Crosslinkable polymeric contrast agent for high-resolution X-ray imaging of the vascular system

Article abstract of DOI:10.1039/c9cc09883f

A contrast agent for X-ray micro computed tomography (μCT), calledXlinCA, that combines reliable perfusion and permanent retention and contrast properties, was developed forex vivoimaging. The new imaging agentXlinCAfeatures high molecular weight, low viscosity and a high iodine content.

Full text of DOI:10.1039/c9cc09883f

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DOI: 10.1039/C9CC09883F
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Crosslinkable polymeric contrast agent for high-resolution X-ray
imaging of the vascular system
Ngoc An Lea,+, Willy Kuob,c,d,+, Bert Müllerd, Vartan Kurtcuoglub,c,*, Bernhard Spinglera,*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A contrast agent for X-ray micro computed tomography (µCT), Ex vivo high-resolution X-ray imaging is not limited by
called XlinCA, that combines reliable perfusion and permanent movement during respiratory and cardiac cycles5, anaesthetic
retention and contrast properties was developed for ex-vivo tolerance or the dose of ionizing radiation.6 In addition, organs
imaging. The new imaging agent XlinCA features high molecular can be extracted and imaged at smaller fields of view, resulting
weight, low viscosity and a high iodine content.
in a corresponding increase of resolution in cone-beam µCT. The
density difference between blood and soft tissue is, however,
too small to be captured with standard absorption contrast
using laboratory sources. Radiopaque X-ray contrast agents
featuring heavy atomic elements have to be injected into the
vasculature to provide the necessary contrast.7
Currently available contrast agents have various limitations
when used for ex vivo vascular imaging. Standard clinical
angiography contrast agents, such as iopamidol and iohexol, are
small molecular compounds capable of passing through blood
vessel walls, which also leads to a loss of contrast between the
vascular lumen and the surrounding tissue within a few
minutes.8 This issue can be addressed by the use of blood pool
contrast agents, which are large nanoparticles or polymeric
compounds that can achieve vascular circulation times on the
scale of hours.9 In an ex vivo setting, blood pool contrast agents
tend to sediment and aggregate, and can slowly leak out of the
vasculature as well.10 These limitations make both types of
angiography contrast agents unsuitable for capillary resolution
ex vivo vascular imaging.
For such application, the current gold standard consists of
vascular casting with plastic resins, e.g. Microfil (Microfil, Flow
Tech, Carver, MA), PU4ii (vasQtec, Zurich, Switzerland) and
µAngiofil (Fumedica AG, Muri, Switzerland).11 These
hydrophobic resins are incapable of passing through the
hydrated endothelial cell layer and polymerize after injection.
They are thus permanently retained within the vasculature and
provide excellent contrast. Perfusion of these resins is,
however, technically challenging. The plastic resin may
polymerize prematurely before the vasculature has been fully
perfused, leading to incompletely filled blood vessels. Water
inclusions and gas bubble formation are frequently occurring
artefacts that cause disconnected vessel segments.11a, 12 Well-
optimized injection techniques, closure of blood vessels via
Qualitative and quantitative assessment of vascular physiology,
pathology and angiogenesis under various circumstances such
as cancer, myocardial infarction, stroke, atherosclerosis,
vasculitis, and inflammation requires accurate three-
dimensional (3D) structural data.1 A large number of different
3D imaging techniques can provide morphometric parameters
including vessel volume, connectivity, number, thickness,
thickness distribution, separation, and degree of anisotropy.1b,
1d,
2
These parameters are not only useful for investigating
diseases that affect the vasculature, but are also crucial for
proper evaluation of pro- and anti-angiogenic therapies in
preclinical models.3
Micro computed tomography (µCT) allows for non-destructive
3D imaging with isotropic quality and micrometer resolution.
The geometric magnification used in cone-beam µCT allows for
continuously variable effective pixel sizes and for hierarchical
imaging on low-resolution animal scale and high-resolution
organ scale in a single device.4 Whole small animals and organs
can be imaged in their entirety in their native hydrated state,
minimizing sample distortion through sample preparation
artefacts, dehydration, realignment artefacts or optical
distortion.
a. Department of Chemistry, University of Zurich, 8057 Zurich, Switzerland. Email:
spingler@chem.uzh.ch
b. Institute of Physiology, University of Zurich, 8057 Zurich, Switzerland. Email:
vartan.kurtcuoglu@uzh.ch
c. National Centre of Competence in Research, Kidney.CH, 8057 Zurich, Switzerland.
d. Biomaterials Science Center, Department of Biomedical Engineering, University of
Basel, 4123 Allschwil, Switzerland
+ and *: These authors contributed equally to this work respectively.
Electronic Supplementary Information (ESI) available: synthesis of XlinCA and
further experimental details. See DOI: 10.1039/x0xx00000x
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ligation in order to divert all flow to the organ of interest and added through reaction with acrylic anhydride and a catalytic
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high perfusion pressures are, therefore, required to obtain amount of sulfuric acid. The key step in DthOiIs: 1s0y.1n0t3h9e/Csi9sCwC0a9s8t8h3eF
consistent perfusion results.11a While some of these problems polymerization of 2 to give polymer 3 by reversible addition-
can be attributed to the high viscosity of the polymerizing fragmentation chain transfer (RAFT) polymerization.17
plastic resins, they are inherent limitations shared by Trithiocarbonate
7
was chosen as RAFT agent for
hydrophobic casting materials. polymerization of 2 due to the reported compatibility with
While plastic resins can provide reasonable vascular filling, various acrylamide derivatives.18 We found that
extensive practice is required to reliably prevent frequent dimethylformamide (DMF) is the most suitable solvent for RAFT
sample preparation failures and incomplete vessel filling.13 polymerization of 2. It provides high solubility of both starting
Reliable vessel filling is, however, absolutely required for the materials as well as polymeric products and allows a high
quantitative characterization of pathological processes and the degree of conversion of the monomers. The optimal reaction
comparison of vascular phenotypes, as the resulting structural temperature was 70 °C. The concentrations of monomer, RAFT
data is otherwise dominated by sample preparation artefacts agent and AIBN as radical initiator were optimized (Table S2).
and not representative of the true vascular structure.
The average molecular weight of the synthesized polymer
In this paper, we address the shortcomings of current X-ray increases with the ratio of monomer to RAFT agent.17d The
contrast agents in high resolution ex vivo imaging of the optimal ratio of monomer : RAFT agent : AIBN was found to be
vascular network with µCT. We have developed a crosslinkable 400 : 2 : 1. Lower ratios of RAFT and AIBN to 2 led to higher
polymeric contrast agent, XlinCA, that combines the simple and conversion degrees, but lowered the molecular weight of 3,
reliable perfusion of the hydrophobic angiography contrast while higher ratios led to precipitates of both starting material
agents with the permanent retention and high contrast of the and product. The molecular weight of 3 could not be measured
hydrophobic vascular casting resins. We demonstrate its with gel permeation chromatography (GPC) even in warm DMF
application for capillary-level imaging of a brain hemisphere and due to low solubility and the tendency of the polymer towards
vascular imaging of an entire mouse with a non-optimized aggregation. However, the molecular weight and polydispersity
transcardial perfusion. XlinCA was designed to fulfil a specific index of 3 could be estimated indirectly from the GPC result of
set of criteria to resolve issues encountered in ex vivo vascular the final product 6, which is water soluble and stable against
imaging with current contrast agents:
- Highly water-soluble, so as to avoid incomplete vascular filling monomers and the average molecular weight of 6 (Fig. S6), we
and interrupted vessel segments. calculated the average molecular weight of 3 to be 30400 Da.
- Higher molecular weight than 65 kDa, for the prevention of Ethylenediamine was added to the polymer 3 after activation of
the immediate leakage through blood vessel walls. the carboxylic groups with oxalyl chloride. The obtained 5
aggregation. Based on the ratio of the molecular weights of the
- Crosslinkable with free primary amine groups as targets for dissolved easily in dilute hydrochloric acid solution to give the
glutaraldehyde fixation, which enables crosslinking via imine contrast agent XlinCA (6), which is water soluble and can be
formation14, in order to prevent leakage over time.
crosslinked with aldehydes due to the presence of the amine
- High X-ray attenuation coefficient with a high iodine groups.
content15, for reducing the required contrast agent The ability to be crosslinked not only enables polymerization of
concentration, viscosity and osmolarity.
XlinCA after injection into the vasculature, but also facilitates
After considering all the above criteria, we designed the the increase of the molecular weight of the contrast agent by
crosslinkable polymeric contrast agent XlinCA represented pre-crosslinking with glutaraldehyde (Fig. S7). In order to obtain
schematically in Figure 1. The theoretical iodine content is the desired molecular weight of 65 kDa, required to prevent
49.5 m/m %, which is comparable to standard small molecule diffusion through the blood vessel walls, XlinCA was pre-
angiography iodine contrast agents and considerably higher crosslinked with different amounts of 25 % glutaraldehyde prior
than what could be achieved by other typical approaches, such to perfusion into the mouse. The highest amount that could be
as linkage to polyethylene glycol (PEG).16
introduced without inducing gelation was found to be 30 L of
25 % glutaraldehyde solution per 1 g of XlinCA dissolved in 4 mL
water. This ratio led to a limited increase in viscosity through an
increase in the molecular weight of the polymer by pre-
crosslinking. The pre-crosslinked contrast agent was dialysed
with a 100 kDa dialysis membrane to remove all compounds
below the size threshold and lyophilised. In this form it should
be used within a week, as its solubility reduces over time. After
the contrast agent was administered into the vasculature,
further crosslinking of the contrast agent with glutaraldehyde
led to gelation of the contrast agent, preventing any leakage
and loss of contrast over time (Fig. S7).
H2
R
C
R'
O
I
NH
I
O
O
NH
I
HN
NH3Cl ClH3N
n
Figure 1. Chemical formula of the crosslinkable polymeric X-ray
contrast agent XlinCA.
Mice perfused with vascular casting resin PU4ii and polymeric
contrast agent XlinCA were compared by using low-resolution
µCT with a voxel size of 80 µm. In the PU4ii-perfused mouse,
The contrast agent was synthesized through a multi-step
process, see Fig. S1. Starting from the commercially available 5-
amino-2,4,6-triiodoisophthalic acid 1, the acryloyl group was
2 | J. Name., 2012, 00, 1-3
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numerous water inclusions and gas bubbles could be observed injection, and thus not due to any contrast agent-related
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DOI: 10.1039/C9CC09883F
in the descending aorta, vena cava and larger vessels in the property.
kidneys and the liver. Correspondingly, one liver lobe, the The brain of the XlinCA-perfused mouse was removed and the
adrenal glands and part of the kidneys were only partially filled right brain hemisphere was scanned with a voxel size of 4.4 µm,
with PU4ii. Certain large blood vessels, such as the naso-frontal as the smaller field of view allowed for scans with
vein and anterior vein, remained completely devoid of PU4ii approximately twice the resolution compared to a full brain.
(Fig. 2A), while their counterparts in the mouse perfused There were no large areas of missing vasculature as has been
reported for optimized Microfil perfusions.11a Neither vessel
discontinuities through insufficient filling nor gas bubbles could
be identified (Figure 3). Independent of the choice of vascular
casting agent, non-optimized transcardial perfusion
Figure 3. 3D rendering of the brain hemisphere vasculature
perfused with XlinCA. A: Outside view. B: Inside view. C:
Magnified view of the region indicated by the black square in
Fig. 4A. Diameter of blood vessel indicated by the white arrow:
70 µm. D: Magnified view of Fig. 4B. Diameter of blood vessel
indicated by the white arrow: 15 µm.
Figure 2. Conventional µCT images of mice heads perfused with
PU4ii (A) and XlinCA (B). In the PU4ii-filled mouse, the
supraorbital vein (white arrow) is partially filled up to the
bifurcation to the naso-frontal vein and anterior facial vein,
techniques cannot entirely outperform optimized perfusion
techniques in specific organs. On equal terms, however, XlinCA
provided more complete and reliable filling of multiple organs
in an entire mouse with a simple transcardial injection, without
requiring clamping or ligation of the descending aorta and vena
cava (Fig. S11). The procedure is thus much simpler to perform
and allows multiple organs to be harvested and used in the
analysis of the vasculature, which reduces the number of
animals required and the variance in multi-organ correlation
studies.
Factors such as injection volume and flow rate did not have to
be optimized, as crosslinking can be initiated at any time after
perfusion is complete. In contrast, Microfil polymerizes within
approximately 20 minutes11a, limiting the perfusion volume
before viscosity and thus resistance to flow increases. With the
time constraints removed, flow rate and thus perfusion
pressure are no longer factors that require optimization.
Perfusion pressure in our experiment was 150 mmHg, which is
consistent with the pressure used in the transcardial perfusion
reported by Chugh et al.12b, but lower pressures can be used
without risk of premature polymerization and resulting
which do not appear. In contrast, these vessels, along with the
anterior facial vein, are completely filled in the XlinCA-perfused
mouse. C: Maximum intensity projection of the higher
resolution XlinCA-perfused whole mouse dataset. Voxel size: 20
µm, scale bar: 1 cm. D: Virtual section of the µCT dataset shown
in C. Intestine (I), kidney (K), adrenal gland (AG) liver (L) and
brain (B) are clearly visible. Scale bar: 1 cm.
with XlinCA were filled in their entirety (Fig. 2B and S8). In order
to evaluate completeness of the vessel filling further, the
XlinCA-perfused mouse was scanned with a voxel size of 20 µm.
Brain, heart, lungs, liver, kidneys and adrenal glands appeared
well-perfused, see Fig. 2C, 2D, S9 and S10. No discontinuous
vessel segments could be found, as expected for a water-
soluble compound. Vascular filling with XlinCA was not entirely
complete. Small vessels posterior to the kidneys were ill-
defined. Parts of the capillaries were missing in the renal
medulla of the kidney (Fig. S10) and the spleen. Leftover blood
seen in the histological examination indicated that this was
caused by incomplete flushing of blood prior to contrast agent
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incomplete filling. Higher perfusion pressures are still of
advantage in the prior perfusion steps required for flushing the
vasculature of remaining blood, however.
In conclusion, we have shown that our new imaging agent
XlinCA circumvents the limitations of current vascular casting
with plastic resins. As XlinCA is a water-soluble compound,
water inclusions are inherently avoided. The contrast agent is
free of microparticles, thereby preventing blockage of
capillaries by solid aggregates. Crosslinking can be initiated after
perfusion is complete, allowing perfusion to be conducted for
any desired period of time without an increase in viscosity
limiting the amount of contrast agent volume that can be
injected. The flow rate does not need optimization, and
perfusion pressure ceases to be an important factor for contrast
agent filling.
Vascular casting with XlinCA thus requires not only considerably
less technical expertise and time, but also permits perfusion of
both the brain and the whole mouse in a single procedure. We
envision that these features will enable hierarchical imaging of
organs in their original spatial context and identification of
areas with vascular abnormalities, which can then be excised for
higher resolution imaging. The vasculature of more than one
organ of the same animal could be analysed, which would
reduce the number of animals required and reduce the variance
in multi-organ correlation studies.
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X-Ray Tomography XII, San Diego, California, United States, 2019,
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This work was financially supported by University of Zurich and
the Swiss National Science Foundation through NCCR Kidney.CH
and grant 205321_153523. We thank Dr. Thomas Fox for
recording the 1H-NMR of XlinCA (6).
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J. Roentgenol., 2007, 188, 832-838; b) B. P. Chugh, J. P. Lerch, L.
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Conflicts of interest
A patent application for the polymeric contrast agent described
in this publication has been filed by the University of Zurich.
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