Fluorescent and Water Dispersible Single-Chain Nanoparticles: Core–Shell Structured Compartmentation

Article abstract of DOI:10.1002/anie.202015179

Single-chain nanoparticles (SCNPs) are highly versatile structures resembling proteins, able to function as catalysts or biomedical delivery systems. Based on their synthesis by single-chain collapse into nanoparticular systems, their internal structure is complex, resulting in nanosized domains preformed during the crosslinking process. In this study we present proof of such nanocompartments within SCNPs via a combination of electron paramagnetic resonance (EPR) and fluorescence spectroscopy. A novel strategy to encapsulate labels within these water dispersible SCNPs with hydrodynamic radii of ≈5 nm is presented, based on amphiphilic polymers with additional covalently bound labels, attached via the copper catalyzed azide/alkyne “click” reaction (CuAAC). A detailed profile of the interior of the SCNPs and the labels’ microenvironment was obtained via electron paramagnetic resonance (EPR) experiments, followed by an assessment of their photophysical properties.

Full text of DOI:10.1002/anie.202015179

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 7820–7827
International Edition:
German Edition:
doi.org/10.1002/anie.202015179
doi.org/10.1002/ange.202015179
Polymer Nanoparticles
Fluorescent and Water Dispersible Single-Chain Nanoparticles:
Core–Shell Structured Compartmentation
Justus F. Hoffmann, Andreas H. Roos, Franz-Josef Schmitt, Dariush Hinderberger, and
Wolfgang H. Binder*
Abstract: Single-chain nanoparticles (SCNPs) are highly
versatile structures resembling proteins, able to function as
catalysts or biomedical delivery systems. Based on their
synthesis by single-chain collapse into nanoparticular systems,
their internal structure is complex, resulting in nanosized
domains preformed during the crosslinking process. In this
study we present proof of such nanocompartments within
SCNPs via a combination of electron paramagnetic resonance
(EPR) and fluorescence spectroscopy. A novel strategy to
encapsulate labels within these water dispersible SCNPs with
hydrodynamic radii of ꢀ 5 nm is presented, based on amphi-
philic polymers with additional covalently bound labels,
attached via the copper catalyzed azide/alkyne “click” reaction
(CuAAC). A detailed profile of the interior of the SCNPs and
the labelsꢀ microenvironment was obtained via electron para-
magnetic resonance (EPR) experiments, followed by an
assessment of their photophysical properties.
chemical functionalities into the final SCNPs to allow
applications of SCNPs in the areas of catalysis,[2] drug
delivery[3] or imaging technologies[3b,4] such as photoacoustic
imaging.[5] Crucial thereto is the placement of specific
chemical functionalities within the SCNP, further stimulated
by its nanosized dimension, excellent dispersion and internal
compartmentation. The formation of nanosized compart-
ments within such SCNPs has been intensely discussed,[1a,6]
proposing their use as functional units to generate micro-
environments for catalysis,[7] photophysics,[8] or subsequent
embedding of chiral elements.[9] As the formation of SCNPs is
based on the collapse of single polymer chains at low
concentration via covalent or non-covalent intramolecular
bonding interactions,[10] it “freezes-in” the conformational
state of the polymer chain into the final, crosslinked SCNP.[11]
However, dynamics of the polymer chains often is at least
partially preserved depending on the degree of crosslinking
and solvent effects during the crosslinking process.[6] Thus
recently a strong solvent dependency on the final size of
single-chain nanoparticles has been observed using comb-
shaped polymers, bearing for example, polyisobutylene (PIB)
sidechains with a direct correlation between solvent quality
and the final SCNP size.[8b] Strong photophysical effects as for
example an increasing rate of photoinduced dimerization has
been observed within pre-folded SCNPs via an increase of
quantum yields by the confinement-effects within the
SCNP.[8b] Structural dynamics can lead to a modulation of
the band structure of SCNPs and the excited states of bound
dye molecules on the sub-ns timescale which determines the
achieved fluorescence quantum yield and both, the absorp-
tion and fluorescence spectra of the dyes.[12]
Although the formation of nanosized compartments
within the SCNPs has been often proposed and observed
indirectly, a direct proof has not been accomplished as many
of the used methodologies provide an only indirect measure
of compartmentation. Thus monitoring the molecular dy-
namics of water molecules around SCNPs via Overhauser
dynamic nuclear polarization (ODNP) hinted to supramolec-
ular organized hydrophobic benzene-1,3,5-tricarboxamides
(BTA) into chiral folds within the SCNP, resembling their
self-assembly in solution.[13] Also the solution behavior of
polymers within SCNPs has been shown to deviate from those
of the respective uncrosslinked polymers due to confinement
within nanosized compartments, leading to significant
changes in their lower critical solution (LCST) behavior.[14]
We here probe the formation of compartments within
synthetic SCNPs based on amphiphilic polymers, equipped
with an active spin label, via continuous wave electron
Introduction
Among many nanosized carriers single chain nanoparti-
cles (SCNPs) are the most versatile, both in molecular design,
structural diversity and embedded functionalities.[1] Derived
from single polymer chains by covalent or noncovalent
crosslinking they directly link the vast synthetic space of
controlled polymer synthesis with their function, embedding
[*] J. F. Hoffmann, Prof. Dr. W. H. Binder
Macromolecular Chemistry, Institute of Chemistry, Faculty of Natural
Science II (Chemistry, Physics and Mathematics), Martin Luther
University Halle-Wittenberg
von-Danckelmann-Platz 4, 06120 Halle (Germany)
E-mail: wolfgang.binder@chemie.uni-halle.de
A. H. Roos, Prof. Dr. D. Hinderberger
Physical Chemistry, Institute of Chemistry, Faculty of Natural Science
II (Chemistry, Physics and Mathematics), Martin Luther University
Halle-Wittenberg
von-Danckelmann-Platz 4, 06120 Halle (Germany)
Dr. F.-J. Schmitt
Institute of Physics, Faculty of Natural Science II (Chemistry, Physics
and Mathematics), Martin Luther University Halle-Wittenberg
von-Danckelmann-Platz 3, 06120 Halle (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015179.
ꢀ 2020 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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paramagnetic wave resonance (CW EPR), allowing to probe
mers,[21] and the dye labels[22] were synthesized according to
the internal compartments of the formed SCNPs. Addition-
ally, fluorescent dyes, appropriate for use in pump-probe
photoacoustic imaging[15] are embedded into small (rh <
10 nm) SCNPs to allow sufficient permeation for cell-
incorporation.[16] Thus the SCNPs act as a barrier against
defense mechanisms of the targeted tissues and stabilize the
dye against external photobleaching. Important in our
endeavor was the quest to position the dye within the
hydrophobic core of the SCNP, as such its accessibility is
reduced and thus a proposed protection effect of the SCNP
could be expected to be most effective.
With the here reported single-chain nanoparticles one can
therefore generate a scaffold for encoding the required
different functionalities stimuli-response, for example, ther-
mo- or pH-responsivity,[3c,17] and biocompatibility,[18] by using
three types of monomers to adjust both, the required
amphiphilic balance for the single-chain collapse, and the
dye attachment.
literature with small adaptions (see Supporting Information).
For the polymerization the molar fractions of the three
monomers were set as 0.8, 0.12, and 0.08, respectively, to
achieve an appropriate number of crosslinking groups[23] and
to retain residual attachment sites for modification with the
labels after the single-chain collapse (polymer I: Mn =
36.1 kDa, PDI = 1.7, DP = 129). Furthermore, this composi-
tion allows to induce thermoresponsivity together with
sufficient dispersibility and hydrophobic packaging of the
labels. Final composition and structural integrity of the
resulting polymer was proven by NMR-spectroscopy, allow-
ing to adjust the desired functionalities along the polymer
chain in relation to the initially used amounts of the three
monomers (see Supporting Information, including the final
copolymer-compositions). Subsequently, single-chain collapse
of polymer I and labelling (see Scheme 1) was accomplished
in a two-step/one-pot reaction, where the alkyne group was
first deprotected by tetra butyl ammonium fluoride in water,
followed by slow addition of the resulting polymer solution to
an aqueous CuSO4/NaAsc solution via a syringe pump to
adjust low concentrations (cpolymer < 10À6 M) for the required
single-chain folding. The amphiphilic polymer thus is adopt-
ing a preorganized nanoparticle by forming an intramolecular
core–shell structure with the hydrophilic PEG sidechains in
the shell and the reactive azide-groups in the core, resulting in
sole intrachain crosslinking of the reactive groups to form
SCNP II via CuAAC-reaction (see Scheme 1).[24] For further
labelling of SCNP II, the catalyst was reactivated by adding
additional sodium ascorbate and a label-alkyne solution in
water (with the help of Kolliphor EL as detergent for the
Results and Discussion
The required poly(poly(ethylene glycol) methacrylate)
(PEGMA)-copolymer (polymer I) was synthesized by RAFT
copolymerization of poly(ethylene glycol) methacrylate
(Mn = 300 Da, n = 4.5), azidopropyl methacrylate, and 3-
(trimethylsilyl)propargyl methacrylate in DMF, using AIBN
as initiator and cyanoisopropyl dithiobenzoate (CPDB) as
chain transfer agent (CTA), followed by the removal of the
CTA using an excess of AIBN.[19] The CTA,[20] the mono-
Scheme 1. Synthesis of the precursor polymer I by RAFT polymerization followed by the formation of the unlabeled SCNP II and labelling to obtain
the labelled SCNP IIIa-c.
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Table 1: The hydrodynamic radii (rh) and cloud point temperatures (Tcp)
of the precursor polymer I, the unlabeled SCNP II and labelled
SCNPs IIIa-c, bearing the different labels TEMPO (IIIa), RhoB (IIIb) and
aBOD (IIIc).
2,2,6,6-tetramethylpiperidine oxide (TEMPO)-label and aza-
BODIPY (aBOD)). This allows a reaction of the still present
residual terminal azido moieties in the hydrophobic core to
attach the two fluorescent dyes Rhodamine B (RhoB) and
aBOD as a NIR-fluorescent dye. Especially aBOD is
attractive as it displays a tumor targeting functionality via
deprotonation, resulting in intramolecular charge transfer,
and thereby suppression of fluorescence.[22d,25] The EPR-label
2,2,6,6-tetramethylpiperidine oxide (TEMPO) was attached
for subsequent EPR investigations of the SCNPꢀs inner core
(see Scheme 1).
Progress of the crosslinking was followed by attenuated
total reflection infrared (ATR-IR) spectroscopy (see Fig-
ure S1a and b) indicating a full disappearance of the alkyne
band at 2178 cmÀ1 in the SCNP II, and a partial and complete
removal of the azide band at 2100 cmÀ1 for SCNP II and the
SCNPs III, respectively. The new bands of the triazole ring
are overlapped by the bands of the polymer backbone and the
PEG sidechains and therefore not visible in the spectrum. 1H-
NMR spectroscopy proves a complete reaction of the reactive
groups under formation of triazole rings (see Figure S1d,
removal of the TMS-protecting group at 0.2 ppm, absence of
the alkyne proton at 2.6 ppm). A crosslinking density of ꢀ 9
crosslinks per SCNP was roughly calculated from the
stoichiometric ratios, leaving ꢀ 4 remaining attachment sites
per SCNP for the labels. The so obtained SCNPs are water
dispersible and can be redispersed after freeze-drying.
Diffusion-ordered NMR spectroscopy (DOSY-NMR) and
dynamic light scattering measurements of polymer I and
SCNP II (see Figure 1 and Table 1) independently show
a reduction of the hydrodynamic radius after the CuAAC
from 5.2 nm to 4.2 nm. This proves the single-chain collapse in
good agreement with literature, where a reduction of the
hydrodynamic radius is an indication of a single-chain
collapse.[26] Upon further labelling of the SCNP II yielding
SCNPs IIIa-c, both methods show the trend of an increased
[a]
[b]
[c]
[d]
DDOSY
rh,DOSY
rh,DLS
Tcp
[”10À7 cm2 sÀ1
]
[nm]
[nm]
[8C]
Polymer I
SCNP II
SCNP IIIa (TEMPO)
SCNP IIIb (RhoB)
SCNP IIIc (aBOD)
3.3
4.9
3.4
3.4
3.7
5.3
3.6
5.2
5.2
4.7
5.2Æ1.9
4.2Æ0.4
4.9Æ1.0
4.5Æ0.6
4.6Æ0.4
48
54
65
69
75
[a] Diffusion coefficient measured by DOSY-NMR in D2O. [b] Calculated
using the Stokes–Einstein equation (rh =(kB T)/(6phD) with D=DDOSY
,
hD O =1.25 MPas). [c] Measured by DLS in H2O. [d] Cloud point
2
temperature measured by turbidimetry, Tcp =T at 96% transmittance
(see Figure S3b).
hydrodynamic radius of ꢀ 1.4 nm (DOSY-NMR, see Table 1)
and of ꢀ 0.5 nm (DLS) indicating the successful attachment of
the respective labels, which lead to an expansion of the
measured volume, as the dye-labels are comparably large in
dimension. Atomic force microscopy (AFM) further proved
the formation of SCNPs with an average height of 7.5 Æ
1.5 nm (see Figure 1c,d and Figure S2). This underscores the
formation of distorted particles due to the surface/SCNP-
interaction between the polar mica-surface and the hydro-
philic PEG-shell, also indicative of the high responsivities of
the SCNPs during adsorption, inline with previous observa-
tions.[27]
Size exclusion chromatography (SEC) measurements in
water further prove the covalent attachment of the labels to
the SCNPs via an overlap of the R.I. and UV/Vis track for the
dye-labelled SCNPs IIIb and IIIc (see Figure S4 d and e).
During single-chain collapse, the hydrophobic reactive groups
(azide/alkyne) are proposed to be located in the inner core of
the nanoparticles, hidden from the aqueous surroundings. We
therefore observe a polarity shift in SEC, inline with an
increased hydrophobic collapse (Tcp) after the single-chain
collapse and with further modification moving from the
native polymer I (Tcp = 488C) to SCNP II (Tcp = 548C) to
SCNP IIIa-c (Tcp = 658C, 698C and 758C, respectively) (see
Table 1 and Figure S3). It should be noted that this is the first
observation of such a consequent increase of Tcp, initially only
observed for noncrosslinked, dynamic random copolymers.[14]
Additionally, this behavior explains the decrease of retention
time in SEC moving from polymer I to SCNP II, with
concomitantly increasing Tcp. Labelling SCNP II with the dyes
to yield SCNPs IIIa-c leads to a further decrease of the
retention time and a further increase of Tcp.
To achieve a deeper understanding of the microstructure
and the distribution and location of the individual polymer
and label segments within the SCNPs, CW EPR measure-
ments were conducted. For the protective effect in SCNPs to
be effective, the exact location (spatially and in chemical
environments) of the labels within the SNCPs and the desired
location within the hydrophobic core is crucial. Moreover,
a rough quantification of the number of labels in one SCNP is
needed. With the CW EPR spectra of the attached TEMPO
label it is possible to specify the chemical environment of the
Figure 1. a) DOSY-NMR and b) DLS measurements of the precursor
polymer I and the unlabelled SCNP II at concentrations of 1 mgmLÀ1
in water and D2O, respectively. c) 2D and d) 3D AFM topography
image of SCNP IIIb (1”1 mm).
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nanostructure surrounding the probe within a radius of
mixtures with the free TEMPO probe. The three identical
ꢀ 1 nm.[28] For comparison of the TEMPO-labelled SCNP
IIIa with an equally labelled linear polymer, the TEMPO-
labelled polymer I’a was synthesized analogously to polymer
I, but without the alkyne moieties (see Supporting Informa-
tion).
Upon covalently binding TEMPO as a label (Figure 2b)
to polymer I’ yielding polymer I’’a, a significant broadening of
the EPR peaks is observed, caused by the hindered rotation of
the label attached to the large polymer. This effect is even
stronger in the TEMPO-labelled SCNP IIIa, indicating
a reduction of the mobility of the label upon attachment to
the SCNP. Figure 2a in comparison shows the CW EPR
spectra of polymer I and SCNP II in water in pure physical
normalized spectra indicate free TEMPO in water, proving
the absence of specific, mostly hydrophobic interactions of
the amphiphilic TEMPO probe with the polymer and the
SCNP, respectively, as we usually and regularly observe it with
amphiphilic (LCST-type) polymers.[28a] Simulated spectra of
the samples quantify the mobility reduction and the low
polarity of the environment as expected for water-depleted,
polymer-rich regions (see Figure 2c). For SCNP IIIa, but not
for the respective polymers an exchange coupling of 6 MHz
had to be included to the calculation, indicating high local
concentrations and direct contact of two or more TEMPO
labels with distances < 1 nm. Even though a certain amount of
TEMPO was probably deactivated by reduction during the
labelling process because of sodium ascorbate present in the
reaction mixture, the degree of labelling was approximated to
be 1.3 active labels per particle (see Figure S10). This,
together with our spectra, leads to the conclusion that the
SCNPs bear one to two chemically attached active spin labels
and even more EPR-inactive labels.
Concentration dependent measurements of SCNP IIIa
in Figure 3a,b indicate that the label is hidden from the
Figure 2. CW EPR spectra of a) native TEMPO, polymer I with a TEM-
PO probe, and SCNP II with a TEMPO probe; b) native TEMPO,
polymer I’a, and SCNP IIIa; c) simulated spectrum of SCNP IIIa. The
spectra were all normalized to the center peak. All measurements were
conducted in water at 208C. The concentrations of the polymers and
SCNPs were 1 mgmLÀ1. The concentrations of the TEMPO probes
were 100 mM.
Figure 3. CW EPR spectra of SCNP IIIa at different concentrations:
a) unnormalized, b) normalized to the center peak. All measurements
were conducted in water at 208C. c) Schematic depiction of the CW
EPR results from the simulation (left side) and the concentration
dependence (right side).
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environment outside the nanoparticle as expected for the
specific topology and compartmentation in the SCNPs. All
normalized spectra show the same shape, apart from small
differences in the signal-to-noise ratio. Even at high concen-
trations the surroundings of the labels remain the same. Thus,
the label seems to be completely unaffected by external
influences outside of the particle, even though collision or
agglomerates with other particles at high concentrations
might occur (see Figure 3c). Summing up the EPR results, the
up to two active labels per nanoparticle are covalently bound
in SCNP IIIa in a confined space surrounded by the non-polar
polymer backbone, which is reflected in the lower hyperfine
splitting of labels on SCNPs, in the nanoparticles core,
altogether as a strong proof for the formation of such
a compartment. External influences do not affect the label,
indicating its embedding in the SCNPꢀs core. Considering that
TEMPO is more hydrophilic than the hydrophobic dyes
embedded for optical applications, the SCNPs are therefore
able to protect a photoactive label from photooxidation or
other degradational processes.
The optical properties of dyes can be constructively tuned
by binding to SCNPs.[12,29] Figure 4 and Table 2 show the
absorption and fluorescence spectra and wavelengths of the
RhoB and aBOD labelled SCNPs in water and aqueous
phosphate buffer (pH 6.0), respectively, spectra of the free
dyes can be seen in Figure S5. In comparison to free RhoB-
alkyne, the RhoB-labelled SCNP IIIb shows a red shifted
absorption at 559 nm and a blue shifted fluorescence at
582 nm (stokes shift reduced to 23 nm). In the aBOD-labelled
SCNP IIIc, resonances are blue shifted by 6 nm in comparison
to the free aBOD dye in aqueous phosphate buffer, with
a stokes shift of 27 nm for the free aBOD dye and SCNP IIIc,
respectively. In line with the results from EPR measurements,
those shifts occur by a different solvation of the dyes in the
particleꢀs hydrophobic core, well separated from the sur-
rounding bulk water phase, again indicative for the compart-
mented nanostructure of the SCNP.
The near neighborhood of two or more bound dye
molecules leads to interactions and, dependent on their
distance and orientation, possible excitonic coupling between
the dyes which changes the fluorescence quantum yield, the
wavelength maximum and FWHM of the fluorescence
emission.[12] In addition internal relaxation processes on the
sub-ns time scale and the interaction of the dye with the
polymer at different sites and possible distributions of the
molecular structure modulate the fluorescence emission and
determine the fluorescence lifetime of excited states.[30] Time
resolved fluorescence spectroscopy enables the analysis of
sub-band structures, dynamics of interaction between excited
states and the environment and possible heterogeneous decay
channels resulting from excitonic coupling and/or compart-
mentation that contribute to the integral fluorescence spec-
trum. Just like in the EPR measurements we synthesized
a linear aBOD-labelled polymer (polymer I’c see Supporting
Information) to compare it to SCNP IIIc in view of their
excitational behavior and heterogeneity.
Figure 5 reveals how an excited state heterogeneity that
might either result from molecular coupling and/or a struc-
tural heterogeneity, possibly with subsequent relaxation
dynamics after light absorption in the bound dyes, leads to
a split of the formerly homogeneous excited singlet states of
different molecules as indicated in Figure 5d. While the free
aBOD quickly decays with a time constant of 110 ps in water
due to quenching of the surrounding aqueous medium the
fluorescence lifetime and fluorescence quantum yield signifi-
cantly rises after binding the dye to a polymer or SCNP. For
polymer I’c one can see a strong heterogeneity in the decay
associated spectra (DAS, Figure 5b) with a spectral separa-
tion of two states with distinct different lifetimes of 1.2 Æ
0.2 ns (red curve, energetically lower level) and 2.7 Æ 0.2 ns
(blue curve, energetically higher level) additionally to a fast
decay component that is similar to the free dye (black curve,
220 Æ 50 ps) with contributions from molecular interaction
(see below). This is caused by a weak and dynamic compart-
mentation, as depicted in Figure 5e, with partially folded
regions in which the dye molecules can have direct contact to
each other and free unfolded regions without direct contact
between dye molecules that can be quenched by the aqueous
surrounding. This heterogeneity between the bound dye
molecules also explains the strong inhomogeneous broad-
ening of the absorption band in Figure S6. The absorption is
therefore even broader as compared to SCNP IIIc because
the elongated polymer has a larger degree of freedoms as
compared to the SCNP. As the fluorescence maxima of all
components in the DAS are similar the observed heteroge-
neity refers to structural differences rather than excitonic
interaction of the different dye molecules.
Figure 4. Absorption and fluorescence spectra of a) RhoB-labelled
SCNP IIIb in water (c=0.5 mgmLÀ1, lEx =559 nm, slit=5 nm,
V
detector =600 V), and b) aBOD-labelled SCNP IIIc in aqueous phosphate
buffer (pH 6.0), c=0.5 mgmLÀ1, lEx =694 nm, slit=5 nm,
detector =600 V).
V
Table 2: Absorption and fluorescence maxima of the fluorescent dyes
For SCNP IIIc a stronger compartmentation can be
observed from the DAS (Figure 5c). The decay components
redistribute with distinct different maxima, indicating that
absorbed light energy potentially is transferred between
strongly coupled dye molecules. Slight spectral separation of
the two states which were also observed in polymer I’c can be
seen. The 1.2 Æ 0.2 ns component (red curve) seems to be
and the dye-labelled SCNPs in water.
RhoB-alkyne
SCNP IIIb
aBOD
SCNP IIIc
lAbs [nm]
lFl [nm]
Stokes shift [nm]
pKa
558
596
38
559
582
23
700
727
27
694
721
27
–
–
6.8
7.6
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The spectroscopic properties are strongly determined by
the exact localization of the states at the binding sites of the
aBOD molecules in SCNP IIIc. Specific configuration for the
bound molecules by specific molecular design of the binding
sites in the SCNP would allow for the creation of highly
individual optical properties of the coupled aBOD dyes and
allow for the fine tuning of color and fluorescence yield
lifetime of the bound molecules.
The location of the aBOD dye in the SCNPꢀs core also
effects its pKa and thereby its pH-responding functionality. As
depicted in Table 2 and Figure S7 the fluorescence of the label
is strongly changed by the pH value of the solvent, with a pKa
of 6.8 for the free aBOD and 7.6 for SCNP IIIc. Changing the
pH of the SCNP IIIc dispersion from 6.0 to 8.6 strongly
decreases its fluorescence, by 92%. The pH-responsivity has
no strong influence on the fluorescence lifetime (see Fig-
ure S8 and S9). The lifetimes in both, the free aBOD dye and
SCNP IIIc, stay nearly unchanged over the whole pH range,
prospective for a proper tumor tissue targeting. These data
are in compliance with the overall fluorescence yield shown in
Figure S7 with stronger amplitude reduction observed for
SCNP IIIc (see Figure S9, Figure S7d) as compared to aBOD
in solution (Figure S8, Figure S7b).
Conclusion
Figure 5. Decay associated spectra measured on a) aBOD in water
(c=25 mM), b) polymer I’c in water (c=0.1 mgmLÀ1, dye concentra-
tion ca. 5–10 mM) and c) SCNP IIIc in water (c=0.1 mgmLÀ1, dye
concentration ca. 5–10 mM) after Fit with 3 exponential functions
(excitation wavelength 632 nm, see SI for further information). d) Ex-
citonic interaction in SCNP IIIc and resulting split of the S1 states and
qualitative band structure due to excitonic splitting and or interaction
with the environment of the first excited singlet state (S1). The green
arrow indicates the excitation from the ground state (GS) and the blue
arrow fluorescence from the higher excitonic level with a time constant
of 2.9 ns while fluorescence from the lower level decays with 1.2 ns.
e) Schematic interpretation of the time resolved fluorescence spectro-
scopic data of polymer I’c (left side) and SCNP IIIc (right side).
In summary we could observe the formation of internal
compartments within single chain nanoparticles (SCNPꢀs) via
EPR- and fluorescence spectroscopic methods. The SCNPs
are synthesized by using a clickable PEGMA based copoly-
mer synthesized by RAFT polymerization, followed by an
intramolecular CuAAC within its hydrophobic core. DLS and
DOSY-NMR measurements proved single-chain collapse
with hydrodynamic radii of 4–5 nm. The water dispersible
SCNPs all displayed a Tcp higher than that of the free chain of
the native polymer I, indicative for the formation of core–
shell structured nanoparticles with the hydrophilic PEG
chains in the shell and the hydrophobic groups with the
embedded labels located inside the core. The inherent
dynamics of the SCNPs allows for a medium-responsive
nanostructure as indicated by their thermoresponsivities, and
the formation of contacts between two or more labels in one
SCNP. Definite proof of this compartmented model consisting
of a core–shell structure was accomplished via EPR measure-
ments, proving the presence of at least two covalently
attached labels within a confined space with distances below
1 nm, surrounded by the non-polar polymer backbone and
shielded from outer influences by the particleꢀs shell. Based
on the quite unique combination of thermal, EPR and
photophysical measurements we could prove the nanocom-
partmented structure of such SCNPs, consisting of the
hydrophobic labels embedded within the corresponding
hydrophobic compartments, with the short PEG-chains gen-
erating the outer shell. Coupling between dye molecules and
the local environment strongly influences their optical
properties giving rise to a strategy that allows for specific
molecular design of the optical properties. Due to the so
enabled shielding of the fluorescent dyes inside the SCNPs,
emitted from the energetically lower level while the 2.9 Æ
0.2 ns component (blue curve) results from the energetically
higher level. The fastest component slows down further as
compared to polymer I’c and was measured 300 Æ 50 ps with
strong spectral asymmetry and negative value at 730 nm
which is typical for an excitonic relaxation from strongly blue
shifted states with an emission around 700 nm to the observed
fluorescence maximum around 720–730 nm.
The possible band structure in the material is described
schematically in Figure 5d. The decay associated spectra in
Figure 5c indicate that excitation from the ground state (GS)
is followed by radiative decay from the higher state with
a time constant of 2.9 ns while fluorescence from the lower
state decays with 1.2 ns. Light absorption and relaxation of
the exciton induce a complex dynamic into the surrounding
polymer and SCNP environment that might cause an
apparent relaxation time of about 300 ps possibly accompa-
nied by rearrangements of different compartments or molec-
ular dynamics/ conformation changes of the polymer.[30]
Angew. Chem. Int. Ed. 2021, 60, 7820 –7827
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7825

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the fluorescent SCNPs IIIb and IIIc prospect their application
as contrasting agents in photoacoustic imaging, still main-
taining their targeting properties uninfluenced by their
encapsulation in the SCNPs.
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Acknowledgements
WHB acknowledges funding form the DFG (Deutsche
Forschungsgemeinschaft) project BI1337/14-1, the SFB
TRR 102, TP A03 Project ID 189853844, the TP B2 in the
DFG-GRK 2670. We thank Matthias Hoffmann for the AFM
measurements. Open access funding enabled and organized
by Projekt DEAL.
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Conflict of interest
[16] A. B. Benito, M. K. Aiertza, M. Marradi, L. Gil-Iceta, T. S.
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The authors declare no conflict of interest.
Keywords: amphiphiles ·decay associated spectra ·
EPR spectroscopy ·fluorescence spectroscopy ·nanostructures ·
single-chain nanoparticles
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Manuscript received: November 13, 2020
Revised manuscript received: December 13, 2020
Accepted manuscript online: December 29, 2020
Version of record online: February 25, 2021
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7827