Harnessing Nanotechnology to Expand the Toolbox of Chemical Biology

Abstract

While nanotechnology often addresses biomedical needs, nanoscale tools can also facilitate broad biological discovery. Nanoscale delivery, imaging, biosensing, and bioreactor technologies may address unmet questions at the interface between chemistry and biology. Currently, many chemical biologists do not include nanomaterials in their toolbox, and few investigators develop nanomaterials in the context of chemical tools to answer biological questions. We reason that the two fields are ripe with opportunity for greater synergism. Nanotechnologies can expand the utility of chemical tools in the hands of chemical biologists, for example through controlled delivery of reactive/toxic compounds or to signal binding events of small molecules in living systems. Conversely, chemical biologists can work with nanotechnologists to address challenging biological questions that are inaccessible to both communities. This perspective aims to introduce the chemical biology community to nanotechnologies that may expand their methodologies while inspiring nanotechnologists to address questions relevant to chemical biology.

Graphical Abstract

Introduction

Chemical biologists leverage chemical tools to interrogate, manipulate, and perturb a system for biological discovery. While chemical biologists primarily reply on chemical principles to address biological questions, nanotechnology focuses on the manipulation of nano-scale synthetic materials in biological systems¹. Nanomaterials are unique due to their tunable and distinct physical, chemical, and biological properties compared to bulk materials (Figure 1). Chemical biology and nanotechnology, due to their complementary goals and toolkits, can potentially be explored in a collaborative, synergistic manner. Synthetic nanomaterials can interact with biological processes on cell surfaces, inside living cells, and even within specific intracellular compartments. The size, physicochemical control, and resulting biological interactions of nanomaterials have facilitated abundant investigative directions. These include chemical-, cell-, and mechano-biology to study a wide range of biomolecules and cellular processes. Multiple broad classes of nanomaterials, from inorganic to biodegradable polymers, have been developed for molecular delivery, enzymatic catalysis, molecular imaging, and molecular sensing. In addition, a number of nanomaterial applications have reached the clinic². The unique features of nanomaterials can be of great interest to chemical biologists, although most have yet to be extensively implemented for biological discovery. We envision the expansion of nanomaterial utility as or together with chemical tools for broad biological applications (nanochemical biology). There are also existing chemical biology challenges that nanotechnologists can collaboratively address, as described herein.

Figure 1.

Major classes of potential contributions of nanotechnology to chemical biology. Potential contributions of nanotechnology to chemical biology is classified into four modules: nanocarriers of bioactive chemicals or delivery (upper-left red quadrant); enzymatic nanoreactors (bottom-left blue quadrant); nanoparticle-based molecular imaging (upper-right yellow quadrant); nanoscale sensors (bottom-right green quadrant).

Nanocarriers of Bioactive Chemicals

While chemical biologists have developed highly potent modulators (both agonists and antagonists) of biomolecules, many may not exhibit ideal functionality in vivo due to limited solubility, stability, biocompatibility, poor pharmacokinetics, and off-target activity³. Nanotechnologists have developed tools to deal with the delivery and pharmacokinetic issues of otherwise-poorly-behaved molecules via encapsulating active cargos and targeting specific tissue/cell/organelle types. Specifically, nanomaterials are developed to “deliver” these chemicals via encapsulation and controlled release within subcellular organelles or specific organs/tissues in live animals (Figure 1, red quadrant).

While these benefits of nanomaterials have typically been applied for medical needs, the principles are expected to be readily transformed to deliver bioactive chemicals to interrogate biological systems. In addition, pharmacokinetic properties of delivered molecules, such as their half-life, can be enhanced by nanocarrier loading. This strategy allows significant accumulation and functional modifications of encapsulated molecules at nanoparticle-targeted sites compared to free diffusion⁴. For general consideration of chemical biologists, three important aspects of formulating nanocarriers are: to encapsulate bioactive molecules, to target them to desired intra/extracellular loci, and to control their temporal and spatial release.

A wide range of materials have been explored as nanocarriers of bioactive cargos (Table 1). These include lipids, polymers, metals, and other inorganic materials. Liposomes, highly-organized hollow lipid bilayer nanoparticles, were first described in 1965 and are currently used in the clinic to encapsulate doxorubicin (Doxil), daunorubicin (DaunoXome)^2,5, and other drugs. The amphiphilic structure makes liposomes suitable for delivery of various compounds. Less-ordered lipoplexes such as Lipofectamine may form a complex with nucleic acids to result in nanoparticles for transfecting cells in culture. Other materials include biodegradable polymers such as poly(lactic-co-glycolic acid) (PLGA) and polycapralactone (PCL), which form nanoparticles with a hydrophobic core^2,5. Solid inorganic nanoparticles, often composed of gold, iron oxide, various metals, or carbon nanotubes, can deliver surface-adsorbed or covalently conjugated small molecules^6,7. Nanoparticles formulated through protein-drug interactions, such as albumin bound paclitaxel (Abraxane), have also resulted in clinical success^2,8. The choice of nanocarrier material is important for chemical biologists in considering the cargo type, efficiency of loading and release, organelle/tissue targeting, and other biological interactions.

Table 1.

General classes of nanomaterials.

Type of Material	Examples	Benefits	Potential Drawbacks
Lipids2,5,46	phospholipids synthetic lipids, many varieties lipid bilayers (liposomes) solid lipid nanoparticles other forms	biocompatible (in most cases) increases solubility well-studied adaptable to many cargoes	cargo leakage particle stability/solubility reactivity of lipids immune response low drug loading efficiency
Polymers2,5,10,29	poly-lactic-co-glycolic acid (PLGA) polycaprolactone (PCL) polymeric micelles (block copolymers)	biocompatible (in many cases) well-studied facilitates controlled release increases solubility adaptable to many cargoes	some polymers have toxic degradation products may be difficult to scale-up potential immune response low drug loading efficiency
Aggregates2,4,8,20,21	excipients include proteins (albumin) dyes polysaccharides other drugs	Solubilization drug loading efficiency generally biocompatible	rapid degradation potential for low stability
Inorganic2,6,7,51,71	quantum dots MRI contrast agents carbon nanotubes	unique optical or magnetic properties do not degrade potential for simultaneous delivery and imaging	potential toxicities generally poor drug loading efficiencies

To associate pharmacologically active molecules with nanoparticle carriers, multiple strategies are available. Liposomal formulations can be assembled with hydrophilic or hydrophobic small molecules loaded into the aqueous core or the lipid bilayer, respectively. Crystalline formulations represent another strategy, such as with doxorubicin in the approved drug Doxil^5,9. Hydrophobic drugs are often encapsulated within a hydrophobic polymeric matrix via co-precipitation¹⁰, allowing controlled release. Alternatively, chemical conjugation methods often involve a labile linker, such as a pH-responsive hydrazine-carbonyl condensation bond¹¹. Solid or hydrogel matrix nanoparticle systems may also be conjugated with drugs via dithiol linkages to facilitate release¹². Layer-by-layer assembly¹³ is another strategy to load cargoes into a polymeric nanoparticle. Porous metal-organic frameworks are also loaded via adsorption and trapping of drug molecules¹⁴.

Nanoparticles often enter cells via endocytosis, sequestering particles in late endosomes/lysosomes. The physicochemical properties of nanocarriers can be modified to control their localization within specific subcellular compartments, allowing delivery of active cargoes to specific organelles, which can be useful with non-specific inhibitors. For example, to facilitate escape of nanoparticles from late endosomes/lysosomes into the cytosol, particles may be engineered to swell, fuse with the endosome membrane, destabilize the membrane, or cause an increase in osmotic pressure¹⁵. Targeting nanomaterials to the nucleus has been achieved via the attachment of natural or synthetic nuclear localization signals¹⁶. Localization to the endoplasmic reticulum and Golgi apparatus can occur by receptor-mediated retrograde trafficking, while mitochondrial localization has been achieved by cationic surface functionalization of nanoparticles^17,18. In addition, methods such as electroporation, mechanical cellular manipulation via microfluidics, and direct injection enable the insertion of nanoparticles into the cytoplasm¹⁹. While intracellular uptake via endocytosis is the rule rather than the exception, alternative uptake may be accomplished by designing surface coatings which minimize protein adsorption, such as polyethylene glycol-functionalized nanoparticles. While available nanomaterials can only selectively target a subset of intracellular compartments, direct collaboration may better fulfill the needs of chemical biologists to improve subcellular localization of biologically active compounds. For example, more triphenyl-phosphonium or poly-guanidinium-containing chemical bulding blocks can be designed to coat nanoparticles for subcellular targeting to mitochondria and nucleus, respectively^17,18.

In animals, nanocarriers can abrogate toxicities and side effects associated with systemic administration. Nanocarrrier-mediated delivery of chemotherapeutic agents has been shown to reduce toxicities compared with systemic administration of carrier-free therapies²⁰. Even molecularly-targeted small molecules, such as kinase inhibitors, exhibit suppressed toxicity profiles when encapsulated in site-targeted nanocarriers²¹. The administration of active compounds via nanoparticles for the purpose of extending chemical biology investigations in vivo is largely underutilized.

Controlling the size, shape, and surface chemistry of nanocarriers can target them to specific organs or tissues. A majority of nanoparticles exhibit significant localization to the liver and spleen due to their size (< 200 nm) and opsonization (serum protein adsorption), resulting primarily hepatobiliary clearance²². Polyethylene glycol and zwitterions can reduce but often not completely abrogate opsonization-induced liver localization²³.

The majority of nanoparticle passive targeting strategies have focused on tumor targeting. In tumor-bearing animals, nanoparticles exhibit some tumor localization via leakiness of tumor vasculature, termed the enhanced permeability and retention (EPR) effect²⁴. This is often achieved by controlling particle size of up to 200 nm²⁵. There is evidence, however, that this effect may not translate well to many human cancers²⁶. Additional targeting strategies have been described elsewhere²⁷. Ultra-small nanoparticles (<10 nm) exhibit renal clearance from the body²⁸, although mesoscale nanoparticles (~400 nm) and quasi-one dimensional carbon nanotubes exhibit renal tubular localization and retention^6,29–31. Other particles within a defined particle size range (10–80 nm) can achieve localization to the glomeruli of the kidneys³¹. Microparticles often localize to the lungs through intravenous or inhaled administration^32,33. Inhaled administration has also been used to target nanoparticles into the brain³⁴. Despite these strategies, most particle systems still exhibit some level of liver accumulation and hepatobiliary clearance.

Nanoparticle size and shape are thus key parameters that influence their interaction with biology. Size has been controlled by speed-controlled nano-emulsion, centrifugation, and sonication methods, among others³⁵. While many nanoparticles are spherical, non-spherical particles such as fibrils, discs, and rods can exhibit altered uptake kinetics, sub-cellular localization, and pharmacokinetic profiles³⁶. Methods to control nanoparticle shape include synthesis by deposition of materials into template molding (Particle Replication In Non-wetting Templates: PRINT)³⁷.

A more specific targeting strategy, called “active” targeting, uses molecular recognition elements to bind to cells or tissues of interest. These targeting moieties can include antibodies, peptides, aptamers, and small molecules that are conjugated to the nanoparticle surface to enhance uptake in target tissues³⁸, with potential for valency effects³⁹. The targeted delivery of cargoes to particular cell subsets within a target organ could enhance drug therapeutic index in vivo⁴⁰. However, the biodistribution of particles are not often significantly altered using these methods.

Loading cargoes into nanocarriers can restrict their reactivity, metabolism, and toxicity within cells or an organism⁴¹. This strategy is commonly used for the administration of macromolecular cargoes to effect RNA delivery and gene editing^42,43. However, nanoparticles can allow biologically active compounds, including inhibitors and catalysts, to be used in vivo to address biological questions without the need for substantial chemical modification.

The use of transition metals in living cells is limited. However, transition metal catalysts can be encapsulated within nanoparticles to catalyze biochemical reactions in cells (Figure 2)⁴⁴. A prominent example is polystyrene microspheres crosslinked with a bis-1,ω-acid chloride to entrap Pd²⁺, in turn producing Pd⁰ in situ. (Figure 2b)⁴⁴. Such Pd⁰ nanoparticles can catalyze intracellular Suzuki–Miyaura cross-coupling between arylboronates and aryl triflates for non-enzymatic aryl–aryl bond formation within living cells (Figure 2f). In vitro catalytic activity of nanoparticles was demonstrated via cleavage of bis-allyloxycarbonyl rhodamine (Figure 2d) and allylcarbamate-derivatized amsacrine (Figure 2g). Similarly, catalytic activities of Ru²⁺/Pd²⁺ immobilized on gold nanoparticles⁴⁵ have been demonstrated within live cells as demonstrated by removing the allylcarbamate of bis-N,N′-allyloxycarbonyl rhodamine (Figure 2d) and the propargyl group of N1-propargyl-5-fluorouracil (Figure 2e), respectively, inside living cells. This interaction can be reversible via host–guest chemistry. This exogenous regulation thus provides a modulation mechanism mimicking the allosteric properties of certain enzymes. These approaches thus provide examples to explore metals for live-cell catalysis of diverse organic reactions, potentially facilitating in situ production of bioactive molecules.

Figure 2.

Nanoparticle-based delivery of transition metal catalysts for intracellular chemical reactions. a, Transition metal catalysts are immobilized onto the surface of a nanoparticle to facilitate intracellular chemical transformations. b, Encapsulation of Pd(0) catalyst into the nanoparticle core via stepwise synthesis⁴⁴. c, Encapsulation and modulated accessibility of a metal catalyst⁴⁵. The reversible competitive host-guest interactions of cucurbit[7]uril (grey) with tertiary amine moieties surface-coated on nanoparticles (dark blue) or 1-adamantylamine (brown) could modulate the accessibility of transition metal catalyst to inactive substrate (light blue). Intracellular reactions catalyzed by transition-metal-encapsulated nanoparticles including: d, Activation of Rhodamine 110⁴⁴. e, Activation of 5-FU⁴⁵. f, Intracellular Suzuki–Miyaura cross-coupling to generate a mitochondria-localized fluorescent compound⁴⁴. g, Activation of Amsacrine⁴⁴.

Synthetic small interfering RNA (siRNA) holds strong potential for studying the effects of specific pathways in biological systems, although they are particularly susceptible to nuclease degradation. Nanoformulations of siRNA and mRNA with lipids and polymers have demonstrated the ability to abrogate siRNA degradation and facilitate in vitro or in vivo use⁴⁶. An additional timely example is the recent FDA approval of the first-in-class therapeutic patisiran, comprised of liposomal formulated siRNA to treat a hereditary liver disease⁴³.

Nanotechnologies have also facilitated the delivery of metabolically-active synthetic compounds to specific cell types for use in subsequent bio-orthogonal reactions. Prior work loaded 9-azido sialic acid (9AzSia) into efficiently-internalized folate-coated PEGylated liposomal nanoparticles⁴⁷. The cargo was then available for biosynthesis of cell-surface glycans and fluorescent labeling. RGD-peptide targeted liposomes encapsulating 9AzSia have been used to target xenografted B16–F10 cells in vivo as demonstrated by in vivo copper-free click chemistry. The same group further evaluated additional ligands to address this issue, including the antibody trastuzumab (Herceptin), an aptamer, and glycan ligands to target additional receptors⁴⁸.

Because of the diverse nanocarrier materials available, chemical biologists must choose materials for their specific needs. For a given compound, it is unlikely that a chemical biologist can exhaustively evaluate the appropriate strategy. It will thus be helpful for nanotechnologists to compare nanomaterials in parallel and provide general guidance for chemical biologists. Admittedly, cargoes harboring diverse chemical and physical properties may behave differently upon choosing the best carrier. In lieu of direct collaborations, the availability of general algorithms or plug-and-play solutions would be welcome by the chemical biology community. It would be of interest to nanotechnologists and chemical biologists to make nanocarrier materials available as kits for rapid translation of ideas to the bench.

Enzymatic Nanoreactors

Nanomaterial scaffolds have been applied to reactors for enzymatic catalysis. These materials facilitate the regulation and enhancement of catalyzed reactions controlled by the nanoparticle, enabling potential reaction cascades (Figure 1 blue quadrant). Compared with conventional homogenous assays in solution, enzyme nanoreactors provide the unprecedented ability to constrain the catalysis of enzymes (Figure 3). This technique enhances stability and activity of enzymes, allowing enzymatic reactions to be conducted in new contexts⁴⁹. Moreover, enzyme nanoreactors can compartmentalize multiple enzymes to control biological cascades⁵⁰.

Figure 3.

Representative examples of enzymatic nanoreactors. a, A pH-sensitive nanoreactor for interphase hydrolysis of cellulose to glycose catalyzed by cellulase⁵¹. b, Encapsulation of superoxide dismutase and catalase into Aluminum-based metal-organic frameworks (PCN-333) enhances the active duration and resistance to acidic stress/proteases in the intracellular environment⁵². c, Co-expression of small and large subunits of [Ni-Fe] hydrogenase EcHyd-1 with scaffold protein and coat protein in E. coli leads to the in vivo self-assembly of EcHyd-1 encapsulated in bacteriophage P22 capsids⁵³. d, A supramolecular self-assembling nanoreactor for DNA-mediated dynamic recruitment and regulation of β-lactamase in cells⁵⁴. BTA: benzene-1,3,5-tricarboxamide; BLIP: β-lactamase inhibitor protein. e, A DNA-origami based multi-enzyme cascade nanoreactor, which is switchable between two metabolic pathways⁵⁵.

Enzymatic stability and spatial control are significant enhancements imported by nanotechnology. A nanoscale “enzymogel” reactor for cellulase-catalyzed hydrolysis, consisting of an inorganic core and polymer shell, has been reported (Figure 3a)⁵¹. The flexible poly(acrylic acid) shell is negatively-charged and thus readily binds to positively charged enzymes such as cellulase. Cellulase loading was reversible and its mobility maintained within the shell, facilitating hydrolysis at the boundary between shell and cellulose substrate. Another example demonstrated the utility of metal-organic frameworks (MOFs) as an encapsulation platform for antioxidative enzymes (Figure 3b)⁵². This work found that superoxide dismutase and catalase can be co-trapped within the MOF nanoparticle. Compared with free enzymes, encapsulation maintained their activities and chemical resistance, as demonstrated in cell culture. Another study found that enzymatic stability and protection were improved by bacteriophage-based enzyme caging (Figure 3c)⁵³. In this work, the [Ni-Fe] hydrogenase interaction with a scaffold protein in E. coli allowed self-assembly into bacteriophage P22 capsids, exhibiting a significant increase in activity.

Enzymatic nanoareactors can also allow temporal and multiplexed control over enzymatic reactions. A DNA-mediated self-assembled nanoreactor (Figure 3d)⁵⁴ has exhibited programmable and reversible enzyme recruitment, allowing spatiotemporal reaction control. Another DNA-mediated nanoscaffold has demonstrated the ability to switch between malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) (Figure 3e)⁵⁵. The authors immobilized the enzymes glucose-6-phosphate dehydrogenase (G6pDH), and a cofactor (NAD+) on single-planar DNA-based nanoparticle. The position of the cofactor NAD⁺ could be anchored in proximity with either MDH or LDH in a switchable manner and could be controlled by the DNA-based nanotechnology.

In addition to the above examples, various studies show that nanoparticles alone could mimic the activity of enzymes and catalyze diverse biochemical transformations following Michaelis-Menten kinetics. Such systems are termed “nanozymes” and include⁵⁶: (1) cerium oxide nanoparticles mimicking superoxide dismutase, catalase, and oxidase to scavenge reactive oxygen species generated in radiotherapy; (2) Fe₃O₄ magnetic nanoparticles, iron chalcogenides, single-walled carbon nanotubes, and graphene oxide functioning as peroxidases; (3) zinc-anchored gold nanoparticles mimicking the activity of phosphodiesterase; and (4) molybdenum trioxide nanoparticles that demonstrate sulfite oxidase-like activity. Nanozymes display high catalytic efficiency, resistance to denaturing, and tunable activities derived from the redox potentials of metal ions and programmable acidity, charge, and exposed coordination sites⁵⁷. Chemical biologists may find benefit in broadening these technologies to additional enzyme families while using similar examples to address biological questions.

Nanoparticle-Based Molecular Imaging

Nanotechnologists have developed many biological imaging tools (Figure 4). “Molecular Imaging” tools on the nanoscale often employ the unique physicochemical properties of nanoscale materials, for a diverse array of imaging modalities, for use either in vitro (including within live cells) or in vivo (Figure 1, yellow quadrant).

Figure 4.

Nanotechnologies for imaging applications. This application can be classified into four modules: (a) fluorescent dye-loaded/conjugated nanoparticles, (b) intrinsically fluorescent nanoparticles, (c) nanoparticles for several radioiamaging modalities, and (d) dye-bound gold nanoparticle for Raman scattering-based imaging. Scale bar is approximate.

Nanoscale imaging agents may be used to increase photostability, decrease systemic toxicity, and improve multiplexing in imaging studies (Figure 5). Nanoscale probes may also enable single-molecule and multi-modality imaging capabilities⁵⁸. They fall into two broad categories: 1) incorporating small molecules, such as radiotracers or fluorescent dyes, into a nanostructure, and 2) nanoscale materials that have measurable signal themselves. While a handful of nanoimaging agents are used clinically, and several are commercially available as research tools, many nascent technologies are currently at the initial demonstration stage. As a result, there is a surfeit of underutilized molecular probe technology that has not been examined for non-clinical, in-depth biological investigations.

Figure 5.

Examples of optical properties unique to certain nanomaterial imaging agents. a) A large Stokes shift compared to organic fluorophores. b) A narrow full-width half-maximum (FWHM) compared to organic fluorophores. c) Unique photostability as compared to organic fluorophores.

Loading fluorophores into nanoparticles can increase signal, improve biodistribution profile, allow for improved targeting, and protect dyes from photobleaching⁵⁹. Due to the widespread use of fluorescence imaging, and the advantages of fluorescent nanoparticles, they are available commercially. In addition to simple fluorescence output, more complex nanostructures may be designed for sub-cellular targeting, similar to drug delivery. One example is silica nanoparticles, which may be easily loaded with a wide range of fluorophores, conjugated with modular ligands, and even co-loaded with radioimaging probes^58,59. Sol-gels may be doped in a similar manner and hydrophilic polymers such as cellulose, or hydrophobic polymers such as polystyrene may be used to encapsulate hydrophilic and hydrophobic molecules respectively⁶⁰.

Nanomaterials that function as imaging agents, such as quantum dots and carbon nanostructures, may be used for similar applications as fluorescent dyes, and may confer additional advantages. These structures can exhibit tissue-transparent near-infrared emission, high photostability, single-molecule resolution, and narrow emission bandwidths that can confer multiplexing capabilities⁶¹. Quantum dots (QDs) are fluorescent particles with diameters of a few nanometers that exhibit high quantum yield and photochemical stability. The absorption and emission profiles of QDs are both composition- and size-dependent⁶² with typically narrow, symmetrical, and tunable bands. Single-walled carbon nanotubes (SWCNTs) also exhibit inherent, photostable, tissue-penetrant near-infrared fluorescence between 800 nm and 1600 nm^{63–65,66,67}. Carbon dots are 2–8 nm carbon structures that exhibit tunable excitation and emission in the visible-to-near-infrared range. Their emission is stable and exhibit strong biocompatibility⁶⁸. Fluorescent imaging via nitrogen vacancy centers of nanodiamonds is also gaining utility through surface functionalization⁶⁹, similar to other nanomaterials in this class.

Magnetic resonance imaging (MRI) is a powerful tool for noninvasive clinical diagnosis due to its soft tissue contrast, spatial resolution, and depth of penetration⁷⁰. Magnetic contrast agents are used to improve signal and image analysis, though conventional magnetic contrast agents can be resolution-limited. Iron oxide and lanthanide nanoparticles are used to enhance the signal-to-noise ratio (SNR) of conventional MRI⁷¹ via shortened relaxation times (T1 and T2). Thus, it is unsurprising that these probes have found commercial and clinical use⁷². Computed tomography (CT) contrast agents are largely based on iodinated molecules that absorb X-rays. Nanoparticles composed of gold or other metals have been developed that allow greater X-ray absorption⁷³ compared to traditional iodinated contrast agents. Positron emission tomography (PET) imaging is often performed in addition to MRI and CT. Nanomaterials have been synthesized that incorporate radiolabels, such as ⁸⁹Zr, to image tumors⁷⁴. Several nanoparticles simultaneously incorporate multiple imaging modalities, such as fluorescence or MRI capabilities, into single particle formulations⁷⁵. A notable example due to its clinical translation is a silica nanoparticle technology (C-dots) encapsulating fluorescent dyes and incorporating PET imaging agents⁵⁹.

Raman scattering is a vibrational phenomenon that imparts chemical information and can be measured via spectroscopic and imaging methods. Although relatively weak, it can be amplified by nanomaterials⁷⁶. Benefits include high photostability and narrow spectral bands facilitating multiplexed imaging⁷⁷. Raman probes have been used in vivo to detect and image nucleic acids, lipids, proteins, and other biomolecules of interest⁷⁸. Metallic nanoparticles are common, as they produce relatively strong Raman signals, and surface-enhanced Raman scattering probes (SERS) are commonly used bioimaging agents.

Upconverting nanoparticles exhibit increased depth of penetration as this property results from two low-energy excitation photons causing the emission of a single higher-energy photon from the material. These materials are of interest for use in optogenetic manipulation, where targeted upconverting nanoparticles may add enhanced targeting and precision to optogenetic therapies, in addition to their imaging modality⁷⁹. Additionally, they may be designed to produce signal only in the presence of a target, such as microRNA inside of a cell⁷⁹.

Nanoscale Sensors

Nanoscale biosensors enable quantitative measurements of diverse analytes, often within living biological systems (Figure 6). Similar to molecular imaging, “sensing” with nanoscale materials relies on the unique physicochemical properties of the material to detect analytes through various detection modalities, both in vitro (including within living cells) and in vivo (Figure 1, green quadrant). Analytes can include metabolites, such as lipids, metal ions, nucleic acids, proteins, intracellular environments (such as pH, reactive oxygen species), and others. Signal transduction can be electrical, optical, mechanical, or via other mechanisms (Figure 6) using materials including carbon nanostructures, metals, semiconductors, nucleic acid-based nanostructures, and others (Table 2). Often new materials are studied, as opposed to studying biological applications of existing materials. Strong potential exists to harness nanosensor technologies to interrogate biological systems.

Figure 6.

Examples of nanoscale sensor strategies. Among representative nanoscale sensor strategies are: (a) ‘turn-on sensors’ triggered by fluorescence de-quenching, (b) a silicon nanowire-based electronic sensor, (c) a solvatochromic single-walled carbon nanotube-based sensor, and (d) a FRET (Förster Resonance Energy Transfer) sensor.

Table 2.

Examples of nanosensors and their characteristics.

Nanoscale Sensor Material	Context	Common Analytes	Performance
Dye-functionalized nanostructures79–83	mostly in vitro and in cells some in vivo examples	nucleic acid sequences - including single-base mutations proteins enzymes	strong sensitivity signal amplification subject to false positives/negatives light penetration into biological specimens dependent on the dye
Carbon nanostructures84–87,89–94	in vitro in cells in vivo	small molecules proteins DNA/RNA	Single-molecule sensitivity signal penetration into tissues multiplexing specialized instrumentation often needed
Silicon nanowires88	primarily in vitro	ions/pH proteins nucleic acids	very high sensitivity usually requires electrical contacts preventing use within cells
Quantum dots/silica nanoparticles58,59,61,62	in cells in vivo	ions/pH	bright signals subject to potential biocompatibility/toxicity issues

Nanosensors may be conjugates or aggregates of organic dyes programmed to quench or de-quench, enabling triggered signal changes in fluorescence emission. Fluorescence restoration from due to nanomaterial decoupling is a common technique demonstrated in detection of DNA sequences and single-base mutations using self-assembled gold nanoparticle-oligonucleotide hybrids⁸⁰. In cells, imaging of enzyme-triggered self-assembly of nanofibers from designed chemical precursors allowed the detection of inhibitors for those enzymes, potentially forming the basis of a screening assay⁸¹. Larger biomolecules, like microRNA, can also be detected in live cells using self-assembled nanoparticles⁷⁹. In vivo examples include self-assembled magnetic “nanogrenades” and peptide-functionalized gold nanoparticles were used to detect intra-tumoral pH and proteolytic activity of trypsin, respectively^82,83.

Carbon nanostructures are frequently employed as sensors, including carbon dots, graphene, and SWCNTs. In vitro, SWCNTs have been used to detect molecules such as dopamine⁸⁴ and protein cancer biomarkers⁸⁵, while thrombin has been detected with graphene based aptasensors⁸⁶. Significant progress has been made in developing carbon-based nanosensors to detect nucleic acids in vitro, reviewed elsewhere⁸⁷. Silicon nanowires can have high sensitivity due to depletion or accumulation of charge carriers, with a specific example being femtomolar detection of nucleic acid⁸⁸. Carbon nanostructures have also been applied for sensing applications in living cells and in vivo. These include examples such as an graphene-functionalized aptasensor which detects nucleolin overexpression in cancer cells⁸⁹. Tissue-transparent SWCNT photoluminescence is exquisitely sensitive to its local environment, allowing intracellular sensors for lipids⁹⁰ and nucleic acids¹⁶ via hyperspectral microscopy⁹¹. Recent in vivo SWCNT applications include detecting reactive oxygen species⁹², exogenous microRNA⁹³, lipids⁹⁴, and endogenous protein cancer biomarkers⁹⁵ in mice.

While nanoscale sensors have been developed to examine important bioanalytes, few have been applied to address fundamental biological questions. Small molecule fluorescent probes have long been used by the biological community, but certain drawbacks remain, including difficulties with producing quantitative measurements, low photostability which often limits transient measurements, and limited in vivo use. The unique properties of nanosensors (Table 2) can potentially address these weaknesses if properly developed in collaboration with chemical biologists. Among the successful examples are nanoscale sensors for subcellular lipids⁹⁰. Few are available commercially, making them relatively inaccessible to the chemical biology community. Furthermore, there are numerous bioanalytes of interest to chemical biologists such as short-chain fatty acids as building blocks of reversible lysine posttranslational modifications, but only a small fraction can be probed with nanosensors. Such technology gaps could be addressed more rapidly through collaborative efforts.

Nanomaterial Biocompatibility

Nanomaterials interact with biological systems in novel ways, which can be leveraged to develop new tools, though these interactions may also perturb the system. Nanomaterials necessarily interact with the cell during uptake, intra-cellular transport, and clearance. These processes are modulated by nanomaterials parameters such as size, surface chemistry, and biodegradability³⁷. Depending on the mode of interaction, toxicities can occur via different molecular mechanisms, including metallic particles generating oxidative stress within the mitochrondria, long multi-walled carbon nanotubes mechanically damaging the lysosomes, or quantum dots leaching selenium and cadmium from their cores⁶². Various strategies such as surface passivation exist to mitigate these issues and have been successfully demonstrated for the different classes of nanomaterials reviewed in this work²³ However, close collaboration between nanotechnologists and chemical biologists is thus essential to effectively leverage the capabilities of nanomaterials to ensure that deleterious interactions do not interfere with biological measurements. For example, nanomaterials consisting of iron (III) as delivery cargos⁷¹ should be avoided when exploring biological questions involved with reactive oxygen species.

Future Outlook

Nanotechnologists have developed tools of potential interest for chemical, cell, and disease biology research. Biocompatible carriers have been developed to solubilize and stabilize compounds, and to target locations in live cells and animals while attenuating toxicities. Nano-imaging agents are capable of high-resolution snapshots of biological processes in real-time at single-molecule resolution, in live cells, and in vivo. Nanosensors can detect wide ranges of bioanalytes within live cells and in vivo, allowing researchers to better interrogate chemical or biological perturbations. Therapeutic delivery and stable signal transduction from biocompatible nanoreporters, along with high resolution imaging modalities, can be used to gain insights into important biological processes. While nanotechnology holds great promise for chemical biology and other fields, there are necessary advancements to be made. Areas of future progress include addressing biocompatibility/toxicity questions associated with certain materials, improving in vivo and subcellular localization strategies, increasing drug encapsulation/conjugation, and additional physicochemical control. More broadly, the field of nanotechnology must improve communication with chemical biologists, make their materials available to the community, and work to address fundamental biological questions, which we hope to inspire herein. In addition, chemical biologists will benefit from quantitative guidance upon comparing and choosing nanomaterials among various choices to meet context-specific needs. Despite these areas for improvement, nanotechnology holds promise for expanding cutting-edge science and innovation at the interface of chemistry and biology.

Chemical biologists have also made substantial contributions that have advanced nanotechnology by providing access to useful molecules and molecular tools. Some notable examples include development of remarkable biorthogonal reactions such as “copper-free click chemistry” that allows metal-free chemical conjugation in biological systems⁹⁶, as well as Diels-Alder reactions⁹⁷. Further, materials manipulation techniques, such as with synthetic peptides and DNA, were developed for chemical biology. Direct research collaborations between scientists in chemical biology and nanotechnology can thus improve the engineer’s toolkit as well as that of the biologist.

Many nanoscale delivery, imaging, and sensing tools have not been applied to address biological questions. With relatively few exceptions, outstanding tools and technologies developed in nanotechnology laboratories have remained investigative and have yet to be used to solve unmet biological problems. Many nanotechnology groups appear to focus on materials development or clinical applications. In contrast, the current generation of chemical biologists focuses on tool development and carefully consider utilities of these chemical tools in relevant biological contexts. Synergistic collaboration between nanotechnologists and chemical biologists may leverage these perspective strengths, with additional likelihood for clinical technology translation. We envision that this perspective may inspire not only nanotechnology experts to realize potentially promising applications in chemical biology and contribute to the existing toolbox, but also that chemical biologists may increasingly take advantage of advancements in nanotechnology.