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Online Program Overview Session: PW-06

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New Probes | MRI and Multimodal I

Session chair: Alexei Bogdanov - Worcester, USA; Mónica Carril - San Sebastián, Spain
 
Shortcut: PW-06
Date: Thursday, 22 March, 2018, 11:30 AM
Room: Banquet Hall | level -1
Session type: Poster Session

Abstract

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# 060

[68Ga]Ga-Sienna+ PET-MRI as a Preoperative Imaging Tool for Sentinel Lymph Node Biopsy: Synthesis and Preclinical Evaluation in a Metastatic Breast Cancer Model (#126)

H. Savolainen1, A. Volpe1, A. Phinikaridou2, M. Douek3, G. O. Fruhwirth1, R. T. M. de Rosales1

1 King's College London, Imaging Chemistry and Biology, London, United Kingdom
2 King's College London, Biomedical Engineering, London, United Kingdom
3 King's College London, Research Oncology, London, United Kingdom

Introduction

Accurate identification of sentinel lymph node (SLN) location and health status at the whole-body level by an imaging method prior to biopsy is of interest in order to prevent unnecessary (>70%) LN excisions. Superparamagnetic iron oxide nanoparticles (SPIO) are taken up by macrophages in healthy LNs but not by cancer cells in metastatic LNs and serve as contrast agents in MRI.1,2 We hypothesized that radiolabeled SPIOs would be a useful tool to locate SLNs (PET) and evaluate their metastatic status (MRI). Here we describe our preclinical results in a metastatic breast cancer mouse model.

Methods

Clinically approved SPIO Sienna+ was radiolabeled with 68Ga without a chelator in pH 5 at 100 °C in 10 minutes (Fig 1A). The stability of [68Ga]Ga-Sienna+ was evaluated in human serum at 37 °C and cell uptake was compared between MTLn3E Δ34-CXCR4-GFP hNISTagRFP breast cancer cells and macrophage cell lines J774A.1 and RAW264.7.GFP. NSG-mice (n=6) were inoculated with breast cancer cells into mammary fat pad. LN metastasis was analysed by SPECT-CT with 99mTcO4-, which is a substrate for hNIS expressed in the cancer cells (Fig 1B). [68Ga]Ga-Sienna+ was injected into front paws and mice were scanned by PET-MRI. Excised LNs were analyzed by histopathology by staining for cancer cells (GFP), macrophages (Iba1) and iron (Perl’s Prussian blue).

Results/Discussion

[68Ga]Ga-Sienna+ was produced with high radiochemical purity (93%) without need for purification. Radiolabel stability of [68Ga]Ga-Sienna+ in serum was sufficient (84% after 4 h). Uptake of [68Ga]Ga-Sienna+ in the macrophage cells was significantly higher than in the cancer cells. SPECT-CT imaging confirmed metastasis in the left axillary lymph nodes and lungs (Fig 1B). In PET-CT, significantly higher [68Ga]Ga-Sienna+ uptake was seen in the healthy axillary LN than in the metastatic one. In MRI, [68Ga]Ga-Sienna+ uptake in healthy LNs was observed by decreased MR signal in T2/T2* weighted sequence, whereas metastatic LN appeared unchanged. Histological analysis confirmed the imaging results (Fig 2).

Conclusions

[68Ga]Ga-Sienna+ PET-MRI can locate and distinguish healthy SLNs from the metastatic ones and could be a useful preoperative imaging tool to guide SLN biopsy.

References

1) Johnson, L et al. Histopathology 2013, 62, 481–486. 2) Wunderbaldinger, P et al. Magnetic Resonance in Medicine 2002, 47, 292–297.

Figure 1.
A) Chelator-free 68Ga-radiolabeling of Sienna+ B) Examples of SPECT-CT, PET-CT and MR images.
Figure 2.

Histological analysis. Comparison of appearance in fully metastatic, partially metastatic and healthy lymph nodes. Scale bar represents 100 µm.

Keywords: chelator-free labeling, PET-MRI, sentinel lymph node biopsy, SPECT, SPIO
# 061

CEST-MRI studies of cells loaded with Lanthanide Shift reagents (#432)

G. Ferrauto1, E. Di Gregorio1, D. Delli Castelli1, S. Aime1

1 University of Torino, Dept of Molecular Biotechnologies and Health Sciences, Torino, Italy

Introduction

Magnetic resonance Imaging(MRI) has been extensively used to track in vivo implanted cells that have been previously labelled with relaxation enhancers. However,this approach is not suitable to track multiple cell populations. The use of Chemical Exchange Saturation Transfer(CEST) agents allows overcoming this drawback. Upon encapsulating paramagnetic Lanthanide Shift Reagents(SR) one may shift the absorption frequency of the intracellular water resonance(dIn) thus generating frequency-encoding CEST cells that can be visualized in the MR image by applying the proper radiofrequency.1,2

Methods

Eu, Dy and Tm-HPDO3A have been used as Shift Reagents for labeling murine breast cancer cells (TS/A) and murine macrophages (J774A.1) by hypotonic swelling and pinocytosis. CEST-MR images have been acquired at 7T. dIn value and Saturation Transfer (ST%) effect have been measured. Different dilutions of cells have been analized to quantify the detection thresold. In vitro experiments of cells proliferation have been carried out with TS/A and J774A.1 cells. Finally, TS/A cells have been subcutaneously injected in mice and MR images have been acquired to calculate the proliferation index in vivo

Results/Discussion

The entrapment of the paramagnetic SRs into cells leads to the onset of a CEST effect in the 3-8 ppm range, depending on the used metal. The intracellular water resonance (dIn) is proportional to the the effective magnetic moment (meff) of the metal. Therefore, the highest shift is observed with Dy-HPDO3A (meff =10.6) and the lowest with Eu-HPDO3A (meff =3.5) (Fig.1).

Moreover, dIn depends on the intracellular localization of the SRs. When they are loaded inside endosomes  (by using macropinocytosis),dIn is higher. When they are loaded in the cytoplasm (by using hypotonic swelling3), dIn is lower.

Dy-labelled cells are detectable when they are less than 10% of the total numer of cells in the analysed sample.

Cells proliferation index has been assessed (in vitro and in vivo) by evaluating the reduction of dIn in the days after the labeling. In fact, with mitosis, cells diluite their content of SRs, thus reducing dIn. An example of in vivo proliferation rate has been reported in Fig.2

Conclusions

Ln-labelled Cells can be visualized by CEST-MRI by exploiting the large ensemble of intracellular shifted water. A higher performance is obtained when the complexes are entrapped inside the endosomes. The dIn value is strongly correlated to the chemical nature of the SR, to its concentration and to cellular localization. Two applications of this method have been reported, i.e. i) for in vivo cells visualization and ii) for monitoring the cellular proliferation process as it is accompanied by a change in dIn that, therefore, may be exploited as longitudinal reporter of the proliferation rate.

References

1) Ferrauto G, et al. J. Am. Chem. Soc. 2014;136:638-41

2) Ferrauto G. et al. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2016; 8 :602-18

3) Di Gregorio E. et al. Contrast Media Mol Imaging. 2013;8:475-86

Acknowledgement

G.F. and E.D.G. gratefully acknowledge Fondazione Italiana Ricerca sul Cancro (FIRC-AIRC) for their fellowships

Figure 1

A) Comparison among ST profiles of cells labelled by pinocytosis with Dy- (blue), Eu- (red) or Tm-(green) HPDO3A at the same intracellular concentration. B) Relationship between the induced chemical shift and the effective magnetic moment of the Lanthanide. The number of Ln-HPDO3A/Cell is maintained constant for all the cellular samples to ca. 1.5x1010 Ln/cell.

Figure 2

A) In vivo visualization of Dy-labelled TSA cells (yellow arrow) 24h post injection by irradiating at 4.8 ppm. Unlabelled TSA cells have been injected as control (white arrow) ; B) Decreasing of chemical shift inside tumor region during the days post injection of Dy-labelled TS/A cells.

Keywords: MRI, cell labeling, CEST, lanthanide complexes, cell tracking
# 062

L-ferritin: a natural theranostic agent for MRI visualization and treatment of breast cancer cells (#296)

V. Bitonto1, D. Alberti1, S. Geninatti Crich1, S. Aime1, J. C. Cutrin1

1 University of Turin, Molecular Biotechnologies and Health Sciences, Torino, Italy

Introduction

The altered regulation of iron in cancerous cells compared to normal cells, along with the potential for iron mis-regulation to selectively cause oxidative stress and cell death, makes iron overload an attractive idea for the treatment of cancer. Here we propose the use of horse spleen ferritin (HoS-Ferritin) as a natural theranostic agent that can be used for MRI visualization and treatment of breast cancer cells. Properly, due to its high percentage of L-chains (85%), HoS-Ferritin is mainly endocytosed by SCARA5 receptor-specific pathway, that appears to be up-regulated in some tumor cells.

Methods

Murine 4T1 and TS/A mammary carcinoma cell lines and a healthy mouse mammary gland NMuMG cell line were used. After 24h of incubation with 2 μM of HoS-Ferritin cells were placed in agar phantom to acquire MR images at 7 T. Cell viability was determined by the MTT assay.

In order to evaluate the biodistribution of HoS-Ferritin in vivo and if the amount of HoS-Ferritin taken-up by tumors is sufficient to visualize tumor lesions, a group of female BALB/c mice were inoculated subcutaneously with 6x104 4T1 cells. When tumors reached a dimension of approximately 0,1 cm3, mice were intravenous injected with HoS-Ferritin (0,2 mmol kg-1 of iron).

SCARA5 expression was evaluated ex vivo by immunohistochemistry in 4T1-derived tumor and in normal breast tissue.

Results/Discussion

4T1 and TS/A incubated with ferritin displayed markedly lower signal intensity when compared to untreated cells, while only small changes in signal intensity were observed in NMuMG incubated in the absence or in the presence of ferritin, respectively. The T2-weighted RARE image confirmed that the ferritin-induced contrast is markedly more efficient in 4T1 cells. 

Internalized HoS-Ferritin triggered an iron-uploading dependent cell death pathway. The half maximal inhibitory concentration (IC50) was 2.5 μM and 4.2 μM for TS/A and for 4T1 cells, respectively.

In all 4T1 tumour-bearing mice, contrast enhancements upon intravenous injection of HoS-Ferritin were successfully observed. Contrast enhancement in tumor, spleen and liver was already visible 3h after the injection, while the maximum signal was recorded after 6h.

Neoplastic cells resulted positive for the expression of the SCARA5 receptor, while in the normal tissue a mild positive reaction was visible only in some stromal cells.

Conclusions

This theranostic system was able to selectively target breast cancer cells over-expressing L-ferritin receptors, while at the same time the superparamagnetic ferrihydrite (5Fe2O3Ÿ9H2O) crystal, contained in the protein internal cavity, accelerated the transverse NMR relaxation rate (R2) of solvent water protons, causing a negative contrast well detectable in the corresponding MR images. Moreover, from the MTT tests results we speculate that iron overloading could be used as a novel strategy to kill cancer cells.

References

 

  1. S.P. Foy, V. Labhasetwar, Biomaterials, 2011, 32, 9155-9158.

  2. S. Geninatti Crich, M. Cadenazzi, S. Lanzardo, L. Conti, R. Ruiu, D. Alberti, F. Cavallo, J.C. Cutrin and S. Aime, Nanoscale, 2015, 7, 6527-6533.

In vitro evaluation of HoS-Ferritin uptake

T2-Weighted MRI image (7T) of an agar phantom containing unlabeled NMuMG (A), TS/A (B) and 4T1 (C) cells; NMuMG (D), TS/A (E) and 4T1 (F) cells incubated for 24 h with 2 μM HoS-Ferritin. 

In vivo biodistribution of HoS-Ferritin

In vivo MRI on BALB/C mice inoculated with 6x104 4T1 cells. T2-weighted image (tumor mass) acquired before and 6h after the administration of HoS-Ferritin at a Fe dose of 0.2 mmol kg-1

# 063

Development of light-responsive MRI contrast agents for imaging and theranostics (#214)

F. Reeßing1, 2, M. C. A. Stuart2, R. A. J. O. Dierckx1, B. L. Feringa2, W. Szymanski1, 2

1 University Medical Center Groningen, Radiology, Groningen, Netherlands
2 University of Groningen, Stratingh Institute for Chemistry, Groningen, Netherlands

Introduction

MRI is an outstanding anatomical imaging technique, due to its non-invasiveness and excellent resolution. However, the limited functional information obtained by MRI is a major drawback. Thus, contrast agents are needed that respond to the local changes in biochemical processes.1-2

In our efforts to boost the sensitivity of MRI using new, out-of-the-box approaches, we aim to develop MRI contrast agents responsive to light, envisioning the use of light-emitting targeting moieties accumulating in the disease tissue. This strategy would lead to significant signal amplification.

Methods

We investigated different designs of photoactivatable contrast agents, amongst them T1 and paraCEST agents. The main focus of the talk will lie on the development of an agent (Scheme 1A) decreasing T1 relaxation time, which - in its intact from - can be incorporated into liposomes. Photocleavage leads to the conversion of a macromolecular to a small, quickly tumbling contrast agent (Scheme 1B), causing a change in relaxivity.3-5 The Passerini multicomponent reaction (MCR) is used to synthesize photoactivatable contrast agents, employing a precursor for the photoresponsive moiety as one of the reactants (Scheme 1A).6 CryoTEM and an FFC relaxometry were used for the visualization of liposomes and acquisition of NMRD profiles.

Results/Discussion

Compound 1 was synthesized starting with the photoresponsive core structure, after which the hydrophobic tail functioning as an anchoring group for liposomes and the Gd3+ ligand were introduced. Subsequently, 1 was successfully incorporated into liposomes with DOPC, as confirmed by cryoTEM (Scheme 2A). The accumulation of Gd3+ on liposomes and absence of unbound Gd3+ was confirmed by EDX analysis. NMRD profile curves show about 40% decrease in T1 relaxation rate upon irradiation with light (λ = 400 nm) with a substantial decrease already after 10 min irradiation time. As expected, with progressing irradiation time, the NMRD profile converges from the one of a macromolecular contrast agent (increase of relaxation rate at high field strength) to the one of a small molecule following the same kinetics as observed in the UV Vis analysis of the photocleavage. A permeation assay using Calcein indicates the disintegration of liposomes upon irradiation.7

Conclusions

A potential contrast agent has been designed, synthesized and evaluated in terms of relaxation rate. A substantial change in relaxation rate is observed upon irradiation with λ = 400 nm. The observed permeability of the liposomes after photocleavage gives rise to the possibility of using the system for theranostics.9-8 Having shown the principle of photo-responsive contrast agents, the next step is to design new molecules that are responsive to red light and show faster photocleavage, thus improving their suitability for in vivo applications.

References

1. G. Davies, et al. Chem.Commun. 2013, 49, 9704

2. J. R. Morrow, et al. Inorg. Chem., 2017, 56, 6029–6034

3. S. Lacerda, et al ChemMedChem,  2017, 12, 883-894

4. E. Boros, et al. Dalton Trans., 2015, 44, 4804–4818

5. . V. Catanzaro, et al. Angew. Chem. Int. Ed., 2013, 52, 3926-3930

6. W. Szymanski, et al., Angew. Chem. Int. Ed. 2014, 53, 8682–8686.

7. T. Shimanouchi, et al. Biochimica et Biophysica Acta, 2007, 1768, 2726-2736

8. T. Lammers, et al. Acc. Chem. Res., 2011, 44, 1029–1038

9. D. Alberti, et al. ChemMedChem 2017, 12, 502-509

Acknowledgement

The financial support from the Dutch Organization for Scientific Research (NWO VIDI grant no. 723.014.001 for W.S.) is gratefully acknowledged.

Synthetic strategy, molecular design and schematic represenation of photoactivation
A) A multicomponent reaction is used to synthesize compound 1 bearing the Gd3+-ligand and an anchoring group for liposomes connected via a photocleavable group; B) The macromolecular, slowly tumbling contrast agent is expected to have a higher relaxivity than the small Gd3+-complex released from the liposome upon irradiation
cryoTEM image and NMRD profiles
A) cryoTEM picture of liposomes formed of 50% DOPC and 50% compound 1; B) NMRD profiles of intact liposomes (blue rhombs) and products of photocleavage after given irradiation times.
Keywords: responsive MRI probes, light activation, photocleavage, theranostics, multi component reaction
# 064

Stable Mn(II)-complexes with high relaxivity for MRI applications (#444)

S. Ghiani1, G. Tircsò2, Z. Baranyai1, Z. Garda2, I. Toth2, M. Botta3, A. Maiocchi1

1 Bracco Imaging SpA, CRB, Colleretto Giacosa, Torino, Italy
2 University of Debrecen, Department of Inorganic and Analytical Chemistry, Faculty of Science and Technology, Debrecen, Hungary
3 University of Eastern Piedmont, Department of Science and Technological Innovation, Alessandria, Italy

Introduction

The biogenic Mn(II), with its 5 unpaired electrons and a high magnetic moment, is one of the most promising candidates to substitute Gd(III) for a new generation of CAs. However, due to the lack of ligand-field stabilization, the symmetric d5 electron configuration system of the Mn(II) ion results in thermodynamically less stable complexes than those of the other transition metals [1]. With the aim to overcome the above limitation we have developed a new class of Mn(II) complexes with both higher kinetic inertia to transmetallation reactions and improved thermodynamic stability.

Methods

New Mn(II) complexes based on bis-amides derivative of CDTA were synthesized and fully characterized. Physico-chemical parameters such as thermodynamic stability constant, kinetic inertness, redox stability, relaxivity, albumin binding were studied. In order to predict the potential in vivo dissociation of the new Mn(II) complexes incubation experiments in human plasma were extensively performed.

Results/Discussion

The promising physico-chemical parameters of the Mn(CDTA)2-, attributed to the rigid backbone of the ligand, can be further improved by the replacement of two acetate pendant arms for amides based on the structure shown in Figure 1.[2] The effect of the amide substituents on the stability and relaxivity properties of the derived Mn(II) complexes was systematically explored. Although the substitution of the acetate arms with the amide groups slightly decreases the thermodynamic stability of the complexes, the enhanced structural rigidity induced by the substituents in R and R1 and the reduced propensity for proton assisted dissociation reactions, result in a positive balance and provide Mn(II) complexes with suitable property profiles for in vivo applications.

 

Conclusions

We have performed the synthesis and full in vitro characterization of a new class of Mn(II) complexes showing good thermodynamic stability, high kinetic inertness, and ideal water-exchange rates that, in turn, allows to obtain a property profile close to the linear Gd(III) complexes currently used in clinics.

References

[1] K. G. Toft, S. O. Hustvedt, D. Grant, I. Martinsen, P. B. Gordon, G. A. Friisk, Å. J. Korsmo, and T. Skotland, Acta Radiologica 38 (1997) 677-689. Drahos, B.; Kotek, J.; Hermann, P.; Lukes, I.; Toth, E. Inorg. Chem. 2010, 49, 3224−3238.

[2] WO2016135234 Ethylenediaminetetraacetic acid bis(amide) derivatives and their respective complexes with Mn(II) ion for use as MRI contrast agent.

The general structure of bis-amide derivatives of CDTA
# 065

GRPr-Targeted Gold Nanoparticles for Multimodal Imaging (#367)

S. Lacerda1, F. Silva2, L. Gano2, A. Pallier1, S. Meme1, E. Toth1, R. Kannan3, A. Paulo2, M. P. Campello2

1 Centre de Biophysique Moleculaire - CNRS, Orleans, France
2 Instituto Superior Técnico, Universidade de Lisboa, Center for Nuclear Sciences And Technologies, Bobadela, Portugal
3 University of Missouri-Columbia, Department of Radiology, Columbia, United States of America

Introduction

Gold nanoparticles (AuNPs) can play a pivotal role in the design of new cancer theranostic tools, due to their appealing properties for medical application such as, biocompatibility, easy functionalization with molecular vectors and good biological half-life.1 Here we report on the coordinating capability of a AuNP platform with two medically relevant metals, Gd3+ and 67Ga3+, targeted with a bombesin (BBN) analogue, to assess their potential bimodal MR/SPECT imaging nanoprobe. BBN has affinity to the gastrin releasing peptide receptor (GRPr), overexpressed in a variety of human cancer cells.

Methods

Gd-containing AuNPs with (BBN-Au-Gd) and without (Au-DOTA-Gd) a BBN analog conjugated2, were synthesised and successfully radiolabeled with 67Ga (Figure 1). Relaxivity (2x10-4 to 1.9T) and 9.4T-MRI phantom studies were performed on cold Gd-AuNPs, and in vitro stability and cellular uptake studies were done with 67Ga-labeled congeners.

In vivo stability and biodistribution studies in normal mice and in prostate cancer PC-3 xenografts were performed by 67Ga gamma-counting measurements for AuNPs with1 and without Gd. ICP-OES analyses to determine the Au and Gd content on selected organs for 67Ga labeled AuNP-Gd particles (post decay of 67Ga).

Results/Discussion

The nanoprobes can be efficiently labeled with Gd3+ and 67Ga3+ (30 min and 70°C: yield > 99%). The relaxometric properties of BBN-Au-Gd particles are good for a potential T1- and T2-weighted MR application: r1 = 10.19 mM-1s-1; ratio r2/r1 = 4.5 at 9.4T. The biodistribution results show good profile, with some liver retention 24h post injection (intravenously: 13.3 ± 1.7, intraperitoneally: 1.4 ± 0.4,1 and intratumorally: 0.23 ± 0.03). The BBN-decorated AuNPs have a reasonable tumor uptake and retention 24h post injection (intravenously: 3.7 ± 0.5, intraperitoneally: 0.95 ± 0.03,1 and intratumorally: 76.8 ± 23.3). The in vivo integrity of the 67Ga labeled BBN-Au-Gd multifunctional nanoconstructs, as corroborated by the ICP-OES measurements.

Conclusions

Multimodal MRI/SPECT AuNPs-BBN nanoplatforms have a significant tumor accumulation in PC-3 xenografts, in contrast with the congener AuNPs without the BBN analogue. These promising results confirm the feasibility of specific in vivo targeting of tumors (GRPr-mediated) for this class of AuNPs.

References

  1. Yeh Y.C., Crevan B., Rotello V.M. Nanoscale, 2012, 4, 1871.
  2. Silva F., Zambre A., Campello M.P.C., Gano L., Santos I., Ferraria A.M., Ferreira M.J., Singh A., Upendran A., Paulo A., Kannan R. Bioconj. Chem., 2016, 27, 1153.

Acknowledgement

This work was supported by Fundação para a Ciência e Tecnologia (projects EXCL/QEQ-MED/0233/2012 and UID/Multi/04349/2013).

Figure 1

Schematic representation of the BBN functionalized AuNPs, coordinated to Gd3+ and 67Ga3+.

# 066

Magnetic Particle Imaging: an emerging field for superparamagnetic iron oxide nanoparticle research (#169)

J. M. Gaudet1, P. Pandit1, P. W. Goodwill1

1 Magnetic Insight, Alameda, California, United States of America

Introduction

Magnetic Particle Imaging (MPI) is an emerging molecular imaging technique that can non-invasively detect and linearly quantify superparamagnetic iron oxide nanoparticles (SPIOs) with high sensitivity1. MPI directly detects SPIOs with positive contrast, unlike MRI which can struggle to quantify negative contrast signal voids.  Factors such as the nanoparticle iron (Fe) core size and crystal structure all contribute to the observed imaging properties.2 In this study, we investigate the performance of seven nanoparticles, some clinically approved, on a field-free line MPI scanner.

Methods

Different nanoparticle formulations were compared to Ferucarbotran, considered the standard in MPI. Phantoms were prepared by diluting stock solutions with deionized water over a 1-100% range.  At each concentration a point source (1 μL) phantom was imaged and the signal measured. Imaging was performed with the MOMENTUM MPI system (Magnetic Insight Inc., CA, USA).  This system produces a 6 T/m field gradient, a 45 kHz drive field, and uses x-space MPI reconstruction to generate 2D projection images.3 A linear regression model from the peak MPI signal at each concentration was used to assess the signal per μg of Fe.  Resolution was estimated using the measured full-width, half-maximum (FWHM) generated by a single point source of signal.

Results/Discussion

The MPI properties of the seven nanoparticle formulations are compared in Table 1. All nanoparticles had a linear signal relationship with iron concentration, R2 > 0.98. In general, we observed an improved signal efficiency from the monodispersed nanoparticles, compared to the multicore aggregrates.  However, this signal improvement did not correlate with a change in estimated resolution as assessed by the FWHM of the point spread function.  Nanoparticle 2 stood out, with more than twice the signal per μg of Fe and almost two-times improved estimated resolution of the next best sample.  MPI utilizes the flexibility of the SPIO core as a backbone for different applications, such as cell labeling and vascular imaging. Nanoparticle can also be functionalized to cater to specific applications by altering their surface coatings.4 Table 2 summarizes potential applications of the seven SPIOs studied here.

Conclusions

Development and assessment of novel SPIO nanoparticles remains an important area of research for the field of MPI. Optimization of the nanoparticles is necessary to increase sensitivity and resolution, in addition to consideration of the biological application. Ultimately, clinical translation of MPI will depend on the biocompatibility, safety, sensitivity, and consistency of SPIO tracers.

References

1. Gleich et al. Nature. 435:1214-1217. (2005)

2. Wang et al. Quant Imaging Med Surg. 1(1): 35-40. (2011)

3. Goodwill et al. IEEE Trans Med Imaging. 31:1076-1085. (2012)

4. Matuszewski et al. Radiology. 235(1): 155-161. (2005)

Acknowledgement

Research reported in this publication was supported by NIBIB of the NIH under award number R43EB020463.

Table 1: Comparison of nanoparticles with MPI
Table 2: Influence of surface coating on in vivo Applications
Keywords: Magnetic Particle Imaging, Superparamagnetic Iron oxide nanoparticles, SPIO, MPI
# 067

Novel Bifunctional MRI Probe: Iron-Quercetin complex induced differentiation of circulating mononuclear progenitor cells and therapeutic impact in cardiovascular diseases. (#73)

J. Kantapan1, N. Dechsupa1

1 Chiang Mai University, Department of Radiologic Technology, Research Unit of Molecular Imaging Probes, Chiang Mai, Thailand

Introduction

Patient-specific therapy is a highly promising treatment in cardiovascular diseases. However, due to the rarity of progenitor cells in blood circulation, and the lack of consistent data about the fate of stem cells in vivo, remains challenging, hampering the success of this approach.Magnetic resonance imaging with contrast agents is an excellent tool for studying the fate of transplanted stem cells. We investigated the potential use of the novel MRI probes to determine if it could proliferate and differentiate the circulating progenitor cells and monitor transplanted cells by clinical MRI.

Methods

Peripheral blood mononuclear cells (PBMCs) obtained from healthy donor blood were cultured in the presence of an Iron-Quercetin complex compare with conventional culture. Differentiated cells were characterized by immunostaining and proteomic analyses, and their angiogenic functions were evaluated in vitro. The intracellular uptake of complex and visualizing of magnetically labeled cells by in vitro MRI were investigated by a clinical 1.5T MRI.

Results/Discussion

Iron-Quercetin complex significantly increased the proliferation and differentiation of circulating mononuclear progenitor cells into the spindle-shape like cells, expressing both haematopoietic and stromal cell markers and the expansion increased number of colony-forming unit (CFU). The expanded cells secreted pro-angiogenic factor and matrix modified protein. Co-culture of expanded cells and human umbilical cord endothelial cells increasing the tube formation under growth factor- reduce conditions.  Preliminary findings suggest that intracellular uptake of Iron-Quercetin complex increases in a time-dependent manner and the magnetically labeled cells demonstrated an increase of MRI signal intensity in the T1-weighted image when compared to the signal of unlabeled cells.

Conclusions

Our study demonstrates that ex vivo expansion of circulating progenitor cells derived from peripheral blood mononuclear cells and cellular tracking by a novel bifunctional paramagnetic agent Iron-Quercetin complex might be a viable alternative to autologous cell based therapy for regeneration-inductive therapies.

References

  1. Young PP. and Schäfer R. Cell-based therapies for cardiac disease: a cellular therapist's perspective. Transfusion, 2015; 55(2):441-51.
  2. Cesselli D, Beltrami AP, Rigo S, Bergamin N, D'Aurizio F, Verardo R, et al., Multipotent progenitor cells are present in human peripheral blood. Circ Res, 2009; 104(10):1225-34.
  3. Kang KT, Lin RZ, Kuppermann D, Melero-Martin JM, Bischoff J. Endothelial colony forming cells and mesenchymal progenitor cells form blood vessels and increase blood flow in ischemic muscle. Sci Rep, 2017;7(1):770.
  4. Azene N, Fu Y, Maurer J, Kraitchman DL. Tracking of stem cells in vivo for cardiovascular applications. J Cardiovasc Magn Reson. 2014; 16(1):7.

       5. Kantapan J, Dejphirattanamongkhol S, Daowtak K, Roytrakul S, Sangthong P, Dechsupa N. Ex-vivo expansion of EPCs derived from human peripheral             blood mononuclear cells by Iron-Quercetin complex. Biomedical Research (2017); 28(6):2730-2737.

Acknowledgement

-

Figure 1
Representative diagram of Iron-Quercetin complex application in Patient-Specific cell based therapy.
Keywords: Cell-Based therapy, MRI contrast agent, cardiovascular diseases, Circulating progenitor cells, Iron-Quercetin complex.
# 068

A novel mannan-based probe for multimodal imaging of cancer and inflammation (#96)

A. Gálisová1, M. Jiratova1, M. Rabyk2, M. Hrubý2, M. Hájek1, D. Jirák1

1 Institute for Clinical and Experimental Medicine, MR Unit, Prague, Czech Republic
2 Academy of Sciences, Institute of Macromolecular Chemistry, Prague, Czech Republic

Introduction

Mannan exploits promising properties for cancer and inflammation theranostics due to preferential uptake by immune cells via DC-SIGN receptors1. Its nanosize allows preferable accumulation in solid tumors by Enhanced Permeability and Retention effect. Here, mannan was modified by a gadolinium chelate for MRI and by a label for fluorescence imaging (FLI). Modification with oxazoline was tested for slowing down the elimination rate and increased accumulation in tumors. Mannan without (MN) and with oxazoline (MNOX) were characterized and in vivo distribution was assessed on tumor-bearing mice.

Methods

Hydrodynamic radius of the probes was measured by dynamic light scattering. Properties of the probes were assessed by r1 and r2 relaxometry, MRI and FLI. For in vivo monitoring, 50 μL of the probes (3.5 mg Gd3+/mL) was administered into the calf muscle of right hind leg of Balb/c mice with induced orthotopic tumors (4T1 cells in the 5th mammary gland). T1-weighted MR images of animals were acquired by a turbo spin echo sequence: TR=339 ms, TE=12 ms, TF=2, resolution 0.16x0.16x0.70 mm3, scan time 4.5 min. In vivo FLI images (60 s exposure, excitation 745 nm, emission 810-875 nm) of mice and ex vivo images of excised organs were acquired at various time points after agent injection (0-3 days). Commercial contrast agent gadoterate meglumine (GM) was used as control.

Results/Discussion

The hydrodynamic radius of mannan conjugates was 3.3 nm (MN) and 3.7 nm (MNOX). Both MN-based agents have higher r1 and r2 relaxivites, MRI and FLI signal compared to GM. All agents were visualized at the injection sites with decreasing trend 2 hours after agent application and with higher signal of MNOX compared to MN (Fig.1C,D). FLI signal originating from the liver was higher in case of MN than MNOX (Fig.1A,C,D). Higher signal of MNOX in muscle and lower in the liver confirm slower elimination process due to oxazoline modification. Multimodal imaging revealed higher uptake of MN in lymph nodes compared to MNOX and GM (Fig.1B, Fig.2D). Importantly, accumulation of MN and MNOX was higher in sentinel lymph nodes than in distant lymph nodes (Fig.1B). MN exploited higher imaging signal in tumors compared to MNOX and GM, what could be caused by lower uptake of oxazoline by the tumor-associated macrophages (Fig. 2C). Ex vivo fluorescence of organs confirmed the in vivo results (Fig.1C,D).

Conclusions

We present novel mannan-based conjugates with targeting properties to solid tumors and sentinel lymph nodes. According to our pilot results in mice, oxazoline modification slowed down the elimination rate; however it restricts accumulation of the probe in tumors. Nevertheless, mannan conjugate without oxazoline exploited superior tumor- and sentinel lymph node-targeting properties. Easily modified chemical structure allows incorporation of drugs and makes this platform a suitable drug delivery system for theranostics of cancer, metastasis and inflammation in future applications.

References

[1] Cui Z. et al. Drug Dev Ind Pharm, 29(6), 2003

Acknowledgement

Supported by MH CR-DRO (Institute for Clinical and Experimental Medicine IKEM, IN00023001) and the Ministry of Health, Czech Republic (grant #15-25781A).

Fig. 1 Fluorescence imaging.

Changes of fluorescence signal from the liver (A) and from the lymph nodes (LN) (B) of mice with injected MN and MNOX. Representative fluorescence images of mice acquired 4 hours after injection of MN (C) and MNOX (D). Arrows point the liver and the muscle. The smaller images on right show ex vivo fluorescence images of excised organs on day 1 – liver, tumor and sentinel lymph node (SLN).

Fig. 2 Magnetic resonance imaging.
Representative MR images of mice acquired at 4 hours after injection of MN (A) and MNOX (B). Arrows point tumor, sentinel lymph node (SLN) and a lymph node at the opposite site. Accumulation of the probes in the SLN was detected. Quantification of signal-to-noise ratio (SNR) of tumor (C) and sentinel lymph node (D) at various time points after probe injection.
Keywords: mannan, theranostics, MRI, fluorescence, sentinel lymph nodes, cancer, tumor
# 069

Highly fluorescent and superparamagnetic nanosystem for biomedical applications (#27)

C. F. G. C. Geraldes1, 2, M. P. Cabrera3, 4, P. E. Cabral Filho3, C. M. C. M. Silva3, M. M. C. A. Castro1, 2, B. F. O. Costa1, 5, M. S. C. Henriques5, J. A. Paixão5, L. B. Carvalho Jr.6, B. S. Santos7, F. Hallwass4, A. Fontes3, G. A. L. Pereira4

1 University of Coimbra, Department of Life Sciences, Coimbra, Portugal
2 University of Coimbra, Coimbra Chemistry Center, Coimbra, Portugal
3 Universidade Federal do Pernambuco, Biophysics and Radiobiology Department, Recife, Pernambuco, Brazil
4 Universidade Federal do Pernambuco, Department of Fundamental Chemistry, Recife, Pernambuco, Brazil
5 University of Coimbra, CFisUC, Department of Physics, Coimbra, Portugal
6 Universidade Federal do Pernambuco, Biochemistry Department and Laboratory of Immunopathology Keizo Asami, Recife, Pernambuco, Brazil
7 Universidade Federal do Pernambuco, Department of Pharmaceutical Sciences, Recife, Pernambuco, Brazil

Introduction

The development of magnetic/fluorescent bimodal nanoparticles (BNPs) is of increasing interest in molecular imaging research.1 The present work reports on the preparation,  characterization and cell labeling studies of new highly fluorescent and superparamagnetic BNPs obtained by a simple and efficient method, as probes for fluorescence analysis and/or contrast agents (CAs) for MRI.

Methods

CdTe QDs functionalized with mercaptosuccinic acid (MSA) (MSA-CdTe QDs) were synthesized as described in the literature.2,3 They were covalently coupled with superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with APTES in the presence of EDC and Sulfo-NHS, as shown by FTIR.3 The QDs were characterized by UV/Vis and emission spectroscopy. The BNPs were characterized by EDX, TEM, XRD, TGA, atomic absorption spectrometry (AAS), Mössbauer spectroscopy (MS) and NMR relaxometry. The HeLa cell labeling with the BNPs was studied using flow cytometry and confocal microscopy, showing efficient labeling without significant changes in their morphology. The Resazurin assay indicated a relative viability of ca. 80% at the BNP concentration applied to cell labeling.3

Results/Discussion

The BNPs with small dimensions (ca.17 nm) were obtained by successful coupling of SPIONs-APTES with covalently bound to CdTe QDs (ca. 3 nm), as shown by FTIR. The chemical structure of the magnetic part of BNPs is predominantly magnetite, with minor goethite and maghemite contributions, as shown by MS, which is compatible with the X-ray diffraction data. Their size evaluation by different techniques showed that the SPIONs derivatization process producing the BNPs, does not lead to a large size increase. The BNPs saturation magnetization, when corrected for the organic content of the sample (obtained by TGA), is ca. 68 emu g-1, which is only slightly reduced relative to the bare nanoparticles. The experimental magnetization curves were fit using a uniform model and a core–shell model. The SPIONs surface functionalization does not change considerably their magnetic properties. The BNPs aqueous suspensions are stable, highly fluorescent, with a high r2 relaxivity (r2/r1 ratio of 25).

Conclusions

The properties of the BNPs described are appropriate for their in vitro and potentially in vivo applications as fluorescent probes and negative T2-weighted MRI CAs. Their potential magnetic response could also be used for fast bio-separation applications.

 

References

1. O. Chen et al, 2014, Nature Commun. 5, 5093

2. P.E. Cabral Filho, et al,  BBA General Subjects, 2016, 1860, 28-35.

3. M. P. Cabrera, et al, Nanotechnology, 2017, 28, 285704

Acknowledgement

We thank CAPES (Brazil) and FCT (Portugal) support through the Transnational Brazil-Portugal Cooperation Program CAPES/FCT (331/2012), CNPq, FACEPE, the National Institute of Photonics (INFo) and the Nanotechnology Laboratory Net (LARNANO/UFPE) (Brazil).

Keywords: bimodal nanosystems, magnetic nanoparticle, quantum dots, fluorescence analysis, MRI contrast agents
# 070

Multifunctional nanoparticles for targeted MRI and therapy (#470)

J. Wilton-Ely1

1 Imperial College London, Department of Chemistry, London, United Kingdom

Introduction

Through the attachment of gadolinium (Gd) units to the surface of gold nanoparticles, these assemblies lead to dramatically improved contrast in MR imaging due to the increase in mass and large number of Gd units in a localised area. However, these studies1,2 have all been based on flexible thiol(ate) tethers, which undermine the relaxivity enhancement through the rapid rotation of the individual Gd units, in contrast to the beneficial slow tumbling of the nanostructure.3 Thiolate units are also susceptible to loss from the surface, illustrating the need for new attachment methodologies.

Methods

New gadolinium chelates based on the clinically-approved DOTA scaffold were synthesised and characterised by multinuclear NMR, FTIR spectroscopy and mass spectrometry. The functionalised nanostructures were characterised by UV-vis, ICP-OES, TGA, DLS, EDS, Zetapotential and TEM and found to be stable over a wide pH range and in the presence of high concentrations of zinc ions. Cytotoxicity assays and cell uptake studies confirmed the compatibility of the assemblies in cellular media. Relaxivity enhancement was measured using NMRD profiles, while a 1.5 T medical scanner was used to confirm the effectiveness of the materials under clinical conditions.

Results/Discussion

Our early studies on the surface functionalisation of nanoparticles4 with metal units were then extended to the attachment of gadolinium units through a hexadentate chelate.5 Recently, these investigations have led to a range of octadentate macrocycles (q = 1), which feature a dithiocarbamate-based moiety for anchoring to the gold surface. This novel approach uses a rigid linker which dramatically enhances relaxivity per Gd unit and allows multiple additional surface units to be introduced in a modular fashion to deliver biocompatibility, targeting and therapy. Robust, non-cytotoxic multifunctional nanoparticles have been successfully prepared by this route. These materials display a remarkable 5-fold relaxivity enhancement per Gd unit over the clinical standard DotaremTM at 1.5 T. Additional surface units improved uptake across a range of cell lines while the addition of a photosensitizer suited to photodynamic therapy (PDT) led to 80% cell death in 30 mins on irradiation.

Conclusions

This versatile approach6 allows the straightforward preparation of robust, targeted, non-cytotoxic Gd-functionalised nanoparticles which allow enhanced contrast MR imaging. This allows their location to be easily determined through the high payload of Gd units before photoswitchable therapeutic action (PDT) is initiated, providing an effective theranostic combination.

References

1. C. Alric, J. Taleb, G. Le Duc, C. Mandon, C. Billotey, A. Le Meur-Herland, T. Brochard, F. Vocanson, M. Janier, P. Perriat, S. Roux and O. Tillement, J. Am. Chem. Soc., 2008, 130, 5908–5915.

2. L. Moriggi, C. Cannizzo, E. Dumas, C. R. Mayer, A. Ulianov and L. Helm, J. Am. Chem. Soc., 2009, 131, 10828–10829.

3. P. Caravan, Chem. Soc. Rev., 2006, 35, 512−523.

4. S. Naeem, S. Serapian, A. Toscani, A. J. P. White, G. Hogarth, J. D. E. T. Wilton-Ely, Inorg. Chem., 2014, 53, 2404–2416.

5. S. Sung, H. Holmes, L. Wainwright, A. Toscani, G. J. Stasiuk, A. J. P. White, J. D. Bell and J. D E. T. Wilton-Ely, Inorg. Chem., 2014, 53, 1989.

6. N. Chabloz, M. Wenzel, J. D. E. T. Wilton-Ely, manuscript under consideration

Acknowledgement

We thank the Wellcome Trust and the EPSRC for funding and the KCL-ICL CDT in Medical Imaging for provision of a relaxometer.

Schematic image of multifunctional nanoparticles
Keywords: Nanoparticles, gadolinium, imaging, MRI, therapy