An albumin coupling strategy improves aptamers' blood circulation and tumor targeting

Introduction: Aptamers, oligonucleotides with specific three-dimensional structures, have the broad prospect for clinical translation because of their excellent affinity with the target. However, their rapid clearance from the kidneys greatly limits their tumor targeting in vivo, which impedes potential clinical translation. Some albumin-binding groups, such as Evans Blue, have been employed to extend aptamers' circulation for cancer diagnosis. But the difficulty of chemical modification and the uncertain biotoxicity of small hydrophobic molecules urges better methods. Here, we report a more convenient and biocompatible method of site-specific albumin coupling for tumor imaging.

Methods: The albumin-aptamer complex consists of two components: human serum albumin (HSA) and AS1411, an aptamer targeting the nucleolin overexpressed on cancer cell membrane. We purified HSA-AS1411 by anion exchange chromatography and determined the successful synthesis and purity by gel electrophoresis and MALDI-TOF. Using the same method, we synthesized HSA-random sequence (RS) as a control. The serum stability and target affinity were characterized by gel electrophoresis and Bio-Layer Interferometry (BLT), respectively. We constructed the 4T1 subcutaneous tumor model with high nucleolin expression, and synthesized four 99mTc labeled probes: ^99mTc-HSA-AS1411, ^99mTc-HSA-RS, ^99mTc-AS1411, ^99mTc-RS to evaluate their tumor imaging performance. Through the SPECT/CT imaging at different time points and the biodistribution at 36 hours, we systematically evaluated blood circulation, tumor retention, and biodistribution profiles of the aptamer alone and albumin-modified aptamer.

Results: We confirmed the successful synthesis of HSA-AS1411 and HSA-RS by gel electrophoresis and MALDI-TOF. Compared with RS, AS1411 and HSA-AS1411 are more stable in fetal bovine serum. Similar to AS1411, HSA-AS1411 has a much higher affinity to nucleolin than RS and its albumin conjugates in vitro. In SPECT/CT images, the two ^99mTc-labeled albumin-aptamer complex showed long blood circulation, and the blood pool signal was 15%ID/g at 12 h, about 15 times than AS1411, and still higher than 5%ID/g at 36 h. Because ^99mTc-AS1411 was quickly cleared from the blood and excreted via hepato-intestinal pathway. The tumor uptake of ^99mTc-HSA-AS1411 was 17.2%ID/g at 36 h, which was significantly higher than the other three probes (^99mTc-HSA-RS was 9.9%ID/g, ^99mTc-AS1411 and RS were averaged at 2%ID/g).

Conclusions: The albumin coupling strategy has greatly overcome the poor pharmacokinetics and unsatisfied tumor uptake of AS1411. Given the excellent biocompatibility of HSA, we strongly believe the strategy can improve the tumor targeting/therapeutic effect of aptamers and suggest excellent clinical application prospects.

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