A Translocation Pathway for Vesicle-Mediated Unconventional Protein Secretion.

Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.

Membrane Domain Anti-Registration Induces an Intrinsic Transmembrane Potential.

Plasma membrane segregation into various nanoscale membrane domains is driven by distinct interactions between diverse lipids and proteins. Among them, liquid-ordered (Lo) membrane domains are defined as "lipid rafts" and liquid-disordered (Ld) ones as "lipid non-rafts". Using model membrane systems, both intra-leaflet and inter-leaflet dynamics of these membrane domains are widely studied. Nevertheless, the biological impact of the latter, which is accompanied by membrane domain registration/anti-registration, is far from clear. Hence, in this work, we studied the biological relevance of the membrane domain anti-registration using both all-atom molecular dynamics (MD) simulations and confocal fluorescence microscopy. All-atom MD simulations suggested an intrinsic transmembrane potential for the case of the membrane anti-registration (Lo/Ld). Meanwhile, confocal fluorescence microscopy experiments of HeLa and 293T cell lines indicated that membrane cholesterol depletion could significantly alter the transmembrane potential of cells. Considering differences in the cholesterol content between Lo and Ld membrane domains, our confocal fluorescence microscopy experiments are consistent with our all-atom MD simulations. In short, membrane domain anti-registration induces local membrane asymmetry and, thus, an intrinsic transmembrane potential.

Molecular View on the Impact of DHA Molecules on the Physical Properties of a Model Cell Membrane.

Docosahexaenoic acid (DHA) is a ω-3 polyunsaturated fatty acid, which can be uptaken by cells and is essential for proper neuronal and retinal function. However, the detailed physical impact of DHA molecules on the plasma membrane is still unclear. Hence, in this work, we carried out µs-scale coarse-grained molecular dynamics (MD) simulations to reveal the interactions between DHA molecules and a model cell membrane. As is known, the cell membrane can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) membrane domains due to the differential interactions between lipids and proteins. In order to capture this feature, we adopted the three-component phase-separated lipid membranes and considered both anionic and neutral DHA molecules in the current work. Our results showed that DHA molecules can spontaneously self-assemble into nanoclusters, fuse with lipid membranes, and localize preferably in Ld membrane domains. During the membrane fusion process, DHA molecules can change the intrinsic transmembrane potential of the lipid membrane, and the effects of anionic DHA molecules are much more significant. Besides, the presence of DHA molecules mainly in the Ld membrane domains could regulate the differences in the lipid chain order, membrane thickness, cholesterol preference, and cholesterol flip-flop basically in a concentration-dependent manner, which further promote the stability of the intraleaflet dynamics and inhibit the interleaflet dynamics (or promote membrane domain registration) of the membrane domains. In short, the impact of DHA molecules on the physical properties of a model cell membrane on the molecular level revealed in our work will provide useful insights for understanding the biological functions of DHA molecules.

Evaluation of Interactions between SARS-CoV-2 RBD and Full-Length ACE2 with Coarse-Grained Molecular Dynamics Simulations.

Compared to all-atom models, coarse-grained models enable the investigation of the dynamics of simulation systems on a much larger length scale and a longer time scale, which makes them suitable for studying macromolecular systems. Hence, in this work, we performed multiple µs-scale Martini coarse-grained molecular dynamics simulations to reveal the interaction details between SARS-CoV-2 RBD and full-length human ACE2. Besides, the key coarse-grained systems were backmapped into the corresponding all-atom system for the display of structural details. Our results indicated that the plier structure in two ends of the binding interface plays a key role in the binding process of SARS-CoV-2 RBD with ACE2. Furthermore, we also found that when there is no B0AT1 in the simulation system, the N-terminus of ACE2 is more likely to approach the cell membrane, which has a strong correlation with the subsequent fusion of the virus with the cell membrane. These binding details of SARS-CoV-2 RBD and the ACE2 protease domain (PD) as well as the membrane orientation thermodynamics can promote the development of therapeutic drugs and preventive vaccines against SARS-CoV-2.

Single-Molecule Study of Peptides with the Same Amino Acid Composition but Different Sequences by Using an Aerolysin Nanopore.

Nanopores are original sensors employed for highly sensitive peptides/proteins detection. Herein, we describe the use of an aerolysin nanopore to identify two similar model peptides, YEQYEQQDDDRQQQ (YEQ2Q3) and QDDDRQQQYEQYEQ (Q3YEQ2), with the same amino acid composition but different sequences. All-atom molecular dynamics (MD) simulations reveal that YEQ2Q3 possesses fewer hydrogen bonds and a more extended conformation than Q3YEQ2. These two peptides, which fold differently, exhibit obviously distinct mass-independent current blockades with characteristic dwell times when entering the aerolysin nanopore. Typically, at +60 mV, the statistical dwell time of 0.630±0.018 ms for peptide Q3YEQ2 is four times longer than the value of 0.160±0.001 ms for peptide YEQ2Q3, and yet peptide YEQ2Q3 induces ∼1.9 % larger blockade current amplitude than peptide Q3YEQ2. The obtained results show the remarkable potential of aerolysin nanopore for peptides/proteins identification, characterization, sequencing and also demonstrate that the mass identification of nonuniformly charged peptides/proteins by using the nanopore technique could be complicated by their folded structure and complex analyte-pore interaction.

Transmembrane potential of physiologically relevant model membranes: Effects of membrane asymmetry.

Transmembrane potential difference (Vm) plays important roles in regulating various biological processes. At the macro level, Vm can be experimentally measured or calculated using the Nernst or Goldman-Hodgkin-Katz equation. However, the atomic details responsible for its generation and impact on protein and lipid dynamics still need to be further elucidated. In this work, we performed a series of all-atom molecular dynamics (MD) simulations of symmetric model membranes of various lipid compositions and cation contents to evaluate the relationship between membrane asymmetry and Vm. Specifically, we studied the impact of the asymmetric distribution of POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine), PIP2 (phosphatidylinositol 4,5-bisphosphate), as well as Na+ and K+ on Vm using atomically detailed MD simulations of symmetric model membranes. The results suggest that, for an asymmetric POPC-POPC/POPS bilayer in the presence of NaCl, the presence of the monovalent anionic lipid POPS in the inner leaflet polarizes the membrane (ΔVm < 0). Intriguingly, replacing a third of the POPS lipids by the polyvalent anionic signaling lipid PIP2 counteracts this effect, resulting in a smaller negative membrane potential. We also found that replacing Na+ ions in the inner region by K+ depolarizes the membrane (ΔVm > 0). These divergent effects arise from variations in the strength of cation-lipid interactions and are correlated with changes in lipid chain order and head-group orientation.

A Review on Applications of Computational Methods in Drug Screening and Design.

Drug development is one of the most significant processes in the pharmaceutical industry. Various computational methods have dramatically reduced the time and cost of drug discovery. In this review, we firstly discussed roles of multiscale biomolecular simulations in identifying drug binding sites on the target macromolecule and elucidating drug action mechanisms. Then, virtual screening methods (e.g., molecular docking, pharmacophore modeling, and QSAR) as well as structure- and ligand-based classical/de novo drug design were introduced and discussed. Last, we explored the development of machine learning methods and their applications in aforementioned computational methods to speed up the drug discovery process. Also, several application examples of combining various methods was discussed. A combination of different methods to jointly solve the tough problem at different scales and dimensions will be an inevitable trend in drug screening and design.

Lipid Acyl Chain cis Double Bond Position Modulates Membrane Domain Registration/Anti-Registration.

Inter-leaflet coupling is critical to control the dynamics of membrane domain registration/anti-registration, which is important in maintaining proper biological functions. Factors such as lipid acyl chain inter-digitation and membrane remodeling have been found to be able to regulate the inter-leaflet coupling. However, detailed molecular mechanisms that dominate the inter-leaflet coupling are still far from clear. Here, we revealed that lipid acyl chain cis double bond position can regulate the inter-leaflet coupling according to our coarse-grained and all-atom molecular dynamics simulations. The farther the double bond is away from the lipid tail terminal, the weaker the inter-leaflet attractive interactions between unsaturated lipids. Therefore, the relative motions of membrane domains in two membrane leaflets become more obvious (membrane domain anti-registration). Generally, our simulations validated a direct indicator for the inter-leaflet coupling strength, which provides physical insights into the molecular mechanisms of membrane domain registration/anti-registration.

Poly(amidoamine) Dendrimer as a Respiratory Nanocarrier: Insights from Experiments and Molecular Dynamics Simulations.

Pulmonary drug delivery is superior to the systemic administration in treating lung diseases. An optimal respiratory nanocarrier should be able to efficiently and safely cross the pulmonary surfactant film, which serves as the first biological barrier for respiratory delivery and plays paramount roles in maintaining the proper mechanics of breathing. In this work, we focused on the interactions between poly(amidoamine) (PAMAM) dendrimers and a model pulmonary surfactant. With combined Langmuir monolayer experiments and coarse-grained molecular dynamics simulations, we studied the effect of environmental temperature, size, and surface property of PAMAM dendrimers (G3-OH, G3-NH2, G5-OH, and G5-NH2) on the dipalmitoylphosphatidylcholine (DPPC) monolayer. Our simulations indicated that the environmental temperature could significantly affect the influence of PAMAM dendrimers on the DPPC monolayer. Therefore, results obtained at room temperature cannot be directly applied to elucidate interactions at body temperature. Simulations at body temperature found that all tested PAMAM dendrimers can easily penetrate the lipid monolayer during the monolayer expansion process (mimicking "inhalation"), and the cationic PAMAM dendrimers (-NH2) show promising penetration ability during the monolayer compression process (mimicking "expiration"). Larger PAMAM dendrimers (G5) adsorbed onto the lipid monolayer tend to induce structural collapse and inhibit normal phase transitions of the lipid monolayer. These adverse effects could be mitigated in the subsequent expansion-compression cycle. These findings suggest that the PAMAM dendrimer may be used as a potential respiratory drug nanocarrier.

Differentiation of intrapancreatic accessory spleen from small hypervascular neuroendocrine tumor of the pancreas: textural analysis on contrast-enhanced computed tomography.

BACKGROUND: Intrapancreatic accessory spleens (IPASs) are usually misdiagnosed as pancreatic neuroendocrine tumors (PNETs). Texture analysis is valuable in tumor detection, diagnosis, and staging. PURPOSE: To identify the potential of texture features in differentiating IPASs from small hypervascular PNETs. MATERIAL AND METHODS: Twenty-one patients with PNETs and 13 individuals with IPASs who underwent pretreatment dynamic contrast-enhanced computed tomography (CT) were retrospectively analyzed. The routine imaging features-such as location, size, margin, cystic or solid appearance, enhancement degree and pattern, and lymph node enlargement-were recorded. Texture features, such as entropy, skewness, kurtosis, and uniformity, on contrast-enhanced images were analyzed. Receiver operating characteristic (ROC) analysis was performed to differentiate IPASs from PNETs. RESULTS: No significant differences were observed in margin, enhancement degree (arterial and portal phase), lymph node enlargement, or size between PNETs and IPASs (all P > 0.05). However, IPASs usually showed heterogeneous enhancement at the arterial phase and the same degree of enhancement as the spleen at the portal phase, both of which were greater than those of PNETs (69% vs. 35%, P = 0.06; 100% vs. 29%, P = 0.04). Entropy and uniformity were significantly different between IPASs and PNETs at moderate (1.5) and high sigma values (2.5) (both P < 0.01). ROC analysis showed that uniformity at moderate and high sigma had the highest area under the curve (0.82 and 0.89) with better sensitivity (85.0-95.0%) and acceptable specificity (75.0-83.3%) for differentiating IPASs from PNETs. CONCLUSIONS: Texture parameters have potential in differentiating IPASs from PNETs.

Protein Partitioning into Ordered Membrane Domains: Insights from Simulations.

Cellular membranes are laterally organized into domains of distinct structures and compositions by the differential interaction affinities between various membrane lipids and proteins. A prominent example of such structures are lipid rafts, which are ordered, tightly packed domains that have been widely implicated in cellular processes. The functionality of raft domains is driven by their selective recruitment of specific membrane proteins to regulate their interactions and functions; however, there have been few general insights into the factors that determine the partitioning of membrane proteins between coexisting liquid domains. In this work, we used extensive coarse-grained and atomistic molecular dynamics simulations, potential of mean force calculations, and conceptual models to describe the partitioning dynamics and energetics of a model transmembrane domain from the linker of activation of T cells. We find that partitioning between domains is determined by an interplay between protein-lipid interactions and differential lipid packing between raft and nonraft domains. Specifically, we show that partitioning into ordered domains is promoted by preferential interactions between peptides and ordered lipids, mediated in large part by modification of the peptides by saturated fatty acids (i.e., palmitoylation). Ordered phase affinity is also promoted by elastic effects, specifically hydrophobic matching between the membrane and the peptide. Conversely, ordered domain partitioning is disfavored by the tight molecular packing of the lipids therein. The balance of these dominant drivers determines partitioning. In the case of the wild-type linker of activation of T cells transmembrane domain, these factors combine to yield enrichment of the peptide at Lo/Ld interfaces. These results define some of the general principles governing protein partitioning between coexisting membrane domains and potentially explain previous disparities among experiments and simulations across model systems.

Membrane potential and dynamics in a ternary lipid mixture: insights from molecular dynamics simulations.

Transmembrane potential (Vm) plays critical roles in cell signaling and other functions. However, the impact of Vm on the structure and dynamics of membrane lipids and proteins, which are critical for the regulation of signaling, is still an open question. All-atom molecular dynamics (MD) simulation is emerging as a useful technique to address this issue. Previous atomistic MD simulations of pure or binary model membranes indicated that both ion imbalance and electric field can be used to generate Vm, but both approaches failed to yield structural changes in lipids with statistical significance. We hypothesized that a possible reason for this could be oversimplified membrane composition or limited sampling. In this work, we tested if and how Vm modulates the structure and dynamics of lipids in a physiologically relevant model membrane. Using a detailed side-by-side comparison, we first show that while both ion imbalance and electric field generate Vm in our complex membranes, only the latter could produce physiologically relevant Vm. We further show that double bonds in lipid acyl chains have a relatively large sensitivity to Vm. A single-bilayer model with an electric field showed the highest sensitivity in simulations under the isothermal-isobaric (NPT) ensemble, reproducing expected responses of head-group dipoles to Vm and suggesting that this approach may be more suitable for studying the structural effects of Vm. Our findings also shed light on the relationship between the macroscopic Vm and its atomic-level underpinnings.

Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order.

The plasma membrane (PM) serves as the functional interface between a cell and its environment, hosting extracellular signal transduction and nutrient transport among a variety of other processes. To support this extensive functionality, PMs are organized into lateral domains, including ordered, lipid-driven assemblies termed lipid rafts. Although the general requirements for ordered domain formation are well established, how these domains are regulated by cell-endogenous mechanisms or exogenous perturbations has not been widely addressed. In this context, an intriguing possibility is that dietary fats can incorporate into membrane lipids to regulate the properties and physiology of raft domains. Here, we investigate the effects of polyunsaturated fats on the organization of membrane domains across a spectrum of membrane models, including computer simulations, synthetic lipid membranes, and intact PMs isolated from mammalian cells. We observe that the ω-3 polyunsaturated fatty acid docosahexaenoic acid is robustly incorporated into membrane lipids, and this incorporation leads to significant remodeling of the PM lipidome. Across model systems, docosahexaenoic acid-containing lipids enhance the stability of ordered raft domains by increasing the order difference between them and coexisting nonraft domains. The relationship between interdomain order disparity and the stability of phase separation holds for a spectrum of different perturbations, including manipulation of cholesterol levels and high concentrations of exogenous amphiphiles, suggesting it as a general feature of the organization of biological membranes. These results demonstrate that polyunsaturated fats affect the composition and organization of biological membranes, suggesting a potential mechanism for the extensive effects of dietary fat on health and disease.

Reversible Effects of Peptide Concentration and Lipid Composition on H-Ras Lipid Anchor Clustering.

Dynamic clusters of lipid-anchored Ras proteins are important for high-fidelity signal transduction in cells. The average size of Ras nanoclusters was reported to be independent of protein expression levels, and cholesterol depletion is commonly used to test the raft-preference of nanoclusters. However, whether protein concentration and membrane domain stability affect Ras clustering in a reversible manner is not well understood. We used coarse-grained molecular dynamics simulations to examine the reversibility of the effects of peptide and cholesterol concentrations as well as a lipid domain-perturbing nanoparticle (C60) on the dynamics and stability of H-Ras lipid-anchor nanoclusters. By comparing results from these simulations with previous observations from the literature, we show that effects of peptide/cholesterol concentrations on the dynamics and stability of H-Ras peptide nanoclusters are reversible. Our results also suggest a correlation between the stabilities of lipid domains and Ras nanoclusters, which is supported by our finding that C60 penetrates into the liquid-disordered domain of the bilayer, destabilizing lipid domains and thereby the stability of the nanoclusters.

Cholesterol affects C60 translocation across lipid bilayers.

Cholesterol plays an important role in regulating the structural properties of phospholipid membranes and further influences the permeability of molecules and nanoparticles. However, nanoparticles' translocation across phospholipid membranes in the presence of cholesterol on the molecular scale is rarely studied. Here, we performed coarse-grained molecular dynamics simulations to probe the translocation of C60, one of the most popular nanoparticles, across dipalmitoylphosphatidylcholine bilayers with different concentrations of cholesterol molecules (0-50 mol%). The results reveal that the presence of cholesterol molecules induces lower area per lipid, larger bilayer thickness, and more ordered orientation of lipid tails. The higher the concentration of cholesterol molecules, the more significant is the condensing effect of lipid bilayer as just mentioned. Besides, dynamic processes, free energy profiles and permeability coefficients further indicate that the permeability of C60 decreases with increasing cholesterol concentration, which can be explained by the condensation effect and reduced free volume. Our researches provide an explicit description of the impact of cholesterol on C60 translocation across lipid bilayers.

A dual role of ERGIC-localized Rabs in TMED10-mediated unconventional protein secretion.

Cargo translocation across membranes is a crucial aspect of secretion. In conventional secretion signal peptide-equipped proteins enter the endoplasmic reticulum (ER), whereas a subset of cargo lacking signal peptides translocate into the ER-Golgi intermediate compartment (ERGIC) in a process called unconventional protein secretion (UcPS). The regulatory events at the ERGIC in UcPS are unclear. Here we reveal the involvement of ERGIC-localized small GTPases, Rab1 (Rab1A and Rab1B) and Rab2A, in regulating UcPS cargo transport via TMED10 on the ERGIC. Rab1 enhances TMED10 translocator activity, promoting cargo translocation into the ERGIC, whereas Rab2A, in collaboration with KIF5B, regulates ERGIC compartmentalization, establishing a UcPS-specific compartment. This study highlights the pivotal role of ERGIC-localized Rabs in governing cargo translocation and specifying the ERGIC's function in UcPS.

Neuroinflammation catching nanobubbles for microglia-neuron unit modulation against epilepsy.

Epilepsy is a common neurological disease caused by synchronous firing of hyperexcitable neurons. Currently, patients with epilepsy are typically treated with antiseizure medicines that work by interrupting the hyperexcitability or hypersynchrony of localized neurons or by inhibiting excitatory neurotransmission. However, these drugs do not treat the underlying causes of epilepsy, and nearly one-third of patients have seizures that cannot be controlled by these medications. Animal and clinical evidence suggests that inflammation caused by neuronal and non-neuronal cells within the epilepsy lesion could play a central role in seizure disorders. Here we report a gas-filled nanobubble (NB) conjugated with diammonium glycyrrhizinate (DG) drugs and sphingosine-1-phosphate (S1P) molecules (S1P@DG-NBs) on the lipid shell for targeted therapy and real-time ultrasound visualization applications against neuroinflammatory injury. Affinity of S1P@DG-NBs for the S1P receptor endows these NBs with enhanced targeting capability to the neuroinflammatory microenvironment of epilepsy, where the DG drugs modulate endothelium-microglia-neuron inflammation by inhibiting high-mobility group box 1 molecules and downregulating the Toll-like receptor 4 signaling pathway, resulting in anti-inflammatory M2 microglia that exert anti-epilepsy effects. Our results show that this technology can enhance visualization of epileptic brain and deliver drugs with anti-inflammatory and immunomodulatory properties to ameliorate seizures symptoms.

Bio-inspired peptide-conjugated liposomes for enhanced planktonic bacteria killing and biofilm eradication.

Developing new antimicrobial agents has become an urgent task to address the increasing prevalence of multidrug-resistant pathogens and the emergence of biofilms. Cationic antimicrobial peptides (AMPs) have been regarded as promising candidates due to their unique non-specific membrane rupture mechanism. However, a series of problems with the peptides hindered their practical application due to their high toxicity and low bioactivity and stability. Here, inspired by broadening the application of cell-penetrating peptides (CPPs), we selected five different sequences of cationic peptides which are considered as both CPPs and AMPs, and developed a biomimetic strategy to construct cationic peptide-conjugated liposomes with the virus-like structure for both enhancements of antibacterial efficacy and biosafety. The correlation between available peptide density/peptide variety and antimicrobial capabilities was evaluated from quantitative perspectives. Computational simulation and experimental investigations assisted to identify the optimal peptide-conjugated liposomes and revealed that the designed system provides high charge density for enhanced anionic bacterial membrane binding capability without compromised cytotoxicity, being capable of enhanced antibacterial efficacy of bacteria/biofilm of clinically important pathogens. The bio-inspired design has shown enhanced therapeutic efficiency of peptides and may promote the development of next-generation antimicrobials.

Molecular dynamics simulation insights into the cellular uptake of elastic nanoparticles through human pulmonary surfactant.

The interaction between inhaled nanoparticles (NPs) and the pulmonary surfactant (PS) monolayer has drawn significant attention due to its potential in drug delivery design and application for respiratory therapeutics in active and passive cellular uptake pathways. Even though much attention has been given to explore the interaction between NPs and the PS monolayer, the effects of the NP elasticity on the translocation across the PS monolayer have not been thoroughly studied. Here, we performed a series of coarse-grained (CG) molecular dynamics simulations to study active or passive cellular uptake pathways of three NPs with different elasticities through a PS monolayer. The differences between active and passive pathways underly the enhanced targeting ability by ligand-receptor interaction (L-R interaction). In the active or passive cellular uptake pathways, it is found that the increase in stiffness level leads to a higher penetrability of NPs at the same time range. The soft NP has always been withheld inside the PS monolayer due to the lowest level of elasticity, while the other two types of NPs penetrate through the PS monolayer as the simulation progresses toward the end. The NPs in the active cellular uptake pathways take a longer time to penetrate the PS monolayer, resulting in a longer average penetration distance of approximately 40.55% and a higher average number of contacts, approximately 36.11%, than passive cellular uptake pathways, due to the L-R interaction. Moreover, it demonstrates that NPs in active cellular uptake pathways have a significantly higher targeting ability with the PS monolayer. We conclude that the level of NP elasticities has a substantial link to the penetrability in active or passive cellular uptake pathways. These results provide valuable insights into drug delivery and nanoprobe design for inhaled NPs within the lungs.

The stiffness-dependent tumor cell internalization of liquid metal nanoparticles.

The properties of nanoparticle (NP) carriers, such as size, shape and surface state, have been proven to dramatically affect their uptake by tumor cells, thereby influencing and determining the effect of nanomedicine on tumor theranostics. However, the effect of the stiffness of NPs on their cellular internalization remains unclear, especially for circumstances involving active or passive NP targeting. In this work, we constructed eutectic gallium indium liquid metal NPs with the same particle size, shape and surface charge properties but distinct stiffness via tailoring the surface oxidation and silica coating. It has been found that the softer NPs would be endocytosed much slower than their stiffer counterparts in the presence of specific ligand-receptor interaction. Interestingly, once the interaction is eliminated, softer NPs are internalized faster than the stiffer ones. Based on experimental observations and theoretical verification, we demonstrate that this phenomenon is mainly caused by varying degrees of deformation of soft NPs induced by ligand-receptor interactions. Such a finding of the stiffness effect of NPs implies great potential for fundamental biomedical applications, such as the rational design of nanomedicines.