Cell-based studies revealed that the µMax20 analog is cell-permeable. We anticipated that inserting the double hArg mutations in µMax20 would facilitate its cellular uptake compared with the native analog. Furthermore, we anticipated that the selective incorporation of peptide staples at strategic positions in µMax20 would further enhance its cell permeability. To generate a stapled version of the µMax20 analog, we inserted two aromatic staples through late-stage Cys alkylation using 2,6bis(bromomethyl)pyridine at specific positions (Cys32-Cys36 and Cys81-Cys85) to obtain a double-stapled version of µMax20 tagged with TAMRA fluorophore (T-2s-µMax20) (SI section 5). pronounced nuclear localization at higher concentrations (Figure ). T-2s-µMax20 exhibited the highest efficiency, achieving notable nuclear localization at 1 μM and a brighter fluorescence signal compared to other analogs (Figure ). Brightfield images confirmed that cells retained their characteristic elongated morphology and strong attachment, suggesting intact membrane integrity and low toxicity after incubation with Max analogs (SI section 10). This is particularly notable for T-2s-µMax20, given its high permeability, which could risk membrane disruption, underscoring its potential biocompatibility. Fluorescence patterns for internalizing analogs revealed a punctate distribution (Figure ), especially at lower concentrations, indicative of endosomal localization and suggesting an endocytosisdriven uptake mechanism. To confirm this distribution, we treated cells with MiTMAB, 31 an endocytosis inhibitor, which blocked the internalization of T-WT Max and T-µMax20 (SI section 11). However, the T-2s-µMax20 uptake was significantly reduced but not completely inhibited (SI section 11), indicating a dual mechanism of internalization involving both endocytosis and direct translocation. These observations align with known behaviors of stapled peptides that internalize through dual mechanisms. Overall, these findings highlight the power of the inserted novel transformations in µMax20 and 2s-µMax20 to enhance cellular internalization and nuclear localization. We envision that the discovery of potent µMax20 variants with exceptional intrinsic cell permeability will pave the way for expanding the power of engineered TFs for gene expression regulation without the need for further engineering with a delivery sequence. 33