Phosphate-Dependent Colloidal Stability Controls Nonendocytic Cell Delivery of Arginine-Terminated Nanoparticles

Abstract

Although arginine-rich polymers and peptides are extensively used as delivery carriers for drugs/proteins/nanoparticles, their cell delivery mechanism is not clearly understood. Recent studies show that arginine-terminated nanoparticles can enter into a cell via a nonendocytic approach that involves direct membrane penetration. However, poor colloidal stability of arginine-terminated nanoparticles under physiological conditions restricts their application potential. Here, we show that the nonendocytic cell delivery of arginine-terminated nanoparticles is controlled by their colloidal stability in the presence of phosphates. We have designed arginine-terminated quantum dots (QDs) of 10–15 nm hydrodynamic size, which enter into the cell via a nonendocytic approach, provided that they are colloidal and dispersed during cellular uptake. We have demonstrated that arginine-terminated QDs rapidly precipitate in the presence of monophosphates or polyphosphates, and polyphosphates have a stronger effect than monophosphates. Introducing polyethylene glycol at the QD surface can improve the colloidal stability against phosphates. Control experiments show that amine/ammonium-terminated cationic QDs of similar sizes do not have such a type of phosphate-dependent precipitation issue. We propose that arginine-terminated colloidal nanoparticles have a unique advantage over amine/ammonium-terminated nanoparticles as they can bind with the cell membrane phosphate via guanidinium–phosphate salt bridging. Bulk phosphate provides reversibility in this binding interaction so that nonendocytic cell uptake occurs via charge compensation of cationic nanoparticles without membrane damage. The developed surface chemistry approach and the proposed mechanisms can be adapted to other nanoparticles for efficient cell delivery and for designing delivery carriers.

Rapid Mitochondria Targeting by Arginine-Terminated, Sub-10 nm Nanoprobe via Direct Cell Membrane Penetration

“…Second, nanoprobes are terminated with a guanidinium groups that offers a specific binding interaction with the cell membrane via phosphate-guanidinium salt bridging. This type of interaction is responsible for direct membrane penetration of the nanoprobe . Third, the nanoprobe is cationic in nature due to guanidinium and primary amine groups.…”

“…So, lipophilicity and cationic charge are required for mitochondria targeting. , Guanidinium groups have a very high cationic charge that helps it to attach to the highly anionic mitochondrial membrane. Additionally, guanidinium can form a strong salt bridge interaction with the cell membrane phosphate groups that offers nonendocytic and direct membrane penetration . Such an uptake mechanism leads to very high cell uptake efficiency, bypassing the lysosomal/endosomal trafficking, and helps for enhanced and faster subcellular targeting.…”

“…Additionally, guanidinium can form a strong salt bridge interaction with the cell membrane phosphate groups that offers nonendocytic and direct membrane penetration. 32 Such an uptake mechanism leads to very high cell uptake efficiency, bypassing the lysosomal/endosomal trafficking, and helps for enhanced and faster subcellular targeting. So, we expect that guanidinium functionalization offers nonendocytic cell uptake that enhances the subcellular targeting, and their strong electrostatic attraction with phosphate groups of mitochondrial cardiolipin offers mitochondria targeting.…”

Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer

“…We have recently shown that arginine-terminated hard nanoparticles of <10 nm enter via direct membrane penetration, but endocytosis dominates as the size increases . This size limit of direct membrane penetration is increased up to 100 nm for soft and non-ionic nanoparticles. , We have also shown that arginine-terminated nanoparticles rapidly precipitate in the presence of phosphates (present in biological media) via guanidinium-phosphate salt bridging that restricts their cellular entry …”

“…29,30 We have also shown that arginineterminated nanoparticles rapidly precipitate in the presence of phosphates (present in biological media) via guanidiniumphosphate salt bridging that restricts their cellular entry. 31 Here we have designed a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of proteins and DNA. We show that direct cytosolic delivery efficiency is sensitive to the size and colloidal property of the nanoassembly formed between the carrier and protein/DNA, and <200 nm size with colloidal property is critical for efficient cytosolic delivery.…”