PH-Sensitive, N-ethoxybenzylimidazole (NEBI) bifunctional crosslinkers enable triggered release of therapeutics from drug delivery carriers

Article abstract of DOI:10.1039/c0ob00228c

This paper presents a pH-sensitive bifunctional crosslinker that enables facile conjugation of small molecule therapeutics to macromolecular carriers for use in drug delivery systems. This N-ethoxybenzylimidazole (NEBI) bifunctional crosslinker was designed to exploit mildly acidic, subcellular environments to trigger the release of therapeutics upon internalization in cells. We demonstrate that an analog of doxorubicin (a representative example of an anticancer therapeutic) conjugated to human serum albumin (HSA, a representative example of a macromolecular carrier) via this NEBI crosslinker can internalize and localize into acidic lysosomes of ovarian cancer cells. Fluorescence imaging and cell viability studies demonstrate that the HSA-NEBI-doxorubicin conjugate exhibited improved uptake and cytotoxic activity compared to the unconjugated doxorubicin analog. The pH-sensitive NEBI group was also shown to be relatively stable to biologically-relevant metal Lewis acids and to serum proteins, supporting that these bifunctional crosslinkers may be useful for constructing drug delivery systems that will be stable in biological fluids such as blood.

Full text of DOI:10.1039/c0ob00228c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
pH-Sensitive, N-ethoxybenzylimidazole (NEBI) bifunctional crosslinkers
enable triggered release of therapeutics from drug delivery carriers†
Alice Luong, Tawny Issarapanichkit, Seong Deok Kong, Rina Fong and Jerry Yang*
Received 8th June 2010, Accepted 27th July 2010
DOI: 10.1039/c0ob00228c
This paper presents a pH-sensitive bifunctional crosslinker that enables facile conjugation of small
molecule therapeutics to macromolecular carriers for use in drug delivery systems. This
N-ethoxybenzylimidazole (NEBI) bifunctional crosslinker was designed to exploit mildly acidic,
subcellular environments to trigger the release of therapeutics upon internalization in cells. We
demonstrate that an analog of doxorubicin (a representative example of an anticancer therapeutic)
conjugated to human serum albumin (HSA, a representative example of a macromolecular carrier) via
this NEBI crosslinker can internalize and localize into acidic lysosomes of ovarian cancer cells.
Fluorescence imaging and cell viability studies demonstrate that the HSA-NEBI-doxorubicin conjugate
exhibited improved uptake and cytotoxic activity compared to the unconjugated doxorubicin analog.
The pH-sensitive NEBI group was also shown to be relatively stable to biologically-relevant metal
Lewis acids and to serum proteins, supporting that these bifunctional crosslinkers may be useful for
constructing drug delivery systems that will be stable in biological fluids such as blood.
Here, we evaluate
a novel, acid-sensitive, bifunctional
Introduction
crosslinker, based on an N-ethoxybenzylimidazole27 (NEBI) plat-
form, for conjugation of drugs to a protein carrier. We designed
this bifunctional NEBI crosslinker (1, Fig. 1A) on one side to
carry a carboxylic acid functionality for conjugation to amine-
or alcohol-containing drugs through standard amidation or
esterification conditions,28,29 and on the other side to carry an azide
functionality for, e.g., conjugation to macromolecules through
“click” reactions.30,31 We evaluated the utility of this acid-sensitive
linker in DDSs by analyzing the uptake and activity of an analog of
doxorubicin (an FDA-approved cancer therapeutic32) on human
ovarian cancer cells, when the doxorubicin analog was conjugated
to human serum albumin (HSA). In this work, we used HSA
as a model drug delivery carrier since this protein is known to
enter cancer cells through a endocytotic mechanism.33 HSA is also
currently used as an FDA-approved drug carrier for paclitaxel for
the treatment of breast cancer, and therefore is an excellent model
for these studies.34
Bifunctional crosslinkers are widely used for modification of natu-
ral and synthetic macromolecules.1,2 A missing tool in bioconjuga-
tion chemistry, however, is a bifunctional crosslinker that exhibits
controlled hydrolytic properties in mild acidic environments. Such
crosslinkers could be useful for generating novel acid-responsive
materials for a range of applications including drug delivery. A
common characteristic of many macromolecular- or nanoparticle-
based drug delivery systems (DDSs), for instance, is the rapid
internalization and intracellular localization of the DDS into
acidic endosomes (pH 5.5–6.0) and lysosomes (pH 4.5–5.0) of
cells upon arrival to the targeted tissue.3,4 Due to this common
pathway of cellular uptake, there has been significant interest in
methods to engineer acid-triggered responses from DDSs for rapid
intracellular release of therapeutics.4–15 Acid-sensitive groups, for
instance, have been previously explored for covalent conjugation
and controlled release of specific drugs from macromolecular
carriers for various drug delivery applications.12,16–24 Although
many previously reported acid-sensitive groups exhibit accelerated
hydrolysis in acidic solutions, only hydrazones have thus far made
it into clinical applications in an FDA-approved drug as a pH-
sensitive linker for conjugating a hydrazine-containing drug to a
ketone-containing antibody.25,26 In order to broaden the use of
acid-sensitive linkers in DDSs, several challenges remain to be
addressed including: (1) stability of the linker in biological fluids,
(2) synthetic accessibility of DDSs comprising acid-cleavable
linkers, and (3) capability of using the linkers for conjugating a
broad range of therapeutics to drug delivery carriers under mild,
biocompatible conditions.
Results and discussion
Fig. 1 shows the structure of an acid-sensitive bifunctional
crosslinker (1) derived from a NEBI platform. In previous work,
we demonstrated that this class of molecules undergoes accelerated
hydrolysis in mild acid (i.e., at pH 5.5) compared to solutions at
physiological pH of 7.4.27 Fig. 1 outlines the procedure used to
conjugate doxorubicin to HSA via NEBI linker 1. We incorporated
alkyne functionalities on the lysines of HSA by reacting the
NHS ester of 4-pentynoic acid with HSA in 0.1 M HEPES
buffer (pH 8.3)35 to afford HSA-alkyne 2.36 After coupling of
the amine of doxorubicin to the carboxyl functionality on 1
using standard amide coupling conditions, we reacted this NEBI-
doxorubicin conjugate with 2 under copper-mediated “click”
conditions to afford HSA-NEBI-doxorubicin conjugate 3. This
procedure resulted in a loading of ~1 doxorubicin molecule
Department of Chemistry and Biochemistry, University of California, San
Diego, 9500 Gilman Drive, MC 0358, La Jolla, CA, 92093-0358, USA.
E-mail: jerryyang@ucsd.edu; Fax: (+1) 858-534-4554; Tel: (+1) 858-534-
6006
† Electronic supplementary information (ESI) available: Details of the
experimental procedures. See DOI: 10.1039/c0ob00228c
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ovarian cancer cells. Fig. 3B illustrates that incubation of cells
with 3 (red) for 24 h resulted in significant cellular uptake.
Subsequent treatment of these cells with Lysotracker Blue revealed
the location of the lysosomes (blue regions in Fig. 3C). Fig. 3D
shows that a significant fraction of 3 co-localized (green) with
Lysotracker Blue in these cells, suggesting that 3 indeed localized
primarily within the lysosomes of these cells. This result is in
agreement with previous reports for the uptake of HSA into the
cells via an endocytotic mechanism.33 Since these imaging studies
confirm that HSA-NEBI-doxorubicin conjugate 3 internalizes and
localizes in acidic compartments of cancer cells, and since NEBI
groups exhibit accelerated hydrolysis in mild acidic solutions,27 we
hypothesized that incubation of these cells with 3 will result in
intracellular release of doxorubicin analog 6, resulting in reduced
viability of the cells.
Fig. 1 Structure of a pH-sensitive bifunctional crosslinker and of two
doxorubicin-human serum albumin (HSA) conjugates. (A) Synthetic
scheme for the synthesis of HSA-doxorubicin conjugate 3 compris-
ing a pH-sensitive NEBI crosslinker 1. (B) Structure of an HSA–
doxorubicin conjugate 4 comprising acid-stable amide and triazole
groups. EDC = 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, TCEP =
tris(2-carboxyethyl)phosphine, TBTA = tris(benzyltriazolylmethyl)amine.
Fig. 3 Fluorescence imaging studies for the internalization and localiza-
tion of HSA-NEBI-doxorubicin conjugate 3 in human ovarian carcinoma
2008 cells. (A) Differential interference contrast image of a human ovarian
cancer 2008 cell. (B) Fluorescence micrograph from a z-slice through
the cell showing the location of 3 (red) inside the cell. The subcellular
distribution of 3 was determined by monitoring the intrinsic fluorescence
of doxorubicin. (C) Fluorescence micrograph from the same z-slice through
the cells as shown in (B), except showing only the location of the lysosomes,
which were stained with Lyostracker blue. (D) A merged fluorescence
micrograph of (B) and (C) indicating areas of co-localization of 3 and the
Lysotracker Blue. Scale bar = 5 mm.
per HSA protein (see the ESI† for details of the synthesis and
characterization of 3). In order to provide a comparison for the
utility of acid-sensitive linker 1 for drug delivery applications, we
also synthesized an HSA-doxorubicin conjugate 4 (Fig. 1B), which
did not comprise an acid-labile group.
Previous studies showed that a NEBI group carrying dox-
orubicin 5 hydrolyzes in mildly acidic solutions to produce
doxorubicin analog 6 (Fig. 2), with a half-life at pH 5.5 of
◦
55 h at 37 C.27 At pH 7.4, however, 5 hydrolyzed with a much
slower half-life of 1150 h at 37 ◦C. These results suggest that
doxorubicin molecules conjugated to drug delivery carriers via
NEBI linkers would hydrolyze with accelerated rates inside of
cells if these doxorubicin-NEBI conjugates are internalized and
localized within the acidic compartments of the cells.
In order to assess whether 3 exhibits cytotoxic activity to cancer
cells, we determined the viability of human ovarian carcinoma
2008 cells 38 that were exposed to HSA-NEBI-doxorubicin conju-
gate 3. Indeed exposure of cells to increasing concentrations of 3
resulted in reduced cell viability with an IC50 of 1.6 mM (Fig. 4A).39
As expected, control experiments revealed that native HSA and
HSA-alkyne conjugate 2 were not toxic to cells within the same
protein concentration range (Fig. 4B). Additionally, Fig. 4A shows
that conjugate 3 was ~4 times more toxic than doxorubicin analog
40,41
6 alone
(i.e., the doxorubicin analog expected to accumulate
Fig. 2 Schematic illustration for the hydrolysis of NEBI-doxorubicin
conjugate 5 in mildly acidic solutions.
in cells after hydrolysis of the NEBI group, IC50 = 5.8 mM).
The HSA-NEBI-doxorubicin conjugate 3 was also 10 times more
potent than HSA-doxorubicin conjugate 4 (IC50 = 16 mM), which
lacked a pH-sensitive linker.42 These results from cytotoxicity
experiments demonstrated that a drug conjugate comprising
To determine whether the HSA-NEBI-conjugate 3 could in-
ternalize and localize within acidic compartments of cells, we
examined the uptake and subcellular localization of 3 in human
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Table 1 Hydrolysis of NEBI groups in buffered solutions containing
biologically-relevant Lewis acids or FBS
Additive
Total concentration in blood/mM
k/h-1
krel
None
N/A
0.107
0.016
1.56
8.00
1.51
85.6
41.5
N/A
0.0211
0.0241
0.0249
0.0222
0.0228
0.0238
0.0222
0.0221
0.0221
1
Fig. 4 Cytotoxicity studies of HSA, HSA conjugates, and doxorubicin
analog 6 on human ovarian carcinoma 2008 cells. (A) Graph of the percent
Zn(II)
Cu(II)
Mg(II)
Fe(III)a
Ca(II)
Na(I)
1.14
1.18
1.05
1.08
1.13
1.13
1.05
1.05
viability of cells upon exposure to HSA-NEBI-doxorubicin 3 (IC50
=
1.6 mM), HSA-doxorubicin conjugate 4 (IC50 = 16 mM), and doxorubicin
analog 6 (IC50 = 5.8 mM). (B) Control experiments showing the viability
of cells upon exposure to HSA or HSA-alkyne 2. In all experiments, cells
were exposed to the samples for 3 days prior to analysis for viability using
a standard SRB cell viability assay37 and compared to the viability of
untreated cells.
K(I)
10% Serum
a [FeCl3] used in these experiments was 0.01 mM due to solubility
limitations of FeCl3 in buffer.
pH-sensitive NEBI linkers was more potent compared to a
conjugate that did not comprise an acid-labile group (4) or
compared to free 6 alone.
doxorubicin conjugated with HSA via the NEBI linker correlated
well with the observed increased toxicity of conjugate 3 compared
to doxorubicin analog 6 (Fig. 4A). Additionally, to attempt to
gain some insight for the origin of the observed toxicity from
conjugate 3, we incubated the ovarian carcinoma 2008 cells with 3
for 72 h. The cell lysate was then analyzed by LC-MS. This analysis
revealed a complex mixture of doxorubicin-derived species, with
at least three known metabolites44,45 of doxorubicin identified
by MS (see Fig. S3 in the ESI† for details).46 These combined
results support that conjugating doxorubicin to an HSA carrier
via the pH-sensitive NEBI crosslinker can result in improved
uptake and toxicity compared to unconjugated molecule, with
cytotoxic activity originating from the release of active derivatives
of doxorubicin in the cells.
We hypothesized that the observed improved toxicity of HSA-
NEBI-doxorubicin 3 compared to free 6 was due to the capability
of 3 to exhibit improved uptake of doxorubicin in the cells
through an HSA-mediated, endocytotic process.33 To support this
hypothesis, we incubated the ovarian cancer cells with conjugate
3 or doxorubicin analog 6 (with an equal concentration of
total doxorubicin in both experiments). After 3 h,43 the cells
were washed to remove excess extracellular molecule 3 or 6
and the cells were examined for the uptake of doxorubicin by
deconvolution microscopy (Fig. 5A and B). This analysis revealed
that cells incubated with conjugate 3 had an average of ~5.5 times
the amount of doxorubicin within the cells compared to cells
incubated with molecule 6 (Fig. 5C). This increased uptake of
Finally, since it is important for a pH-sensitive linker in DDSs
to be stable in biological fluids, we examined the hydrolysis of
the parent NEBI (7) in solutions containing biologically relevant
factors (other than protons) that are present in blood. For instance,
since protons are Lewis acids that catalyze the hydrolysis of NEBI
groups in solution (Fig. 2),27 we tested the hydrolytic stability
of NEBI groups in the presence of common metal Lewis acids
found in the body. Table 1 shows the measured rate constants
(k) for the hydrolysis of 7 in buffered solutions (pH 7.4, 37 ◦C)
containing various concentrations of biologically-relevant metal
Lewis acids.47 In these studies, we used metal ion concentrations
corresponding to their reported total concentration in blood,48
although it is expected that the concentrations of free metal ions
in the blood are much lower than these values since the metal ions
are likely associated with various biomolecules under physiological
settings. These hydrolysis studies revealed that the metal Lewis
acids at these high concentrations did not significantly affect the
rate of hydrolysis of 7 relative to hydrolysis in the absence of these
metal ions (krel). Table 1 also shows that the rate constant for
hydrolysis of 7 in a solution containing 10% fetal bovine serum
Fig. 5 Analysis of the uptake of HSA-NEBI-doxorubicin (3) and
doxorubicin analog (6) by human ovarian carcinoma 2008 cells. (A) and
(B) are micrographs of representative z-slices through cells that were
incubated with molecule 3 (A) or molecule 6 (B) for 3 h. Here, the
intrinsic fluorescence of doxorubicin is shown in red. Scale bar = 15 mm.
(C) The bar graph indicates that there is approximately 5.5 times the
amount of doxorubicin in the cells incubated with molecule 3 compared
to cells incubated with molecule 6. Average total fluorescence per cell was
determined from analysis of 10 cells in each sample image. Analysis of the
data in (C) revealed a p-value of 3.2 ¥ 10-9.
◦
(FBS) and 90% phosphate buffered saline (PBS, pH 7.4, 37 C)
remained relatively unchanged compared to hydrolysis of 7 in
solutions of pure buffer. These results collectively demonstrate
that pH-sensitive NEBI groups are relatively stable to biological
fluids such as blood.
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Cytotoxicity studies
Conclusion
Human ovarian carcinoma 2008 cells were plated into each well
of a 96 well plate (3000 cells/well) using RPMI-1640 media with
10% FBS. The cells were incubated for 24 h at 37 ◦C with 5%
CO2. After the incubation period, cells were dosed with various
concentrations of HSA, 2–4, 6, or 4-carboxybenzaldehyde. The
cells were allowed to incubate with the molecules for 72 h.
After incubation, cells were washed 3 times with 200 mL of PBS
(Mediatech, 46-013CM) pH 7.4. Cells were fixed with 200 mL
of PBS and 50 mL of 50% trichloroacetic acid for one hour at
This work demonstrates that a new pH-sensitive, bifunctional
NEBI crosslinker (1) can be employed for facile modification
of macromolecules with small molecules. Here, we present one
application for such an acid-sensitive crosslinker by demonstrating
its use for conjugating an anticancer agent (here, doxorubicin ana-
log 6) to a protein-based drug carrier (here, HSA). The resulting
DDS (3) exhibited improved cellular uptake and cytotoxic activity
in cancer cells compared to free molecule 6 alone. Conjugate
3 also exhibited 10-fold improved cytotoxic activity compared
to a conjugate that did not comprise an acid-labile group (4),
supporting that the cleavable, pH-sensitive NEBI group can have
a strong influence on the potency of therapeutics conjugated to
HSA. Although several factors may contribute to the observed
cytotoxic activity of 3 in ovarian cancer cells (Fig. 4A), the
results presented here are consistent with the hypothesis that the
activity of 3 may be due to the accelerated hydrolysis of the NEBI
crosslinker upon uptake and intracellular localization of 3 in the
acidic lysosomes of cancer cells.
Bifunctional crosslinkers based on NEBI groups such as 1
possess several attractive features as a tool for generating acid-
responsive materials. For instance, they afford facile methods to
attach a broad range of amine- and alcohol-containing molecules
to macromolecules through standard amide or ester bond-forming
reactions.12,28,29,49–51 Additionally, the ability to incorporate func-
tional groups on NEBI crosslinkers with orthogonal reactivity
to amide or ester bond-forming reactions (e.g., alkyne or azide
functional groups for “click” reactions) facilitates conjugation
of small molecules to a range of natural and synthetic macro-
molecules. Moreover, the previously reported capability to tune
the rates of hydrolysis of NEBI groups under mild aqueous
acidic conditions27 makes it possible to optimize the rates of
release of small molecules for specialized applications. Finally,
these bifunctional NEBI crosslinkers are attractive since they are
synthetically accessible from readily available and cheap starting
materials. These advantageous features make it possible, for
instance, to rapidly synthesize a range of DDSs carrying a variety
of therapeutic agents that can be released from drug delivery
carriers at controlled rates within acidic compartments of targeted
cells.
4
◦C. After fixation, cells were washed 5 times with water and
allowed to dry. A 0.4% sulforhodamine B (SRB, Sigma Aldrich,
S1402) solution in 1% acetic acid was added to the cells and
incubated for 15 min at room temperature. The cells were washed
3 times with 1% acetic acid and the 96 well plate was allowed to
dry. Tris base solution (10 mM, 200 mL) was added to the wells
for 15 min prior to measuring the absorbance at 515 nm. All data
points represent the UV absorbance of SRB relative to a sample
of cells that were fixed at time zero of the incubation period (i.e.,
24 h after introduction of cells to the wells of the 96 well plate).
Ref. 37 reports a similar protocol for estimating cell viability.
Measurement of hydrolysis of N-ethoxybenzylimidazole 7
Solutions containing 0.5 mM N-ethoxybenzylimidazole 7 was
incubated in 0.1 M HEPES (pH 7.4) buffer and incubated at
37 ◦C. The hydrolysis of 7 in the presence of various metal Lewis
acids was monitored by observing the formation of benzaldehyde
product by UV absorbance at 235 nm. For measurement of the
hydrolysis of 7 in the presence of FBS, FBS was dialyzed against
the solution of 0.5 mM 7 (in 0.1 M PBS buffer, pH 7.4) during
the course of the hydrolysis experiment. The dialysis chamber
was removed only during measurement of the formation of
benzaldehyde product by UV spectroscopy; the dialysis chamber
was necessary for these studies since the UV absorption of serum
proteins in FBS interfered with the analysis of the hydrolysis of 7.
Acknowledgements
This work was partially supported by the American Cancer
Society (RSG-07-024-01-CDD) and the NSF (CHE-0847530).
We also acknowledge the NSF for support of the NMR and
mass spectrometry facilities at UCSD (CHE-0741968 and CHE-
0116662). We thank Kersi Pestonjamasp from the UCSD Moores
Cancer Center for help with fluorescence imaging experiments.
The ovarian cancer cells used in this research were a gift from
Prof. Stephen Howell from the UCSD Moores Cancer Center.
The TBTA was a gift from Dr. Jordan Meier in the Department
of Chemistry and Biochemistry at UCSD.
Experimentals
Measurement of cellular uptake and subcellular localization of 3
by fluorescence microscopy
Human ovarian carcinoma 2008 cells were plated with Roswell
Park Memorial Institute-1640 (RPMI-1640) phenol red free
media supplemented with 10% fetal bovine serum (FBS) on
35 mm glass bottom dishes (MatTek Co., Ashland, MA) and
incubated overnight. A solution containing 10 mM of HSA-NEBI-
doxorubicin (3) was added to the cells and allowed to incubate for
24 h. Lysotracker Blue (Invitrogen, L7525) was added to the cells
and incubated for 30 min. The remaining living cells were washed
three times with phenol red free RPMI-1640 media without FBS
and then immediately imaged with a Delta Vision Deconvolution
Microscope System (Applied Precision, Issaquah, WA).
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two analytical methods.
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The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 5105–5109 | 5109
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