Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo

Article abstract of DOI:10.1021/acschembio.9b00439

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel Split Luciferin Peptide (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

Full text of DOI:10.1021/acschembio.9b00439

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Article
Real-time imaging and quantification of peptide uptake in vitro and in vivo
Hacer Karatas, Tamara Maric, Pier Luca D'Alessandro, Aleksey
Yevtodiyenko, Thomas Vorherr, Gregory J. Hollingworth, and Elena A. Goun
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00439 • Publication Date (Web): 09 Sep 2019
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Universal assay for peptide uptake imaging
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Real‐Time Imaging and Quantification of Peptide Uptake In Vitro
and In Vivo
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Hacer Karatas , Tamara Maric , Pier Luca D'Alessandro , Aleksey Yevtodiyenko , Thomas
2
2
1*
Vorherr , Gregory J. Hollingworth , Elena A. Goun
1
Laboratory of Bioorganic Chemistry and Molecular Imaging, Institute of Chemical Sciences and Engineering (ISIC),
Swiss Federal Institute of Technology Lausanne (EPFL), 1015 Lausanne, Switzerland
2
Novartis Pharma AG, Werk Klybeck Postfach, 4002 Basel, Switzerland
*
e-mail: elena.goun@epfl.ch
ABSTRACT: Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological and neu‐
rodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages
of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability.
Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide‐
based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the
need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an
inability to provide kinetic information on the internalization of a compound or distinguish between internalized and
membrane‐bound compounds. In addition, many of these methods are based on endpoint assays and require multiple
sample manipulation steps. Herein, we report a novel “Split Luciferin Peptide” (SLP) assay that enables the real‐time non‐
invasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence
readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a D‐cysteine
tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening
peptide libraries and can thus become an important tool for preclinical peptide drug development.
Keywords: Cell penetrating peptides, bioluminescent imaging, drug discovery
on its ability to penetrate the cellular membrane. However,
INTRODUCTION
Peptides have evolved as promising therapeutic agents in
the treatment of many human diseases, including cancer, car‐
diovascular disorders, and diabetes.1, 2 Therapeutic peptides
possess a number of advantages over small‐molecule drugs
and other biopharmaceuticals, such as high specificity and af‐
finity for the biological target, excellent safety and tolerability,
predictable metabolism, low cost of production, and shorter
most peptides are naturally highly hydrophilic, which signifi‐
cantly hampers their cell permeability (i.e., their uptake into
3
cells). This characteristic of peptide‐based compounds has
been recognized as a major challenge in peptide‐based drug
discovery. Therefore, there is a need for the development of
robust assays that provide accurate and quantifiable readouts
of real‐time peptide uptake and internalization kinetics in
both living cells and animals. In principle, such an assay could
also be used for the optimization of amino acid sequences in
focused peptide libraries and for the development of novel
peptidic drug formulations with enhanced cellular or tissue
uptake.
3
time to market. As a result, increasing numbers of peptide‐
based drugs have been introduced into the clinic over the last
decade, with approximately 60 approved peptide drugs cur‐
rently on the market.3, 4 Since most biological targets are in‐
tracellular, the efficacy of a therapeutic peptide is dependent
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Currently, optical imaging remains the most com‐
readers).22 While the first applications of BLI were dependent
on the use of biological reporters, many recently developed
applications are based on chemical caging of the substrate D‐
luciferin. In these applications, the amount of light produced
is proportional to the activity of the biological process of in‐
terest. Examples of such applications include real‐time in vitro
and in vivo imaging of enzymatic activity, metabolite absorp‐
tion, and small molecule fluxes.23‐26 Caged luciferin technology
has also been previously used for real‐time quantification of
intracellular peptide internalization and cell‐permeable pep‐
tide (CPP) uptake both in living cells and in whole animals
and, to the best of our knowledge, remains the only available
tool for the real‐time kinetic assessment of these important
parameters.27‐29
monly used method to study peptide uptake in the field of
drug discovery. One of the classic methods is based on conju‐
gation of a large fluorophore (e.g., FITC or TAMRA) to the
peptide of interest followed by confocal microscopy or flow
cytometry.5, 6 However, nonspecific binding of the fluorophore
to the cell membrane and fluorescence quenching can con‐
found interpretation of the results.7, 8 In addition, covalent at‐
tachment of hydrophobic fluorescent dyes may significantly
alter the physicochemical properties and permeability of the
original peptide molecule.9‐11 To circumvent this problem,
functionalization of peptides with small click handles fol‐
lowed by secondary staining with fluorescently labeled rea‐
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gents has recently been proposed. However, while modifica‐
tion of the original peptide remains minimal, this technique
requires multiple washing steps to minimize the background
signal due to the unreacted fluorescent dye. Additionally,
none of these available methods provide information on the
real‐time internalization kinetics of the peptides of interest.
While the caging of a biomolecule with luciferin is a power‐
ful technology, it still requires rather large modifications to
the original molecule that might be difficult to adapt for low‐
molecular weight compounds such as therapeutic peptides.
To circumvent this problem, we recently developed a split lu‐
ciferin reaction, which relies on in situ luciferin formation via
a facile cyclization reaction between D‐cysteine (D‐Cys) and
2‐cyano‐6‐hydroxy benzothiazole (CBT) (Figure 1A, Movie 1).
This reaction occurs efficiently in various physiological solu‐
tions as well as in living cells and animals.21, 25, 30, 31 Similar to
caging strategies that have been previously reported for the
full D‐luciferin scaffold, the split luciferin reaction provides
the opportunity to cage one or both of the reaction compo‐
nents (i.e., D‐Cys and/or CBT). Thus far, this approach has
been successfully used for imaging and quantification of the
activity of various proteases, such as caspase 3/7, caspase 8,
and dipeptidyl peptidase 4 (DPP‐4), both in vitro and in vivo.25,
Similarly, other methods used for the evaluation of peptide
uptake, such as MRI,13, 14 PET imaging,15, 16 or cell lysate pull‐
down assays followed by MALDI‐TOF‐MS analysis,17, 18 are not
readily amenable to high‐throughput screening, are unable to
provide real‐time kinetic results of peptide uptake into cells,
and require expensive capital equipment. Similar to fluores‐
cence techniques, the in vivo imaging of uptake processes in
live animals is limited by the inability to distinguish between
internalized, membrane‐bound, or blood‐circulating peptides
and can require multiple sample manipulation steps (e.g., MS‐
based methods). Therefore, there remains a clear need for
novel assay technologies that provide sensitive and quantifia‐
ble real‐time information both in vitro and in vivo and require
only minimal modification of the original peptide.
30, 32, 33
However, this novel tool has never been used to assess
the cellular uptake of biomolecules.
Bioluminescent imaging (BLI) is characterized by a number
of superior features over existing imaging modalities, includ‐
ing high sensitivity, biocompatibility, an extremely low back‐
ground signal, low cost, and the ability to provide kinetic in‐
formation both in vitro and in vivo.19‐21 Bioluminescence is
based on the principle that firefly luciferase oxidizes the small
molecule D‐luciferin to yield yellow‐green light that can be
sensitively detected with widely available optical imaging sys‐
tems (e.g., charge‐coupled device (CCD) cameras and plate
Here, we report a novel assay, the “Split Luciferin Peptide”
(SLP) assay, for the sensitive real‐time imaging and quantifi‐
cation of peptide internalization both in vitro and in vivo. The
approach is based on an easy to perform, stable modification
of the peptide of interest. Although we demonstrate in this
work the application of this assay for imaging and quantifica‐
tion of peptide internalization, the same technology could be
easily adapted for uptake studies on other important biomol‐
ecules such as proteins, therapeutic antibodies, nanoparticles,
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and small‐molecule drugs.
Imaging with the plate reader: Bioluminescence was quanti‐
fied with an acquisition time of 1 ms at 1 min intervals. Statis‐
EXPERIMENTAL SECTION
Real‐time imaging of peptide uptake in cells with the
tical analysis was performed using GraphPad Prism 6.
Real‐time imaging of peptide uptake in vivo. Female
FVB‐Luc+ mice34 (FVB‐Tg(CAG‐luc, GFP)L2G85Chco/J) (Jack‐
son Laboratory) were used for the in vivo studies. All experi‐
ments were approved through license 2849 from the Swiss
Cantonal Veterinary Office Committee for Animal Experimen‐
tation according to the Swiss National Instituitional Guide‐
SLP assay. Cells were plated in a 96‐well flat‐bottom
black/clear tissue culture plate (BD Falcon) at the indicated
densities specific for each cell line (described in detail in the
Supporting Information) and were cultured in 200 µl of cell
culture medium supplemented with 10% FBS and 1% penicil‐
lin/streptomycin for 48 h. Then, the medium was removed,
and the cells were washed once with 250 µl of HBSS (supple‐
mented with 25 mM glucose). Next, we added 140 µl of the
same HBSS buffer followed by addition of 5 µl of CBT solution
and incubation at 37 °C. Then, 5 µl of the test SLP compound
in HBSS buffer was added immediately before the biolumines‐
cence was read with an IVIS Spectrum system (PerkinElmer),
an IVIS 100 Imaging System (PerkinElmer) or a plate reader
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lines. The r
8
C‐c peptide was desalted before use. The mice
were divided into 3 groups (n=4 (CBT), n=6 (r
8
C‐c), and n=6
(E4c‐c)) and all of them received an i.p. injection of 100 µl of 1
mM CBT in sterile PBS containing 2% DMSO before being
8
placed back into the cage to rest for 3 min. Then, “r C‐c” and
“E4c‐c” groups received i.p. injections of 100 µl of 1 mM of
their corresponding SLP solutions in sterile PBS buffer. The
mice were anesthetized via inhalation of isoflurane (Atta‐
neTM), placed immediately in the imaging chamber on a heat‐
ing pad (37 °C) and kept under anesthesia for the duration of
the imaging session. Bioluminescence was recorded over an
hour using an IVIS Spectrum system with a 30 s exposure du‐
ration. ROI analysis of the bioluminescent signals was per‐
formed using Living Image Software 4.4.
(Tecan). The MDA‐MB‐231‐luc cell line was preincubated with
50 µM CBT for 30 min. U87MG‐luc and BT474‐luc cells were
preincubated with 70 µM and 50 µM CBT, respectively, for 60
min.
Imaging with the IVIS systems: Images were obtained every
5 min with automatic settings or manual settings (exposure
time: 30 s, binning: large, f‐stop: 1). Alternatively, images were
obtained at 10 min intervals (exposure time: 10 min, binning:
large, f‐stop: 1) for the experiments to determine the assay sen‐
sitivity (Figure 3C,D). Region of interest (ROI) analysis of the
bioluminescent signals was performed using Living Image
Software 4.4 (Caliper Life Sciences). The data were plotted us‐
ing GraphPad Prism 6 Software.
Statistical analysis. Statistical analysis was performed using
GraphPad Prism 6. The error bars in all the in vitro (cell‐free
and cellular) experiments represent the ± SD. The error bars
for the in vivo experiments represent the ± SEM. Unpaired
two‐tailed Student's t‐tests were used for the statistical analy‐
sis in GraphPad Prism 6. * P < 0.05, ** P < 0.01, and *** P <
0
.001.
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Figure 1. Design strategy for noninvasive real‐time imaging and quantification of peptide uptake using the “Split Luciferin Peptide”
(
SLP) assay. (A) An in situ split luciferin reaction between D‐cysteine (D‐Cys) and 6‐hydroxy‐2‐cyanobenzothiazole (CBT) results
in firefly D‐luciferin formation and photon generation in the presence of the firefly luciferase enzyme. This reaction proceeds
efficiently in test tubes, live cells and living animals. (B) Working principle of the SLP assay in cells. The cells are first treated with
CBT; followed by the addition of the SLP of interest. Subsequent internalization of the SLP into the cytosol of live cells leads to
the cleavage of the disulfide bond between the peptidic side chain and D‐Cys by the high concentration of glutathione (GSH),
resulting in the release of free D‐Cys. The in situ split luciferin reaction between D‐Cys and CBT yields D‐luciferin; in the presence
of the luciferase enzyme, this reaction produces an amount of light proportional to the amount of peptide internalized into the
cell.
is known to be an efficient reporter of the extracellular envi‐
RESULTS AND DISCUSSION
Design of the “Split Luciferin Peptide” (SLP) assay
ronment due to its stability in the extracellular space and effi‐
cient cleavage in a reducing cytosolic environment enriched
The overall design of the SLP assay is based on the split lu‐
with glutathione (GSH). Indeed, GSH concentrations differ
ciferin approach and is summarized in Figure 1 and Movie 1.
significantly between the intracellular (1‐10 mM)35, 36 and ex‐
In this assay, we caged one of the components of the split lu‐
tracellular (10 µM)37 environments. Cleavage of the disulfide
ciferin reaction, D‐Cys, by covalently linking it to the peptide
bond in the cytosol results in the uncaging of D‐Cys that is
of interest via a disulfide bond. This can be achieved by con‐
proportional to the amount of peptide entering the cell. Treat‐
necting D‐Cys with a suitable thiol‐containing linker, such as
ment of cells with the other D‐luciferin precursor, CBT, ena‐
L‐cysteine (L‐Cys) or mercaptopropanoic acid, appended to
bles a reaction between the free D‐Cys and CBT to form D‐
the peptide chain. We selected the disulfide linker because it
luciferin. This process results in light production that can be
quantified in real‐time in a sensitive, noninvasive manner
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both in cells and in whole animals expressing luciferase (e.g.,
signals from the three different reagents (P > 0.1) (Figure
transgenic reporter mice and luciferase‐expressing tumor xen‐
S2B), suggesting that intracellular GSH concentrations are
sufficient to quantitatively uncage D‐Cys from the SLPs.
ograft models).
Synthesis of split luciferin peptides (SLPs)
Imaging and quantification of peptide uptake in live
cells using the SLP assay
The first set of experiments was focused on validating the
SLP assay using well‐characterized CPPs, such as poly‐argi‐
nine, Penetratin, and TAT peptides, which are frequently used
as transporters to deliver impermeable molecules (e.g., pro‐
teins and nanoparticles) across cell membranes.38, 39 In these
studies, two cysteines were conjugated to the C‐terminus of
the peptide (Figure 2A). A first, L‐Cys was introduced via a
peptide bond to the peptide C‐terminus, while D‐Cys was con‐
jugated to L‐Cys via a disulfide bond. Two other SLPs were de‐
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In the next step, we tested whether SLP uptake could be di‐
rectly measured in a cell culture. Breast cancer cells stably
transfected with firefly luciferase (MDA‐MB‐231‐luc) were in‐
itially washed with Hank’s Buffered Salt Solution (HBSS), pre‐
incubated with CBT (50 µM in 150 µl of HBSS buffer) and then
treated with 10 µM r
8
C‐c peptide or with a 10 µM concentra‐
tion of a corresponding control peptide, such as r
4
C‐c or E C‐
4
c. Then, the bioluminescent signal was quantified over three
hours using an IVIS Spectrum imaging system. No washing
steps were performed before the peptides were added to the
4
veloped as controls: a tetra‐D‐arginine derivative (r C‐c),
which has been reported to have significantly reduced cell per‐
meability,40 and a highly negatively charged tetra‐glutamate
8
cells. The signal from r C‐c increased gradually over 1 h before
4
derivative (E C‐c) that acts as a nonpermeable control to de‐
reaching equilibrium, producing a total bioluminescent signal
that was 60‐fold greater than that of the background signal
(Figure 2C and Figure S2C). Furthermore, the tetra‐arginine
termine the extent of any extracellular disulfide bond reduc‐
tion. The chemical structures of the SLPs are shown in Figure
S1. The synthesis and characterization of the SLPs are de‐
scribed in detail in the Supporting Information (Schemes S1‐
control peptide (r
indeed demonstrated significantly lower light output than
C‐c. An even lower bioluminescent signal resulted from the
nonpermeable control peptide E C‐c (Figure 2C and Figure
S2C). As expected, the r C peptide without the D‐Cys modifi‐
4
C‐c) that is known to have reduced uptake
2
).
Cell‐free evaluation of the SLP assay
To determine whether D‐Cys linked to the SLP via a disul‐
r
8
4
8
cation showed only a background signal identical to that of
the HBSS control (Figure S2D). These data demonstrate that
the SLP assay can be successfully used for imaging and quan‐
tification of peptide uptake in vitro and that the light output
is proportional to the amount of internalized peptide.
fide bond could be uncaged by common reducing reagents
naturally found in eukaryotic cells, we performed an in vitro
cell‐free assay with GSH. CBT and a mixture of luciferase en‐
zymes with cofactors such as ATP and MgSO
4
were sequen‐
tially added to a solution of the r C‐c peptide in the presence
8
Optimization of the SLP uptake assay to reduce back‐
or absence of 0.5 mM GSH (Figure S2A), and the biolumines‐
cent signal was then detected using a plate reader.In the pres‐
ground signals
ence of GSH, the bioluminescent signal of r
8
C‐c was 118‐fold
During our initial studies, we observed significant back‐
ground bioluminescence when cell culture medium contain‐
ing L‐Cys or L‐cystine was used. It has been previously re‐
ported that L‐Cys, which is found in high concentrations in
cell culture medium, can react with CBT to form L‐luciferin,
which can then undergo racemization in the presence of fire‐
fly luciferase and produce light in the presence of the enzyme
(Figure S3).41 Replacement of the cell culture medium with
higher than that of the negative control (not treated with
GSH) (Figure 2B). To predict the efficiency of the intracellular
disulfide cleavage reaction, the bioluminescence output was
measured from the cells treated with equimolar concentra‐
8
tions of r C‐c or free D‐Cys. The resulting signal was compared
to the light produced by the cells treated with half the concen‐
tration of D‐cystine (since D‐cystine is the dimer of D‐Cys).
No significant differences were observed among the overall
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HBSS or L‐Cys‐free medium significantly reduced the back‐
living cells. This background signal reduces the sensitivity of
ground bioluminescence, but a significant signal was still ob‐
served, most likely due to the L‐Cys naturally present inside
the SLP assay. To minimize the background signal, we decided
to preincubate cells with an excess of CBT to scavenge
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Figure 2. In vitro validation of the SLP uptake assay. (A) Structures of the SLPs (r8C‐c, r4C‐c and E4C‐c) used for the in vitro
validation studies. All peptides were conjugated to D‐Cys through a disulfide bond. (B) Quantification of the efficiency of the SLP
uncaging in the presence and absence of the reducing agent glutathione (GSH) in cell‐free conditions. *** P < 0.001; unpaired two‐
tailed Student's t‐tests were used for statistical analysis. (C) Imaging and quantification of real‐time SLP peptide uptake in live
breast cancer cells stably expressing luciferase (MDA‐MD‐231‐luc). Bioluminescent signals were acquired with an IVIS Spectrum
system for a period of 3 h immediately following the addition of the SLP peptide. (D) Representative images of cells 20 min after
peptide addition. The error bars represent the ± SD.
most of the L‐Cys prior to the addition of the SLP assay rea‐
gents. CBT was added at concentrations ranging from 10 to 50
µM to live cells maintained in HBSS medium, and the result‐
ing bioluminescent signal was measured over 1 h (Figure 3A).
Notably, CBT is a nontoxic reagent, and no toxicity has been
reported even at much higher concentrations in vitro or in
vivo.25, 30 The resulting signal reached saturation at 40 µM CBT,
suggesting that this concentration was sufficient to quench
the endogenous L‐Cys background signal. In addition, we ob‐
served that the signal resulting from this treatment reached
the background of the instrument within 30‐60 min, indicat‐
ing that 30‐60 min is an optimal duration to achieve complete
suppression of the background signal. Therefore, in all future
experiments, live cells were preincubated with an excess of
CBT at 40‐50 µM for 30‐60 min before the SLP of interest was
added and the signal was acquired.
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Assessment of SLP uptake assay sensitivity, quantifia‐
ranging from 0.63 µM to 10 µM, and the signal was acquired
bility, and compatibility with common imaging plat‐
over 1 h with an IVIS Spectrum system. We observed a linear
increase in the total photon flux that was proportional to the
forms
8
r C‐c concentration (Figure 3B, Figure S4). These data
To determine whether there is a linear correlation between
the total bioluminescent signal and the extracellular concen‐
tration of the SLP probe, MDA‐MB‐231‐luc cells were first pre‐
incubated with 40 µM CBT for 1 h to eliminate the background
demonstrate that the total bioluminescent signal correlates
linearly with the exogenous concentration of the octaarginine
peptide, which is consistent with previously reported results.42
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signal from L‐Cys. Then, r C‐c was added at concentrations
Figure 3. In vitro optimization and investigation of the kinetic parameters of the SLP uptake assay. (A) Bioluminescent signal
produced by MDA‐MB‐231‐luc cells upon incubation with various concentrations of CBT. (B) Linear dependence of a total biolu‐
minescent signal from MDA‐MB‐231‐luc cells incubated with various concentrations of r C‐c. Each point on the graph represents
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the total photon flux of the corresponding real‐time kinetic curve in Figure S4. The error bars represent the ± SD. (C) Biolumines‐
cent signal production from MDA‐MB‐231‐luc cells upon treatment with lower concentrations of r C‐c to determine assay sensi‐
tivity. Cells were preincubated with 40 µM CBT for 1 h to eliminate the background signal from L‐Cys; then, r C‐c was added in
increasing concentrations, followed by immediate signal acquisition for 3 h with an IVIS Spectrum system. (D) Area under the
curves depicted in (C). ***P < 0.001 and *P < 0.05; unpaired Student's t‐tests were used for statistical analysis. (E) Investigation of
the kinetic parameters of the uptake of different CPPs, including octaarginine (r C‐c), TAT (TatC‐c), and Penetratin (PenC‐c). The
8
graph shows the real‐time bioluminescent signals resulting from MDA‐MB‐231‐luc cells preincubated with 40 µM CBT for 1 h to
eliminate the background signal from L‐Cys and then subjected to treatment with the three different SLPs at a concentration of
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0 µM. The signals were acquired over 1 h with an IVIS Spectrum system. (F) Total bioluminescent signals over 10 min as calculated
from the areas under the curves in (E). The error bars represent the ± SD.
To assess the SLP uptake assay sensitivity, MDA‐MB‐231‐luc
with those of a previous report in which the uptake of these
cells were preincubated with 40 µM CBT for 1 h to eliminate
three peptides was compared by flow cytometry.43 Interest‐
ingly, after one hour of signal acquisition, the total Penetratin
uptake exceeded both octaarginine and TAT peptide uptake
(Figure S6). These data are also consistent with previously
published data from a study by Sagan et al. that utilized an
endpoint assay based on MALDI‐MS quantification.18 These
results further demonstrate the importance of the real‐time
readout capability of cell internalization assays and show that
the SLP assay is suitable for simultaneously providing both
types of readouts: real‐time uptake kinetics and an endpoint
readout that can yield information about how much of a pep‐
tide of interest is taken up at any given time point.
8
the background signal from L‐Cys, and r C‐c was then added
at concentrations ranging from 25 nM to 200 nM. A significant
signal‐to‐background ratio (P < 0.05) was detected in cells in‐
8
cubated with the lowest concentration of r C‐c (Figure 3C,D).
To further demonstrate the versatility of the SLP uptake as‐
say, we compared the in vitro uptake of several SLPs (r C‐c,
C‐c and E C‐c) using two common imaging systems: the IVIS
8
r
4
4
Spectrum system and a standard plate reader. Very similar re‐
sults were obtained with both instruments under identical ex‐
perimental conditions (Figure S5). These data indicate that a
more advanced CCD camera is not required to utilize this as‐
say; rather, a simple plate reader can be used to run this assay
with a comparable level of sensitivity.
Next, we used the SLP assay to evaluate the uptake of oc‐
taarginine, Penetratin, and TAT cell‐penetrating peptides in
other cell lines, namely, the U87MG‐luc glioblastoma and
BT474‐luc breast ductal carcinoma cell lines, that stably ex‐
press luciferase. In this experiment, all of the tested peptides
yielded results similar to those observed for the MDA‐MB‐231‐
luc cell line (Figure S7).
Investigation of the real‐time kinetic parameters of the
SLP assay
To further validate our SLP assay, we investigated the ki‐
netic uptake parameters of octaarginine, Penetratin, and TAT
cell‐penetrating peptides that have been extensively studied
in the literature over the past decade.5, 6, 8, 18 We therefore syn‐
Application of the SLP assay for the assessment of ther‐
apeutic peptide cellular permeability
thesized octaarginine (r
8
C‐c), Penetratin (PenC‐c), and TAT
(
TatC‐c) peptides; their structures are illustrated in Figure S1,
To further validate our assay, we evaluated the uptake of two
therapeutic peptides and analyzed the correlations between
the permeability and cellular activity of these peptides. We se‐
lected two previously published p53‐HDM2 protein‐protein
interaction (PPI) peptide inhibitors known to have high affin‐
ities for their targets in cell‐free assays but different cellular
activities in live cells. In particular, we evaluated a fluorinated
and the synthetic procedures are summarized in the Support‐
ing information. We then investigated the kinetics and total
uptake of these peptides at different time points in breast can‐
cer cells (MDA‐MB‐231‐luc) using the SLP assay (Figure 3E, F
and Figure S6). We observed that during the first 10 min after
SLP addition, the octaarginine peptide (r
8
C‐c) demonstrated
D
5‐ and 3‐fold higher light output than the corresponding TAT
analog of the linear peptide PMI‐γ (HDM2‐LP) that is known
and Penetratin analogs, respectively (Figure 3F). The rate
constants that represent the rate of uptake calculated from the
curves are summarized in Table S1.These data are consistent
to have poor cellular uptake, and a stapled peptide, sMTide‐
02A (HDM2‐SP), known for its remarkable cellular activity.44,
45
To test these peptides using our SLP assay, both peptides
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were labeled with D‐Cys through a mercaptopropionate linker
at the N‐terminus, which is a part of the molecule that is easily
accessible for derivatization (HDM2‐LP‐c and HDM2‐SP‐c,
Figure S1). The cellular permeability of these D‐Cys‐tagged
peptides at two different concentrations (1 and 20 µM) was
then evaluated using an SLP assay with the SJSA‐1‐luc cell line
previously used for p53‐HDM2 PPI inhibitor activity studies44,
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45.
As described previously, the cells were first pretreated with
50 µM CBT and subsequently incubated with the peptides of
interest at the two different concentrations, 1 and 20 µM. The
total signal output was calculated from the area under the
curve (AUC) values of the corresponding kinetic curves 24 h
after incubation (Figure S8). Interestingly, the biolumines‐
cent signal for HDM2‐SP‐c was approximately 2‐fold higher
than that of HDM2‐LP‐c after incubation at a concentration
of either 1 or 20 µM. These data are fully consistent with the
activity profiles and previous data on the cellular permeability
of these two therapeutic peptides, suggesting a greater cellular
response should be expected for the stapled peptide.44, 45
Noninvasive real‐time imaging and quantification of
peptide uptake in vivo using the SLP assay
Figure 4. Noninvasive real‐time imaging and quantification
of peptide uptake in vivo using the SLP assay. (A) FVB‐Luc+
luciferase reporter mice were divided into 3 groups. The CBT
To investigate the feasibility of the SLP uptake assay in live
+
group (n=4) received an i.p. injection of CBT. The r
group (n=6) received the same CBT injection followed by an‐
other i.p. injection of the r C‐c peptide 3 min later. The E4C‐
8
C‐c+CBT
animals, we utilized FVB‐Luc luciferase reporter mice that
ubiquitously express firefly luciferase under the β‐actin pro‐
moter.46 The mice were divided into 3 groups (n=4 (CBT), n=6
8
c+CBT group (n=6) received the same CBT injection followed
by i.p. injection of the E4C‐c peptide 3 min later. All mice were
anesthetized, and the bioluminescent signals were recorded
over 1 h using an IVIS Spectrum system. (B) The total photon
flux as calculated from (A) over a period of 1 h. The error bars
represent the ± SEM. ***P < 0.001; unpaired Student's t‐tests
were used for statistical analysis.
(r
jection of 100 µl of 1 mM CBT compound. Then, “r
E4C‐c” groups received i.p. injections of 100 µl of 1 mM of
8
C‐c), and n=6 (E4C‐c)) and all of them received an i.p. in‐
8
C‐c” and
“
their corresponding SLP solutions 3 min later. The mice were
then anesthetized, and the bioluminescent signal was rec‐
orded over 1 h using an IVIS Spectrum system. The data de‐
CONCLUSIONS
The SLP uptake assay represents a simple and flexible pro‐
picted in Figure 4A demonstrate the rapid uptake of r
8
C‐c,
cedure for real‐time monitoring of peptide uptake in live cells
and living animals. While this novel approach is fully comple‐
mentary to existing methods, it offers many advantages. Un‐
like other methods in which peptides are directly conjugated
to fluorescent dyes or MRI probes, the SLP assay requires only
a small modification with one amino acid (D‐Cys) that also
maintains the peptidic character of the original molecule.
Since the readout is dependent on the intracellular release of
D‐Cys, which occurs only in the reducing environment of the
cytosol, false positives from noninternalized or cell mem‐
brane‐bound peptides are fully eliminated. Importantly, this
which resulted in the production of a 3‐fold greater biolumi‐
nescent signal than that of the CBT‐only control (Figure 4B).
As expected, no significant difference was observed between
the signal from CBT alone and that from the nonpermeable
4
E C‐c peptide (Figure 4B). These results are in full agreement
with the data obtained for the cultured cells (Figure 2B,C).
Additionally, these results demonstrate that the SLP uptake
assay translates to in vivo studies in which important factors
such as the clearance, biodistribution and metabolic stability
of a peptide of interest need to be considered.
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probe design provides kinetic information in addition to in‐
from test‐tube and live cell experiments all the way to live an‐
formation on the total amount of peptide taken up inside the
cell at a given time point without the need for multiple sample
processing steps (such as those in MS‐based techniques). This
feature is very important for the comparison of different ther‐
apeutic lead candidates. For example, several previously re‐
ported studies have compared the performance of CPP pep‐
tides such as TAT, octaarginine and Penetratin and have re‐
ported conflicting results because the uptake of these peptides
was measured at different time points post peptide addition.18,
imal experiments, offering the additional advantage of screen‐
ing therapeutic leads in relevant in vivo disease models. Our
data demonstrate that the SLP uptake assay can be success‐
fully used to study peptide uptake in living animals, taking
into account the clearance, biodistribution, and metabolic
stability of a peptide.
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In conclusion, we present a novel, simple, sensitive method
to monitor the real‐time intracellular uptake of peptides both
in vitro and in vivo. Overall, we believe that the SLP uptake
assay will accelerate efforts towards the development of novel
peptide therapeutics and help to elucidate the often rather
complex internalization mechanisms of these peptides. In this
work, we have demonstrated the applications of this approach
to the imaging and quantification of peptide uptake. The same
method can be used for studies on other important biomole‐
cules, such as small‐molecule drugs, siRNAs, and nanoparti‐
cles (Movie 1). Several of these applications are currently on‐
going in our laboratory.
33, 47
Importantly, the uptake of these peptides, as determined
using the SLP assay, was consistent with that in previous re‐
ports at comparable time points.
Another important advantage of this assay is that SLP syn‐
thesis is based on standard synthetic protocols and is robust
and cost‐effective with high yields. The D‐Cys moiety can be
readily conjugated to existing Cys residues or introduced at
the C‐terminus or the N‐terminus of the peptide chain,
providing great flexibility and applicability of our method to a
wide range of therapeutic peptides or proteins. To increase as‐
say sensitivity, our efforts focused on lowering the high back‐
ground noise resulting from endogenous L‐Cys.28, 48 Despite
the fact that a considerable number of studies have utilized
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
the split luciferin approach for in vitro and in vivo applica‐
These data include supplementary methods and materials,
structures of the peptides used in the uptake studies, in vitro
validation of the SLP uptake assay, data from the MDA‐MB‐
tions,17, 32, 49, 50 this problem had not yet been addressed.50 We
found that quenching the background signal with excess CBT
prior to treatment with SLP yields remarkable signal‐to‐noise
ratios, with signals up to 60‐fold greater than background lev‐
els even at low nanomolar concentrations of the peptide. Im‐
portantly, we also observed a linear correlation between the
exogenous concentrations of the tested peptides and the re‐
sulting bioluminescent signals, indicating that the SLP uptake
assay is highly quantitative. Indeed, the SLP assay results for
linear and stapled versions of p53‐HDM2 PPI inhibitors were
in full agreement with previously published data suggesting
that a greater cellular response should be expected for the sta‐
pled peptide.44 Since the cellular uptake of a number of pep‐
tides can be assayed simultaneously on the same assay plate
and since the assay is based on a very simple procedure, the
SLP assay can be easily adapted for high‐throughput screening
2
8
31‐luc cells treated with r C‐c, data on SLP uptake imaging
comparison with the two different readouts, data on the total
uptake of various peptides at different time points, data on the
uptake of different cell permeable peptides in cells, data on
the cellular permeability of investigated peptide inhibitors
and supplementary synthesis schemes of various peptides
used in a study.
AUTHOR INFORMATION
Corresponding Author
*E‐mail: elena.goun@epfl.ch
Notes
The authors declare no competing financial interests.
Funding
Author H.K. received funding from Foundation Leenaards
grant 3699 and Intrace Medical, Switzerland; A.Y received
funding from IFNC Microbiome grant 14210 (EPFL, Switzer‐
land); T.M. received funding from NCCR chemical biology
(Swiss National Foundation; grant number 51NF40_185898);
(
HTS). Importantly, the assay can be carried out using com‐
mon, readily available plate readers and CCD cameras with
comparable results. Finally, only very few assays can translate
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T.V. G.J.H. and P.L.A. were supported by Novartis, Switzer‐
land.
16. Wuest, M., Perreault, A., Kapty, J., Richter, S., Foerster, C.,
Bergman, C., Way, J., Mercer, J., and Wuest, F. (2015) Radio‐
pharmacological evaluation of 18F‐labeled phosphatidylser‐
ine‐binding peptides for molecular imaging of apoptosis,
Nucl. Med. Biol. 42, 864‐874.
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