Fluorescent Inorganic-Organic Hybrid Nanoparticles

Abstract

Inorganic‐organic hybrid nanoparticles (IOH‐NPs) with a general composition [ZrO]2+[RDyeOPO3]2‐, [Ln]3+n/3[RDye(SO3)n]n‐, [Ln(OH)]2+n/2[RDye(SO3)n]n‐, or [LnO]+n[RDye(SO3)n]n‐ (Ln: lanthanide) are a novel class of nanomaterials for fluorescence detection and optical imaging. IOH‐NPs are characterized by an extremely high load of the fluorescent dye (70‐85 wt‐%), high photochemical stability, straightforward aqueous synthesis, low material complexity, intense emission and high cell uptake at low toxicity. Besides full‐color emission, IOH‐NPs are suitable for multimodal imaging, singlet‐oxygen generation as well as drug delivery and drug release. This focus review presents the material concept of the IOH‐NPs as well as their synthesis and characterization. Their characteristic features are illustrated by selected in vitro and in vivo studies to initiate application in biology and medicine.

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Hybrid Materials
Fluorescent Inorganic-Organic Hybrid Nanoparticles
B. Lilli Neumeier,[a] Mikhail Khorenko,[a] Frauke Alves,[b] Oliver Goldmann,[c] Joanna Napp,[b]
Ute Schepers,[d] Holger M. Reichardt,[e] and Claus Feldmann*[a]
Dedicated to Professor Bernt Krebs on the occasion of his 80th birthday
1ChemNanoMat 2018,4,1–23  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
Focus Review
DOI: 10.1002/cnma.201800310
Wiley VCH Freitag, 19.10.2018
1899 / 121529 [S. 1/23] 1

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Abstract: Inorganic-organic hybrid nanoparticles (IOH-NPs)
with a general composition [ZrO]2+[RDyeOPO3]2, [Ln]3+n/3[RDye
(SO3)n]n, [Ln(OH)]2+n/2[RDye(SO3)n]n, or [LnO]+n[RDye(SO3)n]n
(Ln: lanthanide) are a novel class of nanomaterials for
fluorescence detection and optical imaging. IOH-NPs are
characterized by an extremely high load of the fluorescent
dye (70–85 wt-%), high photochemical stability, straightfor-
ward aqueous synthesis, low material complexity, intense
emission and high cell uptake at low toxicity. Besides full-
color emission, IOH-NPs are suitable for multimodal imaging,
singlet-oxygen generation as well as drug delivery and drug
release. This focus review presents the material concept of
the IOH-NPs as well as their synthesis and characterization.
Their characteristic features are illustrated by selected in vitro
and in vivo studies to initiate application in biology and
medicine.
1. Introduction
Medicine and molecular biology belong to the most fascinating
and most challenging areas of nanoparticle application.[1] Here,
nanoparticles generally address two different subjects: Diag-
nosis and therapy.[2] Aiming at diagnosis, nanoparticles can
serve as contrast agents for all kinds of imaging techniques
including optical imaging (OI), photoacoustic imaging (PAI),
magnetic resonance imaging (MRI), ultrasonic imaging (US),
computed tomography (CT), scintigraphy (SC), or positron
emission tomography (PET).[3] In regard of therapy, nano-
particles are promising carriers for drug delivery and release.
For instance, this includes analgesic, anti-tumor, anti-inflamma-
tory, antibiotic or antiviral agents and allows to address a wide
range of disease patterns.[4]
In the recent decade, imaging techniques have made
tremendous progress due to the technological development of
imaging devices (i.e. hardware), enormous advancements in
the evaluation and processing of great amounts of data (i. e.
software),[3] and finally due to powerful additives – so-called
contrast agents – that allow enhancing contrast, significance,
reliability and specificity.[3] Whereas the first areas belong to
engineering and computer science, contrast agents are the
domain of natural science. In general, the role of contrast
agents is to visualize whole organisms, specific organs and
tissue, or even single cells in animals (e. g., mice, rats) and
humans.[1,5] Certain contrast agents are long used in the clinics
for almost all imaging techniques.[3,5] Prominent examples
comprise, for instance, Gd complexes for MRI,[6] BaSO4for CT,[7]
or 99Tc and 131I compounds in nuclear medicine.[8]
Imaging techniques are widely used in clinical practice and
suitable for obtaining detailed information at high resolution.
However, they also require cumbersome equipment and time-
consuming data acquisition.[3] In this regard, especially OI offers
new opportunities for non-invasive diagnosis and in vivo
observation of complex vital functions.[9] OI is fast and easy to
operate and requires comparably cheap equipment as well as
uncomplex data manipulation. Tremendous efforts have been
already made to unveil organ distribution with deep tissue
information and to improve optical contrast and spatial
resolution. However, OI essentially requires suitable fluorescent
contrast agents. Even more interesting than single-modality
contrast agents is the option of multimodal detection to
combine the specific assets of different imaging techniques for
the visualization of various types of tissue on different scales of
resolution, for the translation from preclinical to clinical
imaging, or the translation of preoperational to intraoperative
imaging.[10]
This review is specifically focused on fluorescent nano-
particles as powerful tags for fluorescence detection and OI. In
general, various requirements are prerequisite to contrast
agents for any application in basic medical research and life
sciences, including: i) Low toxicity and sufficient biocompati-
bility; ii) Easy detectability with standard hardware equipment;
iii) Highly specific signals to prevent optical overlap with
autofluorescence from cells and tissue; iv) Deep tissue pene-
tration of the irradiation used for excitation and emission; v)
Straightforward synthesis of contrast agents with low material
complexity. In this regard, three types of fluorescent nano-
particles have been yet applied most often: i) Semiconductor-
type quantum dots (Q-dots);[11] ii) Up-converting nanopar-
ticles;[12] iii) Immobilized organic dyes.[13]
Aiming at fluorescent nanoparticles, Q-dots beyond doubt
represent the most prominent and most widely applied class of
inorganic nanoparticles.[11] They are designated by unrivalled
brightness, intense size-depending emission, likewise in the UV
to IR spectral regime, superior photostability, and low photo-
[a] B. L. Neumeier, M. Khorenko, Prof. Dr. C. Feldmann
Institute of Inorganic Chemistry
Karlsruhe Institute of Technology (KIT)
Engesserstraße 15, 76131 Karlsruhe, Germany
E-mail: claus.feldmann@kit.edu
[b] Prof. Dr. F. Alves, Dr. J. Napp
Translational Molecular Imaging
Max-Planck-Institute for Experimental Medicine
Hermann-Rein-Straße 3, 37075 Gçttingen, Germany
[c] Dr. O. Goldmann
Helmholtz-Center of Infection Research
Inhoffenstraße 7, 38124 Braunschweig, Germany
[d] Prof. Dr. U. Schepers
Institute of Toxicology and Genetics
Karlsruhe Institute of Technology (KIT)
Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Ger-
many
[e] Prof. Dr. H. M. Reichardt
Institute for Cellular and Molecular Immunology
University Medical Center Gçttingen (UMG)
Humboldtallee 34, 37073 Gçttingen, Germany
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cnma.201800310
©2018 The Authors. Published by Wiley-VCH Verlag GmbH
&
Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial NoDerivs License, which permits use and dis-
tribution in any medium, provided the original work is properly cited, the
use is non-commercial and no modifications or adaptations are made.
2ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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bleaching.[14] Since the emission of Q-dots relates to a
quantum-confinement effect – i.e. a size-depending band gap,
and thus, a size-depending emission – precise size control is
essential to guarantee the quantum-confinement effect (i. e.
B. Lilli Neumeier studied chemistry at the
Karlsruhe Institute of Technology (KIT). She
did her bachelor’s thesis in Polymer Science
in 2013 and received her master’s degree in
2015. She is currently a PhD student in Claus
Feldmann’s group at the Institute of Inor-
ganic Chemistry, KIT. Her research includes
novel inorganic-organic hybrid nanomateri-
als for medical application.
Mikhail Khorenko studied molecular science
at the University Erlangen-Nuremberg (Ger-
many, 2011–2016) and completed his master
thesis at the Imperial College London (UK,
2016). Since October 2016 he is a PhD
student in Claus Feldmann’s group at the
Karlsruhe Institute of Technology (Germany).
His research interests focus on the synthesis
of novel multimodal inorganic-organic hy-
brid nanoparticles for biomedical applica-
tion.
Frauke Alves studied medicine at the Uni-
versity of Go
¨ttingen. In 2009, she received her
professorship in Internal Medicine. As a
clinician she developed a thorough bench to
bedside approach in the cancer field by
heading a tandem research group at the
University Medicine Center (Dept. of Hema-
tology and Medical Oncology and Institute of
Diagnostic and Interventional Radiology) and
the Max-Planck-Institute for Experimental
Medicine, Go
¨ttingen. The focus of her “Trans-
lational Imaging Group” is the investigation
of mechanisms of tumor progression, angio-
genesis and inflammation as well as the
development of novel therapeutic ap-
proaches and diagnostic tools in nanomedi-
cine and oncology.
Oliver Goldmann studied biology at the
Technical University of Braunschweig and did
his doctorate on host genetic influence on
infection diseases caused by the human
pathogen Streptococcus pyogenes in the
department of microbial pathogenesis (Singh
Chhatwal) at the German Research Centre of
Biotechnology, Braunschweig. He continued
his work on microbial pathogenesis and the
influence of host genetic on bacterial infec-
tions in the Infection Immunology Group
(Eva Medina) at the Helmholtz Center for
Infection Research (HZI, Braunschweig). His
research interests are focused on bacterial
persistence and host pathogen interactions
of Gram-positive cocci.
Joanna Napp studied biology at the Nicolaus
Copernicus University in Torun, Poland. She
did her PhD at the Max-Planck-Institute (MPI)
of Experimental Medicine and the European
Neuroscience Institute in Go
¨ttingen. In 2006,
she joined the Translational Molecular Imag-
ing Group of Frauke Alves, with a combined
position at the University Medical Center
Go
¨ttingen (UMG) and at the MPI in Go
¨ttin-
gen. In 2015, she started her junior research
group Image-based Nanotherapy and Diag-
nostics at the UMG, supported with a guest-
scientist position at the MPI. She has speci-
alized in the in vivo imaging of animal
disease models, particularly focusing on
development and evaluation of novel tumor
therapies.
Ute Schepers studied chemistry (University of
Bonn) and did her doctorate in Biochemistry
of brain diseases under Konrad Sandhoff.
After post-doctoral studies with Tom Kirch-
hausen (Harvard Medical School, Boston),
she started her junior group at the LIMES
Institute in Bonn on organ-targeted drug
delivery. In 2009, she became group leader at
the Karlsruhe Institute of Technology (KIT),
where she habilitated in 2011. In 2015, she
was appointed at KIT as leading scientist and
received a professorship for Chemical Biology
and Biochemistry. Her research interests ad-
dress organ-targeted drug delivery.
Holger M. Reichardt studied biochemistry in
Germany (University of Tu
¨bingen) and Swit-
zerland (University of Fribourg) and did his
doctorate in molecular biology at the Ger-
man Cancer Research Center (DKFZ) in
Heidelberg. After post-doctoral training in the
laboratories of Gu
¨nther Schu
¨tz (DKFZ) and
John Mullins (University of Edinburgh), he
became Professor of Molecular Immunology
at the University of Wu
¨rzburg, where he
worked on T cells and macrophages and
developed new techniques to genetically
manipulate the rat genome using lentiviruses
and RNA interference. In 2007, he was
appointed Professor of Experimental Immu-
nology at the University of Go
¨ttingen. The
main focus of his current research is the
pathomechanism of chronic inflammatory
diseases and the design of new therapeutic
concepts and improved immunosuppressive
strategies.
Claus Feldmann studied chemistry (Univer-
sity of Bonn) and did his doctorate in solid-
state chemistry under Martin Jansen. After
post-doctoral studies with Hans-Georg von
Schnering (Max Planck Institute of Solid-State
Research, Stuttgart), he moved to industry
(Philips Research Laboratories, Aachen/Eind-
hoven), where he was engaged in lumines-
cent materials. Simultaneously, he habilitated
at the RWTH Aachen on nanomaterials. In
2003, he was appointed at the University of
Karlsruhe, the present Karlsruhe Institute of
Technology. His research interests address
solid-state chemistry and functional nano-
materials.
3ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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mean particle diameter <10 nm) and the color purity of
emission (i.e. maximum deviation from the mean diameter
0.5 nm).[15] Moreover, high crystallinity and high purity are
necessary to exclude defect-driven loss processes. The latter
requirement also demands core-shell structures (e. g.
CdSe@ZnS) with a luminescent core (e. g. CdSe) that is coated
by a non-luminescent shell (e. g. ZnS) to eliminate the solid-to-
liquid surface of the luminescent core.[16] The above require-
ments – size control, low defect level, core-shell structure – lead
to high efforts for chemical synthesis. The applicability of Q-
dots, finally, suffers from additional inherent drawbacks such as
harmful elements (e.g. Cd2+), sensitivity to hydrolysis, and the
hydrophobic properties of the as-prepared material (e. g. due to
alkyl-terminated surfaces).[17] All in all, advanced strategies of
synthesis and surface conditioning are needed to obtain state-
of-the-art water-dispersible core-shell Q-dots showing intense
emission.[18] As an alternative class of fluorescent nanoparticles,
moreover, carbon dots (C-dots) came up recently and turned
out as very promising due to their improved biocompatibility.[19]
Similar demands regarding crystallinity, purity, and surface
conditioning with core-shell structures also apply to up-
converting nanoparticles (e.g. NaYF4:Er,Yb@NaYF4) to avoid all
kinds of defects, which otherwise reduce the emission inten-
sity.[12] Up-conversion – meaning the absorption of two or more
photons of lower energy (e.g. IR) followed by the emission of
one photon at higher energy (e.g. green) – is established by
photon-cascade processes on the precisely defined energy
levels of rare-earth-metal ions (e.g., Er3+,Yb
3+).[20] Up-conver-
sion-based fluorescence guarantees low background and
excellent tissue penetration. On the other hand, up-conversion
materials suffer from poor absorption and high photon density.
The latter is needed for sufficient excitation (e. g. by mono-
chromatic laser light) of the quantum-mechanically forbidden f-
ftransitions on rare-earth-metal ions.
Besides inorganic nanoparticles, such as Q-dots and up-
converting materials, fluorescent organic dyes in aqueous
solution are, of course, most often used as tags for fluorescence
detection and optical imaging.[13] Thus, a great number of
fluorescent dyes showing emission from the blue to the
infrared spectral regime has been discovered since the
beginning of fluorescence microscopy in the early 20th century
to analyze all kinds of biological probes.[21] However, fluorescent
organic dyes often do not meet the demands on high emission
intensity, sufficient photostability, and chemical resistance
under the conditions of investigation.[2g,22] In particular, low
intensity and rapid photobleaching are severe limitations of
many fluorescent organic dyes. To evade these intrinsic
weaknesses, fluorescent organic dyes – most often including
derivatives of rhodamine, cyanine, squaraine, boron-dipyrrome-
thene, porphyrin, phthalocyanine, etc.[23] – were attached to or
encapsulated in inorganic matrices (e. g., silica, calcium
phosphate),[24] metalorganic frameworks and coordination poly-
mers,[25] organic polymers (e.g., polyglycolic acid/PGA, polylactic
acid/PLA, poly(lactic-co-glycolic) acid/PLGA, polycaprolactone/
PCL, chitosan),[13a,26] as well as liposomes and dendrimers.[27]
However, surface-attached dyes hold the risk of abrasive
debonding. Encapsulation of fluorescent organic dyes in a
matrix, on the other hand, is typically performed via micro-
emulsion techniques, which limits the amount of available
material.[28] Moreover, the concentration of the fluorescent dye
in relation to the inert matrix is usually low (5 wt-%). The
limited number of fluorescent centers per nanoparticle volume,
however, reduces the emission intensity and increases the
threat of photobleaching.
Although fascinating chemical compositions and material
structures of fluorescent nanoparticles were realized, aspects
such as the emission intensity, photostability, biocompatibility,
or biodegradability still need further improvement. The com-
plexity and great number of constituents of certain nano-
particular architectures, in fact, can be a limitation in itself since
synthesis and material are becoming more and more complex
and expensive. Aiming at medical application, furthermore, all
individual constituents as well as all their combinations might
need clinical approval.[29] Based on the above discussed state-
of-the-art, we present inorganic-organic hybrid nanoparticles
(IOH-NPs) as a novel concept and class of fluorescent nano-
particles for biomedical issues. We have explored these IOH-NPs
since 2008,[30] with the intention to identify uncomplex, low-
cost nanoparticles showing intense emission and high bio-
compatibility.
2. Fluorescent Inorganic-Organic Hybrid
Nanoparticles (IOH-NPs)
The concept of the IOH-NPs is illustrated with phosphate-based
IOH-NPs and sulfonate-based IOH-NPs as examples. This
includes the chemical synthesis, the material composition as
well as the luminescence properties with the specific focus on
biomedical application.
2.1. Material Concept
Aiming at uncomplex, low-cost nanoparticles for biomedical
use, we intended to perform the synthesis in water only, since
the addressed area of application – for obvious reasons – is
limited to water. Consequently, the aspired nanoparticles need
to be insoluble in water. In terms of biocompatibility,
phosphates seemed reasonable as they are essential for the
energy metabolism of almost all cells.[31] In this regard, the most
insoluble metal phosphate in water is zirconium phosphate,
which is well-known in qualitative analysis to prove the
presence either of zirconium or phosphate.[32] The precipitation
is highly indicative since zirconium phosphate is the only
insoluble metal phosphate even in hydrochloric acid at low pH
(i.e. pH2).
Although the qualitative test reaction is described in many
textbooks, the chemical composition of the aqueous precipitate
at room temperature is still not clear. Typically, the composition
is denoted as Zr3(PO4)4or Zr(HPO4)2H2O.[32,33] With aqueous
conditions at room temperature and at moderate pH (4 to 9), in
fact, neither Zr4+nor PO43are likely as dissolved species due
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to their high acidity or alkalinity, respectively. The expected
species rather are [ZrO]2+and [HPO4]2or [H2PO4]. Aiming at
nanoparticles, a composition [ZrO]2+[HPO4]2is indeed formed
upon injection of an aqueous solution of ZrOCl2into an
aqueous solution of Na2(HPO4) (Figure 1a). It needs to be noted
that [ZrO]2+[HPO4]2– similar to all IOH-NPs presented in the
following – is amorphous and does not show any Bragg peak.
This complicates the determination of composition and
structure, hence requiring different independent tools to prove
the adopted composition (e.g. Zr:P ratio of [ZrO]2+[HPO4]2
determined to 1:1; see 2.4 for details).
Although not showing any luminescence, [ZrO]2+[HPO4]2is
the initial point of all IOH-NPs and the origin of a platform of
compounds with different compositions.[34] To obtain fluores-
cent IOH-NPs, the simple hydrogen phosphate anion is replaced
by a phosphate with a fluorescent organic dye (RDye) bound via
aPOC ester bond, resulting in a general composition [ZrO]2+
[RDyeOPO3]2(Figure 1b).[34,35] With this composition, it is obvious
that RDye, in principle, can comprise a great number of different
fluorescent dyes. Essentially, the fluorescent organic dye needs
to contain a phosphate functionality to obtain insoluble nano-
particles in water.[34,35]
Phosphate-based IOH-NPs [ZrO]2+[RDyeOPO3]2are com-
posed of [ZrO]2+as an inorganic cation and [RDyeOPO3]2as a
fluorescent organic anion.[36] Due to charge neutrality, cation
and anion are available in 1 : 1 ratio (in the case of identical
charges) and in molar quantities. Such saline composition is
similar to simple sodium chloride that consists of equimolar
amounts of Na+cations and Clanions. The saline composition
also accounts for a specific advantage of the IOH-NPs, which is
an unprecedentedly high dye load (70–85 wt-%). Due to the
intermixing of the inorganic part (the cation) and the organic
part (the anion) on the molecular level, the nanoparticles are
designated as “inorganic-organic hybrid nanoparticles”, accord-
ing to the Latin origin: “hybrid”: crossbreed.[34,35]
The predominant role of [ZrO]2+in the IOH-NPs is to
guarantee their insolubility in water, which is prerequisite for
obtaining nanoparticles as well as for the intended water-
limited biomedical application.[34,35] A comparable approach was
yet only reported for bisphosphonates, which serve as organic
bridging ligands in coordination polymers.[25b,37] Zirconium as a
metal cation is known for its low toxicity and has been clinically
approved in the US and the EU, for instance, in the form of
sodium zirconium cyclosilicate (Lokelma, AstraZeneca) for the
treatment of hyperkalemia[38] or in the case of different
zirconium complexes as antiperspirants.[39] The fluorescent
organic anion [RDyeOPO3]2entails the fluorescence features of
the IOH-NPs.[34,35] In regard of the above considerations, [ZrO]2+
[RDyeOPO3]2IOH-NPs already offer the following advantages: i)
Uncomplex aqueous synthesis; ii) Low material complexity; iii)
High dye load leading to intense emission; iv) Wide variability
of fluorescent organic anions.
2.2. Phosphate-based IOH-NPs
Phosphate-based IOH-NPs with a general chemical composition
[ZrO]2+[RDyeOPO3]2are here introduced with the exemplary
system [ZrO]2+[(HPO4)1x(FMN)x]2(x=0–1) containing the in-
organic cation [ZrO]2+as well as the anions [HPO4]2and
[FMN]2.[34] FMN represents the fluorescent organic anion flavin
mononucleotide and is a derivative of vitamin B2(Figure 2a).[40]
Already in 2008,[30,34] [ZrO]2+[(HPO4)1x(FMN)x]2, and in partic-
ular [ZrO]2+[FMN]2, attracted our attention in regard of several
aspects: i) Its insolubility in water that supports nucleation and
growth of nanoparticles; ii) The chemical inertness of zirconium
phosphates; iii) The good biocompatibility of all components
(e.g. lethal intake of ZrCl4>1 g/kg);[41] iv) The replaceability of
[HPO4]2and [FMN]2in variable ratios.
Due to the low solubility of [ZrO]2+[(HPO4)1x(FMN)x]2in
water, straightforward aqueous synthesis is possible, avoiding
expensive precursors, multistep procedures, and complex
structures. Specifically, the synthesis of [ZrO]2+[(HPO4)1x
(FMN)x]2IOH-NPs comprises the injection of an aqueous
solution of ZrOCl28H2O to an aqueous solution of Na2(FMN)
and Na2(HPO4) (Figure 2a).[34] For controlling particle nucleation
and particle growth and for obtaining uniform nanoparticles
and colloidally stable suspensions, general aspects of colloid
chemistry need to be considered as expressed by the LaMer-
Dinegar model.[42] Thus, the injection was performed whilst
Figure 1. Scheme illustrating the synthesis of: a) [ZrO]2+[HPO4]2NPs and b)
fluorescent inorganic-organic hybrid nanoparticles (IOH-NPs) with a general
composition [ZrO]2+[RDyeOPO3]2.
Figure 2. [ZrO]2+[FMN]2IOH-NPs (FMN: flavin mononucleotide) with: a)
Scheme of synthesis; b) Particle size according to SEM; c) Aqueous
suspensions at daylight and with blue-light excitation; d) Excitation and
emission spectra; e) Optical microscopy, f) fluorescence microscopy imaging
in cells, and g) in mice after intradermal injection of nanoparticles (Cy5-NHS
intravascular vessel stain), (modified reproduction from ref. [34]).
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vigorously stirring at slightly elevated temperatures (55 8C).
Moreover, the dye anion was added with 5–10 mol-% excess to
guarantee anion-terminated particle surfaces. Subsequent to
the synthesis, the IOH-NPs were washed by repeated redis-
persion and centrifugation in/from water and/or ethanol to
remove remaining salts and starting materials. After the
purification, the [ZrO]2+[(HPO4)1x(FMN)x]2IOH-NPs can be
easily suspended in polar solvents (e. g., water, ethanol,
diethylene glycol) or biological buffers (e. g., HEPES, aqueous
dextran). The as-prepared suspensions are colloidally stable
over several months and can contain up to 10 wt-% of the IOH-
NPs.[34]
Suspensions of [ZrO]2+[(HPO4)1x(FMN)x]2IOH-NPs are
transparent and – depending on their concentration – exhibit a
yellow to orange color (Figure 2c). Upon blue-light excitation
(LED with lmax =465 nm), bright green emission (480-650 nm) is
observed with its maximum at 530 nm (Figure 2d).[34] According
to dynamic light scattering (DLS), scanning electron microscopy
(SEM), and transmission electron microscopy (TEM), the as-
prepared [ZrO]2+[(HPO4)1x(FMN)x]2IOH-NPs have a mean
hydrodynamic diameter of 39(12) nm and a primary particle
diameter of 25–40 nm, respectively (Figure 2b).[34] The size
distribution indicates that all particles are below 100 nm. In this
regard it needs to be taken into account that the synthesis was
performed in water without any specific surface agent for
controlling nucleation and growth. The determination of the
chemical composition of the IOH-NPs is generally challenging,
and hence, described in detail in a separate chapter (see 2.4).
The fluorescence of [ZrO]2+[(HPO4)1x(FMN)x]2– as ex-
pected – originates from the FMN anion. Quantum size effects
are naturally not involved. Thus, excitation and emission of the
[ZrO]2+[(HPO4)1x(FMN)x]2IOH-NPs (in suspension) can be
directly compared to free FMN in solution (Figure 2d).[34] The
quantum yield of FMN even in solution is comparably low
(about 30%).[43] Interestingly, the quantum yield of [ZrO]2+
[(HPO4)1x(FMN)x]2with 282% is identical when considering
the experimental significance. Although the [FMN]2anions –
and thus, the fluorescence centers – are in close proximity, no
concentration quenching was observed. This finding can be
ascribed to the amorphous nature of the IOH-NPs that do not
exhibit any periodic packing and long-ranging order. Actually,
this random distribution of [FMN]2guarantees intense emis-
sion. In particular for [ZrO]2+[FMN]2, the enormous dye load of
81 wt-% and the resulting quasi-infinite reservoir of fluorescent
centers per volume of each single nanoparticle leads to intense
spotlight-type emission.[34] Even certain photobleaching does
not noticeably reduce the emission intensity since a great
number of fluorescence centers still remains intact (see 2.3:
Figure 6c).
Since the IOH-NP concept – including aqueous synthesis,
uncomplex material composition and structure, high load of
FMN per nanoparticle (81 wt-%) – aims at biomedical applica-
tion, [ZrO]2+[(HPO4)1x(FMN)x]2, and especially [ZrO]2+[FMN]2,
were evaluated in in vitro and in vivo studies regarding
fluorescence and imaging performance. Indeed, [ZrO]2+[FMN]2
IOH-NPs show massive uptake into cells. They exhibit high
biocompatibility and show intense green emission (Fig-
ure 2f,g).[34] In cells or mice the green fluorescence is typically
stable over several hours and disappears after 2–3 days with
complete dissolution of the IOH-NPs. Toxic or allergic effects –
even after a period of two months – were not observed.[34]
According to TEM and electron-energy loss spectroscopy (EELS),
the [ZrO]2+[FMN]2IOH-NPs localize exclusively in vesicles
(Figure 2e). They do not colocalize with mitochondria or nuclei.
Thus, [ZrO]2+[FMN]2IOH-NPs appear to be a suitable tool for
staining viable structures.
In spite of the discussed advantages of the [ZrO]2+
[(HPO4)1x(FMN)x]2IOH-NPs, FMN-related green emission is less
favorable for biomedical application. On the one hand, cells
and tissue show green autofluorescence themselves so that a
considerable background is present in addition to the green
emission of the IOH-NPs. Moreover, green emission and – even
more important – blue light, required for excitation, exhibit low
tissue penetration.[9b,44] Biomedical issues and optical imaging-
based applications in animal models and in humans, therefore,
require long-wavelength emission rather in the far-red and
near-infrared range, since the light absorption by water and
hemoglobin is minimal in this spectral range, resulting in
optimal tissue penetration. Taking the composition [ZrO]2+
[RDyeOPO3]2and the concept of the IOH-NPs as a general
strategy, phosphate-functionalized fluorescent organic dyes
with other luminescence properties as FMN are needed.
Indeed, the IOH-NP concept allows using further fluorescent
organic anions and creating a platform of materials. Here, we
show the expansion of the concept to [ZrO]2+[PUP]2, [ZrO]2+
[MFP]2, [ZrO]2+[RRP]2, and [ZrO]2+[DUT]2with PUP: phenyl-
umbelliferon phosphate, MFP: methylfluorescein phosphate,
RRP: resorufin phosphate, and DUT: DY-647 uridine triphosphate
(Figure 3a).[35,36] All these additional phosphate-based IOH-NPs
are insoluble in water, too, and can be prepared via aqueous
synthesis. They exhibit mean particle diameters of 20–40 nm at
high colloidal stability (Figure 4a). [ZrO]2+[PUP]2, [ZrO]2+
[MFP]2, [ZrO]2+[RRP]2, and [ZrO]2+[DUT]2show full color
emission in the blue (380–600 nm, lmax =458 nm), green (460–
700 nm, lmax =518 nm), red (550–700 nm, lmax =584 nm) and
near-infrared (630–780 nm, lmax =675 nm) spectral regime (Fig-
Figure 3. [ZrO]2+[PUP]2, [ZrO]2+[MFP]2, [ZrO]2+[RRP]2, and [ZrO]2+[DUT]2
IOH-NPs with: a) Structure of fluorescent dye anions; b) Excitation and c)
emission spectra (modified reproduction from ref. [35]).
6ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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ure 3b,c).[35] Due to the quasi-infinite number of fluorescence
centers (dye content up to 85 wt-%), all these phosphate-based
IOH-NPs again show intense spotlight emission in aqueous
suspensions.
To illustrate the biomedical performance, [ZrO]2+[MFP]2,
[ZrO]2+[RRP]2and [ZrO]2+[DUT]2were incubated in vitro with
murine alveolar macrophages (MHS) cell lines (50 mg IOH-NPs
per mL of cell culture medium) to analyze uptake and
fluorescence. After incubation (5 h at 378C), the internalization
of the IOH-NPs is clearly demonstrated and becomes even
more evident after 24 h (Figure 4b).[35] The granularly structured
fluorescence in the macrophages indicates the presence of the
IOH-NPs, which was also confirmed by EELS, showing a similar
granular structure for the localization of zirconium in the
macrophages. Controls with macrophages incubated with the
IOH-NPs at 48C at reduced metabolic activity as well as
macrophages cultivated without IOH-NPs do not show any
comparable fluorescence. Furthermore, no relevant toxic effects
of [ZrO]2+[MFP]2, [ZrO]2+[RRP]2and [ZrO]2+[DUT]2were
observed with concentrations up to 250 mM.[35] After subcuta-
neous injection in nude mice, the emission is also clearly visible
in vivo (Figure 4c). Thus, the capability of phosphate-based IOH-
NPs for in vitro optical imaging is confirmed as a proof-of-the-
concept.
2.3. Sulfonate-based IOH-NPs
Although phosphate-based IOH-NPs [ZrO]2+[RDyeOPO3]2already
stand for a broad platform of materials with different
fluorescent organic anions [RDyeOPO3]2, the number of com-
mercially available fluorescent organic dyes containing a
phosphate functionality is limited. Moreover, phosphate-func-
tionalized dyes, and in particular those showing red and
infrared emission, are often extremely expensive (up to 500 E
per 1 mg).[45] In contrast, almost all conventional fluorescent
organic dyes are commercially available with sulfonate func-
tions. Functionalization of aromatic organic molecules by
sulfonate groups is synthetically straightforward and often used
to make such dyes soluble in water.[21] As a consequence,
almost all organic dye systems – such as coumarins, rhod-
amines, oxazines, cyanines, etc. – are available with one or
more sulfonate group. Introducing such sulfonate-based fluo-
rescent anions [RDye(SO3)n]ninto the IOH-NP concept could, of
course, dramatically broaden the platform of fluorescent nano-
materials.
In contrast to phosphate-based fluorescent anions
[RDyeOPO3]2, however, sulfonate-based fluorescent anions
[RDye(SO3)n]ndo not form any insoluble compound upon
addition of [ZrO]2+in water. Taking binary metal sulfates as
most suitable reference systems, BaSO4and PbSO4are actually
known as the most insoluble sulfates in water.[32] Both cations
Ba2+and Pb2+, however, are also known as severely harmful to
animate beings.[46] Anyway, both cations are not suitable
though they also do not form insoluble compounds with
sulfonate-based fluorescent anions [RDye(SO3)n]n. This can be
rationalized based on the monovalent charge of the sulfonate
group in comparison to the divalent sulfate ion, which reduces
the Coulomb interaction significantly. Since the charge of a
sulfonate group is fixed, the only option is to choose a cation
having a comparable radius as Ba2+/Pb2+(149/133 pm)[47] but a
higher charge. In this regard, La3+(117 pm)[47] is promising and
indeed results in the formation of insoluble compounds
together with sulfonate-based fluorescent anions
[RDye(SO3)n]n.[48] Due to the similarity of the lanthanides, in
principle, this holds for all rare-earth-metal ions Ln3+(Ln3+:La
3+
to Lu3+). From all these ions, La3+is interesting due to its low
cost. Aiming at novel contrast agents, however, Gd3+is even
more interesting as it opens the option of multimodal imaging
via the fluorescence of the dye anion and the paramagnetism
of Gd3+.[6]
Sulfonate-based IOH-NPs with a general composition M3+n/3
[RDye(SO3)n]n, [M(OH)]2+n/2[RDye(SO3)n]n, or [MO]+n[RDye(SO3)n]n
with M: rare-earth metal, and most preferentially with La3+or
Gd3+, can indeed contain a great number of different sulfonate-
based dye anions.[36,48] As an illustrative example, we take M3+
[AMA]3(M=La, Gd) with amaranth red (AMA) as the sulfonate-
based fluorescent dye anion (Figure 5a),[36] which is also known
as E123, C.I. 16185, Acid Red 27, C-Red 46, Echtrot D, or Food
Figure 4. [ZrO]2+[PUP]2, [ZrO]2+[MFP]2, [ZrO]2+[RRP]2, and [ZrO]2+[DUT]2
IOH-NPs with: a) Aqueous suspensions at daylight and with excitation; b)
Emission after uptake by MHS macrophages; c) Emission after subcutaneous
injection in nude mouse (modified reproduction from ref. [35]).
Figure 5. Gd3+[AMA]3IOH-NPs (AMA: amaranth red) with: a) Scheme of
synthesis; b) Particle size according to SEM; c) Particle size according to DLS
in DEG and in water; d) Zeta potential in water (modified reproduction from
ref. [48]).
7ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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Red 9.[49] AMA is widely used, for instance, in food industry, and
can be considered as a less harmful fluorescent dye in
comparison to many azo-dyes.
The synthesis of M3+[AMA]3(M =La, Gd) is comparable to
the synthesis of the phosphate-based IOH-NPs and performed
by injecting an aqueous solution of LaCl37H2O or GdCl3
6H2O to an aqueous solution of Na3(AMA). Again, general
aspects of colloid chemistry according to LaMer’s model[42]
need to be considered to control particle nucleation and
particle growth and to obtain nanoparticles and colloidally
stable suspensions (Figure 5c). Hence, injection was again
performed whilst vigorously stirring at slightly elevated temper-
ature (558C). Moreover, the dye anion was used with 10 mol-%
excess in relation to the cation to guarantee anion-terminated
particle surfaces. After synthesis and purification, M3+[AMA]3
IOH-NPs can be easily suspended in solvents, such as water,
ethanol, diethylene glycol, or biological buffers like HEPES or
aqueous dextran. According to the chemical composition, La3+
[AMA]3and Gd3+[AMA]3contain extraordinarily high dye
loads of 79 wt-% and 77 wt-% [AMA]3, respectively.[48]
The particle diameter of the as-prepared M3+[AMA]3IOH-
NPs was at first measured in DEG, which is known for excellent
stabilization of nanoparticles via surface coordination.[50] Here, a
mean hydrodynamic diameter of 68 10 nm with narrow size
distribution was observed (Figure 5c). In water, nanoparticles
generally show larger hydrodynamic radii due to a rigid layer of
adsorbed solvent molecules. Based on the high polarity and
extensive hydrogen bonding networks, this rigid solvent layer is
largely expanded in water.[51] Thus, a mean hydrodynamic
diameter of 10530 nm was obtained (Figure 5c). Finally,
overview SEM images show uniform spherical particles with a
mean diameter of 4710 nm, which was calculated by
statistical evaluation of 130 nanoparticles (Figure 5b). Zeta
potential analysis of La3+[AMA]3and Gd3+[AMA]3show
negative charging at 12.5 mV in the biologically most relevant
pH range of pH 4 to 8 (Figure 5d).[48] The resulting electrostatic
stabilization is beneficial for both controlling the particle size as
well as suppressing agglomeration.
Suspensions of La3+[AMA]3and Gd3+[AMA]3exhibit a
brilliant red color (Figure 6a) and an intense red emission upon
excitation by green light (glass fiber with green filter, lmax =
555 nm) (Figure 6a).[48] Fluorescence spectroscopy indicates
strong absorption at 400 to 650 nm and emission at 650 to
800 nm peaking at lmax =700 nm (Figure 6b). Such lumines-
cence features are ideal for biomedical application in terms of
low background from autofluorescence and deep penetration
of tissue.[44]
M3+[AMA]3IOH-NPs show higher photostability than
dissolved AMA (Na3(AMA)) in solution at identical concentration
(33 mmol/mL AMA). UV-irradiation of M3+[AMA]3suspensions
and of dissolved AMA (lexc =310 nm, 15 h) displays continuous
photobleaching with only 27% of pristine emission intensity
remaining for dissolved AMA (Figure 6c). In contrast, M3+
[AMA]3suspensions show almost constant emission intensity
over the complete period of irradiation.[48] The photostability of
the IOH-NPs is even more remarkable when compared to
conventional nanoparticle systems with fluorescent organic
dyes encapsulated in inorganic or polymeric matrices (Table 1).
They all show considerable photobleaching even on short
timescales. The high photostability of the M3+[AMA]3IOH-NPs
can be attributed, on the one hand, to the extremely high dye
load so that certain photobleaching at the particle surface
leaves the emission intensity more-or-less unaffected. Despite
of strong absorption in the visible (450–700 nm), on the other
hand, the IOH-NPs exhibit a high reflectivity in the UV spectral
regime (<450 nm).[48] In particular, the absorption of high-
energy light is lower, resulting in a reduced formation of
reactive oxygen species (ROS), and therefore, a higher photo-
stability. SiO2or Ca3(PO4)2, as widely applied inorganic matrices
to encapsulate fluorescent organic dyes, are much less UV-
reflective, resulting in a significant photobleaching at daylight
(Table 1).
Besides the AMA-based fluorescence, Gd3+-related para-
magnetism of seven unpaired electrons is expected for Gd3+
[AMA]3. Indeed, powder samples of the IOH-NPs can be
already attracted by a bar magnet (Figure 7a). Magnetic
measurements quantify the magnetic properties of the as-
prepared Gd3+[AMA]3to an effective magnetic moment of
meff =6.83(1) mBper Gd atom and a Weiss constant of qp=4.3(5)
K (Figure 7b).[48] These data are comparable to Gd3+-based MRI
contrast agents, such as the coordination complexes Gd-DPTA
(DPTA: diethylenetriaminepentaacetate) and Gd-DOTA (DOTA:
1,4,7,10-tetraazacyclododecane-N,N’,N’’,N’’’-tetraacetic acid),[55]
which are clinically applied with about 0.1 mmol Gd3+per kg of
body weight.[56] An equal Gd3+content would require about
70 mg of Gd3+[AMA]3per kg of body weight. In comparison to
standard Gd-DPTA and Gd-DOTA, it must be noted that Gd3+
Figure 6. Gd3+[AMA]3IOH-NPs with: a) Aqueous suspension at daylight and
with green light excitation (glass fiber with green filter, lmax =555 nm); b)
Excitation and emission spectra; c) Photostability of Gd3+[AMA]3(suspen-
sion) in comparison to free AMA (solution of Na3(AMA)), all in water,
33 mmol/mL AMA, lexc =310 nm for 15 h), (modified reproduction from ref.
[48]).
8ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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[AMA]3IOH-NPs also show fluorescence so that they become
suitable for multimodal imaging (OI, MRI).
To further evaluate the applicability of Gd3+[AMA]3as a
contrast agent, the IOH-NPs were incubated with MHS macro-
phages.[57] Similar to [ZrO]2+[FMN]2, a massive uptake of the
IOH-NPs was demonstrated by fluorescence microscopy after
5 h of incubation at 37 8C (Figure 8a), whereas internalization is
strongly reduced at 48C. The viability of the macrophages is
unaffected by the uptake of the Gd3+[AMA]3IOH-NPs (Fig-
ure 8b). Only at high concentrations (200 mg/mL), the cell
viability is decreasing, which, in fact, can be related to a
reduced concentration of culture medium. This finding points
to a good biocompatibility of the Gd3+[AMA]3IOH-NPs.[48]
As discussed for phosphate-based IOH-NPs [ZrO]2+
[RDyeOPO3]2,M
3+[AMA]3(M=La, Gd) can be also considered as
first representative of a wider platform of sulfonate-based IOH-
NPs with a general composition [M]3+n/3[RDye(SO3)n]n, [M(OH)]2+
n/2[RDye(SO3)n]n, or [MO]+n[RDye(SO3)n]n.[58] Additional examples
are, for instance, [Gd(OH)]2+2[CSB]4, [Gd(OH)]2+2[DB71]4,
[Gd(OH)]2+[NFR]2, [Gd(OH)]2+[AR97]2, and [Gd(OH)]2+2[EB]4
containing the sulfonate-based fluorescent anions Chicago Sky
Blue ([CSB]4), Direct Blue 71 ([DB71]4), Nuclear Fast Red
([NFR]2), Acid Red 97 ([AR97]2), or Evans Blue ([EB]4) (Fig-
ure 9a). All these fluorescent dyes are commercially available
and used in solution for staining in cell biology and
histology.[3,9,10] In particular, this holds for Chicago Sky Blue,[59]
Table 1. Photobleaching of conventional nanocomposites with fluorescent organic dyes encapsulated in inorganic or polymer matrices.
Encapsulated
fluorescent dye
Matrix
material
Time of UV
irradiation
Intensity after
irradiation
Half-lifetime
of emission
Ref.
Cy3
Cy3
Ca3(PO4)2
SiO2
300 s
7h
97%
98%
Not measured
Not measured
[24f] [52]
Cy5 SiO2200 s 25% 60 s [24g]
Tetramethyl-rhodamine isothio-cyanate/TRITC SiO230 min 88% Not measured [53]
Fluorescein iso-thiocyanate/FITC SiO230 min 70% Not measured [24d]
Indocyanine green/ICG Ca3(PO4)2633 s 50% 633 s [24h]
Indocyanine green/ICG
Nile red
PLGA[a]
PVK[a]
60 min
55 min
52%
87%
60 min
Not measured
[26b] [54]
M3+[AMA]3
(M: La, Gd)
Hybrid
(no matrix)
15 h 100% Infinite[b] [48]
[a] PLGA: poly(lactic-co-glycolic acid); PVK: poly-N-vinylcarbazole. [b] As no photobleaching and decrease of intensity was observed for M3+[AMA]3IOH-NPs
on a timescale of 15 h, the formal half-lifetime of emission is infinite.
Figure 7. Magnetic properties of Gd3+[AMA]3IOH-NPs: a) Powder sample
upside down with a bar magnet attracting the nanoparticles; b) Magnet-
ization at 70 K and 300 K in dependence of the external magnetic field
(modified reproduction from ref. [48]).
Figure 8. Gd3+[AMA]3IOH-NPs incubated with MHS macrophages: a)
Fluorescence microscopy showing incubated Gd3+[AMA]3(24 h after
incubation with 50 mg/mL); b) Viability of MHS cells 24 h and 48 h after
incubation with different concentrations of the IOH-NPs (scale bar: 20 mm),
(modified reproduction from ref. [48]).
Figure 9. [Gd(OH)]2+2[CSB]4, [Gd(OH)]2+2[DB71]4, [Gd(OH)]2+[NFR]2, [Gd(O-
H)]2+[AR97]2, and [Gd(OH)]2+2[EB]4IOH-NPs with: a) Structure of fluores-
cent dye anions; b) Aqueous suspensions at daylight and with excitation
([Gd(OH)]2+2[CSB]4, [Gd(OH)]2+2[DB71]4, [Gd(OH)]2+[AR97]2excited via UV-
LED; [Gd(OH)]2+[NFR]2excited via halogen lamp with green glass filter;
[Gd(OH)]2+2[EB]4excited via white light halogen lamp); c) Excitation and d)
emission spectra (normalized on maximum intensity for direct comparison),
(modified reproduction from ref. [48]).
9ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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Nuclear Fast Red,[60] and Evans Blue.[61] Similar to M3+[AMA]3,
the sulfonate-based fluorescent dye anions can be made
insoluble upon addition of rare-earth-metal ions such as La3+or
Gd3+to form IOH-NPs with unprecedentedly high dye loads
(Table 2).[58]
All these sulfonate-based IOH-NPs have mean particle
diameters of 40–50 nm at narrow size distribution (Table 2).
Additional stabilizers are not required. Moreover, the sulfonate-
based IOH-NPs exhibit intense absorption leading to the blue
color of [Gd(OH)]2+2[CSB]4and [Gd(OH)]2+2[EB]4as well as to
the orange to red color of [Gd(OH)]2+[NFR]2, [Gd(OH)]2+
[AR97]2and [Gd(OH)]2+2[DB71]4(Figure 9b).[58] The absorptive
color of the IOH-NPs is similar to the pure organic dyes and
again very intense due to the quasi-infinite number of dye
anions in each IOH-NP. Accordingly, the IOH-NPs can be also
interesting for staining in cell biology and histology as a
promising alternative to molecular dyes in solution.
Sulfonate-based IOH-NPs show intense emission upon
excitation with visible light (e. g. blue-light LED). Whereas
[Gd(OH)]2+2[CSB]4and [Gd(OH)]2+2[DB71]4exhibits emission
in the blue spectral regime (Table 3, Figure 9d), [Gd(OH)]2+
[NFR]2and [Gd(OH)]2+2[AR97]4emit yellow and red light.
[Gd(OH)]2+[EB]2shows deep red to infrared emission (Table 3,
Figures 9d).[58] Most interestingly, fluorescence was yet only
reported for NFR, CSB, and EB in the case of the molecular dyes
(in solution),[62] whereas an emission of DB71 and AR97 was not
reported before. Again, the great number of fluorescent centers
per nanoparticle not only guarantees intense light absorption
but also sufficient emission to be detected by the naked eye or
via fluorescence microscopy. All in all, the fluorescent sulfonate-
based IOH-NPs can be very interesting for optical imaging in
cell biology and histology but also for in vitro and in vivo
studies.[5d,9,63] Due to the quasi-infinite number of fluorescence
centers per nanoparticle – similar to La3+[AMA]3and Gd3+
[AMA]3– low photobleaching is observed (see 2.3: Figure 6c).
2.4. Chemical Composition
Proving the chemical composition of the IOH-NPs is challeng-
ing. First of all, it needs to be noticed that all IOH-NPs are non-
crystalline. They do not show any specific Bragg peak in X-ray
diffraction or electron diffraction experiments.[34,35,48,58] Conse-
quently, crystal structures of the compounds are unknown. On
the other hand, the absence of periodically ordered arrays is
advantageous, if not essential in regard of the fluorescence of
the IOH-NPs. In the case of crystalline structures with periodi-
cally aligned fluorescent organic anions, severe concentration
quenching would have been expected that could eradicate the
emission of the IOH-NPs partly or completely.[13]
In order to elucidate the chemical composition of the IOH-
NPs, different analytical methods need to be involved to gain
insights at different levels of priority. Obviously, it is the highest
priority to prove the presence of the fluorescent organic anion
[RDyeOPO3]2or [RDye(SO3)n]n. Besides fluorescence spectroscopy,
here, Fourier-transformed infrared (FT-IR) spectroscopy is
indicative, as exemplarily shown for Gd3+[AMA]3(Fig-
ure 10a).[48] A comparison with the starting material Na3(AMA)
as reference shows all characteristic vibrations of AMA,
including n(OH): 3600–3000 cm1,n(N=N): 1370 cm1,n(CN=
Table 2. Average particle size (obtained from SEM), zeta potential (in water
at pH 7.0), and dye load of sulfonate-based IOH-NPs.
IOH-NP composition Particle size
from SEM (nm)[a]
Zeta potential
(mV)
Dye load
(wt-%)
Gd3+[AMA]347 10 13 77
[Gd(OH)]2+2[CSB]438526 74
[Gd(OH)]2+2[DB71]437742 73
[Gd(OH)]2+[NFR]244928 70
[Gd(OH)]2+[AR97]247 619 79
[Gd(OH)]2+2[EB]44210 42 77
[GdO]+[ICG][b] 49 827 81
La43+[TPPS4]34[c] 68834 83
Gd43+[AlPCS4]34[c] 47726 81
[a] Statistical average based on >100 nanoparticles. [b] See 3.1; [c] See 3.2.
Table 3. Excitation and emission of sulfonate-based IOH-NPs.
IOH-NP composition Excitation range
(nm)
Emission range
(nm)
Emission
lmax (nm)
Gd3+[AMA]3400–650 650–800 700
[Gd(OH)]2+2[CSB]4240–460 400–550 437
[Gd(OH)]2+2[DB71]4320–440 400–550 444
[Gd(OH)]2+[NFR]2400–580 520–740 578
[Gd(OH)]2+[AR97]2550–730 550–640 592
[Gd(OH)]2+2[EB]4350–640 700–880 782
[GdO]+[ICG][a] 700–820 780–840 810
La43+[TPPS4]34[b] 380–600 540–700 585
Gd43+[AlPCS4]34[b] 250–400, 550–720 650–770 686
[a] See 3.1; [b] See 3.2.
Figure 10. Chemical composition of as-prepared Gd3+[AMA]3IOH-NPs with:
a) FT-IR spectra (Na3(AMA) as a reference); b) TG (Na3(AMA) as a reference),
(modified reproduction from ref. [48]).
10ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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NC): 1230 cm1,n(SO3): 680–420 cm1.[64] Certain broadening
of the vibrations in the case of the IOH-NPs originates from
their non-crystallinity. In addition to the fluorescent dye anion,
the presence of the inorganic metal cation is important and
qualitatively proven by energy dispersive X-ray spectroscopy
(EDXS). Quantification is usually not possible via EDXS since
both electron absorption and X-ray emission of the heavy
elements (e.g., La, Gd) are too different from the light elements
(C, H, N, S, O) for reliable determination.[48]
After proving the presence of the inorganic cation and the
fluorescent organic anion, their ratio becomes most relevant in
order to determine the chemical composition of the IOH-NPs.
In this regard, first of all, the charge of the inorganic cation and
the fluorescent organic anion should be considered. In addition,
the analysis of the total organics content is indicative, especially
since inorganic cation and organic anion are of comparable
molecular weight. Taking Gd3+[AMA]3again as an example,
the charges of cation and anion already suggest a cation-to-
anion ratio of 1:1, which is validated by performing thermo-
gravimetry (TG) to obtain the total organics content.[48] Prior to
TG, the as-prepared Gd3+[AMA]3IOH-NPs were dried in vacuo
at room temperature for 8 h to remove all adsorbed volatiles
(e.g. water). Thereafter, TG shows a total weight loss of 72% up
to a temperature of 1000 8C, which corresponds well to the
calculated weight loss of 69% of total organics combustion for
the assumed composition Gd3+[AMA]3(Figure 10b). Moreover,
the thermal remnant of TG analysis was identified via XRD as
Gd2O2(SO4). Accordingly, the thermal combustion reaction can
be rationalized as follows:[48]
2Gd3þ½C20H11 N2O10S33þ531
=
2O2!
Gd2O2ðSO4Þþ40CO2þ11H2Oþ2N
2þ5SO2
In addition to TG analysis, the composition of Gd3+[AMA]3
can be independently verified by elemental analysis (EA)
resulting in C/H/N/S contents of: 32 wt-% C, 3 wt-% H, 4 wt-%
N, and 12 wt-% S. Within the experimental error, these data are
well in accordance with the calculated values: 36 wt-% C, 2 wt-
% H, 4 wt-% N, and 14 wt-% S. Taking all analytical data
together (FT-IR, EDX, TG, EA), the chemical composition of Gd3+
[AMA]3is reliably substantiated.[48] With similar investigations
the chemical composition of other phosphate- or sulfonate-
based IOH-NPs was determined, as well (Tables 4, 5).
For different reasons the determination of the chemical
composition of the IOH-NPs still remains challenging. Thus, the
thermal decomposition is complicated in the case of the
phosphate-based IOH-NPs due to the encapsulation of the
organic content within the metal phosphate formed during
thermal decomposition. Actually, such effect is well-known for
flame retarding materials. They often contain phosphates to
encapsulate organic materials, and thereby, to increase the
ignition temperature.[65] For analysis of the IOH-NPs, slow
heating needs to be performed to guarantee the total
combustion of all organic constituents via TG and EA.
For all IOH-NPs, and in particular for sulfonate-based IOH-
NPs, oxide atoms and/or hydroxide and water molecules could
be coordinated to the metal cation. This is another option to
vary the chemical composition of the IOH-NPs as expressed by
the formula [M]3+n/3[RDye(SO3)n]n, [M(OH)]2+n/2[RDye(SO3)n]n,or
[MO]+n[RDye(SO3)n]n. Due to its low weight (relative to the
inorganic cation and the fluorescent dye anion), the coordina-
tion of charged species like O2or OHto the metal cation
cannot be reliably evidenced based on the above described
analytical techniques. On the other hand, the presence of O2
or OHwould naturally change the net charge of the inorganic
cation, and thereby also the ratio of inorganic cation and
fluorescent dye anion. This molar ratio, however, can be
precisely determined based on TG and EA. Finally, non-charged
H2O molecules could be coordinated to the inorganic cation. As
they do not influence the net charge of the cation or the
charge neutrality of the IOH-NPs, and as they only have low
weight, such H2O coordination (most probably of 1–2 H2O
molecules per formula unit) cannot be excluded.
Although non-crystalline, finally, the IOH-NPs can be
assumed to exhibit layer-type structures. Especially in the case
of zirconium phosphates, such as Zr(HPO4)2H2O, layered
arrangements have been described.[66] Layered structures were
also often observed for zirconium in combination with organo-
phosphates, and especially, organophosphonates.[67] Actually,
these compounds are highly relevant as flame retardants.[68]
Table 4. Chemical composition of phosphate-based IOH-NPs according to
EA (C,H,N,S content), EDX (Zr :P ratio), and TG (total organic combustion).
Compound[a] C con-
tent
(%-wt,
EA)
(calcd)
H con-
tent
(%-wt,
EA)
(calcd)
N con-
tent
(%-wt,
EA)
(calcd)
Zr:P ra-
tio
(EDX)
(calcd)
Weight
loss
(%-wt,
TG)
(calcd)
[ZrO]2+[RRP]226 (34)3(2)3(4) 1.2:1
(1:1)
43 (48)
[ZrO]2+[FMN]234 (36)/ 9(10) 1.0:1
(1:1)
61 (64)
[ZrO]2+[MFP]242 (47)4(3)0(0) 1.1:1
(1:1)
62 (64)
[ZrO]2+[PUP]232 (42)3(2)0(0) 1.3:1
(1:1)
50 (54)
[a] Note that the composition of [ZrO]2+[DUT]2was not studied in detail
due to the very high cost of [DUT]2(about 500 Efor 1 mg).
Table 5. Chemical composition of sulfonate-based IOH-NPs according to
EA (C,H,N,S content) and TG (total organic combustion).
Compound C con-
tent
(%-wt,
EA)
(calcd)
H con-
tent
(%-wt,
EA)
(calcd)
N con-
tent
(%-wt,
EA)
(calcd)
S con-
tent
(%-wt,
EA)
(calcd)
Weight
loss
(%-wt,
TG)
(calcd)
Gd3+[AMA]332 (36)3(2)4(4)12(14)72(69)
[Gd(OH)]2+2[CSB]430 (33)4(2)6(7)9(10)68(64)
[Gd(OH)]2+2[DB71]426 (27)3(2)5(8)7(10)59(66)
[Gd(OH)]2+[NFR]235 (33)3(2)3(3)8(6)62(56)
[Gd(OH)]2+[AR97]246 (48) 4 (3) 6 (7) 8 (8)75(73)
[Gd(OH)]2+2[EB]431 (33)4(2)6(7)9(11)67(64)
[GdO]+[ICG][a] 55 (56) 5(5)3(0)7(7)83(78)
La43+[TPPS4]34[b] 46 (47) 3(2)5(5)10(11)76(76)
Gd43+[AlPCS4]34[b] 36 (35) 3(1)13(10)8(11)79(78)
[a] See 3.1; [b] See 3.2.
11ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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Nevertheless, it must be stated that the structural character-
ization of zirconium phosphates is still lacking, in general.
3. Specific Properties of Inorganic-Organic
Hybrid Nanoparticles (IOH-NPs)
Subsequent to the illustration of the material concept of the
IOH-NPs and after having shown their feasibility for
fluorescence detection and optical imaging, we now address
more specific functionalities of selected IOH-NPs. This includes
[GdO]+[ICG](ICG: indocyanine green) for multimodal imag-
ing,[69] Gd43+[AlPCS4]34(AlPCS4: aluminium(III) chloride phthalo-
cyanine tetrasulfonate) showing singlet-oxygen generation,[70]
as well as the dissolution of the IOH-NPs and their use for drug
delivery and drug release.
3.1. Multimodal Imaging
To combine the specific assets of different imaging techniques
(e.g., resolution, imaging of different types of tissue) and/or to
translate preoperative to intraoperative imaging (and vice
versa), suitable contrast agents for multimodal imaging are
highly interesting.[10] In this regard, materials with different
functionalities were often integrated with high virtuosity into
complex nanoarchitectures. This includes, for instance, super-
paramagnetic iron oxide nanoparticles (SPIONs) and inorganic
fluorescent nanoparticles (e.g., Q-dots, lanthanide-doped ox-
ides) or molecular fluorescent dyes (e.g., coumarins, rhod-
amines, oxazines, cyanines) that were encapsulated in or
attached to inorganic or organic matrices (e.g., SiO2, calcium
phosphate, polymers, liposomes, dendrimers).[3,10] As discussed
before, the complexity of the resulting contrast agents and the
sheer number of constituents can be a restriction in itself as all
constituents and combinations must be verified individually for
clinical approval. In practice, in vivo application becomes the
more prohibitive the greater the complexity and the more
multi-component the employed materials.[29] Moreover, the
amount of active contrast agent can be very low in an inert
matrix as majority component, which reduces the detection
limit.[24–27]
In regard to the IOH-NPs, we already discussed optical
absorption and fluorescence (see 2.2, 2.3), and we also pointed
to Gd3+as an optional cation in sulfonate-based IOH-NPs (see
2.3) to implement the IOH-NPs in regard of OI and MRI. Besides
imaging, optical absorption, fluorescence, and magnetism can
be also used to locate the IOH-NPs in cells and tissue and to
determine their in vitro and in vivo behavior and dissolution. A
most interesting type of IOH-NP for multimodal imaging, in this
regard, is [GdO]+[ICG], which consists of equimolar amounts
of paramagnetic gadolinium as the inorganic cation and [ICG]
as the organic fluorescent dye anion (Figure 11a).[69]
Again, an aqueous synthesis was applied to prepare [GdO]+
[ICG]using GdCl36H2O and Na(ICG) as the starting materials.
[GdO]+[ICG]contains 81 wt-% [ICG]and can be easily
suspended in polar solvents (e. g., water, ethanol, diethylene
glycol) or biological media (e.g., HEPES, aqueous dextran) with
concentrations up to 10 mg/mL. The analytical characterization
regarding particle size and chemical composition was per-
formed as described before (see 2.3/2.4: Tables 2, 5). Accord-
ingly, SEM and DLS show mean particle diameters of 498nm
and 509 nm, respectively, with narrow size distribution (Fig-
ure 11b) and a zeta potential of 20 to 35 mV at pH 6–8
(Figure 11c).[69]
Aiming at OI, ICG is optimal for biomedical application in
many aspects. On the one hand, its strong visible absorption
(700–820 nm) and its NIR emission (780–840 nm) are ideal for
deep-tissue penetration minimizing the absorbance by water
and hemoglobin (Figure 11d).[9b,44] Moreover, ICG is well-toler-
ated (LD50: 50–80 mg/kg), approved for clinical use, and already
widely used in the clinic for histology.[71] Furthermore, ICG is
cheap (about 50 Eper 1 g)[72] in comparison to many alter-
native commercial red- and infrared-emitting dyes that are
conventionally used for OI.[13,21,45] On the other hand, ICG as a
dissolved molecule has several weaknesses such as: i) Rapid
binding to human serum albumin and high-density lipoproteins
causing agglomeration and rapid clearance via the liver; ii) Very
short circulation time (half-life of only 2–4 min in mice); iii) Low
fluorescence quantum yield (only about 5% in water); iv) Low
chemical stability under physiological conditions due to fast
biodegradation; v) Rapid photobleaching under light expo-
sure.[71,73] Similar to other fluorescent organic dyes, ICG was also
often encapsulated in stabilizing matrices (e.g., organic poly-
mers, liposomes, micelles, silica) to overcome these limitation-
s.[71a,84] Moreover, ICG has been intercalated in layered double
hydroxides (LDHs).[75] Again, such inert matrices reduce the
available amount of ICG per nanoparticle and lower the
intrinsically weak emission intensity even further.
In contrast to ICG solutions, suspensions of [GdO]+[ICG]
IOH-NPs show intense emission due to the high ICG load
(81 wt-%) and the great number of fluorescent centers per
nanoparticle (see 2.3: Table 3). Naturally, [GdO]+[ICG](in
suspension) exhibits identical fluorescence features as ICG (in
solution).[69] Accordingly, the dark green aqueous suspensions
show deep red emission upon visible light excitation (Fig-
Figure 11. [GdO+][ICG]IOH-NPs (ICG: indocyanine green) with: a) Scheme
of synthesis; b) Particle size according to SEM; c) Zeta potential in water; d)
Excitation and emission spectra (free ICG in solution as reference); e)
Aqueous suspension at daylight and excited with white light (modified
reproduction from ref. [69]).
12ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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ure 11e). Similar to Gd3+[AMA]3(see 2.3: Figure 6c), [GdO]+
[ICG]IOH-NPs show a higher photostability as well as a greater
storage stability and a higher emission intensity as ICG
solutions (at identical ICG concentration).[69] These features are
highly relevant to practical handling since exactly those
weaknesses of ICG (in solution) are addressed, which limit its
use for fluorescence detection and OI.[71,73]
In vitro studies with MHS macrophages show an excellent
uptake of [GdO]+[ICG]as indicated by their intense emission
(Figure 12a). Massive uptake of IOH-NPs was again observed
upon incubation at 378C, whereas only minimal uptake was
observed at 48C. This observation indicates an active acquis-
ition of [GdO]+[ICG]by the macrophages. It is to be noticed
that the IOH-NPs were coated by dextran to improve the
membrane permeability and cell uptake.[76] Despite massive
internalization of [GdO]+[ICG]IOH-NPs, the metabolic activity
and viability of the MHS cells – according to cell proliferation
assays – remain almost unaffected (Figure 12b).[69] When
comparing [GdO]+[ICG](in suspension) with ICG (in solution)
or Gd-DOTA/Gd-DTPA (in solution) as references, the viability of
MHS cells treated with the IOH-NPs turned out as only slightly
lower as compared to dissolved ICG and significantly higher
than for the standard MRI contrast agents Gd-DOTA/Gd-DTPA
(Figure 12b).[69] Naturally, this comparison was performed at
similar concentration of ICG and/or Gd. In regard of its
performance as a contrast agent, finally, it must be noted that
only [GdO]+[ICG]is multimodal and suitable as contrast agent
for OI and MRI.
Whereas free ICG (in solution) is typically not suitable for
fluorescence detection due to its low emission,[71,73] it is one of
the most promising absorptive contrast agents for PAI.[9a,77]
Therefore, an evaluation of [GdO]+[ICG]IOH-NPs in regard of
its feasibility for PAI seemed obvious. The performance of
[GdO]+[ICG](in suspension) indeed turned out as similar to
ICG (in solution) at identical ICG concentration (Figure 12c).
Blood vessel phantoms, chicken-breast phantoms, and dead-
mouse phantoms showed promising signal intensity and depth
of detection, indicating the feasibility of [GdO]+[ICG]IOH-NPs
also for PAI.[69]
Besides in vitro studies, the multimodal features of [GdO]+
[ICG]in terms of MRI and OI were evaluated in vivo. In
response to increasing concentrations, the IOH-NPs clearly
result in a reduction of the T1-relaxation (Figure 13a).[69] The
specific relaxivity (r1) per gadolinium at 7 Tesla was determined
to 8.00.4 mM1s1for dextran-coated [GdO]+[ICG]. In fact,
the relaxivity is even higher than for Gd-DTPA or Gd-DOTA (3–
5mM
1s1).[6,55] In vivo, mice were imaged before and 5 h after
intravenous injection of [GdO]+[ICG]IOH-NP suspensions
resulting in T1-relaxation heat maps with a noticeable decrease
in relaxation time in the gall bladder and liver (Figure 13b).
Both results indicate that [GdO]+[ICG]IOH-NPs can be a
promising MRI contrast agent.
In parallel with MRI, mice were also imaged in vivo with
fluorescence molecular tomography (FMT) and post mortem –
after exposing the organs – using fluorescence reflectance
imaging (FRI) (Figure 14). Again, [GdO]+[ICG]IOH-NPs can be
clearly detected.[69] Detection via FMT is even possible over a
time range of several hours. In combination, FMT and FRI
demonstrate that [GdO]+[ICG]IOH-NPs are suitable as multi-
modal contrast agents in vitro as well as in vivo for OI, PAI and
MRI. Straightforward synthesis and low material complexity of
[GdO]+[ICG]IOH-NPs are additional assets in comparison to
many contrast agents discussed in the literature. In the clinics,
[GdO]+[ICG]IOH-NPs could allow to combine the presentation
of different types of tissue, for instance, soft tissue via MRI,
blood vessels via PAI, and single cells via OI.[3,5,55]
Figure 12. In vitro studies with [GdO]+[ICG]IOH-NPs: a) Fluorescence
images of [GdO]+[ICG](10 mg/mL of medium) after 24 h incubation with
MHS macrophages at 37 8C; b) Metabolic activity of MHS macrophages after
0, 24, 48 and 72 h cultivation with [GdO] +[ICG]suspensions (dextran-
coated); ICG, Gd-DOTA and Gd-DTPA solutions as references (0-0.200 mmol/
mL of [GdO]+[ICG], ICG, Gd-DOTA, Gd-DTPA); c) PAI with [GdO]+[ICG]
(suspension) and ICG (solution) at identical concentrations in a dead-mouse
phantom (modified reproduction from ref. [69]).
Figure 13. [GdO]+[ICG]IOH-NPs (dextran-coated) as MR contrast agent: a)
Maps of T1-relaxation time calculated for varying concentrations of dextran-
coated IOH-NPs; b) Mice imaged before and 5 h after injection with
[GdO]+[ICG]IOH-NPs. Images show T1-relaxation time heat maps with a
noticeably reduced relaxation in the gall bladder and liver (by 35%),
(modified reproduction from ref. [69]).
13ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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3.2. Singlet Oxygen Production
In addition to multimodal imaging, specific IOH-NPs are also
suitable for photoactivated singlet-oxygen (1O2) generation. For
this purpose, the sulfonate-based anions aluminium(III) chloride
phthalocyanine tetrasulfonate ([AlPCS4]4, Figure 15a) and tetra-
phenylporphyrin sulfonate ([TPPS4]4, not shown) were applied
as functional organic anions.[70,78] The resulting IOH-NPs have a
composition Gd43+[AlPCS4]34and La43+[TPPS4]34. Both contain
extremely high photoactivator concentrations with 81 wt-%
[AlPCS4]34in Gd43+[AlPCS4]34and 83 wt-% [TPPS4]34in La43+
[TPPS4]34. The synthesis was again performed in water using
GdCl36H2O or LaCl36H2O and H4AlPCS4or H4TPPS4as
starting materials and resulted in transparent greenish blue
Gd43+[AlPCS4]34and brownish La43+[TPPS4]34suspensions (Fig-
ure 15c). Gd43+[AlPCS4]34and La43+[TPPS4]34exhibit mean
diameters of 47 and 56 nm, respectively (Figure 15b; see 2.3:
Table 2).[70,78] Their chemical composition was validated by FT-IR,
TG, EDXS and EA (see 2.4: Table 5). Both Gd43+[AlPCS4]34and
La43+[TPPS4]34also show visible emission, which, in principle, is
also sufficient for fluorescence detection and OI (see 2.3:
Table 3).
In fact, phthalocyanines as well as porphyrins are well-
known for efficient photoactivated 1O2generation. Both are
already discussed for applications such as the selective
oxidation in organic synthesis (e. g., cycloadditions, Diels-Alder
reactions, Ene reactions, heteroatom oxidations),[79] the degra-
dation of organic molecules and germs (e. g. for water
purification),[80] and photodynamic therapy (PDT) (most often
used for tumor therapy).[81] Certain porphyrins and phthalocya-
nines are already approved in the clinics.[81c,d,82] Aiming at PDT,
molecular photosensitizers in solution, however, have certain
disadvantages, such as low solubility in water and rapid
aggregation under physiological conditions. Both reduce the
efficiency of 1O2production and result in limited membrane
permeability and poor cell uptake.[83] Again, it was suggested to
immobilize the molecular porphyrins and phthalocyanines on/
in nanosized/nanoporous substrates, such as silica and gold
nanoparticles as well as carbon nanotubes,[81b,83,84] which again
leads to only low amounts of the active molecular photo-
catalyst (typically <10 wt-%). Moreover, metalorganic frame-
works containing porphyrin linkers were presented.[85] For
medical application, in particular, encapsulation in vesicles and
liposomes or functionalization with hydrophilic capping ligands
was established.[86] However, these measures also enhance the
material complexity, and any encapsulation/capping, as a
matter of fact, blocks the active sites of the photocatalysts.
The feasibility of Gd43+[AlPCS4]34and La43+[TPPS4]34for 1O2
production was validated by the DPBF method (DPBF: 1,3-
diphenylisobenzofuran)[87] and the iodide method.[88] DPBF is
oxidized in the presence of 1O2as indicated by the vanishing
characteristic red color of DPBF (Figure 15d). The decreasing
absorption at 420 nm can be easily monitored by UV-Vis
spectroscopy and results in a quantum yield of 33% for 1O2
production of Gd43+[AlPCS4]34IOH-NPs (in suspension). This
value matches very well with molecular H4AlPCS4(35%, in
solution).[87] This finding is even more interesting, since
H4AlPCS4is known for rapid photodegradation in solution. In
contrast, Gd43+[AlPCS4]34IOH-NPs show good photostability
without any considerable concentration quenching.[70] Both can
be explained, on the one hand, by the great phthalocyanine/
porphyrin reservoir, and, on the other hand, by the non-
crystallinity of the IOH-NPs that avoids any ordered alignment
of the phthalocyanine anions. The quantum yield of 1O2
generation of La43+[TPPS4]34cannot be performed via the
more common DPBF method since the absorption of [TPPS4]34
overlays the DPBF absorption band. Instead, the iodide method
Figure 14. [GdO]+[ICG]IOH-NPs (dextran-coated) as OI contrast agent: a) In
vivo and post mortem fluorescence images acquired of mice 0, 5 and 24 h
after [GdO]+[ICG]injection using fluorescence molecular tomography
(FMT) and fluorescence reflectance imaging (FRI). Organs were removed and
the fluorescence distribution quantified for (b) [GdO]+[ICG](modified
reproduction from ref. [69]).
Figure 15. Gd43+[AlPCS4]34IOH-NPs (AlPCS4: aluminium(III) chloride phthalo-
cyanine tetrasulfonate) with: a) Scheme of synthesis; b) Particle size
according to SEM; c) Excitation and emission spectra with aqueous
suspensions at daylight and with excitation (blue-light LED); d) Determi-
nation of the quantum yield (fD) for 1O2production via the DPBF method
(modified reproduction from ref. [70]).
14ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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was applied here,[88] resulting in a quantum yield of 49% for
La43+[TPPS4]34(in suspension), which is again close to the value
of molecular H4TPPS4(51%, in solution).[88]
To determine the photocatalytic performance of Gd43+
[AlPCS4]34and La43+[TPPS4]34(Figure 16a), both were first
conceptually evaluated in regard of the photocatalytic degrada-
tion of Eriochrome Black T (EBT). EBT was chosen as a model
dye since its absorption (lmax =525 nm) does not overlap that
of Gd43+[AlPCS4]34(lmax =670 nm) and La43+[TPPS4]34(lmax =
425 nm). The photocatalytic degradation was studied with
simulated daylight (halogen lamp) as well as with red-light
illumination (glass filter with l>610 nm) by comparing Gd43+
[AlPCS4]34and La43+[TPPS4]34(in suspension) with H4AlPCS4
and H4TPPS4(in solution) as references.[70] Despite of identical
concentrations of the photoactive phthalocyanine/porphyrin,
the IOH-NPs (in suspension) show significantly higher photo-
activity and faster EBT degradation than the dissolved refer-
ences (Figure 16b,c). This higher performance can be rational-
ized when considering the negative charges of all sulfonate-
based anions at neutral pH (i.e., [AlPCS4]34, [TPPS4]34, [EBT])
leading to a stronger electrostatic repulsion of the dissolved
species than to the non-charged IOH-NPs.[89] Moreover, the local
absorption intensity of the IOH-NPs (in suspension) is higher
due to the great number of absorbing centers per volume of
each nanoparticle compared to the widely separated H4AlPCS4
and H4TPPS4molecules (in solution).
With the above described features and performance –
including the extremely high phthalocyanine/porphyrin load
and the promising photocatalytic effect with daylight illumina-
tion – the Gd43+[AlPCS4]34and La43+[TPPS4]34IOH-NPs can be
highly interesting not only for photocatalytic dye degradation
but also for PDT.[78] PDT is generally considered as a useful
addition to the armory against cancer since it is minimally
invasive and non-damaging to healthy tissue. Specifically, PDT
is known for treatment of near-surface tumors (e. g., skin,
esophagus or intestinal cancer)[90] and intensely discussed for
post-surgery treatment to kill cancer cells that may remain after
extraction of the solid primary tumor.[91] In view of deep-tissue
penetration, the long-wavelength excitation of Gd43+[AlPCS4]34
(550–720 nm) seems most interesting to PDT and was therefore
studied in vitro and in vivo. Gd43+[AlPCS4]34IOH-NPs are even
more interesting since H4AlPCS4(in solution) is already
approved for PDT.[92] Based on the paramagnetism of Gd3+and
the deep red emission of [AlPCS4]34, furthermore, Gd43+
[AlPCS4]34IOH-NPs are also suitable for multimodal imaging
including MRI and OI. With these features, Gd43+[AlPCS4]34can
be an advantageous addition to existing nanoparticulate
photosensitizers for PDT. So far, this includes inorganic nano-
particles such as TiO2or ZnO as well as rare-earth based up-
converters,[93] which suffer from UV-activation (i.e., TiO2, ZnO)
being harmful to cells[93a,b] or narrow-line laser-type excitation
(i.e. up-conversion via f-f transitions on rare-earth metals).[93c,d]
The feasibility of Gd43+[AlPCS4]34IOH-NPs was demon-
strated in vitro with human liver carcinoma (HepG2) cells and
human cervix carcinoma (HeLa) cells.[78] The incubation of cells
with dextran-coated Gd43+[AlPCS4]34IOH-NPs proves good
cellular uptake as indicated by an intense red fluorescence
(Figure 17a,b). Moreover, efficient photoactivated 1O2genera-
tion and ROS production upon illumination (lexc =635 nm) was
evidenced with DCFDA-treated (DCFDA: profluorescent 2’,7’-
dichlorofluorescein diacetate) cells, which show bright green
fluorescence due to 7’-dichlorofluorescein (DCF) produced in
the presence of ROS (Figure 17c,d). Furthermore, a comparison
of the Gd43+[AlPCS4]34IOH-NPs (in suspension) with free
H4AlPCS4(in solution) at identical phthalocyanine concentration
(20 mM) indicates the Gd43+[AlPCS4]34-treated cells to be much
more active in generating photoinduced ROS.[78]
The phototoxic effect of the Gd43+[AlPCS4]34IOH-NPs (in
suspension) was quantified by MTT toxicity assays and
compared to molecular H4AlPCS4(in solution) at identical
Figure 16. Photocatalytic dye degradation of Eriochrome Black T (EBT,
monitored at 528 nm, c(EBT) =0.03 mmol/L) with: a) Scheme of photo-
catalytic degradation with structure of EBT; b) Comparison of
Gd43+[AlPCS4]34(in suspension) with H4AlPCS4(in solution); c) Comparison
of La43+[TPPS4]34(in suspension) with H4TPPS4(in solution). Illumination
with simulated daylight (halogen lamp) and red light (halogen lamp with
red filter, l>610 nm). Gd43+[AlPCS4]34,La
43+[TPPS4]34,H
4AlPCS4, and
H4TPPS4compared at identical phthalocyanine/porphyrin concentration
(8 mMGd
43+[AlPCS4]34/La43+[TPPS4]34;24mMH
4AlPCS4/H4TPPS4), (modified
reproduction from ref. [70]).
Figure 17. In vitro evaluation of Gd43+[AlPCS4]34IOH-NPs (20 mM) in HepG2
cells after 24 h of incubation: a) Prior to illumination in daylight and b) after
10 min of illumination with red fluorescence indicating the IOH-NP uptake
(nuclei stained with Hoechst 33342); c) Cells treated with DCFDA prior and
d) after illumination with green fluorescence of DCF indicating ROS
generation. Illumination performed by scanning slides 4-times for 13 min
using scan cycles at 670 nm (Pearl Imager, LI–COR Biosciences; N: nucleus;
scale bar: 20 mm), (modified reproduction from ref. [78]).
15ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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phthalocyanine concentrations (1–20 mM) (Figure 18).[78] Meta-
bolically active cells reduce the yellow tetrazolium compound
3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide
(MTT) to a purple formazan, which can be monitored photo-
metrically. First of all, the dark toxicity was assessed and
showed slightly higher toxicity of the IOH-NPs in comparison to
H4AlPCS4in solution (Figure 18a). Thereafter, the phototoxicity
subsequent to light exposure (2 5 min, 670 nm) was deter-
mined and results in a significantly higher cytotoxic effect with
Gd43+[AlPCS4]34-treated cells (in suspension, LD50 <5mM) com-
pared to cells treated with molecular H4AlPCS4(in solution,
LD50 >20 mM) (Figure 18b). Moreover, a suppressed angiogene-
sis of microcapillary networks was detected after treatment of
endothelial cells with Gd43+[AlPCS4]34in vitro and subsequent
illumination.[78] Altogether, these results validate the phototoxic
activity and performance of the Gd43+[AlPCS4]34IOH-NPs and
their advantage over dissolved H4AlPCS4.
Finally, the phototoxic effect of the Gd43+[AlPCS4]34IOH-
NPs was evaluated in an in vivo zebrafish tumor model.
Zebrafishes are generally known as a suitable platform for
testing and refining therapies in the preclinical phase of drug
development.[94] They are easier to handle than mice and allow
a direct readout by OI due to their small size and transparency.
In our study, Gd43+[AlPCS4]34IOH-NPs were first incubated with
HeLa-GFP cells expressing green fluorescent proteins (GFP).
Thereafter, the IOH-NP-pretreated HeLa-GFP cells were injected
into the cardinal vein of zebrafish larvae to induce tumors
(Figure 19a). The Gd43+[AlPCS4]34IOH-NP-pretreated HeLa-GFP
cells could be easily detected in the zebrafish larvae by their
red and green emission (Figure 19b). Upon light exposure over
a certain period of time (10 min, 670 nm), the green emission of
the HeLa-GFP cells is significantly decreased, which indicates
their reduced viability. Finally, only the red emission of the
Gd43+[AlPCS4]34IOH-NPs remains at the position of cellular
debris and necrotic tumors (Figure 19b).[78] HeLa-GFP cells
without Gd43+[AlPCS4]34IOH-NPs were injected into zebrafish
larvae as a control and did not show any effect and vanishing
of the green emission at all (Figure 19a). As a result, the
phototoxic performance of the Gd43+[AlPCS4]34IOH-NPs is
clearly shown in vivo. Specific optimization of parameters –
such as the concentration of the IOH-NPs, the duration of
illumination, certain repeat treatments – is of course needed to
reliably explore the optimal treatment efficiency and to
establish therapy protocols.
3.3. Dissolution of IOH-NPs
In cell and mouse studies of all IOH-NPs, and in particular of the
phosphate-based [ZrO]2+[RDyeOPO3]2IOH-NPs, the fluorescence
was observed to vanish on a timescale of several hours to some
days.[34,35] Typically, no fluorescence and no nanoparticle
remains could be identified after 3–4 days. This finding points
to a slow dissolution of the IOH-NPs, which can be rationalized
upon hydrolysis of the POC phosphorus acid ester bond,
resulting in the dissolved species [ZrO]2+, [HPO4]2and RDyeOH
(Figure 20). Such dissolution can be triggered by acid or base
catalysis as well as by ubiquitous phosphatases in cells and
tissue.[95] In fact, such dissolution is ideal in terms of biocompat-
ibility and biodegradability, especially if there is no specific
toxicity of the dissolved species and if all species are completely
released from cells, tissue and body after certain period of time.
Figure 18. In vitro phototoxicity of Gd43+[AlPCS4]34IOH-NPs as indicated by
MTT assays: a) Cell s treated with Gd43+[AlPCS4]34IOH-NPs (green bars) and
with dissolved H4AlPCS4(black bars) after 72 h of incubation in darkness; b)
Cells treated similarly and with illumination at 670 nm for 25 min
(statistical error bars calculated from n=6; significance determined accord-
ing to student’s t-test with p<0.05; N: nucleus), (reproduction from ref.
[78]).
Figure 19. In vivo phototoxicity and imaging of Gd43+[AlPCS4]34IOH-NP-
treated HeLa-GFP cells in zebrafish larvae after NIR illumination. Larvae were
xenografted with GFP expressing and Gd43+[AlPCS4]34IOH-NPs (5 mM)
pretreated HeLa cells. After 24 h, the larvae were illuminated for 10 min at
670 nm and thereafter imaged using fluorescent confocal microscopy: a)
GFP expression exited at 488 nm (Argon laser) and the emission detected at
498–540 nm; b) Gd43+[AlPCS4]34IOH-NPs excited at 635 nm and the
emission detected at 644–786 nm. Upon illumination the cells were losing
GFP expression and the tumors were reduced in size (N: nucleus; scale bar:
200 mm), (modified reproduction from ref. [78]).
Figure 20. Model reaction for the slow metabolic dissolution of
[ZrO]2+[RDyeOPO3]2IOH-NPs (modified reproduction from ref. [35]).
16ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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Various in vitro and in vivo studies in the meantime have
proven that no specific toxicity or allergic reaction is caused by
the IOH-NPs.[34,35,48,58,69,78] In particular, this holds for the cations
[ZrO]2+and Gd3+/[Gd(OH)]2+/[GdO]+. Even for the latter, a
lower toxicity as compared to the standard MRI contrast agents
Gd-DOTA and Gd-DTPA was observed.[48,58,69,78] A detection of
the dissolved species after their release from the IOH-NPs,
however, is not straightforward. On the one hand, immediate
dilution of the dissolved species in the volume of cells, tissue
and body leads to only low concentrations remaining, and
thereby hampers the detection. Analyzing [HPO4]2and RDyeOH
is difficult anyway due to the ubiquitous physiological avail-
ability of phosphate and the rapid metabolic decomposition of
the fluorescent dye anion. Here, radio-labeling could be a useful
option. Tracking of [ZrO]2+is easier due to its absence in
animate beings. In vivo studies indeed show that the pristine
amount of injected zirconium in [ZrO]2+[RDyeOPO3]2IOH-NPs
can be typically retrieved from urine on a time scale of 2–4
days.
To verify the dissolution of the IOH-NPs, we have initiated
several test reactions with suitable model compounds. A first
example relates to [ZrO]2+[UFP]2IOH-NPs (UFP: umbelliferone
phosphate).[96] [ZrO]2+[UFP]2exhibits a typical particle size of
479 nm and shows characteristic, but weak blue emission
(lmax =455 nm) of UFP upon UV excitation (lexc =366 nm).
Although less interesting for imaging purposes (see 2.2), [ZrO]2+
[UFP]2is very interesting since the emission intensity of free
umbelliferone (UF) in solution is considerably higher compared
to the solid IOH-NPs (in suspension). Thus, UF release from the
IOH-NPs, and thereupon, their dissolution can be directly
monitored by fluorescence spectroscopy.[96] Whereas the UF
release in aqueous suspensions at pH 7 and 37 8C is very slow, it
can be significantly accelerated upon addition of acid phospha-
tase (Figure 21). Accordingly, the emission intensity rises
continuously after phosphatase addition over a period of 10 h
indicating the release of UF from the [ZrO]2+[UFP]2IOH-NPs
via hydrolytic cleavage of the POC ester bond (Figure 21). On
the one hand, this verifies the dissolution of the IOH-NPs, and
on the other hand, this specific reaction can also serve as a
fluorescent probe of the presence of phosphatases.[96] To
classify the timescale of the release reaction, it must be noticed
that the hydrolysis of POC ester bonds is particularly fast if
the phosphate group is directly linked to an aromatic system
POCaromatic.[97] In the case of aliphatic systems POCaliphatic,
the release is significantly slower and moves on a timescale of
several days.[35]
A second example to illustrate the dissolution of the IOH-
NPs is [ZrO]2+[AAP]2, containing the analgetic prodrug acet-
aminophen phosphate (AAP).[98] Here, the dissolution of the
IOH-NPs and the release of acetaminophen (AA) were evaluated
based on two different approaches. First, the carbon content of
[ZrO]2+[AAP]2was determined by EA (Figure 22a). Second, the
fluorescence of mixed-anion [ZrO]2+[(AAP)0.9(UFP)0.1]2IOH-NPs
containing 90 mol-% of [AAP]2and 10 mol-% of fluorescent
[UFP]2was monitored.[98] Whereas EA is indicative for the AAP-
related carbon content in the residual solid IOH-NPs, the
fluorescence intensity refers to the released amounts of AA and
UF in the solution. During the experiments, the IOH-NPs were
continuously stirred in aqueous HEPES buffer at neutral pH and
258C. After certain periods of time, a defined aliquot of the
suspension was extracted and centrifuged to obtain the IOH-
Figure 21. Monitoring the dissolution of [ZrO]2+[UFP]2IOH-NPs:
Fluorescence of aqueous suspensions prior (left cuvette) and after (right
cuvette) the addition of acid phosphatase (lexc. =366 nm), (modified
reproduction from ref. [96]).
Figure 22. Monitoring the dissolution of [ZrO]2+[AAP]2and [ZrO]2+[(AAP)0.9
(UFP)0.1]2IOH-NPs with: a) Determination of carbon content of the residual
solid IOH-NPs via EA; b) Fluorescence detection of released UF (in solution)
via fluorescence spectroscopy (48 h, pH 7, 25 8C), (modified reproduction
from ref. [98]).
17ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
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NPs and to perform EA. [ZrO]2+[(AAP)0.9(UFP)0.1]2was treated
similarly with repetitive measurements of the emission intensity
(Figure 22b). Both measures – determination of carbon content
and of emission intensity – indicate a continuous release of AA
and UF with the as-expected exponential slope. Over 18 and
48 h, a total release of about 60 and 80% was observed,
respectively. All in all, the release data stemming from carbon
content and fluorescence detection show good coincidence.[98]
Again, the timescale of release is comparably fast, since both
AAP and UFP exhibit direct bonding of the phosphate group to
an aromatic system (POCaromatic).[97]
3.4. Drug Release and Delivery
Slow dissolution of IOH-NPs is not only relevant in terms of
biocompatibility and biodegradability, but it is also the key to
transfer the IOH-NP concept to drug release and drug delivery.
Hence, the material platform and the feasibility of the IOH-NPs
can become even broader. [ZrO]2+[AAP]2, containing the
analgetic prodrug acetaminophen phosphate, can be consid-
ered as a very first example for drug delivery and release that
combines uncomplex synthesis in water with very high drug
load of 68 wt-% AAP.[98]
Actually, the material concept has been already expanded
to drug delivery and drug release, especially in the case of
phosphate-based IOH-NPs. In this regard, numerous
phosphate-functionalized pharmaceutical agents are available.
Similar to the fluorescent [ZrO]2+[RDyeOPO3]2IOH-NPs, a general
composition [ZrO]2+[RDrugOPO3]2can be derived for drug-
containing IOH-NPs with a phosphate-functionalized pharma-
ceutical anion [RDrugOPO3]2.[99] Illustrative examples comprise,
for instance, [ZrO]2+[FdUMP]2, [ZrO]2+[BMP]2and [ZrO]2+
[CLP]2that contain the cytostatic agent 5’-fluoro-2’-deoxyur-
idine 5’-monophosphate (FdUMP),[35,100] the anti-inflammatory
agent betamethasone phosphate (BMP),[35,101] and the antibiotic
agent clindamycin phosphate (CLP) (Figure 23).[102] Similar to
fluorescent IOH-NPs, [ZrO]2+[RDrugOPO3]2also show excellent
uptake into cells at high biocompatibility.[35,100–102] In difference
to [ZrO]2+[UFP]2and [ZrO]2+[AAP]2(see 3.3), the phosphate
group in [ZrO]2+[FdUMP]2, [ZrO]2+[BMP]2and [ZrO]2+[CLP]2
is linked to an aliphatic system (POCaliphatic) that only shows
slow hydrolysis and drug release (i.e. 5–10% release of total
drug content after 48 h).[35,100–102]
The anti-proliferative potential of [ZrO]2+[FdUMP]2(in
suspension) with 75 wt-% load of active FdUMP was exempla-
rily shown on human mammary carcinoma cells and compared
to non-active [ZrO]2+[UMP]2IOH-NPs (in suspension) as
negative control (UMP: uridine monophosphate) as well as to
the clinically applied 5-FU (in solution) as positive control (5-FU:
5-fluorouracil).[100] Whereas [ZrO]2+[UMP]2(negative control)
had no effect on the cell viability, 5-FU (positive control) and
[ZrO]2+[FdUMP]2IOH-NPs show considerable cytostatic effects.
Interestingly, the anti-proliferative activity of the [ZrO]2+
[FdUMP]2IOH-NPs is even higher than of 5-FU (positive
control), although applied with identical concentration of the
active agent.[100] [ZrO]2+[BMP]2– as the second example –
contains 81 wt-% of the glucocorticoid BMP and shows
excellent anti-inflammatory response in vitro (MHS macro-
phages, primary mouse macrophages, human peripheral blood
monocytes).[35,101] In vivo studies, furthermore, indicate a promis-
ing therapeutic efficiency in a mouse model of multiple
sclerosis with a strongly increased cell-type specificity for
macrophages compared to conventional free glucocorticoids
(in solution).[101] [ZrO]2+[CLP]2IOH-NPs, finally, represent a
novel nanoparticle-based strategy to treat persisting and
recurrent Staphylococcus aureus-caused infections. [ZrO]2+
[CLP]2also contains an extremely high amount of 82 wt-% of
the clinically approved antibiotic clindamycin phosphate and
shows high uptake at low toxicity. In comparison to the free
drug in solution, most interestingly, the [ZrO]2+[CLP]2IOH-NPs
(in suspension) result in a 70 to 150-times higher drug uptake
into cells, although both – free drug and IOH-NPs – were
administered in identical concentrations.
Besides [ZrO]2+[FdUMP]2, [ZrO]2+[BMP]2and [ZrO]2+
[CLP]2and their application for tumors, inflammation and
infection, we could realize phosphate-based [ZrO]2+
[RDrugOPO3]2IOH-NPs with about 50 different pharmaceutical
agents, which illustrates the feasibility of the concept as a
general platform of materials. Drug delivery and drug release,
however, are not a subject of this review and therefore only
conceptually discussed as an additional option. Similar to
[ZrO]2+[(HPO4)1x(FMN)x]2(see 2.2), the pharmaceutical anion
[RDrugOPO3]2can be also partially exchanged by a fluorescent
dye anion [RDyeOPO3]2. In order to maintain maximum drug
load, the fluorescent dye anion is available only in low
concentrations of 0.005 to 0.05 mol-%. Specifically, this results
in [ZrO]2+[(FdUMP)0.95(ICG)0.05]2, [ZrO]2+[(BMP)0.95(FMN)0.05]2or
[ZrO]2+[(CLP)0.995(DUT)0.005]2, which show drug release and
which can be also detected via their fluorescence (see 2.2,2.3,
3.1, Figure 23).[100–102]
Figure 23. IOH-NPs for drug release with fluorescence labelling exemplarily
shown for: a) [ZrO]2+[(FdUMP)0.95(ICG)0.05]2, b) [ZrO]2+[(BMP)0.95(FMN)0.05]2,
and c) [ZrO]2+[(CLP)0.995(DUT)0.005]2(modified reproduction from ref. [100–
102]).
18ChemNanoMat 2018,4,1–23 www.chemnanomat.org  2018 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA
Focus Review
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4. Conclusions
Phosphate- and sulfonate-based IOH-NPs with a general
composition [ZrO]2+[RDyeOPO3]2, [Ln]3+n/3[RDye(SO3)n]n,
[Ln(OH)]2+n/2[RDye(SO3)n]n, or [LnO]+n[RDye(SO3)n]n(Ln: lantha-
nide) are presented as a novel platform of functional nano-
particles for fluorescence detection and optical imaging.
Specifically, the here discussed IOH-NPs include [ZrO]2+
[HPO4]2, [ZrO]2+[(HPO4)1x(FMN)x]2, [ZrO]2+[FMN]2, [ZrO]2+
[MFP]2, [ZrO]2+[RRP]2, [ZrO]2+[DUT]2,La
3+[AMA]3,Gd
3+
[AMA]3, [Gd(OH)]2+2[CSB]4, [Gd(OH)]2+2[DB71]4, [Gd(OH)]2+
[NFR]2, [Gd(OH)]2+[AR97]2, [Gd(OH)]2+2[EB]4, [GdO]+[ICG],
Gd43+[AlPCS4]34,La
43+[TPPS4]34, [ZrO]2+[UFP]2, [ZrO]2+[AAP]2,
[ZrO]2+[FdUMP]2, [ZrO]2+[(FdUMP)0.95(ICG)0.05]2, [ZrO]2+
[BMP]2, [ZrO]2+[(BMP)0.95(FMN)0.05]2, [ZrO]2+[CLP]2, and [ZrO]2+
[(CLP)0.995(DUT)0.005]2. Although already comprising a great
number of compounds, these IOH-NPs stand as representatives
for a much greater number of nanomaterials and an even
broader platform of materials.
Besides the variability of the chemical composition, the
IOH-NPs exhibit several features that differ from alternative
fluorescent nanomaterials, including: i) Straightforward aqueous
synthesis; ii) Low material complexity; iii) Extraordinarily high
load of fluorescent dye and/or pharmaceutical drug (70–85 wt-
% per nanoparticle); iv) Use of many approved fluorescent dyes;
v) High biocompatibility and high biodegradability. All these
aspects are highly relevant to medicine and clinical practice.
In addition to full-color emission, the IOH-NPs can feature
even more functionalities. With Gd3+as the inorganic cation,
for instance, the IOH-NPs are magnetic and suitable for MRI.
IOH-NPs such as [GdO]+[ICG]are multimodal and applicable
for OI (due to the emission of ICG), PAI (due to the absorption
of ICG) and MRI (due to the paramagnetism of Gd3+). Moreover,
IOH-NPs like Gd43+[AlPCS4]34or La43+[TPPS4]34show photo-
induced ROS generation (singlet oxygen) and become suitable
for photodynamic therapy. Finally, the phosphate- and/or
sulfonate-based fluorescent anion can be replaced by pharma-
ceutical anions to realize IOH-NPs showing drug delivery and
drug release. All these different features can be available in a
single IOH-NP by combining two or more functional organic
anions. All in all, the combination of inorganic cations and
functional organic anions, like from a construction kit, allows
realizing multimodal and multifunctional IOH-NPs with many
more compositions and functions, which, in fact, is the most
relevant feature and advantage of the IOH-NPs.
Acknowledgements
The authors are grateful to additional partners from biology
and medicine for excellent scientific cooperation, including
Prof. U. Schaible, Research Center Borstel (tuberculosis); Prof. S.
Boretius, Leibnitz Institute of Primate Research Go
¨ttingen (MRI);
Prof. T. Lammers, RWTH Aachen University Clinic (OI, PAI); Prof. I.
Hilger, University Hospital Jena (MRI, magnetothermal heating);
Prof. H. Garritsen, University Hospital Braunschweig (allergy).
C.F. is grateful to Henriette Gro
¨ger and Nicole Klaassen for
accurate preparation and reproduction of IOH-NP samples.
The authors acknowledge the German Research Society (DFG)
for funding and equipment. Moreover, this work was supported
by the DFG Research Training Group 2039 and the DFG
graduate School “Karlsruhe School of Optics and Photonics
(KSOP)” at the KIT, as well as by the German Federal Ministry of
Education and Research (BMBF) within the joint research
project ANTI-TB. Finally, M.K. acknowledges the Studienstiftung
des deutschen Volkes for scholarship.
Conflict of Interest
The authors declare no conflict of interest.
Keywords: inorganic-organic hybrids ·phosphates ·
sulfonates ·fluorescence ·imaging
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