Clinical toxicities of nanocarrier systems

Available online at www.sciencedirect.com

Advanced Drug Delivery Reviews 60 (2008) 929 – 938 www.elsevier.com/locate/addr

Clinical toxicities of nanocarrier systems ☆ Karina R. Vega-Villa a , Jody K. Takemoto a , Jaime A. Yáñez a , Connie M. Remsberg a,1 , M. Laird Forrest b , Neal M. Davies a,⁎ a

b

Pharmacology and Toxicology Graduate Program, and Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534, USA College of Pharmacy, Department of Pharmaceutical Chemistry, The University of Kansas, Simons Labs, 2095 Constant Ave, Rm. 136B, Lawrence, Kansas 66047-3729, USA Received 17 April 2007; accepted 19 November 2007 Available online 7 February 2008

Abstract Toxicity of nanocarrier systems involves physiological, physicochemical, and molecular considerations. Nanoparticle exposures through the skin, the respiratory tract, the gastrointestinal tract and the lymphatics have been described. Nanocarrier systems may induce cytotoxicity and/or genotoxicity, whereas their antigenicity is still not well understood. Nanocarrier may alter the physicochemical properties of xenobiotics resulting in pharmaceutical changes in stability, solubility, and pharmacokinetic disposition. In particular, nanocarriers may reduce toxicity of hydrophobic cancer drugs that are solubilized. Nano regulation is still undergoing major changes to encompass environmental, health, and safety issues. The rapid commercialization of nanotechnology requires thoughtful environmental, health and safety research, meaningful, and an open discussion of broader societal impacts, and urgent toxicological oversight action. © 2008 Elsevier B.V. All rights reserved. Keywords: Nanoparticle; Toxicity; Nanomedicine; Nanocarrier; Polymeric micelles; Dendrimer; Nanosphere; Nanopharmaceutics

Contents 1.

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Targets of nanocarrier systems . . . . . . . . . . . 1.1. Physiological principles . . . . . . . . . . . 1.2. Macrophages . . . . . . . . . . . . . . . . 1.3. Endothelium . . . . . . . . . . . . . . . . 1.4. Tumors . . . . . . . . . . . . . . . . . . . Biodistribution of nanocarrier systems . . . . . . . Toxicity of nanocarrier systems . . . . . . . . . . 3.1. Important routes of exposure . . . . . . . . 3.1.1. Skin . . . . . . . . . . . . . . . . 3.1.2. Respiratory tract . . . . . . . . . . 3.1.3. Gastrointestinal tract . . . . . . . . 3.2. Toxicological effects of nanocarrier systems 3.2.1. Physicochemical determinants . . . 3.2.2. Molecular determinants . . . . . . 3.3. Genotoxicity and antigenicity . . . . . . . .

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This review is part of the Advanced Drug Delivery Reviews theme issue on “Clinical Developments in Drug Delivery Nanotechnology”. ⁎ Corresponding author. Tel.: +1 509 335 4754; fax: +1 509 335 5902. E-mail address: [email protected] (N.M. Davies). 1 Connie M. Remsberg was supported by an AFPE student fellowship on this paper.

0169-409X/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2007.11.007

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4. Environmental and regulatory issues . . . . . 5. The future of nanotechnology in drug delivery 6. Conclusions . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Targets of nanocarrier systems Nanotoxicology refers to the biokinetic evaluation of engineered nanostructures and nanodevices [1,2]. The need for this area of investigation became apparent after the intensive expansion of nanotechnology, which in the last two decades has been widely used in the pharmaceutical industry, medicine, and engineering technology [3,4]. Particle toxicology and the consequent adverse health effects of asbestos fibers and coal dust, serve as a historical reference points to the development of nanotoxicologic concepts [2,4] (Fig. 1). However, due to the great differences between products, the generalization of potential toxicological effects is extremely difficult [3,5–7]. Nanomaterials may have different chemical, optical, magnetic, and structural properties; and consequently, differential toxicity profiles [8,9]. Nanoparticles are substantially smaller than eukaryotic or prokaryotic cells; their size is comparable to that of an antibody or virus (1 nm = 10− 9 m) [1,3]. In the area of medicine, the field of nanomedicine is defined as the monitoring, repair, construction, and control of human biological systems at the molecular level, using engineered nanodevices and nanostructures [2,3,10] (Table 1). In comparison to unintentional nanosized particles that are polydispersed and chemically complex, intentionally designed nanoparticles used in nanomedicine are monodispersed, precisely engineered, and in solid form [2,11]. However, the same

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toxicological principles apply to unintentionally and intentionally engineered nanoparticles [2]. In spite of the difficulty in classifying nanoparticles, two important unifying features have been recognized [11]. Firstly, drug solubility issues are often overcome by making nanoparticles. The pharmacokinetic profiles of different nanoparticles vary widely, as the particles are designed to be rapidly soluble or conversely slowly dissolving. Secondly, nanoparticles can be targeted to specific cells or locations in the human body. These physicochemical characteristics can enhance detection sensitivity, improve therapeutic effectiveness or decrease unwanted side effects [3,11,12]. Conversely, the advantageous properties of designing nanoparticles may also lead to harmful side effects [1–3,8,10,11,13,14]. 1.1. Physiological principles Nanocarrier systems can also be referred to as nanosized materials, or nanosized particles [2]. They differ from ultrafine particles in that nanoparticles are anthropogenic and often purposely-engineered materials, whereas ultrafine particles encompass both natural and anthropogenic particles that are not produced in a controlled manner [2,11,15]. Nanocarrier systems behave differently to comparable bulk materials; their physical and chemical properties also vary due to their

Fig. 1. Complex array of issues surrounding toxicity of nanocarrier systems.

K.R. Vega-Villa et al. / Advanced Drug Delivery Reviews 60 (2008) 929–938 Table 1 Biological and medical application of some nanoparticles and carriers Nanoparticle/nanocarrier

Application

Reference

Superparamagnetic iron oxide crystals Quantum dots

In vivo and in vitro diagnostic procedures Optical coding in gene expression studies, high throughput screening, in vivo imaging Delivery of therapeutic and diagnostic agents Systemic delivery of water-insoluble xenobiotics Specific delivery of xenobiotics Specific delivery of xenobiotics Delivery of xenobiotics and antigens

[11,76]

Transfection, gene therapy, gene transfer

[11,83]

Dendrimers Polymeric micelles Liposomes Nanospheres Aquasomes (carbohydrate-ceramic nanoparticles) Polyplexes/lipolyplexes

[11,77]

[11,78]

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Macrophages are considered helpful for disease detection [10,11]. For example, when iron oxide crystals were injected intravenously, lymph nodes bearing tumors appear dark due to iron oxide crystal accumulation in macrophages, when compared to surrounding normal tissues as detected by magnetic resonance imaging [19]. This imaging diagnostic approach has been helpful in detecting very small metastases within normal sized lymph nodes in patients with prostate cancer [11,19].

[11,79] [11,80] [11,81] [11,82]

nanometer-scale metrics [3,8]. Indeed, Garnett and Kallinteri [1] proposed that nanosized materials behave differently to drugs of low molecular weight, and their biological properties rely mainly on the physiology and anatomy of the human body. Nanoparticles enter cells via endocytotic processes including clathrin-mediated endocytosis, potocytosis, pinocytosis, and patocytosis [1,8,10]. Following endocytosis, the engulfed material is delivered to the endosome and subsequently ends up in a degradative compartment, the lysosome [1]. In the lysosome, materials are exposed to hydrolytic enzymes that are active on proteins, polysaccharides, and nucleic acid components [1]. 1.2. Macrophages The macrophage is a specialized host defense cell found in the reticuloendothelial system [1,10]. Macrophages are described to have a predisposition for rapid recognition and clearance of particulate matter, and are therefore, well recognized as great nanocarrier targets [10,11,16]. In addition to macrophages, dendritic cells are also known to play an important role in the generation of an adequate immune response upon presentation of invasive materials [2,11]. For instance, macrophagal lysosomes and/or cytoplasm are essentially the intracellular home for most microorganisms. For this reason, macrophages and dendritic cell receptors that affect immunogenicity or adjuvanticity are considered useful in the design of nanocarrier systems [11]. Several examples demonstrate the use of macrophages as nanoparticle targets. Veerareddy et al. demonstrated that encapsulated microbial agents can passively target nanoparticle vehicles to infect macrophages [17]. In this study, lipid-based nanosystems presented amphotericin B (Amp-B) for treating visceral leishmaniasis or specific fungal infections [16]. In other studies, nanocarrier-mediated macrophage suicide was accomplished using endocytic delivery of macrophage toxins in order to remove unwanted macrophages for clinical conditions like autoimmune blood disorders [18] and T-cell mediated autoimmune diabetes [16].

1.3. Endothelium The endothelium consists of thin specialized epithelial cells that line the inner surface of serous cavities, lymph vessels, and blood vessels [11]. Endothelial cells control the passage of materials and have a gate keeping role. The role of the endothelium has been described in pathological processes including cancer, inflammation, oxidative stress, and thrombosis [11,15,20,21]. Unfortunately, detailed scientific evidence on how well nanomaterials can cross certain tissues currently remains limited, but some studies have begun to resolve this [1–3,11]. For instance, administration of nanoparticles via intradermal or subcutaneous injection demonstrated differential distribution depending on the presence or absence of coating [1,3]. When free nanoparticles were injected intro the extracellular matrix they remained in the site of injection, whereas poly(ethylene) glycol (PEG)-coated particles try to reach the lymphatic vessels and eventually the circulatory system [22]. Even the degree of coating can make a difference in the distance that nanoparticles can reach [3,9,11]. If nanoparticles are covered by a thin coating, they remain in lymph nodes; however, if they are surrounded by a thick coating, nanoparticles will reach the lymph nodes and continue their way through systemic circulation [2]. These circulating nanoparticles will eventually be engulfed by macrophages in the liver [2,23], which is the organ most likely affected regardless of the method of preparation [1]. Nanoparticles can also accumulate in the spleen, the gut, the bone marrow, the lymph nodes, the colon, the lungs and the brain [1–3,11,24–26]. Endothelial, vascular, and lymphatic cells have molecular markers which could be used to improve the targeting of these cells with nanoparticles [11,19]. For example, integrins αvβ3, αvβ5, and α5β1 are up-regulated in angiogenic endothelial cells and have been described to play a role in angiogenesis [19,27]. A synthetic analog of integrin αvβ3 has been used to target cationic nanoparticles carrying therapeutic genes to endothelial cells associated with tumors, working as agents to the vasculature of solid tumors [11,19,27]. Integrins are also known to bind sequences containing the Arg-Gly-Asp (RGD) motif. A complex of cyclic nonapeptide RGD-4C coupled with doxorubicin yielded a xenobiotic more effective than the cytotoxic antibiotic alone [27]. Also, cell adhesion molecules (CAM), specifically two forms: intercellular cell adhesion molecule-1 (ICAM) and platelet endothelial cell adhesion molecule-1 (PECAM-1) are targets for therapeutic drug delivery by nanoparticles [11,28]. Endocytosis of anti-ICAM-1 and anti-PECAM-1 nanoparticles successfully delivered a variety of xenobiotics to the pulmonary and cardiac endothelium in vivo.

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However, antibodies against ICAM-1 and PECAM-1 are not taken up by endothelial cells [11]. 1.4. Tumors As previously described, some examples of the application of nanocarrier systems in targeting tumors include for example iron oxide crystals [19] and integrin analogs [27]. Iron oxide crystals applied to magnetic resonance imaging have been helpful in detecting very small metastases in patients with prostate cancer via macrophages [19]. In the same way, integrin αvβ3 analogs coupled to nanocarrier systems have been used for gene therapy in tumors based on the role of integrins in endothelial cells [27]. Diffusion of macromolecules and nanoparticles in tumors varies widely with tumor type, anatomical location, and extracellular matrix composition [11]. Perfusion heterogeneity, for example, refers to the abnormalities of blood and lymphatic vessels at the structural or functional level that impede efficient delivery [11]. This might be overcome by decompressing the vessels to improve therapy efficiency, but this theoretically can increase metastasis as well [11]. Nanoparticles can leave the circulation through a variety of routes such as paracellular and transcellular egress. Paracellular egress use tight junctions, with diameters b 2 nm [3]. Tight junctions are normally too small to allow nanoparticles to slip between cells; however, under certain conditions, like in some cancers [3] and during inflammation [8], the endothelium becomes more porous, making the barrier leaky allowing the passage of nanoparticles. Transcellular egress involves receptor-mediated endocytosis or pinocytosis, a non-specific endocytic process [1–3,8]. In this case, specific coatings or chemical modifications on the surface of nanoparticles allow their passage through the endothelium to target specific cells [9,29]. Ideal carriers must have high drug loading capacity and low drug loss [11]. For example, long circulating liposomes carrying doxorubicin have been approved for treatment of cancers due to their favorable pharmacokinetic disposition profile [3,11]. Doxorubicin-containing liposomes, known as Doxil®, present an area under the curve (AUC) ∼ 300-fold greater than doxorubicin alone, a clearance (CL) value ∼ 250-fold greater than free doxorubicin, and a volume of distribution (Vd) ∼60fold greater than free doxorubicin [11]. The mechanism of action of Doxil® is still unclear but it is known that a nonspecific chemical disruption or collapse of the liposomal pH gradient triggers the release of liposomal content [30]. Liposomeencapsulated doxorubicin and doxorubicin loaded bacterial magnetosomes have also been described to have fewer side effects than free doxorubicin [30].

al to be more efficiently engulfed by macrophages — occur under certain conditions for nanoparticles depending on size and surface characteristics [3,11]. This means that differential opsonization translates into differences in clearance rates and macrophage sequestration of nanoparticles [11]. Therefore, the suppression of opsonization events is necessary to enhance passive retention of nanocarrier systems at desired sites or anatomical compartments. For example, hydrophobic particles in the body are coated by serum and then opsonized, which leads to clearance by specialized cells in the reticuloendothelial system such as the spleen, the liver, and the lymphatic system [1]. If hydrophobic particles were coated with poly(ethylene) glycol (PEG), hydrophilicity would increase, hence greater time spent in the systemic circulation [1]. Coated materials are expected to remain in the vasculature until they are gradually eliminated by scavenger mechanisms [1]. When inhaled, nanoparticles are found to be distributed to the lungs, liver, heart, kidney, spleen and brain [15,21,25,33]. Nanoparticles are cleared in the alveolar region via phagocytosis by macrophages facilitated by chemotactic attraction of alveolar macrophages to the deposition site [1–3]. The average half-life (t1/2) for nanoparticles in the respiratory tract is ∼700 days in humans [2]. Nanoparticle clearance from the lungs involves a combination of physical and chemical processes [1–3,8,10]. Physical clearance processes in addition to macrophage phagocytosis include mucociliary movement, epithelial endocytosis, interstitial translocation, lymphatic drainage, blood circulation translocation, and sensory neuron translocation [2]. Chemical clearance processes include dissolution, leaching, and protein binding [2]. Some clearance processes show particle size-dependent differences, and nano-selective effectiveness [2,7,31]. After intraperitoneal injection, nanoparticles have been found to cross the trans-placental membrane or across the peritoneal cavity into the uterus. This affected the embryos cranial development and even caused embryo death. Further research in the effects of nanoparticles in reproductive areas is necessary. After oral exposure, nanoparticles distribute to the kidneys, liver, spleen, lungs, brain and the gastrointestinal tract [25]. Few studies have looked at clearance of nanoparticles from the gastrointestinal (GI) tract [8]. Some nanoparticles can pass through the GI tract and are rapidly eliminated in feces and in urine, indicating that they can be absorbed across the GI barrier and into the systemic circulation [2,3]. However, some nanoparticle systems can accumulate in the liver during first-pass metabolism [2]. 3. Toxicity of nanocarrier systems

2. Biodistribution of nanocarrier systems Nanoparticles have been found to be distributed to the colon, lungs, bone marrow, liver, spleen, and the lymphatics after intravenous injection [25,31,32]. Distribution is followed by rapid clearance from the systemic circulation, predominantly by action of the liver and splenic macrophages [11]. Clearance and opsonization — the process that prepares foreign materi-

Natural nanosized particles serve as models for the description of a possible toxicological profile for nanocarrier systems [2,3,8,10,11]. It is worth noting that toxicological research has been mainly conducted using occupational and environmental data that involves natural nanomaterials. This can be applied to the study of man-made nanoparticles since the same toxicological principles apply to both types of nanosized particles [3,8].

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Indeed, certain workplace conditions can generate nanosized particles that reach higher concentrations than typically found in the environment [2,3,34]. To our knowledge, there has not been extensive research conducted to analyze the toxicity of nanocarriers per se [1–3,8,10,11,14,34,35]. However, Curtis et al. [3] hypothesized possible mechanisms of toxicity for nanoparticles: 1) Toxicology of bulk materials is relatively well defined as in the case of heavy metals as this toxicity is quite facile to quantify [3]; 2) The electrical properties of nanoparticles differ from bulk material. Nanomaterials can create and/or scavenge reactive oxygen species (ROS) and free radicals [2,3,8,15,20,21]; 3) Toxicity of nanoparticles may be linked to their size as suggested by studies of ultrafine particles in the respiratory tract [3]. The toxicological effects of ultrafine particles are determined by their size and their propensity to agglomerate. They are also known to pass biological barriers, like skin, vascular endothelium and the blood brain barrier; therefore, affecting absorption, distribution, and excretion of these particles [1–3,8,11,25]; 4) Shape may also be a factor that determines toxicity, as is the case of carbon nanotubes (CNTs) [2,3,8,11,29,36,37]; 5) It has not been clearly delineated how nanoparticles can trigger immune responses; however, there is a growing concern about their role in possible allergic reactions [3,8,11]. 3.1. Important routes of exposure Mammalian systems have been used as the main in vivo model to test toxicity of nanocarrier systems; in particular, several studies describe the exposure of the respiratory system to airborne ultrafine particles in order to test the hypothesis that they cause significant health effects [1,3,8,10,11,15,20,21,23,25,29,37–40]. Other exposure routes, such as skin and GI tract, have not been considered as extensively as the respiratory tract as portals of entry for nanocarrier systems [2,8,10,21,25,30,40]. 3.1.1. Skin It has been hypothesized that dermal exposure might be the most significant route of exposure [3]; however, few literature reports are available that refer to the absorption and effects of nanoparticles in the skin [2,3,8,41]. Contact with nanoparticles through the skin can occur due to occupational exposure during the manufacturing of solvents, pesticides, or pharmaceuticals [2]. Skin exposure to nanoparticles can also occur during nonoccupational situations from the use of cosmetics and in the intentional application of topical creams and other drug treatments [2,3,25]. Initial studies of nanoparticle absorption through the skin are inconclusive; some demonstrate little penetration into the epidermis while others using more complex flexing protocols show deep absorption [25,42,43]. Due to their unique physicochemical properties, nanoparticles are rendered more biologically active than structures of the same chemical make-up, which is apparent by the inflammatory, oxidant, and anti-oxidant capacities described for nanocarrier systems [2]. Evidence of mitochondrial distribution and oxidative stress also exists for nanocarrier systems [8]. Broken skin represents a readily available portal of entry even for large (0.5–7.0 µm) micron size particles [2]. Even intact

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skin, when flexed, makes epidermis permeable to nanoparticles [2,3,8,25,44]. A recent study demonstrated that fluorospheres (0.5–1 µm) can penetrate the epidermis and reach the epidermis employing a skin flexing protocol that is likely to be representative of physiological conditions [42]. Once in the epidermis, nanoparticles reach the lymphatic system and regional lymph, and from there they can translocate to the systemic vasculature [2]. Nanoparticles can also reach sensory skin nerves; as reported after the injection of nanoparticles in the tongue and facial muscles of mice [2]. Cationized nanoparticles can reach cell bodies of facial neurons, highlighting the importance of electric charge on nanoparticle incorporation and disposition into axons [2,45]. To better understand dermal absorption of nanoparticles more research on regular skin, dry skin, and damaged skin is necessary [25,46]. 3.1.2. Respiratory tract Exposure to nanosized materials has increased since new anthropogenic sources developed around three decades ago [11]. Inhalation nanoparticles are deposited in all regions of the respiratory tract, however, larger particles may be filtered out in the upper airways, whereas smaller particles reach distal airways [3,25]. The respiratory tract can be divided into three regions: nasopharyngeal, tracheobronchial, and alveolar regions [11]. Significant amounts of certain particle size ranges can deposit in each region, for example, 90% of nanoparticles of 1 nm in diameter deposit in the nasopharyngeal region, whereas only 10% of these nanoparticles deposit in the tracheobronchial region and almost none reach the alveolar region [11]. In comparison, 15% of nanoparticles of 20 nm in diameter deposit in the nasopharyngeal region, 15% in the tracheobronchial region, and approximately 50% in the alveolar region [11]. After absorption across the lung epithelium nanoparticles can enter the blood and lymph to reach cells in the bone marrow, lymph nodes, spleen, and heart [2,25]. Nanoparticles can even reach the central nervous system and ganglia following translocation [2,21,25,47]. Epidemiologic analysis and controlled clinical trial studies in humans have been used to describe the toxicology of airborne natural nanosized materials [2]. These nanosized materials often have cardiovascular and respiratory effects that result in significant morbidity and mortality in susceptible segments of the population [2]. Subjects with asthma or chronic obstructive pulmonary disease show greater deposition of natural nanosized materials in the respiratory tract than healthy individuals [2]. The presence of natural nanosized particles is associated with the formation of blood markers of coagulation, has effects on the systemic inflammation and pulmonary diffusion capacity, and increases the risk of ventricular disrhythmias [2,16]. A correlation between the size of particles and general health effects has been proposed to exist [3]. Ultrafine particles are described to be more toxic than larger particles with the same chemical make-up due to their large surface area, causing cytotoxicity, allergic response or inflammation [2,8,29,37]. Further studies are needed to investigate the toxic effects and fate of nanoparticles after their deposition in the respiratory tract [1,3,8,23,36,37,39,48].

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Translocation, the transport of dissolved materials within the body, has been proposed as a mechanism for nanosized particles to reach extrapulmonary sites and then other target tissues [2,39]. Nanoparticles can access the systemic vasculature directly or via lymphatic transfer by transcytosis, crossing the epithelia of the respiratory tract into the interstitium, phagocytosis, endocytosis or some other transmembrane process [2,25,39,49]. A second target after translocation is suggested to be the sensory nerve endings embedded in the airway epithelia, followed by translocation to ganglia and the central nervous system via axons [2,45]. In addition to epidemiological and controlled clinical studies, the effects of nanoparticles in the respiratory tract have been studied through inhalation and instillation studies in rodents and in vitro cell culture systems [2]. In rodents, ultrafine particles cause mild pulmonary inflammatory responses and have effects on extrapulmonary organs [2,3,8]. Dosing with both natural and anthropogenic nanosized particles, in in vitro studies showed pro-inflammatory and oxidative stress related cellular response [2,3,8]. Interpretation of in vitro studies needs to be prudently evaluated to consider differential chemical disposition, different cell types and targets, and the use of high doses and consequent levels [2,35]. In other words, cautious interpretation needs to be undertaken with concentrations that are orders of magnitude higher than those predicted from relevant ambient exposures [2]. Shape and structure of nanoparticles may also predispose them to inhalational toxicity [1,2,8]. For example, carbon nanotubes (CNTs) have distinct pulmonary effects as compared to carbon black and graphite, which are larger structures of similar chemical make-up [50]. CNTs are arranged helically into cylindrical structures, and they form single-walled (SWCNTs), or multi-walled (MWCNTs) carbon nanotubes [2,51]. The usual shape is elongated and 0.4 nm in diameter to hundreds of nm in length [50,51]. When toxicity of CNTs was compared to that of carbon black after intratracheal instillation in mice, CNTs proved to be significantly more harmful [2]. Carbon black was ingested by macrophages in the alveolar region and resided predominantly at this site. In comparison, macrophages that ingested CNTs migrated to centrilobular locations and caused interstitial granulomas [2,3]. Pharyngeal aspiration of SWCNTs caused increased inflammation and cell damage. Two patterns of lung remodeling were present depending on whether SWCNTs aggregate (granuloma) or distribute (interstitial fibrosis) through the lung space [2,8]. Either pro-inflammatory (TNF-α, IL-1β) or anti-inflammatory profibrogenic cytokines (TGF-β, IL-10) are expressed in tissue affected by nanoparticles [10,20,21,26]. When SWCNTs and MWCNTs were compared to C60 fullerenes they showed greater cytotoxicity to alveolar macrophages (SWCNT NN MWCNT NN C60) [10]. C60 fullerenes are allotropic forms of carbon that are arranged in clusters and are also used as nanocarrier systems [2,3,10]. 3.1.3. Gastrointestinal tract Nanoparticles can reach the GI tract after mucociliary clearance from the respiratory tract through the nasal region, or can be ingested directly in food, water, cosmetics, drugs, and drug delivery devices [2,21,25,52]. The utility of biodegradable nano-

particles in the delivery of oral vaccines has been proposed for antigens known to be susceptible to proteolysis [53]. Few studies have looked at toxicity of nanoparticles following oral ingestion [3]. Acute toxicity of copper particles and nanocopper was measured in mice; LD50 for nanocopper is 413 mg/kg compared to N 5000 mg/kg for copper [3]. Nanocopper was also reported to cause pathological damage to the liver, the kidney, and the spleen. Muller and Keck [54] noted that recrystallisation is possible in a supersaturated nanosuspension which can occur during dissolution in the GI tract. New studies that can overcome recrystallisation issues will be helpful to accurately assess toxicity of nanoparticles in the GI tract. Further studies on gastrointestinal lymphatic uptake and transport, and direct toxicological effects on the GI tract are required. 3.2. Toxicological effects of nanocarrier systems 3.2.1. Physicochemical determinants Due to their size, nanoparticles have a large specific surface area [8,11,29,37]. This may translate into increased biological activity, due to different contact interactions with cells and its components, and variable biokinetics [8]. Physicochemical properties of nanoparticles vary widely from the properties of bulk materials [1–3,8]. The stability of nanoparticles requires further detailed investigation; however, the possibility of Oswalt ripening and agglomeration exists [13,55,56]. Few studies have looked specifically at the stability of nanoparticles, for example, amphiphilic β-cyclodextrin nanosphere suspensions with and without poloxamer, a stabilizing agent, demonstrated good physical stability after 3 years of storage at room temperature due to small size and structural organization of the nanoparticles [55]. Additionally, Liu et al. reported for the first time the effect of Ostwald ripening in nanoparticle formulations [56]. The addition of antisolvent results in the reduction of the ripening rate for β-carotene nanoparticle dispersions by dramatically decreasing bulk solubility. It has also been reported that using dispersant layers can increase the stability of nanoparticles in aqueous solutions and avoid agglomeration [9]. Using a pyrogallol-PEG architecture to adsorb on the surface of alumina nanoparticles can also counterbalance van der Waals forces that are responsible for agglomeration [9]. 3.2.2. Molecular determinants Nanoparticles favor the formation of pro-oxidants, especially under exposure to light, ultraviolet (UV) light, or transition metals; thereby, destabilizing the balance between the production of reactive oxygen species (ROS) and the biological system's ability to detoxify or repair the system [3,57]. Nanoparticles can modify mitochondrial function, as well as cellular redox signaling [3,57]. ROS can also be produced by the NADPH oxidase in phagocytic cells or as a product of P450 cytochrome metabolism [3,57]. Oxidative stress induced by nanoparticles is reported to enhance inflammation through upregulation of redox-sensitive transcription factors including nuclear factor kappa B (NFκB), activating protein 1 (AP-1), extracellular signal regulated kinases (ERK) c-Jun, N-terminal kinases, JNK, and p38 mitogen-activated protein kinases pathways [3,57].

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3.3. Genotoxicity and antigenicity Gene therapy investigations have recently discovered and incorporated the advantages of using nanocarrier systems to deliver genes more efficiently [3,57,58]. Gene transfer is based on the destabilization of internalized vesicles via the surface charge effect [3,57,58]. For example, DNA, antisense oligonucleotides, and small interfering RNAs are condensed into nanostructures that facilitate internalization into the cell via endocytosis [3,57]. Poly(ethylenimine) induces membrane rupture by attracting protons, and facilitates the release of polycationnucleic complexes into the cytoplasm [3,57,59,60]. Differential gene expression after delivery of micelles-carrying cisplatin has been reported in certain cells which resulted in induced cell death via apoptosis or necrosis [3]. Cationic formulations have been described to affect cell proliferation, differentiation, and pro-apoptotic genes in human epithelial cells [3,60,61]. For example, synthetic polycationic non-viral gene transfer systems are proposed to improve the poor success of human gene therapy that utilizes viruses to transport the therapeutic genes [3,60]. The polycationic nature of these delivery systems induces cytotoxicity by necrosis and apoptosis [3,60]. Necrosis occurs when cationic components of the carrier and cell surface proteoglycans or proteins in the cytoskeleton of the target cell interact, disestablishing the membrane and causing the formation of pores [3,11,60]. In comparison, apoptosis occurs in Jurkat cells via cytochrome c release from the mitochondria of target cells due to Bcl-2-sensitivity [3,11,60]. It is known that the use of different cationic materials initiates apoptosis at variable chronology through a variety of different pathways [3,57,60]. It is suggested that carriers may exacerbate, soothe, or mask the effects of delivered nucleic acids [3,11]. In spite of the current application of nanomaterials in gene therapy and gene delivery in pre-clinical research, few studies have focused on the toxigenomic responses [57]. Therefore, assessment of the toxicity of nanomaterials used in these areas of research is fundamental to maximize future clinical outcomes. As stated before, ultrafine particles are described to be more toxic than larger particles with the same chemical make-up, causing cytotoxicity, an allergic responses or inflammation [2,8]. Further studies of the antigenicity of nanoparticles need to be extended to determine when nanoparticles are recognized by the immune system, and whether or not they cause specific immune responses with antigen formation [3,8]. PEG-grafted liposome infusion was described to trigger non-IgE-mediated signs of hypersensitivity [11]. In comparison, peptide-functionalized CNTs form immunogenic complexes, enhancing the antibody response [3]. Based on this research and the growing body of scientific evidence of nanoparticles, possible vaccine creation has been proposed [3]. 4. Environmental and regulatory issues The lack of toxicology data on nanocarrier systems hinders governmental regulation [2,3,11,34,62–64]. Currently, no regulatory requirement to test nanoparticles for health, safety, and environmental impacts has been formalized [65]. The Food and

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Drug Administration (FDA) was the main governmental entity entitled to regulate nanocarrier systems through the Center for Drug Evaluation and Research (CDER), the Center for Biologics Evaluation and Research (CBER), and the Center for Devices and Radiological Health (CDRH) since their appearance in the late 1990s [62]. The Environmental Protection Agency (EPA), the Department of Labor through the Occupational Safety and Health Administration (OSHA), the National Institute for Occupational Safety and Health (NIOSH), and the FDA have also participated since 2000 in the National Nanotechnology Initiative (NNI) approved by President Bill Clinton who nearly doubled governmental funding for nanotechnology [3,8,63,64]. The NNI is a federal research and development programme established in 1996 to coordinate governmental multiagency efforts in nanoscale science, engineering, and technology [3,8,65]. From 2000 to 2004, the NNI coordinated 3.2 billion dollars in grant money among the National Science Foundation (NSF), the Department of Defense (DOD), the Department of Energy (DOE), the National Institute of Health (NIH), the National Aeronautics and Space Administration (NASA), the National Institute of Standards and Technology (NIST), OSHA, FDA, EPA, and NIOSH for nanotechnology research [3,8,65]. The NNI budget request for 2008 is of 1.5 billion dollars, almost triple the estimated 464 million dollars spent in 2001 [3,8,65]. A substantial portion of the funding will go to the American Competitiveness Initiative (ACI), of which NSF, DOE, and NIST are all part of [3,8,65]. Currently, 26 federal agencies participate in the NNI, 13 of which have budgets for nanotechnology research and development coordinated through the Nanoscale Science, Engineering, and Technology (NSET) subcommittee of the National Science and Technology Council (NSTC) [65]. In addition to the departments and agencies previously listed, the Consumer Product Safety Commission (CPSC), the Department of Agriculture (USDA) through the Cooperative State, Research, Education, and Extension Service (CSREES), the Department of Commerce's Bureau of Industry and Security Technology Administration (BISTA), the Department of Education, the Department of Homeland Security (DHS), the Department of Justice (DOJ), the Department of Labor (DOL), the Department of State (DOS), the Department of Transportation (DOT), the Department of Treasury, the Intelligence Community, the International Trade Commission (ITC), the Nuclear Regulatory Commission (NRC), and the Patent and Trademark Office (PTO) participate in the NNI [3,8,65]. In 2006, the NNI invested about 4% (42 million dollars) of the total budget (1054 million dollars) in research and development (R&D) that addresses the potential risks caused by nanotechnology to environment, health, and safety (EHS) [65] (Fig. 2). Three main areas of research focus in understanding the effects of nanotechnology in the environment, health, and safety [65]. They are 1) basic research to understand the behavior of nanomaterials in the environment and the human body, 2) research to develop instruments and methods to measure, characterize, and test nanomaterials and to monitor exposure, and 3) research to assess safety of technology that use nanoparticles [3]. But governmental funding is not the only source for

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Fig. 2. Projection of the environment, health, and safety (EHS) research and development (R&D) funding from the National Nanotechnology Initiative (NNI) for the year 2008. Abbreviations — National Science Foundation (NSF), Department of Defense (DOD), Department of Energy (DOE), National Institute of Health (NIH), National Institute of Standards and Technology (NIST), National Aeronautics and Space Administration (NASA), Environmental Protection Agency (EPA), Cooperative State, Research, Education, and Extension Service (CSREES), National Institute for Occupational Safety and Health (NIOSH), Department of Agriculture Forest Service(USDA/FS), Department of Education, the Department of Homeland Security (DHS), Department of Justice (DOJ), Department of Transportation (DOT).

nanotechnology [3,8,65]. The private sector has taken advantage of nanotechnology due to the tremendous economic benefits [34,66]. Nanotechnology is expected to become a trilliondollar industry by 2015 [3,41]. On the other hand, the Action Group on Erosion, Technology, and Concentration (ETC) calls for a moratorium on the use and manufacture of nanotechnology that uses self-assembly and selfreplication due to its biological, environmental, and social implications [34,66]. ETC is an international organization that “researches and organizes for democratic and transparent technology assessment, greater cultural and biological diversity, and strengthened human and Farmer's Rights in the framework of Food Sovereignty” [34]. In addition to the assessment of toxicity of nanoparticles per se the ETC proposes the evaluation of social implications of all nanotechnologies and a partial moratorium [34,66]. Other groups, like the Natural Resources Defense Council, the American Federation of Labor and Congress of Industrial Organizations, Beyond Pesticides, the Center for Environmental Health, the Center for Food Safety, Corporate Watch, Edmonds Institute, the Institute for Agriculture and Trade Policy, the International Center for Technology Assessment, the Project on Emerging Nanotechnologies, and the Environment Defense criticize industry influence in the decisionmaking process and ensure that as nanotechnologies advance, possible risks are minimized, public and consumer engagement remains strong, and the potential benefits of these new technologies are realized [66]. Academia, industry and regulatory governmental agencies need to consider the unique biological properties of nanoparticles, and the related potential risks that may differ from bulk

material of the same chemistry [3,8,35]. Multidisciplinary studies are encouraged to establish a nanoparticle classification design and testing procedures, including toxicology, material science, medicine, molecular biology, and bioinformatics [3,8]. On this matter, multinational collaboration is critical due to the impact that the manufacture of nanoparticles has or will have at the local, national, and international level [3,8]. The American National Standards Institute (ANSI), the International Council on Nanotechnology (ICON), and the International Organization for Standardization (Geneva, Switzerland) made the first move towards multinational collaboration [3,8]. On these matters it is also important to consider arguments such as those presented by the ETC to evaluate not only the environmental and occupational issues, but to also include the social implications of the development of nanotechnology [34,66]. Some opponents to nanotechnology have compared the promises made by the development of genetically modified organisms (GMOs) to those made by nanotechnology, hence propose a careful approach to the development of the latter [2]. 5. The future of nanotechnology in drug delivery Toxicity studies are critical to establish the full in vivo potential of nanotechnology and nanomedicine in particular [1,2,8,10,11,34,35,63–65]. Understanding the physicochemical, molecular, and physiological processes of nanoparticles is imperative for nanomedicine to become a reliable and sustainable treatment modality [1,2,8,10,11,34,65]. Further studies are needed to determine the biodistribution of nanoparticles after skin and GI tract exposure. Many pre-clinical studies have demonstrated a reduced toxicity profile when incorporating and delivery of drugs such as immunosupressants (i.e. rapamycin and cyclosporine) as well as a variety of anti-cancer drugs (i.e. paclitaxel, geldanamycin) into nanocarrier systems in rodent studies [12,67–73]. In spite of the scientific knowledge gained in recent years in nanotoxicology, scientists still are not able to precisely anticipate the behavior and biokinetics of nanoparticles. Medical, academic, and regulatory communities both at the national and the international level need to determine the potential threats in the workplace for manufacturers and the environment [1–3,8,10,11,21,34,65]. The need for separate registration of nanoparticles from conventional substances is grounded in the evidence of changes in physicochemical properties that may modify their inherent toxicity profile [1– 3,8,11,13,35,62,63,65,74]. Nanotechnology is growing at an exponential rate and will undoubtedly have both beneficial and toxicological impact and consequences on health and the environment. However, the rapid commercialization of nanotechnology requires focused environmental, health, and safety research, meaningful and open discussion of broader societal impacts, and urgent oversight [34,66,75]. 6. Conclusions A wide-array of complex issues involving physiological, physicochemical, and molecular processes needs to be considered

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