Biochemical Pharmacology 92 (2014) 112–130
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Review - Part of the Special Issue: Metabolism 2014 – Alterations of metabolic pathways as therapeutic targets
Pharmacological potential of bioactive engineered nanomaterials Fanny Caputo a,b, Milena De Nicola a,b, Lina Ghibelli a,* a b
Dipartimento di Biologia, Universita’ di Roma Tor Vergata, via Ricerca Scientifica, 1 00133 Roma, Italy Dipartimento di Scienze e Tecnologie Chimiche, Universita’ di Roma Tor Vergata, via Ricerca Scientifica, 1 00133, Roma, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2014 Accepted 15 August 2014 Available online 28 August 2014
In this study we present an overview of the recent results of a novel approach to antioxidant and anticancer therapies, consisting in the administration of intrinsically active nano-structured particles. Their particulate (as opposed to molecular) nature allows designing multifunctional platforms via the binding of molecular determinants, including targeting molecules and chemotherapy drugs, thereby facilitating their localization at the desired site. The intrinsic activity of nanomaterials with pharmacological potential include peculiar trans-excitation reactions that render them able to transform radiofrequency, UV, visible or infrared radiations into cytocidal reactive oxygen species or heat, thereby inducing local cytotoxity in selected areas. The use of such devices has been shown to improve the efficacy of antitumor chemo- and radio-therapies, increasing the selectivity of the cytocidal effects, and reducing systemic side effects. In addition, catalytic nanomaterials such as cerium oxide nanoparticles can perform energy-free antioxidant cycles that scavenge the most noxious reactive oxygen species via SOD- and catalase-mimetic activities. A vast body of in vivo and in vitro studies has demonstrated that they reduce the damage induced by environmental stress and ameliorate an impressive series of clinically relevant oxidation-related pathologies. Similar effects are reported for carbon-based materials such as fullerenes. Overall, great improvements are expected by this novel approach. However, caution must be posed due to the poor knowledge of possible adverse body reactions against these novel devices, thoroughly analyzing the biocompatibility of these nanomaterials, especially concerning the biokinetics and the problems potentially caused by long term retention of nonbiodegradable inorganic nanomaterials. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Nanopharmacology Antioxidant therapy Photodynamic therapy Cerium oxide nanoparticles Radio-sensitization
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineered nanoparticles as novel antioxidants. . . . . . . . . . . . . . . . . . . . . . Cerium oxide nanoparticles: a novel self-regenerating antioxidant . 2.1. Mechanism of action and biological properties . . . . . . . . . 2.1.1. Pharmacological potential . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Platinum nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.
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Abbreviations: BBB, blood brain barrier; C60, fullerene; Cat, catalase; CNPs, cerium oxide nanoparticles; CNTs, carbon nanotubes; DDR, DNA damage response; GNPs, gold nanoparticles; GSHpx, glutathione peroxidase; GSH, glutathione; GSSG, oxidized glutathione; MF, magnetic fileds; MWCNTs, multi-walled carbon nanotubes; NF–kB, nuclear factor-kappa B; NIR, near infrared radiation; NOS, nitric oxide synthase; NPs, nanoparticles; PDT, photodynamic therapy; PNP, platinum nanoparticles; PS, photosensitizer; QDs, quantum dots; RNS, reacting nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; SPIONs, super-paramagnetic iron oxide nanoparticles; SPR, surface plasmonic resonance; SWCNTs, single-walled carbon nanotubes; TGF beta1, transforming growing factor beta1; UV, ultraviolet radiation; VEGF, vascular endothelial growth factor; Zx, atomic number of the element X. * Corresponding author. E-mail address: [email protected] (L. Ghibelli). http://dx.doi.org/10.1016/j.bcp.2014.08.015 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
F. Caputo et al. / Biochemical Pharmacology 92 (2014) 112–130
3.
4. 5.
Engineered nanoparticles for cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct killing of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Hyperthermia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Nanoparticles as co-adjuvant in radiation therapy . . . . . . . . . . . . . . . . . . . . . . 3.2. Biological bases of radio-sensitization . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Modulation of the ‘‘absorbed radiation dose’’ by nanoparticles . . . . 3.2.2. Gold nanoparticles as a paradigm for NPs radio-sensitizing studies. 3.2.3. 3.2.4. The ‘‘smart’’ selectivity of cerium oxide nanoparticles . . . . . . . . . . . Modulation of tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Reduction of tumor neo-angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. 3.3.2. Potentiation of the immune response . . . . . . . . . . . . . . . . . . . . . . . . Modulation of tumor cells–stroma interaction . . . . . . . . . . . . . . . . . 3.3.3. Biocompatibility of pharmaceutical nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The prevalent current pharmacological approaches for the treatment of diseases consist of synthetic, semisynthetic or natural molecular compounds with intrinsic activity that interact with biomolecules and signaling pathways to restore the organism homeostasis compromised by pathological or stress conditions. Many potentially active molecular pharmaceutical agents display problems of body retention, solubility, stability and selectivity that hinder their pharmacological usefulness. Nanomedicine is primarily aimed at overcoming such problems, providing nanosized solid structures that can target the drug to the desired body district, at the same time protecting, retaining, and masking the drug until the correct final destination is reached. The most studied category of nanomedicine tools consists of nanoparticles acting as carriers of more than one molecular determinants; they are linked to the same structure, behaving as a cohort allowing the simultaneous delivery of multiple determinants. Therefore, these platforms may carry at the same time delivery molecules (e.g., antibodies against tissue specific integrins or protein over-expressed in tumor cells), active drug(s), sensitizing agents, diagnostic molecules for tissue imaging. In these instances, the core of the nanomedicine tool is an inert carrier that serves as a focal binding site for the functional molecules. Such approaches are revolutionizing chemotherapic and diagnostic procedures, combining them in the novel ‘‘theranostic’’ approaches, and have been extensively reviewed in the recent literature [1–3]. Materials at the nanosize acquire different properties with respect to larger structures, for example the noble materials such as gold and platinum, essentially inert in the bulk, acquire reactive catalytic functions when in the nanoscale [4–6]. The threshold of the nanosized features is generally 100 nm in at least one dimension, therefore this limit has a physicochemical meaning and is not merely a convention. Nanomaterials offer therefore unique characteristics that render them very much appealing for many types of applications, including catalysis, electrical conductance and many other industrial applications, and as a consequence the field of nanotechnology is rapidly expanding, including also nanomedicine. In this instance, nanomaterials are not merely a platform for drug delivery, but their intrinsic physicochemical features are exploited for their reactivity, such as, e.g., the electrochemical potential that allow the fine control of fundamental redox reactions occurring in living matter. The physico-chemical features that nanomaterials display depend on their elemental constitution as well as on their size, shape and surface morphology, parameters that deeply influence reactivity and must be taken into account when projecting a
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biomedical device. Nanoparticle (NP) surface can be modified by the addition of molecular determinants (functionalization) for many purposes, including fluorescent molecules to facilitate detection in biological environments, lipids to provide hydrophobicity, determinants that change superficial charge to control aggregation/agglomeration and favor the colloidal stability, and also molecular drugs and determinants for targeting, since their particulate nature allow to combine reactivity with drug delivery. Some of the terms mentioned are briefly defined in Box 1. The reactivity of nanoparticles that are considered for their pharmacological potential may be either an intrinsic basal feature (e.g., antioxidant effect of cerium oxide nanoparticles [7]) or a feature that is transiently induced by a specific treatment, such as the prooxidant effect that a set of nanoparticles exert upon irradiation [8]. The administration of nanoparticles for pharmacological purposes arises unprecedented safety issues; the biocompatibility of nanoparticles indeed is a very critical aspect, and nanotoxicology, the novel branch of toxicology aimed at studying the specific toxicological problems deriving from exposure to nanoparticles, needs to face unprecedented effects that require novel paradigms to be correctly addressed. For example, it must be considered that nanoparticles made of the same element and possessing similar crystal structures may exert very different effects according to their size or shape. It is plain that molecular drugs are effective only if they interfere with metabolism, and may be beneficial or detrimental according to the circumstances; therefore, the totally safe drug is a nonsense. Likewise, when the drug is a nanoparticle, the biocompatibility must be conditional to the situation, and adverse effects such as, e.g., cytotoxicity, turn into appreciated features when tumor cells need to be killed. A big advantage of nanoparticles over molecular drug relies in that they can be much more easily targeted to, and confined in, the pathological tissue, where the antitumor cytotoxic effect may take place in restricted areas with reduced damage for the organism. A survey of the specific literature identifies two specific fields of utilization of intrinsically active nanoparticles as pharmacologic agents, namely oxidative-related pathologies and cancer. Redox active nanoparticles have been shown to ameliorate many clinically relevant pathological disorders that implicate oxidative stress, reducing the oxidative burden and alleviating many important symptoms, as revealed by in vitro, ex vivo and in vivo studies; importantly, some treatments are already undergoing clinical trials for perspective wide utilization in the clinical practice. These nanoparticles act either in a catalytic way
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Box 1. Nanoglossary.
resembling the action of antioxidant enzymes such as catalase and superoxide dismutase, or they act as activating surfaces to facilitate reactions between the aqueous environment and the reactive oxygen species present at high level in the pathological tissues. These mechanisms will be thoroughly discussed in the following chapters. In anticancer therapies, metal and metal oxide nanoparticles are being used experimentally to directly kill tumor cells e.g., allowing extremely localized thermo-ablation by transforming applied magnetic fields into strong hyperthermia, or performing efficient photodynamic therapies able to reach even the internal tissues by transforming in loco a penetrating infrared radiation into visible light inside the tumor. In addition, nanoparticles made of heavy atoms are able to react to irradiation producing cytocidal reactive oxygen species, thereby increasing the pro-apoptotic effects of radiotherapies once correctly localized at the site of the
tumor. Also in this case, clinical trials are on the way. A schematic representation of the main nano-pharmacological strategies is shown in Fig. 1, and in the following chapters these approaches will be analyzed in detail.
2. Engineered nanoparticles as novel antioxidants Reactive oxygen species (ROS) are at the basis of many clinical disorders (a scheme is shown in Fig. 2). ROS are physiologically produced by living organisms by enzymatic and non-enzymatic processes. In particular, superoxide are produced by a set of oxidase enzymes (e.g. xanthine oxidase, monoamine oxidase and many others) for metabolic and signaling purposes [9,10] or as byproducts of enzymatic metabolic reactions, such as, e.g., leukotriene and prostaglandin synthesis by lipoxygenases and
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115
Fig. 1. Nanoparticles exploitation in biomedicine.
Fig. 2. Oxidative stress related diseases.
cyclooxygenases enzymes, respectively, or still by non enzymatic processes such as accidental leakage from the electron transport chain [9,10]. Moreover different types of ROS (e.g. nitric oxide, hydrogen peroxide) are secreted during macrophages oxidative burst processes [9,10]. Basal ROS are controlled by endogenous antioxidant defenses, including enzymes such as superoxide dismutase, catalase, the glutathione system, and nutritional antioxidant adjuvants such as vitamins and flavonoids present in food. The dynamics of ROS production and scavenging is
schematically depicted in Fig. 3A. A basal level of ROS is necessary to maintain cellular homeostasis and to perform cellular signaling tasks [9,10]. Environmental stress (e.g. ionizing or UV radiation, heavy metals, pollutants) or deranged metabolism [9,10] produce excessive amount of ROS that may overwhelm basal antioxidant defenses; these conditions are defined as oxidative stress, a state that causes damage to DNA, proteins, organelles and membranes, leading to improper cell signaling, mutations or apoptosis [9,11]. For this reason oxidative stress is associated with many
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Fig. 3. Antioxidant nanoparticles: mechanisms of actions. (A) Engineered nanoparticles possessing SOD and catalase mimetic activity (e.g. platinum, fullerenes and cerium oxide) support endogenous antioxidants defenses, ameliorating oxidative stress conditions. GSHpx: glutathione peroxidase; GSH: reduced glutathione, GSSG: oxidize glutathione. (B) Cerium oxide nanoparticles (CNPs) redox regenerative cycle: catalytic mechanism. (C) Fullerene antioxidant activity: direct scavenging of free radicals R, (left); fullerene SOD-like activity (right).
pathological conditions such as cancer, neurodegenerative disorders, autoimmune syndromes, cardiovascular diseases, diabetes, arthritis, infertility, endometriosis and many others [9,12], and with aging, a process characterized (and determined) by high oxidative conditions. A sensible strategy to overcome the deleterious effects of oxidative stress is reinforcing cellular defenses by supplementation of exogenous antioxidants. To this purpose, natural or synthetic molecular antioxidants are used as remedies during stress or pathological conditions. However, the advantages of such treatments have been deceiving due to lack of specificity, improper delivery or rapid decay of canonical molecular antioxidants. In the last decade the administration of synthetic nanoparticles with antioxidant action have been proposed as a valid alternative to molecular antioxidants. These engineered nano-antioxidants include inert nanoparticles covalently modified (functionalized) with molecular antioxidants to improve their delivery and stability, and nanoparticles that possess antioxidant activity per se, due to their physicochemical composition. The first approach has been extensively reviewed [13], therefore in this chapter we point to analyze the features, activity and administration of
nanoparticles possessing intrinsic redox activity, focusing on cerium oxide and platinum nanoparticles (PNPs) and fullerenes, which presently appear as the most promising nanomaterials for antioxidant pharmacological applications. These concepts are depicted in Fig. 3B and C. 2.1. Cerium oxide nanoparticles: a novel self-regenerating antioxidant 2.1.1. Mechanism of action and biological properties Cerium ions switch between two valence states (Ce3+ and Ce4+), which coexist in the surface of cerium oxide nanoparticles (CNPs). The charge deficiency due to Ce3+ is compensated by the formation of oxygen vacancies; these ‘‘surface defects’’ confer to CNPs intrinsic antioxidant properties [14–16]. The mechanism of action of CNPs resembles that of naturally occurring metallo enzymes, which use transition metals cofactors such as Fe, Zn, Cu or Mn to scavenge ROS in cells and tissue. It has been demonstrated that CNPs mimic superoxide dismutase, when the Ce3+ form reacts with superoxide turning into Ce4+ while reducing superoxide into hydrogen peroxide [17,18]. Likewise, oxidation of Ce3+ to Ce4+ ions allows scavenging other noxious reactive species such as hydroxyl
F. Caputo et al. / Biochemical Pharmacology 92 (2014) 112–130
radicals [19], NO [20] and peroxynitrite (ONOO ) [21]. On the other hand, Ce4+ can be reduced to Ce3+ while oxidizing hydrogen peroxide to molecular oxygen, similarly to catalase enzyme [22]. Therefore, the Ce3+/Ce4+ couple can reversibly switch back and forth while scavenging superoxide and peroxides [14– 16,23]. Considering the catalase- and the SOD-mimetic activities together, it has been proposed that CNPs undergo a complete redox cycle while scavenging two superoxide and one hydrogen peroxide [14] as depicted in Fig. 3B. This would be an elegant way to eliminate in a sequential set of energy-free reactions both superoxide and hydrogen peroxide [14], thereby freeing cells from the most noxious and abundant ROS. This CNPs selfregenerating antioxidant property is potentially an invaluable pharmacological tool, which renders this nanomaterial a unique and very attractive ROS biological scavenger. In CNPs, the Ce3+/Ce4+ ratio changes according to the synthesis methods: for example, the hexamethylenetetramine or base (sodium hydroxide or ammonium hydroxide) co-precipitation methods allow to obtain 21–30% surface concentration of Ce3+ ions, whereas higher concentrations (55–65%) can be obtained using hydrogen peroxide as a synthesis cofactor [16]. Therefore, CNPs ability to preferentially react with different free radicals may be modulated varying the method of synthesis and the Ce3+/ Ce4+ratio. CNPs with higher Ce4+ (70–80%) concentration show a better catalase mimetic action [16,22], whereas CNPs possessing higher amounts of Ce3+ ions exert a better SOD-like activity. Accordingly, the SOD-like action is completely lost if the Ce3+ surface concentration is reduced to 1 year) tumor free survival in mice carrying intra-cerebral malignant gliomas [178]. Many other in vivo studies were published, demonstrating the efficiency of GNPs as radio-sensitizers, as reviewed in [166,167,172,176]. The efficiency of GNPs in absorbing ionizing radiation producing free radicals, was estimated by theoretical models and compared with their effects in actually enhancing tumor cells killing: it clearly emerges that the pro-oxidant ability of irradiated GNPs does not fully explain the extremely strong radio-sensitizing effects actually observed in cells and animals [166,167,172,176]. This suggests that an additional (biological?) mechanism should be involved [166]. This was demonstrated by a pioneering study showing that GNPs internalized into breast cancer cells (MDA-MB-231) hardly increased X-rays induced DNA damage, but nonetheless increased apoptosis [165]. The same study reports that GNPs were able to potentiate the effect of bleomycin, a radiomimetic redox active agent that induces DNA damage via ROS production. This suggests that in the presence of DNA lesions caused by different sources (i.e., ionizing radiation and bleomycin), GNPs enhance the DNA damage response towards a more stringent, pro-apoptotic outcome, and consequently induce an additional radio sensitizing activity [146,166]. This hypothesis is supported by the evidence that GNPs have the ability to chemosensitize cancer cells to 5-fluorouracil by enhancing cell cycle arrest in the G2 phase [146,165,168]. The complex mechanisms of NPs sensitization to ionizing radiation is summarized in Fig. 6C. In addition to GNPs, other metal nanoparticles capable of increasing the radiation absorbed dose, such as silver [179,180], platinum [173], bismuth [1173] and semiconductor quantum dots (QDs) [122] have been tested in biological systems for their radiosensitizing ability. Platinum nanoparticles were shown to sensitize cancer cells to hadron therapy [181] increasing by twofold the DNA strand breaks induced by fast ions irradiation. Any possible additional biological effects downstream to DNA damage have not been considered yet. It is known that treatment with exogenous SOD enhances the arrest in the G2 phase of the cell cycle in the
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Fig. 6. Mechanisms of nanoparticles radio-sensitization (A) Nanoparticles composed by heavy atoms absorb X-rays or gamma rays emitting electrons that react with water generating ROS. (B), nanoparticles increase the radiation absorption dose inside the tumor mass reducing tumor cells viability. In the absence of NPs, tumor cells are metabolically active (deep violet) while in the presence of NPs they become apoptotic (light violet). (C) Nanoparticles radio sensitize tumor cells to ionizing radiation by increasing DNA damage via ROS production (physical mechanism) and by enhancing the DNA damage response of tumor cells (biological mechanism).
presence of DNA damage [11]. Interestingly, PNPs have a superoxide dismutase mimetic activity [74] that may possibly influence the DNA damage response. 3.2.4. The ‘‘smart’’ selectivity of cerium oxide nanoparticles CNPs are a novel and very interesting material for radiation therapy, possessing the ‘‘smart’’ ability to selective induce the death of irradiated cancer cells [40], while protecting the surrounding tissue from radiation induced damage and oxidative stress [37,39]. Therefore, CNPs have the unique feature of acting as radio-protecting as well as radio-sensitizing agents at the same time. Cerium oxide nanoparticles interact with ionizing radiation with two antithetic mechanisms that produce opposite effects. On the one hand, being cerium a high Z element (ZCe = 58), CNPs may physically produce free radicals in response to radiation, peaking in the 50–60 keV energy range (dose enhancement effect) [182]. On the other hand, the Ce3+/Ce4+ redox couple on CNPs surface is able to scavenge the ROS produced by radiation. As shown by Wason [40], the resulting effects depend on the balance between these two mechanisms, which is influenced by the material properties, that is, Ce3+/Ce4+ ratio, the size, aggregation and surface coating, and by the energy of the radiation applied. Interestingly enough, it was shown that CNPs irradiated with X-Rays at 160 keV, an energy where the physical dose enhancement effect of cerium is low, exert an additional ‘‘smart’’ biological activity that depends on the cell line tested, that is, tumor vs normal cells [40]. CNPs selectively increased oxidative stress and apoptosis in irradiated cancer cells, while
protecting normal tissues [37–40]. Wason et al. hypothesized that the selective toxicity of CNPs against cancer cells is due to the inhibition of CNPs catalase-like activity occurring in acidic (pH 4.3) environments [23]: in the presence of superoxide produced by the ionizing radiation, the SOD like activity, which is maintained even at low pH, would lead to H2O2 accumulation, incrementing radiation toxicity [40]. The hypothesis is based on the assumption that pH of cancer cells is acidic [40]. In fact, the cytosol of cancer it is slightly more alkaline (pH > 7.4) than normal cells, whereas the extracellular tumor microenvironment is slightly acidic due to the Warburg effect, and pH decreases from 7.1 (normal tissues) to 6.7 [183,184]. However, catalase inhibition requires much more severe pH derangements ( 2–3 nm with proteins present in body fluids and cells (and also in the media of tissue cultures), has posed a question on this issue. Proteins are essentially zwitterionic, and when they are absorbed on the NPs surface they strongly attenuate the charge of the NP core (zeta potential), which thus becomes close to neutrality [213]. More studies are therefore required to solve this key point of biocompatibility of NPs. Another important issue is NPs dissolution, that is, the release of molecules or atoms from their crystalline structure. A well-known example, partly mentioned above, deals with the conditional toxicity of cerium oxide nanoparticles. CNPs administration in vivo is well tolerated, and does not induce death or tissue histological alteration [32,33,58,214]. However, CNPs may release Ce ions at acidic pH (
Contents lists available at ScienceDirect
Biochemical Pharmacology journal homepage: www.elsevier.com/locate/biochempharm
Review - Part of the Special Issue: Metabolism 2014 – Alterations of metabolic pathways as therapeutic targets
Pharmacological potential of bioactive engineered nanomaterials Fanny Caputo a,b, Milena De Nicola a,b, Lina Ghibelli a,* a b
Dipartimento di Biologia, Universita’ di Roma Tor Vergata, via Ricerca Scientifica, 1 00133 Roma, Italy Dipartimento di Scienze e Tecnologie Chimiche, Universita’ di Roma Tor Vergata, via Ricerca Scientifica, 1 00133, Roma, Italy
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2014 Accepted 15 August 2014 Available online 28 August 2014
In this study we present an overview of the recent results of a novel approach to antioxidant and anticancer therapies, consisting in the administration of intrinsically active nano-structured particles. Their particulate (as opposed to molecular) nature allows designing multifunctional platforms via the binding of molecular determinants, including targeting molecules and chemotherapy drugs, thereby facilitating their localization at the desired site. The intrinsic activity of nanomaterials with pharmacological potential include peculiar trans-excitation reactions that render them able to transform radiofrequency, UV, visible or infrared radiations into cytocidal reactive oxygen species or heat, thereby inducing local cytotoxity in selected areas. The use of such devices has been shown to improve the efficacy of antitumor chemo- and radio-therapies, increasing the selectivity of the cytocidal effects, and reducing systemic side effects. In addition, catalytic nanomaterials such as cerium oxide nanoparticles can perform energy-free antioxidant cycles that scavenge the most noxious reactive oxygen species via SOD- and catalase-mimetic activities. A vast body of in vivo and in vitro studies has demonstrated that they reduce the damage induced by environmental stress and ameliorate an impressive series of clinically relevant oxidation-related pathologies. Similar effects are reported for carbon-based materials such as fullerenes. Overall, great improvements are expected by this novel approach. However, caution must be posed due to the poor knowledge of possible adverse body reactions against these novel devices, thoroughly analyzing the biocompatibility of these nanomaterials, especially concerning the biokinetics and the problems potentially caused by long term retention of nonbiodegradable inorganic nanomaterials. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Nanopharmacology Antioxidant therapy Photodynamic therapy Cerium oxide nanoparticles Radio-sensitization
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Engineered nanoparticles as novel antioxidants. . . . . . . . . . . . . . . . . . . . . . Cerium oxide nanoparticles: a novel self-regenerating antioxidant . 2.1. Mechanism of action and biological properties . . . . . . . . . 2.1.1. Pharmacological potential . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Platinum nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.
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Abbreviations: BBB, blood brain barrier; C60, fullerene; Cat, catalase; CNPs, cerium oxide nanoparticles; CNTs, carbon nanotubes; DDR, DNA damage response; GNPs, gold nanoparticles; GSHpx, glutathione peroxidase; GSH, glutathione; GSSG, oxidized glutathione; MF, magnetic fileds; MWCNTs, multi-walled carbon nanotubes; NF–kB, nuclear factor-kappa B; NIR, near infrared radiation; NOS, nitric oxide synthase; NPs, nanoparticles; PDT, photodynamic therapy; PNP, platinum nanoparticles; PS, photosensitizer; QDs, quantum dots; RNS, reacting nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; SPIONs, super-paramagnetic iron oxide nanoparticles; SPR, surface plasmonic resonance; SWCNTs, single-walled carbon nanotubes; TGF beta1, transforming growing factor beta1; UV, ultraviolet radiation; VEGF, vascular endothelial growth factor; Zx, atomic number of the element X. * Corresponding author. E-mail address: [email protected] (L. Ghibelli). http://dx.doi.org/10.1016/j.bcp.2014.08.015 0006-2952/ß 2014 Elsevier Inc. All rights reserved.
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3.
4. 5.
Engineered nanoparticles for cancer therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Direct killing of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Hyperthermia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Nanoparticles as co-adjuvant in radiation therapy . . . . . . . . . . . . . . . . . . . . . . 3.2. Biological bases of radio-sensitization . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Modulation of the ‘‘absorbed radiation dose’’ by nanoparticles . . . . 3.2.2. Gold nanoparticles as a paradigm for NPs radio-sensitizing studies. 3.2.3. 3.2.4. The ‘‘smart’’ selectivity of cerium oxide nanoparticles . . . . . . . . . . . Modulation of tumor microenvironment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Reduction of tumor neo-angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. 3.3.2. Potentiation of the immune response . . . . . . . . . . . . . . . . . . . . . . . . Modulation of tumor cells–stroma interaction . . . . . . . . . . . . . . . . . 3.3.3. Biocompatibility of pharmaceutical nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The prevalent current pharmacological approaches for the treatment of diseases consist of synthetic, semisynthetic or natural molecular compounds with intrinsic activity that interact with biomolecules and signaling pathways to restore the organism homeostasis compromised by pathological or stress conditions. Many potentially active molecular pharmaceutical agents display problems of body retention, solubility, stability and selectivity that hinder their pharmacological usefulness. Nanomedicine is primarily aimed at overcoming such problems, providing nanosized solid structures that can target the drug to the desired body district, at the same time protecting, retaining, and masking the drug until the correct final destination is reached. The most studied category of nanomedicine tools consists of nanoparticles acting as carriers of more than one molecular determinants; they are linked to the same structure, behaving as a cohort allowing the simultaneous delivery of multiple determinants. Therefore, these platforms may carry at the same time delivery molecules (e.g., antibodies against tissue specific integrins or protein over-expressed in tumor cells), active drug(s), sensitizing agents, diagnostic molecules for tissue imaging. In these instances, the core of the nanomedicine tool is an inert carrier that serves as a focal binding site for the functional molecules. Such approaches are revolutionizing chemotherapic and diagnostic procedures, combining them in the novel ‘‘theranostic’’ approaches, and have been extensively reviewed in the recent literature [1–3]. Materials at the nanosize acquire different properties with respect to larger structures, for example the noble materials such as gold and platinum, essentially inert in the bulk, acquire reactive catalytic functions when in the nanoscale [4–6]. The threshold of the nanosized features is generally 100 nm in at least one dimension, therefore this limit has a physicochemical meaning and is not merely a convention. Nanomaterials offer therefore unique characteristics that render them very much appealing for many types of applications, including catalysis, electrical conductance and many other industrial applications, and as a consequence the field of nanotechnology is rapidly expanding, including also nanomedicine. In this instance, nanomaterials are not merely a platform for drug delivery, but their intrinsic physicochemical features are exploited for their reactivity, such as, e.g., the electrochemical potential that allow the fine control of fundamental redox reactions occurring in living matter. The physico-chemical features that nanomaterials display depend on their elemental constitution as well as on their size, shape and surface morphology, parameters that deeply influence reactivity and must be taken into account when projecting a
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biomedical device. Nanoparticle (NP) surface can be modified by the addition of molecular determinants (functionalization) for many purposes, including fluorescent molecules to facilitate detection in biological environments, lipids to provide hydrophobicity, determinants that change superficial charge to control aggregation/agglomeration and favor the colloidal stability, and also molecular drugs and determinants for targeting, since their particulate nature allow to combine reactivity with drug delivery. Some of the terms mentioned are briefly defined in Box 1. The reactivity of nanoparticles that are considered for their pharmacological potential may be either an intrinsic basal feature (e.g., antioxidant effect of cerium oxide nanoparticles [7]) or a feature that is transiently induced by a specific treatment, such as the prooxidant effect that a set of nanoparticles exert upon irradiation [8]. The administration of nanoparticles for pharmacological purposes arises unprecedented safety issues; the biocompatibility of nanoparticles indeed is a very critical aspect, and nanotoxicology, the novel branch of toxicology aimed at studying the specific toxicological problems deriving from exposure to nanoparticles, needs to face unprecedented effects that require novel paradigms to be correctly addressed. For example, it must be considered that nanoparticles made of the same element and possessing similar crystal structures may exert very different effects according to their size or shape. It is plain that molecular drugs are effective only if they interfere with metabolism, and may be beneficial or detrimental according to the circumstances; therefore, the totally safe drug is a nonsense. Likewise, when the drug is a nanoparticle, the biocompatibility must be conditional to the situation, and adverse effects such as, e.g., cytotoxicity, turn into appreciated features when tumor cells need to be killed. A big advantage of nanoparticles over molecular drug relies in that they can be much more easily targeted to, and confined in, the pathological tissue, where the antitumor cytotoxic effect may take place in restricted areas with reduced damage for the organism. A survey of the specific literature identifies two specific fields of utilization of intrinsically active nanoparticles as pharmacologic agents, namely oxidative-related pathologies and cancer. Redox active nanoparticles have been shown to ameliorate many clinically relevant pathological disorders that implicate oxidative stress, reducing the oxidative burden and alleviating many important symptoms, as revealed by in vitro, ex vivo and in vivo studies; importantly, some treatments are already undergoing clinical trials for perspective wide utilization in the clinical practice. These nanoparticles act either in a catalytic way
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Box 1. Nanoglossary.
resembling the action of antioxidant enzymes such as catalase and superoxide dismutase, or they act as activating surfaces to facilitate reactions between the aqueous environment and the reactive oxygen species present at high level in the pathological tissues. These mechanisms will be thoroughly discussed in the following chapters. In anticancer therapies, metal and metal oxide nanoparticles are being used experimentally to directly kill tumor cells e.g., allowing extremely localized thermo-ablation by transforming applied magnetic fields into strong hyperthermia, or performing efficient photodynamic therapies able to reach even the internal tissues by transforming in loco a penetrating infrared radiation into visible light inside the tumor. In addition, nanoparticles made of heavy atoms are able to react to irradiation producing cytocidal reactive oxygen species, thereby increasing the pro-apoptotic effects of radiotherapies once correctly localized at the site of the
tumor. Also in this case, clinical trials are on the way. A schematic representation of the main nano-pharmacological strategies is shown in Fig. 1, and in the following chapters these approaches will be analyzed in detail.
2. Engineered nanoparticles as novel antioxidants Reactive oxygen species (ROS) are at the basis of many clinical disorders (a scheme is shown in Fig. 2). ROS are physiologically produced by living organisms by enzymatic and non-enzymatic processes. In particular, superoxide are produced by a set of oxidase enzymes (e.g. xanthine oxidase, monoamine oxidase and many others) for metabolic and signaling purposes [9,10] or as byproducts of enzymatic metabolic reactions, such as, e.g., leukotriene and prostaglandin synthesis by lipoxygenases and
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Fig. 1. Nanoparticles exploitation in biomedicine.
Fig. 2. Oxidative stress related diseases.
cyclooxygenases enzymes, respectively, or still by non enzymatic processes such as accidental leakage from the electron transport chain [9,10]. Moreover different types of ROS (e.g. nitric oxide, hydrogen peroxide) are secreted during macrophages oxidative burst processes [9,10]. Basal ROS are controlled by endogenous antioxidant defenses, including enzymes such as superoxide dismutase, catalase, the glutathione system, and nutritional antioxidant adjuvants such as vitamins and flavonoids present in food. The dynamics of ROS production and scavenging is
schematically depicted in Fig. 3A. A basal level of ROS is necessary to maintain cellular homeostasis and to perform cellular signaling tasks [9,10]. Environmental stress (e.g. ionizing or UV radiation, heavy metals, pollutants) or deranged metabolism [9,10] produce excessive amount of ROS that may overwhelm basal antioxidant defenses; these conditions are defined as oxidative stress, a state that causes damage to DNA, proteins, organelles and membranes, leading to improper cell signaling, mutations or apoptosis [9,11]. For this reason oxidative stress is associated with many
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Fig. 3. Antioxidant nanoparticles: mechanisms of actions. (A) Engineered nanoparticles possessing SOD and catalase mimetic activity (e.g. platinum, fullerenes and cerium oxide) support endogenous antioxidants defenses, ameliorating oxidative stress conditions. GSHpx: glutathione peroxidase; GSH: reduced glutathione, GSSG: oxidize glutathione. (B) Cerium oxide nanoparticles (CNPs) redox regenerative cycle: catalytic mechanism. (C) Fullerene antioxidant activity: direct scavenging of free radicals R, (left); fullerene SOD-like activity (right).
pathological conditions such as cancer, neurodegenerative disorders, autoimmune syndromes, cardiovascular diseases, diabetes, arthritis, infertility, endometriosis and many others [9,12], and with aging, a process characterized (and determined) by high oxidative conditions. A sensible strategy to overcome the deleterious effects of oxidative stress is reinforcing cellular defenses by supplementation of exogenous antioxidants. To this purpose, natural or synthetic molecular antioxidants are used as remedies during stress or pathological conditions. However, the advantages of such treatments have been deceiving due to lack of specificity, improper delivery or rapid decay of canonical molecular antioxidants. In the last decade the administration of synthetic nanoparticles with antioxidant action have been proposed as a valid alternative to molecular antioxidants. These engineered nano-antioxidants include inert nanoparticles covalently modified (functionalized) with molecular antioxidants to improve their delivery and stability, and nanoparticles that possess antioxidant activity per se, due to their physicochemical composition. The first approach has been extensively reviewed [13], therefore in this chapter we point to analyze the features, activity and administration of
nanoparticles possessing intrinsic redox activity, focusing on cerium oxide and platinum nanoparticles (PNPs) and fullerenes, which presently appear as the most promising nanomaterials for antioxidant pharmacological applications. These concepts are depicted in Fig. 3B and C. 2.1. Cerium oxide nanoparticles: a novel self-regenerating antioxidant 2.1.1. Mechanism of action and biological properties Cerium ions switch between two valence states (Ce3+ and Ce4+), which coexist in the surface of cerium oxide nanoparticles (CNPs). The charge deficiency due to Ce3+ is compensated by the formation of oxygen vacancies; these ‘‘surface defects’’ confer to CNPs intrinsic antioxidant properties [14–16]. The mechanism of action of CNPs resembles that of naturally occurring metallo enzymes, which use transition metals cofactors such as Fe, Zn, Cu or Mn to scavenge ROS in cells and tissue. It has been demonstrated that CNPs mimic superoxide dismutase, when the Ce3+ form reacts with superoxide turning into Ce4+ while reducing superoxide into hydrogen peroxide [17,18]. Likewise, oxidation of Ce3+ to Ce4+ ions allows scavenging other noxious reactive species such as hydroxyl
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radicals [19], NO [20] and peroxynitrite (ONOO ) [21]. On the other hand, Ce4+ can be reduced to Ce3+ while oxidizing hydrogen peroxide to molecular oxygen, similarly to catalase enzyme [22]. Therefore, the Ce3+/Ce4+ couple can reversibly switch back and forth while scavenging superoxide and peroxides [14– 16,23]. Considering the catalase- and the SOD-mimetic activities together, it has been proposed that CNPs undergo a complete redox cycle while scavenging two superoxide and one hydrogen peroxide [14] as depicted in Fig. 3B. This would be an elegant way to eliminate in a sequential set of energy-free reactions both superoxide and hydrogen peroxide [14], thereby freeing cells from the most noxious and abundant ROS. This CNPs selfregenerating antioxidant property is potentially an invaluable pharmacological tool, which renders this nanomaterial a unique and very attractive ROS biological scavenger. In CNPs, the Ce3+/Ce4+ ratio changes according to the synthesis methods: for example, the hexamethylenetetramine or base (sodium hydroxide or ammonium hydroxide) co-precipitation methods allow to obtain 21–30% surface concentration of Ce3+ ions, whereas higher concentrations (55–65%) can be obtained using hydrogen peroxide as a synthesis cofactor [16]. Therefore, CNPs ability to preferentially react with different free radicals may be modulated varying the method of synthesis and the Ce3+/ Ce4+ratio. CNPs with higher Ce4+ (70–80%) concentration show a better catalase mimetic action [16,22], whereas CNPs possessing higher amounts of Ce3+ ions exert a better SOD-like activity. Accordingly, the SOD-like action is completely lost if the Ce3+ surface concentration is reduced to 1 year) tumor free survival in mice carrying intra-cerebral malignant gliomas [178]. Many other in vivo studies were published, demonstrating the efficiency of GNPs as radio-sensitizers, as reviewed in [166,167,172,176]. The efficiency of GNPs in absorbing ionizing radiation producing free radicals, was estimated by theoretical models and compared with their effects in actually enhancing tumor cells killing: it clearly emerges that the pro-oxidant ability of irradiated GNPs does not fully explain the extremely strong radio-sensitizing effects actually observed in cells and animals [166,167,172,176]. This suggests that an additional (biological?) mechanism should be involved [166]. This was demonstrated by a pioneering study showing that GNPs internalized into breast cancer cells (MDA-MB-231) hardly increased X-rays induced DNA damage, but nonetheless increased apoptosis [165]. The same study reports that GNPs were able to potentiate the effect of bleomycin, a radiomimetic redox active agent that induces DNA damage via ROS production. This suggests that in the presence of DNA lesions caused by different sources (i.e., ionizing radiation and bleomycin), GNPs enhance the DNA damage response towards a more stringent, pro-apoptotic outcome, and consequently induce an additional radio sensitizing activity [146,166]. This hypothesis is supported by the evidence that GNPs have the ability to chemosensitize cancer cells to 5-fluorouracil by enhancing cell cycle arrest in the G2 phase [146,165,168]. The complex mechanisms of NPs sensitization to ionizing radiation is summarized in Fig. 6C. In addition to GNPs, other metal nanoparticles capable of increasing the radiation absorbed dose, such as silver [179,180], platinum [173], bismuth [1173] and semiconductor quantum dots (QDs) [122] have been tested in biological systems for their radiosensitizing ability. Platinum nanoparticles were shown to sensitize cancer cells to hadron therapy [181] increasing by twofold the DNA strand breaks induced by fast ions irradiation. Any possible additional biological effects downstream to DNA damage have not been considered yet. It is known that treatment with exogenous SOD enhances the arrest in the G2 phase of the cell cycle in the
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Fig. 6. Mechanisms of nanoparticles radio-sensitization (A) Nanoparticles composed by heavy atoms absorb X-rays or gamma rays emitting electrons that react with water generating ROS. (B), nanoparticles increase the radiation absorption dose inside the tumor mass reducing tumor cells viability. In the absence of NPs, tumor cells are metabolically active (deep violet) while in the presence of NPs they become apoptotic (light violet). (C) Nanoparticles radio sensitize tumor cells to ionizing radiation by increasing DNA damage via ROS production (physical mechanism) and by enhancing the DNA damage response of tumor cells (biological mechanism).
presence of DNA damage [11]. Interestingly, PNPs have a superoxide dismutase mimetic activity [74] that may possibly influence the DNA damage response. 3.2.4. The ‘‘smart’’ selectivity of cerium oxide nanoparticles CNPs are a novel and very interesting material for radiation therapy, possessing the ‘‘smart’’ ability to selective induce the death of irradiated cancer cells [40], while protecting the surrounding tissue from radiation induced damage and oxidative stress [37,39]. Therefore, CNPs have the unique feature of acting as radio-protecting as well as radio-sensitizing agents at the same time. Cerium oxide nanoparticles interact with ionizing radiation with two antithetic mechanisms that produce opposite effects. On the one hand, being cerium a high Z element (ZCe = 58), CNPs may physically produce free radicals in response to radiation, peaking in the 50–60 keV energy range (dose enhancement effect) [182]. On the other hand, the Ce3+/Ce4+ redox couple on CNPs surface is able to scavenge the ROS produced by radiation. As shown by Wason [40], the resulting effects depend on the balance between these two mechanisms, which is influenced by the material properties, that is, Ce3+/Ce4+ ratio, the size, aggregation and surface coating, and by the energy of the radiation applied. Interestingly enough, it was shown that CNPs irradiated with X-Rays at 160 keV, an energy where the physical dose enhancement effect of cerium is low, exert an additional ‘‘smart’’ biological activity that depends on the cell line tested, that is, tumor vs normal cells [40]. CNPs selectively increased oxidative stress and apoptosis in irradiated cancer cells, while
protecting normal tissues [37–40]. Wason et al. hypothesized that the selective toxicity of CNPs against cancer cells is due to the inhibition of CNPs catalase-like activity occurring in acidic (pH 4.3) environments [23]: in the presence of superoxide produced by the ionizing radiation, the SOD like activity, which is maintained even at low pH, would lead to H2O2 accumulation, incrementing radiation toxicity [40]. The hypothesis is based on the assumption that pH of cancer cells is acidic [40]. In fact, the cytosol of cancer it is slightly more alkaline (pH > 7.4) than normal cells, whereas the extracellular tumor microenvironment is slightly acidic due to the Warburg effect, and pH decreases from 7.1 (normal tissues) to 6.7 [183,184]. However, catalase inhibition requires much more severe pH derangements ( 2–3 nm with proteins present in body fluids and cells (and also in the media of tissue cultures), has posed a question on this issue. Proteins are essentially zwitterionic, and when they are absorbed on the NPs surface they strongly attenuate the charge of the NP core (zeta potential), which thus becomes close to neutrality [213]. More studies are therefore required to solve this key point of biocompatibility of NPs. Another important issue is NPs dissolution, that is, the release of molecules or atoms from their crystalline structure. A well-known example, partly mentioned above, deals with the conditional toxicity of cerium oxide nanoparticles. CNPs administration in vivo is well tolerated, and does not induce death or tissue histological alteration [32,33,58,214]. However, CNPs may release Ce ions at acidic pH (
