Non-Covalent Active Mixing as a Viable Alternative to Covalent Conjugation for the Intracellular Delivery of Biologics: The Case of Glucarpidase.

Glucarpidase or carboxypeptidase G2 (CPG2) is an antidote for methotrexate (MTX) toxicity, but cannot penetrate cellular membranes. This study aims to clear intracellular MTX reservoirs and inhibit the MTX rebound effect. Therefore, two intracellular drug delivery approaches using the Tat-penetrating peptide, in the form of a Tat-CPG2 covalent conjugate and a Tat: CPG2 non-covalent complex, were compared. The native cpg2 gene from Pseudomonas sp. and the plasmid encoding tat-cpg2 conjugate were heterologously expressed in E. coli and purified using Ni-NTA. Tat: CPG2 complexes were formed by mixing 1 µM purified CPG2 with 0.5-80 µM Tat peptide. The HEK-293 cells were treated with both formulations to compare transduction efficiency by intracellular enzyme activity measurements at 320 nm and functional rescue of cells using 1-100 µM MTX. Molecular docking by ClusPro 2.0 revealed a distinct anionic patch on CPG2, facilitating electrostatic interactions through several salt bridges with the cationic Tat peptide. The highest transduction efficacy of the complex was observed at a Tat: CPG2 ratio of 20:1. While both formulations successfully delivered functional CPG2 into the cells, Tat-CPG2 conjugate displayed higher intracellular specific activity and retention, reaching approximately 2.6 ± 0.3 U/mg at 60 min compared with 1.9 ± 0.25 U/mg for the complex. In cytotoxicity assays, both forms protected HEK-293 cells from MTX; however, the conjugate provided statistically superior viability (94.8% vs. 68.5%) at high MTX concentrations (100 µM). While covalent conjugation remains the gold standard for maximal potency and stability, non-covalent complexation offers a rapid and cost-effective alternative for intracellular protein delivery.