**4. Toxicity of AuNPs**

One major concern regarding AuNPs application in medical field relies in its toxicity in the biological systems, i.e. the production of a general toxicity response not only in cancer cells but also reaching healthy cells at the vicinity [78]. Taken into account the size, surface modi‐ fications and solubility in promoting biocompatibility of the nanovectorization systems, they can be safer to apply in the medical field to the treatment of cancer [130]. In fact, nanoparticles size is an important feature because it turns possible to circumvent the immune response and renal clearance, which maintains the therapeutic capacity of such systems [5].

Toxicity of AuNPs is generally accepted to be dependent on particle size, shape, and surface charge and chemistry [131-134]. However, it is thought that once AuNPs have a smaller size, approximately the size of biomolecules, it can be taken like one and then evade cellular barriers, with access to different tissues, and in the end can lead to the disruption of cell biological processes [75, 135]. A control of the size dependent cytotoxicity of AuNPs, revealed that AuNPs with a 1-2 nm size represents more toxicity towards four cancer cells lines compared to AuNPs with 15 nm that do not display any toxicity (Pan *et al.*, 2007). Additionally, the main organs affected by AuNPs are the liver and the spleen (Sun *et al.*, 2011). Also, very small particles (1.4 and 5 nm in diameter) seem to be capable to enter the nucleus, where they can interact with DNA and cause molecular disturbance [136, 137]. Larger particles (16 nm and 33 nm) are retained in endosomes and accumulate in the periphery of the nuclear region [138, 139]. At least three different studies reported that cellular uptake of AuNPs reach maximum levels for a particle size of about 50 nm [140-142]. Also, surface functionalisation seem to be capable of inducing higher level of apoptotic cell death, probably related to increased cell uptake when compared to unmodified 40 nm AuNPs [141]. According to data from in vitro studies, AuNPs' toxicity is believed to result mainly from the induction of oxidative stress [143-145]. Indeed, up-regulation of stress related genes was found to result from cell exposure to AuNPs, which also promoted the down-regulation of cell cycle related genes [145-147]. Nevertheless, most of these studies paid little attention to genome damage, such as DNA strand breaks and nuclear abnormalities, or characterization of protein markers for toxicity. An integrated toxicology evaluation encompassing DNA damage, stress related enzymes and a proteome profiling approach showed no significant cytotoxicity of PEGylated AuNPs and no up-regulation of proteins related to oxidative damage [148]. Nevertheless, previous studies using metallic nanoparticles showed acute toxicity, mainly by the introduction of damages to the DNA molecule and also by oxidative damage [146, 149, 150].

binding cassette (ABC) membrane transporters, such as P-glycoprotein (P-gp), and others efflux pumps such as BCRP, which imply these as potential targets of silencing for cancer therapy [122, 123]. Cancer stem cells (CSCs) can also express these membrane proteins which confer to this subpopulation resistance to the current chemotherapeutic agents [123, 124]. Thus, the implication of a silencing strategy towards these cancer related genes evolve in order to minimize cancer resistance barriers to the actual therapy and then obtain an efficient response towards the chemotherapeutic agents applied [115, 125]. It was demonstrated by a system of lipid-modified dextran nanoparticles bearing siRNAs towards *ABCB1* gene (P-gp), that this approach can efficiently deliver the siRNA molecule and reduces the expression of P-gp although at the same order of greatness as the siRNA alone [126]. This reveals the necessity to continuously develop nanoparticles systems that can target and silencing these genes and

Another multidrug resistance mechanism is associated to the capacity of cancer cells to evade apoptotic response, when resistance induced by efflux pumps is not seen [127]. Apoptosis is the major cellular process induced by chemotherapeutic agents, so cancers bearing apoptosis defects cannot be efficiently treated by those agents, then discovery of the molecular basis of such system can formulate novel therapeutic approaches [127, 128]. For example, the antiapoptotic protein Bcl2 is considered a proto-oncogene, and nano-based vector delivery systems

One major concern regarding AuNPs application in medical field relies in its toxicity in the biological systems, i.e. the production of a general toxicity response not only in cancer cells but also reaching healthy cells at the vicinity [78]. Taken into account the size, surface modi‐ fications and solubility in promoting biocompatibility of the nanovectorization systems, they can be safer to apply in the medical field to the treatment of cancer [130]. In fact, nanoparticles size is an important feature because it turns possible to circumvent the immune response and

Toxicity of AuNPs is generally accepted to be dependent on particle size, shape, and surface charge and chemistry [131-134]. However, it is thought that once AuNPs have a smaller size, approximately the size of biomolecules, it can be taken like one and then evade cellular barriers, with access to different tissues, and in the end can lead to the disruption of cell biological processes [75, 135]. A control of the size dependent cytotoxicity of AuNPs, revealed that AuNPs with a 1-2 nm size represents more toxicity towards four cancer cells lines compared to AuNPs with 15 nm that do not display any toxicity (Pan *et al.*, 2007). Additionally, the main organs affected by AuNPs are the liver and the spleen (Sun *et al.*, 2011). Also, very small particles (1.4 and 5 nm in diameter) seem to be capable to enter the nucleus, where they can interact with DNA and cause molecular disturbance [136, 137]. Larger particles (16 nm and 33 nm) are retained in endosomes and accumulate in the periphery of the nuclear region [138, 139]. At least three different studies reported that cellular uptake of AuNPs reach maximum levels for

has been establish with great efficacy towards this molecule [127, 129].

renal clearance, which maintains the therapeutic capacity of such systems [5].

proteins.

**4. Toxicity of AuNPs**

138 Application of Nanotechnology in Drug Delivery

AuNPs are however generally considered a system that do not cause acute or adverse toxicity, and then are been taken as safer systems for therapeutic use [135]. AuNPs demonstrate to be a safe system due to their easy of functionalization [151]. This ideal is based in the assumption that gold nanoparticles do not lead to any effect in the cell, and instead, the function moiety in its surface promote the cytotoxic effect expected [139]. In the other way, expression studies revealed an overexpression of stress and inflammation related genes after AuNPs treatment, being associated to the action of AuNPs in oxidative stress induction [75]. A decrease in cell cycle genes expression was simultaneously observed, which symbolizes an irreversible damage that leads to cell death by necrosis [75].

Nanoparticles surface composition is another relevant point when talking about toxicity of nanoparticles systems [5]. The ligands and surface capping agents of AuNPs as the first line of contact with the different actors in the cell pathways can promote toxicity that in the end represents the overall toxicity associated to these nanoconjugates [5]

Also, both positive and negatively charged AuNPs were found to be similarly more cytotoxic against human keratinocytes (HaCaT cells) when compared to neutral AuNPs, with LD50 values of roughly half of those determined for the latter [152]. Despite the disruption in cell morphology and the dose-dependent toxicity observed for all three types of AuNPs, both anionic and cationic AuNPs induce mitochondrial stress and apoptosis in opposition to the necrotic cell death caused by neutral particles [152]. Another in vitro study comparing positive and negatively charged AuNPs reported that cationic NPs were far more toxic to Cos-1 cells, human red blood cells and E. coli than anionic NPs, possibly as a result of cell lysis, as shown by a dye leakage technique [133]. However, Alkilany and co-workers clearly showed that serum proteins become readily adsorbed to the surface of charged NPs, inducing an inversion of surface charge in particles that were originally cationic [153]. This would reduce electrostatic interaction between the original positive NPs and the negative cell membrane, the first step towards cell lysis mediated toxicity of cationic NPs [133].

Regarding in vivo experiments, several studies have demonstrated that AuNPs of 50 nm and larger were non-toxic to mice, conversely to what has been observed for AuNPs <40 nm [54, 55]. In fact, there are concordant data from different studies on the biodistribution and accumulation of AuNPs in mice showing that most of the intravenously injected nanoparticles are retained in the liver, regardless of their size [156-158]. There is also an agreement in that AuNPs have the ability to transpose the blood-brain barrier and thus reach the brain, with a cut-off limit in diameter of around 20 nm [159], and that smaller particles have the most widespread organ distribution [156-158]. Organ distribution seems to be ruled by a more or less complex relationship with nanoparticle size. For instance, it is known that renal excretion of AuNPs is maximized for a narrow size range of 6-8 nm, resulting in an accelerated clearance rate [160]. Despite the valuable use of animal models, the effect of size on the toxicity of AuNPs in humans is difficult to predict since the size of endothelial cells' fenestrae is highly variable between individuals, thereby affecting nanoparticle clearance [75]. Therefore, more consistent data on the toxicological profile of AuNPs in vivo is necessary. For a more complete review on biodistribution, encompassing earlier studies and administration routes other than intravenous injection, see Khlebtsov and Dykman [159]. Furthermore, core size, charge and surface chemistry of AuNPs seems to correlate to toxicity on the development of zebrafish embryos, with positive and negatively charged AuNPs causing mortality and malfunctions to the embryos, respectively [161]. Adverse effects were also found in the model system Droso‐ phila melanogaster after exposure to citrate-capped AuNPs, which were shown to reduce fertility in a dose-dependent manner and also the life span [144, 162].

What is more, many tumors become resistant to drugs, requiring that novel strategies involv‐ ing drug targeting vehicles that deliver high concentrations of combinatorial therapeutics to the selected targets. For this to happen, it is crucial that these nanoconjugates are capable to withstand the body's clearance and reaction to non-self particulates. The robustness of AuNPs as target delivery platforms will be achieved when reticuloendothelial system clearance is avoid and occur an enhance of the endothelial penetration, once the first one can lead to a longer time in circulation and the second leads to an increase of targeting and drug accumu‐

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The use of multiple nanoparticles that can be used together may overcome current limitations of each individual nanoformulation alone. For example, AuNPs have proven to be outstanding vectorisation systems for gene delivery and can be used to target molecular pathways, including those involved in drug resistance and in survival of cancer cells. These NPs may be used in combination with any other polymeric and/or metallic nanoparticles in therapeutic approaches that include drug and thermal ablation, selective delivery via out of the boy

All of these applications of AuNPs in therapeutics still lack enough toxicology and pharma‐ cology studies and data that can support the effective translation into the clinics. However, the efficacy in fighting cancer cells shows that the effort to push forward with the needed regulatory requirements and compliance is worth pursuing since the enhanced properties allow for outstanding improvements to biocompatibility, circulation and therapeutic response.

The authors acknowledge Fundação para a Ciência e Tecnologia (FCT/MEC) for funding:

1 Department of Life Sciences, Faculdade de Ciências e Tecnologia, Universidade Nova de

3 CIGMH, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Univer‐

CIGMH (PEst-OE/SAU/UI0009/2011); PTDC/BBB-NAN/1812/2012.

\*Address all correspondence to: pmvb@fct.unl.pt

sidade Nova de Lisboa, Campus de Caparica, Portugal

, Alexandra R. Fernandes1,2 and Pedro V. Baptista1,3\*

2 Centro de Química Estrutural, Instituto Superior Técnico, Lisboa, Portugal

lation (Kumar *et al.*, 2013).

triggering (light source).

**Acknowledgements**

**Author details**

Joana Silva1

Lisboa, Portugal

Nonetheless, long-term studies in higher organisms are necessary to further characterise the safety of AuNPs as therapeutic agents, so they can be safely administrated to humans without concerns about late toxicity symptoms.
