Page 84 - Read Online

Gupta et al. Extracell Vesicles Circ Nucleic Acids 2023;4:170-90 https://dx.doi.org/10.20517/evcna.2023.12 Page 178

sinusoidal endothelial cells (SECs), which orchestrates with various plasma proteins, such as complement
[121]
systems, to regulate nanoparticle clearance from the blood . In addition, the microarchitecture of the liver
and spleen is designed in a way that enhances the interaction of nanoparticles with the MPS system to
enhance the clearance [121-123] [Figure 3]. As the nanoparticles move from the peripheral circulation to the
liver, the velocity of nanomaterial is reduced from 10-100 cm/sec to 0.2-0.8 cm/sec [124-126] . This reduction in
blood flow by 100-1,000 folds allows nanoparticles to interact with the MPS system in the liver more
efficiently. The blood in the liver enters through portal triads, which are bordered by B and T cells. The
decrease in blood flow allows the uptake of the nanoparticles by both B- and T-cells [122,123] . The nanoparticle,
which escapes the first line of defence, then enters the liver sinusoids. The sinusoids are enriched with SECs
and Kupfer cells, a specialized tissue-associated macrophage [121,123] . These cells collectively recognize various
surface molecules as a trigger for the phagocytosis of nanoparticles. Importantly, this trigger is different for
different nanoparticles ranging from size, shape, core properties, surface composition, and charge . The
[127]
residual population of nanoparticles exits through the central vein into the systemic circulation and is
ultimately carried back again in the liver or to another MPS system such as the spleen or bone marrow .
[122]
This repetitive process primarily governs the nanoparticle clearance by the liver. The same phenomenon
also seems to be responsible for the short half-life and hepatic uptake of EVs. Various studies have shown
that the MPS system is primarily responsible for the clearance of EVs, similar to other nanoparticle-based
drug delivery vectors, as EVs injected in animals with impaired innate immune- and complement systems
have much slower plasma clearance and liver accumulation [87,119] . These results are also supported by various
studies where either blockade of scavenger receptors on macrophages or the sequestration of exposed
phosphatidylserine (PS), one of the primary triggers for macrophage-mediated clearance on the surface of
EVs, prevented rapid clearance and liver uptake [102,128,129] . Notably, a recent study investigated the protein
corona on the surface of EVs in plasma and identified apolipoprotein and complement association with the
EVs . The EV protein corona had a high overlap with viruses and synthetic nanoparticles [130,131] .
[130]
Importantly complement opsonization of the nanoparticle surface enhances phagocytosis through
complement receptor-mediated uptake on macrophages. Interestingly, tumour cell-derived EVs enriched in
surface-bound Factor H, one of the negative regulators of complement opsonization, showed lower
[132]
phagocytic uptake and had higher metastatic potential . This clearly implies that EVs are opsonized by
complement proteins and are taken up by monocytes and macrophages in the liver (Kupffer cells and/or
SECs). Therefore, for enhancing the in vivo biodistribution of EVs to extrahepatic tissues, EV engineering
strategies to augment MPS clearance are an attractive approach.

Extracellular matrix
After penetrating the endothelial barrier, the EV enters the dense and complex environment of the tissue
extracellular matrix. Extracellular matrix (ECM) is a non-cellular component of tissue and possesses a
mesh-like structure through crosslinking of collagen, fibronectin, elastin, proteoglycans (Hyaluronic acid,
decorin, aggrecan, and perlecan, etc.) and glycosaminoglycans . ECM is composed of gel-like components
[133]
and provides structural support for the cells in the tissue. Due to the gel-like properties of ECM, the high
[134]
viscosity limits the fluid flow speed (to 0.1-4 µm/sec) . This significantly impairs the Brownian diffusion
of the nanoparticles across the ECM . In addition, the mesh-like structure of ECM generates steric
[135]
[136]
hindrance for larger nanoparticles such as EVs and partially restricts their diffusion across the tissue .
Apart from steric hindrance, nanoparticles also experience electrostatic repulsions due to highly polar and
negatively charged proteoglycans in ECM . These physical forces impose a significant barrier for any
[137]
nanoparticulate to diffuse into ECM and achieve cellular targeting. Hence, the majority of the delivery by
nano particulates is restricted to the periphery of the tissues as the rigidity of ECM inhibits deep tissue
penetration .
[138]