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European Journal of Cardio-Thoracic Surgery 50 (2016) 6–16 doi:10.1093/ejcts/ezw002 Advance Access publication 2 February 2016 Cite this article as: Hofferberth SC, Grinstaff MW, Colson YL. Nanotechnology applications in thoracic surgery. Eur J Cardiothorac Surg 2016;50:6–16.

Nanotechnology applications in thoracic surgery Sophie C. Hofferbertha, Mark W. Grinstaffb and Yolonda L. Colsona,* a b

Division of Thoracic Surgery, Department of Surgery, Brigham and Women’s Hospital, Boston, MA, USA Departments of Biomedical Engineering, Chemistry, and Medicine, Boston University, Boston, MA, USA

* Corresponding author. Division of Thoracic Surgery, Brigham and Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA. Tel: +1-617-7326648; fax: +1-617-7302853; e-mail: [email protected] (Y.L. Colson). Received 31 August 2015; received in revised form 1 November 2015; accepted 16 November 2015

Summary Nanotechnology is an emerging, rapidly evolving field with the potential to significantly impact care across the full spectrum of cancer therapy. Of note, several recent nanotechnological advances show particular promise to improve outcomes for thoracic surgical patients. A variety of nanotechnologies are described that offer possible solutions to existing challenges encountered in the detection, diagnosis and treatment of lung cancer. Nanotechnology-based imaging platforms have the ability to improve the surgical care of patients with thoracic malignancies through technological advances in intraoperative tumour localization, lymph node mapping and accuracy of tumour resection. Moreover, nanotechnology is poised to revolutionize adjuvant lung cancer therapy. Common chemotherapeutic drugs, such as paclitaxel, docetaxel and doxorubicin, are being formulated using various nanotechnologies to improve drug delivery, whereas nanoparticle (NP)-based imaging technologies can monitor the tumour microenvironment and facilitate molecularly targeted lung cancer therapy. Although early nanotechnologybased delivery systems show promise, the next frontier in lung cancer therapy is the development of ‘theranostic’ multifunctional NPs capable of integrating diagnosis, drug monitoring, tumour targeting and controlled drug release into various unifying platforms. This article provides an overview of key existing and emerging nanotechnology platforms that may find clinical application in thoracic surgery in the near future. Keywords: Nanotechnology • Lung cancer • Imaging • Theranostics • Drug delivery • Nanoparticles

INTRODUCTION The past decade has seen dramatic growth in the field of nanotechnology, with development of nano-sized materials that have application across several fields, including chemistry, engineering and medicine. The basic premise is that nanoscale materials ( particles, shallow spheres, rods etc.), ranging in size from 1 to 1000 nm, can be created with unique physical and biochemical properties that differ from discrete molecules or bulk materials [1–3]. Moreover, these nanoscale materials can be strategically enhanced to be responsive to various stimuli ( pH, temperature, ultrasound, light etc.), and thus provide a mechanism to achieve selective drug delivery or on-demand control of a response [4, 5]. To this aim, numerous nanotechnology platforms are described that have potential to improve the care of cardiothoracic surgical patients, particularly in the field of thoracic oncology. Importantly, several nano-sized materials and devices may offer solutions to existing physiological challenges in the detection, diagnosis and treatment of lung cancer. Emerging nanoparticle (NP)-based imaging technologies may improve the likelihood of achieving surgical cure in patients with thoracic cancers. Recent advances could facilitate intraoperative tumour localization, lymphatic mapping and accurate oncological resection in the near future. Moreover, nanocarrier-based therapeutics are beginning to impact adjuvant lung cancer therapy. For

example, chemotherapeutic drugs commonly used in the treatment of lung cancer such as paclitaxel, docetaxel and doxorubicin are now formulated within various nanotechnology platforms, including micelles, liposomes, NP-bound drug conjugates and polymeric NPs [2]. These drug delivery systems demonstrate promising early results, predominantly in the preclinical setting. These nanotherapeutic systems also offer opportunities to achieve targeted in vivo delivery of a therapeutic agent for cancer treatment [6]. Several investigators describe stimuli-responsive NPs that ‘trigger’ chemotherapeutic drug release from the nanocarrier under specific biological conditions [5, 7, 8], whereas others have utilized nanotechnology to expand targeted intratumoural small molecule-based therapies and anticancer gene delivery platforms [9]. In addition, NP-based imaging technologies show enhanced detection and monitoring of tumour microenvironments. These successes are fuelling advances in the field of molecularly targeted cancer therapy [10]. Although these nanotechnology platforms are exciting, the next significant advances in lung cancer therapy will likely involve the evolution of ‘nanotheranostic’ multifunctional nanocarriers that are capable of integrating diagnosis, stimuliresponsiveness, drug monitoring, targeted delivery and controlled drug release into a single unifying platform [11]. The purpose of this review is to provide an overview of existing and emerging nanotechnology platforms that may find clinical application in cardiothoracic surgery in the future.

© The Author 2016. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

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Recent advances in nanotechnology promise to improve the surgical management of lung and oesophageal cancers. NP-based therapeutics and imaging platforms have been evaluated in the preclinical arena, and early results suggest these technologies have potential to improve rates of curative resection in thoracic malignancies. Interestingly, multiple NP-based imaging platforms have recently emerged that could enable thoracic surgeons to perform real-time intraoperative tumour localization, sentinel lymph node (SLN) mapping and image-guided nanosurgery to achieve complete oncological resection of thoracic malignancies in the near future.

Lymphatic mapping Patients with Stage I lung cancer have a recurrence rate of nearly 40% and the overall 5-year survival rate is just 52% [12]. The high rate of disease recurrence suggests that these patients are frequently understaged and, subsequently, undertreated. By identifying lymph nodes at highest risk for tumour metastasis, SLN mapping helps identify patients who could benefit from adjuvant therapy. Several NP-based molecular imaging techniques have been explored for potential in the mapping of tumour-draining lymph nodes in human cancers [13, 14]. Quantum dots (QDs) have been utilized as lymphatic mapping agents in a porcine model of non-small-cell lung cancer (NSCLC), with favourable properties including appropriate size for lymphatic migration and visible histological fluorescence (Fig. 1) [15]. More recently, superparamagnetic iron oxide (SPIO) NPs were coupled with magnetic resonance imaging (MRI) to create ultrasensitive nanoprobes for cellular and molecular imaging of various cancers. Notably, SPIO-enhanced MRI technology has been shown to detect tumour metastases in the SLNs of breast, bladder and prostate cancer patients [16, 17]. In a 2011 study of 102 breast cancer patients with clinically negative axillary nodes, Motomura et al. [16] demonstrated that SPIOenhanced MRI achieved almost 90% accuracy in the detection of SLN metastases. These promising results warrant the upcoming investigation of SPIO-enhanced MR imaging as an SLN mapping strategy for lung and oesophageal cancer patients. The ability to accurately identify and treat SLNs would be a significant advance in the current treatment of thoracic surgical patients, since metastatic nodal disease is frequently missed with current lymphadenectomy and histological processing. Several studies have analysed SLNs, deemed negative by haematoxylin and eosin (H&E) staining, with subsequent SLN sectioning and immunohistochemistry (IHC). For example, in breast cancer patients, 14–19% of ‘reportedly negative SLNs’ harboured micrometastatic disease [18, 19]. Similarly, SLN processing and IHC analysis of lymph nodes previously staged as pN0 and pN1 by H&E staining in lung cancer patients ‘missed’ positive nodal disease in an additional 15.7–16% of patients [20, 21]. Furthermore, Liptay et al. in an analysis of 104 lung cancer patients, and Takizawa in a series of 157 lobectomies demonstrated that the SLN was the only node with metastatic disease in 36–37% of node-positive patients [22, 23]. Until now, it has not been possible to accurately identify the SLN for focused histological analysis and possible targeted lymphatic drug delivery to all lymph nodes. However, several nanotechnologies demonstrate the capability of lymphatic mapping for improved diagnosis, and specific lymphatic-based targeted drug delivery for therapeutic benefit in lung cancer.

Image-guided nanosurgery NP-based technologies possess several properties that enhance imaging of biological targets, including the ability to amplify contrast signal, unique physiochemical characteristics such as magnetic, thermal and pH-responsive phase changes, and the ability to modify pharmacokinetics via alterations in surface chemistry [10]. The development of targeted near-infrared fluorescent (NIRF) and surface-enhanced Raman scattering imaging probes for the evaluation of surgical margins and the real-time intraoperative identification of residual disease is a major focus of recent investigation. These technological advances have been translated to patient care with excellent initial results. Currently, NIRF-image-guided surgery utilizing 5-aminolevulinic acid (5-ALA) generates tumour fluorescence and better tumour visualization in patients with malignant glioma. 5-ALA precursor in the haemoglobin synthesis pathway elicits the accumulation of fluorescent porphyrins in various epithelia and cancerous tissues. Oral administration of 5-ALA leads to preferential accumulation of fluorescent porphyrins within glioma tissue, enabling more complete oncological resection and improved progression-free survival [24]. Ongoing research efforts are focused on the clinical translation of various Raman NPs that can be specifically utilized for NIRF imaging of various solid tumours, including lung cancer. It is anticipated that this technology will improve intraoperative guidance of surgical resection with negative margins in the future. Several investigators have developed triple-modality NP imaging probes for use with a number of cancer imaging modalities, including PET, NIRF and MRI, to allow for preoperative tumour localization and surgical planning. One of the best characterized strategies utilizes MRI-photoacoustic silica-coated Raman NPs [25]. Kircher et al. reported the intravenous injection of Raman NPs into glioblastoma-bearing mice, which leads to intratumoural accumulation and retention for several days. Triple-modality MRI-based photoacoustic imaging was then used to guide intraoperative resection and accurately delineate tumour margins (Fig. 2) [25]. Ultra-sensitive Raman imaging can be utilized to detect and remove residual microscopic tumour burden. Coupling of highresolution tissue-penetrating photoacoustic imaging and tumourhoming Raman NPs has potential to enable localization and accurate resection of non-palpable deep parenchymal lung lesions. Such approaches highlight the potential for translation of emerging nanotechnology platforms to thoracic surgery, particularly with the focus of enabling more accurate tumour imaging and improve oncological resection in patients with thoracic cancers.

NANOPARTICLE-BASED DRUG DELIVERY PLATFORMS IN LUNG CANCER NPs are colloidal particles ranging in size from 1 to 1000 nm synthesized from a variety of materials, including lipids, polymers, metals and ceramics. NP-based adjuvant therapy is an emerging therapeutic strategy in medical oncology that improves drug efficacy and decreases toxicity [19]. The limited solubility and unfavourable pharmacokinetics of many novel, potentially efficacious antineoplastic agents hinder clinical application. Encapsulation of drugs within NPs increases drug solubility, improves pharmacokinetics through sustained release, alters biodistribution, protects sensitive drugs from low pH environments or enzymatic alteration/ degradation and facilitates targeting of the drug to desired tissues [7, 26].

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Figure 1: Lymphatic mapping of a porcine lung utilizing NIR fluorescent QD technology. Representative NIR fluorescence images depicted from top to bottom of the surgical field over the following timepoints: (i) pre QD injection (autofluorescence), (ii) time of QD injection, (iii) 45 s post QD injection (lung retracted), (iv) 1 min post QD injection, (v) post sentinel lymph node resection. For each timepoint, colour video (left), NIR fluorescence (middle) and colour-NIR (right) images are demonstrated. White arrow indicates rapid QD localization to the SLN. Absence of fluorescence in the nodal basin after resection confirms complete removal of the sentinel nodal tissue (Reprinted from ref. [15], with permission from Elsevier, Fig. 2A). NIR: near-infrared; QD: quantum dot; SLN: sentinel lymph node.

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Figure 2: Schematic illustration of image-guided surgery using triple-modality MPR nanoparticle. Top: MPRs are injected intravenously into a mouse bearing an orthotopic brain tumour. As nanoparticles enter the blood stream, they diffuse through the disrupted blood–brain barrier and are sequestered and retained by the tumour. MPRs are too large to cross the intact blood–brain barrier and therefore cannot accumulate in healthy brain. Bottom: Proposed use of MPR technology in the clinical setting. Preoperative MRI allows for tumour localization and surgical planning. Only a single injection is necessary as the probe is retained allowing for intraoperative detection in the tumour several days post injection. Photoacoustic imaging with its relatively high resolution and deep tissue penetration is then able to guide bulk tumour resection intraoperatively. Raman imaging with its ultra-high sensitivity and spatial resolution can then be used to remove residual microscopic tumour burden. Resected specimen can subsequently be examined with a Raman probe ex vivo to verify clear margins (Reprinted from Ref. [25], with permission from Nature Publishing Group, Fig. 1). MPR: MRI-photoacoustic-Raman; MRI: magnetic resonance imaging.

Figure 3: Schematic illustration of nanotechnology-based drug delivery platforms.

Despite the survival advantage seen with adjuvant chemotherapy in patients with advanced lung cancer, overall benefits of systemic treatment in early-stage lung cancer are often outweighed by systemic side effects of chemotherapeutic agents [27, 28]. Paclitaxel and docetaxel are two of the most common chemotherapeutic drugs utilized in the treatment of late-stage lung cancer; however, both agents are hydrophobic and can be difficult to deliver to lung tissue due to poor solubility. Therefore, the current clinical formulation of paclitaxel is delivered in Cremophor EL ( polyethoxylated castor oil and ethanol mixture). However, many of the toxic side effects associated with paclitaxel treatment are attributed to this carrier rather than the drug itself. Furthermore,

drug distribution within lung parenchyma is often suboptimal as systemically administered chemotherapeutics are generally cleared quickly, leaving only a small percentage of the total dose locally available in the lung. NPs, however, have the potential to target drug delivery directly to the lungs following intravenous injection, oral delivery or inhalation, as well as deliver a higher dose of drug directly to the tumour for a significantly longer duration [29]. Several NP-based drug delivery platforms are described for the treatment of NSCLC to improve tissue-specific delivery, decrease toxic side effects, increase bioavailability and prevent drug resistance secondary to enhanced cellular efflux (Fig. 3). This section outlines specific drug delivery platforms that are already in

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clinical use or that show particular promise as viable adjuvant therapies in patients with thoracic malignancies. We will focus on the technological advantages, current challenges to clinical translation and specific characteristics of each nanotechnology platform that show promise to improve the care of thoracic surgical patients.

Nano-based lung cancer therapy: Preclinical studies Nanocrystals. Nanocrystals are pure chemotherapeutic drugs processed down to nanometre sizes. The extremely small particle size of nanocrystals increases drug surface area, thereby greatly enhancing drug solubility. As a general solubilization technology, nanocrystals serve as a ‘universal’ multifunctional formulation approach to enhance oral and IV dose-bioavailability and improve intratumoural absorption and penetration of poorly soluble drugs. Importantly, the surface properties and in vivo behaviour of nanocrystals can easily be altered. Various surface coatings can be applied to facilitate absorption, deliver substantial drug payload ratios and enable the encapsulation of multiple nanocrystals [30]. Recent studies utilizing nanocrystal formulations of paclitaxel to target in vivo murine models of lung cancer demonstrated enhanced drug loading and antitumour activity, with less systemic toxicity [31]. Nevertheless, despite the significant therapeutic advantages of nanocrystals, assessment of in vivo biodistribution and tumour accumulation after nanocrystal treatment is limited. A further advance is the development of hybrid paclitaxel nanocrystals that contain fluorescent molecules [32]. This in vivo theranostic platform shows promise to further elucidate existing knowledge gaps and enhance the clinical application of nanocrystal drug suspensions.

Polymeric nanoparticles. The advantages of encapsulating chemotherapeutic agents in polymer NPs include the ability to: (i) encapsulate a wide variety of drugs and release them over prolonged periods in response to different biological triggers, (ii) modify surfaces with multiple targeting ligands and (iii) exhibit excellent stability, both in vitro and in vivo [33]. While poly(lactic acid) and poly(lactic-co-glycolic acid) (PLGA) are the most widely studied due to availability, biocompatibility and their use in FDA-approved products, natural and other synthetic polymers have also been used to prepare polymeric NPs. Limitations of using PLGA NPs for local drug delivery included the rapid ‘burst’ release of the encapsulated drug, regardless of NP location, with potential drug release and delivery of the payload extracellularly and/or via direct drug transfer to contacting cells [34]. Therefore, studies focused on modifying PLGA NP surfaces to improve tumour-targeting efficiency. For example, paclitaxel-loaded PLGA NPs modified with covalently bound wheat germ agglutinin demonstrated superior antiproliferation activity against A549 human NSCLC cells in vitro compared with conventional paclitaxel [35]. Likewise, surface attachment of YIGSR (laminin receptor binding peptide) enhances NP targeting to metastatic lung cancer cells, as laminin expression is up-regulated in metastatic cells [36].

Liposomes. Liposomes are spherical lipid bilayers that are
100– 200 nm in size. Liposomes provide a hydrophobic environment within the lipid bilayer that enables transport of insoluble drugs, while soluble drugs are contained within the internal aqueous compartment inside the liposome. The unique properties of liposomes alter the pharmacokinetics and biodistribution of the

native drug, thereby facilitating delivery of different drug payloads. Limitations of liposomal delivery systems include inadequate drug loading capacity, premature drug expulsion and drug leakage. A recently discovered curcuminoid, CLEFMA, was found to have potent antiproliferative properties and induce autophagic cell death in lung cancer cells; however, the chemotherapeutic utility was limited due to significant drug hydrophobicity [37]. Importantly, the development of a drug-in-cyclodextrin-in-liposome formulation greatly increased the in vivo bioavailability of CLEFMA and demonstrated more potent in vitro antiproliferative activity against lung adenocarcinoma H441 cells compared with naturally occurring curcumin [38]. More recent advancements include a dual-functional liposome system with extracellular pH response and mitochondrialtargeting properties that facilitate drug accumulation within mitochondria and trigger apoptosis of drug-resistant lung cancer cells [39]. Jiang et al. observed increased cytotoxicity against naïve and drug-resistant lung cancer A549 cells when paclitaxel was encapsulated within a dual-functional liposome compared with free paclitaxel and paclitaxel-loaded traditional liposomes.

Micelles. A micelle is defined as a collection of amphiphilic surfactant molecules that spontaneously aggregate in water into a spherical vesicle. The micelle centre is hydrophobic and therefore can sequester hydrophobic drugs until they are released through a drug delivery mechanism. The major disadvantage for most micelle carriers is their rapid clearance from circulation. Copolymeric micelles have proved to be simple, biodegradable, effective and controllable drug delivery carriers for paclitaxel, creating a sustained drug release system that significantly decreases tumour burden compared with free drug in in vitro models of human NSCLC [40]. Zhang et al. showed that paclitaxel-loaded mixed micelles exhibit superior antiproliferative activity against human lung adenocarcinoma A-549 cells compared with free paclitaxel. New frontiers for lung cancer treatment include the development of a dual-drug delivery system that combines a thermosensitive cisplatin-containing hydrogel with paclitaxel-loaded micelles [41]. Early results suggest this delivery system has potential to enable in situ treatment of lung cancer via minimally invasive injection methods. Dendrimers. A dendrimer is a synthetic polymeric molecule composed of multiple symmetrical, branched monomers that emerge radially from a central core. Dendrimers possess unique molecular architectures that facilitate drug delivery; including structural and molecular uniformity, monodispersity and a capacity to bypass efflux transport. Moreover, the high ratio of surface groups to molecular volume makes dendrimers a promising synthetic vector for gene delivery [42]. To date, the clinical utility of such dendrimers is limited by toxicity. Interaction of surface cationic charged dendrimers with negatively charged biological membranes often leads to membrane disruption, manifesting as haemolytic and haematological toxicity. Development of biocompatible dendrimers and the use of surface engineering to overcome toxicity are two key strategies that are being employed to enhance the clinical application of dendritic polymers. Additionally, surface modification utilizing various targeting moieties enables site-specific chemotherapeutic drug delivery and minimal systemic toxicity. Recent technological advances include inhalable drug–dendrimer delivery systems that improve exposure of lung tumour to cytotoxic drug. Kaminskas et al. showed inhalation of doxorubicin-conjugated dendrimers reduces lung tumour burden by >95% compared with IV doxorubicin solution which achieves only 30–50% of tumour burden reduction [43]. Furthermore, a poly(amidoamine) dendrimer nanocarrier system recently facilitated siRNA delivery directly to lung

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epithelium via an aerosol-based formulation [44]. The authors performed in vitro studies to demonstrate that siRNA–dendrimer complexes efficiently target lung alveolar A549 cells and induce gene silencing.

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Quantum dots. QDs are light-emitting semiconductor nanocrystals with a core–shell structure and a diameter that ranges from 2 to 10 nm. They have emerged as a new class of fluorescent labels for cellular analysis, molecular imaging and biodiagnostics [45]. However, the toxicity of QDs as a function of heavy metal content remains a barrier to clinical translation. To reduce potential toxicity, QDs have been encapsulated in polymers with varying surface chemistries and successfully used as imaging probes both in vitro and in vivo [45]. Another important advance is the development of multiplexed QD-antibody conjugates to map the molecular, cellular and glandular heterogeneity of human cancers [46]. Multiplexed QD mapping can provide new molecular and morphological information that is not detected by traditional histological tissue stains, especially at complex and suspicious disease foci. Specific to lung cancer, antibody-labelled QDs can detect micrometastatic disease in peripheral blood through identification of circulating tumour cells enriched by pan-cytokeratin antibody-labelled magnetic NPs [47]. Furthermore, as outlined previously, novel QDs have potential for real-time image-guided localization of SLNs during oncological resection [15, 48, 49]. Carbon nanotubes. Carbon nanotubes (CNTs) are an allotrope of carbon that forms cylindrical carbon molecules. Due to a high surface area, superior chemical stability and rich electronic polyaromatic structure, CNTs are able to adsorb (or be conjugated to) a wide variety of therapeutic molecules, including drugs, proteins, antibodies, DNA and enzymes. Nanotubes are a promising vehicle for drug delivery as they are capable of direct cell penetration and can prevent drug metabolism during transport through the body [50]. A significant concern, however, is the risk of systemic toxicity, including the potential for carcinogenesis. Therefore, significant research effort is focused on surface functionalization to solubilize CNTs and increase biocompatibility. Moreover, laser-responsive intratumoural cargo delivery has been shown to be achievable through utilization of CNTs’ unique optical properties and recent studies describe the utility of CNTs as an antitumour vaccine delivery tool, or local antitumour hyperthermia therapy upon excitation with near-infrared light [51]. An exciting recent finding is that oxygen– CNTs significantly increase the chemotherapeutic effect of paclitaxel on breast cancer cells [52]. Therefore, CNTs may have potential as a chemosensitizer for other malignancies, including lung cancer.

Stimuli-responsive nano drug delivery systems It has long been the goal to achieve selective delivery of therapeutic agents to target tissues to maximize therapeutic potential while simultaneously minimizing local and systemic side effects. An exciting advance is the design of stimuli-responsive nanocarriers, i.e. materials that change in response to an external stimulus and actively participate in the optimization of therapy. Stimuli-responsive nanocarriers, including liposomes, polymeric NPs, copolymer micelles and dendrimers, can be ‘tuned’ to obtain a desired stimuli-responsive phenotype. These nanocarriers are most attractive when the stimuli are unique to the disease pathology, allowing the nanocarrier to respond specifically to a pathological ‘cancerspecific’ trigger. Examples of biological stimuli that are being explored for targeted drug and gene delivery include pH,

Figure 4: Prevention of lung tumour growth in an in vivo model using paclitaxel-loaded expansile nanoparticles. (A) Paclitaxel-loaded expansile nanoparticles prevent tumour growth in vivo. Paclitaxel-loaded non-expansile nanoparticles, empty expansile nanoparticles and paclitaxel exhibit inferior antitumour efficacy. †P < 0.0005 vs control. (B) Tumour growth depicted over time for animals undergoing various treatments. Data displayed as mean (SEM. Day 11 *P < 0.05 vs control and Day 14 †P < 0.0005 vs control (reprinted from ref. [7], with permission from American Chemical Society, Fig. 5). SEM: standard error of the mean.

temperature, light, oxidative/reductive environments and electromagnetic radiation [5, 53]. One of the biologically triggered systems being explored for clinical application in lung cancer are pH-responsive nanocarriers. These responsive NPs enter tumour cells via endocytosis, with exposure of the NP to the acidic environment of the endosome, resulting in cleavage of the pH-responsive ‘protecting group’, thereby triggering NP expansion and subsequent intratumoural drug release. In a lung cancer model designed to mimic the microscopic disease that can remain at the surgical margin after resection, Griset et al. [7] locally applied these pH-responsive paclitaxel-loaded expansile nanoparticles (Pax-eNPs) in a subcutaneous murine model of rapidly growing lung cancer cells. Pax-eNP prevented the initial in vivo growth of these tumours, whereas conventional paclitaxel-loaded non-expansile NPs, empty expansile NPs or even a 10-fold higher dose of paclitaxel alone exhibited inferior efficacy (Fig. 4). Subsequent studies using a more clinically relevant murine model of tumour recurrence demonstrated that local delivery of Pax-eNPs immediately following resection of established NSCLC implants delayed, but did not prevent, local recurrence [54].

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Pathological conditions such as inflammation and cancer promote certain environmental conditions within the cell. As such, these tumour-specific triggers have been utilized to activate nanocomplexes for imaging and/or drug delivery [55, 56].

Combined drug and gene delivery systems Chemotherapy is the mainstay of advanced-stage lung cancer treatment. Treatment failures secondary to multidrug resistance and toxic side effects could potentially be overcome with active tumour targeting using nanocarriers coupled with co-delivery of chemotherapeutic agents. Nanostructured lipid carriers (NLCs) are an example of such a nanotherapeutic approach. NLCs demonstrate increased chemical stability, higher drug loading capacity and controlled drug release [57]. Uniquely, this novel system co-delivers gene and chemotherapeutic drug via the same nanocarrier [58]. The concept behind this co-delivery mechanism is to enhance the efficacy of a given cytotoxic agent by simultaneously targeting antiapoptotic genes or multidrug resistance-related genes within the tumour. Han et al. recently developed surfacemodified, co-encapsulated solid lipid NPs containing green fluorescence protein plasmid and doxorubicin to actively target the human A549 lung cancer cell line both in vitro and in tumourbearing mice in vivo [59]. This model enhanced therapeutic effect with improved drug delivery and gene transfection, culminating in increased antitumour cytotoxicity. Similarly, Shao et al. describe a novel dual-targeted therapy for the treatment of NSCLC via co-delivery of transferrin-decorated paclitaxel and DNA-loaded NLCs. Antitumour cytotoxicity was increased with a 4-fold dose advantage over free paclitaxel against lung cancer cells, while also demonstrating improved gene transfection efficiency [58]. Another important advance is the use of a complex tumour targeted mesoporous silica NP-based drug delivery system in inhaled lung cancer therapy. Taratula et al. recently developed a multifunctional system capable of simultaneous tumour targeted inhaled delivery of chemotherapeutic agents (doxorubicin and cisplatin) combined with siRNA inhibition of specific mRNA responsible for pump and non-pump cellular resistance in NSCLC tumours [60]. Using an in vivo murine model, the authors directly delivered the nanocarrier system to the lungs, thereby preventing release into the systemic circulation and minimizing toxic side effects [60]. Suppression of tumour resistance was achieved by delivering siRNA inside NSCLC cells, which subsequently enhanced the cytotoxic efficacy of the co-delivered anti-cancer agents. These results suggest that multifunctional nanocarrier co-delivery models have significant potential to improve current approaches to lung cancer treatment.

NANOTHERANOSTICS: THE NEXT FRONTIER IN THORACIC CANCER THERAPY The term ‘nanotheranostics’ refers to the simultaneous integration of diagnostic and treatment modalities via a single, unified nanotechnology platform [11, 61]. The advanced capabilities of multifunctional nanotheranostic systems hold great promise in the advancement of thoracic cancer care (Fig. 5). Unique properties of theranostic nanosystems include the ability to reach the systemic circulation, evade host defences, promote stimuli-responsive drug release, enable synergistic and combination therapy and deliver multimodal diagnostic and therapeutic agents to the target site,

thereby facilitating simultaneous diagnostic and therapeutic interventions at the cellular and the molecular level [62, 63]. To date, much of the focus employs polymer-based nanomaterials for the construction of multifunctional theranostic formulations. Polymer-based theranostic platforms generally consist of three major components: (i) a polymer that provides biocompatibility and stabilization of the system; (ii) a therapeutic agent such as a small-molecule drug, siRNA or DNA vector etc.; and (iii) an imaging agent which is commonly an MRI contrast agent, radionuclide, fluorophore etc. Many theranostic formulations also include targeting ligands designed to enhance specific delivery to the tumour site [63]. Each component may be arranged in a variable fashion, depending on the specific delivery platform being utilized. The focus of this section is on emerging theranostic technological innovations that hold potential to significantly impact the surgical management of lung and oesophageal cancer patients in the future.

Selective tumour ablation Recent advances in nanotechnology are also enabling the evolution of several techniques in NP-mediated selective tumour ablation. Complex NP formulations possessing unique physical and chemical properties are created with the capacity to interact with a variety of external energy sources. Exposure of these NPs to radiofrequency, ionizing radiation, light or ultrasound energy sources mediates thermal, chemical and mechanical effects and ultimately allows for the selective ablation of tumour tissue. Particularly relevant is the discovery that the use of heat to activate the release of drug payloads from nanocarriers leads to increased antitumour cytotoxicity. This approach has shown clinical applicability in the treatment of several different tumour types, including breast, ovarian and hepatocellular cancers [64, 65]. Thermosensitive nanomaterials exploit the inherent or induced hyperthermia of the tumour cell environment [66]. One such advance is the recent development of the magnetically activated release system [67]. Thomas et al. combined zinc-doped iron oxide nanocrystals within a surface-modified mesoporous silica framework. Application of an oscillating magnetic field induces local heat generation by the nanocrystals, causing the molecular machinery to disassemble and release drug (Fig. 6). Treatment of breast cancer cells with doxorubicin loaded into this system demonstrated cell death following exposure to an oscillating magnetic field. This approach holds promise as a non-invasive, externally controlled drug delivery system and warrants further study as a future adjunctive therapy in lung cancer patients. Another example is ultrasound-triggered drug delivery vehicles that consist of cationic drug-loaded liposomes encapsulated within microbubbles. Intriguingly, exposure of the tumour to ultrasound leads to local mechanical attenuation of the microbubble-liposomal formulation, resulting in rapid drug release at the tumour site [68]. Lastly, the combination of photothermal–chemotherapeutic properties within a single construct or radiofrequency-activated drug can be released from thermosensitive liposomal-based nanocarriers [65]. The ability of these highly functional, minimally invasive, NP-based formulations to enhance tumour-specific drug delivery and facilitate targeted in vivo tumour ablation achieves the dual goals of increased antitumour cytoxicity and reduced systemic toxicity. These rapid advances highlight that NP-based biomedical imaging approaches will play an important role in the evolution of personalized treatment strategies for thoracic cancers in the future.

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Figure 5: Schematic illustration of multifunctional nanoparticles (reprinted from ref. [11], with permission from Annual Reviews, Inc., QD: quantum dot; GNP: gold nanoparticles; HfO; hafnium oxide nanoparticles; MNP: magnetic nanoparticles; UCNP: upconversion nanoparticles.

CLINICAL TRANSLATION OF NANOTHERAPIES IN LUNG CANCER Over the past decade, nano-based therapeutics have undergone extensive in vitro and preclinical characterization. More recently, a number of clinical studies have evaluated nanotherapies in thoracic diseases, particularly lung cancer [55]. The first nanomedicine translated to the clinic was albumin-bound paclitaxel, nabpaclitaxel (Abraxane). Approved by the FDA in 2012, this combination therapy has been utilized in the treatment of patients with advanced-stage NSCLC. Recent phase I/II clinical trials in latestage NSCLC patients demonstrate that nab-paclitaxel is a safe and well-tolerated therapeutic option [69, 70]. Furthermore, the FDA-approved liposomal formulations of doxorubicin (Doxil) and cisplatin (lipoplatin) have demonstrated clinical efficacy as combination therapies in patients with advanced-stage NSCLC [71, 72]. Notably, these nanotherapeutic compounds exhibited equal or better antitumour cytotoxicity compared with chemotherapeutic drug alone, however with significantly less systemic toxicity. Patlakhas et al. combined liposomal doxorubicin with the doublet regimen of docetaxel/gemcitabine, in chemotherapy naïve patients with advanced NSCLC. They demonstrated equivalent efficacy to Phase II studies using docetaxel/gemcitabine alone, and

there was zero incidence of clinically significant neutropenia/ thrombocytopenia and no cases of interstitial pneumonia [71]. In comparison, previous docetaxel/gemcitabine trials reported haematological toxicity and interstitial pneumonia rates of up 25 and 18% of patients, respectively [73, 74]. Furthermore, a Phase III clinical trial comparing liposomal cisplatin/paclitaxel versus cisplatin/paclitaxel in non-squamous NSCLC patients demonstrated significantly higher response rates and reduced gastrointestinal and nephrotoxic side effects in patients receiving the liposomal cisplatin/ paclitaxel regimen [75]. Recently, a polymeric micelle formulation of paclitaxel (Genexol-PM) was combined with cisplatin and gemcitabine to treat advanced-stage NSCLC patients. Phase II clinical trials demonstrated improved antitumour efficacy for both micelle-drug combinations [76, 77]. Nonetheless, significant systemic toxicity was observed (sensory neuropathy, bone marrow suppression), therefore limiting the clinical utility of this nanotherapy. To date, several nano-based lung cancer therapies have been successfully translated to clinical application, albeit only as simple nanocrystal, liposomal or polymeric formulations of FDA-approved drugs. Nevertheless, several sophisticated preclinical innovations discussed in this review have already leveraged these early successes and demonstrated significant clinical potential to have real impact in the care of patients with thoracic malignancies in the future.

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Figure 6: Non-invasive, remote-controlled drug delivery in vitro using magnetic actuation of mechanized nanoparticles. (1 and 2) Zinc-doped iron oxide nanocrystals are synthetically positioned within a core of mesoporous silica nanoparticles. (3) A newly created molecular machine is attached to the surface of the nanoparticle. (4) Drug is loaded into the particle and capped. (5) Drug release is achieved using remote heating via the introduction of an oscillating magnetic field. The particles and machines are not drawn to scale (reprinted from ref. [67], with permission from American Chemical Society, Scheme 1).

CONCLUSION Current approaches to thoracic cancer treatment are limited to surgical resection, radiation and chemotherapy. These therapies are often highly invasive or non-specific, with associated morbidity and toxic side effects. The evolving field of nanotechnology shows significant potential to overcome many of the current challenges associated with the diagnosis and treatment of lung and oesophageal cancer at all stages. Recent technological advances have resulted in minimally invasive NP-based therapies with the ability to remotely control drug delivery via external energy sources. Furthermore, multifunctional nanotheranostic systems hold the promise of integrating detection, diagnosis, imaging, targeted delivery and controlled release of therapeutic cargo all within a single, unifying platform. Fortunately, many of these novel nanotechnology platforms are directly applicable to thoracic oncology. The ability to integrate novel imaging techniques, drug delivery strategies and minimally invasive ablative and image-guided surgical approaches offers real hope to those patients diagnosed with thoracic cancers.

Funding This work was supported in part by Brigham and Women’s (BWH) Hospital Center for Surgical Innovation, Boston University, Center for Integration of Medicine and Innovative Technology, National Science Foundation grant (DMR-1006601), National Institutes of

Health (CA R01CA149561) and Boston University’s Nanomedicine Program and Cross-Disciplinary Training in Nanotechnology for Cancer, NIH R25 CA153955 and BWH Advanced Training in Surgical Oncology, NIH T32 CA009535. Conflict of interest: none declared.

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