New Wine in Old Bottle: Crown Ether-Functionalized Digital Polymer toward Efficient MALDI-TOF MS/MS Decoding via a Classical Supramolecular Interaction.

Digital polymers (DPs), which serve as promising molecular-level storage media, have increasingly garnered interest. Their application significantly depends on the efficiency of the information writing (synthesis) and reading processes (sequencing). For reading, rational incorporation of weak bonds in the main chain was applied in most cases in order to improve readability of the tandem mass spectra (MS/MS), which would limit the chain length of DPs, thus reducing the information storage capacity. In this study, the introduction of commercially available crown ether (CE) at the terminus of digital oligo(γ-butyrolactone)s (DOBLs) significantly enhances the predictability and fidelity of matrix-assisted laser desorption/ionization time-of-flight tandem mass spectra (MALDI-TOF MS/MS), thus improving the decoding process. The use of crown ether, leveraging a well-established supramolecular interaction with alkali cations known since 1967, offers a strong affinity between ionization agents and CE motifs, to form a selective effect of the desired fragments during the tandem MS. This method is particularly effective for long-chain DPs, extending up to 32-mer, and allows for customizable fragmentation patterns. The incorporation of CE at the DP chain end presents a novel and efficient strategy for enhancing MS/MS readability and amplifying the information storage capacity of polymers.

Endocytosis-Independent and Cancer-Selective Cytosolic Protein Delivery via Reversible Tagging with LAT1 substrate.

Protein drugs targeting intracellular machineries have shown profound therapeutic potentials, but their clinical utilities are greatly hampered by the lack of efficient cytosolic delivery techniques. Existing strategies mainly rely on nanocarriers or conjugated cell-penetrating peptides (CPPs), which often have drawbacks such as materials complexity/toxicity, lack of cell specificity, and endolysosomal entrapment. Herein, a unique carrier-free approach is reported for mediating cancer-selective and endocytosis-free cytosolic protein delivery. Proteins are sequentially modified with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl carbonate as the H2 O2 -responsive domain and 3,4-dihydroxy-l-phenylalanine as the substrate of l-type amino acid transporter 1 (LAT1). Thus, the pro-protein can be directly transported into tumor cells by overexpressed LAT1 on cell membranes, bypassing endocytosis and endolysosomal entrapment. In the cytosol, overproduced H2 O2 restores the protein structure and activity. Using this technique, versatile proteins are delivered into tumor cells with robust efficiency, including toxins, enzymes, CRISPR-Cas9 ribonucleoprotein, and antibodies. Furthermore, intravenously injected pro-protein of saporin shows potent anticancer efficacy in 4T1-tumor-bearing mice, without provoking systemic toxicity. Such a facile and versatile pro-protein platform may benefit the development of protein pharmaceuticals.