The Why and How of Ultrasmall Nanoparticles

Abstract

In this Account, we describe our research into ultrasmall nanoparticles, including their unique properties, and outline some of the new opportunities they offer. We will summarize our perspective on the current state of the field and highlight what we see as key questions that remain to be solved. First, there are several nanostructure size-scale regimes, with qualitatively distinct functional biological attributes. Broadly generalized, larger particles (e.g., larger than 300 nm) tend to be more efficiently swept away by the first line of the immune system (for example macrophages). In the "middle-sized" regime (20-300 nm), nanoparticle surfaces and shapes can be recognized by energy-dependent cellular reorganizations, then organized locally in a spatial and temporally coherent way. That energy is gated and made available by specific cellular recognition processes. The relationship between particle surface design, endogenously derived nonspecific biomolecular corona, and architectural features recognized by the cell is complex and only purposefully and very precisely designed nanoparticle architectures are able to navigate to specific targets. At sufficiently small sizes (<10 nm including the ligand shell, associated with a core diameter of a few nm at most) we enter the "quasi-molecular regime" in which the endogenous biomolecular environment exchanges so rapidly with the ultrasmall particle surface that larger scale cellular and immune recognition events are often greatly simplified. As an example, ultrasmall particles can penetrate cellular and biological barriers within tissue architectures via passive diffusion, in much the same way as small molecule drugs do. An intriguing question arises: what happens at the interface of cellular recognition and ultrasmall quasi-molecular size regimes? Succinctly put, ultrasmall conjugates can evade defense mechanisms driven by larger scale cellular nanoscale recognition, enabling them to flexibly exploit molecular interaction motifs to interact with specific targets. Numerous advances in control of architecture that take advantage of these phenomena have taken place or are underway. For instance, syntheses can now be sufficiently controlled that it is possible to make nanoparticles of a few hundreds of atoms or metalloid clusters of several tens of atoms that can be characterized by single crystal X-ray structure analysis. While the synthesis of atomically precise clusters in organic solvents presents challenges, water-based syntheses of ultrasmall nanoparticles can be upscaled and lead to well-defined particle populations. The surface of ultrasmall nanoparticles can be covalently modified with a wide variety of ligands to control the interactions of these particles with biosystems, as well as drugs and fluorophores. And, in contrast to larger particles, many advanced molecular analytical and separation tools can be applied to understand their structure. For example, NMR spectroscopy allows us to obtain a detailed image of the particle surface and the attached ligands. These are considerable advantages that allow further elaboration of the level of architectural control and characterization of the ultrasmall structures required to access novel functional regimes and outcomes. The ultrasmall nanoparticle regime has a unique status and provides a potentially very interesting direction for development.

Figure 1:

¹³C-NMR spectra of ultrasmall gold nanoparticles (1.8 nm), functionalized with ¹³C-1,2,3-labelled cysteine (top) and of dissolved ¹³C-1,2,3-labelled cysteine (bottom). Note the peak shift and the peak broadening of cysteine after binding to the nanoparticles. The split of the β-carbon peaks indicates at least three different magnetic environments for cysteine on the gold surface. Adapted with permission from ref. . Copyright 2019 American Chemical Society.

Figure 2:

When particles (gold sphere) become ultrasmall, their interactions with ambient biomolecules become sufficiently short-lived, and limited in variety (gold sphere with proteins in the environment), so that the particles can interact directly with cells and cellular machinery. Since they do not exist as long-lived complexes between particles and biomolecules from the environment, they are not recognized in a biological sense by the various receptors (red) and other components that trigger complex biological defenses of living organisms. In principle this can allow ultrasmall particles to cross membranes, biological barriers, and evade immune responses, in much same ways as some small molecule drugs with appropriate physiochemical properties. Reprinted (adapted) with permission from ref. . Copyright 2020 American Chemical Society.

Figure 3:

Schematic reaction pathway for the conversion of amino groups of glutathione on the gold nanoparticle surface to azide groups. A covalent coupling of alkyne-terminated ligands (represented by a blue ball) to the gold nanoparticle surface is possible via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Adapted with permission from ref. . Copyright 2022 Royal Society of Chemistry.

Figure 4.

a) Surfactant-like ligands bind and denature ChT. b) TEG disrupts protein binding. c) Appending a carboxylate to the outside of the TEG layer provides ChT binding without denaturation. d) Gold nanoparticles (2 nm core) with anionic amino acid termini. Quantification of e) nanoparticle-protein and f) protein-protein interactions by ITC. Overlap of entropy-enthalpy compensation plots shown in the inset. Adapted with permission from ref.. Copyright 2007 American Chemical Society.

Figure 5.

a) Protein complexation is observed by DLS with AuNP_TTMA but not with AuNP_TZwit. b) Large aggregates are observed in serum (5%) with AuNP_TTMA but no larger-sized assemblies were observed with AuNP_TZwit. c) Dilution after incubation in 55% human serum showed no irreversible corona formation with either AuNP_TZwit or AuNP_TTMA, where size increase corresponds to a simple monolayer of protein around the particle. Adapted with permission from ref. Copyright 2014 American Chemical Society.

Figure 6:

Top: Schematic view of the modified sequence of the protein CRaf, conjugated to a gold nanoparticle (1.55 nm) by a central cysteine, drawn to scale. Bottom: Schematic view of CRaf on a gold nanoparticle interacting with the homodimeric protein 14-3-3σ. Only the amino acids that interact with the protein-binding region are shown; the other part of the chain is shown as dashed line. Adapted with permission from ref.. Copyright 2021 Wiley-VCH.

Figure 7.

Uptake of 2, 4, and 6 nm core gold nanoparticles by HeLa cells. a) Nanoparticle monolayers. b) HeLa cell uptake of gold nanoparticles as quantified by ICP-MS. c) Observed uptake pathways for gold nanoparticles. Adapted with permission from ref. Copyright 2015 American Chemical Society.

Figure 8:

Confocal laser scanning microscopic images (z-stacks across the cells) to demonstrate the uptake of FAM-labelled fluorescing ultrasmall gold nanoparticles (2 nm) by HeLa cells (24 h). The particles easily entered the cell and the cell nucleus. 1·10⁴ cells were plated per well, and 2.09∙10¹⁴ ultrasmall gold nanoparticles were present in each well. Scale bar: 20 μm. Red: Actin staining of the cell cytoskeleton; blue: Nuclear staining by Hoechst33342; green: FAM-labelled gold nanoparticles. Adapted with permission from ref. Copyright 2019 American Chemical Society.