows the
UV−HPLC chromatograms of bare PMO-assisted drug
metabolism after 30 min for comparison, where the surface of
the unmodified PMO is negatively charged. The results showed
that bare PMO could also improve the efficiency of drug
metabolism compared with that of the in-solution reaction but
not as well as the NH2-PMO-assisted protocol. The existence of
positive amino groups on the NH2-PMO surfaces contributes
to the extraction of negatively charged microsomes and
NADPH into the mesoporous material.
existence of dehydronifedipine. The formation of the
metabolized product of nifedipine at different reaction times
is shown in Figure S4 (Supporting Information). The same
conclusion can be obtained from the differences in metabolic
efficiencies with and without nanoreactors as from the UV−
HPLC experiments.
Enhanced Metabolic Reaction Kinetics with the NH2-
PMO−Microsome Nanoreactor. To characterize more
precisely the performance of NH2-PMO−microsome nano-
reactors for drug metabolism, conversion ratios were calculated.
As the microsomes and drug molecules were preloaded into the
NH2-PMO, the reaction was initiated as soon as the NADPH
was added and terminated when it was quenched for the
collection of products. As demonstrated in Figure S1c
(Supporting Information), the adsorption of NADPH is very
fast and cannot limit the overall reaction kinetics. Therefore,
the reaction kinetics was analyzed on the basis of the classic
Michaelis−Menten kinetics.22 It was found that the overall
reaction kinetics followed pseudo-first-order reaction with
respect to the substrate. The metabolic reaction rates based
on the NH2-PMO−microsome nanoreactors and bulk solution
were found to be 0.14 and 0.0051 min−1, respectively (Figure
5b and Table 1). As a result, the NH2-PMO−microsome
nanoreactors improved the metabolic reaction rate by about 27-
fold compared to that of the in-solution metabolism. Figure 5a
and Table 1 also display the conversion rate of nifedipine. As an
example, a much higher conversion of ∼75% can be obtained
The metabolites of nifedipine were characterized by tandem
MS. Figure 4a shows the mass spectra of nifedipine and its
Figure 4. (a) Mass spectra of nifedipine and its metabolites from
oxidation catalyzed by human liver CYP3A4 microsomes with and
without the assistance of NH2-PMO at 10 min. Peaks of
dehydronifedipine: m/z = 345.1 [M + H]+, m/z = 367.1 [M +
Na]+, and m/z = 711.3 [2M + Na]+. Peaks of nifedipine: m/z = 347.1
[M + H]+, m/z = 369.1 [M + Na]+, and m/z = 715.4 [2M + Na]+. (b)
CID spectrum of the precursor ion at m/z = 345.1.
metabolites generated under the catalysis of human liver
CYP3A4 microsomes with and without the assistance of NH2-
PMO after 10 min of reaction. The ion peaks at m/z = 345.1
[M + H]+, m/z = 367.1 [M + Na]+, and m/z = 711.3 [2M
+Na]+ correspond to the oxidized nifedipine product. The
collision-induced dissociation (CID) spectrum of the precursor
ion at m/z = 345.1 was obtained to study the structure of the
oxidized nifedipine as shown in Figure 4b. The predominant
ion with m/z 284.1 represents the loss of CH3−COOH from
the metabolite, and the fragment at m/z 224.1 represents the
loss of another CH3−COOH from the metabolite. The other
fragments correspond to the loss of −N or −O from the related
fragments. Both the ion peaks and fragments indicated the
Figure 5. Metabolism reaction profiles of the experimental data with
fitted curves: (a) conversion ratio of nifedipine and (b) reaction
kinetics of nifedipine metabolism with and without the assistance of
the NH2-PMO nanoreactor. [nifedipine]0 = the initial concentration of
nifedipine, and [nifedipine]t = the concentration of nifedipine at time
t.
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center.27,39 As a consequence, the metabolic reaction time is
greatly reduced, while the conversion rate is largely enhanced.
NH2-PMO−Microsome-Nanoreactor-Based in Vitro
Metabolism of Testosterone. To further confirm the
capabilities of such a nanoreactor approach for the analysis of
complex drugs, testosterone was employed for metabolism
profiling. Testosterone is a male sex hormone that is important
for sexual and reproductive development and plays a vital role
in carbohydrate, fat, and protein metabolism. CYP3A4 is
involved in the oxidation of testosterone, catalyzing oxidation at
the 2β, 6α, 6β, 7α, 15β, 16α, and 17 positions, with 6β-
hydroxytestosterone being the primary oxidation product of
testosterone (Scheme 2b).33,41 Similar to nifedipine oxidation,
this reaction has also been used as a model for CYP3A4-
catalyzed metabolism studies. Figure 6 shows the UV−HPLC
chromatograms of testosterone oxidation activated by NADPH
with and without the assistance of the NH2-PMO−microsome
nanoreactor at different times. Corresponding mass spectra of
testosterone oxidation are shown in Figure S5 (Supporting
Information). As illustrated in Figure 6, several isometrical
metabolites of testosterone (m/z = 305.2 [M + H]+, 327.1 [M
+ Na]+, and 631.4 [2M + Na]+) were observed at tR ≈ 15, 16,
17 and 18 min, and the substrate (m/z = 289.2 [M + H]+ and
599.4 [2M + Na]+) was seen at tR ≈ 22 min. Except the peak
eluted at 17 min, which did not correspond to any of the
available metabolites from early work, the others have been
identified.40,41 In good agreement with the nifedipine oxidation,
most of the testosterone was metabolized in a short time using
the NH2-PMO−microsome nanoreactors, whereas the in-
solution metabolic reaction proceeded slowly, resulting in a
lower conversion until 120 min of reaction. It is noteworthy
Table 1. Conversion Ratio of Nifedipine Metabolism after
120 min and Calculated Apparent Rate Constants (k) with
and without the Assistance of the NH2-PMO Nanoreactor
metabolism strategy
conversion ratio/%
k/min−1
NH2-PMO nanoreactor
in-solution reaction
77
25
0.14
0.0051
within 20 min when NH2-PMO−microsome nanoreactors are
added to the metabolic reaction system, whereas the in-solution
reaction achieved a conversion of only less than 15% within the
same time. Such a conversion rate derived from the NH2-PMO
protocol is very high compared with that of other developed
strategies.8 The difference in metabolism efficiency between the
nanoreactor-assisted and bulk solution reactions is always
significant for all the examined metabolic reactions.
Both the qualitative and quantitative results validate that the
metabolic reaction based on the NH2-PMO−microsome
nanoreactor is significantly enhanced compared with the in-
solution method and the reaction with the assistance of pure
PMO nanoreactors, stemming from the amphiphilic property
and positively charged surface of NH2-PMO for the rapid
enrichment of microsomes and substrates. Furthermore,
hydrophilic and negatively charged NADPH can easily partition
into the pores. The fast enrichment not only dramatically
increases the concentration of multiple enzymes, drugs, and
dissolved oxygen in the nanoreactor, but also provides a
suitable environment for retaining their native stability and
catalytic activity. This, in fact, offers abundant catalytic sites for
the metabolic reaction while also facilitating the access of
enzymes and substrates, allowing fast charge transfer via
CYP450 reductase from NADPH to the enzyme’s iron heme
Figure 6. UV−HPLC chromatograms of testosterone oxidation by human liver CYP3A4 microsomes with and without the assistance of the NH2-
PMO nanoreactor monitored at 244 nm at different times: (a) 2 min, (b) 10 min, (c) 30 min, (d) 120 min. The testosterone substrate was observed
at tR ≈ 22 min. The metabolites of testosterone were observed at tR ≈ 15, 16, 17 (red star labeled), and 18 min, respectively.
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presence of NH2-PMO−microsome nanoreactors, demonstrat-
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PMO−microsome nanoreactor. Therefore, the metabolic
reaction based on the nanoreactor approach may provide
more information on the drug metabolism compared to the
conventional bulk solution in vitro metabolic reaction, which is
of great value in identifying reactive metabolites of drugs to
predict possible toxicity in the early stage of drug development.
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CONCLUSIONS
■
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ASSOCIATED CONTENT
* Supporting Information
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Detailed description of the LC−MS experiments, data for the
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nifedipine sustained-release tablet metabolism, and mass
spectra of nifedipine and testosterone metabolism. This
material is available free of charge via the Internet at http://
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AUTHOR INFORMATION
Corresponding Author
*E-mail: bhliu@fudan.edu.cn. Fax: (+86) 21-6564-1740.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (NSFC) (Grants 21175028 and
21375022) and 863 Project 2012AA020202.
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