Luis Javier Cruz1, Ivo Que1, Markus Aswendt3, Eric Kaijzel1, Alan Chan2, Mathias Hoehn1,3, Clemens Löwik1. 1Deparment of Radiology, Leiden University ...
Targeted Nanoparticles for the Non-invasive Detection of Traumatic Brain Injury by Optical Imaging and 19F MRI Luis Javier Cruz1, Ivo Que1, Markus Aswendt3, Eric Kaijzel1, Alan Chan2, Mathias Hoehn1,3, Clemens Löwik1 1Deparment of Radiology, Leiden University Medical Center, Leiden, The Netherlands 2 Percuros, Enschede, The Netherlands 3In-Vivo-NMR Laboratory, Max Planck Institute for Neurological Research in Cologne, Cologne, Germany
Results Design, preparation and characterization of targeted bimodal PLGA NP SO3Na SO3
SO3Na O
PFCE
800CW
N
N
SO3Na HO
O
800CW 800 nm 200 nm
NIR 700 nm NIR 700nm 19F N
H3C
N
CH3
I
Physicochemical characterization of PLGA NPs PLGA NPs with and without targeting moiety
PLGA NPs size ± S.D. (nm)
Zeta potential ± S.D. (mV)
PLGA-NP (PFCE)Not
214.0 ± 12.3
-25.4 ± 1.60
250 ug
----
----
PLGA-NP (PFCE)800CW
216.2 ± 10.5
-41.5 ± 3.25
250 ug
----
0.25 ug
PLGA-NP (NIR700 + PFCE)Not
212.6 ± 12.5
-22.7 ± 4.6
240 ug
25 ug
----
PLGA-NP (NIR700 + PFCE)800CW
215.0 ± 11.5
-38.6 ± 3.9
240 ug
25 ug
0.3 ug
Fig. 1. Schematic diagram of bimodal PLGA NP (NIR700 + PFCE) targeted with 800CW-specific ligand towards the dead cell. NPs were generated containing NIR and PFCE for optical imaging and 19F MRI purposes. The PLGA NP was shielded by a combination of PEG-lipid and amine functionalized PEG-lipid layers. The PEG-lipid prevents nonspecific interactions and the amine functionalized PEG-lipid allows introduction of 800CW-NHS on the PLGA surface for targeting purposes. TEM image of representative PLGAs. Image analysis revealed the presence of the PEG-lipid layer surrounding the NPs. (Scale bar, 200 nm; magnification, 60000 x).
In vivo imaging of targeted PLGA NP A
C
B
Targeted vs non-targeted NP to the traumatic brain injury
700 nm
800 nm
6000
3h
6h
and the CTMM project Cancer Vaccine Tracking (03O-302). Dutch Organization for Scientific Research
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hours
In vivo 19F MR imaging over time of brain lesion in mice with targeted PLGA NP
1000
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NP (700 + PFCE)-800CW NP (700 + PFCE)-Not
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*** 200
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Fig. 2. In vivo optical imaging over time of brain lesion in mice with targeted and non-targeted PLGA NP +/- 800CW coating. Whole body fluorescence imaging at 3 h, 6 h, 24 h and 48 h post induction of cryo-lesion and subsequent i.v. injection of NP(NIR700 + PFCE)-800CW and NP(NIR700 + PFCE)-Not (A, left panels at 800 nm channel) and (B; right panels at 700 nm channel). C; Quantification of the total integrated intensity of NP(NIR700 + PFCE)-800CW/-Not signals in the brain lesion over time at the 700 nm and 800 nm channels respectively. Results are the total integrated intensity ± SD of a total of 3 mice from two separate experiments. ***, p < 0.001.
Biodistribution by optical imaging and 19F MRS Biodistribution of targeted vs non-targeted NP after 48 hrs
*** 1000 NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
800
60000
ID/g at 800 nm
D
NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
ID/g at 800 nm
A
40000
600
400
20000 200
0
Brain
Heart
0
Liver Spleen Kidney Lung
Brain
E NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
8000 6000 4000
NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
80
60
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****
100
Brain
Heart
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Liver Spleen Kidney Lung
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F NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
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ns
Brain
Heart
Liver
Spleen
Kidney
Lung
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3
0 -85
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ppm
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0
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NP(700 + PFCE)-800CW NP(700 + PFCE)-Not
4
Integrated SNR/g
15000
5
SNR
ID/g at 700 nm
10000
ID/g at 700 nm
B
Integrated SNR/g
Fig. 3. In vivo 19F MR imaging over time of brain lesion in mice with targeted PLGA NP. 19F MR images of a mouse at 4 h, 24 h and 48 h post induction of cryo-lesion and subsequent i.v. injection of NP(NIR700 + PFCE)800CW. A) Coronal slices through the mouse brain at different times after surgery. Anatomical 1H MR images shown in gray, 19F MR images overlaid in false color. The lesion can clearly be depicted on anatomical images as a bright area, which co-localizes well with areas of significant 19F signal. B) 3D representations of MR images further illustrate accumulation of particles inside the lesion. Brain surface shown in transparent yellow, lesion in blue, significant 19F signal intensity in red and a coronal anatomical slice in gray.
1- F.G. Blankenberg. Current pharmaceutical design, 14 (2008) 2974-2982. 2- S.L. Wolters, et al. European journal of nuclear medicine and molecular imaging, 34 Suppl 1 (2007) S86-98. 3- E.M. Laufer, et al. The quarterly journal of nuclear medicine and molecular imaging : official publication of the Italian Association of Nuclear Medicine, 53 (2009) 26-34. 4- Z. Medarova, et al. Diabetes, 54 (2005) 1780-1788. 5-B.A. Smith, et al. Bioconjugate chemistry, 23 (2012) Fig. 4. Biodistribution of targeted and non-targeted 1989-2006. PLGA NPs in TBI. Biodistribution of targeted and nontargeted PLGA NPs after 48 h of i.v. injection and optical imaging (A) at 800 nm and (B) 700 nm, and (C) 19F MRS. The authors wish to thank the technicians of the LUMC Quantification of brain lesion-specific signal after 48 h of Radiology Department for assistance. This work was NP injection (D) at 800 nm (E) at 700 nm, and (F) 19F financially supported by the VIDI personal grant (project MRS. Representative brain images are shown next to each number 723.012.110), the FP7 European Union Marie Curie graph. Data is shown as total intensity ± SD from one IAPP program, BRAINPATH, under grant number 612360 experiment using 2-3 mice per group. ***, p < 0.001.
Acknowledgments
4000
0
48h
References
NP (700 + PFCE)-800CW
0
Conclusions The results showed that both targeted and nontargeted NPs reach the TBI within 1 h after i.v injection. This suggests that the majority of NPs diffuse through the disrupted blood-brain barrier. However, the retention and the amount of NPs depend on the ligand on the NP surface. In organs, no differences were observed in routing between targeted and non-targeted NPs, but there was clear evidence of enhanced retention of targeted NPs within lesion area. The ability to target NPs to the lesion is a valuable tool to study necrosis by in vivo imaging.
Targeting moiety 800CW ug/mg PLGA) (w/w)
Entrapment Entrapment efficiency of efficiency of PFCE (ug/mg NIR 700 nm PLGA) (ug/mg PLGA) (w/w) (w/w)
Total Intensity 800 nm
In a clinical context, cell death-specific non-invasive imaging techniques are highly relevant as a diagnostic tool and the evaluation of therapeutic responses to assess optimal treatment regimes. Numerous previous studies used nuclear imaging (e.g. positron emission tomography, PET) to target specifically dead cells in order to monitor treatment responses in cancer [1], to localize tissue damage in myocardial infarction [2] and to identify vulnerable plaques in atherosclerosis [3]. In addition, optical imaging, e.g. using Cy5.5-labeled annexin V, enabled tracing of diabetes-associated β-cell death [4]. However, while there has been considerable progress over the last five years, in vivo cell death imaging remains one of the most important unsolved problems in clinical molecular imaging. To be successful in the clinic, many technical challenges must be overcome. Central to such ongoing research and clinical efforts is the need for imaging technologies that can locate and identify cell death highly specifically [5]. Here, we report a novel NIR-PFC dual-mode agent for highly specific in vivo detection of necrotic cell areas found within traumatic brain lesions. To our knowledge, this is the first report on a targeted PLGA 19F MRI/optical dual-mode agent applied to in vivo imaging of the traumatic brain injury (TBI). We show specific binding to dead cells in vitro by a cell assay and in vivo in a cryo-lesion mouse model of TBI. Ex vivo imaging and histological evaluation of these findings suggest that our bimodal PLGA NP(NIR700 + PFCE)-800CW strongly and specifically targets necrotic cells in the cryo-lesion area of the brain. Our bi-modal PLGA NP can potentially be used in the clinic for the non-invasive detection of TBI using optical and MR imaging and to deliver regenerating drugs.
Total Intensity 700 nm
Introduction
0 Brain
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