ABSTRACT
Many of the conventional therapies can be improved using drug delivery systems (DDS). They are designed mainly to modify the pharmacokinetics and biodistribution of small molecular drugs. This is of special importance in the case of anticancer therapies in which a widespread distribution of small molecular chemotherapeutic drugs is often limiting treatments. In this context, nanotechnology emerges as a disruptive technology to design carriers that improve the delivery of drugs to their target organs. The composition of these nanocarriers comprises a wide range of materials such as polymeric nanocapsules, lipidic liposomes, or metallic nanoparticles. Gold nanoparticles (Au NPs) are of special interest due to its demonstrated biocompatibility, their tunable surface chemistry, and their special optical and electronic
Joan Comenge, PhD,a Francisco Romero, PhD,b Aurora Conill, MS,c and Víctor F. Puntes, PhDaaInorganic Nanoparticles Group, Catalan Institute of Nanotechnology, Barcelona, SpainbMolecular Science Institute, University of Valencia, Valencia, SpaincNanotargeting SL, Barcelona, Spain Keywords: nanotechnology, nanopharmaceutical, nanomedicine, targeting, gold, nanoparticles, cancer, drug delivery, enhanced permeability and retention effect, surface plasmon resonance, nanoparticle biodistribution, radiosensitizer, radiotherapy, photothermal therapy
properties that allow its use not only as carriers but also as effectors. Hence, Au NPs are perfect candidates to be used for treatment of cancer in the clinics thanks to the capacity to be used as scaffolds to attach drugs or targeting molecules, as imaging agents, and as effectors themselves. 44.1 Historical Perspective on the Medical Use
of GoldGold in different forms has been used for health in humans since ancient times. The synthesis of Au NPs has been in the spotlight since Faraday discovered in 1857 the mechanism of formation of pure gold colloids. This synthesis has been the keystone of a large amount of chemical routes to obtain Au NPs with controlled size, shape, and surface chemistry. Today, scientists have a wide catalog of Au NPs available, which can be used as excellent model systems to investigate the nano-bio interface. In the 1950s, the first use as contrast agents for radiotherapy was reported. Since the 1970s, Au NPs have been used in combination with antibodies (Abs) or other proteins to visualize specific cellular compartments, proteins, and receptors. Another well-known application of Au NPs is their use as probes of biomarkers in the pregnancy test (e.g., First Response®, marketed in the 1990s). This test is based on the specific recognition of human chorionic gonadotropin (hCG), a hormone produced during pregnancy, by Au NPs conjugated with an antihCG Ab. Nowadays, advanced NP bioconjugate chemistries allow scientists to tailor NPs for much higher sophisticated purposes, such as orchestrating chemical reactions inside cells and manipulating cell response. The successful development of these challenging tasks relies on intelligent surface structure design and the ability to synthesize NP bioconjugates with the desired architecture, which is also paramount in obtaining a well-defined and reproducible behavior. 44.2 Gold in the ERA of Nanotechnology:
New Properties for a Known MaterialPersonalized health care, rational drug design, and targeted drug delivery are some of the proposed benefits of a nanomedicine-
based approach to therapy. The progress of the drug development is nowadays limited since most of the delivery methods are based mainly on oral or injection delivery routes, which strongly determines the formulation of the drugs. Precise drug release into highly specified targets involves miniaturizing the delivery systems to become much smaller than their targets. Nanoparticle DDS, due to their small size, can penetrate across the barriers through capillaries into individual cells to allow efficient accumulation at the targeted locations in the body. A wide variety of engineered NPs has been extensively used or is currently under investigation for drug delivery, imaging, biomedical diagnostics, and therapeutic applications. Among those, Au appears as one of the most use and most promising medical nanoparticles for diagnosis, therapy, and theranostics. The employment of NPs for the delivery of pharmaceuticals can result in higher concentrations than possible with other drug delivery methods, which could enhance the drug bioavailability or dosing at the targeted site as well as the overall efficiency of the used drug. For example, the involvement of stable conjugates of Au NPs coated with antibiotic molecules for therapy increases the efficiency of drug delivery to target cells in some studied cases.Au is a biocompatible inert material that can be produced with extreme size control and monodispersity; its functionalization and derivatization are very well developed; and it has a very high electron density, which can be exploited as contrast agents for X-ray imaging or X-ray radiotherapy, or as contrast agents for imaging in the near infrared (a much weaker highly penetrating photon wavelength) and as photoablation (hyperthermia) agents. 44.3 Synthesis of Gold NanoparticlesWhen using Au NPs for biological applications, special care has to be taken in the synthesis step since the size and size distribution play an important role in some biological responses such as biodistribution, tumor accumulation, and penetration, time of circulation, and immune response among others. Moreover, the nanoparticles surface should be ready for further chemical modifications in order to link the drug of interest.The synthesis of colloidal gold was introduced by Michael Faraday in the 1850s [1], but it was not until 1951 when the most
usual synthetic methodology to obtain gold nanoparticles was exhaustively described by Turkevich [2]. This approach, based on the reduction of a gold salt by citrate, results in the production of 20 nm Au NPs with a relatively narrow size distribution. This methodology, as well as subsequent modifications [3], is based on supersaturation of the monomer in solution that induces homogenous nucleation followed by growth of these nuclei without additional nucleation events. This “burst nucleation” is a necessary condition to obtain highly uniform nanoparticles [4]. Nevertheless, the range of Au NP sizes available to obtain using these methods is small and goes from 7 to 25 nm. Frens proposed to decrease the citrate/HAuCl4 ratio in order to increase the nanoparticle size up to 150 nm [5]. However, it has been recently demonstrated that this approach do not follow the “burst nucleation” mechanism and consequently monodispersity becomes poor, and also the spherical shape of the Au NPs is partially lost [6]. To overcome these limitations, a great number of synthetic protocols to achieve better control over the size and shape of nanoparticles have been developed during the past years [4]. Among them, the seeding-mediated strategies based on the temporal separation of nucleation and growth processes are considered very efficient methods to obtain monodisperse NPs [7-9]. In this strategy, small particles are synthesized first and later used as seeds (nucleation centers) to grow larger NPs. For example, Murphy and coworkers proposed the use of a weak reducing agent (ascorbic acid), which reduces Au3+ to Au+ and prevented the total reduction to Au0 unless seeds are present [10]. By using this methodology, additional nucleation that would lead to polydispersity is avoided. However, the use of CTAB as capping agent restricts the possibilities of further functionalization since their replacement by thiols is difficult to achieve [11]. This condition is especially important in nanomedicine, where the ability to render a biological functionality to inorganic nanostructures is one of the cornerstones of this emerging field [12]. In this context, citrate-stabilized Au NPs appear as unique candidates since the loosely bound capping layer provided by the sodium citrate can easily be exchanged by thiolated molecules that pseudo-covalently bind (~45 kcal/mol) to the gold surface [13]. However, the control of the size is complicated since the energy needed to form Au nuclei is not much higher than the needed to grow the NPs and therefore to separate nucleation
Figure 44.1 Representative TEM images of Au NPs after every growth step in the kinetically seeded growth approach. Monodispersity and absence of secondary populations are maintained all over the process. The morphology of the Au NPs is quasi-spherical in all cases avoiding the formation of elongated Au NPs.from growth is not trivial. Obviously, the formation of new nuclei during growing stages would lead to a broadening of the size distribution and should be avoided in any seed growth approach. Recently, our group proposed a kinetically controlled seeded growth approach that allows obtaining citrate-capped Au NPs with a perfect control of the size from 8 to 200 nm [8]. This methodology, which avoids the use of any surfactants, is based on the reduction of HaAuCl4 by sodium citrate (as in the classic synthesis of Au NPs). Here, the separation of nucleation and growth stages is achieved by manipulating the reaction conditions: pH, temperature, and ratio seed/monomer are key factors to avoid
secondary nucleation that would have led to a broadening of size distribution (Fig. 44.1).Although spherical Au NPs are the most used in biomedical applications, nanoparticle synthesis is an extremely active field of research. In recent years, protocols allowing the synthesis of Au NPs of exotic shapes have been described. The special physico-chemical properties of anisotropic Au NPs, hollow structures or core-shell Au NPs make them appealing to be used in biomedicine as contrast agents, for cell labeling or as effectors for photothermal therapy. Anisotropic gold nanorods (Au NRs) can be synthesized following also a seeding growth approach proposed by El Sayed’s and Murphy’s groups [14, 15]. Au seeds (4 nm) are synthesized with a strong reducing agent (NaBH4) in the presence of cetyltrimethylammonium bromide (CTAB). They are immediately used as nucleation centers for growing Au NRs. It is in this growth stage that the control of the size, and therefore the tuning of the Au NRs aspect ratio, is possible. The growth stage is based on the use of a weak reducing agent (ascorbic acid) to avoid secondary nucleations, and the presence of a micelle-forming surfactant (CTAB and/or benzyldimethylhexadecylammonium chloride, BDAC) and Ag ions to allow the growth in only one direction. 44.4 Gold Nanoparticles to Target CancerIn cancer, the rapid growth of tumor results in leaky vessels. Macromolecules and NPs are able to permeate through the fenestrations in tumor vessels. In addition, they are retained due to the lack of a functional lymphatic system. This effect (Enhanced Permeability and Retention effect, EPR) is widely reported in the literature [16, 17] and has been exploited to passively accumulate nanocarriers in tumors [18]. This passive accumulation of NPs into the tumor may be even further improved if a ligand that is recognized by a receptor overexpressed in tumors is also attached to the NP. Examples include epidermal growth factor (EGF) [19], folate [20], and transferrin [21]. However, this point is still controversial and it is not clear if the addition of a ligand may change the final biodistribution of the NPs. It has been proposed that NPs with tumor-specific ligands are more rapidly taken up
by tumoral cells rather than affect the biodistribution which is mainly influenced by the physicochemical properties of the NP (size and surface charge) and composition (e.g., presence of stealthing agents) [22]. Long-time circulating NPs maximize the EPR effect since the changes to permeate through the tumor vessels are greater. Related to this, the size of NPs plays an important role in determine tumor accumulation and distribution: 60 nm Au NPs showed a greater accumulation than 20 nm Au NPs likely due to a slower clearance. However, larger Au NPs are accumulated in the perivascular region of the tumor failing to penetrate deeper, while 20 nm Au NPs were still found significantly at 50 μm from the blood vessel [23].
