Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation

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A quantitative view on multivalent nanomedicine targeting

Although the concept of selective delivery has been postulated over 100 years ago, no targeted nanomedicine has been clinically approved so far. Nanoparticles modified with targeting ligands to promote the selective delivery of therapeutics towards a specific cell population have been extensively reported. However, the rational design of selective particles is still challenging. One of the main reasons for this is the lack of quantitative theoretical and experimental understanding of the interactions involved in cell targeting. In this review, we discuss new theoretical models and experimental methods that provide a quantitative view of targeting. We show the new advancements in multivalency theory enabling the rational design of super-selective nanoparticles. Furthermore, we present the innovative approaches to obtain key targeting parameters at the single-cell and single molecule level and their role in the design of targeting nanoparticles. We believe that the combination of new theoretical multivalent design and experimental methods to quantify receptors and ligands aids in the rational design and clinical translation of targeted nanomedicines

Single-Particle Functionality Imaging of Antibody-Conjugated Nanoparticles in Complex Media

The properties of nanoparticles (NPs) can change upon contact with serum components, occluding the NP surface by forming a biomolecular corona. It is believed that targeted NPs can lose their functionality due to this biological coating, thus losing specificity and selectivity toward target cells and leading to poor therapeutic efficiency. A better understanding of how the biomolecular corona affects NP ligand functionality is needed to maintain NP targeting capabilities. However, techniques that can quantify the functionality of NPs at a single-particle level in a complex medium are limited and often laborious in sample preparation, measurement, and analysis. In this work, the influence of serum exposure on the functionality of antibody-functionalized NPs was quantified using a straightforward total internal reflection fluorescence (TIRF) microscopy method and evaluated in cell uptake studies. The single-particle resolution of TIRF reveals the interparticle functionality heterogeneity and the substantial differences between NPs conjugated with covalent and noncovalent methods. Notably, only NPs covalently conjugated with a relatively high amount of antibodies maintain their functionality to a certain extent and still showed cell specificity and selectivity toward high receptor density cells after incubation in full serum. The presented study emphasizes the importance of single-particle functional characterization of NPs in complex media, contributing to the understanding and design of targeted NPs that retain their cell specificity and selectivity in biologically relevant conditions

A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines

Valency and affinity control of aptamer-conjugated nanoparticles for selective cancer cell targeting

Nanoparticles (NPs) are commonly functionalized using targeting ligands to drive their selective uptake in cells of interest. Typical target cell types are cancer cells, which often overexpress distinct surface receptors that can be exploited for NP therapeutics. However, these targeted receptors are also moderately expressed in healthy cells, leading to unwanted off-tumor toxicities. Multivalent interactions between NP ligands and cell receptors have been investigated to increase the targeting selectivity towards cancer cells due to their non-linear response to receptor density. However, to exploit the multivalent effect, multiple variables have to be considered such as NP valency, ligand affinity, and cell receptor density. Here, we synthesize a panel of aptamer-functionalized silica-supported lipid bilayers (SSLB) to study the effect of valency, aptamer affinity, and epidermal growth factor receptor (EGFR) density on targeting specificity and selectivity. We show that there is an evident interplay among those parameters that can be tuned to increase SSLB selectivity towards high-density EGFR cells and reduce accumulation at non-tumor tissues. Specifically, the combination of high-affinity aptamers and low valency SSLBs leads to increased high-EGFR cell selectivity. These insights provide a better understanding of the multivalent interactions of NPs with cells and bring the nanomedicine field a step closer to the rational design of cancer nanotherapeutics

Multicolor Super-Resolution Microscopy of Protein Corona on Single Nanoparticles

Nanoparticles represent a promising class of material for nanomedicine and molecular biosensing. The formation of a protein corona due to nonspecific particle-protein interactions is a determining factor for the biological fate of nanoparticles in vivo and strongly impacts the performance of nanoparticles when used as biosensors. Nonspecific interactions are usually highly heterogeneous, yet little is known about the heterogeneity of the protein corona that may lead to inter- and intraparticle differences in composition and protein distribution. Here, we present a super-resolution microscopic approach to study the protein corona on single silica nanoparticles and subsequent cellular interactions using multicolor stimulated emission depletion (STED) microscopy. We demonstrate that STED resolves structural features of protein corona on single particles including the distribution on the particle surface and the degree of protein internalization in porous particles. Using multicolor measurements of multiple labeled protein species, we determine the composition of the protein corona at the single-particle level. We quantify particle-to-particle differences in the composition and find that the composition is considerably influenced by the particle geometry. In a subsequent cellular uptake measurement, we demonstrate multicolor STED of protein corona on single particles internalized by cells. Our study shows that STED microscopy opens the window toward mechanistic understanding of protein coronas and aids in the rational design of nanoparticles as nanomedicines and biosensors

A structurally diverse library of glycerol monooleate/oleic acid non-lamellar liquid crystalline nanodispersions stabilized with nonionic methoxypoly(ethylene glycol) (mPEG)-lipids showing variable complement activation properties

Pluronic F127-stabilized non-lamellar liquid crystalline aqueous nanodispersions are promising injectable platforms for drug and contrast agent delivery. These nanodispersions, however, trigger complement activation in the human blood, where the extent of complement activation and opsonization processes may compromise their biological performance and safety. Here, we introduce a broad family of nanodispersions from glycerol monooleate (GMO) and oleic acid (OA) in different weight ratios, and stabilized with a plethora of nonionic methoxypoly(ethylene glycol) (mPEG)-lipids of different PEG chain length and variable lipid moiety (monounsaturated or saturated diglycerides or D-α-tocopheryl succinate). Through an integrated biophysical approach involving dynamic light scattering, synchrotron small-angle scattering, and cryo-transmission electron microscopy, we examine the impact of nonionic mPEG-lipid stabilization on size, internal self-assembled architecture, and gross morphological characteristics of nanodispersions. The results show how the nonionic mPEG-lipid type and concentration, and dependent on GMO/OA weight ratio, can variably modulate the internal architectures of nanoparticles. Assessment of complement profiling from selected nanodispersions with diverse structural heterogeneity further suggests a variable modulatory role for the lipid type of the nonionic mPEG-lipid in the extent of complement activation, which span from no activation to moderate to high levels. We comment on plausible mechanisms driving the observed complement activation variability and discuss the potential utility of these nanodispersions for future development of injectable nanopharmaceuticals

Mapping Antibody Domain Exposure on Nanoparticle Surfaces Using DNA-PAINT

Decorating nanoparticles with antibodies (Ab) is a key strategy for targeted drug delivery and imaging. For this purpose, the orientation of the antibody on the nanoparticle is crucial to maximize fragment antibody-binding (Fab) exposure and thus antigen binding. Moreover, the exposure of the fragment crystallizable (Fc) domain may lead to the engagement of immune cells through one of the Fc receptors. Therefore, the choice of the chemistry for nanoparticle-antibody conjugation is key for the biological performance, and methods have been developed for orientation-selective functionalization. Despite the importance of this issue, there is a lack of direct methods to quantify the antibodies’ orientation on the nanoparticle’s surface. Here, we present a generic methodology that enables for multiplexed, simultaneous imaging of both Fab and Fc exposure on the surface of nanoparticles, based on super-resolution microscopy. Fab-specific Protein M and Fc-specific Protein G probes were conjugated to single stranded DNAs and two-color DNA-PAINT imaging was performed. Hereby, we quantitatively addressed the number of sites per particle and highlight the heterogeneity in the Ab orientation and compared the results with a geometrical computational model to validate data interpretation. Moreover, super-resolution microscopy can resolve particle size, allowing the study of how particle dimensions affect antibody coverage. We show that different conjugation strategies modulate the Fab and Fc exposure which can be tuned depending on the application of choice. Finally, we explored the biomedical importance of antibody domain exposure in antibody dependent cell mediated phagocytosis (ADCP). This method can be used universally to characterize antibody-conjugated nanoparticles, improving the understanding of relationships between structure and targeting capacities in targeted nanomedicine.</p

Artificial Antigen-Presenting Cell Topology Dictates T Cell Activation

Nanosized artificial antigen-presenting cells (aAPCs), synthetic immune cell mimics that aim to activate T cells ex or in vivo, offer an effective alternative to cellular immunotherapies. However, comprehensive studies that delineate the effect of nano-aAPC topology, including nanoparticle morphology and ligand density, are lacking. Here, we systematically studied the topological effects of polymersome-based aAPCs on T cell activation. We employed an aAPC library created from biodegradable poly(ethylene glycol)-block-poly(d,l-lactide) (PEG-PDLLA) polymersomes with spherical or tubular shape and variable sizes, which were functionalized with αCD3 and αCD28 antibodies at controlled densities. Our results indicate that high ligand density leads to enhancement in T cell activation, which can be further augmented by employing polymersomes with larger size. At low ligand density, the effect of both polymersome shape and size was more pronounced, showing that large elongated polymersomes better activate T cells compared to their spherical or smaller counterparts. This study demonstrates the capacity of polymersomes as aAPCs and highlights the role of topology for their rational design