Intracellular fate of bioresponsive poly(amidoamine)s in vitro and in vivo

Abstract

Linear poly(amidoamine)s (PAAs) have been designed to exhibit minimal non-specific toxicity, display pHdependent membrane lysis and deliver genes and toxins in vitro. The aim of this study was to measure PAA cellular uptake using ISA1-OG (and as a reference ISA23-OG) in B16F10 cells in vitro and, by subcellular fractionation, quantitate intracellular trafficking of 125 I-labelled ISA1-tyr in liver cells after intravenous (i.v.) administration to rats. The effect of time after administration (0.5-3 h) and ISA1 dose (0.04-100 mg/kg) on trafficking, and vesicle permeabilisation (N-acetyl-b-D-glucosaminidase (NAG) release from an isolated vesicular fraction) were also studied. ISA1-OG displayed~60-fold greater B16F10 cell uptake than ISA23-OG. Passage of ISA1 along the liver cell endocytic pathway caused a transient decrease in vesicle buoyant density (also visible by TEM). Increasing ISA1 dose from 10 mg/kg to 100 mg/kg increased both radioactivity and NAG levels in the cytosolic fraction (5-10 fold) at 1 h. Moreover, internalised ISA1 provoked NAG release from an isolated vesicular fraction in a dose-dependent manner. These results provide direct evidence, for the first time, of PAA permeabilisation of endocytic vesicular membranes in vivo, and they have important implications for potential efficacy/toxicity of such polymeric vectors.

Polyamidoamine Nanoparticles as Nanocarriers for the Drug Delivery to Malaria Parasite Stages in the Mosquito Vector

“…injection [42], others circulate in the bloodstream for an extended period [44], showing a tendency to localize in tumours due to the enhanced permeability effect. This long blood residence time is an important feature to consider when selecting candidates for the design of drug delivery systems, since an increased circulation will facilitate interactions of polymers with the target cell, which usually internalizes them via the endocytic pathway [45]. PAAs were first tested for their ability to form polyelectrolyte stable complexes with heparin, in order to neutralise its anticoagulant activity [34,46], results later extended to PAA-crosslinked resins, which have also been assayed for metal ion complexation [47].…”

“…It is a rather weak polymeric base with, on average, 0.55 positive charges per unit at pH 7.4. All these polymers had been reported as vectors for the intracellular delivery of nucleic acids [56,61,71], whereas ISA1 and ISA23 had been also studied for protein delivery [45,62,71] and as anticancer drug carriers [51,53]. ISA23 in particular has been proven to be endowed with stealth-like properties without selectively concentrating in the liver [44], while a significant portion of AGMA1 did show hepatic localization after intravenous injection in mice [42].…”

“…In intracellular compartments where the pH decreases to 6.5 (endosomes) and then to 5.0 (lysosomes), PAAs become prevailingly cationic and display endosomolytic properties [72]. Fluorescence microscopy revealed colocalization of ISA1 and ISA23 with Lysotracker, a marker for lysosome and late endocytic structures, and ISA1 also colocalized with the Early Endosomal Antigen 1 that accumulates in early endocytic structures [45]. Atomic force microscopy images (Figure 4) revealed a globular conformation for AGMA1, ISA23 and ISA1 adsorbed on mica substrates, showing a homogenous polymer size distribution with a hydrodynamic radius between 6 and 7 nm according to size exclusion chromatography analysis [70].…”

Poly(amidoamine)s synthesis, characterisation and interaction with BSA

“…Although poly(amidoamine)s have been used to promote intracellular delivery, 5,11,14,15 the interactions between the polymers and the protein and their effect on the protein stability are poorly understood. Protein-polyelectrolyte complexation may results from different types of interactions, including ionic and hydrophobic interactions, hydrogen bonding or coulombic forces.…”

“…4,5 The complexes formed from self-assembly of the biomacromolecules with the polymers have generally an overall cationic charge and are able to bind to the cell surface. Alternatively, the carrier will target a specic receptor on the cell membrane and internalisation will be done via receptor mediated endocytosis (RME).…”