A versatile platform for intracellular delivery of various macromolecules using a pH-responsive, biomimetic polymer

The physical barrier posed by the plasma membrane greatly restricts the potential of intracellular delivery of macromolecules. Currently available delivery methods suffer from various limitations, including low delivery efficiency or high cytotoxicity. To overcome these issues, stimuli-responsive polymers such as the bio-inspired, pH-responsive PP50 polymer, comprising a poly-L-lysine isophthalamide backbone with hydrophobic L-phenylalanine grafts can be used. In mildly acidic environments, PP50 can permeabilise the cell membrane overcoming the problem of payload entrapment in the endosomes and allowing for efficient delivery of molecules into the cell interior. The work presented herein demonstrates that PP50 is capable of delivering various macromolecular payloads in vitro, such as different-sized dextrans, green fluorescent protein and the apoptotic peptide Bim by simple co-incubation with the desired cargo at pH 6.5. The delivery process was fast, non-toxic and compatible with multiple cell lines tested, including adherent and suspension cell lines, primary human mesenchymal stem cells as well as cells grown as spheroids. In addition, peptide delivery by co-incubation with PP50 was at least 3 times more effective than delivery using other common delivery methods, including poly(ethyleneimine), cell penetrating peptides and electroporation. In addition, novel conjugates of PP50 and different model and functional payloads were developed using a cleavable crosslinker to enable in vivo delivery and release. Model fluorescent payloads such as a peptide-sized PEG and green fluorescent protein were delivered to HeLa cells following conjugation with PP50. Finally, Bim conjugated to PP50 was shown to retain its apoptotic effect in vitro and was demonstrated to be non-immunogenic and well tolerated in a mouse model and exhibited preferential tumour accumulation. Our findings suggest that PP50-mediated payload delivery is a versatile method allowing delivery of various payloads to many different cell lines and can find many potential uses both in vitro and in vivo.