Controlled synthesis and assembly of ultra-small nanoclusters for biomedical applications

Qiyue Wang abc, Shuying Wang a, Xi Hu a, Fangyuan Li ab and Daishun Ling *abc
aInstitute of Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China. E-mail: lingds@zju.edu.cn
bHangzhou Institute of Innovative Medicine, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
cKey Laboratory of Biomedical Engineering of the Ministry of Education, College of Biomedical Engineering & Instrument Science, Zhejiang University, Hangzhou 310027, China

Received 27th September 2018 , Accepted 12th November 2018

First published on 13th November 2018


Inorganic nanomaterials have been studied extensively in recent years because of their unique physical and chemical characteristics. Moreover, ultra-small nanoclusters (USNCs), which are less than 3 nm in size, exhibit extraordinary properties different from those of larger-sized nanoparticles. For example, ultra-small iron oxide nanoclusters (NCs) show paramagnetic properties; ultra-small noble metal NCs exhibit bright fluorescence; most USNCs gain in vivo renal clearance capability to guarantee safety. Therefore, USNCs could be promising safe materials for biomedical applications. In this mini-review, we summarize recent advances in the controlled synthesis, assembly, and biomedical applications of USNCs. We also discussed future challenges and perspectives in the development of USNC-based nanomedicine.


image file: c8bm01200h-p1.tif

Qiyue Wang

Qiyue Wang received her B.S. (2017) in the School of Materials Science and Engineering from Zhengzhou University. She is now a doctoral candidate in the College of Pharmaceutical Sciences at Zhejiang University under the guidance of Prof. Daishun Ling. She currently focuses on nanoparticle functionalization and assembly for theranostic applications.

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Shuying Wang

Shuying Wang received her B.S. (2018) in the West China School of Pharmacy from Sichuan University. She is now a doctoral candidate in the College of Pharmaceutical Sciences at Zhejiang University under the guidance of Prof. Daishun Ling. She currently focuses on functional assembly of nanoparticles for diagnostic and therapeutic applications.

image file: c8bm01200h-p3.tif

Daishun Ling

Daishun Ling received his Ph.D. (2013) from School of Chemical and Biological Engineering at Seoul National University. Later, he worked as a senior researcher at the Center for Nanoparticle Research, Institute for Basic Science. He joined the faculty of the College of Pharmaceutical Sciences at Zhejiang University in 2014 through “Zhejiang University 100 Talent Professor” and “National 1000 Young Talent Program”. He currently focuses on the synthesis and assembly of functional nanoparticles for biomedical applications.


1. Introduction

Inorganic nanoparticles (NPs) with sizes of 1–100 nm have been extensively studied in recent decades due to their unique chemical, magnetic, optical, and electrical properties compared with their bulk counterparts.1 In particular, ultra-small nanoclusters (USNCs), composed of several tens to hundreds of atoms, have recently attracted a great deal of research interest.2 The percentage of atoms exposed on the surface of nanoclusters (NCs) dramatically increases as their size decreases. For example, the percentage of surface atoms with respect to the total atoms is up to 76% for 1.2 nm palladium (Pd) clusters, whereas that for 3.4 nm Pd clusters is only 35%.3 Interestingly, the increase in the surface atom ratio of USNCs caused by the reduced size has a significant impact on the physicochemical properties of the materials, which can be beneficial for high-performance biomedical imaging and therapy, and also the inherent small size of USNCs can benefit the efficient body elimination for safety (Fig. 1). For example, ultra-small noble metal NCs, e.g. gold (Au) and silver (Ag) NCs, possessing sizes close to the Fermi wavelength of electrons, can give rise to attenuated surface plasmon resonance, discrete electronic states, and molecule-like optical properties.4 Ultra-small iron oxide NCs (<3 nm) exhibit nearly paramagnetic properties because almost all spins are canted in this size range.5 In addition, the increased proportion of atoms on the surfaces of certain USNCs, such as platinum (Pt) and Ag NCs, endows them with high surface reactivity, causing efficient ion leaching.6,7 However, independent USNCs do not always meet the requirements for therapy and biosafety simultaneously. Accordingly, researchers have been studying the self-assembly of USNCs required to achieve complex nanostructures in a controllable manner, which could yield USNCs with multiple functions and collective properties of differently sized inorganic nanomaterials, facilitating their potential applications.
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Fig. 1 Schematic illustration of the characterization and biomedical applications of USNCs. Examples emphasize the extraordinary intrinsic properties of USNCs and illustrate the feasibility of USNC-based nanomedicine for efficient and safe biomedical applications.

In this review, we summarize the methods and principles for the controlled synthesis and assembly of USNCs. Then, we focus on the recent progress of USNCs and their assembly in biomedical applications. Finally, we provide a brief overview of the challenges and potential opportunities for future USNC research in the biomedical field.

2. Controlled synthesis of USNCs

The synthesis of monodisperse USNCs requires a deep understanding and sophisticated control of the synthetic process. Solution-phase colloidal methods, including techniques for reduction, thermal decomposition, and sol–gel process, have been widely used due to their simplicity and controllability in the synthesis of monodisperse USNCs.8,9 Depending on the polarity of the reaction solution, solution-phase colloidal methods can be divided into aqueous phase synthesis and organic phase synthesis. In this section, instead of summarizing all achievements in USNC synthesis, we will focus on the solution-phase synthetic methods of USNCs for biomedical applications, and some representative examples are listed in Table 1. More synthetic methods for USNCs can refer to some previously published reviews.1,2,10–12
Table 1 Synthetic methods for USNCs
  Nanocluster Ligands Reaction temp. Size Method Ref.
Aqueous phase synthesis Au Bovine serum albumin 37 °C 25 gold atoms Reduction 15
Au Poly-(amidoamine) dendrimers R.T. 1–2 nm Reduction 20
Au Pentaerythritol tetrakis(3-mercaptopropionate)-terminated polymethacrylic acid R.T. 1.1–1.7 nm Reduction 21
Au Polyethylenimine R.T. 8 gold atoms Etching 22
Ag Dihydrolipoic acid R.T. 4/5 silver atoms Reduction 24
Ag Mercaptosuccinic acid R.T. 7/8 silver atoms Etching 25
Ag DNA sequences R.T. 3 nm Reduction 27
Fe3O4 Poly(acrylic acid) 80 °C 2.2 nm Coprecipitation 28
Pt Peptide sequences R.T. 1.7, 2.7, 3.5 nm Reduction 29
Pt Amine-terminated dendrimer R.T. 0.93 ± 0.22 nm Reduction 30
Organic phase synthesis γ-Fe2O3 Oleic acid 200 °C/250 °C 1.5, 2.2, 3.1 nm Thermal decomposition 5
γ-Fe2O3 Long-alkyl-chain amine R.T. 2.8 nm Organometallic approach 33
Pt Oleic acid, oleylamine 170 °C 2.5 nm Thermal decomposition 6


2.1 Aqueous phase synthesis

USNCs synthesized by aqueous phase methods exhibit excellent water dispersibility without further surface modification. Many different methods for synthesizing USNCs in the aqueous phase have been reported. For example, the approach to produce ultra-small Au NCs in aqueous solutions can be generally divided into reduction-based13 and etching-based strategies.14 The mechanisms of these two strategies have not been fully elucidated yet, but some hypotheses have been proposed to explain the synthesis principles. For the reduction-based strategy, reducing ligands strongly binding with metal precursors allowed the reduction of entrapped ions to obtain Au NCs in situ through regulating the pH value of the reaction system to approximately 12.15 However, because NCs tend to aggregate in aqueous solutions, the reduction of metal ions will yield larger NPs rather than NCs.2 Consequently, using appropriate stabilizers to prevent the aggregation of NCs is extremely important for synthesizing ultra-small Au NCs. Thiol-containing molecules, such as glutathione,16 tiopronin,17 and phenylethylthiolate,18 are widely used as stabilizers for synthesizing ultra-small Au NCs because of their high affinity to gold.19 In addition to thiol-containing molecules, other stabilizers, such as dendrimers,20 polymers,21 peptides, and proteins,18 have also been reported. Alternatively, ultra-small Au NCs can be prepared using an etching-based strategy. For example, Duan et al.22 reported a ligand-induced etching process for obtaining ultra-small Au NCs via using polyethylenimine in exchange for the ligand on preformed Au NPs. To explain the mechanism of the etching-based strategy, Muhammed et al. proposed two possible theories:23 (1) Au atoms on the surface are removed by excessive ligands, and Au/ligand complexes then aggregate via aurophilicity to form Au NCs; and (2) the sizes of Au NPs gradually decrease by etching the surface of Au atoms, resulting in the generation of Au NCs.

Similar approaches for synthesizing ultra-small Au NCs can also be used to prepare ultra-small Ag NCs. Adhikari et al.24 used dihydrolipoic acid as a stabilizer and sodium borohydride as a reducing agent to obtain ultra-small Ag NCs. The thiol-capped ultra-small Ag NCs could also be prepared through an interfacial etching reaction at the aqueous/organic interface.25 Moreover, DNA oligonucleotides can be used as an alternative stabilizing agent because Ag+ exhibits tight binding with cytosine.26,27

In addition, appropriate ligands have been shown to facilitate the synthesis of ultra-small iron oxide nanoclusters (USIONs) in aqueous solutions through forming layers around the NP surface to prevent crystal growth. For example, Wang et al.28 reported the synthesis of water-dispersible ∼2.2 nm USIONs via a one-step coprecipitation approach using poly(acrylic acid) (PAA) as both the crystal grain growth inhibitor and stabilizer. A PAA-capped layer formed on the surface of USIONs, limiting their further growth because the carboxyl groups on the PAA chains had strong binding interactions with ferric ions.

The approach combining suitable ligands as stabilizers and inhibitors of crystal growth has also been developed to obtain ultra-small Pt NCs. Li et al.29 prepared monodisperse ultra-small Pt NCs in aqueous solution with controllable sizes ranging from 1.73 to 3.54 nm using peptides as the stabilizer. The peptide sequence Thr-Leu-His-Val-Ser-Ser-Tyr, which was selected using a phage display technique, showed specific binding to Pt NCs based on the interaction between hydroxyl and/or polar residue groups and the Pt surface. The peptide was adopted as the stabilizer to regulate the crystal nucleation and growth processes of Pt NCs and eventually control their morphology and size. Another strategy to achieve the desired Pt NCs is to restrict the crystallization of inorganic precursors in the small pores of macromolecules. For example, an amine-terminated dendrimer (G2NH2) could trap Pt precursors, such as PtCl62−, as a cage, leading to the formation of ultra-small Pt NCs (∼0.93 ± 0.22 nm).30

2.2 Organic phase synthesis

Organic phase synthesis methods have recently been developed to obtain USNCs with higher crystallinity and monodispersity than those obtained by aqueous phase synthesis methods.9 Hyeon and coworkers reported ultra-large-scale synthesis of monodisperse iron oxide nanoparticles (IONPs) in organic solutions using inexpensive and nontoxic Fe(oleate)3 as the precursor via the thermal decomposition method, in which the size of IONPs could be fine-tuned from 5 to 22 nm.31 Recently, they further synthesized monodisperse USIONs with diameters of approximately 1.5, 2, and 3 nm by decreasing the ratio of oleyl alcohol to oleic acid and increasing the reaction temperature.5 In general, during the nucleation process, smaller nuclei tend to dissolve back into the solution as a result of the decrease in the supersaturation level.32 However, in this reaction mixture, oleyl alcohol is able to decrease the reaction temperature by reducing Fe(oleate)3, whereas the low temperature inhibits smaller nuclei from dissolving, resulting in an increase in the number of nuclei in solution. Given the fixed amount of iron atoms, the decreased level of supersaturation could suppress the growth of the NP. In addition to the thermal decomposition reaction, USIONs stabilized by long-alkyl-chain amines (octylamine, dodecylamine, or hexadecylamine) have also been successfully prepared at room temperature in an organic solvent (tetrahydrofuran or toluene) following the hydrolysis and oxidation of an organometallic precursor, Fe[N(SiMe3)2]2.33

The thermal decomposition methods have also been applied to synthesize ultra-small Pt NCs. Xia et al. regulated the amounts of Pt(acac)2, oleic acid, and oleylamine to tune the sizes of Pt NCs. A high concentration of oleylamine (as both the surfactant and the solvent) was exploited to achieve colloidal uniformity. To obtain an ultra-small size, oleic acid and superhydride were added to the reaction system, yielding Pt NCs with a size of around 2.5 nm.6

High temperature organic phase synthesis methods could also be used for synthesizing ultrasmall-sized Gd3+-doped upconversion nanoparticles (UCNPs), which exhibited higher r1 relaxivity than the commercially used gadopentetate dimeglumine.34,35

3. Controlled assembly of USNCs

Controlled assembly makes use of a chemical design to achieve the regulation and enhancement of USNC performance, which plays a significant role in biomedical applications. For example, the controlled assembly of USIONs can “turn on” T1 magnetic resonance (MR) imaging when they disassemble in tumor tissues for the early stage diagnosis of tumors.36 pH-Sensitive Pt NC assemblies can accelerate Pt2+ release in cancer cells, thus improving the safety of the materials and overcoming drug resistance and heterogeneous stemness of hepatocellular carcinoma.6 Ultra-small ceria nanocrystal-decorated mesoporous silica nanoparticles (MSNs) not only restrict reactive oxygen species (ROS) generation but also have high tissue adhesion strength, which could be used for wound healing.37 In this section, we will introduce the controlled assembly of USNCs directed by polar molecule-mediated interactions, including hydrophobic interactions, hydrogen-bonding interactions and dipole–dipole interactions, as well as by template-based methods (Fig. 2).
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Fig. 2 Examples illustrate the controlled assembly methods of USNCs including polar molecule-mediated interactions and template-based methods.

3.1. Assembly directed by polar molecule-mediated interactions

3.1.1. Hydrophobic interactions. Hydrophobic interactions play important roles in the controlled assembly of USNCs, which are realized by grafting USNCs to or encapsulating USNCs in amphiphilic polymers based on structural alterations of amphiphilic polymers in aqueous solutions. Furthermore, polymers can be easily tailored to achieve the stimulus-responsive assembly of USNCs, which serve as a platform for activating the diagnostic or therapeutic functions of assembly at the targeting site. For example, pH changes are widely used as effective triggers for the controlled stimulus-responsive assembly of USNCs. The pH stimulus-responsive assembly can be prepared by grafting some pH-sensitive functional groups on polymers, such as imidazole or tertiary amines, which become protonated and switch to a hydrophilic state under acidic pH conditions. Wang et al.38 designed triblock copolymer-treated tertiary amines as pH-sensitive ligands for the assembly of paclitaxel and quantum dots (QDs). Our group has designed a pH-sensitive polymer for controlled dynamic assembly of USNCs.36 Initially, imidazole groups and chlorin e6 grafted poly(ethylene glycol)-poly(β-benzyl-L-aspartate) were synthesized as a pH-sensitive platform ligand. Then, catechol groups were added to the platform ligand to produce ligand A, facilitating the controlled assembly of USIONs. Next, 3-phenyl-1-propylamine was engineered on the platform ligand to fabricate ligand B, aiming at tuning the ligand hydrophobicity so that a suitable phase transition could be achieved to realize the following pH-triggered disassembly inside cells. As such, a type of pH-sensitive magnetic nano-assembly based on USIONs, ligand A and ligand B was fabricated via a dual solvent-exchange method and could be a promising agent for the early-stage diagnosis and treatment of tumors.
3.1.2. Hydrogen-bonding interactions. DNA molecules have also been widely used for nano-assembly owing to their sequence specificity and structural versatility.39 Based on the principle of complementary base pairing, the hydrogen-bonding interaction allows the spontaneous and controllable assembly of DNA-functionalized USNCs. For example, our group designed a novel iron oxide nano-assembly with the diameter of around 120 nm assembled by ∼3 nm USIONs through DNA linkers.40 All USIONs were initially functionalized using single-strand anchor DNAs, followed by a mixture of i-motif DNA linkers containing complementary sequence regions of anchor DNAs. Eventually, the nano-assembly was formed via hydrogen-bonding interactions. Notably, the DNA-programmable nano-assembly was much more flexible and changeable because the interaction between NPs and DNA molecules could be tuned by changing the DNA sequence and length, the presence or absence of nonbonding single-base DNA sequences inside the linkage, and changes in the surrounding environment.41 In the case mentioned above, the i-motif structure of the DNA linkage was single-stranded with no bonding when in neutral pH but transformed to a quadruplex structure while the pH changed from 7.4 to 5.5, thereby allowing the changeable structure of the USION-based nano-assembly.
3.1.3. Dipole–dipole interactions. The controlled assembly of USNCs can also be achieved through the surface modification of light-responsive polymers, such as azobenzene, which could be isomerized under light to induce self-assembly. For example, Manna et al. synthesized 2.5 nm Au NPs functionalized by thiolated 4-(dimethylamino)azobenzene in a non-polar solvent.42 Under irradiation with blue light (420 nm), the azobenzene-derived polymer would undergo trans to cis isomerization, inducing molecular dipoles of the azobenzene units. As a result, the self-assembly process was initiated through the dipole–dipole interaction of cis-azobenzene moieties.43 Moreover, the next exposure to near-UV light (365 nm) could reverse the conformational change, i.e., switch from cis to trans and consequently cause the disassembly of clustered Au NPs.

3.2. Assembly directed by template-based methods

In addition to the polar molecule-mediated interactions described above, templates can also direct the controlled assembly of USNCs. MSNs are one of the most common templates for directing the assembly of USNCs due to their good biocompatibility and easy surface functionalization. Our group prepared the assembly of ultra-small iron oxide nanocrystals and ceria nanocrystals onto the surface of MSNs for combinational therapy in patients with Alzheimer's disease. The nanocrystals were modified with bromo-2-methylpropionic acid and subsequently anchored to MSNs modified with 3-aminopropyltriethoxysilane via the reaction between bromine and amino groups.44 Wu et al.45 used hollow mesoporous silica spheres (HMSSs) as a template for the assembly of ZnO QDs on the outer surface via disulfide-conjugated two-amide linkages, and the anticancer drug doxorubicin (DOX) was encapsulated within the HMSS cavities. When the disulfide bonds were broken following induction by the relatively high concentration of glutathione in cancer cells,46 ZnO QDs would be separated from HMSSs, subsequently leading to massive release of DOX.

In addition to using inorganic nanomaterials with rigid geometries as templates, biological materials, such as DNA scaffolds47 or viruses,48 can also be applied for the controlled assembly of USNCs. For example, Fischler et al.47 developed a type of chain-like assembly of azide-functionalized Au NCs (1.6 nm in diameter) on alkyne-modified DNA templates via the copper(I)-catalyzed Huisgen cycloaddition. Although biological material-based templates may be difficult to obtain assemblies with well-defined shapes, they can provide fixed binding sites for USNCs and thus controllably arrange the number and spatial distribution of USNCs in the assembly.

4. Biomedical applications

The physicochemical properties of USNCs are distinct from those of large-sized nanoparticles. For example, USIONs exhibit paramagnetism; ultra-small noble-metal NCs are fluorescent;49–51 other types of USNCs show high surface reactivity52,53 and outstanding renal excretion properties.54,55 Controlled stimulus-responsive assembly technology can endow USNCs with improved performance (for example, multifunctionality and long blood circulation time) and control their functionality via biological stimuli (for example, controllable drug release and “turn on/off” adjustment of imaging). USNCs and their assemblies have attracted significant attention in biomedical fields based on their unique properties and exhibited great potential in diagnostic and therapeutic applications.

4.1. Biomedical imaging

4.1.1. Magnetic resonance imaging (MRI). IONPs have been widely used as T2 MR contrast agents (CAs).56,57 Nevertheless, T2-weighted MRI generates dark signals, which may cause misdiagnosis.58 In contrast, clinical diagnosis is more successful when using T1-weighted imaging with bright signals. However, United States Food and Drug Administration (FDA)-approved gadolinium (Gd)-based T1 CAs may have long-term toxicity resulting from the leakage of Gd3+ ions59 or the accumulation of Gd in the bone60 and the brain.61 Therefore, safer and more efficient T1 CAs need to be exploited. USIONs have been reported to have great potential as T1 CAs.5,62 The superiority of USIONs for T1 MRI can be explained by the spin canting effect, in which atoms on the surface of IONPs are magnetically disordered, generating a disordered layer of approximately 0.9 nm thickness.63 Therefore, the proportion of magnetically disordered atoms increases sharply as the size decreases, resulting in dramatically reduced saturation magnetization (Ms). Hence, the Ms of USIONs with diameters of 1.5, 2.2, and 3 nm is much lower than that of 12 nm-sized IONPs.5 Except for the influence of Ms, r1 relaxivity arising from the inner-sphere water, which directly coordinates with CAs, is also affected by the hydration number (q) and residence time (τm).64
image file: c8bm01200h-t1.tif

In the equation, rIS1 is the inner-sphere relaxivity, q is the hydration number, T1m is T1 in the inner sphere, and τm is the residency time of water. The r1 relaxivity can be improved by either increasing q or decreasing τm or T1m. Among these factors, the hydration number increases as the surface-to-volume ratios of NPs are augmented. Accordingly, USIONs have better T1 imaging effects than large-sized IONPs.

Kim et al.5 demonstrated the potential for the utilization of USIONs as efficient T1 MR CAs in rats. USIONs with diameters around 3 nm possessed a great number of Fe3+ ions with five unpaired electrons on the surface, thus exhibiting high r1 relaxivities of 4.78 mM−1 s−1 and decreasing the r2/r1 ratios to less than 6.2. In vivo analyses further proved their excellent performance in high-resolution blood pool imaging, which allowed clear observation of 0.2 mm blood vessels. Furthermore, Lu et al.65 tested polyethylene glycol (PEG)-coated USIONs as T1 CAs on large animal models, including beagles and macaques. USIONs produced by the heat-up method with a uniform size of approximately 2 nm were stabilized by phosphine oxide-PEG (Fig. 3a). The results showed that USIONs could be successfully used for high-resolution MR angiography and detecting cerebral ischemia in macaques (Fig. 3b–d). They also exhibited relatively higher sensitivity and lower toxicity than FDA-approved gadolinium-based CAs. The large-scale synthesis of iron oxide nanoclusters, with good biocompatibility and excellent imaging performance, shows great clinical potential to produce next-generation T1 MR CAs.5,65


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Fig. 3 (a) Transmission electron microscopy image of PEG-coated USIONs in aqueous solution. (b) High spatial resolution MR angiography of macaques using USIONs as CAs. Cerebral susceptibility-weighted images of the brain before (c) and after (d) injecting the PEG–IONCs in the left cerebral ischemia model of a macaque. Reprinted with permission from ref. 65, Copyright 2017, Springer Nature.

The controlled dynamic assembly of USIONs can realize the “on–off” adjustment of MRI in vivo. As illustrated in Fig. 4, after pH-responsive ultra-small magnetic nanosystems disassemble in the tumor microenvironment, the MR T1 contrast would be “turned on” at the tumor site due to the reduction of Ms as well as the improvement of interactions between magnetic NCs and water molecules.


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Fig. 4 Schematic illustration of the “on–off” adjustment of MR T1 imaging in vivo realized by pH-responsive magnetic nano-assemblies.

Accordingly, our group reported a pH-sensitive dynamic assembly structure to improve the contrast between the normal liver and hepatocellular carcinoma (HCC) tumors. The assemblies were constructed by linking USIONs with i-motif DNA-derived pH-responsive linkers. Upon encountering acidic conditions in the pH range from 5.5 to 7.0, the i-motif DNA could undergo sensitive structural changes, resulting in the formation of a quadruple-helical structure and consequently inducing disassembly into well-dispersed USIONs (∼3 nm; Fig. 5a–c). The manipulation of the aggregation state of USIONs by pH-responsive linkers successfully achieved the transformation of MRI mode from T2 to T1, enhancing the detection sensitivity for early-stage HCCs (Fig. 5d and e).40


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Fig. 5 (a) Schematic illustration of the formation of pH-responsive USION assemblies. TEM images of (b) USION assemblies at pH 7.4 and (c) USION assemblies at pH 5.5. (d) Relaxivity of USION assemblies under different pH conditions. (e) T1-Weighted MRI in orthotopic HCC mice. Reprinted with permission from ref. 40, Copyright 2018, American Chemical Society.
4.1.2. Fluorescence imaging. Ultra-small noble metal NCs showed molecule-like optical properties because their sizes were close to the Fermi wavelength of electrons.1 For Aun NCs, the crystal structure and the optical absorption transition occur when the n ranges from 144 to 187. When the n is less than 144, the Aun clusters show non-face-centered cubic and discrete electronic structures.66 Due to the discrete electronic structure, ultra-small noble metal NCs exhibit strong photoluminescence rather than surface plasmon resonance49,67 and can therefore be exploited in fluorescence imaging.

For example, 2.6 nm protein-stabilized Au20NCs have strong fluorescence emission at 620 nm with a high fluorescence quantum yield up to approximately 15%, and could be used as luminescent probes for optical imaging. Their excellent active targeting ability, biocompatibility, water dispersibility, and photostability make them ideal candidates for active tumor-targeted imaging (Fig. 6a).68 Moreover, Chen et al.69 found that highly ROS (hROS) could quench the fluorescence of Au NCs; accordingly, they developed AuNC-decorated silica particles with dual-emission fluorescence for live-cell hROS detection. In addition, Ju et al.70 constructed nanocomposites of Au NCs and carbon dots with high stability and good biocompatibility. This design enabled the highly sensitive detection of ROS in a local ear inflammation model (Fig. 6b). In addition to the ROS, fluorescent USNCs also provide a platform for detecting other biomolecules, such as nucleic acids71,72 and proteins.73,74 Enkin et al. applied luminescent Ag NCs for DNA detection by hybridizing nucleic acid-stabilized Ag NCs and quencher-modified nucleic acids with hairpin DNA scaffolds. Apparently, once the target DNA triggered the opening of the hairpin, Ag NCs would spatially separate from quencher units, triggering the turned-on luminescence.72


image file: c8bm01200h-f6.tif
Fig. 6 (a) Au20NCs for active tumor-targeted imaging. (1) TEM and high-resolution TEM images of the Au20NCs. (2) Matrix-assisted laser desorption ionization time-of-flight mass spectrum of the Au20NCs. (3) Distributions of FA–Au20NCs in A549 tumor-bearing nude mice. Reprinted with permission from ref. 68, Copyright 2014 Royal Society of Chemistry. (b) Nanocomposite of Au NCs and carbon dots for the detection of hROS. (1) High-resolution TEM image of C-dots-AuNC. (2) The function of fluorescence intensity ratios changed with hROS concentrations. (3) In vivo imaging of hROS. Reprinted with permission from ref. 70 Copyright 2014, American Chemical Society.

4.2. Therapeutic activity

Some USNCs possess therapeutic activity and can act as active antitumor elements. As described in section 1, there are more atoms on the surfaces of USNCs in contact with the external environment, leading to the increased release of ions. For Pt NCs30,75 and Ag NCs,7 the release of toxic Pt2+ ions and Ag+ ions with significant DNA affinity is the key factor in their therapeutic effects. Chien et al. designed caged Pt NCs with excellent performance in cancer treatment through controlling the size of Pt NCs at the atomic level (0.93 ± 0.22 nm). Accordingly, Pt NCs could easily undergo oxidative dissolution and consequently lose their intrinsic chemical inertness, which allowed the corrosive Pt NCs to be subjected to further dissolution inside the targeted cells and release toxic Pt2+ for DNA platination.30 Moreover, the formation of Pt–DNA adducts would induce members of pro-apoptotic pathways and consequently lead to caspase activation.76 In addition, ultra-small Ag NCs also have promising applications in the biomedical field; in particular, the ultra-small size endows them with excellent antibacterial activity in contrast to their bulk counterparts because ultra-small Ag NCs possess a large number of available Ag atoms on the surface for further ROS production or oxidative dissolution to release active Ag+.77 For example, Xie et al. synthesized molecule-like Ag NCs with a core size of less than 2 nm. This feature facilitated the inclusion of more atoms to induce the generation of ROS and generated a better interface to interact with components inside cells. Therefore, they packaged Ag NCs with daptomycin (a commercial antibiotic) and finally achieved desirable synergetic antibacterial activity.7 Recently, Porto et al. found that Ag NCs of three atoms (Ag3-AQCs) could make chromatin more accessible to DNA-acting drugs, such as cisplatin, thus increasing their cytotoxic effect on tumors.

Specifically, Ag3-AQCs can dissociate single-nucleosome preparations only during DNA replication in proliferating cells, thereby enhancing the amount of cisplatin bound to DNA. Notably, the action of Ag3-AQCs only presented in proliferating cells and remained quiescent in normal cells. Therefore, the enhancement of the cytotoxic effects induced by the coadministration of cisplatin and Ag3-AQCs was more significant in tumors than in healthy tissues.78 Similarly, 1.4 nm Au55 NCs have also been reported to be much more toxic than larger gold NPs due to the distinct interactions between Au NCs and DNA.79

USNC assembly can further improve the therapeutic accuracy and efficacy. For example, Xia et al. synthesized a pH-sensitive polymer-wrapped ultra-small Pt NC assembly with modification of the SP94 peptide as an HCC targeting ligand (Fig. 7a–c). These Pt nano-assemblies (Pt-NAs) enabled prolonged blood circulation time, HCC targeting ability, and disassembly in an acidic intracellular microenvironment. After uptake in HCC cells, Pt-NAs disassembled into monodisperse Pt NCs with large specific surface areas, and released excess toxic Pt2+ accelerated by acidic pH. This Pt-NA strategy significantly suppressed HCC cells and overcame cisplatin resistance and heterogeneous stemness (Fig. 7d and e).6


image file: c8bm01200h-f7.tif
Fig. 7 (a) TEM image of ultra-small Pt NCs. (b) TEM image of Pt nano-assemblies. (c) DLS size of Pt nano-assemblies at pH 6.0 and 7.4. (d) Bioluminescence signals of orthotopic HCC tumors at the therapeutic endpoint of different treatments. (e) Survival conditions of mice in the four therapeutic groups. Reprinted with permission from ref. 6, Copyright 2016, American Chemical Society.

4.3. Bioelimination

Large-sized NPs (>10 nm) may accumulate in the reticuloendothelial system (e.g., liver or spleen), resulting in long-term toxicity.80 Renal clearable nanomaterials may have applications in clinical transformation associated with their rapid excretion for safety. However, only small-sized NPs (<6–8 nm) can rapidly clear via the kidney.81 Accordingly, the available excretion of USNCs, such as Au NCs82 and QDs,83via urine reduces the potential side effects, and this feature is critical for further development of nanomaterials in the biomedical field and in their clinical translation. However, rapid excretion also results in a short blood circulation time and low tumor retention rates, which may reduce the effectiveness of USNC-based therapy. The controlled dynamic assembly of USNCs can be used to realize not only high tumor retention rates, but also rapid excretion after therapy to avoid long-term toxicity in vivo. Our group reported the controllable assembly of ultra-small bismuth subcarbonate NCs (BNCs) into bismuth subcarbonate nanotubes (BNTs) for tumor-targeted computed tomography (CT) imaging and chemoradiotherapy (Fig. 8a).53 Drug-loaded BNTs facilitated effective tumor enrichment and CT imaging (Fig. 8b). Moreover, these BNTs disassembled into BNCs in an acidic tumor microenvironment, leading to drug release and enhancement of chemoradiotherapy effects (Fig. 8c). Additionally, the disassembly of BNTs induced rapid renal clearance and prevented potential long-term toxicity (Fig. 8d and e).
image file: c8bm01200h-f8.tif
Fig. 8 (a) TEM images of (BiO)2CO3 nanoclusters (BNCs), (BiO)2CO3 nanotubes (BNTs), and disassembly of BNTs at pH 5.5. (b) Reconstructed three-dimensional CT images demonstrating effective tumor enrichment of drug-loaded BNTs. (c) The combined chemoradiotherapy could inhibit tumor growth. (d) Schematic illustration of the disassembly of BNTs for renal clearance. (e) Renal clearance after i.v. injection of BNTs in tumor-bearing mice. Reprinted with permission from ref. 55 Copyright 2018, American Chemical Society.

5. Conclusions and perspectives

The aim of this mini-review was to provide an overview of recent development in the synthesis, assembly, and biomedical applications of USNCs. Immense progress has been made in this area owing to the unique chemical, magnetic, optical, and electrical properties of these nanomaterials. Moreover, the controlled assembly of USNCs may be the basis of future studies of nanomaterial synthesis. Nano-assemblies utilizing USNCs as building blocks may possess different morphologies and structures for realizing diverse functions, and could retain unique physicochemical properties originating from the primary building blocks when stimulated to disassemble. Hence, controlled assembly of USNCs may be one of the most promising future directions for USNC development, and reports to date have indeed demonstrated some applications of USNC-based nano-assembly in the biomedical field.

USNCs and their assemblies exhibit huge clinical translation potential. In practice, the clinical interventional therapy relies heavily on multiple imaging modalities to diagnose, to treat diseases and to do follow-up.84 USNCs on the one hand show low toxicity and could rapidly clear via the kidney, and on the other hand exhibit efficient imaging. Therefore, they have great potential in interventional image guided nanomedicine. These and other clinical potential uses of USNCs would greatly motivate the development of USNC-related nanosystems. However, despite great progress in biomedical applications, certain problems still remain to be solved. First, the road towards clinical translation is still long, and there are many obstacles that need to be overcome. The problem of insufficient manufacturing infrastructure and poor production technology is particularly worthy of attention. It is essential to precisely control the atomic quantity per particle for each batch, and to find suitable techniques to characterize USNCs, because differences in a few atoms may influence the performance, biodistribution, and elimination of USNCs. In addition, from the standpoint of biosafety, the US FDA claims that all injected imaging nanomaterials must be capable of being biodegraded or excreted via the renal pathway to avoid long-term retention.85 Although their extremely small size endows USNCs with the capacity to be excreted via the renal route, they are prone to aggregation due to their high surface energy and could be covered by biomolecules, such as proteins, in the body, leading to an increase in the apparent radius and hindering of the intended performance. Therefore, it is essential to improve our understanding of the stability, retention time, distribution, and excretion of USNCs in the body. Finally, despite many advances, the controlled assembly of USNCs is still in its preliminary stages. To date, few nanosystems have been studied thoroughly; those that have been are limited to several inorganic materials, and the existing new structures of nano-assembly may be unstable in the body, resulting in the disassembly or transformation of the configuration and causing loss of primary function. Therefore, there are still several challenges that need to be addressed. Given that the assembly occurs according to the surface modifications of USNCs, further studies are needed to evaluate the optimal arrangement and stoichiometric control of functional ligands on the USNC surface for self-assembly, as the foundation for the subsequent directional and spatial control of the assembly process. From a synthetic perspective, a systematic assembly approach is urgently needed to achieve quality uniformity. With further research in this area, we believe that these problems will be overcome, and that USNCs will be found to have extensive applications in clinical practice.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (2016YFA0203600), the National Natural Science Foundation of China (31822019, 51503180, 51703195, and 51611540345), the Fundamental Research Funds for the Central Universities (2018QNA7020), and “Thousand Talents Program” for Distinguished Young Scholars.

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Footnote

These authors contributed equally to this work.

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