Diverse Applications of Nanomedicine

Diverse Applications of Nanomedicine Beatriz Pelaz, † Christoph Alexiou, ‡ Ramon A. Alvarez-Puebla, §,∥ Frauke Alves, #,¶,□ Anne M. Andrews, ■,○,Φ Sumaira Ashraf, † Lajos P. Balogh, ● Laura Ballerini, △ Alessandra Bestetti, ▲ Cornelia Brendel, ▽ Susanna Bosi, ▼ Monica Carril, ⬡,⬢ Warren C. W. Chan, $ Chunying Chen, ∞ Xiaodong Chen, ⊗ Xiaoyuan Chen, ∫ Zhen Cheng, ⧧ Daxiang Cui, ℏ Jianzhong Du, ∀ Christian Dullin, ¶ Alberto Escudero, †,ƛ Neus Feliu, ∂ Mingyuan Gao, ∮ Michael George, ∏ Yury Gogotsi, ◑ Arnold Grunweller, ⊕ Zhongwei Gu, & Naomi J. Halas, ¤ Norbert Hampp, ⌽ Roland K. Hartmann, ⊕ Mark C. Hersam, ⌀ Patrick Hunziker, ⟠,◊ Ji Jian, ⧫ Xingyu Jiang, ∞ Philipp Jungebluth, ◨ Pranav Kadhiresan, $ Kazunori Kataoka, ⊡ Ali Khademhosseini, †† Jindrich Kopecek, ‡‡ Nicholas A. Kotov, §§ Harald F. Krug, ⊥⊥ Dong Soo Lee, ¶¶ Claus-Michael Lehr, □□,■■ Kam W. Leong, ○○ Xing-Jie Liang, ∞,●● Mei Ling Lim, ∂ Luis M. Liz-Marzan, ⬡,⬢,△△ Xiaowei Ma, ●● Paolo Macchiarini, ▲▲ Huan Meng, ■,▼▼ Helmuth Mohwald, ▽▽ Paul Mulvaney, ▲ Andre E. Nel, ■,▼▼ Shuming Nie, ⬡⬡ Peter Nordlander, ¤ Teruo Okano, ⬢⬢ Jose Oliveira, $$ Tai Hyun Park, ∞∞,⊗⊗ Reginald M. Penner, ∫∫ Maurizio Prato, ▼,⬡,⬢ Victor Puntes, ∥,⧧⧧,ΦΦ Vincent M. Rotello, ℏℏ Amila Samarakoon, $ Raymond E. Schaak, ∀∀ Youqing Shen, ƛƛ Sebastian Sjoqvist, ∂ Andre G. Skirtach, ▽▽,∂∂ Mahmoud G. Soliman, † Molly M. Stevens, ∮∮ Hsing-Wen Sung, ⊕⊕ Ben Zhong Tang, ∏∏ Rainer Tietze, ‡ Buddhisha N. Udugama, $ J. Scott VanEpps, §§ Tanja Weil, &&,◑◑ Paul S. Weiss, ■,○,¤¤ Itamar Willner, ⌽⌽ Yuzhou Wu, ◑◑,⌀⌀ Lily Yang, $$ Zhao Yue, † Qian Zhang, † Qiang Zhang, ⟠⟠ Xian-En Zhang, ◊◊ Yuliang Zhao, ∞ Xin Zhou, ⧫⧫ and Wolfgang J. Parak* ,†,⬡ † Fachbereich Physik, ▽ Fachbereich Medizin, ⊕ Fachbereich Pharmazie, and ⌽ Department of Chemistry, Philipps Universitat Marburg, 35037 Marburg, Germany ‡ ENT-Department, Section of Experimental Oncology & Nanomedicine (SEON), Else Kroner-Fresenius-Stiftung-Professorship for Nanomedicine, University Hospital Erlangen, 91054 Erlangen, Germany § Department of Physical Chemistry, Universitat Rovira I Virgili, 43007 Tarragona, Spain ∥ ICREA, Pg. Lluís Companys 23, 08010 Barcelona, Spain # Department of Haematology and Medical Oncology, ¶ Department of Diagnostic and Interventional Radiology, University Medical Center Gottingen, 37075 Gottingen Germany □ Department of Molecular Biology of Neuronal Signals, Max-Planck-Institute for Experimental Medicine, 37075 Gottingen, Germany ■ California NanoSystems Institute, ○ Department of Chemistry and Biochemistry and Φ Department of Psychiatry and Semel Institute for Neuroscience and Human Behavior, ▼▼ Division of NanoMedicine and Center for the Environmental Impact of Nanotechnology, and ¤¤ Department of Materials Science and Engineering, University of California, Los Angeles, Los Angeles, California 90095, United States ● AA Nanomedicine & Nanotechnology Consultants, North Andover, Massachusetts 01845, United States △ International School for Advanced Studies (SISSA/ISAS), 34136 Trieste, Italy ▲ School of Chemistry & Bio21 Institute, University of Melbourne, Parkville, Victoria 3010, Australia ▼ Department of Chemical and Pharmaceutical Sciences, University of Trieste, 34127 Trieste, Italy ⬡ CIC biomaGUNE, Paseo de Miramon 182, 20014, Donostia - San Sebastian, Spain ⬢ Ikerbasque, Basque Foundation for Science, 48013 Bilbao, Spain $ Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada ∞ CAS Center for Excellence in Nanoscience and CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, China Received: September 7, 2016 Published: March 14, 2017 Nano Focus www.acsnano.org © 2017 American Chemical Society 2313 DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes. ⊗ School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 ∫ Laboratory of Molecular Imaging and Nanomedicine, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland 20892, United States ⧧ Molecular Imaging Program at Stanford and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California 94305, United States ℏ Institute of Nano Biomedicine and Engineering, Department of Instrument Science and Engineering, School of Electronic Information and Electronical Engineering, National Center for Translational Medicine, Shanghai Jiao Tong University, 200240 Shanghai, China ∀ Department of Polymeric Materials, School of Materials Science and Engineering, Tongji University, Shanghai, China ƛ Instituto de Ciencia de Materiales de Sevilla. CSIC, Universidad de Sevilla, 41092 Seville, Spain ∂ Department of Clinical Science, Intervention, and Technology (CLINTEC), Karolinska Institutet, 141 86 Stockholm, Sweden ∮ Institute of Chemistry, Chinese Academy of Sciences, 100190 Beijing, China ∏ Nanion, 80636 Munchen, Germany & College of Polymer Science and Engineering, Sichuan University, 610000 Chengdu, China ◑ Department of Materials Science and Engineering and A.J. Drexel Nanomaterials Institute, Drexel University, Philadelphia, Pennsylvania 19104, United States ¤ Departments of Physics and Astronomy, Rice University, Houston, Texas 77005, United States ⌀ Departments of Materials Science and Engineering, Chemistry, and Medicine, Northwestern University, Evanston, Illinois 60208, United States ⟠ University Hospital, 4056 Basel, Switzerland ◊ CLINAM, European Foundation for Clinical Nanomedicine, 4058 Basel, Switzerland ⧫ Department of Polymer Science and Engineering and ƛƛ Center for Bionanoengineering and Department of Chemical and Biological Engineering, Zhejiang University, 310027 Hangzhou, China ◨ Thoraxklinik Heidelberg, Universitatsklinikum Heidelberg, 69120 Heidelberg, Germany ⊡ The University of Tokyo, Bunkyo, Tokyo 113-8654, Japan †† Havard University, Cambridge, Massachusetts 02139, United States ‡‡ Biomedical Polymers Laboratory, University of Utah, Salt Lake City, Utah 84112, United States §§ Emergency Medicine, University of Michigan, Ann Arbor, Michigan 48019, United States ⊥⊥ EMPA, Federal Institute for Materials Science and Technology, CH-9014 St. Gallen, Switzerland ¶¶ Department of Molecular Medicine and Biopharmaceutical Sciences and ∞∞ School of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea □□ Department of Pharmacy, Saarland University, 66123 Saarbrucken, Germany ■■ HIPS - Helmhotz Institute for Pharmaceutical Research Saarland, Helmholtz-Center for Infection Research, 66123 Saarbrucken, Germany ○○ Department of Biomedical Engineering, Columbia University, New York City, New York 10027, United States ●● Laboratory of Controllable Nanopharmaceuticals, Chinese Academy of Sciences (CAS), 100190 Beijing, China △△ Biomedical Research Networking Center in Bioengineering Biomaterials and Nanomedicine, Ciber-BBN, 20014 Donostia - San Sebastian, Spain ▲▲ Laboratory of Bioengineering Regenerative Medicine (BioReM), Kazan Federal University, 420008 Kazan, Russia ▽▽ Department of Interfaces, Max-Planck Institute of Colloids and Interfaces, 14476 Potsdam, Germany ⬡⬡ Emory University, Atlanta, Georgia 30322, United States ⬢⬢ Tokyo Women’s Medical University, Tokyo 162-8666, Japan $$ SMALL, Wiley-VCH, Weinheim, German ⊗⊗ Advanced Institutes of Convergence Technology, Suwon, South Korea ∫∫ Department of Chemistry, University of California, Irvine, California 92697, United States ⧧⧧ Institut Catalade Nanotecnologia, UAB, 08193 Barcelona, Spain ΦΦ Vall d’Hebron University Hospital Institute of Research, 08035 Barcelona, Spain ℏℏ Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, United States ∀∀ Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, United States ∂∂ Department of Molecular Biotechnology, University of Ghent, B-9000 Ghent, Belgium ∮∮ Department of Materials, Department of Bioengineering, Institute for Biomedical Engineering, Imperial College London, London SW7 2AZ, United Kingdom ∏∏ Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, Hong Kong, China ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2314 ⊕⊕ Department of Chemical Engineering and Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu City, Taiwan, ROC 300 && Institut fur Organische Chemie, Universitat Ulm, 89081 Ulm, Germany ◑◑ Max-Planck-Institute for Polymer Research, 55128 Mainz, Germany ⌽⌽ Institute of Chemistry, The Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel ⌀⌀ School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, 430074 Wuhan, China ⟠⟠ School of Pharmaceutical Science, Peking University, 100191 Beijing, China ◊◊ National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, 100101, China ⧫⧫ Key Laboratory of Magnetic Resonance in Biological Systems, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, China ABSTRACT: The design and use of materials in the nanoscale size range for addressing medical and health-related issues continues to receive increasing interest. Research in nanomedicine spans a multitude of areas, including drug delivery, vaccine development, antibacterial, diagnosis and imaging tools, wearable devices, implants, high-throughput screening platforms, etc. using biological, nonbiological, biomimetic, or hybrid materials. Many of these developments are starting to be translated into viable clinical products. Here, we provide an overview of recent developments in nanomedicine and highlight the current challenges and upcoming opportunities for the field and translation to the clinic. Medicine is the science, engineering, and practice of diagnosing, treating, curing, monitoring, predicting, and preventing diseases. Most people associate nanomedicine with pharmaceutical formulations, where soft or hard particles of nanometer dimensions are injected into humans for diagnosis and treatment. However, this field covers a broader range of research and development. Nanomedicine differs from other types of medicine in that it involves the development and application of materials and technologies with nanometer length scales to function in all the ways described below. 1−5 Properties of nanoscale objects are transitional between molecular and bulk regimes. Nanoscale properties exist for all materials, regardless of whether they are found in nature or are synthetic. However, only synthetic objects are typically considered part of “nanoscience and engineering”, whereas the study of biological nanoscale structures is often thought as part of characterization without considering biological properties. Because of the transitional nature of “nanoscale”materials, it is difficult to limit a material’s reach and define its borders by strict definitions and solid numbers (e.g., larger than atoms or small molecules, but smaller than 100 nm). More importantly, nanoscale particles can demonstrate new properties that can be exploited for the design of new therapeutic effects and diagnostics. Nanomedicine is an interdisciplinary field, where nanoscience, nanoengineering, and nanotechnology interact with the life sciences. Given the broad scope of nanomedicine, we expect it eventually to involve all aspects of medicine. Moreover, nanomedicine, like medicine, can enter the clinics and can be part of conventional clinical practice assuming all aspects of translation are satisfied, including safety, regulatory, and ethical requirements. It is expected that nanomedicine will lead to the development of better devices, drugs, and other applications for early diagnoses or treatment of a wide range of diseases with high specificity, efficacy, and personalization, with the objective being to enhance patients’quality of life. In this Nano Focus, we do not attempt to define nanomedicine but rather to provide an overview of recent achievements, materials, and technologies belonging to nanomedicine. Nanoparticles (NPs) are key components of nanomedicine, and currently, a large variety of nanoparticle types exist. However, no standardized nomenclature exists in the literature; therefore, terms such as engineered nanomaterials, nonbiological complex drugs (NBCDs), nanomedicals/nanomedicines, etc. are used freely. Many nanomaterials can replicate some functions of globular biological macromolecules. 6 Examples are lipid micelles, 7 different polymeric nanostructures, 8 protein constructs, 9 ribonucleic acid (RNA) NPs (RNPs), 10 carbon dots (C-dots), 11 nanodiamonds (NDs), 12 carbon nanotubes (CNTs), 13 graphene, 14 as well as inorganic materials such as mesoporous silica NPs (MSNP), superparamagnetic iron oxide NPs (SPIONs), 15 quantum dots (QDs), 16 plasmonic NPs, 17 gold nanoclusters (GNCS), 18 upconverting NPs (UCNPs), 19 etc. Many of these nanoscale materials have unique size- and shapedependent optical, electronic, and magnetic properties, and these properties are dependent upon methods to synthesize, to purify, and to characterize them. 20−23 Many researchers note that small changes in size and shape can significantly affect the properties of the NPs. Precision syntheses are therefore necessary to produce samples with tightly focused distributions in order to achieve the targeted functions specifically and to correlate observed functions with specific NP characteristics. Detailed characterization of NP samples that are used in a medical application is also critical because one must know and understand what is being injected into the body. A sample of NPs may be heterogeneous with distinct subpopulations after synthesis. 24,25 Microscopic imaging is conventionally used, but this technique may be insufficient ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2315 because it is limited to a small number of NPs that may or may not be representative of the whole sample. Thus, microscopic imaging may not provide sufficient information about surface functionalization, composition, and other property-determining features. Other techniques that are starting to become part of the characterization scheme of NPs prior to use in humans are dynamic light scattering, transmission electron microscopy, gel electrophoresis, and ζ-potential analysis. However, there are no standardized characterization requirements of NPs 26 prior to use in humans, and this must be a focus for nanomedicine applications. The main reason is that the biodistribution and interaction of NPs with proteins is strongly size- and surface-dependent, and thus, in a heterogeneous sample, many NPs will distribute differently and may exhibit undesired effects or even toxicity. In addition to characterization, there is also a need to develop new and improved methods of NP separation and purification to produce optimal samples for nanomedical applications and for studying NP behavior inside the body 27,28 (which is important to design optimal NP formulations for medical use). Despite the need to standardize characterization methods, NPs are expected to improve the detection and diagnosis of diseases. First, smart NPs can be designed to provide contrast at the zone of interest and report information about the local environment after administration into the body. This information can aid in imaging the anatomical fine structures of organs and labeling tissues with certain markers and enables local readout of the concentrations of molecules of interest, which helps to analyze diseases directly inside the human body. Second, NPs are key components of many high-throughput diagnostics machines that can analyze extracted samples (such as blood, tissue, etc.) outside of the body for rapidly detecting biological makers and molecular alterations. The ability to analyze multiple biomarkers simultaneously may improve diagnostic precision. Moreover, multifunctional or theranostic NPs that can simultaneously diagnose, treat, and even monitor therapeutic efficacy are being engineered. 29 Nanoparticles are also being developed for the treatment of disease; NPs are used as delivery vehicles for pharmaceutical agents, 30 as bioactive materials, or as important components in implants. 31 In the case of delivery, NP-based carrier systems have a unique ability to cross biological barriers. Thus, NPs can enter tumors via their localized leaky vasculature and are retained due to poor lymphatic drainage in the tumor microenvironment. 32 This passive targeting is called the enhanced permeation and retention (EPR) effect. 33 There is an ongoing debate in the literature about the effectiveness of active, i.e., ligand/receptormediated targeting, versus passive targeting, but any carrier has to be delivered to the designated site before it can bind to cell surface receptors or be retained by other effects. 34 In addition to using nanomedicine to diagnose and to treat diseases, it is also important to establish NPs’efficacy and safety in biological systems. After the NP has functioned as designed after administration into the body, what happens to the carrier particle when the drug has been delivered or the tissue imaged? Elimination of the particles can potentially occur via renal or hepatobiliary clearance. If they do not get cleared, the long-term fate of the NPs is not clear. These particles may degrade and get cleared renally because they are small enough to transport through the kidney’sfiltration slits, 35−37 or they may accumulate in different organs and interact with off-target cells. The in vivo fate of NPs can potentially be a dynamic process, and thus, there is a need to understand nanobiokinetics (nanopharmacokinetics and pharmacodynamics), which may relate to unique and interesting toxicological responses of NPs. Nanomedicine is not limited to colloidal materials and technologies to evaluate them for in vivo applications. Nanomedicine developments go beyond the “magic particulate bullet” concept. 38 Nanomedicine could involve the design of new scaffolds and surfaces for engineering sensors or implantable systems and electronics to aid in the regeneration of tissues (i.e., regenerative medicine). Many of these concepts are still at the early stages of development, but some have already reached clinical practice. This Nano Focus article is organized into four subsections that are focused on specific applications of materials or systems with nanoscale properties: (a) in vivo diagnostics, (b) in vitro diagnostics, (c) in vivo therapeutics, and (d) implantable nanomaterials. IN VIVO DIAGNOSIS (“SMART IMAGING”) A key focus in nanomedicine involves the use of nanomaterials as contrast agents for anatomical and functional imaging. Using nanomaterials as contrast agents enables visualization of structures inside the human body and helps clinicians to delineate healthy from diseased tissues and to recommend proper treatment. Nanoparticles can be engineered with different contrast properties. The most common modalities are computed tomography (CT); magnetic resonance imaging (MRI); imaging of radioactivity, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT); fluorescence imaging; and photoacoustic imaging. For all these techniques, material development is crucial because the NPs are contrast agents that enable visualization of biological tissues. For this application, NPs can be engineered to localize in specific tissues and potentially produce high contrast. Computed Tomography. X-ray-based imaging enables high-resolution anatomical and, in the case of CT also threedimensional (3D), imaging of mostly skeletal tissues at unlimited depth in human applications. Computed tomography imaging is the work-horse in clinical diagnostics due to the simplicity of the technique, the comparable low demands on infrastructure, the rapid image generating, and the low costs for a standard examination. In recent years, around 250 out of every 1000 people in the United States underwent CT imaging. 39 Classical X-ray imaging harnesses the tissue-specific attenuation of X-ray energy to generate contrast in the recorded radiographs. Therefore, bones generate more contrast than soft tissue because of the larger relative electron density of bone. In order to boost the low contrast of soft tissue, contrast agents with elements such as iodine and barium characterized by a high-electron density are typically applied to visualize blood vessels in gastrointestinal (GI) tract, tumors, and other soft tissues. In contrast to typical functional imaging techniques such as PET and SPECT, in CT, the photons/X-rays are produced outside the body and only modulated by the tissues through which they travel. Thus, the large photon flux enables a high signal-to-noise ratio, leading CT to outperform other 3D imaging techniques in terms of spatial A key focus in nanomedicine involves the use of nanomaterials as contrast agents for anatomical and functional imaging. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2316 resolution. However, the weak interaction between tissue and the incident X-ray beam results in comparably limited sensitivity of CT, which might explain why specifically targeting contrast strategies are virtually non-existent. This poor sensitivity has encouraged nanomedicine researchers to develop nanoparticles as contrast agents for X-ray imaging. Gold nanoparticles (AuNPs) are central to this development. Due to its high atomic number and electron density (79 and 19.32 g/cm3, respectively, versus the typical X-ray contrast agent, iodine, with values of 53 and 4.9 g/cm3, respectively), AuNPs have higher attenuation coefficients and can be used as contrast agents for X-ray imaging, CT, and micro-CT. 40 The AuNPs are typically coated with targeting molecules such as folic acid so that they can highlight distinct tissue structures. Gold nanoclusters (NCs), which are smaller structures than AuNPs, are also being developed as CT contrast agents. 18,40 Folic-acid-conjugated AuNCs with silica shells were demonstrated to exhibit good biocompatibility and could actively target the folic acid receptor (+) in vitro of MGC-803 cells and in vivo gastric cancer tissues with 5 mm diameter in nude mice models. The researchers showed that the use of NCs exhibited excellent contrast for CT imaging. In addition to CT imaging, these NCs can also be used for molecular imaging since red fluorescence emission was observed. 41,42 These NCs also penetrated the tumor and were retained, but their small size enabled them to be cleared renally. 41,42 Thus, these NPs are promising candidates for future clinical use. Besides gold, NPs composed of other materials with a high atomic number are also suited for CT. One example is NaGdF4- based UCNPs. Besides providing contrast for CT, these NPs can be imaged optically (see Figure 1). In addition to their ability to provide contrast for CT, potential toxicity of NPs plays an important role toward translation to clinics (vide infra). In parallel to the development of new CT contrast agents, there has also been progress in instrumentation relating to the preparation and detection of the agents. Novel X-ray sources such as synchrotrons provide tremendously more flux and a higher degree of coherence and therefore enable the exploitation of the wave nature of X-rays for imaging purposes. These novel X-ray sources have led to new applications like phase contrast CT, diffraction enhanced imaging, and holotomography, to name a few. 44−46 For example, phase contrast CT provides 10−200 times more contrast in soft tissue applications than classical absorption-based CT. 47,48 This gain in contrast directly translates into an increase in sensitivity. By coupling this increased sensitivity with the use of target-specific contrast agents, submicrometer-scale 3D spatial resolution can be achieved. This technique can be applied to gather structural Figure 1. (I) In vitro CT images of (a) lanthanide-doped NaGdF4upconversion “nanoclusters”(<5 nm) suspended in aqueous solution. (b) CT attenuation plot (given in Hounsfield units, HU) of NaGdF4NPs in dependence of the concentration of each sample from 0.2 to 10 mg/mL to further investigate the CT contrast effect. (II) Images of a control group before injection of NPs: (c) photograph of a nude mouse loaded with gastric cancer MGC-803 cells; (d) X-ray image, and (e−g) CT images of nudemice from the controlgroup. (III) (h−k) CT images and (IV) (l−n) MRI images of mice after intravenous injection with NaGdF4UCNPs, making use of passive targeting (EPR effect). The pulse sequence: electromagnetic conversion (EC) = 1/141.7 kHz; repetition time (TR time) = 2000; echo time (TE time) = 65.6/Ef (echo frequency). Parameters of transverse plane: the pulse sequence, EC = 1/141.7 kHz; TR time = 2000; TE time = 43.8/Ef. It took about 6 h to acquire one image. The relaxivity value of NaGdF4UCNPs at 1.5 T is about 4.5 mMs−1. Adapted with permission from ref 43. Copyright 2015 Royal Society of Chemistry. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2317 information indicative of asthma within the lung of a mouse. 49 It can also be applied in the visualization of macrophages loaded with barium sulfate after intracheal instillation. 49 In a study by Dullin et al., the researchers achieved 9 μm resolution, which enabled the visualization of macrophage function within the local 3D environment. 46 The spatial resolution could be further improved to 400 nm by using holotomography (see Figure 2). 50 The NP-based CT imaging technologies can potentially change the way we perform CT-based clinical diagnosis. 51 Currently, soft tissue imaging via CT requires a large dose of iodine- or bariumcontaining contrast agents to overcome poor sensitivity. However, the high dose may not be well tolerated by patients. Furthermore, it is difficult to design traditional contrast agents to target specifically and to bind to cellular biomarkers or accumulate in tissues of interest such as sites of inflammation or primary tumors and metastasis. Combining targeted AuNPs with the recent development of energy-resolved detectors, 52 the discrimination of a multitude of different materials in a single CT scan should be possible in the near future. Therefore, the challenge facing the field is to design and to engineer small NP probes of high-atomic-number materials like iodine, gold, or barium, conjugated to targeting moieties that specifically label certain cell types in vivo, similar to probes used in optical imaging. Moreover, since novel CT techniques do not focus on X-ray attenuation but on phase shift or scattering as sources of contrast, other types of NPs like hollow spheres 53 or NPs with X-ray scattering properties will become increasingly important. Despite the negative effects of the applied X-ray dose, CT is the most applied technique in clinical diagnosis (e.g.,∼50% of imagingbased diagnostics performed in Germany in 2013 were by CT). 54 Improved detectors and the implementation of novel algorithms, especially newly developed reconstruction techniques, have already dramatically lowered X-ray doses. 55 Techniques like limited angle reconstruction and zoom tomography will further aid in this development. 50,56 Magnetic Resonance Imaging. Magnetic resonance imaging is widely used for in vivo applications, due to its safety, spatial resolution, soft tissue contrast, clinical relevance, and ability to record anatomical and functional information about soft tissues and organs. Notably, MRI-responsive contrast agents provide physiological information that complements routine anatomical images. Since the technology is based on the interaction of nuclei with surrounding molecules in a magnetic field, MRI has no need for ionizing radiation and possesses unlimited depth of penetration and unparalleled soft tissue contrast. 57 However, MRI has relatively poor sensitivity in comparison to nuclear and optical modalities, thus leading to longer acquisition time and use of large amounts of contrast agents. Although the introduction of higher magnetic fields (higher than 4.7 T) could increase the signal-to-noise ratio, thereby permitting higher resolution and faster scanning, the safety of high static magnetic field strengths on human health is of great concern. 58,59 The development of hyperpolarized MRI (i.e., polarization of the nuclear magnetic moments far beyond thermal equilibrium conditions) 60 has improved the sensitivity of the desired nuclei and enables quantitative in vivo imaging and real-time metabolic profiling using stable isotope precursors. 60 Current contrast agents such as paramagnetic agents or SPIONs play important roles in enhancing contrast in T1or T2images, which provide higher MRI sensitivity and accuracy for imaging living subjects. These agents accelerate the rate of T1and T2 relaxation, thereby enhancing local contrast. 61 Paramagnetic agents (e.g., gadolinium-based agents) principally accelerate longitudinal T1recovery, generating positive contrast, while superparamagnetic agents (e.g., iron oxide-based agents such as SPIONs) primarily increase the rate of dephasing or transverse T2decay, resulting in negative contrast effects. 61 However, both types of agents have been associated with toxic effects, which needs to be taken into account for their use on humans and in veterinary medicine (vide infra). 62,63 Conventional T1contrast agents such as paramagnetic complexes (based on Gd3+,Mn 2+,orFe 3+) are small molecules that leave the vascular system within minutes and are renally cleared. The short circulation time makes it difficult to acquire a high-resolution image of desired sites. Biocompatible NP-based T1contrast agents have been developed because they have a number of advantages over conventional T1contrast. 64,65 Researchers can tailor the size, shape, and composition; circulation time; target cells and tissues; and optical and physical properties to meet the biological requirements for optimizing imaging. For example, Gd(III)−nanodiamond conjugates enable contrast enhancement with a much smaller amount of Gd compared to other agents used. 66 For example, melanin has been intensively studied as a target for melanoma imaging and was recently a major focus for designing a multimodal imaging nanoplatform. Melanin NPs with diameters of 4.5 nm not only retain their unique optical properties but also have natural binding capability with various metal ions. After chelation with Fe3+ ions and subsequent conjugation with cyclic arginineglycine-aspartic acid (RGD) molecules, melanin NPs serve as T1 contrast agents for targeted MRI of U87MG glioblastoma. 67,68 In another example, a noncytotoxic asymmetrical cancertargeting polymer vesicle based on R-poly(L-glutamic acid)- block-poly(ε-caprolactone) [R is folic acid or diethylenetriaminepentacetatic acid (DTPA)], as shown in Figure 3, was reported as a T1MRI contrast agent with enhanced sensitivity, and it also served as a delivery vehicle for cancer chemotherapy. 69 Such asymmetrical vesicles have a cancer-targeting outer corona Figure 2. Three-dimensional localization of labeled macrophages in a 500 μm thick lung section of a healthy mouse, scanned by holotomography. Three orthogonally oriented slices are shown, together with automatically labeled barium clusters (green), representing macrophages loaded with barium sulfate and alveolar walls in a small region of interest (ROI, yellow). A part of a blood vessel has been marked semi automatically (purple). Adapted with permission from ref 50. Copyright 2015 Nature Publishing Group. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2318 coupled with a Gd(III)-chelating and drug-loading-enhancing inner corona. Liu and colleagues demonstrated that such vesicles exhibited an extremely high T1relaxivity (42.39 mM−1s−1, 8-fold better than that of DTPA-Gd) and anticancer drug loading efficiency (52.6% for doxorubicin hydrochloride, DOX·HCl). 69 Moreover, the DOX-loaded vesicles exhibited 2-fold better antitumor activity than free DOX. 69 As T2contrast agents, SPIONs establish a substantial locally perturbed dipolar field to shorten proton relaxation of the surrounding tissues significantly. The magnetism of NPs under normal magnetic field strengths is dependent on the magnetocrystalline anisotropy and the size of the NPs. In comparison to their spherical counterparts, for example, iron oxide NPs with an octapod shape exhibit an ultrahigh transverse relaxivity value, and dramatically increase the sensitivity of MRI for early stage cancer detection, largely due to their effective radius and the local field inhomogeneity of the magnetic core. 70 Because of the negative contrast effect and magnetic susceptibility artifacts, it is still a major challenge to distinguish the region of signals induced by iron oxide NPs from low-level background magnetic resonance (MR) signals originating from adjacent tissues such as bone, vasculature, calcification, fat, hemorrhage, blood clots, etc. As a combination of paramagnetic and SPIONs, e.g., in core/ shell structures, 71 dual-mode T1/T2contrast agents have been developed for dual T1and T2mode MRI because they help validate reconstruction and visualization of the data in an accurate and reliable way. However, interference inevitably occurs when both T1and T2contrast agents are integrated into a single nanostructure in close proximity. The magnetic field induced by the T2contrast material perturbs the relaxation process of paramagnetic T1contrast materials, leading to undesirable quenching of the T1signal. Core/shell structures can efficiently reduce their magnetic coupling by introducing a separating layer between the superparamagnetic core and paramagnetic shell. Such magnetically decoupled dual-mode T1/T2contrast agents not only provide superior MR contrast effects in both modes, but also enable self-confirmation of images and essentially function as an “AND logic gate”to reduce susceptibility artifacts from the raw images to enhance accuracy of the MRI. 72,73 In contrast to core/shell structures, dumbbell hybrid nanostructures combine T1and T2contrast agents together in a nanocomposite via a bridge NP, which spatially separates two contrast agents at a certain distance to reduce their magnetic coupling. 74 Unlike core/shell structures where T2contrast agents are sequestered within a supporting matrix, dumbbell nanostructures allow both T1and T2contrast agents to be exposed to their immediate environment without compromising their magnetic properties. Moreover, both surfaces of T1and T2contrast agents are available for subsequent surface coating for targeting molecular imaging. Besides being “mere”contrast agents for MRI, 75,76 activatable or “smart”NPs can respond to a change in tumor microenvironment to instigate the therapeutic and diagnostic mechanism. 77,78 The most common triggers for activatable NPs are stimuli such as pH, temperature, redox reactions, 79 metabolites, ions, proteases, ultrasound, and light. 80 The tumor microenvironment regulates tumor progression and the spread of cancer in the body. Responsive agents capable of reporting diagnostically relevant physicochemical properties of the microenvironment in which the contrast agent distributes have gained tremendous attention. Various nanocarriers have been tested in combination with a myriad of imaging contrast agents, payload drugs, and targeting moieties, leading to the formulation of theranostic NPs capable of delivering therapy concomitant with diagnosis. In the case of MRI, different “smart”NP probes have been demonstrated. The design of pH-responsive probes is of great interest, since it is an important physiological parameter and pH dysregulation can be a cancer marker. Gao and colleagues reported a smart pH-activatable 19F-probe for detecting specific and narrow pH transitions ranging from 5.5 to 7.4 in biological systems. 81 They developed micelles composed of fluorinated polymers with tertiary amines having different pKavalues. The protonation of such amines at pH below their pKaresults in micelle disassembly and 19F-MRI/nuclear magnetic resonance spectroscopy (NMR) signal activation. They were able to determine the pH through the identification of the corresponding activated 19F-reporter. In another related example, hypoxia in the tumor microenvironment results in lactic acid production and, hence, acidic conditions. Zhou and colleagues fabricated pH-triggered NPs for 19F-MRI and fluorescence imaging (MRI-FI) of cancer cells. 82 19F was attached to AuNPs by acid-labile hydrazone linkers. Fluorescence imaging revealed highly selective uptake of these NPs by lung cancer cells. The “trick”to these NPs involves selective release of 19F-MRI contrast agent after the NPs have reached the tumor environment. In the acidic intracellular compartments of cancer cells the 19F detached from the AuNPs and appreciably enhances the intracellular 19F-MRI signal, as shown in Figure 4. Temperature differences between tissues is a common feature in pathological conditions, especially in tumors. Thus, a second class of “smart”NP probes for MRI uses hypersensitive detection of temperature changes in different tissues. 83 Langereis and colleagues 84 reported a combined temperature-sensitive liposomal 1H chemical exchange saturation transfer (CEST) 85 and 19F magnetic resonance (MR) contrast agent as a potential carrier system for MRI-guided drug delivery. The liposomes contain both a chemical shift agent as well as a highly fluorinated compound. Upon reaching the melting temperature of the agents’lipid membrane, the lipo-CEST contrast enhancement vanishes, due to the release of the chemical shift agent. Simultaneously, the 19F-MRI probe is freed from the influence of the paramagnetic shift agent causing an appearance of a 19F-MRI signal. The combined CEST and 19F-MR temperaturesensitive liposomal carrier provides CEST-based contrast enhancement, which can be switched on and off, to localize the liposomes before release, while the 19F-MR signal can potentially quantify local drug release of drugs with MRI. Redox reactions play crucial roles in biological processes, and abnormal redox reactions are implicated in various conditions including liver damage and human immunodeficiency virus (HIV). A third class of “smart”NP probes for MRI uses redox Figure 3. Illustration of multifunctional asymmetrical polymer vesicles for ultrasensitive T1MRI and effective cancer targeted drug delivery. Adapted from ref 69. Copyright 2015 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2319 reactions in the tumor microenvironment for cleavage of atoms from the NP structure. Kikuchi and co-workers designed redox activatable NPs to image reducing environments. 86 Gd3+ complexes were attached to the surface of 19F containing NPs by disulfide linkers. Vicinity of the Gd3+ to 19F reduces the transveral relaxation time T2of the fluorine compounds by the paramagnetic relaxation enhancement effect, which attenuates the 19F NMR/MRI signal (see Figure 5). When the disulfide bonds enabling attachment of Gd3+ are reduced, the Gd3+ complexes are cleaved from the NP surface. Then, the T2 NMR/MRI signal of the encapsulated 19F compounds increases, indicating the presence of the NPs in redox active environment. Imaging Radiolabels. There has been rapid progress in the development of NP-based radiolabels. 87 Chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid are commonly used to label diagnostic positron emitters, e.g.,68Ga or 64Cu, or therapeutic beta-emitters, e.g.,177Lu or 90Y. The sequential use of different poly(ethylene glycol) (PEG) -coated emitters, e.g., 68Ga and 177Lu enable multiplexed labeling, as well as improved circulation time. For this purpose, micelle encapsulation was introduced for combining a mixture of amphiphiles comprising chelators bearing the radiolabels, ligands for targeting, and PEG for surface passivation with originally hydrophobic NPs. 88 The same method could successfully be applied for different types of NPs, such as SPIONs, plasmonic NPs, and core/shell UCNPs. Sometimes the NPs can be labeled postsynthetically without the use of chelators for radiometals. 89 The resulting radiolabeled NPs enable researchers to trace their biodistribution when administered systemically, and to study fundamental NP in vivo process (e.g., determining the contribution of active versus passive tumor targeting in mouse models). 90 In fact, radiolabels are particularly suited for quantitative biodistribution studies. γ-emission is barely absorbed by tissue, and radioactivity is independent of the probe’s local environment, i.e., there are no substantial quenching effects such as in the case of fluorophores, which often exhibit pH-dependent emission. Using radiolabels produces excellent signals and, therefore, a low dose can yield sufficient image quality to make a diagnostic interpretation. One additional advantage is the ability for multiplexed read-out of radiolabels with different γ-emission energies in parallel. This has been recently used for recording the biodistribution of several compounds of NPs independently. 37 Most NPs are hybrid structures composed of their functional part (i.e., contrast for imaging) and an organic surface coating that provides colloidal stability and targeting capability, as well as the corona of adsorbed proteins. 91,92 By adding radiolabels of different energies to different parts of the NPs, the NPs could disintegrate after in vivo administration, i.e., the organic surface capping may come offthe functional particle core. 37 Such studies in the future will enable imaging of the different NP components to elucidate the fate of the different parts of the NP. This will be important for understanding the biodegradability of all different parts of a NP and their elimination. This is decisive for guiding the design of NPs for use in in vivo delivery applications. However, when different labeling methods and radioisotopes are used to label NPs to study their fate, it is crucial to ensure that the radioisotopes are incorporated onto the NPs with little impact on their original biological behavior. Fluorescence Imaging. Fluorescence is a useful imaging modality because the emission of the probes after excitation can be visualized by the naked eye or at higher resolution with optical microscopy. There is an abundance of available fluorophores that can be tailored to specific applications. Many traditional organic fluorophores suffer from aggregation-caused quenching (ACQ), thereby limiting design schemes where specific interactions promote fluorophore localization. This has forced researchers to use dilute solutions (often at the nanomole level) of fluorophores. Given the minuscule amount of fluorophore, they can be easily photobleached, which imposes a limit on the achievable contrast. Contrary to ACQ, aggregation-induced emission (AIE) is where increased aggregation yields a stronger fluorescent signal. 93 This makes AIE fluorogens (AIEgens) good candidates for bioimaging applications. 94 The accumulation of AIEgens at regions of interest results in the formation of nanoaggregates (AIE dots), which intensifies the fluorescence signal. The AIE dots are resistant to photobleaching, enabling high-quality bioimaging over a wide time window. They are suitable for longterm tracking of theranostic processes such as tumor-metastasis Figure 4. (A) Synthesis of AuNPs capped with folic acid and 19F contrast agent. Folic acid with a high tumor affinity due to the overexpression of its receptors on the cancer cells was conjugated onto the surface of the AuNPs. 19F contrast agent was covalently conjugated onto the AuNPs via acid-labile hydrazone linkers. (B) pH-triggered release of the 19F contrast agent from the AuNPs to the cytosol via the selective removal of the pH-labile cap in the acidic intracellular compartments of cancer cells. Adapted with permission from ref 82. Copyright 2014 Royal Society of Chemistry. Figure 5. Design of redox activatable NPs. Adapted with permission from ref 86. Copyright 2015 Wiley-VCH Verlag GmbH & Co. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2320 monitoring, drug delivery/release, and stem-cell therapy. 95 Organic AIE dots enjoy almost all advantages of inorganic QDs, 96,97 yet are free of the disadvantages of the latter (e.g., cytotoxicity 98,99 ). In addition, AIE molecules and nanoaggregates can be decorated by functional groups with binding specificity to particular receptors that enables active targeting. 94,96,97 Integration of an RGD unit with the AIEgen structure, for example, has generated integrin-specificfluorescence turn-on bioprobes for tumor cell targeting, image-guided drug delivery, in situ monitoring of therapeutic effect, etc. 100,101 Two decades ago, fluorescent semiconductor QDs were introduced for the fluorescent labeling of biological molecules and cells. 102,103 Due to their narrow emission bands, QDs are suited for multiplexed read-out, as detailed below. 104,105 Semiconductor QDs are also bright and show reduced photobleaching, which makes them suitable for long-term imaging at the single-molecule level. 106−108 However, semiconductor QDs can be cytotoxic toward cells due to the release of metal ions 98,99 and many researchers take this fact into consideration in designing QDs for their long-term imaging studies. 109−111 For example, CdSe QDs can be coated with ZnS polymer shells or SiO2layers to suppress the dissolution of possibly toxic semiconductor core materials. 99 Details about the mechanism for potential toxicity are still under discussion. Rare earth (RE)-based nanophosphors, which consist of a host inorganic matrix doped with luminescent lanthanide (Ln) cations, constitute another type of luminescent materials. Their main advantages are their low toxicity, high photostability, high thermal and chemical stability, high luminescence quantum yield, and sharp emission bands. 112 However, they normally show low luminescence intensity, which is caused by the low absorption of the parity-forbidden Ln3+ 4f−4f transitions. 113 The optical properties of such NPs can be further modified by doping, as shown recently in the case of Eu3+,Bi 3+ codoped REVO4 (RE = Y, Gd) NPs. Intense red luminescence was achieved by using indirect excitation of the Eu3+ cations via the vanadate anions. The incorporation of Bi3+ into the REVO4structure resulted in a shift of the original absorption band corresponding to the vanadate toward longer wavelengths, yielding nanophosphors excitable by near-ultraviolet and visible light. 114 Carbon dots are another class of fluorescent NPs. Carbon dots are zero-dimensional carbon nanomaterials and possess the advantageous characteristics of QDs over organic fluorophores, such as high photostability, tunable emission, and large twophoton excitation cross sections. 11 Moreover, C-dot fluorescence does not blink. They are water-soluble, can be functionalized, can be produced economically, and have excellent cell permeability and biocompatibility, making them attractive for intracellular sensing. 11,115,116 Also, C-dots without heavy metal content are environmentally friendly and can be much safer for biological applications than traditional Cd-containing QDs. 11,115,116 In a recent study, C-dots were subjected to a systematic safety evaluation involving acute toxicity, subacute toxicity, and genotoxicity experiments (including a mouse bone marrow micronuclear test and a Salmonella typhimurium mutagenicity test). 117 Carbon dot dosages of 51 mg/kg weight body showed no significant toxic effects, i.e., no abnormality or lesions, and no genotoxicity in the organs of the animals. Nanodiamonds, another group of carbon NPs, are receiving growing interest for drug delivery and other biomedical applications because they have good biocompatibility. 118 Intravenously administered ND complexes at high dosages did not change serum indicators of liver and systemic toxicity. 119 Nanodiamond powder produced by detonation (5−6 nm, see Figure 6) or other methods (20−50 nm) are readily available in commercial quantities at moderate cost and are among the most promising carbon nanomaterials for drug delivery, imaging, and theranostic applications. Fluorescence of NDs due to nitrogenvacancy (NV) centers (see Figure 6) that are composed of a substitutional nitrogen and a next neighbor vacancy enables biomedical imaging. 120,121 Fluorescent NPs can also be produced by linking or adsorbing various fluorophores onto NDs. For example, bright blue fluorescent NDs were produced via covalent linking of octadecylamine to carboxylic groups on the ND surface (ND-ODA). 122 In biomedical imaging, fluorescent NDs combine the advantages of semiconductor QDs (small NP size, high photostability, and bright multicolor fluorescence) with biocompatibility, lackof toxicity, and tunable surfacechemistry. Therefore, they are promising for in vivo imaging. Since the sizes of ND particles can be smaller than 5 nm NPs, they can be removed by renal excretion without leaving any toxic residue in the body. 123,124 Figure 6. (A) Schematic model of a 5 nm ND with a fluorescent NV center and a variety of surface terminations after oxidative purification is shown. The diamond core is covered by a layer of surface functional groups that stabilize the NP by terminating its dangling bonds. The surface can also be stabilized by the conversion of sp3carbon to sp2carbon. Adapted with permission from ref 118. Copyright 2012 Nature Publishing Group. (B) Plot showing the strength of binding (KL)versus the monolayer capacity (Amax) based on a Langmuir model for DOX, polymyxin B, tetracycline, and vancomycin adsorbed on detonation-produced ND from two sources (ZH and ND) with different surface chemistries: the as-received surface, like in the right picture, carboxylated (−COOH) and aminated (−NH2) surfaces. Adapted with permission from refs 125 and 126. Copyright 2016 Elsevier and copyright 2013 American Chemical Society, respectively. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2321 Similarly, a nanosensor capable of monitoring intracellular glucose levels is depicted in Figure 16B). Polyacrylamide NPs were implemented as a carrying matrix for glucose oxidase, GOx, and the Ru(II)-tris-bipyridine, Ru(bpy)32+, transducer. While the Ru(bpy)32+ fluorescence is quenched by intracellular O2levels, the O2-driven GOx-catalyzed oxidation of glucose depletes the O2levels, resulting in the triggered-on fluorescence of the Ru(bpy)32+ luminescent probe. As the degree of O2depletion by the biocatalytic process is controlled by the concentration of glucose, the resulting fluorescence of the transducer provided a quantitative measure for the concentration of glucose. 187 The use of composite polymer NPs carrying enzymes and optical transducers as bifunctional sensing elements is particularly attractive since the products generated by many enzyme-driven processes may activate optical (fluorescent) transducers. The use of such intracellular nanosensors should be implemented with caution, however, since intracellular environmental conditions, e.g., changes in pH, might alter the enzyme activities, thus perturbing the intracellular nanosensor performances. Screening Based on Surface Plasmon Resonance. Plasmonic NPs are also designed for optical read-out, 188−192 in particular, in the form of colorimetric responses. The aggregation of AuNPs shifts the absorption maximum to longer wavelengths. Aggregation can be induced by the selective binding of analyte molecules to the functionalized surfaces of AuNPs. 193 The most popular example in this research direction is DNA detection, as developed by Mirkin and co-workers. 136,194,195 There are now many similar assays. For example, Stevens and co-workers used AuNPs to detect the enzyme phospholipase A2at a concentration of 700 pM. 190 A more complex scheme was used by Pompa and co-workers to detect cancer-related point mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene. 189 In this case, AuNP aggregates form when the target gene hybridizes with the complementary single-stranded capture probes functionalized to AuNPs. Subsequently, the AuNP− target-gene complex undergoes a secondary binding event with DNA-functionalized magnetic Fe3O4microparticles. In addition to nucleic acid detection, AuNPs functionalized with capture antibodies and patterned onto dielectric surfaces can be used to fabricate a protein biosensor. 192 The characteristic surface plasmon resonance (SPR) of such AuNPs is shifted by the binding of target proteins, and this spectral shift can be detected using conventional SPR imaging spectrometers. Tamiya and co-workers demonstrated a 300-channel version of this device, capable of label-free detection of targets down to 100 pg/mL. 192 Figure 15. (A) Chemically modified boronate ester-functionalized BPAN NPs for intracellular fluorescence probing H2O2. (B) Fluorescence spectra changes of the modified polymer NPs upon interaction with increasing amounts of H2O2: 0, 20, 40, 60, and 80 μM. Adapted from ref 185. Copyright 2012 American Chemical Society. Figure 16. Bifunctional recognition/dye polymer NPs for sensing: (A) Sensing of H2O2via the HRP-driven oxidation of Amplex-Red to the fluorescent Resorufin product. (B) Sensing of glucose by the depletion of O2by the GOx-mediated oxidation of glucose, using Ru(bpy)32+ as auxiliary fluorescent probe. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2328 Taking advantage of the high cross-section absorbance of plasmonic NPs, their SPR can also be used in plasmonic-driven thermal sensing. Qin et al. presented an improved lateral flow immunoassay using AuNPs, which improved the analytical sensitivity of the method 32-fold, achieving similar sensitivity to an enzyme-linked immunosorbent assay (ELISA). 196 Furthermore, Polo et al. reported a SPR-based detection of carcinoembryonic antigen (CEA) with a sensitivity 3 orders of magnitude better than standard ELISAs using anisotropic AuNPs. 197 One of the emerging techniques related to plasmon resonance that demonstrated high sensitivity and versatility is the chiroplasmonic method developed by Kotov and Xu. 198−200 This technique is based on the giant polarization rotation characteristic of nanoscale assemblies highly polarizable metallic nanoparticles. The chiroptical effects in these structures are several orders of magnitude higher than in small organic molecules due to high polarizability of the inorganic nanomaterials and larger dimension. Notably, the physics of chiroplasmonic detection differs from that of red-blue plasmon coupling assays or screening with Raman scattering based (see below). Polarization rotation in NP assemblies is primarily based on interactions of the electromagnetic field with asymmetric nanostructures rather than on the formation of so-called ‘hot spots’or plasmon coupling. 201 This difference is essential for biomedical diagnostics because it enables detection of long strands of DNA and large proteins. For instance, translation-inspiring sensitivity was obtained for prostate specific antigen using this method. 202 Most recently, this method in combination with UCNPs also enabled dual detection of one of the most promising diagnostic targets micro RNAs (miRNA) at the levels sufficient for its application in cancer diagnostics. 203 Screening based on surface-enhanced Raman scattering. Surface-enhanced Raman scattering (SERS) is another class of NP-based molecular detection. 204,205 Surface-enhanced Raman scattering is an ultrasensitive molecular spectroscopy technique that benefits from the electromagnetic fields generated upon excitation of plasmons in nanomaterials. Despite the Figure 17. (A) Scheme showing prion mutation and prion ultradetection in human blood. Surface-enhanced Raman spectra (SERS) of (a) natural and (b) spiked human blood; (c) natural and (d) spiked human plasma; (e) spiked human plasma after spectral subtraction of the matrix (human plasma); (f) the scrambled prion. Adapted with permission from ref 205. Copyright 2011 National Academy of Sciences. (B) (a) Optical spectra and SERS (mapped at 1548 cm−1, as marked with the arrow below) images of 3T3 cells in the presence of capsules. The SERS spectra (bottom) show the signals for the colored circles in the image (top). (b) Optical images and intracellular NO formation over time (obtained through the I1583/(I1583 +I1548) relation) for three different samples upon NO induction with hydrogen peroxide (H2O2). A control sample without the presence of H2O2is also shown for comparison. Representative normalized SERS spectra obtained at different times are shown. The SERS dashed (blue) and dotted (red) spectra represent the reference vibrational pattern for aminobezenethiol and hydroxybenzenethiol, respectively. Adapted with permission from ref 212. Copyright 2013 John Wiley & Sons, Inc. (C) (a) Outline of the c-Fos/c-Jun dimerization on the metal surface and the resulting deformation of the Raman label structure. (b) Details of the 1000−1100 cm−1spectral regions of the SERS of the molecular spring (benzenethiol) interfacing the NP and the protein c-Fos.(c) Spectral shift of the benzenethiol band at ca. 1075 cm−1as a function of c-Jun concentration (logarithmic scale) in HEPES buffer. Adapted from ref 215. Copyright 2013 American Chemical Society. (D) (a) In vivo cancer targeting and SERS detection by using ScFv-antibody-conjugated AuNPs that recognize the tumor biomarker epidermal growth factor receptor (EGFR). Top: Photographs showing a laser beam focusing on the tumor site or on the anatomic location of liver. Bottom: SERS spectra obtained from the tumor and the liver locations by using (a) targeted and (b) nontargeted NPs. Two nude mice bearing human head and neck squamous cell carcinoma (Tu686) xenograft tumors (3 mm diameter) received 90 mL of ScFv EGFR-conjugated SERS tags or PEGylated SERS tags (460 pM). The NPs were administered via tail vein single injection. SERS spectra were taken 5 h postinjection. In vivo SERS spectra were obtained from the tumor site (red) and the liver site (blue) with 2 s signal integration and at 785 nm excitation. The spectra were background-subtracted and shifted for better visualization. The Raman reporter molecule was malachite green, with distinct spectral signatures as labeled. Adapted with permission from ref 216. Copyright 2008 Nature Publishing Group. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2329 impressive detection limits of SERS, down to the single molecule level, 206 its application in diagnosis has been restricted until recently by the inherent complexity of biological samples. Thus, direct SERS detection of disease markers have typically been carried out either in lab samples, where the marker was diluted in a controlled solution, or in biological samples, where the marker is characterized by an extraordinary affinity for the plasmonic surfaces, such as the use of SERS detection for Creutzfeldt-Jakob prionic diseases (see Figure 17A). 205 The use of chemoreceptors, intermediary molecular species with selective affinity for the molecular target (i.e., the disease marker), 207 has notably increased the applicability of SERS as an efficient diagnostic tool. Surface-enhanced Raman scattering has therefore been applied to ex vivo detection of drugs, 208 proteins, 209 metabolites indicative of disease, 210 or even toxic ions, 211 but also to in vitro monitoring of relevant small inorganic molecules such as nitric oxide 212 or pH levels 213 inside living cells (see Figure 17B). In this approach, SERS detection is achieved indirectly as the spectral changes for the chemoreceptor are monitored before and after reaction with the target analyte, rather than directly detecting the analyte. Ideally, the chemoreceptor needs to show a recognizable spectral change, either structural or electronically, upon conjugation with its target. Although this property can be found easily in small aromatic molecules, such as aromatic thiols and amines, porphyrins, and dyes, macromolecules such as peptides and proteins usually do not present spectrally discernible differences. In such cases, however, the use of molecular springs as an interface between the metallic surface and the protein chemoreceptor can be employed to monitor the interactions with the analytical target. This strategy has been demonstrated successfully in the quantification of the oncogenic protein c-JUN in cell lysates by using the transcription factor FOS 214 coupled with mercaptobenzoic acid as the molecular spring (see Figure 17C). 215 An alternative approach to the use of SERS for diagnostics and bioimaging relies on the highly intense signals that can be obtained from plasmonic NPs labeled with molecules featuring large SERS cross sections. This strategy, pioneered by Shuming Nie, has been demonstrated extensively for bioimaging of tumors in mice (see Figure 17D) and for use as a contrast agent for the multiplex labeling in tissue preparations. 217 The use of different Raman labels in combination with different antibodies can further expand the multiplexing capability of these systems. Moreover, a variety of membrane receptors can be targeted specifically to obtain pertinent spatial information. 218 Surface-enhanced Raman scattering NP tags have also been used for multiplexed detection of circulating tumor cells and cardiovascular protein biomarkers in blood samples. 219 Nie and co-workers developed a sandwich-type assay involving magnetic capturing beads and SERS tags for measuring a panel of four cardiac protein biomarkers (sVCAM-1, suPAR, HSP70, and CRP). The magnetic beads enable easy purification while the SERS tags enable simultaneous quantification. Due to multiplexing, high sensitivity, and the large dynamic range of SERS, multiple biomarkers can be assessed over a wide concentration range in a single tube. Since this system removes many purification and enrichment steps, SERS-based assays can provide better positive and negative predictive values in high-risk patients. In the context of coronary arterial disease, a major clinical problem is the prediction of sudden cardiac events such as plaque rupture and myocardial infarction. Hence, it is important to develop assays to distinguish high-risk populations for plaque rupture that require immediate intervention and treatment. Screening Based on Electronic Read-Out. Nanowires 220−229 are enabling a novel class of biosensors that are capable of detecting disease markers in blood, saliva, and urine, and able to convert the detection events into electronic signals. Semiconductor nanowire field-effect transistors (FETs) make use of nanowires that are sensitive to surface-binding events. 230 This sensitivity is derived from the proximity of the binding sites to the charge carriers within the nanowire. 231−233 Nanoengineered FETs differ considerably from conventional, microlithography-based FETs for solution-based detection. 234−236 For example, nanoengineering enables miniaturization and also enables the detector to be close to the target. Silicon nanowires can detect proteins at 10 fM concentrations in low ionic strength buffers; however, the detection of analyte molecules by nanowire FETs in high ionic strength (>mM) solutions, including all physiological solutions of interest, has posed problems. In such solutions, the Debye length is reduced to 0.2−2.0 nm and the screening of analyte charge by the electrolytes either reduces sensitivity or, more often, prevents detection of the analyte altogether. 221,237 Andrews, Weiss, and Yang showed that aptamer-functionalized FETs can operate in full ionic strength buffer, presumably because of the proximity of the charged backbone to the gated semiconductor, and thus show promise for in vivo measurements. 238,239 A recent study demonstrated 230 that backfilling antibody-functionalized silicon nanowires with a PEG layer extends the Debye length, enabling the detection of prostate-specified antigen at a concentration of 10 nM in 100 mM phosphate buffer. 230 Nanowires composed of conductive polymers (e.g., polyaniline 240−243 )havealsobeen adapted for electrical biosensing using either FET-based transduction 244,245 or chemoresistive transduction. 228,229 In these systems, the binding sites are either antibodies or virus particles displaying peptide motifs, both of which are capable of recognizing and binding analyte molecules. Conveniently, either of these two “receptors”can be entrained in the polymer from the polymerization solution during nanowire fabrication. In contrast to nanowire FETs, transduction of analyte binding to polymer nanowires is not completely understood. Carbon nanotubes have been also used as transducers for biosensing. 246 All these biosensors are “label-free”capable of providing a signal correlated to the concentration of analyte by immersion in a solution without added reagents or process steps. Thus, these devices are ideally suited for use in point-of-care disease diagnostics. An electrically responsive NP sensing scheme requires electrical contact. One approach described by Gadopadhyay et al. 240 involves the incorporation of AuNPs into polyaniline films on electrode surfaces. 240 In this case, AuNPs facilitate the attachment of single-stranded DNA (ssDNA) capture strands to the nanowire, using a thiol-based immobilization scheme. Binding of target DNA to the ssDNA capture strands impedes the redox responsiveness of polyaniline and reduces peak currents by providing an insulating layer to the electrode surface. 240 Gadopadhyay et al. demonstrated that target nucleotide concentrations down to 10−18 M can be detected by monitoring changes in peak currents. Similarly, QDs have been used as transducer in electrochemistry based detection schemes. 247−254 Biological molecules can also act as transducers that directly convert binding events into electrical signals. This action can be in the form of ion channels, which open in response to binding events and subsequently result in a signal cascade. 255 Olfactory receptors, for example, can be used for the development of sensitive and selective odor-sensing biosensors. 256−258 These biosensors can screen numerous volatile compounds generated ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2330 by the human body, such as exhaled breath and body odors, to detect diseases and health conditions. 259,260 Olfactory receptors have also been combined with various transducers such as quartz crystal microbalance, 261−263 SPR sensors, 264,265 microelectrodes, 266,267 NPs, 268 graphene, 269,270 and nanotubes 271−277 to generate different types of read-outs. For example, a biosensor with an electrical read-out was constructed by immobilizing olfactory receptors on nanotubes. 271,272 Similarly, a nanovesiclebased olfactory biosensor, where nanovesicles with surface olfactory receptors were immobilized on nanotubes, was successfully used for cancer diagnosis. 263,276,277 In addition to olfactory receptors, others, such as hormone receptors, 268 taste receptors, 278,279 and peptides 280 can also efficiently be used in biosensors that can detect components of saliva, tears, blood, urine, cerebral spinal fluid, mucous, and even tissues. 281,282 Similarly, whole cells can be used as transducers, whereby electric currents of electrically excitable cells are recorded. 283 Primary cells 284 and stem cells 285 from patients have become interesting targets that can be used as potential tools for personalized medicine. In addition to whole cells, specific ion channels can also be used as transducers. Ion channels are involved in electrical signal conduction (such as in the nervous or cardiac systems) as well as in other disease areas such as oncology, 286 cystic fibrosis, 287 diabetes, 288 and infection. 289 The gold standard for monitoring ion channel currents is the patch clamp technique, 290 which provides highly accurate functional recordings from single or multiple ion channels in cells. However, the low throughput and the need for highly trained operators exclude the classical patch clamp technique from many screening applications. This situation changed with the development of the planar patch clamp technique. 291,292 The latest generations of planar patch clamp devices like the SyncroPatch 384PE (2013 by Nanion Technologies) enable recordings of up to 768 cells at a time, and can be integrated into high-throughput screening cascades of the pharmaceutical industry. Nanotechnology has had a leading role in shaping these technologies by providing nanopores responsible for sealing the cell and the detector surface. Such nanopores can be used not only for electrophysiological measurements of cells, but also have been used in the context of DNA detection. 293 In most cases, electrophysiological detection is used for monitoring overexpressing ion channels, which has implications in primary drug targeting or for eliciting dangerous side effects. One prominent example is the cardiac herG channel that plays an important role in heart arrythmia and QT-prolongation. 284,285,294−296 At the tissue and organ levels, indicators like electrophysiological signals have been widely employed for evaluation of organ function. However, conventional electrodes are confronted with the mismatch between rigid/planar electrodes and soft/curvilinear tissues. By designing gold nanobelts with sinusoidal structures on flexible tripod substrates, one is able to create stretchable biointegrated electrodes for direct recording of electrophysiological signals of rat cerebral cortex with desirable sensitivity and stability. 297 The transfer of thin, flexible electronics onto polymers also enables conformal placement and measurements. 298 Given the abundance of material candidates and nanofabrication techniques, more novel and smart tools capable of monitoring function and quantity would emerge and boost the development of nanomedicine and its transition into practical applications. Biomechanical Assays. Nanoenabled tools can be designed for diagnostics and therapies based on the mechanical properties of cells. 299 At the subcellular and cellular levels, many biophysical properties have recently emerged as indicators of cell physiology and pathology, as complementary or regulatory alternatives for disease development. Cell migration, for example, can be traced by removing the trail of adherent cells left on a substrate coated with AuNPs 300,301 or with QDs. 110 The morphology of the trails correlates with the metastatic potential of the cells. 302 The force that adherent cells exert on a substrate can also be measured. For example, cell-traction-force microscopy based on the deformation of the polymer substrates on which cells are grown is able to spatiotemporally map cell traction force as precise as a nanonewton. 303−305 It can also be used to uncover previously hidden details of drug−cell interactions and mechanical contributions during cell migration. 303−305 For example, previously retarded cell migration upon NP uptake was attributed to cytoskeleton disruption. However, the uptake of the NPs actually transforms cells from the motile phenotype into an adhesive phenotype, as revealed by the increased cell traction force and altered patterns of cell traction force. 306 More importantly, ultrasensitive cell traction force microscopy could be competitive to traditional cell biology methods such as the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assays, 307 WB, and flow cytometry, since early changes can be readily observed under cell-traction- force microscopy. IN VIVO TREATMENT (“PARTICLE-BASED DELIVERY”) Nanoparticles have been designed to treat many diseases, but the most prominent disease focus has been in cancer. The literature is replete with large sets of in vivo data obtained from animals, and some NP formulations for cancer treatment have already been approved by regulatory agencies and have reached hospitals, showing much reduced adverse effects compared to bare drugs. 308 However, their therapeutic efficacies are most often not or only marginally improved. 309,310 Thus, there are ongoing efforts to develop systems with high therapeutic efficacies. 311 The key in such developments is to improve the design of the nanodelivery system by precisely controlling the functions/ properties of the materials. Nanoparticles as Anticancer Drugs. The cancer drug delivery process to a solid tumor consists of five critical steps, termed the CAPIR cascade: circulation in blood, accumulation and penetration into the tumor, cellular internalization, and intracellular drug release. Thus, the overall therapeutic efficiency of a nanomedicine is determined by its efficiency in each step (see Figure 18). 312 A nanomedicine efficiently accomplishing the whole CAPIR cascade should have a high therapeutic index. Correspondingly, such a nanomedicine should have 2R2SP properties, the abbreviation of “drug retention vs release (2R)”, “surface stealthy vs sticky (2S)”and “tumor penetration (P)”. 313 The 2R indicates the required properties of the nanomedicine in terms of the loaded drug, i.e., it must tightly retain the drug without burst release during the transport in blood compartments and tumor tissues, while efficiently releasing the drug at the intracellular target to exert its pharmaceutical action. Similarly, the 2S gives the needed properties in terms of the NP surface, which should be stealthy while in blood circulation for Nanoparticles have been designed to treat many diseases, but the most prominent disease focus has been in cancer. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2331 passive tumor accumulation, whereas it should become sticky once nearby the tumor cells to interact with them for efficient cellular uptake. Besides the 2R2S properties, the nanomedicine should be able to penetrate in tumor tissues to reach remote tumor cells from blood vessels. Only if a nanomedicine possesses 2R2SP properties will it be able to accomplish the CAPIR cascade successfully and to deliver active drugs at the right time and place to provide an overall high therapeutic efficacy and favorable prognosis. 312,313 Therefore, the current focus on developing nanomedicines of high therapeutic index lies on tailoring the basic physiochemical properties of NPs, i.e.,“size, surface properties, and stability (3S nanoproperties)” to achieve 2R2SP properties to accomplish the CAPIR cascade efficiently. A demonstration of such a 2R2SP nanomedicine is a dendrimer core−lipid shell nanoassembly mimicking a “clusterbomb”, which shows enhanced efficacy, as demonstrated by Sun et al. 312 The nanocomplex is able to cruise to a tumor, shed “bomblets”to penetrate into the tumor, and then “fire”at tumor cells to accomplish the CAPIR cascade. A sixth-generation nontoxic degradable polyaminoester dendrimer with a diameter of 5 nm was chosen as the “bomblet”because of its ability to carry anticancer drugs and, more importantly, its pH-dependent 2-(N,N-diethylamino)ethyl termini. 312,314,315 The lipid shell was composed of fusogenic DOPE phospholipids, PEGylated DSPEPEG lipids, and cholesterol in order to keep the nanoassembly stable and to provide a stealth surface in the blood compartment. The 45 nm NPs contained ∼27 5 nm dendrimers surrounded by a PEGylated lipid layer. The NPs could circulate in blood and accumulate in the tumor via the EPR effect. Once in the tumor, the fusion of the lipid layer with the cell membrane released the dendrimers intracellularly or extracellularly. The nearly neutral dendrimers were shown to penetrate into tumor tissues easily due to their size, where the extracellular pH is acidic (pH ∼6−7). The dendrimers were then protonated and became positively charged, efficiently triggering fast cellular uptake, and releasing the drug inside the cells. The dendrimer−lipid nanoassembly efficiently accomplished the CAPIR cascade and thus exerted strong anticancer activity in DOX-resistant tumor compared with typical PEG−PCL micelles. 312 Nanoparticulate Delivery Vehicles. A vast number of classes of nanoparticulate delivery vehicles have been reported. 316−351 As demonstrated by Huynh et al., 317 drug-loaded delivery vehicles are attractive because they can be passively or actively targeted to cancer tissues to improve anticancer drug selectivity and thereby reduce severe side effects. Liposomes 316 are perhaps one of the most advanced delivery vehicles concerning clinical translation, with several formulations approved by the U.S. Food and Drug Administration (FDA). 316 Liposome-based systems encapsulating drugs are already used in some cancer therapies (e.g., Myocet, Daunoxome, Doxil). However, liposomes have some significant drawbacks: they have a low capacity to encapsulate lipophilic drugs, they are manufactured through processes involving organic solvents, they are often leaky and are unstable in biological fluids and aqueous solutions. 317 Polymeric micelles embedded with drugs can also reduce undesirable side effects. Micelle formation involves the selfassembly of amphiphilic molecules in which, upon separation of hydrophilic and hydrophobic moieties, hollow capsules are formed. In water, the interior of those micelles forms the hydrophobic container, whereas the outside imposes the hydrophilic interface. Encapsulation of anticancer drugs within micelles can reduce toxicity and improve circulation. 318,352,353 For example, nephrotoxicity is significantly reduced by loading cisplatin into polymeric micelles. 318 Shielding the cisplatinloaded core with PEG improves longevity in blood circulation by reducing acute kidney accumulation of polymeric micelles. In this regard, the secondary structure of the micelle core also plays a critical role in stabilizing polymeric micelles in diluted conditions in the blood. For example, since the core-forming segment in this formulation is poly(L-glutamic acid) (PLG), cisplatin binding to the side chain of the PLG segment induces α-helix formation. Regular assembly of cisplatin-bound helical bundles in the core is key to keeping the micellar structure intact during blood circulation. 319 Five different formulations of polymeric micellar drugs based on poly(ethylene glycol)-poly(amino acid) block copolymers, developed by Kataoka and colleagues, are already in clinical translation in Asia and North America. 320 Specifically, a polymeric micellar drug loaded with paclitaxel is close to completing its Phase III clinical trial, and is expected to be up for approval soon. Polymeric micelles thus provide a mature platform with numerous advantages to improve patient compliance appreciably, including outpatient treatment without hospitalization. Micelles can also be jointly fabricated with polymers as well as lipids. Microfluidic chips can be used for the controlled production of these micelles for the delivery of therapeutic agents. 321,322 By adopting a microfluidic platform (see Figure 19), NPs of varying water content and rigidity but with the same chemical composition, size, and surface properties can be synthesized. It is important to be able to vary the properties of these micelles efficiently since cellular uptake can be entirely dependent on these characteristics. For example, rigid or “hard” NPs, with less interfacial water between the polymeric core and lipid shell, can enter cells more easily than those that are flexible (“soft”). In contrast, bigger particles have poor accumulation in cells. 128,323 In this regard, a hollow-structured rigid nanovesicle (RNV) was fabricated to entrap various hydrophilic reagents effectively by multistage microfluidic chips in one step, without complicated synthesis, extensive use of emulsifiers or stabilizers, or laborious purification procedures. The RNVs contain a hollow water core, a rigid poly(lactic-co-glycolic acid) (PLGA) shell, and an outermost lipid layer. The entrapment efficiency of Figure 18. (A) Five-step CAPIR cascade in targeted cancer drug delivery. Reprinted with permission from ref 312. Copyright 2014 John Wiley & Sons, Inc. (B) Needed properties of a nanomedicine capable of accomplishing the cascade. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2332 hydrophilic reagents such as calcein, rhodamine B, and small interfering ribonucleic acid (siRNA) inside the hollow water core of RNVs was approximately 90%. 42 Unlike micelles, polymer capsules provide a delivery platform for encapsulating hydrophilic drugs. Polymer capsules can be formed with entirely hydrophilic polymers, for example, the LbL approach can form these capsules by templating particles. 324,325,354 While such capsules are often on the micrometer scale, smaller polymer NPs can be obtained from polymer capsules by thermal shrinkage. 327 In this approach, hollow capsules “filled”with a desired medicine with an even polyelectrolyte layer number are subjected to elevated temperatures (above the temperature of the glass transition temperature of the polyelectrolyte complex, i.e.,∼40 °C). At temperatures above 60 °C, polymeric NPs are formed. In contrast to the aforementioned NPs, where lab-made synthesis is required, exosomes are naturally occurring nanosized-vesicles endogenously secreted by cells. 328 They are involved in intercellular and tissue-level communication by transferring biological material (i.e., microRNAs [miRNAs], siRNA) among cells. Exosomes exhibit great potential as nanocarriers for a wide range of therapies, including inflammatory diseases and cancer, 329 as well as for diagnosis. 330 Nanocarriers are not always in the form of vesicles; there are numerous other types of delivery vehicles including water-solu- ble polymers, dendrimers, and even polysaccharides. 331,334,340,341 Water-soluble polymers in a random coil conformation can be efficient carriers of anticancer drugs. 324,331 Recently, backbone degradable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer−drug conjugates synthesized by reversible addition− fragmentation chain transfer copolymerization demonstrated high efficacy in treating solid tumors. These conjugates contain a degradable oligopeptide sequence (GFLG) in the backbone and drug in the backbone and at the termini. This design permits the use of high molecular weight, long-circulating polymer carriers without impairing their biocompatibility (the carrier will degrade into fragments that will be eliminated from the body). 355 Consequently, as a result of maintaining the concentration gradient (blood to tumor) for an extended period of time, the EPR effect is considerably more effective: the drug can accumulate in solid tumor to a higher extent. Conjugates with epirubicin, 332 DOX, 333 and a combination of conjugates with gemcitabine (GEM) and paclitaxel 8 were highly effective in the treatment of experimental ovarian cancer using this platform. Dendrimers 334 are another form of polymeric NPs, which have the advantage of being nearly monodisperse, in contrast to polymerization products, while having a defined structure and composition. 334,335 Remarkably, dendrimers with adequate hydrophilicity, unlike liposomes and polymer micelles, do not have a critical micelle concentration. 336 Therefore, dendrimers should not “in principle”fragment upon dilution. 337 Additionally, the number of functional groups grows exponentially as the number of generations increases. These features make dendrimers a powerful platform for drug delivery. Drugs can be loaded into the dendrimers in different ways, such as by encapsulation or by covalent binding of the drug. Drug encapsulation can be realized in unimolecular micelles or in supramolecular assemblies. 338 This approach enables encapsulation of the most widely used chemotherapeutic agents, e.g., DOX, methotrexate, paclitaxel, and campthothecin. 335 The covalent binding of drugs is traditionally used to overcome the encapsulation problems, and the drugs are linked to the dendrimer surface using groups that will be cleaved following cellular uptake (e.g.,cis-aconityl or acyl hydrazine groups, ester groups or disulfide groups). 335 All these characteristics distinguish these materials as potential carriers for different therapeutic applications. 339 Another class of carrier vehicles is based on polysaccharides, such as chitosan, which are promising carriers for oral delivery of therapeutic proteins. 356 The oral route is considered to be the most convenient and comfortable means of drug administration for patients. 357 Chitosan is a biocompatible polysaccharide derived from the shells of crustaceans. It is a mucoadhesive agent and a permeation enhancer that is able to mediate the opening of tight junctions between epithelial cells reversibly. 358 A NP carrier system formed by self-assembly of positively charged chitosan and negatively charged poly(γ-glutamic acid) in an aqueous milieu has been developed for delivering protein drugs orally. 340,341 The as-prepared chitosan NPs are pH-responsive in the intestines, because the pKaof chitosan is approximately 6.5. The working mechanism for this oral delivery system is that the mucoadhesive chitosan NPs adhere to and infiltrate the mucus layer on the intestinal surface. The infiltrated NPs can transiently open the tight junctions between epithelial cells while becoming unstable and disintegrated, owing to their pH-responsiveness, and releasing the loaded protein. The released protein then permeates the paracellular pathway via opened tight junctions, ultimately absorbing into the systemic circulation. 342 However, the enhanced absorption of therapeutic proteins, as delivered by chitosan NPs, occurs mainly in the duodenum, most likely because chitosan is insoluble in a neutral/basic pH environment in the jejunum and ileum. 343 Additionally, these chitosan NPs are not able to protect the released protein from degradation by the proteases present in the mucus layer. One strategy of inhibiting intestinal protease activities is to remove essential metal ions such as Ca2+ from the enzyme structure using chelating agents such as ethylene glycol tetraacetic acid (EGTA). Furthermore, the use of chelating agents for removing extracellular Ca2+ can disrupt intercellular junctions and increase paracellular permeability. According to reported experiments, EGTA-conjugated chitosan NPs can significantly reduce enzymatic degradation by the specific removal of extracellular Ca2+ from the intestinal lumen. This enhanced stability causes intracellular internalization of junctional components, and thereby increases the Figure 19. Scheme of a two-stage microfluidic platform for assembling polymer−lipid hybrid NPs with varying amounts of water. (a) Nanoparticles composed of the lipid shell and PLGA core are produced by injecting the PLGA solution in the first stage and lipid−PEG solution in the second stage (mode 1, P−L NPs). (b) Nanoparticles composed of the lipid shell, interfacial water layer, and PLGA core are produced by injecting the lipid−PEG solution in the first stage and the PLGA solution in the second stage (mode 2, P−W−L NPs). Adapted with permission from ref 322. Copyright 2015 John Wiley & Sons, Inc. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2333 absorption of protein throughout the small intestine. 344 A recent large animal study performed with beagle dogs revealed that EGTA-conjugated chitosan NPs could successfully deliver proteins into the plasma. 345 Although chitosan-based NPs are promising for oral delivery of therapeutic proteins, further preclinical studies are required to test their efficacy and safety. Proteins can themselves form the basis of another class of drug carriers. 346 The abundant plasma protein, serum albumin, has been successfully converted into a drug transporter and was launched to the market in 2005 for delivering the anticancer drug paclitaxel (Abraxane). 347 Serum albumin is a nontoxic, nonantigenic, and biodegradable material. In fact, albumins are excellent charge-reversible materials. Albumins have characteristic isoelectric points, due to the existence of both carboxylic acid and amino groups. When the environmental pH is higher than the pI, albumins have more negative charge on their surface, otherwise albumins are more positively charged. In addition, the pI of albumins can be adjusted by changing the ratio between the carboxylic acid and amino groups through chemical modifications. By tuning charge, albumin NPs have been successfully used to deliver negatively charged nucleic acids as biomolecules for gene therapy. 359 Recent approaches broadened the applicability of this successful platform and there is further development of novel features that aims to advance intelligent drug delivery. Boronic-acid-rich albumin NPs have been equipped with targeting peptides cRGD on the surface and DOX encapsulated inside the NPs. 348 The boronic acid groups enhance the recognition of NPs to cancer cells, since the boronic acids reversibly interact with overexpressed sialic acid residues. Encouraged by the success of albumin NPs, other endogenous proteins are being investigated such as milk casein, 349 or the iron-transport protein ferritin. 350 Proteins in their denatured form reproducibly enable the attachment of PEG chains as well as drug molecules to improve circulation times and solubility. In this way, polypeptide−PEG copolymers of human serum albumin have been obtained that can form small micelles. These micelles were able to transport and to release multiple copies of the drug DOX in leukemia cells via a two-step release mechanism. 351 Similar to protein carriers, ribonucleic acid (RNA) nanotechnology has emerged as a potential platform for drug delivery applications. This use is largely due to the ability to design structures with high thermodynamic stability, and favorable and distinctive in vivo attributes. 360 RNA can act both as a delivery vehicle as well as an active therapeutic agent. As an example, Qiu et al. combined the bacteriophage phi29 DNA packaging motor pRNA three-way junction (3WJ) with folic acid, a near-infrared (NIR) marker, and BRCAA1 (breast cancer associated antigen 1, AF208045) siRNA (see Figure 20). 43 These RNA NPs comprise functionality for targeting, imaging, delivery, gene silencing, and regression of gastric cancer. In vitro assays revealed that the RNA NPs specifically bind to gastric cancer MGC803 cells. The siRNA incorporated in the RNA NPs thereby significantly silences the BRCAA1 gene. The apoptosis of gastric cancer cells was observed as a result of silencing the antiapoptosis factor BCl-2 and up-regulation of the proapoptosis factor Rb and Bax expression. Animal trials using a gastric tumor-bearing nude mice model confirmed that these RNA NPs could be used to image gastric cancer in vivo, while showing little accumulation in crucial organs and tissues several hours postsystemic injection. The growth of the gastric tumor noticeably decreased during the course of treatment. No damage to important organs by the RNA NPs was detected. All results show that RNA nanotechnology can overcome some conventional cancer therapeutic limitations and open new opportunities for specific delivery of therapeutics such as siRNA, miRNA, and/or chemotherapeutic drugs to stomach cancer. In recent work, siRNA attached to glucosemodified AuNPs (AuNP@PEG@glucose@siRNA) were applied in vitro to a luciferase-CMT/167 adenocarcinoma cancer cell line and in vivo to the lungs of B6 albino mice. 361 The siRNA-bearing NPs induced the expression of pro-apoptotic proteins such as Fas/CD95 and caspases 3 and 9 in CMT/167 adenocarcinoma cells in a dose-dependent manner, independent of the inflammatory response. Moreover, in vivo pulmonary delivered siRNA- bearing NPs were capable of targeting c-Myc gene expression (a crucial regulator of cell proliferation and apoptosis) via in vivo RNA interference (RNAi) in tumor tissue. This led to an ∼80% reduction in tumor size without associated inflammation. Overall, RNA NPs can improve therapeutic efficacy while reducing toxicity and side effects and therefore provide an avenue for use in clinical tumor therapy in the near future. Nanodiamonds are among the most promising carbon nanomaterials for drug delivery, as well as imaging and theranostic applications. Nanodiamonds that are ∼5 nm have a large accessible surface and tailorable surface chemistry, combined with unique optical, mechanical, and thermal properties. 118 In drug delivery, the rational surface modification of NDs allows for enhanced adsorption and chemical binding of the drugs for sustained or triggered drug release. Both the amount of drug adsorbed and the strength of adsorption can be controlled by the surface chemistry. By attaching drugs to the surface of NDs and by achieving controlled release, it is possible to renew the potency of anticancer chemotherapeutics. 125 Similarly, the development of NDs for targeted delivery of antibiotics and other drugs may also restore the drug efficiency and reduce systemic toxicity. Currently, NDs are being investigated for the delivery and sustained release of anticancer chemotherapeutics, 119,363 Figure 20. Global structure of therapeutic RNA NPs with BRCAA1 siRNA. (A) Design of the RNA NPs. The left image shows the NP structure as used in animal trials. On the right, an extended structure imaged by atomic force microscopy (AFM) is shown. (B) AFM image of extended 3WJ RNA NPs. The RNA complex as shown in (A) on the left is estimated to be around 10 nm in size. Due to convolution of the tip size (∼10 nm in diameter) in AFM images, features smaller than the size of the tip cannot be resolved. To characterize the structure of the RNA constructs, the 3WJ NPs were extended by 39−60 base pairs (in red color, A, on the right), which is within the persistence length of dsRNA and will not affect the 3WJ folding as described before by Shu et al., 362 to generate the AFM image as shown. Adapted with permission from ref 360. Copyright 2015 Nature Publication Group. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2334 nucleic acids, 364 and insulin. 365 Nanodiamonds have also been demonstrated for covalent binding of proteins, sometimes enhancing their activity. 366 Similarly, inorganic NPs are frequently used as delivery vehicles. Among the inorganic nanocarriers, mesoporous silica nanoparticles (MSNPs) have emerged as a multifunctional platform that enables the development of custom-designed treatment modalities for disease-specific applications. One example is the use of MSNPs to address cancer-specific challenges such as the dysplastic stroma of pancreatic ductal adenocarcinoma (PDAC). The stroma serves as a treatment barrier due to inaccessible tumor blood vessels as well as the expression of cytidine deaminase, which is responsible for rapid degradation of the first line chemotherapeutic agent, GEM. It is possible to use a variety of MSNP designs to address the stromal barrier, including drug delivery to decrease the stromal abundance or negotiating vascular access by interfering with the action of pericytes that closely adhere to vascular fenestrations in the stromal blood vessels (see Figure 21A). 367−370 In one formulation, a MSNP carrier was developed to deliver a small-molecule inhibitor of the TGF-βreceptor kinase, which is responsible for providing the cellular signal for the adherence of pericytes to endothelial cells. 371 This was accomplished by placing a polycationic polymer on the NP surface to interact with the small molecule inhibitor via hydrogen bonding. Under acidic conditions, the small molecule inhibitor was rapidly released in the tumor stroma. When used as a first-wave carrier, this NP construct opened vascular fenestrations within 1−2 h, thereby making it possible for a second-wave carrier (e.g., a liposome) to deliver GEM to the tumor site (see Figure 21B). 371 Another formulation included the development of a dual delivery carrier, where a lipidbilayer-coated MSNP (LB-MSNP) was used to codeliver a synergistic GEM/paclitaxel combination to the site of orthotopic human tumor implants in the pancreas of a murine model. 372 This dual-delivery MSNP carrier was ∼10×more potent than Abraxane, which recently received FDA approval in combination with GEM. Not only did the ratiometric-designed carrier suppress the tumor stroma, but it also decreased the expression of cytidine deaminase, enabling increased uptake of activated GEM at the cancer site (see Figure 21C). 372 The LB-MSNP could also be used with considerable efficacy to deliver irinotecan to the pancreatic cancer site. This result can be achieved using a remote loading method in which the drug is imported for high loading efficiency across the lipid bilayer through the use of a proton gradient. 373 This carrier was more effective than a liposomal equivalent that was recently approved by the FDA, but it also improved safety because of its more stable drug retention. Together, these examples demonstrate the use of multifunctional MSNPs for the treatment of pancreatic cancer. Naturally there are many more types of NPs used for delivery applications, such as CNTs, graphene, and also the already mentioned inorganic NPs such as SPIONs, plasmonic NPs, etc. However, due to their sheer number, it is impossible to describe them all in this Nano Focus. Targeting. Targeting of NPs refers to delivery to specific tissues and cells in vivo. Nanoparticles can be designed to be stealthy to escape immune clearance and avoid nonspecific cell uptake but should also be capable of being sticky to target tissues and interacting with and/or be taken up by desired cells. Nanoparticles can passively or actively accumulate in the desired tissue via transport through leaky vessels and unique intraorgan pressures, or adhere to specific biological structures in the targeted tissue via molecule recognition to surface-bound ligands. 374 Recently, researchers designed smarter NPs that can turn from stealthy to sticky. Switching between these two properties may enhance the biodistribution and targeting of NPs to diseased tissues. Researchers have found inspiration in nature by using principles of viruses, transport proteins and their interactions with cell membranes, to achieve “stealthy and sticky”properties. Mimicking envelope-type viruses, different envelope-type 375,376 or shell-sheddable nanocarriers 377,378 have been explored to enhance the distribution of NPs in target tissue. Polymers with strong antifouling properties, such as PEG or zwitterionic polymers, were tethered onto NPs by cleavable anchor groups that are responsive to different stimuli, e.g., pH, redox, and proteolytic enzymes. The NPs with the stealthy shell had prolonged circulation times and enhanced accumulation in solid Figure 21. (A) Schematic to show the barriers and challenges that are responsible for failed chemotherapy in PDAC. This includes abundant dysplastic stroma, which serves as a physical and biological barrier, interference in vascular access and the presence of a high local concentration of deaminase activity, which leads to in activation of GEM. Trichrome staining of human PDAC is also shown: blue staining is collagen deposition. (B) Two-wave approach for PDAC treatment. PEI/PEG-MSNP binds to the TGFβinhibitor, LY364947. The complexation is highly stable in the physiological conditions, but can be disrupted in the acidic stromal environment. NIR-labeled particles retention was increased 10-fold prior to injecting the TGFβi carrier into mice with BxPC3 xenografts (circle), with significantly decreased pericyte coverage on endothelial cells in tumor blood vessel. Reprinted from ref 371. Copyright 2013 American Chemical Society. (C) Schematic describing GEM trapping in MSNP pores, which are sealed offby the LB that contains a subtoxic dose of paclitaxel (PTX). Ratiometric codelivery of GEM/PTX by LB-MSNP inhibits PANC-1 orthotopic tumor growth through increased delivery of GEM at the tumor site. GEM/PTX loaded LB-MSNP leads to CDA inhibition in parallel with increased oxidative stress. Adapted from ref 372. Copyright 2015 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2335 tumors due to the EPR effect, i.e., passive targeting. The unique stimuli within the targeting tissue enable the NP to release the shell, thus converting from stealthy into sticky. In a similar direction, zwitterionic surfaces containing mixed positive and negative charges have received great attention due to their unique stealthy properties and their similarity to proteins. Many proteins behave stealthily to most cells and they can have long circulation times in vivo. However, they can alternate between “stealthy and sticky”under even a slight stimulus such as a minor change in pH. 379 Although the detailed mechanism of this change has not been elucidated and might indeed be complex, the zwitterionic properties of amino acids and the mixed-charge structures of proteins might contribute to this feature. By exploiting pH changes in different structures (e.g., in tumor tissues, 6.5−6.8; in endosomes, 5.5−6.8; and in lysosomes, 4.0−5.0), zwitterionic polymer-based NPs, capable of altering their charge densities in different tissues, can be prepared. This responsiveness involves introducing a surface moiety sensitive to tumor extracellular acidity (pH ∼6.8) 379 as the anionic part of the zwitterionic polymer (see Figure 22). Nanoparticles can also be covered by neoglycoconjugates, or sugar-like functional groups and other functionalities such as thiol groups, to convert the NPs from stealthy to sticky. Though typically uncharged, such glycan-based ligands provide extremely high stability in biofluids, while minimizing nonspecific cell internalization. Whereas much research has been carried out with nanometer-size AuNPs, 381 these moieties have only recently been shown to work for larger AuNPs with various morphologies, including nanorods. 375,382 Interestingly, even though the stealthiness was shown to be superior to that of PEG ligands, specific protein-binding was retained, enabling the AuNPs to target cell types that overexpress certain protein receptors. The NPs thus exhibit prolonged circulation times due to the pronounced antifouling properties of the zwitterionic polymer. However, when the NPs are in an acidic tumor environment, surface charges change from negative to positive, leading to enhanced cellular uptake and accumulation of the NPs within tumor tissue. Compared with the NPs that switch their charge states by cleavage of the responsive covalent bonds, charge conversion of weakly electrolytic groups at different pH proceeds rapidly and better mimics the changes in charge density of amino acid segments within proteins. Surface engineering of inorganic NPs via mix-charged ligands can enhance NP retention in tumors by modulating the aggregation of NPs. 380,383,384 The mixed-charge modified AuNPs present not only a transition from stealthy to sticky under the acidic extracellular pH of solid tumors, but also a significant aggregation of NPs with much higher tumor accumulation, retention, and cellular internalization. Aggregation of AuNPs can also shift the surface plasmon band to the near-infrared wavelengths for photothermal therapy. 385 Moreover, recent studies on 3D spheroids of liver cells made in inverted colloidal crystals demonstrated that targeted nanotubes display unusually fast passive diffusion in the tissues. 386 Such an effect originates from the effective twodimensional (2D) translation of nanotubes over the surface of the cells in the tissues when the attractive forces between nanotubes are balanced with electrostatic repulsion. This finding suggests that nanoscale design of drug delivery vehicles can result in deep and effective penetration into the tumors. In addition to tailoring the surface properties of NPs, optimizing the sizes of NPs is also particularly important for tumor accumulation, penetration, and finally treatment efficacy, 387 particularly in targeting stroma-rich tumors. 388 By using micelles, ranging from 20 to 300 nm with the same chemical structure and physical properties, Wang et al. found that the blood circulation time and tumor accumulation of micelles increased with an increase in their diameters; with optimal diameters ranging from 100 to 160 nm. However, higher tumor accumulation of the large micelles (100 nm) did not result in significantly improved therapeutic efficacy, because larger micelles had poorer tumor penetration than did smaller micelles (30 nm). An optimal size that balances drug accumulation and penetration in tumors is critical for improving the therapeutic efficacy of nanoparticulate formulations. 387 The size effect is more pronounced in pancreatic tumors, which usually have thicker stroma and hypovascular characteristics that limit NP penetration. In the same direction, Kataoka and co-workers discovered that tuning the size of polymeric micelles in the range below 50 nm significantly improved the penetrability into stroma-rich pancreatic tumor and consequently improved antitumor efficacy. 367 In addition, 30 nm polymeric micelles loaded with cisplatin have proceeded onto Phase III clinical trials for the treatment of pancreatic cancer patients by extending survival times in Phase I/II compared to standard treatment using GEM. Apart from passive targeting, as described above, there are also exciting recent developments in actively targeting NPs. As one of the early studies in designing integrin-targeted nanomedicines, RGD-modified stealth liposomes achieved higher intracellular levels of DOX. 389 In recent years, more novel ligands against various tumor targets have been employed by making use of different targeting motifs, including alpha-conotoxin ImI, PHSCNK, lanreotide, octreotide and chlorotoxin for alpha 7 nAChR, integrin α5β1, somatostatin receptors, and chloride channels. 390−394 However, receptor redistribution induced by ligand binding decreases receptor affinity for ligands. A special dimeric RGD with proper distance between two RGD motifs was used to circumvent this problem. 395 On the other hand, an electrically neutral but hydrophobic penetration peptide was introduced to modify NPs against different breast cancer cell phenotypes irrespective of their receptor expression, displaying lasting accumulation and better inhibition of the tumor. 396 Still, there are ongoing discussions about the benefits of active versus passive targeting. 34,397 Besides ligand-mediated active targeting, active physical targeting has also been demonstrated. Magnetic focusing can Figure 22. Interchange between “stealthy and sticky”for zwitterionic polymer micelles (left) and the mix-charged AuNPs (right). Adapted with permission from ref 379 and 380. Copyright 2012 and 2014 John Wiley & Sons, Inc. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2336 enrich iron oxide NPs in the tumor region. Materials most commonly used for magnetic drug delivery contain metal or metal oxide NPs, such as SPIONs. Superparamagnetic iron oxide NPs consist of an iron oxide core, often coated with organic materials such as fatty acids, polysaccharides, or polymers to improve colloidal stability and to prevent separation into NPs and carrier medium. 398,399 For some applications, a permanent magnet is placed in the targeted region prior to the administration of magnetic NPs. This has been done for ferric steel implants, which were placed in the subarachnoid space in an in vitro human spine model prior to NP administration into the spine. 400 Similarly, dilute microferrimagnetic wires implanted within blood vessels in combination with an externally applied magnetic field can provide specific enrichment of simultaneously administered ferromagnetic NPs. 401 The challenge of NP enrichment in deeper body regions was addressed by He and co-workers, who deployed Halbach-like magnet arrays. 402 Nacev et al. also developed a sophisticated approach based on fast magnetic pulses on ferromagnetic rods that led to reversing the sign of the potential energy term in Earnshaw’s theorem. This process enabled a quasi-static, stable trap between magnets, resulting in the concentration of ferromagnetic rods on a central target. 403 These concepts are still far away from clinics. Magnetic drug targeting (MDT), based on intra-arterial administration of drug-loaded SPIONs, is closer to application in patients. This technology involves enriching SPIONS via a strong external magnetic field. A proof-of-principle preclinical study was conducted to demonstrate successful application of SPIONs for cancer treatment. 404 Simultaneous injection of Mitoxantone−SPIONs into the tumor-supplying vessel in rabbits and the application of a strong external magnetic field over a VX2 squamous cell led to complete tumor remissions without side effects. By applying only 5−10% of the conventional chemotherapeutic dose, complete tumor remissions were achieved. The distribution profile after MDT displayed 57.2% of the total recovery of administered drug: with 66.3% of the particles localized in the tumor region with magnetic targeting, compared to less than 1% of drug and NPs reaching the tumor region during conventional intravenous application. Magnetic drug targeting enables better enrichment of drug formulations and, consequently, enables more specific treatment. The majority of magnetic material is based on magnetic NPs either encapsulated or coated by different inorganic core/shell coatings 405 or by various polymer-based matrices, which are from natural 406−408 or artificial origin. 409,410 To translate these magnetic nanoparticles for clinical applications, the development of methods for largescale synthesis is required. A promising approach to synthesizing fatty acid-coated SPIONs, shielded with albumin and functionalized with adsorptive mitoxantrone, was developed with easy scale-up synthesis pathways in order to obtain antitumoreffective magnetic NPs. 411 Nanoparticles derived from this process have proven to be stable under appropriate storage conditions. 412 Delivery of Macromolecular Biopharmaceuticals across Biological Barriers. Nanomedicines are also being developed for diagnosing and treating infectious, neurodegenerative, or cardiovascular diseases. 413 Anticancer drugs are typically small organic molecules; however, the number and impact of macromolecular biopharmaceuticals (i.e., polypeptides and polynucleotides) are increasing enormously. Their size and complexity impose significant challenges to their use as safe and effective medicines, including fast elimination and poor metabolic stability. Furthermore, this class of compounds notoriously suffers from limited permeability across biological barriers, such as cellular membranes or epithelial tissues (see Figure 23). 414 On the other hand, their medical use offers not only the ability to ameliorate symptoms, but also the opportunity to cure or to prevent diseases, either by directly correcting hormonal disorders (e.g., insulin in diabetes) or by correcting, inhibiting, or reprogramming genetically disordered cells. The delivery of macromolecular biopharmaceuticals, preferentially via noninvasive (“needle free”) approaches, is therefore another important future application of nanomedicines. This approach takes advantage of the fact that NPs can both stabilize and protect the therapeutic cargo, while translocating across epithelial tissue barriers like those of the gastrointestinal (GI) tract, skin, or lungs. 415 Similar to the EPR effect, which occurs at the endothelium of tumor blood vessels, NPs can also accumulate in inflamed mucosal areas. This phenomenon was demonstrated recently by Figure 23. Cellular and noncellular barriers of the lung: after landing on lung lining fluids, (1) the drug (or eventually entire NPs) must cross the pulmonary epithelium (2) in order to reach the underlying tissue or the systemic circulation, respectively. Besides, inhalation, nanopharmaceuticals must also overcome effective clearance processes (3) provided by either mucus in the central lung (bronchi) or by macrophages in the peripheral lung (alveoli). Reprinted with permission from ref 414. Copyright 2014 Elsevier. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2337 tumor sphere formation and abrogation of tumor initiation and metastasis, probably due to its selective toxicity to regular tumor cells only but not to CSCs. 520 However, the nontoxic Gd@C82(OH)22 NPs selectively target CSCs but not regular tumor cells under hypoxic conditions. Such effects have not been observed in normal stem cells. The direct use of nontoxic nanomedicines with new acting mechanisms like “caging cancer” instead of killing cancer can be considered a potentially revolutionary shift in cancer therapeutics in clinical applications. 521 In the future, such strategies based on shutting down the tumor infrastructure will gain more importance due to their reduced side effects. Despite the broad medical utility of this NP system, the use of Gd-based nanomedicine has recently faced controversy related to potential toxicity issues. 522,523 In another example, Cha et al. proposed ZnO NPs as a therapeutic agent since these NPs can be engineered to specific shapes that act like biomimetic enzyme inhibitors. 524 The ZnO NPs were able to inhibit the activity of beta-galactosidase reversibly in a shape-dependent manner (see Figure 28A). That inhibition was not the result of protein denaturation, but rather a subtler conformational frustration of the enzyme that more closely resembles the mechanism of naturally occurring enzyme inhibitors. Furthermore, Cha et al. demonstrated that the NP-based enzyme inhibition can be characterized using the classical Michaelis−Menten and Eadie-Hofstee formalisms (see Figure 28C−E). As many traditional pharmacological therapeutics are enzyme inhibitors, this work opens the door for the design of a new class of degradation-resistant inorganic NP enzyme inhibitors that can be particularly suitable for antibiotic resistant bacteria exemplified by methicillin-resistant Staphylococcus aureus (MRSA, see below). Another approach used to design NP-based drugs to induce apoptosis in malignant cells is based on the cross-linking induction of the surface noninternalizing receptors. Receptor cross-linking involves the biorecognition of high-fidelity natural binding motifs with cell receptors or with side chains of watersoluble polymers. The general design concept of drug-free macromolecular therapeutics isshown in Figure29. An important feature of these designs is the absence of low-molecular-weight cytotoxic compounds. The concept was validated for the treatment of non- Hodgkin lymphoma (NHL) using nanoconjugates that target CD20 (noninternalizing) receptors on B cells. The system is composed of two nanoconjugates: (a) an anti-CD20 Fab′fragment covalently linked to a biological moiety (oligopeptide or oligonucleotide) and (b) a polyHPMA grafted with multiple copies of the complementary oligopeptide/oligonucleotide. Exposure of human NHL Raji B cells to anti-CD20 Fab′-Motif 1 decorates the cell surface with peptides/oligonucleotides. Further exposure of the decorated cells to graft copolymer P-(Motif 2)xresults in heterodimerization or hybridization and triggers the cross-link between CD20 receptors and initiating apoptosis in vitro and in a NHL animal model in vivo. 525−527 Magnetic resonance imaging and flow cytometry analysis indicated that no residual cancer cells were detectable in longterm survivors (125 days). 527 Multimodality imaging studies investigating the interaction between cellular membranes and nanoconstructs confirmed that self-assembly plays a critical role in this highly specific therapeutic system. 528,529 This new paradigm demonstrated activity in the treatment of cells isolated from patients with chronic lymphocytic leukemia and mantle cell lymphoma. 530,531 This interdisciplinary strategy has potential to overcome rituximab-related resistance and become a new drug development pipeline for the treatment of NHL and other B-cell-derived hematological neoplastic diseases and autoimmune disorders. 532 Killing Cells with Inorganic NPs upon External Stimuli. Inorganic NPs can respond to external stimuli to elicit tumor cell death. In the following section, we describe three common examples of external stimuli-driven NP therapies, including photodynamic therapy (PDT), photothermal therapy (PTT), and magnetic hyperthermia. 533−535 In its simplest form, PDT relies on the interaction between a photosensitizer (PS), a light source, and oxygen. A PS is a nontoxic compound capable of absorbing light and producing ROS upon de-excitation, producing ROS. Photosensitizer excitation is usually achieved via one-photon transition between its ground state S0and a singlet excited state, S1. At this point, the molecule may return to its ground state via emission of a photon (fluorescence), but the excited PS may also generate a triplet state T1by intersystem crossing. The lifetime of T1is longer (microseconds) than S1(nanoseconds), enabling the system to react in one of two ways, called the Type I and Type II mechanisms. The Type I mechanism involves electron transfer between the PS and a substrate, yielding free radicals that can then react with oxygen to form ROS, such as superoxide radical anions. In the Type II mechanism, singlet oxygen is generated via an energy transfer between the PS and triplet oxygen. 536 Desirable characteristics of PSs are (i) high absorption coefficient in the spectral region of the excitation light, preferably in the NIR emission range if PSs are to be used in PDT of cancer states; (ii) a triplet state of appropriate energy (ET > 95 kJ mol−1) to enable efficient energy transfer to ground state oxygen; (iii) high quantum yield (QY) for formation of the triplet state and long triplet state lifetimes (τ>1μs); and (iv) high photostability. Organic dyes and aromatic hydrocarbons are one class of molecules that possess these properties. They include dyes such as rose bengal, eosin, and methylene blue that are effective PSs because they possess triplet states of appropriate energy. They have high singlet oxygen yields, with rose bengal Figure 29. Schematic of overall design and possible mechanism of drug-free macromolecular therapeutics for treatment of NHL. Adapted from ref 527. Copyright 2014 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2344 reaching a value of Φ= 0.71, 537 and strong absorption bands in the green region of the spectrum. Aromatic hydrocarbons such as napthalenes, anthracenes, and biphenyls have also been investigated as PSs. Studies found that the efficiency of energy transfer to triplet oxygen is greatly affected by the substituents on the biphenyl ring and by the polarity of the solvent. Finally, quinones are good PSs, with QYs in the range of 0.11−0.76, 538 but sometimes they lack strong absorption bands at wavelengths shorter than 600 nm. Porphyrins, phthalocyanines, and related tetrapyrroles are another class of molecules that were identified as excellent PSs. Porphyrins and their analogues are ideal PS candidates because they occur naturally in biological systems and therefore generally lack cytotoxicity in the absence of light. These compounds have high QYs and properties that can be easily tuned by changing the substituents on the macrocycle, but they are susceptible to photobleaching. Also worth mentioning are naphthalocyanines, which bear a second benzene ring on the primary phthalocyanine ring. This addition is responsible for an attractive red-shift in absorption from 680 to 770 nm. Chlorins, derived from the porphyrin structure through saturation of one or two double bonds, also have absorbance spectra red-shifted into the therapeutic spectral window. The most studied member of this class is hematoporphyrin, which has high singlet oxygen production QY of 0.65 but unfortunately only a weak absorption band at 630 nm (ε= 3500 M−1cm−1). Some transition metal complexes have been identified as efficient photosensitizers, having singlet oxygen QY comparable to the well-studied organic compoundsmethylene blue and rose bengal. Ru(II)tris-bipyridine and [Ru(bpy)3]2+, in particular, are effective singlet oxygen producers with a QY of 0.73 in oxygen-saturated deuterated methanol and 0.22 in water. 539 Lastly, little attention has been paid to semiconductorNPs as PSs. However, singlet oxygen production and subsequent cytotoxicity from photoexcited semiconductors such as TiO2and ZnO have been reported. 540 In addition to the aforementioned requirements, the application of PDT to the treatment of cancer or other pathological conditions places other requirements on potential PSs. First, the compound should have low dark toxicity (i.e., it should not be toxic to the organism in the absence of light). Second, it should selectively accumulate into the affected tissue in order to minimize side effects and maximize cytotoxic efficiency. In fact, the half-life of singlet oxygen in biological systems is <0.04 μs, translating into a radius of action of <0.02 μm. 541 Finally, strong absorbance in the red-NIR portion of the spectrum would be advantageous for the PS, as these wavelengths are able to penetrate the deepest into biological tissue (see Figure 30). The ability to excite a PS in this region would increase ROS production efficiency, lowering the number of scattered photons, and achieve maximum attainable treatment depth. Keeping in mind these requirements, PSs for PDT of cancer have been classified into three generations. (i) The first generation consists of hematoporphyrin derivative (HpD) and its analogues. Beginning in the 1970s, this compound was the first tested and authorized for clinical use. HpD (stage 1) is obtained from hematoporphyrin and is a mixture of ∼10 components. Descending directly from HpD and now one of the most widely used PSs, porfimer sodium (Photofrin, Sinclair) is HpD stage 2, which is HpD purified from its monomeric fraction via high performance liquid chromatography. Despite being the most studied, this group of PSs suffers from major drawbacks; their selectivity is poor and only 0.1−3.0% of injected PS is found in tumor tissue. 542 The cutaneous tissue can take up and retain these compounds for up to 10 weeks after intake, thus causing photosensitivity in the patient. Furthermore, their absorbance in the NIR optical window is weak, limiting the efficiency of light uptake. (ii) The second generation has been designed with the aim of overcoming previous limitations and includes structurally distinct compounds with long-wavelength absorption and high singlet oxygen QYs. Part of this generation is hematoporphyrin derivatives with enhanced solubility properties for faster clearance from the organism and chlorins. Meso-tetrakis(mhydroxyphenyl)chlorin (m-THPC/Temoporfin, commercialized as Foscan, Biolitech Pharma), for example, causes less photosensitization than Photofrin but is 100 times more photoactive. Other members of the group are purpurines, which have the disadvantage of being highly hydrophobic, and phthalocyanines. (iii) The third generation focuses on PSs with additional properties through the use of composite structures. Better selectivity toward the tumor tissue is often achieved by conjugation of PSs to monoclonal antibodies or other ligands that bind specifically to the surface of cancerous cells. Also, the use of delivery vehicles like proteins or NPs leads to improved overall hydrophilicity and higher PS loads. (iv) Emerging PDT systems target the development of mesoscale carriers, which can incorporate large numbers of sensitizer molecules. These mesoscale particles are surface modified to minimize removal by immunogenic response mechanisms and reduce nonspecific binding. Specific antibodies attached to these larger functional delivery particles offer advantages over simpler PSs, which have low absorption cross sections and which do not localize specifically to tumors. There are three main mechanisms by which PDT mediates tumor destruction: direct killing, vascular damage, and activation of an immune response against tumor cells. In the first case, the ROS generated by PDT can kill tumor cells directly. However, complete eradication is not always realized because of the nonhomogeneous distribution of the PS within the tumor, nonhomogeneous penetration of light, and the possibility of limited oxygen availability within the tissue due to the photodynamic process itself. Vascular damage, on the other hand, targets the tumor vasculature system and, therefore, the concentration of nutrients and oxygen that reach it. In this case, the precise action Figure 30. Relative absorbance of body tissue components such as water, hemoglobin (Hb), oxygenated hemoglobin (HbO2), and melanin. The window that will allow the deepest penetration is the 600−1200 nm region, in the red-NIR. Adapted from ref 547. Copyright 2001 Nature Publishing Group. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2345 mechanism is not yet fully understood, but a biphasic vascular reaction following PDT has been suggested, 543 namely, a first immediate response consisting of vasoconstriction and, after 3 h, a second long-term response characterized by thrombus formation. Both of these effects were correlated with delay or inhibition of tumor growth. Depending on the kind of pathology to be treated, the interval between the sensitizer administration and light exposure might be a deciding factor for one mechanism over the other. When a short interval (minutes) is allowed between drug and light administration, the sensitizer predominantly accumulates in the vascular compartment, thus favoring indirect tumor killing by vascular damage. Conversely, a longer interval (hours) between intravenous drug injection and light administration results in localization of the PS within the extravascular compartment of the tumor and subsequent direct tumor-killing action. Therefore, administration of PSs at multiple time intervals before light activation (fractionated drug-dose PDT) is found to be the most effective way to target both tumor blood vessels and tumor cells. More recent studies in the late 1980s and early 1990s reported infiltration of lymphocytes, leukocytes, and macrophages into PDT-treated tissue, indicating activation of the immune response. Differences in the nature and intensity of the inflammatory reaction between normal and cancerous tissues could contribute to the selectivity of PDT-induced tissue damage. Photodynamic therapy is a reliable tool for treatment of a variety of diseases, whose clinical use is common and well established. This process involves two major steps, the administration of a PS and the delivery of light. The administration of a PS can be topical or intravenous, depending on the pathology to be treated and the ease of access to the area: skin diseases that allow for topical application will require drugs with different characteristics than will diseases for which an intravenous approach is required. Along with vascular permeability and interstitial diffusion, chemical properties like molecular size, configuration, charge, and the hydrophilic or lipophilic character of the compound will all impact transport and retention of the PS. Conventionally, PDT is administered as a single treatment using the highest PS dose that avoids systemic toxicity and skin photosensitivity, and the highest light fluence rate that avoids tissue heating and photochemical depletion of oxygen. However, for reasons discussed above, there have been a few studies about fractionated PDT, in which the PS administration is spread over time. Preliminary studies have shown improved responses to this kind of metronomic PDT compared to single doses, most likely due to the simultaneous action of direct tumor cell killing and blood vessel damage. Light can be delivered by surface or interstitial irradiation. Surface irradiation includes cases where the light source is external to the body (e.g., treatment of skin lesions), or is placed within and illuminates the inner surface of a body cavity (e.g., a hollow organ or a surgical resection space). Interstitial treatments are usually carried out by placing an optical fiber that carries the light directly into the tissue. Typically, 1−5 W of usable power are required in the 630−850 nm range at irradiances of up to several hundred mW·cm−2in order to deliver treatments in tens of minutes. For this reason, there are three main classes of clinical PDT light sources: lasers, light-emitting diodes (LEDs), and filtered lamps (see Figure 31). Lasers are powerful and efficient but they are expensive single-wavelength devices where a separate unit is required for each photosensitizer. In the past few years, LEDs have become a viable technology for PDT, particularly for irradiation of easily accessible tissue surfaces. The main advantages over (diode) laser sources are the low cost and ease of configuring arrays of LEDs into different irradiation geometries. On the other hand, the relatively poor electrical-to-light conversion efficiency (less than 15%) remains an obstacle, because the generated heat must be removed. Finally, a number of lamp systems can be spectrally filtered to match any photosensitizer and they allow flexible geometries. Their drawback is that they are not suitable for endoscopic use because they can be efficiently coupled only into optical fiber bundles or liquid light guides (5−10 mm diameter). 544 Photodynamic therapy design is still a relatively young field and substantial improvements could come from better PSs: higher absorption in the optical window, larger extinction coefficients, more efficient singlet oxygen production, and better chemical and physical properties. Interesting new directions that could overcome some of the intrinsic limitations of current compounds are currently being explored. (i) In two-photon PDT, the PS ground state is able to absorb two photons of NIR light simultaneously, which can be administered in short pulses: the total energy absorbed is the same as that from one-photon absorption at λNIR/2 and the resulting photophysical and photochemical processes are the same. This two-photon absorption enables a much wider choice of PS absorbing at high extinction coefficients in the visible, while retaining the possibility of using light in the biological optical window. Also, the quadratic dependence of the two-photon absorption probability can be exploited to give more precise spatial confinement of the PDT effect by high-numerical-aperture focusing of the activating laser beam. One of the drawbacks of this technique is that, in order for the photons to be absorbed simultaneously, the light pulse must typically be shorter than ca. 100 fs, a factor that substantially increases the cost of light sources. (ii) Another concept currently being investigated is the use of molecular beacons to improve target selectivity. Molecular beacons comprise a targeting moiety, a PS, and, optionally, a quencher. In their simplest form, the targeting group is responsible for accumulation of a higher number of PSs in the cancer cells and therefore higher selectivity. Alternatively, a target-labile linker keeps a PS and a quencher in close proximity, so that FRET prevents activation of the PS. Upon interacting with the target, the linker is either broken or opened so that the PS and quencher are separated, enabling FRET-driven PDT action to occur. (iii) Finally, NP technologyprovides several Figure 31. Surgeons delivering laser light through an optical fiber for the treatment of an internal tumor. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2346 different novel approaches to PDT. A first approach is the use of NPs as PS carriers, a method already applied to many other imaging contrast agents and therapeutic compounds. The advantages are that the loading of PS molecules per NP can be high and that the NPs can impart new desirable qualities to the compound, such as greater hydrophilicity or targeting ability. Examples include the incorporation of PS into modified silica NPs or into lipoprotein NPs with antibody targeting capabilities, the decoration of AuNPs with PS, or the use of NPs that are themselves photoactive. Another approach is the use of NP−PS conjugates, where the NP serves as the primary light absorber that, through FRET, activates the PS. An issue with all these NP strategies, besides potential toxicity, is the delivery of the NPs or NP−PS conjugates to the target tissues, because the pharmacokinetics are strongly dependent on the NP properties. In contrast to PDT, which kills cells by the generation of ROS, hyperthermia kills cells by heat. Tumor treatment by heat is one of the oldest ways to cure cancer. The application of pyrogens induces endogenous hyperthermia, where heat is generated by a substance or agent that reacts with the human immune system. This was the standard treatment for inoperable tumors until the middle of the 20th century. After administration of various bacterial toxins (or later on, vaccines) and the subsequent feverish reaction, a significantly higher survival rate was observed. Because of the inability to control the induced fever and the insufficient outcome, it is now applied only rarely. 545 Modern hyperthermia can be performed using plasmonic NPs and light, as well as magnetic NPs and alternating magnetic fields. Inorganic NPs act as local heat sources, triggered by light or alternating magnetic fields. Hyperthermia can be used as a stand-alone approach, or in combination with certain chemotherapeutics. One of the most promising therapeutic strategies in nanomedicine is that of photothermal cancer therapy. 546 In this drug-free therapy, these NPs absorb energy at a wavelength of 700−1000 nm 547 and generate heat. They can burn offthe tumor when the irradiation occurs after the particles have accumulated in the tumor. Intensity of the incident laser irradiation, the nature of the NPs (i.e., the localization of the surface plasmon resonance), and the size of NPs are the factors that will determine the efficiency of the localized heating. It was shown at an in vitro level that such localized heating can be applied to kill cancer cells. 459,548−552 In vivo, the highly localized hyperthermic response induced by resonant laser irradiation results in tumor remission for subcutaneous tumors with high efficacy (see Figure 32). 553 Researchers have demonstrated both passive and active uptake of nanoshells, a type of heat-generating NPs, into tumors. 554,555 In addition to nanoshells, other plasmonic NPs such as gold nanorods and gold nanocages have been shown to be useful for hyperthermia applications. 549,556 In preparation for transition to clinical trials, the toxicity of nanoshells was investigated extensively. No toxic effects were found in this study. 557 Recent improvements in nanoshell design can enhance their clinical utility. The addition and integration of imaging functionalities with the therapeutic function of these NPs resulted in NIR-enhanced fluorescence and NIR-enhanced imaging of tumors. 558−560 Smaller diameter, multilayer NPs known as “nanomatryoshkas”, which also have NIR-absorptive responses, can enhance NIR fluorescence for emitters encased between their inner and outer gold layers. 561 These NPs have shown increased uptake in tumors relative to nanoshells and outstanding remission rates with active targeting of aggressive triple negative breast cancer tumors. 562 Local heat can also be produced by magnetic NPs upon exposure to alternating magnetic fields. 564−567 The basic precondition for effective magnetic heating is that the NPs have adequate magnetization values. Iron oxides (magnetite/ maghemite) are particularly preferred since they have been used as contrast agents in radiology for many years, e.g., in the detection of cancers in the liver and spleen. The generation of heat is based on NP morphology as well as on the energy dissipation during the NPs magnetization process. 568−570 This means that the magnetization reversal of NPs with core diameters larger than 100 nm is governed by the motion of magnetic domain walls. In contrast, cores that are smaller than 100 nm behave like single-domain NPs where no domain walls exist. Even smaller NPs behave as if they are superparamagnetic and the magnetization reversal proceeds either by rotation of the magnetic moment inside the core (Neel relaxation) or by mechanical rotation of the whole NP (Brown relaxation) during exposure to an alternating magnetic field. It has been demonstrated that sufficient thermal energies can be obtained with NPs with core sizes between 10 to 50 nm. 571−573 Concerning NP synthesis, the current challenge is the stabilization of NPs with a One of the most promising therapeutic strategies in nanomedicine is that of photothermal cancer therapy. Figure 32. Evaluation of tumor response to PTT by bioluminescence imaging. The bioluminescence signal is generated only in living cancer cells, as a result of luciferase activity. (A) Representative mice of each experimental group showing the luciferase activity in the tumor. The mice injected with nanomatryoshkas or nanoshells and treated with laser experienced loss of bioluminescence in the area illuminated by the laser, as seen after therapy. Mice were euthanized when tumor volume reached 1500 mm3or if the tumor persisted at 60 days after treatment. (B) Luciferase activity in the tumor. The luciferase signal was normalized to the signal before treatment. Adapted from ref 563. Copyright 2014 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2347 relatively larger core size distribution. 574 Promising materials in this context are magnetosomes derived from magnetotactic bacteria, which were successfully deployed in a mouse breast cancer model. 575 To enhance treatment efficacy, coapplied chemotherapeutics or drug-carrying magnetic NPs are included. Cisplatin, in combination with magnetic heating, exhibits additive cytotoxic effects, particularly when it reaches its target site of action where it forms DNA adducts after its chloride ions have been displaced by hydroxyl groups. 576 Apromising NP formulation with cisplatin payloads was developed by Unterweger et al. 406 Most magnetic hyperthermia developments for treating diseases are in the preclinical stage with a few used in the clinic. Biodegradation. There is increasing awareness of the potential threats of using exogenous nanomaterials in vivo. In particular, the development of increasingly sophisticated nanocarriers further increases the risk of undesired side effects, discussed below. Therefore, it is highly desirable that drug delivery vehicles exert only minimal effects on the biological system after their cargo has been delivered, where the vehicles should ideally be eliminated after delivery to target tissues. Consequently, in vivo degradation with well-defined rates and with the formation of nontoxic and degradable byproducts that can be excreted becomes increasingly important. 577,578 Currently, there are a number of organic NPs that can be degraded, which will be discussed below. Water-soluble polymeric drug carriers are one such class of compounds that can be designed to be biodegradable. For these carriers, molecular weight and molecular weight distribution are important factors in their design, impacting both their therapeutic efficacy and their biocompatibility. The higher the molecular weight of the polymer carrier, the higher the accumulation of polymer−drug conjugate in the tumor tissue with concomitant increase in therapeutic efficacy. 331,579 However, the renal clearance threshold limits the molecular weight of nondegradable polymeric carriers (such as HPMA-based) to below 50 kDa. 580 This issue lowers the retention time of the conjugate in circulation with simultaneous decrease in pharmaceutical efficiency. Consequently, higher molecular weight drug carriers with nondegradable backbones deposit and accumulate in various organs, impairing biocompatibility. These concerns can be addressed by the incorporation of degradable polymer backbones, such as those seen in backbone degradable (second generation) HPMA copolymer carriers. These carriers have long circulation times while preserving biocompatibility and are based on the combination of controlled/living radical polymerization and click chemistry. 355,581,582 The backbone degradable HPMA copolymer− drug conjugates provide long-circulating water-soluble therapeutics that effectively accumulate in tumor tissue. They are eliminated from the body after enzymatic cleavage of the oligopeptide segments in the backbone (producing polymer fragments below the renal threshold) and in side chains (releasing the drug). The synthesis procedures proposed are versatile; they provide a platform for the preparation of a large variety of polymer−drug conjugates with tailor-made properties, such as predetermined circulation time and composition. Controlled degradationnonspecific cargo release from slow hydrolysis in physiological fluids like the bloodstreamremains a challenge. Therefore, design of biodegradable polymers requires incorporation of fast and efficient polymer degradation at the target site while retaining high stability in circulation and at off-target sites. Polypeptides offer great potential in this aspect, since their stability can be adjusted based on their monomer sequence, their molecular weight, or by attaching stabilizing or degradable side chains along the backbone. The most commonly used hydrophilic polypeptides have protonated amino side chains, e.g., poly(lysine), poly(arginine), poly(glutamate), and poly(aspartate), which ensure excellent water solubility and increase the interaction with the negatively charged cell membrane to facilitate cellular uptake. Amphiphilic block copolymers of two or three homopolypeptides have been prepared with hydrophobic segments such as poly(leucine) and hydrophilic segments such as poly(lysine) that form polymersomes and encapsulate hydrophobic drugs. However, highly positively charged polypeptides often reveal uncontrolled protein absorption, immediate immune responses, opsonization by macrophages, or rapid clearance from the bloodstream by renal or hepatobiliary excretion. To overcome these limitations while taking advantage of the aforementioned properties, innovative materials in biological systems that have a degradable but peptidic nature are highly desirable. Polymers based on the body’s own amino acid sarcosine (N-methylated glycine) have been obtained and are one of the first polymers synthesized by living ring-opening polymerization. 583 The polypeptoid polysarcosine offers high water solubility, nonimmunogenicity, 584−586 and protein resistance 587 due to its special characteristics, e.g., weak hydrogen bond acceptor with the lack of hydrogen donor capabilities. 588 Polysarcosine is a nonionic and highly water-soluble polymer that is similar to PEG but potentially offers in vivo degradability as suggested from in vitro studies under physiologically relevant conditions. 589 The synthesis of block copolymers based on polysarcosine and polylysine, polycysteine, polyglutamic acid, or derivatives thereof, by ring-opening polymerization of the corresponding α-N-amino acid carboxyanhydrides in a controlled fashion, offers high end-group integrity, precise control over molecular weight, and low dispersities of around 1.1. The amphiphilic block copolypeptides are nontoxic up to concentrations of 3 mg/mL and stabilize hydrophilic/hydrophobic interfaces in drug formulations and mini emulsion techniques. 589 The deprotected polymers polysarcosine-block-polylysine enabled the formation of core/shell polyplexes for efficient in vitro transfections. 590 Degradable polypeptide copolymers have been achieved from natural protein precursors. Unlike synthetic polypeptides, which consist of repeating amino acid sequences, the polypeptide backbone of proteins provides a higher diversity of reactive functional groups, which enables further chemical modifications. Moreover, depending on the precursor protein chosen, these polypeptides offer distinctive and adjustable molecular weights, precisely defined sequences, low cytotoxicity, and full degradability. They have been applied for coating fluorescent NPs as well as for the delivery of cytotoxic drug molecules. A challenge when using inorganic NPs is the ability of the animal to degrade and to eliminate them. Sub-5 nm NPs such as NDs can be eliminated from the body renally without biodegradation and without leaving toxic residues. 591 However, larger inorganic NPs are more difficult to clear from the body. Studies have shown that AuNPs reside in macrophages after uptake and do not appear to be degraded; 592 ZnS-CdSe QDs have also been shown to remain stable in mice and rats after several months. 593,594 There is evidence to suggest that some inorganic NPs (e.g., CdSe QDs and hollow AuNPs) can break down by oxidation or other chemical processes. This process is slow but there is evidence to show they are excreted through the ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2348 kidneys. 35,595,596 The chemistry and design of the inorganic NPs can influence the degradation pattern. Although the core composition is difficult to degrade for most inorganic NPs, the organic surface coating has been shown to degrade in vivo. This effect was demonstrated by using multiplexed radiolabels that tag the inorganic core and surface coating with an organic molecule. This labeling strategy enabled the measurements of differential temporal biodistribution of all different parts of a NP after in vivo administration. It was discovered that inorganic NPs stabilized by an amphiphilic polymer shell via amide bonds lose parts of the organic shell upon enzymatic cleavage in vivo. 37 Taking the concept further, a number of researchers have started to use organic molecules to assemble inorganic NPs into complex systems for in vivo medical applications. For example, DNA can assemble inorganic NPs into a complex core−satellite system that can target tumors, but these NPs can subsequently enter liver macrophages. 36 The enzymes in these cells degrade the DNA linker and release the inorganic NPs back into the bloodstream to be eliminated through the kidneys. Linker molecules to build nanosystems are not limited to DNA but can also be polymers that can potentially be responsive to enzymatic degradation. 597,598 The clinical translation of inorganic NPs may depend on whether they are cleared from the body, as the longterm toxicity of metal-containing NPs remains unclear. This research area will continue to be active from the fundamental (i.e., understanding the clearance mechanism of inorganic NPs) to the design (i.e., using assembly techniques to build degradable nanosystems containing inorganic NP components) perspectives. LABORATORY-BASED MATERIAL PRODUCTION FOR TREATMENT (“IMPLANTS MADE WITH NANOTECHNOLOGY”) While some nanomaterials are designed to deliver a drug or an action to diseased tissue and programmed to degrade afterwards, researchers are also synthesizing nanomaterials that can be introduced into the body with the intention “to stay”. Surface modeling on the nanoscale can provide new functions and properties to implants, etc. 599 Incorporation of nanomaterials with key features for driving tissue engineering is a useful means to achieve unprecedented performance. 600,601 In particular, carbon nanostructures (e.g., single- or multiwall CNTs (SWCNT/MWCNT), graphene, carbon nanohorns, NDs, fullerenes, etc.) can provide mechanical strength and useful electronic properties, thanks to their chemical structures. 602 They have been shown to be biocompatible with and to support the growth and proliferation of many classes of cells. Despite the issues associated with CNT use in medicine, 603 there is a growing body of work showcasing their utility in nanomedicine after appropriate chemical functionalization. 604 For example, CNT addition to hydrogels are biomaterials that can be designed to respond to diverse stimuli, e.g., for drug release applications. 605 Some selected examples and applications are detailed below. Nano- and Microengineered Implants. Nanodiamond− polymer composites can be used in tissue engineering and regenerative medicine, particularly showing potential for restoring damaged tissue. Excellent mechanical properties of NDs in combination with fluorescence, delivery of drugs and biologically active molecules, and biocompatibility are used for reinforcement of implantable polymers to create multifunctional tissue engineering scaffolds. Octadecylamine-modified NDs incorporated into poly L-lactic acid (PLLA) provided fluorescence for monitoring the implant biodegradation (see Figure 33). While biodegradable polymers such as PLLA are not mechanically robust, the incorporation of NDs brings the composite properties close to those of human cortical bone. It increased the composite’s mechanical strength and strain to failure, and enabled the use of the composite as a bioresorbable bone-growth scaffold for manufacturing bone fixation devices. 31 In terms of toxicity, murine 7F2 osteoblast morphology and proliferation were unaffected when cultured with the ND-PLLA. These composites possess clinically relevant properties while being nontoxic and highly scalable to produce. 606 Naturally, for such implants the interface between the implanted devices and surrounding cells and tissues is important. Selection of the geometry offers a useful handle in this direction. Elongated objects, such as nanoneedles, for example, have suitable properties for the delivery of bioactive agents to cells and tissues. This field of nanoneedles for cell-interfacing, which is still emerging, is now growing rapidly and shows great promise. A number of interesting nanoneedles have been developed recently with a multitude of capabilities. Some of these capabilities include transferring biological information to many cells in parallel, 607 sensing intracellular biological signaling such as the presence of enzymes, 608 and measuring intracellular pH at many points across individual cells. 609 The nanoneedles can deliver to the cell cytosol and yet, due to their nanoscale dimensions, are not destructive to the cells. These nanoneedles represent a highly promising technology for intracellular delivery, probing, and interfacing. Porous silicon-based nanoneedles were developed and utilized to deliver DNA and siRNA to cells in vitro efficiently and showed transfection efficiencies greater than 90% while silencing (of GAPDH expression) was quantified as 80% effective, 610 encouraging for in vivo applications. The authors showed that they could achieve neo-vascularization in the muscles on the backs of mice by using porous silicon nanoneedles to deliver human VEGF165 plasmid DNA. 610 This finding is of interest when considering the potential to prevascularize sites at which tissue engineered implants might be transplanted in vivo. It also has the added advantage that nucleic acids can simply be loaded into the nanoneedles and no covalent modifications are required. The nanoneedles are a platform technology that could potentially be used to deliver a wide range of nucleic acids and other bioactive agents for all types of tissues. Toward Interfacing Electrically Active Tissues. Electrical signals can be used to control the interactions of nanomaterials with tissues. This strategy to control tissue function may be important for many applications such as in the design of neuroprostheses. Below, we describe a number of these applications. Figure 33. Bone fixation screw made of a biodegradable polymer (PLLA) reinforced with octadecylamine (ODA)-funtionalized fluorescent nanodiamond NPs (ND-ODA) that provide reinforcement, monitoring of biodegradation, and, eventually, drug delivery. Adapted with permission from ref 606. Copyright 2011 Elsevier. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2349 First attempts for electrically interfacing cells were based on microstructured devices, 611−613 but more recently, nanostructured devices such as nanowires have been used. 614 Two criteria for building successful devices are “wiring”and size, where “wiring”involves the connection of the recording sites to the outside, 615 and size is essential for brain implants to avoid local inflammation. 616 Neuroprosthetic implants made from LbL biomimetic nanocomposites investigated by Kotov and coworkers provide a unique combination of properties that enable manufacturing of smallest possible brain implants that can be safely integrated within brain tissues. 617 Some of these properties include high strength, conductivity, toughness, and biocompatibility. 618,619 Additionally, 615−617 brain implants have been made with gold nanowires. 620 A major step forward in this field has emerged by exploring the interactions of CNTs with neuronal systems. Indeed, the presence of CNTs influences the electrical activity of neurons, especially in terms of synaptic events. In the past decade, it has been established that CNT-based substrates are able to impact neuronal physiology profoundly from the functional (electrical) point of view. With their exceptional properties of small size, flexibility, strength, inertness, electrical conductivity, and ease of combination with various biological compounds, CNTs are the excellent candidates for interfacing with damaged neuronal tissues. In 2000, it was found that neurons deposited on functionalized CNTs were not only surviving, but also elongating their neurites in all directions. 621 Since then, the study of these materials as functional components of composites for the support of the regeneration of neural tissue has been set up by many research groups. Carbon nanotubes have high electron mobility, have an affinity for neurons, and can integrate with cell membranes at both dendrites and somas. As a result of these tight interactions, not only neurons, but also entire 3D slices of spinal cord (SC) tissue exhibit desirable behavior. 622−625 The most important results obtained can be summarized in three points: (i) neuronal signaling is enhanced when brain circuits are cultured interfaced to CNT films, (ii) CNT networks are able to induce the formation of a higher number of new synaptic contacts with respect to reference substrates, and (iii) CNT interfaces promote nerve fibers regrowth, potentiating growth cone activity and synaptic responses in entire SC slices:this is an efficient translation from single cell to complex tissues such as SC explants. It has been found that neonatal hippocampal neurons, laid over a film of CNTs, increase their spontaneous electrical activity. Thereafter high-resolution electron microscopy elucidated the intimate contacts between regrown neuronal processes and CNTs where the boundary line between organic and inorganic matter is ambiguous. 623,625 The most outstanding results concerning the interface between CNTs and neurons are related to the effects on the electrical activity of neuronal networks. Lovat et al. compared the electrical activity of hippocampal neuronal networks directly grown on a MWCNT mat with that of networks grown on control substrate by means of the patch-clamp technique. 622 The frequency of spontaneous events (postsynaptic currents) in networks cultured on CNTs was strongly increased (approximately 6-fold) compared with controls. The balance between inhibitory and excitatory components in the neuronal network was not affected. 622 By means of single-cell electrophysiology techniques, electron microscopy analysis and theoretical modeling, it was hypothesized that CNTs can provide a kind of shortcut between the proximal and distal compartments of the neuron. 624 This hypothesis, supported by the observation that neuronal membranes establish tight contacts with the CNT substrate, was further corroborated by other experiments. When cells were forced to fire trains of action potentials, reverberations were detected following depolarization potentials and this reverberation occurred more frequently on CNT-deposited cells than on those grown on inert glassy supports. This type of back-propagating action potential represents a regenerative ability that neurons exhibit in cellular processes, such as the tuning of synaptic activity, the expression of spike-timing-dependent plasticity, the release of modulatory messengers and the modulation of synaptic plasticity. Another interesting observation concerns the impact of CNTs on the synaptic activity of neuronal networks. The probability of finding synaptically connected pairs of neurons is almost doubled in the presence of the CNT substrate. The synaptic plasticity was also affected because cells grown on CNTs demonstrate potentiated short-term synaptic conditions instead of normal depression in response to afferent presynaptic spike trains. This activity mimicked the high frequency flow of information. All this gain of function is entirely attributable to the peculiar features of conductivity and physiochemical properties of CNTs that affect the network activity and spike propagation. 624 The ability of CNT substrates to have an effect on central nervous system tissue has been tested by coculturing embryonic spinal cord and dorsal root ganglia multilayered explants chronically interfaced to a film of purified MWCNTs. 625 With respect to the controls, spinal explants cultured on CNTs displayed higher numbers of longer neuronal processes growing in tight contact with substrates bearing higher numbers of growth cones at their tips. These neuronal processes seemed to be slack on the CNT carpet that increased their contact surface but were less stiffthan in the control, apparently integrating CNTs as their own supports or exoskeletons. The overall interactions of the spinal tissue with the substrate appear to be intimate and similar to the results reported for isolated cell cultures. When sensory afferent pathways, preserved in spinal explants differentiated in vitro were exogenously activated, the resulting synaptic responses recorded from single neurons not in direct contact with the CNT layer were strongly increased. This result indicates that the boosting effects of CNTs at the interface were transferred from the layers of neurons directly exposed to the CNTs to those functionally connected yet physically far from the interface. In order to improve the biocompatibility of unconstrained CNTs, groups have tried to incorporate CNTs into polymeric scaffolds. In addition to improved biocompatibility, they aim to produce 3D structures that are able to be colonized by neuronal cells and to foster the communication among them, where the CNTs play strengthening and electrically functional roles. To this end, CNTs have been integrated into various biopolymerbased hydrogels as collagen, chitosan, 626 and agarose. 627 In general, all these substrates are good supports for neuronal cells, able to sustain their growth and their ability to extend neurites and growth cones without a remarkable toxicity. None of these scaffolds have yet been tested in vivo, however. Similar results have been achieved by CNT composites based on synthetic polymers, mostly polyester polymers such as electrospun fibers of PLGA 628 and of poly(L-lactic acid-co-caprolactone). 629 Bosi et al. have recently produced a nanocomposite system where CNTs are integrated into a polymeric matrix that can be modeled into different porosities, stiffnesses, and shapes. This nanocomposite consists of a microporous, self−standing, elastomeric scaffold whose skeleton material is made of PDMS, while the ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2350 micrometric cavities are generated by dissolving a sugar template embedded in PDMS. 630 This 3D structure showed viability as a supporting scaffold allowing neuronal colonization and 3D synaptic network reconstruction, but was soft enough to match the viscoelastic nature of native hippocampal neural tissue. 631 The PDMS porous scaffold surfaces was then enriched of MWCNTs, endowing the structure with nanofeatures and implementing its electrical conductivity. Because of their peculiar electrical features, CNTs are useful as interfaces with other electrically active tissues such as cardiac tissue. Martinelli et al. 632 discovered outstanding effects on cardiac cells cultured on CNT substrates. Neonatal rat ventricular myocytes (NRVM) were able to interact with nonfunctionalized MWNTs deposited on glass coverslips by forming tight contacts with the material (see Figure 34). Cardiac myocytes modify their viability, proliferation, growth, maturation, and electrophysiological properties when interacting with CNT scaffolds. Two opposing effects on the development appear to take place. Cells prolong the proliferative state, which maintains some cells in an undifferentiated state. They accelerate the maturation of the differentiated cardiac myocytes in terms of a more negative NRVM resting potential compared to the control so that the cells become more “adult-like”. By transmission electron microscopy it has been observed that CNTs establish irregular tight contacts with the membranes, in a way that is morphologically similar to neurons cultured on MWNTs. 622 It is not excluded that other modifications indirectly brought about by MWNTs, such as the deposition of the extracellular matrix (ECM) or the cell contact driven cytoskeletal dynamics, are ultimately responsible for the positive effect. Such an effect holds promise to solve the issue of arrhythmias arising from nonconductive scaffolds, as exemplified also by a chitosan-based hydrogel, where the inserted CNTs act as nanobridges that electrically connect cardiomyocytes. 633 Another interesting cardiac construct has been produced by Shin and colleagues: 634 CNTs were embedded into photo-cross-linkable gelatin methacrylate (Gel-MA) hydrogels, resulting in ultrathin 2D patches where neonatal rat cardiomyocytes were seeded. These cells showed strong spontaneous and stimulated synchronous beating. In addition, a protective effect against DOX (cardiotoxic) and heptanol (cardio-inhibitor) was observed. When released from glass substrates, the 2D cardiac patches (centimeter size) formed 3D soft actuators with controllable linear contractile, pumping, and swimming actuation behaviors. For heart tissue engineering, CNTs can also constitute a templating guide for the electrospinning of hybrid hydrogel nanofibers that form a superb biomimicking scaffold that is capable of matching the orientation index of the left ventricular tissue, thus promoting cardiomyocyte alignment and synchronous beating. 635,636 The high potential of CNTs in the field of excitable tissue engineering stems from their exceptional nature and the desire to develop synthetic materials with appropriate nanostructure and cell-recognition properties that are able to regulate cell adhesion, survival, and growth. The in vivo application of nanotechnology tools and of CNT-based platforms, in particular, is largely directed to basic research applications. However, the extraordinary effects of these materials hint at developing safer devices to promote tissue reconstruction. A major challenge for biomedical applications of CNTs is to overcome the current limitations of CNT degradation time, elimination, and/or safety of use. Biological Cell-Based Implants. Implants used for regenerative medicine can also be of biological origin. Cellbased therapies offer great potential. For example, stem cells delivered to injured tissue have the potential to regenerate defective tissue. 637,638 One general problem with cell-based therapies, however, is that individually delivered cells have limited retention times in the target tissue. In order to improve retention times, cells can, for example, be delivered in already connected form (so-called cell patches), or they can be seeded into matrices to which they have high binding affinity. Treatment based on connected cell patches instead of isolated cells thus may offer decisive advantages. However, it is not trivial to cultivate cell patches, as at one point connected cells need to be isolated from the culture substrate. Cell adhesion typically can be suspended by enzymatic treatment (e.g., with trypsin) or by depletion in calcium. However, while this enables the collection of cells from culture substrates it also interferes with intracellular contacts. Nanotechnology has helped create culture substrates that enable switching on and offadhesive properties to cells. For example, smart surfaces enable switching from hydrophobic to hydrophilic properties upon changes in temperature. In this way, whole cell patches can be detached from the culture substrate and can be used for transplants. 639 Also, plasmonic heating may be a useful technique in this direction. 640 Such advanced culture methods pave the way for future cell and tissue-engineering therapies. Cell sheet patch therapies were clinically applied to human patients suffering from diseases such as cornea epithelium deficient disease, recovery from endoscopic submucosal dissension surgery for esophageal epithelial cancer, cardiomyopathy, 641,642 periodontal regeneration, 643 and osteoarthritis. 644 While the number of cell layers grown on top of each other within one cell patch is limited as transport of nutrients and oxygen to the cells needs to be warranted, 645 subsequent transplantation of several cell patches of up to ∼100 μm thickness is possible and vascularization of the transplants has been demonstrated. 646,647 Cells can also be seeded into matrices resembling replicas of cell surfaces, 648 into decellularized matrices, 649 or even into completely synthetic scaffolds to improve their biocompatibility and engraftment. 650,651 There have been some attempts for translation to the clinic, 652,653 but results have been and are still heavily scrutinized. 654−656 The basic requirements such transplants need to fullfill involve vascularization, in order to warrant continuous blood supply, 657 and, in case of exposure to the environment (e.g., air, food), antimicrobial coatings. Severe skepticism toward the use of these technologies in general is reported. 658 Figure 34. Left: Scanning electron microscopy (SEM) image of a spinal explant peripheral neuronal fiber on a MWCNT substrate. Note the tight and intimate contacts (red arrows) between the neurite membrane and the MWCNTs. Scale bar: 500 nm. Reprinted from ref 625. Copyright 2012 American Chemical Society. Right: SEM image of cardiac myocyte deposited on a layer of MWNT. Adapted from ref 636. Copyright 2013 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2351 Toward Artificial Organs. A current need in medicine is with the construction of whole organs in vitro to address the issue of shortages in organ donation. 659 Nanotechnology is emerging as a powerful tool that can help to fulfill this need by assisting in the engineering of artificial organs for regenerative medicine as well as organs-on-a-chip applications. 660,661 The need for nanoscale architecture is derived from the ability of cells to sense the nanoscale structures in their microenvironment. 662 In particular, many ECM molecules that surround cells form nanofibrous structures that help to organize cellular architecture and to induce directional migration and alignment. 663 As outlined in the case of implants, one approach to engineer such nanoscale structures is through the generation of 3D-fibrous scaffolds. 663−665 Techniques like electrospinning and microfluidic-based fiber fabrication have emerged as methods to engineer 3D scaffolds for inducing cellular organization and alignment. 666−669 Furthermore, self-assembling peptides have been used to form nanofibrous structures, which mimic the complex nanoscale architecture of native ECM molecules. Utilization of biomimetic composite coatings made using LbL assembly on inverted colloidal crystal scaffolds on ceramics, 670 gels, 671,672 PLGA polymer 673 enabled, for instance, the creation of ex vivo models of bone marrow, 674 thymus, 675 and liver. 676 One of the advantages of the ex vivo organs is that they enable multifaceted evaluation of the toxicity of NPs in human tissues in the cellular constructs replicating real organs. 677 In addition, nanomaterials can be integrated into hydrogels to direct cellular behavior. For example, the integration of nanoscale silicate materials can induce the directed differentiation of mesenchymal stem cells (MSCs) into osteogenic fates. 678 Polymeric nano- and microparticles that release growth factors and cytokines in a controlled manner can be integrated into scaffolds to regulate the surrounding biology. For example, controlled release of angiogenic factors, such as VEGF and PDGF, through polymeric NPs can induce the formation of blood vessels. Other types of nanomaterials such as CNTs and graphene NPs can be incorporated into hydrogels to change their mechanical and electrical properties, as described above. The resulting gels have been shown to enhance the formation of cardiac and skeletal muscle tissues. 634,679,680 Nanoscale tools also provide means to form tissues with controlled microarchitectures. For example, cell-laden hydrogels can be induced to assemble into particular structures that better mimic the microarchitecture of tissues in the body through the use of nanoscale “programmable glues”. 681 In this approach, the various sides of microgels of controlled shapes were coated with adhesive, long, single-stranded DNA where the function of the strands is to induce programmed assembly. It was demonstrated that the resulting DNA strands form nanoscale objects on the surface of gels that uniquely recognized their complementary base-paired strands on other gels to induce assembly. Such structures could 1 day provide scalable ways of engineering tissue structures with predefined architectures. These and many other examples have shown the power of nanotechnology to direct cellular behavior for inducing tissue formation. It is anticipated that with our increased knowledge of how nanoscale objects interact with cells along with our ability to engineer more controllable nanomaterials will lead to a new era of nanomedicine for regenerative medicine applications. Antibacterial Coatings. Biofilm formation and dissemination of antibiotic-resistant bacteria by indirect transmission through contaminated surfaces is becoming a major global public health threat. Steel and aluminum surfaces, commonly present in hospital settings as knobs, buttons, frames, taps, etc. possess limited or very low antimicrobial activity. A promising approach for limiting indirect transmission is the realization of stable and effective antimicrobial surfaces obtained by nanostructured coatings. Similarly, infections associated with medical devices (catheters, implants, etc.) have become a major concern. The adhesion of bacteria to the devices’surfaces during the initial 24 h is believed to be a decisive period for implantassociated infections and the design of optimal antiadhesion of microbes in this period is key. Multilayer coating by LbL selfassembly provides a robust method to design multifunctional dynamic antibacterial surfaces. For example, a multilayer of (heparin/chitosan)10−(polyvinylpyrrolidone/poly(acrylic acid))10 [(HEP/CHI)10−(PVP/PAA)10] provides the properties of contact killing of bacteria due to the underlying (HEP/CHI)10 while the top (PVP/PAA)10 multilayer film shows a top-down degradable capability in the determined period. This activity leads to almost no adhesion of bacteria within 24 h. The combination of the adhesion resistance and the contact-killing properties shows enhanced antibacterial capability through targeting the “decisive period”of implantation and may have potential for applications in medical implants, tissue engineering, etc. 682 Surfaces with immobilized antimicrobial peptides present high and broad antimicrobial activity. 683 Similarly, a topic worth exploring involves immobilizing antimicrobial ligands onto NPs to enhance their antimicrobial activity. A quaternary ammonium cationic surfactant, didodecyldimethylammonium bromide (DDAB), was immobilized onto silica NPs to develop antimicrobial NPs. These NPs revealed much lower minimal inhibitory concentrations against bacteria and fungi than the soluble surfactant. The electrostatic interaction of the DDAB with the NP is strong since no measurable loss of antimicrobial activity was observed after suspension in aqueous solution for 60 days. This result means that the antimicrobial activity of the NP does not require the leaching of the surfactant from the surface of the NPs. More interestingly, the NP properties expanded to virucidal activity. The versatility, relative facility in preparation, low cost, and large antimicrobial activity of those NPs makes them attractive as coatings for biomedical devices. 684 Suspensions of ZnO NPs are effective against a broad spectrum of microbial organisms 685−687 but there are many misconceptions about their mechanism(s) of killing bacteria. 524 Generation of ROS was often considered to be the source of antibacterial activity. 688 However, according to analysis of the mechanism of bacterial inhibition by ZnO NPs of different shapes, 524 at least part of the antibacterial activity originates from enzyme inhibition. Anisotropic ZnO and potentially other NPs can mimic biological inhibitors by partially matching their geometries with those of the protein inhibitors. Such activity originates from the similarity of the surface chemistry and size of water-soluble NPs and those of many globular proteins. 6 Applerot et al. used ultrasound irradiation to generate ZnO NP-coated surfaces that resist bacterial biofilm formation. 689 Layer-by-layer coatings of ZnO NPs reduce biofilm growth of MRSA by 95%. Silver in bulk and nanomaterial forms has been A current need in medicine is the construction of whole organs in vitro to address the issue of shortages in organ donation. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2352 used extensively as medical device coatings but these devices have not lived up to the promise of their in vitro data. 690 Silver’s antimicrobial function is based on the concentration of silver ions and their associated toxicity. 691,692 Unfortunately, the therapeutic window between efficacy and toxicity for silver is quite narrow, which has led to disappointing clinical effectiveness of silvercoated medical devices. 690 ZnO is significantly less expensive than silver and has selective toxicity for bacteria over mammalian cells. 693,694 ZnO is generally recognized as safe by the FDA. Nanoparticles can be designed with antibacterial coatings to circulate in the blood to target bacterial infections post-tumor surgery. During chemotherapy, some patients may be more susceptible to bacterial infections. 695,696 This susceptibility cannot be addressed using antibiotics alone, however, as some antibiotics may lead to serious side effects for some patients postsurgery. Abuse of antibiotics has caused the emergence of more resistant and virulent strains of pathogens. As a result, in some cases, clinicians need to prescribe multiple antibiotics in addition to anticancer drugs before and after the surgery. Particles can be engineered with antibacterial properties to target bacteria in vivo specifically, which can potentially minimize resistance. For example, a polymer vesicle antibacterial drug carrier has been developed recently. The chitosan-based carrier is grafted with an antibacterial polypeptide and demonstrated to kill both Gram-positive and Gram-negative bacteria (see Figure 35). This polymer vesicle has excellent blood compatibility and low cytotoxicity, which may benefit patients after tumor surgery. 697,698 The enhanced antibacterial efficacy of polymer vesicles compared with an individual antibacterial polymer chain results from the high concentrations of local positive charges in the polymer vesicles. IN VIVO COMBINATION OF DIAGNOSIS AND TREATMENT (“THERANOSTICS”) Theranostics involves the combination of both diagnosis and treatment. Image-guided tumor resection is one such application that uses theranostics. The diagnostic use of NPs to label cancer cells, in particular tumor borders and small metastatic regions, can be used to guide the surgeon for subsequent therapeutic surgical removal of the tumors. 699 These diagnostic NPs could also be loaded with therapeutic agents for treating postsurgery. Another interesting application of theranostic agents is NPs that can deliver therapeutic agents into tumors while tracking NP drug delivery. 700 These agents enable noninvasive imaging to provide information on the fate of the drug once they are administered in vivo. Theranostics research is an exciting area of research as many different combinations of diagnostic and therapeutic strategies can be built into NP systems. Image-Guided Surgery. Recently, first-in-human pilot studies have successfully used both passive and active targeting agents for image-guided surgery of human tumors. 701 The specific tumor types studied include breast, lung, ovarian, and pancreatic cancers. Although still preliminary, the results have helped clarify two important issues: (i) most human tumors (but not all) have moderately leaky vasculatures, so the EPR effect provides a general means for passive delivery of imaging agents (5−6 nm albumin-bound indocyanine green) to a broad range of human tumors; (ii) fluorescent dyes such as fluorescein can be conjugated to targeting ligands such as folic acid for specific targeting and high-contrast imaging of human tumors. In the first approach, passive fluorescent agents do not involve targeting ligands, but rely simply on the biodistribution of the free fluorophore. Singhal and co-workers conducted a small trial on nontargeted NIR dyes, enrolling 5 patients undergoing surgery for removal of their cancers (3 lung nodules, 1 chest wall mass, and 1 anterior mediastinal mass). 701,702 Two surgeons reached a consensus about the clinical stage and operative approach prior to surgery. All enrolled patients were thought to have limited disease, be amenable to surgery, and have no metastases (i.e., potentially curable). Patients were injected with indocyanine green (ICG) prior to surgery. 703 At the time of surgery, the body cavity was opened and inspected. The results demonstrated that NIR imaging can identify tumors from normal tissues, provides excellent tissue contrast, and facilitates the resection of tumors. 701 In situations where there is significant peritumoral inflammation, NIR imaging with ICG is not helpful. This suggests that nontargeted NIR dyes that accumulate in hyperpermeable tissues will have significant limitations in the future, and receptor-specific NIR dyes may be necessary to overcome this problem. In contrast to passive agents, active fluorescent agents are linked to targeting ligands that are designed to recognize specific receptors at the target tissue. Near-infrared-dye-labeled antibodies (MW∼150 kDa) that are currently used in the clinic for targeted cancer therapy, such as VEGF antibody (bevacizumab), EGFR antibody (cetuximab), and HER2 antibody (trastuzumab), have been evaluated as targeted optical imaging probes in preclinical animal tumor models. 704−706 Several antibody-based imaging probes are approved for clinical trials for image-guided surgery. 707 Potential limitations for antibody-based optical imaging probes include increased background fluorescence resulting from relatively long blood circulation times, as well as low efficiency in penetrating through tumor tissues. 704 To address these issues, smaller antibody fragments, such as affibodies (MW∼7−8 kDa) against HER2 or EGFR, were labeled with NIR dyes for targeted optical imaging. Specificity and sensitivity of those affibody optical imaging probes have been demonstrated in various animal tumor models, 704,708−710 with several groups in the process of conducting clinical trials. For intraoperative imaging of tumor margins, a class of protease activatable optical imaging probes can detect tumor edges since many proteases are highly expressed in the invasive tumor areas that are close to the tumor edge. 711 The first-in-human results have been reported from intraoperative tumor-specificfluo- rescence imaging by using fluorescein-conjugated agents to target the folate receptors in both ovarian cancer and lung Figure 35. Antibacterial polypeptide-grafted chitosan-based vesicles capable of delivering anticancer and antiepileptic drugs simultaneously. Reprinted from ref 697. Copyright 2013 American Chemical Society. ACS Nano Nano Focus DOI: 10.1021/acsnano.6b06040 ACS Nano 2017, 11, 2313−2381 2353 1036672 (to N.A.K.), Canadian Institute of Health Research (grant to W.C.W.C.), and Natural Sciences and Engineering Research Council of Canada (grant to W.C.W.C.). S.A. and B.P. acknowledge a fellowship from the Alexander von Humboldt Foundation. N.F. acknowledges the Lars Hiertas Minne Foundation. M.C. acknowledges Ikerbasque for a Research Fellow position. X.C. acknowledges the Intramural Research Program (IRP), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH). B.Z.T. acknowledges the Innovation and Technology Commission of Hong Kong (ITC-CNERC14SC01). The Pancreatic Cancer research of A.E.N. and H.M. was funded by the U.S. National Cancer Institute, NIH grant #U01CA198846. A.E. acknowledges Junta de Andalucía (Spain) for a Talentia Postdoc Fellowship, co-financed by the European Union's Seventh Framework Programme, grant agreement no 267226. 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