64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

doi:10.1016/j.nucmedbio.2013.08.003

Nuclear Medicine and Biology

Volume 40, Issue 8, November 2013, Pages 1018-1024

https://doi.org/10.1016/j.nucmedbio.2013.08.003 Get rights and content

Abstract

Introduction

We synthesized and evaluated ⁶⁴Cu-labeled tetraiodothyroacetic acid (tetrac)-conjugated liposomes for PET imaging of tumor angiogenesis, because tetrac inhibits angiogenesis via integrin α_Vβ₃.

Methods

Tetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized and formulated with other lipids into liposomes. The resulting tetrac/DOTA-liposomes were labeled with ⁶⁴Cu at 40 °C for 1 h and purified using a PD-10 column. ⁶⁴Cu-DOTA-liposomes were also prepared for comparison. Human aortic endothelial cell (HAEC) binding studies were performed by incubating the liposomes with the cells at 37 °C. MicroPET imaging followed by tissue distribution study was carried out using U87MG tumor-bearing mice injected with tetrac/⁶⁴Cu-DOTA-liposomes or ⁶⁴Cu-DOTA-liposomes.

Results

HAEC binding studies exhibited that tetrac/⁶⁴Cu-DOTA-liposomes were avidly taken up by the cells from 1.02 %ID at 1 h to 11.89 %ID at 24 h, while ⁶⁴Cu-DOTA-liposomes had low uptake from 0.47 %ID at 1 h to 1.57 %ID at 24 h. MicroPET imaging of mice injected with tetrac/⁶⁴Cu-DOTA-liposomes showed high radioactivity accumulation in the liver and spleen. ROI analysis of the tumor images revealed 1.93 ± 0.12 %ID/g at 1 h and 2.70 ± 0.36 %ID/g at 22 h. In contrast, tumor ROI analysis of ⁶⁴Cu-DOTA-liposomes revealed 0.54 ± 0.08 %ID/g at 1 h and 0.52 ± 0.09 %ID/g at 22 h. Tissue distribution studies confirmed that the tumor uptakes of tetrac/⁶⁴Cu-DOTA-liposomes and ⁶⁴Cu-DOTA-liposomes were 1.75 ± 0.03 %ID/g and 0.36 ± 0.01 %ID/g at 22 h, respectively.

Conclusion

These results demonstrate that tetrac/⁶⁴Cu-DOTA-liposomes have significantly enhanced tumor uptake compared to ⁶⁴Cu-DOTA-liposomes due to tetrac conjugation. Further studies are warranted to reduce the liver and spleen uptake of tetrac/⁶⁴Cu-DOTA-liposomes.

Introduction

Nanoparticles have been utilized for molecular imaging due to their unique characteristics that allow delivery of bioactive molecules to target tissues by carrying them inside or conjugating them to a large surface area. Nuclear imaging provides highly sensitive and quantitative information on biochemical and physiological changes in vivo. The benefits of combining these properties have led to increased uses for nuclear imaging using radiolabeled nanoparticles including liposomes, metals, quantum dots, carbon nanotubes, dendrimers and others [1], [2].

Liposomes are nanoparticles composed of phospholipids and have long been used for drug delivery. For the last three decades, liposomes have been used in nuclear imaging by encapsulating radioisotopes or labeling radioisotopes on their surfaces [3]. ^99mTc, ¹¹¹In, ⁶⁷Ga, ¹⁸F, and ⁶⁴Cu-labeled liposomes were used to study pharmacokinetics or to visualize tumors, infection, or cardiovascular abnormalities by SPECT or PET imaging [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. Among them, ¹¹¹In-loaded or ¹¹¹In-DTPA-PEG-labeled liposomes showed potential for the in vivo imaging of various human tumors [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17].

Liposomes are also excellent candidates for molecular imaging because of easy modification with various functionalities and, perhaps most importantly, their biocompatibility. It has been reported that liposomes could preferentially penetrate into neovasculature of tumor tissues and retain in the tissues [18], which is termed as an enhanced permeation and retention (EPR) effect. Furthermore, distribution of liposomes to tumor tissues can be enhanced by conjugation with molecules such as antibodies, proteins, peptides, or small ligands with high binding affinities to antigens or receptors which are over-expressed in certain tumors [19], [20], [21], [22], [23], [24]. As such, small anti-angiogenic molecules with high binding affinity to the integrin α_Vβ₃ can be good candidates for the targeted delivery of liposomes to integrin α_Vβ₃-expressing tumor tissues.

Angiogenesis is associated with integrin α_Vβ₃, which mediates adhesion, migration, proliferation, differentiation, and survival of endothelial cells and is found to be over-expressed on tumor endothelial cells [25], [26]. Because of this important role, integrin α_Vβ₃ has been the key molecular imaging target for tumor angiogenesis imaging. Cyclic Arg-Gly-Asp (RGD), an integrin α_Vβ₃ antagonist, has been developed as nuclear imaging probes for monitoring the expression of the integrin α_Vβ₃, and some of them showed promising outcomes in animal models and patients [27], [28]. It was also reported that integrin α_Vβ₃ has a binding site for thyroid hormone (T₄), which was supported by the strong binding of T₄ to integrin α_Vβ₃ (K_d = 333 pM) as well as T₄-induced phosphorylation of MAPK and angiogenesis [29]. The binding of T₄ to integrin α_Vβ₃ was inhibited by 3,3′5,5′-tetraiodothyroacetic acid (tetrac), RGD peptide, and α_Vβ₃ antibody, suggesting that the T₄ binding site may be located at or near the RGD recognition site [29]. Tetrac, a thyroid hormone derivative, was shown to have anti-angiogenic activity by binding to the cell surface receptor for T₄ on integrin α_Vβ₃ [30]. It was also shown that tetrac inhibited tumor growth and tumor angiogenesis in renal cell carcinoma xenografted mice and in human medullary thyroid carcinoma xenografted mice [31], [32]. Moreover, tetrac-conjugated nanoparticles consisting of poly(lactide-co-glycolide) were reported to provide antitumor activity similar to tetrac in animal models [31], [32]. A recent study demonstrated that tetrac-conjugated liposomes inhibited tumor growth and thus increased survival of A375 tumor-bearing mice compared to control liposomes when edelfosine, an alkyl lysophospholipid-based anticancer drug, was loaded into both liposomes [33]. Thus, this previous study supports tetrac's key role in enhancing drug delivery to tumors.

The diagnosis of tumor tissues with the high sensitivity and specificity of PET imaging is crucial for the early and accurate detection of tumors. The PET-based detection of tumor tissues, however, still suffers from the lack of probes that can differentiate tumors from normal tissues. Given that tetrac may serve as a ligand that provides enhanced drug delivery to tumors, we hypothesized that tetrac-conjugated liposomes could be useful for PET imaging of tumors.

In the present study, we prepared ⁶⁴Cu-labeled tetrac-conjugated liposomes and evaluated them for tumor angiogenesis imaging using PET (Fig. 1).

Section snippets

Materials and equipment

1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA)-NHS ester was purchased from Macrocyclics (Dallas, TX, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG-NH₂), DSPE-N[methoxy(polyethylene glycol)-2000] (DSPE-mPEG), egg l-α-phosphatidylcholine (PC), egg l-α-phosphatidyl-dl-glycerol (PG), and cholesterol (Chol) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ⁶⁴CuCl₂ and U87MG human glioblastoma cells were kindly

Synthesis of tetrac/DOTA- and DOTA-conjugated liposomes

Tetrac-PEG-DSPE and DOTA-PEG-DSPE were synthesized (Fig. 2A and B) and identified by MALDI-TOF mass spectrometry, which showed repeating patterns of molecular ions by 44 mass units, a characteristic mass distribution of PEG (Fig. 2C and D) [35]. The unreacted DSPE-PEG-NH₂ (molecular weight: 2772.7711 Da) was not detected based on the mass spectrometric analysis data (Fig. 2C and D). Further HPLC analysis showed that purity of tetrac-PEG-DSPE and DOTA-PEG-DSPE was 87.9% and 89.1%, respectively.

Discussion

Recent studies showed that radiolabeled liposomes containing tumor-targeting molecules provide superior tumor uptake compared to control liposomes, indicating an important role of the tumor-targeting molecules in liposomes. ¹¹¹In-labeled 2C5 antibody-PEG-liposomes had faster and higher tumor uptake than control PEG-liposomes or IgG-PEG-liposomes in 4T1 and LLC tumor-bearing mice, with higher tumor to non-target uptake ratios (tumor/muscle = 16.3 vs. 6.8–7.4; tumor/liver = 0.45 vs. 0.24–0.26) [8].

Conclusion

This study showed that tetrac/⁶⁴Cu-DOTA-liposomes had higher uptake by HAECs, clear in vivo visualization of tumors in microPET images, and significantly enhanced tumor uptake in tissue distribution, compared to ⁶⁴Cu-DOTA-liposomes. Moreover, the in vitro endothelial cell binding result was consistent with the in vivo microPET imaging and tissue distribution data of U87MG tumor-bearing mice. Although tumor uptake of tetrac/⁶⁴Cu-DOTA-liposomes is comparable to that of [¹⁸F]galacto-RGD, the most

Acknowledgments

We thank Mr. Hunnyun Kim for operating the microPET imaging system. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2011-0030164).

References (45)

O.C. Boerman et al.
Radiolabeled liposomes for scintigraphic imaging
Prog Lipid Res
(2000)
I. Ogihara-Umeda et al.
Development of a liposome-encapsulated radionuclide with preferential tumor accumulation – the choice of radionuclide and chelating ligand. Int J Radiat Appl Instrum Part B
Nucl Med Biol
(1992)
J. Marik et al.
Long-circulating liposomes radiolabeled with [¹⁸F]fluorodipalmitin ([¹⁸F]FDP)
Nucl Med Biol
(2007)
N. Oku et al.
PET imaging of brain cancer with positron emitter-labeled liposomes
Int J Pharmaceut
(2011)
J.W. Seo et al.
Positron emission tomography imaging of the stability of Cu-64 labeled dipalmitoyl and distearoyl lipids in liposomes
J Control Release
(2011)
A.L. Petersen et al.
⁶⁴Cu loaded liposomes as positron emission tomography imaging agents
Biomaterials
(2011)
N. Yagi et al.
Synthesis and evaluation of a novel lipid–peptide conjugate for functionalized liposome
Bioorg Med Chem Lett
(2007)
X. Li et al.
Targeted delivery of doxorubicin using stealth liposomes modified with transferrin
Int J Pharmaceut
(2009)
H. Shmeeda et al.
Delivery of zoledronic acid encapsulated in folate-targeted liposome results in potent in vitro cytotoxic activity on tumor cells
J Control Release
(2010)
S. Lee et al.
Tetraiodothyroacetic acid-tagged liposomes for enhanced delivery of anticancer drug to tumor tissue via integrin receptor
J Control Release
(2012)

J.C. Klock et al.

Cholesterol, phospholipids, and fatty acids of normal immature neutrophils: comparison with acute myeloblastic leukemia cells and normal neutrophils

J Lipid Res

(1979)

X. Li et al.

Targeted delivery of doxorubicin using stealth liposomes modified with transferrin

Int J Pharm

(2009)

H. Hatakeyama et al.

A multifunctional envelope type nano device (MEND) for gene delivery to tumours based on the EPR effect: a strategy for overcoming the PEG dilemma

Adv Drug Deliv Rev

(2011)

K. Maruyama

Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects

Adv Drug Deliv Rev

(2011)

Y.H. Bae et al.

Targeted drug delivery to tumors: myths, reality and possibility

J Control Release

(2011)

M.J. Welch et al.

The advantages of nanoparticles for PET

J Nucl Med

(2009)

W. Cai et al.

Nanoplatforms for targeted molecular imaging in living subjects

Small

(2007)

D.J. Hnatowich et al.

Labeling of preformed liposomes with Ga-67 and Tc-99m by chelation

J Nucl Med

(1981)

L.E. Williams et al.

Possible application of phospholipid vesicles (liposomes) in diagnostic radiology

J Nucl Med Allied Sci

(1984)

I. Ogihara et al.

Tumor uptake of ⁶⁷Ga-carrying liposomes

Eur J Nucl Med

(1986)

T.A. Elbayoumil et al.

Enhanced accumulation of long-circulating liposomes modified with the nucleosome-specific monoclonal antibody 2C5 in various tumours in mice: gamma-imaging studies

Eur J Nucl Med Mol Imaging

(2006)

N. Kitamura et al.

Biodistribution of immunoliposome labeled with Tc-99m in tumor xenografted mice

Ann Nucl Med

(2009)

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64Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

Abstract

Introduction

Methods

Results

Conclusion

Introduction

Section snippets

Materials and equipment

Synthesis of tetrac/DOTA- and DOTA-conjugated liposomes

Discussion

Conclusion

Acknowledgments

Prog Lipid Res

Nucl Med Biol

Nucl Med Biol

Int J Pharmaceut

J Control Release

Biomaterials

Bioorg Med Chem Lett

Int J Pharmaceut

J Control Release

J Control Release

J Lipid Res

Int J Pharm

Adv Drug Deliv Rev

Adv Drug Deliv Rev

J Control Release

The advantages of nanoparticles for PET

J Nucl Med

Nanoplatforms for targeted molecular imaging in living subjects

Small

Labeling of preformed liposomes with Ga-67 and Tc-99m by chelation

J Nucl Med

Possible application of phospholipid vesicles (liposomes) in diagnostic radiology

J Nucl Med Allied Sci

Tumor uptake of 67Ga-carrying liposomes

Eur J Nucl Med

Enhanced accumulation of long-circulating liposomes modified with the nucleosome-specific monoclonal antibody 2C5 in various tumours in mice: gamma-imaging studies

Eur J Nucl Med Mol Imaging

Biodistribution of immunoliposome labeled with Tc-99m in tumor xenografted mice

Ann Nucl Med

⁶⁴Cu-Labeled tetraiodothyroacetic acid-conjugated liposomes for PET imaging of tumor angiogenesis

Tumor uptake of ⁶⁷Ga-carrying liposomes