Intracellular Delivery Of Biomacromolecules Research Articles

Split luciferase-based estimation of cytosolic cargo concentration delivered intracellularly via attenuated cationic amphiphilic lytic peptides

Intracellular delivery of biomacromolecules is challenging as these molecules are taken up by cells and encapsulated into vesicular compartments called endosomes, and the fraction of molecules that are translocated to the cytosol are particularly important to obtain desired biological responses. This study aimed to estimate the cytosolic concentrations of intracellularly delivered peptides and proteins to aid the design of novel and effective biopharmaceutical delivery systems. To this end, we employed the split NanoLuc luciferase system, using the 11-residue HiBiT peptide segment as a probe for the delivered molecules in cells expressing the complementary LgBiT protein segment. The efficacy in cytosolic HiBiT delivery was determined by measuring the resultant luciferase activity when the HiBiT segment delivered into the cytosol forms a complex with LgBiT. Mean cytosolic HiBiT concentration was calculated using cell number and cell volume analysis. L17E and HAad peptides, developed in our laboratory for intracellular protein delivery, yielded approximately 6-fold cellular HiBiT concentrations than that obtained in their absence.

Improved cytosolic delivery of macromolecules through dimerization of attenuated lytic peptides

Intracellular delivery of biomacromolecules is a challenging research field in chemical biology and drug delivery. We previously reported a peptide named L17E, which successfully delivered functional proteins, including antibodies, into cells. However, relatively high concentrations of L17E and proteins are needed. In this study, we prepared dimers of L17E and its analog L17E/Q21E. Dimerization of L17E increased cytotoxicity leading to reduced intracellular delivery compared with L17E. On the other hand, the dimers of the L17E analog, L17E/Q21E, especially when tethered at the N-termini, yielded a comparable level of intracellular delivery with L17E at decreased amounts of delivery peptides and cargoes.

膜透過ペプチドの生体膜透過機構と高分子の細胞内導入

One of the great obstacles in developing therapeutic agents based on biomacromolecules, including peptides, proteins, and nucleic acids, should be their low membrane permeability due to their high hydrophilicity and large molecular sizes. On the other hand, there is a growing research interest in intracellular delivery of biomacromolecules using arginine-rich cell-penetrating peptides (CPPs). Chemical conjugation of bioactive macromolecules with these CPPs allows efficient translocation of these molecules into cells. Successful intracellular delivery of various nano-particles using CPPs have also been reported. Involvement of macropinocytosis has been reported as an internalization mechanism of arginine-rich CPPs. Accumulation of arginine-rich CPPs and their conjugates on cell surfaces by the interaction of membrane associated proteoglycans and the eventual induction of macropinocytosis can be a rationale to explain the high efficacy of the intracellular delivery.