Artificial Cells, Nanomedicine, and Biotechnology, 2014; Early Online: 1–13 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 2169-1401 print / 2169-141X online DOI: 10.3109/21691401.2014.955107

Application of gold nanoparticles in biomedical and drug delivery Artificial Cells, Nanomedicine, and Biotechnology Downloaded from informahealthcare.com by University of North Carolina on 09/25/14 For personal use only.

Hadis Daraee2, Ali Eatemadi2, Elham Abbasi1, Sedigheh Fekri Aval1,2, Mohammad Kouhi3 & Abolfazl Akbarzadeh1,2 1Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz,

Iran, 2Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran, and 3Department of Physics, College of Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran

quintessence—“quinta essentia auri,” which he detected by the reduction of gold chloride by vegetable dig outs in alcohols or oils. He used the “potable gold” for healing a number of mental diseases and syphilis. His contemporary, Giovanni Andrea, utilized “aurum potabile” as a therapy for patients with leprosy, plague, epilepsy, and diarrhea. In 1583, the alchemist, David de Planis-Campy, who served, when the doctor to Louis XIII of France recommended, his “longevity elixir,” a colloidal solution of gold in water. Nanoparticles based on gold chemistry have attracted significant research and practical consideration lately. They are flexible agents with a selection of biomedical applications including use in highly sensitive analytical assessments, ablation thermal and radiotherapy development, as well as drug and gene delivery (Chiu and Rana 2003, Soutschek et al. 2004, Jeynes et al. 2013, Prigodich et al. 2009, Dhar et al. 2009, Horisberger et al. 1975). For biomedical uses, external functionalization of gold nanoparticles (GNPs) is necessary in order to target them to specific disease areas and allow them to selectively interact with cells or biomolecules. The resulting GNP have unique properties, such as size- and figure-dependent visual and electronic characteristics (Qian et al. 2008, Niemeyer et al. 2003, Wheeler et al. 1999, Javier et al. 2008, Huang et al. 2009, Loo et al. 2005), a high surface area to amount ratio, and surfaces that can be readily modified with ligands containing functional groups such as thiols, phosphines, and amines, which display affinity for gold surfaces (Stamatoiu et al. 2012). By using these functional groups to fix the ligands, additional moieties such as proteins oligonucleotides, and antibodies can be used to impart even greater functionality (Wang et al. 2010, Pavlov et al. 2004). The broad range of application for GNP is based on their unique physical and chemical properties. In particular, the visual properties of GNP are determined by their plasmon resonance, which is associated with the combined excitation of conduction

Abstract Nanoparticles are the simplest form of structures with sizes in the nanometer (nm) range. In principle any collection of atoms bonded together with a structural radius of ⬍ 100 nm can be considered nano particles. Nanotechnology offers unique approaches to probe and control a variety of biological and medical processes that occur at nanometer scales, and is expected to have a revolutionary impact on biology and medicine. Among the approaches for exploiting nanotechnology in medicine, nanoparticles offer some unique advantages as sensing, image enhancement, and delivery agents. Several varieties of nanoparticles with biomedical relevance are available including, polymeric nanoparticles, metal nanoparticles, liposomes, micelles, quantum dots, dendrimers, and nanoassemblies. To further the application of nanoparticles in disease diagnosis and therapy, it is important that the systems are biocompatible and capable of being functionalized for recognition of specific target sites in the body after systemic administration. In this review, we have explained some important applications of gold nanoparticles. Keywords: biocompatible, drug delivery, gold nanoparticle, immunoassay, photothermal

Introduction Gold is one of the first metals to have been discovered; the history of its study and application spans at least some thousand years. The initial information on colloidal gold can be found in treatises by Arabian, Chinese, and Indian scientists, who tried to attain colloidal gold as early as in the fifth-fourth centuries BC. They utilized it for medicinal (Indian “liquid gold,” Chinese “golden solution”) and other functions. In the Middle Age, in Europe, colloidal gold was studied and used in alchemist laboratories. Paracelsus wrote about the curative properties of gold

Correspondence: Dr. Abolfazl Akbarzadeh, Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Tel/Fax: ⫹ 984113355789. E-mail: [email protected] and Dr. Mohammad Kouhi, Department of Physics, College of Science, Tabriz Branch, Islamic Azad University, Tabriz, Iran. Tel/Fax: ⫹ 984113844395. E-mail: [email protected] (Received 31 July 2014; revised 8 August 2014; accepted 12 August 2014)

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electrons and localized in the broad region, from the visible to the infrared (IR) region, depending on the particle size, shape, and structure (Thiruppathiraja et al. 2011). Taking into account the large volume of data published and the high speed at which they are updated, our review aims to take a broad view of the outcomes obtained over the past several years in the most promising directions in the use of GNP in modern medical and biological studies (Horisberger et al. 1975) (Figure 1). Figure 2. Gold nanoparticles in biosensor.

Application in biomedical Biomolecule-directed nanoparticles organization: Nanoparticles as biolabels

Protein-based recognition systems: Enhanced immunosensing

The dimensions of the metal nanoparticles are similar to those of biomolecules such as proteins (enzymes, antigens, antibodies) or DNA whose dimensions are in the range of 2–20 nm (Wang et al. 2010, Neely et al. 2009, Kamnev et al. 2002). Immobilization of biomolecules onto nanoparticles to give new hybrid nanobiomolecules has been achieved by a variety of techniques including electrostatic binding, physical adsorption, covalent coupling, and specific recognition. Under appropriate conditions, non-covalent bonding is a general strategy to bind colloidal gold and macromolecules, with little or no change in the specific activity of the bound macromolecule.

Though, a large number of complementary binding pairs are available, nucleic acid based conjugation might offer advantages over protein based assembly (Dykman and Khlebtsov 2011). These biomolecule based coupling systems were useful in various diagnostic applications and for generating inorganic nanoparticle networks (Neely et al. 2009). The conjugation of proteins on colloidal gold nanoparticles is achieved by the electrostatic interactions between negatively charged citrate on surfaces of gold nanoparticles and positively charged groups of the proteins (Zharov et al. 2003). The experimental gold nanoparticle-protein conjugate architectures involve either direct binding of antigen-gold nanoparticles bioconjugates to an antibody modified surface or the exposure of an antibody derived surface to free antigen and then to a secondary antibodygold nanoparticles conjugate. Biosensors for immunoassays in human serum have been developed (Huang et al. 2008).

DNA-gold nanoparticles assemblies and sensors Negatively charged DNA was found to substitute citrate ions around gold nanoparticles to form a DNA-nanoparticle probe, which was confirmed by electrophoresis and fluorescence (Russier-Antoine et al. 2008). DNA functionalized gold and semiconductor nanoparticles have been prepared using the n-alkylthiolated DNA and also using DNA containing several adenosylphosphothioate residues at their ends (Neely et al. 2009). DNA is used as a template to prepare nanocrystal chains consisting of two or three 1.4-nm particles on a single oligonucleotide strand. Conjugates of gold nanoparticles-oligonucleotides are of great interest for detection of DNA hybridization because of its application in the diagnosis of pathogenic and genetic diseases. Most of the DNA hybridization techniques utilize fluorescent, chemiluminescent, and radioactively labelled probes that require special instrumentation or both (Figure 2).

Figure 1. Near Infrared window.

Drug delivery Nanoparticles can easily enter cells although the mechanism(s) involved are not well understood. The nanoparticle influx occurs by endocytosis; the particles are inserted and diffused through the lipid bilayer of the cell membrane. Furthermore, these nanoparticles were shown to be able to enter the cells even after linkage to proteins such as antibodies. Nanoparticles conjugated with antibodies against exclusive cancer cell surface receptors have been used to specifically bind with cancerous cells; the functionalized nanoparticles have also been used for targeted entry into cells. Phthalocyanine-stabilized gold nanoparticles have been shown to be a potential delivery

Figure 3. Colloidal gold nanoparticles.

Gold nanoparticles in biomedical and drug delivery vehicle for photodynamic therapy (Doubrovsky et al. 2010). Gold nanoparticles with a size of 20 nm have been conjugated to various cellular targeting peptides to provide functional nanoparticles that penetrate the biological membrane and target the nucleus. Various nanoparticles have also applied as targeted biomarkers and drug-delivery agents for diagnosis and medical treatment of cancer.

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Cytochemical labels and other applications Colloidal gold nanoparticles prepared in sizes from 1 to 25 nm are electron dense due to the high atomic number of the gold atoms, and this makes them ideal for electron microscopy. Site-specific labeling of biological macromolecules finds use in histological applications. Colloidal gold nanoparticles are used as cytochemical labels for the study of macromolecules with transmission and scanning electron microscopy, light microscopy and freeze-etch electron microscopy and also to enhance the signals of both surface enhanced Raman spectroscopy and surface Plasmon resonance. A further advantage of using the colloidal gold marker is that the colloidal gold nanoparticles can be easily counted and thus the cytochemical signal may be evaluated quantitatively. Thus, gold nanoparticles serve as large surface area platforms for organo-functional groups that interact with the capillary surface, the analytes, or both. The use of gold nanoparticles in conjunction with chip-based capillary electrophoresis to improve the selectivities between solutes and to increase the efficiency of the separation has been reported (Akbarzadeh et al. 2012a). In summary, gold nanoparticles that are functionalized with proteins have long been used as tools in the biosciences. Gold nanoparticles were utilized both as color reporting agents and also as the signal amplification probes for the detection of the antigen (Figure 3).

Photothermal cancer cell therapy in near infrared region using Anti-epidermal growth factor receptor antibody conjugated gold nanorods Reducing a material’s size to the nanometer length scale (which is the length scale of the electronic motion that determines the material’s properties) makes it sensitive to further reduction in size or a change in shape. In semiconductor nanoparticles, the property change results from quantum detention of the electronic shift. Recently, photothermal therapy using the absorption properties of antibody conjugated gold nanoshells (Akbarzadeh et al. 2012c) and solid gold nanospheres have been demonstrated to selectively kill cancer cells leaving the healthy cells unaffected. In order to use long wavelength laser irradiation that penetrates tissue optimally (can be over 10 cm in penetration depth depending on tissue types) for in vivo photothermal treatment (650–900 nm), (Akbarzadeh et al. 2012c) the absorption band of the nanoparticles has to be tuned by adjusting the ratio of the thickness of the gold shell to the diameter of the silica core (about 120 nm in diameter) and thus enabling photothermal therapy in this region. Carbon

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nanotubes absorb naturally in this region and have recently been proposed as near-infrared therapy agents. It is important to mention that a surface plasmon field is applied in drug delivery and gene therapy (Akbarzadeh et al. 2012d).

Gold nanoparticles in biosensor applications The basic principle involved in the design of a biosensor based on gold nanoparticles is that the GNP are functionalized or capped with a thiolated biomolecule which upon identifying the complementary biomolecule causes change in the optical absorption of GNP (Ebrahimnezhad et al. 2013). GNP functionalized with antigen (antibody) aggregate when matching antibody (antigen) binds causing a shift in the plasmon absorption (Alimirzalu et al. 2014, Hosseininasab et al. 2014). Significant efforts have focused on the binding of the oligonucleosides to metal surfaces and colloids for a variety of applications, including a multiplexed DNA detection technology, rapid sequencers based on surface-enhanced Raman spectroscopy or surface-enhanced Raman scattering (SERS) from single DNA bases (Alimirzalu et al. 2014, Hosseininasab et al. 2014), and the real-time DNA detection methodology. In the present project, we have developed a new SERS substrate based on gold nanoparticles in an agarose matrix that provides better enhancement in the Raman signal of DNA nucleosides than that with commercially available gold nanoparticles.

Bioconjugation of GNP Hybrid gold nanoparticles are produced by the interaction of highly reactive nascent GNP with chemical functionalities present on specific molecules of biological interest (including peptides and proteins). The conjugation protocols that are applied for production of radiolabel led bioconjugates, traditionally used for cancer diagnosis and therapy (Rezaei-Sadabady et al. 2013), can be extended to labeling nanoparticles of gold and other metals with tumor specific peptides. The biomolecule chosen for bioconjugation with GNP is the seven amino acid truncated bombesin analogue (BBN8–14) that is known to target gastrin releasing peptide (GRP) receptors that are overexpressed in a variety of neoplasma including small cell lung, prostate, breast, gastric, pancreatic, gastrointestinal carcinoid, and colon cancers (Nejati-Koshki et al. 2013, Ghasemali et al. 2013). S-S group undergo oxidative addition to GNP and the reaction is very selective, even in the presence of thiol groups. Thioctic acid, a biological antioxidant (Mollazade et al. 2013) believed to exhibit metal chelating properties (Akbarzadeh et al. 2014) contains disulfide and carboxylic acid groups to conjugate

Figure 4. Design strategy for bioconjugate/hybrid gold nanoparticles.

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to peptide. S-S group acts as a chelating moiety to hold the GNP and five carbon atoms act as a space between S-S and the biomolecule. The thioctic acid modified bombesin is used for GNP bioconjugation (Figure 4).

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Application of gold nanoparticles for immunosensors Immunosensors are important analytical tools based on the detection of the binding event between antibody and antigen. Among types of immunosensors, electrochemical immunosensors are attractive tools and have received considerable attention because they are easy and economical for mass production, are robust, and achieve excellent detection limits with small analyte volumes. A novel and sensitive electrochemical immunoassay for immunoglobulin G (IgG) is using a colloidal gold label via anodic stripping voltammetry technology. Furthermore, a novel electrochemical immunoassay based on the precipitation of silver on colloidal gold labels has been reported (Figure 5). The enhancement in sensitivity for an electrochemical immunoassay is achieved by the autocatalytic deposition of Au3⫹ onto GNP. After the interfacial competitive immunoreactions, the formed HRP labeled immunoconjugate showed good enzymatic activity for the oxidation of o-phenylene diamine by H2O2. The technique is mainly based on the detection of a change in physical properties as a result of antibody–antigen complex formation. The direct determination of immunospecies by detecting the change of impedance caused by immunoreactions has been demonstrated. A simple and sensitive label free electrochemical immunoassay electrode for detection of carcinoembryonic antigen (CEA) is obtained as follows: CEA antibody (CEAAb) is covalently attached on glutathione (GSH) monolayermodified GNP and the resulting CEAAb-GNP bioconjugates were immobilized on Au electrode by electrocopolymerization with o-aminophenol (OAP).

cells (Alizadeh et al. 2014). Their study demonstrates that, in a HeLa cell model, the amount of time that the citrate particles remain internalized is independent of the particle size when they have diameters between 14 and 74 nm. However, the size does affect the overall number of nanoparticle conjugates internalized throughout the experiment. By using inductively coupled plasma atomic emission spectroscopy (ICP-AES) to determine the intracellular gold content, these researchers determined that citrate-capped gold nanoconjugates with diameters of 50 nm are most readily internalized by HeLa cells (Figure 6). The mechanism by which the citrate-capped gold nanoconjugates enter cells were shown by recorded transmission electron microscopy images of internalized “bare” citrate nanoconjugates and showed that the particles were mainly localized within vesicles inside of the cells (Pourhassan-Moghaddam et al. 2014). They correlated cell uptake with the nonspecific adsorption of proteins to the citrate-capped nanoparticle surfaces. The negatively charged citrate surface presents a suitable scaffold to attach positively charged proteins such as transferrin, which is expected to make possible and get better entry into cells. The images obtained suggest vesicle formation at the cell surface and nanoconjugate internalization through endocytosis (Anganeh et al. 2014, Davoudi et al. 2014). Many investigations in cells use citrate-capped GNP as important precursors of covalent conjugates with additional functionality, because further derivatization has been shown to boost uptake capability (Akbarzadeh et al. 2012b), alter intracellular localization (Valizadeh et al. 2012, Akbarzadeh et al. 2013b), or impart functionality that can be used to affect a cellular response (Pourhassan-Moghaddam et al. 2013, Kouhi et al. 2014). Indeed, citrate-coated particles are generally not ideal structures for investigations and internalization studies on cells.

Amines Citrate and transferrin Citrate-functionalized gold nanoparticles can be prepared on a relatively large scale. These methods allow for the synthesis of citrate-capped spherical nanoparticles with diameters ranging from 5 to 250 nm (Akbarzadeh et al. 2013a, Ahmadi et al. 2014). This well-established synthesis and the ability to finely control size have contributed to citrate-functionalized nanoconjugates forming the basis of recent investigations of the uptake of gold nanoparticles by

This method has been developed for production of gold nanoparticles. The Brust–Schiffrin technique allows for production of monodisperse gold nanoparticles ranging from 1 to 3 nm in diameter (Russier-Antoine et al. 2008). The resultant nanoparticles are stabilized by a monolayer of alkanethiolates. The composition of the monolayer can be changed from the beginning to the end through a substitution reaction to consist of specific functionalities, depending on the intended use of the nanoparticles (Eatemadi et al. 2014). Accordingly, gold nanoconjugates functionalized with a monolayer of amine-terminated alkanethiolates (hereafter referred to as amine-functionalized) have been prepared for various biological applications.

Gene transfection

Figure 5. Gold nanoparticles for immunosensors.

The ability to induce control over biological systems at the genetic level is a fundamental concept in experimental biology, and embraces huge promise for developing novel treatments of disease (Huang et al. 2009). The search for the best method for controlling gene expression is ongoing.

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Gold nanoparticles in biomedical and drug delivery

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Figure 6. Transmission electron microscopy imaging and measurements of gold nanoparticles in cells. A) Graph of number of gold nanoparticles per vesicle diameter for various nanoparticle sizes. B)–F) TEM images of gold nanoparticles with sizes of 14, 30, 50, 74, and 100 nm, respectively, trapped inside vesicles of a HeLa cell.

Amine surface groups are positively charged at physiological pH values, and thus amine-functionalized nanoconjugates electrostatically act together with negatively charged nucleic acids. Actually, these nanoconjugates are talented to transfect these cells with a superior efficiency than the universally used cationic polymer transfection agent polyethylenimine (PEI, 60 kDa). Building on these observations, these researchers have recently shown that gold nanoparticles functionalized with lysine moieties are highly effective at delivering DNA plasmids, and outperform a profitable vector by a factor of 28 (Abbasi et al. 2014a). The concentration of PEI was used to control the size of the functionalized nanoparticles from 2.3 to 4.1 nm in diameter. The resultant nanoconjugates deliver plasmid DNA to COS-7 cells more efficiently than PEI alone.

Stability In addition to providing functional groups, surface-bound ligands also contribute to the constancy of the GNP. The constancy of the nanoconjugates is an significant consideration for their impending use as therapeutic means because they must preserve their constancy under inconsiderate conditions such as in the cell or in the bloodstream. It was found that increasing the net positive charge on the nanoparticle surface caused a more rapid dislocation of ligands, whereas more negatively charged nanoconjugates did not display measurable dislocation of surface-bound ligands (Abbasi et al. 2014a). This result is consistent with studies by our research group on the stability of 13 nm oligonucleotide/gold nanoparticle conjugates which found that the negatively charged thiolated oligonucleotide ligands are not easily displaced in intracellular environments or by small molecules such as glutathione (Kouhi et al. 2011).

Oligonucleotides Over the past decade, our research group and others have synthesized, characterized, and applied polyvalent DNA functionalized gold nanoconjugates (DNA-GNP). Indeed, the optical, catalytic, and binding properties of DNAGNP have been used for a variety of colorimetric (Wang et al. 2010, Pavlov et al. 2004, Thiruppathiraja et al. 2011), electronic, scanometric (Kamnev et al. 2002), and Raman-based detection strategies, some of which have recently been commercialized and approved by the FDA.

Synthesis Nanoconjugates densely functionalized with synthetic oligonucleotides are prepared by mixing alkanethiolterminated oligonucleotides and citrate-capped GNP. Oligonucleotide ligands dislocate the citrate from the GNP through creation of a gold–thiol bond. Methods have been optimized for functionalizing particles with diameters ranging from 2 to 250 nm (Vahedi et al. 2013). This polyvalent material has a number of emergent properties that are unique compared to the properties of the oligonucleotides or the GNP alone (Figure 7).

Properties One unusual but now fairly well understood property of DNA-GNP is their ability to bind complementary nucleic acids with a high affinity. Additionally, the oligonucleotides on the GNP surface are close enough such that the counterions associated with one oligonucleotide also act to screen negative charges on adjacent oligonucleotides. In the context of cellular applications, it was theorized and subsequently it makes obvious that the higher binding constant of the DNA-GNP would cause better intracellular binding of the target molecule, thus raising the helpfulness of antisense gene regulation. Nucleic acids are often hampered

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Figure 7. The synthesis of the oligonucleotide gold nanoconjugates: Alkanethiol-terminated oligonucleotides are added to citrate-stabilized GNP, thereby displacing the capping citrate ligands through formation of a gold–thiol bond. Subsequent addition of a salt shields repulsion between the strands, thus leading to a dense monolayer of oligonucleotides.

in biological investigations by enzymatic hydrolysis, which directs to degradation and cause them inactive. Another emergent property of DNA-GNP is resistance to degradation by enzymes such as DNase I. Two explanations have been proposed as the origin of this enhanced stability: The local Na⫹ concentration is the dominant factor that contributes to the improved constancy of DNA. The resistance of DNA-GNP to enzymatic degradation is an important property that renders these structures extremely promising candidates for introducing nucleic acids into cells, where oligonucleotide degradation has historically been a major challenge.

Cellular uptake Perhaps the most surprising property of DNA-GNP is their ability to enter a wide variety of cell types. Indeed, because of their high negative charge, most researchers at the time would have predicted that the nanoparticles would not enter cells. At DNA surface loadings of greater than about 18 pmol cm⫺2, cellular uptake can exceed one million DNA-GNP per cell. The importance of the polyvalent arrangement of

oligonucleotides to cellular uptake can be further emphasized when comparing DNA-GNP to other types of GNP. For instance, HeLa cells internalize only a few thousand citrate-coated gold particles, compared to over one million DNA-GNP under nearly identical conditions. Importantly, fluorescence spectroscopy studies reveal that the thiolated oligonucleotides remain bound to the GNP after cellular internalization (Figure 8). Interestingly, biophysical characterization of DNA-GNP after exposure to serum-containing media reveals changes in the charge and size of the nanoconjugates. The interaction of polyvalent nanoparticle conjugates with proteins provides a possible mechanism of recognition and subsequent internalization of these highly negatively charged particles, the details of which are still under intensive investigation.

Applications in cells Methods based on nucleic acids for detecting and controlling gene expression have had a significant impact on fundamental studies of gene pathways and functions. Methods for controlling gene expression include the use of antisense oligonucleotides and small interfering RNA (siRNA).

Figure 8. Fluorescent microscopy images of C166-EGFP cells incubated for 48 h with gold nanoconjugates functionalized with dual fluorophorelabeled oligonucleotides (3′-Cy3 and 5′-Cy5.5) only reveal fluorescence from Cy5.5 (706–717 nm, upper left). Negligible fluorescence is observed in the emission range of Cy3 (565–615 nm, upper right). Transmission and composite overlay images are shown in the lower left and lower right quadrants, respectively. The arrows indicate the location of the cell.

Gold nanoparticles in biomedical and drug delivery

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Table I. Cell types that internalize polyvalent DNA gold nanoconjugates. Cellular internalization was determined using mass spectrometry and cell-associated fluorescence measurements. Cell type Designation or source Breast Brain Bladder Colon Cervix HeLa Skin Kidney Blood Leukemia Liver Ovary Macrophage

SKBR3, MDA-MB-321, AU-565 U87, LN229 HT-1376, 5637, T24 LS513 SiHa C166, KB, MCF, 10 A MDCK, 293T Sup T1, Jurkat K562 HepG2 CHO RAW 264.7

An ideal gene regulation system—from a search standpoint—should feature high uptake efficiencies across all cell types, strong binding affinity for target nucleic acids, high intracellular stability, and very low toxicity. Recently, DNA-GNP were used as agents to alleviate several of the challenges that are commonly associated with the application of nucleic acids in cells (Table I).

Antisense gene control As a demonstration of this concept, DNA-GNP were extended, which target the mRNA sequences that code for enhanced green fluorescent protein (eGFP) expressed in mouse endothelial cells. An antisense sequence complementary to an internal coding region of the mRNA for eGFP was used in the proposal and creation of “antisense nanoparticles”. Initial experiments make obvious a silencing of approximately 20%, but further optimization of the experimental parameters and conjugate structure has

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increased the gene silencing capability to greater than 75% (Figure 9). Indeed, GNP can be encoded with a suite of designer oligonucleotides that confer improved properties, ranging from amplified target specificity to catalytically improved biological processing. GNP densely functionalized with LNA form remarkably stable duplexes (Russier-Antoine et al. 2008) with complementary nucleic acids, and can be easily manipulated and governed under biologically relevant conditions. For function in cells, the use of LNA-modified GNP enlarges the efficiency of gene knockdown compared to analogous DNA-modified GNP.

Intracellular detection and imaging Oligonucleotide-based probes to visualize and detect intracellular RNA, including those used for in situ staining, molecular beacons, and fluorescence resonance energy transfer (FRET) probes are important biological tools to measure and quantify biological activity in living systems. Recently, our research group has developed novel intracellular detection probes termed “nanoflares” that take advantage of the properties of DNA-GNP (Lin et al. 2008). Nanoflares are oligonucleotide-functionalized gold nanoparticles that are hybridized to short, fluorophore labeled complements designed to provide an intracellular fluorescence signal that correlates with the concentration of a specific nucleic acid or molecular goal. In the lack of a target, the fluorophore is near to the nanoparticle surface, which quenches its fluorescence. Target binding releases the fluorophore, thereby generating a signal that can be detected inside a live cell. Nanoflares can distinguish between different cell types on the basis of the expression profile, and give a semi-quantitative real-time readout of gene expression in a living sample (Figure 10). Several problems commonly

Figure 9. A) Representative Western blots showing the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in HeLa cells treated with various concentrations and compositions of the gold nanoconjugates. GAPDH expression is reduced in a dose- and sequence-dependent manner. α-Tubulin is shown as the loading control. B) Relative decrease in GAPDH expression in HeLa cells. α-Tubulin was used as a loading control and for subsequent normalization of GAPDH knockdown. The error bars represent the standard deviation from at least three Western blots.

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Figure 10. “Nanoflares” are gold nanoconjugates functionalized with oligonucleotide sequences complementary to a specific nucleic acid target (messenger RNA) hybridized to short fluorescent sequences. In the absence of a target, the nanoflares are dark because of quenching by the gold nanoparticle. In the presence of a target, binding displaces the short flare through the formation of a longer (more energetically favorable) duplex. The result is a fluorescence signal inside the cell, which indicates the target has been detected. Scale bar: 20 mm.

associated with intracellular RNA detection, including toxicity, intracellular instability, and the difficulty associated with cell entry, are obviated as these nanoparticles are densely functionalized with oligonucleotides.

shift in the loss spectra, thus providing a direct recite of target binding.

RNA interference

The targeting portions of many proteins are short stretches of oligopeptides. Peptide-based nuclear localization signals have been employed to increase efficacy of conjugated biomolecules and modify the intracellular localization.

Recently, we determined that RNA-GNP can be synthesized and subsequently introduced into cells without the use of transfection agents. Traditional RNAi uses molecular RNAs, which have very short half-lives as a consequence of the instability of ribonucleotides to RNase-type enzymes, thus limiting their efficacy (Chiu and Rana 2003, Soutschek et al. 2004). In the case of RNA gold nanoconjugates, a dense monolayer of surface-immobilized RNA increases the protection from nonspecific degradation both in cell culture media and in the intracellular environment.

Cellular detection In addition to intracellular applications, a colorimetric assay has been developed that uses DNA-GNP for the detection of cancer cells or tissues. In particular, GNP were functionalized with a monolayer of aptamers selected to have a high affinity for surface receptors expressed by a cancer cell line (CCRF-CEM). The aptamer-functionalized nanoconjugates accumulate on the cell surfaces, which causes their surface plasmon resonances to interact. This results in a red

Peptides

Peptide nanoconjugates Conjugation of peptides to gold nanoparticles is achieved through attachment to bovine serum albumin (BSA) and subsequent electrostatic association. The resulting nanoconjugates enter the nucleus of HepG2 cells in culture. Fascinatingly, only nanoconjugates functionalized with peptides containing both nuclear localization signal (NLS) and a receptor-mediated endocytosis (RME) are able to enter the nucleus of these cells (Figure 11).

Peptide/DNA-gold nanoparticle conjugates We recently prepared gold nanoconjugates functionalized with both antisense oligonucleotides and NLS or HIV Tat peptides (Jeynes et al. 2013). Our synthetic strategy uses thiolated oligonucleotides and cysteine-terminated peptides to functionalize the GNP external surface. As the oligonucle-

Figure 11. Images of nanoparticle–peptide complexes incubated with HepG2 cells for 2 h. Complexes were: A) nuclear localization peptide, B) receptor-mediated endocytosis peptide, C) adenoviral fiber protein, and D) both nuclear localization and receptor-mediated endocytosis peptides.

Gold nanoparticles in biomedical and drug delivery otides and oligopeptides are conflictingly charged, the addition of salt is necessitated to monitor conflictingly charged biomolecules during synthesis.

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Multifunctional and multi component DNA nano conjugates Recently, our research group has demonstrated that nanoflares can be adapted for both intracellular mRNA detection and gene knockdown (Prigodich et al. 2009). These nanoflares enter cells and bind mRNA in a location suitable for gene knockdown, so decreasing the relative wealth of mRNA, whereas simultaneously releasing a fluorescent flare. Here, the nanoflare gives a read-out of gene regulation within the cell. In addition, one can, in principle, create all sorts of cell-sorting genetic screening assays by using the nanoflare approach. Other therapeutic nanoconstructs have been designed to take advantage of the uptake of DNA-GNP by cells. These constructs, similar to their canonical DNA counterparts, deliver the drug payload effectively to cells (Dhar et al. 2009). Future work in this area will examine regulating gene expression to chemo sensitize the cells while delivering drugs and materials. Such multi-component conjugates should reduce the total of chemotherapeutic agent required for therapeutic usefulness while simultaneously reducing systemic toxicity.

Antibodies Antibody-labeled gold nanoconjugates have been used in immunohistochemistry for almost 40 years (Horisberger et al. 1975). Synthetic methods to produce antibody-gold nanoconjugates include adsorption (Horisberger et al. 1975), N-hydroxysuccinimide (NHS) ester chemistry (Horisberger et al. 1975, Qian et al. 2008), and oligonucleotide-directed immobilization (Niemeyer et al. 2003). Antibodies can adsorb onto GNP through hydrophobic and ionic interactions, or through chemisorption of native thiol groups present in their chemical structure (Wheeler et al. 1999). However, conjugates synthesized with this method have limited stability because the proteins are easily desorbed (Javier et al. 2008). GNP functionalized with monolayers containing NHS esters can be reacted with the primary amine groups of the antibody to form more stable organizations. Alternately, DNA GNP can be hybridized with antibodies that have been conjugated to complementary oligonucleotides (Javier et al. 2008).

Imaging GNP modified with antibodies specific to cancer associated proteins have been used to image cancerous cells. Light microscopy experiments show that conjugates bind to cancerous cells with six-times greater affinity than the noncancerous controls, thus making this method potentially helpful for the recognition of cancer cells (Huang et al. 2009).

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that use antibody-coated surfaces to hone on cancerous cells. These cells were then irradiated with near-IR light at a frequency that is resonant with the surface plasmon resonance of the nano-shell. Light absorption directs to heating, which causes cell death (Loo et al. 2005). Nano-shells conjugated to control antibodies did not demonstrate this influence because of the lack of nano-shell binding on the cell exteriors. These conjugates are also being expanded as materials that merge photothermal therapy with near-IR imaging capabilities (Stamatoiu et al. 2012).

Lipids Recently, lipids have joined oligonucleotides, peptides, and antibodies as biomolecules used to modify GNP. In this synthesis, thiolated lipids or alkane thiols along with apolipoprotein A1 (APOA1), a protein component of HDL, are adsorbed onto the surface of GNP. Next, a second lipid is adsorbed onto the GNP surface through hydrophobic interactions between the thiolated species and lipid tails. Trouble-free methods for synthesizing HDL with control over the shape, composition, and size had not been revealed prior to these studies. It is being increasingly realized that shape, size, and chemistry of HDL has an impact on its in vivo physiology, and these structures might confirm usefulness as therapeutics and imaging agents (Russier-Antoine et al. 2008).

Therapeutics Natural HDL is critical for transporting cholesterol from macrophages in atherosclerotic plaques and from the body, and increasing the HDL levels may give an improvement to reversing or preventing atherosclerosis. To that end, our research group synthesized HDL mimics called HDLGNP whose size as well as protein and lipid contents are like those of natural HDL (Figure 12). To the best of our knowledge, this is the first measured binding constant for any form of HDL and a cholesterol derivative. This is important as it presents a key data point from which to assess future constructs and their capability to bind cholesterol as well as their potential as novel therapeutic candidates.

Imaging In addition to cholesterol transport, HDL-GNP mimics have been employed to image macrophage cells in vivo. Macrophage density is indicative of high-risk atherosclerotic plaque, thus making it a beautiful imaging target. Tomography images of the mice aortas demonstrated a build-up of HDL-GNP, thus indicating that the nanoparticles possibly will be applied to atherosclerotic imaging.

Gold nanoparticles in diagnostics

Photothermal therapy

Visualization and bioimaging

Gold nanorods and nanoshells conjugated with antibodies are being developed as photothermal therapy agents

Gold nanoparticles have been in active use in the identification of chemical and biological agents The visualiza-

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Figure 12. Templated synthesis of spherical HDL nanoparticles through use of thiol-terminated peptides and the protein (APOA1). Adapted from Ref. [111], with permission from the American Chemical Society; Copyright 2009.

tion methods with the use of GNP and optical microscopy (Wang et al. 2010), in particular, confocal laser microscopy, have gained increasing popularity in medical and biological research. The methods for obtaining confocal images include fluorescence detection (confocal fluorescence microscopy) or resonance elastic or two-photon (multiphoton) light scattering by plasmon nanoparticles (resonance scattering confocal microscopy or two-photon luminescence confocal microscopy). These methods are based on detecting micro-objects using an optical microscope in which the object’s luminescence is excited because of the simultaneous absorption of two (or more) photons; the energy of each of them being inferior than that required for fluorescence excitation. The main advantage of this technique is that the strong reduction in the background signal results in the contrast being enhanced.

Raman scattering (Lin et al. 2008), have been used to enhance the sensitivity of the analytical homophase reaction (Figure 13).

Dot immunoassay At the early stages of the development of immunoassays, preference was given to liquid phase methods, in which the unbound antigen or the bound antibodies deposited were removed using dextran-coated activated coal. The solidphase methods have lately been widely used (first used in radioimmunoassay of proteins), since they supply the possibility to considerably make simpler the analysis procedure and shrink the background signal. The mainly widespread solid-phase carriers are polystyrene plates and nitrocellulose membranes. Enzymes (peroxidase, alkaline phosphatase, etc.) and radioactive isotopes (125I, 14C, 3H) are extensively used as a label in membrane tests (dot and blot

Analytic methods for diagnostics Homophase methods This method is based on two principles: 1) the color and absorption spectrum of a sol vary little upon biopolymer adsorption on individual particles (Thiruppathiraja et al. 2011), 2) when particles approach a distance that is less than one-tenth of their diameter, the sol’s red color alters into purpuric; the absorption range broadens and shifts into the red region (Thiruppathiraja et al. 2011). These changes in the absorption spectrum can be easily detected either spectrophotometrically or visually, this method was subsequently used for performing immunoassay of the antigens of schistosomes and rubella viruses and for the quantitative determination of immunoglobulins, for determining thrombin (using aptamers) and glucose, for the direct detection of cancer cells and leptospira cells in urine, and for determining markers of Alzheimer’s disease (Neely et al. 2009) and protease activity (Neely et al. 2009). The simultaneous use of conjugates of gold nanorods and nanospheres with antibodies for detecting tumor antigens was described. The data on the determination of the hepatitis B virus in blood using gold nanorods conjugated with specific antibodies were published. Various optical methods, including different versions of IR Fourier (Kamnev et al. 2002) and UV-vis beam absorption or deflection spectroscopy, hyper-Rayleigh, differential static and dynamic light scattering, as well as surface-enhanced

Figure 13. Sol-particle immunoassay: a scheme of conjugate aggregation caused by binding to target molecules (a) and corresponding changes in the sol color and absorption spectra.

Figure 14. Dot immunoassays of a normal rabbit serum (1 by using 15-nm GNPs and silica/gold nanoshells (180-nm-core diameter and 15-nm gold shell) conjugated to sheep’s antirabbit antibodies. The IgG quantity equals 1 μg for the first (upper left) square and is decreased by two-fold dilution (left to right). The bottom rows (2) correspond to a negative control (10 μg BSA in each square).

Gold nanoparticles in biomedical and drug delivery

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analyses). In 1984, four studies were separately published in which colloidal gold was employed as a label for solid-phase immunoanalysis. The use of GNP conjugates in solid-phase analysis is based on the fact that the strong red coloration of a gold-containing marker permits one to determine visually the results of a reaction that was carried out on a solid carrier. “Immuno-gold techniques” in a dot blot assay are better than the other types of assays (e.g., immunoenzyme assay) in sensitivity (Beckmann et al. 1994), cost, speed, and simplicity (Figure 14).

Immunochromatography Immunochromatographic assay (Dykman and Khlebtsov 2011) is based on eluent motion along the membrane (lateral diffusion), resulting in the formation of specific immune complexes that are detected as stained bands on different membrane regions. Enzymes, stained latexes, and quantum dots are used as labels in these systems; but, in the overwhelming bulk of cases, gold nanoparticles are employed. The sample under investigation migrates along the test strip due to capillary forces. If an illustration contains the desired compound or immunologically close ones when the sample passes through the absorbing device, a reaction with specific antibodies labelled with colloidal gold occurs, accompanied by the formation of an antigen–antibody composite. The colloidal preparation is involved in the reaction of competitive binding with the antigen immobilized in the test zone (haptene conjugated with a protein carrier is usually used for immobilization in the detection of low-molecular-weight compounds) (Figures 15 and 16).

Figure 16. Scheme for detection of target molecules with a BIA core™ device based on a total internal reflection prism covered by a thin gold layer. Adapted from Ref.

Gold nanoparticles in therapy Photothermal therapy using gold nanoparticles In 2003, GNP were applied for the first time as agents for photothermal therapy (Soutschek et al. 2004, Zharov et al. 2003); it was latter proposed to refer to this kind of therapy as plasmonic photo thermal therapy (PPTT). A new method for selective damage of target cells, which is based on the use of 20–30 nm gold nanospheres radiated by 20-ns laser pulses (532 nm) in order to create Tumor local warming-up, has been described (Pitsillides et al. 2003). The sandwich technology consisting of labeling T-lymphocytes with GNP conjugates was used for the pulse photothermy in the model experiment.

Photodynamic therapy using gold particles Plasmon resonance biosensors New unique technologies are currently being used for the design of biosensor devices, including monolayer selfassembly of metal particles, nano lithography, and vacuum evaporation. These properties of metal clusters served as the basis for the design of new promising plasmon resonance biosensor systems (SPR-biosensors) based on the conversion of biospecific interactions into an optical signal. GNPbased biosensors are applied not only in immunoanalysis, but also for the detection of nucleotide sequences. A record sensitivity was achieved for these sensors in pioneer studies in the zepto-molar range based on the recording spectra of resonant scattering from individual units. This unlocked the gate for the registration of intermolecular interactions at the stage of individual molecules.

The photodynamic method (Doubrovsky et al. 2010, Akbarzadeh et al. 2012a, 2012c, 2012d, Ebrahimnezhad et al. 2013) is applied in the therapy of oncological diseases, certain dermal or infectious diseases, and is based on the use of light-sensitive agents—photosensitizers (including dyes) and, typically, visible light of a certain wavelength. It is well-known that metal nanoparticles are efficient fluorescence quenching agents. Thus, gold nanostructures with plasmon resonance show promise for the selective PPTT of oncological and other diseases.

The use of gold nanoparticles as therapeutic agents Gold nanoparticles are increasingly actively employed not only in cell photo thermolysis experiments and diagnostics, but also for therapeutic reasons. Accumulation of GNP in the tumor is attested by the modification in the color of the tumor; the tumor obtains a bright red/purple color (the color typical of colloidal gold and its aggregates), which coincides with the maximum of tumor-specific activity of the TNF (Alimirzalu et al. 2014, Hosseininasab et al. 2014, Rezaei-Sadabady et al. 2013, Nejati-Koshki et al. 2013, Ghasemali et al. 2013, Mollazade et al. 2013, Akbarzadeh et al. 2014, 2013a, Ahmadi et al. 2014).

Immunologic properties of gold nanoparticles Figure 15. Positive (1) and immunochromatography assay.

negative

(2)

results

of

an

A significant number of studies devoted to the use of GNP in designing DNA vaccines with gene constructions encod-

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ing proteins, to which antibodies had to be produced, have been published, Gold nanoparticles can stimulate antibody synthesis in rabbits, rats, and mice if a lower dose of the antigen is used in comparison with the amount that is required when using a number of conventional adjuvants. Gold nanoparticles used as antigen carriers were shown to stimulate the phagocytic activity of macrophages and affect the functioning of lymphocytes, which most likely are accountable for their immune-modulating effect. The fact is that gold nanoparticles act both as a carrier and an adjuvant. We believe that the detection of adjuvant properties in GNP creates favorable conditions for the development of a new generation of vaccines (Alizadeh et al. 2014, Pourhassan-Moghaddam et al. 2013, 2014, Anganeh et al. 2014, Davoudi et al. 2014, Akbarzadeh et al. 2012b, 2013b, Valizadeh et al. 2012).

Conclusions It is now widely accepted that GNP conjugates are excellent labels for solving the problems of bioimaging, which can be implemented using various optical technologies, including resonance scattering dark-field microscopy, confocal laser microscopy, different variants of two-photon luminescence of GNP, optical coherence tomography, acoustic tomography, etc. GNP conjugates have found application in analytic studies that can be based both on modern instrumental methods (surfaceenhanced Raman spectroscopy, LISNA, IR Fourier spectroscopy, etc.) and the use of simple solid-phase or homo phase procedures (dot analysis, immune chromatography). Finally, there is a necessity to continue and broaden studies on the biodistribution and the toxicity of GNP. First of all, a coordinated program is required, which would reveal the correlations between particle parameters (size, shape, functionalization with various molecular probes), experimental parameters (model, doses, method, and administration scheme, observation duration; organs, cells, subcellular structures under study, etc.), and the observed biological effects. Coordinated efforts in the introduction of standards for the particles and methods used for the testing of nanomaterial toxicity are also required (Kouhi et al. 2014, Sadat Tabatabaei Mirakabad et al. 2014, Eatemadi et al. 2014, Abbasi et al. 2014a, 2014b, Kouhi et al. 2011, Malekynia et al. 2010, Vahedi et al. 2013).

Authors’ contributions AA conceived of the study and participated in its design and coordination. MK participated in the sequence alignment and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgments The authors thank Department of Medical Nanotechnology, Faculty of Advanced Medical Science of Tabriz University for all supports provided.

Declaration of interest The authors report no declarations of interest. The autors alone are responsible for the content and writing of the paper.

This work is funded by 2014 Drug Applied Research Center Tabriz university of Medical Sciences Grant.

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