Controlled Supramolecular Assembly Inside Living Cells by Sequential Multistaged Chemical Reactions

Article abstract of DOI:10.1021/jacs.0c05261

Synthetic assembly within living cells represents an innovative way to explore purely chemical tools that can direct and control cellular behavior. We use a simple and modular platform that is broadly accessible and yet incorporates highly intricate molecular recognition, immolative, and rearrangement chemistry. Short bimodular peptide sequences undergo a programmed sequence of events that can be tailored within the living intracellular environment. Each sequential stage of the pathways beginning with the cellular uptake, intracellular transport, and localization imposes distinct structural changes that result in the assembly of fibrillar architectures inside cells. The observation of apoptosis, which is characterized by the binding of Annexin V, demonstrates that programmed cell death can be promoted by the peptide assembly. Higher complexity of the assemblies was also achieved by coassembly of two different sequences, resulting in intrinsically fluorescent architectures. As such, we demonstrate that the in situ construction of architectures within cells will broaden the community's perspective toward how structure formation can impact a living system.

Full text of DOI:10.1021/jacs.0c05261

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Article
Controlled Supramolecular Assembly inside Living
Cells by Sequential Multi-staged Chemical Reactions
Michaela Pieszka, Shen Han, Christiane Volkmann, Robert Graf, Ingo
Lieberwirth, Katharina Landfester, David Y.W. Ng, and Tanja Weil
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c05261 • Publication Date (Web): 19 Aug 2020
Downloaded from pubs.acs.org on August 19, 2020
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Controlled Supramolecular Assembly inside Living Cells by
Sequential Multi-staged Chemical Reactions
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Michaela Pieszka,1,2 Shen Han,1 Christiane Volkmann,1 Robert Graf,1 Ingo Lieberwirth,1 Katharina
Landfester,1 David Y.W. Ng1,* and Tanja Weil.1,2,*
1Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany
2Institute of Inorganic Chemistry I, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany
Supramolecular Chemistry, Self-Assembly, Nanoscale Architectures, Boronic Acids, Peptides
ABSTRACT: Synthetic assembly within living cells represents an innovative way to explore purely chemical tools that can
direct and control cellular behaviour. We use a simple and modular platform that is broadly accessible and yet incorporates
highly intricate molecular recognition, immolative and rearrangement chemistry. Short bi-modular peptide sequences un-
dergo a programmed sequence of events that can be tailored within the living intracellular environment. Each sequential
stage of the pathways beginning with the cellular uptake, intracellular transport and localization imposes distinct structural
changes that result in the assembly of fibrillar architectures inside cells. The observation of apoptosis, which is character-
ized by the binding of Annexin V, demonstrates that programmed cell death can be promoted by the peptide assembly.
Higher complexity of the assemblies was also achieved by co-assembly of two different sequences, resulting in intrinsically
fluorescent architectures. As such, we demonstrate that the in-situ construction of architectures within cells will broaden
the community’s perspective towards how structure formation can impact a living system.
from a chemotherapeutic agent or singlet oxygen produc-
tion by metal complexes.10, 11 In contrast, specific biological
responses, like programmed cell death, driven purely by
the formed self-assembled nanostructures are less known.
We envisioned that the assembly of non-functional con-
stituents into functional architectures directly in a living
cell would not only be a significant milestone in nanobi-
otechnology, but also provide a platform to integrate syn-
thetic chemistry with living processes.
Herein, we report the construction of two peptide se-
quences designed to undergo a multi-stage transformation
that results in the assembly of fibrillar architectures inside
cancer cells (Figure 1). The first stage, comprising the
cellular entry process, is gated by a pH-dependent boronic
acid-salicylhydroxamate complexation.12 This chemistry
links a transporter “TAT” sequence (trans-activator of
transcription), derived from the human immunodeficiency
virus (HIV),13 together with a pro-assembling sequence
(henceforth referred as depsipeptide). As such, upon suc-
cessful endocytosis, the acidification within the intracellu-
lar vesicles releases the pro-assembling sequence. Here,
the second and third stage is incorporated into the pro-
assembling sequence, with the second stage guarded by an
immolative boronic acid cage sensitive towards elevated
or endogenous H2O2 within cancer cells.14-16 Upon the im-
molation of the cage, the third stage is triggered by the
O,N-acyl rearrangement of the depsipeptide that generates
the linear isoleucine-serine-alanine (ISA) self-assembling
motif.17, 18 The production of ISA promotes the final stage
of self-assembly into fibrillar architectures and in the pro-
cess triggers apoptosis.
INTRODUCTION
Supramolecular interactions govern core aspects of cellu-
lar life where they are omnipresent in every biological
pathway. On the molecular level, non-covalent forces guide
structure formation and biomolecular interactions, which
can be seen within the DNA double helix, the secondary to
quaternary structures of proteins, and the dipoles of lipids.
Systematically, the individual assemblies propagate into
interconnecting systems to perform DNA replica-
tion/transcription, protein folding/receptor interactions
and shuttling molecules in and out of cells.1 The resulting
dynamics between these biological processes would thus
define the fundamental elements of life (i.e. proliferation,
homeostasis, metabolism).
As a whole, it is critical to realize that many of these as-
semblies elicit their function at the nanometer level, while
their separate constituents are seemingly non-functional
(i.e.
nucleotides/DNA,
fatty
acids/vesicles,
rRNA/ribosome).1 Therefore, instead of using intrinsically
bioactive components like proteins or DNA, the impact of
nanoscience towards biology can also be realized through
structure formation. Application wise, there has been a
growing interest in methods to enrich and accumulate
drug molecules within cells to circumvent efflux based
drug resistance.2, 3 As the rate of efflux of molecules is di-
rectly dependent on size,4, 5 significant efforts have been
made to direct drug/imaging molecules to form aggregates
within cells6, 7 and with promising in vivo results.8, 9
Nonetheless, the bioactivity of these systems often origi-
nates from known small molecule interactions such as
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Figure 1: Intracellular co-assembly of peptides. a, Depsipeptides (kinked arrows) are uptaken by cells due to dynamic covalently
bound salicylhydroxamate-TAT (SHA-TAT, red strang, step A). After hydrolysis of the complex in acidic environments (step B), the
boronic acid head group of the depsipeptides is cleaved by intracellular hydrogen peroxide (step C). The subsequent O,N-acyl shift
forms the linear co-assembling peptides (step D). The linear peptides (straight arrows) form fibrillar networks inside A549 cells
(step E), which is visible by transmission electron microscopy (TEM, scale bar 500 nm). b, Chemical reactions that lead to cellular
uptake, peptide linearisation and peptide co-assembly of Fmoc (green) and Coumarin 343 (blue) functionalised ISA.
In essence, the design comprises three modular compo-
nents: (1) the pro-assembling depsi unit and its pH-
reversible functionalisation with TAT, (2) the peroxide-
triggered cleavage of the boronic acid masking group and
the (3) pH-controlled O,N-acyl rearrangement to generate
the self-assembling peptide sequence. In this way, intracel-
lular transport, release and supramolecular assembly into
peptide fibrils is individually and sequentially pro-
grammed inside different cellular compartments by con-
secutive chemical reactions (Figure 1).
Additionally, we demonstrate co-assembly as a strategy to
increase the level of functionality by imparting fluores-
cence into the fibrillar structures to allow imaging.19, 20 Co-
assembly, i.e. the assembly of more than a single compo-
nent, is prevalent in Nature and important examples in-
clude the assembly of α-/β-tubulin in microtubules21, cho-
lesterol/phospholipids in membranes22 or the Arp2/3
complex in actins.23 While Nature uses highly specific pro-
teins to transport and program these assemblies, synthetic
methods are advantageous as they can be bioorthogonal
and also be specifically tailored. By incorporating sophisti-
cated chemical designs into a simple bi-modular peptide
sequence, we demonstrate that synthetic architectures can
be formed directly within living systems using natural
triggers.
RESULTS AND DISCUSSION
Solid phase peptide synthesis using fluorenylmethoxycar-
bonyl (Fmoc) chemistry was conducted with alanine pre-
loaded Wang resin (Scheme 1). Fmoc-serine was added as
the second amino acid using (benzotriazol-1-
yloxy)tripyrrolidinophosphonium
hexafluorophosphate
(PyBOP) and N,N-diisopropylethylamine (DIPEA).24 Im-
portantly, Fmoc-serine was used without a protecting
group on the hydroxyl group in order to create an ester
bond with isoleucine later in the synthesis. After Fmoc
deprotection of serine, the modification of the N-terminus
using 4-(nitrophenyl)phenylboronic acid pinacol ester was
performed. Fmoc-isoleucine was coupled on the amino
acid
side
chain
of
serine
(DIC)
using
and
N,N′-
diisopropylcarbodiimide
4-
(dimethylamino)pyridine (DMAP), forming the ester and
therefore the so called depsipeptide.17 The peptide was
removed from the solid support, and the boronic acid was
deprotected simultaneously using a cleavage cocktail
based on trifluoroacetic acid (TFA). After purification by
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high-performance liquid chromatography (HPLC), the pure was found in the sample. Incubation of Depsi(C343-I)pba-
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peptide 1a was characterised by electrospray ionisation
mass spectrometry (ESI-MS, Figure S6). In order to synthe-
sise the fluorescent Depsi(C343-I)pba-SA (Depsi(Coumarin
343–isoleucine)phenylboronic acid–serine–alanine), the
Fmoc group was removed using piperidine, and the pep-
tide was modified with Coumarin 343 using PyBOP/DIPEA
for activation before cleavage of the peptide from the solid
support. The identity of the product 1b was confirmed
after HPLC purification by ESI-MS (Figure S9) which also
showed that the peptide was isolated in high purity.
SA 1b in NH4HCO3 buffer without H2O2 led to formation of
only 3% of the linear peptide 3b, which proves the stability
of the peptide under these conditions (Figure 2 d).
TEM measurements of Depsi(Fmoc-I)pba-SA 1a in phos-
phate buffer pH 7.4 after 24 h incubation with and without
hydrogen peroxide showed the oxidation-triggered self-
assembly of the peptide due to formation of the linear
fibrillating sequence Fmoc-ISA 3a, while the correspond-
ing boronic acid modified depsipeptide 1a did not form
peptide fibers (Figure 2 f-g). In order to determine the
critical fibrillation concentration of Fmoc-ISA 3a, which is
important for intracellular fiber formation, Fmoc-ISA was
incubated at different concentrations ranging from 2 mM
to 15 µM for 24 h in phosphate buffered saline (PBS). In
TEM measurements, the lowest detectable concentration
of peptide fibers was 62.5 µM (Figure S26).
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The fluorescent peptide Depsi(C343-I)pba-SA 1b was
synthesized in order to enable live cell imaging of the pep-
tide fibers, by co-assembly of both intracellularly rear-
ranged peptides. While incubation of Depsi(C343-I)pba-SA
1b in phosphate buffer did not lead to fiber formation,
addition of H2O2 led to the appearance of amorphous ag-
gregates in TEM (Figure 2 h-i). Co-incubation of both PBA
depsipeptides upon hydrogen peroxide treatment at a
ratio of 5:1 of 1a:1b led to a mixture of fibres and some
aggregates (Figure 2 j). Preliminary confirmation of co-
assembly was accomplished by fluorescence microscopy,
demonstrating the co-localization of the coumarin 343
signal with Proteostat, which detects cross-β-sheet con-
taining peptide fibers (Figure 2 k). 19, 20, 24, 25
Next, we elucidated the secondary structure of Fmoc-ISA
as it is the primary driving force for fiber formation. To
address this question, we performed Fourier-transform
infrared spectroscopy measurements (Figure S29). The
results indicated formation of β-sheets, due to the appear-
Scheme 1: Synthesis of depsipeptides Depsi(Fmoc-I)pba-SA
1a and Depsi(C343-I)pba-SA 1b by solid phase peptide syn-
thesis. i) Piperidine, ii) Fmoc-Serine, PyBOP, DIPEA, iii) 4-
(nitrophenyl)phenylboronic acid pinacol ester, iv) Fmoc-
isoleucine, DIC, DMAP, v) TFA, triisopropylsilane, H2O, vi)
piperidine, vii) Coumarin 343, PyBOP, DIPEA.
ance of a maximum at 1634 cm1 Another maximum at
.
1688 cm1 is usually assigned to antiparallel β-sheets in
proteins but recently has been reported to derive from the
carbamate bond of the Fmoc group. A shoulder which
appeared at 1653 cm1 in the FT-IR spectrum might be
assigned to either α-helices or disordered structures.26-29
13C (1H) CP-MAS NMR spectra (cross polarisation-magic
angle spinning nuclear magnetic resonance) confirmed the
formation of three different competing structures by show-
ing two sharp peaks and one broad peak for the Fmoc
carbonyl, as well as several overlapping peaks for the C=O
groups of the amino acids (Figure S30).
For additional proof of co-assembly of both peptides, circu-
lar dichroism spectroscopy, was used to visualize changes
in the secondary structure (Figure 2 l). The CD spectrum of
Fmoc-ISA 3a in water showed a maximum at 218 nm,
which can be attributed to a n->π*-transition and a shoul-
der peak at ~209 nm was observed in the CD spectrum, as
well as a minimum at 190 nm.30 Notably, a strong positive
cotton effect was observed with a maximum at 267 nm,
which corresponds to the π->π*-transition of the Fmoc
groups in the self-assembled peptide fibers.18 In contrast,
the CD spectrum of C343-ISA 3b revealed a maximum at
195 nm and a minimum at 215 nm, with opposite elliptici-
ty compared to 3a. In proteins these signals are attributed
to β-sheets and correspond to π->π* and n->π*-transitions
In order to prove the hydrogen peroxide induced removal
of the PBA protecting group and the subsequent O,N-acyl
shift in solution outside cells, Depsi(Fmoc-I)pba-SA 1a was
incubated in NH4HCO3 buffer pH 7.4 with and without
hydrogen peroxide (Figure 2 a-c, Figure S15). The peptide
(RT = 15.6 min) was stable in the absence of H2O2 and led
to only 3% conversion into Fmoc-ISA 3a (RT = 14.6 min)
within 45 h, due to slow hydrolysis of the carbamate bond.
In contrast, addition of hydrogen peroxide led to a yield of
94% Fmoc-ISA 3a in the same time. The intermediate
Depsi(Fmoc-I)-SA 2a could also be observed by the ap-
pearance of a peak at 14.0 min, which started to decrease
after 8 h, while the product peak of 3a increased. The same
study was performed using Depsi(C343-I)pba-SA 1b and
the results showed that both – the removal of the phenyl-
boronic acid by H2O2 as well as the O, N-acyl shift – were
successful for the Coumarin derivative of the peptide (Fig-
ure 2 e, S16). After 4 h, Depsi(C343-I)pba-SA 1b (RT =
6.9 min) was no longer present, while the peaks for
Depsi(C343-I)-SA 2b (RT = 5.0 min) and the linear peptide
C343-ISA 3b (RT = 6.2 min) increased. After 24 h only 3b
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Figure 2: Hydrogen peroxide induced peptide fiber formation. a, Phenylboronic acid removal of Depsi(R*-I)pba-SA peptides 1a and
1b and subsequent O,N-acyl shift to give R*-ISA 3a and 3b (R*=Fmoc or Coumarin 343). b, HPLC spectra at different time points
showing the stability of Depsi(Fmoc-I)pba-SA 1a (15.6 min) in NH4HCO3 buffer and reference spectrum of Fmoc-ISA 3a (14.6 min).
c, H2O2 (2 mM) induced removal of the boronic acid to give Depsi(Fmoc-I)SA 2a (14.0 min) and linearisation of the peptide to
Fmoc-ISA 3a (14.6 min). d, Stability of Depsi(C343-I)pba-SA 1b (6.9 min) in buffer and HPLC spectrum of C343-ISA 3b (6.2 min). e,
H2O2 (1 mM) induced PBA removal and O,N-acyl shift of Depsi(C343-I)pba-SA 1b (6.9 min) to first give Depsi(C343-I)SA 2b (5.0
min) and finally C343-ISA 3b (6.2 min). TEM images of: f, Depsi(Fmoc-I)pba-SA 1a, g, Fmoc-ISA 3a, h, Depsi(C343-I)pba-SA 1b, i,
C343-ISA 3b, j, coincubation of Fmoc-ISA 3a and C343-ISA 3b 5:1. Scale bars 500 nm. k, Fluorescence microscope images of Fmoc-
ISA:C343-ISA 3a:3b 5:1. Left: Coumarin 343 channel (cyan), middle: Proteostat stained peptide fibers (yellow), right: merged Pro-
teostat and Coumarin 343 channel showing the overlay of the fluorescent dyes (green). Scale bars 20 µm. l, Circular dichroism
spectra of Fmoc-ISA (orange), C434-ISA (red) and their differently prepared assemblies in H2O (co-assembled: green, mixed: blue.)
Fmoc/C343-ISA (calculated, dashed purple line) is the sum of the separately recorded spectra of Fmoc-ISA and C343-ISA at a ratio
of 5:1 to match the experimental conditions. The region of interest is highlighted in gray.
respectively.31 For co-assembly, the study was accom-
plished using two different sample preparation methods –
1) the individual peptides (3a and 3b) were first mixed
before triggering the co-assembly in H2O, and 2) the sepa-
rate peptide assemblies are pre-formed in H2O and the
resultant nanostructures are combined. Mixing of the pre-
formed nanostructures leads to appearance of an addition-
al shoulder peak at ~235 nm and overall decrease in signal
intensities. This effect is larger than expected from the
spectral sum of a 5:1 mixture of 3a:3b, which represents
the hypothetical spectrum in absence of interactions be-
tween 3a and 3b. Hence, the results indicate interactions
at the nanostructure level, causing a decrease in chirality
of the assemblies. Upon coincubation of both peptides to
induce co-assembly, the intensities of maxima and minima
are further decreased significantly, which also includes
signals attributed to electronic transitions of Fmoc groups.
This is especially visible in the change of peak proportions
at 218 and at 206 nm where the latter was previously
observed as a shoulder at ~ 209 nm. We conclude that
upon co-assembly of 3a and 3b, the overall structural
chirality of the peptide assemblies is decreased leading to
a comparable CD spectrum of 3a due to the five-fold excess
but with distinct differences in ellipticity.32-34
In order to provide cellular uptake of depsipeptides 1a/b a
salicylhydroxamate functionalized TAT peptide 12 was
needed. TAT which has the amino acid sequence
YGRKKRRQRRR, was synthesised by standard SPPS meth-
ods and was modified with 4-pentynoic acid on its N-
terminus to first give molecule 11 (Figure S11). After a
copper-catalysed azide–alkyne cycloaddition (CuAAC) with
trityl (Trt) and (2-methoxyethoxy)methoxy (MEM) pro-
tected 4-azido salicylhydroxamate 10, which was synthe-
sised in seven synthetic steps (Figure S1), the peptide was
cleaved from the solid support and purified by HPLC. After
isolation of the pure peptide SHA-TAT 12, it was character-
ised by MALDI-TOF mass spectrometry (matrix-assisted
laser desorption/ionisation - time of flight, Figure S13).
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Figure 3: Complexes of SHA-TAT 12 and the boronic acid depsipeptides 1a and 1b. a, Chemical structure of dynamic covalently
bound SHA-TAT and boronic acid modified depsipeptide conjugates 13a and 13b. b, MALDI spectra showing the successful for-
mation of complexes of SHA-TAT 12 with Depsi(Fmoc-I)pba-SA 1a (top) and Depsi(C343-I)pba-SA 1b (bottom). [M‘] corresponds
to the molecular weight after loss of OH. [M‘]=[MOH]. c, Determination of the KD of the SHA-TAT complex with Depsi(C343-
I)pba-SA (13b) by fluorescence quenching.
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The attachment of the transporter peptide TAT was ac-
complished by boronic acid/salicyl hydroxamate chemis-
try. The formation of the dynamic covalent bond between
the boronic acid functionalised depsipeptides 1a and 1b
and SHA-TAT 12 in phosphate buffer pH 7.4 was proven by
MALDI-TOF measurements for both Fmoc and C343 modi-
fied peptides 1a and 1b. Both products were formed by the
condensation of PBA and SHA and multiple cation adducts
were observed in the spectra (Figure 3 a-b). Fluorescence
quenching experiments with Depsi(C343-I)pba-SA 1b and
SHA-TAT 12 gave an KD of 2.67 µM, which was expected
due to previously reported results for SHA-PBA complexes
(Figure 3 c).35 Reversibility of the complexation was ac-
complished at pH 5, which results in the recovery of fluo-
rescence (Figure S31). The binding and release were con-
firmed by previous reports of our group to transport pro-
teins into cells.36, 37
Alongside the acidification induced release of the pro-
assembling sequence 1 a/b from the TAT complexes 13
a/b inside endosomes, the H2O2 stimulus is required for
the assembly. The average concentration of H2O2 within
A549 cells was assayed using the Intracellular Hydrogen
Peroxide Assay Kit from Sigma-Aldrich and found to be
1.64 ± 0.16 µM (Figure S 57). Stimulation of H2O2 produc-
tion can be performed with 100 nM of phorbol-12-
myristat-13-acetate (PMA)38 affording a 49% increase to
2.45 ± 0.37 µM. Using these conditions, the TAT-complexed
depsipeptides 13 a/b were incubated with A549 cells and
the successful cellular uptake was shown by confocal mi-
croscopy (Figure 4 a). The concentration of the depsipep-
tides was adjusted to be 150 µM in total to meet the
requirements of the critical fibrillation concentration. The
depsipeptides were used at a ratio of 5:1 (13a : 13b) in
order to receive a sufficient fluorescence signal by Couma-
rin 343 inside cells, while maintaining an excess of the
Fmoc peptides to direct the fiber formation. Incubation of
cells with only the depsipeptides 1a and 1b did not show
internalization of the peptides, which proves the necessity
of SHA-TAT 12 for cellular uptake (Figure 4 a top row).
The increase in fluorescence intensity of Coumarin 343 in
the presence of PMA suggests local accumulation of fluo-
rescent 3b upon fiber formation indicating that the co-
assembly was more pronounced at higher concentrations
of H2O2, although as shown in Figure S47 and S48, peptide
fiber formation also occurs without PMA. Significant cell
deformation and nuclear condensation imply that cell
viability is significantly affected (Figure S38, S39, S41).
Incubation of the peptide samples with the cells for 2 h
showed less cellular uptake, while extending it to 6 h led to
more internalisation of the peptides (Figure S34). Peptide
treated cells were also examined under higher magnifica-
tion, where fibrillar structures inside the cell were visible
in the Coumarin 343 channel (Figure S35). A striking ob-
servation was the inefficient staining of the nucleus in cells
where the postulated assembly has occurred. This phe-
nomenon has already been described in literature, where
formation of peptide fibers inside cells prevented the
staining of nuclei as nuclear stains were trapped inside
fibrillar networks.39
We subsequently explored the mechanisms involved in
each step of intracellular transport using time-lapsed
Förster resonance energy transfer (FRET) and
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Figure 4: Cellular uptake and intracellular co-assembly of peptides. a, Confocal laser scanning micrographs of A549 cells treated for
4 h with only the depsipeptide mixture of 1a and 1b (top row) and treated with the addition of SHA-TAT 12 to induce cellular
uptake without (middle row) and with PMA (bottom row). Scale bars 20 µm. b, Förster resonance energy transfer studies on A549
cells with 15 a/b using Coumarin343 (donor, cyan) and 5-FAM (acceptor, red) as FRET pairs. Scale bars: 20 µm, λex = 405 nm.
co-localization studies. TAT was labelled with 5-FAM
(compound 14) serving as the FRET acceptor for Coumarin
343 (Figure S14). Using this FRET labelled variant of 13
a/b, named 15 a/b, we performed confocal microscopy at
1 h and 4 h time points (Figure 4 c). At 1 h, uptake of 15
a/b can be visualized as punctuated spots characteristic of
vesicle based intracellular transport. A significant propor-
tion of these vesicles exhibit FRET signals (red) suggesting
that most of the uptaken 15 a/b have not undergone dis-
sociation. Nonetheless, a first indication of release into the
cytosol was observed for a small proportion of these vesi-
cles. At 4 h, among cells that are still intact, assembly has
been accomplished in large areas of the cells. In these re-
gions, FRET signals are absent, suggesting that 15 a/b has
been mostly dissociated into 1 a/b and TAT 14 followed
by the peroxide driven cascade. However, remaining
amounts of undissociated 15 a/b could be observed to
proceed further along the TAT transportation pathway
into the nuclear region. Here, the complex remains bound
as the pH within the nucleus is no longer acidic.40 The
transport pathway through TAT-promoted endocytosis
was confirmed using early/late endosome studies with
CellLightTM, which saw major co-localization (Figure S37).
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Figure 5: a, Apoptosis assay using Annexin V-FITC/propidium iodide on 13 a/b and PMA treated A549 cells over 2 h and 4 h. Bind-
ing of Annexin V (green) towards the cell membrane was observed prominently at 2 h, demonstrating cells undergoing apoptosis
due to the inversion of the phosphatidylserine motifs. Membrane collapse at 4 h was detected with the entry of propidium iodide
(red) into the nucleus. Scale bars 20 µm. b, Cell viability of depsipeptides 1a and 1b and/or TAT 12 treated A549 cells determined
by CellTiter-Glo Luminescent Cell Via-bility Assay. Cells were treated for different incubation times from 2-6 h with either only the
depsipeptides 1a and 1b, only SHA-TAT 12 or both to create 13a and 13b at a concentration of 150 µM. A significant cytotoxic
effect was observed for A549 cells treated with the depsipeptide-TAT complexes 13a and 13b. All samples were co-incubated with
100 nM PMA.
In addition, endosomal escape could also be visualized as
diffused Coumarin 343 signals in the cytosol. Furthermore,
nuclear transport of undissociated peptides 13 a/b,
demonstrated by SYTOTM RNASelectTM Green, was found to
be localized inside the nucleolus, most likely due to the
electrostatic attraction between TAT and DNA/RNA or
perhaps hydrophobic interactions with the depsipeptides
(Figure S40).41 A previous report showed that self-
assembling peptides might interact with RNA located in-
side the nucleoli.42 In order to ascertain that the H2O2 driv-
en assembly is specific towards the cytosol, we monitored
the fiber formation using TEM at pH 5.0, 6.0 and 7.4 (Fig-
ure S23, S25). At both acidic pH values, corresponding to
the early and late endosomes as well as lysosomes, H2O2
does not possess enough oxidative strength to initiate the
reaction cascade for assembly. This pH dependent activity
of H2O2 was reported in the literature.43 As such, the acidic
conditions within the endosomes specifically trigger the
dissociation of 13 a/b, while the cytosolic environment
provides the condition for the oxidative cascade to initiate
fiber formation. To demonstrate the molecular rearrange-
ment of the depsipeptides inside cells, we analyzed cell
lysates by HPLC and showed the H2O2 induced conversion
of 1 b to 3 b (Figure S49).
At 2 h, Annexin-V was found to bind prominently to the cell
membranes, indicating that affected cells are undergoing
apoptosis. At this time frame, the integrity of the cell mem-
brane still remains intact as propidium iodide failed to
enter the cells. In contrast, at 4 h, propidium iodide signals
were observed in the nuclear region due to its affinity to
DNA.44 This observation represented the membrane per-
meabilization process associated with late stage apoptosis.
Analysis of cytotoxicity was accomplished using CellTiter-
Glo Luminescent Cell viability assay, which is based on
quantification of adenosine triphosphate (ATP) and there-
fore actively metabolising cells.45 No significant toxic ef-
fects were observed after incubation of 1 a/b at a concen-
tration of 150 µM for 6 h with A549 cells (Figure 5 b).
However, upon complexing with TAT to form 13 a/b, sig-
nificant impact towards cell viability was observed. Only
14% of cells were viable after 6 h, while incubation for 4 h
or 2 h led to a viability of 34% and 66%, respectively. As
the A549 cancer cells do produce intrinsic H2O2, transfor-
mation of 1 a/b into 3a/b still occurs in cells which are not
treated with PMA and therefore the cell viability without
PMA was 40% after 4 h (Figure S56), which is similar to
the cell viability with addition of PMA. As the increase of
hydrogen peroxide concentration by PMA stimulation
(49%) does not cause a significant difference in cell viabil-
ity, we conclude that non-stimulated cells already offer
enough H2O2 to generate sufficient peptide fibers leading to
effective apoptosis. Additionally, as known from β-amyloid
structures, we believe that shorter fibers/protofibrils can
already contribute to the apoptotic effects.46
The biological response of the cells to the intracellular
assembly was subsequently investigated by Annexin-
V/propidium iodide assay (Figure 5 a). Annexin-V is a
protein that binds to phosphatidylserine, which is located
on the external leaflet of the membrane structures exclu-
sively during apoptosis.44 The assay was conducted after
2 h and 4 h on A549 cells treated with 13 a/b.
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Figure 6: Intracellular peptide fiber formation. a, b, TEM micrographs of peptide fibers inside A549 cells (marked with dashed
lines) after co-assembly of intracellularly generated Fmoc-ISA 3a and C343-ISA 3b and images of a cell, which were received after
stitching TEM images together. Scale bars 500 nm. c, Fluorescence microscopy images of Proteostat stained peptide fibers after
formation inside cells and extraction from the cells by lysis. Scale bars 5 µm. d, TEM micrographs of peptide fibers, which were
received after lysis of depsipeptide TAT complex 13a and 13b treated A549 cells. Scale bars 500 nm.
Lastly, to visualize the intracellular fiber formation direct-
ly, 13 a/b treated cells were fixed by high pressure freez-
ing and subsequently freeze substitution was performed
using acetone. After infiltration with epoxy resin and its
polymerisation, sample blocks were sectioned into slices.
The slices of the cells were examined in TEM (Figure 6 a
and b). The formation of many dense peptide fiber net-
works was clearly visible inside the cells. Peptide fibrils
were distributed inside the cell and were also observed to
form next to the nucleus and mitochondria. To further
show the formation of fibers, cells were lysed after treat-
ment with the sample and the cell lysate was analysed by
TEM and fluorescence microscopy (Figure 6 c and d).
Fibers were observed in TEM, which further proved the
successful assembly of 3 a/b inside cells. Furthermore,
Proteostat staining of the cell lysate clearly showed for-
mation of amyloid, cross-ß-sheet structures to which Pro-
teostat is known to bind and coumarin343 fluorescent
fibers could also be observed in the lysate (Figure S45,
S46). Due to the resolution in the fluorescence microscope,
only very thick fibers could be found in the cell lysate. Cell
lysates of cells which were not treated with PMA but only
with the complexes 13a/b also contained fibers which
shows that enhancing the intracellular H2O2 concentration
with PMA is not necessary to induce peptide assembly, as
cells naturally already produce hydrogen peroxide (Figure
S47, S48).33 TEM images of cells as well as cell lysate of
A549 cells, which were not treated with the depsipeptide-
TAT complexes 13a and 13b, did not show fibrillar struc-
tures, which demonstrates that fibers derived from the co-
assembly of the intracellularly rearranged peptides Fmoc-
ISA 3a and C343-ISA 3b (Figure S44, S54 and S55).
CONCLUSIONS
In summary, we have designed a peptide sequence that
provides synthetic components for controlling cellular
entry, intracellular dissociation and supramolecular as-
sembly. The reactivity of each synthetic component within
the peptide sequence is dictated by the intracellular locali-
zation where its immediate environment defines its chem-
istry and subsequent transport pathway. Consecutive reac-
tions are initiated in a controlled fashion to afford co-
assembling sequences that form fibrillar structures within
the cytosol. The formation of these superstructures was
imaged by fluorescence and electron microscopy and led to
programmed cell death accompanied by nuclear fragmen-
tation, actin disruption and membrane collapse. Further-
more, co-assembly features the potential of the nanosys-
tem to dynamically customize functions and/or compo-
nents to tune additional biological behavior. Collectively,
the platform provides a broad accessibility and expands
the domain of nanotechnology to directly impact living
systems through structure formation.
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Journal of the American Chemical Society
W.; Gabelli, S. B.; Goutsias, J.; Inoue, T., Intracellular
ASSOCIATED CONTENT
Supporting Information. Materials and methods as well as
extensive supplementary figures and data are available free of
charge via the Internet at http://pubs.acs.org.
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production of hydrogels and synthetic RNA granules by
multivalent molecular interactions. Nat. Mater. 2018,
17 (1), 79-89.
9.
S. S.;
Ye, D.; Shuhendler, A. J.; Cui, L.; Tong, L.; Tee,
Tikhomirov, G.; Felsher, D. W.; Rao, J.,
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Bioorthogonal cyclization-mediated in situ self-
assembly of small-molecule probes for imaging caspase
activity in vivo. Nat. Chem. 2014, 6 (6), 519-526.
AUTHOR INFORMATION
Corresponding Author
* david.ng@mpip-mainz.mpg.de; weil@mpip-mainz.mpg.de
10.
Blunden, B. M.; Stenzel, M. H., Incorporating
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ruthenium into advanced drug delivery carriers – an
innovative generation of chemotherapeutics. J. Chem.
Technol. Biotechnol. 2015, 90 (7), 1177-1195.
ACKNOWLEDGMENT
The project received funding from the Max Planck-Bristol
Centre for Minimal Biology and the Volkswagen Foundation
under project number 89943. The authors would like to thank
Dr. Anke Kaltbeitzel for help with planning of the experiment
and Christoph Sieber for sample preparation for TEM of cells,
Dr. Gönül Kizilsavas for assigning NMR peaks, Jutta Schnee for
measuring MALDI spectra and Jessica Wagner for measuring
an APCI spectrum. We thank the reviewers for their efforts in
stimulating additional experiments that improved the quality
of the manuscript.
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Cheng, Z.; Cheng, Y.; Chen, Q.; Li, M.; Wang, J.;
Liu, H.; Li, M.; Ning, Y.; Yu, Z.; Wang, Y.; Wang, H., Self-
assembly of pentapeptides into morphology-adaptable
nanomedicines for enhanced combinatorial chemo-
photodynamic therapy. Nano Today 2020, 33, 100878.
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Shin, S. B. Y.; Almeida, R. D.; Gerona-Navarro,
G.; Bracken, C.; Jaffrey, S. R., Assembling ligands in situ
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Biol. 2010, 17 (11), 1171-1176.
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Herce, H. D.; Garcia, A. E., Molecular dynamics
simulations suggest a mechanism for translocation of
the HIV-1 TAT peptide across lipid membranes. Proc.
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