ABSTRACT
Protein transduction domains (PTDs) are powerful nongenetic tools that allow intracellular delivery of conjugated cargoes to modify cell behavior. Their use in biomedicine has been hampered by inefficient delivery to nuclear and cytoplasmic targets. Here we overcame this deficiency by developing a series of novel fusion proteins that couple a membrane-docking peptide to heparan sulfate glycosaminoglycans (GAGs) with a PTD. We showed that this GET (GAG-binding enhanced transduction) system could deliver enzymes (Cre, neomycin phosphotransferase), transcription factors (NANOG, MYOD), antibodies, native proteins (cytochrome C), magnetic nanoparticles (MNPs), and nucleic acids [plasmid (p)DNA, modified (mod)RNA, and small inhibitory RNA] at efficiencies of up to two orders of magnitude higher than previously reported in cell types considered hard to transduce, such as mouse embryonic stem cells (mESCs), human ESCs (hESCs), and induced pluripotent stem cells (hiPSCs). This technology represents an efficient strategy for controlling cell labeling and directing cell fate or behavior that has broad applicability for basic research, disease modeling, and clinical application.
Subject(s)
Cell-Penetrating Peptides/metabolism , Drug Delivery Systems , Glycosaminoglycans/metabolism , Amino Acid Motifs , Animals , Cell Differentiation/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Proliferation/drug effects , Cell-Penetrating Peptides/chemistry , Detergents/pharmacology , Endocytosis/drug effects , Genome , Homeodomain Proteins/metabolism , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/drug effects , Human Embryonic Stem Cells/metabolism , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/metabolism , Integrases/metabolism , Mice , Mouse Embryonic Stem Cells/cytology , Mouse Embryonic Stem Cells/drug effects , Mouse Embryonic Stem Cells/metabolism , Muscle Development/drug effects , MyoD Protein/metabolism , NIH 3T3 Cells , Nanog Homeobox Protein , Nanoparticles , Nucleic Acids/metabolism , Protein Structure, Tertiary , Solubility , Trypsin/metabolismABSTRACT
Many cell therapy approaches aim to deliver high-density single-cell suspensions to diseased or injured sites in the body. Long term clinical success will in part be dependent on the cells that remain viable and that assume correct functionality post-administration. The research presented in this paper focuses on the potential of cell aggregate delivery to generate a more supportive environment for cells than single cell suspensions. An in vitro model of injection delivery of C2C12 myoblast cells showed a significant difference in cell function and phenotype between adhesive collagen and non-adhesive alginate, indicating that in vitro assays based on this approach can discriminate between cell-cell/cell-matrix interactions and could be valuable when assessing cell therapy systems. Contrary to single cells, aggregates maintain viability, cellular activity, and phenotype beyond that of single cells, even in non-adhesive matrices, enabling delivery of higher cell densities with enhanced proliferative and differentiation capacity.
