gold and nano-gold in medicine: overview, toxicology and ...potential catalytic applications in...

Journal ofAPPLIEDBIOMEDICINE

J Appl Biomed7: 75–91, 2009

ISSN 1214-0287

REVIEW

Gold and nano-gold in medicine: overview, toxicology andperspectivesNagender Reddy Panyala1, Eladia María Peña-Méndez2, Josef Havel1, 3

1Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic2Department of Analytical Chemistry, Nutrition and Food Chemistry, Faculty of Chemistry, University of La Laguna, La Laguna, Tenerife, Spain3Department of Physical Electronics, Faculty of Science, Masaryk University, Brno, Czech Republic

Received 21st March 2009.Revised 23rd May 2009.Published online 9th June 2009.

SummaryNano-technology has entered the field of medicine in recent decades and many of the nanomaterialsdeveloped have already had a high impact on health care. Among nanomaterials, gold nanoparticles (GNPs)and gold quantum dots (QDs) are receiving significant attention because their unique physical, chemical andbiological properties are quite different from the bulk of their counterparts. In this article, after a briefhistorical overview, the use of gold and nano-gold in medicine is reviewed, analyzed, and discussed. Thereview particularly deals with the use of GNPs and bio-conjugated GNPs in cancer treatment, drug or genedelivery, DNA detection, biomedical imaging including that of brain activity, enhancement of gene regulation,the detection of toxic metals, immuno-assays, disease detection and diagnostics, therapy and also the toxicityof gold and GNPs, etc. A number of novel applications of GNPs in medicine and perspectives of nano-golduse in medicine are also discussed.

Key words: gold nanoparticles; gold quantum dots; nanomedicine; biomolecules; toxicity

INTRODUCTION

Nanotechnology is a rapidly growing science whichdeals with structures with at least one dimension ofthe size of one hundred nanometres or less andinvolves producing materials and devices of that size.Nanotechnology is focused on the integration offields such as physics, biology, engineering,chemistry, computer science and others.

Nanomaterials have been extensively studied in thelast few years. For example, in our laboratory we haverecently studied nanodiamonds (NDs) and variousclusters (Houška et al. 2008, 2009a, b). Among thesenanomaterials, nanoparticles (NPs) are now playing acrucial role in the field of nanotechnology. Over a fewyears the use of NPs has brought a new era to the fieldof nanomedicine and has altered the foundations ofdisease diagnosis, treatment and prevention based ontheir properties. Furthermore, there is a wide array offascinating nanoparticulate technologies capable oftargeting different cells and extra cellular elements inthe body to deliver drugs, genetic materials, anddiagnostic agents specifically to these locations(Brigger et al. 2002, Paciotti et al. 2004, Jain 2005,Moghimi et al. 2005, Mukherjee et al. 2005, Cuencaet al. 2006, Kam et al. 2006, Zhang et al. 2006,Bhattacharya et al. 2007). The strong optical

Josef Havel, Department of Chemistryand Department of Physical Electronics,Kotlářská 2, 611 37 Brno, Czech [email protected]+420 549 494 114+420 549 492 494

Havel et al.: Gold and nano-gold in medicine

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absorption of GNPs, their scattering properties, andlow or complete lack of toxicity have made them apromising class of nanomaterials for the developmentof novel biomedical applications.

Within nanotechnology, GNPs have attractedgreat attention due to their unique electronic, optical,thermal, chemical, biological properties and theirpotential catalytic applications in various fields suchas biology, medicine, physics, chemistry, materialscience and other interdisciplinary fields (Salata et al.2004). Biocompatible GNPs labelled with specifictargeting biomolecules/drugs, in site specific delivery,are playing a key role in the diagnosis and therapy ofseveral incurable diseases, for example, in cancertreatment. The physical, chemical and biologicalproperties of materials at the nano-scale differ fromcorresponding bulk counterparts. For example, GNPsare typically wine red in colour whereas metallic goldis yellow in colour. The colour of nanoparticles varieswith their size and shapes. Gold atoms can aggregatethemselves under various conditions and can formGNPs by sequential aggregation. Gold clusters can beformed by various methodologies. For example,charged gold (Aun

+) clusters are formed by laserdesorption/ionization (LDI) of Au(s) or Au salts suchas auric acid (HAuCl4) where Aun

+ clusters with n upto 1–25 were detected (Peña-Méndez et al. 2008). Anexample of mass spectra concerning the formation ofgold clusters is given in Fig. 1.

Fig. 1. Part of Laser Desorption Ionisation TOF massspectrum showing the formation of gold clusters Aun

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(n = 1–25). Conditions: commercial MALDI TOF MSinstrument (Axima-CFR. Shimadzu Biotech, U.K.) with apulsed nitrogen laser of a wavelength of 337 nm; linearpositive ion mode, laser energy 100 arbitrary units (A.U.).

GNPs are also synthesized from the reduction ofgold salts by various reducing agents such as gallicacid, H2O2, hydrazine, etc. (Panda et al. 2007, Wang

et al. 2007). GNPs represent several different shapes,i.e. gold nanorods, gold nanospheres, star-like GNPs,etc. The shape of GNPs plays a major role in theuptake of GNPs into cell lines. Chithrani et al. (2006)investigated the intracellular uptake of GNPs withdifferent size and shape into mammalian cells. Theyhave concluded that kinetics and saturationconcentrations are highly dependent upon the physicaldimensions of the nanoparticles. This finding willhave implications in the chemical design ofnanostructures for biomedical applications. Theoptical properties of GNPs are also dependent on theirshape (Nehl et al. 2006, 2008).

Quantum dotsQuantum dots are semi-conducting nanocrystalswhich have quantum optical properties due to theconfinement of their excitons in all three spatialdimensions. These non-fluorescent compoundsdevelop intense, long-lasting colours excitable by UVand visible light-emitting diodes, lasers, etc. due totheir incredible small size (2–100 nm). The coloursproduced are a function of the particle size and thisfunction depends on the diameter of the nanocrystals.The fluorescence is due to a phenomenon calledquantum confinement.

QDs have ideal properties and their applications asbiological tags in ultra sensitive biological detectionhave been extensively investigated (Bruchez et al.1998, Chan et al. 1998, 2002). They are preferable tothe conventional organic fluorophores due to theirstrong, narrow emission spectrum, and photochemicalstability. They also display a wide range of sizetunable colours. So it is possible that a series ofdifferent coloured dots can be activated at the sametime using a single laser. The surface modification ofQDs for various bio-applications has been studied(Tan et al. 2005). Applications of QDs inphotodynamic and radiation therapies for cancer havebeen recently reviewed extensively (Juzenas et al.2008).

Although several reviews have been publishedconcerning the applications of GNPs in medicine, theaim of this particular review is to overview the mostrecent papers concerning new applications of GNPs inmedicine, and to summarize and/or discuss variousaspects of these applications not sufficiently discussedin the literature and also to overview the possibletoxic effects of GNPs on human health.

Forms of goldAlthough gold is a noble metal and a commonly usedmaterial because of its resistance to oxidation and

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interesting electrical, magnetic, optical, and physicalproperties, it forms many and diverse compounds.The oxidation state of gold in its compound rangesfrom –1 to +5, but Au(I) and Au(III) dominate.Gold(I), referred to as the ‘aurous ion’, is the mostcommon oxidation state and reacts with “soft” ligandssuch as thioethers, thiolates, and tertiary phosphines.Only salts and radioisotopes of gold are ofpharmacological value, as elemental (metallic) goldis inert to all chemicals it encounters inside the body.Pure gold is non-toxic and non-irritating wheningested (http://www.espi-metals.com/msds%27s/gold.htm) and is sometimes used as a food decorationin the form of gold leaf. Gold is even approved as afood additive in the European Union.

While metallic gold is non toxic, gold salts are;e.g. gold chloride is a neurotoxin and known to causeperipheral neuropathy. Soluble gold salts such aspotassium gold cyanide, used in gold electroplating,are toxic to the liver and kidneys. There are rare casesof lethal gold poisoning from potassium gold cyanide.Gold toxicity can be ameliorated with chelationtherapy with an agent such as Dimercaprol (Wright etal. 1986, Wu et al. 2001). Even if metallic gold inliving organisms is considered non-toxic and nooxidation reactions are known up to now, thisoxidation problem requires attention.

Gold as a medicine: Historical overviewThe use of GNPs has been known throughout historyand alchemists referred to them as a possible “Elixirof Life”. Gold was already being used by the Chineseas a medicine in 2500 B.C. In India, colloidal redgold was used in the form of ayurvedic medicine forrejuvenation and revitalization during old age, andwas called “Swarna Bhasma” (Mahdihassan 1971,1981). Cinnabar-gold, also known as“Makaradhwaja” (Mahdihassan 1985) is used as adrug for youthful vigour in India. Gold was for a longtime used as a drug, called “nervin” for therevitalization of people who are suffering fromnervous disorders (Fricker et al. 1996). In the 16th

century, gold was recommended for the treatment ofepilepsy (Daniel et al. 2004) for which Paracelsuscreated a potion called Aurum Potabile. Colloidalgold was used to colour glass yellow or red and thecolour of the famous bohemian ruby glass is alsoobtained from nano-gold. Herschel used colloidalgold to record images on paper in 1842. However, themodern scientific evaluation of colloidal gold did notcommence until 1857, when Michael Faraday learnedby experiment that the colour of gold solutions wasdue to the small size of gold particles. In thebeginning of the 19th century, gold was used as a drugfor the treatment of syphilis (Daniel et al. 2004). The

therapeutic use of gold was established for the firsttime when Robert Koch discovered the bacteriostaticeffect of gold cyanide on Mycobacteriumtuberculosis, following which the medicinal use ofgold for the treatment of tuberculosis was introducedin the 1920s (Daniel et al. 2004). Gold and goldcompounds are mainly applied as a drug for thetreatment of rheumatic diseases including psoriasis,palindromic rheumatism, juvenile arthritis and discoidlupus erythematosus (Felson et al. 1990 and Shaw1999). Rheumatoid arthritis (RA) is a form of arthritisthat causes pain, swelling, stiffness and loss offunction in joints. It can affect any joint of the bodybut is common in the wrist and fingers. The mostimportant drugs for the treatment of RA are goldthiolates, and prime examples of these are sodiumaurothiomalate and aurothioglucose. Following thebody’s extensive exposure to these gold thiolatedrugs, the gold can diffuse to various organs such asthe liver, kidney and spleen (Bhattacharya et al.2008). The adsorption of gold in the kidney causesnephrotoxicity. In addition, several adverse effectssuch as skin irritation, mouth ulcers, liver toxicity andblood disorders have been associated with prolongedexposure to gold compounds (Bhattacharya et al.2008). To avoid these poor pharmacokinetics andtoxicity, a safe and superior second generation drug,auranofin (AF) was introduced for the treatment ofarthritis. A critical case study was carried out on theefficacy and toxicity of AF by Glennås et al. (1997),which found that AF can be more lipophilic and hasbetter retention in the circulation due to the presenceof phosphine-ligands. Also renal toxicity has beenreduced by the use of AF in comparison with theparental gold (Bernhard 1982). Subsequently,phosphines containing gold drugs received greatattention and are considered as suitable drugs for thetreatment of cancer, as they exhibit potentialanti-tumour properties; however, when used inclinical trials they have caused cardiovasculartoxicity. The mechanism of action for these drugs hasbeen poorly understood. However, it is believed thatunder biological conditions, gold (I) and gold (III)species can be reduced to gold (0). Recently,Bhattacharya et al. (2008) reported, for the first time,the anti-angiogenic properties of gold. Even thoughthere are many papers published concerning goldapplications in medicine (Mukherjee et al. 2007, Caiet al. 2008) this paper discusses the more recent andmost important applications of GNPs and attempts toput them in perspective.

The role of gold and/or GNPs in medicinalapplications as a direct medicine (active medicine)and as an additive to drugs or biomolecules (passivemedicine) will be discussed in individual chapters.


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Any discussion of the medical applications of GNPscannot be dealt with in discrete chapters and therewill inevitably be some overlapping of topics as newperspectives are brought into play.

GOLD AS AN ACTIVE MEDICINE

Gold, designated as an active medicine, has been usedfor the treatment of various autoimmune diseases andfor RA. In this section, selected examples of variousdrugs containing gold and their medicinalapplications are discussed, while their potential toxiceffects are also considered.

Gold has been used for the treatment of RA forseveral years. The cause of RA is still unknown andautoimmunity plays a pivotal role in its chronicityand progression. RA is a chronic, systemicinflammatory disorder, which mainly attacks thejoints producing an inflammatory synovitis that oftenleads to the destruction of the articular cartilage andankylosis of the joints. In addition, RA can alsoproduce diffuse inflammation in the lungs,pericardium, pleura, sclera, and nodular lesions arethe most common symptoms in subcutaneous tissue.Although gold and gold compounds had been usedfor some time in the treatment of disease it was notuntil 1927 that aurothioglucose was used for the firsttime in the treatment of RA by Landé (1927). Goldthiopropanol sodium sulfanate was used in more than550 cases of RA with beneficial results by Forestierin 1935. Subsequently, several gold compounds weredeveloped and clinical trials confirmed Forestier’sresults (Forestier 1935).

Gold therapy is available in two forms: injectionand the oral route. Clinical benefits have beenachieved from treatment with one of these twotherapies. Both therapies have highly significantside-effects and should be considered as toxic but theoral preparation appears to have a better safety profilethan the injectable compound (Davis 1988).

Gold therapy with auranofinThe treatment of certain diseases, especially RA, withgold compounds is known as Chrysotherapy.Auranofin (AF) is the name of an effective goldcompound which has been used for the treatment ofrheumatoid arthritis. The exact mechanism of actionis still unknown, but it probably acts viaimmunological mechanisms and the alteration oflysosomal enzyme activity. Although AF has limitedlong term clinical experience, its efficacy appears toapproach that of sodium aurothiomalate (an injectableform of gold). AF is suitable for most RA patients,

but patients with diarrhoea, skin rash, and pruritus aresometimes troubled, and thrombocytopenia andproteinuria are potentially serious adverse effectswhich may occur during the therapy. Mucocutaneousside effects are more frequent with injectable goldcompounds and gastrointestinal reactions are the mostcommon adverse effects seen with AF. Auranofin andsodium aurothiomalate have the same frequency ofside effects, but they are generally less severe withauranofin. Although there are adverse effects, sucheffects can be controlled by a reduction in dosagealthough temporary or permanent withdrawal of AFmay be necessary. A combination of AF withdisease-modifying potential agents is presentlyavailable for the treatment of rheumatoid arthritis.This therapy will generate significant interest as itacquires a better place in therapy through additionalwell-designed investigations and wider clinicalexperience (Chaffman et al. 1984).

AF is also showing potential antimalarial activitybeyond its anti-arthritic properties. Recently theanti-malarial activity of auranofin has been reported(Sannella et al. 2008); it seems that it acts as apotential inhibitor of mammalian thioredoxinreductases which cause oxidative stress. The authorsconclude that it exhibits potential antiplasmodicactivity, which means that AF could inhibit thegrowth of Plasmodium palsifarum, the pathogenwhich causes the malaria disease. The combination ofartemisinin (an anti-malarial drug) and AF showedadditional antimalarial effects (Sannella et al. 2008).

Gold therapy with aurothioglucoseIt is thought that an immune system response maycontribute to arthritis. Gold may affect this responseand decelerate the progression of the disease.Aurothioglucose (ATG, Solganal®)), also known asgold thioglucose, is a glucose derivative used to treatRA. It reduces the inflammation and swelling due toarthritis. Aurothioglucose is used to treat only theearly stages of adult or juvenile (childhood)rheumatoid arthritis and it is not effective in treatingadvanced cases of arthritis. Although ATG exhibitsanti-rheumatic activity, it also presents adverse effectssuch as blood in urine, mouth sores, fever, skin rashor itching, unusual tiredness, swelling of eyes andface, etc. (http://www.uamshealth.com/HealthLibrary,viewed on 21st March 2009).

HIV therapyNF-κB (nuclear factor kappa-light-chain-enhancer ofactivated B cells) is a protein complex that acts as atranscription factor, which is a potent cellularactivator of HIV-1 gene expression. Traber et al.(1998) reported that ATG significantly inhibited


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TNF-α induced HIV-1 replication in latently infectedcells and also reported its efficacy as an inhibitor ofHIV replication in latently infected OM10.1 andAch2 cells. The tumour necrosis factor(TNFα)-induced HIV-1 replication in OM10.1 [ahuman immunodeficiency virus type-1(HIV-1)-infected promyelocytic cell line, designatedas OM, derived from HL-60 cells] or Ach2 cells(Ach-2 is a clone that survived infection and thatconstitutively produces low levels of reversetranscriptase) was significantly inhibited bynon-cytotoxic doses of ATG (>10 µM in OM10.1cells and >25 µM in Ach2 cells). The effect of ATGon NF-κB-dependent gene expression was confirmedby a transient CAT assay (An enzyme assay. CATstands for chloramphenicol acetyl transferaseenzyme). Electron microscopic examinations, as wellas specific staining, revealed the accumulation ofmetal gold in the cells, indicating that gold ions couldblock NF-B-DNA binding by a redox mechanism(Traber et al. 1998). It was concluded that themonovalent gold compound ATG is a potentiallyuseful drug for the treatment of patients infected withHIV.

GNP AS A PASSIVE MEDICINE

Due to their unique properties, GNPs have been usedas an additive to various drugs, and are in this respectdesignated as a passive medicine; We will discusshere the recent novel applications of GNPs as apassive medicine.

GNPs in cancer treatmentConventional strategies for the treatment of cancerare surgery, radiation therapy and chemotherapy butcancer nanotechnology is a fascinating field withbroad applications in the fight against cancer, whichinclude molecular imaging, molecular diagnosis, andtargeted therapy. GNP applications in cancertreatment have been investigated recently, because ofthe unique properties of GNPs. Laser irradiated goldnanorods, nanospheres, nanoshells, and nanocagescan kill bacteria and cancer cells (Zharov et al. 2006,Chen et al. 2007). According to Gannon et al. (2008),lasers have been used in clinical diagnosis to treatmalignant tumours, but the procedure presents anumber of difficulties (Bilchik et al. 2000, Bleicher etal. 2003, Curley et al. 2004, Haemmerich et al. 2005).These limitations include: 1) laser ablation is aninvasive treatment because it requires insertion ofneedle electrodes directly into the tumours; 2) thetreatment is non-specific as it causes thermal injury to

normal tissues together with malignant tissues; 3)there is the possibility of incomplete tumourdestruction; and 4) invasive laser ablation treatment oftumours is limited to a few organ sites (liver, kidney,breast, lung, and bone) (Gannon et al. 2005, Raut etal. 2005). Although laser ablation methods havelimitations, GNPs have been used extensively todetect and destroy cancerous cells.

GNPs can act as chemical carriers to deliver drugsto a targeted location to destroy a cancer cell or theycan be used together with radiation. Radio frequenciesare used to heat GNPs. The heated nanoparticles inturn heat, and destroy the cancer cell. These radiowaves might not harm the healthy cells. Infrared lightwaves can be used in place of radio waves to heat upthe cancer cells for destruction, but, in reality, thelaser mediated destruction of tumour cells requiresintracellular and intra-tumoral agents that release heatunder the influence of a radio frequency field. Alsorequired are suitable agents that have little or nointrinsic cellular or tissue toxicity to malignanttumour cells and not harmful to healthy cells.Potentially these non-invasive techniques for thetreatment of cancers, with minimal or no toxic effects,could be highly beneficial (Gannon et al. 2008). Aninstance of this, according to Patra et al. (2007), isGNP induced apoptosis (cell death) in the humancarcinoma lung cell line A549. For comparison, theprocess was checked with another two cell lines, i.e.,BHK21 (baby hamster kidney) and HepG2 (humanhepatocellular liver carcinoma), and no effects inBHK21 and HepG2 cell lines were observed. Theseresults demonstrated that the specificity of inductionfor the death response in A549 cells indicated thatGNPs do not target all cell type indiscriminately, butare specific to a few cell lines. Flow cytometricstudies have shown a dose dependent threshold effectin this death response (Patra et al. 2007).

Recently, Bhattacharya et al. (2008) reviewed theuse of GNPs in cancer therapy. So called coatedGNPs with specific antibodies represent a promisingalternative as probes for the detection of antigens onthe surfaces of cells. The interaction between GNPsand proteins has been known for some time(Krasovskii et al. 2007, Dobrovolskaia et al. 2008)and has been applied in the immunogold labellingtechnique in which GNPs labelled with antibodies areused to mark specific cells and act as a probe for thetransmission of the scanning electronic microscopyobservations (Faulk and Taylor 1979). By electronmicroscopy observation, the immunogold labelsappear as extremely dense round spots at the positionof the antigen in ultra thin sections of tissues (Roth etal. 1981).

Lu et al. (2009) used tiny hollow gold spheres


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with a highly targeted peptide to hunt down andpenetrate deep inside melanoma cells destroying themusing heat converted from infra red light. Thistechnique utilized tiny gold spheres, 40 to 50nanometres in diameter, their hollowness allowingthem to penetrate the cancer cells. The hollow goldspheres have a strong but narrow and tunable abilityto absorb light at the visible and near-infrared end ofthe spectrum, whereas other metal nanoparticles donot. Photo thermal ablation, a minimally invasivetreatment in which the target tissue is destroyed byirradiating the target area with light, was applied tolive lab mice. The light is converted to heat anddestroys the surrounding tissue. However, it is noteasy to treat melanomas using this method due to thedifficulty in aiming nanoparticles and differentiatingbetween healthy and cancerous tissue. To avoid thisproblem, a peptide, was embedded in the goldnanospheres. The peptide was highly target specificand it could only bind to the melanocortin type 1receptor, which is excessively abundant in melanomacells (Lu et al. 2009).

When gold nanosphere treated melanoma cellcultures are irradiated, the majority of cellscontaining the targeted nanospheres die, and nearlyall those left are damaged beyond repair. Whenuntargeted nanospheres are used, only a very smallfraction of treated cells die (Lu et al. 2009).

Laser induced explosion of GNPs have been usedfor the selective nanophotothermolysis of cancerouscells. Letfullin et al. (2006) demonstrated that whenGNPs are irradiated with short laser pulses, theirtemperature rises very rapidly and it leads to theformation of nonlinear effects such as micro bubbleformation, acoustic and shock wave generation andthe abnormal cancerous cell are destroyed. Byengineering laser wavelength, laser pulse duration,nanoparticles size and shape, this method can providehighly localized cancer cell damage in a controlledmanner without damaging the surrounding healthytissue. Thermal explosion is realized when heat isgenerated within a strongly absorbing target morequickly than the heat can diffuse away. It isdemonstrated that on the basis of simple energybalance, the threshold energy density of a single laserpulse required for the thermal explosion of a solidgold nanosphere is ~ 40 mJ/cm2 (Letfullin et al.2006). A schematic presentation of the laser inducedexplosion of GNPs for the treatment of cancer isshown in Fig. 2.

GNPs as drug or gene carriers or vectorsThere is great interest in finding pharmaceuticallyactive particles to mediate the targeting of cell types.Monoclonal antibodies have been used for the

Fig. 2. Schematic presentation of the laser inducedexplosion of GNPs for the treatment of cancer.

A Areas of thermo mechanical damage.B Au atoms and clusters with high velocity.C Shock waves with supersonic expansion.D High pressure layers.

treatment of various illnesses, especially in thetargeting of cytotoxic drugs and radio nucleotides todiseased cells in the human body. However, the use ofmonoclonal antibodies presents limitations related totheir immunogenicity and targeting specifics.Photodynamic therapy (PDT) is another type oftreatment, in which cancer cells are destroyed usinglight activated dyes. When dye-loaded cells aretreated with a laser, the dye interacts with oxygen toproduce singlet state oxygen, which is highly toxic tocells. However, residual dye molecules can migrate toother normal tissues such as the skin and eyes,resulting in sensitivity to daylight (Salata 2004,El-Sayed et al. 2006).

Recently the use of gold colloids and GNPs hasbecome an attractive platform in biomedicalapplications. GNPs have had a great impact on themedical community, are used for cancer diagnosistreatment, and as drug delivery vectors for biologic orpharmacological agents (Hirsch et al. 2005, Huang2006, Niidome et al. 2006, Ding et al. 2007,Mukherjee et al. 2007). Biomedical applications ofplasmon resonant metal nanoparticles were reportedby Liao et al. (2006). The use of GNPs as drugcarriers in diagnostic and medical fields is based oncertain specific qualities such as: 1) they are easilyfabricated and 2) the binding capacities of moleculeswith GNPs to target cancer cells, antibodies,carbohydrates and pharmacological agents, is higherthan other nanoparticles (Hirsch et al. 2005, Huang2006, Niidome et al. 2006, Mukherjee et al. 2007,Ganon et al. 2008). An illustration of the use of GNPsas drug carriers is shown in Fig. 3.

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Fig. 3. Schematic illustration of nanogold used as a drugcarrier for drug delivery.

GNPs have been used also as effective genedelivery vectors for the efficient transfection of DNAinto the nucleus by tuning the surface charge densityof aminoacid-functionalized GNPs. Transfection isthe process of introducing nucleic acids into cells bynon-viral methods. Transfection of animal cellsinvolves opening transient pores or ‘holes’ in the cellplasma membrane, to allow the uptake of material.Genetic material (supercoiled plasmid DNA orsiRNA constructs), or proteins such as antibodies,may be transfected into the cell plasma membrane.G h o s h e t a l . ( 2 0 0 8) fu n c t i o n a l i z e da m i n o a c i d - c o n j u g a t e d t h i o l s w i t h1-pentaanethiol-protected gold clusters (Au-C5, corediameter ~ 2 nm). The functional head grouptransfection efficacy was explored using threedifferent vectors (GNP-LysG1, GNP-Lys, andGNP-Gly) with differing densities of cationic sites.Aminoacid-functionalized gold colloids provide ascaffold for effective DNA binding with consequentcondensation. The research concluded that particlesfunctionalized especially with lysine and lysinedendron formed compact complexes and that genedelivery efficiency was high without any observablecytotoxicity. GNPs functionalized with firstgeneration lysine dendrons (GNP-LysG1) showed~ 28-fold greater efficacy than polylysine in reportergene expression (Ghosh et al. 2008).

Multiple drug therapy has been proven to be anefficient treatment for complex diseases, but thedifference in chemical properties andpharmacokinetics of different drugs causes seriousconcerns in terms of loading, delivering, andreleasing multiple drugs. Wijaya et al. (2009)demonstrated the load and selective release of twodifferent DNA oligonucleotides from two differentgold nanorods, where DNA was loaded on the goldnanorods via thiol-conjugation. Ultra fast laserirradiation was used at the nanorods’ longitudinalsurface plasmon resonance peaks to melt gold

nanorods for selective release. This particular type ofgold nanorod can be selectively melted by theexcitation at one specific wavelength after which itselectively releases one type of DNA strand. Therelease of DNAs was laser fluence tunable and itsefficacy was 50–80%. Released oligonucleotides werestill functional (Wijaya et al. 2009).

GNPs enhance gene regulationSmall interfering RNA (siRNA), also known assilencing RNA, is a class of 20–25 nucleotide-longdouble-stranded RNA molecules which is involved inthe RNA interference (RNAi) pathway, where itinterferes with the expression of a specific gene.GNPs can be used as a shield to protect siRNAs fromdegradation and they can enhance the regulation ofgenes involved in cancer. Consequently, thisstraightforward method is receiving significantattention for the delivery of potent gene-regulatingagents into targeted cells.

The therapeutic use of siRNA molecules has faceda number of problems among which is the degradationof siRNA molecules in the presence of the nucleases.These potentially therapeutic siRNA molecules arehighly unstable, particularly if trace levels of enzymescalled nucleases that break down nucleic acids arepresent. GNPs have been examined to avoid thedegradation of siRNA molecules. For thisexamination, Giljohann et al. (2009) used a harshstripping method to ensure that the surface of theGNPs was free of any nucleases. Surprisingly, thistreatment did not have any effect on the optical orphysical properties of the nanoparticles. Without theapplication of this pre-treatment, it was not possibleto add any RNA molecules to nanoparticles. Usingtheir developed pre-treatment, the resulting13-nanometer GNPs held an average of 34 siRNAmolecules each. In the confocal microscopicalexamination, the entry of nanoparticles into culturedtumour cells was also visible and inside the cell thesiRNA was able to escape from the nanoparticlesurface and inactivate its gene target. The efficacy ofgene silencing achieved with the siRNA-nanoparticleconstruct was double compared with cells which weretreated with siRNA alone. In conclusion, a boost intherapeutic efficacy arises because of improvedsiRNA stability when associated with GNPs.

DNA-FUNCTIONALIZED GNPs IN TOXICMETAL DETECTION

The application of GNPs to the detection of toxicmetals such as mercury in the human body and in the

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environment (rivers, streams, lakes and oceans)represents a new goal. Industrial pollution caused bythe release of many potentially toxic metals into theenvironment is a major world problem. Methylmercury, a potential neurotoxin, is dangerous toyoung children and pregnant women when ingestedin for example methyl mercury-contaminated fish orshellfish.

Lee et al. (2007) reported a novel colorimetricmethod which is sensitive, accurate, and capable ofdetecting mercury ions at 100 nanomolar level inaqueous samples. This is a procedure for monitoringmercury levels in lakes and rivers which is instrumentfree, highly selective for GNPs and, unlike other Hg2+

detection methods, does not require organic solventsor co-solvents. GNPs-DNA conjugates exhibit apurple colour, as they are held with oligonucleotidesat a a critical distance. The key to the method is thata purple to red colour change occurs at 46 °C if thesolution has no mercury, and occurs at a highertemperature if mercury is present. Mercury bindsstrongly to the T-T mismatch site and the GNPs arenow held together even more tightly. The mercurycreates a stronger bond that requires a highertemperature to break apart the DNA strands andexhibit the purple colour. When we heat this solutionabove 46 °C, DNA strands will automatically beseparated and the GNPs are no longer in closeproximity to each other. The colour turns to to brightred, indicating how much mercury is present at thehigh temperature.

Based on previous knowledge of the capacity ofmercury to bind to the bases of thymidine-thymidine(T-T) mismatch, Lee et al. (2007) designated eachstrand of DNA containing thymidine-thymidinemismatchto be attached to GNPs. If there is mercuryin the test solution, then it is tightly bounded tothymidine-thymidine mismatch.

So, in the test solution, the detection of mercuryion traces can be made easily by following thechanges in the colour of the solution from purple tobright red. This method is selective for Hg2+ andinsensitive to Mg2+, Pb2+, Cd 2+, Co2+, Zn2+, Ni2+, andother metal ions.

Recently scientific results reveal that GNPs can beused to trap toxins, bacteria, viruses used bybioterrorists. GNPs act as very effective detectors ofbiological toxins. Solutions of the GNPs are a strongred colour but instantly they change to blue when thetarget substance is present. The particles reveal thepresence of toxins faster and with a detection limitl o w e r t h a n e x i s t i n g t e c h n i q u e s(http://news.bbc.co.uk/2/hi/technology/4872188.stm,viewed on 18th February 2009).

OTHER MEDICAL APPLICATIONS

GNPs in DNA detectionNanotechnology is achieving better results in allresearch areas related to/and connected with DNAdetection – sensitivity, selectivity, ease of use, costand speed. Park et al. (2002) developed a novel DNAdetection technique by using gold nanoprobes. Thetechnique is more accurate, less expensive and fasterthan conventional methods. The technique applied asimple electrical signal for the detection of targetDNA. These new DNA detection techniques replaceclassical DNA detection techniques such as forexample, the polymerase chain reaction (PCR), usingconventional fluorescent probes in clinicaldiagnostics. Different DNA samples can be placed ona glass slide. Each sample is made up of single strandsof synthesized DNA or oligonucleotides, with asequence designed to bind with its complimentarytarget DNA. These oligonucleotides are then placedbetween a pair of electrodes. The gold nanoparticleprobes, each covered with oligonucleotide strands,latch on to these pairings. Only perfect matches willremain, and this allows silver-coated-gold nanoprobesto carry a measurable electrical signal across the gapbetween the electrodes. The detection limit of thetarget DNA is 500 femtomolar concentrations and apoint mutation selectivity factor of 100,000:1 wasfeasible by this method (Park et al. 2002). Thismethodology may be applied to the detection ofhundreds of pathogenic agents, simultaneously. Afteroptimization, the method may be used to quickly andeasily detect biological weapons such as anthrax, andsmall pox and also to diagnose many pathogenic andgenetic diseases, cancer, HIV and other sexuallytransmitted diseases. Obtaining results would takeonly a matter of minutes instead of days (Park et al.2002).

The development and application of hybridmaterials which include the conjugation of inorganicnanoparticles, such as GNPs with biomolecules (i.e.proteins, polynucleotides), have been reported byDubertret et al. (2001). A 25-base syntheticoligonucleotide modified with primary amine at its 3’end and a disulfide at its 5’ end was selected for theexperiment. This oligonucleotide acquires ahairpin-loop structure as its extremities. The patternof sequence provides a hairpin-loop structure whichis very stable at room temperature. The combinationof the highly specific properties of biomolecules withthe unique optical properties of nanoparticles, makethem highly suitable for use in the field of bio-diagnostics. Dubertret et al. (2001) demonstrated thathigh sensitivity is reached when hybrid


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dye-DNA-nanoparticle materials such as nucleic acidprobes to detect specific nucleotide sequences areapplied. The structure of the synthetic oligonucleotideopens easily on hybridization of the loop to its target.Initially, an amine reactive dye was conjugated to theprimary amine at the 3’ end of the oligonucleotide.Disulfide at the 5’ end of the oligonucleotide wascleaved after purification and free sulfhydryl wascovalently attached to 1.4 nm diameter gold cluster.This hairpin structure, with a gold nanoparticle at oneend and fluorescent dye at the other end presents astrongly suppressed fluorescence. When present insolution of specific strands of DNA, the linker isunzipped, and the dye begins to shine as much as1000 times brighter than before (Fig. 4).

Fig. 4. Hybrid dye-DNA-nanoparticle materials can beused as nucleic acid probes to detect specific nucleotidesequences with incredible sensitivity.

The newly developed GNP-DNA nanoconjugatesare used to measure the nuclease activity and forDNA foot printing (Liu et al. 2006) (Fig. 5A, B).

Fig. 5A. DNA conjugate with a single GNP.

Fig. 5B. GNP-DNA nanoconjugate for measuringnuclease activity and DNA footprinting.

GNPs in bioimagingOptical imaging is a technique which encompassesthe elucidation of morphology, molecular function,and metabolism of tissue. GNPs have intense plasmonresonance driven absorption and scattering properties.The plasmon resonance condition of GNPs dependson their size, shape, structure, and refractive index ofthe embedding medium. Due to these specialproperties, they are used as contrast agents for opticalimaging techniques. Cellular imaging using goldnanorods was also reported recently by Murphy et al.(2008). Rayavarapu et al. (2007) applied thebio-conjugated GNPs (nanospeheres, nanorods) forimaging, where the conjugation was achieved byusing a combination of electrostatic and hydrophobicbinding interactions. The particles selected for thisconjugation were 25 nm citrate-capped gold spheres.Gold nanorods conjugated with HER81 antibodiesbind with high efficiency to Her2 receptors expressedby SKBR3 breast carcinoma cells (Rayavarapu et al.2007).

Using multifunctional GNPs, Kumar et al. (2007)described the intracellular molecular imagingplatform. The multifunctional GNPs incorporatingboth cytosolic delivery and targeting moieties arenamed as “nanosensors”. These nanosensors can beeffectively delivered into the cytoplasm and bind totheir target providing a strong molecular specificoptical signal. For developing the nanosensors,monodisperse water soluble 20 nm GNPs andanti-actin and anti-biotin antibodies were used. TheGNPs were attached to antibodies via a heterofunctional linker that consisted of an alkalineterminating in a dithiol tether and an amide-bondedadipic hydrazide (Kumar et al. 2007). Finally, thesenanosensors will bind strongly to the actin or biotinand give the optical signal (Fig. 6).

Au

FluorophoreGold nanoparticle

+


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Fig. 6. Multifunctional plasmonic nanosensor forimaging intra-cellular biomarkers in live cells. Aschematic representation of gold nanoparticle basednanosensor with both targeting and delivery components.

GNPs in brain activity imagingThe human brain is a complicated structure,consisting of trillions of interconnections betweentens of billions of neurons. The aim of modernneuroscience is to develop creditable goals for thefundamental understanding of these circuits forneurological and general health care reasons. Metalnanoparticles, such as GNPs have a localized surfaceplasmon (LSP) resonance property and can be usedfor optical sensing. The optical properties of GNPsmake them most significant as they can provideenhanced functionality for several biologicalapplications. Some optical properties of star shapedGNPs have also been reported (Nehl et al. 2006). Dueto their bright, stable scattering signal, GNPs arebeing used as labels for microscopic imaging. Liningup tiny electrodes within or near single neurons toprobe their electrical activity is one of the ways to‘walk around’ the brain’s microcircuit system.Although it is a well established technique, it hassome limitations due to it being an invasive methodand noisy, because of the background electricalactivity in the brain. Many techniques have been triedto investigate the neuronal structures of the brain, forexample by using optical probes that can detectneuronal activity with light. However, these methodsoften require labelling neuronal cells with electricallysensitive dyes that may be toxic to neurons in humanbrain (Zhang et al. 2009). Avoiding the use of toxicdyes, a new type of dye-free optical probe, that candirectly sense naturally occurring neural activity, wasdeveloped (Zhang et al. 2009). This methodologyincludes the embedding of GNPs into brain tissuecultures, followed by measurement of the electricalactivity of live neurons. Zhang et al. (2009) reportedthe optical detection of brain cell activity by using

plasmonic GNPs. An electrostatic field sensingtechnique has been used to detect mammalian braincell activity, by optically measuring the cellularpotential induced shift in the surface plasmonresonance mode of an adjacent planar goldnanoparticle array. Hippocampal brain neural cellswere cultured onto the GNPs template. When thechemically triggered neurons switched their potential,optical cellular level individual transient signals weredetected. The neuronal activity modulates the electrondensity at the surface of the nanoparticle, giving as aresult, an observable spectral shift that can bemonitored. Zhang et al. (2009) compared theexperimental data with calculations using the Drudemodel for the dielectric response of gold and the Sternmodel for the metal-electrolyte junction and they werefound to be in close agreement with theoreticalcalculations.

GOLD QDs APPLICATIONS

Gold QDs in immuno assaysDuring the last decade, fluorescence based assaysh a v e t a k e n a p r i o r i t y p o s i t i o n i nimmunochemistry-based in vitro diagnosis and in vivoimaging assays. There are a number of advantages inusing radio chemical labels and chromophores: highersensitivity, their potential for easy multiplexing, theiroptical properties, including narrow absorption bandwidth, broad excitation, size dependent controllableemission spectra, high molar absorptivity andpotential stability against photo bleaching, and it isthought that semiconductor QDs will replace thetraditional organic or organometallic fluorophores.Triulzi et al. (2008) synthesized gold QDs usingpolyamidoamine (PAMAM) dendrimers as cappingligands. The gold QDs show size- dependent narrowemissions, narrow excitation and excellentbiocompatibility and are suitable for biologicalapplications. PAMAM dendrimers have proven to beexcellent cell transfect ion agents due to the presenceof hydroxyl or amine groups at their end groups. So,with the combination of a biologically inert materialsuch as gold, along with biological compatibility, itpenetrates to the cell membranes. This combinationcould be efficiently conjugated to variousbiomolecules as DNA, RNA, proteins andcarbohydrates. PAMAM dendrimers can becovalently or electrostatically conjugated to DNA(Choi et al. 2004, 2005), RNA (Kang et al. 2005),carbohydrates (Tsvetkov et al. 2002) and proteins(Lee et al. 2004, Wang et al. 2007, Triulzi et al.2008).

Targetting Ab

Anti - biotin Ab and

biotinylated delivery

peptide

PEG-SH

Hetero functional

linker

Gold nanoparticle


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Triulzi et al. (2008) reported the use of gold QDsin a human IgG immunoassay. The gold QDs wereelectrostatically conjugated to a goat-derivedanti-human IgG polyclonal antibody to detect humanIgG. The conjugate of gold QDs-PAMAMdendrimers-antibody complex was treated withspecific antigens such as IgG, rabbit IgG, and anumber of unrelated proteins such as bovine serumalbumin and β-casein. Encapsulated gold QDs inPAMAM dendrimers with the combination ofspecific antigens like anti-human IgG were verystable. The different systems exhibited a linearluminescence quenching response over themicromolar to nanomolar human IgG concentrationrange with little or no interference from BSA orβ-casein and a slight interference from rabbit IgG,which indicated the specificity of anti-human IgGantibody. A strong influence of specific antigen(human IgG) concentration on the luminescence ofgold QDs was observed and no influence wasobserved on BSA or β-casein. A linear decrease inthe luminescence of the gold QDs was seen with anincrease in the concentrations of human IgG over therange of concentration from micromolar tonanomolar. It followed that the mechanism ofantibody-induced selectivity in competitive surfacequenching is mainly dependent on the size of thequantum dots. This lends great credence to the usageof different varieties of ligand specific assays fordiagnostics and imaging applications in nanomedicine(Triulzi et al. 2008).

Gold Quantum dots-Protease complexes as “smartnanoprobes” for disease detectionImaging for the detection of diseases is a longstanding problem in medicine. The use of GNP-basednanostructures allows ‘illumination’ of the diseaselocation, making it possible to distinguish betweenthe signals due to “cancer is here”, and so called‘background’ light (arising from nanostructures notspecifically bound to their molecular targets).Actually, QDs make it possible to solve this problem.Emissive nanoparticles as QDs exhibit a uniqueproperty: they give off light in the near-infrared, arare portion of the spectrum that has no backgroundcomponents in biomedical imaging. Near-infraredlight also passes harmlessly through muscle, cartilageand skin. So these new QDs probes can alertresearchers to tumours and other diseases localizeddeeply in the human body. There is no need for abiopsy and/or invasive surgery to detect the locationsof these diseases. The ‘smart nanoprobes’ developedconsist of gold tethered quantum dots, which are ahundred times smaller than a human cell and areasprogrammed to light up when they are activated by

specific proteases. This altered expression of specificproteases is a hallmark of cancer, arthrosclerosis andmany other diseases (Chang et al. 2005).

The major mechanism in the construction of these‘smart nanoprobes’ is the use of QDs which have aunique property of luminescence. These probes areprepared by a special technique called “quenching”.Quenching involves binding a gold nanoparticle to thequantum dot to inhibit its luminescence. TheseQD-peptide-GNPs are used for detection ofproteolytic activity proteases. When a specificprotease cleaves peptide in the QD-peptide-GNPscomplex, the GNPs are released and these GNPsexhibits luminescence, and by following thisluminescence, the exact disease location can be found(Chang et al. 2005).

GOLD AND GNPs TOXICITY

Although gold and gold compounds have been usedas a potential drug for the treatment of RA, someadverse effects, such as skin irritation, dermatosis,stomatitis, contact allergy, and hypersensitivityreactions were associated with over exposure to goldand gold compounds.

Gold compounds such as gold sodium thiosulphate (GSTS) have been used as a dental goldalloy. Vamnes et al. (2000) reported two critical casesof the adverse effects of dental gold alloys. In the firstcase study, a healthy 34-year-old woman was affectedby dental gold and demonstrated symptoms whichincluded itching in the mouth, loss of taste, andburning sensation in the oral mucosa. Non-specificdermatitis was also observed on her face. Hersymptoms persisted and increased over some months(Vamnes et al. 2000). In the second case studydescribed by Vamnes et al. (2000), a healthy 50-year-old man was affected by the wearing of agold-containing crown; the symptoms were itchylichenoid dermatitis, which was symmetricallydistributed on the trunk and thighs.

According to McKenna et al. (1995), 278consecutive patients with suspected contact dermatitiswere patch tested with GSTS. Around 13 patients(4.6%) were affected with a positive allergic response.The patients were female, with an average age of37 years. The most affected sites of eczema (genericterm for inflammatory conditions of the skin) werehead and neck (62%); 46% of people had eczema onthe limbs and 15% had a perianal rash.

Räsänen et al. (1999) examined thehypersensitivity reactions involved in dermatosisinduced by gold sodium thiomalate (GSTM).


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Svensson et al. (2002) examined the clinical andbiochemical effects of a low starting dose for goldtherapy in RA patients with a contact allergy to gold.Four out of 19 patients were found to be affected bycontact allergy to gold. In comparison with thegold-negative patients (starting dose: 10 mg GSTM),a large increase of TNF-α, sTNF-R1, IL-1 ra(interleukin-1 receptor antagonist) was observed inthe serum of gold-allergic patients. Räsänen et al.(1999) concluded that it is better to minimize the doseof GSTM to 5 mg to reduce the risk of adversereactions. Fleming et al. (1997) examined 8 patientsto detect gold contact hypersensitivity. Two out ofeight patients were shown to be positive to goldallergy which is mainly caused by wearing goldjewellery.

GNPs cytotoxicityNanoscale engineering is one of the most attractivetechnologies and includes knowledge of variousfields such as electronics, physics, biology andmedicine. However the use of nanoparticles could belimited because of toxic effects on health. (Lewinskiet al. 2007). It has recently been reported that silvernanoparticles can have hazardous effects on humanhealth and the environment (Panyala et al. 2008).GNPs also represent some hazardous effects onhuman health. The few papers which have beenpublished on the cytotoxicity of GNPs are referred toin the following discussion.

According to Pan et al. (2007) the cytotoxicity ofGNPs was checked by including them with variousincubating cell lines; for example, HeLa cervixcarcinoma epithelial cells (HeLa), SK-Mel-28melanoma cells (SK-Mel-28), L929 mouse fibroblasts(L929) and mouse monocytic/macrophage cells. Itwas observed in transmission electron microscopicalstudies that the cells were swollen and lost theirsubstrate contact. Membrane blebbing and vesicleformation at the plasma membrane were observed.After 12 hours incubation with GNPs, many cells hadlost cell-to-cell and cell-to-substrate contact. A fewcells showed cytoplasmic disintegration, membraneblebbing and nuclear fragmentation indicatingapoptosis and secondary necrosis. Pan et al. (2007)described the cell death by gold clusters in the celllines, finding two types of cell death in the cell lines.Firstly, there is a rapid cell necrosis caused by strongphysical stress, such as freezing, boiling, shearing,and rupture of the cell membrane. The productsreleased by this necrosis process are also highlyinflammatory so they can cause inflammation in thehuman body. The second type of cell death – slowacting cell death – also called apoptosis, doesn’tinvolve membrane damage and inflammation. In this

apoptosis or programmed cell death, cells undergo anenergy-dependent sequence of events, which leads tothe fragmentation of nuclei and cytoplasmicorganelles into small apoptotic bodies eliminated byphagocytes. Apoptosis is a default process in whichdestroyed particles and dead cells are scavenged bythe macrophages in our body (Pan et al. 2007).

Connor et al. (2005) examined the GNPs uptakeinto human cell lines and their cytotoxic levels byculturing K562 human leukaemia. As a result it wasconcluded that GNPs and functionalized GNPs haveno potential toxic effects on human leukaemia celllines. Moreover, the results confirmed that a fewfunc t iona l ized GNPs such as CTAB(cetyltrimethylammonium bromide)-modified GNPshave potential toxic effects on human cell lines.CTAB also shows toxicity in human cell lines.Goodman et al. (2004) reported cationic and anionicfunctionalized GNP toxicity and they concluded thatcationic functionalized GNPs have moderate toxicitywhen compared with anionic functionalized GNPs.

Pernodet et al. (2006) examined the adverseeffects of citrate coated GNPs on human dermalfibroblast cells. The presence of GNPs led to majorchanges in cell growth, cell shape and alteration ofactin fibres. It was shown that 14 nm GNPs can easilypass through the cell membrane and accumulate intovacuoles. The presence of GNPs is responsible forabnormal actin filaments and extra cellular matrixconstructs in dermal fibroblasts. It leads to a decreasein cell proliferation, adhesion and motility.

Wiwanitkit et al. (2009) examined the in vitrotoxic effects of 9 nm size GNPs on the humanspermatozoa. A fresh semen sample was cultured with500 µL of GNPs and motility and morphologicalchanges were studied after 15 minutes by clinicalmicroscopy. The same changes were studied as wellfor the blank sample which was not cultured with theGNPs. Microscopic examinations revealed that 25%of sperm cells were not motile and penetration ofGNPs into the sperm head and tail was observed. Inaddition, the fragmentation of the sperm was visible.

Cho et al. (2009) studied the in vivo toxic effectsof 13 nm size PEG-coated GNPs on mice. Thenanoparticles were seen to induce acute inflammationand apoptosis in the liver. They accumulated in theliver and spleen for up to 7 days after injection andhad long blood circulation times. In addition,transmission electron microscopical examinationsrevealed that numerous cytoplasmic vesicles andlysosomes of liver Kupffer cells and spleenmacrophages contained the PEG-coated goldnanoparticles. Because PEG-coated GNPs are widelyused in biomedical applications these effects haveobvious clinical implications.


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Chen et al. (2009) also studied the in vivo toxicityof GNPs on mice. Naked GNPs ranging from 3 to 100nm were injected intraperitoneally into mice at a doseof 8 mg/kg/week. GNPs of 3, 5, 50, and 100 nm didnot show any toxic effects; however, GNPs rangingfrom 8 to 37 nm induced severe sickness in mice andmice injected with GNPs in this range showedfatigue, change of fur colour, loss of appetite, andweight loss.

DISCUSSION AND CONCLUSIONS

From ancient time, gold and gold compounds havebeen used as a medication as it was believed that goldhas the power to cure a number of diseases, amongthem rheumatoid arthritis. Recent developments innano-science have also made a number of applicationbased upon the use of nano-materials available tomedicine. The most important of these materials for‘nanomedicine’ is nano-gold.

GNPs show entirely different properties incomparison with their bulk counterparts. GNPs havea capability of binding strongly to bio molecules likeproteins, peptides, antibodies, oligonucleotides, andpathogens such as bacteria, viruses, etc. GNPs couldbe used as biomarkers to detect diseases and todeliver a suitable drug to cure the disease. However,even though gold and gold salts such as auranofin,aurothioglucose have been used as active medicine inthe treatment of, for example, RA they could havetoxic effects on human health.

The use of functionalized GNPs as biomarkersand their binding capacity to biomolecules is openingnew feasibilities, for example, attenuation of thefunction of the disease causing proteins. Muchconcern is paid to the investigation of GNPinteraction studies such as type of binding, structureof biomolecules, and influence of charge on theinteraction. The interaction of biomolecules withGNPs needs to be even more deeply investigated.There are still many problems and questions to beanswered. For example, is interaction of biomoleculeswith GNPs a size dependent process or are there otherfactors influencing the interaction? Are we able todesign GNPs that will attenuate the function ofdisease causing proteins with more efficiency andmore specificity? There are many questions similar tothese.

It is shown in the literature that there are a numberof potential adverse effects of gold and GNPs onhuman health. It is evident that the toxic andcytotoxic effects of GNPs have not been sufficientlyinvestigated to date, and a critical examination is

needed of GNP cytotoxicity and the symptoms ofover-exposure to GNPs. Also it is necessary toinvestigate the feasibilities of minimizing thecytotoxic levels of GNPs to use them in variousbiomedical applications without any hazardous effectson human health. The design of less toxic andtherapeutically significant GNPs might open newways of approaching incurable diseases. Generally,the use of gold and GNPs applications in medicine isgrowing so rapidly that great progress and perhapssurprising new developments can be expected in thenear future.

ACKNOWLEDGEMENTS

Support from the Ministry of Education, Youth andSports of the Czech Republic, projectsMSM0021622411 and LC 06035 and Academy ofSciences of the Czech Republic, project KAN101630651 are greatly acknowledged. E.M.P-M andJ.H. acknowledge the partial support from Universityof La Laguna, La Laguna, Tenerife, Spain.

REFERENCES

Bernhard GC: Auranofin therapy in rheumatoidarthritis. J Lab Clin Med 100:167–177, 1982.

Bhattacharya R, Patra CR, Earl A, Wang S, KataryaA, Beng LL, Kizhakkedathu JN, Yaszemski MJ,Greipp PR, Mukhopadhyay D, Mukherjee P:Attaching folic acid on gold nanoparticles usingnoncovalent interaction via different polyethyleneglycol backbones and targeting of cancer cells.Nanomed Nanotechnol Biol Med 3:224–238,2007.

Bhattacharya R, Mukherjee P: Biological propertiesof “naked” metal nanoparticles. Adv Drug DelivRev 60:1289–1306, 2008.

Bilchik AJ, Wood TF, Allegra D, Tsioulias GJ,Chung M, Rose DM, Ramming KP, Morton DL:Cryosurgical ablation and radiofrequency ablationfor unrespectable hepatic malignant neoplasms: aproposed algorithm. Arch Surg 135:657–662,2000.

Bleicher RJ, Allegra DP, Nora DT, Wood TF, FoshagLJ, Bilchik AJ: Radiofrequency ablation in 447complex unrespectable liver tumours: lessonslearned. Ann Surg Oncol 10:52–58, 2003.

Brigger I, Dubernet C, Couvreur P: Nanoparticles incancer therapy and diagnosis. Adv Drug DelivRev 54:631–651, 2002.


88

Bruchez MJ, Maronne M, Gin P, Weiss S, AlivasatosAP: Semiconductor nanocrystals as fluorescentbiological labels. Science 281:2013–2016, 1998.

Cai W, Gao T, Hong H, Sun J: Applications of goldnanoparticles in cancer nanotechnology.Nanotechnol Sci Appl 1:17–32, 2008.

Chaffman M, Brogden RN, Heel RC, Speight TM,Avery GS: Auranofin. A preliminary review of itspharmacological properties and therapeutic use inrheumatoid arthritis. Drugs 27:378–424, 1984.

Chan WCW, Nie S: Quantum dot bioconjugates forultrasensitive nonisotopic detection. Science281:2016–2018, 1998.

Chan WCW, Maxwell DJ, Gao X, Bailey RE, HanM, Nie S: Luminescent quantum dots formultiplexed biological detection and imaging.Curr Opin Biotechnol 13:40–46, 2002.

Chang E, Millar JS, Sun J, Yu WW, Colvin VL,Drezek R, West JL: Protease-activated quantumdot probes. Biochem Biophys Res Commun334:1317–1321, 2005.

Chen J, Wang D, Xi J, Au L, Siekkinen A, WarsenA, Li Zhi-Yuan, Zhang H, Xia Y, Li X: Immunogold nanocages with tailored optical properties fortargeted photo thermal destruction of cancer cells.Nano Lett 7:1318–1322, 2007.

Chen YS, Hung YC, Liau I, Huang GS: Assessmentof the in vivo toxicity of gold nanoparticles.Nanoscale Res Lett, 2009, in press.

Chithrani BD, Ghazani AA, Chan WCW:Determining the size and shape dependence ofgold nanoparticle uptake into mammalian cells.Nano Lett 6:662–668, 2006.

Cho WS, Cho M, Jeong J, Choi M, Cho HY, Han BS,Kim SH, Kim HO, Limc YT, Chung BH, Jeong J:Acute toxicity and pharmacokinetics of 13nm-sized PEG-coated gold nanoparticles. ToxicolAppl Pharmacol 236:16–24, 2009.

Choi Y, Mecke A, Orr BG, Holl MMB, Baker JR Jr.:DNA-directed synthesis of generation 7 and 5PAMAM dendrimer nanoclusters. Nano Lett4:391–397, 2004.

Choi Y, Thomas T, Kotlyar A, Islam MT, Baker JR:Synthesis and functional evaluation ofDNA-assembled polyamidoamine dendrimerclusters for cancer cell-specific targeting. ChemBiol 12:35–43, 2005.

Connor EE, Mwamuka J, Gole A, Murphy CJ, WyattMD: GNPs are taken up by human cells but donot cause acute cytotoxicity. Small 1:325–327,2005.

Cuenca AG, Jiang H, Hochwald SN, Delano M,Cance WG, Grobmyer SR: Emerging implicationsof nanotechnology on cancer diagnostics andtherapeutics. Cancer 107:459–466, 2006.

Curley SA, Marra P, Beaty K, Ellis LM, Vauthey JN,Abdalla EK, Scaife C, Raut C, Wolff R, Choi H,Loyer E, Vallone P et al.: Early and latecomplications after radiofrequency ablation ofmalignant liver tumours in 608 patients. Ann Surg239:450–458, 2004.

Daniel MC, Astruc D: GNPs: assembly,supramolecular chemistry, quantum-size relatedproperties, and applications toward biology,catalysis, and nanotechnology. Chem Rev104:293–346, 2004.

Davis P: Gold therapy in the treatment of rheumatoidarthritis. Can Fam Physician 34:445–452, 1988.

Ding Y, Liu J, Wang H, Shen G, Yu R: Apiezoelectric immunosensor for the detection ofα-fetoprotein using an interface ofgold/hydroxyapatite hybrid nanomaterial.Biomaterials 28:2147–2154, 2007.

Dobrovolskaia MA, Patri AK, Zheng J, Clogston JD,Ayub N, Aggarwal P, Neun BW, Hall JB, McNeilSE: Interaction of colloidal gold nanoparticleswith human blood: effects on particle size andanalysis of plasma protein binding profiles.Nanomed Nanotechnol Biol Med, 2009, in press.

Dubertret B, Calame M, Libchaber AJ:Single-mismatch detection using gold-quenchedfluorescent oligonucleotides. Nat Biotechnol19:365–370, 2001.

El-Sayed IH, Huang X, El-Sayed MA: Selective laserphoto-thermal therapy of epithelial carcinomausing anti-EGFR antibody conjugated GNPs.Cancer Lett 239:129–135, 2006.

Faulk W, Taylor G: An immunocolloid method forthe electron microscopy. Immunochemistry8:1081–1083, 1979.

Felson DT, Anderson JJ, Meenan RF: Thecomparative efficacy and toxicity of second-linedrugs in rheumatoid arthritis. Results of twometaanalyses. Arthritis Rheum 33:1449–1461,1990.

Fleming C, Forsyth A, Mackie R: Prevalence of goldcontact hypersensitivity in the west of Scotland.Contact dermatitis 36:302–304, 1997.

Forestier J: Rheumatoid arthritis and its treatment bygold salts. J Lab Clin Med 20:827–40, 1935.

Fricker SP, Buckley RG: Comparison of twocolorimetric assays as cytotoxicity endpoints foran in vitro screen for antitumour agents.Anticancer Res 16:3755–3760, 1996.

Gannon CJ, Curley SA: The role of focal liverablation in the treatment of unrespectable primaryand secondary malignant liver tumours. SeminRadiat Oncol 15:265–272, 2005.

Gannon CJ, Patra CR, Bhattacharya R, Mukherjee P,Curley SA: Intracellular GNPs enhance


89

non-invasive radiofrequency thermal destructionof human gastrointestinal cancer cells. JNanobiotechnol 6:2–10, 2008.

Ghosh PS, Kim Chae-Kyu, Han G, Forbes NS,Rotello VM: Efficient gene delivery vectors bytuning the surface charge density ofaminoacid-functionalized GNPs. ACS Nano2:2213–2218, 2008.

Giljohann DA, Seferos DS, Prigodich AE, Patel PC,Mirkin CA: Gene regulation with polyvalentsiRNA-nanoparticle conjugates. J Amer ChemSoc 131:2072–2073, 2009.

Glennås A, Kvien TK, Andrup O, Clarke-Jenssen O,Karstensen B, Brodin U: Auranofin is safe andsuperior to placebo in elderly-onset rheumatoidarthritis. Br J Rheumatol 36:870–877, 1997.

Goodman CM, McCusker CD, Yilmaz T, RotelloVM: Toxicity of GNPs functionalized withcationic and anionic side chains. BioconjugateChem 15:897–900, 2004.

Haemmerich D, Laeseke PF: Thermal tumourablation: Devices, clinical applications and futuredirections. Int J Hyperthermia 21:755–760, 2005.

Hirsch LR, Halas NJ, West JL: Whole-bloodi m m u n o a s s a y f a c i l i t a t e d b y g o l dnanoshell-conjugate antibodies. Methods MolBiol 303:101–111, 2005.

Houška J, Alberti M, Havel J: Laser ablationsynthesis of phosphorus sulphides, selenides andternary PpSqSer clusters from various precursors.Rapid Commun Mass Spectrom 22:417–423,2008.

Houška J, Panyala NR, Peña-Méndez EM, Havel J:Mass spectrometry of nanodiamonds. RapidCommun Mass Spectrom 23:1125–1131, 2009a.

Houška J, Peña-Méndez EM, Kolář J, Frumar M,Wágner T, Havel J: Laser ablation of AgSbS2 andcluster analysis by time-of-flight massspectrometry. Rapid Commun Mass Spectrom23:1715–1718, 2009b.

H u a n g S H : G o l d n a n o p a r t i c l e - b a s e dimmunochromatographic test for identification ofStaphylococcus aureus from clinical specimens.Clin Chim Acta 373:139–143, 2006.

Jain KK: Nanotechnology-based drug delivery forcancer. Technol Cancer Res Treat 4:407–416,2005.

Juzenas P, Chen W, Sun YP, Coelho MAN,Generalov R, Generalova N, Christensen IL:Quantum dots and nanoparticles forphotodynamic and radiation therapies of cancer.Adv Drug Deliv Rev 60:1600–1614, 2008.

Kam NW, Liu Z, Dai H: Carbon nanotubes asintracellular transporters for proteins and DNA:an investigation of the uptake mechanism and

pathway. Angew Chem Int Edn Engl 45:577–581,2006.

Kang H, DeLong R, Fisher MH, Juliano RL:Tat-conjugated PAMAM dendrimers as deliveryagents for antisense and siRNA oligonucleotides.Pharm Res 22:2099–2106, 2005.

Krasovskii VI, Nagovitsyn IA, Chudinova GK,Savranskii VV, Karavanskii VA: Interaction ofgold nanoparticles with bovine serum albumin.Bull Lebedev Phys Inst 34:321–324, 2007.

Kumar S, Harrison N, Richards-Kortum R, SokolovK: Plasmonic nanosensors for imagingintracellular biomarkers in live cells. Nano Lett7:1338–1343, 2007.

Landé K: Die gunstige, Heinflussing schleichenderDauerinfectionen surch Solganal. Munch MedWchschr 74:1132–1139, 1927.

Lee JS, Han MS, Mirkin CA: Colorimetric Detectionof Mercuric Ion (Hg2+) in Aqueous Media usingDNA Functionalized GNPs. Angew Chem46:4093–4096, 2007.

Lee SC, Parthasarathy R, Botwin K, Kunneman D,Rowold E, Lange G, Klover J, Abegg A, Zobel J,Beck T, Miller T, Hood W et al.: Biochemical andimmunological properties of cytokines conjugatedto dendritic polymers. Biomed microdevices6:191–202, 2004.

Letfullin RR, Joenathan C, George TF, Zharov VP:Laser induced explosion of gold nanoparticles:potential role for nanophotothermolysis of cancer.Nanomedicine 1:473–480, 2006.

Lewinski N, Colvin V, Drezek R: Cytotoxicity ofnanoparticles. Small 4:26–49, 2007.

Liao H, Nehl CL, Hafner JH: Biomedical applicationsof plasmon resonant metal nanoparticles.Nanomedicine 1:201–208, 2006.

Liu GL, Yin Y, Kunchakarra S, Mukherjee B, GerionD, Jett SD, Bear DG, Gray JW, Alivisatos AP,Lee LP, Chen FF: A nanoplasmonic molecularruler for measuring nuclease activity and DNAfootprinting. Nat Nanotechnol 1:47–52, 2006.

Lu W, Xiong C, Zhang G, Huang Q, Zhang R, ZhangJZ, Li C: Targeted photo thermal ablation ofmurine melanomas with melanocyte-stimulatinghormone analog-conjugated hollow goldnanospheres. Clin Cancer Res 15:876–886, 2009.

Mahdihassan S: Colloidal gold as an alchemicalpreparation. Janus 58:112–118, 1971.

Mahdihassan S: The tradition of alchemy in India.Am J Chin Med 9:23–33, 1981.

Mahdihassan S: Cinnabar-gold as the best alchemicaldrug of longevity, called Makaradhwaja in India.Am J Chin Med 13:93–108, 1985.

McKenna KE, Dolan O, Walsh MY, Burrows D:


90

Contact allergy to gold sodium thiosulfate.Contact dermatitis 32:143–146, 1995.

Moghimi SM, Hunter AC, Murray JC:Nanomedicine: Current status and futureprospects. FASEB J 19:311–330, 2005.

Mukherjee P, Bhattacharya R, Wang P, Wang L,Basu S, Nagy JA, Atala A, Mukhopadhyay D,Soker S: Antiangiogenic properties of GNPs. ClinCancer Res 11:3530–3534, 2005.

Mukherjee P, Bhattacharya R, Bone N, Lee YK,Patra CR, Wang S, Lu L, Charla S, Banerjee PC,Yaszemski MJ, Kay NE, Mukhopadhyay D:Potential therapeutic application of GNPs inB-chronic lymphocytic leukaemia (BCLL):enhancing apoptosis. J Nanobiotechnol 5:4, 2007.

Murphy CJ, Gole AM, Stone JW, Sisco PN, AlkilanyAM, Goldsmith EC, Baxter SC: GNPs in biology:Beyond toxicity to cellular imaging. Acc ChemRes 41:1721–1730, 2008.

Nehl CL, Liao H, Hafner JH: Optical properties ofstar-shaped GNPs. Nano Lett 6:683–688, 2006.

Nehl CL, Hafner JH: Shape-dependent plasmonresonances of gold nanoparticles. J Mater Chem18:2415–2419, 2008.

Niidome T, Yamagata M, Okamoto Y, Akiyama Y,Takahashi H, Kawano T, Katayama Y, NiidomeY: PEG-modified gold nanorods with a stealthcharacter for in vivo applications. J ControlRelease 114:343–347, 2006.

Paciotti GF, Myer L, Weinreich D, Goia D, Pavel N,McLaughlin RE, Tamarkin L: Colloidal gold: anovel nanoparticle vector for tumour directeddrug delivery. Drug Deliv 11:169–183, 2004.

Pan Y, Neuss S, Leifert, Fischler M, Wen F, SimonU, Schmid G, Brandau W, Jahnen-Dechent W:Size-dependent cytotoxicity of GNPs. Small3:1941–1949, 2007.

Panda BR, Chattopadhyay A: Synthesis of Aunanoparticles at “all” pH by H2O2 reduction ofHAuCl4. J Nanosci Nanotechnol 7:1911–1915,2007.

Panyala NR, Peña-Méndez EM, Havel J: Silver orsilver nanoparticles: a hazardous threat to theenvironment and human health? J Appl Biomed6:117–129, 2008.

Park SJ, Taton TA, Mirkin CA: Array-basedelectrical detection of DNA with nanoparticleprobes. Science 295:1503–1509, 2002.

Patra HK, Banerjee S, Chaudhuri U, Lahiri P,Dasguptha AK: Cell selective response to GNPs.Nanomedicine: Nanotech Biol Med 3:111–119,2007.

Peña-Méndez EM, Hernández-Fernaud JR, NagenderR, Houška J, Havel: The chemistry of goldclusters in plasma generated with MALDI, Laser

desorption ionization and laser ablation fromvarious precursors. Chem Listy 102:s1394–s1398,2008.

Pernodet N, Fang X, Sun Y, Bakhtina A,Ramakrishnan A, Sokolov J, Ulman A,Rafailovich M: Adverse effects of citrate/GNPs onhuman dermal fibroblasts. Small 2:766–773, 2006.

Raut CP, Izzo F, Marra P, Ellis LM, Vauthey JN,Cremona F, Vallone P, Mastro A, Fornage BD,Curley SA: Significant long-term survival afterradiofrequency ablation of unrespectablehepatocellular carcinoma in patients withcirrhosis. Ann Surg Oncol 12:616–628, 2005.

R ä s ä n e n L , K a i p i a n e n - S e p p ä n e n O ,Myllykangas-Luosujärvi R, Käsnänen T, Pollari P,Saloranta P, Horsmanheimo M: Hypersensitivityto gold sodium thiomalate-induced dermatosis. BrJ Dermatol 141:683–688, 1999.

Rayavarapu RG, Petersen W, Ungureanu C, Post JN,Leeuwen TGV, Manohar S: Synthesis andbioconjugation of GNPs as potential molecularprobes for light-based imaging techniques. Int JBiomed Imaging 2007:29817–29827, 2007.

Roth J, Bendayan M, Carlemalm E, Villiger W,Garavito M: Enhancement of structuralpreservation and immunocytochemical staining inlow temperature embedded pancreatic tissue. JHistochem Cytochem 29:663–671, 1981.

Salata O: Applications of nanoparticles in biology andmedicine. J Nanobiotechnol 2:3, 2004.

Sannella AR, Casini A, Gabbiani C, Messori L, BiliaAR, Vincieri FF, Majori G, Severini C: New usesfor old drugs. Auranofin, a clinically establishedantiarthritic metallodrug, exhibits potentantimalarial effects in vitro: Mechanistic andpharmacological implications. FEBS Lett582:844–847, 2008.

Shaw IC: Gold-based therapeutic agents. Chem Rev99:2589–2600, 1999.

Svensson A, Möller H, Björkner B, Bruze M, LedenI, Theander J, Ohlsson K, Linder C: Rheumatoidarthritis, gold therapy, contact allergy and bloodcytokines. BMC Dermatol 2:2–6, 2002.

Tan WB, Zhang Y: Surface modification of gold andquantum dot nanoparticles with chitosan forbioapplications. J Biomed Mater Res A 75:56–62,2005.

Traber KE, Okamoto H, Kurono C, Baba M, SaliouC, Soji T, Packer L, Okamoto T: Anti-rheumaticcompound aurothioglucose inhibits tumournecrosis factor-a-induced HIV-1 replication inlatently infected OM10.1 and Ach2 cells. IntImmunol 11:143–150, 1998.

Triulzi RC, Micic M, Orbulescu J, Giordani S,Mueller B, Leblanc RM: Antibody-gold quantum


91

dot-PAMAM dendrimer complex as animmunoglobin immunoassay. Analyst 133:667–672, 2008.

Tsvetkov DE, Cheshev PE, Tuzikov AB, ChinarevAA, Pazynina GV, Sablina MA, Gambaryan AS,Bovin NV, Rieben R, Shashkov AS, Nifant’evNE: Neoglycoconjugates based on dendrimerpoly(aminoamides). Russ J Bioorg Chem28:470–486, 2002.

Vamnes JS, Morken T, Helland S, Gjerdet NR:Dental gold alloys and contact hypersensitivity.Contact Dermatitis 42:128–133, 2000.

Wang W, Chen Q, Jiang C, Yang D, Liu X, Xu S.One-step synthesis of biocompatible goldnanoparticles using gallic acid in the presence ofpoly-(N-vinyl-2-pyrrolidone). Colloid Surface A301:73–79, 2007.

Wang X, Inapagolla R, Kannan S, Lieh-Lai M,Kannan RM: Synthesis, characterization, and invitro activity of dendrimer-streptokinaseconjugates. Bioconjugate Chem 18:791–799,2007.

Wijaya A, Schaffer SB, Pallares IG,Hamad-Schifferli K: Selective release of multiple

DNA oligonucleotides from gold nanorods. ACSNano 3:80–86, 2009.

Wiwanitkit V, Sereemaspun A, Rojanathanes R:Effect of gold nanoparticles on spermatozoa: thefirst world report. Fertil Steril 91:e7–e8, 2009.

Wright IH, Vesey CJ: Acute poisoning with goldcyanide. Anaesthesia 41:936–939, 1986.

Wu ML Tsai WJ, Ger J, Deng JF, Tsay SH, YangMH: Cholestatic hepatitis caused by acute goldpotassium cyanide poisoning. Clin Toxicol39:739–743, 2001.

Zhang Z, Yang X, Zhang Y, Zeng B, Wang S, Zhu T,Roden RB, Chen Y, Yang R: Delivery oftelomerase reverse transcriptase small interferingRNA in complex with positively charged singlewalled carbon nanotubes suppresses tumourgrowth. Clin Cancer Res 12:4933–4939, 2006.

Zhang J, Atay T, Nurmikko AV: Optical detection ofbrain cell activity using plasmonic GNPs. NanoLett 9:519–524, 2009.

Zharov VP, Mercer KE, Galitovskaya EN, SmeltzerMS: Photo thermal nanotherapeutics andnanodiagnostics for selective killing of bacteriatargeted with GNPs. Biophys J 90:619–627, 2006.

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