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Alkaline phosphatase (ALP) enables intracellular targeting by peptide assemblies, but how the ALP substrates enter cells remains elusive. Here we show that nanoscale phosphopeptide assemblies cluster ALP to enable caveolae-mediated endocytosis (CME) and endosomal escape. Specifically, fluorescent phosphopeptides undergo enzyme-catalyzed self-assembly to form nanofibers. Live cell imaging unveils that phosphopeptides nanoparticles, coincubated with HEK293 cells overexpressing red fluorescent protein-tagged tissue-nonspecific ALP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME. Further dephosphorylation of the phosphopeptides produces peptidic nanofibers for endosomal escape. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Decreasing the transformation to nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of nanoscale assemblies for cellular uptake, this work illustrates an effective design for enzyme-responsive supramolecular therapeutics and provides mechanism insights for understanding the dynamics of cellular uptake of proteins or exogenous peptide aggregates.
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Self‐Complementary Zwitterionic Peptides Direct Nanoparticle Assembly and Enable Enzymatic Selection of Endocytic Pathways
Abstract Supramolecular self‐assembly in biological systems holds promise to convert and amplify disease‐specific signals to physical or mechanical signals that can direct cell fate. However, it remains challenging to design physiologically stable self‐assembling systems that demonstrate tunable and predictable behavior. Here, the use of zwitterionic tetrapeptide modalities to direct nanoparticle assembly under physiological conditions is reported. The self‐assembly of gold nanoparticles can be activated by enzymatic unveiling of surface‐bound zwitterionic tetrapeptides through matrix metalloprotease‐9 (MMP‐9), which is overexpressed by cancer cells. This robust nanoparticle assembly is achieved by multivalent, self‐complementary interactions of the zwitterionic tetrapeptides. In cancer cells that overexpress MMP‐9, the nanoparticle assembly process occurs near the cell membrane and causes size‐induced selection of cellular uptake mechanism, resulting in diminished cell growth. The enzyme responsiveness, and therefore, indirectly, the uptake route of the system can be programmed by customizing the peptide sequence: a simple inversion of the two amino acids at the cleavage site completely inactivates the enzyme responsiveness, self‐assembly, and consequently changes the endocytic pathway. This robust self‐complementary, zwitterionic peptide design demonstrates the use of enzyme‐activated electrostatic side‐chain patterns as powerful and customizable peptide modalities to program nanoparticle self‐assembly and alter cellular response in biological context.
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- PAR ID:
- 10361722
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Materials
- Volume:
- 34
- Issue:
- 1
- ISSN:
- 0935-9648
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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