Microbubble‐encapsulated Cobalt Nitrato Complexes for Ultrasound‐triggerable Nitric Oxide Delivery

Abstract

Cobalt complexes exhibit versatile reactivity with nitric oxide (NO), enabling their utilization in applications ranging from homogeneous catalysis to NO‐based modulation of biological processes. However, the coordination geometry around the cobalt center is complex, the therapeutic window of NO is narrow, and controlled NO delivery is difficult. To better understand the complexation of cobalt with NO, we prepared four cobalt nitrato complexes and present a structure‐property relationship for ultrasound‐triggerable NO release. We hypothesized that modulation of the coordination geometry by ligand‐modification would improve responsiveness to mechanical stimuli, like ultrasound. To enable eventual therapeutic testing, we here first demonstrate the in vitro tolerability of [Co(ethylenediamine)2(NO)(NO3)](NO3) in A431 epidermoid carcinoma cells and J774A.1 murine macrophages, and we subsequently show successful encapsulation of the complex in poly(butyl cyanoacrylate) microbubbles. These hybrid Co‐NO‐containing microbubbles may in the future aid in ultrasound imaging‐guided treatment of NO‐responsive vascular pathologies.

Full text

Microbubble-encapsulated Cobalt Nitrato Complexes for
Ultrasound-triggerable Nitric Oxide Delivery
Patrick Koczera,*[a] Fabian Thomas,[b] Elena Rama,[a] Sven Thoröe-Boveleth,[c]
Fabian Kiessling,[a] Twan Lammers,*[a] and Sonja Herres-Pawlis[b]
Cobalt complexes exhibit versatile reactivity with nitric oxide
(NO), enabling their utilization in applications ranging from
homogeneous catalysis to NO-based modulation of biological
processes. However, the coordination geometry around the
cobalt center is complex, the therapeutic window of NO is
narrow, and controlled NO delivery is difficult. To better
understand the complexation of cobalt with NO, we prepared
four cobalt nitrato complexes and present a structure-property
relationship for ultrasound-triggerable NO release. We hypothe-
sized that modulation of the coordination geometry by ligand-
modification would improve responsiveness to mechanical
stimuli, like ultrasound. To enable eventual therapeutic testing,
we here first demonstrate the in vitro tolerability of [Co-
(ethylenediamine)2(NO)(NO3)](NO3) in A431 epidermoid carcino-
ma cells and J774A.1 murine macrophages, and we subse-
quently show successful encapsulation of the complex in
poly(butyl cyanoacrylate) microbubbles. These hybrid Co-NO-
containing microbubbles may in the future aid in ultrasound
imaging-guided treatment of NO-responsive vascular patholo-
gies.
Introduction
Nitric oxide (NO) is grouped among the gaseous signaling
molecules and has captured interest in the research community
due to its ability to modulate central biological processes like
blood perfusion, inflammation or cell survival.[1] In addition to
this, immune cells like granulocytes or macrophages leverage
excess levels of NO for exploiting the antibacterial effects of
reactive nitrogen species. In the body, production of NO is
mediated by the enzyme group of nitric oxide synthetases
(NOS) via the degradation of the amino acid arginine, or by
reduction of nitrite/nitrate during the entero-salivary
circulation.[2] Generation of NO by NOS is controlled by localized
expression of different NOS isoforms, as the constitutively
expressed neuronal and endothelial NOS balance NO levels for
modulation of blood perfusion and neuronal activity. The
inducible NOS of inflammatory cells produces excess levels of
NO for antibacterial activity and immunomodulation.[3]
Due to this biological role, exogenous administration of NO
via inhalation or via NO releasing molecules is clinical routine
for treatment of e. g., pulmonal hypertension or ischemic heart
disease. NO releasing molecules are utilized as “prodrugs”,
which release the active ingredient NO.[4] Besides nitroglycerin,
the complex sodium nitroprusside, Na2[Fe(CN)5NO], is probably
the most prominent example, for which the gas NO is
complexed with iron and cyanide.[5] However, the drugs do not
enable the local and temporal control of NO levels as compared
to the tightly controlled biological balance. In addition, use as
compressed gas for inhalation is limited by concerns regarding
safety and applicability.[6]
This problem could be overcome by chemical modulation
of the metal complexes to tailor properties, such as responsive-
ness to stimuli, e.g. by mechanical forces for triggered release
mechanisms. Cobalt complexes exhibit versatile reactivity with
NO and coordination of NO to cobalt centers influences its
redox chemistry.[7] Ligand substitution dynamics govern the
binding affinity of cobalt for NO. The electronic structure of the
ligands modulates NO activation mechanisms as cobalt‘s d-
orbitals facilitate π-backbonding with NO. Altering the ligand
environment around the cobalt center can change the
electronic structure and steric properties, thereby affecting the
complex‘s affinity for NO. Additionally, adjusting conditions
such as temperature, pressure, and solvent can influence the
kinetics and thermodynamics of NO binding to cobalt. More-
over, the introduction of external stimuli such as light, electric
fields, or other chemical agents can trigger reversible changes
in the reactivity of the cobalt complexes.[8] Hence, cobalt
complexes offer a possibility for triggered release of NO.
Microbubbles (MB) used for contrast-enhanced ultrasound
imaging have recently been explored for triggered delivery of
medical gases like O2, NO, CO, Xe and H2S, for use in cardio/
neuro-protection and in models of myocardial infarction, and
for enhancing drug delivery to pancreatic tumors and breast.[9]
[a] P. Koczera, E. Rama, F. Kiessling, T. Lammers
Institute for Experimental Molecular Imaging, Medical Faculty, RWTH
Aachen University, Aachen, Germany
E-mail: pkoczera@ukaachen.de
tlammers@ukaachen.de
[b] F. Thomas, S. Herres-Pawlis
Institute of Inorganic Chemistry, RWTH Aachen University, Aachen,
Germany
[c] S. Thoröe-Boveleth
Institute for Occupational, Social and Environmental Medicine, Medical
Faculty, RWTH Aachen University, Aachen, Germany
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202400232
© 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial License, which permits use, distribution and re-
production in any medium, provided the original work is properly cited and
is not used for commercial purposes.
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The MB consist of a gas core, which is responsible for the
detectable echo in ultrasound imaging, and a stabilizing shell,
creating spheres in the size range of 1–10 μm. Different shell
materials like phospholipids, polymers or proteins were devel-
oped in order to improve stability and increase circulation
time.[10] Among these polymers poly(butyl cyanoacrylate)
(PBCA) excels with biocompatibility and drug-loading, as the
thick shell enables efficient encapsulation of drug molecules.
Drug-loaded MB exposed to ultrasound waves give rise to
different mechanical phenomena, namely stable or inertial
cavitation, microstreaming and jet formation in combination
with drug release. This leads ultimately to the permeation of
biological barriers (referred to as sonopermeation) and ultra-
sound-mediated drug delivery.[11]
To explore the responsiveness of cobalt- and NO-containing
complexes to ultrasound, we evaluated four previously devel-
oped cobalt complexes for NO release. After identification of
one complex capable of ultrasound-induced NO release and in
preparation for therapeutic evaluation we explored its bio-
logical tolerability and feasibility for encapsulation in ultrasound
responsive MB with the shell composed of PBCA.
Results and Discussion
Cobalt nitrato complexes show potential for controlled release
of NO. To investigate the release of NO, we synthesized four
candidates exhibiting a low-spin d8CoI-NO+couple and differ-
ing in their ligands. In short, cobalt (II) salts were dissolved in
acetonitrile and purged with N2using standard Schlenk
techniques. The corresponding ligands were added to the
solution and N2was replaced by NO2-free NO. It was previously
described that N2or argon purge can release NO from the
complex in solution, which is indicated specifically by colour
change. Therefore, we re-examined this observation and ex-
plored the effect of mechanical triggering with ultrasound.
Table 1 shows the results of the evaluation of NO release in
addition to structural parameters of the evaluated complexes as
described before.[12]
Sonication of the crystals did not induce any changes, and
sonication of complexes in situ and after solvation in methanol
also showed no impact on the complexes, except for [Co-
(ethylenediamine)2(NO)(NO3)](NO3). For this complex, sonifica-
tion in situ or after solvation in methanol induced a colour
change from dark red-brown to pale-red, accompanied by a
light white/pink precipitate (Figure 1). Quantification of absorb-
ance before and after sonication revealed a prominent decline
between 350 and 410 nm and between 410 and 600 nm. The
most prominent decline of absorbance in the visible range was
at 496 nm (Figure S1). This colour change is specific to the
release of NO, as previously described for releasing NO by argon
purge.[12a] Besides this, also changes in complexation by ligand
exchange with e.g. water or OH, or in the oxidation state of
cobalt, could explain the observed colour change.
As ultrasound-induced NO release was not described before,
we evaluated the available structural information of the
complexes for understanding the response. Notably, [Co-
(en)2(NO)(NO3)](NO3) stands out due to its longest distance of
cobalt to nitrogen (1.820 Å). Longer cobalt to nitrogen distances
seem to indicate weaker bond strength, for which energy
Table 1. NO release properties and structural information of the evaluated complexes. [Co(en)2(NO)(NO3)](NO3) has the longest Cobalt-to-NO-nitrogen bond,
and a rather trigonal-bipyramid to vacant-octahedron coordination sphere, as compared to the more square-pyramid sphere of the other complexes. 2-
aepyrr=N-(2-aminoethyl)pyrrolidine, fpin=perfluoropinacolate(2), 2-aemor =N-(2-aminoethyl)morpholine, dmen =N,N-dimethyl-ethylenediamine, en=
ethylenediamine.
Complex [Co(2-aepyrr)(fpin)(NO)] [Co(2-aemor)(fpin)(NO)] [Co(dmen)(fpin)(NO)] [Co(en)2(NO)(NO3)](NO3)
NO-release by Argon – slow – –
NO-release by Ultrasound – – – yes
NO [Å] 1.176(3) 1.169(3) 1.165(4) 1.173(3)
CoN [Å] 1.787(2) 1.788(2) 1.799(3) 1.820(3)
CoNO [°] 123.8(2) 125.1(2) 126.6(3) 121.3(2)
S TBPY-5 3.5 3.7 5.6 7.7
S SPY-5 1.5 1.0 0.8 1.9
EOS(Co) EOS(NO) +1+1+1+1
EFO σ/π1 0.434 0.433 0.431 0.473
EFO π/π2 0.246 0.247 0.259 0.204
Figure 1. Four previously described cobalt nitrato complexes were successfully resynthesized.[12a] Ultrasound-induced release of NO was detected for
[Co(en)2(NO)(NO3)](NO3) by colour change.
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deposition by ultrasound is sufficient for bond disruption. In
addition, the shape of the coordination polyhedron of the
central cobalt atom in the complex differs from coordination
polyhedron shapes in other complexes. Based on their X-ray
structure, the complexes can be positioned on the Alvarez's
shape map for pentacoordinate species, which is used in
evaluation of continuous shape measures.[12b] While the position
of [Co(en)2(NO)(NO3)](NO3) on this map indicates a shape of a
vacant octahedron, other complexes rather cluster around a
position indicating shapes in the transition from a trigonal-
bipyramid to a vacant octahedron via Berry pseudo-rotation.
These characteristics indicate a structure-property relationship,
in which a long cobalt-to-NO-nitrogen bond and a vacant
octahedron shape of the coordination sphere favour ultra-
sound-induced NO release.
Based on our findings that [Co(en)2(NO)(NO3)](NO3) showed
ultrasound-triggered release of NO, we aimed to assess its
tolerability for potential in vivo applications. Although cobalt is
a trace element and essential in form of cobalamines as
coenzyme for methionine synthase and methylmalonyl-CoA
mutase, the cobalt ion is typically trapped within a stable corrin
ring and not readily released. Soil bacteria are the natural
source of cobalamines, and humans rely on its intake by animal
or fortified foods.[13] Exogenous administration of cobalt has
been shown to exhibit toxicity similar to other transition metals,
impacting multiple organ systems and leading to conditions
such as cardiomyopathy, interstitial lung disease and hearing
loss. Pathophysiologically, Co2+competes with Ca2+and Mg2+
ions, disrupting cellular homeostasis and inhibiting enzymes
involved in protein and RNA synthesis.[14] To evaluate the
tolerability of [Co(en)2(NO)(NO3)](NO3), we incubated J774A.1
and A431 cells with various concentrations of the compound
and assessed toxicity using LDH release and XTT testing as
indicators of cell death via membrane permeation and metabol-
ic disruption. Our study focused on A431 as robust neoplastic
human epidermoid carcinoma cell line, which exhibits high
proliferation, and the macrophage-derived J774A.1 cell line,
which as inflammatory cell line can modulate inflammation by
NO production. The results, depicted in Figure 2, showed no
observable toxic effects at concentrations ranging from 0.1 to
100 μM, defined by cellular metabolism remaining above 80 %
of controls and cytotoxicity below 15 %. Exemplary images of
the in vitro viability analyses are provided in Figure S2.
Statistical evaluation of the obtained results did not indicate
significant differences between the evaluated complex concen-
trations and cell types (Figure 2).
Our next step involved the synthesis and assessment of MB
loaded with [Co(en)2(NO)(NO3)](NO3) for contrast enhancement
in ultrasound imaging. MB are commonly used as contrast
agent for ultrasound imaging. Moreover, polymeric MB with
PBCA as shell material show potential as drug carriers. Their
thicker shell, ranging from 100–200 nm, allows for better
encapsulation of drug molecules compared to standard
phospholipid MB with a thinner shell of 1–4 nm.[15] MB respond
to ultrasound by oscillation. By incorporating [Co-
(en)2(NO)(NO3)](NO3) into the MB shell, we aimed to enhance its
interaction with ultrasound waves. The embedded complex
within the shell would experience mechanical strain during
oscillation, leading to MB destruction and cavitation.
Polymerization of the shell for MB synthesis was achieved
through anionic emulsion polymerization in the presence of
OHions.[15a] [Co(en)2(NO)(NO3)](NO3) was added during poly-
merization alongside the monomer BCA for encapsulation in
the MB shell. Mechanical agitation resulted in an air-in-water
solution where polymerization of butyl cyanoacrylate (BCA)
occurred at the interphase, yielding an encapsulated gas core
surrounded by a polymeric shell (Figure 2A). We labeled these
newly synthesized complex-loaded microbubbles as “NO Co
MB”. The size distribution of particles is depicted in Figure 3B,
showing narrow size distribution between 1.43 μm to 2.43 μm
peaking at 1.93 μm for the NO Co MB. In comparison to
unloaded MB (mean diameter of 2.30 �0.62 μm), complex
incorporation had no relevant effect on size distribution within
the favorable range of 1–10 μm, which is a crucial factor
regarding response to ultrasound and intravascular distribution
after injection.[10]
To quantify the loading of NO Co MB with the cobalt
complex, we assessed cobalt content via Inductively Coupled
Figure 2. Toxicological evaluation of [Co(en)2(NO)(NO3)](NO3) in vitro. A431-epidermoid carcinoma cells (A) and J774A.1 murine macrophages (B) were treated
with the complex and assessed for toxicity by LDH release and XTT testing, which indicate membrane permeation and metabolic disruption. The results are
normalized to control conditions. Values represent mean�SD, n=3. No significant differences were detected comparing cell types and complex
concentrations by two-way ANOVA.
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Plasma–Mass Spectrometry (ICP-MS) analysis. Successful loading
was evidenced by a complex concentration of 0.14 (�
0.003) μg/mL derived from cobalt content at a MB concen-
tration of 6.8×108MB /mL (Figure 3C). Besides encapsulation
within the polymeric shell of the MB, a fraction of the charged
complex could also be adhering to the shell surface due to
static interactions. In comparison to the concentration of metal
carbonyl complexes in PBCA MB, the concentration of encapsu-
lated [Co(en)2(NO)(NO3)](NO3) was low and could be explained
by the rather hydrophilic character of the cobalt complex.
Nonetheless, the concentration of encapsulated complex aligns
with previous drug loading studies.[16,15b] The structure-property
relationship we discussed earlier could be used to tailor the
ligands in complexes, not just to enhance release character-
istics, but also to improve encapsulation in PBCA MB.
To evaluate whether the NO Co MB could be detected by
contrast-enhanced ultrasound imaging, we prepared in vitro
gelatin phantoms. A clear contrast enhancement was demon-
strated. In comparison to background levels without MB, NO Co
MB enhanced the signal intensity in nonlinear contrast mode
significantly, by a factor of 279 (�74), which was also the case
for unloaded MB (by a factor of 224�85). No relevant differ-
ences in backscatter signals upon inverted nonlinear ultrasound
pulses were detected comparing complex-loaded and standard
unloaded MB. After application of a high intensity ultrasound
sequence, nonlinear signal intensity decreased to background
levels without MB, confirming MB fragmentation (Figure 4). In
MB, backscatter signals depend on different factors like size,
shell thickness, gas core and drug encapsulation. NO Co MB did
not exhibit inferior performance compared to standard PBCA
MB.[17] In fact, both formulations were equally fragmented by
high-intensity ultrasound pulses, indicating feasibility for sono-
permeation to enhance drug delivery. These results suggest
that [Co(en)2(NO)(NO3)](NO3) did not negatively impact acoustic
properties and that NO Co MB could be used for contrast
enhancement in ultrasound imaging similarly to unloaded
PBCA MB. However, higher encapsulation by tailored cobalt
complexed could also affect the response of these loaded MB
to incident ultrasound waves, therefore encapsulation of each
new complex requires new evaluation of ultrasound responsive-
ness of the MB.
Taken together, we synthesized four previously described
cobalt nitrato complexes, detected the ultrasound-induced
release of NO from [Co(en)2(NO)(NO3)](NO3), and presented a
structure-property relationship for the mechanochemical re-
sponsiveness of the NO-Co bond. Towards exploring therapeu-
tic applications, we demonstrated tolerability of [Co-
(en)2(NO)(NO3)](NO3) in A431 human epidermoid carcinoma
cells and J774A.1 immortalized murine macrophages, and
showed successful encapsulating of the complex in PBCA MB,
which we coined “NO Co MB”.
The understanding of binding properties of ligands in cobalt
nitrato complexes and confirmation of structure-property
relationship helps to tailor ligand chemistry for controlling and
manipulating the behavior of the cobalt-NO interaction. This
approach not only offers opportunities for temporally and
spatially improving NO release for biomedical applications, but
also for tailoring the application of cobalt complexes in electro-
chemical sensing, as well as for catalysis in e. g. cross-coupling,
hydrogenation and cycloaddition reactions.
While these results are encouraging, there are notable
limitations to the current study that must be addressed.
Primarily, the proposed structure-property relationship ob-
served requires additional validation. A broader range of cobalt
nitrato complexes should be investigated to substantiate these
initial observations. NO release was here only verified qualita-
tively; a more precise and quantitative detection method would
allow for a better comparison of how these complexes respond
to ultrasound. For this purpose, a refinement in detection
methodologies is needed, particularly for in vivo translational
Figure 3. Synthesis of [Co(en)2(NO)(NO3)](NO3)-loaded PBCA microbubbles (NO Co MB). (A) One-step synthesis of loaded MB was achieved by co-emulsifying
an aqueous solution of n-butyl-cyanoacrylate (BCA) and the complex with 1 % Triton X at pH 2.5 at 10 000 RPM for 1 hour, yielding an off-white emulsion with
a final concentration of 6.8×108NO Co MB/mL. (B,C) NO Co MB had a mean diameter of 1.93 (�0.50) μm containing 0.14 (�0.003) [Co(en)2(NO)(NO3)](NO3)μg/
mL. Values represent mean�SD, n=3.
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research, where standard NO quantification techniques like the
Griess reaction may cross-react with nitrites and nitrates present
both within the cobalt complex [Co(en)2(NO)(NO3)](NO3) and in
various biological fluids. Future studies could address this issue
by implementing needle electrodes for electrochemical NO
detection, or by employing electron paramagnetic resonance
spectroscopy.
In terms of biocompatibility, our in vitro analysis demon-
strated good tolerance by J774A.1 and A431 cell lines, yet these
preliminary findings need further verification. Testing with
primary cells from different organs, organoids, as well as
systemic toxicity studies in rodent models would provide a
more comprehensive understanding essential for moving
towards translational applications. For eventual clinical trans-
lation, an ideal complex would exhibit non-toxicity while
maintaining a delicate balance: it should be responsive enough
to ultrasound-triggered NO release without being disrupted by
the conditions within the body, such as reductive and oxidative
environments, or unintended ligand exchange with halide ions,
water molecules, hydroxide ions, or amino acids. Encapsulation
within MB could offer a solution by confining the complex
within the MB shell, thereby minimizing interactions with
biological fluids, and allowing NO with its low molecular weight
to diffuse rapidly into tissues following ultrasound-induced
release.
Given NO’s pivotal role in modulating tissue perfusion
through vasodilation, future research should furthermore also
focus on validating image-guided ultrasound-facilitated NO
delivery therapies both in vitro and in vivo. For vascular
pathologies characterized by constricted vessels leading to
diminished organ perfusion and function, initial investigations
might utilize endothelial cell cultures to monitor increases in
intracellular cyclic guanosine monophosphate (cGMP), a key
mediator in NO-driven smooth muscle relaxation and conse-
quent vasodilation. Subsequent steps could include studying
vessel dilation in explanted rat or mouse arteries perfused with
complex-loaded MB for contrast-enhanced ultrasound-imaging
and ultrasound-triggered NO release. Final preclinical validation
could come from utilizing a rodent stroke model emblematic of
the vessel narrowing seen post-subarachnoid hemorrhage. In
such setups, ultrasound imaging could facilitate both detection
of constricted vessels and localized NO release from complex-
loaded MB, with the latter pharmacologically alleviating tissue
hypoxia and damage in-situ, and thereby contributing to
enhanced neural cell survival and brain function.
Methods
Synthesis and Characterization of the Cobalt Nitrato Complexes
The synthesis of the cobalt nitrato complexes was performed
according to the protocol of previously reported by Popp
et al..[12a] For evaluation of ultrasound-induced release of NO,
1.5–3.5 mg of the four different complexes were mixed with
200 μL of methanol in tubes and placed into a sonication bath
at 35 kHz. For 15 minutes qualitative assessment of changes
was performed. Optical absorbance of [Co(en)2(NO)(NO3)](NO3)
Figure 4. NO Co MB exhibited contrast enhancement in ultrasound imaging using B- and Contrast-mode. (A) Schematic illustration of the phantoms used for
ultrasound imaging. (B) Contrast enhancement at 18 MHz was compared before and after ultrasound-mediated MB fragmentation in both B- and Contrast
mode. (C) Quantification of MB-induced nonlinear signal enhancement in contrast mode showed no difference between NO Co MB and unloaded MB. Values
represent mean�SD, n=3. *indicates p <0.0001 and n.s. non-significance (i.e., p >0.05), evaluated by two-way ANOVA and Tukey’s multiple comparisons
test.
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before and after sonication was measured using a Tecan Infinite
200 spectrophotometer (Tecan, Männedorf, Switzerland).
Cell Culture
The XTT assay and LDH release assay were used to evaluate
cellular metabolic activity and toxicity as per manufacturer’s
protocol. Two different cell lines were evaluated: A431
epidermoid carcinoma cells were obtained from ATCC and
J774A.1 murine macrophages from Sigma-Aldrich. Five thou-
sand cells were seeded in 96-well plates for overnight attach-
ment in RPMI (A431 cells) or low-glucose DMEM (J774A.1 cells)
supplemented with 10% fetal bovine serum (v/v) and 1% (v/v)
penicillin/streptomycin maintained at 37°C with 95% air and
5% CO2. After overnight attachment, cells were incubated with
fresh medium containing different concentrations of [Co-
(ethylenediamine)2(NO)(NO3)]NO3(0.1, 0.5, 1, 5, 10, 50 and
100 μM) in aqueous DMSO (2 % (v/v)). Cells were incubated for
4 hours. The colorimetric assays were quantitatively measured
using the Tecan spectrophotometer.
Microbubble Synthesis
NO Co MB were synthesized based on a previously established
‘one-step’ loading protocol.[15a] 200 mg of [Co-
(en)2(NO)(NO3)](NO3) was dissolved in DMSO (4 mL) and added
dropwise to aqueous solution (300 mL) of BCA and Triton-X
(1% (v/v)) at pH 2.5. An Ultra-Turrax T-50 basic (IKA Werke,
Germany) was used for agitation and the resulting mixture was
emulsified at 10 000 RPM for 1 hour at room temperature.
Residual [Co(en)2(NO)(NO3)](NO3), DMSO and polymer fragments
were centrifuged at 500 RPM for 20 minutes and the MB were
washed 3 times with aqueous Triton-X (0.02 % (v/v)) at pH=7.
Similarly, standard unloaded PBCA MB were synthesized with-
out [Co(en)2(NO)(NO3)](NO3). The MB formulations were stored
in aqueous Triton-X (0.02% (v/v)). Using a Multisizer-4 Coulter
Counter (Beckman Coulter, Germany) MB size and concentration
were measured. For this, 2 μL of MB solution were mixed with
20 mL of ISOTON®II (Beckman Coulter) and readings were
obtained in triplicate.
Inductively Coupled Plasma – Mass Spectrometry (ICP-MS)
Analysis
Approx. 200 mg of the samples were taken and weighed exactly
into microwave pressure digestion vessels. In addition to 1 mL
of internal standard (rhodium, 1 mg/L, Merck, Darmstadt,
Germany as 10 mg/L), 2.5 mL of nitric acid (suprapure, Merck,
Darmstadt, Germany), 2.5 mL of hydrochloric acid (suprapure,
Merck, Darmstadt, Germany), 2.5 mL of hydrogen peroxide
(suprapure, Merck, Darmstadt, Germany) and 2.5 mL of MilliQ
water were added to the sample. The sample mixtures were
then digested in the Ethos.lab microwave pressure digestion
system (MLS GmbH, Leutkirch, Germany). For this, the samples
were first heated to 210 °C within 45 minutes. This temperature
was hold for 15 minutes and then cooled to room temperature,
diluted 1:10 and measured on the ICP-MS (8900-QQQ, Agilent,
Waldbronn, Germany).
Ultrasound Imaging
In vitro custom-made gelatin phantoms were used for inves-
tigation of contrast enhancement of MB in response to ultra-
sound. Both types of MB were mixed in aqueous gelatin (2 %
(w/v) in 4.5 mL) (final concentration =30 MB/μL) and embedded
in gelatin (10% (w/v)) to set overnight. Ultrasound imaging was
performed in non-linear contrast mode at 18 MHz frequency
and 4% power using the preclinical VEVO 3100 ultrasound
system (Visualsonics) and a linear-array based MS-250 trans-
ducer. For this, a pre-defined imaging sequence of total 200
frames in both B-mode and nonlinear contrast-enhanced ultra-
sound mode was recorded. For the first 5 seconds, a cine loop
at 4% power (low mechanical index) was acquired. Then a
destructive pulse with 100 % power (high mechanical index)
was applied. To calculate nonlinear signal intensity three
images per phantom were collected and three regions of
interest per image were drawn. Signal intensity in nonlinear
contrast mode before and after MB fragmentation was
quantified.
Statistical Analysis
All samples were measured in triplicate (n =3). Descriptive
statistics were used for obtained results and data are presented
as means�SD. Normal distribution was evaluated with the
Shapiro-Wilk normality test. For small sample sizes, normal
distribution was assumed. For evaluation of cytotoxicity in cell
culture, values were normalized to control conditions, i.e.
medium without probe for cytotoxicity. Viability in cell culture
and nonlinear signal intensity in contrast-enhanced ultrasound
imaging were evaluated by two-way ANOVA (analysis of
variance) and Tukey’s multiple comparisons test. In cell culture
differences between cell types and between concentrations
were evaluated and in ultrasound imaging signal intensities of
the MB formulations, and before and after ultrasound-mediated
fragmentation were compared. Statistical significance was
considered at p<0.05. GraphPad Prism 10 by GraphPad
Software was used for statistical testing. Image analysis was
performed with Imalytics Preclinical 2.1 by Gremse-IT and Fiji.[18]
Acknowledgements
This work was funded by the European Research Council (ERC
CoG 864121, Meta-Targeting), German Research Foundation
(DFG: GRK 2375 (grant 331065168), SFB 1066, and LA2937/4-1)
and the Federal Ministry of Education and Research (CLIMBING
CROHN, 01EK2201A). Open Access funding was enabled and
Wiley VCH Freitag, 09.08.2024
2416 / 356517 [S. 107/108] 1
ChemMedChem 2024,19, e202400232 (6 of 7) © 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH
ChemMedChem
Research Article
doi.org/10.1002/cmdc.202400232

organized by Projekt DEAL. Open Access funding enabled and
organized by Projekt DEAL.
Conflict of Interests
The authors declare no conflict of interest.
Data Availability Statement
The data that support the findings of this study are available
from the corresponding author upon reasonable request.
Keywords: Cobalt ·Nitrogen oxides ·Microbubbles ·
Ultrasound ·Ultrasound imaging
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Manuscript received: March 30, 2024
Revised manuscript received: May 9, 2024
Accepted manuscript online: May 15, 2024
Version of record online: June 20, 2024
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ChemMedChem 2024,19, e202400232 (7 of 7) © 2024 The Author(s). ChemMedChem published by Wiley-VCH GmbH
ChemMedChem
Research Article
doi.org/10.1002/cmdc.202400232