Acid-labile traceless click linker for protein transduction

Area of Science:

• Bioconjugation Chemistry
• Cellular Biology
• Drug Delivery Systems

Background:

• Intracellular delivery of active proteins is crucial for research and therapy.
• Existing protein delivery methods face challenges with stability and endosomal entrapment.
• Novel strategies are needed to improve protein transduction efficiency.

Purpose of the Study:

• To develop a novel traceless click linker with pH-sensitive reversible bonds for protein transduction.
• To create an effective protein delivery shuttle system utilizing this linker.
• To demonstrate efficient intracellular protein delivery and endosomal escape.

Main Methods:

• Synthesis and characterization of the azidomethyl-methylmaleic anhydride (AzMMMan) linker.
• Bioreversible protein modification using click chemistry with dyes, PEG, and a carrier.
• Evaluation of linker stability at physiological and acidic pH.
• Assessment of protein delivery efficiency using nlsEGFP and ß-galactosidase as cargo proteins.
• Comparison of pH-reversible versus irreversible linkage for protein delivery.

Main Results:

• The AzMMMan linker demonstrated compatibility with various click chemistries and bioreversible protein modification.
• Linkages were stable at physiological pH but reversible at acidic pH found in endosomes/lysosomes.
• The pH-reversible shuttle system facilitated efficient cellular uptake, endosomal escape, and nuclear import of nlsEGFP.
• Irreversible linkage resulted in cytosolic trapping of nlsEGFP, hindering nuclear delivery.
• Successful intracellular delivery of bioactive ß-galactosidase was achieved using the pH-controlled shuttle.

Conclusions:

• The developed traceless click linker offers a pH-sensitive mechanism for controlled protein release.
• The pH-reversible protein transduction shuttle system enhances intracellular protein delivery, overcoming endosomal entrapment.
• This technology holds significant promise for advancing protein-based therapeutics and research applications.