A light-responsive liposomal agent for MRI contrast enhancement and monitoring of cargo delivery

Article abstract of DOI:10.1039/c9cc05516a

Medical magnetic resonance imaging (MRI) produces high-resolution anatomical images of the human body, but has limited capacity to provide useful molecular information. The light-responsive, liposomal MRI contrast agent described herein could be used to p

Full text of DOI:10.1039/c9cc05516a

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A light-responsive liposomal agent for MRI
contrast enhancement and monitoring of
Cite this:DOI: 10.1039/c9cc05516a
cargo delivery†
Received 17th July 2019,
Accepted 14th August 2019
F. Reeßing,ab M. C. A. Stuart,
D. F. Samplonius,c R. A. J. O. Dierckx,a
b
ab
ab
B. L. Feringa,
W. Helfrichc and W. Szymanski
*
DOI: 10.1039/c9cc05516a
rsc.li/chemcomm
Medical magnetic resonance imaging (MRI) produces high-resolution can be readily visualized6–10 – targeted imaging of less abundant
anatomical images of the human body, but has limited capacity to receptors or other proteins that are associated with certain
provide useful molecular information. The light-responsive, liposo- pathological conditions, remains challenging.
mal MRI contrast agent described herein could be used to provide an
This problem has been previously addressed through the
intrinsic theranostic aspect to MRI and enable tracking the distribu- development of responsive CAs, that show increased contrast
tion and cargo release of drug delivery systems upon light-triggered enhancement upon activation by enzymes, ions, neurotrans-
activation.
mitters or that take advantage of changes in e.g. pH, temperature
or redox potential.4,10–16 Even though the effectiveness of this
In the clinic, Magnetic Resonance Imaging (MRI) is widely used strategy has been proven for these targets, certain limitations to
as a non-invasive medical imaging technique that provides this approach remain: for instance, the untimely and/or off-target
anatomical information with excellent resolution, without activation, as the conditions for the activation of the responsive
exposing the patient to ionizing radiation.1 The contrast in CAs are frequently also present outside the lesion(s) in normal,
MRI stems from the difference in local densities and relaxation healthy tissues.
times of protons in tissues. In B30 million clinical scans
In this respect, local activation of a CA with light could be
performed annually worldwide, the contrast is further enhanced used as a general strategy for improved MRI contrast enhance-
by the administration of paramagnetic contrast agents (CAs), ment. Of note, the use of photons as CA activators would not
such as gadolinium(III) complexes, which significantly shorten interfere with endogenous physiological processes.17 Moreover,
the T1 relaxation time of surrounding protons.2–4 This causes a light can be delivered with high spatiotemporal resolution and
higher intensity in the T1-weighted MR image and enables the is biocompatible within a broad wavelength range.18,19 Due to
visualization of the distribution of the CA in the human body.
these advantages, the research fields focusing on the use of
Tissue-specific CAs are currently available to image structures light for biomedical applications, e.g. photopharmacology,20
that are barely distinguishable on a regular scan, such as the photodynamic therapy (PDT),21 or optogenetics,22 are expanding
vascularization of the brain.5 However, due to the low sensitivity very quickly fueled by promising results. As compared to other
of MRI, the requirement of relatively high (40.01 mM) local stimuli, such as ultrasound or heat, the use of light also presents
concentrations of CAs for effective signal enhancement presents specific challenges, including the limited penetration depth and
a major limitation, especially regarding the development of CAs potential toxicity.
for the imaging of disease-specific biomarkers that are present at
The research presented here aims to establish a general
much lower concentrations. Therefore – while structures that are strategy for signal amplification in contrast-enhanced MRI, which
highly abundant in the human body, such as fibrin or collagen, could be used for selective imaging of low-concentration targets.
This strategy envisions the use of targeted light-emitting systems
that locally activate the MRI CA, resulting in signal amplification.
A key advantage of this approach is that the use of light for
activation provides a CA that is readily adaptable to various
targets by changing the light-emitting system, in contrast to the
systems that are limited to one specific target.
a Department of Radiology, Medical Imaging Center, University of Groningen,
University Medical Center Groningen, Hanzeplein 1, 9713GZ Groningen,
The Netherlands. E-mail: w.szymanski@umcg.nl
b Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4,
9747 AG Groningen, The Netherlands
c Translational Surgical Oncology, Department of Surgery, University of Groningen,
University Medical Center Groningen, Hanzeplein 1/BA44, 9713GZ Groningen,
The Netherlands
As a key step towards this general goal, we describe here
the synthesis and evaluation of a photoactivated MRI CA that
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc05516a changes its relaxivity in response to irradiation with blue light.
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as well as a change in the hydration state, since the liberated
carboxylate moiety may coordinate to the gadolinium ion
replacing one water molecule from the complex. Since it is
generally preferred to obtain an increase in signal upon activa-
tion, we envision a ratiometric approach, analyzing the T1 and T2
relaxation time, for future applications, following the example of
Aime et al.27
To achieve an efficient, short and high-yielding synthesis, we
used a Passerini multicomponent reaction (MCR) for creating
the photoactive scaffold28 that could in subsequent transforma-
tions be modified with a liposome-anchoring group and a chelator
for gadolinium yielding compound 1 (Fig. 3).
The liposomes were prepared with an equimolar mixture of
compound
1
and 1,2-dioleoyl-sn-glycero-3-phosphocholine
Fig. 1 Design principle for light-activated MRI contrast agents for imaging
(A) and theranostics (B). The Gd(III)-complex for T1-signal enhancement is
incorporated into the bilayer of liposomes. Upon irradiation with l = 400 nm
light, the complex is released, causing a decrease in T1 relaxivity. (A) A
targeting moiety (here an antibody) binding to the target tissue bears a light-
emitting system that leads to the release of the gadolinium complex from
the lipid bilayer of the liposomes. (B) The liposomal CA can be used for site-
selective drug delivery using local irradiation as a stimulus to release the
liposome cargo. Upon light irradiation, the liposomes concurrently release
the Gd(III) complex and the payload incorporated in their aqueous lumen.
(DOPC) in TBS buffer (pH 7.5). By adding Gd(III) to the pre-
formed liposomes, we assume to form the complex with the
ligands facing to the outside only, resulting in the photo-
triggered release of the Gd(III) complex solely to outside and
not the lumen. After removal of unselectively bound Gd(III) ions
by dialysis, cryoTEM (Fig. S1, ESI†), dynamic light scattering
analysis (Fig. S7, ESI†) and EDX spectroscopy (Fig. S1, ESI†)
confirmed the formation of small unilamellar vesicles and
accumulation of gadolinium in them. The concentration
of gadolinium in the sample, determined by ICP-OES, was
Furthermore, we show how this liposomal CA can simulta- 0.95 mM, indicating that the complex was formed with 76%
neously be used as a responsive cargo delivery system (Fig. 1).23 of all available ligands, assuming that the complex was only
For the successful design of a photoactivatable CA, it is formed with the ligands facing outside.
crucial to consider the molecular characteristics influencing its
We further used the FFC NMR relaxometry to confirm the
relaxivity,24 such as (i) tumbling time, and (ii) number and (iii) synthetic reproducibility, stability over up to 4 weeks and
residence time of water molecules coordinated to the gadolinium photoresponsiveness of the liposome formulation (Fig. 4B,
complex.8 Control over of the first two features is straightforward C and Fig. S2, ESI†). Next, we examined the effect of exposure
and was used for the design of responsive CAs.25 In line with the to light (l = 400) nm on the relaxation rate. Irradiation results in
enzyme-based approach by Aime and co-workers,26 we designed a a marked decrease in relaxivity within 1 h of irradiation
CA that, upon light-activation, converts from a relatively large (Fig. 4A). Already after 10 min, a change DT1 of 21% (measured
nanoscopic complex to a small molecule, resulting in a significant at 10 MHz) was observed, which is comparable to values reported
change in relaxivity.
for other light-switchable paramagnetic metal complexes.29–31
The design relies on linking a gadolinium complex, via a Moreover, the decrease in relaxivity coincided with a change in
photocleavable group, to a lipophilic alkyl chain, which functions the shape of the NMRD profile from the one characteristic for
as an anchoring group for liposomes (Fig. 2). We hypothesized macromolecular or nanoscopic contrast agents with an increase
that irradiation of such liposomes would induce photocleavage
and subsequent release of the gadolinium complex with an
additional free carboxylic acid group (Fig. 2). This process leads
to a lengthening of the T1 relaxation time. The reasons behind
the signal change include the modulation of the tumbling time,
Fig. 3 Summary of key synthetic steps in the synthesis of compound 1:
Passerini MCR for the synthesis of the photoactive scaffold, an azide–
Fig. 2 Molecular structure of the gadolinium complex of compound 1
(Gd-1) and its photo-product Gd-2.
alkyne cycloaddition for attachment of the alkyl chains and a nucleophilic
substitution for introduction of the gadolinium ligand.
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Fig. 5 Evaluation of the effect of photocleavage on liposome integrity.
Fluorescence intensity of 50% DOPC/50% compound 1 liposomes, loaded
with calcein at self-quenching concentration (0.1 M), measured as a
technical triplicate. Upon irradiation, membrane integrity is reduced as is
evident from an increase in fluorescence due to calcein release.
Fig. 4 Stability and photochemical analysis of the liposomes containing
Gd-1. (A) Average NMRD profile curves of samples 1–3 before and after
irradiation with light (l = 400 nm) for indicated times. The results show the
average of three measurements of independently prepared samples. (B)
The relaxation rates measured at 10 MHz of three independently prepared
samples (1–3). The relaxation rate of sample 3 did not change substantially
for up to 28 days of storage. (C) Average decrease in relaxivity recorded at
10 MHz compared to decrease in absorbance at l = 365 nm.
whether cleavage of compound 1 destablizes the lipid bilayer
and thereby promotes the release of the liposome cargo. Calcein,
a fluorescent dye, was encapsulated into the aqueous lumen of
liposomes decorated with 1 at high, self-quenching concentra-
tions (0.1 M). Only upon irradiation with l = 400 nm light, a clear
increase in fluorescence was observed (Fig. 5), due to the release
and dilution of calcein35–37 indicating the destabilization of
at higher field strength (47 MHz) to the one of a small molecule the bilayer. Unfortunately, it was not possible to determine the
CA,26 thus indicating the successful uncaging of the gadolinium exact release rate due to calcein photobleaching.38 DLS showed
complex from the liposome. With prolonged irradiation (for re-organization of the liposomes, leading to a net decrease in
60 min in total), the decrease in relaxivity could be further size (Fig. S7, ESI†).
enhanced to 49% of the initial value (from 10.7 mMÀ1 sÀ1 to
5.2 mMÀ1 À1). Likewise, the relaxation rate at 4.7 T, which is CA with intrinsic capability for drug delivery, offering prospects
closer to the operating field of (pre-)clinical MRI scanners, for diagnostics and image-guided therapy. We developed and
decreased by 61% after 60 min irradiation (Fig. S4, ESI†). evaluated a light-responsive liposomal gadolinium complex
We present here the proof of principle for an activated MRI
s
Correlation of the kinetics of the relaxivity decrease, measured and we demonstrated that its exposure to light results in a
by FFC relaxometry, and the uncaging process, followed by UV- marked decrease in relaxation rate, indicating the conversion of
vis spectroscopy, affirms that the change in relaxivity stems from a nanoscopic object into a small molecule.
the photocleavage of compound Gd-1 docked into the liposomal
bilayer (Fig. 4C).
The increase in the permeability of the liposomes upon
light exposure opens new possibilities to employ this CA for
A major concern of the application of Gd-based CAs is the theranostic applications. To date, there are only few examples
instability of the Gd(III) complex, as free Gd(III) has long-term of agents combining MR-imaging with pharmacotherapy,23,39
toxic effects on the human body.3 While cyclic complexes are including thermo-sensitive release of MRI CA and therapeutics
generally considered to be stable,4,32,33 we nevertheless inves- from liposomes and a combination of Gd(III) complexes with
tigated the stability of our Gd(III) complex upon irradiation, porphyrins for PDT.40,41 Our strategy, however, stands out due
employing a photometric assay in which xylenol orange is used to the prospect of using internal light-emitting targeting moieties
as a sensitive indicator for the presence of free Gd(III).34 We for activation, which makes the drug release system unbiased and
found no substantial increase in free Gd(III) concentration after independent of external stimuli.
1 h of irradiation with blue light (Fig. S5, ESI†).
In further perspective, we envision to use a two-step
To further validate the applicability of the presented CA in a approach in which the patient is first injected with a disease-
biological setting, we evaluated the toxicity of the liposome specific antibody (or derivate thereof) equipped with a biolumines-
formulation and its photo-products towards human umbilical cent enzyme–substrate system. After its injection, the conjugate is
vein endothelial cells (HUVEC), human normal epithelial cells allowed to selectively accumulate in the lesion(s). Subsequently,
and M1 macrophages. No cytotoxic effect of liposomes that the corresponding substrate of the light-producing enzyme is
contained Gd-1 in their bilayer and that were either kept in the injected and converted at the site of the lesion only, resulting in
dark or pre-irradiated for up to 60 min, as compared to medium the localized generation of photons. In turn, these photons locally
control, were detected on the cell lines (Fig. S8, ESI†).
enhance MRI contrast of the light-activatable CA. Deliberate
Next, we explored the possibility of using the liposomal timing of the scans and stepwise administration of the respective
CA for MRI-guided drug delivery. To this end, we evaluated components would result in distinguished resolution. Possible
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12 M. Lepage, W. C. Dow, M. Melchior, Y. You, B. Fingleton,
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are luciferin/luciferase systems, which have been optimized for
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which has been expressed in mammalian cells and explored for
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treatment of localized malignant disease.
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tissue penetration and reduce light-associated toxicity.18 Recent
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With regards to the use of our system for MRI guided drug
delivery, a bathochromic shift in activation wavelength would
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systems,49–51 commonly used in PDT or photothermal therapies,
for triggering the drug release.
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The financial support from the Dutch Organization for
Scientific Research (VIDI grant no. 723.014.001 for W. S.), the
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