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Inorganic nanomedicine—Part 1
POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 516 – 522
Review Article
Inorganic nanomedicine—Part 1
www.nanomedjournal.com
Bhupinder S. Sekhon, PhD⁎, Seema R. Kamboj, BPharm PCTE Institute of Pharmacy, Ludhiana, India Received 26 March 2010; accepted 9 April 2010
Abstract Inorganic nanomedicine refers to the use of inorganic or hybrid nanomaterials and nanosized objects to achieve innovative medical breakthroughs for drug and gene discovery and delivery, discovery of biomarkers, and molecular diagnostics. Potential uses for fluorescent quantum dots include cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. Biocompatible quantum dot conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Magnetic nanowires applications include biosensing and construction of nucleic acids sensors. Magnetic cell therapy is used for the repair of blood vessels. Magnetic nanoparticles (MNPs) are important for magnetic resonance imaging, drug delivery, cell labeling, and tracking. Superparamagnetic iron oxide nanoparticles are used for hyperthermic treatment of tumors. Multifunctional MNPs applications include drug and gene delivery, medical imaging, and targeted drug delivery. MNPs could have a vital role in developing techniques to simultaneously diagnose, monitor, and treat a wide range of common diseases and injuries. From the Clinical Editor: This review serves as an update about the current state of inorganic nanomedicine. The use of inorganic/hybrid nanomaterials and nanosized objects has already resulted in innovative medical breakthroughs for drug/gene discovery and delivery, discovery of biomarkers and molecular diagnostics, and is likely to remain one of the most prolific fields of nanomedicine. © 2010 Elsevier Inc. All rights reserved. Key words: Inorganic nanoparticles; Nanomedicine; Magnetic nanoparticles; Quantum dots; Nanomaterials
In this two-part article we describe some aspects of inorganic nanomedicine that bridge the areas of nanotechnology, inorganic chemistry, and medicine. Part 1 describes general aspects of inorganic nanomaterials, techniques for commonly used nanoprobes in biological environments, quantum dots (QDs), magnetic nanoparticles (MNPs), magnetic cell therapy, multifunctional MNPs, and their applications. In biology, biomolecules such as amino acids, proteins, DNA, and viruses are measured in nanometers (1 nm = 1 billionth of a meter). Nanoscale devices smaller than 50 nm can easily enter most cells, whereas those smaller than 20 nm can transit out of blood vessels. Nanodevices are similar in size to large biological molecules such as enzymes and receptors.1 Nanoparticles (NPs) sized in dimensions of roughly 1–100 nm are of immense scientific interest, because they act effectively as a bridge between bulk materials and atomic or molecular structures (Figure 1).
No conflict of interest was reported by the authors of this article. ⁎Corresponding author: PCTE Institute of Pharmacy, Jhande, Near Baddowal Cantt., Ludhiana 142021, India. E-mail address: [email protected] (B.S. Sekhon).
Nanotechnologists have created a variety of inorganic nanoparticles (INPs) and nanomaterials (INMs) with unique physicochemical properties (e.g., ultra-small size, large ratio of surface area to mass, and high reactivity).2 A natural combination of nanotechnology and medicine exists because life itself is a nanoscale phenomenon, and nanomedicine is the key subdiscipline of nanotechnology. Nanomedicine focuses on the application of nanotechnology to medicine, particularly with its promise of improved therapy and diagnostics3,4 and reduction in side effects.5 The pharmaceutical industry already uses INPs. At the local drugstore one can purchase a pregnancy test kit that relies on gold NPs (AuNPs) to reflect red light and thus reveal the presence of pregnancy-related hormones (human chorionic gonadotropin) in a woman's urine. Iron oxide NPs are improving the effectiveness of magnetic resonance imaging (MRI). Gadolinium-containing NPs have high visibility in MRI scans. Nanosilver is a potential antibacterial agent recommended as a preservative in personal care and consumer products. Zinc oxide and titanium dioxide are made more effective in sunscreens when used in the form of NPs. The porous nature of ceramic NPs makes them suitable for drug delivery, particularly in cancer treatment. Recently, INPs and INMs that interact with biological
1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.04.004 Please cite this article as: B.S. Sekhon, S.R. Kamboj, Inorganic nanomedicine—Part 1. Nanomedicine: NBM 2010;6:516-522, doi:10.1016/j. nano.2010.04.004
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Figure 1. General range of dimension of nanomaterial.
systems have attracted widespread interest in imaging, targeting, drug or gene delivery, biology, and medicine.6-10
Inorganic nanomedicine The field of inorganic nanomedicine requires a range of scientific knowledge from inorganic chemistry and microorganisms involved in the preparation and characterization of the NPs through physics, biochemistry, and medical science to allow for their functionalization, and engineering and technology for various clinical applications. Inorganic nanomedicine refers to the use of man-made nanosized (typically b100 nm) INPs and INMs for medical and biomedical applications suited to their unique properties, and nanosized objects in clinical medicine for drug and gene discovery and delivery, the discovery of biomarkers, molecular diagnostics and therapy.
been described. Methods for preparation of bioimaging NPs having high dispersibility in an aqueous solution, biocompatibility, and targetability with a high yield by early introduction of an irregular structure have been reported.15 Furthermore, the use of microorganisms,16 ionic liquids,17 and supercritical fluid technology with reference to the processing of biomedical materials18 has been reported. Chemically modified nanoparticulate vectors of drugs capable of crossing the blood-brain barrier have been reported, and an ideal theoretical therapeuticsdelivery NP system for brain cancer has been suggested.19 INPs are likely to be the cornerstones of current nanomedical devices to be used for drug discovery and delivery, discovery of biomarkers, and molecular diagnostics, which can be designed to (1) target diseased tissues, (2) control the release of therapeutic concentrations over a prolonged period of time, and (3) facilitate administration by various routes. A fundamental understanding regarding the properties of INPs in relation to (1) drug delivery, (2) diagnostic products, (3) biomarker discovery, and (4) regenerative nanomedicine has been reported.20 In general, the different steps in the INMs (NPs and nanocomposites among others) pipeline approach include production of NPs, their functionalization (via coating with, for example, stabilizers, hydrophilic or hydrophobic substances, or biomolecules), their incorporation into nanocomposites, and finally, NP applications.
Inorganic nanoparticles and nanomaterials Applications of inorganic nanomaterials Nanostructured materials are processed forms of raw nanomaterials that provide special shapes or functionality, for example, metals, metal oxides, metal alloy, metal phosphide, metal chalcogenide; metal oxide ceramics (e.g., zinc oxide, cadmium oxide), nonoxide ceramics (e.g., hydroxyapatite ceramics), silicates, nanowires (nanorods), semiconductor nanocrystals (QDs), nanoshells, nanorods, nanowires, inorganic fullerenes, inorganic-organic hybrid or bionanohybrid systems, and silica are representative examples of nanoscale INMs, although some do not yet have commercial applications. For INPs (e.g., metal and semiconductors), two forms are of particular interest in the size range between 2 and 100 nm: nanoclusters in the size range of 1 to 10 nm, and nanocrystals, typically between 2 and 100 nm. When gold, silver, or other metals and even semiconductors are made small enough, they no longer behave in ways that we are used to visualizing. For example, gold no longer has the familiar bright yellow color of jewelry or coins. Instead AuNPs are purple or even black in color. Semiconductors such as cadmium selenide (CdSe) or cadmium telluride (CdTe) normally look black. By contrast, when they are made nanosized their colors turn yellow, orange, and red.11 An overview of the progress made in integrating INPs (including luminescent QDs, MNPs, magnetic nanocrystals, inorganic-organic hybrid nanomaterials) with biology, as well as some of the basic aspects associated with the synthesis, and physical and chemical characteristics of these nanosystems has been reported.12,13 Recent advances in methods to synthesize, stabilize, passivate, and functionalize diverse NPs from metals, metal oxides, semiconductors, polymers, organics, and biomolecules have
Common applications In Japan, many patients have received totally artificial hip joints consisting of a titanium alloy that consists of a nanoscale sodium titanate hydrogel layer on a titanium metal surface.21 Certain formulations of nanoscale powders possessing antimicrobial properties are made of simple, nontoxic metal oxides such as magnesium oxide (MgO) and calcium oxide in nanocrystalline form,21 carrying active forms of halogens (e.g., MgOCl2 and MgOBr2). Nanoscale MgO exhibits high activity against bacteria, spores, and viruses after adsorption of halogen gases.22 Silver NPs incorporated into Acticoat bandages (Smith & Nephew Wound Management, Largo, Florida) are highly toxic to pathogens in wounds. Silicon dioxide NPs combined with zinc or silver have shown antibacterial properties.23 The antimicrobial effect of nanometric bioactive glass 45S5 used as a disinfectant in dentistry has been reported.24 Detection methods for cell tracking probes Technology has made immense strides to enable visualization, identification, and quantitation in biological systems on the nanometer scale to create new materials with enhanced properties. Various techniques relevant to inorganic nanomedicine for commonly used nanoprobes in biological environments include fluorescence emission–scanning microscopy, resonant Raman spectroscopy, fluorescence Raman microscopy, Rayleigh light-scattering microscopy, MRI, computed tomographic (CT) imaging, and x-ray absorption. Techniques to improve contrast in ultrasound and MRI images are being further developed.
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Clinical MRI cell tracking is likely to become an important tool at the bedside once (stem) cell therapy becomes mainstream.25-27 Superparamagnetic iron oxide (SPIO) was used to label green fluorescent protein (GFP) cells effectively with no effects on cell function and GFP expression. The tracking of GFP, a gene marker, with the SPIO-loaded cells using MRI suggests that this technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker to enhance the understanding of cell-based and gene-based therapeutic strategies.28 Neural stem cells labeled with SPIO were tracked by MRI in vitro and in vivo after implantation.29 Fluorine (19F) cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. Researchers demonstrated that 19F MRI of cells labeled with different types of liquid perfluorocarbon NPs produced unique and sensitive cell markers distinct from any tissue background signal.30 The overall usefulness of perfluorocarbons nanoemulsions as reagents for 19 F cell tracking has been reported.31 The use of colloidal fluorescent semiconductor QDs as highly luminescent stains has expanded the use of fluorescence microscopy as a potential tool for fast and precise diagnosis of different kinds of cancer and other pathologies.32 The potential of resonant Raman spectroscopy from vibration modes for the analysis of embedded monolayers and QDs has been demonstrated.33,34 A new type of ultrasound (US) contrast agent, consisting of microbubbles incorporating a gas core, a layer of SPIO, and oil in water in the outermost layer, has the following advantages compared to gas-encapsulated microbubbles without SPIO inclusion: (1) The SPIO-inclusion microbubbles provide better contrast for US images; (2) they generate a higher backscattering signal (the mean gray scale is 97.9, which is 38.6 higher than that of microbubbles without SPIO); and (3) because SPIO can also serve as a contrast agent for MRI in vitro, they can be potentially used as contrast agents for doublemodality (MRI and US) clinical studies.35 Researchers demonstrated hyper-Raman scattering on biological structures such as single cells after incubation with AuNPs.36,37 Scientists showed that resonant Raman imaging of flavocytochrome b558 at 413.1 nm excitation in QD-labeled neutrophilic granulocytes or nonresonant Raman imaging of proteins and lipids at 647.1 nm excitation in QD-labeled macrophages can be integrated with linear one-photon excitation and nonlinear continuous-wave two-photon excitation fluorescence microscopy of QDs, respectively. The enhanced information content of these two hybrid Raman fluorescence methods provided new multiplexing possibilities for single-cell optical microscopy and intracellular chemical analysis.38 Aminoterminal fragment-conjugated iron oxide NPs were able to specifically bind to and be internalized by urokinase-type plasminogen activator (uPA) receptor-expressing tumor cells, suggesting thereby, that uPA receptor-targeted amino-terminal fragment-conjugated iron oxide NPs have potential as molecularly targeted, dual-modality imaging agents for in vivo imaging of breast cancer.39 Spectral and 3-dimensional tracking of single-molecule AuNP interacting with living mouse fibroblast cells has been studied by Rayleigh light-scattering microscopy.40 Two-photon
Rayleigh scattering (TPRS) properties of gold nanorods can be used for rapid, highly sensitive and selective detection of Escherichia coli bacteria from aqueous solution. TPRS intensity increased 40 times when nanorods conjugated to antibody specific to E. coli were mixed with various concentrations of E. coli O157:H7 bacterium.41 Diagnostics of single base-mismatch DNA hybridization on AuNPs has been achieved using TPRS.42 The use of plasmonic NPs as highly enhanced photoabsorbing agents has introduced plasmonic photothermal therapy (PPTT). The synthetic tunability of the optothermal properties and the biotargeting abilities of the plasmonic gold nanostructures make the PPTT method even more promising. Scientists discussed the development of the PPTT method using gold nanospheres coupled with visible lasers and gold nanorods and silica-gold nanoshells coupled with near-infrared (NIR) lasers.43 AuNPs protected by thermosensitive diblock copolymers with tunable collapse temperature have potential uses for biomedical applications.44 AuNPs (1.9 nm in diameter, ∼50 kDa) as a CT contrast agent exhibited good stability, high x-ray absorption, a good safety profile, long blood half-life, and enhanced CT contrast of the vasculature, kidneys, and tumor in mice.45 Chithrani et al.46 showed that AuNPs between 14 and 74 nm in diameter entered cultured HeLa tumor cells in medium that contained 10% serum and were trapped in vesicles in the cytoplasm. Gold nanoprobes selectively and sensitively targeted tumor-selective antigens while inducing distinct contrast in CT imaging.47 SPIO NPs coated with a lipid bilayer induce cells to acquire magnetic activity following uptake into cells, and biotinconjugated MNPs functionalized with fluorescent tag (streptavidin–fluorescein isothiocyanate) conferred both magnetic activity and fluorescence labeling.48 The appropriate use of QDs and dendrimers enabled the simultaneous tracking of cancer cells within draining lymphatics.49 Applications for specific nanocarriers Development of different types of INPs has led to novel health-care and medical applications,50,51 some of which are already commercially available. At present only NP-based magnetic fluid hyperthermia to treat brain cancers52 (Magforce, Berlin, Germany) and the use of Tat peptide–derivatized MNPs for stem cell therapy for cardiac diseases53 have proceeded to clinical trials. Examples of various medical or biomedical applications are here selected from various nanocarriers such as QD nanocrystals, MNPs, metal NPs, silica NPs, inorganicorganic hybrid nanomaterials, hybrid dendrimer nanomaterials, bioinorganic nanohybrid materials, and other nanomaterials related to bone implants and regenerative nanomedicine. Upcoming nanomaterials for in vivo imaging: an alternative to QDs Lanthanide-doped upconversion nanocrystals have been developed as a new class of luminescent optical labels.54,55 Functionalized yttrium(III) oxide–based upconverting nanomaterial is a promising platform for in vivo optical-based diagnostic imaging, and the new particles avoid potential interference from
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tissue autofluorescence. In addition, the NPs are composed of less toxic materials than are QDs.56 Rare-earth doped β-NaYF4:Yb,Er upconversion nanoparticles (UCNPs) with strong UC fluorescence were coated with a thin layer of silicon dioxide (SiO2) to form core-shell NPs, which were further modified with amino groups, and subsequently, rabbit antibodies to CEA8 were covalently linked to the UCNPs to form antibody-UCNP conjugates. The antibody-UCNP conjugates results indicated that the amino-functionalized UCNPs can be used as fluorescent probes in cell immunolabeling and imaging.57 Individual lanthanide-doped UCNPs— specifically, hexagonal-phase NaYF4 (β-NaYF4) nanocrystals with multiple Yb3+ and Er3+ dopants—emit bright anti-Stokes visible upconverted luminescence with exceptional photostability when excited by a 980-nm continuous-wave laser. Furthermore, these UCNPs proved ideally suited for single-molecule imaging experiments.58 QD nanocrystals QDs are inorganic semiconductor nanocrystals having typical diameters between 2 and 8 nm, and have emerged as a new class of fluorescent labels with better brightness, resistance against photobleaching, and multicolor fluorescence emission.59 They are generally composed of atoms from groups II and VI elements (e.g., CdSe and CdTe) or groups III and V elements [e.g., indium phosphide (InP) and indium arsenide (InAs)] of the periodic table. In general, QDs consist of a semiconductor core, overcoated by a shell to improve optical properties, and a cap allowing improved solubility in aqueous buffers. Among the range of available QDs, the ones composed of CdSe cores overcoated with a layer of zinc sulfide (ZnS) are produced by a range of well-developed synthetic routes.60 These QDs become highly fluorescent and feature such attractive optical properties as high quantum yield, large absorption cross section, and high photostability.61 Various QDs solubilization strategies have been developed over the past decades.62-64 QDs have been systematically tried in virtually all fluorescence-based assays and in vivo imaging procedures.59,61,63 NP water solubility is an essential requirement for in vivo imaging. In this regard, various approaches have used ligand exchange of the original hydrophobic surfactant ligands by hydrophilic molecules. The most often applied molecules are thiol-containing compounds, such as mercaptoacetic acid (dihydrolipoic acid derivatives), or more sophisticated compounds such as alkylthiol-terminated DNA, thioalkylated oligoethyleneglycols, D,L-cysteine, poly(ethylene glycol [PEG])terminated dihydrolipoic acid having significant in vitro and in vivo stability.65-67 Another strategy for solubilization is based on encapsulation into a layer of amphiphilic diblock or triblock copolymers, phospholipid micelles, silica shells, or amphiphilic polysaccharides, polymer shells, oligomeric phosphine coating, or by phytochelatin-peptides coating or histidine-rich proteins.68-70 Glutathione-capped CdTe, zinc selenide (ZnSe), and Zn1 – xCdxSe QDs were successfully synthesized in aqueous solution with fluorescence emissions tunable between 360 and 700 nm, having a quantum yield of up to 50%.71 The glutathione-capped CdTe QDs were successfully conjugated with bioprobes for
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cell-imaging applications. QDs can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids, or small-molecule ligands for use as biological labels in molecular and cellular imaging.73,74 Biological applications of QDs include fluorescence resonance energy transfer analysis, gene technology, fluorescence labeling of cellular proteins, cell tracking, pathogen and toxin detection, and in vivo animal imaging.75-81 One-pot synthesis, encapsulation, and solubilization of highquality QDs based on the use of amphiphilic multidentate polymer ligands and short PEGs at high temperatures allowed for in situ growth of an inorganic passivating shell (CdSe) on the QD core, with a large dynamic range for QD emission from the visible to the NIR.82 Multiple QD fluorescence in situ hybridization probes with different emission spectra were used in a one-step hybridization-detection experiment to visualize, in situ, two subchromosomal regions of highly condensed chromatin structure within the centromere.83 The most fruitful inorganic materials–based optical contrast agents are QDs. First, in vivo studies with QDs in cell lineagetracing experiments with frog embryos,84 and then, their use to image cell signal transduction,85 cancer markers,86 and tumors in living animals87 were reported. QD-conjugated probes to specific biomarkers are powerful tools that can be applied in a multiplex manner to single tissue sections to measure expression levels of multiple biomarkers.88 QDs coated with zinc(II)dipicolylamine coordination complexes selectively allowed optical detection in a living mouse leg infection model.89 QDs have the potential to function as multimodal imaging platforms in vivo and the ability to detect an optical NP preoperatively with clinical imaging modality offered a distinct advantage to clinicians engaged in image-guided surgical applications.90,91 QDs, in addition to being excellent fluorescent probes, can be used as photoacoustic and photothermal contrast agents and sensitizers, thereby providing an opportunity for multimodal high-resolution (300 nm) photoacousticphotothermal fluorescent imaging as well as photothermal therapy.92 Applications of QDs at the cellular level, including immune labeling, cell tracking, in situ hybridization, fluorescence resonance energy transfer, and in vivo imaging, have been reported.93 QDs have potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, longterm in vivo observation of cell trafficking, tumor targeting, and diagnostics.94,95 Human metaphase chromosomes from transformed lymphocyte cultures and breast cancer cell line SK-BR-3 were analyzed by fluorescence in situ hybridization based on streptavidin-linked CdSe QDs.96,97 The use of streptavidincoupled QDs for live-cell imaging has also been reported.98 Cadmium sulfide–cadmium hydroxide QDs maintained high levels of luminescence as well as high photostability in cells and tissues.99 Noninvasive visualization of blood vessels was observed using QDs over time.100 Strongly luminescent copper indium disulfide–ZnS core-shell nanocrystals functionalized with dihydrolipoic acid to the aqueous phase were used as fluorescent labels for in vivo imaging.101 Dextran-coated nanoworms particles composed of a linear aggregate of 5–10 iron oxide cores and a total length between
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∼50 and 80 nm allowed a larger number of interactions between peptides conjugated to the nanoworm and cell surface receptors to take place relative to nanospheres of similar composition.102 Stable PEGylated QDs capped with D-mannose, D-galactose, and D-galactosamine provided selectivity and accumulated in the liver but not in other parts of the body. These could be used to deliver anticancer drugs to one organ, without causing the body-wide side effects that occur with currently existing cancer drugs.103 The promising clinical potential of QDs, their limitations for clinical use, and the recent progress in abrogating their inherent toxicity have been reported.104 Glutathionemediated release of functional plasmid DNA from positively charged CdTe QDs has been demonstrated, thereby suggesting potential applications of these positively charged QDs in selective unpacking of payload in living cells.105 InP-ZnS QDs can be used to replace CdSe-based ones (unsuitable due to toxicity) for almost any biomedical application.106 However, InP-ZnS QDs must be covered with other materials, allowing dispersion and preventing leaking of the toxic heavy metals in case of biological applications.107 Multivalent metal cations have been used to remove water-soluble CdTe QDs capped with thioglycolate ligands.108 The optical properties of QDs, the biofunctionalization strategies, and focus on their biosensing and in vivo imaging applications have been reported.109 Researchers have used QDs linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neuoverexpressing live cells successfully, thereby indicating that composition based on QDs and scFv antibody can be successfully used for cancer cell visualization.110 Both QDantibody and QD-IgG probes were successfully used to label HeLa cells using carboxyl-functionalized CdSe QDs, thereby suggesting practicability of CdSe QDs as attractive fluorescent labels for biological applications such as protein probes and cell imaging.111 QD-gelatin nanocomposites exhibited increased QD luminescence efficiencies, and the nanocomposites penetrated the cell membrane and illuminated the cytoskeleton of macrophage cells.112 Furthermore, live-cell labeling of cytoplasmic structures, including actin microfilaments, through the incorporation within the nanocomposite core of QDs conjugated with antibody to actin was demonstrated.113 QDs are also an attractive choice for regenerative therapy114 and were used to identify the presence and monitor the progression of respiratory syncytial virus infection over time by labeling the F and G proteins, suggesting that QDs may provide a method for early, rapid detection of viral infection.115 The cytotoxicity of green and red mercaptopropionic acid–capped CdTe QDs was significantly enhanced in human pancreatic carcinoma cells (PANC-1) under ultraviolet illumination.116 Multifunctional QDs In vivo targeting studies of human prostate cancer growing in nude mice indicated that the QD probes can be delivered to tumor sites, achieving sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions.117 The conjugation of QDs with photosensitizers and targeting agents provided a new class of versatile multifunctional NPs for both diagnostic imaging and therapeutic applications.118 QD-apata-
mer-doxorubicin conjugate, a multifunctional NP system, can deliver doxorubicin to the targeted prostate cancer cells and sense the delivery of doxorubicin by activating the fluorescence of QD, while allowing for simultaneous imaging of the cancer cells.119 Researchers have demonstrated a simple electrostatic conjugation scheme to conjugate QDs to hyaluronic acid (HA) to produce stable and size-tunable (50 to 120 nm) HA-QD conjugates that showed cancer specificity. Furthermore, these HA-QDs can be also used to visualize lymphatic vessels by fluorescence staining, because they can be delivered into lymphatic endothelial cells through receptors such as LYVE-1.120 Hydrophobic QDs incorporated into the bilayer membrane of lipid vesicles are capable of fusing with live cells, thereby selectively staining the cell's plasma membrane with QDs and either transferring the vesicle's cargo into the cell or transferring it into the cytoplasm of live cells.121 QDs as photosensitizers in photodynamic therapy have provided a novel use as an anticancer therapy.122 Multifunctional high-density lipoprotein NPs have been developed by the incorporation of gold, iron oxide, or QD nanocrystals for CT, MRI, and fluorescence imaging, respectively.123 Magnetic nanoparticles Magnetic inorganic nanomedicine is emerging rapidly, and the nanomaterials used are MNPs, which include nanowires, nanospheres, nanotubes, and magnetic thin films. Diagnostic uses of MNPs include detection of malignant tissues or pathogenic bioaggregates. Use of MNPs has advanced MRI, guided drug and gene delivery, MFH cancer therapy, tissue engineering, cell tracking, and bioseparation. Integrative therapeutic and diagnostic (i.e., theragnostic) applications have emerged with MNP use, such as MRI-guided cell replacement therapy or MRI-based imaging of cancer-specific gene delivery.124 Great progress has been made in the applications of MNPs in biomedicine.125-128 SPIO NPs (15– 60 nm) can be coated with dextran, phospholipids, or other compounds to inhibit aggregation and passive or active targeting agents.129 Upon controlled surface functionalization and coupling with fragments of DNA strands, proteins, peptides, or antibodies, SPIO NPs can be used for drug delivery, magnetic separation, MRI contrast enhancement, and MFH.130 Cationic MNPs enter into cells with high effectiveness and remain localized in endosomes; they are easily detected inside cells by optical microscopy, are retained for relatively long periods of time, and do not induce cytotoxicity.131 A nonspecific labeling method based on anionic MNPs can predict uptake efficiency by all cell types including tumor cells. In addition, the same label provided sufficient magnetization for MRI detection and distal manipulation.132 Fluorescence-modified chitosancoated MNPs for high-efficiency cellular imaging have been developed.133 SPIO NPs are used for MRI and noninvasive cell tracking.134,135 SPIO NPs have the unique capability as contrast agents for MRI due to their ability to shorten T2⁎ relaxation times of the gastrointestinal tract, liver and spleen, lymph node, blood pool, and atheromatous plaque.136 Researchers have summarized the recent progress to develop NP-based T1 contrast agents in inorganic NP-based MRI contrast agents.137
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MNPs can be used in a wide variety of biomedical applications such as contrast agents for MRI (to differentiate healthy and pathological tissues), to combine imaging with hyperthermia treatment or imaging with drug delivery, and to visualize various biological events inside the body.138-141 Scientists have described a novel strategy for NP delivery for treating the hypoxic regions of tumors.142 Scientists have fabricated MRI probes by tuning NPs magnetism by varying their size or composition.143,144 Iron oxide MNPs have received U.S. Food and Drug Administration (FDA) approval to be used as MRI signal enhancers. Recent advances in the development of targeted iron oxide NPs for tumor imaging and therapy have been reported.145 Cellular labeling with ferumoxides (Feridex I.V.; Advanced Magnetics, Inc., Cambridge, Massachusetts) SPIO NPs can be used to monitor cells in vivo by MRI.146 Ferumoxides (Feridex I.V.), dextran-coated SPIO NPs, were approved by the FDA as an MRI contrast agent. Ultra-small SPIO NPs such as ferumoxtran-10 and ferumoxytol have particle diameters of 20 to 50 nm and have longer plasma half-lives of 14–30 hours.147 A family of calcium indicators for MRI formed by combining a powerful SPIO-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets has been reported.148 SPIO NPs measuring 2–3 nm have been used in conjunction with MRI to reveal small and otherwise undetectable lymph node metastases, and ultrasmall SPIO enhances MRI for imaging cerebral ischemic lesions. Dextran-coated iron oxide NPs enhance MRI visualization of intracranial tumors for more than 24 hours.149 SPIO nanocrystals encapsulated inside mesostructured silica spheres, labeled with fluorescent dye molecules, and coated with hydrophilic groups, prevented aggregation. Furthermore, water-insoluble anticancer drugs were delivered into human cancer cells, and surface conjugation with cancerspecific targeting agents increased the uptake into cancer cells relative to that in noncancerous fibroblasts.150 The ability of coated SPIO to be rapidly taken up and distributed into lymphoid tissues demonstrated the feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.151 Advances in MNP design, in vitro and animal experiments with MNP-based drug and gene delivery, and clinical trials of drug targeting have been reported.152 The targeted detection technique can be used for detecting extremely early signs of disease.153 Biocompatible iron oxide MNPs coated with PEG and size-dependent accumulation in murine tumors following intravenous injection showed enhanced MRI contrast of the larger MNPs in the tumor.154 Multifunctional MNPs Two strategies are utilized to fabricate MNP-based multifunctional nanostructures.155 The first, molecular functionalization, involves attaching antibodies, proteins, and dyes to the MNPs, whereas the other integrates the MNPs with other functional nanocomponents such as QDs or other MNPs. Hybrid nanostructures, which combine MNPs with other nanocomponents, exhibited paramagnetism alongside features such as fluorescence or enhanced optical contrast, and thus could
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provide a platform for enhanced medical imaging and controlled drug delivery.155 Photonic explorers for biomedical use with biologically localized embedding have been successfully applied for significant MRI contrast enhancement and photodynamic therapeutic efficacy with targeted nanoplatforms in a rat 9L gliosarcoma in vivo model, thereby, demonstrating significant promise for their use as a combined therapeutic and diagnostic tool for cancer.156 Dexamethasone conjugated to rhodamine B gum arabic–modified MNPs by photosensitive linker showed significant release of dexamethasone by phototriggered response on exposure to radiation having a wavelength in the NIR region, thereby suggesting an on-demand release of the drug by this nanocarrier.157 The excellent passive tumor-targeting efficiency of thermally cross-linked SPIO NPs allowed detection of tumors by MRI and at the same time exhibited anticancer activity.158 Multicolored highly fluorescent superparamagnetic nanoparticles employing hydrophobic multicolor QDs and hydrophobic Fe3O4 (MNPs) fabricated via ultrasonic emulsification have potential for serving as a hybrid of QDs and MNPs in bioanalysis communities.159 Magnetic nanowires Most magnetic nanowires are compatible with living cells and can be functionalized with biologically active molecules. Many efforts have been made to explore the applications of singlesegment and multiple-segment magnetic nanowires in biomedicine.160 Compared to nanospheres, multifunctionality can be more easily realized on multisegment nanowires.161 Magnetic nanowires used in biomedicine are metal cylindrical electrode(s) positioned in nanoporous templates162 and have shown potential for use in biosensing applications and construction of nucleic acids sensors.163 Functionalization with biomolecules involving two-segment nickel-gold nanowires served as synthetic gene delivery systems.164 The binding of pcDNA3 to the nickel segment of the nanorod provided a strong immunostimulatory adjuvant effect to the antigen bound on the gold segment.165 Separation of heterogeneous cell mixtures based on the differences in physical size of the cells has been achieved with magnetic nanowires.166 Magnetic cell therapy Magnetic cell therapies are applied worldwide at hospitals and university clinics on a case-by-case basis. Repairing blood vessels with cell therapy is a very important concept that can be realized with magnetic targeting. Intravascular gene targeting can be combined with positioning of the transduced cells via MNPs, thereby combining gene-based and cell-based therapies.168 Magnetic fluid hyperthermia In MFH, SPIO NPs used for hyperthermic treatment of tumors are incorporated by the tumor cells much faster than by healthy cells and become concentrated in the tumor cells. Upon applying an alternating-current magnetic field, the particles increase in heat anywhere from 43°C (110°F) up to 70°C (158°F) inside the tumor, thus destroying the tumor cells, while leaving normal cells unharmed.169,170
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MFH led to a significant growth inhibition in this orthotopic model of the aggressive MatLyLu tumor variant. Additionally, intratumoral deposition of magnetic fluids was found to be stable, allowing for serial MFH treatments without repeated injection.171 The results showed evidence of a localized effectiveness and with only minor to moderate therapyassociated side effects.172 The development of NP-based hyperthermic methods was spearheaded by the German startup company Magforce, with the only hyperthermia setup suitable to treat humans developed at the Charité Medical School, Clinic of Radiation Therapy in Berlin. NP-based MFH therapy against brain tumors is under investigation in a phase II study. Conclusions and perspectives Inorganic nanomedicine holds great promise in diagnostics, drug and gene delivery, sensing and biosensing, and in vivo imaging under the present scenario. Smartly engineered inorganic NPs can boost drug efficacy and can improve drug targeting to specific areas within the body, therefore making treatment less toxic and invasive. Fluorescent QDs serve as credible alternatives to commonly used fluorophores and genetically encoded fluorescent proteins. Silica-based nanomaterials are easily functionalized, which allows for the conjugation or encapsulation of important biomolecules, and have shown promise for cell-selective drug delivery and bioimaging. Different multifunctional agents encompassing both imaging and therapeutic capabilities can manage simultaneous monitoring and treatment of diseases. Scientists argue that applications with targeted NPs are expected to revolutionize molecular imaging and cancer therapy. Undoubtedly, QDs, MNPs, and gold NPs, nanospheres,
nanorods, and nanowires will be an integral part of our imaging toolbox. DNA nonviral vectors in gene therapy represent biomedical applications of bionanohybrids. Inorganic-organic bionanohybrids help in bone tissue regeneration and controlled delivery. Integration of targeting ligands, imaging labels, therapeutic drugs, photodynamic therapy, and other functional moieties into the NP conjugate is highly desirable for effective molecular imaging and molecular therapy of otherwise fatal diseases. Future developments relating to the new contrast agents should enable diagnosis of neurological disorders such as Alzheimer's disease, Parkinson's disease, and stroke. Computational tools might help in predicting the various binding sites for conjugation of NPs to cells, and therefore, theoretical and computation modeling can accelerate visualization of the biological environment of the NPs and assist in the design of nanomaterials for use in medicine. Keeping in view the diversity of engineered NPs, their several possible side effects should not be overlooked. Further research is required on the interaction of nanomaterials with biological systems from the perspective of safe use of nanomaterials. Experts are of the opinion that inorganic nanomedicine has the potential to provide groundbreaking methods for the prevention, diagnosis, and treatment of some otherwise fatal diseases.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nano.2010.04.004.
Review Article
Inorganic nanomedicine—Part 1
www.nanomedjournal.com
Bhupinder S. Sekhon, PhD⁎, Seema R. Kamboj, BPharm PCTE Institute of Pharmacy, Ludhiana, India Received 26 March 2010; accepted 9 April 2010
Abstract Inorganic nanomedicine refers to the use of inorganic or hybrid nanomaterials and nanosized objects to achieve innovative medical breakthroughs for drug and gene discovery and delivery, discovery of biomarkers, and molecular diagnostics. Potential uses for fluorescent quantum dots include cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. Biocompatible quantum dot conjugates have been used successfully for sentinel lymph node mapping, tumor targeting, tumor angiogenesis imaging, and metastatic cell tracking. Magnetic nanowires applications include biosensing and construction of nucleic acids sensors. Magnetic cell therapy is used for the repair of blood vessels. Magnetic nanoparticles (MNPs) are important for magnetic resonance imaging, drug delivery, cell labeling, and tracking. Superparamagnetic iron oxide nanoparticles are used for hyperthermic treatment of tumors. Multifunctional MNPs applications include drug and gene delivery, medical imaging, and targeted drug delivery. MNPs could have a vital role in developing techniques to simultaneously diagnose, monitor, and treat a wide range of common diseases and injuries. From the Clinical Editor: This review serves as an update about the current state of inorganic nanomedicine. The use of inorganic/hybrid nanomaterials and nanosized objects has already resulted in innovative medical breakthroughs for drug/gene discovery and delivery, discovery of biomarkers and molecular diagnostics, and is likely to remain one of the most prolific fields of nanomedicine. © 2010 Elsevier Inc. All rights reserved. Key words: Inorganic nanoparticles; Nanomedicine; Magnetic nanoparticles; Quantum dots; Nanomaterials
In this two-part article we describe some aspects of inorganic nanomedicine that bridge the areas of nanotechnology, inorganic chemistry, and medicine. Part 1 describes general aspects of inorganic nanomaterials, techniques for commonly used nanoprobes in biological environments, quantum dots (QDs), magnetic nanoparticles (MNPs), magnetic cell therapy, multifunctional MNPs, and their applications. In biology, biomolecules such as amino acids, proteins, DNA, and viruses are measured in nanometers (1 nm = 1 billionth of a meter). Nanoscale devices smaller than 50 nm can easily enter most cells, whereas those smaller than 20 nm can transit out of blood vessels. Nanodevices are similar in size to large biological molecules such as enzymes and receptors.1 Nanoparticles (NPs) sized in dimensions of roughly 1–100 nm are of immense scientific interest, because they act effectively as a bridge between bulk materials and atomic or molecular structures (Figure 1).
No conflict of interest was reported by the authors of this article. ⁎Corresponding author: PCTE Institute of Pharmacy, Jhande, Near Baddowal Cantt., Ludhiana 142021, India. E-mail address: [email protected] (B.S. Sekhon).
Nanotechnologists have created a variety of inorganic nanoparticles (INPs) and nanomaterials (INMs) with unique physicochemical properties (e.g., ultra-small size, large ratio of surface area to mass, and high reactivity).2 A natural combination of nanotechnology and medicine exists because life itself is a nanoscale phenomenon, and nanomedicine is the key subdiscipline of nanotechnology. Nanomedicine focuses on the application of nanotechnology to medicine, particularly with its promise of improved therapy and diagnostics3,4 and reduction in side effects.5 The pharmaceutical industry already uses INPs. At the local drugstore one can purchase a pregnancy test kit that relies on gold NPs (AuNPs) to reflect red light and thus reveal the presence of pregnancy-related hormones (human chorionic gonadotropin) in a woman's urine. Iron oxide NPs are improving the effectiveness of magnetic resonance imaging (MRI). Gadolinium-containing NPs have high visibility in MRI scans. Nanosilver is a potential antibacterial agent recommended as a preservative in personal care and consumer products. Zinc oxide and titanium dioxide are made more effective in sunscreens when used in the form of NPs. The porous nature of ceramic NPs makes them suitable for drug delivery, particularly in cancer treatment. Recently, INPs and INMs that interact with biological
1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2010.04.004 Please cite this article as: B.S. Sekhon, S.R. Kamboj, Inorganic nanomedicine—Part 1. Nanomedicine: NBM 2010;6:516-522, doi:10.1016/j. nano.2010.04.004
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Figure 1. General range of dimension of nanomaterial.
systems have attracted widespread interest in imaging, targeting, drug or gene delivery, biology, and medicine.6-10
Inorganic nanomedicine The field of inorganic nanomedicine requires a range of scientific knowledge from inorganic chemistry and microorganisms involved in the preparation and characterization of the NPs through physics, biochemistry, and medical science to allow for their functionalization, and engineering and technology for various clinical applications. Inorganic nanomedicine refers to the use of man-made nanosized (typically b100 nm) INPs and INMs for medical and biomedical applications suited to their unique properties, and nanosized objects in clinical medicine for drug and gene discovery and delivery, the discovery of biomarkers, molecular diagnostics and therapy.
been described. Methods for preparation of bioimaging NPs having high dispersibility in an aqueous solution, biocompatibility, and targetability with a high yield by early introduction of an irregular structure have been reported.15 Furthermore, the use of microorganisms,16 ionic liquids,17 and supercritical fluid technology with reference to the processing of biomedical materials18 has been reported. Chemically modified nanoparticulate vectors of drugs capable of crossing the blood-brain barrier have been reported, and an ideal theoretical therapeuticsdelivery NP system for brain cancer has been suggested.19 INPs are likely to be the cornerstones of current nanomedical devices to be used for drug discovery and delivery, discovery of biomarkers, and molecular diagnostics, which can be designed to (1) target diseased tissues, (2) control the release of therapeutic concentrations over a prolonged period of time, and (3) facilitate administration by various routes. A fundamental understanding regarding the properties of INPs in relation to (1) drug delivery, (2) diagnostic products, (3) biomarker discovery, and (4) regenerative nanomedicine has been reported.20 In general, the different steps in the INMs (NPs and nanocomposites among others) pipeline approach include production of NPs, their functionalization (via coating with, for example, stabilizers, hydrophilic or hydrophobic substances, or biomolecules), their incorporation into nanocomposites, and finally, NP applications.
Inorganic nanoparticles and nanomaterials Applications of inorganic nanomaterials Nanostructured materials are processed forms of raw nanomaterials that provide special shapes or functionality, for example, metals, metal oxides, metal alloy, metal phosphide, metal chalcogenide; metal oxide ceramics (e.g., zinc oxide, cadmium oxide), nonoxide ceramics (e.g., hydroxyapatite ceramics), silicates, nanowires (nanorods), semiconductor nanocrystals (QDs), nanoshells, nanorods, nanowires, inorganic fullerenes, inorganic-organic hybrid or bionanohybrid systems, and silica are representative examples of nanoscale INMs, although some do not yet have commercial applications. For INPs (e.g., metal and semiconductors), two forms are of particular interest in the size range between 2 and 100 nm: nanoclusters in the size range of 1 to 10 nm, and nanocrystals, typically between 2 and 100 nm. When gold, silver, or other metals and even semiconductors are made small enough, they no longer behave in ways that we are used to visualizing. For example, gold no longer has the familiar bright yellow color of jewelry or coins. Instead AuNPs are purple or even black in color. Semiconductors such as cadmium selenide (CdSe) or cadmium telluride (CdTe) normally look black. By contrast, when they are made nanosized their colors turn yellow, orange, and red.11 An overview of the progress made in integrating INPs (including luminescent QDs, MNPs, magnetic nanocrystals, inorganic-organic hybrid nanomaterials) with biology, as well as some of the basic aspects associated with the synthesis, and physical and chemical characteristics of these nanosystems has been reported.12,13 Recent advances in methods to synthesize, stabilize, passivate, and functionalize diverse NPs from metals, metal oxides, semiconductors, polymers, organics, and biomolecules have
Common applications In Japan, many patients have received totally artificial hip joints consisting of a titanium alloy that consists of a nanoscale sodium titanate hydrogel layer on a titanium metal surface.21 Certain formulations of nanoscale powders possessing antimicrobial properties are made of simple, nontoxic metal oxides such as magnesium oxide (MgO) and calcium oxide in nanocrystalline form,21 carrying active forms of halogens (e.g., MgOCl2 and MgOBr2). Nanoscale MgO exhibits high activity against bacteria, spores, and viruses after adsorption of halogen gases.22 Silver NPs incorporated into Acticoat bandages (Smith & Nephew Wound Management, Largo, Florida) are highly toxic to pathogens in wounds. Silicon dioxide NPs combined with zinc or silver have shown antibacterial properties.23 The antimicrobial effect of nanometric bioactive glass 45S5 used as a disinfectant in dentistry has been reported.24 Detection methods for cell tracking probes Technology has made immense strides to enable visualization, identification, and quantitation in biological systems on the nanometer scale to create new materials with enhanced properties. Various techniques relevant to inorganic nanomedicine for commonly used nanoprobes in biological environments include fluorescence emission–scanning microscopy, resonant Raman spectroscopy, fluorescence Raman microscopy, Rayleigh light-scattering microscopy, MRI, computed tomographic (CT) imaging, and x-ray absorption. Techniques to improve contrast in ultrasound and MRI images are being further developed.
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Clinical MRI cell tracking is likely to become an important tool at the bedside once (stem) cell therapy becomes mainstream.25-27 Superparamagnetic iron oxide (SPIO) was used to label green fluorescent protein (GFP) cells effectively with no effects on cell function and GFP expression. The tracking of GFP, a gene marker, with the SPIO-loaded cells using MRI suggests that this technique holds promise for monitoring the temporal and spatial migration of cells with a gene marker to enhance the understanding of cell-based and gene-based therapeutic strategies.28 Neural stem cells labeled with SPIO were tracked by MRI in vitro and in vivo after implantation.29 Fluorine (19F) cell tracking is a useful technology because of the high specificity for labeled cells, ability to quantify cell accumulations, and biocompatibility. Researchers demonstrated that 19F MRI of cells labeled with different types of liquid perfluorocarbon NPs produced unique and sensitive cell markers distinct from any tissue background signal.30 The overall usefulness of perfluorocarbons nanoemulsions as reagents for 19 F cell tracking has been reported.31 The use of colloidal fluorescent semiconductor QDs as highly luminescent stains has expanded the use of fluorescence microscopy as a potential tool for fast and precise diagnosis of different kinds of cancer and other pathologies.32 The potential of resonant Raman spectroscopy from vibration modes for the analysis of embedded monolayers and QDs has been demonstrated.33,34 A new type of ultrasound (US) contrast agent, consisting of microbubbles incorporating a gas core, a layer of SPIO, and oil in water in the outermost layer, has the following advantages compared to gas-encapsulated microbubbles without SPIO inclusion: (1) The SPIO-inclusion microbubbles provide better contrast for US images; (2) they generate a higher backscattering signal (the mean gray scale is 97.9, which is 38.6 higher than that of microbubbles without SPIO); and (3) because SPIO can also serve as a contrast agent for MRI in vitro, they can be potentially used as contrast agents for doublemodality (MRI and US) clinical studies.35 Researchers demonstrated hyper-Raman scattering on biological structures such as single cells after incubation with AuNPs.36,37 Scientists showed that resonant Raman imaging of flavocytochrome b558 at 413.1 nm excitation in QD-labeled neutrophilic granulocytes or nonresonant Raman imaging of proteins and lipids at 647.1 nm excitation in QD-labeled macrophages can be integrated with linear one-photon excitation and nonlinear continuous-wave two-photon excitation fluorescence microscopy of QDs, respectively. The enhanced information content of these two hybrid Raman fluorescence methods provided new multiplexing possibilities for single-cell optical microscopy and intracellular chemical analysis.38 Aminoterminal fragment-conjugated iron oxide NPs were able to specifically bind to and be internalized by urokinase-type plasminogen activator (uPA) receptor-expressing tumor cells, suggesting thereby, that uPA receptor-targeted amino-terminal fragment-conjugated iron oxide NPs have potential as molecularly targeted, dual-modality imaging agents for in vivo imaging of breast cancer.39 Spectral and 3-dimensional tracking of single-molecule AuNP interacting with living mouse fibroblast cells has been studied by Rayleigh light-scattering microscopy.40 Two-photon
Rayleigh scattering (TPRS) properties of gold nanorods can be used for rapid, highly sensitive and selective detection of Escherichia coli bacteria from aqueous solution. TPRS intensity increased 40 times when nanorods conjugated to antibody specific to E. coli were mixed with various concentrations of E. coli O157:H7 bacterium.41 Diagnostics of single base-mismatch DNA hybridization on AuNPs has been achieved using TPRS.42 The use of plasmonic NPs as highly enhanced photoabsorbing agents has introduced plasmonic photothermal therapy (PPTT). The synthetic tunability of the optothermal properties and the biotargeting abilities of the plasmonic gold nanostructures make the PPTT method even more promising. Scientists discussed the development of the PPTT method using gold nanospheres coupled with visible lasers and gold nanorods and silica-gold nanoshells coupled with near-infrared (NIR) lasers.43 AuNPs protected by thermosensitive diblock copolymers with tunable collapse temperature have potential uses for biomedical applications.44 AuNPs (1.9 nm in diameter, ∼50 kDa) as a CT contrast agent exhibited good stability, high x-ray absorption, a good safety profile, long blood half-life, and enhanced CT contrast of the vasculature, kidneys, and tumor in mice.45 Chithrani et al.46 showed that AuNPs between 14 and 74 nm in diameter entered cultured HeLa tumor cells in medium that contained 10% serum and were trapped in vesicles in the cytoplasm. Gold nanoprobes selectively and sensitively targeted tumor-selective antigens while inducing distinct contrast in CT imaging.47 SPIO NPs coated with a lipid bilayer induce cells to acquire magnetic activity following uptake into cells, and biotinconjugated MNPs functionalized with fluorescent tag (streptavidin–fluorescein isothiocyanate) conferred both magnetic activity and fluorescence labeling.48 The appropriate use of QDs and dendrimers enabled the simultaneous tracking of cancer cells within draining lymphatics.49 Applications for specific nanocarriers Development of different types of INPs has led to novel health-care and medical applications,50,51 some of which are already commercially available. At present only NP-based magnetic fluid hyperthermia to treat brain cancers52 (Magforce, Berlin, Germany) and the use of Tat peptide–derivatized MNPs for stem cell therapy for cardiac diseases53 have proceeded to clinical trials. Examples of various medical or biomedical applications are here selected from various nanocarriers such as QD nanocrystals, MNPs, metal NPs, silica NPs, inorganicorganic hybrid nanomaterials, hybrid dendrimer nanomaterials, bioinorganic nanohybrid materials, and other nanomaterials related to bone implants and regenerative nanomedicine. Upcoming nanomaterials for in vivo imaging: an alternative to QDs Lanthanide-doped upconversion nanocrystals have been developed as a new class of luminescent optical labels.54,55 Functionalized yttrium(III) oxide–based upconverting nanomaterial is a promising platform for in vivo optical-based diagnostic imaging, and the new particles avoid potential interference from
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tissue autofluorescence. In addition, the NPs are composed of less toxic materials than are QDs.56 Rare-earth doped β-NaYF4:Yb,Er upconversion nanoparticles (UCNPs) with strong UC fluorescence were coated with a thin layer of silicon dioxide (SiO2) to form core-shell NPs, which were further modified with amino groups, and subsequently, rabbit antibodies to CEA8 were covalently linked to the UCNPs to form antibody-UCNP conjugates. The antibody-UCNP conjugates results indicated that the amino-functionalized UCNPs can be used as fluorescent probes in cell immunolabeling and imaging.57 Individual lanthanide-doped UCNPs— specifically, hexagonal-phase NaYF4 (β-NaYF4) nanocrystals with multiple Yb3+ and Er3+ dopants—emit bright anti-Stokes visible upconverted luminescence with exceptional photostability when excited by a 980-nm continuous-wave laser. Furthermore, these UCNPs proved ideally suited for single-molecule imaging experiments.58 QD nanocrystals QDs are inorganic semiconductor nanocrystals having typical diameters between 2 and 8 nm, and have emerged as a new class of fluorescent labels with better brightness, resistance against photobleaching, and multicolor fluorescence emission.59 They are generally composed of atoms from groups II and VI elements (e.g., CdSe and CdTe) or groups III and V elements [e.g., indium phosphide (InP) and indium arsenide (InAs)] of the periodic table. In general, QDs consist of a semiconductor core, overcoated by a shell to improve optical properties, and a cap allowing improved solubility in aqueous buffers. Among the range of available QDs, the ones composed of CdSe cores overcoated with a layer of zinc sulfide (ZnS) are produced by a range of well-developed synthetic routes.60 These QDs become highly fluorescent and feature such attractive optical properties as high quantum yield, large absorption cross section, and high photostability.61 Various QDs solubilization strategies have been developed over the past decades.62-64 QDs have been systematically tried in virtually all fluorescence-based assays and in vivo imaging procedures.59,61,63 NP water solubility is an essential requirement for in vivo imaging. In this regard, various approaches have used ligand exchange of the original hydrophobic surfactant ligands by hydrophilic molecules. The most often applied molecules are thiol-containing compounds, such as mercaptoacetic acid (dihydrolipoic acid derivatives), or more sophisticated compounds such as alkylthiol-terminated DNA, thioalkylated oligoethyleneglycols, D,L-cysteine, poly(ethylene glycol [PEG])terminated dihydrolipoic acid having significant in vitro and in vivo stability.65-67 Another strategy for solubilization is based on encapsulation into a layer of amphiphilic diblock or triblock copolymers, phospholipid micelles, silica shells, or amphiphilic polysaccharides, polymer shells, oligomeric phosphine coating, or by phytochelatin-peptides coating or histidine-rich proteins.68-70 Glutathione-capped CdTe, zinc selenide (ZnSe), and Zn1 – xCdxSe QDs were successfully synthesized in aqueous solution with fluorescence emissions tunable between 360 and 700 nm, having a quantum yield of up to 50%.71 The glutathione-capped CdTe QDs were successfully conjugated with bioprobes for
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cell-imaging applications. QDs can be covalently linked with biorecognition molecules such as peptides, antibodies, nucleic acids, or small-molecule ligands for use as biological labels in molecular and cellular imaging.73,74 Biological applications of QDs include fluorescence resonance energy transfer analysis, gene technology, fluorescence labeling of cellular proteins, cell tracking, pathogen and toxin detection, and in vivo animal imaging.75-81 One-pot synthesis, encapsulation, and solubilization of highquality QDs based on the use of amphiphilic multidentate polymer ligands and short PEGs at high temperatures allowed for in situ growth of an inorganic passivating shell (CdSe) on the QD core, with a large dynamic range for QD emission from the visible to the NIR.82 Multiple QD fluorescence in situ hybridization probes with different emission spectra were used in a one-step hybridization-detection experiment to visualize, in situ, two subchromosomal regions of highly condensed chromatin structure within the centromere.83 The most fruitful inorganic materials–based optical contrast agents are QDs. First, in vivo studies with QDs in cell lineagetracing experiments with frog embryos,84 and then, their use to image cell signal transduction,85 cancer markers,86 and tumors in living animals87 were reported. QD-conjugated probes to specific biomarkers are powerful tools that can be applied in a multiplex manner to single tissue sections to measure expression levels of multiple biomarkers.88 QDs coated with zinc(II)dipicolylamine coordination complexes selectively allowed optical detection in a living mouse leg infection model.89 QDs have the potential to function as multimodal imaging platforms in vivo and the ability to detect an optical NP preoperatively with clinical imaging modality offered a distinct advantage to clinicians engaged in image-guided surgical applications.90,91 QDs, in addition to being excellent fluorescent probes, can be used as photoacoustic and photothermal contrast agents and sensitizers, thereby providing an opportunity for multimodal high-resolution (300 nm) photoacousticphotothermal fluorescent imaging as well as photothermal therapy.92 Applications of QDs at the cellular level, including immune labeling, cell tracking, in situ hybridization, fluorescence resonance energy transfer, and in vivo imaging, have been reported.93 QDs have potential for the study of intracellular processes at the single-molecule level, high-resolution cellular imaging, longterm in vivo observation of cell trafficking, tumor targeting, and diagnostics.94,95 Human metaphase chromosomes from transformed lymphocyte cultures and breast cancer cell line SK-BR-3 were analyzed by fluorescence in situ hybridization based on streptavidin-linked CdSe QDs.96,97 The use of streptavidincoupled QDs for live-cell imaging has also been reported.98 Cadmium sulfide–cadmium hydroxide QDs maintained high levels of luminescence as well as high photostability in cells and tissues.99 Noninvasive visualization of blood vessels was observed using QDs over time.100 Strongly luminescent copper indium disulfide–ZnS core-shell nanocrystals functionalized with dihydrolipoic acid to the aqueous phase were used as fluorescent labels for in vivo imaging.101 Dextran-coated nanoworms particles composed of a linear aggregate of 5–10 iron oxide cores and a total length between
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∼50 and 80 nm allowed a larger number of interactions between peptides conjugated to the nanoworm and cell surface receptors to take place relative to nanospheres of similar composition.102 Stable PEGylated QDs capped with D-mannose, D-galactose, and D-galactosamine provided selectivity and accumulated in the liver but not in other parts of the body. These could be used to deliver anticancer drugs to one organ, without causing the body-wide side effects that occur with currently existing cancer drugs.103 The promising clinical potential of QDs, their limitations for clinical use, and the recent progress in abrogating their inherent toxicity have been reported.104 Glutathionemediated release of functional plasmid DNA from positively charged CdTe QDs has been demonstrated, thereby suggesting potential applications of these positively charged QDs in selective unpacking of payload in living cells.105 InP-ZnS QDs can be used to replace CdSe-based ones (unsuitable due to toxicity) for almost any biomedical application.106 However, InP-ZnS QDs must be covered with other materials, allowing dispersion and preventing leaking of the toxic heavy metals in case of biological applications.107 Multivalent metal cations have been used to remove water-soluble CdTe QDs capped with thioglycolate ligands.108 The optical properties of QDs, the biofunctionalization strategies, and focus on their biosensing and in vivo imaging applications have been reported.109 Researchers have used QDs linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neuoverexpressing live cells successfully, thereby indicating that composition based on QDs and scFv antibody can be successfully used for cancer cell visualization.110 Both QDantibody and QD-IgG probes were successfully used to label HeLa cells using carboxyl-functionalized CdSe QDs, thereby suggesting practicability of CdSe QDs as attractive fluorescent labels for biological applications such as protein probes and cell imaging.111 QD-gelatin nanocomposites exhibited increased QD luminescence efficiencies, and the nanocomposites penetrated the cell membrane and illuminated the cytoskeleton of macrophage cells.112 Furthermore, live-cell labeling of cytoplasmic structures, including actin microfilaments, through the incorporation within the nanocomposite core of QDs conjugated with antibody to actin was demonstrated.113 QDs are also an attractive choice for regenerative therapy114 and were used to identify the presence and monitor the progression of respiratory syncytial virus infection over time by labeling the F and G proteins, suggesting that QDs may provide a method for early, rapid detection of viral infection.115 The cytotoxicity of green and red mercaptopropionic acid–capped CdTe QDs was significantly enhanced in human pancreatic carcinoma cells (PANC-1) under ultraviolet illumination.116 Multifunctional QDs In vivo targeting studies of human prostate cancer growing in nude mice indicated that the QD probes can be delivered to tumor sites, achieving sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions.117 The conjugation of QDs with photosensitizers and targeting agents provided a new class of versatile multifunctional NPs for both diagnostic imaging and therapeutic applications.118 QD-apata-
mer-doxorubicin conjugate, a multifunctional NP system, can deliver doxorubicin to the targeted prostate cancer cells and sense the delivery of doxorubicin by activating the fluorescence of QD, while allowing for simultaneous imaging of the cancer cells.119 Researchers have demonstrated a simple electrostatic conjugation scheme to conjugate QDs to hyaluronic acid (HA) to produce stable and size-tunable (50 to 120 nm) HA-QD conjugates that showed cancer specificity. Furthermore, these HA-QDs can be also used to visualize lymphatic vessels by fluorescence staining, because they can be delivered into lymphatic endothelial cells through receptors such as LYVE-1.120 Hydrophobic QDs incorporated into the bilayer membrane of lipid vesicles are capable of fusing with live cells, thereby selectively staining the cell's plasma membrane with QDs and either transferring the vesicle's cargo into the cell or transferring it into the cytoplasm of live cells.121 QDs as photosensitizers in photodynamic therapy have provided a novel use as an anticancer therapy.122 Multifunctional high-density lipoprotein NPs have been developed by the incorporation of gold, iron oxide, or QD nanocrystals for CT, MRI, and fluorescence imaging, respectively.123 Magnetic nanoparticles Magnetic inorganic nanomedicine is emerging rapidly, and the nanomaterials used are MNPs, which include nanowires, nanospheres, nanotubes, and magnetic thin films. Diagnostic uses of MNPs include detection of malignant tissues or pathogenic bioaggregates. Use of MNPs has advanced MRI, guided drug and gene delivery, MFH cancer therapy, tissue engineering, cell tracking, and bioseparation. Integrative therapeutic and diagnostic (i.e., theragnostic) applications have emerged with MNP use, such as MRI-guided cell replacement therapy or MRI-based imaging of cancer-specific gene delivery.124 Great progress has been made in the applications of MNPs in biomedicine.125-128 SPIO NPs (15– 60 nm) can be coated with dextran, phospholipids, or other compounds to inhibit aggregation and passive or active targeting agents.129 Upon controlled surface functionalization and coupling with fragments of DNA strands, proteins, peptides, or antibodies, SPIO NPs can be used for drug delivery, magnetic separation, MRI contrast enhancement, and MFH.130 Cationic MNPs enter into cells with high effectiveness and remain localized in endosomes; they are easily detected inside cells by optical microscopy, are retained for relatively long periods of time, and do not induce cytotoxicity.131 A nonspecific labeling method based on anionic MNPs can predict uptake efficiency by all cell types including tumor cells. In addition, the same label provided sufficient magnetization for MRI detection and distal manipulation.132 Fluorescence-modified chitosancoated MNPs for high-efficiency cellular imaging have been developed.133 SPIO NPs are used for MRI and noninvasive cell tracking.134,135 SPIO NPs have the unique capability as contrast agents for MRI due to their ability to shorten T2⁎ relaxation times of the gastrointestinal tract, liver and spleen, lymph node, blood pool, and atheromatous plaque.136 Researchers have summarized the recent progress to develop NP-based T1 contrast agents in inorganic NP-based MRI contrast agents.137
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MNPs can be used in a wide variety of biomedical applications such as contrast agents for MRI (to differentiate healthy and pathological tissues), to combine imaging with hyperthermia treatment or imaging with drug delivery, and to visualize various biological events inside the body.138-141 Scientists have described a novel strategy for NP delivery for treating the hypoxic regions of tumors.142 Scientists have fabricated MRI probes by tuning NPs magnetism by varying their size or composition.143,144 Iron oxide MNPs have received U.S. Food and Drug Administration (FDA) approval to be used as MRI signal enhancers. Recent advances in the development of targeted iron oxide NPs for tumor imaging and therapy have been reported.145 Cellular labeling with ferumoxides (Feridex I.V.; Advanced Magnetics, Inc., Cambridge, Massachusetts) SPIO NPs can be used to monitor cells in vivo by MRI.146 Ferumoxides (Feridex I.V.), dextran-coated SPIO NPs, were approved by the FDA as an MRI contrast agent. Ultra-small SPIO NPs such as ferumoxtran-10 and ferumoxytol have particle diameters of 20 to 50 nm and have longer plasma half-lives of 14–30 hours.147 A family of calcium indicators for MRI formed by combining a powerful SPIO-based contrast mechanism with the versatile calcium-sensing protein calmodulin and its targets has been reported.148 SPIO NPs measuring 2–3 nm have been used in conjunction with MRI to reveal small and otherwise undetectable lymph node metastases, and ultrasmall SPIO enhances MRI for imaging cerebral ischemic lesions. Dextran-coated iron oxide NPs enhance MRI visualization of intracranial tumors for more than 24 hours.149 SPIO nanocrystals encapsulated inside mesostructured silica spheres, labeled with fluorescent dye molecules, and coated with hydrophilic groups, prevented aggregation. Furthermore, water-insoluble anticancer drugs were delivered into human cancer cells, and surface conjugation with cancerspecific targeting agents increased the uptake into cancer cells relative to that in noncancerous fibroblasts.150 The ability of coated SPIO to be rapidly taken up and distributed into lymphoid tissues demonstrated the feasibility of macrophage-targeted nanoformulations for diagnostic and drug therapy.151 Advances in MNP design, in vitro and animal experiments with MNP-based drug and gene delivery, and clinical trials of drug targeting have been reported.152 The targeted detection technique can be used for detecting extremely early signs of disease.153 Biocompatible iron oxide MNPs coated with PEG and size-dependent accumulation in murine tumors following intravenous injection showed enhanced MRI contrast of the larger MNPs in the tumor.154 Multifunctional MNPs Two strategies are utilized to fabricate MNP-based multifunctional nanostructures.155 The first, molecular functionalization, involves attaching antibodies, proteins, and dyes to the MNPs, whereas the other integrates the MNPs with other functional nanocomponents such as QDs or other MNPs. Hybrid nanostructures, which combine MNPs with other nanocomponents, exhibited paramagnetism alongside features such as fluorescence or enhanced optical contrast, and thus could
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provide a platform for enhanced medical imaging and controlled drug delivery.155 Photonic explorers for biomedical use with biologically localized embedding have been successfully applied for significant MRI contrast enhancement and photodynamic therapeutic efficacy with targeted nanoplatforms in a rat 9L gliosarcoma in vivo model, thereby, demonstrating significant promise for their use as a combined therapeutic and diagnostic tool for cancer.156 Dexamethasone conjugated to rhodamine B gum arabic–modified MNPs by photosensitive linker showed significant release of dexamethasone by phototriggered response on exposure to radiation having a wavelength in the NIR region, thereby suggesting an on-demand release of the drug by this nanocarrier.157 The excellent passive tumor-targeting efficiency of thermally cross-linked SPIO NPs allowed detection of tumors by MRI and at the same time exhibited anticancer activity.158 Multicolored highly fluorescent superparamagnetic nanoparticles employing hydrophobic multicolor QDs and hydrophobic Fe3O4 (MNPs) fabricated via ultrasonic emulsification have potential for serving as a hybrid of QDs and MNPs in bioanalysis communities.159 Magnetic nanowires Most magnetic nanowires are compatible with living cells and can be functionalized with biologically active molecules. Many efforts have been made to explore the applications of singlesegment and multiple-segment magnetic nanowires in biomedicine.160 Compared to nanospheres, multifunctionality can be more easily realized on multisegment nanowires.161 Magnetic nanowires used in biomedicine are metal cylindrical electrode(s) positioned in nanoporous templates162 and have shown potential for use in biosensing applications and construction of nucleic acids sensors.163 Functionalization with biomolecules involving two-segment nickel-gold nanowires served as synthetic gene delivery systems.164 The binding of pcDNA3 to the nickel segment of the nanorod provided a strong immunostimulatory adjuvant effect to the antigen bound on the gold segment.165 Separation of heterogeneous cell mixtures based on the differences in physical size of the cells has been achieved with magnetic nanowires.166 Magnetic cell therapy Magnetic cell therapies are applied worldwide at hospitals and university clinics on a case-by-case basis. Repairing blood vessels with cell therapy is a very important concept that can be realized with magnetic targeting. Intravascular gene targeting can be combined with positioning of the transduced cells via MNPs, thereby combining gene-based and cell-based therapies.168 Magnetic fluid hyperthermia In MFH, SPIO NPs used for hyperthermic treatment of tumors are incorporated by the tumor cells much faster than by healthy cells and become concentrated in the tumor cells. Upon applying an alternating-current magnetic field, the particles increase in heat anywhere from 43°C (110°F) up to 70°C (158°F) inside the tumor, thus destroying the tumor cells, while leaving normal cells unharmed.169,170
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MFH led to a significant growth inhibition in this orthotopic model of the aggressive MatLyLu tumor variant. Additionally, intratumoral deposition of magnetic fluids was found to be stable, allowing for serial MFH treatments without repeated injection.171 The results showed evidence of a localized effectiveness and with only minor to moderate therapyassociated side effects.172 The development of NP-based hyperthermic methods was spearheaded by the German startup company Magforce, with the only hyperthermia setup suitable to treat humans developed at the Charité Medical School, Clinic of Radiation Therapy in Berlin. NP-based MFH therapy against brain tumors is under investigation in a phase II study. Conclusions and perspectives Inorganic nanomedicine holds great promise in diagnostics, drug and gene delivery, sensing and biosensing, and in vivo imaging under the present scenario. Smartly engineered inorganic NPs can boost drug efficacy and can improve drug targeting to specific areas within the body, therefore making treatment less toxic and invasive. Fluorescent QDs serve as credible alternatives to commonly used fluorophores and genetically encoded fluorescent proteins. Silica-based nanomaterials are easily functionalized, which allows for the conjugation or encapsulation of important biomolecules, and have shown promise for cell-selective drug delivery and bioimaging. Different multifunctional agents encompassing both imaging and therapeutic capabilities can manage simultaneous monitoring and treatment of diseases. Scientists argue that applications with targeted NPs are expected to revolutionize molecular imaging and cancer therapy. Undoubtedly, QDs, MNPs, and gold NPs, nanospheres,
nanorods, and nanowires will be an integral part of our imaging toolbox. DNA nonviral vectors in gene therapy represent biomedical applications of bionanohybrids. Inorganic-organic bionanohybrids help in bone tissue regeneration and controlled delivery. Integration of targeting ligands, imaging labels, therapeutic drugs, photodynamic therapy, and other functional moieties into the NP conjugate is highly desirable for effective molecular imaging and molecular therapy of otherwise fatal diseases. Future developments relating to the new contrast agents should enable diagnosis of neurological disorders such as Alzheimer's disease, Parkinson's disease, and stroke. Computational tools might help in predicting the various binding sites for conjugation of NPs to cells, and therefore, theoretical and computation modeling can accelerate visualization of the biological environment of the NPs and assist in the design of nanomaterials for use in medicine. Keeping in view the diversity of engineered NPs, their several possible side effects should not be overlooked. Further research is required on the interaction of nanomaterials with biological systems from the perspective of safe use of nanomaterials. Experts are of the opinion that inorganic nanomedicine has the potential to provide groundbreaking methods for the prevention, diagnosis, and treatment of some otherwise fatal diseases.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.nano.2010.04.004.