Nuclear
Localization Signal Enhances the Targeting
and Therapeutic Efficacy of a Porphyrin-Based Molecular Cargo: A Systemic
In Vitro and Ex Vivo Evaluation
posted on 2024-03-13, 11:34authored byNaveen Kumar, Amit K. Sharma, Mohini Guleria, Sandeep B. Shelar, Avik Chakraborty, Sutapa Rakshit, Soumi Kolay, Drishty Satpati, Tapas Das
The objective of the present work was to evaluate the
potential
of a nuclear localization signal (NLS) toward facilitating intracellular
delivery and enhancement in the therapeutic efficacy of the molecular
cargo. Toward this, an in-house synthesized porphyrin derivative,
namely, 5-carboxymethyelene-oxyphenyl-10,15,20-tris(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation
with the NLS sequence [PKKKRKV]. The three compounds synthesized during
the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS,
and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer
cell lines (HT1080), wherein all exhibited minimal dark toxicity.
However, during photocytotoxicity studies with DOTA-Lys-UTriMA and
DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited
significantly higher light-dependent toxicity compared to the former.
Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy
cell line (WI26VA4) was found to be significantly lower than that
observed in the cancer cells. Fluorescence cell imaging studies carried
out in HT1080 cancer cells revealed intracellular accumulation for
the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated
porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic
effects of the synthesized conjugates, all three compounds were radiolabeled
with 177Lu, a well-known therapeutic radionuclide with
high radiochemical purity (>95%). During in vitro studies, the
[177Lu]Lu-DOTA-UTriMA-Lys-NLS complex exhibited the highest
cell binding as well as internalization among the three radiolabeled
complexes. Biological distribution studies for the radiolabeled compounds
were performed in a fibrosarcoma-bearing small animal model, wherein
significantly higher accumulation and prolonged retention of [177Lu]Lu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24
h p.i.) in the tumorous lesion compared to [177Lu]Lu-UTriMA-Lys-DOTA
(2.3 ± 0.13% IA/g at 24 h p.i.) and [177Lu]Lu-DOTA-Lys-NLS
complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The
results of the biodistribution studies were further corroborated by
recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice
administered with [177Lu]Lu-DOTA-UTriMA-Lys-NLS at different
time points. Tumor regression studies performed with [177Lu]Lu-DOTA-UTriMA-Lys-NLS in the same animal model with two different
doses [250 μCi (9.25 MBq) and 500 μCi (18.5 MBq)] resulted
in a significant reduction in tumor mass in the treated group of animals.
The above results revealed a definite enhancement in the targeting
ability of molecular cargo upon conjugation with NLS and hence indicated
that this strategy may be helpful for the preparation of drug-NLS
conjugates as multimodal agents.