Poroelastic Attributes: A Real-time Determinant Membrane Nanorheology for Receptor Dependent Endocytosis of Targeted Gold Nanoparticles

BackgroundEfficacy of targeted drug delivery using nanoparticles relies on several factors including the uptake mechanisms such as phagocytosis, macropinocytosis, micropinocytosis and receptor mediated endocytosis. These mechanisms have been studied with respect to the alteration in signaling mechanisms, cellular morphology, and linear nanomechanical properties (NMPs). Commonly employed classical contact mechanics models to address cellular NMPs fail to address mesh like structure consisting of bilayer lipids and proteins of cell membrane. To overcome this technical challenge, we employed poroelastic model which accounts for the biphasic nature of cells including their porous behavior exhibiting both solid like (fluid storage) and liquid like (fluid dissipate) behavior ResultsIn this study, we employed atomic force microscopy to monitor the influence of surface engineering of gold nanoparticles (GNPs) to the alteration of nonlinear NMPs such as drained Poisson’s ratio, effective shear stress, diffusion constant and pore dimensions of cell membranes during their uptake. Herein, we used pancreatic cancer (PDAC) cell lines including Panc1, AsPC-1 and endothelial cell HUVECs to understand the receptor-dependent and -independent endocytosis of two different GNPs derived using plectin-1 targeting peptide (PTP-GNP) and corresponding scrambled peptide (sPEP-GNP). Compared to untreated cells, in case of receptor dependent endocytosis of PTP-GNPs diffusion coefficient altered ~1264-fold and ~1530-fold and pore size altered ~320-fold and ~260-fold in Panc1 and AsPC-1 cells respectively. Whereas for receptor independent mechanisms, we observed modest alteration in diffusion coefficient and pore size, in these cells compared to untreated cells. Effective shear stress corresponding to 7.38±0.15 kPa and 20.49±0.39 kPa in PTP-GNP treatment in Panc1 and AsPC-1, respectively was significantly more than that for sPEP-GNP. These results demonstrate that with temporal recruitment of plectin-1 during receptor mediated endocytosis affects the poroelastic attributes of the membrane. ConclusionThis study confirms that nonlinear NMPs of cell membrane are directly associated with the uptake mechanism of nanoparticles and can provide promising insights of the nature of endocytosis mechanism involved for organ specific drug delivery using nanoparticles. Hence, nanomechanical analysis of cell membrane es using this noninvasive, label-free and live-cell analytical tool can therefore be instrumental to evaluate therapeutic benefit of nanoformulations.

Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes

The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of dAmnionless and two cubilin paralogues Cubilin and Cubilin-2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Noteworthy, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD-positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes argue for a possible unexpected conserved role of this endocytic receptor in vertebrate SD remodelling.

Morphological control of receptor-mediated endocytosis

Receptor-mediated endocytosis is the primary process for nanoparticle uptake in cells and one of the main entry mechanisms for viral infection. The cell membrane adheres to the particle (nanoparticle or virus) and then wraps it to form a vesicle delivered to the cytosol. Previous findings identified a minimum radius for a spherical particle below which endocytosis cannot occur. This is due to the insufficient driving force, from receptor-ligand affinity, to overcome the energy barrier created by membrane bending. In this paper, we extend this result to the case of clathrin-mediated endocytosis, which is the most common pathway for virus entry. Moreover, we investigate the effect of ligand inhibitors on the particle surface, motivated by viral an- tibodies, peptides or phage capsids nanoparticles. We determine the necessary conditions for endocytosis by considering the additional energy barrier due to the membrane bending to wrap such inhibiting protrusions. We find that the density and size of inhibitors determine the size range of internalized particles, and endo- cytosis is completely blocked above critical thresholds. The assembly of a clathrin coat with a spontaneous curvature increases the energy barrier and sets a maximum particle size (in agreement with experimental observations on smooth particles). Our investigation suggests that morphological considerations can inform the optimal design of neutralizing viral antibodies and new strategies for targeted nanomedicine.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid–base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

Cubam receptor-mediated endocytosis in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni

Nutrient transfer from mother to the embryo is essential for reproduction in viviparous animals. In the viviparous teleost Xenotoca eiseni belonging to the family Goodeidae, the intraovarian embryo intakes the maternal component secreted into the ovarian fluid via the trophotaenia. Our previous study reported that the epithelial layer cells of the trophotaenia incorporate a maternal protein via vesicle trafficking. However, the molecules responsible for the absorption were still elusive. Here, we focused on Cubam (Cubilin-Amnionless) as a receptor involved in the absorption, and cathepsin L as a functional protease in the vesicles. Our results indicated that the Cubam receptor is distributed in the apical surface of the trophotaenia epithelium and then is taken into the intracellular vesicles. The trophotaenia possesses acidic organelles in epithelial layer cells and cathepsin L-dependent proteolysis activity. This evidence does not conflict with our hypothesis that receptor-mediated endocytosis and proteolysis play roles in maternal macromolecule absorption via the trohotaenia in viviparous teleosts. Such nutrient absorption involving endocytosis is not a specific trait in viviparous fish. Similar processes have been reported in the larval stage of oviparous fish or the suckling stage of viviparous mammals. Our findings suggest that the viviparous teleost acquired trophotaenia-based viviparity from a modification of the intestinal absorption system common in vertebrates. This is a fundamental study to understand the strategic variation of the reproductive system in vertebrates.

Interplay of Nanoparticle Properties during Endocytosis

Nanoparticles (NPs) have been widely applied as drug carriers in drug delivery, due to their unique physical and structural properties. To achieve the drug delivery purpose, receptor-mediated endocytosis is a primary explored mechanism to internalize NPs into tumor cells. During the endocytosis process, properties of NPs, including size, shape, and surface functionality, play an important role in determining the final drug delivery efficacy. Many of these NP properties have been extensively explored individually. However, the multiple NP properties naturally interplay with each other in the endocytosis process to determine the internalization efficiency together. Therefore, it is significantly important to understand the interplay of different NP properties to improve the NP’s final delivery efficacy. In this review, we focus on the interplay of NPs properties on the endocytosis process to summarize the relevant experimental observations and physical mechanisms. Particularly, three different aspects are discussed in detail, including the interplay between size and shape; size and elasticity; shape and elasticity. We have summarized the most recent works and highlighted that building up systematic understandings for the complex interplay between NP properties can greatly help a better design of NP platforms for drug delivery.