Safety and toxicity concerns of orally delivered nanoparticles as drug carriers.

Review

Safety and toxicity concerns of orally delivered nanoparticles as drug carriers 1.

Introduction

2.

Oral route: advantages and disadvantages

3.

Different nanoparticulate

Francisca Ara ujo, Neha Shrestha, Pedro L Granja, Jouni Hirvonen, Helder A Santos & Bruno Sarmento† †

Universidade do Porto, INEB -- Instituto de Engenharia Biom e dica, Biocarrier Group, Porto, Portugal

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Otago on 12/25/14 For personal use only.

systems 4.

Conclusion

5.

Expert opinion

Introduction: The popularity of nanotechnology is increasing and revolutionizing extensively the drug delivery field. Nanoparticles, as carriers for oral delivery of drugs, have been claimed as the perfect candidates to overcome the poor bioavailability of most of the drugs by improving their solubility and/or permeability across biological barriers. However, this is still a promise to be fulfilled. Areas covered: In this review, several nanosystems used as oral drug carriers are described along with their toxicological profiles. A number of nanoparticles based on different types of materials such as polymers, lipids, silica, silicon, carbon and metals are reviewed. Both in vitro and in vivo-based toxicological studies are discussed in this paper. Expert opinion: Toxicological concerns have been raised in the past few years regarding the safety of the developed nanosystems. Assuming that most of the materials used are biocompatible and biodegradable, the toxicity caused by them when formulated into nanoparticles is usually neglected by the scientific community, existing only a few number of studies that approach the toxicity of the nanosystems. This is particularly important, because the materials that composed of the nanoparticles as well as their features such as size, charge and surface properties, will influence their pharmacokinetics after oral administration. Keywords: carbon, gold, lipids, mesoporous silicon, nanoparticles, oral, polymeric, silica, toxicity Expert Opin. Drug Metab. Toxicol. [Early Online]

1.

Introduction

In the past decades, the use of nanoparticles for oral administration of drugs has been increasing tremendously. However, some concerns are being raised regarding their safety and potential toxicity, which is not only about the nanoparticles themselves but also their components, when administered to humans [1,2]. It is certain that the nanoparticles are able to enhance the drugs’ therapeutic effect but, on the other hand, they can also increase the toxicity associated with the nanosystems. Some examples of nanoparticles used in oral drug delivery are shown in Figure 1 [3,4]. This review aims to discuss about the safety and toxicity profiles of different orally delivered nanoparticulate systems such as polymeric, lipid, silicon, silica, metallic and carbon nanoparticles. 2.

Oral route: advantages and disadvantages

The oral route is the most widely accepted and preferred administration route for pharmaceuticals. It is mainly due to the possibility to self-medication with oral 10.1517/17425255.2015.992781 © 2014 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

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Owing to the advantages such as enhancement of the solubility and permeability of the drug, nanoparticles have been used vastly for oral drug delivery. There have been several toxicological concerns regarding the safety of the nanoparticulate systems. Different characteristics of the nanoparticles can have an influence in the toxicity such as its components, size, surface charge and surface chemistries. Very limited numbers of toxicological studies of orally delivered nanoparticles are available. The available studies give minimal information regarding the safety of such systems, indicating the need of increasing the focus and awareness regarding the importance of this area of study.

This box summarizes key points contained in the article.

dosage form which makes it highly convenient and patient friendly. Moreover, compared to the parenteral route of administration, the cost-effectiveness and the noninvasive nature of the oral route make it very convenient for chronic therapy and significantly increase patient compliance [5,6]. Regardless of the various advantages, the oral route can have several limitations for both small molecules and large macromolecules such as instability and poor solubility in the gastrointestinal tract (GIT), mucosal barrier, poor intestinal permeability, intestinal transit and interactions with the gastric contents/secretion [7,8]. However, these barriers can be overcome by designing nanoparticles to enhance the oral bioavailability and also by taking the advantage of the unique characteristics of the GIT [6], as discussed next. GIT and its interaction with nanoparticles The oral route takes advantage of the highly absorptive intestinal epithelium, which has a total absorptive surface in the GIT of 300 -- 400 m2 [9]. The intestinal epithelium is composed of microvilli which are composed of absorptive enterocytes, mucus producing goblet cells and lymphoid regions, known as Peyer’s patches, covered with M cells for antigen sampling that has high transcystolic activity [6,10]. Taking into consideration the physiology of the GIT, it is expected that the uptake of the nanoparticles occurs in the small intestine. However, the nanoparticles have to overcome different biological barriers in the small intestine before they can be translocated through the intestinal epithelium such as the harsh conditions in stomach, mucus layer and tight junctions (TJs) between the intestinal epithelial cells. The next subsections will discuss the different biological properties that affect the translocation of nanoparticles across biological barriers and how the translocation is influenced by the various properties of the nanoparticles. 2.1

Mucus layer Mucus layer is present on the surface of the intestinal epithelium to protect the exposed cells by lubricating it along with 2.1.1

2

removing the pathogens adherent/adherence to it. Owing to the nature of the mucus layer and its continuous secretion and turnover, it acts as a major barrier for penetration of nanoparticles across the intestinal tract [8,11]. The mucus layer is composed of a firmly adhered mucus gel layer attached to the intestinal cells and an additional loosely adhered mucus layer. The thickness of the two layers varies along the GIT, with the thinnest firmly adherent mucus layer present in the small intestine region, thereby making it the major site for absorption and translocation [8]. Modified nanoparticles that have mucoadhesive (targeted and nontargeted) [12], mucuspenetration [13] and/or mucolytic [14] properties have been developed and proposed to overcome this barrier. The use of mucolytic nanoparticles disrupts the natural mucus barriers, exposing the intestinal surface. This effect enhances the uptake of nanoparticles and also enhances the bacterial attachment and translocation, which can lead to infections [15]. Moreover, the absence of mucus layer also exposes the cell surface to the harsh conditions of the intestinal tract leading to their further damage. Another important parameter is the size of the nanoparticles. For example, Lehr et al. were able to show a size-dependent mucoadhesion, with significantly improved mucoadhesion with nanosized particles [16]. Route of nanoparticle transport The intestinal epithelial cells are connected to each other by TJs that form a physical barrier and act as a gatekeeper, selecting what can/cannot pass through it in order to reach the bloodstream [17,18]. This control is mainly dependent on the characteristics of the mucosal layer and on the nanoparticles’ physicochemical properties such as size, charge and lipophilicity [7,19]. The nanoparticles can cross the epithelium either by transcellular or paracellular pathway as shown in Figure 2. In the transcellular pathway, nanoparticles pass through the cells while in the paracellular pathway this passage is made through the TJs between the cells [7,19,20]. Within the transcellular mechanism, the uptake process can occur through several endocytic mechanisms where the nanoparticles are taken up at the apical cell membrane, transported through the cells within vesicles and released at the basolateral side of the epithelial cells [21]. Phagocytosis is a receptor-mediated process, ATP-dependent, where the cellular membrane protrudes to engulf the nanoparticles. Among the intestinal epithelial cells, this mechanism is restricted to M cells, which have features such as lack of mucus layer and the presence of a scant glycocalyx that favor the nanoparticles transport. Hence, the transport through the M cells is higher than the transport through the enterocytes and the cut-off point for this process has been estimated to be from 20 to 500 nm [19,21,22]. When these cellular membranes projections engulf the fluid-containing nanoparticles without any receptor-mediated mechanism and in any cell type, the process is known as micropinocytosis [23]. In contrast to these mechanisms, other ATP-dependent mechanisms exist, but instead of the cellular membrane projections, the cellular membrane forms pits forming small vesicles. In a 2.1.2

Expert Opin. Drug Metab. Toxicol. (2014) 11(3)


Safety and toxicity concerns of orally delivered nanoparticles as drug carriers

A.

B.

C.

D.

E.

F.

Figure 1. Schematic representation of the nanoparticles used in drug delivery applications. (A) Polymeric nanoparticles, (B) solid lipid nanoparticles, (C) carbon nanotubes, (D) carbon nanohorns, (E) porous silicon nanoparticles and (F) gold nanoparticles. Reproduced from [3,4] with permission from Elsevier. 2008, 2014.

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Figure 2. Schematic representation of the transport of nanoparticles across the intestinal epithelial cells. Particles can be transcytosed by normal enterocytes (1) as well as by M cells (4). They can reach the basal pole by passive diffusion (2) or by paracellular transport (3). Reprinted with permission from [20] with permission from Elsevier. 2006.

very simplistic way, it is possible to say that when it is a receptor-mediated uptake the mechanism is called clathrinmediated endocytosis, while when it is not receptor-mediated the mechanism is called caveole-mediated endocytosis [24]. Regarding the paracellular pathway, due to the very limited space between the cells (3 to 10 A˚ of diameter) and the small surface area of < 1% of the total intestine, it is commonly accepted that this pathway is not the main mechanism for the nanoparticles’ transport [6,19,25]. However, the use of some absorption enhancers and/or cell-penetrating mediators can increase the paracellular space and facilitate the transport through this mechanism [6]. Effect of the nanoparticle characteristics on translocation

2.1.3

The translocation of nanoparticles across the intestinal epithelium is not only governed by the biological obstructions, but also by the properties of the nanoparticles, such as size,

composition, shape, surface charge and presence of targeting ligands, which can also have comparable influence [26]. In the case of particle size, as the particle size decreases to submicron level, the uptake of the particles increases. Particles with hydrodynamic diameter of 3 µm are taken up by Peyer’s patches but they stay there. Also, the surface charge can influence the uptake/translocation of particles; however, the influences of the shape of the particles are not well known. Another important property is the presence of targeting ligands, which can ensure cellular uptake, but translocation of the nanoparticles is not guaranteed. The physical and chemical stability of the nanoparticulate systems are also important and they govern in what state the nanoparticles will be presented in the intestinal lumen. For example, poor physical stability can lead to aggregation of particles and chemical instability in intestinal tract can lead to loss of ligand or other surface modification [26].

Toxicological concerns with nanoparticles The oral toxicity of the nanoparticles can occur both locally, when in contact with the intestinal cells or systemically, after they are translocated to the blood stream. The local toxicity can be caused by direct interaction of the nanoparticles with the intestinal cells, which can be influenced by different properties of the nanoparticles such as size and charge. In the case of systemic toxicity, all the characteristics of the nanoparticles that influence both their translocation and interaction with different tissues must be considered, as discussed above. In this section, the features of the nanoparticles that cause toxic effects by interacting with different organs at the cellular level are described. The nanosize of the particles is one of the main characteristic features that are closely correlated with the toxicity of the system. This is mainly because of the large surface area-to-volume ratio associated with the size of the nanoparticles. The charge and the surface properties of the nanoparticles are other examples of the properties of the 2.2


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nanoparticles that can lead to harmful interactions with the biological systems, modifying their cellular uptake and pharmacokinetic profile through the GIT [27,28]. Toxicity studies are most of the times neglected considering the fact that most of the materials used to produce the nanoparticles are claimed to be biocompatible and biodegradable. Nonetheless, the nanoparticles composed of biodegradable materials can lead to severe cellular toxicity due to the intracellular changes caused by their accumulation inside the cells. Moreover, the properties of the materials may change completely upon some chemical modifications, and besides the toxicity of the materials, also the products of their degradation are a concern. Furthermore, the reagents used during the nanoparticles production should be as less toxic as possible and should be quantified on the final product in order to be in agreement with the FDA limits [27,29]. For example, long-term use of absorption enhancers and surfactants can lead to the damage of the intestinal epithelium with an increased possibility of promoting the passage of pathogens and toxins through the GIT [30]. This absorption enhancement can also take place as a result of the use of some biomaterials that can induce structural reorganization in the TJs or chelate the calcium inducing the TJs disruption [6,31-33]. Often, the toxicity is only associated with the materials that are part of the nanoparticles but it is well known that the pharmacokinetic properties of a drug or excipient can change considerably when incorporated in a nanoparticulate system [34,35]. 3.

Different nanoparticulate systems

Polymeric nanoparticles With the increasing knowledge about polymers and their properties, polymeric nanoparticles have been extensively used in the drug delivery field. These polymers may have a natural, synthetic or semisynthetic source and they can be used either separately or combined with each other [36]. Among all the polymers, chitosan is the most studied and widely used for oral drug delivery applications. Chitosan is a natural polymer with mucoadhesive properties, produced by the alkaline deacetylation of chitin and composed by N-acetyl-D-glucosamine and D-glucosamine [37-39]. It is considered a nontoxic and biocompatible polymer, and has been approved by the FDA for wound dressing [34]. However, despite all the work developed with chitosan, it is still not approved by the FDA for any product in the drug delivery field, and as a consequence, very few companies are using this material for drug delivery applications [27]. Toxicity tests are still needed to answer some safety concerns in order to include chitosan as an excipient in new drug delivery formulations [34]. As example, one of the concerns is the biodistribution of chitosan after its oral administration that has not been well investigated so far. Chitosan polymers are not absorbed by the GIT and are unlikely to show biodistribution, while chitosan oligosaccharides may be absorbed to some 3.1

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extent [40,41]. Some of the studies showed that chitosan’s absorption in Caco-2 cells and followed oral administration in rats, is dependent on its molecular weight [40]. Small-molecular-weight oligomers (3.8 kDa, 88.4% deacetylation degree [DD]) showed absorption with an increase in their concentration in plasma, unlike the high-molecular-weight chitosan (230 kDa, 84.9% DD) that indicated almost no absorption [40]. Other studies have also reported the absorption of oligomers of chitosan [42,43] and chitosan derivatives such as trimethyl chitosan (TMC) [41] and N-acetylglucosamine [44,45]. In addition to the biodistribution, the molecular weight together with the DD also influences the toxicity of chitosan [46,47]. Several in vitro tests using Caco-2 and HT29 cell lines, as well as ex vivo studies using jejunum from rat have been performed. These studies showed that at high DD, the chitosan toxicity is related to the molecular weight and the concentrations of the polymer; however, at lower DD the toxicity is less marked and is less associated to the molecular weight [48-50]. Arai et al. found that the lethal dose 50% (LD50) of chitosan when orally administrated to mice was 16 g/kg, which is a value comparable with sucrose or household salt [27,51,52]. When orally administrated in humans in a dose up to 4.5 g/ day, no side effects were reported. However, when taken regularly for 12 weeks, some symptoms such as mild nausea and constipation appeared [27,34,53]. Regarding the derivatives of chitosan, Yin et al. showed that TMC conjugated with cysteine in solution presented toxicity when the polymer size was 500 kDa [54]. However, when forming as nanoparticles, they presented lack of toxicity even when the polymer used had a size of 500 kDa [54]. Another study performed by Zheng et al. showed that at high doses, TMC nanoparticles can be slightly toxic causing light diarrhea, which was relieved by discontinuing the administration [41]. Sonaje et al. performed toxicity studies for nanoparticles made of poly-g-glutamic acid and chitosan in the presence of MgSO4 and sodium tripolyphosphate [55]. The nanoparticles were well tolerated after oral administrations for 14 days, lacking toxicity even when the administrated dose was 18 times higher than the dose used for pharmacodynamic/pharmacokinetic studies. Neither significant differences in clinical signs nor in body weight were observed between the experimental and control groups. No relevant pathological changes were observed in kidney, liver and intestine and no inflammatory reactions were also observed. In addition, the toxicity of decanoic acid-grafted oligochitosan nanoparticles has also been assessed in rats [56]. Animals were sacrificed after a few hours after the administration and the histopathology studies showed no significant differences between the experimental and the control groups. The villus structure of the intestinal epithelium was normal with no disruption observed and there was also no significant presence of inflammatory cells. Moreover, nanoparticles of alginatedextran sulfate core complexed with a bilayer of chitosan




and PEG coat, followed by an albumin coat were well tolerated [29] with a viability between 90 and 100% of the rat intestinal tissue after 4 h of incubation with the nanoparticles [57]. Studies after chronic administration (twice daily over 15 days) of these nanoparticles in rats were also performed and there was no signs of inflammatory reactions in tissues like liver, spleen, pancreas, kidney and intestinal sections, and the effects were similar to the animals receiving no treatment [58]. Besides the natural polymers, also the use of synthetic polymers has been a promising strategy toward oral administration of drugs. However, similar to the natural polymers, the toxicity of synthetic polymers has also not been addressed in most of the studies. Poly(lactic-co-glycolic acid) (PLGA) is one of the most used synthetic polymers to produce nanoparticles for oral drug delivery applications, mainly due to its biodegradability and biocompatibility, as well as sustained drug-release profiles. Some in vitro studies performed using the intestinal cell lines Caco-2 and HT29 showed that PLGA nanoparticles presented no toxicity either alone or coated with chitosan [12,59]. Moreover, Semete et al. evaluated the coating of PLGA nanoparticles with chitosan and PEG [60]. In this study, the cytokines expression profiles were assessed and it was shown that the amounts of proinflammatory cytokines IL-2, IL-6, IL-12p70 and TNF-a in the plasma and in the peritoneal lavages were maintained at low concentrations even after 24 h of oral administration [60]. The toxicity of PLGA nanoparticles using a quaternary ammonium salt didodecyl dimethylammonium bromide after oral administration was also evaluated [61]. Through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, it was possible to observe that the formulation was safe in the cell cultures at concentrations < 33 µM. When orally administered to rats, the nanoparticles were as effective as the administration of the drug by the intravenous route at 50% lower dose [61]. When compared to other materials such as zinc oxide, PLGA nanoparticles had higher viability (75%) in vitro. In vivo, the biodistribution of the PLGA nanoparticles was analyzed 7 days after oral administration and it was possible to detect the presence of the fluorescence in the brain, heart, lungs and spleen with highest amounts in the liver (40.04%) and in the kidney (25.97%). No damages were observed in the histopathological evaluations. Thus, the administration of PLGA nanoparticles was not related with the toxic effects observed with various industrial nanoparticles [62]. However, there is a possibility that the particles detected in the different organs were just the free fluorescent molecule that was detached from the PLGA nanoparticles. A study made by Jain et al. showed that tamoxifen when encapsulated within PLGA nanoparticles showed significant reduction in the hepatoxicity in comparison with tamoxifen in solution [2]. The liver section of rats treated with PLGA tamoxifen nanoparticles presented normal histopathological appearance in contrast with the liver sections of rats administrated with tamoxifen in solution that presented edema and

swelling of hepatocytes, necrosis, hyperplasia of Kupffer cells and apoptosis [2]. Dendrimers is another kind of system that has been extensively used for drug administration and only in some extent for oral drug delivery. Dendrimers are nanoparticles that exhibit a micelle-like structure, that is, a hydrophobic core and a hydrophilic periphery, constituted by polymeric branches [63]. One of the most used polymers in dendrimers is the poly(amidoamine) [64]. Thiagarajan et al. studied the effect of the dendrimers charge, length and termination on their toxicity. They reported that the cationic dendrimers are more toxic than the anionic ones that were tolerated at 10 times higher doses. Moreover, larger dendrimers were shown to be more toxic, causing hemobilia and splenomegaly, in comparison with the smaller ones. It was also shown that masking cationic residues with noncharged groups improved their safety and uptake by the epithelial cells [64,65]. Lipid-based nanoparticles Similar to the polymeric nanoparticles, the characteristics of the lipid nanoparticles also have a huge influence in their toxicity. More than the toxicity of the lipids used, the surfactants, co-surfactants and co-solvents used to develop these systems may raise some toxic concerns [66]. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLC) are the most common examples of lipid nanoparticles used in the oral administration of drugs. Several lipids (Imwitor 900K, Imwitor 491, Dynasan 118 and Dynasan 116, Softisan 142, Witepsol E85, Compritol 888 ATO, Lipocire, Cetyl Palmitate, Precirol 5 ATO, Gelucire 39/01 and Gelucire 43/01 and Gelucire 50/13) have been used to produce SLNs. All of the SLNs constituted of the different lipids showed 90% cell viability in Caco-2 cells [67]. Also, SLNs composed of tristearin lipids were well tolerated by the macrophages isolated from the rat peritoneum with viability above 90% [68]. In vivo toxicology of SLNs was studied by Cho et al. that showed that 8 h after oral administration of SLNs there was no evidence of damage of the intestinal epithelium such as villi fusion, occasional epithelial cell shedding, and congestion of the mucosal capillary with blood and focal trauma [69]. SLNs are also able to protect from the toxicity caused by some drugs. One example is the Tripterygium (TWHF), an anti-inflammatory and immunosuppressive drug, with reported GIT toxicity [70]. In vivo assays in rats with arthritis showed that TWHF-SLN can significantly reduce rat paw volume at 60 mg/kg with no significant elevation of the liver enzymes. Histopathology observations found that free TWHF caused more serious damage to the liver than TWHF-SLN [70]. Similarly, the intestinal toxicity of tripterine formulated in the NLCs has also been investigated. According to the result, the tripterine-loaded NLCs could greatly decrease the cytotoxicity of the drug when compared to tripterine solution [71]. Moreover, the use of self-emulsifying drug delivery systems, liposomes and micelles as oral drug carriers has been very 3.2


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limited. It is mainly used for poorly water-soluble drugs, especially highly lipophilic ones, to enhance their bioavailability [66]. However, due to the characteristics of the GIT, such as the degradation of the lipid contents by the bile salts, they are not considered as a promising choice for oral administration of drugs [72]. Lv et al. used a self-double-emulsifying drug delivery system (SDEDDS) to improve the absorption of hydroxysafflor yellow A. When in vitro tests in Caco-2 cells were performed, no toxicity was shown. However, the histopathologic studies of the rat intestine showed that the villi were compromised presenting shortened height with their top lost. This means that SDEDDS can cause mucosal damage to a certain degree of toxicity [73]. Another study conducted by Buyukozturk et al., showed how important the formulation design is for the safety of the emulsion-based formulations. The main aspects that need to be considered to develop safe systems are the oil structure, surfactant hydrophilic--lipophilic balance (HLB) values and surfactant-to-oil ratio [74]. When some of these parameters are unbalanced, toxic effects may occur. For instance, Tween 80 is a surfactant with high HLB that may loosen the TJs of the epithelial intestinal cells [74]. Therefore, each and every component must be crucially selected and tested to achieve safe formulations. Carbon nanomaterials The carbon nanomaterials that are used for drug delivery applications are namely carbon nanotubes (CNT), carbon nanohorns (CNH) and graphene oxide (GO). CNT belongs to the family of fullerenes. CNT are cylindrical structures formed by rolling of single layer (single-walled CNT) or multiple layers (multiple-walled CNT) of graphene sheets [3]. The cylindrical structures are capped at the ends by carbon networks. Since its discovery in 1991 [75], CNTs have been greatly explored as drug nanocarriers due to their advantageous properties such as high surface area, conductivity, high tensile strength and potential higher absorption capabilities [76]. The presence of hollow monolithic structure of CNT allows the incorporation of drug molecules for controlled and site-specific delivery [77]. Moreover, the outer surface of CNTs can be functionalized to enhance their biocompatibility and biodegradability [76]. There have been several toxicological studies after oral administration of CNTs. However, the results from the different studies are contradictory to each other with some studies showing acute toxicity and genotoxicity with CNTs, and other studies showing no toxic influence of the CNTs. Table 1 summarizes some of the examples reported in the literature of the toxicological studies of CNTs after oral administration. Another type of carbon nanomaterials are CNHs, which are single-walled carbon nanomaterials made of graphene sheets (2 -- 3 nm of diameter) forming cone-shaped hornlike structures. CNHs are usually present as aggregates forming spherical particles of diameter of 80 -- 100 nm [76,78]. 3.3

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Miyawaki et al. observed that after the peroral administration of single-walled CNH with a dose of 2000 mg/kg to healthy rats, no signs of abnormalities were observed with normal body weight gain [79]. Also, all the rats were able to survive the 2-week test period. Regarding the immunological reactions of CNTs, it was previously shown that CNT had some effects; however, it was later observed that the effects were seen merely due to the metallic impurities and contaminants present in the CNTs [80]. CNHs, on the other hand, are prepared in the absence of metal catalyst, thereby having high purity with minimal possibility of toxic effects [79]. GO is a modified form of graphene, produced by the harsh oxidation of crystalline graphite, which results in single atom thick carbon sheets with pH-dependent properties [81]. There is an increasing number of studies that explores GO as drug delivery systems, and recently GO has also been investigated as an oral delivery platform for quercetin by Rahmanian et al. [82]. The oral toxicity of GO was studied by Yang et al., where they administered GO and PEG to mice at a dose of 4 mg/kg. After 4 h of oral administration, large quantities of both unmodified and modified GO were detected in the stomach and intestine, and not in other organs. After day 1 of administration, low amounts of GO were detected in other major organs, but after 1 week no traceable amounts were detected. In addition, histological studies were also performed by administering a dose of 100 mg/kg to mice and analyzing after 30 days. This study showed that the PEGylated GO was not adsorbed to the main organs. This showed that GO is not toxic at the tested doses, which can be attributed to its poor adsorption in the GIT. However, more studies with high GO doses and long-time exposure must be performed to better assess the toxicity of GO in biological barriers [83]. Silica and silicon nanoparticles Silica (silicon dioxide) and silicon-based nanoparticulate systems are one of the emerging drug delivery systems for different routes [84,85]. The biocompatibility, ease of fabrication, chemically inert properties and possibility to tailor the physicochemical properties make these nanoparticulate systems an optimal alternative for drug delivery applications. The drugs can either be loaded into the pores of the mesoporous silica/ silicon or they can be physically adsorbed on the surface of nonporous silica nanoparticles [86,87]. Moreover, compared to carbon nanomaterials, silicon-based nanomaterials are biodegradable owing to weak Si--Si and Si--O bond compared to C--C bond, making it more biocompatible and a more optimal option for drug carriers [88]. Although a lot of effort has been made to use these materials for developing oral formulations for different drugs, there is still little known about the toxic effects of these particles when administered via the oral route. 3.4

Nonporous silica nanoparticles The major prevalence of nonporous silica is as food and pharmaceutical additive; however, few studies have been 3.4.1



Table 1. Cytotoxic and genotoxicity studies of CNT. CNT

Type of study


COOH- SWCNT In vitro cellular study in Caco-2 cells (24 h exposure study)

COOH-SWCNT

In vitro cellular study

SWCNT

In vivo study in Fisher 344 rats (single dose genotoxicity study)

Ultra-short and full length SWCNT

In vivo study in Swiss mice (single bolus dose)

SWCNT

In vitro and in vivo genotoxicity assays

Characteristics of CNT

Dosing

Individual SWCNT: 1.4 ± 0.1 nm Bundle dimensions = 4 -- 5 nm 0.5 -- 1.5 µm

5 -- 1000 µg/ml

Results of the study

Cell viability reduced to 20% at concentration above 500 µg/ml Inhibitory response of neutral red uptake and tetrazolium salt metabolization at concentration > 100 µg/ml Differentiated Caco-2 showed higher sensitivity to cytotoxicity Diameter: 1.58 50 -- 150 µg/ml Genotoxic effect observed at all doses ± 0.20 nm DNA damage, cytotoxicity, reactive Length: 0.76 oxygen species production and heat ± 0.70 µm shock protein 70 induction observed Individual size 0.064 and 0.64 mg/kg Elevated levels of 8-oxo-7,8-dihydro-2’= 0.9 -- 1.7 nm body weight deoxyguanosine in lungs and liver SWCNT generated oxidative damaged DNA in liver and lung cells by a gastrointestinal route Diameter = 1 nm 1000 mg/kg of body No animal death and abnormalities Length ranging from weight observed 20 nm to 2 µm No acute oral toxicity observed regardless of the length, surface area and surface interactions Diameter: 3.0 ± 60 and 200 mg/kg The high purity and well-dispersed 1.1 nm sample of SWCNTs showed no Length: < 1.2 µm genotoxic effects in both in vitro and in vivo experiments

Ref.

[111]

[112]

[113]

[114]

[115]

CNT: Carbon nanotube; SWCNT: Single-walled carbon nanotube.

performed to evaluate its potential as drug nanocarriers [89,90]. van der Zande et al. studied the subchronic toxicity of two silica nanoparticles, synthetic amorphous silica (SAS) and nanostructured silica (NM-202), after daily oral dosage. SAS of size of 7 nm was administered in doses of 100, 1000 or 2500 mg/kg of body weight, and NM 202 of size of 15 -- 25 nm was administered in doses of 100, 500 and 1000 or 2500 mg/kg of body weight for 28 days and the highest doses were continued until 84 days [91]. At the end of 24 days, no significant rise in the silica levels were observed in different tissues; however, after 84 days the silica accumulation was observed in spleen with SAS nanoparticles. Also, for NM-202 nanoparticles after 84 days of exposure showed increased incidences of liver fibrosis with significant increase in the fibrosis-related genes in liver samples [91]. These studies did not show many toxic effects of these nanoparticles at such high doses, but more studies to evaluate the reason for accumulation in spleen and liver fibrosis are still needed. Contradictory to the results from Zande et al. [91] a recent research article from Hassankhani et al. showed that silica nanoparticles have toxic effects in different tissues such as liver, lung, kidney and testis after oral administration [92]. In this study, silica nanoparticles with diameters of 10 -- 15 nm were given as oral gavage at doses of 333 mg/kg/day for 5 days. One mouse was dead at the end of the test periods with the others showing symptoms of vomiting, loss of

appetite and severe lethargy. Moreover, changes in albumin, cholesterol, triglyceride, total protein, high-density lipoprotein and low-density lipoproteins were observed, indicating the severe toxic effects of silica nanoparticles just after 5 days of exposure [92]. Mesoporous silica nanoparticles Since its first use in 2001, mesoporous silica nanoparticles (MSNs) have shown a great potential to be developed as smart multifunctional drug delivery systems [93]. In a study by Fu et al., the absorption, distribution, excretion and toxicity of MSNs in mice were studied [94]. After the oral administration of MSNs at different doses 50, 500 and 5000 mg/kg of body weight, no deaths of the mice were observed after 24 h of incubation without any clinical signs of loss of appetite, weight loss and passive behavior. It was observed that after 24 h of administration, the MSNs were absorbed through the portal vein ending up in the liver. The major excretion route for MSNs was the feces where > 80% of the initial dose of MSNs was found. In conclusion, it was showed that the oral route is a safe route of MSN delivery due to its good tissue biocompatibility [94]. 3.4.2

Mesoporous silicon nanoparticles Mesoporous silicon (PSi) has been successfully used as nanocarriers for both small molecules and macromolecules. 3.4.3


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The surface of PSi can be easily modified to make it hydrophilic or hydrophobic, or can be modified by chemically conjugating to biopolymers such as chitosan [87,95,96]. There are a number of studies that have studied the cytotoxic effect of PSi on intestinal cell lines such as enterocytes like Caco-2 and mucus-producing goblet cells HT29 [12,97,98]. In these studies, it has been investigated the different surface chemistries of the PSi such as thermally oxidized, thermally hydrocarbonized and undecylenic acid-modified particles ranging from 150 to 200 nm. It was observed that thermally oxidized PSi was the least toxic to the cells, which maintained the cell viability up to 80% even after 24 h exposure to both Caco-2 and HT29 cell lines. However, thermally hydrocarbonized PSi nanoparticles showed slight cytotoxicity at particle concentrations > 100 µg/ml [97]. In the case of undecylenic acidmodified PSi nanoparticles, the cellular viability remained above 80% for both time periods and both cell lines for all concentration, except after incubation with Caco-2 cells for 12 h at all concentrations tested. However, a significant improvement in the cell viability was observed for both cell lines and both incubation time periods after chitosan modification of the undecylenic acid-modified PSi nanoparticles [12]. These studies showed that the cytotoxic effect of the PSi nanoparticles is highly dependent on the surface properties of the particles. In a similar study, Bimbo et al. observed that the thermally hydrocarbonized PSi nanoparticles did not have a significant effect on the cytotoxicity of Caco-2 cells at concentrations up to 250 µg/ml [99]. It was also showed that the in vitro cytotoxicity of Caco-2 cells was independent of the size in the range of 97 -- 188 nm. Furthermore, the in vivo biodistribution studies of 18F-labeled thermally hydrocarbonized mesoporous nanoparticles after oral delivery showed that the nanoparticles remained in the GIT, which was proven by the negligible amount of radioactivity detected in systemic circulation [99]. Metallic nanoparticles Metal nanoparticles include different systems such as gold nanoparticles (AuNPs) and supramagnetic metal oxides (iron oxides: Fe2O3 or Fe3O4). Besides their application as drug delivery systems, they are also widely used for imaging [100]. 3.5

Gold nanoparticles Among these metallic nanoparticles, AuNPs are the most widely used systems, mainly because of ease of preparation with desired shape, size and surface functionalities. However, the use of these nanoparticles as oral delivery systems is very limited with limited amount of toxicological studies. In 2001, Hillyer et al. studied the fate of the AuNPs with different sizes (4, 10, 28 and 58 nm) after oral ingestion in mice. They showed that 4 nm AuNPs were able to cross the GIT more readily with accumulation in kidney, liver, spleen, lungs and brain in significantly higher amounts compared to other sizes [101]. 3.5.1

8

Pokharkar et al. investigated the acute and subacute oral toxicity of chitosan reduced AuNPs in rats for 28 days [102]. No subacute toxicities such as changes in clinical signs, body weight, food consumption, hematological parameters, organ weights and histopathological changes were observed. Moreover, they found out that the median lethal dose was > 2000 mg/kg. Zhang et al. compared the toxicity of different doses (137.5 -- 2200 µg/kg) of AuNPs with a diameter of 13.5 nm after oral delivery [103]. They found that at lower doses, the nanoparticles showed no appreciable toxicity with any significant decrease in the body weight. However, at higher doses, a decrease in the red blood cells (RBCs) count was observed with increased accumulation in spleen at the end of 28 days. Thus, factors such as size, surface coating and the dose of AuNPs must be carefully considered when developing nanoformulations for oral delivery. Supramagnetic metal oxides Supramagnetic metal oxides nanoparticles are extensively used as contrast agents for MRI; however, they can also be used in developing magnetic field responsive drug delivery systems [104]. Kumari et al. studied the size, dose and timedependent acute oral toxicity of iron oxide (30 nm; Fe2O330) and iron oxide bulk (Fe2O3) on female Wistar rat model [105]. The bulk Fe2O3 did not show any significant changes in the biochemical markers. However, Fe2O330 showed inhibition of acetylcholinesterase in RBCs and brain, along with the activation of hepatotoxic marker enzymes (e.g., aspartate and alanine aminotransferase) in liver and serum. Thus, this indicates that the oral exposure to nanosized Fe2O3 can lead to adverse effects in the biochemical profile. Similar toxicity studies with Fe2O3-30 and Fe2O3 have been carried out by Singh et al., where they evaluated the genotoxicity of these iron oxide nanoparticles after oral exposure to female Wistar rats at doses of 500, 1000 and 2000 mg/kg [106]. Fe2O3-30 showed a size- and dose-dependent biodistribution in different organs and tissues, but the accumulated nanoparticles did not show any significant genotoxicity. Overall, the number of studies regarding the toxicity of these nanoparticles is very limited. Moreover, the toxicity studies are not extensive enough lacking chronic exposure studies or lethal dose studies. Most of the studies are only focused on the biodistribution of the nanoparticles and the interaction of these nanoparticles with the tissues is also missing. Therefore, there is a need of extensive studies with these nanoparticles to have a better picture of their safety profile in biological barriers. 3.5.2

Protein nanoparticles Protein-based systems are composed of a protein or combination of proteins that self-assemble to generate different structures such as cages, microspheres, nanoparticles, hydrogels, films, minirods and minifilms [107]. A number of proteins such as transferrin, whey, b-caesin and b-lactoglobulin have been used to prepare these protein-based systems for oral 3.6




drug delivery. The biocompatibility and biodegradability along with low toxicity make them ideal for drug delivery [108,109]. Recently, Golla et al., developed protein nanoparticles composed of apotransferrin and lactoferrin for the oral delivery of the anticancer drug doxorubicin [110]. The study showed decreased liver and heart toxicity of doxorubicin when delivered using transferrin nanoparticles. Although these drug delivery systems are considered safe due to the biocompatible of the proteins and polypeptides used, detailed both in vivo and in vitro toxicity studies must be performed for multiple chronic therapies to completely ensure the safety of these nanosystems. 4.

Conclusion

Nanotechnology has emerged as a boon in the field of drug delivery and diagnostics. Despite of a large number of advantages and possible applications of the nanoparticles, the number of marketed products based on nanoparticulate systems is minimal. One possible reason for this could be the lack of toxicity and safety information related to these nanosystems that are needed to surpass the regulatory requirements. Although the oral route is considered the safest administration route, the ability of the nanoparticles to overcome the intestinal barrier to reach the systemic circulation poses another threat to its safety profile. The understanding of the fate of the nanoparticles in the biological systems and the interaction between them is negligible and this is mainly due to the minimal effort put into this field of study. Different nanosystems based on different materials are unique and each system requires a case-specific study. A basic and conceptual understanding of the interactions of the nanosystems with the biological systems is needed in order to have safe and effective nanosystems for improved drug delivery applications. Overall, the information regarding the toxicological evaluation of the inorganic nanoparticles is still very limited, which makes it difficult to draw any conclusions regarding the safety of these nanoparticles for oral drug delivery. 5.

Expert opinion

Nanoparticulate technology has been greatly explored for drug delivery applications and this field has been undergoing a tremendous development in the past decades. The nanoparticulate systems are developed aiming at solving the limitations and drawbacks of conventional dosage forms. The same idea is applied in the case of nanoparticles for oral drug delivery systems, where the aim is to overcome the limitations of the drug solubility, stability and permeability across biological barriers. Despite of the rigorous amount of research involving oral nanoparticulate systems, the amount of toxicity and safety assessments of these nanosystems is still minimal, which is the biggest hurdle to develop a safe and efficacious drug delivery system.

There is a strong need for understanding the potential toxicities of such nanomaterials, which would give us crucial information to develop safer and more efficient nanoformulations. It must be kept in mind that the safety profiles of the materials used in the preparation of the nanosystems cannot be directly translated to the final nanoparticles. Moreover, the understanding of how each property (such as size, charge, surface chemistries) of the nanoparticles system influences the toxicity must be studied. Each system must be thoroughly investigated to establish detailed toxicity safety profiles, which will help in fulfilling the stringent requirements by the regulatory authorities and will give faster acceptance. The limited amount of information available on safety and toxicity of nanoparticles can be overcome by establishing a strong collaboration between formulation scientists and toxicologists. This unification of ideas and expertise between different fields can help in developing standardized toxicity studies. A more systematic toxicological study should also be developed, initiating with suitable in vitro cell-based toxicity studies such as cell viability and interaction studies followed by in vivo toxicity studies in both normal animal models and diseased animal models. Moreover, efforts toward incorporation of these toxicity studies from the beginning of the formulation development stages can be beneficial in developing safer formulations in the future.

Acknowledgement F Ara ujo and N Shrestha contributed equally to this work.

Declaration of interest This work was financed by European Regional Development Fund (ERDF) through the Programa Operacional Factores de Competitividade -- COMPETE, by Portuguese funds through FCT -- Fundaçaõ para a Cieˆncia e a Tecnologia in the framework of the project PEst-C/SAU/LA0002/2011, and co-financed by North Portugal Regional Operational Programme (ON.2 -- O Novo Norte) in the framework of project SAESCTN-PIIC&DT/2011, under the National Strategic Reference Framework (NSRF). F Ara ujo would like to thank to Fundaçaõ para a Cieˆncia e a Tecnologia (FCT) for financial support (SFRH/BD/87016/2012). HA Santos acknowledges financial support from the Academy of Finland (decision nos. 252215 and 256394), the University of Helsinki Funds, Biocentrum Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007--2013) grant no. 310892. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript other than those disclosed.


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large doses of ultrashort and full-length single-walled carbon nanotubes after oral and intraperitoneal administration to swiss mice. ACS Nano 2010;4:1481-92 115. Naya M, Kobayashi N, Mizuno K, et al. Evaluation of the genotoxic potential of single-wall carbon nanotubes by using a battery of in vitro and in vivo genotoxicity assays. Toxicol Lett 2011;205:S286-6

Affiliation Francisca Ara ujo1,2,3, Neha Shrestha3, Pedro L Granja1,2, Jouni Hirvonen3, Helder A Santos3 & Bruno Sarmento†1,4 † Author for correspondence 1 Universidade do Porto, INEB -- Instituto de Engenharia Biomedica, Biocarrier Group, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal E-mail: [email protected] 2 University of Porto, ICBAS -- Instituto Cieˆncias Biomedicas Abel Salazar, Porto, Portugal 3 University of Helsinki, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, FI-00014 Helsinki, Finland 4 IINFACTS -- Instituto de Investigaçaõ e Formaçaõ Avançada em Cieˆncias e Tecnologias da Sa ude, Instituto Superior de Cieˆncias da Sa udeNorte, Department of Pharmaceutical Sciences, CESPU, Rua Central de Gandra, 1317, 4585-116 Gandra, Portugal

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Uptake carriers and oncology drug safety.

Ceramic nanoparticles: Recompense, cellular uptake and toxicity concerns.

Orally delivered cocaine as a reinforcer for rhesus monkeys.

Synthesis of β-cyclodextrin modified chitosan-poly(acrylic acid) nanoparticles and use as drug carriers.

Aptamers as both drugs and drug-carriers.

Ultra-small BaGdF5-based upconversion nanoparticles as drug carriers and multimodal imaging probes.

Therapeutic implications of orally delivered immunomodulatory oligonucleotides.

Inhalable chitosan nanoparticles as antitubercular drug carriers for an effective treatment of tuberculosis.

131I-Traced PLGA-Lipid Nanoparticles as Drug Delivery Carriers for the Targeted Chemotherapeutic Treatment of Melanoma.

Optimizing superparamagnetic iron oxide nanoparticles as drug carriers using an in vitro blood-brain barrier model.

Doxorubicin-conjugated core-shell magnetite nanoparticles as dual-targeting carriers for anticancer drug delivery.

Silver nanoparticles: therapeutical uses, toxicity, and safety issues.

Preparation of solid lipid nanoparticles as drug carriers for levothyroxine sodium with in vitro drug delivery kinetic characterization.

Charge-reversal nanoparticles: novel targeted drug delivery carriers.

Microparticulates as drug carriers for pediatric use.

Toxicity of colloidal silica nanoparticles administered orally for 90 days in rats.

Effects of feeding condition on orally delivered ethanol as a reinforcer for rhesus monkeys.

Dual role of nanoparticles as drug carrier and drug.

Polymeric micelles as drug carriers: their lights and shadows.

Polymeric nanoparticles based on chitooligosaccharide as drug carriers for co-delivery of all-trans-retinoic acid and paclitaxel.

Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides.

Poly(lactic acid) microspheres as immunological adjuvants for orally delivered cholera toxin b subunit.

Therapeutic Efficacy of Orally Delivered Doxorubicin Nanoparticles in Rat Tongue Cancer Induced by 4-Nitroquinoline 1-Oxide.

Mesoporous manganese silicate coated silica nanoparticles as multi-stimuli-responsive T1-MRI contrast agents and drug delivery carriers.