There are an abundance of nanoparticle technologies being developed for use as part of therapeutic strategies. This review focuses on a narrow class of metal nanoparticles that have therapeutic potential that is a consequence of elemental composition and size. The most widely known of these are gold nanoshells that have been developed over the last two decades for photothermal ablation in superficial cancers. The therapeutic effect is the outcome of the thickness and diameter of the gold shell that enables fine tuning of the plasmon resonance. When these metal nanoparticles are exposed to the relevant wavelength of light, their temperature rapidly increases. This in turn induces a localized photothermal ablation that kills the surrounding tumor tissue. Similarly, gold nanoparticles have been developed to enhance radiotherapy. The high-Z nature of gold dramatically increases the photoelectric cross-section. Thus, the photoelectric effects are significantly increased. The outcome of these interactions is enhanced tumor killing with lower doses of radiation, all while sparing tissue without gold nanoparticles. Silver nanoparticles have been used for their wound healing properties in addition to enhancing the tumor-killing effects of anticancer drugs. Finally, platinum nanoparticles are thought to serve as a reservoir for platinum ions that can induce DNA damage in cancer cells. The future is bright with the path to clinical trials is largely cleared for some of the less complex therapeutic metal nanoparticle systems. WIREs Nanomed Nanobiotechnol 2015, 7:428–445. doi: 10.1002/wnan.1322
The promise of nanoparticle technology lies in the idea that the composition and physical size of the material bestows properties that are unique from that observed in the other forms of the materials. Nanoparticle research has come a long way toward that in the last two decades. There is a burgeoning field of therapeutic nanoparticle gene delivery research that appears to grow by the day.[1-4] This research is clinically relevant as many clinical trials focused on gene therapy are ongoing or even completed.
However, the most heavily researched nanoparticle field is drug delivery.[5-9] There are a multitude of approved formulations that are commercially available now and no doubt many more on the way. The motivations for this research are improved pharmacokinetics, targeting, and stability. The outcomes are some of the most sophisticated and well-characterized nanoparticle technologies in play today.
While these fields are producing effective reagents and making poorly soluble drugs useful through encapsulation, controlled release, and targeting, the nanoparticles themselves are usually not therapeutic. They are mostly utilized for other properties, such as platforms into which functionality is engineered. Some exceptions are the sunscreen nanoparticles ZnO and TiO2. These nanoparticles selectively scatter harmful UV light to help prevent DNA damage that can prematurely age the skin and ultimately cause skin cancer. The sizes of these preventative nanoparticles are tuned to remain transparent to visible light, meaning improved cosmetic outcomes, while efficiently scattering a range of UV light.
Researchers witnessed and participated in the generation of significant promises and speculation of the impact nanoparticles would have on medicine that have largely been realized through commercialized drug delivery applications. Along with this progress, there were some exciting discoveries like the optical properties of quantum dots that are still finding their way into commercial applications.[10-13] More recently, up-converting nanoparticles have stolen the lime light as they redefine what we know about fluorescence.[14-16] These steps toward diagnostic and theranostic applications are the earliest on a very long road. For a review on the medical application and impact of engineered nanoparticles, readers are referred to Ref .
When examining the literature, it was evident that the metal nanoparticle field contained well-researched examples where the nanoparticles themselves were uniquely used for therapeutics. These are not drug delivery devices per se, they are the drug. This is the focus of this review. We follow the development and trends of therapeutic metal nanoparticles from their inception to the most recent developments. A summary of the types of nanoparticles and their applications described in this manuscript are shown in Table 1. The therapeutic properties of metals have been used for ages. Silver and copper have been known for their antimicrobial properties since ancient times. Gold has been used in elixirs and colloids for medicinal properties for thousands of years. Gold nanoparticles have optical properties that are not observed with larger particulates. Indeed, gold nanoshells have become a heavily researched area with a focus on photothermal cancer tissue ablation and are an excellent example of a unique nanoscale phenomena developed for a therapeutic application.
|Nanoparticle Composition||Biological Target Site||Model System||Application||Reference||Year|
|Gold sulfide, 50-nm nanoshell||NA||NA||Plasmon resonance with ∼520–900 nm light||Zhou et al.||1994|
|Silica–gold nanoshells||NA||NA||Plasmon resonance with broad wavelength light range||Oldenburg et al.||1998|
|Silica–gold nanoshells||Cancer||Human breast carcinoma cells (in vitro) and transmissible venereal tumor (in vivo)||Photothermal therapy||Hirsch et al.||2003|
|Silica–gold nanoshells conjugated with anti-HER2 antibody||Breast cancer||HER2+ breast carcinoma cells||Photothermal therapy||Loo et al.||2005|
|Superparamagnetic iron oxide nanoparticles coated with silica–gold nanoshells||Head and neck cancer||Head and neck cancer cell lines overexpressing EGFR||Photothermal therapy||Melancon et al.||2011|
|Silica–gold nanoshells embedded with magnetic Fe3O4 nanoparticles, conjugated with anti-HER2 antibody||Head and neck cancer||Head and neck cancer cell lines overexpressing EGFR||Photothermal therapy||Kim et al.||2006|
|Gold-coated iron oxide nanoparticles; silver-coated Fe3O4@Au nanoparticles, and hollow-type gold nanoshells containing gold-coated iron oxide nanoparticles||Breast cancer||HER2+ breast carcinoma cells||Imaging and photothermal therapy||Lim et al.||2007|
|Silica–gold nanoshells and gold nanorods||NA||Tissue-simulating phantoms||Photothermal therapy||Pattani and Tunnel||2012|
|Silica–gold nanoshells||Brain tumor||Mouse brain metastatic tumor xenografts||Photothermal therapy||Choi et al.||2012|
|Silica–gold nanoshells||Cancer||In vitro model using head and neck squamous cell carcinoma cells||Photothermal therapy||Trinidad et al.||2014|
|Gold-branched shell nanostructures PLGA/doxorubicin-core functionalized with a human serum albumin/indocyanine green/folic acid complex||Cancer (human breast adenocarcinoma)||HeLa or MDA-MB-231 cells (in vitro) and MDA-MB-231-injected immunodeficient BALB/c nude mice||Imaging and photothermal/chemotherapeutic therapy||Topete et al.||2014|
|Gold nanoparticles||Cancer||Chinese hamster ovary (CHO-K1), mouse tumor (EMT-6) and human prostate cancer cells (DU-145)||Radiosensitizer||Herold et al.||2000|
|Gold nanoparticles targeted with conjugated antibodies||Cancer||Human colon carcinoma cells (LSl80) or control melanoma cells (WM164)||Radiosensitizer||Hainfeld et al.||1990|
|Gold nanoparticles||Breast cancer||Mouse mammary EMT-6 tumors||Radiosensitizer||Hainfeld et al.||2004|
|Gold nanoparticles||Cancer||In silico dose estimation||Radiosensitizer||Cho||2005|
|Gold nanoparticles||Cancer||In silico dose estimation||Radiosensitizer||Cho et al.||2009|
|Gold nanoparticles||Cancer||Prostate cancer cells||Radiosensitizer||Roa et al.||2009|
|Gold and iron oxide nanoparticles capped with glutathione||Cancer||CT26 colon cancer cell lines injected into mice||Radiosensitizer||Kim et al.||2010|
|Gold nanoparticles capped with glucose||Cancer||Implanted breast MCF-7 adenocarcinoma in mice||Radiosensitizer||Roa et al.||2012|
|Gold nanoparticles capped with PEG||Cancer : glioblastoma multiforme||Mouse model of glioblastoma multiforme||Radiosensitizer||Joh et al.[37, 38]||2013|
|Gold nanoparticles||Cancer||Rat hepatoma model using JM-1 cells||Radiofrequency ablation||Cardinal et al.||2008|
|Silver nanoparticles||Antimicrobial||Human adipose-derived stem cells||Toxicity evaluation||Samberg et al.||2012|
|Silver nanoparticles||Antimicrobial||Escherichia coli, Ag-resistant E. coli,Staphylococcus aureus, methicillin-resistant S.aureus (MRSA), andSalmonella sp.||Antimicrobial assessment||Sabella et al.||2011|
|Silver nanoparticles||Antimicrobial||Skin and in vitrokeratinocytes||Toxicity evaluation||Samberg et al.||2010|
|Silver nanoparticles||Antimicrobial||A431 (human skin carcinoma) and HT-1080 (human fibrosarcoma)||Antimicrobial||Arora et al.||2008|
|Silver nanoparticles||Antimicrobial||Zebrafish||Health and environmental impact||Asharani et al.||2008|
|Silver nanoparticles capped with starch||Antimicrobial||Normal human lung fibroblast cells (IMR-90) and human glioblastoma cells (U251)||Toxicity evaluation||Asharani et al.||2009|
|Silver nanoparticles-grafted dressings||Wound healing||Normal and diabetic mice||Therapeutic evaluation||Tian et al.||2007|
|Silver nanoparticles||Wound healing||Full-thickness excisional wound mouse model||Therapeutic evaluation||Liu et al.||2010|
|Silver nanoparticles||Skin||Franz diffusion cell with intact/damaged human skin||Skin penetration evaluation||Larese et al.||2009|
|Silver nanoshell with a carbon core||Cancer||Prostate adenocarcinoma cell line model||Photothermal ablation or radiation-enhanced therapy||Kleinauskas et al.||2013|
|Silver nanoparticles capped with polyvinylpyrrolidone encapsulated in polymer nanoparticles||Brain cancer||Human glioblastoma-astrocytoma epithelial-like cell line (U87MG) (in vitro); Swiss mice and severe combined immunodeficiency mice bearing U87MG tumors (in vivo)||Therapeutic evaluation||Locatelli et al.||2014|
|Platinum nanoparticles||Cancer||Human colon carcinoma cells (HT29)||Toxicity evaluation||Gehrke et al.||2011|
|Platinum nanoparticles||Cancer||Human colon carcinoma cells (HT29)||Toxicity evaluation||Pelka et al.||2009|
|Platinum nanoparticles||Cancer||In vitro||Therapeutic evaluation||Porcel et al.||2010|
|Platinum nanoparticles capped with polyvinyl alcohol||Brain cancer||Human lung fibroblasts (IMR-90) and human glioblastoma cells (U251)||Toxicity evaluation||Asharani et al.||2010|
|Au@Ag/Au nanoparticles||Cancer||A549 lung cancer cells in vitro and injected into mice||Imaging and photothermal therapy||Shi et al.||2014|
|Ag@Au nanoparticles||Cancer||HeLa and HepG2 cells||Imaging and photothermal therapy||Yang et al.||2012
It appears that therapeutic metal nanoparticles, such as gold, are similar to chemotherapeutics because even though they can be therapeutic, metal nanoparticles can also be toxic.[41, 57, 58] The presence of this double-edged sword is common knowledge with small molecules but the concept also applies to metal nanoparticles. The topic of gold nanoparticle toxicity is ongoing therefore it is critical to keep in mind that both therapeutic and toxic properties of these nanoparticles need to be simultaneously studied. Thus, it is critical to keep in mind that both therapeutic and toxic properties of these nanoparticles need to be simultaneously studied. This also highlights the need to design metal nanomaterials for therapy that do not adversely affect the biological systems where they will be used. Indeed, sophisticated targeting, signaling, feedback, and therapeutic systems have been developed with the unique properties of metal nanoparticles as enabling technologies.
von Maltzahn et al. described a multistep nanoparticle system that described a nanoparticle communication system to amplify tumor targeting and treatment. The study utilized gold nanorods for photothermal activation of the coagulation pathway and magnetofluorescent iron oxide nanoworms for imaging. This complex feedback system shows that complex multicomponent systems enable new paradigms for treating disease. The unique characteristics that some metal nanoparticles have can be used in quite sophisticated ways. Indeed, the future of medicine is bright and future therapeutics have the potential for multicomponent activity that should vastly improve healthcare.
PHOTOTHERMAL ABLATION WITH GOLD NANOSHELLS
The first report describing gold nanoshells was of a Au2S nanoparticle core surrounded by a gold shell published in 1994 by Zhou et al.The critical feature of these nanoparticles was the capacity to tune the plasmon resonance to longer light wavelengths with larger diameters (Figure 1). The limitation of the Au2S core was that 50 nm was the diameter limit, which meant that the tunable range was likewise limited to approximately 520–900 nm. Simply put, the kinetics of the nanoparticle core and shell synthesis prevented controlled growth of each. This synthesis limitation was overcome by the development of a silica core nanoparticle coated in gold by the Halas laboratory at Rice University, published in 1998. This approach was founded on the capacity to coat relatively large silica nanoparticles (120 nm) with smaller gold nanoparticles (1–2 nm) in a controlled manner to produce shells with defined thicknesses (14–30 nm). This was advantageous because the plasmon resonance could be finely tuned. In 2003, the Rice University collaborative group headed by Halas and West published a landmark report on the use of this technology for near-infrared thermal therapy of tumors. This report described leveraging the relative transparency of biological tissue in the near-infrared (820 nm) with the tuneable gold nanoshells that would absorb this light and increase in temperature by approximately 37°C in 5 min. The system was tested in vitro and in a mouse tumor model (Figure 2). The nanoshells were composed of a 55-nm silica core and a 10-nm thick coating of 1–2 nm gold nanoparticles. The nanoparticles were then coated with PEG-5000 and injected directly into the tumor. The authors described localized cell death in vitro andin vivo that was limited to the treatment area. This report marked the beginning of a steep increase in the number of gold nanoshell publications that have only started to level off in the last few years. Since then there have been many different variations on this theme and many have observed similar results in other models.
In 2005, Loo et al. demonstrated the use of antibody-targeted gold nanoshells for cancer imaging and therapy applications. They used 120-nm diameter silica nanoparticles to achieve peak absorption efficiencies in the near-infrared. Then either anti-HER2 (breast cancer biomarker) or a nonspecific antibody (anti-IgG) was conjugated to the gold nanoshell surface. These antibody-capped gold nanoshells were added to HER2-positive SKBR3 breast adenocarcinoma cell cultures and incubated for 1 h. They interpreted these experiments as a proof-of-concept for simultaneous molecular imaging of HER2 expression and photothermal therapy in vitro. The results showed that anti-HER2 nanoshells presented significantly increased light-scattering-based optical contrast compared with the two control cell groups. Photothermal therapy was also applied by using near-infrared laser for 7 min (820 nm, 0.008 W/m2). The result showed that cell death was observed only in cells treated with anti-HER2 nanoshells, thus illustrating specific molecular targeting. Additional dose-response experiments with different incubation times suggested no cytotoxicity of anti-HER2 nanoshells to SKBr3 cells without near-infrared light exposure. This study showed that immunotargeted gold nanoshells can provide light scattering contrast for imaging. At the same time, they can also exhibit sufficient absorption to be effective as a photothermal therapy.[21, 60]
An aqueous solution of superparamagnetic iron oxide nanoparticles coated with silica–gold nanoshells was developed to aid in magnetic resonance imaging (MRI) during photothermal ablation treatment. Cell cultures treated with these multifunctional aggregate particles were irradiated with 810 nm continuously for 15 min. The temperature was increased by 16°C at a concentration of 7.5 × 1012 particles/mL, thus killing all the cells in the treatment area. The increase in temperature was concentration-dependent.
Similarly, in 2006, Kim et al. used same SKBR3 breast cancer cell culture model as Loo et al. but with fabricated magnetic gold nanoshells consisting of gold nanoshells embedded with magnetic Fe3O4 nanoparticles, 7 nm stabilized with oleic acid, and conjugated with anti-HER2 antibody. This time, SKBr3 cells were exposed to a femtosecond laser pulse with 800 nm and a beam of 1 mm for 10 seconds. They also tested various power of the laser ranging from 20 to 80 mW. From this study, it showed the embedded magnetic Fe3O4 nanoparticles provided high contrast in MRI images and cancer cells incubated with magnetic gold nanoshells conjugated with anti-HER3 were rapidly destroyed upon short exposure to femtosecond laser pulse with an near-infrared wavelength and a low power.
In 2007, a similar magnetic gold nanoshell that contained iron oxide nanoparticles (Fe3O4, 9–11 nm), as described above by Kim et al., was reported by Lim et al. Subsequently, they synthesized gold-coated magnetic nanoparticles coated with a silver nanolayer. These hollow gold shells contain magnetic nanoparticles in the hollow center that can provide supermagnetic characteristics for strong MRI contrast. In addition, composite nanoparticle-treated SKBr3 cells were damaged within 3 min of near-infrared laser at the relatively low exposure of 12.7 W/cm at 808 nm.
However, despite these successful in vitro models of gold nanoshells, in the next few years there was a noticeable lack of reports focused on the efficacy of gold nanoshells in vivo after systematic administration. This is due to the number of biological barriers inhibiting nanoparticle circulation and targeting that are commonplace in oncology, but not in cell culture. The challenges include reticuloendothelial removal, leakage into extravascular fluid space, and low nanoparticle accumulation into the tumor matrix.
In 2012, Pattani and Tunnel conducted a comparative study of heating between gold nanoshells and gold nanorods. They measured the heat generated by both types at the same optical density to determine the photothermal transduction efficiency. They found that owing to the larger size of nanoshells, their absorption cross-section was much larger than nanorods. As a result, gold nanoshells produced more heat than gold nanorods. But when they compared the effectiveness of converting light radiation into thermal energy (photothermal efficiency), the nanorod shape was two times more efficient. This study brought to light the need to consider not only the amount of heat generated per particle but also the size of the particle needed to target the tumor. The nanorods generated less heat, per particle, but have the potential to be more effective at targeting owing to their smaller size.
In the same year, Choi et al. reported the successful in vivo model of delivering gold nanoparticles to brain metastases of breast cancer by using macrophages known as ‘a cellular Trojan horse’ system. They synthesized silica-core gold nanoshells by the hydrolysis of tetraethylorthosilicate. Their idea was to load macrophages with nanoparticles and use these cells to traverse the blood–brain barrier without damaging it. Because the blood–brain barrier is a major impediment to the delivery of therapeutics to brain, this was and still is a critical unmet clinical need. Choi et al. used mice to establish human breast metastatic tumor xenografts in the brain. Macrophage cultures were incubated with gold nanoshells for 3 days. The resulting gold nanoshell-loaded suspension was then injected into the systemic circulation via the tail vein. These gold nanoshell conjugates were also fluorescently labeled so that the loaded macrophages could be tracked. The result shows that gold nanoshell-loaded macrophages were able to cross blood–brain barrier following injection into systemic circulation. They could also observe macrophages loaded with nanoparticles in lung and liver. Choi et al. suggested that this indicates a cancer treatment advantage where lungs and liver are frequent site of metastatic diseases and not a targeting issue. Moreover, this ‘Trojan Horse’ delivery system became a successful active gold nanoshell delivery strategy, and this system could enable the delivery of other nanovectors such as nanorods, hollow nanospheres in the sub-100 nm and sub-150 nm. Since this report, others have utilized this Trojan Horse approach to deliver gold nanoshells in other experimental cancer models.
One of the examples was reported by Trinidad et al. in 2014. They investigated that the combination therapy of macrophages with gold nanoshell enabled photothermal ablation therapy in an in vitro model using head and neck squamous cell carcinoma (HNSCC) cells. Common treatments for HNSCC include hyperthermia and photodynamic therapy. However, hyperthermia therapy can cause unwanted toxicity to normal tissue around tumor. To avoid this side effect, they used 120-nm silica core with a 12–15 nm gold shell to investigate the combined effect of photothermal therapy and photodynamic therapy in vitro. For photothermal therapy, the cells were irradiated with λ = 810 nm for 5 min and for the photodynamic therapy experiment, λ = 670 nm for 5 min. The result from this study showed that macrophages loaded with gold nanoshells could enhance the effect of photodynamic therapy because of photothermal properties of gold nanoshells.HNSCCs have been found to contain significant quantities of macrophages. Therefore, these tumors should be good candidates for macrophage-mediated therapy. This Trojan horse concept has promise for nanotherapeutics to overcome the challenge of tumor microenvironment. Many publications report investigations on other types of gold nanoshell conjugates that further expand therapeutic applications of this technology.
Topete et al. hypothesized that drug targeting could be combined with gold nanoshell technology for a multimodal therapeutic. They developed a multifunctional nanoplatform that consisted of a biodegradable polylactic-co-glycolic acid (PLGA) matrix loaded with the anticancer drug doxorubicin. This system could provide chemotherapy, phototherapy, and thermotherapy with fluorescence imaging for diagnosis under near-infrared light capabilities. They used a cell culture model to test cytotoxicity of this nanoplatform on tumor cells and used an in vivo model to investigate the ability of the nanoplatform as fluorescence imaging. The results from both models are promising. In vitro, the combination of chemotherapy and hyperthermia and reactive oxygen species from the irradiation caused enhanced cell toxicity. In addition, in vivo model showed that the nanoplatform was localized and retained in the tumor region for a long period of time.
One of the major issues with therapeutic nanoparticle applications is getting a clinically relevant dose to the tumor. Thus, there are numerous reports beyond what is possible to describe herein that focus on different targeting strategies to maximize tumor dose, for instance. In this review, we have tried to cover the most relevant strategies, including PEG capping, antibody targeting, and the Trojan horse approach. These and related strategies are limited by a number of factors. First, scale up production and quality control of commercial quantities is difficult at best. Second, incorporating antibodies into the equation is extremely expensive. Finally, one can only guess that working an antibody-targeted, multilayered nanoparticle through the regulatory maze would be daunting, despite device or drug classification. Nevertheless, the large number of academic publications containing supporting data for this strategy underlines the commitment of funding bodies and scientists to this area. In searching http://clinicaltrials.gov, there was only one trial, NCT01270139, that the terms ‘gold nanoshell OR nanoshell OR gold nanoparticle’ returned. This trial utilized the Trojan horse technique (with allogeneic stem cells) to traffic the gold nanoshells (60/15- and 70/40-nm silica core/gold nanoshell) to atherosclerotic plaques in 60 patients per group. The primary outcome was atheroma volume, or the volume of the degenerative plaque on the inner wall of the artery. The nanoshell group contained the least number of serious adverse events compared with stenting control group. The gold nanoshell group also had the lowest atheroma volume (108 mm3), where a normal 53-year old would have a volume of 188 mm3 and the stent group had 178 mm3. Overall, the trial appeared to be successful with the follow up showing that the Trojan Horse nanoshell group had a significantly lower risk of cardiovascular death than the other groups tested. This study is certainly promising. There are likely to be other studies completed or ongoing that may not be registered with this database. In any event, it is possible to attain clinically relevant outcomes with therapeutic metal nanoparticles despite the complex nature of nanoparticle fabrication.
The shape of the gold nanoparticle, i.e., sphere, nanoshell, or nanorod, is thought to be useful for different types of photothermal therapy. At the heart of this hypothesis is the goal of achieving maximum absorption efficiency. This can be calculated using Mie theory or discrete dipole approximation. Kessentini and Barchiesi calculated the absorption efficiency of nanorods, nanoshells, and hollow nanospheres that approximated the current state-of-the-art with gold nanoparticles used for photothermal therapy. They observed that shallow cancer therapy would be most efficient with the nanoshells. The nanoshells and nanorods were reported to have similar absorption efficiencies for deeper treatment. The hypotheses generated from these data will need to be validated in animal models.
ENHANCED RADIOSENSITIZATION WITH GOLD NANOPARTICLES
Radiotherapy is used to treat cancer with great success in some scenarios and less in others (for a recent review, see Ref ). The concept of using high-Z materials to enhance radiotherapy began in the 1980s with iodine.[64-66] This research was based on the established use of iodine as a contrast agent. The rationale for investigating these agents was to improve the therapeutic ratio of radiotherapy to effect lethal damage to the tumor target with sparing of adjacent normal tissue. For a review on the adverse effects of radiotherapy on normal tissue, see Ref . It was appreciated that low-energy X-ray absorption dose was enhanced by iodine. The question that Iwamoto et al. explored was whether this enhanced absorption translated to improved brain tumor treatment. They delivered 15 Gy of 120 kVp X-rays to brain tumors in a rabbit model. Computerized tomography scans were used to estimate the treatment enhancement with iodine at 30%. Similarly, the median days of survival improved with iodine compared with the controls with just radiotherapy and no treatment at 38.5, 25.5, and 3 days, respectively.
Herold et al. made an important intellectual step by exploring the use of 1.5–3.0 µm diameter gold particles to enhance radiotherapy in a mouse tumor model. Ten thousand EMT-6 mouse tumor cells were injected in C.B17/Icr SCID mice. Experimental groups were subjected to three intra-tumor injections of a 1% gold microparticle solution in fetal bovine serum of 100 μL each. Irradiation was subsequently carried out in a chamber with 8 Gy of 200 kVp X-rays, a little more than half of the dose used by Iwamoto et al. described above. A number of outcomes were examined in both in vitro and in vivo models. Herold et al. reported that the average dose increase with the microparticles was 42–43%. This effect was quantified primarily by in vitro plating efficiencies of the EMT-6 cells derived from treated tumors. Thus, direct comparison between the Iwamoto et al. and Herold et al. data is not possible. However, we speculate that they both strongly support the hypothesis that high-Z materials can significantly enhance radiotherapy in the dose range used as these doses are much higher than that used in standard radiotherapy. Herold et al. showed that gold microparticle treatment enhanced the therapeutic effect to a similar degree as Iwamoto et al., with X-rays of higher energy but at a much lower dose. This helps support the idea that gold is an excellent candidate for this approach.
Herold et al. pointed out that smaller gold nanoparticles hold promise in terms of targeting tumors, while also enhancing radiotherapy. This combined with the earlier efforts of Hainfeld in immunotargeting gold nanoparticles to tumors form the foundation for today’s research into targeted gold nanoparticles for enhanced radiotherapy. Hainfeld et al. suggested using radioactive gold nanoparticles labeled with antibody fragments targeted to tumors as improved therapeutics in 1990. In the two decades that followed, this metal nanoparticle therapeutic technology has evolved, but has not moved too far from these founding concepts.
In 2004, Hainfeld et al. published ‘The use of gold nanoparticles to enhance radiotherapy’ with studies like Herold et al. and Regulla et al.forming a strong foundation on which to build. This report detailed two major experiments based on a mouse mammary carcinoma model. The tumor-bearing mice received a single injection of small (1.9 nm) gold nanoparticles. The doses ranged from 1.35 to 2.7 g gold nanoparticles per kilogram injected via the tail vein. Both tumor volume and survival were monitored, with survival data exceeding 1 year (Figure 3). The resulting data strongly supported the hypothesis that gold nanoparticle-enhanced radiotherapy was a viable therapeutic strategy for treating cancer. These data showed dose enhancement and even ‘cured’ some animals. The radiation dose was 26 or 30 Gy using 250 kVp X-rays. Hainfeld et al. used atomic absorption spectrometry to quantify the mass of gold in various tissues, including the tumor. These results revealed that 4.9 ± 0.6% of the injected dose was present in the tumor 5 min after injection and that the majority of the dose was in the kidney at that stage. The most favorable tumor-to-tissue ratio was 3.5 when comparing tumor-to-muscle that had 1.4 ± 0.1% of the injected dose. It must be mentioned that the nanoparticles used in this study were just gold, without any targeting moieties. The targeting strategy was to passively use the enhanced permeability and retention effect, but the time course for injection and treatment were only separated by 2 min. In the end, this was a seminal report that was very exciting and confirmed earlier speculation that gold nanoparticles could be effectively used to enhance radiotherapy. However, the mechanism of action and modeling to predict the optimal conditions were unknown.
In 2005, Cho et al. reported phantom-based estimations of the dose enhancement when using gold nanoparticles for enhanced radiotherapy. They put forth a two-part rationale for gold nanoparticle radiotherapy enhancement. First, gold has a high-Z number (Z = 79) compared with the previous elements evaluated for dose enhancing with iodine (Z = 53) and gadolinium (Z = 64). This translates to an increased photoelectric cross-section which is founded on photoelectric effects, or more simply the interactions of photons and atoms. This relationship is based on the atomic mass and energy. The corollary is that that the photoelectric effects increase sharply with atomic mass. Therefore, gold would be a much more suitable element in this context than iodine or gadolinium. As it happens, gold also has a long history of being nontoxic in living systems. Secondly, the size of the particle was critical for escaping the tumor vasculature using the enhanced permeability and retention phenomena (for a recent review, see Ref ). Data support the minimal cutoff of 40 kDa in terms of molecular weight or 10 nm in diameter, a maximal diameter of 400 nm, weakly negative to neutral surface charge, and a long circulation time. Cho et al. focused on only a handful of possible variable states using phantoms irradiated with either 140 kVp X-rays, 4 or 6 MV photon beams, or 192Ir γ rays. The most effective scenario was with the 140 kVp X-rays on a superficial target loaded with 30 mg gold nanoparticles per gram tumor resulted in a dose enhancement factor of 5.6. This level of gold loading may not be within realistic ranges, but it does illustrate that enhancement gains are possible. Cho et al. also reported that the photon beam approach did not result in any appreciable improvements in dose enhancement. Given the much higher energy of the photon beams this is not a surprise as they may pass through the high-Z material without the photoelectric or Compton interactions. There was, however, a maximal dose enhancement of 1.3 when using 192Ir γ rays. This exposure level is less energetic and likely to interact with the high-Z material given that the γ emission is in the range of 0.2–0.6 MV. This dose enhancement increased toward the edge of the target before falling off completely outside the target. Again the dose enhancement was closely tied to the amount of gold present, the more gold, the more the dose was enhanced. The overall conclusion was that the amount of gold per gram of tumor was the most important factor. They suggested that 30 mg gold per gram of tumor would be key for clinically relevant dose enhancement with 192Ir γ rays. The varied dose enhancement with differing energies of X-rays and γ rays supports the hypothesis that energy of the photon is a critical factor for achieving a clinically relevant response.
The potential for enhanced radiotherapy with much lower doses was explored by Cho et al. in 2009 through a Monte Carlo-based study where they simulated several low-energy approaches. Their simulations included 125I, 50 kVp, and 169Yb sources (250–350 keV). The estimates showed promising dose enhancement ratios for each source based on 7 and 18 mg gold nanoparticles per gram tumor. These ratios were most favorable for the 169Yb source at 2.1 at the center of the tumor for the 18 mg gold nanoparticles per gram tumor dose. These and the authors’ previous publication supported the overall use of gold nanoparticles in the context of radiotherapy enhancement, but importantly also suggested possible dose goals and sources for the elusive ‘clinically relevant dose’.
The term ‘clinically relevant dose’ is a critical concern and a topic of considerable debate within this field. The progress of gold nanoparticle-enhanced radiotherapy technology has gone through two major shifts in focus. The above reports are focused on establishing the basic feasibility of gold nanoparticle-enhanced radiotherapy. The work was conducted on both theoretical and biological fronts. It is clear from these relatively early reports that there is indeed the potential for gold nanoparticle-enhanced radiotherapy. In order to achieve the ‘clinically relevant dose’, the variables need to be optimized. A practical way to optimize this approach would be to systematically optimize the energy and dose photon and the nanoparticle dose. The second focus in this field is on targeting nanoparticles in hopes of achieving a more favorable tumor to nontumor dose ratio and of course elevating the tumor nanoparticle dose. If successful, this would enable lower radiotherapy doses to have higher impact on tumor killing. The nanoparticle-targeting strategies reported to date are all based on attaching targeting or cell entry moieties onto the surface of gold nanoparticles, which is a relatively straightforward approach.
Hainfeld et al. had foreshadowed targeted gold nanoparticle approaches in their 1990 manuscript entitled ‘Radioactive gold cluster immunoconjugates: potential agents for cancer therapy’ Targeted gold nanoparticle approaches are now being sought as a means to improve the mass of gold present in tumors as was suggested by Cho et al. One approach reported was to utilize glucose-capped gold nanoparticles by Roa et al. The hypothesis was that the increased metabolic rate of tumors would facilitate uptake of these nanoparticles relative to the normal tissue. The 10-nm gold nanoparticles were capped with 6-deoxy-6-fluoro-1-thio-d-glucose via a reductive reaction with the thiol group and the gold nanoparticle. These nanoparticles showed improved uptake in a prostate carcinoma cell line (DU-145) compared with a fibroblast cell line. The treated cells (15-nm gold nanoparticles) were irradiated with a 137Cs (1.176 MeV that is slightly higher energy than the previously discussed experiments) source for a 2-Gy dose. Roa et al. monitored both surviving fractions and metabolic activity with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. They also evaluated cell cycle perturbations with flow cytometry. The results showed that the targeted nanoparticles alone had no serious effects, but when combined with 2 Gy irradiation there was increased cell death and a G2/M delay attributed to the inhibition of cyclin A. The authors suggested that this could help to synchronize the cell cycle of tumor cells making them an easier target for therapy. The dose enhancement factor for these in vitro experiments appeared highest at 6 h post-treatment at a 1.38.
Kim et al. reported using both targeted and diethylenetriamine pentaacetic acid (DTPA) with cysteine-conjugated gold nanoparticles. The authors explored the use of these and similar iron oxide nanoparticles within in vitro and in vivo cancer models. The models were focused on survival outcomes as previously discussed. They used 1.9 (as in Ref ) or 13-nm gold nanoparticles with the larger ones capped with DTPA (glutathione) for improved uptake. The in vitro doses ranged from 0.2 to 2.0 mg/mL. Cellular uptake was reported to range from 3.6 to 176.6 µg gold/106 cells for both the 1.9- and 13-nm gold nanoparticles without much difference between the two. The cells received a 100-Gy dose, which is a surprisingly high dose and would be expected to kill all exposed cells calling into question the study design and interpretation. Metabolic function, as measured by MTT assay, decreased in a dose-dependent manner in all nanoparticle-treated cells but was surprisingly unchanged without irradiation. Data for irradiated cells without nanoparticles were not presented, making interpretation difficult. In the animal tumor model, mice were injected with CT26 (colon cancer cell line) cells and tumors allowed to grow to 6–11 mm. Tail vein injections of nanoparticles were done at 100 or 300 mg/kg body weight. Nanoparticle distribution was assessed 30 min postinjection by inductively coupled plasma. Unfortunately, a breakdown of gold and tissue was not presented. The only values given were of the tumor (0.28%) for the highest dose of gold nanoparticles which is 10 times less than that observed by Hainfeld et al. using the same 1.9-nm uncapped gold nanoparticles. These divergent results illustrate the inherent difficulty in animal models. This highlights that there is little understanding of the physical interaction of the photons, range of energies used, and the concentration and size of the gold nanoparticles.
Physical interactions are likely to be a significant confounder in these biological systems. Obviously, the biology of one tumor model will be different from the next, but importantly, there could be even greater clinical differences. The other notable difference between these studies was that Hainfeld et al. used 1.35 g/kg and Kim et al. used a maximum of 0.3 g/kg. It is not clear why Kim et al. used a 4.5 times lower dose, but the results clearly show that there is a far less effective response. Together these data show two approaches, one with a high dose of untargeted particles and another with a far lower dose of capped particles. It appears that the latter has an effect, but not anywhere near the former despite using three times more irradiation (100 Gy in Kim et al. and 30 Gy in Hainfeld et al.).
More recently, Roa et al. showed significant improvements in tumor uptake with the addition of PEG-6000 capping to their glucose-coated gold nanoparticles to the naked, citrate-stabilized nanoparticles (Figure 4). Noncancerous tissue uptake was 0.043 µg/g and the maximal tumor intake was 0.198 µg/g. Joh et al. also reported a PEG-capped gold nanoparticle that was tested as a means to enhance radiosensitization in a mouse model of glioblastoma. The authors found an in vitro dose enhancement of 1.3 that agrees well with previous reports. They also found that the radiotherapy (20 Gy, 7–14 days before nanoparticle injection) increased brain blood vessel permeability to gold nanoparticles and proposed that this phenomenon could help passive brain tumor targeting. The amount of gold found in this tissue was reported to be approximately 1.5% (increased from ∼0.4% with radiotherapy pretreatment) of the injected mass (0.4 g/kg) per gram of tissue. So, approximately 12 mg would have been injected into a 30 g mouse, resulting in 0.18 mg/g tissue. In this study, the mean mouse survival significantly increased from 18.3 ± 8.4 days with radiotherapy alone to 27.2 ± 6.5 days with gold nanoparticles and radiotherapy.
There is still a long way to go to reach the levels projected by Cho et al. at 7 and 18 mg/g tumor. Only Hainfeld et al. have reported doses approaching those suggested as achievable by Cho et al.[32, 33] at 6.5 mg/g in the periphery of one tumor. The lack of additional data showing this level of tumor-specific gold can be due to many factors. It appears that recent reports are using relatively low concentrations of gold nanoparticles in their models and some do not specifically measure gold content in tumors. These studies also appear to be reporting dose enhancement factors in the range of 1.5 despite a variety of models, nanoparticle capping agents, and endpoint assays. One clear trend is that the irradiation dose range has settled in at 2–10 Gy. This is a clinically relevant dose of radiotherapy in the treatment of many cancers.
The statement that is clearly repeated throughout the literature is that this approach is expected to be most effective in superficial tumors and that the mode of nanoparticle targeting needs to be tuned to the disease. It appears that this approach has an upward trajectory, but is mired in animal models owing to a lack of physical modeling of the interactions needed to predict more relevant conditions. Interestingly, an early gold foil report by Regulla et al. showed significantly higher dose enhancement (up to 114 using mGy doses) than more recent nanoparticle-based reports using much higher photon doses. The low toxicity of gold and the correspondingly low irradiation dose needed to examine possible enhancement lend itself to further testing in volunteers. The question is whether nanoparticles, microparticles, or some other modality will be optimal. Perhaps, each type of cancer will have an optimal enhancement technology? Regardless, volunteer studies are a necessity. Only then will we know the true potential for gold nanoparticle-enhanced radiotherapy.
RADIOFREQUENCY ABLATION WITH GOLD NANOPARTICLES
Radiofrequency fields can be used to induce gold nanoparticle thermal ablation in a manner similar to that of photothermal and radiosensitization therapies. Essentially, the gold nanoparticle is hypothesized to function as a tiny resistor during radiofrequency exposure, heating the nanoparticle to induce thermal damage. For example, Cardinal et al. used a solid-state radiowave to transmit at 13.56 MHz into cultured HepG2 cells and a rat hepatoma model. Gold nanoparticles were used to treat the cultures and the animals at 4 nmol/L in vitro and a 0.5-mL injection of gold nanoparticles (13 nmol/L). There was significant thermal increases in the gold-treated groups that resulted in thermal injury. The data were promising but additional targeted studies are needed to support the further development of this strategy for human use.
ANTIMICROBIAL SILVER NANOPARTICLES
Silver nanoparticles represent the most common man-made nanomaterials used in commercial medical and consumer products including household antiseptic sprays and antimicrobial bandages. For a review, see Rizzello and Pompa. Silver nanoparticles have been used as powerful antimicrobial agents[73, 74] and the toxicity of silver nanoparticles has been well documented.[40, 75, 42] Silver ions appear to block the respiratory enzyme pathways and alter microbial DNA and the cell wall, resulting in antimicrobial effect. Silver ions from silver nanoparticles exhibit toxicity; however, there is still no conclusive evidence to show whether metallic nanoparticles themselves exert the particle-specific toxicity. In fact, some studies showed that silver nanoparticles were generally more toxic than gold nanoparticles by showing that cell exposure to silver nanoparticles actually enhanced cytotoxicity.[43-45] Silver nanoparticles have been shown to act as antifungal, antiviral, antiprotozoal, and antiarthropod, indicating a potential treatment and control of infectious disease. There is still a large amount of research required in the field of therapeutic application of silver nanoparticles. One commonly described application is the use of silver nanoparticles to improve wound healing.
In 2007, Tian et al. demonstrated that topical delivery of silver nanoparticles promoted wound healing and reduced scar appearance in a dose-dependent manner in mice. They used a murine thermal injury model and compared silver nanoparticles to silver sulfadiazine, which has been the standard treatment for burns (Figure 5). In this study, silver nanoparticles were spherical, with diameters of 14 ± 10 nm. The concentration of 1 mM in the presence of citrate as a stabilizer agent was used. This study also showed that silver nanoparticles not only worked as the efficient antimicrobial property but also suppressed both local and systemic inflammation in vivo.
Later in 2010, there was another study done by Liu et al., examining skin wound healing in vivo with silver nanoparticles. They specifically investigated the mechanical would healing process by focusing on two different cell types: keratinocytes for re-epithelialization and fibroblasts for would contraction. The mean silver nanoparticle diameter was 10 nm (ranging from 5 to 15 nm) and final concentration of 1 mM containing sodium citrate was used in this experiment. They found that silver nanoparticles could increase the rate of wound closure by promoting the proliferation and migration of keratinocytes. Moreover, silver nanoparticles appeared to drive the differentiation of fibroblasts into myoblasts, thereby promoting wound contraction.
Around the same time in 2009, there was a study shown for the first time that silver nanoparticles could penetrate into human stratum corneum and the outermost surface of the epidermis. This has opened up another possibility of using a topical application of silver nanoparticles compared with the zinc oxide or titanium dioxide particles, which are often accumulated on the external surface of the stratum corneum. The size of silver nanoparticles ranged from 25 to 49 nm and the stabilization was achieved by coating nanoparticles with polyvinylpyrrolidone. The final concentration of 0.14 wt% in ethanol was used in this experiment. They used excised human abdominal full thickness skin and used an electrical conductometer at 300 Hz to differentiate between intact and damaged skin. The results showed that a median amount of 0.46 ng/cm2 of silver nanoparticles could penetrate through intact human skin. On the other hand, a median amount of 2.32 ng/cm2 of silver nanoparticles could pass through damaged human skin. Also smaller than 30-nm silver nanoparticles were able to reach to the deepest layer of the stratum corneum and the outermost surface of the epidermis by passive permeation. These results suggest that silver nanoparticles could at least adhere to the skin surface and give relatively long-term therapeutic benefit.
Silver, like gold, is a high-Z element that has potential for both enhancing photothermal ablation or radiation-enhanced therapy. In 2013, Kleinauskas et al. reported the synthesis of a silver nanoshell with a carbon core. They tested the capacity of these silver nanoshells for photothermal ablation or radiation-enhanced therapy. An in vitro prostate adenocarcinoma cell line model was utilized to evaluate the killing effects of each therapeutic modality. Carbon-core silver-shell nanorods were prepared in an aqueous solution from PEGylated carbon core particles (26 ± 12 nm) that had a mean diameter of 60 nm. They tested this composite nanoparticle at a final concentration of 1.5 mM in the cells that were subsequently exposed to either UV light (375–410 nm for 5–7 J/cm2) or 160 kV X-rays (0–25 Gy). The results from this study showed that composite nanoparticles were significantly cytotoxic to the cells for both types of radiation compared with the cells irradiated only with ionizing/nonionizing radiation. The dose enhancement ratios for the radiotherapy were in line with the reports above with the highest dose (25 Gy) showing a dose enhancement ratio of 1.3. The dose enhancement of the UV exposure was greater at approximately 15. This complex report showed that these silver nanoshells are capable of enhancing both UV and radiotherapy, but interpretation is limited by the in vitro nature of the models investigated.
Recently, to aim to develop multifunctional tumor-targeted silver nanoparticles, Locatelli et al. synthesized polymeric nanoparticles with smaller silver nanoparticles embedded within in addition to a cytotoxic drug, alisertib. This composite nanoparticle was also coated with a chlorotoxin-derived peptide for targeting glioma cells that express matrix metalloproteinase-2 (MMP-2). The core nanoparticle, PLGA-block-PEG-carboxylic acid (PLGA-b-PEG) copolymer, was previously reported by Ding et al. The nanocomposite evaluated by Locatelli et al. was smaller in size at 112 nm compared with Ding et al., which was ranged between 150 and 180 nm. Brain cancer cells often overexpress MMP-2, and therefore, they hypothesized that peptide targeting should be specific in the glioma cell line model they used. They also injected radiolabeled composite nanoparticles into immunodeficient mice bearing glioblastoma. The results from in vitro study indicated that there was little to no cytotoxicity from composite nanoparticle treatment up to 72 h incubation. In vivo, tumor size reduction only occurred after day 48. Also, there were detectable concentrations of the radiolabel in the mouse tumors, but the stability studies showed that at 6 h only a fraction of the complete nanocomposite remained. It is encouraging that a six-component nanocomposite can be synthesized and used in vivo with a positive outcome (−34 ± 12% change in tumor size). There does appear to be a clear disconnect between the stability data with the 48 day onset of tumor regression. There is also the need to understand the effects silver nanoparticles have on cellular biochemistry versus that of the silver ions. It is well known that silver nanoparticles have been widely used as novel therapeutic agents, including uses in anti-inflammatory or would healing. A very promising prospect of silver nanoparticles is its use in cancer therapy. The potential of multitargeted silver nanoparticles can be possible by developing variety systems.
Gold and silver nanoparticles are not mutually exclusive therapeutics. They can also be combined within one nanoparticle to provide improved functionality. For example, Shi et al. recently reported the use of Au@Ag/Au nanoparticles that were investigated for use in image-guided photothermal therapy for cancer. They used an aptamer with a quencher as a fluorescent sensor probe specific for A549 tumor cells. Shi et al. used mice injected with A549 cells as the in vivo model. The mice were injected with 0.1 mL containing 2.2 × 109nanoparticles. The results showed that the targeted photothermal therapy resulted in necrosis in the area where the tumor was injected. The outcome of this study showed that even a complex theragnostic system is possible using Au@Ag/Au nanoparticles.
Interestingly, Ag@Au nanoparticles can also be used for photothermal therapy. Yang et al. investigated the photothermal killing potential for Ag@Au@phenol formaldehyde resin nanoparticles in vitro with HeLa and HepG2 cells. The cells were treated with 130 µg/mL of as-synthesized nanoparticles for 6 h. There were significant increases in temperature with 808-nm irradiation. Cell viability fell to less than 5% in the two highest nanoparticle dose groups. The Ag@Au nanoparticles have tuneable optical properties that increase the potential for multifunctional applications. In summary, many different combinations of materials can be explored, but the potential for translation to clinical application decreases with increasing complexity of the material used. Therefore, it may be the simpler metal nanoparticle therapies that reach the clinic first.
DNA DAMAGE WITH PLATINUM NANOPARTICLES
In 1965, Rosenberg et al. published a report on the inhibition of Escherichia coli division, not grown, by platinum electrolysis products.Four years later, Rosenberg et al. revealed that a square planar form of platinum, what we now know as cisplatin, was highly effective against rat sarcomas. The drug entered clinical trials (1971) and was finally approved for use by the USA Food and Drug Administration in 1978.
However, cisplatin is not a nanoparticle. Platinum nanoparticles have been shown to possess the capacity to enter cells. There is also evidence for DNA damage and antioxidant response changes associated with platinum nanoparticle treatment in vitro. Pelka et al. observed that DNA integrity was compromised and that cellular glutathione was decreased at 1 ng/cm2 platinum nanoparticles. Their data showed that there was an increase in toxicity with a decrease in nanoparticle diameter where <20-, <100-, and >100-nm particles were evaluated in vitro using a human colon carcinoma cell line (HT29). Finally, the authors observed that the toxic effects did not appear to be due to reactive oxygen formation, leading to the hypothesis that platinum ions could be the cause. Platinum ions from nanoparticles could then be used as anticancer therapies with a similar strategy to cisplatin. Asharani et al. observed that cultured cells treated with platinum nanoparticles showed activated p53 and subsequent p21 activation that was hypothesized to be stimulated by genotoxic stress. Porcel et al. suggested that platinum nanoparticles could be used for enhanced radiosensitization. They evaluated this hypothesis in vitro with promising results. Together, these data present a picture of a potential cancer therapeutic. The development of a therapeutic platinum nanoparticle is far earlier than that of gold, but is promising none the less.
Metal nanoparticles are being used in a wide array of applications that reach far beyond therapeutics. However, in the therapeutic arena it is clear that gold and silver nanomaterials are the most promising agents. This is largely owing to their unique interactions with light and radiation. The exception is of course the interesting effects of silver nanoparticles have in wound healing and potentially for amplifying the effects of anticancer drugs. It can be expected that the next decade will reveal clinical effectiveness of the more robust but less complicated systems. Overall, there is real potential for metal nanoparticles to cross the regulatory barrier into clinical use as effective therapeutics.
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