Intracellular Delivery of Native Proteins Facilitated by Cell‐Penetrating Poly(disulfide)s

Abstract

Intracellular delivery of therapeutic proteins is highly challenging and in most cases requires chemical or genetic modifications. Herein, two complementary approaches for endocytosis-independent delivery of proteins to live mammalian cells are reported. By using either a "glycan" tag naturally derived from glycosylated proteins or a "traceless" tag that could reversibly label native lysines on non-glycosylated proteins, followed by bioorthogonal conjugation with cell-penetrating poly(disulfide)s (CPDs), we achieved intracellular delivery of proteins (including antibodies and enzymes) which, upon spontaneous degradation of CPDs, led to successful release of their "native" functional forms with immediate bioavailability.

Disulfide‐Containing Macromolecules for Therapeutic Delivery

“…Moreover, replacing lipoic acid with asparagusic acid monomers lead to significant loss in cellular uptake of the resultant polymers. The polymerization system has also been further applied to facilitate intracellular delivery of small‐molecule drugs and proteins …”

Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers

“…Intact KLAK peptides are released upon applying a reductive trigger . f) Cell‐penetrating poly(disulfide)s (CPDs) mediate the intracellular uptake of protein conjugates, which undergo self‐immolative degradation and release native proteins …”

“…[47] f) Cell-penetrating poly(disulfide)s (CPDs)m ediate the intracellular uptake of protein conjugates, whichu ndergoself-immolative degradation and releasen ative proteins. [48] Caruso et al [47] reported the fabrication of nanoparticles directly from mitochondria-disrupting KLAK peptides by using self-immolative hetero-bifunctionall inkers (NDBC and NDEC) to cross-link peptides and eight-armed PEG-SH.T hese nanoparticles disassemble into native KLAK peptides through disulfide cleavage and self-immolative cascaded egradation (Figure 5e). In another report, Yaoe tal.…”

“…developeds elf-immolativel inkers (pNP-SS-Nor and pNP-SS-TCO) that enabled the convenient bridging of CPDs with native proteins/antibodies, andi ntracellularly triggered releaseo fc onjugatedp roteins was achieved (Figure 5f). [48] 3. Fluorogenic Bifunctional Linkers…”

Glycosylated Cell‐Penetrating Peptides (GCPPs)

“…acids were optimal. [41][42][43][44] After these structural and functional discoveries, the potential of CPPs has led to the development of myriadp enetrating peptides equences fort he cellulari nternalization of different payloads. [14,16] Similaru ptake properties were found for the enantiomeric TAT [49][50][51][52][53][54][55][56][57] peptidea nd in penetrating peptoidsw ith guanidiniums ide chains anchored at the nitrogen of the amide group.…”