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Introduction
For malignant lesions, the combination of a local temperature increase induced by High Intensity Focused Ultrasound (HIFU) and temperature sensitive liposomes
(TSL) enables a triggered drug release confined to the disease site, thereby limiting side effects. Co -release of an MR contrast agent (CA) allows for indirect imagin g
of the drug release with MR, providing an indirect assessment of the HIFU treatment.1 However, liposome encapsulation of commonly used Gd -based MR-CA leads
to prolonged retention times in liver and spleen, increasing the risk on nephrogenic systemic fibrosis. 2 In this study an Fe-deferoxamine derivative is proposed as a
safe alternative T1 - CA for TSL encapsulation.
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Materials & Methods:
In vitro characterization: TSLs were prepared using DPPC, DSPC, cholesterol, DPPE-PEG2000 (61:14:15:3 molar ratio). They were either actively loaded with
doxorubicin (dox) or passively loaded with Fe(III) N-succinyl deferoxamine (Fe-SDFO)3 . TSLs were characterized by Dynamic Light Scattering, Differential
Scanning Calorimetry and Inductive Coupled Plasma-Optical Emission Spectroscopy. Release assays were carried out in Fetal Bovine Serum (FBS) at 37, 40 and
42˚C either fluorometrically (dox) or by measuring the longitudinal relaxivity r1 at 1.41T (Fe-SDFO).
In vivo proof-of-concept study: 9L glioma tumors were inoculated
subcutaneously on the hind limb of Fisher 344 rats. The tumor-bearing
animals were placed into a rat MR-HIFU setup in a clinical Philips 3T
Sonalleve® MR-HIFU system4 . Animals were divided into a MRHIFU treatment group (n = 4) and a non-treated control group (n = 3).
All animals were injected i.v. with a mixture of TSLs encapsulating
dox and Fe-SDFO (5 mg/kg and 79 μg/kg respectively) just prior to
MR-HIFU or control treatment. The MR-HIFU treatment consisted of
two sonication periods of 10-15 minutes each (acoustic power 9 – 15
W, acoustic frequency 1.44 MHz) to reach and maintain a tumor
temperature of 42˚C. Tumor temperature changes were monitored by
proton resonance frequency shift MR thermometry. T 1 maps were
acquired before and after the two hyperthermia treatments or at
corresponding time points for the control animals, using a single slice
Look Locker sequence.
Results
The characteristics of both dox TSLs and Fe-SDFO TSLs are
summarized in Table 1 and were considered comparable and suitable
for in vivo application. Both TSLs showed a fast release at 42˚C
(Figure 1). 90 ± 4 % of the dox was released within 2 min, compared
to a release of 80 ± 4% of the Fe-SDFO. At 37 ˚C, the dox TSLs
showed no release over 1.5 h, while the Fe-SDFO TSLs showed a
slight release of 15 ± 11%. At 1.41T, unheated Fe-SDFO TSLs
displayed an r1 of 0.80 ± 0.01 mM -1 s -1 . After 60 min at 42 ˚C, r1
increased to 1.35 ± 0.02 mM -1 s -1 . Tumor T1 maps obtained before
and at various time points after TSL injection are shown in Figure 2.
All treated tumors showed an average contrast change upon the heat
treatment (ΔR1 = 0.25 ± 0.14 s -1 ). In the non-treated tumors, the
average contrast change was smaller (ΔR1 = 0.027 ± 0.009 s -1 ).
Strikingly, the contrast change across the treated tumors was
homogeneous in some (animal 2, animal 4), while inhomogeneous in
others (animals 1 and 3), suggesting untreated tumor areas in the
latter.
Fe-S DFO TS L
Dox TS L
63 ± 2
64 ± 3
(PDI 0.05)
(PDI 0.05)
41.3 ± 0.2
41.7 ± 0.1
Tm [˚C]
Fe-DFO* to phosphate ratio (molar) [-]
0.55 ± 0.03
N.A.
Dox to phosphate ratio (w/w) [-]
N.A.
1.1 ± 0.2
Table 1. Overview of relevant liposomal characteristics for both Fe-SDFO TSL as well as Dox
TSL
Hydrodynamic radius [nm]
Figure 1. Release curves of the TSLs at several temperatures in FBS. A) Fe-SDFO release as deduced
from the increase in relaxation rate upon release of Fe-SDFO. B) Dox release derived from its change
in fluorescence upon release.
Discussion and conclusion
Dox-loaded TSLs and Fe-SDFO-loaded TSLs were characterized in
vitro, showing the potential of this combined system for image guided triggered drug release, even though the r1 of the proposed CA
Figure 2. T 1 maps obtained at various time points throughout the treatment for HIFUis around a factor three lower than that of more commonly used Gd treated animals (left panel) and non-treated control animals at corresponding time points
based CA.1 An in vivo proof-of-concept study was conducted to
(TP, right panel), overlaid onto an anatomical fast field echo image.
assess the feasibility of monitoring drug release using the newly
designed drug/CA loaded TSL systems. Treated tumors showed an increase in R1 while the R1 of the untreated control tumors stayed relatively constant . Moreover,
the pattern of R1 change could elucidate the pattern of drug release across the tumor. Future work aims to correlate the dox delivery to the tumors with the observed
R1 changes.
References 1. Smet, M. De, et al. J. Control. Release 150, 102–110 (2011), 2. MacNeil, S. et al. Invest. Radiol. 46, 711–7 (2011),
K. a. et al. Magn. Reson. Med. 22, 88–100 (1991). 4. Hijnen, N. M. et al. Int. J. Hyperthermia 28, 141–55 (2012).
3. Muetterties,
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*Kneepkens, E., Fernandes, A., Nikolay, K. & Grüll, H (Abstract PP-005, ISMRM Benelux 2016) Iron-based
T1 MRI contrast agent for MR guided drug delivery from temperature sensitive liposomes.