Gold nanocages (AuNCs) are gold nanoparticles of size 20-500 nm, with a hollow cubic structure and porous walls. They can be synthesized by reacting silver nanoparticles with chloroauric acid (HAuCl₄) in boiling water.^[1] Gold nanocages have been suggested for use in drug delivery, photothermal therapy, and as contrast agents.^[2]

Gold nanocages were first created in 2002 by a group at Washington University, Saint Louis, led by Younan Xia. According to Xia, he developed the idea for the synthesis based on a coincidence: he happened to be teaching galvanic replacement in a general chemistry course at the same time as a method for silver nanocube creation via polyol reduction was being developed.^[2]

Since the invention of AuNCs, one major branch of research has focused on the development of alternative synthesis strategies for more precise tuning of nanocage structure and properties.^[3][4][5] Another significant area of nanocage development has been the investigation of their biomedical applications. In 2007, gold nanocages were first demonstrated as agents for photoacoustic tomography and photothermal cancer therapy.^[2] In 2009, the first research was published demonstrating the use of AuNCs for controlled drug delivery.^[2] Subsequent work has investigated further applications of gold nanocages in biomedicine and attempted to combine multiple applications for more comprehensive treatment.^[6]

The fundamental reaction in the preparation of gold nanocages is a galvanic replacement reaction between chloroauric acid (HAuCl₄) and “sacrificial templates” made of Ag nanostructures.^[5] Galvanic replacement syntheses are reactions in which a salt precursor to a certain element undergoes reduction by a substrate, leading to the deposition of that element onto the substrate surface.^[7] The driving factor in the galvanic replacement syntheses of AuNCs from Ag is the electrochemical potential difference between the two metals: Ag/Ag⁺ has an electrochemical potential of 0.80 V, which is less than the electrochemical potential of Au/AuCl4− (1.00 V). Due to this difference, Ag can be oxidized by the chloroauric acid:^[5]

3Ag_(s) + HAuCl_4(aq) → Au_(s) + 3AgCl_(s) + HCl_(aq)

The reaction can be performed as a titration, in which HAuCl₄ is added to a boiling Ag-nanostructure solution.^[5] As the reaction proceeds, the gold in HAuCl₄ is reduced to atomic gold and is deposited epitaxially on the surface of the Ag nanostructures, resulting in Au/Ag alloy shells with a core of unreacted Ag.^[6] As more HAuCl₄ is added, Ag is oxidized and dealloyed, leading to the creation of porous, hollow nanocage structures composed of an Au-Ag alloy dominated by Au.^[5][6] Dealloying is halted before only pure Au remains, as complete dealloying has been shown to fragment the nanocage structure.^[5]

Silver nanotemplates (often nanocubes) can be synthesized via a polyol reduction in which ethylene glycol is oxidized by atmospheric oxygen to form glycolaldehyde. Glycolaldehyde can then be used to reduce Ag⁺ into elemental Ag.^[2][8]

2Ag⁺_(aq) + HOCH₂CHO_(aq) + H₂O_(l) → 2Ag_(s) + HOCH₂COOH_(aq) + 2H⁺_(aq)

Polyol reduction has been used to synthesize nanoparticles since its introduction by Fievet, Lagier, and Figlarz in 1989.^[9] In traditional polyol reductions, elemental Ag is produced using AgNO₃ as a precursor.^[3] However, initial attempts at forming Ag nanocubes using AgNO₃ found that the silver yield was extremely sensitive to various reaction conditions (like impurities and O₂ amount).^[3] As of 2011, silver trifluoroacetate (CF₃COOAg) has been presented as an alternative precursor that has shown higher Ag yield and less sensitivity when compared to AgNO₃.^[6]

AuNCs can be formed from different silver nanostructures, including nanocubes with sharp or truncated corners, single-crystal octahedrons with truncated corners, and polycrystalline quasispheres.^[5]

When Ag nanocubes with sharp corners are used as a precursor, Ag dissolution begins at a high-energy site on one of the Ag nanocube faces, usually a hole or defect in the cube structure. This dissolution creates a “pinhole” that serves as the anode in the galvanic replacement reaction, where elemental silver is oxidized and Au in AuCl₄ is reduced, leading to gold deposition on the nanocube surface. As the reaction proceeds, Au deposition and/or mass diffusion lead the "pinhole" to eventually reseal.^[1]

In contrast, when Ag nanocubes with truncated corners are used as a precursor, Ag dissolution begins at all corners of the template. This effect is due to the presence of the stabilizing polymer poly(vinyl pyrrolidone) (PVP) in the reaction mixture. While PVP interacts equally with all surfaces of a sharp-cornered nanocube, in a synthesis with truncated-corner nanocubes, PVP has a much stronger interaction with the {100} faces over the {111} corners. As PVP acts as a stabilizing agent, the lack of interaction at the truncated corners leads Ag dissolution to begin at these sites. This effect leads to the creation of gold nanocages with porosity present at all corners.^[1]

Seed-mediated growth followed by selective etching has been proposed as a more precise alternative to the traditional template-based galvanic reaction: in the traditional synthesis, the simultaneous reduction of AuCl₄ and oxidation of Ag in the galvanic reaction can lead to difficulties in controlling nanocage structure (like the thickness of cage walls). In this alternative synthesis, a strong reductant like NaOH is added to the reaction mixture, reducing Au³⁺ ions faster than the galvanic replacement reaction. Patches of Ag₂O are formed at the corners of the Ag nanocubes; these patches can then be selectively etched using a weak acid that also dissolves the center of the cube, producing a gold nanocage.^[10] While offering control of cage wall thickness down to one atomic layer, seed-mediated growth and etching necessitates further reaction steps and more precise reaction conditions when compared to the traditional synthetic method.^[4]

The synthesis and development of gold nanocages at various stages can be visualized using common electron microscopy techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM).^[1] X-ray ptychography and scanning wide-angle nanoprobe diffraction (WAXS) have also been used to image the galvanic reaction process, allowing for differentiation between different compounds in the developing nanocages and visualization of their crystalline structure.^[11]

The synthesis of AuNCs produces structures that can range in size from 20-500 nm, with wall thicknesses that can be tuned in the range of 2-10 nm (with accuracy up to 0.5 nm).^[2] Being bio-inert and nonreactive, AuNCs are ideal for in vivo biomedical applications.^[2][5] Their hollow centers increase surface area and functionality, as well as allowing them to hold high payloads for drug delivery.^[3]

Much of the advantageous optical properties of gold nanocages derive from the phenomenon of LSPR (localized surface plasmon resonance). When light hits the gold nanocage structure, free-floating electrons begin to collectively oscillate, leading to the absorption and scattering of specific resonant wavelengths of electromagnetic radiation.^[2][12] In smaller gold nanocages, (<45 nm edge length), light is overwhelmingly absorbed, while in larger AuNCs, light is predominantly scattered.^[1]

This LSPR effect leads to the observation that suspensions of gold nanocages can appear to be various colors. The apparent color of the colloidal solution is a function of a particular oscillation frequency, which can be tuned by changing the thickness and porosity of the nanocage walls.^[13]

LSPR is not unique to gold nanocages, and instead is a property of various classes of metal nanostructures. However, conventional (spherical, solid) gold nanoparticles exhibit LSPR peaks that are restricted to the visible light region of the electromagnetic spectrum.^[2] The hollow nature of AuNCs lead to increased surface area, resulting in a significantly higher absorption cross-section than traditional nanoparticles.^[3] This allows for LSPR that can be tunable to 600-1200 nm, in the near-infrared (NIR) region.^1,7 This “tunability” can be achieved by modifying the size of AuNCs and the thickness of their walls, effectively altering the ratio of wall thickness to the overall size of the nanocage.^[1][13][14]

To harness the properties of AuNCs for theranostic applications, it is essential to deliver gold nanocages precisely to targeted areas within the body. The addition of uncoated, bare nanocages to the biological system triggers the body’s immune response, leading to protein deposition on the nanocages and the removal of AuNCs via the bloodstream.^[14] To circumvent this response, nontoxic coatings like polyethylene glycol (PEG) can be applied to the nanocage surface to “disguise” the nanoparticles, allowing them enough circulation time to collect in tumors.^[14] AuNCs can then be directed to malignant cells via passive or active targeting.^[5]

Passive targeting exploits the abnormality of tumor vasculature systems to allow AuNCs to infiltrate the cancer cell. The blood supply system of a tumor is characterized by irregular branching and diameters, as well as leaky, penetrable vessel surfaces. Passive targeting of AuNCs harnesses this morphology as nanocages enter through these openings and accumulate in tumors.^[5][14]

Following the development of surface functionalization of AuNCs, active delivery has also been presented as a nanocage targeting method. Active targeting relies on the interactions between ligands added to nanoparticle surfaces and receptors that are overexpressed in cancer cells.^[5] To chemically functionalize nanocage surfaces, various targeting moieties such as antibodies and peptides can be conjugated post-synthetically to the PEG chains making up the surface coating of the nanocage.^[5] The large surface area of AuNCs means that a large number of ligands can be attached to the nanocage.^[6] Once in the body, ligand-receptor interactions lead to the adhesion and subsequent internalization of AuNCs in tumors.^[5]

AuNCs have shown promise as biosensors: they can be engineered with artificial antibodies to detect biomarkers, or serve as electrochemical transducers for plasmonic sensing.^[15][16] These biosensing capabilities have afforded improved detection of kidney disease and lung cancer in laboratory studies.^[15][16]

As the transparent window for soft tissue and blood lies between 650-900 nm, the ability of gold nanocages to exhibit LSPR in the NIR region gives them a particular advantage as biomedical contrast agents.^[3] They are ideal contrast agents for photoacoustic tomography (PAT), a technique that combines optical and ultrasonic sensing to provide high resolution, penetration, and sensitivity. In PAT, the irradiation of tissue with a laser beam creates ultrasound signals, which are then compiled to provide a three-dimensional model of the targeted area. As the amplitude of the ultrasound signals increases with increasing optical absorption, tunable plasmonic nanoparticles like AuNCs are particularly good contrasts agents for PAT.^[5] They have been shown to have greater effect than gold nanoshells, which is attributed to the smaller size and larger absorption cross-sections of gold nanocages.^[1]

AuNCs are also promising contrast agents for optical coherence tomography (OCT) and spectroscopic optical coherence tomography (SOCT).^[1] The optical coherence tomography system is based on the backscattering of light from a given sample with the interference signals of a reference material. Traditional OCT relies on tissues’ natural scattering and absorption of light, which can be amplified by the inclusion of AuNCs in the system.^[1]

Additionally, gold nanocages functionalized with iron oxide (Fe₃O₄) nanoparticles have been proposed as an ideal candidate as contrast agents for multimodal MRI/CT imaging. Multimodal, noninvasive imaging approaches such as MRI/CT have become increasingly crucial in various diagnostic methods, necessitating the improvement of contrast agents that can enhance their imaging resolution. Gold nanocages coupled to Fe₃O₄ demonstrated improved simultaneous MRI and CT contrast both in vivo and in vitro.^[17]

AuNCs have also been modified to emit Cherenkov radiation, which is emitted as visible and NIR light when radioactive gold-198 undergoes beta decay. Tumor markers are traditionally imaged by exciting the targeted region, which can also illuminate healthy tissue and therefore decrease resolution. By using AuNCs with gold-198 incorporated into the nanocage structure, tumors can be imaged without an external excitation source.^[18]

AuNCs are also promising candidates for photothermal therapy, in which heat is used to selectively kill cancer cells via hyperthermia.^[1] When a laser hits electrons in the nanocage, these electrons become excited; they then undergo electron-phonon coupling with the surrounding lattice and transfer energy to their environment in the form of heat.^[5] The large absorption cross-section of AuNCs makes this conversion incredibly efficient, taking place on timescales of 10-100 picoseconds (ps).^[5][19] The absorption cross-sections of AuNCs are nearly five orders of magnitude higher than a common NIR dye, indocyanine green, leading to superior conversion efficiency.^[2] This large absorption cross-section also leads to superior performance over nanorods: gold nanocages were shown to require 18.4 times less radiation than gold nanorods to increase heat by the same amount.^[19] When nanocages are able to be delivered to and taken up by tumor cells, subsequent exposure to NIR radiation can lead to heat-induced cell death.^[1] Photothermal therapy can be paired with other therapeutic approaches, like the use of immune checkpoint inhibitors, to provide a multi-pronged attack on cancer cells.^[20]

The photothermal properties of AuNCs can be combined with their hollow interiors to provide a novel method of drug delivery and controlled release.^[21] Traditional controlled release systems rely on photolysis with ultraviolet (UV) light to release drug payloads; however, this methodology can damage healthy tissue and is only applicable for in vivo use.^[22] AuNCs can circumvent this drawback due to their absorption of light in the NIR region of the electromagnetic spectrum.^[22] For drug delivery, nanocages can be filled with a specific drug payload and then sealed with a temperature-sensitive polymer. When the nanocages are exposed to NIR radiation, the nanocages’ photothermal effect raises the polymer above its melting point. As the polymer changes phases, the pores of the nanocage are exposed and the drug is released. When the radiation source is removed, the polymer re-seals the cages, allowing for control over drug release.^[14] This “smart” drug delivery system has been demonstrated in various in vitro and in vivo studies, in which researchers have demonstrated the ability of AuNCs to deliver chemotherapy drugs to targeted cells.^[21][23]

• Colloidal gold
• Gold nanorods
• Gold nanoparticles in chemotherapy