**2. Nanoparticles as delivery systems**

2

Nanotechnology is the manipulation of matter on an atomic, molecular, and supramolecular scale involving the design, production, characterization and application of different nanoscale materials in several key areas providing novel technological advances mainly in the field of medicine (so called Nanomedicine) [6, 18-20]. The development and optimization of drug delivery approaches based in nanoparticles concerns the early detection of cancer cells and/or specific tumor biomarkers, and the enhancement of the efficacy of the treatments applied [21]. The most important biomedical applications of nanoscale materials can be organized as shown

> Biomedical applications of nanotherapeutics

These nanotherapeutics' potential in cancer relies on i) passive targeting due to the enhance of the permeability and retention (EPR) effect promoted by angiogenic vessels with defective vasculature and improper lymphatic flow surrounding the tumor [18] that can be reinforced by ii) specific targeting based on multifunctional nanomaterials that bypass the biological barriers and reach cancer cells [4]. Nanotechnology for drug vectorization provides for new and more specific drug targeting and delivery platforms that can reduce toxicity and other side effects and also maintain or improve the therapeutic index [9, 22, 23]. In fact, the devel‐ opment of targeting delivery systems is the ultimate goal in cancer therapy, which has been

Here, we will discuss recent applications on AuNPs as platforms for anticancer therapy, emphasizing strategies for targeted delivery for gene silencing focusing on the optimal pathways to test these therapeutics *in vitro* and *in vivo*. Also, an overview of the toxicological

taking the lead in what concerns overcoming the MDR problem [9, 13, 24, 25].

Drug delivery Targeted

 Nanotechnology is the manipulation of matter on an atomic, molecular, and supramolecular scale involving the design, production, characterization and application of different nanoscale materials in several key areas providing novel technological advances mainly in the field of medicine (so called Nanomedicine) [6, 18-20]. The development and optimization of drug delivery approaches based in nanoparticles concerns the early detection of cancer cells and/or specific tumor biomarkers, and the enhancement of the efficacy of the treatments applied [21]. The most important biomedical applications of nanoscale materials can be organized as shown

therapy

Detection and Diagnosis

Gene delivery

drug delivery systems (DDSs) and also gene delivery systems [3, 4, 14-17].

Cancer is one of the leading causes of death worldwide, occupying the second place in developing countries, and showing a growing incidence over time [1]. Current cancer therapy strategies are based in surgery, radiotherapy and chemotherapy, being the chemotherapy the one that shows the greater efficiency for cancer treatment, mainly in more advanced stages [2, 3]. Despite of this great response, anticancer agents are administrated at higher amounts in order to provide a final suitable concentration to the target tissues or organs, and this procedure is repeated in each cycle of chemotherapy [4]. Introduction of new agents to cancer therapy has greatly improved patient survival but still there are several biological barriers that antagonize drug delivery to target cells and tissues, namely unfavorable blood half-life and physiologic behavior with high off-target effects and effective clearance from the human organism [2, 5, 6]. Moreover, in cancer, there is a small subset of cancer cells - cancer stem cells (CSC) - that, like normal stem cells, can self-renew, give rise to heterogeneous populations of daughter cells, and proliferate extensively [7, 8]. Standard chemotherapy is directed against rapidly dividing cells, the bulk of non-stem cells of a tumor, and thus CSC often appear relatively refractory to those agents [7-9]. The development of side effects in normal tissues (e.g. nephrotoxicity, neurotoxicity, cardiotoxicity, etc) and multidrug resistance (MDR) mechanisms by cancer cells leads to a reduction in drug concentration at target location, a poor accumulation in the tumor with consequent reduction of efficacy that may associate to patient relapse [9-13]. To overcome these issues and still improve the efficiency of chemotherapeutic agents there is a demand for less toxic and more target specific therapies towards cancer cells, i.e. novel drugs,

in Figure 1.

in Figure 1.

128 Application of Nanotechnology in Drug Delivery

Biomarker mapping

> Molecular Imaging

**Figure 1.** Biomedical application of nanotherapeutics (adapted from [6]).

aspects of these materials will be provided.

Introduction

1. Nanomedicine for cancer

Nanoparticles have been developed as effective target specific strategies for cancer treatment, acting as nanocarriers and also as active agents [4, 6, 5, 26]. Over the last decades, different types of nanoparticles have been developed based on various components, including carbon, silica oxides, metal oxides, nanocrystals, lipids, polymers, dendrimers, and quantum dots, together with increasing variety of newly developed materials [4, 27-34]. These nanomaterials are capable to provide a high degree of biocompatibility before and after conjugation to biomolecules for specific function so as to translate into nanomedicines and clinical practice. Nanomaterials provide for a favorable blood half-life and physiologic behavior with minimal off-target effects, effective clearance from the human organism, and minimal or no toxicity to healthy tissues in living organisms [35, 36].

In fact, the protection from adsorption to plasma proteins and/or degradation by circulating nucleases allows for an increased availability of effector molecule at site of interest. This is further enhanced by the considerable decrease to clearance from the organism that conjugation to nanoparticles confers. The modulation of pharmacokinetic and pharmacodynamics parameter constitutes a key factor when modifying the mode of administration (and vehicle and route of administration associated) that is usually neglected when compared to the ability of therapeutic nanoconjugates to offer the possibility of enhanced targeting (active and/or passive) and cell uptake. When considering nanoparticles for therapeutics one should also evaluate the effect on cellular metabolism and fate that can be attained via optimal conjugation with (bio)molecules of interest.

DDSs can improve the properties of free drugs by increase their *in vivo* stability and biodis‐ tribution, solubility and even by modulation of pharmacokinetics, promoting the transport and even more important the release of higher doses of the drug in the target site in order to be efficient [18, 22, 37, 38].

DDSs can be constructed by direct conjugation with the drugs and further surface modifica‐ tions can lead to a better delivery for such systems, promoting a targeted delivery to specific types of cells and reaching cell compartments such as nucleus and mitochondria [15, 39]. As far as drug delivery is concerned, the most important nanoparticle platforms are liposomes, polymer conjugates, metallic nanoparticles (for example AuNPs), polymeric micelles, den‐ drimers, nanoshells, and protein and nucleic acid-based nanoparticles (for a more complete review see [40-42].

Among a wide variety of nanosystems, only a few nanomedicines, such as Doxil® (Janssen Biotech Inc., Horsham, PA, USA), DaunoXome® (Galen US Inc., Souderton, PA, USA), Depocyt® (Pacira Pharmaceuticals Inc., San Diego, CA, USA), Genexol-PM® (Samyang Biopharmaceuticals Corporation, Jongno-gu, Seoul, Korea), Abraxane® (Celgene Corporation, Inc., Berkeley Heights, NJ, USA), Myocet® (Sopherion Therapeutics Inc., Princeton, NJ, USA) and Oncaspar® (Enzon Pharmaceuticals Inc., Bridgewater, NJ, USA), are approved for use in the treatment of cancer (for a review see [6]).

The implementation of nanoparticles towards cancer treatment can be based in certain characteristics as their size, surface properties and the possibility of a variety of specific ligands in their surface [18]. The high surface properties and other physicochemical features of nanoparticles can be modulated for the development of valuable systems that detect tumor cells either qualitatively or quantitatively [10, 19].

tics. The main process for nanoparticles development requires chemical administration of capping agents that adsorb in the surface of nanoparticles ([55] and references therein). AuNPs can be synthesized with different sizes through the reduction of gold with different agents such molecules bearing a thiol group, an aliphatic chain and a charged end group, and that can avoid particle aggregation [37]. Furthermore, this dense layer of stabilizing agent promotes a general change in the surface charge of AuNPs allowing ligand exchange with several molecules, promoting AuNPs functionalisation and then an increase in particle stability in physiological environments [55, 56]. AuNPs deliver systems can be formulated based in their capacity to bearing different functional groups, once it can be involved in covalent and noncovalent bindings by a thiol-linker [37, 55]. In fact, robust AuNPs appear by the stabilization with thiolates once the bond between Au and the thiol (S) is very strong [57]. This process enhances the affinity of the AuNPs surface for several types of ligands such as polyethylene glycol (PEG) molecules, nucleic acids (DNA and RNA), peptides, antibodies, and also small

Application of Nanotechnology in Drug Delivery

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**Figure 2.** Multifunctional NP-based systems for tumor targeting, delivery and imaging. These innovative NPs comprise a targeting moiety, a silencing moiety and anticancer drug molecules for delivery to the target tissue. Depending on the targeting mechanism, they can be on the surface or inside the NPs. Multifunctional systems can carry reporter mol‐

ecules tethered to the particle surface and employed as tracking and/or contrast agents.

drug molecules (Figure 2) [9, 13, 37, 47, 52, 56, 57].

Targeting the cancer cells occurs via two different strategies: passive targeting and active targeting [4, 43, 44]. The passive targeting of tumor cells by nanoparticles depends upon an EPR effect promoted by angiogenic vessels with defective vasculature and improper lymphatic flow, reaching a higher accumulation in tumor cells compared to normal cells [15]. The increased accumulation of a drug in the tumor *interstitium* achieved by nanoparticles can be more than ten times higher compared to the drug alone [4]. This type of deliver is based in nanoparticle's half-time of circulation on the bloodstream, size and surface properties, and even depends on the degree of angiogenesis [45]. Despite the increased drug accumulation inside the tumor, this strategy rise some concerns about the targeting specificity of such mechanism based in the controversial influence of the EPR effect on drug externalization, which promotes a widespread distribution all over the tumor [4, 46]. The lack of specificity of such targeting led to further innovation with the implementation of an active targeting, which is achieved by the functionalization of nanoparticle's surface with a plethora of functional moieties such as antibodies and other biomolecules that recognized the specific surface antigens or specific biomarker of tumor cells [4, 44]. The targets choice depends on its high abundance in cell surface and its unique expression, and consequently the capacity of inter‐ nalization of the nanoconjugate [4, 47, 48]. Although it is considered that active targeting does not have a direct association to the total nanoparticles accumulated within the tumor, it will influence the uptake of nanoparticles via receptor-mediated internalization and improve the efficiency of anti-tumor agents that have intracellular targets [49, 50]. Active targeting can be the potential way of polymeric nanoparticles to deliver chemotherapeutic drugs to cancer cells and is, therefore, one of the main vectors of DDS development at present involving tailoring of nanoparticles to deliver the effective cargo without compromising the selective targeting.
