1 A versatile polymer-based platform for intracellular delivery of macromolecules Michal Kopytynski 1 , Siyuan Chen 1 , Sandrine Legg 2 , Ralph Minter 2 , Rongjun Chen 1, * 1 Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, United Kingdom 2 Department of Antibody Discovery and Protein Engineering, AstraZeneca, Milstein Building, Granta Park, Cambridge CB21 6GH, UK *Corresponding author: Email: rongjun.chen@imperial.ac.uk Keywords: pH-responsive polymer, drug delivery, macromolecular delivery, apoptotic peptide, spheroids Abstract The plasma membrane barrier greatly restricts intracellular delivery of macromolecules. Currently available methods suffer from various limitations, including low delivery efficiency, high cytotoxicity or incompatibility with a wider range of macromolecules or cell types. To overcome these issues, stimuli-responsive polymers such as the bio-inspired, pH- responsive poly(L-lysine isophthalamide) grafted with L-phenylalanine at a stoichiometric ratio of 50% (PP50) can be used. In mildly acidic environments, the pseudopeptidic polymer can permeabilize the plasma membrane overcoming the problem of payload entrapment in the endosomes and allowing for efficient intracellular delivery. We demonstrate that PP50 was capable of intracellular delivery by simple co-incubation at pH 6.5 with various macromolecules, including different-sized Dextrans, green fluorescent protein (GFP) and an apoptotic peptide. The delivery process was fast, non-toxic and compatible with multiple cell types, including adherent and suspension cell lines, primary human mesenchymal stem cells,