Radiolabeling and in vitro evaluation of 67Ga-NOTA-modular nanotransporter – A potential Auger electron emitting EGFR-targeted radiotherapeutic

Abstract

Introduction Modular nanotransporters (MNTs) are vehicles designed to transport drugs from the cell surface via receptor-mediated endocytosis and endosomal escape to nucleus. Hence their conjugation to Auger electron emitters, can cause severe cell killing, by nuclear localization. Herein we evaluate the use of MNT as a platform for targeted radiotherapy with 67Ga. Methods EGF was the targeting ligand on the MNT, and NOTA was selected for its radiolabeling with 67Ga. In the radiolabeling study we dealt with the precipitation of MNT (pI 5.7) at the labeling pH (4.5–5.5) of 67Ga. Cellular and nuclei uptake of 67Ga-NOTA-MNT by the A431 cell line was determined. Its specific cytotoxicity was compared to that of 67Ga-EDTA, 67Ga-NOTA-BSA and 67Ga-NOTA-hEGF, in A431 and U87MGWTT, cell lines, by clonogenic assay. Dosimetry studies were also performed. Results 67Ga-NOTA-MNT was produced with 90% yield and specific activity of 25.6 mCi/mg. The in vitro kinetics revealed an increased uptake over 24 h. 55% of the internalized radioactivity was detected in the nuclei at 1 h. The cytotoxicity of 67Ga-NOTA-MNT on A431 cell line was 17 and 385-fold higher when compared to non-specific 67Ga-NOTA-BSA and 67Ga-EDTA. While its cytotoxic potency was 13 and 72 – fold higher when compared to 67Ga-NOTA-hEGF in the A431 and the U87MGWTT cell lines, respectively, validating its nuclear localization. The absorbed dose, for 63% cell killing, was 9 Gy, confirms the high specific index of 67Ga. Conclusion These results demonstrate the feasibility of using MNT as a platform for single cell kill targeted radiotherapy by Auger electron emitters.

Preparation, cytotoxicity, and in vivo antitumor efficacy of ¹¹¹In-labeled modular nanotransporters

“…This is accomplished by its endosomolytic module, which increases its hydrophobicity to provide penetration through the endosomal membrane in response to the decreased pH of acidifying endosomes. 26 The ability of MNTs to escape from acidifying endosomes as well as the necessity of tDTox inclusion into MNT molecule have been demonstrated by us previously, 26 along with their ability to accumulate efficiently in the nuclei of target cancer cells in vitro 13,16,17 and in vivo.…”

“…In at neutral pH as well as the increased hydrophobicity of these MNTs at slightly acidic pH, attempts to label the NOTA-MNT or DOTA-MNT conjugates with 111 In either in the acetate buffer routinely used for 111 In labeling of proteins 27 or using a protocol that was successful for 67 Ga labeling of NOTA-MNT 17 were futile. Based on the studies by Brom et al 28 and Morfin and Toth, 29 we used HEPES and citrate; we also added a small amount of SDS (10 times below the critical micelle concentration) to the reaction mixture in an attempt to shield the hydrophobic sites that are known to be exposed on the MNT molecule at mildly acidic pH.…”

“…Moreover, MNTs possess interchangeable component domains, permitting relatively facile exchange of receptor recognition domains, which ultimately might be adapted for different diseases or might help improve homogeneity of drug delivery by targeting several tumor-specific molecules for personalized medicine. MNTs reactive with a variety of tumor-associated receptors have shown promise for delivering short range of action therapeutics including photosensitizers, 13 Ga,17 as they were demonstrated to colocalize in the nuclei of target cancer cells in vitro (up to 60% of internalized found in nuclei) 13,14,16,17 and in vivo, 14 as well as enhance significantly cytotoxicity of all the above-mentioned cargoes in vitro 13,[15][16][17][18] and the therapeutic potential of different photosensitizers in vivo.…”

“…All buffers for chelator conjugation and labeling procedures were first passed through a Chelex-100 resin (200-400 mesh, Bio-Rad Laboratories Inc.) to minimize adventitious heavy metal ion contamination. Briefly, 1-16 mg of each MNT was incubated with a 10-fold molar excess of the chelator in 1× pH 8.6 conjugation buffer 17 for 24 h at RT with a final concentration of MNT $1.5 mg/mL. The chelator-MNT conjugate was concentrated and separated from the excess chelator by five cycles of ultrafiltration with an Amicon Ultracel-30K unit.…”

“…This Specific activity has been shown to be important for maximizing the therapeutic potential of a radiolabeled molecule even when labeled with α-emitters that only require ,10 atoms bound per cell to achieve effective cell kill. 34 Because the number of Auger electron emitters needed to accomplish a similar degree of cytotoxicity is two to three orders of magnitude higher, 16,17 maximizing the specific activity is a critical parameter for Auger emitter radiotherapy. Indeed, our studies with where about 12% of MNT was labeled.…”