Smart Shell-by-Shell Nanoparticles with Tunable Perylene Fluorescence in the Organic Interlayer

Article abstract of DOI:10.1002/chem.202003232

A new series of shell-by-shell (SbS)-functionalized Al₂O₃ nanoparticles (NPs) containing a perylene core in the organic interlayer as a fluorescence marker is introduced. Initially, the NPs were functionalized with both, a fluorescent perylene phosphonic acid derivative, together with the lipophilic hexadecylphosphonic acid or the fluorophilic (1 H,1 H,2 H,2H-perfluorodecyl)phosphonic acid. The lipophilic first-shell functionalized NPs were further implemented with amphiphiles built of aliphatic chains and polar head-groups. However, the fluorophilic NPs were combined with amphiphiles consisting of fluorocarbon tails and polar head-groups. Depending on the nature of the combined phosphonic acids and the amphiphiles, tuning of the perylene fluorescence can be accomplished due variations of supramolecular organization with the shell interface. Because the SbS-functionalized NPs dispose excellent dispersibility in water and in biological media, two sorts of NPs with different surface properties were tested with respect to biological fluorescent imaging applications. Depending on the agglomeration of the NPs, the cellular uptake differs. The uptake of larger agglomerates is facilitated by endocytosis, whereas individualized NPs cross directly the cellular membrane. Also, the larger agglomerates were preferentially incorporated by all tested cells.

Full text of DOI:10.1002/chem.202003232

Accepted Article
Accepted Article
Title: Smart Shell-by-Shell Nanoparticles with Tunable Perlyene
Fluorescence in the Organic Interlayer
Authors: Andreas Hirsch, Lisa M. S. Stiegler, Stefanie Klein, Winfried
Neuhuber, and Carola Kryschi
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202003232
Link to VoR: https://doi.org/10.1002/chem.202003232
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1/2020

Chemistry - A European Journal
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Smart Shell-by-Shell Nanoparticles with Tunable Perylene
Fluorescence in the Organic Interlayer
[
a]
[b]
[c]
[a]
Lisa M. S. Stiegler , Stefanie Klein , Winfried Neuhuber and Andreas Hirsch*
[
a]
Lisa M. S. Stiegler and Prof. Dr. Andreas Hirsch
Department of Chemistry & Pharmacy
Chair of Organic Chemistry II
Friedrich-Alexander University of Erlangen-Nuremberg
Nikolaus-Fiebiger-Straße 10, 91058 Erlangen (Germany)
andreas.hirsch@fau.de
[
[
b]
c]
Dr. Stefanie Klein and Prof. Dr. Carola Kryschi
Department Chemistry and Pharmacy
Physical Chemistry I and ICMM
Friedrich-Alexander University of Erlangen-Nuremberg
Egerlandstraße 3, 91058 Erlangen (Germany)
Prof. Dr. Winfried Neuhuber
Department of Anatomy
Chair of Anatomy I
Friedrich-Alexander University of Erlangen-Nuremberg
Krankenhausstraße 9, 91054 Erlangen (Germany)
Supporting information for this article is given via a link at the end of the document.
Abstract: A new series of shell-by-shell (SbS)-functionalized Al
2
O
3
inorganic core.[2] In the next step, an attachment of amphiphiles
around first-shell functionalized NPs follows, which is based on
the self-assembly provided by solvophobic interactions of the
apolar moiety of the amphiphiles interdigitating into the first ligand
shell of the NPs. The polar moieties pointed outwards and create
water-dispersible character of the corresponding hybrids.[3]
nanoparticles (NPs) containing a perylene core in the organic
interlayer as a fluorescence marker is introduced. Initially, the NPs
were functionalized with both, a fluorescent perylene phosphonic acid
derivative, together with the lipophilic hexadecylphosphonic acid or
the fluorophilic (1H,1H,2H,2H-perfluorodecyl)phosphonic acid. The
lipophilic first-shell functionalized NPs were further implemented with
amphiphiles built of aliphatic chains and polar head-groups. However,
the fluorophilic NPs were combined with amphiphiles consisting of
fluorocarbon tails and polar head-groups. Depending on the nature of
the combined phosphonic acids and the amphiphiles, tuning of the
perylene fluorescence can be accomplished due variations of
supramolecular organization with the shell interface. Because the
SbS-functionalized NPs dispose excellent dispersibility in water and
in biological media, two sorts of NPs with different surface properties
were tested with respect to biological fluorescent imaging applications.
Depending on the agglomeration of the NPs, the cellular uptake differs.
The uptake of larger agglomerates is facilitated by endocytosis,
whereas individualized NPs cross directly the cellular membrane. Also,
the larger agglomerates were preferentially incorporated by all tested
cells.
Scheme 1. General structure of SbS-functionalized NPs.
Introduction
This field of NP functionalization was extended recently, with new
implications and applications. First, this functionalization principle
Over the last years, our group established for the first time a highly
hierarchical structured functionalization concept for metal oxide
NPs (scheme 1), which is based on a combination of two
of metal oxide NPs was used to generate
a switchable
dispersibility behavior of NPs in fluorocarbons, hydrocarbons, and
water and further allows the tuning of surface energies of NP
surfaces, for example, from hydrophilic to superhydrophobic, from
lipophilic to lipophobic, from fluorophilic to fluorophobic.[4] We
have also established a method, in which the fluorescence of a
functionalization steps.[1] In the first step,
a wet-chemical
functionalization process takes place, where a metal oxide NP
surface is treated with phosphonic acid derivatives, resulting in
covalent binding between the organic molecules and the
1
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1
Scheme 2. Hydrocarbon and fluorocarbon based SbS-architectures consisting of aluminum oxide NP cores, the fluorescence marker PAR , the spacing phosphonic
acids PAR2 and PAR , as well as the hydrocarbon-polar fused amphiphiles SDBS, SDS, DTAB, HC-PEG and the fluorocarbon-polar fused amphiphiles PFUnDA,
FC-PB, FC-PEG (see also scheme 3).
3
Scheme 3. Building blocks for the SbS-functionalization of aluminum oxide NPs with I) a mixture of PAR1 and PAR with the amphiphiles SDBS, SDS, DDPB,
2
DTAB, HC-PEG (left column) and II) PAR and PAR3 with PFUnDA, FC-PB, FC-PEG (right column).
1
2
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pyrene core coupled to a phosphonic acid attached covalently to
amphiphiles containing
a
pyridinium head-group. The
a Al
2
O
3
NP surface, could be quenched and turned on and off.[5]
hydrocarbon analogy of the particles that were functionalized with
SDBS or DTAB (contrary surface charge), namely [(Al
(PAR110% PAR290%)]SDBS and [(Al -(PAR110% PAR290%)]DTAB,
were further used in fluorescence microscopy of the non-
cancerous human umbilical vein endothelial cells (HUVEC) and
breast epithelial cells (MCF-10 A) as well of the cancerous A549
(lung cancer) and MDA-MB-231 (breast cancer) cells. Further
their biocompatibility, cellular uptake pathway and the intracellular
Therefore, the spacing phosphonic acids and amphiphiles were
carefully selected in order to control the intermolecular interaction
of the NP surface. By introducing an amphiphile with a pyridinium
head-group, the fluorescence of the pyrene moiety was turned off
and by removing the amphiphile from the SbS-structure, the
fluorescence was switched on, again. Compounds involving
pyridinium units cause a decrease in the fluorescence of alternant
2 3
O -
2
O
3
polycyclic aromatic hydrocarbons (PAHs), like pyrene or perylene, NP content was studied.
in polar solvents such as water and acetonitrile.[6] Responsible for
the quenching is a photo-induced electron-transfer (PET) from the
LUMO of the PAHs excited state towards the LUMO of the
Results and Discussion
positively charged nitrogen atom of the pyridinium unit, that has a
lower energy level.[7] Recently, we demonstrated that the type and
nature of spacing phosphonic acids influences the aggregation of
Synthesis of the parent compounds
2 3
perylene bisimides (PBIs) onto Al O NP surfaces and thus the
The stepwise synthesis of the so far unknown phosphonic acid
optical properties of the PBIs.[8] Whereas the rigid fluorocarbon
phosphonic acids separate the PBI units very effectively,
interactions of PBIs with hydrocarbon phosphonic acids are less
hindered. The highly flexible glycol phosphonic acids lead to the
weakest hindered interaction causing strong aggregation. We
also reported on SbS-functionalized NPs with electronic
1
PAR is outlined in scheme 4. Whereas compounds 1 and 2 were
[5]
1
known , synthetic access to 3 and PAR had be established. The
precursor molecule 1 was synthesized by a Michaelis-Arbuzov
reaction of 1,12-dibromododecane with triethylphosphite and
isolated as a pure compound in 26% yield. Compound 2 was
generated by a nucleophilic substitution of the bromide in 1 with
sodium azide in 96% yield. The azide functionality of the
phosphonate-ester was subsequently subjected to a “click”
reaction with 3-ethynyl perylene leading to the formation of 3 in
[9]
communication between the first and second-layer interface. In
lego-type principle, electron-donors were combined with
a
electron-acceptors, which resulted on one hand in electronical
communications between the chromophores with different
electron demand and on the other hand, the optical properties of
the NPs changed. Because of the excellent water dispersibility
and stability of SbS-functionalized NPs with interdigitating
amphiphiles containing polar head-groups, also some biological
applications were already reported.[10] Initially coated hydrophobic
4% yield.[5,
9, 13]
3
Final deprotection of 3 afforded the target
1
molecule PAR in 80% yield.
Au-Fe
acid, were encapsulated in a self-assembled bilayer shell
formation with 1-octadecylpyridinium, or 4-
3 4
O nanoheterodimers (NHDs) that were capped with oleic
dodecylbenzenesulfonate, to provide a positive or negative
surface charge onto the NP surface. We recognized, that the
surface charge as well as the surface architecture of the NHDs
influenced the mechanism and the efficiency of the cellular uptake
pathway, cellular localization, and the toxicity of human tumor
(
MCF-7) and healthy epithelial (MCF-10 A) cells.[10]
Herein, we report on a series of SbS-functionalized Al
2
3
O NPs,
involving a perylene core as a fluorescence marker in the first
ligand shell in combination with spacing phosphonic acids and
amphiphiles of different nature resulting in diverse mutations of
the initial perylene fluorescence. We created a hydrocarbon as
well as a fluorocarbon analogue of Al
2
O
3
NPs, by combining 10%
1
2
-(PAR110% PAR290%) NPs,
PAR with 90% PAR , resulting in Al
or in the case of combining 10% PAR with 90% PAR , Al
PAR110% PAR390%) NPs were formed. The hydrocarbon analogy
2 3
O
1
3
2
3
O -
(
was further implemented with amphiphiles built of aliphatic chains
and polar head-groups (SDBS, SDS, DDPB, DTAB, HC-PEG),
generating lipophilic environment around the perylene units. The
fluorocarbon analogy was further implemented with amphiphiles
consisting of fluorocarbon tails and polar head-groups (PFUnDA,
FC-PB, FC-PEG), generating fluorophilic pockets. This allowed
for tailoring the perylene fluorescence, from drastic increase of
fluorescence by disaggregation of perylene excimers[11] onto the
NP surface up to fluorescence quenching by forcing the perylene
units in its excimeric form[12]. Drastic decrease of fluorescence
was observed by combining the perylene functionalized NPs with
Scheme 4. Synthesis of the phosphonic acid PAR1.
Whereas the amphiphiles SDBS, SDS, DTAB, and PFUnDA are
commercially available, we synthesized DDPB, HC-PEG, FC-PB,
and FC-PEG according to literature procedures.[4a, 5, 14] The
syntheses of the amphiphiles are outlined in scheme 5. DDPB
was synthesized by implementation of n-dodecyl bromide with
3
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pyridine and was received as a pure compound in 57% yield.[14a]
Assembly of the hydrocarbon SbS-functionalized NPs
FC-PB was generated via a two-step synthesis. In the first step,
a
Steglich esterification of 11-bromoundecanoic acid with
heptadecafluorodecan-1-ol was performed, resulting in molecule
in 43% yield. Subsequently, 4 was refluxed in pyridine, resulting
in 40% yield of FC-PB.[5]
HC-PEG was afforded by
The optical properties of the hydrocarbon SbS-functionalized
Al
-(PAR110% PAR290%) NPs during the formation of a lipophilic
pocket among the perylene units caused by the assembly of
amphiphiles, were studied (scheme 2, 3). The Al NPs are
commercially available. As obtained from BET measurements
they have an average diameter size of 14 nm. DLS
measurements of the pristine NPs in IPA show a hydrodynamic
diameter of < 50 nm whereas the SbS-functionalized NPs in
deionized water reach a hydrodynamic diameter of around 100
2
O
3
4
2
O
3
implementation of lauric acid chloride with triethylene glycol, the
reaction was catalyzed by triethylamine. The product was
obtained in 61% yield. Towards FC-PEG, firstly, perfluoro-n-
undecanoic acid was converted to the corresponding perfluoro-n-
undecanoic acid chloride 5 by the help of thionyl chloride.
Subsequently, 5 reacted with triethylene glycol, catalyzed by
triethylamine, resulting in pure FC-PEG in 13% yield.[4a]
nm. Additional information on the Al O NP core is given in the
2
3
supporting information. For the functionalization procedure, 7.5
mL 0.15wt% Al NP dispersions in isopropanol were mixed with
mL of a phosphonic acid solution in MeOH/toluene (1:1),
resulting in a total concentration of the phosphonic acid mixture of
mM. Subsequently, 30 min. of ultrasonication and repeated
2
O
3
5
5
washing with isopropanol/toluene/isopropanol followed, to
achieve homogeneous surface functionalization and to remove
excess of phosphonic acid. The value of 5 mM has already been
proven for providing a full surface coverage of such metal oxide
NPs with phosphonic acids.[
measurements, grafting densities of 4.29 nm-2 for the Al
PAR110% PAR290%) hybrids and 3.02 nm-2 for the Al -(PAR110%
O
2 3
9, 15]
With the results of TGA
2
3
O -
(
PAR290%) hybrids were obtained (see figure S3). The fact, that
phosphonic acid mixtures can be stoichiometrically displayed
onto metal oxide surfaces, was already corroborated in recent
studies.[4b,
5,
8-9, 15-16]
TGA measurements of Al
O
NPs
2
3
3
1
2
functionalized with different ratios of PAR and PAR /PAR were
shown in the supporting information (figure S3) and displayed the
differences in the grafting densities as well as in the DTG curves.
The coverage with PAR1 and PAR2 provides a lipophilic
surface.[4a, 17] The second ligand shell was assembled through the
non-covalent interdigitation of the amphiphiles fused of a
hydrocarbon moiety and a polar head-group, namely sodium
dodecylbenzenesulfonate (SDBS), sodium dodecylsulfate (SDS),
1
-dodecylpyridinium
bromide
(DDPB),
(1-
dodecyl)trimethylammonium bromide (DTAB), or 2-(2-(2-
hydroxyethoxy)ethoxy)ethyl dodecanoate (HC-PEG). The
amphiphiles comprising the second ligand shell caused a polarity
2
umpolung from lipophilic (PAR ) to hydrophilic causing water-
dispersibility.[4a] The formation of the second shell was monitored
via fluorescence measurements. Therefore, 2 mL of 0.015wt%
2 3
O
-(PAR110% PAR290%) were filled in a cuvette and were titrated
Al
with 0.015wt% [Al
O
3
-(PAR110% PAR290%)]20 mM amphiphile
2
dispersions, resulting in a concentration range from zero up to 10
mM amphiphile with a constant 0.015wt% concentration of first-
shell functionalized NPs. The constant concentration of first-shell
functionalized NPs is crucial for excluding dilution effects during
titration, so that the changes in the fluorescence spectra can be
assigned exclusively to the addition of amphiphile. Exactly the
same titration procedure was applied for all other examples
(
scheme 2, 3).
Figure 1 a, b, c shows the fluorescence and UV-Vis spectra of
Al
-(PAR110% PAR290%) NP dispersions, that were titrated with
Al
-(PAR110% PAR290%)]20 mM SDBS dispersions (scheme 6).
The NPs Al
-(PAR110% PAR290%) without SDBS give raise to
weak fluorescence signals with weak and undefined 0-0* and 0-
* transition bands and a missing 0-2* transition band. During
titration with SDBS, the overall fluorescence intensity of the
2
O
3
[
2 3
O
2
O
3
Scheme 5. Synthesis of the amphiphiles DDPB, FC-PB, HC-PEG, and FC-PEG.
1
4
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perylene unit increased. The intensity of the 0-0* transition at 469
nm increased strongest, then the intensity of the 0-1* transition at
1000
800
600
400
200
0
fluorescence at 469 nm
fluorescence at 499 nm
b
4
99 nm, and also the signal of the 0-2* transition at 537 nm
appeared. By titrating SDBS to the Al
2
O
3
-(PAR110% PAR290%) NPs,
of course, the intensities of the absorption bands of the benzene
moieties of SDBS at 255 and 261 nm increased, whereas the
characteristic absorption signals of the perylene core at 392, 421,
and 448 nm stayed unaffected. Because of an unchanged
concentration of perylene during titration, the molecular density of
the perylene core stayed constant (see figure 1 c). The same
2 3
trend of intensifying fluorescence was observed for Al O -
(
(
PAR110% PAR290%) NPs, that were titrated with SDS or DTAB
figure S5 and S8, SI). The UV-Vis spectra of those two systems
0
2
4
6
8
10
concentration of SDBS (mM)
stayed unaffected during the titration process, because of the
optical inactive amphiphiles.
0 mM SDBS
.10 mM SDBS
4
c
0
0
0
0
0
1
1
1
2
2
3
4
6
8
1
.20 mM SDBS
.39 mM SDBS
.58 mM SDBS
.86 mM SDBS
.13 mM SDBS
.48 mM SDBS
.82 mM SDBS
.22 mM SDBS
.61 mM SDBS
.33 mM SDBS
.00 mM SDBS
.67 mM SDBS
.57 mM SDBS
0.0 mM SDBS
3
2
1
0
3
00
400
500
600
700
800
wavelength (nm)
Figure 1. (a) Fluorescence spectra, (b) trend of the fluorescence spectra, and
c) UV-Vis spectra of Al
-(PAR110% PAR290%) titrated with SDBS in DIW (λext
408 nm).
(
2 3
O
=
2 3
Further experiments on the optical properties of Al O
-(PAR110%
PAR290%) NPs focussing the perylene fluorescence, were done by
using DDPB as a second-shell amphiphile (see scheme 7). Figure
2
a and b show that already after the first titration step with DDPB,
resulting in a concentration of 0.10 mM, the fluorescence intensity
of the perylene core drop. After the second titration step, the
fluorescence intensity decreased more moderate and stayed
constant at the same level during further titration with DDPB (see
figure 2 b). The UV-Vis spectra in figure 2 c show an increase of
the pyridinium absorption signal at 259 nm and constant perylene
2 3
O
-(PAR110% PAR290%) with
-(PAR110%
2 3
O
absorption bands during titration of Al
DDPB.
Scheme 6. Schematic representation of the formation of [Al
PAR290%)]SDBS NPs.
0
mM SDBS
a
1
000
0.10 mM SDBS
0
0
.20 mM SDBS
.39 mM SDBS
8
6
4
2
00
00
00
00
0
0.58 mM SDBS
0
1
.86 mM SDBS
.13 mM SDBS
1.48 mM SDBS
1
2
.82 mM SDBS
.22 mM SDBS
2.61 mM SDBS
3.33 mM SDBS
4.00 mM SDBS
6.67 mM SDBS
8.57 mM SDBS
10.0 mM SDBS
4
50
500
550
600
650
700
wavelength (nm)
5
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8
6
4
2
0
0
0
0
mM DDPB
.10 mM DDPB
.20 mM DDPB
c
0.39 mM DDPB
0.58 mM DDPB
0.68 mM DDPB
1.13 mM DDPB
3
00
400
500
600
700
800
wavelength (nm)
Figure 2. (a) Fluorescence spectra, (b) trend of the fluorescence spectra, and
c) UV-Vis spectra of Al
-(PAR110% PAR290%) titrated with DDPB in DIW (λext
408 nm).
(
2 3
O
=
2 3
O
-(PAR110%
The differences in the optical properties of [Al
PAR290%)]SDBS and [Al -(PAR110% PAR290%)]DDPB NPs can
2
O
3
also be seen from the visual appearance of the corresponding
dispersions themselves. Figure 3 a show the NP dispersions
2 3
Scheme 7. Schematic representation of the formation of [Al O
-(PAR110%
PAR290%)]DDPB NPs with insights into the electronical behavior of the structure.
2 3 2 3
O O -
-(PAR110% PAR290%)]SDBS (right side) and [Al
[
(
Al
PAR110% PAR290%)]DDPB (left side) under day-light conditions,
both dispersions dispose of equivalent yellow colors. Under UV-
light conditions (see figure b), the [Al
PAR290%)]SDBS NPs show a strong yellowish-green fluorescence
color, whereas the [Al
-(PAR110% PAR290%)]DDPB NPs show a
quenched fluorescence with a slightly orange fluorescence color.
2 3
O
-(PAR110%
3
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
0
0
1
mM DDPB
a
.10 mM DDPB
.20 mM DDPB
.39 mM DDPB
.58 mM DDPB
.68 mM DDPB
.13 mM DDPB
2
O
3
4
50
500
550
600
650
700
wavelength (nm)
5
4
4
3
3
2
2
1
1
0
5
0
5
0
5
0
5
0
fluorescence at 469 nm
fluorescence at 499 nm
b
2 3
O
-(PAR210% PAR190%)]DDPB (left side)
Figure 3. Optical appearance of [Al
and [Al
-(PAR210% PAR190%)]SDBS (right side) under (a) day-light and (b) UV-
light conditions.
2
O
3
In a next step, the nonionic amphiphile HC-PEG, was used to
generate another family of SbS-functionalized NPs, namely
0,0
0,5
1,0
1,5
concentration of DDPB (mM)
-(PAR110% PAR290%)]HC-PEG (see scheme 8). Figure 4 a
[
Al
show the fluorescence measurements of Al
PAR290%) NPs that were titrated with HC-PEG and in figure 4 b
2 3
O
O
3
-(PAR110%
2
2 3
the trend of the fluorescence intensity of the Al O
-(PAR110%
PAR290%) NPs in dependence on the HC-PEG concentration is
shown. After the first titration step, resulting in a concentration of
0
.10 mM HC-PEG, the fluorescence band of the 0-0* transition
(
392 nm) strongly increased, also the fluorescence band of the 0-
6
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1
* transition (421 nm) increased, but more moderately compared
to the 0-0* transition. Additionally, the fluorescence signal of the
-2* transition (448 nm) arise. Extra amount of HC-PEG had no
130
120
110
100
b
0
further influence on the fluorescence properties of the NPs, the
fluorescence signals of the perylene core stayed constant after
2 3
the first titration step. In figure 4 c, the UV-Vis spectra of [Al O -
9
8
7
6
5
4
0
0
0
0
0
0
1
2
(
PAR 10% PAR 90%)]HC-PEG NPs during increasing amount of
HC-PEG, were shown. From a concentration range from 0 mM to
.20 mM HC-PEG, the initial UV-Vis spectrum stayed unaffected
0
by increasing the amount of HC-PEG in the NP dispersion. During
further titration with amphiphile, beginning at 0.29 mM HC-PEG,
the overall absorption of the NPs increased and cloudy
dispersions were formed.
fluorescence at 469 nm
fluorescence at 499 nm
0
1
2
3
4
concentration of HC-PEG (mM)
3,0
2,5
2,0
1,5
1,0
0,5
0,0
0
0
0
0
0
0
1
mM HC-PEG
c
.10 mM HC-PEG
.20 mM HC-PEG
.39 mM HC-PEG
.58 mM HC-PEG
.86 mM HC-PEG
.13 mM HC-PEG
3
00
400
500
600
700
800
wavelength (nm)
Figure 4. (a) Fluorescence spectra, (b) trend of the fluorescence spectra, and
2 3
O
-(PAR110% PAR290%) titrated with HC-PEG in DIW
(
c) UV-Vis spectra of Al
ext = 408 nm).
(λ
Assembly of the fluorocarbon SbS-functionalized NPs
2 3
O
-(PAR110% PAR290%) NPs, the fluorinated
As an alternative to Al
pendants were manufactured. The functionalization procedure
2
3
was the same, but instead of using 90% PAR , 90% PAR was
used as a spacing phosphonic acid, resulting in Al
PAR3 ) NP hybrids. The predominant coverage with PAR
2
O
3
-(PAR110%
3
9
0%
provides a fluorophilic surface (scheme 2, 3).[4a, 17] For the second
functionalization step, amphiphiles with a fluorocarbon tail were
used, for providing a successful assembly of the second layer
around first-shell functionalized NPs. For this purpose we
-(PAR110%
2 3
Scheme 8. Schematic representation of the formation of [Al O
PAR290%)]HC-PG NPs with excess of the HC-PG amphiphile forming NP-
independent micelles
selected
perfluoroundecanoic acid (PFUnDA), 1-(11-
(
(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)oxy)-
1
1-oxoundecyl)pyridin-1-ium bromide (FC-PB), or 2-(2-(2-
0
0
0
0
0
0
1
mM HC-PEG
hydroxyethoxy)ethoxy)ethyl 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,
1,11,11-henicosafluoroundecanoate (FC-PEG).
In comparison to the hydrocarbon analogy Al
PAR290%), the Al -(PAR110% PAR390%) NPs showed a better
refined fluorescence spectrum with a more intense 0-0* transition
458 nm) compared to the 0-1* transition (484 nm). But the 0-2*
transition band is also hidden. Figure 5 a and b represent the
fluorescence and UV-Vis measurements of Al
PAR390%) NPs that were titrated with PFUnDA (scheme 9). After
the first titration step of Al
-(PAR110% PAR390%) with PFUnDA,
a
1
00
.10 mM HC-PEG
.20 mM HC-PEG
.39 mM HC-PEG
.58 mM HC-PEG
.86 mM HC-PEG
.13 mM HC-PEG
1
O
3
-(PAR110%
2
8
6
4
2
0
0
0
0
0
2
O
3
(
O
3
-(PAR110%
2
2
O
3
4
50
500
550
600
650
700
resulting in a concentration of 0.10 mM, the perylene fluorescence
was quenched and during further titration, the fluorescence band
resulted in a broad undefined band that was shifted to higher
wavelength (nm)
7
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wavelength. Starting from a concentration of 0.58 mM PFUnDA,
the fluorescence spectrum did not further change. In the UV-Vis
spectra of the [Al O -(PAR 10% PAR 90%)]PFUnDA NPs, shown in
2 3
figure 5 b, it can be seen, that the optical density of the perylene
core was unaffected during the titration experiment with PFUnDA.
2,0
0
0
0
mM PFUnDA
.10 mM PFUnDA
.20 mM PFUnDA
1
3
b
1,5
1,0
0,5
0,0
0.39 mM PFUnDA
0.58 mM PFUnDA
0
1
.86 mM PFUnDA
.13 mM PFUnDA
1.48 mM PFUnDA
.82 mM PFUnDA
1
3
00
400
500
600
700
800
wavelength (nm)
2 3
O
-(PAR110%
Figure 5. (a) Fluorescence spectra, (b) UV-Vis spectra of Al
PAR390%) titrated with PFUnDA in DIW (λext = 408 nm).
2 3
Next, [Al O
-(PAR110% PAR390%)]FC-PB NPs were manufactured
involving FC-PB as a special type of amphiphile within the second
shell (see scheme 10). FC-PB consists of a fluorocarbon moiety
on one molecular end, the other end consists of a polar pyridinium
bromide head-group generating the water-dispersibility. The
fluorocarbon moiety and the polar head-group were bridged
through a hydrocarbon spacer. That extraordinary type of
amphiphile caused special fluorescence variations of Al
PAR110% PAR390%) NPs during the titration course with FC-PB.
Figure 6 a, b, and c show detailed fluorescence measurements.
The first titration step with the amphiphile applied in
2 3
O -
(
a
concentration of 0.10 mM FC-PB, lead to a decreased perylene
fluorescence. In the later course of the titration, the fluorescence
of the perylene unit increased stepwise with increasing amount of
FC-PB. Noticeable is also the fact, that the fluorescence band of
2 3
the 0-0* transition of [Al O
-(PAR110% PAR390%)]FC-PB is shifted
Scheme 9. Schematic representation of the formation of [Al
2
O
3
-(PAR110%
around 12 nm to higher wavelength, compared to the system
PAR390%)]PFUnDA NPs, whereas PFUnDA formed a fluorophilic pocket around
-(PAR110% PAR390%
Al
2
O
3
) without amphiphile. Figure 6 d
the perylene units.
2 3
represent the UV-Vis spectra of the NP system Al O
-(PAR110%
PAR390%) during titration with amphiphile FC-PB. The absorption
signal of the pyridinium unit at 259 nm increased, whereas the
optical density of the perylene core was constant during the
titration experiment.
4
3
2
1
0
0
0
0
0
0
0
0
mM PFUnDA
.10 mM PFUnDA
.20 mM PFUnDA
a
0.39 mM PFUnDA
0.58 mM PFUnDA
0.86 mM PFUnDA
1.13 mM PFUnDA
1.48 mM PFUnDA
.82 mM PFUnDA
1
4
50
500
550
600
650
700
wavelength (nm)
8
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2
2
2
1
1
8
4
0
6
2
8
fluorescence at 470 nm
fluorescence at 489 nm
c
0
2
4
6
8
10
concentration of FC-PB (mM)
d
0 mM FC-PB
5
4
3
2
1
0
0
0
0
0
0
.10 mM FC-PB
.20 mM FC-PB
.39 mM FC-PB
.58 mM FC-PB
.86 mM FC-PB
2 3
Scheme 10. Schematic representation of the formation of [Al O
-(PAR110%
PAR390%)]FC-PB NPs with insights into the electronical behavior of the system.
3
00
400
500
600
700
800
wavelength (nm)
3
3
2
2
1
1
5
0
5
0
5
0
5
0
1
2 nm
0 mM FC-PB
.10 mM FC-PB
0.20 mM FC-PB
a
Figure 6. (a), (b) Fluorescence spectra, (c) trend of the fluorescence spectra,
0
-(PAR110% PAR390%) titrated with FC-PB in DIW
and (d) UV-Vis spectra of Al
(λext = 408 nm).
2 3
O
0
0
.39 mM FC-PB
.58 mM FC-PB
0.86 mM FC-PB
1.13 mM FC-PB
1.48 mM FC-PB
1.82 mM FC-PB
2.22 mM FC-PB
2.61 mM FC-PB
3.33 mM FC-PB
4.00 mM FC-PB
As a third example to study the optical properties of Al
PAR110% PAR390%) NPs during assembly of a second shell, the
nonionic amphiphile FC-PEG was used (see scheme 11). In figure
a and b the monitoring of the fluorescence and UV-Vis spectra
of [Al
-(PAR110% PAR390%)]FC-PEG NPs in dependence on the
2 3
O -
(
6.67 mM FC-PB
8
1
.57 mM FC-PB
0.0 mM FC-PB
7
2
O
3
amphiphile concentration is displayed. During titration with FC-
PEG, the overall perylene fluorescence decreased. The
fluorescence band of the 0-0* transition (458 nm) decreased
stronger than that of the 0-1* transition (484 nm) and beginning
with a concentration of 1.48 mM FC-PEG, there is just the leftover
of a broad undefined fluorescence band. By looking at the UV-Vis
spectra (see figure 7 b), no changes in the optical density of the
perylene core were observed, because of a stable concentration
4
50
500
550
600
650
700
wavelength (nm)
3
3
2
2
1
1
5
0
5
0
5
0
5
0
0
0
mM FC-PB
.1 mM FC-PB
b
2 3
O
-(PAR110% PAR390%) during titration with the optical
of Al
inactive amphiphile FC-PEG.
4
50
500
550
600
650
700
wavelength (nm)
9
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Assembly of hydrocarbon SbS-functionalized NPs including a
mixed second ligand shell
In a next step of complexity, a hydrocarbon SbS-architecture was
generated including a mixed second ligand shell of DDPB and
SDBS. That system is of high importance for comparative studies,
because theoretically, it combines a fluorescence quencher and
a fluorescence intensifier in one system. For this purpose, the
2 3
first-shell functionalized hydrocarbon analogue Al O
-(PAR110%
PAR290%) was mixed with the second-shell ligands DDPB and
SDBS in a ratio of 1:1 (scheme 12), 1:2, or 2:1). It can be assumed,
that the amphiphiles were attached roughly stoichiometrically
onto the first-shell functionalized nanoparticles, as indicated by
zeta-potential measurements of the mixed second-shell systems
(
see supporting information, table S4). The fluorescence
measurements showed in all three cases the same trend. There
is one significant quenching step which could be matched to
DDPB (figure 8 a, b) and in the further course of the titration, the
fluorescence increased, initiated by SDBS (see figure 8 a and c,
S6 and S7). Beginning at a total concentration of 1.13 mM DDPB
and SDBS (1:1), the NPs formed flaky dispersions indicating that
the electrosteric stabilization of the NPs could no longer be
provided. Regarding the UV-Vis spectra, starting from
a
concentration of 0.86 mM DDPB-SDBS mixture, the absorption
signal at 260 nm stayed constant.
2 3
Scheme 11. Schematic representation of the formation of [Al O
-(PAR110%
PAR390%)]FC-PEG NPs, whereas FC-PEG formed a fluorophilic pocket around
the perylene core.
3
2
2
1
1
0
5
0
5
0
5
0
0
0
mM FC-PEG
.10 mM FC-PEG
a
0.20 mM FC-PEG
0.39 mM FC-PEG
0.58 mM FC-PEG
0.86 mM FC-PEG
1.13 mM FC-PEG
1.48 mM FC-PEG
.82 mM FC-PEG
1
4
50
500
550
600
650
700
wavelength (nm)
0
0
0
0
0
0
mM FC-PEG
2
2
1
1
0
0
,5 b
.10 mM FC-PEG
.20 mM FC-PEG
.39 mM FC-PEG
.58 mM FC-PEG
.86 mM FC-PEG
,0
,5
,0
,5
,0
1.13 mM FC-PEG
1
1
.48 mM FC-PEG
.82 mM FC-PEG
2
00 300 400 500 600 700 800
wavelength (nm)
-(PAR110%
2 3
Scheme 12. Schematic representation of the formation of [Al O
PAR290%)]SDBS&DDPB NPs including a mixed second layer consisting of
SDBS and DDPB in a one-to-one ratio.
2 3
O
-(PAR110%
Figure 7. (a) Fluorescence spectra, and (b) UV-Vis spectra of Al
PAR390%) titrated with FC-PEG in DIW (λext = 408 nm).
1
0
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1
50
Comparative analysis and theoretical interpretation of the
obtained fluorescence studies during SbS-functionalization
0
0
0
0
0
0
1
mM DDPB & SDBS, 1:1
a
.10 mM DDPB & SDBS, 1:1
.20 mM DDPB & SDBS, 1:1
.39 mM DDPB & SDBS, 1:1
.58 mM DDPB & SDBS, 1:1
.86 mM DDPB & SDBS, 1:1
.13 mM DDPB & SDBS, 1:1
First of all, the fact, that both starting materials, the lipophilic
1
00
-(PAR110% PAR290%) and the fluorophilic Al -(PAR110%
Al O O
2
3
2
3
3
PAR 90%) NPs vary in their fluorescence spectra, is striking.
Whereas Al
-(PAR110% PAR290%) NPs give rise to undefined
transition bands of the perylene core, Al
-(PAR110% PAR390%
NPs displayed more defined fluorescence signals. The undefined
transition bands of Al NPs are
characteristic for excimer formation of the perylene
2
O
3
5
0
0
2
O
3
)
2
3
O )
-(PAR110% PAR290%
[12]
units.
4
50
500
550
600
650
700
Fluorocarbon moieties are more rigid than hydrocarbons[ and
are thus more suitable for separating the perylene cores onto the
NP surface and prevent excimer formation, resulting in more
18]
wavelength (nm)
5
4
3
2
1
0
0
0
0
0
0
0
0
mM DDPB & SDBS, 1:1
.1 mM DDPB & SDBS, 1:1
b
-(PAR110% PAR390%
structured fluorescence signals of the Al
2
O
3
)
NPs. This has already been seen for pyrene and perylene
bisimide functionalized Al NPs by using either hydrocarbon or
fluorocarbon spacing phosphonic acids.
2
O
3
[5, 8]
The higher hierarchically assembled SbS-system [Al
PAR110% PAR290%)]SDBS displayed an increasing fluorescence
-(PAR110% PAR290%) during addition of SDBS,
intensity of Al
with refined 0-0*, 0-1* and 0-2* transition bands (figure 1 a and b).
Very similar results were observed for the titrations of Al
PAR110% PAR290%) with SDS and DTAB (see figure S5 and S8,
2 3
O -
(
2
O
3
2
3
O -
4
50 500 550 600 650 700 750
(
wavelength (nm)
SI). During all three titration experiments, the form of the
fluorescence spectra aligned to the reference fluorescence
spectrum of the monomeric compound 3 in THF (figure S4 a). This
is a proof, that the lipophilic moiety of SDBS (or SDS/DTAB) was
incorporated into the first ligand shell, and lead to disaggregation
of the perylene excimers[11] onto the NP surface.
fluorescence at 469 nm
fluorescencee at 499 nm
c
1
1
20
00
8
6
4
2
0
0
0
0
By doing the same experiment with DDPB instead of SDBS, the
2 3
fluorescence of the Al O
-(PAR110% PAR290%) NPs was strongly
quenched (figure 2 a and b). Reason for that phenomenon is an
electronic communication between the pyridinium and the
perylene unit. Pyridinium quenches the fluorescence of the
perylene through a photo-induced electron-transfer (PET) from
the excited state of the perylene, that was formed during
excitation with light of 408 nm, towards the nitrogen atom of the
pyridinium, that has a lowered LUMO energy level compared to
0
,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 1,6
concentration of DDPB & SDBS (mM)
5
0
0
0
0
0
0
1
mM DDPB & SDBS, 1:1
d
.10 mM DDPB & SDBS, 1:1
.20 mM DDPB & SDBS, 1:1
.39 mM DDPB & SDBS, 1:1
.58 mM DDPB & SDBS, 1:1
.86 mM DDPB & SDBS, 1:1
.13 mM DDPB & SDBS, 1:1
[6a, 6b, 6d, 6e, 19]
that of the perylene.
4
3
2
1
0
By creating mixed second ligand shells, a combination of the
amphiphiles SDBS and DDPB in a ratio of 1:1, 1:2, or 2:1, was
2 3
titrated to Al O ) NPs, while a new
-(PAR110% PAR290%
combination of effects has been arisen (figure 8 a and b, S6 and
S7). After the first titration step, the fluorescence was quenched
which could be matched to the DDPB that generated a PET from
the perylene core to the pyridinium unit. By further titration, the
effect of SDBS was predominant and the perylene fluorescence
increased again, initiated by disaggregation of the perylene units
onto the NP surface.
3
00
400
500
600
700
800
wavelength (nm)
Figure 8. (a), (b) Fluorescence spectra, (c) trend of the fluorescence spectra,
and (d) UV-Vis spectra of Al
-(PAR110% PAR290%) titrated with DDPB & SDBS
1:1) in DIW (λext = 408 nm).
-(PAR110% PAR290%)]HC-PEG NPs lead to
2 3
Manufacturing of [Al O
2 3
O
an increased fluorescence intensity with well resolved
fluorescence signals (figure 3 a and b), merely after the first
titration step of Al O
2 3
-(PAR110% PAR290%) with HC-PEG, resulting
(
in a HC-PEG concentration of 0.10 mM. This fluorescence
spectrum is already comparable to the monomeric fluorescence
1
1
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FULL PAPER
spectrum of reference compound 3 in THF (figure S4 a). Further
addition of HC-PEG had no further influence on that fluorescence
spectrum. These observations proof the successful formation of a
lipophilic pocket among the perylene units and following
separation of excimers. The single-step increase of the
fluorescence and the formation of cloudy dispersions by further
addition of HC-PEG indicate that pure micelles of the amphiphile
were formed without interaction to the NP surface. This
explanation could be strengthened by taking into account, that
those of [Al
2
O
3
-(PAR110% PAR390%)]PFUnDA. This is also a
successful proof for the formation of fluorophilic pockets around
the perylene units causing stack formation.
Biological experiments
Fluorescent dyes and proteins are used in immunofluorescent
assay, live cell or in vivo animal imaging. Long observation times
and cell autofluorescence in the visible spectrum leads to the
search for new fluorescent markers with high quantum yields,
large saturation intensities and good biocompatibility. Because of
4
tetraethylene glycol monododecyl ether (C12EO ), an amphiphile
with comparable molecular length and polarity to HC-PEG, has
also a relatively low critical micelle concentration of around cmc =
2 3
O
-(PAR110%
their excellent fluorescence properties, the NPs [Al
PAR290%)]SDBS, PAR290%)]DTAB,
and [Al
.046 mM.[20]
2 3
O
-(PAR110%
0
functionalized with PAR and PAR2 in the first shell and
amphiphiles fused of hydrocarbons and polar head-groups with
contrary surface charge, were tested for their application in
fluorescence imaging in different cell lines (A549-lung cancer
cells, HUVEC-endothelial cells, MCF-10 A-breast epithelial cells,
and MDA-MB-231-breast cancer cells). These NPs are excellent
dispersible in water. Both kinds of SbS-functionalized
nanoparticles exhibit a hydrodynamic diameter of 100 nm (table
1, figure S12 and S15) in water. Compared to the diameter size
of 14 nm obtained by BET and TEM measurements (figure S1 and
S2), the size is quite enlarged. The hydrodynamic diameter
obtained by DLS measurements of the pristine NPs are below 50
nm (figure S11). This indicating already some clustering of the
pristine NPs. Therefore, it is possible that polynuclear clusters
form the center of the SbS-functionalized NPs. However,
biological fluids have a different ionic strength compared to the
ultrapure water and is additionally enriched with proteins and
other biological substances that can form the so-called protein
corona on the surface of the NPs. Supplementary to the DLS
measurements of a 0.015wt% NP dispersion in water, the same
measurements were conducted with NPs in cell culture medium
enriched with 10% fetal calf serum (FCS) as a protein source. The
1
Coming to the fluorocarbon system, the first titration experiment
was performed with Al
-(PAR110% PAR390%) and PFUnDA.
During the formation of the SbS-system [Al
-(PAR110%
2
O
3
2
O
3
PAR390%)]PFUnDA, it could be observed that the perylene
fluorescence was quenched, already beginning at a concentration
of 0.10 mM PFUnDA (see figure 5 a, b). Further addition of
PFUnDA resulted in a broad undefined fluorescence band that
[
12a,
was shifted to higher wavelengths, characteristic for excimers.
2
1]
At a concentration of 0.58 mM PFUnDA, the fluorescence
spectrum stayed constant, at this point, the surface seems fully
covered with amphiphile. Reason for the changes in the
fluorescence spectra was a formation of a fluorophilic pocket
around the perylene cores through incorporation of the
fluorocarbon moiety of PFUnDA into the mainly fluorinated first-
shell of the NPs (see scheme 9). During this process, a phase
1
separation of the hydrocarbons (PAR ) from the fluorocarbons
3
[22]
(
PAR /PFUnDA) onto the NP surface took place, which leads
to a stronger interaction of the perylene units forming stacked
complexes, so called excimers of the perylene[12], resulting in a
reduced fluorescence intensity with a limited resolution of the
characteristic fluorescence bands of the perylene.
2 3
O
-(PAR110% PAR290%)]SDBS
hydrodynamic diameter of the [Al
decreases in cell culture medium, whereas the diameter of the
[(Al
-(PAR110% PAR290%)]DTAB NPs increases (table 1). With
hydrodynamic size of around 46 nm in cell medium, the attached
biomolecules provide an even better dispersibility of [Al
(PAR110% PAR290%)]SDBS NPs in medium than in water. The
[Al
-(PAR110% PAR290%)]SDBS NPs with the protein shell
2 3
O
-(PAR110% PAR390%)]FC-PB, the
During the formation of [Al
fluorescence of Al
-(PAR110% PAR390%) was quenched after
addition of 0.10 mM FC-PB, while the fluorescence band of the 0-
* transition of [Al
-(PAR110% PAR390%)]FC-PB is shifted
around 12 nm to higher wavelength, compared to the system
Al
-(PAR110% PAR390%) without amphiphile. The quenching
2
O
3
2 3
O
0
2
O
3
2 3
O -
2
O
3
2 3
O
effect as well as the shift of the fluorescence band are signals for
an electronical communication between the perylene core and the
pyridinium unit. The forthgoing titration displayed a stepwise
increase of the fluorescence with increasing amount of FC-PB. It
is thus comparable to the hydrocarbon SbS-functionalized system
including a mixed second ligand shell of SDBS and DDPB. When
the fluorocarbon moiety of FC-PB was incorporated into the
become single particles or very small aggregates (figure S20).
After incubation of the NPs in serum enriched buffer solution,
proteins or other biomolecules of the solution became attached to
the surface. To determine the change in the surface potential, the
NPs were collected by centrifugation and re-dispersed in water.
The zeta potential is reduced but still negative. The hydrodynamic
2 3
diameter of [(Al O
-(PAR110% PAR290%)]DTAB NPs is around 149
fluorinated first-shell of the Al
O
2 3
-(PAR110% PAR390%) NPs, the
nm with a broad size distribution in the DLS measurements (figure
S21). Since the diameter of the NPs are much smaller and it is
very unlikely, that the attachment of the coating and protein
corona expands a single particle to this size, these NPs form
hydrocarbon spacing unit of the amphiphile FC-PB was
positioned around the perylene core and separated the perylene
units onto the NP surface which goes hand-in-hand with an
increased fluorescence intensity of the perylene signals. The
absent formation of a broad undefined excimer band signals that
in this case no fluorophilic pocket was formed among the perylene
core.
2 3
larger agglomerates than the [(Al O
-(PAR110% PAR290%)]DTAB
NPs. The zeta potential turns from positive to negative charge,
which indicates that the surface charge is determined by the
charge of proteins and biomolecules attached. Since the zeta
potential of both particles after protein attachment is similar, only
the surface attached proteins of the serum define now the zeta
potential of the NPs. In a next step the amount of protein at the
NPs surface was measured. Both NPs were incubated in a PBS
2 3
During the SbS-formation of [Al O
-(PAR110% PAR390%)]FC-PEG,
the fluorescence signals changed into a broad undefined
excimeric fluorescence band. These results are very similar to
1
2
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solution with 1% FCS overnight and afterwards the amount of
proteins on the surface was determined. The proteins are getting
adsorbed on the surface of single particles, smaller or larger
aggregates forming a small shell around these. Since both NP
a
b
c
solutions contain the same concentration, the larger
1
agglomerates of the [Al
2
O
3
-(PAR 10% PAR290%)]DTAB NPs form
fewer particles than the smaller sized aggregates of the [Al
PAR110% PAR290%)]SDBS NPs and therefore a lesser amount of
proteins is needed to surround these NPs as can be seen in table
2 3
O -
(
1
.
Table 1. DLS (intensity in number) and zeta-potential measurements of the
Al
-(PAR110% PAR290%)]SDBS and [Al -(PAR110% PAR290%)]DTAB NPs in
[
O
2 3
2 3
O
water and cell culture medium with 10% serum and amount of proteins attached
onto the particle surface.
DLS
water)
ζ
DLS
(medium)
ζ
mass of
protein
(
(water)
(water)
/1 mg NP
[
Al
2
O
3
2
-(PAR110%
100.89
± 6.80
nm
- 58.0
±2.46
mV
46.13 ±
4.00 nm
- 26.4
± 1.89
mV
0.80 mg
protein/mg
NP
PAR 90%)]SDBS
[
Al
2
O
3
2
-(PAR110%
100.89
± 6.80
nm
+ 33.8
± 2.87
mV
149.27 ±
12.93 nm
- 32.8
±
0.651
mV
0.38 mg
protein/mg
NP
PAR 90%)]DTAB
The size of the NP aggregates also determines the way NPs were
taken up by cells and their localization inside the cells[23] (figure 9
a - c). The large agglomerates of the NPs enter the cells via
endocytosis most likely macro-pinocytosis. Macro-pinocytosis
involves the formation of actin filament driven plasma membrane
protrusion, which finally covers the large agglomerates of the NPs
(
see figure 9 a). Finally a large vesicle, the micropinosome,
loaded with these NPs is formed (see figure 9 b).[24] The well
dispersed [Al
-(PAR110% PAR290%)]SDBS can be found in small
NP clusters or single particles distributed in the whole cytoplasm
figure 9 c). These particles are not found inside of vesicles like
the [Al
-(PAR110% PAR290%)]DTAB. The absence of vesicles,
which are built during endocytosis, indicates a different uptake
2
O
3
(
2
O
3
mechanism like
a
direct diffusion through the plasma
membrane.[ Both NPs were tested for their biocompatibility via
7]
the neutral red assay in all different cell lines. The [Al
(
2 3
O -
PAR110% PAR290%)]SDBS NPs show nearly no cytotoxic effects
on the non-cancerous as well as the cancerous cell lines, when
cultivated for 24 h in cell culture medium containing 50 μg/mL NPs
2 3
O
-(PAR110% PAR290%)]DTAB NPs
(
figure 9 d). In contrast, the [Al
are more toxic for the A549, MDA-MB-231 and MCF-10 A cells
than the [Al
-(PAR110% PAR290%)]SDBS NPs.
2
O
3
1
3
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d
1
5 0
a
c
b
d
1
0 0
5
0
0
M C F -1 0 A
H U V E C
A 5 4 9 M D A -M B -2 3 1
2
1
[
[
A l2 O 3 -(P A R
P A R
)]D T A B
10%
90%
1
2
A l2 O 3 -(P A R
P A R
)]S D B S
10%
90%
Figure 9. TEM images of the A549 cells with (a, b) [Al
2
O
3
-(PAR110%
PAR290%)]DTAB, (c) [Al
O
2 3
-(PAR 10% PAR290%)]SDBS and (d) biocompatibility
1
2 3
O
-(PAR110%
of the NPs to the different used cell lines.
Figure 10. Fluorescent images of the A549 cells with (a) [Al
PAR290%)]DTAB, (b) [Al -(PAR110% PAR290%)]SDBS and HUVEC cells with
-(PAR110% PAR290%)]DTAB and (d) the [Al -(PAR110%
2 3
O
(
c) [Al
2
O
3
2 3
O
2
PAR 90%)]SDBS NPs.
The different uptake mechanisms are also reflected in the
fluorescent images of the cells. To ensure, that the attached
protein shell and the retention time of the NPs in cell culture
medium, does not change the absorption and fluorescence of the
NPs, long term stability UV-Vis and fluorescence measurements
were conducted (figure S10). After 58 h no change in the UV-Vis
absorption or fluorescence can be detected. The fluorescent
-(PAR110%
2 3
To test if the fluorescent signal of [Al O
PAR290%)]SDBS is to low inside the cells or the concentration of
these NPs is really much smaller inside the HUVECs, the
concentration of the NPs inside the cells was detected. The
fluorescent intensity was correlated with the concentration of the
different NPs and normalized to the cell number. For the
2 3
O
-(PAR110% PAR290%)]DTAB NPs build large green spots,
[
Al
which surround the blue stained cell nucleus of the A549 cells
figure 10 a) and HUVECs (figure 10 c). The large green spots
represent the agglomerates of NPs concentrated inside the
vesicles. The [Al
-(PAR110% PAR290%)]SDBS NPs appear as
-(PAR110%
fluorescent images the concentration of [Al O
2 3
(
PAR290%)]DTAB NPs is clearly greater inside all cell lines than
-(PAR110% PAR290%)]SDBS NPs (figure 11 a), but
that of the [Al
compared to the non-cancerous HUVECs and MCF-10 A cells the
-(PAR110% PAR290%)]SDBS is still much higher
amount of [Al
O
2 3
2
O
3
small green dots or a blurry shell around the cell nucleus of the
A549 cells (figure 10 b). These NPs are evenly distributed
2
O
3
inside the cancerous A549 and MDA-MB-231 cells. To test if
these results reflect the actual concentration of the NPs inside the
cells, the amount of aluminum was determined via ICP-AAS and
also normalized to the cell number. For a better comparability the
aluminum concentration of the NPs was measured and so the NP
concentration per cell was stated (figure 11 b). The determination
of the mass of NPs via ICP-AAS shows very similar results to the
measurements via the fluorescent imaging. The smaller
throughout the whole cytoplasm. The concentration of the [Al
PAR110% PAR290%)]DTAB NPs inside of the macropinosomes
lead to a higher fluorescent signal inside the cells than the evenly
2 3
O -
(
1
2
distributed [Al
fluorescent can be seen inside the cytoplasm of the HUVECs
figure 10 d). A similar behavior can are recognized for the MDA-
MB-231 and MCF-10 A cells (figure S22).
2 3
O -(PAR 10% PAR 90%)]SDBS NPs. No green
(
2 3
aggregates or single NPs of the [Al O
-(PAR110% PAR290%)]SDBS
dispersion are hardly penetrate the cellular membranes of the
non-cancerous cells, in contrast to the cancerous cells. The
structure and composition of the plasma membrane as well as its
biophysical status regulates the intracellular uptake of different
substances. The rapid proliferation of cancer cells leads to a
frequently need of membrane lipids and proteins. Therefore, their
biosynthetic pathways are modified and up-regulated in cancer
cells, which is followed by a continuous production of alterations
in lipids or proteins. The nature of these changes determines the
properties and uptake mechanics of the cancer cell membrane,
which differs from the uptake behavior of healthy cells like HUVEC
and MCF-10 A.[25]
1
4
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Chemistry - A European Journal
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dispersible NPs, we selected two systems with excellent
fluorescence features and opposite surface charges, namely
1
5 0
a
-(PAR110%
PAR290%)]SDBS
-(PAR110%
[
Al
2
O
3
and
[Al O
2 3
2
PAR 90%)]DTAB, for biological applications. These NPs differ in
their dispersibility in the cell culture medium. The [Al O
2 3
-(PAR110%
1
0 0
PAR290%)]DTAB NPs form large agglomerates surrounded by a
protein corona that change their surface charge. These large
particles were taken up by all used cells to a large extend via
5
0
0
2 3
macro-pinocytosis. The [Al O
-(PAR110% PAR290%)]SDBS NPs
are well dispersible forming individualized NPs or very small
aggregates surrounded by a still negatively charged protein shell.
These NPs cross the membranes of the cancer cells by
penetration and are afterwards evenly distributed in the cytoplasm.
M C F -1 0 A
H U V E C
1
A 5 4 9 M D A -M B -2 3 1
2
[
[
A l2 O 3 -(P A R
P A R
)]D T A B
10%
90%
In fluorescent imaging the large agglomerates of the [Al
(PAR110% PAR290%)]DTAB NPs inside vesicles can be easier
spotted than the single, widespread [Al
-(PAR110%
2 3
O -
1
2
A l2 O 3 -(P A R
P A R
)]S D B S
10%
90%
2
O
3
PAR290%)]SDBS NPs. In upcoming studies of our laboratory these
excellent fluorescent probes will be developed for their use in
multicellular tumor spheroids. With the help of the fluorescent
signal, the pathway and penetration depth of NPs inside this
several micrometers large tumor spheroids will be followed.
2
0 0
5 0
0 0
b
1
1
Acknowledgements
5
0
0
We thank the Cluster of Excellence “Engineering of Advanced
Materials” (EAM), as well as the SFB 953 „Synthetic Carbon
Allotropes” funded by the German Research Council (DFG) and
the Graduate School Advanced Materials and Processes (GS
AMP), for financial support. We also thank the Chair of Chemical
Reaction Engineering (CRT) in Erlangen for the BET
measurement. We thank Tobias Weißenberger (Institute of
Chemical Reaction Engineering, University Erlangen-Nuremberg)
for the ICP-AAS measurements.
M C F -1 0 A
H U V E C
1
A 5 4 9 M D A -M B -2 3 1
2
[
[
A l2 O 3 -(P A R
P A R
)]D T A B
10%
10%
90%
1
2
A l2 O 3 -(P A R
P A R
)]S D B S
90%
Figure 11. Determination of the intracellular NP content either by (a) fluorescent
intensity or by (b) ICP-AAS measurements.
Keywords: shell-by-shell (SbS)-functionalization • perylene
chromophore • amphiphiles • hydro-, lipo-, fluoro-philic/phobic •
fluorescent imaging
Conclusion
We reported on a series of SbS-functionalized Al
2 3
O NPs
involving perylene terminated phosphonic acid as
a
a
fluorescence marker in the first ligand shell in combination with a
hydrocarbon or a fluorocarbon terminated phosphonic acid as
well as amphiphiles in the second ligand shell. Already the
combination of the perylene phosphonic acid with the matrix
phosphonic acids leads to different fluorescence features of the
perylene core. For the second-shell functionalization, amphiphiles
consisting of a hydrocarbon or fluorocarbon moiety were used.
The interdigitation of the amphiphiles into the first ligand shell
leads to unique changes in the fluorescence properties of the NP
hybrids. The changes in the fluorescence are associated to
disaggregation or aggregation of the perylene units through
formation of a lipophilic or fluorophilic pocked among the perylene
units. An increase in fluorescence and creation of more refined
fluorescence signals was observed for disaggregation of the
perylene units, whereas a decrease of fluorescence sets in when
the perylene units are forced to aggregate. Furthermore, the
perylene fluorescence of first-shell functionalized NPs was
reduced by implementation of amphiphiles involving a pyridinium
head-group, due to the establishment of a photo-induced
electron-transfer from the perylene towards the pyridinium
building block. After studying a series of examples of water-
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Table of Contents
Adjustable fluorescence features of a perylene unit in the organic interlayer of SbS-functionalized Al
2
O
3
NPs were described. A perylene
terminated phosphonic acid is attached covalently onto the NP surface, amphiphiles generated the second ligand shell. Depending on
the type of amphiphile, changes in the initial fluorescence properties of the perylene were observed, that could be matched to
aggregation, disaggregation, or electronic effects.
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