Cell Membrane-Based Nanoreactor To Mimic the Bio-Compartmentalization Strategy of a Cell

Abstract

Organelles of eukaryotic cells are structures made up of membranes, which carry out a majority of functions necessary for the surviving of the cell itself. Organelles also differentiate the prokaryotic and eukaryotic cells, and are arranged to form different compartments guaranteeing the activities for which eukaryotic cells are programmed. Cell membranes, containing organelles, are isolated from cancer cells and erythrocytes and used to form biocompatible and long-circulating ghost nanoparticles delivering payloads or catalyzing enzymatic reactions as nanoreactors. In this attempt, red blood cell membranes were isolated from erythrocytes, and engineered to form nanoerythrosomes (NERs) of 150 nm. The horseradish peroxidase, used as an enzyme model, was loaded inside the aqueous compartment of NERs, and its catalytic reaction with Resorufin was monitored. The resulting nanoreactor protected the enzyme from proteolytic degradation, and potentiated the enzymatic reaction in situ as demonstrated by maximal velocity (V_max) and Michaelis constant (K_m), thus suggesting the high catalytic activity of nanoreactors compared to the pure enzymes.

Figure 1

Transmission electron microscopy (TEM) photos of (A, B) cancer cell derived vesicles and (C–F) empty NERs, obtained using only the sonication/extrusion techniques. The thickness and polydispersity of the cancer cell derived vesicles (≈ 4 nm) and NERs (≈11 nm) were analyzed using the TEM images and further compared. Vesicles obtained from (A, B) cancer cell membrane derivatives are broad size distributed, whereas (C, D) NERs are size distributed. (E) The zoom of NERs allows appreciating the narrow size distribution and the spherical shape of nanovesicles. (F) NERs preserved the characteristic discoidal biconcave shape of RBCs. The TEM photos are representative of three different and independent analyses.

Figure 2

Dynamic light scattering analysis of size and polydispersity index (PDI) of empty nanoerythrosomes (NERs) and horseradish peroxidase loaded-nanoerythrosomes (HRP-NERs) before (red) and after (blue) purification through bag dialysis. Empty NERs were not purified before the analysis. The dialysis was carried out only for the HRP-NERs to remove the untrapped enzyme. The PDI of HRP-NERs decreased below 0.1 due to the removing of enzyme which is adsorbed on the surface of NERs and is not loaded from the nanovesicles. The resulting PDI shows a narrow sized distribution of HRP-NERs. The analysis was carried out using a Zetasizer Nano ZS (Malvern, UK) and represents the average ± standard deviation (S.D.) from at least three independent measurements.

Figure 3

Nanoparticle tracking analysis (NTA) of (A) empty nanoerythrosomes or NERs and (B) horseradish peroxidase-loaded nanoerythrosomes or HRP-NERs: D10, D50, and D90 represent the average diameter of the 10, 50, and 90% of the nanoparticles. The analysis represents the average ± standard deviation (S.D.) from at least three independent measurements.

Figure 4

Transmission electron microscopy (TEM) images of horseradish peroxidase loaded-nanoerythrosomes (HRP-NERs). HRP-NERs (A, B) before and (C, D) after dialysis purification do not modify significantly their size and shape compared to the empty vesicles (see Figure 1). The TEM images are representative of three different and independent analyses.