Nanomaterials for Biotechnology: Synthesis, Properties, and ApplicationsView this Special Issue
Recent Advances in the Synthesis, Properties, and Biological Applications of Platinum Nanoclusters
Noble metal nanoclusters (M NCs), defined as an aggregation of a few to tens of atoms, are considered a borderline between atoms and metal nanoparticles (M NPs), which tends to exhibit molecule-like behaviours such as discrete electronic state and size-dependent fluorescence. In the past decades, gold and silver nanoclusters (Au NCs and Ag NCs) have been massively explored and utilized in the field of industrial catalysis, optoelectronic devices, biological imaging, environmental detection, clinical diagnoses, and treatment. The analogue of Au and Ag NCs and platinum nanoclusters (Pt NCs), especially their biological applications, is relatively and rarely discussed. This review firstly investigates the synthetic methodology of Pt NCs including template-assisted and template-free approaches and then introduces their unique optical, catalytic, and thermal properties. Particular importance here is the biological applications of Pt NCs such as the bioimaging of various cells as a preferred fluorophore in contrast to traditional fluorescent markers (e.g., organic dye, semiconductor quantum dots, and fluorescent proteins), the usage of Pt NCs-based antitumour drugs as a new class chemotherapeutics for malignant tumour therapy, and the utilization of antibacteria as an alternative of Ag-based antibacterial agent. On the whole, the development of Pt NCs has already gained delectable progress; however, the study of ultrafine Pt NCs is at the beginning stage and there are still plenty of challenges like synthesis of near-infrared (NIR) fluorescent Pt NCs, the explicit signal pathway of cell apoptosis, and attempt in diverse biological applications that need to be urgently tackled in future.
Noble metal like rhodium (Rh), palladium (Pd), silver (Ag), platinum (Pt), and gold (Au) is one kind of modish and desired material, according to their inherent resistance to oxidation and corrosion even in the moist environment [1–3]. Its physical and chemical properties appear to be entirely change as the size of metal continuously decreases into nanoscale because of the quantum size effect, surface effect, small size effect, and macroquantum tunnel effect . For example, noble metal nanoparticles (M NPs) which are defined as the particle size ranged from 1 to 100 nm have the high surface-to-volume ratio and electrodynamic interaction, leading to emerge distinct electronic, magnetic, and optical properties in contrast to bulk counterparts or individual atoms [5, 6]. In view of freely moving delocalized electrons in the conduction band, metals in a bulk state are good optical reflectors and electrically conducting (Figure 1). As for M NPs, a specific size-dependent plasma absorption will be presented when the size is smaller than the average free path length of conduction electrons (i.e., <20 nm) based on Mie’s theory [7, 8]. If the M NPs are irradiated by light, strong optical absorption and/or scattering phenomenon will happen forcefully relied on their size, morphology, and dielectric environment, which is recognized as localized surface plasmon resonance (LSPR) [9–11]. Consequently, M NPs show the intense colours owing to the collective oscillation of conduction electrons upon interaction with light and this particular property has been widely developed in catalysis, optoelectronics, sensing, and surface-enhanced Raman scattering (SERS) [12–16]. Further declining the size of metal nanomaterials into around 0.1-2 nm, M NPs turn into metal nanocluster (M NC) region . M NCs as a borderline between M NP and atoms were firstly discovered by Cotton and Haas in 1964 . On this length scale, the electronic band structure of M NCs is broken down into discrete energy levels under the condition of free electrons’ size near Fermi wavelength (i.e., <2 nm), resulting in the acquisition of molecule-like behaviours like the discrete electronic state [19–21]. Moreover, M NCs exhibit the intense light absorption and emission by the interaction between NCs and light via electronic transitions between energy levels. This unique electronic properties of M NCs are potently depended on their size, morphology, metal oxidation state, and surrounding ligands [22, 23]. Thus, a plenty of efforts focused on the preparation of desired and versatile M NCs by precise control of their sizes or shapes through meticulously choosing stabilized ligands or templates and the usage of NCs in an optical device [24, 25], chemical detection [26–29], catalytic conversion , and especially in biological applications .
Platinum (Pt), as one of the representative noble metal, has the physicochemical stability and remarkable resistance to corrosion even at high temperature based on its steady electrical structure . Its physical and chemical inertness makes Pt widely employ in the fundamental industrial fields such as electrodes, dentistry equipment, Pt resistance thermometers, and catalytic converters [2, 32]. The fabrication and application of Pt materials on the subnanoscale became a research hotspot in the past few decades, especially in the catalysis and medicine. The most common utilizations of Pt nanomaterials are the catalysis of chemical reaction according to their high surface activity . Based on their scarcity and preciousness, the research priorities of Pt materials are aimed at developing high-performance Pt-based materials through enhancing the catalytic efficiency as well as decreasing the usage amount. Undoubtedly, Pt’s size and morphology play a critical and indispensable role . On the other hand, Pt-based antineoplastic agents (like cisplatin, oxaliplatin, and carboplatin) have been universally used in the clinical chemotherapy against multiple cancers . For example, Pt(II) anticancer drugs could induce the crosslinking of DNA, leading to the inhabitation of DNA synthesis or repair in tumour cells .
Up to now, typical Pt NPs demonstrated their applicable and prospective capacities in enormous areas such as gas detection, fuel cells, biosensors, and chemotherapeutics . Besides, the analogues of Pt NCs, namely, Au NCs [38, 39], Ag NCs , Pd NCs  and Cu NCs , have been intensively investigated for their catalytic abilities, biological behaviours, and electrical and optical properties. By comparison of well-studied Pt NPs or congeneric NCs, the investigation of Pt NCs is just at an early stage in the last few decades; herein, this review summarizes the recent advance in the synthetic method of Pt NCs and special physicochemical properties, especially their fascinated biological applications.
2. Synthetic Method of Platinum Nanoclusters
In comparison to metal bulk or M NPs, the preparation of Pt NCs refers to the precise control and rigorous synthetic conditions due to their extreme small size . There are many classification standards of the preparation method (Figure 2) [44–47], like physical and chemical method and one-pot and etching method. We generally divided the synthetic protocols of Pt NCs into two aspects: template-assisted method and template-free method.
2.1. Template-Assisted Method
Template-assisted method is based on the presence of various templates during the synthetic process, which plays a role as a protecting agent, a stabilizer, a capping agent, and even as the restrict space provider. The usual templates impart the M NCs’ new unique features or specific structure by means of three main ways : (1) decline of the surface energy to prevent the NCs’ aggregation via electrostatic interaction, chemical bonding, and space steric effect; (2) accurate control and tailor of M NCs’ size, dispersity, and morphology, which will profoundly influence, determine, and enhance their inherent functions; and (3) decoration and modification of the M NCs’ surface to endow some reactive functional groups in order to achieve the further applications. The ideal templates always include electron-rich atoms (e.g., N, P, S, and O) or certain functional groups (e.g., -COOH, -NH2, -OH, and -SH).
Common templates utilized during the synthesis of M NCs are small organic molecules like representative surfactant, thiol compounds, organophosphorus compounds, and amino compounds [48–51]. Polymer ligand including nonionic and ionic polymers (such as acrylic polymer, amine polymer, polyethylene glycol, poly(N-vinyl-2-pyrrolidone), polypyrrole, and dendrimer) is another widely used template materials [52–54]. A polymer template stabilizes the NCs by chemical bonding and electrostatic effect, as well as steric effect due to large spatial configuration. Compared to small organic molecules, polymer has the easier modification, better controlling ability, and lower toxicity [55, 56], making them a preferred option for synthesis of M NCs. Furthermore, a biomacromolecule template, such as DNA, protein, oligonucleotides, and enzymes, is a kind of prevalent materials used to manufacture the medical and biological metal nanomaterials [57, 58]. Biomacromolecule is always relative to a specific biomolecular recognition function, multifunctional group (-SH, -COOH, -OH, and -NH2), and excellent biocompatibility , showing a promising potential in the development of various biofunctional M NCs. In general, a template cannot reduce the metal precursor to form the M NCs without adding any other chemical redundant or with the help of physicochemical means such as the γ-radiolysis method , microwave-ultrasonic method , sonochemical method , photoreduction method , and electrochemical method .
Chemical etching method also called ligand-induced exchange etching involves two processes: the larger-sized M NPs are formed firstly under the stabilized template with a weak interaction and secondly, ligand-induced etching of larger-sized NPs occurs under the existing excess ligands by a strong interaction between ligands and metal atoms to produce smaller size NCs. Highly blue fluorescent Pt NCs with two peaks at 410/436 nm were synthesized by phase transfer through electrostatic interactions under an etching environment . The presynthesized glutathione- (GSH-) protected Pt NPs were transferred into organic solvent with the support of cetyl trimethyl ammonium bromide (CTAB) to secondly form the fluorescent small Pt NCs. However, this method is always related to a time-consuming and complicated process, which is not suitable for the production of M NCs at a large scale.
Direct reduction in the present of templates and extra chemical reductant is a classical and extensive way to acquire the small size NCs. Atomically precise Pt NCs which consist of 11 atoms (Pt11 NCs) were obtained by a direct chemical reduction using small molecule 4-(tert-butyl)benzyl mercaptan (BBS) as the template and sodium borohydride (NaBH4) as the reducing agent . The structure of NCs was defined as Pt11(BBS)8 by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) and electrospray ionization (ESI) technology. Moreover, special octahedral Pt NCs were successfully synthesized employing glucose as the reducing agent and CTAB as the shape-control templates via one-pot hydrothermal process . The formation mechanism of octahedral NCs is that glucose reduces Pt ions into atoms and then atoms grow to octahedron by the precise control of CTAB, namely, the synergetic effect both of CTAB and glucose. Cho et al. put forward the sol-gel polymerization protocol of poly(2-hydroxyethyl-2-mercaptoethyl aspartamide) (PHMA) capped Pt NCs . PHMA as a polymer template could control the morphology of NCs and organize their structure association, based on binding Pt procurers via amine functional groups and particles via thiol functional groups. For another instance, dendrimer, as a favourable template, has a uniform structure which can supply a predetermined formation environment to accurately control the NCs’ size and morphology [68–71]. A linear structural Pt NCs with 4-8 atoms were fabricated inside of polyamidoamine dendrimer by UV irradiation at 254 nm (Figure 3) . The tools of resonance Raman spectra, ultraviolet-visible (UV-Vis) spectroscopy, X-ray photoelectron spectroscopy (XPS), and high-resolution transmission electron microscopy (HR-TEM) were employed to clarify that the assemble of Pt nanocrystals is owing to an oriented attachment mechanism.
Physicochemical technique can assist the preparation of ultrafine Pt NCs, instead of using chemical reductants. Microwaves, as the electromagnetic waves, could obtain monodispersed M NCs by the fast and homogeneous heating . Microwaves heat polar molecules rapidly at the high temperature without heating the glass container, leading to the formation of colloidal metal nanomaterials. The synthesized M NCs are always related to the high quality and narrow size distribution. Tu and Liu employed microwave irradiation to prepare the poly(N-vinyl-2-pyrrolidone)-supported Pt NCs with an average size at . This method could continuously produce the uniform Pt NCs even at a large scale, satisfying industrial applications. Photoreduction is a simple, feasible, and nontoxic approach avoiding the usage of additional reducing agents . The reduction mechanism is due to the energy transfer under the condition of light irradiation to generate the reductive hydration electrons or reactive radicals , which is frequently utilized to explore the origination of photoluminescence because no other compounds are introduced. The Pt NCs with the size ranged from 1.0 to 2.2 nm were prepared with the aid of UV light under the alkaline environment . The presynthesized NCs have a face-centred cubic spatial structure, and the author inferred that the UV light could achieve the nucleation and growth of NCs, not by the thermal reduction. Finally, electrodeposition is a usual and effective method to control size and shape of metals and decorate the substrate surface by adjusting deposition parameters, involving a plenty of distinct advantages such as low cost, rapid producing rates, and precious controllability [76, 77]. Qian et al. firstly modified the four-generation poly(amidoamine) dendrimer (G4-NH2) onto indium-doped tin oxide (ITO) and then electrodeposited Pt NCs on the surface of G4-NH2 dendrimer to form larger-sized NPs near . The size and morphology could be tailored by polyelectrolytes or the types of PAMAM ligands. These physicochemical techniques mentioned above are environmentally friendly, less time-consuming, and convenient, which are beneficial to investigate the formation process and fluorescence mechanism and meet the demand of green industrial fabrication.
In addition, the defined templates during the formation of Pt NCs can be extended to a broad range involving multicomponent materials like multimetallic alloy [79–83] and doped substrate material [84–87]. For instance, bimetallic or multimetallic alloy is designed by a combination concept of different metallic compounds in order to obtain composite performance. Pure Al, Co, and Pt were melted and then dealloyed in alkaline solution at certain temperature to form Al85Co14Pt1 ternary alloy . Even the amount of Pt was quite small, the electrocatalytical activity was improved dramatically and the ampere-metric determination limit of sodium nitrite (NaNO2) was 0.067 μM (). On the other hand, a doped substrate method is allowing Pt NCs doped or dispersed into the substrate materials such as polymer film [89–92], inorganic substrate [93–96], metal organic framework (MOF) , carbon nanotubes (CNTs) [98, 99], and graphene [100–102], which could easily adjust and enhance the pure NCs’ chemical and physical performance. Pt NCs with an average diameter of were deposited on SmMn2O5 (SMO) mullite-type oxides by an atomic layer deposition method (ALD), showing the efficient ability to solve the CO poisoning problem for the Pt-based catalyst . This catalytic activity even under low temperature originated from O2 dissociation at the bifunctional interface structure. Lee et al. put forward that monodispersed Pt NCs () were loaded onto three-dimensional graphene-like carbon (3D GLC) which was employed in the electrochemical oxidation reaction . These Pt NCs-doped 3D GLC catalysts possess near superficial area and exhibit excellent glycerol oxidation reaction (GOR) activity and extreme stability via firm adhesion of glycerol on the Pt NCs’ surface. Recently, Pt precursor solution was added into poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) to assemble polyelectrolyte multilayer (PEM) films and then the Pt NCs were in situ yielded with various sizes from 1.2 to 2.3 nm only by tailoring the salt concentration and reduction time, instead of the reduction temperature .
2.2. Template-Free Method
Template-free protocol is a method avoiding the introduction of extra substance and has extensive advantages such as effortless postprocessing and pure product . Kawasaki’s group proposed a surfactant-free synthetic approach to obtain Pt NCs consisting of 4 to 6 atoms with blue fluorescence in N,N-dimethylformamide (DMF) solution . These NCs showed extreme stability against strong ionic and variable acid-alkali conditions. Subsequently, Duchesne and Zhang employed X-ray absorption near-edge spectroscopy (XANES) and extended X-ray absorption fine structure (EXAFS) techniques which revealed the details of surfactant-free-synthesized Pt NCs’ local structure and oxidation states . The local structure of Pt is primarily due to the changes in the metal-ligand coordination, not the Pt-Pt bonding. The oxidation of Pt species is a combination of Pt(IV) and Pt(0), indicating that nonmetallic Pt NCs are responsible for their fluorescent properties. Meanwhile, this surfactant-free method synthesized Pt NCs were used to sensitively sense the aqueous Fe3+ ion solution and the limit of detection was 4 ppm (15 μM) under the concentration range of Fe3+ ions from 0.007 to 0.530 mM .
3. Properties of Platinum Nanoclusters
The properties of Pt NCs are the consequence of their distinct electronic and structural properties, which have access to NC’s size, morphology, and surface surroundings inseparably. Herein, we summarize the classical features of Pt NCs including optical properties, catalytic properties, and other properties.
3.1. Optical Properties
Optical properties benefit for offering an insight to understand the electronic and geometric structures of M NCs in depth . The applications of M NCs strongly depend on their optical properties, and there are a lot of research reported the NCs’ unique optical phenomenon such as steady-state absorption and fluorescence, temperature-dependent fluorescence, ultrafast fluorescence and transient absorption, fluorescence enhancement, and electrochemiluminescence [21, 111–113]. This part, we will introduce about the theoretical and practical progress of size-dependent fluorescence for Pt NCs.
Unlike M NPs which possess apparent surface plasmon resonance (SPR) absorption, Pt NCs lost this particular property, replaced by the size-dependent fluorescence ranging from the visible to near-infrared (NIR) region. Generally, this fluorescence of Pt NCs generated from the electronic transitions between the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Their finite size is a critical point for molecule-like electronic transitions between HOMO and LUMO energy levels. Energy transitions can be expressed as Equation (1) based on the jellium model [3, 114]: where represents the energy level spacing, is the Fermi energy, and stands for the number of atoms in NCs. The of free electron is only related to the metals’ Wigner-Seitz radius () or the electron density (ρ0) because the free electrons are piled up with constant electron density. Owing to equals to , Equation (1) transfers to Equation (2) using the emission/excitation frequency () and NCs’ radius () expressed as follows:
Equation (2) is suitable for the M NCs when is smaller than 20, which is well depicted by a spherical harmonic potential. Hamiltonian for an electron in a single-particle 3D harmonic oscillator can be described as follows: where represents the single-electron momentum and is the coordinate operators. The small anharmonic distortion term should be taken into consideration under the condition of . Defining the distortion parameter () as a constant value (0.033), the correlated transition energy spacing () is shown as Equation (4) using angular momentum () and shell number () expressed as follows: where represents the angular momentum of excited state and is the angular momentum of ground state. Based on the Equations (2) () and (4) () displayed above, this dependency can be defined as the large size of M NCs is related to the small energy level spacing with longer emission wavelength fluorescence emitted and vice versa (Figure 4(a)).
Besides of the size-dependent fluorescence effect described above, surrounding ligands or templates also play a significant role on the fluorescent properties through ligand-metal charge transfer (LMCT) (Figure 4(b)) [115–118]. Not only the enhancement or quenching of fluorescence could be realized by the collaboration between NCs and ligands but also the fluorescent intensity and maximum peak of emission wavelength can be adjusted. Thus, the mechanism of M NCs’ fluorescence is ascribed to the intrinsic electronic transitions between HOMO-LUMO energy levels and the electronic transitions between NCs’ surface and surrounding ligands via LMCT.
Until now, fluorescent Au and Ag NCs have been deeply researched and utilized in various fields [119–125]. As for the fluorescent Pt NCs, the number of synthetic methods, usable templates, and optical applications is significantly fewer than Au or Ag counterparts and most reports focused on the blue-, green-, and yellow-emitting fluorescence (Figure 5). The longer emission wavelength, especially near-infrared (NIR) region, is rarely mentioned. NIR fluorescence has a plenty of merits such as easy detection, less autofluorescence interference, and more use safety, making it attractive in both biological sensing and labelling analysis . Furthermore, the fluorescent Pt NCs have the specific advantage than Ag and Au NCs, such as higher photoluminescence quantum yield (QY) and instinct properties like anticancer . Therefore, great efforts payed out to study on the facile and repeatable synthesis of fluorescent Pt NCs with longer emission wavelength in the past decades.
Bovine serum albumin (BSA) supported blue fluorescent Pt NCs (emission wavelength region from 350 to 500 nm) which were fabricated by an easy NaBH4-reduced method. These NCs could achieve the selective examination of hypochlorite among the concentration range from 12 to 240 mM via the visible fluorescence quenching due to the formation of oxidized Pt . A GSH-induced etching process were employed to produce the yellow fluorescent Pt NCs (maximum emission wavelength at 570 nm) with the fluorescent QY at 17% . Most Pt NCs are in the Pt(I) state, and the optimized molar ratio between Pt and GSH is determined at 1 : 10. Afterwards, blue  and green  fluorescent Pt NCs were precisely synthesized by employing the template of fourth-generation polyamidoamine dendrimer, short for PAMAM (G4-OH), and their absolute QYs reach to 18% and 28% in water, respectively. We previously reported that various aqueous Pt NCs from blue to yellow fluorescence were prepared by a facile method using hyperbranched polyethyleneimine (PEI) as a stabilizing agent and environment-friendly L-ascorbic acid as a reductant . Their optical properties can be tailored by adjusting the molar ratio between Pt ions and protecting ligands. Moreover, these Pt NCs have the ability of quantitative and selective detection for Co2+ ions and the limit of detection is up to 500 nM. Meanwhile, Xu et al. used the same method to prepared blue fluorescent Pt NCs and applied them for sensing nitroimidazoles (MTZ) with the limit concentration of 0.1 μM . As for longer fluorescent emission, García’s group demonstrated that red fluorescent Pt NCs in aqueous solution could be obtained by a chemical reduction method using lipoic acid (LA) as a capping agent . The synthesized NCs with the maximum emission wavelength at 680 nm have a relatively high QY at 47% and present excellent stability towards pH media and high ionic solution. Moreover, hemoglobin-protected Pt NCs appeared to have two fluorescence regions: emission wavelength at 450 nm which is contributed by Pt6 NCs at zero oxidation state and emission wavelength at 760 nm that is due to the formation of aggregated Pt(II)-Hb complexes caused by LMCT effect from N/O to Pt atoms (the structure is assigned to Pt16 NCs) . They also indicated that the NIR fluorescence is too weak to observe clearly because of a lower proportion of Pt(II) on the NC’s surface (only near 13.68%). To our knowledge, it is the longest emission fluorescence reported for Pt NC materials, even for their poor fluorescent intensity.
In spite of the progress discussed above, the synthesis of fluorescent Pt NCs is still in its initial stage. There remains some challenge that needs to be solved, including the acquisition of highly longer emission fluorescent Pt NCs to realize the practice application, clarification of fluorescent mechanism to provide guidance, and large-scale production of Pt NCs to meet industrial demand.
3.2. Catalytic Properties
M NCs have been widely investigated for their catalytic properties from the theory to practical application [32, 136, 137]. Pt-based catalysts are pervasively applied in the development of the cost-effective proton exchange membrane fuel cells (PEMFCs) involving electrochimerical oxygen reduction reaction (ORR), methanol oxidation reaction (MOR), ethanol oxidation reaction (EOR) [138–142], and the catalysis of different chemical reactions like hydrogenation reactions [143–146]. It is well known that the catalytic reactivity of Pt varies by their sizes, morphology, and dispersion .
There are two common views for Pt-based catalysts. One is that Pt NPs (near 2 nm) are thought to be at the limit of their catalytic performance due to too strong Pt-O binding energy on the smaller clusters [148, 149]. That is to say, large Pt NPs with a face-centred-cubic (FCC) structure (near 2 to 3 nm) have the best activity and the ultrasmall Pt NCs are considered to have lower or even no catalytic ability, especially for ORR. However, a recent study rejected this traditional concept and their applicability . It was found that Pt NCs with the size less than 2 nm also emerged a better catalytic performance. Accordingly, the catalytic capacity is no longer dependent on the size effect. Vajda et al. proved that small Pt8-10 NCs loaded on a high-surface area template showed 40-100 times higher catalytic activity for the oxidative dehydrogenation than common Pt-based catalysts and extreme selectivity to produce the propylene . Considering about the hydrogenation of methyl acrylate, the catalytic capacity of Pt-based materials depended on their size ranged from 2.4 to 3.0 nm which could be controlled by the amount of poly(N-vinyl-2-pyrrolidone) (PVP) . Subsequently, Lan’s group modified the counter electrode by PVP-protected Pt NCs for the dye-sensitized solar cell (DSSC) and found that this modified electrode exhibited light soaking durability and high conversion efficiency (near 9.37%) in a highly volatile electrolyte system . Based on the results above, the “uniqueness of size effect” issue for Pt-based catalysts is now reexamined and no longer exist.
The other common view is that certain morphology of Pt NCs like topological magic number structure (e.g., Pt13 and Pt55) with high symmetry could exhibit the higher catalytic ability [148, 154]. Imaoka et al. compared the ORR catalytic capacity between Pt12 NCs with less symmetric structure prepared using a phenylazomethine dendrimer with a tetraphenylmethane core (DPA-TPM) and Pt13 NCs with high symmetry obtained using phenylazomethine dendrimer with a triphenylpyridylmethane core (DPA-PyTPM) (Figure 6) . One interesting finding is that misshapen structure Pt12 has a double catalytic activity compared with that of the topological stable Pt13 with the high symmetry. This distinction is mainly caused by two reasons: (1) less symmetric structure of Pt12 with a smaller number of internal Pt-Pt bonds and (2) structural transition of Pt12 makes the size further decreases against the smallest limit of icosahedral Pt13 NCs. Besides, this group further explored the atomicity-specific catalytic activity of Pt NCs within a significant small atomicity (), revealing Pt17 and Pt19 exhibited higher performance than other series . As a result, the atomic coordination structure is completely different from that of the larger-sized FCC nanomaterial and the catalytic activities for the ORR are significantly altered by the spatial arrangement and atomicity. Hence, the fact discussed above proved the idea that the catalytic activity has weak access to the topological magic number of Pt on the nanoscale.
In summary, the emergence of Pt NCs has already reversed the traditional idea on Pt-based catalysts. The further investigations about the precise control of atomic number, seeking of different steric topological structures, and catalytic application of diverse chemical reactions need to be comprehensively and deeply studied in future.
3.3. Other Properties
Except for the optical and catalytic properties, Pt NCs also have unique physical characteristics, like thermal properties . The phase stability of Ptn NCs (, 147, 309, and 561 atoms) under various temperature conditions was surveyed by the molecular dynamics (MD) simulation combined with an embedded atom scheme (EAM) . Furthermore, Akbarzadeh and Parsafar discussed the melting and thermal physical properties of Ptn NCs in a larger size ( to 8788 atoms) by means of molecular dynamics simulations employing quantum Sutton-Chen (QSC) potential . Both for the larger and relative smaller NCs, the melting temperature goes up as the NCs’ size increased and that of Pt8788 NCs approaches to the Pt bulk limit.
4. Biological Applications of Platinum Nanoclusters
Pt NCs consisting of few to tens of atoms own plenty of outstanding features and possess a great potential in the various applications, e.g., catalysis , sensing , and cancer therapy . This part, we focus on the Pt NCs’ biological applications which are strongly in accordance with their size-dependent effects and the coordination between NCs and functional surrounding ligands.
4.1. Biological Imaging
In the past decades, fluorescent biological imaging technology which is the process of light emission in living organisms  became an indispensable and visualized tool for the drug delivery system [162, 163], gene therapy , and cancer diagnoses [165, 166]. The used fluorophore is a key point for successful bioimaging of cells, which concerns about their safety, sensitivity, and wide applicability. Organic dyes [167–169], semiconductor quantum dots (QDots) [170, 171], fluorescent proteins [172, 173], lanthanides [174, 175], and carbon dots (CDots) [176, 177] as the common fluorophores have been already explored for practical imaging and extensively presented their merits and drawbacks. For example, organic dyes possess the high fluorescence QY; however, the dramatic cytotoxicity severely handicaps their practical applications . Besides, QDots have unique features such as tunable colours, great photostability, narrow emission spectra, and broad excitation spectra . The disadvantages like large size (>3 nm), on-and-off blinking behaviour, and low biocompatibility are important issues that need to be solved [180, 181]. As an alternative to organic dyes and QDots, M NCs have a crowd of strike features like ultrasmall size, water solubility, high fluorescent efficiency, large Stokes shifts, excellent photostability, and low cytotoxicity , making them become the safe and nontoxic clinical fluorescent contrast agents. In comparison to well-studied Au and Ag NCs [31, 183–185], relatively little research investigated the bioimaging application of fluorescent Pt NCs. In general, the imaging way of Pt NCs can be divided into two parts: (1) direct labelling without any other materials and (2) combination of certain biomolecule (e.g., proteins and DNA) to targeted imaging. Our previous work reported that fluorescent Pt NCs stabilized by polyamine ligands (average size near 1.4 nm) could accomplish the biostaining of the suspension hematopoietic cell system . The ligand-capped Pt NCs could selectively enter into K562 and BV173 cancer cells compared to the normal peripheral blood mononucleated cells (PBMCs) from healthy donors (Figure 7). This distinction gives an opportunity to achieve the specific labelling of hematopoietic cells during the disease diagnosis. Recently, we extended this fluorescent probe to label the lung cancer . The classical human lung adenocarcinoma cells were chosen to examine their biological imaging ability (Figure 8). Both A549 (normal cells) and A549/DDP (cisplatin-resistant cells) cells exhibit the red fluorescence signal that is emitted from Pt NCs-based drugs, while the cell nuclei are stained by 4,6-diamidino-2-phenylindole (DAPI) exhibiting the blue fluorescence. Most interesting finding is that Pt NCs preferably enter almost cell nuclei in the cisplatin-resistant A549/DDP cell groups, compared to the A549 cell group where the Pt NCs are observed evenly distributed in both cell nuclei and cytoplasm. As a result, Pt NC nanomaterial could realize the visual imaging individually as a fluorophore on the account of the fluorescence effect.
The aim of conjugating the biomolecule is to achieve the deliberate target of the specific tissue. Antibody is a suitable and effective choice. An antibody belonging to proteins has a lot of functional groups (e.g., -NH2 and -COOH) which could feasibly react with those groups on the surrounding ligands of NCs by chemical reaction, and then, the presynthesized Pt NC-antibody complex is delivered to express on the certain targeted position via antigen-antibody reaction. This approach could complete the targeted imaging of lesion location. For example, after bounding to the antichemokine receptor antibody (anti-CXCR4-Ab) through a conjugated protein A, blue fluorescent mercaptoacetic acid- (MAA-) capped Pt NCs were observed on the cell membranes where the receptors are expressed (Figure 9) . In order to check this specific combination of antibody-modified Pt5(MAA)8-protein A-anti-CXCR4-Ab complex and chemokine receptor, Chinese hamster ovary (CHO-K1) cells were selected as a control group due to their negative behaviours against the chemokine receptor. The result indicated that the Pt NC complex cannot stain the CHO-K1 cells, proving the success in the targeted imaging. Simultaneously, the same work was also done for green-emitting Pt NCs . Most importantly, these reported that Pt NCs have the considerably low cytotoxicity and excellent biocompatibility, demonstrating enormous potential in the tracking, imaging, and labelling of cancer cells or other kinds of cells as an alternative fluorescently labelled probe.
Similarly, yellow fluorescent PEI-stabilized Pt NCs (Pt NCs@PEI) could effortlessly conjugate with an antichemokine receptor antibody and then successfully realized the double staining of HeLa cells using DAPI-stained nuclei and Pt NCs@PEI expressed on the cell membrane (Figure 10) . To achieve targeted expression on the cell membrane, a simple glutaraldehyde method was used to conjugate Pt NCs@PEI to the anti-CXCR4-Ab. Confocal fluorescence images show HeLa cell nuclei in blue color (DAPI stained) and cell membranes as yellow color, demonstrating the evidence that the usage of Pt NCs@PEI will not be affected by any other fluorophores, simultaneously. Furthermore, the relationship between NCs and PEI ligands was also checked and the results elucidate that these Pt NCs are stabilized mostly by primary amine. Based on this discovery, the fluorescence of Pt NCs may be originated via two pathways, that is, the electronic transitions between HOMO-LUMO energy levels of Pt NCs and the NCs’ surface surrounding ligands through LMCT.
4.2. Antitumour Drugs
Pt-based antitumour drugs are one of the most effective tools for the treatment of different tumours, and Food and Drug Administration (FDA) authorized the Pt as the effective antitumour drugs for various cancer therapies in 1978 [127, 189–192]. Cisplatin in the Pt(II) state as a representative drug emerged a few deficiencies which influences the therapeutic efficiency. For instance, it could have side effect like myelosuppression, nephrotoxicity, and neurotoxicity in the course of medicine treatment [193–195]. On the other hand, typical breast, colorectal, and prostate cancers exhibit less sensitive to cisplatin [196, 197]. More serious is that testicular and ovarian cancers intrinsically resist to cisplatin treatment after several cycles of therapy, even though it is efficient at the beginning stage . These drawbacks including the systemic toxicities and poor specificity impede their anticancer efficiency; therefore, developing a new-type Pt-based antitumour drug with little side effect and excellent specificity could afford a powerful supporting technique for diagnosis and treatment of diverse malignant tumours.
In the current years, Pt NPs and NCs have been used to develop the latest Pt-based anticancer nanomedicine and found their preferable ability of inducing the apoptosis of several cancer diseases [160, 199, 200]. Chien et al. reported a low-generation dendrimer-caged Pt NCs (CPN) with 0.93 nm diameter . After attaching to the cleavable polyethylene glycol (PEG) corona and targetable iRGD (CRGDKGPDC), this complex achieved the targeting of human breast cancer cell line MDA-MB-231 and release of toxins against malignant cells by affecting tumour-inside activation for anticancer chemotherapeutics (Figure 11). By means of subcutaneous breast cancer xenograft in mice, the therapeutic effect of CPN was examined via intratumoural injection in vivo and the result indicated that this kind of chemotherapeutics has the same efficacy compared to cisplatin.
Fluorescent GSH-capped Pt NCs were prepared by a green and simple chemical method and employed to biolabel the HepG2 cells . It is worth noting that the synthesized Pt NCs could obviously kill the HeLa cells under the irradiation by infrared (IR) light, while it was not happened under UV light condition. The killing mechanism of cancer cells is contributed to heating effect instead of free radical effect. Xia et al. presented an approach to package the Pt NCs with polypeptide and targeting peptide SP9443 to form the assembled Pt NAs. These Pt NAs could damage DNA through targeting disseminated hepatocellular carcinoma (HCC) tumour-initiating cancer stem-like cells (CSLCs) to achieve inhibiting proliferation of tumours . Gene expression profile analysis proved that ABCG2 and CD24, which expressed highly in the sorted SP+CD24+ cells, could be adjusted by Pt NAs, while the cisplatin could not downregulate. Furthermore, real-time quantitative polymerase chain reaction (RT-qPCR) analysis also demonstrated that Pt NAs induced the downregulation of CCNB1, CDK1, and TOP2A, leading to DNA damage and modulation of the cell cycle (Figure 12). This study verified that the prepared ultrafine Pt NAs have the ability to accelerate the release of toxic Pt ions and overcome the cisplatin-resistant problem for HCC CLSCs.
In a previous study, we used the dual-functional Pt NCs-based anticancer materials to biologically image the blood system suspension cells as the fluorescent markers. Meanwhile, the selective inhibition of hematopoietic K562 and BV173 cancer cells was investigated as well . The relative cell apoptotic rate for K562 and BV173 cancer cells is three times higher than hematopoietic normal cell (PBMCs) via induction of the expression of p53, PUMA, and cleaved caspase-3 proteins (Figure 13). These Pt NCs manifest the evident apoptosis efficacy possibly due to the inherent characteristic of Pt and exhibit a great potential in effective treatment of hematopoietic system disease, especially acute myeloid leukaemia and lymphoma. Currently, the cisplatin-resistant-non-small-cell lung cancer (NSCLC) was chosen as the targeted object because the lung cancer incidence is increasing continually owing to the environment deterioration and smoking. The problem of drug resistance seriously affects the chemotherapy efficiency and survival rate of patients during the treatment with chemotherapy drugs due to multiple mechanism, such as the lack of effective drug concentration in tumour cells, reduction of drug activity, cell apoptosis changes, and DNA repair pathways. The experimental results illustrated that Pt NCs-based anticancer drug could achieve the excellent induced apoptosis in both cisplatin-resistant A549/DDP and non-cisplatin-resistant A549 cells . More interesting is that cisplatin-resistant A549/DDP showed the superior inhibitory and apoptotic effects than non-cisplatin-resistant A549 cells by the way of activating p53 protein and the related signalling pathway, which could be proved through the apparent endocytosis behaviour by the nucleus of cisplatin-resistant A549/DDP cells. As for NSCLC, the synthesized Pt NCs-based anticancer drugs could overcome the toxic side effects and drug resistance to enhance the clinical therapeutic effect.
In contrast to the well-known cisplatin resistance mechanism concerning about antiapoptotic factors that counteract caspase activation (Figure 14(a)), the mechanism for Pt NCs-based nanomedicine is still inconclusive. Some researches assume that ultrafine Pt subnanomaterials possess extreme tiny size approximately 1 nm, leading to near 90% of Pt atoms exposed on the NC’s surface. This kind of high surface-active Pt NCs is affected by intracellular acidic organelles like endosomes and lysosomes and then rapidly decomposed to form oxidation states of Pt (Figure 14(b)) . These corrosive Pt trends to combine with DNA or proteins and then destroy the DNA consequently, resulting in the apoptosis of cancer cells. In addition, ultrafine Pt NCs have an ability to anchor onto the grooves of DNA double helix to further damage the DNA. Thus, the reasonable and receivable mechanism for the Pt NCs-based chemotherapeutics may be summarized as the synergistic effect of both Pt NCs and Pt ions causing the damage of DNA to kill the cancer cells.
The usage of noble metal (Ag and Au) as antimicrobial agents was largely investigated, especially for Ag-based nanomaterials . The mechanisms of antibacteria are related to the DNA damage, membrane damage, and production of some active radicals (e.g., reactive oxygen species (ROS)). Because of the ultrafine size of NCs, Ag NCs with higher surface-to-volume ratios and abundant surface atoms express higher antimicrobial efficiency. However, the antibacterial feature of Pt NCs is rarely studied. Subramaniyan et al. put forward the green synthesis protocol employing phytoprotein obtained from spinach leaves as a ligand to gain spherical Pt NCs with the average size of 5 nm and self-assembled species at the size range from 100 to 250 nm . These protein-stabilized Pt NCs have the excellent Salmonella typhi-inhibiting ability, and the minimum inhibitory concentration (MIC) was determined at 12.5 μM (Figure 15). The inhibition effect was proved as the damage of established biofilms, confirmed by scanning electron microscopy (SEM) and fluorescence microscopy. Moreover, intracellular ROS generated by Pt NCs was also the ancillary killer to Salmonella typhi via oxidative injury against the antioxidant defence.
5. Conclusions and Outlook
Conclusively, Pt NCs containing few to dozens of atoms exhibit unique physicochemical properties due to their molecule-like behaviours such as discrete electronic state and size-dependent fluorescence. The synthesis of Pt NCs can be divided into two ways: template-assisted approach that is related to designed properties, controllable size, and specific morphology and template-free protocol which has access to the feasible posttreatment process and pure product. Subsequently, the optical, catalytic, and thermal properties of Pt NCs were introduced and these features have a strong relationship with the distinct electronic and structural characteristics, as well as the various surrounding ligands. Breaking the traditional concepts, ultrafine Pt NCs exhibit the favourable catalytic abilities even in the form of less symmetric topological structure. Most importantly, the diverse biological applications of Pt NCs were summarized in detail. Fluorescent Pt NCs have already bioimaged different kinds of tumours like HeLa cells, hematopoietic K569 and BV173 cells, NSCLC A549 cells, and HepG2 cells, as a preferred fluorophore in contrast to traditional fluorescent labels. Moreover, Pt NCs were employed as new class chemotherapeutics in the diagnoses and treatment of hematopoietic, lung, and hepatocellular malignant tumours, exhibiting excellent therapy effect, especially overcoming the problem of cisplatin resistance. Finally, Pt NCs were identified to possess a good antibacterial capacity which could be used as an alternative of the Ag antibacterial material.
Despite these exciting and promising progress of Pt NCs mentioned above, the study of ultrafine Pt NCs is at the beginning stage and there still remains a great challenge as follows: (1) synthesis of NIR fluorescent Pt NCs with outstanding optical features, (2) evident clarification of the apoptosis pathway and mechanism of Pt NCs for hematopoietic tumour and cisplatin-resistant NSCLC, (3) valid combination of Pt NCs with other materials to endow multifunctionality, and (4) comprehensive utilization of Pt NCs in diverse biological applications, not only for the different tissue systems (like osteocarcinoma and pancreatic carcinoma) but also the application types that need to be extended such as gene therapy, DNA sensing, and protein detection.
Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.
This research was funded by the National Natural Science Foundation of China (21807121), Key Scientific Research Projects of High Education of Henan Province (18A430005), and Project for Fundamental Research Funds of Zhongyuan University of Technology (K2018YY020). Dr. X.H. gratefully acknowledges the Collaborative Innovation Centre of Textile and Garment Industry, Henan Province, for their assistance and the support from the 2019 Youth Talents Promotion Project of Henan Province, the 2018 Backbone Teachers of Zhongyuan University of Technology, and the Program for Interdisciplinary Direction Team in Zhongyuan University of Technology, China.
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