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Ligand-Receptor Interaction Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics Junshi Liang, Pengyu Chen, Bojun Dong, Zihan Huang, Kongyin Zhao, and Li-Tang Yan Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00241 • Publication Date (Web): 06 Apr 2016 Downloaded from http://pubs.acs.org on April 8, 2016
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Ligand-Receptor Interaction Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics
Junshi Liang, †,‡ Pengyu Chen,†,‡ Bojun Dong,‡ Zihan Huang, Kongyin Zhao,§ and Li-Tang Yan*,‡
‡
Key Laboratory of Advanced Materials (MOE), Department of Chemical Engineering, Tsinghua University, Beijing 100084, P. R. China
§
State Key Laboratory of Separation Membranes and Membrane Processes, Tianjin Polytechnic University, Tianjin 300387, China
*
Corresponding Author:
[email protected] †
These authors contributed equally.
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Abstract: Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better applications in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane, and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding on some recent experimental observations, and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.
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1. Introduction Dendrimer-like soft nanoparticles (DSNPs), featured by unique ability to control molecular architectures, size, and structural properties, have shown great promise in the biomedically relevant applications.1-5 In order to target, deliver, image, or otherwise mediate interactions with the biological environment, many biomedical applications of this class of nanoparticles rely on the conjugated small molecule ligands.6-8 Conjugation to DSNPs, whether by covalent attachment or by physical adsorption, results in changes to the structure of this nanoparticle-based platform. Typically, the number of ligands attached per nanoparticle has been determined to present obvious heterogeneous distribution.9,10 However, studies in the field often use the mean value of the ligand/dendrimer ratio as the accepted level of characterization of DSNPs, which grossly underestimates the effects of the heterogeneous ligand-nanoparticles distributions on the cellular internalization mechanism and cytotoxicity of these materials.6 Indeed, experimental data indicate that the actual distribution of ligand-dendrimer components is much more heterogeneous than is commonly assumed.10 Scientific understanding and commercial translation call for a thorough molecular understanding of the effects of the ligand distribution on the cell interaction of DSNPs. However, to understand these effects usually requires direct measurements in a single cell and is thus very difficult and more challenging. When experiments encounter difficulties, tailored computer simulations as well as theoretical analysis offer alternative approaches to identify the process of interest.11-16 In fact, the mechanism that allows nanoparticles to across the cell membrane has been the subject of extensive simulation and theoretical research because understanding and control of such uptake 3
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processes is a key step in nanomedicine development.14-19 Particularly, the receptor-mediated endocytosis is found to be the major internalization pathway for ligand-conjugated nanoparticles, where the interaction between membrane receptors and nanoparticle ligands dominates the thermodynamic force driving this passive endocytosis.17,20-22 However, it should be pointed out that almost all current simulation and theoretical studies of the receptor-mediated endocytosis focus on the “hard” nanoparticles free of elastic deformation. The influence of ligand-receptor interactions on the cell interactions of “soft” nanoparticles with obviously elastic deformation and various molecular architectures, such as DSNPs, has yet remained to be definitely elucidated. Two points are emphasized here for the difference in the transmembrane transport mechanism and kinetics behavior between hard nanoparticles and DSNPs. The first is that the elastic deformation of soft nanoparticles may result in the change of the spatial distribution of ligands and thereby affect ligand-receptor interactions and the following endocytosis. Actually the full wrapping of a softer nanoparticle has been theoretically demonstrated to experience a higher energy barrier than a harder one.23 The second is that the complex molecular topology of DSNPs can significantly complicate the internalization mechanism and uptake kinetics.2,4,24 The impact of this factor is of undoubted importance for the receptor-mediated transmembrane transport of dendrimer nanoparticles conjugated with ligands, which, however, remains poorly understood. In this paper, we present the first simulation studies on receptor-mediated cellular uptake of DSNPs conjugated with ligands, which systematically accounts for the underlying mechanism and kinetic behaviors mediated by the receptor-ligands interactions. We predict a phase diagram of interactions states with varying ligand densities and membrane tensions. Two novel 4
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endocytosis mechanisms are identified for dendrimer nanoparticles, i.e., the direct wrapping and the penetration-extraction vesiculation, featuring the interplay between the ligand-receptor interaction and the unique molecular architecture of such soft nanoparticles. The results gain insight into the diffusivity of receptors and dendrimers in the membrane plane, and reveal that the ligand density can evidently influence the receptor diffusion that is crucial for the uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. Our findings regarding various effects on receptor-ligand interaction mediate cellular uptake suggest principles of dendrimer-based therapeutics with optimized cellular targeting.
2. Model and methods Full technical details on the simulation method and the molecular models of lipid, receptor and dendrimers are presented in Supporting Information. Briefly, we use a mesoscopic simulation technique, dissipative particle dynamics (DPD),25 that is a coarse-grained method, includes explicit solvent particles, and faithfully reproduces all key properties of self-assembling fluid bilayer membrane and the interactions between membrane and nanoparticles on the time scale of tens µs and the length scale of tens nanometers meaningful for the nanoparticle-membrane interactions.21,26,27
Here, the model of the dendrimer-like nanoparticle is mapped from the polyamidoamine (PAMAM) like dendrimer. An effective coarse-grained methodology of dendrimers is used in the
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modeling, which has been proved its validity and efficiency in the simulations of their conformational behaviors and interactions with lipid bilayers.24,26 Only the terminal beads of the dendrimer model are hydrophilic and carry the charge of +1. The other moiety of the dendrimer is uncharged and hydrophobic. To preserve charge neutrality, the randomly selected solvent beads, with the same number of the charged terminal beads of the dendrimer, are changed into the counterions with a charge of -1, and the concentration of these counterions is equal at the two sides of the membrane due to the periodic boundary condition.38
As the present work focuses
on the effects of the ligand distribution and the ligand-receptor interaction, the seventh dendrimer (G7) is used as the sample of DSNPs in the whole simulations (Figure S1a). Unless otherwise explicitly mentioned, the ligands conjugated to a dendrimer are modeled as single solid beads randomly attached to the terminal beads of the dendrimer, following the stochastic synthesis techniques (Figure S1b). Our simulations also reveal that both obvious shape deformation and unique molecular architecture of DSNPs can weaken the effects of the initial surface ligand distributions on the interaction state and the wrapping kinetics of dendrimers, in contrast to the hard nanoparticles. The model of the amphiphilic lipid is constructed by a head group with three hydrophilic beads and two tails consisting of three hydrophobic beads. The head group contains three connected hydrophilic beads and the top two of them carry the charges of +1 and -1 respectively (Figure S1c). The receptors are modeled as the same as the lipids except having a different ligand-receptor attractive head group (Figure S1d), which has been widely used in the simulation of receptor-mediated nanoparticle-membrane interaction.17,28 As the generic aspects of molecular mechanism and kinetics behaviors for ligand-receptor 6
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interaction mediated transmembrane transport of DSNPs are concerned in the present work, we do not focus on the specific types of ligands and receptors. The specific receptors that can selectively bind with ligand-conjugated dendrimers include, for example, folate receptor.29 Based on partial experimental information, the receptor density in a cell membrane should be in the range of 50 to 500 receptors per square µm.30,31 However, once the contact between ligands and receptors starts, the receptor density within the contact area is raised to the level of ligand density on the nanoparticle surface through an in-plane diffusion of receptors. Fundamentally, the directional diffusion of receptors is driven by a local reduction in free energy caused by ligand-receptor binding and will be investigated in the present work. To simplify matters, we focus on the case where the number of receptors is relatively high in order to study the transmembrane process that is not receptor limited, as adapted by some previous simulations.17,28 Thus, the receptor density of the membrane, fR, is set as fR=0.33 except where noted otherwise. fR is defined as the ratio of the receptor number to the total number of both receptors and lipids within a membrane. 5,616 lipids and receptors with various densities self-assemble into a lipid bilayer membrane spanning the simulation box.
The interaction parameters between different species are similar to those provided by the MARTINI force field,32,33 and have been successfully used in our previous works.26,34 Particularly, to capture the highly specific ligand-receptor binding, the interaction parameter (the maximum repulsion parameter in the conservative force, aRL) between ligands and head groups of receptors is set to zero which has been widely used in the simulation study of ligand-receptor 7
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interaction for hard nanoparticles.22,28 Note that such an interaction parameter between beads i and j, aij, has a linear relationship with Flory-Huggins parameter: χ ij ≈ ( aij − aii ) / 3.27 , where aii is usually chosen to 25 for interactions between like species.25 Clearly, if aij 1.0 kBT/rc2) in which the ligand density has a trivial effect on the interaction state especially at fL0.6. As denoted by the red, pink, and violet circles, the other three regions are characterized by the full wrapping state where the dendrimer can be completely wrapped by the membrane, resulting in a dendrimer-encased vesicle (Figure 1c and d).41 Note in real cellular endocytosis, the fully wrapped object can be further internalized through membrane fission accelerated by external force, e.g., actin polymerization or Sar1.42-44 In fact, recent experiments have demonstrated that the formation of the full wrapping state plays a crucial role in the endocytosis of DSNPs.41,45 What is interesting is that the kinetics pathways of the vesiculation events in these three regions are absolutely different. At bottom left (σ
