Strategies for the biofunctionalization of gold and iron oxide nanoparticles.

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Invited Feature Article

Strategies for the biofunctionalization of gold and iron oxide nanoparticles Raluca M Fratila, Scott G. Mitchell, Pablo del Pino, Valeria Grazu, and Jesús M. De La Fuente Langmuir, Just Accepted Manuscript • Publication Date (Web): 09 Jun 2014 Downloaded from http://pubs.acs.org on June 17, 2014

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Strategies for the biofunctionalization of gold and iron oxide nanoparticles Raluca M. Fratila,1 ,2 Scott G. Mitchell,1 Pablo del Pino,3 Valeria Grazu,1 ,4 Jesús M. de la Fuente1,5,6* 1.

Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, C/ Mariano Esquillor s/n, 50018 Zaragoza, Spain.

2.

Fundación ARAID, C/ María de Luna 11, Edificio CEEI Aragón, 50018 Zaragoza. Spain

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CICBiomaGUNE, Paseo Miramon 182, 20009 San Sebastián. Spain

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Nanoimmunotech SL, Edificio Cero Emisiones, Avenida de la Autonomía 7, 50003 Zaragoza. Spain

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ICMA-CSIC. C/ Pedro Cerbuna 12, 50.009 Zaragoza (Spain)

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Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and Microfabrication Technology of the Ministry of Education, Research Institute of Translation Medicine, Shanghai Jiao Tong University, Dongchuan Road 800, 200240 Shanghai, People’s Republic of China.

KEYWORDS: Nanoparticles, Biofunctionalization, Antibodies, Peptides, DNA, Carbohydrates.

ACS Paragon Plus Environment

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ABSTRACT: The field of nanotechnology applied to medicine (nanomedicine) is developing at a fast pace and is expected to provide solutions for early diagnosis, targeted therapy and personalized medicine. However, designing nanomaterials for biomedical applications is not a trivial task. Avoidance of the immune system, stability in physiological media, control over the interaction of a nanomaterial with biological entities such as proteins and cell membranes, low toxicity and optimal bioperformance are critical for the success of the designed nanomaterial. In this Feature Article we provide a concise overview of some of most recent advances concerning the derivatization of gold and iron oxide nanoparticles for bioapplications. The most important aspects relating to the functionalization of gold and iron oxide nanoparticles with carbohydrates, peptides, nucleic acids and antibodies are covered, highlighting the recent contributions from our research group. We suggest tips for the appropriate (bio)functionalization of these inorganic nanoparticles in order to preserve the biological activity of the attached biomolecules and ensure their subsequent stability in physiological media.

1. Introduction Recent developments in the field of colloidal science have extended our ability of fine-tuning the physicochemical properties of nanomaterials in order to achieve enhanced or novel magnetic, optical and electronic properties when compared to their bulk counterparts. These features enable engineered nanomaterials to operate at the biomolecular level, with functions ranging from contrast for (multimodal) molecular imaging1 to more complex tasks such as drug delivery, targeting or therapy.2,3 Multiple tasks can be also combined in one single “smart” multifunctional nanomaterial, resulting in the development of theranostic nanoprobes for simultaneous detection and therapy of diseases.4,5 The field of nanotechnology applied to medicine (also called


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nanomedicine) has witnessed significant advances during the last decade and holds great promise for early diagnosis, targeted therapy and personalized medicine. However, several important challenges need yet to be overcome in order to make nanomedicine widely available to patients: avoiding the immune system, controlling the interaction of nanomaterials with biological entities such as proteins and cell membranes, anticipating their toxicity and ensuring an optimal bioperformance.6,7 The aim of this Feature Article is to give a concise overview of the most recent advances in the field of derivatization of smart nanomaterials for bioapplications, with emphasis on the importance of their appropriate (bio)functionalization in order to ensure the stability in physiological media and to preserve the biological activity of the attached biomolecules. Due to the overwhelming diversity of nanoparticles (hereafter referred to as NPs) and biologically relevant molecules, we will focus solely on gold and iron oxide NPs, the two types of inorganic nanomaterials that showed the most promising in vivo applications so far. The most important aspects of their functionalization with carbohydrates, peptides, nucleic acids and antibodies will be covered, highlighting the recent contributions from our research group. First, we will provide a brief overview of the methods employed for a primary coating of the NPs in order to improve their colloidal stability in physiological media. We will then address their functionalization with molecules of biological relevance and discuss the most promising biomedical applications of the resulting nanoconstructs. We will conclude with a critical outlook of the field, identifying the main challenges and possible future directions. We certainly hope that we will provide an interesting and useful reading for researchers in the fields of nanomedicine and nanobiotechnology, but also for a broader readership.


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a O Au NPs

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Figure 1. a) Types of nanoparticles discussed in this review and biomolecules of increasing complexity used for their functionalization. b) Functionalization steps necessary for biomedical applications. 2. Primary coating of nanoparticles Physiological media are highly complex fluids, containing salts, carbohydrates, lipids, proteins, amino acids and enzymes that can destabilize nanoparticles and cause their aggregation through van der Waals interactions. Therefore, the colloidal stability of the NPs in the biological milieu is a must for a successful biomedical application. Ideally, the NPs should also be able to overcome biological barriers and to avoid recognition by the immune system, which would lead to their unspecific accumulation in the liver and spleen and compromise their envisaged application. The primary immune response of the body to foreign entities such as NPs consists in


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the absorption of serum proteins (called opsonins) onto their surface. As a result of the opsonization, NPs circulating freely in the body are recognized by phagocytes and cleared from the bloodstream.6 An appropriate coating is therefore of crucial importance for obtaining robust NPs with high colloidal stability, preserving the physicochemical properties of the inorganic core and defining the interaction of NPs with their biological environment. Coating can also provide a flexible surface chemistry for further functionalization with biomolecules and allows for the outer layer of the NP to be engineered for specific biological interactions. The most common strategies for engineering the primary coating of NPs include ligand exchange, coating with silica, polymer wrapping and encapsulation (Figure 2).1,8

Coating with amphiphilic polymers

Ligand exchange Hydrophobic nanoparticle

Silica coating

Layer-by-layer (LbL) coating

Liposomes Lipospheres

Figure 2. Primary coating strategies for engineering the surface of nanoparticles. The ligand exchange of the original surfactant with hydrophilic ligands is the most straightforward method to derive water colloidally - stable NPs and relies on the ability of the chosen ligand to replace the original bound or adsorbed molecules. In this approach, the chemical affinity of the ligand for the material and the presence of ligand excess are key parameters for achieving a densely packed ligand shell. The choice of the ligand depends on the


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composition of the inorganic core, a particularly prolific approach involving the use of thiolated chains to form self-assembled monolayers on the surface of gold nanoparticles.9,10 We have used different types of heterofunctional thiolated poly(ethylene glycol) linkers as exchange ligands for citrate or sulfate to yield stable spherical Au NPs11–13 or Au nanoprisms,14 respectively. The function of these linkers can go well beyond their primary stabilization task; for instance, carboxylated linkers provide anchoring points for further covalent functionalization with amineterminated biomolecules, while azide-containing spacers ensure a positive ζ-potential that is crucial for cellular uptake. Coating with silica yields robust water-soluble NPs with good colloidal stability and biocompatibility. Other important advantages of this versatile coating alternative include easy control of the coating process, optical and magnetic transparency, low cost, controlled porosity and the possibility for subsequent functionalization of the NPs via well-established siloxane chemistry. For an overview of various synthetic approaches to silica-coated nanomaterials, we refer the reader to the review of Liz-Marzán and coworkers,15 while details regarding the biofunctionalization of silica-coated NPs can be found in the excellent review by Erathodiyil and Ying.1 Polymer coating has been also widely employed to increase the stability of nanomaterials in water and to increase their blood circulation by reducing their nonspecific uptake. Typical polymers include poly(ethylene glycol) (PEG), dextran and chitosan derivatives, which are highly biocompatible and, conveniently, often possess additional functional groups necessary for further conjugation with biomolecules. Parak and coworkers have developed a versatile methodology to transfer hydrophobic inorganic NPs coated with hydrocarbon chains to water by using amphiphilic polymers.16 This approach exploits the nonspecific hydrophobic interactions


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between the surfactant chains of hydrophobically capped NPs and the alkyl chains of the polymer. Upon cross-linking of the polymer chains around each nanoparticle and hydrolysis of the unreacted anhydride groups, the NPs become water-soluble and can be further functionalized with the biomolecules of interest. Our group has successfully employed this method for the transfer of magnetic iron oxide nanoparticles to aqueous media using poly(maleic anhydride-alt1-octadecene) (PMAO).17 The main advantage or our improved version is the synthetic efficiency: only carboxylic groups are generated on the surface of the NPs, thus facilitating their subsequent functionalization; moreover, the cross-linking step is eliminated, shortening and simplifying the synthetic protocol. Another important advantage concerns the easy purification of the stabilized NPs. As opposed to previously reported purification strategies that used gel electrophoresis, size exclusion chromatography or sucrose gradients to remove the excess of polymer, in our method a simple centrifugation at high speed was sufficient for this purpose. Encapsulation of inorganic NPs in organic structures can also be used to engineer hybrid nanostructured materials. Del Pino et al. employed lipospheres functionalized with magnetic nanoparticles as magnetic vectors to deliver siRNA to cells under fluidic conditions mimicking the blood flow.18 Polymersomes loaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO) and doxorubicin have been reported as proof-of-concept of magnetochemotherapy, in which the drug is released from its container under local magnetic hyperthermia conditions. Moreover, these hybrid self-assemblies have shown a significant contrast enhancement in MRI with a subnanomolar limit of detection.19 The layer-by-layer (LbL) encapsulation strategy was used to coat Au NPs by the sequential deposition of oppositely charged polyectrolyte solutions - poly(ethylene imine) (PEI) and siRNA - to yield well-defined nanocarriers for siRNA delivery.20


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Of course, the choice of one or another of the aforementioned coating strategies is strongly influenced by the nature and original coating of the inorganic nanomaterial and the ultimate biological application envisioned for the nanomaterial in question.

3. Functionalization of nanoparticles with biologically relevant molecules Once the NPs have an adequate colloidal stability and the desired chemical moieties, after the coating of the inorganic core, the second step to obtain bioactive NPs is to select the correct biofunctionalization strategy. This is not an easy task, for no universal methodologies are available so far to cover the wide variety of nanomaterials and biomolecules available. A functionalization protocol that works well for one material may not work for another, since they could be very different in terms of size, surface area, colloidal stability, density and type of reactive groups, etc. Furthermore, the biomolecules to be conjugated to the surface of the material could have significantly different size, chemical composition, 3D complexity, location of the biological active site, and so forth. In the absence of standard functionalization protocols or procedures, each particular case (nanomaterial + biomolecule) requires a careful optimization, always keeping in mind the final application.

3.1. Functionalization of nanoparticles with low molecular weight biomolecules (carbohydrates and peptides) Carbohydrates and especially their bioconjugates with peptides, proteins and lipids (so-called glycoconjugates) are involved in many normal and pathological biological processes, such as cell-cell recognition, protein folding, and inflammatory events. Therefore, a great deal of research is being devoted towards understanding the role of carbohydrates in these processes.


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However, the study of biological interactions involving sugars is not trivial because of their low affinity that in Nature is usually compensated by a multivalent presentation of the ligands. An excellent tool to mimic this multivalency in synthetic systems consists in the attachment of carbohydrates to inorganic nanoparticles. The concept of “glyconanotechnology” developed by the group of S. Penadés in the early 2000s had a significant impact for the study of biologically relevant carbohydrate interactions;21–23 herein, we highlight some recent examples of carbohydrate-functionalized nanoparticles for diagnostic and therapeutic applications. The functionalization of NPs with carbohydrates is relatively easy when compared to more complex biomolecules (see the next sections on nucleic acids and antibodies). The chemistry used for the sugar-NP conjugation itself involves classical EDC-promoted amide coupling and Au-S bond formation. Moreover, the chemical modification of the carbohydrate molecules prior to their conjugation to the NPs is quite straightforward, and a wide variety of functionalized sugars are nowadays commercially available. However, there are some important aspects to be taken into account when designing glyconanoparticles for biomedical applications. Carbohydrate density and presentation on the NP surface are of particular importance as they can affect the molecular recognition behaviour of NPs. A low carbohydrate density can lead to unspecific biological interactions of the NPs, including undesired protein absorption, while a too high surface coverage can compromise recognition events due to steric hindrance.23 As mentioned in the previous section, obtaining NPs with a controlled size and high stability in biological media represents one of the most challenging goals for biotechnological applications. Our group reported the first examples of the use of carbohydrates as alternative to PEG for the coating of magnetic Au-Fe and Fe3O4 nanoparticles.17 We have shown that monosaccharides have the ability to inhibit unspecific interactions of the NPs with proteins, providing stable,


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water-soluble nanoparticles. The density of the attached molecules was found to be crucial in order to prevent protein adsorption. We found out that although a low density of glucose moieties was sufficient to inhibit adsorption of large proteins due to steric hindrance, it could not impede adsorption of very small and positive proteins such as lysozyme. Different monosaccharides present on the surface of the NP elicit a different cellular response, suggesting that tailoring the nanoparticle coverage could open up the way to control cell-nanomaterial interactions. In addition to the passivation of the NP surface, derivatization with sugars provides a valuable tool for studying carbohydrate-protein interactions. The specific interaction between glucose and Concanavalin A (ConA) has been demonstrated for the glucose-functionalized NPs by aggregation studies of the NPs in presence of the ConA (Figure 3a). The clustering of the NPs could also be verified by NMR relaxometry and magnetic resonance imaging (MRI), since aggregated nanoparticles show a significant shortening of the transverse relaxation time values (T2) of water protons (see Figure 3b).


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Figure 3. a) Interaction of glucose- and galactose- functionalized magnetic NPs with Concanavalin A. b) T2-weighted MR images of Glu-NPs (top) and Gal-NPs (bottom) incubated with various concentrations of ConA. Reproduced from ref. 17 with permission from The Royal Society of Chemistry. c) Cell internalization of glucose NPs via the lipid raft uptake mechanism. Reproduced with permission from ref 24. Copyright (2012) American Chemical Society. We have performed the first detailed investigation of the cell-internalization route of carbohydrate-functionalized NPs using Vero cells.24 Our findings showed that the cellular uptake and distribution depend on the type and density of the grafted molecules. Glucose-NPs entered throughout the cell, while galactose-NPs were mainly found at the cell periphery or remained


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attached to the cell membrane. Given the fact that glucose- and galactose-functionalized NPs shared the same type of inorganic core, hydrodynamic diameter and ζ-potential, this clearly different cellular distribution can only be attributed to the carbohydrate moiety. This finding reinforces the importance of the appropriate derivatization of NPs in order to achieve the desired biofunctionality. Glucose-NPs were internalized mainly by a caveolar/lipid raft uptake, with the clathrin-mediated endocytosis pathway commonly acknowledged for NPs smaller than 200 nm having only a minor contribution in the cell internalization (Figure 3c). An extensive cytotoxicity evaluation including cell viability assays on different cell lines, reactive oxygen species (ROS) production, cell cycle disruption and activation of the complement system showed that these carbohydrate-modified NPs are nontoxic, thus reinforcing the potential of glyconanoparticles for in vivo applications. Penadés and coworkers have reported the use of mannose-functionalized Au nanoparticles as inhibitors for the C-type lectin DC-SIGN, a carbohydrate-binding protein involved in the HIV trans-infection.25 The multivalent presentation of the sugar moieties on the surface of NPs was correlated to the efficient inhibitory activity of this synthetic system, in the nanomolar concentration range. Early detection of inflammatory processes associated with neurological disorders such as multiple sclerosis and stroke is extremely difficult because conventional imaging techniques rely on the increased permeability of the blood-brain barrier (BBB) at later stages of these diseases. Van Kasteren et al. have reported the first example of a nontoxic, MRI-visible iron oxide glyconanoparticle acting as a highly efficient T2-contrast agent and allowing for the presymptomatic in vivo detection of brain diseases.26 The interaction of the tetrasaccharide sialyl LewisX (sLeX) with carbohydrate-binding transmembrane proteins CD62E (E-selectin) and


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CD62P (P-selectin) regulates the initial tethering and rolling of leucocytes in the inflammatory cascade. The authors achieved the aforementioned multivalent affinity enhancement by designing nanoparticles decorated with a large number of copies of sLeX, which allowed for the detection of CD62E by MRI in clinically relevant models of multiple sclerosis and stroke. Noteworthy, this method enables the detection of a biomarker present in the blood side of the BBB, while being indicative of a pathological situation present on the brain side, therefore eliminating the challenge of crossing the BBB, one of the most restrictive barriers present in the body. Frigell et al. recently addressed the problem of BBB targeting with multifunctional Au glyconanoparticles decorated with small neuropeptides as vectors for BBB passage and

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complexes as reporters for in vivo positron emission tomography (PET) imaging.27 Targeted glyconanoparticles bearing a Leu-enkephalin peptide showed improved brain uptake when compared to their non-targeted counterparts, confirming the importance of the appropriate functionalization of nanomaterials for achieving favorable properties for biomedical applications.

Figure 4. Glyconanoparticles for neuroimaging and blood-brain barrier targeting. a) Magnetic NPs decorated with sLeX allow for the detection of brain inflammation using MRI. Adapted with


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permission from ref 26. b) Multifunctional Au NPs for PET imaging. Functionalization with glucose and neuropeptides improves the brain uptake. Reproduced with permission from ref 27. Copyright (2014) American Chemical Society. Dually functionalized Au NPs have been very recently explored by Vargas-Berenguel and coworkers as targeted drug-delivery vehicles.28 Multiple copies of β-D-lactose units were incorporated on the surface of the NPs as targeting ligands for human galectin-3 (Gal -3), a carbohydrate-specific protein overexpressed on cancer cells surface, while β-cyclodextrin moieties were used as containers for the anticancer drug methotrexate. Although the NPs were not tested on cell cultures or animal models, both carbohydrate-protein affinity studies and drug loading ability tests revealed their potential as a site-specific drug delivery system. In order to perform their in vivo function, nanomaterials must first be able to avoid the immune system and reach their target tissue in a specific and highly efficient fashion. At the desired site, NPs face a second challenge: cellular uptake, which is crucial for their therapeutic or imaging function. Peptides are particularly well-suited for targeting and cell delivery, since they are small, non-immunogenic molecules with low toxicity, relatively easy to synthesize and to modify with the desired chemical functionality.29,30 Moreover, the multivalent presentation on the NP surface can enhance the generally moderate affinity of the targeting peptides to their receptors. Depending on the chemical functionalities present on the NP surface, peptides can be covalently linked using their primary amine (N-end) or carboxylic acid (C-end) groups via classical peptide coupling methods. Peptides containing specific amino acids such as lysine or aspartic acid possess extra amino or carboxyl groups and in these particular cases special attention has to be paid to the conditions chosen for coupling in order to promote the reaction via the terminal amine or carboxylic acid. For instance, if the peptide contains lysine residues the pH of the coupling


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reaction must be carefully adjusted in order to direct the coupling through the terminal amine group (pKa ~7-8) and to avoid reaction of the ε-amine group (pKa ~10.5-10.7). Another way to direct the coupling reaction through a terminal group when several reactive (CO2H and NH2) moieties are present in the peptide sequence is the incorporation of an extra terminal cysteine residue. If amine groups are present on the surface of the NP, any amine-to-sulfhydryl heterobifunctional crosslinkers could be used to perform the covalent binding of the peptide to the NP, including succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), or N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Cell-penetrating peptides (CPPs) or “Trojan horse” peptides are small peptides with cell membrane-translocation properties and have been used in the last decade to facilitate the cellular uptake of different cargo entities, from small molecule drugs to large biomolecules and nanoparticles. Tat peptide and penetratin are the CPPs most commonly employed for the transport of nanomaterials into cells and they successfully delivered magnetic and gold NPs both in vitro and in vivo. However, cell-penetrating peptides are not cell-specific and in order to perform targeted delivery functions they must be further “engineered” or used together with a targeting strategy. We have conducted the first “proof-of-concept” study on the combined use of a magnetic field and penetratin for the delivery of magnetic NPs to cells cultured in a collagen gel matrix acting as a three-dimensional model equivalent of a tissue.31 Our results revealed that the presence of the magnetic field indeed increases the penetration depth of the NPs. The cellpenetrating peptide enhances the cellular uptake, but penetratin-functionalized NPs are not able to move away from the surface of the gel in the absence of the magnetic field, stressing out the importance of the combined approach. However, further studies are required in order to develop


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an efficient strategy for in vivo delivery, and one must be aware that the external magnetic fields can cause alterations in the gene expression when applied to cell cultures, as we have observed in one of our initial studies of the combined use of magnetofection and Tat-functionalized NPs.32 One of the most promising approaches to improve the specificity of NPs for the target tissue is their functionalization with so-called “homing” peptides. Active targeting of tumors is based on the recognition of over-expressed receptors present on the cancer cell surface by the homing peptide and has been employed both for imaging and targeted delivery purposes using nanoparticles. Chlorotoxin (CTX), a 36-amino acid peptide with strong affinity for the vast majority of neuroectodermal tumors, has been conjugated to iron oxide nanoparticles incorporating a near-IR fluorophore to yield multimodal imaging nanoprobes for the specific in vivo targeting of brain tumors33 and gliosarcomas34 in mice. Moreover, CTX-modified magnetic nanoparticles have demonstrated their superiority over free CTX in terms of cellular uptake and invasion inhibition rate of glioma cells.35 An interesting example of a cooperative nanosystem comprised of Au nanorods plus iron oxide nanoworms (NWs) or doxorubicin loaded liposomes was reported for targeting and therapy of MDA-MB-453 carcinomas in mice.36 Au nanorods accumulate in tumors and were used to induce local heating of the tumor tissue by absorbing near-infrared radiation (Figure 5). This local heating allows for a more efficient recruitment and cell internalization of the second component, NWs decorated with a cyclic nine-amino acid peptide (LyP-1, Cys-Gly-Asn-ArgThr-Arg-Gly-Cys) that binds the p32 receptors overexpressed on the surface of tumor cells undergoing stress, or liposomes carrying the anticancer drug doxorubicin.


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Figure 5. Cooperative two-component nanosystem. The first component consists of gold nanorods (NR), which act as a photothermal sensitizer. The second component consists of either magnetic nanoworms (NW), or doxorubicin-loaded liposomes (LP). Irradiation of the NR with a NIR laser induces localized heating that stimulates changes in the tumor environments. The NW or LP components decorated with LyP-1 tumor targeting peptides bind to the heat-modified tumor environments more efficiently than to the normal tumor environments. Reproduced with permission from ref 36. Tumor-homing peptides containing the RGD motif (Arg-Gly-Asp) are known to bind αvβ3 and

αvβ5 integrins expressed in tumor vasculature and involved in cell adhesion processes. When coadministered together with chemotherapeutic drugs, RGD peptides enhance their efficiency, while reducing the adverse effects.37 Iron oxide nanoworms (NWs) decorated with a tumor vascular marker (CGKRK, Cys-Gly-Lys-Arg-Lys) and the proapoptotic peptide D[KLAKLAK]2 that induces cell death by disrupting the mithocondrial membrane were employed by Ruoslahti and coworkers as a potential therapy for glioblastoma.38 This system eradicated lentiviralinduced brain tumors in mice, while increasing significantly the survival period of mice bearing more aggressive human U87 glioblastomas, especially when co-administered together with the


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tumor-penetrating peptide iRGD. This is a representative example of a successful design of multifunctional nanomaterials: i) the peculiar elongated geometry ensures more effective targeting because of the multivalent presentation of the ligands; ii) CGKRK has the ability not only to target the tumor vasculature, but also to act at subcellular level by targeting mithocondria; iii) the tumor-penetrating peptide iRGD allows the NPs to reach the tumor cells, in addition to the tumor blood vessels.

3.2. Functionalization with nucleic acids Nucleic acid-based biomolecules have played an integral part in the bionanotechnology for close to two decades and have facilitated the design of a range of materials possessing unique properties, from sensors39 to imaging40 and delivery systems.41,42 The predictable - and programmable - self-assembling properties of DNA by recognition of their complementary molecular motifs, has significantly advanced the directed assembly of nanoscale materials43,44 and distance-dependent physical properties such as heat, electron and energy transfer have also been studied using DNA as a rigid spacer. Consequently, a variety of procedures now exist for linking DNA to surfaces using both covalent and non-covalent approaches, although the selection of an appropriate method is highly dependent on both the type of nanomaterial and its subsequent applied role, either in vitro or in vivo. Perhaps unsurprisingly so, Au NPs occupy a substantial portion of the published literature and thus deserve special attention. Specifically for Au NPs, the most crucial step - and indeed the most widely used method - involves the covalent attachment of a thiol-terminated DNA to the gold surface using a salt-aging protocol to ensure the stability of the functionalized particles under physiological conditions;45 however, new protocols are published regularly. To illustrate this point, two recent related reports describe new


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methods towards rapid and quantitative multiple adsorption of DNA at predetermined ratios using a pH 3.0 citrate buffer, thus providing new approaches to modulate the interactions between both thiolated46 and non-thiolated47 DNA and Au NPs. One area where the controlled functionalization of NPs with DNA becomes especially relevant is the fabrication of DNA-based nanoconstructs as sensing devices. NP-based DNA sensors can detect pathogens and genetic diseases by binding to target disease-related DNA strands and providing a quantitative signal to determine how much DNA is present. This technology has made significant use of Au NPs functionalized with primer strands that bind to the DNA of interest. When the primers link to the targets changes in the UV-visible absorbance of the NPs provide a quantifiable signal. However, sensors employing Au NPs randomly functionalized with single-stranded DNA (ssDNA) can lead to primer strand cross-binding and agglomeration which reduces the number of primer strands available for binding to the target DNA, leading to inaccurate changes in absorbance signals. For example, triangular gold nanoprisms, densely functionalized with oligonucleotide ligands, hybridize to complementary particles with an aﬃnity that is several million times higher than that of spherical nanoparticle conjugates functionalized with the same amount of DNA and exhibit association rates that are two orders of magnitude greater than those of their spherical counterparts.48 The authors attribute this discrepancy to the ability of the large flat extended surfaces of non-spherical AuNPs to support a larger number of AuNP-ligand interactions than spherical counterparts; while at the same time relieving the conformational stresses imposed on AuNP-bound ligands on AuNPs possessing curved surfaces. This has the knock-on effect of increasing the effective local concentration of terminal DNA nucleotides that are available to mediate ssDNA hybridization. However, despite the apparent limitations of spherical AuNPs in this respect, monofunctionalized spherical Au


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NPs can be prepared with just one short complementary ssDNA primer strand of just ten base pairs from Enterococcus faecalis for subsequent DNA detection/hybridization.49 The short primer strands displayed reduced steric and electrostatic repulsions, decreasing the tendency for agglomeration, forming double-stranded (dsDNA)–Au NP complexes which involve either tailto-tail or head-to-head alignment. Since the formation of randomly agglomerated AuNPs can result in non-hybridized primer-DNA cross binding with AuNPs trapped within a sterically crowded network, thereby producing inaccurate shifts in UV/visible absorption and shifts, monofunctionalized Au NPs such as these offer the possibility of achieving clear quantifiable spectroscopic signals via the formation of discrete dimeric (hybridized) dsDNA–Au NP complexes. Oligonucleotide-functionalized Au NPs have also been employed in an ultrasensitive and simple dynamic-light-scattering (DLS)-based method for sequence-specific recognition of dsDNA.50 In this instance, DLS was used to monitor the average change in diameter of hybridized oligonucleotide-modified Au NP probes due to induced aggregation with the target dsDNA - which formed triplex DNA - increasing the average diameter accordingly. The ultrahigh extinction coefficient (108−109) of the surface plasmon resonance (SPR) absorption of Au NPs of course also lends itself to the development of highly sensitive colorimetric assays for sensing applications. Indeed, the construction of a powerful amplification platform to produce large amounts of Au NP aggregates with traces of target DNAs is one of the grand challenges in this area. Recently, a ultrasensitive colorimetric DNA assay was developed whereby the detection of the product of a ligation chain reaction (LCR) was coupled with the use of Au NPs as signal generators.51 Most importantly, this colorimetric assay can be used to quantify and monitor DNA-related Au NP ligation processes in real time, either by visual inspection or with


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high-selectivity using UV-vis spectrophotometry. Breaking from the traditional self-assembly of NP arrays that merely exploit Watson−Crick base pairing of single-stranded DNA sequences, a report by Graham and co-workers describes programmed Au NP aggregation directed by the recognition of dsDNA sequences using minor-groove-binding pyrrole-imidazole polyamide-Au NP conjugates.52 The selectivity and reversibility of this assembly/disassembly strategy was demonstrated in the presence of fully matched dsDNA sequences relative to dsDNA sequences containing one- and two-base-pair mismatches (Figure 6).

Figure 6. Schematic overview whereby the 5′-end of a DNA strand is attached to a different Au NP (GNP2) via thiol−gold linkages. The addition of the complementary ssDNA strand forms a dsDNA duplex on the Au NP surface (i.e., GNP2 or GNP4). (a) Sequence PA 1 exemplifies this


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hypothesis, as its core is known to bind to the seven-base-pair DNA sequence 5′WWGWWCW (W = A/T) with a nanomolar binding constant; (b) Scheme of 1 and ssDNA-functionalized Au NPs and their subsequent aggregation upon addition of DNA matching sequences. Reproduced with permission from ref 52. Copyright (2012) American Chemical Society. Yet despite the obvious applicability of Au NPs, they have the particular issue of being ultraeﬃcient quenchers of light exciting fluorophores, meaning that fluorophores and Au NPs should ideally be separated by bulky spacers, including DNA strands or antibodies, for sufficient fluorescence to be observed. Fluorescently labeled hairpin DNA probes of different lengths attached to Au NPs of varying size experience intimate contact between the fluorophore and the gold, resulting in an efficient energy transfer (quenching); however, hybridization of the hairpin DNA-Au NPs with complementary DNA strands causes stretching and yields a strong increase (dequenching) in fluorescence signal.53 This DNA hybridization approach demonstrates the potential of Au NPs for fluorescent label-free DNA sensing. Magnetic NPs (MNPs) act to lower DNA detection limits as a result of efficient magnetic separation. A complex cocktail of magnetic microparticles and Au NPs coupled with sulfidebased NP tracers has been used as a NP-based bio-barcoded DNA sensor with detection limits as low as 0.2 ng/mL, suggesting potential applications in multiplexed detection of bioterrorism threat agents.54 However, progressive advances in the synthesis of MNPs17 have enabled substantial improvements in their surface functionalization, which now means that magnetic NPs can be used in biomolecule-based sensing applications in their own right.24,55 The assessment of local temperature on (or near to) the surface of nanoparticles in comparison with the global temperature of the medium has proven to be a challenging task, made especially difficult due to temperature gradients associated with distance from NP surface. Recent


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approaches have employed the use of fluorescently labeled azo-functionalized iron oxide NPs to determine the absolute temperature at distances below 0.5 nm from the surface of the NPs.56 However, DNA-functionalized NPs have also been shown to be extremely useful in the sensing of local temperature environments. Determination of local temperatures on the surface of NPs down to 3 nm was achieved by the use of polymer-coated 12 nm iron oxide NPs functionalized with fluorescent-labeled hybridized DNA (Figure 7).57 Under alternating magnetic field (AMF) heating, the dehybridization of the DNA can be monitored by fluorescence spectroscopy due to the presence of fluorophores on the ssDNA. Since chains of hybridized DNA of varying lengths denature at different temperatures, local temperature created by AMF heating could be followed by monitoring the concentration of fluorophore-labeled DNA released from the surface of the iron oxide NP. In summary, during AMF heating, when the global temperature of the media reached a desired temperature the amount of DNA released could be measured and correlated to the local temperature created on the surface of the particle as a result of the heating. The temperature on the surface of the NP was found to be several degrees Celsius higher than the global temperature of solution. For example, during AFM heating, when the global temperature reached 30 ºC, at a distance of 5.6 nm from the particle surface the temperature was found to be 6.1 ºC higher (36.1 ºC), while at 5.3 nm and 5 nm from the surface the temperatures were found to be 7.8 (37.8 ºC) and 8.3 (38.3 ºC) higher, respectively, showing that significantly more heat is produced near to the particle surface.


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Figure 7. Iron oxide NPs functionalized with ssDNA and hybridized with fluorophore-modified ssDNAs: 6-carboxyfluorescein (F), hexachlorofluorescein (H) and sulforhodamine 101 acid chloride (T), which have 10, 11 and 12 base pairs, respectively. Thus the total distance between the 5’-end of the DNA and the NP surface is estimated to be 5, 5.3 and 5.6 nm, respectively, since DNA double helix is considered to be a rigid structure. Under AMF heating, temperature gradients could be determined as a function of the distance of the 5’-end of the DNA strands from the NP surface. For a solution temperature of 30 ºC this corresponded to +8.3, +7.8 and +6.1 ºC for F, H, and T, respectively. Reproduced with permission from ref 57. Copyright Wiley-VCH. Nanomaterials capable of binding DNA in a reliable manner can also be exploited for use as gene delivery systems to treat and prevent disease. RNA can be used as a therapeutic agent by delivering small-interfering RNA (siRNA) to bind cellular mRNA in order to inhibit the translation processes of proteins in the cytoplasm and therefore has an important role in the down-regulation of gene expression. The use of RNA interference (RNAi) to specifically block gene function is dependent on the concentration of siRNA delivered to site of interest - and


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nucleic acids suffer from notoriously poor cellular penetration58 - meaning that the mode of delivery is critical in order to achieve maximum therapeutic effect. Furthermore, delivery agents must circulate unaltered throughout the blood steam, undetected by the immune system. Both magnetic and Au NPs are model ‘non-toxic’ (i.e. passive) carriers for gene therapy due to their reduced immunogenicity and the location of such therapeutic NPs can be monitored by fluorescence techniques, and in the case of MNPs via Magnetic Resonance Imaging (MRI). However, Au NPs in particular have been used with some success to improve siRNA delivery and have been investigated without unearthing negative immune or off-target toxicity.59–61 Recently, our group designed a hierarchical comparative approach to produce a library of smart multifunctional nanostructures as versatile tools for efficient RNA interference in three eukaryotic systems.12 Nanocarrier delivery vectors based on poly(ethylene glycol)-coated Au NPs demonstrated functionality both in vitro (HeLa cultured cells) and in vivo (invertebrates: Hydra vulgaris - freshwater polyps and mammals: mice) for RNA interference therapy (Figure 8). This proof-of-concept study demonstrated that the type of surface functionalization of the NP was critical for efficient siRNA delivery to each eukaryotic system in order to achieve maximum therapeutic effect. More specifically, ionic linking of siRNA to the Au NP was efficient for controlling gene expression in cells and in Hydra; but covalent bonds were required for siRNA delivery in mice. Inclusion of the cell-adhesion peptide RGD on the Au NP surface further enhanced their activity as RGD is recognized by integrins (cell-surface receptors known to mediate cell adhesion and proliferation). This comprehensive chemical and biological strategy enabled the optimum design of an RNAi nanocarrier for therapeutic purposes by studying the effect of cell penetration and adhesion peptides. This functional screening approach was then expanded by taking the most active Au NPs found for RNAi delivery in the in vivo studies to


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treat lung cancer in orthograft mouse tumor models.13 The results demonstrated delayed tumorcell growth and enhanced mouse survival related to an enhanced inflammatory response when compared to the healthy lungs of control mice. RGD-functionalized Au NPs with covalently bound siRNA were effective in targeting LA-4 cancer cells leading to down-regulation of c-myc oncogene expression in both orthotopic mouse models, illustrating the need for specific tailoring of Au NP-based RNAi delivery system.

Figure 8. A functional screening approach to in vivo gene silencing through NP-mediated RNAi. The nanocarrier is an Au NP functionalized with multiple biomolecules: PEG, cell penetration and cell adhesion peptides, and siRNA. Two different approaches were employed to conjugate the siRNA to the Au NP: (1) covalent approach, use of thiolated siRNA for Au-thiol binding to the NP; (2) ionic approach, interaction of the negatively charged siRNA to the modified surface of the Au NP through ionic interactions. A hierarchical scheme employed three biological


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systems of increasing complexity: cultured cells (HeLa), invertebrates (freshwater polyp Hydra vulgaris), and vertebrates (C57BL/6j mice) to screen the synthesized nanocarrier library and select the ideal engineered Au NP for the delivery of siRNA to block gene function (in this case Au NPs@PEG@RGD@siRNA). Bioluminescent imaging of B6 albino mice injected with luciferase-CMT/167 adenocarcinoma cells was used to assess the tumour size in each mouse. A: control lung cancer mouse with no treatment; B: treated with Au NPs@PEG@RGD and C: with Au NPs@PEG@RGD@siRNA. The results indicated a tumor regression equivalent to ca. 8090% for the group treated with the optimum nanocarrier, Au NPs@PEG@RGD@siRNA. Adapted with permission from ref 12 and 13. Copyright (2012) American Chemical Society (ref 12) and Elsevier (ref 13).

3.3 Functionalization with antibodies Antibody (Ab) immobilization on nanostructured materials is an essential process for the development of most immune-based assay nanosystems ranging from biosensors and antibody arrays to cellular targeting systems. Among the five major classes of antibodies, immunoglobulin G (IgG) is the most abundant in human serum and therefore the most widely used for biofunctionalization of nanomaterials. Given their high 3D complexity as proteins comprising 4 polypeptide chains and having a molecular weight of 180 kDa, we feel that some general considerations regarding Ab conjugation to nanomaterials are appropriate before moving to some representative examples (for a comprehensive discussion, see the recent review of Montenegro et al.62). The basic unit of IgG consists of two identical light chains and two identical heavy chains that are held together by non-covalent interactions as well as disulfide bonds (Figure 9a). The four protein chains are assembled into a specific “Y-shaped” geometry, with two identical


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antigen-binding sites localized at the end of the arms of the Y and called Fab fragments (antigen binding fragment, in green). The stem of the Y is known as the Fc fragment (crystallizable fragment) and ensures that the antibody generates an appropriate immune response for a given antigen by triggering effector functions. The position of the antigen binding sites clearly indicates that the final molecular orientation of the Ab on the surface of a nanomaterial affects antigen binding. Thus, there are four possible spatial orientations upon conjugation to the surface of a nanostructure (Figure 9b).

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Figure 9. a) Schematic cartoon showing the Y-shaped structure of an antibody. The light chains (variable regions) and the heavy chains (constant regions) are colored in violet and red respectively. The antigen-binding sites are drawn in green, while groups that can be used for attachment to NPs are drawn in yellow. b) Possible orientation of antibodies immobilized on surfaces. Adapted with permission from ref 62. Copyright Elsevier. Clearly, antibodies immobilized through their Fc region (“end-on orientation”) would be properly oriented with their antigen binding sites well exposed to the solution phase. However, if the Ab-functionalized nanomaterial is intended for in vivo therapeutics, it is important to keep in mind that this orientation may compromise or sacrifice the powerful effector functions of the Fc fragment. Recently, it has been demonstrated that “flat-on” orientations (all three fragments attached to the surface) also preserve the antibody’s antigen-binding activity. Thus, strategies that allow “end on” or “flat on” orientations of the Ab upon immobilization would yield Abfunctionalized nanomaterials with higher antigen binding capacities compared to random (“head-on” and “sideways-on”) orientations. As a matter of fact, the steric hindrance of antigenrecognition sites in the case of a randomly immobilized Ab can even cause a 1000-fold decrease in the binding affinity when compared to its soluble counterpart.63 This directly affects the targeting efficiency of Ab-functionalized NPs and the limit of detection achieved in nanoparticle based immuno-biosensing devices, as it has been reported that a good orientation of the immobilized Ab molecule can significantly improve the sensitivity of the biosensor.64 There are several strategies available for the site-specific oriented immobilization of antibodies on gold and iron oxide nanoparticles. The oriented direct covalent binding of the Ab takes advantage of the site-specific location of carbohydrate chains64 or disulfide bridges65 of antibodies far away of the antigen-recognition sites (Figure 10a). However, this method requires


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chemical modification of antibodies such as carbohydrate oxidation and disulfide bond reduction, prior to the immobilization, and in the case of sugar oxidation, an additional limitation is the necessity for the Ab molecule to be glycosylated. Polyclonal Abs are usually glycosylated, but other Ab preparations may not present carbohydrate moieties, such as recombinant Abs fermented in bacteria, or some monoclonal Abs which may not suffer post-translational glycosylation. Other oriented functionalization strategies use adapter biomolecules such as protein A and G that specifically target the Fc region of an antibody (Figure 10b).66,67 In this case Ab modification is not required for the immobilization, therefore there is no risk that bound antibodies could have inferior antigen binding abilities due to a chemical modification process. Other adapter proteins widely used are avidin/streptavidin, which can both interact strongly with biotinylated Abs via near irreversible interactions. In order to obtain an oriented immobilization using these proteins, the biotinylation of the Ab must be site-specific within its Fc region.62 A major drawback associated with this strategy could be the additional immobilization process of the antibody-binding protein on the surface of the nanomaterial prior to the immobilization of the Ab. However, unlike natural antibodies, these adapter proteins can be genetically engineered and easily prepared in large quantities providing many options for their immobilization.

a

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Figure 10. Different strategies for functionalization of nanoparticles with antibodies: a) covalent binding via amine groups on the Ab; b) use of adapter biomolecules (Protein G; streptavidin–


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biotin); c) Two steps strategy involving ionic adsorption plus covalent binding. Adapted with permission from ref 62. Copyright Elsevier.

All these strategies directing Ab immobilization through a fully active “end-on” orientation have the added advantage of generality of use among IgGs recognizing different antigens. This is possible because the Fc region of Abs with different specificities share more than 95% homology in the amino acid sequence of Abs of the same isotype and host species as the major differences in the amino acid composition and structure are mostly localized in the antigen recognition sites and the hinge region. Despite the advantages of oriented binding strategies, protocols that result in a random Ab immobilization are still used by a high number of researchers. For instance, Hinterwirth et al. used a direct covalent binding strategy involving the primary amine groups of the Abs to immobilize anti-oxidized low-density lipoprotein antibodies on the surface of Au NPs, resulting in a novel Au NP-Ab conjugate for the specific screening of biomarkers of oxidative stress.68 This approach is rather popular due to the fact that primary amines are among the most available groups on the Ab surface. Moreover, they show high reactivity towards a wide variety of reactive groups placed in the nanomaterial surface, without the necessity of their previous activation. However, it requires several steps including the introduction of functional groups on the NP surface and/or the use of chemical linkers to activate them in order to achieve the covalent binding of the amine groups of the antibody. Another aspect to take into account is the high reactivity of all the amine-binding linkers on the NP surface, which makes them very unstable at alkaline pH values. This imposes the requirement of mild pH conditions (below pH 8.0) for the coupling step. At this pH value the most reactive amine moieties are the terminal amine groups


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of each of the four polypeptide chains belonging to the Ab, located within the antigen recognition sites. Consequently, using this strategy can result in interference with the antigen detection capability if the antibody is immobilized via amine groups of or adjacent to the antigen-binding site. However, it has been demonstrated recently that a “flat on” orientation of Ab molecules can be obtained via a two-step immobilization process - ionic pre-adsorption followed by covalent attachment - rather than direct covalent binding through amine groups (Figure 10c).62,69 As ionic adsorption processes are faster than covalent reactions, if the pH of incubation of the Ab with the NPs is below its isoelectric point, the Ab would be positively charged and its ionic adsorption will be favored over its direct covalent reaction. The antibody would adopt a “flat-on” orientation, as this plane of interaction with the nanoparticle is the one with the greatest number of positive charges. Next, site-specific covalent binding to the NP would occur only via the amine groups located far away from the antigen-binding zone. We have demonstrated the versatility and general applicability of this approach for the immobilization of three Abs with very different isoelectric points (antiperoxidase, carcinoembryonic antigen, and human chorionic gonadotropin hormone) on magnetic NPs (Figure 11a).55 The antigen binding efficiencies obtained for the three Abs were similar to the ones of NPs functionalized in a oriented way via the sugar moieties of the Ab. Besides, this method was used for the development of a new antibody - Au NP conjugate for immunosensing applications (Figure 11b).70 The Au NPs were modified with carboxyl groups-containing PEG, in order to achieve the ionic adsorption of the Ab. In the next step, the stable negative intermediate interacted with the major plane of the antibody to form the covalent peptide bond. A magnetosandwich immunoassay revealed a limit of detection nearly one order of magnitude higher than that of a similar random-oriented conjugate. This two-step immobilization strategy has been also


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successfully applied in our group for the development of a high-sensitivity plasmonic-driven thermal sensing approach for the detection of tumoral markers (Figure 11c).69 a

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Figure 11. a) Top: Two-step immobilization mechanism proposed when using bifunctional MNPs that bind the Ab through its most positively charged region. Bottom: Relative biological activities of MNPs functionalized with anti-peroxidase (anti-HRP), anti-carcinoembryonic antigen (anti-CEA), and anti-human chorionic gonadotropin hormone (anti-hCG) using random


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orientation and our two-step strategy. Results are normalized to the biological activity of MNPs functionalized with Abs through its sugar moieties (positive control) Adapted with permission from ref 55. Copyright (2011) American Chemical Society b) Random vs. oriented immobilization of antibodies on gold nanoparticles (left) and scheme of the electrochemical magnetosandwich immunoassay for the detection of HIgG using (right). The graph presents the results obtained for the random-orientation and fixed-orientation antiHIgG/AuNP conjugates. SPCE: screen-printed carbon electrode. Adapted with permission from ref 70. Copyright (2013) American Chemical Society. c) Thermal signals developed after NIR illumination for CEA detection using anti-CEA derivatized NPRs using our two-step strategy in real patient serum samples with a visual limit of detection in the attomolar range. Adapted from ref. 69 with permission from The Royal Society of Chemistry. It is clear that the oriented binding of Abs is a key feature to obtain highly bioactive nanomaterials. However, a correct surface packing density of the oriented Ab molecules is also important in order to achieve high antigen binding capacity. Despite oriented antibody binding, the antigen binding efficiency tends to drop drastically if nanomaterials are functionalized with a high density of Ab molecules, due to the steric hindrance of the antigen binding sites when neighboring antibodies are too close.55

Conclusion and perspectives In summary, there is an ever-growing number of chemists, cell biologists, molecular biologists, geneticists and physicists who are currently working with medical experts with the aim of developing novel sensing and diagnostic techniques as well as gene and drug delivery methods based on functionalized NPs. These nanoparticles - when used in combination with


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proteins, peptides, cell-integration molecules and fluorescent and/or magnetic resonance imaging probes – represent powerful nanocarriers capable of realizing real advances in nanotherapy and nanodiagnostics. However, researchers must be careful to accurately characterize and identify the exact functionalization of the NPs being developed since the type of functionalization has a dramatic effect on the end use of the nanocarrier. In fact, accurate characterization and precise quantification of the extent of functionalization, together with reproducibility and scale-up problems are the main problems to be solved in the near future in order to allow a more successful implementation of nanomaterials in biomedicine. While we certainly gained a significant understanding on the synthesis and characterization of inorganic nanomaterials, thanks to modern analytical techniques such as electron microscopy, there are many aspects to be addressed with respect to the (bio)functionalization of nanomaterials and the toxicity of the resulting nanoconstructs.

AUTHOR INFORMATION Corresponding Author * [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT The present work was supported by MAT2011-26851-C02-01 (Spanish Government), Shanghai 1000People Plan, European Regional and Social Development Funds, Aragón


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Autonomous Government (DGA) through Research Groups and ERC-Starting Grant 239931NANOPUZZLE. R.M.F. and J.M.F. acknowledge the financial support from ARAID. S.G.M thanks the People Programme (Marie Curie Actions) FP7/2007-2013 for the REA grant agreement 328985. REFERENCES (1)

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Conde, J.; Tian, F.; Hernández, Y.; Bao, C.; Cui, D.; Janssen, K.-P.; Ibarra, M. R.; Baptista, P. V; Stoeger, T.; de la Fuente, J. M. In Vivo Tumor Targeting via NanoparticleMediated Therapeutic siRNA Coupled to Inflammatory Response in Lung Cancer Mouse Models. Biomaterials 2013, 34, 7744–7753.

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Pelaz, B.; Grazu, V.; Ibarra, A.; Magen, C.; del Pino, P.; de la Fuente, J. M. Tailoring the Synthesis and Heating Ability of Gold Nanoprisms for Bioapplications. Langmuir 2012, 28, 8965–8970.

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Moros, M.; Pelaz, B.; López-Larrubia, P.; García-Martin, M. L.; Grazú, V.; de la Fuente, J. M. Engineering Biofunctional Magnetic Nanoparticles for Biotechnological Applications. Nanoscale 2010, 2, 1746–1755.

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Del Pino, P.; Munoz-Javier, A.; Vlaskou, D.; Rivera Gil, P.; Plank, C.; Parak, W. J. Gene Silencing Mediated by Magnetic Lipospheres Tagged with Small Interfering RNA. Nano Lett. 2010, 10, 3914–3921.

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De la Fuente, J. M.; Barrientos, A. G.; Rojas, T. C.; Rojo, J.; Cañada, J.; Fernández, A.; Penadés, S. Gold Glyconanoparticles as Water-Soluble Polyvalent Models To Study Carbohydrate Interactions. Angew. Chemie Int. Ed. 2001, 40, 2257–2261.


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Marradi, M.; Chiodo, F.; García, I.; Penadés, S. Glyconanoparticles as Multifunctional and Multimodal Carbohydrate Systems. Chem. Soc. Rev. 2013, 42, 4728–4745.

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Moros, M.; Hernáez, B.; Garet, E.; Dias, J. T.; Sáez, B.; Grazú, V.; González-Fernández, Á.; Alonso, C.; de la Fuente, J. M. Monosaccharides versus PEG-Functionalized NPs: Influence in the Cellular Uptake. ACS Nano 2012, 6, 1565–1577.

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Martínez-Avila, O.; Bedoya, L. M.; Marradi, M.; Clavel, C.; Alcamí, J.; Penadés, S. Multivalent Manno-Glyconanoparticles Inhibit DC-SIGN-Mediated HIV-1 TransInfection of Human T Cells. ChemBioChem 2009, 10, 1806–1809.

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Van Kasteren, S. I.; Campbell, S. J.; Serres, S.; Anthony, D. C.; Sibson, N. R.; Davis, B. G. Glyconanoparticles Allow Pre-Symptomatic in Vivo Imaging of Brain Disease. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 18–23.

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Frigell, J.; García, I.; Gómez-Vallejo, V.; Llop, J.; Penadés, S. 68Ga-Labeled Gold Glyconanoparticles for Exploring Blood-Brain Barrier Permeability: Preparation, Biodistribution Studies, and Improved Brain Uptake via Neuropeptide Conjugation. J. Am. Chem. Soc. 2014, 136, 449–457.

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Aykaç, A.; Martos-Maldonado, M. C.; Casas-Solvas, J. M.; Quesada-Soriano, I.; GarcíaMaroto, F.; García-Fuentes, L.; Vargas-Berenguel, A. Β-Cyclodextrin-Bearing Gold Glyconanoparticles for the Development of Site Specific Drug Delivery Systems. Langmuir 2014, 30, 234–242.

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Berry, C. C. Intracellular Delivery of Nanoparticles via the HIV-1 Tat Peptide. Nanomedicine (Lond). 2008, 3, 357–365.

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Ruoslahti, E. Peptides as Targeting Elements and Tissue Penetration Devices for Nanoparticles. Adv. Mater. 2012, 24, 3747–3756.

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Child, H. W.; Del Pino, P. a; De La Fuente, J. M.; Hursthouse, A. S.; Stirling, D.; Mullen, M.; McPhee, G. M.; Nixon, C.; Jayawarna, V.; Berry, C. C. Working Together: The Combined Application of a Magnetic Field and Penetratin for the Delivery of Magnetic Nanoparticles to Cells in 3D. ACS Nano 2011, 5, 7910–7919.

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Smith, C.-A. M.; de la Fuente, J.; Pelaz, B.; Furlani, E. P.; Mullin, M.; Berry, C. C. The Effect of Static Magnetic Fields and Tat Peptides on Cellular and Nuclear Uptake of Magnetic Nanoparticles. Biomaterials 2010, 31, 4392–4400.

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Veiseh, O.; Gunn, J. W.; Kievit, F. M.; Sun, C.; Fang, C.; Lee, J. S. H.; Zhang, M. Inhibition of Tumor-Cell Invasion with Chlorotoxin-Bound Superparamagnetic Nanoparticles. Small 2009, 5, 256–264.

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Park, J.-H.; von Maltzahn, G.; Xu, M. J.; Fogal, V.; Kotamraju, V. R.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Cooperative Nanomaterial System to Sensitize, Target, and Treat Tumors. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 981–986.

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Sugahara, K. N.; Teesalu, T.; Karmali, P. P.; Kotamraju, V. R.; Agemy, L.; Greenwald, D. R.; Ruoslahti, E. Coadministration of a Tumor-Penetrating Peptide Enhances the Efficacy of Cancer Drugs. Science 2010, 328, 1031–1035.

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Agemy, L.; Friedmann-Morvinski, D.; Kotamraju, V. R.; Roth, L.; Sugahara, K. N.; Girard, O. M.; Mattrey, R. F.; Verma, I. M.; Ruoslahti, E. Targeted Nanoparticle Enhanced Proapoptotic Peptide as Potential Therapy for Glioblastoma. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 17450–17455.

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Liu, J.; Cao, Z.; Lu, Y. Functional Nucleic Acid Sensors. Chem. Rev. 2009, 109, 1948– 1998.

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Jones, M. R.; Macfarlane, R. J.; Lee, B.; Zhang, J.; Young, K. L.; Senesi, A. J.; Mirkin, C. A. DNA-Nanoparticle Superlattices Formed from Anisotropic Building Blocks. Nat. Mater. 2010, 9, 913–917.


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Auyeung, E.; Cutler, J. I.; Macfarlane, R. J.; Jones, M. R.; Wu, J.; Liu, G.; Zhang, K.; Osberg, K. D.; Mirkin, C. A. Synthetically Programmable Nanoparticle Superlattices Using a Hollow Three-Dimensional Spacer Approach. Nat. Nanotechnol. 2012, 7, 24–28.

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Cutler, J. I.; Auyeung, E.; Mirkin, C. A. Spherical Nucleic Acids. J. Am. Chem. Soc. 2012, 134, 1376–1391.

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Zhang, X.; Servos, M. R.; Liu, J. Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a pH-Assisted and Surfactant-Free Route. J. Am. Chem. Soc. 2012, 134, 7266–7269.

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Zhang, X.; Liu, B.; Dave, N.; Servos, M. R.; Liu, J. Instantaneous Attachment of an Ultrahigh Density of Nonthiolated DNA to Gold Nanoparticles and Its Applications. Langmuir 2012, 28, 17053–17060.

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Jones, M. R.; Macfarlane, R. J.; Prigodich, A. E.; Patel, P. C.; Mirkin, C. A. Nanoparticle Shape Anisotropy Dictates the Collective Behavior of Surface-Bound Ligands. J. Am. Chem. Soc. 2011, 133, 18865–18869.

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Chak, C.-P.; Lai, J. M. Y.; Sham, K. W. Y.; Cheng, C. H. K.; Leung, K. C.-F. DNA Hybridization of Pathogenicity Island of Vancomycin-Resistant Enterococcus Faecalis with Discretely Functionalized Gold Nanoparticles in Organic Solvent Mixtures. RSC Adv. 2011, 1, 1342–1348.

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Miao, X.-M.; Xiong, C.; Wang, W.-W.; Ling, L.-S.; Shuai, X.-T. Dynamic-LightScattering-Based Sequence-Specific Recognition of Double-Stranded DNA with Oligonucleotide-Functionalized Gold Nanoparticles. Chem. Eur. J. 2011, 17, 11230– 11236.

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Shen, W.; Deng, H.; Gao, Z. Gold Nanoparticle-Enabled Real-Time Ligation Chain Reaction for Ultrasensitive Detection of DNA. J. Am. Chem. Soc. 2012, 134, 14678– 14681.

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Krpetić, Ž.; Singh, I.; Su, W.; Guerrini, L.; Faulds, K.; Burley, G. A.; Graham, D. Directed Assembly of DNA-Functionalized Gold Nanoparticles Using Pyrrole–Imidazole Polyamides. J. Am. Chem. Soc. 2012, 134, 8356–8359.

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Cheng, Y.; Stakenborg, T.; Van Dorpe, P.; Lagae, L.; Wang, M.; Chen, H.; Borghs, G. Fluorescence Near Gold Nanoparticles for DNA Sensing. Anal. Chem. 2011, 83, 1307– 1314.

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Zhang, D.; Anderson, M. J.; Huarng, M. C.; Alocilja, E. C. Nanoparticle-Based Biobarcoded DNA Sensor for the Rapid Detection of pagA Gene of Bacillus Anthracis. Nanotechnology, IEEE Transactions on, 2011, 10, 1433–1438.


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Puertas, S.; Batalla, P.; Moros, M.; Polo, E.; del Pino, P.; Guisán, J. M.; Grazú, V.; de la Fuente, J. M. Taking Advantage of Unspecific Interactions to Produce Highly Active Magnetic Nanoparticle−Antibody Conjugates. ACS Nano 2011, 5, 4521–4528.

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Riedinger, A.; Guardia, P.; Curcio, A.; Garcia, M. A.; Cingolani, R.; Manna, L.; Pellegrino, T. Subnanometer Local Temperature Probing and Remotely Controlled Drug Release Based on Azo-Functionalized Iron Oxide Nanoparticles. Nano Lett. 2013, 13, 2399–2406.

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Dias, J. T.; Moros, M.; del Pino, P.; Rivera, S.; Grazú, V.; de la Fuente, J. M. DNA as a Molecular Local Thermal Probe for the Analysis of Magnetic Hyperthermia. Angew. Chemie Int. Ed. 2013, 52, 11526–11529.

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Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. What Controls the Melting Properties of DNA-Linked Gold Nanoparticle Assemblies? J. Am. Chem. Soc. 2003, 125, 1643– 1654.

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Lee, J.-S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.; Anderson, D. G. Gold, Poly(β-Amino Ester) Nanoparticles for Small Interfering RNA Delivery. Nano Lett. 2009, 9, 2402–2406.

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Conde, J.; de la Fuente, J. M.; Baptista, P. V. In Vitro Transcription and Translation Inhibition via DNA Functionalized Gold Nanoparticles. Nanotechnology 2010, 21, 505101.

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Conde, J.; Rosa, J.; de la Fuente, J. M.; Baptista, P. V. Gold-Nanobeacons for Simultaneous Gene Specific Silencing and Intracellular Tracking of the Silencing Events. Biomaterials 2013, 34, 2516–2523.

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Montenegro, J.-M.; Grazu, V.; Sukhanova, A.; Agarwal, S.; de la Fuente, J. M.; Nabiev, I.; Greiner, A.; Parak, W. J. Controlled Antibody/(bio-) Conjugation of Inorganic Nanoparticles for Targeted Delivery. Adv. Drug Deliv. Rev. 2013, 65, 677–688.

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Tajima, N.; Takai, M.; Ishihara, K. Significance of Antibody Orientation Unraveled: Well-Oriented Antibodies Recorded High Binding Affinity. Anal. Chem. 2011, 83, 1969– 1976.

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Puertas, S.; Moros, M.; Fernández-Pacheco, R.; Ibarra, M. R.; Grazú, V.; de la Fuente, J. M. Designing Novel Nano-Immunoassays: Antibody Orientation versus Sensitivity. J. Phys. D. Appl. Phys. 2010, 43, 474012.

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Wang, Z.; Yue, T.; Yuan, Y.; Cai, R.; Niu, C.; Guo, C. Preparation of Immunomagnetic Nanoparticles for the Separation and Enrichment of Alicyclobacillus Spp. in Apple Juice. Food Res. Int. 2013, 54, 302–310.


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Gabrovska, K. I.; Ivanova, S. I.; Ivanov, Y. L.; Godjevargova, T. I. Immunofluorescent Analysis with Magnetic Nanoparticles for Simultaneous Determination of Antibiotic Residues in Milk. Anal. Lett. 2013, 46, 1537–1552.

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Arenal, R.; De Matteis, L.; Custardoy, L.; Mayoral, A.; Tence, M.; Grazu, V.; De La Fuente, J. M.; Marquina, C.; Ibarra, M. R. Spatially-Resolved EELS Analysis of Antibody Distribution on Biofunctionalized Magnetic Nanoparticles. ACS Nano 2013, 7, 4006– 4013.

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Hinterwirth, H.; Stübiger, G.; Lindner, W.; Lämmerhofer, M. Gold NanoparticleConjugated Anti-Oxidized Low-Density Lipoprotein Antibodies for Targeted Lipidomics of Oxidative Stress Biomarkers. Anal. Chem. 2013, 85, 8376–8384.

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Polo, E.; del Pino, P.; Pelaz, B.; Grazu, V.; de la Fuente, J. M. Plasmonic-Driven Thermal Sensing: Ultralow Detection of Cancer Markers. Chem. Commun. 2013, 49, 3676–3678.

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Parolo, C.; de la Escosura-Muñiz, A.; Polo, E.; Grazú, V.; de la Fuente, J. M.; Merkoçi, A. Design, Preparation, and Evaluation of a Fixed-Orientation Antibody/Gold-Nanoparticle Conjugate as an Immunosensing Label. ACS Appl. Mater. Interfaces 2013, 5, 10753– 10759.


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Author Biographies Dr. Raluca M. Fratila obtained her PhD in Chemistry at University Politehnica Bucharest (Romania) in 2005. She accomplished postdoctoral stays at the University of Basque Country, San Sebastian, Spain (2006– 2008), and the University of Twente, Enschede, The Netherlands (2009– 2013). Since November 2013, she is ARAID-EU researcher at the Institute of Nanocience of Aragón (INA). Her research interests include bioorganic chemistry, microfluidics, NMR spectroscopy, magnetic resonance imaging (MRI) and magnetic nanoparticles for biomedical applications.

Dr. Scott G. Mitchell obtained an M.Sci. in Chemistry with Medicinal Chemistry (2005) followed by a Ph.D. (2010) from the University of Glasgow (Scotland). His postgraduate studies were carried out under the supervision of Prof. Lee Cronin and focused on the self-assembly of polyoxometalate framework materials. After 18 months as a postdoctoral researcher in Glasgow he joined the Institute of Nanocience of Aragón (INA) in 2011 to work with Dr. Martinez de la Fuente, first as a “Juan de la Cierva” researcher and then in 2012 as a Marie Curie Fellow. His current academic interests involve engineering hybrid inorganic nanomaterials as functional devices for a variety of biomedical purposes.

Native of Uruguay, Dr. Valeria Grazú received her PhD in Science from Autonomous University of Madrid in 2006. She was a postdoctoral fellow in the Nanotherapy and Nanobiosensors Group (GN2) at the Aragon Institute of Nanoscience (INA) from 2006-2013 and currently holds the position of Research & Development Director at Nanoimmunotech S.L. Her research interests include the development of novel mono/multifunctionalization strategies of nanomaterials with different biomolecules (enzymes, peptides, antibodies, carbohydrates, and so forth) for their use in diagnosis and therapy.

Dr. Pablo del Pino graduated in Physics from the Universidad de Sevilla in 2002 and obtained his Ph.D. degree at the Technische Universität München (Germany) in 2007. He then joined the group of Wolfgang Parak as a postdoctoral fellow at the Ludwig-Maximilians-Universität München (Germany). From 2009 to 2013, he was a scientist at Institute of Nanoscience of Aragón (INA) at the Universidad de Zaragoza (Spain), firstly, as postdoctoral researcher in the group of Dr. Jesús M. de la Fuente and in 2013, as an ARAID junior researcher. In November 2013, he joined CIC biomaGUNE as senior postdoc in the Biofunctional Nanomaterials unit. His current research interests focus on synthesis and bioapplications of nanostructured materials.


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Dr. Jesus M. de la Fuente (Barakaldo, 1975) started his PhD work in 1999 on the evaluation of carbohydrate-carbohydrate interactions using gold nanoparticles (IIQ-CSIC, Seville, Spain). After obtaining his PhD he was funded by the Spanish Ministry of Science to work in the Centre for Cell Engineering at The University of Glasgow (UK) to develop a research project involving the biological application of nanoparticles. In June 2007, he gained a permanent position in the Institute of Nanoscience of Aragón (INA) belonging to the University of Zaragoza (Spain) as Senior Researcher supported by ARAID. He currently leads the research group specialized in the Biofunctionalization of Nanoparticles and Surfaces at ICMA-CSIC. His research interests are based on the development of general and simple strategies for the functionalization of nanoparticles and surfaces for biomedical and biotechnological applications. He has 100 published articles with more than 2600 citations and 5 international patents. From 2014 he is a Visiting Professor at the Shanghai Jiao Tong University (P.R. China) under the “1000 People Plan Program”.


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Strategies for the biofunctionalization of gold and iron oxide nanoparticles Raluca M. Fratila, Scott G. Mitchell, Pablo del Pino, Valeria Grazu, Jesus M. de la Fuente TOC


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Trapping Iron Oxide into Hollow Gold Nanoparticles.

The effect of gold and iron-oxide nanoparticles on biofilm-forming pathogens.

Targeted gold-coated iron oxide nanoparticles for CD163 detection in atherosclerosis by MRI.

The One Year Fate of Iron Oxide Coated Gold Nanoparticles in Mice.

Nitric oxide donor superparamagnetic iron oxide nanoparticles.

iron oxide composite and iron oxide nanoparticles.

Targeted iron oxide nanoparticles for the enhancement of radiation therapy.

DNA-length-dependent quenching of fluorescently labeled iron oxide nanoparticles with gold, graphene oxide and MoS2 nanostructures.

Multi-domain short peptide molecules for in situ synthesis and biofunctionalization of gold nanoparticles for integrin-targeted cell uptake.

Multifunctional iron oxide nanoparticles for diagnostics, therapy and macromolecule delivery.

Gemcitabine and Chlorotoxin Conjugated Iron Oxide Nanoparticles for Glioblastoma Therapy.

Cardioprotective activity of iron oxide nanoparticles.

Engineering of radiolabeled iron oxide nanoparticles for dual-modality imaging.

Washing effect on superparamagnetic iron oxide nanoparticles.

Stem cell tracking using iron oxide nanoparticles.

Iron oxide nanoparticles can cross plasma membranes.

gold plasmonic nanoparticles for magnetic targeted chemo-photothermal treatment of rheumatoid arthritis.

Cytotoxicity and antibacterial activity of gold-supported cerium oxide nanoparticles.

Heterobifunctional PEG ligands for bioconjugation reactions on iron oxide nanoparticles.

New carboxysilane-coated iron oxide nanoparticles for nonspecific cell labelling.

Polymer-iron oxide composite nanoparticles for EPR-independent drug delivery.

Nanoparticle strategies for cancer therapeutics: Nucleic acids, polyamines, bovine serum amine oxidase and iron oxide nanoparticles (Review).

Biofunctionalization strategies on tantalum-based materials for osseointegrative applications.

Labeling mesenchymal cells with DMSA-coated gold and iron oxide nanoparticles: assessment of biocompatibility and potential applications.