Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells

Abstract

For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly( n -butylcyanoacrylate) (PBCA) nanocapsules as a shell and separately an oligonucleotide (20 mer) as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides) reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.

Shear Stress-Enhanced Internalization of Cell Membrane Proteins Indicated by a Hairpin-Type DNA Probe

“…The fluorescence recovery might be due to alteration of the DNA conformation during the interaction or the fluorophores dissociation when trapped in the intermembrane space of mitochondria. Red fluorescence signals were shown in mitochondria after long-time (>5 h) cell incubation with dissociative oligonucleotides. , However, in our experiments, under the effect of shear stress, red fluorescence appeared in mitochondria in as little as 10 min. Because of the strong linkage between the cell membrane proteins and DNA probes, it could be deduced that shear stress enhanced cellular internalization of membrane proteins, thereby leading to the cellular entry of the membrane protein-bound DNA probes.…”

Cellular uptake and intracellular degradation of poly(alkyl cyanoacrylate) nanoparticles

“…PACA NP-assisted drug delivery in vivo can also be indirectly linked to degradation rates. To the best of our knowledge, however, [ 11 ] remains the only work where the fate of PBCA nanocapsules was observed intracellularly using dual labeling of the nanocapsule shell and the cargo. That study, however, was performed with only one type of PACA NPs and mostly concerned the release and intracellular fate of the payload, while in the present work we studied the intracellular fate of different PACA nanocarriers.…”

“…Therefore, uptake profile and intracellular degradation of PACA NPs are the two key factors affecting intracellular drug availability once the NPs have reached the tumor cells. The uptake of NPs in cells, including PACA NPs, demonstrates different uptake efficacy by different cell types and organs [ 10 , 11 ]. Degradation of PACA NPs mainly occurs by surface erosion [ 12 , 13 ] into water-soluble poly(cyanoacrylic acid) and a primary alcohol following hydrolysis of the ester [ 14 ].…”

“…To the best of our knowledge, however, degradation and drug release for PACA NPs have been mainly studied in solution using physicochemical techniques [ 12 , 17 – 19 ]. One recent study described the intracellular payload release from poly(butyl cyanoacrylate) NPs [ 11 ], but knowledge of the intracellular degradation of different PACA NPs remains scarce.…”

A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

“…Mice were euthanized by intraperitoneal injection of sodium pentobarbital to obtain the lungs, livers, kidneys, spleens, and hearts for imaging and measuring their fluorescence value. The relevant radiant efficiency (P s −1 cm −2 sr −1 ) (μW −1 cm 2 ) was then calculated by Living Image 4.4 software ( 56 , 71 , 72 ).…”