controlled drug release by using gold nanoparticles

Recent Patents on Nanomedicine, 2012, 2, 000-000 1

1877-9123/12 $100.00+.00 © 2012 Bentham Science Publishers

Smart Delivery and Controlled Drug Release with Gold Nanoparticles: New Frontiers in Nanomedicine

Valerio Voliani*,1,2

, Giovanni Signore2, Riccardo Nifosí

2, Fernanda Ricci

1, Stefano Luin

1 and

Fabio Beltram1,2

1NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR,

2Center for Nanotechnology Innovation @NEST,

Istituto Italiano di Tecnologia, Piazza San Silvestro 12, I-56127 Pisa, Italy

Received: 22 November 2011; Revised: 01 February 2012; Accepted: 07 February 2012

Abstract: Discoveries in the field of nanotechnology are triggering a revolution in medicine, by providing a profusion of

potential and actual applications of nanosized objects in the diagnosis and treatment of several diseases. This trend is also

evident in the increasing number of patents recorded annually. In particular, nanoparticles able to find a target and release

their payload upon a specific stimulus are highly attractive for the diagnostics and therapeutics, for example, of cancer and

gene diseases. In this view, gold nanoparticles (AuNPs) stood out as suitable platforms for the development of efficient

delivery and release systems. AuNPs are biocompatible, and can be easily synthesized and functionalized. The capability

of tuning their size and geometry allows manipulating their optical and physical properties. Engineering their coating

controls the stability, targeting and release features. In this review several patents concerning release strategies based on

gold nanostructures are reported, together with a discussion about their operating processes and their mutual advantages

and disadvantages.

Keywords: Biocompatibility, controlled release, drug delivery, gold nanoparticles, nanomedicine, photochemistry.

INTRODUCTION

Recently, considerable effort has been focused on the creation of Drug Delivery nanoSystems (DDSs), in particular for therapy and diagnostic of tumors or genetic diseases [1-3]. Conventional treatments against cancer include surgery, radiotherapy and chemotherapy; while surgery is an invasive treatment, often not sufficient for a final cure, radiotherapy and chemotherapy may have deleterious effects on the organism [4]. Thus, the research on novel drug-delivery systems with increased cell specificity and reduced adverse effects is an active challenge. In this view, nanotechnology applied to the development of DDSs able to recognize a target in the organism and to promote controlled release of drugs in terms of quantity, location and time is expected to maximize the therapeutic results while minimizing side effects [2, 5].

Nanoparticles (NPs) are structures with dimensions of 5-100nm composed by metals, semiconductors or amorphous materials; the ones used in medicine and biophysics are usually synthesized by wet chemistry. Their novel chemical, physical, and biological features, correlated with their small size, can be exploited for their use in theranostics and in basic research. An example of useful biological property is the enhanced permeability and retention (EPR) effect, i.e. the fact that NPs tend to accumulate into tumor tissues when inserted in the bloodstream [6]. Examples of interesting chemical-physical features of NPs are their possible superior fluorescence properties with respect to organic dyes [7] or

*Address correspondence to this author at the Center for Nanotechnology

Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12,

I-56127 Pisa, Italy; Tel: +39 050 509453; Fax: +39 050 509417;

E-mail: [email protected]

their localized surface plasmon resonances (LSPRs) in the interaction with light [8]. Many nanometric systems have been developed in order to improve delivery and release of payloads directly in specific cells, but therapeutic and diagnostic applications of most nanosystems are often seriously limited by several factors such as the potential aggregation of NPs in physiological media or their short circulation time in organisms, due to elimination from the blood stream by macrophages or by significant uptake by the liver before they reach their targets [9, 10]. Other important drawbacks for potential applications include a low loading capacity, a lack of site-specific targeting or limited release of the payload [11].

In order to obtain a truly efficient nanosystem for delivering drugs to tissues, it should fulfill the following criteria: a) good synthetic control of size and geometry (i.e. nanometric size- and shape-monodispersity), b) high stability in physiological media in order to prevent aggregation, c) stealthiness for the organisms (i.e. the ability to deceive the immune system and thus avoid being removed from blood circulation), d) adequate drug-loading capacity in order to reach adequate drug concentration in target cells, e) effective drug release, f) effective active or passive targeted delivery. In this view, gold nanoparticles (AuNPs) are very appealing building blocks [12, 13] for the development of DDSs, because they exhibit low cytotoxicity [14] and good cell permeability [15], and offer high drug-loading efficacy thanks to their intrinsically-high surface-to-volume ratio stemming from their nanometric size [16]. Moreover, it is possible to exploit the excitation of LSPRs; this can, for example, lead to enhanced local oscillating fields favoring superlinear light interactions such as multiphoton absorptions in molecules linked to the nanoparticles (thus making these effects accessible even at relatively low

2 Recent Patents on Nanomedicine, 2012, Volume 2, No. 1 Voliani et al.

excitation intensity [17]) or induce local heating by photothermal processes, which are markedly significant for AuNPs with particular geometries such as rods, shells, etc. Noticeably, the LSPR is tunable throughout the visible and near infrared regions of the electromagnetic (EM) spectrum by changing the size and geometry of the nanoparticles [18].

Generally, drugs are loaded on nanosystems by covalent conjugation or non-covalent interactions [19], both with the coating or directly with the metallic surface (Fig. 1). The non-covalent approach employs active drugs while the covalent attachment in general requires intracellular processing of a prodrug [20]. The release of the attached payloads can be triggered by internal (endogenous) or external (exogenous) stimuli, such as high concentration of glutathione (GSH) [21] or shining with light [1, 22, 23], respectively. This step is usually well controlled in the case of covalent binding, while drugs loaded by adsorption may suffer from non-specific release or other types of interactions [20].

Fig. (1). Three different types of interaction and release of payloads

from gold nanospheres [13].

GSH-mediated release represents a strategy for the selective intracellular release and subsequent activation of prodrugs which are linked to AuNPs by a thiol moiety. This methodology relies on the drastic difference between intracellular GSH (1–10 mM) and extracellular thiol concentrations (cysteine 8 μM, GSH 2 μM) [24, 25]; the high level of intracellular GSH is used to release payloads from AuNPs either because of place-exchange reactions at the metallic core or via disulfide exchange. On the other hand, maybe the most used exogenous controlled release mechanism relies on the use of light, which provides a highly orthogonal external stimulus, enabling spatiotemporal control of payload release. To this end it is possible to categorize current photo-releasing systems using gold nanostructures in three classes: i) systems where nanoparticles are used as carriers only, ii) systems exploiting nanoparticles photothermal properties, and iii) systems where nanoparticles induce multiphoton effects. In the first case the payloads are linked to nanostructures by specific intrinsically photo-cleavable groups, such as o-nitrobenzyl ester or carbamate moieties [1, 26], and the nanostructures are merely used as carriers for cellular uptake. In the second type, the metallic nanostructures (usually rods, cages or very

small spheres) release the cargo(s) thanks to photothermal effects [27] induced by excitation with infrared light; indeed, 800-1200nm light can cause local heating in suitably designed nanostructures. Examples of this methodology are nanoparticles encapsulated in thermally-sensitive hydrogels [28], or nanostructures coated with thermally-responsive polymers, which change conformation, and therefore release the load, upon temperature increase [29, 30]. In the third and more recent release process, the nanosystem is multicomponent and synergistic, i.e. it entails a cooperative effect of nanoparticle and cleavable moiety. The cargo release is based on multiphoton interactions enhanced by LSPR, allowing highly improved time- and space-control.

This review addresses the recent patents involving nanotechnology in the field of drug delivery and controlled release of payloads. We focus the discussion on gold-based nanostructures, which are the most used in nanomedicine thanks to their versatility and low cytotoxicity. In the following the endogenous and exogenous release mechanisms will be presented and the exciting perspectives in therapy, biosensing, and diagnostics will be exposed in the summary. A rapid overview on the processes to synthesize gold nanoparticles and on their biocompatibility is also given.

SYNTHETIC PROCESSES

One of the prominent features which make AuNPs particular appealing building blocks for the development of nano(bio)probes is the relative easiness in synthesizing gold nanostructures with controlled size and geometries. Optical and biological properties [18, 31-35] of colloids result from a delicate balance among material, size and shape of the nanostructures. In order to reach these results, in the last decade many synthetic processes for the desiderate nanomaterials were explored. In this section we shortly present the most common methods to produce by wet chemistry gold nanospheres, which are the well-studied nanoparticles. We invite to read more specific references for the general protocols for the production of gold nanoparticles with different sizes and shapes [36-41].

The reaction processes are usually based on the reduction of salts of the metal of interest in the presence of reducing and surfactant agents in aqueous or organic media. By changing reactants, relative molar concentrations, reaction temperature and/or stirring velocity, it is possible to control the nucleation and growth processes, achieving colloids with the desired properties. The fine control of the reaction is a tight requisite to achieve the desired size and shape of the particles, and, thus, to finely tune their physical-chemical properties.

The strategy proposed by Brust37

(Brust method) consists in growing the metallic clusters with the simultaneous attachment of self-assembled thiol monolayers on the growing nuclei. In order to allow the surface reaction to take place during metal nucleation and growth, the particles are grown in a two-phase system. In this method, AuCl4

- is

transferred from aqueous solution to toluene using tetraoctylammonium bromide as the phase-transfer reagent and reduced with aqueous sodium borohydride in the presence of dodecanethiol (C12H25SH). The outcome of the

Smart Delivery and Controlled Drug Release with Gold Nanoparticles Recent Patents on Nanomedicine, 2012, Volume 2, No. 1 3

reaction is determined by the ratio of thiol to gold. This single reaction yields a surface-functionalized gold colloid in the 2-8nm diameter range with a dispersion of about 4-6%.

The method proposed by Turkevich [36] (Turkevich method) is based on the reduction of tetrachloroauric acid (HAuCl4) with sodium citrate in water at 90-100 °C. This is the most commonly used process to synthesize gold nanospheres (AuNSs) due to its fairly simple and environmentally benign procedure and its ability to theoretically tune the size of nanospheres from 10 to 150nm by varying the molar ratio of citrate to HAuCl4. Unfortunately, the nanospheres obtained by the Turkevich method have usually a broad distribution of size and shape. Nanoparticles are generally produced in the diameter range of 12-60nm with a relative size distribution of 10-15% and usually with a non-uniform and irregular shape, such as quasi-spheres, ellipsoids, and triangles. This result is attributed to the fact that the chemical mechanism of the nucleation and of the crystal growth of AuNS is governed by many factors: pH, concentration of reactants, reducing agents and surfactants, stirring speeds and temperature. Several improvements of the Turkevich method [38, 39, 42-44] were made in order to obtain better dispersion in size, shape, and ability of functionalization, due to the relevance to produce colloids in water for biological use.

It is important to notice that stability, reactivity and biocompatibility of AuNPs depend on the coating of the metallic surface [45]. For these reasons, many specific coating methods for water-synthesized nanoparticles were reported [46-49]. The ability to improve the flexibility of AuNPs through stable coatings is another point that increases dramatically their use in nanomedicine. We do not discuss further this subject since it is not the main topic of this review.

ENDOGENOUSLY TRIGGERED RELEASE

Nanocarriers able to respond to biological stimuli are very appealing because they could allow the nanosystems to be triggered specifically by some pathological event. Such biological stimuli could include pH, temperature, and redox microenvironment [50]. For instance, extracellular pH in the organic tissue of solid tumour is more acidic (5.5 to 6.5) than in physiological conditions [51] (about 7.4). Also, the intracellular glutathione (GSH) level in tumour cells are 100-1000 fold higher than in normal tissues and, usually, the temperature of tissues is higher if tumour, inflammation and infection processes are present [51]. Several biologically-triggered nanosystems based on gold nanoparticles (AuNPs) encapsulated by PolyEthylenGlycol (PEG) or liposome were developed and tested in living cell applications, and they generally show low cytotoxicity [15, 52].

Rotello and colleagues1 developed an efficient tool for

gene delivery in mammalian cells thanks to the use of cationic gold clusters [53] (Mixed Monolayer Protected Gold Clusters, MMPCs). The cationic clusters interact electrostatically with DNA and give rise to an MMPC-DNA complex (Fig. 2), which is internalized by mammalian cells. The release mechanism relays on the unpackaging of DNA by competition of the cationic coating with intracellular GSH

[21]. Anionic GSH binds to the cationic gold core via disulfide exchange reactions on the MMPC-DNA complex in intracellular environment, reducing the overall positive charge and consequently decreasing the DNA affinity for the MMPC. The mechanism was ascertained by the significant reduction in the zeta potential of MMPC systems in presence of GSH (from 26 ±1 to 2 ±4 mV at 0 and 10 mM GSH, respectively) and by in vitro experiments attesting the recovery of T7 polymerase activity (Fig. 2). Moreover, GSH-mediated release was also observed in living cells [21]. For this purpose (Fig. 3A) gold nanoparticles were covered by a monolayer of tetra(ethylene glycol)lyated cationic ligands (TTMA) and thiolated Bodipy dyes (HSBDP); the TTMA ligands generate a cationic surface thereby enhancing cellular uptake. These nanosystems are not fluorescent, because the Bodipy fluorescence is quenched by the proximity of the AuNP surface. When glutathione-ethyl-ether (GSH-OEt) was added to the medium, it was internalized by the cells and metabolized to produce GSH. This increase of intracellular GSH produced a fast release of HSBDP, which turned fluorescent and promptly diffused in the entire cytoplasm, as observed by fluorescence microscopy (Fig. 3B).

Fig. (2). Mechanism of Glutathione (GSH)-triggered transcription

recovery. GSH binding to cationic gold core decreases the cationic

charge of the AuNPs and leads to the reduction of DNA affinity for

the NPs. As a consequence, T7 polymerase can interact with the

DNA [1].

Decher and co-workers [4, 54] developed a release system triggered by enzyme activity. In this approach the gold core is covered by a polyamine polymer and functionalized with a “terpolymer” composed by three different monomeric units by covalent layer-by-layer (LbL) deposition technology [55, 56], based on the alternating adsorption of charged species onto an oppositely charged substrate. The outer monomer, N-methacryloyl-glycyl-DL-phenylalanyl-leucyl-glycyl doxorubicin (Ma-Y-Dox), contains a nontoxic pro-drug form of doxorubicin (Dox), a potent DNA intercalation agent used in cancer chemotherapy. The pro-drug was incorporated in a


Fig. (3). Glutathione (GSH)-triggered drug release in living cells.

A) Scheme of the AuNP carrier and of the GSH-mediated surface

monolayer exchange reaction, which releases the payload. B)

Schematic representation and fluorescence images when using

glutathione-ethyl-ether (GSH OEt) as an external stimulus to

release HSBDP from AuNPs. The GSH OEt concentrations are 0,

5, and 20 mM in panels a, b, and c, respectively [50].

polymeric drug carrier system based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers carrying an oligopeptide spaced (Y) Dox side groups. The spacer was designed for specific cleavage by the lysosomal enzyme Cathepsins (Fig. 4). The specificity of the release mechanism relies on the exclusive release of the Dox through the enzymatic digestion of the spacer, occurring in the endocytotic pathway (Fig. 4).

In a recent study, Gong et al. [57] have designed a doxorubicin releasing system based on pH-responsive AuNPs. Gold nanoparticles are stabilized by encapsulation with 1:1 molar ratio of thiolated methoxy (poly)ethyleneglycol (mPEG-SH) and methyl thioglycolate (MTG), and Dox is covalently conjugated to the coated AuNPs by a reaction between the hydrazine moiety of the MTG domain and the ketonic groups of Dox. The resulting hydrazone bond is an acid-cleavable moiety, which provides a pH-responsive cleavable nanoparticle (Dox-AuNPs) with a high solubility in physiological medium thanks to the presence of mPEG-SH. They observed an increase in Dox releasing rates [57] when Dox-AuNPs were incubated in more acidic medium: i) at pH 5.3, 80% of Dox was released in 5h, and ii) at pH 7.4, only 10% of Dox was released in 5h. The group of Gong also demonstrated the release of the drug from Dox-AuNPs in living cells and the low toxicity of the system they proposed. The Dox fluorescence ( ex= 485 nm) was localized in the perinuclear region of the cells after 28 hours of incubation of the cells with Dox-AuNPs, confirming an uptake based on an endocytotic pathway.

Fig. (4). Multifunctional AuNPs obtained by the layer-by-layer (LbL) deposition method. A) From left to right: 13nm sized gold

nanoparticles (AuNPs) coated with five layers of poly(allylamine) (PAH) and poly(stryrenesulfonate) (PSS). The five-layer architecture is

denoted as Au5+. Au5

+ further coated with an external layer of terpolymer (F-HPMA) yielding multifunctionalized AuNPs (MFNP). The red

circles represent doxorubicin moieties (Dox). B) Cathepsin B induced release of Dox from MFNPs by cleavage of a specific tetrapeptide

(Y=Gly-Phe-Leu-Gly) spacer between Dox and the F-HPMA terpolymer backbone. The control with a spacer Y=Gly-Gly is not cleaved

[54].


In summary, endogenous systems have attracted great attention because of their peculiar ability to respond directly to a specific molecular or biological stimulus. Unfortunately, in these systems, despite the steric shielding effect of the encapsulating monolayer several processes can occur before the target is reached, for example unexpected release or thiol-disulfide exchange phenomena, e.g. with blood proteins, potentially creating a carrier with altered bioavailability and pharmacokinetic profiles [50]. These drawbacks are caused by the difficulty of taking into account every variable inside a living organism, and the possibilities to overcome them are subject of intense studies.

PHOTOTHERMAL RELEASE

Photothermal release from gold nanoparticles is perhaps the most mature and widely applied delivery method for in vivo use, owing to the simple and straightforward application of infrared radiation, which can effectively penetrate several centimeters in living tissues. Indeed, gold nanoparticles have been widely used for years in photothermal therapy (PTT). PTT is based on absorption of radiation of suitable wavelength, which is transformed into heat, causing irreversible damage to the targeted tissue. Gold nanostructures have been used in this regard to enhance the absorption of light at their LSPR. Generally, the absorbed radiation can be converted efficiently giving rise to a considerable temperature increase in the surroundings of the nanostructure (up to 50

oC) under good spatio-temporal

controlled conditions [29]. Photothermal therapy through effectively targeted nanoparticles can be used to cause, e.g., the selective death of malignant cells [58].

Besides the application to direct photothermal therapy, local heating of AuNPs can lead to a controlled release of dyes and therapeutic agents previously bound to the nanocarrier. Indeed, when no covalent bond is established between nanosystems and (bio)molecules, the temperature increase often causes significant desorption of the less-stably adsorbed molecules. This feature can be conveniently exploited in the development of light-activated photothermal release systems. The working principles for controlled release of therapeutic agents by interaction with IR electromagnetic radiation can be divided in two main classes: 1) direct desorption from the nanoparticle; 2) use of smart, thermally responsive envelops, such as polymer coatings of liposomes. In the following we will discuss mechanisms, advantages, and disadvantages of these methods.

Thermal Desorption of Ligands from Gold Nanoparticles

Desorption of molecular payloads by heating nanoparticles is a well-studied and common strategy for controlled release. The method resides on the establishment of weak interactions between the molecular payloads and the metallic nanoparticle. Upon the heating up of the system (e.g. due to photothermal effects promoted by irradiation), the energetic barrier for desorption is overcome and the release of the active principles takes place. The most used model is the thiol-gold bond, owing to the easy synthetic access to thiol-coated nanoparticles. For instance, a system based on gold nanoparticles decorated with glutathione

(GSH) was recently patented for the controlled release of GSH in living cells [59]. GSH plays a critical role in many metabolic pathways [41], and acts as a protecting group against free radical oxidation of cells. Controlled release of GSH in specific domains would be of extreme interest in metabolic research. According to what reported in the cited patent [59], 3nm-diameter gold nanospheres (AuNSs) can be loaded with varying amounts of the active principle (glutathione) and of an excipient (mPEG-SH), in order to modulate their cellular uptake and residence time in the organism. GSH is then desorbed by irradiating with light at wavelengths near the plasmon resonance of the AuNS (i.e. 520nm) with power of some watts. Although the reported procedure enables release in living systems, the use of visible light at relatively high powers is impaired by limited penetration in tissues, thus severely hampering the in vivo applicability.

Despite the attractiveness of direct desorption of ligands from nanoparticles by cleavage of thiol-metal bonds, only few (bio)molecules present thiol groups, severely limiting the applicability of direct thiol desorption. Also, coating with thiols is essential for the stability of gold nanoparticles, and unwanted precipitation or agglomeration of AuNPs can occur upon thiol desorption. Thus, different approaches have been developed which rely on the release of active molecules from the coating of the nanoparticles, rather than on desorption of potentially harmful thiols. Among the proposed systems, it is worth citing the controlled release of oligonucleotides linked by cationic coatings or coating with complementary sequences. One of the advantage of this delivery systems is that DNA degradation, one of the major issues in in vivo gene delivery, is inhibited by the tight packing which occurs on the surface of the nanoparticle; this effect induces considerable steric crowding, thus preventing to a large extent enzymatic degradation [11].

As an example, delivery of negatively charged residues such as short interfering RNAs (siRNA) can be favored by their adsorption on positively charged AuNPs, e.g. nanoparticles encapsulated with polymers such as poly-L-lysine. SiRNA are bound on the surface of the coated nanoparticle by electrostatic interactions between the negatively charged oligonucleotide and the positively charged polylysine coating. Actually, AuNPs can be coated with a multilayer system [60]; in this case AuNPs are firstly covered with a positively charged (poly)lysine layer. Oligonucleotides can be efficiently packed on the positively encapsulated AuNPs owing to electrostatic interactions, thus allowing efficient packing of the siRNA; finally, an outermost layer of positively charged peptides allows efficient targeting and increased protection from enzymatic degradation. Upon irradiation with NIR laser, the temperature increase induced by photothermal effect leads to the destabilization of the lysine-oligonucleotide interaction and consequently to the disruption of the outer charged shell. This in turns leads to the desorption of the therapeutic payload into specific tissues. Conversely, the higher thermal stability of the thiol-gold bond, together with the use of low-power radiation, preserves the stability of the inner polylysine coating. The efficiency of this approach was tested by silencing the GFP-coding gene through one specific siRNA delivered in this way, while nothing happened when using a scrambled RNA sequence [61]. A


closely related strategy was exploited in the selective delivery of oligonucleotides by denaturation of antisense oligonucleotide pairs. In this approach, local heating upon laser irradiation induces destabilization and release of the DNA or siRNA, because of the thermal sensitivity of the interactions in oligonucleotide double strands. Low NIR laser-powers were involved in these experiments because of the relatively weak non-covalent interaction between DNA sequences [62]. Indeed, this kind of releasing systems requires irradiation with lower powers than the ones based on direct desorption of thiol groups from nanoparticles, resulting in less cellular or tissutal damages.

Use of Photothermally Sensitive Polymers and Nanocages

As stated above, the direct desorption of ligands from AuNPs is a straightforward and simple approach for photothermal release of molecules; however, it suffers from intrinsic limitations, such as the necessity of appropriate functional groups on the payloads for their assembly on nanoparticles. Indeed, the payload should contain either a thiol functional group or a multiply charged residue (as in the case of oligonucleotides). Many molecules of interest in diagnostics and nanomedicine do not possess suitable anchoring groups. New approaches without this drawback are based on the inclusion of the molecules of interests inside micro or nanomaterials containing nanoparticles, or inside nanoparticles themselves (Fig. 5). In these cases, the release is triggered by some sort of phase transition driven by a local mild increase of temperature caused by the photothermal effect promoted by AuNPs irradiated close to their LSPR.

A first example is the use of nano- or microparticles formed by polymers with melting point near 40

oC. These

materials, which solidify upon cooling, can incorporate both AuNPs and the drugs of interest. Photothermal effects promoted by AuNPs under near infrared irradiation triggers the delivery of the drugs by simply melting the solid matrix. A remarkable example of this approach employs 1-tetradecanol [63]. The main disadvantage of this approach is the uncontrolled leakage of the payload from the polymeric matrix. Indeed, defects in the crystallization of the polymeric support can lead to considerable desorption of the drug even without external irradiation.

Thermally responsive polymers constitute a valid choice for the controlled release of non-functionalized small molecules as well. In particular, polymer chains that undergo a phase transition increasing their hydrophobicity shrink at rising temperature; this shrinkage creates pores in the polymeric matrix, allowing therefore the trapped drugs to escape. Exploiting this mechanism, it is possible to load, and release, active drugs directly in, and from, hollow or porous nanostructures (see the example reported in Fig. (5), where a polymer coating closes the apertures of a gold nanocage only when it is in its extended state). Among the thermally responsive polymers which have been successfully adopted [64, 65], it is worth citing poly(N-isopropylacrylamide)/ polyAcrylamide copolymer, which undergoes a phase transition at temperatures tunable anywhere in the range 32-50

oC [29, 66]. The advantage over the previously cited

thermally-sensitive systems is the controlled release of payloads by reversible conformational changes in the

polymer, not involving melting of the solid matrix. This enables multiple controlled release-caging cycles.

Alternatively, liposomes were employed as thermally-triggered carriers for drug delivery. Analogously to what previously seen for copolymers, liposomes can effectively shield the active payload and avoid diffusion in the surroundings but can be reversibly destabilized by local heating above their phase transition temperature. As pointed out for other polymer-based caged systems, proximity of metal nanoparticles enhances the effect of laser irradiation. Regarding the release mechanism, the effect of the temperature increase is not completely understood: both destabilization due to phase transition [67] and boiling followed by collapse of the water network around the liposome [68] were proposed but not investigated in details. However, the small temperature change in the surroundings of the liposome strongly suggests that a phase transition in the lipid actually takes place, in analogy with other systems [67].

PHOTOCHEMICAL RELEASE

Even a mild local temperature increase could be adverse in many applications of carrying-and-releasing nanosystems; therefore, it could be advantageous to use AuNPs with almost no-photothermal effects when irradiated at the frequencies of their LSPR (the dependence of the physical features of AuNPs on size and geometry are discussed elsewhere [35]). For this reason, other methodologies based on the interaction between light and nanosystems were developed to achieve site- and time-specific exogenous control [22] of the release of payloads from nanoparticles (Fig. 6). In this kind of systems a caged drug (prodrug) is generally covalently linked to a nanoparticle, which in some cases can merely act as a carrier. In this way the activity of the drug is suppressed by attaching it to a blocking element through a photo-removable protecting group. The photoactive part of the bound molecule interacts with UV light (usually between 250 and 380 nm) and releases the active principle. Nakanishi et al. (Fig. 6A) proposed this approach to develop photo-responsive nanocarriers for amines [26]. Using 10-1000-ms pulses of laser irradiation at 365 nm with intensity of 100 mW/cm

2, a carbamate linkage

could be dissociated by photocleavage of the 2-nitrobenzyl group. In an experiment where histamine was caged within a nanosystem of the afore-described type, they found biological activity of histamine only after irradiation of the nanosystems. Also Rotello et al. developed a gold nanosystem (Fig. 6B) with a photocleavable o-nitrobenzyl ester moiety that dissociates upon light irradiation, resulting in a light-controlled release of the anti-cancer drug 5- fluorouracil [69]. AuNSs were coated with photocleavable moieties and zwitterionic ligands, in order to improve solubility and decrease cellular uptake and, therefore, cytotoxicity. In their proof-of-concept experiment they used irradiation at 365 nm for cleaving 5-fluorouracil from the AuNSs decorated with orthonitrobenzyl groups, and measured the effects of the released drugs by monitoring cell deaths. No significant cell death was observed, instead, in cells treated with AuNSs and irradiated in the same conditions, in absence of 5-fluorouracil conjugation.


Fig. (5). Schematic illustration of gold nanocages encapsulated with

a thermal-responsive polymer. On exposure to a near-infrared laser,

the light is absorbed by the nanocage and converted into heat,

triggering the collapse of the polymer. Thus, the pre-loaded

effectors can escape from the nanocage. When the laser is turned

off, the polymer chains will relax back to the extended

conformation and terminate the release. Reproduced by Xia et al.

[29].

Despite the great potential of photocleavable nanosystems as diagnostic and therapeutic tools, several unsolved problems hinder their widespread clinical use [70]. One of the most prominent issue is the radiation frequency required for photorelease [71], which usually belongs to the UV–violet region of the spectrum and it is consequently toxic for living cells and marred by low penetration depth in tissues. To red-shift the radiation frequency necessary for release, Pop et al. proposed the irradiation of gold nanostructures on their plasmonic resonance to trigger the release of glutathione [59]: the vibrations of the nanostructures (supposedly induced by irradiation) could turn into heat or “other types of radiations” leading to an unloading of the glutathione molecules adsorbed on the metallic surface. Unfortunately they have not reported experiments validating their idea.

Voliani et al. invented the first example of a modular biocompatible nanosystem (Fig. 6C) designed for the photorelease of molecular payloads [72] by irradiation with yellow-green light. These systems, based on AuNSs, can effectively release its payload by low-intensity focused irradiation with cw-laser light at 561 nm, through the

Fig. (6). Photochemical induced release systems. A) Capture and release of amines (NH2-R) from Gold NanoParticles (GNP) having a

photocleavable succinimidyl ester. B) Photochemical reaction at 365 nm of Au_PCFU (Gold nanosphere PhotoCleavable 5-FluoroUracil)

and delivery of the payload to cell. C) Multiphoton release of fluorescein from gold nanospheres in living cells induced by 561 nm cw laser

cleavage of 1,2,3-triazole. Reprinted with permission from Refs [23, 26, 69].


cleavage of UV-absorbing triazole rings [23]. The modular system they describe is composed by four interacting components: i) 30-nm-diameter gold nanospheres, ii) peptidic coating [73], iii) triazolic rings, and iv) probes to be released (in their proof of concept, fluoresceinazide). These carriers were designed to exploit multi-photon excitation of the 1,2,3-triazolic rings triggered by the local EM-field enhancement [17] induced by AuNSs. Thanks to the enhancement in the effective multiphoton absorption cross section for the whole system, it is possible to cleave UV-photolabile bonds by irradiating the system with light at longer wavelengths (visible or near-IR, Fig. 6C), hence allowing deeper penetration in tissues. The click-chemistry formation of the 1,2,3-triazole ring linkage between the AuNS and the payload is another advantage of the described system, which opens the possibility to covalently bind (and release) almost the totality of (bio)molecules of interest.

In summary the photo-triggered cleavable systems are very promising tools able to release payloads and avoid, for example, the hazardous balance between the delivery and the thermal toxicity of the systems exploiting the photothermal effect. Further efforts to red-shift the irradiation to the biological-transparency window (800-1200nm) are necessary in order to make these systems really efficient in organisms.

ULTRASOUND-TRIGGERED RELEASE

As a final example of the use of AuNPs in controlled release, we describe some applications in which they are employed to regulate the ultrasound-driven rupture of multilayered microcapsules carrying the payload in their interior. Ultrasound irradiation triggering offers the advantage of deeper tissue penetration with respect to laser radiation even in the IR range [74-76]. The mechanism of release is the disruption of the encapsulation layers due to morphological alteration of the wall by ultrasound waves. The systems are produced via LbL deposition of polyelectrolytes, with the possible addition of AuNPs, on calcium carbonate microparticles with the payload entrapped in their pores. The carbonate core is then solubilized in low pH water, leading to hollow microcapsules filled with the payload. In principle, any molecular species can be

incorporated into the porous template. However, this approach works when diffusion through the capsule walls is inhibited thanks to a sufficiently high molecular weight of the entrapped species.

In a demonstrative example [75], a dye (FITC-dextran) is incorporated in the CaCO3 microparticles by co-precipitation. A first type of LbL coating consists solely of polyelectrolytes, namely poly(styrene sulfonate) (PSS) and poly(allylamide hydrochloride) (PAH), while in a second type a hybrid coating of PAH and gold nanoparticles is used. The disruption of the wall of the hollow microcapsule is accomplished by ultrasound excitation at 20 kHz with power of 20-100 W and duration of 1-10 s. 1 s sonication at 20 W is sufficient to break a large number of particles and at 100 W almost no intact capsule was observed. The microcapsules with AuNPs appear more stable mechanically, because for times shorter than 10 s less than one-half of the microcapsules with AuNPs were broken with respect to those with polyelectrolytes only.

The same concept brought to controlled protein release [76] by using lower power irradiation (< 3.2 W) at higher frequency (850 kHz). In this setup, the presence of AuNPs in the microcapsule shell enhanced the release efficiency by up to four times. Payload release occurred also before the complete rupture of the microcapsule, presumably because of increased permeability induced by the creation of small defects in the multilayer barrier.

The issues of microcapsule uptake, toxicity and biodegradability have been investigated [77], and Kreft et al. used laser light to open polyelectrolyte microcapsules functionalized with AuNPs inside living cells [78]. On the other hand, to the best of our knowledge, no demonstration of microcapsule opening by sonication in vivo or in living cell has been provided.

BIOCOMPATIBILITY

According to many reports, gold nanoparticles have been found to be non-toxic for living cells and organisms [10, 79]. On the other hand, despite the literature on bio-toxicity of AuNPs is relatively abundant, it is mostly controversial

10.

Fig. (7). Schematic representation of encapsulation and release of species in/from polyelectrolyte capsules by sonication. Calcium carbonate

microparticles with macromolecules (light gray lines) in their pores are coated with polyelectrolytes (black lines) and nanoparticles (black

dots) using the layer-by- layer (LbL) deposition technique (A). After dissolution of the calcium carbonate, hollow capsules are obtained (B).

Before ultrasound irradiation, the capsules, which consist of polyelectrolytes and nanoparticles, form a closed structure that keeps high-

molecular-weight species encapsulated. Ultrasound irradiation (C) causes membrane rupture and subsequent release of encapsulated species

[75].


The conflicting results could arise from the variability of the used toxicity assays, cell lines, nanoparticles samples and size/geometry dispersion, coatings, etc. Many recent papers report discussions about this topic [10, 79, 80] and in this section we report only some prominent results on the delivery and toxicity of AuNPs.

Delivery of AuNPs into cells usually involves the adsorption of the nanostructure onto the cell surface followed by internalization through endocytosis, facilitated eventually by the presence on the nanoparticle coating of internalization molecules such as cell-penetrating peptides or antibodies. The geometry (size and shape) of the nanostructure greatly influences the cellular uptake and the vitality of any cell involved. For example, the uptake of 10 to 80 nm gold nanospheres was recently investigated in HeLa cells [15]: the uptake kinetics varied with the nanoparticle diameter, and the main internalization was achieved by spheres with diameter of about 50 nm. This putative optimal size for efficient nanomaterial uptake into cells probably arises from a so-called “wrapping effect”[15]. The effect of nanoparticle shape on its internalization was also examined: spherical particles of similar size were internalized 5 times faster than rod-shaped particles, probably because of the longer membrane wrapping time required for elongated particles. Also gold nanocubes or nanocages seem to be uptaken with more difficulty than gold nanospheres, probably for the different initial surface contact between the nanostructures and the cell membrane [81, 82]. The inherent polydispersity of any batch of nanoparticles may cause unpredictable events, hence different batches of the same nanomaterial may display different results in cell studies; in this view the monodispersion in nanosphere diameters is a key-goal for developing useful reproducible nano-probes.

The dependence on AuNPs geometry of their uptake and toxicity in living-cells can be strongly influenced by their encapsulation with organic molecules [81]. In fact, the coatings of nanoparticles can change the colloidal stability and/or the interactions with macromolecules or cell membrane. As an example, the presences of neutral functional groups on the encapsulated nanoparticles such as ethers are excellent in preventing unwanted interactions between nanomaterials and biological matter. Most charged functional groups, such as amines for the positive charged nanoparticles and carboxylic acid for the negative ones, are responsible for active interactions with cells. Generally, neutral and negatively charged nanoparticles are less adsorbed on the negatively charged cell-membrane surface for ionic repulsions, and consequently show lower levels of internalization as compared to the positively charged particles [83]. Several studies reported that the internalization of negatively charged nanoparticles may occur by two processes: i) nonspecific binding of the particles on cationic sites on the plasma membrane followed by their endocytosis [52], and/or ii) adsorption of serum proteins [84] on the surface of nanoparticles through electrostatic and hydrophobic interactions, which allowed the nanoparticle to interface with the cell membrane. The cellular toxicity of negatively charged nanoparticles is strongly dependent on the type of surface coating [85]; usually, peptide-coated ones do not interfere with cell viability [49]. On the other side, cationic particles are known

to bind to negatively charged groups on the cell membrane [86] and translocate across the plasma membrane. However, despite cationic AuNPs are the most effective in crossing cell-membrane barriers and in localizing in the cytosol or nucleus, usually they show greater cytotoxicity than negative or neutral nanoparticles.

A whole organism is much more complex than a single cell; therefore there is a need for studies on nanoparticles bio-distribution and on general health indicators such as behavioral abnormality, weight loss, percentage of mortality, and average life span. It was found that an organism can absorb AuNPs by all the ways generally used for drugs [9, 87]: oral, intestinal, intravenous, etc. After 24 to 48 hours, AuNPs accumulate in all the secondary target organs, with a strong dependence on their size and a weak one on their charge [9]. Indeed AuNPs with diameter >50 nm usually accumulate in liver, kidney and spleen, while the ones with diameter <20 nm could accumulate also in brain, heart, lung, and blood [9, 10, 87]. In general the results are on spherical nanoparticles and no final long-term studies on the fate of AuNPs have been reported to our knowledge. Nanoparticles are more resistant to elimination routes such as metabolism and renal excretion, therefore the topic of the elimination should be seriously considered before their clinical use.

CURRENT AND FUTURE DEVELOPMENTS

In recent years, research in nanoscience and nanotechnology established the basis for the development of smart nano-machines able to address specific targets and convey theranostics effects in organisms. In this view, the ability to control both spatially and temporally the release of active (bio)molecules is essential. Many releasing nanostructures were proposed and developed but we are still far from the commercialization of completely life-safe and functional systems. In this direction the strengthening of the existent systems in terms of loading capacity of drugs and control of the release is needed. Also the ability to use common external triggering source, such as continuum wavelength laser, maybe at low powers and in the “biological transparent window” of irradiation is another important issue. On the other hand, we think that another important subject is to understand the toxicity of the nanosystems in organisms and in long time studies. The collective results in the literature show controversy about these issues, but if proper attention will be paid on encapsulation and dosage of nanoparticles, the full potential of AuNPs for biomedical application can be exploited.

CONCLUSION

In summary, gold nanoparticles are key materials for achieving functional release nanosystems, thanks to their stability, tunability, functional flexibility, good cell permeability, and low cytotoxicity. As described in this review, the types of patented release nanosystems can be categorized by the triggering stimulus, which can be endogenous or exogenous. The systems responding to endogenous stimuli are very specific and show an intrinsic control of the payloads delivery, thanks to internal triggering caused by enhanced concentration of target molecules, changes in pH, etc. These systems are, however, difficult to


synthesize and in most cases can suffer from uncontrolled cargo release. The second type of systems is triggered by external stimuli, such as light or ultrasounds, which interact with the nanostructures leading to unbinding or uncaging of the payloads. By using these systems, it is possible to load drugs or prodrugs on nanoparticles, wait for the internalization in the targeted compartment of the organism, and then release the (bio)molecules only when the region is irradiated. In some cases, the ability to deliver payloads by light irradiation resides in the intrinsic physical and chemical features of gold nanoparticles, as in the case of photothermal- or multiphoton-triggered nanosystems. These systems, which are under intensive study, display very intriguing properties, such as the precise control of release. However, despite their great potential, additional investigations will be required to fully understand their pharmacokinetics, their interactions with the immune system, and the extent of cytotoxicity due to the surface and the geometry of the gold nanoparticles.

ACKNOWLEDGEMENT

This work was partially funded by the Italian Ministry of Research (MiUR) under FIRB project RBIN048TSE and by Fondazione Monte dei Paschi di Siena. The authors declare no conflict of interest.

CONFLICT OF INTEREST:

Declared none.

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