Effects of receptor properties on particle internalization through receptor-mediated endocytosis.

Receptor-mediated endocytosis (RME) is a highly complex process carried out by bioparticles, such as viruses and drug carriers, to enter cells. The discovery of both clathrin-dependent and clathrin-free pathways makes the RME process even more intriguing. Numerical models have been developed to facilitate the exploration of the process. However, the impacts of the receptor properties on RME have been less studied partially due to the oversimplifications of the receptor models. In this paper, we implement a stochastic model to systematically investigate the effects of mechanical (receptor flexure), geometrical (receptor length) and biochemical (ligand-receptor cutoff) properties of receptors, on RME with and without the existence of clathrin. Our simulation results show that the receptor's flexural rigidity plays an important role in RME with clathrin. There is a threshold beyond which particle internalization will not occur. Without clathrin, it is very difficult to achieve complete endocytosis with ligand-receptor interactions alone. A shorter receptor length and longer ligand-receptor reaction cutoff promote the formation of ligand-receptor bonds and facilitate particle internalization. Complete internalization can only be obtained with an extremely short receptor length and long reaction cutoff. Therefore, there are most likely some additional mechanisms to drive the membrane deformation in clathrin-free RME. Our results yield important fundamental insights into RME and provide crucial guidance when correlating the simulation results with experimental observations.

Effect of Slp4-a on Membrane Bending During Prefusion of Vesicles in Blood-Brain Barrier.

Vesicle exocytosis is a promising pathway for brain drug delivery through the blood-brain barrier to treat neurodegenerative diseases. In vesicle exocytosis, the membrane fusion process is initiated by the calcium sensor protein named synaptotagmin-like protein4-a (Slp4-a). Understanding conformational changes of Slp4-a during the prefusion stage of exocytosis will help to develop vesicle-based drug delivery to the brain. In this work, we use molecular dynamics (MD) simulations with a hybrid force field coupling united-atom protein model with MARTINI coarse-grained (CG) solvent to capture the conformational changes of Slp4-a during the prefusion stage. These hybrid coarse-grained simulations are more efficient than all-atom MD simulations and can capture protein interactions and conformational changes. Our simulation results show that the calcium ions play critical roles during the prefusion stage. Only one calcium ion can remain in each calcium-binding pocket of Slp4-a C2 domains. The C2B domain of calcium-unbound Slp4-a remains parallel to the endothelial membrane, while the C2B domain of calcium-bound Slp4-a rotates perpendicular to the endothelial membrane to approach the vesicular membrane. For the calcium-bound case, three Slp4-a proteins can effectively bend lipid membranes at the prefusion stage, which could later trigger lipid stalk between membranes. This work provides a better understanding how C2 domains of Slp4-a operate during vesicle exocytosis from an endothelial cell.

Electrokinetic actuation of an uncharged polarizable dielectric droplet in charged hydrogel medium.

Electrokinetic transport of an uncharged nonconducting microsized liquid droplet in a charged hydrogel medium is studied. Dielectric polarization of the liquid drop under the action of an externally imposed electric field induces a non-homogeneous charge density at the droplet surface. The interactions of the induced surface charge of the droplet with the immobile charges of the hydrogel medium generates an electric force to the droplet, which actuates the drop through the charged hydrogel medium. A numerical study based on the first principle of electrokinetics is adopted. Dependence of the droplet velocity on its dielectric permittivity, bulk ionic concentration, and immobile charge density of the gel is analyzed. The surface conduction is significant in presence of charged gel, which creates a concentration polarization. The impact of the counterion saturation in the Debye layer due to the dielectric decrement of the medium is addressed. The modified Nernst-Planck equation for ion transport and the Poisson equation for the electric field is considered to take into account the dielectric polarization. A quadrupolar vortex around the uncharged droplet is observed when the gel medium is considered to be uncharged, which is similar to the induced charge electroosmosis around an uncharged dielectric colloid in free-solution. We find that the induced charge electrokinetic mechanism creates a strong recirculation of liquid within the droplet and the translational velocity of the droplet strongly depends on its size for the dielectric droplet embedded in a charged gel medium.

Substrate-dependent transport mechanism in AcrB of multidrug resistant bacteria.

The multidrug resistance (MDR) system effectively expels antibiotics out of bacteria causing serious issues during bacterial infection. In addition to drug, indole, a common metabolic waste of bacteria, is expelled by MDR system of gram-negative bacteria for their survival. Experimental results suggest that AcrB, one of the key components of MDR system, undergoes large scale conformation changes during the pumping due to proton-motive process. However, due to extremely short time scale, it is difficult to observe (experimentally) those changes in the AcrB, which might facilitate the pumping process. Molecular simulations can shed light to understand the conformational changes for transport of indole in AcrB. Examination of conformational changes using all-atom simulation is, however, impractical. Here, we develop a hybrid coarse-grained force field to study the conformational changes of AcrB in presence of indole in the porter domain of monomer II. Using the coarse-grained force field, we investigated the conformational changes of AcrB for a number of model systems considering the effect of protonation in aspartic acid (Asp) residues Asp407 and Asp408 in the transmembrane domain of monomer II. Our results show that in the presence of indole, protonation of Asp408 or Asp407 residue causes conformational changes from binding state to extrusion state in monomer II, while remaining two monomers (I and III) approach access state in AcrB protein. We also observed that all three AcrB monomers prefer to go back to access state in the absence of indole. Steered molecular dynamics simulations were performed to demonstrate the feasibility of indole transport mechanism for protonated systems. Identification of indole transport pathway through AcrB can be very helpful in understanding the drug efflux mechanism used by the MDR bacteria.

Mechanical characterization of vesicles and cells: A review.

Vesicles perform many essential functions in all living organisms. They respond like a transducer to mechanical stress in converting the applied force into mechanical and biological responses. At the same time, both biochemical and biophysical signals influence the vesicular response in bearing mechanical loads. In recent years, liposomes, artificial lipid vesicles, have gained substantial attention from the pharmaceutical industry as a prospective drug carrier which can also serve as an artificial cell-mimetic system. The ability of these vesicles to enter through pores of even smaller size makes them ideal candidates for therapeutic agents to reach the infected sites effectively. Engineering of vesicles with desired mechanical properties that can encapsulate drugs and release as required is the prime challenge in this field. This requirement has led to the modifications of the composition of the bilayer membrane by adding cholesterol, sphingomyelin, etc. In this article, we review the manufacturing and characterization techniques of various artificial/synthetic vesicles. We particularly focus on the electric field-driven characterization techniques to determine different properties of vesicle and its membranes, such as bending rigidity, viscosity, capacitance, conductance, etc., which are indicators of their content and mobility. Similarities and differences between artificial vesicles, natural vesicles, and cells are highlighted throughout the manuscript since most of these artificial vesicles are intended for cell mimetic functions.

Overcoming blood-brain barrier transport: Advances in nanoparticle-based drug delivery strategies.

The Blood-Brain Barrier (BBB), a unique structure in the central nervous system (CNS), protects the brain from bloodborne pathogens by its excellent barrier properties. Nevertheless, this barrier limits therapeutic efficacy and becomes one of the biggest challenges in new drug development for neurodegenerative disease and brain cancer. Recent breakthroughs in nanotechnology have resulted in various nanoparticles (NPs) as drug carriers to cross the BBB by different methods. This review presents the current understanding of advanced NP-mediated non-invasive drug delivery for the treatment of neurological disorders. Herein, the complex compositions and special characteristics of BBB are elucidated exhaustively. Moreover, versatile drug nanocarriers with their recent applications and their pathways on different drug delivery strategies to overcome the formidable BBB obstacle are briefly discussed. In terms of significance, this paper provides a general understanding of how various properties of nanoparticles aid in drug delivery through BBB and usher the development of novel nanotechnology-based nanomaterials for cerebral disease therapies.

Electrophoretic transport and dynamic deformation of bio-vesicles.

Study of the deformation dynamics of cells and other sub-micron vesicles, such as virus and neurotransmitter vesicles are necessary to understand their functional properties. This mechanical characterization can be done by submerging the vesicle in a fluid medium and deforming it with a controlled electric field, which is known as electrodeformation. Electrodeformation of biological and artificial lipid vesicles is directly influenced by the vesicle and surrounding media properties and geometric factors. The problem is compounded when the vesicle is naturally charged, which creates electrophoretic forcing on the vesicle membrane. We studied the electrodeformation and transport of charged vesicles immersed in a fluid media under the influence of a DC electric field. The electric field and fluid-solid interactions are modeled using a hybrid immersed interface-immersed boundary technique. Model results are verified with experimental observations for electric field driven translocation of a virus through a nanopore sensor. Our modeling results show interesting changes in deformation behavior with changing electrical properties of the vesicle and the surrounding media. Vesicle movement due to electrophoresis can also be characterized by the change in local conductivity, which can serve as a potential sensing mechanism for electrodeformation experiments in solid-state nanopore setups.

On electrophoresis of a pH-regulated nanogel with ion partitioning effects.

The electrophoresis of a polyelectrolyte nanoparticle, whose charge condition depends on the salt concentration and pH of the suspended medium as well as the dielectric permittivity difference, is analyzed. The present nonlinear model for the electrophoresis of this pH-regulated polyelectrolyte (PE) particle is based on the consideration of full set of governing equations of fluid and ion transport coupled with the equation for electric field. The Born energy of the ions are incorporated to account for the difference in the dielectric permittivity of the PE and the electrolyte. The governing equations are computed numerically through a control volume approach. The nonlinear effects are highlighted by comparing with the existing linear model as well as results based on the first-order perturbation analysis valid for a weak applied field. The ion partitioning effect arising due to the difference in self energy of ions between the two media, have a strong impact on the mobility of the PE. The ion partitioning effect attenuates the penetration of counterions in the PE, which enhances the electric force and hence, results in a larger mobility of the PE. The nonlinear effects due to the double layer polarization and relaxation are intensified due to the ion partitioning effect. The ion partitioning effect influences the association/dissociation of PE functional group by tuning the hydrogen/hydroxide ions. Present study shows that the ion partitioning effect is profound for higher salt concentration and/or higher volume density of PE functional groups.

Mechanical characterization of HIV-1 with a solid-state nanopore sensor.

Enveloped viruses fuse with cells to transfer their genetic materials and infect the host cell. Fusion requires deformation of both viral and cellular membranes. Since the rigidity of viral membrane is a key factor in their infectivity, studying the rigidity of viral particles is of great significance in understating viral infection. In this paper, a nanopore is used as a single molecule sensor to characterize the deformation of pseudo-type human immunodeficiency virus type 1 at sub-micron scale. Non-infective immature viruses were found to be more rigid than infective mature viruses. In addition, the effects of cholesterol and membrane proteins on the mechanical properties of mature viruses were investigated by chemically modifying the membranes. Furthermore, the deformability of single virus particles was analyzed through a recapturing technique, where the same virus was analyzed twice. The findings demonstrate the ability of nanopore resistive pulse sensing to characterize the deformation of a single virus as opposed to average ensemble measurements.

Entry modes of ellipsoidal nanoparticles on a membrane during clathrin-mediated endocytosis.

The membrane wrapping and internalization of nanoparticles, such as viruses and drug nanocarriers, through clathrin-mediated endocytosis (CME) are vitally important for intracellular transport. During CME, the shape of the particle plays crucial roles in the determination of particle-membrane interactions, but much of the previous work has been focused on spherical particles. In this work, we develop a stochastic model to study the CME of ellipsoidal particles. In our model, the deformation of the membrane and wrapping of the nanoparticles are driven by the accumulation of clathrin lattices, which is stimulated by the ligand-receptor interactions. Using our model, we systematically investigate the effect of particle shape (ellipsoids with different aspect ratios) on the CME. Our results show three entry modes: tip-first, tilted, and laying-down modes, used by ellipsoidal nanoparticles for internalization depending on the aspect ratio. Certain ellipsoids are able to take multiple entry modes for internalization. Interestingly, the prolate ellipsoid with an aspect ratio of 0.42 can be internalized with a significantly reduced number of ligand-receptor bonds. Particles which can be internalized with fewer bonds are excellent candidates for transcellular drug delivery. Moreover, our results demonstrate that internalization of ellipsoids with intermediate aspect ratios is easier than that of particles with low and high aspect ratios. Our model and simulations provide critical mechanistic insights into CME of ellipsoidal particles, and represent a viable platform for optimal design of nanoparticles for targeted drug delivery applications.

Review: Electric field driven pumping in microfluidic device.

Pumping of fluids with precise control is one of the key components in a microfluidic device. The electric field has been used as one of the most popular and efficient nonmechanical pumping mechanism to transport fluids in microchannels from the very early stage of microfluidic technology development. This review presents fundamental physics and theories of the different microscale phenomena that arise due to the application of an electric field in fluids, which can be applied for pumping of fluids in microdevices. Specific mechanisms considered in this report are electroosmosis, AC electroosmosis, AC electrothermal, induced charge electroosmosis, traveling wave dielectrophoresis, and liquid dielectrophoresis. Each phenomenon is discussed systematically with theoretical rigor and role of relevant key parameters are identified for pumping in microdevices. We specifically discussed the electric field driven body force term for each phenomenon using generalized Maxwell stress tensor as well as simplified effective dipole moment based method. Both experimental and theoretical works by several researchers are highlighted in this article for each electric field driven pumping mechanism. The detailed understanding of these phenomena and relevant key parameters are critical for better utilization, modulation, and selection of appropriate phenomenon for efficient pumping in a specific microfluidic application.

Stochastic simulations of nanoparticle internalization through transferrin receptor dependent clathrin-mediated endocytosis.

BACKGROUND: Receptor dependent clathrin-mediated endocytosis (CME) is one of the most important endocytic pathways for the internalization of bioparticles into cells. During CME, the ligand-receptor interactions, development of clathrin-coated pit (CCP) and membrane evolution all act together to drive the internalization of bioparticles. In this work, we develop a stochastic computational model to investigate the CME based on the Metropolis Monte Carlo simulations. METHODS: The model is based on the combination of a stochastic particle binding model with a membrane model. The energetic costs of membrane bending, CCP formation and ligand-receptor interactions are systematically linked together. RESULTS: We implement our model to investigate the effects of particle size, ligand density and membrane stiffness on the overall process of CME from the drug delivery perspectives. Consistent with some experiments, our results show that the intermediate particle size and ligand density favor the particle internalization. Moreover, our results show that it is easier for a particle to enter a cell with softer membrane. CONCLUSIONS: The model presented here is able to provide mechanistic insights into CME and can be readily modified to include other important factors, such as actins. The predictions from the model will aid in the therapeutic design of intracellular/transcellular drug delivery and antiviral interventions.

Iron transport kinetics through blood-brain barrier endothelial cells.

BACKGROUND: Transferrin and its receptors play an important role during the uptake and transcytosis of iron through blood-brain barrier (BBB) endothelial cells (ECs) to maintain iron homeostasis in BBB endothelium and brain. Any disruptions in the cell environment may change the distribution of transferrin receptors on the cell surface, which eventually alter the homeostasis and initiate neurodegenerative disorders. In this paper, we developed a comprehensive mathematical model that considers the necessary kinetics for holo-transferrin internalization and acidification, apo-transferrin recycling, and exocytosis of free iron and transferrin-bound iron through basolateral side of BBB ECs. METHODS: Ordinary differential equations are formulated based on the first order reaction kinetics to model the iron transport considering their interactions with transferrin and transferrin receptors. Unknown kinetics rate constants are determined from experimental data by applying a non-linear optimization technique. RESULTS: Using the estimated kinetic rate constants, the presented model can effectively reproduce the experimental data of iron transports through BBB ECs for many in-vitro studies. Model results also suggest that the BBB ECs can regulate the extent of the two possible iron transport pathways (free and transferrin-bound iron) by controlling the receptor expression, internalization of holo-transferrin-receptor complexes and acidification of holo-transferrin inside the cell endosomes. CONCLUSION: The comprehensive mathematical model described here can predict the iron transport through BBB ECs considering various possible routes from blood side to brain side. The model can also predict the transferrin and iron transport behavior in iron-enriched and iron-depleted cells, which has not been addressed in previous work.

Mathematical Model for Tissue-Level Hypoxic Response in Microfluidic Environment.

Availability of essential species like oxygen is critical in shaping the dynamics of tumor growth. When the intracellular oxygen level falls below normal, it initiates major cascades in cellular dynamics leading to tumor cell survival. In a cellular block with cells growing away from the blood vessel, the scenario can be aggravated for the cells further inside the block. In this study, the dynamics of intracellular species inside a colony of tumor cells are investigated by varying the cell-block thickness and cell types in a microfluidic cell culture device. The oxygen transport across the cell block is modeled through diffusion, while ascorbate (AS) transport from the extracellular medium is addressed by a concentration-dependent uptake model. The extracellular and intracellular descriptions were coupled through the consumption and traffic of species from the microchannel to the cell block. Our model shows that the onset of hypoxia is possible in HeLa cell within minutes depending on the cell location, although the nutrient supply inside the channel is maintained in normoxic levels. This eventually leads to total oxygen deprivation inside the cell block in the extreme case, representing the development of a necrotic core that maintains a dynamic balance with growing cells and scarce supply. The numerical model reveals that species concentration and hypoxic response are different for HeLa and HelaS3 cells. Results also indicate that the long-term hypoxic response from a microfluidic cellular block stays within 5% of the values of a tissue with the basal layer. The hybrid model can be very useful in designing microfluidic experiments to satisfactorily predict the tissue-level response in cancer research.

Exploration of conformational changes in lactose permease upon sugar binding and proton transfer through coarse-grained simulations.

Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, and large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the binding of a ß-d-galactopyranosyl-1-thio- ß-d-galactopyranoside (TDG) molecule to a wild-type LacY. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Transitions from inward-facing to outward-facing conformations upon TDG binding and protonation of Glu269 have been achieved from ∼5.5 µs simulations. Both the opening of the periplasmic side and closure of the cytoplasmic side of LacY are consistent with double electron-electron resonance and thiol cross-linking experiments. Our analysis suggests that the conformational changes of LacY are a cumulative consequence of interdomain H-bonds breaking at the periplasmic side, interdomain salt-bridge formation at the cytoplasmic side, and the TDG orientational changes during the transition.

Steady-state protein focusing in carrier ampholyte based isoelectric focusing: Part I-Analytical solution.

The determination of an analytical solution to find the steady-state protein concentration distribution in IEF is very challenging due to the nonlinear coupling between mass and charge conservation equations. In this study, approximate analytical solutions are obtained for steady-state protein distribution in carrier ampholyte based IEF. Similar to the work of Svensson, the final concentration profile for proteins is assumed to be Gaussian, but appropriate expressions are presented in order to obtain the effective electric field and pH gradient in the focused protein band region. Analytical results are found from iterative solutions of a system of coupled algebraic equations using only several iterations for IEF separation of three plasma proteins: albumin, cardiac troponin I, and hemoglobin. The analytical results are compared with numerically predicted results for IEF, showing excellent agreement. Analytically obtained electric field and ionic conductivity distributions show significant deviation from their nominal values, which is essential in finding the protein focusing behavior at isoelectric points. These analytical solutions can be used to determine steady-state protein concentration distribution for experiment design of IEF considering any number of proteins and ampholytes. Moreover, the model presented herein can be used to find the conductivity, electric field, and pH field.

Steady-state protein focusing in carrier ampholyte-based isoelectric focusing: Part II-validation and case studies.

In this study, we systematically investigate the validity and applicability of an analytical model developed for carrier ampholyte-based isoelectric focusing (IEF). Three different IEF cases are considered in order to evaluate the efficacy of the approximate analytical results by comparison with high-resolution computer simulations. In the first case, three proteins are separated in a narrow pH range (6-9) by using 50 carrier ampholytes. In the second and third cases, the separation of proteins is studied in broad pH range (3-10) IEF by using 100 carrier ampholytes. Results obtained from the approximate analytical models are in very good agreement with the numerical results for IEF separation of cardiac troponin I, albumin, and hemoglobin in both narrow and broad pH ranges. The sensitivity of the analytical model is also tested for different initial mass ratios of proteins to ampholytes. No appreciable differences are observed between the approximate analytical and numerical results within the mass ratio range studied. The effect of a nominal electric field and/or a nominal pH gradient on protein focusing is also examined to demonstrate the effectiveness of the analytical model. Our results indicate that the use of both nominal electric field and pH gradient will result in erroneous peak concentrations for proteins. Finally, we describe the limitations of the approximate analytical solutions.