Remote Control and Modulation of Cellular Events by Plasmonic Gold Nanoparticles: Implications and Opportunities for Biomedical Applications Jiayang Li, Jing Liu, and Chunying Chen* CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety and CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology of China, Beijing 100090, China ABSTRACT: Compared to traditional hyperthermia methods, gold nanoparticles (Au NPs) successfully achieve sitespecific incremental temperature in deep tissues. By virtue of near-infrared (NIR) laser-mediated photothermal treatment, Au NPs have been widely explored in clinical and preclinical applications, including cancer therapy and tissue engineering. In this issue of ACS Nano, Suzuki, Ciofani, and colleagues demonstrate how gold nanoshells can remotely activate striated muscle cells via inducing myotube contraction and modulating related gene expression by mild heat stimulation under NIR irradiation. This Perspective provides a brief overview of the current developments and future outlook for multifunctional platforms based on Au NPs for cancer treatment, tissue engineering, and regenerative medicine.
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photophysical properties of Au NPs, such as efficient absorption of X-rays and luminescence properties have been extended into X-ray-based imaging techniques (e.g., computed tomography) and two/three-photon luminescence imaging techniques. Therefore, Au NPs are being developed as platforms for multimodal imaging contrast agents in clinical theranostics.3 Based on their strong light absorption and subsequent nonradiative energy dissipation, Au NPs have emerged as potential tools in plasmonic photothermal therapy.4 Figure 1 illuminates
s a quintessential noble metal, gold has been used in money, electronics, jewelry, and the decoration of artifacts for thousands of years. Because of its relative rarity, inertness, and easy fabrication, gold has been endowed with great value throughout the development of human society. During the past two decades, engineering gold nanoparticles (Au NPs) with different sizes, shapes, and surface modifications has rapidly developed. As promising candidates for biomedical applications, Au NPs have attracted tremendous attention in nanobiotechnology and nanomedicine.1 In this Perspective, we briefly overview the current developments and future outlook for multifunctional platforms based on Au NPs for cancer treatment, tissue engineering, and regenerative medicine. Gold NPs exhibit extraordinary physicochemical properties, which have led to applications in many imaging techniques and thermal treatments. One of the most significant and wellknown properties of Au NPs is localized surface plasmon resonance (LSPR), which consists of coherent localized oscillations of free conduction-band electrons. Upon being resonant with the wavelengths of incident light, the surface plasmons of Au NPs support the enhanced light scattering and absorption, and the subsequent photon confinement may generate strong electromagnetic fields at the surface of gold, demonstrating various optical phenomena and applications.2 For example, light scattering by Au NPs has been applied in imaging techniques such as optical coherence tomography and dark-field microscopy, while the optical absorption of Au NPs may help photoacoustic tomography imaging. More interestingly, other © XXXX American Chemical Society
Figure 1. LSPR and subsequent photothermal conversion in gold nanospheres by the collective oscillation of conduction-band electrons on the Au NP surface via irradiation at resonant wavelengths.
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cube, flower, prisms, star, and so on) endow different physicochemical properties to obtain expected biological characteristics, which further benefit their subsequent applications in personalized theranostics. As shown in Figure 2, anisotropic gold-based NPs have been explored to achieve optimized properties for various clinic applications. As one of the most commonly used NPs, gold nanospheres (Figure 2A) have the smallest surface area-tovolume ratio as well as the highest stability compared to other shapes. They always have a single LSPR peak at about 520 nm, which red shifts slightly with increasing diameter. Gold nanoclusters consist of ultrasmall gold nanospheres (smaller than 5 nm) and may exhibit strong fluorescent properties.6 Gold nanovesicles (Au NVs, Figure 2C), which are also assembled by ultrasmall gold nanospheres, have great potential in bioapplications because they present ultrastrong plasmonic coupling between adjacent gold nanospheres and, consequently, enhance their LSPR absorption in the near-infrared (NIR) region.7 Another common and broadly investigated nanostructure of Au NPs is gold nanorods (Au NRs, Figure 2G).8
the photothermal heating process in Au NPs. After exciting free electrons in the plasmon band of Au NPs by laser photons, nonthermal electron distribution relaxes via electron−electron scattering and is followed by electron−phonon coupling and heat transferring from the electrons to the gold lattice. Finally, heat dissipation occurs from the crystal lattice to the surrounding environment by phonon−phonon interactions. Therefore, Au NPs can efficiently convert absorbed light to heat, which manifestly benefits medical applications of Au NPs in photothermal therapy as well as thermo-responsive drug release. Given the advantages of multivalent surface effects and the high surface areas of Au NPs, we are able to facilitate precise surface modification for controlled release and targeted delivery.5 More importantly, we are able to tune Au NPs’ plasmonic properties, such as efficient light scattering and absorption, increased conversion of light into heat, and enhancement of local electromagnetic fields near surfaces of Au NPs, by tailoring their sizes, and structures, as well as composites. Different nanostructures of Au NPs (sphere, cluster, rod, shell, bowl, cage,
Figure 2. Electron microscopy images of various gold nanostructures: (A) Gold nanospheres. Reprinted with permission from ref 2. Copyright 2016 Wiley-VCH Verlag GmbH & Co. (B) Gold nanoshells. Reprinted from ref 14. Copyright 2007 American Chemical Society. (C) Gold nanovesicles. Reprinted with permission from ref 7. Copyright 2013 Wiley-VCH Verlag GmbH & Co. (D) Gold nanostars. Reprinted with permission from ref 2. Copyright 2016 Wiley-VCH Verlag GmbH & Co. (E) Gold nanocages. Reprinted with permission from ref 10. Copyright 2011 American Chemical Society. (F) Gold nanoprisms. Reprinted with permission from ref 12. Copyright 2006 Wiley-VCH Verlag GmbH & Co. (G) Gold nanorods. Reprinted with permission from ref 9. Copyright 2012 Wiley-VCH Verlag GmbH & Co. (H) SiO2coated Au NRs. Reprinted with permission from ref 20. Copyright 2014 Wiley-VCH Verlag GmbH & Co. (I) Gold nanocrosses. Reprinted from ref 13. Copyright 2011 American Chemical Society. B
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facile and controllable synthesis procedure. In the wavelength range of 700−1100 nm, water absorption is minimal, while tissue and blood present maximum transmissivity. Light can penetrate tissue by as much as 10 cm, depending on the tissue type. Gold NS-based photothermal therapy is already undergoing human clinical trials for metastatic head and neck tumors and for prostate cancer, which has been approved by the U.S. Food and Drug Administration.
Gold NRs not only have a transverse LSPR band at about 520 nm but also exhibit distinctive longitudinal LSPR bands, which vary in the range from visible to NIR by increasing the aspect ratio (length/width) of Au NRs; Au NRs also present higher photothermal conversion efficiencies and scattering contrast than gold nanospheres. In addition, coating the surface of Au NRs with mesoporous materials such as SiO2 (Figure 2H) makes them suitable for applications in effective drug/gene delivery and heat-induced drug release in tumor therapy.9 In pioneering research, Xia’s group synthesized gold nanocages (Au NCs) as a class of gold nanostructures in 2002. Silver nanocubes were used as templates to synthesize Au NCs by inducing the growth of small gold spheres around the core and subsequently building a mesoporous shell (Figure 2E).10 Precise control of the thickness and porosity of the shell as a result of increasing gold source usage induces a red-shift of the longitudinal LSPR band from 400 to 1200 nm. In addition, adjusting the size of Au NCs from 20 to 500 nm can influence their cellular uptake and biodistribution, hence advancing their clinical applications. In order to obtain high photothermal conversion efficiency and good imaging in terms of clinical performance, various gold nanostructures have recently been developed that are endowed with strong LSPR scattering or absorption in the NIR region, which is useful for the deeper tissue penetration and minimal tissue damage. Therefore, Au NPs with high aspect ratio, sharp tips, or heavy branching have attracted much interest. For instance, gold nanostars (Figure 2D),11 gold nanoprisms (Figure 2F),12 and gold nanocrosses (Figure 2I),13 which have multiple nanoscale sharp corners, edges, and branches, all exhibit strong LSPR in the NIR and mid-IR regions. Gold nanoshells (Au NSs), which are principally deposited onto silica colloidal cores, were recognized as the first example of Au NP-mediated cancer thermotherapy in 2003 by Halas and her co-workers (Figure 2B).14 The plasmon resonance of Au NSs can be tailored to any desired wavelength within a large range of the visible and infrared spectrum by modulating their ratio of shell thickness to core radius, or the surface topography. Based on these unique properties, the longitudinal LSPR peak of Au NSs can be moved into the NIR region through a
In this issue of ACS Nano, Suzuki, Ciofani, and colleagues report an innovative approach for muscle cell stimulation that is temperature driven and based on excitation by NIR radiation in the presence of polyvinylpyrrolidone (PVP)-wrapped Au@SiO2 NSs. Au@SiO2 NSs can remotely activate striated muscle cells by producing mild heat stimulation under NIR irradiation.15 This work suggests that the wireless stimulation technique based on remote control of NIR irradiation and localized plasmonic feature-based photothermal properties will have great potential for muscle tissue engineering, bionics, and regenerative medicine (Figure 3).
THERMAL THERAPY VIA GOLD NANOPARTICLES CAN REALIZE SITE-SPECIFIC AND PRECISE THERMAL EFFECTS COMPARED TO TRADITIONAL HYPERTHERMIA METHODS Traditional hyperthermia can be traced back to the early 1900s and is usually carried out with externally electromagnetic fields or ultrasound. Because cancer cells cannot endure temperatures above 42 °C, traditional hyperthermia would heat the tumor region up to 45 °C to help eradicate plenty of cancer types in clinical treatment. Among various photothermal nanomaterials, the plasmonic properties of gold nanostructures are
Figure 3. Au NP-mediated mild heat stimulation undergoes unexpected biofunctions, which can remotely modulate the cell behavior and events, e.g., gene expression, in both cancer and normal cells. Encouraged by the development of multifunctional platforms based on Au NPs, there should be broad prospects for their applications in cancer treatment, tissue engineering, and regenerative medicine. C
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In addition to thermal ablation tumor therapy, mild heat mediated by Au NPs is also endowed with more unexpected biological functions. Zhao et al.18 have developed a nanoplatform to achieve deeper penetrated tumor theranostics, based on the mild heat effect of remote NIR irradiation. From their previous work, NIR photothermal conversion of AuNR@ MSN@polymer thermally triggers drug release and subsequently performs the synergistic effects of chemotherapy and hyperthermia.19 In a more recent study, a moderate thermal effect at ∼42 °C was generated by Gd-hybridized plasmonic Au-nanocomposites upon laser irradiation and dramatically altered the tumor interstitial transient permeabilization and improved their accumulation up to 3.9-fold. With a high payload of gadolinium and an anticancer drug, the Au nanocomposite increased the intratumoral accumulation and permeability of imaging agents and drugs, which afforded highsensitivity imaging and improved cancer therapeutic efficacy. More interestingly, Au NRs also triggered moderate heat to evoke high expression of heat shock factor trimers, while depressing the expression of P-glycoprotein and mutant p53 in tumor cells.20 This function could be used as a strategy for overcoming drug-resistant effects in chemotherapy. Inspired by investigations of tumor hyperthermia, applications of Au NP-triggered thermal treatment for tissue engineering have been attracting increasing interest.21 In clinical tissue engineering applications, researchers have explored active tools by various biocompatible polymers to control the behavior and fate of cells that properly develop target tissue. Recently, increasing numbers of nanomaterials have been adapted to perform specific biofunctions for tissue engineering.22 With more understanding of the interface between nanomaterials and biomolecules, we begin to recognize that a number of factors, including nanoscale structures, shapes, stiffness, sizes, and surface modifications, can significantly affect the fate of cells. Therefore, Au NPs, which can easily be tailored to afford specific physicochemical properties, have been investigated as biocompatible materials in the field of tissue engineering. Performing as surface modulators or releasing agents, AuNPs can be explored to the fabrication of tissue scaffolds. For example, Heo et al. recently demonstrated in vivo osteoclastogenic inhibitory effect by curcumin-conjugated AuNPs,23 which is a type of therapeutic agent for treating and preventing osteoporosis. Hung et al. investigated the performance of Au NP-treated mesenchymal stem cells (MSCs),24 which may promote MSCs for the proliferation, adhesion, migration, and cell differentiation.25 Shevach et al. successfully achieved the elongation and alignment of cardiac cells by Au NP composites with polycaprolactone−gelatin fibers.26 In addition, gold nanowires have been shown to expedite the structural and functional assembly of cardiac tissues growth more efficiently than traditionally engineered cardiac patches for treating damaged heart tissues, due to the improved electrical communication between adjacent cardiac cells. Mild thermal treatments handled by Au NPs have also been explored in the field of neuronal tissue engineering. Gold NRs also exhibit interesting functions in the stimulation of neuronal tissue, as reported by Stoddart et al.27 As an extrinsic absorber, silica-coated Au NRs can induce significant increases in electrical activity in rat primary auditory neurons by using NIR laser irradiation at 780 nm.28 Therefore, silica-coated Au NRs have the potential to improve the efficiency and increase the penetration depth of treatments compared to traditional methods. Furthermore, upon excitation of the NIR laser,
outstanding for their high and fast photothermal conversion efficacy upon NIR excitation. Compared to traditional hyperthermia methods, Au NPs successfully achieve site-specific incremental temperature in deep tissues with NIR irradiation. Using Au NPs for hyperthermia also results in improved and controllable heat stimulation in terms of the amplitude and duration due to faster heat diffusion from the nanostructures, which behave as small heat sources for the surrounding materials. This technique takes advantage of the photothermal conversion phenomenon, and it is currently being adopted in several clinical and preclinical applications, including cancer therapy and tissue engineering.
MULTIFUNCTIONAL GOLD NANOPARTICLES ARE CONSIDERED AS PROMISING CANDIDATES FOR NANOMEDICINES IN TUMOR THERANOSTICS DUE TO THEIR UNIQUE OPTICAL PROPERTIES AND SUPERIOR THERAPEUTIC INDEX WITH HIGH DRUG-LOADING EFFICIENCY They not only behave as multimodal imaging contract agents for cancer diagnosis but also perform the synergistic therapy effects of hyperthermia and chemotherapy. For example, Halas et al. have demonstrated the application of Au NSs as therapeutic oligonucleotide delivery vehicles that may release therapeutic genes and anticancer drugs for synergistic effects with irradiation by a NIR laser.16 Chen et al. have developed a series of biodegradable Au NVs that are based on dense packing of ultrasmall gold nanospheres in PEG-b-PCL block copolymer.7 With strong NIR absorption and remarkably high photothermal conversion efficiency, these nanocomposites exhibit photoacoustic imaging and enhanced photothermal therapy. In addition, the assembled hollow structure of Au NVs can be disbanded and evacuated with external stimulation by NIR light, which benefits the drug’s controlled release and biodegradation. Xia and his lab have been working on utilizing Au NCs with hollow interiors and porous shells as promising carriers for drug delivery since 2002. Because of the excellent photothermal effect of Au NCs, heating via NIR irradiation could accelerate the release of anticancer drugs, thus achieving more effective tumor therapy.10
With a large porous surface or hollow cavity in their structure, Au NPs, such as Au NSs, Au NCs, Au NVs, and Au NRs, are being explored as potential nanocarriers for their high drug-loading capabilities. By virtue of the photothermal conversion effect, Au NPs have been employed to construct artificial tissue stents for thermotherapy to treat esophageal cancer.17 Although researchers have studied anticancer drug-loaded stents by coating a nitinol stent with a polymer or hydrogel shell for esophageal cancer, a Au NS-coated stent with effective photothermal conversion was first developed and applied in the photothermal therapy for esophageal cancer. Irradiated with a NIR laser, the Au NS-coated stent efficiently increases the temperature of pork and porcine intestines. Considering the excellent stability and biocompatibility of the Au NS-modified stent, this work provides an opportunity for the clinical use of this newly functionalized stent in esophageal cancer therapy. D
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1455 nm laser irradiation were able to induce the contraction of cardiomyocytes by directly promoting the cross-bridge formation between actin and myosin without Ca2+ transients, which is different from electrical stimulation.32 Possible advantages for remotely controlling the muscle cell contraction may be more biocompatible, which did not induce Ca2+dependent cell apoptosis.33 Mild heat stress induces mitochondrial biogenesis in C2C12 myotubes, while the beneficial effects of mild heat stimulation, such as improved insulin sensitivity, may be associated with mitochondrial biogenesis. In this work, chronic remote photothermal stimulations significantly increased the mRNA transcription of genes encoding heat shock proteins (HSPs) and Sirt1, a protein that can induce mitochondrial biogenesis.15 The reduced level of cellular energy can activate AMP-activated protein kinase (AMPK) and Sirt1, both of which further upregulate PGC-1α and the downstream mitochondrial biogenesis program. The following molecular mechanism study of this research also demonstrated that mild heat generating from the synergistic NIR+NS stimulation was able to upregulate the expression of different HSP genes significantly, such as Hspa1a, Hspa1b, Hspb1, and Sirt 1. HSPs are known to play fundamental roles in promoting myocyte mitochondriogenesis and in protecting the skeletal muscle tissues against apoptotic damage.15 The HSP genes encode proteins belonging to the chaperone family. Their transcription is mainly activated by heat shock transcription factor 1 (HSF1) in response to heat stimulus. Similarly, the mild photothermal effect generated from Au NR-based mesoporous silica nanocarriers (Au@SiO2) in combination with a pulsating laser causes high and long-term expression of HSF-1 trimers in breast cancer drug-resistant cells (MCF-7/ADR). Further, the HSF-1 trimers can repress the NF-κB pathway, which depresses MDR-1/P-glycoprotein (Pgp) and mutant p53 expression. Thus, both sensitivity to drugs and drug retention in cancer cells are greatly improved. This strategy is quite interesting if contrasted with common photothermal strategies for hyperthermia ablation.20 When NG108-15 neuronal cells were cultured with Au@SiO2 and irradiated with a lowpower laser device, enhanced neuronal outgrowth was found, such as neurite length, the number of neurites per neuron, and the percentage of neurons with neurites. The use of a NIR laser at 780 nm to stimulate cultured rat primary auditory neurons that were pretreated with silica-coated Au NRs showed significant increases in electrical activity. Together, these initial results open up new opportunities for future biomedical applications such as muscle tissue engineering, peripheral nerve regeneration treatments, regenerative medicine, and cancer treatment. Biocompatibility and Challenges in Clinical Translation. Future studies should be dedicated not only to the translation of this approach in muscle tissues in vivo but also to the investigation of the role of chronic photothermal stimulations on mitochondrial biogenesis and on myocyte differentiation. However, risk assessment of the adverse effects of Au NPs on biological systems is becoming urgent and necessary for clinical applications. Currently, only Au NSs have been approved for hyperthermia therapy (i.e., AuroLase Therapy), and in preclinical studies, they have shown highly selective and rapid tumor destruction with minimal damage to surrounding healthy tissue and no detectable systemic toxicity. There have been two human clinical trials for photothermal treatment using Au NSs. The first trial consisted of a single dose NP treatment for five patients with refractory and/or recurrent tumors of the head and neck,34 and the second clinical trial was
Au NRs have been demonstrated to increase the neurite length up to 25 μm and possibly induce intracellular calcium transients in NG108-15 neuronal cells. This strategy provides potential opportunities for peripheral nerve regeneration treatments and, hence, could be a useful approach to repair central nervous system axons following spinal cord injury. Carvalho-de-Souza et al. reported the temperature-dependent excitation of neurons in the presence of Au NPs that absorb 532 nm light and convert this energy into heat using a pulsed laser.29 The researchers employed a cell-targeting anchor to localize the AuNP photosensor at the membrane for photothermal cell activation; this method represents a potentially widely applicable advance in the technology of neuronal optical stimulation. To explore the future potential in vivo, Bianco et al. used laser-triggered remote bioexcitation of a frog (Xenopus laevis) paw in a living model.30 One possible application of Au NP-enabled neuronal photostimulation is that of human therapeutics. Although the use of Au NPs in tissue engineering has received increasing attention, investigations have not previously been reported on striated muscle cells. In this issue of ACS Nano, Suzuki, Ciofani, and colleagues developed remote activation of striated muscle cells mediated by NIR light and Au NSs, affording applications in tissue engineering and bionics.15 They adopted C2C12 cells, a line of murine myoblasts, as in vitro models, because they can be differentiated toward multinucleated myosin-positive myotubes, which show the peculiar properties of contractile skeletal muscle cells. C2C12 cells tolerated the NSs well up to high concentrations, showing a slight, yet significant, decrease of metabolic activity at a dose of 500 μg/mL after 2 days of incubation. C2C12 cells treated with NSs at a lower concentration of 50 μg/mL were able to differentiate toward myotubes efficiently and without any apparent toxicity. Transmission electron microscopy showed extensive uptake and the presence of these NPs in C2C12 cells, either enclosed in pinosome membranes or distributed extensively in the cytoplasm. We know from the literature that local temperature increases of approximately 5−6 °C are able to evoke cardiomyocyte contraction without the generation of calcium transients, and Suzuki, Ciofani, and colleagues found that the mild NIR+NS stimulation was able to induce remarkable contraction mediated by actin−myosin interactions in the C2C12 myotubes via the Ca2+-independent pathway. Based on these results, we are encouraged to extend the applications of Au NPs in tissue engineering in the future.
OUTLOOK AND CHALLENGES Cellular Responses to Mild Heat Stimulation. The main strategy for using photothermal inorganic NPs as a heat source by converting NIR light to heat is to provide a method for simultaneous chemotherapeutics and hyperthermia, the latter of which has been used to kill tumor cells directly at >45 °C (i.e., ablation).31 However, in this issue of ACS Nano, Suzuki, Ciofani, and colleagues provide evidence that remote stimulation of muscle cells is realized based on mild heating (to ∼42 °C) generated by NIR radiation in the presence of Au NSs, demonstrating the effective induction of myotube contraction in the absence of calcium fluxes.15 One of the most striking results of this research is that combined NIR+NS stimulation can induce relevant myotube contraction due to the photothermal effect of a local temperature increase of approximately 5 °C (from 36.5 °C) without apparently adverse effects. This result is consistent with the authors’ previous findings that the temperature increments obtained with focused E
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for the treatment of primary and/or metastatic lung tumors, and patients were given a systemic infusion of Au NSs followed by escalating doses of NIR irradiation.35 A number of studies have been carried out to evaluate the biosafety of Au NPs in vitro (primary and cultured cells) and in vivo. The major problem encountered with the use of Au NPs in biomedical applications is their quick entrapment by the reticuloendothelial system, which results in their high accumulation and retention in the liver and spleen. The clearance and/or degradation of delivered Au NPs and their nanocomposites probably limit treatment doses and usage frequency.36 Large Au NPs (between 10 and 100 nm) will rapidly be removed from the bloodstream and delivered to the liver and spleen, while, conversely, small Au NPs (
