Review on gold nanoparticles and their applications

Minakshi Das1, Kyu Hwan Shim1, Seong Soo A. An1 & Dong Kee Yi1

1Department of BioNano Technology, Gachon University,Seongnam 461-701, KoreaCorrespondence and requests for materials should be addressedto S. S. A. An ([email protected]),D. K. Yi ([email protected])

Received 6 December 2011Accepted 21 December 2011DOI 10.1007/s13530-011-0109-y©The Korean Society of Environmental Risk Assessment and Health Science and Springer 2011

Abstract

Gold nanoparticles are widely used in many fields aspreferred materials for their unique optical and physicalproperties, such as surface plasmon oscillations forlabeling, imaging, and sensing. Recently, many ad-vancements were made in biomedical applicationswith better biocompatibility in disease diagnosis andtherapeutics. Au-NPs could be prepared and conju-gated with many functionalizing agents, such as poly-mers, surfactants, ligands, dendrimers, drugs, DNA,RNA, proteins, peptides and oligonucleotides. Thisreview addressed the use of gold nanoparticles andthe surface functionalization with a wide range ofmolecules, expanding and improving gold nanopar-ticles in targeting drugs for photothermal therapy withreduced cytotoxic effcts in various cancers, genetherapy and many other diseases. Overall, Au-NPswould be a promising vehicle for drug delivery andtherapies.

Keywords: Gold nanoparticles, Functionalization, Cancer,Drug delivery, Cytotoxicity

Introduction

Nanotechnology emphasizes materials in 10-9 meterscales, involving biotechnology, material sciences,computer sciences, medicines, pharmacy and engineer-ing1. Nanoparticles (NPs) in crystalline and amorphousforms received many attentions worldwide for theiruses in many commercial applications, and fueled

many research centres to devote in developing andexpanding various nano-applications2-4. Korea investedtrillions of wons in nanotechnology and received numer-ous proposals for opening research centres and facilitieswith international networks and supports from variousagencies5. Many nanomaterial applications came outwith high expectations from diverse fields, which weregrown and diversified into medical areas, includingfield of clinical trials for their unique optical and phys-ical properties1.

Nanotechnology originally came into existence in9th century by Mesopotamian people for giving lustr-ous effect in pots. For the first time in 1857, MichaelFaraday discovered the ruby gold nanoparticles (Au-NPs), which became the foundation for the modernnanotechnology6-8. Forty years later, Zsigmondy mergedhis technology with Faraday’s discovery and introducedthe procedure called, ‘seed mediated method’, whichwould still be used in the present day for the synthesisof various NPs9. Zsigmondy also invented an ultrami-croscope for characterizing the structure, shape, and sizeof NPs9,10. Another scientist, Svedberg introduced ultra-centrifuge and showed that the motion of macromole-cules (colloids) depended on their shape and size11-13.In the similar period, G. Mie tried to find out the reasonfor various colors of gold (Au) colloids14. Recently,the applications of Au-NPs expanded into various bio-medical fields, such as biosensors, clinical chemistry,immunoassays, genomics, photothermolysis of cancercell, microorganisms detection and control, targeteddrug delivery, optical imaging and monitoring of bio-logical cells and tissues by exploiting resonance scat-tering, or in vivo photo acoustic techniques15-24.

Biomedicine adopted NPs in developing many appli-cations. For past few years, Au-NPs focused in explor-ing their unique properties for imaging and therapeuticapplications. Recently, engineered NPs were used asnano-platforms for the effective and targeted drug de-livery in bio imaging areas by labelling NPs with bio-logical, biophysical, and biomedical carriers1. Severalreviews on their basic physical, chemical and opticalproperties were also published25-31. Au-NPs conjugatedwith drugs created an interesting theme, which fuelledmany interests, especially for their binding propertywith vast range of organic and biological molecules,their low toxicity and strong absorption spectrum.Hence, Au-NPs played the major role as a carrier for

Review on Gold Nanoparticles and Their Applications

drugs and vaccines to the targeted cells or tissues. Ingeneral, the conjugation with drugs or biomoleculeswas performed by modifying the surface of Au-NPs.In case of NPs for drug delivery, higher concentrationsof drugs with the NPs would be required to increasethe efficiency of drugs to kill the pathogens32. By uti-lizing unique physical, chemical and photo thermalproperties of Au-NPs, the conjugation and release ofdrugs into the cells could be controlled33,34. Releaseof drug would involve two processes, such as internalstimuli operated system, which could occur in a bio-logically controlled manner, or external stimuli, ope-rated by the support of stimuli-generated processes31,35.

At present, focus is on the structure divergent of syn-thesize Au-NPs, starting from general colloidal Aunanospheres, nanorods, or silica coated Au nanoshells,nanocubes, nanorice, nanostars, to nanocages36-39. Fin-ally, surface functionalization protocols were developedto be continuously used in various biomedical applica-tions, such as conjugation between Au-NPs and mole-cular probes, including polyclonal and monoclonalantibodies (Ab), DNA oligonucleotides, enzymes, anddrugs40-42.

Starting from ancient Chinese medicine to modernmedicine, Au was used for medicinal purposes43. Rheu-matoid arthritis was treated with Au salts, but its mech-anism was still unresolved. Intense researches on Au-NPs were undertaken, utilizing advantages of its uniqueproperties, but few adverse toxic effects of Au-NPsbecame to known, which would need further investi-gations.

Historical Background forExistence of Au Colloids

Michael Faraday was astonished by the ruby colorof Au colloids. He was mainly interested in the inter-actions of light with metal particles, but the paper fo-cused mostly on several aspects of the formation, pro-perties and nature of ruby Au. The modern colloidalchemistry was born from these studies and interpreta-tions, which led the development combining nanosci-ence and nanotechnology44. Faraday knew about rubyglass from its usage in making staining glass windowsfrom centuries back. Since last seventeenth century,Purple of Cassius could be made by adding tin intoAu solution, which was further applied in coloringglass and enamels44.

Faraday showed that Au-chloride could be reducedupon heat treatment, due to the side reactions withmany reagents, such as organic compounds and phos-phorus6. Based on some physical and chemical under-standings, Faraday emphasized that metallic Au was

dispersed uniformly in both ruby glass and ruby fluids.When particles were smaller than the wavelength oflight, then the particles in various sizes would showdifferent colors from their original color8. Around 100years later, the Ruby colored colloids, prepared by Fara-day, were stable, and their sizes were determined tobe in ranges between 2-6 nm from electron microscope6.

Zsigmondy became interested in colloids and beganhis investigations for the color and opacity of the rubyglass. Since Faraday first produced Au colloids andother compounds of ruby glasses through reductionmethods, Zsigmondy tried to find different ways forreproducing Au colloids. Afterwards, Au sols becamehis major work of interest9,10. Great invention of Zsig-mondy was the ultramicroscope for visualizing indivi-dual particles. Moreover Zsigmondy could determentexact sizes of the particles, and he noticed interestingproperties of the particles, based on their sizes andmotion, especially with NPs. The passage of light thr-ough the hydrosol of Au could provide an understand-ing of the motion of Au particles in the solution. Hence,Zsigmondy investigated the changes in colors of Auby adding salts into the Au sols, in order to assess theeffects from the addition of protective agents, such asgelatine and gum arabic. By using the ultramicroscope,he explained the reason for changes in colors mightbe due to the coagulated particles in different sizes,and the action of protective agents could inhibit coa-gulation. Zsigmondy developed another useful ma-chine to perform ultrafiltration, for the investigationof colloidal systems45.

Another scientist named Theodor Svedberg also be-came interested in the properties of colloidal sols, hencehe focused and built an ultracentrifuge, which couldgenerate forces over 100,000 times of gravity46,47. Sved-berg developed his first low-speed ultracentrifuge andthen the high-speed ultracentrifuge to determine theshape and size of the protein particles48. Svedberg de-veloped another way of determining the proteins mol-ecular weight by modifying the methods and demon-strated that the molecular weight of hemoglobin wasaround 67,00049,50.

Ostwald also played a major role of contributing inthe synthesis of Au sols by various experimental andtheoretical methods. He also stated that “if you dipyour finger into the solution, reduction of organic sub-stances present in skin will occur and it will be stainedbluish violet due to the formation of Au colloids”51.Ostwald tried to focus on the importance of size forparticle dispersion. He showed several examples toexplain the phenomenon of Au sols and other colloids.Later, he also explained the mechanism for the forma-tion of Liesegang rings during diffusion reaction, whendrops of colloidal Au rod solutions evaporated52.

194 Toxicol. Environ. Health. Sci. Vol. 3(4), 193-205, 2011

Synthesis of Various Types ofAu Colloids

Au-NPs could be categorized depending on the shape,size, and physical properties. The first achievement inthe field of Au-NPs was Au nanospheres, althoughthey were not exactly spherical. Later, various otherforms were obtained, such as nanorods, nanoshells, andnanocages as shown in Figure 1. Another types of Au-NPs were also produced with great surface enhancedRaman scattering properties, named as, “SERS NPs”.The synthesis techniques were continuously developedfor many years. As result, the many simple syntheticprotocols became available, and their sizes and shapescould be wellcontrolled and creative, such as nano-cages1.

Au Nanospheres

The other name of Au colloid is “Au nanospheres”.The diameters could vary from 2 nm to100 nm, whichcould be synthesized by reducing aqueous HAuCl4

solution with addition of various reducing agents underdifferent parameters and conditions. The reducing agent,where most commonly used was the citrate, producedmonodisperse Au nanospheres53,54. Lesser the amountof citrate would yield the greater amount of nanos-pheres. The size of nanospheres could also be controlledby changing the ratio of citrate and Au. The majordisadvantage of the above method was for the low yieldof Au nanospheres and the restricted water usage, asa solvent. Two-phase method of synthesising nanos-pheres with tetraoctylammonium bromide as phasetransfer reagent was used by Faraday in 1857, whichproduced air and thermal stable Au nanospheres withreduced dispersity55,56. Monodispersed Au nanosphereswould be produced, if the reactants were added at avery faster rate to the cooled solution1.

Hence, many diverse methods for nanosphere syn-thesis were experimented using other reducing agentsor ligands57-59. Interestingly, dendrimers could be usedas stabilizers or templates for synthesis of Au nano-sphere preparation60-65. The shape and size of Au nano-

spheres would depend on controlling the synthesisparameter, such as, concentrations of the reactants,HAuCl4, and blocked co-polymers. The absorptionpeak of Au nanosphere ranged from 510 nm to 550 nm.As the particle size increased, the absorption peakshifted to a longer wavelength, and the width of thepeak indicated the range of size distribution. Interest-ingly, several investigators tried to grow Au nanos-pheres in human cells66.

Au Nanorods

Several strategies were tested for synthesizing Aunanorods. Synthesis of Au nanorods was performedusing the template method, based on the electrochemi-cal deposition of Au within the pores of nanoporouspolycarbonate or alumina template membranes67,68.The diameter of Au nanorod could be pre-determinedby the diameter of the pores of the template membrane.The length of Au nanorod could be controlled by theamount of deposited Au within the pores of the mem-brane. On the other hand, the major disadvantage ofabove method would be the low yields of Au nanorod,since only a single layer of nanorods could be prepared.Electrochemical synthetic method for the productionof Au nanorods was also reported, where the length ofthe nanorod could be determined, affecting the aspectratio of longer diameter over the shorter diameter69-71.

The most common way of synthesis of Au nanorodwould be the “Seed-mediated synthesis”, as it resultedhigher aspect ratios in comparison to other methods72,73.Au seed solution was generally made in the presenceof a strong reducing agent, like NaBH4, for reducingthe Au chloride. These seeds would act as the site ofnucleation for nanorods. If they were would continueto grow in the presence of Au chloride growth solutionalong with a weak reducing agent, like ascorbic acidand hexadecyltrimethylammonium bromide1. By regu-lating the Au seed solution with respect to Au precur-sor, the aspect ratio of Au nanorods could be controlled.Moreover, if AgNO3 was added to the solution, thenthe yield of nanorods would increase dramatically74,75.Apart from the seed mediated method, there were sev-

Gold Nanoparticles: Applications 195

Au

Au

SilicaAucore

Unique SERS reporter

Silica encapsulation

Sphere Rod Shell Cage SERS

Figure 1. Schematic representation of various types Au nanomaterials.

eral other methods and approaches were reported, suchas bio-reduction, growing Au nanorods on the surfaceof mica, and synthesis by photochemical method76-78.

Au Nanoshells

Nanoshell referred as a type of spherical nanoparti-cle with a dielectric core, which was covered by a thinmetallic shell (usually Au)79. These nanoshells involveda quasi-particle, called plasmon, produced from col-lective excitation or quantum plasma oscillation, wherethe electrons could simultaneously oscillate with re-spect to all ions. The simultaneous oscillation was alsoknown, as Plasmon hybridization, which was associatedwith the hybridization of outer and inner shells to pro-duce higher or lower energy levels. The lower energylevel strongly would combine with the incident light,whereas the higher energy would not bind and couldcombine rather weakly with the incident light. Hence,the interaction of plasmon hybridization on thin shelllayers would be stronger, and the shell thickness andoverall particle radius, as coupled together, could deter-mine the wavelength of the light emitted80. Due to highreflective optical and chemical properties of the nano-shells, its major application was with biomedical opti-cal imaging, fluorescence enhancement of weak mole-cular emitters, therapeutic applications, surface enh-anced Raman spectroscopy and surface enhanced in-frared absorption spectroscopy. Optical imaging couldhave inferences from the deflection of emitted lightfrom laser or infrared source in investigating their struc-ture, texture, anatomic and chemical properties of sam-ples. In near-infrared region between 700-900 nm, ab-sorbance levels of all the bimolecular could reach aminimum, providing a clear window for optical imag-ing1,81.

Au nanoshells could also be synthesized by changingthe composition and dimensions of layers, which couldbe fabricated with Surface Plasmon Resonance (SPR)with peaks between visible and NIR regions82. Bychanging the core size ratio to its shell thickness, SPRpeak of Au nanoshell could be tuned for a given com-position. By coating Au nanoshells with silica or poly-mer beads, Au nanoshells could be prepared with SPRpeaks in the NIR region83. The growth of silica coreswas carried out by reducing Tetra-ethyl Ortho Silicatein Ethanol, using the Stober process. Silica NPs werecoated with Au solution by using the seed mediatedmethod1. Other approaches exhibited the attachmentprocess of small Au nanospheres to the silica core withdiameter of 2-4 nm. Amine-terminated silane was usedas a linear molecule, until the seed particle combinedto be integrated into one layer of shell by reducing

additional Au84. The diameter of the silica core woulddetermine the diameter of the Au nanoshell. The shellthickness could be controlled by the amount of depo-sited Au on the core surface. Synthesis of Au nanoshellscould be performed by situ formation of Au nanopar-ticle from thermo sensitive core-shell particles, as tem-plate. Microgel could be used as a core, which couldreduce the particle aggregation and help in controllingthe thickness of Au nanoshells from Au plating. Wecould obtain cores with smaller diameters, approxi-mately 80 nm, with narrower size distribution thansilica85.

Au Nanocages

In 2006, Au nanocages, consisted controllable poreson the surface, were synthesized by the galvanic replace-ment reaction of truncated silver nanocubes and aqueousHAuCl4. Furthermore, it was observed that the gene-rated morphologies of the Silver nanostructures couldbe controlled through Pylol reduction. Here, ethyleneglycol reduced AgNO3 to generate silver atoms, andfurther reduction yielded nanocrystals or seeds. Desirednanostructures were produced through addition of extrasilver atoms and by simultaneously controlling thesilver seed crystalline structures with addition of poly-vinylpyrrolidone, which posed a potential of selectivebinding to the surface86.

The silver nanostructures could be used as a sacrifi-cial template, which could be metamorphosed into in-ternal hollow space within Au nanostructures throughthe galvanic replacement86,87. By adjusting the molarratio of silver to HAuCl4, the dimension and wall thick-ness of the resultant Au nanocages could be preciselycontrolled1. Au nanocages could provide some majoradvantages, such as: (i) their surface Plasmon resonancepeaks could be tuned by changing the ratio betweenthe Ag nanocubes and HAuCl4. This could also coverthe entire spectral region from 500 to 1200 nm; (ii) bycontrolling the number of truncated corners and voidsizes, their absorption coefficients could be varied; (iii)the Au nanocages could still exhibit resonance peaksin the near-IR region with extremely small size of about(⁄50 nm); and (iv) surface modifications could beperformed and applied in various biomedical applica-tions87.

Stimuli-responsive Surface-enhancedRaman Scattering (SERS) NPs

Stimuli-responsive surface-enhanced Raman scatter-ing (SERS) is an optical technique with many advan-


tages over the common traditional technologies. Thesignificant merits of SERS would be the fluorescenceand chemiluminescence, higher sensitivity, high levelsof multiplexing, robustness, and superior performancein blood and other biological matrices1,88,89. In 2002,Au nanospheres (~13 nm in diameter) were modifiedwith Cy3-labeled alkylthiol capped oligonucleotidestrands, as probes to monitor the presence of a specifictargeted DNA strands90. Cy3 group was chosen for itslarge Raman cross-section1. In 2008, modified 60 nmAu nanospheres with Raman reporter were stabilizedthrough a process with a layer of thiolated polyethyleneglycol91. In 2008, another kind of SERS nanoparticlewas constructed with a Raman-active molecular layeron silica coated Au core92. The silica coating ensuredthe physical robustness and inertness to various envi-ronmental conditions, but also simplified the surfacemodification through silica chemistry. Conjugation ofthiol groups were introduced onto the silica shell, andthe maleimide activated PEG chains improved biocom-patibility1.

Application of Au-NPs forDrug Delivery

Numerous reports demonstrated the versatility ofAu-NPs as a vital drug delivery agent. Drug deliverybecame a very efficient in transferring the drug with-out any safety and efficacy issues. Few common routesof delivering drugs would include skin, nasal, mouth,ocular, rectal, buccal and inhalation. Several biomole-cules, such as proteins, antibodies, peptides, genes andvaccines, were unable to be delivered through the abovemethods due to the organisms’ potential enzymaticdegradations. These biomolecules would not be abs-orbed in the circulation easily due to their molecularsize. Hence, above reasons would be the major difficul-ties for protein and peptide based drugs to be deliveredwith nanoneedle array. Therefore, many different drugdelivery methods were developed to increase the repro-ducibility, reliability, sensitivity, and specificity intothe targeted areas. Here, few methods would be listed.Thin film drug delivery method used rapidly dissolvinghydrophilic polymers, which could be easily absorbedupon contact with buccal cavity. The self-micro emul-sifying drug delivery system used micro emulsion forspecial ouzo effect. The neural drug delivery systemscould target the drugs into the specific injured nervoussystem. Lastly, acoustic targeted drug delivery reliedon ultrasound to transfer energized molecules throughor into the tissues. Drug delivery systems became astrong motivation in the field of “nanomedicines”.

Au nanocolloids became one of the vital and fre-

quently used materials in the field of nanomedicine anddrug delivery. The motive of using Au-NPs was toenhance the targeted drug delivery, especially in can-cer therapies. Tumor necrosis factor-alpha (TNF-α), acytokine, was proven as an excellent anticancer agentfor therapeutic application due to its toxic effects aga-inst cancer cells93. Later, a nanoparticle drug deliverysystem was designed with TNF-α conjugated PEGcoated Au nanoparticle, which efficiently increaseddamages to tumor cells with and decreased the toxicityfrom TNF-α94. From above study, the combination oftemperature and TNF-α conjugated PEG coated Aunanoparticle resulted in enhanced treatment results incomparison with the treatment with TNF-α alone.TNF-α conjugated PEG coated Au nanoparticle, givenintravenously with a proper dose and time, delayedthe tumor growth. It also suppressed the blood flowto the tumor and killed tumor cell by anti-angiogenesisapproach. Even though the particles were administratedintravenously, no accumulation was observed in theorgans of healthy animals95. Another mechanism ass-aulted cryoinjury by employing vascular-targetingmolecules in ice ball to kill tumor cells completelywithout any toxic effect96. Another Au based conju-gate, named as CYT-6091, was created and distributedinto the blood stream in delivering TNF-α to solidtumors successfully97.

Methotrexate (MTX), an inhibitor to dihydrofolatereductase, was used as a chemotherapeutic agent fortreating various cancer types98. A hybrid material ofMTX-Au nanoparticle was prepared to examine theantitumor and toxic effects in vitro and in vivo. In acomparative study, MTX-Au hybrid suppressed thetumor growth, whereas equal amount of free MTX didnot show any antitumor effect99. Interestingly, Au nano-shells with encapsulated horseradish peroxide (HRP)enzyme in form of hydro gels into hollow space weredeveloped by soft chemical method for the photo ther-mally modulated drug delivery100. Au nanoshells all-owed HRP to remain active inside the hollow Au-NPs.

The intracellular uptake of Au-NPs could be depend-ed on their physical dimensions, such as sizes andshapes, especially when Au-NPs were conjugatedwith ligands101. Hence, the conjugation between theligand and Au-NPs needed to be stable and reliable.Since thiolated DNA strands could be conjugated ontoAu-NPs through Au-S bond, the femtosecond pulseexcitation of Au-NPs at 400 nm wavelength couldeasily break Au-S bond by increased temperature ofthe particles from absorbing the energy102. Next, inorder to improve the stability of Au-NPs bioconjugates,PEG/Mixed peptides/PEG monolayer were createdon the nanoparticle surface. The stability of bioconju-gates was checked in high ionic strength media, as a


function of nanoparticle size, PEG length, and mono-layer composition. The stability of NPs increased withincreasing PEG length. The diameter of Au nanopar-ticle was smaller, and higher mole fractions of PEGwere observed. Above surface modified Au-NPs withoptimal size of PEG chain increased the half life tofew hours, which would be most efficient for cancertherapy103. Therefore, Au-NPs in drug delivery shouldbe specific, reliable, well targeted and directed to thesite of interest without any hindrance.

Role of Conjugated Au-NPs inDrug Delivery

Au nanoparticle conjugates with drugs or gene couldbe delivered in both ‘active’ and ‘passive’ targets withuse of antibiotics25,104-107. MTX, being an equivalentof folic acid, could perturb the folate metabolism forits cytotoxic effects in cancer therapy. MTX was con-jugated to 13 nm colloidal Au and revealed effectiveresults108. Furthermore, when ampicillin, streptomycin,or kanamycin were directly conjugated to the non-functionalized spherical Au-NPs of about 14 nm dia-meter, they could efficiently inhibit the growth of ba-cteria with greater stability104.

Role of Surface Modified Au-NPs inDrug Delivery

Surface modification of Au-NPs would present fourmajor benefits, as indicated in Figure 2. (i) It wouldincrease the circulation lifetime of conjugates by pre-venting or slowing their removal by Reticulo-Endothe-lial System (RES). (ii) Drug molecules could specifi-cally locate and attach to targeted cells. (iii) The sta-bility would be improved, and (iv) the non-specificcytotoxicity would be reduced by capping Au-NPs.Coating Au-NPs with PEG for the surface modificationprovided promising results25. PEG also strongly incr-eased the efficiency of cellular uptake in comparisonto unmodified Au-NPs109,110. PEG could also preventaggregations111,112. When a comparative study of bio-distribution of Au nanorods with and without PEG

modifications in mice, less toxicity and accumulationsin major organs were observed with PEG modifiedAu nanorods113. Furthermore, ‘layer by layer’ techni-que was introduced, which increased the stability andefficiency of the Au-NPs114. Recently, Au-NPs couldbe targeted nucleus of cells with payloads115.

Since Au is photo sensitive, many investigationsfocused in using external stimulating light to releasethe drug from Au-NPs. First successful photo-activateddrug release by plasmon activated particles was repor-ted in 2000, followed with a related US patent116,117.These works exploited a polymer-gel, permeated withAu nanoshells to control the drug release. Shortlythereafter, Caruso et al. used spherical Au-NPs with acomparable role to achieve the light-induced burst oflysozyme for destroying Micrococcus lysodeikticus118.Since body tissues would be more transparent at NIRwavelengths, more complex NPs, like Au nanorods ornanoshells, could be valuable for in vivo therapies119,120.The release of bound PEG (mPEG5000-SH) moleculeson Au nanorods could be well controlled with irradia-tion with pulsed laser121. In addition, NIR laser couldcause rapid shrinkage of the hydro gels and release thedrug122,123. Hence, such conjugates onto Au-NPs couldbecome the basis of an effective light-mediated, con-trolled release system to treat selected conditions25.

Use of Au-NPs as a Drug DeliveryAgent in Cancer

Role of Au-NPs in Drug Delivery of PancreaticCancer

Even though the targeted drug delivery was expectedto show very minimal side-effects, its applications werelimited due to the unavailable technologies to validatethe results. Hence, the biomedical nanotechnologycould provide the proper assessment in advancingmedical science through implementing new forms ofdisease treatment. Au-NPs for their unique physico-chemical properties are mostly involved in targeteddelivery of drug. These Au-NPs have been used inpancreatic cancer as a drug delivery agent to increasethe effect of chemotherapy124.

Recently, chemotherapeutic agents were combined


Au NP

Polymer chain

DrugAu NP

Figure 2. Schematic representation of surface modified Au-NPs.

with other compounds to improve their affectivity formany solid tumors rather than applying single agent.Two agents, cetuximab and gemcitabine, were combinedand used in preclinical models, and the toxicity of thesehybrid agents was very mild, such as fatigue, feverand skin rash. Above results received much attentionfrom researchers and medical professionals, whichprompted them to design an alternative and better tar-geted drug delivery system, based on nanotechnologyagainst cancers, especially for pancreatic cancer125-127.Such drug delivery system was finally developed withfollowing design. Anti-epidermal growth factor recep-tor (EGFR) antibody, called cetuximab (C225), wasused as targeting agent, anti-cancer drug was gemci-tabine and the drug delivery vehicle was Au-NPs inFigure 3. This delivery system inhibited the growthof pancreatic tumor cells in vitro and orthotropic pan-creatic tumor growth in vivo128.

Since EGFR existed on the cell surface, composedof an extracellular ligand-binding domain, a hydropho-bic transmembrane domain and an intracellular tyrosinekinase domain, C225 could specifically bind and acti-vate EGFR129-133. Nest, phosphorylation would occuras the ligand bound with EGFR, leading to the receptorhomo/hetero dimerization, followed by activation ofthe signaling cascade towards apoptosis129-130. The overexpression of enzyme tyrosine kinase, such as EGFR(ErbB-1), could lead to pancreatic cancer. Gemcitabinecould block the receptor of tyrosine kinase and helpto cure pancreatic cancer134. In the same manner,gemcitabine could be used as an anti-cancer drug forother cancers, such as neck, head, breast and ovariancancers135-137. The most challenging approach wouldbe to transfer multiple drugs to the metastasized sitesand to monitor the drug efficacy without destroying

healthy cells or tissues. Hence, the targeted drug deliv-ery system with NPs may reduce the dose of antican-cer drugs with increased efficacy, specificity and lowtoxicity124.

Role of Au-NPs in Drug Delivery of LungsCancer

Cisplatin, Carboplatin, and Oxaliplatin were theimportant and most used platinum-based anticancerdrugs in chemotherapy. Cisplatin was approved in1971, since then it was used for treating cancers138.The drug cisplatin completely cured testicular cancer.Cisplatin, Carboplatin and Oxaliplatin could bind tothe DNA and prevent the transcription and replication,thereby inducing cellular apoptosis139,140. The reductionin the uptake of drug, tolerance of drug DNA adductsand increases of tripeptide glutathione in intracellularlevels could inactive cisplatin138. In a recent literature,another possible reason for the resistance could be theelevated intracellular chloride concentrations141. Useof Cisplatin also had adverse effects, which could leadto the neurotoxicity, ototoxicity, and nephrotoxicity138.

Few years ago, only oxaliplatin was used worldwidefor treating a vast range of cancers, but now it wasonly used for the colorectal cancer. Use of oxaliplatindecreased due to nausea, vomiting and neurotoxicity138.The reasons for such adverse effects were from the nonspecific attacks towards rapidly dividing cells. Hence,platinum based chemotherapy could be enhanced withbetter drug delivery system142. The enhanced permea-bility and retention effects of platinum drugs couldimproved with carriers, like aptamers, peptides, anti-bodies or other cancer related ligands, to target solidtumors and leukemia’s143-147. Hence, Au-NPs were usedas scaffolds for the platinum based drug delivery, sincethey would be nontoxic & nonimmunogenic148-151. Au-NPs were modified with several linkers, like thiols,dicarboxylates, or amine groups, to be conjugated withseveral drugs, such as cisplatin or BBR3464. TheseAu-NPs with platinum based drug were attached withtargeting groups, such as aptamers or peptides, in orderto increase the specificity towards leukemia or lungcancer, respectively. This approach could suggest anew direction in the future for the platinum based can-cer treatments. The Platinum based Au-NPs producedbetter results than oxaloplatin, where they were speci-fically able to penetrate into the nucleus of lungs can-cer cells, causing cytotoxicity142.

Au-NPs for Cytotoxicity Studies

The main reason for cytotoxicity depended on thesize of Au-NPs. The recent in-vitro studies with very


Au

Figure 3. Schematic representation of Antibody conjugatedAu-NPs.

small Au-NPs around 1.4 nm revealed necrosis of thecells from the induction of oxidative stress and mito-chondrial damage152. On the other hand, whereas lesstoxicity was observed from 3.7 nm NPs, even thoughthey both entered into the nucleus of cells153. Cytotox-icity of Au-NPs showed direct correlation with theconcentrations of Au-NPs. At low concentration (1ppm) of Au-NPs in range from 2-20 nm in size werenot toxic to murine macrophage cell line, whereas con-centrations higher than 10 ppm induced apoptosis ofcells and upregulation of pro-inflammatory genes154.Since the particles were produced by physical methodswithout residual ions or stabilizers, no toxic effect wasobserved by the cells. With surface modifications, thecytotoxic effects of NPs could be avoided155,156. When15 nm Au-NPs were exposed to an artificial epithelial-airway, con\sisting of A549 lung carcinoma cell line,human monocyte-derived macrophages, and dendriticcells, no oxidative stress or inflammation were obser-ved157. Moreover, synergistic or suppressive effectswere not observed, which suggested that Au-NPs didnot cause immune reactions. In other cases, IL-6, IL-βand TNF-α were secreted from murine macrophagesfrom addition of peptide conjugated Au-NPs, inducingimmune responses from the peptide158. Peptide conju-gated NPs were recognized by murine bone marrowmacrophages, whereas alone peptides or NPs were notrecognized. Above studies suggested the potentials forvarious nanoparticle conjugates and their therapeuticapplications in cancer and autoimmune diseases159. NPswith plasma proteins attracted much attention for theirentry into circulation system.

Endocytosis of Au-NPs would be influenced bytheir size, surface properties and aspect ratio160. Sphe-rical Au-NPs (50 nm) were taken up easily by cancercell lines, whereas particles less than 50 nm needed tobe functionalized with cell penetrating peptides160-163.PEG coated NPs seemed to avoid phagocytosis andmacrophage recognitions164,165. However, in recent invivo study, 13 nm PEG-Au-NPs, injected intravenous-ly into mice, were deposited mainly in the liver, kuf-fer cells and spleen macrophages, and resulted anacute inflammation in liver upon biopsy166. When bio-distribution studies were performed with 1.4 nm and18 nm sized particles intravenously injected into rats,18 nm Au-NPs seemed to be completely removed fromblood within 24 hrs and were accumulating in liverand spleen. On the other hand, 1.4 nm sized Au-NPswere excreted through kidney with low accumulationin liver and spleen167. Au-NPs in 1.4 nm size couldpass through respiratory tract easily, whereas 18 nmparticles were completely trapped in the lungs. Fur-thermore, the hydrodynamic size of Au nanoparticlewould be depended with binding interactions with

coagulation factors, creating protein corona in humanplasma. When 30 and 50 nm sized colloidal Au wereincubate in human plasma, the hydrodynamic size ofNPs increased. Interestingly, 30 nm particles with pro-tein corona from plasma had larger hydrodynamicsize in comparison to 50 nm sized particles. Accumu-lation of 69 different proteins was observed on thesurface of Au-NPs168. However, the interactions withplatelet aggregation, plasma coagulation time, thecomplement activation, and protein binding conse-quences are being investigated.

Conclusions

Au-NPs could be prepared and conjugated withmany functionalizing agents, such as polymers, surfac-tants, ligands, dendrimers, drugs, DNA, RNA, proteins,peptides and oligonucleotides. Au-NPs showed excel-lent optical properties due to its surface Plasmon absorp-tion, which were utilized for labeling, imaging, andsensing. SERS, as a recent spectroscopic technique,could provide large Raman signals. SERS combinedelastically scattered visible light from Au-NPs, whichcould be imaged using a dark-field optical microscope.Inelastic SERS effect from adsorbed molecules couldresult a Raman spectrum, leading to the identificationof biomolecules. Hence, Au-NPs, as a biosensor, couldhelp to diagnose cancer, Alzheimer, HIV, and Tuber-culosis. Therefore, Au-NPs would be a general vectorfor the drug treatments.

Toxicity of Au-NPs occurred from the presence ofCTAB, which required in stabilizing Au-NPs duringsynthesis process. The noticeable toxicity could beavoided with surface modification methods as men-tion above. Many biocompatible ligands includingthiolated-PEGs were non-toxic. Overall, Au-NPswould be much less toxic than other types of NPs, andoptimizing for the potential applications could be easi-ly modified.

In conclusion, Au-NPs were biocompatible and easilyconjugated with biomaterials for detection, imaging,diagnostics, and therapeutic applications in variouscancers, gene therapy and many other diseases169. Au-NPs were proven to be a promising vehicle for drugdelivery, especially with Au nanorods. Nevertheless,their biosensor applications also would hold muchattention in miniaturized device applications, such asnano-barcodes. In spite of all potential applications,cytotoxicity of Au nanomaterials should always beverified, especially from the cationic surfactant CTAB,and the toxicity should be overcome with various sur-face modification strategies, maintaining Au-NPs’unique optical, physical and chemical properties.


Acknowledgements

We most gratefully acknowledge the financial sup-ports, granted from the GRRC program of Gyeonggiprovince.

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