BioShuttle mobility in living cells studied with high-resolution FCS & CLSM methodologies.
ABSTRACT With the increase in molecular diagnostics and patient-specific therapeutic approaches, the delivery and targeting of imaging molecules and pharmacologically active agents gain increasing importance. The ideal delivery system does not exist yet. The realization of two features is indispensable: first, a locally high concentration of target-specific diagnostic and therapeutic molecules; second, the broad development of effective and safe carrier systems. Here we characterize the transport properties of the peptide-based BioShuttle transporter using FFM and CLSM methods. The modular design of BioShuttle-based formulations results in a multi-faceted field of applications, also as a theranostic tool.
- SourceAvailable from: Rüdiger Pipkorn[Show abstract] [Hide abstract]
ABSTRACT: The personalized medicine, also documented as "individualized medicine", is an effective and therapeutic approach. It is designed to treat the disease of the individual patient whose precise differential gene expression profile is well known. The trend in the biomedical and biophysical research shows important consequences for the pharmaceutical drug and diagnostics research. It requires a high variability in the design and safety of target-specific pharmacologically active molecules and diagnostic components for imaging of metabolic processes. A key technology which may fulfill the highest demands during synthesis of these individual drugs and diagnostics is the solid phase synthesis which is congenial to automated manufacturing. Additionally the choice of tools like resins and reagents is pivotal to synthesize drugs and diagnostics in high quality and yields. Here we demonstrate the solid phase synthesis effects dependent on the choice of resin and of the deprotection agent.International journal of medical sciences 01/2013; 10(3):331-7. · 1.55 Impact Factor
Int. J. Med. Sci. 2012, 9
I In nt te er rn na at ti io on na al l J Jo ou ur rn na al l o of f M Me ed di ic ca al l S Sc ci ie en nc ce es s
2012; 9(5):339-352. doi: 10.7150/ijms.4414
BioShuttle Mobility in Living Cells Studied with High-Resolution FCS & CLSM
Kiyoshi Nokihara5, Wolfhard Semmler1, Jürgen Debus6, Waldemar Waldeck2
, Marcel Beining2, Manfred Wiessler1, Twan Lammers3, Rüdiger Pipkorn4, Ute Hennrich1,
1. German Cancer Research Center, Dept. of Imaging and Radiooncology, INF 280, D-69120 Heidelberg, Germany.
2. German Cancer Research Center, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany.
3. Utrecht University, Dept. of Pharmaceutics, Sorbonnelaan 16, NL-3584 Utrecht, The Nederlands.
4. German Cancer Research Center, Division of Biophysics of Macromolecules, INF 580, D-69120 Heidelberg, Germany.
5. HiPep Laboratories, Nakatsukasa-cho, 486-46, Kamigyo-ku, Kyoto 602-8158, Japan.
6. Heidelberg University Hospital, Dept. of Radiation Oncology, INF 400, D-69120 Heidelberg, Germany.
Corresponding author: Klaus Braun, Ph.D. German Cancer Research Center (DKFZ), Dept. of Imaging and Radiooncol-
ogy, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Phone: +49 6221-42 2495 Fax:
+49 6221-42 3326 e-mail:
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2012.03.29; Accepted: 2012.06.18; Published: 2012.07.01
With the increase in molecular diagnostics and patient-specific therapeutic approaches, the
delivery and targeting of imaging molecules and pharmacologically active agents gain increasing
importance. The ideal delivery system does not exist yet. The realization of two features is
indispensable: first, a locally high concentration of target-specific diagnostic and therapeutic
molecules; second, the broad development of effective and safe carrier systems. Here we
characterize the transport properties of the peptide-based BioShuttle transporter using FFM
and CLSM methods. The modular design of BioShuttle-based formulations results in a mul-
ti-faceted field of applications, also as a theranostic tool.
Key words: Drug Delivery, BioShuttle, Confocal Laser Scanning Microscopy-CLSM, Fluorescence
Correlation Spectroscopy-FCS, Fluorescence Fluctuation Microscopy-FFM, Ligation chemistry,
Non-Viral Carrier Systems, Spinning Disc Microscopy–SDM.
The fields of molecular imaging, drug targeting,
bioinformatics and biomedical research are currently
merging in to the expanding field of theranostics [1-3].
The theranostic trends change increasingly from sys-
temic towards patient-specific strategies [4-6]. Instead
of native nucleic acids whose use as a drug is ham-
pered by their nuclease sensitivity, the use of
amine-polymers functionalized with the potential of
Watson-Crick binding lacking nuclease sensitivity can
produce relief [7-10]. The cell membrane however,
forms an almost impassable obstacle. This basic
problem of nucleic acid-based active molecules, re-
sponsible for their poor uptake into living cells and
tissues, remains to be solved [11-14]. Great efforts
resulted in multifaceted methods of viral [15-17] and
non-viral carrier [18-22] solutions. Peptide-based
molecules which harbour cell membrane penetrating
properties are documented as CPP (cell penetrating
peptides) [23-27]. In our hands, one approach to cir-
cumvent this hurdle and enter cells is based on func-
tional peptides which are modularly composed de-
pending on their conceptual formulation, hereafter
called “BioShuttle” : BioShuttle-based formula-
tions employ amphiphilic peptides, ligated to their
Int. J. Med. Sci. 2012, 9
functional groups via disulfide bridge formation, for
transport across the cell membrane. By the use of dif-
ferent cell immanent mechanisms, based on reductive
conditions located in the cytoplasm, the disul-
fide-bridges can be cleaved. As a consequence, the
CPP-part of the BioShuttle is separated after passage
across the cell membrane from a nuclear localization
sequence (NLS) -based address moiety [28-30], ena-
bling the delivery of imaging molecules and/or
pharmacologically active components into the nucle-
us [11, 31]
To provide an experimental proof-of-principle
for these features, three BioShuttle constructs were
tested, which are illustrated schematically in Figure 1:
A. The CPP-Rd110 consists of the pAntp peptide
fragment which is labeled with Rhodamine 110 at the
N-terminus. The pAntp’s (CPP) amino acid sequence
is KKWKMRRNQFWIKIQRC which facilitates the
passage of molecules across the cell membrane. All
constructs are able to pass through cell membranes
into the cytosol via the CPP.
B. The double-labeled BioShuttle construct (At-
to647N)-CPP-SS-NLS-(Atto488) comprises pAntp
(CPP) (KKWKMRRNQFWIKIQRC) conjugated to a
NLS (VKRKKP) module by a disulfide linker. Both
modules are conjugated to fluorescent dyes (coupled
by a peptide bond to the N-terminus or to lysine, re-
spectively): the CPP is labeled with Atto647N and the
NLS sequence is labeled with Atto488. After crossing
the cell membrane, the linker between the modules
should be cleaved and the NLS should be transported
into the nucleus. Hence, cytoplasm and nucleus will
be labeled differently. After cleavage, the residual
fluorescently labeled NLS can diffuse freely and can
actively enter the cell nucleus.
C. The CtsB-PNA construct’s structure is more
: The CPP is coupled to a peptide nucleic acid
(PNA) sequence by a disulfide linker which can be
cleaved by intracellular disulfide reductases. The
PNA sequence (in italic) acts as an antisense sequence
molecule and should hybridize specifically with the
mRNA transcript of cathepsin B, which is a peptidase
overexpressed in many (metastatic) cancer cell lines.
A short peptide sequence (GFGRK) inserted between
the complementary PNA and the nuclear localization
sequence (NLS) module acts as a substrate, enabling
specific cleavage by cathepsin B . Nuclei of cells
with normal Cathepsin B expression without an active
gene product should be fluorescence-identifiable
barely by this construct. This experiment should
demonstrate the potential of FFM for imaging of gene
expression in diagnostics. Here we used as an exam-
ple the CtsB gene encoding the matrix metal protein-
ase CtsB whose activity is associated with tumor ma-
lignancy [34, 35].
Whereas functional modules associated with
transmembrane-transport facilitating properties, like
the cell penetrating peptides[12, 15, 23, 36, 36-39], nu-
clear localization sequences [28, 40-46] and the anti-
sense molecules [47-53] have been broadly character-
ized, the intra- and extracellular dynamics and the
subcellular distribution of modular carrier materials
containing such moieties have not been evaluated in
great detail. Here, the mobility of the three above-
mentioned BioShuttle constructs was investigated by
fluorescence correlation spectroscopy (FCS). In FCS,
dynamic parameters of a fluorescent sample are de-
rived from statistical fluctuations in fluorescence in-
tensity in a small detection volume of a confocal mi-
croscope . These fluctuations are mainly induced
by the diffusion of the fluorescent probe through the
observation volume. The high spatial resolution and
the applicability of FCS also in vivo are powerful tools
for measuring the local concentration and the diffu-
sion coefficient in different subcellular compartments
. In addition, BioShuttle-facilitated transmem-
brane transport, nuclear targeting and intracellular
localization were investigated using an adapted con-
focal laser scanning microscopy (CLSM) protocol.
Figure 1. illustrates schematically the investigated Bioshuttle-conjugates.
Int. J. Med. Sci. 2012, 9
Material & Methods
Cells lines [HeLa and HeLa S3 (suspension cul-
ture), MCF7 and TP366 cells] were maintained in
DMEM (RPMI1640 for HeLa S3) medium without
phenol red and with 10 % fetal calf serum at 37°C in a
humid atmosphere with 5 % CO2 and passaged twice
in a week. Two days before the measurements, about
1 105 cells were transferred into Lab-TekTM Number
Fluorescence Fluctuation Microscopy
Briefly, to investigate the molecular diffusion
and localization, a custom-made  Fluorescence
Fluctuation Microscope (FFM), combining an FCS
unit and a CLSM, was used. More details are pub-
lished by the Langowski group . The cus-
tom-made FFM software allowed taking pictures of
the cells and measuring diffusion at selected loci in
the cells. A diffusion model was fitted to the data to
calculate 1) the diffusion coefficient, 2) the number of
particles, and 3) the triplet states, using the cus-
10626451.html). Data with large fluctuations due to
aggregated fluorescent molecules were filtered out.
The FFM was calibrated every day with standard
fluorophores (i.e. Alexa Fluor® 488 for the 488 nm
laser; and Cy5 for the 647 nm laser [Life Technologies,
Germany]. Both were used at a concentration of
PerkinElmer Spinning Disc confocal micros-
copy (SDM) – PerkinElmer Ultra View
SDM was carried out with a PerkinElmer Spin-
ning Disc confocal microscope (Nikon Ti-inverse mi-
croscope). It produces high gray scale quality pictures
due do the specific construction type and the rotating
pinhole disc which allows the generation of high
speed pictures of cells (exemplarily HeLa S3 cells).
They exhibit a xyz-scan by use of an Ar/Kr laser
(488/568/647 nm). The detection was carried out with
a Hamamatsu EM-CCD camera (high-sensitive-grey-
The excitation and emission spectra of fluores-
cent substances were measured with a fluorimeter
(Aminco SLM 8100 SLM, Urbana, USA). As a stand-
ard, a highly concentrated Rhodamin B (RdB) solution
was used. All spectra were recorded at room temper-
Absorption spectra measurements
The absorption spectra were recorded with a
Cary 4E spectrophotometer (Varian, Mulgrave, Aus-
tralia) at room temperature. It is suitable for wave
lengths of 175-900 nm with an accuracy of 0.001 opti-
cal density (OD). The molar extinction coefficients of
the dyes were obtained from the manufacturer's web-
Synthesis of BioShuttle conjugates
(KKWKMRRNQFWIKIQRC) and the NLS (VKRKKP)
as well as of the PNA (cagcgctgcag), we employed the
Fmoc-strategy [57, 58] in a fully multiple automated
synthesizer Syro II (MultiSyn Tech, Germany). Pep-
tide chain assembly was performed using in situ ac-
tivation of amino acid
hexafluorophosphate (HBTU). The PNA sequences
were synthesized on a PetiSyzer®  in the HiPep
Laboratories Kyoto Japan and details are described
elsewhere . The different markers [Atto488, At-
to565 and Atto647N (Atto-TEC, Germany); Rhoda-
mine 110 (Santa Cruz Biotechnology, USA) were in-
troduced as free acids or succinimidyl esters. A disul-
fide bridge between the N-terminal cysteine of dro-
sophila’s antennapedia derived peptide fragment
“pAntp” CPP and the N-terminal cysteine residue of
the NLS was formed by the activation of one cysteine
using 2,2’-dithiopyridine and coupling to the cysteine
of the other module. The coupling reaction was car-
ried out in an ethanol / water buffer at 60 °C. The
individual components and the complete conjugates
(A, B, C) were validated using LCMS (Schimadzu
LC-10 and LCQ electrospray Finnigan). The purity
levels were generally >90%.
phase synthesis of the CPP
building blocks by
Purification of samples
To separate free dye from BioShuttle-conjugated
dye, we used Vivaspin 500 3.000 MWCO centrifugal
units (Sartorius, Göttingen, Germany) in TE buffer
(Tris/EDTA) containing 0.02 % of the detergent
Nonidet P40 (NP40) (Roche, Mannheim, Germany).
The concentration was assessed using the absorption
spectrometer (in silanized glass cuvettes). Two hun-
dred µl of the sample was pipetted into the Vivaspin
centrifugal unit and centrifuged at 11.000 rpm for 10
min as described by the manufacturer. The filtrate
was measured again.
Int. J. Med. Sci. 2012, 9
Optimizing Fluorescence correlation spec-
troscopy (FCS) measurements
Determination of the stability of measurements
For the determination of the stability of the FCS
measurements, n = 60 measurements of Alexa Fluor®
488 were conducted. Figure 2a shows the au-
to-correlation functions (ACFs) of these measure-
ments. It is evident that all ACFs describe the identical
particle with low divergence.
Calibration measurements with Alexa Fluor® 488
In order to correlate diffusion with molecular
mass, we compared small (Atto565) and large mole-
cules (A: CPP-RD 110) (Figure 1). We measured dif-
fusion coefficients of A under normal conditions
(5 µW laser power, room temperature, calibrated FCS,
dimmed room, etc.) (Figure 2). The standard deviation
of 4.2 % verifies stable measurement, accuracy and
precision. Arithmetic average and standard devia-
tions were calculated (
24.59 µm2/s as shown in Figure 2c.
) and resulted in 586.13
Influence of Alexa Fluor® 488 concentration on the stability
of measurements in solution
The influence of the concentration on the diffu-
sion rate was determined with an Alexa Fluor® 488
dilution series from 0.2 up to 200 nM (n = 4 for each
concentration; 30 s). Figure 2b shows the inverse
proportionality of the decline of the amplitude while
increasing the molar concentrations of the fluorescent
Influence of measurement temperature on the diffusion
In order to optimize the influence of the meas-
urement temperature on the diffusion behaviour of
the samples in solution, we first tested an Alexa Flu-
or® 488 calibration solution at 11 °C, 25 °C, and 37 °C,
respectively (n = 4 test runs; 20 s; see Figure 2c).
Figure 2. a) represents the auto-correlation functions (ACFs) of the performed Alexa Fluor® 488 measurements (n = 60). b) illustrates
the decline of the autocorrelations under increasing concentrations of Alexa Fluor® 488 dye. Colour-coded curves of the dilution series:
0.2 nM (blue), 2 nM (red), 10 nM (yellow), 20 nM (magenta), 50 nM (black), 100 nM (brown), 200 nM (green). c) shows the graphs of the
diffusion coefficients (ordinate) and the temperature profile (abscissa).
reference values. d) shows the plots of the corresponding diffusion times [µs] (red ordinate) and the focus amplitude (blue ordinate), the
abscissa represents the laser output [µW]. The standard deviations of the diffusion times decreased (
and in parallel the focus amplitude (excitation volume) increased (
, , are equivalent to one and four runs and represents
) when the laser output increased,
)] (n = 6).
Int. J. Med. Sci. 2012, 9
Influence of laser intensity on diffusion time
In order to receive adequate correlation func-
tions, sufficient fluorescence signal intensities are
needed. An increased laser power illuminates the ef-
fective focus volume more intensely. Therefore the
influence of the laser output on the calculated focus
amplitude as well as on the precision of the diffusion
times was investigated using a standard solution of
Alexa Fluor® 488 at several laser outputs (n = 6 test
runs; 30 s). The resulting diffusion times were calcu-
lated using Quickfit 2 software and graphically illus-
trated as shown in Figure 2d.
Estimation of fluorophore diffusion properties
The diffusion characteristics of the fluorophores
used in the study were determined by FCS at 25 °C in
TE buffer (6 runs; 30 s). Table 1 provides an overview
of the measured values. As reference dyes for the de-
termination of the actual focus aperture, Alexa Fluor®
488 (488 nm), Rhodamin B (Rd B, 568 nm) and Cy5
(647 nm) were investigated. Except for the Atto674N,
the measured diffusion coefficients oscillate around
the corresponding literature values during the cali-
bration procedure [60-62].
Table 1. demonstrates the conclusion of the FCS charac-
teristics. For a better understanding, the dyes are arranged
according to the increasing MW.
diffusion coefficient [µm²/s]
502,760 ± 18,963
418,308 ± 56,748
392,243 ± 13,927
414,579 ± 14,295
546,647 ± 36,881
370,070 ± 9,640
448,956 ± 32,455
*) Fluka (Sigma-Aldrich), Switzerland.
Diffusion measurements of Atto565 mixed
with CPP-Rd110 in vitro - calibration of the
Fluorescence fluctuation microscope (FFM)
For investigation in more detail, we compared
the Atto565 fluorescent dye and the conjugate
CPP-Rd110 (A) at a final concentration of 20 mM dis-
solved in TE buffer (Tris/EDTA). A further
CPP-Rd110 probe mixed with 0.02 % NP40 detergent
was evaluated to avoid sticky effects. The FFM meas-
urements were accomplished in µ-slides as detailed
in , and the resulting graphs are shown in Figure
Figure 3. displays the dependency of different FFM parameters on
the measuring depth with Atto565 [20 mM]. The calculated par-
ticle number (black) and count rate (blue) feature a near identical
run of the curves approximating the glass surface whereas the
diffusion time (red) remains relatively constant. The graph of the
averaged ACF (brown /) shows a higher diffusion time slightly
increasing near the glass surface.
Impact of construct A and Atto565 on glass
cuvette surfaces - measurements of the laser
intensity and diffusion time dependent on FFM
calibration in vitro.
Up to 4 µW of laser power, the focus amplitude
increased nearly linearly whereas at higher laser
powers, it was saturated (Figure 2d). The data show a
clear proportionality between the count rate and the
calculated particle number, suggesting a concentra-
tion gradient. The constant diffusion time (32 µs) re-
veals neither an aggregation nor a structural adhesion
(Figure 3). The FFM measurements with CPP-Rd110 A
and Atto565 with and without NP40 are shown in
The probe treated with NP40 clearly offers a re-
duced adsorption, which is confirmed by the constant
count rate as well as by the constant particle number.
The distance from the surface between 4 and 20 µm as
well as the slight increase near the measuring cham-
ber’s basement was taken into account. Graph B rep-
resents the diagrams of the probe treated with NP40
and shows an increase of the diffusion time under a
distance of 3 µm.
Int. J. Med. Sci. 2012, 9
Figure 4. the graphs display the dependency of different FFM parameters on the measuring depth with CPP-Rd110 A [20 nM] without (A)
and with NP40 detergent (B) [0.02 %]. The abscissa represents the spatial distance of the measured molecule from the glass’s surface
[µm]. Graph A demonstrates diagrams of the particle number (black), the counting rate (blue) [kHz], and the diffusion time (red) [µs]
similar to the Atto565 diagrams (Figure 3).
In vitro test of the cleavage of the disulfide
bridge of B using β-mercaptoethanol and di-
An important step for the nuclear transport of
the cargo by the BioShuttle, is its separation from the
pAntp module (CPP) after reaching the cytosol. Since
the pAntp module is linked to the rest of the construct
via a disulfide bridge linker, it should be cleaved in-
tracellulary by cytosolic reductases. In vitro, this mol-
ecule could be cleaved using strong reducing agents
such as β-mercaptoethanol (2-Me) and dithio-threitol
(DTT). The separation was validated using FCS, re-
vealing an increased diffusion coefficient after cleav-
age in both cases (Table 2). The system can be used in
a pH range of 6.5-9.0. In order to analyze the separa-
tion of the modules in TE buffer, the (At-
to488)-CPP-SS-NLS-(Atto647N) B BioShuttle conju-
gate was treated with 2-Me or DTT. The BioShuttle
construct had a final concentration of 40 nM. 2-Me
was used in a concentration between 50 mM and
250 mM, DTT at 50 mM.
BioShuttle applications - Addition of BioShut-
tle conjugates to cells
Low volumes of BioShuttle constructs dissolved
in buffers were used for application to cells in order to
avoid side effects of solvents such as NP40 and ace-
tonitrile. To obtain a homogenous distribution of the
molecules, 200 µl cell culture medium were mixed
with 0.5-3 µl BioShuttle conjugate and then distrib-
uted on the cells and incubated for 24 h or overnight
depending on the experiment. In live imaging ex-
periments, BioShuttle administration was directly
performed at the microscope stage after starting the
Fluorescence Imaging Studies in living cells
In HeLa and MCF7 cells, fluorescence imaging
studies were carried out. We investigated the effects
of CPP-Rd110 A on the phenotypical change of HeLa
and MCF7 cells. Single pictures and real-time shots
were generated by use of FFM and SDM microscopes,
equipped with highly sensitive CCD cameras or
Quantitative fluorescence analysis in HeLa S3
Two samples with 0.56 ml of HeLa S3 suspen-
sion cells and two samples with 0.56 ml of cell me-
dium (positive control) were treated with 50 µl (At-
to647N)-CPP-SS-NLS-(Atto488)-BioShuttle B to ob-
tain a final concentration
to647N)-CPP-SS-NLS-(Atto488)-BioShuttle B. As a
negative control, an untreated HeLa S3 cell suspen-
sion was used. All samples were mixed, incubated for
1 h at 37 °C and centrifuged at 800 rpm for 5 min. The
supernatant was analyzed in the absorption spec-
trometer to calculate the residual amount of (At-
to647N)-CPP-SS-NLS-(Atto488)-BioShuttle B. Glass
cuvettes were silanized before use.
of 1.46 µM (At-
Fluorescence analysis of Cathepsin B activity in
HeLa, MCF7 and TP366 cells
The status analysis of the CtsB enzyme activity
was performed with the BioShuttle conjugate C
(1.46 µM). The data were obtained by FFM method-
ologies under identical treatment conditions over 24 h
as described above (BioShuttle applications - Addition
of BioShuttle conjugates to cells).
Int. J. Med. Sci. 2012, 9
Contemplation of the BioShuttle measure-
The summarized parameters achieved by the in-
struments’ calibration procedures can be considered
as a complex but helpful analysis platform. It is
needed for a deeper characterization of the dynamics
of functional molecules, investigation of the transport
of active components and imaging of molecules after
crossing biological membranes.
Three different shuttle molecules were con-
structed and investigated. Molecule A was used to
analyze the transport into cells, molecule B to detect
the transfer into the nucleus after cleavage of the di-
sulfide-bridge in the cytoplasm, and molecule C to
examine the status of the activity of the Cathepsin B
Fluorescence fluctuation microscopy – FFM in
For the evaluation of effects of CPP-Rd110-
BioShuttle A in the FF microscope HeLa and MCF7
cell lines were used. The fluorescence micrographs
were taken 2.5 h after incubation with A in a final
concentration of 100 nM.
In the pictures
CPP-Rd110-BioShuttle A is located inside of cyto-
plasmic vesicles and compactly aggregated in a size
range between 0.5 and 3 µm. The nucleus is free of
in Figure 5, the
Spinning Disc Microscopy studies – SDM
In the investigated HeLa S3 cells, the confocal
equipment of the SDM (Perkin Elmer) realizes pic-
tures of good quality. The low sensitivity of the
available sensors, however, needed a high laser power
to get noise-free pictures, which resulted in dramatic
cell death. Therefore SDM was inappropriate for the
creation of real time pictures and monitoring of the
CPP-Rd110 BioShuttle’s A passage across the cell
membrane, but the SDM is ideally suited for Z-scans
of BioShuttle construct A to visualize it inside of the
cells (Figure 6).
The results obtained in Figure 5 are confirmed
here by scanning microscopy in the z-direction. A 3D
reconstruction is visualized as a movie by the fol-
lowing link and illustrates the spatial distribution of
the cytoplasm-located fluorescence signals whereas
the cell nucleus area appears empty. (The software
used was allocated at http://fiji.sc/wiki/index.php/
Design of the double-labeled Shuttle for
transport into the nucleus
In order to examine whether the construct can
reach the nuclei, we built an extended shuttle labeled
with Atto647N which contains a nuclear localization
sequence (NLS) labeled with Atto488 (schematic Fig-
ure 1, B). The specific non-interacting spectra are
shown in the atto-tec’s website http://www.atto-
tec.com. For excitation of the fluorescent dye, the
measurements with the (Atto647N)-CPP-SS-NLS-
(Atto488)-BioShuttle B required 488/647 nm wave
Figure 5. the confocal FFM micrographs exhibit the intracellular distribution of CPP-Rd110 A in HeLa cells (picture ) and in MCF7 cells
(picture ) 2.5 h after application. The red fluorescence signals are located almost exclusively in vesicle like structures inside of the
Int. J. Med. Sci. 2012, 9
Figure 6. illustrates the Z-scan through a HeLa S3 cell 1 h after incubation with CPP-Rd110 BioShuttle A [200 nM]. The series of pictures
show optical longitudinal sections [300 nm] taken from bottom to top. The total high of the stack amounts to 9.3 µm.
Quantitative fluorescence analysis in HeLa S3
The relative amount of the BioShuttle in (At-
B) was analyzed by assessing the intracellular fluo-
rescence intensity of BioShuttle treated and subse-
quently washed HeLa S3 suspension cells after de-
fined time periods. First, fluorimetric studies with
HeLa S3 cells were conducted as described in the
methods part for estimation of the relative amount of
the free fluorescent dyes originating from the (At-
Int. J. Med. Sci. 2012, 9
(Figure 1, B). As a negative control the conjugate
without fluorescent dyes and as a positive control
medium plus fluorescent dyes were used. The result-
ing spectra, as illustrated in Figure 7, confirm the
chemical stability of the construct. The negative con-
trol values were subtracted. Compared to the positive
controls (A488 and A647N, separately dissolved in the
medium), the intensities of the fluorescence signals
originating from the
(Atto488) B are lower.
Figure 7. shows the absorption spectra of the cells’s supernatants
1h after incubation with (Atto647N)-CPP-SS-NLS-(Atto488)-
BioShuttle B. The curves are colour-coded as follows: initial
concentration (black), probe 1 (blue), probe 2 (dark blue), su-
pernatant of the Hela S3 probe 1 (brown), supernatant of the Hela
S3 probe 2 (red).
Transmembrane transport, cleavage of the
S-S-bridge of BioShuttle B and nucle-
A CLSM-experiment for 60 min provided addi-
tional clarity about the transmembrane transport.
HeLa cells were examined with a Nikon A1 micro-
scope and after the
to647N)-CPP-SS-NLS-(Atto488)-BioShuttle B (100
nM), a time series was generated (10 s interval). As
shown in the left hand panel of Figure 8 upper part, 30
s after incubation, the cells 1 and 2 are visible as
non-fluorescent black areas. Sixty min after incuba-
tion as shown in the middle part and the right hand
picture shows a membrane-located strong red fluo-
rescence and inside of the cells 1 and 2 a clear green
fluorescence is seen. The spatial separation of the flu-
orescence signals proofs the cleavage of the disulfide
application of (At-
FCS diffusion studies of BioShuttle B in living
Two cells were selected (cells 1 and 2; high-
lighted in the center of the left panel of Figure 8) and
their intracellular red and green fluorescence signal
intensities were recorded over time (Figure 8). The
intracellular fluorescence intensities of the separated
BioShuttle B could be visualized. An interesting ob-
servance is given in the zoomed version of the cells of
interest (ROI): Inside of the cell (2) particular signals
with a mixed fluorescence are detectable (accentuated
by arrows). Cell cycle phenomena as well as spill over
effects can not be excluded and a background sub-
traction was not intended. Based on the long-term
experiment it is shown that the dynamics of the
transport of (Atto647N)-CPP-SS-NLS-(Atto488)-
BioShuttle B could be better highlighted by FCS
In order to deepen the knowledge of the diffu-
sion properties, the probe was measured with FCS in
different solutions at 25°C (at 488 nm and 647 nm).
The results are outlined in the Table 2. The measure-
ments offer intact B molecules with a slightly different
diffusion rate (line 2 and 4), wherein large molecules
behave slowly and smaller molecules diffuse much
faster (labelled cargo, line 1 and labelled CPP, line 3).
The measurements of B at 488 nm and 647 nm, rep-
resent the decelerated diffusion (42.179 and 46.247
µm²/s, respectively) of the intact (i.e. uncleaved) Bi-
oShuttle B conjugate (lines 2 and 4). The in living cells
cleaved conjugate B showed diffusion coefficient
values of 347.495 and 239.685 µm²/s, (lines 1 and 3)
which are lower but closer to the corresponding coef-
ficient values (414.579 and 546.667 [µm²/s]) of the pure
fluorescent dyes Atto647N and Atto488 (as shown in
Table 1, lines 4 and 5). Table 1 also reflects the com-
ponents of BioShuttle B after DTT mediated disulfide
cleavage (Table 2, lines 5 and 6). The separated com-
ponents (modules) of the double-labelled BioShuttle
B are shown.
The separated probes treated in vitro with DTT,
enabling disulfide bond cleavage, show diffusion co-
efficient values of 185.499 and 223.168 µm²/s respec-
tively, indicating the retarded diffusion behaviour of
both components after cleavage of the disulfide bond
(Table 2, lines 5 and 6). The divergent coefficients
demonstrate an independent diffusion.
Amplification cytoplasmic retention and
cleavage of CtsB-PNA-BioShuttle C
In order to achieve specific enrichment in areas
of enhanced CtsB gene expression, a peptide-nucleic
Int. J. Med. Sci. 2012, 9
acid (PNA) complementary to CtsB mRNA was used
in the design of BioShuttle C, to serve as an anchor
inside treated cells by formation of CtsB mRNA/
CtsB-PNA hybrids. Twenty four hours after incuba-
tion with CtsB-PNA-BioShuttle C (final concentration
100 nM), HeLa, MCF7 and TP366 cells were investi-
gated using FFM. All three cell lines show clear fluo-
rescence signals in the cytoplasm.
Figure 8. The upper part illustrates the identical cross section of pictures at the time points of 30 sec (left) and 60 min (right) of a
long-time uptake-experiment during 60 min with (Atto647N)-CPP-SS-NLS-(Atto488)-BioShuttle B into HeLa cells. The pictures rep-
resent a superimposition of the red and the green channels. The cells, marked 1 and 2 (left picture), were used for FCS measurements. The
zoom into the image reveals particular signals with mixed fluorescence (3 arrows in the right picture). The graphs in the lower part of
the figure visualize the intracellular fluorescence intensities relating to the separated transporter (Atto647N)-CPP-SS-NLS-(Atto488) B.
The two arrays of curves indicate the intensity of the respective dye. The upper curves represent the fluorescence of Atto488, the lower
array curves that of Atto647N in two selected cells during the time series under the Nikon A1Rsi. No saturation of the signal intensity is
Table 2. lists the diffusion characteristics of (Atto647N)-CPP-SS-NLS-(Atto488) B. The data confirm high quality puri-
fication and a cleavable disulfide bridge. The probe was measured in 100 mM TE-buffer, containing DTT.
Int. J. Med. Sci. 2012, 9
Figure 9. shows confocal FFM images of HeLa (left), MCF7 (middle), and TP366 (right) cell lines 24 h after incubation with
CtsB-PNA-BioShuttle C. All investigated cell lines show the successful uptake of CtsB-BioShuttle C as shown by the cytoplasm located
fluorescence signals. In HeLa cells (left picture) the fluorescence signal is identified perinuclearly inside of the cell; however, no signal is
shown inside of the nucleus. The middle picture illustrates MCF7 cells whose nuclei show band like fluorescence signals near the nuclear
envelope and inside of the nuclei. TP366 glioma cells (right picture) show band like fluorescence signals inside of the cell nucleus.
The picture in the middle also presents clear cy-
toplasm located signals inside of the MCF-7 cells.
Additionally, vesicularly structured areas with high
fluorescence signals arranged perinuclearly are
shown which indicate CtsB gene expression.
It is notable that particular vesicle-shaped fluo-
rescence signals are depicted inside of the nucleus
near the nuclear envelope of MCF-7 cells. In TP366
glioma cells we detected clear fluorescence signals
spreading over the cytoplasm but also areas with a
much stronger fluorescence in the nuclei. i) PNA
trapping in cells with active CtsB enzyme resulted in
clear fluorescence signals inside of the cell nucleus
(Figure 9; right picture). Ii) Starting inside of the nu-
clear envelope to the core of the nucleus we could
identify lamellarly shaped fluorescence signals as a
network. Iii) This proofs accumulation of CPP by
separation (disulfide-bridge cleavage), retardation of
the molecule and a final nuclear transport.
The efforts in transporter or vehicle development
led to a myriad of viral vectors and non-viral pep-
tide-based carrier systems with cell penetrating
properties. Additionally, small functional peptides,
also responsible for subcellular targeting, like nuclear
localization sequences have been recognized and
used. Also different physico-chemical uptake charac-
teristics, like membrane-fusion and endocytotic
mechanisms as well as immunological effects of the
different membrane transport-facilitating molecules
were documented [16, 63-70]. However, despite pro-
gress in the diagnostic and therapeutic fields, the tar-
geted transport of imaging components and (nucleic
acid-based) pharmacologically active agents remains
A solution could be a combination of both: de-
livery and targeting systems by ligations of imaging
molecules as a cargo with peptides harbouring bio-
logic address informations which in turn are con-
nected to a cell penetrating peptide via disulfide
bridge formation. These can be selectively decom-
posed by enzymes, for instance by cytosolic disulfide
reductases. Sophisticated specifications for safe and
efficient delivery by such transporters are beyond
dispute [71-79]. The compilation of the transporter
specifications, however, needs specific parameters
which can account for their characterizations’ refine-
ment. Therefore, we here – as an example – investi-
gated the modular peptide-based BioShuttle drug
delivery and targeting system in more detail using
physical techniques. We used fluorescent molecules to
demonstrate the enzymatic cleavage of the two subu-
nits of the Atto488 and Atto647 double-labelled Bi-
oShuttle B conjugate (Figure 8). After cleavage of the
disulfide bridge, the NLS module labelled with
ATTO488 reaches the nucleus, whereas the mem-
brane-transport facilitating module (CPP = cell pene-
trating peptide), labelled with ATTO647N raises the
red fluorescence signals, membrane-located or in the
direct environment. The depiction of the processes is
documented [11, 80]. The FCS diffusion studies with
this BioShuttle conjugate B, indicate that the FCS
methodology turns out to be a suitable technique for
analyzing BioShuttle intactness and separation. The
variation in the diffusion behaviour can be considered
Int. J. Med. Sci. 2012, 9
as a meaningful parameter, as proven in Table 2.
Moreover, the data can confirm a high quality and
purification after solid phase peptide synthesis SPPS
and an intact cleavable disulfide bridge.
In addition, the correlation coefficients’ calibra-
tion procedures indicated measured values differing
from the literature values (Table 1). This phenomenon
is likely caused by physical interactions with the sur-
face of the glass cuvettes of the molecules functional-
ized with fluorescent dyes due to their physicochem-
ical properties . Each measurement step was fol-
lowed by a loss of probes caused by adhesion to pi-
pette tips and reaction chamber surfaces. This per-
ception is critical and demands intensive investiga-
tions on developing exigent standards not only for
proper chemical synthesis methodologies, but also for
the high quality of the educts and the intermediates
and for the surface coating, e.g. silanizing, of reaction
vessels, measurement chambers, and lastly for the
packing materials. The development of standards
according to pharmacopoeia principles, eligible for
pharmaceutical use in e.g. personalized medicine,
therefore is highly important.
A further interesting and important aspect in this
context is the use of enzyme activities (here CtsB),
which correlate with the invasive potential and be-
haviour of tumors. The BioShuttle’s modular compo-
sition is detailed in , and allows the insertion of a
peptide-based cathepsin B (CtsB) cleavage site, acting
as a substrate for the active CtsB enzyme [81-84]. The
molecular mechanism of C is documented . As a
further improvement, this BioShuttle variant C was
tested on cell lines with different CtsB status and was
visualized in Figure 9. Here the extent of the Atto488
fluorescence intensity and its subcellular localization
could be observed depending on the CtsB gene ex-
pression level and on the corresponding CtsB enzyme
activity status respectively [34, 85-87].
It is important to note that pre-treatment of cells
is critical for the CLSM and FCS studies. Therefore, to
avoid cellular artefacts which could distort the results,
only living and non-fixed cells were used. The com-
bined use of both methodologies may give insight into
new aspects of theranostics via physicochemical
characterization and can be consulted as a helpful
concept for the directly comparable characterization
of carrier and targeting constructs [88-90].
Care should be taken regarding unforeseen be-
haviour, like the adsorption of molecules at the cu-
vettes’ surfaces which can influence measuring results
as observed during fluorescence correlations spec-
troscopy (FCS) (Figure 3 and Figure 4). These diffi-
culties and unexpected proceedings should be defi-
nitely considered. Furthermore, physical and equip-
ment specific parameters in CLSM like laser intensi-
ties as well as sensitivity of the different sensors can-
not be ignored in the interpretation of the results
(Figure 2). It is generally agreed that the choice of
technical equipments deriving from different manu-
factures can impact the outcome of measurements, as
shown in the calibration studies. As shown here the
characterization of such complex conjugates is possi-
Strong fluorescence signals are located inside of
vesicular compartments indicating an internalization
of the BioShuttle conjugates into vesicular particles.
These phenomena were already observed and docu-
mented in previous BioShuttle publications  .
Their similarity to the structures documented as light
granulation triggered by physical interference reac-
tions of optical raggedly surfaces is evident . This
indicates that our observations in the CLSM meas-
urements as well as in the magnetic resonance imag-
ing (MRI) (not shown here) resemble the data derived
from the laser speckle contrast imaging studies (well
reviewed by Boas and Dunn in 2010) . In this re-
gard, BioShuttle carrier variants functionalized with
fluorescent dyes , and/ or with MR contrast
agents  can be considered as eligible tools in fluo-
rescent speckle microscopy and in molecular imaging
for studying intracellular metabolism dynamics in
vivo, as documented by the Keating group . Worth
mentioning is also a BioShuttle conjugate which can
be ligated to ultrasound contrast agents, as a tool in
the field of acoustically modulated speckle imaging
All in all, despite the modular BioShuttle’s drug
delivery platform multi-faceted applications, the
competent knowledge and the choice of many critical
physical parameters as qualified measurement
methods is pivotal. It is important for its further spec-
ification development, efficient drug delivery tools
and for a hopeful outlook to further applications in
the patient-specific medicine, not restricted to imag-
ing. The observations focused on the physi-
co-chemical properties of the imaging components
and of the functional molecules should be discussed
with respect to: 1) lipophilic/hydrophilic characteris-
tics, 2) distribution kinetics and dynamics within
dispersoids 3) for use as a theranostic tool in the per-
This work was supported by the Deutsche
Krebshilfe, D-53004 Bonn; Grant Number: 106335. The
authors are thankful to Gabriele Müller, Biophysics of
Int. J. Med. Sci. 2012, 9
Macromolecules Department, DKFZ and Mario Koch,
Central Peptide Synthesis Unit, DKFZ for their en-
The authors have declared that no competing
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