Changjin Huang

Role of nanoparticle geometry in endocytosis: laying down to stand up.

Nanoparticles (NPs) hold great promises for targeted disease diagnosis and therapy. Despite considerable progress in biomimetic design of NP-bioconjugates, the roles of NP size and shape in endocytosis are still not fully understood. Using an efficient coarse-grained molecular dynamics (CGMD) model, we simulate receptor-mediated endocytosis of NPs of various sizes and shapes. Our simulations demonstrate that both NP size and shape modulate the kinetics of endocytosis. For spherical NPs, there exists an optimal size at which endocytosis takes the shortest time. For a spherocylindrical NP with the initial upright docking position on the membrane plane, endocytosis proceeds through a laying-down-then-standing-up sequence. A free energy analysis reveals that NP size primarily determines whether endocytosis can complete, while NP shape breaks the symmetry of curvature energy landscape and hence dictates the endocytic pathway and the angle of entry. The findings shed light on the rational design of NP-based diagnostic and therapeutic agents with improved cellular targeting.

Multiple stiffening effects of nanoscale knobs on human red blood cells infected with Plasmodium falciparum malaria parasite

Significance Our coarse-grained molecular dynamics (CGMD) simulations show that the deposition of nanoscale knobs, rather than spectrin network remodeling, is the primary cause of the dramatically increased stiffness of the Plasmodium falciparum (Pf)-infected red blood cell (RBC) membranes. Our analyses further reveal that the knobs stiffen the RBC membrane in a unique manner by simultaneously harnessing composite strengthening, strain hardening, and knob density-dependent vertical coupling effects. In addition to providing a fundamental understanding of the stiffening mechanism of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies. During its asexual development within the red blood cell (RBC), Plasmodium falciparum (Pf), the most virulent human malaria parasite, exports proteins that modify the host RBC membrane. The attendant increase in cell stiffness and cytoadherence leads to sequestration of infected RBCs in microvasculature, which enables the parasite to evade the spleen, and leads to organ dysfunction in severe cases of malaria. Despite progress in understanding malaria pathogenesis, the molecular mechanisms responsible for the dramatic loss of deformability of Pf-infected RBCs have remained elusive. By recourse to a coarse-grained (CG) model that captures the molecular structures of Pf-infected RBC membrane, here we show that nanoscale surface protrusions, known as “knobs,” introduce multiple stiffening mechanisms through composite strengthening, strain hardening, and knob density-dependent vertical coupling. On one hand, the knobs act as structural strengtheners for the spectrin network; on the other, the presence of knobs results in strain inhomogeneity in the spectrin network with elevated shear strain in the knob-free regions, which, given its strain-hardening property, effectively stiffens the network. From the trophozoite to the schizont stage that ensues within 24–48 h of parasite invasion into the RBC, the rise in the knob density results in the increased number of vertical constraints between the spectrin network and the lipid bilayer, which further stiffens the membrane. The shear moduli of Pf-infected RBCs predicted by the CG model at different stages of parasite maturation are in agreement with experimental results. In addition to providing a fundamental understanding of the stiffening mechanisms of Pf-infected RBCs, our simulation results suggest potential targets for antimalarial therapies.

Substrate Stiffness Regulates Cellular Uptake of Nanoparticles

Nanoparticle (NP)-bioconjugates hold great promise for more sensitive disease diagnosis and more effective anticancer drug delivery compared with existing approaches. A critical aspect in both applications is cellular internalization of NPs, which is influenced by NP properties and cell surface mechanics. Despite considerable progress in optimization of the NP-bioconjugates for improved targeting, the role of substrate stiffness on cellular uptake has not been investigated. Using polyacrylamide (PA) hydrogels as model substrates with tunable stiffness, we quantified the relationship between substrate stiffness and cellular uptake of fluorescent NPs by bovine aortic endothelial cells (BAECs). We found that a stiffer substrate results in a higher total cellular uptake on a per cell basis, but a lower uptake per unit membrane area. To obtain a mechanistic understanding of the cellular uptake behavior, we developed a thermodynamic model that predicts that membrane spreading area and cell membrane tension are two key factors controlling cellular uptake of NPs, both of which are modulated by substrate stiffness. Our experimental and modeling results not only open up new avenues for engineering NP-based cancer cell targets for more effective in vivo delivery but also contribute an example of how the physical environment dictates cellular behavior and function.

Cooperative Transmembrane Penetration of Nanoparticles

Physical penetration of lipid bilayer membranes presents an alternative pathway for cellular delivery of nanoparticles (NPs) besides endocytosis. NPs delivered through this pathway could reach the cytoplasm, thereby opening the possibility of organelle-specific targeting. Herein we perform dissipative particle dynamics simulations to elucidate the transmembrane penetration mechanisms of multiple NPs. Our simulations demonstrate that NPs’ translocation proceeds in a cooperative manner, where the interplay of the quantity and surface chemistry of the NPs regulates the translocation efficiency. For NPs with hydrophilic surfaces, the increase of particle quantity facilitates penetration, while for NPs with partly or totally hydrophobic surfaces, the opposite highly possibly holds. Moreover, a set of interesting cooperative ways, such as aggregation, aggregation-dispersion, and aggregation-dispersion-reaggregation of the NPs, are observed during the penetration process. We find that the penetration behaviors of multiple NPs are mostly dominated by the changes of the NP-membrane force components in the membrane plane direction, in addition to that in the penetration direction, suggesting a different interaction mechanism between the multiple NPs and the membrane compared with the one-NP case. These results provide a fundamental understanding in the underlying mechanisms of cooperative penetration of NPs, and shed light on the NP-based drug and gene delivery.

Virus-inspired design principles of nanoparticle-based bioagents.

The highly effectiveness and robustness of receptor-mediated viral invasion of living cells shed lights on the biomimetic design of nanoparticle(NP)-based therapeutics. Through thermodynamic analysis, we elucidate that the mechanisms governing both the endocytic time of a single NP and the cellular uptake can be unified into a general energy-balance framework of NP-membrane adhesion and membrane deformation. Yet the NP-membrane adhesion strength is a globally variable quantity that effectively regulates the NP uptake rate. Our analysis shows that the uptake rate interrelatedly depends on the particle size and ligand density, in contrast to the widely reported size effect. Our model predicts that the optimal radius of NPs for maximal uptake rate falls in the range of 25–30 nm, and optimally several tens of ligands should be coated onto NPs. These findings are supported by both recent experiments and typical viral structures, and serve as fundamental principles for the rational design of NP-based nanomedicine.

Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages

Significance This study provides, to our knowledge, the first ultrastructural and dynamics analysis of the host red blood cell membrane of Plasmodium falciparum gametocytes, revealing reversible expansion of the spectrin–actin skeleton, accompanied by reversible modulation of skeletal coupling to the plasma membrane. We use the measured physical parameters to inform a computationally efficient coarse-grained model. This model shows that restructuring the skeletal meshwork can fully account for the observed deformability changes. We reveal a critical role for actin remodeling in driving this reversible biomechanical host cell subversion. This work provides fundamental insights into the molecular changes that underpin gametocyte survival in the circulation. The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer’s clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.

The role of substrate topography on the cellular uptake of nanoparticles.

Improving targeting efficacy has been a central focus of the studies on nanoparticle (NP)-based drug delivery nanocarriers over the past decades. As cells actively sense and respond to the local physical environments, not only the NP design (e.g., size, shape, ligand density, etc.) but also the cell mechanics (e.g., stiffness, spreading, expressed receptors, etc.) affect the cellular uptake efficiency. While much work has been done to elucidate the roles of NP design for cells seeded on a flat tissue culture surface, how the local physical environments of cells mediate uptake of NPs remains unexplored, despite the widely known effect of local physical environments on cellular responses in vitro and disease states in vivo. Here, we report the active responses of human osteosarcoma cells to fibrous substrate topographies and the subsequent changes in the cellular uptake of NPs. Our experiments demonstrate that surface topography modulates cellular uptake efficacy by mediating cell spreading and membrane mechanics. The findings provide a concrete example of the regulative role of the physical environments of cells on cellular uptake of NPs, therefore advancing the rational design of NPs for enhanced drug delivery in targeted cancer therapy.

Dynamic shape transformations of fluid vesicles

We incorporate a volume-control algorithm into a recently developed one-particle-thick mesoscopic fluid membrane model to study vesicle shape transformation under osmotic conditions. Each coarse-grained particle in the model represents a cluster of lipid molecules and the inter-particle interaction potential effectively captures the dual character of fluid membranes as elastic shells with out-of-plane bending rigidity and 2D viscous fluids with in-plane viscosity. The osmotic pressure across the membrane is accounted for by an external potential, where the instantaneous volume of the vesicles is calculated via a local triangulation algorithm. Through coarse-grained molecular dynamics simulations, we mapped out a phase diagram of the equilibrium vesicle shapes in the space of spontaneous curvature and reduced vesicle volume. The produced equilibrium vesicle shapes agree strikingly well with previous experimental data. We further found that the vesicle shape transformation pathways depend on the volume change rate of the vesicle, which manifests the role of dynamic relaxation of internal stresses in vesicle shape transformations. Besides providing an efficient numerical tool for the study of membrane deformations, our simulations shed light on eliciting desired cellular functions via experimental control of membrane configurations.

Coupled vesicle morphogenesis and domain organization

Protein-enriched membrane domains with distinct biophysical properties have been thought as the basic organizing units of cellular membrane. Using particle dynamics simulations, here we study phase separation dynamics and domain formation in binary vesicles. Our simulations clearly show the intimate coupling between domain compositions, lateral domain organization, and vesicle shape transformation. Our simulation results provide insights into the essential role of membrane domains in the coordinated remodeling of cellular membranes and in protein and lipid sorting.

Formation and size distribution of self-assembled vesicles

Significance Small unilamellar vesicles formed via self-assembly of phospholipids or block copolymers have been investigated in the context of human physiology and biomedical research. Here, we present both energetics and thermodynamics analyses that incorporate nonlinear elasticity to predict, in a unique manner, the limiting size and size distribution of vesicles as well as to identify the conditions for the formation of stable open vesicles, disks, and closed spherical vesicles. In addition to providing a comprehensive understanding of vesicle formation, the framework presented here may be adapted to develop tools for in vivo liquid biopsies and to elucidate the biophysical features of extracellular vesicles, thus suggesting new approaches to diagnostics and therapeutics for cancer and other diseases. When detergents and phospholipid membranes are dispersed in aqueous solutions, they tend to self-assemble into vesicles of various shapes and sizes by virtue of their hydrophobic and hydrophilic segments. A clearer understanding of such vesiculation processes holds promise for better elucidation of human physiology and disease, and paves the way to improved diagnostics, drug development, and drug delivery. Here we present a detailed analysis of the energetics and thermodynamics of vesiculation by recourse to nonlinear elasticity, taking into account large deformation that may arise during the vesiculation process. The effects of membrane size, spontaneous curvature, and membrane stiffness on vesiculation and vesicle size distribution were investigated, and the critical size for vesicle formation was determined and found to compare favorably with available experimental evidence. Our analysis also showed that the critical membrane size for spontaneous vesiculation was correlated with membrane thickness, and further illustrated how the combined effects of membrane thickness and physical properties influenced the size, shape, and distribution of vesicles. These findings shed light on the formation of physiological extracellular vesicles, such as exosomes. The findings also suggest pathways for manipulating the size, shape, distribution, and physical properties of synthetic vesicles, with potential applications in vesicle physiology, the pathobiology of cancer and other diseases, diagnostics using in vivo liquid biopsy, and drug delivery methods.